THE ROLE OF INSECTS IN THE EPIDEMIOLOGY 0F CUCUMBER MOSAEC VERUS Thesis {or flue Degree 6* pk. D. MICHIGAN STATE UNIVERSITY Robert Joseph McCIanahan 1961 ”\‘L‘ "349'. ‘3 MW“ WWWWWW 1 L— 3 293 01095 3432 This is to certify that the thesis entitled THE ROLE OF INSECTS IN THE EPIDEIVIIOLOGY OF CUCUMZBER MOSAIC VIRUS presented by Robert J. McClanahan has been accepted towards fulfillment of the requirements for 1% degree in/é/f’KW/Jzz'f/ A r /'7 ,/ ’, A / ' / / // / ' f fl’W/fl .. Ail/[fab Major professor Date/(Z: /g/I€7/‘r/ I B R A R Y ’ Michigan State University I. vw‘v 0-169 ~—.__. ,7— PLACE IN RETURN BOXto monthl- emckomtrom you: record. TO AVOID FINES Mum on or baton date duo. DATE DEE DATE DUE DATE DUE MSU Is An Affirmative ActionlEqud Opponunlty Intuition W pus-9.1 ABSTRACT THE ROLE OF INSECTS IN THE EPIDEMIOLOGY OF CUCUMBER MOSAIC VIRUS by Robert Joseph McClanahan Entomological aspects of the epidemiology of cucumber mosaic virus (CMV) were studied in Michigan. Insects on cucumbers and other CMV hosts were identified and their abundance, activity, and seasonal distribution were related to the introduction and spread of CMV in cucumbers. Laboratory studies were conducted to determine the efficiency of various insects as vectors of CMV. The activity of alate aphids was measured by the numbers trapped in yellow water pans. Collections of aphids indicated host relationships and degree of colonization. Trans- mission studies were made with laboratory reared insects and plants grown in the greenhouse or a growth chamber. Anti- serum for the detection of CMV was produced by inoculating rabbits with partially purified virus. In the serological experiments, clarified plant sap was used in precipitin tests. The first infection of cucumbers with CMV in 1961 was related to yellow pan catches of alate Mzzus persicae Robert Joseph Mc Clanahan (Sulz.). A period of gradual increase in the incidence of the virus coincided with a period of low aphid activity. In August there was a rapid spread of CMV within the fields. Alate aphids generally increased at that time and m gossypii Glover activity reached a peak. Sequential exposure of small cucumbers to field infections indicated that most plants were infected late in the growing season. In 1961 CMV appeared in cucumbers two weeks later than it had in 1960, and growers harvested most of their slicing cucumbers before virus incidence reached 20 per cent. Similarly, infection of peppers with CMV was not as extensive in 1961 as it was in 1960. Cooler weather prevailed in the spring of 1961, when aphids were moving to secondary hosts. Tobacco ring-spot virus was not found in cucurbits in 1960 or 1961, although it was present in 1959. Wild host plants of OMV in Michigan were mainly pokeweed, milkweed and wild cucumber. The latter also carried squash mosaic virus. Plants grown from seed of various CMV—infected plants showed no evidence of the disease. The wild cucumbers were infected in late June from an overwintering virus source such as milkweed. Robert Joseph McClanahan Myzus persicae and Aphis gossypii were efficient vectors and transmitted the virus between the common wild hosts and cucumber, and Macrosiphum euphorbiae (Thomas) transmitted the virus from wild cucumber to cucumber. An aphid, Myzocallis asclepiadis (Monell), found: only on milkweed, was shown to be a new vector of CMV, with transmissions between milkweed. Neither the grasshOpper, Melanoplus differentialis (Thomas), nor the striped cucumber beetle, Acalymma vittata (Fab.), transmitted the virus in limited trials . The peak concentration of CMV in tobacco was demonstrated on the sixth day after inoculation. Leaves harvested at this time were used as a virus source. Six lots of partially purified virus injected into a rabbit's ear at four-day intervals resulted in an antiserum with a titer of 1/256. Positive precipitin reactions were given with clarified plant sap of CMV—infected cucumber, pokeweed, and wild cucumber. Saline macerates of lots of four or more apterous melon aphids from an infected tobacco leaf gave a positive serological test . Robert Joseph Mc Clanahan Control of CMV by application of systemic insec- ticides was not successful under conditions of the experiment. Granular phorate applied in a band around the seed proved to be phytotoxic when heavy rains followed. Early planting in weed-free fields minimized early infection. ‘THE ROLE OF INSECTS IN THE EPIDEMIOLOGY OF CUCUMBER MOSAIC VIRUS By Robert Joseph Mc Clanahan A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Entomology 1961 ACKNOWLEDGMENTS These studies were conducted while on leave of absence from the Canada Department of Agriculture Research Station at Chatham, Ontario. Thanks are extended to Mr. G.F. Manson, Officer—in-Charge of the Chatham Laboratory, and other officers of the Department, whose recommendations made this leave possible. The guidance and encouragement given by Dr. G.E. Guyer are greatly appreciated. Professor R. Hutson and Dr. Guyer were responsible for establishing the research program on an interdepartmental basis which aided greatly in its completion . Dr. D.J. deZeeuw, of the Botany and Plant Path- ology Department, spent much time discussing the virus studies and suggesting possible experiments, as well as giving help with techniques of virus transfers and serology. The technical assistance of R.A. Crum and A.L. Wells is gratefully ack— nowledged . The financial aid of Gerber Products Company, who provided travelling expenses, made it possible to have plots in southwestern Michigan in the slicing cucumber grow— ing area. fi The competent secretarial assistance of my wife Erla was invaluable in completing this thesis. Her support and understanding throughout these studies have been of great help. iii TABLE OF CONTENTS Page INTRODUCTION...................................... 1 LITERATURE REVIEW 3 Overwintering of CMV. 5 Insect Transmission of CMV. 8 Aphid Sampling 10 Serological Studies 12 SEROLOGY 11+ Assay of Virus in Tobacco.........................11+ Purification of CMV17 Preparation of Antisera........................... 19 Serological Tests21 VIRUS STRAINS AND INCIDENCE OF INFECTION. 27 Methods...........................................27 Observations...................................... 29 APHIDS ON HOST PLANTS AND MIGRATIONS..... 1+3 MethodsbB Observations...................................... 1+6 INSECT TRANSMISSION OF CUCUMBER MOSAIC VIRUSOOOCOOOOOOOO0.00.00.00.00...0.0.0....56 MethOdSOOOOCOOOOOOOOOOOO00......OOOOOOOOOOOOOOOOOO56 iv TABLE O F CONTENTS Page Observations...................................... 58 A. Field Transmission........................ 58 B. Greenhouse and Growth Chamber Studies.. 61 APHID CONTROL AND CUCUMBER MOSAIC VIRUS PREVENTION...0.0.0.000...IOCOOOOOOOOOOOO.68 Methods...........................................68 Observations...................................... 70 DISCUSSION ’75 Serology as aTool in CMV Investigations...........'75 Strains of CMV in Michigan........................7'7 Aphids on Host Plants and Migrations.............. 79 Experimental Virus Transmission. . . . . . . . . . . . . . . . . . . 81 Probable Vector Relationships...................... 83 Control of CMV81+ LITERATURE CITEDOOOOO.0.OOOOOOOOOOOOOOOOOOOOOO.O.90 Table 10 11 LIST OF TABLES Page Numbers of local lesions on cowpea half— leaves produced by CMV from tobacco on successive days after inoculations of the tObaCCOOOCOOOOOOOOOOOOOOOOOOOOO0......0.......0. Reactions of CMV antiserum after four intra- venous injections of partially purified CMV. . . . . . . Precipitin reactions of CMV antiserum and clarified plant sap from CMV—infected tobacco... Precipitin reactions of CMV antiserum with clarified sap of healthy and diseased pokeweed, wild cucumber and tobacco....................... Symptoms of severe and mild strains of CMV on small plants of cucumber, tobacco, pumpkin and watermelon...I.OOOOOOOOOOOOOOOOOOODOOOO0.0.0... Tests for seed transmission of CMV in various hostSOOOOOO...OOOOOOOOOOOOCOOOOOO...0.0.0.000... Aphids found in colonies on various CMV host plants in central and southwestern Michigan during the summers of 1960 and 1961............ Species of alate aphids from various CMV host plants in central and southwestern Michigan..... . Distribution of aphids trapped in water pans in consecutive three-day periods between May 16 and August 24, 1961 at East Lansing, Michigan.. Incidence of CMV in cucumber plants exposed for five-day intervals to field conditions at East LanSingOCCCOOOOCOOOOOCOOOCOOOOIOOOOO0.0... Transmission of CMV from cucumber to cucumber by single Myzus persicae with various starvation and acquition periOds.OOOOOOOOOOOOOOOOOOOOOOO... vi 16 23 21+ 26 30 1+2 2+7 1+8 50 59 63 Table 12 13 LIST OF TABLES Page Per cent CMV infection in treated and un- treated cucumbers at East Lansing on August 5, 196000.00...OOOOOOOOOOOOOOOOOOOOOO... 72 Number of plants infected with CMV after various spray treatments and subsequent inoculation with CMV, in the greenhouse at East Lansing, Michigan......................... 73 vii LIST OF FIGURES Figure Page 1 Modified hypodermic syringe used to deliver and mix 0.1 ml. aliquots in serological tubes. 22 2 Severe strain of CMV on National Pickling cucumberOOCOOOOOOOOOOOOI....0...OOOOOOOOOOOOOOOO31 3 Severe strain of CMV on Small Sugar pumpkin... 31 1... Incidence of CMV in three plots of cucumbers in196000.00...0.0.0....OOOOOOOOOOOOOOOOOOOOOO.0.31+ 5 Severe squash mosaic virus mechanically trans- mitted from wild cucumber to watermelon........ 38 6 Cucumber mosaic virus mechanically transmitted from cucumber to wild cucumber. . . . . . . . . . . . . . . . . 38 7 Stand of flowering wild cucumber in Allegan Co. , MiChigan’ August17, lgélooooooooooooooooooooooo1+0 8 Wild cucumber naturally infected with CMV. . . . . . . 1+0 9 Water pan aphid trap in a weedy field. . . . . . . . . . . . 1+5 10 Plastic dish used for aphid trap in a plot of piCRlingcucumberSOOOOOCOOOOOIOOOOO000......0..0.1+5 11 Transmission of CMV between various hosts by Myzus persicae..................................65 12 Transmission of CMV between various hosts by Aphis gossypii....................................65 \riii INTRODUCTION Cucumbers were established as a commercial crop in Michigan between 1910 and 1915 when the acreage of pickling cucumbers rose from 7,061 to 32,2h0 acres. Cucumber mosaic disease was discovered in Michigan in 191b,. It became serious by 1918 and has continued to be a limiting factor in cucumber yields in Spite of the development and widespread use of resistant varieties. Cucumber mosaic virus has numerous crop hosts in addition to cucurbits. Peppers are seriously affected in Michigan and many ornamental plants are often infected with cucumber mosaic virus. Both gladioluses and dahlias serve as overwintering hosts as well as being important in the distribution of the virus through shipment of infected corms and tubers . Considerable research has been done on cucumber mosaic, but there are a number of points associated with the disease which remain obscure. Although insects have been shown to be vectors under experimental conditions, those responsible for the spread of the virus to cucumbers under Michigan conditions have not been ascertained. Infected perennial wild hosts are found associated with fields of diseased cucumbers, but their role as primary sources of the virus is uncertain. Secondary dispersal within cucumber fields has been evident but the mechanism poorly understood . This study was undertaken to investigate the basic principles of virus-vector-host relationships of cucumber mosaic virus with emphasis on the entomological aspects. It was postulated that a study of the epidemiology of cucumber mosaic virus might disclose where control measures against one or more vectors could be applied effectively, either on the primary or secondary host. LITERATURE REVIEW Cucumber mosaic virus (CMV), although not recog- nized as such, was probably the organism which caused the disease of cucumbers in Ohio described by Selby (1903). Stone (1910) reported calico or mosaic disease of cucumber in Massachusetts, and Ruggles and Stakman (1911) mentioned wart disease of pickling cucumbers in Minnesota. The infective nature of cucumber mosaic disease was first recorded by Gilbert (1916) when he described preliminary work done since 191b, by Doolittle in Michigan and Jagger in Ohio. Doolittle's first paper (1916) discussed symptoms, mechanical transmission by various methods and transmission by melon aphids. Jagger (1916) reported on the mechanical transmission of CMV to cucumbers, squash, pumpkin and gourds. A second mild strain was described by Jagger (1917) from crookneck squash and muskmelons, which did not affect the fruit. In 1918 an extensive report on spinach blight and its transmission by aphids was published by McClintock and Smith (1918). Some of the characteristics of this virus were different from those later described for CMV, which is now known to cause spinach blight. They were unable to infect peppers, eggplants or potatoes with the spinach blight virus . Doolittle (1920) published a bulletin "The Mosaic Disease of Cucurbits", in which he gives the results of his early studies on the epidemiology of this disease. On the basis of the type of