Some New Bacterial Diseases of Legumes and The Relationship of the Organisms Causing the Same THESIS ERESENTED STO. THE FACULTY -OF THE GRADUATE- SCHOOL OF THE UNIVERSITY OF PENNSYLVANIA IN PARTIAL FUL- FILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY - BY THOMAS F* MANNS PHILADELPHIA 1914 - BULLETIN NO. 108 APRIL, 1915 Delaware College Agricultural Experiment Station Some New Bacterial Diseases of Legumes and the Relationship of the Organisms Causing the Same BY THOMAS F. MANNS Newark, Delaware a oe ae ve. Babb ssctin gk bir _anetaAO: od} ase a | ccank sit ul UE ed : ei 4, a Se Some New Bacterial Diseases of Legumes and The Relationship of the Organisms Causing the Same” By THOMAS F. MANNS INTRODUCTION The history of phytopathology in relation to bacterial diseases is of comparatively recent years. We are indebted to Burrill, (1877 & 1878) for the first contribution giving definite experimental evi- dence that bacteria may and do cause injuries to plants of a patho- logical nature. His work was done upon the twig blight of pear (1878- 1883). Following closely upon the work of this investigator we find others demonstrating what some considered an impossibility, viz. that plants could be subject to bacterial diseases (See p. 27, Lehrbuch, Har- tig. 1882),. Among Burrill’s contemporaries who were at work on bacteria that are active pathogens of plants may be mentioned Pril- lieux, (his first publication in 1879), Comes , (1880), Sorauer, (1886), Savastano, (1887), Arthur, ( 1886), Beyerinck, (1888), and Wakker, (1883). Erwin F. Smith,, 1899-1901, has been active in removing the doubt existing on the possibility of bacteria being active producers of disease in plants. His painstaking and exact work on bacteria as causes of plant disease will stand as a monumental contribution to the science of plant pathology. Within the last decade the number of exact workers in the bacterial pathology of plants has increased rap- idly ; it is beyond the scope of this paper to mention their names. It is sufficient to state that we must admit that certain diseases which were at different times assigned to higher fungi as casual organisms are now known to be caused by bacteria. This bulletin deals with a discase of the sweet pea (Lathyrus odor- atus) which has variously been stated to be due to different parasitie fungi. It describes algo similar diseases upon the clovers (‘Trifolium pp.), culinary beans (Phaseolus spp.), and soy bean (Soja spp.), which are here proved to be bacterial in nature. *Submitted for publication June, 1913. Presented before the graduate school of the University of Pennsylvania as a thesis in partial fulfillment of the re- quirements for the degree of Doctor of Philosophy, June, 1913. + DEVELOPMENT OF PROBLEM Mr. J. J. Taubenhaus,, having taken up in 1911 the project of ‘“Some Fungous Diseases of the Sweet Pea’’ for thesis work at the University of Pennsylvania, met a disease of the sweet pea known generally in England and less in the United States as ‘‘streak.’’ Spee- imens of diseased plants from Boston, Mass., and from England were referred to the writer. The appearance of the disease suggested to him that it was of bacterial origin. Preliminary isolation work for the parasite readily revealed an abundance of bacteria in the beginning lesions. Attempts at infection of sweet peas with pure cultures gave positive results in ten days. The symptoms of this ‘‘streak’’ disease of the sweet pea were so similar to those of a disease we had previously observed on red clover that it occurred to us that the latter was possibly of bacterial origin and related to the former. A search in the fields in the vicinity of Newark, Del., revealed the presence of the formerly observed disease, in red, alsike, and mammoth clovers. The ‘‘streak’’ disease was also prevalent on several species of Lathyrus, the latter of forage types and upon the numerous varieties of sweet peas with which Mr. Tauben- haus was working. Platings of beginning lesions from the clovers, the different forage species of Lathyrus, and the different varieties of sweet peas indicated the causative organism to be a bacterium. HISTORY OF “‘STREAK’”’ The disease was first observed, according to a letter in 1912 from Mr. T. A. Weston of Orpington, England, by H. J. R. Digges of Dub- lin in 1904 or 1905. Mr. Weston states that, in the fall of 1906, he had a note of the disease in ‘‘The Gardener’’ describing it as ‘‘a new disease under the title of ‘streak’.’’ In 1908 George Massee,, in a letter to a correspondent who had sent in diseased specimens replied, ‘‘the disease is of a physiological nature.’’ As far as we have been able to obtain literature, it appears that ou.ly since the summer of 1908 has any definite work been done upon the disease. Chittenden,, in England during 1908, 1909 and 1910 carried out inoculation work with the supposed parasite, viz., Thie- lavia basicola, obtaining during the first two seasons negative results. In this work Chittenden,, gave a good description of the disease. He found in 1910 that by excessive watering he was able to get infec- tions from Thielavia, but he did not indicate that they were the typ- 3D ical ‘‘streak.’’ He writes, ‘‘To sum up, as far as our experiments go, the ‘‘Streak’’ disease is brought about by the attack of the fungus Thielavia basicola on plants that have received some check at the root.’’ During the season of 1912 Massee,, again took up the disease, and attributed it to Thielavia basicola. Another worker in England, a Mr. Dyke,, of St. Margarets found Macrosporium solani constantly associated with the disease, and _ be- heved it to be the cause of the trouble. The serious nature of the disease may be seen by noting the num- erous popular articles which have appeared in various horticultural and gardeners’ journals of England. Citations from several of the more important of these articles are as follows: In the Fruit-Grower, Fruiterer, Florist and Market Gardener, p. 155 (England) of Sept. 5, 1907, Mr. T. A. Weston in the ‘‘ Kent Notes’’ writes: “‘T regret to note how the stripe disease, Peronospora viciae, in- creases every year, and this season it has been very prevalent every- where. One always knows the tell-tale streaks in the foliage and vine, the stripes appearing in the flowers in due course.’’ In the Oct. 24, 1907 number of the same journal Mr. Weston again writes upon the same disease, partly quoting a letter from a friend in Ireland. ‘‘Mr. B— has the same thing in freshly broken up grazing land, no manure of any kind being used. Haulm 7-8 in. wide on healthy plants on same ground.’’ He goes on to say that he sent a couple of plants to Mr. Massee, of Kew, who gave it as Peronospora trifoliorum. That was two years ago. This year he has had an expert watching haulm and fohage under a microscope since the disease showed itself, the plants at that time being 6 in. Whole plants 6 ft. have been lifted and grown under conditions that would have brcught fungoid disease out if it existed, yet no trace of such has been discovered either in the tissues or exter- nally. The roots of diseased plants all showed very poor root develop- ment, few fibres and total absence of nitrogenous nodules. Whole batches of some sorts were affected, while with others a plant here and there showed the effects. Some showed the trouble before a flower ap- peared, others gave some beautiful flowers and then went wrong. ‘‘So far, my friend. Now, this is as I myself find it. A lot of 6 my plants are going off, and I am sending you samples of growth, roots and flowers. Is it an atmospheric disease, soil disease, eelworm, or carried over in the seed? A friend of mine near London also reports an awful time this year, and he cannot get any really useful advice. I’ve seen journal experts referring to it as stated by Massee, but I am beginning to doubt it very much. My Irish friend has sprayed with copper without avail. Kent T. A. W.”’ In ‘The Gardener’’ for Nov. 16, 1907, p. 585, Mr. Weston gives a somewhat more detailed statement of the disease. The article is here quoted in full: THE STREAK DISEASE OF SWEET PEAS ‘*T fully expected that this steadily increasing trouble would have been discussed by the many able exponents of Sweet Pea culture con- nected with The Gardener. ‘‘My note on p. 419 brought me several letters from a gentleman well known by name to the sweet pea world, and I venture to quote a few extracts, as they tend to show how serious the trouble is. The writer, an amateur by the way, says, ‘I have had the disease in my garden for years, and it has been worse than ever this year, although most of my Sweet Pea plots have been replaced with fresh top spit loam from grazing land. I put 20 tons of this soil in a 60-feet row. A friend also broke up a new piece of pasture, giving nothing in the way of manure, not even artificials, and his plants were equally as bad as my own. Plants that escaped the disease were tremendously vigorous. I am of the opinion that the Peronospora theory is played out, for this year I had many plants carefully tested under the micro- scope, both root and branch, throughout the season, and not a trace of fungus was to be found. Some plants showed the disease, or what- ever it is, before any flowers developed, others gave a few blooms and then went wrong. I am so disheartened that I think of giving up Sweet Peas altogether.’ ‘“The foregoing tends to show that there is danger in this disease. As I have previously stated, I have seen the trouble for years, but never looked upon it as serious until two years ago, when it prevented my cutting any quantity of King Edward VII. This year I found it decidedly serious, more so when a gentleman visiting the National show told me that nearly all his plants were ruined by it. ‘“With us two or three clumps showed the disease early in the season, while single plants in other clumps also gave evidence of the 7 trouble, and so seriously were they affected that I pulled up the plants. One I particularly noticed, because it was the only one remaining of the variety. This showed a brown mark a few inches above the ground, the plant then being about 1 yard high. In a few days the brown mark had extended and had totally encircled the main stem, and also one of the laterals adjoining, while other laterals also showed signs of attack. The badly affected lateral went off hmp, and on eutting it away I found the tissues, where affected by disease, quite dead. So alarmed was I that I scraped with a knife the outer covering of the main stem where affected, and could not find any live or green tissue, although the plant was still healthy. On close examination I found a plant of another variety similarly affected, and in utter hopelessness I painted the affected parts with strong Bordeaux mixture. The single plant got no worse, and eventually became a giant specimen, the other collapsed. ‘‘Later on came the streaked foliage amongst many plants, while streaked, starved looking flowers were produced from all affected plants. The disease did not confine itself to any one variety, nor to every plant of one variety. Nigger, Midnight, Lord Nelson, Hetty Green, Helen Pierce, Frank Dolby, Enchantress, John Ingman, Cod- sall Rose, and Lady Pollock all gave us streaked flowers, and although the affected plants continued to grow and flower right up to October, they never recovered normal health; in fact, as the season advanced the flowers got worse, until it was impossible to find a flower worth a second glance. It has been declared to be the Vetch disease, Perono- spora trifolorum, and sulphide of potassium, or sulphurated potas- sium has been given as the remedy. A local grower found this of no value whatever, whilst the writer of the letters referred to has tried everything possible without result; and the fact of his being unable to discover disease spores in any shape or form shows it to be useless to use spray fluids. Another well known grower, however, informed me that after spraying with ‘sul. potass.’ his plants sent out new growth unaffected by the trouble. ‘‘A peculiarity about the diseased plants is their poor root de- velopment, nodules being totally absent. Personally I am doubtful as to the trouble being due to Peronospora. Unlike the Sweet Pea blight, the disease under notice does not confine itself to the foliage at the start, but seems to attack the whole plant at once. The stems show brown streaks, as do the leaves, whilst the tops of the growths 8 are curled and twisted. The flowers are distorted and look quite out of character. Is the disease a product of California? Is it a bac- terial disease? Has it any affinity to curl disease in Potatoes? If it comes through the seed, why are not all the plants from one packet affected? Advice is urgently needed by growers both in England and Ireland. Who can aid us? T. A. W.”? | In the February (1908) issue of the ‘‘Amateur Gardening”’ (England) p. 643, the ‘‘Streak’’ disease is again described as fol- lows: “STREAK IN SWEET PEAS”’ ‘‘Sweet pea growers in many parts of the country have had to contend with a comparatively new disease called the ‘‘Streak,’’ which threatens to considerably hamper the successful growth of this popu- lar flower. ‘‘This disease appears in the form of greyish or brownish streaks, either on the plants when young or when in flower. The effect of an attack is the loss of natural color in the foliage and a sickly diseased appearance. In some cases whole groups of rows of plants have been quite ruined by the disease. ‘So far its origin is more or less of a mystery, and all attempts to find a satisfactory remedy for it have resulted in failure. A pecul- larity of the disease, apart from the streaks on the foliage, is the ab- sence on attacked plants of the usual bacterial nodules on the roots. This naturally leads to the inference that a cold and wet season like that of last year may have prevented the nitrogen-fixing bacteria doing their work. ‘“The special appearance of the foliage caused by this disease must not be confounded with the grey and streaky results following an attack of thrips, or by sun sealds. The former is accompanied on the under side of the leaves by black shining dots; the latter is only partial. The Peaman’”’ In the Aug. 19th (1911) number of the ‘‘ Amateur