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Interactions of cowpea strains of southern bean mosaic...
Interactions of cowpea strains of southern bean mosaic virus and of tobacco mosaic virus in cowpea and… Molefe, Thandie Leagajang 1979
Title Interactions of cowpea strains of southern bean mosaic virus and of tobacco mosaic virus in cowpea and pinto bean
Creator Molefe, Thandie Leagajang
Description Double infection by cowpea strains of southern bean mosaic virus (CP-SBMV) and of tobacco mosaic virus (CP-TMV) caused additive growth reductions in California blackeye cowpea. Plant height, weight and numbers of seed and pods were significantly reduced by double infection and by CP-TMV single infection compared to healthy and CP-SBMV-sing 1 y infected plants. Singly and doubly inoculated California blackeye cowpea plants developed CP-SBMV symptoms on the primary leaves, but CP-SBMV symptoms in doubly infected trifoliates were masked by CP-TMV symptoms. CP-TMV symptoms did not mask CP-SBMV symptoms in systemically infected trifoliate leaves of another cowpea variety, V45-Bots. CP-TMV infection conditioned systemic infection of V45~Bots by CP-SBMV, as indicated by infectivity,serology and analytical sucrose density gradient centrifugation. CP-TMV also induced susceptibility of Pinto to infection by CP-SBMV, as ascertained by infectivity, immunodiffusion and electron microscopy. Analytical sucrose density gradient centrifugation measurements demonstrated that in doubly inoculated primary leaves of California blackeye cowpea CP-SBMV and CP-TMV were synthesized less than in the same leaves singly inoculated. CP-SBMV synthesis in trifoliate leaves, following simultaneous inoculations of primary leaves, was enhanced 5 times that in singly infected trifoliate leaves, whereas CP-TMV synthesis was not greatly affected. When CP-TMV preceded CP-SBMV in the primary leaves by 2k and 72 hr CP-SBMV synthesis was enhanced more in trifoliate leaves that were undifferentiated at the time of inoculation than in those of plants simultaneously inoculated. When CP-TMV preceded CP-SBMV into preformed 3rd trifoliate leaves by 22 hr, the ratio of CP-SBMV concentration in doubly infected tissue to that in singly infected tissue was 2.7 versus 1.9 when both viruses arrived simultaneously at these leaves. When either virus preceded the other by 72 hr into preformed 3rd trifoliate leaves the synthesis of the challenging virus was greatly retarded. CP-SBMV synthesis was also enhanced by CP-TMV infection under differential temperature synchronous system of infection. Although virions of both viruses were detected in the same cell no genomic masking was detected by infectivity neutralization test. It is theorized that CP-TMV infection predisposes the host cells to infection by CP-SBMV and thus the enhanced synthesis of CP-SBMV. The effect of CP-TMV infection on CP-SBMV synthesis in cowpea seems to be a physiological one. CP-SBMV, but not CP-TMV, was transmitted through planted seed and decontaminated embryos of California blackeye cowpea. Buffer extracts made from decontaminated embryos also were infectious for CP-SBMV. Seed coats contained both viruses. Double infection of California blackeye cowpea decreased seed transmission of CP-SBMV from 13-5 to 7.6%. Buffer extracts of healthy seed were inhibitory to infectivity of both viruses. Germination of seed reduced infectivity of CP-SBMV in the seed coats, but not of CP-TMV. It is also concluded that seed transmission of CP-SBMV is a result of embryo infection rather than contamination with virus in the seed coats.
Subject Mosaic diseases
Tobacco mosaic virus
Kidney bean --Diseases and pests
Bean common mosaic virus
Program Plant Science
831-UBC_1979_A1 M64.pdf [ 10.03MB ]
INTERACTIONS OF COWPEA STRAINS OF SOUTHERN BEAN MOSAIC VIRUS AND OF TOBACCO MOSAIC VIRUS IN COWPEA AND PINTO BEAN 9 1 by THANDIE LEAGAJANG MOLEFE B.Sc., The U n i v e r s i t y o f Botswana, L e s o t h o and S w a z i l a n d , 1 9 7 0 B . S c , The U n i v e r s i t y o f C a l i f o r n i a , D a v i s , 1971 M . S c , C a l i f o r n i a P o l y t e c h n i c S t a t e U n i v e r s i t y , San L u i s O b i s p o , 1 9 7 3 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (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 the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September 1 9 7 9 Q T h a n d i e Leagajang M o l e f e , 1 9 7 9 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 a n 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 m a k e 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 m a y b e g