disease described by Doolittle, Johnson (1927) classified the virus as cucumber virus 1. Johnson (1930) distinguished three separate strains of cucumber virus 1 on tobacco. Other strains are described by Bhargava (1951), Brierley and Travis (1958), Doolittle and Webb (1960), Price (193b,, 1937, 191.1) and Sinclair and Walker (1956). The physical properties of CMV were determined by Doolittle (1920) and Walker (1926) and summarized by K.M. Smith (1957). There are hundreds of references to cucumber mosaic virus; however, it is only feasible to review those pertinent to this study. Literature covering the various aspects of overwintering, insect transmission, aphid sampling and serological studies will be treated accordingly. Overwintering of CMV The possibility of CMV overwintering in the soil was considered by Doolittle (1920). Extensive experiments gave no evidence that soil or plant residues therein could harbor the virus overwinter under Michigan conditions. An early note by McClintock (1916) described severe epidemics of mosaic in fields planted with seed obtained from infected cucumbers. The plants had been grown in a partly open cold frame, and the possibility remained that the small plants were infected by an early aphid infestation . Doolittle and Gilbert (1918) found only one diseased cucumber in 5,500 grown in the greenhouse from seed of infected plants. Doolittle's (1920) conclusion that "it is doubtful whether cucumber mosaic is seed- borne to any considerable extent, but it seems possible that it may occur in rare cases" was further substantiated in later work (Doolittle and Walker, 1925) when they found one case of infection in approximately 22,000 cucumbers. The report of Bewley and Corbett (1930), relat- ing to seed transmission of cucumber mosaic, is probably based on infections by cucumber mosaic 2 because he refers to "aucuba mosaic". Kendrick's (1931+) paper on cucurbit mosaic transmitted by muskmelon seed seems to deal with squash mosaic virus, according to his illustration. The wild cucumber, Echinocystis lobata, was reported by Doolittle and Gilbert (1919) as frequently infected by CMV through seed transmission. Recent work by Lindberg, Hall and Walker (1956) indicated that there were two strains of squash mosaic virus and CMV infect- ing wild cucumbers in \Afisconsin. Two per cent of the plants grown from wild cucumber seed were infected by either the severe strain of squash mosaic virus or CMV. In the northern states, CMV overwinters in perennial hosts. Doolittle (1920) suspected that the mosaic of pokeweed, milkweed, ragweed, pepper and sumac was caused by CMV but he was unable to obtain mechanical transmission to cucumber . He later (1921) was able to transmit CMV from cucumber to milkweed (Asclepias syriaca), pepper (Capsicum annuum ) and Martynia louisiana, and from these hosts back to cucumber. Only milkweed is perennially infected. Doolittle and Walker (1922) noted that "mosaic milkweeds" were found in the neighborhood of cucumber fields and further implicated pokeweed (Phytolacca decandra) when they obtained typical mosaic symptoms by transmission with aphids from diseased cucumbers . Two perennial species of ground-cherry, Physalis heterophylla and E. subglabrata_, were found naturally infected in the vicinity of infected cucumber (Walker, 192b,). Further work by Walker (1925, 1926) proved Physalis mosaic was caused by CMV. Catnip is mentioned as a perennial weed which carries CMV overwinter (Hardenburg 2:: _a_l_., 19149 and Boswell _e_t 31;, 1952). Cultivated perennial flowers sometimes serve as overwintering hosts of CMV. Phlox is one of the more important overwintering hosts in Wisconsin according to Faan and Johnson (1951). In England, Smith (1957) has observed delphinium, columbine, geranium and Euphorbia splendens with mosaic and transmissions to tobacco produced cucumber mosaic symptoms. Smith (1955) reported CMV in Buddleia sp. in England, and Bouwman and Noordam (1955) found this shrub to be a host in Holland. No indication is given of the importance of this host in the epidemiology of CMV in cucumbers. B05 and van der Want (1958) report that CMV is present in alsike clover in Holland. The source of CMV in gladioluses in Oregon was thought likely to be adjacent red clover fields (McWhorter, 1957). Insect Transmission of CMV The most serious pests of cucurbits in the northern states are aphids and cucumber beetles. They were the first insects that were evaluated as vectors of cucumber mosaic disease. Doolittle (1920) recalled that he had noted an interval of 10 days between appearance of the melon aphid (m gossypii Glover) and the disease, when he first worked on this problem in 1914. He later proved that melon aphids transmitted the virus when moved from infected plants to caged healthy plants (Doolittle, 1916). At the same time Jagger (1916) obtained a high proportion of positive trans- missions with the melon aphids in greenhouse trials in Ohio. The earliest record of the green peach aphid, Myzus persicae (Sulzer), as a vector of CMV, is by Hoggan (1929). She obtained 3L6 transmissions in 370 tests between six hosts. More details of the vector-virus relationship of M; persicae and CMV were given by Hoggan (1933), Watson and Roberts (1939), and Hidaka and Nakazawa (1955). Many other aphids have been reported to transmit CMV. Swenson and Nelson (1959) list no aphid species that have been recorded as vectors of CMV or related strains. The details of these transmissions indicate CMV is a non- persistant virus. Doolittle and Walker (1928) used an acquisition period and a feeding period of five minutes each. Later workers, Semal (1955) for example, used acquisition periods of one to two minutes and a feeding period of 2A hours. Both the striped cucumber beetle, Acalymma vittata (Fab.), and the Spotted cucumber beetle, Diabrotica undecimpunctata howardi Barb., were mentioned as vectors by Doolittle and Gilbert (1918) and details of the experiments were given by Doolittle (1920) . Freitag (1941) found that the western striped cucumber beetle, Acalymma trivittata (Mann.), and the western Spotted cucumber beetle, Diabrotica undecimpunctata undecimpunctata Mann., did not transmit CMV. In Japan, Komuro and Asuyama (195A) found that CMV was transmitted by aphids but not by a beetle Aulacophora femoralis. Although many reports state that the eastern Species of cucumber beetle transmit cucumber mosaic virus, Doolittle's experiments have not been repeated. 10 Aphid Samplig Aphid sampling has been accomplished by many different methods. Shands 2E a_l. (1952+) made direct counts of aphids on a certain number of basal leaflets or half-leaves of potato. The response of aphids to certain yellow colors was utilized by Moericke (1951) in designing water traps to catch alate aphids. Muller (1953) found that this type of trap caught aphids which were in active flight in search of food plants. Species of aphids differed in their response to various colors of yellow. A/L persicae showed a strong preference for a bright, clear yellow (zinc chromate) paint (Lamb, 1958). \Alindvane traps have not been used extensively, but Shands 21; $1.: (1955) reported fair catches in potato fields. Suction traps, developed by Johnson (1950a) and Taylor (1951) provide a better measurement of aerial popu- lations, but mask the behavior which is better estimated by color traps according to Kennedy and Stroyan (1959). Schreier (1953) and Robertson g 11; (1958) used the sticky board traps originally developed by Kaloostian and Yeomans (191m) for trapping pear psylla. Johnson (1950b) 11 found that suction traps caught more aphids than sticky traps. Heathcote (1957) also reported that water traps and Johnson suction traps were equally effective and were superior to flat or cylindrical sticky traps. Information on the natural controls operating against aphid populations can be obtained at the same time as direct sampling counts are made. MacGillivray and Spicer (1953) studied the parasites of various aphids on potato during in- vestigations of aphid distribution. They found three species of Aphidius (Aphidiinae, Braconidae) parasitizing Myzus persicae. Other parasites which they reared from this host were Diaeretus £3233 (Curtis), Asaphes lucens (Provancher), Pachyneuron Sighonophorae (Ashmead), Praon flSmith and E: occidentalis Baker. In Oklahoma Walton (1951+) also found Aphidius sp. to be the most important parasite in the control of the green peach aphid on Spinach. A chalcid, Aphelinus semiflavus Howard, has been recorded in Kansas (Simpson 232 2.1:, 1959) as a parasite of Myzus persicae and other aphids. The braconid, Aphidius testaceipes (Cresson), para- sitizes both the green peach aphid and the melon aphid and occurs throughout North America (Sekhar, 1957). Another parasite of the melon aphid is Pachyneuron siphonophorae Ashmead (Watts, 1936). 12 Aphid predators are mentioned as controlling factors of green peach aphid populations (Walton, 1951+). Coccinellid larvae and adults, Hippodamia convegens Guerin—Méneville, for example, are important predators (Schopp and Landis, 1959). Many predators check infestations of Pills gossypii on cotton. Among them are lacewings, Chrysopa oculata Say, _C_. plorabunda Fitch and 9_. rufilabris Burmeister, studied by Burke and Martin (1956), and beetles, Collops vittatus (Say) and £3; balteatus Leconte reported by Walker (1957). Occasionally diseases of aphids become epidemic and control a population, Wolcott (1955) found m persicae eliminated by the fungus Acrostalam (Empusa) aphidis Oud. after a period of high humidity. Lawson (1958) cites evidence for a pathogenic factor Operating against the green peach aphid when temperatures reach 90° F. for several days in succession. Serological Studies The literature of plant virus serology has been collected and reviewed by Matthews (1957) in a book on the subject. Cucumber mosaic virus was included in a number of viruses studied by Birkland (1931+) . He found that antiserum prepared by injecting rabbits with cucumber mosaic—diseased plant juice reacted to give a positive precipitin test only with 13 sap from plants infected with CMV. Birkland (1936) found that there was no serological relationship between aucuba mosaic and cucumber mosaic viruses. Chester (1937) included CMV in a serological study of a number of plant viruses. The antiserum prepared by Roland (1955) with CMV infected tobacco plants gave a positive precipitin test with juice from diseased tobacco or tomato, but no reaction with diseased dahlia or cucumber. Hall (1956) was success- ful in preparing an antiserum for viruses of the squash mosaic group and for a mosaic of wild cucumber. The same procedure was unsuccessful with melon mosaic virus and common cucumber mosaic. Recently Tomlinson g}; 21: (1959) were able to purify cucumber mosaic virus by differential centrifugation. Partially purified virus was used to develop an antiserum which gave a positive test against unpurified sap from CMV—infected tobacco. SEROLOGY Assay of Virus in Tobacco The instability of CMV made it necessary to use techniques which ensured the highest concentration possible in plants used for virus purification. An assay of the build— up of CMV in tobacco (Nicotiana tabacum var. xanthi) was made using cowpeas as test plants. Several passages of the virus through tobacco provided a pure strain. The tobacco plant used for the assay had 20 leaves and was 10 in. high. It was dusted with car- borundum before inoculation with plant sap from tobacco infected a week previously. The inoculum was applied undiluted with a small muslin pad to all but the smallest leaves of the plant. This was followed a few minutes later by a light wash- ing of the plant. The assays were conducted each day for eight days at about 10:30 a.m., the same time as the inoculation. A sharpened piece of copper tubing was used to cut leaf discs 3 mm. in diameter. One lot of five leaf discs was cut from five leaves towards the top of the plant, which excluded older leaves and the tip leaves. The discs of this replicate were 11+ 15 taken alternately from the right and left halves of the leaves. A second replicate of five discs was taken from the same leaves, from the side not used for the first replicate. Each lot of five leaf discs was ground in a small drop of water on a glass slide with a ground glass spatula. The COWpea seed for the assay was from an inbred selection of the variety Black which displayed particularly clear local lesion reactions on inoculation with CMV.1 The cotyledons of 10-day old plants were inoculated by half-leaves. The inoculum was applied to the carborundum dusted leaves by five strokes of the glass spatula. Four cowpea plants, providing 16 test areas, were used for the first day's assay and five plants each day thereafter. On each plant the first replicate was put on the left half of one cotyledon and the right half of the other, then the second replicate was put on the alter- nate half leaves. This design minimized position error in applying the inoculum. Lesion counts were made after 1+8 hours. The number of lesions per half-leaf are given in Table 1. These results indicated an increase in concentration of CMV up to the third day after inoculation, a slight drop 1The seed was supplied by Dr. D.J. deZeeuw, who had made the selection for local lesion reaction. 