Gardening’’ the seriousness of the disease is clearly brought out as follows: “SWEET PEA DISEASE” ‘“ As stated in a recent note, the streak disease has once again put in an appearance, and I am fain to confess that it has proved more virulent and destructive than ever before. In a season like last, the disease was somewhat slow in accomplishing its deadly work; in fact 9 the agony was long drawn out, for the plants continued to live for varying lengths of time, although incapable of producing satisfactory flowers. This season, however, there has been no mistaking its action, and one can only assume that the great heat has materially assisted the disease to accomplish its purpose. During the eight years that I have been acquainted with this disease, I have never known it to work such havoe as it has done this season, and now that five-sixths of my plants are wiped out beyond recall I feel I must record a few impres- sions. ‘‘T do not for one moment suppose that I am relating a solitary ease. More than one expert grower informed me that they were un- able to show at the National owing to the disease. One of these gen- tlemen has won championship cups in previous years, and he assured me his plants had collapsed by the cartload. This tale was told me at the Olympia Show. At the Sweet Pea Show, no less than six ex- pert gardeners from one district in Kent were upon my heels for a remedy. The disease was worse with them than ever before. At the sweet pea outing one or two efforts were made to secure some informa- tion, but the end of it all was nothing. ‘‘What does it mean? Does it mean total annihilation of sweet peas? Plants affected by the disease do not produce seed. The re- sults of my planting upon manured soil suggests that the disease comes to overfed plants, but the fact remains that streak can and does affect some plants on unmanured soil. It was visible at the N. S. P. S. trials, where no coarse growth is encouraged. I have seen it in the fields where sweet peas are growing for seed. It is not infrequently seen in America, for this much I elicited from one of our recent Ameri- ean visitors.’’ ‘The Peaman.”’ Thruout the season of 1911 and 1912 discussions of a popular nature upon the ‘‘streak’’ disease of the sweet pea are found in many of the gardening and horticultural journals. The articles in the Ama- teur Gardening, The Gardeners’ Chronicle and the Annual Reports of the National Sweet Pea Society, throw much light upon the nature of the disease. The writer,, presented a paper illustrated by lantern slides before the American Phytopathological Society at Cleveland on Jan. 3 of this year giving a preliminary report on his findings about this dis- ease. The causal organism was announced as a newly described path- ogen under the name Bacillus lathyrt. ; 10 Directly following the above paper, W. Bateson read a paper by Dorothy M. Cayley,, before the Royal Society (London) under the caption ‘‘A Preliminary Note on a New Bacterial Disease of Pisum sativum’’ in which is described a disease having symptoms resembling in many respects those of the ‘streak’? disease of sweet pea. I quote from her paper the following: ‘‘Tnvestigations have been carried out this year at 'the John Innes Horticultural Institution to elucidate the nature of a disease which affects culinary peas (Pisum sativum). ‘‘The disease, in this district at all events, is a serious one, kill- ing a large proportion of the crop, but I have no information as to its prevalence in other parts of the country. I have succeeded in proving that the disease in culinary peas is caused by a large bacillus which exhibits a peculiar feature, inasmuch as it is transmitted in the inte- rior of the seeds of the plant. As far as I am aware no analogous in- stances are known. ‘“The general symptoms are as follows: In mild cases after germ- ination the shoot can develop normally, but in bad eases it is frequently abortive, brown and dead at the tip, and laterals grow out prematurely to take the place of the main shoot. Quite early in the development of the plant, when the plumule is from half an inch and upwards in length, light brown longitudinal streaks can be seen on the stem and root, and the first leaves are often brown at the tip. These streaks develop later into slits. In very bad eases little or no germination takes place. After this stage no further definite signs are noticeable till about the flowering period. Then the development of the disease depends a good deal on external conditions. If the weather is warm and dry, and the plants are growing vigorously, the disease develops rapidly, and in a few days the plants become unhealthy and change colour. The stem turns slightly brown, and looks somewhat water- soaked. Brown longitudinal streaks appear at the base of the petioles on either side of the rib of the stem, which is continuous with the mid-rib of the leaf. The streaks split open and dry out. The collar may be badly disorganized. The leaves become spotted, streaked and yellowish in color, and if the disease is progressing rapidly the younger portions of the plant show discoloration, and fail to develop properly. ‘‘Except in bad cases the plants grow to full height, and can flower and set a certain amount of seed, but on examination the coty- ledons of the seeds of a diseased plant show brown discoloration, which 11 may be limited to a mere spot in the centre of each cotyledon, or, on the other hand, nearly the whole of the cotyledon may be involved. In the latter case there is often a cavity in the centre of the cotyledon. ‘“Sections of the diseased cotyledon show large numbers of bacilli in various stages of development in the cells and intercellular spaces. ‘“The bacillus works it way into the intercellular spaces and then breaks into the cells. There the nucleus is often attacked, the cyto- plasm destroyed, and the cells collapse, thus forming rents in the tis- sues . ‘“There is considerable evidence to show that the bacillus passes up the plant through the tissues above mentioned, through the funicle, and probably the micropyle into the young developing seed. If one pea is diseased all the other peas in the same pod are diseased to an equal extent. The disease is chiefly spread by the seed, but fresh in- fection may take place through the soil. ‘‘Tnoculation experiments were carried out in the open, but little stress can be laid on the results, as the disease was so prevalent throughout the experimental plot. Pea plants grown in heated soil in boxes, and inoculated just above the ground, when the plants were about 1 foot in height, showed no disease, whereas, in the open, seven out of ten inoculations on the stem just below the youngest unfolding leaf were successful. | ‘‘Hurther inoculation experiments are necessary, but the above results tend to show that the bacillus can only penetrate very young tissue. This is supported by the fact that large numbers of the bacilli have been found in the inner tissues of the radicle when only about half an inch long. ‘‘FKurther investigations are in progress. ‘‘In many respects the symptoms resemble those of the formid- able disease of sweet pea (Lathyrus odoratus) known as ‘‘streak.’’ This disease has been held to be due to Thielavia basicola, but, in view of these observations, that conclusion seems very doubtful, and I may add that, in the stem of diseased sweet peas, I have already found bac- teria like those here described.’’ The fact that Miss Cayley has isolated bacteria from the stems of sweet pea affected with ‘‘streak’’ has caused her to question the conclusions of Massee and Chittenden, viz. that Thielavia basicola is the cause of ‘‘streak.’’ Beginning with the issue of Apr. 5 (1913) of the Gardeners’ 12 Chronicle, the writer and his associate Mr. J. J. Taubenhaus published several articles giving a review of the diseases of the sweet pea (Lathy- rus odoratus) to date. The first article in the issue above noted describes and illustrates the ‘‘streak’’ disease. SYMPTOMS OF THE ‘‘STREAK”’ DISEASE On Sweet Pea. Like the Bacteriosis of beans, it makes its ap- pearance in the season of heavy dew. On the sweet pea the disease usually appears just as the plants begin to blossom; it is manifested by light reddish brown to dark brown spots and streaks (the older almost purple) along the stems, having its origin usually near the ground, indicating distribution by spattering rain and infection through the stomata (See Pl. 3). The lesions which at first are sepa- rate and distinct soon confluently meet, causing the streaked appear- ance. The disease becomes quickly distributed over the more mature stems until the cambium and deeper tissues are destroyed in continuous areas when the plant prematurely dies. Occasionally petioles and leaves show infection, the latter show the watersoaked spots common to the bacterial leaf blight of beans. The disease is not a vascular infection; it confines its attack to the mesophyll, the cambium and deeper parenchymous tissues; the lesions on the stems gradually enlarge and deepen till they come together. On clovers (Trifolium spp.) the disease first appears in August and Sep- tember on the young seedling plants, when often it is particularly se- vere, vying with Bain’s Anthracnose,, in its activity. In young clover it causes leaf spot (See Plates 7-12), water soaked as in the bean, and it also attacks the petioles and crown. In more mature clover prob- ably the most severe attack takes place in the petiole and sheath at the union with the stem; in this case the entire leaf dies and the lesion extends down into the stem. The blackening of the stems and the spotting and water soaking of areas on the leaves are common with the clovers (See Plates 6, 7, & 8). It is not uncommon to find the blackened lesiouis on the stems of clover so overlapped as to cause the entire stem to darken. Soy Bean. The disease was particularly severe upon one variety of soy bean, in which the lower lesions girdled the stem or penetrated so deeply that the plants blew over, suggesting the black leg of pota- toes (See Plates 13, 14, and 15). The lesions were common on the 13 upper branches and even the pods, the latter showing very conspicu- ous blackened spots (Plate 14). Bean (Phaseolus spp.) On several varieties of beans a stem lesion, which was supposed to be caused by Pseudomonas phaseoli, showed upon culturing an organism similar to that met with in the sweet peas, the clovers, and the soy beans. These lesions usually were small, elongated, rusty brown areas one-fourth to several inches in length ; occasionally the lesion was slightly sunken (Pl. IV). In order to compare this organism with that of Pseudomonas phaseoli of bean blight some 350 isolation plates were made from be- ginning lesions on bean stem and leaves. Instead of Ps. phaseoli in the stem lesions, in most cases this new organism was found. From watersoaked pods and leaves a Pseudomonas was obtained which an- swered closely to Ps. phaseoli (Pl. 5). In order to determine the position of our work, extensive isola- tions, cross infections, and systematic studies were started. ISOLATION AND MORPHOLOGICAL STUDIES Over 1500 plate cultures of beginning or young lesions were made from the several hosts. The organism may be taken almost invariably in abundance in pure culture from the beginning lesions in the stems of sweet peas when the surface is properly sterilized. Some difficulty was experienced at first in taking the organism from the clovers. This we attributed to non selection of young lesions, to too severe surface sterilization and to the ease with which the lesions in the thin eambium of the clover dry out, thereby causing the death of the organism. The isolation work clearly indicated that the parasite was bac- terial; a yellow organism which grows luxuriantly upon all the nut- rient media and especially rapid upon nutrient media containing sugars. On standard nutrient glucose agar the colonies appear within 24 to 36 hours. The center becomes granular and the colonies have a marked tendency to become stellate or auriculate. Morphological studies showed the organism to be a comparatively small rod-shaped bacillus, which in fresh cultures is never found in chains, and seldom even united in twos or fours. The flagella are not easily demonstrated. (See Fig. 1). 14 INOCULATION EXPERIMENTS Preliminary inoculation work was carried out Aug. 9, 1912 with sweet peas using two cultures, viz. sources No. 1 and No. 3 (see p. 17). The former came from sick sweet peas at the Experiment Station farm at Newark, Del., and the latter from sweet peas affected with ‘‘streak”’ from Boston, Mass. Inoculations were carried out by atomizer spray- ings with 48 hour cultures, after having first thoroughly wet the plants. The sprayings were applied in the evening on all parts of the plants above ground. ‘Typical ‘‘streak’’ infections showed on the 9th day. On the 10th day lesions were cut out, surfaces sterilized, washed, erushed, and cultured with the result that the typical organism used in the inoculation work was recovered. Inoculations were repeated on Aug. 22, 1912, this time using cultures of source No. 8 from sweet peas affected with ‘‘streak’’ sent here by T. A. Weston from Orpington, England, and source No. 13a from Red Clover. The inoculations were this time both sprayed on certain plants with atomizer, while others were pricked with hypodermic needle. In both cases, infection was obtained in from 7 to 10 days. The spots pricked by the needle were the first to show infection, these appearing in seven days. The red clover cultures gave equally strong infections as did the sweet pea. Subsequent platings of these infections gave an organism identical with that used in the inoculation work. Several of the sources used in the cultural and biological studies were those secured from this preliminary infection work, viz. cultures No. 4, No. 5, and No. 7 and No. 10 E. Following the above preliminary inoculation experiments a series. of cross infection work (Sept. 5) was tried out in the field with only partial success. Tall moist chambers were placed over small young plants of clover, alfalfa, cowpeas and soybeans, using cultures from sweet pea and from red clover. The red clover showed infections from the bacteria taken from Lathyrus