Historic, archived document Do not assume content reflects current scientific knowledge, policies, or practices. ‘UNITED STATES DEPARTMENT OF AGRICULTURE BULLETIN No. 879 Contribution from the Bureau of Plant Industry WM. A. TAYLOR, Chief Washington, D. C. PROFESSIONAL PAPER November 15, 1920 THE MOSAIC DISEASE OF CUCURBITS By S. P. DOOLITTLE, Assistant Pathologist Office of Cotton, Truck, and Forage Crop Disease Investigations CONTENTS Page scopeof tne, Investigations <5. ssi Ea he eee 1 The Mosaic Disease Se Dd TC EO EN hg Kae” ol) Seer e oe L. Mere oie ee sensed foe oe Ft Se SOP NB ho Se ae ivechous. Nature ofthe, Disease.” i o> 69 es i ae 29 MURS arte e Ea SIR ISSIOME Ss bos ice em Pe gM hab tae ah | ahr 40 Wiethodror AUServwinterime ss ars Mr, Sd ee a et ol Bee 47 Control Measures - - - - - + + += + + + + = «+ 68 Summary = He does Oe ee Bee Ma eee a OE erin) Brak 64 Literature Cited De hil Bat Se. cop P Paes cal Kiar) 1 See (ame OO ee ae oe a 68 WASHINGTON _ GOVERNMENT PRINTING OFFICE / 1920 HE INVESTIGATIONS of the cucumber mosaic reported in this paper were begun by the writer in 1914 at Hamilton, Mich., while a graduate student at the Michigan Agricultural College. In 1915 and 1916 they were continued at Big Rapids, Mich., and in 1917-18 at Madison, Wis., under cooperative relations between the Bureau of Plant Industry of the United States Department of Agriculture, the experiment stations of Michigan and Wisconsin, and certain interested pickle growers, | under the joint direction of Dr. E. A. Bessey, Dr. L. R. Jones, and Mr. W. W. Gilbert. Laboratory facilities and material were furnished jointly by the cooperating parties and land, tools, and labor by a local pickle company. The writer wishes especially to thank Messrs. Bessey, Jones, and Gilbert for assistance and supervision throughout the course of the work. This paper was presented to the faculty of the University of Wisconsin in partial fulfillment of the requirements for the degree of doctor of philosophy. UNITED STATES DEPARTMENT OF AGRICULTURE gy, BULLETIN No. 879 Contribution from the Bureau of Plant Industry Sw? TS. WM. A. TAYLOR, Chief Washington, D. C. PROFESSIONAL PAPER November 15, 1920 THE MOSAIC DISEASE OF CUCURBITS. By 8. P. Doourrriz, Assistant Pathologist, Office of Cotton, Truck, and Forage Crop Disease Investigations. CONTENTS. Page. | Page Scope of the investigations..................- ie Method foverwinteringe s. .--osccecseseoese 47 ALINE MATOSAleIGISCASC Scan a> ese oe ccc ee ss 1a Controlem Caswneste=s-5-- se neene eae see eee 63 CWaUSCOMENEIGISCASCH se oe an eee eas leas LSE ENS LETITIA Teyis stars ovale cry eer a en eae en 64 Infectious nature of the disease.............-. 20M PlnTLenatuGe cited saaee seen eae eet eye ee 68 Mosaic transmission.......... AR e Ee Seen Eee 40 SCOPE OF THE INVESTIGATIONS. | ae the last four years the more important diseases of the cucumber have been studied by various workers in coopera- tion with the Bureau of Plant Industry of the United States Depart- ment of Agriculture. One of the most significant facts developed in the early work was the occurrence of a mosaic disease, heretofore practically unrecognized, which is probably at the present time the most widespread and destructive disease of cucumbers. The disease, however, is not confined to the cucumber but affects to a greater or less extent most of the cucurbits grown_in this country. The present bulletin deals with the nature, transmission, and overwintering of the disease in relation to cucurbits. The greater portion of the work has been done with the cucumber as grown for pickling purposes, in an attempt to develop field control measures. THE MOSAIC DISEASE. HISTORY. Although mosaic has been reported in the field and greenhouse for some years, it is only recently that the disease has received detailed attention. Selby (26)! in Ohio in 1902 and Stone (29) in Massa- chusetts in 1909 recorded a mosaic. disease on the leaves of green- house cucumbers, and Clinton (8) in 1908 noted a chlorosis of musk- melon leaves in Connecticut. It is not certain, however, that all —— 1 The serial numbers in parentheses refer to ‘‘ Literature cited’’ at the end of the bulletin. 185118°—20——1 2 BULLETIN 879, U. S. DEPARTMENT OF AGRICULTURE. these were true mosaic, as the disease was not proved to be infectious in any case and no mention was made of symptoms on the fruit. Selby (27) stated in 1910 that cucumber mosaic was transmitted like that on tobacco. There is no early record of the disease in the field, but it is certain that it had caused serious losses in the Central States for some time prior to 1914, and from evidence obtained from pickle men in the vicinity of Chicago it is practically certain that the mosaic had been a serious disease in that section for 10 to 15 years and was responsible for forcing at least one pickle company out of business. Ruggles and Stakman (25) in 1911 reported the mosaic as occurring in Minnesota and used the name wart disease in describing it. Coons (10) in 1915 reported that this disease was then causing serious losses in the field in Michigan and had been present in some localities for seven years. He described the chief symptoms and stated that the progress of the disease indicated that 1t was infectious. The first proof of the infectious nature of the disease and the methods by which it is transmitted was presented in the papers of Gilbert (14), Jagger (17), and Doolittle (11) in 1916. Jagger (18) later reported the appearance of another type of cucumber mosaic, affecting only the leaves of the plant, and in a further paper (19) mentioned a third type as occurring on Summer Crookneck squash. Stakman and Tolaas (28) mentioned nubbin or wart disease of cucumber as an infectious disease occurring in both the field and the greenhouse in Minnesota. McClintock (22) in 1916 observed possible cases of transmission of the disease through the seed and described its occurrence in the district near Norfolk, Va. Clinton (9) also gave data as to the severity of the disease in Connecticut. Freiberg (13) in 1917 reported the disease on various cucurbits in Missouri and described inoculation experiments. Jagger (20) in 1918 also reported numerous cross-inoculations from cucumber to other cucurbits and showed that cucumber mosaic could be trans- mitted to plants outside the Cucurbitacee. These papers include all the available material on cucumber mosaic to the present, with the exception of an abstract by Doolittle and Gilbert (12) which dealt with certain phases of the work here considered in greater detail. GEOGRAPHICAL DISTRIBUTION. The general occurrence of cucurbit mosaic in the field was not recognized until after these studies were begun, but investigation has shown that it is widely distributed on Cucurbitacez throughout the United States. Doubtless the extensive cultivation of cucumbers makes it seem more common on that crop, but it may occur on prac- tically all cucurbits in localities where it is found on cucumbers. The disease is probably most widespread and serious in Wisconsin, Michigan, Indiana, northern Illinois, and on Long Island, It is re- ae THE MOSAIC DISEASE OF CUCURBITS. 3 ported to have caused serious losses in Illinois as early as 1908 and was present about as early in Michigan and Wisconsin. The center of the pickle-growing industry is located in these States, which devote a large acreage to the crop. Here the disease is present to some extent in almost all localities, and in most cases there is infec- tion every year. The disease occurs commonly in New York and is becoming serious in the seed-growing districts of Ohio and Iowa. It has been found quite prevalent in the fields around Rocky Ford and Greeley, Colo., causes severe losses in the trucking region about Norfolk, Va., and does much injury in many sections of Florida and Louisiana. Recent Inspections of the principal city markets of the Western and Pacific Coast States have shown the mosaic disease to be present in practi- cally all of them, and in many cases it was very prevalent. Itis also reported in the field from Maine, Massachusetts, Minnesota, Ver- mont, Connecticut, West Virginia, New Jersey, Pennsylvania, Georgia, Texas, California, Nebraska, and Ontario, Canada. In the greenhouse it has caused serious injury for several years in Michigan, Wisconsin, Illinois, and New York and has also been reported from Mississippi, Ohio, Indiana, Minnesota, Pennsy]- vania, Louisiana, and Kansas. Up to the present the disease has been found only in the United States and Canada, but the fact that it has not been reported from other parts of the world may be due to its not having been extensively studied elsewhere. ECONOMIC IMPORTANCE. Cucurbit mosaic is the most serious disease of cucumbers in the Middle West, and yearly it increases in severity in districts where this crop is grown on a large scale. The disease is not confined to the cucumber, but causes considerable loss on the muskmelon and to a less extent on squash and pumpkin. The greater losses on the cucumber are due partly to the special severity of the disease on the fruits of this host, but more especially to the fact that the acreage of cucumbers far exceeds that of any other cucurbit. The increase in the pickling and trucking industries in certain sections has localized much of this crop in restricted areas, and thus furnished conditions very favorable to the spread of such a highly infectious disease. The other cultivated cucurbits ordi- narily are grown on a smaller scale, and thus the loss is distributed among many small growers, so that it is less noticeable. Cucumber plants infected with mosaic are practically worthless, owing to the great reduction in yield and to the fact that the fruits produced are so mottled and deformed that they are usually refused by pick- ling companies and are of little value for market purposes. The disease spreads very rapidly, and many fields may become 50 to 75 per cent diseased almost before picking has begun. As a result of 4 BULLETIN 879, U. S. DEPARTMENT OF AGRICULTURE. the losses from mosaic, many growers, especially those in certain sections of the Middle West, have ceased to grow cucumbers, despite increased prices (Pl. I, C). In the case of forcing cucumbers, growers occasionally lose an entire crop, because, in addition to the reduced yield and deformed fruit, mosaic often causes the sudden wilting and death of the plants under glass. An additional factor in the loss occasioned by mosaic is the fact that affected table stock sells for about one-fourth the price obtained for sound fruits, owing entirely to the effect on their appearance. Of the other cultivated cucurbits squash and muskmelon seem most susceptible to the mosaic, but neither of these plants is injured as severely as the cucumber. The muskmelon vines are stunted and bear only a few fruits, which are inferior in quality but only occasionally show mosaic symptoms. Squashes are similarly affected and in addition sometimes have warty and deformed fruits, the Summer Crookneck variety being most often and most severely attacked. The disease is less common and of minor importance on the pumpkins, gourds, and ornamental cucurbits, which are of less commercial! value. CUCURBIT HOSTS. Most of the species of the family Cucurbitaceze appear to be sus- ceptible to mosaic. Field observations have proved that most cultivated species are commonly affected, and that the disease also occurs on wild species. The host range has been further extended by cross-inoculation experiments which included most genera and species of cucurbits found in this country and many varieties of the commoner cultivated species. Most of the field inoculation tests were made during 1916 and 1917. All moculated plants were kept under insect-proof cages, and reciprocal oculations were made in most cases. The various species were usually moculated in stems or leaves with the expressed juices of mosaic cucumber plants, but many successful inoculations were made between other species, leaving no doubt that the disease is identical on the various cucurbit hosts. From 10 to 50 successful inoculations have been made with all the species noted below. In the case of cucumber, Cucumis sativus L., 15 varieties were inoculated successfully, and all seemed equally susceptible. Mosaic was also produced on the West Indian gherkin, C. anguria L., on a closely related variety, C. grossulariaeformis Hort., and on C. metul- liferus Mey. Inoculations on 11 varieties of muskmelon, Cucumis melo L., have all produced the disease, including the pomegranate melon, C. melo var. dudavm Naudin. Seven varieties of squash and four varieties of pumpkin, Cucurbita pepo L., C. moschata Duchesne, and C. maxvma Duchesne, have also been infected. cs Bis a0 lito , “ Fs is THE MOSAIC DISEASE OF CUCURBITS. 