16 Table 1. Numbers of local lesions on cowpea half-leaves produced by CMV from tobacco on successive days after inoculation of the tobacco. Days after inoculation Repli- COWpea half—leaf number cats 1 2 3 b, 5 6 7 8 9 10 Mean 1 12 1+ 5 3 0 1 2 0 5 8 2 22 32 2 7 0 7 1+ 0 3 3 1 67 55 69 82 38 61 68 126 29 29 3 h9.6 2 11 21 33 3h 2h #7 59 86 19 3h 1 23 25 55 18 50 61+ 36 36 17 29 2+ 2907 2 20 1+3 21+ 11+ 32 57 Ll- 11+ 15 l9 1 45 11 A9 8A 100 73 6a 72 1u5 75 5 86.6 2 215 83 46 102 116 87 75 97 112 82 1 126 183 71+ 1+9 83 115 85 225 33 1+8 6 109.9 2 2+2 203 165 10b, 81+ 160 66 152 78 123 1 20 31+ 17 102 30 81 65 61 63 hi 7 39.7 2 9 23 1+8 32 6 32 20 51 39 21 1 87 69 1+ 71 6 12 85 96 70 56 8 60.)+ 2 2+8 123 1).). 19 10 22 101+ 87 112+ 111 _L_.l_________.l_ 17 on the fourth day and a high peak on the Sixth day. This agrees with the work by Tomlinson gt _a_1_. (1959) in which they found two peaks of virus concentration, the first at three days and the second at six days, but their peak con- centrations were equal. In the present studies plants were harvested six days after inoculation for purification of the virus . Purification of CMV The deveIOpment of an antiserum which would be specific for CMV required that the virus be partially purified to eliminate most of the normal plant proteins so that anti- bodies would not be formed that would react with normal plant juice extracts. Fortunately, a purification method had been recently developed by Tomlinson 2.)}. EL. (1959) and the necessary equipment was available. Since a few changes were made in the procedure it is outlined below: 1. Between 25 and 35 g. of leaves were stripped from the tobacco plant stems and weighed. 2. For each 10 g. of leaf tissues, 1+0 m1. 18 of 0.5M phosphate buffer of pH 7.51, containing 0.1% thioglycollic acid, were used in homogenizing the leaves with a Waring Blendor for two minutes. 3. The homogenate was filtered through a Buchner funnel and the filtrate was measured in a graduated cylinder. 1+. Butanol was added to give 8.5% butanol in the mixture; it was stirred by a magnetic stirrer in a corked flask for 30 minutes, in a cold room at 2+° C. 5. After centrifugation at 5,000 rpm for 10 minutes, in the cold room, the mixture was again filtered through a Buchner funnel and the residue discarded. 6. The light green filtrate was centrifuged at 25,000 rpm for two hours in a refrig- erated Spinco Ultracentrifuge. 7. The filtrate was discarded and the pellet was resuspended in 5.0 m1. 0.05M phosphate buffer with a small magnetic stirrer, for two hours at 2+° C. lPhosphate buffer of pH 7.5 was prepared by adding 9 parts of 0.5M KBHPOII to 1 part of 0.5M KHBPOA. l9 8. A centrifugation at 5,000 rpm for 10 minutes in the cold room gave a super- natant which was partially purified virus. This partially purified virus solution was colorless and slightly opalescent. It was kept in a screw—top vial packed in chipped ice. A new lot of virus solution was made for each injection . Preparation of Antisera The antiserum was prepared by injections of par- tially purified CMV into the ear vein of a six pound New Zealand rabbit. In addition, normal serum was obtained by bleeding a rabbit before any injections were made, and a control antiserum was prepared by several injections of clarified sap from healthy tobacco plants . Rabbit No. 1 was given four injections of virus at four-day intervals. Ten days after the fourth injection the rabbit was bled from the ear for 10 ml. and a fifth injection given in the other ear. The small sample of blood was re- frigerated overnight and the clear serum poured off. This was used to test the titer of the antiserum. A final injection was given five days after the fifth injection. 20 The rabbit was bled on the Sixth and eighth day after the last injection of virus. The serum was pooled, partitioned into four-ml. screw-top vials and frozen. A total of 72 ml. of antiserum (designated CMV As 1) was obtained from this rabbit. Rabbit No. 2 was bled for normal serum from the ear. Thirty m1. of blood were taken and this provided 12 ml. serum. Unfortunately, this rabbit twisted his back and lost the use of his hind legs. Since it could not be cured easily, it was replaced by a third rabbit. Rabbit No. 3 was injected with three lots of clarified plant sap from healthy tobacco leaves. The healthy tobacco leaves were treated in the same manner as a virus purification, up to the point where the filtrate was ready for high-speed centrifugation. The last step of the clarifi- cation was dialysis for two hours against distilled water. The rabbit was injected with 5 ml. of the liquid three times at four-day intervals. Fifty ml. of blood were taken after 10 days and 21+ m1. serum were recovered. This control serum was put in vials and frozen for storage. The same rabbit was used two months later for the production of more CMV antiserum. Six injections of 5 ml. 21 partially purified virus were given at three-day intervals. The virus was freshly purified for each injection. Heart punctures were made with the rabbit anesthetized with Nembutol (0.25 ml./lb. rabbit), on the eighth and twelfth day after the last injection. A total of 100 ml. blood was removed and the rabbit survived. The resulting antiserum was frozen in small vials and labelled as CMV As 2. Serological Tests The preliminary test of antiserum from Rabbit No. 1 after four injections of CMV gave positive results. In this test healthy tobacco leaves and leaves infected with CMV were separately macerated in 20 ml. distilled water and centrifuged at 5,000 rpm for 10 minutes. The anti- serum was made up in two-fold dilutions with isotonic saline.1 This was done by placing 0.1 ml. of saline in each of a number of micro test tubes (6 mm. x 50 mm.) with the modified hypodermic syringe shown in Fig. 1. Then 0.1 ml. of anti- serum was placed in a tube and mixed with the saline by several strokes of the syringe plunger. For the next dilution 0.1 ml. of the first dilution was withdrawn and mixed with 1 Isotonic saline prepared by dissolving 0.9 gm. NaCl in 100 ml. distilled water. 22 / HY PODERMIC / SYRINGE RUBBER TUBING GLASS TUBING O.| ML. MARK Fig. 1. Modified hypodermic syringe used to deliver and mix 0.1 ml. aliquots in serological tubes. 23 0.1 ml. saline, and so on until there were six dilutions. When 0.1 m1. of the plant sap was added to the first dilution the concentration of antiserum was one part in four, so that tube was labelled 1/11, the next dilution 1/8, and so on to 1/128. Two sets of six dilutions were made up for this experiment. In each tube of one set, 0.1 m1. of plant sap from healthy tobacco was mixed, and similarly plant sap from infected tobacco was added to the other set of tubes. The tubes were incubated in a water bath at 37° C. for 20 minutes. Table 2. Reactions of CMV antiserum after four intravenous injections of partially purified CMV. Antiserum dilution 114 118 1116 1/32 1/6u 11128 Healthy plant extract + — — .. .. .. Diseased plant extract ++++ +++ ++ - - .— The results are given in Table 2. From these results it can be seen that an antiserum specific for CMV had been built up in the rabbit and its titer was 1/16. A serology test was conducted on March 6, 1961 with the first lot of antiserum (CMV As 1) against undiluted 21+ partially purified virus. There was no reaction with antiserum diluted 1/2, positive reactions with antiserum dilutions 1/11, 1/8 and 1/16 and no reactions with further dilutions. Since there was a heavy precipitate at 1/16 dilution the negative results with higher dilutions were considered to be caused by a virus excess 0 The same antiserum was tested in a more extensive experiment, involving virus dilutions as well as antiserum dilu- tions, and gave the results shown in Table 3. Table 3. Precipitin reactions of CMV antiserum and clarified plant sap from CMV-infected tobacco. Plant sap Antiserum dilutions cfihflfions* 1/2 1/u 1/8 1/16 1/32 1/6u 11/128 1/256 1/2 _ ++ +++ ++ + i — _ 1/b, - + + ++ ++ + + _ 1/8 - - - i + ++ + + 1/16 — — - - - _ - - *Plant sap dilutions were made with isotonic saline plus 0.1% thioglycollic acid. 25 The characteristics of this lot of antiserum, in the terminology of Matthews (1957), were a fairly narrow reaction range, an oc optimum of 11:1, and a large area of antiserum excess. The titer was concluded to be 1/256. Rabbit No. 3 was injected with partially purified CMV in an attempt to procure an antiserum having a higher titer than the first lot. Experiments with CMV As 2 included a block test of antiserum dilutions and clarified infected plant sap dilutions. However, the results were invalidated by precipitin reactions occurring in the micro— tubes of antiserum and clarified healthy tobacco sap. An experiment was conducted to investigate the possibility of using CMV antiserum to detect CMV in pokeweed (Phytolacca americana) and wild cucumber (Echinocystis lobata) as well as tobacco. The plant leaves were ground in a mortar alone, or with a little isotonic saline if necessary. The sap was clarified by centrifugation at 5,000 rpm for two minutes. Aliquots of 0.1 ml. were added to 0.1 m1. of CMV As 2, and the tubes incubated at 37° C. in a water bath, for 20 minutes. The reactions are given in Table Ll. Apparently this antiserum was reacting with the tobacco sap per Egg. The rabbit used to get this antiserum 26 Table 2+. Precipitin reactions of CMV antiserum with clarified sap of healthy and diseased pokeweed, wild cucumber and tobacco. Healthy Diseased Pokeweed - + Wild cucumber - + Tobacco ++ ++ was the same one which had been injected two months before with clarified healthy tobacco for control antiserum. Anti- bodies may have persisted from the first series of injections. Matthews (1957) states that, "The same rabbit after a rest period of a few weeks can then be given a further course of injections with the same virus if more serum is required". The possibility of using CMV antiserum to detect the virus in aphids was investigated by several experiments. There was a reaction only when at least four apterous melon aphids were removed from an infected tobacco leaf and immediately macerated in a drop of saline for the precipitin test. Single apterous or alate aphids gave negative results. Complement fixation tests were not attempted. VIRUS STRAINS AND INCIDENCE OF INFECTION Methods From June 1 to August 15, 1960 samples of plant material suspected of being infected with CMV were collected and mechanical transfers of the virus were made to cucumber. Further transfers were made to an S strain of COWpea to detect tobacco ringspot virus (deZeeuw and Ballard, 1958). Strains of cucumber mosaic virus were differentiated by symptom expression on National Pickling cucumber, Nicotiana tabacum var. xanthi, Small Sugar pumpkin and watermelon. For comparative purposes, cucumber and tobacco were infected with known virus isolates. The incidence of cucumber mosaic in field plantings was determined by counting the number of infected plants in three rows of 100 plants each. When cucumber plants had run together in the rows the per cent infection was determined by counting infection foci in three strips of 65 feet of row. The incidence of virus in wild host plants was estimated by checking each plant in a strip six feet wide and long enough to include 100 plants. 27 28 Virus build-up in several fields of Slicing cucumbers in the southwestern part of Allegan Co., Michigan was followed through the season. Planting dates, the cucumber variety, treatments and harvest dates were also obtained for these fields, along with observations on CMV weed hosts in and around the fields, and aphid infestations. In the study of cucumber mosaic symptoms, it was necessary to grow certain weed host plants in the greenhouse. The main problem with some seeds was to break the dormant stage. The late Dr. G.P. Steinbauer advised germinating pokeweed seeds by using light, an alternating temperature (8 hours at 30° C., 16 hours at 20° C.), and a source of nitrogen (0.1 per cent solution of KNOB). The seeds were placed in petri dishes on blotting paper moistened with the nutrient solution. When the cotyledons had expanded, the seedlings were transplanted to small pots in the greenhouse. Wild cucumber seeds required a period of storage with high humidity and low temperature. After three months in moist Sphagnum at Li° C. the seeds germinated rapidly. The seedlings grew well in a soil mixture of one part muck, one part loam and one part rotted manure. A similar cold period gave the best germination of milkweed seed, but only two weeks were required to give 85 per cent germination. 29 Observations Two related strains of CMV were isolated from cucumbers collected in the field. They could not easily be distinguished in the field but on small, uniform cucumbers in the greenhouse symptoms of each strain were distinctive. The two strains were designated as severe and mild CMV. On cucumbers in the second true-leaf stage both strains of CMV produced mosaic patterns. The symptoms caused by the two virus strains on various hosts are summarized in Table 5. About ten per cent of the fruit of plants infected with the mild strain of CMV were severely "warted" and dwarfed. The remainder of the infected fruit was not dis— torted but had light and dark green areas in a diffuse mosaic. The fruit infected with the severe strain of CMV had a similar diffuse mosaic . The experimental plots were not sprayed for pow- dery mildew, caused by Erjsiphe cichoracearum, and this disease became severe towards the end of the season. Con— sequently, mosaic symptoms were hard to distinguish in mature plants. Blumer 2}; El: (1955) reported that infection with powdery mildew fungus inhibited systemic invasion by CMV, probably by metabolic interference. Table 5 . 