spp. while, on the other hand the cowpea, soy bean and alfalfa showed no infection whatever. On Sept. 15, this series was again tried out with only evidence of infection on the red clover. It would seem that alfalfa, and cowpea as well as soy bean in the young stages are not readily susceptible to this parasite. CROSS INOCULATION IN THE LABORATORY About the first of October (1912) some two dozen red clover plants were transplanted in eight inch pots and taken into the labora- tory where they made excellent growth. All evidence of infection was. 1) removed and the leaves were thoroughly sprayed with potassium per- manganate to surface sterilize them; the disinfectant was later care- fully washed off; the clover plants were then placed under moist jars for several days to note whether any natural infection would follow. The sterile plants were then thoroughly covered by atomizer, first with sterile water and then sprayed with young cultures of the streak dis- ease bacterium from ten different sources, viz., one from red clover and one from alsike clover, two from sweet pea, one from another Lathyrus sp., two from soy bean, one from Lima bean, one from al- falfa, and one from infected soil. The following table shows the amount of infection on the clover leaves and stems. Table Showing Amount of Artificial Infection on Red Clover 1. Red Clover on Red Clover 10% of infection 2. Sweet Pea (American) on Red Clover 12% ‘‘ a 3. Lathyrus sp. Pe dee Lac Te i 4. Soy Bean Ne cs a 4% ‘* pe 5. Soy Bean c¢ ceé ce 60% (a5 a4 6 Sweet Pea (English) ‘“ “ is: 50% ‘‘ ui 7. Alsike ih iad a 15% *' ? 8. Lima Bean spr ms Siti 40% ‘‘ a 9. Alfalfa tn a 89% * : 10. Soil ce «¢ c¢ 90% ce ce The results of these infections are shown in Plates 10, 11 and 12. Infection began in 3 to 7 days. The leaf lesion is a typical water- soaked area, quite similar to that of the bean blight disease. These experiments were carried out again beginning Nov. 11, 1912, along with some additional inoculations. Pseudomonas phaseoli of bean blight was tried on red clover but it failed to produce any infec- tion. Three sources of the ‘‘streak’’ disease organisms were used from sweet pea,‘and two sources from forage species of Lathyrus. Infee- tion was evident in three days. Two sources of the ‘‘streak’’ organism B. lathyri from beans were used getting definite infection in three to five days. From this work it was quite apparent that the organism from the several different hosts was capable of producing the disease on red clover identical with that in the field. Many different attempts were made to get infection by spraying on clover and sweet peas and not paying any attention to keeping the 16 plants moist. Invariably the results were negative. All attempts to infect young sweet pea plants have been failures. The disease will not develop till the plant is about to blossom. Just what physiological changes take place in the plant just prior to and at the time of blos- soming has not been determined, but it is evident that there must be a radical change, as needle inoculations or sprayings will make no ad- vance whatever. The writer is of the opinion that along with a possible change in the physiology of the plant, owing to the fact that the flowering period falls in the time of heavy dew, there is a turgescence in the plant throughout the nights which probably favors the entrance and growth of the organism into the interior of the plant. THE RELATIONSHIP OF THE CASUAL ORGANISMS The series of apparently similar organisms taken from such a va- riety of hosts offered a splendid opportunity for studying the possible eultural and biochemical variation. Having proved that red clover could be infected with the organisms from the different sources (see p. 14) these same sources, together with several morphologically sim- ilar organisms from other hosts were used for a detailed study of variation in cultural, physical and bio-chemical features. In order to be certain that the media were standard in their properties and reac- tions, several well known organisms were run as checks. For this pur- pose Bacillus coli, Pseudomonas compestris and Pseudomonas phaseolt were selected. The cultures used in this study were taken from begin- ning lesions which appeared free from contamination. The isolation of bacteria from lesions was carried out by following a method which has been used by us,, for a number of years. This consists in cutting out the lesions somewhat beyond the area of infection, pieces being small enough to be placed in a culture tube. If the lesions are on small stems, as that of clover or sweet pea, sec- tions of the stem, including lesions are cut out one-half to three-fourths of an inch long. If the lesions are on the leaf, the youngest ‘infections are selected and taken out entire. ‘The surface sterilization is carried out in a clean test tube, upon several pieces at a time, using enough of a 50% aleoholic solution containing one gram of bichloride of mercury to the liter, to cover the infected pieces of plant tissue at a depth of one inch. After placing the plug in the tube and shaking the disinfectant to all parts of it, the solution is allowed to stand on the material from 15 seconds to two minutes, depending on the nature of the plant tis- 17 sue. Thin leaves, lightly infected, will hardly stand more than 10 seconds, while deeply infected stems and tubers, or root tissues will stand two minutes, or even much more when the disinfectant does not penetrate too deep. At the required time, the disinfectant is poured off and the material is washed three times with 12 to.15 ee. of sterile water for each washing. This washing is carried out in the same tube in which the surface sterilization is done. Sterilized water for this purpose is kept in stock at all times in the laboratory, stored in tubes containing 12 to 15 ee. After flaming the plug and mouth of the tube carrying sterile water, the contents is poured directly upon the mate- rial to be washed and the flamed plug is used to close the tube. The water is thoroughly shaken to all parts of the tube in order to wash away every trace of the disinfectant. This water is then poured off and the process repeated, each time using the new cotton plug from the sterile water tube, until three washings have been given, when the material is gathered near the mouth of the tube. Here the indi- vidual pieces are picked out with sterile forceps and each is crushed with the same forceps in the mouth of a tube containing a medium properly coo'ed for growth. The crushed lesion is washed down by the medium, sub-cultures are made and poured into sterile. plates. The writer has had excellent success with this method, succeed- ing almost invariably in taking out pure cultures of the pathogen, whenever it is not associated deeply in the lesion with contaminating organisms. The following is a list of the organisms isolated and used in the cultural studies: No. 1. From Sweet Pea Stem, showing typical ‘‘streak’’ obtained July 20, 1912 from the Experiment Station farm at New- ark, Del. No.