SB 608 .C885 D6 Copy 1 ^ITED STATES DEPARTMENT OF AGRICULTURE BULLETIN No. 879 Contribution from tlie Bureau of Plant Industry WM. A. TAYLOR, Chief Washington, D. C. PROFESSIONAL PAPER November 15, 1920 THE MOSAIC DISEASE OF CyCURBITS By S. P. DOOLITTLE, Assistant Pathologist OflBce of Cotton, Truck, and Forage Crop Disease Investigations CONTENTS Page Scope of tlie Investigations 1 Tlie Mosaic Disease 1 Cause of tJie Disease 18 Infectious Nature of tlie Disease 29 Mosaic Transmission 40 Method of Overwintering 47 Control Measures - 63 Summary 64 Literature Cited 68 WASHINGTON GOVERNMENT PRINTING OFFICE 1920 P^> VP^ THE INVESTIGATIONS of the cucumber mosaic reported in this paper were begun by the writer in 191 4 at Hamilton, Mich., while a graduate student at the Michigan Agricultural College. In 1915 and 1916 they were continued at Big Rapids, Mich., and m 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 c(jmpany. The writer wishes especially to thank Messrs. Bessiey, Jones, and Gilbert for assistance and supervision throughout the course of the work. This paper was presented to th<^ faculty of the University of Wisconsin in partial fulfilhnent of the requirements for the degree of doctor of philosophy. n. 4€^ Cf ^ UNITED STATES DEPARTMENT OF AGRICULTURE ^ BULLETIN No. 879 i Contribution from the Bureau of Plant Industry WM. A. TAYLOR, Chief S>Js^<^^u Washington, D. C. PROFESSIONAL PAPER November 15, 1920 By S. THE MOSAIC DISEASE OF CUCURBITS. p. DooLiTTLE, Assistant Pathologist, Office of Cotton, Truck, and Forage Crop Disease Investigations. CONTENTS. Page. Scope of the investigations 1 The mosnie disease 1 Cause of the disease 18 Infectious nature of the disease 29 Mosaic transmission 40 Page. M ethod of overwintering 47 Control measures 63 Summary 64 Literature cited 68 SCOPE OF THE INVESTIGATIONS. DURING 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 Ignited States Depart- ment of Agricidture. 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 bidletin deals with the nature, transmission, and overwintering of the disease in relation to cucurbits, llie 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 > The serial numbers in parentheses refer to " Literature cited " at the end of the bulletin. 185118°— 20 1 2 HULLKTIN H7l>, U. S. DI•:^AUT^ll:^'T OF AURICULTUIU:. those wrro truo mosaic, as the discMiso was not proved to !)(> infectious in any ease and no mention was made of symptoms on tin' fruit. Selhy (27) stated in 1910 that cucumber mosaic was transmitted like flinl on tol>acco. 'Hiere is no early record of the disease in the field, l)ul i( is certain that it liad caused serious losses in the ( 'entral 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 aiul was responsible for forcing at least one pickle company out of business. Ruggles and Stakman (25) in 1911 reported the mosaic as occurring in Miiniesota 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, lie described the chief symptoms and stated that the j)rogress of the disease indicated that it 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 (Jilbert (14), Jagger (17), and Doolittle (11) in 1916. Jagger (18) later reported the appearance of another type of cucumber mosaic, afTecting oidy the leaves of the plant, and in a furtlier paper (19) mentioned a third type as occurring on Summer Crookneck s(juash. Stakman and Tolaas (28) mentioned luibbin or wart disease of cucumber as an infectious disease occurring in both the field and the greenhouse in Minnesota. McCIintock (22) in 1916 observed possible cases of transmission of tlie disease through the seed and described its occurrence in tlu« district near Noi-folk, 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 191S also reported numerous cross-inoculations from cucund)er lo other cucurbits and showed that cucumber mosaic could be trans- mitted to plants outside the Cucurbitaceie. These papers include all the available nuiterial on cucund)er mosaic to the present, with the exception of an abstract by Doolittle and Gilbert (12) which dealt with certain phases of the work hero considered in greater detail. GEOGKAPHICAL DISTKIBHTION. The general occurrence of cucurbit mosaic in the field was not recognized until after these studies were l)egun, but investigation has shown that it is widely distributed on Cucurbitacctv throughout the Cnited States. Doubtless tlie extensive ciJtivation of cucumbers makes it seem more common on that crop, i)ut it may occur on prac- tically all cucurbits in localities where it is found on cucumbers. The disease is probably most widespread and s(>rious in Wisconsin, Michigan, Indiana, northern Illinois, and on Long Lshuul. It is ro- THE MOSAIC DISEASE OF CUCURBITS. 6 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, wliich devote a largo acreage to the crop. Hero tlio disease is present to some extent in almost all localities, and in most cases there is infec- tion eveiy 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. It is 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 injur^^ for several years in Michigan, Wisconsin, Illinois, and New York and has also been reported from Mississippi, Ohio, Indiana, Minnesota, Pennsyl- 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. Tlie 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 higlily 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, OA\dng 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 BlLLl/riN 87!>, U. S. UEPAUTMENT i)V AGRICULTURE. tlio losses from iiiosuic, inany <;ro\v('rs, osjx'cially tliosc in certain sections of the Middle West, have ceased to grow cucuiiibei-s, despite increased prices (PI. I, ('). In the case of forcin<;j cucuinhei-s, growers occasionally lose an entire croj), because, in addition to the reduced yield and deformed fniit, 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 all'ected tabic stock sells for about one-fourth the price obtained for sound fruits, owing entirely to the effect on their ap|>earance. Of the other cidtivated cucurbits squash and muskmelon seem most susceptible to the mosaic, but neither of these plants is injured as severely as the cucumber. 'Hie muskmelon vines are stunted and bear only a few fruits, which arc inferior in qujility 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 pumi>kins, gourds, and ornamental cucurbits, which are of less commercial value. CUCURBIT HOSTS. Most of the species of the family C'ucurbitacea^ appear to be sus- ceptible to mosaic. Field o])servations have proved that most cultivated species are commonly affected, and that the disease also occurs on ^^ild 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 cidtivated species. Most of the field inoculation tests were made during 1916 and 1917. All inoculated plants were kept under insect-proof cages, and reciprocal inoculations were made in most cases. The various species were usually inoculated in stems or leaves with the expressed juices of mosaic cucumber plants, but many successful uioculations 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, Cucuniis sath^us L.. 15 varieties were inoculated successfully, and all seemed equally susceptible. Mosaic was also produced on the West Indian gherkin. C. nnguria L.. on a cl(jsely related variety, C. grossulariatforntis Ilort., and on C. nietul- lijerus Mey. Inoculations on 11 A'arieties of muskmelon, Cucuims mrlo L., have all produced the disease, including the pomegranate melon, C. melo var. dudaim Naudin. Seven varieties of squash and four varieties of pum|)kin, Curiirhiid jxpo L., C. itutschata Duchesne, and C. ifi/ijcliiKi Duchesne, have also been infected. THE MOSAIC DISEASE OF CUCURBITS. On the gourds infection occurred on 12 varieties, including Cucur- hita pepo L., Luffa cylindrica Roehm, L. acutangula Roxbg., Lagenaria vulgaris Sor., and L. leucantlia Rusby. Mosaic has also been produced by inoculation on the following species: Momordica involucrata E. Meyer, M. cliarantia L., Ecballium elaterium A. Rich., Benincasa Mspida Cog., and Trichosanthes anguina L. The wild species, Micrampelis lohata (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 havmg obtamed 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-INOCULATIONS TO OTHER 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 louisiana Mill, appeared in one of the cucumber plats, the seed having been accidentally included with tli^t 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 m.osaic tj^pe 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 Cucurbitacefp, 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 BULLKTIN 879, U. S. DEPARTMENT OF AGRICULTURE. The motliods used in tliis iiloculation work were very similar to those used in the other (•ueum])er-mosiue studies. Wliere fiehl inoc- ulations were made, the i)hints were protected with insect-proof ca«;es wlierever possihle, and iti tho g^reenhouso all plants used in surli inocuhvtions were kept isolated from other mosaic experiments. The inoculum was prepared in much the same way as in other cucumher-mosaic inoculations, either the expressed juice or crushed tissues of mosaic ])lants heinj? used. Wiiere the juice of a mosaic plant was employed, tiie parts of the plant used as inoculum were passed throu{j:h a sterilized food chopper and tho juice expressed and filtered tlirou<;h filter paper. Inoculations were made at from three to five points in each of the younger leaves, a drop of the exjiressed juice heinj; pricked into the leaf with a sterile needle. The stem usually was inoculated at one or more points, a slight incision being made with ii sterile scalpel carrying a drop of tlH> inoculum. Taiuk I. - Rtcurd itj inncuUitiom from viosair-diseascd cucumber plants to plants oj other families. Dateinoai- lated. Aug. Mar. Mav Oct. Nov. Aug. Aug. 4,1916 _ 11.1917 Apr. 27,l"Jl7 Mav 8, 1917 20,1917 1,1917 6,1917 4,191G ,. 18,1916 Feb. 2.3,1917 Feb. 30,1917 Apr. .5,1917 Oct. 8,1917 Dec. 8,1917 Aug. 4,19Ki Do Feb. 1.5,1917 Mar. 24,1917 Sept. 7,1916 Aug. 15,1916 Aug. 16,1915 Mar. 22,1916 Aug. 1,1917 Aug. 5,1917 Plant inoculated. Tobacco do do do do do do Tomato do do do ...do ...do ...do Wax bean I-inia bean Red kidney beau , do Martynia louixiana Potato Pokeweed (Phytolacca dt- randra.) Petunia A mbroxia trifida ....do Preparation ofinoculum. Expresi^ed juices do do do Cru.shed leaf tis-^ues . do E.\ pressed juices do ....do ....do ....do Crashed leaf tis.sues. ....do E xpressed j uices do Number of plants inocu- lated. .do. .do. -do. .do. .do. -do. .do. .do. .do. Results. Number of mosaic planus Date last observed. Aug. 17,1916 Mar. 26.1917 Mav 28,1917 Do. June 14,1917 Oct. 30,1917 Dec. 1,1917 Aug. 17,1916 Aug. 31,1916 Mar. 20,1917 2, 1917 8.1917 19. 1917 12. 1918 Aug. 17,1916 Do. Mar. 10,1917 Apr. 12,1917 t^ept. 23, 1916 Do. Sept. 30, 1915 Apr. 10,1916 Sept. 1,1917 Do. Mav May Nov. Jan. Where the crushed tissues of tho mosaic i>lant 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 slight in(isi(»iis made at one or two points in tho stem of the ])lant hioculated aiul other ])i('ces sometimes pricked into tho young leaves. The results of all the inoculations from mosaic cucumber to ])lantfi outside the cucurbits have been negative,' as shown by Table I. ' Rwent experiment? (1919) indicate that cucumlK>r mosaic may be tninsmltted to ilarifnia louitiana by mcims of uphills lukcii from ni(is;ilc cuciimbrr plants. Inoculations from martynia pliujts infected In this miuiucr have also produced tho disease oa tho cucumber. 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 Lobeliaceae {Lobelia erinus L. var. gracilis) and to one of the Compositse {Helianthus dehilis Nutt.)- It is thus evident that in some cases the disease may pass from the cucumber to hosts out- side the Cucurbitaceae. Table II. — Record of inoculations of cucumber plants with infectious mosaic diseases of noncucurbitaceous plants and with suspected mosaic plants of other families. Pl.vnts Having Mosaic Diseases Known to be Infectious Used as Source of Inoculum. Date inocu- lated. All jr. Sept. Dec. Feb. Aup. Sept. Feb. Feb. Dec. Feb. Dec. Aug. Feb. Mar. May Nov. Jan. Sept. Sept. July Aug. Mar. Sept. Sept. Aug. Sept. 25. 1915 S,1915 21,1915 4,1916 19,1916 7,1916 18, 1917 20,1917 2,1917 15,1918 21,1915 29,1916 17,1917 30, 1917 20,1917 10,1917 11,1918 :i, 1915 10,1915 15. 1916 30, 1916 5,1918 3.1915 in, 1915 23,1916 3, 1915 Sept. 10,1915 Aug. 23,1916 Aug. 30,1916 Mar. 19,1916 Apr. 1,1916 Sept. 3,1915 Sept. 10, 1915 July 20,1916 Sept. lO; 1915 Aug. 30,1916 Plant used as source of inoculum. Mosaic tomato. ....do do do do do do do do do Mosaic tobacco do do do do do do Mosaic bean do do do do Mosaic potato do do Mosaic pokeweed (Phyto- lacca aecandra). .do .do .do Mosaic petunia do Peach with yellows (leaves and twigs). do.... do Little peach leaves and twigs. do Preparation of inoculum. Expressed juices do do do Crushed leaf tissues . Expressed juices Crushed leaf tissues. Expressed juices do Crushed lea f tissues . Expressed juices do do do do Crushed leaf tissues . Expressed juices do .do. .do. .do. .do. -do. .do. .do. .do. do do Crushed leaf tissues. Expressed j uices do do .do. -do. -do. .do. Number of plants inocu- lated. Results. Number of mosaic plants. Date last observed. Mar, Jan. Mar, Jan. Sept. 10,1915 Sept. 20, 1915 Jan. 22,1916 Feb. 27,1916 Sept. 5,1916 Sept. 23, 1916 Mar. 3, 1917 21,1917 16,1918 1,1918 22, 1916 Sept. 23, 1916 Mar. 18,1917 Apr. 20,1917 June 14,1917 Dec. 1, 1917 Jan. 30,1918 Sept. 20, 1915 Do. Aug. 17,1916 Sept. 23, 1916 Mar. 23,1918 Sept. 20, 1915 Sept. 23, 1916 Do. Sept. 20, 1915 Do. Sept. 15,1916 Sept. 23, 1916 Apr. 1,1916 Apr. 22,1916 Sept. 20, 1915 Do. Aug. 17,1916 Sept. 20, 1915 Sept. 23, 1916 Plants HA^^NG Diseases of the Mosaic Type of Suspected Infectious Natube Used as Source OF Inoculum. Sept. 10, 1915 .July 20,1915 .\nfi;. 2.5,1916 Sept. 10, 1916 July 20,1916 Sept. 7,1916 Aug. 10,1917 Sept. 8,1916 Aug. 18,1917 Aug. 30,1917 Aug. 30,1916 Mosaic mi\kweQA(.Asclepias syriaca). do do, Mosaic red clover ( Trifo- lium jrratense). do Mosaic mart ynia {Afartynia louisiana) do Mosaic pepper {Capsicum annuum). Mosaic ragweed {Ambrosia iTifida). do Mosaic sumac {Rhus ty- phina). Expressed juices. .do. -do. .do. .do. -do. Crushed leaf tissues . E.xpressed juices .do. Crushed leaf tissues. E xpressed j uices 9 5 7 10 5 8 6 3 7 8 8 Sept. 20,1915 Aug. Sept. Sept. Aug. Sept. Sept. Sept. 17, 1916 23,1916 20, 1916 17, 1916 23. 