A 51'qu or erus DISEASES or R . STRAWBERRY, IN MICHIGAN 7, Thesis ’0!“ the Degree “Ph D" _ 1 wcamm STATE cones: -~ ' Rgbm Harryfultgnji‘. _ 1954‘ ” 7TIH “Knit-:15 This is to certifg that the thesis entitled A STUDY OF VIRUS DISEASES OF STRAWBERRY IN MICHIGAN presented by Robert. Harry Fulton has been accepted towards fulfillment of the requirements for _Bh‘D_._ degree in Mhology Major professor Date W9 /X; /if‘/ O~169 A STUDY OF VIRUS DISEASES OF STRAWBERRY IN’MICHIGAN By Robert Harry Fulton A DISSERTATION Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology l95L THESIS ACKNOWLEDGELENTS The writer will be forever indebted to Mr. Donald Cation, at whose suggestion this study was initiated and under whose guidance the project was conducted. Nhile performing his duties as major professor, he has instilled a stimulating and exacting philosophy of individual research which the writer has endeavored to gain. A deep appreciation is also felt for the following: Directors of the Michigan Agricultural Experiment Station for their encouragement and support during these investigations; Plant Pathologist Emeritus J. B. Demaree for his suggestions and professional aid in the field of strawberry virology; Doctor F. L. wynd for his helpfulness and en- thusiasm during the course of the physiological studies; the staff and graduate students of the Department of Botany and Plant Pathology and the Department of Horticulture for their suggestions and interest through- out the period of study; to my friend, William J. Young, whose generous donation of time aided in the preparation of the photographic work; and the Bureau of Plant Industry, Iichigan State Department of Agriculture, who by their support and cooperation during these studies have aided in crystallizing many of the problems encountered. Finally, an expression of thanks to my wife, Lorraine, for her understanding cooperation during the course of studies in attaining this degree and expecially for her help in preparing this manuscript. A STUDY OF VIRUS DISEASES OF STRAWBERRY IN MICHIGAN By Robert Harry Fulton AN ABSTRACT Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 195A Approved MM!“ @1; W Robert H. Fulton Virus investigations in Michigan showed that the Type 2 virus was universally present in all cultivated varieties to the extent of 75 per cent of 575 samples. Type 1 virus was present in 15 per cent and combin- ations of the two types in 3 per cent. The type 1 virus was found only on plants tracing to out-of—state origin. Only 6.8 per cent of the 575 samples were found virus-free and as a result a virus-free foundation planting was established. The leaf roll and witches'-broom producing viruses were also reported. Three innovations were developed which improved graft unions. They were enlargement of the stolons, creation of additional grafting surface and prevention of "tongue" curling. Fragaria fliginiana, Frzgaria platnetala and Fragaria bracteata were used for the first time in comparison with Fragaria vesca for Type 1 virus and Type 2 virus symptom expression. ‘3. virginiana was symptomless to both virus types. The other two species, E. platypetgla and E. bracteata displayed varying degrees of sensitivity to virus- infection. The cultivated varieties Dunlap and Robinson were symptomless to both virus types. Catskill reacted with transient flecking to Type 1 virus and interveinal chlorosis to Type 2 virus infection. All plants showing leaf roll virus symptoms also carried the Type 2 virus as demonstrated on 30.122230 Reddening of the petioles was a characteristic symptom for the leaf roll disease on E. zgggg. Catskill and Dunlap showed leaflet chlorosis and mottling with extreme marginal rolling when infected with the leaf roll virus. Robinson and - Gem were mildly affected. Witches'-broom disease, leaf roll and Type 2 viruses were trans- Robert H. Fulton mitted by dodder, Cuscuta'campegtris; the first time this dodder species was used for transmission of these strawberry virus diseases. Through the dodder technique, Potentilla argentea, Potentillglregga and Potentilla anserina were found to be latent carriers of the Type 2 virus. The viruses investigated were nontransmissible mechanically, through seed, pollen, soil, plant fragments or the root knot nematode in these studies. Transmission tests with Aphis forbesii, Aphrophorg sp., Tarsonemus pallidus, Tetranyghug sp. and Magggpsis trimaculata were negative. The Type 2 virus was inactivated in vizg with drybheat ranging be- tween 36° to #00 C. for varying exposure periods, with 38° C. for eight days giving the best results. Zinc salts also inactivated the Type 2 virus _i_n_ 1119.0 Virus-free Robinson strawberry plants showed A per cent more total and amino nitrogen than Type 2 virus-infected plants. The Type 2 virus- infected plants showed a 58 per cent reduction in tranSpiration rate. It was demonstrated in these studies that Thornberry's * variegated clones with viruslike 'particles in the plant extracts were also infected with the Type 1 virus, which may have been responsible for his results with electron microscopy. *Thornberry, H. H., A. E. Vatter and D. M. Beeson. 