PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINE return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE MW FEB ‘9 3 3, DD? APHID TRANSMISSION OF BLUEBERRY SHOESTRING VIRUS AND SEASONAL POPULATIONS OF ITS VECTOR ILLINQ1§.EEEEEBI (MACGILLIVRAY) By Kathryn Margaret Morimoto A THESIS Submitted to Michigan State University in partiai fu1fi11ment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and P1ant PathoIogy 1984 ABSTRACT APHID TRANSMISSION OF BLUEBERRY SHOESTRING VIRUS AND SEASONAL POPULATIONS OF ITS VECTOR .ILLINQIA.EEEEEBI (MACGILLIVRAY) By Kathryn Margaret Morimoto The quantity of biueberry shoestring virus (BBSSV) taken up by its known aphid vector lllinoia‘peppeni (MacGiiiivray) reached a threshoid with an acquisition access period (AAP) of 24 hr. Transmission occurred with a 24 hr AAP and a 1 hr inocuiation access period. Field popu'lations L penned were monitored weekiy from May through Septem- ber. Popuiations aiatae and apterae were greatest in June. Apterae were found throughout the growing season; few aiatae were observed after mid-Juiy. Individuai 1;.peppeni were tested for BBSSV with radioimmunoassay (RIA). Percentages of viruiiferous aphids ranged between S and 15% throughout the season. There was wide variabiiity in the quantity of virus detected in individuais with up to 250 ng BBSSV detected per aphid. Field transmission of BBSSV to biueberry trap piants occurred throughout the season; however. incidence of infection was highest in May and June when the 1. pepper: popu'iations were greatest. ACKNOWLEDGMENTS I sincerely thank Dr; Don Ramsdell. my graduate advisor. for his ample guidance. support. and encouragement throughout this course of study. I thank also Dr. John Lockwood. Dr. Mark Whalon. and Dr. Jim Hancock. members of my graduate committee. for their advice and encouragement. I thank John Nelson. Research Director of the Michigan Blueberry Growers Association. who provided invaluable information and assistance to this research project. I acknowledge the support of the Michigan Blueberry Growers Association. whose efforts made possible the USDA special grant that funded this research. Special thanks are extended to Dr. Bill Chaney and Dr. Walt Esselman for their expertise and generous use of equipment. Deep appreciation is extended to Jerri Giliett. Adeie Childress. Nancy Schulte. and Maureen Petersen for all of the support. assistance. and information shared. And to Bob Kriegel. Terry Davis. and Erwin Elsner. many thanks for teaching me about blueberry aphids. TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . LIST OF FIGURES . . . . . . . . . INTRODUNION I O C O O C O O O O 0 LITERATURE REVIEW . . . . . . . . Blueberry Shoestring Disease and Biology of the Blueberry Aphid . Plant Virus Transmission by Aphid Virus and Vector Sources . . . . Vector Dispersal . . . . . . . . Monitoring Populations . . . . . METHODS AND MATERIALS . . . . . . Virus Purification . . . . . . . Antiserum Production . . . . . . Enzyme-Linked Immunosorbent Assay Gamma Globulin Purification . the S Causal Virus Conjugation of Alkaline Phosphatase1x>Gamma Globulin Assay Procedure . . . . . . . . . . . . . . . . . . Radioimmunoassay . . . . Iodination . . . . . Assay Procedure . . Virus-Vector Studies . Aphid Culture . . . Acquisition Access Period Studi Inoculation Access Period Studi Location of Field Studies . . . Source of Overwintering Blueberry Aphids Alate Blueberry Aphid Activity . . . . . Alate Aphid Movement Outside the Field Alate Aphid Activity Within the Field Alate Aphids on Screen Cages . . . . . Seasonal Trap Plant Infection . . . . . . . . Seasonal Blueberry Population Dynamics and 95 65 Bush-to-Bush Movement of Blueberry Aphids . . . . . . . . . . . . . Page viii RESULTS . . . . . . . Virus Purification Serology . . . . . ELISA . . . . . . RIA . . . . . . . . BBSSV Acquisition by Blueberry Aphids Virus Transmission by Blueberry Aphids. Source of Overwintering Blueberry Aphids Alate Blueberry Aphid Activity . . . . . Alate Aphid Movement Outside the Field Alate Aphid Movement Within the Field Alate Aphids on Screen Cages . . . . . . Seasonal Apterous Blueberry Aphid Populatios Bush Movement Source Plants Trap Plants Viruliferous Apterous Blueberry Aphids . . Source Plants Trap Plants Seasonal Alate Blueberry Aphid Populations Source Plants Trap Plants Viruliferous Alatae Source Plants . . Trap Plants Quantity of BBSSV in Individual Seasonal Trap Plant Infection DlstSION O O O O O O O I O O I O O I O oooooeooooeoeoaoeooeoooooe O O O O O O O O O O O I O 0 g C O O O O O O O O O U ad B O O O O O O O O O O C O O O c O C O O O O O O O O O APPENDICES O O O O O O O O O O O O O O O C O O C O O O A. 1982 BLUEBERRY APHID POPULATION AND PERCENTAGE V IRUL IF ERCXJS DATA B. 1981 FIELD STUDIES OF THE SEASONAL POPULATIONS ILLINQIA.EEEEERI AND THE SPREAD OF BLUEBERRY SHOESTRING VIRUS O O O C C O C O O O C I C C O O m o o 3’. o o o o o o O o o o I oofioeeeoeooooo OF REFERENCES 0 O O O O O O O O O O O O O O O O O O O C O O O O 104 105 118 131 Table Page A9. The Proportion of Viruliferous Apterous Blueberry Aphids on Blueberry Trap Plants Touching Blueberry Shoestring Virus (BBSSV)-Infected Source Plants in the Field . . . 110 A10. Analysis of Variance of the Proportion of Viruliferous Apterous Blueberry Aphids on Blueberry Trap Plants Touching Blueberry Shoestring Virus-Infected Source Plants in the Field . . . . . . . . . . . . . . . . . . 110 All. The Proportion of Viruliferous Apterous Blueberry Aphids on Blueberry Trap Plants Not Teuching Blueberry Shoe- string Virus (BBSSV)-Infected Source Plants in the Field . . . . . . . . . . . . . . . . . . . . . . . . . 111 A12. Analysis of Variance of the Proportion of Viruliferous Apterous Blueberry Aphids on Blueberry Trap Plants Not Touching Blueberry Shoestring Virus-Infected Source Plants in the Field . . . . . . . . . . . . . . . 111 A13. Alate Blueberry Aphid Populations on Blueberry Shoe- string Virus-Infected Blueberry Source Plants in the F181d O O O O O O I O O O O O O O O O O I O O O O O 112 A14. Analysis of Variance of Alate Blueberry Aphid Popula- tions on Blueberry Shoestring Virus-Infected Blueberry Source Plants in the Field . . . . . . . . . . . . . . . 112 A15. Alate Blueberry Aphid Populations on Blueberry Trap Plants Touching Blueberry Shoestring Virus-Infected Blueberry Source Plants in the Field . . . . . . . . . . 113 A16. Analysis of Variance of Alate Blueberry Aphid Popula- tions on Blueberry Trap Plants Touching Blueberry Shoestring Virus-Infected Blueberry Source Plants in the Field . . . . . . . . . . . . . . . . . . . . . . 113 A177. Alate Blueberry Aphid Populations on Blueberry Trap Plants Not Tbuching Blueberry Shoestring Virus- Infected Blueberry Source Plants in the Field . . . . . 114 A18. Analysis of Variance of Alate Blueberry Aphid Popula- tions on Blueberry Trap Plants Not Touching Blueberry Shoestring Virus-Infected Blueberry Source Plants in the Field . . . . . . . . . . . . . . . . . . . . . . . 114 A19. The Proportion of Vi ruliferous Alate Blueberry Aphids on Blueberry Shoestring Virus (BBSSV)-Infected Source Plants in the Field . . . . . . . . . . . . . . . 115 v1 Table Page A20. Analysis of Variance of the Proportion of Viruliferous Alate Blueberry Aphids on Blueberry Shoestring Virus- Infected Source Plants in the Field . . . . . . . . . . 115 A21. The Proportion of Viruliferous Alate Blueberry Aphids on Blueberry Trap Plants Not Touching Blueberry Shoestring Virus (BBSSV)-Infected Source Plants in the Field . . . . . . . . . . . . . . . . . . . . . . 116 A22. Analysis of Variance of the Proportion of Viruliferous Alate Blueberry Aphids on Blueberry Trap Plants Not Touching Blueberry Shoestring Virus-Infected Source Plants in the Field . . . . . . . . . . . . . . . . . . 116 A23. The Proportion of Viruliferous Alate Blueberry Aphids and Blueberry Trap Plants Touching Blueberry Shoe- string Virus (BBSSV)-Infected Source Plants in the Field . . . . . . . . . . . . . . . . . . . . . . . 117 A24. Analysis of Variance of the Proportion of Viruliferous Alate blueberry Aphids on Blueberry Trap Plants Touching Blueberry Shoestring Virus-Infected Source Plants in the Field . . . . . . . . . . . . . . . . . . ll7 Bl. Apterous Blueberry Aphid Populations on Blueberry Shoestring Virus-Infected Blueberry Source Plants in the F161d O O O O O O O O C O O O O O O O O O O O O O 125 82. Apterous Blueberry Aphid Populations on Blueberry Trap Plants Touching Blueberry Shoestring Virus- Infected Blueberry Source Plants in the Field . . . . . 126 83 . Apterous Blueberry Aphid Populations on Blueberry Trap Plants Not Touching Blueberry Shoestring Virus-Infected Blueberry Source Plants in the Field . . 127 EH4. TheeProportion of Viruliferous Apterous Blueberry Aphids on Blueberry Shoestring Virus (BBSSV)- Infected Blueberry Source Plants in the Field . . . . . 128 E§5.. The Proportion of Viruliferous Apterous Blueberry Aphids on Blueberry Trap Plants Touching Blueberry Shoestring Virus (BBSSV)-Infected Source Plants in the Field . . . . . . . . . . . . . . . . . . . . . . 129 5K5. ‘The Proportion of Viruliferous Apterous Blueberry Aphids on Blueberry Trap Plants Not Touching Blue- Berry Shoestring Virus (BBSSV)-Infected Source Plants in the Field . . . . . . . . . . . . . . . . . . 130 vii 'IO. LIST OF FIGURES Seasonal Life Cycle of the Blueberry Aphid. 1111agia.peppeni (MacGillivray) . . . . . . . . . . . . ELISA Absorbance Values (405 nm) of a Twofold Dilution Series of a Purified Preparation of Blueberry Shoe- string Virus (BBSSV) in ELISA Extraction Buffer . . . RIA Counts Per Minute (cpm) of a Twofold Dilution Series of a Purified Preparation of Blueberry Shoe- string Virus (BBSSV) in ELISA Extraction Buffer . . . Acquisition Kinetics of Blueberry Shoestring Virus (BBSSV) Uptake by Blueberry Aphids Which Fed on BBSSV-Infected Plant Tissue for 10 min. 1. 