1916 5, 1917 23. 1917 Sept. 12, 1917 Sept. Sept. 24, 1917 23, 1916 8 BULLKTIN 87f), U. S. I)i:rAi;TMKNT OF AtiUlCUl/rrilK. INOCULATIONS THOU MOSAIC PI-ANTS OF OTHKU FAMILIES TO CUCUMBER. Attempts have nlso boon made to transmit mosaic disoasos found on plants outsido tlio Cuciirhitacoay to tho oucumbor. A considorable numbor of inociihitions liavo boon mado from tomato, tobacco, bean, and other plants liaving infectious mosaic diseases and also from plants whicii showed diseases of the mosaic type but whose infectious nature was not proved, such as popper {Capsicum annuum L.), sumac {Rhus tijphina L.), milkweed {Asclepias syriaca L.), red clover {Tri folium priitcnse L.), and others. Inoculations in most cases were made with the expressed juice of tho mosaic plant in the manner already described. All the results of these inoculations have been negative, as shown in Ta])le 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. Tho profrross of tho mosaic disease in tlio plant is characterized by distinct morpliologic and physiologic chan.<;es, wliicli are most markod in tlio loaves and fruits. Those consist principally of an abnormal mosaic coloring, accompanied by certain malformations and dwaifmg of growth. Tho symptoms vary somowliat according to tlio ago of tlio plant and its vigor of growth at tho time of infection, but tho general characters aro tho same in all stages of growth. There is also a variation in tlio symptoms on tho different species of Cucurbitacoa? which aro susceptible to tho disease. Tho loaves of different species show many diverse forms of mosaic coloring, and the fruits may or may not bo afToctod, although usually they show markod s^iiiptoms soon after tho plant becomes diseased. Tho symptoms on tho cu- cumber have been described in greater detail, as it is the most im- portant host and has been most closely studicnl, but the general characteristics will apply to all otlior hosts except for the variations mentioned later. riAXT SYMI'TOMS. Siimpfoutft on (iffeclcd .see(Za>? 7.9.— Seedling cucuml^oi-s nns rarely affected with mosaic either in tho field or in tho greenhouse. The disease may aj)poar on seedlings under certain conditions, ]iow(>V(?r. as is shown by the fact that a very general and serious mosaic inf(H'- tion occurred on the first true leaves of cucuml)er seedlings at Big Rapids, Mich., in tho summer of lOlS. The seed was plantcMJ bite in August and when the plants came up the re^st of the iield was already rather badly infected with the disease. When such infection does occur, the first symptoms appear as a yellowing and wilting of tho cotylcHJons, accompanied b}^ a slight mottling of t!u> young loaves. A sudden chocking of growth soon follows and tho latci leaves remain small, wrinkled. mihI distort. m! iind have a distinct Bui. 879, U. S. Dept. of Agriculture. Plate I. iH dp ' ■ '■.* ' '^9 M ss9 1 1^' s^iik^yo ;».<•%•*» »>■'■ s;^ •^•^^:^- ^^ -^ i^ 4j.? ^V^^ 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 savoycd ellect on the smaller leaves: Big Uapids, Mich., 1910; C, a cucumber field at Holland, Mich., on August 19, 191.5, showing severestunting and loss to the late-planted crop due to infection w hile the plants were small; theearly planted portion in the background was not so badly allected. Photographed by ^\. W. Gilbert. Bui. 879. U. S. Dept. of Ai:rirulture. Plate II. Mosaic Cucumber Plants. A , Mosair cuciimldT plant infcctwl whili' voiiiik, sliowlnR ilwarCmc and curling of the Iravos and shortcJi- iiiK of tlio st.in'.; (ir.-clcy, Colo., SoptcmlM-r, 1'.I17. rhot(i>:niplii'd by W. \V. (iillxTt. H. Cuciimhcr mosjiic frrnii iiiiMiilulion "with apliiils from ini>saic plants, the fruit svmplonis Im-Uik most marked: Hig JCaplds, Mich., I'.il... rtiotov;raphi"il l.y \V. \V. (iillx-rt. r, .Mosaic plants in late stanc showing death of till' l)ii.sal leaves, whitening of the main stem, uud the dwurliug and curling of the tij) loaves. Photo- graphed by M. \V. Gardner, September, lUlO. 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 1 2 inches. Symptoms on 'plants infected wTien 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 to run. 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 loaves 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 smaller 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 sUghtly 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. II, A). All growth subsequent to infection is mucn dwarfed, the stem intornodes are shortened, the leaves attain only about one-half nor- mal size, and the petioles are reduced in length. As the plant be- comes older the wrinkled and savoyed character of the leaf is more marked, but the motthng is generally less conspicuous. Plants infected at this stage blossom sparingly and set few fruits. Thoy send out few runners and have a bunched and bushy habit of growth, with the leaves Ijmg close to the ground in a rosettelike clump. Siimptom,s 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 Ion ves turn yellow and wilt, their edges becoming brown and withered, and all the leaves of a shoot may tlius wilt within a few days. Ordi- narily, however, only three or four leaves at the growing point are afi"ectod in this way, and the runner soon sends out a new shoot just b;u^k of the withered tip, which shows the ordinary mosaic sj'mptoms. 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. II, B). Frequently the most pronounced 10 Bl'LLI-TIN 879, U. S. DKPAKT>rKNT OF AGRICULTURE. leaf syiiij)t(>iiis app(»ar on new swoudary shoots put out from tli(^ axils of the leaves near tho haso of tho ])lants. Tli<» wilt symptoms are often noted on tho cucumber and pumpkin and occasionally on the Summer C'looknix k scjuasli and muskmelon In till* t,Teenhouso tliis typo of icsponso is much more intens<», and it is conmion to have tho vine suddenly develop a yollowinnj and wiltini;, wliieh extends to the entire plant within two to three days, the older leaves usually sliowini; yellowinu; hut wiltinj; very rapidly. This wilting and death of the plant occur only occasionally in the fi(»ld and never prot^ressso rapidly there as they do in tho <;re(^nlious»v Aside from these diirerences, 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 ])ronounced and the mottling is of the same tyj)e as that on younger plants. The shortening of the stem internodes and the petioles gives the ruimers a fhittenod appearance, and the leaves lie close to the ground. f 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 basal lobes. The tissues of these j'ellowed portions rapidly turn brown and die, l)eginning at the edges in the angle formed i by the lobes and working toward the base of the lateral veins. (PI. ' III.) Tlie 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. Tliis is a characteristic symj)tom 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 still attached, but terminating in a cluster of dwarfed, dark-green, mottled, and ^v^inkled leaves wliich lie 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 ftymptoinfi on cneunihers. — On the (•ucuml>er (Cunimi'i sativus) the mosaic mottling is usually most pronounced and tyjiical on the young leaves of plants which are growing rapidly (PI. IV, A). Tho green portions of these leaves are very dark in color and are raised slightly ai)ove the surrounding surface, thus giving the leaf its savoyed appearance. In most cases the green areas predominate, being intermingleil with spots of light greenish yellow. These yellow sj)ots are irre«riilar in outline and are limitetl bv the small veins (PI. IV, li). THE MOSAIC DISEASE OF CUCURBITS. 11 In some cases tlie green and yellow areas are both small and about equal in number (PI. Ill) , being scattered irregularly over the leaf 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 j^oung leaves is usually very noticeable, but there is less contrast in color. Tlie 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 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 wliich 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 w^here such a mottling occurs, producing a peculiar wilted appearance in the, lobe of the leaf 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. Tliis 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 l)ase 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. Ill) . 12 BriJ.KTIN «70, IT. S. DF,rART>rKNT OF AGRICULTURE. Another syiMj)t()in on tlie oMi'i- lojivcs which is nioiv common in the grponhouso than in the fichl, consists of a yellowing which alTects nearly all the leaf at the same time. In such leaves the tissues ailjoining the larger veins retain their color longest and outline these veins against the yellow of the remainder of the leaf. These symp- toms are usually associated with the wilting type of tht disease. Leaf sijmptoms on inusk melons. — 'Hie leaf symptoms on the musk- melon {Cucumw melo) are very similar to those on the cucumber. The lii*st 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 (PI. 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. Tlie older leaves gradnallv become yellow, but show little or none of the tendency to die early which appears in mosaic cucumber leaves of the same age. Leaf symptoms on squashes. — In the case of the squash (Cucurbita spp.) the symptoms of the disease are much the same on all varieties. The 3'ounger loaves 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 3x4lowdsh green and tend to coalesce and form a few large blotches rather than the smaller and more numerous spots found on the cucund)er (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 for some time, the symptoms of the disease being confined to the younger leaves. The most marked symptoms have been found on the Summer Crookueck and Cocozelle bush varieties {Cucurbita pepo Yar-condensa), those of the Hubbard type (Cucurbita maxima) showing the disease in a less extreme form. Leaf symptoms on pumpTcins. — 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 of ten show a rapid yellowing and wilting which corre- sponds very closely to that found on the cucumber in the gi(>cn house. Leaf s-ijvipfonis on oilier cuciirhits. — The general symptoms on the gourds and (»|Ium' ornamental cucurbiisi are very similar ((► those on the squash. The chi(>f points of difTerence occur in the patterns of the mottled Icaxcs. In the case of the (^ucurbita i;(»urds the surface Bui. 879, U. S. Dept. of Agriculture. PLATE III. AiloenLCoBallrn MOSAIC LEAVES, FRUITS, AND BRANCH OF CUCUMBER. Three fruits illustrating cominon types of mosaic effect; two leaves, the smaller showing pro- noimced mottling, comroon to yoiing leaves, the larger showing dying of older leaves on plants infected when nearly mature ; tip of plant with dwarfed and dying leaves. Bui. 879, U. S. Dept. of Agriculture. PLATE IV. Mosaic Cucumber Leaves. A , Small cucumber leaf, showing extreme mottling and wTinkling 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. 13 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 Luffa and Lagenaria gourds, the spots in the latter species being larger and very nearl}^ circular in form. (PI. V, A.) On the wild cucumber {MicrampeUs 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 (5f light yellow which are similar to those noted on the Cucurbita gourds. Tliese 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 composed 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 shoMang 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 other 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 tlie case of the cucumber. FRUIT SYMPTOMS. Finiif sympfoms on cucamhers. — 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 lU'LLKTlN 87S), U. S. DKPARTMENT OF AGRICULTURK. fruit fii-st l)o<'om«'s mottlod with yellowish groen, this mottled char- artor giadiiiilly sjnoadiiij^ ovc^r tho oiitiro fruit. As tiiis pros^rcsses tliobod}' of tho fruit ordinarily hecomos a li<;^ht yellowish jrreen, intor- niinp;l(»d with spots of a much darker groon 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. Till* numhei- and size of these protuberances vary greatly, and we find many tj-pes of affected fruits (PI. VIII). In some cases, particu- larly in tho greenhouse, the mottled character is very marked, the fruit being coverotoms, hut these symptoms have not as ye heen noted on any other variety of i)umpkin susceptihle to the disease Tlie young fruits are distinctly mottled ^vith light yellow, and tin surface is covered with large dark-green warts. (PI. V, C.) At matur ity the fruits are irregular in form, deeply furrowed, and the wart] chtu'acter is very pronoiniced. The fruits of most of the squashes, gourds, and the ornamental CU' curhits seem to he little affected hy the disease, and on many of thes< hosts no symptoms have as yet heen noted. The fruits of the mo saic wild cucumher { }f'i cramp elis JohaUi), however, are usually dwarfed, distorted in shape, and smoother than the fruits of normal plants, the spines l)eing fewer in numher and scattered unevenly ov the surface. In a few cases fruits have heen noted which devel oped rather large irregular swellings. The epidermis of the fruii eventually splits open at these points and the tissues helow push through to form dark-green sw^ollen areas, which are somewhat similar to those found on the cucumher. (See PI. VII, D and /''.) These symptoms apparently do not occur on all the mosaic fruits of this host, hut they have heen noted on a numher of plants. ITie fruits of the l-seeded hur cucumher (Sicyos nngnlatus) , on the other hand, do not seem to show any evidence of the disease. BLOSSOM SYMPTOMS. Mosaic cucumher flowers are not streaked or variegated as are those of tohacco plants affected with mosaic. Tliey are greatly reduced ini numher, however, especially the pistillate flowei-s. 