:OJ :ru - Bacteria in Relation to Plant Diseases BY ERWIN F. SMITH In charge of Laboratory of Plant Pathology, Bureau of Plant Industry, U. S. Department of Agriculture VOLUME THREE VASCULAR DISEASES (CONTINUED) )t4, photographed <>ct. 30 bv transmitted light. The organism grew slowly (temperature about 23° C). COBB'S DISEASE OF SUGAR-CANE. 21 ETIOLOGY. This disease is caused by Bacterium vascularum (Cobb) Greig Smith, a honey-yellow, one-flagellate organism, which forms the yellow slime always present in the vessels of diseased plants. It is a short rod occurring singly, in pairs, fours, or eights (end to end), and it often exists in practically pure culture in the fibro-vascular bundles of the diseased sugar-cane. The constant presence of this particular yellow organism in the diseased cane goes a long way toward establishing a causal relation, or would if it could be shown that nothing else is constantly or usually present. The writer knows, however, from personal observation, that other bacteria are sometimes present in the vessels, and Dr. Cobb's paper leads one to suppose that "traces" of other organisms are usually present in some part of each diseased cane. There are many parts of the cane, however, where Bad. vascularum exists in pure culture (Cobb, EFS.), and the fact that no other organism is necessary to the production of the disease has been proved conclusively by the writer's successful pure- culture inoculations. In October 1902, the writer received from the Richmond River district, Australia, through the courtesy of the Commissioner of Agriculture of New South Wales, three diseased canes, from two of which he had no difficulty in obtaining pure cultures of the organism by means of Petri-dish poured plates. No attempt was made to get cultures from the most badly decayed of these canes, because in places the bacterial ooze was nearly white, indi- cating an extensive mixture of organisms in the stem.* Nine Petri-dish poured plates were made from the interior of each of the other canes. One set of plates gave in addition to Cobb's organism a mixture of other things, mostly white bacteria of several sorts, but also colored colonies and molds; the other set of plates yielded practically pure cultures of Cobb's organism (fig. 14). Cuttings from healthy canes were obtained in November 1902 from southern Georgia and were planted in a hot-house in Washington. Before planting, the canes were shortened at each end and inspected critically by the writer. All were free from Cobb's disease and otherwise sound. Shoots from these canes were inoculated early in February 1903, with slime from potato subcultures of the yellow organism derived from the Australian cane. These inoculations succeeded beyond expectation, the canes developing local signs in a few days. Stripes extended up and down from the pricked part of the leaves and in one variety constitutional signs of a pronounced and typical sort appeared a few weeks later, the same being preceded by the enormous multiplication of the honey-yellow bacterium, first in the bundles of the inoculated leaves and later in those of the main axis of the cane and those of the uninoculated leaves. From the interior of the stem of several of these inoculated plants the organism was again obtained in pure culture. Inoculations made with sub- cultures of the organism thus isolated from the artificially infected cane were likewise successful, and the bacterium was again isolated from the diseased cane in pure cultures. INOCULATIONS. SERIES I, 1903. The first set of inoculations (12 in all) was made on February 6, into common green cane. The inoculations were made by means of needle-pricks on the leaf blades about 1 to 2 feet from the stem. Two leaves were inoculated on each plant, those selected being about the tenth or eleventh from the base of the plant. About 20 to 30 fine pricks were *Respecting these canes Dr. Cobb makes the following statement in his Third Report: "There can not be the slightest doubt as to the identity of the organism cultivated by Dr. Smith and inoculated by him into American canes. * * * The second shipment was attended to personally by myself after it had been forwarded to me by my friend Mr. A. C. Barry, manager of the Broadwater sugar-mill, Richmond River, who at my request selected the canes and forwarded the same to Sydney. They were there unpacked, and sealed up with sealing wax and repacked at my laboratory. I examined the gum. The organism was the same species as that described in 1893 and reported on subsequently as the cause of the newly discovered disease for which I proposed the name " Gumming of the sugar-cane." 22 BACTERIA IN RELATION TO PLANT DISEASES. made on each leaf, some in the midrib, but the majority in the tissue on either side of it. The pricked area covered about 4 sq. cm. Three subcultures from as many separate yellow colonies on the plates poured from the Australian cane were used. Many leaves were pricked as checks, but these never developed any disease. Until nightfall the inoculated leaves were protected from the light by paper, but the air was not shut off. A semitropical temperature was maintained in the greenhouse and the plants grew rapidly, an important factor. Three weeks after the bacteria were introduced by the needle-pricks the inoculated leaves showed white stripes, in which reddish or brown dead spots and stripes soon appeared. These stripes had their beginning in the inoculated part of the leaf and slowly spread upward and downward. For some weeks there were no further signs of infection; then white stripes appeared on other leaves (uninoculated ones on the same shoots) and, somewhat later, the red or brown stripes, with shriveling of the leaf-parenchyma. At this time the plants were considerably dwarfed, and this nanism finally became very marked (fig. 15). The condition of these plants at the end of two months (April 3) was as follows: (1) The leaf 1 b is dead from the punctures down to the stem on each side of the midrib. The distance from the pricked area to the top of the sheath is 19 inches. The length of the sheath itself is about 1 foot. The leaf is dried out on the margins and brown above the pricked area for a distance of 2.5 feet. The terminal one foot of the leaf is bright green, and the midrib is green throughout its whole length. The signs on leaf / a, which is about the same length as the other, are the same, namely, dry brown margins about 0.25 inch or more in width extending down to the sheath and upward to within a foot of the tip. The only marked difference in the upper end is that the extreme tip has lost its chlorophyl and a portion of the leaf inside of the dry brown margins is also yellow. The midrib is green throughout. The yellow bacterial gum is oozing out of the green midrib about 9 inches above the top of the sheath, i. e., from the pricked portion. Two or three little, bright yellow gummy masses are present on the inner face of the leaf at this point. This leaf was severed from the stem 3 inches above the sheath. Photograph made. The stem of the plant 1.5 feet from the ground is about an inch in diameter. It has 17 leaves and the height of the plant is about 7 feet. The secondary signs of the disease are conspicuous on most of the upper leaves. They have ap- peared as white or yellowish-white longitudinal stripes, which in some cases have already be- come reddish-brown, not unlike the signs I obtained some years ago by inoculating Bad. hyacinthi into amaryllis leaves. They extend in many cases from the base of the leaf-blade, in some instances half-way up, and in others entirely to the apex. This striping shows on five of the upper leaves. One of the most conspicuously striped leaves was cut off and photographed (fig. 16). (2) This plant is about 6 feet high and bears 13 leaves. The check-leaf is normal; that is, only the tissues in the immediate vicinity of the punctures have turned white. The tissue in the pricked area as a whole is bright green and normal in appearance. This is the plant from which a pricked (inoculated) leaf was cut off some weeks ago and found to contain the organism. That leaf (the foot long stub) now has dry-shriveled nearly to the sheath. This is 2 a. The other inoculated leaf (2 b) is dried out and has become brown on the margins from the pricked area down to the sheath and up to within 1.5 feet of the tip on one side, and entirely to the Fig. 15. 'Pig. 15. — Row of inoculated badly dwarfed sugar-cane (at X) with a row of taller, sound, uninoculated cane of same age to either side. Hot-house, Washington, D. C, 1903. Drawn from a broken negative. COBB S DISEASE OF SUGAR-CANE. 23 tip on the other side. The midrib is bright green the whole length. The needle-punctures were made about a foot from the leaf-sheath. Secondary signs are visible on three of the upper leaves of this plant as long yellowish stripes, interrupted in one of the leaves. In places on one leaf the stripe is dead and reddish-brown for a distance of 8 inches and a breadth of one-sixteenth to one-eighth inch. On another there is a marginal dead stripe 2 feet long. (3) Leaf 3 a is dead entirely from the tip down for a distance of 3 feet, and the rest of the way it is dead on the margins, but the midrib is green. It was pricked about 18 inches from the top of the leaf-sheath. The leaf-sheath itself is about a foot long. Leaf 3 J is dead at the tip and down for a distance of 3 feet and the midrib is dead a foot farther down, but the margins are green. In the lowest foot both midrib and margins seem to be normal. There are no secondary signs on this plant, which is 6 feet tall and bears 13 leaves. (4) Leaf 4 a is dead from the apex down a distance of about 3 feet, while for an additional 2 feet it is dead on the margins but green in the midrib. Punctures made about 9 inches from leaf-sheath. Leaf-sheath is about 9 inches long. Leaf 4 b is dead from the apex down a distance of about 4 feet. A foot farther down it joins the stem. The lowest foot of the leaf is dead on both margins but green in the midrib. The plant itself is about 5 feet high and shows conspicuous secondary signs; 18 inches from the ground the diameter of the stem, including the leaf-sheaths, is about half an inch. It bears 12 leaves. Two of the upper leaves are dead almost all the way down and two more show brown striping on one side. The upper two are flabby. Plant brought in and photographed. It is the larger of the two of this date. Bad. vascularum was plated from the stem of this plant. A white gas-forming organism was also obtained from two places in this stem, 8 inches apart. (5) This plant is about 4 feet high and is rather small. Diameter of the stem a foot from the ground is a little more than 0.25 inch. Both inoculated leaves are dried up throughout. The plant bears 10 leaves, not counting some small basal ones which are dried out. It shows distinct secondary signs, which consist of whitening and striping of the leaves. (6) Plant 6 is about 6 feet high. At a foot from the ground the diameter of the stem is nearly an inch. It bears 14 leaves. Inoculated leaf -blade 6 a, which is about 5 feet long, has dried out throughout, except about a foot at the extreme base, and even here there are brown spots on the margins running down to the leaf-sheath. The midrib, how- ever, is bright green. Leaf-blade 6 b is dead throughout, except about 9 inches of the mid- rib at the base. The sheath itself is also drying out. This plant shows also conspicuous white, yellow, and brown striping on 3 of the upper leaves (secondary signs). (7) Plant 7 is about 6 feet high. A foot from the ground the stem is 0.75 inch in diameter. It bears 13 leaves. Leaf 7 a, which is about 5 feet long (or 6 feet if the leaf-sheath be included), is dead throughout, including everything except the base of the leaf-sheath. Leaf-blade 7 b, which is like 7 a in size, is also dead throughout, including most of the leaf-sheath. There is distinct etiolation of the margins of two of the uninoculated upper leaves, and there are also red lines and specks in these etiolated parts for a distance of a foot in one leaf near the base and a less distance on the other. (8) The diameter of the stem of this plant a foot from the ground is about 0.625 inch. It bears 13 leaves. The check-leaf pricked on March 2 with more than 100 needle punctures at 6 inches from the stem, is bright green in the pricked area, the only dead parts being the tissue immediately around the punctures, which is white. Leaf 8 a, which is about 4 feet long (5 feet with sheath), is dead throughout, except extreme base of sheath. Leaf-blade 8 b is dead for a distance of 4 feet down. The basal 1 foot is dead on the margin but green in the midrib. There are distinct signs of secondary infection on the upper leaves which are Fig. 16.* *Fig. 16. — Middle part of one of the uninoculated higher leaves of the common green cane, showing etiolation due to secondary infection with Bacterium vascularum: Margins green and normal in appearance, midrib green. To either side of the midrib are white stripes with red-brown dead spots and stripes in them. The white striping continued to the apex of the leaf. In this stage of striping bacteria are usually present in some of the bundles of the affected part of the leaf. Drawn from a broken negative. 24 BACTERIA IN RELATION TO PLANT DISEASES. whitening in stripes along the margin. Four leaves show these signs and a fifth shows median white striping with dead brown patches toward the base, in the stripes. (9) Plant 9 is about 4 feet high and about half an inch in diameter at 18 inches above the ground. It bears 14 leaves. Leaf g a, which is about 5 feet long, is dead throughout, except possibly the extreme base of the sheath. Leaf 9 b, which is about 5 feet long, is dead throughout, except a small portion of the basal part of the midrib and a part of the sheath. The plant shows distinct secondary signs. One entire upper leaf, which is about 6 feet long, is dead throughout. Another large leaf is drying out on one margin for a distance of 3 or 4 feet and the apex is flabby. A third leaf is dry from the base upward to the tip on one side. A fourth small central leaf is flabby. (10) No. 10 is a small plant. Height 3.5 feet; diameter a foot from the ground 0.25 inch; number of leaves 11. Inoculated leaf 10 a is dead throughout, including all the visible part of the leaf-sheath. Leaf 10 b is dead throughout, including all the visible part of the leaf-sheath. The top of the plant is also dead ; that is, three leaves. Three additional upper leaves are dying in stripes. Photograph made. It is the smaller of the two plants photographed to-day (April 3). (11) No. 11 is nearly an inch in diameter at 1 foot from the ground. It is about 5 feet high. It bears 14 leaves. Leaf 1 1 a is dead throughout, unless it be a portion of the leaf-sheath. Inocu- lated leaf 11b, which is about 6 feet long, is dead and dry throughout, except the basal 1 foot of the midrib and a portion of the middle part of the sheath. This plant also shows conspicuous secondary signs. Of the upper leaves, five are distinctly striped and the two uppermost are flabby. One long leaf is dead throughout, except the basal 1 foot, and even this part is dead on the margin, the midrib being the only live part. There is conspicuous etiolation of the margins of some of these leaves. This sign precedes the drying out. There are also distinct, narrow, brown or red-brown interrupted stripings in the basal portion of the etiolated part. One of these upper leaves was pricked with about 150 needle-pricks (sterile) at 3 feet from the leaf-sheath on March 2, as a check: No striping has resulted. The needle-punctures themselves have a narrow white margin around them but the tissue between the pricks, which are set in close together, is still bright green. I mean by this that the pricked area is not over 1.25 inches broad (width of the leaf) by 1.5 inches long, and yet with all this number of punctures in it there has been no general death of the tissue, and no up or down striping such as took place within a few days in case of every one of the inoculated leaves. (12) Plant 12 is about 3.5 feet high; the diameter at 1 foot from the ground is about 0.5 inch; it has 12 leaves. Inoculated leaf 12 a is dead from the tip down a distance of about 3 feet. From there to the sheath, a distance of another foot, the midrib is green, but the margins are dead. Inoculated leaf 12 b, which is 4 feet long, is dead in the upper 1 foot and dead on both margins all the rest of the way down to the sheath, but the midrib in this part is green. This plant also shows distinct secondary signs on five leaves. One uninoculated leaf is dead from the apex down a distance of 3 feet. Another is dead on both margins for a long distance down and also has reddish-brown, narrow, interrupted stripes in the green part farther down, and a third has conspicuous etiolation near the midrib on one side, and there are a great number of narrow interrupted reddish-brown spots in this etiolated part. The two rows of cane to either side of this row are at least 3 feet higher and are altogether thriftier, showing no signs whatever comparable to those just described (fig. 15). Neither of these vigorous rows to either side are outer rows, but they are at the same time not of the same variety, one of them being Purple Striped cane and the other Striped Green cane. At the end of 2.5 to 3 months all of the inoculated leaves and some other large leaves were shriveled and the tip of the terminal bud had gone over into a bacterial decay. All of the inoculated plants succumbed. The leaves at this time were not all dead and the well- developed lower part of the stem was green and sound externally. The internodes were shortened and the basal buds were either much swollen or beginning to push. The plants stood in a dry house and had not received an excessive amount of water. During the pro- gress of the disease some shoots and roots pushed out from the nodes at the base of the cane. When the first signs appeared on the inoculated leaves one was removed and thin cross- sections were examined under the microscope for the presence of the bacteria. These were found in great numbers in the bundles about 18 em. above and for some distance (10 cm., perhaps) below the pricked area. Later the bacteria became very abundant in the stems. They were also found in bundles in the etiolated parts of the uninoculated leaves. Plates of nutrient agar poured from several plants and from different heights in the stem yielded PLANT BACTERIA. VOL. 3. 'V- PLATE 2. Cobb's disease of sugar-cane. Plant No 6. inoculated on the blades of two leaves by needle-pricks. Feb. 6, 1903, with a pure culture of Bacterium vascularum derived from Australian cane. The vessels of the stem from its base nearly to the terminal bud were filled with the yellow bacteria. The leaf-sheaths on the inside were also very gummy from the presence of bacterial ooze. Photographed May 1903. Variety. Common Green cane. COBB S DISEASE OF SUGAR-CANE. 25 in pure culture great numbers of the same yellow organism which had been isolated from the Australian cane (fig. 1 7). The bacteria were present in very great numbers and usually in pure culture in the yellow bundles of these stems. The extent of this stem infection (resulting, be it remembered, from a few needle-pricks on leaf-blades several feet removed from the nodes) will be better appreciated if transcripts are inserted from notes made at the time the canes were cut. For this purpose some observations made at the end of the third month will suffice. Brought in to-day (May 6, 1903) 4 sugar-cane plants, inoculated Feb. 6. They are all badly diseased. Photographs were made of No. 6 (plate 2) and of Nos. 8 and 12 together (plate 3). No. 6. — The height of this plant is about 70 inches to the curve of the top leaves. The height to the youngest leaf is 40 inches, i. e., the plant is small for its age (6 months, nearly). Both of the inoculated leaves are dead and dried, including the leaf-sheaths. Of the leaves lower down one is dead and dry and has fallen off, and three others are partly dead — that is, dead at the tips and along one margin but green in the midrib and on the other margin. Farther up, three large leaves are dead throughout, except the sheath. Four others are dead at the tips and in the case of one for 4 feet downward. The small terminal leaf is also dead at the tip. The diameter of the stem at the surface of the earth is 0.68 inch. The diameter of the stem a foot from the ground is 1 inch, including the leaf- sheaths. At the base of this plant there are three young shoots which are healthy in appearance, and five buds in various stages of pushing, one of them being an inch long. Signs of disease in the shape of bacterial slime are present under the leaf-sheaths a few inches from the base of the stem, the inner surface being gummy and wet. The inter- nodes are short; the longest ones are only about 2 inches in length, but the surface of the lower in- ternodes looks healthy. Gum- ming of the inner surface of the sheaths continues up a distance of several feet, and on the inner surface of one sheath the bacte- ria are abundant enough to look yellowish; the organisms have also broken through from the interior of the stem in three places and come to the surface as yellow slime on as many internodes. The sheaths are more or less rusty-brown spotted, especially in the gummy parts on the inner surface. There is no indication of rottenness, but a considerable quantity of gum covers the inner face of many of the leaf-sheaths, while the main axis is still green. Aphides are not present. Most of this bacterial slime has come from the interior of the leaf-sheath, but some from the interior of the stem. There is a tendency in this, just as in two other plants brought in to-day, for the terminal bud to push out sidewise. In the interior of the plant above the terminal bud there is much red-brown spotting of the tissues and much bacterial gumming. This rusty-red spotting of the inner, rolled-up leaves — those that are protected from the light and from surface contaminations — is very conspicuous. Terminal bud seems alive, but is crowded down on itself (zigzag) by the drying up and gumming together of the sheaths above it. The base of the uppermost young leaf is alive and wraps around another smaller leaf, the top of which is dead and gummy. Fig. 17.* *Fig. 17. — A pure agar culture of Bacterium vascular um poured from the interior of plant No. 9. at 20° to 280 C. Age 8.5 days 26 BACTERIA IN RELATION TO PLANT DISEASES. On cutting across the basal secondary shoots an inch from the stem their tissues seem to be sound (hand-lens). The main axis was then cut open and examined. The stem was first cut crosswise in the middle of an internode about 6 inches from the base and then in other places. Some of the vessels are gummy (yellow) and others are distinctly red stained. This red staining, as in the stems previously dissected, is much more conspicuous immediately below a node than it is above a node. A few minutes after cutting, the yellow bacterial ooze was very distinct from many of the bundles. There are cavities in the upper part of the stem. In other places, as well as those first recorded, the red stain is greater just below the node than it is above. The middle 3 inches of the stem was slit longitudinally and found to contain a closed bacterial cavity which is 0.75 inch long (estimated) and 0.1875 hieh wide in the widest part, full of pale yel- low slime. Slit the basal 3 inches longitudinally and found the disease in the base in the shape of reddened vascular bundles and yellowed ones from which ooze the yellow bacteria. The longitudinal section shows marked red-browning in the vicinity of the nodes and some red vessels in the internodes and, quite interestingly, there is in many cases a red and yellow stain in the same vascular bundle — red in one place and yellow in another, showing clearly that the red stain is closely associated with the yellow slime. This material was saved in alcohol and may be identified by the fact that it is cut in the form of thin slabs. No. 8. — The height of this plant is about 58 inches to the curve of the top leaves. It is badly diseased, both of the inoculated leaves being dead to the stem and also all of the lower leaves, while the upper leaves are dead at the tips and dried up along one or both sides, but with the midrib living. The terminal leaflet is flabby. The stem is very short. This plant is especially interesting from the fact that 6 inches above the ground the main axis is doubled and twisted and wrinkled crosswise in a swollen mass, the terminal growth being stopped by a drying and gumming of the tissues above the terminal shoot. Lower down the terminal shoot zigzagged and tried to push out sidewise in the same manner as shown in fig. 8. Around this swollen, bulged portion the leaf-sheaths are rusty- brown or pale green and very gummy, the bacterial slime occurring in sufficient quantity to show itself distinctly yellow. The gum does not string up much, but it is sticky to the fingers and so copious that both of my hands are covered with it. The interrupted rusty-brown striping is con- spicuous in the leaf-sheaths that are buried deep in the interior of the shoot, and some very interesting and conspicuous examples of this have been saved in alcohol. The terminal bud is not killed — only gummy and glued up and squeezed together (endwise) by the effect of the disease on the sheaths outside of it. There is an abundant yellow bacterial ooze from cross-sections of the stem of No. 8. Some of the vascular bundles are also reddened. There is distinctly more reddening of the vascular bundles immediately under the nodes than there is immediately above the nodes. These red vessels are mixed in with those giving rise to the yellow ooze. Slit the stem longitudinally. Some of the vessels are bright yellow from the presence of the bacteria and others are red. These are mixed in. Specimens put into alcohol. I am inclined to think that the reddening of the bundles is a later stage in the disease than the yellowing. Three cross-sections were cut from No. 6 and one from No. 8 (smaller) and a photograph (X3) made of them together, designed to show the yellow bacterial ooze and also the red bundles (plate 1). A photograph was also made of a longitudinal section of No. 6, showing the bacterial cavity (fig. 4). On the same negative is a longitudinal section of No. 12 (fig. 11), showing red stain in the nodes, and red and yellow bundles in the internodes. No. 12. — This plant is a little smaller than No. 8. The two inoculated leaves are dead and dry and all the upper leaves are dead, including the terminal one. Lower down there are two partially green leaves. Both, however, are dead at the tip and have dead stripes on the margin. This plant has been allowed to stand too long and some of the outer, dead leaf-sheaths are moldy, which was not the case with either of the others. The inner sheaths are rusty-red spotted, as in case of the others, and there is much bacterial gumming between them. In some cases the spotting is quite brilliant — almost a vermilion red. The gumming here also is abundant enough to stick up ray fingers very markedly. About 6 inches from the ground there is the same effort of the shoot (terminal bud) to wrinkle, swell, and push out sidewise. Above this swelling (6 inches to a foot) the sheaths surrounding the young leaves are very gummy, and there can be no doubt that this sidewise bulging is due to the gumming and pressure of the outer leaves around the terminal bud. Unwrapping the youngest leaf visible, which was dead at the tip, I find inside of it a younger leaf, also dead at the tip, and the terminal bud is in worse condition than in either of the other plants. The basal buds to the number of seven have begun to push. These three stems (6, 8, and 12), stripped of their leaves, were photographed together (fig. 9), and 8 and 12 were photographed together before removal of the leaves (plate 3). PLANT BACTERIA, VOL. 3. PLATE 3. Cobb's disease of sugar-cane. Plants 8 and 12, inoculated Feb. 6, 1903, with a pure culture of Bacterium vascidarum plated out of sugar-cane received from New South Wales. Inoculated by needle-pricks only, each plant in two leaf-blades at some distance from the stem. Most of the leaves dead and dry when photographed, stem bundles filled with the honey-yellow slime. The terminal bud pushing out sidewise may be seen near the base of each cane. Photographed May 1903. About one-twelfth natural size. Com- mon Green cane. COBB'S DISEASE OF SUGAR-CANE. 27 On cutting open the stem of No. 12, red bundles were found to the extreme base. These are mixed in with yellow ones, from which there is a distinct bacterial ooze. Slit the basal 4 inches longitudinally. Here again the stem shows a series of red cross-stripes corresponding to the nodes. In the internodes some of the bundles are bright yellow, others are red, and still others are mixed (mottled) red and yellow, and there are bacterial cavities. The red stain is evidently a later stage, just as the black stain is a later stage in sweet corn attacked by Bacterium stewarti. No. 5. — This plant is a little smaller than No. 12. All the basal leaves are dead. Both of the inoculated leaves are dead, including the sheaths. The terminal leaf is flabby and all the other leaves are more or less diseased, being dead at the tips and with dead margins for a long distance down. The base of the plant for a foot up inside the sheaths is very wet and gummy, just like the others. These sheaths are rusty spotted. There is a peculiar smell to the disease not unlike that of some bacterial cultures, but I am not able to define it any closer. The inner sheaths are green and some of them are badly rusty spotted. The color of the spots is a brick red. Cut open the stem at the base; the vascular system is stained a reddish-brown. This reddish- brown staining is very conspicuous. The terminal bud appears to be normal. The tissues in its vicinity appear to be sound in places, diseased in others. Other plants in this series gave similar results. These were examined in April or else later on, in May and June (plate 4). January 5, 1904, only one good ratoon cane has come up from the stools. In addition there are two small canes and a shoot a foot high. The large cane is free from signs of disease at the base, or nearly free. The little shoot is free. Had these stools been healthy they would have given many tall shoots. SERIES II, 1903. Fourteen shoots of Louisiana cane, Variety No. 74, were inoculated February 9, 1903, from two potato cultures 11 days old by making needle-pricks on two leaves of each plant, as in case of the first series of inoculations. At the end of 5 weeks all showed local signs of the disease and six of the shoots also developed constitutional signs, but on the most of them these were not pronounced. The greater number of these plants proved very resistant, i. e., subsequently they grew out of the disease, although they were somewhat dwarfed by it. At the date of inoculation these canes were the most vigorous ones in the patch, but when they were cut out for exam- ination, on July 20, the largest and best canes were the sixteen uninoculated ones. These big canes were small and inferior at the time the others were inoculated. From the inoculated canes dwarfed cuttings, containing red vascular bundles, were saved for planting, but no yellow slime was present in the bundles of these stems ; at least none that could be detected with the hand-lens, and only one reddish-yellow bundle was seen. SERIES III, 1903. Eight shoots of common purple cane planted in the greenhouse on November 15, 1902, were inoculated with Bad. vascularum on May 4, 1903. The organism used for these inoculations was from cultures 7 days old on potato cylinders kept at room-temperatures. These were subcultures made from colonies on Petri-dish plate No. 9, poured April 16, 1903, from plant No. 11 of the common green cane inoculated February 6, 1903. The plate was absolutely a pure culture of Bad. vascularum. The inoculations were made by spreading three or four 3 mm. loops of the organism over about 6 or 8 sq. cm. of leaf-surface, generally about a foot above the top of the leaf- sheath, and then making about 100 to 150 needle-pricks through this area. After the pricks were made, two or three more loops of the organism were rubbed over the pricked area. Two leaves on each plant were inoculated. Leaves near the top of the plant were chosen when possible. The canes were quite tall at this time — averaging perhaps 6 feet, and many of the leaves reached to the top of the greenhouse (10 ft.?). The inoculated areas were protected from the sun for a day by cuffs of manila paper. Five checks were made. The 28 BACTERIA IN RELATION TO PLANT DISEASES. inoculated plants were given numbers from 27 to 34 inclusive, and the inoculated leaves were labeled 27a, 276; 28a, 28b, etc. For these inoculations tubes Nos. 1 and 3 of April 27, 1903, were used. On May 5, 1903, finished inoculating the row of common purple cane. Four plants, Nos. 35, 36, 37, and 38, were inoculated in just the same way as those of preceding day, with one exception; in this case the plant was inoculated in the stem and on one leaf instead of two leaves. This was plant No. 38. Tube No. 4 of April 27, 1903, from poured plate No. 6, of April 16, 1903, was used for these four plants. On May 14, 1903, all these plants showed the effects of the inoculation. A distinct discoloration about the punctures was noticed three days earlier and to-day the punctured area is dead, entirely or in great part, on at least eight of the inoculated leaves. All the other inoculated leaves show a marked reddish-brown streaking or spotting in the punctured areas. The check leaves show no such discoloration. The punctures on these leaves show simply as small white points. On June 13, 1903, common purple cane No. 33, inoculated May 4, 1903, from tube No. 3, April 27, was broken over by accident and brought in for examination. It is a shoot about 9 or 10 feet high, with a thick stem and large green leaves, but the basal ones are mostly dead (normal) . The inoculations were on the blades of two leaves about midway down the stem, and the pricks were about 8 inches from the sheath on both leaves. The midrib of each inoculated leaf is alive throughout. To either side of the midrib on one leaf are dead areas for a distance of 2 to 3 feet. The other leaf is much the same, except that on one side the dead portion is not so long, perhaps 18 inches. Above this, however, in the green part are yellowish and rusty- yellow dead stripes for a distance of another 1 8 inches. The outer edges of the sheaths are also dead nearly or quite to the nodes. The ends of both leaves are wholly alive. Made cross-section of the sheath at the extreme base and examined it under hand-lens for bac- teria. In one they seem to be present in small quantities, but nothing very definite in either one under the hand-lens. These must be looked at under the microscope to determine the presence of bacteria in the vessels of the inoculated leaves. Cut the stem 6 inches above the base. No evidence under the hand-lens of bacteria in the bundles. Cut 6 inches higher up and examined again. No bacteria visible in the stem when examined under the hand-lens. Cut another 6 inches up. Here, also, the stem appears to be perfectly sound under hand-lens. Cut another 6 inches up. The whole stem appears here also to be sound. We are now getting into the vicinity where the inoculated leaves join on to the stem. The whole stem appears to be sound. vSlit the 6-inch segment below the last longitudinally and examined. No evidence of disease. Cut another 6 inches up (cross-section). One red bundle in the internode, which is indicative of the disease. Slit this segment longitudinally and examined: Distinct reddening at both the included nodes, but very slight in comparison with the effect on the common green cane. At each node there are a dozen or more red bundles. No ooze. This is a very resistant variety. Cut another 6 inches up. No evidence of disease. Slit the stem longitudinally. No evidence. The whole upper part of the stem for a distance of several feet was now slit longitudinally and examined for the presence of bacteria. No stain in the bundles; no evidence of disease. The disease is confined to the two inoculated leaves, to the two nodes which bear these leaves, and to the immediate vicinity of these two nodes. On subsequent examination there seems to be a very light red stain on one side of two nodes next farther up. These also were put into alcohol for further study. A microscopic examination showed the bacteria to be present in the bundles of the midrib of the inoculated leaf-blades and in the bases of the leaf-sheaths. On August 8, 1903, two of the plants of common purple cane which were inoculated with Bad. vascularum, May 5, 1903, broke off with their own weight. The two plants are Nos. 37 and 38. No. 37 was inoculated in two leaves, while No. 38 was inoculated in one leaf (386); and through various leaf-sheaths into the stem (38a). On examination the conditions were as follows: PLANT BACTERIA. VOL. 3. PLATE 4. M , Rot of sugar-cane due to Bacterium vascular um . Leaves removed and top of cane split longitudinally to show advanced bacte- rial decay. This decay at the top includes all but the surface of the cane: Nodes red, cavities in the internodes full of yellow bacteria. A little far- ther down the cavities disappear. Base of cane sound. Inoculated Feb. 6, 1903. Photographed June 15, 1903. cobb's disease of sugar-cane. 29 Externally, plant 38 shows much more disease than 37. Plant 38 is about 7 feet high and is almost entirely dead as regards leaves. The basal part is devoid of leaves for a distance of 2 feet from the ground and the stem is about 1 .5 inches in diameter. The first leaf is partially green, but is beginning to die along the margin. The next leaf is entirely dead. The sheath of this leaf was pricked through when the stem was inoculated. The next two leaves are about half dead, while the next (which is the inoculated one, 38 b) is about three-fourths dead. Two or three of the leaves higher up show traces of green, but the entire terminal bud, com- prising about 15 leaves, is entirely dead and dry. All these leaves are rotted at the base, so that the whole mass of them came loose from the stem. On removing the leaves from the stem, 10 or 1 1 of the uppermost buds are seen to be well started, some of the upper shoots being 4 inches long. Half of the internode into which pricks were made has a water-soaked appearance and there is a large black wound at one end of it. The outside of this internode is sticky to the touch. None of the internodes above this point are healthy. They show water-soaked areas all along one side. In the lower foot of this stem there are no red bundles, but above that point one or two red bundles appear. The number of diseased bundles increases from this point up, until at a distance of 4 feet from base of stem there are at least 30 diseased bundles. Some are black, others red, while still others are yellow, and after a few minutes standing show a yellow ooze from the cut surface. The upper 1.5 feet of this stem was photographed in toto to show development of shoots (fig. 10). The diseased bundles in the stem are all on one side, which corresponds to the needle pricks in the stem higher up. In the internode next below the pricked one there is a large group of diseased bundles, black, red, and yellow. Yellow ooze shows quite distinctly from about 20 of these. There are at least 100 bundles affected in the inoculated internode. About half of these are black and a few red, but there are a large number which show the yellow bacterial ooze. All of the internodes above this point, 12 in number, are very badly diseased. About one-quarter of the bundles are entirely black in these, and nearly all the others show yellow ooze. There is a small cavity along one side of the stem in three of these internodes, which contains yellow slime. The plates poured from the first internode below the inoculated one miscarried. I did not split the stem longitudinally, as I wished to save it for planting. On sectioning: Two cuttings apparently not diseased; four cuttings slightly diseased; five cut- tings badly diseased. Plant 37 is about 8 feet high and externally looks to be a very healthy cane. Both inoculated leaves are entirely dead, but with one or two exceptions all the other leaves seem all right except one or two of the upper, which were torn in the ventilator. The basal part of the stem is 1.5 inches in diameter. On cutting the cane, the first red bundle appeared about 2 feet above the base of the stem. In the cut a foot higher there seemed to be no diseased bundles. None showed farther up at all. Cuttings saved for planting: 3 very slightly diseased (1 or 2 bundles red) ; 6 not at all diseased. On January 5, 1904, the remainder of these canes were cut out, with the following results: 27 a and b. — Very large cane, no trace of red bundles, nor other signs of disease in either of the nodes into which inoculated leaves ran. Another cane, apparently inoculated, is sound. 34 a and b. — Excellent cane about 10 feet long. A few red bundles run in at two nodes, which probably bore the inoculated leaves. 35 a and b. — No evidence of the starting of buds in this or the other canes examined. A large cane, at least 10 feet tall and 1.25 inches in diameter. No ooze nor red bundles in the internode close below the lower inoculated leaf. In internode above the upper inoculated leaf there are 4 red bundles, from one of which comes a yellow ooze. This was examined under the compound microscope and found to consist of bacteria. In the red bundles of this stem there were comparatively few bacteria, each separate in the field, mostly in pairs and very distinct, the feeble motion probably Brownian. Cover and slide stained. Saved for poured plates. j6 a and b. — Cane about 10 feet. Plain evidence of red staining. In one place 1.5 feet from the ground there are at least 12 red bundles. A little lower down only 1 or 2. One foot farther up there are 7 red bundles and 1 yellowish one. Another foot higher up and immediately under an inoculated leaf are 15 affected bundles; 1 is black, some are red, and some yellow. Apparently an ooze from some. Cutting down from top of stem, about 14 inches above last previous cut, there are about a dozen bundles stained and apparently bacterial ooze from 1 bundle at least. Cut 8 inches lower down, same phenomenon. Slight disease here as compared with that of last year in the Common Green variety. This cane is the most badly-diseased in this lot. Intend to plate out to find if the 30 BACTERIA IN RELATION TO PLANT DISEASES. organism is alive in cane after having been inoculated 8 months. Split longitudinally, bundles red in some places, yellow in others, and black in still others. Material saved for plates. Cane perfectly sound externally for many feet in the region of the diseased bundles; the affected portion is in the middle of the cane — not at top or bottom. Longitudinally split, it shows bundles both yellow and red. Up towards the top the cane is free from stain, healthy and sound. One large cane apparently inoculated, but labels are gone ; at least 10 feet long and 1.5 inches in diameter; not badly affected. Only a few red bundles at the nodes; they do not run down into the internodes at all. Largest cane we have cut. Has been no interference with growth. Red staining in only 2 nodes — probably those to which inoculated leaves ran, but these have fallen. Upper inter- nodes perfectly sound and white. Another cane without labels but with leaf with label 306 lying near its base. Cane 10 feet high ; diameter 1 to 1.25 inches. Perfectly sound outside, and well grown. At height of 5 feet from ground 3 red bundles, 1 of which shows a yellow ooze. Extreme base of cane sound inside and out. Buds are well developed, but they are not pushing. Leafy portion cut away, leaving 5 or 6 feet; sound at cut. Stem shortened 1 foot, beginning at top. Two bundles red. Another foot, there is increase in number of red bundles, about ten, all in a group in the center of the cane. Yellow bac- terial ooze from some. Shortened another foot, same diseased area continues. Bundles red and brownish in perfectly sound tissue. Basal 3 feet of cane split longitudinally. Disease runs out about 20 inches from the base. Excellent evidence that the bacteria got in through the inoculated leaves. 2Qa and b. — Cane 12 feet long, 1.5 inches in diameter; it has fallen down and rooted in places and sent up some shoots, which are healthy. Top of cane sound. Base of cane, two red bundles in sound tissue. Exterior of cane sound from base to top. At 4 inches above the base there are no red or yellow bundles; 9 inches above base cane sound; 12 inches above base cane sound; 2 feet above base cane has one red bundle, which continues down the stem 8 inches and there disappears. At 1 foot higher, i. c, in the internode below one of the inoculated leaves, cane is sound. Location of other inoculated leaf not known. Signs of disease are very slight, as there is discoloration in only one internode. A cane about 10 feet high, 1 inch in diameter, no labels. Sound at top and base; probably an inoculated cane, but can not tell. In middle part of cane there are three red bundles in sound tissue. Outside of the cane sound. Trouble is confined to one node which looks sound outside. No stain in flesh. Another cane 10 feet high, 1.25 inches in diameter. Top and base sound; 8 inches up sound; 1 foot up sound; 18 inches up sound; 2 feet up sound; entire cane sound. There were 5 large canes besides those above noted. All were sound at the top and base, and none had labels. About an hour after cutting, four or five little yellow cirri pushed out from the ends of cut bundles of cane No. 36. These ranged from 1 to 5 mm. long and were about 0.25 mm. in diameter. The red stain present in the bundles was carried out on these cirri so that they were in some places red and in others yellow. Looked at under the microscope, these cirri were simply masses of bacteria, enormous numbers, in what seemed to be a pure growth. They were short rods, single, in pairs, fours, or eights. Most of them were paired, i. e., rods with a plain constriction mid-way. None were motile. Stained a cover and a slide. From the other end of this cane there were also drops of ooze which were masses of bacteria. The cutting from No. 30 also shows drops of yellow and red ooze, which under the microscope are seen to be made up of masses of bacteria. They are like those in 36, but more of them are in small clumps (pseudozoogloeae) . The plates which were poured from plants 30 and 36 were made as follows: A tube of bouillon was inoculated abundantly by putting into it a good-sized piece of the inner tissue containing vessels determined by the microscopic examination to be filled with the bacteria, and letting it stand one hour. This tissue was then mashed with a sterile glass rod. As already stated, these plants had not suffered materially from the disease, but some of the vessels showed im- mense numbers of bacteria. The tissue was cut out with a cold, sterile knife. Neutral beef agar poured at 41° to 380 C. was used; seven plates were poured direct and seven from the first dilution. ( )f those poured directly from tubes in which the tissue was mashed, one received six 3 mm. loops of the bouillion and two others received each three 3 mm. loops, i. e., an enormous number of the bacteria. Result: All dead. Not a colony developed on the agar plates. Canes, therefore, may have this disease and recover from it. SERIES IV, 1903. The field experiment miscarried, owing, perhaps, to defective soil (a barren sand) on which the canes from series 2 and 3 were set. 00 A, PLANT BACTERIA, VOL. 3. PLATE 5. Cross-sections of stem of sugar-cane plant No. 40, inoculated June 22, 1904. Cut and fixed in alcohol Sept. 27, 1904. Photographed under alcohol Jan. 23, 1905* Nearly every bundle full of the bacterial slime. This plant was inoculated by needle pricks in the stem. The organisms used were descendants of colonies plated from plant No. 7, inoculated Feb. 6, 1903, and cut out Apr. 22, 1903, for examination, cultures, and photo- graphs. COBB S DISEASE OF SUGAR-CANE. 3 1 SERIES V, 1904. The fourth set of inoculations was made June 22, 1904, on canes planted in December, 1903. All inoculations were by needle-pricks through the leaf-sheaths into the stem. The cultures used were strains plated out of plants Nos. 7, 9, and 11, inoculated February 6, 1903. Only cultures from plant No. 7 gave positive results, and it is probable that the two subcultures from plant No. 9 and the one from plant No. 1 1 were made from non-infectious yellow colonies resembling Bacterium vascularum on agar, but not identical. It was thought at the time of inoculation that these might be different, as they browned the agar in a way the true organism does not. The plants inoculated from these suspected tubes have starred numbers. In making the inoculations the surface bloom and the prickles were first rubbed off the lower part of the selected leaf-sheath ; the surface was then rubbed with a few loops of the yellow organism scraped from the slant agar, and eight or ten needle-pricks were made through the sheaths into the center of the stem. Check-pricks were made on each variety. These are the inoculations referred to at the end of my German paper. On September 27, 1904, the condition of the sugar-cane inoculated June 22, 1904, was as follows: Striped Green, f No. 3Q*. — A large-sized cane, 8 feet long and 1.5 inches in diameter. The inoculated internode is about 2 feet from the base of the cane. No signs of infection in the internodes directly above or below the inoculated internode. Splitting the inoculated internode longitudinally, the tissues about the needle-pricks are discolored, but the discoloration does not spread for more than 0.125 incn in either direction. All the leaves are green in this cane, and from the top to the bottom it seems to be perfectly healthy. On dissecting out the terminal bud, however, the very youngest leaves are dead, but this injury, I think, has been done by the ventilating apparatus in the greenhouse. No. 40. — This cane is 7 feet long and about 1.25 inches in diameter. The basal 15 leaves are entirely dead and brown, while the upper leaves are gradually dying from the tip toward the base. The terminal bud is entirely dead and brown. There is a large quantity of yellow gummy ooze exuding from the base of this stem, which has been cut about 2 hours. It is 10 internodes below the one inoculated. This cane was carried to the laboratory in loto, where it was dissected. Laboratory note. — The yellow ooze from many bundles has coalesced at the basal cut, so that the surface is just a mass of yellow gum. All of the leaves are in an unhealthy condition, some being entirely dried up, while others are drying from the tip down and along the margins. The leaves enveloping the terminal bud are all dead. On sectioning there is abundant bacterial ooze from prac- tically all bundles in all the internodes below the inoculated internode. The ooze does not show much in the upper internodes, however. It runs out completely in the fifth above the point of inocu- lation. Plates were poured from this cane. Sections of the stem were saved in alcohol and photo- graphs were made (plate 5). Inoculation No. 41 could not be found. The label was lying on the ground. No. 42. — This is a cane about the same size as No. 39. It seems to be very healthy. The inter- nodes are from 6 to 8 inches long. The upper leaves are all healthy except the topmost ones, which have been torn by the ventilating apparatus. At the basal cut there are at least 15 or 20 discolored bundles. These run all the way up to the top of the stem — apparently as many at the top as there are at the base. The discoloration (red stain) of the vascular system is much more marked at the nodes than in the internodes. When the stem was split longitudinally, some of the bundles were observed to be red, while others were yellow. A large number of the bundles in this stem, when examined microscopically, showed enormous masses of bacteria. Plates were poured from this stem. No. 4 j*. — A medium-sized cane, about 5 feet high and 1.25 inches in diameter at the base. There is no sign of discoloration in the tissues in any of the basal nodes. From top to bottom the stem seems to be perfectly sound. The inoculation has not taken at all. No. 44*. — This is a very large cane, about 9 feet in height and 1.25 inches in diameter at the base. The cane externally seems to be perfectly healthy. All the leaves are green from the bottom to the top. The terminal bud is perfectly sound. On sectioning the inoculated internode, the discolor- fThis row of cane was labeled "Common Green," but it looks very much like "Striped Green." The labels may have been mixed in the transplanting. 2,2 BACTERIA IN RELATION TO PLANT DISEASES. ation of the tissue does not extend for more than o. 1 25 inch either way from the needle-pricks. There is no sign that the inoculation has been effective. No. 45*. — This is an excellent cane, being about 9 feet long and 1.25 inches in diameter. The lower internodes are about 6 inches long, while some of the upper ones are at least 8 inches in length. The whole stem, with the exception of the inoculated internode, is perfectly white within, there being no discolored bundles. In the inoculated internode a red discoloration has extended in one or two cases about an inch from the needle-pricks. There is no sign of a general infection, however. Check. — The stem containing the check-pricks is rather small, as it has been crowded by the larger canes in the stool. There are no discolored bundles, however. The needle-pricks present much the same appearance as they did in canes Nos. 44, 45, etc. No. 46. — This is a small cane, about 4 feet in length and 0.75 inch in diameter. It has not made much growth. Some of the leaf -sheaths in the upper part of the stem are brown and dead. The terminal bud seems to be healthy. There are very many discolored bundles, however, in all the leaf- sheaths which envelop the bud, and in the upper part of the stem much of the vascular system is dis- colored, there being at least 100 red and yellow bundles. The portion of the stem below the inoc- ulated internode, however, seems to be perfectly white, the infection having run up rather than down the stem. There seems to be a little yellow ooze from many of the bundles in an internode which is at least 4 feet above the one inoculated. This cane was taken to the laboratory for microscopic examination. Laboratory note. — This stem shows abundant bacterial ooze in the ninth internode above the point of inoculation. It comes from at least 100 bundles. Plates were poured from this stem. No. 4J. — This is a very small, stunted cane. It has made scarcely any growth since it was inoc- ulated. The basal leaves are dead, but the upper ones are green and seem to be perfectly healthy. There are five or six discolored bundles in the basal part of the stem. In the upper part of the stem there are at least 20 discolored bundles. This cane has very little juice in it. It seems to have entirely dried up. Common Green. f Xo. 48. — This is a very small and stunted plant. The terminal bud and terminal leaflets are dead and shriveled. The basal three or four leaves are green along the midrib, but the leaf-sheath and edges of the leaves are dead. The cane has made scarcely any growth since it was inoculated. In the basal internode there are ten or fifteen yellow bundles. In the uppermost internodes the num- ber of discolored bundles has increased. Whether or not the condition of this stem is due to bacterial infection is uncertain, for there seems to be no yellow ooze.t The terminal bud seems to have been killed from some external cause rather than an internal one. No. 4Q. — This cane is quite short, being about 3 feet in height. It is very thick at the base, however. The terminal bud is entirely dead and the surrounding leaflets brown and dried up. The basal internodes are very short, some of them being not more than an inch in length. The tissue throughout, however, is white, except in the inoculated internode and the one immediately above it. In the latter there is one red bundle, which extends throughout its entire length. There is quite a little discoloration within the inoculated internode, but it has not spread far in either direction from the needle-pricks. There are no cavities in this internode. The tissue seems to be intact throughout. A few of the bundles are discolored for two or three inches. This is the only sign of infection. Xo. 50. — This is a very long cane, being about 9 feet in height. Many of the basal leaves are brown, while some of the upper ones are dying at the tips and along the margins. The topmost leaves have been crushed by the ventilating apparatus. The terminal bud is dead and dried up. The cane at the base is about 1.5 inches in diameter and externally seems to be very healthy through- out its entire length. The only discoloration in the tissues of the stem occurs in the inoculated inter- node and the two adjacent ones. Other than this, the cane is perfectly healthy. The central part of the tissue in the inoculated internode has a water-soaked appearance and contains one or two red bundles. Laboratory notk. — A few of the bundles in the first internodes above and below the point of inoculation show the presence of bacteria. In two of them, little yellow cirri about 0.25 inch long have been pushed out, and these, when examined microscopically, were found to be made up of a solid mass of bacteria. No. 51. — This is another long cane, which has made an exceptionally good growth. Some of the internodes near the base are 10 inches long. The leaves are all green and perfectly sound, with the exception of those at the to]), which have been crushed by the ventilator. The stem in no portion fWhether or not this is "Common Green" is a question. It may be "Striped Green." The latter is the more likely hypothesis, as the rest of the row is of this varii 1 \ ^Although a microscopic examination was not made it is probable that the cane was bacterially diseased. COBB S DISEASE OF SUGAR-CANE. 33 shows any discoloration in the interior except in the immediate vicinity of the needle-pricks. The discoloration has not extended either up or down into any of the internodes. Caledonia Queen. No. j2. — This is a fairly good cane. It is about 8 feet in length. The basal leaves are brown and shriveled, but the upper ones are all perfectly healthy. The stem is about 1.5 inches in diameter at the base. There is no discoloration in the stem except in the inoculated internode, where it does not extend at all from the needle-pricks. The cane seems to be perfectly healthy throughout its entire length. No. 5 j. — This cane is about 5 feet in length and has much the same general appearance as the preceding one. The cane is perfectly healthy throughout. No. 54. — Presents the same external appearance as the two preceding canes and is about the same size. Running through the four internodes above the point of inoculation is a small number of yellow bundles. Whether or not these contain bacteria could not be determined with the hand-lens. The cane has made a very good growth and is about the same size as the uninoculated canes of this variety. Laboratory note. — The section taken from this cane showed a distinct yellow ooze from a small group of bundles in the center of the stem. This was made up of masses of bacteria. Plates were poured from this stem. No. 55. — This cane is about the same size as the others and presents the same general appear- ance. In three of the basal internodes there are a few red bundles, but these soon run out, not extend- ing as high as the point of inoculation. The only other discoloration in the stem is in the inoculated internode. Presents exactly the same character as that in check. Is simply a local oxidation. Crystalina. No. $6*. — This is a cane which has made a fairly good growth, although it is of a rather small diameter. It is perfectly healthy throughout its entire length. No. 57*. — This is an exceedingly small cane. It is very sickly looking. Although the inocu- lation was made in very young internodes, it has given no result. The stunting of the cane may be due to the fact that some of the pricks pierced the terminal bud. Jamaica. No. 58*. — This is a cane about 6 feet in length. The basal leaves are all dead. This is probably due to the fact that they have been strangled by the wire with which the label was fastened on. The majority of the upper leaves are beginning to brown at the tips, while the terminal bud is entirely dead and the surrounding leaves are covered with fungi. The cane internally is perfectly healthy throughout. No. 59*. — This cane is about 7 feet long and seems to be perfectly healthy, with the exception of the uppermost leaves enveloping the terminal bud, which are dead and brown. This injury, however, is due to the ventilating iron. Perfectly healthy throughout internally. No. 60*. — This cane is much like No. 59, except that it is not quite as long. Externally it seems to be very healthy. The tissue of this cane is perfectly healthy throughout. Check. — The check presents exactly the same appearance as all the other canes of this variety. General Note. — The distribution of the bacteria from the point of inoculation in the diseased canes can not be judged accurately, however, in any of these cases, as the needle- pricks were made through many leaf -sheaths as well as through the stem. In all cases where bacteria were found in the stem the organism used was of the same parentage. At the time of inoculation it was known that there were in our cultures at least two kinds of yellow organisms, some of the stock tubes behaving differently from the others. Cane-plants 40, 42, and 46, inoculated June 22, 1904, and cut September 27, 1904, yielded from the interior of the stems numerous colonies of Bacterium vascularum in prac- tically pure culture (14 poured plates). I do not find any record respecting No. 54. SERIES VI, 1905. Inoculations of Sugar-cane in Hot-house No. 4. On May 22, 1905, two assistants, Miss Florence Hedges and Miss Harriett Thomson, were set at work inoculating sugar-cane with Cobb's organism. I planned to inoculate several shoots on every other row, 2 leaves to be selected and 30 or 40 punctures to be made 34 BACTERIA IN RELATION TO PLANT DISEASES. on each one at a distance of 6 inches to a foot from the stem, that is, on the basal portion of the blade of the leaf. None to be pricked into the stem. The rows extend east and west. The rows selected are the first row on the north, third row from the north, fifth row from the north, etc., until we reach the south end. This leaves as checks, beginning at the north, the second, fourth, sixth, etc., rows. Each variety of cane is represented in the bed by two rows, and there are eight varieties. This cane was planted in the autumn of 1904 and had grown very slowly. This statement should be borne in mind, as it was evidently the govern- ing factor in what followed. One-half the sugar-cane was inoculated May 22, using five potato cultures marked # made May 17 from an old indigo-blue litmus-milk-culture # of March 25, descended from inoculated cane-plant No. 40. The other one-half was inoculated May 23 from six potato cultures called X, made May 1 7 from an old indigo-blue litmus-milk-culture X, made March 25 and descended from cane plant No. 40 (pi. 11, fig. 12). All of these potato cultures were covered with a bright yellow growth and were in good condition for use. Transfers from them were made to as many more tubes of potato before beginning. The inoculations were made as follows: The potato cultures were shaken until the yellow surface growth was washed off into the fluid. Loops of this fluid were then spread on the surface of the leaf, and thirty or forty delicate needle-pricks were made through this wet surface into the leaf. Generally four 3 mm. loops of the cloudy fluid were placed on the surface of each leaf, one to the right of the midrib, one to the left, and two along the groove of the midrib over an area of about 1 .5 to 2 inches in length. Then the pricks were made through this fluid. Afterward about three 3 mm. loops of the fluid were rubbed over the pricked surface on the under side of the leaf, that previously mentioned being put on the upper surface. As soon as the leaf was pricked it was covered from the bright light by pinning a piece of manila paper around it. Two leaves were inoculated on each shoot. All were inoculated on the blade of the leaf at a distance of 6 to 12 inches from the leaf-sheath. The varieties inoculated, and the num- ber of shoots on each variety, beginning at the north end, are as follows: Cinta, 61 to 67 (7 shoots); Caledonia Queen, 68 to 77 (10 shoots); Louisiana No. 74, 78 to 82 (5 shoots); Common Green, 83 to 87 (5 shoots); Blanca, 88 to 90 (3 shoots); Jamaica, 91 to 96 (6 shoots); Striped Green, 97 to 99 (3 shoots); Crystalina, 100 to 105 (6 shoots). Each leaf has a wooden label bearing its inoculation number. On June 13, 1905, the condition of the cane leaves inoculated May 22 was as follows, all of the uninoculated leaves being sound: Cinta. 61 A. Begins to show white stripes in and above the pricked area. The stripes are visible 6 inches above the pricks. 61 B. Stripes are visible 9 inches above the pricks. 62 A. Begins to be white; bleached in pricked area and above it for a few inches. 62 B. White stripes in the pricked area and extending downward some inches. 63 A. White stripes in the pricked area, and up and down for a short distance (2 to 3 inches). 63 B. Shows white striping in pricked area. 64 a. White striping in the pricked area, extending upward 2 or 3 inches. 64 B. Same as 64 A. 65 a. Same as 64 a. 65 B. While striping, and a red stain in the midrib above and below the pricks. 66 A. Faint striping in the pricked area and extending upward 4 inches, downward I or 2 inches. 66 B. Paint white striping in the pricked area. 67 A. Pricked area dead to cither side of the midrib, and white around the dead area. 67 B. While striping in the pricked area and red stain in the midrib for about an inch. Caledonia Queen. 68 A. White stripes in the pricked area, extending upward 4 inches and downward about 3 inches. There are little brown dead specks in some of these white stripes, not only on this plant but on the preceding plants. 68 B. Slight signs in the pricked area. 69 A. Same as 68 B. 69 B. Yellow and red striping in the pricked area. 70 A. White striping in the pricked ana. 70 b. Same as 70 A. cobb's disease of sugar-cane. 35 71 A. Same as 70 A. 71 B. Same as 70 A. 72 A. Same as 70 A. 72 B. White and red striping in the pricked area, and a white stripe with a few little brown dead patches on it 4 inches above the pricked area. 73 A. White striping in the pricked area. 73 B. White and red striping in the pricked area 2 inches above and 2 inches below. 74 A. Dead on both sides of the midrib in the pricked area, and white striping, which extends 4 or 5 inches above on either side. 74 B. White striped or dead in the pricked area. 75 A. White striping in the pricked area, extending above 6 inches, and below 4 inches. Occasionally, small rusty patches in the white stripes. 75 B. Like 75 A. White striping extends at least 4 inches below pricked area, and as far above on both sides of midrib. 76 A. Dead in the pricked area on both sides of the midrib White striped for several inches above the pricks and a few inches below. There are some stains in the midrib. 76 b. Dead in pricked area on both sides of the midrib. White striped for several inches above the pricks and a few inches below. 77 A. White in the pricked area, running up about an inch. 77 b. Brown dead spots in the pricked area. White stripes run about 2 to 3 inches up and down. Louisiana No. 74. 78 A. Faint white striping in the pricked area, red striping in the midrib, and a rusty stain along one edge of the leaf for a distance of 8 inches, beginning in the pricked area. 78 B. Conspicuous red stain in the pricked part of the midrib, extending upward for 2 inches and downward for 1 inch. 79 A. Slight white striping in the pricked area. Faint red stain in the midrib. 79 B. Slight white striping in the pricked area. Faint red stain in the midrib. 80 a. Conspicuous red stain in the midrib in the pricked part, also 1 inch above and 2 inches below. 80 B. Like 80 A, but also some white dead stripes in the blade to either side of the midrib (pricked part). 81 A. No signs. 81 B. Slight red stain in the pricked part of the midrib. 82 A. No distinct signs. 82 B. Same as 82 a. Common Green. 83 a. Dead in the pricked area to either side of the midrib, and yellowish stripes with brown specks in them extending upward 3 inches on one side and 6 inches on the other. 83 b. Same as 83 A, but signs more extensive, and traceable above the pricked area for about 8 inches, and downward a lesser distance. 84 A. Distinct white striping in the pricked area, with red specks in the white stripes. These run interruptedly to a distance of a foot above the pricked area on one side, and about 4 or 5 inches on the other side. 84 B. Same as 84 A. 85 a. White striping in the pricked area. Some of the tissue dead. White striping, with brown specks in it, runs up interruptedly to a distance of a foot on one side of the midrib above the pricked area, and nearly as high up on the other side. 8.5 B. Same as 85 A. The striping is not so extensive. 86 a. Dead in the pricked area in places, and white stripes extending upward about 6 inches. 86 b. Same as 86 A. 87 A. White stripes in the pricked area and for 10 inches above; 2 inches below. Red specks in the white stripes. 87 b. White striping in the pricked area for 10 inches above and 2 inches below. A few red spots in the white stripes, and red striping in the pricked area of the midrib. Blanca. 88 a. White striping in the pricked area, extending upward 4 inches on one side; downward a lesser distance. 88 b. Signs less conspicuous than in the others. 89 A. White striping in the pricked area, running up interruptedly 6 inches, and down a lesser distance. 89 b. Same as 89 A. .,...,,., 90 A. White striping in the pricked area. Some of the tissue dead. On one side it extends upward from the pricks 5 inches, with little rusty brown specks in the white. 90 B. Same as 90 a, only striping more conspicuous. The stripes extend downward a lesser distance than upward, and this remark is true of all so far examined. The checks appear to be sound. Jamaica. 91 A. A little red stain in the midrib of the pricked part. 91 b. Same as 91 A. Signs not conspicuous. 92 A. Red stain in the midrib of the pricked part about 0.06 inch wide and about 1 inch long. 92 B. Same as 92 a, but red stain in the midrib 2 inches long. No other signs. 93 a. Slight red stain in the pricked part of the midrib about 0.5 inch long and o. 125 inch wide. No other signs. 9-5 B. Less signs than in 93 a. . 94 A. Slight signs in the midrib, that is, a red stain 2 inches long and a little of the tissue dead. 94 B. Same as 94 a. 95 A. Same as 94 a. 95 B. Same as 94 a. 96 a. Signs slight. Red in the midrib for 2 to 3 inches. 96 B. Same as 96 A. The signs on this variety are inconspicuous. 36 BACTERIA IN RELATION TO PLANT DISEASES. Striped Green. 97 A. White striped in the pricked area. Small strip of dead tissue on either side of the midrib. White stripes run up and down 5 inches. Slight red stripes. 97 b. White stripe in the pricked area, extending upward 3 inches. 98 A. Shows distinct white stripes in the pricked area and some dead tissue on one side of the white stripe, extending upward 8 inches above the pricks. 98 B. Conspicuous red stain in the pricked part of the midrib, extending downward 2 inches below the pricks. Some dead tissue to either side in the pricked part. Interrupted white stripe on one side extends upward 8 inches and downward about 6 inches. 99 A. Dead tissue in the pricked area. Red stain in the midrib of that part, extending upward interruptedly 4 inches above the pricks. White stripe to either side of the midrib, extending upward about 4 inches above the pricks. 99 B. Some dead tissue in the pricked area. Conspicuous narrow white stripe to either side, extending upward 4 or 5 inches. Crysfalina. 100 A. Slight signs confined to the pricked area. Some red stain in the midrib. 100 B. Signs less than in 100 A. No red stain. 101 a. Slight signs in the pricked area. 101 B. Slight signs in the pricked area. Plant evidently quite resistant. 102 A. Slight signs in the pricked area. 102 B. Very inconspicuous signs in the pricked area. None above or below. 103 A. Very inconspicuous signs in the pricked area. 103 B. Same as 103 A. 104 A. Slight striping in the pricked area. Very faint red in the midrib. 104 B. More conspicuous striping in the pricked area; runs upward 2 or 3 inches; downward 4 or 5 inches. More con- spicuous red in the midrib. 105 A. Slight white striping in the pricked area; runs upward 2 inches. 105 B. White striping in the pricked area ; runs upward 3 to 4 inches; downward 2 inches. Very slight red mark in midrib. General Remark. — The disease is taking quite generally, but the signs are not yet very conspicuous. On June 20, the signs were progressing slowly. I could see increased striping since June 13, but no secondary signs. On June 28, 1905, the inoculations of May 22, on the leaves of the different varieties of sugar-cane in this house, were progressing finely. The signs of the disease on the inoculated leaves had become more pronounced in most cases than they were when the last records were made. One inoculated leaf on the Common Green cane had now entirely dried out, and many of the inoculated leaves on several varieties showed long yellowish-white stripes, much longer than at the last writing. The Crystalina, which appeared at first to be quite resistant, now showed marked striping on its inoculated leaves. The most resistant varie- ties at this time appeared to be Jamaica and Louisiana No. 74. On July 7, 1905, the condition of the sugar-cane, inoculated May 22 in hot-house No. 4, was as follows : Cinta. 61 A. Leaf plainly diseased for a distance of 1 foot below the pricks and 2 feet above. Yellowish-white stripes with small red-brown spots on them. No secondary signs. 61 B. Whole leaf diseased. Dying from the apex back, and yellow striping toward the base. 62 A. Signs on the leaf now extend down to the base of the blade and up a distance of 4 feet. No secondary signs. 62 B. Same as 62 A. 63 A. Whole leaf diseased. Dead striping extends up at least 4 feet. No signs on the uninoculated leaves. 63 B. Signs extend to the base of the blade and up a distance of several feet. 64. No record. 65 A. The striping is visible on this leaf for a distance of 3 feet upward from the pricks, and downward nearly to the base of the blade. No constitutional signs. 65 B. Whole leaf diseased; nearly all of it dried out. 66 A. White striping for about 2 feet. No general signs. 66 n. White striping extending to the base of the leaf from the pricks, and upward 2 feet. 67 A. White striping extending from the base 2.5 feet above the pricks. Dead on one side for a long distance. 67 B. White striping extending from the base about 2 feet up. Dead on one side for about 10 inches. No secondary signs. Caledonia Queen. 68 A. Inoculated leaf. White striping from base to within a few feet of the top. Half of the blade dead. 68 b. White striping the whole length of the leaf. No secondary signs. 69 A. White striping the whole length of the leaf. One edge of the leaf dead to within about a foot of the top. 69 B. White striping extending above the pricks 2.5 feet and only about 1 inch below. No secondary signs. cobb's disease of sugar-cane. 37 70 A. White striping extending from the base of the leaf to within about a foot of the top. Both edges of the leaf dead from the base to 2 feet above the pricks. No secondary signs. 70 B. White striping extending from the base of the leaf to within a foot of the top. One edge dead to within 2 feet of the top. 71 A. Comparatively little white striping; extends only about 10 inches above the pricks and to the base. No second- ary signs. 71 B. Diseased to within 2 feet of the top. Diseased downward to the leaf-sheath. 72 A. Leaf diseased the whole length; a good part of it on one side, for a distance of 3 or 4 feet. No secondary signs. 72 b. Dead, including the sheath. 73 A. Entire leaf dried out ; not only the blade, but the sheath as well. No secondary signs, i. e., as yet none of the uninoculated leaves are involved. 73 B. Can not locate the inoculated leaf. The label is on a leaf which appears not to have been inoculated. Pricked leaf may have fallen. 74 A. Diseased to the apex. Dried up along both sides toward the base, and yellow on one side almost to the apex. Diseased condition extends down into the sheath. 74 B. Inoculated leaf badly diseased on both sides, nearly to the apex. Diseased condition extends nearly to the base. No distinct constitutional signs. 75 A. Signs extend up a distance of 2 feet above the pricks and a few inches below. 75 B. Narrow yellow stripes extend up a distance of more than 2 feet above the pricks and downward nearly to the stem. No secondary signs. 76 A. Entire inoculated leaf dead and dried out. No constitutional signs. 76 B. Entire inoculated leaf dead and dried out. 77 A. Entire inoculated leaf dead. No secondary signs. 77 B. Not found. Possibly fallen off, or removed by the gardeners. Louisiana No. 74. Very little white striping through the pricks. Some red stain. Tissue is white around the pricks. No general signs. Rzd stain around the pricks in the midrib and about 6 inches above. Small white stripes running through the pricks in the blade. No other signs. Red stain around the pricks in the midrib. White stripes through the pricks in the blade. No secondary signs. The check-pricks on these plants show no signs of disease. Red stain in the midrib, passing through the pricks and extending about 2 inches above and below. Tissue white around the pricks in the blade. No other signs. Red stain around the pricks in the midrib and white stripes through the pricks in the blade. No other signs. No signs. Slight red stain in the pricked area of the midrib. Slight red stain in the pricked area of the midrib. Tissue white around the pricks in the blade. No other signs. Same as 82 A. Dead stripes in the pricked area, extending upward a foot or two and downward to the sheath. No record. Common Green. Signs confined mostly to the pricked area and a foot or so above it. Entirely dead, including most of the sheath. The leaves immediately below this and immediately above are alive. Dead stripes in the inoculated leaf, extending upward a distance of 2 feet. Dead stripes in the inoculated leaf, extending to the leaf-sheath and upward a distance of about a foot. Dead stripes in the pricked area, extending down to the sheath and upward a distance of a foot. Slight yellow striping further out. Signs less conspicuous, but dead in pricked area, and yellow stripes above and below. No constitutional signs. Dead striping in the pricked area, and yellow stripes with small reddish specks in them, extending upward above the pricked area 15 inches. 87 B. Much like A, but rather more diseased. No secondary signs. Broke off examinations here owing to the extreme heat. No constitutional signs on any of these varieties. These notes are given in full because of what followed. This lot of cane has been examined from time to time all the summer and autumn, but has shown no secondary signs of the bacterial disease, or at least none which could be attributed to it beyond doubt. Now (October g, 1905), quite a good many of thecaneshave lost many of their lower leaves, and some of them have sprouted out at the base, but there is not any distinct white striping of the uninoculated leaves which can be ascribed to the disease. The canes are all tall and jammed up against the roof of the house, which is too low for them. My impression is that the inoculations were made when the canes were too old, and particularly after they had made a slow growth through the whole of the winter. which would undoubtedly result in hardened tissues in the basal nodes, so that the bacteria would have difficulty in passing from the inoculated leaves into this part of the stem and into the upper and softer tissues. It was a new hot-house and it was not known that there would be great inequalities in the temperature. As a matter of fact we selected the cold end for the cane, but did not know it until it was too late to transplant it. 78 A. 78 B. 79 A. 79 B. 80 A. 80 B. 8l A. 8l B. 82 A. 82 B. 83 A. 83 B. 84 A. 84 B. 85 A. 85 B. 86 A. 86 b. 87 A. 38 BACTERIA IN RELATION TO PLANT DISEASES. The following final observations were made January 13, 1906: These canes, planted in the fall of 1904 and inoculated May 22, 1905, grew very slowly through the first winter season and were rather old and woody at the base when inoculated at the end of May. The primary signs developed first on the inoculated leaves in the vicinity of the needle-pricks and ran down to the stem, but secondary signs have not been visible. The canes which have been inoculated 8 months are large and have been large and leafy all summer, too tall in fact for the house in which they were grown. They are still quite leafy, although a good many basal leaves have died. I looked repeatedly through the summer and autumn for dis- tinct signs of white striping in the uninoculated leaves and could not satisfy myself that there were any. Some of the labels have dropped off, and it is now hardly possible in many instances to know to which cane of the row the label belonged. I cut out the inoculated rows first and dissected all of the canes, dictating results to the stenographer from time to time as I cut and made examinations under the hand-lens : Cinta. First row to the north (inoculated row): Removed a large stem, which is leafy to the base. Find it sound within. From its soft texture it is pn ibably a younger shoot which has developed since the date of the inoculations. vSplit the base carefully and examined for presence of yellow stain or red stain in the stem; there is none. Removed another stem, basal leaves of which are dead. It has not reached nearly the diameter of the preceding. It appears to be an older stem. The top looks sound, and there are no indications of disease in the stem. Neither of these canes had any label on them. Removed a cane that is labeled 62b. It is a large old cane which has lost its leaves nearly to the top. As before remarked, I do not have much confidence in the labels now being on the particular canes which were inoculated, since in some instances at least the gardeners picked up fallen labels and wired them to any convenient place. No disease in the upper part of the cane. Cross-sectioned every internode; no disease in any part of the cane. Large leafy cane, upper part of which is quite soft, showing that it has grown since the date of the inoculation. Sound within. No recent sprouts from the base of this clump. Next clump : Removed 2 young sprouts which may be about 6 feet high. They are both sound at the base, and also further up. These have grown within the last 2 or 3 months. A very large cane ; extremely thrifty leaves, except the basal ones, which are dead. This also is a cane which has grown since the sprouts were inoculated. Tissue soft and immature (in the lower part). Perfectly sound. There remain in this clump 2 old canes; these are leafy at the top, leafless for the lower 4 feet or so. Stems small, firm. First cane sound in the upper part; also in all other parts. No yellow slime in the vessels, and no red stain. The second old cane is like the first, but has lost more leaves. It is leafless to a height of 6 feet. Top part sound. In the basal part I find a brown stain in one group of vessels, which, however, is very slight, and soon runs out. Third clump on this row: Label 63a. This is a cane dating from the time of inoculation. It is leafless for a height of 6 feet. It is sound at the top. It has red bundles in the stem at the base. There are a dozen or fifteen of these. The stain in the vessels disappears 1 5 inches higher up. There is no reasonable doubt that the inoculation has taken slightly in this stem. The staining seems to be most extensive in the vicinity of the basal half dozen nodes. The cane is sound externally. Under the hand-lens I see bacterial ooze from some of the red-stained vessels. On splitting the stem some portions of the bundles are seen to be red and other portions to be pale yellow, the stain being irre- gularly distributed in them, just as it was in the set of canes previously inoculated. This third clump lias quite a number of young shoots coming out of the ground. These are sound at the base. Old cane. It dates from the time of inoculation and is leafless for a distance of 6 feet. Sound throughout. ( lid cane, leafless for a height of 6 feet. Sound throughout. A large cane, leafless for the first 4 feet. Very green above. Upper part sound. Middle por- tion rather undeveloped. Cane has probably grown since the last inoculation. Basal part sound. All the rest sound. The next clump, same row: Voting shoot. .Sound at base and all the rest of the way. Old shoot, leafless for a distance of 5 feet. Whole cane sound. These canes are purple-striped. cobb's disease; of sugar-cane;. 39 Next clump : 3 young shoots. Sound. Older shoot, very leafy nearly to the base. Must have grown since the date of inoculation. Sound throughout. Old shoot, bears label of 67a and 676. It is leafless to a height of 6 feet. It is sound throughout. My custom is to section top and base and every internode between before deciding. Old cane, leafless for first 6 feet. Sound throughout. Another clump on same row: 3 young shoots. Sound at the base and above. Old shoot, leafless for a distance of 6 feet. Sound at the top; sound at the base. In the second internode above the base there is a red stain in one bundle, which soon runs out; plant sound otherwise. General Remark. — All that precedes relates to first (north) row of Cinta. Only one plant on this row (No. 63) shows any distinct indications of bacterial infection, and in this the signs are slight and confined to five internodes. They are here, however, beyond any dispute. Having waited some little time there is now a distinct, yellow, bac- terial ooze from the diseased vessels of this stem, which for the most part are central vessels rather than peripheral ones. The nodal infection is most abundant in two of the nodes, which probably correspond to the two inoculated leaves. Mr. Johnston made plates from No. 63, and got numerous, round, yellow colonies of typical appearance. They came up rather slowly. Cultures were saved from six colonies on January 24, and subcultures from three of them were used to make inoculations 106 to 140 of February 9, 1906. On January 17 I continued my examination of canes. (Labels misplaced in some instances, no doubt.) Caledonia Queen. North (inoculated) row: Two recent shoots. Very green; leafy; sound inside. Five more young shoots from the same clump, all about 6 to 8 feet high. Sound both externally and within. One more young shoot of the same character. No signs of disease. There are three old canes in the clump. The first one bears label No. 77a. It is a tall cane, leafless for the first 8 feet, leafy the next 4 feet. The cane is greenish colored on the surface. The upper leaves do not show any white striping. Some undeveloped leaves around the terminal bud are dead. Shortening the cane several internodes; these dead leaves continue, and are surrounded at this level by five healthy leaves and by the inner living bud. Shortened another 2 feet. Stem sound. Stem continues sound in every internode for a distance of more than 9 feet. There is no indication of disease anywhere in the cane beyond a slight red stain in two of the vascular bundles near the base. Another large cane, like the last in external appearance. Terminal bud dead. Some red stain in the central tissues under the terminal bud. This soon runs out and does not look like the bacterial cane disease. Shortened the stem in every node for many feet. There is no indication of yellow or red staining in the vessels, except close to the ground, where two or three bundles have a slight red stain. No disease. Third cane: This is also an old cane, not labeled. Sound externally. Leafy only at the top. First red stain in the bundles was met at a distance of 4 feet from the ground. Runs only a short distance. No other stained bundles were encountered anywhere in the stem, which was sound. The next clump : Two young vigorous shoots. Very leafy. Sound externally and internally. Three old canes. One bears label 75a, and is leafless for a distance of about 6 feet. This one has two or three red bundles in an internode close to the ground ; 2 feet higher up the leaves are dying at the margin, but are not white striped. Sound at the top within; continues sound for many feet. Every internode cross-sectioned. Continues sound down to the third node from the ground, which has the before-mentioned red stain in a few of the vascular bundles. In the node above this and the node above that one also there is a little red stain. No bacterial ooze. Next cane : A few red bundles in the third internode from the ground. The first three inter- nodes are very short. This is an old cane. The leaves are all dead up close to the top. Terminal bud dead. Leafy under the terminal bud. Sectioned every internode from top to base. Sound down to within 3 inches of the ground. At about this height there is a small cavity in the center of the stem and slight red staining of some bundles. Injury very slight. This cavity is in no way con- nected with the surface of the stem at this level, and is due, I think, to the inoculations. Third old cane from same clump : No red stain at the base. This is a large cane, naked up to a height of about 10 feet, and has only a few good leaves. Sound within from the top downward a distance of many feet, in fact entirely to the base. 40 BACTERIA IN RELATION TO PLANT DISEASES. As noted the other day, the labels are lost in many cases, and we can not tell whether this was an inoculated cane or not. Next clump: Young shoot, very leafy and green. Sound within. An old shoot. Sound at the base. Cane destitute of leaves for the first 9 feet. Sound within at the top; sound all the way down. Next clump: Small old cane. Sound at the base. Stem naked for the first 8 feet. Leaves above not striped. Terminal bud sound. Stem sound within all the way down. Another old cane. Three red bundles at the base on cross-section. Stem naked nearly to the top. No white striping of the foliage. The gardener topped this cane some weeks ago in order to shut the ventilators of the house ; the result is that there are purple-red bundles under this topped part for a distance of about a foot down. Six inches under they are 40 in number ; 6 inches further they are reduced to 3 in number; below the next node they are reduced to 1 ; under the next node this one disappears. These are clearly due to topping the cane. The cane from this point is sound down- ward for many feet, until within a few inches of the ground (the last 5 or 6 nodes). In these there are a few bundles stained yellowish-red, and from these on cross-section there is a slight yellow bac- terial ooze. Another old cane: Sound at the base, except that there are three red-stained bundles. Desti- tute of leaves for the first 4 feet ; leafy the rest of the way, but some of the leaves dead on one margin. No distinct white striping. Sound at the top on cross-section, and for many feet, that is, down to the extreme base. Another clump : Two young shoots about 6 feet high. Very leafy, sound within. Old shoot. Very woody at the base and hard to cut. Stem much larger 2 feet above the base. Buds pushing slowly. Naked the first 4 feet; very leafy the rest of the way. No external signs of disease. Cross-sectioned from the top downward. Sound at the top; continues so for many feet, that is, entirely to the ground. Old cane. Destitute of leaves for the first 6 feet. Sound at the top on cross-section, and all the way down. Another old cane. Nearly destitute of leaves. Sound at the top on cross-section; continues sound for many feet. No indications of disease anywhere in the interior of the stem. The remark about labels remains in force, that is, they have fallen off in most cases. Another old stem leafless for many feet. Sound at the base. Top leaves not white-striped; terminal bud dead. Sound under the terminal bud. Stem continues to be sound on cross-section for a distance of many feet down. First sign of red bundles about 6 inches from the ground. These are very few in number and soon run out. No signs of any yellowing of the vessels, or of bacterial ooze. Another old stem ; leafless nearly the whole length. Sound at the ground ; also at the top and for many feet down. I find no red stain or yellow slime anywhere in the vascular system. Another old stem; leafless nearly the whole length. Sound at the ground, with the exception of a red stain in one or two bundles. Upper leaves not white-striped. Sound within at the top; con- tinues sound for many feet. No evidence of disease in any part. Young shoot. Dwarfed and tough at the ground, expanded above. Very leafy at the top. vSound externally; sound within. Another clump. Young shoot. About 6 feet high. Very leafy. Sound externally. Sound within. Old shoot. Sound at the base. Leafless nearly to the top. Upper leaves not white-striped. Terminal bud dead. Sound just under the terminal bud. Stem continues sound for many feet. The only signs of disease are near the ground, where there are a few red bundles beginning in one of the nodes. Old cane. Sound at the ground. Leafless for 6 feet. Upper leaves not white-striped. Cross- sectioned from top downward; sound within all the way down. Another old cane, last one of this row. Sound at the base. Leafless for many feet. Upper leaves not white-striped. Sound at the top on cross-section. Continues sound all the way down, except in one node and internode near the base, where there are a few reddened bundles, and very slight evidence of bacterial occupation. General Remark on this Variety. — There is no doubt whatever that the bacteria entered the stem in some of these plants, but they were not able to make their way through these stems so as to induce any general disease. If one did not know that they had been inocu- lated and did not search critically for the presence of the red bundles and the bacterial ooze, he would never suspect that anything had been done to the canes. cobb's disease of sugar-cane. 41 Louisiana No. 74. Inoculated row of tall cane (same remarks about loss of labels apply as before) : Cut out from first clump 3 young, very leafy shoots, varying from 3 to 6 feet in height. Sound externally and within. Observed also 2 or 3 buds just pushing out of the ground. Old shoot, sound at the base, leafless for about 4 feet. Very leafy above; leaves green. Cross- sectioned from the top downward. Stem sound within all the way down. Another old shoot. A few red bundles at the base of the stem on cross-section. Stem leafless for lower 6 feet, and most of the leaves above are only partly alive. Terminal bud dead, evidently due to injury by jamming against the top of the house, i. e., cut by closing the ventilators, as in the other cases of dead terminal leaves and buds. Sound immediately under the terminal bud. Stem continues sound on cross-section for a distance of many feet. First evidence of a reddened vascular bundle about a foot from the ground. This stain runs out, and no others are visible. Stem sound. Another old cane. Sound at the base. This bears label 82a. Leafless for the first 6 feet; leafy above. Leaves not white-striped. Cross-sectioned from the top downward. Stem sound within for a distance of many feet. No evidence anywhere of anything wrong. The next cane (apparently an old one) is rotted, nearly to the ground. .Sound under the rotted part. The rot, I believe, is not due to any inoculation ; in fact, I think the cane grew after the date of the inoculation ; it is hardly tall enough to have dated from so long back. Next cane, an old one. Sound at the base; naked for a distance of 5 feet. Upper leaves green, battered somewhat, and red-striped in some places along the midrib. Sound within from the top downward a distance of many feet. Next clump: Five young shoots of varying heights (3 to 5 feet). All very leafy and healthy looking. Sound within. Old shoot. Sound at the base. This bears label 80a. Small diameter. Leafless for the first 5 feet. Most of the upper leaves dead. Sound at the top on cross-section; continues sound for a distance of many feet. No internal evidence of disease anywhere in the stem. Another old stem. This is a large cane, leafless nearly to the top. Top dead and rotted. Stem sound under the rotted portion. Continues sound for a distance of many feet. Sound at the base. Within about one foot of the ground a red bundle appears, and a little lower down there are several others. These continue through the next internode, but there are not many of them. I believe these particular red bundles are due to the bacterial inoculation ; at least they have all appearances of being that type of disease. They are few in number and the stem as a whole is sound. The bundles have the characteristic of being red and yellow by turns. Another old cane. Large cane leafless nearly to the top. This has been topped within the last few weeks to shut the house, and some of the bundles under this top part have red stains in them. A little lower down this red stain disappears. Stem continues sound on cross-section for many feet. The first indications of anything wrong are about 2 feet from the earth. There are some dead cells in the center and red stain in a very few bundles. This runs out about 6 inches lower and the stem from that point down is sound. Another clump : Some very young shoots ; these are sound. An old cane leafless nearly to the top. Has two labels, Nos. 97a and 976. Sound at the earth and within from the top downward a distance of many feet. First red stain about 2 feet from the ground, restricted to a few bundles in the middle of the stem. This disappears a little further down, that is, it is restricted to one node and two internodes. Another old stem, naked nearly to the top. Upper leaves not white-striped. Stem at the top sound on cross-section; continues sound all the way down. Another clump : Four young shoots of varying ages, 2 to 5 feet high, and very leafy. Sound within. Shoot about 8 feet high. Very leafy, except near the base; leaves very healthy. Sound within. This undoubtedly grew after the inoculation. Old, large shoot, naked for the first 4 feet, top leaves green. On cross-section the top is sound and the stem continues sound for a distance of many feet. No indications anywhere of bacterial ooze or red stain in the vessels. Another old stem. Leafless for the first 4 feet. Green leaves above. Sound at the base. Cross-section of the upper part of the stem sound. Stem continues to be sound on cross-section all the way down. Another old stem. Leafless to the top. Top part rotted. The rot is not like the bacterial rot ; it is a soft, pale brown uniform rot. It stops quite suddenly at a certain node, and from this on the stem is sound on cross-section clear to the base. Old stem. Sound at the base. Leafless to the top. Top part soft-rotted. It had been short- ened to get it inside of the house, and like some others that have rotted at the top, it may have been 42 BACTERIA IN RELATION TO PLANT DISEASES. frosted somewhat before the house was shut, i. e., it projected through the ventilators. The rot ceases suddenly at a distance of about 7 feet from the ground, and the rest of the way the stem is entirely sound. Remark.- -This ends the first (north row) of the Louisiana No. 74. There are no indi- cations of bacterial disease in it beyond the very slight ones mentioned under particular canes. I >n January 19, examination of the inoculated cane was continued, as follows: Common Green. North (inoculated) row: Six young shoots from the base in a rather unhealthy condition ; height 2 to 3 feet. The leaves are not white-striped, but many of them are dead on the margins. 1. Center dead. No red or yellow stain in the bundles. 2. Center dead. No red or yellow stain in the bundles. 3. Two middle leaves dead. Brown, water-soaked. No sign of the gum-disease. 4. Outer leaves dead. Center of the stem sound. Further up the terminal bud is dead and soft- rotted, but there are no signs of the yellow gum-disease. 5. Central bud rotted out. I see no evidence of Cobb's disease. 