STUDIES ON TRANSMISSION OF POTATO Y

by

NIKOLAOS KATIS (B .S c.)

Thesis submitted to the University of London for the degree of Doctor of Philosophy

Plant Pathology Department Rothamsted Experimental Station Harpenden, Herts* AL5 2JQ

November 1984 2

ABSTRACT

Two strains of potato virus Y (PVY) are common in potato crops. The ordinary stra in (PVY^) induces leaf drop streak in many potato cultivars followed by severe mosaic and vein clearing in tobacco, and the tobacco veinal necrosis strain (PVY N ) induces mild mosaic in almost all potato cultivars and veinal necrosis in tobacco. PVYN did not affect the concentration of PVY° but PVY^ supress- ed PVY N in doubly infected of tobacco cv. White Burley and potato cv. King Edward, especially in plants kept at 30°C. At this temperature, no PVY N was recovered from doubly infected plants, which developed a slight mosaic although veinal necrosis appeared in two days on transfer to a cool glasshouse. The cereal aphid Rhopalosiphum padi (L.) transmitted both strains of PVY from and to both tobacco and potato, although much less frequently than did Myzus persicae (Sulz.) even when making single probes were compared. R, padi and M. persicae retained PVY for similar duration. Both apterae and alatae of R. padi transmitted infrequently as also did progenies of individual R. padi that had transmitted PVY. M. persicae frequently inoculated both PVY 0 and PVY N after a single probe into a doubly infected . Inefficient vectors (M. euphorbiae (Thomas) and R. padi) inoculated both strains much less frequently than M. persicae suggesting that they inoculated less virus. Access of M. persicae to oil treated, doubly infected leaves resulted in some preferential reduction in transmission of both PVY 0 and PVY N m . comparison with the untreated ones only 24-36 h after application, 3

M. persicae which probed a PVY N- or a beet mosaic virus (BMV)- infected leaf either before or after access to a PVY0-infected leaf were less likely to transmit PVY° than aphids which probed uninfected leaves. Transmission of BMV was similarly inhibited by aphids prob- ing a PVY N -infected leaf. However, transmission of PVY N was unaffect­ ed by whether or not M, persicae also probed a PVY°- or a BMV-infected leaf. Transmission of PVY 0 and PVY N was also unaffected by whether or not M. persicae also probed leaves infected with the non-aphid transmissible virus tobacco mosaic. Retention of PVY ,0 PVY N and BMV by M. persicae was similar as was the effect of temperature on retent- ion of PVY 0 and PVY N by the same aphid species. In laboratory tests with glasshouse-grown potato plants cv. King Edward, M. persicae acquired PVY as readily from plants inoculat* ed 20 days previously as from secondarily-infected ones. With PVY° this was also the case when plants were inoculated young. Twenty days after inoculation. M. persicae acquired PVY 0 and PVY N from plants of five potato cultivars differing in resistance to PVY and also from eight other isolates of PVY 0 and PVY N. Field potato plants of cvs. Record and King Edward inoculated with either PVY 0 or PVY N early in the growing season acted as foci for further spread although initially they were poorer sources than plants grown from infected tubers. No PVY was detected in any wild plant growing in or near a plot of PVY-infected potatoes. In 1984, alate aphids were trapped downwind of a plot of PVY- infected potato plants at Rothamsted. A total of 3969 alates were trapped, 2.57. of which infected test plants with PVY. Brachycaudus 4 helichrysi (Kltb.), flying early in the growing season, accounted for ca. 787. of transmissions and was probably the most important vector of PVY at Rothamsted that year. From early to mid-July M. persicae, and from mid-July to 8 August Phorodon humuli (Schrank) and Aphis spp., transmitted PVY most frequently. In this experiment,

Metopolophium festucae (Theobald), Myzaphis rosarum (Kltb.), My 2us myosotodis (Borner) and Sitobion fragariae (Wlk.) were identified as vectors of PVY for the first time. 5

Contents

Page ABSTRACT 2 Contents 5 List of Tables 9 L ist of Figures 12 List of Plates 13 List of virus abbreviations 15 ACKNOWLEDGEMENTS 17 1. GENERAL INTRODUCTION 19 2. REVIEW OF THE LITERATURE 24 2.1 Nonpersistent transmission of plant by aphids 24 a. Introduction 24 b. Characteristics of nonpersistent transmission 25 c. Virus groups transmitted nonpersistently 28 d. Physical properties of nonpersistent viruses 29 e. The role of helper component 29 f. Sites of virus acquisition and inoculation in plant 32 g. Sites of transmitted virus in/on the aphid 33 h. Vector specificity 36 2.2 Transmission of by non-colonizing aphids 37 3. GENERAL MATERIALS AND METHODS 46 3.1 Virus isolates 46 3.2 Plants 46 3.3 Aphids 46 3.4 Techniques 51 a. Virus acquisition and transmission by aphids 51 6

Page b. Manual transmission of viruses 51 c. Immunosorbent electro n microscopy (ISEM) 51 d. Enzyme-linked immunosorbent assay (ELISA) 52 e. Electron microscopy 53 3.5 Statistical analysis 53 EXPERIMENTAL WORK 54 4. INTERACTION OF PVY° AND PVYN IN DOUBLY INFECTED PLANTS 54 4.1 Interaction of PVY 0 and PVY N in glasshouse-grown tobacco plants* 54 4.2 Interaction of PVY0 and PVY N in glasshouse-potato p lan ts. 55 4.3 Interaction of PVY0 and PVY N in tobacco plants grown at different temperatures. 55 5. TRANSMISSION OF PVY° AND PVYN FROM DOUBLY INFECTED PLANTS 63 5.1 Acquisition of PVY 0 and PVY N by M. persicae after a single probe on a doubly infected tobacco leaf. 63 5.2 Acquisition of PVY 0 and PVY N by M. persicae> M. euphorbiae and R. padi from doubly infected potato p lan ts. 66 5.3 Transmission of PVYN by M. persicae and M. euphorbiae that initially fail to transmit. 73 5.4 Effect of oil on acquisition of PVY 0 and PVY N from doubly infected tobacco. 76 6. EXPERIMENTS TESTING FOR INTERFERENCE BETWEEN VIRUSES OR VIRUS STRAINS DURING APHID TRANSMISSION 85 6.1 Experiments testing for interference between PVY0 and PVYN during sequential transmission by M. persicae. 85 6.2 Experiments testing for interference between BMV, PVY 0 and PVY^ during sequential transmission by M. persicae. 89 6.3 Experiments testing the ability of BMV from N. clevelandii and sugarbeet to inhibit transmission of PVY^ during sequential transmission by M. persicae. 94 7

Page 6*4 Experiments testing for interference between PVY , PV\fN and TMV during sequential transmission by M. persicae. 100 6.5 Cross-protection tests 101 6.6 Comparison of electrophoretic mobility of PVY0 , PVY N and BMV by gel-electrophoresis. 104 VIRUS RETENTION BY M. PERSICAE 114 7.1 The retention period of PVY^, PVY^ and BMV by M. persicae. 114 7.2 Effect of temperature on the retention of PVY° and PVYn by M. persicae. 118 ROLE OF WEEDS AS PVY SOURCES 121 PRIMARILY-INFECTED PLANTS AS SOURCES OF PVY 127 9.1 Acquisition of PVY O and PVY N by M. persicae from primarily infected potato cv. King Edward inoculated at different ages. 131 9.2 Acquisition of PVY O and PVY N by M. persicae from primarily infected potato plants of different c u ltiv a rs. 132 9.3 Acquisition of different isolates of PVY O and PVY N by M. persicae from primarily infected potato cv. King Edward plants. 135 9.4 Acquisition of PVY 0 and PVY N by M. persicae from different leaves of primarily infected potato plants cv. King Edward 137 9. 5 Field experiment 139 TRANSMISSION OF POTATO VIRUS Y BY CEREAL APHIDS 146 10.1 Transmission of PVY by R. padi, M. dirhodumt S. avenae and M. persicae. 147 10.2 Duration of acquisition access. 148 10.3 Transmission of PVY by apterae and alatae R. padi. 148 10.4 Transmission of PVY^ and PVY^. 150 10.5 Transmission of PVY to and from potato and tobacco. 150 8

Page 10.6 Retention of infectivity by R. padi and M. persicae. 151 10.7 Transmission of PVY by R. padi and M. persicae after a single probe. 151 10.8 Transmission of PVY by progeny of known vector R. padi. 154 11. SPREAD OF PVY BY APHIDS 158 12. GENERAL DISCUSSION 177 13. APPENDIX 185 14. REFERENCES 189 9

List of Tables

Table Page 1. Transmission of potyviruses by non-colonizing aphids. 39 2. Test plants used in experiments for virus identif­ ication and assay. 47 3. Transmission of PVY 0 and PVY N by M. persicae or sap from singly and doubly infected tobacco cv. White Burley plants. 57 4. Transmission of PVY 0 and PVY N from singly and doubly infected potato cv. King Edward plants by manual inoculation of sap diluted to 10"^. 58 5. Sap transmission of PVY0 and PVY N from singly and doubly infected tobacco cv. White Burley plants kept at different temperatures. 61 6. Numbers of plants infected with PVY 0 and/or PVY N by M. persicae allowed a single probe into doubly infected tobacco cv. White Burley. 65 7. Transmission of PVY 0 and PVY N from doubly infected potato cv. King Edward by different aphid species. 70 8. The analysis of variance of numbers of plants infected w ith PVY0 , PVYN or PVY0 + PVYN by M. persicae, M. euphorbiae and R. padi. 71 9. Transmission of PVYN by aphids that either transmitted (A) or failed to transmit (B) in the previous test. 75 10. Transmission of PVY 0 and PVY N from oil-treated and untreated doubly infected tobacco by M. persicae allowed a single probe 30 mins after oil treatment. 79 11. Statistical analysis of the 'fit1 of each model to transmission of PVY0, PVYN and PVY0 + PVYN 30 mins after oil treatment. 80 12. Transmission of PVY 0 and PVY N from oil-treated and untreated doubly infected tobacco by M. persicae allowed a single probe 24-36 h after oil treatment. 81 13. Statistical analysis of the 'fit' of each model to transmission of PVY0, PVYN and PVY0 + PVYN 24-36 h after oil treatment. 82 10

Table Page 14. Numbers of plants infected with PVY by viru- liferous aphids also probing a tobacco leaf with or without PVYN. 87 15. Numbers of plants infected with PVY N by virulifer- ous aphids also probing a tobacco leaf with or without PVY0. 88 16. Numbers of plants infected with PVY° by virulifer- ous aphids also probing a sugarbeet leaf with or without BMV. 91 17. Numbers of plants infected with PVY N by viruliferous aphids also probing a sugarbeet leaf with or without BMV. 92 18. Numbers of plants infected with BMV by viruliferous aphids also probing a tobacco leaf with or without PVY0 or PVYn. 93 19. Numbers of plants infected with PVY° by viruliferous aphids also probing N. Cleveland!! or sugarbeet with or without BMV. 97 20. Numbers of plants infected with BMV by viruliferous aphids also probing a tobacco leaf with or without PVY0. 98 21. Relative concentration of BMV in Nicotiana Cleveland!! and sugarbeet cv. Hilleshog Monotri. 99 22. Numbers of plants infected with PVY° by viruliferous aphids also probing a tobacco leaf with or without TMV. 102 23. Numbers of plants infected with PVY N by viruliferous aphids also probing a tobacco leaf with or without TMV. 103 24. Numbers of tobacco cv. White Burley plants infected with challenging strain of PVY when sap-inoculated. 105 25. Numbers of tobacco cv. White Burley plants infected by M. persicae with the challenging strain of PVY. 106 26. Numbers of plants infected with PVY and PVY by M. persicae following different periods of post-acquisit­ ion fastin g . 117 27. Numbers of plants infected with PVY0 or PVYN by M. persicae following different periods of fasting at different temperatures. 120 11

Table Page 28. Some non-crop plants susceptible to PVY. 122 29. Non-crop plants tested for PVY. 125 30. Numbers of plants infected by M. persicae given access to PVY0 and PVY^ primarily-infected plants. 133 31. Numbers of tobacco plants infected by batches of ten M. persicae allowed access to potato cultivars inoculated with PVY0 or PVYN. 136 32. Numbers of tobacco plants infected by batches of ten M. persicae allowed access to potato cv. King Edward inoculated with different isolates of PVY° and PVYN. 138 33. Transmission of PVY 0 and PVY N by M. persicae from different leaves of primarily-infected potato cv. King Edward plants 20 days after inoculation. 140 34. Numbers of tobacco plants infected in laboratory tests with PVY° or PVYN by M. persicae from primarily- infected King Edward and Record potato plants. 141 35. Proportion of infected tubers from plants near to primarily or secondarily infected fieldpotato plants. 142 36. Proportion of infected tubers from plants close to infectors. 143 37. Transmission by aphids given different acquisition access periods. 149 38. Transmission of PVY O and PVY N by R. padi using different source and test plants. 152 39. Transmission of PVY after different post-acquisition fasting periods. 153 40. Aphid species identified as vectors of PVY. 160 41. Number of each aphid species caught and, in parentheses, number which infected test plants withPVY. 166 42. Relative abundance, transmission efficiency and overall transmission of the major vector species trapped in the n et. 170 43. A comparison of the percentages in catches of the major vector species in the net and in the Rothamsted suction trap (trapping at 12.2 m). 173 12

List of Figures

Figures Page 1. Histograms showing transmission of PVY N , PVY^ and BMV by single aphids to successive plants. 116 2. Plan of the field trial. 129 3. Plan of a plot. 130 4. Numbers of aphids caught in nets close to a PVY-infected plot at Rothamsted, June 8 - July 11, 1984. 171 5. Numbers of M. persicae, M. euphorbiae and B, helichrysi caught in nets (open bars) and the numbers that transmitted PVY (solid bars). 174 13

List of Plates

Plate Page 1. Particles of PVY in a purified preparation stained with sodium phosphotungstate (magnification x 100.000) 20 2. Tobacco plants cv. White Burley showing vein clearing ten days after inoculation with PVY® (right),veinal necrosis after inoculation with PVYN (centre) or inoculated with phosphate buffer (left) 21 3. Leaves of tobacco plants, showing vein clearing ten days after inoculation with PVY® (left), veinal necrosis after inoculation with PVYN (centre), or no symptoms after inoculation with buffer (right). 21 4. Primary 'leaf-drop streak* symptoms, 3 weeks after inoculation, in King Edward potato plants inoculated with PVY® (right), compared with infection with PVYN (centre) and healthy (right). 22 5. Symptoms of PVY in leaves of King Edward potato plants, a, healthy; b, primarily infected with PVY®; c, prim­ arily infected with PVY®; d, secondarily infected w ith PVY0. 23 6. Systemic necrosis in Physalis floridana three weeks after inoculation with PVYU (left) and mild mosaic after inoculation with PVYN (right). 48 7. Chlorotic local lesions in Chenopodium amaranticolor, eight days inoculation with PVY® (left) and no lesions after inoculation with phosphate buffer (right). 48 8. Sugarbeet cv. Hilleshog Monotri showing mosaic, ten days after inoculation with BMV (left), and healthy control (right). 49 9. Local lesions in Chenopodium amaranticolor, six days after inoculation with BMV (left) and no lesions when inoculated with buffer (right). 49 10. Nicotiana clevelandii showing mottling, two weeks after inoculation with BMV (left), and healthy control (right). 50 11. Leaves of N. Cleveland!! showing mottling 2 weeks after inoculation with BMV (right), or uninoculated control (left). 50 14

Plate Page 12. Tobacco plants showing veinal necrosis (right) or slight mosaic (left) 2 weeks after inoculation with PVYn and incubation at 15°C and 30°C respectively, 59 13. Leaves of tobacco plants inoculated with PVY N and incubated after inoculation at 15°C (right) or at 30°C (left) showing veinal necrosis or slight mosaic respectively. 59 14. Electrophoresis of PVY0 , PVY N and BMV in polyacrylamide and agarose gels, using a tris-borate buffer, pH 8.3. Electrophoresis was carried out at 3mA/gel for 2 hours. Migration was toward the anode. 110 15. View of the net and suction trap used for trapping aphids. 164 16. Tobacco cv. White Burley plants used to test whether trapped aphids carried PVY. 164 15

List of virus abbreviations

AMV Alfalfa mosaic virus BCMV Bean common mosaic virus BCTV Beet curly top virus BidMV mottle virus BMV Beet mosaic virus BYDV Barley yellow dwarf virus

BYMV Bean yellow mosaic virus BYNV Beet yellow net virus BYV Beet yellows virus CarLV Carnation latent virus CMV Cucumber mosaic virus HMV Henbane mosaic virus LMV mosaic virus MDMV Maize dwarf mosaic virus OYDV yellow dwarf virus PAMV Potato aucuba mosaic virus PeaSV Pea streak virus PeMotV Pea mottle virus PeVMV Pepper veinal mottle virus PLRV Potato leaf roll virus PMV Pea mosaic virus PPV Plum pox virus PSbMV Pea seedbome mosaic virus PVA Potato virus A PVC Potato virus C 16

PVM Potato virus M PVS Potato virus S PVY Potato virus Y RCVMV Red clover vein mosaic virus SoyMV Soybean mosaic virus TAV Tomato aspermy virus TBV Tulip breaking virus TEV Tobacco etch virus TLV Turnip latent virus TMV Tobacco mosaic virus TuMV Turnip mosaic virus TVHV Tobacco veinal mottle virus WMV mosaic virus 17

ACKNOWLEDGEMENTS

I am greatly Indebted to Dr. R.W. Gibson, my supervisor, for his interest and encouragement throughout both the period of exp­ erimental work and the preparation of this thesis and to my super­ visor at Imperial College, Dr. R.H.A. Coutts for his advice and constructive criticism. I also thank Dr. R.A.C. Jones for provid­ ing strains of potato virus Y and Dr. L. Torrance for helping me with ELISA. Thanks are also due to Dr. R. Harrington (Entomology Department, Rothamsted) for the identification of aphid species and Dr. J.M. Carpenter for doing the electrophoresis. Within Virology, I wish to thank all staff but particularly Mr. E. Lester and Dr. R.T. Plumb for providing facilities in their department for this work, Dr. D.A.Govier for helpfull discussions and for supplying antisera of PVY, Dr. A.C. Cockbain for his advice and helpful d iscussions, Mr. R. Woods for electronm icros- copical help, Mrs. Sheila Roberts and Mr. G. Higgins for providing photographs, Mrs. E.L. Lennon, R. Gutteridge and Miss J. Isger for occasional assistance and Mr. D.W. Roberts and his colleagues for growing the plants for experiments. I wish also to thank Mr. R.W. Payne and A.D. Todd (Statistics Department, Rothamsted) for help with the statistical analyses. 18

I THANK THE GREEK STATE SCHOLARSHIP FOUNDATION FOR A POSTGRADUATE STUDENTSHIP, DURING THE TENURE OF WHICH THIS WORK WAS DONE 19

GENERAL INTRODUCTION

The potato originated from South America and is a major World food crop. It is vegetatively propagated and so viruses are readily perpetuat­ ed from one growing season to the next through the tubers. One of the most

particles 730 x 11 nm (Plate 1) and is serologically distantly related to several members of the group, including tobacco etch virus (TEV), henbane mosaic virus (HMV), potato virus A (PVA), pepper veinal m ottle virus (PeVMV), and bidens m ottle virus (Bid MV) (De Bokx & H uttinga, 1981). PVY can be transmitted by sap inoculation but most spread in the field is by aphids. Over 25 aphid species, not all of which colonize potato, can transmit PVY (Kennedy et al.t 1962 ; Van Hoof, 1980 ). Aphids transmit PVY nonpersistently, virus being acquired and transmitted in a few seconds by aphids probing mainly epidermal cells. However, infectious aphids become non-infective within a few hours. There are two strains of PVY which are common in potato crops.

Potato virus Y° (P v P ) (ordinary strain). This initially induces leaf drop streak in many potato cultivars followed by severe mosaic, and vein clearing in tobacco (Plate 3, 4).

Potato virus (PVY ^ ) (tobacco veinal necrosis strain). This induces mild mosaic in almost all potato cultivars and veinal necrosis in tobacco (Plate 3, 5). This thesis reviews the literature on nonpersistent transmission of plant viruses and describes studies on the mechanism of aphid transmission and the epidemiology of PVY. 20

Plate 1. Particles of PVY in a purified preparation stained with sodium phosphtungstate (magnification x 100.000)* 21 A r

S

Plate 2, Tobacco plants cv. White Burley showing vein clearing ten days after inoculation with PVY^ (right), veinal necrosis after inoculation with PVYN (centre) or inoculated with phosphate buffer (left).

Plate 3. Leaves of tobacco plants, showing vein clearing ten days after inoculation with PVY° (left), veinal necrosis after inoculation with PVYN (centre), or no symptoms after inoculation with buffer (right). 22

Plate 4. Primary 'leaf-drop streak* symptoms, 3 weeks after inoculation, in King Edward potato plants inoculated with PVY^ (right), compared with infection with PVY^ (centre) and healthy (ri^ht). (Z flr) 23

Plate 5. Symptoms of PVY in leaves of King Edward potato plants. a, healthy; b, primarily infected with PVY^; c, primarily infected with PVY0; d, secondarily infected with PVY0. 24

2. REVIEW OF THE LITERATURE

2.1 Nonpersistent transmission of plant viruses by aphids

2.la Introduction Two main classification systems have been used to categorize virus transmissions by aphids. One is based on how long vectors retain a virus, the other on where and how virus is carried by the vector. The former system, the older of the two, was proposed by Watson & Roberts (1939 ), According to this system the viruses transmitted are classified as (i) nonpersistent, (ii) semipersistent (Sylvester, 1956 ), or (iii) persistent depending on whether virus is readily retained for (i) minutes, (ii) several hours to days, or (ili) for weeks to life and through a moult. This method of classification has the advantage of utilizing an easily determined property, but its value is lessened somewhat by the fact that virus retention can vary with factors such as ambient temperature and vector probing activity before or after virus acquisition. The second system was proposed by Kennedy et al. (1962 ), They classified viruses as stylet-borne or circulative. The stylet-borne viruses include all those familiar as nonpersistent, together with a few semipersistent and even persistent ones; the circulative viruses include the bulk of the persistent viruses. The term circuteive refers to a process in which virus is imbibed in infected sap, absorbed through the gut wall, transferred to the salivary gland and eventually inoculated into plants in virus-laden saliva. Circulative viruses which also multiply in their vectors were described as circulative-propagative (Smith, 1965 ). This system of classification was widely adopted, but it suffers from a lack of evidence that the so-called stylet-borne viruses are actually transferred via the stylets. There is good evidence for some persistently- 25

transmitted viruses that virus circulates through the vector and more recently it has been proposed that the terra circulative should be retain­ ed but that stylet-borae should be replaced by the noncirculative (includ­ ing nonpersistent and semipersistent subcategories) (Harris, 1976 , 1977a, 1977b, 1979 , 1981 ). However, the assignment of most viruses to one or other category is still based on persistence in the vector and in this thesis, the terms non-, semi- and persistently-transmitted viruses have been retained. General reviews on nonpersistent virus transmisstion are (Sylvester, 1962 ; Bradley, 1964 ; Smith, 1965 ; Pirone, 1969 ; Watson & Plumb, 1972 ; Garrett,1973 ; Pirone & Harris, 1977 ; Harris, 1983 ).

2. lb Characteristics of nonpersistent transmission Effect of preacquisition fasting. The transmission rates of nonpersistent viruses are increased by fasting the aphids prior to acquisiton (Watson, 1938 ) even for periods as brief as 15 min. Only slight increases occur with fasting periods longer than 1 h and the effect is cancelled out if fasted aphids are allowed to probe a leaf for more than a few minutes

(Watson, 1938, 1972 ; Watson & Roberts, 1939 , 1940 ). Different hypotheses have been postulated in order to explain this phenomenon Watson (1938 ) and Watson & Roberts (1939 ) postulated that transmission by nonfasted aphids is hampered by a virus-inactivating substance(s) which the aphids secrete during feeding presumably in the saliva. Keeping aphids off plants for some time arrests or slows down the production of this inactivator, and the virus transmission by fasted aphids remains relatively unaffected until probing and feeding activity again stimulate the production of inactivator (s). However, aphids fasted in cellophane-covered petri dishes salivate considerably while probing against the side of the container and through the cellophane top 26 but subsequent virus acquisiton is still enhanced (Hashiba & Misawa, 1969 ). Fasting also affects aphid feeding behaviour. Thus, when fasted aphids are put on a leaf they almost always make one or more brief probes during which they may be sampling sap to test its host-status whereas unfasted aphids often make long probes to initiate feeding. Accordingly, the few unfasted aphids that made brief probes within two minutes of being placed on a virus source plant transmitted almost as well as fasted aphids (Bradley, 1961 ). During these brief, presumed sap-sampling, probes aphids may be alternately imbibing and egesting sap (Harris, 1977a) and this behaviour may ensure good acquisition of virus. Van der Want (1954 ) and Bradley (1952 , 1964 ) have also suggest­ ed that virus acquisition may be good during these brief probes because aphids do not secrete salivary sheaths, but this now seems unlikely

(Mclean & Kinsey, 1964 , 1965 ; Hodges & Mclean, 1969 ; Hashiba & Misawa, 1970 ; Harris & Bath, 1973 ). Whatever the cause cf enhanced virus transmission by fasted aphids, epidemiologically it may be important as it makes aphids migrating into a crop likely to be more efficient vectors than those bred on the crop (Broadbent & Martini, 1959 )• Duration of acquisition probes. Aphids can acquire virus during probes as brief as 5 sec (Swenson, 1968 ; Harris, 1977a). However, uninterrupted probes of 15-60 s are generally optimal, and longer access to source leaves results in poor acquisition. Thus, only 5 Myzus persicae (Sulz.) of 45 tested transmitted PVY after 4 h acquisiton access whereas 25 transmitted after 2 min (Watson & Roberts, 1939 ). Hodges & Mclean (1969 ), using electrical conductivity to accurately measure the duration of probes, demonstrated that the pea aphid, Acyrthosiphon pisum (Harris), acquired bean yellow mosaic virus (BYMV) during probes of 16 + 4 s. Acquisition and inoculation thresholds were 4.50 s and 4.25 s respectively. 27

As probes last beyond 1 min, there is a decrease in acquisition (Watson, 1940 , 1946 ; Hashiba & Misawa, 1969 )• Latent period. An aphid is infectious immediately after it has probed an infected plant. Thus, unlike persistently-transmitted viruses which require time for the virus to circulate through the aphid, nonpersistently- transraitted viruses have no latent period and this is part of the evidence that viruses are carried in the region of the mouthparts or foregut. In practice, this is important because it allows nearby plants to be infected (Doncaster & Gregory, 1948 ; Duncan et al., 1956 ). Duration of inoculation probes. The minimum time required for inoculation is slightly shorter than that for acquisition (Sylvester, 1950 ; Hamlyn, 1953 ; Sylvester, 1955 ; Hodges & Mclean, 1969 ) but unlike acquisition, long probes can give high rates of inoculation (Bradley, 1964 ). However, viruliferous aphids generally become non-viruliferous following prolonged probes on healthy plants (Hashiba & Misawa, 1969 )• Retention period. Following acquisition, the chance of an aphid transmit­ ting a nonpersistently-transmitted virus diminishes gradually with time. At about 20°C, an aphid usually remains viruliferous for no more than a few hours following access to a source leaf, although this varies with particular virus-vector combination. For example M.persicae retains maize dwarf mosaic virus (MDMV) for up to 30 min (Thongmeearkom et al., 1976 ), but peanut m ottle virus (Pe MotV) fo r a t le a s t 12 h (Paguio & Kuhn, 1976 ). Aphids remain infective somewhat longer off than on a healthy leaf; for example, when aphids carrying PVY probed into healthy tobacco plants, they usually ceased to be viruliferous within 1 h whereas some aphids kept in a glass tube remained viruliferous up to 4 h (Bradley, 1959 ). The rate at which they become noninfective is similar on leaves of plants that are, and are not, susceptible to the virus (Bradley, 1959 ). At low temperatures aphids may remain infective for longer (Kassanis, 1941 ; Bradley, 1954 ; 28

Sylvester, 1954 ; Cockbain et al., 1963 ). Nonpersistent viruses are not retained through ecdysis and this characteristic provides a clear-cut means of distinguishing non- and persistently-transmitted viruses. A common reason for attempting to determine how long aphids retain the ability to infect is in relation to the spread of virus in the field. In most experiments, conditions have not been particularly close to those that might exist under field conditions but Cockbain et al. (1963 ) attempted to simulate field conditions by allowing tethered aphids to fly for various time in an air current. The infectivity of alatae of M.persicae and Aphis fabae Scop, carrying either pea mosaic virus (PMV) or beet mosaic virus (BMV) diminished at about the same rate regardless of whether they were flying or kept fasting in a glass container. Few aphids transmitted these viruses if they were held at temperatures above 30°C fo r 30 rain.

2.1c Virus groups transmitted nonpersistently Potyviruses. Most viruses that are transmitted in the nonpersistent manner are potyviruses. The virions are flexuous rods with modal length from about 680 to 900 nm. Those thus far characterized have helical symmetry and contain about 5 7. single-stranded RNA (Shepherd, 1977 ). Common members of this group are PVY, BYMV, BMV, PeMotV, p’lum pox virus (PPV), TEV, PVA, turnip mosaic virus (TuMV), lettu c e mosaic virus (LMV), and soybean mosaic virus (SoyMV). Cucumoviruses. This group includes cucumber mosaic virus (CMV) and tomato aspermy virus (TAV). The virions of members of this group are isometric particles of about 30 nm. Those characterized contain about 18 7. single-stranded RNA in four different-sized particles (Shepherd, 1977 ). Alfalfa mosaic virus. This is a raonotypic group for which no formal name has been proposed. At least four types of virions occur, of which 29

three are bacilliform, 58, AS, and 36 nm in length and 18 nro in diameter, and one is spheroidal, about 18 nm in diameter; all contain about 16 7. single-stranded RNA. A number of strains of alfalfa mosaic virus (AMV) have been described (Pirone & Harris, 1977 ). Carlaviruses. Common members of this group are potato virus S (PVS), potato virus M (PVli), carnation latent virus (CarLV), red clover vein mosaic virus (RCVMV) and pea streak virus (PeaSV). Viruses are slightly flexuous rods with modal lengths of 620-690 nm. Those characterized have helical symmetry and contain about 6 7. single-stranded RNA, Not all members of this group have been shown to be aphid transmissible (Shepherd, 1977 ). Potexviruses. Typically members of this group are transmitted only by sap, although aphids may be able to transmit them in artificial conditions

(Pirone & Kassanis, 1975 ). However, potato aucuba mosaic virus (PAMV) although a potexvirus (Matthews, 1982 ), is naturally transmitted by aphids but only when it is in association with certain potyviruses (Kassanis & Govier, 1971a).

2.Id Physical properties of nonpersistent viruses, Nonpersistent viruses are relatively stable and are generally easily sap transmissible. They are inactivated by drying, but not completely by freeze drying, by heating for 10 min at 60°C, and by treatment with acid or alcohol. Long­ evities in vitro (minutes to days) and dilution end-points vary consider­ ably depending on the virus (Harris, 1977a).

2.le The role of helper component. In 1936, Clinch et al. reported that they were unable to transmit tuber blotch virus (PAMV) by aphids from infected potatoes unless the potatoes were also infected with PVA. The problem was reinvestigated by 30

Kassanis (1961 ). None of twelve strains of PAMV was transmitted by M. perslcae from plants Infected with PAMV alone but they were transmit­ ted from plants which were also infected with either PVA or PVY. Kassanis suggested several explanations for the phenomenon: phenotypic mixing leading to the protein of PAMV containing material from PVY or PVA which conferred aphid transmissibility, increase in PAMV concentration in certain tissues, a change in the position of PAMV in the infected cells so that it becomes available to the aphids, and a mechanism by which the helper virus may cause the particles of PAMV to aggregate with each other or with those of the helper viruses and thus form larger virus units, which can attach to aphid mouthparts. Exchange of genetical determinants has also been postulated to explain the transmission of potato virus C (PVC), a virus not normally aphid-transmissible, from plants also infect­ ed with PVY (Watson, 1960 ). However, Kassanis & Govier (1971a) showed that PAMV and PVC are transmitted by M. persicae not only from plants also infected with PVY, but from plants infected with PAMV and PVC alone, provided the aphids fed first on plants infected with PVY, However, when the order was reversed they were not transmitted. Pirone & Megahed (1966 ) transmitted both CMV and AMV by aphids probing into purified preparations. However, neither they nor Watson jet al. (1967 ) could obtain transmission of purified potyviruses such as HMV, PVY, TuMV. However, lik e PAMV, p u rified PVY could be transmitted by aphids which had first fed on a PVY-infected leaf, the leaf having been irradiated with ultraviolet light to inactivate the virus in the leaf (Kassanis & Govier, 1971b). It was further demonstrated that a virus-free extract from PVY-infected leaves promoted the transmiss­ ion of purified PVY and PAMV (Govier & Kassanis, 1974a, 1974b). The active principle in these extracts was termed helper component. Character­ ization studies indicated that helper component is a protein of between 31

100.000 and 200.000 daltons, which is serologically distinct from PVY coat protein or inclusion protein (Govier et al.t 1977 ). The evidence available suggests that aphids in order to transmit possibly all potyviruses must acquire helper component normally induced in plants as a result of infection by these viruses. Purification separates virus from its helper component so aphids cannot transmit the virus. Nontransmissible isolates of potyviruses, as have been reported for PVY, certain strains of PeMotV, TuMV and TEV probably induce no or defective helper component as they can be transmitted if aphids acquire helper component induced in the same plant by another potyvirus or from another infected plant (Kassanis & Govier, 1971a; Paguio & Kuhn, 1976 ; Simons, 1976 ; Sako, 1980 ; Mossop, 1982 ). In most research, M. persicae was used as a vector; however Aphis gossypii Glov. also requires helper component to transmit (Pirone, 1981 ). A requirement for helper component has been demonstrated in many poty­ viruses including PVY, TEV, tobacco vein mottling virus (TVMV), watermelon mosaic virus (WMV), BYMV, and TuMV, so helper component-dependency may be general for all aphid - potyvirus-combinations. Serologically-distinct helper component proteins are produced in response to specific potyvirus infection suggesting that helper component is virus coded (Thombury & Pirone, 1983 ; Hellmann et al., 1983 ) and that induced by one virus may not be (as) effective for transmitting another (Pirone, 1981 ; Sako & Ogata, 1981 ). There is also indirect evidence that the particular helper component induced by a virus is involved in determining whether or not an aphid species is a vector (Sako, 1981 ). However, transmissibility of at least TEV is a function of the virus particles as well as of the helper component (Pirone & Thornbury, 1983 ). So far, the existence of helper component regulating transmission of nonpersistent viruses has been documented only for potyviruses; CMV (Pirone & Megahed, 1966), AMV (Pirone, 32

1964 ; Pixone & Megahed, 1966 ), and two carlaviruses (Weber & Hampton, 1980 ) retain transmissibility after purification. The mode of action of helper component has yet to be determined. Recent hypotheses are that it may act either by enabling virus to bind to receptor sites in the aphid from which it subsequently can be releas­ ed, by affecting the ability to ingest virus, preventing the breakdown of aggregation of particles or preventing virus from being bound to parts of the alimentary tract (Govier & Kassanis, 1974b; Pirone, 1977 ; Pirone & Harris, 1977 ; Lopez-Abella et al., 1981 ).