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 hlossoms. On other cucurhits the symp- toms are of the same character, thoujrh the dwarfinj]: is usuallv leas pronounced except in the ciusc of the muskmelon, the hlossoms of which are usuallj' reduced in size and numher and are a much lighter yellow than those of normal plants. , I STEM SYMrrOMS. (I The stems in the case of the cucumher are shortened, as noted ahovc (PI. II, .4), and where the older leaves have died the ejiidermis ^ Bui. 879 U. S. Dept. of Agriculture. Plate V. Mosaic Melon and Gourd Leaves and a Pumpkin. A, Mosaic leaf of a Lagenaria gourd, inoculated from cucumber; Madison, Wis., 1017. B, Mosaicleaf of a cantaloupe, natural infection; Big Kapids, Mich., 1U17. Photographed by W. W. Gilbert. C, Mosaic fruit of pumpkin, showing large dark-green swellings on a yellow background; Madison, Wis., 1919. Bui. 879. U. S. Dept. of Agriculture. Plate VI. LcAF AND Fruits of Mosaic Summer Crookneck Squash. '^k}^\l"i '/■'"'l'' •"'""""•■'■ V""'5"r'''' •'^'l'>n'='>. Mii'lison, Wis., 1917. B an.l C, VomiK fruits of mosaic .'*iimrn.T < rrM.kiHNk s-iunsli o^ ^^'^P^"'"^ "f »'"' ''iseiuso; B, from Irondequoit, N.Y. (photographed by W.W. (iillKTt),an\![h' r'' ^^r''%fJ(!"''""''''''l ( " ^"Z" '. -^IV""''"^ ™^'"'^'''^' mottling, malformation , and wrinkling. dSSiF Fn tsof l^'.r™ Y,"."?'?' ''■''^ "^ -U>cramiHlisiohafa. for comparison with' mosaic leaves. r^,■r:t,\uLu^ of l/;rrampf/;s/o6a?a from a mosaic plant, sliowiiig tlie irreRular shape and the wartliJ^e protuberances combined with the splitting of the outer integument; Madison, Wis. wartlike Bui. 879, U. S. Dept. of AErirulture. Plate VIM. Mosaic Cucumber Fruits. '*f.,«"Jmnnir.'.'"''n''.'!'T''i^ ^'''"r'' ""'"t';-l>''>irli-Kr..,.n warts a.i.iinimcr- w"k m. " • nt U JhlnBio, "'/"c ) "^ ^"^ "^ ^^ ^*^''-'" '*'**^''- ^^ ^^ ^' P''"t, V. S. DKPARTMENT OF AGRICULTURE. iKMirlv isodiaincJric and loss in ininilxM- per nnit aron than in the normal leaf. The sj)on}j:y |)aroncliyma of these yellowed areas is also more compact and tlie intercellular spaces smaller than in the green portions. The chloroplasts of the cells of the yellowed portions arc decidedly smaller than in normal cells and are often ])ressed 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. 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 occm' in the palisade cells of the dis- eased leaves. In the raised green areas, these cells are slightly I longer and narrower than in normal fruits, and the chloroplasts are larger and crowded closely together. In the 3'ellow portions of the diseased fruits these subej^idermal 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 hj'pertrophy. CAUSE OF THE DISEASE. The cause of cucurbit mosaic, like that of all other infectious chlorotic diseases, is unknown. WTiile 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 cucumbei-s in the lield have shown that the physical or chemical nature of the soil a])par- ently has no relation to the origin of the disease. Mosaic \vill de- velop with equal rapidity on clay, muck, or sandy soils, and a|)|)lica- tions of lime, wood ashes, stable manure, or commercial fortilizere have little appreciable effect. It has also been impossible to con- nect environmental conditions with the disease in any way which indicates that it origimites from any unfavorable condition of soil, temperature, or humidity.' ' Recent InvestiRutlons iinlicate that envlronmontal conditions, particularly temperaturp, may affect the Buaceplibillty of the plant to mosaic, ami it is therefore probable that the rapidity with which the dUeose spreads is partially determined by the conditions of enviroumont. I 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 attemjjting 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 grow^th 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 from the 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 OP 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 inosaic 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 tlie 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 wath the 20 BULLETIN 879, U. S. DEPARTMENT OF AdRHTLTURE. virus of tho mosaic of cucurbits havo boon Ic^s oxtonsivo, thoy indi- cMto tliat it is similar in many rospocts to tliat of tol)acco and other j)ljnits. Tho ])owcr of infection may bo destroyed in each case and is (h'linitely connected with tomperaturo, moisture, age, and other factors. EFFECT OF IIKAT oN TIIK I'OWKK (»K INFECTION. The expri>ssed juices of mosaic cucuml)er plants lose their powor of infection when lieated above 70° C. Tliis temperature has proved tho limit in all exporinients, as is shown in Table III. In thase tests tlie ]>lant jnictvs were expressed and filtered throuf^h filter paper b(»foro u.s«v Small test tubes were used and 5 c c. portions of tho juice taken to insure ra])id heating to the desired temperature. A constant-tempivaturo water bath was used and the tubes heated 10 minutes and tlieii immediatt^ly cooled to room temperature. Inocu- lations were made, from the different tubes to healthy cucumber plants, inoculating into the stem, at the tip and base, and also into ■ two of the 3'oung leaves. These plants were protected from possible outside infection by covering them with insect-proof cages. Table III. — Effect of heat, on power of infection of expressed juices of mosaic cucumber plants. Temperature (°C.). Number of plants inocu- lated. Results. Date. Number of masaic plants. Date la. infectious material present in mosaic plants, since in these ca.ses the period of incubation was no longer than when the undiluted juices were used. KFFEer OF VAHKdS CHKMICALS ON THK VIRUS. Experiments with the effect of various chemicals on the virus of cucurbit mosaic show that the power of infection is easily destroyed by c<^>mmon 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. c. portions and 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 sho\\T[i 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 j)lants were caged from the beginning of the season. Cliloroform in a 10 per cent mixture destroys the activity of the virus, but a 5 per cent cliloroform 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. Table V. — -Effect of chemicals on the power of infection of the expressed juices of mosaic cucumber plants. Date inoculated. Chemical and strength used. Number of plants inocu- lated. Results. Number of mosaic plants. Date ob- served. Series I (field experiments): Aug. 10, 1917 Do Do Do Do Do Do Do Series II (greenhouse ex- periments): Oct. 25, 1917 Do..." Do Do Do Do Do Do Series III (greenliouso ex- periments): Mar. 28, 1918 ^ Do Do Do Do Do Do Do Formaldehyde 1 per cent.. Phenol 1 per cent CUSO4 1 per cent HgClo 1:1,000 Chloroform 10 per cent Toluene 10 per cent Untreated juice Distilled H2O (control).... Formaldehyde 1 per cent.. Phenol 1 per cent CUSO4 1 per cent HgCls 1:1,000 Chloroform 10 per cent Toluene 10 per cent Untreated juice Distilled H2O (control).... Formaldehyde 0.5 per cent Phenol 0.5 per cent CuSOj 0.5 per cent HgCl. 1:2,000 Chloroform 5 per cent Toluene 5 per cent Untreated juice Distilled HjO (control) . . . . Aug. 20, 191- Do. Do. Do. Do. Do. Do. Do. Oct. 31,1917 Do. Do. Do. Do. Do. Do. Do. Apr. 20, 1918. Do. Do. Do. Do. Do. Do. Do. 24 BULUCTIN 87!>, V. S. DEPARTMENT OF AGRICULTURE. ( IIKMK'AI.S AS VIUUH IiISINKKCTANTS K»K TIIK HANDS. In addition to tiio work with clicmicnls in direct combination witli mosaic juices, further tests were made U> determine tlie value of various solutions as disinfectants for the hands after handling mosaic plants. In these tests the hands were smeared witli the juices of a mosaic cucunil)er 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. Taum-: \ I. — Value of rcrious chemicah as hand disin/crfauls o/lrr handling mosaic ciiruiiihcr plants. l)atc inoai- Chomicols and strength ^ used as disinfectants. Number of plants handled. Aup. 17,1916 Formaldehyde 1 per cent. Do Formaldch.vdo 2 i>er cent. 1)0 Phenol 2 [lorcciit Do I'hciiol.l percent Do HgClj 1:1,000 Do Soap and water , Do Dbtilled water Do I'ntrcatod mosaic juices.. Au^'. IS, 1917 i Formaldehyde 1 percent. Do I'heiiol 1 p«'rcent Do ('uSO< 1 percent , Do HuCl, 1:1,000 Do Soap and water Do , Distilled water Do Untreated mosaic juices.: Results. Numlier of mosaic plants. Date ob- served. Aue. 26, 1916 Do. Do. Do. Do. Do. Do. Do. Ann 2S, 1917 Do. Do. Do. Do. Do. Da 4 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 liigh j)ercentage of infection when no wash was used. EI-FECT OF FILTRATION' ON THE VIUUS. lirrl-tfthl Jiltfr. — The presence of n liitcrahh' \ iius as tlje causal factor in cuciiibit mosaic lias ah-cady been th'monstratcd. lioth Jjij^ger (17) and the writer (1 1) have shown that the juices of mosaic cucumber plants retain their power of infection after passing through a lierkefeld filter. The work on this phase of tlie problem lias been continued by the writer, using both the lierkefeld and tli(> Chainberland types of liltei"S. In the tests with the lierkefeld filler the juices of mosaic cucumbers were expressed and passed lliidiigh double lilter i)aper. .Vfter this THE MOSAIC DISEASE OF CUCURBITS. 25 filtration the juices were at once passed through a sterile normal Berkefeld bougie, the bougie, receiving flask, 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 removed 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. Tliese 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. Table VII. -Effect of fiUrafion through a Berkefeld (normal) filter on the infectnity of the expressed juice of mosaic cucumber plants. Date inocu- lated. Aug. 31,1915 Do Do Sept. 6,1916 Do Do May 12,1917 Do Do May 18,1917 Do Aug. 30,1917 Do Do Treatment. Filtered mosaic juice Unfiltered mosaic juice Unfiltered healthy juice (control) Filtered mosaic juice Unfiltered mosaic juice Unfiltered healthy juice (control) Filtered mosaic juice Unfiltered mosaic juice Unfiltered healthy juice (control) Filtered mosaic juice Unfiltered mosaic juice Filtered mosaic juice Unfiltered mosaic juice Unfiltered healthy juice (control) Results. Number of plants inocu- lated. Niunber ofmosaic plant.s. Date last observed. 6 4 Sept. 15, 1915 6 5 Do. 6 Do. 8 5 Sept. 14,1916 / 5 Do. 8 Do. S 3 May 21, 1917 S 6 Do. 8 Do. 10 5 May 31, 1917 10 6 Do. 5 2 Sept. 12,1917 5 3 Do. 5 Do. Chamherland 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- land filters, but no positive results have been obtained. Tlie 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 Lll. The L2 and L3 grades were supposed to be permeable to the more minute forms of bacteria, and the other 26 BITLLETIN 87f>, IT. S. DEPARTMENT OF AGRICUT.TURE. types wore supposedly of suHicuMit density to prevent the pjtssjige of visible organisms. It Wfts hoped thiit a eompurison of (lie iiltrfttcs from these (hlferent grades of filters woidd ])erhaps indicate the size of the particla^ responsible for infection, but the results did not warrant any con- clusion alonj^ this line, as no infection has occurred in a total of more than foo 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 at mom- eter. A fdter made of a layer of powdered talc from seven-eightlis to li inches thick also gave a noninfectious filtrate. Similar talc filters have been used in experiments with cucumber mosaic, the layer of talc varying from three-fourths of an inch to 1 inch in thick- nass. Three trials have been made with these filters, and a total of 37 plants have hcon inoculated from the fdtrates. Tlie 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 e^'ident 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 tj'pes. ENZYTIS IN RELATION TO THE DISEASE. Since many writers have advan^^^ed the theory that enzyms, par- ticularly oxidases, arc in some way connected with the mosaic of tobacco, a few attempts were made by the writer to isolate a pos- sible causal enzym from the Juices of mosaic cucumber ])lants. The fact that the juices of such plants usually lose their })ower of infection ^v•itl^n 24 to 48 houi-s has proved an almost insurmountable obstacle hi such work,. and so little has been done that it is not fcjusible to draw any definite conclusions. Tests of the juices of healthy and di.seiLsed 1)1 ants wlu, U. S. DEPARTMENT OF AGRICULTURE. regard. 'IMic juices of both luosiiic tobacco and cucumber ])lant8 may be diluted to 1 : 1,000 \\'ith(»ut aHectin*^ the power of infection, and dilutions oi 1 : 10,000 will also ])roiluce the disease, ^ivinj?, how- ever. H lower j^ercenta^e of infected j)lants. Tln' tobacco \'irus will pass through both the normal Berkefel filters and those of the Chamberlantl type, while the cucunil^er virui passes only tlu'ough the former. This diderence is probably of mino: importance, however, as Allard (4) has shown that the juices ol tobac<-o mc^saic are also rendered noninfectious if passed through i j)orous clay atnionieter or through u :{-inch layer of powdered talc so that the beliavior of the Airus of both diseases is essentially tht same as regards filtration. POSSIBLE NATURE Or 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 (."^2), Koning (21), and more recently Freiberg (\'S) and Chapman (7) have held that enzjTns, particularly oxidases, peroxi- dases, and catalase, are in some way connected with the cause of tobacco mosaic. iUlard (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 t accord better with the theory advanced by ^\llard than with th enzymic h>T>othesis. 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 supj)ort 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. Tlie 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 th(» plant, regai-dless 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 whicli seems to support tlie tlieory of an organism as the cause of mosaic is th<' ability of tlie juices of mosaic plants to prudiicr infection in a dilution of 1 : 10,000. IVo or three drops THE MOSAIC DISEASE OF CUCURBITS. 29 of such a dilution when pricked into the leaf of a health}^ plant wiJl produce the disease as rapidly and with as intense symptoms as when the undiluted juices are used. 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 thi'ee 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 enzyras. The behavior of the virus in filtration may be used to support the theory, although the fact that the fmer 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. Tlie epiphytotic character of cucumber mosaic and its rapid spread to plants adjoining 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 ehminated 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 BUIJ.KTIN H7!), V. S. nF.PARTMKNT OF AGRICULTURE. I •aircS cage as a partial shield (lurin, U. S. DKrARTMliNT OF ACKICULTURK. This method is proforrod chiefly hocause the preparation of the expressed juice re(iiiires time, and such extracts, unlike those from mosaic tobacco plants, lose their pathogenicity rapidly, usually haviu}^ to be prepared fresh every day to insure success. In the case of the second method, fresh material is constantly availa])le and its preparation takes little time. Table IX f2;ives results which are ty])ieul of inoculations by this method, but represent only a small fraction of the inoculations actu- ally niiid(\ Table IX. — Results of inoculations with the crushed tismes of mosaic cucuviber plants. Drtfe inoculated. Trentmcnt. Inoculum. Point of inoculation. Number of plants inocu- lated. Rosolts. Number of masaic plants. Pate lust observed. .