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Purification and properties of a new carlavirus from dandelion Johns, Lois 1979

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PURIFICATION AND PROPERTIES OF A NEW CARLAVIRUS FROM DANDELION by L o i s Joanne J o h n s B.Sc. (Hon. B i o l . ) , C o n c o r d i a U n i v e r s i t y , 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (The Department o f P l a n t S c i e n c e ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1979 (c) L o i s Joanne J o h n s , 1979 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t n f P l a n t S c i e n c e T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 D a t e A p r i l 2 0 . 1 9 7 9 ABSTRACT A c a r l a v i r u s was i s o l a t e d from n a t u r a l l y - i n f e c t e d dandelions i n the Okanagan Valley, B.C. In t o t a l , 31 plant species belonging to 12 families were tested as possible hosts for the dandelion v i r u s . In only four f a m i l i e s (Amaranthaceae, Chenopodiaceae, Compositae, Solanaceae) were susceptible species found. The v i r u s was contained as l o c a l lesions i n Gomphrena globosa and Datura stramonium and became systemic i n Chenopodium amaranticolor, C. quinoa and Taraxacum  o f f i c i n a l e . The c a r l a v i r u s , for which the name Dandelion Virus S (DVS) i s proposed, has s l i g h t l y curved p a r t i c l e s with normal length 637 nm and width 12-13 nm. A p u r i f i c a t i o n scheme was developed that yielded 20-30 mg of v i r u s per kg of C. quinoa leaf and stem t i s s u e . P a r t i a l l y p u r i f i e d virus preparations had a s i n g l e nucleoprotein component i n rate zonal sucrose and cesium c h l o r i d e density gradient c e n t r i f u g a t i o n . The UV absorption spectrum has a maximum at 259 nm and a minimum at 245 nm. The ratio, of A m a x / A m - L n i s approximately 1.1; of A 2 6 0 / A 2 8 0 , 1 . 4 . In sap from in f e c t e d C. quinoa, DVS had a thermal i n a c t i v a t i o n point of 75-80°; an i n f e c t i v i t y -5 -6 d i l u t i o n end point of 2 x 10 to 2 x 10 ; a longevity i n v i t r o of 4-5 days at 23°, 28-56 days at 4° and at lea s t 16.5 months i n a l y o p h i l i z e d state at 23°. An antiserum against DVS was prepared by four intramuscular i n j e c t i o n s of 1 mg each and the maximum homologous t i t r e was 40,960. Two c a r l a v i r u s e s with s i m i l a r symptoms i n C. quinoa, Peru v i r u s S (PeVS) and Helenium v i r u s S (HVS) were p u r i f i e d f o r antisera production and comparative s e r o l o g i c a l t e s t i n g . Antisera to other c a r l a v i r u s e s were also used to determine i f s e r o l o g i c a l r e l a t i o n s h i p s existed with DVS and other members of the group. S e r o l o g i c a l l y , DVS i s r e l a t e d to potato virus S (PVS) and PeVS, and d i s t a n t l y r e l a t e d to chrysanthemum v i r u s B (CVB), Helenium v i r u s S (HVS) and narcissus l a t e n t v i r u s (NLV). iv, TABLE OF CONTENTS PAGE TITLE PAGE ABSTRACT i i TABLE OF CONTENTS i v LIST OF FIGURES v i i LIST OF TABLES X ACKNOWLEDGEMENT x i INTRODUCTION 1 MATERIALS AND METHODS 7 a. F i e l d Occurrence 7 b. Host Range and Symptomatology 7 c. Properties i n Crude Sap 8 d. Seed Transmission 9 e. Aphid Transmission 10 f. P u r i f i c a t i o n 10 g. Electron Microscopy - P a r t i c l e Size 12 h. I n f e c t i v i t y of Gradients 13 i . Absorption Spectrum 13 j . Serology 13 1. Antiserum production 13 2. Agar g e l d i f f u s i o n serology 14 3. Tube p r e c i p i t i n serology 15 4. Sodium dodecyl s u l f a t e (SDS) agar 15 gel serology V . TABLE OF CONTENTS (cont'd) PAGE 5. Latex agglutination serology 17 6. Enzyme-linked immunosorbent assay 17 7. Transmission electron microscope 18 serology RESULTS 19 a. F i e l d Occurrence of DVS 19 b. Host Range and Symptomatology 20 c. Properties i n Crude Sap 26 d. Seed Transmission 26 e. Aphid Transmission- 27 f. P u r i f i c a t i o n 27 g. Electron Microscopy - P a r t i c l e Size 34 h. I n f e c t i v i t y of Gradients 37 i . Absorption Spectrum 40 j . Serology 41 1. Antiserum production 41 2. Agar gel d i f f u s i o n serology 43 3. Tube p r e c i p i t i n serology 44 4. Sodium dodecyl s u l f a t e (SDS) agar 45 gel serology 5. Latex agglutination serology 47 6. Enzyme-linked immunosorbent assay 47 7. Transmission electron microscope 50 serology DISCUSSION 54 TABLE OF CONTENTS (cont'd) SUMMARY LITERATURE CITED BIOGRAPHICAL INFORMATION LIST OF FIGURES FIGURE 1. Symptoms of DVS i n f e c t i o n i n C. quinoa. A. healthy plant B. i n f e c t e d plant showing vein c l e a r i n g and epinasty a f t e r 14 days C. l o c a l lesions on inoculated l e a f a f t e r 10 days D. healthy leaf ( l e f t ) and progression of symptom development i n 4 top leaves of inoculated p l a n t 2. A. Electron micrograph of semi-purified DVS used as inoculum f o r host range studies (bar = 500 nm) Symptoms induced on G. globosa B. healthy plant C. inoculated plant with n e c r o t i c l o c a l l e s i o n s Symptoms induced on C. amaranticolor D. healthy plant E. inoculated plant with systemic vein c l e a r i n g as pointed out by arrows F. l o c a l lesions on inoculated l e a f a f t e r 14 days 3. Absorbance scan patterns of sucrose density gradients showing e f f e c t s of combinations of extraction buffers and additives on p u r i f i c a t i o n of DVS 4. Absorbance scan patterns of sucrose density gradients showing e f f e c t s of ce n t r i f u g a t i o n time and addition of EDTA 5. Absorbance scan patterns of sucrose density gradients showing e f f e c t s of pH f c e n t r i f u g a t i o n time and addition of EDTA to 0.01M. Extra c t i o n buffer was 0.2M borate, pH 8.0 LIST OF FIGURES (cont'd) PAGE 6. Absorbance scan patterns of sucrose density 31 gradients showing e f f e c t s of pH, c e n t r i f u g a t i o n time and addition of EDTA to 0.01M. Extraction buffer was 0.2M borate, pH 8.5 7. Absorbance scan patterns of sucrose density 32 gradients showing e f f e c t s of pH, c e n t r i f u g a t i o n time and addition of EDTA to 0.01M. Extraction buffer was 0.2M borate, pH 9.0 8. Absorbance scan patterns of sucrose density 33 gradients showing e f f e c t s of pH, c e n t r i f u g a t i o n time and addition of EDTA to 0.01M. Extraction buffer was 0.2M borate, pH 9.5 9. Absorbance scan patterns comparing the 35 sharpness of the v i r u s peak (indicated by an arrow)obtained with A. sucrose B. cesium c h l o r i d e density gradient c e n t r i f u g a t i o n at 38 000 rpm for 90 min and 32 000 rpm f o r 18 h, r e s p e c t i v e l y i n an SW 41 rotor 1 0 . Flow diagram of the standard p u r i f i c a t i o n method 36 1 1 . E f f e c t of p u r i f i c a t i o n methods on the p a r t i c l e 37 length d i s t r i b u t i o n of DVS as determined by measurement of p a r t i c l e s i n electron micrographs 1 2 . Absorbance scan pattern of DVS a f t e r sucrose 38 density gradient c e n t r i f u g a t i o n for 90 min at 38 000 rpm i n an SW 41 rotor 1 3 . Electron micrographs of various f r a c t i o n s 39 c o l l e c t e d from a sucrose density gradient 1 4 . Absorbance scan pattern of DVS, from 230-340 nm: 40 uncorrected (-•—•—»-) and corrected (—•--•—•-) for l i g h t s c a t t e r i n g 1 5 . Antiserum production against DVS antigen, as 41 determined by tube p r e c i p i t i n serology. Arrows i n d i c a t e i n j e c t i o n s of antigen 1 6 . R e l a t i v e amounts of p r e c i p i t a t e formed with 43 tube p r e c i p i t i n reactions I X , LIST OF FIGURES (cont'd) PAGE 17. Sodium dodecyl s u l f a t e (SDS) agar gel serology. 46 A. E f f e c t of treatment steps on r e l a t i v e strength of p r e c i p i t i n l i n e s B. Determination of DVS antiserum t i t r e C. Determination of r e l a t i o n s h i p between DVS and PeVS 18. Absorbance pattern obtained by passing concen- 48 trated antiserum from a r a b b i t immunized with DVS ( ) and not immunized ( ) through a DEAE-22 Sephadex column 19. Determination of optimum conditions for detection 49 of DVS by ELISA. A. shows i n t e n s i t y of enzyme substrate re a c t i o n with the d i f f e r e n t concentrations of coating IgG and conjugated IgG i n a m i c r o t i t r e p l a t e . B. shows corresponding concentrations of coating IgG and conjugated IgG 20. Standard concentration curves for DVS determined 51 by enzyme-linked immunosorbent assay with A. sap from i n f e c t e d dandelion and i n f e c t e d C. quinoa B. d i l u t i o n s e r i e s of p u r i f i e d DVS from 10 pg/ml to 2 ng/ml 21. Electron micrographs of the s e r o l o g i c a l reactions 52 obtained by TEMS , showing the extent, of coating and aggregation of DVS caused by varying d i l u t i o n s of antiserum 22. Ele c t r o n micrographs of the s e r o l o g i c a l reactions 53 obtained by TEMS using combinations of three viruses and two anti s e r a X . LIST OF TABLES -TABLE PAGE 1. Results of indexing f i e l d c o l l e c t i o n s of 19 dandelions from various areas of Canada fo r natural i n f e c t i o n with DVS 2. Results of the experimental host range studies 24 as determined by symptoms i n the inoculated host and indexing the inoculated (I) and systemic (S) leaves by ELISA and back in o c u l a -t i o n to C. quinoa 3. Tube p r e c i p i t i n t i t r e and end point determin- 42 ation of DVS using varying concentrations of antigen and antiserum. The contour l i n e s j o i n readings of equal i n t e n s i t y 4. P r e c i p i t i n end points i n two way tube p r e c i p i t i n 44 tests including four antisera and t h e i r respective antigens at 80 jug/ml 5. P r e c i p i t i n end points i n two way tube p r e c i p i t i n 44 tests including four antisera and t h e i r respective antigens at 20 ;ug/ml x i . ACKNOWLEDGEMENT The a u t h o r w i s h e s t o e x p r e s s h e r g r a t i t u d e t o t h e members of h e r committee: D r . M. W e i n t r a u b and Dr. R. S t a c e - S m i t h , A g r i c u l t u r e Canada, R e s e a r c h S t a t i o n , V ancouver; D r . A . J . Hansen, A g r i c u l t u r e Canada, R e s e a r c h S t a t i o n , Summerland; Dr. V.C. R u n e c k l e s and D r . R . J . Copeman ( P r o f e s s o r and Chairman and A s s o c i a t e P r o f e s s o r , r e s p e c t i v e l y ) , Department o f P l a n t S c i e n c e , U n i v e r s i t y o f B r i t i s h C o l u m b i a , f o r t h e i r g u i d a n c e , c r i t i c i s m and s u g g e s t i o n s p e r t a i n i n g t o t h i s t h e s i s . The a u t h o r a l s o w i s h e s t o thank Ms. B. S c h r o e d e r and Mr. F. S k e l t o n f o r a s s i s t a n c e w i t h e l e c t r o n m i c r o s c o p y , Mr. W. Mac-D i a r m i d f o r p h o t o g r a p h y and t h e o t h e r members o f t h e s t a f f a t A g r i c u l t u r e Canada, R e s e a r c h S t a t i o n , Vancouver f o r t h e i r a s s i s t a n c e and encouragement. 