r a n t e d b y t h e H e a d o f my D e p a r t m e n t o r b y 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 b e 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 2 0 7 5 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 V 6 T 1W5 D a t e K. D E - 6 B P 75-5 I \ E i i ABSTRACT Double i n f e c t i o n by cowpea s t r a i n s of southern bean mosaic v i r u s (CP-SBMV) and of tobacco mosaic v i r u s (CP-TMV) caused a d d i t i v e growth reductions in C a l i f o r n i a blackeye cowpea. Plant height, weight and numbers of seed and pods were s i g n i f i c a n t l y reduced by double i n f e c t i o n and by CP-TMV s i n g l e i n f e c t i o n compared to healthy and CP-SBMV-sing 1 y infe c t e d p l a n t s . S i n g l y and doubly inoculated C a l i f o r n i a blackeye cow-pea plants developed CP-SBMV symptoms on the primary leaves, but CP-SBMV symptoms in doubly infected t r i f o l i a t e s were masked by CP-TMV symptoms. CP-TMV symptoms did not mask CP-SBMV symptoms in s y s t e m i c a l l y infected t r i f o l i a t e leaves of another cowpea v a r i e t y , V45 -Bots. CP-TMV i n f e c t i o n conditioned systemic i n f e c t i o n of V45~Bots by CP-SBMV, as indicated by i n f e c t i v i t y , s e r o l o g y and a n a l y t i c a l sucrose d e n s i t y gradient c e n t r i f u -gation. CP-TMV a l s o induced s u s c e p t i b i l i t y of Pi n t o to i n f e c t i o n by CP-SBMV, as ascertained by i n f e c t i v i t y , immunodiffusion and e l e c t r o n microscopy. A n a l y t i c a l sucrose d e n s i t y gradient c e n t r i f u g a t i o n measure-ments demonstrated that in doubly inoculated primary leaves of C a l i f o r n i a blackeye cowpea CP-SBMV and CP-TMV were synthesized less than in the same leaves s i n g l y inoculated. CP-SBMV synthesis in t r i f o l i a t e leaves, f o l l o w i n g simultaneous i n o c u l a t i o n s of primary leaves, was enhanced 5 times that in s i n g l y i n f e c t e d t r i f o l i a t e leaves, whereas CP-TMV synthe-s i s was not g r e a t l y a f f e c t e d . When CP-TMV preceded CP-SBMV in the primary leaves by 2k and 7 2 hr CP-SBMV synthesis was enhanced more in t r i f o l i a t e leaves that were u n d i f f e r e n t i a t e d at the time of i n o c u l a t i o n than in those of plants simultaneously inoculated. When CP-TMV preceded CP-SBMV into preformed 3 r d t r i f o l i a t e leaves by 2 2 hr, the r a t i o of CP-SBMV concentration in doubly infected tissue to that in singly infected tissue was 2 . 7 versus 1 . 9 when both viruses arrived simultaneously at these leaves. When either virus preceded the other by 7 2 hr into preformed 3 r d t r i f o l i a t e leaves the synthesis of the challenging virus was greatly retarded. CP-SBMV synthesis was also enhanced by CP-TMV infection under d i f f e r e n t i a l temperature synchronous system of in f e c t i o n . Although v i -rions of both viruses were detected in the same c e l l no genomic masking was detected by i n f e c t i v i t y n e u t r a l i z a t i o n test. It is theorized that CP-TMV infection predisposes the host c e l l s to infection by CP-SBMV and thus the enhanced synthesis of CP-SBMV. The e f f e c t of CP-TMV infection on CP-SBMV synthesis in cowpea seems to be a physiological one. CP-SBMV, but not CP-TMV, was transmitted through planted seed and decontaminated embryos of C a l i f o r n i a blackeye cowpea. Buffer extracts made from decon-taminated embryos also were infectious for CP-SBMV. Seed coats contained both viruses. Double infection of C a l i f o r n i a blackeye cowpea decreased seed transmission of CP-SBMV from 1 3 - 5 to 7.6%. Buffer extracts of healthy seed were inh i b i t o r y to i n f e c t i v i t y of both viruses. Germina-tion of seed reduced i n f e c t i v i t y of CP-SBMV in the seed coats, but not of CP-TMV. It is also concluded that seed transmission of CP-SBMV is a result of embryo infection rather than contamination with virus in the seed coats. Supervisor's signature i v TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES x i LIST OF FIGURES x v ACKNOWLEDGEMENTS x v i i i INTRODUCTION 1 LITERATURE REVIEW 4 I. Mixed p l a n t v i r u s i n f e c t i o n s 4 A. E f f e c t on p l a n t growth 5 B. E f f e c t on c o n c e n t r a t i o n and d i s t r i b u t i o n o f v i r u s e s ... 