6. A lew red bundles at the extreme base. Further up the whole stem is dead and soft-rotted ; not Cobb's disease; it seems to be a brown soft rot which has run in from the terminal bud. Old large cane. This bears label 83a. All the lower leaves are dead, that is, up to a height of 8 feet. Sound at the base. Sound at the top on cross-section; continues sound on cross-section for many feet. The onlv evidence of disease in the vascular system is about a foot from the ground and ci m lined to one node, a bundle of which is stained brownish, and to another node about 6 inches lower, where there is a red-stained bundle ; that is, disease absent or extremely slight. No internodal stains, so far as I can see. Another old stem. Sound at the base. Basal buds well developed. Very leafy and green for many feet. It is undoubtedly a shoot which grew after the date of inoculation. Sound at the top on cross-section, and throughout. Young shoot from the next clump. Heart rotted out. No evidence of yellow bacterial ooze in this stem. Another young shoot. Heart rotted out in the same way. Old shoot, very tall. Leafless for the first 6 feet. Upper part of the shoot has sound, green leaves. The length of this cane is about 10 feet (that is, of the stem itself). On cross-section not a trace of disease either in the nodes or internodes. Another old cane. This bears label No. 846. All of the leaves are dead, except a few at the top. It is a large cane, and mature for a distance of at least 8 feet. Cross-sectioned each internode from the top downward. Cane sound for many feet. Basal buds well developed. At about a foot from the ground there is one internodal bundle stained red-yellow, and the node below has several vessels in it which are stained red and yellow. Signs of disease very slight indeed. They seem to be re- stricted to this node and internode, and are scarcely worth mentioning. Large ripe old stem. It bears label 85a. It is sound at the base. The basal buds are well devel- oped. The leaves are dead nearly to the top. It is a tall cane, and sound externally. Cross- sectioned from the top downward. No signs of disease anywhere. Old stem. Sound at the base. Basal buds well developed. Leafless except for a few green leaves at the top. Cross-sectioned from the top downward in every node : No signs of disease in any part of the cane. Old cane. Sound at the base. Basal buds well developed. Stem sound externally. Leafy toward the top, leaves healthy looking. Cross-sectioned : Interior of the stem healthy throughout. Healthy young shoot, about 5 feet high. Stem sound on cross-section. Old cane, leafless nearly to the top. Sound at the base. Basal buds well developed. This bears label S6b. The diameter of this cane is about 1.5 inches. On cross-section, one or two red ves- sels in the stem 6 feet from the ground; these soon run out to appear again a little lower down; they extend through several nodes, and a larger number appear at a level of about 3 feet, but are not numerous even there. General appearance of the stem is good. Red stain runs out again a little [i m 1 r d( >\vn. Basal 2 feet of the stem entirely sound. Laid aside a piece of this stem to see if there would be any bacterial ooze. Cane about 8 feet high, evidently developed since the inoculation. Sound externally and within. Old cane, quite leafy at the top, naked below. Basal buds well developed. Sound at the base and within its whole length. COBB S DISEASE OF SUGAR-CANE. 43 Old mature stem. Sound at base. Basal buds well developed. Upper part leafy. Cross- sectioned every internode. One red bundle about 4 feet from the ground. The stain passes from the node into the internode below and runs out; it is slight, and confined to 2 or 3 bundles. Down 2 internodes there are two bundles with red stain ; this also runs through the node into the upper por- tion of the next internode and then ceases. The lower 2 feet of the cane has occasional red bundles, very few and scattered. Old cane. Bears label 87a. Sound at the base. Basal buds well developed. Only a few living leaves. Top of cane dead and a sort of water-soaked brown rot (frost?). No bacterial rot. This condition runs out about 4 feet from the ground. From this point down the cane throughout is sound within. No evidence of infection with Cobb's disease. General Remark. — Under the hand-lens I can see no indications of bacterial ooze from these stained bundles. No distinct indication of Cobb's disease in these plants. This completes observations on the inoculated row of the Common Green cane. Blanca. North (inoculated) row of this variety: Old cane. Sound at the base. Leafy in upper half of the stem, the leaves quite green and of good appearance. On cross-sectioning the stem, found no signs of disease, with the exception of one red bundle about 4 feet from the ground, which soon runs out. Old stem. Sound at the base. Bears labels 90a and 906. Leafless nearly to the top. Basal buds well developed. Stem looks sound externally and ripe. Made cross-sections and found the whole interior sound. A very juicy, sweet cane. A large old cane. Sound at the base. Basal buds well developed. Leaves on the lower half dead. Stem looks sound externally. Cross-sectioned the stem and found the interior sound in every internode from top to base. Old cane. Sound at the base. Bears label 896. Only upper leaves living. Cane is sound externally and appears to be mature. Basal buds well developed but not pushing. Cross-sectioned stem in every internode and found the interior sound. Old shoot. Sound at the base. Leafy only near the top. Stem well developed. Sound exter- nally. Basal buds well developed, but not pushing. Cross-sections of stem show interior to be sound throughout. Old stem. Sound throughout. Old mature stem. Only the upper leaves living. Bears label 88a. Basal buds well developed but not pushing. Looks healthy. Cross-sectioned every internode ; stem free from disease. A very juicy, sweet cane. Old stem. Sound on cross-section at the base. Upper part leafy and leaves healthy. Basal part of the cane looks healthy also. Cross-sectioned every internode and found the whole plant healthy. There are 4 or 5 young shoots from this stool, but all look so healthy that I have refrained from cutting them out. General Remark. — No evidence of Cobb's disease in this row. This is the end of the inoculated Blanca. Jamaica. The two rows of Jamaica have sent out a great many suckers, which now bear green leaves and are about 4 or 5 feet high. The notes are on the north (inoculated) row. Old cane. Sound at base. Rotted at the top, but not with Cobb's disease. Another old cane. Label 9 1 a and 9 1 b. Leafy above. Leaves green. Cane looks sound exter- nally. Basal buds well developed. Made cross-sections of stem from top downward in every inter- node and found cane sound until about 4 feet from the ground, when a few red bundles appeared, which soon ran out. These red bundles showed no bacterial ooze. Sound at base. No certain indi- cations of Cobb's disease. Cane very tender, juicy, and sweet. Old cane. Sound at base. Basal buds well developed. Cane mature. Top bears green leaves. Made cross-sections from the top downward. Found the interior sound for a number of feet, then came to an internode about 5 feet from the ground with some red bundles. These continue through several internodes, becoming more numerous at about 3 feet from the ground. At this level there are about 1 5 of these unsound bundles. Disease continues in the next internode below and the one below that, but runs out in the next lower one. Throughout this region the exterior of the cane appears to be almost perfectly sound, there being only a trace of reddening here and there in the nodes and inter- 44 BACTERIA IN RELATION TO PLANT DISEASES. nodes. Examined these red bundles under the hand-lens, and thus far find no distinct evidence of bacterial ooze. Most evidence of disease in this stem of any examined this morning, but it is not seriously diseased. The numerous young shoots look so healthy that I will not cut them out. Old shoot. Bears label 926. Sound at base. Cane well matured. Sound on the surface. Leafy toward the top and leaves healthy looking. Buds well matured. Cross-sections of the stem from the top downward show the upper portion to be sound. At a distance of about 6 feet from the ground stained vessels appear (3 or 4 in number) although the surface of the cane is sound in that internode. Stain runs out in the next internode and appears in the third one below. Here again it is confined to the middle of the stem and the middle of the internode, 3 or 4 bundles being reddened. It appears again in the next internode, and runs out in the next below that. Appears again with a greater number of vessels stained in the node below that. All the way down the exterior of the stem is perfectly sound. The basal 2 feet of the stem appears to be free from stain. The cane is very sweet and juicy. Examined these stained internodes under the hand-lens. I find no distinct evi- dence of bacterial ooze; will look at it under the microscope later. Old cane. Sound at the base. Basal buds well developed. Healthy looking, well-developed, mature cane. LTpper leaves green and healthy. On cross-section, find top perfectly healthy. About 6 feet from the ground I begin to get red-stained vessels in the middle of the stem (a few only) ; these increase in number in the next internode, run out in the one below that, appear again in the next one, are still more abundant in the one below that (always in the middle of the cane), run out in the one below that, appear again in the next one, appear again in the next three, and disappear about 15 inches from the ground. The external portion of this part of the stem appears to be sound. The interior of the basal portion of the stem is sound. Examined these red vessels under the hand-lens. No immediate bacterial ooze. Old cane. Sound at the base. Good size. Sound externally. Leafy at the top only. Upper leaves healthy looking. Cross-section of the upper part of the stem perfectly sound. Stem con- tinues to be sound until one reaches a level of about 6 feet, when a few red bundles appear; these run out a little lower down. Stem very tender, juicy and sweet. The red bundles appear again in the third internode. They appear again in the next one sparingly, and in the next one below that; I am not sure that it may not be due to the age of the cane. The cane is fully ripe, if not over ripe. Basal part of the cane sound. Old cane. Sound at base. Buds well developed. Stem sound externally. Leafy at the top and leaves healthy. Only the lower 4 feet of the cane is naked. Cross-sections at the top show the interior of the stem to be perfectly healthy. This state continues downward the whole length of the cane. I believe this cane grew after the plants were inoculated. Old rather small cane. Sound at the base. Basal buds well developed. Stem sound exter- nally. Upper part very leafy and perfectly healthy. Evidently grew after the inoculations. It bears 17 healthy leaves. Cross-sections at the top perfectly sound. Whole interior of the cane sound. ( >ld cane. Bears labels 94a and 946. Sound at the base on cross-section and externally. Basal buds well developed; not pushing. Basal portion of stem naked. Only the upper part bears healthy green leaves, the middle portion of the cane being covered with dead leaves. This cane is well devel- oped and mature for a distance of more than 6 feet. On cross-section perfectly sound at the top and for several feet, then a few red bundles begin to appear; these run out in the next internode, appear again in the second one, continue in the third and fourth one, are more abundant in the fifth one, and still more in the sixth one. Much less in the seventh and eighth one, and run out in the ninth one at a distance of 16 inches from the ground. The rest of the stem is free from internal stain until the fifth short node from the ground, which also has a few red-stained bundles; these run out in the next inter- in ide under. Examined under the hand-lens: No distinct evidence of bacterial ooze. Old cam-. Sound at the base. Basal buds well developed. Very leafy at the top, and leaves healthy. I believe the plant grew after the inoculations were made. Cross-section at the top per- fectly healthy; continues so For the whole length of the stem. Large old cane. Sound at the base. Leafy only near the top. Extreme upper leaves green. Stem healthy within for a distance of many feet, that is, until one reaches a height of about 4 feet from the ground, when some red bundles appear in several internodes. These continue until one comes to about a foot from the ground, where they disappear. Basal part of the stem perfectly healthy. Cane very tender, juicy, and sweet. Examined the red bundles under the hand-lens: No distinct evidence of bacterial ooze. 1 >ld cane. Sound at the base. Basal buds well developed. Cane mature. Bears label 956. Upper leaves well developed and green. < >n section of the stem upper part sound. About 5 feet from the ground red stain begins in the middle of the stem and continues through several internodes, COBB'S DISEASE OF SUGAR-CANE- 45 getting worse and worse. In the third one down there are 30 or 40 stained bundles. The surface of this internode looks perfectly sound. The next node below contains fewer stained bundles; the next one down is very bad, mostly in the middle; the next one below that is also very badly stained; the next one below that shows less stain; the next one below that is free from stain, except one or two bundles. The next one (about a foot from the ground) shows no stain. From this point down the cane seems to be perfectly sound. Examined these red vessels under the hand-lens; result: no dis- tinct bacterial ooze, except possibly in one place, and it will take the compound microscope to settle this. A large old cane, 2 inches in diameter at the base. Basal buds well developed. Stem naked up to about 6 feet. Cross-section at the top of stem sound ; continues sound for many feet. The cane is so soft on cutting that I believe it grew since the date of inoculation. It is sound throughout. Old cane. Bears label 766. Basal buds well developed. Cross-section at base sound. Leafy only in the upper part. Leaves are not white-striped. Stem sound on cross-section for many feet. First signs of red stain in the middle of the stem at a level of about 3 feet; slight in the next internode, and in the next two. It seems to run out in the next one about a foot from the ground. From here downward the stem shows no signs of disease, except a bright red bundle in one of the basal internodes. Examined under the hand-lens. No evidence of bacterial ooze. Staining comparatively slight in this plant. I have left all of the numerous young shoots, they look so healthy. General Remark. — There is more red stain in these stems than any other lot thus far examined, but whether it means that there is a slight bacterial infection or only that the stems are very old and going naturally over into a condition preceding decay is what I do not know. The general appearance of the pale reddened bundles is non-typical for Cobb's dis- ease as I have seen it in other varieties. Examined the suspected stems microscopically, and was not definitely certain of bacteria in any of them. On February 9, 1906, the examination of canes inoculated in house No. 4, May 22, 1905, was continued as follows: Striped Green. North (inoculated) row: Several old canes. Well developed ripe buds; large. No external signs in canes either at top or bottom. The canes were now cut open (every internode). One cane showed traces of red and yellow bundles in the basal part : Confined to a few bundles in a few nodes and internodes. Signs so slight that only the use of the microscope will settle it. Doubtful under microscope ; seem to be some bacteria. Johnston thought them bacteria. The young healthy shoots not cut out. Crystalina. North (inoculated) row: Sixteen large canes, most of them fully mature. Young healthy shoots not cut out. All canes large and sound looking. Free on cross-section at top and bottom from any signs of gum disease. A very few red bundles in one cane — a node near the base. One cane (less mature than the other) shows distinct red and yellow bundles (a few) in some of the basal nodes and internodes, and what looks like bacterial slime is oozing from some of the bundles, yellow slime from some and red slime from others. Will examine microscopically. Three other canes show a trace of red and yellow- bundles in one or two basal nodes (very slight, hardly to be called diseased). Laboratory note. — Under the microscope the one most affected showed great quantities of bacteria (short rods, single or paired) in the yellow ooze, and also in the red ooze. Stained and mounted the latter (fig. 18). Mr. Johnston has made plates from this stem. *Fig. 18. — Contents of red slime from a vascular bundle in the interior of the stem of a sugar-cane (variety Crys- talina) 9 months after inoculations with Bad. vascularum. Surface of stem sound. Slide ss, Feb. 9, 1906. Stained with carbol fuchsin. Drawn with a Zeiss 3 mm. 1.40 n. a. oil-immersion objective. No. 12 compensating ocular, and Abbe camera. 46 BACTERIA IN RELATION TO PLANT DISEASES. February 14. Johnston's plates miscarried (probably too thinly sown). Miss Hedges then poured plates from Johnston's second bouillon dilution, which had been saved and was now moder- ately clouded, and got Bacterium vascularum in pure culture. Probably by far the larger part of the bacteria in this stem were dead, as in case of canes Nos. 30 and 36. February 19, 1906. The plates poured January 13 from inoculated plant No. 63 (Cinta) showed nothing for quite a number of days, so that we thought they had been inoculated too thinly. After- wards numerous yellow colonies came up, but it required a week or more. General Remark. — When I finished cutting out the cane on February 9 a stalk of Crys- talina was selected which showed yellow ooze from some bundles, and dark purple-red ooze from others. Two sets of plates were poured: one from the red ooze, one from the yellow ooze. Just as in the previous set of plates there was for a number of days no indication of colonies, but afterwards the plates showed numerous typical colonies of Bacterium vascu- larum. The plates poured from the red ooze showed no red colonies, but only yellow ones. This indicates two things: First, that the dark red ooze was due not to red-colored bacteria, but to a red reaction of the host plant; and, second, that when the bacteria have lain dormant for many months in old canes they are not resuscitated as early as from fresh growths. The same remark applies to other bacteria causing plant diseases. The results from this series of inoculations indicates also very clearly that it is difficult to obtain secondary (stem) infections on old canes by means of leaf-inoculation unless such canes have grown rapidly. The primary signs on these plants were entirely satisfactory, the constitutional ones so scanty that I had great reason to be thankful that it was not my first series of inoculations. As it was, the experiment proved more instructive than if it had been an exact duplicate of the first one. SERIES VII, 1906. On February 9, 1906, thirty-five young green shoots (second growth) were inoculated with Bacterium vascularum by means of hypodermic injections. To each of the slant agar cultures (1 to 3, February 7, descended from three separate colonies on Mr. Johnston's plate 2, January 13, poured from artificially infected sugar-cane No. 63) 10 c.c. of autoclaved redistilled water were added. The tubes were shaken until most of the pale yellow slime was washed off and this milky fluid was injected into the plants in 2 to 4 c.c. quantities. Most of the inoculations were made in or above the heart (2 to 4 pricks in each plant), but some of each variety were pricked in the leaves only with 6 to 8 pricks. The varieties inoculated were as follows: 106-110, Caledonia Queen, pricked in stem; m-112, Caledonia Queen, leaves pricked. 113-115, Louisiana No. 74, stem pricked; 116-117, Louisiana No. 74, leaves pricked. 1 18-1 19, Common Green, stem pricked; 120, Common Green, leaves pricked. 1 21-123, Jamaica, stem pricked; 124, Jamaica, leaves pricked. 125, Striped Green, stem pricked; 126, Striped Green, leaves pricked. i27_I33. Crystalina, stem pricked; 134-135, Crystalina, leaves pricked. 136-138, Jamaica, stem pricked; 139, Jamaica, leaves pricked. 140, Common Green, stem pricked. Local signs appeared on some of these plants within a few weeks, but constitutional ones never developed, at least not so as to be conspicuous, and the plants after making a tall growth and standing for 18 months were finally cut down to make room for other things, without being examined internally. GENERAL REMARKS ON THE INOCULATIONS. In all, 140 canes have been inoculated in the greenhouses in Washington, 26 with Bad. vascularum, plated from the Australian cane, the remainder (except as noted under Series V) with descendants of this strain isolated from stem-bundles of the artificially infected plants at long distances from the point of inoculation (3 to 5 feet). The results have varied according to the variety and the condition of the plants at the time of inoculation. Canes, even of sensitive varieties, which had grown very slowly or were old and woody at the time of inoculation, were not susceptible to general infection. cobb's disease of sugar-cane. 47 Of all varieties tested Common Green has shown itself most susceptible. Louisiana variety No. 74 and Common Purple were very resistant, and all the others under the con- ditions of the tests were more or less so. Unfavorable conditions of growth rendered the variety test unsatisfactory, and the writer does not consider the results trustworthy. ACID CANES LESS SUSCEPTIBLE. The question of immunity is perhaps the most interesting one. To what is the im- munity of resistant varieties due? or of susceptible varieties under special conditions? It is a very interesting and perhaps a very complex problem, but also perhaps one relatively simple. Only a few observations will be noted here. Before making the first inoculations on Common Green cane, Common Purple cane, and Louisiana variety No. 74 (Feb. 1903), the freshly expressed juices of well-grown, sound canes of the three varieties were titrated with phenolphthalein and sodium hydroxide to determine their acidity. According to these titrations the sap of the Common Green cane was the least acid. Bacterium vascularum also grew very much better on steamed cylinders of this variety than on those made from the other two varieties. The cane used for this latter purpose was pared, cut into cylinders, put into test-tubes containing an amount of distilled water sufficient to cover less than half the cylinder, and heated a few minutes on three successive days in the steam sterilizer. The cut surface of the cane was then streaked copiously and the amount of growth noted. It was these unexpected results which led the writer to choose Common Green cane for his first inoculations. He believed it would be more susceptible than the other varieties, and such proved to be the case. The others were a hundred, yes, a thousand times more resist- ant to infection. The majority showed only local signs on the inoculated leaves or stems. Signs were not observed on the uninoculated canes from these stools, not even after many months. A few of the inoculated canes of these resistant varieties showed dwarfing with secondary signs on the leaves, but after 6 months, when the plants were cut down, there were to be seen for the most part only a few red bundles without distinct bacterial ooze, although in two stems there were a very few bundles containing yellow bacterial slime. The red bundles appeared only in the nodes giving rise to the inoculated leaves and in the adjacent internodes; they were not to be found in the greater part of the well-developed stems either above or below. These facts can be tabulated as follows: Table 4. — Relation of Bacterial Growth to Acidity oj Cane Juice. Variety. Acidity of sap (^ NaOH per liter). Growth of bacteria on sterilized cane cylinders. Susceptibility. C.C. I9.OO 32.00 31 .00 Good. Poor. Poor. Very great. Very slight. Do. It may be noted here also that the Common Green cane is the one which is most cul- tivated for eating, which would not be the case if the juice were very acid. On steamed cylinders of Striped Green cane and Striped Purple cane, which titrated + 20 and +32 respectively, the organism also grew very feebly. Another titration experiment was made in April 1906 (by Mr. Johnston and Miss Hedges) on the nine varieties of uninoculated canes which had been growing (very slowly) in the greenhouse since the winter of 1904-5. Eight of these canes were from the lot used for a variety test in the inoculations of May 22, 1905, which resulted rather unsatisfactorily even on the Common Green cane, due, no doubt, to the condition of the plants at the time of inoculation (slow growth, hard nodes). The ninth was a Cuban variety which had been growing in the greenhouse about the same length of time. The old uninoculated canes 48 BACTERIA IN RELATION TO PLANT DISEASES. were cut out on March 21, and the central or lower portions saved for planting. The juice titrated was taken from canes belonging to these cuttings. Between the date of cutting and the time of extraction of the juice for titration a period of 17 days elapsed, during which the canes were kept in the ice-box, the cut ends sealed with paraffin. The titration was performed in the same manner as previously. Two titrations were made of each variety. The results were as follows (Fuller's scale) : Cinta +25 Crystalina +33-5 Caledonia Queen + 24 Striped Green +25 Blanca +30.5 Common Green +40 Jamaica +19-5 A Cuban variety +20 Louisiana No. 74 +35 The writer has had no opportunity to repeat these tests on fresh canes. A sugar chemist, with whom the writer discussed the matter after the first titration, did not believe that well-grounded conclusions could be drawn from single canes ; the next titration might give very different results, as, in fact, proved to be the case (but so also did the inoculations). He said that in order to determine the comparative acidity of different varieties of sugar-cane, the mixed sap of many canes of each variety must be titrated. This I am ready to concede. So much depends on varying factors, e. g., soil, manure, sunshine, rain, time of year, degree of maturity, lodging, etc. The writer does not know whether any chemist has tested the comparative acidity of the sap of different varieties of sugar-cane. From a pathological standpoint, he believes that it would be very interesting. Many bac- teria do not thrive in acid culture-media and often a strong antiseptic influence is exerted. That there should be an average higher degree of acidity in some varieties of cane than in others is in accord with many facts in plant physiology, and perhaps the great susceptibility of some varieties is due to weak acidity, i. e.,low total acidity, or else to a smaller amount of some particular acid unfavorable to the growth of the organism and more abundant in resist- ant varieties. It is conceivable that the cane may contain mixed acids in varying propor- tions, one more antiseptic than the other, and this fact would not be brought out by ordinary titrations, but might be fundamental. This might explain why Jamaica was resistant to the disease when from its low total acidity I had anticipated that it would be sensitive. The difference in immunity is evident, whatever the explanation may be. The acidity of the cane-juice explains, furthermore, to the writer at least, why the bacteria tend to avoid the parenchyma and inhabit by preference the vascular system, the juices of which (Greig vSmith) are only slightly acid or neutral. They do finally make cavities in the parenchyma, especially in late stages of the disease and in the upper part of the stem, but in this part of the plant there is an excess of water, favoring rapid growth of the bacteria, and with this more vigorous growth there would be produced sufficient alkali to neutralize the inhibiting acids. Another reason, perhaps, why the bacteria are more abundant in the vessels is the better aeration, i. c, presence of more free oxygen in the vessels than in the parenchyma. CONDITIONS FAVORING THE SPREAD OF THE DISEASE. The writer has seen nothing to indicate that the disease is very infectious. In very bad cases, younger canes from the same stools sometimes showed etiolation, which the writer believed to be due to the secondary effect of the bacteria in the mother stem underground, but no signs were observed among canes growing from stools bearing no inoculated canes, although often these were for months in close proximity to infected canes. So far as it goes, and it must be admitted this is not a very great way, since insect enemies of the cane were absent and water did not rest long on the foliage, this indicates that the disease generally originates with the sets. We can well imagine, however, that under ordinary field condi- tions, with an abundance of dew or rainfall, and plenty of insect depredators, diseased plants might readily infect neighboring healthy ones, especially when young. Wet soil would tend to waterlog the plant, and would also favor the deposit of dew, in both ways favoring infection. COBB S DISEASE OF SUGAR-CANE. \<> According to Cobb, an excess of moisture seems to favor the development of this dis- ease. The conditions below Maclean, on the Clarence River ( New South Wales), where the disease is very prevalent, differ from those above Maclean, where the disease is rare, in the following ways: There is a greater rainfall down the river; the banks of the river are lower; the surface drainage is not rapid; and the nearly impervious clayey subsoil is within a few- feet of the surface. He says also: In the diseased fields it is apparent that the better drained parts suffer less than the others, though all the conditions except this one of drainage are the same. F.g. 19.* THE RED STAIN. In 1904, R. Greig Smith investigated the red vascular bundles of gummed cane and reached the conclusion that the stain must be due to the symbiotic action of an undeter- mined pvenidia-bearing fungus and a white slime-producing bacillus, named by him *Fig. n). — Bat If riii m vascularum in stem of sugar-cane received in 1902 from New South Wales The figure i< pre sents a bundle in cross-section. The ground tissue, endodermal sheath, and phloem are siill free, also a pari of the xylem, including Uk- two l>i.s pitted vessels Sectioned from paraffine ami stained with Flemming's triple slam, the contrast being not exaggerated. Slide 121 (9. 5° BACTERIA IN RELATION TO PLANT DISEASES. B. pseudarabinus. This is peritrichiate and non-sporiferous, liquefies gelatin slowly, and does not stain by Gram. He says of the fungus: In glucose-gelatin the mould produced a brilliant crimson-scarlet color, and it undoubtedly was the agent which was primarily responsible for the color of the strings. But from the presence of gum in the vessels I was of the opinion that the mould was accompanied by a slime bacterium, and that the complete phenomenon of red gum was brought about by the simultaneous growth of two organisms, — a mould and a bacterium. This view was confirmed during the research. It may, however, be men- tioned here that every portion of red vascular bundle that was taken did not contain the mould, but did contain slime-forming bacteria ; and from this we must conclude that the mould does not accom- pany the gum along the whole length of the string, but colors the gum which is carried along the ves- sels, perhaps by sap-pressure, perhaps by bacterial growth, or that the rapid growth of the bacteria starves out the mould after the color has been pro- duced. At any rate two things are certain: (i) The mould can, under certain conditions, produce the color and cannot produce the slime, and (2) the bacteria do produce the slime. Greig Smith did not reproduce this disease by in- oculations into living cane. The brilliant red pigment produced on glucose-gelatin did not form on levulose agar. On this medium it seldom produced a trace of color, but, on sowing the middle of a plate of nutrient levulose agar with the fungus and then the margin of one side of the same plate with his Bacillus pseudarabinus, a brilliant red color appeared as soon as the two organisms fused, and this crimson color developed not only through- out the colony, but in the neighboring region. He did not get the same result when Bad. vascularum was substituted for B. pseudarabinus. A colony of Bad. sacchari developed a foxy -red color at the side toward the mould, and the medium was faintly stained the same color. This bacterium grew upon fresh, sterilized portions of sugar-cane as white slime, while the mould during its growth upon [other samples of] the same substratum produced practically no color [except upon old portions of sugar-cane, where a red color is said to have developed], the older cultures only showing spots of pinkish aerial hyphse, but when both bacterium and mould were grown upon the [same] cane a deep crimson color developed upon the outside of the cane where the gum was form- Fig. 20. *Fig. 20. — Cobb's disease of sugar-cane. Introduced for comparison with fig. 19. The section is from the same stem, but in this bundle all the vessels of the xylem are occupied by the bacteria. The thin-walled tissue immediately above the vessels is phloem. Slide 121 (7. COBB S DISEASE OF SUGAR-CANE. 51 ing. Upon cutting the cane across many of the vascular strings were colored ; and finally the cotton wool upon which the cane rested also became crimson from the red gum which had flowed down the vessels of the strings. This experiment is, in my opinion, very significant as regards the combined (symbiotic) action of the mould and bacterium in producing a red gum in the large vessels of the vas- cular strings of the sugar-cane. Apparently the sugar-cane had been sterilized before inoculation. To the writer this means simply that the red pigment is produced by the fungus when grown in an acid substratum and not when grown in a neutral or alkaline substratum. Noth- ing is said about the production of acids by the Bacillus, but it is stated to be a gas-producer, and from this we might infer the production of sufficient acids for this purpose. The phe- Fig. 21.* nomenon is not peculiar to this fungus, the writer having pointed out similar changes in color 15 years ago in connection with his studies of species of Fusarium, some of which remained white on alkaline media, but developed the most brilliant reds and purples on acid media. f The writer observed no fungus in the red strands of his inoculated cane plants and obtained on poured-plate cultures made therefrom, as already stated, only Bacterium vas- *Fig. 21. — Cross-section of cane-stem (inoculated plant No. 6), showing three vessels occupied by Bacterium vas- cularum. Other vessels (at left) and connective tissue free. One normal nucleus and what appear to be two distorted nuclei are present. Fixed in 95 per cent alcohol. Slide 310 (2. tSee Wilt Disease of Cotton, Watermelon, and Cow-pea. Bull. No. 17, Div. Veg. Path, and Phys., U.S. Dept. of Agriculture, Washington, 1899, especially pp. 13 to 30. 52 BACTERIA IN RELATION TO PLANT DISEASES. cularum, exception being made of inoculated plant No. 4, which was allowed to stand too long. vSince the appearance of Greig Smith's paper I have gone over slides made from these inoculated plants to see if I could have overlooked such a fungus. The red bundles contain an amorphous granular substance, and sometimes bacteria, but no fungi were found. RELATION TO SEREH. Is this disease identical with Sereh, the Javanese "curse" of the sugar-cane? The signs are for the most part the same, i. c, red bundles, dwarfing, shortening of the internodes, etiolation, sprouting of the buds, transmission through infected sets, distorted terminal buds, etc. The writer has never seen the disease and so can make no personal observations. Janse and Kriiger consider Sereh a bac- teriosis of the bundles, but speak only of red gum and do not mention yellow slime in the bundles, at least not in any of their papers read by the writer. Valeton mentions yellow gum, how- ever, and states that this precedes the red stain, and he appears to have given more attention to histology than any of his colleagues. Wakker and Went are opposed to this hypothesis. Went's observations in the West Indies, how- ever, did not confirm him in his earlier belief that a fungus, Hypocrca sacchari, is the cause of Sereh, for there he ob- served the same fungus on the cane but Sereh was absent (verbal commu- nication to the writer). Only this somewhat suggestive fact seems estab- ished, that Sereh is not confined to Java (according to Kriiger, it occurs in Mal- akka, Borneo, and Bangka),and Cobb's disease is found not only in Australia but also, it is said, in Java, Mauritius, and Brazil. It is very desirable that a good bacteriologist should make a careful study of Sereh. Perhaps two or more different diseases are now united under this name, one of which may be Cobb's disease. Query: Does Sereh occur in Australia? Went is inclined to believe that it does. The subject of Sereh will be treated in the following chapter (p. 72). MORBID ANATOMY. There are no hyperplasias caused by this disease, but only occasionally roots out of place, premature development of axillary buds, and certain distortions of the terminal bud which might be taken for such. The disease is for the most part confined to the vascular system, the bundles of which are gradually filled with the bacterial slime. Figs. 19 and 20 show this very well. They were made from the Australian cane already mentioned as sent to me for study. In one of these bundles all the vessels of the xylem are occupied ; in the other the two larger vessels are free. The lysigenetic cavity is also filled. The phloem is not affected in either, nor the tissue surrounding the bundles. Such, however, is not always the case. In the upper part of the stem, just below the terminal bud, where the tissues are soft, the parenchyma is also attacked and numerous bacterial cavities are formed in it. In the Fig. 22. *Fig. 22. — Bacterium vascularum occupying xylem part of bundle. Like fig. 21, but cut longitudinally. Section from inoculated cane-plant No. 6 at the end of three months. Slide 310 (20, second section from left. COBB S DISEASE OP SUGAR-CANE. 53 writer's inoculated plants some of these closed cavities contained as much as a teaspoonful of the yellow slime (plate 4, and fig. 4). The organism also breaks out of the foliar bundles, especially when the leaves are immature, occupies the intercellular spaces very generally, making passageways between the cells, and comes to the inner surface of the leaf-sheaths and on the softer inner sheaths to the outer surface also, as a copious slime. Whether it exudes solely through stomata, or also sometimes makes openings of its own, is not known. I have found it oozing from the stomata on leaf-sheaths so commonly as to make it unneces- sary to consider any other method of reaching the surface (figs. 5, 6, 7). Figure 21 shows in cross-section an early stage in the occupation of the bundle and figs. 22, 23, in longi- tudinal section, show later stages, with much disorganization of the tissues of the bundle, all three taken from inoculated plant No. 6, at the end of 3 months. When we reflect that the bacteria were not intro- duced into this stem directly, but that the plant was inoculated solely by means of a few needle-pricks on the blades of two leaves, and con- sequently that the bacteria had to travel or grow through the vascular bundles a distance of 4 or 5 feet to reach the tissues here figured, the short time required for the general infection and the enormous multi- plication of the organism are aston- ishing. For a longitudinal section through a slightly diseased cane see fig. 24. The red stain, as already men- tioned, is believed to be an oxidation product closely connected with the presence of the bacteria. Its greater abundance in the bundles occurring in the nodes is accounted for by the falling away of the diseased leaves and the entrance of air in larger amounts than would be possible on the falling away of normal leaves, the diseased leaf- traces being unable to heal over so as to exclude air. That the stain should also occur in much greater amount in the inter- nodal bundles immediately below the nodes than in those immediately above the nodes is accounted for by the downward movement of the leaf-traces as they enter the stem. For evidence of the close connection between the red stain and the presence of the bacteria consult figs. 12, 13, 25, and various statements under Etiology. *Fig. 23. — Longitudinal section showing the xylem part of a bundle wholly destroyed by Bad. vascularum. From plant No. 6. Slide 310 (13, left-hand section. For a detail see fig. 27. 54 BACTERIA IN RELATION TO PLANT DISEASES. THE PARASITE. Bacterium vascularum (Cobb) Greig Smith* is a honey-yellow organism, occurring in the bundles of diseased sugar-cane as short rods, single, in pairs (mostly), fours, or eights, joined end to end (figs. 26, 27). Sometimes the rods are clumped in the stem, i. c, in the form of pseudozoogloeae. The organism is motile by means of a single polar flagellum (RGS., EFS.f). This is easily demonstrated according to Greig Smith by using the night-blue method and agar cultures (fig. 28). They were stained in the writer's labora- tory by L6 wit's method, using young agar cultures (fig. 29) . The motility is more evident in young cultures than in old ones. The organism is generally non-motile when crowded in the bundles of the stem. This restricted motility is, however, observed under similar conditions in many other bacteria (see Bad. campestre, vol.11, p. 3 16). Rods stained and embedded in balsam have an average measurement of 0.4 X im (RGS.). Fig. 30 is from a young culture on agar. The writer's measurements are as follows: Bacterium vascularum from tissues of inoculated plant No. 4, stained with carbol fuchsin; slide 466 h 10; average size 0.9X0.4^; another slide 0.88 to 1.0/1X0.3 to 0.35/i. Plant No. 6, tissues stained with earbol fuehsin; average size, 0.65 to i.2ji X 0.3 too.5/j. Plant No. 10, tissues stained with carbol fuchsin; average size, 0.6 to 1.4/u X 0.3 to 0.4M. Plant No. 35, smear stained with carbol fuchsin; average size, 1 to 1.4/j X 0.35 to 0.5*1. Agar culture, 8 days old, stained with Lowit's flagella stain, slide 8, January 1 1, 1906; average size, 1.2 to 1.5/i X 0.45 to 0.55/i. No spores have been observed (EFS., RGS.). It is stained fairly well by Gram's method, 4 min. stain, 2 min. iodine (it does not stain by Gram, according to Greig Smith) . Carbol violet followed by dilute alcohol produces the best films, while the blues stain it but feebly (RGS.). The organism stains well with carbol fuchsin. The writer had poor success with Loefner's alkaline methylene blue. Bacterium vascularum is easily isolated from diseased canes (when not too old) by means of Petri-dish poured plates. On plates of standard nutrient agar at 250 C. the colonies come up rather slowly, i. e., in 7 to 10 days, so that often for the first few days well-sown plates will ap- pear sterile, or only sparsely spotted with intruding colo- nies. This will be better appreciated if I give transcripts from notes (made on the fourth, sixth, and eleventh days) of nine Petri-dish poured plates made from the interior of inoculated cane No. 1 1 : Fourth day. — Many of the organisms must have been dead. There are as yet no growths on the plates, except one or two colonies which are not Bad. vascularum. Sixth day. — Plate I, one colony which has become yellowish, but is much too opaque for Bad. vascularum. Plate II, one small white colony. Plates III and IV, nothing. Plate V, one Fig. 24.| *Synonyms: Bacillus vascularum Cobb; Pseudomonas vascularum (Cobb) EFS. tRGS. = R. Greig Smith; EFS. = Erwin F. Smith. JFig. 24. — Longitudinal section through cane No. 30 (common purple) inoculated May 5, 1903. Only slightly- diseased (the red bundles show here as dark stripes) and disease confined to the vicinity of the nodes which bore the inoculated leaves. Plates were poured. Photographed January 5, 1904. COBB S DISEASE OF SUGAR-CANE. 55 fungus and one small whitish bacterial colony. Plate VI, nothing. Plate VII, nothing. Plate VIII, 2 white colonies. Plate IX, nothing. The plates were now put into the incubator at 30° C. Eleventh day. — Plate I, one large opaque yellow colony, and hundreds of small yellow colonies of Bact.vascularum. A pure culture, wTith exception of the large colony. Plate II, one white colony and hundreds of the Bad. vas- cularum; a pure culture, with exception of the one white colony. Plate III, a pure culture of Bad. vascularum. Plate con- tains several hundred colonies and those which have broken through to the surface are circular, quite transparent, and 1 to 5 mm. in diameter (fig. 31). Plate IV, hundreds of colonies of Bad. vas- cularum, a pure culture. Plate V, one fungus colony and one white bac- terial colony; with these exceptions a pure culture ; hundreds of minute yel- low colonies of Bad. vas- cularum. Plate VI, a pure culture with at least 100 colonies, transfers. Plate VII, a pure culture. Plate VIII, same, with exception of two intruding colonies. Plate IX, a pure culture. Colonies on VII to IX too close together to estimate their number; 2,000 at least on each plate. Fig. 25.* The colonies appear sooner on plates made from young cultures. >omm X100 mm Fig. 26. f Fig. 274 F'8- 28§ The well-developed surface colonies in uncrowded agar plates are 4 to 6 mm. in diameter (occasionally 8 mm.), pale-yellow, wet-shining, rather flat, with sharp margins and no dis- tinct appearance of granulation under a hand-lens magnifying 6 times (fourteenth day). *Fig. 25.— Cobb's disease of sugar-cane: Cross-section of a small bundle from an inoculated plant, showing the bacteria, and red stain (the dark parts). Slide 299 A 2, lower left section. fFic. 26.— Bacterium vascularum: A detail from slide 310 (17; i. e., from fig. 12, near location of micrometer scale. JFig. 27. — Bacterium vascularum: Detail from the cavitv shown in fig. 23. §Fig. 28.— Flagella of Bacterium vascularum stained by the night-blue method. After R. Greig Smith. 56 BACTERIA IN RELATION TO PLANT DISEASES. They give a decidedly alkaline reaction to moist litmus paper and are not noticeably viscid (fourteenth day) . On opening plates of this age they had a peculiar smell common to many bacteria (trimethylamin ?). The buried colonies are elliptical-pointed or spindle-shaped, and small. On slant peptonized beef- agar the organism makes a thin, rather scanty, wet-shining, pale yellowish growth which is sometimes scarcely distinguishable from the agar itself. In some streaks 5 days old the penholder was visible through three superposed tubes. The streak is finely granular under the hand-lens. A small amount of pale yellow precipitate forms in the fluid in the V and numerous prismatic crystals are sometimes present in the agar; 200 or more of these crystals were observed in a single tube. In beef-agar stab-cultures there was a variable growth in the stab, often fairly good the whole length, but always thinning out below and sometimes scanty. There was a good, wet-shining, pale yellow surface growth. In one set of tubes inoculated from slant agar and described the third day, the nail-head was 4 to 5 mm. in diameter, wet-shining, and maize yellow. The slime of very old cultures may be buff-yellow. The agar is not stained. Fig. 29.* Fig. 30. t On agar with cane-sugar or fruit-sugar there is slow growth at 300 C. or under (RGS.). Greig Smith describes the streak on slant glycerin agar as "a thin, broad, translucent white, moist, glistening growth, with turbid condensed water. The color deepens to a prim- rose-yellow." On agar containing 10 per cent saccharose and 0.1 per cent peptone there is a thin, white fluid growth, which gravitates into the condensed water, in which there is a yellow sediment (RGS.). A very good medium is a solution of 4 per cent agar added to an equal amount of neu- tralized cane-juice containing 0.5 per cent peptone. With a greater per cent of peptone there is better growth at first, but ultimately less (RGS.). The writer obtained a very copious growth on slant tubes of sugar-beet agar, as much growth as on cane-juice agar. This medium was made as follows: Juice of sugar-beets 300, *Fig. 29. — -Flagella of Bacterium vascularum stained by Lowit's method. Selected samples from a cover-glass preparation (young agar culture in sterile water). Slide B, mordanted one-half minute, stained 5 minutes, Jan. 9, 1906. fFiG. 30. — Rods of Bacterium vascularum from a young agar culture stained by Lowit's method. Mordanted 0.75 minute, stained 2 minutes. A few flagella show feebly in some places. Slide A, January 9, 1906. COBB S DISEASE OF SUGAR-CANE. 57 Witte's peptone 15, cane-sugar 15, agar flour 4.5, autoelaved for 15 minutes at 1 io° C. The surface growth was wet-shining and about a cubic centimeter of pale yellow slime accu- mulated in the V of each tube. The growth on potato-agarwas somewhat better than on beef-agar; there was a smooth, wet-shining, yellow streak (fig. 32), varying from scanty to abundant. On litmus-laetose-agar there is a very scanty to moderate growth, with slow but dis- tinct bluing and no reduction of the litmus. No acid is formed. On Hunger's agar (Mr. Johnston's notes) there is a very noticeable difference in the growth, depending on whether the monobasic or dibasic phosphate is used. When the monobasic (acid) salt is used there is a growth rather light yellow in color, while with the dibasic salt it is rich yellow with paler edges, and is much more luxuriant, although not heaped up as in the case of Bacterium phascoli. Greig Smith gives the fol- lowing description of the organ- ism in glucose-gelatin plates: The colonies develop slowly. In 7 days at 220 C. they are 1 mm. in diameter, and appear as small, raised, viscid drops. When mag- nified 60-fold [see fig. 33] they appear round and uniformly gran- ular, like a thin yeast colony, the deep colonies like those upon the surface. In 20 days the colonies reach a diameter of 4 to 8 mm. and look like drops of yellow beeswax. The medium shows no signs of liquefaction, but when the colony is scraped or washed off a pit is revealed. Growth in the absence of glucose is similar but much slower (RGS.). In cane-juice gelatin-stabs there is a smooth, piled-up, restricted yellow surface growth. There is no liquefaction (possibly in some instances there was an unobserved slight liquefaction, ;'. c, a sinking in of the nail-head), no production of gas, no stain, and no formation of crystals. Greig Smith reports slow growth on gelatin in the presence of cane-sugar or fruit-sugar at a temperature of 300 C. or under. In a gelatin stab (stock i.ooof), inoculated from a young culture on litmus lactose agar and kept mostly at 220 to 240 C, there was at the end of two days a yellow, wet-shining, restricted (piled-up) surface growth and a needle-track best developed in the top layers of the gelatin. There was no liquefaction in 20 days. The surface growth remained piled up and restricted. Separate colonies were visible only in the lower part of the stab. No crystals appeared, no stain, no gas. In six tubes of nutrient gelatin composed of 100 distilled water, 10 gelatin (Nelson's photographic No. 1), 0.5 Witte's peptone, 0.5 dipotassium phosphate, and 0.02 malic acid, Fig. 31.* *Fig. 31.— An agar-poured plate of Bacterium vauularum (a pure culture) 1 1 days old. Plated from the interior of plant No. 11. Photographed April 27, 1903. |A +15 gelatin containing beef-broth, Witte's peptone, 10 per cent Nelson's No. 1 gelatin, and sodium hydrate. 58 BACTERIA IN RELATION TO PLANT DISEASES. inoculated from young potato cultures, there was in 7 days a very feeble growth restricted mostly to the upper part of the stab and yellow only at the surface. The cultures were fol- lowed for 43 days, but there was no increased vigor of growth; no crystals, liquefaction, or staining of the gelatin occurred. In six tubes of the same gelatin with addition of 1 per cent cane-sugar, growth at the end of 43 days was about double. These tubes were inoculated at the same time from the same cultures as the preceding, and were subject to the same conditions. Even in this medium, growth was rather feeble and there was no liquefaction. Probably the medium was too acid. This inference was confirmed by subsequent experiments in the same medium neutral- ized with sodium hydrate. There was a comparatively good growth at the end of 10 days, but no liquefaction. At the end of a month there was a strong growth the whole length of the stab, thinning out below, whitish also below (where air was less abundant) and pale yellow in the upper half. The surface growth was paler yellow and more ex- tensive than in the same medium without the alkali ; the nail-head was also sunken into the gelatin a little, as if there had been slight liquefaction. Experiments with gel- atin, to which filtered cane- juice had been added, yielded interesting results. In streaks on stock 727 the organism made a fairly good but not a very vigorous growth. On the forty-third day the growth was dis- tinctly yellow (in contact with the air) and at least ten times as abundant as in the gelatin containing peptone, dipotassium phosphate, malic acid, and cane-sugar. The surface growth was restricted (piled up), smooth, wet- shining, and Naples yellow in color. There was no liquefaction. The gelatin was not stained and no crystals were present. Growth started off slowly. The temperature varied somewhat, averaging about 200 C. This stock was prepared as follows : juice of Common Purple cane 250 c.c, distilled water 750 c.c, Nelson's No. 1 gelatin 100 grams. On the same stock rendered slightly alkaline (to phenolphthalein) by the addition of NaOH (stock 741) there was only the least trace of growth in 5 out of 6 streak cultures. On the sixth streak, in course of 2.5 months, the organism succeeded in getting a start, but piled up locally in a very striking manner. There were many of these bacterial masses, most of them approximately circular, 1 to 8 mm. in diameter, and some of them 2 mm. high. Their color was about Ridgway's maize yellow. There was no liquefaction of the gelatin. Six stab Fig. 32.! *Fig. 32. — Streak-cultures of Bacterium vascularum on potato agar. Subcultures from three different plants (7, 9, and n), inoculated Feb. 6, 1903. Streaks made Nov. 2, 1903. Photographed after several days. COBB S DISEASE OP SUGAR-CANE. 59 cultures made at the same time yielded no better results. There was a trace of growth in the mouth of the stab, but none below the surface and no spreading over the surface, not even in a month's time. This failure is probably attributable to excess of sodium hydroxide, since when this substance was reduced one-half by mixing equal volumes of 727 and 741 the organism finally grew much better, although the growth was scanty at first. On gelatin made in the same way but with juice of the Common Green cane (stock 750) the growth was scanty for the first two weeks, then better, but by no means copious. This growth (surface) was at first rough granular, then smooth. There was no staining or lique- faction (70 days). There was a beaded stab, most growth being on the surface. Streak cultures developed in a similar slow manner and there was no liquefaction in them. When 1 per cent Witte's peptone was added to this same stock there was from the start distinctly more growth (several times as much), although not very copious. Subsequently there was a copious growth. This was the only one of the cane-juice gelatins on which there was what might be styled a good growth. The growth in the stab was better developed and the sur- face growth was smooth from the start. There was no lique- faction, but eventually some browning of the gelatin. In the streak cultures the growth was at first reticulate-roughened, then smooth, with tear-drop formations sliding down into the V, where at least 0.75 c.c. of straw-yellow slime collected in course of a month, but without liquefaction (fig. 34). On long standing there was a feeble browning of the more exposed parts of this gelatin. The character of the growth was quite similar in all of the gelatins. Sometimes, especially when the medium was not well adapted to g r o w t h , the sur- jglSSSSISSS face was reticu- late-roughened at first, but the reti- culations them- selves, as seen under the hand- lens, were smooth. Later the surface growth became smooth. In tubes placed in an up- right position the slime collected in places along the streak and piled up at the bottom of the slant in a characteristic manner, very well described by Greig Smith as a "tear-drop" formation (fig. 35). Some of these piles were 2 mm. high. There was very little depth of slime on the rest of the streak. A feeble browning of the more exposed parts of the gelatin (751) took place after long stand- ing (9 weeks). In most of the gelatins there was no liquefaction. In one set of streak-cultures the record states that there was moderate liquefaction on the forty-fourth day, but none on the twenty-fifth day; and in one set of stab cultures the nail-head settled into the gelatin slightly after some weeks. Fig. 33.' Fig. 34. f *Fig. 33. — Colony of Bacterium vascularum on glucose gelatin. X 85. After R. Greig Smith. fFic. 34. — Streak-cultures of Bacterium vascularum after 21 days at 18° to 23°C. on cane-juice gelatin with and without peptone (250 c.c. expressed juice, 750 c.c. distilled water, 10 per cent Nelson's shredded photographic gelatin). Tube No. 1 received in addition 1 per cent Witte's peptonum siccum, and contained 100 times as much growth as tube No. 2. On this peptone gelatin the organism showed a strong tendency to pile up and run down into the V (see fig. 35). The color of the slime was straw yellow (Ridgway). There was no liquefaction. 6o BACTERIA IN RELATION TO PLANT DISEASES. fzza Greig Smith states that there is very slow liquefaction both of eane-gelatin and ordinary gelatin. This is seen usually only as a slight depression of the medium under the streak. On ordinary gelatin, according to Greig Smith, the growth is scanty and ivory white in color. By this he means, perhaps, the pale yellow of old ivory. The organism I have studied is not pure white on any medium, although for the first few days it might be thought to be so. In streak-cultures the slime slowly gravitates to form a yellowish-white mass at the bottom of the slant (RGS.). On glucose-gelatin the growth is less luxuriant and less deeply colored than on cane-gelatin (RGS.). There was a striking difference in the amount of growth on steamed cylinders of Com- mon Green cane and that made on Striped Green, Common Purple, and Striped Purple cane. On the first-named variety there was at least 50 times as much growth as on the others. On the last three there was little growth, while on the other growth was good though not copious. Greig Smith obtained no growth on slices of cane (variety Rappoe ?) inocu- lated with Bad. vascularum. The organism makes a much better growth on potato-cylinders and on coconut-cylinders. The growth on potato is generally better than on agar (see pi. 1 1, fig. 13). It is moder- ately abundant, i. e., about like that of Bad. hyacinthi, but with rather more slime in the water (fig. 36) ; surface growth smooth, wet- shining, and yellow (between Ridgway's canary yellow and lemon yellow), occasion- ally it is Naples yellow or between lemon yellow and gamboge yellow (3 days). The palest yellow (4 days) was between primrose yellow and straw yellow. Growth is some- times raised and restricted, sometimes flat and watery, spreading over the surface (RGS.). Growth on potato is good rather than copious, and ceases early, indicating that the organism has only a small ability to obtain its food from the potato-starch. Re- peated tests showed much starch present, even in old cultures. Mashed cylinders became deep bluish-purple or wine-purple in iodine water. The undisturbed surface layer became dirty purple or brown-red. The growth is 'mostly out of the water. There is no such rapid destruction of starch and piling up of the bacteria in the water as occurs in cultures of Bacterium campestre. The slime is distinctly yellow on all potatoes, but bright yellow on some and paler on others. The potato is moderately grayed, but mostly out of the water. Old cultures (43 days) were alkaline to litmus. Young cultures are also alkaline. On beet-cylinders (acid medium) there is only a small amount of growth, but on steamed onion-bulbs and cauliflower-cylinders there is a good growth of the organism. On carrot and turnip there is a raised, slimy, yellow growth, at first restricted, but eventually covering the surface and gravitating (RGS.). There is a good yellow growth on coconut-cylinders which remain unstained. Bacterium vascularum makes a good, though not copious, growth in +15 peptonized beef-bouillon. There is moderate clouding of the fluid, beginning usually within 24 to 48 hours, and when the tube is shaken there are distinct rolling clouds. Sometimes the cloud- 9 s^^b!" m MB ■■'■- 'jK M If . Jjw K Fig. 35.' Fig. 36. t *Fig. 35. — Tear-drop formation of Bacterium vascularum on cane-juice gelatin. After R. Greig Smith. tFic. 36. — Streak-cultures of Bacterium vascularum on potato cylinders (the lower part in water) after 8 days at room-temperature. Potato grayed out of the water. The darkest part is the wet-shining yellow slime. Each tube inoculated from a single poured-plate colony. COBB S DISEASE OF SUGAR-CANE. 61 ing is much slower (4 to 7 days) . The fluid is never what might be called turbid, but pseudo- zoogloeae are sometimes present in small numbers, and there is a small amount of pale yellow precipitate (Ridgway's primrose yellow or a little deeper, to his maize color or Naples yellow). A 3 mm. loop from a feebly clouded bouillon culture 24 hours old clouded feebly 10 c.c. of + 15 standard beef-bouillon on the third day at 25°C. ; a 2 mm. loop of well-clouded bouillon 6 days old did the same at the end of about 24 hours. After 7 days' growth in peptonized bouillon one could usually see a pencil through 70 mm. of the cloudy fluid (4 tubes, one behind another). Notes of December 5, 1902, on six tube-cultures of Bacterium vascidarum, made November 12, 1902, in standard peptonized bouillon (stock 712), as checks for thermal death-point experiments: Fluid moderately cloudy in each one ; rolling clouds on shaking ; no pellicle, and in most no trace of rim; in others the merest outline of a whitish rim. Can not detect pseudozoogloeae even with the hand-lens. Precipitate distinctly yellow and 5 mm. broad. Pencil easily visible through 2 of the tubes (in series), less plainly so through 3, barely visible through 4, not visible through 5. These 5 tubes make a diameter of 9 cm. Organism not a copious grower in this bouillon. After 2 to 3 months the fluid becomes clear, but is not browned (once slightly) . There is no rim, or only a faint whitish one which may be 2 to 3 mm., or more, in width. In only one set of transfers was a pellicle noted; this was thin and easily shaken down. Prismatic crystals are often present. After 70 days about 7 to 14 mm. breadth of the pale yellow, fine-grained precipitate may be present on the bottom of the test tube. Experiments with bouillon of different degrees of acidity and alkalinity, i. e., titrating + 25, +15, o, and —20 with phenolphthalein, have given the following results: The -f-15 bouillons clouded first and in the end gave most precipitate. At the end of 9 days the + 15 showed the best growth, *. c, 8 or 10 times as much precipitate as in neutral bouillon; the neutral bouillon the next best, then + 25 ; the — 20 bouillon was very feebly clouded with almost no precipitate. Six days later the order was : Neutral, densely clouded, i. e., more so than +15, but less precipitate; +15, well clouded, heaviest clouding in the uppermost centi- meter; + 25, as well clouded as lower part of +15 and uniformly, but much less precipitate than in +15; —20, feebly clouded. In a later experiment, using — 6, —4, neutral, +26, + 28, and +30 bouillons (Mr. Johnston's notes), the best growth was in the acid bouillon; -f-26, +28, and +30 were very heavily clouded, the neutral bouillon well clouded (probably corresponding to E- F. Smith's "moderately clouded"), and one of the —6 tubes lightly clouded. The other —6 tube and both —4 tubes were clear. All of the cultures were transfers from the same tube — a beef-bouillon culture 8 days old. The acid was that of beef-juice. Greig Smith obtained no growth in acid fluids such as wort and cane juice, and scanty growth in neutral fluids, but does not state what fluids nor what indicator was used. The organism grew in beef-bouillon with phenolphthalein, with or without the addition of potassium formate, but in neither case did the fluid become red. There is a moderate clouding of nitrate bouillon, but no reduction of nitrates to nitrites (RGS., EFS.). At the end of a week in this medium, inoculating from cultures on slant agar, all of 6 tubes were uniformily clouded, free from pellicle and rim, no pseudozoogloeae were visible to the naked eye in the unshaken tubes, and the penholder was distinctly visible through three tubes, and barely so through four, placed one behind another. Similar results were obtained by inoculating from six potato cultures. Litmus-milk-cultures (pi. 11, figs. 10, 11, 12) gradually become a deep indigo-blue (30 days, 54 days, 74 days, 6 months). Some tubes 38 days old are recorded as deep blue, near Saccardo's atrocyaneus (sapphire of Standard Dictionary, nearly). The bluing begins to be noticeable about the fourth or fifth day. No acid is produced. The milk was lavender or lilac when inoculated. There is no reduction of the litmus or precipitation of the casein, the milk remaining perfectly opaque and fluid. The milk does not become viscid (45 days, 75 days). A distinct yellow precipitate was formed, but in most tubes there was very little 62 BACTERIA IN RELATION TO PLANT DISEASES. evidence of growth at the surface ; in a few there was a distinct yellow rim and pellicle (six- teenth day). Old litmus-milk cultures (7 months) were dried down to about one-eighth of the original volume : The liquid was then thick, syrupy, or gelatinous, and very dark-colored. There were no tyrosin crystals, not even in very old cultures. According to Greig Smith, milk cultures give a neutral reaction and the fluid remains unaltered. I attribute this state- ment to incomplete observations, i. c, of first stages only of growth in milk. In Uschinsky's solution there is sometimes a feeble clouding, sometimes none at all. Out of six attempts to grow the organism in this medium three failed utterly, although the transfers were made from cultures known to be alive and the inoculations were copious. The following is a note on two sets which did cloud : Tubes of Uschinsky's solution, inoculated from young agar streaks, were feebly clouded at end of the first day, and thinly clouded at end of the seventh day with a scanty yellowish precipitate, and without rim, pellicle, or pseudozoogloese. These tubes were followed for 16 days longer, but there was little change. The organism did not grow wrell in this medium (stock 982). In another set of six tubes (stock 738) inoculated each with a 3 mm. loop from bouillon cultures 2 days old, all were clear on the sixteenth day. On the thirty-ninth day two were clouded with a small amount of Naples yellow precipitate. Twelve days later the other four tubes clouded. To test the action of the organism on cane-sugar, cultures were made in water contain- ing Witte's peptone and 1 per cent cane-sugar (stock 809). The fluid became uniformly thinly clouded (penholder barely seen behind four tubes, each 16 mm. in diameter). A yellowish- white rim and a pale yellow precipitate appeared. On the twenty-second day the cultures were tested with Soxhlet's solution. There was a distinct reduction of the copper sulphate on boiling half a minute. 5 c.c. of the alkaline fluid and 5 c.c. of the solution of copper sulphate in 50 c.c. of distilled water were used in each case, the fluid brought to a boil in a clean porcelain capsule, and then the cultures added. It required about 15 c.c. of the cloudy culture-fluid to reduce all the copper in 5 c.c. of the copper sulphate solution, boiling 2 minutes. There was a reducing substance in the cultures, and it is reasonably certain to have come from the breaking-up of the cane-sugar into simpler sugars. The uninoculated stock was tested as a check, but there was no such reaction : The fluid became and remained a hyacinth blue. Previous to this an old cane-juice gelatin culture (stock 751) had been tested in a similar manner, but there was very little reaction while the uninoculated cane stock showed an abundance of reducing sugar in it on boiling 2 minutes with Soxhlet's solu- tion. There was more reduction in the two uninoculated cane-juice gelatins (750 and 751) than in the 4-months-old culture tested. This probably means that the reduced sugars had been consumed as food. Greig Smith on the contrary says that the organism does not secrete invertase. In his experiment saccharose-agar cultures were melted, dissolved in water, and treated with basic lead acetate, etc. (Linn. Soc. Proc, 1902, p. 44.) Only 3 per cent of the saccharose had been inverted to fruit-sugar, a quantity which he thinks the hydrolytic action of the small amount of acid in the medium might easily have produced. Cobb is also of the opinion that the organism does not act to any appreciable extent on cane-sugar. To determine the action of Bacterium vascularum on cane-sugar he put 457 mg. of the air-dried gum obtained from diseased canes with 1 gram of pure cane-sugar into 10 c.c. of water and allowed it to stand 3 days. The solution was then tested. It was exactly like the check-tube, i. e., there was no reduction of the amount of cane-sugar present. The experiment was repeated with the same result. From these two experiments the conclusion is drawn that Bacterium vas- cularum does not act to any appreciable extent on cane-sugar. To the writer this does not appear to be a necessary conclusion. It is too broad an inference since (1) cane-sugar may perhaps be reduced only when the organism is alive and growing; (2) the organisms in the carefully dried gum may have been killed by light or by dry air in the process of drying; (3) the organism, although alive when put into the sugar solution, may not have found in it the cobb's disease of sugar-cane. 63 elements necessary for growth, i. e., nitrogen or some other substance may have been lack- ing; or (4) the enzyme presumably present in the gum may have been destroyed in some inadvertent way or rendered inactive. All it proves beyond question is that the "gum" did not act on the cane-sugar under the conditions of the experiment. Boname's analyses show that diseased canes have a smaller sugar-content than sound ones, and so do those cited from Cobb's paper. Some analyses made by Miiller and published by Tryon show only about 1 per cent difference between gummed cane and cane supposed to be free from gum, but this 1 per cent is on the side of the healthy cane. Growth is not inhibited by sodium chloride in small quantities, e. g., there is feeble growth in peptone water with 0.5 per cent and 1 per cent NaCl, but larger amounts (1.5 per cent, 2 per cent, and 2.5 per cent) prevent growth. Greig Smith also found that 2.5 per cent sodium chloride was a distinct poison. In cultures in 1 per cent peptone-water with 0.1 per cent oxalic acid there is no clouding of the fluid, but a finely granular and flaky or stringy precipitate is formed. The following acid agars gave negative results: Beef-agar with 0.2 per cent oxalic acid (1687) ; beef-agar with 0.1 per cent malic acid (1682) ; 2 per cent beef-agar with 0.2 per cent oxalic acid (1776) ; 2 per cent beef-agar with 0.2 per cent citric acid (1777) ; 2 per cent beef- agar with 0.2 per cent malic acid (1787). There was no growth whatever on any of these agars. Greig Smith reports the following results of experiments made to determine the reaction of the medium best suited to the bacterium. The medium used was water containing 0.5 per cent peptone, 5 per cent saccharose, 0.5 per cent [di ?] potassium phosphate and 2 per cent agar, to which were added various quantities (0.0 1 per cent, 0.02 per cent, 0.03 per cent, 0.05 per cent, 0.08 per cent) of solutions containing 10 per cent tartaric acid and 10 per cent sodium carbonate. He says: The effect of slight differences of acid or of alkali in the culture media is very pronounced. In an absolutely neutral medium [neutral to what?] the bacteria grow very slowly, while, when the reaction is faintly acid, the growth is quick and luxurious. Alkalies prohibit the multiplication of the microbe . With 0.02 per cent the stroke was slow to show itself, was always scanty, and finally, after ten days' incubation, it dried up. A series of experiments made for me by John R. Johnston to determine from what com- pounds the organism could obtain its nitrogen and carbon resulted as follows: Nitrogen-free medium (1664*) with 0.5 p. ct. asparagin. — Well clouded with thin film on the surface. Nitrogen free (1664) with 0.5 p. ct. sodium asparaginate. — Moderately clouded; a slightly yellowish more or less gran- ular film on surface. Nitrogen-free (1664) with 0.5 p. ct. ammonium citrate. — Heavy flocculent clouding, heavy precipitate. Nitrogen-free (1664) with 0.5 p. ct. ammonium lactate. — Moderately clouded. Nitrogen-free (1664) with 0.5 p. ct. ammonium tartrate. — Moderately clouded. Fischer's mineral solution (nitrogen-free).f — Very slight growth. Fischer's mineral solution with 1 p. ct. cane-sugar. — Very slight growth. Fischer's mineral solution with 1 p. ct. KNOt. — Very slight growth. Fischer's mineral solution with 1 p. ct. KNOs and 1 p. ct. cane-sugar. — Very slight growth. Fischer's mineral solution with 1 p. ct. KNOj and 1 p. ct. dextrose. — Very slight growth. Fischer's mineral solution with 1 p. ct. Witte's peptone. — Thinly clouded; small precipitate. Fischer's mineral solution with 1 p. ct. Witte's peptone and 1 p. ct. dextrose. — Moderate clouding; small, yellow pre- cipitate. Fischer's mineral solution with 1 p. ct. Witte's peptone and 1 p. ct. glycerin. — Moderate clouding. Fischer's mineral solution with 1 p. ct. Witte's peptone and 1 p. ct. cane-sugar.— Heavily clouded; yellow precipitate, white surface pseudozoogloeae. Fischer's mineral solution with 1 p. ct. asparagin and 1 p. ct. dextrose. — Good growth. Fischer's mineral solution with 1 p. ct. ammonium tartrate and 1 p. ct. dextrose. — No growth. Water with 2 p. ct. asparagin and 1 p. ct. dextrose. — Thinly clouded, growth much retarded. Water with 1.2 p. ct. asparagin and 1 p. ct. Witte's peptone. — Well clouded, blackish (?) precipitate. "Stock 1664: fFischer's mineral solution: Cane-sugar 9 . 00 Dipotassium phosphate 2.0 Dipotassium phosphate 900 Magnesium sulphate 0.4 Magnesium sulphate 0.90 Calcium chloride 0.2 Sodium chloride 1 .80 Distilled water 2,000.0 Calcium chloride 0.45 Distilled water 1,800.00 64 BACTERIA IN RELATION TO PLANT DISEASES. From these results it is probable that the organism can obtain its nitrogen from aspa- ragin and ammonium salts but not from KN03, and that it can use cane-sugar, dextrose and glycerin (?) as carbon foods. Greig Smith says glycerin can not be utilized by the organ- ism. Further experiments should be made. Greig Smith makes the following deductions from experiments made to test the influence of salts upon the growth of the organism : (i) The bacterium has a preference for phosphate and is indifferent to the other acid radicals; (2) the acid potassium phosphate, on account of its acidity, checked the growth; (3) potassium may be replaced by calcium, magnesium, or ammonium ; (4) sodium is a distinct poison. The medium used was a faintly acid one containing 0.5 per cent peptone, 10 per cent dextrose, 2 per cent agar, and 2.5 per cent of the various salts to be tested. Greig Smith's conclusions regarding the influence of carbon foods are as follows : Either dextrose, levulose or saccharose are absolutely necessary for the free growth of the organ- ism and the production of gum. The bacteria did not grow in the presence of the other sugars and carbonaceous matters (glycerin,* starch, dextrin, maltose,* lactose*), from which we infer that these cannot be utilized. Of the three sugars, levulose and saccharose are more easily assimilated than dextrose. The medium was a faintly acid one containing 0.5 per cent peptone, 0.2 percent sodium phosphate, 0.5 per cent potassium chloride, 2 per cent agar, and 5 per cent of the various carbon substances. Cohn's solution : I can not find that we made any tests in Cohn's solution. Greig Smith states that he obtained a faint indol reaction in nutrient bouillon. So far as tested Bacterium vascularum is a strict aerobe (EFS., RGS.) . No gas is formed in any of the common culture media, nor is there any clouding of the closed end of fermenta- tion-tubes containing grape-sugar, cane-sugar, milk-sugar, maltose, mannit, or glycerin in 1 per cent quantities in water containing 2 per cent Witte's peptone, although all were moder- ately clouded in the open end and outer part of the U- Cultures were repeated in 1 per cent peptone-water containing 1 per cent maltose and the same with 1 per cent mannit, but with the same results. In twice-distilled water containing 2 per cent glycerin, 1 per cent dex- trose, and 1 per cent sodium asparaginate there was no growth. This medium was alkaline to litmus paper. The organism did not grow on steamed potato or in peptonized beef-bouillon in an atmosphere of carbon dioxide (14 days) . One test only. The check tubes behaved properly . The thermal death-point lies between 490 and 500 C. when exposures are made from young bouillon cultures as described in vol. I of this monograph. It is a little higher when made from potato cultures. Growth is inhibited by a temperature of 37. 50 C. (RGS., EFS.), and a temperature of 350 C. much retards it. Two out of three tubes of bouillon continued to be thinly clouded after 27 days at 350 C. and were living, as shown by streak cultures on agar, but the streaks developed colony-wise at first, indicating that the bouillon was then thinly stocked with living organisms. Bouillon-tubes inoculated copiously and placed at 37. 50 C. for 7 days not only remained clear but refused to cloud when subsequently placed at room tempera- tures ( 1 2 days) . Agar plates poured from these three tubes also remained free from colonies. The three check-tubes clouded promptly. There is more rapid growth at 300 C. than at room-temperature (180 to 230 C), about 3 times as much in the first 48 hours, but at the end of 4 days not much cloudier than the checks. "That strain of Bacterium vascularum with which I have experimented has moderately clouded peptone-water containing glycerin, maltose, and lactose, but in the light of Greig Smith's statements I am not prepared to say that it would not have clouded the peptone-water to the same extent without these substances. Additional tests should be made. I can not make them myself, because I have lost all my cultures of this organism. COBB S DISEASE OF SUGAR-CANE. 65 No pellicle, no pseudozoogloeae, very scant whitish rim (6 tubes), precipitate yellowish, 2 mm. broad, growth feeble. The optimum temperature is probably 30°C, or a little below. Greig Smith reports growth at 300 C. much better than at 250 C, and also considers 30° C. the optimum temperature. Greig Smith found the most suitable medium for the growth of this organism to be neu- tral cane-juice gelatin (p. 34, Proc. Linn. Soc, 1902 ; but on p. 38 he says that very faintly acid cane- juice gelatin is best), and the next best, ordinary glucose-gelatin, and slices of potato. He describes its growth on suitable solid media as occurring in the form of "raised yellowish patches which have the appearance and consistency of softened yellow beeswax. When treated with water the culture slowly mixes, forming what appears to be a partial sus- pension and partial solution." Another excellent medium is: 0.5 peptone, 5.0 saccharose or levulose, 0.5 [di?] potas- sium phosphate, 2.0 agar, 100 tap-water. Acidity of 10 c.c. =0.14 c.c. tenth-normal acid [ + 1.4 Fuller's scale]. On this medium the organism grew most luxuriantly (RGS.). Table 5. — Thermal Death- point Experiments. Stock cultures. 6 bouillons, 5 days old, each from a separate colony. Exposures. 6 tubes, 1 loop each. Do Do 2 bouillons, 3 days old. 4 bouillons, 10 days old, each from a separate colony. 6 bouillons, 28 days old, each from a separate colony. 6 potato cultures, 13 days old, each from a separate colony. 2 bouillons, 2 days old. 1 bouillon, 6 days old. 1 bouillon, 1 day old. 1 bouillon, 4 days old (faintly clouded). 1 potato culture, 4 days old. ....Do ...Do 4 tubes, one 2 mm. loop. 4 tubes, 1 loop each. . . . 6 tubes, one 3 mm. loop. 6 tubes, 1 loop each. . . . 4 tubes, one 2 mm. loop. 4 tubes, one 2 mm. loop. 4 tubes, one 3 mm. loop. 4 tubes, one 3 mm. loop. Checks. 4 tubes. None; all exposed tubes clouded when reinocu- lated 18 days later from same stock tubes ....Do ....Do 2 tubes, clouded 2d day. 2 tubes, clouded 5th day 6 tubes, which clouded 3d to 5th day. 6 tubes, 5 clouded 3d day, 1 on 4th day. 2 tubes, clouded in 48 hours. 2 tubes, clouded next day. 2 tubes, clouded 3d day. 2 tubes, clouded 7th day. 2 tubes, clouded 4th day. 10-mmute tempera- tures. °c. 5J 52 5'-4 50.8 50.7 50.7 50 49 49 48 48 Results. All killed. Do. Do. Do. Do. Do. Retarded, but did not kill.* All killed. All clear till 5th day, 1 clouded on 5th, 2 on 7th, 1 on 8th day. All killed. Do. Faintly clouded on 7th day. ♦Portions of solid cultures put into bouillon and heated might be protected, perhaps, to a slight but sufficient degree by non-conducting air inclusions. The vitality on culture-media varies greatly, of course, with the medium and the temper- ature. The length of time the organism can live in the stem is not known. The first sugar- cane received by the writer from Australia (1891) was 5 months on the way (detained in a custom-house) and contained no living yellow organisms when it reached me, although the bundles were full of the yellow slime ; but in a second shipment, which arrived from Australia in a much fresher condition, the organism was alive. The ends of these canes were sealed with sealing wax, and the canes were en route not more than 6 weeks. The organism was dead in two inoculated stems at the end of 8 months. These were plants 30 and 36, purple canes, which had not suffered from the disease. Some of the vessels of the stem showed immense numbers of bacteria, however, and a good-sized piece of this tissue was cut out with a cold knife, thrown into 10 c.c. bouillon for an hour, then mashed with a sterile glass 66 BACTERIA IN RELATION TO PLANT DISEASES. rod and 14 plates poured at 410 to 380 C, after inoculating very copiously — six 3 mm. loops in one instance. In other canes it was found alive at the end of a year. Tubes of bouillon inoculated November 9, 1903, and kept at room-temperatures were still cloudy on March 17 (129 days), but a transfer from one of them did not cloud bouillon. In beef-agar stabs kept in the refrigerator at io° to i5°C.the cultures were alive at the end of 7 months 10 days. Under similar conditions another culture was dead at the end of 7 months 18 days. Here the temperature may have been a few degrees higher, i. e.,\2° to 1 6° C. Another tube inoculated 14 days later and subject to the same conditions was alive at the end of 6 months 21 days. Two other tubes of the same lot were dead at the end of 6 months 12 days. Three other stab-cultures were dead at the end of 7 months 3 days. The organism was alive in cultures on potato- agarattheendof 7 months, but they were probably in the cold box. The organism was alive in 6 potato cultures at the end of 35 days (room- temperature about 25°C), but probably a large part were dead, as the transfers to fresh potato-cylinders grew slowly. On March 21, 1905, ten cultures on potato in the refrigerator at io° to 1 40 C. since October 6, 1 904 ( 1 66 days) were tested for vitality, transfers being made to potato-agar (streaks) . Two grew well ; two feebly in small portions of the streak ; and the rest did not grow. Of the two which grew well, one came from cane plant No. 40 and the other from cane plant No. 42. The organism has been found alive in milk cultures 54 days old; also in some 75 days old. Milk is a suitable culture medium for this organism. The organism is killed by sunlight (fig. 37). Fig. 37.* RESUME OF SALIENT CHARACTERS. POSITIVE. Parasitic in sugar-cane, clogging the vascular bundles with a bright yellow slime and forming cavities in the soft parenchyma ; frequently comes to the surface of the inner leaf- sheaths as a viscid slime. Surface colonies on +15 standard nutrient agar pale-yellow, *Fig. 37. — Bacterium vascularum in an agar-poured-plate after exposure of right side to bright sunlight (on ice) for 30 minutes. Plate poured and exposed January 19, 1906. Photographed January 26, 1906. The few bacteria which grew into colonies on the exposed side of the plate were sheltered, it may be presumed, from the direct action of the sun by other overlying bacteria. cobb's disease; of sugar-cane. 67 smooth, glistening, rather small, round, rather flat with sharp margins, no distinct appear- ance of granulation, alkaline to moist litmus paper, not noticeably viscid, coming up slowly at 250 C. (about 5 to 10 days when poured from cane) ; rods short, measuring on an average 0.4 X im when stained, but sometimes plumper and also longer forms occur; single, in pairs (mostly), fours, or eights, end to end; pseudozoogloese ; motile, single polar flagellum; cap- sules ( ?) ; stains fairly well by Gram (see negative) ; strictly aerobic (so far as known) ; optimum temperature about 300 C; growth retarded by temperature of 350 C; thermal death-point 49 ° to 500 C; growth on +15 peptonized beef-agar inhibited by addition of small amounts of vegetable acids; growth on potato-agar somewhat better than on beef- agar; scanty growth on litmus-lactose-agar, with slow but distinct bluing; good growth on neutral or slightly acid peptonized cane-juice gelatin, streaks slide down in characteristic "tear-drop" formation; occasional very slight liquefaction of gelatin; fairly good growth on steamed cylinders of susceptible varieties of sugar-cane ; growth on potato cylinders and coconut-cylinders good but not copious, moderate graying of the potato cylinder, mostly out of the water; scanty growth on steamed red table-beet cylinders; good growth on steamed onion and cauliflower; fairly good growth on steamed carrot and turnip; +15 peptonized beef-bouillon moderately clouded, pellicle rarely observed, whitish rim some- times formed, prismatic crystals often present ; nitrate bouillon moderately clouded ; good growth in neutral bouillon and in slightly acid beef -bouillon (+25 Fuller's scale) ; feeble growth in beef-bouillon titrating — 20 Fuller's scale ; feeble growth in Dunham's solution, and in Uschinsky's solution (see negative) ; litmus milk is blued, the milk remaining fluid and opaque; the organism can utilize aspa- ragin and ammonium salts as nitrogen food; it can obtain carbon from cane- Fig. 38.* sugar, dextrose, fructose, and glycerin (?); grows best with saccharose or fructose (RGS.); sodium chloride in amounts greater than 1 per cent inhibits growth; slight reducing action on cane-sugar (see negative) ; alkalies prohibit the multiplication of the organism, but when the reaction is faintly acid growth is quick and luxuriant (RGS.) ; the most suitable media are neutral or slightly acid, peptonized cane-juice gelatin, beet-juice agar with addition of peptone and saccharose, litmus milk, and steamed potato. It is sensitive to sunlight. Crystals occur on agar (fig. 38). Group No. 21(^.3332523. NEGATIVE. No spores ; no long chains or filaments ; no pellicle (usually) ; colonies not noticeably viscid; not pure white on any medium; not red, nor orange; no gas in any medium; no reduc- tion of nitrates to nitrites; no acids; no reduction of litmus (lactose-agar, milk) ; no precipi- tation of the casein in litmus-milk cultures ; no liquefaction of gelatin (usually) ; no growth at 37. 50 C.;no piling up of the slime in the water in potato-cultures, as in the case of yellow organisms, such as Bacterium campestre and Bacterium phaseoli; no growth in Uschinsky's solution (frequently) ; no growth in Cohn's solution (?) ; no growth in an atmosphere of car- bon dioxide ; no clouding of closed end of fermentation tubes of peptone-water containing grape-sugar, cane-sugar, milk-sugar, maltose, mannit, or glycerin. No growth (usually) on steamed cylinders of resistant varieties of sugar-cane standing in distilled water. Organism does not use potassium nitrate as nitrogen food. Does not stain by Gram's method (RGS.) . Does not use glycerin as carbon food (RGS.). Does not grow in peptonized beef-bouillon with 1.5 per cent or more sodium chloride. Does not reduce cane-sugar, according to R. Greig Smith. Does not brown agar. *Fig. 38. — Crystals from an agar streak-culture of Bacterium vascularum. More than 200 appeared in some of the tubes. Notes of March 7, 1902, and of February 1904. 68 BACTERIA IN RELATION TO PLANT DISEASES. TREATMENT. Little is known regarding the transmission of this disease from one plant to another except by way of the sets. The planters themselves are believed both by Cobb and Tryon to be largely responsible for the spread of the disease. Dr. Cobb says : It seems evident that gumming is not a disease that is spread to any great extent through the air * * * . This is shown by an array of facts that cannot be for a moment overlooked. Some stalks in a stool may be badly gummed and others in the same stool fairly healthy; part of a crop may be gummed, and the rest remain in good condition; one field may be badly gummed, and an adjacent field perfectly healthy; the Upper Clarence is comparatively free from gumming, while the Lower Clarence has suffered severely for several seasons. All these facts are incompatible with the idea that the disease is very infectious. If healthy plants easily caught the disease by receiving the germs of it from elsewhere, borne on the wind, such a case as a healthy crop standing alongside a badly diseased one would be almost an impossibility. [The writer has made precisely similar observations on the black rot of the cabbage. In this connection see Bacterium campestre, vol. II, p. 306.] On the other hand, the above facts are in harmony with the idea that the disease originates with the seed — the sets. * * * I was able to discover three cases on the Lower Clarence in which the crops were almost a total failure on account of gumming, where the planters, now that they know the nature and injuriousness of gumming, can recollect that the sets were badly gummed. They noticed the gum in the sets, which, when bad, is indeed very conspicuous, but not then knowing its nature, went ahead and put in the sets notwithstanding. These three cases are those of very intelligent farmers, to converse with whom was to be convinced that they were quite right in their observations. In another case a farmer purposely took sets from diseased plants in order to see whether they would reproduce the disease. The disease was reproduced. I had an opportunity to examine the resulting plants, and can certify to the result. Tryon speaks no less emphatically. According to his observations in Queensland the disease sometimes occurs spontaneously in patches of cane planted in low places onundrained land, but much more often it can be traced directly to the planting of cane-sets taken from diseased fields. I quote from his paper, published in 1895, as follows: It is an established fact in connection with this gumming disease that pieces of affected cane, when used for "sets, "give rise to it in the resulting crop. The malady may be sooner or later in mani- festing its presence, but, as a rule, its occurrence may be depended upon. Should the sets be badly gummed, however, no crop at all is obtained, as, though they may emit slender sprouts, these soon cease to be further developed. These facts have been demonstrated experimentally both at Winder- mere and Fairymead, and they explain in great measure the present distribution of the disease. * * * . Similarly, when a plantation was discovered in which no disease occurred * * * it was found that drainage had been resorted to, the crops from which the seed cane had been procured were perfectly healthy, or that no seed cane had been used except such as had been yielded by local healthy crops. Many instances could be adduced of this method of propagating the disease, and even just prior to my visit a considerable amount of unhealthy cane had been distributed from one Mary- borough center to be used for starting fresh cane plots. In many cases having observed a stand of diseased cane, and not discovering any explanation of its occurrence in local conditions of growth, a visit was afterwards made to the plantation whence the seed cane from which this was raised had been derived, when it was invariably found that the original stock was also diseased, and that other stands of cane which it had also served to originate were similarly affected. * * * When the disease appeared upon dry ridges, and no cane plants had been procured from beyond the plantation for sev- eral years, it was generally found that it was also present in some low-lying spots, where it had evi- dently originated spontaneously at an earlier date, and that its occurrence in one situation had led through the use of local sets, to its manifestation in the other. My own experience tends to confirm Cobb's statement respecting transmission of the disease. It is not readily spread from diseased to healthy plants, at least not in the hot- house. Cobb stated to the writer that he had recommended the use of certain varieties whose resistance to the disease he had observed, and wherever his advice had been followed the disease had disappeared. cobb's disease of sugar-cane. 69 The remedies advised by Cobb are: (1) selection of healthy sets; (2) good drainage; (3) burning of the trash; (4) rotation of crops, or bare fallow once every few years; (5) produc- tion of new varieties of cane by raising seedlings; (6) improvement by selection; (7) introduc- tion of new sorts from outside Australia: (8) the fostering of nurseries where (5), (6), and (7) are to be carried out. At the close is the following pertinent paragraph, which, be it remem- bered was written in 1893: Selection of disease resistant sorts. This is a subject that needs an essay by itself. I am con- vinced that one of the greatest improvements destined to be made in agriculture is in the line of secur- ing pest-resisting varieties. We stand as yet but on the threshold, yet we can clearly see the alluring prospect. What we now possess in a few cases, having obtained them almost by accident, shows how on the alert we should be to discover varieties as little subject to disease as possible. Cuttings designed for planting should be inspected very critically and those showing any signs of the yellow bacterial ooze on the cut surface must be rejected. Even very care- ful inspection at the time of cutting is not sufficient, however, for the removal of all dis- eased canes. Queensland planters, according to Tryon, are in the habit of covering the piles of cut canes for about three days with trash upon which water is thrown ; this induces a sweating process which greatly facilitates the detection of unsound cane. In this way "tons of bad cuttings have been picked out which otherwise would have spoiled the stand of the cane. " The Fiji method of detecting slightly diseased cane-cuttings is by steaming. The poisonous action of sodium salts on the organism suggested to Greig Smith that common salt might be used to check the disease unless the amount necessary to inhibit the growth of the bacterium would injure the sugar-cane. On inquiry, he found that in Fiji healthy crops of cane were grown on soil containing 1 per cent or less of salt, but any greater percentage was more or less injurious. Reports differ as to the comparative amount of this disease among crops raised on the seashore. One instance is given of a man whose farm was on an island in the Lakes, Clarence River, who was able to grow a comparatively sound crop of Mauritius Ribbon cane long after other growers had abandoned it because of its sus- ceptibility to this disease. On the other hand, there is the opinion on the part of some that canes grown on salty soil are more liable to be affected. This, Greig Smith thinks, may be traced to defective drainage. He agrees with Cobb, however, in regard to growing disease- resistant varieties, and mentions one well-known variety, Tanna, which has never been known to develop this disease. The sugar-content is less high than in some of the more sus- ceptible varieties, but Tanna is a stout, heavy cane, and when planted in fields which have yielded badly diseased crops produces perfectly sound canes. Table 6. — Susceptible and Resistant Varieties of Sugar-Cane. SUSCEPTIBLE. RESISTANT. Common Green (EFS.) Tanna or Elephant (Tryon, RGS.) La Canne Bambou (Boname) Louisiana No. 74 (EFS.) Louzier or White Bamboo (Tryon) Common Purple (EFS.) Rappoe or Rose Bamboo (Tryon) Malabar or Green Tanna (Tryon) Djioenig-Djioenig (Tryon) Daniel Dupont or Bambou Brancheuse Rayee (Tryon) Striped Singapore (Tryon) Cheribon or Outamite (Tryon) Mauritius or Striped Guingham (Tryon) China (Tryon) Meera (Tryon) Moore's Purple (?) Lahina (McGuigan) Green Dupont or Bamboo Brancheuse Blanche (Tryon) Kewensis (Knox) Australian Creole, a cane resembling Meera (Tryon) According to Tryon, the canes most subject to the gum-disease are just those varieties richest in sugar. "It may be remembered, however," he says, "that the canes that have been found to be free from disease in Queensland are included in both the poor-in-sugar and rich-in-sugar categories," e. g., Daniel Dupont, rich, and Elephant, poor. Tryon noted also that the disease was most pronounced in canes grown upon the best lands. Most of the immune varieties have hard canes and the susceptible ones soft canes, but this does not appear to be a rule without exceptions, since the Outamite or Cheribon is 70 BACTERIA IN RELATION TO PLANT DISEASES. rather soft. As a rule, also, the immune canes are fibrous and the susceptible ones other- wise. Susceptible canes such as Rappoe, Meera, and Djioenig-Djioenig are very intolerant of wet and cold. Resistant canes such as Black Tanna, Green Tanna, or Malabar, and China withstand cold weather and wet soils very well. In dealing with cultivation and its influence on the occurrence and virulence of the gumming dis- ease considerable stress is laid on the question of drainage, it being shown that the disease arises spon- taneously (generally, if not exclusively) on ill-drained areas. This proposition, therefore, gains sup- port from the experience that those canes which can tolerate wet land are not subject to the disease, whilst it especially prevails amongst varieties which are not endowed with this character (Tryon). According to Tryon soils, as such, exert no influence on this disease. It is equally prevalent on rocky, stony, stone-free, sandy, and clay lands. Lack of drainage, however, has a marked influence. When not directly traceable to affected sets, the disease is almost always most prevalent in low places or flat wet lands. This was observed by Tryon so often that it seems established beyond reasonable doubt. I may quote a few lines from his report : Proceeding again from the Bingera estate proper toward Kolan, patches of affected cane were here and there found, and in every instance their occurrence marked the site of areas of surface depres- sion and the convergence of watersheds, and the presence of a wet soil and subsoil. Again, in almost every other case where the disease was met with, both in the Wide Bay and Burnett districts, it was decidedly more pronounced on flats and hollows and where the ground was wet and undrained, a con- clusion not only founded on extended personal observation on my part, but also agreeing with the general experience of the planters themselves. This applies of course only to spontaneous outbreaks, not to infection due to planting diseased canes. To recapitulate: Select cuttings only from perfectly sound canes; discard supersensitive varieties; and on soils already much infected plant only very resistant varieties. PECUNIARY LOSSES. In countries where this disease has gained a foothold it is very destructive. The heavi- est losses have been reported by Cobb and Tryon in Australia, and by Dranert in Brazil. In many cases the crops have been almost entirely destroyed. Cobb said in 1893 that there was scarcely a farm on the Lower Clarence, in New South Wales, where the gumming was not abundant. In 1895, Tryon also regarded it as a very serious disease in Queensland, plots containing scores of acres being rendered valueless. Dranert speaks in similar terms of the Brazilian disease. It had been destructive in Bahia for 6 years. The Argentine disease suspected by me to be this malady is also serious (see page 85). According to Mr. Clark, a former cane-inspector of Queensland, thoroughly familiar with the disease as it occurs in Australia, and now an inspector in Fiji, this disease occurs in the Fiji Islands to a serious extent. It has been present some years. At first the planters did not know what it was, but now they are familiar with it and are taking restrictive meas- ures. I have this information through Dr. Cobb, who says another man from Fiji told him the same thing. If the disease is identical with Sereh, as the writer suspects, then the losses from it in Java alone are to be reckoned in hundreds of thousands of dollars, Sereh having at one time nearly put an end to the sugar industry of that great island. Enough has been said to show that the disease is a dangerous one, and planters should avoid the introduction of canes from affected districts even when they are guaranteed to be sound, lest they inadvertently introduce the disease on some of the canes. It is much easier to keep out this disease, as they are now endeavoring to do in the Sandwich Islands, than to fight it when it has gained a foothold. Planters in Louisiana and in Cuba, Porto Rico, and other West Indian islands should in particular be on guard against its introduction. As Cobb remarks, it is a disease peculiarly liable to be transmitted from one country to another in cane-cuttings. cobb's disease of sugar-cane. 71 LITERATURE. 