5 On the gourds infection occurred on 12 varieties, including Cuecur- bita pepo L., Luffa cylindrica Roehm, L. acutangula Roxbg., Lagenaria vulgaris Ser., and L. leucantha Rusby. Mosaic has also been produced by inoculation on the following species: Momordica wnvolucrata KE. Meyer, M. charantia L., Hcballiwm elaterium A. Rich., Benincasa hispida Cog., and Trichosanthes anguina LL. The wild species, Micrampelis lobata (Michx.) Greene, is common in many parts of the country both as an ornamental vine and grow- ing wild and is often diseased. The identity of this disease with that on the cucumber as well as with that which occurs on the less common wild species, Sicyos angulatus L., has been proved by repeated inoculation. — From data so far collected, the watermelon, citron, and other species of Citrullus seem partially resistant to the disease. _No successful inoculations have been made by the writer on any plants of this genus with the exception of the green-seeded citron, Citrullus vulgaris Schrad., and watermelons grown in the field have never been found infected although often intertwined with mosaic-diseased cucurbits. Jagger (19) reported having obtained a single case of mosaic on watermelon by inoculation, but was not able to repeat such infection. He also noted the disease on the green-seeded citron, but not on the red-seeded variety. Freiberg (13), at the Missouri Botanical Gardens, also reported mosaic on the citron and showed photographs of the disease. CROSS-INCCULATIONS TO CTHER PLANTS. INOCULATIONS FROM MOSAIC CUCUMBERS TO PLANTS OF OTHER FAMILIES. Certain field observations have at times suggested that cucurbit mosaic might possibly be transmitted to plants of other families. An example of such a case occurred at Madison, Wis., in 1916. A few plants of Martynia lowsiana Mill. appeared in one of the cucumber plats, the seed having been accidentally included with that of the cucumber. A large number of the cucumber plants became infected with mosaic early in the season, and a few of the martynia plants’ also developed a disease of the mosaic type later in the summer, the circumstances suggesting a possible transfer- ence of the infection from the cucumber. Inoculations were made from the mosaic martynia to healthy cucumber plants and also from mosaic cucumber to healthy martynia, but all the inoculations gave negative results. Most of the inoculations from cucumber to plants outside the Cucurbitacez, however, have been with plants known to be subject to infectious mosaic diseases, such as tomato, tobacco, bean, petunia, and pokeweed (Phytolacca decandra L.), although a few inoculations have been made on plants subject to unknown chlorotic diseases. 6 BULLETIN 879, U. S. DEPARTMENT OF AGRICULTURE, The methods used in this inoculation work were very similar to those used in the other cucumber-mosaic studies. Where field inoc- ulations were made, the plants were protected with insect-proof cages wherever possible, and in the greenhouse all plants used in such inoculations were kept isolated from other mosaic experiments. The inoculum was prepared in much the same way as in other cucumber-mosaic inoculations, either the expressed juice or crushed tissues of mosaic plants being used. Where the juice of a mosaic plant was employed, the parts of the plant used as inoculum were passed through a sterilized food chopper and the juice expressed and filtered through filter paper. Inoculations were made at from three to five points in each of the younger leaves, a drop of the expressed juice being pricked into the leaf with a sterile needle. The stem usually was inoculated at one or more points, a slight incision bemg made with a sterile scalpel carrying a drop of the inoculum. TaBLE I.—Record of inoculations from mosaic-diseased cucumber plants to plants of other families. Results. Number Paes Plant inoculated. Preparation ofinoculum. of MSts NiGReE ee Date last lated. | of mosaic see plants. ia i Ano AS19167 |e LObaeCO steer aan se aoe Bapressed juices sheave 3 0 | Aug. 17,1916 Maree losses 2 : GOR cee ease Ae ale eG. Os Sere eee aarreetee 4 0 | Mar. 26,1917 APES 27,1910) ~ 2: dOe S52 hte eee aes aa t le ope en sae eee 4 0 | May 28, 1917 May 281917 2% OLS soa re Se See ts ie CORE eee eee en eee 8 0 Do. May 20,1917 |...-- OES Oe Ae es Crushed leaf tissues... --- 20 0 | June 14, 1917 OGt ea O fae GOSH eh ote sar oe See oe O02 See eee eee 18 0 | Oct. 30, 1917 INOW D LOR. |ee-- - dOS22 eee Se eee Expressed juices. ---.---- 21 0 | Dec. 1, 1917 Ae 4 S191 Gel ROMATO=. cee ne eee meee ee 0 0 eee ee Sis 5 Fe 3 0 | Aug. 17,1916 Aug. 18,1916 |-.--- (6 C0 eae re ee aie race eee dO eee ease eee 6 0 | Aug. 31,1916 Keb. 231907 foo =2 GO Sees as sees Sosa aoe C9 10 EP per ne a ee 4 0 | Mar. 20,1917 Feb. 30,1917 |----- COn ea sor a nen Sees ea bearers CO (0 ee ie tn se 2 ee 6 0} May 2,1917 Apr. 5,1917 0 1 ER eee ar eT ea Crushed leaf tissues... --- 15 0} May 8,1917 Oct. 8,1917 (3 Co ae eeeuee es a ate oe |e Oe a O25 Se a eae 12 0 | Noy. 19,1917 Deer. 85 19175 |5o- 52 dOs=s55se 0 Se ee ee Hepes Bene eee 15 0} Jan. 12,1918 (AUS At OLE Pe Wiak Deal aces oaeerts 2 Sea = Scie OO seein ee nae ere 9 0 | Aug. 17, 1916 Dome Ibime Deane see ae eee AG So Beas Nee ee 6 0 Do. Feb. 15,1917 | Red kidney bean..........|--..- Gosia ace steer oe 15 0 | Mar. 10,1917 Mar. 24,1917 |..... COs yas see i sece eeeeoteeee Oe Soa ss aso eee ee 10 0 | Apr. 12,1917 Sept. 7,1916 | Martunia lowisiana.......-|-.-.- GO Bae Se ees 12 0 | Sept. 23,1916 ANTS; 09165) (eOLatO aap ee eae er | eee Oso a sesenns eee 8 0 Do. Aug. 16,1915 | Pokeweed (Phytolacca de- |..... GOs. 82 seeee aes ee 5 0 | Sept. 30,1915 candra.) Mar 2219163) sbetuniaes =- == oe Se eee Oe Feet eae 6 0 | Apr. 10,1916 ATIC ea OT | DA MUTOSIM ER2{LO Cees aa es | GO nee cases eee 12 0 | Sept. 1,1917 PAIS sof ON ee ae COIs S.. Sse eee SSeS GO Seeseks aoa eee 10 0 Do. Where the crushed tissues of the mosaic plant were used, a frag- ment of one of the young leaves was crushed in a sterile dish with sterile instruments and small portions of this crushed material in- serted in shght incisions made at one or two points in the stem of the plant inoculated and other pieces sometimes pricked into the -young leaves. The results of all the inoculations from mosaic cucumber to plants outside the cucurbits have been negative,! as shown by Table I. 1 Recent experiments (1919) indicate that cucumber mosaic may be transmitted to Martynia louisiana by means of aphids taken from mosaic cucumber plants. Inoculations from martynia plants infected in this manner have also produced the disease on the cucumber. c* IS THE MOSAIC DISEASE OF CUCURBITS. 7 Jagger (20), however, states that the white-pickle type of mosaic disease, which is the one studied by the writer, has been transmitted to one of the Lobeliaceze (Lobelia erinus L. var. gracilis) and to one of the Compositz (Helianthus debilis Nutt.). It is thus evident that in some cases the disease may pass from the cucumber to hosts out- side the Cucurbitacee. TasBLE II.—Record of inoculations of cucumber plants with infectious mosaic diseases of noncucurbitaceous plants and with suspected mosaic plants of other families. PLANTS HAVING Mosaic DISEASES KNOWN TO BE INFECTIOUS USED AS SOURCE OF INOCULUM. Renee Results. ete ad Plant used = ies eee of | Preparation of inoculum. oeuiaats Number] potelast lated. Onin observed. Aug. 25,1915 | Mosaic tomato.......---..- Expressed juices.....-.-.-- | 5 0 | Sept. 10,1915 Sept. 3,1915 |....- Oe eee a So Me oa = dO 2a See ee ee 9 0 | Sept. 20, 1915 ee PANE) a5 eee a ae Be SER OR tea aes ae ena Ee a oeeenetmaemeaeerss 3 0 ia 22,1916 e ASLGIGE | a ose Ones ad ae eee Pasa OR Ram eae Sercictsiocr osc 0 | Feb. 27,1916 Aug. 19,1916 |_.... Oma od ee cee ce GHihell leaf tissues-.--.- 6 0 | Sept. 5,1916 Sept. 7,1916 |..... GO sae ees eee Expressed juices-......-- 8 0 | Sept. 23, 1916 Heb: 18) 1917 |e: GOR a ee at Soe Gee Crushed leaf tissues. .--.- 8 0 | Mar. 3,1917 BRebse201Oifa| ta. (LO se ee ee eee ee Expressed juices.......-.- 12 0 | Mar. 21,1917 Decree 2191 a Oe et aa nd Oe Gees GOS aes ae ere ae 15 0| Jan. 16,1918 Feb. 15,1918 |....- Osea ie Se Crushed leaf tissues. .--.. 24 0 | Mar. 1,1918 Dees 21-1915) | Mosaic tobacco: --..+- =. . = Expressed juices.......-- 5 0 | Jan. 22,1916 Aug. 29,1916 |_.... Onno ees oe re ere ease GOES es ee eee: 6 0 | Sept. 23, 1916 Feb. 17,1917 |_.-... COGS S ERs en ees Se ee GO eee eee 6 0 | Mar. 18,1917 Mar. 30,1917 |....- LO ee a ee dont: Sees aes 18 0 | Apr. 20, 1917 May 20,1917 |..... (OK acpi Sear Not eee aoe ae ee! GOSS Ae ans 20 0 | June 14,1917 Nov. 10,1917 |..... Osea ese ase eee Crushed leaf tissues... -.- 35 0} Dec. 1,1917 Samia i OTS! GOs eer eee ee Expressed juices........- 25 0 | Jan. 30,1918 Sepie co Olas Mosaic beaten. 5s 2ee a5 |o sane GOs ae 9 0 | Sept. 20, 1915 Sept. 10,1915 |..... OSS Seperate ee cA Sess GOS Ree eee 10 0 Do. Silivct S916) ee = Ona eee se see eae’ GOSS nase | 5 0 | Aug. 17, 1916 Ane = 30-1916. S22 GOP Sere ee ee Seas Oe ee ete 4 0 | Sept. 23, 1916 Mar. 5,1918 |..... CLO Gane ee ae eeaeear sl See COS 5a ease es 18 0 | Mar. 23,1918 Sepissoe! los eMosaic PotatOse s-15-2 ee loon COSE aS ee ae 9 0 | Sept. 20,1915 Sept. 10,1915 |-.-..- Oa ee seo ee SRE Ones le aaee 10 0 | Sept. 23, 1916 ATI S23. 1916) |aece COPA Sees ees eee GO neers eee eee 8 0 Do. Sept. 3,1915 | Mosaic pokeweed (Phyto- |..-.- GOSS ae ee 9 0 | Sept. 20, 1915 lacca decandra). Sept. 10,1915 |..... Oe ssa eee ees Ses GO ee ee 10 0 Do. Aus 23; 1916: |5 =; SO) Sees oa aaa ne ae ee ee GOSS ee ee a ais 6 0 | Sept. 15,1916 Aug. 30,1916 |....- CLO ee a ee ae Crushed leaf tissues. .---- 4 0 | Sept. 23, 1916 Mar. 19,1916 | Mosaic petunia........__-- Texpressed juices.--....--- 5 0| Apr. 1,1916 AG ace 21 1916: | 2 ORs eile eee GORS eee ese 8 0 | Apr. 22,1916 Sept. 3,1915 “ond ae per ucexes eae 3 Oe Seas ee ese 3 0 | Sept. 20,1915 and wigs SKF TWO = TE SE Ss eat Vo See eee ee eee et eed be a GONE Eee ess 17 0 Do. July 20,1916 |..... aC Sos eae ee eee ee dOe=s esse Be ess 5 0 | Aug. 17,1916 Sept. 10,1915 fate peach leaves and |...-- COs Se sa ee a a 17 0 | Sept. 20,1915 wigs. Aug. 30,1916 |..... GOSS 5 5E eee eee | ae Ca (Oe ee Pee rane 8 0 | Sept. 23, 1916 PLANTS HAVING DISEASES OF THE MOSAIC TYPE OF SUSPECTED INFECTIOUS NATURE USED AS SOURCE OF INOCULUM. Sept. 10,1915 | Mosaic pele need Aucieris Expressed juices......... | 9 0 | Sept. 20,1915 syriaca). : iahy 20-1915 rs so MOAR ee eee eee ee | Se Og GOS Senet eee 5 0 | Aug. 17,1916 ANTS S251 916: | So: = OER pan et ees [ee Goes sree ee 7 0 | Sept. 23,1916 Sept. 10,1916 | Mosaic red clover ( Trifo- |--.-.-.- COs se esees a er 10 0 | Sept. 20,1916 lium pratense). July 20,1916 |-.... CLO Rei ae ee eases oak CO Sascee eee eee 5 0 | Aug. 17,1916 Sept. 7,1916 | Mosaicmartynia (Mariynia |..-.-.- GOS: eet aateret ane 8 0 | Sept. 23,1916 louisiana) Aug. 10,1917 |..... CO see es ania e Crushed leaf tissues... ... 6 0 | Sept. 5,1917 Sept. 8,1916 | Mosaic pepper (Capsicum Expressed juiices..-....-- 3 0 | Sept. 23,1917 1} annuum). Aug. 18,1917 Soe ragweed (Ambrosia |..--- GO atu Hoes sees 7 0 | Sept. 12,1917 trifida). Aug. 30,1917 |_...- Ossett oe ee Crushed leaf tissues... ... 8 0 | Sept. 24,1917 Aug. 30,1916 | Mosaic sumac (Rhus ty- | Expressed juices.......-- 8 0 | Sept. 23,1916 phina). ie) BULLETIN 879, U. S. DEPARTMENT OF AGRICULTURE. INOCULATIONS FROM MOSAIC PLANTS OF OTHER FAMILIES TO CUCUMBER. Attempts have also been made to transmit mosaic diseases found — on plants outside the Cucurbitacez to the cucumber. A considerable number of inoculations have been made from tomato, tobacco, bean, and other plants having infectious mosaic diseases and also from plants which showed diseases of the mosaic type but whose infectious nature was not proved, such as pepper (Capsicum annuum L.), sumac (Rhus typhina L.), milkweed (Asclepias syriaca L.), red clover (Trifolium pratense L.), and others. Inoculations in most cases were made with the expressed juice of the mosaic plant in the manner already described. All the results of these inoculations have been negative, as shown in Table II and there is as yet no evidence that any of the diseases on the hosts tried are identical with the mosaic disease on the cucurbits. MOSAIC SYMPTOMS. The progress of the mosaic disease in the plant is characterized by distinct morphologic and physiologic changes, which are most marked in the leaves and fruits. These consist principally of an abnormal mosaic coloring, accompanied by certain malformations and dwarfing of growth. The symptoms vary somewhat according to the age of the plant and its vigor of growth at the time of infection, but the general characters are the same in all stages of growth. There is also a variation in the symptoms on the different species of Cucurbitacez which are susceptible to the disease. The leaves of different species show many diverse forms of mosaic coloring, and the fruits may or may not be affected, although usually they show marked symptoms soon after the plant becomes diseased. The symptoms on the cu- cumber have been described in greater detail, as it is the most im- portant host and has been most closely studied, but the general characteristics wil] apply to all other hosts except for the variations mentioned later. PLANT SYMPTOMS. Sumptoms on affected seediings.