6. 12. 249 48: 30d 74 hr . e . . e e e . e e e o e . o o o 0 Acquisition Kinetics of Blueberry Shoestring Virus (BBSSV) Uptake by Blueberry Aphids Which Fed on Purified Preparations of BBSSV in Sachets . . . . . . Acquisition Kinetics of Blueberry Shoestring Virus (BBSSV) Uptake by Blueberry Aphids Which Fed on ZSI-IODBIBO BBSSV 1n saChe‘tS o o o e o e o e e o o o The Relationship Between Acquisition Access Period and Number of Viruliferous Blueberry Aphids Which Fed on Purified Blueberry Shoestring Virus (BBSSV) in Sachets or BBSSV-Infected Tissue . . . . . . . . . The Percentage Distribution of RIA Counts Per Minute (cpm) of 30 Individual Late-Instar and Adult Apterous Blueberry Aphids Which Fed for 24 hr on BBSSV- infected Tissue . . . . . . . . . . . . . . . . . . . Percentage Distribution of RIA Counts Per Minute (cpm) of 30 Individual Late-Instar and Adult Apterous Blueberry Aphids Which Fed for 24 hr on Purified BBSSV in Sachets . . . . . . . . . . . . . . Transmission of Blueberry Shoestring Virus (BBSSV) by Blueberry Aphids. Experiment One . . . . . . . . . viii Page 33 36 38 4O 42 45 47 49 52 Figure Page 10. Transmission of Blueberry Shoestring Virus (BBSSV) by Blueberry Aphids. Experiment One . . . . . . . . . . 52 11. Transmission of Blueberry Shoestring Virus (BBSSV) by Blueberry Aphids. Experiment Two . . . . . . . . . . 54 12. Seasonal Apterous Blueberry Aphid Populations on Caged and Uncaged BBSSV-Infected Source Plants . . . . 56 13. Seasonal Alate Blueberry Aphid Populations on Caged and Uncaged BBSSV-Infected Source Plants . . . . . . . 58 14. Map of Locations and Dates of Viruliferous Blueberry Aphids Caught in Yellow Pan Traps . . . . . . . . . . . 61 15. Seasonal Distribution of Apterous and Alate Blue- berry Aphids Caught in 10 Yellow Pan Traps . . . . . . 63 16. Seasonal Distribution of Apterous Blueberry Aphids on Blueberry Trap Plants Touching BBSSV-Infected Source Plants . . . . . . . . . . . . . . . . . . . . . 67 17. Seasonal Distribution of Apterous Blueberry Aphids on Blueberry Trap Plants Not Touching BBSSV-Infected source Plants I O O O O O O O O O O O O I O O O O O O O 69 18. Seasonal Distribution of Viruliferous Apterous Aphids on Caged and Uncaged BBSSV-Infected Source Plants . . . 72 19. Seasonal Distribution of Viruliferous Apterous Aphids on Blueberry Trap Plants Touching BBSSV-Infected Source Plants . . . . . . . . . . . . . . . . . . . . . 75 20. Seasonal Distribution of Viruliferous Apterous Aphids on Blueberry Trap Plants Not Touching BBSSV-Infected source P1ants O O O I O C O O O O O O O I O O O O O O O 77 21. Seasonal Distribution of Alate Blueberry Aphids on Blueberry Trap Plants Touching BBSSV-Infected Source P1 ants O O O O O O O O I O O O O I O O O O C O O O O O 80 22. Seasonal Distribution of Alate Blueberry Aphids on Blueberry Trap Plants Not Teaching BBSSV-Infected source P1ants O O I O C O O O O O O O I I O O O O O O O 82 253. Seasonal Distribution of Viruliferous Alate Blueberry Aphids on Caged and Noncaged BBSSV-Infected Source P1 ants O O I O O O O O O O I O O O I O O O O O O O O O 86 Figure 24. 25. 27. Seasonal Aphids Source Seasonal Aphids Source Seasonal Page Distribution of Viruliferous Alate Blueberry on Trap Plants Touching BBSSV-Infected P1ants O O O O I O O O I O O O O O O O O O O O O 88 Distribution of Viruliferous Alate Blueberry on Trap Plants Not Touching BBSSV-Infected P1ants O O I O O O I O O O O I O O O O O O O O O 90 Blueberry Trap Plant Infection After 4-Week Exposure Period to BBSSV-Infected Source Plants Within the F161d O O O O O O O O O O O O O O O O O O O 92 The Relationship Between the Incidence of BBSSV- Infected Trap Plants and the Mean Numbers of Apterous Blueberry Aphids on Noncaged Source Plants and Trap Plants . . . . . . . . . . . . . . . . . . . . 95 INTRODUCTION Blueberry shoestring disease. caused by blueberry shoestring virus (BBSSV). is an economically important virus disease of highbush blueberry. W W. L. In Michigan. the nation's leading producer of highbush blueberries. blueberry shoestring disease is the most widespread virus-caused disease of highbush blueberries. Infected bushes have decreased vigor and are eventually debilitated by the disease.~ The only known vector of BBSSV is the blueberry aphid. 1]]jngja peppenj (MacGillivray). In the past. sole control of the disease consisted of rogueing infected bushes to remove the source of inoculum. Only recently. growers have begun to spray insecticides to control the aphid vectors in addition to removing the diseased bushes. Prior to this work there had been no epidemiological studies of blueberry shoestring disease. The aphid-vector relationship. aphid dispersal. aphid population dynamics. and aphid-mediated transmission all needed to be studied in order to develop better control measures for the disease. The first research objective was to determine the optimal times for BBSSV acquisition and inoculation by blueberry aphids. The virus- vector relationship plays an important role in determining whether or not insecticidal sprays may be effective in preventing the spread of an aphid-vectored plant virus. The second research objective was to determine if blueberry aphids overwinter within the blueberry field. At the time this research project was started it was unknown whether blueberry aphids over- wintered within the blueberry field or immigrated into the field from an outside source. It was suspected. however. that the aphids over- wintered within the field. The third objective was to monitor the movement of alate blueberry aphids inside and outside an isolated blueberry field with yellow pan traps and to determine the percentage of alatae which were viru- liferous. In western Michigan there are often blueberry fields adjacent to each other. Winged (alate) blueberry aphids could probably easily fly to adjacent blueberry fields and spread the disease. Recently developed ultrasensitive serological assays would be used to determine if the vectors carried virus. The fourth objective was to determine the seasonal blueberry aphid population trends within the field and to determine the percentage of wingless (apterous) blueberry aphids that were Viruliferous. These data would provide information for timing control measures. Lesney et a1. (1978). using van der Plank's model. which tests for randomness of spread of a plant disease (van der Plank. 1944). determined that shoestring disease spreads down the row. Within the field. blueberry bushes usually touch and overlap adjacent bushes. This provides a natural avenue for walking aphids to move to adjacent plants and transmit virus. The fifth objective was to determine whether or not BBSSV is as likely to be transmitted to adjacent trap plants not touching BBSSV-infected source plants as trap plants which touch source plants. The final objective was to determine when during the season BBSSV- infection occurs within the field and at what levels relative to aphid populations. This study would provide information for timing control measures. All of these objectives were directed toward having a better understanding of the spread of blueberry shoestring disease. which would eventually lead to the development of better control measures. L ITERATURE REV I EW WWI-Lamas Blueberry shoestring virus (BBSSV) disease was first reported in New Jersey on highbush blueberry. 1mm mm L.. by Varney (1957). Since that time. shoestring disease has been reported in Michigan (Stretch 8. Hilborn. 1970). Washington State (P. Bristow 8. D. Ramsdell. unpublished data). North Carolina (R. Milholland. personal communication. 1983). and Nova Scotia (Lockhart & Hall. 1962). The probable mode of spread of shoestring disease of blueberry to these areas was through infected nursery stock which could be traced back to New Jersey (J. Nelson. personal communication. 1983). In Michigan. shoestring disease is the most widespread virus- caused disease of highbush blueberry. A 1983 Michigan Department of Agriculture survey of Ottawa County. which produces 39% of the state's blueberry crop. identified 2435 shoestring diseased plants on the basis of symptomatology (H. Marlow. personal communication. 1983). The most common symptom on shoestring diseased plants is elongated reddish streaking on current and l-year-old shoots. Severely affected leaves are crescent or strap-shaped. It is this strap-11 ke ”shoestring" symptom that is the basis for the descriptive shoestring disease name. Other common symptoms of the disease include red vei nbanding or red oak leaf patterns and a red to purple cast to immature berries. In addition. berry production progressively decreases as the infected bushes decline in vigor. Hartmann. Bath. and Hooper (1973) found virus-like particles (VLPs) in epidermal. palisade. spongy mesophyll. and xylem parenchyma. They did not. however. find VLPs in the phloem vascular tissue. In addition. crystalline arrays of VLPs were found in leaf epidermal cells and root xylem cells. with larger masses of VLPs in the roots. Transmission studies by Lockhart and Hall (1962) and localization studies by Hartmann. Bath. and Hooper (1973) indicated a virus-like causal agent of shoestring disease. It was not until later that Lesney et a1. (1978) showed that shoestring disease is caused by a virus--blueberry shoestring virus (BBSSVL Blueberry shoestring virus is a spherical single stranded (55) RNA virus that is 28 nm in diameter (Ramsdell. 1979a). It is not sero- logically related to viruses with similar physical and chemical proper- ties (Lesney et al.. 1978). but it does have physical and chemical properties that are similar to those of members of the southern bean Inosaic virus group (Ramsdell. 