\ug. 24, 1915. Do Jan. 22,1916 •N'ov. 3,1916 Do Feb. 17,1917 Do .Mar. 11,1917 Do Mar. 15,1917 Do Do Mar. 29,1917 . Do May 19,1917 Do Jime 20,1917 Do Inoculated. Control Inoculated. do Masaic .stem... Healthy stem. Mosaic leaf do Base of stem . ....do do do Control Inoculated. ("onlrol Inoculated. Control Inoculated, .do. Control Inoculated . Control Inoculated . Control Inoculated. Control Health V leaf.. Mosaic leaf... Healthy leaf.. Mosaicleaf... Healthy leaf.. Mosaic stem.. Mosiio fruit .. lloallhy fruit. Mosaicleaf... Ilcalliiy leaf.. Mosaic leaf. .. Healthy leaf.. Mosaic leaf. . . Healthy leaf.. do Tip of stem. ,do. I$a.se and tip of stem.! do I Base of stem do do Base and tip of stem do do do Tip of stem do Sept. 2,1915 Do. .Tan. 31,191f. Nov. 10, 191t) Do. Feb. 2S, 1917 Do. Mar. 19,1917 Do. Mar. 22,1917 Do. Do. Apr. 9, 1917 Do. May 30,1917 Do. Julv 4, 1917 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 wounds produced by the removal of a leaf are ordinarily more effectivo than at other points, possibly because the virus comes more directly in contact \vith the vascular bundles at such points. LEAP INOCULATIONS. Inoculation of any green leaf will produce mosaic infection, but the inoculation of young leaves gives a much higher percent.age of mosaic plants than the inoculation of loaves that have reached their full development. Th(^ 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 (he trichomes. He hivs shown that infection iiiiiy occur if the trichoiiu-.s of healthy plants aii' cut or pinched with instruments which have previously been dipptMl in the juices of a mosaic phint or when tlui mosaic juices jire pjiinted on the trichomes. Similar experiments have been made with the cucum- Bui. 879, U. S. Dept. of Agriculture. PLATE IX. ■■--«■*. ■-^ fb '^'■%t ^^ 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 niaking inoculations Photographed l^y W. W. Gilbert. B, Soil overwintering test at Madison, Wis S^tember 19 1917. This land grew a mosaic crop in 1916. The few large healthy plants were grown imde?cages' All the uncaged plants became diseased early and were dead or severely stunted. An insect hibernation cage is seen at the left. Bui. 879. U. S. Dept. of Agriculture. Plate X. Mosaic and Healthy Cucumber Plants from Commercial Seed. A, Mosaic plant ^rown f rom com iiiiTcial seed in aKreoiiluniscat Mailison. Wis.,in 1917. Note the dwarfing tt.scointmrtsted that old(>r ])lants may contain the virus but show the symptoms in so mild a form that they can not be detected. However, this has aj)parently been disproved by making inocu- lations from old plants which had been previously inoculated with I Tho term virus Is used here In tho commonly aooopt«d sonso of the lofooUvo agent o(a diaeaso with whicl< iiu visible organism hus boon iissocIuUhI, p THE MOSAIC DISEASE OF CUCURBITS. 35 mosaic but showed no symptoms of the disease. No such plant has ever shown evidence of containing the virus, as all inoculations from them gave negative results, and it is probable that a definite differ- ence in susceptibility occurs, depending on the age of the plant. LOCATION OF THE VIRUS IN THE PLANT. The virus of curcubit mosaic is distributed throughout practically all parts of the plant, with the possible exception of the roots. The data given in Tables VIII to X show that the juices of stem, leaves, and fruits of mosaic cucumber plants are all capable of producing infection. All the living leaves of a mosaic plant contain the virus, regardless of age or the presence of actual mosaic symptoms, its thorough distribution in the leaf cells being shown by the results of inoculation by contact of healthy and mosaic trichomes. VIRUS IN THE FLOWER PARTS. The disease can also be produced by inoculation with the flower parts of diseased plants. Inoculations were made with petals, anthers, and stigmas of mosaic cucumber plants by carefully dis- secting out with sterile instniments the portion of the flower desired without breaking other parts and crushing the niaterial in a sterile dish with a few drops of sterile water. This crushed mass was then used for stem inoculations, the results being positive in each case, as shown in Table XII. Allard (3) has shown that the virus of tobacco mosaic is also present in the anthers, filaments, and even in the placental column and integument of the ovule of infected plants. Table XII. — Results of inoculations with the crushed tissues of flower parts of mosaic cucumber plants. Treatment. Inoculum. Number of plants inocu- lated. Results. Date inoculated. Number of mosaic plants. Date observed. Sept. 7,1916 Do Inoculated .' .. . do . . Corolla of mosaic blossom 3 3 3 10 8 8 8 10 8 3 3 2 3 5 6 Sept. 23,1916 Stigma of mosaic blossom Do. Do.. . . do Do. Mar 12 1917 do do Mar. 21,1917 Do . Control Anthers of healthv blossom Do. Do Inoculated Do. Do StlRtna of healthy blossom Do. Do Inoculated Do. Do... Corolla of healthy blossom Do. VIRUS IN THE FRUITS. Inoculations made with the crushed tissues of mosaic cucumber fruits have shown that the virus is apparently present in fruits of all ages. The inoculum was prepared by removing small i)ortions of the tissues close to the ovule with a sterile scalpel and crushing them in a sterile dish, the inoculations being made in the stems of 36 BULLKTIN S7!», U. S. DEPART.MKNT OP' AGRICULTURE. licalthy cucuiiihrr plants by ihc liu-thod clt'scribcd on panics 30 lo 32, Tho fruitij used as inocula were of various a^es, mcludinj; young fruita about ]\ inclios in length, large, partially niatui-e fruits that were beginning to turn yellow, and yellow and matured fruits such as art collected for seed. The very young and the partially mature fruitj showed the presence of the vims in all cases (Table XIII), and whiU the inoculations from the mature fruits were not so constantly suc- cessful they produced the disease in a number of plants. The pres- ence of the virus in the ovule itself has not been demonstrated, as if is diilicult to remove the ovules from a watery fleshy fruit like the cucumber without carrying traces of tho juices of other parts of the fruit. Tahi.k XIII. — licsttlls n/ itioi-u lotions with crushed tissues of mosaic rucvmhcr fruits of various ages. r>atc inocu- lated. Treatment . Inoculum, cmshctl tissues of— Number Point of inocula- of plants tion. inocu- lated. Results. NumbCTJ Pate last Sept. 10, 1917 Do Do Inoculated . do -do. Do Sept. 20, 1917 Do Control Inoculated. do Do. Do. do.. Control . Young mosaic fruit Partly mature mosaic fruit. Mature, yellow mosaic fruit. Ilealthy fruit Vouni; mosaic fruit Partly mature mosaic fruit. Mature, yellow mosaic fruit. Healthy fruit Base and tip of stem. do -do. ..do. ..do. ..do. -do. .do. Sept. 21, 1917 Do. Do. Do. Oct. 14, 1917 Do. Do. Do. VIRU.'^ IM THE M.\TUKK SEKI). Although the young fruits and even fruits at maturity may have the virus present, as is shown in Table XIII, it has never been de- tected in any portions of the seed which have been removed from the fruit and subsequently dried. The drying process probably accounts for its absence from the seed coat, since the juice of mosaic cucumber plants soon loses its infectious quality if subjected to desiccation. Tests have been made of both seed coat and embr^'o in 37 inocula- tions, the material being crushed in sterih* distilled water and both the water extract and the crushed tissue used for inoculation, but no infec- tion has ever occurred. Allard (3) has found that the virus of tobacco mosaic may bo present in the mature dry seeds of mosaic tobacco plants, and this is consistent with the fact that the virus of tobacco mosaic is more resistant to drying than that of cucurbit mosaic. At the present time, however, there is ratherdefinite proof that the mosaic disease of cucumlxM- may be carried over in the seed. (PI. I, A.) If such is the case, it is possible that the virus is occasionally present in the seed, altliough perliaps so rarely that a large number of inocula- tions would be necessary to demonstrate its j)resence. THE MOSAIC DISEASE OF CUCURBITS. 37 VIRUS IN THE ROOTS. Inoculations from the roots of mosaic plants have so far given negative results, and it is doubtful whether the virus is present in these parts as in the remainder of the plant. Allard (1) notes that the roots of mosaic tobacco plants contain the virus in many cases. In the case of cucumber mosaic, inoculations have been made from the roots of plants of various ages, using either the expressed juice of the root tissues or crushed fragments of the tissues themselves. Inoculations have been made into the stems, leaves, or roots of 58 plants, but no infection has occurred. VIRUS DISTRIBUTION IN THE PLANT. RAPIDITY OF SPREAD OF THE VIRUS. In all inoculations with cucurbit mosaic the earliest signs of infec- tion appear in the youngest leaves. This is also the case with to- bacco mosaic and other diseases of this type, the leaves which are in process of development seeming to respond most rapidly to the disease stimulus. The time required for the development of these symptoms is usually the same, regardless of the point of inoculation, indicating that the virus is rapidly distributed to all parts of the plant. The youngest leaves of the cucumber develop mosaic sjinp- toms as rapidly when inoculation is made at the base of the stem as when made in the leaf itself. This does not prove that the virus is im- mediately distributed throughout the plant, however, for the infective principle is present in the cells for some time before visible S3anptoms occur and it is probable that the virus is present in greater amount near the point of inoculation for at least a short time after inocula- tion. The older leaves, however, do not show definite signs of the disease for some time, and no external indication of the disease ap- pears until the young leaves develop mosaic symptoms. The accu- mulation of the virus at the point of inoculation earlier than at the growing tip has been demonstrated in large cucumber plants where inoculation was made at the base of the stem about 3 inches below a small lateral shoot which was 6 inches in length. The leaves of such a shoot showed the symptoms of the disease 12 hours before it appeared in the young leaves at the tip of the main runner, which were 30 inches beyond the point of inoculation. This occurs occa- sionally in large vines, but in most cases there is no appreciable dif- ference in time between the development of s}Tnptoms at various pomts, and the distribution of the vii-us through the plant must be comparatively rapid. Experiments have shown that the juice of an inoculated plant may be infectious 18 to 48 hours previous to the appearance of any definite mosaic symptoms and also, as already indicated, that the juice from leaves near the point of inoculation may become infec- 38 BULLETIN 870, U. S. DEPARTMENT OF AGRICULTURE. tious 12 hours earlier than that frorii h'avos at more distant points. In those tests cucumber j)lants were inoculated cither at the l)ase or tip of the stem, and other ])lants were then successively inoculated with fragments of leaf tissue from various j)oints on the plant first inoculated. These later inoculations were made every 1 2 or 24 hours. The results of this work are given in Tabic XIV. The fact that mo- saic diseased plants possess the power of infection before the appear-j ance of visible symptoms is a most important factor in its relation taj disease dissemination and control. Efforts to control cucumber mo-l saic in the held by the eradication of all diseased plants as soon they appeared have met with little success, largely because of th( fact that a great number of plants were constantly serving as a sourc< of infection cluring the period wh(>n their juices were infectious, bu( before visible s^onptoms had appeared. Table XIV. — Occurrence of mosaic virus in inoculated cucumber plants prior to the appearance of visible symptoms. ^ Plant No, Date of first inoculation. Point of inocu- lation. Date first symptoms were noted. Part used for secondary inoculations. Date of first successful secondary inoculation. -) Time • virus , was present before first . sjTnp- toms appeared (nours). July 18,1910 do ....do ....do ....do Aug. 8, 1917 Tip of stem.. Uaseofstcm. Tip of stem . . Base of stem . Tip of stem... | Base of stem. July 22,1910 July 24,1916 do ....do ....do July 25,1910 do \ug. 15,1917 Aug. 16,1917 Leaf at tip of stem do do Leaf at base of stem Leaf at tip of stem Leaf at base of stem Leaf at tip of stem Tip leaf of shoot inches from poiut of inocula- tion. Tip leaf of m:un stem 30 inches from point of iuoculatiou. July 22,1910 July 23.1910 do do do. July 24,1910 JiUy 23,1910 Aug. 13, 1917, 6.45 a. m. Aug. 13, 1917, 4 p. m. METHOD OF DISTRIBUTION OF THE VIRUS. The distribution of the virus tliroughout the plant is probably effected either by dilfusion through the parenchymatous colls or by conduction through the vascular elements. While the que-stion is diflicult to determine definitely, it seems more probable, in the writer's estimation, that the vascular system is most important, although diffusion may also be a factor. When inoculation is made at the base of the stem, the fii-st symptoms, which invariably appear in the young leaves, develop as rapiilh' as when inoculation is made directly into these leaves. It thus appeai-s that the distribution is rapid, since the young leaf tissue may contain the virus within tliroe days after inoculation in sudicient amount to produce infection in other plants. It would seem that dilfusion alone would be too slow to allow the virus to reach all parts of the plant in as short a time as THE MOSAIC DISEASE OF CUCURBITS. 