—Seedling cucumbers are rarely affected with mosaic either in the field or in the greenhouse. The disease may appear on seedlings under certain conditions, however, as is shown by the fact that a very general and serious mosaic infec- tion occurred on the first true leaves of cucumber seedlings at Big Rapids, Mich., in the summer of 1918. The seed was planted late in August and when the plants came up the rest of the field was already rather badly infected with the disease. When such infection does occur, the first symptoms appear as a yellowing and wilting of the cotyledons, accompanied by a slight mottling of the young leaves. A sudden checking of growth soon follows and the later leaves remain small, wrinkled, and distorted and have a distinct Bul. 879, U. S. Dept. of Agriculture. PrATeaie MOSAIC ON YOUNG CUCUMBER PLANTS AND A BADLY DISEASED FiELD. A, A single mosaic plant obtained from seed from mosaic plants in a test at Big Rapids, Mich., 1917; B, a young mosaic cucumber plant, showing the curling and savoyed effect on the smaller leaves; Big Rapids, Mich., 1916; C,a cucumber field at Holland, Mich., on August 19, 1915, showing severe stunting and loss to the late-planted crop due to infection while the plants were small; the early planted portion in the background was not so badly affected. Photographed by W. W. Gilbert. Bul. 879, U. S. Dept. of Agriculture. PLATE II. MOSAIC CUCUMBER PLANTS. A, Mosaic cucumber plant infected while young, showing dwarfing and curling of the leaves and shorten- ing of the stems; Greeley, Colo., September, 1917. Photographed by W. W. Gilbert. B, Cucumber mosaic from inoculation with aphids from mosaic plants, the fruit symptoms being most marked; Big Rapids, Mich., 1915. Photographed by W. W. Gilbert. C, Mosaic plants in late stage, showing death of the basalleaves, whitening of the main stem, and the dwarfing and curling of the tip leaves. Photo- graphed by M. W. Gardner, September, 1916. THE MOSAIC DISEASE OF CUCURBITS. 9 mottling of yellowish green. Such plants seldom produce fruit and are usually short lived, rarely reaching a length of more than 12 inches. Symptoms on plants infected when young.—-The period of most general infection begins when the plants are about 6 weeks old and growing vigorously. At this time they have from six to eight leaves and are commencing torun. ‘The first symptoms in all cases appear in the young leaves which are still in the process of development and usually are not easily recognized by an untrained observer. The youngest of the leaves develop small greenish yellow areas, often not “more than a millimeter or two in diameter, occasionally circular, but more often limited in outline by the smailer veins of the leaf. These spots are slightly more translucent than the remainder of the leaf and are often scarcely visible except by transmitted light. Less commonly the normal green of the leaf changes to a peculiar yellow at the tip and the sharply defined yellow areas do not appear. Accompanying these symptoms there is a gradual downward curling of the edges of the leaf, and the surface presents a finely wrinkled appearance, the tissue between the small veins becoming shghtly raised so as to form minute convex surfaces (Pi. I, B.) Following these early symptoms, the gross mosaic characters develop rapidly, and the leaves become strikingly mottled with green and yellow. Such leaves are wrinkled and savoyed in appearance and may be somewhat distorted and curled (Pi Ts A): All growth subsequent to infection is mucn dwarfed, fhe stem internodes are shortened, the leaves attain only about one-half nor- mal size, and the aenelles are reduced in length. As the plant be- comes older the wrinkled and savoyed character of the leaf is more marked, but the mottling is generally less conspicuous. Plants infected at this stage blossom sparingly and set few fruits. They send out few runners and have a bunched and bushy habit of growth, with the leaves lying close to the ground in a rosettelike clump. Symptoms on plants infected when nearly mature.—In the case of large plants the general symptoms are similar to those described above. The first signs of the disease appear in the youngest leaves, where the yellowing and curling before noted are the usual indica- tions of infection, while all the older leaves appear normal and may remain so for some time. In some cases, however, the youngest leaves turn yellow and wilt, their edges becoming brown and withered, and all the leaves of a shoot may thus wilt within a few days. Ordi- narily, however, only three or four leaves at the growing point are affected in this way, and the runner soon sends out a new shoot just back of the withered tin, which shows the ordinary mosaic symptoms. Symptoms may also appear very early on the young fruits, and in the first stages of the disease they are often more marked than those which occur on the leaves (PI. If, B). Frequently the most pronounced 10 BULLETIN 879, U. S. DEPARTMENT OF AGRICULTURE. leaf symptoms appear on new secondary shoots put out from the axils of the leaves near the base of the plants. The wilt symptoms are often noted on the cucumber and pumpkin and occasionally on the Summer Crookneck squash and muskmelon. In the greenhouse this type of response is much more intense, and it is common to have the vine suddenly develop a yellowing and wilting, which extends to the entire plant within two to three days, the older leaves usually showing yellowing but wilting very rapidly. This wilting and death of the plant occur only occasionally in the field and never progress so rapidly there as they do in the greenhouse. Aside from these differences, the older plants in the field show the same symptoms as plants infected at an earlier stage in their devel- opment. The dwarfing and wrinkling of the younger leaves are very pronounced and the mottling is of the same type as that on younger plants. The shortening of the stem internodes and the petioles gives the runners a flattened appearance, and the leaves lie close to the ground. The older leaves of mature plants are later more severely affected, and the greater portion gradually die off. Portions of the leaf turn yellow, those at the base of the stem being affected first. This may occur as a gradual yellowing of the entire leaf, or more characteris- tically as a V-shaped yellowed area along one of the large lateral veins of one of the basallobes. The tissues of these yellowed portions rapidly turn brown and die, beginning at the edges in the angle formed by the lobes and working toward the base of the lateral veins. (PI. III.) The entire leaf eventually becomes yellow, this yellowing grad- ually extending to the leaves farther up the stem. Such leaves finally wither and die, leaving the basal portion of the stem bare. This is a characteristic symptom in late stages of the disease, and by the end of the season many plants show 1 to 3 or more feet of bare stem with the dead leaves stili attached, but terminating in a cluster of dwarfed, dark-green, mottled, and wrinkled leaves which le close to the ground. (PI. II, C) LEAF SYMPTOMS. The pattern of coloring which the leaves of mosaic plants assume varies with the species, the age of the plant at the time of infection, and the stage of development of the individual leaves. Leaf symptoms on cucumbers.—On the cucumber (Cucumis satiwus) the mosaic mottling is usually most pronounced and typical on the young leaves of plants which are growing rapidly (PI. IV, A). The green portions of these leaves are very dark in color and are raised slightly above the surrounding surface, thus giving the leaf its savoyed appearance. In most cases the green areas predominate, being intermingled with spots of light greenish yellow. These yellow spots are irregular in outline and are limited by the small veins (Pl. LV, B). t THE MOSAIC DISEASE OF CUCURBITS. 11 In some cases the green and yellow areas are both small and about equal in number (PI. III), being scattered irregularly over the leat and sometimes coalescing to form larger areas, with the green portions rather sharply defined, owing to their raised character. More often, _ however, there are a few isolated patches of yellow from 3 to 5 milli- , meters in diameter, the greater portion of the leaf being dark green with the typical savoyed character. On other leaves the yellowing may take the form of large blotches which tend to follow the large veins, the margins not being sharply defined but blending gradually into the green portions of the leaf. In such leaves the savoyed appearance is much less marked. In the case of older plants the wrinkling of the young leaves is usually very noticeable, but there is less contrast in color. The raised parts of the leaf are dark green, while the remainder is merely a lighter shade of green than that found in normal leaves, the borders y of these light spots not being sharply defined. In such plants the base of the leaf will occasionally develop a sharply contrasted mosaic mottling, while the tips of the leaves will remain normal in color for some time. In the older leaves the symptoms are somewhat different in nature. The savoyed character does not appear in many cases, and the yellow- ing tends to include the entire leaf in a short time. In leaves which are nearly full grown the yellowing very often extends along the net- work of the larger veins of one of the basal lobes. The portions of the leaf inclosed within these veins may remain green for some time and have a slightly convex appearance. A pronounced drooping or curling of the leaf often follows in the parts where such a mottling occurs, producing a peculiar wilted appearance in the lobe of the Aleaf affected. In other cases certain portions of the leaf turn yellow and the re- mainder retains its green color for a considerable time. The yellow portions in such leaves often include a few small, isolated, dark-green areas, producing an odd, checkered appearance in these portions of the leaf. This is quite distinct from the ordinary mosaic mottling in that the green tissues show no tendency to be elevated above the surrounding leaf surface. This type of yellowing occurs most com- monly in the angle formed by the junction of the large veins at the base of the leaf, but also appears at the tips of the lobes. In such _. leaves the green portions retain their color for some time. One of * the most common symptoms in the older leaves of mature plants commences as a yellowing which appears along the large lateral veins of one of the basal lobes, extending their entire length. These yellow areas are widest at the edge of the leaf and narrow toward the juncture of the veins, producing V-shaped areas which have already been described (PI. III). 103) BULLETIN 879, U. S. DEPARTMENT OF AGRICULTURE. Another symptom on the older leaves which is more common in the greenhouse than in the field, consists of a yellowing which affects nearly all the leaf at the same time. In such leaves the tissues adjoining the larger veins retain their color longest and outline these veins against the yellow of the remainder of the leaf. These symp- es toms are usually associated with the wilting type of the disease. Leaf symptoms on muskmelons.—The leaf symptoms on the musk- melon (Cucumis melo) are very similar to those on the cucumber. The first signs of the disease appear on the youngest leaves, which turn light yellow in color and develop a sharp downward curl. Later, all the younger leaves of the plant show typical mosaic characters (Pl. V, B). The light yellow portions of the leaf are of irregular outline and about the same size and color as those on the young leaves of the cucumber. The dark areas are more definitely outlined on the muskmelon, and the leaves show a very pronounced curling. The characteristic-symptoms found on the older leaves of the cucum- ¢ ber are much less common on the muskmelon. The older leaves gradually become yellow, but show little or none of the tendency to die early which appears in mosaic cucumber leaves of the same age. Leaf sympioms on squashes.—In the case of the squash (Cucurbita spp.) the symptoms of the disease are much the same on ail varieties. The younger leaves usually develop an extremely savoyed appear- ance, the darker parts of the leaf being much more definitely raised above the leaf surface than in the case of the cucumber. The light spots on the leaf are pale yellowish green and tend to coalesce and form a few large blotches rather than the smaller and more numerous spots found on the cucumber (PI. VI, A). The older leaves often become yellow and gradually die off, as in the case of the cucumber, _ but in many plants these leaves will retain their normal color forgs some time, the symptoms of the disease being confined to the younger leaves. The most marked symptoms have been found on the Summer Crookneck and Cocozelle bush varieties (Cucurbita pepo var. condensa), those of the Hubbard type (Cucurbita maxima) showing the disease in a less extreme form. Leaf symptoms on pumpkins.