1979a.bL. The host range of BBSSV is quite limited. ‘The only known host plants are highbush blueberry. 1. Wm (Varney. 1957) and lowbush blueberry. L angusjjjgjjm (Lockhart & Hall. 1962). The virus can be transmitted between blueberry plants by chip budding (Lockhart 8. Hall. 1962; Schulte. 1983) and rub-inoculation using purified virus (Lesney et al.. 1978). Attempts to transmit purified BBSSV to herbaceous plants have been unsuccessful (Lesney et al.. 1978). Blueberry shoestring virus has been shown to be vectored by the blueberry aphid. 11.1mm penned (MacGillivray). The virus was transmitted by blueberry aphids having acquisition access periods of 2 min and inoculation access periods of 100 hr (Ramsdell. 1979b). W The blueberry aphid. 1.. mm. is commonly found in areas of blueberry production in Michigan (Giles. 1966; Elsner. 1982). A general life cycle of .1. pepper; is shown in Figure l (Elsner. 1982). The egg stage overwinters on or underneath the blueberry bush. Apterous (wingless) female aphids emerge from the eggs and produce second-generation viviparous females that reproduce parthenogenically. Many of the second-generation aphids develop into alate (winged) adults that migrate to other blueberry bushes where they produce apterous young. Some colonies produce alate aphids at a constant rate (approximately 2%) throughout the season (M. Whalon. personal communi- cation. 1984). Newly colonized bushes subsequently support several generations of apterous females during the growing season. As the blueberry leaves age physiologically toward the end of the season. the aphid population declines. Very late in the season the few remaining viviparous aphids produce oviparous females. These oviparous females produce the overwintering eggs. Giles (1966) noted that the blueberry aphids preferred the upper surface of the blueberry leaves as the primary feeding site. Elsner (1982) reported. however. that the blueberry aphids prefer to feed underneath tender leaves. on succulent growing shoots. and on swelling buds of the blueberry plant. The feeding aphids are sessile unless Figure l.--Seasonal life cycle of the blueberry aphid. IJ_1_i_n.o_i_a penned, (MacGillivray). (From Elsner. 1982.) _ ocam_m ¢w02w>oz icmmokoo noon 95.25333 22.me ><2 09.022 5.395“. mmmehamm 5:82.00 :2: IkflDOD< \ beau..— / V. 332 E2: 62268 = use: 082.2 620:; ‘III\ mzas 62a? 32 >42. 6 5203242 35355.3 3:528 crowded conditions or poor food quality cause them to moveriElsner. 1982); once disturbed. however. the aphids readily move. Blueberry aphids have been observed feeding and reproducing on woody plants other than M..cgnymbgsum (Elsner. 1982). ‘These plants included Quencus.nubna (red oak). Nyssa.sylxatjga (black gum). Age: ‘Lubnnm,(red maple). 11ex.xent19111a1a (winterberry holly). and Enunus Spp. They did not. however. seem to be significant alternate hosts (El sner. 1982). WWW Plant viruses must be able to disperse to new plants in order to reproduce. Common modes of plant virus transmission include trans- mission through seed. pollen. and infected propagation stock. The most common mode of plant virus transmission in nature. however. is by insect vectors. ‘The insect order Hgmgptena contains the largest number of plant virus vectors. Included in this order are the aphids (sub- order Stenngnnhyncha). which vector approximately 200 different viruses (Harris. 1981; D'Arcy 8. Nault. 1982). There are three classifications of plant virus transmission by aphids: nonpersistent. semi-persistent. and persistent based upon the length of time the virus is retained by its vector. Nonpersistent virus transmission was characterized by Watson and Roberts (1939) as having very short acquisition and inoculation threshold times on the order of minutes. Both acquisition and inoculation of virus occur durVing the brief periods that aphids probe or sample the host plants. In addition. virus is retained in its vector for very short intervals. Other characteristics of nonpersistent viruses are that they are not IO retained through a molt. there is no latent period after acquisition before virus can be transmitted. and there is increased efficiency of virus transmission with preacquisition fasting of the aphids. Kennedy. Day. and Eastop (1962) suggested that the term "stylet- borne" be used instead of nonpersistent. They believed that the virus is carried on the vector's stylet. These nonpersistent viruses are said to have a low vector specificity (Sylvester. 1969) because they can be transmitted by many different aphid species. Examples of viruses that.are transmitted in a nonpersistent manner are those that are members of the following virus groups: potyviruses. cucumoviruses. carlaviruses. caulimoviruses. and alfalfa mosaic virus. Watson and Roberts (1939) also described persistent transmission. Persistent transmission is characterized by very long acquisition and inoculation time thresholds. The term "persistent" relates to the long length of time (days) that these viruses are retained by their vectors. Black (1959) called this type of virus-vector relationship "circula- tiveJ' These circulative viruses are believed to pass through the vector's gut lining into the hemolymph. where they circulate and bathe the internal organs. Eventually the virus passes into the salivary glands from where the virus is inoculated into the host plant. There is a characteristic latent period between acquisition and inoculation that corresponds to the time it takes the virus to reach the salivary glands of the vector. In addition. since the virus is associated with the hemolymph and internal organs. it is retained through a molt. Luteoviruses. which include barley yellow dwarf virus. beet western yellows virus. and potato leafroll virus. and two other 11 nonluteoviruses. lettuce necrotic yellows virus and sowthistle yellow vein virus. are examples of viruses transmitted by aphids in a persistent manner. Those viruses which are acquired and inoculated after intermediate acquisition and inoculation times (hours to days) were termed semi- persistent by Sylvester (1956). Day and Venables (1961) suggested that these viruses are stylet-borne. but with different physical properties and in different distributions in the host plant tissues. Semi-persistent viruses are retained by the vectors for 1 to 2 days. There is no requisite latent period before the vector is able to transmit the virus. Closteroviruses such as citrus tristeza virus. beet yellows virus. and beet yellow stunt are semi-persistent viruses. Currently. the terms nonpersistent. semi-persistent. and persistent are most commonly used in virus-vector relations studies. Ramsdell (1979b) reported transmission of BBSSV to blueberry seedlings by l..peppeni having acquisition access periods (AAPs) of 2 min and inoculation access periods (IAPs) of more than 100 hr. Transmission did not occur with AAPs of 1 or 24 hn. Viruses that are taken up and transmitted with short AAPs on the order of minutes are nonpersistent viruses. Optimal IAPs for nonpersistent viruses. how- ever. are also short. being on the order of minutes or a few hours rather than several days. The results of the BBSSV transmission test indicate that BBSSV does not clearly fit into any of the virus-vector relationship classifications. Additional experiments need to be 12 conducted to determine what type of virus-vector relationship BBSSV has with its aphid vector. WWW: Crop plants. weeds. and seeds are common sources of plant viruses. Many virus diseases of potato are perpetuated through infected seed pieces which provide virus sources for aphid dissemination. Potato virus diseases that are transmitted by aphids include potato virus Y (PVY). potato virus A (PVA). potato virus M (PVM). potato aucuba mosaic virus (PAMV) (transmitted with helper virus PVA). alfalfa mosaic virus (AMV). cucumber mosaic virus (CMV). and potato leafroll virus (PLRV) (Beemster & Rozendaal. 1972). All of these viruses are nonpersistently transmitted except for PLRV. which is persistently transmitted. Overlapping crops often serve as virus sources. Mangold clamps in England are virus sources of beet yellows virus. beet mild yellowing virus. and beet mosaic virus (Broadbent et al.. 1949). all of which infect nearby sugar beet fields. The clamps also serve as protective sites where the virus vector. the green peach aphid Esz s pensicae (Sulz)] overwinters. The beet crop itself is the main source of beet yellows virus in both the United States (Duffus. 1963) and in Europe (Broadbent et alu 1949). Shepherd and Hills (1970) have reported that beet western yellows virus overwinters in the first-season beet plants and suggested that new beet fields be planted up to 20 miles away from overwintering beet fields. Perennial crops are important as continuous virus sources. Viruses of perennial plants are often spread through infected 13 propagation stock. Blueberry shoestring disease is a prime example of this. Many of the BBSSV-infected fields in Michigan were planted with infected nursery stock which originated from New Jersey (J. Nelson. personal communication. 1983L Duffus (1971) has reviewed the role of weeds in the incidence of virus diseases. Quite importantly. weeds may be a reservoir of both viruses and their aphid vectors. Certain aphid species are dioecious; that is. they have alternate hosts. These aphids overwinter on a primary host (a woody plant) usually in the egg stage. In the spring. winged (alate) aphids migrate to secondary hosts. .Myzus.pensicae is an example of a dioecious aphid. The aphid will usually overwinter as eggs on peach trees or other ‘Enunus species. In warmer climates or during mild winters. the green peach aphid will overwinter as adults in weeds or field crops. Potato storage sheds. greenhouses. and bedding plants also serve as over- wintering sites to parthenogenic EL 29:51:19 (Whalon. 