2.If Sites of virus acquisition and inoculation in plant Uninterupted probes of 15-60 sec are, in general, optimal for acquisition of nonpersistent viruses (p. 26). During these probes the stylets penetrate only a few ( ( ten) micrometers into the epidermis, and this distance is less than the depth of an epidermal cell (Bradley, 1964 ; Nisawa & Hashiba, 1967 )• As acquisition probes last beyond one minute transmission rates decline (Watson, 1940 , 1946 ; Hashiba & Misawa, 1969 ) and this is about the time it takes an aphid's stylets to penetrate to subepidermal tissues (Roberts, 1940 ; Misawa & Hashiba, 1967 )• This suggests that virus is not acquired from sub-epidermal cells. To explain this Bawden et al. (1954 ), Bradley (1954 ) and Watson (1958 ) have postulated that virus in sub-epidermal cells is less abundant or in a less aphid-transmissible form than virus in the epidermis, but BYMV is actually less abundant in the epidermis than in the underlying tissue, aphids allowed acquisition probes into exposed subepidermal tissue transmitting the virus at three times the rate of aphids probing into stripped epidermis (Hashiba, 1970 ). Other studies with leaves that have been stripped of epidermis have also shown that virus in mesophyll cells is at least as available for acquisition as virus in the epidermis 33

(Van Hoof, 1958 • Nambra, 1962 ; Normand & Pirone, 1968 ). Whether brief probes are made into or between epidernaL cells remains unclear. Thus stylet insertion has been reported to be intercellular (Yoshii, 1966 ), intracellular (Swenson, 1962 ) or both (Roberts, 1940 ; Bradley, 1952 ; Hashiba, 1969 ). These differences may result from the use of different aphid-plants systems. However, 90 % of the initial brief probes of fasted M. persicae on were intracellular (Hashiba, 1969 ) and it was these probes that acquired BYMV (Hashiba & Misawa, 1970 ). Lopez-Abella & Bradley (1969) found that 50 7. of the probes that were initiated intercellularly by M. persicae eventually became intracellular as the stylet tips penetrated an epidermal cell and they also considered that acquisition of CMV by M. persicae was intracell­ ular (Lopez-Abella & Bradley, 1969 , 1970 ). Intracellular acquisition would also seem to agree more with the high rates of transmission observed for many nonpersistent viruses since, if acquisition occurs between cells, seemingly inefficient mechanisms, such as acquisition from broken plasmodesmata (Yoshii, 1966 ), must be postulated. Fewer studies have been made on the sites of stylet insertion during inoculation probes, but the sites involved may include the sub-epidermal tissues as well as epidermal as both long and brief probes are effective at virus inoculation.

2.Ig Sites of transmitted virus in/on the aphid Currently, there are two main contenders for the site of transmiss­ ible virus in/on the aphid. The stylet-borne hypothesis. There are several indications that nonpersistent viruses may be transmitted on aphid stylets. Thus, transmission occurs during brief probes, infectivity is lost when aphids 34 shed their stylets along with the exoskeleton, fore and hint-gut during ecdysis and otherwise persists for only a few hours or less if aphids feed. Although Gamez & Watson (1964 ) failed to get transmission by artificially inserting aphid stylets first into HMV-infected, then into healthy leaves, Barnett & Pirone (1966 ) obtained transmission of CMV, although at a low rate, after dipping the stylets of anaesthetized aphids into capillary tubes containing purified virus. Furthermore, treating stylets with formalin or exposing them to ultraviolet (UV) irradiation rendered aphids carrying PVY nonviruliferous (Bradley & Ganong, 1955 , 1955 ). However, UV irradiation of the aphid stylets before access to infected leaves also prevented aphids transmitting PVY suggesting that it is the aphid that was affected rather than the virus (Bradley, 1964 ). Also, exposing the stylets of viruKferous aphids to other antiviral agents such as 8-azaguanine, ribonuclease, milk and saliva did not diminish virus transmission (Bradley, 1959 ; Simons & Ross, 1963 ; Nishi, 1969 ; Hashiba & Misawa, 1970 ). Aphid stylets possess ridges and grooves on the mandibles that might function to hold virus particles (Van Hoof, 1958 ). However, examinations of the stylets of ten aphid species, revealed no substantial differences in stylet structure (Schmidt et al., 1974 ), although they differ in vectoring ability (Kennedy et al., 1962 ). Direct evidence that virus might be stylet-borne has been sought by electron microscopy. Taylor & Robertson (1974 ) found a few virus-like particles lining the distal 20 pm of the maxillary food canal in transverse sections of the stylets of M. persicae which had probed leaves infected with TEV. Lim & Hagedom(1977 ) detected pea seedbome mosaic virus (PSbMV) or its protein on the inner surfaces of the mandibles of Macrosiphum euphorbiae (Thos.), using scanning electron microscopy and labelled antibody. However, detection of stylet-associated virus is 35 not proof that this virus would be transmitted. The ingestion-egestion hypothesis. The concept that nonpersistent viruses were acquired by ingestion, carried into the anterior portion of the alimentary canal where they might adhere and were inoculated by regurgitation has long been favoured by Watson and co-workers (Watson & Roberts, 1940 ; Gamez & Watson, 1964 ; Watson & Plumb, 1972 ). Aphids feeding on artificial diets can be observed to ingest and egest carbon black particles (Harris & Bath, 1973 ) and when they probed for less that 10 min on plants labelled with P 32 , they contained considerably 32 less P after 6-8 min probes than after probes that lasted 3-5 min, suggesting that the tracer has been regurgitated (Garrett, 1973 ). There was a correlation between the number of M. persicae which ejected 570 U>m 3 or more of P 32 labelled sap and the number that transmitted CMV. This sap volume exceeds by ten times the volume of the food canal of the stylets suggesting that egested sap was from the fore-gut where CMV is concentrated (Gera e ta l., 1979 ).

At first sight, the stylet-borne hypothesis accounts well for the short retention and the brief acquisiton and inoculation thresholds which typify nonpersistent transmission. However, virus-vector specific­ ity might better be explained by the ingestion-egestion hypothesis via some form of selective attachment to the membranes lining the aliment­ ary canal which are biologically active tissues rather than to the stylets. The ingestion-egestion hypothesis is also attractive because the explanation of the preacquisition fasting effect based on increased frequency of sap-sampling probes by fasting aphids fits well and because 36 it provides for the uptake of relatively large volumes of virus contain­ ing sap into protected areas and its subsequent inoculation into a plant. However, at present there is no clear experimental evidence discriminating between whether nonpersistent viruses are transmitted via the stylets or ingested into the pharynx or foregut.

2.lh Vector specificity Nonpersistent viruses are usually transmitted by more than one aphid species, for example, at least sixteen aphid species transmit PVY (Kennedy et al.» 1962 ), but vectors of one virus are not necessarily vectors of another. Furthermore, some species, such as M. persicae, are vectors of many viruses whereas others transmit few if any (Kennedy et al., 1962 ). Even within an aphid species, there may be large differ­ ences in vectoring ability between aphid seasonal forms (Paine & Legg, 1953 ; Orlob, 1962 ), growth stages, morphs (Sylvester, 1955 ; Broadbent, 1960 ; Cockbain et al., 1963; Thottappilly et al., 1972 ; El Kady et al., 1973 ), clones and biotypes (Simons, 1959 , 1966 ; Sohi & Swenson, 1964 ; Kvicala, 1968 ; Upreti & Nagaich, 1971 ; Jurik et al., 1980 ; Singh ejt al., 1983 ). Different hypotheses have been proposed to explain virus-vector spec­ ificity. Thus, Van der Want (1954 ) proposed a mechanical-surface adherence hypothesis in which specificity was an expression of differ­ ences in the structure and surface adherence properties both of the stylets of different aphids and of particles of different viruses. How­ ever, there does not appear to be relationship between stylet-tip morph­ ology and vector specificity (Proeseler et al., 1972 ; Forbes, 1977 ). For example, Schmidt et al. (1974 ) found no substantial differences in stylet morphology on ten aphid species, which included M. persicae, a good vector of PVY and Rhopalosiphum padi (L.), a poor vector of PVY (Van Hoof, 1980 ). However, there is evidence for differences in the surface 37 adherence of the stylets. Thus, large amounts of labelled virus were detected on the inner surface of mandibles of a biotype of M. euphorbiae that transmitted PSbMV efficiently whereas only small amounts were on those of a biotype that transmitted PSbMV inefficiently (Lim et al., 1977 )• Some virus isolates have become non-transmissible by some aphid species whilst still being transmitted by others (Badami, 1958 ) and others are not transmissible by the normal range of vectors (Lucas & Hill, 1980 ). Helper component has also been suggested as a factor which may play a role especially for potyviruses, in determining fector specificity (Sako, 1981 ). In CMV, which does not require a helper component, the coat protein of the virus particle may determine rates of transmission (Gera et al,, 1979 ). Virus inactivating properties of saliva have also been suggested as an explanation of vector specificity (Bradley, 1952 ; Day & Irzykiewicz, 1954 ; Sylvester, 1954 ; Van der Want, 1954 ; Hashiba & Misawa, 1969 ; Nishi, 1969 ). Saliva may well play a role in restrict­ ing virus transmission; for example, limiting persistence in the aphid (Loebenstein & Raccah, 1980 ), as it can diminish virus infectivity (Nishi, 1969 ; Pirone, 1970 ) but attempts to demonstrate selectivity of saliva on viruses have failed (Pirone, 1970 ).

2,2 Transmission of potyviruses by non-colonizing aphids Although there is some evidence that aphids might be attracted by plant odour (Chapman et al., 1981 • Brandley & Anderson, 1982 ), gener­ ally flying aphids cannot recognize their host plants (Swenson, 1968 ) and aphids may alight equally readily on non-host and host plants (Kennedy et al., 1959a, 1959b). Aphids which have alighted generally probe at least briefly to determine host status. However, such brief probes of only a few seconds would allow acquisition and transmission of nonpersis tently 38

transmitted viruses such as potyviruses (Hoilings & Brunt, 1981 ) and many non-colonizing aphids are capable vectoring of nonpersistently- transmitted viruses (Table 1), The laboratory evidence available suggests that efficiency of transmission of a particular virus is related to the virus-aphid vector combination rather than to host or non-host status of the aphid to the plant. Thus, some non-colonizing aphids transmit maize dwarf mosaic virus (MDMV) (Messieha, 1966 ) more efficiently than the colonizing ones whereas PVY (Kostiw, 1979 ; Van Hoof, 1980 ) and LMV (Dickson & Laird, 1959 ) are transmitted more efficiently by M. persicae, a colonizing aphid. WMV (Adlerz, 1974 ), BYMV (Cockbain, 1970 ), PeliotV (Bechcken, 1970 ) are transmitted efficiently by both non-colonizing aphids and colonizing ones whereas only non-colonizing aphids transmitted tulip breaking virus (TBV) (Brierley & Smith, 1944 ). So, although the cause of differences in transmission efficiency is not understood, it seems to be unrelated to whether or not the aphid colonizes the crop. Amongst the winged aphids available and capable of transmitting a virus, those species also colonizing the crop might even be behaviouraly less effective as vectors compared with species not colonizing the crop since the former are less inclined to fly away again soon after alight­ ing and probing (Kennedy, 1950 ). Furthermore, it is likely that more non-colonizers than colonizers visit most crops, indicating a possible dominance of non-colonizing aphids in the epidemiology of nonpersistent viruses. So, breeding for resistance to infestation by aphids should not be expected to result in resistance to infection by nonpersistent viruses (Gibson & Plumb, 1977 ; Rizvi & Raman, 1983 ; Atiri et al., 1984 ) al­ though in a few, apparently unusual situations it has (Lecoq et al*,

1979 ; Lecoq et al., 1980 ; Gunenc & Gibson, 1980 ; Maison & Massonie, 1982 ). Table 1. Transmission of potyviruses by non-colonizing aphids

Virus Crop Aphid species References Bean common mosaic Bean Acyrthosiphon pisurn (Harris) Zaumeyer & Kearns (1936) Aphis spp. (4)4* Brachycolus atriplicis (L.) Brevicoryne brassicae (L.) Dactynoctus ambrosiae (Thos.) Hyadaphis erysimi pseudobrassicae Davis Macrosiphum euphorbiae (Thomas) Mygus persicae (S u lz .)

Bean yellow mosaic Bean Acyrthosiphon spp. (2) Swenson (1957) Aphis spp. (3) Brachycaudus spp, (2) Chaetosiphon fragaefolli (Cock.) crataegi (Kltb.) Macrosiphum spp. (3) Myzus spp. (2) Nasonovia spp. (2) Acyrthosiphon pisurn (Harris) Gumb & McWharter (1948) Red clover Brachycaudus helichrysi (Kltb.) Cockbain (1970)

+ Number in parentheses denotes the number of vector species. Table 1#- continued

Virus Crop Aphid species References Beet mosaic Spinach Brachycaudus helichrysi (Kltb.) Cockbain (1970) Celery mosaic Celery Acyrthosiphum pisum (Harris) Severin&Freitag (1938) Brevicoryne brassicae(L •) Hyadaphis erysimi pseudobrassicae Davis Hyalopterous pruni (Geoffr.) Macrosiphum rosae (L.) Hyzus cerasi (F.)

Leek yellow stripe Leek Aphis fabae Scop, Verhoyen & Harvat (1973) o Myzus persicae (S ulz,) Bos e t a l . (1978)

Lettuce mosaic Lettuce Aphis gossypii Glov. Dickson & Laird (1959) Nasonovla lactucae (L.)

Maize dwarf mosaic Maize Acyrthosiphum pisum (Harris) Messieha (1966), Shaunak & Pitre (1971) Aphis gossypii Glov, Messieha (1966) Myzus persicae (S u lz ,) Messieha (1966)

Onion yellow dwarf Onion Acyrthosiphum spp, (2) Drake et al. (1933) Amphorophora spp, (2) Anoecla sp. Table 1. - continued

Virus Crop Aphid species References Aphis spp, (10) Brachycaudus cardui (L.) Brachycolus atriplicis (L.) Brevicoryne brassicae (L.) Calaphis betune11a Walsh Chaitophorus spp. (2) Cryptomyzus ribis (L.) Dactynotus spp, (5) Drepanaphis acerifoliae (Thos.) Hoplochaitophorus quercicola (Mon.) Hyadaphis.erysimi pseudobrassicae Davis Hyalopterus pruni (Geoffr.) Hysteroneura setariae (Thos.) Kakakia purpurascens (Oestl.) Macrosiphum spp. (3) Monellia spp. (2) Myzocallis spp. (2) Myzus spp. (2) Neoceruaphis vibumicola (Gill.) Ovalus monardae (W ills.) Periphyllus negundinis(Thos.) Table 1. - continued

Virus Crop Aphid species References Prociphilus corrygatans (Sirr.) Pterocomma salicis (L.) Rhopalosiphum padi (L.) Sitomyzus rhois (Mon.) Theriophis trifolii (Mon.) Thripsaphis ballii (Gill.)

Papaya ringspot Papaya Myzus persicae (S u lz .) Is h ii (1972)

Passionfruit ringspot Passionfruit Aphis spp. (2) De Wijs (1974)

Pea mosaic Pea Brachycaudus helichrysi (Kltb.) Kvicala (1965) Cryptomyzus ribis (L.)

Peanut mottle Peanut Aphis gossypii Glov. Behncken (1970) Hyperorayzus lactucae (L.) Myzus persicae (S u lz .) Rhopalosiphum padi (L.) Rhopalosiphum maidis (Fitch) Highland et al. (1981)

Potato virus A Potato Brachycaudus helichrysi (Kltb.) Cockbain (1970) Table 1. - continued Virus Crop Aphid species References Potato virus Y Potato Acyrthosiphon pisum (Harris) Ryden (1979) Aphis spp. (2) Van Hoof (1980) Brachycaudus helichrysi (Kltb.) Edwards (1963, 1965), Bell (1983) hippophaes (Wlk.) Van Hoof (1980) Metopolophium albidum Hille Ris Lambers Metopolophium dirhodum (Wlk.) Phorodon humuli (Schrank) Karl (1971), Van Hoof (1980) Rhopalosiphum padi (L.) Kostiw (1979)

Pepper Aphis citricola Van der Goot Raccah (1983)

Soybean mosaic Soybean Aphis craccivora Koch Halbert et al (1981) Dactynotus ambrosiae (Thos.) Abney et al. (1976) Hysteroneura setariae (Thomas) Costa Lima Neto & Costa (1978) Macrosiphura euphorbiae (Thomas) Abney et al. (1976) Myzus persicae (S u lz .) Halbert et al. (1981) Rhopalosiphum spp. (2) Schizaphis graminum (Rond.) Costa Lima Neto & Costa (1978) Table 1. - continued

Virus Crop Aphid species References

Tobacco etch Pepper Acyrthosiphon pisum (Harris) Laird & Dickson (1963) Aphis spp. ( 2) Macrosiphum euphorbiae (Thomas)

Tulip breaking Lily Aphis gossypii Glov. Brierley & Smith (1944) Macrosiphum euphorbiae (Thomas) Nyzus persicae (Sulz.)

\~a terme lon mosaic Nelon Aphis spp. (2) Adlerz (1974)

~1yzus persicae (Sulz.) Anuraphis middletonii (Thomas) Adlerz (1978) Acyrthosiphum pisum (Harris) Dickson et al. (1949) Aphis spp. (2) Macrosiphum euphorbiae (Thomas) Nyzus persicae (Sulz.) Brevicoryne brassicae (L.) Coudriet (1962) Hysteroneura setariae (Thomas) Myzus persicae (Sulz.) Rhopalosiphum maidis (Fitch) 45

Evidence that non-colonizing aphids are actually important in spreading potyviruses in crops rather than laboratory evidence that they are merely capable of being vectors is more difficult to obtain.

However, trapping of live aphids in American soybean crops has shown that spread of SoyMV was entirely dependent on non-colonizing aphids, no aphids colonizing the crop being found in the area (Lucas, 1978;

Halbert et al., 1981). In China, where the soybean aphid Aphis glycines

Hats. occurs alates of H. persicae and M. euphorbiae although not col­ onizing soybean were also the more important vectors of SoyMV (Halbert, et al., 1983). Similarly, onion yellow dwarf virus (OYDV) (Drake et al.,

1933) and bean common mosaic v~rus (BCMV) (Zaumeyer & Kearn, 1936) were transmitted by non-colonizing aphids in laboratory tests and these aphid species might be responsible for the spread of these viruses since aphids seldom colonized crops of onion and bean. Non-colonizing aphids may not only be important in virus spread if they are very efficient vectors or in cases where no colonizing aphids found. In contrast, catches of live aphids showed the spirea aphid Aphis citricola (Kirk.), although it does not colonize peppers and transmitted less efficiently than the colonizing

N. persicae and 1'1. euphorbiae, was the most important vector of PVY in peppers largely because of its abundance (Raccah, 1983 ).

Although transmission tests in the laboratory give valuable inform­ ation of the vectors and non-vectors of a particular virus only infect­ ivity tests of trapped live aphids such as carried by Highland et al.,

1981 , Halbert et al., 1981 and Raccah (1983 ) reveal the importance of each aphid species under field conditions and will determine the actual virus transmitting potential of each aphid species be it a colonizer or a non-colonizer. This information is essential to improve our under­ standing of the epidemiology of potyviruses and our ability to predict their spread from the numbers of aphids of each species involved. 46

3. GENERAL MATERIALS AND METHODS

3.1 Virus isolates* One isolate of PVY 0 and one of PVY N , both from naturally infected potato cv. Kind Edward, were used in all experiments unless otherwise stated. Both strains were propagated in tobacco cv. White Burley grown in an aphid-free glasshouse with daylength during winter increased to 16 h by artificial light. One isolate of BMV from naturally infected sugarbeet was also used. Sugarbeet cv. Hilleshog Monotri was used as propagation host.

3.2 Plants. For both glasshouse and field experiments, the potato cultivars Kind Edward, Record, Maris Piper, Desiree and Pentland Crown were grown from tubers. Test plants (Table 2 ) used for the identific­ ation and assay of PVY, BMV and TMV were grown from seeds. For tests with aphids, N. tabaccum cv. White Burley and sugarbeet cv. Hilleshog Monotri were grown in boxes (24 plants/box). Other plants were grown in individual small pots. Plants giving local lesions were used to assay virus concentration, numbers of lesions on inoculated leaves being correlated with concentration (Bawden, 1964 ; Roberts, 1964 ).

3.3 Aphids. The aphid species, M. persicae, was reared on Chinese cabbage (Brassica pekinensis L.) or sugarbeet cv. Hilleshog Monotri. M. euphorbiae was reared on potato cv. Pentland Crown. The cereal aphids Rhopalosiphum padi (L.), Metopolophium dirhodum (Walker) and Sitobion avenae (Fabr.) were reared on oats cv. Blenda. The cultures originated from Rothamsted. 47

Table 2. Test plants used in experiments for virus identification and assay

T est p lan t Symptoms+ Viruses

Chenopodium amaranticolor local lesions PVY°, BMV Coste & Reyn Nicotiana clevelandjij Gray mosaic BMV N icotiana tabacum L. cv. vein clearing PVY0 White Burley veinal necrosis PVY Nicotiana tabacum L. cv. mosaic Tobacco mosaic virus Xanthi (TMV) Physalis floridana Rydb. systemic necrosis PVY° systemic mottling PVYN Sugarbeet cv. Hilleshog Monotri mosaic BMV

+ (Hollings, 1959b; Beiss, 1963 ; Russell, 1971 ; Zaitlin & Israel, 1975 ; De Bokx St Huttinga, 1981 ) 48

inoculation with PVY° (left) and mild mosaic after inoculation with PVYN (right).

Plate 7, Chlorotic local lesions in Chenopodium amaranticolor, 8 days after inoculation with PVY^ (left) and no lesions after inoculation with phosphate buffer (right). 49

Plate 9* Local lesions in Chenopodium amaranticolor, six days after inoculation vith BMV (left) and no lesions when inoculated with buffer (right). 50

Plate 10. Nicotiana clevelandii showing mottling, two weeks after inoculation with BMV (left), and healthy control (right).

Plate 11. Leaves of N. clevelandii showing mottling 2 weeks after inoculation with BMV (right), or uninoculated control (left). 51

3.4 Techniques

3.4a. Virus acquisition and transmission by aphids. Apterous adults aphids were used for virus acquisition and transmission tests unless

otherwise stated. They were fasted for 2-4 h, usually given 2 h min access to infected source leaves and then confined for 24 h on the test plant with small glass tubes. Afterwards, plants were sprayed with an insecticide (Pirimicarb) to kill the aphids, and plants were kept in an aphid-free glasshouse, for 2-3 weeks, to allow symptons to develop.

3.4b. Manual transmission of viruses. Inocula were prepared by grind­ ing leaves from infected plants in 0.01 M phosphate buffer (pH 7.0), the sap being diluted 1/50 or 1/100. Before inoculation, plants were lightly dusted with 600-mesh carborundum (abrasive) and then inoculated by gently rubbing the upper surface of leaves with the fore-finger wet with inoculum. Inoculated leaves were washed with tap water and placed in a glasshouse.

3.4c. Immunosorbent electron microscopy (ISEM). Freshly prepared carbon- coated grids were floated film side down on 20 Jjll drops of antiserum diluted 1:1000 in 0.06 M Sorensen's phosphate buffer at pH 6.5. Prelim­ inary tests indicated that this dilution of PVY antiserum was optimal for attachment of virus particles to the grids. The grids were kept on the drops for Ca. lh at 35°C and then washed by floating for two successive periods of 10 min on phosphate buffer. The grids were drain­ ed by touching to filter paper. Two grids were prepared for each 52

test sample and 25 JU drops of sap from each sample (diluted 1/10 with phosphate buffer) were placed on waxed slides in Petri dishes containing moist filter paper. Grids freshly coated with antiserum were then floated on these drops overnight at 5°C. Grids were washed with 20 drops of buffer stained with 17. PTA (potassium phosphotungstate) and examined with an electron microscope.

3.4d. Enzyme-linked immunosorbent assay (ELISA). The procedures used for preparing antibody globulin, conjugating globulin with alkaline phosphatase and performing ELISA in microtitre plates (Dynatech Ltd.; type M129A) were based on those of Clark & Adams (1977 ). However, fractionation on a DE 22 cellulose column was omitted when preparing the antiserum globulin. Concentrations of serological reagents suitable for use in the tests were determined by experiment, to maximise the reaction with PVY and to minimize the effect of other components of leaf sap. The micro­ titre plates were coated with globulin at 2 |Ag/ml and enzyme-conjugated globulin was used at 1:500. The tissue (Ca.lg) was triturated in 0.02 M phosphate buffer (2 ml/g tissue), pH 7.4, containing 0.15 M Nad, 0.057. Tween 20 and 2% polyvinyl pyrrolidone (PSTP). Wells were coated with globulin for 2-3 h at 25°C and exposed for 12-16 h at 4°C to the samples being tested; conjugate was added for 3-4 h at 25°C and freshly prepared substrate was added for 60 min at 20°C. Absorbance readings were then recorded at 405 nm with a Titertek Multiscan colorimeter (Flow Laboratories Ltd.) Duplicate wells were used for each sample and the mean of their A^q^ value was calculated after subtracting the absorb­ ance given by substrate in wells not exposed to tissue extracts. The reaction was considered to be positive when this corrected figure 53

exceeded by more than 0.05 the figure given by an extract of virus-free tissu e . For both ISEM and ELISA, antiserum prepared against PVY° by Dr. D.A. Govier was used.

3.4e. Electron microscopy. Samples were negatively stained with 27. PTA (potassium phosphotungstate), sprayed from an air brush onto filmed grids (Hall, 1964 ) and examined with an electron microscope with Mr. R. Woods.

3.5 Statistical analyses. Unless otherwise stated, results from transmission tests were logit-transformed and analyzed by generalised linear models (Nelder & Wedderbum, 1972 ; Gibson et al., 1982 ). The results were prepared for analysis on a computer by R.W. Payne and G.L. Smith (Statistics Department, Rothamsted). 54

EXPERIMENTAL WORK

4. INTERACTION OF PVY° AND PVYN IN DOUBLY INFECTED PLANTS

Introduction

Although PVY 0 and PVY N can co-exist in the same plant, there is evidence that PVY N is suppressed in potato plants infected with PVY 0 (Bawden & Kassanis, 1951 ) and PVY^ was transmitted more frequently than PVY N by M. persicae from tobacco plants infected with both strains (Richardson, 1958 ). The following experiments further investigate interactions of PVY 0 and PVY N in tobacco and potato plants.

Materials and Methods

4.1 Interaction of PVY0 and PVY N in glasshouse-grown tobacco plants Tobacco plants were inoculated with either PVY°, PVYN or PVY° + PVY N ; the inoculum consisted of 0.1 g of leaf infected with the relevant strain ground up in 5 ml of phosphate buffer pH 7.0. Two weeks later, batches of 24 M. persicae were allowed min access to infected leaves after which aphids were caged singly on tobacco test plants. The leaves which had been used as sources for aphid transmiss­ ion tests were assayed for virus concentration by sap transmission to tobacco test plants at close to the dilution end point. Leaf material weighting 0. lg were ground up in 10 ml phosphate buffer pH 7.0 and then further diluted to 10 -4 . This was manually inoculated to 24 tobacco test plants and the numbers of plants infected were recorded. The experiment was replicated eight times. 55

4. 2 Interaction of PVY0 and PVY N in glasshouse-grown potato plants One fully expanded leaf from each of five plants of the potato cultivar King Edward grown from tubers infected either with PVY ,0 PVY N or PVY 0 + PVY N were assayed as above by sap transmission.

4, 3 Interaction of PVY 0 and PVY N in tobacco plants grown at different temperatures Tobacco plants were inoculated manually as before, with PVY°, PVY N or PVYON + PVY and then two of each treatment were grown in controlled environment cabinets (Fisons plant growth cabinets, Fisons

Scientific Apparatus, Loughborough, England) (16 h day/ 8 h night) at 15°C and at 30°C (humidity about 907.). One leaf of each plant was

detached 2 weeks after inoculation and virus concentration was assayed, as above, by manually inoculating 24 plants with sap at 10 .4 dilution. The experiment was replicated seven times.

PVY 0 induces vein clearing in tobacco whereas PVY N induces veinal necrosis (Klinkowski & Schmelzer, 1960 ). However, tobacco plants with both strains are indistinguishable from PVY -infectedN ones. To test if seedlings showing veinal necrosis were also infected with PVY^, they were sap-inoculated to Physalis floridana which gives systemic necrosis only with PVY^ (De Bokx & Huttinga, 1981 ). Subsequently, it was discovered that the isolates of PVY^ used induces local lesions in Chenopodium amaranticolor(Kurpa, 1983 ) whereas that of PVY N gives no symptoms, and the test plant was also used to test for double infectio n s 56

R esults

Interaction of PVY0 and PVY N in glasshouse-grown tobacco plants Vein clearing appeared in all treatments eight days after inoculation. Two weeks after incoulation, plants infected with PVY N or PVY 0 + PVY N had developed veinal necrosis and appeared indistinguish­ able# Rates of sap and aphid transmission of PVY° were similar from singly and doubly infected plants (Table 3 ) suggesting that virus concentration was similar# However, transmission of PVYN by both

aphid and sap was less from doubly infected plants (P ( 0#05) suggest- ing that the concentration of PVY was less in doubly infected plants. About 407# of the aphids that transmitted from doubly infected plants inoculated both strains.

Interaction of PVY 0 and PVY N in glasshouse-grown potato plants Plants infected with PVY°, PVYN and PVY° + PVY^ all appeared similar# Rates of sap transmission of PVY° were similar from singly and doubly infected plants whereas sap transmission of PVY N was less from doubly infected plants (Table 4 ) (P { 0.01).

Interaction of PVY 0 and PVY N in tobacco plants grown at different temperatures In plants kept at 30°C, vein clearing developed four to five days after inoculation but plants infected with PVY N or PVY ON + PVY did not develop veinal necrosis even a month after inoculation (how­ ever, when transferred to a cool glasshouse, veinal necrosis appeared in two days). In plants kept at 15°C, vein clearing appeared nine to ten days after inoculation and plants infected with PVY N or PVY ON + PVY 57

Table 3, Transmission of PVY 0 and PVY N by M. persicae or sap from singly and doubly infected tobacco cv. White Burley plants.

Virus strain Means of transmission Sources of Virus transmitted Aphid Sap PVY° PVY° 100/zioo i*(-0-074K).130)^ 32/216(-l.405+0.189)

PVY11 pvyn 120/ol.(Zlo 0.123+0.131) - 78/ 2i 6(-°. 754+0.141.)

PVY° 84 /2 i 6(-°.236+0.134) PVY° -f PVYN 44/ 2l 6(' U 198± °' 167) pvyn 69/_zio *(-0.398+0.140) - 35/-.,(-1.348+0.183)zio “

+ Numbers in parantheses are mean logits + S.E. * Different from rate for singly infected plants at P ( 0,05 58

Table 4 . Transmission of PVY0 and PVY N from singly and doubly infected potato cv. King Edward plants by manual inoculation of sap diluted to 10 .