39 it does when inoculation is made in the stem tissues. The fact that the pores of a Chamberland filter can hold back the particles of infectious material also leads to a belief that such particles may bo colloidal in nature and not likely to pass readily through the cell membranes. Allard (5) has shown in the inoculation of older leaves of tobacco plants that severing the lateral veins from the midrib or cutting the base of the midrib itself does not appreciably increase the time usually required for the virus to reach the stem and pass to the young leaves. He believes that in such cases "multiplication and diffusion of the virus from cell to cell, aided perhaps by the fine anastomosing lateral veins, would sooner or later allow the virus to reach the petiole and pass to the rest of the plant." The writer is inclined to believe from the results of work with cucumber aphids that conduc- tion through the fine veins is a more important means of transference than diffusion. These insects apparently attack only the small veins of the leaf, as shown both by observation and by stained microtome sections of aphids killed and embedded while still attached to the leaf. In all such sections the sucking apparatus of the insect extended into the small leaf veins. Further support of this fact is given by Woods (31) in his work on stigmonose of carnation, in which he shows drawings and photomicrographs which indicate that the aphis attacks the vascular elements. Since aphids are the most consist- ently successful agency of inoculation for cucurbit mosaic yet dis- covered, it seems likely that their high percentage of infection is due to the introduction of the virus at a point where it is most rapidly carried to all parts of the plant. Stem inoculations made by cutting off a petiole and pricking the juices of mosaic cucumber plants into the region about the bundles of the leaf trace give about double the percentage of infection that occurs when the virus is pricked into the stem at random, indicating again that the vascular system is concerned. The portion of the vascular system especially concerned is still rather indefinite, but the work with aphids again furnishes a clue. Woods's work before mentioned (31) indicated that the soft bast parenchyma cells of the phloem were the ones particularly attacked. Other observers have also recorded the same facts, and the \\Titer's work along this line, although not extensive, indicates that the cucumber aphis punctures the cells of the phloem. If this is true it may be possible that the pliloem is at least as important as the xylem in the distribution of the virus, since the inoculation work with aphids is so universally successful. Beijerinck (6) is of the opinion that the phloem is the means of distribution of tobacco mosaic, since inoculation of the older leaves first produces symptoms in the young leaves, and he therefore concludes that the virus is carried in the descending sap flow. 40 BXTLLKTIN 87f), U. S. DEPARTMENT OV AGRICULTURE. All tho nl)<)V(> conclusions arc Tncicly tentative, liowcvcr, particu- larly in rcf]jar(l to the rOlc of tho ])hl()em in tho distrihution of tho virus. The vascular elements, however, seem to offer the most pro])ablo channel for the rapid distribution of infectious material through the plant. INCUBATION PERIOD. Tiio incul)ation period of cucurbit mosaic varies somewhat with the age of the plant inoculated. In young cucumber plants which ai'o growing rapidly the lirst visil)le signs of the disease may appear within 4 to 5 days following inoculation, and the incubation period in such plants vnW rarely exceed H or 9 days. In the case of older plants which are growing less rapidly, the period of incubation is often longer and the first symptoms may not develop for 12 or 141 days, although these plants generally do not require over 10 days] for the appearance of the symptoms. ' Tlie incubation period for plants of the same age and vigor of growth is quite constant, and where several plants are inoculated at the same time those which are infected show the signs of the disease within 3 to 4 days after the earliest symptoms appear. Plants which do not develop mosaic sj-mptoms \vithin this period usually remain healthy. The incubation period of the other cucurbits is very nearly the same as that of the cucumber and varies within approximately the same limits. In the case of large scjuash and gourd })lants the incu- bation period is slightly longer, ordinarily, than that of most other cucurbits, and usually extends over 12 or 15 days. On the younger plants of all species, the first symptoms will normally develop within 7 to 9 days after inoculation. Tlie general incubation period may be said to lie between 4 and 15 daj's, with the average period varying from 7 to 12 days for all species. MOSAIC TRANSMISSION. I Many mosaic diseases are of such a nature that field operations, in which, healthy and diseased j)lants are handled in succession, tend to further disseminate the disease. This has been demonstrated by Hunger (15) for tobacco mosaic and is particularly true with the mosaic on cucumber, the method of growing and harvesting the crop being such as to favor the spread of infection throughout the season. As infection may occur whenever the juices of a mosaic plant come in contact with slight wounds in healthy vines, any thinning, training, or other handling of mosaic and healthy j)lants may jiroduce infec- tion. That nieic handling of the ])lants may ri'snlt in disease trans- mission has been j)r<>ved by bruising the leaf of a mosaic vine between the fingers and then handling the leaves of healthy i)lants in a simi- THE MOSAIC DISEASE OF CUCURBITS. 41 lar manner, but without crushing them. The results, shown in Table XV, indicate the ease with which infection may be brought about by this method. Table XV. — Infection resulting from successive handling of mosaic and healthy cucumber plants. Trealmont. Number of plants. Results. Date. Number ofmosaic. plants. Date observed. Aug. 24,1910 Po Aug. 11,1917 Do Mar. 12,1018 Do Leaves of diseased and healthy plants handled in suc- cession. Leaves of two healthy plants handled in succession (control). Leaves of diseased and healthy plants handled in suc- cession. Leaves of two healthy plants handled in succession (control). Leaves of diseased and healthy plants handled in suc- cession. Leaves of two healthy plants handled in succession (conirol). 5 4 6 6 10 8 3 4 Sept. 4,1916 Do. Aug. 30,1917 Do. Mar. 28,1918 Do. TRANSMISSION DUE TO CULTURAL OPERATIONS. TRAINING AND REMOVAL OF INTERTWINED PLANTS. The evidence reviewed above shows that handling may spread infection at any time, but it is more common later in the season when the plants have made a heavy growth. As the vines lengthen it is necessary constantly to train them back from the center of the row, and in picking they are also lifted and pulled about. Tliese opera- tions produce many small abrasions, as the adjacent vines are usually interlaced, and when a mosaic and healthy plant occur together these slight wounds very often result in infection. It is commonly observed where few insects are present that the disease spreads along the row to the plants adjacent to the first isolated cases of infection. That this is due to such means as those mentioned above was strikingly demonstrated at Big Rapids, Mich., in 1916. The mosaic appeared at several points in a small field, and during the remainder of the season, in an effort to control it, all plants showing the disease were removed as soon as the first definite symptoms were observed. It was noted that, 10 days after the removal of the diseased individuals, plants in the same row and immediately adjacent to them began to show symptoms of mosaic, the disease progressing down the rows in both directions from the original diseased plants. In this experiment the vines were removed carefully, with the idea of avoiding infection, but slight injuries were unavoidable, and it is evident that such infection is constantly occurring throughout the season. It is probable that the factor of trichome infection is of special importance in cases of tliis sort, since the injuries need be but slight to produce infection through this channel. I 42 BULLETIN 87r>, IT. S. DEPARTMENT OF AGRICULTURE. INFECTION UY WAl.KINO ON PLANTS. In many cases the ^'inos gi-ow so tliicldy that a certain amount of injury from treading on the ])lants during ])i(king is unavoitlnble, particularly where tlie rows are narrow. This ])robably has ])roduced infection in many cases, one of whicli was noted by IMr.- W. W. Gillx'rt in 1915 at Muscatine, Iowa. A party of seAenil persons had crossed a field of seed cucumbers where the growth was rank and many mosaic plants were present. In so doing, vines were necessarily crushed under foot. A field of healthy ])lants in the neighborhood was ^'isited soon after. The growth was hca\y in this secontl field also, and further tram])ling of the ])lants occurred. Three weeks later the ])ath taken through the second field was distinctly marked by mosaic infection, wliilo the remainder of the field was still free from mosaic. This doubtless was an extreme cjise, but it is likely that some infection takes ])hicc in this way in many fields. INFECTION UY PICKINfJ. In harvesting i)ickling cucumbers, they arc picked every day or every other day, in order to secure small fruits. The fruits are broken off l)y hand in rapid succession and the juices, which soon cover the fingers, furnish an ideal and rapid means of mosaic trans- mission throughout the season. This has been demonstrated, a>> shotvn in Table XVI, by alternately picking fruits from diseased and healthy vines. The fruits were merely broken off Mith the thumb nail and no effort was made to produce any greater wouncUng or ad- herence of juices to the fingers than is common in field operations. || Table "KYI.— Experiments to -prove mosaic infection of cucumber plants due to picking. Treatment. Number of fruits picked pr plant. Number of plants inocu- lated. Results. Dale. Number ^ ^ , » Aug. 5,1910 Do Oct. 25,1910 Do Nov. 24, 1917 Do Fruits picked from healthy plant after picking mosaic fruits. Fruits picked from healthy plantsonly(control). Fruits ijjekod from healthy plant after picking mosaic fruits. Fruits picked from liealthyplantsonly (control). Fruits picked from hcaltliy plant after picking mosaic fruits. Fruits picked from healthy plants only(control). 2 2 2 2 1 1 4 4 5 5 7 7 3 3 4 Aug. 21,1916 Do. Nov. 15,1917 Do. Dec. 8,1917 Do. WOUND.S NECE.'«SAUY FOR CONTACT INFECTION. In all the above work, the wound factor must be emphasized, since infection lias never been knoNMi to occur wliere diseased and liealthy \iuc^s were in undisliirbed contact. Hundreds of cases have been observed where only a portion of the plants in a cage would THE MOSAIC DISEASE OF CUCURBITS. 43 develop mosaic symptoms after inoculation, and these healthy and diseased plants would remain intertwined for several weeks without further cases of mosaic appearing, no insects being present to cause infection. INSECT TRANSMISSION. While dissemination of mosaic often occurs by the means already noted, a large part of the infection in the field is often due to insects attacking the cucumber. TRANSMISSION BY APHIS GOSSYPH. The importance of insects as a source of infection has been demon- strated by both Jagger (17) and Doolittle (11) in the case of the melon apliis {ApMs gossypii Glover), and field observations have repeatedly shown that this insect may be instrumental in the production of epidemics of cucumber mosaic. At Hamilton, Mich., in 1914, a severe attack of aphids occurred over a wide teriitory during the latter part of July, when mosaic was present in a number of fields but had not become widely distributed throughout the district. As the apliids spread from field to field and increased in numbers, it was noted that a severe outbreak of the disease usually occurred about ten days after the appearance of the insects. Within a month most of the fields about Hamilton showed from 60 to 90 per cent of mosaic infection, and the crop was practically destroyed. Similar results of aphid infestation were noted in Michigan in 1916 and in Wisconsin in 1917, but over less extended areas. Table XYII.— Transmission of cucumber mosaic by Aphis gossypii. Treatment. Source ofaphids. Number ofaphids placed on each plant. Number of plants inocu- lated. Results. Date. Number of mosaic plants. Date ob- served. Aug. 6,1915 Aug. 23,1915 Do.. Aug. 24,1915 Do Aug. 30,1915 Do Inoculated do Mosaic cucumber leaves do 25 to 30 15 30 10 20 5 3 15lo25 10 8 6 6 3 2 8 5 3 1 10 8 8 8 9 6 7 6 10 6 9 12 8 8 8 8 6 6 6 8 8 8 9 7 6 6 9 9 7 6 2 5 1 Aug. 21,1915 Sept. 3,1915 Control Inoculated Control Inoculated do Healthy cucumber leaves. . Mosaic cucumber leaves Healthy cucumber leaves. . Mosaic cucumber leaves do Do. Do. Do. Sept. 10,1915 Do. Do Aug. 4,1916 Aug. 10,1916 Aug. 5,1917 Aug. 17,1917 Do Control Inoculated do Healthy cucumber leaves. . Mosaic cucumber leaves do Do. Aug. 14,1916 Aug. 18,1916 do .. .. .do Aug. 16,1917 do.... ...do Aug. 29,1917 do do Do do .. .. .do Do. Do Sept. 1,1917 Do Control Inoculated do Healthy cucumber leaves. . Mosaic cucumber leaves .do Do. Sept. 10, 1917 Do. Do do do Do. Do Control Healthy cucumber leaves . . Do. The agency of the insect in transmitting mosaic has been proved by experiments in which aphids taken from a mosaic cucumber plant were placed on healthy plants imder cages. In nearly all cases, 44 BULLETIN 87f>, U. S. DEPARTMENT OF AGRICULTURE. tliis has resultod in tlio infoction of all tlio ])lants on wliich the insect woro ])Iaco(l (PI. II, /?). Aphids transfcrrcMl from one healthy planl to another liave never ])ro(luce(l the disease. I The liif^h ])er(enta<;je of mosaic infection obtained from inoculatioi by means of a])liids from diseiised ])lants (Table XVJI) is probabli due to the fact that, bein«^ a sucking insect, it introduces the \inii into tliose tissues which will cUstribute it most rapidly throughouj the ])lant. The number of a])liids necessary to produce infectioa is small, infection liaving occuiTcd where only three of the in.sect^ were placed on each plant, but where only one to three individuals are used the ]>ercentage of infection is often lowered. TRAN'SMISSION BY DIABKOTKA VITTATA. Although the cucumber aphis may be responsible for severe epil demies of mosaic, the striped cucumber beetle {Diahrotica vitiai Fabr.) is probabl)^ the most important insect agent in the transmis- sion of the disease. AMiilc the aphis can cause extensive and rapid mosaic dissemination when present in large numbers, it appears only at intervals in most districts and is then often confined to a few fields. The striped beetle, however, is common to most cucumber- growing sections and usually occurs in considerable numbers tluough- out the season. It is a very active and voracious insect and attacks practically all parts of the plant, but more particularly the leaves. While apliids will nearly always j)roducc infection when transferred from mosiac to healthy plants, the percentage of infection is much lower in the case of the beetle. Being a chewing insect, it trans- mits the disease only when mosaic plant juices are carried on its mouth parts to wounds in healthy plants and are then distributed throughout the tissues. ]Man3' of these insect wounds are slight, however, and are often located in the blossoms and at other points which are not favorable to the rapid distribution of the virus in the plant, so that it is probable that (lr}nng out often prevents infection. This low percentage of infection is offset by the fact that the beetles are immerous and active, constantly feeding first on one plant, then on another, ^\■\{\\ the result that the chances for infection are many. In localities where beetles have been numerous the disease lias spread very rapidly and usually over a wide territory, as the msect often travels considerable distances. i}xj)erimental work has given definite proof of the agency of beetles in mosaic transmission. Beetles captured directly from mosaic Anncrt and placed on healthy plants under cages have produced the disease in numerous cases. I'sually a considerable number of in- sects were placed in each cage, owing to the lower chances of infection before iiKMitioncil, but the numbers were no <:reatcr than were com- THE MOSAIC DISEASE OF CUCURBITS. 