1. INTRODUCTION It i s becoming i n c r e a s i n g l y important to recognize the p o t e n t i a l r e s e r v o i r of plant pathogens contained i n the weed population, e s p e c i a l l y when control or eradication programs are being implemented to maximize y i e l d s of economically important crops. V i r u s - i n f e c t e d weeds, volunteer plants and natural vegetation i n or near crops, or even at remote distances may contribute to crop i n f e c t i o n by harbouring p a r t i c u l a r viruses and/or t h e i r vectors, as well as increasing the number of i n f e c t i v e f o c i i n the case of seed-transmitted v i r u s e s . The weed hosts are often ignored i n crop surveys as they usually show no symptoms when inf e c t e d (Bos, 1978). Taraxacum o f f i c i n a l e Weber, the common dandelion, i s one such p o t e n t i a l r e s e r v o i r of v i r u s . I t has become one of the most common weeds i n Canada due to i t s e f f i c i e n t seed d i s p e r s a l , i t s long flowering period and i t s a d a p t a b i l i t y to a wide range of environmental conditions (Gilkey, 1957; Mulligan, 1976). Although dandelion i s widespread throughout the world, and i s frequently associated with a g r i c u l t u r a l crops, i t s capacity as a virus r e s e r v o i r does not adversely a f f e c t the majority of crops. The a v a i l a b l e l i t e r a t u r e suggests that dandelion i s not a host for many viruses that are known to have wide natural host ranges, except those that are transmitted by nematodes. The following nepoviruses have been reported to i n f e c t dandelion n a t u r a l l y : arabis mosaic (Harrison, 1958); cherry rasp leaf (Hansen et a l - , 1974); peach r o s e t t e mosaic 2. (Ramsdell and Myers, 1978); tobacco ringspot (Tuite, 1960; L i s t e r and Murant, 1967); tomato bla c k r i n g (Harrison, 1957) and tomato ringspot (Dias, 1977). In a l l cases, the nepoviruses were seed-transmitted at a low l e v e l i n dandelion seedlings and the seedlings remained symptomless. I t i s because of seed transmission and the ubiquitous nature of dandelions that they are implicated i n the spread of nepoviruses over larger ranges than i s possib l e simply by movement of infected nematodes through the s o i l . There are few reports of other viruses i n f e c t i n g dandelion n a t u r a l l y : chrysanthemum lat e n t (Hollings, 1957); dandelion yellow mosaic (Kassanis, 1944); potato virus Y (Lytaeva, 1971) and tobacco streak (Fulton, 1948). Although rod-shaped viruses: Bidens mottle ( P u r c i f u l l et a l . , 1976), Cassava common mosaic (Costa and Kitajima, 1972), plum pox (Kegler and Schade, 1971) and tobacco mosaic ( Z a i t l i n and I s r a e l , 1975), and b a c i l l i f o r m viruses: l e t t u c e n e c r o t i c yellows (Francki and Randies, 1970), potato yellow dwarf (Black, 1970) and sowthistle yellow vein (Peters, 1971), do i n f e c t Compositae, few reports have so f a r been published on the natural occurrence of ca r l a v i r u s e s i n dandelion. This probably r e f l e c t s the low l e v e l of i n t e r e s t on the part of the a g r i c u l -t u r a l industry i n virus/weed ecology i n r e l a t i o n to crops, rather than a true i n d i c a t i o n of one incidence, as reported here, of a c a r l a v i r u s n a t u r a l l y i n f e c t i n g dandelion. When tomato bushy stunt v i r u s (TBSV), ( M a r t e l l i et a l . , 3. 1971), was found i n the Okanagan V a l l e y , the p o t e n t i a l source of i n f e c t i o n was sought by Dr. A.J. Hansen and Ms. L. Green. During t h e i r survey, (1972-74), they indexed a v a r i e t y of indigenous plants growing i n and around the i n f e c t e d orchards. Some of the index plants inoculated with dandelion f i e l d material showed symptoms of virus i n f e c t i o n that were not t y p i c a l of TBSV i n f e c t i o n i n those p a r t i c u l a r hosts. A pre-liminary examination of a leaf dip from these hosts revealed s l i g h t l y flexuous rod-shaped v i r u s p a r t i c u l e s ca. 650 nm (Hansen, personal communication). Since there was no evidence that the v i r u s from dandelion occurred i n any tree f r u i t s i n the Okanagan, no further work was done with the v i r u s at the time. In 19 77, when I was considering possible projects for a t h e s i s , Dr. Hansen suggested that t h i s problem warranted further i n v e s t i g a t i o n and that he would be pleased to provide me with a culture of the v i r u s . Based on p a r t i c l e s i z e and morphology, the v i r u s was almost c e r t a i n l y a member of the c a r l a v i r u s group (Harrison e_t a l . , 1971). Whether i t was a new v i r u s , or whether i t was c l o s e l y or d i s t a n t l y r e l a t e d to a recognized virus was not known. For these reasons, t h i s study was undertaken to determine i f the virus from dandelion was a previously-undescribed c a r l a v i r u s occurring i n dandelion or i f dandelion was an unrecognized host for a known c a r l a v i r u s . The International Committee on the Taxonomy of Viruses (ICTV) has approved the use of various s i g l a to denote virus 4. groups and has attempted to c l a s s i f y the groups accordingly (Harrison ejt a l . , 1971; Fenner, 1976). Carnation latent virus i s the type member of the c a r l a v i r u s group and when the group was o r i g i n a l l y described, nine other viruses were included: cactus v i r u s 2; chrysanthemum vi r u s B (CVB); cowpea mild mottle v i r u s ; l i l y symptomless v i r u s ; p a s s i f l o r a l a t e n t v i r u s ; pea streak virus (PSV); potato v i r u s M (PVM); potato virus S (PVS) and red clover vein mosaic v i r u s (RCVMV). Possible members included: chicory blotch v i r u s ; cole latent v i r u s ; cynodon mosaic v i r u s ; elderberry v i r u s ; f r e e s i a mosaic v i r u s ; hop l a t e n t v i r u s ; muskmelbn vein necrosis v i r u s ; narcissus l a t e n t v i r u s (NLV) and poplar mosaic v i r u s (PMV) (Harrison et a l . , 1971; Fenner, 1976). Since the group has been established, at l e a s t seven ad d i t i o n a l c a r l a v i r u s e s have been reported: a l f a l f a l a t e n t virus (Veerisetty and Brakke, 1977); Chinese yam necrotic mosaic v i r u s (Fukomoto and Tochihara, 1978); Daphne virus S (Forster and Milne, 1978); eggplant mild mottle v i r u s (EMMV) ( K h a l i l and Nelson, 19 7 7); Helenium v i r u s S (HVS) (Kuschki et a l . , 1978); l i l a c mottle v i r u s (Waterworth, 1972) and s h a l l o t l a t e n t virus (Bos et a l . , 1978). Complete character-i z a t i o n has not been reported for a l l of these viruses and i n the case of Daphne v i r u s S, further studies are warranted as the s i z e of the p a r t i c l e (704-716 nm) does not f i t well within the range f o r ca r l a v i r u s e s (620-690 nm). Carlaviruses are characterized by s l i g h t l y flexuous rod-5. shaped p a r t i c l e s , 620-690 nm long, containing about 6% s i n g l e stranded RNA. In general, the longevity i n sap at room temperature i s a few days and the thermal i n a c t i v a t i o n point i s between 55° and 70°. The concentration i n sap i s 20-100 mg/1, but because of a tendency to aggregate s i d e - t o - s i d e and end-to-end, the y i e l d of p u r i f i e d v i r u s i s much lower (Varma et a l . , 1970; Luiso n i e_t a_l., 1976; Veerise t t y and Brakke, 1978) . The members of t h i s group i n f e c t narrow host ranges both n a t u r a l l y and experimentally, causing no symptoms or only mild ones. They are r e a d i l y transmitted by sap in o c u l a t i o n , except when natural plant i n h i b i t o r s are present and i n t e r s p e c i f i c transfers are being attempted. In those cases where a vector has been found, i t has proven to be an aphid that transmits the v i r u s non-persistently (Harrison et. al., 19 71; Fenner, 19 76; C h r i s t i e and Edwardson, 1977). S e r o l o g i c a l r e l a t i o n s h i p s e x i s t between some members of the c a r l a v i r u s group, although i t i s not e s s e n t i a l that a v i r u s be s e r o l o g i c a l l y r e l a t e d to a described member of the c a r l a -viruses i n order to be included i n the group (Gibbs et. a l . , 1966). For example, poplar mosaic v i r u s i s considered a ca r l a v i r u s even though i t i s not s e r o l o g i c a l l y r e l a t e d to any other known members of t h i s group (Brunt et a l . , 1976; Luisoni et a l . , 1976). The main c r i t e r i o n i s that p a r t i c l e s i z e and shape must be i n the range of values set down for members of t h i s group and on t h i s b a s i s , planned s e r o l o g i c a l 6. i n v e s t i g a t i o n s can be undertaken t o determine r e l a t i o n s h i p s among v i r u s e s . As the study p r o g r e s s e d , i t became obvious t h a t i n o r d e r to determine whether i n f a c t the v i r u s from d a n d e l i o n was an u n d e s c r i b e d c a r l a v i r u s or a s t r a i n o f a known v i r u s , i t was e s s e n t i a l t o o b t a i n c u l t u r e s o f s i m i l a r v i r u s e s and to under-take d i r e c t comparisons under s t a n d a r d c o n d i t i o n s . The l i t e r a t u r e was reviewed t o determine which o t h e r c a r l a v i r u s e s c o u l d produce s y s t e m i c symptoms i n Chenopodium q u i n o a W i l l d . , s i m i l a r t o those produced by the d a n d e l i o n v i r u s . From the d e s c r i p t i o n s i n the l i t e r a t u r e , the v i r u s t h a t seemed most l i k e l y t o be r e l a t e d was one t h a t had been i s o l a t e d from Mantaro p o t a t o e s i n Peru, and was c o n s i d e r e d t o b e a s t r a i n o f p o t a t o v i r u s S ( H i n o s t r o z a - O r i h u e l a , 1 9 7 3 ) . A c u l t u r e o f t h i s v i r u s was o b t a i n e d from Dr. A.M. Lekeu, Lima, Peru and the n e c e s s a r y comparisons were made. In o r d e r t o a v o i d c o n f u s i o n w i t h p o t a t o v i r u s S (PVS) i n the t e s t s , the Peru i s o l a t e was r e f e r r e d t o as Peru v i r u s S (PeVS). While these s t u d i e s were underway ohLthe r e l a t i o n s h i p w i t h PeVS, another c a r l a v i r u s was r e p o r t e d from Helenium  amarum h y b r i d s i n Germany, t h a t a l s o caused s y s t e m i c symptoms i n C. q u i n o a (Kucshki e_t a l . , 1978). T h i s v i r u s appeared s i m i l a r t o the d a n d e l i o n v i r u s and warranted i n c l u s i o n i n the comparative s t u d i e s . A c u l t u r e o f Helenium v i r u s S was o b t a i n -ed from Dr. R. Koenig, Braunschweig, Germany and comparative t e s t i n g was s t a r t e d . 7. Carnation latent v irus also causes systemic i n f e c t i o n of C. quinoa (Kemp and High, 1979), but i n view of i t s r e l a t i v e l y mild symptoms and the d i f f i c u l t y with which i t i s transmitted from carnation to other host species, i t was not considered as a p o t e n t i a l l y close r e l a t i v e of dandelion v i r u s . Consequently, no cultu r e of carnation la t e n t v i r u s was obtained. When t h i s project was started, the object was to describe some of the b i o l o g i c a l , physiochemical and s e r o l o g i c a l properties of dandelion v i r u s . (The abbreviation DVS w i l l be used throughout t h i s paper for dandelion virus.) For reasons outlined above, i t has evolved in t o a comparative study of three c a r l a v i r u s e s from three geographically i s o l a t e d and d i s t i n c t areas: Canada, Germany and Peru, as well as a de s c r i p t i o n of some of the properties of DVS. MATERIALS and METHODS F i e l d occurrence of dandelion v i r u s was determined by indexing dandelions from various s i t e s i n B r i t i s h Columbia and to a les s e r extent from other parts of Canada. The c o l l e c t -ed samples were indexed by enzyme-linked immunosorbent assay (ELISA) (Clark and Adams, 1977) and by rub in o c u l a t i o n to C. quinoa. Host Range and Symptomatology Leaves from i n f e c t e d dandelions were ground i n in o c u l a t i n g buffer, (0.1M KP0 4, pH 7.4 with 1% p o l y v i n y l p y r r o l i d i n e (PVP) and 1% n i c o t i n e ) , and rubbed onto carborundum-dusted C. quinoa 8. leaves. The systemically infected leaves provided the source of inoculum for the host range study. To reduce the e f f e c t of Chenopodium spp. i n h i b i t o r s , the sap was c l a r i f i e d by t r e a t -ment with calcium phosphate and the virus was concentrated twofold by p e l l e t i n g through 20% (w/v) sucrose (Veerisetty and Brakke, 1978). The v i r u s p e l l e t was resuspended i n i n o c u l a t i n g b u f f e r . Plants from 12 f a m i l i e s were l i g h t l y dusted with carborun-dum and the inocululum was applied gently with a foam pad. A negative check was included for each t e s t by rubbing a second plant with i n o c u l a t i n g buffer only. Immediately a f t e r i n o c u l a t i o n , the plants were rinsed with tap water. The t e s t plants were assayed 2 wk afte r i n o c u l a t i o n by back i n o c u l a t i n g onto C. quinoa and by ELISA. Properties i n Crude Sap The longevity i n v i t r o of DVS i n C. quinoa sap at 4° and 23° was determined over time. Systemically i n f e c t e d C. quinoa leaves were ground 1:2 (w/v) with i n o c u l a t i n g b u f f e r and centrifuged at 5,000 rpm for 5 min i n a S o r v a l l SS 34 c e n t r i -fuge. The supernatant was dispensed i n 1 ml aliq u o t s and stored at 4° or 23° f o r the appropriate length of time before i n f e c t i v i t y was tested. Inoculating buffer served as the negative check i n each case, and symptoms were recorded 3 wk after i n o c u l a t i o n . L y o p h i l i z e d t i s s u e was stored at room temperature and the i n f e c t i v i t y was checked about every 4 months by inoc u l a t i o n to C. quinoa. 