30 Symptoms of severe and mild strains of CMV on small plants of cucumber, tobacco, pumpkin and watermelon. Hosts Symptoms Severe strain of CMV Mild strain of CMV National Pickling cucumber Nicotiana tabacum var . xanthi S mall Sugar pumpkin New H amp— shire water- melon Prominent mosaic pattern with variegated leaf tissue both darker and fighter than healthy tissue. Leaves dwarfed and blistered (Fig. 2). Large areas of the leaf lighter colored than normal, leaves distorted and slightly blistered. Sharp distinction between fight and dark areas. Prominent mosaic pattern with many shades of green. Leaves narrow with reduced lamina. Veins extend past the leaf margin in a filiform effect (Fig. 3). Many small, pale yellow spots, most noticeable when viewed by transmitted fight. Light mosaic pattern composed of normal colored leaf areas and lighter green diffuse regions. Same color pattern as the severe strain. Not usually any blistering on the leaves. Prominent mosaic pattern. Less leaf distortion than the severe strain and no filiformity. Same as the severe strain. _______.l_________L__________ 31 Fig. 2. Severe strain of CMV on National Pickling cucumber. Fig. 3. Severe strain of CMV on Small Sugar pumpkin. 3 2' All virus isolates from diseased cucurbit plants gave only local lesion reactions with the S strain of COWpea. If tobacco ring-spot virus had been present, the COWpeaS would have shown large local lesions on the inoculated primary leaf, followed by systemic infection and necrosis of the stem and leaves above the primaries (deZeeuw and Ballard, 1958). The incidence of cucumber mosaic in some typical plantings of cucumbers, at intervals during the growing season and harvest period of 1960, is Shown in Fig. 1+. The per cent infection followed a Similar pattern in all fields. A low incidence of CMV infection through June and most of July was followed by a rapid increase and nearly all plants were diseased by mid-August. In 1961 only one plot of National Pickling cucumbers at East Lansing was followed closely. No disease was noted up to July 13, but on July 20 five plants in 300 (1.7%) were showing symptoms. This proportion did not change until August 19, when 7% of the plants had mosaic. A week later 99% were infected. The rapid spread of the virus in this experimental plot, which was weeded and harvested carefully to avoid mechanical transmission, closely followed a rapid increase of melon aphids. 33 LEGEND Figure A A - Long Ashley Slicing cucumbers. Planted May 20, South Haven, Michigan. B — Long Ashley slicing cucumbers. Planted June 15, South Haven, Michigan. C - National Pickling cucumbers. Planted June 11, East Lansing, Michigan. .ooma E mnonEdoSo mo muofi. omngu. 5 >20 mo mocmpmocH .4 .mmrm 5.3.25 32. . 9.2. mm ON a. o. w 0» mm ON 2 o. m 0» mm Cu m. o. d . 3 ON 8 . 3 3 N I... .oe 3 . W A row .N. 4.. 3 . 3 l o ......... o 0 ho: nlu .om N on ...... -10 m scum o e a scam . :2 35 In Allegan Co. in 1961 most fields of slicing cucumbers were free of CMV infections until the first picking on July 23 to 27. Foliage symptoms were absent, but from 1 to 3% of the fruit had a mosaic mottle. This infection had not increased appreciably by mid—August. One exception was a field found with 21% of the plants showing prominent leaf mosaic symptoms on July 20. The next time the field was visited on August 17, it was apparent that every plant was infected, and senescence was much more rapid in this field than in those with Slight infection. Cucumber mosaic virus in hosts other than cucur- bits was frequently observed but not studied extensively. In some cases samples of plant material were used to inoculate cucumber and tobacco seedlings to check the identity of the virus . Peppers (Capsicum annuum) were heavily infected with CMV in 1960. The large acreage of processing peppers in Macomb Co., Michigan, was surveyed on September 20. The incidence of CMV in eight fields ranged from an estimated 5 to 20%. Infection with tobacco mosaic was high in most fields. A plant moved to the greenhouse proved to be in- fected with the severe strain of CMV. It remained infected 36 for over a year in the greenhouse, through several cycles of vegetative and reproductive growth. At Sodus, Berrien Co., Michigan, infection was high in 1960, but not in 1961. The populations of green peach aphid on peppers were not appreciably different in the two years. In 1L. stands of pokeweed, Phytolacca americana, mosaic was observed on most plants in ten stands, with the other four stands healthy. This perennial plant is an over- wintering host of CMV. Tubers from a stand near East Lansing were brought into the greenhouse in November, and the developing shoots showed the familiar mosaic pattern. The identification of the virus was made by serological methods. In preliminary tests green peach aphids transmitted the virus from pokeweed grown from field collected tubers to small poke- weed plants that had been grown from seed. Aphid trans- mission tests from pokeweed to cucumber were negative. This might have resulted from reluctance of the aphids to feed on cucumber after pokeweed . Wild cucumber, Echinocystis lobath, was found to be infected with two different viruses in the East Lansing area. The observations made after the wild cucumber plants had two or three true leaves disclosed that 6.9 per cent had mosaic symptoms. This mosaic pattern tended to become 37 diffuse as the leaves aged, and in the new leaves formed later the symptoms were small chlorotic spots which also became diffuse. Wild cucumber seed collected in the fall and planted in the greenhouse produced three plants in 18 with the same virus disease. Inoculation to watermelon produced symptoms of melon mosaic. The leaves were small, with deep lobes and reduced lamina. A slight mosaic pattern was observable by transmitted light (Fig. 5). The virus is considered identical to one of those found by Lindberg _e__i_;_ _a_1_. (1956) on wild cucumber in Wisconsin. They called this seed- transmitted strain severe squash mosaic virus. The second virus found in wild cucumber proved to be cucumber mosaic virus. The symptoms were much like those on cucumber, and included varying degrees of mottling but no definite leaf distortion (Fig. 6). Plants inoculated in the greenhouse showed symptoms in 15 days. The new leaves at the growing points showed a distinct mosaic pattern, and this remained in the leaves as they matured. Cucumber mosaic virus was not present in 1+0 plants grown in the green— house from seed obtained the previous autumn from areas later observed to be heavily infected. Lindberg e2 a_l. (1956) found 12 per cent of one lot of seed carried cucumber mosaic virus, and suggested that seed from diseased plants might give 70 per cent infected seedlings. 38 '1 ‘I’l‘l" Fig. 5. Severe squash mosaic virus mechanically transmitted from wild cucumber to watermelon. Fig. 6. Cucumber mosaic virus mechanically transmitted from cucumber to wild cucumber. 39 In the field the first observation of cucumber mosaic virus disease in wild cucumber was made June 111, 1961 when the majority of plants had five to six true leaves. By that time the melon mosaic symptoms were limited to chlorotic spots on some of the older leaves. No examples of double infection with the two viruses were noted. A survey on June 28 in the same marshy area found eight plants in 130 with CMV symptoms. The entire area was estimated as one acre, and the possible number of CMV infected wild cucumbers about 100. This concentration of viruliferous plants, Situated approximately one mile from Michigan State University Horticultural Farm, was likely a source of infection for the cucumbers grown there. One particular site in the southwest portion of Allegan Co. (107th Ave. between 70th and 7lst St.) was literally covered with wild cucumber (Fig. 7), and on August 17, 1961 an estimated 25 per cent of the vines showed symptoms of cucumber mosaic (Fig. 8). Nearby cucumber fields suffered heavy damage from cucumber mosaic in 1960, but the per cent infection was much lower in 1961. Wild cucumber plants seem to spread most readily in bushy areas along creek banks or in areas that are inun- dated in the spring. Apparently in these sites a good 1+0 Stand of flowering wild cucumber in Allegan Co., Michigan, August 17, 1961. 1+1 percentage of the seed germinates each spring. This would explain the annual occurrence of cucumber mosaic virus, even if it is only carried in a small percentage of the seed. Another perennial wild host plant of CMV found in the Lansing area and in Allegan Co. was milkweed. Infected plants were identified as Asclepias syriaca. Other less common Species were present and probably would carry the virus. Symptoms were not well defined and consisted of a mottle on the leaves and stunting of the plant. Mechanical transmission of the virus from milkweed collected near South Haven, Michigan to cucumber, pumpkin and watermelon gave symptoms of mild cucumber mosaic virus. Around one cucumber field 1+6 out of 100 plants appeared infected. Only one example of infected phlox (5.2.1.93. sublata) was observed in 1960, in the six plantings examined in the cucumber growing area of Allegan Co. The symptoms, ob- served July 26, were an obvious mosaic pattern and general chlorosis of the leaves in contrast to adjacent healthy plants. No insects were present on the phlox at that time. Experiments on the transmission of CMV through the seeds of host plants gave negative results. To indicate the extent of these tests Table 6 gives the source of the 1+2 Table 6. Tests for seed transmission of CMV in various hosts. Observation Seeds Period Infected Seed Source Germinated in weeks Seedlings Cucumber Infected fruit 500 3 0 Commercial 11,000 3—6 0 Milkweed Pods from infected plant 288 1+ 0 Pokeweed Infected plant 1,0h5 6 0 Wild Infected plant 25 3 0 cucumber General collection 218 3 0 Pepper Infected fruit 63 6 0 seeds, the number germinated and the length of the observation period after germination . The significance of these negative findings can be judged from the fact that in 1,000 observations there is a five per cent chance of obtaining no seed transmission in the sample, even when the true incidence of transmission is two per cent. APHIDS ON HOST PLANTS AND MI GRATIO NS Methods Collections of aphids on host plants of CMV were made in various regions of Michigan during the summers of 1960 and 1961. These collections were qualitative, but notes were kept on the relative abundance of the aphids at the time of collection . A few of the alate aphids from collections of colonies of aphids were fixed and made up as permanent mounts. The method of clearing was one described by Essig (191.8). The aphids were heated at 49° C. in a small dish of a solution composed of 11,5 parts lactic acid, 5 parts acetic acid, 30 parts ethyl alcohol, 5 parts water saturated phenol, and 15 parts water. The heating time varied from 30 minutes to 6 hours depending on the color and length of time in the alcohol solution prior to clearing. The aphids were mounted in Canada balsam . Identificatiors of the aphids were made from mounted material using the keys of Palmer (1952) and Hottes and Frison 1+3 Ab. (1931). In many cases original descriptions were checked for verification . The method of measuring the activity of migrating aphids in 1961 was by yellow pan water traps. Four traps, .12 in. x 12 in. x 2 in., painted with "Canary Yellow Effecto Enamel" (Pratt 8 Lambert Co.), were obtained from Dr. J.T. Medler, University of Wisconsin. Yellow polyethylene circular pans, 1b, in. diameter and 5 in. deep, were painted with the same color. They proved as effective as the metal pans. Four trapping stations were selected as sites where aphids might overwinter and increase in the spring, before migrating to new hosts. A fifth station was situated in cucumbers. The stations were located as follows: 1. In a field where topsoil had been removed and various weeds were growing (Fig. 9). 2. In grass between rows of ornamental crab- apples, Malus spp. and 50 yards from a permanent woodlot, predominately oak. 3. In a swampy area of trees and bushes where there were many wild cucumber plants. 1+. In a mixed field of wheat and purple vetch. 5. Between two rows of National Pickling 1+5 Fig. 10. Plastic dish used for aphid trap in a plot of pickling cucumbers. 