45 monly found on an equal number of plants in the same field. Checks were not run with the insects, as in field experiments it was impossible to determine with certainty whether or not the beetles had previously fed on mosaic vines. The results of these tests, shown in Table XVIII, indicate that the beetle must be an important agent of transmission in most fields. Table XVIII. — Transmission of cucurbit mosaic bt/ caging Diabrotica vittata over healthy cucumber plants, as shown by expcrinienis made at Big Rapids, Mich., and Madison. Wis. Num- ber of bee- tles per cage. Source of the l^eelles. Method of infec- tion of beetles. Num- ber of plants per cage. Total num- ber of plants. Results. Location and date beetles were caged. Num- ber of mosaic planets. Date last observed. Big Rapids, Mich.: July 28, 1916... Aug. 4,1916.... Aug. 5,1916.... Aug. 11,1916... Aug. 14,1916... Madison, Wis.: Aug. 11,1917... Do 8 12 8 10 8 35 32 28 30 30 30 30 30 20 22 27 100 40 38 45 Madison, Wis Big Rapids , Mich . . . .do Natural infection.. Fed on mosaic plants in cage. do 2 2 3 3 2 8 4 7 9 7 5 5 6 6 5 6 40 IS 15 15 16 2 12 12 10 8 4 7 9 7 5 5 6 6 5 6 40 IS 15 20 3 1 3 4 3 1 3 5 3 1 3 2 1 2 1 Aug. 12,1916 Aug. 17,1916 Aug. 21,1916 Madison, Wis Big Rapids, Mich. .. Madison, Wis do Natural infection.. Fed on mosaic plants in cage. Natural infection.. .. .do Sept. 5, 1916 Do. Aug. 21,1917 Do. Do do do Do. Do .do do Do. Aug. 23,1917.. Do do . . . .do Sept. 4,1917 .do.... do Do. Do .do do Do. Do do... ... .do Do. Sept. 1. 1817... Do .do do Sept. 12, 1917 .do do Do. ' Do do . .do do Do. Oct. 15, 1917 (Cucumber vines killed by frost.) do Oct. 30,1917 Oct. 22 1917 do.... Nov. 17,1917 Oct 27 1917 do . .do Nov. 21,1917 Nov 10 1917 do.. .. do Do. It wdll be noted that no beetles collected on or after October 15, 1917, produced infection. All cucumber plants were killed by frost about September 25, and all beetles collected in cucumber fields after that date had fed on other plants or on squash and cucumber fruits which still lay in the fields and had not fed on mosaic cucumber vines for three or more weeks prior to collection. In those tests plants were placed m a large cage in the greenhouse w^iich held 40 plants in small pots, and the beetles were allowed to feed on the young cucumber plants for several days. The results indicate that this insect can transmit the disease for only a short time after feeding on mosaic plants. UIABROTICA DUODECIMPUNCTATA AS A CARRIER OF THE DISEASE. The 12-spotted cucumber beetle {Biahrotica duodedmpundaia Oliv.) is also an agent of transmission of cucurbit mosaic in the field. This hisect is so similar to the striped cucumber beetle that I 46 BULLETIN 875), V. S. DEPARTMENT OF ACRK'ULTl'hK. all stutonu'iits rt'pirdiiig one insect Avill pro})nl>ly api)!^ cquiilly well to the other. The work with IHahrotica duode dm punctata was done in the same maimer as that wilii />. littata, and tho results were similar, as is shown in Table XIX. Taui.k XlX.— TrdHsmmion of cucumber mosaic bij Diahrolini ihifxhciinpiiminlii in experiments at Madison, Wis. DatGl>ee(lps were caged. AiiR. 16,1917 Do Do Aug. 21,1U17 Do Do Do Do Number of beetles per cage. Source of lK>ctIps. Madison, \Vis. ....do ....do ....do ....do ....do ....do ....do Method of infection of I)cctles. Natural infection. do do do do do do do Number of plants per cage. Results. Number p , , , , Aug. 30,1917 Do. Do. Sept. 20,1917 Do. Do. Do. Do. TRANSMI.SSION UY OTHER CUCUMBER INSECTS. It is quite possible that other insects attacking the cucumber may also transmit the disease, but they are probably of less importance.. Tho tarnished plant bug (Lygus 2>ratensis L.), being a sucking msectJI very probably carries the disease to some extent, but the few testal made with this insect have given negative results. Tlirips (ThripS"^ tabaci Lhid.), the red spider {Tetranych'us teJarins L.), and the white fly (Aleyrodes vaporanomm Westw.) have shown no indications ofi behig carriers of the disease, although greenhouse observations haveJ included a largo number of cjises where the insects were constantlyl feeding on adjacent healthy and mosaic ])lants. This is in agreo^ ment with the work ofAllard (5), who found that neither the white" fly nor the red spider acted as carriei-s of tobacco mosaic in the greenhouse. TKANSMISHIOM UY IJEi;S. Tlio cucumber and other cucurbits which blossom heavily thnmgh- out the season are constantly frequented by great numbers of bees. These insects might therefore be important agents in disease dis- semination, ])rovi(led they carry the infection. However, tho evi- dence thus far secured indicates that little, if any, infection is so carried. To test this ])()ssibility bees were taken directly from the blossoms of mosaic plants and then caged with healthy plants for some time. Infection has never been known to occur, altiiough trials have been made with 53 plants. As stated on page 33, all attempts at artificial inoculation through the blossoms have so far failed, and it is likely that if infection occurs at this pomt it is THE MOSAIC DISEASE OF CUCURBITS. 47 rather rare. Field observations, moreover, give little support to the theory that bees are a factor in mosaic dissemination, since in many cases where few other insects were present the spread of the disease would in all probability still have been rapid if the bee were a carrier, whereas in such cases the increase of infection was relatively slow. POLLEN AS A CARRIER OF INFECTION. The work with bees has also brought up the question of possible infection through the fertilization of flowers of healthy plants with pollen from the blossoms of mosaic plants. This has been tested by artificial fertilization of healthy cucumber flowers with pollen from mosaic vines, the operation being performed with a camel's-hair brush to avoid wounding the flower parts. Although many fruits were thus produced, no mosaic infection has ever been found. In these experiments care was taken to protect the blossoms from outside pollen and to make certain that the fruits set were pollinated only with pollen from mosaic plants. METHOD OF OVERWINTERING. FIELD OBSERVATIONS. Since the factors concerned in the field transmission of the disease are sufficiently well established to account in large measure for its rapid dissemination, the problem of its overwintering and reappear- ance each year is perhaps most important, aside from the actual cause, in relation to possible methods of control. As before stated, the origin of the disease seems unrelated to soil or climatic conditions, and its appearance is dependent on some specific infection. For the past four years it has appeared in Michigan, Indiana, and Wisconsin at approximately the same time, the first reports of its occurrence each year having been somewhere between July 7 and July 20, at a time when the plants were from 5 to 6 weeks old and had from 8 to 10 leaves. Infection of very young plants is rare and has usually appeared in cases where the seed was planted very late in the season, at a time when infection was already present on older plants in the same field. The regularity in the appearance of the disease extends to most localities in the States mentioned, and a survey of the cucumber- growing districts of Michigan or Wisconsin about the middle of July usually shows the disease developing almost simultaneously in most localities where it was severe the year previous. In many cases the first infections are found on a few plants scattered tlu-ough the field, or perhaps on plants in a single center, including six to eight vines within a short distance of one another, and from these it s])reads gradually to neighboring plants. This manner of appearance and 48 BULL.KTIN 87<>, U. S. DKPARTMKNT OF AGRICULTURK. , subsoqiiont dovoiopincnt is most roimnon, but in some instnnros the tlis(>as(> has suddenly dcvolopod in ('j)i])liytotic foiTn, u striking ox- lunplo of which occurred at Madison, Wis., in 101 (>, followed bj a second and more spvcro case in 1917. During 191G the writer waj not at >radison, but Dr. M. W. Crai'dner reported that the disease appeared early in July and in a short time in many fields had affected '2r^ to 50 per cent of the plants. During 1917, fre(iuent observations were made on the experimental fields at Madison from the beginning of the season. The fields under observation consisted of six plats within a radius of 1^ miles and varied in size from one-half to 1^ acres. These were practically the only large cucurbit fields in the locality, Avith the exception of a few plantings of muskmelon and squash. T'^p to July 19 no mosaic had aii])eared on any cultivated cucurbits in the locality so far as known. On this date, however, two of the six plats showed a number of cases of mosaic m its earliest stages. On July 21 definite sj^mptoms of mosjiic were noted on 25 per cent of the plants in these two plats and on a few plants in each of the remaining four plats. On eTuly 20 a least 50 per cent of the plants in the fii"st two plats and from 3 to 1 per cent of the plants in the other plats were affected. A survey o; gardens in the vicinity also showed a few cases of the dise^use o July 2(3. The plants noted between July 19 and 2G were all in th early stages of the disease, and so far as could be determined all the? infection must have occurred within seven or eight days. Such a sudden development of the disease would seem to indicate that some agent of dissemination was present which produced rapid and widespread infection at a definite period. | The most j)robable factors in overwintering would seem to be: (1) .Soil containing refuse from mosaic vines of previous seiisons; (2) the use of seed from diseased plants; (3) possible wild cucurbit hosts; (4) the presence of an identical disease on plants of another family; or (5) some insect agency. Most of the work has therefore been conducted with these possibilities in juind. SOIL AS A SOURCE OF INFECTION. Tt has been shown that the expressed juices of mosaic ]dants remain infectious for only a short period and that ilried tissues of such plants have never shown the presence of the virus. This indi- cates that soil containing tlie remains of mosaic plants is not likely to j)rove a sourc-e of infection to further crops. (iUEKNIIOrSK KXI'KUIMKNTS. During (li{> winter of 191(», 40 ])lants were grown in the gi'eenliouse in soil from a field at IljOnilton, Mich., where mosaic had been seri- ous foi- tiiree successive yeai"s. The plniils W(>re transplanted to THE MOSAIC DISEASE OF CUCUEBITS. 49 insure root injury as a point of possible infection. As controls, 40 plants were transplanted into the same soil after it had been steril- ized with steam at 15 pounds' pressure for three hours. Plants grown in this soil, whether sterilized or not, remained healthy in all cases. During the month of August, 1910, a large number of mosaic cucumber vines were buried about G inches deep in sandy loam at Big Kapids, Mich., and left until the following year. In July, 1917, some of this soil containing the remains of the mosaic vines was taken to Madison, Wis., and used in the following experiments: (1) Cucumbers were gi'own in the soil directly from seed, and small seedlings were also transplanted into pots of the same soil. In each case, 25 plants were used, together with 30 check plants grown in the same soil after steam sterilization for thi'ee hours at 20 poxmds' pressure. All plants remained healthy. (2) A 50-gram portion of the soil, containing a number of fragments of dried stems and leaves, was mixed with 50 c. c. of sterile distilled water and allowed to stand for 12 hours. This extract was then filtered and the filtrate used to inoculate 10 healthy cucumber plants, inoculations being made into stems and young leaves. No infection occurred on any of the plants thus inoculated. (3) The remains of the vines present in the soil were removed, ground through a meat chopper, and enough sterile distilled water added to soak the material into a soft mass. Fragments of this were then inserted in incisions in the roots and stems of 10 healthy cucumber plants, but no infection resulted. FIELD TESTS WITH CAGED PLANTS. While this work gave only negative results, more conclusive series of experiments were conducted in the field during the summer seasons of 1915, 1916, and 1917. Observations during 1914 at Hamilton, Mich., where mosaic had been severe for some time, showed that the disease seemed to occm- as early and severely on new land as on fields where mosaic cucumbers had been grown for two or three years in succession. An attempt was made therefore, to determine whether fields that had previously grown mosaic plants furnished a source of infection for crops the following season. Hamilton, Ilich., 1915. — One of the fields wliich had been under observation for two years was selected for work in 1915. The plat was one-eighth of an acre in size, had grown cucumbers for three con- secutive years, and nearly every plant had been diseased each year. This plat was again planted to cucumbers in 1915, and as soon as the plants appeared 18 cheesecloth cages were distributed over it, each cage covering two plants. The cages were lifted only once during the season, to allow weeding and thinning, and every precaution was taken to avoid outside infection. Mosaic appeared in the plat about Jul}^ 20, and on September 6 every uncaged plant showed mosaic symptoms. The cages were lifted on this date and the entire 36 plants were found free from disease. 