9. To determine the d i l u t i o n end point, systemically infected C. quinoa leaves were ground 1:5 (w/v) with i n o c u l a t -ing buffer and the r e s u l t i n g sap was d i l u t e d i n te n f o l d increments to 10~^. One ml of each d i l u t i o n was inoculated to f i v e C. quinoa leaves and the symptoms were recorded at 14 and 21 days afte r i n o c u l a t i o n . Inoculating b u f f e r served as the negative check. The thermal i n a c t i v a t i o n point was determined with sap from systemically i n f e c t e d C. quinoa leaves, ground 1:2 (w/v) with i n o c u l a t i n g b u f f e r . The sap was dispensed i n 1 ml aliquots i n t o thin-walled b o r o s i l i c a t e t e s t tubes (10 x 75 mm) and incubated i n a Thelco Model 81 water bath f o r 10 min a f t e r the sap had reached the treatment temperature. The aliquots were kept on i c e before and a f t e r treatment. The cooled aliquots were inoculated onto C. quinoa, with i n o c u l a t i n g buffer as the negative check. Symptoms were recorded 14 and 35 days a f t e r i n o c u l a t i o n . Seed Transmission Mature £. quinoa seeds were c o l l e c t e d 10 wk a f t e r i n o c u l a -t i o n and a f t e r a 4-wk aging period, 100 seeds were germinated on moist f i l t e r paper. The seedlings and seed coats were separated and ground i n inoc u l a t i n g buffer for indexing on healthy C. quinoa. Seedlings from 100 seeds germinated i n s o i l were trans-planted to i n d i v i d u a l 10 cm pots 10 days a f t e r germination. The seedlings that survived t h i s t r a n s f e r were indexed at the s i x leaf stage by in o c u l a t i o n to healthy C. quinoa and by ELISA. Dandelion seeds were c o l l e c t e d from i n f e c t e d dandelions and allowed to age 6 months before t e s t i n g . Of the 64 seed-li n g s tested by ELISA, 13 were also indexed onto C. quinoa. The rate of germination was determined by p l a c i n g the seeds from healthy and i n f e c t e d plants on s o i l or moist f i l t e r paper and incubating them at room temperature. Aphid Transmission A colony of Myzus persicae (Sulz.) was established on healthy C. quinoa and maintained at 10-15° with a day length of 16 h. The colony was allowed to adapt to C. quinoa before vector studies were undertaken, i n order to reduce the e f f e c t of anomalous feeding behaviour. Aphids were c o l l e c t e d into a glass P e t r i d i s h and starved for 1 hr. Access times from 0-45 min at 5 min i n t e r v a l s were tested with 25 aphids per t e s t , except at time 20 and 35 min, when 50 aphids were used. A f t e r exposure to i n f e c t e d C. quinoa leaves, the aphids were transferred to healthy C. quinoa f o r 18 h r a f t e r which time they were removed and the plants trans-ferred to the greenhouse. Symptoms were recorded 10 days after i n o c u l a t i o n . P u r i f i c a t i o n An e f f i c i e n t and inexpensive method of p u r i f i c a t i o n was developed to prepare concentrated v i r u s . Systemically i n f e c t e d C. quinoa leaves and stems were homogenized f o r 3 min i n a Waring blendor with 2 v o l (w/v) col d 0.2M sodium borate bu f f e r , pH 9.0, containing 1% mercaptoethanol and 0.2% sodium diethyldithiocarbamate. The homogenate was pressed through nylon c l o t h and the sap was centrifuged at 10,000 rpm for 20 min (Sorv a l l GSA r o t o r ) . The supernatant was c o l l e c t e d through a layer of Miracloth and l e f t at 4° overnight, then centrifuged at 10,000 rpm for 20 min. The r e s u l t i n g supernatant was centrifuged at 26,000 rpm for 90 min (Beckman NO. 30 r o t o r ) . The p e l l e t s consisted of a gelatinous bottom layer containing the v i r u s with a top layer of green debris. The top green layer was allowed to s l i d e o f f the v i r u s p e l l e t by i n v e r t i n g the centrifuge tubes f o r about 10 min. D i s t i l l e d water was used to r i n s e the remaining green debris o f f the vi r u s p e l l e t . The v i r u s was resuspended i n a small amount of 0.02M borate buffer, pH 8.5 for 1-2 h at 4°. The suspension was c e n t r i f u g -ed at 5,000 rpm for 5 min (Beckman NO. 40 rotor) and the supernatant was applied to sucrose density gradients. Continu-ous or l i n e a r log gradients were made i n polyallomer tubes with 10-40% (w/v) sucrose i n 0.02M borate bu f f e r , pH 8.5. The loaded gradients were centrifuged at 38,000 rpm for 90 min (Beckman SW 41 rotor) and then scanned at 254 nm with an ISCO density gradient monitor. A l l centrifugations were c a r r i e d out at 4° while a l l other manipulations were at room temperature, except as noted otherwise. Cesium chloride gradients were also run using a modified procedure developed f o r potato v i r u s Y p u r i f i c a t i o n (Hiebert 12 and McDonald, 1973). The high speed p e l l e t s obtained by the standard procedure f o r p u r i f i c a t i o n of DVS were resuspended i n 5 ml 0.02M KPC>4 buffer, pH 8.5, and layered onto 7 ml cesium c h l o r i d e , density 1.2858 gm/cc, i n 0.02M KPO^ buffer, pH 8.5. The gradients were centrifuged at 32,000 rpm for 18 h (Beckman SW 41 rotor) and the v i r u s band was c o l l e c t e d e i t h e r by puncturing the c e l l u l o s e n i t r a t e tube and c o l l e c t i n g the opalescent zone by drops or by f r a c t i o n a t i n g the column through the ISCO density gradient monitor at 254 nm. Changes i n concentration and pH of extraction buffer, the use of additives and solvents for c l a r i f y i n g or concentrating the v i r u s preparation and the duration of the i n i t i a l high speed c e n t r i f u g a t i o n were also investigated. The effectiveness of the various procedures was determined by scanning the sucrose density gradients at 254 nm with the ISCO monitor and comparing the r e l a t i v e y i e l d and p u r i t y of the preparations. Electron Microscopy - P a r t i c l e Size Leaf dips were prepared by mincing systemically i n f e c t e d C. quinoa leaves i n 2% phosphotungstic acid (PTA), pH 6.7, and placing a drop of the extracted sap onto a carbon-fronted, collodion-coated copper g r i d (200 mesh) for 1 min. The g r i d was r i n s e d with 2% PTA and the excess s t a i n removed with a f i l t e r paper wedge. A f t e r a i r drying for a few minutes, the grids were scanned with a P h i l i p s EM 200 or EM 300 and photo-graphs taken as required. P u r i f i e d and p a r t i a l l y p u r i f i e d virus preparations were applied d i r e c t l y to the grids and then the same procedure was followed as for le a f d i p s . Varying numbers of p a r t i c l e s were measured f o r the various p u r i f i c a t i o n methods as well as for le a f d i p s . I n f e c t i v i t y of Gradients A p a r t i a l l y p u r i f i e d DVS preparation was layered onto continuous sucrosec.density gradients formed with 10-40% (w/v) sucrose i n 0.0165M disodium phosphate and 0.0018M trisodium c i t r a t e b uffer, pH 9.0. After c e n t r i f u g a t i o n at 38,000 rpm for 90 min (Beckman SW 41 r o t o r ) , the gradients were scanned at 254 nm and fr a c t i o n a t e d with an ISCO density gradient moni-tor and f r a c t i o n c o l l e c t o r . Selected f r a c t i o n s were checked by EM and a l l f r a c t i o n s were inoculated to C. quinoa. Symp-toms were recorded at 12, 14 and 18 days a f t e r i n o c u l a t i o n . Absorption Spectrum P u r i f i e d v i r u s preparations were scanned i n a Beckman DU spectrophotometer over the u l t r a v i o l e t range from 230-340 nm at 5 nm i n t e r n a l s . The values were p l o t t e d and corrected f o r l i g h t s c a t t e r i n g (Noordam, 19 73). Both the corrected and uncorrected values were used i n determining the absorbance c h a r a c t e r i s t i c s of DVS. Serology Antiserum production A young white New Zealand r a b b i t was immunized with four intramuscular i n j e c t i o n s of p u r i f i e d DVS (1 mg/ml) emulsified 1:1 with Freund's complete adjuvant. The f i r s t and second 14. i n j e c t i o n s were 2 wk apart, with two booster i n j e c t i o n s at 10 and 14 wk. A f t e r the second i n j e c t i o n , bleedings were done at weekly i n t e r v a l s over the next 19 wk and tested by the tube p r e c i p i t i n t e s t with the appropriate d i l u t i o n s of both a n t i -serum and antigen to determine the t i t r e of the antiserum and the end point of the antigen, i e . the minimum concentration of antigen to produce a v i s i b l e p r e c i p i t a t e . The r e l a t i v e amount of p r e c i p i t a t e formed f o r each r e a c t i o n was evaluated on a seven u n i t scale: 4=very dense; 3=dense; 2=moderate; l = s l i g h t ; t=trace; s = v i s i b l e with hand lens and -=no r e a c t i o n . S i m i l a r schedules were followed for production of a n t i s e r a to PeVS and HVS. A l l antisera were stored at 4° with a few c r y s t a l s of chlorobutanol as preservative. Agar g e l d i f f u s i o n serology Healthy and i n f e c t e d leaves of C. quinoa were ground 1:2 (w/v) with 0.85% NaCl and 0.2% sodium azide i n d i s t i l l e d water. The sap was c o l l e c t e d through Miracloth and then divided: 1 ml sap plus 1 ml grinding buffer; 1 ml sap plus 1 ml 5% p y r r o l i d i n e . The samples were incubated at room temperature for 5 min and then placed i n the appropriate wells i n the agar g e l s . The antiserum was prepared at twofold d i l u t i o n s from 1:80 to 1:10,240 and then added to the appropriate wells i n the agar ge l s . The agar gels were prepared with 0.9% Ionagar, 0.8% NaCl and 0.2% NaN^ i n d i s t i l l e d water; heated for 20 min i n a b o i l i n g water bath and while the mixture was s t i l l hot, dispensed i n 2 ml aliquots onto collodion-coated microscope s l i d e s . The t e s t s l i d e s were incubated at room temperature for 48 h i n a moist chamber and then checked for p r e c i p i t i n l i n e s . Tube p r e c i p i t i n serology In tube p r e c i p i t i n reactions, homologous and heterologous tests were done with d i l u t i o n s of DVS, PeVS and PVS i n combin-ation with d i l u t i o n s of t h e i r a n t i s e r a . A n t isera to DVS and PeVS were produced i n the course of t h i s study, while a n t i -serum to PVS from the Plant Virus and Antiserum Bank, A g r i c u l -ture Canada, Vancouver Research St a t i o n , had been stored i n g l y c e r o l (1:1) and frozen since 19 72, and was not as good q u a l i t y . This d i f f e r e n c e was taken into consideration when preparing the d i l u t i o n ranges of antiserum to be tested. Antiserum to HVS was from the f i r s t bleeding as HVS had not been included i n the comparative studies u n t i l r e c e n t l y . A l l of the antigens at 80 and 20 ug/ml, were tested against the appropriate antisera d i l u t i o n s and the endpoints determined. P u r i f i e d DVS was tested against antisera to other c a r l a -viruses obtained from the Plant Virus and Antiserum Bank, Ag r i c u l t u r e Canada, Vancouver Research Station, f o r which homologous pure antigen preparations were not a v a i l a b l e . Antisera d i l u t i o n s were used from 1:20 to 1:320, at twofold i n t e r v a l s against 80 ug/ml DVS. Sodium, dodecyl s u l f a t e agar g e l serology Since flexuous rod-shaped viruses do not migrate e f f e c t i v e l y through an agar gel media, unless the p a r t i c l e s are i n some way degraded into shorter, faster moving compon-ents, agents such as SDS were used to fracture the p a r t i c l e s ( P u r c i f u l l and Batchelor, 1977). Sap extracts were obtained by grinding l e a f t i s s u e 1:1 (w/v) with water and subjecting t h i s extract to low speed c e n t r i f u g a t i o n , a f t e r which the supernatant was c o l l e c t e d through Miracloth. SDS was added to a f i n a l concentration of 1% and the extract was heated for 90 sec i n b o i l i n