1951. Viruslike particles in strawberry plants with foliar variegation. Phytopath. 1.1: 35 (Abst.). Robert Harry Fulton candidate for the degree of Doctor of Philosophy Final Examination: May 18, 195h Dissertation: A Study of Virus Diseases of Strawberry in Michigan 'Outline of Studies: Major Subject: Plant Pathology Collateral Subjects: Entomology, Horticulture Biographical Items: Born: November 22, 1925. Chicago, Illinois. Undergraduate Studies: Michigan State College, East Lansing, Michigan, l9h6-l950. Graduate Studies: Michigan State College, East Lansing, Michigan, 1950-195A. Experience: Biological aide, United States Department of Agriculture, 1949. Fieldman for stone fruit virus research projebt, 1950- 1952. Instructor (Research), Department of Botany and Plant Pathology, Michigan State College, small fruit pathology, 1952- l95h. Member of Society of Sigma Xi, American Phytopathological Society, and Sem. Bot. TABLE OF CONTENTS CHAPTER PAGE I 0 INTRODUCTION 0 O O O O O O 0 O O O O O O O O O O 0 0 O O O O 1 II. RWIEW OF LITERATURE O O 0 O O O O O O C O O O O O O O O O O 3 A. Strawberry Virus Diseases in the British Isles . . . . . 3 B. Strawberry Virus Diseases in Western United States and British Columbia, Canada . . . . . . . . . . . . . A C. Strawberry Virus Diseases in Eastern United States andOntario,Canada..................6 III . MATERIAIS AND IETHODS O O O O O O O O O O O O O O O O O O O O 7 A. Virus-Sensitive Indicator Species. . . . . . . . . . . . 7 B. Culture of Indicator Species and Cultivated Varieties Under Test . . . . . . . . . . . . . . . . . 8 C. Method of Indexing . . . . . . . . . . . . . . . . . . . 9 IV. SYMPTOMATOLOGY OF TYPE I AND TYPE 2 VIRUSES ON ' INDICATOR SPECIES AND CULTIVATED VARIETIES . . . . . . . . IL ' A. Type 1 Virus Symptoms. . . . . . . . . . . B. Type 2 Virus Symptoms. . . . . . . . . . . C. Combinations of Virus Types 1 and 2. . . . . 18 V. VIRUS DISEASES AND OTHER VIRUSLIKE ABNORMALITIES OF THE STRAWBERRY IN MICHIGAN. . . . . . . . . . . . . . . 2A A. Virus Content in Cultivated Varieties. . . . . . . . . . 2A B. Virus Content in Fragaria virginiana in Southern Michigan. . . . . . . :'. . . . . . . . . . . 28 C. Other Strawberry Virus Diseases and Abnonmalities with Viruslike Symptoms. . . . . . . . . . . . . . . . 30 VI. TRANSMISSION OF STRAWBERRY VIRUSES . . . . . . . . . . . . . LO A. Dodder Transmission Studies. . . . . . . . . . . . . . LO B. Seed Transmission Studies. . . . . . . . . . . . . . . . A7 C. Soil and Nematode Transmission Studies . . . . . . . . 51 D. Insect Transmission Studies. . . . . . . . . . . . . . . 53 E. Mechanical Transmission Studies. . . . . . . . . . . . . 55 VII. STUDIES ON THE INACTIVATION OF THE TYPE 2 VIRUS IN VIVO. . . 61 A. Heat Treatment Studies . . . . . . . . . . . . . . . . . 61 B. Chemical Treatment Studies . . . . . . . . . . . . . . . 65 CHAPTER PAGE VIII. PHYSIOLOGICAL STUDIES ON THE TYPE 2 VIRUS. . . . . . . . . . 70 A. Hydrogen-ion Concentration . . . . . . . . . . . . . . . 70 B. Nitrogen Metabolism. . . . . . . . . . . . . . . . . . .'7l C. Transpiration Rate . . . . . . . . . . . . . . . . . . . 74 IX. SUMMARY. . . . . . . . . . . . . . . . . . . . . . . . . . . 75 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 APPENDIX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Summary of the Virus-Free Strawberry Certification Program.in.Michigan . . . . . . ... . . . . . . . . . . . .'. 89 Analytical Procedures . . . . . . . . . . . . . . . . . . . . . 91 LIST or TABLES TEXT TABLES PAGE I. Comparison of Type 1 Virus Symptomatology on Indicator Species. . . . . . . . . . . . . . . . . . . 17 II. Comparison of Type 2 Virus Symptomatology on Indicator Species. . . . . . . . . . . . . . . . . , . 20 III. Sample of Cultivated Strawberry Varieties in Michigan for Virus Content 1950.53 . . . . . . . . . . 25 IV. Type 2 Virus Content in Fragaria virginiana in Southern Michigan. . . . . . . . . . . . . . . . . . . 30 V. Symptanatology of the Leaf Roll Complex on Several Cultivated Strawberry Varieties. . . . . . . . . . . . 