Number o f tobacco Source of virus Virus strain transmitted plants infected

PVY° PVY0 31/ 120(' 0, 531i 0 ,m ) + pvyn pvyn 55/ 120(" 0#084i 0,150) PVY° 38/i20(-«>.387±O.16l) PVY° + PVYN ** pvyn 30/ 120("0,553^0, 173)

+ Numbers in parentheses are mean logits + S.E. ** Different from rate for singly infected plants at P ^ 0.01 59

Plate 12, Tobacco plants showing veinal necrosis (right) or slight mosaic (left) 2 weeks after inoculation with PVY^ and incubation at 15°C and 30°G respectively.

Plate 13, Leaves of tobacco plants inoculated with PVY N and incubated after inoculation at 15°C (right) or at 30°G (left) showing veinal necrosis or slight mosaic respectively. 60 developed veinal necrosis two weeks after inoculation.

Rates of sap transmission of PVY^ was similar from singly and doubly infected plants grown at 15°C and 30°C (Table 5 ). However, transmission of PVYN was less from doubly infected plants grown at either temperature, especially at 30 o C, when no transmission occurred.

D iscussion Rates of Transmission of PVY°from singly and doubly infected tobacco and potato plants were similar suggesting that the concentrat- ion of PVY 0 was unaffected by the presence of PVY N in either host. However, transmission of PVY N was less from doubly than from singly infected tobacco and potato plants, confirming previous results (Richardson, 1958 ) and suggesting that the concentration of PVY N was depressed by the presence of PVY0 • Transmission of PVY N was especially depressed at 30o C, no transmission of PVY N being reported although tests demonstrated that the source plants were infected. This was not a direct effect of temperature as in the absence of PVY 0 , PVY N was abundant in plants kept at 30°C; at 30°C, perhaps PVY^ occupied most sites of virus multiplication. The result that less frequent transmission of PVY N by aphids from doubly infected plants was paralleled by less frequent sap transmiss ion, incidentally confirms that transmission of PVY by M. persicae is correlated with concentration (Bradley, 1962 ; De Bokx et al., 1978 ) and suggests that the less frequent transmission of PVY N from doubly infected plants by li. persicae was neither because it preferred to probe at sites where PVY 0 was localized nor that PVY 0 was more readily available to aphids when both strains were present in the same plant (Richardson, 1958 ). Table 5 Sap transmission of PVY0 and PVY N from singly and doubly infected tobacco cv. White Burley plants kept at different temperatures

Temperature Virus source PVY° pvyn PVY° + pvyn Virus strain transmitted PVY° pvyn PVY° pvyn k 15°C 58/192 + 64/192 58/192 44/192 (-0.428+0.079) (-0.355f0.077) (-0.428+0.079) (-0.620+0.087) 30°C 62/192 35/192 78/192 0 /192 (-0.445P0.172) (-0.753+0.202) (-0.191+0.159)

+ Numbers in parentheses are mean logits + S.E. Different from rate for singly infected plants at the same temperature at P ( 0.05. 62

The ability of M. persicae to transmit PVY 0 and PVY N simultaneously has also been demonstrated in these experiments. In line with these results, Schroeder et al. (1959) found that A. pisum could transmit a combination of two nonpersistently-transmitted virus (RCVMV + BYMV). However, individual M. Persicae transmitted only one strain of CMV or AMV from plants infected with two strains (Castillo & Orlab, 1966 ). In the case of AMV, a low concentration of one strain in the doubly infected plants might explain this. In conclusion, the isolate of PVY° used suppressed multiplication of PVY N in both tobacco and potato. The direct practical implications of this are not clear even if other isolates of PVY 0 and PVY N behave similarly, as having enough PVY°-infected plants in a crop to control PVY would itself be a disaster. However, a knowledge of the mechanism(s) by which PVY 0 diminishes the concentration of PVY N might suggest novel means of control 63

5. TRANSMISSION OF PVY° AND PVYN FROM DOUBLY INFECTED PLANTS

5.1 Acquisition of PVY 0 and PVY N by M. persicae after a single probe on a doubly infected tobacco leaf.

Introduction In the previous experiment (p. 56 ), about 407. of M. persicae

that transmitted when allowed 2\ min access to a doubly infected tobacco leaf, transmitted both PVY 0 and PVY N • It is. unclear whether closely- related strains of the same virus co-exist in the same cell (Benda, 1956 ; Siegel, 1959 ; Cassells & Herrick, 1977 ) and acquisition of both strains by a single aphid could have resulted from two or more probes, one in a region containing PVY0 and another in a region containing PVY N , from a probe between cells one containing PVY 0 and the other PVY N , or from a deep probe penetrating several cells. Alternatively, a single tobacco cell in a doubly infected plant may contain both PVY 0 and PVY N • To investigate this the following experiment was carried out.

Materials and Methods Tobacco plants cv. White Burley were inoculated with PVY^ 4* PVY N ; the inoculum consisted of a mixture of 0.1 g of leaf infected with PVY 0 and 0.1 g of leaf infected with PVY N ground up in 5 ml of phosphate buffer. Two weeks after inoculation, non-inoculated leaves of these plants were used as virus sources. M. persicae observed individually under a binocular micro­ scope were allowed a single probe into an infected leaf. The durat­ ion of this probe was recorded. A probe was considered to have begun 64

as soon as the rostrum came in contact with the leaf surface (Swenson, 1967 )• Aphids were then confined individually on tobacco test plants. Two weeks later the numbers of plants with symptoms of PVY^ and PVY N were recorded. Tobacco plants showing veinal nocrosis were tested for double infections as described previously (p.55 )• Twenty- four aphids were used for each replicate and the experiment was replicated nine times.

R esults Several M. persicae transmitted both strains following a

single acquisition probe (Table 6 )• Probes resulting in transmission of PVY°, PVYN and PVY° + PVYN or no transmission were of similar duration and one 9 s probe resulted in transmission of both strains whereas one 120 s probe resulted in transmission of only one strain.

D iscussion As mentioned in the Literature Review, aphid stylets penetrate only a few ( (. ten) micrometers into the epidermis during brief probes and this distance is less than the depth of an epidermal cell (Bradley, 1964 ; Nault & Gyrisco, 1966 ; Misawa & Hashiba, 1967 )• In my experiment, there was no indication that probes acquiring both strains were of longer duration, and perhaps therefore deeper than probes acquiring only a single strain and one 9 s probe resulted in transmission of both strains. Whether these brief probes are made into a cell or between cells is unclear (Pirone & Harris, 1977 ). Stylet insertion has been reported to be intercellular (Yoshii, 1966 ), intracellular (Swenson, 1962) or a mixture of the two (Roberts, 1940 ; Bradley, 1952 ; Hashiba, 1969 ). So, aphids could have acquired both 0 N Table 6 . Numbers of plants infected with PVY and/or PVY by M. perslcae allowed a single probe into doubly infected tobacco cv. White Burley

S train (s) transmitted PVY° pvyn PVY° + PVYN No transmission

68/240 21/240 38 / 240 113/240

Mean duration of probes (s) 22.4+3.42 25.8+5.15 19.39+2.44 25.07+2.38 66 strains from a single cell or by probing between cells each infected with a different strain. However, if acquisition occurred between cells, seemingly inefficient mechanisms, such as acquisition from broken plasmodesmata (Yoshii, 1966 ), must be postulated. Alter­ n ativ ely , M. persicae acquired the two strains of PVY from a single cell infected with both strains. This is in line with recent evidence that related strains of viruses can co-exist in the same cell (Hull & Plaskitt, 1970 ; Dawson et al., 1975 ; Otsuki & Takebe, 1976a, 1976b;

C assels & Herrick, 1977 ; Barker & Harrison, 1978 ). If so, one can also ask how some aphids acquired only a single strain or failed to acquire. It could be because many of the cells are infected with only a single strain or one uninfected. Alternatively all epidermal cells were infected with both strains but aphid probing into them often failed to acquire and transmit- any virus, or transmitted only one or a few particles. The evidence presented here cannot distinguish between these hypotheses. Attempts, in collaboration with Biochemistry Department to test whether or not all cells were infected with both virus strains by growing plants from isolated protoplasts from doubly infected tobacco have been unsuccessful so far because plants have not^regenerated from protoplasts fjpertnoSr infected plants,iy \ it . u ^

5.2 Acquisition of PVY 0 and PVY N by M. persicae, M. euphorbiae and R. padi from doubly infected potato plants.

Introduction Although it is well-documented that different aphid species or clones of a species may differ considerably in their ability to transmit a particular nonpersistent virus (p. 36 ), the cause(s) of 67 these differences is not fully understood. In the previous exper­ iment, H. persicae, an efficient vector of PVY often transmitted two strains of PVY simultaneously. The following experiment was carried out to test whether less efficient vectors of PVY such as M, euphorbiae and R, padi can transfer sufficient virus to transmit two strains simultaneously.

Materials and Methods Potato cv. King Edward infected with both PVY 0 and PVY N were used as virus source. The same source leaf was used for all species in each replicate, M, persicae and M, euphorbiae were given 2h min access to infected leaves and then caged singly on tobacco cv. White Burley. R, padi were given 10 min access and batches of 20 were then caged on each tobacco test plant. From the number of tobacco plants R, padi infected, an estimate was made of the per­ centage of plants that would have become infected had single aphids been used (i.e. the percentage of infective aphids) (Gibbs & Gower, 1960 ). Twenty-four tobacco test plants were used for both M, euphorbiae and R, padi, whereas twelve were used for M. persicae. Tobacco test plants developing veinal necrosis were tested for the additional presence of PVY° as before (p. 55). The experiment was replicated eighteen times. Two mathematical models were tested* to see if they fitted the transmission data obtained in these experiments . The models were:

+ R.W. Payne, Statistics Department, Rothamsted. 68

Model 1 Aphid species Strain transmitted PVY° pvyn PVY+PVY0 N M. persicae + pi P2 P3 M. euphorbiae P4P1 P4P2 P4P3 R. padi P5P1 P5P2 P5P3 Model 2 Aphid species Strain. transmitted PVY° PVY PVY U +PVY N M. persicae P lU -P 2) P2U -P l) P1P2 M. euphorbiae P3U -P 4) p4

R esults Calculations from data in Table 7 show that, on a single

aphid basis, 68 . 07., 7. 57. and 2.0Z of M. persicae, M. euphorbiae and R. padi respectively transmitted PVY. Of those that transmitted, 24.07., 4.07. and 1.07. of H. persicae, H, euphorbiae and R, padi

(batches of 20) respectively transmitted both strains simultaneously. Transmission data of PVY^, PVYN and PVY^ + PVYN by the different

species were significantly different from those predicted by model 1

(Table 8 ) (P ^0.001) but not from those predicted by model 2 (P > 0.05).

D iscussion The above results confirm that M. euphorbiae and R. padi both transmit PVY less frequently than M. persicae (Van Hoof, 1980). This might be either because a few individuals in the populations of M. euphorbiae and R. padi are capable of acting as vectors of PVY and the rest are non-vectors (Bawden & Kassanis, 1947) or alternatively all individuals in the population are equally inefficient as vectors of PVY. In the first case, where the populations of M. euphorbiae and R. padi each consist of a mixture of a small proportion of efficient vectors and a high proportion of non-vectors, the efficient vectors will transmitt PVY 0 + PVY N at rates similar to M. persicae and model 1 should describe the results. On the other hand, if all individuals in the population are equally inefficient as vectors of PVY and transmission of PVY 0 is independent of transmission of PVY N then the probability of a single aphid transmitting both PVY 0 + PVY N will be the probability of any one aphid in the whole population transmitting PVY° X the probability of any one aphid in the whole Table 7 . Transmission of PVY° and PVY*1 from doubly infected potato cv« King Edward by different aphid species

Expected transmission strain(s) transmitted of PVY°+ PVYn by model Aphid species PVY° pvyn PVY 0 + PVY N i+ **»% *4- in 00 M. persicae o “ 'w o 30'l8 0 30/180 32/180

M. euphorblae 8 ^ 6 0 18/360 L/360 8 /360 °- 5 /360

R. pad! 42/360 46 L/360 28/360 5*5/360

0 N O N 4- Ratio of transmission of PVY and PVY : PVY + PVY remains as for M. persicae -H- Probability of simultaneous transmission equals probability of transmitting PVY° X probability of transmitting PVYN, assuming independent transmission of the two stra in s . 71

Table 8 • The analysis of variance of numbers of plants infected with PVY0 , PVT "n or PVY o + PVY N by M. persicae, M. euphorbiae and R« padi

Source of variance d .f. S.S. M.S. F P

Model 1 166 392.1459 12.565 5.95 < 0.001

Model 2 165 350.2719 2.7954 1.32 NS

R esidual 162 341.8857 2.1104 72 population transmitting PVYN • This situation is described by model 2* Transmission data for the three species did not differ signif­ icantly from model 2. This suggests that all individuals of M. euphorbiae and R. padi are similarly inefficient vectors and their inability to frequently cause double infections may be because they seldom inoculate sufficient virus particles to be likely to simul­ taneously inoculate both strains. Extrapolating from this, their inability to frequently transmit even one virus strain could also be caused by their inability to inoculate frequently sufficient virus particles to the test plants. This is in accord with previous evidence that inefficient vectors acquire less virus. Thus, PVY was detected by enzyme linked immunosorbent assay (ELISA) only in M. persicae, an efficient vector of PVY, whereas attempts to detect it in three other species, less efficient than M. persicae, were unsuccessful1 (Carlebach et al.# 1982 ). Similarly, Lim et al. (1977) observed large amounts of labelled virus on the inner surface of mandibles of an aphid bio­ type of M. euphorbiae that transmitted PSbMV efficiently whereas only a small amount was observed on the mandibles of a bio type that transmitted PSbMV inefficiently. Aphid transmission is probably more efficient than sap transmission in terms of number of virus particles needed to produce an infection site (Bawden, 1950 ; Walker & Pirone, 1972 ). It has been calculated that the mouthparts of M. persicae can contain about 1000 CMV particles (Walker & Pirone, 1972 ) and 100 tobacco severe etch virus (TSEV) particles (Taylor & Robertson, 1974 ). However, M. persicae is capable of causing more than one infection with TSEV, a potyvirus, with the virus charge obtained from a single acquisition 73 probe (Taylor & Robertson, 1974 )v so the actual number of virus poi-ct inoculated by a single probe is presumably less* These results suggest that inefficient vectors such as R. padi and M. euphorbiae inoculate even fewer virus particles.

5.3 Transmission of PVY N by M. persicae and M. euphorbiae that initially fail to transmit

Introduction Rates of transmission of nonpersistent viruses by single aphids of any species seldom exceed 807. (Watson, 1938 ; Sylvester, 1949 ; Bradley & Rideout, 1953 ; Simons, 1958 ) even when aphids are known to have probed on both source and test plants (Sylvester, 1952 ; Bradley & Rideout,1953 )• The following experiment investigated this further by testing whether aphids which initially do not transmit are efficient vectors when retested few hours later.

Materials and Methods

Both M. persicae and M. euphorbiae were given 2h min access to a PVYN -infected tobacco leaf and were then caged singly on tobacco cv. White Burley test plants. After 5 h the aphids were removed from each test plant and kept singly in glass vials for 2-3 h, after which each aphid was given a further 2*s min access to a PVY N -infected leaf and transferred singly to tobacco test plants. The 7-8 h between the first and second access were to ensure that aphids did not carry virus from the previous test (retention of PVY in both species is less than 8 h) (Proeseler & Weidling, 1975 ). In each test, twenty- four aphids of each species were used and the experiment was replicated nine times. 74

R esults As indicated in Table 9 47.57* of the M* persicae that did not transmit in the first test did transmit in the second test and this did not differ significantly (P > 0.05) from the 44.07. of aphids that transmitted in both tests. Similarly 12.07. of the M. euphorbiae that did not transmit in the first test did transmit in the second, again not significantly (P > 0.05) different from the 21.57. that transmitted in both tests.

D iscussion The results of these experiments show that both M. persicae and M. euphorbiae which failed to transmit PVY** during a first transmission test readily did so when retested a few hours later. Absence of transmission during the first test may therefore be due to failure of these aphids either to acquire the virus or to transmit it but was unlikely to be because the aphids were incapable of being vectors. Failure to acquire the virus might be because the aphids probe into virus-free regions. For example, intercellular probes may seldom acquire virus (Pirone & Harris, 1977 ) and some epidermal cells contain no virus (Hashiba, 1970 ; Hashiba & Misawa, 1970 ). However, a probe by a viruliferous aphid does not necessarily result in virus infection as shown by sequential testing of a viru­ liferous aphid on a series of test plants (Sylvester, 1955 ; Garrett, 1971 ; Taylor & Robertson, 1974 ) and aphids may not always introduce virus in a site suitable for establishment. 75

Table 9 . Transmission of PVY N by aphids that either transmitted (A) or failed to transmit (B) in the previous test

Aphid species A+ B-H-

53^120 57/120 M* persicae (-0.169J0.075) (-0.043J0.075)

6 /28 25/212 M. euphorbiae (-1.1704-0. 272) (-1.428+0.126)

+ nominator: number of aphids that transmitted both in the first and second test*denominator: number of aphids transmitted in the first test. ++ nominator: number of aphids that transmitted only in the second test* denominator: number of aphids that did not transmit in the first test* 76

5.4 Effect of oil on acquisition of PVY^ and PVY^ from doubly infected tobacco

Introduction Inhibitory effects of oil on aphid transmission of PVY were first reported in 1962 by Bradley et al. and this has since been confirmed for many other nonpersistent viruses (Vanderveken, 1977 ; Simons & Zitter, 1980 ; Simons, 1982 )• Despite 20 years having passed, the mechanism by which oil prevents aphids transmitting is still not understood. Bradley (1963) suggested that oil may either remove virus particles from the stylets or cause virus particles to adhere firmly to the stylets. Since then, Loebenstein & Raccah (1980) have reported that CMV content in A* gossypii given access to oil-treated leaves, as determined by ELISA, was less than that of aphids given access to untreated leaves, and they suggested that oil prevents virus attachment to the stylets. If so, oil treatment of leaves infected with both PVY 0 and PVY N would prevent aphids acquiring much virus, perhaps insufficient to inoculate two virus strains at a time. The following experiment investigated this.

Materials and Methods A mixture (0.1 g of each strain diluted in 5 ml of phosphate buffer pH 7.0) of the virus strains PVY 0 and PVY N was used to sap- inoculate tobacco plants. Leaves were detached from these plants, two weeks after inoculation, and cut along the midribs to fo'rm two half-leaves. JMS stylet-oil (Simons & Zitter, 1980 ) which contains an emulsifier was blended with water for 1 min in a Sorral omnimixer at full speed. Oil was brushed over the upper surface of one half­ 77

leaf with a paint-brush and the other half was untreated (control). The leaves were used as sources either as soon as they dried off (about 30 min after oil application) (first experiment) or 24-36 h later (second experiment). In the first experiment, oil was used at 0.27. and in the second 0.17. M. persicae observed individually under a binocular micro­ scope were allowed a single probe into either a treated or untreated half-leaf. Twenty-four aphids were allowed a single probe into oil- treated leaves and eight into an untreated one (control) for each replicate. The aphids were then confined individually on tobacco test plants. The numbers of test plants which were not infected, showed a mosaic symptom (PVY ) or veinal necrosis (PVY or PVY + PVY ) were observed two weeks later. Plants showing veinal necrosis were tested for double infections as described previously (p. 55 ). The first experiment was replicated 18 times whereas the second one was rep licated 69 tim es. -J- Three different mathematical models were tested to see if they fitted the transmission data obtained in those experiments. The models were: Treatment Strain(s) transmitted PVY° pvyn pvy0+pvy'

Model 1 non-oil P1 P2 P3 o il P4P1 P4P2 P4P3

Model 2 non-oil P l( l'P 2) P 2U -P l> P 1P2 o il pl p3(1-p2p3> P2P3(1'P1P3) P1P3P2P3

Model 3 non-oil pl P2 P3 o il p4 P5 P6

4- R.W. Payne, Statistics Department, Rothamsted. 78

Model 1 assumes that the probabilities of transmission of PVY°, PVYN and PVY° + PVY^ are p^, p^ and respectively, and oil modifies these probabilities uniformly by a factor p^. In model 2 it is assumed that transmission of PVY 0 and PVY N occur independently of each other. Therefore the probabilities of PVY0, PVY N and PVY O + NPVY , derived by the binomial theorem, are p^(l-p2), p2(l-p^) and PjJ?2 respectively. Oil is assumed to modify the probabilities p^ and p^ by the same factor p^ and so the probability of both PVY 0 and PVY N being transmitted is modified by (p^) 2 . In model 3, different probabilities are fitted for PVY O , PVY N and PVY O + NPVY for both the oil-treated and untreated situat­ ion and provides the basic model against which model 1 and 2 were tested .

R esults Fewer than 107. of aphids given access to oil-treated leaves transmitting PVY compared to 507. of those given access to untreated leaves (Table 10, 12). Transmission data from the first experiment were significantly different from those predicted by model 2 (Table 11) (P ^0.0Bj)but did not differ from model 1 (Table 11 ) (P > 0.05) whereas results from the second experiment differred significantly (Table 13) (P { 0.05) from both models tested, being intermediate between model 1 and model 2.

D iscussion As expected (Vanderveken, 1977 ; Simons & Zitter, 1980 ; Simons, 1982 ) aphids allowed access to oil-treated leaves seldom 79

Table 10. Transmission of PVY 0 and PVY N from oil-treated and untreated doubly infected tobacco by M. persicae allowed a single probe 30 mins after oil treatment

Strain(s) transmitted Treatm ent No PVY° pvyn pvy°+pvyn transmission

Untreated (co ntro l) 46/160 18/160 26/160 70/160

Oil-treated 19/480 9/480 8/480 444/480 80

Table 11. Statistical analysis of the "fit* of each model to transm ission o f PVY0, PVYN and PVY° + PVYN 30 mins after oil treatment

Source of variance d.f. S.S. M.S. F P

Model 1 116 212.4946 0.3936 0.212 NS

Model 2 117 244.4656 10.919 5.87 ( 0.001

Residual 114 211.7074 1.8571 81

Table 12. Transmission of PVY 0 and PVY N from oil-treated and untreated doubly infected tobacco by M. persicae allowed a single probe 24-36 h after oil treatment

Strain(s) transmitted Treatm ent No PVY° pvyn PVY 0 + PVY N transmission

U ntreated (co ntro l) 10° / 568 39/568 59^568 370/568

3il-treated l9 /1704 5/1704 2/1704 l678/1704 82

Table 13. Statistical analysis of the 'fit* of each model to transm ission of PVY°» PVY^ and PVY^ + PVY^ 24-36 h after oil treatment

Source of variance d.f• S.S. M.S. F P

Model 1 422 600.0083 4.6493 3.30 ^0.05

Model 2 423 633.7837 14.538 io.209 <; 0.001

Residual 420 590.7097 1.4064 83

transmitted PVY, The average content of CMV in A. gossypii allowed access to oil-treated leaves and which gave transmission rates similar to those obtained in my experiments was less than that of aphids hav­ ing access to untreated leaves (Loebenstein <& Raccah, 1980)* How­ ever, this does not distinguish whether this is because (a) all aphids having access to oil-treated leaves acquired less virus or because (b) the few that transmitted acquired normal amounts of virus and the majority, that did not transmit, acquired less or no v iru s. If all aphids acquired less virus, the chance of any one aphid carrying enough virus to carry both PVY 0 and PVY N might be

diminished as described by model 2 whereas if the few that transmitted all acquired normal amounts of virus, the proportion of transmitting both PVY 0 and PVY N would remain constant as described by model 1. Results from the first experiment, in which aphids probed leaves about 30 min after oil-application, were not significantly different from those predicted by model 1 , indicating that oil diminished transmission of PVY O , PVY N and PVY O + NPVY uniformly. Scanning electron microscopy has shown that it takes 24-36 h for oil to spread to a film over a leaf (Kulps, 1969 ) and aphids which did not transmit in this experiment may have been those probing through oil droplets, the few which transmitted being those probing between droplets. If so, the amount of virus acquired by these few aphids would not change, explaining how the proportion transmitting PVY 0 + PVY N apparently remained constant. 84

When leaves with the same concentration of oil as in the first experiment were used as virus sources 24-36 h after applicat­ ion, none out of 480 test plants was infected (unpublished prelim­ inary results) confirming that oil had spread more evenly after 24-36 h. For this reason, in order to get some transmission from leaves 24-36 h after treatment, oil concentration was halved in the second experiment* Results of this second experiment did net fit either of the two models tested but were intermediate between them. This suggests that oil did reduce transmission of PVY 0 + PVY N preferentially but not as much as predicted by model 2. It is possible that this was caused by a combination of some of the aphids which transmitted being those whbh probed between oil droplets therefore acquiring undiminished amounts of virus as may have occurred in the first experiment and same being those which probed through the oil, which 24 h later was now a film thin enough to allow some acquisition. 85

6. EXPERIMENTS TESTING FOR INTERFERENCE BETWEEN VIRUSES OR VIRUS STRAINS DURING APHID TRANSMISSION

0 N 6.1 Experiments testing for interference between PVY and PVY during sequential transmission by M. persicae

Introduction When an aphid or leafhopper acquires two viruses either in sequence or simultaneously, the transmission of either virus may be enhanced, unaffected or diminished by the presence of the other. The term "interference" has been ascribed to the last situation (Gildow & Rochow, 1980 ). There was no interference between isolates of beet curly-top (BCT) virus in leafhoppers (Bennett, 1967 ) and .none s y n o n y w A i between potato leaf ro lL virus (PLRV) and turnip la te n t virus (TLVV when loQjQX, OPSTCrx* aphids acquired the viruses either in sequence or simultaneously (MacKinnon, 1960 ). Likewise, there was no interference between beet yellow virus (BYV) and beet yellow net virus (BYNV) (Sylvester, 1956 ) or between mild and severe isolates of PLRV (Harrison, 1958 ). However, interference between two isolates of barley yellow dwarf virus (BYDV) in Macrosiphum a venae (Fabr.) has been reported (Gildow & Rochow, 1980 ; Rochow et al., 1983 ). The above reports relate mainly to persistent viruses, and little is known about interactions between strains or viruses transmitted nonpersistently except when one relies on the other for helper component. Transmission of one strain of CMV or AMV by M, persicae was independent of a second strain (Castillo & Orlob, 1966 ) whereas interference occurred between CMV and PVY (Garrett, 1971 )• This work describes experiments testing whether PVY 0 and PVY N interact during transmission by M, persicae. 86

Materials and Methods

PVY 0 and PVY N were sap-inoculated to separate plants of Nicotina tabacum cv. White Burley, Tobacco leaves of similar size were detached for use as source leaves 12-15 days after inoculation.

Batches of 24 M. persicae were given 2h min access to two source leaves in one of six sequences and then were caged singly on tobacco cv. White Burley. The sequences were: Sequence 1 PVY N -infected leaf first then PVY 0 -infected Sequence 2 Healthy leaf first then PVY^-infected Sequence 3 PVY N-infected leaf first then healthy leaf Sequence 4 PVY 0 -infected leaf first then PVY N -infected Sequence 5 PVY^-infected leaf first then healthy leaf N Sequence 6 Healthy leaf first then PVY -infected The experiment was replicated six times. Tobacco plants showing veinal necrosis were tested for double infections as described prev­ iously (p.55 ). R esults Fewer aphids transmitted PVY0 if they had access to a PVY N - infected leaf either before or after acquisition of PVY^, than if they had probed on a healthy leaf (Table 14) (P ^ 0.001). Thus 547. of test seedlings were infected with PVY^ when aphids first probed a healthy leaf whereas only 337. were infected when aphids first probed a PVY N - infected leaf. When aphids probed first on a PVY^-infected leaf and then on a healthy or a PVY N -infected one, 557. and 377. respectively transmitted PVY 0 (Table 14). In contrast, transmission of PVY N was unaffected by whether or not an aphid probed a PVY^-infected leaf (Table 15) (p > 0.05). 87

Table 14. Numbers of plants Infected with PVY by viruliferous aphids also probing a tobacco leaf with or without PVYN

Treatment sequence (page 86).

Sequence 1: m N f i r s t 56/t ^ ( - ° . 303t 0- 073) 118/336( 0.086+0.051) Sequence 4: pvyn second 62/l6Q (-0* 358+0.07 2)

Sequence 2: Healthy first 90/168( °*099±°*069) L82/336(“0*33°M)*046) Sequence 5: Healthy second 92/l68( 0.074+0.069)

Tobacco first 146/336(-0.103+0.049) (PVYN or healthy)

Tobacco second 154/336(-0.142+0.049) (PVYn or healthy) }

*** Difference significant from appropriate control at P ( 0.001 + Numbers in parentheses are mean logits + S.E. 88

Table 15. Numbers of plants infected with PVY by viruliferous aphids also probing a tobacco leaf with or without PVY*

Treatment sequence (page 86).

Sequence 4: PVY° first 92/168(0.097+0.058 )+' 181/336(0#079i 0#040) Sequence 1: PVY° second 89/168(0'061±0#058)

Sequence 6s Healthy first 90/168^0'012i 0#058) 185/336(0*042£0#040) Sequence 3: Healthy second 85/168(0#073-°'057) '

Tobacco firs t 18 2/336(0.055+0.040) (PVY° or control)

Tobacco second 174/338(0.067+0.040) (PVY° or control)

+ Numbers in parentheses are mean logits + S.E, 89

0 N 6,2 Experiments testing for interference between BMV, PVY and PVY during sequential transmission by M. persicae

Introduction

In the previous experiments, PVY N inhibited transmission of PVY 0 • The following experiments tested whether another potyvirus, BMV, could affect transmission of PVYO and NPVY , and vice-versa. PVY O and NPVY are serologically closely related (Bawden & Kassanis, 1951 ; Bartels, 1957 ; De Bokx et al. , 1975 ; Makkouk & Gurapf, 1976 ) whereas BMV is serologically distantly related (Bercks, 1960 ; Fujisawa et al., 1983).

Materials and Methods

N, tabacum cv. White Burley were sap-inoculated either with PVY 0 or with PVY N and leaves of similar size were detached for use as virus sources, 12-15 days later. Infected sugarbeet cv. Hilleshog Monotri were used as sources of BMV, 2-4 weeks after inoculation. H. persicae were given min access to two source leaves in one of ten sequences. The sequences were: Sequence 1: BMV-infected leaf first then PVY°-infected Sequence 2: Healthy sugarbeet leaf first then PVY°-infected Sequence 3: BMV-infected leaf first then healthy tobacco Sequence 4: PVY^-infected leaf first then BMV-infected Sequence 5: Healthy tobacco leaf first then BMV-infected

Sequence 6 : PVY^-infected leaf first then healthy sugarbeet Sequence 7: BMV-infected leaf first then PVY N -infected N Sequence 8 : Healthy sugarbeet leaf first then PVY -infected Sequence 9: PVY N -infected leaf first then BMV-infected Sequence 10:PVY N -infected leaf first then healthy sugarbeet 90

Batches of 24 aphids were used in sequences 1, 4, 7 and 9 of which 12 were singly transferred to sugarbeet and 12 to tobacco* Batches of 12 were used in the other sequences. The experiment was replicated twelve times. To test whether PVY0 and PVY N infect sugar- beet and whether BMV can infect tobacco, 10 sugarbeet seedlings were sap-inoculated with PVY 0 and 10 with PVY N , and 10 tobacco seedlings were sap-inoculated with BMV. As controls tobacco plants were sap- inoculated with PVY° and PVYN, and sugarbeet with BMV.

R esults

Fewer aphids transmitted PVY° if they had access to a BMV- infected leaf, either before or after acquisition of PVY0, then if they had probed on a healthy leaf (Table 16) (P ^ 0.001). Thus, 45% of test seedlings were infected with PVY° when aphids probed first on healthy whereas only 257. were infected when aphids first probed on a BMV-infected leaf (P ^ 0.001). Similarly, 477. of test seedlings were infected with PVY° when aphids probed first on a PVY°-infected leaf, and then on a healthy whereas 357. were infected when aphids probed first on a PVY°-infected leaf and then a BMV-infected one (P ^ 0.05). In contrast transmission of PVY N seemed unaffected by whether or not an aphid probed a BMV-infected leaf (Table 17) (P y 0.05). Transmission of BMV was unaffected by whether or not an aphid probed a PVY 0 -infected leaf but it was less if aphids probed a PVY N - infected leaft (Table 18) (P ( 0.05). Thus, 277. of test seedlings were infected with BMV when aphids probed first on healthy compared with 177. when aphids probed first on a BMV-infected leaf. When aphids probed first on a BMV-infected leaf and then on a healthy or a PVY N - 91

Table 16. Numbeisof plants infected with PVY° by viruliferous aphids also probing a sugarbeet leaf with or without BMV.

Treatment sequence (page 89)

Sequence Is BMV f i r s t 39/l56(-°«59 3+0.089)

93/312("0,469£0#059) Sequence 4s BMV second 5 4156 /,*(-0.352+0.081)

Sequence 2: Healthy f i r s t 71/156(“0,111-0,077) 145/312("0,091t°*055) Sequence 6: Healthy second 74/,e,(156 0.070+0.077)

Sugarbeet first 110/312(-0.346+0.057) (BMV or healthy)

Sugarbeet second 128/^2(-0. 213+0.055) (BMV or healthy)

Difference significant from appropriate control * = P ^ 005; *** * P < 0.001 + Numbers in parentheses are mean logits + S.E. 92

Table 17. Numbers of plants infected with PVY N by viruliferous aphids also probing a sugarbeet leaf with or without BMV.