—On the pumpkin (Cucurbita pepo) the leaf symptoms are very nearly identical with those found on the squash. The younger leaves develop with the same extreme mottled and wrinkled appearance, the general color of the leaf being a lighter yellow than that of a normal plant. On the pumpkin, however, the older leaves very often show a rapid yellowing and wilting which corre- he sponds very closely to that found on the cucumber in the greenhouse. Leaf symptoms on other cucurbits—The general symptoms on the gourds and other ornamental cucurbits are very similar to those on the squash. The chief points of difference occur in the patterns of the mottled leaves. In the case of the Cucurbita gourds the surface Bul. 879, U. S. Dept. of Agriculture. PLATE Ill. AHoen& Cn Baltimare_ MOSAIC LEAVES, FRUITS, AND BRANCH OF CUCUMBER. Three fruits illustrating common types of mosaic effect; two leaves, the smaller showing pro- nounced mottling, common to young leaves, the larger showing dying of older leaves on plants infected when nearly mature; tip of plant with dwarfed and dying leaves. Bul. 879, U. S. Dept. of Agriculture. PLATE IV. MOSAIC CUCUMBER LEAVES. A, Small cucumber leaf, showing extreme mottling and wrinkling characteristic of a severe mosaic attack; Madison, Wis., 1919. B, Cucumber leaf, showing simple mottling often seen in the earlier stages of mosaic attack; Big Rapids, Mich., 1915. Photographed by W. W. Gilbert. THE MOSAIC DISEASE OF CUCURBITS. LS of the leaf is finely wrinkled and the yellow areas are very small but present in large numbers, so that the leaf appears to be covered with fine dots of yellow, which in some cases bear a close resemblance to insect injury. This type of mosaic symptoms is also common on the _ Inffa and Lagenaria gourds, the spots in the latter species being > larger and very nearly circular in form. (PI. V, A.) On the wild cucumber (Micrampelis lobata) there is usually a marked variegation and deformity in the leaves of mosaic plants. In most cases the symptoms are similar to those on the cucumber with the exception that the lighter colored portions of the leaf pre- dominate. These are a light yellowish green with an occasional almost colorless area which is in sharp contrast to the surrounding tissue. The other parts of the leaf are a deep green and are raised above the surrounding surface. As a result the savoyed character is usually pronounced, and the leaves are deeply wrinkled and curled. » There is also considerable dwarfing and deformity in mosaic leaves, the lobes being irregular in size and outline. In some leaves, how- ever, the difference in color in the various parts of the leaf is less marked, and in such cases the savoyed character does not appear, although the leaf may be distorted in shape. (See Pl. VII, A, B, and C, for comparison.) While the above types of symptoms occur most commonly, there are often cases in which the greater part of the leaf remains almost a normal green but develops numerous small circular areas of light yellow which are similar to those noted on the Cucurbita gourds. These yellow spots are about 1 millimeter in diameter and are slightly depressed. In other leaves we find a number of small, dark-green areas scattered over the surface, the center of each of them being ‘weomposed of a small yellow spot similar to those above described. ‘The green portion of these areas is raised, while the yellow centers are slightly sunken, producing a peculiar pitted appearance. The older leaves of mosaic plants gradually turn yellow, some of them showing the V-shaped yellow patches which occur on the older leaves of the cucumber. This yellowing of the leaves eventually affects most of the plant and is a distinguishing character of the later stages of the disease. The symptoms on the othér wild host, Sicyos angulatus, are much like those on micrampelis. The mottling of the young leaves is seldom extreme, but the older leaves tend to yellow and die off rapidly, *the dead leaves remaining attached to the stem as in the case of the cucumber. FRUIT SYMPTOMS. Fruit sympioms on cucumbers.—The symptoms on the fruits of the cucumber are extremely marked in most cases and when present make the disease easily recognized. The stem end of the young 14 BULLETIN 879, U. S. DEPARTMENT OF AGRICULTURE. fruit first becomes mottled with yellowish green, this mottled char- acter gradually spreading over the entire fruit. As this progresses the body of the fruit ordinarily becomes a light yellowish green, inter- mingled with spots of a much darker green color. These dark por- tions are usually raised above the surrounding surface in such a way ~ that they form wartlike projections and often produce more or less distortion of the fruit. The number and size of these protuberances vary greatly, and we find many types of affected fruits (PI. VIII). In some cases, particu- jarly in the greenhouse, the mottled character is very marked, the fruit being covered with mingled blotches of light and dark green, but witb little or no wart production or distortion of shape. Such fruits are common in the field, as also those in which the stem end is the only part to show a well-defined mottling. More commonly, however, the dark-green portions of the surface are distinctly raised, the projections varying from 1 millimeter up to 2 centimeters in diameter and from 1 to 6 millimeters in height. The general appearance of all such fruits is the same, but the minor char- acters are subject to great variation. In some cases the warts are small, sharply outlined, isolated, and scattered over the surface in varying numbers (PI. VIII, A to C). On other fruits they are larger, coalesce to some extent, and form irregular raised patches, which are sometimes slightly elevated and do not greatly affect the shape of the fruit, while in still other specimens they are so large as to produce an extremely rough and irregular form (PI. VIII, D, E). In some cases there may be one or two large warts, often at the stem end, the remainder of the fruit being yellowish white or mottled with yellowish green, but nearly as smooth as normal. In general, however, the fruits are decidedly mottled in appearance, « and show swellings of all sizes, some isolated and others merging” into one another in such a way as to produce a very irregular form, the symptoms often being well defined on fruits which are but an inch in length. The fruits of this type have given rise to the names “wart disease” and ‘‘nubbin,”’ which have been applied to the disease by growers in some parts of the country. | In the later stages of the disease the vines occasionally produce - fruits which are smooth, pale whitish green in color, and rather more blunt at the ends than normal fruits of the same age. In most cases these fruits are mottled with fine spots of yellowish green, and a few dark-green projections appear here and there on the surface. (PL. é VIII.) These are usually small, but occasionally fruits are found © which have a single large dark-green swelling near the stem end, producing a most unusual appearance. These white fruits are responsible for the older common name of the disease, ‘‘ white pickle,’ which was the term applied to the trouble ¥ THE MOSAIC DISEASE OF CUCURBBITS. 15 by the growers in Michigan and Wisconsin for some years. The more common green and distorted fruits were classed with the imper- fect types which are often produced under unfavorable soil or climatic conditions, as the name “‘nubbin”’ in use in Minnesota indicates. All these names, however, neglect the mottled character of both leaves and fruits, which is the symptom most typical of the disease. Since the term mosaic had already been applied to a disease of _tobacco and other plants in which the symptoms and general char- acter were much like those on the cucumber, it seemed best to adopt the same name in this case as being more descriptive of the real nature of the disease than those already in use by the growers. On vines which are old and stunted another type of fruit often occurs which is abnormally dark green, with little or no mottling. Such fruits have a smooth surface, but are much distorted and swollen In appearance and are found only at the end of the season on vines which have nearly ceased to set fruit. Vines occasionally appear to recover from the effects of the disease and toward the end of the season produce fruits with no visible mosaic symptoms. Many mosaic fruits show a tendency to become russeted while still green, and in the case of the whitish fruits before described this tendency is very marked. As they turn yellow and ripen, the fruits which show less marked mosaic characters may be almost. indis- tinguishable from those of healthy plants, but in general they retain a misshapen and dwarfed appearance. Seed is usually produced, but in the case of fruits set during the later stages of the disease, the seed is slow to mature and often small and shrunken. Mosaic fruits may also have a slightly bitter flavor, particularly those from plants which have had the disease for some time, but this is not a common Symptom. Fruit symptoms on other cucurbits.—Although the leaves of all the cucurbits susceptible to mosaic show symptoms which have the same general character, the fruits in some cases show no evidence of the disease aside from a reduction in size. In general, however, the fruits of other mosaic cucurbits have the same mottled and warty appearance that is found on those of the cucumber, although these symptoms are often less marked or almost obscured as the fruit reaches maturity. The young fruits of mosaic muskmelon plants are often mottled, and a few dark-green warts may appear, but as the fruits mature the symptoms of the disease become less pronounced and the fruits are nearly normal in appearance. In the case of the Hubbard squash the young fruits of mosaic plants are sometimes mottled and lighter in color than those of healthy plants, but these symptoms are apparently obscured at maturity by the natural wartiness of the fruit. On the Summer Crookneck squash, however, the fruits are 16 BULLETIN 879, U. S. DEPARTMENT OF AGRICULTURE. distinctly mottled, and the warty character is even more pronounced than on the cucumber. The symptoms differ from those on most cucurbits, however, in that the raised portions of the fruit are lighter in color than the surrounding surface. The contrast is often very pronounced, the warts being bright orange-yellow and the remainder a dark green (PI. VI, B, C). The fruits of the Large Cheese pumpkin (Cucurbita moschata) show very marked mosaic symptoms, but these symptoms have not as yet: been noted on any other variety of pumpkin susceptible to the disease. The young fruits are distinctly mottled with light yellow, and the surface 1s covered with large dark-green warts. (PI.V,C.) Atmatur- ity the fruits are irregular in form, deeply furrowed, and the warty character is very pronounced. The fruits of most of the squashes, gourds, and the ornamental cu- curbits seem to be little affected by the disease, and on many of these hosts no symptoms have as yet been noted. The fruits of the mo- saic wild cucumber (Jfcrampelis lobata), however, are usually dwarfed, distorted in shape, and smoother than the fruits of normal plants, the spines being fewer in number and scattered unevenly over the surface. In a few cases fruits have been noted which devel- oped rather large irregular swellings. The epidermis of the fruit eventually splits open at these points and the tissues below push through to form dark-green swollen areas, which are somewhat similar to those found on the cucumber. (See Pl. VII, D and F) These symptoms apparently do not occur on all the mosaic fruits of this host, but they have been noted on a number of plants. The fruits of the 1-seeded bur cucumber (Sicyos angulatus), on the other hand, do not seem to show any evidence of the disease. BLOSSOM SYMPTOMS. Mosaic cucumber flowers are not streaked or variegated as are those of tobacco plants affected with mosaic. They are greatly reduced in number, however, especially the pistillate flowers. Blossoms pro- duced in the later stages of the disease are dwarfed, the corolla often measuring not over three-fourths of an inch in diameter, and are slightly paler than normal blossoms. On other cucurbits the symp- toms are of the same character, though the dwarfing 1s usually less pronounced except in the case of the muskmelon, the blossoms of which are usually reduced in size and number and are a much lighter yellow than those of normal plants. STEM SYMPTOMS. The stems in the case of the cucumber are shortened, as noted above (Pl. II, A), and where the older leaves have died the epidermis Bul. 879 U.S. Dept. of Agriculture. PLATE V. ; MOosAic MELON AND GOURD LEAVES AND A PUMPKIN. A, Mosaic leaf of a Lagenaria gourd, inoculated from cucumber; Madison, Wis., 1917. B, Mosaic leaf of a cantaloupe, naturalinfection; Big Rapids, Mich., 1917. Photographed by W. W. Gilbert. C, Mosaic fruit of pumpkin, showing large dark-green swellings on a yellow background; Madison, Wis., 1919. Bul. 879, U. S. Dept. of Agriculture. PLATE VI. LEAF AND FRUITS OF MOSAIC SUMMER CROOKNECK SQUASH. A, Leaf of mosaic Summer Crookneck squash, Madison, Wis., 1917. B and C, Young fruits of mosaic Summer Crookneck squash exhibiting pronounced symptoms of the disease; B, from Irondequoit, N. Y. (photographed by W. W. Gilbert), and C,from Madison, Wis., 1917. Bul. 879, U. S. Dept. of Agriculture. PLATE VII. Mosaic WILD CUCUMBER LEAVES AND FRUITS. A, B,and E, Mosaic leaves of Micrampelis lobata, showing marked mottling, malformation, and wrinkling. Compare with C. C, Healthy normal leaf of Micrampelis lobata, for comparison with mosaic leaves. D and F, Fruits of Micrampelis lobata from a mosaic plant, showing the irregular shape and the wartlike protuberances combined with the splitting of the outer integument; Madison, Wis. Bul. 879, U. S. Dept. of Agriculture. MOSAIC CUCUMBER FRUITS. A, Small cucumber showing typical ‘‘white-pickle’’ symptoms, the color being yellowish white with a few smallraisedislands of normal green. Band C, Fruits with green wartlike protuberances larger than in A and morenumerous. D and £, Half-grown fruits with several large dark-green warts and numer- ous small ones, the rest of the fruit being yellow to greenish white. (A to D, photographed at Madison, Wis., and £, at Washington, D. C.) ee THE MOSAIC DISEASE OF CUCURBITS. 