1869. Dranert, Friedr. M. Bericht iiber die Krank- heit des Zuckerrohres. Zeitschrift fur Para- sitenkunde, Bd. 1, pp. 13-17, and Tafel 11, Jena, 1869. 1869. Dranert, F. M. Weitere Notizen iiber die Krankheit des Zuckerrohrs. Zeitschrift fur Parasitenkunde, Bd. 1, p. 212, and Tafel IV, fig. 58, 1869. 1893. Cobb, N. A. Gumming of cane. Dept. Agric, N. S. W. 1893.pp.8-10. Reprinted in Kew Bulletin of Miscellaneous Information, No. 85, Jan. 1894, pp. 1-4. 1893. Cobb, N. A. Plant diseases and their remedies. Diseases of sugar-cane. I. Microbe diseases of the sugar-cane; Gumming of cane {Bacillus vascularum). Agric. Gazette of New South Wales. Sydney, vol. iv, part 10, Oct. 1893, PP- 777-798, with 14 figures. Also a reprint, 8vo, 56 pp. The two half-tone figures from photomicrographs illustrating cross-sections of portions of diseased canes are reproduced so badly that the presence of the bacteria can not be made out. 1894. Anonymous. Cane disease in Pernambuco. The Sugar Cane . Manchester, 1 894, vol . xxvi, PP- 377-379- 1894. Editorial. The Sugar Cane. Manchester, 1894, vol. xxvi, p. 505. The insults the scientific man must sometimes endure from the so-called "practical man" are well illustrated in the fol- lowing citation: "The peculiarity of the disease here referred to in attacking the fibro-vaseular bundles is characteristic also of the disease known as "gumming" (described by Dr. Cobb) in New South Wales, and of the "gomziekte" (described by Kruger, Janse, et al.) in Java. The disease is not new. But how does it arise? We notice that an editorial in the Revue Agricole, of Mauritius, mentions that the bacillus is known. This statement, so often made respecting diseases in men. animals, and plants, seems to partake^ somewhat of the nature of a solemn joke. Of how many diseases can it be said that the knowledge of the bacillus has led to the cure of the disease, or even that the bacillus causes the disease? Up to now whatever kills the bacillus kills the patient also. It is high time for scientists to get off the stilts of mere technical assertion, and find us a remedy for disease, whether they can find a bacillus or not." 1894. Palmares. Cane disease in Pernambuco. The Sugar Cane. Manchester, 1894, vol. xxvi, P- 555- 1894. Boname, Ph. Rapport sur la maladie de la canne. Revue Agricole et Journal de la Chambre cTAgriculture de l'ile Maurice, 8 ann. No. 8, Aout, 1894. Port Louis, Maurice, pp. 178-187. A translation of this paper may be found in The Sugar Cane, Manchester, England, vol, XXVI, 1894, pp. 589-593 and 621-625. 1895. Cobb, N. A. The cause of gumming in sugar- cane. Agric. Gazette of New South Wales, Sydney, vol. vi, part 10, Oct. 1895, pp. 683- 689, with two figures. 1895. Tryon, Henry. Gumming of cane; being a report of an inquiry into the origin and nature of a disease affecting the sugar-cane in the Wide Bay and Burnett districts. Annual Report Dept. Agric. (Queensland) for 1894- 95- Brisbane, 1895. Paged separately, pp. 1-64. 1898. Wakker, J. H., and Went, F. A. F. C. Die Ziekten van het Suikerriet op Java. Deel 1, Ziekten, die niet door Dieren veroorzaakt worden. Mit 25 Tafeln, Leiden, 1898. 1898 (?) Dodson, W. R. Bacteriological notes on "red cane," in "sugarcane," etc., by Wm. C. Stubbs, vol. 1, State Bureau of Agric. and Im- migration, La. No date, but a copy was re- ceived at the U. S. Dept. of Agric, Feb. 14, 1898, pp. 173-178. Author thinks one or more bacterial forms always accompany red cane, but can not be said to have proved his case. Paper has no special relation to Cobb's disease. 1898. Raciborski, M. Voorloopige Mededeelingen omtrent eenige Rietziekten. Overgedruckt uit hef'Archief voor de Java-Suikerindustrie, Kagok Tegal, 1898. 1900. Went. Krankheiten des Zuckerrohres. Verh. vom Pariser Kongress, Juli, 1900. cf. Zeit- schr. f. Pflanzenkr., 1901, p. 297. 1 901. Smith, Erwin F. The cultural characters of Ps. hyacinthi, etc. Bulletin 28, Div. Veg. Phys. and Path., U. S. Dept. Agric, Aug. 6, 1901, p. 153. Name of organism changed to Pseudomonas vascularum, on account of polar flagellum. 1902. Smith, R. Greig. The gummosis of the sugar- cane (Bacterium vascularum Cobb). Proc. Linn. Soc. N. S. W., 1902, part 1, Apr. 30, 2 plates, pp. 31-47. Issued Aug. 22, 1902. Also a separate. See also Centr. f. Bakt., 2te. Abt., IX Bd., 1902, pp. 805 and 806, which adds nothing to the preceding. 1904. .Smith, R. Greig. The red string of the sugar- cane (Bacillus pseudarabinus n. sp.). Proc. Linn. Soc. N. S. W., 1904, vol. xxix, part 3, No. 115, issued December 16, 1904, pp. 449- 459- Sydney, N. S. W. 1904. Smith, Erwin F. Ursache der Cobb'schen Krankheit des Zuckerrohrs. Centralb.f. Bakt., 2 Abt., xiii Bd., 1904, pp. 729-736. Also a separate. For an English translation of this paper see pp. 12 to 22 of the next reference, :'. 5 3 24 No record. No record. 80 No record. t54 Cosmopolitan 12 rows *30 per cent, if the missing plants are included as diseased. tOne only of the six strains of this variety, and the one showing least disease in July, were not examined in August. The others October ig, igo 3.— Tenny and Rorer cut out and examined every plant in the 14 rows of Old Colony sweet corn planted on July 29 at the Arlington Farm. The corn at the lower (west) end and in the first two rows (south side next the fence row) was about 3 or 4 feet high and well tasseled out and looked fairly well. In the upper (east) and central part of the plot, however, the corn had made a very poor growth, many of the plants being only about a foot high and less than half an inch in diameter (due to poor soil) . The examination resulted as follows : Old Colony. Stalks found to be healthy, 6,235; stalks found to be diseased, 65. Total, 6,300; diseased, 1 per cent. Many of the diseased stalks showed only two or three yellow bundles, while only a few were very badly diseased. Some of the most badly affected were put into alcohol. October 24and2j, igoj. — The other varieties of corn were cut and examined by the writer, assisted by Messrs. Tenny, Rorer, and Deane B. Swingle, with the following results: Cosmopolitan. Stalks found to be healthy, 1,905; stalks found to be diseased, 366. Total, 2,271; diseased, 16 per cent. It appeared on the start that there were many more cases in the lower moister land, where the corn had made the best growth, than on the side hill or the level land on the top. When classified in this way the result shown in table 1 1 was obtained : Table i i . — Effect of Slow Growth on General Injection. Kind of ground. Healthy stalks. Diseased stalks. Total. Per cent, diseased. 754 605 546 233 58 75 987 66i 24 (3) Flat • at top of hill Owing to the labor involved and the bad weather the entire plot was not counted, but only aver- age portions of it. The same method was followed with the remaining varieties; the only variety counted in full being Old Colony. Country Gentleman. Healthy stalks, 881; diseased stalks, 32. Total, 913; diseased, 3.5 per cent. STEWART'S DISEASE OF SWEET CORN (MAIZE). 123 The variations in the plot were not great, as may be seen from selected portions (table 12). Table 12. — Showing that Kind of Soil Makes no Difference when Seed is not Infected. Kind of ground. Healthy stalks. Diseased stalks. Total. Per cent, diseased. 445 17 462 451 3.68 3 33 (2) North side; middle of field, better growth than to 436 is Crosby's Early. Healthy stalks, 2,171; diseased stalks, 437. Total, 2,608; diseased, 17 per cent. Arranged in three groups according to amount of soil-moisture and vigor of the plants, the same results follow as in the case of the Cosmopolitan : Table 13. — Showing that Moist Soil is Favorable to Development of the Disease when the Organism is Present. Kind of ground. Healthy stalks. Diseased stalks. Total. Per cent. diseased (1) Middle of field (east and west) on hill-slope (2) West side, moist end (3) North side, near west end, i. e., in lowest, moistest part of the field 1,145 456 570 165 76 196 1,310 532 766 12.6 14.3 25.6 This corn was larger than any other variety except the south two rows of Old Colony. Potter's Excelsior. Healthy stalks, 2,227; diseased stalks, 44. Total, 2,271; diseased, 2 per cent. Arranged in groups according to soil-moisture and size of plants, the above count gave prac- tically the same results as the Country Gentleman : Table 14. — Showing that Moist Soil has no Injurious Influence in Absence of the Parasite. Kind of ground. Healthy stalks. Diseased stalks. Total. Per cent, diseased. (1) Lower, moister part of field (west), near badly dis- 764 920 543 21 12 I 1 785 932 554 2-7 i-3 2.0 (2) About 8 rods from west end. (These plants about same size as neighboring portion of Crosby's Early, (3) Upper (east) end of field; higher ground. (Plants averaged only 1 . 5 feet in height; stand not very good) SUMMARY. The above facts may be summarized as follows : Table 15. — Per cent of Cases by Varieties and Soil. Variety. Irrespective of nature of soil. On moist parts of field. Old Colony 1 2 16 3-5 »7 No record. 2-7 23.6 3-7 25.6 124 BACTERIA IN RELATION TO PLANT DISEASES. The results at Arlington agree substantially with those obtained from the trial-rows on the other farm. In both Cosmopolitan and Crosby's Early they point unmistakably to infection of the plants from the seed-corn. In the other three varieties the cases are so few that they may have arisen from proximity to the two diseased varieties. It is not diffi- cult to understand how this could have taken place in blocks of healthy plants sandwiched in between diseased ones, provided some of the latter developed the disease early in the growing season. If the disease was actually derived from the seed-corn there probably would have been some cases during the seedling stage, and fragments of these soft plants full of the bacteria would have been blown upon neighboring plots, or dragged by cultivators, or carried on the feet of men and horses, or bitten into by insects, or washed about by rains and dews. There are ways enough to account for the dissemination of the bacteria and the infection of a few plants where the distance is only a matter of a few feet. When plants are well past the seedling stage there is much less danger of infection, even though the infectious material is then much more abundant. That a larger number of cases did not occur in the Crosby's Early and the Cosmopolitan is attributable to the poor soil and slow growth as compared with that on the Flats and probably also to the gradual dying out of the bacteria on the seed-corn between spring, when the first plantings were made, and midsummer, when the last plantings were made. The experiment is equally interesting, however, if we assume all of the seed-corn to have been free from Bad. stcwarti. In such event the organism causing the disease must have been present already in the soil of the field. If for the sake of argument such a supposition be granted (and it can be granted for no other reason), it certainly stretches the limits of probability to assume also that the organism was prevalent only in those narrow strips where Crosby's Early and Cosmopolitan corn happened to be planted. If the organism was present in the soil it is likely to have been there a long time and to have been pretty uniformly distributed, just as it is in the infected soils on Long Island. Granted these premises, then we may assume that 3 of the 5 varieties tested are very resistant to the disease, and are, therefore, desirable varieties to plant on infected land, and important to use as one parent in originating resistant varieties by cross-breeding. The other two varieties, on the contrary, should never be planted on infected land. Subsequent experiments showed that an abundance of moisture stimulating rapid growth is very favorable both to primary infection during the seedling stage, and also to the general distribution of the bacteria through the stem of the plant later on. On the contrary, dry soil and dry weather following planting interferes with infection, and any decided check in growth later in the season interferes with the movement of the organism through the plant by hardening the basal nodes and thus rendering it difficult for the organism to get past them. In infected plants, however, the disease shows sooner, accord- ing to Stewart, in periods of drouth than in rainy weather. The writer has observed the same thing in diseased Cucurbitaceae (see this monograph, Vol. II, pp. 216, 284). EXPERIMENTS OF 1908. In 1908, Golden Bantam sweet corn attacked by Bacterium stewarti was sent to me from a garden in Falls Church, Virginia, with the statement that the seeds were obtained from a Philadelphia seedsman, who was believed by the planter to have sold him bad seeds, because nearly all the plants of this variety were badly diseased, while other varieties obtained elsewhere showed no disease or only very little. I asked the dealer where he procured the seed of this variety and he said it came from Mr. — — , Wakeman, Ohio, the man to whose farm I traced the disease in 1903. This interesting fact led me to procure a half-bushel of this particular lot of seed (all the dealer had left) for experiment. The first endeavor was to isolate Bacterium stcwarti from the surface of the kernels. In this effort we failed. The organism may have been STEWART'S DISEASE OF SWEET CORN (MAIZE). 1 25 present, but not alive after so many months. Several kinds of yellow bacteria were obtained in abundance from the numerous poured-plates, but none corresponded exactly to Bacterium stewarti. Many resembled it in the agar-poured-plates, but all of the hundred or more colonies selected as hopeful differed in some particular, i. e., either they grew differently on potato, or coagulated milk, or liquefied gelatin, or clouded Cohn's solution, or refused to grow in Uschinsky's solution, or formed gas, or rapidly reddened or blued litmus milk, etc. They were checked up against three strains of Bacterium stewarti obtained from as many different localities. One form in particular, designated as "Nv" which appeared as though it might be Bacterium stewarti (before additional cultural tests were made which threw it out) was pricked into the leaves of young plants of this variety of sweet-corn copiously, but no disease resulted.* This failure to obtain Bacterium stewarti believed to be present on the kernels is prob- ably to be attributed to the presence in abundance of non-parasitic yellow bacteria on the surface of the corn, while the right organism occurred in a viable form in very small numbers or not at all, so that on the poured-plates it either did not appear or occurred in such small numbers as to be overlooked, the problem of finding it on agar-poured-plates under such conditions being a difficult one. Probably if we had cultured from crushed kernels we would have obtained the organism. This conclusion is borne out by the following experiments. SERIES XVI TO XIX, 1908. On July 23, 1908, a center bed extending the whole length of one of our large hot-houses was planted to this corn. Hitherto corn had never been planted in this house. Orange trees of large size stood in this earth and had done so for many years. The trees were grubbed out, the soil was spaded, a little bone meal was added, and then the suspected corn was planted thickly in rows crosswise of the bed, at a distance apart of about 18 inches. The bed was divided crosswise into four portions as follows: (1,2) two plots planted with selected sterilized seed; (3) one plot planted with untreated seed just as it came from the original sack; (4) one plot planted with the most inferior looking kernels in the sack (also untreated) . The crop was harvested on October 6 to 9. Cross-sections of every stem were examined under the hand-lens by the writer. Some were also examined under the compound micro- scope and many were put into alcohol for future use. The results were as follows: (1) Selected (hand-picked) good-looking seed, soaked 15 minutes in 1:1000 mercuric chloride water, rinsed in tap-water, and immediately planted (11 rows) : Number of healthy plants, 1,090; number of diseased plants, 13. Total, 1,103; per cent diseased, 1.2. Remarks. — None of the affected plants were badly diseased. The record is as follows : Eleven slightly diseased, 2 of which had open wounds on the stem above. Only one bundle visibly infected in most of these. Bacteria visible only in the basal part of the stem. The twelfth had 3 diseased bundles, and the thirteenth, which contained several bundles occupied by yellow bacteria, had one whole internode above soft-decayed. Possibly those with bruised or wounded stems should have been omitted from the record. None had yet reached the stage of shriveling leaves. (2) Selected (hand-picked) good-looking seed, soaked 10 minutes in 1 : 1000 mercuric chloride water, rinsed in tap-water, and immediately planted (12 rows): Number of healthy plants, 1,277; number of diseased plants, 23. Total, 1,300; per cent diseased, 1.8. Remarks. — Two plants with 3 bundles infected; 19 slightly diseased (a few bundles in the lower part of the stem) ; 2 badly diseased, both having many bundles occupied by the organism and the leaves dried out. In one, the stem was infected the whole length, and toward the base nearly every bundle was occupied by the yellow slime. *In 1904, Miss Florence Hedges, of my laboratory, obtained similar negative results on a much larger scale, using a yellow organism supposed at first to be Bad. stewarti, but afterwards found to behave differently on certain media. Three hundred sweet-corn plants were inoculated very thoroughly on the leaf tips when the seedlings were extruding water, but no disease resulted. 126 BACTERIA IN RELATION TO PLANT DISEASES. Assuming the kernels to have been the carriers of the organism, the failure of the relatively long soaking in mercuric chloride water would seem to indicate either that the bacteria escaped destruction because they were inside the kernels, or else because they were protected from the action of the germi- cide by being massed together in dried-down hard crusts, the former being the more probable explana- tion. Certainly all those freely exposed on the surface of the kernels must have been destroyed. (3) Unselected, unsterilized seed (20 rows) : Number of healthy plants, 1,548; number of diseased plants, 156. Total, 1,704; per cent diseased, 9.0. Remarks. — Of these plants 24 were slightly diseased; 25 moderately diseased; and 52 badly dis- eased. No remarks on the remainder. Comments on some of the badly diseased will be of interest as showing the extent of infection : One badly diseased: Bacteria as far up as the cob, and also far above the cob in the top of the stem. One badly diseased: Bacteria have oozed through and formed water-soaked yellowish stripes or spots on the pedicel of the cob, and have also oozed through the husks, forming many water-soaked and yellow patches on at least half a dozen inner husks. The bacterial slime was on the inner surface of these husks and also on the kernels. Had photograph made of the husks (fig. 53). A similar oozing through the husks was found yesterday in a plant removed from this same plot to send to Prof. H. H. Whetzel, of Cornell University. Miss Lucia McCulloch examined the slime on the kernels with the compound microscope and found it to be made up of bacteria of the typical sort. These are just as good examples of disease of the cob as any I obtained on the Potomac Flats in 1902. Two badly diseased: Every bundle apparently occupied. Infection extends up to male inflorescence, a distance of 3.5 feet from the base, and out into the husks, which are sticky to the touch from the extruded bacterial slime. Two badly diseased: Husks badly spotted by the bacteria, and bacterial slime abundant in the stem far above the husks. One badly diseased: I find the bacterial ooze bright yellow and viscid on the inside of some of the leaf-sheaths in masses sufficient to scrape up with the knife. It has also oozed out in certain places on the internodes. These places are long, water-soaked, yellowish stripes. Until yesterday I had never seen it forming water-soaked stripes on the sur- face of the internodes. The ears of this plant are also diseased, there being many water-soaked places and many yellow spots on the inner husks. One badly diseased: Here the bacteria have formed pockets in the leaf-sheaths, and have oozed out on the surface of the stem as a conspicuous bright yellow slime, which is viscid enough to string out in cobwebby threads. I have just stretched them out 6 inches. One badly diseased: Here again the bacteria have oozed from the inner surface of the leaf-sheaths, especially the lower ones, and have formed a great number of yellow pockets in the inner husks. The bacteria have oozed onto the inner surface of the husks in large yellow masses which are sticky. The infection is very striking. Seeing such ears one readily appreciates how impossible it is for the surface of the kernels to remain free from infection. One badly diseased: Nearly every bundle at the base of the stem is occupied. The cob is not visibly infected, but the stem near the cob is infected. Two badly diseased : The smaller one has no ears, but the inner face of the leaf-sheaths contains water-soaked and yellow spots, and there is a copious yellow bacterial ooze on the inner face of at least three such leaf-sheaths. The larger plant shows no ooze from the leaf -sheaths, but the ear is infected in the same way as those previously described, i. e., there are numerous yellow spots with bacterial ooze on the inner face of the husks. One diseased badly: There are water-soaked stripes on the surface of the internode, but no bacteria have actually come through. Four diseased badly: Every bundle is occupied in each stem. One diseased badly: Every bundle at the base of the plant is occupied by the yellow slime and bacteria are visible in the bundles at least 3 feet from the ground, probably all the way up to the male inflorescence. Yellow ooze is visible also on the inside of some of the leaf-sheaths and also on cross-section from the base of the cob. One badly diseased: Every bundle of the stem is occupied. The bacteria have oozed to the inner surface of some of the leaf-sheaths. One diseased badly : In the husks as well as in the stem. One badly diseased: Bacterial pockets in lower leaf-sheaths. The husks of the ear are diseased. One badly diseased: Here for the first time in my study of this disease I find evidence of bacterial ooze from the pedicel of the cob (surface). It is water-soaked and yellow and there is ooze from a half-dozen or more stomata in the form of short cirri. One of these cirrus threads is many times as long as broad. This material is so good that I will put it into alcohol by itself (fig. 64 a). One badly diseased : Yellow bacterial ooze on inside of leaf-sheaths. Three badly diseased: In the largest one of the three there are water-soaked stripes on the internodes, and also a badly diseased ear, the husks containing numerous yellow bacterial pockets from which slime has oozed to the inner face of the husk. The lower leaf-sheaths are also crusted with yellow bacterial slime on the inner faces. One badly diseased: Every bundle of the stem occupied. Four badly diseased: Nearly every bundle occupied at the base of the stems. Also spots on the inner face of the leaf-sheaths where the bacterial growth is bright yellow in patches, also many bacterial patches on the inner husks. One badly diseased : No ears. One badly diseased: Husks of the ear spotted with water-soaked and yellow places. One very badly diseased: Like most of these it has no living leaves. A badly infected ear. Many yellow spots and much bacterial ooze on the inner husks. Nearly every bundle is occupied at the base of the plant and I can count at least a dozen bundles full of bright yellow slime at a distance of 6 feet from the ground and about 4 inches under the male inflorescence. One badly diseased: Every bundle of the stem occupied. No ear. One badly diseased: Every bundle apparently is occupied at the base of the stem. Bacterial ooze from inside of some of the leaf-sheaths and on the husks. One moderately diseased: Has perhaps 20 or 25 bundles occupied. STEWART'S DISEASE OP SWEET CORN (MAIZE). 127 One very badly diseased: Husks and cob included. One slightly diseased : 3 or 4 bundles show yellow ooze and four are browned. Two badly diseased: Cobs infected. Hundreds of little yellow spots on the husks. One diseased : About 20 or 30 bundles infected at base. One badly diseased: No ears. One badly diseased: The husks as well as the stem. The leaf-sheaths show bright yellow bacterial ooze on their inner surface. (4) Selected, bad seed, untreated (six rows) : Number of healthy plants, 284; number of diseased plants, 29. Total, 313; per cent diseased, 9.3. Remarks. — No notes on the first 25, some of which were badly diseased. The twenty-sixth, twenty-seventh, and twenty-eighth were slightly diseased. In the twenty-ninth four bundles were observed to be infected. GENERAL REMARKS ON SERIES XVI TO XIX. July 28. — The germinated plants are 1 to 2 inches high. The best germinations are in the plot treated with mercuric chloride 1 : 1000 for 10 minutes. The next best are those treated in the same way 15 minutes. The untreated ones (unselected seed) are decidedly poorer. The poorest plot is that planted with the selected bad seed. October g. — The experiment gave about 8 times as many cases on the untreated as on the treated. The contrast in the amount of infection in individual plants on the untreated plots and on those treated was very striking. Many stems of the former had nearly every bundle occupied to the top of the plant, and also many ears involved, and all of the leaves were dried out, whereas in the latter there were only a very few plants which showed any infected bundles except near the base of the plant, and most of the leaves were yet green. The total bacterial multiplication in the plants grown from unsterilized seed was several thousand times as much as in those grown from the sterilized seed. The infection of the ears was as abundant and striking in this experiment (untreated plots) as in any I have ever made. Several of these ears were put into alcohol. They had a very large number of yellow spots on the husks (fig. 53) and the bacterial slime had oozed from some of them and was sticky to the touch. In one it was so viscid that the slime was stretched out a distance of 31 inches before the cobwebby thread ruptured. In the untreated plots I did not find a single row that had no diseased plants in it. This experiment indicates to me quite clearly that the seed corn was the carrier of this disease. Probably if I had extended the soaking in mercuric chloride to 20 minutes, the check plots would have been entirely free from the disease instead of nearly free. I feared to do this lest I might destroy the germinating power of the corn. The soil was the same, the corn was the same, the water was the same, the experiment was controlled in the hothouse, and the only known variable factor was the germicide. Moreover, the corn was already under suspicion because of its source and of its behavior at Falls Church, Virginia. The experiment serves to establish more firmly the conclusion drawn from an earlier experiment respecting the existence of infected fields at Wakeman, Ohio. It further empha- sizes the wrong done by seedsmen to the general public in selling corn grown on fields infected by this organism, since it serves to distribute the disease broadcast over the country. Wherever such corn is planted it not only gives a poor crop, but also serves to cause disease in a succession of crops on the same land as a result of soil infection. The time between planting and the first appearance of cases was about 60 days. The plants were crowded in the rows much like fodder corn and grew spindling, many of the stems being brittle. Had the plants grown uncrowded in a moist field the number of cases would undoubtedly have been tripled or quadrupled, as a rapid watery growth greatly favors the multiplication of the bacteria inside the stems, whereas stunted, hard basal nodes often prevent the organism from getting out of the base of the plant. Probably a spring planting would also have given more cases than the midsummer planting, since the dried-out bacteria gradually perish with lapse of time. 128 BACTERIA IN RELATION TO PLANT DISEASES. The effect of stunting in preventing general infection is shown quite clearly in Series III to VII (1902) and in the field experiments at Arlington in 1903. It is also shown in the following experiment. SERIES XX TO XXIV, 1908. This experiment was begun July 14-15, 1908, on another part of the grounds of the U. S. Department of Agriculture, out of doors, in a vacant cold frame between two hot- houses. The same Golden Bantam sweet corn was planted as in Series XVI to XIX, but the soil was different, and the other conditions were such that it was known from the start that the plants would be badly dwarfed. In fact, the experiment was made for this very purpose, so that the effect of slow growth might be compared with more rapid growth such as was anticipated and obtained to some extent in the preceding experiment. The corn was planted rather thickly in new 6-inch pots. Good hot-house soil was used. This was prepared from well-rotted sods thoroughly mixed with old cow manure which had been on the Department grounds in compost heaps for the previous 9 months. The cold frame was divided into five compartments and planted as follows: (1) Two hundred and seventy-five pots, planted with selected good seed, about 6 seeds per pot, more rather than less, soaked 8 minutes in 1 : 1000 mercuric chloride water, and then washed in sev- eral waters. On July 21, 621 plants were visible. About one-half the seeds germinated. (2) Two hundred and seventy-five pots, planted with selected good seed, about 6 seeds per pot, more rather than less, soaked for 30 minutes in 1 : 200 formalin water. The formalin delayed the germination. On July 21, 481 plants were visible. About one-third of the seeds germinated. The above two plots were planted on July 14 and the following next day. (3) Two hundred and seventy-five pots, planted with selected good seed, about 6 seeds per pot, more rather than less, soaked for 1 hour in 1 : 2 hydrogen peroxide water. On July 21, 603 plants were visible. About one-half germinated. (4) Six hundred pots, planted with unselected, untreated seed, about 8 seeds per pot, more rather than less. On July 21, 875 plants were visible. Less than one-fourth germinated. (5) Three hundred and eighty-five pots, planted with selected bad seed, about 12 seeds per pot. On July 21, 502 plants were visible. An average of about 1 in 6 finally germinated. The pots stood close together on clean sand. The average height of the plants when harvested was between 2 and 3 feet, i. e., they were much stunted by lack of room for the roots. Many of the seedlings showed, soon after germinating, what were regarded at first as suspicious signs, and plants in 74 of the pots were marked, but nothing developed later. The suspicious signs were water-soaked streaks on the young leaves. These turned out afterwards to be due to excessive pumping out of water through the water pores and were not infections. Some of the leaves recovered promptly; others retained yellowish stripes for a long time, but none contracted Stewart's disease. On September 1 (forty-seventh day) a careful examination of all the plants showed no cases. The corn had begun to tassel. It was at that time also badly dwarfed and yellowish from lack of room for the roots. The tallest and greenest plants were next to the hot-house, where they were somewhat shaded from the morning sun, and did not, therefore, dry out as quickly. The first cases were observed at the end of about 60 days. The plants were harvested September 29. The per cent of cases by plots is shown in table 16. Table 16. — Effect of Slow Growth on Development of the Disease. No. of plot and kind of seed. Treatment. Healthy plants. Diseased plants. Total. Per cent, diseased. Soaked 8 minutes in i: 1000 mercuric chloride water. 1 : 200 formalin, one-half hour. i: 2 hydrogen peroxide, one hour. Untreated 736 620 721 1.533 811 3 4 3 8 9 739 624 724 i,54i 820 0.4 0.7 0.4 0.5 1 .2 (2) Do (3) Do (4) Average kernels out of bag. (5) Worst kernels in sack. . . . Do STEWART'S DISEASE OF SWEET CORN (MAIZE). 129 Here the overmastering influence was the lack of room to make a vigorous juicy growth Many of the stalks were brittle as pipe-stems when cut. SERIES XXV, 1912. In the spring of 191 2, Lucia McCulloch, of my laboratory, inoculated sweet corn and field corn in the hot-house by spraying on a water suspension of Bad. stewarti when the plants were about a foot high. Soon after, they were set out on the grounds of the U. S. Department of Agriculture, where they grew well and showed but slight signs of disease. When cut in September the field corn was free from disease. Of the sweet corn about one-half of the stems were infected, but most of them only slightly. Of the whole number (about 50) only 5 or 6 were badly diseased. The season was Fig. 54.* Fig. 55. f a growing one and the organism was infectious. The feeble results, therefore, are to be attributed, I believe, either to resistance on the part of the variety used or to the lateness of the inoculation, i. c, after the plants had passed out of the seedling stage. *Fig. 54. — Cross-section of the base of a sweet-corn kernel, showing a single spiral vessel filled with Bacterium stewarti, the result of an inoculation made about 2 months earlier by placing a trace of a pure culture on the tips of the leaves of the seedling plant. Material fixed in strong alcohol, embedded in paraffin, sectioned on the microtome, stained with carbol-fuchsin, differentiated in 50 per cent alcohol, and mounted in Canada balsam. Figure drawn under the microscope with the aid of an Abbe camera. Slide 235 A 8. For orientation see fig. 47. tFiG. 55. — Cross-section of internodal bundle of a sweet-corn stem showing Bacterium stewarti occupying a single vessel. Material obtained on Long Island, New York, July 16, 1902, fixed in 95 per cent alcohol, embedded in paraffin, sectioned on the microtome, and stained with carbol-fuchsin. Slide 249 C 2. 13° BACTERIA IN RELATION TO PLANT DISEASES. MORBID ANATOMY. The lesions in this disease are strikingly like those induced by other organisms of this group, e. g., Bacterium hyacinthi and Bad. vascularum. The vascular system is occluded by the bacteria to an astonishing degree. So far as I know, scarcely anything comparable to it in extent and severity occurs in the animal body, the nearest approach, perhaps, being certain septicaemias (figs. 54, 55,56). There is this astonishing difference, however, due to the very different character of the circulation in plants and animals, that while in many cases the general septicaemias of the animal body are rapidly fatal, a few days sometimes sufficing, this disease of maize progresses slowly in spite of the presence of enormous numbers of the bacteria, and destroys the plant apparently only when the water-conducting tissues (vascular Fig. 56.* Fig 57-t bundles) have become blocked up by the invading organism to such an extent that transpiration greatly exceeds water-supply. An early death as the result of the action *Fig. 56. — Cross-section of an internodal bundle in sweet-corn, showing restriction of Bacterium stewarti to the xylem part of the bundle. The result of a pure-culture inoculation made in the seedling stage by placing the bacteria on the tips of young leaves. Material collected and fixed in October 1902. The entire stem at this level contains 287 vascular bundles, of which all but 10 are occupied by the bacteria. In every instance (?) the bacteria are still confined to the xylem part of the bundle. Slide 248 A 6. fFio. 57. — Diagrammatic longitudinal section through lower half of a sweet-corn kernel attacked by Bacterium stewarti, the areas occupied by the bacteria being drawn in solid black (lower left-hand side). 5, envelope of the grain; St, starchy part; E, scutellum; P, plumule; R, radicle; B, base of kernel. The starchy part was very soft when embedded and is squeezed together and upper part torn away. A detail from X is shown in fig. 58, A. Slide 485 (7. X 14. From Golden Bantam corn grown from diseased seed in the summer of 1908. STEWART'S DISEASE OF SWEET-CORN (MAIZE). 131 of toxic substances does not occur. Stewart's disease is preeminently a vascular disease, but is not confined to the vessels exclusively, as he supposed. The disease, like many other bacterial diseases, begins in the parenchyma and ends in the parenchyma. In the earliest stages of the disease, in order to enter the vascular system the organism must first penetrate -.y- it X A * * '/too •t 0 § • Fig. 58.* the epithem or multiply in the cellular tissues under the substomatic chamber (vol. I, fig- 75)! only in the rare event of infectious wounds made directly into the vessels can it be otherwise. In the late stages of the disease, also, as already noted under "Etiology," one very frequently finds conspicuous bacterial pockets in the soft parenchyma adjacent to the bundles. The histological character of these pockets is shown in figs. 48, 49. Their small size and their relative abundance can be seen in fig. 53 and plate 9. The writer has not observed in sweet corn any cavities at all compar- able to the large ones frequently seen in sugar-cane attacked by Cobb's disease. Many organs of the plant are subject to invasion by this organism, i. c, roots, nodes, internodes, leaf-sheaths, leaf-blades, male inflorescence, female inflores- cence; but there are no hyperplasias. Occasionally small shoots push from the base of the plant (plates 6, 8, 10), but these are not very numerous, nor frequent. In the ears the bacterial slime occurs in the cob, in the kernels (sparingly), and in the husks, often very abundantly in the latter, as may be seen by consulting my notes under "Etiology." From the husks the surface of the kernels is readily infected. *Fig. 58. — A. A detail from fig. 57, at X, showing cavities in the vascular region of the outer layers of a corn kernel occupied by Bacterium stewarti. Stained with methyl green and acid fuchsin. B. Similar infection, but from another kernel and in cross-section. The slides prepared contain numerous serial sections and were numbered from the base of the kernel upwards, i, 2, 3, etc. The sections on slides 1 to 12 contain bacteria, i. e., to a point above the level of the base of the radicle and close to the scutellum. Besides the central bacterial cavity, spiral vessels in the upper part of the drawing are also sparingly occupied. The heavily shaded cells are part of the enveloping sclerenchyma. Slide 485 A 1 (6, second row from top, second section from left, stained with pyronine and methyl violet. The bacteria are deep red, the sclerenchyma is blue. fFic. 59. — A. Bacterium stewarti drawn unstained from the margin of a hanging drop. Organism grown in +15 bouillon for 52 hours. B. Bacterium stewarti from yellow slime on a potato culture 3 days old at 30° C. Rods motile, many dividing. Drawn unstained from a hanging drop of water. Fig. 59. f 132 BACTERIA IN RELATION TO PLANT DISEASES. The slime finds its way to the inner surface of the husk more readily than to the outer surface, but sometimes it reaches the latter also. It generally comes to the surface through stomata (figs. 49, 50, 51, 52). The only conspicuous parts in which the slime has not been seen are the floral organs and the silicified outer parts of the stem. Even the latter state- ment must be modified somewhat as a result of the observations recorded in 1908, since rarely the bacteria may be seen issuing from small cracks in the siliceous covering of the internodes, especially where protected by the leaf-sheaths. Probably the stigmas (silks) are never infected, they are so fugitive, but the male flowers might well be infected, since the writer has traced the infection well up into the main axis of the male inflorescence a number of times. As yet we do not know how the wall of the vessel is pierced. It is probable that cellu- lose is not destroyed. THE PARASITE. Bacterium steward EFS.* as it occurs in the vessels of the maize plant and in young cultures on ordinary culture media is a short rod under in in diameter and generally less than 4(jl in length, with rounded ends (fig. 59). It occurs singly, in pairs, or fours, joined end to end. According to Stewart the organism usually occurs in pairs with a plain con- striction. "A pair varies in length from 2.5(1 to 3.3^ and in width or diameter from 0.65/1 to 0.85^." My own measurements do not differ materially. The following are average measurements taken from organisms grown in various media : Measurements made July 21, 1909, from potato-cultures 4 days old gave the following results: Amyl Gram stain gave 1.3 to 2.1/1X 0.4 to o.6/<; carbol-fuchsin gave 0.9 to 2.0/1X0.3 to 0.7/1. On some of the slides the carbol-fuchsin seemed to have contracted the protoplasm into small lumps. Agar streak-cultures 2 days old gave the following results : Amyl Gram, 0.9 to 2.0/1 X 0.4 to 0.7/1; carbol-fuchsin, 0.9 to 1.7/1 X 0.4 to 0.6/1, Taken direct from the vessels of the maize plant, the following measurements were obtained : Bacterium stewarti, stained in the tissues (material from Jamaica, Long Island), measured 1.1 to 1.5/1 X 0.5/1. A smear preparation from the Long Island sweet-corn stained by carbol-fuchsin measured 0.9 to 1.4 /1 X 0.4 to 0.55/1- Stained in section from a corn husk taken from the Flats experiment, slide 477 c 1, the organism measured 1 to 1 .4/1 X 0.4 to 0.5/1. The general appearance of the organism is shown in figs. 59 and 60. The organism on media is slightly viscid at times and undoubtedly possesses a capsule, although no efforts have been made to demonstrate it by means of special stains. The only very viscid masses seen are those mentioned under examination of diseased plants in 1908. The organism is motile, especially in young cultures, and the writer has demonstrated a polar flagellum (fig. 60). Usually, at least, only one flagellum is present on the end of each rod. Long chains or filaments have not been observed. So far as known the organism does not produce endospores. Small clumps and bunches of the bacteria (pseudozoogloeae) occur in various media as follows: Uschinsky's solution, nitrate bouillon. The organism stains readily with various anilin dyes. The writer has tried the follow- ing with fairly good results: Carbol-fuchsin, Loeffler's alkaline methylene blue, amyl Gram. Gram's stain gave negative results or at least not a deep stain. The rods were visible with wide-open diaphragm, but were only a pale blue. When cultivated in salted peptone water containing methylene blue the bacterial pre- cipitate was stained a deep blue, the pigment in the fluid remaining unreduced and bright blue or greenish-blue in color. Repeated in 1908 with same result, i. e., bacteria distinctly blue both to the naked eye and under the microscope. *Syn. Pseudomonas stewarti EFS. STEWART'S DISEASE OF SWEET-CORN (MAIZE). 133 x \ Fig. 60.' This organism grows slowly on gelatin without liquefaction. In a stab-culture in nutrient gelatin No. 478 at the end of 41 days, at 170 to 220 C, there was a thin line of growth along the needle-track, best toward the top, and a dense, rather dry, slightly rough- ened, bright, buff -yellow surface growth 7 mm. in diameter. Fig. 61 a shows its appearance in gelatin stabs. In +10 nutrient gelatin, in rather thin sowings in Petri-dish poured-plates, at the end of 7 days, at io° to 200 C, the surface colonies of Bacterium stewarti under the Zeiss 16 mm. objective and 12 comp. ocular were small, not perfectly circular, ^ not uniformly granular, more or less finely fissured, the margins not entire and sometimes even more or less decid- edly notched ; the buried colonies were globose-lobulated and less than 1 mm. in diameter. This may not be a con- stant phenomenon. In streaks on Loeffler's solidified blood-serum Bact. stewarti made a good bright buff -yellow growth, but without liquefaction. The cultures were under observation for 32 days. On agar plates it grows slowly, forming small round yellow colonies, the buried ones being very small. In agar streak-cultures it gives a smooth trans- lucent yellow slime which is usually paler than that of Bact. hyacinthi (fig. 62), and frequently lobed on the margins. Old streak-cultures are usually thin and somewhat dry, and the older bacterial layer generally baars numerous small raised, wet-shining, yellow colo- nies on its surface. Jagged X-shaped crystals and short prismatic crystals are not uncommon (fig. 63). In agar stab-cultures at the end of 16 days there was a moderate growth the whole length of the needle- track and a distinct surface growth some millimeters in diameter. Even where there were several stabs in one tube the growths did not cover the entire surface of the agar or coalesce ; the color was between saffron- yellow and deep chrome (Ridgway). At the end of 5 weeks the surface growth was a deep yellow and not at all viscid ; there was a good growth the whole length of the stab; the agar was unstained, and there were prismatic crystals in the upper part of it. At the end of an additional 9 weeks, during which the cultures were in the refrigerator, the surface growth was chrome yellow. In stab-cultures in nitrate agar (stock 718) the organism made a good growth, the surface layer being at first whitish. At the end of 24 days the agar was not stained, and no gas had been formed in the agar. A few crystals were present in the uppermost layers of the agar. The stab had made a good growth the whole length, but it thinned out slightly toward the bottom. There was a very distinct Fig. 61. f *Fig. 60. — Flagella of Bacterium stewarti. Stained by van Ermengem's method from dilute Uschinsky's solution. Slide *, August 7, 1904. fFiG. 61. — a. Gelatin stab-cultures of Bacterium stewarti after 10 days at about 200 C. Gelatin not liquefied. b. Gelatin stab-cultures of a non-pathogenic yellow liquefying organism from the surface of sweet-corn kernels. Sum- mer of 1908. Two- thirds to three- fourths liquefied. 134 BACTERIA IN RELATION TO PLANT DISEASES. surface growth, which was plainly yellow and rough granular (possibly ? from the presence of the crystals). It covered only a part of the surface and was between Ridgway's buff- yellow and deep chrome in color. A month later there was a very good growth, the surface was wet-shining and not then granular, but covered with little denser masses of the bacteria, resembling colonies growing on the older growth. In stab-cultures in 6 per cent glycerin agar the organism grew nearly or quite the whole length of the stabs, but best toward the top. The surface growth increased slowly, so that finally most of the surface was occupied. The growth was pale yellow, particularly at first. vSurface smooth or slightly irregular. Streak-cultures also grew. On silicate jelly (see vol. I) Bad. stewarti made a very feeble growth. The streak- cultures were under observation 2.5 months. In 2 days there was a very scanty, rough, yellowish growth along the line of the streak which did not increase much. A number of organisms were tested. The only other one that failed eventually to make a good >_a growth on this I^k medium was Bad. vascularum, al- though quite a good many had difficulty in start- ing off, and had made little or no visible growth at the end of 6 days. Bact. stewarti behaves on potato much like Bad. hyacinthi, i. e., out of the water it makes a moderate amount of yellow growth which Fig. 62.' Fig. 63. t soon passes its maximum (see this monograph, vol. II, plate 17, fig. 2). The fluid around the cylinder never becomes thickened by the continued development of the organism, and the surface growth ceases for the most part after a week or two, owing to the exhaustion of the small amount of sugar and other soluble foods and the inability of the organism to take its food readily from starch. No gas bubbles appear. The litmus reaction is alkaline. The iodin- starch reaction is always strong in potato-cylinders on which this organism has grown, even when tested immediately under the bacterial layer, although it must have a slight action on starch, since the reaction is generally a deep purple-blue rather than a pure blue, such as the check-tubes yield. The cell-walls of the potato are not softened. The following are notes on a series of 8 cultures (from as many plants) on potato-cylinders in test-tubes : *Fig. 62. — a. Streak-culture of Bacterium stewarti on a +15 agar slant 3 days at 27° C, September 1902. b. the same on +15 agar slant 8 days at 260 C, October 1908. fFiG. 63. — Crystals formed in old nutrient agar-culture of Bacterium stewarti. Photographed down upon the surface (nail-head) of the culture. The dark body in the center is the denser growth along the needle-track. Crystals confined to the surface. X4. The culture had been at a temperature of about 14° C. for many weeks. STEWART'S DISEASE OF SWEET-CORN (MAIZE). 135 August 24, 1902. — Inoculated. August 29. — All the cultures resemble each other closely. The potato out of the water is slightly to distinctly grayed, and bears a thin, decidedly yellow slime. In one tube it was recorded as bright yellow. September. — The potato-cylinders are grayed to a greater extent than they were and the organism is yellower in some tubes than in others (variability in potato-cylinders). When compared with the check-tubes there is an incon- spicuous pink tint in the potato (?). Repetitions in 1909 and 1913 (held 5 months) failed to give this tint. Fig. 64 shows appearance on potato contrasted with that of Bacterium campestre. On coconut cylinders Bact. stewarti made only a moderate amount of growth. Its color was buff -yellow and crystals were formed. Repeated in 19 13 the following results were obtained : Moderate to good growth ; pale buff -yellow slime and precipitate ; no crys- tals. In the milk of the coconut there was a moderate growth with yellow rim and precipi- tate, and subsequently the fluid became yellow (checks remained colorless). When old there was a copious yellow growth with a wide yellow rim; the fluid was pale yellow. Though acid when inoculated it was now feebly alkaline. There were no crystals. On cylinders of yellow turnip there was a thin buff-yellow slightly iridescent growth. At the end of a week this was decidedly less than in corresponding tubes of Bact. campestre. Growth ceased, practically, after the second week. There was a buff-yellow precipitate in the fluid surrounding the cylinders, but no thickening of the water with bacterial slime. There was no browning or softening of the substratum. The culture was alkaline to litmus at the end of the fifth and ninth weeks. Repeated in 19 13 with same results, except recorded as not iridescent. When old the growth was orange-ochraceous, feebly alkaline, wet-shining, not viscid, moderate in amount, and free from crystals. The appearance of this organism when grown on rutabaga was much the same as on yellow turnip. On the seventh day the growth was about one-tenth as much as that of Bact. hyacinthi and one-twentieth to one-thirtieth as much as that of Bact. campestre. On the eighteenth day streak-cultures yielded a thin buff-yellow growth covering the whole surface exposed to the air ; this was not smooth and not dense enough to hide the slight irregularities of the substratum. Its surface was slightly iridescent, and when examined under the hand-lens fine striae were visible. The water contained a moderate amount of buff-yellow precipitate, but was not filled with a solid slime. In old cultures there was no increased growth, no brown stain, and no softening of the tissues. The iridescence persisted. The cultures were alkaline, at least after a time (thirty-fourth and sixty-fourth days), and had a feeble, peculiar smell. On the thirty- fourth day the thick slime would not wet litmus paper until water was added. When tried in 1913 on white turnip cylinders standing in water (stock 5778), there was a moderate to good, pale yellow, flaky, iridescent surface growth, and a pale yellow precipi- tate. When old the slime was ocher yellow, slightly alkaline, not viscid, wet-shining, not copious, and free from crystals. On radish cylinders (tests of 1913), there was a thin, whitish, iridescent surface growth with a scanty yellowish precipitate and either no browning or slight. Steamed cylinders of sugar-beet standing in distilled water proved a very good medium for Bact. stewarti. It grew on this substratum abundantly and without retardation. The color of the growth on the cylinders was a deep buff-yellow, and there was a copious buff- yellow precipitate in the fluid at the bottom of the tube. *Fig. 64. — Bacterium stewarti (a), and Bacterium campestre (b), after 30 days on cooked potato at room-temper- ature. The former grew only during the first few days, i. e., was not able to use the starch as food ; the latter continued to grow for weeks, owing to its diastasic action on starch. Bact. stewarti shows a little slime on the upper part of the potato (dark part) and a small amount of precipitate in the water (at right). Bact. campestre has covered the potato and filled the water solid. Photographed November 2, 1908. Fig. 64. 136 BACTERIA IN RELATION TO PLANT DISEASES. In tubes of peptonized beef bouillon at the end of about 1 8 days (room-temperatures of spring) there was a feeble, whitish rim, a well-clouded fluid, about 10 mm. breadth of pale yellow precipitate, and no distinct pellicle (stock 791). Repeated in 1908 in stock 3398. At end of 10 days, when notes were taken, the tubes were well clouded with a moderate amount of pale yellow flocculent precipitate. There was also a well-defined but rather scanty whitish rim. In a fluid prepared by grinding green cabbage-leaves, taken from old, slow-growing hot- house plants, and extracting the juice under pressure without addition of water, there was a very prolonged and copious growth without retardation. A portion of this fluid was steril- ized by forcing it through a Chamberland filter and the rest by discontinuous steaming. The reaction of each was +40 on Fuller's scale. Bad. phaseoli and Bad. campestrc refused to grow in this fluid, but when it was solidified by adding agar-flour, Bad. campestre grew upon it very copiously and for a long time, although it was started with much difficulty. Bad. stewarti was retarded at first, but afterward made a prolonged and copious growth in three out of four tomato juices undiluted with water. In the fourth it refused to grow. In stocks 333 and 334, obtained respectively by squeezing green full-grown or nearly full-grown fruits, and small green fruits (one-twentieth to one-quarter grown) there was a good growth on the fifth day. The acidity of these two stocks was respectively +55 and +59 of Fuller's scale. In stock 33 1, which was the steamed juice of ripe fruits, there was a greater retardation, the tubes becoming clouded between the eighth and fifteenth day. The acidity of this med- ium was +64. Stock 332, in which the organism would not grow, was +68, i. lpin Ravn, of Copenhagen, heads of Dactylis glomcrata (collected in Denmark), attacked by a bacterial disease which appears to be identical with that described by Rathay. Dr. Ravn's note is as follows: As samples without value I send you some specimens of Dactylis glomcrata, infected by bacteria. The disease is reported in Kirchner: Krankheiten und Beschadigungen, 2 Aufl., p. 163, but has — so far as I know — not been studied more closely. Last summer my assistant, Mr. F. Find, dis- covered the disease in this country and found that it is widely spread and is of some significance for the growing of seed of Dactylis. Mr. Lind continues his field studies, but we would be much obliged to you if you might have time to report to us your opinion as to the identity of the bacterium after cultural characters and comparison with others. The heads of these plants are dwarfed, distorted, and gummy-yellow from the presence of enormous masses of surface bacteria, and often imprisoned within the leaf-sheaths which are stuck together (fig. 71a). The glumes, which are badly attacked, are lemon yellow or water-soaked, and the sheaths and stems are also involved more or less. On some spike- lets only a portion of the glumes are attacked and these not in all parts. (For an early and late stage of the disease on the spikelets see plate 41, figs. 9 and 10.) The yellow slime is also abundant between the upper unfolded leaves and the stem (B, B of fig. 7 id). This yellow slime reddens blue litmus paper. Microtome sections show extensive occupation of the floral organs (pi. 1 1 a) . The bac- teria are present mostly between and surrounding these organs, as in the gum-bud disease of carnations (see this monograph, vol. II, fig. 4). In many cases they have been seen occupying intercellular spaces (figs, yie, 7 if), and more rarely vessels. The organism most abundant on these plants is a yellow, non-motile, non-sporiferous schizomycete, which so far has refused to grow readily on any sort of agar, but which grows 158 BACTERIA IN RELATION TO PLANT DISEASES. promptly and very well on cooked potato where it is viscid after some days. We have tried to grow it with negative or unsatisfactory results on standard + 1 5 beef peptone agar, Fig. 71*.t Fig. 71a.* rig. l\c.% corn meal agar, potato agar, banana agar, carrot agar, Loeffler's malt extract agar, and standard beef peptone agar with oxalic acid (prepared for fungi). Neither have we been *Fig. 71a. — Stems of Dactylis glomerata from Denmark, showing dwarfing and distortion due to Rathay's disease. fFio. Tib. — Stem of Dactylis glomerata from Austria, photographed in 1913 from alcoholic material collected by Rathay. Leaves stuck together above and culm below pushing side-wise out of the sheath. Original in Museum of Lehr Anstalt at Klosterneuberg. JFig. 71c — A. Cross-section of stem of Dactylis glomerata. showing bacterial layer (white mass) entirely sur- rounding the culm, and separating it from the enveloping leaf-sheath. B. Cross-section from another plant, showing partial separation of an inner and outer leaf-sheath by the bacteria (white line at the top). Both were cut and photo- graphed in Washington from Rathay's material. rathay's disease of orchard grass. 159 able to grow it in +15 beef peptone bouillon. At the end of 16 days small circular yellow colonies appeared on some of the agar plates, and transfers grew fairly well on the surface of agar stab-cultures. It also grew slowly on agar-stabs when intro- duced along with the white organ- ism mentioned below. It did not grow readily in nitrate bouillon. In the plant it is quite inclined to be associated with a white lique- fying organism which grows as a very short rod usually in pairs or fours with a plain constriction. This white organism grows in agar, where it is motile in early stages of growth on the margin of the colo- nies. It was not clearly motile when taken from a 5 -day potato culture, and here T's and Y's were seen. It stains by Gram. The yellow organism is alcohol fast, but not acid fast. It is Gram positive, and during the viscid stage Fig. 71e.f Fig. 7W Fig 7I/.J on potato possesses a capsule. The yellow slime from a 7-day culture on potato was feebly alkaline to neutral litmus paper. The color on potato resembles that of Aplano- bacter michiganense (pi. 11, figs. 8, 9) and the substratum is grayed. *Fig. 7id. — Cross-section of stem similar to those shown in fig. 71a. At B, B are dense bacterial masses separat- ing the immature leaves which are still nearly free from infection. At X bacteria may be seen occupying the sub- stomatic chamber and intercellular spaces. Slide 940 F 5. |Fig. 712. — Cross-section of a leaf of Dactylis glomerata, showing occupation of some of the intercellular spaces by A planobacter rathayi. Section of material received from F. K(/>lpin Ravn, of Denmark, summer of 1913. Slide 940 G 2. {Fig. 71/. — Detail of bacterial occupation of the intercellular spaces of leaf of Dactylis glomerata. From the same material as fig. 71c Figs. 71c and 71/ drawn by Katherine Bryan. §Fig. -jig. — Bacteria from a 20-day agar plate culture of Rdthay's organism stained by Ribbert's capsule stain. Dec. 29, 1913. **Fig. 71/j. — A detail from fig. 71c A, showing one inner epidermal leaf-cell with some of the adherent bacterial layer. Drawn from a free-hand section by Katherine Bryan. The fine granules are an effort on the part of the artist to represent bacteria out of focus. 160 BACTERIA IN RELATION TO PLANT DISEASES. It grew slowly or not at all in peptone water with i per cent potassium nitrate. In one tube, which clouded, no nitrite was detected in 13 days. It grew slowly in a mixture of grass broth, cane-sugar water, and peptone nitrate water, clouding the fluid, but there was no production of nitrite (9 days). Doubtful growth in Dactylis broth with 1 per cent peptone (10 days). There was no growth in potato broth with 1 per cent potassium nitrate. Subsequently in fermentation tubes of peptone bouillion with 1 per cent potas- sium nitrate there was no reduction of the nitrate (17 days). When tested the tubes were clear in the closed end, well-clouded in the open end, with an ample yellow precipitate and no pellicle. There was very slow growth during the first week. The check tube gave a bright blue reaction with diphenylamin. In first 1 1 days (at 260 C.) no change in milk except the formation of a narrow yellow rim. In litmus milk no change during first week, then (11 days) slightly bluer than check. On nutrient gelatin at the end of 19 days at 200 C. there was a distinct pale yellow surface growth, colony-form, with some buried colonies. No liquefaction for some weeks; at end of 7 weeks 5 mm. liquefied; at end of 5 months about one-third liquefied. I have named this organism Aplanobacter raihayi in honor of the late Emerich Rathay (Science). It is related to Aplanobacter michiganense perhaps too closely, but so far inocula- tions into tomato have yielded only negative results (see p. 165). It is a yellow non-flagellate rod with bluntly rounded ends, and on the plant is about twice as long as broad, measuring when stained with carbol fuchsin 0.6 to 0.75 X0.75/X to 1.5/1. On culture media (2-day potato) stained with carbol fuchsin, they measure 0.5 to 0.75/i X0.95 to 1.3M. It occurs most frequently singly or in pairs joined end to end. Capsules occur (fig. 71 g). It grows in milk with formation of a yellow rim, but no visible change in color or consistency (11 days); subsequently the rim widened and a large amount of chrome yellow precipitate formed and there was separation of the casein. Litmus milk is slowly blued : subsequently (4 months) the milk became a uniform purplish and occasionally there was some reduction of the color (pi. 23). Nitrates are not reduced. It does not grow in Cohn's solution. Grape sugar is split and acid is formed, but not gas. It grows slowly on nutrient agar and gelatin, and in other culture media such as peptone water with potassium nitrate, milk, and litmus milk; in milk it makes a long-con- tinued and very copious yellow growth.* Saccharose and lactose are fermented slowly. On agar plates some strains of the organism grow only when sown thickly or when cultivated with the associated white organism (pi. 11B). Curiously the yellow colonies appeared only on the buried white colonies, not on the surface ones. The organism is undoubtedly diseminated on the seeds. It retains its vitality on dried spikelets for a long time, probably a year or more, judging from the fact that it grew readily in our tests at end of 7, 9, 10 and 1 1 months To insure growth inoculation into most media must be heavy. Group No. 21 1.2222522. LITERATURE. 1899. Rathay, Emerich. Ueber eine Bakteriose von Dactylis glomerata L. Sitz. Ber. der Wiener Akad. 1 Abth. Bd. cvm, pp. 597-602. Also a separate. 1913. Smith, Erwin F. A New Type of Bacterial Disease. Science, N. S., Vol. XXXVIII, No. 991, Dec. 26, 1913, P- 926. *After the above was in type the writer visited Klosterneuberg and through the courtesy of Dr. Linsbauer, Director of the Lehr-Anstalt, had opportunity to examine 15 jars of alcoholic material left by Rathay, and to satisfy himself that the Austrian disease is identical with the Danish (see figs. 71b, c, and h, made from this material). Rathay's manuscript was not left in the Lehr-Anstalt but in his house, where it came into possession of one of his daughters, who finally destroyed it as valueless. From this sacrifice to the goddess of good-housekeeping there escaped only some photographs and drawings which I had hoped might be reproduced here and to that extent help to keep alive the memory of a brilliant man called away too early from the scene of his activities. They were promised but not received. That this disease still occurs on the Kahlenberg is shown by the fact that I collected it there myself (Nov. 2,1913) in the oak woods on the east side of the railroad track, a few hundred yards below the uppermost station. Moved to a hot house in Washington, one of these diseased plants produced 15 flowering shoots in 1914, all of which were healthy. PLANT BACTERIA, VOL. 3. /to* PLATE MB. (i) Photographs of Petri-dish poured plates, showing behavior of Aplanobader rathayi in +15 peptonized beef agar. A, plate sown heavily— growth slow in developing but finally good; B, plate sown thinly — growth retarded; C, plate sown more thinly — growth still more retarded; D, plate sown still more thinly, but with quite a good many bacteria — no growth. Plates 18 days old at room temperature (250 C.±). Growth viable on A after about 9 days. (2) Portion of an agar poured plate, showing at B a buried colony of the white organism frequently associated with Aplanobader rathayi, in which (but nowhere else on the plate) are growing the small yellow (black in photo) colonies of Ap. rathayi. which was sown copiously. Time 24 days. (3) Like 2, but from another sowing: B. A buried white colony containing the yellow (white in photo) colonies of A planobacter rathayi; S, S, two surface colonies of the white schizomycete in which no yellow colonies were visible. The plate was sown heavily with the yellow organism, but none developed, except in the body of the buried white colonies. Time 26 days. THE GRAND RAPIDS TOMATO DISEASE. The writer received (July 27, 1909), from Grand Rapids, Michigan, diseased tomato plants taken from a field of 112 acres, said to be badly affected by a new disease. PRELIMINARY STUDIES. There was no surface indication of the cause of the disease, but some of the vessels, espe- cially those next to the pith, were packed full of a short bacterium which appeared to have no self-motility when examined in water. It was a short rod, single or in pairs, termo-like. There was very little brown stain in the wood, but yet some. In morphology it was closely like Bad. solanacearum from this part of the country, i. % a WJ C f/i ,d £ In *n a j3 9 o n rt ._ = fs .-. ^ CJ £ o U PLANT BACTERIA, VOL. 3. PLATE 22. 110 •n-a >. a j fl v a c c o-r o S <_ ° _ - J= 3 _ 3 u s « s s 2 E -C ^ ■» S SJJ5 05 is S 2f A .-5 z rt -..' - ■d n ■0 a — -. 3 u o Z - ■ c a u p X. CB - _rt •a c OJ rt o -, C id - 0 7 a a >> 0 — = rt -c C V Cfi ft 3 O rt O en ,, , -a 0 3 3 - ej 0) sd CO rt - in t/J (/] VASCULAR DISEASES OF BANANA. 171 tions, reisolations, and reinoculations have been made by him, both on the Moko and on other varieties. Manila hemp plants were also inoculated, but proved quite resistant. From a lack of the necessary bacteriological apparatus and supplies but little has been done on the biology of the organism, but it has always been recognized in cultures by the fact that it quickly becomes black when grown on potato cylinders. It is also very short lived and loses its pathogenic power quickly, especially on media which contain starch. In these respects it is similar to Bacillus solanacearum. Inoculations have been made with it several times on solanaceous plants, but so far they have been unsuccessful. The writer is giving the name Bacillus musae to the organism and will at some future time give the complete bacterio- logical chart. Although many of the signs of the bac- terial disease are like those exhibited by the Panama disease, they are not the same. The two diseases are quite distinct. Though the vascular bundles of plants attacked by the Panama disease are discolored and frequently filled with bacteria, B. musae has never been isolated from such plants. The bacteria in the vessels are usually rapid growing gas formers, and at least, so far as the writer's experience goes, are not pathogenic. The longitudinal splitting of the leaf- sheaths which is a characteristic of the Panama disease [and also of the Cuban disease — E. F. S.] is not found in plants attacked by the [bacterial] "moko" disease. Moreover the Gros Michael plant, which is very susceptible to the former dis- ease, seems to be naturally resistant to the latter. A species of Fusarium was isolated in Novem- ber, 1909, from a diseased Gros Michael plant and since that time the same fungus has always been found associated with the Panama disease both in Trinidad and in Surinam. Evidence seems to point to the fact that this fungus is the cause of the disease, as was pointed out last year by Dr. Erwin F. Smith. The writer received pure cultures of B. musae from Mr. Rorer and inoculated them by needle-pricks into banana shoots, but unsuc- cessfully. Portions of diseased plants were also received from him, and examined micro- scopically, the vessels being free from fungi and swarming with bacteria. One of the fruit stalks which was green and sound externally extruded from a cross-section numerous small drops of a white bacterial ooze (fig. 76), and on making direct inoculations from such drops into petioles, the disease was reproduced on banana in one of the Washington hot-houses (fig. 77), but not on young tomato shoots, into which it was also inoculated. The appearance of ten-day-old gelatin stab-cultures of this organism is shown in fig. 78. Fig. 79 shows the manner in which the bacteria sometimes flood out of the vascular bundles Fig. 79.* *Fig. 79. — Longitudinal section through a banana fruit-stalk from Trinidad showing some parenchyma cells occupied by Bacillus musae and others free. 172 BACTERIA IN RELATION TO PLANT DISEASES. into the parenchyma filling the interior of certain of these cells, while at other times they occupy only the intercellular spaces and wedge apart the parenchyma cells to form cavities. Fig. 80 B shows Bacillus mitsae filling one of the spiral vessels of a bundle. Fig. 80.* THE PANAMA DISEASE. In 1904, Dr. R. E. B. McKenney discovered a banana disease in Central America which had destroyed whole fields and which threatened to destroy the industry over large areas. During the next 4 years he found the disease on every banana farm in Costa Rica, meaning by this every plantation where bananas are grown in quantity for market. He also observed the disease as far south as the canal region of Panama, and from reports made to him he believes that it occurs as far north as British Honduras. The disease has probably existed in Central America for a long time, but has been destructive only within the last 10 years. During this time many large fields of bananas belonging to the United Fruit Com- pany have been entirely destroyed. Up to the summer of 1909 he believed the disease to be of bacterial origin, and thought that he had some evidence of this from pure-culture inocu- lations made in Costa Rica. But inoculations made in the hot-houses of the U. S. Depart- ment of Agriculture with his bacterial cultures have yielded only negative results. This disease first attacks portions of the rhizome. The rhizome is so large, however, that many months usually elapse before the whole of it is destroyed. The most conspicuous signs of this disease are : first, the yellowing of the leaves ; then the sudden wilting and shriveling of the entire foliage. The rhizome now sends up new shoots, which appear to be healthy at first, but after a time these also perish with the same signs. In a badly diseased field one sees very few tall banana plants, but multitudes of low shoots, healthy or in various stages of the disease. When the banana leaves are full grown, or nearly so, the signs are yellowing and wilting. In younger leaves the yellowing, which begins at the terminal portion, is accompanied by the downward curvature of the apical 2 to 3 feet of the leaf in a peculiar and characteristic manner. *Fig. 80. — A. Longitudinal section of spiral vessel of a banana bundle attacked by Smith's Cuban disease. Petiole sound externally. Section 7 feet 4 inches below point of inoculation. Bundle browned and occupied sparingly by Fusarium cubense, which is producing internal conidia. Section drawn after being cleared in 10 per cent potash over night. Inoculations of 1909. For comparison with B. B. Longitudinal section of banana fruit-stalk (fig. 76) showing two spiral vessels of a bundle: One occupied by Bacillus mitsae, the other empty. Rorer's Trinidad disease. VASCULAR DISEASES OF BANANA. 173 There are no external indications as to the cause of this disease, but when one makes a section of the stem, particularly toward the base, the vascular bundles are found to be very fully occupied by bacteria and stained first yellow, then a dark color (brownish-violet). This bacterial occupation of the vessels may be traced out into the affected leaves, to a lesser extent. A vascular bundle, here and there, may be occupied, but there is not conspicuous brown staining of the veins such as occurs in the black rot of the cabbage. It remains to be determined whether this Central American disease is the same as that seen by Earle in Jamaica and whether it is really due to bacteria. I believe not.* In recent years several persons, including Dr. McKenney, have searched for this disease in Jamaica in the locality indicated by Earle without finding it, which only means, however, that the growers took Earle's advice seriously, and grubbed out the diseased plants very thor- oughly. The writer is inclined to ascribe the Panama disease to his Fusarium cubense. In 191 3 the disease was reported by Ashby from Jamaica, identification being made first in 191 2 by Mr. Goldsmith Williams, an agent of the Fruit Company. The Cavendish or Chinese banana and the Congo are resistant to this disease. SOUTH AMERICAN DISEASES. Rorer has shown that the so-called " Panama disease, " both in Trinidad and in Surinam, is associated with a Fusarium in the vascular bundles, and this corresponds to the writer's observations on the Cuban disease. The most recent and longest paper is by Drost, but he does not cite earlier literature. It is possible, of course, that the Fusarium found also by Drost in the diseased bundles of the banana is the conidial stage of the Leptospora also found by him, but the evidence of this is very inconclusive. Essed's papers seem to me negligible. OLD WORLD DISEASES. Basu has seen a banana disease in Bengal which resembles the Surinam disease and associated with which he has seen a fungus bearing Ccphalosporium and Fusarium conidia. LITERATURE. 1903. 1910. 1910. Earle. F. S. Banana leaf-blight, in "Report on a trip to Jamaica." Jour. N. Y. Bot. Garden, vol. iv, Jan. 1903, pp. 7-8. Rorer, James Birch. A bacterial disease of bananas and plantains. Society paper No. 412 in Proc. Agric. Society of Trinidad and Tobago, vol. x, part 4, April 1910, pp. 109-113. Rorer, James Birch. Diseases of bananas. Bull. Dept. Agric, Trinidad, Port of Spain, vol. ix, No. 65, July 1910, p. 157. Separate. 1910. Smith, Erwin F. A Cuban banana disease. Science, n. s., vol. xxxi, No. 802, May 13, 1910, pp. 754-755- Also a separate. Organism called Fusarium cubense. McKenny, R. E. B. The Central American banana blight. Science, n. s., vol. xxxi, No. 802, May 13, 1910, pp. 750-751. Separate. Rorer, James Birch. A bacterial disease of bananas and plantains. Phytopathology, vol. 1, April 17, 191 1, 4 pis., pp. 45-49- 1910. 191 1. 1911 1911 Basu, S. K. Report on the banana disease of Chinsurah. Quart. Journ. Dept. Agric, Ben- gal, vol. iv, No. 4, April 1911, pp. 196-198. Essed, E. The Panama disease of bananas. Annals of Botany, vol. xxv, pp. 343_36i- London and Oxford, April 191 1. The disease both in Panama and in Surinam is said to be due to a new species of fungus, Uslilagiuoidella musaperda. 1912. Drost, A. W. De Surinaamsche Panamaziekt. in de Gros Michel bacoven. Bull. No. 26, Paramaribo, Maart, 1912, pp. 45, with 11 plates. Disease ascribed to Leptospora muse. "Besides perithecia it produces spores of a Cephalosporium and of a Fusarium type. " 1912. 1913 PittiER, H. La Enfermedad del Banano y su Causa. La Hacienda, Agosto 1912, pp. 343- 346, 3 figs. Ashby, S. F. Banana Diseases in Jamaica. Bull. Dep. Agric. Jamaica, N. S., vol. 2, No. 6, January 1913, PP- 95~i°9- Fusarium is present in the bundles. * Since this was in type Mr James B. Rorer visited Central America for the United Fruit Company and wrote me as follows under date of March 16, 1914: " We had a very pleasant trip to Bocas del Toro and I was able to get a good deal of work done * * * I made many cultures from diseased bananas— Gros Michael— and got Fusarium every time from the Panama disease. I did not see the bacterial disease there at all even in the susceptible varieties. BROWN ROT OF SOLANACEAE. Synonyms: Tomato-wilt, Potato-rot, Blight of Egg-plant, Pepper-blight, Granville Wilt of Tobacco, etc. DEFINITION. This is a specific communicable disease of potatoes, tomatoes, egg-plants, etc., char- acterized by a sudden wilting and shriveling of the foliage, with drooping of the softer shoots and a brown stain in the vascular bundles, which are filled with a great number of bacteria. It also causes more or less disorganization of the parenchyma, especially of the pith. Young watery plants are much more subject to general infection than old and woody ones. The internal browning is frequently visible on the surface of stems, etc., as dusky patches or streaks. In woody plants the leaves may become yellow and die without wilting. In potato tubers it rots the region of the vascular ring with a brown stain (pi. 23, figs. 8, 10). HOST-PLANTS. This disease has been observed in the United States in potatoes (Solanum tuberosum), a wild plant obtained from Montevideo, Uraguay, and supposed to be Solanum commersoni (pi. 24, fig. 2), egg-plant (5. melongena) , tomato (S. lycopersicum), and in tobacco. Hunger has reported its occurrence on tomatoes, tobacco, and peppers in the Dutch East Indies and Honing in a variety of non-solanaceous plants — see History and also a separate chapter on Tobacco-wilts (p. 220) and on a disease of peanuts (p. 151). The disease has been successfully inoculated by the writer into potatoes and tomatoes many times over, and also into the following: Nicotiana tabacum (recent experiments), Solanum nigrum, Datura stramonium, D. metclloides, Physalis crassifolia, P. philadelphica, and Petunia sp. (hybrid). In heliotrope, Ricinus communis, Vigna catjang, and Portulacca oleraceae, the organism lived for some weeks in the inoculated area, but no general disease of the plant was devel- oped. Inoculations into the following plants were unsuccessful: Cucurbita foetidissima, Cucumis sativus (stem and fruit*) , Nicotiana tabacum (1895, 1896, 1901, 1905!), Capsicum annum, Solanum muricatum, S. carolinense, S. dulcamara, Pyrus communis, Elcusine indica, Abutilon avicennae, and Pelargonium zonule. Beyond one unsuccessful trial no attempts have been made to inoculate it into egg- plants, because none happened to be at hand when the writer was making his experiments, but he obtained numerous infections on tomato and potato with bacteria taken from the egg-plant, and Rolfs has inoculated into the latter very successfully. Inoculations into the stems of cucumber plants caused no general sickening, but in some cases (not all) there was a local enlargement of the inoculated part. The surfaces of these stems were not, however, sterilized before inoculation (by needle-puncture), and only two cases of this kind occurred. Local enlargements have been very frequent on tomato stems inoculated with old isolations and feebly virulent strains. Inoculations in the summer of 1903 showed Datura metclloides (vol. I, fig. 4) to be very susceptible. Datura fastuosa and D. comucopiae were stunted, but managed to overcome the disease. A specimen of Datura tatula inoculated in 1896 resisted a virulent strain of the organism, i. c., one that destroyed D. stramonium and other plants. "Inoculations in 1896 into full-grown, green cucumber fruits removed from the parent plant and placed under bell- jars led in several cases to a soft watery rot involving the whole interior of the fruit and similar to that sometimes observed in the field in ripening cucumbers. Inasmuch, however, as plate-cultures were not made from the bacteria swarming in the interior of these fruits, the possibility of this phenomenon being due to other bacterial organisms was noted at the time as not excluded. This experiment was repeated in the summer of 1901 on six ripe and ripening cucum- bers, in the open air, attached to the vine, with entirely negative results. Each fruit was inoculated copiously by means of a dozen or more deep needle-punctures. The check-plants, consisting of the growing shoots of tomatoes and pota- toes, contracted the disease promptly, but the cucumbers showed no signs. fFor later successful inoculations, see Will-Diseases of Tobacco. 174 PLANT BACTERIA-VOL. 3. \lCf PLATE 23. TOBACCO WILT. POTATO DISEASE. RATHAY'S DISEASE. (1) Litmus-milk culture, 12 days old, of Bacillus solanisaprus I. Painted March 19, 1907. (la) Litmus-milk Culture 12 days, old, of Bacillus phytophthorus. Inoculated and painted at same time as fig. 1. (2) Old browned colonies on the surface of an agar plate of the N. C. tobacco organism, poured August 1, 1905, painted October 14; agar stained brown. (3) Litmus-milk culture of Appel's Bacillus phytophthorus. Tube inoculated November 13; 1906. Painted November 26. (4) Uninoculated (check) tube of litmus-milk. (5) Bacillus solanisaprus Harrison I. An old litmus-milk culture kept in the refrigerator (14°C.) and partly dried out. Inoculated December 10, 1906; painted March 4, 1907. (6) Old potato-culture of Bacillus phytophthorus Appel. Stock 1975, painted March 5, 1907. (6«) Potato-culture of Bacillus solanisaprus Harrison II. Inoculated March 7, 1907, and painted March 19. The colorclose, but the organism wet-shining. (7) An old litmus-milk culture of Hart, solanacearum, Florida potato, tube inoculated June 2, 1905, painted August 28 and equally representative of an old culture of the N. C. tobacco organism. (8, 10) Cross-sections of 9 and 11, showing the characteristic bacterial decay. (9, 11) Internally brown rotted potato tubers from Portsmouth, Va., painted October 18-19, 1905. Infection by way of the vessels of the rhizome; the surface of the tuber not ruptured: Bad. solanaccarum very abundant in the vascular system of rhizomes and tubers. (12, 13) Aplanobaeter rathaui; milk and litmus-milk, 5 months. BROWN ROT OF SOLANACEAE. 175 GEOGRAPHICAL DISTRIBUTION. This disease occurs in Colorado (?) Arizona (?), New Mexico (?), Texas, Mississippi, Alabama, Florida, Georgia, South Carolina, North Carolina, Maryland, Virginia, New Jersey, and apparently as far north as middle New England. It probably occurs also in Ohio and in Illinois and other States in the middle and far west, but its northern and its western distribution have not been worked out. Clinton has reported it from Connecticut. It is found in America as far south at least as Porto Rico, the writer having received it from that island in tomatoes and egg-plants. This disease, or one suspiciously like it, has been reported from near St. Petersburg, Russia, and from various parts of Great Britain, France, and Italy. In all probability the disease occurs in many parts of Europe. Hunger has reported it from Java and Sumatra. Tryon has also stated (1899) that this disease is the same as one found by him in Australia (see Appendix p. 207) . What appears to be the same disease occurs, it is said, on potatoes and tomatoes in Umtali, Rhodesia, South Africa. The disease probably occurs in Japan. SIGNS OF THE DISEASE. Both the sleepy disease of tomatoes (Fusarium), and the Grand Rapids disease (Aplanobacter), cause tomato plants to wilt suddenly, and therefore they might be confused easily with this disease. On potato C3 )/ — \\\V -\ both here and in Europe it might be confused with some forms of the "black leg" or "schwarzbeinigkeit" described by Appel as due to Bacil- lus phytophthorus, by van Hall as due to Bacillus atrosepticus, by Pril- lieux and Delacroix as due to B. caulivorus, and by Pethybridge and Murphy as due to Bacillus melan- ogeiics. There is also a potato disease in France attributed by Delacroix to Bacillus solanincola (seep. 214). The foliage becomes prema- turely yellow and dies gradually (pi. 24, fig. 3), or wilts suddenly without loss of green (pi. 25), and in large leaves of the tomato the main axis of the leaf is also often bent downward in a characteristic way (plates 26, 27,28); the stems droop and shrivel when not too woody (pi. 24, fig. 1) ; and there is usually a decided brown stain in the vascular system in advance of the death of the external parts. The vessels of such stems are filled with enormous numbers of the small termo-like bacteria, which are not sticky and which ooze out of the stem on cross- section as a dirty white or brownish-white slime. The bacteria pass up and down the stems considerably in advance of the shriveling, and the accompanying brown stain can often be seen through the younger and more translucent stems and petioles, especially of Fig. 81. *Fig. 81. — Root of a diseased egg-plant from Porto Rico, sound externally but browned within and showing in cross-section three vessels of the vascular system occupied by bacteria. Received June 8, 1905, and drawn with the Abbe camera, unstained, from a free-hand section, the bacteria dragged over by the knife being omitted. The root was about 5 mm. in diameter: Its actual diameter is shown by the small circle at the left. 176 BACTERIA IN RELATION TO PLANT DISEASES. the potato, as long brownish streaks, although the surface of these parts still appears to be normal. After a time, especially in soft and rapidly growing plants, the pith and bark are both involved more or less; and in the pith large cavities may arise, these being occupied by the bacteria and by detritus of the tissues which they have disorganized (see vol. II, fig. i). Tomato plants have been seen in which nearly the whole pith had been converted into a watery slime. The localization of the signs in the form of large, open wounds at the surface of the earth (see Basal Stem-Rot) is not characteristic of this disease (see pis. 24, 25). In the tomato and the egg-plant, so far as observed by the writer, this is pri- marily a disease of the vegetative part of the plant rather than of the fruit, which is itself subject to a special dis- ease (the Point-rot). The writer obtained, however, a local infection in the green fruits of the tomato by inocu- lation. The inoculated side as a whole ripened sooner than the opposite side, but the tissue immediately border- ing on the punctures remained green for a long time. The bacteria multiplied considerably in the inner tissues and there was a brown stain, but not what might be mis- taken for a soft rot, or the Point- rot. The roots as well as the stems are subject to this disease (fig. 81). In good-sized, stocky potato plants, even when the vascular sys- tem is badly dis- eased (browned and occupied by the bacteria), the exterior of the stem is often green and normal in ap- pearance, except the leaves and ex- Fig. 82.* Kg. 83. t *FiG. 82. — Vertical section through external part of a potato tuber from plant No. 14. which was inoculated with Bacterium solanacearum on June 15, 1896, by needle-pricks on the upper part of the stem. Material collected and fixed in strong alcohol on July 27. Tuber sound externally. This represents a very early stage in the rot of the tuber. The bacteria entered the tuber from the vascular system of the stem, and were still confined to the vicinity of the vascular ring ( V). Below this region is the central starch-bearing parenchyma; above it is the cortical parenchyma, ending at the top of the drawing in cork (potato skin) ; the oval dark bodies are starch-grains; there are very few in the vicinity of the bundle, but this is not due to their destruction by the bacteria (see figs. 104, 105). The bacteria lie within and between the cells, and have already eroded a considerable cavity. Drawn under the Abbe camera from material which was cut on the microtome after infiltrating with paraffin. Cells shriveled by the strong alcohol. Slides 156(2 and 156(6. fFiG. 83. — Cross-section of a small developing potato tuber diseased by Bacterium solanacearum, collected at Portsmouth, Virginia, October 1905. Tuber infiltrated with paraffin and cut on the microtome; section stained with carbol-fuchsin. The skin and subjacent layers are unbroken, the infection having taken place by way of the under- ground stem. The unshaded parts denote absence of starch and the dark blotches the presence of the bacteria. (For details at .4, B, see figs. 107, 108.) The most crystal-sand cells are on the left side. There are a few starch-bearing cells at X. For a detail of this part see fig. 106. Slide 349(15. PLANT BACTERIA, VOL. 3. PLATE 24. «S _rt __ > >. B O C - S°< ■g-oo 1* M V > = $ u'io « now .s = >. = '" j3 a- | i 6 si a ; ? ~ £!° Isi 3 Q 1== o — tn O ■a io" ^ 0 M |0-s 3 2 E fill PLANT BACTERIA, VOL. 3. PLATE 25. Two potato-plants badly diseased by Bad. solanacearum (anatural infection). Portsmouth, Virginia, Oct. 1905. All of the tubers were internally brown rotted, i. e.. in the vicinity of the vascular ring. The stems were free from superficial ulcers and also the tubers, but in some of the latter the internal brown stain shows through, as in the front tuber and lower tuber of fig. 2. BROWN ROT OF SOLANACEAE. 177 treme tips of the growing shoots, which are flabby or shriveled (plate 30) . In less woody plants the branches will be shriveled, or flaccid, as shown in plate 24. In the potato plant the organisms pass down through the vascular system of the stems into the underground parts and cause an internal brown rot of the tubers. This rot of the tuber begins in the vascu- lar system at the stem end and gradually extends to the oppo- site end by way of the vessels, rot- ting the tuber from within and causing the appearance of dusky patches on the smooth surface of the tubers in advance of any rupture of the superficial cork- layer (pi. 23, figs. 9, n). On cross-section these patches are seen to be due to the internal brown stain centered in the vas- cular ring and showing through the more superficial layers of white tissue. In later stages of the disease the cork-layer is ruptured and the rot continues as a mixed infection due to the entrance of other organisms di- rectly from the soil. In tubers less far advanced in the rot, cross-sections often show the de- cay pretty closely restricted to the vascular ring (figs. 82, 83), from which there is always a gray-white or dirty white bac- terial ooze (vol. I, plate 24, top). In yet earlier stages of the dis- ease the brown stain and bac- terial decay are found only in the stem end of the tuber (vas- cular ring) or only in the vascular bundles of the underground stem leading to the tuber, the surface of this rhizome and the whole of the attached tuber being entirely sound (figs. 84, 85). On plants attacked with great virulence and very early no tubers are formed. The tubers on plants infected in the middle of the growing season are small and few and are found at harvest time in all stages of decay (pis. 24, 25). Often on such plants, as first noted by Dr. Halsted, tubers not larger than a pea, together with their rhizome, will be found sound superficially but brown-rotted in the vascular ring; in other cases, as already noted, the Fig. 84.' /too M M Fig. 85. f *Fig. 84. — Cross-section of underground stem of a potato-plant leading to a tuber sound on the surface but bac- terially rotted in the vascular system, i. c, a tuber such as that shown in fig. 83. Rhizome fixed in Carnoy's fluid, embedded in paraffin, and sectioned on the microtome. Slide 348 a 1, stained with carbol-fuchsin. The bark and pith are entirely free from bacterial occupation. The bacteria are confined principally to the vessels of the woody part of the bundle, but there is a cavity in the outer phloem (at the top) and there are various small bacterial foci in the inner phloem. The V-shaped marks in parenchyma-cells (use lens) denote crystal-sand. Material collected at Portsmouth, Virginia, October 1905. Drawn with a Zeiss 16-mm. apochromatic objective, No. 4 comp. ocular, and the Abbe camera. (For general appearance of the bacteria in these vessels see fig. 85.) fFio. 85. — Bacterium solanacearum from one of the vessels in fig. 84 (cross-section rhizome, Portsmouth, Virginia, potato). Slide 348 Ai. 178 BACTERIA IN RELATION TO PEANT DISEASES. entire tuber will be sound and only the rhizome affected — this through the whole length of its vascular system or only at that end farthest from the tuber. On the other hand, when plants are attacked late in the growing season, the tubers are more numerous and larger, and the majority of them, especially if borne on long rhizomes, may escape infection or be diseased only slightly at the stem end when dug. In tubers of this sort the rot is apt to continue after digging, but exceptionally it might, perhaps, remain dormant, or nearly so, until the season for planting. In plants attacked by this disease the juice of the stems will be found to have an alkaline reaction to litmus paper and, if examined microscopically, will be seen to be swarming with bacteria, which are very easily cultivated. On tomato shoots this organism causes the early development of great numbers of incipient roots in the form of small nodules. Hunger was the first to point out this sign and his statements have been confirmed repeatedly by the writer (see pi. 27). Sometimes swelling of the inoculated parts of the tomato stem and the appearance of these incipient roots, with dwarfing, and the changed position of the leaves are the only external signs of disease in inoculated plants (pi. 28) . These nodules are visible in the first figure of an inocu- lated tomato published by the writer (in 1896) and were observed repeatedly in the field in 1895, but inasmuch as they develop naturally on sound stems under very moist conditions, e. g., when a stem lies for some time on the earth, the influence of this disease in stimulating their premature development was overlooked. Anyone may satisfy himself easily, how- ever, that Hunger's statements are correct. For instance, if he will select a young, rapidly growing tomato plant, the stem of which is entirely free from such developments, and will inoculate one of two nearly equal branches, that one which was inoculated will promptly develop the roots, while the other will remain free (pis. 26, 27) or will develop them only much later as the result of a general infection of the stem. This is true even when the younger of the two shoots has been the one purposely selected for inoculation. Moreover, these roots always appear first in parts of the inoculated stem nearest the punctures. Wounds made by a sterile needle do not cause them to appear. ETIOLOGY. The cause of this disease is a dirty white or brownish-white schizomycete which the writer named Bacillus solanaccarum in 1896. Further studies have shown that it is usually motile by means of one polar flagellum. It should be classified therefore as Bacterium sola- nacearum or Pseudomonas solanacearwm, if one follows Aligula's system. This organism was first isolated and described by the writer, unless Burrill's statements relate to this species (see History and Literature), and the statements here given rest chiefly upon his own observations. The disease is sometimes very readily induced in susceptible species by simple needle-punctures, without hypodermic injection. At other times the writer has experienced many failures, using in some instances, at least, what appeared to be equally genuine material.* In recent years the irregular behavior of plants inoculated with this organism has been a source of much perplexity. Some have contracted the disease with great rapidity ; others not at all; others have shown some signs of the disease and then recovered, or have been seriously injured only in the inoculated shoots, or only after a great length of time (pi. 29). The reason for these marked differences has not been made out clearly. It seems to depend partly, at least, on the amount of water in the stem. Frequently the punctured areas have been separated from the rest of the plant by the development of a protective cork-layer. In general, infections have been more successful in young, rapidly growing, soft, watery plants than in slow-growing, wroody ones or in mature ones ; infections have been more suc- *One labors under difficulties in the isolation of this organism. On agar plates its colonies are best distinguished from other white colonies by waiting a week or two for the appearance of a brown stain in the colony, and yet this treatment seems to weaken the virulence of the organism. PLANT BACTERIA, VOL. 3. PLATE 26. Tomato-plant No. 17 (1903), showing two equally developed young, smooth shoots, one of which was inoculated by needle-pricks with Bacterium solanacearum (South Carolina strain) immediately after photographing (see plate 27, for condition 9 days later.). Photographed and inoculated July 16, 1905. PLANT BACTERIA. VOL. 3. PLATE 27. Tomato plant No. 17, nine days after inoculation; leaves of the inoculated branch reflexed, and incipient roots well developed. Check branch at right side normal. hi PLANT BACTERIA, VOL. 3. PLATE 28. Tomato-plant inoculated with Bacterium solanacearum (District of Columbia strain) on September 6, 1904, and photographed September 30, at two-thirds natural size. No wilt resulted. Observe swollen stem, shoots pushing, reflexed leaves and incipient roots which are absent above and below. The needle-pricks were made in the middle (swollen) portion of the stem. PLANT BACTERIA, VOL. 3. PLATE 29. Tomato plant two-thirds natural size. Inoculated on Sept. 6, 1904, with Bacterium solanacearum (District of Columbia strain) which had been grown in the thermostat at 37. 