g water. SDS agar was prepared with 0.6% Ionagar, 0.2% SDS, 0.7% NaCl and 0.1% NaN 3 i n 0.1M Tris-HCl buffer, pH 9.0 and 1 ml aliquots were dispensed into small (35 x 10 mm) P e t r i dishes. Once the gels had set, wells were cut using a template made from discharged 0.22 c a l i b r e brass cartridges (Wright and Stace-Smith, 1966) and the agar plugs were aspirated out j u s t p r i o r to f i l l i n g the wells with the t e s t samples. To t e s t i f DVS antiserum had been produced against whole virus and/or subunits, antigen wells were f i l l e d with sap extracts that had not been treated with SDS or heat; with sap extracts that contained 1% SDS, but not heated; and with sap extracts that contained 1% SDS and that had been heated for 90 sec i n b o i l i n g water. SDSaagar gel d i f f u s i o n i s frequently used to detect r e l a t i o n s h i p s among rod-shaped viruses and DVS, (1 mg/ml and 0.5 mg/ml) and PeVS, (2 mg/ml), were tested against f u l l strength DVS antiserum to determine i f a spur would form at the i n t e r s e c t i o n of the homologous and heterologous reactions. Latex agglutination serology Antiserum with a t i t r e of 40, 960 was prepared at twofold d i l u t i o n s from 1:100 to 1:12,800 and conjugated with latex beads (Phatak, 1974). About '3-ul of antiserum-latex d i l u t i o n was drawn i n t o the c a p i l l a r y tube; the t i p was wiped clean and 8 i l l of t e s t sample were drawn up from the same end of the c a p i l l a r y tube; the t i p was wiped dry and the tube was taped to a microscope s l i d e . When the range of antiserum d i l u t i o n s had been used, the microscope s l i d e s were taped to an i n c l i n e d r o t a t i n g wheel and the contents of the tubes: mixed for 15 min at room temperature. The r e s u l t s were read under a stereo-microscope with top l i g h t i n g of the c a p i l l a r y tubes. P o s i t i v e r e s u l t s were indicated by an agglutination of the latex beads. Enzyme-linked immunosorbent assay (ELISA) The ELISA technique (Clark and Adams, 1977) was adapted for use with DVS. Minor modifications were made to the stand-ard procedure: the immunoglobulin G was concentrated from crude antiserum by two cycles of ammonium s u l f a t e p r e c i p i t a t i o n instead of one cycle and the f i r s t major peak eluted through the DEAE-22 Sephadex column was c o l l e c t e d ; the conjugate was stored at 4° without the addition of bovine serum albumin and the coated m i c r o t i t r e plates (Cooke-Dynatech) were stored at 4° overnight or longer p r i o r to use. A moist environment was maintained throughout the assay by enclosing the p l a t e i n a small p l a s t i c bag held shut by an e l a s t i c band. Test samples 18. were incubated overnight at 4°, rather than at 37° for 4-6 h. The colour r e a c t i o n was i n i t i a l l y assessed spectrophoto-m e t r i c a l l y at 405 nm with a twofold d i l u t i o n of the reaction mixture. However, f o r assay of f i e l d material or host range, v i s u a l observation was s u f f i c i e n t to d i s t i n g u i s h p o s i t i v e and negative r e s u l t s . Black and white p r i n t s and colour s l i d e s were taken of the plates about one hour aft e r the substrate had been added, so that when no absorbance readings were taken there was s t i l l a permanent record of the t e s t . Transmission electron microscope serology (TEMS) A drop of antigen was placed on a wax pl a t e and a drop of antiserum was added. The antiserum was used at d i l u t i o n s over the range 1:10, 1:20 and 1:40. The mixture was incubated at room temperature for 3 min and then a drop of 4% PTA, pH 7.2 w was added. After 1 min, a g r i d was f l o a t e d on the drop, co l l o d i o n side down, fo r 5 min. The g r i d was d r i e d with a f i l t e r paper wedge and scanned. Various combinations of antisera and antigens were used to study the DVS and PeVS reactions i n an attempt to d i s t i n g u i s h these two c a r l a v i r u s e s by electron microscopy. Tobacco mosaic virus (TMV) was included as a c o n t r o l . 19. RESULTS F i e l d Occurrence of DVS A more extensive survey of dandelions i n and around the o r i g i n a l c o l l e c t i o n s i t e near Kelowna was undertaken. A t o t a l of 109 plants from Kelowna, Naramata, Summerland and Westbank were indexed and of these, 71 were in f e c t e d (Table 1). Dandelions growing i n other areas of B r i t i s h Columbia, including Vancouver Island, the Fraser Valley, the C h i l c o t i n Table 1. Results of indexing f i e l d c o l l e c t i o n s of dandelions from various areas of Canada for natural i n f e c t i o n with DVS Area S p e c i f i c S i t e Number of Dandelions Indexed P o s i t i v e Vancouver Island Fraser V a l l e y Okanagan V a l l e y Kootenay V a l l e y C h i l c o t i n Alberta Saskatchewan Manitoba Ontario Saanichton 14 Abbotsford; Langley; 134 Vancouver Manning Park 13 Kelowna; Naramata; 109 Summerland; Westbank Castlegar; C h r i s t i n a 25 Lake; Creston; Grand Forks Williams Lake 5 Calgary 20 Cut Knife; Maple Creek 108 B i r d s ' H i l l Park; 75 Winnipeg Ottawa 15 518 0 0 0 71 0 0 0 0 0 _0 71 20. area and the Kootenay V a l l e y were indexed and found to be negative for DVS. S i m i l a r l y , dandelions c o l l e c t e d from other parts of Canada, as f a r east as Ottawa were also negative. In t o t a l , 518 dandelions were indexed from 44 c o l l e c t i o n s i t e s and of these only 71 were found to be n a t u r a l l y - i n f e c t e d with DVS. In Kelowna, 70 of the 92 dandelions indexed were infected, while 1 out of 5 dandelions indexed from Naramata was i n f e c t e d with DVS. Host Range and Symptomatology Infected dandelions i n the f i e l d exhibited no symptoms by which they could be r e a d i l y i d e n t i f i e d compared with other noninfected dandelions. S i m i l a r l y , dandelion seedlings rub-inoculated with DVS, and back indexed to C. quinoa to prove i n f e c t i o n , showed no symptoms. Some var i a t i o n s i n l e a f s i z e and shape were evident i n seedlings from both healthy and infected plants but t h i s was a t t r i b u t e d to inherent seedling v a r i a t i o n rather than to expression of disease symptoms. C. quinoa was chosen as an assay host because of i t s obvious symptoms r e s u l t i n g from i n f e c t i o n with DVS. C h l o r o t i c l o c a l l e s i o n s developed 5-7 days a f t e r i n o c u l a t i o n of plants at the 8-10 leaf stage, and with increasing time, these became more noticeable as n e c r o t i c spots on the senescent inoculated leaves. At 10-14 days a f t e r i n o c u l a t i o n , systemic c h l o r o s i s became evident, progressing from mild vein c l e a r i n g to complete ch l o r o s i s and epinasty; a x i a l shoots also exhibited c h l o r o s i s ( F i g . 1A-D). Inoculation of C. quinoa at the 6-8 l e a f stage 21. . 1. Symptoms of DVS i n f e c t i o n i n C. quinoa. A. healthy plant; B. inf e c t e d plant showing vein c l e a r i n g and epinasty a f t e r 14 days; C. l o c a l l e s i o n s on inoculated l e a f a f t e r 10 days and D. healthy l e a f ( l e f t ) and progression of symptom development i n 4 top leaves of inoculated plant r e s u l t e d i n m i l d s t u n t i n g . F l o w e r i n g and seed s e t o c c u r r e d s u c c e s s f u l l y o n l y i f the p l a n t was i n o c u l a t e d a t an o l d e r age and t h e r e f o r e d i d not c o l l a p s e c o m p l e t e l y p r i o r t o f l o w e r i n g . The inoculum used f o r host range s t u d i e s was c l a r i f i e d and c o n c e n t r a t e d i n o r d e r t o d e c r e a s e the i n h i b i t o r e f f e c t o f Chenopodium sap, the r e b y i n c r e a s i n g the chance o f t r a n s m i s s i o n to o t h e r h o s t s . The p r e p a r a t i o n was checked by e l e c t r o n microscopy to determine the amount of breakage t o the p a r t i c l e s . Few p a r t i c l e s were found t o be broken ( F i g . 2A) and the procedure f o r c l a r i f i c a t i o n and c o n c e n t r a t i o n d i d not a f f e c t the i n f e c t i v i t y of the v i r u s i n C. q u i n o a . The e x p e r i m e n t a l h o s t range s u r v e y i n c l u d e d p l a n t s from 12 f a m i l i e s , 4 of which had members s u s c e p t i b l e t o i n f e c t i o n by DVS. D. stramonium developed some p i n p o i n t n e c r o t i c l o c a l l e s i o n s ; G. g l o b o s a developed some l a r g e n e c r o t i c l o c a l l e s i o n s ( F i g . 2B,C); T. o f f i c i n a l e showed no symptoms o f i n f e c t i o n ; C. a m a r a n t i c o l o r d e v e l o p e d many c h l o r o t i c l o c a l l e s i o n s t h a t were emphasized by a r e d h a l o as the l e a f senesced, and sy s t e m i c v e i n c l e a r i n g , e p i n a s t y and s t u n t i n g ( F i g . 2D,E,F). The C. q u i n o a developed t y p i c a l symptoms as d e s c r i b e d above. V i r u s i n f e c t i o n or l a c k t h e r e o f , i n both i n o c u l a t e d and u n i n o c u l a t e d l e a v e s was assayed by the ELISA t e c h n i q u e and by back i n o c u l a t i o n to C. quinoa f o r a l l p l a n t s i n the ho s t range ( T a b l e 2 ) . T e s t i n g w i t h ELISA d e t e c t e d v i r u s from the i n o c u l a t e d l e a v e s of a l l the s u s c e p t i b l e h o s t s and a l s o from the s y s t e m i c l e a v e s o f C. a m a r a n t i c o l o r , C. q u i n o a and d a n d e l i o n . F i g . 2. E l e c t r o n m i c r o g r a p h of s e m i - p u r i f i e d DVS u sed as i n o c u l u m f o r h o s t range s t u d i e s (A. b a r = 500 nm) and symptoms i n d u c e d on G. g l o b o s a (B. h e a l t h y p l a n t ; C. i n o c u l a t e d p l a n t w i t h n e c r o t i c l o c a l l e s i o n s ) and on C. a m a r a n t i c o l o r (D. h e a l t h y p l a n t E. i n o c u l a t e d p l a n t w i t h s y s t e m i c v e i n c l e a r i n g as p o i n t e d o u t by arrows and F. l o c a l l e s i o n s on i n o c u l a t e d l e a f a f t e r 14 d ays) 24. Table 2. Results of the experimental host range studies as determined by symptoms i n the inoculated host and indexing the inoculated (I) and systemic (S) leaves by ELISA and back i n o c u l a t i o n to C. quinoa Family Species Symptoms ELISA C. quinoa I S I S Amaranthaceae Gomphrena + + - - -globosa L. Apocynaceae Vinca rosea L. - - - -Caryophyllaceae Dianthus - - - -barbatus L. Chenopodiaceae Chenopodium + + + + + amaranticolor Coste & Reyn. Chenopodium quinoa + + + + + W i l l d , Compositae Cruciferae Helianthus annus L. -Lactuca s a t i v a L. -var. capitatum Taraxacum o f f i c i n a l e -Weber Verbesina  encelioides (Car.) Benth. & Hook. Zinni a elegans Jacq. -Brassica juncea (L.) Coss Brassica pekinensis (Lour.) Rupr. var Petsai + + Cucurbitaceae Gramin eae Leguminosae Cucumis sativus L. Hordeurn vulgare L. Phaseolus  v u l g a r i s L. var Bountiful var Pinto Pisum sativum L. var Perfection V i c i a faba L. Vigna sinensis E r d l , var Black eye Table 2. (cont'd) Family Species Symptoms ELISA C. quinoa I S Plantaginaceae Plantago lanceolata L. Plantago major L, Rosaceae Fragaria vesca L, var Alpine Solanaceae Capsicum frutescens L. var grossum Datura stramonium L. + + -Lycopersicum - - -esculentum M i l l . var Rutgers var Subarctic - - -Nicotiana - - -c l e v e l a n d i i Gray Nicotiana debneyi - - -Domin. Nicotiana - - -qlu t i n o s a L. Nicotiana r u s t i c a L. - - -Nicotiana tabacum L. - - -var Haranova var Havana - - -var"Samsun - - -var S i I v e s t r i s - - -var White Burley - - -var Xanthi - - -Petunia hybrida Vilm.