17 becomes whitish gray in appearance (Pl. Hi, C.). This leafless and whitened condition of the basal parts of the stem is one of the most easily recognized symptoms in the late stages of the disease on the cucumber. Such stems seem more brittle than those of normal plants and break more easily. The whitening is due apparently to the loss of leaves and resultant exposure to the weather, since it occurs on healthy plants if the lower leaves are removed. The cu- cumber stem is not mottled, but it often develops a yellowish green color, somewhat lighter than the dark green of normal stems. Some cucurbits, particularly the squash and pumpkin, occasionally show a slightly mottled appearance of the stem in addition to the shortening of the internodes found on all cucurbits. This mottling consists of pale-green circular patches, about a centimeter in diameter, which occasionally have a slightly sunken appearance. ROOT SYMPTOMS. The roots of mosaic cucumber plants show no external evidence of the disease except in its later stages, when the number of finer rootlets is much reduced and the larger roots tend to show a faint yellow color accompanied by an unusual brittleness. Other cucur- bits examined have shown no differences between the roots of healthy and diseased plants except for a reduction in the number of finer rootlets. PATHOLOGICAL ANATOMY. ROOTS AND STEMS. Neither the roots nor stems of mosaic cucurbits, so far as examined, show any internal symptoms of the disease. There are no lesions, and sections of these parts have the same appearance as those of normal root and stem tissues. LEAVES. The leaves of mosaic cucurbits exhibit many variations in their external symptoms, the size of the yellow areas varying with different hosts, but the internal symptoms are essentially the same in all cases. The dark-green portions are slightly thicker than the normal leaf tissue, and this fact accounts for the blistered and distorted appearance which is very marked in the leaves of cucumbers and squashes. The intervening yellow areas are somewhat thinner than the green portions, but are of nearly the same thickness as the tissue of normal leaves. When stained sections of the leaf are examined, the palisade cells of the green areas are found to be crowded closely together and to be somewhat narrower and longer than those of the normal leaf. The palisade cells of the yellowed portion, on the other hand, are more 185118°—20——2 18 BULLETIN 879, U. S. DEPARTMENT OF AGRICULTURE. nearly isodiametric and less in number per unit area than in the normal leaf. The spongy parenchyma of these yellowed areas is also more compact and the intercellular spaces smaller than in the green portions. The chloroplasts of the cells of the yellowed portions are decidedly smaller than in normal cells and are often pressed so closely to the walls of the cell as to be almost invisible. In contrast to this the chloroplasts in the dark-green areas are so large and numerous as to seem to fill the cells. The vascular elements of mosaic leaves show little variation from the normal. FRUITS. On the fruits, the dark-green warty growths and the abnormally light yellowish green of the surrounding surface are the chief external symptoms. Here, the cells directly beneath the epidermis assume the same abnormalities that occur in the palisade cells of the dis- eased leaves. In the raised green areas, these cells are slightly longer and narrower than in normal fruits, and the chloroplasts are larger and crowded closely together. In the yellow portions of the - diseased fruits these subepidermal cells are more nearly square in longitudinal section and their chloroplasts are much reduced in size. The other tissues of the fruit, including the vascular elements, appear to be normal, the raised character of the green tissues being evidently a result of hyperplasia rather than hypertrophy. CAUSE OF THE DISEASE. The cause of cucurbit mosaic, like that of all other infectious chlorotic diseases, is unknown. While the infectious character of the trouble is readily proved, there is no definite indication of the nature of the infective principle or its origin. RELATION TO SOIL AND WEATHER CONDITIONS. Extended observations on the mosaic of cucumbers in the field have shown that the physical or chemical nature of the soil appar- ently has no relation to the origin of the disease. Mosaic will de- velop with equal rapidity on clay, muck, or sandy soils, and applica- tions of lime, wood ashes, stable manure, or commercial fertilizers have little appreciable effect. It has also been impossible to con- nect environmental conditions with the disease in any way which indicates that it originates from any unfavorable condition of soil, _ temperature, or humidity. 1 Recent investigations indicate that environmental conditions, particularly temperature, may affect the susceptibility of the plant to mosaic, and it is therefore probable that the rapidity with which the disease spreads is partially determined by the conditions of environment. Sacened tar QAR epee A Ss \ THE MOSAIC DISEASE OF CUCURBITS. 19 NO VISIBLE CAUSAL ORGANISM DISCOVERED. CULTURAL STUDIES. No parasitic organism has been found to be constantly associated with mosaic plants, although many cultures have been made from roots, stems, leaves, and fruits. In attempting to isolate a causal organism a great variety of media of all types, both vegetable and synthetic, were employed, particular use being made of those con- taining the juices of the cucumber plant itself. Tissue cultures from all parts of the plant remained sterile in most cases, and where growth occurred it resulted only from contaminations, no single organism being constantly present. Variations in temperature and oxygen supply and in acidity of the media were also tried, but these experiments were equally unsuccessful. The possibility of an increase of the virus in the culture medium without visible growth has been tested in many cases by inoculation fromthe medium itself, but no infection has ever appeared. The juices of mosaic plants may produce infection after passing through a Berkefeld filter, and attempts were therefore made to make cul- tures from these filtrates on various media, but without success. Similar filtrates of the juices of healthy plants when inoculated from infectious filtrates have never shown evidence that the virus could increase on such a medium. All attempts to cultivate the virus from the filtered juice of mosaic plants are also complicated by the fact that the juice loses its infectious character within two to three days after it is expressed from the tissues. ABSENCE OF ORGANISM IN DISEASED TISSUES. The absence of a visible causal organism is further supported by the fact that stained sections made from all parts of mosaic plants have given no indication of the presence of any parasite in the tissues. No differences have been detected between the stems and roots of healthy plants and those affected with the mosaic disease. The leaves and fruits show some morphologic differences, but no lesions of any sort are present and no visible organisms have been found in either the fruit or leaves of mosaic plants. All data so far collected, therefore, indicate that the disease is highly infectious, but no causal factor can as yet be associated with infection. On the other hand, the expressed juices of mosaic plants possess definite properties related to their power of infection. NATURE AND PROPERTIES OF THE MOSAIC VIRUS. The study of the nature of the infective principle of mosaic dis- eases has been most extensive in the case of tobacco, many workers having contributed to a knowledge of the properties and character of the virus concerned. While the writer’s experiments with the 20 BULLETIN 879, U. S. DEPARTMENT OF AGRICULTURE. virus of the mosaic of cucurbits have been less extensive, they indi- cate that it is similar in many respects to that of tobacco and other plants. The power of infection may be destroyed in each case and is definitely connected with temperature, moisture, age, and other factors. | EFFECT OF HEAT ON THE POWER OF INFECTION. The expressed juices of mosaic cucumber plants lose their power of infection when heated above 70° C. This temperature has proved the limit in all experiments, as is shown in Table IIT. In thase tests the plant juices were expressed and filtered through filter paper before use. Small test tubes were used and 5 c. c. portions of the juice taken to insure rapid heating to the desired temperature. A constant-tempecature water bath was used and the tubes heated 10 minutes and then unmediately cooled to room temperature. Inocu- lations were made from the different tubes to healthy cucumber plants, inoculating into the stem, at the tip and buse, and also into two of the young leaves. These piants were protected from possible outside infection by covering them with insect-proof cages. TaBLe II1.—Lffect of heat on power of infection of expressed juices of mosaic cucumber plants. lts. Number Results ‘ ° of plants Date. Temperature (°C.). amGone pues Darciect lated. plants, observed. Field tests: SED ES ral OIG Hier eal certie pon aus = ra Reape anyale 45 3 2 | Sept. 18,1916 UT) eas aN aso la biatity eI ise ag ct ae 50 3 2 Do. Ti) Qs ee ra aie piney ea CUR CI ME rs en oe 55 3 3 Do. ELD) sce ees tenn AAs Bee ee ride Milne die 60 3 0 Do. TD Yoo se ee Res eM Die lade SMa tly, WaeeS Ab Ba 65 3 2 Do. I DY oye se thee ate nee cna panes cae toa Sich enh 70 3 2 Do. I) a aie ete MGA Rei lesen, aig och aa 75 3 0 Do. NB) epee ain hate enna Oa eter age 80 3 0 Do. DY ayes cy aad See Me ara ga hit Mpa ae ea St AS Unheated. 3 3 Do. ISHS COVA TCT Mabie le ed SUELO ee ey ary 60 4 3 | Aug. 21,1917 TD YG Sedaris Stee na toca aad ry mam AL aoe tLe lar 65 4 2 Do. NBD Yo OMAN taal Se ola ate cer aL AISLE hy 70 4 4 Do. HED) QO ee BR Nas aU eal a a Ee eae 75 4 0 Do. DON Oe PEERS Gone ih MUR ares ah stan 80 4 0 Do. AD YoVe ssh esha Stall pa vast Me ch yin ee A Ser 8 85 4 0 Do. dD Yoysestie ia Wa Sehy aN, ak SEs: la et hea 90 4 0 Do. Doe OW cree Nene IKER UNCON MRN TL GUAlsy A ac ehe 95 4 0 Do. OEE Ue PGE cE LST ae Cn i ed Unheated. 4 4 Do. Greenhouse tests: . OCERS OO Le eee eR ear ay Mer SN 55 6 4 | Nov. 10,1917 TTS) eae ae Lee a a pre ern nda esa 60 6 5 Do. TD) OSES 0 ones NL Cae edge ela ae 65 6 2 Do. 1 DX oy pee Aa ENGI caine UU Gol Cae ac aN He 70 6 3 Do. A DY G SERA Re SU aA TRAN CRC SBS Me ce east) 75 6 0 Do. AD Yo yetniy ees date ca tall aaa a A ps A TA Se my 80 6 0 Do. Dy Aaa Maoh gaye, MCN prey Ae caveat ae oe Smee 85 6 0 Do. TD) eh SOU SB cA apis ee Re ae 90 6 0 Do. TB Yo ects av eh Pa PEIN NRI ful MN eR at Gk A Ht Unheated. 