50 C. Photographed Sept. 30; pricked part swollen and nodular, leaves reflexed but not wilted, has wilted, shrivelled, and fallen off. The extreme top BROWN ROT OF SOLANACEAE. I 79 cessful in hot wet weather than in cool dry weather, and with cultures recently plated from diseased plants sent in from various parts of the United States than with organisms which have been longer removed from the plant, i. e., cultivated in the laboratory on various nutri- ent media for 6 months or a year. One of two inferences seems to be warranted: (i) the hosts vary enormously from plant to plant and time to time, or condition to condition, in their ability to resist this disease; or (2) the organism readily loses its virulence, for reasons yet unknown, when subjected to ordinary cultural conditions. Both inferences may be true. The subject requires for its elucidation more time than the writer has been able to devote to it. That the failures noted were due to accidental substitution of similar-looking non-infectious organisms was sought as a first explanation and might answer for a single failure, but hardly, one would think, for repeated failures, since the same care was exercised as in the case of other organisms where no such phenomena appeared. Descendants of the most virulent strain the writer ever had in the laboratory (vol. I, pi. 26) lost all infectious power in the course of a year, although apparently preserving the same cultural peculiarities. This strain was isolated from the plant photographed on plate 30. When virulent cultures are used on susceptible plants the time elapsing between the puncture and the first appearance of the disease is usually not more than 8 to 10 days and is sometimes as short as 2 or 3 days on watery shoots in very hot weather. All of the success- ful inoculations of 1895 were made with the organism taken from tomato plants grown in Mississippi. The very successful inoculations of 1896 were made with cultures derived from an egg-plant grown at Charleston, South Carolina. In both years infections were obtained on tomatoes and potatoes, the resultant disease being identical with that observed in the field. The organism was demonstrated in the vessels of the plants in enormous numbers at long distances from the points of inoculation (fig. 86, about 15 inches, time 18 days; fig. 82, about 3 feet, time 42 days). Pure cultures were isolated from the interior of such plants and the disease was again produced by means of these cultures. Needle-punctures on leaf- lets of potato produced the disease as readily as those into the stems, the only difference being that the organism had a longer distance to travel and consequently the tubers were not reached and destroyed so quickly. Sometimes the organism was observed to pass out into the under portion of the midrib of the potato leaf, blackening it for a distance of several inches in advance of the staining of the upper surface or the wilting of the foliage; at other times it ran out in long, narrow black lines on the upper surface of the leaf. The most rapid downward movement of the bac- teria in inoculated potato stems was observed during hot weather in July 1896 : In 16 days from the date of an inoculation by needle-pricks on the upper part of a stem the bacteria had passed downward a distance of rather more than 2 feet and caused signs of wilt in another stem from the same root. In another tall shoot pricked lower down, i. c, in the more woody tissues 6 inches from the earth, the signs on other shoots from the same root were slower to appear, i. e., they did not develop until after the twentieth day. Signs of the disease were observed to pass up and down stems on the pricked side much faster than sidewise. In 1 895 a tomato plant which was of large size when inoculated showed local signs after a few weeks, but general signs developed only after a long time. On then making sections, the bacteria were found in the vascular system in enormous numbers and at long distances from the point of inoculation. A similar case occurred in 1903, another in 1905, and still another in 1909. The latter was 12 feet high and full of fruit when it finally wilted. It was inoculated on a terminal shoot when about 4 feet high. It grew well for 3 months after inoculation, showing at first only a feeble wilt of a few leaves, from which it soon recovered. When dissected the upper 6 feet of the stem was free from bacteria. They were very abun- dant, however, in the vascular bundles of the lower 6 feet of the stem, including some of the roots. Cavities had formed in the tissues only near the point of inoculation. The stem was sound externally. The wood was stained brown in the part occupied by the bacteria. i8o BACTERIA IN RELATION TO PLANT DISEASES. In 1 901 the writer again obtained successful infections. The plants inoculated were potato, tomato, etc. The source of infection was a tomato plant received that summer from South Carolina. The inoculations were by means of needle-pricks on leaves and the upper parts of growing shoots. The infectious material was fluid from the bottom of slant agar- cultures (the first subcultures from colonies on poured plates). The period of incubation varied from 3 to 7 days. vSuccessful inoculations were made in the summer of 1903 with pure cultures obtained from a diseased potato plant gathered at Norfolk, Virginia. Inoculations were carried out successfully in 1904 on tomatoes, potatoes, etc., with pure cultures obtained from a potato- plant growing in the District of Columbia, near Washington. Partially successful inocu- lations were carried out in 1905 on potatoes with pure cultures obtained from a tomato stem received from Florida (vol. I, pis. 24 and 25), and on tomatoes with subcultures of colonies plated from Florida pota- toes. For details concerning these results and for the various failures see synopsis of the in- oculations. Plants in the field are attacked in all stages of growth. There seem to be, how- ever, varietal dif- ferences in suscep- tibility as well as great individual differences. The writer succeeded in pro- ducing the disease by allowing the Colorado potato- beetle (Doryphora 10-Uncata) to feed on diseased plants and then transferring them for a few hours to healthy plants. The beetles were obtained from healthy potato fields where the disease did not afterwards appear, and the only possible source of infection was the diseased potato leaves and stems on which they were fed, and which were the result of pure-culture inoculations. The interior of these leaves and stems swarmed with bacteria and it was impossible that the mouth-parts of the beetles should not have become contaminated. The infected plants did not contract the disease immediately, but signs appeared in from 7 to 9 days as narrow brown streaks extending downward rapidly inside of the stems and leaf-stalks. There were numerous infections on each plant and all of them appeared to originate in slight wounds made by the jaws of the Fig. 86.' *Fig. 86. — Cross-section of a small portion of a potato-stem, showing bacteria in vessels, and also three bacterial cavities in the outer phloem. From plant No. 14, May 27, 1895, inoculated by needle-pricks, using a pure culture of Bacterium solanacearnm. The activity of the organism is shown by the following facts: The distance (downward) from the needle-pricks to the level of this cross-section was about 15 inches; the time between date of needle-punctures and fixing (in alcohol) of material for sections was only 18 days; all of the leaves had shriveled when the stem was fixed. Drawn from a photomicrograph, X circa 100. Slide 171 M. (For a highly magnified detail, see fig. 101.) PLANT BACTERIA, VOL. 3. PLATE 30. Potato plant which yielded Bacterium solanacearum (District of Columbia strain). Stems externally sound but flabby at the tips; leaves wilted or shriveling; vascular system browned and occupied by the bacterial slime. No superficial ulcers. From a potato field at Woodndge, D. C, July 8, 1904. BROWN ROT OF SOLANACEAE. 181 beetles. In course of a few weeks the plants were destroyed (fig. 87), and the tubers were rotted from within in the same way as when the plants were inoculated by needle-punctures. From this it was concluded that the disease depended for its dissemination very largely on insect-carriers — an inference needing further verification. All of the writer's successful experiments having been made on leaves and stems, it is likely that in his bulletin too much stress was laid on the possibilities of infection through parts above ground. At that time the writer certainly regarded these as the most vulner- able parts. Burrill's observations led him to the same conclusion. Hunger has since shown that when tomatoes are grown in infected soil, or water, they contract the disease readily if the roots are wounded but not otherwise. His field observations led him to the same Fig. 87. conclusion. Observations on tomatoes in Mississippi led Earle to believe that underground infections were very common. Dr. Van Breda de Haan's observations on the slime-disease of tobacco in Sumatra led him to the same conclusion. I have also obtained underground infections on both tomato and tobacco, using the North Carolina tobacco-wilt organism. We may conclude, therefore, that the bacterium can enter readily through wounds made on any part of the plant either above ground or below. In such event the manner of infection would vary from plant to plant and field to field, according to the prevalence of particular insects or other sources of injury. Whether the organism can enter the plant in the absence of abrasions must be left an unsettled question. Burrill thought it could. Hunger states that it can not, but his experi- *Fig. 87. — Potato plant (left) promptly destroyed by Bacterium solanacearum as the result of the gnawings of infected beetles. (See text, p. 184.) Control plant on the right. 182 BACTERIA IN RELATION TO PLANT DISEASES. ments were not very numerous, and, especially, were not very long-continued. Basing our conclusions on Hunger's experiments with tomato roots, we may assert with some degree of confidence that an unwounded root is impervious to this organism, but it does not appear to be equally certain that the disease can never begin in the sub-stomatal chamber. So far, however, as we yet know definitely, infection takes place only through wounds. In one of the writer's hothouse experiments, a potato plant grew to maturity in health in the same pot with one which became badly diseased (in leaves, stems, and tubers) as the result of a pure- culture inoculation, but here insects were kept off. Exclusive of variable degrees of virulence on the part of the parasite and of individual or varietal resistance on the part of the host, the progress of the disease on attacked plants in the field must vary to a large extent with fluctuating temperature and rainfall. Rainy weather favors the disease, beyond question. So does moist soil. Under the equator, with rain every day, one might expect the disease to be much more rapidly fatal than in a cooler, drier climate, and such appears to be the case. According to Mr. Irons, of Porto Rico, Solatium mammosum is resistant to this disease. The Seed and Plant Introduction No. 24650, supposed to be this plant, bears small yellow gourd-like fruits having a white, tough, inedible flesh and small brown seeds. The specimen fruit seen by the writer was globose and about 2 inches in diameter. It is thought that the egg-plant and tomato might be grafted upon it, but I believe we shall find eventually more practical methods of dealing with this disease. SYNOPSIS OF INOCULATIONS. The following is a summary of all inoculations made by the writer and his assistants with Bacterium solana- cearum, so far as records were kept and are now available, except certain ones mentioned under "Wilt Diseases of Tobacco:" Fis-88* May 27, 1S95. — Tomato and potato plants were inoculated with slant agar culture No. 2 of May 22 (from slant agar, May 16, which was direct from the interior of a green, turgid, odorless stem of tomato from Ocean Springs, Mississippi). Fourteen inoculations were made: In fruit, rather woody part of stem, tip of stem, leaves and petioles of tomato, and on young shoots and leaflets of potato. *Fig. 88. — Cross-section of a single bundle from the hypocotylof Datura stramonium, plant No. 23, inoculated June 8, 1895, from tube I, June 4, with Bacterium solanaccarum by needle-pricks on hypocotyl and fixed in strong alcohol on June 20. The upper part of the drawing represents the outer part of the bundle. In the lower part there is a bac- terial cavity. In the lower left corner the knife passed through a crystal-cell. A few nuclei are visible. The alcohol caused some shrinkage of the bacterial masses. Several other bundles of this hypocotyl are partially occupied; the remaining bundles, together with the tissues between them and on the periphery, are free from bacteria. The foliage of this small plant was badly collapsed when the material was cut for fixing. Drawn by Alice C. Haskins under the Abbe camera from a stained section. Slide 192(1. The organism inoculated was a culture from a tomato plant from Ocean Springs, Mississippi. BROWN ROT OF SOLANACEAE. 183 Result. — Successful; the first signs appeared on the fifth day; weather exceedingly hot for last 4 days. The diseased stems of potato were swarming with short motile rods. Illustrated in Bull. 12, Division of Vegetable Phy- siology and Pathology, 1896, pi. I, fig. 1. Tune 8. — Fifteen inoculations were made on pear tree (Japan), pepper, pepino, Solatium nigrum, Datura stramonium, tobacco, tomato, potato, using slant agar No. 1 of June 4, from culture used for inoculations of May 27. Each plant received many pricks. Result. — Succeeded on tomato, potato, Solanum nigrum, and Datura stramonium (fig. 88). The organisms in the diseased Daturas were short, motile bacteria. June 15. — Sixteen inoculations into Pelargonium zonale. Datura stramonium, Solanum carolinense, potato and tomato, were made with cultures derived from the same source as those used for the inoculations of June 8 and May 27. Result. — Failed, except on potato, the stem and tubers of which finally rotted. There was only a local multiplication of the bacteria in 2 green fruits of the tomato. June 21. — The plants used for these inoculations were 40 strong, growing tomato vines 10 inches high in 4-inch pots. Nos. 46 to 66 were pricked in middle of stem, and Nos. 67 to 85 in the tender tip of stem; many deep pricks were made in each case. The culture used was slant agar No. 3 of June 18, whose history is as follows: Made from a single colony on poured plate No. 3, June 14, from slant agar No. 1, June n, which was made directly from interior of stem of potato No. 13, inoculated May 27. Result. — Failed; at least there were no signs for 30 days. Possibly a wrong colony was used; at any rate the method was defective, as plate cultures should have been made direct from plant No. 13. July 5. — Egg-plants, cucumbers, tobacco, potato, Datura stramonium were used for these inoculations (30 in all). The leaves and stemswere pricked. The culture used was slant agar No. 1, June 18, from another colony on plate No. 3, June 14. The bacteria covered nearly the whole surface of the slant with a smooth, wet-shining, whitish, copious growth, crenate at margins, not sticky, possessing a moderately bad smell; the agar was not stained. Result. — Failed? No records found. Probably a wrong colony was used. A ugust — . — Tomatoes in the field on James Island, South Carolina, were inocu- lated direct from slime in stem of egg-plant from Charleston, S. C. Fifteen terminal, rather woody shoots of large plants were selected. Result. — Successful; all of the inoculated plants con- tracted the disease, the wilt appearing slowly. None of the check-plants became infected. mm May 7, Result. June 1 Result June 75 Rainy weather. i&q6. — Tomato, Datura stramonium, and potato (2 watery shoots) were inoculated from tube No. 2, May 5, from interior of tomato stem from Lawrence, Massachusetts. The plants were healthy and 12 to 15 inches high, except the Datura, which was half as large and old. There were 7 inoculations. —No record found. The inoculations probably failed. — Six plants, part tomato, part potato, were inoculated from peptonized beef-broth culture No. 3 May 29, made direct from the interior of stem of egg-plant from Charleston, South Carolina (1896). The inoculations were made into petioles, tender shoots, and leaflets. —Very successful (Bull. 12, pi. 1, figs. 3, 4, 5). The organism in diseased tissues is a motile, short rod (see Centralblatt f. Bakt., 2 Abt., vn Bd., 1891, Tafel xi, figs. 41, 42). . — Eight young plants (each bearing 6 leaves), part tomato, part potato, were inoculated in soft tissues in active growth by means of needle-pricks. The culture used was a peptonized beef-broth, tube No. 6, June 13, from No. 2, June 12 (broth-culture), from a colony on poured plate No. 6, June 4, from peptone water 1, May 29, which was a companion tube to No. 3, May 29, used on June 1. *FiG. 89. — Cross-section of stem of Solanum nigrum, showing vessels occupied by Bacterium solanacearum. Plant No. 34, 1896, inoculated from bouillon July 11, in upper part. Collected July 24. Source of organism, South Carolina egg-plant. All of the leaves were wilted when the stem was fixed in strong alcohol. Bacteria are confined to the heavily shaded parts of the xylem, and inner phloem. Slide 1 19*. A few of the infected vessels are shown highly magnified in fig. 90. 184 BACTERIA IN RELATION TO PLANT DISEASES. Result. — Very successful (Bull. 12, pi. 1, figs. 6, 7, and pi. 2, fig. 2). First signs appeared on third or fourth day; weather hot, 280 to 320 C. The organism swarming in tissues of affected plants was a short, motile rod. June 23. — Three old potato tubers and 3 cucumber fruits were used for these inoculations (4 inoculations, 2 checks). The cucumbers were 7 inches long by 2 inches thick, green and firm. The potato tubers were large, smooth and white, probably Burbank, kept under bell-jars. Same culture used as for inoculations of June 15. Result. — The cucumber fruits rotted, but no plates were poured. In the worst injured potato the bacteria, which were numerous along the line of the punctures, extended scarcely 1 mm. beyond; starch intact. July 11. — Two cucumber fruits under a bell-jar were inoculated from tube No. 4, July 8. Result. — One of the fruits rotted, but no plates were poured. The bacteria filling the interior were short, actively motile rods (see footnote on p. 174). July II. — The following plants were inoculated: Potato (7 plants), tobacco (Connecticut seed leaf, 2 plants), cucumber (4 plants), petunia (2 plants, 6 shoots), Solatium nigrum (1 plant). Datura stramonium (2 plants). Datura tatula (?) (1 plant), Physalis crassifolia (1 plant, several shoots). Twenty-five inoculations were made by needle-pricks on a very hot, sunny day. The inoculated parts were shaded. The cultures used were tubes of beef-broth Nos. 1, 2, 3, 4, 5, 6, and 8 of July 8, from sugared peptone-water in fer- mentation-tubes (descended from egg-plant from Charleston, S. C). The potatoes were tall shoots, half-grown in 8-inch pots, and each plant was inoculated from a separate tube (about 30 pricks in all). Numerous check-plants were held. The tobacco was inoculated (100 pricks) in a big leaf. The Datura tatula (?) was a young, vigorous plant. The plants of D. stramonium were old and stunted. Result. — Very successful on potato (Bull. 12, pi. I, fig. 8), Solatium nigrum. Physalis crassi- folia. and Datura stramonium. Feeble infection in petunia. The inoculation on D. tatula failed. There was pos- sibly some local disease on one of the tobaccos. Saved in alcohol. All the potato plants con- tracted the dis- ease. The first signs were ob- served on the third day, and on the sixth day there was de- cided wilt of the leaves. The weather was very hot. Ex- amined micro- scopically, the stems of 5. nigrum swarmed (figs. 89, 90, 91, 92) withsmall to medium-sized short rods, single or in pairs, with a constriction; a tremulous motion. July 23, 24, 25, 27. — Potato plants (4, each with several shoots) were used for this experiment. The inoculations were made by potato-beetles which were fed for some hours on tips of potato-vines badly wilted and blackened by Bad. solanacearum (inoculations of July 1 1), then colonized on the sound vines for some hours. *Fig. 90. — Cross-section of small portion of stem of black nightshade, showing Bacterium solanacearum in vessels and connective.tissue. This figure is a detail from one of the bundles shown in fig. 89 introduced to give an idea of the size and shape of the bacteria. The bacterial masses are somewhat contracted from the walls of the vessels owing to the use of strong alcohol as a fixative. The cells marked X belong to a medullary ray. Slide 1 19(14. fFiG. 91. — Longitudinal radial section of a portion of the vascular ring of the stem of Solanum nigrum, showing location of the bacteria. The right-hand side faces the inner phloem. A cavity is visible in the bundle. Plant inocu- lated with Bacterium solanacearum by needle-pricks on July 11, 1896; stem fixed in strong alcohol July 24. A detail from another section in the same series is shown in fig. 92. The entire stem in cross-section is shown in fig. 89. Drawn with the help of the Abbe camera, 16 mm. objective and 12 ocular. From slide 1 19(9. BROWN ROT OF SOLANACEAE. I85 Result. — Very successful: The first signs appeared on the seventh to ninth day, beginning as brown streaks in many bitten places. On August 4, 49 separate infections were counted. August 14. — Twelve plants were inoculated: Potato, tomato, cucumber, and heliotrope. Potato culture No. 1, August 12, direct from the interior of plant No. 16, inoculated July 1 1, was used. Result. — Very successful on potato; less so on tomato. On cucumber a swollen stem appeared (the pricked internode) ; this contained living (motile) bacteria on September 8. Heliotrope (?). August 17. — In this experiment 8 plants were inoculated: Portulaca oleraceae, tomato, Physalis ph iladelphica, heliotrope, Cucurbita foelidissima, Eleusine indica, Ricinus communis, and Vigna catjang. Many pricks were made in each plant, introducing potato culture No. 1, August 12, which was direct from interior of plant No. 16, inoculated July 1 1 . The tomato was punctured on a thrifty shoot near the top. Result. — Very successful on Physalis philadelphica, the first sign appearing about the tenth day. For effect on Physalis see figs. 93, 94. The infection of tomato was slow — no wiltup to August 28. The other plants failed to contract the disease. August 20. — Ten inoculations were made on this date, using Solanum carolinense for the experiment. Many needle-pricks carried in large quantities of the bacterial culture (tube No. 1, August 12, as above), some of the pricks being made into leaves, others into the upper part of rather woody stems. Result. — All failed. Perhaps the inoculations were made too late in the season. The stems were woody. September o. — Nine plants, part tomato, part potato, were inoculated from a fluid culture made directly from the swollen cucumber stem of August 14. Large numbers of bacteria were introduced by means of many needle-pricks. Result. — Negative. No records after September 21. Possibly the wrong organism was isolated — the method is not commendable. Fig. 92. Fig. 93. t September 16. — Six inoculations were made on potato, from cultures Nos. 1 and 2, September 14, from Nos. 1 and 3, August 29, which clouded after heating 10 minutes at 51° C. (culture descended from egg-plant from Charleston, S. C). Result. — Failed. Summer of 1001 (before July iq). — Ten plants, part tomato, part potato, were inoculated. Many needle-pricks were made, introducing a pure culture from the interior of a tomato stem from South Carolina (descendant of poured-plate colony). Result. — Very successful. All contracted the disease. *Fig. 92. — A single pitted vessel of Solanum nigrum, plant 34, occupied by Bacterium solanacearum as the result of a pure culture inoculation made some inches above and 13 days previous; two unoccupied spiral vessels at the right. fFic. 93. — Cross-section of small part of the stem of Physalis philadelphica, showing bacteria in the spiral and pitted vessels, and the beginning of a small cavity; epidermis at top and pith at bottom of the figure. Bacteria in the cavity more abundant and with denser stain than is here shown. Plant No. 57 inoculated with Bacterium solanacearum August 17, 1896; collected and fixed in strong alcohol September 23. Drawn with the Abbe camera from a section cut on the microtome and stained with carbol-fuchsin. Slide i2o(*. Crystal-sand not present. (For a longitudinal section see fig. 94.) i86 BACTERIA IN RELATION TO PLANT DISEASES. July 19. — Thirty-five inoculations were made into the following plants: Tomato (15 plants), cucumber (6 fruits), potato (6 plants), Solatium dulcamara (8 plants). The cucumbers and part of the tomatoes were in the open air, the rest in the hothouse (temperature 90° to ioo° F.). The cultures used were 6 agar streaks from as many colonies on poured plate from the interior of a tomato plant inoculated earlier in the summer (see pre- ceding set). Result. — Successful on tomato and potato, the first signs appearing on the fourth day (in one potato on the third day). Only slight local signs in Solatium dulcamara. Negative on cucumber. (For appearance of cross-section of petiole of potato at end of the sixth day see fig. 95.) November 5. — The following plants were inoculated: Tobacco (6 plants, 1 foot high), tomato (6 plants), potato (6 plants). Two leaves on each tobacco plant were inoculated by needle-pricks (20 to 30 pricks each). The plants were in the hothouse at 8o° to 90° F. All were very healthy and growing well. Two potato cultures were used, one 7 days old or more and browning, the other 72 hours old and from the preceding, descended from agar-tube No. 13, July 18, from inoculated tomato. The first potato culture was inoculated from an old browned agar culture, which had been in the ice-box all summer. Result.— All failed. June, 1902. — Potato and various weeds (Abutilon, etc.) were inoculated by hypodermic injection from cultures obtained from diseased tomato received from Georgia (1902). Thiswas a large field experiment with various fertilizers. Result. — The disease was produced on the potato, but it appeared slowly and in comparatively few of the many inoculated plants. June — . — A duplicate of the above ex- periment, except that the cul- tures used were obtained from a diseased tomato received from Porto Rico. Result. — As above. July 9. — Another duplicate of the experi- ments of June. Cultures used were from diseased tomato (?) from Maryland. Result. — As in June. June 4, 1903. — Six tomato plants were inoculated with cultures of June 1 (1 to 3 potato, 4 to 6 slant agar), which were isolated from a tomato plant received from Texas, all from plate 17, May 28, poured from interior of green and recently diseased petiole. The plants were rather far ad- vanced in disease when received and contained numerous in- truders. Six colonies were used. The slime was viscid and dis- tinctly yellow in some tubes. Result. — No wilt within 5 weeks. Hot- house experiment. Probably wrong organism isolated.* July 6. — One plant of Datura stramonium was inoculated by means of forty needle-pricks in leaf-blade, using slant agar-tube No. Result. — No record. Probably failed. July 7. — Tomato (6 plants), Datura stramonium (2 plants), Datura fastuosa (1 plant), Datura cornucopiae (1 plant), were inoculated from agar-slant tubes Nos. 1, 3, 4, July 2, from potato cultures which had become black (descended from tomato stems from Spartanburg, South Carolina). The agar-tubes were the second sub- cultures from poured-plate colonies. Result. — Everyone of the ten plants contracted the disease. One tomato plant (No. 7) was perfectly healthy in appear- ance July 21, but succumbed August 3. For appearance of plant No. 10 at end of 7 days see fig. 96. July J. — Tomato (4 plants), Datura metalloides (1 plant), were inoculated by needle-pricks (12 into each stem), using tubes Nos. 3 and 4, July 10 (from potato stem, Norfolk, Virginia). Result. — All became diseased. For result on Datura see vol. 1, plate 4. July 16. — Tomato plant No. 17 was inoculated from slant agar No. 1, July 13, the third subculture from a colony from tomato (South Carolina). The plant divided into two equal branches. Result. — Successful. The first signs of infection were visible on the third day. Several leaves wilted on July 21. Numerous adventitious roots pushed out along stem in the two inoculated internodes, while the surface of the check-internodes was perfectly smooth. Roots were also pushing out of the two internodes below the lowest point of inoculation. Several photographs were made of this plant at different stages (consult plates 26 and 27). Fig. 94.| July 2 (descended from tomato from South Carolina). *I now suspect this Texan disease may have been due to Aplanobacter michiganense (see page 161). fFiG. 94. — Radial longitudinal section through xylem part of the stem of Physalis philadelphica, showing Bacte- rium solanacearum confined to the spiral vessels. The pith is toward the left. The pitted vessel x lies not between the spirals but just below them. This section was made from the same piece of stem as fig. 93, i. e., the plant was No. 57 inoculated August 17, 1896, by needle-pricks on the upper part. The material used for this section was taken on Sep- tember 23 about 6 inches below the pricked part. The plant was infected with a pure culture derived from an egg- plant. Drawn from slide 120(3. BROWN ROT OF SOLANACEAE. I87 July 28.- July 14. — Early Rose potatoes in the field (plants Nos. 1 to 120) were used for this experiment. The cultures used were 5 slant agar-tubes of July 1 1 , from No. 4, March 1 (South Carolina ) ; slime washed off into distilled water (150 c.c). The inoculations were made near the top of the plants with a hypodermic syringe, 0.2 c.c. of the liquid being put into each plant. The stems were very tender and soft. Result. — First recorded signs on July 23, when 77 per cent showed signs, but they were slight for 9 days' incubation. Jul v 16. — Burbank potatoes in the field (plants Nos. 121 to 240) were inoculated in the same manner as the above, using 6 slant agar-tubes of July 14, from No. 1, March 1 ; slime washed off into distilled water (150 c.c). Result. — On July 25, 76 per cent showed signs of disease. July 22. — Potatoes in the field (plants Nos. 241-480) were inoculated in the same manner as those of July 14. using 6 slant agar-tubes of July 17, descended from No. 1, March 1 ; slime washed off into distilled water (150 c.c). Result. — On July 27, 60 per cent showed signs of disease; almost no wilting. July 22. — Potatoes in the field, half Early Rose, half Burbank (plants Nos. 1 to 240), were inoculated from 6 slant agar- tubes of July 20, from No. 6, July 10 (descended from potato-stem from Norfolk, Virginia), employing the method of inoculation used on July 14. Result. — On July 27, 96 per cent of the Early Rose and 89 per cent of the Burbank showed signs of disease. July 22. — Early Rose and Burbank potatoes in the field — half of each variety — were inoculated (using plants Nos. 241- 480) . The cultures were 6 slant agar-tubes, July 2 1 . from slant agar of July 20 from Virginia. The bacteria were put on outside of stem with a hypodermic syringe, which was kept protected from the light; the needle was then passed through this liquid, and entirely through the stem, 10 pricks being made into each plant near the top. No injection. Result. — First signs on July 25. On August 3, 100 per cent showed signs of disease; many were wilting and falling over. There were also many attacked by Fusarium oxysporum (see Smith and Swingle, Bull. 55, Bureau of Plant In- dustry, U. S. Dept. of Agric, Feb. 16, 1904). -Potatoes in the field (plants 481 to 720), half Early Rose and half Burbank, were in- oculated from 6 slant agar-tubes of July 26; slime washed off in dis- tilled water. A little liquid was drawn up with a hypodermic syringe wrapped in black paper to protect it from the light. This liquid was put on the stems and 10 needle- pricks made through it into the stems, with- out injection. In all of these large field tests Mr. Deane B. Swingle did the inocu- lations and made the counts. -On August 22, 96 per cent of Early Rose and 94 per cent of the Bur- bank were diseased as a result of the inocu- lations. July 21. — Two tomato plants were inoculated from culture from No. 1 , July 1 (from tomato, South Carolina) with a hypodermic syringe (0.2 c.c). Result. — The experiment was followed for many weeks. Adventive roots pushed out but the plants failed to wilt. They grew luxuriantly after repotting, and bore fruit. March 77, IQ04. — Twelve plants of tomato (6 varieties), were inoculated from potato-cultures Nos. 2, 5, 8, March 14 (Virginia, isolation of 1903). The plants when inoculated were 4 weeks old, about 6 inches high, and had 3 to 4 leaves. Made 15 to 20 needle-pricks in leaf-blades. Result. — No wilt ; plants grew well. The experiment was followed until May 10. April 16. — Eight potato plants, 8 to 16 inches high, were inoculated from potato cultures Nos. 1 to 3 of April 13 (Vir- ginia, 1903). Young internodes were pricked. Result. — No wilt. The stems became very badly swollen and discolored (see vol. II, fig. 27). Result.- Fig. 95. *Fig. 95. — Cross-section of base of petiole of a potato-leaf, showing restriction of the bacteria (solid black masses in center at right) to the vascular part. Plant No. 23, 1901, inoculated from a pure culture of Bacterium solanacearum, July 19, by needle-pricks. Material collected and fixed in strong alcohol on July 2.5, at which time the blade of the leaf was badly shriveled. The shading between the cells in the outer part of the section represents collenchyma and not bacterial occupation. Drawn under the Abbe camera with 8 mm. apochromatic objective and No. 1 compensating ocular, from a paraffin section which was stained with carbol-fuchsin and washed in 50 percent alcohol. Slide 123*. BACTERIA IN RELATION TO PLANT DISEASES. A ugust 8.- May 21. — Tomato plants (6) were used for this experiment. The inoculations were made from potato-cultures Nos. 4 and 5, May 12, descended from colony on plate poured after exposure to liquid air (from Virginia, 1903). They were made on shoots of plants which had been cut back and repotted. Result. — No wilt. Pricked area became blackened and somewhat swollen. May 24. — Tomato plants (6 vines about 9 to 12 inches high), were inoculated from beef-bouillon Nos. 19, 21, 22, of June 9 (1903?), and potato No. 26, June 9 (1903?). Result. — No wilt (June 17). The stems became swollen and put out adventitious roots. July 5. — Eleven tomato plants were inoculated with potato-cultures Nos. 1 to 6 of July 2, from plate colonies, from potato stem from Woodridge, D. C. (see pi. 30). Made 10 to 12 pricks in each stem with a No 12 needle. Result. — Very successful. All but 2 showed the disease July 8, and one of these two was inoculated with a gas-forming organism. (Consult fig. 97 for appearance of an inoculated plant at end of third day.) -Potato plants (8 pots) of the Green Mountain variety, received about 36 inoculations on leaves and young shoots. The plants were stocky and only a few inches high. Needle-punctures varied in number from 1 to 20. The cultures used were slant agar subcultures, Nos. 3 and 4, August 6, from potato stem from Woodridge, D. C. Result. — Very successful ; first signs on third day. (See pi . 3 1 . ) August 25. — Potato plants (3 pots) were inoculated with potato-cultures Nos. 1 and 2, August 19, and litmus- potato-agar No. 2, August 15 (from potato-stem from Woodridge, D. C). Part of the plants were inoculated with descendants of culture after passing through the thermostat (37. 5° C), and part with descendants of culture held at room temperature. Made 10 to 12 needle-pricks in upper internodes. Result. — The plants were too old. None contracted the dis- ease. All of them died down from thrips or other causes. September 6. — Fourteen tomato plants were inoculated from subcultures in tubes of litmus-lactose-agar, Nos. 3, 5, 7, 15, and 17, September 1 (Woodridge, D. C, organism). Part of the plants were inoculated from culture grown at 37. 50 C, part from descend- ants from culture grown at 37. 5° C. (pi. 29), part inoculated from descendants from culture grown in ice-box, and part inoculated with organism grown at room temperature since its isolation (pi. 28). The inoculations were made on young shoots coming up from plants cut back. Result. — No rapid wilt. For the most part only swollen stems and slow progress of the disease. October 6. — Six tomato plants of as many varieties were inocu- lated from slant agar Nos. 1 and 2, October 3, from No. 2, August 29 (Woodridge, D. C, organism from potato). The stems were pricked 3 to 4 inches from the top. -No wilt. The stems became somewhat swollen and pushed out adventitious roots; they also became slightly black around the pricks. October 11. — This experiment was made with 5 tomato plants, inoculating them near the tips in tender tissues in most cases, and directly from tomatoes which were inoculated October 6, and which had not wilted but showed pricked area swollen and considerably discolored (Woodridge, D. C, organism from potato through tomatoes 102 and 103). The plants inocu- lated were 10 to 18 inches high. Result. — None of the plants ever wilted. Slight swelling and discoloration in the pricked area — less than in the preceding. October 24. — Tomato (2 plants) of the varieties Red Pear and Honor Bright, were inoculated direct from tomato No. 109, inoculated October 11, which had swollen and discolored at pricked part more than the others (Woodridge, D. C, potato through tomato twice). Result. — Negative. October 26. — Six tomato plants of as many varieties (1.5 to 2 feet high, not yet branched) were inoculated on stems 3 to 6 inches from the top with potato cultures Nos. 2 to 5, October 22, from agar-stabs Nos. 7 to 11, August 26 (Woodridge, D. C, potato). Result. — No wilt. The stems became considerably swollen and adventitious roots were formed, but further than this the bacteria had no effect upon the plants. Result.- Fig. 96. *Fig. 96. — Tomato-plant inoculated July 7, 1903, with Bacterium solanacearum (South Carolina strain), graphed July 14. Plant "inoculated]by"needle-pricks from a pure culture 5 days old. Photo- PLANT BACTERIA, VOL. 3. PLATE 31. Potato-shoots inoculated by needle-pricks August 8, 1904, with a pure culture of Bad. solanacearuni plated from the stem of the Woodridge, D. C, potato shown in plate 30. Photographed Aug. 16. Notice particularly the dark stripes extending down inside the stems. The left-hand shoot was pricked in a petiole (the black one at the left midway). The right-hand shoot received one needle-puncture in an upper internode. The middle shoot received 10 needle-pricks in an upper internode. BROWN ROT OF SOLAN ACEAE. 189 June 27, 190$. — Potato plants (2 varieties) were inoculated from tubes Nos. 4, 5, 6, June 22, from as many colonies (stem of tomato, Hastings, Florida). Made 85 inoculations on shoots and leaves on 26 plants. Result. — Positive in part; slow. On July 13 the Blush showed fewer cases than the Early Rose. Plants on this date 2 to 2.5 feet high. The difference in resistance appeared to be lodged in particular plants. The Blush was more resistant than Early Rose. Each culture was virulent. (See vol. I, plate 24. The source of the culture is there stated to be potato, but the potato organism from Hastings appears to have been tried only on tobacco except as below, unless certain of the notes have been lost.) July 8. — Eight tobacco plants were inoculated in stem and leaf with potato-cultures A and B.July 6 (from two separate colonies, descended from potato tuber, Hastings, Florida). Result. — Failure. The experiment was followed until September 6, when 6 plants were examined microscopically. Bacterial multiplication and brown stain slight and confined to pricked parts. July 8. — Thirty-four young tomato plants 12 to 16 inches high were used in this experiment (variety test). Potato cultures A and B, July 6 (from 2 separate colonies descended from potato tuber, Hastings, Florida), were used. Fig. 97.* Result. — Most of the plants grew to be 6 to 9 feet long, fruited well, and were alive 4 months after the inoculations. They doubled in size within 6 days after the inoculations, and the inoculated shoots had elongated about 12 inches. The plants continued to grow rapidly. On July 25 they were 3 times as large as on July 8. A few cases developed very slowly, most failed, except that the inoculated parts were swollen and browned and adventive roots pushed out. Of many varieties, Livingston's Dwarf Aristocrat was most susceptible In October a plant with large pear-shaped fruits contracted the disease and the bacteria were found filling vessels of stem and branches 4 feet from the needle-pricks. September 23. — Tobacco (24 large plants) was used for this experiment. The plants were inoculated by needle-pricks in the stem, introducing potato-cultures A to D, September 21 (potato tuber, Hastings, Florida.). Result. — Slow progress of disease; pith blackened. No distinct wilt. September 23. — Tomato plants were inoculated with potato cultures A to D, September 21 (potato tuber, Hastings, Florida). Result. — Adventive roots developed and some shoots were swollen and stunted; wilt developed not at all or very slowly. September 30. — Tomatoes (20 plants, 10 to 12 inches high) were inoculated by needle-pricks with beef-bouillon cultures A to D, September 20 (potato tuber from Hastings, Florida). Result. — Failure. *Fig. 97. — Tomato plants pricked with a delicate needle at X, X on July 5, 1904. When used on the left-hand plant the needle was sterile, when used on the other (No. 64) it was flamed and then dipped into a 3-day-old culture of Bacterium solanacearum (District of Columbia strain). Photographed July 8. 190 BACTERIA IN RELATION TO PLANT DISEASES. September 30. — Tobacco (24 plants) was inoculated by needle-pricks with beef-bouillon cultures A to D, September 20 (potato tuber from Hastings, Florida). Result. — Failure. October 26. — Tomatoes (3 plants about 18 inches high) were inoculated with potato cultures Nos. 1 and 2, October 20 (growth black on the potato) ; alive October 23. The organism was not plated out, but descended from potato cultures Nos. 1 and 2, October 19, which were streaked directly from diseased tuber from Portsmouth, Vir- ginia. It was probably the right organism — it showed the typical jet-black growth on potato. Result. — Failure. October 26. — Tobacco plants were inoculated with same cultures used for above. Result. — Failure. October 28. — Tobacco plants were inoculated with potato cultures Nos. 3 and 4, October 23 (from Portsmouth, Vir- ginia, tuber); alive October 27. Result. — Failure. (For successful inoculations on tobacco see Wilt Diseases of Tobacco, p. 227.) October 30. — Tomatoes (2 vines, 18 inches high), were inoculated with potato culture No. 2, October 23 — jet-black (from Ports- mouth, Virginia, tuber). Result. — Failure. November 24. — Tomatoes (5 vines) were inocu- lated with potato cultures Nos. 1 to 5, November 10, each from a separate colony on plates of October 19 (potato tuber, Portsmouth, Virginia). Result. — Failure. The cultures were alive; the transfers made from them on date of inoculation grew well. A single needle-prick introduc- ing into a susceptible plant a small quantity of a virulent culture is suffi- cient to produce this disease, as shown on plate 31, right-hand figure. MORBID ANATOMY. The premature and excessive de- velopment of roots on tomato shoots as a result of this disease has been mentioned under "Etiology." For a section through such incipient roots, showing their origin and relation to infected tissues see fig. 98. An en- larged view of bac- teria from this section is shown in fig. 99. In rapidly fatal cases there are no hyperplasias in con- nection with this dis- ease other than the adventive roots on tomato stems, but in stems of tomato and potato inoculated with feebly virulent cultures there may be considerable enlargement in the vicinity of the punctures. Such stems Fig. 98.* Fig. 99. | *Fig. 98. — Cross-section of a small portion of stem of tomato plant (No. 26) inoculated with Bacterium solana- cearum on June 8, 1895, and fixed in strong alcohol on July 3. The figure shows vessels occupied, the formation of small bacterial cavities, and a well-developed incipient root (R), the growth of which has been stimulated by the pres- ence of the bacteria at a distance. Several much more rudimentary roots are depicted in the upper portion of the fig- ure (Rr). At the left, above the lower cavity, the bacteria fill the intercellular spaces around a few cells (X). The bast fibers are not distinct in the section itself, which was stained with special reference to the bacteria. There are no bac- teria immediately under or in the budding root (R). Slide 1 1 1 (2. For further illustrations of these budding roots see various plates. fFlG. 99. — Bacterium solanacearum from inoculated tomato plant No. 26, 1895. An optical plane from one of the cavities shown in fig. 98. BROWN ROT OF SOLANACEAE. 191 frequently attain a diameter twice that of the stem a few inches above or below the pricked area (pis. 28, 29). In such enlarged areas there is usually developed around the punctures a considerable area of protective cork-tissue, and an excessive amount of bundle-tissue is formed. This malady is chiefly a disease of the vascular bundles (figs. 81, 84, 90, 100, 101, 102, 103), but the organism is not so strictly confined to the bundles as in case of the wilt of cucurbits and the brown rot of the cabbage. There is, however, the same enormous multi- plication of the parasite inside of the vessels. The walls of the vessels are dissolved in their thin places or else are ruptured, and cavities are produced in the surrounding tissues, the bacteria being found in the in- tercellular spaces but also often inside of cells without one's being able to make out clearly how they got there (see vol. II, p. 76). The parenchyma, especially in soft parts, such as the pith of young potato shoots and tomato shoots, succumbs quite readily, a large portion of the interior of such stems break- ing down into a soft, wet rot which sometimes oozes through to the surface. The bacteria first invade the intercellular spaces, which are generally occupied quite fully in advance of the complete destruction of the cells. The middle lamella is then dissolved. The sepa- rated cells are squeezed into a variety of shapes by the multi- plication of the bacteria and finally are crowded aside alto- gether, leaving behind cavities partly or fully occupied by the parasite (figs. 86, 88, 91, 93, 98). In woody parts such as stems of old potato plants or tomato plants, and of well- grown egg-plants, there is usually a marked browning of the lignified tissues and an enormous multiplication of the bacteria in the vessels and the adjacent parts, but there is no such general collapse as in case of soft stems. Lignified tissues are not dissolved. In the potato tuber, brown-walled cavities beginning in the rudimentary vascular tissue are numerous. These cavities, which are full of bacteria and remnants of the broken-down tissue, gradually enlarge and coalesce until all of this part of the tuber is involved. Subsequently they reach the surface (pi. 23, portions of figs. 8 and 10) and open up the tuber to all sorts of external destructive agents. The starch in the Fig. 100.' *Fig. 100. — Longitudinal section of a potato stem, No. 5, 1896, inoculated June 1, collected June 11, showing Bacterium solanacearum restricted to a single spiral vessel. Drawn from a photomicrograph. The fibers at the left polarize in the same way as the spiral thickenings. Slide 138, Vernier readings on Zeiss photomicrographic stand 13.4X2.0. Stained with carbol-fuchsin and washed in 50 per cent alcohol. X210. 192 BACTERIA IN RELATION TO PLANT DISEASES. tubers resists well and appears to be but slowly acted upon by this organism (figs. 104, 105, 106). In young tubers, however, such as that one shown in figs. 83, 107, 108, there may be considerable areas of the starch-bearing tissue in which no starch occurs. These are most extensive on that side of the tuber longest occupied by the bacteria, and the only explanations which seem to fit the case are that (1) the potato plant has withdrawn the starch preparatory to the formation of a cork-barrier as Appel has described for B. phytoph- thorus in potato, or that (2) excreted products of the bacteria (enzymes or other sub- stances) have killed these cells or have so paralyzed their activities that the plant has not been able to make use of them for the storage of starch. A very little is sometimes found (figs. 105, 106). That the starch was once stored and then dissolved by diastasic action of the bacteria is contrary to all that we know of its behavior, both in vitro and in older tubers. In the inoculated plants of the Jamestown weed (fig. 88), of the black nightshade (fig. 91), and Physalis Philadelphia! (fig. 94), there was the same enormous multiplication of the bacteria in the vascular system, even at long distances from the point of inoculation, and the tissues were destroyed in the same way, with the formation of extensive bacterial cavities. Several observers have reported finding an excess of crystal-sand in plants at- tacked by this organism. This substance occupies particular cells and consists of numerous discrete tetrahedral crystals of calcium oxalate (fig. 88). It occurs naturally in a large part of the Solanaceae, the amount varying with particu- lar organs and with the age of the parts; it is, therefore, not easy to determine whether there is an excess of it in dis- eased tissues, but there seems to be. In most of the draw- ings crystal-cells are depicted diagrammatically by minute arrow-marks ( < < ). In the Solanaceae we Fig. 101.* have a bicollateral bundle, the two phloems being flanked or subtended by thick-walled bast-fibers, as shown very clearly in the cross-section of a potato-rhizome (fig. 84). In the plants which I have examined there is also usually a large pith and considerable subepi- dermal collenchymatic tissue, which is usually somewhat shrunken and torn on fixing in alcohol and does not always show distinctly as collenchyma in the accompanying draw- ings, but may be seen very clearly in the cross-section of a potato petiole shown in fig. 95. Both phloems are frequently invaded by the bacteria and numerous cavities are produced in this tissue, as shown in the figures already referred to. In later stages of the disease, cavities are also common in the bark-parenchyma and in the pith. Indeed, the disorganization has proceeded very far in such stems as that shown in cross-section in fig. 1 of vol. II. *Fig. 1 01. — Cross-section of small part of same potato stem as fig. 86, showing vessels occupied by Bacterium solana- cearum, and surrounding tissues mostly free, except as they have flooded out of the vessels during the preparation of the section. These latter bacteria occur only on under side of section and are omitted from the drawing. Drawn from a photomicrograph by the writer. Section stained with carbol-fuchsin and differentiated in 50 per cent alcohol. The lignified connective tissue is represented by solid black walls and that of the vessels by fine dots. X800. Slide 171. BROWN ROT OF SOLAN ACEAE. 193 THE PARASITE. Bacterium solanacearum EFS.* is a medium-sized rod with rounded ends. It multi- plies by fission. It often occurs in pairs with a plain constriction (fig. 109). It is usually 1.5 to 3 times as long as broad. When crowded in the plant it may be so short as to some- what resemble a micrococcus. It stains readily. When taken from young cultures in pep- tonized beef-bouillon and stained with methyl violet many of the rods are 1.5^ by 0.5^. Longer and shorter rods occur and thicker and thinner ones, the measurements depending to a considerable extent upon the age of the culture and the kind of stain employed (fig. 109). It is motile at times both in the plant and in cultures, and is often very actively so in young cultures. Owing to imperfect preparations it was at first supposed that the flagella were per- ipheral, but further studies of these slides and of others subse- quently prepared indicate that the or- ganism is usually motile by means of a single polar flagel- lum, which is much longer than the rod (fig. 109). Pseudo- zoogloeae are com- mon in fluid cultures and inclined to ac- cumulate in the upper layers. Spores have not been ob- served. Long chains and filaments have not been observed. On August 8, 1904, two 3 -mm. loops of the District of Columbia organism taken from a slant potato-agar culture 3 days old (tube 2, Aug. 5) were put into 15 c.c. of sterile water, and then the faintly clouded fluid was studied in a hanging drop under high powers of the microscope. The organism was actively motile. It was a short rod, single or in pairs, rarely in fours, the doublets with a plain constriction in the middle. The length of each element of the doublet was not more than 1.5 to 1.8 times its breadth. In old potato cultures examined in 1904, the organism was a short rod with rounded ends, single or in pairs, with a plain constriction. Involution forms were not -• -: x Fig. 102.f *Syn. Bacillus solanacearum EFS.; Pseudomonas solanacearum EFS. fFlG. 102. — Cross-section of stem of potato (No. 5, 1896) inoculated with Bad. solanacearum June i ; fixed in alco- hol June 11, after being badly wilted for 3 days. The culture used was a tube of peptone-water inoculated directly from the interior of an egg-plant stem. The heavily shaded parts are the ones occupied by the bacteria. There are 16 crystal-cells marked by arrow heads (use lens). The parts of the stem not heavily shaded are free from the bacteria, viz, all the bark, most of the pith, and considerable portions of the woody ring. Slide 166(2. Figure somewhat diagrammatic, especially in the collenchyma and phloem. Compare with fig. I, vol. II, which shows cross-section of a potato stem in a later stage of the disease. The vessel at X is shown highly magnified in fig. 103. 194 BACTERIA IN RELATION TO PLANT DISEASES. observed. This has been the general form of the organism seen nearly every summer since 1895 in tomato stems and potato stems collected in various southern States, and in cultures made therefrom. In glycocoll solutions Honing has seen involution forms (p. 253). This organism is often not so pure a white as Bacillus amylovorus or Bacillus trachei- philus. In the host plant and on certain media it may be described as pure white at first, but soon grayish-white, dirty white, or brownish-white, becoming brown, since it produces a soluble brown pigment which modifies its appearance. On steamed potatoes this stain is usually developed to such an extent that the cultures soon become brown or even nearly black (3 to 10 days); the water also is browned around potato cylinders. This pigment, which is produced in the dark as well as in the light and which on potato is not infrequently as black as tar (pi. 23, figs. 8, 10, and pi. 41, fig. 4), is soluble in water and glycerin and slightly in methyl alcohol on long stand- ing; it is insoluble in ethyl alcohol (exp. 1896, 1905*), ether, chloroform, xylol, and carbon bisulphide. The browning did not occur in the absence of sugar and alkali. The pigment is not destroyed by weak acids or alkalies. It behaves in some re- spects like a humous compound, i. e., it does not dialize readily and is precipitated by compounds of iron and calcium. This organism grows well in pepton- ized beef-bouillon, with or without the addition of sodium carbonate, and yields much more precipitate than similar cul- tures of B. tracheiphilus; a brown stain appears, and this was especially noticeable in case alkali was added; tendency to form pellicle rather slight. Old peptone- water cultures do not brown. In +15 peptonized beef-bouillon the District of Columbia organism at the end of 20 days showed good clouding with an enormous number of small pseudozoogloeae, but with no rim or pellicle ; the dirty white or pale brownish precipitate covered a breadth of 13 mm. on the bottom of the test-tube. In Dunham's solution cultures of the same age were well clouded, but less so than the bouillons, with only about half as much of the dirty white precipitate. Cultivated in milk there is no precipitation of the casein, but after about 3 to 7 weeks the fluid becomes transparent (fig. 1 10, a). Milk was intensely alkaline to litmus paper as early as the twentieth day. The organism is able to live in milk for at least 48 days. The casein of the transparent milk is thrown down by hydrochloric acid (1896, repeated in 1905), and by strong solutions of sodium chloride or copper sulphate. In litmus milk no acid is developed, but after 2 or 3 days the fluid becomes slightly bluer, and this alkalinity increases gradually from day to day until the milk is an intense deep blue. On the twenty-eighth *Bact. solanacearum (District of Columbia strain) in gelatin-stab. On adding water to the alcohol the pigment was slowly soluble. fFic. 103. — Cross-section of a very small portion of a potato-stem (a detail from fig. 102), showing a single vessel occupied by Bacterium solanacearum, the surrounding tissue being free from the bacteria. The lignified wall of the vessel is indicated by fine dots. Slide 166(2. Zeiss photomicrographic stand, vernier readings 14.7 X2. 8. Plant inocu- lated with pure peptone-water culture made direct from the interior of a diseased egg-plant. Fig 103. t BROWN ROT OF SOLANACEAE. 195 day such milk was approximately Ridgway's indigo blue ; after 3 months it was dark hya- cinth blue (pi. 23, fig. 7) ; but the fluid was neither viscid nor gelatinous. Litmus in milk is sometimes a little reduced (white) in the bottom of the tubes. The bacterial precipitate is white. In another set of tubes of litmus milk examined at the end of the seventh week the color was recorded as a clear intensely dark blue. All these milks were cream-free. Litmus-laetose-agar streaked with this organism becomes slowly a deeper blue. There is never any reddening, but sometimes the litmus is partially reduced and the agar finally browns. The behavior in cream-free litmus milk is like that of the culture figured on pi. 41. The streak of the District of Columbia organism on slant litmus-lactose-agar after 10 days at about 270 C. was white, wet-shining, and there was a copious whitish precipitate in the V; the upper half of the agar was blued very decidedly, in the lower half the litmus was reduced. The surface of the slant beyond the streak was iridescent. The agar was not then browned, but later (seventh week) it was browned decidedly and the surface growth was a dirty brownish-white ; the extreme upper part of the agar continued bluer than the check-tube, but in the lower part the formation of the alkali was masked by the brown stain and the reduction processes. The organism does not liquefy gelatin (experiments of 1895-96, repeated in 1904 and in 1905), at least not when made as described in vol. I of this monograph. The surface colonies in gelatin are small, circular, thin, thin-edged, smooth, white, wet-shining; the buried col- onies are globose, yellowish or brownish, and smooth, with well- defined margins. In stab-cultures (fig. in) the upper part is best de- veloped, but growth was not rapid at 240 C. and ceased at io° C. The Virginia organism grown in gelatin- stabs for 7 days at 220 to 250 C, and then 2 days at 150 C, developed a thin, white, wet- shining surface growth which was roundish and about 3 to 4 mm. in diameter. Growth was visible the whole length of the stab, but was best at the top. There was no stain, no gas, no liquefaction, no development of crystals. The District of Columbia organism grown for 10 days in gelatin-stabs at 250 C. was similar in all respects to the above. The + 10 gelatin in old cultures* becomes dusky or browned, especially in the upper part of the stab. In agar the buried colonies remained small and were irregularly round or oblong, with a roughened margin. The surface growth was white or gray-white at first, then brownish. The general form of the surface colonies after some days on +15 nutrient agar at 250 C. is shown in figs. 112, 113. As a rule the agar was stained brown by growing this organism on it (pi. 23, fig. 2, and pi. 41, fig. 8, from tobacco, will answer for potato and tomato). In thin sowings on peptonized beef agar the organism from the South Carolina tomato, used for successful inoculations in 1901, formed colonies about 2 to 3 mm. in diameter at the *Fig. 104. — Cross-section of a few cells of a potato tuber rotted by Bacterium solanacearum. Plant No. 14, 1896, inoculated June 15. A detail from fig. 82 made under x, showing cells occupied by the bacteria. The starch-grains which lie buried in the bacterial mass appear to be uncorroded; they polarize, but I was unable to detect any rings of light and dark substance in them. Section stained with carbol-fuchsin. Drawn with the Abbe camera. Slide 156(2. 196 BACTERIA IN RELATION TO PLANT DISEASES. end of a week (temperature 2 8° C). days later they were brownish and the after the first 10 days. On steamed potato this organism was white at first, then a dirty yellowish-white, and finally brown or even nearly black. The poorest agar-streaks, with perhaps one exception, were on al- kaline agar containing 6 per cent glycerin. In agar-stabs the growth was best on the surface and in the , upper part of the needle-track. No gas-bubbles or acid reac- tions were observed as the result of I cultivation in any of the ordinary culture-media, but in each case there was a gradually increasing alkalinity. Grown in fermenta- tion-tubes in 2 per cent alkaline peptone-water with addition of grape-sugar, fruit-sugar, cane- sugar, milk-sugar, galactose, mal- tose, or dextrine, there was no gas production, no detected acid de- velopment, and no clouding of the These were circular, white, and wet-shining, and 9 agar was also browned. They did not enlarge greatly Fig. 106.f have been tried and in all of them it requires free oxygen for respiration Fig. 105.* closed end of the tubes. The same result was obtained with the juice of potato tubers diluted with water and sterilized in fermentation-tubes (1909 organism from tobacco) . Culture-fluids containing grape- sugar, fruit-sugar, and cane-sugar browned decidedly after some weeks. In 1904, using the District of Columbia organism, clouding was heaviest with cane-sugar. All of the writer's numerous experiments go to show that Bacterium solanacearum is a strict aerobe (the fermentation-tube experiments were repeated with the same results in 1904). If it is ever capable of growing in the absence of air we do not know on what media or under what cir- cumstances. All of the common media *Fio. 105. — Border of a small cavity in the vascular region of a potato tuber attacked by Bacterium solanacearum showing 7 uncorroded starch-grains lying in one cell embedded in a mass of bacteria. Slide 156(3. Vernier readings on Zeiss photomicrographic stand, 12. 5X8.1. Plant No. 14, 1896, inoculated on the stem June 15. Drawn by polar- ized light. The dark patches are masses of bacteria. fFiG. 106. — Detail from fig. 83 at x, showing uncorroded starch-grains embedded in a cell occupied by Bacterium solanacearum. The dotted parts of the starch-grains are the only ones stained by the carbol-fuchsin. Light and dark rings can not be made out in these grains, but they polarize the same as grains in the middle of the tuber, remote from the bacteria. Drawn with 2 mm. 1.30 n. a. Zeiss apochromatic objective, 12 compensating ocular and Abbe camera, Slide 349(i5. BROWN ROT OF SOLAN ACE AE. 197 The organism is not sticky, or only slightly so occasionally on agar, and diffuses out of the vessels of the host-plants readily in 75 per cent alcohol, but not, according to Hunger, when 1 per cent nitric acid is added to the alcohol. It possesses a decided odor when grow- ing on potato, i. c, a smell like that of some rotting potatoes, and likened by one person to the odor of sour bran. The starch of steamed potatoes is not destroyed to any great extent, but a little of it is converted into amylodextrine, i. e., yields a red reaction with iodine. There was no growth, or very slight, in Cohn's solution; very little growth appeared on silicate jelly (with Fermi's solution) at the end of 6 days. The : 1 - ss v>j Fig. 107/ Fig. I08.f *Fig. 107. — Portsmouth, Virginia, potato tuber. A detail from fig. 83 at A, showing more extensive bacterial occupation than in fig. 83 at B, and complete absence of starch on the periphery. A few starch-grains occur inside of cells at Si. (.center). Only four cells containing crystal-sand are present Cavities have begun to form and additional ones are present beyond C (right margin), where also are a few cells containing starch-grains (see fig. 106). In various places, as at X (use lens), some change has taken place in the walls of the cells and they are made out with difficulty. In other places, as on the left side, cells which appear to have unbroken walls are occupied by the bacteria. The walls of the cells destitute of starch seem to take the stain somewhat heavier than those of cells in which it is present. Camera drawn, but slightly diagrammatic. Slide 349(15. fFic. 108. — A detail from fig. 83 at B, showing character of the tissues and absence of the starch in the vicinity of the bacterial foci (at the center). Tissues fixed in Carnoy's solution. Camera drawn, but slightly diagrammatic t. e., it is probable if the slide were taken and compared cell by cell with the drawing, some omissions and some errone- ous lines would be detected. 198 BACTERIA IN RELATION TO PLANT DISEASES. organism reduces potassium nitrate to nitrite in bouillon; does not produce indol in peptone-water; and does not stain by Gram's method, i. c, is only slightly blue after washing (diaphragm wide open). Cultures were easily obtained from a bouillon-culture after exposing it for 20 minutes to minus77°C.,but quantitative experiments with liquid air show that a large proportion of the rods are killed by a single freezing (pi. 32). The minimum temperature for growth is about io°C; optimum temperature 350 to 370 C. (?); maximum temperature not determined (about 410 C.*); the thermal death-point (South Carolina organism, 1896) is above 510 C. and below 530 C, being probably about 520 C. In 1904 all 10-minute exposures of the Dis- trict of Columbia organism at 520 C. remained sterile but not those at 510 C. The organism grew readily and for a long time in the thermostat at 370 C, i. e., in peptonized beef-bouillon held at 370 C. the South Carolina organism was alive in one test after 3 weeks' exposure, and in another after 7 weeks' exposure. At 380 to 400 C. the District of Columbia organism -^\^ B J-P Vioo mm 1 Fig. I09.f clouded +15 peptonized beef -bouillon in 48 hours and grew well, but not so freely as at room temperature. After 10 days' exposure in the thermostat at this temperature, streaks to slant agar gave only a discrete growth (separate colonies), indicating that the cloudy bouillon was then only thinly occupied by living bacteria. It also lives for a considerable time in peptone-water and in bouillon at 200 to 250 C. This bacterium does not live for many weeks ♦Exposed in Feb. 1904 in the thermostat at 39.4° C. the Virginia organism clouded peptonized beef-bouillon of the following reactions: +25. +15. °. and refused to cloud —20 bouillon, Dunham's solution, or Uschinsky's solution. The experiment was twice repeated with the same results. fFiG. 109. — (a) Bacterium solanacearum (District of Columbia strain). From a slide (2a) of August 9, 1904. Flagella stained by Lowit's method. (B)Bad. solanacearum (D. C. strain) showing flagella stained by van Ermengem's silver nitrate method. Slide of August 12, 1904. (c) Freehand sketches of flagella from three slides of Bad. solana- cearum, stained in 1896 by van Ermengem's silver nitrate method. Slides X, XX, and XXX (South Carolina organ- ism. No. XXX taken from inoculated Physalis No. 57, the others from cultures). The numbers correspond to the fol- lowing vernier-readings on Zeiss photomicrographic stand 32532 used by the writer: (1) 12.8X5.35; (2) 12.2X6.8+; (3) 14.4X7-3; (4) 20.1 X7.3; (5) 20+X7-2; (6) 18. 5X7-15; (7) 13-6X5.6; (8) 20X5; (9) 19-3X6.75; (10) 18X4-4; (") 173X0.9; (12) 15.4X6.6; (13) 16.1X6.9; (14) 15-6X5.75; (15) 138X5-6 artefact (?); (16) 16.35X11.35; (17 and 18) 14.17 X6.35. Numerous flagellate rods are visible on slide XXX at 14.17 X6. 35 and all seem to be one-flagellate, but it requires a high power and a bright light to make them out as all are feebly stained: One might also add, keen eyesight. (d) Cover-glass preparation of Bad. solanacearum (D. C. strain) stained August 12, 1904, by van Ermengem's method from a young agar culture. Flagella wanting or not visible. This organism was extremely pathogenic at first (see pis. 30, 31, and vol. 1, pi. 26), but afterwards lost much of its virulence. The bacteria are thicker than when stained without mordanting. PLANT BACTERIA, VOL. 3. PLATE 32. Four Petri-dish poured-plates of Bacterium solanacearum (Virginia strain), showing the effect of freezing. ,4 and B, plates poured before freezing; C and D, corresponding plates inoculated with the same carefully measured amount of the bouillon culture, but after freezing, i e., as soon as the culture was thawed. The plates were incubated for some days at 300 C. The large dark colonies are on the surface, the large pale colonies are between the bottom of the dish and the agar, the dot-like growths are colonies buried in the body of the agar. Photographed Feb. 20, 1904. BROWN ROT OF SOLANACEAE. 199 ^ A a - B 3 Fig. !I0.! in steamed potato cultures, especially if the brown stain is well developed, and the well- browned colonies on agar are usually dead. Two weeks is about the limit of vitality on cooked potato ; sometimes cultures were dead at the end of one week. An actively motile bouillon-culture frozen in liquid air for 20 hours was still motile upon thawing out. Exam- ined in a hanging drop within 20 minutes hundreds of the rods were active. This was not a Brownian movement, but a rapid darting motion which often carried the rods out of the field. However, a distinctly less number seemed motile than before the exposure and poured plates (Vir- ginia organism) demonstrated 50 per cent to be dead. The organism grew well in acid bouillon ( + 33, acid of beef juice), but less rapidly at first than in nitrate bouillon (+15). After 3 months the bouillon was stained brownish. There was then an interrupted, dirty, gray-white pellicle, a dirty, brownish-white precipitate, and numerous small crystals. The Virginia organism did not grow well on No. 602, a rather acid agar made from the juice of sugar-beets diluted with water. The Virginia organism after growing in peptonized Uschinsky's solution for from 9 to 14 days had developed no pellicle, rim, or pseudozoogloese, but only a thin clouding, with a small amount of brownish-white precipitate. Around the surface growth, in the agar in contact with the air, a white amorphous sub- stance develops. This substance is finely granular under the microscope and dissolves in acetic acid. There appear to be many degrees of virulence, and possibly there are several strains of the organism. RESUME OF SALIENT CHARACTERS. POSITIVE. Cause of a vascular disease in solanaceous plants — potato, tomato, egg-plant, etc. — and from recent studies by Honing and others it would seem also of a disease in plants of several other families. vShort rod, often termo-like, motile, flagella polar; organism in the plant often easily mistaken for a coccus; dissolves middle lamella, cellulose (?); plugs vessels, attacks phloem, forms numerous closed cavities in parenchyma of hosts; causes pre- mature development of adventive roots in tomato stems. The feebly virulent strains induce slight local enlargements ; surface colonies on agar rather slow-growing, roundish, white at first, then brownish; clouds bouillon with formation of numerous flocculent particles which accumulate in top layers; culture fluids containing grape-sugar, fruit-sugar, or cane-sugar brown decidedly after some weeks ; growth in acid bouillon ( + 33, acid of beef-juice) is feeble at first in comparison with that in +15 bouillon, *Fig. 1 10. — Drawing designed to show the clearing action of Bacterium solanacearum on milk (a sub-culture from colony B, Florida potato): A, tube of milk inoculated July 6, 1905, and drawn August 4; B, uninoculated check-tube. A has become translucent slowly without a previous precipitation of the casein; B is opaque. fFiG. in. — Gelatin stabs of Bacterium solanacearum (Virginia organism) after about 16 days at room temperature. No liquefaction. Inoculated Feb. 2, 1904. Photographed Feb. 18. Fig. 111. t 200 BACTERIA IN RELATION TO PLANT DISEASES. but afterwards excellent; produces a brown stain in host, on agar, gelatin, etc.; pigment soluble in water and glycerin; streaks on silicate jelly (with Fermi's solution) feeble at end of sixth day (250 C.) ; grows readily on steamed potato cylinders, producing a pale brown to dark brown stain and an alkaline reaction ; odor on potato feebly disagreeable ; has slight action on potato starch ; slowly converts milk into an alkaline translucent fluid ; gradually changes the lilac color of litmus-milk to a deep indigo or hyacinth blue ; litmus- lactose-agar slowly becomes a deeper blue ; slight reducing power on litmus ; aerobic ; reduces nitrates to nitrites ; portion of rods killed by freezing, others retain vitality and motility ; injured by acids (Hunger, EFS.) ; thermal death-point 520 C, approximately; minimum temperature io° C, approximately; optimum 370 Cor below; maximum 41 ° C, approxi- mately; alive in milk after 48 days; browned growths are usually dead; dies early on steamed potato, usually the first or second week. Chains occur (Honing, p. 247). Group No. 21(1.3333823. NEGATIVE. long Spore-formation ; capsules ; chains and filaments (so far as observed) ; liquefaction of gelatin (at least during first weeks), blood-serum ; starch-de- struction (or feeble) ; precipita- tion of casein from milk; acid from milk; gas-formation (all media) ; anaerobism (so far as known) ; acids from sugars and alcohols (so far as known) ; dendritic growth; green fluo- rescence; vile odors; wrinkled growth on potato; disintegra- tion of cooked potato-cylinders ; action on pigment of dilute acids (10 per cent) and alkalies (5 per cent) ; pigment insoluble in ethyl alcohol (absolute), sul- phuric ether, chloroform, turpentine, benzine, xylol, benzole, carbon bisulphide; cultures not viscid, or only exceptionally and slightly so; growth in Cohn's solution absent or feeble; Gram's stain; indol. Non-motile (Hutchinson). Any organism which produces spores, stains well by Gram, gives a decided pink reaction in old peptone-water cultures with sulphuric acid and sodium nitrite, evolves gas, liquefies gelatin readily, acidifies cream-free milk, develops a lab ferment, grows abundantly in Cohn's solution, grows well anaerobically in bouillon with grape-sugar or cane-sugar, has a thermal death-point above 530 C, or fails to blue (cream-free) litmus milk and to brown steamed potato on long standing, may be set down at once as something else. This organism may be distinguished from Bacillus phytophthoms and its allies by the fact that it does not liquefy gelatin, at least for several weeks, does not redden cream-free litmus Fig. 112.* *Fig. 112. — Colonies of Bacterium solanacearum on +15 standard nutrient agar after 8 days at 230 to 27° C. Plate poured June 18, 1903, from the interior of a diseased tomato stem, received from South Carolina. Natural size. BROWN ROT OF SOLAN ACEAE. 20I milk (compare pi. 23, figs. 1, la, 3, 5 with fig. 7); or grow in closed end of fermentation tubes in peptone- water with common sugars. There are other differences, e. g., behavior on cooked potato (compare pi. 23, figs. 6, 6a with pi. 41, fig. 4) ; appearance of colonies in thin-sown gelatin plates; and, finally, behavior in the plant. TREATMENT. Fields on which this disease has appeared should be planted for some years to crops which are not subject to it. Inasmuch as the organism appears to be a wound-parasite often disseminated by insects, etc., prompt attention should be given to the destruction of these pests. Land subject to nematodes should not be planted to Solanaceae if this bacterial organism also occurs in the soil. When transplanting to such soil is necessary, it should be done early and with great care not to injure the roots. Plants with broken roots should not be set on such land. In the transplanting of large plants many roots are certain to be broken. The prompt removal of diseased plants is also recommended in early stages of the disease. Such a course reduces to a minimum the amount of infectious material sub- ject to insect depredations and liable to contaminate the soil further, a precau- tion which must always be an important matter in all diseases disseminated in this way. It is probable that the dis- ease is sometimes spread from field to field in "seed" potatoes. For this rea- son potatoes designed for planting should be sorted over several times at intervals of some weeks with the greatest care, all suspicious tubers being rejected. There should also be a final inspection at planting time. It would be still better to secure tubers for plant- ing from fields known to be free from this disease. The tubers from infected localities should be cut across the stem- end at planting time and a further re- jection made of all suspicious ones. The sound ones should now be exposed before planting to dry air for a day or two, i. WFr ' #•" ¥ ■ " • • • -ij^Hk^^B - lM * '■•„.'-* ■ ; • y - • •■•• v • ■'• "*i #-JS ■ > • • • >'' m &£. * ■■■*&*■•■'■ <^Pfl Agk* ?z3f& Fig. 113/ 202 BACTERIA IN RELATION TO PLANT DISEASES. until rainy weather has set in. The cardinal rules should be: Dig early, dry thoroughly, store in a cool, dry place after removing all suspicious-looking tubers, and use as soon as pos- sible. The crop can not rot from this disease if stored at 8° C. (46+°F.) or under. Moist land should be underdrained or planted to other crops. Tomatoes and other plants subject to the disease should either be germinated where they are to stand or else transplanted quite young and with the greatest care to avoid crushing or breaking the roots. Seedlings having the galls of eel-worms on their roots should be rejected ; also over-grown seedlings, the latter because their roots are likely to be broken in transplanting. To recapitulate : Inspect tubers at the stem-end by cutting some days before planting and reject all that show any trace of this disease. If possible procure seed tubers from localities where this disease does not occur. Plant on land not infected by this organism. If infected land must be used, select that not subject to root-nematodes, and in transplanting seedlings to such land do it early in their growth, and break the roots as little as possible. Practice rotation, but not of one susceptible species after another. Drain the moister portions of the field. Burn over or steam the soil selected for a seed bed. Potatoes grown on infected land should be sold in the summer or autumn, and always stored in a cool dry place. Destroy insect enemies. Be on the lookout for resistant varieties. Read what is said under "Wilt-Diseases of Tobacco" {p. 238). PECUNIARY LOSSES. This disease has destroyed a great many fields of tomatoes and potatoes in the South, and has put an end to commercial tomato-growing in certain sections, e. g., southern Missis- sippi, southern Alabama, and parts of Florida. The following letter, dated May 25, 1905, from a tomato grower in North Florida, is like many others received by the U. S. Department of Agriculture from various parts of the South in recent years: Under separate cover I send you a diseased tomato plant. Out of a crop of 30,000 plants I have lost fully one-third, and many more seem to be becoming affected. The first thing noticeable is the tender tips of the plant wilting, but nothing can be seen outwardly or even by cutting into the plant at the top, but by cutting into the roots, or stem, for a short distance above the ground, the wood immediately under the bark and the interior of the roots are found to be discolored and bad. The disease has spread with great rapidity during the past few weeks and is still increasing, and it seems about to wipe out the entire crop. It is not confined to any one field and is equally distributed on soils of widely different character, some of it high, light, and some of it low, black soil. Also, the disease is equally distributed on fields where tomatoes were never grown till this year. Another man writes as follows under date of May 16, 1903, respecting this disease: Our truck farmers around this section, Tyler, Texas, who have hundreds of acres of tomatoes, are complaining that the blight is destroying some of their fields, thus entailing a loss of thousands of dollars. One planter at Norfolk, Virginia, lost 3,000 barrels of potatoes by this rot in 1908. Additional statements respecting losses may be found in the chapter dealing with the history of this disease. If Bad. solanacearum also plays any considerable part in the frequent and widespread outbreaks of potato-rot in the northern United States, as now seems likely, but has not been proved, then the annual losses can be reckoned only by hundreds of thousands of dollars. The northern distribution of this organism is unknown. The subject of potato-rots is still surrounded with a good deal of uncertainty. The fungus Phytophthora infestans undoubtedly causes great losses in the cool, moist regions of Europe and occasionally in the more northern parts of this country, where the writer has seen BROWN ROT OF SOLANACEAE. 203 the foliage of whole fields destroyed in a week, but there is no basis for the assumption that this is the only or the principal cause of the rot of potatoes either in this country or abroad. Species of the fungus form-genus Fusarium also cause, in the United States as well as in Europe, serious diseases of potatoes, one of which begins in the field as a vascular disease of the stems and continues in cellars as a dry rot of the tubers.* The great bulk, how- ever, of the soft rot of potatoes, in this country and also in Europe, is due to bacterial organisms, either acting independently, which is often the case, or following Phytophthora and Fusarium. Several are known to induce decay and very likely a dozen or more dis- tinct kinds of schizomycetes will be found capable of destroying the tubers in wet seasons when the lenticels open in the soil. When this happens all sorts of soil bacteria gain an easy entrance to the tubers under conditions likely to be very favorable to their destruction. This attack of the tubers in a badly aerated, wet soil, with the lenticels wide open, is a very different affair from the active development in the parts above ground, and under normal conditions, which is manifested by Bacterium solanacearum, or by Bacillus phytophthorus, which causes the only other bacterial decay of potatoes which has thus far been described with any great degree of accuracy. Since this was written, Harrison, in Ontario, has described Bacillus solanisaprus, an organism strongly suggestive of Appel's organism but not absolutely identical (see Basal Stem-rot in vol. IV), and Pethybridge and Murphy in Ireland have also described their Bacillus melanogenes, so that in general it may be said that our knowledge of the bacterial diseases of the potato is much further advanced than it was ten years ago, when the rough draft of this chapter was written. HISTORY. According to Hunger this disease was first recognized by Comes in Italy in 1 882. There is much uncertainty, however, concerning this. Professor Comes undoubtedly observed a serious bacterial disease of tomatoes in the vicinity of Naples in 1884 or earlier and recog- nized it as such; but his descriptions of it, and of the fluid cultures made therefrom, do not enable one to speak with any degree of certainty either concerning the signs of the disease or its exact cause. It may have been due to B. phytophthorus or to some other organism. His name, if retained, should apply to a grape bacterium. The disease formed brown or black cankers on the stems near the earth, the whole plant being finally destroyed. Bark and pith were disorganized and granular; the vessels of the wood with their surrounding cells, but not the medullary rays, were occupied by a yellowish or brownish granular mass. "In the disorganized tissues, " says Comes, "and in the gummy granulations was observed the presence of myriads of microbes, similar to those of Bacterium gummis, found by me always in tissues of woody plants affected by gummosis. " In 1890, before the Society for the Promotion of Agricultural Science, Prof. T. J. Burrill, of the University of Illinois, described very briefly a disease of potatoes which had attracted his attention as something new, and which was probably this disease. The examinations were made on tubers shipped from the south in June, many of which were rotting. Only bacteria were found. Several species of these were isolated, and suc- cessful infections were obtained with the one presumed to be the potential factor in the rot, but these experiments are not described. The organism causing the rot was a termo-like bacterium actively motile, oval to short cylindrical in outline, occurring single, in couples, or rarely in chains of 3 or 4 links, its rods measuring about 0.7 X 1 to 1 .5/1; it was non-sporifer- ous, non-liquefying, and formed non-characteristic zoogloese in the potato. How the organisms gain access to the subcortical tissues without some previous puncture of the skin of the tuber has not been ascertained, but some observations seemed to show that the same bac- terium infests the leaves and culms, causing injury and death to these parts and possibly sometimes reaching the tubers through them. *See Smith and Swingle, Bull. 55, Bureau of Plant Industry, U. S. Dept. Agric, Feb. 16, 1904. 204 BACTERIA IN RELATION TO PLANT DISEASES. In 1891, Dr. Byron D. Halsted reported a widespread and destructive decay of pota- toes in south New Jersey, part of which appeared to be due to bacteria. The potato tubers, it is said, Developed well-formed ulcers, and slices of these quickly turned to almost a coal black, especially near the surface beneath the skin, and over all rapidly grew [oozed?] an almost velvety layer, consist- ing entirely of masses of bacteria. This entry probably belongs here, or if not here, then most likely under Basal Stem-rot. Early in 1892, Dr. Halsted published a bulletin from the Mississippi Experiment Sta- tion giving an account of the Southern tomato-blight, as observed by him in that State in 1 89 1. This disease he ascribed without hesitation to bacteria, basing his conclusions on numerous microscopic examinations. Dr. Halsted noted the wilting of young leaves and growing tips of the stem, the water-soaked appearance of certain tissues, the watery greenish disorganized pith, the brown stain in the vascular system, and under the microscope in freshly gathered material the presence of large numbers of bacteria, while all traces of fungi were absent. Inoculations into growing plants failed. A blight of potatoes was also observed and studied by Dr. Halsted at Ocean Springs, Mississippi. The above-ground signs were similar to those on the tomato. Below ground the stem is more or less darkened, in patches brown, and occasionally almost black. The old "seed" potato is a soft rotten mass, and the few new potatoes usually small and decayed, invariably at the stem end and almost always at the eye, appearing at first "watery" and afterward brown. It is an interesting fact to be stated in passing that the earth adheres very closely to the surface of the decaying portions. It seems evident from the extended study of these diseased potato plants, hundreds being examined, that the rot passes from the main stem to the tubers, and probably comes originally from the "bud." Often a lateral underground branch including its minute potatoes, as large as peas, is entirely softened. Upon making halving sections of the tubers it is seen that the most diseased portion is in a circle which includes the base of each eye. This portion is a soft, grow- ing layer, rich in nitrogenous substances, and furnishes the avenue through which the disease spreads from one eye to another. From this decayed tract a milky juice quickly accumulates upon the sec- tion that is made up very largely of bacteria and contains no other form of fungus [consult this Monograph, vol. I, pi. 24]. Dr. Halsted believes the disease is transmitted to the growing plant from the mother- tuber. Cross-inoculations from potato to tomato in the field failed. The tomato-plants were then full-grown. Direct infections under bell-jars in moist air succeeded. These were made many times over and the checks usually remained sound. For this purpose Dr. Halsted used slices of tomato-fruits and potato-tubers and also short pieces of tomato- stems and potato-stems. The decay was rapid. The milky ooze from the potato rotted tomato, and that from the tomato rotted potato, but under the same conditions melon- tissues were also rotted when inoculated with this material, and Bacillus phytophthorus, Bacillus melonis, or some similar organism must have been present. The disease as observed in the field must, however, I think, have been due to Bacterium solanaccarum. Several attempts were made to infect seedling tomatoes by planting them on soil con- taining diseased material, by soaking seeds in it before planting, and by spraying infected fluid on the plants, but as the results were not conclusive it is not necessary to make further mention of them. According to a note by Director S. M. Tracy prefaced to this paper the losses from this disease in Mississippi had been serious for several years, amounting in some cases to from a quarter to half the plants. In 1890 the losses were widespread and severe. In 1 89 1 the disease appeared at Ocean Springs, Mississippi, in the tomato fields on May 1. (In 1914, Rolfs found it for me on potato shoots at Gainesville, Fla. April 15.) In 1892, in Garden and Forest Dr. Halsted reported occurrence of this southern tomato disease in the north. The material was received from F. L. Stevens, Syracuse, New York, where 3 fields were affected. In the field most seriously injured about 3 per cent of the plants were dead, 8 per cent badly affected, and many more showing traces when the BROWN ROT OF SOLAN ACEAE. 205 inspection was made. This may have been, however, the Grand Rapids disease, due to Aplanobacter michiganense (see p. 161). The same year, in two of the Cornell University Experiment Station bulletins, Bailey and Corbett made a brief mention of a disease which was serious upon some of their tomatoes that year. Specimens were submitted to Dr. Halsted, who thought the disease was "prob- ably identical with the Southern one" (Bull. 45), and "distinct" (Bull. 43). Professor Bailey adds: Apparently the same disease has been reported to me from three localities in this State during the last two seasons and in two cases it had practically ruined the crop. Probably this was the Grand Rapids disease due to Aplanobacter michiganense. In 1893, Professor Burrill published another note on the new potato disease, the most important part of which is here cited : The present note is simply to substantiate as correct what was simply surmised before. The micro-organisms taken from the different portions named of the plant not only appear alike and grow alike in artificial cultures, but the results are identical when cross-inoculated into the living tissues. When the aerial stems are infected, pure artificial cultures are as easy to make as from the tuber itself, and wTe have had abundant opportunity to do it. [These cultures and inoculations are not de- scribed.] It now appears that in most instances at least the tubers in the ground are infected by way of the rhizomes and aerial stems. * * * The leaves are primarily infested and very commonly first at the tips or margins, then are progressively destroyed, becoming first watery, or semi-trans- lucent, then brown, and afterward dry and curled. The supporting petioles and stems are in turn invaded, but initial spots may also be found on these. It has not been ascertained whether or not previous mechanical rupture of the epidermis is necessary, but all observed facts indicate the contrary. In 1895, Dr. Halsted wrote as follows about conditions in New Jersey: Bacteriosis of the tomato prevails in many parts of the State, and is not, as yet, well understood. In 1895-96, the writer isolated the organism causing this disease and studied it many months. He obtained typical infections on tomato and potato plants, inoculating from pure cultures by means of needle-pricks on stems and leaves; he determined by cross- inoculations the identity of the disease as it occurs on potato, tomato, and egg-plant, obtained successful inoculations on various weeds {Datura, etc.), failed to obtain the disease in tobacco or peppers, perhaps because these plants were not growing rapidly, determined experiment- ally that the organism was non-infectious to the cucumber and some other plants, and demonstrated that the disease could be conveyed by insects. As a result of these investi- gations he described the disease and figured it, named the organism causing it, and for the first time described the latter according to well-recognized bacteriological methods. According to Selby, the disease is serious in parts of Ohio. One tomato-grower in Clermont County stated on July 23, 1896, that "in my three acres there is scarcely a sound plant." In 1898, P. H. Rolfs, at Lake City, Florida, published short notes on this disease in two of the Florida Experiment Station bulletins. The disease was known to be present in Florida in several fields. It was inoculated successfully into tomatoes, which were all destroyed. Egg-plants appeared to be more resistant than tomatoes, but suffered quite severely. Peppers seemed to be free from this disease. Some potatoes on the Experiment Station were carefully treated with fungicides, but no good result could be seen from the use of this material. In Bulletin 47, of the Florida Station, it is said: That the disease is not indigenous to Florida I think is evident from the fact that it rarely occurs in new portions of the State until after vegetables have been grown for two or three years. The loss 206 BACTERIA IN RELATION TO PLANT DISEASES. from this disease varies from a few plants to at times every plant in small areas — ofttimes amounting to as much as 80 per cent to 90 per cent of the whole field. There seems to be no special preference for either high or low land. The same year, in Proceedings Florida Horticultural Society, Rolfs made some observa- tions on the distribution of the disease in Florida and again reported successful inoculations. Last summer at the Experiment Station [Lake City] several plots were set aside for studying the effects of this disease. A plot of egg-plants, another plot of tomatoes and a plot of peppers, each one- hundredth of an acre, were planted contiguously and the disease [was] started in the corner of the egg- plant field, this corner being the most remote from the tomatoes and the peppers. The disease mani- ested itself in less than a week on the plants inoculated. For a time it was confined to this corner but before long sporadic cases occurred throughout the plots of egg-plants and tomatoes, until within the course of about three weeks the entire plots of egg-plants and tomatoes were destroyed. In no case was a pepper plant hurt. * * * Soon after the egg-plants and tomatoes had all been destroyed the field was cleared of all these plants and of all the debris left from the plots. The plots were then kept free from vegetation for two weeks, when they were again planted to egg-plants and tomatoes just as before. The disease now appeared in various spots over the entire field. We then took out the plants as soon as they showed signs of the blight and the place occupied by the plant was filled by another one, so that it gave us a full field, all of it being in plants that showed no blight, but neverthe- less the blight continued to appear until cold weather cut short the growth of both egg-plants and tomatoes. * * * During last winter these plots were planted first to radishes, and next to lettuce. None of these plants showed any indications of the disease. As soon as the weather permitted, egg-plants and tomatoes were planted in the same plots as last year. These have now begun to show signs of blight- ing which makes it seem quite probable that the disease can remain over winter in the field. In 1900, F. S. Earle, then of the Experiment Station at Auburn, Alabama, also wrote upon the disease as follows : This serious disease of the tomato has so far only been observed in the southern part of the State. It is very destructive in Mobile and Washington counties. * * * Each succeeding crop suffers worse than the last. * * * As the result of rather wide experience with it in Mississippi I am of the opinion that direct underground infections do take place as suggested by Dr. Smith. * * * Contagion carried by winged insects may well be the means by which the disease first becomes introduced to new fields, but this method of infection can hardly account for the spread of the disease from year to year in somewhat regular concentric circles from such new centers, especially as it usually takes almost every plant in its path. Insect infection would not either account for the facts reported by me in the 6th Annual Report of the Mississippi Station, pp. 53-61, where, in a large tomato field that was under observation, the disease was very largely confined to a narrow strip of wet, seepy land, running diagonally through it, while the drier land on either side was nearly exempt. As the disease is thus so markedly a soil disease, the possibility of soil treatment as a remedy at once suggests itself. Very few experiments are recorded in this direction. In the Mississippi experi- ments mentioned above in one case heavy applications of kainit seemed beneficial and in another case there was apparent benefit from the use of lime. Marked benefit also seemed to follow the use of lime in an experiment at Deer Park, Ala. (see Ala. Bull., 92 : 109). These experiments, however, need confirmation. Sulphuring the soil does not seem to have been tried. Spraying the plants and the surface of the ground with Bordeaux mixture gives no result. In his report for 1907-8 Stevens, of the North Carolina Experiment Station, stated that the wilt disease of tomatoes due to Bad. solanacearum was widely prevalent in North Caro- lina and constantly spreading into new territory. The newer Dutch East Indian studies are reported under Wilt-Diseases of Tobacco (pp. 224, 244). APPENDIX. In conformity to the writer's custom of keeping things separate until it has been defi- nitely established that they are identical, the following diseases are put by themselves, although it is not unlikely that some of them are due to Bacterium solanacearum. There is the more reason for suspending judgment in some of these cases, because in recent years through the labors of Dr. Appel, in Berlin, we have come to recognize a widespread disease of potatoes similar in some respects to the one described by the writer, as due to an entirely different organism (see Basal stem-rot of potato, vol. IV), and also because a full description of Bad. solanacearum has not been available. [Recently, I have ceased to have doubt concerning identity of the American and Dutch East Indian Disease. See Wilt-Diseases of Tobacco.] THE NEW ZEALAND DISEASE. Kirk is authority for the statement that Bad. solanacearum causes a disease of potatoes in New Zealand. It is not widespread. THE AUSTRALIAN DISEASE. In 1894, the entomologist, Mr. Tryon, reported the appearance in Queensland of a new and destructive disease of potatoes and tomatoes, ascribed to a bacterial parasite which fills the vascular system, wilts the plant, and destroys the tubers. The leaflet, which is only a brief excerpt from an unpublished MS. report of Mr. Tryon to the Department of Agriculture at Brisbane, mentions no inoculation experiments and contains no description of the organism. The reader is given no information concerning it, other than what may be inferred from the following statements: "A small, living microbe, having an average length of less than one ten-thousandth of an inch, resembling in appear- ance the bacillus of chicken cholera and other organisms." (The italicizing is mine.) In the tubers the disease first appears as "an indistinct, translucent line running parallel to the outer margin" of the tuber when cut across. "They soon, however, commence to rot, decay starting in the more superficial portions of the tissue, and the potato substance is eventually converted into an offensive, odorous, tenacious, whitish slime." (Italics mine.) This leaflet deals chiefly with suggestions for treatment and does not materially help the pathologist or bacteriologist. In June 1895, Mr. Tryon stated that he had had an opportunity of reinvestigating the new potato disease and that the microbes are "scarcely distinguishable from those which are met with in diseased sugar-cane. " "They are found clogging up the vessels of stems, roots and rhizomes, and, in the initial stages of the disease, nowhere else." The disease here referred to is Cobb's gumming of sugar-cane, due to a honey-yellow bacterial organism. Without further description the organism is therefore designated Bacillus vascularum solani. In 1895, Helms reported on several potato diseases occurring in the Clarence River dis- trict in New South Wales, in south latitude 290 to 300. The paper is agricultural rather than bacteriological. Judging from the plates, one of these diseases (No. 1) is like Prillieux's potato disease ascribed to Bacillus caulivorus, and another (No. 3, p. 328) is possibly like that due to Bad. solanacearum. No cultures or inoculations are mentioned, and positive or even presumptive identification from the figure is impossible. If it were infected with Bad. solanacearum, the tuber in longitudinal section might be expected to show decay at the stem end, but it is not so represented. In 1899, Tryon identified his Australian disease as that described by me and intimates that his name, Bacillus vascularum solani, should have been used in my bulletin on "The brown rot of tomatoes, egg-plants, and potatoes," published in 1896. I would have used 207 208 BACTERIA IN RELATION TO PLANT DISEASES. Mr. Tryon's name, or some portion of it, had I at that time had any means of knowing what he meant by it. This I did not know, and do not yet know. His organism certainly can not be like three very different organisms. It would appear that Mr. Tryon had written a considerable manuscript on this disease and had submitted it to the Department of Agri- culture of Queensland, but that unfortunately they allowed it to remain unpublished. He now makes citations from this unpublished paper, but these are not sufficient to clear away the uncertainties. For all we know from anything he has published, this Australian disease may be due to Bacillus phytophthorus, or to some undescribed species. Mr. Tryon has never published a proper (exact) description either of the disease or of the organism causing it. Certain statements of his do, indeed, make the reader think of the brown rot, but there are others which certainly can not be applied to Bact. sola- naccarum, e. g., the "sticky, tenacious" slime which is said to choke up the vessels; the "froth-like" viscid exudate from the eyes of the tuber; the "foetid odor," the opening of the lenticels; the gradual decay of the stems from the base upward ; the frequent destruction of the organism by the temperature of the Queensland summers, etc. Mr. Tryon is either describing mixed infections, or else a different disease. Why not "Schwarzbeinigkeit" due to Bacillus phytophthorus? We shall never know the specific cause of this Australian potato disease until some bacteriologist takes hold of the problem, isolates and describes the organism in ways recognized as proper, and demonstrates his ability to reproduce the disease with one particular organism by means of pure- culture inoculations. A few words as to Mr. Tryon's name. In my judgment the name Bacillus vascularum solani is unusable because it can not be attached, by means of anything he has yet published, to any particular organism. It is a trinomial, it was published with- out description, without isolation of the organism in pure culture, without proof that the bacteria actually under observation and named by him had anything to do with the disease, with- out proof that the disease itself was actually due to bacteria, and finally without careful description of the disease, e. g., such descriptions as would have enabled anyone to identify it with the North American disease. His name, therefore, for reasons sufficiently set forth in volume I of this monograph, can be regarded only as a nomen nudum. Professor McAlpine, of Melbourne, has sent me a photograph (fig. 114) of a potato- tuber decaying at the eyes, this disease being known in Australia as "sore eye" and "spewey eye." This disease is associated with bacteria and probably due thereto. Some years it causes great losses, it is said. Perhaps it is the disease observed by Tryon. Living material sent to me was too badly decayed when received to make anything out of it. It appeared to be a brown bacterial rot (only a little Fusarium was present), and the vicinity of the vascular ring was stained darker than any other part. Fig. 114.