185118°— 20 4 50 BULLETIN 871), U. S, DEPARTMENT OF AGRICULTURE. Hamilton, Mich., 1.916. — The snmo pint was usod in 1016 and 30 cages sot out on July 10, covorini; GO plants. Tlio un(a<;;o(l plants bocamo afToctod oarly in the soason and on August 22 each of them showed the disease in severe form. The caged vinc^ liad growTi so raj)idly that it was necessary to remove tho cages at this time, and it was found that all had remained healthy. Biff liajnds, Mich., 1916. — A test similar to that at Hamilton was conducted at Big Rapids, Mich., on a quarter-acre plat that had been badly diseased the previous year. Thirty-five cages were pul^ dcmn on June 20, live on Jime 29, and five on July 7. However, the disease was not severe in the imcagcd portions of the field, and only 10 per cent of the plants were found to have mosaic at the end of tho season, so that tho results were not as conclusive as in the other tests. All caged plants remained healthy throughout the season. Madison, Wis., 1917. — In 1917 the work was transferred to Madi- son, Wis, and the experiment was repeated on a f-acro plat where all plants had mosaic in 1916. The cages in this case were set out on Juno 26, as soon as the seed germinated, 18 cages being used, covering 36 plants. Beetles wer pre«sont in tho field in gi'eat numbers after July 10, and a ft appeared in some of the cages after a heavy rain which had washe the earth away from the edges of the cages. The plants in these cages were removed on July 9 and new seed planted. The cages were watched carefully; no more insects appeared until August 18, when l)eotlos were found working in nearly every cage, but as there scwmed to be no means of entry from tho outside it was suggested })y entomologists who were consulted that they were from tho second ])rood and had emerged from tho soil. No effort was made to remove them, and the cages were left until September 13. The uncaged plants had been very badly infected with mosaic early in the season, 25 per cent of the plants developing tho disease witliin five days after July 19, when tho first cases of mosaic were foimd. By September 13, when all the uncaged plants had been infected for some time and the vines were much stunted, tho caged plants still appeared perfectly healthy and normal with the exception of injuries from the beetles noted above. (PI. IX, B.) During the same season an additional test was carried on in a small garden belonging to the department of plant pathology. Here 12 cages were used to cover 24 vines which were transplanted from the greenhouse early in June. Fifty additional plants were transplanted near by and 100 more were grown from seed, both lots beiug left uncagi^d. All the uncaged vines became diseased by August 20, and when the cagear soon after. Mosaic ])lants of (lie wild cucuiiibcr {Micrampehs lohata) may also be ]>reseii(, being often oviMlooked l)ocause of their loca- THE MOSAIC DISEASE OF CUCURBITS. 5S tion in out-of-the-way places. All such infection is so easily possible and so difhcult to trace that field observations are always subject to doubt as proof of seed transmission of the disease. TESTS WITH SEED FROM MOSAIC PLANTS. In order to obtain more definite data in regard to seed transmission, a number of tests have been made with seed saved from mosaic plants. These trials were conducted both in the greenhouse and in the field, the field tests being at Big Rapids, Mich. Tests of 1915. — Seed collected from mosaic plants at Hamilton, Mich., during the fall of 1914 was tested in the greenhouse of the department of botany at the Michigan Agricultural College at East Lansing, Mich., during the winter of 1914-15. The seed from indi- vidual plants was not kept separate in these trials, most of it bemg very immature, and only 90 plants were obtained from the entire lot. These were grown in pots on sterilized soil for five weeks, but no signs of mosaic developed. Tests of 1916. — During the fall of 1915, seed was collected from mature fruits of mosaic cucumber plants, using only those fruits which actually showed evidence of the mottling and deformity charac- teristic of the disease. The seed from each fruit was removed sepa- rately, and in planting these separate lots w^ere kept distinct. ' A preliminary test was made during the winter of 1915-16 in the greenhouse at East Lansing, Mich., 500 plants being grown in pots on sterilized soil at a temperature of 28° C. (82° F.) and kept under close observation for seven weeks. No evidence of mosaic appeared on any of the plants during this trial. An extensive field experiment was made during the siunmer of 1916 at Big Rapids, Mich., on land which had not grown cucumbers previously and in a district nearly free from the disease. The seed was from the lot used in the greenhouse test during the winter and represented seed collected from mosaic plants in Michigan, Indiana, Wisconsin, Iowa, and Ontario, Canada. The seeds from each fruit were planted in separate rows 3 feet apart, with the seeds about 4 to 8 inches apart in the row. Approximately 100 fruits were represented , which gave about 4,500 plants for observation. The germination was very mieven, but this seemed to result from the immaturity of some of the seed rather than from any effect of the disease, since all seed which germinated produced plants of healthy appearance. Observations were made practically every day in the early part of the season and at least once every two days in August and September. All plants remained healthy and normal in appearance and no disease appeared in the adjoinnig plats until July 28. On this date mosaic appeared on micaged plants in a plat close to the seed test, tlu'ough the accidental escape of aphids used in other experiments. In spite 54 BULLKTIN 871), U. S. DEPARTMENT OF AGRICULTURE. of (haslif ofTorts for its eradication, the disease si)rea(l <;rudually through the phit until it reached the rows adjoining tiie seed test where it first appeared on August 8. After this date, it worked slowly through tlie plat, although all mosaic vines were removed as soon as noted. The plants had remained healthy for seven weeks, however, and the disease which appeared was so clearly a result of outside infection that the evidence of seed transmission may safely he called negative. Tests of 1917. — \i\ 1917 a further trial of mosaic seed was again conducted at Big Rapids, Mich., using seed collected from Michigan, Indiana, and Wisconsin. In this work the seed from 119 fruits was planted on new land, located at some distance from that used in former experiments and on considerably higlier ground. The plat was about 1 acre in size and was between two other blocks of cucurbits, principally cucumbers. No other experiments with mosaic were made during the season in any of the plats. About 5,500 plants were under observation and 1,150 of these were covered with cheesecloth cages to prevent accidental infection by insects. Use was made of 250 cages, each covering from 3 to 15 plants. They WQTQ set out as soon as the seed was planted and were not lifted until late in July, except where repairs were necessary. The writer was absent from Big Rapids most of the time, and the planting and early inspection work was done under the supervision of Mr. W. W. Gilbert. The plants were left un thinned as long as possible and were inspected at frequent intervals. The writer examined the plants, beginning July 30, and lifted all cages for the purpose of inspection. Great care was taken to avoid outside infection from insects, and the cages were replaced at once. A few striped beetles were present in the field, but only one cage was found to contain insects. All uncaged plants in the seed test and in the adjoining plats were free from mosaic, and all caged plants were healthy with a single exception. This plant was abnormally dwarfed, its lower leaf was j^oUow and wilted, and the four other leaves were peculiarly dark green in color and showed shght indications of a yellow mottling. (PL I, A.) All of the remain- ing j)lants which were in the same cage were of normal size and appear- ance, without the least trace of insect injury. This cage had no de- fects in its covering and had not been lifted after the seed was planted. The writer again visited the field on August 18 and inspected the plants. No case of mosaic was fomid in the plat with the exception of the suspected plant found on July 30. The appearance of this was so unmistakably mosaic that 12 healthy plants were inoculated from the juice of one of its leaves and covered with cages. Most of these develoj)e(l typical mosaic symptoms on leaves and fruits, and there is no doubt that the plant in question was mosaic. Mr. Gilbert reported that no further cases of mosaic a|)peare(l on cucuinbei's in '^1 THE MOSAIC DISEASE OF CUCURBITS. 55 any plat during the season, but two plants of Hybrid Casaba melon in another plat did later develop typical mosaic symptoms. These plants were pulled as soon as found, and no other cases occurred. The conditions at Big Rapids during 1917 were unusually favorable for a test of this kind, since the district was practically free from mosaic, and insects were not unusually niunerous. The single case of mosaic that occurred in the seed test can hardly be attributed to outside agencies, since soil and insects were eliminated almost beyond question and there was apparently no source of infection in the dis- trict adjacent to the experimental field. Furthermore, the very nature of the disease symptoms was such as might be expected in cases of seedling infection, the plant being much dwarfed and the first leaf showing signs of having been affected almost as soon as it appeared. Another case has oocmTed which is very similar to the above and lends support to the theory of seed as a means of overwinterhig. Seed was planted m the greenhouses of the plant-pathology depart- ment of the Univ^ersity of Wisconsm in the fall of 1917, immediately after the houses had been thoroughh^ cleaned and fumigated and fresh soil placed in the benches. The plantmg was made on Septem- ber 12, using ordmary commercial cucumber seed, and the plants were under observation daily after they appeared. On Septem- ber 23, one plant out of 65 showed defuiite mosiac mfection, the cotyledons were yellowed and wilted, and the fu'st true leaf, which was v^ery small, was mottled and distorted. (PL X, A.) The plant was proved to be infected by mosiac by the successful moculation of several healthy plants from it. It was kept for several Aveeks in a separate house for observation and later developed the same peculiar \\Tinklmg and yeliowmg observed m the case at Big Rapids. It remained much stunted and deformed. All the other plants grew vigorously and showed no signs of the disease. (PI. X, B.) In this case the factor of insect transmission can not be enthely eliminated, smce insects were present m the near-by fields until some time m November, but the facts that no injury was visible on any plant in the house and no insects could be found partly removed this objection, since slight injury of this kind is usually very noticeal)le on young seedlmgs. The results so far, tJierefore, show that out of 10,000 plants groAni from seed from mosiac vmes, only one has developed a case of mosaic that could not definitely be attributed to outside hifeotion. Together with this is the case above noted, which appeai-ed in the greenhouse mider conditions that rendered outside hifection very unhkely. The shigle case of hifection w^hich appeared in the open field at Big Rapids, Mich., m 1915, is most easily explahied on the basis of seed trans- mission, shice no other mfection developed in the field aside from that resulting from artificial hioculation. The field data aside from tliis 56 BULLETIN 870, U. S. DEPARTMENT OF AGRICULTURE. aro of rather doubtful value as evidenee of tno oecurrenee of the | disease from the seed. Whiles these data seem overwhelmingly' negative at first glance, the single case of mosaic which developed from the seod of mosaic vines was of sueli a charneter that it is difiicult to cx]>lain its aj)])earance on any basis other than tliat of ' seed transmission. It is dou}>tful whether cucumber mosaic is seed borne to any considerable extent, but it seeuLs possil)l(^ that it may occur in rai'e cases, and the ri«ults so far obtained have l(>ft tlie ])rol)- lem still open, warrajituig further mvestigation. In general, the (Usease a])peai's every year in most infested districts, and in a manner that would j)reclude any belief in seed transmission as the sole cause of its outbreak. In districts where the disease had not a]>peared, however, a single case of seed transmission in several thousand plants would be suOicient to hitroduce the disease, and the many agents of dissemination present Avould hisure its rapid spread to other jilants and fields. INSECTS IN RELATION TO OVERWINTERING. The amount of cucumber mosaic present each year in \arious localities has frequently been in direct relation to the numl)er of cucum])er insects in the fields. This would be expected in view of their im})ortance as agents of dissemination. It has been found, also, that the first appearance of the disease in many localities seems closely related to the number of insects present, as far as tlio amount of the early infection is conoenied. During 1916 and 1917 there was an unusually small number of cucum})er bisects in southwestern Michigan, and the disease aj^pcared hi a much less severe form than in ])revious seasons. Durhig the same period, cucumber hisects at MaiHson, Wis., and hi adjacent localities were present in unusually large numbers hi June and July, particularly the Diabrotica s])ecios. The disease developed there with great severity both years, several fields hi 1917 having 15 to 25 per cent of the plants affected withhi five days from the tune the first case was noted. These observations led to tlie bohef that hisects might bear some part in tlie overwintering of mosaic. THE RELATION OF APHIDS TO OVERWINTERING. During 1915 and 1916 an attempt was made to overwijiter a])hids takcin from mosaic vhies, but no insects a])]>eared in the s])ring and, as little is known of the method by which this insect passes the winter, the work was finally abandoned. As tho cucumber aj)liis (Aphis goHfiHini) has always a|)])eared rather late in the season add not until some time after the disease developed, it is ])n)])able that this insect is not a factor in tlie lirst ap])earance of tlie disease. This is further incHcated by the fa<-t that a]>hi(ls are not ])resent every yeai'in locali- ties where l\w, diseju>e appears each season. THE MOSAIC DISEASE OF CUCURBITS. 