Treatment sequence (page 89)

Sequence 7: BMV first 59/156(“0#260t°-09°) 102/312(“0#378t 0,066) Sequence 9: BMV second 43/l56(-0.504^0.098)

Sequence 8 : Healthy f ir s t 52/,I JO (-0.362+0.093) 104/312(“0#363^0,065) Sequence 10:Healthy second 52/156(“0,362-0#093)

Sugarbeet f i r s t 111/312("0#310i°* 06A) (BMV or control) $

Sugarbeet ") f second 95/312(“0,43L-°*067) (BMV or control) J

+ Numbers in parentheses are mean logits + S.E, 93

Table 18# Numbers of plants infected with BMV by viruliferous aphids also probing a tobacco leaf with or without PVY 0 or PVY N.

Treatment sequence (page 89)

i Sequence 4: PVYPVY° f i r s t 61/l^ 8(-0. 229^0. 103) -> l* 104/ 31t (" 0' 359t 0' 075) Sequence Is PVYPVY° second 43/l56(-0.500^0.112) -

Sequence 9: PVYpvyn f i r s t 27/156(“0,807-0#133) 1 > 63/312(-0.710+0.088) Sequence 7: PVYpvyn second 36/156("0*623t0' U9) ^ Healthy f i r s t 42/156(-0.517+0.113) I > 94/312(-0.435^0.077) Healthy second 52/156(“0#359^0#107)

Tobacco first 130/468(-0.504+0.064) (PVY or healthy)

Tobacco second 131/48 q(-0.498+0.064) (PVY or healthy)

* Difference from appropriate control significant at P ( 0.05. + Numbers in parentheses are mean logits + S.E. 94

infected one, 337. and 237. respectively transmitted BMV. No symptoms developed on sugarbeet seedlings inoculated either with PVY 0 or PVY N , and back inoculations to tobacco indicated that they were healthy. Similarly, tobacco plants showed no symptoms after inoculation with BMV and back inoculations to sugarbeet indicat­ ed they were healthy. These results are consistent with those of Bennett (1949).

6.3 Experiments testing the ability of BMV from N. clevelandii and sugarbeet to inhibit transmission of PVYU during sequential transmission by M. persicae.

Introduction In the previous experiment, aphids which had been given access to a BMV-infected sugarbeet leaf transmitted PVY° less frequently than aphids given access to a healthy leaf. Preliminary electron- microscopical observations suggested that N. clevelandii, a host plant of BMV (Bennett, 1949 ; Hoilings, 1959b), contained more BMV than sugarbeet and the following experiment was done to test whether such a source was more inhibitory.

Materials and Methods N. tabacum cv. White Burley used 12-15 days after sap inoculat­ ion with PVY°. Sugarbeet cv. Hilleshog Monotri and N. clevelandii were used 2-4 weeks after sap-inoculation with BMV. H. persicae were given 2% min access to each of two source leaves in one of six sequences. The sequences were: 95

Sequence Is PVY°-infected tobacco leaf first then BMV-infected sugarbeet Sequence 2s PVY°-infected tobacco leaf first then healthy sugarbeet Sequence 3: Healthy tobacco leaf first then BMV-infected sugarbeet Sequence 4s PVY^-infected tobacco leaf first then BMV-infected N. clev elan dii Sequence 5s PVY^-infected tobacco leaf first then healthy N. clevelandii

Sequence 6s Healthy tobacco leaf first then BMV-infected N. clevelandii Batches of 4S M. persicae were used in sequences 1 and 4, of which 24 were caged singly on sugarbeet cv. Hilleshog Monotri and 24 on tobacco cv. White Burley. Batches of 24 were used in the other sequences and aphids were transferred to either sugarbeet (sequences

3 & 6) or tobacco (sequences 2 & 5). The experiment was replicated eleven times. The concentration of BMV in these source leaves was estimated by the following two methods. Local lesion assay. Chenopodium amaranticolor gives local lesions when sap-inoculated with BMV (Hoilings, 1959a; Beiss, 1963 ) and in each replicate eight plants were pruned to four leaves to give four * units' per plant. Plants were kept in the dark 24 h before use to increase susceptibility (Bawden & Roberts, 1948 ). Four inocula were prepared from leaves ground in a pestle and mortar with 0.01 M phosphate buffer pH 7.0. (A) 0.1 g BMV-infected N. clevelandii + 0.1 g healthy sugarbeet in 9.8 ml phosphate buffer pH 7.0 (dilution 1 /^ q0^# (B) 1 ml of A in 2 ml phosphate buffer (dilution (C) 1 ml of B in 2 ml phosphate buffer (dilution I/^ qq)* (D) 0.1 g BMV-infected sugarbeet + 0.1 g healthy N. clevelandii in 9.8 ml phosphate buffer (dilution 96

These were inoculated to carborundum-dusted leaves, each inoculum being applied once on each plant and once at each leaf position in each group of 4 plants. Lesions were counted 8 to 10 days after inoculation. Counting virus particles using electron microscopy 0.1 g of N. clevelandii or sugarbeet were ground up in 1 ml of distilled water. This was negatively stained with one drop of 27. P.T.A. (potassium phosphotungstate) and sprayed from an air-brush onto filmed grids (Hall, 1964 ). The number of particles that could be counted in 1 min was recorded (Gibson & Heard, 1979 ). Statistical analyses. The relative concentration of BMV in N. clevelandii and sugarbeet plants was estimated from the lesion counts by a statistical method described by Kleczkowski (1968). Aphid transmission results were logit transformed and analysed by generalized linear models (Nelder & Wedderburn, 1972 ; Gibson et al., 1982 )•

R esults When aphids probed a BMV-infected sugarbeet or N. clevelandii leaf, transmission of PVY° was decreased by 357. (Table 19) (P ^ 0.01) and 267. (Table 19) (P ^ 0.05) respectively. In accord with previous results (p.90), transmission of BMV, either from N. clevelandii or from sugarbeet, seemed unaffected by whether or not an aphid had probed a PVY°-infected leaf (Table 20). BMV was transmitted more frequently from sugarbeet than from N. clevelandii (Table 20) (P ^ 0.05). However, concentration of BMV, as measured by both methods, was 3 to 7 times higher in N. clevelandii than in sugarbeet in 9 out of 11 experiments (Table 21). 97

Table 19, Numbers of plants infected with PVY° by viruliferous aphids also probing N. clevelandii or sugarbeet with or without BMV

Treatment sequence (page 95)

BMV-infected sugarbeet second 56/256 (-0.697+0.074)+

Healthy sugarbeet second 86/256 (-0.387+0.064) . * BMV-infected N. clevelandii second 51/256(-0.759+0.077)

Healthy N. clevelandii second 69/256(-0. 5534-0.069)

Total BMV-infected leaf 107/tfo(-0.729+0.053)512 Healthy leaf 155/_.512 ,,(-0.469+0.047)

Differences significant from appropriate control * = P ^ 0.05, ** » P < 0.01, *** = P < 0.001

+ Numbers in parentheses are mean logits + S.E. 98

Table 20 . Numbers of plants infected with BMV by viruliferous aphids also probing a tobacco leaf with or without PVY°

Treatment sequence (page 95)

Infected sugarbeet as BMV source

Sequence Is PVY^ first 563+0.093)+

Sequence 3: Healthy first 660+0.098)

Infected N. clevelandii as BMV source

Sequence 4:, PVY o first 24/264(-l.174^0.139)

Sequence 6 : Healthy first 23/264(-l.197+0.143)

Total PVY°-infected leaf 90/r528("°'862-°'075) Healthy leaf 90/528(“0#936t 0#079)

+ Numbers in parentheses are mean logits + S.E, 99

Table 21. Relative concentration of BMV in Nicotlana clevelandii and sugarbeet cv. Hilleshog Honotri

Exp. no. BMV in sugarbeet / BMV in N. clevelandii local lesion assav electron microscopy minimum"*' mean maximum"1"

1 0.42(38/89)++ 0.43 0.57 0.75

2 0.15(18/120) 0.10 0.14 0. 20

3 0.23(24/1q4) 0. 26 0.39 0. 58 4 0.17(17/^0) 0. 31 0.41 0. 53 5 0.29(32/109) 0.14 0. 21 0.30

6 0.80(92/U5) 0.83 1.19 1.72 7 0.31(33/106) 0.25 0.33 0.42 8 0.31(25/80) 0. 26 0.37 0.52 9 0.72(62/85) 0.66 0.91 1.27

10 0.27(32/^2o> 0.21 0.33 0.53

11 0.12(18/148) 0.23 0.33 0.46

+ 957. confidence limits -H* nominator: numbers of virus particles in sugarbeet denominator numbers of virus particles in N. clevelandii. 100

6.4 Experiments testing for interference between PVY 0 » PVY N and TMV during sequential transmission by M. persicae.

Introduction Results of the previous experiments show that there is inter- ference between PVY 0 , PVY N and BMV during aphid transmission. How­ ever, all these viruses are aphid-transmissible and potyviruses. The following experiment was carried out to test if tobacco mosaic virus (TMV), a non-aphid-transmissible tobamovirus (Gibbs, 1977), inter- fered with transmission of PVY 0 and PVY N .

Materials and Methods Detached leaves of N. tabacum cv. White Burley inoculated either with PVY0 or PVY N and N. tabacum cv. Xanthi inoculated with TMV were used as virus sources, 12-15 days after inoculation. Batches of 24 M. persicae were used for transmission tests. They were given 2^ rain access to two source leaves in one of eight sequences. The sequences were: Sequence 1: TMV-infected leaf first then PVY0-infected Sequence 2: Healthy tobacco leaf first then PVY°-infected Sequence 3: PVY°-infected leaf first then TMV-infected Sequence 4: PVY°-infected leaf first then healthy control Sequence 5: TMV-infected leaf first then PVY N -infected N Sequence 6: Healthy tobacco leaf first then PVY -infected Sequence 7: PVY N -infected leaf first then TMV-infected N Sequence 8: PVY -infected leaf first then healthy control The experiment was replicated five times 101

R esults Transmission of PVY0 and PVY N was unaffected by whether or not an aphid probed a TMV-infected leaf before or after acquisition of PVY° or PVY^ (Table 22-23).Thus, 50% and 527. of test plants were infect ed with PVY^ when aphids probed first on TMV or on a healthy leaf. Similarly, 587. and 537. of test plants were infected with PVY N when aphids probed first on TMV or on a healthy leaf. When aphids probed first on a PVY°-infected leaf and then a TMV-infected or a healthy lea f 487. and 497. resp ectively transm itted PVY°. When aphids probed first on a PVY N and then a TMV-infected or a healthy leaf 567. and 587. respectively transmitted PVYN • None of the test plants were infected with TMV.

6. 5 Cross-protection tests

Introduction PVY 0 either does not cross-protect against PVY N (Bawden & Kassanis, 1951 ) or, if so, only partially (Munro, 1955 ; Klinkowski & Schmelzer, 1957 ; Richardson, 1958 ; Schmelzer et al., 1960 ; Todd, 1961 ; Ramirez et al., 1964 ). In partial protection, the challenging PVYN caused either milder symptoms or symptoms appeared later in PVY 0 - infected plants than in healthy plants, or it infected only some of the PVY^-infected plants. Also in tobacco cv. Havana 425, which is infected systerrifically by PVY° but gives local lesions with PVY^, fewer lesions were produced following inoculation with PVY N in plants already infected with PVY^ than in healthy (Makkouk & Gumpf, 1976 ). Therefore, experiments were done to test whether there was cross- protection between the isolates of PVY0 and of PVY N used in the previous experiments. 102

Table 22. Numbers of plants infected with PVY^ by viruliferous aphids also probing a tobacco leaf with or without TMV.

Treatment sequence (page 100)

Sequence 1: TMV first 73/ < 0.013£0.189)+

142/288("0-016i ° '133) Sequence 3: TMV second 69/144^0*044i°'189)

Sequence 2: Healthy first 75/144< 0.041+0.189) 146/288( 0-013t0*133) Sequence 4: Healthy second 71/144(”0#016t°-189>

Tobacco f i r s t l ^ / 288( 0.027+0.133) (TMV or healthy)

Tobacco second l^O^ggC-0.030£0.133) (TMV or healthy)

+ Numbers in parentheses are mean logits + S.E, 103

Table 23* Numbers of plants infected with PVYN by viruliferous aphids also probing a tobacco leaf with or without THV

Treatment sequence (page 100) CO <* Sequence 5 : TMV first n*l 0*114+0,084)

164/288(0-144t0#059) Sequence 7 s THV second 80W 0.173+0.084)

Sequence 6 : Healthy f i r s t ?6/ 144( 0.158+0.083) 159/288(0#107t°*059) Sequence 8 : Healthy second 8 3 / i 44( 0.057+0.084)

Tobacco f i r s t 160/288( 0.136+0.059) (TMV or control) ^

Tobacco 1 second 163/288( 0.115+0.059) (TMV or control)

+ Numbers in parentheses are mean logits + S.E, 104

Materials and Methods

Two methods were used to test for cross-protection. In the first method, 24 tobacco plants infected with PVY 0 or PVY N were sap-inoculated after 8 days with the respective challenging strain. As a control, 24 healthy tobacco plants were sap-inoculated with only the challenging strain. In the second method, 24 tobacco 0 N plants infected with PVY or PVY were aphid-inoculated after 8 days with the respective challenging strain. Controls were aphid- inoculated with only the challenging strains. This experiment was replicated four times. When PVY^ was the challenging strain, its presence was checked by sap-inoculation to Physalis floridana.

R esults Although all plants were infected with the challenging strain when sap-inoculated, irrespective of the strain used (Table 24), a delay in appearance of necrosis was observed. However, when the challenging strain was aphid-inoculated fewer of plants already infected were super-infected with the challenging strain (Table 25).

0 N 6.6 Comparison of electrophoretic mobility of PVY , PVY and BMV by gel-electrophoresisT^

Introduction Although there is no direct evidence that virus particles are attached to receptor sites in aphid mouthparts, indirect evidence suggest that electrical charge differences between aphid mouthparts

+ work done by J.M. Carpenter 105

Table 24* Numbers of tobacco cv» White Burley plants infected with the challenging strain of PVY when sap"inoculated

First inoculated with PVY° Healthy pvyn Healthy Challenge strain pvyn pvyn PVY° PVY°

24/24 24/24 24/24 106

Table 25. Numbers of tobacco cv. White Burley plants infected by M. persicae with the challenging strain of PVY

First inoculated with PVY° Healthy pvyn Healthy Challenge strain pvyn pvyn PVY° PVY°

13/120 58/120 U / 120 56/120 107 and virus particles might enable nonpersistent viruses to bind temporarily to the receptor sites (Govier & Kassanis, 1974b), Thus, a correlation between aphid transmissibility of five isolates of BYMV and their electrophoretic mobility has been reported (Morales, 1981) and a reduction of the electrophoretic mobility of a virus by oils has been suggested as an explanation of reduced aphid trans­ mission of nonpersistent viruses from oil treated leaves (Zschiegner et al., 1974). In the previous experiments, PVY N inhibited trans­ mission of both PVY^ and BMV, BMV inhibited transmission of PVY^ and PVY N was not affected. The following experiment was carried out in order to test whether*power of interference' is related to virus charge as demonstrated by electrophoretic mobility.

Materials and Methods Purification of BMV. BMV was cultured in glasshouse-grown Nicotiana clevelandii. Two to four weeks after inoculation, 75 g of leaves were blended with 225 ml berate buffer pH 7.0 containing 0.5 M borate, 0.05 EDTA and 0.02 M DIECA in a Sorval Omnimixer four times at full speed for 15 seconds. The extract was filtered through muslin and centrifuged at 25.000 g for 5 rain to remove cell debris. The supernatant was stirred with 150 ml carbon tetrachloride for 30 min and centrifuged again (5 min at 10.000) to break the emulsion. The aqueous layer was then centrifuge! for 1^ h at 60.000 g. The supernatants were discarded and the pellets were dispersed in a few drops of borate buffer pH 8.0 and combined into one tube. The extract was centrifuged 5 min at 25.000 g and the pellet extracted twice more with a few ml of buffer. The combined supernatant was centrifuged 108

fo r 2 h at 60.000 g (this time onto a 3 ml cushion of 257. sucrose). The pellets were resuspended in borate - EDTA buffer and centrifuged at low speed as before. The extraction was repeated once more with buffer and twice with water. The combined extracts was given a third high speed centrifugation again onto sucrose and the pellet taken up in about 0.25 ml water and clarified. 20-50 g virus was obtained. All the above operations were done at approx. 5°C. Further purification was by centrifugation in a MSE 2 x 25 ml swing-out rotar for Vi h at 15°C and 60.000 g on 10-40 sucrose gradients. Virus containing fractions were diluted about 4 x with distilled water and the virus removed by centrifugation as before.

Purification of PVY 0 and PVY N . This procedure was similar to that described by Govier & Kassanis (1974b). Tobacco cv. White Burley infected with either PVY 0 or PVY N were blended with borate buffer pH 7.1 containing 0.02 M EDTA and 0.02 M DIECA. The filtered and clarified extract was made 27. in Triton X-100, stirred for 30 rain, then centrifuged at high speed. Pellets were resuspended in borate buffer pH 7.1 with 0.02 M DIECA and given another cycle of low and high speed centrifugations. This time the pellets were resuspended in 0.01 M borate buffer containing 0.5 H urea pH 8.3 and again centrifuged at low speed. Virus was further purified in 10-407. sucrose gradients.

Preparation of composite gels containing 0.57. w/v agarose and 17. or 1. 57. w/v acrylamide. 0.15 g agarose (BDH Ltd.) was dissolved in 15 ml water by boiling under reflux. Separately, 3 ml tris borate buffer pH 8.3 109

(ten times working strength stock contained 108 g tris base, 55 g boric acid and 9,2 g disodium ethylenediamine tetra-acetic acid in 1 litre of water), 1.5 or 2.25 ml 207. acrylamide (19 g acrylamide and

1 g bis-acrylamide dissolved in water, made up to 100 ml and filter through No 54 paper) and water to 12 ml were mixed. The two solutions were brought to 50°C, then were mixed and 3 ml ammonium persulphate and 25 pi tetramethylene ethyledeniamine added. Then the mixture was poured into 100 mm long x 5 mm glass tubes closed at the lower end with dialysis membrane and left on the bench at least one hour to polymerise.

Electrophoresis. Virus preparations containing 50-100 ^ig/ml were mixed with an equal volume of solution containing 0.5 H urea and 107. sucrose in double working strength borate buffer. Gels were loaded with 5-50 pi of the above mixture, and a current of 3mA/gel applied at approximately 100V for 2 h. The gels were then removed and stained overnight in 0.01% (W/V) coomassie brilliant blue R (in methonol. acetic acid: water, 5:1:5), and destained for 3-4 h in the same solvent. The gels were stored in 77. acetic acid.

R esults All those viruses migrated toward the anode, PVY N having the highest electrophoretic mobility and BMV the lowest (it hardly moved). (Plate 14).

D iscussion These results show that PVY N inhibited transmission of PVY 0 • Similarly, PVYW inhibited transmission of BMV and BMV inhibited 110

Plate 14. Electrophoresis of PVY0 , PVY N and BMV in polyacrylamide and agarose gels, using a tris-borate buffer, pH 8.3. Electrophoresis was carried out at 3mA/gel for 2 hours. Migration was toward the anode. Ill transmission of PVY 0 • However, BMV did not affect transmission of PVY N and PVY 0 did not affect transmission of either BMV or PVY N • TMV interfered with the transmission of neither PVY 0 nor PVY N • At least two general mechanisms might explain the observed PVYN

- PVY^, PVY*1 - BMV and BMV - PVY^ interference during sequential trans­ mission by M. persicae. One is cross-protection in the recipient test plant, a common virus interaction in plants that involves interference in replication of one virus strain by another strain previously estab- lished in the host, PVY 0 and PVY N did partially cross-protect when the challenging strain was inoculated by aphids although not when sap- inoculated, incidentally confirming that cross protection may depend on the method of challenging inoculation (Kassanis, 1963 )• However, the protecting strain was already established in the test plant and the results were similar whether PVY 0 or PVY N was used as the challeng- ing strain. In the sequential transmission tests, PVY 0 and PVY N , al­ though aphid-transmitted, were inoculated in the host plant simult­ aneously and neither strain had already established itself in the test plant. Since the test plants used for BMV is immune to PVY and vice-versat cross-protection cannot explain interference between PVY^ - BMV and PVY N - BMV. There was also interference only between aphid- transmissible viruses and not with TMV, which is normally not aphid- transmissible (Kennedy et al., 1962 ; Orlob, 1963 ; Smith, 1965 ). Thus, it seems that the interference observed occurs in/on the aphid. In both experiments PVY N had the greatest "power of inter­ ference", inhibiting transmission of both BMV and PVY^ whereas BMV could only inhibit transmission of PVY^ and PVY^ inhibited neither. As already mentioned (p. 31 )» potyviruses such as PVY and BMV require 112

a helper component in order to be aphid-transmissible. Helper comp­ onent may act by enabling virus to bind to receptor sites in/on the aphid and different viruses may have different helper components (Sako & Ogata, 1981 ). If so, and if the potyviruses share common receptor sites, competition either between the different helper comp­ onents or helper components and the virus for these sites might explain the interference observed. If binding is electrostatic (Kassanis & Govier, 1971a), differences in charge as demonstrated by electrophoretic mobility might explain interference. However, there was no relation between electrophoretic mobility and power of interference. In agree- ment w ith Makkouk & Gumpf (1976) PVY had g reater electro p h o retic mob­ ility but unlike Morales (1981) there was no relationship between electrophoretic mobility and aphid transmission. Thus, although PVY N had greater electrophoretic mobility (plate 14) transmission rates of w P and PVY^ by M. persicae were similar. Although charge interactions between virus-helper component-receptor sites cannot be ruled out by these data they must be more subtle than the total charge of virus p a rtic le s . Aphids carrying PVY^ and subsequently having access to BMV- infected N. clevelandii or sugarbeet transmitted PVY° less frequently than those having access to either healthy N, clevelandii or healthy sugarbeet. Although not significant (P ^ 0.05) inhibition of PVY^ was, if anything less if aphids had had access to a BMV-infected N. clevelandii than to a BMV-infected sugarbeet despite the concentration of BMV being greater in N, clevelandii (Table 19). However, although sugarbeet contained less BMV, aphids acquired BMV more frequently from it than from N. clevelandii and if inhibition occurred in/on the 113 aphid, this could also explain how BMV-infected N, clevelandii was not especially inhibitory* It was unexpected that M* persicae would acquire BMV more readily from sugarbeet than from N* clevelandii since virus concentrat­ ion was less in sugarbeet; however, plants with high virus titres are not always the best sources for aphid transmission (Bar-Joseph

6c Loebenstein, 1973 )• It is possible that virus was less accessible in N* clevelandii to aphids for some physical or chemical reason such as lack of helper component* As probing during acquisition was not monitored, differences in aphid behaviour on sugarbeet and N* clevelandii could also be responsible for differences in transmission* 114

7. VIRUS RETENTION BY MYZUS PERSICAE

7.1 The retention of PVY^, PVYN and BMV by M. persicae

Introduction Long-distance spread of nonpersistent viruses requires retention of virus by aphid vectors. Most work on PVY has dealt with PVY° or PVYN separately (Bradley, 1954 ; Kostiw, 1975 ; Conti et al., 1979 ; Van Hoof, 1980 ) though retention can differ between strains of the same virus (Thongmeearkom et al.t 1976 )• In addition it was thought possible that retention could be related to the ability of a virus to inhibit transmission of others (p.112). The following experiments compared the retention of PVY0 , PVY N and BMV in M. persicae.

Materials and Methods Retention was studied in two ways: Serial plant transfer. M. persicae were given 2^ min access to a PVY0 - or PVY N-infected tobacco cv. White Burley or to a BMV-infected sugarbeet cv. Hilleshog Monotri. Then each aphid was allowed to probe 10 tobacco cv. White Burley or sugarbeet cv. Hilleshog Monotri test plants, successively probing each test plant once. Inoculation probes were artificially ended if they exceeded 40 s. Five aphids were used for each treatment and the experiment was replicated 9 times. Post-acquisition fasting. M. persicae were given 2^ min on PVY°- or

PVY -infected tobacco cv. White Burley and then kept for 0, ht 1, 2

4, 6, 8 or 24 h in glass rials. Afterwards, aphids were caged indiv­ idually on tobacco cv. White Burley test plants. Six aphids were 115 used for each treatment and the experiment was replicated 14 times* The experiment was done at a room temperature of 20 to 22°C. Statistical analysis Results for serial plant transfer were analysed by chi-squared test* The proportion of the first three plants in the sequence infected with each virus or virus strain, was compared with that for the subsequent seven.

R esults Serial aphid transfer* For all three viruses there was a decline in the likelihood of infection of plants from the first to the last plant in the sequence (Fig* 1 )• Thus, 267., 167* and 18% of the aphids transmitted PVYN , PVY 0 and BMV to the first test plants whereas 2,3 and 27* respectively transmitted to the tenth plant* There were no significant differences between the proportions of aphids that trans- mitted PVY0 , PVY N or BMV to the first three plants and those which transmitted to the final seven plants (P y 0*05). Post acquisition fasting* The retention of PVY 0 and PVY N were similar during fasting, declining at approximately the same rates (Table 26)*

None of the strains were transmitted after fasting for 6 h or more*

D iscussion Retention is of obvious importance in the epidemiology of nonpersistent viruses; for example, the longer retention of strain A of MDMV than of strain B may well contribute to the prevalence of strain A in nature (Thongmeearkom et al., 1976). However, my results suggest that there is little difference in the retention of the isolates of BMV, PVY 0 and PVY N tested. M* persicae has been reported 116 Figure 1. Histograms showing transmission of PVY , PVY and BHV z by single aphids to successive plants.

1 2 3 A 5 6 7 8 9 10 plant o

1 2 3 A 5 6 7 89 10

123A56789K) plant 117

Table 26. Numbers of plants Infected with PVY 0 and PVY N by M. persicae following different periods of post-acquisition fasting

Pos t-acquis i tion Virus strain fasting period (h) PVY° pvyn

0 53/90 49/90

h 36/90 31/90

1 16/90 19/90

2 9 /90 H /90

4 4 /90 5 /90

6 0 /90 0 /90

8 0 /90 0 /90

24 0 /90 0 /90 118 to retain PVY up to 17 h (Kostiw, 1975 ) but in my experiments retent­ ion did not exceed 4 h. These differences may be due to different Isolates of PVY or to different clone of M. persicae being used, but both experiments were done at similar temperatures* Previous experiments have shown that PVY N inhibited trans­ mission of both BMV and PVY° whereas BMV could inhibit transmission of only PVY° and PVY0 could inhibit neither. That retention did not differ significantly between PVY 0 , PVY N and BMV suggests that retent­ ion is not related to the ability of a virus to inhibit transmission of o th ers.

7.2 Effect of temperature on the retention of PVY 0 and PVY N by M. persicae.

Introduction In the previous experiment, the retention of PVY 0 and PVY N in apterous M. persicae were compared at room temperature (20-22°C). However, virus retention often decreases with increasing temperature (Kassanis, 1941 ; Bradley, 1954 ; Sylvester, 1954 ; Cockbain et al.» 1963). Therefore, the transmission of PVY0 and PVY N by M. persicae after postacquisition fasting at different temperatures was studied.

Materials and Methods

M. persicae fasted for 2-3 h at 20-22°C, were given 2h min access at the same temperature, either to a PVY 0 - or PVY N-infected tobacco cv. White Burley leaf. These aphids were then caged individ­ ually on tobacco test plants for 24 h either Immediately, or after fasting for 1, 2, 4 and 6 h at 9°C, 18°C and 27°C. Twelve test plants were used for each treatment and the experiment was replicated

ten times.

R esults Aphids fasted after acquiring virus were less infective than unfasted ones (Table 27). Few aphids fasted at 27°C were infective, transmission of both PVY 0 and PVY ‘ N increasing as the temperature de­ creased. Thus, 347. and 187. transmitted PVY° after 1 h at 9°C and 27^C respectively and 27% and 11% transmitted PVYN at 9°C and 27°C.

D iscussion In accord with previous reports, these results show that loss of infectivity was fastest at the highest temperature (Kassanis, 1941 ; Bradley, 1954 ; Sylvester, 1954 ; Cockbain et al., 1963 ). The effect of temperature on retention of PVY 0 and PVY N was similar and thus long-distance spread of the Isolates of PVY 0 and PVY N tested is affected similarly by the ambient temperature. Retention at 18°C and 27^C is of practical importance because these temperatures are in the range at which aphids normally fly (Broadbent, 1949, Johnson, 1952 Broadbent, 1953 ). Therefore, the ability of aphids to remain infect­ ious for long periods may differ with the season; this could be espec­ ially important in areas where there is more than one crop (Srivastava et al., 1971 ; Horio, 1981 ; Marco, 1981 ; Chiu & Chang, 1982 )• 120

Table 27. Numbers of plants infected with PVY^ or PVY^ by M. persicae following different periods of fasti-ng at different temperatures

Pos t-acquisition Temperature (°C) Virus strain fa stin g (h) 9-10 18-19 27-28

PVY° 0 78/132 1 45/132 33/132 14/132

2 26/132 18/132 7/132

4 13/132 6 /132 2 /132

6 5 /132 2 /132 0 /132

pvyh 0 70/132

1 35/132 25/132 15/132

2 22/132 13/132 6 /132

4 15/132 4 /132 L/132

6 4 /132 2 /132 0 /132 121

8. ROLE OF WEEDS AS PVY SOURCES

Introduction

Weeds can be important in the epidemiology of plant viruses (Duffus, 1971 ). However, despite field or laboratory investigations demonstrating that forty-nine plant species from 13 families (al­ though not all occurring in Britain) are susceptible to PVY (Table 28) the role of weeds in the epidemiology of PVY in potato crops has not been established (Doncaster & Gregory, 1948 ; Schmelzer, 1967 ; Ksiazek, 1980.), most spread apparently being within the crop. There­ fore, samples from different weeds and growing close to an infected potato plot were tested for PVY.

Materials and Methods

Samples of different plants, which were next to an infected potato crop were tested for PVY by both aphid and sap transmission to tobacco cv. White Burley. Twenty fasted adult apterae M. persicae were given 2% min access to each sample and then transferred to a single test plant. Each sample was also ground in a pestle and mortar, diluted 1:50 with phosphate buffer pH 7.0 and sap-inoculated to carbonundum-dusted leaves of tobacco. Virus-like symptoms were not observed in ary of samples tested except Mercurial^is perennis which was also tested by electron microscopy (p. 53 ).

R esults

No PVY was detected in any of the 352 plants tested (Table 29)either by aphid or sap transmission tests although the species Plantago lanceolata, P. major and Taraxacum officinale have been reported to be susceptible 122

Table 28 • Some non-crop plants susceptible to PVY Family and species References BORAGINACEAE Anchusa officinalis L. Akhatova et al. (1980) CARYOPHYLLACEAE Spergula arvensis L. Ksiazek (1980) Stellaijih media (L.) Ksiazek (1980) COMPOSITAE Dimorphotheca plurialis (L.) Moench Schmelzer (1967) Dimorphotheca sinuata DC. Schmelzer (1967) Galinsosa parviflora Ca. Ksiazek (1980) Senecio vulgaris L. Schmelzer (1967) Taraxacum officinale agg. Akhatova et al. (1980) Lytaeva (1972) CRUCIFERAE Barbarea arcuata (Opiz) Akhatova et al. (1980) GRAMINEAE Agropyron repens (L.) Akhatova et al. (1980) Ksiazek (1980) HYPERICACEAE Hypericum perforatum L. Akhatova et al. (1980) LOBELIACEAE Lobelia erinus L. Schmelzer (1967) Lobelia hederacea Hort. Kew. ex DC. Schmelzer (1967) PAPILIONACEAE lielilotus indica (L.) All. Schmelzer (1967) Melilotus messanensis All. Schmelzer (1967) Melilotus sulcatus Desf. Schmelzer (1967) Melilotus sp. Lytaeva (1972) Trigonella calliceras Fisch. Schmelzer (1967) Trigonella coerulea (L,) Ser. Schmelzer (1967) Trigonella foenum-graecura L. Schmelzer (1967) PLANTAGINACEAE Plantago major L. Akhatova et al. (1980) Plantago lanceolata L. Akhatova et al. (1980) 123

Table 28. - continued Family and species References POLYGONACEAE Rumex confertus L. Akhatova et al. (1980) PORTULACACEAE Portulaca oleraceae L. Pochard (1979) Datura ferox L. Gracia & Feldman (1972), Feldman & Gracia (1972) Datura metel Linn. Prasad Rao (1976) Lycium chinense Hill. Akhatova et al. (1980) Lycium flex icau le L. Akhatova et al. (1980) Lycium turcomanicum L. Akhatova et al. (1980) Nicandra physaloides (L.) Schmelzer (1967), Suteri et al. (1979) Nicotiana glauca Graham. Laid & Dickson (1963) Petunia hybrida Vilm. Anderson (1959) Physalis angulata L. Anderson & Corbett (1957), Anderson (1959), Lapp & Gooding (1976) Physalis ciliosa Rydb. Anderson (1959) Physalis mendocina Phil. Gracia & Feldman (1972), Feldman & Gracia (1972) Physalis virginiana L. Gooding et al. (1975), Lapp & Gooding (1976) Physalis viscosa L. Anderson (1959), Pontis & Feldman (1963) Physalis sp. Dykstra (1933) aculeatissimum Jacq. Anderson (1959) Solanum cf. atriplicifolium Gill, Pontis & Feldman (1963) ex Nees Solanum atropurpureum Shrank Chagas et al. (1977) Solanum ciliaturn Lam. Kudamatsu et al. (1981) Solanum gracile Link. Simons (1956), Anderson & Corbett (1957) Solanum nigrum L. Anderson & Corbett (1957) Anderson (1959), Ksiazek (1980), Pochard (1979), Carlos Ramalho (1981), Eskarous et al. (1983) Solanum nodiflorum Jacq. Sakimura (1953) Dun Kudamatsu et al. (1981) 124

Table 28 - continued Family and species References Solanum villosum Mill. Dykstra (1933) Solanum xanthocarpum Schad. Suteri et al. (1979) TROPAEOLACEAE Tropaeolum raajus L. Suteri et al. (1979) 125

Table 29• Non-crop plants tested for PVY Plant species Number of plants tested

Acer pseudoplatanus L. 6 Anthriscus sylvestris (L.) 51 Comus sanguinea L* 4 Corylus avellana L* 8 Geum urb.anum L. 17 Mercurial|(is perennis L. 39 Myosotis arvensis (L.) Hill 28 Plantago lanceolata L. 36 Plantago maior L. 19 Ranunculus repens L. 28 Rumex crispus L. 47 Symphoricarpos rivularis Suksdorf 7 Taraxacum officinale agg. 62 126

to PVY (Lytaeva, 1972 ; Akhatova, 1980 ) and even though virus-like particles about 800 nm in length were observed by electron microscopy in 35 samples of Mercuriayfis perennis showing mosaic symptons. No particles were detected in 4 samples without symptoms.