- - -Splanum tuberosum L. - - -var Mirton Pearl Presumably, the v i r u s was capable of multiplying i n the two l o c a l l e s i o n hosts to the extent that the presence of v i r i o n s could be detected by ELISA, but there were not adequate numbers of i n f e c t i v e v i r i o n s to be detected by back i n o c u l a t i o n to C. quinoa. Properties i n Crude Sap I n f e c t i v i t y was retained i n crude C. quinoa sap between 4 and 5 days at 23°; between 28 and 56 days at 4° and more than 16.5 months i n a l y o p h i l i z e d state at 23°. Systemic i n f e c t i o n of C. quinoa was used as the determining factor for the d i l u t i o n end point, as s u f f i c i e n t v i r us was then considered to be present to e s t a b l i s h a t y p i c a l i n f e c t i o n . At a d i l u t i o n _5 of 2 x 10 , the inoculum was s t i l l able to cause systemic i n f e c t i o n , while further d i l u t i o n d i d not produce systemic i n f e c t i o n even a f t e r 21 days, although a few l o c a l lesions were present. The temperature range of i n a c t i v a t i o n f o r DVS i n C. quinoa sap was 75-80°, as determined by the a b i l i t y of the tested aliquots to cause systemic i n f e c t i o n i n C. quinoa 35 days a f t e r i n o c u l a t i o n . • Seed Transmission The germination rate of seeds from healthy and infected C. quinoa on moist f i l t e r paper was 95-100%. Inoculation of the seed coats and the seedlings to C. quinoa produced no symptoms of i n f e c t i o n . These seedlings matured with no symp-toms and no latent i n f e c t i o n was detected by ELISA or by back inoc u l a t i o n to C. quinoa. The germination rate of seeds from healthy and infected dandelions on f i l t e r paper and on s o i l was 64%. No lat e n t i n f e c t i o n was detected by ELISA or by back i n o c u l a t i o n to C. quinoa. Aphid Transmission Myzus persicae established colonies on C. quinoa, a f t e r an adaptation period of about 8 wk, however, the colony became infected with a hymenopteran p a r a s i t e and was discarded. A second colony was established and no i n f e c t i o n by parasites occurred. M. persicae would not colonize dandelions i n the rosette stage. DVS was transmitted to C. quinoa by M. persicae a f t e r 5 min access to i n f e c t e d C. quinoa, but not a f t e r 35 min. No transmission was obtained when aphids were allowed to feed on healthy C. quinoa. P u r i f i c a t i o n Various methods of c l a r i f i c a t i o n and concentration were t r i e d and compared on the basis of t h e i r absorbance scan patterns. In F i g . 3, the e f f e c t s of combinations of extracting buffers and additives are shown: with the standard method, the virus peak i s s u f f i c i e n t l y separated from host contaminants with borate buffer to take advantage of the increased virus y i e l d compared with phosphate buffer extraction ( F i g . 3A); chloroform with e i t h e r buffer d i d not c l a r i f y the preparation and also reduced the v i r u s y i e l d ( F i g . 3B); carbon t e t r a c h l o r i d e had a s i m i l a r e f f e c t to chloroform, except with phosphate buffer no virus peak was obtained ( F i g . 3C); polyethylene g l y c o l d i d not aid i n c l a r i f y i n g or concentrating the virus ( F i g . 3D), nor d i d ammonium s u l f a t e ( F i g . 3E); T r i t o n X-100 e f f e c t i v e l y removed the virus peak ( F i g . 3F), as d i d treatment F i g . 3. Absorbance scan patterns of sucrose density gradients showing e f f e c t s of combinations of extraction buffers and additives on p u r i f i c a t i o n of DVS. The extraction buffers were 0.5M borate, pH 8.0 ( ) and 0.5M phosphate, pH 7.4 ( ). Position of v i r u s peak i s indicated by an arrow. Treatments were as follows: A. standard method as outl i n e d i n F i g . 10; B. 1:1 chloroform; C. 1:1 carbon t e t r a c h l o r i d e ; D. 10% polyethylene g l y c o l and 0.1M NaCl; E. 15% ammonium su l f a t e ; F. 1% T r i t o n X-100; G. 1% bentonite; H. adjustment to pH 5.0; I. 1:1 butanol and J . 1:1 chloroform/butanol 29. with bentonite ( F i g . 3G) , a c i d i f i c a t i o n ( F i g . 3H), butanol (Fi g . 31) and chloroform/butanol ( F i g . 3J). No s i g n i f i c a n t increase i n p u r i t y and y i e l d was obtained with 0.5M' borate buffer ( F i g . 4), compared with 0.2M borate buffer ( F i g . 5), so f o r economy the lower concentration of buffer was adopted as standard. By comparing the c l a r i f i c a -tion achieved by a range of pH's from 8 to 9.5 (Fi g s . 5-8), the optimum pH for extraction was determined to be pH 9.0, with-out readjustment back to pH 9.0 a f t e r homogenization ( F i g . 7C, E). The i n i t i a l c l a r i f i c a t i o n was achieved a f t e r high speed ce n t r i f u g a t i o n by allowing the green p e l l e t to s l i d e o f f the underlying v i r u s p e l l e t and the ease with which t h i s was achieved depended on the duration of the c e n t r i f u g a t i o n : a f t e r 60 min, the v i r u s p e l l e t was loosely packed and a portion of i t tended to s l i d e o f f with the green p e l l e t ; a f t e r 90 min, the v i r u s p e l l e t was firm enough that the green p e l l e t could s l i d e o f f without d i s t u r b i n g the v i r u s p e l l e t ; a f t e r 120 min the p e l l e t s had become too f i r m l y packed to be separated without some force, such as a stream of water, which res u l t e d i n loss of virus (Figs. 5-8). A d d i t i o n a l c l a r i f i c a t i o n was achieved by adding ethylene diamine t e t r a - a c e t i c ; acid (EDTA) to the resuspended v i r u s p e l l e t s , but the advantage was not great enough to o f f s e t the reduction i n virus y i e l d to warrant i n c l u s i o n i n the standard method (F i g s . 5-8 B,D,G). Sucrose density gradients were r o u t i n e l y used as the 30. R E L A T I V E D E P T H » F i g . 4. Absorbance scan patterns of sucrose density gradients showing e f f e c t s of ce n t r i f u g a t i o n time and addition of EDTA. P o s i t i o n of v i r u s peak i s ind i c a t e d by an arrow. Extraction buffer was 0.5M borate, pH 8.0 and treatments were as follows: A. 60 min c e n t r i f u g a t i o n ; B. 90 min ce n t r i f u g a t i o n and C. 90 min cen t r i f u g a -t i o n plus EDTA to 0.01M in • » 1 i c IN t E A N I \ * G R E L A T I V E D E P T H • F i g . 5. Absorbance scan patterns of sucrose density gradients showing e f f e c t s of pH, ce n t r i f u g a t i o n time and addi-^ t i o n of EDTA to 0.01M. P o s i t i o n of v i r u s peak i s indicated by an arrow. Extraction buffer was 0.2M borate, pH 8.0 and treatments were as follows: A. 60 min c e n t r i f u g a t i o n ; B. 60 min c e n t r i f u g a t i o n plus EDTA; C. 90 min ce n t r i f u g a t i o n ; D. 90 min ce n t r i f u g a t i o n plus EDTA; E. readjustment of extract-ed sap to pH 8.0, 90 min c e n t r i f u g a t i o n ; F. 120 min ce n t r i f u g a t i o n and G. 120 min ce n t r i f u g a t i o n plus EDTA 31. * A • B * c < E » G R E L A T I V E D E P T H • F i g . 6. Absorbance scan patterns of sucrose density gradients showing e f f e c t s of pH, c e n t r i f u g a t i o n time and addition of EDTA to 0.01M. Positi o n of the virus peak i s ind i c a t e d by an arrow. Extr a c t i o n buffer was 0.2M borate, pH 8.5 and treatments were as follows: A. 60 min c e n t r i f u g a t i o n ; B. 60 min c e n t r i f u g a t i o n plus EDTA; C. 90 min ce n t r i f u g a t i o n ; D. 90 min cen t r i f u g a t i o n plus EDTA; E. readjustment of extracted sap to pH 8.5, 90 min c e n t r i f u g a t i o n ; F. 120 min c e n t r i f u g a t i o n and G. 120 min ce n t r i f u g a t i o n plus EDTA 32. * A • b \ 0 * E J U 1 * G R E L A T I V E D E P T H » F i g . 7. Absorbance scan patterns of sucrose density gradients showing e f f e c t s of pH, ce n t r i f u g a t i o n time and addition of EDTA to 0.01M. Positi o n of the virus peak i s ind i c a t e d by an arrow. Extraction buffer was 0.2M borate, pH 9.0 and treatments were as follows: A. 60 min c e n t r i f u g a t i o n ; B. 60 min c e n t r i f u g a t i o n plus EDTA; C. 90 min ce n t r i f u g a t i o n ; D. 90 min ce n t r i f u g a t i o n plus EDTA; E. readjustment of extracted sap to pH 9.0, 90 min c e n t r i f u g a t i o n ; F. 120 min ce n t r i f u g a t i o n and G. 120 min c e n t r i -fugation plus EDTA 3 3 . F i g . 8. Abso r b a n c e s c a n p a t t e r n s o f s u c r o s e d e n s i t y g r a d i e n t s showing e f f e c t s o f pH, c e n t r i f u g a t i o n t i m e and a d d i t i o n o f EDTA t o 0.01M. P o s i t i o n o f t h e v i r u s peak i s i n d i c a t e d by an a r r o w . E x t r a c t i o n b u f f e r was 0.2M b o r a t e , pH 9.5 and t r e a t m e n t s were as f o l l o w s : A. 60 min c e n t r i f u g a t i o n ; B. 60 min c e n t r i f u g a t i o n p l u s EDTA; C. 90 min c e n t r i f u g a t i o n ; D. 90 min c e n t r i f u g a t i o n p l u s EDTA; E. r e a d j u s t m e n t o f e x t r a c t e d s a p t o pH 9.5, 90 min c e n t r i f u g a t i o n ; F. 120 min c e n t r i f u g a t i o n and G. 120 min c e n t r i f u g a t i o n p l u s EDTA 34. f i n a l separation step to p u r i f y the v i r u s . The peak obtained was not always sharp, as the dimers and polymers moved much deeper int o the gradients ( F i g . 9) and consequently, the lower portion of the gradient contained v i r u s p a r t i c l e s i n various stages of aggregation. Cesium c h l o r i d e gradients produced a sharp v i r u s band at equilibrium ( F i g . 9), but were not routine-ly used due to the high cost of cesium chloride and the long c e n t r i f u g a t i o n time required. As a r e s u l t , a p u r i f i c a t i o n schedule was developed that required a minimum of chemicals and that was e f f i c i e n t for p u r i f y i n g DVS, PeVS and HVS from C. quinoa ( F i g . 10). Y i e l d s obtained by t h i s method were 20-30 mg/kg of pla n t material. El e c t r o n Microscopy - P a r t i c l e S i z e The e f f e c t of various p u r i f i c a t i o n treatments on p a r t i c l e s i z e was assessed by comparing the d i s t r i b u t i o n of p a r t i c l e length categories. The standard p u r i f i c a t i o n method yielded p a r t i c l e s of normal length 645 nm ( F i g . 11D), chloroform and EDTA ( F i g . H E ) , butanol ( F i g . 11F) , butanol and EDTA ( F i g . 11G) or cesium chloride gradients ( F i g . H I ) were d i s t r i b u t e d around a mean of 630 nm. P a r t i c l e s from preparations treated with T r i t o n X-100 had a.aean of 650 nm ( F i g . HH) . Of the 1 064 p a r t i c l e s measured, the average length was found to be 637 nm ( F i g . 11J), which was approximately that found with leaf dip preparations, i e . 640 nm ( F i g . H A ) . The width of the p a r t i c l e s i n l e a f dip preparations was 12-13 nm. 35 . R E L A T I V E D E P T H — • F i g . 9. Absorbance scan patterns comparing the sharpness of the virus peak (indicated by an arrow) obtained with A. sucrose and B. cesium ch l o r i d e density gradient c e n t r i f u g a t i o n at 38T000 rpm for 90 min and 32,000 rpm for 18 h, r e s p e c t i v e l y i n an SW 41 rotor 3 6 . I n f e c t e d C. quin o a l e a v e s and stems 1:2 (w/v) 0.2M b o r a t e b u f f e r , pH 9.0 +1% mercaptoethanol +0.2% sodium d i e t h y l d i t h i o c a r b a m a t e Residue Sap 10,000 rpm, 20 min (4°, o v e r n i g h t ) 10,000 rpm, 20 min '26,000 rpm, 90 min + 0.02M b o r a t e b u f f e r , pH 8.5 (4°, o v e r n i g h t ) 5,000 rpm, 5 min SDGC, SW 41, 38,000 rpm, 90 min P u r i f i e d V i r u s S: s u p e r n a t a n t P: p e l l e t F i g . 10. Flow diagram of the s t a n d a r d p u r i f i c a t i o n method 37. P A R T I C L E L E N G T H (nm) F i g . 11. E f f e c t of p u r i f i c a t i o n methods on the p a r t i c l e length d i s t r i b u t i o n of DVS as determined by measure-ment of p a r t i c l e s i n electron micrographs: A. l e a f dip preparation; B. standard p u r i f i c a t i o n method as outlined i n F i g . 10; C. 0.01M EDTA; D. 1:1 chloro-form; E. 1:1 chloroform and 0.01M EDTA; F. 1:1 butanol; G. 1:1 butanol and 0.01M EDTA; H. 1% T r i t o n X-100 and I. standard p u r i f i c a t i o n method with cesium chloride density gradient c e n t r i f u g a t i o n . d i s t r i b u t i o n i s obtained when the r e s u l t s from a l l treatments are combined (J) I n f e c t i v i t y of Gradients Fractions from the top quarter of the gradients, as represented on the absorbance scan pattern by the area from the s t a r t i n g meniscus to the end of f r a c t i o n A ( F i g . 1 2 ) , were not in f e c t i o u s and t h i s was correlated with the absence of virus p a r t i c l e s ( F i g . 1 3 A ) . The f r a c t i o n s between A and C ( F i g . 