1+6 cucumbers near the edge of a field (Fig. 10). Other plots of muskmelons, cabbage and cauliflower were within 100 yards. The traps were operated on a three—day schedule. The water was drained through a piece of nylon cloth and the insects were rinsed with water into a small, screw-top jar. In the laboratory, most of the water was siphoned from each jar, and the insects were examined with a binocular microscope at 10X. Aphids were removed and preserved in 80 per cent alcohol and the remaining insects were placed in vials of 70 per cent alcohol. The collections were labelled by collecting station and date. The cucumber plots, situated at the Michigan State University Horticultural Farm, were examined frequently for aphids. In 1961, 100 plants were inspected every two days in July and August. Infected pokeweed, wild cucumber and milkweed were examined as often as possible. Aphids from these wild host plants were collected for identification. Observations In the summers of 1960 and 1961 colonies of aphids were found on various CMV host plants as shown in Table 7. 1+7 l _-__—i Table 7. Aphids found in colonies on various CMV host plants in central and southwestern Michigan during the summers of 1960 and 1961. Host Plants L. <1) '2 s: s. ,8 ii i, 3 $4 .13 :3 E ‘1’ E a) m o :3 3 x o. o 0 Ad 0) Q :3 'U :3 T. :5 (D U‘ '2 o z 2 a. U) f: Aphis 055 ii x x x x Myzus persicae x x x Macrosiphum euphorbiae x x Myzocallis asclepiadis x The colonies of aphids usually included alate forms. Alates of Myzocallis asclepiadis (Monell) and Macrosiphum euphorbiae (Thomas) were more easily disturbed than Myzus persicae or Aphis gossypii. The collections of alate aphids from various plants which were hosts of CMV indicated some associations in addition to those given in Table 7. The relevant collections are listed in Table 8. Table 8 . 2+8 in central and southwestern Michigan. Species of alate aphids from various CMV host plants Aphid Host Plant Date(s) A his fabae cucumber July 19, 1960 _E_ _— Aphi oss ii cucumber July 8 August, 1960 Macrosiphum euphorbiae Myzocallis asclemidis Myzus persicae Rhopalosiphum fitchii milkweed muskmelon dogbane cucumber wild cucumber milkweed cucumber milkweed milkweed July 10 and later, 1960 June 27, July 19, 1961 1961 June 28, 1960 October 8, 1960 May 30, 1961 June 12 1961 October 1960, and later, June 8 July, 1960 8 1961 August 1+, 1960 August A , 1960 The water pan traps were set out at the first four stations on May 15. started on June 25, All traps were discontinued on August 21+, The fifth station , in cucumbers, was when the first true leaves were forming. Problems 1961. that were encountered were evaporation of the water over the three-day intervals or pans in exposed areas blown over. L19 Especially in the latter part of the collecting period birds proved to be a problem since they ate trapped insects and used the pans as bird baths with resulting contamination by feathers and droppings. A total of 1,231 aphids were collected. The station site was influential in determining the number of aphids trapped. The total number of aphids from stations one to five were 399, 200, 1+9, 131 and L152, respectively. Many species of aphids were attracted to the water pan traps and identification of them all was not practical. The total number of aphids collected during each three—day period, and the numbers of certain species in these lots are summarized in Table 9. The station site influenced the catch of aphids. For instance, 80 per cent of the melon aphids were trapped at station five. This species accounted for slightly over half of the total station catch. On the other hand, melon aphids comprised only 11+ per cent of the catch at station three, situated among wild cucumbers. The seasonal cycle of the melon aphid indicates a late migration to cucumber. Alate forms were first trapped 50 Table 9. Distribution of aphids trapped in water pans in consecutive three-day periods between May 16 and August 21+, 1961 at East Lansing, Michigan. Total Aphid Species Date Catch A.g. M.p. A.m. T.b. M.s. M.pi. R.f. T.t. D.a May 16 0 - — - — — _ .. .. .. 19 1 — — - - - - - - — 22 0 — - — - — .. .. _ .. 26 2 — — - .. .. 1 _ .. _ 29 2 - — - - - 1 _ .. .. June 1 13 - — 3 - .. 6 - - 1 1+ 36 - 1 6 - - 9 - 1 3 7 55 - - 18 - 8 12+ 9 3 1 10 121 - - 27 - 3 1 3 3 3 13 116 — 1 19 — 27 29 27 15 3 16 11+ — - - — — — 6 1 2 19 37 - - - - - - - 2 - Key to Aphid Abbreviations A.g. Aphi oss ii Glover, melon aphid M.p. Myzus persicae (Sulzer), green peach aphid A.m. Aphis medicaginis Koch, cowpea aphid T.b. Thripsaphis balii (Gillette), thrips aphid M.s. Myzus solani (Kaltenbach), foxglove aphid M.pi. Macrosiphum Risi (Harris), pea aphid R.f. Rhopalosiphum fitchii (Sanderson), apple grain aphid T.t. Therioaphis trifolii (Monell), yellow clover aphid D.a. Drepangphis acerifoliae (Thomas), painted maple aphid 51 Table 9 . (continued) Total Aphid Species Date Catch A.g. M.p. A.m. T.b. M.s. M.pi. R.f. T.t. D.a. June 22 1+5 - - — — — _ - _ .. 25 21 - - — - .. _ .. .. 3 28 7 - — — — _ _ - - .. thny 1. 8 — _ - - - _ - _ _ u 6 - - - 2 - 2 — 1 — '7 32 - - - - - 2 - - 1 10 30 — 2 - 1 — - — — 1 13 17 — - - .. _ _ .. - _ 16 21 3 2 2 5 - 1 .. 1., - 19 25 — LL 2 13 - 2 - — 1 22 51 1 6 — 1 15 _ _ - 2 25 26 - - 1 — — - _ 1 1 28 113 8 5 2 19 22 2 22 3 1 31 63 18 1 '7 3 - _ _ _ 2 .Aug. 3 38 22 2 3 6 - 1 - - _ 6 111 97 1 .. 5 _ .. .. - 1 9 121+ 91 2 L. 31 — — — - 1 12 7 - - — — _ .. .. _ - 15 23 19 1 _ - - _ _ _ 1 52 Table 9. (continued) Total Aphid Species Date Catch A.g. M.p. A.m. T.b. M.s. M.pi. R.f. T.t. D.a. Aug. 18 1+9 1+5 - - - - — — 1 - 21 1o — _ .. _ .. _ _. _ - 22. L. - .. _ _ .. _ - .. - Species Totals 302. 28 9b, 86 75 72+ 67 35 28 July 16, with a peak flight on August 6. Most of the aphids trapped after this represent intra-crop movement in the cucumbers. Kring (1959) states that Aphis gossypii is a facul- tative migrant and may remain on the primary host for the entire summer. The total number of green peach aphids trapped was only 28; two of these were caught in June and the rest were caught between mid-July and mid-August. The first aphids were likely spring migrants from primary hosts and the majority would be migrants between secondary hosts. Frost and Pepper (1957) caught green peach aphids in ultra- violet light traps in Pennsylvania. Over a four year period the peaks of activity for this aphid were the third week in June and the first week in October. It was interesting to 53 note that only one Aphis gossypii was trapped in this manner compared to 80 Myzus persicae. In Maine, Shands g _al_. (1955) found the peak of fall migration of the green peach aphid to be September 12 in 19115. Very few green peach aph'ds were trapped at stations other than number five. The two aphids caught in June were at station two, which was the most likely site for trapping spring migrants. The aphids caught at station one included most of the total numbers of Aphis medicaginis, Thripsaphis baliL Rhopalosijghum fitchii, Therioaphis trifolii and Drepanaphis acerifoliae . Some of the records which were not included in Table 9 because only small numbers were involved were none— theless interesting. For example: Rhopalosiphum nymphaeae (Linnaeus) - the first two aphids caught. Drepanagyhis granovskyi Smith and Knowlton - several in early June. Macrosiphum euphorbiae (Thomas) - seven on June 10 and June 13. Toxoptera graminum (Rondani) - seven on July 7 and July 10. 51+ Of course, many other insects were attracted to the water pan traps. Thrips, beetles, flies and Hymenoptera were common. Both the striped and spotted cucumber beetles were caught in small numbers at various times. They were mostly caught at station three, near wild cucumbers, and only a few were recovered at station five in the cucumbers. Few beetles were seen in the cucumber plots in 1961. In the surveys of experimental plantings of cucumbers and wild CMV host plants, observations were made on natural control of aphids. One important factor was the weather. After a rain, even in warm weather, there were relatively few aphids flying, judged by aphid trap catches. Apterous aphids were often found dead or crawling around on the ground. Moderately high winds of July 2A., 1961, at East Lansing resulted in a reduction of melon aphids on cucumbers. The population on July 22 was estimated as moderate (5-10 aphids per leaf), and it dropped to light (0-5 aphids per leaf). In his study of the ecology of the green peach aphid, Lawson (1958) states, "The only weather factor that has a significant direct effect on mortality is heavy rainfall accompanied by high winds" . The degree of parasitism of aphids on cucumbers was quite low in the years 1960 and 1961. The greatest 55 proportion of parasitized aphids was observed in late August, after the cucumber plots were harvested. Even then the percentage parasitism by Aphidius sp. was only three or four per cent. Coccinellid adults were fairly active in July when aphid populations were starting to increase. The three main species were Coleomegilla maculata lengi Timb., Coccinella novemnotata novemnotata Hbst. and Adalia bipunctata (Linn.). These species were identified by comparing them with speci- mens identified by the Systematic Entomology Unit, Ottawa, Canada . INSECT TRANSMISSION OF CUCUMBER MOSAIC VIRUS Methods The determination of CMV transmission in the field was made by sequential exposure of small cucumber plants for five-day periods in a field planting of cucumbers and retention of the exposed plants in an insect-free greenhouse to check the degree of infection. The exposed test plants were ex- amined for aphids and sprayed with Systoxl before they were placed in the greenhouse. The periodicity of virus acquisition was then correlated with aphid trap catches. Research on the potential ability of several insects to act as vectors of CMV was conducted in the greenhouse and in a Sherer-Gilette controlled atmosphere growth room. When possible, the colonies of test insects were maintained on hosts which were not susceptible to CMV. They were kept on potted plants in cages in the greenhouse, and fluores- cent lights provided supplemental illumination to give a 111-hour day length. An effort was made to keep the greenhouse at 75° F. This temperature was maintained during the winter but was exceeded during most summer days. The growth 1Registered trade name for diethylthionophosphate of B—oxyethyl-thioethylether. 56 57 chamber was maintained at 77° F. between 8 a.m. and Li p.m., and at 70° F. during the night. Light was maintained at 3,000 ft. candles at the plant level for 12 hours, from 8 a.m. to 8 p.m. Melon aphids, Aphis gossypii Glover, were main- tained on National Pickling cucumbers. Fresh plants were put in the cage once a week. The green peach aphids, Myzus persicae (Sulz.), originally collected from peppers, were maintained on Nicotiana tabacum var. xanthi, with a new host plant about every 15 days. Striped cucumber beetles, Acalymma vittata (Fab.), were field—collected as adults. These adults confined over cucumbers layed fertile eggs in the soil, but attempts to rear the larvae were unsuccessful. Rearing grasshoppers, Melanoplus differentialis (Thomas), to the third instar was accomplished using the methods of Carothers (1923). Adults collected September 16, 1960 oviposited in a preparation dish (11.5 cm. diameter and 5 cm. deep) filled with moist sand. The nymphs emerged in about a month and grew well on a supply of lettuce leaves and grass clippings. 58 Aphid transmission experiments were carried out with apterous viviparous forms. Generally the aphids were used in groups of five, but some tests were with individual aphids. The standard inoculation cycle included 30 minutes starvation, an acquisition period of five seconds after feed— ing commenced on the viruliferous host, and an infection period of one hour on the test plant. The test plants were kept for at least two weeks, for symptom expression, in an insect-free section of the greenhouse. The transmission experiments were based on the use of the severe strain of CMV, after passage through four Nicotiana tabacum var. xanthi plants, and then to the various host plants, by mechanical transmission. In instances where the virus symptoms induced by vector transmission were doubtful, the presence of the virus was further deter- mined by inoculations from the suspected tissue to small cucumber plants . Observations A. Field Transmission The exposure of successive lots of potted cucumber plants to field conditions for five—day periods resulted in natural infection with CMV. Table 10 indicates the exposure 59 mm mm mm moa mm I mm om OH 4 m: mm I ma ma 0 o m: ma I mH ma 0 o m: mH I m 3 o 6 mm m I m .954 3 m H on m 68.4. .. om bum. ma 0 0 an mom I em me o o 1: em I OH SH 0 8 4H OH I 3H ma w m cm +3 I m m m m :m o .. 8 ran somuommsH germ compomMcH mpcflm mpsmrm .wo 6036mm Lmowuosomnht Demo Lona. UmuommcH ($3852 msdmoaxm “coo hmnm .msmmsmd pmmm no mcowumpcoo go; on 38.26605 hmploiw pom oomomxo mpcflm pmnESodo 5 >20 mo cocooHoQH .OH 038nm. 60 period and degree of infection in the groups of exposed plants. The per cent theoretical field infection was calculated on the basis of remaining healthy plants. For example, after the first eight per cent infection, the next infection was eight per cent of 92, or an additional 7.2+ per cent. The experimental planting of cucumbers in the area where the potted plants were placed did not develop symptoms until August 3 when several plants in the 100 foot row were noted to be infected. By August 28 this permanent plot showed 80 per cent infection. Mth an allowance for a 10-day period for symptom development, it was obvious that the actual field infection was greater than the theoretical infection calculated from interval infections. This might have been caused by the continual increase in leaf area of the field plants as opposed to the constant, relatively small size of the indexing plants . Aphids were observed only on the last lot (August 23 — 28) of index plants. Both alate and apterous melon aphids were present on about one half of the plants. At this time these aphids were very plentiful in the experimental planting of National Pickling cucumbers. 