* *Fig. 114. — Potato tuber, showing the bacterial "sore eye" or "spewey eye" of Australia. From a photograph sent by Prof. D. McAlpine, of Melbourne. This is perhaps Tryon's disease. There is a slimy ooze from the eyes and the earth usually sticks to them. BROWN ROT OP SOLANACEAE. 209 THE DUTCH EAST INDIAN DISEASE. A disease of tomatoes prevalent in Western Java and rapidly fatal was identified by Dr. F. W. T. Hunger of Buitenzorg in 1901 as due to B. solanacearum Smith. The follow- ing observations are from Hunger's paper (see Bibliography) : This disease has not been so prevalent as in the United States, but nevertheless whole plantations have been destroyed by it in Java. The course of the disease is rapid. Hunger experimented with diseased and healthy plants, exposing them to the sun with- out water and then watering them. The diseased plants recovered slowly from the wilted condition, and after some days not at all. The undiseased recovered promptly on watering. In two instances he observed buds out of place, namely, on the leaves, and he is inclined to think that these signs were due to the disease. The adventive root-formation occurs so regularly that there can be no doubt as to this being a sign of the disease. The roots develop regularly on the stem and they may appear also on the leaf-stalk. The vascular ring of the stem was stained brown and bacteria were abundant in the vessels. Whenever a tomato plant showed signs of the disease above ground he always found the root-system diseased. These roots were more or less browned, and when well along in the disease were blackish and soft, and filled with bacteria. In early stages the browning was confined to the xylem wedges of the root. Tissues put into alcohol of various strengths gave the result described by Smith, viz, the bacteria diffused out of the vessels regularly in 75 per cent alcohol, and also to a less extent in 90 per cent, but were hindered in their diffusion by absolute alcohol. Hunger found that by adding 1 per cent nitric acid to the 75 per cent alcohol he obtained a solution in which diseased tissues behaved as in absolute alcohol. He found the pith of diseased plants greatly changed, i. c, from white to spotted gray, with formation of bacterial cavities. The whole pith of diseased stems may disappear, the remnants being found on the inner edges of the vascular ring. Groups of tetrahedral crystals, the so-called crystal-sand, are stated to be much more common in diseased than in healthy plants. Hairs are absent from the vicinity of those parts of stems which are sending out adventive roots [the writer has not observed this in the North American disease]. These roots spring from the pericycle. Hunger believes that the formation of tyloses which he observed abundantly in the vessels of diseased plants is induced by the presence of the bacteria. These tyloses are always filled with bacteria. They soften the walls of the vessels in places and then the tyloses are formed; the evidence adduced in proof of this statement is not conclusive, but nevertheless Hunger is probably right (see similar observations by the writer on mulberry shoots attacked by Bad. mori, vol. II, fig. 30, of this monograph). Hunger also notes that in Tryon's paper there is not the least description of his Bacillus vascularum solani. He says: The bacteria found by me as cause of this tomato disease agree in every particular with Bad. solanacearum Smith, so that I hold the two for identical. Hunger believes that he has determined by inoculation with the tomato organism the occurrence of the same disease on Nicotiana tabacum and Capsicum annuwm. He says that he has repeatedly obtained the same signs of disease in these plants as in sick tomatoes. He inoculated it into tobacco, using cultures from tomatoes, and obtained the slime-disease (see Wilt Diseases of Tobacco, p. 222). His inoculation experiments were as follows: Tomato seeds were germinated on filter-paper and the young plants then brought into water- cultures, which were infected with pure cultures of Bad. solanacearum. The young plant grew as well as the check-plant, and a subsequent examination showed that there were no bacteria in its interior. In a similar experiment, varied only by the fact that the root-system was wounded in various ways, viz, by needle-pricks and by teazing out the end of some of the young roots, the plants became infected after a week and the bacteria were demonstrated in the vascular bundles. In one of the plants the bacteria were found in the vessels of the leaf-stalks of two of nearly the youngest leaves. Tyloses had not yet formed in these vessels. 2IO BACTERIA IN RELATION TO PLANT DISEASES. In another series of experiments the young plant was placed in the earth and when growing rapidly was watered daily with water containing a pure culture of Bad. solanacearum. No infection took place. In a similar experiment, varied only in that the roots were purposely injured, the plants became infected after 1 1 days with typical signs and a microscopic examination showed the presence of the bacteria in the tissues, vessels included. In a third experiment a number of leaves of two tomato-plants were plunged into infected water for some days to see if the bacteria would enter through the water-pores. After 3 days the leaves were investigated microscopically. They were not infected and the writer draws the conclusion that they can not be infected in this way, but this seems not strictly warranted, because of an additional state- ment: "Certainly I found rather commonly Bacillus solanacearum in the large, intercellular spaces under the water-pores, but not at all in the corresponding water-vessels." A similar result would not be uncommon at the end of 3 days in case of the cabbage infected through the water-pores by Bacterium campestre, but a like conclusion would be erroneous, and it seems not unreasonable to suppose in this case that after a longer period had elapsed, say 9 days or 1 2 days, Dr. Hunger might have obtained different results and come to an entirely different conclusion, since in the cabbage after that time the bacteria would have entered the vascular system and begun to move downward in the veins of the leaf (see this monograph, vol. II, p. 308). A plunge-experiment similar to the preceding but varied by the fact that the leaves were bruised and wounded more or less before being put into the infected water gave positive results. After 5 days a distinct infection was observed, and a microscopic examination showed the presence of the bacteria even in the stem of the plant, these having passed down through the leaves from the wounded places. In one specimen there was already the beginning of adventive root-formation on the stem. Hunger also smeared portions of leaves and stems with pure cultures of Bad. solanacearum, and placed these plants under large bell-glasses with water, so that the atmosphere was always moist. No infections were obtained; the time, however, was rather short (8 days). The conclusions drawn are that sound tomatoes are not attacked by Bad. solanacearum, and that infections do not take place through natural openings, such as water-pores. According to Hunger, the greatest care should be exercised in planting that the roots be not broken or crushed, and to this end the plants should be removed from the bed and put into the field early, that is, before they have made long roots, which must necessarily be broken in transplanting. He believes that crushing the roots in planting is a common cause of infection. He cites a case in point at Batavia. The disease occurred seriously in the field, and on examination and inquiry he found that the field had been planted not in the ordinary careful manner by an experienced gardener, but that the planting was delayed beyond the proper time and then performed hurriedly. The plants were too large when transplanted ; therefore many roots must have been broken, and they were set out by the gardener with help of two house servants, the coachman, and water-carrier. Hunger often found the root-system attacked by a species of Phytophtlwra, which he says plainly made a passage-way for the bacteria. He is inclined to think that an imper- fectly drained soil is very favorable to the development of this disease. Of this I, too, think there can be no doubt. He made some experiments to determine the effect of animal and plant parasites on the outbreak of the disease. In his first experiment, which consisted of pot-cultures in disinfected earth, the surface of the earth was infected with Phytophtlwra nicotianae from tobacco. The plant was then watered with water which had been infected with a pure culture of Bad. solanacearum. After 7 days the Phytoph- tlwra had overrun the entire surface of the earth and had begun to attack the lower end of the stem. After 26 days the plant developed the bacterial disease, and the infection plainly originated from the lower end of the stem. In a second set of experiments, made in the same way in disinfected earth, a mole-cricket was buried in the soil of each pot, and the plants were regularly watered with infected water. Ten of the 1 1 plants were soon destroyed outright by the insects, the young stem being bitten off underground. One plant remained normal up to the sixteenth day, when it showed the first signs of the bacterial dis- ease; the following day it died. Upon investigation the mole-cricket was found dead in the soil, but the tap-root had been bitten by it, and it is probable that the infection took place through this wound. In a third series of experiments, in sterilized earth, the plants were infected with eel-worms (Heterodera radicicola), by mixing with the earth small pieces of roots which had been attacked by BROWN ROT OF SOLANACEAE. 211 them but which were free from Bad. solanacearum. The plants were watered with ordinary water; they grew well, blossomed, and bore for the most part small fruits. The disease did not develop. In another experiment, identical with the preceding, except that the plant was watered with water containing a pure culture of Bad. solanacearum, the disease developed after 27 days and the adventive root-formation was already under way. The plant died just one month after the' beginning of the experiment, and both a macroscopic and a microscopic examination demonstrated the typical phenomena of the bacterial disease. As already stated, Dr. Hunger believes the disease is induced mostly through the inter- vention of Heterodera. He thinks the bacteria are carried up the vessels by the water- movement, but he does not bring sufficient proof in support of this latter statement . He states that infection is not limited to the vessels, but may occur throughout the entire vascular bundle, and also in the phloem and the parenchyma. The sieve-tubes may be plugged by the bacteria. He thinks the adventive roots are due to the action of the bacteria on the phloem. By placing wet moss around a stem showing these adventive roots, he induced them to develop further and on removing this part of the plant and setting it out in the earth he obtained a new plant, which grew well for some time, but afterwards developed the disease. He obtained his pure cultures used for infection from Petri-dish poured plates on agar. This agar had the following composition: water 1 liter; agar-agar 20 grams; glucose 10 grams; peptone 10 grams; magnesium sulphate 0.5 gram; potassium phosphate, 0.5 gram. Dr. Hunger states in a number of places that the color of the surface colonies on this medium was "leverkleurige," that is, liver-colored. They were small and round, and spread slowly in a concentric manner. In the writer's experiments colonies of Bad. solanacearum were not brown (liver color) until after a week or two. He states that the least amount of acid in the agar exerts an injurious influence on the bacteria, but that the addition of alkali favors their growth, for example, the addition of sodium phosphate. He found the organism sensitive to sunlight, development being abso- lutely hindered by it, and a long exposure destroying it. He states that Bad. solanacearum is early contaminated in the plant by the presence of other organisms. These impurities consist of saprophytic bacteria, and especially a yeast. In size and shape this yeast, which he seems to have found quite common, corresponds exactly to van Breda de Haan's statement respecting his coccus from tobacco plants, viz, round and 8/x in diameter. We may assume either that this was what van Breda de Haan saw in his diseased tobacco, or that his measurement is erroneous. If for the 0.008 mm. of the text we should read 0.0008 mm., then it is easy to understand how the short rod might have been taken for a coccus, especially with objectives of only medium magnification. On plate-cultures Hunger found he could distinguish colonies of this yeast readily from Bad. solanacearum by the fact that the latter appeared bluish-green by transmitted light. There is some discussion of methods of prevention, but no certain ones are pointed out other than avoidance of planting on diseased land. It is suggested that possibly some resistant species of this family may be found upon which the tomato could be grafted. THE MALAY STATES DISEASE. In 1910, Keith Bancroft, Assistant Mycologist, reported on a bacterial disease of potato and tomato occurring in the federated Malay States as follows : Specimens of potato plants have been recently received from Taiping (Perak) exhibiting the well- known " bacteriosis " caused by Bacillus solanacearum E. Smith. The same disease has been known for two or three years to occur in Kuala Lumpur on tomato plants cultivated in vegetable gardens. These two crops are but little cultivated in the Malay States, so that the disease has little more than a passing interest. It is, however, interesting to record its occurrence in this country, and a brief note will be given of the symptoms of the disease and of some methods of treatment which are likely to prove effective. 212 BACTERIA IN RELATION TO PLANT DISEASES. The disease is well-known in the United States of America where it has been carefully studied by Dr. E. Smith on potato, tomato, and egg-plant (Solarium melongena). It has also been recorded on potato in Scotland, in the north of England, and in Mysore, and recently in Ceylon. The symptoms of the disease appear to be constant in the different countries in which it is known to occur, and they may be briefly reviewed thus: Attacked plants show a wilting and shriveling of the leaves; soon afterwards brown streaks appear on the stem and spread downwards to the parts underground. Finally, the whole stem rots. A browning of the affected parts is very characteristic of the presence of the bacterium ; the browning will be found to be first evident in the vascular ring on examining the cut surface of an affected stem. The vascular bundles are first affected, the supply of water to the leaves is checked and the conse- quent wilting and shriveling makes its appearance. The disease in the potato tubers is very well-marked by the presence of a circular, usually incom- plete, brown ring which is situated at some little distance from the surface and which corresponds in position with the vascular ring of the tuber. This ring darkens and expands until the whole of the starch-producing area of the tuber is affected. The tuber decays and the bacteria are liberated in the soil where they can infect other healthy tubers. Dr. E. Smith considers that the rapid spread of an epidemic is due to insects of different kinds feeding alternately on healthy and infected plants. Under these circumstances it is advisable to spray the plants with an insecticide. A vegetal wash, e. g., tobacco wash, should yield good results. This may be made by infusing half a pound of tobacco leaf in water for about six hours, straining off and pressing the tobacco and again infusing ; the extract is added to a solution of half a pound of soft soap in water, the whole being made up to ten gallons. The wash is applied by means of a spray. Any coarse tobacco leaf may be used. Infected plants should be taken up along with their roots and burnt. They must on no account be thrown on to a manure heap. In the case of the potato plant it is advantageous to lift the crop early when the disease has made its appearance ; in this way many of the tubers may be saved. Plants belonging to the potato family should not be planted on soil which has yielded infected plants for at least two years, since the bacterium probably attacks a large number of members of that family (Solanaceae). THE MYSORE RING DISEASE. In 1909, Leslie C. Coleman published a short paper on a "Ring disease" of potatoes (Solanum tuberosum) , of which the following is an abstract: The Bangadi or ring disease of potatoes has been reported from various localities in India. It was first noticed in Bombay Presidency in the vicinity of Poona, and later in other parts of the Presi- dency. In Mysore it is found practically in every locality where potatoes are grown. The extent of its ravages is very much greater than is generally supposed. The disease is readily to be recognized by the sudden wilting of the potato plants in an affected field. Usually one or two leaves first show the wilting, but within a few days the whole plant wilts down and dries up. If the tubers of an affected plant are cut through, some of them will almost cer- tainly show a brown ring a short distance in from the surface. This ring will be found, usually at least, to begin at the point where the tuber is attached to the underground stalk which bears it and to spread from that point around to the other end of the potato. In its first stages, therefore, the ring is not complete, but is rather simply indicated by a more or less distinct brownish streak in the neighbor- hood of the point of attachment of the tuber. If a diseased potato be cut and squeezed slightly, a series of small creamy-white slimy drops are to be seen exuding along the course of the brown ring, and if these drops are examined they are found to contain myriads of bacteria shaped like small and very short rods. If a very thin slice of such a potato is examined microscopically these tiny rods are found chiefly in the vessels of the tuber which they plug quite full. If the stem of a diseased plant is exam- ined it is found also to be plugged with masses of the bacteria. This plugging up of the vessels from the roots up to the leaves causes the wilting of the plant. The questions investigated in regard to this disease are: " (1) Are these minute bacteria the real cause of the disease? (2) If so, how do they get into the potato plants, and how do they increase to such numbers as to block up the vessels?" The reasons given for answering the first question affirmatively are as follows: " (1) They are to be found in large numbers in the vessels of every potato plant suffering from the disease. (2) In the earlier stages of the disease they are the only organisms to be found in the vessels. Later, when the whole plant or tuber begins to rot, other bacteria, as well as certain kinds of higher fungi, are to be found associated with them. (3) It has been proved by direct experiments that these bacteria are BROWN ROT OF SOLANACEAE. 213 capable of causing the disease. These experiments were carried out as follows: The bacteria were removed from diseased potatoes and grown in the laboratory in such a way that they could be sepa- rated from all other forms of bacteria. When by careful testing it was found that we had masses of these bacteria growing quite free from any other form, such masses were used to give the disease to healthy potato plants. Small pricks were made in the leaves and leaf-stalks of healthy plants with a fine needle which had been held in a flame to kill off all bacteria that might be on it. Then masses of these bacteria were stroked over these pricks so that the bacteria could enter into the substance of the plant. After a few days it was noticed that the leaves and plants infected began to wilt and in the course of 2 to 4 weeks the plants were quite dead. Moreover, when the stems of these plants were cut across and examined under the microscope these same bacteria were found inside the vessels. Some of the tubers attached to these plants also showed the characteristic brown ring. We have thus shown clearly that these bacteria are the real cause of the ring disease of potatoes. " Dr. Coleman states that there are at least two common ways of infection as determined by his experiments: (1) Through the planting of seed-tubers which already have the dis- ease, and (2) the presence of these bacteria in the soil of the plot where the potatoes are planted. An experiment was also conducted which showed that potatoes harvested from a plot where the disease was present, even though they did not show the brown ring, would produce the disease if planted in soil unquestionably free from the bacteria — the young plants wilting throughout the plot soon after coming up. A similar, if not identical, disease is found attacking the potato in America. Dr. Erwin F. Smith of the Agricultural Department, Washington, has studied it thoroughly and has come to the conclu- sion that insects have a great deal to do with the spread of the disease. In all my experiments I have kept a very careful watch for any instance of the spread of the dis- ease by insects. * * * On the whole, however, insect attack has been insignificant, and although I have examined very many wilted plants indeed, I have never yet seen one in which the infection could, with any likelihood, be traced to the gnawing of insects. Dr. Coleman states that because in case of the above-mentioned potato -disease in Amer- ica Dr. Erwin F. Smith has found that it also attacks egg-plants (brinjals) and tomatoes, he instituted experiments to ascertain whether tomatoes and brinjals can be given the disease. The results already obtained show that both these plants can be infected with it by means of inoculation with pure growths of bacteria, or with bacteria taken direct from diseased potato-tubers. In one successful experiment with tomato -plants, 8 comparatively young plants were inoculated with pure growths of the bacteria ; all the 8 contracted the dis- ease and within 3 weeks were practically dead. Three larger plants (bearing fruit) inocu- lated at the same time with the same growths of bacteria remained practically unaffected and could not after 3 weeks be distinguished from an uninoculated plant of the same age (see last paragraph on p. 179). Similar results have been obtained with brinjal plants — the younger plants contracting the disease, whereas larger plants resisted it. From the results obtained the following directions should be strictly adhered to, if possible : (1) Only such potatoes should be used for seed as have been grown on a plot which has been quite free from the disease ; (2) where the seed is cut before planting, all pieces that have the least trace of brown spots or a brown ring should be discarded ; (3) if the disease has been prevalent on a certain piece of ground, potatoes should not be again planted there for at least one year. Probably two years would be better, but a definite statement in this con- nection can not be made until the experiments in progress have been completed. Also, brinjals and tomatoes should not be planted on such infected ground. Spraying the plants with a fungicidal or insecticidal spray is a waste of both time and money, as the disease is not transmitted, at least to any appreciable extent, by insects; nor can it be carried through the air from plant to plant. Another and a very effective means of checking the disease would be by the introduction of a disease-resistant variety of potato. Experiments in this direction are in progress. The description of the organism is withheld by Dr. Coleman for a separate paper. 214 BACTERIA IN RELATION TO PLANT DISEASES. THE CEYLON DISEASE. In 1909, Petch reported very briefly on a wilt disease of tomato plants occurring in Cey- lon, during a period of wet weather, and first visible when the plants were in bearing. The wilted leaves decayed and fell off, and finally the stems decayed also. There is very little evidence of disease in the stem when the leaves begin to droop, but if it is cut across near the base the woody part immediately round the pith is found to be brownish, and minute white or yellowish drops of liquid ooze out from this region. These drops consist of enormous numbers of bacteria, and if sec- tions of the stem are examined under a microscope, they are found to issue from the vessels of the wood which are completely filled by them. * * * The bacterium is, as far as can be ascertained without elaborate bacteriological investigation, Bacillus solanacearum. THE AFRICAN DISEASE. The writer's knowledge of the African potato-disease here referred to was obtained from a conversation with Mr. George Milton Odium, a student of scientific agriculture, who for some time had charge of plantations for English capitalists at Umtali, in Rhodesia, where he saw the disease. He described it to me as a bacterial rot, beginning as a black stain in the vascular system; the foliage wilts, the stems shrivel and blacken, or have black stripes in them, and the tubers decay. He has known whole fields to be destroyed quite suddenly. The microscope always shows great numbers of bacteria in the diseased plants. In his opinion the disease is the same as that described by the writer. THE RUSSIAN DISEASE. In 1899, Iwanoff reported the occurrence in 1898, in all of the fields for a distance of 26 miles around St. Petersburg, of a very destructive bacterial disease of potatoes closely resem- bling that due to Smith's Bacillus solanacearum, if not the same. Theorganism present in the stems in the earliest stage of the disease is described as a "small, short, oval-cylindric, actively motile rod of medium size (in general rather variable) 1.5X0.5/1. " Two organisms isolated from the diseased stems in pure culture were not infectious, but direct infections were successful. The tissues were browned, the bacteria passed up and down the stem from the point of inoculation by way of the vessels, the pith was badly disorganized, the starch was not attacked, young plants were killed much sooner than old woody ones, the juice of diseased plants was alkaline, and in the pith and the bark parenchyma there was an increased produc- tion of calcium oxalate. The tubers were small and few, but were not observed to be dis- eased. The disease spread with great rapidity. A field which contained only scattering cases on August 8, showed all plants diseased on August 15, the only green things in the field, and in many similar fields, being weeds. Possibly this should be referred to B. phytophthorus. THE FRENCH DISEASE. In 1901, in two publications, Delacroix stated that Smith's disease of the potato was prevalent in France. I translate as follows : The bacteria are found far up in the stem and in parts apparently still living. They are espe- cially numerous in the vessels. This bacterium seems to me not different from the Bacillus sola- nacearum of Erwin F. Smith. Its cultural characters are the same; the signs of the disease observed in the United States on the potato, tomato, and egg-plant are exactly those I have myself seen. Afterward Delacroix attributed the disease to a green fluorescent bacillus, B. solanincola Delacr., which is different from Bad. solanacearum, but non-pathogenic in my hands. Here again, it is uncertain whether we have to do withBacillus phytophthorus, Bacterium solanacearum, or some third organism. The writer obtained a culture of B. solanincola from Prof. L- R- Jones, to whom it was given in Paris by Delacroix, but either it never possessed BROWN ROT OR SOLANACEAE. 215 any pathogenic properties, which is quite probable, or else had lost them by cultivation. Nothing Delacroix afterward published on this disease served to clear up the question.* In 1902 Gueraud de Laharpe published a note on a French potato-disease said to be due to Bacillus solanincola. He makes the following observations on the subject: For several years the culture of the potato in France has decreased more and more, so that now in some departments it is insignificant or has been entirely abandoned, owing to the extremely difficult conditions. The year 1901 was particularly disastrous. The Central and Western departments suffered most. Within a few days toward the close of July whole fields were destroyed, leaving of the crop only faded leaves and blasted stalks, while the tubers, when formed at all, were no larger than walnuts. The disease appears in general about July 15 upon Jaune de Holland, an early variety, and a little later on late varieties, such as Richter's Imperator. The early varieties suffer most. It is encountered especially in soils which have borne potatoes repeatedly, but it is also found on newly cleared forest or vineyard soil, even on soils which have not borne potatoes within the memory of man. Though it seems to prefer calcareous soil, it causes much injury also in clayey and sandy regions. Tyloses are said to be formed in the stems. Delacroix advises soaking sound seed-tubers for an hour and a half in 1 part of commercial formalin diluted with 20 parts of water. Delacroix's most susceptible varieties are 40-day Halle and Long Yellow Holland. THE ITALIAN DISEASE. In south Italy, according to Comes, a disease of tomatoes due to his Bacterium gummis is common. The stems rot usually from the base and the tissues are rilled with bacteria. Bad. gummis is now regarded as insufficiently described and it is not known to what the name was applied, as the organism is said to occur in a variety of plants. This is probably a disease of late spring, summer, and autumn. The writer searched in vain for it in the market-gardens about Naples in the early spring of 1906. A great many fields of potatoes were seen, but there was no trace of this disease. Voglino has also reported a disease from Turin, which he ascribes to Bad. solanacearum. THE GERMAN RING DISEASE. Appel and Kreitz describe a ring-disease, which possibly is caused by Bacterium solana- cearum, the most striking characteristic being a browning or blackening of the vascular bundles. In severe attacks the potatoes do not come up. On the seed-tuber small tubers appear and a great development of shoots, but these are too weak to break the ground and gradually die. When less severe the shoots come up, but are stunted. On subterranean parts are brown cracks which soon become glossy, black spots appear on the leaves, and the plant dies. In the lightest form the affected plants can not be distinguished from sound ones until harvest. Then the leaves wilt and usually show black spots. Such plants *Fortunately, Delacroix left an abundance of material both dry and in alcohol to illustrate his potato disease. This, through the courtesy of Etienne Foex, the present director of the Station of Vegetable Pathology, in Paris, the writer had opportunity to examine critically in the autumn of 19 13, i. e., since the above text was written. Numerous microscopic examinations were made, but no bacteria were seen either plugging vessels or corroding tissues and cer- tainly nowhere any in numbers sufficient to account for the disease. All of the plants were examined, and each in several places. In the stems and roots of those in alcohol (a large jar of material), nothing was found; but on the shriveled leaves there was a great abundance of Phytophthora infeslans. On the dried material evidently collected at another time and place, there was no Phytophthora, but Alternaria solani was found on the brown shriveled leaves, or at least an Alternaria resembling it; and in the xylem vessels in the basal part of some of the stems, a fungus suggesting Fusarium, i. e, a delicate colorless mycelium abstricting colorless elliptical conidia in small numbers. Whatever Delacroix may have first identified as "Brunissure de la pomme de terre," due to his Bacillus solanincola, the museum material which he selected to illustrate it represents only fungous diseases of the potato. fFiG. 1 14a. — Margin of a hanging drop showing Bacillus solanincola Delacroix. From an agar culture given to Jones by Delacroix in August 1904. 2l6 BACTERIA IN RELATION TO PLANT DISEASES. bear almost a normal number of tubers, but are a menace to the crop, as the disease spreads from the small spots on the harvested tubers. There can be no injury unless the bacteria come into contact with the vascular bundles. Infection often occurs by abnormal growth of the young stem, resulting in cracks reaching even to the pith. Also through wounds made by animals or otherwise the bacteria easily gain access, e. g., the disease is more prevalent when cut seed-tubers are used. If such are employed they should be covered with moist sacks for two days that the formation of a cork-layer may occur. We made agar-poured plates in the laboratory in Washington from the browned vascular bundles of a potato sent from Munchen, Germany, by Mr. W. A. Orton in 191 1, as affected by the bacterial ring-disease, but obtained only a white endospore-bearing motile schizomycete, non-infectious to growing potato stems. In Eriksson's book (Die Pilzkrankheiten der Eandwirtschaftliehen Kulturpflanzen. Aus der Swedischen uebersetzt von Dr. A. Y. Grevillius, Kempen a. Rh., Reichenbachsche Verlagsbuchhandlung, Leipzig, 1913, pp. 8-9) the ring disease is attributed both in the text and in the figure-description to Bad. solanacearum. For further knowledge of this disease we must await Appel's full report. THE ENGLISH DISEASE. There are a number of statements by English agricultural writers to the effect that Bacterium solanacearum Smith causes a disease of potatoes in Great Britain. These statements, however, are too indefinite to furnish any reliable basis for judgment. MALKOFF'S DISEASE OF SESAMUM. In 1903 a bacterial disease of sesamum was described by Malkoff, from Sadovo in Bulgaria. Early in August 1902, in a plot in which the plants had been watered and had made more rapid growth than in a check plot, many leaves showed brown spots and dried up quickly. Sections revealed the presence of many bacteria. After 2 to 3 days the disease was widespread and the stems were also affected . These were dark brown to black, shrunken and covered with a slimy exudate which dried on the stem. This ooze was at first gray- white, but soon became dark brown. In a few days the stems became entirely black, bent over, and finally dried out. Sometimes the whole plant is attacked, sometimes a single shoot (figs. 114 b, c). The disease progresses rapidly and in 3 to 4 days all the leaves are attacked and the stem decayed. It appeared on the early Bulgarian sesamum first, but later occurred on the two Asiatic varieties. The disease was confined to plants which had been in blossom for some time. In the unwatered plot no plants were affected until after a rainfall in September. Inoculations of sound leaves and petioles with the juice of the affected plants, both with and without wounds, produced the characteristic signs in 4 to 7 days. In inoculations on plants growing in an unwatered plot, the disease also appeared (in 7 to 24 days) except in case of inoculations on the stem and under side of leaves without wounds. Two kinds of bacteria were isolated — a short rod and a long one. The first formed yellow col- onies, the second white ones, in 24 to 48 hours at ordinary temperature. Both were motile and non-sporiferous. Growth in bouillon, gelatin, and agar was very luxuriant and the gelatin was soon liquefied. Ten per cent sesamum leaves with peptone and gelatin was an excellent medium. Milk was not curdled by either form, but was peptonized after 8 days by the gray form. Inoculations were made with pure cultures and here and there the disease appeared, but it did not progress rapidly as the tissues were then hard. Those on leaves produced the disease in 7 to 8 days. This disease occurred throughout the sesamum-growing district in 1902, but was less widespread that year because of the dry season. In previous years, according to the growers, the entire harvest had often been lost through its ravages. It occurs principally in southern Bulgaria on the Turkish border. The author believes that the disease is dis- tributed on the seed. BROWN ROT OF SOLANACEAE. 217 Fig.114 e.§ Fig. 114/.** (After Malkoff.) *FlG. 114b. — Bacterial disease of sesamum: 1, normal; 2, partly, and 3, wholly diseased. tFic 114c. — 1, normal sesamum; 4, bacterially diseased sesamum. (After Malkoff.) JFig. ii4<2. — Malkoff's disease of sesamum: 1, Pseudomonas sesami Malk.; 2, Bacillus sesami Malk. (After Malkoff.) Magnification of 1, said to be X 900; of 2, X 1000. Kind of stain not stated. §FlG. 1 14c. — Bacterial disease of sesamum from Austria. Cross-section of petiole, showing cavities and occupa- tion of the vessels. From material in Museum of the K. K. Laboratorium fur Pflanzenschutz, Vienna. Section stained with picric anilin blue, followed by carbol fuchsin, torn somewhat in cutting. Slide 952 G 2, middle section of lower row. Drawn by Katherine Bryan. **Fig. 114/. — Austrian sesamum bacteria from a leaf-blade. Stained with carbol fuchsin. Slide 952 A 2, middle row, second section from left. Torn tissues omitted. Drawn by Katherine Bryan. X 1000. 218 BACTERIA IN RELATION TO PLANT DISEASES. In 1906 Malkoff published another short paper on the sesamum disease. The gray, liquefying, polar flagellate Pseudomonas sesami ineasures 2ju X o.gn; the yellow non- liquefying, peritriehiate Bacillus sesami, 1.2/1 X 0.9/j (fig. 114(f). Tne flagella of the gray form were stained with difficulty (Pepler's stain). While the two organisms can infect separately he thinks there is also a symbiosis. Inoculations in pots of sterile soil gave the following results: (1) When seed from sound plants was used unsterilized, or from diseased plants after treatment with formaldehyde, no disease resulted; (2) When either infected soil or untreated seed from diseased plants was used, the disease appeared. This subject is placed here because Honing in Sumatra has found sesamum subject to Bad. solanacearum, and because in a sample of "bacteriosis of sesamum" given me in 1903 by Dr. Kornauth, of Vienna, and believed to be of Austrian origin, the morphology of the abundant bacteria and their location in the tissues suggest Bad. solanacearum (figs. 1140,/). Two bacterial diseases of sesamum are said to have been recognized in India (Bull. Imp. Inst., vol. ix, No. 3, pp. 264-265, London, 191 1), one due to Ps. sesami and the other to Bacillus sesami, but no author is cited, no one is responsible for the statement, and the above is all the information given. LITERATURE. 1884. Combs O. Sulla malattia del pomodoro (Lyco- persicum esculentutn) denominata Pelagra o Bolla nella Provincia di Napoli e Sulle Crit- togame che l'accompagnano. Atti del R. Istit. dTncoraggiamento di Napoli, vol. in. No. 11, 1884. See also LAgricoltura meri- dionale, Napoli, 1884, No. 16, and Ann. della R. Scuola Sup. dAgricoltura in Portici, vol. v, fase. 2, Napoli, 1885, p. 83. Separates also. Possibly this reference belongs here. The reader may also consult Comes, Crittogamia Agraria, vol. I. Naples, 1891, pp. 498 and 513 et seq. In the spring of 1883 this was a common disease in potatoes and tomatoes in the Vesuvian region. It spread rapidly in moist, hot weather. 1890. Burrill, T. J. Preliminary notes upon the rotting of potatoes. Proc. nth Ann. Meeting Soc. Prom. Agr. Sci., 1890, pp. 21-22. 1891. Halsted, Bvron D. Bacteria, nth Ann. Rep. New Jersey State Agric. Expt. Sta., and 3d Ann. Rep. New Jersey Agric. College Expt. Sta. for 1890, Trenton, N. J., 1891, p. 347. 1892. Halsted, Byron D. The southern tomato blight. Miss. Agric. and Mechan. College Expt. Sta. Bull. No. 19, Jan. 1892, 12 pp. Reprinted in 12th Ann. Rep. New Jersey State Agric. Expt. Sta. and 4th Ann Rep. of New Jersey Agric. College Expt. Sta. for 1891, Trenton, 1892, pp. 267-273. 1892. Halsted, Byron D. Southern tomato blight at the North. Garden and Forest, vol. v, Aug. 10, 1892, p. 379. 1892. Bailey, L. H. and Corbett, L. C. The south- ern orfield blight, in "Tomato Notes for 1892." Bull, xlv, Cornell Univ. Expt. Sta., Oct. 1892, pp. 293-295, 1 fig. (of a diseased plant). 1893. Burrill, T. J. An additional note on the rot of potatoes. Proc. Soc. Prom. Agric. Sci., vol. for 1891-92, Columbus, 1893, p. 29. Paper read at Washington in August. 189 1. 1894. Tryon, Henry. A new potato disease. Ann, Rep. Queensland Dept. Agric. for 1893-94. Brisbane, 1894, Govt. Printer, pp. 2 to 4. This and the following possibly belong here. i894(?). Tryon, Henry. Virulent potato disease. Suggestions for stamping out the disease. Brisbane, 1 page. Not seen. Review by Schimper in Zeits. f. Pflanzenkr., Bd. v, 1895, p. 234. 1895. Tryon, Henry. Ann. Rep. Queensland Dept. Agric. for 1894-95. Brisbane, 1895. Part of a paragraph buried on page 14 in "Gumming of Cane." The name Bacillus vasculantm solani occurs here. 1895. Helms, Rich. Report on an investigation into the potato diseases prevalent in the Clarence River district. Agric. Gaz. of NevvSouthWales, vol. vi, pt. 5, May 1895, pp. 316-329, 2 col. pi. 1895. Halsted, Byron D. Bacteriosis of tomato. 15th Ann. Rep. New Jersey State Agric. Expt. Sta. and 7th Ann. Rep. New Jersey Agric. College Expt. Sta. for 1894, Trenton, N. J., 1895, part n, p. 361 (two lines). 1895. Smith, Erwin F. The southern tomato blight. Abstract in Proc. Am. Asso. Adv. Sci., vol. xliv for 1895, Salem, 1896, p. 191. Also a separate. A brief notice appeared in Bot. Gaz., Sept. 1895, p. 409. 1896. Smith, Erwin F. A bacterial disease of the tomato, egg-plant, and Irish potato (Bacillus solanacearum n. sp.). Bull. 12, Div. Veg. Phys. and Path., U. S. Dept. of Agric, 8vo, 28 pp., 2 pi., 1 colored. Issued Dec. 19, 1896. 1897. Sf.lby, A. D. Investigations of plant diseases in forcing-house and garden. V. Tomato diseases. 7. Bacterial tomato blight. Bull. 73, Ohio Agric. Expt. Sta., Wooster, Ohio, Dec. 1896, pp. 242 and 245. Printed at Norwalk, Ohio, 1897. 1897. Smith, Erwin F. On the nature of certain pigments produced by Bacillus solanacearum. Proc. Am. Asso. Adv. Sci., vol. xlvi, for 1897, Salem, 1898, p. 288. Bot. Gazette, vol. xxiv, 1897, p. 192. A brief note. Substance believed to be a humus-compound. 1898. Rolfs, P. H. Bacterial tomato blight (Bai ilhts solanacearum S.). Report of the Biologist and Horticulturist, Expt. Sta. of Fla. Agric. Col., p. 6. No date, but published after June 1898. 1898. Rolfs, P. H. Diseases of the tomato. Bull. 47, Fla. Agric. Expt. Sta., Sept. 1898, pp. 128-132. 1898. Rolfs, P. H. Bacterial tomato blight (Bacillus solanacearum .Smith) in "Injurious insects and diseases of the year." Reprint from Proc. nth Ann. Meeting Florida State Hor. Soc, 1898, pp. 90-93. BROWN ROT OP SOLANACEAE. 219 1899. Tryon, Henry. Potato disease. Queensland Agric. Journ., vol. v, part 2, July 1899. Also a separate, 7 pp. 1899. Iwanoff, K. S. Ueber die KartofTelbakteriosis in der Umgegend St. Petersburgs im Jahre 1898. Zeits. f. Pflanzenkr., Bd. ix, Heft. 3, July 1899, pp. 129-131. Also a separate. 1900. EarlE, F. S. Bacterial wilt (Bacillus solanace- arum E. F. Smith), in Bull. 108, Tomatoes, April 1900, Ala. Agric. Expt. Sta., Auburn, pp. 25-28. 1900. Selby, A. D. Bacterial blight of tomato, egg- plant, and potato, in "A condensed handbook of the diseases of cultivated plants in Ohio." Bull. 121 of Ohio Agric. Expt. Sta., Sept. 1900, p. 56. "It was destructive at Mt. Carmel, near Cincinnati, in 1896." * * * "To date this disease has been less destruc- tive than the leaf -spot. " 1 90 1. Delacroix, Georges. Sur une maladie bac- terienne de la Pomme de terre. Comptes rendus des se. de TAcad. des Sci., Paris, 1901, tome cxxxiii, pp. 417-419, 26 aout. See also the same, Dec. 9, 1901. Also a separate. 1 901. Delacroix, Georges. Sur une nouvelle mala- die de la Pomme de terre en France. Journal de r Agriculture, Paris, Sept. 7, 1901. Also a separate, 7 pp. See also Revue Hort., 1892, p. 94; and Bull, du Min. de lAgric. 1901, No. 5. 1 901. Battanchon, G. La maladie bacillaire des pommes de terre. Le Prog. Agr. et Vit., tome xxxvi, Montpellier, 1901, pp. 318-321. Observations from the field. May perhaps belong here. Disease very destructive in 1901 in Sadne et Loire on granite and sandy land well adapted to potatoes. Bordeaux mixture did not prevent its development. Identified as due to Bad. solanacearum on strength of Delacroix's first statements. 1901. Smith, Erwin F. Entgegnung auf Alfred Fisher's 'Antwort," etc. Centralbl. f. Bakt., 2te Abt., Bd. vn, No. 5-6, pp. 133-138 and 196-197. Also a separate. Tafeln X and XT, and the accompanying text, relate to Bad. solanacearum. The plates are heliotypes from photomicro- graphs by the author. The text is mostly a translation of parts of Bull. 13. 1 901. Hunger, F. W. T. Een Bacterie-Ziekte der Tomaat. Mededeelingen uit's Lands Planten- tuin XL viii, Batavia, 1901, 8vo, pp. iv, 57, 2 pi. 1902. ■ Bacteriosis of potatoes. Bacillus solan- acearum (E. F. Smith). Reported in various parts of England in Jour. Bd. Agr. (London), vol. ix, 1902, pp. 308-310, 1 pi. An anonymous contribution, describing several potato dis- eases, states also that this bacterial disease "has, unfortunately, been recorded from several localities in this country during the present season [1902]." 1902. Gueraud de LaharpE, S. Une Nouvelle Mal- adie de la Pomme de Terre. Journ. dAgr. prat., n.s., tome in, Paris, 1902, pp. 481-482. 1903. MalkoFF, Konstantin. Eine Bakterienkrank- heit auf Sesamum orientale in Bulgarien. Centralb. f. Bakt., 2te Abt., Bd. xi, pp. 333- 336. 4 figs. 1904. Clinton, G. P. Blight [of tomato]. Bacillus so- lanacearum Smith. Report of botanist, form- ing part iv Conn. Agr. Expt. Sta. Rep., 1903. p. 364. Plate xxvii a-c. New Haven, 1904. Reports occurrence at New Haven. The disease was con- fined mostly to four rows running through middle of field. 1904. Voglino, PiERO. Osservazioni sulle principali malattie crittogamiche sviluppatesi nel anno 1904 sulle piante coltivate nella provincia di Torino e regioni vicine. Ann. d. R. Accad, d. Agric. Vol. quarantesimosettimo. Torino, 1904, pp. 343-344. Also a separate. Bad. solanacearum is said to cause a widespread disease of tomatoes in the market gardens about Turin. These bacteria taken from the browned internal parts measure 1.5/1 Xl-2p. The colonies give a brown color on potato and on agar. 1906. Kirk, T. W. Vascular bacterial disease of potatoes. Ann. Rep. New Zealand Dept. Agric, vol. xiv, 1906, p. 352, 3 figs. Kirk figures sections of 3 potato tubers attacked by a vas- cular bacterial disease, which, he says, is due to Bacillus sola- nacearum. The figures are very plain and show disorganiza- tion and dark stain in the vicinity of the vascidar ring. 1906. Malkoff, Konstantin. Weitere Untersuch- ungen iiber die Bakterienkrankheit auf Sesa- mum orientale. Centralb. f. Bakt., 2 Abt., xvi Bd., 1906, pp. 664-666, 4 plates. 1907. Kirk, T. W. Brown-rot of the potato. Ann, Rep. New Zealand Dept. of Agric, vol. xv, 1907, p. 168. This rot occurs in New Zealand in a few localities only. 1907. Appel, Otto, und Kreitz, Wilh. Die Bakter- ienringkrankheit. Mitt. a. d. Kais. Biol. Anst. f. Land- und Forstwirtschaft. Heft 5, Aug. 1907, s. 17-19, mit ein Text rig. 1907. Mayer, Adolf. Die Ringkrankheit oder Eisenfleckigkeit der Kartoffel. Jour, fiir Landwirts., Bd. 55, Heft 4, pp. 301-304, 1 taf. 1909. Stevens, F. L. Bacterial disease of tomatoes (Bad. solanacearum). Notes on plant dis- eases occurring in North Carolina. Thirty- first Ann. Rep. N. C. Agr. Expt. Sta., West Raleigh, June 1908, p. 82. Issued in 1909. 1909. Coleman, Leslie C. The ring disease of pota- toes. Bull. 1, Myco. Series, Dep. Agr., Mysore State, Bangalore, India, 1909, 15 pp., 15 figs. 1909. Petch.T. Miscellanea. Tropical Agriculturist, vol. xxxm, No. 6, Dec. 1909, p. 521. 1909. Brandl, Johann. Blattrollkrankheit oder Bakterienringfaulc Wiener Landwirtschaft- liche Zeitung, 59 Jahrg., No. 70, pp. 691-693, and No. 71, pp. 701-702. 1909. Zedwitz. WilhElm von. Blattrollkrankheit und Bakterienringfaule. Wiener Landwirt- schaftliche Zeitung, 59 Jahrg., No. 83, pp. 818-819. 1909. SchandER, R. Kartoffelkrankheiten. Fiihlings Landwirtschaftliche Zeitung, 58 Jahr., Heft 8, April 15, 1909, pp. 273-285. 1910. Bancroft, Keith. A bacterial disease of potato and tomato, Agric. Bull. Str. and Fed. Malay States, vol. ix, Dec. 1910, pp. 478-480. (See also Spieckermann, this Monograph, pp. 166-167.) WILT-DISEASES OF TOBACCO. (Sumatran Slime-disease of Tobacco; Granville Wilt; Japanese Stem-rot, etc.) A portion, at least, of this chapter belongs with the preceding. It is here kept separate, partly for the convenience of the reader, partly because it was written up in this form, my own cross-inoculations (up to 1908) not having been as conclusive regarding exact identity as those reported by Dr. Hunger; and partly because of contradictory statements by Uyeda respecting the cause of the Japanese disease. The whole subject of the etiology of these tobacco-rots needs very careful inquiry. Possibly we have to do with several different wilt- diseases.* In 1908 and 1909, the writer obtained conclusive cross-inoculations on tomatoes, using the North Carolina tobacco organism. In 1909, Stevens sent me a photograph showing the tomato-disease occurring in tobacco fields (fig. 115). THE DUTCH EAST INDIAN DISEASE. HISTORY AND ETIOLOGY. In 1892, from studies made in Buitenzorg, on specimens received from Sumatra, Janse correctly attributed to bacteria the tobacco-disease subsequently studied by van Breda de Haan, by Hunger, by Jensen, and by Honing. Janse describes the brown sunken stripes on the surface of the stem and the brown stain in the vessels, and notes the fact that they are plugged with a grayish mass of bacteria which stained readily in picric-acid-anilin-blue. He also examined microscopically the destruction of bark, bast, and pith, and saw closed cavities extending long distances. These cavities were filled with the bacteria in enormous quantities. Similar lesions occupied by the bacteria were seen by him in the leaf-stalk, the midrib, and the side-veins of the leaves. Morphologically, all the bacteria appeared to be of one sort, were quite short, so as more to resemble coccus forms than rods, and measured 0.7^ in length. He did not find the bacterial plugging of the vessels in all of the diseased plants, but this is not to be wondered at, as he did not himself select the material, and a fungous disease appears to have been present also on the plants both in the seed-beds and in the fields. Most of his observations were made on alcoholic material, .and no cultures or inoculations were undertaken. His general conclusion is sufficiently shown by the following paragraph : From these anatomical studies we may conclude, tentatively, that the disease here considered is probably caused by bacteria. These appear to infect the leaf locally (plaatselijk), developing in the cells of the leaf-tissues, which are destroyed, and subsequently extending further into the plant, either by way of the vessels, or from cell to cell. In the first case they enter the stem, and subse- quently appear outside the vessels (possibly by the normal rupture of the primary vessels due to elongation of the stem), and make their way between and into the adjacent cells (bast and pith) where they may develop into great colonies, and by destruction of the tissues form cavities which one finds on examination to be entirely filled with bacterial masses. Mycelium was occasionally found with the bacteria, but Janse is inclined to think it secondary. In 1898, in the Dutch Hast Indian Journal Teysmannia, Dr. van Breda de Haan described a disease of tobacco prevalent in Sumatra. He speaks of it as the slime-sickness, a "specific decay," resulting in the death of the plant. He attributes it to wounds of various kinds followed by the entrance of a specific bacterium. The bulk of the long paper is *Since this was written Honing has cleared up the Dutch East Indian situation. 220 WILT-DISEASES OF TOBACCO. 221 devoted to a description of the signs of the disease and to statements of the numerous ways in which the organism may penetrate the plant, the latter being conclusions derived from field observations. The disease was believed to enter the plant only through wounds, generally of the root or stem under ground, but also sometimes above ground. Many possibilities are described. The organism is stated to be a coccus, measuring about 8yu in diameter. It was not named, nor were many of its biological peculiarities described. It is stated to issue from the plant as a dark brown slime, but to be grayish-white on agar and to be actively motile in the plant. On plate-cultures it formed fused colonies with an irregular wavy border. It stained readily with carbol-fuchsin and alkaline methylene blue. The external signs in the tobacco plant are sudden wilting of the foliage, pale green spotting and then brown spotting of certain foliar portions, especially the "ears" of the leaf, general yellowing and death of the lower leaves and a local black stain in external portions of the stem, especially toward the base. The brown spots on the ears of the leaf are said to be a sure sign of the disease. The internal signs are a brown or black stain in the vas- cular tissues (from which on cross- sec- tion a brown bac- terial slime issues abundantly), and marked staining of the pith, which is usually found to be badly disorganized , so that in stems attacked for some time the whole central portion be- comes a slimy mass. Such stems are easily crushed be- tween the fingers, although externally they may not show any or much indi- cation of disease. The bacteria mul- tiply most rapidly in the large intercellular spaces of the pith, less rapidly in the wide xylem vessels, where the cross-walls act as hindrances. The phloem part of the stem ordinarily is not greatly injured. The amount of starch in the vicinity of attacked tissues appeared to be notice- ably less than in sound cells. The root-system is usually occupied by the bacteria, and frequently stained dark brown. In bad cases the secondary roots are not well developed. The underground part of the stem was often rotted badly. Eel-worms and an insect (larva of the family Tineidse) causing galls on the stem at the surface of the earth were believed to play a considerable part in paving the way for infection. It is also stated that the removal of the lower leaves at the time of planting, and various other practices of the coolies, favor the distribution of the disease. Sometimes the plants set out by one coolie will be badly attacked, while those set out by his neighbors will be free from disease or nearly free. The disease is said to be most serious on heavy lands, while on mellow ground Fig. 115. *Fig. 115. — Tomatoes following tobacco in a North Carolina field. The tobacco contracted the Granville wilt and the tomatoes set in place now show the same disease. (From a photograph by Dr. F. L. Stevens.) 22 2 BACTERIA IN RELATION TO PLANT DISEASES. rich in humus it is seldom seen. Only exceptionally does the disease begin in the seed-beds, and it is not generally observed until some weeks after setting out the tobacco. If the weather is dry the first conspicuous signs are flagging of the leaves. In such plants the base of the stem is diseased and the tap-root, if not other roots. Where a whole field shows this disease it may be assumed to have been very badly planted, i. e., hastily or with too old plants. Rains favor the spread of the disease, and topping, removal of leaves, suckers, etc., should be done, as far as possible, in dry, sunny weather. Up to the time of publication of his paper Dr. van Breda de Haan had secured very few, if any, pure-culture infections. He had reproduced the disease in the field by direct infection, using slime which had just extruded from the vascular system of cut stems, obtaining an abundance of the bacteria, with typical signs of the disease in the pith 26 cm. above the wound. Later, at Buitenzorg, he states that he reproduced the disease by inject- ing a pure culture in bouillon, but surely not with the coccus form 8^ in diameter. His field observations seem to have been numerous, but the laboratory work appears to have been meager. In 1901, in a general paper on tobacco diseases, Dr. Hunger devoted a chapter to this same disease, confirming van Breda de Haan's statements respecting its prevalence in Deli, the signs of the disease, and the great frequency of underground infections through injuries due to eel-worms and other causes. He was not able to confirm de Haan's statement that the disease is due to a Micrococcus.* Dr. Hunger found this disease due to a much smaller rod-shaped micro-organism which he identified as Bacillus solanacearum. He states that he obtained infections with this organism on tobacco and that he could not distinguish it from the organism attacking tomatoes in Java, nor the latter from that described by Smith from potatoes, tomatoes, and egg-plants in the United States. The following statements respecting the Sumatran tobacco disease are translated and condensed from Hunger's Dutch paper: This slime-disease was so named by van Breda de Haan, who first investigated it. It is caused by a bacterium which lives as well in the above-ground as in the under-ground plant, but which was not exactly recognized by the discoverer, as may be seen from his description on page 17 (544). From van Breda de Haan (whose communication is here borrowed in part with certain correc- tions) the bacteria received no name. As the result of an earlier investigation [tomato disease] I came to the conclusion that the slime- disease of tobacco was caused by Bacillus solanacearum Smith, to which same conclusion Raciborski likewise arrived. The disease occurs over all Deli, as well in the seed-beds as in young and old tobacco, chiefly in the latter, because it occurs most seriously during the period of heavy rains. In the attacked seedlings the lower part of the little stem is dark colored and very flabby, so that it often falls over, while the small leaves wilt and in part hang down brown. In large tobacco this disease begins commonly to be noticeable first in dry weather through the interruption of the water-movement in the above-ground parts of the plant. This is shown by the wilting of the leaves. At the same time they lose their fresh green color and there appear upon the ears of the leaf pale green spots, and afterwards also upon the blades themselves, and these speedily become brown and dry out. The harvested tobacco from such slime-diseased plants is often unripe. The lower end of the stem gets then a dark color and perishes within, so that it can be easily squeezed together between the fingers. The lower leaves become quickly yellow and die before ripening. The root-system of the attacked plant is colored brown, the main root is mostly black and rotted at the top. Sometimes also the white, sound, side roots found on healthy plants are here wanting. From the root-system the decay continues slowly upward, by means of which the pith of the stem is finally wholly decayed into a slimyr mass, wherefrom the name of this disease (slime-sickness) was taken. The disease does not remain limited to the stem, but continues in the leaf-stalks and infects in this way also the leaves, causing spots to appear upon the leaf-surface. "Hunger noted the occasional presence of a round yeast 0.008 mm. in diameter in his tomato tissues diseased for some time. WILT-DISEASES OF TOBACCO. 223 In tobacco already topped the slime-disease often shows strikingly on the above-ground plant, so that the top is said to be water-soaked (ingeregend). If one cuts the stem of a slime-sick plant crosswise there then appear quickly on the cut surface little brown drops, which slowly become greater by fusion and occupy just the place of the vascular ring. If one examines the physical condition of these drops they are seen to consist of a slimy sub- stance which may be stretched out iii threads. If one examines microscopically the leaf-spots of the slime-sick tobacco on cross-section, then one sees that the parenchyma of the spot is completely changed. The cell-walls are dissolved and the chloroplasts wholly disorganized, while in their place a mass of bacteria fills the spots of the attacked leaf-blade. If a section is made across an attacked stem we see that the walls of the vas- cular bundle, and also of the woody parenchyma bordering the same, are colored dark brown, some- times even black, while the lumina of the xylem vessels show a partial or total occlusion. This occlu- sion consists partially of the massing of bacteria with their disorganization products and partially of bladder-formed swellings from the surrounding parenchyma cells, that is, tyloses which have grown into the interior of the vessels. This abnormal filling of the water-paths causes naturally a stoppage, on account of which the sap-stream is noticeably hindered and finally interrupted. The necessary consequence of this is that water-need appears, which is shown by the wilting of the part of the plant above ground. The inclination to this formation of tyloses is caused by a bacterium (Bacillus solanacearum Smith), which by means of many sorts of external wounds is given an opportunity to enter into the interior of the plant. When once entered into the xylem vessels the vessel wall is through them in some places partially absorbed, so that the above mentioned tyloses are formed. The slime-disease described here is altogether a secondary phenomenon, which is made possible by external wounds. In by far the most cases the injury of the plant takes place either on the root or on the stem con- cealed underground. In the latter case I mean the stem part which has been covered with earth by heaping up the ground. The woundings may be due to many and very different causes. In the first place should be mentioned wounds due to plant and animal parasites. Among the first named I reckon chiefly the parasitic molds, especially Phytophthora nicotianae. Some of the principal animal parasites are the injurious soil insects, for example, eel-worms (Heierodera radicicola) and thick bellies* [Glechia sola- nella Boisd. (G. tabacella Reg.)]. In the second place are to be mentioned the mechanical injuries which during culture may be brought about by various means, such as pulling up the seedlings, transplanting, hilling, topping, and removal of suckers. Finally, an injurious chemical and physical soil-condition is not least active in making the under- ground plant parts susceptible to infection, the most important of which is poorly cultivated and badly drained ground. This does not allow the roots to develop normally, and on account of the stagnating water they go easily over into decay. Through all the above summed up circumstances the Bacillus solanacearum, present in the ground, is given the opportunity of penetrating into the plant through the injured places, so that the sickness induced thereby is a secondary process for the occurrence of which occasion may have been given in various ways, but always with the same external signs. The spread of this disease in the field is very easily caused during the care of the tobacco, as well by the coolies themselves as by their tools. In the plant itself the spread of the infection is assured as soon as the bacteria have reached the vascular bundles. With the rise of the sap-stream from the root-system the bacteria are carried along [?] to the parts of the plant above ground, where the signs of the disease appear in the various organs. Bacillus solanacearum Smith is elliptical in form, of middle size, ordinarily 1.5 to 3 times as long as broad (i-5m long and 0.5^ broad), but very variable, according to age and cultural condition. Pure cultures are easily made upon agar plates. In the beginning this bacterium forms on the surface of the nutrient substratum small, round, dark brown colonies,! which slowly extend concen- trically. Mixed in with these round colonies come often also lens-formed colonies, but both are made up of Bacillus solanacearum. These variable ways of growing of the self-same bacteria are a conse- quence of the location of the colonies. If they are buried in the agar then they grow slowly toward the surface on account of lack of oxygen. *So called because they produce a swelling or gall on the stem at the surface of the earth. fThis is probably an error, unless we are to assume the organism different from Bacterium solanacearum. Van Breda de Haan says grayish-white on agar, and this, so far as my own observation goes, is always the initial color of B. solanacearum, the colonies of which become brown only after some days or weeks. 224 BACTERIA IN RELATION TO PLANT DISEASES. Dr. Hunger says: "I succeeded repeatedly in artificial infections with Bacillus solanacearum on sound plants, which then showed the typical phenomenon of the slime-disease." This disease in the tobacco is a specific rotting process caused by Bacillus solanacearum Smith, for the appearance of which a wounding of the plant is first necessary, since the bacteria themselves are not able to enter of their own accord to cause the slime-disease. After a preliminary wounding it is possible for the bacteria to penetrate the plant and to find principally in the vascular bundles their seat of activity. The infection takes place chiefly on the underground parts of the plant, although the first notice- able signs of the disease are shown by the wilting of the leaves. Whenever the path along which the sap-stream moves is completely stopped up the plant dies in consequence of lack of water. The interior of the stem is completely disorganized. In later stages of the slime-disease the rot- ting process is hastened by the appearance of several saprophytic bacteria and yeast species. The tobacco from slime-sick plants is often unripe because, for the most part, it must be harvested too early, otherwise the spots on the leaves form very weak places. A great reduction in the spread of this disease would be brought about by avoiding to plant the places where slime-diseased tobacco plants have stood, at least without special precautions. It is to be desired that tobacco from slime-diseased plants should be pulled up and not cut, so as to avoid bringing the rotting stems into the shed. The hollow stems filled with slime should be carefully removed and burned. In 1909, Jensen published some notes on the Dutch East Indian disease in a general paper on tobacco. Some of his conclusions are as follows: Sound and strong tobacco plants are resistant to the slime-disease bacteria. Only when the bacteria are extremely virulent are strong plants made sick and then in many cases these are not killed, but form new sound roots, develop new shoots, and sometimes wholly recover. Serious injuries occur only on fields with less good soil, where the plants in their youth have not been strong enough to resist the bacteria. In his first experiment, begun September 30, 1908, on 10 plants, slime was taken directly from the cut stem of a tobacco plant received from Djoganalan and injected into the exposed roots by means of a scalpel and the needle of a Pravaz syringe. After 9 days all the plants became diseased and the lower leaves showed the characteristic dried-out spots. The 10 plants were afterwards pulled up and dissected. The roots were rotted and there were, on cross-section, the characteristic small black spots in the vascular ring. In the second experiment 70 plants were used. The results were as follows: Table 22. — Jensen s Results. No. of plants. Source of bacteria, etc. Diseased (in varying degrees) . 25 ■ 5 '5 10 5 Checks 0 '5 13 1 0 Inoculated with a young agar culture of slime-bacteria from Inoculated with Bact. No. 1 (air of the laboratory) Inoculated with Bact. No. 2 (air of the laboratory) None of the inoculated plants died. Most grew well and many reached the height of the uninfected control plants. Neighboring plants were not infected. Following a rain the diseased plants recovered their turgor. Jensen's final conclusion is as follows: The slime-sickness, consequently, must be regarded as a true bacterial disease which can develop only under unsatisfactory cultural conditions and which spreads from plant to plant but little or almost not at all. In August 1910, Honing published in Dutch a very interesting preliminary paper on the tobacco-disease of Sumatra, which I summarize as follows : In all stages of the disease bacteria occur, at first only in the roots, later in the stem and also in the veins of the leaves where they are visible as brown spots on breaking the leaves. WILT-DISEASES OF TOBACCO. 225 With exception of a yeast no fungi have been observed. The cause of the disease will remain in the ground in the absence of tobacco cultivation for 8 years or more. Exhaustion of the soil is not an explanation, because in splendid high tobacco on land which has had little or no fertilizer the disease occurs. Bad drainage certainly favors the spread of the disease, but is not the cause of it, and it may occur to a serious extent on the best-drained lands. Degeneration of the Deli tobacco can not be the cause. Plants which on only moderately good ground and often under unfavorable circumstances make stems 2 meters high, blossom, fruit, and set seeds 90 per cent of which will germinate, can not be run out. On the east coast no plant has been suspected more than Albizzia, but this can not be the cause of the tobacco disease.* There are places where Albizzia has persisted, where none or little of the tobacco disease occurs, and on the contrary there are lalangf soils where 80 to 90 per cent has been killed. "In my opinion the slime-sickness is a bacterial disease." The first thing to be done was to determine whether the disease could be reproduced with the bacteria present in all the slime-sick plants. Stems of such plants were chopped fine, mixed with earth, and then placed under 24 young plants, of which 15 contracted the disease, the presence of the bacteria in them being controlled by microscopic examination. Of the 24 control plants none became diseased. The juice of the sound pith of tobacco is slightly acid, that of diseased plants is alkaline and blues red litmus paper, but if the decay continues until the stems become hollow, it may again become acid. If one makes transfers from the nerves of leaves which have recently begun to droop, and as high up as possible on the stems, frequently one may obtain pure cultures of the slime-sickness bacteria. The bacteria occur first in the large cells of the bundle, later in the phloem and water vessels. With bacterial cultures, so obtained, inoculations were made. On May 23, 12 rows of young tobacco plants, 25 in each row, were treated as follows: 1. Control. 2. Plants dipped into a dish containing about 2 liters of water in which a bacterial culture was poured, and then planted. 3. After the planting, the ground around the seedlings was watered with a very dilute culture, about 10 c.c. to a watering pot. 4. Control. 5. Control. Injection into the stem, 2 days after planting, of the same sterile bouillon that served for the bacterial cultures. 6. Before planting each plant was injected at the crown with a pure culture in bouillon. 7. Control. 8. The day after the watering of the soil with a dilute pure culture each plant-hole was watered with 0.4 gr. potassium permanganate dissolved in 0.5 liter of water, and a half hour later the plants were set out. 9. The stems received injection of a pure culture 8 to 10 cm. above the crown, where a leaf had been pulled off for the purpose. 10. Injection of a leaf-nerve by means of a hypodermic needle and higher up near the leaf top, 3 or 4 pricks. 1 1. The leaves, and consequently the earth, were watered with a diluted pure culture. 12. The plant-holes were watered with a dilute pure culture and the plants were set out one day later. The number of diseased plants were as shown in table 23. Table 23. — Result of Honing' s First Inoculations. 1 (check ). 2 3 ■1 (check). 5 (check). 6 7 (check). 8 9 10 II 12 June 10. . . Aug. 1 I 10 2 22 O 22 O 8 0 6 '5 25 1 7 4 '9 24 1 24 8 24 3 25 5 23 "Na deze proeven blijft er, voor mij ten minste, geen spoor van twijfel over aan den bacter- ieelen aard der slijmziekte." The disease is hastened by introducing the bacteria into the plant, but in the end it enters the plant from the soil. The failure of a portion of the checks is attributed to a natural infection of the soil, the location having proved to be a bad one, as shown by results in three rows of tobacco planted near these some- what earlier and a considerable part of which succumbed. This has reference to the fact that a disease of unknown origin has destroyed many thousands of Albizzia trees, formerly extensively used as a shade plant for various cultivated crops, but now generally abandoned. fA coarse grass. 226 BACTERIA IN RELATION TO PLANT DISEASES. Once also, Honing has seen, in a slime-inoculated culture on a slice of a tobacco stem, a yeast corresponding to van Breda de Haan's yeast. No spores were observed in old cultures of the parasite, but the latter is soon followed by sapro- phytes. In the decaying stems five or six kinds of bacteria occur, as may be made out readily, and no doubt careful cultures would show others. The author had not determined the cultural characters of the parasite. Dr. Honing says the preceding contains nothing especially new, but is confirmatory or explanatory of the observations of van Breda de Haan, Hunger, Uyeda, Smith, and Jensen. Extremely interesting, however, and quite new are his observations that the same or a similar disease occurs on many tobacco fields in wild plants, among these several composites. These plants are: Pouzolzia (a kind of nettle), Agcratum conyzoides, Physalis angulata, Spilanthes acmella, and Pluchea indica. Once the disease was found in Pluchea growing on lalang land which had not been in tobacco for 7 years. The relatively small percentage of disease in the weeds (0.5 to 1 per cent or less) does not necessarily show them to be less susceptible than the tobacco. It may be only that they are less subject because their root- system is less broken than that of the transplanted tobacco. Also from two young diseased Mucuna plants a bacterium was isolated which could not be distinguished from that of the slime-disease. Cultures were made from Pouzolzia and from Ageratum and tried on tobacco, June 18. The results on June 29 showed many more cases in three of the inoculated rows than in the controls. Counting two of the inoculated groups with the checks, because, as was afterwards discovered, they were inoculated with cultures containing a spore-bearing form clearly not Bad. solanaceariiin, we have the results given in table 24. Table 24. — Honing' s Results with Pouzolzia and Agcratum. Plat. Remarks. Number of plants inoculated. Diseased at end of Plat. Remarks. Number of plants inoculated. Diseased at end of II days. 44 days. II days. 44 days. I 2 3 4 5 6 7 Check 95 95 25 25 25 25 25 4 9 1 21 1 21 16 48 68 15 25 15 25 25 8 9 10 Dilute culture in plant hole (.4)... Dilute culture in plant hole (P)... Dilute culture in plant hole (A)... 25 17 17 2 21 . .Do Wrong organism. . . Injected (P) Wrong organism. . . Injected (P) Injected {A) 2 0 '7 14 P = Pouzolzia. A = Ageratum. The large number of cases that finally appeared in plats i and 2 are explained as due to the fact that they were worthless rejected plants used as checks only because no others were available. It will be seen that every one of the plants inoculated with the pure cultures by injection contracted the disease and also nearly all of those in which only the ground under the plant was infected. The best results in treatment of the soil were with chloride of lime and potassium permanganate. Copper sulphate proved very injurious. Further experiments must be made to determine what doses of the lime and permanganate must be used to disinfect the soil, also whether all the soil of a field must be disinfected or only that under the plant, whether the quality of the tobacco will be injured, and finally, whether rain musjt be awaited before planting. In some cases Honing used 2 to 2.5 grams chloride of lime (Chloorkalk) in 1 to 2 liters of water for the soil under each plant and the same of potassium permanganate. In other cases he used as high as 6 grams of the chloride of lime per plant, with excellent success WILT-DISEASES OF TOBACCO. 227 in spots from which diseased tobacco had just been pulled out. In field tests in a bad year (1909) on ground where all the untreated tobacco died, 50 per cent of a crop was made, using 4 to 5 grams in each plant hole. The next year the loss was slight on 8 treated fields, while on the neighboring untreated parts it was 50 to 60 per cent. Permanganate of potash will not penetrate the heavier soil and preference is given the chloride of lime, although it is recognized that in its use there may be danger of injuring the burning quality of the tobacco. Honing found great carelessness on the part of the planters in the disposal of diseased plants. In many instances they have thrown the rotting tobacco plants into wells from which afterwards they took water to put on the fields. (For abstracts of more recent papers by Honing see p. 244.) THE NORTH AMERICAN DISEASE. Synonyms. — North Carolina tobacco wilt; Granville tobacco wilt; Florida wilt. HISTORY. In September 1903, Stevens and Sackett, then of North Carolina Experiment Station, distributed a well-illustrated, brief paper, describing a disease of tobacco which had caused a good deal of injury in Granville County, North Carolina, and attributed the same to a schizomycete. The Dutch papers are not mentioned and were probably unknown to them. This disease is stated to be "so destructive that its spread throughout the country would imply annihilation of the industry of tobacco growing." The bulletin devoted itself prin- cipally to stating the fact of the presence of the disease, furnishing figures of it from good photographs, and giving a brief description of the signs, together with some field observa- tions on its prevalence and supposed manner of spread. The root was believed to be the original seat of infection. The organism was not described or named, nor were any proofs furnished that the disease was really due to bacteria, other than the strongly presumptive evidence offered by the microscopic examinations. The same month (September) and a little in advance of the above publication, Dr. R. E. B. McKenney, of the Bureau of Plant Industry, U. S. Department of Agriculture, who had been engaged for some time in a study of tobacco diseases in cooperation with the Bureau of Soils, but not under the writer's direction, published a brief circular describing and illustrating this North Carolina disease. Dr. McKenney studied the disease in the same localities as Stevens and Saekett. His circular states that the disease is due to a fungus (Fusarium) closely related to that described by Erwin F. Smith as the cause of a wilt in watermelons, cowpeas, and cotton. The publication of Dr. McKenney's circular appears to have been premature, since he never obtained any proofs from inoculation that this disease was due to a Fusarium, and the writer was never able to obtain from him any slides or other material showing the presence of the fungus in the affected plants. From Professor Stevens the writer obtained alcoholic material, both of root and leaf, which in section showed the vessels and surrounding tissues to be plugged with bacteria, but wherein no fungi could be demonstrated microscopically (figs. 116, 117). The writer also, in abun- dant material since received directly from the diseased fields, has seen nothing of any disease attributable to Fusarium. Further studies must be made to determine whether the Fusarium seen by McKenney occurred as a saprophyte or a parasite and also to determine its morphology and cultural characters. One of the writer's second set of inoculated plants (fig. 118) was destroyed in the hothouse under the combined action of bacteria and Fusarium, but this is the only case 22; BACTERIA IN RELATION TO PLANT DISEASES. he has seen. Whether the Fusarium alone is sufficient to cause a wilt disease of tobacco remains to be determined by experiment. Delacroix has recently described a Fusarium disease of tobacco, and it is not improbable that two diseases may have been confused. With these facts in mind, the writer suggested to Dr. McKenney in the summer of 191 2 that he visit the tobacco fields of North Caro- lina. This he did, but could then find only the bacterial disease. I still think, however, that a Fusarium disease of tobacco will be found, for, as I pointed out in 1899, this widely disseminated form-genus contains active parasites destruct- ive to a great variety of cultivated plants, a fact now generally recog- nized, but then unknown to science (Scientific American Supplement, No. 1246, page 19981). The preceding statements rep- resent the writer's knowledge of this subject up to the summer of 1905, if we exclude Uyeda's first paper, to be treated a little later. That sum- mer diseased tobacco plants were sent to him from Ouincy, Florida, and from Creedmoor, North Caro- lina. In both cases the cause of the disease appeared to be bacterial, and the signs of the disease corre- sponded quite closely to descriptions of the slime disease as it occurs in Sumatra, and also to the signs men- tioned by Stevens and Sackett. From the North Carolina ma- terial agar poured plates were made a number of times, and always large numbers of one organism were ob- tained from the inner tissues (pi. 37). This was a short, motile bacterium, the small roundish surface colonies being gray-white at first on agar, but afterwards brownish (pi. 23, fig. 2). Usually the plates showed no other organism present, but occa- sionally scattering colonies of other bacteria appeared. They were made, of course, from clean parts Fig. I17.t *Fig. 1 16. — Cross-section of petiole of a tobacco leaf, showing bacterial invasion of vascular system. From a natural infection in North Carolina. Material for sections received in alcohol from Prof. F. L. Stevens. Slide 291 ad. fFiG. 117. — Cross-section of a tobacco stem diseased by Bacterium solanacearum. A detail from slide 291 o 15, section X, i. e., from the same source as fig. 116. A somewhat diagrammatic wash drawing. PLANT BACTERIA, VOL. 3. t-z* PLATE 33. North Carolina tobacco wilt. Tobacco plant inoculated on the stem by needle-pricks from a pure culture of Bacterium solan1 PLATE 41. TOBACCO WILT. DISEASE OF ORCHARD GRASS. (1, 2, 7) The Japanese tobacco disease. After Uyeda. (3) Tube of milk inoculated on August 23, 1905. with N. C. tobacco organism (colony B), painted October 11, by reflected light. (4) Old potato culture of the N. C. tobacco organism. Inoculated July 17, 1905, painted August 27, bacterial growth dark brown, substratum and fluid browned. This will answer equally well for the potato and tomato organism. (5) Check tube of litmus-milk. (6) Tube of litmus-milk inoculated with the N. C- tobacco organism September 25, 1905, and painted October 20, i. e., after 25 days at about 25°C. Bacterial precipitate at bottom. No acid formed ; gradually increasing alkalinity without separation of casein. Illustrates equally well cultures of potato and tomato organism of same age. (8) Agar poured-plate colony of the Granville tobacco wilt organism after 22 days at about 23°C. x 4, painted April 22, 1909. Plated from stem of wilting tobacco-plant (root infection). Central part of the colony darker than the periphery. The brown stain in the agar is somewhat exaggerated. (9, 10) Spikelets of orchard grass showing early and late stage of Rathay's disease. WILT-DISEASES OF TOBACCO. 233 Fig. 124/ much rim. Most of the tubes were still somewhat cloudy and all became very turbid on shaking. Four of the six cultures from as many colonies produced crystals in the bouillon ; two did not. Milk. — Like Bacterium solanaccarum, except that a brown rim formed on old cultures. No acid was formed, and there was no coagulation of the casein ; the fluid became more and more alkaline, and after some weeks was translucent and pale brownish. (See pi. 41, fig. 3.) The milk was browner by transmitted light and so clear that print could be read behind it and that a penholder could be seen very readily behind two tubes when one was placed back of the other ; the brown bacterial rim was not visible until toward the close of the experiment, it having increased very noticeably during the last 2 weeks ; the black rim has not been ob- served in milk cultures of the tomato and potato organism. Litmus milk. — Like Bacterium solanacearum (pi. 41, fig. 6), except that I have not seen any crystals such as shown in fig. 124. Colin s solution. — No growth. Numerous tests. Reduction of nitrate to nitrite. — Copious in bouillon. Thermal death-point. — This is above 48° C. and below 52° C. Minimum temperature. — This is above 8° C. No growth was obtained at 8.20 C. in 6 tubes of +15 peptonized beef -bouillon exposed for 5 weeks. These were inoculated from as many colo- nies. Four out of 6 similar tubes finally clouded at 8.70 C. Gas. — None from any medium. Aerobism. — Strictly aerobic so far as can be determined from general appearance of buried colo- nies, failure to grow in hydrogen, and behavior of stabs in agar and gelatin. ETIOLOGY. EXPERIMENTS OF 1907. On July 29, 1907, three young tobacco plants about 4 to 6 inches high, Nos. 595_597» were inoculated directly with Bacterium solanacearum from the browned bundles of tomato stems from Lanham, Maryland. The stems were squeezed until the juice came out, and this and bits of the browned bundles themselves were pricked into the stems and peti- oles of the tobacco plants. Result. — The leaves finally wilted and the stems were full of bacteria [some vessels probably] ; then the plants recovered. (Notes made by Miss McCulloch.) EXPERIMENTS OF 1908. On July 9, 1908, a dozen large wilted tobacco plants were received from Creedmoor, North Carolina, affected by the vascular bacterial disease (Granville wilt) . These were buried in good soil in rows about 1 5 inches apart in the old rose house. On July 22,1 received a much larger number of wilted tobacco plants (Granville bacterial disease) from the same locality and buried them in rows in the same way in the same house. After the receipt and burial of the second lot of tobacco, I planted tomato plants (large size) between Fig- i25-t the rows of tobacco laid down on July 9 ; and large plants of Datura stramonium between the rows of the more recently buried tobacco. These plant- *Fig. 124. — A crystal complex from a shining small mass on the inside of a test-tube above the fluid, in an old litmus-milk culture of North Carolina tobacco-wilt organism: Colony A, from poured plate iv, made direct from a tobacco plant received from Creedmoor. Tube inoculated June 19. Drawn free-hand Nov. 13 (Zeiss 8 mm. apochro- matic objective, No. 12 compensating ocular) and then reduced somewhat, X50 (?). Contamination (?). tFic 125.— Stem of Datura stramonium showing gray bacterial ooze from the vascular system. The plant stood in a bed where tobacco plants affected by the Granville (North Carolina) tobacco wilt had been buried and the infecbon took place through roots broken at the time of transplanting. (For appearance of the whole plant at this time see pi 43). It grew in the bed, healthy to all appearances, for about 5 weeks and then suddenly wilted. Photographed Aug. 29, 1908. X2. 234 BACTERIA IN RELATION TO PLANT DISEASES. ings were made on July 23. The roots were broken considerably, and purposely, in trans- planting both the tomato and Datura, and the tops were pruned back to correspond. They soon recovered from the transplanting. The results were as follows : August 3. — Five of the tomato plants show wilt to-day. Brought in two for examination. These are the smaller of the 5 and all their leaves have collapsed. No evidence above ground as to the cause of the wilt. They were all right day before yesterday. Cut stems at surface of earth and also about 4 inches above. The vascular bundles in each plant are gorged with a gray-white slime. Examined sections from each stem under the compound microscope — the slime consists of myriads of bacteria of the same morphology as those seen in the tobacco stems planted in the bed. The tomato plants went into the bed July 23 ; they have, therefore, been exposed to the infection just 1 1 days. The tomatoes came from out of doors on our grounds ; part from west of the grapery and part from the north end of the grounds. There are no checks on these, but they were all sound plants when set into this bed. On the Daturas there are hundreds of checks in the northern part of the city, in open ground where they grew. All of the transplanted Daturas are still healthy. August 5. — The weaker stramoniums between the rows of buried tobacco begin to wilt (old rose house) ; brought in 4 and examined. No bacteria : A white mycelium occurs on the roots and a Fusarium in the vessels (internal conidia). This fungus was probably brought into the house with the plants, some of which came from an old chip pile and rubbish heap. The bacteria have scarcely had time to diffuse through the soil and affect these plants. Brought in 4 more wilted tomatoes. The vascular bundles are browned and swarming with a short motile bacterium which is typical for Bad. solanacearum. It occurs in great numbers. Inoculated 4 tobacco plants in the orange house with juice squeezed from these 4 infected tomato stems; 4 places on each plant — middle of two upper leaves (midrib and parenchyma), on stem under an upper leaf and lower down on the scar of a leaf torn off for this purpose; latter just rubbed in, rest by needle-pricks. Thorough. Plants 2.5 feet tall. Not in bloom. August 7. — Brought in one more wilted tomato plant. At 9 inches from the roots the vessels are browned and full of typical bacteria. At level of the ground the inner tissues are worse affected, but there is no external disease. At a foot from the ground the bacteria are also in the vessels, but the brown stain is wanting. August 10. — Brought in from the bed in the old rose house 8 additional wilted tomato plants. Cut each tomato stem cross-wise in several places. Each is badly infected in the vascular system by bacteria. There are millions of these. They are short rods with rounded ends, oozing out of the browned vascular ring as a gray slime. Examined each plant microscopically. The infection in most cases extended from the base of the stem upward a foot or more; the only free vascular part is in the extreme top of the plants. (Did not examine vascular system of leaves.) In one case I found the gray bacterial ooze 3 inches in advance of the brown stain at the top of the plant. The others were too much infected to show this difference. The outside of the plants was sound even at the surface of the earth. Eighteen tomato plants have now gone down with this wilt. August 14. — One of the stramoniums came down to-day with the tobacco wilt. Its vessels are brown and swarming with short, motile, rod-shaped bacteria, which seem to have entered through two broken roots. The bacteria are abundant enough to ooze from a cross-section of the stem 6 inches from the roots, as a gray slime. The organism was found in cross-sections of both the browned roots (examined microscopically) and issuing as a gray ooze. Surface of roots sound; stem also sound on its surface. Plenty of sound roots. A ugust 16. — Brought in another wilted Datura from the bed which had diseased tobacco planted in it. On dissection the vascular bundles were full of the gray bacillus. Third case. August 77. — One additional tomato wilted. It is sound externally and is 55 cm. high. All the foliage is green and freshly wilted, and there is no external indication as to the cause of the trouble. Bacterium solanacearum is present in the vessels of the stem all the way from its base to the top of the plant. The vessels are browned below but not in the top of the stem. Bacteria most abundant below, where on cutting the stem they ooze as a gray-white slime ; entrance apparently through one small wounded root. This contains the bacteria; the others look sound. Bacteria are present in the midribs of all the leaves at their junction with the stem and in all but the top ones half-way out or more. Two top leaves required the use of the microscope to show the presence of bacteria in the vessels, i. e., no ooze was visible. August 26. — Pulled, brought in, examined microscopically, and had photographs made, of the 4 tobacco plants inoculated August 5, with slime from the stems of tomato plants which were infected through broken roots by planting over the buried tobacco in the bed in the old rose house. Best parts put into Carnoy. Vascular system of plants fully diseased from top to base, especially on one side where there are black stripes on the stems. It is the Granville wilt in typical form (pi. 42) . c PLANT BACTERIA. VOL. 3. PLATE 42. Brown stripes on tobacco stems attacked by the North Carolina tobacco wilt. These plants were inoculated from tomato stems (direct transfer of the bacterial slime) on Aug. 5. 1908, by needle-pricks on leaves and stems higher up, the injuries here shown being accomplished from the vascular bundles downward and out- ward the epidermis remaining unbroken. The tomatoes from which the bacterial slime was taken were wilting plants which contracted the disease through broken roots from soil in which diseased tobacco plants had been buried. Photo- graphed Aug. 27. Compare with plate 41. fig. 2. WILT-DISEASES OF TOBACCO. 235 August 29. — Yesterday I saw slight wilt on another Datura stramonium standing in the bed where the North Carolina tobacco was buried. It is a large plant and has been healthy hitherto. This morning I brought it in and had a photograph made (pi. 43). It is badly wilted. Stem sound externally; vessels filled with Bacterium solanacearum. Fusarium not present. Infections took place through broken roots. The bacteria are most abundant in the lower part of the stem and ooze out copiously on cross-section (fig. 125). August ji. — To-day, removed an additional wilted tomato plant from bed in the old rose house. The surface is sound except some small roots. The vessels in the lower half of the plant are filled with gray slime which under the microscope has the characteristic morphology of Bacterium solanacearum. Short rods ; many in pairs (dividing) . The plant is 3 feet long. The vessels are browned only in the lower part. Not many bacteria present in the upper part and no stain there. As one goes toward the root-system bacterial occupation increases. Infection took place unquestionably through the root system. This is the twentieth tomato plant to wilt. October 28. — Old rose house. Removed the remainder of the Daturas this morning. Some were in fruit. They have lost many of their leaves and others are brown spotted, but the cause of the dis- ease is uncertain. None of them except the 4 already mentioned appear to be attacked by the tobacco disease. Also pulled out the remainder of the tomatoes, none of which were wilted. Out of this bed I have removed 21 wilted tomato plants attacked by Bacterium solanacearum, and now these 9 which are not wilted. These 9 plants have sprawled over the ground, covering it with foliage, and have made a growth of 8 or 10 feet altogether, but have borne very little fruit. Only one or two green tomatoes were present, and the gardener says he has not seen fruit on them. It is not certain from this that the plants are resistant to the disease. It may be that they got such a good root start that the disease could not attack them successfully, or it may be they are resistant individuals. With a view of testing this I made cuttings from soft wood at the tops of 8 of these plants and removed all the leaves. I then planted them in the same bed after spading it up. There are 27 of these cuttings. If the plants are not resistant some of the cuttings ought to contract the disease, provided the organ- ism is still living in the soil. I also pulled out all the remaining Daturas and set in place egg-plants, two varieties of peppers, and some Connecticut tobacco. There are three rows of the egg-plants, 12 plants, var. Black Beauty. They were growing vigorously with leaves as large as my hand or larger, and perhaps a dozen such leaves. I pulled off all the leaves and root-pruned each one severely before setting it out. The peppers are Cayenne and Ruby King, 12 plants of each sort. Six of each sort were set out very carefully without disturbing the roots in the least, and 6 of each sort were thoroughly root-pruned and had all the leaves pulled off. Of the tobaccos, I set 6 in one part of the bed and 6 in another part of the bed. Three of each lot were transplanted without injury to the roots; the other 3 had all the leaves pulled off and most of the roots. In addition, I made 3 cuttings from a vigorous growing Datura, and set these out in the bed. Some tomatoes which were set out in a portion of this soil by Mr. Bisset, the gardener, about 2 months ago have made a very uneven growth. As a control I had a similar bed on the other side of the partition planted at the same time with a number of tomato plants, but in soil which had not received the diseased tobacco. These check -plants are very leafy, 3 feet or more in height and healthy-looking. Of the 24 in the diseased soil only 10 plants are good-looking ones ; the others are dwarfed and have the terminal leaflets dead or dying. Nine of the 14 dwarfed ones are very badly dwarfed. These are only about a foot high, and have only about one-fourth as much foliage as the check-plants. I have seen no wilt on them, and I am not yet certain as to the cause of the dying of the terminal leaves and general dwarfing of the plants. November 16. — The rooted tomatoes, now about 3 months old, show no distinct indications of wilt, but they are dwarfed and sickly. They have made not nearly so good a growth as the check plants in similar soil but which had not received diseased tobacco. I am at a loss to know whether this disease is due to the slow action of the tobacco organism on the roots of the tomato or whether it is a distinct disease. The checks are free, although the two beds are separated only by a glass partition and 16 inches of space. The three cuttings from young Datura stramonium have died, i. e., they did not root. The 1 2 transplanted tobacco plants show no indications of disease. The 1 2 pepper plants with un- pruned roots are continuing to grow normally. Of those with heavily pruned tops and roots, 10 appear to be recovering, one is dead, and another is dying. I have just pulled up the latter and find the base of the stem rotten. These plants are not recovering easily from the heavy top and root pruning. The 1 2 egg-plants, which were transplanted after being very heavily root and top pruned, are beginning to recover and are making a little growth. Of the 29 tomato shoots which we attempted to root in this bed, only 5 appear to be living; the others have gradually rotted away. More mature cuttings should have been used. The stems hitherto examined have not shown any definite indications of bacterial infection in the vascular bundles. 