12) were slow to cause symptoms i n inoculated C. quinoa, but were in f e c t i o u s even though broken p a r t i c l e s were c h a r a c t e r i s t i c of thi s area ( F i g . 1 3 B ) . Fractions from C to G ( F i g . 12) were in f e c t i o u s and the p a r t i c l e s ranged from s l i g h t l y broken 38. (F i g . 13C,D) to i n t a c t v i r i o n s ( F i g . 13E,F). Fractions from G to the bottom of the gradient ( F i g . 12) were quick to induce symptoms i n inoculated C. quinoa, and the p a r t i c l e s i n these f r a c t i o n s were found to be polymers or aggregates ( F i g . 13G, H,I). The rate and i n t e n s i t y of symptom development i n £• crui"Q a was presumably, a r e f l e c t i o n of the concentration of i n t a c t i n f e c t i o u s v i r i o n s within the i n d i v i d u a l f r a c t i o n s used as inoculum. R E L A T I V E D E P T H ' F i g . 12. Absorbance scan pattern of DVS af t e r sucrose density gradient c e n t r i f u g a t i o n for 90 min at 38,000 rpm i n an SW 41 r o t o r . Letters i n d i c a t e representative f r a c t i o n s that were c o l l e c t e d and examined by electron microscopy ( F i g . 13) F i g . 13. E l e c t r o n micrographs of v a r i o u s f r a c t i o n s c o l l e c t e d from a s u c r o s e d e n s i t y g r a d i e n t . A-I c o r r e s p o n d to the l e t t e r e d f r a c t i o n s i n F i g . 12. Bar r e p r e s e n t s 500 nm 40. Absorption Spectrum The absorption scan for DVS i s shown i n F i g . 14, with the corrected scan also drawn i n . This pattern i s t y p i c a l for an RNA v i r u s , with a maximum absorption at 259 nm and a minimum at 245 nm. The absorption c h a r a c t e r i s t i c s were s l i g h t l y higher when c a l c u l a t e d from corrected values than from uncorrected values: A m a x 1.13 + 0.04 and 1.09 + 0.04; A ^ ° 1.59 + 0.11 mm — — ' 280 — and 1.41 + 0.09, r e s p e c t i v e l y . 0.8-0.7-0.6-S 0 .5 -z < CO ce S o * -CQ < 0 .3 -0.2-0.1-2 4 0 2 6 0 ' 2 8 0 ' 3 0 0 3 2 0 3 4 0 W A V E L E N G T H ( n m ) F i g . 14.. Absorbance scan pattern of DVS, from 230-340 nm: uncorrected ( ——•—•-) and corrected (—--•—•-) for l i g h t s c a t t e r i n g 4 1 . S e r o l o g y A n t i s e r u m p r o d u c t i o n The homologous t i t r e was d e t e r m i n e d f o r e a ch b l e e d i n g and t h e h i g h e s t t i t r e , 40,960, was o b t a i n e d a f t e r t h e l a s t b o o s t e r i n j e c t i o n and was m a i n t a i n e d u n t i l t h e l a s t b l e e d i n g ( F i g . 15). The n o n s p e c i f i c t i t r e was 8, i n d i c a t i n g t h a t t h e p u r i f i e d v i r u s p r e p a r a t i o n used t o immunize t h e r a b b i t , had o n l y a t r a c e o f c o n t a m i n a t i o n w i t h h o s t p r o t e i n s . A r e p r e -s e n t a t i v e d e t e r m i n a t i o n o f t i t r e i s shown i n T a b l e 3, w i t h a t i t r e o f 40,960 a f t e r 18 h i n c u b a t i o n . Only m i n o r d i f f e r e n c e s were n o t e d between r e a d i n g s a f t e r 2 h i n c u b a t i o n and a f t e r 18 h i n c u b a t i o n , and t h e t i t r e r e mained unchanged. The c o n t o u r l i n e s emphasize th e optimum c o n c e n t r a t i o n s o f a n t i s e r u m and a n t i g e n (Matthews, 1 9 5 7 ) . The amount o f p r e c i p i t a t e formed as 81,920-1 ,40,960-1 I-s £ 5,120-1 < 2,560-j t 0 6 8 10 12 14 I M M U N I Z A T I O N S C H E D U L E ( w k ) 16 18 F i g . 15. A n t i s e r u m p r o d u c t i o n a g a i n s t DVS a n t i g e n , as d e t e r m i n e d by tube p r e c i p i t i n s e r o l o g y . Arrows i n d i c a t e i n j e c t i o n s o f a n t i g e n Table 3. Tube p r e c i p i t i n t i t r e and end point determination of DVS using varying concentrations of antigen and antiserum. The contour l i n e s j o i n readings of equal i n t e n s i t y Time Antiserum (h) D i l u t i o n Reaction r e l a t i v e to antigen concentration (jug/ml) 320 160 80 40 20 10 2.5 0 18 0 10 20 40 80 160 320 640 1 ,280 2 ,560 5 ,120 10 ,240 20 ,480 40 ,960 81 ,920 163 ,840 : 4: very dense; 3: dense; 2: moderate; 1: s l i g h t ; t: trace; and s: v i s i b l e with hand lens the r e s u l t of antigen/antiserum i n t e r a c t i o n i n tube p r e c i p i t i n serology was scored on a r e l a t i v e scale: 4: very dense; 3: dense; 2: moderate; 1: s l i g h t ; t: trace and s: v i s i b l e with hand lens ( F i g . 16). In routine t e s t s , scores as low as 's' were doubtful and, therefore, not considered as meaningful p o s i t i v e r eactions. S i m i l a r tests were c a r r i e d out to determine the t i t r e of PeVS antiserum: the highest homologous t i t r e was 10,280, with a nonspecific t i t r e of 1 and HVS antiserum: the i n i t i a l bleeding had a homologous t i t r e of 1,280. 43. 0 o e © i • r • • F i g . 16. Relative amounts of p r e c i p i t a t e formed with tube p r e c i p i t i n reactions. P r e c i p i t a t i o n was scored as: A. very dense, 4; B. dense, 3; C. moderate, 2 and D. s l i g h t , 1 Agar gel d i f f u s i o n serology No r e a c t i o n was v i s i b l e between the antiserum and healthy sap. Untreated sap from infected C. quinoa reacted with i t s homologous antiserum to a d i l u t i o n of 1:640, whereas the p y r r o l i d i n e treated sap d i d not react under the same conditions. This may have been due to the f a c t that only o l d reagent grade p y r r o l i d i n e was a v a i l a b l e , and i t may have c o n t a i n e d s u f f i c i e n t c o n t a m i n a n t s t o have d e s t r o y e d t h e v i r u s p a r t i c l e s . Tube p r e c i p i t i n s e r o l o g y The a n t i s e r a end p o i n t s d e t e r m i n e d f o r homologous and h e t e r o l o g o u s r e a c t i o n s i n v o l v i n g DVS, PVS, PeVS and HVS a r e summarized i n T a b l e s 4 and 5. From t h e s e r e s u l t s , i t can be seen t h a t 80 pg/ml i s t o o h i g h a c o n c e n t r a t i o n o f a n t i g e n t o d e t e r m i n e t r u e s e r o l o g i c a l r e l a t i o n s h i p s (Table 4 ) , w h i l e 2 0 pg/ml i s c l o s e r t o t h e o p t i m a l c o n c e n t r a t i o n (Table 5) and v a r y i n g degrees o f s e r o l o g i c a l r e l a t i o n s h i p among t h e v i r u s e s T a b l e 4. P r e c i p i t i n end p o i n t s i n two-way tube p r e c i p i t i n t e s t s i n c l u d i n g f o u r a n t i s e r a and t h e i r r e s p e c t i v e a n t i g e n s a t 8 0 pg/ml A n t i g e n A n t i s e r u m DVS PVS PeVS HVS DVS 10,240 5,120 1,280 40 PVS 2,560 2,560 1,280 40 PeVS 320 5,120 1,280 20 HVS 40 20 10 320 T a b l e 5. P r e c i p i t i n end p o i n t s i n two-way tube p r e c i p i t i n t e s t s i n c l u d i n g f o u r a n t i s e r a and t h e i r r e s p e c t i v e a n t i g e n s a t 2 0 pg/ml A n t i g e n A n t i s e r u m DVS PVS PeVS HVS DVS 20,480 5,120 2,560 80 PVS 1,280 10,240 640 80 PeVS 320 5,120 2,560 80 HVS 40 40 20 1,280 are revealed. DVS i s more c l o s e l y related to PVS than to either PeVS or HVS. Antisera to several other carlaviruses were obtained, although no pure preparations of homologous antigens were available, so one-way tests were carr i e d out with a constant amount of DVS (8 0 yug/ml) against the tes t antisera. Antisera to CVB (1:20); NLV (1:20) and PVS (1:80) were s t i l l able to produce a v i s i b l e p r e c i p i t a t e . No reactions were observed with antisera to PMV, PVM or RCVMV. Sodium dodecyl sulfate (SDS) agar gel serology A comparison of p r e c i p i t i n l i n e s for sap with no other SDS treatment, with SDS added to 1% and with SDS added to 1% followed by heating for 90 sec i n b o i l i n g water, against DVS antiserum showed that equal amounts of pr e c i p i t a t e were formed i n the SDS gels regardless of the pretreatment given to the sap (Fig. 17A). Consequently, the antiserum was probably formed against a combination of subunits, fragments and inta c t virus p a r t i c l e s . Greater concentrations of antigen and antisera were required to obtain a v i s i b l e p r e c i p i t i n reaction i n s o l i d phase serology than i n l i q u i d . DVS antiserum had a homologous t i t r e of 1:2 against 250 /ig/ml p u r i f i e d antigen (Fig. 17B) and a 1:1 d i l u t i o n of sap from systemically infected C. quinoa. When DVS and PeVS were reacted against DVS antiserum, the p r e c i p i t i n l i n e s intersected to form a spur (Fig. 17C), indic a t i n g a se r o l o g i c a l r e l a t i o n s h i p . When PVS antiserum was t e s t e d , no spur was formed, however, t h i s may have been due to low t u t r e antiserum t h a t would not d e t e c t d i s t a n t s e r o l o g i c a l r e l a t i o n s h i p s ( P u r c i f u l l and Shepherd, 1964). No f u r t h e r r e l a t i o n s h i p s between DVS and other c a r l a v i r u s e s were d e t e c t e d F i g . 17. Sodium d o d e c y l s u l f a t e (SDS) agar g e l s e r o l o g y . A. E f f e c t of treatment s t e p s on r e l a t i v e s t r e n g t h of p r e c i p i t i n l i n e s i n h e a l t h y (H) and DVS i n f e c t e d (D) sap from C. quinoa. 1. no treatment, 2, SDS added t o 1% and 3. SDS added t o 1% and heated. The c e n t r e w e l l c o n t a i n e d f u l l s t r e n g t h a n t i s e r u m to DVS (AS). B. D e t e r m i n a t i o n o f DVS an t i s e r u m t i t r e the c e n t r a l w e l l c o n t a i n e d 250 jug/ml DVS (DV) and the o u t s i d e w e l l s c o n t a i n e d a t w o f o l d d i l u t i o n s e r i e s o f DVS antiserum from f u l l s t r e n g t h (F) t o 1 ( 8 ) . C. D e t e r m i n a t i o n of r e l a t i o n s h i p between DVS and PeVS. The c e n t r a l w e l l c o n t a i n e d f u l l s t r e n g t h DVS antiserum (AS). O u t s i d e w e l l s were f i l l e d w i t h PeVS a t 2 mg/ml (Pe); DVS a t 1 mg/ml (Dl) and 0.5 mg/ml (D2) . Note f o r m a t i o n of sp u r a t the i n t e r s e c t i o n o f the homologous and h e t e r o l o g o u s r e a c t i o n s by heterologous t e s t i n g i n SDS agar gels with a n t i s e r a to chrysanthemum virus B, eggplant mild mosaic v i r u s , poplar mosaic v i r u s , pea streak virus, potato virus M and red clover vein mosaic v i r u s . Latex agglutination serology The latex agglutination t e s t was quite e f f e c t i v e i n detecting DVS i n crude sap from i n f e c t e d C. quinoa, but because of nonspecific, healthy reactions? i t could not be used r e l i a b l y to detect DVS i n dandelion. This may have been due to a l a t e x - l i k e substance found i n the sap of the dandelion which could be i n t e r f e r i n g with the latex r e a c t i o n . The latex agglutination t e s t was more s e n s i t i v e than the tube p r e c i p i t i n t e s t : the end point for the latex t e s t was a d i l u t i o n of 1:12 800 f o r the l a b e l l e d antiserum against a 1:1 280 d i l u t i o n of the infected C. quinoa sap, while the end point for the tube p r e c i p i t i n r e a c t i o n was a d i l u t i o n of 1:2 560 for the antiserum against a 1:160 d i l u t i o n of the infected C. quinoa sap. Enzyme-linked immunosorbent assay (ELISA) IgG from antiserum was p u r i f i e d through a DEAE-22 Sephadex column; the e f f l u e n t was monitored at 280 nm and the major peak, corresponding to the IgG f r a c t i o n was c o l l e c t e d i n the f i r s t 2 ml of e f f l u e n t ( F i g . 18). For routine t e s t i n g optimum d i l u t i o n s of coating IgG and conjugated IgG were found to be 1:2 000 and 1:800, r e s p e c t i v e l y . With th i s system, a l l 96 wells on the plate could be used without any edge e f f e c t . 48. — i 1 r 1 2 3 E F F L U E N T V O L U M E ( m l ) F i g . 18. Absorbance pattern obtained by passing concentrated antiserum from a rab b i t immunized with DVS ( ) and not immunized ( ) through a DEAE-22 Sephadex column. F i g . 