8 C. E f f e c t on t r a n s m i s s i o n o f v i r u s e s by v e c t o r s 14 D. S t r u c t u r a l i n t e r a c t i o n s between v i r u s e s 16 1 . In v i t r o s t r u c t u r a l i n t e r a c t i o n s 1 7 2. In v i v o s t r u c t u r a l i n t e r a c t i o n s 20 I I . Synchronous systems f o r p l a n t v i r u s r e p l i c a t i o n 2 5 I I I . Seed t r a n s m i s s i o n o f p l a n t v i r u s e s 2 9 MATERIALS AND METHODS 40 I . The v i r u s e s 40 I I . Hosts and t h e i r p r o p a g a t i o n 41 I I I . I n o c u l a t i o n o f h o s t s f o r v i r u s p u r i f i c a t i o n 43 A. P r o p a g a t i o n and i n o c u l a t i o n o f h o s t s 43 B. P u r i f i c a t i o n o f v i r u s e s 44 1 . P u r i f i c a t i o n o f CP-SBMV 44 2. P u r i f i c a t i o n o f CP-TMV 46 Page C. D e t e r m i n a t i o n o f t h e v i r u s c o n c e n t r a t i o n 4 8 1. D e t e r m i n a t i o n o f p u r i f i e d v i r u s c o n c e n t r a t i o n .. 48 2. E s t i m a t i o n o f c o n c e n t r a t i o n o f p u r i f i e d v i r u s ... 4 8 D. P r e p a r a t i o n o f CP-SBMV and CP-TMV a n t i s e r a 50 IV. Mixed i n f e c t i o n s o f CP-SBMV and CP-TMV i n cowpea 51 A. I n t e r a c t i o n s i n C a l i f o r n i a b l a c k e y e and Botswana l o c a l v a r i e t y V 4 5~Bots cowpeas 51 1. I n o c u l a t i o n s 51 2 . Assay 53 B. Movement of CP-SBMV and CP-TMV from i n o c u l a t e d p r i m a r y l e a v e s i n t o 3 r d t r i f o l i a t e l e a v e s o f C a l i f o r n i a b l a c k e y e cowpea 55 1 . Trimming 55 2. I n o c u l a t i o n s 55 C. E f f e c t o f sequence o f a r r i v a l o f each v i r u s i n the p r e -formed 3 r d t r i f o l i a t e l e a f on t h e c o n c e n t r a t i o n o f the o t h e r 56 1 . I n ocul a t ions 55 2 . Assay 55 D. I n t e r a c t i o n s o f CP-SBMV and CP-TMV i n C a l i f o r n i a b l a c k -eye cowpea under synchronous c o n d i t i o n s 57 1 . Trimming 57 2 . I n o c u l a t i o n s 57 3 . Assay 58 v i Page E. A n a l y s i s f o r s t r u c t u r a l i n t e r a c t i o n s i n C a l i f o r n i a b l a c k e y e cowpea d o u b l y i n f e c t e d by CP-SBMV and CP-TMV.. 59 1. Inocu1 a t ions 59 2. Assay 59 V. I n t e r a c t i o n s o f CP-SBMV and CP-TMV i n P i n t o 62 A. I n o c u l a t i o n s 62 1. I n o c u l a t i o n s w i t h i n t a c t CP-SBMV and i n t a c t CP-TMV. 62 2. I n o c u l a t i o n s w i t h CP-SBMV-RNA and i n t a c t CP-TMV ... 63 3 . Assay 64 B. E x t r a c t i o n o f r i b o n u c l e i c a c i d 65 1. E x t r a c t i o n o f RNA from p u r i f i e d v i r u s p r e p a r a t i o n . . 66 2. E x t r a c t i o n o f RNA from crude sap 66 V I . Seed t r a n s m i s s i o n o f CP-SBMV and CP-TMV 67 A. P r o p a g a t i o n h o s t s f o r CP-SBMV and CP-TMV 67 B. D i s t r i b u t i o n o f CP-SBMV and CP-TMV i n seed p a r t s o f C a l i -f o r n i a b l a c k e y e cowpea and 3 Botswana cowpeas 67 1. H a r v e s t i n g 67 2 . D e c o n t a m i n a t i o n p r o c e d u r e s 67 3 . Assay o f embryos and s e e d l i n g s d e r i v e d from seeds produced on p l a n t s i n f e c t e d by CP-SBMV and CP-TMV 68 C. E f f e c t o f g e r m i n a t i o n on CP-SBMV and CP-TMV i n seed c o a t s 69 1. G e r m i n a t i o n o f seed 69 2. I n f e c t i v i t y a s s a y 69 v i i Page D. E f f e c t o f h e a l t h y mature seed e x t r a c t s on i n f e c t i v i t y o f CP-SBMV and CP-TMV 70 1. E x t r a c t ion . 70 2. I n f e c t i v i t y a s s a y 70 V I I . P r o d u c t i o n o f seed f o r y i e l d a n a l y s i s 70 RESULTS 71 I. E s t i m a t i o n o f c o n c e n t r a t i o n o f p u r i f i e d v i r u s by a n a l y t i c a l s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n 71 I I . E f f e c t o f s i n g l e and d o u b l e i n f e c t i o n s on C a l i f o r n i a b l a c k -eye cowpea and Botswana cowpea v a r i e t i e s 71 A. Comparison o f symptoms 71 1. Symptoms i n C a l i f o r n i a b l a c k e y e cowpea 71 2 . Symptoms i n Botswana cowpea v a r i e t i e s 83 B. E f f e c t on p l a n t growth 83 1. P l a n t growth i n C a l i f o r n i a b l a c k e y e cowpea 83 2 . P l a n t growth i n Botswana cowpea v a r i e t i e s 8 4 C. Comparison o f e f f e c t s o f CP-SBMV and CP-TMV on y i e l d and seed c h a r a c t e r i s t i c s 8 4 1. Y i e l d o f C a l i f o r n i a b l a c k e y e cowpea 8 4 2. Seed c h a r a c t e r i s t i c s i n C a l i f o r n i a b l a c k e y e cowpea... 89 3 . Seed c h a r a c t e r i s t i c s i n Botswana cowpea v a r i e t i e s . . 