236 BACTERIA IN RELATION TO PLANT DISEASES. EXPERIMENTS OF 19D9 (1908 CONTINUED). March 22, igog. — None of the cuttings rooted. None of the peppers or egg-plants have con- tracted the wilt. Probably the stems were too woody when transplanted. The heavily pruned ones recovered very slowly. The older rooted tomatoes gradually got worse and worse, but with no definite wilt, or brown stain in the stems, or bacterial occupation of the vessels. The checks finally contracted the same disease. One of the tobaccos (a large plant now) has contracted the bacterial wilt. Its vessels are full of the gray slime, especially on one side. This was most abundant in the lower part of the stem, but was traced out into the leaves and into the top of the plant just under the seed pods, where a few vessels were browned and occupied by the bacteria, as shown by a microscopic examination. In the middle of the stem the bacterial slime was very abundant, but agar poured- plates sowed copiously showed at the end of the seventh day only intruders (a few colonies with finger- like radiations) . The right organism was not recovered with certainty even on a second set of plates made the fifth day from the second dilution tube, which had been kept and was then clouded. The only possible inference is that the parasitic bacteria had been present in this part of the stem for some time and that they were now dead — supplanted by saprophytes. The outside of the stem, on one side over the most badly affected part of the vascular cylinder, bore long dark stripes and sunken places. This tobacco was one of those not root-pruned. From the interior of a basal branch of the same plant which was then wilting, another set of plates was poured on March 30 and these plates yielded the right organism. On May 6, 1909, a second tobacco plant wilted with characteristic signs, and poured plates made from the browned interior yielded the right organism. July 6, igog. — The experiments with tobacco, egg-plants, and peppers were closed out to-day with the following results: (1) Tobacco: 8 plants healthy, 4 diseased. From 2 of the diseased, as already stated, Bad. solanacearum was plated out; the other 2 had similar signs but no cultures were made. (2) Egg-plant: There was no definite wilt on these egg-plants during the whole of the time they stood in the bed; 9 of them are sound, 2 being in fruit and the rest in blossom; 1 is wholly rotted, and 2 are gone, ;'. e., perished earlier. (3) Pepper: There has been no indication of wilt in the peppers during the whole time they have stood in the bed. Like the egg-plants, the root-pruned ones never fully recovered. There are now 9 free-growing peppers bear- ing green and ripe fruits in abundance and 9 stunted peppers (the root-pruned ones) ; six plants perished ear- lier, but with no definite signs of this disease. There is a trace of brown stain in one or two places in the wood at the base of the stem of 3 of the root-pruned ones, and a microscopic examination shows the presence of tyloses in the browned vessels, but no distinct evidence of bacteria. The bed was now spaded up and prepared for large tomato plants. On April 24, 8 good-sized tomato plants were inoculated with the organism plated from the interior of the diseased tobacco on March 30. These inoculations were made from slant agar-cultures of April 21 by means of needle-pricks on the stem, 2 plants being inoculated from each culture, a large amount of material being introduced and 2 internodes of each shoot being pricked. On May 6, six additional plants of the same series were inoculated from a beef -broth culture of April 22 in the same thorough manner. The tomato plants were rapid-growing, rather tall individuals, propagated from cuttings. July 6, igog. — The tomato plants continued to grow rapidly and up to this date none of them have ever shown any trace of wilt, with the exception of one plant, which after a time showed one or two slightly wilted leaves that subsequently recovered their turgor. The tomato was a free-bearing, red, small-fruited, hothouse variety. Final Note. — The organism appears to have died out of the soil which yielded so many diseased plants (tobacco and tomato) in 1908. It was moved to another hothouse and tomato, tobacco, and the Porto Rican spiny weed (p. 182) grown in it for a year, but with wholly negative results. In 1912, Coleman, of Mysore, told me he had observed the same thing at Bangalore in connection with his work on the Indian potato-disease. After about 2.5 years an arti- ficially infected soil was no longer infectious. This is a highly important matter deserving of thorough study. Very likely the growth of certain soil saprophytes may serve to bring about destruction of Bact. solanacearum in certain soils, and if they could be obtained in pure culture, they might be sown to advantage on infected lands. Since this was written Honing has obtained some evidence bearing on this point (see fig. 134). Perhaps, also, a proper course of rotation and cultivation would do much to free the soil. PLANT BACTERIA, VOL. 3. PLATE 43. Shoot of Datura stramonium attacked by the Granville tobacco wilt. The infection took place through broken roots as the result of transplanting to a bed in which diseased tobacco plants had been buried. Tin vessels of this plant, especially in the lower part of the stem, were swarming with the bacteria (see fig. 125J Photographed Aug. 1908. WILT-DISEASES OF TOBACCO. 237 LOSSES; TREATMENT. The Florida tobacco-disease, according to Mr. W. W. Cobey, has been present on a plantation near Quincy, Florida, for several years. The loss in 1905 was estimated at about $4,000, a total of about 4 acres of plants being destroyed. The disease was patchy, but occurred on many parts of the plantation, being much more widely distributed than in 1904. This was probably due to the fact that the refuse tobacco material of 1904 was composted and spread on the land in 1905, the parasite being undoubtedly present in some of the composted tobacco leaves. The losses around Quincy, Florida, in 1908 far exceeded those of 1905. Mr. Shamel, a tobacco expert of the U. S. Department of Agriculture, wrote to the Department as follows respecting the Florida tobacco disease in June 1908: The Granville wilt has appeared to an alarming extent, especially in the fields of the Owl Com- mercial Company [Quincy, Florida]. I have sent plants to Stevens for identification and to Dr. Briggs in connection with some soil samples. The wilt does not attack the plants noticeably until from 3 to 4 weeks after transplanting. Then one leaf or a portion of a leaf begins to wilt, then follows the destruction of the entire plants. In some of the shaded fields on the Owl plantation from 25 to 70 per cent of the plants have been killed. On an affected field of last season, planted in potatoes this year, the potato plants have all been killed. It is the most discouraging factor I have ever seen in the wray of a parasitic disease of tobacco. The writer examined some of the diseased plants from this locality in 1905 and again in 1908, but could find no Fusarium, only bacteria. The losses in North Carolina have been much greater than those reported from Florida. In the Thirtieth Annual Report of the North Carolina Experiment Station for the year ending June 30, 1907, Dr. F. L. Stevens reports that the Granville tobacco wilt- continues to spread and is reported as worse this year, and the loss is said to be 25 to 100 per cent of the crop in the infected region. The loss in one county is estimated this date at $20,000. It is estimated to be 40 per cent more destructive this year than last year. Reports to the writer in 1908 from North Carolina indicated more extensive losses than any previous year. Many planters harvested their tobacco in July, unripe and half- grown, in order to save some portion of it. In June 1908, Mr. Cozart, statistical agent for the U. S. Department of Agriculture, wrote as follows from Creedmoor, Granville County, North Carolina, respecting the pros- pect for a crop of tobacco : In making this report I am at a loss to estimate the tobacco crop. We have a fine growth, good size, and it promises to be a good crop, but we have a serious problem to contend with — the will tobacco. There is hardly a farm in this township free from it. It is very serious and spreading at an alarming rate ; 20 per cent of our tobacco already lost by wilt, and if ratio continues, 30 to 40 per cent will be lost by the time it goes to knife. W. E. Graham, of Church Road, Virginia, reports (October 7, 1908) that the Granville tobacco wilt is getting worse on his place all the time, and further, that it is "confined mostly to gray lands, as I have seen very little on red soils." In his report for 1907-8, Stevens, of the North Carolina Experiment Station, reports the Granville tobacco wilt as occurring in Granville, Durham, Vance, and Ashe counties in North Carolina. According to the experience of Mr. Tunstall, land affected with the wilt left without tobacco for 10 years is still affected so that if tobacco is again planted it will become diseased. It is also said that there came under his personal observation one field in which the tobacco was very badly wilted, and in which, it is stated, tobacco had not been planted for at least twenty-five years. No statement is made, however, respecting 238 BACTERIA IN RELATION TO PLANT DISEASES. the planting on such fields during the interim of other crops which might act as carriers of the disease, or on the condition of the seed-bed from which this field was set. (For infection of a cabbage field with Bad. campestre from a seed-bed see this monograph, Vol. II, p. 329.) From these statements it is evident that under favoring conditions this disease may prove very detrimental to tobacco-culture. The reader will gather from what precedes, to recapitulate: (1) that only sound plants should be set out, and to this end the seed-bed should be on soil free from this organism; (2) that seedlings should be transplanted early and injured as little as possible in the removal; (3) that they should not be watered from infected wells, ponds, or streams; (4) that diseased plants should be removed from the fields as promptly as possible, so that they may not become centers of subsequent infection; (5) that the fingers or knives should not be used on sound plants after use on diseased plants without a preliminary sterilization; and, finally, (6) that as far as practicable, badly infested fields should be devoted to other and non-solanaceous crops. Toma- toes, potatoes, peppers, or egg-plants must not follow the tobacco, nor should peanuts. THE JAPANESE DISEASE. In 1904, Uyeda described a Japanese tobacco disease which seems to belong here, at least so far as signs are concerned. He says: This disease causes much damage in Japan, especially in those cases in which the transplantation from the seed-bed is carried out rather late. Prolonged moist weather with following great heat will cause the development of the disease. The first symptom is the wilting and yellowing of the lower leaves, then follows the blackening of the stem and leaves, and finally of the root. The plants are generally killed in about two weeks after the first symptoms are observed. The natural infection seems to take place generally through the roots, but very frequently also by the wounds caused by topping and suekering the plants toward the end of July. A microscopical examination reveals an immense number of bacteria in almost a state of pure culture in the vascular bundles. The central part of the stem shows a rotten condition and contains a dark brown liquid. Finally the stem becomes hollow and the root is completely destroyed. The organism, it is said, was cultivated pure, apparently in bouillon, and infections are said to have been obtained in about 2 weeks by irrigating healthy tobacco plants in pots and in the field. Infections through the stomata are also said to have been obtained by spraying a pure culture of the organism upon healthy leaves of young tobacco plants about 25 cm. high, but no evidence of this is offered other than that after 8 days a blackening was observed along the veins of the leaves, which extended more and more, and a microscopical examination showed enormous numbers of bacteria in the pitted vessels. Egg-plants which were irrigated in the same way as the tobacco did not contract the disease. This, however, might have been due to old and woody stems (see p. 236). This organism is described as Bacillus nicotianae and is said to have the following characteristics : Agar-stabs and streaks blacken, especially in the parts exposed to air (fig. 127). Growth on gelatin in streak-culture is very slow, forming a uniform pellicle, white at first, gradually blackening, with feeble liquefaction of the gelatin after about 5 weeks. Gas is produced in glucose agar and glucose bouillon, and there is a rancid smell, but only a little acid is formed. Milk is cleared. On potatoes it produces a yellow pigment, gradually turning brown-gray. In colonies on agar the growth is dirty white : gradually the brown pigment is produced, diffusing beyond the colony, which itself remains generally gray and shows concentric rings as it enlarges (fig. 128). Growth is best at 320 C. The organism is facultative anaerobic. It produces no odor in peptonized bouillon. It is 0.6 to o.gfx by 1 to 1.2/j. long. It is said to bear about 8 peritrichiate flagella. WILT-DISEASES OF TOBACCO. 239 The following statements are taken from the more recent paper by Uyeda (1905) : The disease is widespread in Japan and has been known for at least 25 years, having been de- scribed in 1S81 in Ensoroku, a book on tobacco culture. Since that time it has been written upon with special reference to methods of prevention by T. Kugahara in the experiment station of Huku- shima-Ken (1891) and at about the same time by Dr. S. Hori, in the Imperial Central Agricultural Experiment Station at Tokyo. Prof. Y. Kozai, director of the experiment station at Nishigahara, has also isolated a schizomycete, which he holds to be the cause of the disease. This is the same as that described by Uyeda, who did his work at that station. The disease is variously known to the Japanese as "stem-rot, " "black-leg, ' ' and "wilt-disease. " Uyeda 's studies were begun in 1 899 and continued nearly 5 years. The disease is widespread in the province of Hitachi, and occurs also in the province of Sagami and Shinano. Fig. 126.* The signs of the disease are much like those of the Granville wilt. Compare fig. 126 with pi. 33, and pi. 41, figs. 1, 2, 7, with pi. 42, for brown stripes on the green stems. The disease occurs from June to September on plants of all ages. The first sign is the sudden wilt of the foliage, after which the leaves become yellow. The stem then becomes black and the roots decay. When the juice from diseased plants or a pure culture of the bacillus is placed on healthy tobacco leaves, they begin to blacken and to show brown spots within one or two weeks. The principal veins of the leaf are then hollowed out and destroyed. The infection experiments were made on young and old plants at Nishigahara. These are not described. Old leaves sometimes show wavy black spots bordering the veins, where the bacteria are said to have entered by the stomata. The organism often enters also by the roots or through wounds above ground. All the affected parts contain great quantities of bacteria and the reaction of the fluid containing them is alkaline The bark of the stem shrinks considerably as the disease progresses and black lines appear upon the surface. The tissues *Fio. Uyeda.) 126. — Experimental field of tobacco at Ota, Japan, destroyed by bacterial wilt. Season of 1904. (After 240 BACTERIA IN RELATION TO PLANT DISEASES. of the root become separated and disorganized. At first in both stem and leaves the bacilli are found only in the vascular bundles. In the leaves the veins are first blackened and then the parenchyma is destroyed. In very recently diseased plants the vascular bundles are only stained locally, while the rest of the tissues are entirely sound. In the first stage of the disease only one side of the stem is black. Later the entire woody tissue becomes black. Sometimes the black stain does not appear until after the stems are cut. The bacteria gradually pass inward from the woody tissue into the pith and outward into the bark. The bark-parenchyma is badly disorganized; cell-sap, starch, chlo- rophyll, nucleus, and the rest of the cell-contents disappear; only the sclerenchymatic cells are very resistant. There is the same disorganization of the parenchymatic parts of the vascular bundles. .Sometimes a cork-layer is developed between sound and diseased parts. Small cavities filled with an immense number of bacteria often occur in the stem. The veins of the leaf are often hollowed out by the bacteria. These are often also found swarming, it is said, in the leaf-hairs. When the roots are attacked the first sign is the separation of the bast from the wood. When for any reason the attacked plants are not at once destroyed the leaves are dis- torted. The disease causes much damage by its very rapid spread during the rainy season. The spread of the disease is also greatly favored by the high summer temperature. Fig. 128.t The organism said to cause this disease has been named Bacillus nicotianae by Uyeda. He states that it resembles Bacterium solanacearum , but to the writer it appears to differ very materially in its morphology and cultural characters (if we may assume all these statements to relate to one organism), although the signs of the disease are strikingly like those of the Granville (North Carolina) tobacco wilt. Uyeda's description of this organism differs from Bacterium solanacearum, as I have cultivated it, in the following particulars : *Kig. 127. — Agar stab and streak cultures of Bacillus nicotianae Uyeda, showing a brown stain in the upper part, the lower portion of each being unstained. (After Uyeda.) fFic. 1 28. — Bacillus nicotianae Uyeda, from an agar-plate culture 2 weeks old, showing development of the brown stain in and around the colonies. (After Uyeda.) WILT-DISEASES OE TOBACCO. 24 1 Peritrichiate flagella: There are 4 to 8, which are 3 to 4 times the length of the rod. Endospores: These are formed after a month in both fluid and solid media. They are found very often in the top of agar-streak cultures grown for 3 months at room temperatures, but not in the lower part of the streak. They are not described or figured. Early pellicle: This appears the third day on neutral peptonized beef-bouillon with subsequent brown stain of the fluid; the pellicle breaks upon shaking into parti-eolored portions, blue and white. The pellicle becomes gray and rather granular, and after a month there is a black ring. Liquefaction of gelatin: In stab-cultures liquefaction begins to appear the second day. After 4 days there is a distinct funnel-form liquefaction. After about 3 weeks all of the gelatin is liquefied, and gradually stained gray-black. In streak cultures all of the gelatin is liquefied in 2 weeks. This contradicts an earlier statement by Uyeda. Color and rapidity of growth on agar- plates: The colonies appear within 24 hours at August tem- peratures. They assume a reddish color after about a week and then gradually change to a dirty gray and black. Frequently giant colonies appear on the plate cultures and these become ringed concen- trically. Rhomboidal crystals of ammonium magnesium phosphate are frequent in the agar. Disorganization of potato slices: This takes place within one or two weeks. The growth is yellow. Coagulation of milk by the formation of an acid: Milk inoculated with this organism becomes strongly acid and is coagulated. This contradicts an earlier statement. Later it is partially pep- tonized and becomes chocolate-brown and feebly alkaline. Thermal death-point: This is 54° C. At 530 the result was not uniform. In one place 550 is stated to be the "maximum" temperature. Anaerobism: In sugar-agar it grew well in the depths of the stab. In bouillon in fermentation- tubes it clouded the closed end. It also grew very well in an atmosphere of hydrogen. Acids: Acids were developed in various saccharine media. Gas: Scanty development of gas in glucose-agar and glucose-bouillon. Kind of gas not stated. Indol: A weak indol-reaction was detected in young cultures. Stains by Gram. Reduces litmus in milk. Non-infectious to egg-plant and tomato. Other characteristics of the organism are as follows : It is a short rod with rounded ends usually 1 to 1.2 by 0.5 to 0.7/x (fig. 129). In the plant it occurs often isolated, but some- times in pairs. In cultures it is single, in pairs, or fours. Even in cultures 2 to 3 weeks old it rarely forms chains. It varies somewhat in size according to conditions of growth, e. g., in bouillon the rods are somewhat larger than on agar. Capsules occur in old agar cultures. On gelatin-plates, in 1 to 2 days, small colonies appear both on the surface and in the depths. Magnified 50 times the surface colonies are roundish with an irregular edge, some- what granular and darkest in the center. After 4 or 5 days the gelatin around such colonies is liquefied centrifugally and there is a bacterial sediment in the fluid. After 6 days the colony is somewhat beaker-form. After a week the colonies retain their original form, floating in the liquefied gelatin. The buried colonies are ellipsoidal and within a few days they fuse into a very small mass. In agar-plates (fig. 130) the surface colonies are not granular, but very smooth, moist and bright in their center. A membrane is often formed over the giant colonies. Rarely the concentric giant colonies have a serrate margin. At first the surface colonies are white. The buried colonies are elliptical or egg-shaped. They have a high sheen by transmitted light; by reflected light they are bluish-white and somewhat fluorescent. Growth on steamed potatoat July temperatures is rapid, forming after a time a greenish- yellow layer. In course of 1 to 2 weeks the substratum becomes gray-black. This pigment is soluble in water and is due to tyrosinase, an oxydizing enzyme. The black pigment from agar-streak-cultures is readily soluble in water, very little in alcohol or glycerin, and not at all in benzine, ether, or chloroform. This pigment is produced within a few days in agar, bouillon, gelatin, potato, and milk. In August, at 300 C. (room-temperature), the pro- duction of pigment was very rapid. Bouillon and milk are wholly blackened at a high temperature (350 C.) and a black ring is often formed on the wall of the tube at the surface of the fluid. 242 BACTERIA IN RELATION TO PLANT DISEASES. i» *&''' . »»**»» \- # \ • 1 »v %% t ** ^ \t * C" % » -.* ■- N* « ,"• % 1 t fc % i f * ^ 0 \ \ - * Z 1 . A I % ' * » ■' * f t" m \ ' '»" ■S t '-\ >•* .>:, * » »o Growth on cooked carrots looks yellowish at first. After some days there is a dis- agreeable odor. On cooked radish there is a sharp odor in i to 2 weeks and the tissue is then disorganized. The organism makes only a slight growth in Uschinsky's solution. The optimum temperature is about 320 C. Methylene blue (1 per cent) in bouillon is reduced. Litmus is reduced. Nitrate is reduced to nitrite (tested with meta- phenyldiamin, also sulphanilic acid + a-naphthylainin). In young cultures in peptone-water or bouillon, indol was detected in small quantity. In 10 days in 5 per cent peptone-water the cultures became so black that indol could not be detected. Hydrogen sulphide is produced. The organism was found in the earth of a tobacco field to the depth of 3.5 decimeters. Uyeda has the following on enzymes. Invertase is formed (the filtrate from a bouillon-culture passed through a Chamberland bougie inverted saccharose in one day). A very slight quantity of diastase appears to be excreted. Cytase is believed to be produced. Tyrosinase was detected in fresh agar-streak-cultures and in bouillon-cultures by the addition of paraphenyl- lg' endendiamin and /3-naphthol (Spitzer's reagent). With this there developed a pale red stain soon becoming black. If one adds a 1 to 5 per cent solution of tyrosin to a cul- ture of this bacillus the red-black color increases more rapidly than without it. Trypsin is also produced. This was demonstrated in gelatin-cultures several weeks old by adding some drops of chlorine-water (Neumeister's tryptophan reaction), the color changing to a red. In the same way bromine gives a violet color. The organism grew as well (luxuri- antly) in an asparagin-dextrose solution as in a peptone-dextrose solution. Bacil- lus nicotianae did not grow in a mineral solution (KH2P04 0.1, MgS04 0.03, NaCl 0.5 per cent) with dextrose or glycerin, and the following nitrogen compounds in 1 per cent doses : Ammonium tartrate, po- tassium nitrate, ammonium chloride. In the same mineral solution with 1 or 2 per cent asparagin and 1 per cent dextrose there was a good growth and the forma- tion of a pellicle when the medium was alkaline and no growth when it was acid. In the mineral solution with potassium nitrate and glycerin Bad. solanacearum devel- oped normally, it is said. In the writer's experiments it developed feebly as compared with the growth in bouillon. { The pigment formation is independent of magnesium salts. Fig. I30.f *Fig. 129. — Rods of Bacillus nicotianae Uyeda, from a photomicrograph. X1200. (After Uyeda.) fFic. 130. — Bacillus nicotianae Uyeda, from an agar-poured plate 8 days old. (After Uyeda.) IRepeated in 1914 using Medan III with the same results. When inoculated thinly no growth; when inoculated heavily a moderate growth with reduction of the nitrate. WILT-DISEASES OF TOBACCO. 243 Nicotiana rustica. is not attacked, and the following varieties of N. tabacum are not much subject: Ohasama, Taketadate, Mitsuke, Kentucky White, Green River Prior. Inoculations succeeded on Physalis minimum, Capsicum Ion gum, Amaranthus gangeiicus, and Polygonum tinctorum; they failed on Solatium mclongena, Lycopersicum esculentum, and Physalis alkekengi. No details are given as to methods of inoculation, phenomena produced, or number of experiments. Nitrogenous fertilizers predispose to disease; pot- ash salts do not. Early planting is the best protec- tion. Through the courtesy of Dr. Uyeda, who sent alcoholic material, I was enabled to study his dis- ease in serial sections. The bacteria closely resemble Bad. solanacea- rum in general appearance. They are enormously abun- dant in vessels of the wood, in inner and outer phloem, in the cortical parenchyma, and in the pith. I also found them to have made cavities resembling those due to Bad. solanacearum. They stained deep red with carbol fuchsin and were quite uniform in appearance. Spores were not observed. (Compare figs. 131 and 132 with those made from stems of the North Carolina to- bacco, figs. 116 and 117.) Also, with bacteria taken from this Japanese tobacco stem I made several attempts to stain the flagella. At first I got no indications of peritrichiate rods and many faint indications of Fig 132. t Fig. I33.{ polar flagella ; nevertheless no well-stained ones, such as could be relied on for demonstration. *Fig. 131. — Cross-section of a tobacco-stem received from Uyeda, showing character of the bacterial occupation in the Japanese tobacco wilt (compare with fig. 117). The section is taken from the wood midway between pith and bark, the cambium being in the direction of the arrow. The vessels next beyond V, V, V, V, Fare also occupied by the bacteria, as well as many others. Middle section of slide 482, stained with carbol fuchsin. The contrast is greater than here indicated. At X are a few bodies which look like fungus spores. (For an enlarged drawing of the bacteria see fig. 132.) . fFiG. 132. — Schizomycetes from the Japanese tobacco-stem shown in fig. 131. }Fig. 133. — Flagella on bacteria taken directly from the interior of a tobacco-stem attacked by the bacterial wilt of Japan (Uyeda's disease). Out of alcoholic material received from Uyeda. Pitfield's stain. 244 BACTERIA IN RELATION TO PLANT DISEASES. Subsequently I tried again with somewhat better results, using Pitfield's stain (fig. 133). All the flagella appeared to be polar. I am therefore inclined to think that the description of Bacillus nicotianae Uyeda may not have been drawn wholly from one organism. HONING'S SUMATRAN STUDIES. Since completion of the preceding account of the tobacco-diseases, which includes notice of one paper by Honing (p. 224), 11 additional Dutch papers (191 1 to 1913) dealing with the vSumatran disease have been received from Dr. Honing. These papers add much to our knowledge of that disease. They render it probable that it is identical with the Japanese disease and that both are due to Bad. solanacearum, first described by the writer from the United States, although some discrepancies remain to be explained. These Dutch papers are summarized as follows: (I) THE CAUSE OF THE SLIME-DISEASE AND ATTEMPTS TO COMBAT IT.— II. If two bacteria agree culturally, in reaction to stains, to substrata, etc. (making due allowance for variable culture influence), but do not attack the same plants, then we may assume that they are two nearly related but not identical species. Uyeda was warranted in separating his tobacco bac- teria from Bad. solanacearum only on the ground that the latter does not attack tobacco, a supposition which must now be abandoned, as Smith himself has shown and as has been confirmed in Sumatra. Our experiments with different strains of the bacteria have shown a great difference in virulence. The following experiments (table 25) show the infectious nature of the bacteria from different sources and the effect of a previous infection of the soil. Each of the six rows contained 100 plants, of which the first 50 in each were inoculated by soaking the seedlings for a few minutes in water to which a bouillon-culture had been added. Table 25. — Honing' s Field Results with Bacteria from Various Sources. Source of bacteria. No. of inoculations which died. Dead in control. I . Pouzolzia (Urticaceae) 10 28 5 3 18 '3 23 '5 38 10 20 '9 4. Mucuna (Leguminosae) Rows 4, 5, and 6 were on land infected about 4 months before, and row 3, which also showed many cases in the control, was adjacent to the previously infected land ; 2 of these 5 cultures were more virulent than the others. This experiment was repeated in pots with the same variable result, as shown in table 26. Table 26. — Honing s Pot Results with Bacteria from Various Sources. Origin of bacteria. No. inoculated. No. which became diseased. 10 10 20 10 10 29 0 10 20 10 O 0 Control "Why the bacteria from tobacco a were not able to attack the seedlings I do not know. For the bacteria out of Pluchea the case is different, because proof of their identity must yet be obtained." With the bacteria from tobaccos b, c, and Ageratum, the disease was visible in 5 or 6 days. The other plants were entirely sound after 24 days. WILT-DISEASES OF TOBACCO. 245 In another series of inoculations, 99 out of 103 plants contracted the disease, 13 checks remaining sound. These were as shown in table 27. Table 27. — Result of Further Inoculations by Honing. Plants infected. Source of bacteria. Number inoculated. Number slime-sick. Plants infected. Source of bacteria. Number inoculated. Number slime-sick. Ag.* a Ag. 6 Ag. a, Ti . . T. b, T,.... T. c, Ti'.... T. c, Ta.... 10 10 to 10 10 10 IO 10 10 9 9 8 Tobacco r.d T. c, T3. . . . T. c, T,.... T. c, Ti.... IO ■4 12 7 10 M 12 7 Do Solatium melongena: Violet fruited. . . White fruited. . . Pouzolzia Do.. Do Do Do.., *Ag. = Ageratum; T = Tobacco. The small letters denote plant selected. In 7 cases the strain was reinoculated into tobacco and reisolated before using, the sub-figures denoting the particular plant from which reisolated. Time alone can tell whether cultivation, rotation, disinfection, or selection of less susceptible races will prove most serviceable in combating this disease. Some planters have said that the slime-bacteria would not attack plants under ideal food condi- tions, but this can not be admitted. Inoculated potato plants (16 in number) from two sources contracted the disease within a week, although they were obtaining their food directly from the mother tuber. It is likely that any plant of susceptible sort, variety, or race will be attacked, no matter what the food conditions. It is impractical to prove absence of the slime-bacteria from the soil by the ordinary plate method, but cuttings of tobacco plants may be used. In an experiment in which soil-water from pots, which had been infected about 6 weeks before, was diluted with tap water in 5 dishes and 2 cuttings put into each, 9 of the 10 plants contracted the disease. In two tests made in the same way with suspected soil from two fields, each with 9 cuttings, 7 were positive for soil of one field, 2 only for that of the other. If such tests, therefore, are made, a considerable number of cuttings should be used. Because the soft tobacco stems rot easily, Honing substituted the harder stems of Ageratum and Pouzolzia successfully, but then had to wait longer. Although unquestionably the slime-bacteria are killed off in the soil by other organisms (other- wise there would soon be nothing else in the soil), nevertheless the fact remains that they may persist in the soil for years. The continuation of the experiments with chloride of lime and potassium permanganate yielded nothing very hopeful, as may be seen from the following experiment (table 28) on 1,392 plants in 5 similar plots: Table 28. — Effect of Soil Treatment on Sumatran Tobacco-Disease. Substance used. No. dead. No. sound. 297 9 318 2 299 ! 268 3 215 IO 2. Control 3. Potassium permanganate, 3 kg. . . . This was the worst field. None, however, were very hopeful. The best plot gave 1 1 per cent more sound plants than the control. The remainder of this paper is taken up with discussion of morphological and cultural char- acters of the slime-bacteria, which are here omitted because treated more fully in one of the following papers. (2) THE CAUSE OF THE SLIME-DISEASE AND ATTEMPTS TO COMBAT IT— III. In the year 191 1, up to April, Honing received of tobacco seedlings diseased by the slime-bacteria, 44 samples from 24 plantations. Many of these fields were visited and frequently the disease was found showing in the seedlings so slightly that they would have been selected for planting, especially those infected in the leaves. On a plantation where the leaves of the seedlings were supposed to have been injured by guano or Schweinfurth green, the actual cause of the injury proved to be this bacterium, and all stages were 246 BACTERIA IN RELATION TO PLANT DISEASES. observed between slight marginal injury and penetration of the bacteria through the side veins, into the midrib, and even into the vessels of the stem. The general conclusion from this examination may be stated as follows: "So long as the seedlings stand in the row they are more likely to be infected in the leaf than in the root. " Whether the attacked seedling is planted out depends a great deal on the age of infection. If it is somewhat advanced an intelligent coolie would reject it. On some plantations the rule is : "No seedlings shall be planted except those on which the lowermost leaves are still good." But this rule involves the destruction of many good seedlings. Also, it is not always the lowermost leaves that are first infected. The best proof that such leaf-infected seedlings can yield infected plants is the finding of plants in the field with diseased stems and sound roots. Such plants have been seen as early as a month after planting. Diseased seedlings, however, are not the only cause of diseased tobacco in the fields. This was well shown in a field where a great many of the plants died. This field was planted in part, it is true, from an infected seed-bed, but also in part from a sound bed, which on higher land yielded sound plants. Here the soil of the field, which had stood under water, was infected. A similar case was observed on another plantation. The water used for irrigation may be also a source of infection. Up to this time the water of 7 wells used for this purpose has been examined and the slime-bacteria found to a certainty in 4 of them on 3 different plantations. Because of the organism's need of free oxygen these wells are believed to have become superficially infected by the falling into them of infected earth rather than from the ground-water. This is shown also by the fact that where a new well was made the seedlings watered from it remained free, while those watered from the old well became diseased. This and other cases cited tend to show that sound plants set on healthy land may become diseased by the use of infected water. By use of 1 to 1,000 potassium permanganate such water is disinfected and is not too concen- trated for watering transplants, but would burn seedlings in the bed. It has been communicated to Honing that 0.05 per 1,000 (50 grams per cubic meter) does not burn the seedlings. In using this substance it must be remembered that in water rich in organic matter potassium permanganate soon loses its disinfecting power and greater quantities must be used, concerning which no general rule can be laid down. There are some plantations on which it is not possible to grow healthy seedlings, all of the soil being infected. If one must make seedlings on such land, then not only the water used but also the soil of the seed-bed itself must be disinfected. Sound seedlings are of the utmost importance, because a large percentage of the dead plants have come from infected seedlings. Capsicum is less susceptible than Solatium species. In one case a tobacco strain which failed on Capsicum (18 plants) became infectious to Capsicum after passing it through Mucuna. (3) REPORT ON THE SLIME-DISEASE TESTS OF 1911. General infection of the soil is probably less common than has been supposed. The tobacco has not died on all the flooded lands. The disinfections with chlorid of lime and with potassium permanganate yielded no striking results. Water from 2 1 wells was examined, of which 7 on 5 plantations yielded the slime-bacteria. Bacillus solanacearum Smith has now been isolated and cultivated out of seedlings or field tobacco from 17 plantations. While the cause of the slime-disease is the same on all the plantations,the circumstances under which infection takes place are very different and are known only in part. (4) DESCRIPTION OF THE DELI STRAINS OF BACILLUS SOLANACEARUM SMITH. THE CAUSE OF THE SLIME-DISEASE. The most important questions resulting from a study of the literature, the inoculation experi- ments on tobacco and rotation of crops, and from a study of the cultures are the following : (a) Is there to be found a series of foodstuffs, by means of which this organism can be distin- guished from others by growth or absence of growth; also how to separate virulent from non-virulent forms? (b) Is the slime-bacterium in Japan, which has been described under another name (Bacillus nicotianae Uyeda), possibly the same as that in America and in Deli, so that means of combating it elsewhere recommended can be tried here? (c) Is perhaps Bacillus sesami Malkoff in Bulgaria [see p. 216] also the same bacterial sort as that in our tobacco and our Sesamum? MORPHOLOGY. Form and size. — The Deli strain of Bacillus solanacearum is somewhat more variable than Smith and Uyeda have announced (see pp. 193, 238). WILT-DISEASES OF TOBACCO. 247 The greatest length observed after staining with carbol fuehsin and measuring without a cover glass was i.8ju. In old cultures they are shorter, i. e., o.5ju, and then look more like cocci than rods. In nearly all the preparations the bacteria are single or united in pairs. Chain-form colonies, reported by Uyeda in 3-months-old cultures, were seen only in glycocoll-glucose solutions, where arose twisted threads of 20 or more cells, among which were swollen single cells which no longer stained like the others. Motility. — In 1 or 2 day old bouillon-cultures the bacteria often show self-motility which gradu- ally ceases. Agar-cultures appear less favorable for its demonstration. Also, when freshly isolated out of tobacco, the examination is mostly negative. Flagella- staining by Van Ermengem. — More than 3 flagella I have not seen on a free-lying bac- terium, but whenever a pair lie close together there are more than 3. Capsules. — Contrary to Uyeda, I have not been able to find any capsules, not even in agar-cultures 2 to 3 months old stained with carbol fuehsin or by Friedlander's method. Spores. — According to Uyeda, B. nicotianae forms spores in cultures poor in nutrient substances, but B. solanacearum does not. Since Uyeda neglects to say how he demonstrated the spores, speak- ing neither of germination nor of resistance to dry air or high temperature, and not even of staining, he must still show proof (see under Polar staining). For the Deli strain, spore-staining with carbol fuehsin and methylene blue yielded no positive result, not even after mordanting with chromate of potash (Chroomzure Kali). The slight resistance of these bacteria to heat makes the existence of spores very improbable. Tests were made of 5 strains grown for 16 days in glycocoll-glucose medium by exposing them for 2 minutes at 80° C. and then during the next 3 minutes cooling the water down to 400 C. From each of the 5 cultures so treated 3 transfers were made to peptone bouillon, but all remained sterile. The experiment was repeated using 4 strains grown in bouillon 66 days. These cultures were plunged into water at 920 C, which in 2 minutes cooled to S30 C. and showed after 3.5 minutes a temperature of 400 C. Three transfers were then made to fresh media from each of the 4 strains, but the 1 2 remained sterile. The check tubes clouded in 24 hours, showing that each one of these strains was still living. Finally, 15 sterile strips of filter paper (3 from each culture) were wet with fluid from 5 cultures 26 days old, representing 5 different strains, placed in sterile Petri-dishes, and incubated in a thermostat at 360 C. for 45 hours, after which they were transferred with sterile forceps to sterile bouillon. In none of the 15 transfers was there any growth. Moreover, Uyeda's contention that spores develop best in exhausted media is in contradiction to the statements of Schreiber, and also those of Lehmann and Osborne. These authors find bacterial sporulation proceeds better in nutrient-rich material. Polar Staining. — Polar staining is very plain, even on material taken directly from the diseased tobacco. In order to avoid confusing this with occurrence of spores, slides were fixed in the flame and also with alcohol and colored with Loeffler's methylene blue, with Ziehl-Nielsen's carbol fuehsin undi- luted for some seconds, and diluted (1 : 10) during a minute, and with 0.01 per cent watery methylene blue solution during 5 to 30 minutes. The question now arises whether Uyeda mistook polar staining for endospores. Gram's Stain. — The Deli-tobacco bacillus is Gram-negative. There were tested at the same time Bacillus coli communis (negative) and Diplococcus enteritis (positive). PHYSIOLOGY. Generally 10 strains of the tobacco bacteria from different localities were investigated at the same time. After a couple of weeks they were again inoculated into tobacco to keep the grade of virulence and always afford comparative material. Bouillon (1 per cent Liebig's meat extract, 1 per cent Witte's peptone, 0.5 per cent salt, acid or neutral). — This is generally clouded throughout in 12 to 24 hours. Often the growth is plainly strongest above, which appearance disappears after two days. Some strains then develop a pellicle like Uyeda's B. nicotianae, but others behave like Smith's B. solanacearum, forming a pellicle first after 1 or 2 weeks. The Deli strains also stain the fluid brown, but not all of them to the same degree. A series of 36 cultures (3 from each strain) showed typical differences. The 3 cultures from one strain were colored brown after about 3 weeks, and those from 3 other strains became dark-brown during the same time, while cultures of the 8 other strains were only a little stained. On far more than 200 bouillon-cul- tures a black ring around the pellicle was never observed, but in all a gray-white precipitate. Gelatin. — The behavior of the Deli strains on gelatin is somewhat different from that Uyeda described for B. nicotianae and recalls rather that of B. solanacearum; that is, the liquefaction proceeds more slowly. Stab-cultures are funnel-form above and thread-form below, and sometimes a pellicle spreads slowly over the surface. Later short projections develop along the track of the stab, and liquefaction may occur in the clefts of these outgrowths. 248 BACTERIA IN RELATION TO PLANT DISEASES. Liquefaction begins irregularly in the upper layer mostly after 3 to 5 weeks, and slowly continues. Consequently the variability is very great, just as is the browning. Entire liquefaction in 3 weeks occurs very rarely even with weak gelatin, 4 weeks is rapid, and 2 and 2.5 months the rule, and under paraffin oil more than 3.5 months are required. The melting-point of the gelatin exercises a great influence. Always the gelatin with the highest melting-point liquefied most slowly (Non plus ultra of Von Gehe & Co., Dresden), and this stained black, while other gelatins were not colored or only became brown. These tests were made on 58 stab cultures from 15 isolations out of tobacco, 3 stab- cultures from an Acalypha stem, and 2 cultures from an Ageratum stem. In Uyeda's gelatin-cultures liquefaction with B. solanaccarum was less rapid than with B. nicotianae, i. c, 5 to 6 weeks in place of within less than 2 weeks. Because the rapidity of liquefaction is dependent on the melting-point and on the alkalinity of the gelatin, I can not say that I have found any great difference between the behavior of the bacteria investigated by Smith and by Uyeda. Moreover, not only are the different Deli strains unlike in their behavior on gelatin, but there are also distinct differences between sister cultures of the same strain. Because the time of liquefaction is three times as long for some of the Deli cultures as for others, the liquefaction difference between B. nicotianae and B. solanaccarum pointed out by Uyeda is of importance only in case it is constant. Agar. — In the beginning I obtained no blackening of agar-cultures. Later some cultures were obtained which darkened agar. Potato. — As for Smith's organism and Uyeda's, the Deli bacteria blacken slices of potato after about a week. They become, for the most part, violet-black and not gray-black. Sometimes the 4 per cent glycerin solution, above which the potato rested on a short glass rod, was colored brown. Milk. — In Migula it is stated of B. solanaccarum: "Milk becomes soapy, strongly alkaline, the casein is not precipitated. " In his preliminary communication Uyeda says of B. nicotianae: "Sapo- nifies milk," but in his complete work: "Milk first coagulated, but the coagulum is gradually dis- solved and peptonized. " These two statements do not agree. The 20 isolations first investigated behaved for the most part alike, but different from the Ameri- can and Japanese bacteria. Of 95 cultures from 14 isolations out of tobacco, 3 out of Acalypha, 2 out of Ageratum, and 1 out of Physalis, all remained nearly unchanged, there was no precipitation of the casein, but also not much alkali was produced [clearly, or the milk would have become translucent as I have described, but perhaps the cultures were not held long enough. They should have been kept under observation for at least 8 or 10 weeks]. To litmus the cultures remained feebly alkaline, and were not distinctly acid to phenolphthalcin. Only one isolation showed in 4 of the 6 cultures after 7 weeks, precipitation of the casein with weak acidity. Noticeable for the Deli isolations is the behavior in litmus milk. In 7 isolations [which are men- tioned specifically] each one of the 20 cultures rendered the milk alkaline after 3 days, which reaction progressed gradually. After 5 days each one was entirely alkaline. But two days later 16 of the 20 cultures had begun to form acid, first a small edge which became gradually broader, until finally the whole column was colored red and the milk solid. [If this may be taken as the typical action of the Deli tobacco organism and not the effect of undetected intruding saprophytes, or other non-compara- ble circumstances, then we may conclude that it is not the same as Bad. solanaccarum.} Neither in "Natura Milch, " from Bosch & Co., Waren, Mecklenburg, nor in boiled fresh Medan milk was any chocolate-brown color observed, at room-temperature or at 360 C. [see pi. 41, fig. 3]. Cultures were also made in other milks, with similar results. From his table (p. 229) it would appear that most were alkaline at first, and then acid with coagulation. A few remained alkaline, and one of these was an isolation which soon lost its virulence. The results in milk may be summarized as follows : (1) The Deli isolations coagulate and peptonize as a rule the milk of the zebu (except 4 cultures of 1 isolation of the 95 from 20 sources) just as Uyeda's B. nicotianae. (2) With sterilized European milk all the isolations show in the beginning an alkaline reaction, just as B. sola- nacearum Smith (concerning the strength of the reaction without figures nothing can be judged). The term "soapy" I do not like. (3) Some (weakened?) isolations do not proceed any farther, the most begin afterward to ferment the sugar to form acid and to throw down the casein. This is just the opposite of what Uyeda states for B. sotanacearum [and Smith also]. Conclusion. — Because the Deli isolations have given three different results depending on the age of the cultures and on the kind of milk used, the possibility is not excluded that Uyeda actually had another bacterium, but it is just as likely that if he had worked with "Nutricia, " "Landbouw," "Natura," or "Milkmaid" milk, he would have obtained the same results. Taken in connection with the numerous other points of agreement this is very probable [see pp. 263, 264]. Need of Oxygen. — This is plainly shown by the much slower growth of cultures under paraffin oil, by the form of the gelatin-stab-cultures, by the slow clouding of the closed end of fermentation tubes in comparison with the open end, and by its slow growth in an atmosphere of hydrogen in a Botkin's apparatus. WILT-DISEASES OF TOBACCO. 249 Reduction of Methylene Blue. — In 92 bouillon-cultures from 12 isolations, containing 0.002 per cent methylene blue, 9 were bleached after 1 day, 31 after 2 days, 71 after 3 days, 85 after 5 days, and 87 after 14 days, except the uppermost 3 to 4 mm. The bleaching took place after 1 day in glucose bouillon (3 cultures from each of 3 isolations — out of tobacco, Mucuna, and Blumea). Reduction of Litmus in Milk. — Litmus is also wholly or partly reduced [see p. 264]. Reduction of Sodium Selenite. — To obtain reductions the quantity of sodium selenite (Na2SeC>3) used must be small. Growth and reduction was obtained with all strains in 2 days, using 0.01 per cent in peptone bouillon agar, but no growth where 0.1 per cent was used. Reduction of Nitrate. — Nitrate is reduced when the Deli organism is grown in presence of glucose or saccharose, but not when starch is used as the carbon food, as starch is not assimilable by this organism. Diastase Production. — As mentioned above, starch is not acted upon. In this respect the organ- ism agrees with Smith's statements. Tyrosinase. — Each one of 8 isolations grew using tyrosin as carbon-nitrogen food, but less well than in peptone-water. None of the 34 cultures were actually blackened. "When one adds 1 to 5 per cent tyrosin solution to a culture of the bacillus, it takes on a red-black color quicker than with- out this addition" (Uyeda). On repeating this experiment the result was again negative, but the concentration of tyrosin was less. Without more ammonia or alkali Uyeda could not have made a 1 to 5 per cent solution, since according to Erlenmeyer only one part of tyrosin dissolves at 20° in 2,454 parts of water. Indol. — According to Uyeda, B. nicotianae produces a small amount of indol. The same is true of the Deli cultures. With sulphuric acid and potassium nitrite the reaction of 22 bouillon-cultures from 9 isolations was negative or nearly so. Repeating with 25 cultures from 10 isolations, 20 of the cultures, after heating, gave a permanent red color of extremely variable intensity. It also happened that a culture which gave no indol reaction like Uyeda's B. solanacearum, after inoculation into tobacco, gave a positive indol reaction like Uyeda's B. nicotianae. Yet is it doubtful whether indol is really produced. According to Crossonini the indol reaction of Ehrlich is much sharper with para- dimethylamidobenzaldehyd, and there are bacteria known as indol-formers on account of the old reaction, which when tested by the new method show no trace of indol. Following this suggestion, Honing tested 7 strains of the Deli tobacco-bacteria with the paradimethylamidobenzaldehyd in alcohol with hydrochloric acid, but all with negative results. Hydrogen Sulphide. — This substance is formed in very slight quantity, as Uyeda has also shown. Strips of lead-acetate paper exposed for some days remained white or were only slightly blackened. Of 18 cultures [kind not stated] from 9 sources only one gave a positive reaction. After the careful boiling away of 32 cultures (10 isolations) the lead-acetate paper was black in only 10 tubes, and here only a little around the edge, while 22 remained negative. From one and the same source sometimes one culture was positive and another negative. Fat. — Neither with osmic acid nor with Sudan III in alcohol or glycerine could the formation of fat be shown. Glycogen. — This substance is not formed by the bacteria (reagent, iodine-potassium iodide), nor can it be used as a carbon food by the bacteria, as will be shown later. Disagreeable Odor. — This is noticeable from peptone-bouillon cultures, but varies greatly in cul- tures from different sources. Isolation Ts resembled Uyeda's B. nicotianae, while others produced scarcely any odor, like Uyeda's strain of B. solanacearum. Gas Production. — Gas-formation does not occur in the closed end of fermentation tubes with glucose, saccharose, or mannit. For the most part even in the presence of oxygen there is no trace of gas-formation. Once very fine bubbles were detected in 24 glucose-bouillon cultures, 10 inoculated out of tobacco, 7 out of Physalis, 2 out of Ageratum, 4 out of Mucuna, and 1 out of Blumea. This is hardly a specific difference. Acid- and Alkali-formation. — With exception of milk (in the second stage; i. e., after the alkaline reaction), and with a nutrose-substratum, alkali is always formed. In normal growth the original always feebly acid solution became plainly alkaline. In feeble growth the alkalinity was feeblerorthe fluid had not yet reached the litmus neutral point. Acid-formation has not been detected with any single sugar when the source of nitrogen was not casein. Temperature. — Uyeda's statement that heating to 550 C. is fatal and Smith's that exposure to 520 C. for 10 minutes kills, is true of the Deli strains. The tropical climate (Medan lies in -j-30 north latitude) appears not to have increased the maximum temperature. Bouillon-cultures exposed for 10 minutes at 550 C. appeared to be sterilized (5 isolations tested). We do not, with Uyeda, consider 3° C. difference in maximum temperature as specific, since 55° C. (for B. nicotianae) and 520 C. (for B. solanacearum) are not the average of a great number of determinations with comparable material from different sources (see under "Spores"). Resistance to Drying. — This is slight, as already noted under "Spores. " 250 BACTERIA IN RELATION TO PLANT DISEASES. SOURCES OF NITROGEN AND CARBON. As a stock solution for these tests, Honing used Meyer's mineral solution.* All the substances tested were from Merck, and those marked "insoluble in water" were dissolved with as little as possible of Na2C03 or HC1. In this stock solution, adding one substance as source of both nitrogen and carbon, the results given in table 29 were obtained with the Deli tobacco bacteria. The one substance that brought out distinct differences in the isolations was asparagin. All three cultures of Tg and Th showed growth and also one of Td. The rest remained entirely clear, even after 14 days. For reasons given, the auxanographic method could not be used at Medan; the inoculations, therefore, were all made into solutions after steam-heating to ioo° C. on 5 consecutive days. Potassium Nitrate. — With 1 per cent potassium nitrate (in the following solution : KH2P04, o. 1 per cent; MgSO.4,0.03 percent; and NaCl, 0.5, alkaline) Uyeda found B.nicotianac made no development, not even on addition of 1 per cent glucose or 1 per cent glycerin, while B. solanacearum gave a weak Table 29. — Ability of Deli Tobacco Organism in Meyer's Mineral Solution to obtain both Carbon and Nitrogen from One Substance. Substance tested. Albumin Asparagin Fibrin of blood: Alkaline Acid Gluten Glycocoll Guanine: Alkaline Acid Hemialbumose: Alkaline Acid Legumin: Alkaline Acid Leucine Nuclein Witte's peptone Protein Protein (alkaline) Tyrosine Ammonium acetate. . succinate lactate. . . tartrate citrate. . No. of isolations out of — Tobacco. Ageratum. Physalis 3 7 7 9 7 9 6 9 7 7 5 3 4 5 10 10 10 10 10 No. of cultures. 42 4> 42 27 27 3' 40 29 40 30 38 30 42 4> 33 30 3' 35 31 32 33 33 33 No. showing growth. O 7 o o o o o o o o o o o o 33 o o 34 o 32 33 3° 30 growth in presence of the glucose and a normal one in the presence of the glycerin. This is wholly in agreement with the Deli isolations of B. solanacearum with potassium nitrate in glucose and does not show that Uyeda had two kinds of bacteria, since 4 of Honing's strains failed to grow like Uyeda's B. nicotianae, while several others grewlike B. solanacearum. In addition to these differences, Honing found a strain which grew well with potassium nitrate and inuline, while another would not grow. In the same way one strain grew with potassium nitrate and dextrine, while another would not grow; also one strain grew with potassium nitrate and sorbit, while another would not grow; and, finally, one strain grew with potassium nitrate and saccharose, while another would not grow. These facts, Honing thinks, should make one very careful in drawing conclusions from a small number of cultures made from a few strains. Potassium Nitrite. — This substance may also serve as food. Asparagin. — Uyeda's B. solanacearum did not grow with asparagin, while his B. nicotianae made a feeble growth. That the latter, which with asparagin alone made a weak growth, made none what- ever when 1 per cent glucose was added, should have indicated to Uyeda that bacteria are variable ♦Water, 1,000.00; KILPO,, 1.00; CaCl2, 0.10; MgSO« + 7H20, 0.30; NaCl, 0.10; Fe2Cl6, 0.01. WILT-DISEASES OP TOBACCO. 251 like other things, because, in the first place, 1 per cent glucose is not a poison and second, in an alka- line solution the growth of B. solanacearum is favored. With an eye upon the variability of the Deli strains of B. solanacearum, I must ask once more: Is the difference constant that Uyeda found? Of 12 Deli strains, 8 showed no growth with asparagin and glucose; 4 grew badly and for the most part in a portion only of the tubes. Of these 8 strains, 5 were inoculated into tobacco, reiso- lated, and re-tested with asparagin and glucose, whereupon 4 of the 5 grew in part of the cultures. These same 8 glucose negative strains were also saccharose-negative, but after 5 strains had been "freshened" by inoculation into tobacco each one of the 5 grew in a part of the cultures set, and also with potassium nitrate the same thing was shown — more grew with saccharose than with glucose. In a repetition with saccharose the same result was obtained. Table 30. — Deli Tobacco Organism in Meyer's Solution with 0.25 per cent KNOs and Carbon Foods. Carbon source. Arabinose Glucose Levulose Mannose Galactose Erythrite Adonite Sorbit Mannit Dulcit Rhamnose Saccharose Maltose Lactose Raffinose Quercit Inosite Dextrin Starch Glycogen Inulin Lichenin Glycerin Sodium acetate. . butyrate succinate malate. . lactate. . citrate. . Per cent used. No. of isolations from — Tobacco. 9 9 9 9 10 9 9 9 9 2 9 6 9 9 2 9 9 9 9 9 9 9 Ageratum. Physalis. Acalypha. No. of no. or cultures. cultures in which growth occurred. 44 19 45 27 66 0 '5 1 1 ■5 "3 ■5 12 15 5 47 38 44 39 48 46 42 23 62 50 47 33 47 38 52 50 44 "7 '5 >4 43 27 46 0 45 0 48 7 "5 0 5' 44 5' 0* 52 0 39 20 41 '7 40 0 42 24 Ammonia Nitrogen. — With ammonia nitrogen and with a suitable carbon food the Deli strains of B. solanacearum grew well, as shown by the results with ammonium succinate, lactate, tartrate, and citrate; further by 13 of 21 cultures from 7 sources with ammonia and glucose; and also by the urea cultures (46 of the 52 with glucose and 44 of the 49 with saccharose). In acid solution of 1 per cent ammonium chlorid, B. nicotianae grew for Uyeda and B. solana- cearum did not, or on addition of 1 per cent glucose only feebly, B. nicotianae also then did not. With 1 per cent ammonium chlorid and 1 per cent glucose in Meyer's solution, not neutralized, no growth appeared in 17 of the 18 cultures of the Deli tobacco organism from 6 sources, and with 1 per cent glycerin in none of the 21 cultures from 7 sources, although otherwise the growth with glycerin was comparable with that of glucose, but easier. These results harmonize well with Uyeda's when he used acid solutions. Yet after neutralization with Na2C03 (litmus neutral) the growth was very strong, as well with glucose as with glycerin. Even when inoculated from old weakened selenit- agar cultures there was still growth with glycerin but no longer with glucose. It is thus plain that ammonia nitrogen can be used very well by B. solanacearum, something that is not evident from Uyeda's communications. * Using Medan III and Florida potato (1914), the writer obtained similar results with these sodium salts, i. e.,no growth with the lactate, acetate or butyrate, and prompt moderate growth in all of the tubes (8 of each strain), using the malate, tartrate, or succinate. In Meyer's solution with ammonium citrate there was heavy growth, and with am. tartrate and am. succinate, moderate growth (4 days), but none with ammonium lactate (4 days). Both straitis grew in Meyer + urea + grape or cane sugar. 252 BACTERIA IN RELATION TO PLANT DISEASES. Substituting asparagin for potassium nitrate, the results shown in table 31 were obtained. Table 31. — Deli Tobacco Organism in Meyer's Solution with 1 per cent Asparagin and Carbon Foods. Carbon source. Nothing Arabinose Glucose Levulose Mannose Galactose Erythrite Adonite Sorbit Sorbine Mannit Dulcit Rhamnose Saccharose Maltose Lactose Raffinose Quercit Inosite Dextrine Glycogen Inulin Lichenin Glycerin Sodium acetate. . butyrate. succinate malate. . citrate. . lactate. . No. of isolations from — Tobacco. 12 6 14 8 2 2 2 2 6 6 6 6 6 '4 6 6 6 6 2 6 6 6 2 6 6 6 6 6 6 6 Ageratum. Physalis. Acalypha. No. of cultures. 62 21 81 36 '5 >5 15 15 21 21 21 21 21 79 21 21 21 21 15 21 21 21 15 21 21 21 21 21 21 21 No. of cultures in which growth occurred. 7 o 27 4 9 10 10 8 16 18 20 18 10 40 10 9 16 10 10 '3 o '9 o 12 O O 21 21 12 O Glycocoll. — B. solanacearum with glycoeoll alone does not grow, but it grows on addition of a carbon food such as glucose, maltose, or mannit, although badly in a part of the cultures and not at all with levulose. When glycocoll was used as the nitrogen food the results given in table 32 were obtained. Table 32. -Growth of Deli Tobacco Organism in Meyer's Mineral Solution with 0.1 per cent Glycocoll and the Carbon Foods Named. No. of isolations from — No. of cultures. No. of cultures in which growth occurred. Carbon source. Tobacco. Ageratum. Glucose 9 9 9 9 8 38 49 49 49 45 13 O 40 32 26 Levulose Mannit Maltose Also here occurred some typical cases of change in ability to ferment. Always in sets of 3, grew TgTi in lactose o, in maltose o, in mannit 1 ; but TgTiTi in lactose 2, in maltose 3, in mannit 3. Of 3 cultures from AbTiT! none grew with glycocoll and lactose, while of 3 others inoculated some time later from the same tube all developed. In table 33 all the results are united. The figures relate only to the proportion of cultures that grew, not to the intensity of growth: Thus 1 denotes growth in 1 to 20 per cent of the cultures; 2, growth in 21 to 40 per cent; 3, growth in 41 to 60 per cent; 4, growth in 61 to 80 per cent; and 5, growth in 81 to 100 per cent. The fact that these figures differ so widely throws a strong light on the variability of B. solanacearum. WILT-DISEASES OF TOBACCO. 253 Table 33. — Growth of Deli Cultures of B. solanacearum in Meyer's Mineral Solution with Different Carbon and Nitrogen Compounds. Carbon source. Arabinose. Glucose. . . . Levulose. . . Mannose. . Galactose. Erythrite. Adonite. . . Sorbit. Sorbine. . . Mannit. . . Dulcit Rhamnose. Saccharose Maltose. . . Lactose. . . Raffinose. . Nitrogen source. KNO3. Asparagin. Glycocoll. NHi 3 0 3 2 0 1 4 3 5 4 4 4 2 3 5 4 5 5 5 5 5 3 3 5 3 4 3 5 3 5 4 Carbon source. Quercit Inosite Dextrine Starch Glycogen Inulin Lichenin Glycerin (Sodium)* acetate.. butyrate . Ammonium tartrate (Sodium)* citrate. . . lactate. . malate. . succinate Nitrogen source. KNOi. Asparagin. Glycocoll. NHj 3 4 4 o 5 o 3 o o 3 o 5 5 *Or ammonium compound in the fourth column with NHj. Honing summarizes his conclusions as follows: 1. Bacillus solanacearum Smith often loses its virulence quickly. 2. This loss makes its appearance not all at once, but gradually, first toward Capsicum annuum, later toward Nico- tiana labacum, and finally for Solatium melongena and 5. lycopersicum. 3. These circumstances explain the contradiction in the papers of Smith and Uyeda. 4. In Deli up to this time the bacteria have been found by me in: Nicotiana labacum, Physalis angulata, Indigofera arreeta, Arachis hypogaea, Mucuna sp., Acalypha bcehmerioides, Ageratum conyzoides, Blnmea balsam if era, Synedrella nodifiora. 5. As the result of artificial inoculation the following have also become diseased: Sesamum orientate, Solatium tuberosum, S. lycopersicum, S. melongena, and Capsicum annuum. 6. Both in its morphological and in its physiological characters the variability of B. solanacearum is much greater than is at present supposed. 7. In cultures with glycocoll and glucose the bacteria form threads more than 40 cells long, which are mostly crooked, and here and there show strongly swollen, feebly-staining cells without at first showing loss of virulence. By inoculation from these cultures into bouillon the normal growth in the form of single and paired rods returns. 8. Thus far the Deli strains have formed neither capsules nor spores. For the existence of spores Uyeda has not offered the least proof. 9. The Deli strains show polar staining after fixation in alcohol or in the flame, and staining with carbol fuchsin, or water solution of methylene blue. When peptone is used the polar staining becomes indistinct or may disappear altogether. 10. Examined on a slide at the same time with B. coli communis and Dip/ococcus enteritis, the Deli strains are Gram- negative. 1 1 . The reduction of nitrate by B. solanacearum (and probably also by other bacteria) can not be studied using starch as a carbon food. 12. Sodium selenite interferes with the growth to a high degree, or even sometimes stops it altogether on addition of 0.1 per cent in peptone-bouillon-agar, but is less harmful with 0.01 per cent. The selenite is reduced. 13. The different results reached by Smith and by Uyeda when the organism is grown in milk may be partly explained: a, by difference in the bacterial strains; b, by difference in the age of the cultures; c, very probably by differ- ence in the composition of the milk. Because Uyeda in his complete paper announces a different conclusion concerning the milk cultures than in his preliminary communication, and because the behavior of the Deli strains is rather variable, one can ascribe no great worth to the discovered differences. 14. The trials with fresh milk show that coagulation takes place only rarely (4 out of 95 cultures) and that the reaction is feebly alkalin or neutral. With European milk in tins or flasks the reaction after 3 to 7 days is always alkaline. In most of the strains the reaction changed again 3 to 7 days later and the milk curdled. When inoculated from old weak cultures the reaction remained alkalin. 15. The tests with combinations of different carbon and nitrogen compounds sometimes gave constant results and sometimes variable ones, the latter even when inoculated out of one and the same culture. 16. Of the 18 substances containing both carbon and nitrogen sources, growth occurred only with tyrosine, pepton, ammonia, ammonium succinate, ammonium lactate, ammonium tartrate, and ammonium citrate, and in a few cases also with asparagin (7 of the 62 cultures, and the 7 belonging to 3 strains). 17. The following may serve as sources of nitrogen: asparagin, glycocoll, potassium nitrate, potassium nitrite, and ammonia. 18. As sources of carbon in the combinations tested the following are absolutely unusable: glycogen, starch, lichenin, sodium acetate, sodium butyrate. 254 BACTERIA IN RELATION TO PLANT DISEASES. 19. Growth never followed inoculation into cultures with KNO3 and glycocoll with levulose; with asparagin and levulose only in 4 cultures from 2 sources. This is not strange, because also with asparagin alone growth occurred only once. 20. Development in all the cultures containing asparagin, glycocoll, and KNO3 was extremely rare. Very often only one or two of the three cultures showed growth when these were inoculated from the same culture of bouillon, of KNOi or of glycocoll. 21. In general the number of cultures which succeeded with some of the alcohols was greater than with the cor- responding sugars. 22. In a great number of cultures visible growth was not synonymous with strong development. 23. Because the Deli strains of Bacterium solanacearum are parasitic as well for Nicotiana labacum and Capsicum annuum (like Uyeda's B. nicotianae) as for egg-plant and tomato (like Uyeda's B. solanacearum Smith), and moreover because nearly all of Uyeda's designated cultural differences fall away I think that B. nicotianae Uyeda is identical with B. solanacearum Smith. (5) REPORT ON THE ATTEMPTS TO DISINFECT WITH CHEMICALS THE SEED-BEDS ON SLIME-SICK SOILS. On 20 plantations belonging to 1 1 companies tests were carried out, using potassium perman- ganate, formaldehyd, and carbon bisulphide. The seedlings on 6 of the plantations were destroyed by ants and crickets. Report is made of the results on each of the other 14 plantations which show some success with the disinfectants, but not brilliant nor uniform. The entire subject is summed up in the following conclusions: (1) That in spite of the disinfection of seed-bed and of the well-water used, when the seedlings were planted on infected ground infections took place. (2) That this infection, as far as could be made out, took place more through the leaves than through the roots, so that the infection must have been brought to them from without. (3) How this transmission took place (whether by animals or tools) must be followed up and experiments are already under way. (4) It must also be looked into closer whether the disinfection of the whole seed-bed ground gives an important advantage over the disinfection, simply, of the seed-beds, leaving the paths between untreated. (5) That far away, in most cases, the plants were larger and more vigorous on the treated than on the untreated seed-beds. (6) That in all cases where differences appeared after planting out, they were to the detriment of the untreated seed-beds, so that it certainly is desirable to repeat the tests. (6) A CASE OF SLIME-SICKNESS IN DJATTI SEEDLINGS. This was found in Djatta plants [Tectona grandis] standing among diseased tobacco. The signs appeared to be those of the slime-disease. Plate cultures were set and three sorts of bacteria were isolated, one of which agreed culturally with B. solanacearum from slime-sick tobacco. Although the identification by cultural tests left no room for doubt, inoculations were made into 4 tobacco seedlings, all of which became diseased. Out of two of these plants the bacteria were again isolated and served for new inoculations along with a strain isolated from tobacco. With these three strains 15 young tobacco plants, on land not subject to the disease, were inoculated by a prick, 5 from each culture. At the same time 9 young Djatti plants were inoculated, 3 from each culture. All of the tobacco plants showed the disease at the end of a week ; the Djatti did not. Three days later the tobacco was removed and the earth disinfected, although the disease was still confined to the top of the plant, i. c, not yet in the roots. All the Djatti plants were now diseased, not only those inoculated from the Djatti cultures, but also the 3 inoculated from the tobacco strain. No difference in virulence was observed on Djatti plants, but on the tobacco DTi was much the more virulent. The checks were sound, both the Djatti and the tobacco. With other cultures from the same source the inoculation experiment was repeated 4 days later on a trial field on 15 Djatti seedlings with the surprising result that only one of them died, the others being not much affected. Six weeks later, however, two other cases were found in the plants inocu- lated with the Djatti bacteria. The disease was visible soon after a couple of showers and the bac- teria were demonstrated microscopically in the stems more than 2 dm. above the point of inoculation. Later with DTi and T four tobacco plants in pots were inoculated, all of which became diseased in 5 to 7 days. By inquiry Honing learned that on other plantations occasionally diseased Djatti had been observed. (7) ON THE CONNECTION BETWEEN SLIME-SICKNESS IN SEEDLINGS AND IN PLANTED-OUT TOBACCO. In "Cause of the slime-sickness and attempts to combat it — III," were cited some instances of direct connection between disease in the seed-bed and in the field, even in cases where the disease in the seed-beds was only sporadic and sometimes was not known. In "Report on the slime-disease WILT -DISEASES OF TOBACCO. 255 tests in 1911," the number of examples were somewhat increased. Here are added some observa- tions of 191 2, made on tobacco seedlings received from 1 1 sources and set on the trial grounds. The conditions of the experiment and the results are given in table 34, omitting only the dates of planting (February 14 to March 30). Table 34. — Effect of Occurrence of Disease in Seed-bed on Planted-out Tobacco. Planta- tion. 2 3 a 36 4 5 6 a 66 7 8 9 10 1 1 a 11 b 8a 86 Condition of seed bed. No disease. Quite a good many diseased. ....Do Less sick than in 3a A few diseased Many diseased Very few diseased ....Do Quite a good many diseased . ....Do Few diseased ....Do Few diseased (specimens with bacterial stripe). Sound out of the same infected beds. . . . Very few diseased (specimens with bacterial stripe). Sound out of the same sick beds Prediction as to outcome. Few dead A good many dead. Many dead Less dead Not many dead. . . . Many dead Few dead ....Do Quite a good many diseased. .... Do No remarks Few dead All dead No. planted. Few dead All dead.. Many dead. 36 72 54 54 72 72 54 54 54 54 72 72 15 20 21 45 No. Dead 27 12 22 I I 21 42 6 7 21 '5 '4 13 15 4 20 23 P. ct. dead. 75 17 41 20 20 58 1 i '3 39 28 19 18 20 95 Remarks: Predic- tion fulfilled (+) not fulfilled (-). — (Toa-toh and drought). + + + + + + + + Toa-toh bibit + + + + + The plats 116 and 86 are perhaps the most interesting. Naturally, sources of error were not wholly excluded. The factors which may have influenced the result are: (a) The soil was not wholly free from the slime-disease. (6) The persistent drought. (c) A toa-toh plague, especially for 1 to 9; the latter had toa-toh in the seedlings. (d) The fact that the seedlings from all sorts of ground, loose red earth to low white clay, were planted out on the same piece of clay ground. (c) The small number of plants. All things taken together, it is plain that the dying in the field tobacco was more prevalent according as more sick seedlings occurred in the seed-beds. (8) ON THE ASSERTED NON-SUSCEPTIBILITY OF NICOTIANA RUSTICA FOR THE SLIME-DISEASE. Two ways stand open for him who would obtain a race of resistant Deli tobacco : The first looks to the discovery of fine sound specimens on slime-sick soil ; the second to the crossing with less sus- ceptible or wholly resistant varieties of tobacco. Upon the first method more later. In order to cross the Deli tobacco with immune sorts we must first have these, and if obtained, it remains to be seen whether the desired peculiarity is transmitted in full measure to the descendants and is constant in the cross. If this should fall out according to wish, then comes the question whether the type obtained is of a sort that the planters will try, or whether it must be further im- proved, retaining at the same time the desired resistance. Tests were also made on a number of ornamental tobaccos, seed of which was received from Haarlem, viz., Nicotiana afinis, N. atropurpurea grandiflora, N. colossea, N. glauca, N. sanderae (in 6 colored varieties), and N. silvestris. Both the Djatti and the tobacco strains were tried and all of the inoculated plants became diseased. Nicotiana latissima is also subject. The finer sorts of foreign tobacco of the species N. tabacum, so far as tested in Deli, are badly attacked by the slime-sickness, so that the chance of obtaining a reistant variety from these seems not better than from Deli tobacco. Of the coarser sorts it has been announced in literature (Uyeda) that one, namely, the Boer tobacco (N. rustica), is non-susceptible to the slime-disease. There were obtained, therefore, from the Agricultural Institute at Pusa in India, where this tobacco is commonly cultivated, seeds of different varieties to the number of 20, which varieties were tested by inoculation, only one of 200 plants remaining sound. The details are given in table 35. 256 BACTERIA IN RELATION TO PLANT DISEASES. Table 35. — Result of Inoculating B. solanacearum into 20 Types of Nicotiana rnstica. Type. Pot trials, infected 3 days after planting. Field trials, infected 10 days after planting. Slime-sick after infection with bacteria from — Slime-sick in the controls (of each 4 examples). Slime-sick after infection with bacteria from — Slime-sick in the controls (of each 8 examples) . Djatti stem (of each 3 examples). Tobacco stem (of each 3 examples). Djatti stem (of each 2 examples). Tobacco stem (of each 2 examples). I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII XVIII XIX XX 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 0000-000000000000000 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 "The possibility remains that Uyeda possessed another truly immune variety, yet his communi- cations on other infection experiments which have been described earlier, make this highly improbable . ' ' (9) ON THE VARIABILITY OF BACILLUS SOLANACEARUM SMITH. During the search for combinations of carbon and nitrogen foods by means of which the slime- bacteria could be easily recognized, a variable behavior was often observed in media inoculated from the same culture. That this did not depend on faults of technic is shown by the fact that transfers of the bacteria into far more than 200 cultures of bouillon always succeeded; also all grew when inoculated into pep- tone-water (33 cultures), solution of ammonium succinate (32 cultures), ammonium lactate (t,^ cul- tures), asparagin with sodium malate (21 cultures), and sodium succinate (21 cultures). In the test-tubes in which visible growth occurred the originally feebly acid reaction of the solu- tion had become neutral, or alkaline (if growth was strong), while the tubes that remained clear always retained the feebly acid reaction to litmus. Experiments were then made by inoculating into solutions whose acidity or alkalinity had been increased slightly, with the results given in table 36. Table 36. — Growth of B. solanacearum in Meyer's Mineral Solution with 0.2 per cent Glycocoll and Glucose or Mannil with Variable Reactions (Cultures always in Sets of 5). Carbon food. Per cent N/10 sulphuric acid or sodium hydroxide added. Source of bacteria used. No. of tubes clouding. Ai. A,T. A2T. Ti. Glucose, 1 p. ct Do 2.5 H-SO! 3 3 1 2 2 O O 0 O 0 3 3 3 3 2 1 0 3 3 3 3 2 (of 2) 2 (of 2) 3 0 3 3 3 I.25 H.SO4. ., Do Do I.25 NaOH . Do 2.5 NaOH Mannose, 1 p. ct Do 2.5 H-S04 1.25 H2SO4... Do Neutral Do 1.25 NaOH 3 3 Do 2.5 NaOH. WILT-DISEASES OF TOBACCO. 257 The bacteria taken from one species of plant did not vary in media more than those taken from another. Of 27 isolations of B. solanacearum (20 from tobacco, 2 from Acalypha bcehmerioides, 1 from Ageratum conyzoides, 1 from Synedrclla nodi flora, and 3 from Tcctona grandis), all varied more or less strongly. The retardation of growth due to sodium hydroxide is better shown in table 37. Table 37. — Retarding Effect of Alkali on B. solanacearum by Days (Tests in Sets of 3). Glycocoll+glucosc solution. Days. I 2 3 4 s 6 7 8 9 10 With 2.5 p. ct. N/ioH2S04 With 1.2s p. et. N, 10 H2SOj.... Neutral O 0 O O 0 2 1 O 0 O 3 3 0 0 0 0 1 0 0 0 1 1 O 1 2 2 I 2 2 1 2 2 I 2 2 With 1.25 p. ct. N/10 NaOH. . . With 2.5 p. ct. N/10 NaOH. . . . If one of the cultures succeeded, then from that others could be inoculated with a greater degree of success, as shown by table 38. TablR 38. — Results of Further Studies of Sumatran Tobacco Organism in same Acid and Alkaline Media (Always in Sets of 3 Tubes). Glycocoll + glucose solution. Days re- quired for growth in original culture. Days necessary for visible growth in second series. 1 2 3 4 With 2.5 p. ct. N/10 H2SO4 With 1.25 p. ct. N/10H2SO4 2 3 6 A 7 O 0 0 0 0 2 3 3 0 0 3 1 3 3 With 1.25 p. ct. N/10 NaOH With 2.5 p. ct. N/10 NaOH Temperature and light are said to play no role in this variability. The test-tubes had been cleaned with acetic acid, but some soaked in chromic-acid cleaning mixture gave the same result.* With asparagin and potassium nitrate the results shown in table 39 were obtained. Table 39. — Variable Behavior of Different Strains of Sumatran Tobacco Organism to Combinations of Carbon and Nitrogen Foods (Tests always in Sets of 3 Tubes each). Feebly acid solution. Strain of B. solanacearum used. Feebly acid solution. Strain of B. solanacearum used. A! A,T. AiT. •n T2. Al. A,T. AjT. T,. TV Asparagin: 0 0 0 1 0 0 0 3 2 2 1 1 0 2 2 3 3 3 0 0 3 2 3 3 3 0 0 1 1 2 3 2 3 0 3 KNO3: Adonit 2 I 3 0 0 3 I 3 3 2 0 0 3 I 3 3 3 0 0 3 2 3 3 3 0 0 2 0 1 3 3 0 0 0 Erythrite Galactose Erythrit Galactose Levulose Lichenin Mannose Levulose Lichenin Mannose Further studies showed that media which failed to show growth when inoculated with small doses of the bacteria would do so when inoculated with larger quantities, as shown in table 40. Similar results were obtained in neutral asparagin-glycocoll-KN03, or urea solution, with glucose or mannose, the heavier inoculation yielding the better results. When measured quantities of the bacteria were used the results shown in table 41 were obtained. *MedanIII and Florida potato (1914) grew in Witte's psptone water + H2S04 (1:10000), but not in the same acidulated 1 :iooo (3 days test at 27° C). 258 BACTERIA IN RELATION TO PLANT DISEASES. TablB 40. — Growth of Strain A2T in Glycocoll-glncose Solution after Inoculation with a Small and a Large Loop of Culture Fluid, the Latter Carrying about 5 times as many Bacteria as the Former (Tubes in Sets of 3). Glycocoll + glucose solution. Day, and small or large loop. 2 3 4 5 6 10 Small. Large. Small. Large. Small. Large. Small. Large. Small. Large. Small. Large. With 2.5 p. ct. N/ioH2SO„ With 1.25 p. ct. N/10H2SO4.... Neutral 0 0 0 0 0 0 0 0 O 0 0 0 0 0 0 0 3 2 3 1 1 1 I O 0 2 3 3 3 2 I I 1 0 0 2 3 3 3 3 2 3 2 0 O 2 3 3 3 3 3 3 2 1 0 2 3 3 3 3 With 1 .25 p. ct. N/ 10 NaOH . . . With 2.5 p.ct. N/ioNaOH. . .. Table 41- -Time of Clouding in Neutral Glycocoll-glucose Solution when Inoculated with Variable Numbers of Sumatran Tobacco Bacteria (10 Tubes used in each Case). Days required for appearance of growth.. Number of bacteria used for inoculation of each tube. 300 3.000 30,000 2,400,000 6 10 15 20 O O O 0 0 1 I I 1 4 9 10 10 This disease has now been observed in Sumatra in 15 species of plants belonging to five families. The author summarizes his conclusions as follows: 1. With a great number of carbon and nitrogen foods B. solanacearum shows growth only in a part of the cultures inoculated. 2. Light and temperature have no influence, and small variations in alkalinity have slight influence on this variability. 3. More cultures succeeded after a larger quantity of inoculating material was used. Only a very slight number of individuals, one out of many dozens, is in condition to adapt itself to the new nutrient conditions. 4. The phenomena fall under the concept of modification; the acquired peculiarities are not constant, when the bacteria are once more inoculated into tobacco, reisolated, and again brought into the same solution. There is perhaps another explanation for the variable behavior of this organism in unfavorable solutions, viz, accident. On this supposition all the bacteria are equally capable of growth, but only those actually grow which happen to be lodged under the protecting cover of their fellows, i. e., those in the center of small pseudozoogloese masses. If no masses large enough to protect the first stages of growth are introduced then those particular tubes will remain sterile. This is indicated to me by the fact that Honing often observed the first indications of growth in glycocoll solutions "as little points on the wall of the tube which may grow slowly for some days before the fluid begins to be clouded." Glycocoll solution was used because Bad. solanacearum was easily identified in it on account of the development therein of the peculiar chains and involution forms already described. (10) ON THE DECAY BACTERIA FROM SLIME-SICK TOBACCO, DJATTI, AND SOME OTHER OF THE SLIME-SICK SUSPECTED PLANTS. In December 191 2 Honing published another paper, with the above title, in which he reported on the common bacteria found by him in Sumatra associated with the bacteria causing wilt of tobacco and other plants, but themselves not able to cause any disease when inoculated into tobacco. The following sorts were isolated and studied by him and the new species are described : WILT-DISEASES OF TOBACCO. 259 Micrococcus luteus Lehm. and Neum., Micrococcus pyogenes albus (Rosenbach) Lehm. and Neum., Micrococcus pyogenes (M. bicolor Zimmerman), Bacterium medanense n. sp., Bacterium stalactitigenes n. sp., Bacterium langkatense n. sp., Bacterium deliense n. sp., Bacterium shutfneri n. sp., Bacterium zinnoides n. sp., Bacterium sumatranum n. sp., Bacterium patelliforme n. sp., Bacterium aurantium roseum n. sp., Bacterium rangiferinum n. sp., Bacillus mycoides Fliigge, Bacillus mesentericus, and Corynebacterium piriforme n. sp. These generic names are used in the manner of Lehmann and Neumann, some of the forms being non-flagellate, others polar-flagellate, and others peritrichiate. The most interesting discovery recorded is that of the inhibiting action of some of these saprophytes, e. g., B. mycoides, and especially B. mesentericus, on the growth of Bad. solanaccanitn (fig. 134). The following is an abstract of Honing's last paper. (II) HOW SHALL ONE OBTAIN A RACE OF TOBACCO THAT IS IMMUNE TO THE SLIME-DISEASE? Probably the application of some form of selection in tobacco-culture in Deli is as old as the culture itself. The founder of the industry, Nienhuijs, had seed imported from Java, Cuba, and elsewhere, and selected that which gave the best results on the new soil. Obviously he selected with a practised eye and lucky hand : A valuable new variety, the Deli tobacco, being the result. No one knows the age of Deli tobacco in its present form. Perhaps it is the result of one or more accidental crossings among the sorts imported into Deli ; perhaps it originated through a splitting- caused or not by crossings in Deli — whereby arose combinations of char- acters which the first Deli plants really owe to crossings that took place years before in Java or Cuba. Perhaps, also, the new type arose suddenly or by degrees through ex- ternal conditions, which in Deli differ from those of the original habitats. Of this we know nothing, and prob- ably never shall be able to determine. A similar ignorance concerning the time and manner of origin of varieties is, alas, the rule. Of almost all culti- vated plants having a great number of known varieties, the origin lies in the dark, and only of forms which have been isolated in recent years does one know a part of their history. Only this is certain: In Deli a variety has arisen which did not previously occur in the older tobacco lands and is not found there at present, unless introduced from Deli. Wherever Deli tobacco grows outside of Sumatra: in Java, the United States, Cuba, Hawaii, everywhere one speaks of Sumatra tobacco, which would not be the case if, in the older tobacco districts, the Deli form had been recognized as a variety indigenous there. Had such been the case, so much trouble would not have been taken on all sides to make the variety flourish. Thanks to the favorable results in the years 191 1 and 19 12, one hears less said now than was the case three or four years ago, regarding the degeneration of the Deli tobacco. For *FlG. 134. — Drawing of a Petri-dish agar-poured-plate showing the inhibiting action of Bacillus mesentericus on the Sumatra tobacco bacterium. This experiment was made as follows: The letter A was painted on the bottom of the dish with a spore-bearing culture of Bacillus mesentericus. When this was dry it did not show. A tube of melted agar was then inoculated heavily with Bacterium solanacearum and poured into the dish. After some days, the letter A appeared and some surface colonies of Bacillus mesentericus which were derived from bacteria flooded from the letter A at the time the plate was poured. Bacterium solanacearum has also grown, but only at a distance from the former. The legends are: 1, Bacterium solanacearum; 2, Bottom growth of Bacillus mesentericus; 3, Surface growth of Bacillus mesentericus. (After Honing.) Fig. 134.* 260 BACTERIA IN RELATION TO PLANT DISEASES. the benefit of those who still believe in the degeneration of the Deli tobacco, it is perhaps of service to state that the first Deli planter, fifty years ago, lost a whole field through "the death." At that time the tobacco was immune to the slime-disease even less than now. From this we may conclude that the slime-disease bacteria were here earlier than the culture of European tobacco. Always and everywhere goes hand in hand with extensive culture the multiplication of parasites, animal as well as vegetable, as the result of the one-sided activity, and always and everywhere it has cost much labor to keep a crop on the same level and still more to improve it. Therefore, especially in recent years, men have begun to select examples more resistant to disease, and have found these in forms which, in other respects, are unfit for culture, so that they have tried through crossing, followed by systematic selection, to produce the desired combination. In both ways, that is by selection and by crossing, the attempt must be made to produce from the Deli tobacco a less susceptible race. The selection can begin immediately; for the crossing one must wait the discovery of immune varieties, which must not be far re- moved from the Deli tobacco in quality. And because a variety, which elsewhere is little or not at all susceptible, may be extremely sensitive when subjected to the different soils and climatic conditions in Deli, one must not rely upon the literature, but all varieties must be investigated in Deli in regard to their susceptibility. This year tests have been made with 87 sorts or varieties, among which were good varieties from Java, the United States, Cuba, Mexico, Brazil, Paraguay, Santo Domingo, the Philippines, Japan, and Hawaii. In a later report these experiments will be described in detail. The following are the results in brief : (1) All of the 87 sorts or varieties investigated were susceptible to the slime-disease. (2) The 7 strains of Deli tobacco, which were inoculated at the same time as the foreign tobaccos, did not make a bad showing. They grew better after the inoculation in the stem than a great number of foreign sorts. (3) Two sorts of Philippine tobacco and one Japanese variety, although diseased, gave the impression of being less severely attacked, so that it is worth while to repeat the experiment on a larger scale with these three varieties another year. (4) The disease progressed more rapidly when the inoculation was made with a great number of bacteria than with a few. This last-mentioned fact affords perhaps the explanation of the remarkable phenomena which occurred in the disinfection-tests in 191 1 . In the different experiments the results of the disinfection were at first gratifying, but in the end, when the plants were examined, it appeared that not any or only a very slight advantage had been gained. But in the mean- time in one of the experiments on a trial plot which had been kept under longer and closer observation there were on each plant five to eight leaves more ("scrubs" and basal leaves). Thus disinfection was insufficient, but the important lessening of the number of bacteria had been advantageous. Now it appears that the rapidity with which the disease progresses in a plant is depen- dent on the number of invading bacteria [see pi. 31, middle and right figs.]. This affords perhaps the explanation of the above-described phenomena in the disinfection-tests of 191 1. The chance of finding among foreign tobaccos one which is immune in Deli appears to be not very great. However, the fact that among the 119 examined varieties or sorts not a single resistant one was present does not mean that none exists. The investigation, which fortunately does not consume an undue amount of time, must be made with still other sorts. Only, one must not raise too high his expectations of finding among the known varieties one which in Sumatra is immune to Bacillus solanacearum. And this is surely an additional reason for immediately subjecting our Deli tobacco itself to a severely conducted selection. There are various reasons which make it more than probable that the Deli tobacco is not a clearly circumscribed type, but that it consists of a mixture of several types — how WILT-DISEASES OP TOBACCO. 261 many can not be estimated. Apparently there are only a few, and in any event they are not of such a kind that one can go out into the field and select them. The Deli seed is much purer in Sumatra than that, for example, which was grown some years ago in America, when Shamel found 1 1 types in one field. The " Deli seed " with which this field in Connecticut was planted came from Florida and may thus have been mixed with other sorts. Proofs that in the Deli tobacco hereditary differences still appear are but two, namely, the giant plants and the Strain B 1-5, which in 1906 was obtained in a mosaic-disease test, undertaken with Helvetia and a couple of other varieties. These plants, B 1-5, blossomed in 1907 about 10 days earlier than the others, formed thus a premature type, and this year the sixth generation again blossomed 10 to 12 days earlier than the others. Dr. Diem found in 1907 that the average difference in height between B 1-5 and the race B 1-1 was almost half a meter to the disadvantage of B 1-5, which also produced on an average four leaves less. Moreover, these differences, which indeed that one year were somewhat greater than in other years, have persisted, so that B 1-5 is to be considered a separate type. Besides these two examples there are perhaps more. One often finds in the plantations plants which vary. When it is a single example which is distinguished by broad or small leaves, there is no reason to consider it immediately a separate type. On the contrary, when one gets this impression from a whole plot (afdeeling) it proves that two forms, a broad-leaved and a small-leaved type, have been planted side by side. I remember such a case in an upland plantation in 191 1, and I am not the only one who was subject to this impression. Up to the present there have been but few experiments in which selection has been exercised in the sense of pedigree-culture, such as was advised as early as 1907. One gener- ally restricts himself to the maintenance of a fine stock from which the less vigorous plants are removed; thus a form of "mass-selection." Unquestionably this is one means of keep- ing the culture up to the mark, but the chance of making any advance in this way is ex- tremely slight. To be sure by this method the seed of the best plants is always collected and when there happens to be among them a couple of extra good plants which will produce better offspring, the seeds of these are also gathered, and are not lost. But this seed of the uncommonly good plants becomes diluted with the quantity of common seed which out- number them by some hundreds or thousands. On sowing, the descendants of the best plants are lost in the great mass. In case the circumstance should arise that the descend- ants of such a plant with large leaves should grow somewhat less rapidly in their youth, something more happens, namely, these will disappear during the thinning out, with the result that in the crop, the same as the previous year, but few examples of the better type will appear. If now, again, on a good piece of ground a portion of these be selected for seed plants, then is one just as far advanced as the year before — a single plant of the better type here and there in the great mass. Had one, however, kept separate the seed of a couple of extra fine plants, then it would soon be evident whether the wished-for characteristics were hereditary or not. A concrete example will make this still clearer. On two plantations there has been made this year, at my request, a beginning in selection-tests for immunity to the slime- disease. In one plot on one of these plantations, where there had been much damage from the slime-disease, there were in the worst field 40 plants which had remained intact (zich goed gehouden hadden), and which were guarded from cross-pollination by gauze sacks. The plants had the greatest chance of becoming infected, since the mortality on this spot had amounted to at least from 60 to 80 per cent, and the rest, especially toward the last, appeared to be very much diseased. At the present time it (the disease) is not evident on the plants, which appear to the eye to be sound. It is possible that by chance they have not come into contact with virulent bacteria and possibly in case of an infection they might be very susceptible, but there is also the chance that some of them would be somewhat more resistant, and a single one much more resistant than the average, to say nothing of 262 BACTERIA IN RELATION TO PLANT DISEASES. immunity. vSince all this is not evident to the eye, too small a number of plants should not be isolated, and the seed from each must be watched, kept, and sown separately. Suppose the case that one plant of the 40 was by inheritance so much more resistant than the others that under exactly similar circumstances and on the same kind of soil mortality among its offspring, instead of being from 60 to 70 per cent, was only 5 per cent, while all the rest were barely alive as the result of infection. Now, what would happen if the seed of the 40 plants were mixed ? The mixed seed would give a crop which under the same conditions and on the same kind of soil and with the same amount of infection as the year before, would again show 60 to 70 per cent dead plants. The one superior plant has no noticeable influence on this mixture with the 39 others, and the result is that one must say: "This is just as bad as last year. This method amounts to nothing. Away with it!" If one had, on the contrary, kept this seed separate and planted its offspring separately, then it would be evident that of the 40 selected mother-plants there were 39 worthless ones and but one which would be of service for further testing. Thus in two respects the above-described method of work (that is, the method having, as its special purpose, the obtaining of an immune or more resistant race of tobacco) differs from the one customarily employed : (1) The seed-plants are not selected from the best soil, but preferably from the "sick soil" or places where the loss from the slime-disease has been very great. The chance of resistant individuals becoming conspicuous there is much greater than in places where not a single plant has been exposed to the disease, and thus no plant gets the opportunity of showing its ability to withstand adverse conditions. Twenty or thirty plants remaining sound in a field in which there is much disease give more chance of results than a thousand from a field without any disease. (2) The seed from every one of the carefully selected plants is watched and sown separately. Pedigree-culture is introduced. That these methods involve extra labor it is useless to deny, but without work one accomplishes nothing. In great companies it will certainly be desirable to have a separate "seed assistant," but by "seed assistant" must be understood something other than a person who only sees to it that the company obtains the prescribed number of packages or bottles of clean seed from well-cared-for, active plants. In my opinion he must be a man who tries to select the types most suitable for certain kinds of soils. For this is one of the great differences between the modern culture and the old: one chooses the plant-type according to the kind of soil, well knowing that the soil may be improved but not changed in character. Since it is now known that in Europe for clay, sand, and peat soils, or for com- binations of all kinds of soils, definite varieties of grains, potatoes, cabbage, apples, etc., are used, why should not the same principle be applied to Deli tobacco? Only when it is certain that the Deli tobacco is a constant variety will efforts in this direction have no sense. But the complete uniformity of the Deli tobacco is anything but decided. Every planter knows instances of his ability to recognize in the curing-room the tobacco from a certain field by its color, texture (soepelheid), or by almost indescribable differences. .Sometimes variations in rainfall are the cause of the difference, but sometimes there are variations occurring every year, caused by an entirely different composition of the soil. In most cases the crop thus differentiated is less fine. Would it not be more profitable to apply at once a system of strict selection with reference to the soil? A principle that was very much applied in the past and still is followed is this: Use for an upland plantation seed from a lowland plantation, and vice versa. The purpose of this restoration (bloedverversching) has never been clear to me. Different companies have also resumed growing their own seed, which is continually used for the same plantation; a single company does this for every field. That is surely a step in the right direction. But one must, in my opinion, go still further and give attention to the kind of soil rather than to chance boundaries of the plots or experiments, so that one plantation must try WILT-DISEASES OF TOBACCO. 