33 VI. Camparison of Witches'-Broom and Type 2 Viruses as Expressed by Q. campestrig Transmission to E. vesca O O O O O O O O O O O O C O O O O O O O O 0 0 L5 VII. Effect of Drtheat Treatment on the Type 2 ‘ Virus .jfl v1v00 0 0 O O O O O O O 0 O O O O O O O O O O éh VIII. Inorganic Chemical Effects on the Type 2 Virus i_n Vivo. O O O O O O O O O O O ‘0 O O O O O O O O O O O 67 IX. Total Nitrogen in Healthy and Type 2 Virus-Infected Leaves, Expressed as Percentages of Dry Weight . . . . 72 X. Amino Nitrogen in Healthy and Type 2 Virus-Infected ' Leaves, Expressed as Percentages of Dry Weight . . . . 73 XI. Grams of water Vapor Transpired from Healthy and Type 2 Virus-Infected Leaves . . . . . . . . . . . . . 7A TEXT FIGURES 1. 2. 3. h. 10. ll. 12. 13 0 1A. 15. 16. LIST OF TEXT FIGURES Scraping Epidermis of Stolon with Razor Blade to Create New Callus Surface . . . . . . . . . . . . . Method of Making Longitudinal Incision in Indicator Stolon (Left); Completed "Tongue" Incision (Right). Cutting off Tip of "Tongue" to Prevent Curling. . . Inserting Indicator "Tongue" into Stolon of cultivat ed. variety. 0 O O O O O O O O O O O O O O 0 Binding of Tape Around Upper Portion‘of Graft . . . Binding of Tape Around Lower Portion of Graft . . . Binding of Four Inch Tape Around Entire Graft to Prevent "Tongues" from Twisting . . . . . . . . . . Completed "Tongue" Stolon-Graft . . . . . . . . . . Grafted Plants Lined Out in Greenhouse Sand Bench for Virus Observation . . . . . . . . . . . . . . . Blakemore, Symptomless Carrier of Type 1 Virus (Left); Expression of Type 1 Virus on Fragaria vesca (Right) 0 0 O O O O O O O O C O C O 0 O 0 O . Expression of Type 2 Virus on Fragaria vesca (Left); Dunlap, Symptomless Carrier of Type 2 Vi rus (Right ) O I O O O O O O O O O O O O O 0 O O 0 Type 2 Virus Symptoms of Epinasty on Petioles and Leaflets of Fragaria vesca. . . . . . . . . . . . . Comparison of Leaf Size between Healthy and Type 2 Virus-Infected Fraggria vesca (one-quarter inch grid) 0 O C O O O C O C O O O O C O O O C O O O O . Comparison of Size between Healthy and Type 2 Virus-Infected Fragaria vesca Flowers . . . . . . . Comparison of Leaf Size between Healthy and Type 2 Virus-Infected Fra aria platypetalg, Seneca Strain, (one-quarter inch grid) . . . . . . . . . . . . . . Comparison of Leaf Size between Healthy and Type 2 Virus-Infected Fragaria bracteata (one-quarter inch grid)...00900000000000.0000. PAGE 12 12 12 12 13 13 13 13 22 22 22 22 23 23 23 23 TEXT FIGURES 17. i8. 19. 20. 21. PAGE Comparisons of Fragaria vesca Leaf Sizes between Healthy and Type 2 Virus—Infected, and Combina- tion of Type 1 and Type 2 Viruses . . . . . . . . . . 38 Dunlap Variety Infected with the Leaf Roll Virus Complex.......................38 Seedlings Grown from a Variegated Plant Showing Symptoms of Leaf Variegation. . . . . . . . . . . . . 38 Premier Variety Infected with a Witches'-Broom Producing Virus (Left); Normal Premier Plant (Right) . . . . O O . O O C O O . C 0 0 O C O C 0 O C 38 Map of Lower Michigan Showing the Location of the Strawberry Virus Types Found in a Limited Sampling from Certain Counties. . . . . . . . . . . . 39 CHAPTER I INTRODUCTION The weakening of strains in varieties of strawberries accompanied by reduced yield and runner production have been reported through the past years in.Michigan. Many reasons, mostly unsatisfactory, have been proposed for this varietal "running out". The possibility that these disorders were of a‘ViruS nature was not discounted. However, determi- nation of"virus contamination was considered a lengthy and difficult task, but in recent years development of facilities and methods has made this determination feasible. The importance of this problem was not realized until the announce- ment by workers in the United States Department of Agriculture (17) in 1950 that viruses were isolated from strawberry plants in Michigan. Furthermore, importation of the Marshall strawberry from the Pacific Northwest implicated a virus disease unknown to Michigan. These findings were met with concern for Michigan is considered a leading state in straw- berry production. In 1950, through the encouragement and support of Mr. C. A. Boyer, Michigan Bureau of Plant Industry, the writer was sent to the Plant Industry Station of the United States Department of Agriculture in Beltsville,Maryland, to learn the techniques of virus determination. This was accomplished under the skillful guidance of Mr. J. B. Demaree, Emeritus, Senior Plant Pathologist. Soon thereafter a program was initiated to establish primarily a foundation in strawberry virus research which would ultimately serve the strawberry industry in Michigan. The first objective was to sample 2. cultivated strawberries for virus content and distribution in the