19 shows the type of r e s u l t s when a g r i d i s set up to determine optimum conditions: rows 1-4 have been coated with 1:1 000 IgG; rows 5-8 have been coated with 1:5,000; rows 9-12 have been coated with 1:10,000. Rows 1,5 and 9 have conjugated IgG at 1:200; rows 2,6 and 10 have conjugate at 1:800; rows 3, 7 and 11 have conjugate at 1:3,200 and rows 4,8 and 12 have no conjugate. In t h i s instance, coating at 1:1,000 i s best with conjugate at 1:800, as no nonspecific background i s encountered, 49. B 1/DILUTION OF COATING IgG 1000 0 5000 0 10000 0 1 2 3 4 5 6 7 8 9 10 11 12 A Hd B 3 2 1 - 2 1 tr - 2 1 tr - Dd C 4 4 4 - 4 4 3 - 4 4 3 - Dq D tr Hq E Hp F 3 2 - - 2 - - - 2 tr - - PVS G 4 4 3 - 4 3 2 - 4 3 2 - PeVS H 4 4 4 - 4 4 4 - 4 4 4 - DVS 200 800 3200 0 200 800 3200 0 200 800 3200 0 1/DILUTION OF IgG CONJUGATE F i g . 19. Determination of optimum conditions f o r detection of DVS by ELISA. A. shows i n t e n s i t y of enzyme sub-s t r a t e r e a c t i o n with the d i f f e r e n t concentrations of coating IgG and conjugated IgG i n a m i c r o t i t r e p l a t e . B. shows corresponding concentrations of coating IgG and conjugated IgG; numbers within the g r i d are v i s u a l scores assigned r e l a t i v e to the i n t e n s i t y of reaction i n F i g . 19A. Abbreviations to the r i g h t of the chart are as follows: Hd, healthy dandelion; Dd, i n f e c t e d dandelion; Dq, infected C. quinoa; Hq, healthy C. quinoa; Hp, healthy potato; PVS, PVS i n potato; PeVS, PeVS i n C. quinoa and DVS, 1 jug/ml DVS preparation 50. yet lower l e v e l s of v i r u s concentrations are r e a d i l y detectable. Occasionally, healthy C. quinoa sap would react s l i g h t l y depending on the d i l u t i o n of the sap. Sap from o ld dandelion leaves would also produce a s l i g h t background, po s s i b l y due to the nonspecific adsorption of the l a t e x - l i k e substance that was present i n higher concentrations i n o l d l e a f material than i n young l e a f m a terial. When C. quinoa plants and old dandelion leaves were assayed, the background of healthy sap reaction was always considered, whether the plates were scored v i s u a l l y or spectrophotometrically. Sap from in f e c t e d C. quinoa could be d i l u t e d more than 1:150,000, depending on the extent of i n f e c t i o n and was s t i l l detected by ELISA. Sap from i n f e c t e d dandelions had an end point of 1:31,000 ( F i g . 20A). These end points correspond to about 80 ng/ml virus concentration i n the sap ext r a c t s . P u r i f i e d DVS had an end point of about 2 ng/ml ( F i g . 20B). Transmission electron microscope serology A survey of the s e r o l o g i c a l reactions using d i l u t i o n s of antiserum at 1:10, 1:20 and 1:40 showed that s p e c i f i c coating and aggregation occurred at a l l three d i l u t i o n s ( F i g . 21). A clearer background was obtained with the 1:40 d i l u t i o n and this became the standard d i l u t i o n . P a r t i c l e s of DVS, PeVS or TMV prepared f o r scanning by electron microscope by standard negative s t a i n i n g , were r e a d i l y distinguished i n a scan of the g r i d ( F i g . 22 A,E,I). 51. 1 / D I L U T I O N O F S A P x 50 C O N C O F D V S ( n g / m l ) F i g . 20. S t a n d a r d c o n c e n t r a t i o n c u r v e s f o r DVS d e t e r m i n e d by e n z y m e - l i n k e d immunosorbent a s s a y w i t h A. sap from i n f e c t e d d a n d e l i o n (-0—$---0-) , l o c a l l e s i o n s o f C. q u i n o a (-e—©---©•) , m o d e r a t e l y i n f e c t e d C. q u i n o a T • • »- ) and s e v e r e l y i n f e c t e d C. q u i n o a ( • • -•-) . The v a l u e s have been c o r r e c t e d f o r h e a l t h y b a c k g r o u n d and t h e l i m i t o f d e t e c t i o n o f a p o s i t i v e r e a c t i o n was c o n s i d e r e d as t w i c e t h e ab s o r b a n c e o f t h e h e a l t h y c o n t r o l s . B. d i l u t i o n s e r i e s o f p u r i f i e d DVS from 10 pg/ml t o 2 ng/ml When DVS o r PeVS was mixe d w i t h TMV and p r e p a r e d by s t a n d a r d n e g a t i v e s t a i n i n g , i t was d i f f i c u l t t o l o c a t e p a r t i c l e s o f t h e c a r l a v i r u s on t h e g r i d , w i t h o u t m e a s u r i n g t h e w i d t h o f t h e i n d i v i d u a l p a r t i c l e s ( F i g . 22B,F). When TEMS was used w i t h a m i x t u r e o f DVS o r PeVS, i t s homologous a n t i s e r u m and TMV, t h e c a r l a v i r u s was r e a d i l y l o c a t e d as c o a t e d and clumped p a r t i c l e s f o r m i n g l a r g e a g g r e g a t e s on t h e g r i d a g a i n s t a bac k g r o u n d o f n o r m a l l y d i s p e r s e d TMV p a r t i c l e s ( F i g . 22C,G0. The d i s t r i -b u t i o n o f TMV was n o t a l t e r e d by t h e p r e s e n c e o f a n t i s e r u m t o e i t h e r o f t h e c a r l a v i r u s e s ( F i g . 2 2 J , K ) . E x a m i n a t i o n o f t h e r e a c t i o n a t h i g h e r m a g n i f i c a t i o n showed t h e e x t e n t o f s p e c i f i c F i g . 21. Electron micrographs of the s e r o l o g i c a l reactions obtained by TEMS, showing the extent of coating and aggregation of DVS caused by varying d i l u t i o n s of antiserum: A. 1:10; B. 1:20 and C. 1:40. Bar represents 650 nm coating of eg. DVS by DVS antiserum with TMV remaining uncoated ( F i g . 22L). In heterologous reactions between the ca r l a v i r u s e s , the v i r u s p a r t i c l e s d i d not seem as heavily coated with antiserum nor did they aggregate as densely as i n homologous reaction ( F i g . 22D,H). The i n d i v i d u a l c a r l a v i r u s e s could not be distinguished i n a mixture on the basis of the s l i g h t d i f ferences i n t h e i r homologous and heterologous reactions. 52a F i g . 22. Electron micrographs of the s e r o l o g i c a l reactions obtained by TEMS using combinations of three viruses and two antisera: A. DVS p a r t i c l e s without antiserum treatment; B. DVS and TMV are not r e a d i l y distinguished without antiserum treatment; C. TMV i s not a f f e c t e d by DVS antiserum, but DVS p a r t i c l e s are coated and clumped and r e a d i l y d i s t i n g u i s h a b l e i n a mixture; D. DVS p a r t i c l e s treated with PeVS antiserum aggregate l e s s than i n the homologous rea c t i o n . S i m i l a r l y , PeVS (E) i n a mixture with TMV (F) i s r e a d i l y i d e n t i f i e d with homologous antiserum (G). PeVS does not react as intensely with DVS antiserum (H). TMV i s not affected by the antisera: I. no an t i s e r a ; J . DVS antiserum; K. PeVS an t i s e r a . (Bar = 650 nm f o r A-K). The i n t e n s i t y of s p e c i f i c coating i s shown i n L. at a higher magnification of C. (Bar = 300 nm) 53 54. DISCUSSION The dandelion v i r u s i s a t y p i c a l c a r l a v i r u s i n i t s p a r t i c l e s i z e and morphology, b i o l o g i c a l p roperties, ease of detection by electron microscopy, s e r o l o g i c a l r e l a t i o n s h i p s with other c a r l a v i r u s e s , narrow host range and latency i n i t s natural host (Fenner, 1976; Harrison et a l . , 1971). Most of the c a r l a v i r u s e s are present as l a t e n t i n f e c t i o n s i n t h e i r natural hosts and as such suggest a long evolutionary involvement between the virus and the host. I f t h i s i s the case, then DVS would have to be considered as n a t u r a l l y occur-r i n g i n dandelion, rather than having been introduced recently with the advances of a g r i c u l t u r e and the development of a new e c o l o g i c a l balance. I t i s remarkable that DVS had such a l i m i t e d d i s t r i b u t i o n geographically e s p e c i a l l y when i t was so r e a d i l y recovered from dandelions within the o r i g i n a l orchard surveyed. As the demand for v i r u s - f r e e stock material in^r creases, more latent viruses are being discovered and t h e i r e f f e c t s on crops are being evaluated. With increasing reports of "new" viruses, i t i s becoming more important to e s t a b l i s h a working d e f i n i t i o n of what constitutes c l a s s i f i c a t i o n as a new virus vs a new s t r a i n of a known v i r u s . Harrison et a_l. (19 71) devised a system of information c o l l e c t i o n and assessment based on behaviour of the v i r u s i n hosts, vector r e l a t i o n s , p a r t i c l e properties and p a r t i c l e composition. From t h i s information, they were able to c l a s s i f y the well—characterized viruses and define a v i r u s group as "a c o l l e c t i o n of viruses and/or virus s t r a i n s each of which shares with the type member a l l or nearly a l l the main c h a r a c t e r i s t i c s of the group." Although t h i s system aided i n s o r t i n g out the c l a s s i f i c a t i o n and nomenclature tangle for plant viruses, i t d i d not make f i n a l a d i s t i n c t i o n between st r a i n s and species of vi r u s e s . (Shepherd e_t a_l. , 1975; Harrison et a l . , 1971) Differences i n s e r o l o g i c a l reactions, host range, symptomatology and p h y s i c a l properties d i s t i n g u i s h a virus within a group (Brandes et a l . , 1959; Brandes and Wetter, 1959) D i f f i c u l t i e s i n determining symptomatology are encountered with some host plants i n that d i f f e r e n t seed l i n e s and environ-mental conditions during development can a l t e r the symptoms expressed (Bos et a l . , 1960; H i r u k i , 1975). Comparisons based s o l e l y on descriptions i n the l i t e r a t u r e , that are not supple-mented by d i r e c t comparisons, are always open to erroneous conclusions. A study of host range, symptomatology, physiochemical properties of crude sap and p u r i f i e d preparations, s i z e and shape of the p a r t i c l e , s e r o l o g i c a l r e l a t i o n s h i p s , cross protection and ins e c t transmission w i l l help to determine i f the v i r u s i s d i s t i n c t or i f i t i s a s t r a i n (Bos et a l . , 1960). Based on se v e r i t y of symptoms on d i f f e r e n t potato v a r i e t i e s , Kowalska (19 78) d i f f e r e n t i a t e d 20 i s o l a t e s of PVM into 14 s t r a i n s . However, she could not d i s t i n g u i s h s t r a i n s on the same basis with 8 i s o l a t e s of PVS. The i s o l a t e of PVS from Peru (PeVS) produced systemic i n f e c t i o n i n C. quinoa rather than l o c a l l e s i o n s only (Hinostroza-Orihuela, 1973). In both instances, some s e r o l o g i c a l work supported the s t r a i n status of these i s o l a t e s , rather than differences only i n symptomatology. Gibbs (1969) used the unstable or varying c h a r a c t e r i s t i c s of a v i r u s to d i s t i n g u i s h between s t r a i n s of viruses: d e t a i l s of composition of protein: subunits of the p a r t i c l e ; the s e r o l o g i c a l s p e c i f i c i t y ; e lectrophoretic m o b i l i t y ; host range; sev e r i t y of symptoms; vector s p e c i f i c i t y and ease of trans-mission by vector. Myzus persicae (Sulz.) i s commonly used to test t r a n s m i s s i b i l i t y i n the laboratory because of i t s adapt-a b i l i t y to a wide host range. M. persicae d i d not e s t a b l i s h colonies on dandelions i n the r o s e t t e stage,, although i t d i d become well established on C. quinoa. Another aphid species, Dactynotus chondrillae Nevsk. does not e s t a b l i s h colonies on dandelions i n the r o s e t t e stage e i t h e r , but prefers to colonize the flower s t a l k s as n u t r i t i v e substances such as amino acids and sugars occur more abundantly i n the elongating flower stem (Caresche et a l . , 1974). This may have been the reason H* persicae d i d not e s t a b l i s h colonies on dandelion. Uroleucon taraxaci K a l t . , the dandelion aphid (Blackman, 1974), was not a v a i l a b l e for transmission studies at the time; M. persicae was tested with i n f e c t e d C. quinoa, although adaptation of the aphids to C. quinoa was slow. S e r o l o g i c a l tests are an i n d i r e c t measure of the a f f i n i t i e s between amino acid sequences exposed on the surface of the pr o t e i n coat, r e f l e c t i n g the inherent genetic make up of the v i r u s (Gibbs, 1969). S e r o l o g i c a l resemblance means two viruses are re l a t e d ; lack of resemblance does not imply that the virus should be excluded from the p a r t i c u l a r virus group. B i o l o g i c a l properties r e f l e c t differences i n s t r a i n s that might not be detected i n physical or chemical t e s t i n g of the virus p a r t i c l e . Many nat u r a l l y - o c c u r r i n g s t r a i n