1979). ‘This is very important epidemiologically because spring migrants develop earlier on secondary hosts (Duffus. 1971). Not only is the migration period longer when the aphid overwinters on the secondary hosts versus primary hosts (Doncaster & Gregory. 1948). but these aphids are more. likely to carry viruses than aphids that overwinter on primary hosts (Duffus. 1964; Wallis. 1967; Heathcote et al.. 1965). Wallis (1967) has found that there is a greater incidence of beet western yellows virus (BWYV) in sugar beet plants next to ditches where M. m overwinters in the viviparous summer form than next to peach trees where the aphid overwinters in the egg stage. Both early 14 infection and a longer growing season appear to be the reasons for a higher incidence of BWYV near Walla Walla. WA (Wallis. 1967). W The production of alatae in the spring is associated with physical contact between aphids or "crowding" (Lees. 1966). The physiological condition of the host plant (Johnson. 1966a) as well as the effects of temperature and photoperiod (Johnson. 1966b) also affect aphid wing development. Once in the air. alatae are carried primarily by surface winds. Any change in the ground or crop surface causes a change in the air turbulence. which affects deposition of the winged aphids (Lewis. 1965). ‘This change in air movement accounts for the edge effects of primary infections that are seen in many crops (Broadbent. 1957; Doncaster & Gregory. 1948). There is no evidence that alatae recognize fields of host plants and then alight at the edge of the field (Swanson. 1968). Kennedy et a1. (1959) found that flying aphids are just as likely to land on nonhost plants as well as host plants. Later Kennedy (1962) summed up these findings quite concisely by saying that "dispersal takes precedence over host finding." WW Monitoring aphid population is an important aspect of studying the epidemiology of an aphid-transmitted virus disease. Irwin and Goodman (1981) used horizontal colored tiles (HCT) to monitor aphids in soybean field studies. 'These lime-green-colored traps were designed especially 15 for monitoring aphids alighting on soybeans (Irwin. 1980). The incidence of alighting aphids was closely correlated with the incidence of soybean mosaic virus (Irwin & Goodman. 1981). ‘The numbers and species of aphids caught in the HCT traps were very similar to those found on the soybean plants (Irwin & Goodman. 1981). Live vectors can be collected from vegetation to use for infec- tivity tests. Aphids within crops may be collected using suction traps (D-VAC) or nets (Howell. 1794). Different types of traps are used for vectors flying into the field. Suction traps (Plumb. 1971). vertical nets (Halbert et al.. 1981). or water pan traps (Demski. 1981) have been used to trap incoming vectors. Once collected. the live insects are placed onto test plants. Any aphids that transmit virus are then identified. The seasonal spread of virus diseases may be studied by assessing virus incidence in the field either by noting symptoms or by indexing the plants. There are two problems associated with this method of studying the seasonal spread of virus diseases: (1) the long length of time required before test or field plants show symptoms and (2) multiple infections in the field. Broadbent and others (1950) averted this problem of multiple infections by exposing potted potato plants to the potato field containing infection foci for limited time periods. Other researchers have also used this trap plant method for estimating seasonal infection pressures in the field (Schwartz. 1965; Madden et al.. 1983). Although transmission tests are the most reliable ways of determining if a vector is infective. it does take a long time before 16 results are obtained. Ultrasensitive. serological tests have been developed recently which are able to detect virus in vectors within 1 or 2 days. Gera. Loebenstein. and Raccah (1978) were the first to detect a plant virus.(cucumber mosaic virus) in an aphid vector using enzyme- linked immunosorbent assay (ELISA). Since then. other plant viruses have been detected by ELISA in aphid vectors. Potato leafroll virus (PLRV). a persistent virus. was first detected in groups of aphids by Clarke. Converse. and Kojima (1980L Later. Tamada and Harrison (1981) were able to detect PLRV in single aphids and study the seasonal differences of virus content in the vector. Pea enation mosaic virus (PEMV). another persistent virus. can also be detected in individual aphids (Fargette. Jenniskens. & Peters. 1981). ELISA can also detect potato virus Y (PVY). a nonpersistent virus (Carlebach. Raccah. & Loebenstein. 1982). and citrus tristeza virus (CTV). a semi-persistent virus (Cambra et al.. 1981). in groups of aphid vectors. Derrick's (1973) method of serologically specific electron microscopy (SSEM). also known as immunosorbent electron microscopy (ISEM). has been used by Plumb and Lennon (1981) to detect barley yellow dwarf virus (BYDV) in single aphids. Gillett et aL.(l982) compared ISEM to ELISA and radioimmunoassay'(RIA) for detecting BBSSV in its aphid vector. RIA was the most sensitive for this purpose. ISEM was not suitable due to the low virus concentration in the aphids and the insect particulate matter which obstructed viewing. The use of sensitive tests such as ELISA and RIA has made transmission and epidemiological studies of blueberry shoestring 17 disease a practical possibility. Blueberry shoestring virus infection may be latent in the blueberry host plant up to 4 years before symptoms are apparent (Ramsdell et al.. 1980). With ELISA. test plants may be assayed for the presence of BBSSV instead of waiting for symptoms to develop years after infection. In addition. the movement of BBSSV- carrying blueberry aphids may now be studied by using RIA. which is capable of detecting BBSSV in individual aphids (Gillett et al.. 1982). METHODS AND MATERIAL S Winn Blueberry shoestring virus was purified from frozen BBSSV-infected blossoms as described by Ramsdell (1979a). All purification proce- dures were at 0-4 CL One hundred grams of frozen blossoms were homog- enized in a Waring blender with three volumes cold 0.1 M potassium phosphate buffer. pH 7.0. containing 0.01 M 2-mercaptoethanol and (L005 M thioglycolic acid. Triton X-100 [8% (v/v)] was added to the homogenate and the mixture was stirred overnight. The homogenate was strained through two layers of cheesecloth. Chloroform and butanol (5% each. v/v) were added to the solution and stirred for 15 min. The emulsion was centrifuged for 15 min at 2000 g_ in an IEC No. 872 rotor (International Equipment Co.. Needham Hts.. MA 02194). The aqueous phase was pipetted off and adjusted to 8% (w/v) polyethylene glycol (PEG). mol. wt. 6000. and 0.1 M sodium chloride while stirring. The mixture was stirred overnight and then centrifuged for 30 min at 3500 gin an IEC No. 872 rotor. The PEG pellet was resuspended overnight in 10% of the initial aqueous phase volume with 0.05 M phosphate buffer. pH 7.0. containing 0.001 dithiothreitol (P-DTT). The suspension was clarified by a low-speed centrifugation for 30 min at 3500 g in an IEC No. 872 rotor. and concentrated by 18 19 ultracentrifugation for 2 hr at 28000 rpm in a Beckman No. 30 rotor. The pellet was resuspended overnight in 0.2 m1 P-DTT per tube. The preparation was layered onto 0-30% linear sucrose gradients made in P-DTT the previous night. The sucrose gradients were centrifuged for 90 min at 38000 rpm in a Beckman SW 41 rotor. The single virus band was collected using an 1500 density gradient fractionator and UV-analyzer (Instrumentation Specialties 00.. Lincoln. NE 68505). The sucrose fractions containing the virus were diluted threefold with 0.05 M phosphate buffer (PB). pH 7.0. and centrifuged for 3 hr at 38000 rpm in a Beckman No. 40 rotor. The pellet was resuspended in PB overnight. The concentration of the virus preparation was determined using the molar extinction coefficient of 0.1% BBSSV: E260 nm = 5.2. Wu A female New Zealand white rabbit was initially bled from the marginal ear vein to collect preimmune serum. The rabbit was injected intramuscularly with 1.2 mg purified BBSSV emulsified with an equal volume (1.1 m1) of Freund's complete adjuvant (Difco Products 00.. Detroit. MI 48232). Two subsequent intramuscular injections at 7-day intervals consisted of a total of 2.3 mg purified BBSSV emulsified with an equal volume (2.1 ml total) of Freund's incomplete adjuvant. Five days after the final injection. the rabbit was bled from the marginal ear vein at 3- to 6-day intervals for 1 month. The fresh blood was placed in a 37 C water bath for 2 hr and then kept at 4 C overnight to coagulate the red blood cells. The serum fraction was pi petted from the coagulated material and a few crystals of 20 chlorobutanol (Sigma Chemical Co.. St. Louis. MO 63178) were added to the serum as a preservative. The serum was lyophyllized and stored at -20 C. The anti-BBSSV-serum was titered against purified BBSSV (0.1 mg/ml) in an Ouchterlony gel double diffusion test. The agar consisted of 8% agarose (w/v) (Sigma Type I. Sigma Chemical Co.. St. Louis. MO 63178). 