263 to cultivate two, and if need be three, types which are continually kept for the same kind of soil. This is directly opposed to the principle of "blood-refreshing." As it is agreed that in this "blood-refreshing" the habitat and the climate have influence on the charac- teristics of the offspring, something which is not proved but which we grant for the moment, why should one use seed from a hill plantation which always has sufficient, sometimes too much, rain, for cultivation on the coast where the tobacco stands every chance of being obliged to go through a long period of drought; that does not seem to be logical. If from the Deli tobacco several types can be isolated, and this seems probable, never- theless one must not suppose that they will be precisely alike in their ability to endure drought or much moisture. vShould not something be established in this direction on the coast plantations, where all the growers are again complaining? What has been said here about selection in general is also of value in regard to the selection for the purpose of obtaining a race more resistant to the slime-disease. Also in this connection one will do well to take into consideration the kinds of soil. What will happen I can not assert, but the possibility is not excluded that some one will obtain a more resistant race which is of no value for his neighbor who has different soil, and is perhaps still more susceptible than that which he has been accustomed to plant formerly. What makes this selection difficult is the fact that one can never see in a plant what he has there, although always its value as a seed-plant can be judged the following year in the descendants just as leaf-production is best judged in the curing-house. Two plants of similar height, with exactly the same number of leaves of about the same length and breadth, in short, plants which every one would consider equally valuable, may bring forth offspring which differ to an important extent. A plant with 30 leaves may belong to a family which brings forth on an average 30 leaves, but it mav also be an exceptionally fine specimen of a family which produces on an average not more than 25 or 26, or, finally, an inferior example of a family which averages 33 leaves per plant. All this indicates the necessity of sowing separately purely pollinated seed from each plant. In such pedigree-cultures two or three years must elapse before we know whether the selected lines are constant. This affords the great advantage of comparing pure lines with the ordinary crop for two consecutive years, not only in the field but also in the curing-house. AMERICAN STUDIES OF THE SUMATRAN ORGANISM. Through the courtesy of Dr. Honing the writer received cultures of the Sumatran tobacco organism in the summer of 19 13. One was sent direct, another via Amsterdam, where the original tube was retained and a transfer sent on. Later a third came direct. The three cultures reached Washington in good condition and proved infectious to tobaccos, on which the external signs and the tissue disorganization could not be distin- guished from those due to the North American tobacco-wilt organism. Black stripes appeared on the stems, with yellowing and irregular drying-out of the leaves, the vessels in which were stained black and filled with the bacteria, especially those of the mid-rib. In the sections which I saw, the bacteria were confined to the inner wood, in which bacterial cavities had formed. Some of these sections were made 5 or 6 inches from the needle pricks. Up to the end of the sixth week (when the first experiments were broken off) the be- havior of this organism in our tubes of milk and litmus milk corresponded closely to that of the Granville tobacco-wilt organism, i. c, there was no development of acid or separation of the casein, but only a progressive bluing of the litmus.* Subsequently one of our stocks of litmus milk reddened. I thought at first that the reddening of the milk was due to an unsuspected acid-forming anaerobe, which was not killed by the steamings, and which was unable to make its presence visible except under the protecting influence of some active aerobe, such as Bad. solanacearum. With this in mind we made tests in deep tubes of agar in the following way : Stabs were made the whole length of the agar from litmus milk which had been inoculated with the Medan organism and which *Plain milk tube-cultures 7 weeks old were translucent and brownish. In another set a pencil could be seen behind those S weeks old, although the casein was not precipitated. 264 BACTERIA IN RELATION TO PLANT DISEASES. had reddened. On the surface of the agar a greedy aerobe was then sown (Bacillus subtilis) with the idea that if an anaerobe were present in the milk it would develop in the depths of the agar, whereas if only Bad. solanacearum were present the growth would not appear along the line of the stab or would occur only in the top of the stab. The second of these supposi- tions proved to be the true one, indicating that the litmus-milk cultures which reddened contained only an aerobe, viz, the Medan organism. Owing to the fact that the media which reddened had been made from uncentrifuged milk containing cream, whereas our ordi- nary litmus milk is always made with centrifuged cream-free milk, it then seemed likely that the acid must be due to the action of the bacteria upon the cream, not on the milk sugar, and this was borne out by subsequent tests. When we inoculated skimmed (cream-free) lit- mus milk we always got a gradual blueing of the litmus, just as in case of the American organ- ism, and never any subsequent reddening. On the contrary, whenever we added cream to the skimmed milk or prepared the litmus milk out of milk containing cream, we got a red- dening which began always in the layer of cream at the top and gradually progressed until the whole tube became reddened (pi. 44, figs. 2, 3, 4). There can be no doubt I think, therefore, that the discrepancy be- tween Honing's results and my own is not a real one. The Medan organism, like the American one, blues cream-free litmus milk. The Medan one reddens mixtures of cream and milk containing litmus, and probably the American tobacco organism would do the same thing, although we have not tested it.* In repeated tests we have never ob- served any reddening in cream-free litmus milk, using Honing's organism, but al- ways a reddening when cream was added. Coconut oil added to cream-free lit- mus milk is also split by Medan III with the production of an acid, but under the same conditions no acid was produced from olive oil, peanut oil, or cottonseed oil. Whenever a thin layer of sterile cream, paraffin, or any of the above- mentioned oils was placed on the sur- face of the inocu- Fig. I34a.t iated litmus-milk, Fig. 1344.J so as to interfere with the absorption of oxygen, there ensued a distinct reduction of the litmus, but this reduction was not observed when the surface of the litmus milk was freely exposed to the air — then the lavender blue milk became and remained a uniform deep blue. *The only American strain I have tested is the Florida potato organism (1914): This does not redden creamed litmus milk. In each of 4 trials the numerous inoculated tubes became and remained bluer than the checks. At the end of 2 weeks this strain had grown well in Uschinsky's solution and very feebly in Cohn's solution. It is motile. tFic 134a. — Flagella of Sumatran tobacco organism. Culture received from Honing. JFig. 1346. — Young tobacco plant wilted by motile bacteria, a check on Fig. 1340, i. e., from the same culture. PLANT BACTERIA-VOL 3. PLATE 44. TOBACCO WILT. Behavior of Sumatran Bact. aolanacearum (Medan 111) at 25°C. ± in centrifuged litmus-milk to which cream was added. (1) Control in cream-free litmus-milk, inoculated and kept 31 days (compare with pis. 23, fig. 7, and 41. fig. 6): no reduction, .scanty bacterial precipitate at X; (2) the same with addition of cream: tube inoculated 8 days, acid production beginning in cream layer; (3) another tube of same inoculated milk plus cream, after 31 days: distinct reduction, and copious bacterial precipitate at A'; 14) another tube of the same after 55 days. Tubes 5, 7, 8, are cultures of other organisms in same creamy medium introduced for comparison: (5) Aplanobacter michiaam me I N. Y. strain, colony 5), after 12 days: cream not reddened (compare with pi. 11, fig. 6); (6) uninoculated tube for comparison with 7 and 8, the white mass at the top is cream; (7) Bacterium campettre after 28 days: milk slightly reddened and translucent, cream unchanged ; (8) Burt- rium mori (isolated from French mulberry) after 28 days : milk blued, cream not reddened. WILT DISEASES OF TOBACCO. 265 A part of the rods in young agar streak cultures made from Honing's organism (third sending, which is actively pathogenic) were actively motile (darting about when examined in water), and the appearance of some of these rods when stained is shown in fig. 134a, which may be compared with figs. 109 and 133. I can not, therefore, explain Hutchinson's statement that Bad. solanacearum is non-motile unless it be that he did not wait long enough. Frequently when young solid cultures are transferred to distilled water they are not at once motile, but become actively motile in an hour or so. After the above results were obtained one additional test was made with the Sumatran organism. Eight typical looking colonies were subcultured from an agar poured plate. Each one of these 8 cultures was then examined in hanging drop and a portion of the rods in each observed to be actively motile (darting movements). Each one was then used to inoculate (by needle pricks) one young tobacco plant and within 7 days each one of these 8 plants developed typical signs of the tobacco wilt ; the bacterial lesions were demonstrated in the stems, and the Medan organism was plated out again from two of the plants. Sub- sequently polar flagella were demonstrated on a small portion of the rods in two of these cultures, but the cultures proved very refractory to stains (Van Ermengem's, Pitfield's, Lowit's, Morton's night blue, etc.), and no fine slides were obtained. The greatest number of motile rods were observed in the top layers of 10 or 15 c. c. of autoclaved distilled water added to young agar streaks and allowed to stand for some hours. Treated in this way fully one-half the rods were visibly motile. In our nutrient gelatin liquefaction did not occur, at least not during the first seven weeks. Tests of Medan III in 3 fermentation tubes using water, Witte's peptone and cane sugar, gave the following results at the end of a month: well clouded in the open end and the U copious precipitate, no pellicle, fluid in the bowl browned, reaction to neutral litmus slightly alkalin. In the closed end: no clouding, no gas. Using first lactose and then glycerin in place of cane sugar, similar results were obtained, except that with these carbon foods there was less growth, as indicated by the scanty precipitate, and no browning of the fluid, which was alkaline at the close of the experiment in all of the tubes. At the end of a month in two fermentation tubes containing nitrate bouillon there was a scanty stringy growth in the fluid of the open end, which was now clear. There was no gas, no stain, no clouding of closed end, and the fluid was alkaline. Old litmus-milk cultures of Medan III yielded no crystals [see p. 233]. THE RUSSIAN DISEASE. Iwanowski has described a rot of tobacco from Bessarabia, which I have included here somewhat doubtfully. He does not say in so many words that it is due to bacteria or that he has seen bacteria in the tissues, but he does say that none of the higher fungi were present. The signs are decayed spots which enlarge and fuse. These occur on various organs. The bark is first attacked and then the wood and pith, or the reverse. The Russian paragraph dealing with this disease reads about as follows: Rot is the name the author gives to a disease of tobacco, which consists of peculiar decayed spots appearing on the stem, leaves, flowers, seed-capsules — in a word, on all parts of the plant. It is as likely to occur on one part of the plant as another. The spots may group themselves around the stem, girdling the stem, or frequently they group themselves on one side of the stem, on which side the stem bends over. It often occurs that the decayed spots unite with one another in a transverse direction. The rot attacks either the bark alone, or also the cambium, the wood, and the pith, or it may act in the reverse order. In a given spot the wood may be decayed, but the bark be entirely healthy. The root in such a case commonly becomes decayed. The distribution of this disease on the plantation is similar to that of the spot-disease, that is, the diseased plant does not become the center of infection. Unlike the spot-disease, there is almost no reference to this disease in literature. Concerning its cause, it is only clear to the author that it is not of fungous origin ; whether it is caused by bacteria it is not yet possible to say, but nothing stands in the way of this supposition. 266 BACTERIA IN RELATION TO PLANT DISEASES. This paper was discussed by various botanists and Woronin suggested that, owing to the serious nature of these diseases, a commission be appointed to study them further. Query: Was this commission appointed and did it publish any report? THE ITALIAN DISEASE. From a line or two in Comes's " Crittogamia agraria," page 515, it would appear that he has seen in Italy a bacterial rot of tobacco. This he ascribes to his Bacterium gummis. Comes also reports a bacterial rot of seedlings, but this was also associated with a fungus and with eel-worms. THE FRENCH DISEASE. In 1906 Delacroix described several so-called bacterial wilt-diseases of tobacco. His "canker," "collar-rot," and "pith-rot" seem to belong here, or at least may be placed here tentatively. He has, however, ascribed these three diseases to as many species, two of which are green fluorescent schizomycetes. The "canker," which has done much damage in parts of France, particularly in Meurthe-et-Moselle and in Haute-Savoie, is said to be due to Bacillus aruginosus Delacroix. The collar-rot is ascribed to Bacillus tabacivorus Delacroix. The pith-rot is attributed to Bacillus putrifaciens putridus Fliigge. These are described with about the same degree of completeness as his Bacillus solanincola. In particular the account of his isolation and infection experiments leaves much to be desired ; one also feels inclined to suspect that the descriptions of behavior on media may have been drawn from mixed growths. I could not come to any definite conclusion from a hasty examination of his alcoholic material made in Paris in 1913, but a careful examination since made of stained sections of paraffin embedded material (10 sets, two sources) leads to the conclusion that no bacteria are present in the samples examined. These three organisms are said to have the following characteristics : (1) Bacillus aeruginosas. — The bacteria are elongated cylindric with rounded ends, generally occurring isolated or as diplococei, 0.75 to 1 .oX 2 to 3^ (his figure represents a somewhat larger organ- ism). It has no cilia, spores, capsules, or zoogloese, and does not stain by Gram's method. It grows well in culture-media: nutrient agar, nutrient gelatin; aerobic; forms on bouillon a thin, whitish, fugacious pellicle, quite adherent to the walls of the tube; the bouillon clouds rapidly and shows a green fluorescence inclining to olivaceous, the precipitate being first white, then pale yellowish- cream, and finally slightly grayish. After a time the liquid clears, loses its green color, and gradually becomes fawn-brown, old cultures having the smell of incompletely burned tobacco; this odor, how- ever, is not always produced, even in veal-bouillon; gelatin is not liquified, but after some days it acquires a green fluorescence which lasts longer than in bouillon. The isolated colonies are discoidal, 2 to 3 mm. in diameter, smooth, shining on the surface, pale yellow, becoming darker with age; the colonies often fuse. On agar there is a green fluorescence; the surface of the colonies is brilliant, but when they become confluent the surface is duller and mammelonate, with the border flattened out more than that of the single colony, or than the fusions on gelatin ; the margin also extends as the cul- ture ages; they are a little paler than on gelatin, and sometimes a little greenish; on potato the colonies are golden-yellow, slightly granular, quite brilliant, forming by confluence a creamy, opaque mass, which sinks down and becomes brown with age. These bacteria are often mixed with others in the plant, and cultures should be made, for this reason, from recent infections. Inoculation experiments by means of a needle succeeded in the proportion of 3 to 8 ; inoculations in the middle of August, made by placing a drop of the culture on the freshly cut stem (top of the plant) gave 1 out of 3 positive. Signs were visible in a week, but at the end of a month there was only a slight chancre ; wounds are necessary. Cultures of the second generation gave no result when inoculated ; direct infections gen- erally failed. The chancre frequently attacks the Paraguay, more rarely the Auriac. It has not been seen on the Green Dragon. Nicotiana rustica did not contract the disease from inoculations. Bacillus tabacivorus. — The elements are isolated, cylindrical, with rounded ends; spores, cilia, capsules, and zoogloese are absent; rods vary in size from 0.4 to 0.6X0.75 to 1.50/i. Agar, gelatin, and bouillon are not stained; the organism is aerobic; in bouillon the liquid becomes opaque (louche), with a slight pellicle which adheres to the walls of the tube. There is a scanty, whitish precipitate. On gelatin the colonies are opaque, 1.5 mm. in diameter, brilliant metallic-iridescent, with a duller, slightly raised central portion; they are flat, dirty white, with a slightly irregular contour and a surface radially striate; the gelatin is not liquefied. On agar the colonies are noticeably duller and WII/T-DISEASES OF TOBACCO. 267 flatter; on potato the colonies become rapidly confluent, forming a creamy, pale yellowish-white mass, which is distinguishable from the substratum only by its shining appearance. "I have been able to obtain several artificial infections by lacerating the collar and rubbing the wound with a culture of the first generation of the bacteria, obtained pure with the greatest ease, by sowing some of the diseased tissue." This disease is said to be confined to the collar, to be quite rare, and to require wet soil for its development. Bacillus putrifaciens putridus is said to be a cylindrical rod with rounded ends, in short chains of two or three elements when young; 0.5 to 0.7X1.2 to 1. 8m; it is closely related to his B.ceruginosus;* bouillon, gelatin, etc., are stained uranium green. The bouillon is very lightly clouded, without much surface pellicle, and with a white precipitate. Gelatin is not liquefied ; on agar or gelatin the colonies are small, shining, irregular, opaque, white with a very faint rose color. Inoculation experiments appear to have been scanty and unsatisfactory. Under bell-jars, in a humid atmosphere, about one-third of the inoculations showed some effect. No details are given. The lesions were less profound than those in the field. A collar-rot of tobacco occurs in the United States. Specimens were received in 19 12 from Elmira, New York. The bark is killed between root and stem and the plants wilt. Bacteria are present. Poured plates yielded green-fluorescent and white organisms. The writer knows nothing respecting their cultural characters or pathogenicity. A tobacco canker has also been reported from Connecticut by Clinton. THE INDIAN DISEASE. Hutchinson has recently published a paper on an Indian disease resembling the Gran- ville tobacco wilt. Unfortunately, it was received too late for more than a very brief abstract. The disease occurs annually in the Rangpur district in Bengal in northeastern India, and has been present for many years. In the field it is patchy in its distribution. The loss is usually not more than 5 or 6 per cent, but sometimes 20 to 25 per cent. Growers generally consider it to be a disease of moist soils and its local name is "Rasa" or moisture-disease. It is attributed to Bad. solanacearum Smith, which is said to be non-motile (numerous observations covering a period of more than two years), but accompanied almost invariably by a morphologically similar motile form which produces on potato only a yellow pigment (yellow ocher with a greenish tinge), and is not pathogenic. The true parasite measures 0.6 by 1.0 to 1.5 y.; on agar it is white, moist, and smooth, becoming sepia brown with stain of the substratum; on gelatin the colonies are round, thin, white, opalescent, brown under magnification by transmitted light, and without liquefaction; bouillon is turbid on shaking and alkaline in reaction (a good distinguishing trait) ; on potato the growth is watery and colorless at first, then opaque white, becoming brown, and finally bitumen black; in glucose bouillon no acid or gas is formed. In mixed cultures Bad. solanacearum may be distinguished by its tendency to show bipolar staining with methylene blue. This is a common phenomenon on potato cultures.! Successful inoculations were obtained on tobacco and tomato by needle-punctures. Soil inoculations generally failed unless the roots were injured. Marked differences in virulence were observed in cultures, also in their brown pigmentation and a non-pigmented strain was observed. Variations in morphological character and in virulence are attributed to the culture medium used. Promptest results were had from potato-culture inoculations, slowest from bouillon; agar was mid- way in point of time. When mixed with saprophytes, such as Bad. megatherium and Bad. prodigiosus, inoculations often failed. " It was found that a mixed culture of Bad. solanacearum and Bad. prodi- giosus in bouillon depended for its pathogenicity upon a preponderance of the former in the medium sufficient to produce alkalinity." The wilting of the plant is ascribed to the action of bacterial toxins rather than to mechanical plugging of the vessels. This hypothesis is supported by interesting experiments, i. e., (1) a healthy tobacco plant may be cut half through the stem without causing wilt even in the leaves immediately above the cut; (2) an alcohol precipitate from a bouillon culture when *Bacillus ceruginosus Schroter is earlier (1886), and consequently Delacroix's name should be rejected, and in the present stage of our knowledge of the disease it is scarcely worth while to suggest a substitute. Griffon states that Bacillus brassicarvorus Delacroix and Bac illus aruginosus Delacroix are nothing but B. fluorescens putridus; while Bacillus caulivorus Prill, and Delacroix is B. fluorescens liquefaciens. It is with a sigh of relief that one reads: "II n'y a plus lieu de conserver les denominations speeifique de caulivorus, brassicavorus et ceruginosus. " tThe Florida potato organism (191 4) grown on cooked potato, also showed bipolar staining with methylene blue. See also Honing's statement (p. 253). 268 BACTERIA IN RELATION TO PLANT DISEASES. dissolved in sterile water and fed into the vascular system of the stem of a healthy plant causes wilt in course of a few days if used unboiled, but not if boiled. Much difficulty was experienced in keeping cultures alive throughout the year. It was accomplished only by repeated transfers and storage at 20° C, and then they were found to have lost much of their former virulence, but this was restored by passage through a fresh series of plants, but not the pigment production. Solution of the middle lamella and disintegration of the tissues by separation of the cells is a noticeable feature in this disease. The formation of the brown pigment in cultures tends to destroy their virulence. "It was found that when the cultures reached this stage they were no longer pathogenic, and in fact in many cases failed to produce growth when transferred to similar media." [See footnote page 232.] This interesting paper raises the question whether in some of the preceding studies two organisms may not have been confused, i. e., a non-motile, black-pigment-forming species (the true pathogen) and a closely associated non-pathogenic, motile species pro- ducing only a yellowish growth on media. Certainly the writer of this monograph often has failed to observe motility, has frequently seen only a yellowish-brown stain in potato cultures (and these usually are non-pathogenic), and has seen more variations in virulence and had more failures in the way of inoculations than with almost any other organism worked with. As soon as possible therefore after reading Hutchinson's paper additional obser- vations and experiments were made to determine whether the pathogen is motile or non- motile. For this purpose we used cultures of the tobacco organism received from Honing (the only living cultures of this species then in the laboratory). These studies confirmed earlier ones made on the North American organism. The organism examined on the margin of hanging drops was clearly motile, sometimes darting entirely out of the field, and Miss Bryan succeeded in demonstrating polar flagella by means of stains. The actual agar-streak culture which yielded the polar flagella (fig. 1340) was then used for successful inoculations on tobacco (fig. 134&). If the streak was a pure culture, then there can be no question as to the motility of the parasite. It was a subculture from the third sending of Honing which we designated as Medan III. I do not know its history on the other side, but we may suppose that it was the product of a single colony; it certainly behaved in various media (potato, agar, milk, etc.) like a pure culture. We also plated it out on agar and observed no contaminating organisms. The streak used for the flagella staining was not started, however, from a single colony. For this reason the work was done over using subcultures from single colonies with the same results (see p. 265). THE SOUTH AFRICAN DISEASE. In 1906 Mr. C. P. Lounsbury reported on a serious tobacco wilt occurring in South- eastern Cape of Good Hope, South Africa. This paper is a summary of investigations carried on in 1905-6 in regard to "serious and extensive losses in the tobacco fields of the Kat River Valley." A farmer at Balfour describes the disease as "a plague which is devas- tating the tobacco of almost the whole district." Reports of W. R. Dewar, Entomologist of the Eastern Province, Mr. Thomas Bowhill, the Veterinary Bacteriologist, and Mr. Lounsbury constitute the body of the text, together with several excerpts from letters of others as to the cause or signs of the disease. Mr. Dewar's description of the disease is as follows : The farmer can see a plant just beginning to wilt when he is two or three rows away. It is not so noticeable to a casual observer; but, upon closer examination, the farmer is generally correct. A slight wilting is noticeable, which can not be distinguished in healthy plants. As the disease con- tinues, the plant wilts more, the leaves droop considerably, turn yellowish or sickly, and eventually the plant dies. In many cases when a plant is in the advanced stage the roots are soft and black. This has led the growers to say that the disease is in the soil. Sometimes only half or one side of a plant seems affected, and in some cases only one side of one or two leaves on a plant. In many cases I noticed that the stalk had become black and dry in longitudinal wavy lines under the cortex, and that the drying had pulled the cortex down so as to form irregular corrugations. WII/T-DISEASES OF TOBACCO. 269 The disease, from what I could see and learn, was pretty well scattered throughout the district. In a field of transplants there seemed no order in the plants attacked. The plants here and there over the whole field kept going, and one could see plants in all stages of disease. The promiscuity of attack, the general symptons, and the presence of insects, especially hemip- terous, first started me to wonder if there really was a bacterial disease present ; insects play an im- portant part in the dissemination of wilt diseases. Now, perhaps, all this idea about a bacterial disease is "wildfire, " but it seems to me to require investigation. There is one great objection, and that is the general dispersion of the disease in one year, taking it for granted that this is the first year that it has been noticed. Mr. Bowhill made an investigation in the same section visited by Mr. Dewar, concern- ing which he says: I made a careful examination of the diseased plants, and also dissected a great many of them. In all the infected plants examined I found a small white, active larva with a black head. * * * On making a complete bilateral section of an infected plant from the root upwards the ravages of the maggot are easily discerned. The pith in portions is of a brownish colour, and in other parts it is completely honeycombed. * * * Several infected plants were split open with a sterilized knife and cultures instituted on sterile potatoes from the decolourized portions of the pith. * * * One larva was removed with steril- ized forceps and placed in a tube of gelatine in order to observe what, if any, micro-organisms were introduced by them into the pith and other portions of the stem during their meanderings. * * * Regarding the question as to whether there is also a blight disease amongst the tobacco in this area, I am not prepared from the evidence in my possession to either affirm or deny such a contention, but I must, however, state that I believe there is something else in addition to the larva already dealt with. I have personally noticed, as well as having my attention drawn to the fact, that some of the infected plants droop in one night, while others (supposed to be affected by the so-called blight disease) appear to degenerate by degrees. In some instances the roots were found to be in an ad- vanced state of decay. Mr. Bowhill obtained a variety of bacteria and fungi in his cultures, c. g., Bacillus mycoides roseus, Bacillus megaterium, a slender bacillus unidentified, and some Mucors. Regarding these he says: "I am of the opinion that their presence within the stems of infected plants is due to external combinations caused by the destruction of the external cuticle of the plant." Mr. Lounsbury concluded, after personally visiting various sections where the disease prevailed, that the cause was the potato moth, combined in some cases with the action of the gall worm. He refers to McKenney's description of the North Carolina tobacco wait, and also to the Japanese tobacco wilt, and has the following to say concerning the relation of these two diseases to the Kat River wilt : These more serious tobacco diseases may be distinguished from the Kat River wilt, to judge from the descriptions, by the more extensive blackening of the stalks, by black lines in the wood marking the work of the fungus or bacterium in the sap channels, and by the earlier decay of the roots. Perhaps, however, the only clearly distinguishing feature in many plants would be the presence or absence of the causative bacterium or fungus. In all three troubles the pronounced symptom of wilting appears to be due to a gradual stoppage of the flow of sap. Many growers will doubtless believe that they have one or the other of the foreign wilt diseases. I can not deny the possibility of their being correct, but can say that I saw nothing which, in my mind, justifies an opinion that the whole trouble is not due to the potato moth, assisted in a few places by the gall worm. I failed to find evidence that would lead me to suspect fungi or bacteria as the primary source. Although Mr. Dewar at first inclined to the idea of a true plant disease in addition to injury through insects, he failed to substantiate his suspicions by his subsequent laboratory examination of affected plants, and he had not learned at the time of similar trouble, due to potato moth alone, in Rhodesia. Moreover, were the trouble an entirely new fungoid or bacterial disease, it would probably not have appeared simultaneously over a large area, and would probably have caused more uniform destruction wherever it appeared. Illustrations of the American and Japanese diseases show plants in large patches wilting down all at once. However, should the developments of future years in the Kat River Valley show my conclusions to be wrong, I shall have erred on the safe side, since there are no practical remedies for the true wilt diseases. The potato moth and gall worm, on the other hand, are pests that it should pay to combat by the measures discussed, irrespective of the wilt; and growers are therefore urged to take action. [In this connection see account of African potato disease on p. 214.] LITERATURE. 188S. Iwanowski, D. O. On the diseases of the tobacco plant. [Russian.] Arb. d. Petersb. Naturf. Gesellsch., Bd. xix, pp. 19-21. Describes a spot-disease, and a rot of tobacco from Bes- sarabia. The latter is apparently due to bacteria. No fungi present. Brief description in Bot. Centxalbl., Bd. XLI. 1890, P- 363- 1890. Iwanowsky, Dm. und PoloFTZoff. Die Pock- enkrankheit der Tabakspflanze. Mem. Acad. Imp. de St. Petersbourg, T. xxxvn, No. 7, 1890. This disease occurs in Little Russia. Bessarabia, and the Crimea, and is known generally as the "pox disease." Accord- ing to a Dutch review in Teysmannia the authors think the dis- ease is not due to bacteria. They think that the disease arises as a result of a break in the water-supply. This is perhaps the "spot-disease" referred to in the other paper. Original not seen. 1892. JansE, J. M. Nota over eene ziekte der tabak op Sumatra. Teysmannia, in Deel, Batavia, 1892, pp. 653-662. 1893. Comes, Orazio. Mortalita delle piantine di tobacco nei semenzai cagionata da marciume della radice. Atti del R. 1st. d'Incorragg. di Napolt, ser. IV, vol. VI, mem. Nr. 2, 1893. Also a reprint. Attributes a root rot to Bacillus anrylobacler. Bacteria not isolated. Reviewed in Bot. Centralb., 1893, vol. LVI, p. 253. 1897. van Breda de Haan. De Slijmziekte bij de Tabak in Deli. Teysmannia, Deel vin, Arl. 10/11, Dec. 1897, Batavia, Java, pp. 528-549. Also a separate of 22 pages. Disease produced by inoculations with pure cultures. 1899. Smith, Erwin F. The Fungous Infestation of Agricultural Soils in the United States. Sci- entific American Supplement, No. 1246, No- vember 18, 1899, p. 19981 (paper read Aug. 25, 1 899, at Columbus meeting of the American Assoc. Adv. Science). 1901. Hunger, F. W. T. De Slijmziekte Veroor- zaakt door Bacillus solanaccarum Smith in "Overzicht der Ziekten en Beschadigingen van het blad bij Deli-Tabak." Mededee- lingen uit 's Lands Plantentuin xlvii, Batavia 1901, pp. 20-25. 1903. McKenney, R. E. B. The wilt disease of tobacco and its control. U. S. Dept. Agric, Bur. Plant Industry, Bull. No. 51, part 1, Sept. 18, 1903, pp. 4, 1 fig. 1903. Stevens, F. L., and Sackett, W. G. The Granville tobacco wilt; a preliminary bulletin. North Carolina Agric. Expt. Sta. of the Col- lege of Agriculture and Mechanic Arts, Raleigh. Bull. No. 188, Sept. 1903, pp. 81-96. See also Ann. Rep. for that year. 1904. UyEda, Y. On the tobacco-wilt disease caused by a bacteria. Centralb. f. Bakt., 2 Abt., xiii Bd., 1904, pp. 327-329, with 3 figs, of diseased plants. 1905. Smith, Erwin F. Bacteria in relation to plant diseases, vol. 1, pi. 27. 1905. UyEda, Y. Bacillus nicotianae, sp. nov ; die Ursache der Tabakwelk-Krankheit oder Swarzbeinigkeit in Japan. Bulletin Imperial Central Agricultural Experiment Station, Tokyo, Japan, vol. 1, No. 1, 1905. Also a separate, pp. 1 to 19, 5 plates. 1906. Delacroix, Georges. Le chancre bacterien, in "Recherches sur quelques maladies du tabac en France. " Annales de l'lnstitut National Agronomique. 2 se., t. v., fasc. 2. Paris, 1906. Also a separate, pp. 1 to 12. Disease ascribed to Bacillus ceruginosus Delacroix, a green- fluorescent organism. 1906. Delacroix, Georges. Pourritures bacteri- ennes succedant a des plaies d'insectes. Pour- riture du collet du tabac, in "Recherches sur quelques maladies du tabac en France." Annales de l'lnstitut National Agronomique. 2 se., t. v., Paris, 1906, pp. 12-14. Also a separate, pp. 12 to 14. Disease ascribed to Bacillus tabacivorus, a non-sporiferous, non-motile, non-capsulate, non-fluorescent, white organism, which does not stain by Gram. 1906. Delacroix, Georges. Pourritures bacteri- ennes succedant a des plaies d'insectes. Pour- riture de la moelle, in "Recherches sur quel- ques maladies du tabac en France. " Annales de l'lnstitut National Agronomique, 2 se., t. v., fasc. 2, Paris, 1906. Also a separate, pp. 14-16. Rot ascribed to a green-fluorescent organism, Bacillus putri- faciens pulridus Fltigge. 1906. Lounsbury, Chas. P. Tobacco Wilt in Kat River Valley. The Agricultural Journal of the Cape of Good Hope, vol. xxviii, No. 6, Cape Town, June, 1906, pp. 784-803. Included here doubtfully. 1907. Stevens, F. L. Granville tobacco wilt. 30th Ann. Rep. North Carolina Expt. Sta. for year ending June 30, 1907. A brief note. 1908. Smith, Erwin F. The Granville tobacco wilt. U. S. Dept. Agric, Bureau of Plant Industry, Bull. No. 141, part 11, pp. 13-24. Issued August 31, 1908. Washington. 1909. Griffon, Ed. Sur le role des bacilles fluores- cents de Fltigge en Pathologie vegetale. Comptes r. hebd. d se. de l'Acad. des Sci., tome 149, pp. 50-53. Paris, 1909. "Another precaution, much spoken of since the researches of Laurent, consists in the use of a suitable fertilizer which will procure to the plant a certain immunity: little organic nitrogen, much phosphates and potash salts. The results of the experi- ments I have undertaken with this in mind, and of those I have followed here and there, do not seem to me very encouraging." 1909. Stevens, F. L. Granville tobacco wilt. Notes on plant diseases occurring in North Carolina. Thirty-first Annual Report, North Carolina Agric. Exper. Station, pp. 81-82. West Ral- eigh, June 1908. [Issued in spring of 1909.] 1909. Jensen, Hj. Onderzoekingen over Tabak der Voorstenlanden. 1. Slijmziekte. Jaarboek van het Dcpartement van Landbouw in Neder- landsch-Indie, 1908. Batavia. Landsdruk- kerij, 1909, pp. 100-104, w'th 1 plate (plant recovering from an attack). 1910. Honing, J. A. De oorzaak der slijmzieke an proeven ter bestrijding. Mededeelingen van het Deli Procfstation te Medan. 5e Jaarg., ie Aflev. Medan. [Sumatra.] August 19 10, pp. 1-19. 191 1. Honing, J. A. De oorzaak der slijmzieke en proeven ter bestrijding 11. Mededeelingen van het Deli Proefstation te Medan, 5e Jaargang, 6e Aflevering (verschenen Feb. 191 1), pp. 169-185. 191 1. Honing, J. A. De oorzaak der slijmziekte en proeven ter bestrijding hi. Mededeelingen van het Deli Proefstation te Medan, 5e Jaar- gang, ioe Aflevering (verschenen Juli 1911), PP- 343-358. 270 WILT-DISEASES OF TOBACCO. 271 1912. 191 1. Honing, J. A. Verslag over de slijmziekte proeven in 191 1. Mededeelingen van het Deli Proef station te Medan, 6e Jaargang, ie Aflevering (verschenen Sept. 191 1), pp. 1-30. 1 91 2. Honing, J. A. Besehrijving van de Deli- Statnmen van Bacillus solanacearum Smith, de oorzaack der slijmzieke. Mededeelingen van het Deli Proefstation te Medan, 6e Jaar- gang, 7e Aflevering (verschenen April 1912), pp. 219-250. 191 2. Honing, J. A. Verslag over der ontsmettings- proeven van zaadbedden op slijmzieke gronden met eenige chemiealien. Mededee- lingen van het Deli Proefstation te Medan, 7e Jaargang, ie Aflevering (verschenen Juli 1912), pp. i— 11. Honing, J. A. Een geval van slijmziekte in de djattibibit. Mededeelingen van het Deli Proefstation te Medan, 7e Jaargang, ie Afle- vering (verschenen Juli 1912), pp. 12-15; also naschrift, p. 59- 191 2. Honing, J. A. Over het verband tusschen slijmziekte in de bibit en in de uitgeplante tabak. Mededeelingen van het Deli Proef- station te Medan, 7e Jaargang, 2e Aflevering (verschenen Aug. 1912), pp. 65-69. 1912. Honing, J. A. Over de beweerde onvatbaarheid van Nicotiana rustica voor slijmziekte. Mededeelingen van het Deli Proefstation te Medan, 7e Jaargang, 3e Aflevering (verschenen Sept. 1912), pp. 95-98. 1912. Honing, J. A. Over de variabiliteit van Bacil- lus solanacearum Smith. Mededeelingen van het Deli Proefstation te Medan, 7C Jaargang, 5e Aflevering (verschenen Nov. 1912), pp. 196-20S. 1912. Honing, J. A. Over rottingsbacterien uit slijm- zieke tabak en djatti en enkele andere van slijmziekte verdachtc planten. Mededee- lingen van Het Deli Proefstation te Medan. Jaargang vn, 6e Aflevering (verschenen Dec. 1912), pp. 223-253. 1913. Honing, J. A. Uber die Variabilitat des Bacil- lus solanacearum Smith. Centralb. fiir Bakt., Zweite Abt., 36 Bd., No. 19/25, February, 1913, pp. 491-499. 1913. Honing, J. A. Hoe moet men trachten een tabaksras te verkrijgen, dat immuun is tegen slijmziekte? Mededeelingen van Het Deli Proefstation te Medan, Jaargang VIII, ie Af- levering, August 1913. PP- 12-21. 1913. JEN3EM, Hj. Slijmziekte. Mededeelingen van "Het Proefstation voor Vorstenlandsche Tabak." No. 5. Batavia, 1913, PP- 48-60. No infections with Uyeda's B. nicotianae — 60 plants inoculated. 1913. Fulton, H. R., and J. R. Winston. A disease of peanut plants caused by Bacterium solana- cearum. Phytopathology, vol. 3, No. 1, February 1913, pp. 72-73. Mere mention. Through courtesy of Dr. Fulton I have received the following note: In August 1912. peanuts (Arachis hypogaea) of the Spanish variety growing in Granville county. North Carolina, on soil known to be infected with Bacterium solanacearum (Erw. Sin.), were observed to be dying to the extent of 15 per cent of the planting. Examination of stem and root of the affected peanut plants showed discoloration and disorganization of tissues closely similar to those characteristic of Granville wilt of tobacco. The newly affected parts contained numerous bac- teria. Pure cultures were obtained from which both young and old peanut plants were successfully inoculated on several occasions, the disease induced being similar to that observed in the field. The organism was reisolated from such artificially infected plants, and this culture again used for infecting the peanut. Young and old tobacco plants were inoculated with the original cultures from peanut, with the production of dis- ease similar in all respects to Granville wilt of tobacco, and the typical organism was reisolated from these artificially infected tobacco plants. Cultures from naturally infected tobacco and pepper plants produced the disease in peanut. Cultures of three strains of the bacterium from peanut were compared with cultures from tobacco and from pepper on various media, such as potato agar, nutrient gelatin, potato strips, peptone broth, glucose broth, beef broth, potato broth, milk, and litmus milk. The cultural features on these several media were similar for all the strains tested, and agreed with the original descriptions for Bacterium solanacearum. In size and shape, and in reaction to the ordinary stains, these several strains agreed with Bacterium solanacearum. The facts lead to the conclusion that peanut is susceptible to attack by Bacterium solanacearum. 1913. Hutchinson, C. M. Rangpur Tobacco Wilt. Memoirs of the Department of Agriculture in India. Bacteriological Series, vol. I, No. 2. July, 19 13. Agric. Research Institute, Pusa. Calcutta and London, pp. 67 to 83, with 12 plates (5 colored). 1 913. DE Jong, A. W. K. Eenige Opmerkingen over d? Bacterienziekte (Pelepes) bij Katjang Tanah. Dept. van Landbouw, Nijverheid en Handel, Medeelingen van het Agricultuur Chemisch Laboratorium, No. Ill, Buitenzorg, 1913, PP- 56-59. No advantage was obtained from flooding infected fields for some weeks. Very hopeful results were obtained Irom the planting of selected sound seed. [See also Literature under Brown Rot of Solanaccae (p. 218) and Wilt of Peanut (p. 153).] SCHUSTER'S GERMAN POTATO ROT. OBSERVATIONS BY SCHUSTER. In 1912, Schuster described a polar-flagellate, green-fluorescent schizomycete from rotting potato tubers obtained from six different localities in Germany, as Bacterium xantho- chlorum n. sp., and asserted it to be the cause of the disease. The organism when inoculated also blackened, it is said, and destroyed leaves and stems of Vicia faba, and rotted other plants. It is by no means as active an organism on potato as Bacillus phytophthorus. It does not attack the stems of potato, and from Schuster's own account it seems to be a rather weak parasite except at high temperatures (most active, he said, at 36. 50 to38°C.), or under other abnormal conditions, e.g., heavy doses of the bacteria, under bell-jars in very moist air. In dry air at room-temperatures the inoculations soon dried out and the tuber as a whole remained sound. At times it showed a decided tendency to occupy the vessels, e. g., in Vicia faba, and in early stages of the tuber-rot of the potato. All of Schuster's inoculation experiments appear to have been made under abnormal conditions, i. e., in laboratory rooms, under bell-jars, etc., in a saturated air. His Bad. xanthochlorum causes a slow, gray or yellow, wet-rot of the tuber, often proceed- ing from the vascular bundles in or out, and from the margin of this rot it is easy to isolate pure cultures. Often the tubers are not much injured on the surface. The rotted parts smell of ammonia. It is not able to enter the potato tuber through the opened lenticels [another indication of weak parasitism]. In general it is a wound parasite, but it may enter, it is said, the green parts of Vicia faba through stomata. It was inoculated successfully by Schuster into carrot roots, young tobacco stems (300 to 360 C.), Lupinus nanus douglasi, Physalis alkekengi, and Campanula raphunculus. Fodder beets, sugar beets, yellow lupins, tomatoes, and pelargoniums proved insusceptible. This organism, according to Schuster, has the following morphological and biological characteristics : As a rule it is actively motile, but once a non-motile strain was isolated [this may have been some related organism]. The flagellum is polar. Sometimes two flagella are present, rarely three occur. The straight ones are 4.5 to 6/x long, i. e., two to three times the length of the rod. They stained poorly with carbol-fuchsin, but readily with Pepler's stain followed by earbol gentian violet. Hinterberger's modification of Van Ermengem's stain was also used successfully. At temperatures under 50 C. and above 40° C. no flagella formation could be detected. In bouillon motility was observed for 18 days, but on potato at 200 C.it ceased after one day. On agar-streak-eultures motil- ity ceased after one day at 37° C. but was visible after 20 days at 200 C. Under normal conditions of growth the organism is a slender rod with rounded ends, 1.5 to 3 X0.75M, occurring for the most part singly or in pairs. It stains readily with basic anilin dyes but not by Gram. On agar at 360 C. and at higher temperatures, long threads are produced within 24 hours. These long rods may show constrictions, but do not afterwards separate into short elements. Short, plump rods were observed at 20 C. Spores do not occur. Involution forms (plasmoptyse) occur. Pseudozoogloea occur. The yellowish-green fluorescent pigment is soluble in water. After cultivating this organism on potato agar, carrot agar, or wheat-decoction agar for 3 months the color disappeared, but always could be restored by inoculation into alkaline bouillon, even after 6 months of colorless growth on the potato agar. Glycogen was detected in bouillon-cultures at the end of the second day using potassium iodide. No acids are produced; the cultures are always alkaline [see below]. Ammonia, methylamin, and trimethylamin occur. Trypsin, amylase, tyrosinase, and a hemicellulase are produced. A soluble toxine active on the protoplasm is also produced. 272 Schuster's german potato rot. 273 Cultures in bouillon containing methylene blue (0.2 c.c. bouillon-culture: 1 : 1,000 m. b.). lost their virulence on exposure to diffuse daylight for 4 hours, while similar cultures kept in the dark showed an increasing virulence. The methylene blue in 1 per cent quantity in bouillon is reduced in 3 to 4 hours. Indol occurs (distinct in bouillon after 10 days). Hydrogen sulphide is produced slowly. Nitrates are reduced. The organism liquefies gelatin, but not rapidly. It does not liquefy blood- serum. It acts on starch slowly and incompletely. It grows best at 35. 50 C. [see below]. The range of temperature is not known. Schuster says slight growth at 2° C. and at 40° and 440 C. Good growth at 27. 50 C. and pretty good at 36° C. Moderate at 37° C. and at 17.50 to 18.5° C. Growth at all temperatures from 16° to 2° C. is recorded as slight. The organism is aerobic and facultative anaerobic. Schuster gives a table of its action on sugars, alcohols, etc., but I am not able to make much out of it, except that cane-sugar, grape-sugar, milk sugar, arabinose, mannit, peptone, and asparagin favor growth, whereas ammonium chloride, ammonium phosphate, ammonium tartrate, ammonium sulphate, glycerin, caffein, arbutin, potassium nitrate, potassium phosphate, citric acid, potato starch, and cellulose do not. With ammonium chloride and the substances named immediately thereafter, there was a weak growth, with those italicised there was no growth whatever or only the slightest. These experiments were made in flasks in 100 c.c. portions of Arthur Meyer's nitrogen- free mineral solution* to which were added usually 10 grams of the substance to be tested (caffein 0.3 gr., arbutin 3 gr.). Cane-sugar, glycerin, mannit, and asparagin gave pellicles and no precipitate ; grape sugar and arabinose gave pellicles and precipitate; milk sugar and peptone gave clouding and precipitate without pellicle. These flasks were each inoculated with one loop from a bacterial suspension of a 24-hour agar-culture and were kept at about 200 C. Gas is not produced from dextrose. At room temperatures milk is slowly coagulated (eighth day) and the curd is peptonized, but at 340 C. these changes did not occur. At both temperatures the upper layers (Fettschicht) became greenish-yellow. If a trace of sodium selenite is added to gelatin which is then inoculated with this bacterium the culture becomes blood-red in 12 hours. This organism causes in nutrient agar a greenish-yellow fluorescence. On agar-plates the surface colonies are circular, slightly raised, thinning at the edges, fine granular, with a denser center. The buried colonies are smaller and not characteristic. On alkaline agar-strokes the growth is visible in 24 hours; it forms along the needle track a dry, thin, yellow-white to greenish layer, exhibiting a large number of cross-folds forming a wrinkled mem- brane. In thick sowings the folds may be lifted up vertically a millimeter above the surface, and the layer is then moist and watery or slimy [see below]. In agar-stab-cultures there is only a slight growth along the needle line. This is best at the point of entrance where after 2 or 3 weeks there is a thick mulberry-shaped piling up of yellowish bacterial masses while the surface is covered with a white skinny layer. On Appel's slant potato-juice-agar there was a very abundant development of the yellowish-gray bacterial masses within 24 hours which later fused into a thick slimy layer. In the expressed juice of cooked carrots, with 2 per cent agar, there developed along the lower part of the streak a scanty growth in the form of a thin, white, weak, moist layer. On gelatin plates very small colonies in the form of round drops appear in 1 to 2 days. Lique- faction begins in 48 hours as a pit in which the nearly smooth-edged colony floats in the form of small lumpy masses lying one over the other. The center of the colony is considerably thickened and dark brownish. The buried colonies are globose, granular, and liquefy more slowly. In gelatin-stab-cultures near the surface there is a funnel-form liquefaction extending gradually to the walls of the tube and not far downward along the needle track, where growth is slight. Judg- ing from Schuster's figures liquefaction the first day is in the form of a narrow (steep) funnel; that on the eighth day touches the walls and resembles a champagne glass, i. e., the liquefaction is restricted to the upper quarter of the tube. Sometimes, however, after very copious inoculations there appeared along the needle-track liquefaction centers where clumps of bacteria developed, but elsewhere the needle-track was very little changed. In Appel's potato-juice gelatin (Arb. Kais. Biol. Anst. in, p. 396) there was rapid growth in stab- cultures with intense liquefaction, the entire contents of the tubes being liquefied within a few days, the bacteria being heaped up in an irregular globose mass on the bottom of the tube. The non-motile organism already referred to liquefied gelatin more slowly. In neutral nutrient bouillon a strong general clouding takes place within 24 hours. A white pellicle forms on the surface and the fluid is filled with small particles (Krumeln). There is a copious white precipitate which on shaking holds together. Grape-sugar bouillon is very favorable for the growth of this organism. It did not grow well in beer. ♦Distilled water, 1,000; KH2PO„ 1.0; CaCljO, 1.0; MgS04+7 HA 0.3; NaCI, 0.1; Fe2Cb, 0.01. 274 BACTERIA IN RELATION TO PLANT DISEASES. From a liter of alkaline potato juice inoculated 8 days a clear, bacteria-free fluid was obtained by passing it through a Chamberland filter. When double its volume of absolute alcohol was added a copious precipitate was obtained, which was tested after further purification for its effect on cell-walls of potato tubers. The walls became swollen and the typical rot appeared within 36 hours at 370 C. Here again high temperatures were necessary, i. e., such as usually do not occur in the field or in storage. I translate as follows: Experiments with enzyme injection at different temperatures showed after 3 days at 20 to 280 C. no action; at 29° C. beginning of rot; at 300 to 35° C. feeble rot spots; at 36° to 380 C. strong wet rot; at 400 to 450 C. again feeble decay; at 60° C. no decay. At 350 C. the swollen cell walls became twice their normal width in 18 hours and the cells sepa- rated wholly or partly along the line of the middle lamella the third day. After 8 days the starch- grains were also acted upon, resembling the skeletons obtained by exposing starch to saliva at40°C. This remainder, which does not fall apart, is believed to be amylodextrin. Corrosion from the margin did not occur. The enzyme which dissolves the middle lamella is designated Xanthochlorum hemicellulase. When thin sections of a potato tuber are put into infected potato juice and examined under the microscope, the protoplasm can be seen to separate from the walls of the cell after 10 minutes, and after 40 minutes it lies as a dark-brown lump dead in the middle of the cell ; therefore the bacteria excrete a toxin as well as an enzyme. According to Schuster, the action of this organism on the stems of Vicia faba is very rapid even at room-temperatures ; pitch-black spots appear at the place of inoculation and within 24 hours the stems rot and fall over, but it is not clear to the writer whether these inoculations were done on plants under bell-jars, or in the open, i. e., under normal condi- tions.* Schuster's figures 8, 9, and 10 indicate a vascular infection. The rapidity of stomatal infection on leaves of Vicia faba is something hitherto unheard of, viz, penetration of the stomata with brown staining and bacterial occupation of the vessels of the leaflets, of the petiole, and even of the stem, within 48 hours, and would seem to indicate drenching of the leaves with bacteria and exposure to other abnormal conditions, i. c, to air saturated with vapor of water [see below]. The spiral vessels are the first to be attacked; the pitted vessels are then occupied, the bacteria entering them through the pits; subsequently larger or smaller cavities are formed in the vascular bundle. When cultivated from stems of Vicia faba on alkaline potato agar at 300 C, a weak reddish stain appears which soon turns black on addition of paraphenylendiamin and /3-naphthol (Spitzer's reaction). This red reaction is plainer in cultures to which 1 to 5 per cent tyrosin solution has been added. The rot on lupins was white, never black as on Vicia. The plants were injected and kept moist under bell-jars. The plants in the open were never destroyed by the inoculation, the growth of the bacteria soon ceased and the wounds dried out, but here pustule-like intumescences were observed [see below], and these are ascribed to the action of the organism, since the control plants remained free whether pricked or not (see this monograph, Vol. II, fig. 27). In Physalis also the rot was white. EXPERIMENTS IN WASHINGTON. The writer repeated certain of these experiments with a green-fluorescent organism brought in 191 1 from the Biologisches Anstalt in Berlin by Dr. Wollenweber and said to be Bad. xanthochlorum Schuster. This culture, it should be stated, did not come directly from Dr. Schuster, who had then left the laboratory, but was a transfer by another assistant from a stock culture of this organism. The organism which I have thus received and studied liquefies gelatin in the manner described and rots potato tubers as described, only much more slowly at 260 C, and not at all at 37. 50 C, nor will it grow in culture media at that temperature. Also on Vicia faba *Dr. Wollenweber, who was at the Biologisches Anstalt during this period, tells me they were made under bell-jars in the laboratory. SCHUSTER S GERMAN POTATO ROT. 275 exposed to normal conditions in a hothouse it is not parasitic. I could not get any infec- tions either by rubbing it into stomata or by needle-puncture into leaves or stems. The organism grows in milk about as described, but on agar-streaks ( + 15) I did not obtain the cross-folds and am inclined to regard this as an accidental phenomenon due to dry agar or to some other peculiarity of medium. The organism was tried on various sugars and alcohols in the presence of river-water and Witte's peptone (1 per cent). Contrary to Schuster, it ferments grape-sugar and galactose with production of an acid, but a similar solution with cane-sugar gave an alkaline reaction (fermentation tubes, 11 days). The same results were obtained in two repe- titions (twenty-eighth day and later) . The other car- bon foods tested in fermen- tation tubes were fructose, lactose, maltose, mannit, and glycerin. All of these gave an alkaline reaction (eleventh day). In two repetitions the same result was obtained after longer periods. There was no gas formation and no clouding in the closed end. As stated, the organism did not rot potato tubers at 37° C, nor could we get any evi- dence of growth above 34° C. in + 15 peptone-bouillon or on steamed potato. It grew better at 300 C. than at 340 C. but less well than at26°C. It grows at i° C. Its thermal death-point is 510 C, approximately. It grows in bouillon over chlo- roform without retarda- tion. It grows in +15 peptone-bouillon with 4 per cent sodium chloride and slowly with 5 per cent, but not with 6 per cent. It does not grow in Cohn's solution. It grew in pep- tone bouillon from —30 to +25. It grew about as well in — 15 and —30 as in o or +25. It grew well in Uschinsky's solution, forming a copious pellicle and after a time a pale green fluorescence. f It is sensitive to dry air (dead on cover slips at end of 4 days). It liquefied gelatin in the manner described by Schuster but did not liquefy Loeffler's blood-serum, although it grew well upon it. It is quite sensitive to sunlight. Fig. 135.* *Fig. 135. — -Potato tubers inoculated with Schuster's Bacterium xanlhochlorum and exposed for six days to the dry air of the laboratory at 270 C. Photographed Sept. 1 1, 1912, after slicing to show the very slight rot. The specks in the rotted part are needle-punctures. In right lower figure there is rot in vascular ring at top near the pricks. fMiss Clara Jamieson isolated from Bad. xanlhochlorum a non-fluorescent strain which retained this characteristic in subcultures on various media as long as studied (some months). On agar-plates the non-fluorescent colonies looked exactly like the fluorescent ones. 276 BACTERIA IN RELATION TO PLANT DISEASES. Several of the inoculated potato tubers exposed dry at room-temperature (26° C.) showed a slight vascular infection (rot) extending in one instance a distance of 2 cm. from the pricked area, but only visible close to the bundles. The check-pricked tubers remained sound. The experiments were made in August, i. e., on I tubers recently harvested (fig. 135), and part of a lot which rotted quickly when exposed to B. melanogenes. Miss Nellie Brown repeated this experiment for me on several potato tubers with the same result. Dr. Schuster's figures also call for some interpreta- tion or explanation. I am inclined to think they do not represent what actually took place in the tissues; by this I mean that some of them are suspiciously diagram- matic. Schuster's text figure No. 11, for instance, can hardly represent a stomatal infection in Vicia faba. Here the bacteria are represented as forming a branch- ing strand which has entered through the stoma and penetrated to the opposite side of the leaf in a very un- usual way, i. e., by boring its way through cell-walls. It has conspicuously avoided the intercellular spaces, and the surrounding cells are not only unoccupied by the bacteria but also uncollapsed and apparently unin- jured and the leaf has retained its turgor. I am told that Dr. Schuster did not make these drawings, but he ought, at least, to have supervised them, since profes- sional artists seldom have an eye for details of plant structure. In an unusual case like this, a photomicro- graph would have been more convincing. The subject is left in such shape that some careful bacteriologist should repeat all of Schuster's experi- ments, and make others; only in this way shall we finally come to know what weight to give his statements. On a few of the many plants of Vicia faba inocu- lated by me where the needle-inoculations were not far apart, and one above another, the rapidly growing stems cracked open with callus-formation not unlike that figured by Schuster for Dupinus, but there was no evi- dence of disease due to the bacteria. These stems were about 15 inches high when inoculated and were 5 feet tall with numerous vigorous leaves and blossoms when the photograph was made (fig. 136). Fig. 136.' LITERATURE. 191 2. Schuster, Julius. Zur Kenntniss der Bak- terienfaule der Kartoffel. Arbeiten aus der K. Biologischen Anstalt f. Land. u. Forst- wirtschaft, viii Bd., 4 Heft, pp. 452-492. 1 plate, 13 figs. Berlin, 1912. 1912. Smith, Erwin F. Isolation of pathogenic potato bacteria: A question of priority. Phytopathology, vol.11, p. 213, Oct. 1912. *Fig. 136. — Stem of Vicia faba, 2.5 months after inoculating with Schuster's Bacterium xanthochlorum, showing tissue cracked open, with callus formation along the line of needle-pricks. X 1.75. 1913. Brown, Nellie A., and Jamieson, Clara O. A bacterium [Buct. aptatum] causing a disease of sugar-beet and nasturtium leaves. Jour. Agric. Research, U. S. Dept. of Agric, vol. 1, No. 3, Dec, 1913, with 3 pis. (one-colored), pp. 189-210. That part relating to Schuster's organism begins on page 209. THE DUTCH DISEASE OF WALLFLOWER. In the summer of 1900, in Holland, van Hall found a disease of the wallflower (Cheiranthus) which was characterized by the constriction of the upper part of the tap root, followed by yellowing of the leaves, which gradually fell off, first the lowest, then those farther up the stem. Cross-sections of the constricted portion of the tap root showed the wood to be brown or black, and the vessels of the wood to be occupied by bacteria, which were traced upward into the branches. Professor Beijerinck proved, it is said, that all of these were one sort. " Infection experiments have not yet been published, so that this disease must be placed with the incompletely known ones." Later Briosi and Pavarino published on a similar Italian disease of stock (Matthiola), as described in the following chapter. LITERATURE. 1902. Van Hall, C. J. J. Ziekte dor Zomerviolieren, Cheiranthus anyiuus. In " Beijdragen tot de Kermis der Bakterieele plantenziekten." Academisch Proefschrift, Amsterdam, 1902, p. 72. THE ITALIAN DISEASE OF STOCK. In 19 1 2 Briosi and Pavarino described a vascular and parenchymatic disease of the common stock (Matthiola annua L), prevalent in parts of Italy and attributed it to a green- fluorescent bacterium. The following translation gives the substance of their paper: Two different species of crucifers are commonly designated by the name "Violaeciocca:" The Cheiranthus cheiri L, which is the common violaeciocca, and the Matthiola annua L., commonly called quarantina. Of this latter there are some varieties of notable commercial importance, cultivated on a large scale, especially in many sections of the Liguria, where entire fields are covered with them, and furnish abundant and splendid winter flowers for exportation. Some years the Matthiola quarantina shows signs of a grave disease, which spreads rapidly and causes such damage in some localities that it is necessary to suspend its cultivation. The signs of the disease begin with the appearance on the leaves of spots of a pale green color, of indefinite contour, which at first are observed with difficulty. Following this there appear small brown spots of irregular contour, and more or less punctiform, scattered over the petiole. Frequently the leaves, especially the young ones, become more or less deformed, not attaining the normal dimensions, and their margins curl toward the upper surface. The inflorescence is arrested in its development and becomes rachitic, and so the flowers lose their commercial value. The researches were made on plants gathered by us, in various localities such as Loano [on the Riviera, about halfway from Genoa to Ventimiglia], where some years the disease is active and widespread. Morbid anatomy. — The disease is not limited to the leaves, but extends to all the organs of the plant : flowers, branches, stalks, and roots. Sectioning young branches, it is found that the infection invades first the woody vessels, which show yellowing or browning and are more or less altered. The plant forms a zone of corky tissue which twists and circles about the infected vessels, to prevent the spread of the disease in a transverse direction, and limit the diffusion of it in the tissue. Later the disease is able to invade all the wood as far as the pith, which, in some cases, is seen to be strongly corroded. In the stalk the infection appears first in the primary wood, from which it extends interiorly toward the pith, and externally toward the secondary wood, as is clearly shown by the yellowing and browning of the walls of the vessels and of the fiber, and by the yellow, brown, or black coagu- lated substance which fills up the lumen of the cells of the diseased tissues. 277 278 BACTERIA IN RELATION TO PLANT DISEASES. In the roots the infection begins generally in the woody bundles of the central cylinder, and from there spreads to the secondary wood; and the attacked elements (vessels, fiber, etc.) color as usual either yellow or brown, becoming filled at the same time with the clotted, blackish substance. In the leaves the infection manifests itself by the pale spots above described, which are due to the disorganization of the chloroplasts through work of the microorganism, which multiplies rapidly in the cell. With the advance of the disease the protoplasm of the cell contracts and shrinks, caus- ing the collapse of the cell walls; from this time on the tissue decays and there appear in the mesophyl the brown and depressed spots which we have already noted on the petiole in advanced disease. Microscopic examination shows in the cells of the diseased tissues numerous motile microorgan- isms, isolated and united in colonies, but does not reveal any trace of mycelium. We have cultivated this organism in many nutritive media, proceeding in the following manner: We took small pieces of the leaves, branches, stalks, and roots, carefully washed them with water and soap, and afterwards disinfected them with a solution of 1 : 1000 mercuric chloride; they were then put into sterile distilled water and passed finally through alcohol and ether. The pathological pieces thus treated were put into tubes containing nutritive media of our preparation, and from all the infected organs thus treated and sown in the different nutritive media, we obtained always the development of a particular microorganism, which presented the following morphological and cultural characters: Microscopic aspect and colorability. — This microorganism has the form of a small rodlet — length 2 to 4m, width 0.4 to o.6ju, with the ends slightly rounded. It stains well with all the anilin stains, even cold, but especially with gentian violet; it resists Gram completely. In broth cultures we observed vivacious vibratory and rotary movements of the microorganism, which appeared in the form of rodlets or of spherules, according to the position in which observed. Relation to oxygen. — In anaerobic conditions the microorganism does not develop at all, or only with great slowness, which indicates that it is prevailingly aerobic. Behavior in regard to temperature and to nutritive media. — It develops well at room temperature (150 C. circa), but more rapidly in the thermostat in the various nutrient media, and especially in neutral conditions. Gelatin cultures. — In 24 hours at room temperature it forms, in stab-cultures, a cup of initial liquefaction which progresses rapidly in cylindrical form until all the gelatin is liquefied. On the surface of the culture there is then formed a pellicle sufficiently thick that it detaches itself on agita- tion, falling to the bottom as a whitish, mucilaginous precipitate, and the liquid becomes a beautiful light green color in the upper part of the tube. Agar. — On glycerinated streak-agar cultures there develops, after 48 hours of incubation, a whitish surface growth little extended and slightly elevated, not shining. In stab-cultures the development is still more scanty; after 48 hours' incubation, the stab-growth is scarcely visible; growth soon ceases. In plain agar the organism develops rather better. In streaks, in 24 hours, it forms a wet- shining, surface growth, rather elevated, with lobed margin, of a whitish color, and with a tendency to occupy all the free surface of the tube. In stab-cultures the growth extends to the bottom of the tube, at the surface enlarging into the form of a whitish disk, in the center of which appears in time a roundish, yellow spot. Broth-cultures. — In alkaline broth it develops poorly, with slight turbidity and formation of a scanty deposit; in the upper part of the tube the liquid assumes a pale green color. In plain broth the development is more rapid and vigorous; in 48 hours of incubation the entire culture is uniformly clouded, with an abundant dirty white sediment, which in time becomes a pale yellow. At the surface and along the walls of the test-tube a pellicle forms which detaches itself easily; the liquid assumes a light green color. Potato-cultures. — At the temperature of the incubator there develops, in 48 hours, a gray-white growth, wet, rather elevated, with a tendency to spread. With age the culture assumes a granular aspect with a pale yellow color, tending toward brown. Chemical activity. — In the tubes an offensive gas develops, but not hydrogen sulphide, as we were able to verify by exposing above the cultures a small strip of lead acetate paper previously exposed to the vapor of ammonia. Artificial reproduction of the disease. — To prove that the disease was really due to the pathogenic action of the microorganism of our isolation, we infected some vigorous plants of Matthiola, spraying them with broth-cultures, very much diluted. At the end of a few days we obtained the reproduction of the disease on the aerial part of the plants sprayed, with the same external characters and the same anatomical alterations (in the leaves and in the young branches) which we have described in the plant naturally diseased. THE ITALIAN DISEASE OF STOCK. 279 The infection rapidly spreads on the leaves and branches, extending to the inflorescence, which dries up completely. The plants can throw out other floriferous branches, but of poor development and with few flowers. We tried also infection through the roots, bathing with a pure-culture solution the soil of some pots in which were healthy plants, but not a single sign of disease was observed at the end of two months. For the infection of the roots perhaps wounds are necessary. We are able to establish that the more common manner of infection is through the stomata, as plainly shown by the pale yellow coloring which spreads from the stomata to the surrounding cells, in which we observed the usual disorganization of the chloroplasts and of the protoplasm. From the organs infected artificially we repeatedly made cultures in various nutritive media for the identification of the morphological and cultural characters of the microorganism of our preceding isolation and description. This organism must be regarded as a new species, to which is given the name Bacterium mat- thiolae n. sp. The same year Briosi and Pavarino published a second paper (Baeteriosi della Matthiola annua I/.), which is exactly the same as the one just reviewed, except for the following additions: The stab growth in plain agar is described as ciliated (cigliato). .1 gar plates. — (Natural size.) After 48 hours' incubation the surface colonies are round, margins smooth. They are slightly raised, but not transparent, and of a whitish color. The buried colonics may be roundish, but for the most part have the form of a whetstone. Enlarged §0 diameters. — The surface colonies appear roundish, radiate, with transparent contour, and of a yellowish color. The buried colonies may be roundish, but for the most part are like a whet- stone, always smaller and of a darker yellow color. Under plain broth the sediment is described as viscous. Milk-cultures. — At a temperature of 30° C. the coagulation is complete on the third day with decidedly acid reaction to litmus. Specific and practical methods for the cure of this disease are unknown. We made tests with the usual sprays, using copper sulphate as the base (Bordeaux mixture), but the results obtained were negative. Perhaps such treatments might be effective applied as preventives sometime before the flowering period, with additional sprayings afterward. A good practice to follow is that of extirpating the plants as soon as they present the symptoms of the disease. The extirpated plants should not be permitted to accumulate on the land, where when dried they can be easily transported to a distance by the wind or by animals, but should be carried out of the field and destroyed by fire. Likewise, in order to prevent the infection being carried back by means of stable refuse, these extirpated plants must not be thrown on the manure heap. The rotation should be modified so that the culture of the stocks is not repeated on soil where the disease appeared the preceding year. To prevent the disease being transmitted from one year to another, it is rather useful to employ accurate seed selection, using only seed that has come from healthy plants, and from fields remaining immune to the infection. And since it has been demonstrated that germs of bacterial diseases frequently are able to continue to live on the seed during the winter season, it would be useful also in the present case to disinfect the seeds, immersing them for 15 minutes in a solution of 1 :iooo mercuric chloride, or in a solution of formalin 1 .50 for 20 minutes. This practice ought to be completed by the selection, according to the method of Nilsson, in the field of those individuals which show more resistance to the disease. Finally, it would, perhaps, render the plant more resistant to the disease to avoid abuse of organic fertilizers, and to have recourse to use of potassium fertilizers, and especially the superphosphate. The writer has had no opportunity to study this disease at first hand. Some samples of stock supposed to show this disease were received from Italy, but it proved to be some- thing else. LITERATURE. 1912. Briosi, G. and Pavarino, L. Una malattia 1912. Briosi, G. and Pavarino, L. BacLeriosi della batterica della Matthiola annua L. (Bac- Matthiola annua L. {Bacterium matlhiolae n. terium matlhiolae n. sp.). Atti della Reale sp.). Atti dell' Istituto Botanico della R, Aceademia dei Lincei, Rendiconti, vol. xxi, I'niversita di Pavia, Serie II, vol. xv, Milano, Fasc. 3, Rome, August, 1912, pp. 216-220. 1912, pp. 135-141, 2 pis. (.colored). THE BRAZILIAN DISEASE OF MANIHOT. In 191 2 Gregorio Bondar, of the Agronomical Institute at Campinas in Brazil, described a disease of Manihot that appears to belong here. I translate from his Portuguese leaflet, with some hesitation, as follows: The sweet mandioca (Manihot palmatd), varieties white mandioca and aipim mandioca, has been with us subject to a disease which apparently has not been met with in other countries. This rot of the stalks and the shoots (brotos) is caused by bacteria, Bacillus manihotus Arthaud- Berthet. The disease was studied at the Agronomic Institute in Campinas by the [former] Director, Dr. Arthaud-Berthet with our collaboration. The microbiological study was verified in the bac- teriological laboratory by the specialist Dr. A. Perrier. In the Mandioca aipim the effects of the disease are as follows: The young shoots (brotos) rot internally, wither, and die. By the transparency of the bark one can perceive dark subcortical lines which follow the bundles. Removing the bark one observes that these lines start from the sub- terranean stalk, which becomes rotten in a line corresponding to the shoot (broto) or presents the bundles (canaes) black, infected. In the young shoots (brotos), the bundles (canaes) presently decompose (fig. 138, b), injuring the circulation of the sap and causing the withering. The disease may attack the shoot on a single side, but presently gen- eralizes itself in other bundles (canaes). In young planta- tions the disease occasions the death of the plants. In the white Mandioca the signs of the disease differ: The shoots present subcortical swel- lings which are produced in the bundles by the coagulation of the latex (fig. 138,0,6). These swellings are elongate. The subcortical accumulation breaks the bark and the gummy substance exudes, forming small superficial crusts, of a light yellow color (amarello-clara) at the beginning, and afterwards reddish by oxidation. If the diseased shoots were (estiverem) grown on the stalks of the past year the black markings of the disease run through even to the subterranean part without, however, forming gummy swellings. The roots do not manifest any signs of disease. In a planting of ten months we observed that these roots were able to send out the shoots three or four times, which succumbed successively to the bacteria, until the roots were completely exhausted, dying directly afterwards. We made numerous artificial inoculations of the disease. The healthy shoots, inoculated arti- ficially with virus from the diseased plant, presented the same symptoms of disease after 15 or 20 days' incubation of the bacteria. The cuttings, infected with the bacteria before planting, died, rotting in the earth without budding. Of 20 infected cuttings not a single one grew. The experimental work on the disease, as also many observations, were made in collaboration with the Chefe de Culturas do Instituto, Sr. Joao Herrman. Attacking old stalks, with the roots formed, the bacteria bring about exhaustion of the accumu- lated starchy substance. The roots of the diseased stalks are unfit for food, for they are very hard, even when boiled two or three times as long as the ordinary mandioca. The chemical composition varies greatly also. The anaylsis made by Sr. R. Bolliger, in the Agro- nomic Institute in Campinas, revealed a noticeable diminution of the starchy substance: Instead of Fig. 137. *Fig. 137. 280 -Brazilian manihot wilting from bacterial disease. (After Bondar.) THE BRAZILIAN DISEASE OF MANIHOT. 28l 73.13 per cent of starch in the dry material as in the normal mandioca, there was scarcely 63.90 per cent in the diseased mandioca. The observations show that the disease attacks very often the cuttings (mudas) of mandioca, when they are made very long before planting. In this case the tips of the stems begin to rot. As the circulation of the sap occurs even in the cut stems, the conta- gion propagates itself in the bundles (canaes) without leaving external signs. The slips cut from such stems die for the greater part, and on the other hand, are liable to con- taminate others. The disease is grave, contagious, easily propagated, inoculable, and can be transmitted by insects. If care is not taken it may cause con- siderable injury to the cultivation of this plant of ours, which is of general use. With some cultural precautions the malady may be avoided or les- sened in severity. Curative treatment can not be applied, since the disease is internal, and generalizes itself directly in the plant. The preventive measures which we advise, in order to avoid the pro- pagation of the plague, are as follows : 1. Plant resting shoots (mudas) absolutely not suspected, coming from healthy plantations, rejecting all doubtful stalks. 2. Cut the cuttings (estacas) with the greatest care, without tear- ing or wounding the tissue, in such a manner as to reduce the surface and the possibility of contamination. 3. Do not plant the mandioca in soil infected by preceding diseased plantings, but cultivate other things not subject to contamination by the same bacteria. 4. Plant the cuttings as soon as possible, in order to avoid the contamination which occurs easily during the period of slow growth. 5. Plant resistant varieties: Mandioca parda and Mandioca azul resist better than the Mandioca branca and aipim. The bacteria are found in pure colonies under the bark, in the young swellings. The isolation of the microbe, likewise the artificial inoculation of the plant, show, fortunately, that there are present no other bacterial diseases. A more extensive account of this disease was prepared, but its publication has been delayed. LITERATURE. o Fig. 138.= 1912. Bondar, Gregorio. Uma nova molestia bacteriana das hastes da mandioca. No. 4, April 1912, pp. 15-18, 3 figures, Brazil. Chacaras e Quintaes, vol. v. *Fig. 138. — Stem of manihot attacked by bacterial disease, a. Bacterial blisters and exudate, b. Cross-section of stem showing location of the diseased part (at the top), c. Bacillus manihotus A. Berthet, from a diseased manihot plant. X 1,000. (After Bondar.) [The photographs and drawings in this volume, as in Volumes I and II, were made, with a few exceptions, by James F. Brewer. The writer, however, selected the parts to be drawn, or photographed, and finally checked up under the microscope nearly or quite all of the drawings. With exception of figs. 1 and 2 of plate 11, the colored figures were also made by Mr. Brewer. The color chart, referred to in various places as "Ridgway," is the first edition of Robert Ridgway's Nomenclature of Colors for Naturalists.] ADDENDUM. The following observations were made too late for insertion in the body of the text : (i) Aplanobader michiganense vs. Apl. rathayi. — Both liquefy gelatin slowly, but the latter liquefies the more rapidly and the surface growth (streak) floats about in the nearly clear fluid as a twisted ribbon. Apl. michiganense, on the contrary, clouds the fluid. (2) Peanut wilt. — In June, 1914, we obtained bacterial wilt of peanuts using both Medan III and Florida potato (isolation of 19 14). Inoculations were by needle pricks on stems ; the time between inoculation and appearance of disease was about two weeks ; great numbers of bacteria were seen in the tissues. Subsequently the wilt of peanut was also obtained by inoculating from a pure culture of the Creedmore, N. C, tobacco organism. (3) Tobacco wilt. — This was obtained in June, 1914, using Florida potato (19 14), Florida tobacco (1914) and Creedmore, N. C, tobacco (1914). The inoculations were by needle pricks from pure cultures (subcultures from poured-plate colonies) on stems and leaves. The time between inoculation and appearance of disease was about 4 to 10 days. (4) Bad. solanaccarum — Effect of drying. — On cover slips Medan III and Florida potato (1914) were dead at the end of 6 to 9 days, i. e., refused to grow when thrown into peptone-beef bouillon. These cover slips were wetted from young peptone water cultures and were kept in the dark in a covered Petri-dish at room temperatures (of June). Sub- sequently tests with the Creedmore tobacco organism (191 4) and the Florida tobacco organism (1914) showed all dead on the 6th day or earlier. The tests were begun on the 5th day, at which time the organism was alive on a few of the cover glasses, i. c., 1 out of 8 in Creedmore and 2 out of 8 in Florida. Tests on the 6th, 7th, and 9th days showed all dead. The covers (64 in all) were wet from cloudy 24-hour-old peptone water cultures and kept in the dark, in sterile Petri-dishes, in the well ventilated laboratory at room temperatures (of July). The covers were thrown into tubes of + 14 peptone bouillon. (5) Bacterium solanaccarum in litmus milk+cream. — Medan III always reddens this medium. It may therefore be known as var. Asiaticum nov. var. None of the American isolations of 19 14 (Florida potato, Florida tobacco, and Creedmore tobacco) have done so. (Experiments continued 4 to 8 weeks, using subcultures from 15 colonies.) (6) Bacterium solanaccarum in Meyer's solution + Am. lactate. — With Medan III and Florida potato (1914) at the end of 4 weeks there had been no growth. (7) Bacterium solanaccarum in Meyer s solution + Asparagin. — At the end of 4 weeks, 4 of the 8 tubes of Medan III clouded. All of the Florida potato (19 14) remained clear. (8) Bacterium solanacearum in Meyer's solution + KN03 + Sodium acetate; Do. Do. Do. + Sodium lactate. Do. Do. Do. + Sodium butyrate. Neither Medan III nor Florida potato (19 14) would growin these media. Testof 4weeks. (9) Bad. solanacearum inoculated on Livingston's Dwarf Aristocrat tomato. On page 189 (exp. of 1905) the statement is made that of many varieties tested the above named was most susceptible. Fearing this might be an accident, the experiments were repeated in July, 1914, using four strains of the bacteria: Medan III, Florida potato (1914), Florida tobacco (191 4), and Creedmore tobacco (191 4). In all 24 plants were inoculated, 6 from each strain. All except Medan III were wilting or wilted on the third day with enormous multiplication of bacteria in the tissues. The inoculations were made on young plants showing two good leaves (besides the cotyledons) and four when collected. The pricks were made with a fine needle; most of the punctures were made on petioles. Older plants were not available. The check plants remained sound. These plants contained 93 per cent of water. This experiment was repeated a few days later with similar results. (10) Loss of Virulence. Medan III, lost much of its virulence during 12 months culti- vation in the laboratory without reinoculation into plants, i. e., then it was less active on tobacco and on tomato than the recent (1914) isolations from American sources (PI. 45). 282 PLANT BACTERIA, VOL. III. PLATE 45. Loss of Virulence in Bacterium solanacearum. Livingston's Dwarf Aristocrat tomato after infection with Bad. solanacearum. Each plant was inocu- lated on one petiole by means of delicate needle pricks from a 3-day agar culture. Plants in hot- house subject to ± 350 C. (July temperatures) , and alike. A B, end of 5 days ; C D, end of 7 days. Florida potato (1914). Organism on culture media about 2 months, and still actively virulent. There were two more in this set. both badly wilted. Medan III (1913). Organism on culture media about 12 months. It was isolated by Honing 111 the summer of 1913 and was extremely virulent when received, and for several months thereafter, but is now weak (see page 179). Only 2 of the 6 plants showed any trace of wilt — those in center of figure. (3) Creedmore tobacco (1914). Organism on culture media about 5 weeks. In this set were 6 plants, all badly wilted. (4) Same as 2. In each of the 6 plants the inoculated petiole was now wilting. (1) (2) INDEX. Page. African disease of potato 214 Alternaria solani, in French disease of potato (Delacroix) 215 Amaranths, Smith's disease of 148 (See also Bacterium amaranthi.) description 148 geographical distribution 148, 150 literature 15° tissues attacked 149 Aplanobacter michiganense 165, 205 (See also tomato. Grand Rapids disease.) acid, production of 164, 165 acids, sensitiveness to 164, 165 aerobism 165 agar, slow growth on 165 alkali, toleration of 164 colonies 161 comparison with Aplanobacter rathayi 165, 282 cultivation, difficulty of 165 cultural characters 162 dextrose, acid from 164 dissemination 163 drying, resistant to 165 fermentation tube cultures 164, 165 gelatin, slow growth on 165, 282 Gram positive 165 group number 1 65 growth in or on, acid beef-bouillons 165 akaline beef bouillon 164 beef-bouillon 162, 164 Cohn's solution 162, 165 cornmeal agar stabs 162 cream-free milk 162 litmus milk 162, 164 Loeffler's blood-serum 165 milk 162, 164, 165 nitrate bouillon 162 nutrient agar 161, 162, 164, 165 nutrient gelatin 162, 164, 165, 282 potato cylinders 162, 164 sugars 1 64, 1 65 Uschinsky's solution 165 infection, point of 161 through stomata 163 inoculations on potatoes 164 tomatoes 161, 163 isolation of 161, 164 levulose, acid from 164 literature 165 litmus in milk, reduction of 162, 164 milk, copious surface growth on 165 morphology 161 motility, lack of 165 Porto Rican weed subject to 163 potassium nitrate, no reduction of 165 resume of salient characters 164 sodium chloride, sensitiveness to 164 starch, slow action on 164 substomatic chambers, occupied 163 thermal death-point low 165 tissues attacked 161 virulence, loss of 165 viscidity 165 Pagb. Aplanobacter rathayi 160 (See also Orchard grass, Rathay's disease.) acid, effect on 156 agar, slow growth on 156, 158 associated with white organism 159, 160 capsule 156, 160 Cohn's solution, no growth in 160 coloring matter 156 comparison with Aplanobacter michiganense . . 165, 282 cultivation, difficulty of 156, 157, 158 cultural characteristics 156 cultures, separable from substratum 156 dextrose fermented 160 fermentation of sugars 156 gelatin, growth on 156, 165, 282 Gram's stain 156, 159 growth in, or on agar and gelatin, slow 156, 158 beef bouillon 156, 159 cane-sugar water 160 Cohn's solution 160 colonies of associated white organism 160 Dactylis broth plus peptone 160 Dactylis decoction 156 grass broth 160 litmus milk 160 milk 1 60 nitrate bouillon 159 nutrient agars 156, 157, 158, 159 nutrient gelatins 156, 157, 160, 282 peptone water plus potassium nitrate 160 potato 156, 158, 160 potato broth plus potassium nitrate 160 group number 1 60 hydrogen sulphide, production 156 indol production 156 inoculations 156 inoculations into tomato 160 iodine, reaction 156 isolation 156 lactose fermented slowly 160 lemon juice, effect 156 lemon yellow color 156 liquefaction of gelatin, slow 160, 282 Rathay's experiments 156 literature 160 litmus, reaction 160 longevity 160 milk, behavior in 160 morphology 156, 157, 160 motility, lack of 156, 157 nitrates, no reduction of 160 pellicle present in 156 potato, used for isolation 156, 158 pseudozoogloeae 165 resistance to sunlight 156 saccharose fermented slowly 160 seeds, disease disseminated on 160 spores, not seen 156 stains, reaction to 156, 160 sugars, fermentation 156 sunlight, resistance to 156 viscidity 155, 157, 158 vitality, retention of 160 white organism associated with 159, 160 yellow pigment, nature of 156 283 284 INDEX. Appel, Bacillus phytophthorus, formation of cork barrier . 192 "black-leg," cause of 175 ring disease of potato, Germany 166, 215 (See also Solanaceae, brown rot, ring disease of potato, Germany.) Arachis hypogaea, wilt 151, 153, 253, 271 (See Bacterium solanacearum, peanut- wilt organism; peanut wilt, Sumatra; peanut wilt, United States; peanut wilt, van Breda de Haan's.) Arrowing of sugar-cane 19, 20 Arthaud-Berthet, Brazilian disease of Manihot 280 Ashby, Panama disease of banana reported from Jamaica 173 Australian disease of potato and tomato 207 Bacillus aeruginosus (tobacco canker?) 266 (See also Tobacco, wilt diseases, French disease.) cultural characteristics 266 Gram's stain 266 inoculations 266 morphology 266 synonomy 267 Bacillus amylobacter, tobacco root-rot, Comes 270 Bacillus amylovorus, compared with Bact. solanacearum, color 194 growth in beef-broth concentrated and strongly acid 136 growth in potato broth with malic acid 136 Bacillus atrosepticus 175 Bacillus brassicaevorus, synonomy 267 Bacillus caulivorus 175, 207 synonomy 267 Bacillus cloacae from maize 147 Bacillus coli, sodium chloride, toleration for 138 Gram's stain, reaction to 247 Bacillus fluorescens liquefaciens, synonomy 267 Bacillus fluorescens putridus, synonomy 267 Bacillus glagae 75 Bacillus manihotus 280 (See also Manihot, Brazilian disease.) inoculations 280 literature 281 occurrence, pure, in subcortical swellings 281 Bacillus marcescens 87 Bacillus megaterium, isolated from diseased tobacco . 269 Bacillus melanogenes 175, 203, 276 Bacillus mesentericus, associated with Bact. solanacearum in Sumatra . . 259 resemblance to Aplanobacter rathayi 156 Bacillus musae 170 bacterial ooze 171 biological characters 171 cavities formed by 170, 172 growth black on potato cylinders 171 growth on gelatin 171 inoculations on 170, 171 manila hemp 171 solanaceous plants 171 tomato 171 isolation 170 longevity, feeble 171 pathogenicity, quick loss of 171 similarity to Bacillus solanacearum 171 tissues attacked 170, 171, 172 (See also Banana, Rorer's Trinidad disease.) Bacillus mycoides, associated with Bact. solanacearum in Sumatra 259 Bacillus mycoides roseus, isolated from diseased tobacco 269 Bacillus nicotianae Uyeda, (See also Tobacco, wilt-diseases, Japanese disease.) acid, effect of 242 acid-production 238, 241 alkali, effect of 242 Page. Bacillus nicotianae Uyeda — continued. anaerobism 238, 241 Bacterium solanacearum (potato and tomatc-wilt organism), relation to 244, 254 (See also "comparison, etc.") Bacterium solanacearum (Sumatran tobacco-wilt organism), relation to 244, 254 (See also "comparison, etc.") blackening of, agar cultures 238 bouillon 241 milk 241 capsules 241, 247 colonies on agar 238, 241 colonies on gelatin 241 comparison (Honing's) of Sumatran tobacco-wilt organism with Uyeda's description of B. nico- tianae and Smith's and Uyeda's descriptions of Bact. solanacearum .... 247, 248, 249, 250, 251 (See also "similarity, etc.") comparison (Smith's) with Bact. solanacearum Smith 240, 242, 243, 244 (See also "similarity, etc.") crystals, ammonium magnesium phosphate 241 cultural characters 238, 241 comparison with Bact. solanacearum. Smith's observations 240, 242 cytase, production of 242 description, Uyeda's 238 possibly not drawn from one organism 244 diastase, presence of 242 egg-plant, non-infection of 241 endospores 241, 243, 247 enzymes, presence of 241, 242 flagella 238, 241, 243 gas-production 238, 241 Gram's stain, positive 241 growth on or in, agar 238, 241 asparagin-dextrose solution 242 carrot 242 gelatin 238, 241 glucose agar, gas from 238 glucose bouillon, gas from 238 milk 238, 241 mono-potassium phosphate plus magnesium sul- phate, sodium chloride, dextrose, and ammo- nium chloride 242 mono-potassium phosphate plus magnesium sulphate, sodium chloride, dextrose, and ammonium tartrate 242 mono-potassium phosphate plus magnesium sul- phate, sodium chloride, dextrose and aspara- gin 242 mono-potassium phosphate plus magnesium sul- phate, sodium chloride, dextrose, and potas- sium nitrate 242 mono-potassium phosphate plus magnesium sul- phate, sodium chloride, glycerin, and ammo- nium chloride 242 mono-potassium phosphate plus magnesium sul- phate, sodium chloride, glycerin, and ammo- nium tartrate 242 mono-potassium phosphate plus magnesium sul- phate, sodium chloride, glycerin, and potas- sium nitrate 242 peptone-dextrose solution 242 peptone-water, blackened 242 peptonized bouillon 238, 241 potato, yellow on 238, 241 radish, sharp odor on 242 Uschinsky's solution 242 hydrogen sulphide, production of 242 indol production 241, 242 inoculations, Jensen's, failure of 271 INDEX. 285 Page. Bacillus nicotianae Uyeda — continued, inoculations, Uyeda's, on Amaranthus gangeticus 243 Capsicum longum 243 egg-plant 238 Lycopersicum esculentum 243 Physalis alkekengi 243 Physalis minimum 243 Polygonum tinctorum 243 Solanum melongena 243 tobacco 238 invertase, presence of 242 liquefaction of gelatin 238, 241 literature 270, 271 litmus, reduction of 241, 242 maximum temperature 241 methylene blue, reduction of 242 morphology 238, 241 nitrate, reduction to nitrite 242 North American tobacco wilt, not found in 229 odor 238, 242 optimum temperature 238, 242 pellicle, early appearance 241 pigment, solubility 241 pigment-production 238, 241 effect of temperature 241 potato and tomato-wilt organism, relation to. . .244,254 (See also "comparison, etc.") resistant varieties 243 similarity of tobacco-wilt organisms (Sumatra and Japan) and Bact. solanacearum Smith, de- scribed from potato and tomato 244, 254 (See also "comparison, etc.") soil, depth of penetration in 242 spores 241, 243, 247 staining, carbol fuchsin 243 staining, Gram's method 241 Sumatran tobacco-wilt organism, relation to. 244, 254 (See also "comparison, etc.") temperature relations 238, 241 thermal death point 241 tomato, non-infection of 241 tomato and potato-wilt organism, relation to . 