D iscussion

These investigations failed to identify any weed as a source of PVY. By implication, the results confirm that the major sources of PVY are infected potatoes either planted as part of the crop or surviv­ ing as volunteers from unharvested potatoes from previous crops (Doncaster & Gregory, 1948 ; Hill, 1978 ; Thresh,1980 ; Thomas, 1983 ). 127

9. PRIMARILY-INFECTED POTATO PLANTS AS SOURCES OF PVY

Introduction Little is known of the relative importance of current season infection as a source of PVY for further spread in potato crops. According to Kato (1957), aphids transmitted PVY from glasshouse potato plants as soon as they had developed symptoms. In other tests, M. persicae acquired PVY°from leaves of potato 15-20 days after aphid- inoculation (Stevenson, 1959), and PVY N 10 days after sap-inoculation and 14 days after aphid-inoculation (Beemster, 1979). Virus concentrat ion in primarily-infected potato plants reached levels similar to those of secondarily-infected plants two to five weeks after sap- or aphid-inoculation (Gugerli, 1978). These results suggest that primarily infected plants may be important virus sources in crops. However, Heathcote & Broadbent (1961) found that there was little spread of PVY from plants sap-inoculated early in the season, but aphids were rare that year. None of these tests compared spread of PVY0 and PVY N although differences such as the earlier development of mature plant resistance against PVY0 than against * PVY N (Beemster, 1961a, 1961b, 1965 , 1972 , 1976 ) may be epidemiologically important. The follow­ ing experiments investigated the relative importance of primarily- infected potato plants in the epidemiology of PVY0 and PVY N .

Materials and Methods Glasshouse experiments The experiments were carried out in aphid-free glasshouses at temperatures ranging from 20-24^C and with supplementary light (16 h daylength). 128

Single-stemmed potato plants were grown from small tubers in 15 cm diameter pots of compost. All plants were tested by inoculation to tobacco plants for the initial absence of PVY. Virus-free plants were sap-inoculated on the youngest fully developed leaf using carborundum. Tobacco plants cv. White Burley inoculated two weeks earlier with PVY 0 or PVY N were used as inocula. Tobacco plants cv. White Burley were also used as virus test plants.

Field experiment The experiment consisted of sixteen plots each 7.5 m (10 rows) x 10.5 m arranged in two blocks (Fig. 2 ). The distance between the blocks was 10.5 m (14 rows). All plots were planted with King Edward seed potatoes on 15th May 1983. The plots were in two blocks and seven treatments were applied at random to plots in each block. In two plots, a tuber from the centre of each row was removed and replac­ ed with a PVY-infected tuber cv. King Edward (KS). In three plots, the central potatoes were replaced with healthy tubers cv. Record. In these, three plots and in the remaining three plots in which the central King Edward seed potatoes were not removed the potato plants growing from these central tubers were either left untreated (healthy control, R ___ , K ___ ) or were sap-inoculated with PVY^ (RIO, KIO) or PVYN (RIN, KIN) (Fig. 3 ). Plants were inoculated on 18th June 1983 when the shoots were about 15 cm high. Inocula were prepared from tobacco plants inoculated with PVY 0 or PVY N two weeks earlier and ground in a pestle and mortar with phosphate buffer pH 7.0. Carbo­ rundum was used as an abrasive. The cultivars King Edward and Record were chosen because after inoculation with PVY°, leaf drop streak is induced in the first and mosaic in the second; cultivars 129 Figure 2. Plan of the field trial.

Key Cultivar of Treatment central row KS King Edward Secondarily infected KIO King Edward Primarily inoculated wi th PVY, KIN King Edward Primarily inoculated wi th PVY( RIO Record Primarily inoculated w ith PVY RIN Record Primarily inoculated with PVY K King Edward Healthy (control) R Record Healthy (control) 130 Figure 3, Plan of a plot.

10. 5m

The following treatments were applied to the King Edward tuber planted in the centre of each row: (1) replaced with a PVY-infected tuber cv. King Edward (KS). (2) replaced with a healthy tuber cv. Record. Subsequent plant inoculated with PVY° (RIO), PVYN (RIN) or left untreated (healthy control) (R___ ). (3) Not removed. Subsequent plant inoculated with PVY° (KIO), PVY^ (KIN) or left untreated (healthy control) (K ___ ). At harvest one tuber was removed from each of the 1st, 2nd, 3rd and 4th plants in each row for testing for presence of PVY. 131

with mosaic symptoms may be better sources of PVY than cultivars which become necrotic (Bagnall & Bradley, 1958). Originally, about 0.37. of the King Edward seed tubers planted in this experiment were infect­ ed with PVY (Govier, 1983). Infected plants were removed as soon as symptoms were visible. To compare acquisition from secondarily- and primarily-infected plants, leaves were collected from five plants 20 and 30 days after inoculation from each plot in one block. Twelve

M. persicae were given 2% min access to each leaf and then transferred singly to tobacco plant. To assess spread of PVY, one tuber was taken (15-20/9/1984) from each of the four plants on either side of the ten central plants in each plot and stored 4-5° C for three months until dormancy ended. The weight of each sampled tuber was more than 60 g as fewer small tubers are infected than big ones (Beemster, 1967). Since there is no reliable method of detecting PVY in tubers (De Bokx, 1961 ; De Bokx & Maat, 1979 ; Daniel & Hunnius, 1980 ; Vetten et al., 1983 ; Hill & Jackson, 1984 ) they were tested by growing plants from an eye-plug taken from each tuber in a glasshouse. The first batch of plants were checked visually and by the enzyme-linked immunosorbent assay (ELISA) (p. 52 ). Since there were no differences in results, plants were sub­ sequently checked only visually.

Experiments and Results Glasshouse experiments 9. i Acquisition of PVY 0 and PVY N by M. persicae from primarily infected potato cv. King Edward inoculated at different ages Batches of four potato plants cv. King Edward either three, five or seven weeks old, were sap-inoculated either with PVY 0 or PVY N • 132

Plants were placed in a glasshouse in a randomised block design.

Twelve and twenty days later, ten M, persicae were given 2h min access to uninoculated leaves of each plant. As a control, ten other aphids were given access to two 20-30 days old secondarily- infected potato plants cv. King Edward either with PVY0 or PVY N • Aphids were then caged individually on tobacco plants. The experiment was repeated twice. When three and five weeks old plants were inoculated, symptoms of PVY^ appeared usually 2-3 weeks after inoculation as a mottling followed later by leaf-drop streak. In older plants symptoms had not appeared by the end of the experiment (20 days after inoculation). Symptoms of PVYN in primarily-infected plants were very mild and often difficult to see. However, symptoms of both strains were clear in secondarily-infected plants. Twelve days after inoculation, M, persicae acquired both PVY^ and PVY N but only from young inoculated plants and even these were poorer sources than the secondarily-infected plants (Table 30). Eight days later, all plants inoculated with PVY N and the younger plants inoculated with PVY^ were as good sources as secondarily-infected ones but plants inoculated with PVY° when seven weeks old were still poor sources.

9.2 Acquisition of PVY 0 and PVY N by M. persicae from primarily- infected potato plants of different cultivars. In this experiment, primarily-infected plants of cultivars with different levels of resistance to PVY were compared as sources of PVY 0 and PVY In . The following cultivars were used. 133

Table 30 Numbers of plants infected by H. persicae given access to PVY 0 and PVY N primarily-infected plants

Time of Mean height Transmission from Transmission from inoculation (cm) of plants Days after Primarily- Secondarily- (weeks after when infected infected planting) inoculated Inoculation plants p lan ts PVY° pvyn PVY° pvyn 3 21 + 1.16 12 10+ 9+ 4 6 ^ 42*- 20 79 84 33 39 5 58 + 1. 75 12 2 0 46 42 20 68 90 33 39 7 81 + 2.08 12 0 0 46 42 20 24 69 33 39

plants infected of 144 inoculated -H- plants infected of 72 inoculated 134

C ultivar Resistance to PVY+ King Edward E++ Record E Maris Piper C D esiree B Pentland Crown A Four plants of each cultivar were sap-inoculated with PVY* PVYN three weeks after planting (about 20 cm high). To test for

recovery of virus, batches of ten 11. persicae were allowed 2% min access to each of two leaves from each plant and each batch was transferred to a single tobacco test plant. As a control testing that these aphids could readily acquire PVY,24 aphids were allowed 2^ rain access to a PVY-infected tobacco leaf inoculated two weeks earlier and then transferred singly to tobacco test plants. The experiment was repeated twice. Different symptoms appeared in different virus-strain-cultivar combinations. In PVY^-infected plants, leaves became necrotic in King Edward, Maris Piper and Desiree whereas only a mosaic appeared in Record. Inoculation with PVY N gave no readily visible symptoms in any cultivar. Although all plants of King Edward, Record and Maris Piper were infect- ed after sap-inoculation with PVY 0 and PVY N , some plants of the more resistant Desiree and Pentland Crown remained virus-free.

4- Information obtained by the leaflet "Classified list of potato varieties England & Wales 1981/82" published by the National Institute of Agricultural Botany, 11 pp. Printed by E. & E. Plumridge Ltd., Linton, Cambridge. ++ A means that the cultivar is very resistant to PVY whereas E is very susceptible. 135

Twelve days after inoculation, M. persicae could transmit PVY 0 and PVY N only from King Edward and only rarely (Table 31 )# However, 20 days after inoculation aphids acquired both PVY 0 and PVY N from all cultivars. At both dates, 50-607. of single aphids allowed access to an infected tobacco leaf transmitted.

9. 3 Acquisition of different isolates of PVY° and PVY^ by M. persicae from primarily infected potato cv. King Edward plants Seven isolates of PVY 0 and three isolates of PVY N from different cultivars were tested. Isolates of PVY 0 and PVY U were kindly supplied by Dr. R.A. C. Jones.

STRAIN ISOLATED FROM DESIGNATED PVY° Record Y°(standard)+ King Edward Pentland Crown v°2 Cara *5 Pentland Crown < King Edward King Edward pvyk Record Y^(standard)+ King Edward King Edward

Four plants of King Edward were sap-inoculated with each isolate three weeks after planting (about 20 cm high). Recovery of

+ These two were the isolates used in other experiments. Table 31, Numbers of tobacco plants infected by batches of ten M. perslcae allowed access to potato cultivars inoculated with PVY 0 or PVY N

Days after C ultivar Virus strain inoculation King Edward Record Maris Piper D esiree Pentland Crown*

12 2/12 0/12 0 /n °/ 3 °/5 PVY°

20 12 2 12/12 / l 12/12 l/3 136

12 °/7 2/12 0/12 0/12 0/6 PVY°

20 12 2 n / 12 / l 12/12 5/6 7 /7

+ Although twelve plants were inoculated,only three Desiree and five Pentland Crown were infected with PVY 0 whereas six and seven were found infected with PVY N respectively. 137

virus was tested twelve and twenty days after inoculation using two fully-developed uninoculated leaves on each plant. Batches of ten

M# persicae were allowed 2)$ min access to each leaf and each batch was transferred to a single tobacco test plant. As a control, the ease by which aphids could acquire PVY was tested by allowing 24 aphids Ih. min access to a PVY-infected tobacco leaf inoculated two weeks earlier and then transferring aphids singly to tobacco test plants. The experiment was repeated twice. Mosaic was induced by all PVY^ isolates 2-3 weeks after inoculation followed by leaf drop streak. PVY N isolates all produced mild mosaics, The results (Table 32) suggest that isolates N and Y^ Nwere more readily recovered twelve days after inoculation. However, twenty days after inoculation virus was recovered from plants inoculated with all isolates. At both dates, 50-607. of single aphids allowed access to an infected tobacco leaf transmitted.

9.4 Acquisition of PVY 0 and PVY N by M. persicae from different leaves of primarily-infected potato plants cv. King Edward The experiment investigated the availability of PVY 0 and PVY N to M. persicae in different leaves of a potato plant. One plant was inoculated on a bottom and another one a top fully developed leaf with PVY 0 or PVY N three weeks after planting. Twelve and twenty days after inoculation, batches of ten M. persicae were allowed 2’s min access to a leaflet detached from each leaf and each batch was then transferred to a single tobacco test plant. Table 32 . Numbers of tobacco plants infected by batches of ten M. persicae allowed access to potato cv> King Edward inoculated with different isolates of PVY° and PVY^

STRAIN Days After PVY° pvyn Inoculation 1 2 3 4 5 6 1 2

12 3+ 5 4 6 4 4 3 4 9 8

20 12+ 12 12 12 12 12 12 12 12 12

+ Number of plants of 12 inoculated from which virus was recovered. 139

Twelve days after inoculation, only aphids given access to inoculated leaves transmitted. Twenty days after inoculation both strains were recovered from almost all the leaves irrespective of the leaf inoculated or the virus strain (Table 33).

9.5 Field experiment In laboratory transmission tests, leaves from secondarily- infected plants were better sources of PVY than primarily-infected ones twenty days after inoculation of the latter. Thus, 607. of M. persicae transmitted PVY from secondarily-infected King Edward plants whereas only 207. transmitted PVY 0 or PVY N from primarily-infected Record or King Edward (Table 34; P 0.01). However, thirty days after inoculat­ ion there were no significant differences between transmission from primarily- and from secondarily-infected plants (Table 34; P 0.05). In plots without infectors 207. of the tubers collected were infected. Infection was about three times more frequent in tubers taken from plots with primarily-inoculated infectors of either cultivar or inoculated with either strain but was even more frequent in tubers from plots with secondarily-infected plants (Table 35 ; P ^ 0.001). Neither the cultivar inoculated nor the PVY strain used gave significant (P 0.05) differences in virus spread. Tubers from plants adjacent to infectors were significantly (P ^ 0.05) more likely to be infected than those further away (Table 36).

D iscussion Laboratory tests with glasshouse-grown potato plants showed that primarily-infected plants are soon good sources of both PVY^ and PVY N . Thus, 20 days after inoculation 11. persicae acquired PVY N as 140

Table 33. Transmission of PVY 0 and PVY N by M. persicae from different leaves of primarily-infected potato cv. King Edward plants 20 days after inoculation

Virus stra in Leaf number PVY0 PVY° pvyn pvyn 14 (top) + + 13 + 4* + + 12 4" - + + 11 4" 4- + + 10 + 4- + + 9 + + + + 8 4* + + - 7 + + + + 8 + 4- + + 6 + + + 4- 5 + • + - 4 4* • + x 4- 3 - x . 4- 4* 2 • • + - 1 (bottom) X • • x + -

. = leaves were dead - = aphids did not transmit the virus 4- = aphids transmitted x = inoculated leaf 141

Table 34. Numbers of tobacco plants infected in laboratory tests with PVY 0 or PVY N by M. persicae from primarily infected King Edward and Record potato plants

Days after inoculation Primarily - infected Secondarily- of primarily infected Virus infected plants S train Record King Edward King Edward , ** PVY° 10/60 + 10/6o"~ (-0.827+0.175) (-0.827+0.175) 20 35/60 (0.263+0.132) pvyn 9 /6o'" 12/60 (-0.891+0.183) (-0.714+0.163)

PVY° 31/60 33/60 ( 0.033+0.159) ( 0.101+0.160) 30 36/60 (0.206+0.162) pvyn 29/60 30/60 (-0.035+0.159) (-0.001+0.159)

+ Number in parentheses are mean logits + S.E. ** Transmission rates are significantly different from that from secondarily-infected plants at P = 0.01 142

Table 35. Proportion of infected tubers from plants near to primarily or secondarily infected field potato plants

Cultivar of primarily Secondarily Control (no infectors) S train infected plants infected Record King Edward King Edward Record King Edward

VriWr PVY° 98/1160 + 105/160*** (0.229+0.095) (0.290+0.095)

299/'320 34/160 36/160 (1.167+0.067) (-0.671+0.095) (-0.612+0. 095) pvyn 108/160 (0.314+0.095) (0.352+0.095)

+ Numbers in parentheses are mean logits + S.E. *** Proportion of infected tubers is significantly different from that from plots with secondarily-infected infectors and also from control plots (no infectors) at P = 0.001. Table 36 Proportion of infected tubers from plants close to infectors.

Position of plants in relation to infectors 1 2 3 4

126/160 U 1 /160 9 3 /160 8 7 /160 (0. 413+0.157)"r (0.406+0.157) (0.202+0. 157) (0.095P0.157)

+ Number in parentheses are mean logits + S.E. * Proportion of infected tubers is significantly different from that of plants next to infectors at P=0.05. 144

readily from primarily-infected as from secondarily-infected ones. With PVY° this was also the case with plants inoculated three and five weeks after planting although plants inoculated with PVY° when seven weeks old were poor sources of virus. It is noteworthy that potato plants develop mature plant resistance slower against PVYN than against PVY^ (Beemster, 1961b, 1972 , 1976 ) if at all (De Bokx, 1979 ). Both strains were recovered from all leaves of primarily- infected King Edward plants twenty days after inoculation. In cultivars such as King Edward and Maris Piper, the lower leaves drop in plants primarily-infected with PVY°. As a result aphids such as M, persicae that are abundant there ( Taylor, 1955 ; Holling, 1955 ; Gibson, 1971 ), if forced to leave, may be viruliferous. Both PVY 0 and PVY N were recovered soonest from King Edward and Record which were the most susceptible cultivars tested. Differ­ ences in multiplication and translocation rates of PVY in different cultivars (Beemster, 1972 ) might explain this. However, multiplicat­ ion of the virus once it has become established in a plant is not necessarily correlated to susceptibility to infection (Bawden & Kassanis, 1946). It took longer for primarily-infected plants to become good sources under field conditions than in a glasshouse. This is not surprising since potato plants growing at 22^C had more PVY^ and PVY^ earlier than plants growing at 14 or 18^C (De Bokx & Piron, 1977 ). Similarly, a high concentration of PVY^ was observed earlier in glass- house-grown potato plants than in field-grown ones (Singh & Santos- Rojas, 1983 ). However, results of the field experiment show that primarily-infected plants were important sources of PVY, there being 145 considerably more tuber infection in plots with primarily-infected plants than with none. There was even more spread in plots with secondarily-infected plants but this is to be expected since these were sources of infection from the very beginning of the growing season. Spread of PVY from primarily-infected plants can happen only if there are numerous vectors when the plants have become good sources (about a month after inoculation). As aphid populations vary from year to year, spread from primarily-infected plants will also vary and might explain the negligible spread from primarily-infected plants reported by Heathcote & Broadbent (1961), Spread from primar­ ily-infected plants might also be diminished by the development of mature plant resistance. In my experiment, spread from primarily- infected plants must have occurred before mature plant resistance, even against PVY^, developed. However, the development of mature plant resistance also varies from year to year (Schepers & Reestman, 1975 ; Schepers & Beemster, 1976 ; Braber et al., 1982 ). In other regions, such as in seed growing areas, vectors may appear late in the season (Fisken, 1959 ) and spread from primarily-infected plants might be negligible because by the time these plants can act as sources the crop may have developed mature plant resistance or the vectors become rare again. The influence of distance from sources on the spread of PVY, observed in this experiment, is in line with previous work (Doncaster & Gregory, 1948 ; Duncan et al., 1956 ), 146

10. TRANSMISSION OF POTATO VIRUS Y BY CEREAL APHIDS

Introduction As mentioned in the Literature Review, nonpersistently- transmitted viruses are transmitted by brief probes and, for several potyviruses, common vectors are alates of aphid species which do not colonize the crop concerned (Table 1 )• Similarly, it has been suggested that cereal aphids, especially R, padi, are important vectors of PVY in potato crops (Robert, 1978 ; Ryden et al., 1983 ). Hille Ris Lambers (1972) suggested that R. padi is an efficient vector of PVY and Ryden (1979) confirmed that alates frequently transmitted PVY°. Kostiw (1979) and Van Hoof (1980) found that alatae and apterae were respectively inefficient vectors of PVY0 and PVYN whereas Kurppa (1983) could not transmit PVY using R. padi. These previous examples all involve transmission between potato plants but in tobacco Ryden (1979) reported that R. padi could neither acquire nor inoculate PVY° and Van Hoof (1980) found that R. padi could inoculate but not acquire PVY • N However, other aphid vectors such as M. persicae readily acquire from and inoculate tobacco with both PVY 0 and PVY N • The following experiments exam­ ine in detail transmission of PVY by R. padi, Metopolophium dirhodum and Sitobion avenae.

Materials and Methods Only adult apterae were used in tests, except when transmiss­ ion rates of apterous and alate R* padi were compared. 147

In most experiments, cereal aphids were given 10 min acquisit­ ion access, whereas M, persicae was given 2 min, as initial exp­ eriments showed that these times gave near maximum transmission ra te s. Except where stated otherwise, cereal aphids were afterwards transferred and caged on test plants in groups of 50 whereas M, persicae because of the expected high transmission rates were caged singly, Next day, the plants were sprayed with an aphicide and kept in a glasshouse for 3-4 weeks to allow symptoms to develop, The symptoms of PVY were obvious in all combinations except PVY N in potato, and the success of these inoculations was assessed by immunosorbent electron microscopy (ISEM) (p, 51), Transmissions were usually to tobacco cv. White Burley test plants and from potato plants cv. King Edward grown from naturally-infected tubers contain- ing undetermined isolate(s) of PVY. When PVY 0 or PVY N are specified, either a potato plant naturally infected with a single strain had been identified or a tobacco plant had been inoculated with a single strain.

Experiments and Results

10,1 Transmission of PVY by R. padi, M. dirhodum, S. avenae and M, persicae In this experiment, the transmission of PVY by R, padi, M, dirhodum, S, avenae and M, persicae from PVY-infected potato cv. King Edward was compared. All aphid had access to the same source leaf and aphids were then caged either in batches of 50 (the cereal aphids) or singly (M. persicae) on tobacco test plants. IAS

Four test plants were used for each species in each replicate and the experiment was replicated nine times. M. persicae infected 26 of 40 test plants whereas R. padi infected 19, M. dirhodum 13 and S. avenae none of 40 test plants. The proportions of test plants infected suggest transmission rates

by single R. padi and M. dirhodum of ca. 17. (Gibbs & Gower, 1960), compared to 657. transmission for M. persicae.

10.2 Duration of acquisition access It has been reported that R. padi spends a long time walking on potato without probing (Wiktelius, 1982) and therefore long access on the source plant may be more necessary for transmission of PVY by this species. Therefore, in this experiment R. padi and M. persicae were allowed access for 2, 10, 50 and 250 min to a PVY- infected potato leaf. R. padi were then confined on individual tobacco test plants in groups of 20; M. persicae were caged singly. Eight test plants were used for each aphid species in each replicate and the experiment was replicated 8 times. M. persicae transmitted most efficiently when given 2 or 10 min access whereas R. padi transmitted PVY more efficiently after longer access (Table 37),

10.3 Transmission of PVY by apterae and alatae R. padi Although it is easier to use apterae in laboratory transmiss­ ion tests, spread of PVY in the field is mainly by alatae (Broadbent,

1950 ; Broadbent & Tinsley, 1951 ) and this must always be the case 149

Table 37• Transmission by aphids given different acquisition access periods

Aphid species Acquisition access period in min 2 10 50 250

M. persicae 36/72 43/72 3 /72 ° / 72

R. padi 5 /72 l ° / 72 22/?2 26/72 150 for non-colonizing aphids such as R. padi. Therefore transmission of PVY from potato to tobacco by groups of 50 apterae or alatae were compared. Four plants were used in each replicate for either apterae or alatae and the experiment was replicated twenty-one times. In total apterae infected 31 of 88 test plants whereas alatae infected 37.

10.4 Transmission of PVY° and PVY^ Transmission tests by R. padi reported so far involved either PVY° (Ryden, 1979 ; Kostiw, 1979 ) or PVYN (Van Hoof, 1980). How­ ever, differences in transmissibility of different strains of PVY have been reported (Makkouk & Gumpf, 1976) and this experiment compared the transmission of PVY0 and PVY N by R. padi. Potato infected with either PVY 0 or PVY N was used as source and groups of 50 R. padi were then caged on tobacco test plants. M. persicae were caged singly. Five test plants were used in each test for both PVY 0 and PVY N , and the experiment was replicated 9 times. Twenty of 50 test plants were infected with PVY° and 24 of 50 with PVY N by R. padi whereas M. persicae infected 30 plants with PVY° and 26 w ith PVYN.

10.5 Transmission of PVY to and from potato and tobacco Ryden (1979) reported that R. padi could neither acquire nor inoculate PVY° and Van Hoof (1980) found that it could inoculate but not acquire PVY .N In this experiment, the transmission of PVY 0 and PVY N from and to both tobacco and potato was compared. Four test plants of either combination was used and the experiment replicated four times. 151

Inoculation by R, pad! seemed to be independent of the strain of virus or receptor plant but potato may have been a slightly better source plant (Table 38).

10.6 Retention of infectivity by R. padi and M. persicae After acquiring virus from PVY-infected potato, aphids were kept in glass tubes for set times before transferring to tobacco test plants. Groups of 50 R. padi were then caged on tobacco test plants whereas M« persicae were caged singly. The experiment was replicated 9 times, and each time 5 test plants were used for each treatment. R. padi retained and transmitted PVY for at least as long as M. persicae (Table 39 ). The transmission efficiency of R. padi declined to 407. and 207. of its initial value after one and two hours respectively. During the same time periods, the infectivity of M. persicae declined to 327. and 147..

10.7 Transmission of PVY by R. padi and M. persicae after a single probe Throughout previous experiments, R. padi transmitted PVY much less efficiently than M. persicae and so the abilities of a single probe by R. padi and M. persicae for acquiring PVY were compared. Aphids observed individually under a binocular microscope were allowed a single probe into an infected leaf after which they were transferred to tobacco test plants. Ten aphids of each species were used in each replicate and the experiment was replicated six times. The first 70 M. persicae tested all probed within a 15 min 152

Table 38 • Transmission of PVY 0 and PVY N by R. pad! using different source and test plants

Virus source Tobacco Potato T est p lan t Tobacco Potato Tobacco Potato CO o PVY° CN O 5 /20 to 9 /20 pvyn 6^20 4/^20 u / 2o 9 /20 153

Table 39 • Transmission of PVY after different post-acquisition fasting periods

Post-acquisition Aphid species fasting periods (h) M» persicae R« padi

0 28 25' 1 9 10 2 4 5 4 1 3 6 0 2

8 0 0

24 0 0

+ Nunfers of infected plants of 50 tested 154 maximum access period allowed whereas 104 R. pad! had to be tested to obtain 70 which probed. Probes by M. persicae lasted 24.3+2.2 s and 45 of the aphids transmitted. Probes by R. padi lasted 59.5+14.2 s but none transmitted. Thus although R. padi probes less frequently than M. persicae the major reason for the poor vect­ oring ability of R. padi was that aphids which had probed seldom transmitted PVY.

10.8 Transmission of PVY by progeny of known vector R. padi My results suggest that about one in 100 R. padi transmit PVY. Previous results (p.72) suggest this is probably because all R. padi are ineffic­ ient vectors rather than because most are non-vectors but a small proportion are good vectors. To further investigate thisy 120 R. padi were allowed access to PVY-infected leaves and then caged singly on tobacco test seedlings. After 2 h, each aphid was trans­ ferred and cultured singly on an oat seedling. The experiment was repeated once. One aphid transmitted the virus in the first and two in the second experiment. Aphid cultures started from these trans­ mitters are referred to as 'vector' whereas five aphids from those which did not transmit were used to produce the 'non-vectors'. In the first experiment, groups of 50 'vectors' and 'non­ vectors' infected respectively 47 and 42 of 100 test plants; in the second experiment 'vectors' infected 46 and 'non-vectors' 18 of 100 test plants. Thus, despite this selection 'vectors' still trans­ mitted about 17. on a single aphid basis. 155

D iscussion These results confirm that R. padi is a vector of both PVY° and PVY** but unlike Van Hoof (1980) and Ryden (1979), I obtained transmission by R* padi from and to tobacco as well as and to potato* This is of practical importance because the use of bait plants in crops to detect vectors of PVY (Rasocha, 1966 ; Van Hoof, 1977 ) would otherwise be of limited validity* R* padi was a much less efficient vector than M. persicae. Although R, padi probed potato less frequently than did M, persicae, experiments showed that a single probe by R, padi was much less efficient at acquiring PVY than a single probe by M* persicae. This suggests that the main cause of the differences in vectoring efficiency is associated with the association of virus and aphid mouthparts rather than differences in aphid behaviour; it is there­ fore perhaps surprising that R* padi retained PVY as long as did M* persicae* However, the duration of access to an infected leaf for optimum acquisition of PVY was longer for R. padi than for M* persicae* In ray experiments M, persicae acquired PVY optimally when allowed 10 min access to infected leaves, and long feeding

probes are known to be ineffective for acquisition (Watson & Roberts, 1939 ; Bradley, 1954 ). By contrast, acquisition by R, padi was undiminished even by access of several hours* However, R* padi kept on non-host leaf makes only brief probes (Wiktelius, 1982) and even given long periods of access seldom, if ever, make long feed­ ing probes. Ryden (1979) reported that alate R* padi frequently transmitted 156

PVY° whereas Kostiw (1979) reported inefficient transmission. Similarly Van Hoof (1980) reported that apterous R. pad! trans- mitted PVYN inefficiently and my results for both PVY O and PVY N and for alate and apterous R. padi support his conclusions. However, differences in vector efficiencies of aphid clones occur; for example, clones of M. persicae (Upreti & Nagaich, 1971 ) and of M. euphorbiae (Simons, 1966 ) differ considerably in efficiency of transmission of PVY". My low rates of transmission of R. padi could have resulted from the population being a mixture of a few efficient vectors and a majority of non-vectors. However, the rate of transmission of my population of R. padi remained stable and low throught the year transmitting at the constant low rate of ca. 17. and attempts to select efficient clones by breeding from vectors failed. Kurppa (1983) reported that all the attempts to transmit PVY by R. padi were unsuccessful. However, in ny experiments R. padi transmitted PVY0 and PVY N infrequently confirming previous reports (Kostiw, 1979 ; Ryden, 1979 ; Van Hoof, 1980 ). Similar discrep­ ancies have been reported with other aphid species. Thus, no transmission of PVY by B. helichrysl has been reported by Van Hoof (1980) although previous work suggested that it is an important vector of PVY (Edwards, 1963, 1965 ). This might be due to either different virus strain or aphid clone used. Therefore, it must be accepted that laboratory transmission results can give misleading information. Trapping of alive aphids in the field, as is reported in the following section, may more accurately identify the vector species under field conditions.