0.85% sodium chloride (w/v). and 0.15% sodium azide (w/v). W W Anti -BBSSV-gamma globulin was purified by the procedure described by Clark and Adams (1976). The gamma globulin was diluted 1:10 (v/v) in distilled water and added dropwise to 10 m1 saturated ammonium sulfate solution while stirring. After 30 to 60 min stirring. the mixture was centrifuged for 5 min at 6000 rpm in a Beckman No. 30 rotor. The precipitate was collected and dissolved in 2 ml half-strength PBS (0.01 M sodium-potassium phosphate buffer. pH 7.4. containing 0.8% sodium chloride (w/v). and 0.01% sodium azide (w/v). diluted 1:1. (v/v) in water). The gamma globulin preparation was dialyzed three times against 500 ml half-strength PBS then filtered through a 5 cm high bed of DEAE (Whatman DE-22) cellulose in a 10 ml pipette. Hal f-strength PBS was used to pre-equilibrate the column and e1 ute the gamma globulin. Two ml fractions were monitored at A280 nm and the first protein fractions to e1 ute were collected. The gamma globulin preparation was adjusted to 1 mg/ml and stored at ~20 C. 21 Wombats: Win Gamma globulin was labeled with alkaline phosphatase with the method described by Clark and Adams (1976). Two mg alkaline phosphatase (Type VII-S. Sigma Chemical Co.. St. Louis. MO 63178) were centrifuged for 5 min at 6000 rpm in a Beckman No. 40 rotor. The precipitate was dissolved with 1 mg purified gamma globulin preparation and dialyzed three times against 500 m1 PBS. 61 utaral dehyde (electron microscope grade. Sigma Chemical Co.. St. Louis. MO 63178) was added to make a final 91 utaraldehyde concentration of 0.05% (v/v). The solution was thoroughly mixed and kept at room temperature for 4 hr. The 91 utaral dehyde was removed by dialysis. three times against 500 m1 PBS. Bovine serum albumin (BSA) was added to make a final concentration of 5 mg/ml. The conjugate was stored at 4 C. Wu The double anti body sandwich method of enzyme-linked immunosorbent assay (ELISA) (Clark & Adams. 1976) was used to detect BBSSV in blueberry plant tissue. Flat bottom polystyrene microtiter plates (Dynatech Laboratories. Alexandria. VA 22314) were used for ELISA. The plates were coated with 1 m/ml anti-BBSSV-gamma globulin in coating buffer (0.05 M sodium carbonate-bicarbonate buffer. pH 9.6) at a rate of 200 pl per well and incubated for 3 hr at 37 C. Blueberry plant samples were triturated with a Tissumizer homogenizer (Tekmar Co.. Cincinnati. ()1 45222) in 1:10 (w/v) extraction buffer consisting of 0.01 M sodium potassium phosphate buffer. pH 7.4. containing 0.02% sodium azide (w/v). 0.8% sodium chloride (w/v). 0.5% 22 Tween 20 (v/v). 2.0% polyvinyl pyrrolidone (mol. wt. 40000. Sigma Chemical Co.. St. Louis. MO 63178) MN). and 0.2% ovalbumin (grade II. Sigma Chemical Co.. St. Louis. MO 63178) (w/v). After the homogenates were filtered through two layers of cheesecloth. aliquots of the samples were added at a rate of 200 pl per well. 'The plates contain- ing the samples were incubated at 4 C overnight. Enzyme-conjugate. at a dilution of 1:800 (v/v) in extraction buffer. was added at a rate of 200 pl per well and incubated 4 hr at 37 0. Between each step the plates were flooded with PBS-Tween at least three times to remove any loosely or nonadsorbed reactants. One mg/ml enzyme substrate. [enitrophenyl phosphate»(Sigma Chemical Co.. St. Louis. MO 63178). was freshly dissolved in substrate buffer (10% diethanolamine. adjusted to pH 9.8 with HCL) and added to the plates at a rate of 200 pl per well. After 1 hr incubation at room temperature. the A405 nm was measured spectrophotometrically with a microELISA minireader (Dynatech Laboratories. Alexandria. VA 22314). The threshold used for positive reaction for each plate was the mean A405 nm value of healthy samples plus three standard deviations. Samples in each test plate with A 405 nm values greater than the threshold were considered positive. Badmimunmssax Indination Purified anti-BBSSV-gamma globulin from the DEAE cellulose column was iodinated using the method described by Greenwood et a1. (1963). 23 To 50 pl gamma globulin. 150 pl PBS. 1 mCi Na125I and 5 pl chloramine-T (5 lug/ml in water) were added. The contents were thoroughly mixed and incubated 15 min on ice. Sodium metabisul fite (5 mg/ml in water). 5 pl. was added to stop the reaction. Sodium iodide (20 mg/ml in PBS). 25 pl. and 0.5% bovine serum albumin (w/v) in PBS (PBS-BSA). 100 pl. were added to act as carriers for the 1251 and gamma globulin. The mixture was loaded onto a Sephadex G-50 column (10 cm x 1 cm) pre-equilibrated with PBS-BSA. One ml fractions were eluted with PBS- BSA and collected. Aliquots of each fraction were counted in a Beckman Biogamma II gamma counter. The protein fractions were pooled and dialyzed three times against PBS. Wm A double antibody sandwich system similar to that described for ELISA was used for solid phase radioimmunoassay (RLAL Flexible disposable polyvinyl "V" bottom microtiter plates (Dynatech Laboratories. Alexandria. VA 22314) were coated with gamma globulin. 5 ul/ml. in coating buffer. at a rate of 100 p1 per well. and incubated 3 hr at 37 C. Blueberry tissue samples were prepared as previously described. Aphid samples were triturated with a stirring rod in a test tube containing 100 pl extraction buffer. The entire contents of each test tube were transferred with a pasteur pipet to a plate well. Test samples were incubated overnight at 4 C. As with ELISA. the plates were washed at least three times between each step to remove any nonadsorbed reactants. The wash solution for 24 RIA consisted of 0.5-1.0% bovine serum albumin (w/v) in PBS (PBS-BSA) or in PBS-Tween (PBS-Tween-BSA). Approximately 55.000 cpm 1251-anti-BBSSV-gamma globulin diluted in PBS-BSA was added to each well at a rate of 100 p1 per well. After a 4 hr incubation at room temperature. the nonadsorbed gamma globulin was aspirated out of the wells. ‘The plates were then washed four times with PBS-BSA or PBS-Tween-BSA as previously described. The sides of the flexible plates were cut off with scissors and the top was cut off with a hot wire to obtain individual wells. Each well was individually placed into a counting vial and counted by the gamma counter. Samples with counts per minute (cpm) greater than three ti mes the mean of the healthy sample wells plus three standard deviations were considered positive for BBSSV. Woman: AM The blueberry aphid culture used in the acquisition and inoculation access time studies was started from an aphid culture maintained by Erwin Elsner. Department of Entomology. Michigan State University. Gravid apterous (wingless) adult aphids were placed in petri plates containing moist filter paper. ‘The ensuing nymphs were used to establish the virus-free blueberry aphid colony. One-year-old rooted cuttings of highbush blueberry cv. Jersey were used as host plants for the aphid colony. The plants were tested by EL ISA prior to use to ensure that they were not infected with BBSSV. 25 The culture was maintained with an 18 hr day photoperiod with day and night temperatures of 23 C and 18 C. respectively. Wm: To study the acquisition kinetics of BBSSV by the blueberry aphids. late instar nymphs and apterous adults were allowed access to three different sources of BBSSV: l. symptomatic BBSSV-infected leaves on a detached shoot in water. 2. purified BSSV preparation in 20% sucrose contained in a Parafil membrane feeding cage (sachet). 3. 125I-labeled urified BBSSV preparation in 20% sucrose contained in a Parafil membrane feeding cage. Three different sources of virus were used because BBSSV is present in blueberry tissue in low titers. Since it was initially unknown whether or not BBSSV could be detected in its vector. the aphids were allowed access to very high concentrations of virus under membrane feeding conditions. Sachets for feeding aphids were made from plexiglass cylinders (3.8 cm in diameter x 4 cm high). After one end of the cylinder was covered with Parafildfi)and 20-30 aphids were placed inside the sachet. the top of the sachet was covered with a piece of very thinly stretched Parafian Approximately 200 pl of purified BBSSV in 20% sucrose. 1ZSI-BBSSV in 20% sucrose. or a control of 20% sucrose alone was pi petted onto the very thinly stretched Parafi ln® and then enclosed by a second piece of Parafilngg Aphids were allowed access to the BBSSV-infected tissue or the purified BBSSV in 20% sucrose contained in sachets for acquisition 26 access periods (AAPs) of 10 min. 1. 6. 12. 24. 48. and 72 hr. AAPs of 10 min. 1. 6. 12. 24. and 48 hr were used for aphids allowed access to the 1251-Bessv in 20: sucrose. The test aphids that fed on BBSSV-infected tissue of the purified BBSSV in sachets were tested in groups individually with RIA. 'Those that fed on ”SI-BBSSV were counted individually directly by the gamma counter. WSW To determine the optimum IAP. aphids were first allowed access to BBSSV-infected tissue for a constant AAP; Late-instar nymphs and adult apterous blueberry aphids were transferred to a symptomatic BBSSV- infected shoot contained in a vase of water for an AAP of 24 hr. An AAP of 24 hr was chosen because acquisition kinetics studies showed that there was no significant additional uptake of BBSSV with AAPs greater than 24 hr. After the requisite AAP. the aphids were transferred to potted healthy 1-year-old rooted blueberry cuttings cv. Jersey in groups of 15. Inoculation access times of 1. 6. 12. 24. 48. 96. and 192 hr were used. .At the end of each IAP. the aphids were removed and the test pl ants were sprayed with Pirimicarb (5.6-Dimethyl- 2-dimethylamino-4 pyrimidynl dimethylcarbamateh. There were 15 test plant replications per IAP treatment arranged in a randomized complete block experimental design. After 6 months of incubation in the greenhouse. leaf samples of ‘the test plants were tested by ELISA for BBSSV infection. ‘The test plants were then put into a dark. cold room (4 to 6 C) to satisfy a dormant period. After a dormant period of at least 1000 hr. the test 27 plants were moved to the greenhouse. Leaf samples taken from new growth after dormancy was broken were tested for BBSSV infection with ELISA. as previously described. A second IAP was conducted. Aphids were allowed access to symptomatic-BBSSV-infected shoots and leaves for a constant AAP of 24 hr. Groups of 15 aphids were transferred to test plants as before for IAPs of 0. 