6 4 Do. EFFECT OF AGE ON THE POWER OF INFECTION. The expressed juices of mosaic cucumber and other cucurbitaceous plants retain the power of infection for only a short period after their extraction. Repeated tests have shown that the juices of mosaic plants of cucumber, squash, pumpkin, muskmeion, and Micrampelis lobaia are never infectious for more than three to five days and in most cases lose their virulence within 24 to 48 hours. This is in sharp contrast with results secured by Allard (4) with the juices of mosaic 4 s% THE MOSAIC DISEASE OF CUCURBITS. Dat tobacco plants, which often remain infectious for more than a year, even without the addition of preservatives. In the case of cucurbit mosaic the juices undergo rapid fermentation unless a preservative is added, but various chemicals, such as chloroform, ether, toluene, and glycerine, used at different strengths have never served to * increase the time during which the juices remained infectious. Low temperatures have only a slight effect in prolonging the power of infection, and filtrates from Berkefeld filters in which no visible bacterial growth occurs are equally short lived in this regard. The preservatives used with the unfiltered juices were also tried with Berkefeld filtrates, but neither chemical preservatives nor low temperatures have any noticeable effect in prolonging their power of infection. EFFECT OF DRYING ON THE VIRUS. As the juices of mosaic plants rapidly lose their power of infection with age, it would be expected that drying would also remove this infective character. This has been the case with the tissues of mo- saic cucumber plants. The leaves of mosaic plants when dried at room temperature for periods of 10 days to 1 year have always failed to produce the disease. In these experiments the dried leaf tissues were pulverized and allowed to stand from 8 to 24 hours in a small quantity of sterile distilled water. The water extract was then filtered off and inoculations made both by the injection of the water extract into the stem and leaves of healthy plants and also by the insertion of the ground and moistened leaf tissue in wounds in the stems of healthy cucumber plants. A total of 49 inoculations have been made by these methods, but no infection has ever occurred. In addition to inoculations with dried leaf tissue, the dried stems, small fruits, and roots of mosaic plants were used in the same way as inocula in 88 inoculations, but always with negative results. The expressed juices of mosaic plants also lose their power of infection on drying. Experiments were made in which these juices were dried either on glass or on filter paper at room temperature. At intervals of seven days the dried material was taken up in sterile distilled water and the water extract pricked into the stems and leaves of healthy cucumber plants. Ten plants were inoculated from the dried material on glass and also from that on filter paper at the end of every seven days, the experiment being continued for five weeks, but no infection resulted from anv of the inoculations. The result of experiments on soil transmission of the disease (p. 48) as well as the above data all indicate the rapid loss of the infective principle in the dried tissues of mosaic plants. EFFECT OF DILUTION ON THE VIRUS. The power of the virus of cucurbit mosaic to increase rapidly after its injection into the host tissue has been indicated by the rapidity of its distribution through the tissues of the plant inocu- lated. Further evidence of this rapid increase appears in the results 22 BULLETIN 879, U. S. DEPARTMENT OF AGRICULTURE. of inoculations with diluted juices of mosaic cucumber plants. Allard (2) showed that the virus of tobacco mosaic could be diluted to 1:1,000 without reducing its virulence. He also made a number of successful inoculations with the dilutions of 1:10,000, although higher dilutions rarely gave infection. The work of the writer with cucumber mosaic has given similar results, as is shown in Table IV. The juices of mosaic plants were expressed, filtered through filter paper, diluted with sterile distilled water up to 1:100,000, and used for inoculating healthy plants. These experiments show that dilu- tions of 1:1,000 are as potent as undiluted solutions, but while infections may result from those of 1:10,000 they have never taken place at higher dilutions. In all of this work inoculations were made at the base of the stem and in two or three of the younger leaves, the latter being inoculated at two or three points by pricking a drop of the solution into the leaf. Stems were cut slightly and the cut surface was covered with a drop of the diluted virus. TasLe 1V.—E fect of dilution with sterile distilled water on the power of infection of the expressed juices of mosaic cucumber plants. | | Results. eee Date inoculated. Dilution of expressed juices. | Of Plants | Number . : | eet | otros era a saic (dat plants. e). | Series I: APES 1916 ser sise oe he eee Vudituted Een a Seen | 5 3} Apr. 20,1916 IDO VR Rescate se tess se ee Sees SL OO. oer Sea Ge ia say te 5 3 Do. TD) OE aes te see ea ae He eee 11,060 Brie Ue aes eee eee emes 5 3 Do. ID 0 yas — enya eee re See F10:000 223. S343 ee 5 2 Do. Osa See riare Sat a a epee cere ernie te 100, O00t Ree SE eee 5 0 Series IT: S121 1) Pataki CD Ree Aaa ge Ameen Uniilated Bade nae eames 4 3 | Sept. 18,1916 DD OS Se Se eee SS ae Sane ee | tek OO Re reer es eter a ee 4 3 Do. LB Xo ae as a et EO ROS eel ca 11,000 a Na care SIS ena te 4 2 Do. ED) ee RS Fe SE errr re cee 110: 000.23 tee Seno ee sas 4 0 UDO Rea Be ae cee nee eee Saas lig 100, O00: 2c Seed seen 4 0 Series III: May 15 AGI eee Sea eek eee Undinied Speen aben enon sac | 8 5 | May_ 23,1916. DOS ue sees es secon oe naan ae lh L LOO Ree eee ae ee eee 8 6 Do. Tee He een Pahiry Bae Pe {eRog as: oo en ce | 8 7a ee TL) Q ces ee a eee eee 1210: 0003- essen ese 8 2 Do. DD Opes cube ats Cee Seok eine rae 1:100;000:) Lo eeata eae | 8 0 The regularity of infection which follows inoculation with dilutions of 1:1,000, together with the numerous infections at 1:10,000, shows an apparent power of rapid increase in the infectious material present in mosaic plants, since in these cases the period of incubation was no longer than when the undiluted juices were used. EFFECT OF VARIOUS CHEMICALS ON THE VIRUS. Experiments with the effect of various chemicals on the virus of cucurbit mosaic show that the power of infection is easily destroyed by common disinfectants-and antiseptics. In this work the juices of mosaic cucumber plants were expressed and filtered through filter paper. The extract was put into test tubes in 5 ¢. c. portions and Tee Se SO ee 7 aa ee “i THE MOSAIC DISEASE OF CUCURBITS. 23 the desired chemical added in such amount and strength that when combined with 5 c. c. of the plant juices it gave the desired concen- tration. The tubes were then shaken well and allowed to stand for 12 hours. Inoculations were made with these inocula, as in the case of the dilution experiments, into both the stems and leaves of healthy plants. The results shown in Table V indicate that formaldehyde, phenol, mercuric chlorid, and copper sulphate in dilute concentra- tions were all toxic to the mosaic virus. The tests with copper sulphate gave definite mosaic infection in one case, but the writer is of the opinion that this is not an indica- tion that the juices are resistant to its action, since the same and weaker dilutions have destroyed the virus in all the other tests. The results in this single case, however, were apparently not due to out- side infection, as the plants were caged from the beginning of the season. Chloroform in a 10 per cent mixture destroys the activity of the virus, but a 5 per cent chloroform mixture seems to be harmless. Toluene in a 10 per cent mixture has no apparent effect. In the case of these chemicals special care was used to mix the solu- tion thoroughly, and the tubes were kept tightly corked until inocu- lations were made. The 10 per cent solution represents a large excess and the 5 per cent a slight excess beyond the possibilities of absorp- tion by the plant juices. The results of these tests have been consistent in most respects, however, as is shown in Table V, and indicate that the virus is not resistant to ordinary disinfectants. TasLE V.—Lffect of chemicals on the power of infection of the expressed juices of mosaic cucumber plants. Results. Number Date inoculated. Chemical and strength used. cops NGDCE Naas lated. | of mosaic éervad plants. Sas Series I (field experiments): : ATU OSLO SEO Iifeee ee Formaldehyde 1 per cent............---- 4 0 | Aug. 20, 1917. DD Ostne ee ee: Zen OB PEMCeM tne sa sets sy oney eee 4 0 Do. I) OS ee CuSO sl pericentee eee eee 4 3 Do. DOMES = tae = TEC IS esas os oeneed ee eer soBedos 4 0 Do. ID) Obes oe ors ChioroformilOiper centas ee eee 4 0 Do. DO er MolwenotOpercentia= seen eee 4 3 Do. WD) OR ose ane Winimeateds) ui cera, snes ae 4 4 Do. DOS ee Distiledse.O (Control) pe ens ee 4 0 Do. Series II (greenhouse ex- periments): OC 3 IGN fe ee A Formaldehyde 1 per cent..........-.-.-- 5 0 | Oct. 31,1917 IDOE eae ee ee Iphenolelt percent ssa ee 5 0 Do. DO sees sas. as CuS Oper ceniteurese eyes eee 5 0 Do. DOES Hes ee ae Ee @lop l= O00 Se memce ate Neer eae 5 0 Do. ID Oye a See he aera @hioroiormel Ojpencent= 4269-4 = eee 5 0 Do. IDO Se aaa es eae Moluenen Opencentse: sae eae Smee 5 3 Do. De ee ea are Unirestedhiiicerrery sa hose ae see eres 5 3 Do. IDO een pene ae DistilledsEieOu(control) se eee 5 0 Do. Series TIT (greenhouse ex- periments): Mars28 1918-28. 2.52 Formaldehyde 0.5 per cent........-.---- 7 0 | Apr. 20, 1918 DON ees see a Phenol 0.5 per cent........- Saige epeds 7 0 Do. WORE es ae CusO7055 i percentzs= epee wee 7 0 Do. IDO 33st Roe Reneee Ee Clo sls 2: 000 ie. Se 6 come a eae ee eto 7 0 Do. ID O52 2 eS eres eee Chiloroformioipenicentees see e 7 2 Do. DO ee eae ee ee Tolwenceoip cl cent esas eee eee 7 4 Do. TORS Pee eee ane ae Wintreatedijmicel,.s—) een a eee 7 5 Do. I) Qe ate ee Distilled AsO: (Control) pee 7 0 Do. 24 BULLETIN 879, U. S. DEPARTMENT OF AGRICULTURE. CHEMICALS AS VIRUS DISINFECTANTS FOR THE HANDS. In addition to the work with chemicals in direct combination with mosaic juices, further tests were made to determine the value of various solutions as disinfectants for the hands after handling mosaic plants. In these tests the hands were smeared with the juices of a mosaic cucumber plant and then rinsed in the solution to be tested. The healthy plants were then handled in such a way as to bring the hands in contact with slight wounds, the fruits present were picked, small shoots were pinched off, and other wounds made. Formaldehyde, copper sulphate, phenol, and mercuric chlorid were all used in weak solutions, as shown in Table VI, and in addition a strong soap solution and a mere rinsing, of the hands in distilled water were tried. TaBLe VI.—Value of various chemicals as hand disinfectants after handling mosaic cucumber plants. | Results. 2. Number D eas Chemicals and strengths used as disinfectants. of plants | \umpber : handled. | ~ ae Date ob- ofmosaic Sone plants. : Ate h/0 916s) Hormaldehyderk peneCentss-52- eee eee ae anaes | 5 0} Aus. 26, 1916 Dor sseoe = Bormaldehyde: 2 per cCemta: 2 sae oe ee a en | 5 0 Do. BO seas Phenol 2pen Cente essere Ss eee a ere 5 0 Do. Wort. oes IRHen OL Per CONE yaa ee ee en ea ae ar eee 5 0 Do. ID Were HeGis tee 0002 Seta cere ths B52 eee ee 5 0 Do. DOs 2 Soap and: wiaterine4 sce eee aN a ee ee nee 5 0 Do. 1D ncesce Distilled water = 4208s cs Seen OS SN tye e tae ey aes | 5 0 Do. iD O22 seen' | Untreated mosaic juices <2. tere <2 wane a ene ree 5 5 Do. Aug. ae, 1917 | |" Hormatdehyde t percent...) 5) 5 8 ee 6 0 | Aug 28, 1917 elas Phenol tpekCenbe cs = ae es ene Sa eee 6 0 Do. Do WaSeees ECS Ofc l PenCen taco ees one Seen eo eee nee 6 U Do. DO esas. el 8 Wea On RE ea SC ee ae Se ee ee ee ee Oe oe 6 0 Do. DOs 22 | Soap andiwatero@ so smwle ta area eeneee | 6 0 Do. DOs as-a Distilled waters.) 20a eine. ear | 6 0 Do. Do ss2s2=2| | Untreated mosaic juices.....--..---------------------- 6 4 Do. As no infection occurred after any of these treatments, it is probable that the dilution effect is as important as that of chemical action. Hands covered with the juices of mosaic plants, however, gave a high percentage of infection when no wash was used. EFFECT OF FILTRATION ON THE VIRUS. Berkefeld filter—The presence of a filterable virus as the causal factor in cucurbit mosaic has already been demonstrated. Both Jagger (17) and the writer (11) have shown that the juices of mosaic cucumber plants retain their power of infection after passing through a Berkefeld filter. The work on this phase of the problem has been continued by the writer, using both the Berkefeld and the Chamberland types of filters. In the tests with the Berkefeld filter the juices of mosaic cucumbers were expressed and passed through double filter paper. After this THE MOSAIC DISEASE OF CUCURBITS. 25 filtration the juices were at once passed through a sterile normal Berkefeld bougie, the bougie, receiving fiask, and all connections having been previously sterilized with steam for 45 minutes at 15 pounds’ pressure. Owing to the large amount of finely divided material which re- mains in suspension in the expressed juices, the filtration process is very slow and with the Berkefeld filter 12 to 14 hours are necessary to obtain 200 c. c. of filtrate from a bougie 1 by 6 inches. The filtrate was rémoved to sterile test tubes or small flasks by means of sterile pipettes and inoculation made from each lot to beef bouillon, Tubes which proved free from bacteria were used for inoculation. inoculations being made with the unfiltered juice at the same time. These were made by pricking the filtrate into the young leaves at several points and into a wound at the base of the stem. The results given in Table VII show that the filtration of the infectious juices did not destroy their power of infection, most of the filtrate- inoculated plants developing mosaic symptoms as rapidly as those inoculated with the unfiltered juices. TasLe VII.—Effect of filtration through a Berkefeld (normal) filter on the infectivity of the expressed juice of mosuic cucumber plants. Results. Names Date inocu- of plants | lated. Treatment. inocu- | Number] pate last lated. | ofmosaic me sees | plants. o sige Nicol OL5 ||) wilGeredsm OSaiC \UICe =~. aac e-ce sece ee - ssc es oe 6 4 | Sept. 15, 1915 IDOsssehes Winiiltereduam sale qui Gels -os es eeses ese acee eee esee- 6 5 Do. WO ess Unfiltered healthy juice (control) .......