Because of the low rates of transmission of PVY by R. padi 157 and M. dirhodum and their relative rarity at Rothamsted (Taylor et al«t 1982 ) these species might not be important in the epid­ emiology of PVY in this area. However, R« padi is abundant in Scotland (Taylor et al.» 1982) and Sweden (Ryden et al«, 1983) and here it might play an important role in the spread of PVY. 158

11 SPREAD OF PVY BY APHIDS

Introduction As with other nonpersistently transmitted viruses, PVY is transmitted by aphids making brief probes but aphids lose infect- ivity within a few hours. Therefore,the spread of PVY is mainly within a crop from plants either growing from infected tubers (sec­ ondarily infected plants) or inoculated early in the growing season (primarily infected plants). PVY is transmitted mainly by alate aphids (Broadbent, 1950 ; Broadbent & Tinsley, 1951 ; Gabriel, 1961 ; Hille Ris Lambers, 1972 ; Van Harten, 1979 ). Although M. persicae is a very efficient vector of PVY commonly found in potato crops, in Poland, no correlation was found between its spread in potato crops and catches of alate M. persicae (Gabriel, 1961 ) and only a poor correlation in England (Broadbent, 1950 )• In Scotland most spread of PVY occurs before M. persicae is detected either on the crop or in yellow traps (Cadman & Chambers, 1960 ). M. euphorbiae, Aulacorthum solani (Kltb), Aphis nasturtii Kltb..Myzus certus (Ulk.)» Aphis frangulae Kltb., A. gossypii, Myzus ascalonicus Doncaster and Myzus oraatus. Laing also colonize potato crops (Radcliffe, 1982 ) and may spread PVY (Kennedy et al., 1962 ; Van Hoof, 1980). A. nasturtii and A. frangulae are the most important vectors of PVY in Poland (Gabriel, 1958, 1959, 1961 Gabriel et al, 1975), Czechoslovakia and East Germany (Gabriel et al., 1972) where they are abundant and in Norway (Bjornstad, 1948) A. nasturtii has been reported as the main vector. 159

Potatoes are also visited by alatae of some non-colonizing species (Broadbent, 1953 ) such as A. fabae, Brachycaudus helichrysi (Kltb.) and R. padi which can also transmit PVY (Edwards, 1963 ; 1965 Van Hoof, 1980 )• Non-colonizing aphids may be important vectors (p. 154) of PVY in crops, especially when they are abundant early in the growing season (Van Hoof, 1977 ; Sigvald, 1977 ; Robert, 1978 ; Ryden et al., 1983 ) when plants are most susceptible to infection. Non-colonizing aphids might even be more effective vectors than colonizing ones since the latter are less inclined to fly to another plant soon after alighting and probing (Kennedy, 1950 )• Host spread of PVY in Swedish potato crops is probably due to aphid species other than M. persicae (Emilson, 1958 ), and R. padi, a cereal aphid migrating early in the growing season, is an important vector there (Sigvald, 1977 ; Ryden et al., 1983 ), in France (Robert, 1978 ) and in the Netherlands (Van Hoof, 1977 ). However, in N. Ireland the leaf curling plum aphid B. helichrysi is an important vector (Edwards, 1963 , 1965 ; Bell, 1983 ). Laboratory tests have shown that over 25 aphid species can transmit PVY (Table 40). However, in laboratory tests only one or a few aphid biotypes and virus isolates can be tested and results may sometimes be misleading. For example, in laboratory tests B. helichrysi did not transmit PVY (Van Hoof, 1980 ) although it is an important vector in N. Ireland (Edwards, 1963 , 1965 ; Bell, 1983 ). It is also difficult in laboratory tests to assess effects of differ­ ent alighting behaviour on vectoring ability. The technique of trapping alive aphids on nets and suction traps downwind of infected crops and testing for infectivity has been 160

Table 40 • Aphid species identified as vectors of PVY

Aphid species Virus strain References

Acyrthosiphon pisum (Harris) pvyn Volk (1959), Van Hoof (1980) - Laird & Dickson (1963)

Aphis c itric o la Van der Goot - Raccah (1983)

Aphis craccivora Koch PVY° Eskarous et al. (1983)

Aphis fabae Scop* PVY° Eskarous et al. (1983) pvyn Volk (1959), Van Hoof (1980) - Bawden & Kassanis (1947)

Aphis frangulae Kltb. pvyh Volk (1959), Karl & Proeseler (1976)

Aphis gossypii Glov. - Laird & Dickson (1963), Raccah (1983)

Aphis nasturtii Kltb. PVY° Bradley & Rideout (1953), Ryden (1979) Kassanis (1942), Bawden & Kassanis (1947)

Aphis pomi de Geer pvyn Van Hoof (1980)

Aphis spiraecola Patch - Laird & Dickson (1963)

Aulacorthum circumflextira pvyn Volk (1959) (Buckton) - Dykstra & Whitaker (1938), Bawden & Kassanis (1947)

Aulacorthum solani (Kltb.) PVY° Bradley & Rideout (1953) pvyn Van Hoof (1980) - Dykstra & Whitaker (1938), Bawden & Kassanis (1947) 161

Aphid species Virus strain References Brachycaudus helichrysi (Kltb.) PVY* Edwards (1963) PVYN Edwards (1965) PVYC(AB) Bell (1983)

Capitophorus hippophaes (Wlk.) PVYN Van Hoof (1980)

Cavariella pastinacae (L.) Bawden & Kassanis (1947)

Lipaphis erysimi (Kltb,) PVY Heinze (1960)

Macrosiphoniella sanborni (Gill.) Bawden & Kassanis (1947)

Macrosiphum euphorbiae (Thomas) PVYv Bradley & Rideout (1953) PVYN Volk (1959), Van Hoof (1980) Dykstra & Whitaker (1938), Bawden & Kassanis (1947), Laird & Dickson (1963)

Metopolophlum dirhodum (Wlk.) PVYN Van Hoof (1980)

Microlophium primulae (Theobald) PVY H Heinze (1959)

Myzus certu s (W lk.) PVYN Heinze (1960), Van Hoof (1980) MacGillvray & Bradley (1960)

Myzus persicae (S u lz .) PVY Bradley & Rideout (1953), Ryden (1979) PVYN Volk (1959), Van Hoof (1980) - Bawden & Kassanis (1947), Laird & Dickson (1963), Raccah (1983)

Phorodon humuli (Schrank) PVY Van Hoof (1980) Karl (1971)

Rhopalosiphoninus latysiphon Bell (1982) (Davis) 162

Aphid species Virus strain References

Rhopalosiphoninus staphyleae pvyn Heinze (1959, 1960) subsp. tulipaellus (Koch)

Rhopalosiphum pad! (L.) PVY° Kostiw (1979), Ryden (1979) pvyn Van Hoof (1980)

Rhopalosiphum inserturn (Wlk.) pvyn Van Hoof (1980) 163 used for studying the epidemiology of several nonpersistent viruses (Knoke et al., 1977 ; Halbert et al., 1981 ; Highland et al.t 1981 ; Labonne et al.t 1982b; Raccah, 1983 ), although not of PVY in potato crops. Ideally, some measure of host plant susceptibility at the time of year each vector predominates is also required although this may be difficult to obtain. The purpose of this work was, by using such techniques, to determine which aphid species actually transmit PVY in potato crops at Rothamsted and how efficiently they do so.

Materials and Methods A plot 6m(8 rows)x50m of PVY-infected tubers cv. King Edward was planted in May 1984 at Rothamsted. When the plants had emerged, light blue netting (hexagonal mesh, diameter of hexagon ca. 1.5 mm) 1.4m x 4.4ra long was mounted between vertical canes downwind of the plot. As winged aphids were blown onto the windward side of the vertical net, they were collected and immediately caged indiv­ idually on tobacco test plants. (These plants were otherwise kept inside a cage to avoid contamination (Plate 16). Caged aphids were left for at least 2 h on the test plant before they were removed and preserved in ethanol for subsequent identification. From 8 June to 8 August aphids were trapped from ca. 10 am to 5 pm usually once a week. On some dates a suction trap (plate 15), trapping at 1.4 m, was also used. This trap was checked for aphids every five minutes. The results presented in the thesis are for trappings up to 11 July because aphids collected after this date have not yet been identif­ ied. Plants were discarded if aphids could not be found. Test plants were then kept in an aphid-free glasshouse for at least 3 wk, 164

Plate 15. View of the net and suction trap used for trapping aphids.

Plate 16. Tobacco cv. White Burley used to test whether trapped aphids carried PVY. 165

during which time they were Inspected daily for symptoms of PVY infection. Infection with PVY was confirmed by ELISA (p.52 ) in all plants developing symptoms of virus infection. One hundred test plants not exposed to aphids were also kept in the glasshouse each week to test that no PVY was spread there.

R esults A total of 3969 alates were trapped, by net and a suction trap downwind of the PVY-infected potato plot and 98 ( 2. 57.) of these infected test plants with PVY (Table 41). B. hellch.iysi, H. lactucae, M. euphorbiae, M. festucae, M. persicae, P. humuli, R. insertum and S. fragariae accounted for ca. 957. of all transmissions; these same species accounted for 467. of trapped specimens. B. helichrysi alone accounted for 787. of all transmissions although only comprising 247. of the total catch. B. helichrysi. M. euphorbiae, M. persicae and P. humuli had relatively high and similar transmission rates. H. lactucae, M. festucae, R. insertum and S. fragariae had similar low transmission rates (Table 42). At last 104 species were trapped and assayed (Table 41), includ­ ing several species that transmitted PVY once: eg A. nasturtii, C. ballotae, M. ligustri, M. myosotodis and M. tosarum. Some species trapped in relatively large numbers did not transmit PVY: Thus, none of 79 C. aegopodii, of 97 D. platanoidis, of 92 Ii. camosum, of 73 M. cerasi, of 916 S. a venae or of 74 Uroleucon spp. infected test plants with PVY. Fig. 4 shows the total number of aphids caught by net and 166

Table 41. Number of each aphid species caught and> in parentheses, number which infected test plants with PVY

Numbers trapped Aphid species by Net Suction trap Acyrthosiphon loti (Theobald) 1 Acyrthosiphon malvae (Mosley) 2 Acyrthosiphon pisum (Harris) 18 Anoecia corn! (Fabricius) 3 Anuraphis subterranea (Wlk.) 1 Aphis fabae Scop. 21 1 Aphis nasturtii Kltb. 1(1) Aphis pomi de Geer 29 Aphis rum icis (L .) 2 Aphis sambuci (L.) 4 Aphis spp. 56 4 Atheroides serrulatus Haliday 1 Aulacorthum pallustre Hille Ris 4 Lambers Aulacorthum solani (Kltb.) 5 Brachycaudus cardui (L .) 10 1 Brachycaudus helichrysi (kltb.) 943(76) 7 Brachycaudus klugkisti (Bonier) 1 Brachycaudus persicae (Passerini) 4 Brachycaudus rumexicolens (Patch) 2 Brevicoryne brassicae (L.) 4 Capitophorus horni Borher 2 Capitophorus elaeagni (del Guer.) 8 Capitophorus similis van der Goot 4 Cavariella aegopodii (Scop.) 77 2 Cavariella archangelicae (Scop.) 1 Cavariella pastinacae (L.) 5 Cavariella theobaldi (Gillette & Bragg) 7 Ceruaphis eriophori (Wlk.) 1 Chaetosiphon fragaefolii (Cockerell) 1 Cinara spp. 2 167

Table 41 . - continued

Aphid species Numbers trapped by Net Suction trap Cryptaphis poae (Hardy) l Cryptomyzus ballotae Hille Ris 1(1) Lumbers Cryptonryzus galeopsidis (Kltb.) 43 6 Cryptomyzus korschelti Borner 4 Cryptomyzus ribis (L. ) 3 Cryptomyzus spp. 1 Diuraphis muehlei (Bonier) 1 Drepanosiphum platanoidis (Schrank) 87 10 Dysaphis plantaginea (Passerini) 24 Dysaphis spp. 35 Elatobium abietinum (Wlk.) 8 Eriosoma ulmi Riley 6 Eriosoma spp. 1 Eucalipterus tiliae (L. ) 3 Hyadaphis foeniculi (Passerini) 15 1 Hyalopteroldes humilis (Wlk.) 3 Hyalopterns pruni (Geoffr.) 2 Hyperomyrus lactucae (L.) 118(1) 3 Hyperomyzus lampsanae (Bonier) 1 Hyperomyzus pallidus Hille Ris Lumbers 3 Kallistaphis basalis Stroyan 1 Liosomaphis berberidis (Kltb.) 11 Longicaudus trirhodus (Wlk.) 2 Macrosiphonie11a artemisiae (B. de F.) 5 Macrosiphonie11a m illefolii (de Geer) 6 Macrosiphoniella sejuncta (Wlk.) 5 Macrosiphoniella usquertensis Hille 1 Ris Larabers Macrosiphum euphorbiae (Thomas) 61(4) Macrosiphum funestum (Macchiati) 7 Macrosiphum rosae (L.) 36 168

Table 41. - continued

Aphid species Numbers trapped by Net Suction trap Macrosiphum spp. 0 1 Megoura viciae Buckton 4 Metopolophium albidum Hille Ris 1 Lambers Metopolophium dirhodum (Wlk.) 67 Metopolophium festucae (Theobald) 205(1) 4 Metopolophium frisicum Hille Ris 2 Lambers Metopolophium spp. 1 Microlophium camosum (Buckton) 92 Microlophium primulae (Theobald) 1 Myzaphis rosarum (Kltb.) 7(1) Myzocallis spp. 8 Myzus ascalonicus Done. 26 2 Myzus c e ra si (F .) 69 4 Myzus certus (Wlk.) 7 Myzus ligustri (Mosl.) 1( 1) Myzus myosotodis (Borner) 1( 1) Myzus persicae (Sulz.) 227(7) 2 Myzus spp. 1 Nasonovia pilosellae (Borner) 1 Nasonovia ribisnigri (Mosley) 42 2 Ovatus crataegarius (Wlk.) 9 Ovatus insitus (Wlk.) 1 Periphyllus hirticornis (Wlk.) 0 1 Periphyllus testudinaceus (Femie) 24 1 Periphyllus spp. 2 Phorodon humuli (Schrank) 46(2) 2 Pleotrichophorus glandulosus (Kltb.) 3 Protrama ranunculi (del Guercio) 2 Rhopalomyzus poae (Gillette) 1 Rhopalosiphoninus staphyleae (Koch) 7 1 Rhopalosiphum insertum (Wlk.) 115(1) 14 169

Table 41. - continued

Aphid species Numbers trapped by Net Suction trap

Rhopalosiphum padi (L.) 20 Sitobion avenae (Fabricius) 896 20 Sitobion fragariae (Wlk.) 86( 1) 2 Thecabius affinis (Kltb.) 1 Thelaxes dryophila (Schrank) 21 Tubaphis ranunculina (Wlk.) 2 Tuberculoides annulatus (Hartig) 52 3 Tuberculoides borealis (Krzjwec) 6 Uroleucon tussilaginis (Wlk.) 1 Uroleucon spp. 89 Utamphorophora humboldti (Essig) 3 Vesiculaphis theobaldi Takahashi 1 Wahlgreniella nervata (Gillette) 8 Unidentified 1

TOTAL 3.875 94 170

Table 42. Relative abundance, transmission efficiency and overall transmission of the major vector species trapped in the net

Aphid species R elative Transmission Transmission abundance (%) per species (7.) contributed by species B. h elich ry si 24.3 8.0 77.6 H. lactucae 3.0 0.8 1.0 M. euphorbiae 1.6 6.6 4.1 M. fes tucae 5.3 0.5 1.0 M. persicae 5.8 3.0 7.2 P. humuli 1.2 4.3 2.0 R. inserturn 3.0 0.9 1.0 S. fragariae 2.2 1.2 1.0 Subtotal (8 species) 46.4 5.2 94.9 Other species 53.6 0.2 5.1 Total 100.0 2.5 100.0 171 800i

Total numbers of aphids trapped

June July Figure 4. Numbers of aphids caught in nets close to a PVY- infected plot at Rothamsted, June 8 - July 11, 1984. 172

the number that were viruliferous at each date. The proportion of viruliferous aphids caught changed during the season and the total number of aphids caught and the number of viruliferous aphids were not well correlated. Factors such as wind speed and direction might have affected the proportion of aphids caught in the net which had previously alighted on the PVY-infected plot and therefore the proportion of viruliferous aphids trapped. For example, on 14 June only a small proportion of aphids transmitted PVY. However, on this date the wind was across the narrower part of the plot and few aphids may have alighted on the PVY-infected plot. The suction trap was used only in 4 of 8 sampling dates because few aphids were trapped by this means (Table 41). Table 43 shows the proportion of the major vectors of PVY caught near to the PVY-infected plot compared with the proportion of the same aphid species caught by the Rothamsted suction trap (sampling at 12.2 m at a distance 400 m from the infected plot). B. helichrysi was more abimdartin the net than in the Rothamsted suction trap. This trends was also true for other species such as H. lactucae. M. euphorbiae, M. persicae, R. inserturn and S. fragariae. Relative abundance of aphid species differred between the sampling dates (Fig. 5 ). B. helichrysi reached a peak at 14 June and then decreased. M. euphorbiae was rare on all the sampling dates and M. persicae increased after 19 June.

D iscussion Three methods of trapping live aphids have been used to assess infection pressure and detect aphid vectors. These are: 173

Table 43 . A comparison of the percentages in catches of the major vector species in the net and in the Rothamsted suction trap (trapping at 12,2 m )

Method of trapping Aphid species Net Rothamsted Suction

B. helichrysi 24.0 14.0

H. lactucae 3.0 0.38

M. euphorbiae 1.5 0.8 r** CM M. festucae 5.3 •

M. persicae 5.8 2.1

P. humuli 1.2 1.9

R. inserturn 3.4 1.0

S. fragariae 2.1 0.38

1 o c_n o ------1 i—- i—- i ------1 o CO o ------I I 1 1 ------1 o ° o o U1 ------Numbers Numbers of viruliferous • Numbers of helichrvsi B. B.helichrvsi (solid (solid bars) B.helichrvsi 1

------1 ------1 » » » i * -*■ ho U) tn o ui

Numbers Numbers of viruliferous 1 M.euphorbiae bars) (solid M.euphorbiae Numbers of euphorbiae M. ------LOVl

ro to » » i « ____ i ______1______i K) cr> o o o o Numbers of M. persicae Numbers of viruliferous M.persicae M.persicae (solid bars) i ] ] ] ------

Figure 5. Numbers of M. persicae, M. euphorbiae and B. hellchrysi caught in nets (open bars) and the numbers that transmitte PVY (solid bars). 175

suction traps (Plumb, 1976 ; Labonne- et al., 1982b; Raccah, 1983 ), vertical nets (Halbert et al.« 1981 ) and yellow traps (Highland et al., 1981 ). Although Moerlcke yellow traps (Moerlcke, 1951 ) are simple to operate, aphid species are attracted differentially (Eastop, 1955 )• It has been suggested that suction traps are better than yellow traps for monitoring aphid vectors of viruses since they trap more aphids (Labonne et al. t 1982a) although, Raccah (1983) reported the opposite. In this experiment fewer aphids were trapped by suction trap than by the net. The net trap also has the advant­ age that it is independent of electrical power but its performance is affected by wind speed. It has also been suggested that nets might catch disproportionally few alate vectors of colonizing species (Halbert et al.. 1981 ).

The proportions of each aphid species caught in the net and in the Rothamsted suction trap were often different. This may partly be because trapping in the net was usually carried out from 10 am to 5 pm, whereas Rothamsted suction trap was trapping contin­ uously. Alternatively, the net or nearby vegetation attracted some species preferentially whereas the 12.2 m suction traps are that height in order to minimize such preferential attraction (Taylor & Palmer, 1972 ). Several aphid species reported to transmit PVY under laborat­ ory conditions did not do so in this experiment: A. pisum (Volk, 1959 ), A. solani (Bawden & Kassanis, 1947 ; Bradley & Rideout, 1953 ; Van Hoof, 1980 ), A. fabae (Bawden & Kassanis, 1947 ; Volk, 1959 ; Van Hoof, 1980 ), A. pomi (Van Hoof, 1980 ), C. pastinacae 176

(Bawden & Kassanis, 1947 ), M. dirhodum (Van Hoof, 1980 ), M. certus

(Heinze, 1960 ; MacGillvray & Bradley, 1960 ; Van Hoof, 1980 ), R.

staphyleae (Heinze, 1959, 1960 ) and R. padl (Kostiw, 1979 ; Ryden, 1979 ; Van Hoof, 1980 ). Failure to detect any transmission by these species was probably because few of these species were trapped. On the other hand, C. ballotae, M. festucae, M. myosotodis, M. rosarum, M, ligustrl and S. fragarlae are reported for the first time as vectors of PVY. There may be differences in relative transmission rates of aphid species in field and laboratory conditions (Labonne et al.» 1982b; Raccah, 1983 ) and results reported here confirm this. Thus, in the field experiment B. helichrysi contained the greatest prop­ ortion of vectors followed by M, euphorbiae, P. humuli and then M. persicae. However, in laboratory tests (p.69 ) a greater proportion of M. persicae than of M. euphorbiae transmitted. The importance of aphids as vectors of PVY is also dependent on the season at which the main flight occurs relative to crop growth because potato plants develop mature plant resistance (Beemster, 1972 ), and virus spread early in the growing season might give new sources for further spread (p. 144). The majority of early season transmissions was by B. helichrysi. This aphid is probably the most important PVY vector at Rothamsted because it consistently flies early in the growing season, appear in large numbers (Taylor et al., 1982 ) and because it is a rel­ atively efficient vector. Although this species accounted for only 247. of the total catch, it caused nearly 787. of the total number of trans­ missions (Table 42), all early in the season. Later on from early to mid July M. persicae, from mid-July to 8 August P. humuli and various Aphis spp. transmitted PVY most frequently (unpublished results). 177

12. GENERAL DISCUSSION

Nonpersistent viruses are usually transmitted by more than one aphid species. There may be large differences in ability to transmit a particular virus between aphids of different species (Van Hoof, 1980) or even of the same species (Simons, 1959, 1966 ; Sohi & Swenson, 1964 ; Kvicala, 1968 ; Upreti & Nagaich 1971 ; Jurik et al., 1980 ; Singh et al., 1983 ). Saliva could contribute to specificity (Bradley 1959 ; Day & Irzykiewicz, 1954 ; Sylvester, 1954 ; Hashiba & Misawa, 1969 ; Nishi, 1969 ) but although saliva decreases virus infectivity (Nishi, 1969 ; Pirone, 1970 ), attempts to demonstrate selective effects on viruses have failed (Pirone, 1970 ). Van der Want (1954) proposed a mechanical-surface adherence hypothesis in which specificity was an expression of differences in the structure and surface adherence properties both of the stylets of different aphids and of particles of different viruses. There does not appear to be any relationship between stylet-tip morphology and vector specificity (Proeseler et al., 1972 ; Forbes, 1977 ). For example, Schmidt et al. (1979 ) found no substantial differences in stylet morphology of ten aphid species, which included M. persicae, a good vector of PVY and R. padi, a poor vector of PVY (Van Hoof, 1980 ). However, there is evidence for differences in the surface adherence of the stylets. Thus, large amounts of labelled virus were detected on the inner surface of mandibles of a biotype of M. euphorbiae that transmitted PSbMV frequently whereas only small amounts were on those of a biotype that transmitted PSbMV infrequently (Lim et al., 1977 ). The ingest­ ion-egestion hypothesis extends this idea by offering greater opport­ 178

unity for differences such as in the volume of sap ingested or egested and in the sites contacted. For example, uptake of virus into areas such as fore-gut might allow ingested viruses to come into contact with secretions from the hypodermal cells (Pirone £ Harris,1977) and differ­ ential activity of such secretions on viruses could result in specif­ icity (Garrett, 1973 ). In this thesis, experiments investigated this topic further. II, euphorbiae and R. padi transmitted PVY less frequently than M. persicae (p. 69) and a single probe by R, padi was much less likely to acquire PVY than a single probe by M. persicae (p.154). Similarly, a single probe of M. euphorbiae was less likely to acquire PVY than a single probe of M. persicae (Bradley & Rideout, 1953 ). All indiv­ iduals in a population of M, euphorbiae or R. padi seemed to be poor vectors (p.72 ) and attempts to increase transmission by breeding from vectors failed (p.154, Simons, 1959 ). R. padi and M. euphorbiae seldom inoculated two strains of PVY simultaneously (p.69 ) suggesting that they inoculated few virus particles perhaps because they imbibe less sap than good vectors such as M. persicae. Alternatively they may inoculate few virus particles because the particles are either attached poorly or so well that they are seldom released. Differences in area or volume of the virus receptor site(s) in aphid mouthparts might also explain differences in the number of virus particles inoc­ ulated. It has been calculated that the mouthparts of M. persicae can contain about 100 TSEV particles (Taylor & Robertson, 1974 ). However, the charge of TSEV obtained by M. persicae from a single acquisition probe can infect several plants (Taylor & Robertson, 1974 ), so the number of particles inoculated by a single probe is presumably less. 179

PVY^ inhibited transmission of PVY° and BMV; BMV inhibited transmission of PVY^. Being potyviruses, all three require helper component for aphid transmission (p.31 )• Of the several suggestions put forward to explain the role of helper component in mediating transmission of potyviruses (Govier & Kassanis, 1974a; Pirone, 1977 ; Pirone & Harris, 1977 ; Lopez-Abella et al., 1981 ), the suggestion that helper component may act by enabling virus to bind to receptor

sites in the aphid (Govier & Kassanis, 1974a) could fit with the interference described above. If these sites are common for PVY^, PVY N and BMV, competition either between the different helper comp­ onents or helper components and the virus for these sites might explain the interference observed. If binding is electrostatic (Kassanis & Govier, 1971a; Zschiegner et al., 1974 ; Morales, 1981 ), differences in charge as demonstrated by electrophoretic mobility might explain interference. However, comparison of the three viruses by gel electrophoresis revealed no consistent relationship between electrophoretic mobility and power of interference in transmission tests. A correlation between aphid transmissibility and electrophoretic mobility of five strains of BYMV has been reported (Morales, 1981 ). However, although there was a difference in electrophoretic mobility of PVY^ and PVYN (plate 14, Makkouk & Gumpf, 1976 ), no difference in aphid transmission was detected (p. 57). Charge interactions between virus-helper component-receptor sites cannot be ruled out by these data but they must be more subtle than the total charge of virus part- icles. Electrophoresis of helper components induced by PVY 0 , PVY N and BMV might also help to understand the mechanism of interference. Whether PVY N -helper component can interfere with transmission of PVY 0 and BMV should also be tested and tests for interference should also 180 be extended to other virus-vector systems. There was no interference between TMV, a non-aphid transmiss­ ible virus (Gibbs, 1977 ), and either PVY^ or PVY^ (p. 101). However, aphids transmit poly-L-ornithine (PLO)-treated TMV (Pirone & Kassanis, 1975 ), PLO, perhaps binding TMV to receptor sites in aphids (Pirone & Kassanis, 1975 ). It would be interesting to test whether such aphid transmissible TMV interferes. Potyviruses might not spread or spread only slowly in cultivars which produce no or little hdper component when infected. M. persicae acquired BMV more readily from sugarbeet than from N. clevelandii (p. 96 ) although virus concentration was lower in sugarbeet; it seems possible that this is the result of low concentration of helper component in N. clevelandii, and could provide a valuable model to test the feas­ ibility of this approach. In both tobacco and potato the presence of PVY° diminished the concentration of PVY N in doubly-infected plants, especially at 30 oC. Also it is also interesting that PVY N -infected tobacco kept at 30 o C produced no veinal necrosis whereas plants kept at 15°C did (plate 13). Leaves of Nicotiana tobacum cv. Xanthi-nc which have been inoculated with TMV develop local lesions at 22°C but remain symptomless if the plants are kept at 32°C (Weststeijn, 1984 ) when a necrosis-inducing factor is absent. Although the two systems are not identical because TMV produces local lesions at temperatures below 28 o C whereas PVY N , although producing necrotic lesions also is systemic, the mechanism might be similar. Studies on epidemiology of nonpersistent viruses involve monitor­ 181

ing vectors in order to interpret the pattern and sequence of virus spread and this must involve sampling aphids in flight (e.g. suction traps) or on landing (e.g. Moericke yellow trap). Numbers of differ­ ent aphid vector species trapped coupled with relative vectoring ability as determined from laboratory tests are used to estimate the infection pressure of PVY in the seed potato crop in the Netherlands (Van Marten, 1983 ). These are used to establish haulm destruction dates in seed growing areas. However, laboratory transmission tests mainly use one of few clones of apterous aphids whereas field trans­ mission is by many clones and by alates. Laboratory results should not perhaps be expected to be related to field transmission and, in one instance when large numbers of R. insertum, an apparently efficient vector of PVY (Van Hoof, 1980 ), had been flying, PVY spread was much less than predicted (Van Harten, 1983 ). Trapping of live aphids near to virus infected plots by suction traps (Labonne et al., 1982 ; Raccah, 1983 ), vertical nets (Halbert et al., 1981 ) or yellow traps (Highland et al*, 1981 ) and testing for infectivity has recently been used to determine the virus-trans­ mitting abilities of naturally occuring aphid populations. In a similar experiment in 1984 at Rothamsted, B. helichrysi was the most important vector of PVY confirming reports from N. Ireland (Edwards, 1963, 1965 ; Bell, 1983 ). However, this needs to be repeated for more years to check its general validity. B, helichrysi flies early at Rothamsted (Taylor et al., 1982 ) and therefore might expecially important because the lack of mature plant resistance (Beemster, 1972 ) means the crop will be easy to infect and early infections can serve 182 as sources for further spread later in the season (p.144). M. persicae contributed much less than B. helichrysi in overall trans­ missions and appeared later than B. helichrysi* Similarly, P. humuli and Aphis spp. were important from mid-July onwards* If B* helichrysi consistently is the most important vector, a detailed study of its biology might allow us to predict its flights. An analysis of the results of experiments in different parts of England and Wales, including Rothamsted, from 1941 to 1947 on the spread of PVY showed that there was a poor correlation between numbers of alate M* persicae and the spread of PVY (Bradbent, 1950 ). However, the experiment described in this thesis suggest that this is because non-colonizing aphids such as B* helichrysi are more important than M* persicae and there is good correlation between numbers of M. persicae and B* helichrysi and PVY spread at Rothamsted (Govier, personal communication). The role of primarily infected plants has been studied in both laboratory and field experiments. In laboratory tests with glass- house grown potato plants cv. King Edward, M. persicae acquired PVY N as readily from plants inoculated 20 days previously as from second­ arily-infected ones. With PVY^ this was also the case when plants were inoculated when three and five weeks old but plants inoculated with PVY^ when seven weeks old were poor sources. Field potato plants of cvs. Record and King Edward inoculated with either PVY^ of with PVY N early in the growing season acted as sources for further spread. There was no difference between the two cultivars or between PVY 0 and PVY In either because most spread from primarily infected plants have had occurred before mature plant resistance, even against PVY^, developed or because no mature plant resistance against either 183

PVY 0 or PVY N had developed. However, the effect of the date of inoculation on the role of primarily infected plants either with PVY 0 or with PVY N needs further investigation because potato plants develop mature plant resistance quicker against PVY^ than against PVY (Beemster, 1979 ). The importance of primarily infected plants was investigated under field conditions for only the cvs. Record and King Edward which are especially susceptible to PVY. However, con­ centration and therefore transmission of PVY from primarily infected plants, although not necessarily related to resistance to infection with PVY, might differ between cultivars (Bawden & Kassanis, 1947 ) as also does development of mature plant resistance (Beemster, 1976 ). The role of primarily infected plants should therefore also be tested using other cultivars including more resistent ones. Experiments described in this thesis, although not distinguishing between the major hypothesis explaining the mechanism of aphid trans­ mission of potyviruses, have demonstrated for the first time that these virus can interfere with the transmission of each other. The experiments described might also help either to devise new methods of control or to use more successfully conventional control measures against PVY or other nonpersistent viruses. An understanding how the concentration of PVY N is diminished by PVY 0 in doubly infected plants, especially at 30°C, might allow a mechanism to be developed ce by which this can be mimic^d without the dissadvantage of the plants being infected with PVY°. Similarly, finding chemicals which mimic the interference between the viruses might hinder spread in crops. The success of conventional control measures against aphid-borne viruses depends on timely application, and vector monitoring, as 184

described in this thesis will also be helpful in the choice and timing of control measures such as oil and pyrethroid sprays (Gibson et al., 1982 ), and haulm destruction in seed production areas. The finding that plants infected early in the growing season can serve as PVY sources suggests that control early in the season may be particularly important. 185

13. APPENDIX

N. K atis & R.W. Gibson (1984) Transmission of beet mosaic virus by cereal aphids. PI. Path. 33, 425-427 186

Plant Pathology (1984) 33, 425—427

NEW OR UNUSUAL RECORDS Transmission of beet mosaic virus by cereal aphids

N. KATIS and R. W. GIBSON Department of Plant Pathology, Rothamsted Experimental Station, Harpenden, Herts AL5 2JQ, U.K.

Watson & Healy (1953) were unable to correlate the incidence of beet mosaic virum (BMY) in sugar beet crops with catches of alate Myzits persicae (Sulzer) although this aphid colonizes beet and is an efficient vector (Sylvester, 1952). Similarly, Broadbent (1950) could not correlate spread of another potyvirus, potato virus Y (PVY), in potato crops with catches of alate M. persicae but subsequent work suggested that non-colonizing aphids, notably Brachycaudus helichrysi (Kltb.) (Edwards, 1963; 1965) and the cereal aphid Rhopalosiphum padi L (Kostiw, 1979; Ryde'n, 1979; Van Hoof, 1980), may be responsible for much spread of PVY. Although these aphids are inefficient vectors, they are often very numerous in Britain (Taylor e t al., 19S2) and so may be responsible for much of the spread that occurs. Other potyviruses spread by non­ colonizing aphids are onion yellow dwarf (Drake e t al., 1933), bean yellow mosaic (Cockbain, 1970). watermelon mosaic (Adlerz, 1974) and soybean mosaic (Halbert et al., 1981). Like PVY, BM V is known to be transmitted by B. helichrysi (Cockbain, 1970) and we report here its transmission by cereal aphids. Cereal aphids (R. padi, Metopolophium dirhodum (Walk.) and Sitobion avenae (F.)) were reared on oats cv. Blenda, and M. persicae on Chinese cabbage (Brassica pekinensis). Only adult apterae, starved for 2—4 h before use, were used in these tests. Cereal aphids were given 10 min acquisition access to BMV-infected sugar beet cv. Hilleshog monotri and M. persicae was given 2\z min (earlier experiments showed that these times gave good acquisition using these aphids for another potyvirus PVY). In two experiments, cereal aphids were then caged on individual test beet seedlings in groups of 20 or 50; Af. persicae because of known high rate of transmission were caged singly on test seedlings. Next day, the seedlings were sprayed with the aphicide pirimicarb and kept in a glasshouse (c. 20°C, 18 h light) to allow symptoms to develop. A third experiment compared the efficiency of transmission by R . padi and M. persicae allowed single acquisition probes. Aphids observed under a binocular microscope were allowed up to 15 min to make a first probe. The duration of this probe was recorded and aphids were then transferred to a beet test seedling and observed to ensure that they probed at least once. In the first experiment using groups of 50 cereal aphids, 25 of 40 test seedlings were infected by R . pad i,17 by 5. avenae and 12 by M. dirhodum, whereas single M. persicae infected 21 of 40 seedlings. In the second experiment using groups of 20 R . pad i,13 of 50 seedlings were infected, whereas single M. persicae infected 25 of 50. The proportions of test seedlings infected suggest transmission rates by single cereal apluds of 1—2% (Gibbs & Gower, 1960), compared to the 50% transmission by M. persicae. The rates of transmission of BM V by M. persicae. R. padi and M. dirhodum are similar to those obtained with PVY using the same aphid clones (Katis. unpublished) suggesting that the factor(s) that determine rate of transmission of each aphid species may be similar for both viruses. However unlike BMV, PVY is not transmitted b\ 5. avenae (Ryden, 1979; Van Hoof, 1980; Katis, unpublished). All three cereal apluds transmitted B M V much less efficiently than Al. persicae and, to test whether tills was related to probing behaviour, a third experiment compared the efficiency in acquiring the virus by single probes by R. padi and by M. persicae. The first 60 M. persicae tested all probed within the 15 min allowed, whereas 79 R. pad ihad to be tested to obtain 60 which had probed within 15 min. Probes by M. persicae lasted 21 ± 1-6 s and 27 of the aphids 187

426 N. Katis and R. W. Gibson transmitted. Probes by R. pad ilasted 40 ± 8-8 s but none of the apliids transmitted. Thus although R . pad may i probe less frequently than M. pcrsicae the major reason for the inefficiency of R. padi is the small probability that apliids which have probed will acquire and transmit BMV. Despite their low rates of transmission, these cereal aphids could nevertheless be important vectors of BMV. They appear in large numbers and are widely distributed in England during summer (Taylor e t al., 1982) and together with B. helichr}fsi may account for much of the discrepancy reported by Watson & Healy (1953) between incidence of BM V infected plants and trap catches of M. persicae. Flying aphids cannot recognize their host plants (Swenson, 1968) and apliids may alight equally on non-host and on host plants (Kennedy e t al., 1959a, b) making short duration probes to determine its suitability as a host. Brief probes are sufficient for acquisi­ tion of non-persistently transmitted viruses by vector species and mean that non-colonizing aphids can be responsible for much virus spread.