89 D. D i s t r i b u t i o n and c o n c e n t r a t i o n o f CP-SBMV and CP-TMV n u c l e o p r o t e i n s i n cowpea 89 1. D i s t r i b u t i o n and c o n c e n t r a t i o n o f CP-SBMV and CP-TMV i n t h e i n o c u l a t e d p r i m a r y l e a v e s o f C a l i f o r n i a b l a c k -eye cowpea 89 v i i i Page 2. D i s t r i b u t i o n and c o n c e n t r a t i o n i n p r i m a r y l e a v e s o f Botswana cowpea v a r i e t y V*45-Bots 3k 3 . D i s t r i b u t i o n and c o n c e n t r a t i o n o f CP-SBMV and CP-TMV i n t r i f o l i a t e l e a v e s o f C a l i f o r n i a b l a c k e y e cowpea.. 98 h. D i s t r i b u t i o n and c o n c e n t r a t i o n o f CP-SBMV and CP-TMV in t r i f o l i a t e l e a v e s o f Botswana cowpea v a r i e t y V45-Bots 109 D. D i s t r i b u t i o n and c o n c e n t r a t i o n o f CP-SBMV and CP-TMV i n 3 r d t r i f o l i a t e l e a v e s o f C a l i f o r n i a b l a c k e y e cowpea ... 110 1. A r r i v a l o f CP-SBMV and CP-TMV a t 3 r d t r i f o l i a t e 1 eaves 110 2 . T i m e - c o u r s e s y n t h e s i s o f CP-SBMV and CP-TMV i n preformed 3 r d t r i f o l i a t e l e a v e s 111 3 . E f f e c t o f sequence o f a r r i v a l o f each v i r u s on t h e c o n c e n t r a t i o n o f the o t h e r i n t h e preformed 3 r d t r i f o l i a t e l e a v e s 111 E. C o n c e n t r a t i o n o f CP-SBMV and CP-TMV i n t h e preformed 3 r d t r i f o l i a t e l e a v e s o f C a l i f o r n i a b l a c k e y e cowpea s y n c h r o -n o u s l y i n f e c t e d by d i f f e r e n t i a l t e m p e r a t u r e m a n i p u l a t i o n . 116 1. E f f e c t o f synchronous i n f e c t i o n on a c c u m u l a t i o n o f v i r u s i n 3 r d t r i f o l i a t e l e a v e s 116 I I I . A n a l y s i s f o r s t r u c t u r a l i n t e r a c t i o n s between CP-SBMV and CP-TMV i n C a l i f o r n i a b l a c k e y e cowpea 123 1. A n a l y t i c a l s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . . 123 2 . I n f e c t i v i t y n e u t r a l i z a t i o n 126 i x Page IV. I n t e r a c t i o n o f CP-SBMV and CP-TMV i n P i n t o bean 128 A. I n t e r a c t i o n o f CP-SBMV and CP-TMV a f t e r s e q u e n t i a l i n o -c u l a t i o n o f P i n t o 128 1. I n o c u l a t i o n w i t h i n t a c t v i r u s e s 128 2. I n o c u l a t i o n w i t h CP-SBMV-RNA and i n t a c t CP-TMV 130 3. S t a r c h l e s i o n s 130 B. Ti m e - c o u r s e s y n t h e s i s o f CP-SBMV i n t i s s u e a l s o i n o c u -l a t e d w i t h CP-TMV 133 C. C o n f i r m a t i o n o f CP-SBMV i n l o c a l l e s i o n s o f P i n t o p r i m a r y l e a v e s ^38 1. S e r o l o g y 1 3 8 2. E l e c t r o n m i c r o s c o p y 138 D. I n f l u e n c e o f C o r n e l l i s o l a t e o f CP-TMV on CP-SBMV i n f e c -t i o n o f P i n t o 1 41 V. Seed t r a n s m i s s i o n o f CP-SBMV and CP-TMV i n cowpea -\ LL] A. D i r e c t p l a n t i n g o f seed produced on C a l i f o r n i a b l a c k e y e cowpea p.lants.. ^ 1 1. F r e s h l y h a r v e s t e d v i n e - d r y mature seed 141 2. E f f e c t o f seed s t o r a g e d u r a t i o n on v i r u s t r a n s m i s -s i o n 145 3. E f f e c t o f seed m o t t l e on t r a n s m i s s i o n o f CP-SBMV and CP-TMV t h r o u g h p l a n t e d seed 1 45 B. D i r e c t p l a n t i n g o f seeds from s i n g l y and d o u b l y i n f e c t e d Botswana cowpea v a r i e t i e s 145 Page C. D i s t r i b u t i o n of CP-SBMV and CP-TMV in seed parts 1^9 1. D i s t r i b u t i o n of CP-SBMV and CP-TMV in seed parts of d i f f e r e n t developmental stages of seed of C a l i f o r -nia blackeye cowpea 149 2. D i s t r i b u t i o n of CP-SBMV and CP-TMV in seed parts of Botswana cowpea v a r i e t i e s 158 3 . D i s t r i b u t i o n of CP-SBMV and CP-TMV in planted embryos of C a l i f o r n i a blackeye cowpea seed 158 k. Effect of age of C a l i f o r n i a blackeye cowpea seedling on s u s c e p t i b i l i t y to eit h e r CP-SBMV or CP-TMV 163 5. Effect of healthy seed extract on infe c t i o n of CP-SBMV and CP-TMV on GA 21 and N_. glutinosa 165 D. Effect of germination on survival and i n f e c t i v i t y of virus on seed of C a l i f o r n i a blackeye cowpea 167 DISCUSSION 170 I. Interaction in cowpea 170 A. Effects on plant growth 170 B. D i s t r i b u t i o n and concentration of viruses 171 C. In vivo structural interactions 177 II. Interaction in Pinto 180 III. Seed transmission 182 SUMMARY 189 LITERATURE CITED 190 x i LIST OF TABLES TABLE Page I. Composition and properties of CP-SBMV and CP-TMV 3 II. Relationship between CP-SBMV and CP-TMV concentrations and thei r absorbance areas 80 III. E f f ect of single (SI) and simultaneous double (Dl) infections of CP-SBMV and CP-TMV on growth of C a l i f o r n i a blackeye cowpea 86 IV. Effect of single and simultaneous double