state, which in turn would serve to establish a foundation stock of virus-free strawberry varieties to be made available to cooperating nurseries for future grower use.' Other problems considered were modes of virus transmission in the field, amount of \fimnns content in wild strawberry and method of inacti- vating the virus in 1139. Physiological comparisons between infected and healthy plants which have a bearing on plant productivity have been carried out. The results of the investigations on the viruses affecting Michigan strawberries are recorded herewith. CHAPTER II REVIEW OF LITERATURE The development of strawberry virus research has developed in- dependently in the British Isles and in North America. An account of the existing literature on the various virus diseases present in these areas is recorded in the following sections. A. Strawberry Virus Diseases in the British Isles In 1933, Harris (3h) reported that degeneration of the Royal Sovereign variety was of a virus nature. Infected plants were dwarfed and the leaves showed a pronounced marginal chlorosis. The name yellow-edge was given to this disease. Subsequently, Massee (52) demonstrated that the strawberry leaf aphis, Capitophorus fragaefollii Ckll. transmitted the strawberry virus yellowaedge. {asses (53),working with mild and severe crinkle, obtained trans- mission by using the strawberry leaf aphis. In 19A7, Wood and Whitehead 002) reported that plants infected with severe crinkle were suffering from two virus entities, one persistent in the aphid while the non-per- sistent virus is transmitted when the acquisition feeding period is shortened. Recently, Prentice and Harris (7A) and Prentice (75 and 76) presented evidence that yellowzedge was the result of multiple infection by two or more viruses which were separated by varying the feeding periods of the vector. In 1951, Prentice and Woollcombe (77) showed that a period of 10 to 19 days elapsed before 9. fragaefollii became crinkle infective. This is recorded as the longest "latent period" for an aphid-transmitted virus. A. The complexity of strawberry virus diseases was reported by Prentice (78) in 1952. He showed that by varying the acquisition and transfer feeding periods of the vector the yellow-edge and crinkle viruses could be separated into four entities; namely, strawberry mottle virus, mild yellow-edge virus, crinkle virus, and vein chlorosis virus. In 1953, Posnette (73) reported on a new strawberry virus disease which he named "green-petal". The infected plants were dwarfed and the flower petals were green in color. This virus was considered to be of a killer type for the infected plants never survived. The question of whether any of the viruses isolated in the British Isles are identical with any present in North America still awaits con- firmation. B. Strawberry Virus Diseases in Western United States and British Columbia, Canada In 1922, Horne CT?) reported on a disorder of strawberry in California. He presented a pathological description and a list of conditions which supposedly contributed to its cause. In 1926, Plakidas (69) presented a preliminary report on this disorder covering symptomatic and cytological studies. At that time he tentatively applied the term "yellows" to this disorder. A year later he (70) reported on the successful transmission of the disease by means of the strawberry leaf aphid, Capitophorus fragaefollii Ckll., thus proving its virus nature. This is the first known report in the literature on the verification that strawberry may be infected by a virus. The typical symptoms of this disease were yellowing of leaf margins and a marked stunting of the entire plant. The damage caused by yellows was estimated to be a 50 per cent decrease in productive- ness. 5. In 1927, Zeller.ODA) found a witchesLbroom producing virus infecting the Marshall strawberry that was also transmitted by Capitophorus fragae- lggllii. The symptoms recognized in Oregon were long petioles, small leaf— lets and a multiple crown which gave the plant a bushy appearance. Another disease reported in Oregon on the Marshall strawberry was crinkle. Zeller and Vaughn 005) presented a brief description of the disease in 1932. The presence of chlorotic areas in the leaflets caused ' a wrinkled condition and an uneven margin. It was not proved to be of virwis origin until Vaughn (97) in 1933 reported transmission by the strawberry leaf aphid. In the same year, Zeller (1C6) further found that seven cultivated varieties and two wild species of Fragaria were susceptible to crinkle. The yield in these infected plants was reduced more than 50 per cent. In 1951, Fitzpatrick and Mellor (23) found