° 2. From Sweet Pea Stem showing typical ‘‘streak’’ obtained July 25, 1912 from the bata Station farm at New- ark, Del. No. 3. From Sweet Pea Stem showing typical ‘‘streak’’ obtained Aug. 1, 1912 from the noted sweet pea grower, Mr. William Sim of Boston, Mass. No. 4. From Sweet Pea Stem showing ‘typical ‘‘streak,’’ artificial infection in garden of T. F. Manns, Newark, Del., Aug. 18, 1912. | No. No. No. No. No. No. No. 10 e. 12 d’ v2 ih: 4B saa. 147’. 15. 161. 41 K. 18 From Sweet Pea Stem showing typical ‘‘streak’’ by arti- ficial infection in garden of T. F. Manns, Newark, Dei., Aug. 18, 1912. From Sweet Pea Stem showing typical ‘‘streak’’ by arti- ficial infection in garden of T. F. Manns, Newark, Del., Aug. 2h. OM: From Sweet Pea Stem showing typical ‘‘streak’’ obtained Aug. 22, 1912. from T. A. Weston, St. Johns Road, Orping- ton, England. From Sweet Pea Stem showing typical ‘‘streak’’ obtained Aug. 24, 1912 from T. A. Weston, St. Johns Road, Orping- ton, Eng. From Sweet Pea Stem showing typical ‘‘streak’’ from ar- tificial infection Aug. 22, 1912. from T. F. Manns’ garden, Newark, Del. From Sweet Pea Stem showing typical ‘‘streak’’ obtained from J. J. Taubenhaus’ garden, Newark, Del., Sept. 24, 1912. From Sweet Pea Stem showing typical ‘‘streak’’ obtained from J. J. Taubenhaus’ garden, Newark, Del., Sept. 24, 1912. From Sweet Pea Stem, showing typical ‘‘streak’’ obtained from Marshall Manns’ Garden, Oct. 25, 1912, Newark, Del. From Red Clover petiole, showing black lesion in cambium, obtained Sept. 4, 1912 from the Experiment Station Farm, Newark, Del. From Red Clover Stem, showing darkened lesion in cam- bium, obtained Sept. 7, 1912 in vicinity of Newark, Del. From Red Clover Stem, showing black lesion in cambium, from field near Mr. Taubenhaus’ home, Newark, Del., Sept. 24th, 1912. Same source as No. 15. Different plant. From Red Clover Stem showing black lesion in cambium, called ‘‘OK Culture’’ beeaase material so typical, from vicinity of Newark, Del., Sept. 25, 1912. No. No W182’. . 26’. pao £7. . 28. . 30. . 36. noe . 40 g,. 17d’. 19 From Alsike Clover petiole, showing typical blackened le- sion, from field near Red Men’s Home, Newark, Del., Sept. 4, 1912. From Alsike Clover petiole, same origin and date as No. 17 d’ though different plant. From Soy Bean petiole, showing small sunken black lesion, from Experiment Station farm, Newark, Del., Sept. 3, 1912. From Soy Bean Stem, showing beginning black lesion, from Experiment Station farm, Newark, Del., Sept. 7, 1912. . From Soy Bean Stem, showing beginning lesion, from Ex- periment Station farm, Newark, Del., Sept. 24, 1912. . From Soy Bean stem, from beginning black lesion, same source and date as No. 23 h, though different plant. . From Lathyrus Sp. (for forage purposes) showing typical ‘“streak’’ lesions on stem, obtained from Experiment Sta- tion farm, Newark, Del., Sept. 3, 1912. From Lathyrus Sp. (for forage purposes) showing typical ‘““streak’’ lesions on stem, obtained from Experiment Sta- tion farm, Newark, Del., Sept. 27, 1912. From Lathyrus Sp. Same source and date as No. 26 e’ only a different plant. From Cowpea Leaf, sent in from vicinity of Philadelphia, leaves turning brown: Aug. 3, 1912. From Wax Bean seed taken from green pod showing large watersoaked lesion; obtained from Red Men’s Home, New- ark, Del., Sept. 3, 1912. From Tomato fruit showing typical end rot of fruit, from - T. F. Manns’ garden, Newark, Del., July 13, 1912. From Tomato fruit end rot produced artificially by inocula- tion with source 26 above, from infection in laboratory, Newark, Del. Variety of Tomato ‘‘Stone,’’ July 28, 1912. From Climbing Bean pod (green string bean). ‘Typical brown sunken lesions along pod; from Mr. Taubenhaus’ garden, Newark, Del., Sept. 30, 1912. 20 No. 42. From Dwarf Bean, watersoaked pod, plate No. 15, from Mr. Taubenhaus’ garden, Newark, Del., Oct. 27,1912. This organism is Ps. phaseoli and was hence used as a check. No. 45. From Dwarf Bean Pod. Dark brown lesions along pod; from Mr. Taubenhaus’ garden, Newark, Del., Oct. 27, 1912. This organism is also Ps. phaseolt. No. 46. From Dwarf Bean Pod. Typically watersoaked lesion from Mr. Taubenhaus’ garden, Newark, Del., Oct. 27, 1912. This organism is also Ps. phaseoli and used as a check. No. 47. Bacillus coli from human intestine. Used here as a stand- ard check on media and for reactions. CULTURAL AND BIOCHEMICAL STUDIES Extensive cultural and biochemical studies made upon the thirty- two different strains from the several hosts confirm the results from inoculation work, viz. that the causal organisms are identical as far as our present system of classification is concerned. Some slight vari- ations were noticeable in several of the strains, such as absence of pellicle in nutrient broth, or a very meagre pellicle. These differences were just as marked in the different strains from the one host as they were between the strains from the several hosts. This was likewise true for color gradations on the different solid media. 'The shades of yellow varied from a light straw to that of almost a deep orange. Probably the most uniform cultural reaction was that on nutrient gelatin, in which in practically every strain it required somewhat over two months for complete liquifaction, while at the end of six weeks not more than half of the gelatin was liquified. The fact that liquifaction proceeded entirely from above would indicate that the or- ganism is an obligate aerobe. In table I is shown in a brief summary the group number and other features of the thirty-seven organisms compared thruout the’ cultural studies. It is quite apparent that of the 32 strains first con- sidered in this summary and selected from various hosts because of similarity in disease production, or because of similarity in morph- ological and preliminary cultural studies, that there is not variation enough to differentiate one strain from another on any specific cult- ural reaction (See Table I). a1 The introduction into this series of cultures for comparativd studies of sources Nos. 36 and 37 from ‘‘point’’ or ‘‘fruit rot’’ of to- mato was to learn the cultural and other classifactory features of the organism which here is shown to be the active causal agent of point ‘rot. (see Pl. 16). As far as the present system of bacterial classi- fication is applicable, this organism is not to be distinguished from that described herein as Bacillus lathyri n. sp. The writer did not carry out cross inoculation work on sweet pea and clover with the or- ganism from the point rot of tomato. Though the morphological and cultural features of the tomato organism are similar as far as we are able to determine by present methods, yet it is quite possible that the organism may not be able to cause the ‘‘streak’’ disease on sweet pea and other legumes. A short history of the association of bacteria with the ‘‘fruit rot’’ or ‘‘blossom end rot’’ of tomato is as follows: Elizabeth H. Smith,, (1905) and F. S. Earle,, (1900) have found bacteria associated with the fruit rot of the tomato. Both claim typ- ical infections when inoculations were made from pure isolated cult- ures. William A. Stuart,, has also found bacteria associated with the fruit rot of the tomato. The writer in July 