infections by CP-SBMV and CP-TMV on growth of Botswana vari e t y V45"Bots cowpea 87 V. Effect of single and simultaneous double infections of CP-SBMV and CP-TMV on pod and seed y i e l d of C a l i f o r n i a blackeye cowpea 88 VI. Relative amounts of CP-SBMV and CP-TMV nucleoprotein in single (Si) and double (Dl) infections of primary leaves of C a l i f o r -nia blackeye cowpea 95 VII. Nucleoprotein y i e l d of CP-SBMV and CP-TMV in singly (SI) and doubly (Dl) inoculated (simultaneously) primary leaves and systemically infected 3rd and 4th t r i f o l i a t e leaves of Botswana cowpea vari e t y V45 _Bots 99 VIII. I n f e c t i v i t y of CP-SBMV in extracts from primary leaves of cowpea var. V45 -Bots at d i f f e r e n t intervals a f t e r inoculation with CP-SBMV alone or with CP-TMV 100 x i i TABLE Page IX. Concentration of CP-SBMV and of CP-TMV in 3 r d t r i f o l i a t e (undifferentiated at time of inoculation) of C a l i f o r n i a blackeye cowpea a f t e r single and sequential inoculations 105 of primary leaves ." X. Movement of CP-SBMV and of CP-TMV into 3rd t r i f o l i a t e leaves of C a l i f o r n i a blackeye cowpea 112 XI. Concentration of CP-SBMV when CP-TMV arrived before (+) and af t e r (-) CP-SBMV and of CP-TMV when CP-SBMV arrived before (+) and aft e r (-) CP-TMV 117 XII. Relative amounts of CP-SBMV and CP-TMV on GA 21 and 1^ . g l u t i - nosa respectively recovered a f t e r n e u t r a l i z a t i o n of i n f e c t i -v i t y of a r t i f i c i a l and natural mixtures 127 XIII. Relationship between number of local lesions and varying con-centrations of either CP-SBMV or CP-TMV cha11enge-inocu1ated on Pinto primary leaves 129 XIV. E f f e c t of pre-inoculat ion with CP-TMV on i n f e c t i v i t y of CP-SBMV-RNA (Purified) on Pinto primary leaves 132 XV. Transmission of CP-SBMV and CP-TMV from seed produced by singly or doubly infected C a l i f o r n i a blackeye cowpea 1 44 XVI. Effect of storage duration of seed on seed transmission of CP-SBMV to seedlings of C a l i f o r n i a blackeye cowpea 146 XVII. E f f e c t of seed mottle on transmission of CP-SBMV and CP-TMV. 147 XVIII. Seed transmission of CP-SBMV and CP-TMV in planted seed of three Botswana local v a r i e t i e s 148 x i i i TABLE Page XIX. D i s t r i b u t i o n of CP-SBMV and CP-TMV in pooled seed parts of C a l i f o r n i a blackeye cowpea of d i f f e r e n t developmental stages 1 50 XX. Ef f i c a c y of decontaminating CP-SBMV and CP-TMV from a r t i f i -c i a l l y contaminated embryos 152 XXI. D i s t r i b u t i o n of CP-SBMV and CP-TMV in 15-20 day old infec-ted and a r t i f i c i a l l y contaminated green immature seed parts of singly inoculated C a l i f o r n i a blackeye cowpea which were assayed i n d i v i d u a l l y 153 XXII. D i s t r i b u t i o n of CP-SBMV and CP-TMV in 3 0 - 3 5 day; old infec-ted and a r t i f i c i a l l y contaminated immature (dough stage) seed parts of C a l i f o r n i a blackeye cowpea d i r e c t l y assayed i nd i v idua11y 1 55 XXIII. D i s t r i b u t i o n of CP-SBMV and CP-TMV in natura l l y infected (N) and a r t i f i c i a l l y contaminated (A) seed parts of vine dry mature seed of C a l i f o r n i a blackeye cowpea whose seed parts extracts were d i r e c t l y assayed i n d i v i d u a l l y 156 XXIV. D i s t r i b u t i o n of CP-SBMV and CP-TMV in seed parts of singly infected (Si) vine dry mature seed of C a l i f o r n i a blackeye cowpea whose extracts were d i r e c t l y assayed i n d i v i d u a l l y . . . 159 XXV. D i s t r i b u t i o n of CP-SBMV and CP-TMV in seed parts of Botswana cowpea v a r i e t i e s 160 XXVI. Transmission of CP-SBMV in embryos of natura l l y infected seeds and a r t i f i c i a l l y contaminated embryos 161 x i v TABLE Page XXVII. Transmission of CP-SBMV and CP-TMV in embryos of natur a l l y infected seeds and in a r t i f i c i a l l y contaminated embryos of C a l i f o r n i a blackeye cowpea planted a f t e r decontamination in 5% N a 3 P 0 4 162 XXVIII. Transmission of CP-SBMV and CP-TMV when inoculated to healthy cotyledons and or radicle-plumule shoots 164 XXIX. Effect of an extract of healthy seed of C a l i f o r n i a blackeye cowpea on i n f e c t i v i t y of CP-SBMV and CP-TMV 166 XXX. Effect of germination on i n f e c t i v i t y and survival of CP-SBMV and CP-TMV in the seed coats 169 XV LIST OF FIGURES FIGURE Page 1. Absorbance p r o f i l e s following centrifugation of 0 . 2 ml of pu r i f i e d CP-SBMV through sucrose density gradient columns 72 2. Absorbance p r o f i l e s following centrifugation of 0 . 