ACKNOWLEDGEMENTS N. Katis thanks the Greek State Scholarship Foundation for his research studentship and D. A. Govier and R. H. A. Coutts for valuable advice.

REFERENCES Adlerz W.C. (1974) Spring aphid flights and incidence of watermelon mosaic viruses 1 and 2 in . Phytopathology 64, 350—353. Broadbent L. (1950) The correlation of aphid numbers with the spread of leaf roll and rugose mosaic in potato crops. Annals of Applied Biology 37, 58—65. Cockbain A.J. (1970) The importance of leaf curling plum aphid Brachycaudus helichrysi as a common virus vector. Rothamsted Experimental Station Report for 1969, Part l,pp. 235— 236. Drake C.J., Tate H.D., & Harris H.M. (1933) The relationship of aphids to the transmission of yellow dwarf of . Journal of Economic Entomology 26, 841— 846. Edwards A.R. (1963) A non-colonizing aphid vector of potato virus diseases. Nature, London 200, 1233-1234. Edwards A.R. (1965) The leaf curling plum aphid, Brachycaudus helichrysi (Kltb) — a new’ vector of the tobacco veinal necrosis virus of potato. Record of Agricultural Research XIII, 75-79. Gibbs, A.J. & Gower, J.C. (1960) The use of a multiple-transfer method in transmission studies — some statistical points arising in the analysis of results. Annals of Applied Biology 48, 75-83. Halbert S.E., Irwin M.E. & Goodman R.M. (1981) Alate aphid (Homoptera: ) species and their relative importance as field vectors of soybean mosaic virus. Annals of Applied Biology 97, 1—9. Kennedy J.S., Booth C.O. & Kershaw W.J.S. (1959a) Host finding by aphids in the field I. Gyno- parae of Myzus persicac (Sulzer). Annals of Applied Biolog}’ 47,410—423. Kennedy J.S., Booth C.O. & Kershaw W.J.S. (1959b) Host finding by aphids in the field II. A phis fabae Scop, (gynoparae) and Brevicoryne brassicae L. with a reappraisal of the role of host finding behaviour in virus spread. Annals of Applied Biology 47,424—444. Kostiw M. (1979) Transmission of potato virus Y by Rhopalosiphum padi L. Potato Research 22, 237-238. Ryden K. (1979) Harvebladllusen, R h. Padi, kan sprida potatis virus Y. Vaxtskyddnotiser 43, 51-53. Swenson K.G. (1968) Role of aphids in the ecology of plant viruses. Annual Review of Phyto­ pathology 6,351—374. Sylvester E.S. (1952) Comparative transmission of beet mosaic virus by four aphid species. Phytopathology 42, 252—254. Taylor L.R., Macaulay E.D.M., Dupuch M.J. & Nicklen J. (1982) Rothamsted survey thir­ teenth annual summary. Rothamsted Experimental Station Report for 1981Part 2, pp. 1 SO­ NS. 188

New or unusual records 427 Van Hoof H.A. (1980) Aphid vectors of potato virus YN . Netherlands Journal of Plant Patho­ logy 86, 159-162. Watson M.A. & Healy M.J.R. (1953) The spread of beet yellows and beet mosaic viruses in the sugarbeet root crop II. The effect of aphid numbers on disease incidence. Annals of Applied B iology 40, 38—59. 189

14. REFERENCES

Abney, T.S., Sillings, J.O., Richards, T. L. & Broersma, D.M. (1976). Aphids and other as vectors of soybean mosaic virus. J. econ. Ent. 69, 254-256. Adlerz, W.C. (1974). Spring aphid flights and incidence of water­ melon mosaic viruses 1 and 2 in Florida. Phytopathology 64, 350-353. Adlerz, W.C. (1978). Secondary spread of watermelon mosaic virus 2 by Anuraphis middletonii. J. econ. Ent. 71, 531-533. Akhatova, F. Kh., Eliseeva, Z.N. & Katin, I.A. (1980). Reservoirs of potato virus Y. Rev. Plant Pathol. 59, 875. Anderson, C.W. (1959). A study of field sources and spread of five viruses of peppers in centrol Florida. Phytopathology 49, 97-101. Anderson, C.U. & Corbett, M.K. (1957). Virus diseases of peppers in central Florida. PI. Pis. Reptr. 41, 143-147.

Atiri, G.X., Ekpo, E.J.A. & Thottappilly, G. (1984). The effect of aphid-resistance in cowpea on infestation and development of Aphis craccivora and the transmission of cowpea aphid-borne mosaic virus. Ann, appl. Biol. 104, 339-346. Badami, R.S. (1958). Changes in the transmissibility by aphids of a strain of cucumber mosaic virus. Ann, appl. Biol. 46, 554-562. Bagnall, R.H. & Bradley, R.H.E. (1958). Resistance to virus Y in the potato. Phytopathology 48, 121-125. Bar-Joseph, M. & Loebenstein, G. (1973). Effects of strain, source plant, and temperature on the transmissibility of citrus tristeza virus by the melon aphid. Phytopathology 63, 716-720. Barker, H. & Harrison, B.D. (1978). Double infection, interference and superinfection in protoplasts exposed to two strains of rasberry ringspot virus. J. gen. Virol. 40, 647-658. Barnett, C.B. Jr. & Pirone, T.P. (1966). Stylet-borne virus: active probing by aphids not required for acquisition. Science 154, 221- 222. Bartels, R. (1957). Serologische Differenzierungsversuche mit Stammen des kartoffel-Y-virus. In proceedings of the 3rd Conference on Potato Virus Diseases, Lisse-Wageningen, 1956, pp. 13-19. Bawden, F.C. (1950). Plant Viruses and Virus Diseases, 3rd ed. Chronica Botanica Company, Waltham, Mass. Bawden, F.C. (1964). Method of assay. In: Plant Virology (M.K.Corbett 190

and H.D. Sisler, eds.), pp. 365-385. Univ. Florida Press, Gainesville, 572 pp. Bawden, F.C. & Kassanis, B. (1946). Varietal differences in sus­ ceptibility to potato virus Y. Ann.appl. Biol. 33, 46-50. Bawden, F.C. & Kassanis, B. (1947). The behaviour of some naturally occuring strains of potato virus Y. Ann, appl. Biol. 34, 503-516. Bawden, F.C. & Roberts, F.M. (1948). Photosynthesis and predispos­ ition of plants to infection with certain viruses. Ann, appl. Biol. 35, 418-428. Bawden, F.C. & Kassanis, B. (1951). Serologically related strains of potato virus Y are not mutually antagonistic in plants. Ann, appl. Biol. 38, 402-410.

Bawden, F.C. Hamlyn, B.M.G. & Watson, M.A. (1954). The d istrib u tio n of viruses in different leaf tissues and its influence on virus transmission by aphids. Ann, appl. Biol. 41, 229-239. Beemster, A. B.R. (1961a). Een vergelijking tussen het transport van X-virus en twee verchillende stammen van Y-virus in aardappelplanten. Tijdschr. PPZiekt. 67, 278-279. Beemster, A.B.R. (1961b). Translocation of leaf roll and potato virus Y in the potato. In proceedings of the 4th Conference on Potato Virus Diseases, Braunschweig, 1960, pp.60-67. Beemster, A.B.R. (1965). Het transport van Y-virus in aardappelplanten na besmetting door bladluizen. Meded LandbHoogesch. OpzoekStns Gent 30, 1786-1795. Beemster, A. B.R. (1967). Partial infection with potato virus Y N of tubers from primarily infected potato plants. Neth. J. PI. Path. 23, 161-164. Beemster, A.B.R. (1972). Virus translocation in potato plants and mature plant resistance. In "Viruses of Potatoes and Seed Potato Production" (De Bokx, J.A. ed.) , pp.144-151. Center of Agricultural Publishing and Documentation, Wageningen. Beemster, A.B.R. (1976). Translocation of the potato viruses Y N and Y^ in some potato varieties. Potato Res, 19, 169-179. Beemster, A.B.R. (1979). Acquisition of potato virus Y N by Myzus persicae from primarily infected 'Bintje' potato plants. Neth. J. PI. Path. 85, 75-81. Behncken, G.M. (1970). The occurrence of peanut mottle virus in Queensland. Aust. J. agric. Res. 21, 465-472. Beiss, U. (1963). Infectiositat der nukleinsaure vom mosaik virus der riiben. Naturwissenchaften 50, 675. Bell, A.C. (1982). The bulb and potato aphid Rhopalosiphoninus latysiphon (Davidson) as a vector of non-persistent potato viruses. Rec. Agric. Res. (N. Ir.) 30, 1-3. 191

Bell, A.C. (1983), The life-history of the leaf-curling plum aphid Brachycaudus helichrysi in Northern Ireland and its ability to transmit potato virus yC(AB)# Ann, appl. Biol. 102, 1-6. Benda, G.T.A. (1956). Infection of Nicotiana glutinosa L. following injection of two strains of tobacco mosaic virus into a single cell. Virology 2, 820-827. Bennett, C.W. (1949). Some unreported host plants of sugarbfeet mosaic virus. Phy top a thology 39, 669-672. Bennett, C.W. (1967).Apparent absence of cross-protection between strains of the curly top virus in the beet leafhopper, Circulifer tenellus. Phytopathology 57, 207-209. Bercks, R. (I960). Serological relationships between beet mosaic virus potato virus Y, and bean yellow mosaic virus. Virology 12, 311-313. Bjornstad, A. (1948). Virussjukdommer pa Potet i Norge. Nord. FordbrForskn. pp. 586-590. Bokx, J. A. de. (1961). Het toetsen van aardappelknollen op de aanwezigheid van YN-virus. Tijdschr. PlZiekt. 27, 333-342. Bokx, J. A. de. (1979). Determination of infection pressure of potato virus Yn with potato plants. Med. Fac. Landbouww. RUksimiv. Gent. 44, 653-656.

Bokx, J.A. de., Kratchanova, B. & Maat, D.Z. (1975). Some properties of a deviating strain of potato virus Y. Potato Res. 18, 38-51.

Bokx, J.A. de. & Piron , P.G.M. (1977). Effect of temperature on symptom expression and relative virus concentration in potato plants infected with potato virus Y^ and Y°. Potato Res. 20, 207-213. Bokx, J.A. de., Hoof, H.A. van. & Piron , P.G.M. (1978). Relation between concentration of potato virus YN and its availability to Myzus persicae. Neth. J. PI. Path. 84, 95-100. Bokx, J.A. de. & Maat, D.Z. (1979). Detection of potato virus Y N in tubers with the enzyme-linked immunosorbent assay (ELISA). Med. Fac. Landbouww. Rijksuniv.Gent. 44, 635-644.

Bokx, J.A. de. & Huttinga, H. (1981). Potato virus Y. C.M.I./A.A.B. Descriptions of Plant Viruses. No. 242. Commonwealth Mycol. In s t., Kew, England. Bos, L., Huljberts, N., Huttinga, H. & Maat, D.Z. (1978). Leek yellow stripe virus and its relationships to onion yellow dwarf virus: characterization, ecology and possible control. Neth. J. PI. Path. 84, 185-204. Braber, J.M., Bus, C.B. & Schepers, A. (1982). Changes in leaf components and peroxidase activity of potato plants (cv. Bintjie) in relation to mature-plant resistance to PVY^. Potato Res. 25, 141-153. 192

Bradley, R.H.E. (1952). Studies on the aphid transmission of a strain of henbane mosaic virus. Ann, appl. Biol. 39, 78-97. Bradley, R.H.E. (1954). Studies on the mechanism of transmission of PVY by the green peach aphid, Myzus persicae (Sulz.) (Homoptera: Aphidae). Can. J. Zool. 32, 64-73. Bradley, R.H.E. (1959). Loss of virus from the stylets of aphids. Virology £, 308-318. Bradley, R.H.E. (1961). Our concepts: on rock or sand ? Recent Adv. Bot. _1, 528-533. Univ. of Toronto Press, Toronto, Canada. Bradley, R.H.E. (1962). Different areas of tobacco leaves as sources of potato virys Y for aphids. Virology 16, 366-370. Bradley, R.H.E. (1963). Some ways in which a paraffin oil impedes aphid transmission of potato virus Y. Can. J. Microbiol. 369-380. Bradley, R.H.E. (1964). Aphid transmission of sty let-borne viruses. In: Plant Virology (M.K. Corbett, and H.D. Sisler, eds.), pp. 148-174. Univ. Florida Press, Gainseville, 527 pp. Bradley, R.H.E. & Rideout, D.W. (1953). Comparative transmission of potato virus Y by four aphid species that infest potato. Can. J. Zool. 31, 333-341. Bradley, R.H.E. & Ganong, R.Y. (1955a). Evidence that potato virus Y is carried near the tip of the stylets of the aphid vector Myzus persicae (Sulz.). Can. J. Microbiol. 1, 775-782. Bradley, R.H.E. & Ganong, R.Y. (1955b). Some effects of formaldehyde on potato virus Y in vitro, and ability to transmit the virus when their stylets are treated with formaldehyde. Can. J. Microbiol. _1, 783-793. Bradley, R.H.E., Wade, C.V. & Wood, F.A. (1962). Aphid transmission of potato virus Y inhibited by oils. Virology 18, 327-328. Brierley, P. & Smith, F.F. (1944). Studies on lily virus diseases: the mottle group. Phytopatholody 34, 718-746. Broadbent, L. (1949). Factors affecting the activity of alatae of the aphids Myzus persicae (Sulzer) and Brevecoryne brassicae (L.). Ann, appl. Biol. 36, 40-62. Broadbent, L. (1950). The correlation of aphid numbers with the spread of leaf roll and rugose mosaic in potato crops. Ann, appl. Biol. 37 , 58 -65. Broadbent, L. (1953). Aphids and virus diseases in potato crops. Biol. Rev. 28, 350-380. Broadbent, L. (1960). Infectivity of aphids bred on virus-infected cauliflower plants. Ann, appl. Biol. 48, 377-383. 193

Broadbent, L. & Tinsley. T.W. (1951). Experiments on the colonization of potato plants by apterous and alate aphids in relation to the spread of virus diseases. Ann, appl. Biol. 38, 411-424. Broadbent, L. & Martini, C. (1959). The spread of plant viruses. Adv. Virus Res. 6», 93-135. Bromley, A.K. & Anderson, M. (1982). An electrophysiological study of olfaction in the aphid Nasonovia ribls-nigri. Ent. exp, appl. 32, 101-110. Cadman, C.H. & Chambers, C.J. (I960). Factors affecting the spread of aphid-borne viruses in potato in eastern Scotland. Ill Effects of planting date, roguing and age of crop of the spread of potato leaf-roll and Y viruses. Ann, appl. Biol. 48, 729-738. Carlebach, R., Raccah, B. & Loebenstein, G. (1982). Detection of potato virus Y in the aphid Myzus persicae by enzyme-linked immunosorbent assay (ELISA). Ann, appl. Biol. 101, 511-516.

Carlos Ramallo, J., D'elia de diaz Botta, E. & Weht, S. (1981). Solanum chacoense Bitt y Solanum nigrum L., weeds carrying potato virus X and Y. Rev. Plant. Pathol. 60, 1205. Cassels, A.C. & Herrick, C.C. (1977). Cross protection between mild and severe strains of tobacco mosaic virus in doubly inoculated tomato plants. Virology 78, 253-260. Castillo, M.B. & Orlob, G.B. (1966). Transmission of two strains of cucumber and alfalfa mosaic viruses by single aphids of Myzus persicae. Phytopathology 56, 1028-1030. Chagas, C.M., Vicente, M., Alba, A.P.C. & July, J.R. (1977). Solanum atropurpureum Schrank, a natural reservoir of potato virus Y (PVY). Phytopath. 2. 90, 147-151. Chapman, R.F., Bernays, E.A. & Simpson, S.J. (1981). Attraction and repulsion of the aphid Cavariella aegopodii, by plant odours. J. Chem. Ecol, Tj 881-888. Chiu, W.F. & Chang, Y.H. (1982). Advances of science of plant protect­ ion in the people's republic of China. A. Rev. Phytopathol. 20, 71-92. Clark, M.F. & Adams, A.N. (1977). Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J. gen. Virol. 34, 475-483. Clinch, P.E.M., Loughnane, J.B. & Murphy, P.A. (1936). A study of the aucuha or yellow mosaics of potato. Scient. Proc. R. Dubl. Soc. 21, 431-448. Cockbain, A. (1970). The importance of the leaf-curling plum aphid, Brachycaudus helichiysi as a common virus vector. Rep. Rothamsted exp, stn. 1969, p. 235. 194

Cockbain, A.J., Gibbs, A.J. & Heathcote, G.D. (1963). Some factors affecting the transmission of sugarbeet mosaic and pea mosaic viruses by Aphis fabae and Myzus persicae. Ann, appl. Biol, 52, 133-143. Conti, H., Caciagli, P. & Casetta, A. (1981). Differences in the aphid transmission of cucumber mosaic and some potyviruses. Fifth international congress of Virology, Strasbourg, France, August 2-7, 1981. pp.214. Costa Lima Neto, V. da. & Costa, A.S. (1978). Transmissao experimental do virus do mosaico comum da soja com afidios que ocorrem em trigo. Summa Phytopathologies 4, 125-127. Coudriet, D.T. (1962). Efficiency of various insects as vectors of cucumber mosaic and watermelon mosaic viruses in cantaloups. J. econ. Ent. 55, 519-520.

Crumb, S.E. & McWhorter, F.P.M. (1948). Dusting beans against aphid vectors failed to give economic control of yellow bean mosaic. PI. Pis. Reptr. 32, 169-171. Daniel, G. & Hunnius, D. (1980). Nachweis der kartoffelviren M, S, X and Y in pre saften sekundarinfizierter kartoffelpflanzen mit ELISA (enzyme-linked immunosorbent assay). Gesunde Plf. 32, 118-127. Dawson, J.R.O., Motoyoshi, F., Watts, J.W. & Bancroft, J.B. (1975). Production of RNA and coat protein of wild-type isolate and a temperature-sensitive mutant of cowpea chlorotic mottle virus in cowpea leaves and tobacco protoplasts. J. gen. Virol. 29, 99-107. Day, M.F. & Irzykiewicz, H. (1954). On the mechanism of transmission of non-persistent phytophathogenic viruses by aphids. Aust. J. Biol. Sci. 2> 251-273. Dickson, R.C. Swift, J.E., Anderson, L.D. & Middleton, J.T. (1949). Insect vectors of cantaloupe mosaic in California's desert valleys. J, econ. Ent. 42, 770-774. Dickson, R.C. & Laird, E.F. (1959). California desert and coastal populations of flying aphids and the spread of lettuce mosaic virus. J. econ. Ent. 52, 440-443.

Doncaster, J.P. & Gregory, P.H. (1948), The spread of virus diseases in the potato crop. Agricultural Research Council Report Series No. 7. London, 189 pp. Drake, C.J., Tate, H. D. & Harris, H.M. (1933). The relationship of aphids to the transmission of yellow dwarf of onions. J. econ. Ent. 26, 841-846. 195

Duffus, J. E. (1971). Role of weeds in the incidence of virus diseases* A* Rev* Phytopathol* % 319-340* Duncan, J ., Genereux, H. & Couture, G.R. (1956). La dissemination dans le champ de la mosaique et de l'enroulement des feuilles par les pucerons de la pomme de terre. Ann* ent* Soc* Queb* _2, 53-59. Dykstra, T*P* (1933). Weeds as possible carriers of leaf roll and rugose mosaic of potato. J* agric. Res. 47, 17-32. Dykstra, T.P. & Whitaker, W.C. (1938). Experiments on the transmission of potato viruses by vectors. J. agric. Res. 57, 319-334. Eastop, V. F* (1955). Selection of aphid species by different kinds of insect traps. Nature 176, 936. Edwards, A.R. (1963). A non-colonizing aphid vector of potato virus diseases. Nature 200, 1233-1234. Edwards, A.R. (1965). The leaf curling plum aphid, Brachycaudus helichrysi (Kltb.) - a new vector of the tobacco veinal necrosis virus of potato. Rec. Agric. Res. (N. Ir.) 13, 75-79. El Kady, E.A., Omar, R.A. & Kishtah, A.A. (1973). Studies on tobacco veinal necrosis virus (a strain of potato virys Y) and its vector Myzus persicae (Sulz.). (Homoptera: Aphididae). Bull. Soc. ent. Egypte 57, 109-115. Emilson, B. (1958). Studies in the spread of virus diseases in the potato crop in Sweden. Eur. Potato J. JL, 46-47. Eskarous, J.K., Habib, H.M., Kishtah, A.A. & Ismah, M.H. (1983). A strain of potato virus Y isolated from solanum nigrum var. judaicum in Egypt. Phytopath. Medit. 22, 53-58. Feldman, J.M. & Gracia, 0. (1972). Studies of weed plants as sources of viruses. III.Natural infections of some weeds with tobacco mosaic, cucumber mosaic and potato Y viruses. Phytopath. 2. 73. 251-255. Fisken, A.G. (1959). Factors affecting the spread of aphid-borne viruses in potato in Eastern Scotland II. infestation of the potato crop by potato aphids,particularly Myzus persicae (Sulzer). Ann, appl. Biol. 47, 274-286. Forbes, R. (1977). The mouthparts and feeding mechanism of aphids. In: Aphids as Virus Vectors (K.F. Harris and K. Maramorosch, eds.), pp. 83-103. Academic Press, New York. Fujisawa, I., Tsuchizaki, T. & Iizuka, N. (1983). Purification and serology of beet mosaic virus. Ann. Phytopath. Soc. Japan 49, 22-31. 196

Gabriel, W. (1958). Etudes* sur les vecteurs des maladies a virus \ de la pomme de terre en Pologne. Parasitica 14, 119-134. Gabriel, W. (1959). a) Studies of the vectors of virus diseases in potatoes in Poland, b) Preliminary investigations on correlation between the number of potato aphids and spread of virus diseases in the potato crops. Eur. Potato J. 2, 120. Gabriel, W. (1961). L*importance des certaines especes de pucerons pour la propagation des maladies a virus de la pomme de terre en Pologne. In proceeding of the 4th Conference on Potato Virus Diseases, Braunschweig, 1960, p.126. Gabriel, W., Neitzel, K., Rosocha, V., Wojcik, A.R., Debus, R., Nuckowski, S. & Klinkowsky, M. (1972). Die beziehungen zwischen dem auftreten der vektoren und der hohe des virusbesatzes (Blattroll- und Y-virus) bei kartoffeln in mitteleuropa. Ziemniak 27-63. Gabriel, W., Kostiw, M. & Wislocka, M. (1975). Comparison de plusieurs methodes d*estimation de la quantite de pucerons vecteurs de virus, pour la prevision d*infection par virus des tubercules de poimnes de terre. Potato Res. 18, 3-15. Gamez, R. & Watson, M. (1964). Failure of anaesthetized aphids to acquire or transmit henbane mosaic virus when their stylets were artificially inserted into leaves of infected or healthy tobacco plants. Virology 22, 292-295. Garrett, R.G. (1971). The mechanism of transmission of non-persistent viruses by aphids. Ph.D. Thesis, University of Adelaide, Australia. Garrett, R.G. (1973). Non-persistent aphid-borne viruses. Ins Viruses and Invertebrates (A.J. Gibbs, ed,), pp. 477-492. North-Holland Publishing Company, Amsterdam and London. Gera, A., Loebenstein, G. & Raccah, B. (1979). Protein coats of two strains of cucumber mosaic virus affect transmission by Aphis gossypii. Phytopathology 69, 396-399. Gibbs, A.J. (1977). Tobamovirus group. C.M.I./A.A.B. Descriptions of plant viruses. No. 184. Commonwealth Mycol. Inst., Kew, England. Gibbs, A.J. & Gower, J.C. (1960). The use of a multiple transfer method in plant virus transmission studies. Some statistical points arising in the analysis of results. Ann, appl. Biol. 48, 75-83. Gibson, R.W. (1971). A study of the distributions of five aphid species on potato plants. Ph.D. Thesis, University of Bristol, England. 197

Gibson, R.W. & Plumb, R.T. (1977). Breeding plants for resistance to aphid infestation. In: Aphids as Virus Vectors (K. F. Harris and K. Maramorosch, eds.), pp. 473-500. Academic Press, New York. Gibson, R.W. & Heard, A.J. (1979). The resistance of two clones of perennial ryegrass and their progeny to ryegrass mosaic virus from different parts of Britain. Ann, appl. Biol. 91, 101-106.

Gibson, R.U., Rice, A.D. & Sawicki, R.M. (1982). E ffects of the pyrethoid deltamethrin on the acquisition and inoculation of viruses by Myzus persicae. Ann, appl. Biol. 100, 49-54.

Gildow, F.E. & Rochow, W.F. (1980). Transmission interference between two isolates of barley yellow dwarf in Macrosiphum avenae. Phytopathology 70, 122-126. Gooding, G.V.Jr., Young, J.B. & Lapp, N.A. (1975). Physalis virginiana, a perennial weed host of potato virus Y in north Carolina. Proc. Amer. Phytopathol* Soc. 2, 134 (Abstr.) Govier, D.A. (1983). Potato virus diseases at Rothamsted. Rep. Rothamsted exp. stn. 1982, pp.206-207. Govier, D.A. & Kassanis, B. (1974a). Evidence that a component other than the virus particle is needed for aphid transmission of potato virus Y. Virology 57, 285-286.

Govier, D.A. & Kassanis, B. (1974b). A virus-induced component of plant sap needed when aphids acquire potato virus Y from purified preparations. Virology 61, 420-426.

Govier, D.A., K assanis, B. & Pirone, T.P. (1977). Partial purification and characterization of the potato virus Y helper component. Virology 78, 306-314.

Gracia, 0. & Feldman, J.M. (1972). Studies of weed plants as sources of viruses. I. Datura ferox as a source of beet curly top virus. Phytopath. Z. 73, 69-74. Gugerli, P. (1978). The detection of two potato viruses by enzyme- linked immunosorbent assay (ELISA). Phytopath. 2. 92, 51-56. Gunenc, Y. & Gibson R. W. (1980). Effects of glandular foliar hairs on the spread of potato virus Y. Potato Res, 23, 345-351. Halbert, S.E., Irwin, M.E. & Goodman, R.M. (1981). Alate aphid (Horaoptera: Aphididae) species and their relative importance as field vectors of soybean mosaic virus. Ann, appl. Biol. 97, 1-9. Halbert, S.E., Irwin, M.E. & Goodman, R.M. (1983). Dynamics of aphid transmission of soybean mosaic virus. In: Proceedings of the first Chine/U.S.A. soybean Symposium and working group meeting. July, 1982. Edited by B.J. Irwin, J.B. Sinclair and Wang Jin-ling. 198

Hally C. E. (1964). Electron microscopy: Principles and application to virus research. In: Plant Virology. (M.K. Corbett and H.D. Sisler, eds. ) 9 pp.253-266. Univ. Florida Press. Gainesville, 527 pp. Hamlyn, B.M.G. (1953). Quantitative studies on the transmission of cabbage black ringspot virus by Myzus persicae (Sulz). Ann. appl. Biol. 40. 393-403. Harris, K. F. (1976). '*Nature of the importance of supervicial probing to nonpersistent, noncirculative virus transmission by aphids". Presented at Annu. Meet. Entomol. Soc. Am., Honolulu. Harris, K.F. (1977a). An ingestion-egestion hypothesis of noncirculat­ ive virus transmission. In: Aphids as Virus Vectors (K.F. Harris and K. Maramorosch, eds.), pp. 165-220. Academic Press, New York. Harris, K.F. (1977b). "Resumes de la troisieme Conference Internation­ ale de Virologie Comparee, Mont. Gabriel (Quebec)", pp. 35-36. Harris, K.F. (1979). Leafhoppers and aphids as biological vectors: vector-virus relationships. In: Leafhopper Vectors and Plant Disease Agents. (K. Maramorosch and K.F. Harris, eds.), pp. 217- 308. Academic Press, New York. Harris, K.F. (1981). and nemarlode vectors of plant viruses. A. Rev. Phytopathol. 19, 391-426. Harris, K.F. (1983). Sternorrhynchous vectors of plant viruses: Virus-vector interactions and transmission mechanisms. Ady. Virus Res. 28, 113-140. Harris, K.F. & Bath, J.E. (1973). Regurgitation of Myzus persicae during membrane feeding: its likely function in transmission of nonpersistent plant viruses. Ann, ent. Soc. Am. 66, 793-796. Harrison, B.D. (1958). Ability of single aphids to transmit both avirulent and virulent strains of potato leaf roll virus. Virology £, 278-286. Harten, A. Van. (1979). Summaries of lectures given at the "Potato Aphid Identification and Virus Dispersal Course" Izmir, June 5-9, 1978, organised by the International Potato Center, Region IV, Izmir, Turkey. Institut Voor Plantenziektenkundig Onderzoek. I.P.O. Binnenhaven 12 - Wageningen, 33 pp. Harten, A. Van. (1983). The relation between aphid flights and the spread of potato virus Y^ (PVY^) in the Netherlands. Potato Res. 26, 1-15. Hashiba, T. (1969). Studies on the mechanism of aphid transmission of stylet-borne virus. (IV) The insertion site of the stylet related to feeding and probing. Tohoku J. agric. Res. 20, 172-187. 199

Hashiba, T. (1970). Studies on the mechanism of aphid transmission of stylet-borne virus. (V). The relation between the stylet insertion site on the diseased leaf and virus acquisition. Tohoku J. agric. Res. 21, 1-12. Hashiba, T. & Misawa, T. (1969). Studies on the mechanism of aphid transmission of stylet-borne virus. (II). A comparison between probing and feeding for virus transmission. Tohoku J. agric. Res. 20, 97-106. Hashiba, T. & Misawa, T. (1970). Studies on the mechanism of aphid transmission of stylet-borne virus (VI). Effect of the saliva of the aphid. Tohoku J. agric. Res. 21, 73-87. Heathcote, G.D. & Broadbent, L. (1961). Local spread of potato leaf roll and Y viruses. Eur. Potato F. 4, 138-143. Heinze, K. (1959). Beitrag zur ermttlung neuur ubertrager fur phytopathogene viren. Z. Pflkrankh. Pflschutz. 66, 391-395. Henize, K. (1960). Versuche zur ubertragung nichtpersistenter und persistenter viren durch blattlause. Nachrbl. dtsch. PfISchDienst, Stuttgart 12, 119-121. Hellmann, G.M., Thombury, D.W., Hiebert, E., Shaw, J.G., Pirone T.P. & Rhoads, R.E. (1983). Cell free translation of tobacco vein mottling virus RNA. II. Immunoprecipitation of products by antisera to cylindrical inclusion, nuclear inclusion, and helper component proteins. Virology 124, 434-444.