244, 254 (See also " comparison, etc.") trypsin, production of 242 tyrosinase, presence of 241, 242 Bacillus phytophthorus, Australian disease of potato and tomato 208 Bacterium solanacearum, characters distinguishing from 200 cause of Schwarzbeinigkeit 175 formation of cork-barrier 192 French disease of potato 214 growth on slices of raw potato 166 parasitism, active 203 pathogenicity compared with that of Bact. xantho- chlorum 272 Russian disease of potato 214 Bacillus prodigiosus, mixed culture inoculations on tobacco 267 Bacillus pseudarabinus 50 Bacillus putrifaciens putridus (tobacco pith-rot!1). . . 266 (See also Tobacco, wilt-diseases, French disease.) cultural characteristics 267 inoculations 267 morphology 267 Bacillus sacchari Janse, aerobism 76 copper sulphate, effect 76 Debray 's work 76 growth in culture media 76 isolation 74 Janse's work 74, 75 normal sugar-cane and other plants, presence in. . 75 resemblance to B. subtilis 76 Page. Bacillus sacchari Janse — continued. Sereh, causal relation to 75 spores 76 Bacillus sacchari Spegazzini 86 comparison with B. marcescens 87 comparison with Micrococcus prodigiosus Cohn . . 87 cultural characteristics 87 inoculation experiments 87 isolation from Polvillo 86 Janse's name earlier 86 morphology 87 red pigment produced 87 relation to Bact. vascularum 88 resemblance to B. sorghi 87 spores 87 Bacillus sesami 218 Bacillus solanacearum 178 (See Bacterium solanacearum.) Bacillus solanincola 175, 214, 215 Bacillus solanisaprus 203 Bacillus sorghi 87 Bacillus subtilis, growth on agar with Bact. solanacearum from Sumatran tobacco 264 isolation from top-rot of sugar-cane 83 resemblance to B. sacchari 76 Bacillus tabacivorus (tobacco collar-rot?) 266 (See also Tobacco, wilt-diseases, French disease.) cultural characteristics 266 inoculations 267 morphology 266 Bacillus tracheiphilus, compared with Bact. solanacearum, color 194 growth in beef-bouillon 194 Bacillus vascularum 8 (See Bacterium vascularum; Sugar-cane, Cobb's disease.) Bacillus vascularum solani 207, 208, 218 Bacillus vulgatus, resemblance to Aplanobacter rathayi 156 Bacterium amaranthi 148 (See also Amaranths, Smith's disease.) alkali, production 149 cells occupied by 149 color of 149 gas production, not seen 149 growth on, Loeffler's blood-serum 148 potato cylinders 148 sugar-beet cylinders 149 isolation 148 literature 150 morphology 1 49 starch, slight action on 149 tissues occupied 149 Bacterium aurantium roseum, associated with Bact. solanacearum in Sumatra 259 Bacterium campestre, browning of bundles 90 chloroform, marked effect of 143 comparison with Aplanobacter michiganense . 161, 164 growth in or on, beef-broth concentrated and strongly acid 136 cabbage juice 136 distilled water plus dipotassium phosphate, mag- nesium sulphate, ammonium phosphate and sodium acetate 137 Dunham's solution plus rosolic acid, differential . 139 Loeffler's blood serum 148 nitrate bouillon 1 38 potato broth 136 potato broth plus malic acid 136 potato cylinders, copious 67, 148 rutabaga 135 tomato juice 136 286 INDEX. Page. Bacterium campestre — continued. growth on yellow turnip 135 infection through water-pores 210 pigment, brown 142 potato-starch, action on 142 retarding influence of litmus neutral gelatin 8 size compared with that of Cobb's Bacillus vas- cularum 8 starch, strong action on 60 wind, not borne by 68 Bacterium deliense, associated with Bact solanace- arum in Sumatra 259 Bacterium dianthi, growth in potato broth with malic acid 1 36 Bacterium gummis, tobacco-rot, Italy 266 tomato disease, Italy 203, 215 Bacterium hyacinthi, acids, toleration of 138, 144 alkalies, toleration of 138 growth on or in, agar 133 beef-broth concentrated and strongly acid 136 Dunham's solution plus rosolic acid 139 nitrate bouillon 138 nitrogen 138 potato broth 136 potato broth plus malic acid 136 potato cylinders 60, 134, 148 rutabaga 135 lesions produced by 130 min.mum temperature 143 morphology 149 pota.o-starch, slight action on 142 Bacterium langkatense, associated with Baet. sola- nacearum in Sumatra 259 Bacterium matthiolae, (See also Stock, Italian disease.) aerobism 278 colonies 279 gas-production 278 Gram's stain 278 growth on or in, agar 278, 279 alkaline broth 278 broth 278,279 gelatin 278 glycerin-agar 278 milk 279 potato 278 infection, manner of 279 inoculations 278 isolation 278 liquefaction of gelatin 278 literature 279 morphology 278 motility 278 reaction of media most favorable 278 stains, reaction to 278 temperature relations 278 Bacterium medanense, associated with Bact. solana- cearum in Sumatra 259 Bacterium megatherium, renders tobacco bacterium non-infectious 267 Bacterium michiganense 165 (See Aplanobacter michiganense.) Bacterium mori 209 Bacterium patclliforme, associated with Bact. sola- nacearum in Sumatra 259 Bacterium phaseoli, growth in or on, beef-broth concentrated and strongly acid 136 cabbage-juice 136 Hunger's agar, heaped up 57 potato broth 136 Page. Bacterium phaseoli- — continued, growth in or on — continued. potato broth plus malic acid 136 potato cylinders 67 Bacterim pruni, growth in Uschinsky's solution. . . . 137 Bacterium rangiferinum, associated with Bact. sola- nacearum in Sumatra 259 Bacterium sacchari 50 Bacterium shuffneri, associated with Bact. solana- cearum in Sumatra 259 Bacterium solanacearum (Deli strain), [See Bact. solanacearum (Sumatran tobacco-wilt organism).] Bacterium solanacearum (Indian tobacco-wilt organism) 267 (See also Tobacco, wilt-diseases. Indian disease.) bipolar staining 267 culture medium, effect on virulence 267 growth on or in, agar 267 bouillon 267 gelatin 267 glucose bouillon 267 potato 267 inoculations 267 with mixed cultures 267 literature 271 longevity 268 morphological character, variations in 267 morphology 267 motility, denied 267 pigment 267, 268 effect on virulence 268 tissues, effect on 268 virulence, effect of pigment on 268 loss of 268 restoration of 268 variation in 267 Bacterium solanacearum (?) (Japanese tobacco-wilt organism), (See Bacillus nicotianae Uyeda.) Bacterium sclanacearum (Medan), [See Bact. solanacearum (Sumatran tobacco organism).] Bacterium solanacearum (North American tobacco-wilt organism) 231, 234, 235 (See also Peanut wilt, United States; Tobacco, wilt- diseases, North American disease.) aerobism 233 browning on agar 232 browning on potato 232 comparison with Bact. solanacearum (potato and tomato) 230, 231, 234 comparison with peanut and pepper-wilt organ- isms 271 cultural characters 231 drying, effect on 282 Fulton's work on 271 gas-production, not observed 233 growth on or in, acid bouillon (plus 33 muscle acid) 232 beef-agar 231 Cohn's solution 233 gelatin 232 litmus milk 233 litmus milk with cream 282 milk 233 nitrate bouillon 233 potato 232 standard peptonized bouillon 232 incubation period 234 inoculations, (See also Tobacco, wilt-diseases, N. Am. disease.) direct, on tobacco (organism passed through tomato) . . 234 INDEX. 287 Pace. Bacterium solanacearum (N. Am. tobacco) — cont. inoculations, pure culture on peanut 271 tobacco 229 tomato 220, 230, 236 soil, on Datura stramonium 233 egg-plant 235 peppers 235 tobacco 181, 229, 235, 282 tomato 181, 233, 282 isolation 228 literature 270, 271 longevity 232 minimum temperature 233 morphology 231 peanut- wilt, cause of 271 potato and tomato-wilt organism like . . . 230, 231, 234 reduction of nitrates 233 relation to Bact. solanacearum (potato and tomato) 230, 231, 234 similarity to peanut-wilt organism 27 1 pepper- wilt organism 271 Sumatran tobacco-wilt organism, behavior in cream-free litmus milk 264 signs of disease 263 soil, infection through (peanut) 271 soil, persistence of organism in 237 Sumatran tobacco-wilt organism, similarity in cream-free litmus milk 264 Sumatran tobacco-wilt organism, similarity in signs of disease 263 thermal death-point 233 tissues attacked 227, 230 tomato and potato- wilt organism like . . . 230, 231, 234 vessels occupied 227, 230 water-content of host, effect on inoculations ... 282 Bacterium solanacearum (Peanut-wilt organism) . 153, 253.271 (See also Peanut wilt, Sumatra; Peanut wilt, United States; Peanut wilt, van Breda de Haan's.) comparison with tobacco and pepper organisms. 271 cultural characteristics 271 Fulton's work on 271 Honing's report of 153, 253 inoculations, on peanut 271 tobacco 271 (See also Peanut wilt, United States.) isolation 271 literature 271 morphology 271 similarity to, pepper-wilt organism 271 tobacco- wilt organism 271 stains, reaction to 271 Bacterium solanacearum (potato and tomato) 178, 193 (See also Peanut wilt; Solanaceae, brown rot; Tobacco, wilt diseases.) acids, effect of 200, 2 1 1 acid production, not detected 196, 200 aerobism, strict 196 African disease of potato, Odium's observations on 214 agar, browned by 1 95 alkali, production of 194, 196 Australian disease of potato and tomato, Tryon's observations on 207 Bacillus musae, similarity to 171 Bacillus nicotianae, synonym of 254 not wholly like 244 (See also "comparison, etc.") Bacillus phytophthorus, characters separating from 200 Bancroft, Malay States disease 211 bipolar staining of 267 Pagg. Bacterium solanacearum (potato, tomato) — cont. brown stain, accompanying loss of vitality 199, 200 in form of streaks in stems 176, 185 nature of 194 production of 200 casein, no action on 194 cell-wall, action on 191 Ceylon wilt-disease of tomato, Petch's 214 chains, seldom seen 193, 200 Cohn's solution, no growth in 197 Coleman, Mysore ring disease 212 colonies 195, 199 often dead when browned 199, 232 color 194 Comes, Italian disease of tomato 215 comparison with N. Am. tobacco organism. 230,231,234 comparison (Honing's) of Sumatran tobacco-wilt organism with Smith's and Uyeda's descrip- tions of Bact. solanacearum and Uyeda's de- scription of B. nicotianae . . 247, 248, 249,250, 251 (See also "relation, etc.") comparison (Smith's) with B. nicotianae. . . . 240, 241, 242, 243, 244 (See also "relation, etc.") cucumbers not rotted by 174 cultural characteristics 194, 199, 211 culture-media, growth on, effect on virulence. ... 179 Delacroix, French disease of potato 214 dendritic growth, not characteristic 200 description of 193 diffusion from vessels 197 drying, sensitive to 282 Dutch East Indian disease of tomato. Hunger's observations 209 (See Solanaceae, brown rot, Dutch East Indian disease.) English disease of potato 216 enlargements, local, due to 199 entrance into host 181 fermentation-tube experiments 196 field inoculations on large scale with 187 filaments rare 193 flagella 193 Florida potato-wilt organism, bipolar staining 267 drying, effect of 282 growth in, Cohn's solution 264 litmus milk with cream 264, 282 Meyer's solution plus ammonium salts 251, 282 asparagin 282 potassium nitrate plus sodium salts 282 sodium salts 251, 282 Uschinsky 's solution 264 inoculations on peanut 282 tobacco 282 tomato (Livingston's Dwarf Aristocrat) 282 water-content of host, effect on inoculations 282 freezing, effect on 198, 199, 200 French potato disease, Delacroix's observations 214, 215 de Laharpe's observations 215 gas, not produced by 196, 200 Gram's stain, reaction to 198 Grand Rapids tomato disease, not due to 161 green fluorescence, not characteristic of 200 group number 200 growth on or in, acid bouillon 199 beef-agar, peptonized 195, 199 beef-bouillon, peptonized 194, 199 beef-bouillon, peptonized, plus sodium carbonate 194 blood serum, solidified 200 288 INDEX. Bacterium solanacearum (potato, tomato) — cont. growth, on or in — continued. Cohn's solution 197. 264 Dunham's solution 194 gelatin 195. 200 glycerin-agar, alkaline 196 litmus-lactose agar 195. 2°° litmus milk cream free 194. 2°° plus cream (Florida potato organism) . . 264, 282 Meyer's solution plus ammonium salts 251, 282 asparagin 282 potassium nitrate plus sodium salts 282 sodium salts 251, 282 urea and cane sugar 251 urea and grape sugar 251 milk 194.200 nitrate bouillon 198, 1 99 peptone water '94 plus various sugars 19° potato cylinders, steamed 194, 196, 199, 200 potato-juice '9° silicate-jelly with Fermi's solution 197. 200 sugars '90. 199 sugar-beet agar 199 Uschinsky's solution 264 plus peptone '99 Honing's observations, host-plants '99 involution forms '94 Sumatran disease of Sesamum 218, 246, 253 host-plants 174. 199 (See also Peanut wilt; Tobacco, wilt-diseases.) Hunger's observations 181, 197, 209 incubation period '79 indol-production 198 infection, difficulty of producing with strains long-isolated. 1 79 effect of, dependent on, age of plant 178, 180 moisture content 179. 182 rapidity of growth 178 temperature 1 79. ' 82 incubation period 179 through wounds 181, 182 (See also inoculations.) inoculations 174, 178, 179, 182,205,209,210 feebly virulent strains 199 methods 178, 179. 19°. 209, 210 needle-prick, single, result of 19° successful, strains used for 179 synopsis of 1 82 uncertainty of results 178, 179, 268 (See also infections.) inoculations on, Abutilon sp 186 Capsicum annuum 209 cucumber 183, 184, 185, 186 Cucurbita foetidissima 185 Datura cornucopiae 1 86 Datura fastuosa 186 Datura metalloides 186 Datura stramonium 183, 184, 186 Datura tatula (?) 184 egg-plant 183 Eleusine indica 185 heliotrope 1 85 peanut 282 pear-tree (Japan) 183 Pelargonium zonale 1 83 pepino 183 pepper 1 83 petunia 1 84 Physalis crassifolia 1 84 Physalis philadelphica 185 Pags Bacterium solanacearum (potato, tomato) — cont. inoculations, on — continued. Portulaca oleraceae 185 potato. 179, 180,182,183, 184,185,186,187, 188,189 Blush variety 189 Burbank variety 1 87 Early Rose variety 187, 189 Green Mountain variety 188 Ricinus communis 185 Solanum carolinense 183 Solanum dulcamara 1 86 Solanum nigrum 183, 184 tobacco. . . 183, 184, 186, 189, 190, 209, 230, 233, 282 tomato 179, 180, 182, 183, 185, 186, 187,188, 189, 190, 209, 210 green fruits 176 Honor Bright variety 188 Livingston's Dwarf Aristocrat variety. . . 189, 282 Red pear variety 188 Vigna catjang 185 (See also Tobacco, wilt-diseases.) involution forms 193, 194 isolation, difficulties of 178 Italian disease of tomato, Comes' observations 215 Voglino's observations 215 Iwanoff, Russian disease of potato 214 Japanese tobacco-wilt organism, relation to. . 244, 254 (See also "comparison, etc.") killed by dry air 282 Kirk, New Zealand disease of potato 207 lab-ferment 200 liquefaction, gelatin 195, 200 Loeffler's blood-serum 200 literature 218 litmus milk, effect on 194 local enlargements due to 199 longevity, on or in, agar 199 dry air 282 milk 194, 200 steamed potato 198, 200 loss of virulence 1 88 Malay States disease, Bancroft's observations. ... 211 Malkoff, disease of sesamum 216 milk, clearing of 194 morphology 193, 199 motility 193, 264, 268 Hutchinson's observations on 200, 265, 267 movement of bacteria in inoculated plants 179 New South Wales potato diseases, Helms' 207 New Zealand potato disease. Kirk's observations . . 207 Odium, African disease of potato 214 odor 197, 200 oxygen, free, necessary for respiration of 196 peanut, cause of disease in 271, 282 Petch, Ceylon disease of tomato 214 pigment, action of dilute acids and alkalies on 194 produced by 194, 200 solubility of 194, 200 polar staining 267 pseudozoogloeae 193, 194 reduction of nitrates 198 relation to tobacco-wilt organism North America 230, 231, 234 Sumatra and Japan 244, 254 (See also "comparison, etc.") resistant Solanum 1 82 resume of salient characters 1 99 ring disease of potato, Germany 166, 215 (See also Solanaceae, Brown rot, ring disease of potato, Germany.) ring disease of potato, Mysore 212 (See also Solanaceae, brown rot, ring disease of potato, Mysore.) INDEX. 289 Pagb. Bacterium solanacearum (potato, tomato) — cont. Russian potato disease, Iwanoff's observations. . . 214 sesamum, baeteriosis 218 (See also Solanaceae, brown rot, sesamum baeteriosis.) similarity to. Bacillus musae 171 tobacco-wilt organism (N. Am.) 230, 231, 234 tobacco-wilt organisms (Sumatra and Japan) 244,254 (See also "comparison, etc.") lack of, Sumatran tobacco-wilt organism in lit- mus milk with cream (Florida potato strain) . . 264 Spieckermann's potato-parasite, distinct from .... 167 spores, not seen 193, 200 spread of parasite in inoculated plant 179 stains, reaction to 193 starch, slight action on 192, 197 sugared fluids, browning of 196 Sumatran tobacco-wilt organism, relation to. 244, 254 (See also "comparison, etc.") Sumatran tobacco organism, variations from Florida potato organism in milk with cream. . 264 termo-like shape of 175, 199 thermal relations 198, 200 tissues attacked 175, 176, 191, 199 tobacco-wilt of Dutch East Indies, relation to. . . . 222 tobacco-wilt organism (N. Am.), like. . . . 230, 231, 234 tobacco- wilt organism (Sumatra), variation from Florida potato-wilt organism in litmus milk with cream 264 tobacco-wilt organisms (Sumatra and Japan) re- lation to 244, 254 (See also "comparison, etc.") virulence, degrees of 199 effect on, of growth on culture media 179 loss of 179, 188, 268 viscidity, not marked 197, 200 Voglino, Italian disease of tomato 215 water-content of host, effect on inoculations 282 white substance surrounding colonies on agar. . . 199 wrinkled growth absent 200 Bacterium solanacearum (Sumatran tobacco- wilt organism), American studies 263 acid produced from cream in litmus milk 264 browning of culture-media 263, 265 crystals 265 drying, effect of 282 flagella 265, 268 Florida potato-wilt organism, dissimilarity in litmus milk with cream 264, 282 growth on or in, agar with Bacillus subtilis 264 cream-free litmus milk, plus 264 coconut oil 264 cottonseed oil 264 olive oil 264 peanut oil 264 fermentation tubes 265 lactose not fermented by 264 litmus milk 263, 264, 265 litmus milk containing cream 264, 282 Meyer's solution plus ammonium salts 251, 282 asparagin 282 potassium nitrate plus sodium salts 282 sodium salts 251, 282 urea and cane-sugar 251 urea and grape sugar 251 milk 263 mineral solution with KNO3 and glycerin . . 242 nitrate bouillon 265 nutrient gelatin 265 peptone water, plus sugars 265 glycerin 265 Pagb. Bacterium solanacearum (Sumatran tobacco) — cont. American studies — continued, inoculations, on peanut 282 tobacco 263, 265 tomato (Livingston's Dwarf Aristocrat) 282 liquefaction of gelatin 265 litmus milk bluing of 263, 264 reddening of 263, 264 reduction of 264 motility, occurrence of 265, 268 nitrates, reduction of 242 pathogenicity 263, 265 similarity to North American tobacco-wilt or- ganism, cream-free litmus milk 264 similarity to North American tobacco-wilt or- ganism, signs of disease 263 similarity, lack of, to Florida potato-wilt or- ganism in litmus milk with cream 264 similarity, lack of, to North American tobacco- wilt organism in litmus milk with cream. . . . 282 stains, reaction to 265 virulence, loss of 282 water-content of host, effect on inoculations. . . 282 Honing's studies 224, 244 (See also Tobacco, wilt-diseases, Honing's Suma- tran studies.) acid, toleration of 256, 257 acid-production 249 in litmus milk 248 adonite as carbon food 251, 252, 253, 257 aerobism 248 albumin as carbon-nitrogen food 250 alcohols, growth in 249, 251, 252, 253, 254 alkali, production of 249 retarding effect of 257 toleration of 256, 257 alkali-production in litmus milk 248 ammonia-nitrogen used by 251 ammonium salts as carbon food 253 ammonium salts as carbon-nitrogen food . . 250, 253 amount of inoculating material used in making cultures, effect of 258 arabinose as carbon food 251, 252, 253 asparagin as carbon-nitrogen food 250, 253 asparagin as nitrogen food 252, 253, 254, 257 asparagin plus glucose as food 251 asparagin plus saccharose as food 251 associated organisms 258 Bacillus nicotianae, relation to 244, 254 (See also "comparison, etc.") blackening of agar 248 gelatin 248 potato slices 248 browning of beef bouillon 247 capsules 247, 253 carbon, substances used as source of . 250, 25 1,252,253 carbon-nitrogen foods 250 253 chains in medium containing glycocoll and glucose 253 clouding, time of, relation to number of bacteria used in inoculation 258 comparison of Deli strains with Uyeda's descrip- tions of B. nicotianae and Smith's and Uyeda's descriptions of Bacterium solana- cearum 247, 248, 249, 250, 251 (See also "relation, etc.") cultural characteristics 247 cultural differences in strains 250, 251, 252 dextrin as carbon food 251, 252, 253 diastase production 249 drying, resistance to 249 dulcit as carbon food 251, 252, 253 erythrite as carbon food 251, 252, 253, 257 290 INDEX. PAGB. Bacterium solanacearum (Sumatran tobacco) — cont. Honing's studies — continued. fat, formation of 249 fibrin of blood as carbon-nitrogen food 250 filaments in medium containing glycocoll and glucose 253 flagella 247 galactose as carbon food 251, 252, 253, 257 gas production 249 glucose, action on 249 glucose as carbon food 251, 252, 253 glucose plus asparagin as food 251 glucose plus potassium nitrate as food 251 gluten as carbon-nitrogen food 250 glycerin as carbon food 251, 252, 253 glycocoll as carbon-nitrogen food 250, 252 glycocoll as nitrogen food 252, 253, 254 glycogen as carbon source 249, 251, 252, 253 gb'cogen production 249 Gram's stain, reaction to 247, 253 growth in or on, acid glycocoll-glucose solution 257, 258 agar 248 alcohols 249, 251, 252, 253, 254 alkaline glycocoll-glucose solution 257, 258 asparagin-glycocoll-potassium nitrate solution plus glucose 257 plusmannose 257 asparagin-solution plus carbon foods 257 beef bouillon 247 fermentation tubes 248, 249 gelatin 247 hydrogen 248 litmus milk 248 Meyer's solution, plus ammonium chlorid plus glucose 251 ammonium chlorid plus glycerine 251 ammonium salts 250 asparagin plus carbon foods 252, 253 asparagin plus sodium salts 252, 253 ammonia plus carbon foods 253 carbon-nitrogen compounds 250 glycocoll plus carbon foods 252, 253 glycocoll plus glucose with variable reactions. 256 glycocoll plus mannose with variable reac- tions 256 potassium nitrate plus carbon foods. . . 251, 253 potassium nitrate plus sodium salts. . . 251, 253 milk 248, 253 monopotassium phosphate plus magnesium sulphate plus sodium chlorid plus potas- sium nitrate plus dextrin 250 glucose 250 glycerin 250 inuline. 250 saccharose 250 sorbit 250 potassium nitrate solution plus carbon foods. 257 potato 248 sugars 249, 251, 252, 253, 254 urea solution plus glucose 257 plus mannose 257 growth under paraffin oil 248 guanine as carbon-nitrogen food 250 heat, resistance to 247 hemialbumose as carbon-nitrogen food 250 host-plants 226, 253, 254, 255, 282 (See also Inoculations.) hydrogen sulphide, production of 249 indol-production 249 inoculating culture media, number of bacteria used, effect 258 inoculating plants, number of bacteria used, effect 260 inoculations, Ageratum organism on tobacco .... 226, 244, 245 Page. Bacterium solanacearum (Sumatran tobacco) — cont. Honing's studies — continued, inoculations- — continued. Djatti organism on Djatti plants 254 Nicotiana affinis 255 N. atropurpurea grandiflora 255 N. colossea 255 N. glauca 255 N. rustica 255, 256 N. sanderae 255 N. silvestris 255 ornamental and common tobacco 254, 255 Mucuna organism on tobacco 244 Pluchea organism on tobacco 244 Pouzolzia organism on tobacco 226, 244 tobacco organism on Capsicum 246, 253 Nicotiana affinis 255 N. atropurpurea grandiflora 255 N. colossea 255 N. glauca 255 N. rustica 255, 256 N. sanderae 255 N. silvestris 255 ornamental tobaccos 255 tobacco organism (?), on potato 245, 253 tobacco organism, on Pouzolzia 245 tobacco organism (?), on Sesamum orientale. 253 on Solanun lycopersicum 253 tobacco organism, on Solanum melongena 245, 253 on tobacco 225, 244 (?), 245, 255 inoculations, soil: tobacco organism on tobacco 225 inoculations, soil-water, on Ageratum 245 Pouzolzia 245 tobacco 245 inosite as carbon food 251, 252, 253, 257 inulin as carbon source 251, 252, 253 isolation from Ageratum 226 Mucuna 226 Pouzolzia 226 tobacco 225, 246 Japanese tobacco-wilt organism, relation to. 244, 254 (See also "comparison, etc.") lactose as carbon food 251, 252, 253 legumin as carbon-nitrogen food 250 leucine as carbon-nitrogen food 250 levulose as carbon food 251, 252, 253, 254, 257 lichenin as carbon source 251, 252, 253, 257 liquefaction of gelatin 247 literature 270, 271 litmus, reaction to (milk cultures) 248 litmus, reduction of 249 maltose, as carbon food 251, 252, 253 mannit, action on 249 as carbon source 251, 252, 253 mannose as carbon food 251, 252, 253, 257 maximum temperature 249 methylene blue, reduction of 249 morphology 246 variability in 253 motility 247 nitrate, reduction of 249, 253 nitrogen, substances used as source . . 250,251,252,253 nitrogen and carbon from one substance . . . 250, 253 nuclein as carbon-nitrogen food 250 number of bacteria, effect on growth in culture media 258 effect on progress of disease 260 odor 249 peanut wilt, cause of 153, 253 pellicle 247 peptone as carbon-nitrogen food 250, 253 INDEX. 291 PAG8. Bacterium solanacearum (Sumatran tobacco) — cont. Honing's studies — continued. phenolphthalein, reaction to (milk cultures) . . . 248 physiological characters, variations in 253 physiology 247 polar staining 247, 253 potassium nitrate as nitrogen food. 250, 251, 254, 257 potassium nitrate plus glucose as food 251 plus saccharose as food 251 potassium nitrite as nitrogen food 250 potato and tomato-wilt organism, relation to . . 244 (See also "comparison, etc.") protein as carbon-nitrogen food 250 quercit as carbon food 251, 252, 253 raffinose as carbon food 251, 252, 253 relation of tobacco-wilt organisms (Sumatra and Japan) and Bact. solanacearum Smith described from potato and tomato 244, 254 (See also "comparison, etc.") rhamnose as carbon food 251, 252, 253 saccharose, action on 249 as carbon food 251, 252, 253 plus asparagin as food 251 plus potassium nitrate as food 251 saprophytes, associated with 258 inhibiting action 259 similarity of tobacco-wilt organisms (Sumatra and Japan) and Bact. solanacearum Smith, de- scribed from potato and tomato 244, 254 (See also "comparison, etc.") sodium salts as carbon food 251, 252, 253 sodium selenite, reduction of 249,253 sorbit as carbon source 251, 252, 253 spores 247, 253 staining of culture media 247, 248, 249 starch, action on 249 as carbon food 251, 253 strains, variability 244, 250, 251, 252, 257 sugars, growth in 249, 251, 252, 253,254 summary 253, 258 temperature relations 249 thermal death-point 249 tomato and potato-wilt organism, relation to . . 244 (See also "comparison, etc.") transmission of 254 tyrosin as carbon-nitrogen food 249, 250, 253 variability effect of alkalinity on 258 effect of light 258 effect of temperature 258 in carbon and nitrogen-compound tests ... 253, 256,257, 258 in cultural characteristics .250,251,252,253,256,257 in milk 253 in toleration of acid 257 in toleration of alkali 257 in virulence 244 variations, transitory nature of 258 virulence, loss 253 Bacterium solanacearum var. Asiaticum nov. var 282 Hunger's observations 222 Bacterium stalactitigenes, associated with Bact. solanacearum in Sumatra 259 Bacterium stewarti 91, 132 (See also, Corn, Stewart's bacterial disease.) acids, toleration of certain 136, 144 acid fuchsin, effect of 140 acid production 139, 142, 145 aerobism, pronounced 138 agar stabs, growth in 133 agar streaks 1 34 alkali produced 142 alkali, toleration of 138 Pagb. Bacterium stewarti — continued. animals, no experiments with 144 asparagin, not a carbon food 145 cane-sugar favors growth 138 capsule 132 chains 132 chloroform, slight retarding effect of . . . . 143, 144, 145 colonies, appearance of 133 color on media • . '33. 134. '35. 137. 138 comparison with Apl. michiganense on potato .... 164 crystals 133, 142, 144 cultural characteristics 133 cytase, doubtful occurrence 142 description 132 dextrin, no increased growth with 138 dextrose, increased growth with 142 diastase 142, 145 discoloration of bundles 16 discovery of organism 91 dissemination through infected seed 114, 127 dry air, resistance to 144 endospores not seen 132 enzymes 142, 145 ethyl alcohol, no acid from 139 fermentation tubes, growth in 138, 145 filaments 132 fiagella, polar 91, 132 formalin, infected seeds treated with 128 galactose favors growth 138 gas production not seen 138, 145 gases other than air, behavior in 138, 139 gelatin not liquefied 133 germicides 142 glycerin, no acid from 139 no increased growth with 138 some acid from 139 Gram's stain 132 group number 145 growth on or in, acid beef-bouillon 136, 138, 141, 145 agar 133, 142. 144 agar in atmosphere of hydrogen 139 agar plus potassium formate and phenolphthalein 1 43 alcohols 139, 145 alcohol agars 139 alkaline beef-bouillon 138, 141, 144 alkaline beef-bouillon in vacuo 139 ammonium salts 140, 141 asparagin water 141, 144, 145 beef-bouillon in atmosphere of carbon dioxide . . 139 beef-bouillon in atmosphere of hydrogen. . 138, 139 beef-bouillon over chloroform 143, 144, 145 beef-bouillon concentrated and strongly acid. . . 136 beef-bouillon plus ethyl alcohol 139 beef-bouillon plus glycerin 139 beef-bouillon plus peptone 136, 144 beef-bouillon plus sodium chloride 138 cabbage-juice 136, 138, 144 cane-sugar 139, 142 cane-sugar plus asparagin 144 cane-sugar agar plus litmus 139 cane-sugar starch jelly 138 cane-sugar water 140, 141 cane-sugar water plus ammonium citrate 140 cane-sugar water plus ammonium lactate. . 140, 141 cane-sugar water plus ammonium tartrate . . 140, 141 cane-sugar water plus asparagin 140, 141 carbon dioxide 139. 145 carrot cylinders 142 carrot cylinders in vacuo 139 coconut cylinders 135, 142, 144 coconut cylinders in atmosphere of carbon dioxide 139 coconut cylinders in atmosphere of nitrogen 138, 145 coconut cylinders in vacuo 139 Cohn's solution 138, 140, 144 292 INDEX. Page. Bacterium steward — continued, growth on or in — continued. dextrin starch jelly dextrose agar plus litmus distilled water plus dipotassium phosphate, mag- nesium sulphate, ammonium phosphate and sodium acetate Dunham's solution plus methylene blue Dunham's solution plus rosolic acid in atmos- phere of hydrogen Dunham's solution plus rosolic acid and hydro- chloric acid 139, Fermi's solution galactose galactose agar plus litmus galactose starch jelly gelatin 133. '44. glycerin agar glycerin agar plus litmus glycerin starch jelly grape-sugar 139, hydrogen 138, lactose agar plus litmus lactose starch jelly litmus agars plus sugars litmus milk 137, 140, Loeffler's blood-serum 133, 144, maltose agar plus litmus maltose starch jelly mannit mannit agar plus litmus mannit starch jelly milk 137, 142, mulberry agar neutral beef-bouillon in atmosphere of hydrogen, neutral beef-bouillon plus potassium formate . . nitrate agar 133, nitrate bouillon 132, 138, nitrate bouillon plus grape-sugar nitrogen 138, nutrient starch-jelly plus sugars peptone water peptone-water plus acid fuchsin and hydrochlo- ric acid peptone water plus grape-sugar and methylene blue in an atmosphere of hydrogen peptone water plus salt and indigo carmine .... peptone water plus salt and rosolic acid peptone water plus salt and rosolic acid in an atmosphere of hydrogen peptonized beef bouillon 136, potato broth 136, potato broth plus malic acid 136, potato cylinders 134, 142, 144, potato cylinders in an atmosphere of carbon dioxide potato cylinders in an atmosphere of hydrogen . potato cylinders in vacuo radish 135, rutabaga 135, 142, saccharose 139, saccharose agar plus litmus silicate jelly 134, starch jelly plus sugars 138, sugars 138, 139, sugar agars sugar starch-jellies sugar-beet 135, tomato juice 136, 138, 140, 141, 144, turnip. 135, 142, turnip in atmosphere of nitrogen 138, Uschinsky's solution. . . . 132, 137, 140, 142, 143, Uschinsky's solution in atmosphere of hydrogen. vacuo 139, host-plants 89 hydrogen peroxide, seeds treated with 128 37 4i 39 45 38 39 39 38 45 34 39 38 42 45 39 38 39 45 45 39 38 39 39 38 45 36 39 43 44 40 38 45 38 44 40 39 40 39 39 44 44 38 45 39 39 39 42 44 42 39 44 45 45 39 38 44 45 44 45 45 39 45 Page. Bacterium stewarti — continued. hydrogen sulphide, doubtful production of 142 ice box, growth in 143, 144 indigo carmine, effect of 140, 145 indol reaction 142, 144, 145 infected seed, treatment of 125, 127, 128, 146 inoculations 96 method, by needle punctures 113 by spraying 96, 97, 129 through water-pores 96, 1 1 1 , 112 on field corn ' 93, m, 112, 129 on oats 93 on pop-corn 93, 1 1 1 on sweet corn 92, 96, no, 112, 129 Series I and II 96 Series I and II, detailed results 98 Series I and II, tabulated results 109 Series III-VII no Series III-VII, tabulated results in Series VIII and IX in Series X 112 Series XI-XV 121 Series XVI-XIX 125 Series XX-XXIV 128 Series XXV 129 Stewart's 91, 92 inversion of cane-sugar 142 invertase 142, 145 isolation attempted from infected kernels 124 isolation by direct transfer from diseased stems. . . 96 kernels penetrated by bacteria 114, 116, 130, 131 lab ferment 142, 145 lactic acid, toleration of 144 lactose, no increased growth with 138 lesions produced 130 literature 147 litmus agars, behavior on 139 Loeffler's blood serum, not liquefied by 133 malic acid, toleration of 144 maltose, no increased growth with 138 mannit, no increased growth with 138 maximum temperature 143 mercuric chloride, seeds treated with. . . 125, 127, 128 methylene blue, vital staining with 132, 139, 145 milk, changes in 137, 140 minimum temperature experiments 143 mixed cultures 142 mixed infections 142 morphology 132, 144 motility 132 nitrates, not reduced by 145 nitrogen foods 141 nitrogen, no growth in 138 odor 135 organs invaded 131 oxygen, evolution of from hydrogen peroxide. . . . 142 parts of plant attacked 131 phenolphthalein, reaction of formate agar cultures. 143 pigments 142 polar flagella 91, 132 potassium formate, effect of 143 potato starch, slight action on 142 potato streak cultures, appearance 135 pseudozoogloeae 132, 137, 143 reduction of litmus 137 reduction of nitrates 138 resume of salient characters 144 rosolic acid, effect 139, 145 sodium chloride, marked toleration of 138, 145 stain in host-plant 16, 142 stains, reaction to 132, 144 starch, action on 134 sunlight, effect of 144 tartaric acid, toleration of 144 temperature relations 143, 144, 145 INDEX. 293 Bacterium stewarti — continued. thermal death-point 143 tissues attacked 89, 90, 91, 93, 131 trypsin 142, 145 Uschinsky's solution, good growth in 137 virulence, retention of 142, 145 viscidity 103, 126, 127, 132, 137 vitality 142, 145 wind, not borne by 121 yellow pigment, color dependent on air 139, 142 variable appearance of 142 Bacterium sumatranum, associated with Bact. sola- nacearum in Sumatra 259 Bacterium vascularum 54 (See also Sugar-cane, Cobb's disease of.) acid culture media, inhibiting effect on growth ... 58 61, 63, 67 acid potassium phosphate, effect on growth 64 acidity of cane-juice, relation to bacterial growth . 47 aerobism 48, 64 air-hunger 58, 64 agar not stained by 56 alcohol, slime of, insoluble in 7, 14 alkali, effect on growth 58, 59, 61, 63, 67 alkaline reaction of cultures 56, 57, 60, 61 ammonium, effect on growth 64 ammonium salts as nitrogen foods 63 asparagin as nitrogen-food 63 bacterial slime oozing from leaves and stems ... 16, 22, 25,53,66 calcium, effect on growth 64 cane cuttings, disease transmitted by 48, 68 cane slices, steamed, growth on 60 cane-sugar, action of organism on 62 cane-sugar, as carbon food 57, 63 cane-sugar, effect on growth 64 capsules 67 carbon dioxide, effect on growth 64 carbon foods 63, 64, 67 chemical reactions of bacterial slime 7, 13, 14 citric acid, effect on growth 63 Cobb's studies 5, 6, 7, 8, 12 (See also Sugar-cane, Cobb's disease of.) colonies, appearance of 8, 55, 57, 67 color of 54, 60 conditions favoring spread of 48 crystals, production of 56, 61, 67 cultural characters 8, 54, 67 culture media most favorable 65, 67 description of organism 7, 8, 54 dextrin, effect on growth 64 dextrose as carbon food 63 dextrose, effect on growth 64 effect on sugar-cane 3 fermentation-tube cultures 64 flagella, staining 54 gas-production, not observed 57, 64, 67 gelatin, browning of 59 color on 60 growth on 8 liquefaction of 57,59 tear drops on 59, 67 with cane-juice, growth on 65 with fruit-sugar, growth on 57 with glucose, growth on 57, 65 geographical distribution 3 glycerin, effect on growth 63, 64 Gram's stain, reaction to 54 group number 67 growth, best medium for 65 growth best at 300 64, 65 growth inhibited at blood temperature 64 Bacterium vascularum — continued, growth on or in, acid agars 63, 67 acid bouillons 61, 67 acid fluids, Greig-Smith's investigations 61 agar plus cane-sugar 56 agar plus fruit-sugar 56 agar plus peptone, levulose, potassium phosphate and tap-water 65 agar plus peptone, saccharose, potassium phos- phate and tap-water 65 alkaline agars 63 alkaline bouillons 61 , 67 beef-agar 56, 66 beef-bouillon plus peptone 60, 67 beef-bouillon plus potassium formate 61 beet-cylinders 60 beet-juice agar plus peptone and saccharose ... 67 cane cylinders, steamed 47, 60, 67 cane-juice agar plus peptone 56 cane-sugar water plus peptone 62 carrot 60 cauliflower cylinders 60 coconut cylinders 60 Cohn's solution 67 distilled water plus dextrose, glycerin and so- dium asparaginate 64 Dunham's solution 67 Fischer's mineral solution 63 fruit sugar-agar 56 gelatin 8, 57, 59, 60, 65. 67 gelatin plus cane-juice 57, 58, 65 cane-sugar 5 7, 58 fruit-sugar 57 glucose, Greig-Smith's description 57, 65 peptone, dipotassium phosphate and malic acid 57 peptone, dipotassium phosphate, malic acid and cane-sugar 58 peptone, dipotassium phosphate, malic acid, cane-sugar and sodium hydrate 58 glycerin agar 56 Hunger's agar 57 litmus lactose agar 57, 67 litmus milk 61 milk 66 nitrate bouillon 61 nutrient agar 54, 56, 66 poured-plates 54 slants 56 stabs 56 onion bulbs steamed 60 peptone water plus cane-sugar (fermentation tubes) 64 glycerin (fermentation tubes) 64 grape-sugar (fermentation tubes) 64 maltose (fermentation tubes) 64 mannit (fermentation tubes) 64 milk-sugar (fermentation tubes) 64 oxalic acid 63 potato, Greig-Smith's studies 65 potato agar 57, 66, 67 potato cylinders, steamed 60, 66, 67 saccharose agar plus peptone 56 silicate jelly 134 sugar-agars 56, 65 sugar-beet agar 56 turnip 60 Uschinsky's solution 62 gumming, constant association of organism with . 8, 2 1 gum, identity with bacterial slime 13 (See also Sugar-cane, Cobb's disease of.) gum-production, dependent on certain carbon foods 64 host-plants 3 indol reaction, faint 64 294 INDEX. Page Bacterium vascularum — continued. inoculation experiments, Cobb's 8, 12 inoculation experiments, Tryon's 11 inoculation, methods of 21, 27,31,33,46 inoculations, general remarks on 46 inoculations on Blanca cane 34 Caledonia Queen cane 33, 34, 46 Cinta cane 34 Common Green cane 21, 32, 34, 46 Common Purple cane 27 Crystalina cane 33, 34, 46 Jamaica cane 33, 34, 46 Louisiana cane, Variety No. 74 27, 34. 46 Striped Green cane 31, 34, 46 inoculations, pure-culture 11, 21 inoculations, variety tests 31, 33, 46 inversion of cane-sugar 62 isolation, methods of 54 isolation from artificially infected sugar-cane 21, 24, 39, 46 Australian cane 2 1 , 65 red bundles 17,51 red ooze 46 yellow ooze 46 lactose, effect on growth 64 lesions produced 130 levulose, effect on growth 64 liquefaction of gelatin 57, 59, 67 literature 71 litmus, reaction 56, 57, 61, 67 longevity, effect of temperature on 66 in culture-media 65, 66 in the host 65 magnesium, effect on growth 64 malic acid, effect on growth 63 maltose, effect on growth 64 maximum temperature 64 measurements 54, 67 milk, acids not formed from ... 61 casein not precipitated 61, 67 effect of organism on ... . 61, 62 surface growth not conspicuous 61, 62 tyrosin not formed in 62 with litmus becomes indigo blue. ... 61 morphology _ 7, 21, 54, 67 motility, best seen in young cultures 54 motility, often absent in old growths 54 nitrates, not reduced by 61 nitrogen foods 63, 67 one-flagellate 3. 21, 55, 56 optimum temperature 65 oxalic acid, effect on growth . 63 pathogenicity . 8, 2 1 phenolphthalein, reaction of organism to . 61 phosphates, effect on growth 57, 64 potassium, effect on growth 64 potassium nitrate as nitrogen food . . 63 potato, growth on 60, 65 poured-plate cultures, description 54 pseudozoogloeae ^ 6X pure-cultures from diseased cane 21 red bundles, isolation from . ... 17, 51 reddening of bundles ' 1$ (See also Sugar-cane, Cobb's disease of.) reduction of nitrates 61 relation to B. sacchari Spegazzini . 88 resume of salient characters 66 resuscitation from old diseased canes 46 saccharose, action on 62 saccharose, as carbon food .63 saccharose, effect on growth ... 64 salts, effect on growth 64 Pagb. Bacterium vascularum — continued. slime, oozing from leaf-sheaths and stems 16, 22,25,53,66 Smith, R. Greig, studies 57, 60, 61, 62, 63, 64 (See also Sugar-cane, Cobb's disease of.) sodium chloride, effect on growth 63, 64 spores 54 stains, reaction to 54 starch, effect on growth 64 starch, slight action of organism on 60 substitution for B. pseudarabinus in Greig-Smith's experiments on red stain in gummed cane ... 50 sugars as food 63, 64 sunlight, organism sensitive to 66 tear-drop formation on gelatin 59, 67 temperature, effect on longevity ... 66 temperature relations 64, 67 thermal death-point experiments 64, 65 tissues attacked 6, 7, 15, 24, 66 trimethylamin, production of 56 Tryon's studies 1 1 (See also Sugar-cane, Cobb's disease of) Uschinsky's solution, feeble growth in 62 vascular system attacked by 6, 15, 24, 66 vasculin 7 viscidity 56, 61, 66 vitality in stem and on media 65, 66 yellow slime, primrose to lemon color on potato 60 whitish in absence of air 58 Bacterium xanthochlorum, (See also Potato, Schuster's German Rot.) acid, toleration of 275 acid-production 272, 275 aerobism 273 alcohols, growth in 273, 275 alkali, toleration of 275 alkali-production 272, 275 ammonia-production 272 amylase produced 272 anaerobism, facultative 273 biological characteristics 272 blood-serum, non-liquefaction of .. . . ... 273 chloroform, effect of 275 colonies on agar 273 colonies on gelatin 273 disintegration of potato-tuber sections in infected potato-juice 274 drying, sensitive to 275 entrance of organism into tuber 272 enzym, action on starch 274 effect on cell-walls 274 effect of temperature on 274 extraction of 274 injection into potato-tubers 274 fermentation-tube experiments 275 filaments, production of 272 flagella 272 effect of temperature on formation of 272 fluorescence, greenish-yellow 273 galactose, fermentation of 275 gas, non-production 273, 275 gelatin, liquefaction 273, 274 glycogen, production of 272 Gram's stain 272 grape-sugar, fermentation of 275 growth on, or in, acid bouillon 275 agar (plus 15) 275 alkaline agar 273 alkaline bouillons 275 alkaline potato-agar 274 alkaline potato-agar plus tyrosin 274 beer . 273 bouillon over chloroform 275 bouillon plus grape-sugar 273 bouillon, peptonized, plus sodium chlorid 275 INDEX. 295 Page. Bacterium xanthochlorum— continued, growth 011 or in — continued. carrot-juice agar ' Conn's solution ' fermentation tubes gelatin plus sodium selemte __ • Loeffler's blood-serum . . J'-> Meyer's solution, plus, 2?3 273 273 274.275 . 273 275 Bacterium xanthochlorum- optimum temperature -continued. alcohols ammonium salts arbutin 27, asparagin " 2?3 caffein 273 cellulose 273 citric acid 273 glycerin "".'.'.'.'. 273 peptone 2_3 potassium salts potato starch *"SaTS ■■ '.'.'.'. '273. 275 milk ■ ■ • • • ■ : ; • ' 27'? neutral nutrient bouillon ' ^ nutrient agar peptone-water plus 275 cane-sugar 275 fructose 275 galactose 27- glycerin 2?5 grape-sugar .275 lactose 27j maltose 27j mannit 27, potato-juice-agar potato-juice gelatin ' Uschinsky's solution ' growth, optimum temperature . . ■ '* hemicellulase produced host-plants ■ ■ • • • hydrogen sulphide produced incubation period ! 273 indol produced inoculations on, Campanula raphunculus carrot roots 272 fodder beets lupines Lupinus nanus douglasi pelargoniums. . . Physalis alkekengi potato tubers . 272 273 274 272 272 272,274 . .. 272 . . . . 272, ,274,275, 272 274 276 272 sugar beets 27 274.275 272 276 272 273, 273 . 275 274.275 273, tobacco stems tomatoes. Vicia faba yellow lupins ' 2?2 involution forms ' isolation .••■•' V ' ' 2-72 lenticels, inability of organism to er light, effect on virulence light, sensitive to. . liauefaction of gelatin liquefaction of Loeffler's blood-serum, non-occur rence of literature :■■■•-' methylene blue, reduction ot methylamin-production ... milk, coagulation of milk, peptonization of morphology effect of temperature on. . motility effect of culture media on effect of temperature .... nitrates reduced. . . . non-fluorescent strain 275 276 273 272 273 273 272 272 272 272 272 273 275 273 272 parasitism, weak • Compared with that of Bacillus phytophthoms. 272 effect of temperature on ' 272 272 273 273 274 272 275 2 73 272 2 73 276 274 275 pigment, effect of culture media on solubility of potato starch, effect on potato-tuber, disintegration of sections in infected potato juice • 2 rel^on'Xline^otato-agar; Spitzes reaction 274 Schuster's observations .... sodium chlorid, effect of sodium selenite, action on . solubility of pigment solution of middle lamella *i°> spores, non-occurrence of • ' stains, reaction to starch, action on ........••■•■• ■ ■•■■ stomatal infection (Vicia faba) . . rapidity of . sugars, growth in temperature, effect of, on, enzym action flagella-formation milk cultures morphology pathogenicity »£• 274, 275 temperature, relations ■ • • 273. 274. * ia thermal death-point .... • • _ • • • ■ • ■ '= tissues attacked ■ 272- "4. 27b toxin produced ■ trimethylamin-production trypsin produced tyrosinase produced vascular bundles occupied by virulence, effect of light on wounds, means of entrance xanthochlorum hemicellulase Bacterium zinnoides . associated with Bact. solanacearum in Sumatra Bailey, observations on New York tomato disease Banana, Bengal disease Cephalosporium Fusarium literature Surinam disease, resemblance to . .. ■ ■ ■•■•'« Central American disease. (See Banana, Panama disease.) Banana, Costa Rican disease disease.) fiS Banana, Earle's Jamaican disease .... 1°° geographical distribution literature organism isolated Panama disease, relation to signs of disease j£g Banana, leaf-blight .' ' ,■ \ ' ' (See Banana, Earle's Jamaican disease.) Banana, Panama disease bacteria in vessels gas-forming * ' non-pathogenic 1 73 cause .■ I72 Costa Rica, occurrence 111 .. . ' Earle's Jamaican disease, relation to. . . Fusarium in bundles ™ Fusarium isolated ' • ■ ' geographical distribution • '72, ^a 272 273 272 272 272 272 272 273 272 274 259 205 173 173 173 173 Banana, (See Banana, Panama 173 168 173 168 172 173 171 inoculations leaf-curvature literature losses characteristic of . . J72 173 172 296 INDEX. Pags. Banana, Panama disease — continued. parts of plant attacked 172 resistant varieties T73 Rorer's Trinidad disease, differences 171 signs of disease '72 splitting of leaf-sheaths '71 staining of bundles 1 73 Surinam, occurrence in '73 susceptible variety 171 Trinidad, occurrence in 173 vessels sometimes occupied by bacteria 171 wholesale prevalence of 172 Banana, Rorer's Trinidad disease 170 (See also Bacillus musae.) bacterial ooze '71 breaking over of leaves 1 7° cavities, bacterial 170, 172 cells invaded by 171 diseased suckers, planting of 17° distinct from Panama disease 1 7 : inoculations 1 70, 1 7 1 inoculations on Manila hemp 171 solanaceous plants '71 tomato 1 7 1 isolation of parasite 1 7° literature T73 Panama disease, how different from 171 resistant variety 1 7 1 signs of disease '7° splitting of leaf-sheaths, not characteristic 171 staining of bundles '7° tissues attacked 170, 171. 172 varieties attacked 17° vessels occupied by bacteria 170, 171 Banana, Smith's Cuban disease 168 bacteria sometimes present in 168 cause 1 69 comparison with Trinidad disease 171 inoculations 169 isolation of Fusarium 168 literature 173 signs of disease 170 splitting of leaf-sheaths 171 vessels, browning of 168 vessels, occupied by Fusarium 168 Banana, South American diseases 173 Banana, Surinam disease 173 (See also Banana, Panama disease.) Fusarium present ' 73 literature 173 resemblance to Bengal disease 173 Banana, Vascular diseases 168 (See Banana, Bengal disease ; Banana, Earle's Jamaican disease; Banana, Panama disease; Banana, Rorer's Trinidad disease : Banana, Smith's Cuban disease.) literature T 73 Bancroft, rot of potato and tomato, Malay States . . 211 Basu, Bengal disease of banana 1 73 Benecke, Sereh observations 72, 73 Biffen, English disease of mangolds and sugar-beets. 154 "Black-leg" of tobacco • 239 (See Bacillus nicotianaeUyeda; Tobacco, wilt-diseases, Japanese disease.) Blight of Young Rose Shoots 154 Bolliger, Brazilian disease of manihot. 280 Boname, studies on " gummosis " of sugar-cane . 3, 10, 1 5,63 (See Sugar-cane, Cobb's disease.) Bondar, Brazilian disease of manihot 280 Bos, Ritzema, flax stems, bacteria in vessels 154 Bowhill, tobacco- wilt, South Africa 268 Brazilian disease of manihot 280 (See Manihot, Brazilian disease.) Briosi, Italian disease of stock 277 Brown rot of Solanaceae 174 (See Solanaceae, brown rot; Bacterium solanacearum.) Page. Burrill, bacterial disease of potato 203, 205 Bacterium solanacearum 178 brown rot of Solanaceae, observations on 181 Burrill's disease of maize 89 (See Corn, Stewart's bacterial disease.) Butler, E. J., Sereh and cane-gummosis in India. . . 3 Cabbage, brown rot 191 Canker of tobacco 266, 267, 270 (See Bacillus aeruginosus, Tobacco, wilt-diseases, French disease.) Capsicum annum, wilt. (See Pepper, blight.) Cephalosporium, Bengal banana disease 173 Ceylon disease of tomato (Bacterium solanacearum?) 214 Chlorosis of sugar-cane 81 (See also Sugar-cane, Top-rot, and Sugar-cane, Pokkah bong.) Cladosporium herbarium, orchard grass, Rathay's disease 155 Clark, Cobb's disease of sugar-cane, occurrence in Fiji 3 pecuniary losses in Fiji 70 Clinton, brown rot of Solanaceae reported from Connecticut 17s tobacco canker 267 Cobb. disease of sugar-cane 3 (See Sugar-cane, Cobb's disease of) studies on "gummosis" of sugar-cane. (See Sugar- cane, Cobb's disease of). Cobey, tobacco-wilt, losses 237 Coleman, Mysore ring disease of potato 212 (See also Solanaceae, brown rot, ring disease of potato, Mysore.) Collar-rot of tobacco 266, 267, 270 (See Bacillus tabacivorus; Tobacco, wilt-diseases, French disease.) Comes, Bacterium gummis 203, 215, 266 Italian disease of tomato 215 tobacco-rot, Italy 266 Cook, top-rot of sugar-cane in Cuba 3 Corbett, observations on New York tomato disease. 205 Corn, Stewart's bacterial disease 89 (See also Bacterium stewarti.) bacterial occupation of the vascular system. . . 89, 90, 91, 93. 94. 130 bacterial ooze from cut stems, etc 89, 90, 91, 131 bacterial pockets 13 ' bacterial slime, enormous quantity of 126 homogeneity of 95 occurrence in stem 6 feet from point of infection . 126 paler in upper part of stems 94 bundles, stem, anatomy of 129, 130 cavities 91. 95. 131 cellulose not destroyed 13° cirri in 126 cob penetrated by bacteria 114 congressional seed distribution, disease spread by 114, 115 constitutional signs, how soon visible 97 corn kernel, anatomy of 114, 116, 130, 131 crushed kernels, test cultures should be made from. 125 definition 89 dissemination through infected seed 114, 127 drouth, signs conspicuous during 94 Duggar's observations 89 dwarfing, due to 89, 90 influence on 1 1 1 early sweet varieties, most subject to. . . . 1 12, 113, 118 etiolation 9° etiology 91 field corn varieties subject to 112, 114 field observations on 94, 114 frost effects, resemblance 101 INDEX. ?97 Pagb. Corn, Stewart's bacterial disease — continued. fungicides 93, 146 geographical distribution 89, 93, 146 germicides 93, 146 germination of treated seed best . . . 127 history '47 host-plants 89 hot-water treatment of seed 146 husks, anatomy of 1 19, 120 bacterial pockets in 103 spots and patches on 121 hyperplasias absent in 131 infected seed, dissemination through 114, 127 experimental plantings of 115, 121, 125, 128 treatment of 1 46 treatment with formalin 128 treatment with hydrogen peroxide 128 treatment with mercuric chloride 125, 127, 128 infection, accidental, manner of occurrence 112, 124 course of 90, 102 from base upward 96, 125 from top downward 113 insects, not due to 93, 94 means of, Stewart's theory 93 most serious in seedling stage. ... no, 113, 125, 129 slow to appear 127, 128 through seed 1 14, 127 through water-pores 94 time of 89 inoculations 96 (See Bact. stewarti.) inoculations, Stewart's 91 , 92 result of late 113, 129 isolation of Bact. stewarti, attempted from infected kernels 124 by direct transfer from diseased stems 96 kernels, bacterial contamination of surface 114 penetrated by the bacteria 114, 131 leaf-tips, bacteria found in 95 leaves, effect on 89, 90 lesions produced 130 literature 147 local signs, how soon visible 97 losses, pecuniary 1 46 male inflorescence, premature development 89 male inflorescence, whitening of 89, 94 mercuric chloride, effect on infected seed. ... 125, 127 method of obtaining pure cultures from 96 moist soil, effect of 122, 123, 124 morbid anatomy 1 30 nature of, markedly vascular 131 nodes, brown stain in 90, 100, 102 organism causing, description of 132 (See also Bact. stewarti.) organisms other than Bact. stewarti (on kernels) . . 125 parenchyma, bacteria in 91 parts of plant attacked 131 polar flagellate bacterium, cause of 91 pop-corn, experiments 93, 1 1 2 premature development of male inflorescence . . 19, 89 preventive measures 145 pure cultures from 96 resistant varieties 117 root-pressure ceases with frosting of leaves 89 roots, effect on not conspicuous 90, 95, 105 seed, infected, dissemination through 114, 127 seed, treatment 146 shoots, production 131 signs of disease 89, 94 slow growth, hinders development of disease no, 1 11, 127, 128 Pags. Corn, Stewart's bacterial disease — continued. soil conditions, effect 123, 124 specific nature of organism causing 125 sprouting of basal buds 90 staining of tissues 89, 90, 95 stem, effect on 89 Stewart's studies, browning of tissues 90 discovery of Bact. stewarti 91 field observations 91 geographical distribution 89, 93 inoculations 93 means of infection 93 susceptible varieties 118 weather conditions, effect 94 stomata. bacterial ooze through 1 19, 120, 132 susceptible varieties 89, 94, 118 susceptibilitv, effect of slow growth no, 127, 128 time between infection and appearance of 90, 109, 127, 128 tissues attacked 89, 90, 91, 93 toxine, not conspicuous in 130 treatment 93, 145 trial plots, results on 117 uneveness of fields attacked by 91 varietal resistance, question of 117, 124 varieties resistant to 118 vascular infection, generalized 102, 106 vascular system, bacterial occupation of 89, 90, 91,93,94,130 viscidity of slime in 126, 127 water-pores, infection through 94 weather conditions, effect of 94, 124 whitening of male inflorescence 89, 94, 113, 115 yellow bundles 95 yellow ooze 90, 94 variability in color of 94, 95 Corynebacterium piriforme associated with Bact. solanacearum in Sumatra 259 Councilman, blight of young rose shoots 154 Cozart, tobacco-wilt, losses 237 Crossonini, indol reaction with paradimethylamido- benzaldehyd 249 Cucurbit wilt, vascular nature of 191 Dactylis glomerata, bacteriosis of 155 (See Orchard grass, Rathay's disease of.) Debray, Bacillus sacchari 76 De Jong, peanut-wilt, flooding infected fields for 271 planting, selected seed for 271 Delacroix, Bacillus aeruginosus 266 Bacillus caulivorus 175 B. putrifaciens putridus 266 B. solanincola 175, 215 B. tabacivorus 266 French disease of potato 214 Tobacco, Fusarium disease 228 wilt-diseases 266, 270 de Laharpe, French potato disease 215 Deli tobacco organism [See Bact. solanacearum (Sumatran tobacco-wilt organism.)] Dewar, tobacco-wilt, South Africa 268 Diem, Deli tobacco, variations 261 Diplococcus enteritis, Gram's stain, reaction to 247 Doryphora 10-lineata, distribution of brown-rot of Solanaceae by 1 80 Dranert, studies on bacterial disease of sugar-cane . 3, 70 Drost, South American banana diseases 173 Dutch disease of wallflower 277 Dutch East Indian disease of tobacco 220 [See Bacterium solanacearum (Sumatran tobacco-wilt organism) ; Tobacco, wilt-diseases, Dutch East Indian disease: Tobacco, wilt-diseases. Honing's Sumatran Studies.] 298 INDEX. Page. Dutch East Indian disease of tomato 20Q (See Solanaceae, brown rot, Dutch East Indian disease.) Earle. banana leaf-blight 168 brown rot of Solanaceae 181. 206 Egg-plant blight 174 (See Solanaceae, brown rot: Bacterium solanacearum.) Engelberts, Sereh, losses 72 English disease of mangolds and sugar=beets. . 154 English disease of potato 216 Eriksson, Ring disease of potato 216 Essed, banana disease 1 73 Fertilizers, relation to immunity 270 Flax, bacteria in vessels of stems 154 Florida potato organism 2.51, 264, 267 [See Bact. solanacearum (potato and tomato).] Florida tobacco wilt 227 [See Bacterium solanacearum (North American tobacco-wilt organism); Tobacco, wilt-diseases, North American disease.] Fodder-beets, disease described by Kramer 154 French disease of potato 214 French disease of tobacco 266 (See Bacillus aeruginosus, B. putrifaciens putridus, B. tabacivorus: Tobacco, wilt-diseases, French disease.) Fulton, peanut wilt 1 53, 271 Fusarium, color produced on acid media by some species 51 Fusarium sp. seen in Australian disease of potato 208 Bengal banana disease 1 73 Panama disease of banana 173 potato diseases 203 Surinam disease of banana 173 tobacco-wilt 227, 228 Fusarium cubense 169, 173 isolated from Smith's Cuban banana disease 168 Panama disease of banana ascribed to 173 (See also Banana, Smith's Cuban disease.) German Ring disease of potato 166, 215 (See also Solanaceae, brown rot, ring disease of potato, Germany.) Graham, tobacco-wilt 237 Grand Rapids tomato disease 161 (See Aplanobacter michiganense; Tomato, Grand Rapids disease.) Granville wilt of tobacco 174, 220, 227 |See Bacterium solanacearum (N. Am. tobacco-wilt organism) ; Tobacco, wilt-diseases, N. Am. disease.] Griffon, Bacillus aeruginosus, synonymy 267 B. brassicaevorus, synonymy 267 B. caulivorus, synonymy 267 B. fluorescein liquefaciens, synonymy 267 B. fluorescens putridus, synonymy 267 immunity relation of fertilizers to 270 Gumming of sugar-cane 3 (See Sugar-cane, Cobb's disease.) "Gummosis" of sugar-cane 13 (See Sugar-cane, Cobb's disease.) Halsted, observations on bacterial disease of potato and tomato 204, 205 hamawedang 151 (See Peanut wilt, van Breda de Haan's.) Heart disease of sugar-cane 81 (See Sugar-cane, heart disease.) Helms, New South Wales potato diseases 207 Herrman, Brazilian disease of manihot 280 Heterodera radicicola, effect on outbreak of Dutch East Indian disease of tomatoes 210, 211 Honing, Bacterium solanacearum, host-plants . 199 Bacterium solanacearum, involution forms 194 cited 220 peanut wilt 153, 253 sesamum, sumatran disease 218 Pagb. Honing — continued . tobacco- wilt, Sumatran studies 224, 244 (See Tobacco, wilt-diseases; Honing's Sumatran Studies ) Hori, cited 239 Home, top-rot of sugar-cane 3 Humid gangrene of sugar cane 85 (See Sugar-cane, Polvillo.) Hunger, agar used for isolation of Bact. solanacearum .... 211 brown rot of Solanaceae 178, 181, 197, 209 (See Solanaceae, brown rot, Dutch East Indian disease.) cited 203, 220, 226 Dutch East Indian disease of tobacco 222 Dutch East Indian disease of tomato 209 (See Solanaceae, brown rot, Dutch East Indian disease.) Hutchinson, Bacterium solanacearum 267 motility denied 200, 265, 267 tobacco-wilt, India 267 Hypocrea sacchari 52 Immunity, relation of fertilizers to 270 Indian disease of tobacco 267 [See Bacterium solanacearum (Indian tobacco-wilt organism); Tobacco, wilt-diseases, Indian disease.] Irons, Solanum mammosum resistant to brown rot of Solanaceae 182 Italian disease of stock 277 (See Stock, Italian disease.) Italian disease of tomato 215 Italian rot of tobacco 266, 270 Iwanoff, Russian disease of potato 214 Iwanoski, Rot of tobacco, Russia 265 Janse, Dutch East Indian disease of tobacco 220 Sereh 52, 74. 75 Japanese disease of tobacco 238 (See Bacillus nicotianae Uyeda; Tobacco, wilt-diseases, Japanese disease.) Japanese stem-rot of tobacco 220 (See Bacillus nicotianae Uyeda; Tobacco, wilt-diseases, Japanese disease.) Javan top-rot of sugar-cane 3 Jensen, cited 220, 226 experiments with B. nicotianae 271 tobacco wilt, Dutch East Indies 224 Kirk, New Zealand potato disease 207 Kobus, Sereh, geographical distribution 76, 78 Kornauth, cited 218 Kotthoff, Spieckermann's German Potato disease. . 167 Kozai, tobacco-wilt 239 Kramer, disease of fodder- beets 154 Kreitz, ring disease of potato, Germany 215 (See also Solanaceae, brown rot, ring disease of potato, Germany.) Kriiger, Cobb's disease of sugar-cane, occurrence in Borneo . 3 Sereh 52, 73, 75 Kugahara, cited 239 Lehmann, spore-formation, rich medium favorable to, 247 Leptospora in diseased banana 173 Lounsbury, tobacco-wilt, South Africa 268 McAlpine, Australian disease of potato 208 McKenney, Panama banana disease 172 tobacco wilt, North Carolina 227 Maize, bacterial disease of 89 (See Corn, Stewart's bacterial disease of; and Bact. stewarti.) Burrill's disease of 89 (See Corn, Stewart's bacterial disease of; and Bacterium stewarti.) Stewart's disease of 89 (See Corn, Stewart's bacterial disease of; and Bac- terium stewarti.) Malay States disease of potato and tomato. ... 211 INDEX. 299 Page. Malkoff, Bulgarian disease of Sesamum 216 Mangolds, English disease of 154 Manihot, Brazilian disease of 280 (See also Bacillus manihotus.) black bundles 280 chemical composition of diseased plants 280 cuttings attacked 280 gummy exudate 280 inoculations 280 literature 281 parts of plant attacked 280 preventive measures 281 resistant varieties 281 signs of disease 280 starch-content, diminution in diseased plants. . . . 280 swellings, sub-cortical 280 tissues attacked 280 transmission by insects 281 treatment 281 Mean temperature required for growth of sugar-cane. 4 Medan III 251, 268 [See Bacterium solanacearum (Sumatran tobacco-wilt organism), American studies.] Medan tobacco organism 242, 251, 263, 268 [See Bact. solanacearum (Sumatran tobacco-wilt or- ganism), American studies.] Melanospora globosa, cultural characteristics 87 isolation from Polvillo 86 Mesentericus group, forms of, isolated from Appel's ring disease of potato 166 Micrococcus, luteus, associated with Bact. solanacearum in Sumatra 259 prodigiosus Cohn 87 pyogenes (M. bicolor Zimmerman), associated with Bact. solanacearum in Sumatra 259 pyogenes albus, associated with Bact. solanacearum in Sumatra 259 sp., isolation from top-rot of sugar-cane 83 Moko disease 171 (See Banana, Rorer's Trinidad disease.) Muller, sugar content of gummed cane 63 Murphy, Bacillus melanogenes 175 Musa chinensis, Rorer's Trinidad disease 170 (See also Bacillus rausae; Banana, Rorer's Trinidad disease.) Musa paradisiaca, Rorer's Trinidad disease 170 (See also Bacillus musae; Banana, Rorer's Trinidad disease.) Mysore ring disease of potato 212 (See Solanaceae, brown rot, ring-disease of potato, Mysore.) New South Wales potato diseases 207 New Zealand disease of potato 207 North American disease of tobacco 227 |See Bacterium solanacearum (N. Am. tobacco-wilt or- ganism); Tobacco, wilt-diseases, N.Am, disease.] North Carolina tobacco wilt 227 [See Bacterium solanacearum (North American to- bacco-wilt organism): Tobacco, wilt-diseases. North American disease.] Odium, African disease of potato 214 Orchard grass, Rathay's disease 155 (See also Aplanobacter rathayi.) bacterial slime 155, 157 between leaves and stem 158 cultures from 155 in floral organs 157 in leaf-tissues 159 litmus reddened by 155, 157 microscopical examination of 155 odor of 155 organisms composing 1.55 carriers of the disease 157 Page. Orchard grass, Rathay's disease — continued. chlorophyll bodies, effect on 155 Cladosporium, secondary infection 155 cuticle, effect on 155, 156 distortions of stem in 155, 157, 158 dwarfing 155, 157 geographical distribution 1.55, 157 glumes, lemon yellow or water-soaked 157 infection, point of, unknown 156, 157 time of 155, 156 inoculations 156 knee-shaped bendings of stems 155, 158 leaf-sheaths stuck together 157 literature 160 occurrence, in meadows (Denmark) 157 in woods (Austria) 156 on surface first 156 seasonal 156 portions of plant attacked 155, 156, 157 Rathay's work 155 resemblance to Cobb's disease of sugar-cane, signs 157 Stewart's disease of sweet-corn 157 resistance of other grasses 1 55, 156 secondary infection, with bacteria 155, 159 with fungi 155 signs of disease 155, 157 Sporidesmium (secondary infection) 155 tissues attacked 155, 156, 159 Osborne, spore-formation, relation of medium to . . . 247 Pavarino, Italian disease of stock 277 Peanut wilt, Sumatra. [See also Bacterium solanacearum (peanut-wilt organism); Peanut wilt, United States; Peanut wilt, van Breda de Haan's.] Honing's report of 153, 253 Peanut wilt, United States 153, 271 [See also Bacterium solanacearum (peanut-wilt organ- ism); Peanut wilt, Sumatra; Peanut wilt, van Breda de Haan's.] etiology 271 Fulton's work on 153, 271 inoculations, peanut organism on peanut 271 peanut organism on tobacco 271 pepper-wilt organism on peanut 271 potato-wilt organism (Florida) on peanut 282 tobacco-wilt organism (N. Am.) on peanut . 271, 282 tobacco-wilt organism (Sumatra, Medan III) on peanut 282 isolation of parasite 271 literature 271 signs of disease 271 soil, infection through 271 Peanut wilt, van Breda de Haan's 151 [See also Bacterium solanacearum (peanut-wilt organ- ism); Peanut-wilt, Sumatra; Peanut-wilt, United States.] altitude, effect of 152 etiology 153, 253, 271 literature 153, 271 parasite 153 parts of plant attacked 152 signs of disease 152 soil conditions, effect . 152 tissues attacked. . . .' 132 Pepper blight 1 74 (See also Solanaceae, brown rot.) Pernambuco disease of sugar-cane 9 Perrier, Brazilian disease of manihot 280 Petch, Ceylon wilt-disease of tomato 214 Pethybridge, Bacillus melanogenes 175 ;oo INDEX. Phytophthora infestans, French disease of potato (Delacroix) 215 losses caused by 202 Phytophthora nicotianae, effect on outbreak of Dutch East Indian bacterial disease of tomatoes. . . 210 Pith-rot of tobacco 266, 270 (See Bacillus putrifaciens putridus; Tobacco, wilt dis- eases, French disease.) Plantain, Rorer's Trinidad disease 170 (See also B. musae; Banana, Rorer's Trinidad disease.) inoculations 170, 1 7 1 isolation of parasite 170 literature 173 signs of disease 1 70 varieties attacked 170 Pokkah-bong of sugar-cane 3, 81 (See Sugar-cane, Pokkah-bong.) Polvillo of sugar-cane 3,85 (See Sugar-cane, Polvillo.) Potato, African disease 214 Australian disease 207 "black-leg," resemblance to brown-rot 175 brown-rot, (See Solanaceae, brown-rot.) English disease 216 French disease 214 German ring disease 166, 213 (See also Solanaceae, brown-rot, ring disease of potato, Germany.) Malay States disease 211 New South Wales diseases 207 New Zealand disease 207 ring disease, Germany 166, 215 (See also Solanaceae, brown-rot, ring disease of potato, Germany.) ring disease, Mysore 212 (See also Solanaceae, brown-rot, ring disease of potato, Mysore.) Russian disease 214 Schuster's German rot 272 (See also Bacterium xanthochlorum.) blackening of tissues 272, 274 description of diseased, lupins 274 Physalis 274 potato-tubers 272 Vicia faba 272, 274 incubation period 274 inoculations. (See Bad. xanthocbloruin.) isolation of parasite 272 literature 276 odor of rotted tubers 272 parts of plant attacked 272 Schuster's observations 272 stomatal infection (Vicia faba) 272 rapidity of 274 tissues attacked 272, 274, 276 tubers, diseased, appearance 272 Spieckermann's German disease 166 analyses of tubers, stems, etc 167 Aplanobacter michiganense, suspected cause. . . 164 bacteria in diseased plants 166 Bacterium solanacearum, not cause of 167 brown rot of Solanaceae, relation to 167 cause of 167 diseased stems, microscopical appearance 166 diseased tubers, bacteria in vessels 166 diseased tubers, vascular ring, appearance of . . . 166 dissimilarity to Appel's ring disease 167 eyes of the tuber, blackening of 167 Fusarium. secondary infection 166 germination, effect on 167 inoculations 1 67 literature 167 Pagb. Potato — continued. Spieckermann's German disease — continued. parasite, cultural characteristics 167 description of 167 Gram's stain 167 optimum temperature 167 slow growth on media 167 progress of disease 166 signs of disease 166 similarity to Grand Rapids tomato disease. ... 164 slow softening of parenchyma in 166 softening of vascular ring 166 staining of vascular ring 166, 167 tissues attacked 166 tomatoes, occurrence on 167 varieties, many susceptible 167 vascular ring, effect on 166, 167 vessels occupied by bacteria 166 viscid organism cause of 167 wet-rot 166 yellow bacteria cause of 167 Potato-rot 174 (See Solanaceae, brown rot; Bacterium solanacearum.) Potato-rots, causes 203 recent great advance in knowledge of 203 Prillieux, Bacillus caulivorus 175 Prinsen-Geerligs, Sereh, geographical distribution 7S sugar-cane, cellulose, oxidizable substance in . . . . 16 Pseudomonas, amaranthi 148 (See Bact. amaranthi.) sesami 218 solanacearum 178 (See Bacterium solanacearum.) sp., isolated from ring-disease of potato 166 stewarti 132 (See Bact. stewarti.) vascularum. (See Bacterium vascularum.) Raciborski, tobacco-wilt, Dutch East Indies, cause . . 222 Rasa of tobacco 267 (See Tobacco, wilt-diseases, Indian disease.) Rathay's disease of orchard grass 155 (See Orchard grass, Rathay's disease, and Aplanobacter rathayi.) Ratoon-cane, nature of 6 Ravn, Rathay's disease of orchard grass 157 Ring disease of potato (See also Solanaceae, brown rot, ring disease of potato, Germany.) Germany 166, 2 1 5 Mysore 212 (See also .Solanaceae, brown rot, ring disease of potato, Mysore.) Rolfs, P. H., brown rot of Solanaceae 205 Rorer, Panama disease of banana 173 Surinam disease of banana 173 Trinidad disease of bananas and plantains 170 Rose, blight of young shoots 154 Russian disease of potato 214 Russian disease of tobacco 265, 270 (See Tobacco, wilt-diseases, Russian disease.) Sackett, tobacco wilt, North Carolina 227 Schreiber, spore-formation, relation of medium to . . 247 Schuster's German potato rot 272 (See potato, Schuster's German rot.) Schwarzbeinigkeit, resemblance to brown-rot 175 Selby, brown rot of Solanaceae 205 Septogloeum arachidis 15 ' Sereh disease of sugar cane 3, 52, 73 (See Sugar-cane, Sereh.) Sesamum, bacteriosis of 216 (See also Solanaceae, brown rot, Sesamum bacteriosis.) INDEX. 3OI Page. 261 237 Shamel, Deli, tobacco impurity of seed tobacco wilt, losses Smith R.Greig, on bacterial disease of sugar-cane. (See Sugar-cane, Cobb's disease, and Bact. vascularum.) Smith's Cuban disease of banana i°» (See Banana, Smith's Cuban disease.) Smith's disease of amaranths. ■■■■••••• *f° (See Amaranths, Smith's disease and Bact. amarantni.) Solanaceae, anatomy of J92 Solanaceae, brown-rot . . . . • • ■ • • ■ • J74 (Tobacco-wilt diseases are indexed separately.) [See also Bacterium solanacearum (potato and tomato; , Peanut wilt.] adventitious roots, tomato 178, 190, 199, 209 African disease of potato. Odium's observations .214 alkalinity of juice of attacked plants . . .... I7» animal parasites, as disseminators of . . . . 184. 210, 21 Appel, Ring disease of potato. ■•••■••■■ ^ ■ lCT' Australian disease of potato and tomato, Tr>on s observations bacterial ooze ■ • ■ ■ • • • ■ ■ x75 solanacearum, cultural charactens- 194. 215 207 177 Bacterium tics . . . description . . . (See also Bacterium solanacearum (potato tomato.) Bailey's observations on disease of tomato 205 Bancroft's observations on disease of potato and tomato ■ ■ • ■ ; ■ basal stem-rot of potato ("black leg ), how distinguished from liable to confusion with beetles, disease spread by ■ " black leg." (See basal stem rot.) blackening of tissues broken roots, avoidance of, in transplanting browning of tissues 174. '75. 177. I9L 209 Burrill's observations on disease of potato. . . 203, 205 calcium oxalate, excess of, in diseased plants 192 cavities, common in.. . 176, .91- 199. 209 cell-walls, action on 199 193 and 175 181 179 191 Pagb. Solanaceae, brown rot — continued. Dutch East Indian disease of tomato — cont. pith, effect on 2°9 prevalence 2°9 prevention, methods 2l1 roots, diseased 2°9 infection through 2 IO signs of disease 2°9 tyloses 2°9 vascular ring browned 209 vessels occupied by bacteria 209 Dutch East Indian studies of Honing 224, 244 178 197 180 210 cells occupied by bacteria in. ..... . . ■ *9i Ceylon disease of tomato, Fetch's observations. .214 Coleman, Mysore Ring disease of potato 212 Comes' observations on disease of tomato. . . 203, 215 Corbett's observations on disease of tomato. . . . . 205 cork-layer, protective, formation of i7». '9i crop cut short by : • • • • • • ill 111 crystal-sand, excess of, in diseased plants. ... 192, 209 definition ■ i' ''.'.'' ' ' \1. Delacroix's observations on disease of potato 214 de Laharpe's observations on disease of potato. . diffusion of bacteria from cut vessels dissemination by insects drainage, effect on. . . . ■ ■ • • Dutch East Indian disease of tomato adventitious buds 2°9 adventitious roots 2°9- 2" Bact. solanacearum, cause of Bact. solanacearum, cultural characters 211 cavities in tissues . . contaminating organisms crystals in diseased tissues culture-medium used for isolation of parasite diffusion of bacteria from cut vessels in alcohol . drainage, effect on - . ■ ■ ■ ■ • • ■ • • ■ - effect of animal parasites on outbreak of disease effect of Phytophthora on outbreak of disease . Hunger's observations on infection, manner of infection through wounds 2I" inoculation, methods of 2°9. 2I° inoculations on Capsicum annum - » Nicotiana tabacum 2°9 211 209 211 209 210 210 210 209 211 tomato . 209, 210 dwarfing Earle's observations 011 • early inoculations (1895, 1896) 182, 183, 184, 185 early stages, potato '77 English disease of potato entrance of organism into host J8i etiology *? fluids, alkaline in ' ' ° French disease of potato, Delacroix's observations 214 de Laharpe's observations 215 Smith's studies of Delacroix's material 215 geographical distribution *75 (See also Appendix, p. 207.) Grand Rapids disease, may be mistaken for. . 175. i»6 Halsted's observations on disease of potato and tomato 204,205 Helms' observations on disease of potato 207 history 2°3 Honing, „ bacteriosis of sesamum 2 * ° Dutch East Indian studies 224, 244 host-plants '99 host-plants ••■••• 174.199 (See also Peanut wilt; Tobacco, wilt-diseases.) hot weather, increases virulence of .182 Hunger's observations 17S, 181, 197. 209 hyperplasias I9° incubation period 179, i»° infected soil, tomatoes grown in ■ '81 infection, difficulty of producing with organisms grown long in culture media 1 79, 282 effect of age of plant 178, 180 effect of moisture J79. i»2 effect of rapid growth 178, 2»2 effect of temperature x79. lS2 incubation period • • • ■ ■ .- x79 through wounds ■ 181,182,210 underground, in tobacco l underground, in tomato 181 (See also Pact, solanacearum, inoculations.) inoculations. (See Bacterium solanacearum.) insects, distribution by 180, 184, 210, 213 intercellular spaces occupied by bacteria in 191 isolation of parasite 205 difficulties experienced '7° Italian disease of tomato. Comes' observations 2I5 Voglino's observations 2I5 Iwanoff's observations on disease of potato 214 Kirk's observations on disease of potato 207 Kreitz, Ring disease of potato A215 literature ■ : ■ ■ ■ • ■■•••• 2l8'2J? McAlpine's observations on disease of potato .... 208 Malay States disease of potato and tomato, Bancroft's observations 2I1 signs of disease 2I2 treatment • • • 2 ' 2 Malkoff's observations on disease of Sesamum ... 210 middle lamella, solution of '9i effect on 179.182 moisture, morbid anatomy 190 302 INDEX. Page. Solanaceae, brown rot — continued. New South Wales, potato diseases. Helms' obser- vations 207 New Zealand disease of potato, Kirk's observations 207 Odium's observations on disease of potato 214 parts of plant attacked, egg-plant 176 potato 1 76, 1 77 tomato 176 pecuniary losses 202 pepper-blight 174 comparison of pepper, tobacco and peanut-wilt organisms 271 inoculations, Dutch East Indian tomato-wilt organism on pepper 209 Japanese tobacco-wilt organism on pepper . . 243 North American tobacco-wilt organism on pepper 235 pepper-wilt organism on peanut 271 potato and tomato-wilt organism on pepper . 183 Sumatran tobacco-wilt organism on pepper 246, 253 period of incubation 179, 180 exceptionally long 179 very short 282 Petch's observations on disease of tomato 214 pith, effect on 176, 192 plants subject to 174 (See also Peanut-wilt, and Tobacco, wilt-diseases.) Porto Rico, occurrence in 175 potato-tubers, effect on 177 internal brown rot of 177, 191 mixed infection in 177, 191 signs of disease in 177 tissues attacked 177, 191 production of, effect on 177 rains, favorable to 1 82 resistant Solanum 182 result of disease, potato 177 ring disease of potato in Germany 166, 215 Appel's observations 166, 213 bacteria isolated from 166, 216 cultural characteristics 166 dissimilarity to Spieckermann's German potato disease 167 infected seed tubers, use of 166 infected tubers from apparently sound plants . 166 infection, manner of 166, 216 Kreitz's observations 215 Pseudomonas sp. isolated 166 signs of disease 166, 215 vascular ring, browning of 166, 215 ring disease of potato, Mysore 212 Coleman's studies 212 geographical distribution 212 infection, manner of 213 non-relation of insects to 213 inoculations on brinjals (egg-plant) 213 potato 213 tomatoes 213 signs of disease 212 treatment 213 Rolfs' observations 205 root-infection, tobacco 181 tomatoes 1 8 1 roots, developed on stems in 178 wounding necessary for infection 181 rotation of crops advised 201 Russian disease of potato, Iwanoff's observations. 214 secondary signs 1 79 "seed" potatoes, treatment of 201 Page. Solanaceae, brown rot — continued. Selby's observations 205 selection of "seed" potatoes 201 sesamum bacteriosis 216 Austria 218 Bulgaria, cultural characteristics of organisms 216 dissemination of 216 inoculations 2 16, 2 18 isolation of bacteria 216 Malkoff' s observations 216 signs of disease 216 varieties attacked 216 India 218 Sumatra, Honing's observations 218 shriveling of stems 175 signs of disease 174, 175,209 potato tubers 174, 177 sleepy disease of tomato, signs resemble 175 Smith, early studies of 205 Spieckermann's potato disease, not related to ... . 167 starch, removed from infected tissues (fig. 83) . 176, 192 stem showing incipient roots (fig. 98) 190 stems, brown streaks in 1 80 Stevens' observations 206 stomata, infection through, doubtful 182 storage of potatoes at low temperatures advised . . 202 susceptibility, individual differences in 180 varietal differences in 180 temperature, effect on 179, 182 thickening of inoculated stems 178, 191 time of attack, effect, on potato 177, 178 tissues attacked 175, 176, 191, 192, 199 tobacco-wilt diseases, probable identity 220, 244 transplanting, care in 201, 202 treatment 201, 211 Tryon, on disease of potato and tomato 207 tubers, potato, infected by way of rhizome 177 vascular ring first attacked in 177 tyloses 209 underground infections in 181 vascular ring, browning of 174, 177, 209 vascular tissue, excessive 191 vessels occupied by bacteria 174, 175, 191, 209 viscidity, not characteristic of 197 Voglino's observations on disease of tomato 215 water-rich tissues more subject to . . 174, 178, 179, 282 wilting 174, 175 yellowing of foliage * . 174,175 young plants specially sensitive to 174 Solanum mammosum, subject to Apl. michiganense . 163 Soltwedel, Sereh 73. 74. 75. 78 Sorauer, sugar-beet disease in Germany 154 South African disease of tobacco 268 (See Tobacco, wilt-diseases, South African disease.) Spegazzini, Polvillo, geographical distribution 3 observations 85 Spieckermann's German potato disease 166 (See potato, Spieckermann's German disease.) Sporidesmium, Rathay's disease of orchard grass. . . 155 Stem-rot of tobacco 239 (See Bacillus nicotianae, Uyeda; Tobacco, wilt- diseases, Japanese disease.) Stevens, brown rot of Solanaceae 204, 206 cited 220 tobacco-wilt, losses 237 North Carolina 227 Stewart, observations on bacterial disease of sweet corn. (See Corn, Stewart's bacterial disease of.) Stewart's disease of sweet corn 89 (vSee Corn, Stewart's bacterial disease and Bact. Stewarti.) INDEX. 303 Page. Stock, Italian disease 277 (See also Bacterium matthiolae.) browning of tissues 277 infection, manner of 279 inoculations 278 literature 279 losses 277 microscopic appearance of diseased tissues 278 morbid anatomy 277 parasite, description 278 (See Bact. matthiolae.) parts of plant attacked 277 progress of disease 277, 278 seed-disinfection 279 seed-selection 279 signs of disease 277 tissues attacked 277 treatment 279 Sugar-beets, disease described by Sorauer in Germany 154 Sugar-beets, English disease of 154 Sugar=cane, arrowing 19, 20 chlorosis 81 (See also Sugar-cane, Pokkah-bong; Sugar-cane, top- rot.) effect of copper sulphate on 76 gum formation 81 (See also Sugar-cane, Cobb's disease, and Sugar-cane, Sereh.) mean temperature required for growth of 4 Sugar=cane, Cobb's disease of 3 (See also Bacterium vascularum.) acidity of cane, effect on susceptibility 47 acidity of cane-juice, relation to bacterial growth. 47 adventitious roots 52 aeration and pigment stain 16 bacteria dormant or dead in cane . . . 30, 31, 46, 54, 65 bacteria first discovered in connection with disease . 3 bacteria, location in tissues 6, 15, 24, 52 bacterial ooze from leaves and stem . . 16, 22, 25, 53, 66 bacterial ooze, great abundance of 26 bacterial slime, identity with gum 13 (See also slime, gum and vasculin.) Bacterium vascularum, description 54 (See also Bact. vascularum.) Boname's studies, geographical distribution 3 signs of disease 10 reduction of sugar-content 15. 63 bundles, mottled red and yellow 16, 26, 27, 41 cavities formed in soft tissues 5, 11, 15, 26, 52 check pricks, result of 28, 32 cirri from cut bundles 6, 30, 32 clarification of sugar hindered by 3 climate, effect on disease 4 Cobb's studies, (See also Bact. vascularum.) earliest appearance of disease 5 effect of moisture 49 geographical distribution 3, 5 inoculation experiments 8, 12 pecuniary losses in Australia and New South Wales 70 crystallization, effect on 9 decay of terminal bud 3, 5, 15 definition of the disease 3 destruction of bundles in 53 discoloration of tissues 3, 4, 5, 11, 12, 15, 16, 18 disease not air-borne 68 drainage, partial remedy for 49, 68, 70 Dranert's studies in Brazil 3, 70 dwarfing 3, 15, 22, 27 earliest appearance of disease 3,5 early signs of disease 34, 35, 36, 37 etiolation in 3, 10, 18 etiology 21 Page. Sugar-cane, Cobb's disease of — continued. geographical distribution of 3, 4, 5, 9, 10, 1 1, 13, 14 gum, (See also vasculin.) chemical reactions 7, 13, 14 chemical reactions compared with those of bac- terial slime from cultures 13 comparison with the mucins 14 component parts 7 dissemination in tissues 6, 10, 15 identity with bacterial slime. 13 nature and origin of 7, 8, 13 ooze from leaves and stem 16, 22, 25, 53, 66 separation from bacteria by filtration 13 signs of the disease 3, 15 Boname's observations 10 Cobb's observations 5, 6, 8 Dranert's observations 4 Tryon's observations 12 gummed cane, means of detecting 6, 69 microscopic appearance of 6, 24, 52 gumming of machinery 3,6 gumming of terminal shoot 16, 25, 26 gumming, signs of 3, 6, 15 (See also signs of disease.) history 3 host-plants 3 hyperplasias not present 52 immunity, cause of 47 infectious nature 48 intercelluar spaces, occupation by bacteria 53 inoculations 8, 12, 2 1 (See also Bacterium vascularum.) isolation of Bact. vascularum from artificially infected canes 21 naturally infected canes 21 juice of gummed cane, recognition of 6 lime, use of, in clarification 6, 9 literature 71 leaves, pale stripes on 23 local signs not followed by general infection ... 27, 38, 4°, 42, 43, 4° losses due to 4, 6, 70 microscopic appearance of gummed cane ... 6, 24, 52 moisture, effect of 4, 49, 70 morbid anatomy 52 most disease on best land 69 nodes, red pigment most abundant in or under. 16, 26 odor of 27 organisms associated with Bact. vascularum in badly diseased cane 21, 23 parasite, description of 54 (See also Bact. vascularum.) parenchyma, cause of comparative immunity .... 48 pecuniary losses 70 Pernambuco disease 9 crystallization, effect on 9 cultivation of cane, primitive method 9 gum in 9 preventive measures 10 resistant varieties 10 signs of 9 susceptible varieties 9 theories regarding cause 9 weather conditions, effect of 10 pigment, red 1 6, 53 (See also red stain.) plant-cane, signs of gumming in 6 planters responsible for spread of disease 68 polvillo, points of resemblance 85, 88 premature blossoming 20 development of axillary buds 1 1, 12, 16, 25, 52 drying up of attacked cane 9, 15 ripening 20 ratoons, signs of gumming in 6 3 73 "bouquet" Sereh 74 causes suggested 75 cavities containing bacterial slime 79 cellulose, degeneration of 74 chlorophyll, effect on 73 Cobb's disease of sugar-cane, relation to 52 copper sulphate, effect of 79 cuttings, decay of 73 diseased, sound cane from 79 from mountain cane 76 time of appearance of disease in 73 transmission through 73 Pagg. Sugar-cane, Sereh disease of — continued. death of foliage 73 dwarfing 73. 75 Engelberts' observations 72 etiolation 73 etiology 72 fertilizers, unsuitable, effect 75 foliage, death of 73 geographical distribution 52, 72, 78 growth, effect on disease when rapid 74 gum, bacteria-like particles in 74 color of 74 consistency of 74 origin of 78 solution in potash 74 gummed bundles, isolations from 76 gumming, conditions unfavorable to 74 gumming of bundles 73, 74, 75 Heterodera sp 75, 77 Heterodera radicicola 75 history 72 Hypocrea sacchari 75 immunity 78 internodes, shortening of 73 isolations from gummed bundles 76 isolations from parenchyma 74 Janse's observations 52, 74, 75 Java, home of Sereh 72 Kobus' observations, geographical distribution . 76, 78 Kriiger's observations 52, 73, 75 leaves, conspicuous crowding of 73 dwarfing of 73 dying irregularly 73 etiolation 73 persistence 73 striping 73 literature 79 losses 72 moisture, effect on disease 74, 75 mountain cane, used for cuttings 76 nematodes 75 nucleated cells in phloem 74 odor accompanying decay of cuttings 73 oily or resinous drops in parenchyma 74 origin of term 72 parasites given as cause 75 parasitic nature of 77 parenchyma, granular substance in 74 gumming of 74 isolation of Bacillus sacchari from 74 oily or resinous drops in 74 shining, colorless, yellowish or red drops in ... . 74 staining of walls 74 yellow and brown spots 74 persistence of leaves 73 pigment, red 17, 73, 74 solubility of 74 planting, unsuitable methods, effect 75 planting of affected stock 75 Pokkah-bong, resemblance to 82 portion of plant most diseased 73 premature blooming 73 preventive measures 72, 76 primary Sereh 74 Prinsen-Geerligs, observations 78 progress of the disease 72 Pythium 75 red bundles 73, 74, 75 origin of 76 red stain 73. 74 resistant varieties 76 roots of diseased plants 77. 78 root-system, effect on 73 306 INDEX. Page. Sugar-cane, Sereh disease of — continued. secondary Sereh 74 shining, colorless, yellowish or red drops 74 shoots, abnormal production of 73, 75 shortening of internodes 73 sieve-tubes, plugging of 74 signs of disease 73, 76 slime, yellow, in vascular bundles 74 soil-conditions, effect 75 Soltwedel's observations 73, 74, 75, 78 spread of the disease 78 sprouting of buds below ground 73 starch in diseased canes 75 stooling, abnormally strong 73 striping 73 susceptible varieties 76 time of appearance of disease 73 transmissible Sereh 74 transmission through cuttings 73 Treub's observations 75 Tschirch's observations 73 Tylenchus sacchari 75, 77 Valeton's observations 17. 52, 72, 73, 74, 75 vascular bundles, gumming of 73, 74, 75 Wakker's observations 52, 73, 75, 77 water, lack of, effect 75, 77, 78 weather conditions, effect 75, 77 Went's observations 52, 72, 73, 75, 76, 77 yellow gum in vascular bundles 74 yellow slime in vascular bundles 74 Zeijlstra's observations 75, 78 Sugar=cane, top=rot 3, 81 (See also sugar-cane, Pokkah-bong.) Bacillus subtilis isolated from 83 Bacillus vascularum (?) isolated from 83, 84 bacteria, isolation 83 bacteria in diseased tissues 82, 83 cavities 81 Cobb's disease, points of difference 82 etiology, theory regarding 84 glassy appearance of stem 81, 82 growing- point, effect on 81, 82 gum 82 absence of bacteria in 82 infectious nature 84 inoculation experiments 83 isolation of bacteria 83 leaves, effect on 81 literature 84 Micrococcus, sp isolated 83 microscopic appearance 82 odor 81 preventive measures 84 red gum 82 red border-line 81, 82 signs of disease 81 sprouting of buds 81 stem, disorganization 81 susceptible varieties ~ . . . . 81 time of appearance 81 vessels, gum in S2 Wakker's observations 81 weather conditions, effect 84 yellow disease of hyacinths, comparison with ... 83 Sumatran slime-disease of tobacco 220, 222 [See Bacterium solanacearum (Sumatran tobacco-wilt organism); Tobacco, wilt-diseases, Dutch East Indian disease; Tobacco, wilt-diseases, Honing's Sumatran studies.] Sumatran tobacco-wilt organism, American studies. [See Bact. solanacearum (Sumatran tobacco-wilt or- ganism).] Sumatran tobacco-wilt organism, Honing's studies. [See Bact. solanacearum (Sumatran tobacco-wilt or- ganism).] Page. Sweet corn, bacterial disease 89 (See Bacterium stewarti; Corn, Stewart's bacterial disease.) Stewart's disease 89 (See Bacterium stewarti; Corn, Stewart's bacterial disease.) Tobacco, "black-leg" 239 (See Bacillus nicotianae Uyeda; Tobacco, wilt-diseases, Japanese disease.) canker 266, 267, 270 (See Bacillus aeruginosus; Tobacco, wilt-diseases, French disease.) collar rot 266, 267, 270 (See Bacillus tabacivorus; Tobacco, wilt-diseases, French disease.) Dutch East Indian disease 220 [See Bact. solanacearum (Sumatran tobacco-wilt or- ganism); Tobacco, wilt-diseases, Dutch East Indian disease; Tobacco, wilt-diseases, Honing's Sumatran studies.] Florida wilt 227 [See Bact. solanacearum tNorth American tobacco-wilt organism); Tobacco, wilt-diseases, North Ameri- can disease.] Granville wilt 174, 220, 227 [See Bact. solanacearum (North American tobacco- wilt organism); Tobacco, wilt-diseases, North American disease.] Japanese disease 238 (See Bacillus nicotianae Uyeda; Tobacco, wilt-diseases, Japanese disease.) Japanese stem-rot 220 (See also Bacillus nicotianae, Uyeda; Tobacco, wilt- diseases, Japanese disease.) North American disease 227 [See Bacterium solanacearum (North American tobacco-wilt organism); Tobacco-wilt diseases, North American disease.] North Carolina wilt 227 [See Bacterium solanacearum (North American tobacco-wilt organism); Tobacco, wilt-diseases, North American disease.] pith-rot 266, 270 (See Bacillus putrifaciens putridus; Tobacco, wilt- diseases, French disease.) "pox-disease," Iwanowsky and Poloftzoff 270 root-rot, Comes 270 spot-disease, Iwanowski 270 stem-rot 239 (See Bacillus nicotianae Uyeda; Tobacco, wilt-diseases, Japanese disease.) Sumatran slime disease 220, 222 [See Bact. solanacearum (Sumatran tobacco-wilt or- ganism) ; Tobacco, wilt-diseases, Dutch East Indian disease; Tobacco, wilt-diseases, Honing's Sumatran studies.] Wilt=diseases 220 [See also Bacillus nicotianae Uyeda; Bact. solanace- arum (Indian tobacco-wilt organism); Bact. solanacearum (North American tobacco-wilt or- ganism); Bact. solanacearum (Sumatran tobacco- wilt organism).] brown-rot of Solanaceae, probable identity with 220, 244 Dutch East Indian disease 220 [See also Bact. solanacearum (Sumatran tobacco-wilt organism) ; Tobacco, wilt-diseases, Honing's Suma- tran studies.] bacteria, distribution in tissues. . . 220, 221, 222, 223, 225 staining of 220 brown stain 220, 221 cavities 220 cell-walls, action on 223 conditions, favoring 221, 222, 223, 224 INDEX. 307 Page. Tobacco — continued. Wilt diseases — continued. Dutch East Indian disease — continued. dissemination 223 drainage, effect of 223, 225 eel-worms, relation to 221, 222, 223 entrance of parasite into host 221 etiology 220, 222, 224, 244 fungi, presence of 220, 225 Glechia solanella, relation to 223 harvested tobacco, effect on 222, 224 Heterodera radicicola, relation to. . . 221,222,223 Honing's observations 224, 244 (See Honing's Sumatran studies.) host-plants 226, 253, 254, 255 Hunger's observations 222 infection, manner of 221, 222, 223, 224, 225 inoculations 222, 224, 225, 244, 245 (See also Honing's Sumatran studies.) insects, relation to 221 intercellular spaces occupied by bacteria. ... 221 isolation of parasite 225 Janse's observations 220 Japanese disease, probable identity with .... 244 Jensen's observations 224 literature 270, 271 Micrococcus sp., relation to 222 parasite, description of 221, 223 identity, probable, with Bact. solanacearum Smith 244 inoculations with 224 isolation 225 longevity in soil 225 spores, not seen 226 [See also Bact. solanacearum (Sumatran tobacco- wilt organism).] parts of plant attacked 221, 222, 224 Phytophthora nicotianae, relation to 223 preventive measures 224, 226 reaction of juice of diseased plants 225 resistance to 224 roots, effect on 221, 222 saprophytes present 226 signs of disease 220, 221, 222, 224 soil, effect of 221, 223, 224, 225 persistence of parasite in 225, 245 starch, slight action on 221 " thick bellies," relation to 223 time of attack 222 tissues attacked 220, 221, 222, 223, 225 treatment 224, 226, 245 tyloses 223 van Breda de Haan's observations on 222 vessels occupied by bacteria. . . 220, 221, 222, 223 weather conditions, effect of 222 wounds, relation to 223, 224 yeast present 222, 225, 226 etiology 220, 244 French disease 266 (See also Bacillus aeruginosus; B. putrifaciens putridus; B. tabacivorus.) etiology 266 Fusarium disease, Delacroix 228 inoculations 266, 267 literature 270 susceptible varieties 266 Honing's Sumatran studies 224, 244 [See also Bacterium solanacearum (Sumatran tobacco-wilt organism).] Albizzia, relation to disease 225 bacteria, parts of plant occupied by 224 persistence of, in soil 225 crossing tobaccos to obtain resistant race. . . . 260 Deli tobacco, degeneration of 259 origin of 259, 260 variations in 261, 262 Page. Tobacco — continued. Wilt diseases — continued. Honing's Sumatran studies — continued. drainage 225 etiology 244 field observations 245 food conditions, effect of 245 fungi 225 host-plants 226, 253, 254, 255 immune race of tobacco, how obtained 259 immune varieties of tobacco 255 immunity tests 255 infection, channels of, in plant 254 source of 246, 254 transmission of 254 inoculations, [See Bact. solanacearum (Sumatran tobacco-wilt organism).] irrigation-water, disinfection of 246 irrigation- water, infection of 246 isolation of parasite 225 literature 270, 271 parts of plant occupied by bacteria 224 planted-out tobacco, effect on, of occurrence in seed-bed 255 progress of disease, number of invading bacteria govern 260 reaction of juice of diseased plants 225 resistance, effect of soil 263 resistant tobacco.method of obtaining. 255,260,263 resistant varieties 255 seed-bed, disinfection of 246, 254 seedlings, infected, use of 246, 254 selection, in tobacco-culture 259, 260 selection-experiments 261 selection-methods 262 soil, persistence of parasite in 225, 245 test of, for tobacco organism 245 treatment of 226, 245, 246, 254 soil-exhaustion 225 soil-infection, effect of 244, 246, 254 extent of 246 spread of disease, conditions favoring 225 susceptible varieties 255 transmission of disease 254 trap-plants for detecting Bact. solanacearum in soil 245 treatment 226, 245, 246, 254 variety tests 255, 260 wells, Bact. solanacearum found in 246 Indian disease 267 [See also Bacterium solanacearum (Indian tobacco- wilt organism).] etiology 267 inoculations 267 literature 271 losses 267 middle lamella, solution of 268 organisms associated with 267 parasite, description of 267 tissues, disintegration of 268 toxins 267 wilting, cause of 267 Italian disease 266 literature 270 Japanese disease 238 (See also Bacillus nicotianae Uyeda.) blackening of bundles 240 of stem 238, 239 cavities 243 duration of disease 238 Dutch East Indian disease, probable identity. 244 entrance of bacteria into host, manner. . 238, 239 3o8 INDEX. Page. Tobacco — continued. Wilt diseases — continued. Japanese disease — continued. etiology 240, 244 fertilizers, effect of 243 geographical distribution 239 history 238 incubation period 239 infection, manner of 238 inoculations 238, 243, 271 (See Bacillus nicotianae Uyeda.) literature 270 losses 238 microscopic appearance 243 parasite, cultural characteristics 238, 241 (See Bacillus nicotianae.) description 238 morphology 238,241 protective measures 243 resistant varieties 243 signs of disease 238, 239 tissues attacked 238, 240, 243 Uyeda 's observations 238 vascular bundles, bacterial occupation of . . . . 238 weather conditions favoring 238, 240 wounds, infection through 238 literature 270 North American disease 227 [See also Bacterium solanacearum (North American tobacco-wilt organism).] adventitious roots >*« 230 browning of tissues 229, 230 cavities 230 etiology 233 Fusarium 227, 237 incubation period, tomato 234 infected soil, effect 229, 233, 235, 237, 271 infection through wounded roots. . . 229, 234, 235 inoculations, peanut organism on tobacco 271 potato organism on tobacco 230, 282 tomato organism on tobacco 233 tobacco organism on tobacco 229, 282 [See also Bacterium solanacearum (North Ameri- can tobacco- wilt organism).] isolation of parasite 228, 229 literature 270, 271 losses due to 227, 237 McKenney's observations 227, 228 preventive measures 238 roots, infection through 229, 234, 235 Sackett's observations 227 signs of disease 229 similarity of peanut-wilt to 271 soil, infection through. . . . 229, 233, 235, 237, 271 persistence of parasite in 237 Stevens' observations 227, 237 tissues attacked 227 treatment 237 vessels occupied by bacteria 227, 230 wounds, infection through 229, 234, 235 Russian disease 265 etiology 265 literature 270 signs of disease 265 South African disease 268 Bacillus sp. isolated 269 Bacillus megaterium isolated 269 Bacillus mycoides roseus isolated 269 etiology 269 gall worm, relation to disease 269 isolations from diseased plants 269 larva present in diseased plants 269 literature 270 Mucors isolated 269 potato moth, relation to disease 269 signs of disease 268 Page. Tomato, Australian disease 207 brown-rot (See Solanaceae, brown-rot). Ceylon disease 214 Dutch East Indian disease 209 (See Solanaceae, brown rot, Dutch East Indian disease.) Grand Rapids disease of 161 (See also Aplanobacter michiganense.) bacteria on surface of diseased stems 163 bacterial occupation of substomatic chamber. . 163 brown rot, how unlike 163 leaves not reflexed (pi. 14) 162 mistaken for 175 much less brown stain 161 stems less inclined to form roots (pi. 14) ... . 162 cracking of stems 163 geographical distribution 164 infection, point of 161, 163 through stomata 163 infectiousness, high degree of 163 inoculations on, potatoes 164 tomatoes 161, 163 irregular drying of leaflets in 165 isolation of parasite 161 literature 165 meristematic tissue attacked 164 Porto Rican weed attacked by 163 sieve tissue attacked by 164 signs of disease 161 staining of tissues 161 stomatal infection, common in 163 tissues attacked 161 treatment 163 Italian disease 203, 215 Livingston's Dwarf Aristocrat, sensitive to brown rot 282 Malay States disease 211 sleepy disease 175 water-content 282 Tomato-wilt 1 74 (See Solanaceae, brown rot; Bacterium solanacearum.) Top=rot of sugar cane 3, 81 (See Sugar-cane, Top-rot.) Trap-plants 24s Treub, Sereh 75 Tryon, Australian disease of potato and tomato 207 Bacillus vascularum solani 207, 208 brown rot of Solanaceae in Australia 175, 207 studies on bacterial disease of sugar-cane, (See Sugar-cane, Cobb's disease.) Tschirch, Sereh, stumpy roots in 73 Tunstall, tobacco-wilt, persistence of parasite in soil. 237 Tylenchus sacchari 75, 77, 78 Tyrosin, solubility in water 249 Uyeda, cited 220, 226 tobacco wilt, Japan, observations on 238 Valeton, bacteriological investigations of healthy sugar-cane 75 Sereh, observations 52, 72, 73, 74, 75 Sereh. red pigment in, late stage of 17 van Breda de Haan cited 226 Dutch East Indian disease of tobacco 220, 222 peanut wilt 151 tobacco, diseased, coccus from 211 slime disease, inoculations 270 slime disease, underground infections 181 van Hall Bacillus atrosepticus 175 Dutch disease of wallflower 277 Vasculin 7 Voglino, Italian disease of tomato 215 INDEX. 309 Page. Wakker, Sereh 52. 73. 75. 77 toD-rot of sugar-cane 81 Wallflower, Dutch disease 277 Went, Cobb's disease of sugar-cane, occurrence in Java 3, 14 heart disease of sugar-cane 81 sereh 52, 72, 73. 75. 76, 77 top-rot of sugar-cane 83 Wiesner, Rathay's disease of orchard grass 155 Pagb. Williams, Goldsmith, Panama disease of banana reported from Jamaica . 1 73 Wilt-disease of tobacco 220 [See Bacterium solanacearum (North American to- bacco-wilt organism) ; Bacterium solanacearum (Sumatran tobacco-wilt organism); Tobacco, wilt- diseases.] Yellow disease of hyacinths 83 Zeijhtra, Sereh 75, 78 ERRATA. Page 16. Line 28, read Geerligs. Page 91. Last paragraph, first line, read caused by. Plate 26. Last line under the plate, for 1905 read 1903. Page 150. Under the cut for 67, read 67a. Page 228. Seventh line, after North Carolina add and Virginia. Page 256. Table 36. Second line of heading, for mannit read mannitol. Plate 44. 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