British Sovereign, the commercial strawberry variety of British Columbia, to be a latent carrier of yellows. When Fragaria vesca was grafted to yellows-infected British Sovereign the reaction was rapid and severe.' The young runner tips hooked back, the newly developed leaves were minute and yellow at the margins, and the plants eventually died. Later that year, the same investigators (56) showed that yellows was a complex of at least two component viruses. They described the general symptoms of these individual components on E- lease. In 1953, Frazier (30) presented evidence on a graft transmissible latent virus found in Fragaria vesca. The symptoms were expressed on the leaves as a mild mottle. Information concerning the distribution of this virus in indicator and cultivated strawberries is not known. 6. Recent investigations by Frazier and Thomas (31) show that western aster yellows producesphylloidy of flowers on the Lassen strawberry variety in California. This is the first record of a virus that infects host plants other than Fragaria occurring naturally in strawberries. C. Strawberry Virus Diseases in Eastern United States and Ontario, Canada The presence of virus diseases in strawberry fields in eastern United States was not recorded until 1931. At that time Mook (66) found witches'-broom to be quite abundant in plantings in New York, Minnesota, Wisconsin and Illinois. In l9h2, Berkeley and Plakidas ( 8 ) reported on a new strawberry virus disease found in New York and Ontario which they named'fleaf rolIL They considered leaf roll to be of minor importance in both localities. In l9h6, Demaree (16) stated that a virus disease of the yellows type was present east of the Rocky Mountains. He considered it to be the most prevalent and destructive type common in eastern strawberry plantings. Later Demaree and Marcus (19) reported on a survey of eastern cultivated strawberries. Their data showed that most of the strawberry plants tested were infected with one or more viruses. They described the viruses without given names but designated them as numbered types according to the symptoms expressed on indicator plants. - In 1952, Fulton (24) showed that one of the tobacco necrosis viruses was transmitted mechanically from contaminated E. Egggg roots. This repre- sents one of the few cases of"virus transmission from strawberry by mechanical means. CHAPTER III iATERIALS AND METHODS Increased investigationsof strawberry virus diseases have disclosed that various Egggagia species differ markedly in their reaction to such diseases. Four Fragaria species were used by the writer in these studies to differentiate between virus types and to determine which species could be used as a standard indicator plant in all subsequent work. A brief history of each is given. A. Virus-Sensitive Indicator Species a. Fragaria virgégiana L. The first report on the use of a Fragaria species for virus determination was that of Harris and Hildebrand (35). Their data, published in 1937, proved that Fragaria virginiana was extremely susceptible to the yellowbedge complex. In 19Al, working with leaf roll, Berkeley and Plakidas ( 8 ) demonstrated that this species could be successfully used as an indicator for this virus disease. b. Fragaria vesca L. (East Malling Strain). Progress in determining virus infection in the cultivated strawberry has been greatly increased in recent years due to the discovery of Harris and King (36). In 1942, they reported that a selection of Fragaria vesca collected near East Malling, England, was highly sensitive to strawberry virus diseases. In 1950, Demaree (18) showed that this selection would be useful in the United States as a virus- sensitive indicator plant. As a result of these findings this species strain has been used throughout North America as the principal indicator for determining the strawberry viruses. c. Fragariagplatypetala Rydb. (Seneca Straini Methods of transmitting strawberry"viruzses.have improved and consequently there has been an in- crease in the isolation of new virus-sensitive Fragaria species. In 1951, — —_ -I 7 I - 7—7.; ...:—.-I—-|.' _.‘.