1912 found the rot quite genera! in his garden, and having seen Miss Smith’s report of a ‘“baeterial rot’’ of the tomato took the opportunity of culturing young lesions from affected fruit. Careful surface sterilization was prac- ticed; only beginning lesions were cultured in the first series and the extreme margins of the lesions were used. The result was that in every plate a yellow bacterium came out. In several of the plates there were associated occasionally a Fusarium and an Alternaria. Both of these fungi as we!l as the bacterium were isolated in pure eulture.on an artificial medium. Inoculation work was carried out with each of the organisms. The bacterium gave excellent infection both :n the field and in the laboratory during a period of dry weather, when spreyed with atomizer or inoculated by needle prick. During the same time there was also some evidence of infection when the Fu- sarium was inoculated into the flower end of the fruit by inserting mycelium beneath the epidermis with a sterile scalpel. The Alter- naria, however, made no progress at all. The check sprayings with sterile water and the check incisions gave no infections. The above experiments were duplicated during a period of rainy, cloudy weather with very little progress whatever in the production of rot. The Fu- 23 sarium and Alternaria showed no evidence of infection, while the bac- terium produced only small watery lesions. Drought is a great factor in furthering the disease. Heavy rains with moist atmosphere entirely checked the disease in the experimental work. The writer is inclined to infer that this disease is of bacterial origin and that its progress is very closely associated with the water supply of the plant. (Since writing the above in June 1913 the writer has been privil- eged to review Brooks’ work on the ‘‘Blossom-end Rot of Tomatoes,’’ Phytopathology Vol. VI, No. 5, Oct. 1914. He still holds the view that the disease is of bacterial origin, and is distributed by rain or insects at blossoming time. The very beginning lesions give pure cultures of the bacterium. ) ’ DESCRIPTION OF THE CAUSAL ORGANISM* Bacillus lathyri n. spp. Manns & Taubenhaus I. MorpHoioey 1. Vegetative Cells. When grown upon nutrient agar for 24 hours at 25° 'to 28° C. and stained with aqueous solutions of methylene blue, gentian violet or fuchsin the organism is shown to be a compar- atively small rod-shaped bacillus, with rounded ends which is rarely found in two or fours. 'The stain in the organism from such cultures is evenly distributed thruout the cytoplasm. In older cultures, two to three months, some take a denser polar stain. The organism mea- sures from .75¥ to 1.54 X .6% to .854 the majority being 1.40 X .75u. 2. Sporangia. No sporangia have been observed. 3. Endospores. Cultures on various media carried for ten months show no endospores. 4. Flagella. When stained by Loeffler’s, Pitfield’s or Van Er- mengen’s methods the flage'la may be demonstrated, though not easily. They are shed so readily that usually no more than from two to five may be shown attached, though it is not uncommon to find many de- tached thruout the field. However, when the material is carefully selected, fixed and stained, the flagella ™ay ‘be demonstrated to be well distributed peritrichially, and to number eight or even more. (See Hig) 1): *The thirty-seven descriptive charts used in tabulating the morphological, cult- ural, physical and biochemical features of the different organisms, are not printed herewith. 24 pera +)/+/4+ salto au BEER EEEEREEEE ee || | Hs ae SSCaEREMESE: ae r+ +/+[+]+] ] ee ace +]+|+]e|-[+][+[-|-| fey S 2 AT ee FEO PENTY SESE: Ry By Logue ny/ dv Ot +) OF? PIAZSA] YIX , (ee a a ll ise earns Se eS SEES Cea ee PPE BLATT TPE Ee Ee tp E227 ACS EES SES 6S et EES CSC = 2 PRES ES ES EES EES Ras ES ES EAESESE- Hee +-|-| ee + aR aE aE +|-}4+ ta a Rn nec llc Mel etal gee ME AES SESE SSeS (ae LEAS SESS Cd STS SI eee fie gpa ‘(amelie 2 ES CESSES ES REESE eS MR) cE ESE ES ES ES ES EES ESE pepo eet tl ee pe ee Fes 1 A 8 ie Engw piesa cal features slow eet te | + + RM SS SEVERE eH EGHHE CE PERSE EERE IEEE cea le Be S| ++ + te) S| Saree ic DE ee 1) Ee A EE ee po ET Pie) aE ES C36 CES CS EIESESESESESESES OM Saeco Ea Ev EES SESS EE ES ESS CSS ET J Reid EES EES ES ee BS EES ESE EES eS ese (ee a (eee eet Ed = Dalila Boas Le AD ESE] ATs a FS a Tp Caer alec E7Ea = coves eS cds ess ppg ype ae Do a a ‘me ed A A HO 3S De ee SEF ES bbs Rae Eee es Sey Ses eS ee DR Bik ES a Se ee SESS Seas UST aerate Fats eee erat Hae three months cllon of starch very ung rh Weal t¢ Yote dest Slaw re Gf faction ver Broth, gu sparag: ” es le Grains Number and other Clase, actor f « Fluorescentin @ t Star) signi ee = a O 2ll2222522 Bean Source 0- Wax Bean-Pod 26 In hanging drop or under cover shp the organism in 24 to 48 hour cultures is very active, taking a motion somewhat slower but otherwise similar to that of B. typhosus. 5. Capsules. No capsules have been demonstrated. 6. Zoogloea. No zoogloea has been formed on the ordinary media, but in asparagin broth, and in Uschinsky’s solution a ropiness is com- mon; on starch jelly containing Uschinsky’s solution a pseudozoog- loea is present. 7. Involution forms. Individuals somewhat longer and broader than usual are met with in old cultures four to six months; bipolar staining, and denser regions of cytoplasm are commonly seen with ordinary stains. Some individuals show granulation, though this is not common. 8. Staining reactions. The organism, though staining readily with the ordinary stains. loses these quickly when washed with alcohol. 9. Gram’s Stain. The organism is gram negative. 10. Loeffler’s. Methylene Blue. With Loeffler’s Methylene blue the cytop!asm shows no granulation and is evenly stained thruout from a 24 hour culture. 11. Neisser’s Spore Stain. No evidence of spores present with Neissers’ stain. II. CuLTuRAL FEATURES 1. Agar Stroke—Nutrient Agar. Growth, at 25 to 28° rapid in 24 hours, and abundant in 72 hours. No growth at 37° C. Form of Growth, filiform, usually smooth at margin but occasionally undulate, or even slightly echinulate. Elevation of Growth, slightly convex. Luster, glistening. Topography, usually smooth; surface occasionally very shghtly granulate. Optical Characters, opaque. Chromogenests, light to deeper yellow in different strains. Odor, absent. Consistency, butyrous. Sub medium, shows no change of color. 27 2. Potato. Growth, quite rapid in 48 hours. Form of Growth, filiform becoming more or less irreg- ular. Elevation of Growth, slightly convex. Luster, glistening. Topography, smooth. Chromogenesis, at first a light yellow, later becoming somewhat deeper. Odor, absent. Consistency, butyrous and slightly viseid. Medium, not changed. 3. Loeffler’s Blood Serum. (not used) 4. Agar Stab. Growth, best at top-surface growth quite rapid in 48 hours, abundant after 3 days. Organism a rather closely restricted ‘aerobe. Line of Puncture, filiform, but quite restricted, in growth deep in the medium. Chromogenesis, yellow. Medium, not changed in color. 