2 ml of p u r i f i e d CP-TMV through sucrose density gradient columns 74 3. Relationship between CP-SBMV concentration (mg/ml) and absor-bance area of CP-SBMV in sucrose density gradient columns 76 k. Relationship between CP-TMV concentration (mg/ml) and absor-bance area of CP-TMV in sucrose density gradient columns 78 5. Third t r i f o l i a t e leaves of C a l i f o r n i a blackeye cowpea singly and doubly infected by CP-SBMV and CP-TMV 82 6. C a l i f o r n i a blackeye cowpea plants singly and doubly infected by CP-SBMV and CP-TMV 85 7. Relative absorbance p r o f i l e s following c e n t r i f u g a t i o n , through sucrose density gradient columns, of 0 . 2 ml of c l a r i f i e d ex-tracts of primary leaves of C a l i f o r n i a blackeye cowpea QQ 8. Relative absorbance p r o f i l e s following c e n t r i f u g a t i o n , through sucrose density gradient columns, of 0 . 2 ml of c l a r i f i e d ex-trac t s of 3rd t r i f o l i a t e leaves 92 9. Relative amounts of CP-SBMV and CP-TMV nucleoproteins (mg/g f r . wt.) in primary leaves of C a l i f o r n i a blackeye cowpea afc xv i FIGURE Page 10. Relative amounts of CP-SBMV and CP-TMV nucleoproteins (mg/g f r . wt.) in the 1 s t , 2 n d , 3 r d and 4 t h t r i f o l i a t e leaves of C a l i f o r n i a blackeye cowpea 102 11. Electron micrographs of c e l l s of primary and t r i f o l i a t e leaves of C a l i f o r n i a blackeye cowpea doubly infected by CP-SBMV and CP-TMV 106 12. Time-course r e p l i c a t i o n of CP-SBMV and CP-TMV as indicated by t h e i r respective nucleoprotein contents 114 13. Relative i n f e c t i v i t y of CP-SBMV in 3 r d t r i f o l i a t e leaves of C a l i f o r n i a blackeye cowpea, infected synchronously singly by CP-SBMV and doubly by CP-SBMV and CP-TMV 119 14. Relative i n f e c t i v i t y of CP-TMV in 3 r d t r i f o l i a t e leaves of C a l i f o r n i a blackeye cowpea, infected synchronously singly by CP-TMV and doubly by CP-SBMV and CP-TMV 121 15. Nucleoprotein accumulation of CP-SBMV and CP-TMV in C a l i f o r n i a blackeye cowpea 3 r d t r i f o l i a t e leaves synchronously infected . .-124 16. Primary leaves of Pinto singly and doubly inoculated with intact CP-SBMV and intact CP-TMV 431 17. Time-course r e p l i c a t i o n of CP-SBMV in Pinto primary leaves singly and doubly inoculated with intact CP-SBMV and intact CP-TMV 134 18. Time-course r e p l i c a t i o n of CP-SBMV in Pinto primary leaves singly and doubly inoculated with CP-SBMV-RNA and intact CP-TMV 136 xv i i FIGURE P a g e 19- Immunodiffusion reactions of crude sap from Pinto primary leaves singly and doubly inoculated with CP-SBMV and CP-TMV.. 139 20. Electron micrographs of CP-SBMV-like p a r t i c l e s (arrows) pu-r i f i e d from Pinto primary leaf lesions 142 21. Pinto primary leaves inoculated singly with intact CP-TMV (Cornell isolate) and doubly with intact CP-SBMV and intact CP-TMV (Cornell isolate) 143 22. Relative i n f e c t i v i t y of CP-SBMV in seed coat extracts of germinated and ungerminated seeds derived from C a l i f o r n i a blackeye cowea plants singly infected by CP-SBMV 168 ACKNOWLEDGEMENTS My gratitude is expressed to the following members of the s t a f f of A g r i c u l t u r e Canada Research Station, Vancouver. Dr. Marvin Weintraub, Director, for use of the excellent f a c i l i t i e s at his st a t i o n ; Dr. H.W.J. Ragetl i for allowing me extended use of his growth chamber for experi-ments on synchronous i n f e c t i o n ; Bea Schroeder, for assistance with elec-tron microscopy of sections; Connie Nichols for assistance in electron microscopy and serology; Wes McDiarmid for the excellent photographic work; Al Mosher for supplying styrofoam material and for making e l e c t r i -cal f i t t i n g s in the styrofoam chamber; Dr. J.H. Tremaine for invaluable suggestions; the Library s t a f f for obtaining reference materials unavai-lable l o c a l l y ; and Drs.Bryan D. Frazer and J.W. Hall for advice on sta-t i s t i c a l ana l y s i s . I am g r a t e f u l l y indebted to Drs. B.B. Brantley and CW. Kuhn, Georgia Experiment Station and Department of Plant Pathology and Plant Genetics, respectively, University of Georgia, Athens, for seed of Georgia 21 cowpea; Dr. Milton Z a i t l i n , Department of Plant Pathology, Cornell University, Ithaca, for the Cornell i s o l a t e of cowpea s t r a i n of tobacco mosaic v i r u s ; Nat Mahlatjie, Seed M u l t i p l i c a t i o n Unit, Department of Agr i c u l t u r a l Research, Gaborone, Botswana, for seeds of Botswana cowpea var iet ies. I am