-_I .. .hii-i—- _ Miller (59) showed that a variant of E. vesca (now identified as E. platypetala Rydb. by C. L. Gilly, Michigan State College) was extreme- ly susceptible to the yellows virus. In addition, this species has a stolon of large diameter which is conducive to successful grafting techniques. d. Fragaria bracteata Heller. While investigating insect transmission of strawberry viruses, Frazier (28) also found the bracted strawberry, E, bracteata, to be an excellent indicator plant for yellows. This species appeared to show marked sensitivity to infection the year-round under greenhouse conditions. B. Culture of Indicator Species and Cultivated Varieties Under Test The various indicator species used in this study were grown in four- inch pots. These plants were placed in a greenhouse bench filled six inches deep with sand. During the summer months the plants received partial shade to produce succulent growth. A 16 hour day is essential for the develop- ment of stolons. During the winter months it was necessary to furnish additional artificial light and this was accomplished by suspending dual fluorescent units (no watt std. cool white) two feet above the green- house benches. Every 14 days each plant received 50 milliliters of a stock solution of fertilizer. This solution was prepared by adding two ounces of a 10-52-17 water soluble fertilizer to one gallon of water. Under these conditions the plants tended to develop at a faster rate than those under general maintenance. As the cultivated plants were received from the cooperators they were dipped in a l:hOO solution of nicotine sulfate to kill any aphis present. This is a general practice for control of root aphis. At least 9. four of the most vigorous plants of each lot were set in four-inch pots and given the standard growing conditions described previously. ‘ C. Method of Indexing The idea of stolon-grafting cultivated strawberry varieties to in- dicator species to test for the presence of viruses was first reported by Harris in 1932 (33). A similar method was described by Demaree and Marcus (19) in 1951. Miller (63) described these methods in greater detail summarizing them as the "tongue" graft and the approach graft. The "tongue" stolon-graft method was used throughout these investi- gations. However, certain modifications that further enhance positive transmission of the virus were developed in this study. These modifica- tions were: covering the stolons with sand to enlarge their diameters, scraping the epidermis for additional surface union, and cutting off the tips of each "tongue" to prevent curling. Details of the entire method follow. "Tongue" Stolon-Graft Technique. During the initial phases of these virus studies, the writer used the primary stolon of small diameter for grafting. This resulted in many graft union failures. Subsequent- ly, it was observed that when the stolon tips were buried in moist sand their diameters increased from.0.026 to 0.057 inches. Stolons which had been thus treated were thereafter used for grafting, there- by solving the main objection to most virus-sensitive species of Fragari . Another point considered was the stage of stolon growth best adapted for grafting. Harris (33) showed that successful unions can be made by 10. using stolons which have completed their primary elongation with the terminal leaf bud beginning to unfold. At this time the stolon has grown past the stolon node about four inches. The equipment required for grafting consisted of a single-edge razor blade, one-quarter inch tape (Polyken Industrial, Bauer & Black Go.) out into one and four inch lengths to be readily available for wrapping the joined stolons, and a coded pot label for identification purposes. 1. Procedure The actual graft is made in the terminal two inches of the stolon. Carefully scraping the epidermis in this section with a razor blade creates additional wound surface that may form callus and so unite with a similarly' treated surface of the other stolon when the graft is made. (Figure l). The first cut is made on the side of the test plant stolon in the direction of the plant. This cut is made longitudinally about one- half inch in length slanting towards the center of the stolon. A similar cut is made on the indicator stolon away from the plant. This gives two similar tongue-like slivers of stolon tissue that will be fitted together. Care should be taken in making these cuts, as too deep an incision may result in death of the stolon. (Figure 2). Using the thumb-nail as a backstop, the tip of each "tongue" is cut off with the razor blade (Figure 3) thus preventing curling under of the tips when actual insertions are made. This modification was found to eliminate much time in making the graft and to further insure better contact of the cut surfaces. The "tongue" of the indicator is inserud.into the cut of the variety stolon. (Figure A). 5. 7. 