61 B. Greenhouse and Growth Chamber Studies Studies on the transmission of CMV by insects other than aphids were not extensive. Melanoplus differentialis grasshoppers in the third instar, about 21 days old, were used in one test. After a starvation period of four hours, they were individually fed on a tobacco plant which had been inoculated 10 days previously with CMV. The acquisition period was one minute and then the grasshoppers were moved to individual cucumber test plants for 30 minutes. None of the 80 cucumber plants developed symptoms in an 18—day period. Field collected striped cucumber beetles, Acalymma vittata , were tested as vectors. They were removed from the host cucumbers, starved for two hours, and tested individually. Each beetle was held with forceps on a CMV— infected cucumber leaf until it had taken a bite. Then it was held on a small cucumber until it fed again. The acqui- sition and infection periods varied from one-half to three minutes, and were generally one minute. Sixty-five beetles were used in the experiment and none of them transmitted the virus to the test plants. The green peach aphid proved to be a very effective vector. Transmission tests between cucumbers were 100 62 per cent positive in some cases. Table 11 indicates the effect of varying starvation and acquisition periods on the degree of transmission. The starvation period of 30 minutes was more effective than 15 minutes or none. The acquisition periods of five and ten seconds were equally effective when the aphids had been starved. The tests of green peach aphids as vectors of CMV between various host plants were conducted at different times determined by the availability of vigorously growing viruliferous source plants, and test plants. Aphids in lots of five were transferred from the viruliferous plants to each of the five test plants in a transmission trial between two hosts. The results were indicated by a fraction which had the number of positive transmissions as the numerator and the number of test plants as the denominator. Transmission tests with green peach aphids are indicated in Fig. 11. Green peach aphids colonized on cucumber, milkweed and wild cucumber under greenhouse conditions. They did not colonize on pokeweed; however, the transmission test between small pokeweed plants was positive (5/5). The aphids may also act as vectors of CMV between wild cucumber plants since a transmission test resulted in 2/5 transfers. 63 OH OH OH 0m 0 OH OH m on m m OH OH mH 1H OH OH m mH m H OH OH O N m OH m O H mcowmmHEmcmsrfi mpEan. .Ho mpcoomm E montage E .02 mfifimonH 960,852 pornonH GoHuHmHSUoda poHLonH competencypm pmoH. .mpompmm somfimHSUom can somuwersmpm mdomsme 1.3:,» omommsom mSNNz .2ch can, honfidodo op smnfifiodo Scum >20 mo CommmmfimsmsB .HH 058nm. Figures 11 and 12 /\ Numbers ‘7 6h LEGEND Indicates direction of transmission between different hosts. Transmission between plants of the same species. Number of test plants out of five which were infected by CMV after 21 days. 65 MILKWEED 51 I3 5 PHLOX—4——> CUCUMBER :2 POKEWEED Is 412 4 WILD CUCUMBER <-———) 2 Fig. 11. Transmission of CMV between various hosts by Myzus persicae . <—"——> MILKWEED . 3H4 . PEPPER —“"ECUCUMBER: Poxewsao 5 4115 . WILD CUCUMBER Fig. 12. Transmission of CMV between various hosts by Aphis gossypii . 66 The melon aphid, Aphis gossypii, colonized on cucumber, milkweed and wild cucumber but not on pokeweed. Transmission tests between the wild host plants and cucumber, and also between pepper and cucumber are summarized in Fig. 12. The test for transmission with melon aphids between small milkweed plants was probably influenced by aphid behavior on this plant. The aphids were restless on either leaf surface and only made light feeding probes for 10 or 15 minutes. The melon aphids were more gregarious than the green peach aphids, and in the tests for colonization, the colony was often confined to a Single leaf of the host. Melon aphids on all hosts except pokeweed were not readily disturbed, and when they were moved to another host they soon settled down. Transmission tests with the potato aphid, Macrosiphum euphorbiae, were not extensive. These aphids were collected on wild cucumber and cucumber, but not in large numbers. The vector cycle of one-half hour starvation, five seconds acquisition and one hour on the test plant resulted in li/5 infections from wild cucumber to cucumber, and 3/5 infections in the other direction. 67 One other aphid, Myzocallis asclepiadis, proved to be a vector of CMV. This aphid colonized on milkweed, but not cucumber. Tests gave 3/5 transmissions between small milkweed plants. No infections resulted when the aphids were moved from viruliferous milkweed to cucumber or from cucumber to milkweed. _IL/L asclepiadis was not observed to feed on cucumber although it was common on milkweed plants around cucumber fields . APHID CONTROL AND CUCUMBER MOSAIC VIRUS PREVENTI ON Methods In 1960 an experiment was designed to test two systemic insecticides for aphid control on pickling cucumbers, and at the same time to evaluate the relationship of aphid control and cucumber mosaic virus incidence. One set of plots was established at the Michigan State University Horticultural Farm at Sodus, in southwestern Michigan, and similar plots were put in at the Horticultural Farm at East Lansing, Michigan. Both locations had a past history of severe CMV infections. The National Pickling cucumbers were planted in hills four feet apart. Nine hills in a square formed each of the 20 sub-plots in the randomized block experimental design. There were five treatments including the check, replicated four times. The two planting treatments were granular phorate at four pounds actual per acre in bands around the hills three inches from the seed, and American Cyanamid 18133 granular similarly applied, at two pounds per 68 69 acre. Another treatment consisted of three foliar applications of 1/2 per cent phorate at approximately 10—day intervals, each Spray applied to nearly run-off. Previous reports (unpublished)l had indicated that it was possible to utilize a systemic insecticide as a "carrier" for a resistance factor in certain bean diseases. Treatment four was included in an effort to test this hypothesis with CMV. An extract of a mosaic—resistant (SMR 12) variety of cucumber was prepared for each of the three sprays by macerating 300 gm. of seedlings and pressing the juice through several layers of cheesecloth. The extract was used at a rate of 100 ml. per gallon of 1/2 per cent phorate. Three foliar sprays were applied at 10—day intervals. The plots at East Lansing were observed frequently and a rating was made every two weeks of the aphid popula- tion and virus incidence in each sub-plot. Similar ratings were made at Sodus three times during the growing season. Greenhouse studies were conducted to evaluate the possible interaction of the systemic insecticide phorate and an extract of resistant SMR 12 cucumbers on the development 1 Personal communication from Dr. G.E. Guyer, East Lansing, Michigan. 70 of CMV in inoculated plants. Phorate Sprays made from an 82.6 per cent concentrate were tested to find the maximum concentration that could be applied without appreciable phyto- toxicity. The Sprays were applied with a "Pee-Wee Sprayer"1 which had an aerosol propellant. The main experiment consisted of spraying 16 potted plants with each of the following treatments: 1. Extract from resistant plants, 25 per cent solution. 2. Phorate, one per cent plus 25 per cent extract. 3. Phorate, one per cent. 1+. Water. In each treatment, four plants were inoculated with CMV a few minutes before Spraying, another four three days after treatment, and a third lot eight days after treatment. Observations on the incidence of mosaic symptoms were made two weeks after the inoculations. Observations The plots were planted June 11+ at Sodus and June 16 1 Pee-Wee Sprayer from Thome—Mossman Co., 1111 North Franklin St., Chicago 10, Ill. 71 at East Lansing. In both areas there were heavy rains on June 18. The banding treatments delayed emergence and there was a noticeable retardation in growth for three weeks between those plants and the ones which were not treated at planting time. Aphids were not observed in any of the plots at East Lansing until mid-July. From then on populations of the melon aphid rose rapidly in all plots; however, no signifi- cant differences were found between treatments. At Sodus, aphids were present in the check plots on July 25, but averaged less than one per plant. No aphids were found in any of the treated plots on this date. By August 1.. aphids were equally numerous in all plots and they became very numerous by harvest time. The increase of CMV infection in the two sets of plots during the growing season were similar to those shown in Fig. 1+. Table 12 gives a summary of the counts for the East Lansing plots on August 5, 1960. 72 Table 12 . cucumbers at East Lansing on August 5, Per cent CMV infection in treated and untreated 1960. Treatment Rate Per cent CMV Infection Range Average 1 . Check 2. Phorate Sprays (3) 3. Phorate with resistant extract sprays (3) LI. A.C. 18133 band around seed 5. Phorate granular around seed 1/2% Sprayed to nearly run-off 1/2 % phorate plus 2.7% extract 2 lbs. actual per acre 1:, lbs. actual per acre 89—100 97 37-100 71 77—100 89 67—100 87 71-100 78 Analysis of the original counts of infected plants indicated no significant differences between treatments. Variation among replicates was large. The tests with potted plants in the greenhouse showed that phorate applied to small cucumbers at concen- trations of 1.5 per cent or higher gave severe phytotoxic effects within a 2l+-hour period. The leaves wilted and showed interveinal necrosis and the growing tip of the plant was 73 burned. A one per cent spray gave a Slight burning where the Spray droplets collected; however, the plants grew normally. When cucumber plants were treated with phorate, or the resistant extract, or both, and inoculated with CMV at various intervals, the treatments did not affect the susceptibility of the plants to the virus (Table 13) . W Table 13. Number of plants infected with CMV after various Spray treatments and subsequent inoculation with CMV, in the greenhouse at East Lansing, Michigan. Interval between Treatment and Inoculation Treatment 5 minutes 3 days 8 days Resistant extract 3/5 2/11 li/u Resistant extract plus phorate 2/3 u/u 3/(+ Phorate 3/1+ 3/l+ Z/h None 3/4 3/1+ 2/1+ A few generalizations on cultural practices and their relation to CMV incidence became apparent in the course of this study. 71+ 1. Cucumbers planted in low—lying fields had a greater incidence of mosaic than those on upland ground. The borders around low fields were often covered with weeds and in some places wild cucumbers grew over the undergrowth. 2. Slicing cucumbers in upland fields with no weeds and clean borders had less mosaic than similar fields which were weedy. These differences were most pronounced early in the season. After the first harvest the rapid increase in virus incidence masked this effect . 3. In 1960 when cucumber mosaic caused heavy losses, late— planted fields of pickling cucumbers were 100 per cent infected before they were harvested. A high population of melon aphids developed in these plantings, probably by migrations from earlier cucumbers. DISCUSSION Serology as a Tool in CMV Investigations With a virus as labile as CMV, any purification procedure is greatly facilitated by the use of a local lesion test plant. This is demonstrated by the accurate determina- tions of CMV concentration in tobacco plants by assay on the selected cowpea strain. If the virus concentration changes could have been followed in plants grown under the controlled conditions of the Sherer-Gilette growth room acquired after this portion of the study was complete, a greater uniformity in virus production could have been achieved. The partial purification of CMV was accomplished satisfactorily by the method of Tomlinson 3E 31; (1959). The use of greater quantities of infected tobacco leaves would have given more partially purified virus and this could have been injected into the rabbits in larger amounts. The production of CMV antiserum in rabbit number one was accomplished with six injections of virus over a period of 27 days. The last two injections should have been made on the same four—day interval as the first four, to get a higher titer of antiserum in 20 days. 75 76 The use of the antiserum as a means of detecting CMV in various plants was made impractical in the field by the necessity of a low speed centrifugation of the suspected plant material in order to have a clear precipitin reaction. The detection of CMV in pokeweed, which has a virus inhibitor in the plant sap that makes mechanical transmission tests impossible, was accomplished by precipitin tests. This was considerably easier than making transmissions to a test plant with aphids . The detection of a plant virus in an insect has usually been proven through the transmission of the virus to a healthy test plant by the insect. This may involve long test periods and the necessary plants are not always at hand. A rapid technique for the detection of viruliferous insects could be developed using serological tests. Progress in this direction has been made with positive precipitin tests for CMV in groups of aphids. An antiserum with a higher titer might give reactions with single insects. Techniques, such as complement fixation tests,which are more sensitive than precipitin tests (Matthews, 1957), could be used. This remains an interest- ing avenue for further investigation. 