s of viruses such as TMV, PVS, PVX and PVY have been c a l l e d "new" viruses on the basis of symptomatology alone. Later, as more informa-tion became known, i t appeared more l o g i c a l to c l a s s i f y the so - c a l l e d "new" v i r u s as a s t r a i n of a previously described v i r u s . S e r o l o g i c a l t e s t s may be u s e f u l l y supplemented with cross p r o t e c t i o n tests (Rozendaal and van Slogteren, 1957). However, i f antiserum to the virus i n question i s av a i l a b l e , a greater degree of s e n s i t i v i t y and a more meaningful i n d i c a t i o n of the degree of relatedness can be achieved by serology than with cross protection tests alone. In order to d i s t i n g u i s h r e l a t e d viruses, Matthews (1970) suggested that s t r a i n s are c l o s e l y r e l a t e d v i r u s e s , while serotypes are d i s t a n t l y r e l a t e d v i r u s e s . Viruses with i n t e r -mediate relatedness e x i s t and cannot be c l a s s i f i e d as s t r a i n s and serotypes, thus confusing the problem even f u r t h e r . The s t a b i l i t y of a vi r u s i s usually determined by t e s t i n g i t s longevity i n v i t r o and i t s thermal i n a c t i v a t i o n point. Factors such as storage temperature, presence of o x i d i z i n g 58. agents i n the sap, v i r u s concentration, nature and age of the host, pH and i o n i c strength of the sap can g r e a t l y a l t e r the r e s u l t s . Although DVS had a greater s t a b i l i t y than most c a r l a v i r u s e s , the values were s t i l l close to those expected and no s i g n i f i c a n t d i f f e r e n c e was noted. The d i l u t i o n end point was also within range of that expected f o r a c a r l a v i r u s . No comparisons as to s p e c i f i c i d e n t i t y of a v i r u s can be made because of the extent of variable factors involved i n these i n v i t r o t e s t s , although some i n d i c a t i o n of how well the vi r u s f i t s i n t o the group i s obtained. When two viruses are r e l a t e d but not i d e n t i c a l , only a proportion of the antigenic s i t e s are common to both v i r u s e s . In a tube p r e c i p i t i n t e s t i n v o l v i n g a homologous reaction, an antigen excess i n t e r f e r e s with the phys i c a l formation of the antigen-antibody l a t t i c e responsible for the v i s i b l e p r e c i p i -tate. Consequently, the maximum t i t r e cannot be achieved. In a heterologous t e s t , the antiserum w i l l not react to the same extent as i n a homologous test since only a portion of the s i t e s are r e a c t i v e . A r e l a t i v e l y higher concentration of heterologous antigen i s required to obtain the maximum p r e c i p i -tation at a given d i l u t i o n of antiserum. As outlined i n Table 3, the optimum concentration of antigen to obtain maximum t i t r e s using homologous antiserum was 10-20 ^ ug/ml. I f an excess of antigen was used, the end point was reduced 2-4 f o l d and the true t i t r e was not achieved. In determining heterologous reactions, a two way tube 59 p r e c i p i t i n t e s t was set up using 80 pg/ml antigen. With t h i s high concentration of antigen, the degree of r e l a t i o n s h i p was obscured (Table 4). For example, the t i t r e of DVS antiserum with DVS antigen was 10,240 and with PVS antigen, 5,120, which i s a d i f f e r e n c e of one d i l u t i o n , implying a close s e r o l o g i c a l r e l a t i o n s h i p . Of i n t e r e s t i s that the homologous t i t r e for PVS was 2,560, which i s less than that obtained with DVS or PeVS antiserum. This apparent discrepancy developed by using an excess of antigen f o r homologous reactions. When the same tests were run with 20 pg/ml antigen, the true r e l a t i o n s h i p s were brought i n t o perspective (Table 5). I t i s evident that there are marked s e r o l o g i c a l differences among the four viruses tested: for example, DVS antiserum has a homologous t i t r e of 20,480, and against PVS antigen the end point i s 5,120, implying about 25% common antigenic determin-ants; s i m i l a r l y DVS shares about 6% with PeVS and about 0.4% with HVS. The corresponding homologous reactions showed the highest end point compared with the heterologous reactions. These r e s u l t s c l e a r l y show that DVS shares s l i g h t l y more antigenic s i t e s with PVS than with PeVS and that HVS i s d i s t a n t l y r e l a t e d to each of the other three viruses tested. Based on dif f e r e n c e s i n host range, symptomatology and s e r o l o g i c a l reactions, the dandelion v i r u s i s d i s t i n c t from PVS, PeVS and HVS — the three viruses that were most l i k e l y to show a s t r a i n r e l a t i o n s h i p or i d e n t i t y with the dandelion v i r u s . Whether the dandelion v i r u s should be c l a s s i f i e d as a 60. new vi r u s or as a s t r a i n of PVS or PeVS, i s l a r g e l y a subjec-t i v e d e c i s i o n . In considering a l l the experimental evidence obtained i n the course of t h i s work, i t i s my opinion that there i s s u f f i c i e n t j u s t i f i c a t i o n to consider the dandelion virus as a d i s t i n c t e n t i t y and as such, ai new member of the ca r l a v i r u s group. In considering a s u i t a b l e designation f o r the dandelion v i r u s , I am proposing that i t be c a l l e d dandelion virus S (DVS) i n order to designate the natural host of the v i r u s and to give reference to the group membership. Also, there i s a recent example i n the l i t e r a t u r e concerning a new c a r l a v i r u s from Helenium where the designation Helenium virus S was applied (Kuschki e_t a l . , 19 78), even though HVS i s more c l o s e l y r e l a t e d to PVM than to PVS. Another reason i s that there i s h i s t o r i c a l precedence for considering PVS, rather than CLV as the type member of t h i s group. In a c l a s s i f i c a t i o n of rod-shaped v i r u s -es designed by Brandes and Berckes (1965), viruses with r i g i d to s l i g h t l y f l e x i b l e rods, ca. 650 nm i n length, were c l a s s -i f i e d i n the PVS group. I t was not u n t i l the Plant Virus Sub-Committee of the International Committee on the Taxonomy of Viruses put f o r t h t h e i r c l a s s i f i c a t i o n scheme of 16 virus groups (Harrison et a l . , 1971) and obtained an acceptable s i g l a , that PVS was replaced as the type member by CLV with the " c a r l a " s i g l a . 61. SUMMARY The d a n d e l i o n v i r u s t h a t was t h e s u b j e c t o f t h i s t h e s i s was a s l i g h t l y f l e x u o u s 637 nm r o d and based on i t s p a r t i c l e s i z e and morphology, i t was p l a c e d i n t h e c a r l a v i r u s group. I n c l u s i o n i n t h i s group was f u r t h e r s u p p o r t e d by i t s b i o l o g i c a l p r o p e r t i e s , h o s t range and symptomatology, t r a n s m i s s i o n c h a r a c t e r i s t i c s and s e r o l o g i c a l r e l a t i o n s h i p s . The b i o l o g i c a l p r o p e r t i e s were: t h e r m a l i n a c t i v a t i o n p o i n t 75-80 , d i l u t i o n end p o i n t 2 x 10 and l o n g e v i t y i n v i t r o 4-5 days a t 23° and 28-56 days a t 4°. The h o s t range and symptom-a t o l o g y were l i m i t e d as f o l l o w s : s y s t e m i c i n f e c t i o n o f C. amaran- t i c o l o r and C. q u i n o a , l a t e n t s y s t e m i c i n f e c t i o n o f T. o f f i c i n a l e and l o c a l i n f e c t i o n o f D. stramonium and G. g l o b o s a . The v i r u s was r e a d i l y sap t r a n s m i t t e d and a l s o n o n - p e r s i s t e n t l y t r a n s m i t t e d by M. p e r s i c a e . No seed t r a n s m i s s i o n was o b t a i n e d . I n d e t e r m i n i n g i f t h e d a n d e l i o n v i r u s i s a new v i r u s o r a s t r a i n o f a p r e v i o u s l y d e s c r i b e d v i r u s , s e r o l o g y , h o s t range and symptomatology a r e i m p o r t a n t c r i t e r i a . From t h e l i t e r a t u r e , t h e v i r u s e s most l i k e l y t o be r e l a t e d were PeVS, PVS and HVS. D e t a i l e d s e r o l o g i c a l comparisons r e v e a l e d t h a t t h e d a n d e l i o n v i r u s had a c l o s e r e l a t i o n s h i p w i t h PVS, a more d i s t a n t one w i t h PeVS and a v e r y d i s t a n t one w i t h HVS. As a r e s u l t o f t h e s e s t u d i e s , t h e d a n d e l i o n v i r u s was c o n s i d e r e d t o be a new v i r u s and was d e s i g n a t e d D a n d e l i o n V i r u s S. 62. LITERATURE CITED 1. Black, L.M. 19 70. Potato yellow dwarf v i r u s . C.M.I./A.A.B. Description of Plant Viruses No. 35: 4pp. 2. Blackman, R. 1974. Aphids. Gin and Co., Ltd., London: 100-104. 3. 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Description of Plant Viruses. No. 90: 4pp. 14. Dias, H.F. 1977. Incidence and geographic d i s t r i b u t i o n of tomato ringspot i n De Chaunac vineyards i n the Niagara peninsula. Plant Dis. Report: 61: 24-28. 15. Fenner, F. 1976. C l a s s i f i c a t i o n and nomenclature of viruses . 2nd Report of the Internat i o n a l Committee on Taxonomy of Viruses. S. Karger, Basel. 116pp. 16. F o r s t e r , R.L.S. and Milne, K.S. 1978. Daphne virus S: a c a r l a v i r u s from Daphne. Abstract. Rev. Plant Pathol. 57: 497. 17. Francki, R.I.B. and Randies, J.W. 1970. Lettuce n e c r o t i c yellows v i r u s . C.M.I./A.A.B. Description of Plant Viruses No. 26: 4pp. 18. Fukomoto, F. and Tochihara, H. 19 78. (Chinese yam necr o t i c mosaic v i r u s ) . Abstract. Rev. Plant Pathol. 57: 520. 19. Fulton, R.W. 1948. Hosts of the tobacco streak v i r u s . Phytopathology 38: 421-428. 20. Gibbs, A.J. 1969. Plant v i r u s c l a s s i f i c a t i o n . Adv. Virus Research 14: 263-328. 21. Gibbs, A.J., Harrison, B.D., Watson, D.H. and Wildy, P. 1966. What's i n a v i r u s name? Nature (London) 209: 450-454. 22. Gilkey, H.M. 1957. Weeds of the P a c i f i c Northwest. Oregon State College, C o r v a l l i s : 332-337, 426-427. 23. Hansen, A.J., Nyland, G., McElroy, F.D. and Stace-Smith, R. 1974. O r i g i n , cause, host range and spread of cherry rasp leaf disease i n North America. Phytopathology 64: 721-727. 24. Harrison, B.C. 1958. Raspberry yellow dwarf, a s o i l -borne v i r u s . Ann. Appl. B i o l . 46r 221-229. 64. 25. Harrison, B.C. 1957. Studies on the host range, properties and mode of transmission of beet ringspot v i r u s . Ann. Appl. B i o l . 45: 462-472. 26. Harrison, B.D., Finch, J.T., Gibbs, A.J., H o l l i n g s , M., Shepherd, R.J., Valenta, V. and Wetter, C. 1971. Sixteen groups of plant v i r u s e s . Virology 45: 356-363. 27. Hiebert, E. and McDonald, J.G. 1973. Characterization of some proteins associated with viruses i n the potato v i r u s Y group. Virology 56: 349-361. 28. Hinostroza-Orihuela, A.M. 1973. Some properties of potato v i r u s S i s o l a t e d from Peruvian potato v a r i e t i e s . Potato Research 16: 244-250. 29. H i r u k i , C. 19 75. Factors a f f e c t i n g bioassay of potato virus S i n Chenopodium quinoa. Phytopathology 65: 1288-1292. 30. H o l l i n g s , M. 1957. Investigation of chrysanthemum v i r u s e s . II Virus B (mild mosaic) and chrysanthemum latent v i r u s . Ann. Appl. B i o l . 45: 589-602. 31. Kassanis, B. 1944. A virus attacking l e t t u c e and dandelion. Nature (London) 154: 16. 32. Kegler, H. and Schade, C. 1971. Plum pox v i r u s . C.M.I./A.A.B. Description of Plant Viruses No. 70: 4 pp. 33. Kemp, W.G. and High, P.A. 1979. I d e n t i f i c a t i o n of carnation l a t e n t virus from n a t u r a l l y infected hardy garden Dianthus species i n North America. Plant D i s . 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P u r i f i c a t i o n , serology and properties of a virus from l i l a c , Syringa oblata  a f f i n i s . Plant Dis. Rep;"' 56: 923-926. 58. Wright, N.S. and Stace-Smith, R. 1966. A comparison of the s e n s i t i v i t y of three s e r o l o g i c a l tests for plant viruses and other antigens. Phytopathology 56: 944-948. 59. Z a i t l i n , M. and I s r a e l , H.W. 1975. Tobacco mosaic v i r u s . CM.I./A.A.B. Descriptions of Plant Viruses No. 151: 5pp. 

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