11. While the joined stolons are held in place with one hand, each end of the graft is bound with one inch length of tape. (Figures 5 and 6). Starting one inch beyond the graft at the end nearest the plants, a four inch strip of tape is wrapped spirally around the entire graft._ (Figure 7). This is to prevent the "tongues" from twisting out of position and further reduces strain on.the union. The completed Ftongue" stolon-graft is preSented in Figure 8. The pair of grafted plants is placed in line on a sand bench with the grafted stolons lying on the sand and extending toward the outer side of the bench. (Figure 9). The sand is kept damp to prevent grafts from drying out and to facilitate rooting of the runner plants. . Inspection may be made after a ten day period by removing the tape to examine for callus formation which indicates probable union. If union is not evident then the pair of plants are regrafted later when new stolons develop. The successfully grafted stolons are allowed to remain intact for 60 days to permit establishment of runner plants from each of the grafted plants and for possible symptom expression of virus on the indicator. 'Variety plants found to be infected were discarded. If no symptoms of virus were present after a 60 day period the variety plant was retested by grafting to another indicator plant for confirma- tion. Figure 1. Figure 2. Figure 3. Figure h. Scraping Epidermis of Stolon with Razor Blade to Create New Callus Surface. Method of Making Longitudinal Incision in Indicator Stolon (Left); Completed "Tongue" Incision (Right). Cutting off Tip of "Tongue" to Prevent Curling. Inserting Indicator "Tongue" into Stolon of Cultivated Variety. Figure 5. Figure 6. Figure 7. Figure 8. Binding of Tape around Upper Portion of Graft. Binding of Tape around Lower Portion of Graft. Binding of Four Inch Tape around Entire Graft to Prevent "Tongues" from Twisting. Completed "Tongue" Stolon-Graft. CHAPTER IV SYMPTOMATOLOGY OF TYPE 1 AND TYPE 2 VIRUSES 0N INDICATOR SPECIES AND CULTIVATED VARIETIES Demaree and Marcus (19) showed that two distinct and easily dis— tinguishable types of virus symptoms were expressed on plants of the East'Malling strain of Fragaria vesca; namely, Type 1 and Type 2 viruses. They indicated that Type 1 virus is similar to the crinkle virus predomi- nant west of the Rocky Mountains while Type 2 virus is prevalent only in the eastern section of the United States. They briefly described the general symptom expression of these two virus types on Fragaria 32322. A preliminary sampling of Michigan strawberry varieties for virus content by the writer indicated that Type 1 and Type 2 viruses were evident on Fragaria vesca stolon-grafted plants. This was the first indication of Type 1 virus in.Michigan. Since symptom expression of these virus types had been described on E. Egggg only, it was concluded that a studyd symptomatology of Types 1 and 2 on other Fragaria Species and cultivated varieties would be desirable. (Figures 10 and 11). Stock plants from the preliminary sampling, showing the described symptoms of Types 1 and 2 viruses were maintained and used for inoculation to the virus-free test Species and varieties. In tests where the inoculated plant did not express symptoms it was regrafted to Fragaria vesca for con- firmation of virus transmission. A. Type 1 Virus Symptoms é, Fragaria Virginians L. This species when stolon-grafted to Type 1 Virus-infected plants did not exhibit any marked symptoms, with the ex, ception of a moderate dwarfing in 60 days when compared with healthy Plants. Therefore, 3. virginiana may be regarded as a symptomless l5. carrier of the Type 1 virus. b. Fragaria vesca L. Within 25 days after grafting a healthy plant of this species to the Type I virus-infected plant, the distal runner showed chlorotic mottlihg of young leaflets. The chlorotic spots aver- aged one millimeter in diameter.’ These leaflets differ from healthy ones in the following respects: 1) one or two are about 1/3 normal size or entirely absent, 2) their color is deeper green than normal. Type 1 virus-infected E. 32333 plants produce flowers approximately 1/2 nonmal size and develop a few thin stolons after infection is evident. The symptoms of the Type 1 virus appear on the parent indicator plant about h5 days after grafting. This indicates that the virus entity involved can move in either direction through the stolon. Infected plants maintained for 16 months did not display any new symptoms. . c. Fragaria platypetala Rydb. The initial reaction of this species to the Type 1 virus was evident in about 30 days on the distal runner by chlorotic mottling of the young leaflets. The chlorotic areas on the leaflets were large, averaging.2.5 millimeters in diameter. The leaf symptomatology consisted of the following: 1) leaflets reduced to 1/8 normal size, 2) the entire leaf is deeper green than normal. Type 1 virus—infected E. platypetala plants produce flowers approxi- mately 1/3 normal