77 Strains of CMV in Michigan The pathogenicity tests on several selected hosts distinguished two strains of CMV in Michigan. A more thorough survey would probably reveal other strains. Both strains were isolated from cucumbers. Peppers were infected with the severe strain. Milkweed in Allegan Co. carried only the mild strain. The pattern of infection in 1960 was similar to that experienced in most years, according to growers, and the rapid increase of virus coincided with the second or third picking of Slicing cucumbers. Harvesting operations were considered by Amen and Porter (195b,) to be the major factor responsible for virus Spread when diseased plants were initially present in the field. In this study in Oregon, it was found that the peak migration of winged aphids was early in the season. In Michigan, where melon aphids become numerous near harvest time, it is difficult to apportion the virus trans- mission by: (a) mechanical means (b) normal aphid movement, (0) movement of aphids disturbed by picking. The incidence of CMV in cucumber fields showed similar seasonal patterns in 1960 and 1961, but in 1961 the 78 virus appeared later and the main increase in infection was two weeks later than in 1960. This delay made a great difference in the per cent infection at harvest time. In 1961 growers were able to harvest their fields of slicing cucumbers completely before the virus incidence reached 20 per cent. Although mechanical transmission of the virus probably occurred when picking started, the degree of infection was low enough at this time to minimize spread by this means. Infection of peppers with CMV was not as extensive in 1961 as it was in 1960. The severe strain of CMV was not prevalent in 1961, although pokeweed was infected to the same extent as usual. The low incidence of this strain must have been the result of less transmission from perennial to annual plants by aphids. Evidently the same virus complex existed in wild cucumber in Michigan as was reported for Wisconsin by Lindberg it _al; (1956). The seed-transmitted strain of squash mosaic virus was prevalent in Michigan. Many wild cucumber seedlings were infected and the striped cucumber beetle was apparently responsible for the secondary spread of the virus. The infection of wild cucumbers with CMV occurred in June. It is not certain what the source of virus was, but infected milkweed were found in association with the infected wild cucumber. 79 Tobacco ring-spot virus was not found in cucurbits in the field in 1960 or 1961, even in the fields that had both tobacco ring—spot virus and cucumber mosaic virus in 1959. Very little is known about the epidemiology of TRSV and no explanation is evident for the variations in infection from one year to the next. Aphids on Host Plants and Migrations The water pan aphid traps proved effective in determining what aphid Species were actively flying in certain areas. Enough aphids were caught to give a general indication of aphid activity from mid-May to the end of August. There was a peak of activity about June 10, followed by a period of lesser activity during the remainder of June. During the first week in July very few aphids were flying, but activity again increased during the rest of July and up to August 9. The fairly high catches on July 15 and 18 represented move— ment of melon aphids in the maturing cucumbers at station five. The catches of green peach aphids were low, and this agreed with the scarcity of colonies observed on cucumber. Green peach aphids were more plentiful in the southeastern portion of Michigan than in central or southwestern areas in 80 1961. Peppers in Monroe Co. were heavily infested in mid- June, but very few aphids were seen on peppers in Berrien Co. at that time of the season. In 1960 all areas had high populations of green peach aphids. Aphid species collected in the water pan traps which were reported capable of transmitting CMV or strains thereof (Swenson and Nelson, 1959) were: Aphis fissypii, Macrosiphum pisi, M; euphorbiae (solanifolii), Myzus persicae, _l\_/I_. solani and Rhflialosiphum nymphaeae. Three of these Species, Aphis gossypii, Myzus persicae and _M. solani were collected in fair numbers at station five in the cucumbers, and Macrosiphum pisi were trapped there infrequently. The actual patterns of aphid movements from one site to another were not evident from the catches at the various stations. Melon aphids were (tarapped near wild cucumbers before they were taken in cucumbers. It is questionable just how much of an actual migration this repre- sented. Natural control factors affected the aphid popu- lations on cucumber. The late appearance of cucumber mosaic in 1961 could be related to the delay in apterous aphid move- ment caused by cold Spring weather. Green peach aphids 81 were observed on cucumbers July 1h, 1960 but not until July 25 in 1961. This latter date was in the main flight period for Myz_u_s persicae indicated by the water pan trap catches. The distribution of the total catch of aphids reflected a period of cool weather at the end of June. Predators destroyed some melon aphids through the growing season, and parasites (Aphidius Sp.) were observed during August. However, these enemies were not considered a controlling factor. Experimental Virus Transmission The experimental data on transmission by the green peach aphid showed that it was probably an effective vector in the field since Single aphids transmitted CMV readily. Melon aphids were just as efficient as green peach aphids when they were tested in groups of five. The potato aphid, Macrosiphum euphorbiae, was a vector, but the very low numbers indicated that it was of little importance in the field. Myzocallis asclepiadis was shown to be a vector for the first time. This species was found on milkweed soon after the new shoots emerged (June 12 in 1961) and could be collected from then until October. 82 Hottes and Frison (1931) list only milkweed as a host. Some of the Myzocallis species are found on oak and may overwinter there. Milkweed and related plants, dogbane for example, are most likely the only hosts for both AIL asclepiadis and cucumber mosaic virus. No evidence was obtained for CMV transmission by grasshoppers or the striped cucumber beetle; however, the numbers tested were insufficient to rule out the possibility of a low rate of transmission. The efficiency of the aphids in transmitting CMV was generally higher than that reported by other workers. 1412—113 persicae transmitted CMV 7/20 from cucumber to cucumber in experiments by Swenson and Nelson (1959). Although they used 10 aphids per plant, the interval of in- oculation was five minutes, considerably longer than the optimum for non-persistent viruses. The exposure of small potted cucumbers to field conditions for five-day intervals gave some interesting results. The absence of aphids on these plants when they were removed from the field, and subsequent determination that the plants had been infected, showed that the vectors were alate aphids which did not remain to colonize the cucumbers. This situation 83 was quite analogous to that found by Swenson and Nelson (1959) in the relation of aphids to the spread of CMV in gladioluses. Many aphid species were found on gladioluses. Two of the more efficient vectors, Amphophora sonchi (Oestl.) and Myzus persicae, were never found colonizing on gladioluses. Dickson and Laird (1959) studied the size and com- position of aphid populations during the lettuce-growing season in two areas and their effects on the incidence of lettuce- mosaic virus. m gossypii was about one quarter as efficient as Myzus persicae in transmitting the virus, but very high populations of the former aphid failed to spread the virus in the field. The authors speculated that the flight or feeding pattern of the melon aphid was such that they did not act as effective vectors. Probable Vector Relationships The determination of aphid species found on host plants, whether they can transmit the virus, the measurement of their flight activity and the pattern of infection of the plants with a virus are all discrete factors that can be measured quantitatively. These factors are all interrelated in the epidemiology of the virus, and the real picture of what actually happens in the field can only be postulated from the 81+ circumstantial evidence. The following diSCussion is what the author considers as the most likely plant-vector—virus relation- ship in the field. Cucumber mosaic virus overwinters in perennials, including milkweed, pokeweed and phlox. From milkweed the virus is carried to other milkweed by Myzocallis asclepiadis, and to cucumber by the green peach aphid early in the season. Later, the melon aphid acts as a vector from milkweed to cucumber and between cucumbers. There is also transmission from milkweed to wild cucumber by the green peach aphid. Pokeweed acts as a host of the severe CMV strain and unknown vectors occasionally carry this strain to cucumber. Here it is carried between plants by the green peach aphid and the melon aphid. There is probably some transmission of the virus from cucumber to milkweed in the fall. Control of CMV The control of CMV by the application of systemic insecticides was not successful under the conditions of the experiment. The heavy rainfalls after planting caused leaching of the phorate and resulting phytotoxicity to the germinating cucumbers. The colonization of aphids was Slightly retarded by Spray applications of phorate, but there was evidently no 85 effect on alate viruliferous aphids before they transmitted the virus . Theoretically, the control of CMV by vector control would be complicated by the fact that spray machinery moving through the field would spread the virus mechanically. Early infection might be controlled by systemic treatment under optimum conditions, but further experiments would have to be conducted to find the best treatments. Cultural practices which assisted in minimizing early infection were planting cucumbers on higher ground and keeping the field and margins free of weeds. Early plantings were less severely damaged because the plants were matur- ing when the numbers of infected plants began to rise Sharply. Foliage showed symptoms but a good percentage of the fruit was free from symptoms. SUMMARY A literature survey revealed that considerable work had been done on experimental transmission of cucumber mosaic virus, but there were few investigations of transmission in cucumber fields. The host plants of CMV in Michigan and elsewhere were well known but only a few reports mentioned insect vectors on these hosts. Methods of aphid sampling were reviewed as well as recently developed methods in serological studies . The serological studies included an assay of CMV concentration in tobacco over an eight day period, partial purification of the virus at the peak concentration, preparation of the antiserum in rabbits, and precipitin tests with the anti- serum. Lesion counts on inoculated COWpea leaves indicated two virus concentration peaks, one at three days and a higher one at six days after inoculation. An antiserum was developed in rabbits which had six injections of freshly purified virus at four-day intervals. Clarified plant sap from CMV-infected tobacco at a dilution of 1/8 gave a positive precipitin test with anti- serum diluted 1/256. Infected pokeweed and wild cucumber gave positive tests with no precipitin reaction given with 86 87 healthy plants. The reactions were satisfactory only when clarified plant sap was used (5,000 rpm for two minutes). When four melon aphids from an infected plant were macerated in a drop of saline the homogenate gave a positive reaction. Negative results were given with lots of three or fewer aphids. Virus build-up in several fields of cucumbers at different areas followed a similar pattern. Initial infection occurred early in the Season with an incidence of less than five per cent. There was little increase before the latter part of July when the infection rapidly spread until it involved 80 to 100 per cent of the field. Two strains of CMV were distinguished on the basis of symptoms in various cucurbits. Wild host plants in the vicinity of the East Lansing plots were pokeweed, milkweed and wild cucumber. The latter also carried squash mosaic virus. No instances of seed transmission of CMV occurred in tests with seed from infected host plants. The aphid species on CMV host plants and a measure- ment of their activity was obtained by direct collections from the plants and by the use of yellow water pan traps. Aphi gossypii, Myzus persicae and Macrosiphum euphorbiae colonized on cucumber and some of the other CMV host plants. Another aphid, found only on milkweed, was Myzocallis asclepiadis. 