1. 6. 12. 24. 48. 96. and 129 hr. There were seven replica- tions per treatment set up in-a randomized complete block design. After 6 months of incubation in the greenhouse. leaf samples were collected from the test plants and tested for BBSSV-infection with ELISA. Mummies Experiments were conducted in Ottawa County. west-central Michigan. to study the spread of BBSSV by the blueberry aphid. In 1982 the field plots were set up at the Frank VenRoy blueberry farm. Eastmanville. MI. The cv. Jersey bushes were approximately 20 years old and planted on a 10 x 3 foot spacing. The field was clean cultivated. In 1981 the field was mapped for BBSSV infection by Adele Childress (unpublished data). Any bushes without blueberry shoestring disease symptoms were tested for BBSSV with ELISA. Wanna A caged bush experiment was conducted to determine whether or not blueberry aphids overwinter within the blueberry field. Fourteen BBSSV- infected bushes (hereafter referred to as source plants) at the VenRoy farm were selected and pruned to a uniform size and number of main 28 shoots. Seven of the source plants were each enclosed in a 16 mesh screen cage before bud break. while the other seven were not caged. The source plants were monitored weekly for the presence of blueberry aphids. W W A study was conducted to monitor the movement of alate blueberry aphids outside of an isolated blueberry field using yellow pan traps. The traps were goldenrod-colored plastic dish pans (30 cm x 38 cm x 16 cm) filled with water within 3 cm of the rim. ‘The traps were placed on 2 m high platforms at 100. 200. and 300 m intervals from the east. west. and south edges of the VenRoy blueberry field. Each week blueberry aphids were collected from the traps and the traps were cleaned and refilled with water. The aphids were placed into test tubes containing 100 pl extraction buffer. The tubes were corked and kept at 0 to 4 C until processed for RIA. Two blueberry plants in one-gallon plastic pots were placed at the base of each trap stand outside the field to attract aphids. These trap plants were checked also for blueberry aphids each week. WWW Alate activity within the blueberry field was monitored also with yellow pan traps. The traps were placed on 30 cm boxes and on 2 m high platforms (the height of the canopy) in the corners and center of a block of the VenRoy blueberry field. ‘1..peppe21 were collected 29 weekly from the yellow pan traps. as previously described. and were individually tested for BBSSV with RIA. WW Alate blueberry aphids were collected on ll. 18. 25. and 26 June from the walls of the screen cages enclosing the caged source plants in 1982. Aphids were collected into test tubes as earlier described and then individually tested for BBSSV with RIA. W Blueberry trap plants were exposed to BBSSV-infected source plants in the field for 4-week intervals to determine when BBSSV infection occurs during the growing season. ‘The five time intervals that the trap plants were exposed to the source plants in 1982 were (1) 7 May to 4 June. (2)4 June to 2 July. (3)2 July to 30 July. (4) 30 July to 27 August. and (5) 27 August to 23 September. Two-year-old highbush blueberry cv. Jersey plants in one-gallon plastic pots served as trap plants. ‘The plants were obtained from the John Nelson Blueberry Nursery. South Haven. MI. Prior to placement in the field. the plants were tested for BBSSV infection with ELISA and sprayed with DDVP (2.2-Dichloroviny1 0.0-dimethy1 phosphate). a low residual. contact/fumigant insecticide. .After each 4-week exposure period the trap plants were sprayed with DDVP and kept in isolation outside at Michigan State University. .After a winter dormant period. leaves were sampled and tested for BBSSV infection with ELISA. 30 Wu: Wham: Aphids may move from plant to plant by walking across overlapping branches of adjacent bushes. .A study was conducted to determine if aphids are likely to move to adjacent trap plants whether or not they are touching aphid source plants. The same trap plants used to determine the seasonal BBSSV infection were used in this study. Ten trap plants were placed around each of the 14 source bushes previously described. Five of the 10 trap plants were placed around the source bush to that the trap plants and source plant touched and had overlapping shoots. The other five trap plants were placed around the perimeter of the source plant 0.5 m to 1.0 m away so that the trap plants did not touch the source plants. Alate and apterous (late instar nymphs and adult) blueberry aphid populations on the trap plants and source plants were directly counted at weekly intervals from 7 May to 23 September 1982. Samples of alate and apterous aphid populations were also collected weekly and then tested for BBSSV with RIA to determine if the aphids were Viruliferous. The apterous aphid samples collected from 15 May to 4 June were tested in groups of five. Thereafter the apterous aphids were individually tested for BBSSV. All of the alate blueberry aphids were individually tested. Degree day (base 38 F) accumulation from 1 January to 31 March 1982 for aphid population studies was estimated from National Oceanic and Atmospheric Administration (NOAA) data for Grand Haven. MI. This figure was added to the degree day accumulation (base 38 F) obtained 31 from the agricultural weather observation station at Allendale. M1. for 1 April to 23 September 1982. The aphid populations on the five touching or five nontouching trap plants for each source plant replicate were summed (aggregated) using SPSS (Statistical Package for the Social Sciences. Vogelback Computing Center. Northwestern University. Evanston. IL 60201) run on the Control Data Corporation Cyber 750 computer at Michigan State University. The summed trap plant aphid populations and the source plant aphid populations were then analyzed using BMDP2V. analysis of variance with repeated measures (University of California. Los Angeles. CA). converted for use on the CDC 6000 and Cyber series computers by the Vogelback Computing Center. Northwestern University. Evanston. IL 60201. RESULTS linuLEuLiflcation Yields of the purified BBSSV ranged from 75 to 150 mg of purified virus per 100 g of frozen infected blueberry blossoms. The purified virus concentrations were determined spectrophotometrically 0. using an extinction coefficient of £266an = 5.2 (Ramsdell. 1979a). Sending: Antiserum prepared against purified preparations of BBSSV reacted with purified preparations of BBSSV to a dilution of 1:1024 (v/v) in 0.85% sodium chloride in gel double diffusion tests. There was no reaction of the antiserum with purified healthy blossoms. ELISA ELISA could detect purified BBSSV diluted twofold in extraction buffer at a concentration of approximately 0.5 ng/ml (Figure 2). This was equivalent to approximately 0.1 ng per well. Purified BBSSV diluted in ELISA extraction buffer with extracts of single blueberry aphids (one aphid per 0.2 m1 extraction buffer) was detected at a concentration of 3.0 ng/ml or 0.6 ng BBSSV per single aphid extract (Gillett et al.. 1982). 32 33 Figure 2.--ELISA absorbance values (405 nm) of a twofold dilution series of a purified preparation of blueberry shoe- string virus (BBSSV) in ELISA extraction buffer. The dilution of anti-BBSSV—gamma globulin in coating buffer was 1 mg/ml. while the dilution of enzyme-conjugated gamma globulin was 1:800 (v/v). Each point represents the mean of six replicates. The dashed line represents the threshold of detection determined by the mean value plus three standard deviations of healthy blueberry leaf samples. 3A 0000 000. N mcammu 2535 8:82.88 >mmmm I l I,uL,l 1..L,n . a 1 l n 1 1.4 I n 0.0 o.— 0.. QN (mu 9017) enloA eouquosqv 35 .816 Using RIA. purified preparations of BBSSV diluted twofold in ELISA extraction buffer could be detected at levels down to 0.5 ng/ml (Figure 3). In addition. the assay could detect purified BBSSV diluted twofold in extraction buffer with homogenized single blueberry aphid extracts (one aphid per 0.2 ml buffer) at 0.75 ng/ml (Gillett et al.. 1982). This corresponds to a detection level of 0.15 ng per aphid. Wins Aphids allowed access to BBSSV-infected leaf tissue acquired increasing amounts of virus with increasing AAPs (Figure 4). The first significant (P < 0.05) amount of measurable virus uptake occurred at an AAP of 12 hr. ‘There were no significant differences in amounts of BBSSV acquired with MP5 of 24 hr or more. There was. however. large variability in the amount of BBSSV acquired at the 48 hr AAP. The large variability in the quantity of virus taken up with the 48 hr AAP may have been due to several aphids acquiring very small quantities of virus. This is very likely since the virus is unequally distributed within the plant tissue. Purified BBSSV contained in sachets was acquired by aphids at the greatest rate during the first 24 hr (Figure 5). After 24 hr virus uptake continued to increase. but at a much slower rate. ‘There appeared to be a thresh01d of virus uptake at the 24 AAP. The rate of 125I-BBSSV acquisition from sachets was steady with increasing AAP except for a decrease in virus uptake at 12 hr (Figure 6). This slight decrease at 12 hr may have been due to the greater number of aphids that did not acquire any 125I-BBSSV at that AAP. 36 Figure 3.--RIA counts per minute (cpm) of a twofold dilution series of a purified preparation of blueberry shoestring virus (BBSSV) in ELISA extraction buffer. The dilution of 25I-labeled gamma globulin was approximately 55.000 cpm in PBS-0.5% BSA. Each point represents the mean of six replicates. The dashed line represents the threshold of detection determined by the mean value plus three standard deviations of extraction buffer. m uL:m_u 25358222886 >mmmm 37 oooo ooo. oo. o. _ no o ooo. oooN 3 m ooon m. S oooe w i m oooo m. «U oooo 0005 38 Figure 4.