57 THE RELATIONT OF STRIPED CUCUMBER BEETLES TO OVERWINTERING. The work of Rand (28) on the Diabrotioa species as possible carriers of the bacterial wilt organism {Bacillus traelieiphilus Smith) has already shown that these insects are an important factor in the over- wintermg of one of the diseases of cucurbits and strengthened the hypothesis that they might bear the same relation to mosaic over- whitering- The striped cucumber beetle (Diahrotica vittata) and the 12-spotted beetle (D. duodecimpunctaia) both overwinter in the adult stage, emerging from hibernation h\ the spring, and thus offer a more definite possibility of overwmtering the disease than the aphis, so that the later experiments have all been with the Diabrotica species. Artificial liibernation experiments. — ^Tho work with beetles was done by the wTiter m oooi3eration with Mr. Neale F. Howard, of the Bureau of Entomology, United States Department of Agriculture. Mr. Howard took entire charge of the collection and caging of the beetles and of all work pertainmg to the actual hibernation and life history of the hisects, and the writer conducted the tests relative to disease transmission by these insects. In the fall of 1916 some 8,000 beetles, chiefly Diahrotica viitata, were collected from fields about Madison, Wis., where mosaic was prevalent. The beetles were placed in hibernation cages of various sizes and types (PL IX, B) and fed on mosaic cucumber vines and fruits as long as such material was available. Most of the cages were kept outdoors in various places during the winter, a few being in more sheltered locations than others. The base of each cage contained from 1 to 3 feet of soil and trash, to furnish favorable conditions for hibernation. The number of insects varied with the size of the cage, but at least 200 were placed in each cage. During the winter some of the smaller cages were brought into the green- house at intervals and observed for the possible appearance of the beetles. Only thi-ee emerged dming the winter, however, and no infection resulted when they were placed on healthy cucmnber plants. Aside from these few insects, none appeared from any of the cages either in the early spring or in Jmie and July, so that no data on beetle overwintering of the disease was obtained from these tests. During the faU of 1917 these tests were repeated at Madison, Wis., on a larger scale, about 35,000 insects, both Diahrotica vittata and Diahrotica duodecimpunctata, being used. The beetles were collected during September and October and placed in various outdoor loca- tions, most of the cages being near points where beetles had appeared early that spring. Fifteen cages were used in these experiments, each having from 500 to 5,000 insects, most of them containing from 2,000 to 3,000. The cages were usually made with the base extending deeply into the soil but open at the bottom; the soil 58 BUIXETIN 870, U. S. DEPARTMENT OF AGRICULTURE. within the cage was not disturbed and the natural con ering of grass and loaves was loft intact. The insects wore fed on the fruits and loaves of mosaic cucumber ]>lants as long as those could be secured. Later in the fall seeds of the wild cucumber {M'lcram pells lohata) wore scattered in all the cages, in order to furnish ])lants for an immediate tost of the beetles when they emerged in the si>riiig. On June 17, 1918, two beetles api)eared in each of two cages, but no others wore found in any cages during the rest of the season. The beetles which a])j>oared wore allowed to food on the wild cucum- ber plants present in the cages, but were not othei-wise tested. The writer did not obser\'e these plants after June 22, but other observers roport that no mosaic appeared. So far, therefore, the tests of booties liibernated under artificial conditions have given no evidence that the insects are concerned in the overwintering of the disease. Natural liihernation expenmentin mosaic micninipolis montioiuul l)olo\v. No mosaic syinploins apjwarod on. anv of tho plants. No mosaic was present in the greenhouses in the' \'icinity of Madison, and while striped ])Ootles attacked the vines,, their importiince in overwintering^ the disease is unknown. Wild micrampelis i)lants arc common in all parts of Michii;an and Wisconsin, and in addition to their occurrence in nature the vine is often planted whore a quick-j^-owincr ornamental is desired. After being once plant-od it usually continues to appear each year from _ self-sown seed. | The presence of mosaic on Micrampelis lohata in advance of its appearance on any cultivated cucurbit host, coupled with the fact that the striped beetles feed upon micrampelis as soon as they emerge! from liibernation and go directly from the wild plant to the cultivatedi cucumber when the latter appears, oll'ci-s a most promising cxplana-' tion for the early infections of the cucumber. There is little doubt, apparently, that Micrampelis lohata may be a source of such early infection to the cucumber. The source of primary infection on the wild plant is still uncertain, but much of the work on the overwintering of tho disease on the cul-. tivated cucumber seems equally applical^lo to tho wild species. Soil is probably not a factor and the question of seed as a means of trans- mission is still unsettled, although the appearance of the disease in tho same spot each year would indicate that tho seed may bo the means of overwintering. During the winter of 1917-18, 1,100 plants were gi'own in the greenhouse from seed collected from mosaic micrampelis plants in Michigan, Indiana, Wisconsin, and Colorado. An additional lot of 1 ,000 plants was also gi-own from seed taken from plants supposedly free from mosaic. The conditions under which the plants were gi-own were unfavorable, however, and caused the leaves to develop various abnormal colorings, which made it difficult to determine whether any of the plants were infected with mosaic. Inoculations from suspected plants gave negative results in all cases, but further tests of seed from mosaic plants are in progress under more favorable conditions for observation.* WILD NONCIICURBITACEOIIS HOSTS. Tho work of Jaggor (19), already mentioned, has shown that the mosaic disease of cucurbits may be transfoired to some plants outside the Cucurbitacetr. Although thoie is no definite evidence that the disease overwinters on plants of other familiivs, it is possible that such wild hosts, particularly if they are perennial, might serve as sources of early infection to the cucurbits, especially as cucum)>er insects are often found feeding on plants of other families. AU cross inocula- > Subsequent tc.its hnvo proved that the mosaic disoasc may bo carried over winter lntboae«d, 1919. THE MOSAIC DISEASE OF CUCURBITS. 63 tions made by the wi'iter from plants outside the Cucurbitacese have given negative results, but the large number of wild plants which show various types of chlorotic diseases makes the problem one which will require much inoculation work l)efore final conclusions are justified. CONTROL MEASURES. All efforts to control the mosaic of cucurbits have dealt wnith cucumbers in the field and have been along three lines: (1) The removal of diseased plants as soon as they are detected, (2) the control of insects which spread the disease, and (3) the breeding of a cucumber which would be resistant to the mosaic disease. SANITARY MEASURES. The tests of sanitary measures were made during the season of 1916, a considerable acreage of land being available for this pur- pose at Plymouth, Ind., Madison, Wis., and Big Rapids, Mich. The absence of the disease from all but one field at Big Rapids limited the work to a single plat of about one-third of an acre, where aphids from mosaic experiments escaped by accident and furnished a severe infection. The disease appeared in the outside row of the plat on July 28, the aphids being by that time well disseminated on the rows closely adjoining. All plants in this row were pulled and burned where they lay, the remainder of the field was sprayed with nicotine sulphate, and from that date to the end of the season plants in the plat were inspected daily and all those suspected of mosaic were removed and destroyed. Once started, however, the disease continued to increase, and by August 25 the number of plants had been reduced about 50 per cent. The removal of dis- eased vines after they had become intertwined with healthy plants resulted in contact infection of those adjacent and served to increase rather than diminish the disease. The work at Madison, Wis., was under the direction of Dr. M. W. Gardner during 1916 and included a large acreage of cucumbers. The disease developed rapidly after its first appearance, due prob- ably to the presence of an abnormal number of cucumber insects, and attempts at removing all mosaic plants soon become hopeless, many fields being practically ruined by August 9. Similar results were secured in the experiments at Plymouth, Ind., which were conducted by Dr. George A. Osner. The fact that diseased plants are a source of infection before definite symptoms appear, which had not been proved at that time, adds to the difficulty of control by sanitary measures. While the eradication method is theoretically sound, the practical conditions in the field are such that, except for use early in the season and in the case of slight infections in isolated fields, this method affords little hope of success in stopping the progress of the disease. ( I 64 BUT. T.F.TIN 879, U. S. DEPARTMENT OF AGRICULTURE. INSECT CONTROL. Tlio attempts at insect coiitiol have boeii urulor the l. DOOLITTLE, S. P. (11) 1916. A new infectious mosaic disease of cucumber. In Phytopathology, V. 6, no. 2, p. 145-147. (12) and Gilbert, W. W. 1918. Further notes on cucumber mosaic disease. (Abstract.) In Phyto- pathology, V. 8, no. 2, p. 77-78. (13) Freibero, G. W. 1917. Studies in the mosaic diseases of plant.';. In Ann. Mo. Bot. Gard., V. 4, p. 175-232, pi. 14-17. l$ibliograi)hy, j). 223-225. (14) Gilbert, W. W. 1916. Cucumber mosaic disease. In Phytopathology, v. 6, no. 2, p. 143- 144, pi. 5. (15) IIUNOEK, F. W. T. I'tO.'). Untcrsiichungcn nnd Betrachtungon iihcr die Mosaikkrankheit der Tabak.spflanze. In Ztschr. Pllanzenkrank., Bd. 1"), Heft 5. p. 257- _ 311, 1 fig. Lit^ratur, p. 309-311. (16) IWANOWSKI, I). 1903. librr die Mo.saikkrankheit der Tabaksi)flan/.p. Tn Zts. hr. Pflanzen- krank., Bd. 13, Heft 1, p. 1-41, pi. 1-3. 68 THE MOSAIC DISEASE OF CUCURBITS. 69 Jagger, I. 0. (17) 1916. Experiments with the cucumber mosaic disease. In Phytopathology, V. 6, no. 2, p. 148-151. (18) 1917. Two transmissible mosaic diseases of cucumber. (Abstract.) J/i Phy- topathology, V. 7, no. 1, p. 61. (19) 1918. Hosts of the white pickle mosaic disease of cucujgber. In Phytopath- ology, V. 8, no. 1, p. 32-33. (20) 1918. Mosaic disease of cucurbits. (Abstract.) In Phytopathology, v. 8, no. 2, p. 74-75. (21) KONING, C. J. 1899. Die Flecken- oder Mosaikkrankheit des hollandischen Tabaks. In Ztschr. Pflanzenkrank., Bd. 9, Heft 2, p. 65-80, 2 fig. (22) McClintock, J. A. 1916. Is cucumber mosaic carried by seed? 7/i Science, n. s., v. 44, no. 1144, p. 786-787. (23) Rand, Frederick V. 1915. Dissemination of bacterial wilt of cucurbits. (Preliminary note.) In Jour. Agr. Research, v. 5, no. 6, p. 257-260, pi. 24. (24) Reddick, Donald, and Stewart, V. B. 1918. Varieties of beans susceptible to mosaic. In Phytopathology, v. 8, no. 10, p. 530-534. (25) RuGGLES, A. G., and Stakman, E. C. 1911. Orchard and garden spraying. Minn. Agr. Exp. Sta. Bui. 121, 32 p. Selby, a. D. (26) 1903. Report of committee on vegetable pathology. A peculiar malady of forced cucumbers. In Ann. Rpt. Ohio State Hort. Soc, 1902, p. 109. (27) 1910. A brief handbook of the diseases of cultivated plants in Ohio. Ohio Agr. Exp. Sta. Bui. 214, p. 307^56, 106 fig. Literature, vii p. (28) Stakman, E. C, and Tolaas, A. G. 1916. Fruit and vegetable diseases and their control. Minn. Agr. Exp. Sta. Bui. 153, 67 p., 32 fig. (29) Stone, G. E. 1910. Calico or mosaic disease of cucumber and melon. In Mass. Agr. Exp. Sta. 22d Ann. Rpt. [1909], pt. 1, p. 163. (30) Westerdijk, Johanna. 1910. Die Mosaikkrankheit der Tomaten. Meded. Phytopath. Lab. "Willie Commelin Scholten," Amsterdam, 1, 19 p., 3 pi. (1 col.). Woods, A. F. (31) 1900. Stigmonose. A disease of carnations and other pinks. U. S. Dept. Agr., Div. Veg. Physiol." and Path. Bui. 19, 30 p., 5 fig., 3 pi. (1 col.). (32) 1902. Observations on the mosaic disease of tobacco. U. S. Dept. Agr., Bur. Plant Indus. Bui. 18, 24 p., 6 pi. (3 col.). ADDITIONAL COPIES OF THIS PUBUCATION MAY BE PROCURED FROM THE SUPERINTENDENT OF DOCUMENTS GOVERNMENT PRINTING OFFICE WASHINGTON, D. C. AT 15 CENTS PER COPY V LIBRARY OF CONGRESS 002 811 489 8 J