88 Some 1,200 aphids were trapped in the five water pans between May 16 and August 21+, 1961. A peak of activity occurred June 10 and another about August 9. Melon aphids were trapped from mid—July to mid-August; several green peach aphids were trapped in early June with the majority in the last half of July. Many other aphid species were caught in these traps and the seasonal distribution for some of these was tabulated. Observations were made on aphid parasites and predators, but they did not appear to be a controlling factor of aphids on cucumber. Experiments on insect transmission of CMV were conducted to assess the efficiency of vectors between various host plants. Aphids were mostly tested in groups of five, with an inoculation cycle of 30 minutes starvation, five seconds acquisition and one hour infection. Both the green peach aphid and the melon aphid were highly efficient vectors between all host plants tested. Macrosiphum euphorbiae transmitted the virus from wild cucumber to cucumber, and Myzocallis asclepiadis, acted as a vector between milkweed plants. Neither the grasshopper Melanoplus differentialis nor striped cucumber beetles transmitted the virus. Sequential exposure of small potted cucumbers to field infection at East Lansing in 1961 resulted in infected 89 plants for the periods June 4 to 9, June 9 to 12..., July 29 to August 3 and the two intervals after August 18. The accumulated per cent infection was comparable to that found in the experimental plots. Control of the virus was not accomplished by treatments of granular systemics applied in a band with the seed or phorate sprays applied at 10-day intervals. The failure of the planting treatments might have been caused by heavy rainfall after planting. Certain cultural practices which reduced the damage in cucumber fields were early planting, planting on higher ground and planting in weed-free fields. LITERATURE CITED Amen, C.R. and C.A. Porter 1951+. The spread of cucumber mosaic virus in cucumber plantings during harvesting. Plant. Dis. Reptr. 38: 8111—8113. Bewley, W.F. and W. Corbett 1930. The control of cucumber and tomato mosaic diseases in glasshouses by the use of clean seed. Ann. Appl. Biol. 17: 260-265. Bhargava , K . S . 1951. Some properties of four strains of cucumber mosaic virus. Ann. Appl. Biol. 38: 377. Birkland , J .M. 193b,. Serological studies of plant viruses. Bot. 1936. On the classification of plant viruses. Phyto- pathology 26: 1156-1158. Blumer, S., L.”Stadler and A. Harder 1955. Uber die gegenseitigen Beziehungen zwischen Gurkenmosaik und Gurkenmehltau. Phytopath. Zeitschr. 25: 39-511. Bos, L. and J.P.H. van der Want 1958. Virusziekten van vlinderbloemigen. Landbouw- voorlichting 15: 550-558, 573-587. Boswell, V.R. -e_t 21; 1952. Pepper production, disease and insect control. U.S. Dept. Agr. Farmers' Bull. 2051. Bouwman, L.W.M. and D. Noordam 1955. Komkommermozaikvirus in Buddleia davidii Franch. Tijdschr. over Plantenziekten 61: 79-81. Brierley, P. and R.V. Travis 1958. A virulent strain of cucumber mosaic virus from Easter lily. Plant Dis. Reptr. 1+2: 10311-1036. 90 91 Burke, H.R. and D.F. Martin 1956. The biology of three chrysopid predators of the cotton aphid. Jour. Econ. Ent. 119: 698-700. Carothers, E.E. 1923. Notes on the taxonomy, development and life history of certain Acrididae (Orthoptera). Trans. Amer. Ent. Soc. 119: 7—24. Chester, K.S. 1937. Serological studies of plant viruses. Phyto- pathology 27: 903-912. deZeeuw, D. and J.C. Ballard 1958. A test strain of the black cowpea. Plant Dis. Reptr. 1+2: 898. Dickson, R.C. and E.F. Laird, Jr. 1959. California desert and coastal populations of flying aphids and the spread of lettuce-mosaic virus. Jour. Econ. Ent. 52: MIC-b.16- Doolittle, S.P. 1916. A new infectious mosaic disease of cucumber. Phytopathology 6: 115-1&7. 1920. The mosaic disease of cucurbits. U.S. Dept. Agr. Bull. 879. 69 pp. 1921. The relation of wild host plants to the over- wintering of cucurbit mosaic. (Abstr.) Phytopathology 11:216. Doolittle, S.P. and W.W. Gilbert 1918. Further notes on cucumber mosaic disease. (Abstr.) Phytopathology 8: 77-78. 1919. Seed transmission of cucurbit mosaic by the wild cucumber. Phytopathology 9: 326—327. Doolittle, S.P. and M.N. Walker 1922. Notes on cucurbit mosaic. (Abstr.) Phyto- pathology 12: l+2-Li3. 92 Doolittle, S.P. and M.N. Walker 1925. Further studies on the overwintering and dissemination of cucurbit mosaic. Jour. Agr. Res. 31: 1—58. 1928. Aphis transmission of cucumber mosaic. (Abstr.) PhytOpathology 18: 1A3. Doolittle, S.P. and R.E. Webb 1960. A strain of cucumber virus 1 infectious to blight-resistant Spinach. Phytopathology 50: 7-9. Essig, E.O. 19h8. Mounting aphids and other small insects on microsc0pic slides. Pan-Pacific Ent. 2b: 9-22. Faan, Hwei-Chung and J. Johnson 1951. The overwintering of the cucumber mosaic virus. Phytopathology 1+1: 1001-1010. Freitag, J.H. 19111. A comparison of the transmission of four cucurbit viruses by cucumber beetles and aphids. (Abstr.) Phytopathology 31: 8. Frost, S.W. and J.O. Pepper 1957. Aphids attracted to light traps. Ann. Ent. Soc. Am. 50: 581-583. Gilbert, W.W. 1916. Cucumber mosaic disease. Phytopathology 6: 1143-1114. Hall, Dennis H. 1956. Studies on serology of cucurbit and bean viruses. Ph.D. Thesis, Univ. of Wisconsin (L.C. Card No. Mic 56-3L15) 58 p. Univ. Microfilms, Ann Arbor, Mich. (Dissertation Abstr. 16: 219). Hardenburg, E.V., C. Chupp and R.W. Leiby 191+9. Growing pumpkins and squashes. Cornell Ext. Bull. 776. 8 pp. 93 Heathcote , G. D . 1957. The comparison of yellow cylindrical, flat and water traps, and of Johnson suction traps, for sampling aphids. Ann. Appl. Biol. 1+5: 133-139. Hidaka, Z. and K. Nakazawa 1955. Hoggan, I .A. 1929. 1933 . Hottes, F.C. 1931. Jagger, I .C. 1916. 1917. Johnson, C.G. 1950a. Studies on the transmission of cucumber mosaic virus to tobacco. I. Relation between infection and flight of alate Myzus persicae Sulz. at various times of cutting down the fore crop barley. (In Japanese, English summary). Jubilee Publ. in Commemoration of the 60th Birthdays of Prof. Yoshihiko Tochinai and Prof. Tekichi Fukushi, pp. 259-267. The peach aphid (Myzus persicae Sulz.) as an agent in virus transmission. PhytOpathology 19: 109-123. Some factors involved in aphid transmission of the cucumber mosaic virus to tobacco. Jour. Agr. Res. 117: 689. and T . H . Frison The plant lice, or Aphididae, of Illinois. Ill. Nat. Hist. Survey Bull. 19 (Article 13). hh7 PP- Experiments with the cucumber mosaic disease. Phytopathology 6: 1fi8-151. Two transmissible mosaic diseases of cucumbers. (Abstr.) Phytopathology 7: 61. A suction trap for small airborne insects which automatically segregates the catch into successive hourly samples. Ann. Appl. Biol. 37: 80-91 . 91+ Johnson, C.G. 1950b. The comparison of suction trap, sticky trap and tow net for the quantitative sampling of small airborne insects. Ann. Appl. Biol. 37: 268-285. Johnson, E.M. 1930. Virus diseases of tobacco in Kentucky. Ky. Agr. Expt. Sta. Bull. 306. Johnson, J. 1927. The classification of plant viruses. Ms. Agr. Expt. Sta. Res. Bull. 76. Kaloostian, G.H. and M.S. Yeomans 191m. A sticky board trap used in scouting for pear psylla. U.S. Dept. Agr. Bur. Ent. 8 Plant Quarantine. ET-220. Kendrick , J . B . 1931+. Cucurbit mosaic transmitted by muskmelon seed. Phytopathology 21+: 820-823. Kennedy, J.S. and H.L.G. Stroyan 1959. Biology of aphids. Ann. Rev. Ent. l1: 139-160. Komuro, Y. and H. Asuyama 1951;. Studies on cucumber mosaic virus. I. The symptoms of mosaic disease of cucumber in Japan, and host range and properties of its causal virus. (In Japanese, English summary). Phytopathol. Soc. Japan, Ann. 19(1/2): 18-21-I. Kring, J.B. 1959. The life cycle of the melon aphid, Aphis oss ii Glover, as an example of facultative migration. Ann. Ent. Soc. Am. 52: 2811—286. Lamb, K.P. 1958. Alate aphids trapped in Aukland, New Zealand using Moericke colour traps. New Zealand Jour. Sci. 1: 579-589. Lawson, F.R. 1958. Some features of the relation of insects to their ecosystems. Ecology 39: 515-521. 95 Lindberg, G.D., D.H. Hall and J.C. Walker 1956. A study of melon and squash mosaic viruses. Phytopathology 1+6: 1189—1195. MacGillivray, M.E. and P.B. Spicer 1953. Aphid parasites collected in New Brunswick in 1950. Can. Ent. 85: h23—Ii31. McClintock, J.A. 1916. Is cucumber mosaic carried by seed? Science N.S. 1+4: 786-787. McClintock, J.A. and L.B. Smith 1918. True nature of spinach-blight and relation of insects to its transmission. Jour. Agr. Res. 1h: 1-60. Mci/Ifhorter, F.P. 1957. A localized occurrence of cucumber mosaic virus in gladiolus. Plant Dis. Reptr. 1+1: 11.1—11.2. Matthews, R.E.F. 1957. Plant virus serology. Cambridge Univ. Press, London. 128 pp. Moericke, V. 1951. Eine Farbfalle zur Kontrolle des Fluges von Blattlausen, inbesondere der Pfirsichblattlaus, Myzodes persicae (Sulz.). Nachrichtenbl. deutsch. Pflanzenschutzdienst 3: 23-22+. Miiller, H.J. 1953. Der Blattlaus-Befallsflug im Bereich eines Ackerbohnen und eines Kartoffel-Bestandes. Beitr. Ent. 3: 229—258. Palmer, M.A. 1952. Aphids of the Rocky Mountain region. The Thomas Say Foundation. Vol. V. L152 pp. Price, W.C. 1931+. Isolation and study of some yellows strains of cucumber mosaic. Phytopathology 21+: 7h3-761. Price, W. C. 1937. 19111. 96 Classification of lily—mosaic virus. (Abstr.) Phytopathology 27: 138-139. Classification of Hawaiian Commelina-mosaic virus. Phytopathology 31: 756—758. Robertson, R.S., Jr. and E.O. Klostermeyer 1958. Roland, G. 1955. Ruggles, A. G. 1911. Aphid populations on field beans in Washington. Jour. Econ. Ent. 51: 178-181. Contribution a l'étude du virus de la mosaique du concombre. Parasitica 11: 3-9. and E.O. Stakman Orchard and garden Spraying. Minn. Agr. Expt. Sta. Bull. 121. 32 pp. Schopp, R. and B.J. Landis 1959. Schreier, O. 1953- Sekhar, P. S . 1957- Selby, A. D. 1903- Semal, J. 1955. Fumigation effect of thiodan against the green peach aphid on potatoes. Jour. Econ. Ent. Uber das Auftreten von Blattl'ausen an Kartoffelstauden in Nieder5sterreich im Jahre 1952. Pflanzenschutzberichte 10 (9-10): 129-153 . Mating, oviposition, and discrimination of hosts by Aphidius testaceipes (Cresson) and Praon aguti Smitwh, primary parasites of aphids. Ann. Ent. Soc. Am. 50: 370-375. Report of committee on vegetable pathology. A peculiar malady of forced cucumbers. Ann. Rept. Ohio State Hort. Soc. 1902. p. 109. Quelques transmissions par pucerons de Cucumis virus 1 Doolittle é partir de dahlia. Parasitica 11: 118-123. Shands, W.A. , 1955. Shands , W.A. , 1951+. 97 G.W. Simpson and M. Covell Aphids caught in windvane traps with openings of different sizes. Jour. Econ. Ent. 1.8: 621-625. G.W. Simpson and L.B. Reed Subunits of sample for estimating aphid abundance on potatoes. Jour. Econ. Ent. A7: 102b,-1027. Simpson, R.G., C.C. Burkhardt, F.G. Maxwell and E.E. Ortman 1959. Sinclair, J. B. 1956. Smith, K .M. 1955- 1957. Stone, G.E. 1910. Swenson, K . G. 1959. Taylor, L. R. 1951. A chalcid parasitizing spotted alfalfa aphids and greenbugs in Kansas. Jour. Econ. Ent. 52: 537-538- and J.C. Walker Extent of cross protection among strains of cucumber mosaic virus in cucumber and cowpea. Phytopathology 1+6: 367-371. Some recent work on plant diseases. Sci. A textbook of plant virus diseases. 2nd ed. 652 pp. Little, Brown and Company, Boston. Calico or mosaic disease of cucumber and melon. Mass. Agr. Expt. Sta. 22nd Ann. Rept. (1909). Pt. 1, p. 163. and R.L. Nelson Relation of aphids to the spread of cucumber mosaic virus in gladiolus. Jour. Econ. Ent. 52: L21-1I25 . An improved suction trap for insects. Ann. Tomlinson, J.A., R.J. Shepherd and J.C. Walker 1959- Purification, properties, and serology of cucumber mosaic virus. Phytopathology 1+9: 293-299. Walker, J.K . , 1957. Walker, M. N. 19214». 1925. 1926. Walton, R. R. 1951+. Watson, M.A. 1939. Watts, J.G. 1936. Wolcott, G. N. 1955. 98 Jr. A biological study of Collops balteatus Leo. and Collops vittatus (Say). Jour. Econ. Ent. 50: 395-399. Physalis and cucurbit mosaic intertransmissible.” Phytopathology 11+: 56 . The relation of certain species of Physafis to the overwintering of the mosaic disease of cucumber. PhytOpathology 15: 733—7113. A comparative study of the mosaic diseases of cucumber, tomato and Physalis. Phyto- pathology 16: 1131-1158. Seasonal fluctuations of the green peach and turnip aphids on commercial greens crops in Oklahoma. Jour. Econ. Ent. 1+7: 775—780. and F .M. Roberts A comparative study of the transmission of Hyoscyamus virus 3, potato virus Y and cucumber mosaic virus 1 by the vectors Myzus persicae (Sulz.), E circumflexus (Buckton), and Macrosiphum gei (Koch). Proc. Roy. Soc. London, B, 127: 53h-576. Cotton insects. In Report S.C. Expt. Sta. 1+8: 1+9. Entomogenous fungi in Puerto Rico. Science 121: 875-876. N STQTE V A UNI . LIBRARIES I ll Ill"Hilllllllllllllllll 0953432 29301 ICHIG (Will :31