--Acquisition kinetics of blueberry shoestring virus (BBSSV) uptake by blueberry aphids which fed on BBSSV- infected plant tissue for 10 min. 1. 6. 12. 24. 48. and 74 hr. Aphids were individually tested for presence of BBSSV with RIA. Thirty aphids were used per acqui- sition access period. Bars represent 95% confidence intervals. 39 : 0L:m_u .9505 oEF co_:m_:ao< 2. we on a p o . - 1 q q d oE: :2:£:coo Lea 636232 2:60 0.9..» on l 0? our 00—. CON ova 63.0.2... U309»: 50.... >wmmm .0 :O_u_m_:uo< emugw 18d siunoo 40 Figure S.--Acquisition kinetics of blueberry shoestring virus (BBSSV) uptake by blueberry aphids which fed on puri- fied preparations of BBSSV in sachets. Aphids were individually tested for the presence of BBSSV with RIA. Thirty aphids were used per acquisition access period. Bars represent 95% confidence intervals. 41 m oczm_u 62.2 3 $52: ea: cages—xi Nh wv QN NF 6 .. - q q 000 SN _. 00c w OOON OOQN oE: co_=n_:coa can «33:62 2:66 0.9.3 on muwtomw L 0094. ES. >mmmm 3.32:: oozes... .0 co_._e_=oe< emugw 16d szunoo 42 Figure 6.--Acquisition kinetics of blueberry shoestring virus (BBSSV) uptake by blueberry aphids which fed on 12 I- labeled BBSSV in sachets. Aphids were individually counted directly by the gamma counter. There were 10 aphid replicates per acquisition access period. Bars represent 95% confidence intervals. Counts per Minute Acquisition of 1 25l-BBS€~‘.V from Sachets 200 180 160 140 120 100 80 60 4o 20 ‘43 b 10 single aphid replicates per acquisition time 7 1 I n l 6 12 24 Acquisition Time (hours) Figure 6 44 The effect of AAP on the proportion of viruliferous aphids is shown in Figure 7. In general. there was an increase in the proportion of viruliferous aphids with increasing AAP up to a threshold at 24 hr. This occurred when aphids were allowed to feed on either BBSSV-infected plants or purified BBSSV. The proportion of viruliferous aphids which fed on BBSSV-infected leaves decreased slightly with AAPs greater than 24 hr. ‘This slight decline. which did not occur with those aphids that fed on the purified BBSSV. may have been due to the unequal distribution of virus in the plant tissue. as previously discussed. The percentage distribution of RIA counts per minute (cpm) of individual late-instar and adult apterous blueberry aphids that fed for a 24 hr AAP on BBSSV-infected tissue is shown in Figure 8. The threshold for presence of BBSSV was 85 cpm-~the mean plus three standard deviations of individual aphids that fed on healthy tissue. Forty-three percent of the individual aphids tested for BBSSV contained detectable quantities of the virus. A maximum of 1 ng (250 cpm) of BBSSV per individual aphid was detected. Figure 9 shows the percentage distribution of cpm of the 30 individual late instar nymph and adult apterous blueberry aphids allowed a 24 hr AAP on purified BBSSV in 20% sucrose contained in sachets. All of the individuals were viruliferous as determined by the threshold value of the mean plus three standard deviations of control individuals which fed on 20% sucrose in sachets. ‘The aphids had cpms ranging from 308 to 3265. which corresponded to a range of‘LS to 60 ng of virus. 45 Figure 7.--The relationship between acquisition access period and number of viruliferous blueberry aphids which fed on purified blueberry shoestring virus (BBSSV) in sachets or BBSSV-infected tissue. Aphids were individually tested for the presence of BBSSV with RIA. Each point represents the number of viruliferous aphids out of 30 aphids tested. 46 an 63mm: oo~oo~=7>mmmm Eo: 203336"; 30:03 E >mmmm ooESo nil Ea: coEmSUom n oL:m_u .235 95... 52232 we «a a. o F J - u A h or mp ON mm on spiudv amused VIU :0 JaqwnN 47 Figure 8.--The percentage distribution of RIA counts per minute (cpm) of 30 individual late-instar and adult apterous blueberry aphids that fed for 24 hr on BBSSV-infected tissue. The shaded bars represent the percentage of viruliferous aphids while the nonshaded bar represents the percentage of nonviruliferous aphids. Lie Om N OON m oczm.u 3:55. Lea 3550 Omw III 00.. mm Om hm C1 O.. ON O6 O0. Om efietuemed 49 Figure 9.--Percentage distribution of RIA counts per minute (cpm) of 30 individual late-instar and adult apterous blue- berry aphids that fed for 24 hr on purified BBSSV in sachets. . 50 Comm OOON m 0L:m_u 3:55. Loo 3:300 OOmN OOON OOm .. 000 r oom on O? eBetueoJed 51 Would: Transmission of BBSSV to blueberry plants by blueberry aphids having an AAP of 24 hr occurred after an inoculation access period of 1 hr (Figures 10 and 11). Figure 10 shows the results of the first transmission test with 15 test plant replications per IAP treatment. Infection occurred with IAPs of 1 hr through 96 hr. but not at 6 hr. The results of the second transmission test using a constant AAP of 24 hr and IAPs of 0. 1. 6. 12. 24. 48. 96. and 192 hr are shown in Figure 11. Blueberry plants were infected after all IAPs except for 48 hr in this experiment. WWW Apterous and alate blueberry aphids were first observed on caged source plants on 14 May 1982 (Figures 12 and 13). the same date that blueberry aphids were first observed on noncaged field plants. There were statistically significant differences (3 < 0.001) in the mean aphid population numbers on the caged versus noncaged source plants (Table A-2). The caged aphid populations increased to greater numbers during the season than the uncaged aphid populations. However. both the caged and uncaged populations had the same seasonal patterns. Ame—81W WWW OutsiderLEJsld Only four alatae were trapped outside of the blueberry field; only one caught on 29 May. 100 m east of the field was viruliferous. 52 Figure 10.--Transmission of blueberry shoestring virus (BBSSV) by blueberry aphids. experiment one. Aphids were allowed an acquisition access period of 24 hr on BBSSV-infected tissue. There were 15 test plant replications per inoculation access period treatment with 15 aphids transferred to each test plant. 53 o. ecso_u .2305 oE_._. «mooo< 20.5.3002. «or me we on «P o P e w I m i o. a U I 1. B .. on w . i. .u .. on m. . M B - oe m. . 8 Don .u. .u nu 1.. .u n? 54 Figure ll.--Transmission of blueberry shoestring virus (BBSSV) by blueberry aphids. experiment two. Aphids were allowed an acquisition access period of 24 hr on BBSSV-infected tissue. There were seven test plant replications per inoculation access period treatment with 15 aphids transferred to each test plant. 55 NO-. mm .9505 6E... mmooo< 20323005.. Ow __ etso_u .N N_. O Ow ON ON O? Om OO pezoew| szueid tsel afieiuemed 56 Figure 12.--Seasonal apterous blueberry aphid populations on caged and uncaged BBSSV-infected source plants. Degree day base 38 F. Narrow arrows indicate insecticide spray application of Guthion 2 SC (2 1b ai/gallon). 1 pt/acre. by air blast sprayer. Wide arrows indicate application of Aqua Malathion (8 1b ai/gallon). 2 pt/acre. by air blast sprayer. Eastmanville. MI. 1982. 57 N. e.ae.u .w.a:u mmmouo 02¢ 30.. wwhn sonn mmmw mmvm :ON can. om~_ mew vow - L p p p . p _ _ . c . . r p p _ . . p L Na:m_ mum o:< .2. 2.... >5. UNF!qu )0 JaqwnN 65 trapped 11 June. Fewer than 10 alatae were collected per sampling date after 11 June. and none were collected after 16 July. WNW: Of the 500 alate blueberry aphids collected from screen cage walls enclosing BBSSV-infected source bushes and assayed for BBSSV. 14 were viruliferous (Table 1). Overall. 2.8% of these alate blueberry aphids tested were viruliferous. This figure indicates the proportion of viruliferous aphids that may fly to other bushes. Table l.--Alate blueberry aphids collected from screen cage walls enclosing BBSSV-infected source plants. Sampling Number of Alatae Number of Percent Alatae Date Viruliferous Alatae Assayeda Viruliverous 11 June 2 80 2.5 18 June 1 107 0.9 25 June 2 44 4.5 26 June 3 106 2.8 26 June 6 163 3.7 Total 14 500 2.8 aAphids were individually tested for presence of BBSSV by RIA. Figure 12 shows the seasonal populations of apterous aphids on BBSSV-infected source plants. ‘The points represent the mean numbers of apterous aphids counted for seven caged or seven noncaged source plants. The apterae populations on the caged source plants were much greater over the season than the corresponding populations on the noncaged source plants. The enclosing screen cages provided protection against aphid mortality factors such as rain. wind. parasites. and predators. Although the population numbers of the apterae on the caged versus noncaged source plants were significantly different (:5 < 0.001). the populations followed the same general seasonal pattern. ‘The mean numbers of apterae per source plant were maximunithe last part of June: 320 apterae on 18 June and 71 apterae on 25 June for caged and noncaged source plants. respectively. The populations then decreased to a Ininimum during late July and early August. From mid- to late August ‘there was a slight increase in the mean numbers of apterae on source plants which subsequently decreased and remained very low through .September when the experiments were terminated. W The mean numbers of apterous aphids per trap plant touching and l10t touching source bushes are shown in Figures 16 and 17. I"espectively. As with the apterous populations on the source plants. 67 Figure 16.--Seasonal distribution of apterous blueberry aphids on blueberry trap plants touching BBSSV-infected source plants. ‘Trap plants were or were not enclosed in aphid-proof cages with the source plants. Degree day base 38 F. Narrow arrows indicate insecticide spray application of Guthion 2 SC (2 1b ai/gallon). 1 pt/ acre. by air blast sprayer. Wide arrows indicate application of Aqua Malathion (8 1b ai/gallon). 