5. Gelatin Stab, Growth, best at top, small amount of growth in the lower part of stab. Tine of Puncture, filiform. Iiquifaction, slow, not showing till nearly two weeks old and at end of four weeks only fairly well begun. Not complete till three months. Quite uniform thruout 32 strains. Mediwm, not discolored. 6. Nutrient Broth. Surface Growth, slight pellicle in some eases though not general. Clouding, strong in 24 hours. Odor, absent. Sediment, compact, scant. 28 7. Plain milk...No visible change takes place in two weeks. At the end of three weeks 23 out of the 32 strains showed curdling with- out separation of whey or of digestion of curd, while 9 strains did not show coagulation until heat was apphed. Tests showed that the acidity in the various strains which coagulated milk in three weeks gradually increased from O Fuller’s scale to +6 at end of the third day; to +12 at end of first week, and at time of coagulation, that is three weeks, had reached +36, Fuller’s scale. It seems probable that where coagulation took place before evidence of peptonization of casein set in, that the increased acidity may have in some strains checked the growth of the organism. In the 9 strains noted above some evidence of digestion as well as acid production was showing though the acidity was not great enough to bring about coagulation. At the end of seven weeks 24 strains showed partial or complete di- gestion of casein, while eight remained curded with little or no evi- dence of digestion. In several strains there was evidence of curding from enzymatic action; that is, the production of acid did not appear strong enough to bring about coagulation. 8. Litmus Milk. In most strains there was a gradual and slow increase in acidity thruout the first month. In several of the more rapid digesters of casein the itmus was also digested and re- duced. 9. Gelatin Colonies. Eight days old. Growth, medium. Form, round. Elevation, slightly convex. Edge, smooth. Liquifaction, too slow to show on plate. 10. Agar Colonies, five days old. Growth, rapid at 23 to 28° C.—Yellow colonies, visible to the eye in 24 hours. Form, stellate to ameboid. Surface, smooth, glistening. Elevation, slightly raised. Edge, entire and regular. Internal Structure, granular at center and smooth else- where. ral 12. 14. 15. 16. Ie Ag. 29 Chromogcenesis, yellow. Size, depends on room in plate, much extended. Glycerine agar. Growth, rapid and abundant at 23 to 28° C. Form of Growth, filiform. Elevation of Growth, slightly raised. Luster, glistening. . Topography, smooth. Optical characters, opaque. Chromogenesis, light yellow. Odor, none, or slightly sour. Medium, not changed in appearance. Synthetic agar low in nitrogen. Weak to no growth. 3. Cohn’s solution at 23 to 28° C. Growth, absent. Uschinsky’s solution at 23 to 28° C. Growth, rapid in 24 hours. Flwid, viscid yellow sediment after three days; some- times a pellicle would form and sink, followed by others. No fluorescence. Dunham’s solution at 23 to 28° C, Growth, moderate. Clouding, moderat-, pers'stent, fluid, slightly turbid. Indol formed. Asparagin solution at 23 to 28° C. Growth, abundant. Clouding, moderate, persistent, fluid turbid. Nitrate Broth at 23 ‘to 28° ©.’ Growth, moderate. Clowding, moderate, persistent, fluid shghtly turbid. Nitrates, not reduced. Dextrose Bouillon at 23 to 28° C. Growth, abundant. Clouding, strong, persistent, fluid turbid. Gradually becomes acid from +7 Fuller’s scale to +14 in 7 days. 19. 20. 21. 22. 23. 24. 20. Sugar Free Broth 30 Saccharose Bouillon at 23 to 28° C. Growth, abundant. Clouding, strong, persistent, fluid turbid. Gradually increases in acidity from +3 Fuller’s seale to +9 in 7 days. Lactose Bowllon at 23 to 28° C. Growth, abundant. Clouding, strong, persistent, fluid turbid. acidity. Maltose Bowillon. Growth, abundant. Clouding, strong, persistent, fluid turbid. acidity. Glycerine Bouillon at 23 to 28° C. Growth, abundant. acidity. Manmnite Bowllon at 23 to 28° C. Growth, abundant. Clouding, strong, persistent, fluid turbid. acidity. Growth on Bowllon over Chloroform. Growth, absent. Sodium Chloride in Bouillon. 4% inhibited growth. TABLE IT-A. Clouding, strong, persistent, fluid turbid. Inereases in Increases in Increases in Increases in’ 2. Production of Acid and Alkali (Fullers Seale) plus 1% 0 days 3 days 5th day Dextrose +7 +10 +12 Saccharose +3 + 6 + 8 Lactose +6 a — Maltose +8 — 7 Glycerine +2 — — Mannite +6 -- ~ Degrees of Reaction after 7th day +14 ug Increased. ce a4 ce 3] ‘s]UOMUILIedxe asaq} JNonAYY »29 aCe) - ei 3I [8}0} 9} WlOIF poyoNpep Mseq sey Yooyo oy} Jo AjIploe oy, » »9G »06 WAC YD MSTY Lt | mee 1S°9 ee a »06 T]Oo sny[love = LP »06 »0G ” » IOT »0 »06G pod uesg frend OP 996 | »0G ” y Y9T BS a 20: pog uveg jremq «Gh, Si, 9G | GF i ie UT » OT 06 yoy youeg ‘aseqqey FF} 4,02 | 4,09 i ee a »OT GT poxeossojyeM UvIg FIVMC CP OL GG TOAOTL) PP rset 3 8'8 O° pod urog surquiyy “soF}| ,, 9°9 | 196 ca eee OE »S OL »O02L ”? i} ”) EG »I9L ; »00T | ? ”? uct +) EG ”? GL JOY pug O0FCULOT, 9§€ ” 9°G y ms 08 | ”? ”? »PéL »OO0T »GP pod uvogd XeM 0& »68 »GP | » %0T 18'S 9G Bag MOQ gz], OL OL | pee eas »»0'6 »»0°6 ; ”? i) J9G »9G G9 oe) ”? 8 7021 * G9 ”) ) 996 9G »20E »? ) fb 1 8'8 OL ‘dds snaAygery “pgz} sy, OL | GG yo BD g »08 0S ? 9) UvG re oe ; »GP ” 9 a2 0°01 cd Son melee! | SO | Ore (aoe »0L 7G) 9) 9? GG 269 »G8 | > be) G 09 0'6 | 00 0'9 uvsg £0G §Z 00 0'0 00 G'F | BIg OMG iT Y}OIg ssO1eYISeS| Y}JOI_g 2s01}KOq Y}OIg VSOIreyooRS| YJOI_g aso}, xa(T 39) Q900T Hr YOR OI jsOH pur ainqno jo 12qunN Jo 9900T UT JO 9900T Ut JSOL] pUv daNjI[NO Jo 1equnN pry Q) jo ymry|poy OF jo yay proy QF yo yury|pey Yi jo qury ‘skUp UOASS UL YO osorRyooRG puv WsOI}xXeq Ul yploy Fo uoronporg surmoyg “q-IT ATAVL 32 26. Nitrogen, apparently is not obtained from the atmosphere, but is obtained from all the broths. 27. Best medium for long continued growth. Nutrient glucose agar has given the longest growth in a shake deep tube culture. III. PuHysicaL AND BIOCHEMICAL FEATURES. 1. Gas production. No gas is produced from dextrose, sacchar- ose, lactose, maltose, glycerine and mannite broth in fermentation tubes, very little or no growth takes place in the arm. The limitation of growth was sharply defined at the union of bulb and arm, indicat- ing an obligate aerobe. 3. Production of Ammonia. The production of ammonia has been determined in nutrient broth, Dunham’s peptone solution, asparagin solution and nitrate broth. In all of these solutions some ammonia was present. The ammonification was quantitatively measured in a 1% peptone solution. The following tab‘e (III) shows the amount of N/10 ammonia formed in 100 ec. of above peptone solution in seven days and twelve days respectively by eight different strains of Bacil- lus lathyri from tne following hosts ;—swiet pea (3), Red Clover (2), alsike clover (1), soy bean (1), and wax bean (1). TABLE III Showing Ammonification In Seven Days In Twelve Days IN N N I eos No. of Culture and Total Y, |) NH; 1043 ey 190s ho NES Host NH, in Chects produced NH in check | produced 3 43 C. Sweet Fee | 91ee 16ce 7.5ee 14.6eec 1.9ee 17.7 ce I Ga): eG 56: © 13.25" « ho pies 1Oveo of ey 2 a eG a Oe Ign, queda 4: alee J3a hed *Clover’| ° 6.7°° ol.6"* las | EB BAG 5:6 ** 1 a SOE! AG: 2:3 <4 10:98 26