-- Pile A eats ees | 6 0 | Do- Sept ol Ol6n auilbenedanosale julCOs— = ns5-e5- seca nnos secs s see. | 8 5 | Sept. 14, 1916 MOzeece* Wintil feredarTosalenuicele ses -eeees comer eee aan ae eee ae 7 By Do. DOnccess. Unfiltered healthy juice (control) ..................... 8 0 | Do. NE vaeol Ola peRnitered an osaiG ule sis. ace. oss econ cece cesses ce a 8 3 | May 21, 1917 Doe ks Wmiilterednvgsai cy wicelaese np. ooo ce. eee se 8 6 Do. WOes se Unfiltered healthy juice (control) -.-.........---.....- | 8 0 Do. Morvan Sal Olja eb tered anOSaieilllCes..s25 5 e265 sss. soc nec ceie oi 10 5 | May 31, 1917 ID. Oe scase lWinfileredsmosaic imiGeyena se scoae cn coe ae ae ne Seema see 10 6 Do. Ate 3051 Ola ebalteredsmosaic julCe......-.-22-+2.---4.-25-5s-5--4-0 6 | 5 2 | Sept. 12,1917 DOs WmnnlGeredemosai@juiCesc sao en 5. soe =e c-Se ne cies See see 5 3 Do. ID) Of Saa ee Unfiltered healthy juice (control).....-.--.-.---.-.--- | 5 0 Do. Chamberland filter.—In contrast to the results with the Berkefeld filter, the Chamberland type of porcelain bougie has given filtrates that were noninfectious. A considerable number of inoculations have been made, using the filtrates from different types of Chamber- jand filters, but no positive results have been-obtained. The earlier work was done with filters of the ‘‘F”’ and ‘‘B” types about 1 by 8 inches in size, the ‘‘B” type being supposedly the finer. Later a set of smaller bougies was obtained which gave a gradation in porosity. These were five-eighths by 6 inches and were graded as L2, L3, L5, L7, L9, and Lil. The L2 and L3 grades were supposed to be permeable to the more minute forms of bacteria, and the other 26 BULLETIN 879, U. S. DEPARTMENT OF AGRICULTURE. types were supposedly of sufficient density to prevent the passage of visible organisms. It was hoped that a comparison of the filtrates from these different grades of filters would perhaps indicate the size of the particles responsible for infection, but the results did not warrant any con- _ clusion along this line, as no infection has occurred in a total of more than 100 plants inoculated with these filtrates. The filters of the Chamberland type, being of porcelain, are all denser than the Berke- feld, and filtration is therefore much slower. There is a possibility, therefore, that the infective principle may be held back by absorp- tion, as the suspended material in the plant juices rapidly clogs the filter and covers its surface with a gelatinous layer. Other filters.—Allard (4) reports that the juices of mosaic tobacco plants become noninfectious if passed through a Livingston atmom- eter. A filter made of a layer of powdered tale from seven-eighths to 14 inches thick also gave a noninfectious filtrate. Similar tale filters have been used in experiments with cucumber mosaic, the layer of tale varying from three-fourths of an inch to 1 inch in thick- ness. Three trials have been made with these filters, and a total of 37 plants have been inoculated from the filtrates. The results have been negative in all cases, however, and it is evident that the filtrate is rendered noninfectious, as in the case of tobacco. Iwanowski (16), Koning (21), and Beijerinck (6) have stated that the juices of mosaic tobacco plants do not lose their infectious nature when passed through Berkefeld and Chamberland filters, though Iwanowski found that only the first portion of the Chamberland filtrate was infectious. It is thus evident that the infective principle of both tobacco and cucumber mosaic is of such a nature that it can be removed by filters of the finer types. ENZYMS IN RELATION TO THE DISEASE. Since many writers have advanced the theory that enzyms, par- ticularly oxidases, are in some way connected with the mosaic of tobacco, afew attempts were made by the writer to isolate a pos- sible causal enzym, from the juices of mosaic cucumber plants. The fact that the juices of such plants usually lose their power of infection within 24 to 48 hours has proved an almost insurmountable obstacle in such work, and so little has been done that it is not feasible to draw any definite conclusions. Tests of the juices of healthy and diseased plants which have been passed through filter paper have shown the presence of oxidases and peroxidases in both cases, the eualacum, reaction being used. Very little difference in the intensity was noted, but the juices of diseased plants seem to show a slightly stronger reaction for both enzyms. Similar tests of both healthy and mosaic plant juices after passage through a Berkefeld filter have shown a weaker test for both oxidase and peroxidase in all cases, THE MOSAIC DISEASE OF CUCURBITS. OT the intensity in healthy and diseased juices after filtration being ap- proximately equal. Attempts have been made to precipitate an enzym with 45, 70, and 80 per cent alcohol, using the juices of mosaic _ plants filtered through filter paper. The precipitates gave peroxi- dase reactions in all cases, but inoculations made with solutions of these precipitates were never successful. COMPARISON OF CUCURBIT MOSAIC WITH TOBACCO MOSAIC. The properties of the virus of cucumber mosaic are strikingly like those attributed to the virus of tobacco mosaic. As already indi- cated, the symptoms of the two diseases are very similar, as are also the changes produced in the anatomy of the respective hosts. Also in both hosts the points at which successful inoculations can be made correspond with one exception, i. e., in tobacco root inoculations produce infection. The vigor of growth and age of the plants are important factors in the infection of both hosts. Insects and the handling of mosaic and healthy plants in pruning and picking are responsible for much of the field transmission of tobacco as well as cucumber mosaic. The first symptoms appear in the young leaves, both in tobacco and cucumbers, although the virus is found in all parts of the stem and leaves, regardless of the development of mosaic symptoms. In tobacco the virus is present in the flower parts, in- cluding the placental column and integument of the ovule, as is shown by Allard (8). It is also present in the mature seeds, accord- ing to the same writer. In the case of the cucumber, the virus is present in the flower parts and in the immature fruits, but has never been found in the mature seed. The vascular system may be con- cerned in the distribution of the virus in both cases, at least in the writer’s opinion, since the evidence on which the theory is based is much the same for both hosts. The work on the properties and nature of the virus of tobacco mosaic has been much more extensive than that on cucumber, and furnishes a valuable basis for comparison. The thermal death point as worked out by Beijerinck (6), Woods (382), Iwanowski (16), and later by Allard (4) seems to lie between 80° and 100° C. This is somewhat higher than that of cucumber mosaic, which is rendered nonpathogenic if heated above 70° C. The expressed juices of mosaic tobacco plants will retain their power of infection for a year, or more, in some cases, whether preservatives are used or not, and the dried leaves will remain infectious for relatively long periods. This is different from the disease on cucumbers, where the expressed juices are seldom infectious for more than 48 hours and the dried tissues have never proved infectious. Disinfectants, such as phenol, formaldehyde, mercuric chlorid, and copper sulphate, will destroy the virus in both cases, the virus of tobacco mosaic apparently being slightly more resistant in this 28° BULLETIN 879, U. S. DEPARTMENT OF AGRICULTURE. regard. The juices of both mosaic tobacco and cucumber plants may be diluted to 1 : 1,000 without affecting the power of infection, and dilutions of 1 : 10,000 will also produce the disease, giving, how- ever, a lower percentage of infected plants. The tobacco virus will pass through both the normal Berkefeld filters and those of the Chamberland type, while the cucumber virus passes only through theformer. This difference is probably of minor importance, however, as Allard (4) has shown that the juices of tobacco mosaic are also rendered noninfectious if passed through a porous clay atmometer or through a 3-inch layer of powdered tale, so that the behavior of the virus of both diseases is essentially the same as regards filtration. POSSIBLE NATURE OF THE CAUSAL FACTOR. Since no visible causal organism has been associated with the mosaic diseases, various theories have been advanced as to their nature and origin. Woods (82), Koning (21), and more recently Freiberg (13) and Chapman (7) have held that enzyms, particularly oxidases, peroxi- dases, and catalase, are in some way connected with the cause of tobacco mosaic. Allard (4), on the other hand, has claimed that the disease is due to a specific pathogenic agent, probably an ultra- microscopic organism. Both theories are based principally on work with the mosaic of tobacco, but they apply equally well to the corresponding disease of cucumbers. The evidence so far accumulated, however, seems to accord better with the theory advanced by Allard than with the enzymic hypothesis. The virus of cucumber mosaic, like that of tobacco, seems to pos- sess many of the characteristics of living matter. It loses its power of infection if heated above 70° C., is easily destroyed by chemicals, and will not withstand desiccation. In support of the enzymic theory of the nature of tobacco mosaic it is claimed that similar properties are possessed by enzyms, and this is undoubtedly true to a great extent. The virus of cucumber mosaic, however, does not have such marked enzymic qualities. Unlike that of tobacco, it loses the power of infection within 24 to 48 hours after the juices are expressed from the plant, regardless of the use of preservatives or the temperature at which it is kept, and will not withstand desiccation. It is quite conceivable, however, that an organism might be destroyed rapidly after removal from its natural environment in the plant tis- sues, especially as the juices of the cucumber undergo rapid chemical changes when expressed. Another point which seems to support the theory of an organism as the cause of mosaic is the ability of thejuices of mosaic plants to produce infection in a dilution of 1 : 10,000. Two or three drops S ¥ Jule yu THE MOSAIC DISEASE OF CUCURBITS. 29 of such a dilution when pricked into the leaf of a healthy plant will produce the disease as rapidly and with as intense symptoms as when the undiluted juices areused. We have evidence here that the virus possesses the power of rapid increase, since all parts of the leaves and stem of the inoculated plant contain the virus within three to four days after inoculation. If we adopt the theory of an ultrami- croscopic organism as the causal factor, this increase is readily ex- plained, but examples of similar increase or multiplication are not commonly associated with enzyms. The behavior of the virus in filtration may be used to support the theory, although the fact that the finer filters hold back the virus indicates that the particles composing the virus are probably colloidal in nature and of relatively large size. These qualities, however, neither exclude it from the class of enzyms nor from that of ultra- microscopic organisms. In attempting to prove either of the above hypotheses it must be recognized that a great part of the available data is equally applicable to either theory and that no definite conclusion is as yet possible. It is the writer’s opinion, however, that the causal agent possesses characteristics which tend to place it as an ultramicroscopic organ- ism rather than as an enzym, but no property has yet been discovered which may not be characteristic of either agency. On the other hand, the examples of diseases attributed to a so- called ‘‘filterable virus” or ‘‘ultramicroscopic organism”’ are numer- ous in animal pathology and are generally accepted, while as yet there are no demonstrated examples of diseases due to enzymic causes alone. In the case of tobacco mosaic the recent work of Allard (4) has nearly eliminated the oxidases from consideration, so that we at present can hardly attribute the disease to a definite type of enzym. INFECTIOUS NATURE OF THE DISEASE. The epiphytotic character of cucumber mosaic and its rapid spread to plants adjoiing those first infected at once indicated that it was ‘of an infectious nature, as proved by Jagger (17) and the writer (11). The first inoculation experiments by the writer were begun at Ham- ilton, Mich., in 1914, but as the plants used had no protection from insects and mosaic was already present in the field the results, although positive, did not permit definite conclusions. In 1915 the work was continued at Big Rapids, Mich. This district was practically free from the disease and the experimental fields had never before grown cucumbers. The possibility of outside infection, particularly from insects, was practically eliminated by using cages covered with cheese- cloth to protect all plants in the inoculation