2 pt/ acre. by air blast sprayer. Eastmanville. MI. 1982. 68 m. ecso.. .msa:u wwmuuo om; nmov thn honn OOON OOVN ..ON vmn. OON. ocm VOO — p . P h — b P - p b p h r p p . _ _ b . Nam. sum 22 .2. 22. >4... w .20 N h— o— n 5N ON n— m on nN on a N DN 0.. 2 e mN —N I _ . p e . . . h r P F p r _ . r . .. 0 fl iii a \Tlaiu/ H w J F \ .s a ~ .2. N / \ /.\ . .. m c 8 K ’ I .. my a , . . . H W . . . x» . .9 m. .. .. . M._\ / H. m .25.. .2»: 806 be 2 . I, \ / m Ne w \ i .23.. as: 8062.. 4 . . . \ / \ . m. 3. K a: he. a fin W. —. _\ .\ r’ ' .- -' o: ' ._.‘.~§“ 69 Figure 17.--Seasona1 distribution of apterous b1ueberry aphids on b1ueberry trap piants not touching BBSSV-infected source piants. Trap piants were or were not enciosed in aphid-proof cages with the source piants. Degree day base 38 F. Narrow arrows indicate insecticide spray app1ication of Guthion 2 SC (2 1b ai/ga11on). 1 pt/acre. by air biast sprayer. Wide arrows indi- cate appiication of Aqua Ma1athion (8 1b ai/galion). 2 pt/acre, by air biast sprayer. Eastmanviile. MI. 1982. 70 N. otsm_a msaxu cm; 39. 3.5 53 $3 $5 :8 can. 03. ova vow mwmouo P b P P b L b P p p b p b P p b p p p b _ .mm 22 5.. 22. >3. mum‘mm 3 t S n R ca 3 m on an 2 a a mu 2 c a 8 a I - c _ h p p F . b p b F . . _ . . _ . 0 .III... / z i. W / \e/ \ a wz m ,«\\. .Je / . «W a a / a. v m , a a H. m , , \ i v. 1 a . m s s a H m EsafiEBeé a i \ is a Esanzfiaoéz: 4 ’ w/ ~ . a , \ a , M \ J wm d LP » b5 . M 71 those populations on the plants enclosed within cages were signifi- cantly greater (5 < 0.01) than those on plants not within the cages and exposed to the natural environment. These aphid populations also followed the same seasonal fluctuations. 'The populations on the trap plants were very high the first half of the growing season. through the first week of July. 'The populations were low during late July and then increased again during August before tapering to the low mean apterae numbers found in autunuu The apterae population pattern had two peaks: one very high peak early in the season when the plants were rapidly growing. and another slight peak during August when the plants had new growth after fruiting. The relative decreases in apterae populations found on the trap plants on 11 June. 2 July. 16 July. and 20 August (Figures 16 and 17) were due to insecticide applications. These decreases in populations after pesticide applications are not as apparent on the source plant apterae populations (Figure 12). W: W After the aphid populations were counted. samples were collected and tested for presence of BBSSV using RIA. The incidence of viruliferous apterous aphids on caged and noncaged source plants is presented in Figure 18. There were no significant differences (E'< (L05) in the percentage of viruliferous aphids on caged versus noncaged source plants. The data points for 14 May through 4 June are the results of aphids tested for BBSSV in groups of five. Thereafter. apterae were individually assayed for BBSSV. Through 9 July the mean 72 Figure 18.--Seasona1 distribution of viruliferous apterous aphids on caged and uncaged BBSSV-infected source plants. Degree day base 38 F. Eastmanville. MI. 1982. 73 74 percentages of viruliferous apterous aphids on caged and noncaged source plants were similar; Between 16 July and the end of September these percentages varied widely. 'The large differences in mean percentage viruliferous apterae through the season may have been due to the sample size variation. After 9 July there were very few apterae on the source plants. and there were even fewer apterae that could be collected and assayed. The smaller sample numbers may have resulted in greater differences in the proportions of virus-carrying aphids. W: The mean percentages of viruliferous apterous aphids on trap plants touching and not touching source plants are presented in Figures 19 and 20. respectively. Aphids were tested for BBSSV in batches of five for the data points of the dates 14 May through 4 June. Apterous aphids were individually tested after 4 June. Most of the mean percentages of viruliferous apterous aphids on trap plants were less than 20%; however. these percentages fluctuated throughout the season. This was probably due to the small sample sizes mentioned earlier for the source plants. In addition. the very high percentages found on 16 July for the source plants (Figure 18) and trap plants touching and not touching the source plants (Figures 19 and 20). respectively. may be explained by spurious assay results rather than deviations in field biology. 75 Figure l9.--Seasonal distribution of viruliferous apterous aphids on blueberry trap plants touching BBSSV-infected source plants. Trap plants were or were not enclosed in aphid-proof cages with the source plants. Degree day base 38 F. Eastmanville. MI. 1982. 76 m. oa:m_u sta:u wmmwuo om; nmo¢ wwhn hmnn mwmu OGVN :ON van. Dam. ova vow P F _ P L p p p p b b L F . . - . n p _ _ “gym. twa— 03< .55 23.. >54 wh34 m~.—.® 0.5 to 1.5 28 8 > 1.5 to 5 6 6 > 5 to 15 11 5 > 15 to 50 2 2 > 50 to 100 3 0 > 100 3 2 6Values are based on an RIA standard curve developed using purified BBSSV as test antigen. Winn The percentages of noncaged trap plants touching and not touching infected source plants is presented in Figure 26. There were no significant differences in infection rate between trap plants touching 86 Figure 23.--Seasonal distribution of viruliferous alate blueberry aphids on caged and noncaged BBSSV-infected source plants. Degree day base 38 F. Eastmanville. MI. 1982. 87 MN u.:m_a was “enema no; mace own» 53 comm omcu :ON can. emu. com vow L L L L L L L L L L L L L L L L L L L L L «8. mum 9.2 .55 22. >3. whdo «N C o. n LN on a m on nu o. a N mu 2 I + nu Lu 3 L ELL L L. Li. Lil. _ o LZSQ momnow 805 8 L251 momzow 8052: d .LNV‘Id 338008 83:! SOchN 31W SilOlEdl'IflfllA BOVJNEOEEd NEW 88 Figure 24.--Seasonal distribution of viruliferous alate blueberry aphids on trap plants touching BBSSV-infected source plants. Trap plants were or were not enclosed within aphid-proof cages with source plants. Degree day base 38 F. Eastmanville. MI. 1982. 89 :N mLamLL $3 mucouo mm; nmov was» Lonn mmaw meow =o~ can. omw. com com L L L \L L L L L L L L L L L L L L L L L L New. mum 03< .5... 22. >3. whdo 3.. LL 9 n LN 3 L: m on 3 e N ma 9 LL e mu LN I TLILILILILIL tLiT rltlr/ L _i o / m m \ r / L z \ 5 L25; .5: 898 8 L25; “3:. 8302: 4 j 09 Mei dVHJ. 23d SGlHdV 31W SHOEBJFIOHIA BOVINBOEBd NVBW aberry >urce Mflm 1y base 90 Figure 25.--Seasonal distribution of viruliferous alate blueberry aphids on trap plants not touching BBSSV-infected source plants. Trap plants were or were not enclosed within aphid-proof cages with source plants. Degree day base 38 F. Eastmanville. MI. 1982. 9| LN mL=m_L stazu mwmouo om; nmow thn honn mme OOCN Law '00. CON. atm vow L L L L L L L L L L L L L L L L L L P L L . .meg mum OD< .5... 22. >34 mhdo thLothNONanonnNmLaNMNQLLLLLQNLNVL OI r 02 / \ l 08 25.. .3: 806 a 9 L25“. .3: 8302: 4 .0 We! dVHJ. 83d SGlHdV ELV'N SflOMEdnilHlA BOVLNBOHBd NVBW 92 Figure 26.--Seasonal blueberry trap plant infection after 4-week exposure period to BBSSV-infected source plants within the field. Trap plants were not caged with source plants. Eastmanville. MI. 1982. 93 LN ..=m_a 0.5....— Z. mmm>> mPZjn— ALF—L. 20.P<¢DD QmwMN 034330m >431N mzafiv .03JD...on .>._D..N .mZDfiv .><.2N AAA-A‘AJ-A-A-A mucoE ooLaom 0303:. 9.2038. Loz 3.85 an; - 2cm... 850m “.302... 9.225... 35... 92... m ON CV 00 on 03.1.03le SLNV'Id dVHJ. 39VlN3083d 94 and not touching source plants. The aphids were apparently able to move to and transmit virus to trap plants adjacent to touching plants regardless of physical contact between the bushes. The greatest amount of BBSSV transmission occurred early in the season in May and June. During these infection periods 51% of the nontouching trap plants became infected. while 49% of the touching trap plants became infected. As the season progressed through July and August there was less BBSSV infection. In August only 9% and 6% of the touching and nontouching trap plants. respectively. became infected. The increase of trap plant infection during September corresponds to the slight resurgence in apterous aphid populations during this time. There was no statistically significant difference ([1 < 0.001) between touching and nontouching trap plant infection over time. Figure 27 shows the relationship between noncaged apterous aphid populations on source and trap plants and noncaged trap plant infection through the season. The percentage of trap plant infection seems to correspond with the size of the aphid populations. More infection occurred when the populations were high. Conversely. little infection occurred when the populations were low. as was found in mid-August. 95 Figure 27.--The relationship between the incidence of BBSSV- infected trap plants and the mean numbers of apterous blueberry aphids on noncaged source plants and trap plants. Trap plants were touching or not touching the source plants for 4-week intervals. Degree day base 38 F. Narrow arrows indicate insecticide spray application of Guthion 2 SC (2 lb ai/gallon). l pt/acre. by air blast sprayer. Wide arrows indicate application of Aqua Malathion (8 lb ai/gallon). 2 pt/acre. by air blast sprayer. Eastmanville. MI. 1982. 96 SLNV'ld dVHJ. 0310le-18888 39V1N3083d LN o.=m_a mkflu wwmwwo mm; anLv mNhn Nmnn mwmm mmcm :ON can. OGN. oLVm com L L L F L L L L L L L L L L L L L L L L L mm. mum 02 .55 22. ><1 UNPdO .vN 5.. 0L n RN ON nw m on ON or m N 8 mp LL .v ON LN fir h D 1“ , L L L L - . I D _ u . «iii/z {3 _ \ s - 1 \‘/( \\\. lg“ .. 1 ‘i- \ l m., .a inc .6 ; Z 0 .1 LI 0 .l '''''''''''''''''''''''' II. e. w Jr llllll L fil L. . T m 8 lllll S M 01 A L 10 N 8 . . r m r r W 0 l l O O . mLzSa . m n