grateful to Drs. R.J. Copeman, J.B. Hudson and R. Stace-Smith who, as members of my supervisory committee, gave suggestions in the pre-paration of the thesis manuscript. I am e s p e c i a l l y indebted to Dr. R Hamilton, my thesis supervisor, for his help in choosing the project and for his u n f a i l i n g guidance, patience, invaluable suggestions, en-couragement and support during the three years I spent under his super vi sion. I g r a t e f u l l y acknowledge the scholarship granted to me by the Canadian Commonwealth Scholarship and Fellowship Committee. The generous study leave granted to me by the Botswana Government enabled me to do these studies and I am g r a t e f u l . Last, though not least, I thank my wife A l i c e for typing the f i r s t d r a f t of t h i s thesis. J INTRODUCTION Cowpea (Vigna unguiculata L. Walp) is infected by a number of viruses in i t s natural habitat, including the cowpea strains of southern bean mosaic virus (CP-SBMV) and of tobacco mosaic virus (CP-TMV). CP-TMV was f i r s t reported and isolated in cowpea in Nigeria, A f r i c a ( L i s t e r and Thresh, 1955) and CP-SBMV was isolated from cowpea in North America (Shepherd and Fulton, 1962)- Although CP-SBMV does not infect the French bean (Phaseolus vulgaris) (Shepherd and Fulton, 1962), i t is ser o l o g i -c a l l y related, but not identical (Tremaine and Wright, 1967) to the bean s t r a i n of southern bean mosaic virus (SBMV) described by Zaumeyer and Harter (1943). Recently a s t r a i n of SBMV that infects both bean and cowpea was isolated from Ghana, A f r i c a (Lamptey, 1972; Lamptey and Hamilton, 1974). The Ghana s t r a i n of SBMV is also s e r o l o g i c a l l y related, but not identical to the bean s t r a i n of SBMV. CP-TMV is considered to be simi-lar (Kassanis and Varma, 1975) to the southern sunn-hemp mosaic virus described by Capoor (1962). CP-TMV is also d i s t a n t l y s e r o l o g i c a l l y re-lated to the type s t r a i n of TMV (Bawden and Kassanis, 1968; Kassanis and Varma, 1975)- The chemical compositions and properties of CP-SBMV and CP-TMV are given in Table I for interpretations of the i r behaviour, and e s p e c i a l l y in the section dealing with structural interactions. Both CP-SBMV and CP-TMV are very contagious with i n f e c t i v i t y survival in sap of 20 to 265 days and over 8 years, respectively. These viruses also infect many cowpea v a r i e t i e s and c u l t i v a r s , 2 e s p e c i a l l y the C a l i f o r n i a blackeye type. They also multiply well in this host, thus i t was appropriate to study their e f f e c t s in i t . Obj ect ives: The objectives of this thesis were to compare the ef f e c t s of CP-SBMV and CP-TMV in single and double infections of Vigna  ungu ic u l a t a L. Walp var. Early Ramshorn and in three Botswana cowpea v a r i e t i e s with respect to symptom expression, d i s t r i b u t i o n and concen-t r a t i o n of each of the viruses in various leaves, and d i s t r i b u t i o n in and transmission through seed; and to determine i f structural inter-actions (e.g., encapsidation of v i r a l RNA by heterologous virus coat protein) occur as a consequence of double i n f e c t i o n . Studies of mixed virus infections cannot only o f f e r a better understanding of the l i f e cycles of viruses, but can also o f f e r an understanding of how viruses interact with the host c e l l s . The prac-t i c a l aspect.of such interactions stems from the fact that viruses in a mixture may suppress or accentuate symptoms or a f f e c t transmission of one of the viruses by vectors or through seed. 3 Table |. Composition arid p r o p e r t i e s of CP-SBMV and CP-TMV CP-SBMV3 CP-TMVb P r o p e r t i e s of p a r t i c l e s V i r i o n molecular weight (M.W.) I s o e l e c t r i c point O p t i c a l d e n s i t y ( E ° ^ * 2 6 o n m ) 0 D 2 6 0 / 0 D 2 8 0 Sedimentation c o e f f i c i e n t (S2Q W ) Vector Seed transmission S t r u c t u r e of p a r t i c l e s Shape Length of p a r t i c l e s Diameter Composition of p a r t i c l e s Number of p a r t i c l e components P r o t e i n subunit M.W. Pr o t e i n amino ac i d residues RNA M.W. RNA percentage of v i r i o n 6.6 x 10 6d 3.9 - 6.0 5.85 1,60 115 S leaf beetles equivocal (?) i sometr i c 25 nm one 2.9 x lO^d 270 1.4 x 10 6d 21* 39-4 x 1 0 6 d c 3-5 c 3.2 1.2 187 S unknown none r i g i d rod 40 nm; 300 nm 18 nm two 1.65 x 10 4; 1.81 x 10 4d 161 0.3 x 10 6; 2.0 x 10 6d 5% aData from Shepherd (1971) A l l data from Kassanis and Varma (1975) and Gibbs (1977) except f o r (c) where data are from Z