size and do not form stolons after infection is evident. E. Elatypetala plants infected with the Type 1 virus were usually dead after a period of one year, thus showing this species to be more Sensitive to this virus entity than Fragaria 32222. d. Fragaria bracteata Heller. This species reacted to the Type 1 virus 16. in approximately no days. The mottling of the leaflets_was most pro- nounced on the distal plant of each grafted stolon. The chlorotic spots averaged 1.5 millimeters in diameter. The leaflet became severely asymmetrical because of the presence of only one leaflet per petiole. Type 1 virus-infected E. bracteata plants produced normal flowers and developed normal stolons after infection was evident. The reaction of E. bracteata to the Type 1 virus is manifested by the severe asymmetry of the leaf. The deep green color and abnormal flowers and stolons which develop in E. 33335 and E. platypetala are absent in E. bracteata. e. Cultivated Varieties. Three virus-free cultivated strawberry varieties were used in this study, namely; Catskill, Dunlap and Robinson. These plants were stolon-grafted to plants infected with the Type 1 virus. The varieties, Dunlap and Robinson, displayed no marked symptoms of Type 1 virus infection. Catskill exhibited only slight flecking on young leaflets which disappeared as the leaves matured. These results showed that the varieites used in this test are latent carriers of the Type 1 virus. A comparison of indicator sensitivity when infected with the Type 1 virus is presented in Table l. l7. .eouhmze haoaonpxo magmas .pouoemflv CH .EE m.H mafia Ippofi oapoaoano mmaoflpoa pom peammoa oco kHHmnocom amenoz Heapoz .ono>om hpo> manoeshm< on mpwopoman afiammmaa 68.8% Spams? mucman ”AmpoEmfin ca .55 Tm .mcflfioe 032020 exam Hmanou mouflm Hashes w\Honao>om ceaseloco odoz ones Hwfipcopommwv ouwm om mampmmwumam swummmam 63.8% Spawn... mpcmfln myopmsmwv cw .EE H.mcfiapuos oauoaoano onwm Hmzflo: mawpmemau mwNHm Hmanoc M\Huloamnm mamnnoco Hanan .3om Use mean a“ :oapmwus> mm momo> maymmwpm .nofiapmo mmoa Hafihoz ngnoz leopmahm m we movammmm o . memeHmaw> mensweam sham pcoEQoao>oQ sowpwmom when mofloomm thOHm :oaoum poammog cowsom cowumnzocH mmHommm moa H mmHB mo nomHmdmaoo H mAmnmch mm mpwouomun mmmmmmmm .mpo>om HMHuuoamMMflu ouflm pea defines m\H Immma mmoHOfipoa new pea mHoOHpom myoposmflv Om Immoa so hpmwcwao whopom «Henson N\H Hanan .3om swepo>< mmflmopodno HmCfiw>uopCH mm mwmmmmwmmwm efluemmhm .osflm Heapoc \ 0H\Halamowhposahm was Hmfihoc m\H newness mo>moa moHOflpom mamowoom whmpoEMfiu OOH use poamwoa so mummcflam .Hmspoc N\H Hanan «rem ommno>< mmfimopoano Hmcflo>wopCH Hm momo> mwpmmmnm .uofihpmo mmmH Hespoz Hefihoz ocoz IEOpQShm m we cowgamom o mmmmmmmmmw mmmmmmmm oNHm pcosmoao>on cowuwHoMHHonm seawomom when . mofiommm hosoflm coaopm czouo peaked :owpmnzocH poammog mmHommm moa N mmwh mo zomHMem uses .q on A we oHeow** .msfiaaop poaheoa no Eopmshm Hddmfi> vegan on some wnfipmehm scum asdoo* .osesczoo nous epoa named on» we moflo>eaz .haso scanned Hanan pm nose» peaumoa esp m spoosm sooum same we mefimams ouwmoado um comcflbom haze cowpuom Homes _ as ooaaon hflpzwfiam d neoosm :ooum mama muoaumoa mo magmas: oq see .095» vomenmvaoccsm m mCfiEAOM .nosop pod mcwaopos unwed one no mcwmams H onowsm soaaomunndCoeuo opflmoaao .osopuxm em deacso .aeausoa Ho coauhom eaeotoano Hecamums Hanan ca awauo>o N weakest hapnmfiam one Hmcfio>soucH seaweed mo scamsmz Hm HMfixeaso *amcwpsm wosussm ccwumnoaoo mafiaaom when. mofluofise> hpwho>em a E o a ans 5 m p o H H s e A *oowsom :oamesQCH mmHemHm<> Hmmmm3H9ADo Aqmm>mm zo NMAAZOU 940m m mqmo mesons Hespw>qufi om .pcoew>o no: mcfihozoam “seam Hmcflmwpo co pcoew>o nc3ouo NH woman Haynes: oH\H .mo>moH newness poops mo QdOhm emcee .pmep mo>ema vac owned mm haemeHXopddm coapmhomwaoaa ozone meooho use owpouoano momma Hannah: w\H .eompmze mw>moa meprosm .zoaaoh moaned» mpoauwog “amazonaaoc mo>me eao mo moHOfiuom .camamfi peamsma co 9:02Hana «Hmouoano mowapoEEhnm mpoammoq mowummswae nuofimmoa one noaoapom muse» mass m: .ucopfi>o acoaopn Hamsm mmma pepsfiwpmm.:0fipmaouwaoua ozone esoppxm .osfim amazes: N\H eon pcocfi>o mpoSoam memos am>soH pHo omamq «no haepmsfixopaam coapmpouwaoaa czopo Mona» Heavens w\H .eowpmzu mo>meq .30Haoh measudu ue>moa uao swung MON happeaouqam cogepeufimoud a: o a 0 «poems use cooum pnmwg woman Happens 4\H «pompmxe mo>mea mcwmnoEQ .cooam Hawum mplodeA ”Hensouflhon ue>woa vac mo moaoapom .nwmopoano Hmcfio>uopcw pcwflam masowAposEhn muoaueoq moaumscflne sesameoa pom moaoflaea mcsow when mm when and when 00 whee om £8 on messes? Heaps: weaned COfiuMQSOCH Soonml.monoufi3 .EE m £5 .M 08 onmmHzmz N mafia oz< Soomml.mmmosH3 mo zomHm mqm