experiments. As the cages in most cases had no openings, they were lifted when inoculating the plants, but by exercising proper care and using the 30 BULLETIN 879, U. S. DEPARTMENT OF AGRICULTURE. cage as a partial shield during inoculation insects gained entrance very rarely. It was necessary to inspect all cages frequently to cut the tendrils which penetrated the cloth and to cover any small open- ings with heavy paint. All cages were set out as early in the season as possible, usually immediately after planting or by the time two to three leaves had appeared on the young plants. When plants * were caged after the disease appeared in the field they were left at - least 10 days before being used in inoculation experiments, in order that any previous accidental mosaic infection might have time to develop before inoculations were made. Most of the cages used were of the same general type, the frame being of laths, with the exception of the corner uprights which were made of pieces from 1 to 2 inches square. In the earlier cages extra strips of lath were placed around the lower edge of the frame to allow it to be sunk 2 to 3 inches in the soil, but it was later found advisable to make the lower edges of light 4-inch boards. The cheesecloth & used contained 24 to 30 threads per inch, the lower edge of the cloth being’ wrapped around strips of lath, which were then nailed to the bottom of the frame. Cages were also constructed with an opening in the top, but as they were difficult to build and often failed to remain insect proof, they were later abandoned, except for special purposes. Cages were used in practically all field work and reduced the factor of outside infection, which occurred only in rare cases, to a minimum. The caged plants grew luxuriantly and were only slightly affected by the shading of the cheesecloth. ‘INOCULATION EXPERIMENTS. Inoculation of healthy vines with the juices of mosaic plants has proved that the causal agent of the disease is present in the tissues y~ of the stems, leaves, and fruits of mosaic vines. A light wounding of the plant inoculated is apparently essential for infection, as the virus does not seem to penetrate the unbroken epidermis. The two methods of inoculation used differ chiefly in the form of the inocu- lum, the expressed juice of mosaic plants or their crushed tissues being used. Results by either method have been successful, as is shown below. Control inoculations were made in all cases, using caged plants and inoculating them with the expressed juices or crushed tissues of healthy plants (Pl. IX, A). TESTS WITH EXPRESSED JUICES OF MOSAIC PLANTS. é In the earlier work the expressed juices of mosaic plants were used for inoculation. Portions of a mosaic plant, either leaves, stems, or fruits, were cut finely by means of a sterilized meat chopper, the juice pressed through cheesecloth, and in some cases filtered through ~ filter paper, and the expressed juice used as the inoculum. The THE MOSAIC DISEASE OF CUCURBITS. al inoculations were made in the stems, leaves, and in some cases in the fruits. In the earlier experiments a sterile hypodermic needle was used for inoculation and the inoculum injected rather deeply into the stem. It was found, however, that inoculations made by cutting off a leaf close to the stem and then pricking the inoculum ¥ into the wounded surface gave a higher percentage of infection. In leaf inoculations a drop of the inoculum was pricked into the leaf at various points with a sterile needle. The results of inoculations by these methods are shown in Table VIII. TasLe VIII.—Resuits of inoculations with the expressed juices of mosaic cucumber plants. Results. Inocul ae Date Se, Point of ve) * Treatment. expressed juice F . plants | Num- inoculated. of— inoculation. inocu- | ber of Date last lated. | mosaic | observed plants. ~ Aug. 2,1915 | Inoculated.-..| Mosaic plant.-.-.... Base and tip ofstem. 4 3 | Aug. 17,1915 DORs Controle === Healthy plant.....|..... CO misweseeee acts 3 0 | Aug. 20,1915 Aug. 4, 1915 | Inoculated-...| Mosaic fruit......- ee leaf and base 5 3 | Aug. 14,1915 ofstem. ID OR sae: Controls. = <=. Healthyairuite ce. a\osss- GON Saas ees eee 3 0 Do. Aug. 21,1915 | Inoculated. ..| Mosaic fruit....... iBaselonstem sss - =: 8 8 | Sept. 2,1915 IDO ater Control=- == Healthysirults-- = s|sess5 Ome ere nee See 5 0 Do. Nov. 19,1915 | Inoculated -..| Mosaic leaves... .. Woung leaficrss a=: 8 5 | Nov. 26,1915 Dons see Control...... Healthy leaves.-..-.|....- Cle omartsteercr 6 0 Do. Nov. 28, 1915 | Inoculated. -..| Mosaic plant......|...-- doze 11 5 | Dec. 6,1916 FADES (8.19163). 2 GOS Ean eae GOs Seek eee Base of stem... ..- 7 6 | Apr. 20,1916 Doi: Controlee= =| Sbealthyaplant=s54|5--- GOK a so Ne ae ae 8 0 Do. Sept. 6,1916 | Inoculated.-..} Mosaic plant......|..-.- (as Sat eerescses 7 5 | Sept. 15,1916 Doesnt: Control.....- Healthy plant.....|....- dose ote ae 6 0 Do. TESTS WITH CRUSHED TISSUES OF MOSAIC PLANTS. In later work, experience proved that the easily prepared crushed tissues of mosaic plants formed a more constantly virulent inocu- go than the expressed juices. Infected portions of the plant, ~ either fruit, stem, or leaves, were crushed in a sterile dish after they had been cut into small fragments with sterile scissors or a scalpel. This material was inserted in a small cut in the stem made with a ster- ile scalpel. Such wounds were usually made in the lower portion of the stem, in some cases directly below the oldest leaf, but more commonly a leaf was cut off close to the stem and the incision made in the cut surface. A longitudinal cut, 3 to 5 millimeters long and 2 to 3 millimeters deep, was found sufficient, the small piece of crushed material being inserted in the wound, which healed rapidly. In other cases a small incision was made at the tip of the stem directly §below a young leaf and a fragment of the crushed tissue inserted at this point. Inoculations with crushed material at either of these points, or both, ordinarily gave a slightly greater percentage of infec- tion than was obtained by the injection of the expressed juices. It was found also that moculations at two or three points were more generally successful than where a single inoculation was made. 32 BULLETIN 879, U. S. DEPARTMENT OF AGRICULTURE. This method is preferred chiefly because the preparation of the expressed juice requires time, and such extracts, unlike those from mosaic tobacco plants, lose their pathogenicity rapidly, usually having to be prepared fresh every day to insure success. In the case of the second method, fresh material is constantly available and its preparation takes little time. + Table IX gives results which are typical of inoculations by this method, but represent only a small fraction of the inoculations actu- ally made. TaBLE 1X .—Results of inoculations with the crushed tissues of mosaic cucumber plants. Results. Number Be eee eq, | Treatment. Inoculum. Point of inoculation. Sint Number ; ° imocu- of Date last lated. | mosaic | observed. plants. Aug. 24, 1915.; Inoculaced...| Mosaic stem.....-.- iBaseloistemy ee eees 3 3 Rept. 2, 1915 ‘ IDWOsé6ca6e Control.......| Healthy stem.....|..... COWLES Fie feats 3 0 Jan. 22,1916 | Inoculated...| Mosaic leaf........|..... GOs ieee eee 4 4 | Jan. 2S 1916 Nov. 31916 |..... WO i dowel sn Te ceed ons) ae peeing 6 4| Nov. 10,1916 DOs sees Controle Healilyaleatesesseel eee dose ae ee 6 0 Do. eb. 17,1917 | Inoculated. -..| Mosaic leaf........ Dip of stem ae. mera 5 3 | Feb. 28, 1917 DOR se ee Control.......| Healthy leaf.....-.|...-. dose ee 5 0 Do. Mar. 11,1917 | Inoculated...| Mosaic leaf........ Base and tip of stem. 12 8 | Mar. 19,1917 Doe eee: Control......- .Healthy leaf.......|..-.- OE as ae oe 10 0 Do. Mar. 15,1917 | Inoculated...| Mosaic stem....... Base of stem......-- 12 7 | Mar. 22,1917 ID Yo ia a xia dove ees Mosaic inaitees = seeiseces done. See eee 6 2 Do. IDO ae Control....... Healthy fruit......|..-.. GOs eae 6 0 Do. Mar. 29,1917 | Inoculated...| Mosaic leaf........ Base and tip of stem. if 5| Apr. 9,1917 DOAN ss Controlaes= ealithiy, leatiss. 2245 |e ce Goesca Sener 7 0 Do. May 19,1917 | Inoculated...| Mosaic leaf....-...|..... Goss. Bas 4 4 | May 30, 1917 DYE a Controls Healthy leaf-- SCO: ey ee 4 0 Do. June 20,1917 | Inoculated...| Mosaic leaf........ | Tip Oi GUNS Ss sc5005 8 5 | July 4, 1917 Does. Control....... Healthivsaleadtesses5| seen. GOS ee Rae 8 0 Do. RELATION OF INFECTION TO POINT OF INOCULATION. STEM INOCULATIONS. As shown by the preceding data, stem inoculations are successful, in most cases, regardless of the point of inoculation. Inoculations made in pounce produced by the removal of a leaf are ordinarily more effective than at other points, possibly because the virus comes more directly in contact with the vascular bundles at such points. LEAF INOCULATIONS. Inoculation of any green leaf will produce mosaic infection, but the inoculation of young leaves gives a much higher percentage of mosaic plants than the inoculation of leaves that have reached their full development. The point of inoculation in the leaf, however, seems to make little difference. An interesting fact, already proved by Allard (5) for tobacco mosaic, is that infection can take place through the trichomes. He has shown that infection may occur if the trichomes of healthy plants are cut or pinched with instruments which have previously been dipped in the juices of a mosaic plant or when the mosaic juices are painted on the trichomes. Similar experiments have been made with the cucum- Bul. 879, U. S. Dept. of Agriculture. PLATE IX. CUCUMBER EXPERIMENTAL FIELD AND SOIL OVERWINTERING TEST. A, Experimental field at Big Rapids, Mich., in 1916, showing the various types of insect-proof cages used in making inoculations. Photographed by W. W. Gilbert. B, Soil overwintering test at Madison, Wis., September 19, 1917. This land grew a mosaic crop in 1916. The few large healthy plants were grown under cages. All the uncaged plants became diseased early and were dead or severely stunted. An insect hibernation cage is seen at the left. Bul. 879, U. S. Dept. of Agriculture. : PLATE X. MOSAIC AND HEALTHY CUCUMBER PLANTS FROM COMMERCIAL SEED. £ A, Mosaic plant grownfrom commercialseed in a greenhouse at Madison, Wis.,in 1917. Notethe dwarfing as compared with B, the mottled and curled leaves, the shriveled cotyledons, and the dying of the edges of the first leaf. B, A healthy plant grown from the same seed as A and planted on the same date. THE MOSAIC DISEASE OF CUCURBITS. ao .ber, the trichomes of mosaic and healthy leaves being brought in contact in such a way as to merely break the trichomes without injuring the epidermis of the leaf. Infection has resulted in most of the plants thus inoculated, as is shown in Table X. TaBLE X.—Infection as a result of contact of trichomes of leaves of healthy and mosaic i cucumber plants. Results. RS | esults Date of | Number inoculated. EESEUE Ga plants | of Date last lated. | mosaic observed. "| plants Apr. 20,1916 | Mosaic and healthy leaves in contact ......--......---- 2 | 2) Apr. 28,1916 DOr ces Healthy leaves in contact (control)....2...--......---- 2 0 Do. Aug. 5,1916 | Mosaic and healthy leaves in contact .......-.......... 6 4| Aug. 16,1916 IDS eae Healthy leaves in contact (control)........---...--..-- 4 0 Do. Feb. 21,1917 | Mosaic and healthy leaves in contact ....-.-.--...-.--- 12 9| Mar. 2,1917 Dou Healthy leaves in contact (control)....-.---.---.----.. 12 0 Do. Mar. 22,1917 | Mosaic and healthy leaves in contact ....-..-.--...-.-. 10 5) | Apr. 451917, Dortsta Healthy leaves in contact (control)......-.---.------.. 8 0 Do. June 20,1917 | Mosaic and healthy leaves in contact .......----..--.-. + 2| June 28,1917 IDOE ss: : Healthy leaves in contact (control)......-..----------- 4 0 Do. _ Oct. 24,1917 | Mosaic and healthy leaves in contact............-...-. 12 3 | Nov. 2,1917 4 Dos _-| Healthy leaves ini contact (contro!) = 32-se.- 4. .+-o--e == 6 0 Do. BLOSSOM INOCULATIONS. No evidence has been obtained thus far which indicates infection through the blossoms. Numerous inoculations of male and female flower parts were made by pricking the expressed juice of a mosaic cucumber plant into the petals, stigmas, or anthers of the blossoms of healthy cucumber plants. From 3 to 8 blossoms on each of 38 plants have been inoculated in this way, but no infection has ever occurred. It is still a question, however, whether such infection may not occur, especially as the virus is known to be present in the blos- soms of mosaic plants, as is shown on page 35. FRUIT INOCULATIONS. & Inoculations into small fruits have given infection in a number of plants, as is shown in Table XI, but inoculations of fruits approaching maturity were nearly always unsuccessful. TABLE XI.—Results of inoculation of fruits of healthy cucumber plants. _. {Number Results. Dateinocu- | myoeat t iaocukam ae ED one ms panes NaEanee lated. ene. ne : _ of fruits intedicer inocu- Gemosaie Date last ob- inoculated. plant. iated. plants. served. Mar. 20,1916 | Inoculated. ..| Expressed juice of | 10 days.... 2 é 3 | Apr. 5, 1916 mosaic plant. DORs