Highland, H.B., Demski, J.W. & Chalkey, J.H. (1981). Aphid populations and spread of peanut mottle virus. Peanut Sci. jJ, 99-102. Hill, S.A. (1978). Current virus infection of seed potatoes in Britain. In: Plant Disease Epidemiology (Scott, P.R. and Bainbridge, A., eds.), pp. 229-234. Blackwell Scientific Publications, Oxford. Hill, S.A. & Jackson, E.A. (1984). An investigation of the reliability of ELISA as a practical test for detecting potato leaf roll virus and potato virus Y in tubers. PI. Path. 33, 21-26. Hille Ris Lambers, D. (1972). Aphids: their life cycles and their role as vectors. In: Viruses of Potatoes and Seed Potatoes Product­ ion. (De Bokx, J.A. ed.), pp. 36-56. Center of Agricultural Publishing and Documentation, Wageningen. Hodges, L.R. & Mclean, D.L. (1969). Correlation of transmission of bean yellow mosaic virus with salivation activity of Acyrthosiphon pisum (Homoptera: Aphididae). Ann, ent. Soc. Am. 62, 1398-1401. Hollings, M. (1955). Aphid movement and virus spread in seed potato areas of England and Wales, 1950-53. PI. Path. 4, 73-82. 200

Hollings, M. (1959a). Host-range studies with fifty-two plant viruses. Ann, appl. Biol. 47, 98-108. Hollings, M. (1959b). Nicotiana clevelandii gray as a test plant for plant viruses. PI. Path. <8, 133-137. Hollings, M. & Brunt, A.A. (1981). Potyvirus group. C.M.I./A.A.B. Descriptions of Plant Viruses No. 245. Commonwealth Mycol. Inst. Kew, England. Hoof, H.A. Van (1958). An investigation of the biological transmission of a nonpersistent virus.Onderzoekingen over de biologische overdracht vaneien nonpersistent virus. Doctoral thesis Wageningen Agr. Univ. Meded. Inst, plziektenk., Onderz 161, van Putten and Ortraeijer, Alkmaar, the Netherlands, 96 pp. Hoof, H.A. Van (1977). Determination of the infection pressure of potato virus YN. Neth. J, PI. Path. 83, 123-127. Hoof, H.A. Van (1980). Aphid vectors of potato virus Y N• Neth J. PI. Path. 86, 159-162. Horio, H. (1981). Seed potato production and virus diseases in Japan. Rev. Plant. Protec. Res. 14, 59-72. Hull, R. <& Plaskitt, A. (1970). Electron microscopy of the behaviour of two strains of alfalfa mosaic virus in mixed infections. Virology 42, 773-776. Ishii, M. (1972). Observations on the spread of papaya ringspot virus in Hawaii. PI. Pis. Reptr. 56, 331-333. Johnson, C.G. (1952). The changing numbers of Aphis fabae Scop, flying at crop level, in relation to current weather and to the population on the crop. Ann, appl. Biol. 39, 525-547. Jurik, M., Mucha, V. & Valenta, V. (1980). Intr&species variability in transmission efficiency of stylet-bome viruses by the pea aphid (Acyrthosiphon pisum). Acta virol. Prague 24, 351-357. Karl, E. (1971). Neue vektoren fur einige nichtpersistente viren. Arch. PflSchulz. J7> 337-342. Karl,E. & Proeseler G. (1976).Befahigung van Aphis frangulae beccabungae Koch zur libertragung nichtpersis tenter viren. Arch. Phytopathol. u. Pflanzenschutz, Berlin 12, 135-140. Kassanis, B. (1941). Transmission of tobacco etch viruses by aphides. Ann, appl. Biol. 28, 238-243. Kassanis, B. (1942). Transmission of potato virus Y by Aphis rhamni (Boyer). Ann, appl. Biol. 29, 95. 201 Kassanis, B, (1961), The transmission of potato aucuba mosaic virus by aphids from plants also infected with potato virus A or Y. Virology 13, 93-97*

Kassanis, B, (1963}* Interactions of viruses in plants. Adv. Virus Res. 10, 219-255. Kassanis, B. & Govier, D.A. (1971a). New evidence on the mechanism of aphid transmission of potato C and potato aucuba mosaic viruses. J. gen. Virol. 10, 99-101. Kassanis, B. & Govier, D.A. (1971b). The role of the helper virus in aphid transmission of potato aucuba mosaic virus and potato virus C. J. gen. Virol. 13, 221-228. Kato, M. (1957). Recovery of Y-virus by aphids from infected potato plants. Bull natn. Inst, agric. Sci., Tokyo (Sec. C.) 32, 65-88. Kennedy, J.S. (1950). Aphid migration and the spread of plant viruses. Nature 165, 1024-1025. Kennedy, J.S., Bath, C.O. & Kershaw, W.J.S. (1959a). Host finding by aphids in the field I. Gynoparae of Myzus persicae (Sulzer). Ann appl. B iol. 47, 410-423. Kennedy, J.S., Bath, C.O. & Kershaw, W.J.S. (1959b). Host finding by aphids in the field II. Aphis fabae Scop, (gynoparae) and Brevicoryne brassicae L., with a re-appraisal of the role of host-finding in virus spread. Ann, appl. Biol. 47, 424-444. Kennedy, J.C., Day, M.F. & Eastop, V.F. (1962). A conspectus of aphids as vectors of plant viruses. Commonwealth Inst. Entomol., London. Kleczkowski, A. (1968). Experimental design and statistical methods of assay. In Methods in Virology, vol. 4, pp.615-730. Ed. by K. Maramorosch and H. Koprowski. New York and London, Academic P ress. Klinkowski, M. & Schmelzer, K. (1957). Beitrage zur kenntnis des Virus der tabakrippenbraune. Phytopath. Z. 28, 285-306. Klinkowski, M. & Schmelzer, K. (1960). A necrotic type of potato virus Y. Ann. Potato J. 37, 221-228. Knoke, J.K., Anderson R.J. & Louie, R. (1977). Virus disease epyphy- tology: Developing field tests for disease resistance in maize. In Proceedings of the International Maize Virus Disease Colloquium and Workshop, pp. 116-122. Ed. L.E. Williams, D.T. Gordon & L.R. Nault. August 1976. Ohio Agricultural Research and Development Center. Wooster, Ohio. 145 pp. Kostiw, M. (1975). Investigation on the retention of potato viruses M and Y in two species of aphids (Myzus persicae Sulz. and Aphis nasturtii Kalt.). Potato Res. 18, 637-640. 202

Kostiw, M. (1979)* Transmission of potato virus by Rhopalosiphum padi (L.). Potato Res* 22, 237-238. Ksiazek, D. (1980). Role of weeds in the spread of some selected plant virus diseases* Pol* ecol* Stud* 6, 371-435* Kudamatsu, M., Alba, A.P. C. & Chagas, C.M. (1981)* Determinacao serologica do virus Y da batata em plantas de Solanum cilia turn Lam. e de S. vlraum Dun. naturalraente infectadas. Anq* Inst. Biol* Sao Paulo 48, 45-47. Kulps, G. (1969). Untersuchungen zum Verhalten von Mineralolen im Hinblick auf ihre wirkung bei der virusubertragung durch Blattlasue. Dissertation, Universitat Hohenheim, 85 p. Kurppa, A. (1983), Potato viruses in Finland and their identification. J. scient. agric. Soc., Finland 55, 183-301. Kvicala, B.A. (1965). Additional insect vectors of pea mosaic virus. Phytopath. Z. 54, 94-97. Kvicala, B.A. (1968). Comparative transmission of pea mosaic virus by one strain of Myzus persicae Sulz. and two strains of Acynthosiphon pisum Harris. Biologia PI. 10, 51-57. Labonne, G., Fauvel, C., Leclant, F. & Quiot, J.B. (1982a). Description d*un piege a suction: son emploi dans la recherche des aphides vecteurs de virus transmis sur le mode non persistant. Agronomie _2, 773-776. Labonne, G., Quiot, J.B. & Monestiez, P. (1982b). Role des diverses especes de pucerons vecteurs dans la dissemination du virus de la mosaique du concombre au niveau d'une parcelle de melon dans le Sud-Est de la France. Agronomie 2* 797-804. Laird, E.F.Jr. & Dickson, R.C. (1963). Tobacco etch virus and potato virus Y in pepper, their host plants and insect vectors in Southern California. Phytopathology 53, 48-52. Lapp, N.A. & Gooding, G. V.Jr. (1976). Occurence and sources of inoculum of potato virys Y in tobacco in north Carolina. PI. Pis. Reptr. 60, 1014-1016. Lecoq, H., Coehen, S., Pitrat, M. & Labonne, G. (1979). Resistance to cucumber mosaic virus transmission by aphids in Cucumis melo. Phy top a thology 69, 1223-1225. Lecoq, H., Labbone, G. & Pitrat, II. (1980). Specificity of resistance to virus transmission by aphids in Cucumis melo. Ann Phytopatho1. .12, 139-144. Lim, W.L., Hagedorn, D.J. (1977). Scanning electron microscope local­ ization of virus or virus coat protein on aphid stylets by labeled antibody techniques. Proc. Am. Phytopathol. Soc. .3, 247. 203

Lim, W. L., De Zoeten, G. A. & Hagedom, D.J. (1977). Scanning electron-microscopic evidence for attachment of a nonpersistently transmitted virus to its vector's stylets. Virology 79, 121-128. Loebenstein, G. & Raccah, B. (1980). Control of non-persistently transmitted aphid-borne viruses. Phytoparasitica £, 221-235. Lopez-Abella, D. & Bradley, R.H.E. (1969). Aphids may not acquire and transmit sty let-bome viruses while probing intercellularly. Virology 39, 338-342. Lopez-Abella, D. & Bradley, R.H.E. (1970). The stylet path of aphids that acquire cucumber mosaic virus. Proc. Septieme Congress International de Microscopie Electronique, 1970. Grenoble, France. Is 319-320. Lopez-Abella, D., Pirone T.P., Meraaugh, R.E. & Johnson, M.C. (1981). Effect of function and helper component on the detection of potato virus Y in alimentary tract extracts of Myzus persicae. Phyto­ pathology 71, 807-809. Lucas, B.S. (1978). Studies on some vectors associated with the epidemiology of soybean mosaic virus, M.Sc. Thesis, Iowa State Univ. Lucas, B.S. & Hill, J.H. (1980). Characteristics of the transmission of three soybean mosaic virus isolates by Myzus persicae and Rhopalosiphum maidis. Phytopath,Z, 99, 47-53. Lytaeva, G.K. (1972). Plants-reservoirs of potato virus Y. Rev. Plant* Pathol. 51, 2770. MacGillvray, M.E. & Bradley, R.H.E. (1960). Myzus certus (Ulk.), an efficient vector of potato virus Y. Canad. Entomol. 92, 367-368, MacKinnon, J.P. (I960). Combined transmission by single aphids of two viruses that persist in the vector. Virology 11, 425-433. Maison, P. & Massonie, G. (1982). Premieres observations sur la specific!te de la resistance du pecher a la transmission aphidienne du virus de la sharka. Agronomie 2* 681-683. Makkouk, K.M. & Gumpf, D.J. (1976). Characterization of potato virus Y strains isolated from pepper. Phytopathology 66 , 576- 581. Marco, S. (1981). Incidence of potato virus Y and potato leaf roll virus in autumn potatoes in Israel. Potato Res. 24, 353-356. Matthews, R.E.F. (1982). Classification and Nomenclature of Viruses. Fourth Report of the International Committee on of viruses. Intervirology 17, 11-179.

McLean, D.L. & Kinsey, M.G. (1964). A technique for electronically recording aphid feeding and salivation. Nature 202, 1358-1359. 204

Mclean, D.L. & Kirsey, M.G. (1965). Identification of electrically recorded curve patterns associated with aphid salivation and ingestion. Nature 205, 1130-1131. Messieha, M. (1966). Aphid transmission of maize dwarf mosaic virus. Ph. D. Thesis, Purdue Univ., Lafayette, Indiana. Misawa, T. & Hashiba, T. (1967). Studies on the mechanism of aphid transmission of stylet-borne viruses. Tohoku J. agric. Res. 18, 87-105. Moericke, V. (1951). Eine farbfalle zur kontrolle des fluges von blattlausen insbesondere der pfirsichblattlaus. NachtBl. dt. PfISchutzdienst., Stuttg. 3, 23-24. Morales, F.J. (1981). Electrophoretic properties of the viral capsid protein in relation to the biological transmission of five isolates of bean yellow mosaic virus. Abstracts of the Inter­ national Meeting on Plant Virus Disease Epidemiology, Oxford, 28-30 July, 1981, p. 100. Mossop, D.W. (1982). Potato aucuba mosaic virus - a latent virus of tamarillo (Cyphomanda betacea (Cav.) Sendt.). N.Z. Jl. agric. Res. 25, 449-453. Munro, J. (1955). The reaction of certain solaneceous species to strains of potato virus Y. Can. J. Bot. 33, 355-361. Namba, R. (1962). Aphid transmission of plant viruses from the epidermis and subepidermal tissues: Myzus persicae (Sulzer) - cucumber mosaic virus. Virology 16, 267-271. Nault, L.R. & Gyrisco, G.G. (1966). Relation of the feeding process of the pea aphid to the inoculation of pea enation mosaic virus. Ann ent. Soc. Am. 59, 1185-1197. Nelder, J.A. & Wedderburn, R.W.M. (1972). Generalized linear models. Jl. R. statist. Soc. Series A 135, 370-384. Nishi, Y. (1969). Inhibition of viruses by vector saliva. In: Viruses, Vectors and Vegetation (K. Maramorosch, ed.), Wiley-Interscience, New York, Normand, R.A. & Pirone, T.P. (1968). Differential transmission of strains of cucumber mosaic virus by aphids. Virology 36, 538-544. Orlob, G.B. (1962). Further studies on the transmission of plant viruses by different forms of aphids. Virology 16, 301-304. Orlob, G.B. (1963). Reappraisal of transmission of tobacco mosaic virus by insects. Phytopathology 53, 822-830. 205

Otsuki, Y. & Takebe, I, (1976a)i Interaction of tobacco mosaic virus strains in doubly infected tobacco protoplasts, Ann, Microbiol, (Paris) 127a, 21 (Abstract). Otsuki, Y. & Takebe, I. (1976b). Double infection of isolated leaf protoplasts by two strains of tobacco mosaic virus. In "Bio­ chemistry and Cytology of Plant-Parasite Interaction", pp. 213- 222. Edited by K. Tomiyama, J.M. Daly, I. Uritani, H. Oku & S. Quch. Tokyo: Kodansha. Paine, J. & Legg, J.T. (1953). Transmission of hop mosaic by Phorodon humuli (Schrank). Nature 171, 263-264. Paguio, O.R. & Kuhn, C.W. (1976). Aphid transmission of peanut mottle virus. Phytopathology 66, 473-476. Pirone, T.P. (1964). Aphid transmission of a purified stylet-borne virus acquired through a membrane. Virology 23, 107-108. Pirone, T.P. (1969). Mechanism of transmission of stylet-borne viruses. In: Viruses, Vectors and Vegetation (K. Maramorosch ed.), Wiley-Interscience, New York. Pirone, T.P. (1970). Effect of aphid saliva and extracts of aphid- infected leaves on the infectivity of tobacco mosaic virus and some stylet-borne viruses. Phytopathology 60, 1657-1659. Pirone, T.P. (1977). Accessory factors in nonpersistent virus transmission. In: Aphids as Virus Vectors (K.F. Harris and K. Maramorosch, eds.), pp. 221-235. Academic Press, New York. Pirone, T.P. (1981). Efficiency and selectivity of the helper- component-mediated aphid transmission of purified potyviruses. Phytopathology 71, 9 22-9 24. Pirone, T.P. & Megahed, E. (1966). Aphid transmissibility of some purified viruses and viral . Virology 30, 631-637. Pirone, T.P. & Kassanis, B. (1975). Polyamino acid induced aphid transmission of plant viruses. J. Gen. Virol. 22, 257-266. Pirone, T.P. & Harris, K.F. (1977). Nonpersistent transmission of plant viruses by aphids. Ann. Rev. Phytopathol. 15, 55-73. Pirone, T.P. & Thornbury, D.W. (1983). Role of virion and helper component in regulating aphid transmission of tobacco etch virus. Phytopathology 73, 872-875. Plumb, R.T. (1976). Barley yellow dwarf virus in aphids caught in suction traps, 1969-1973. Ann, appl. Biol. 83, 53-59. 206

Pochard, E. (1979), Study of resistance to European strains of potato virus Y (PVY) in Capsicum. Rev. Plant Pathol. 58, 3588. Pontis, R.E. & Feldman, J.M. (1963). A common weed, Physalis viscosa, new host for potato virus Y. PI. Pis, Reptr. 47, 22. Prasad Rao, R.D.V.J. (1976). Natural occurrence of potato virus Y on Datura metel. Curr. Sci. 45, 467. Proeseler, G., Schmidt, H.B. & Eisbein, K. (1972). Vektoren pflanzenpathogener viren, ihre verbreitung, okologie und bekampfung. Tag, Ber. Akad. Landwirtsch. Wiss. D.D.R. 121, 57-61. Proeseler, G. & Weidling, H. (1975). Die retentionszeit von stammen des kartoffel-Y-virus in verschiedenen aphidenarten und einflub der teraperatur. Arch. Phytopathol. u, Pflanzenschutz, Berlin 11, 335-345. Raccah, B. (1983). Monitoring insect vector populations and the detection of viruses in vectors. In; Plant Virus Epidemiology (R.T.Plumb and J.M. Thresh, eds.), pp. 147-157. Blackwell Scientific Publications, Oxford. Radcliffe, E.B. (1982). Insect pestsof potato. A. Rev. Ent. 27, 173-204. Ramirez, W.R., Gamez, R. & Gonzales, L.G. (1964). Cross protection tests for potato virus Y and potato veinal necrosis virus. Phytopathology 54, 500. Rasocha, V. (1966). Aphids and the spread of virus diseases of potatoes in seed potato growing regions, (In Czechoslovak). Vedecke Prace Vyrzkumneho Ustava Brambo rararskeho v Havlickowe Brode 1966, 101-116. Richardson, D.E. (1958). Some observations on the tobacco veinal necrosis strain of potato virus Y. PI. Path. 7, 133-135.

Rizvi, S.A.H. & Raman, K.V. (1983). Effect of glandular trichomes on the spread of potato virus Y (PVY) and potato leafroll virus (PLRV) in the field. Research for the Potato in the Year 2000, Lima 1982, pp# 162-163. Robert, Y. (1978). Role epidemiologique probably d'especes de pucerons autres que celles de la pomme de terre dans la dissemination intempestive du virus Y depuis 4 ans dans l*ouest de la France. Abst. EAPR Conf. Pap. Warsaw, Poland, 26 June - 1 July 1978, 242-243. Roberts, D.A. (1964). Local lesion assay of plant viruses. In; Plant Virology (M.K. Corbett and H.D. Sisler, eds.), pp. 194-210. Univ. Florida Press, Gainesville, 527 pp. 207

Roberts, F.M. (1940). Studies on the feeding methods and penetration rates of Myzus persicae Sulz., M. circumflexus Bukt. and Macrosiphum gei Koch. Ann, appl. Biol. 27, 348-358. Rochow, W.F., Muller, I. & Gildow, F.E. (1983). Interference between two luteoviruses in an aphid: lack of reciprocal competition. Phytopathology 73, 919-922. Russell, G.E. (1971). Beet mosaic virus. C.M.I,/A.A.B. Descriptions of Plant Viruses. No. 53. Commonwealth Mycol. Inst., Kew, England. Ryden, K. (1979). Havrebladlusen, Rhopalosiphum padi, kan sprida potatisvirus Y. Vaxtskyddnotiser 43, 51-53. Ryden, K., Brishammar, S. & Sigvald, R. (1983). The infection pressure of potato virus and the occurrence of winged aphids in potato fields in Sweden. Potato Res. 26, 229-235. Sakimura, K. (1953). Potato virus Y in Hawaii. Phytopathology 43, 217-218. Sako, N. (1980). Loss of aphid transmissibility of turnip mosaic virus. Phytopathology 70, 647-649. Sako. N. (1981). Different helper factors for aphid transmission of watermelon mosaic, turnip mosaic and potato Y viruses. Fifth international congress of Virology, Strasbourg, France, August 2-7, 1981. p. 212. Sako, N. & Ogata, K. (1981). Different helper factors associated with aphid transmission of some poly viruses. Viroloty 112, 762-765. Schepers, A. & Reestman, A.A. (1975). Mature-plant resistance against PVYN in relation to some morphological characteristics of the potato plant. Abstr. Conf. Pap. 6th. trienn. Conf. EAPR (Wageningen) 100- 102. Schepers, A. & Beemster, A.B.R. (1976). Effect of fertilizers on the susceptibility to virus infection of the potato, with special reference to mature-plant resistance. Proc. 12th, Colloq. Int. Potash Inst. (Izmir, Turkey) 201-210. Schraelzer, K. (1967). Wirte des kartoffel-Y- und des tabakatzmosaik- virus au erhalb der solanaceen. Phytopath. Z. 60, 301-315. Schmelzer, K. Bartels, R. & Klinkowski, M. (1960). Interferenzen swischen den viren der tabakatzmosaik-gruppe. Phytopath. Z, 40, 52-74. Schmidt, H.B., Proeseler, G. & Eisbein, K. (1974). 1st die morphologie der stechborstenspitzen bei blattlausen entscheidend fur die fahigkeit zur Ubertragung nicht persistenter viren ? Biol. Zbl, 93, 227-233. 208

Sclvroeder,W.T., Prowidenti, R. & McEwen, F.L. (1959), Pea streaks naturally incited by combinations of viruses. PI. Pis. Reptr. 43, 1219-1226. Severin, H.H.P. & Freitag, J.H. (1938). Western celery mosaic. Hilgardia 11, 493-558. Shaunak, K.K. & Pitre, H.N. (1971). Seasonal alate aphid collections in yellow traps in northern Mississippi: Possible relationships to maize dwarf mosaic disease. J. econ. Ent. 64, 1105-1109. Shepherd, R. J. (1977). Intrinsic properties and taxonomy of aphid-borne viruses. In: Aphids as Virus Vectors (K.F. Harris & K. Maramorosch, eds.), pp. 121-136. Academic Press, New York. Siegel, A. (1959). Mutual exlusion of strains of tobacco mosaic virus. Virology £, 470-477. Sigvald, R. (1977). Bladlus och virusspredning i potatis. Vaxtskyddsrapporter, Jordbruk JL.» 112-120. Simons, J.N. (1956). The pepper veinbanding mosaic virus in the Everglades area of south Florida. Phytopathology 46, 53-57. Simons, J.N. (1958). Titers of three non-persistent aphid-borne viruses affecting pepper in South Florida. Phytopathology 48, 265-268. Simons, J.N. (1959). Variation in efficiency of aphid transmission of southern cucumber mosaic virus and potato virus Y in pepper. Virology 9^, 612-623. Simons, J.N. (1966). Effects of temperature and length of acquisition feeding time on transmission of nonpersistent viruses by aphids. J. econ. Ent. 59, 1056-1062. Simons, J.N. (1976). Aphid transmission of nonaphid transmissible strain of tobacco etch virus. Phy top a thology 66, 652-654. Simons, J.N. (1982). Use of oil sprays and reflective surfaces for control of insect-transmitted plant viruses. In: Pathogens, vectors, and plant diseases: approaches to control (K.F. Harris and K. Maramorosch, eds.), pp. 71-93. Academic Press, New York. Simons, J.N. & Moss, L.M. (1963). The mechanism of resistance to potato virus Y infection in Capsicum annuum. Phytopathology 53, 684-691. Simons, J.N. & Zitter, T.A. (1980). Use of oils to control aphid- borne viruses. Plant Pis. 64, 542-546. 209

Singh, M.N. Khurana, S.M.P., Nagaich, B.B. & Agrawal, H.O. (1981). Probing behaviour of Myzus persicae clones and transmission of potato virus Y. Fifth international congress of Virology, Strasbourg, France, August 2-7, 1981, p. 214. Singh, M.N., Khurana, S.M.P. & Nagaich, B.B. (1983). Evidence on heredity variations in the virus transmission efficiency of aphid clones. Z. PfKrankh. PflSchutz. 90, 345-351. Singh, R.P. & Santos-Rojas, J. (1983). Detection of potato virus Y in primarily infected plants by ELISA, indicator-host, and visual indexing. Can. Plant Pis. Surv. 63, 39-44. Smith, K.M. (1931). On the composite nature of certain potato virus diseases of the mosaic group as revealed by the use of plant indicators and selective methods of transmission. Proc. Roy. Soc. London, Ser. B. 109, 251-267. Smith, K.M. (1965). Plant virus-vector relationship. Adv. Virus Res. H., 61-96. Sohi, S.S. & Swenson, K.G. (1964). Pea aphid biotypes differing in bean yellow mosaic virus transmission. Ent. exp, appl. 7, 9-14. Srivastava, A.S., Katiyar, S.S.L., Awasthi, B.K., Srivastava, K.M. & Nigam, P.M. (1971). Field assessment of aphid population on potato crop. Z. angew. Ent. 69, 44-48. Stevenson, A.B. (1959). A study of the aphid transmission and field spread of potato virus Y in Wisconsin. Piss. Abstr. 19, 2711-2712. Suteri, D.B., Joshi, G.C. & Bala, S. (1979). Some ornamentals and weeds as reservoirs of potato virus Y and cucumber mosaic virus in Kumaon. Indian Phytopath. 32, 640. Swenson, K.G. (1957). Transmission of bean yellow mosaic virus by aphids. J. econ. Ent. 50, 727-731. Swenson, K.G. (1962). Bean yellow mosaic virus transmission by Myzus persicae. Aust. J. Biol. Sci. 15, 468-482. Swenson, K.G. (1967). Plant virus transmission by aphids. In: Methods in Virology, Vol. 1 (K. Maramorosch and H. Korpowski, eds.), pp. 267-307. Academic Press, New York. Swenson, K.G. (1968). Role of aphids in the ecology of plait viruses. A. Rev. Phytopathol. 351-374. Sylvester, E.S. (1949). Beet-mosaic virus-green peach aphid relation­ ships. Phy top a thology 39, 417-424. 210

Sylvester, E.S. (1950). Transmission of Brasslca nigra virus by the green peach aphid. Phytopathology 40, 743-745. Sylvester, E.S. (1952). Comparative transmission of beet-mosaic by four aphid species. Phy topa thology 42, 252-254. Sylvester, E.S. (1954). Aphid transmission of nonpersistent plant viruses with special reference to the Brassica nigra virus. Hilgardia 23, 53-98. Sylvester, E.S. (1955). Lettuce mosaic virus transmission by the green peach aphid. Phy topa thology 45, 357-370. Sylvester, E.S. (1956). Beet yellows virus transmission by the green peach aphid. J. econ. Ent. 49, 789-800. Sylvester, E.S. (1962). Mechanisms of plant virus transmission by aphids. In: Biological Transmission of Disease Agents (K. Maramorosch, ed.), Academic Press, New York. Taylor, C.E. (1955). Growth of the potato plant and aphid coloniz­ ation. Ann, appl. Biol. 43, 151-156. Taylor, C.E. & Robertson, W.M. (1974). Electron microscopy evidence for the association of tobacco severe etch virus with the maxillae in Myzus persicae (Sulz.). Phytopath. Z. 80, 257-266. Taylor, L.R. & Palmer, J.M.P. (1972). Aerial sampling. In: Aphid Technology (H.F. van Emden ed.), pp. 189-254. Academic Press, London. Taylor, L.R., Macaulay, E.D.M., Dupuch, M.J. & Nicklen, J. (1982). Rothamsted insect survey, thirteenth annual summary. Rep. Rothamsted exp, stn. 1981, part 2, pp. 129-157. Thomas, P.E. (1983). Sources and dissemination of potato viruses in the Columbia Basin of the northwestern United States. Plant Pis. 67, 744-747. Thongmeearkom, P., Ford, R.E. & Jedlinski, H. (1976). Aphid trans­ mission of maize dwarf mosaic virus strains. Phy topathology 66, 332-335. Thornbury, D.W. & Pirone, T.P. (1983). Helper components of two potyviruses are serologically distinct. Virology 125, 487-490. Thottappilly, G., Tsai, J.H. & Bath, J.E. (1972). Differential transmission of two bean yellow mosaic virus strains and compar­ ative transmission of biotypes and stages of the pea aphid. Ann. Ent. Soc. Am. 65, 912-915. 211

Thresh, J.M. (1980), The origins and epidemiology of some important virus diseases. Applied Biology _5, 1-65. Todd, J.M. (1961). Tobacco veinal necrosis on potato in Scotland: control of the outbreak and some characters of the virus. In Proceedings of the 4th Conference on Potato Virus Diseases, Braunschweig, 1960, pp. 82-92. Upreti, G.C. & Nagaich, B.B. (1971). Variations in the ability of Hyzus persicae Sulz. to transmit potato viruses. II Virus Y. Phytopath. Z. 71, 223-230. Vanderveken, J. (1977). Oils and other inhibitors of nonpersistent virus transmission. In: Aphids as Virus Vectors (K.F. Harris & K. Maramorosch, eds.), pp. 435-454. Academic Press, New York.

Verhoyen, M. & Horvat, F. (1973). La striure chlorotique du poireau. I. Identification de 1*agent causal. Parasitica 29, 16-28. Vetten, H.J., Ehlers, U. & Paul, H.L. (1983). Detection of potato viruses Y and A in tubers by enzyme-linked immunosorbert assay after natural and artificial break of dormancy. Phytopath. Z. 108, 41-53. Volk, J. (1959). Zur ubertragung des Y-Virus durch insekten und kontakt. Z. Pflkrankh. Pflschutz. 66, 563-571. Walker, H.L. & Pirone, T.P. (1972). Particle numbers associated with mechanical and aphid transmission of some plant viruses. Phytopathology 62, 1283-1288. Want, J.P.H. van der (1954). Onderzoekingen over Virusziekten van de Boon (Phaseolus vulgaris L.). Doctoral Thesis, Wageningen Agric. Univ. H. Veenmon and Zonen, Wageningen, The Netherlands. (In Dutch). Watson, M.A. (1938). Further studies on the relationship between Hyoscyamus virus 3 and the aphis Myzus persicae (Sulz) with special reference to the effects of fasting. Proc. Roy. Soc. London, Ser. B 125, 144-170. Watson, M.A. (1940). Studies on the transmission of sugar beet yellow by the aphis Myzus persicae. Proc. Roy. Soc. London, Ser. B 128, 535-552. Watson, M.A. (1946), The transmission of beet mosaic and beet yellow viruses by aphids; a comparative study of a nonpersistent virus and a p e rsiste n t virus having host plants and vectors in common. Proc. Roy. Soc. London, Ser. B 133, 200-219. Watson, M.A. (1958), The specificity of transmission of some non- persistent viruses. Proc. 10th Int. Congr. Entomol. 1956, Montreal 3, 215-219. 212

Watson, M.A. (1960). Evidence for interaction or genetic recombin­ ations between potato viruses Y and C in infected plants. Virology 10, 211-232. Watson, M.A. (1972). Transmission of plant viruses by aphids. In: Principles and Techniques in Plant Virology (C.I. Kado and H.O. Agrawal, eds.), pp. 131-167. Van Nostrand Reinhold Company, New York. Watson, M.A. & Roberts, F.M. (1939). A comparative study of the transmission of Hyocyamus virus 3, potato virus Y and cucumber virus 1 by the vectors Myzus persicae (Sulz.), M. circumflexus (Buckton) and Macrosiphum gei (Koch). Proc. Roy. Soc. London, Ser. B 127, 543-576. Watson, M.A. & Roberts, F.M. (1940). Evidence against the hypothesis that certain plant viruses are transmitted mechanically by aphids. Ann, appl. Biol. 27, 227-233. Watson, M.A., Lack, A.J. & Pullen, M.E. (1967). "Artificial feeding"; transmission of non-persistent viruses. Rep. Rothamsted exp, stn. 1966, p. 120. Watson, M.A. & Plumb, R.T. (1972). Transmission of plant-pathogenic viruses by aphids. A. Rev. Ent. 17, 425-452. Weber, K.A. & Hampton, R.O. (1980). Transmission of two purified carlaviruses by the pea aphid. Phytopathology 70, 631-633. Weststeijn, E.A. (1984). Evidence for a necrosis-inducing factor in tobacco mosaic virus-infected Nicotiana tabacum cv. Xanthi-nc grown at 22°C but not at 32°C. Physiological Plant Pathology 25, 83-91. Wijs, J.J. De. (1974). A virus causing ringspot of Passiflora edulis in the Ivory Coast. Ann, appl. Biol. 77, 33-40. Wiktelius, S. (1982). Flight and settling behaviour of Rhopalosiphum padi (L.). Bull. Ent. Res. 72, 157-163. Yoshii, H. (1966). Transmission of turnip mosaic virus by Myzus persicae (Sulz.): mode of stylet insertion and infection site of the virus. Ann. Phytopath. Soc. Japan 32, 46-51. Zaitlin, M. & Israel, H.W. (1975). Tobacco mosaic virus. C.M.I./A.A.R. Descriptions of Plant Viruses No. 151. Commonwealth Mycol. Inst. Kew, England. Zaumeyer, W.J. & Kearns, C.W. (1936). The relation of aphids to the transmission of aphids of bean mosaic. Phytopathology 26, 614-629. Zschiegner, H.J., Eisenbrandt, K. & Fritzche, R. (1974). Beitrag zum Wirkungsmechanisms von Mineralonen bei der Verhinderung der Virusubertragung durch Vectoren. Arch. Phytopathol. u. Pflanzenschutz. B erlin 10, 371-382.