a i t l i n and Isr a e l (1975) f o r the type s t r a i n of TMV; values are assumed to be ca the same f o r CP-TMV. k LITERATURE REVIEW I. Mixed p l a n t v i r u s i n f e c t i o n s . C h a r a c t e r i s t i c s o f t h e i n f e c t i o n o f a host by two v i r u s e s a r e o f t e n d i f f e r e n t from t h o s e induced by t h e i n t e r a c t i n g v i r u s e s i n s i n g l e i n f e c t i o n s . Mixed v i r u s i n f e c t i o n s may a f f e c t y i e l d and growth o f t h e h o s t p l a n t , symptom t y p e o r s e v e r i t y and c o n c e n t r a t i o n o r d i s t r i b u t i o n o f one o r both o f t h e v i r u s e s . Such d o u b l e i n f e c t i o n s may a l s o a l t e r t h e t r a n s m i s s i o n o f one o f t h e v i r u s e s by v e c t o r s o r t h r o u g h seed and i n r a r e i n s t a n c e s one v i r u s may induce s u s c e p t i b i l i t y o f a host o t h e r w i s e r e g a r d e d as immune t o t h e o t h e r v i r u s . I f t h e i n t e r a c t i n g v i r u s e s r e p l i c a t e i n t h e same c e l l s t r u c t u r a l i n t e r a c t i o n s may o c c u r . Such s t r u c t u r a l i n t e r a c t i o n s may l e a d t o encap-s i d a t i o n o f t h e genome o f one v i r u s by t h e p r o t e i n c a p s i d o f the o t h e r v i r u s o r t h e p r o t e i n c a p s i d s u r r o u n d i n g one o f t h e v i r u s e s genome may be made o f mixed p r o t e i n s u b u n i t s donated from both v i r u s e s . The i n t e r a c t i o n s between v i r u s e s i n mixed i n f e c t i o n have been reviewed i n r e c e n t y e a r s . I n t e r a c t i o n s between r e l a t e d and u n r e l a t e d v i r u s e s were review e d by K a s s a n i s (1963) and more r e c e n t l y by Ross (1974). The r o l e o f mixed v i r u s i n f e c t i o n s i n t r a n s m i s s i o n o f v i r u s e s by a p h i d s has been r e v i e w e d (Rochow, 1972) and a r e v i e w o f s t r u c t u r a l i n t e r a c t i o n s between a n i m a l , b a c t e r i a l and p l a n t v i r u s e s , both i n v i t r o and i n v i v o , has been made (Dodds and H a m i l t o n , 1976). Throughout t h e t e x t o f t h i s t h e s i s t h e word " v i r u s " when not q u a l i -f i e d by t h e r e l e v a n t a d j e c t i v e w i l l mean"plant v i r u s " a n d f o r e i t h e r animal 5 or b a c t e r i a l v i r u s t he a p p r o p r i a t e a d j e c t j y e w i l l be used. T h i s t h e s i s i s c o ncerned w h o l l y w i t h p l a n t v i r u s e s and t h e r e f o r e i n t e r a c t i o n s between a n i m a l v i r u s e s and between b a c t e r i o p h a g e s w i l l n ot be d e s c r i b e d e x c e p t where an example may be u s e f u l . E f f e c t s o f d o u b l e v i r u s i n f e c t i o n s on a p l a n t can be m a n i f e s t e d i n s e v e r a l ways. There c o u l d be a r e d u c t i o n i n y i e l d , h e i g h t o r w e i g h t of the d o u b l y i n f e c t e d p l a n t s as compared t o t h a t o f h e a l t h y o r s i n g l y i n f e c t e d p l a n t s ( B e n n e t t , 1949; Holmes, 1956; G a r c e s - O r e j u e l a and Pound, 1957; H a r r i s o n and Gudauskas, 1968; S c h m i t t h e n n e r and Gordon, 1969; Kuhn and Dawson, 1973; S i e v e r t , 1973; Demski and J e l l u m , 1 9 7 5 ) . A. E f f e c t s on p l a n t growth. The h e i g h t o f p l a n t s was found t o be reduced as a r e s u l t o f d o u b l e i n f e c t i o n s by dodder l a t e n t mosaic v i r u s and t o b a c c o e t c h v i r u s ( B e n n e t t , 1 9 4 9 ) , tomato aspermy v i r u s and p o t a t o m o t t l e v i r u s o r TMV (Holmes, 1 9 5 6 ) , CP-SBMV and cowpea c h l o r o t i c m o t t l e v i r u s (CCMV) (Kuhn and Dawson, 1 9 7 3 ) , p o t a t o v i r u s Y (PVY) and TMV ( S i e v e r t , 1973) and CCMV and t o b a c c o r i n g s p o t v i r u s and CCMV and soybean mosaic v i r u s (Demski and J e l l u m , 1 9 7 5 ) . P l a n t s d o u b l y i n f e c t e d by v i r u s e s may a l s o show a r e d u c t i o n i n f r u i t o r seed y i e l d as compared t o t h e y i e l d o f h e a l t h y o r s i n g l y i n f e c t e d p l a n t s . Thus seed y i e l d i n cowpea d o u b l y i n f e c t e d by cucumber mosaic v i r u s and CCMV ( H a r r i s o n and Gudauskas, 1968) and by CP-SBMV and CCMV (Kuhn and Dawson, 1973) was s i g n i f i c a n t l y reduced as compared t o t h a t o f s i n g l y i n f e c t e d p l a n t s . S y n e r g i s t i c y i e l d r e d u c t i o n s were o b s e r v e d i n 6 seed o f soybean d o u b l y i n f e c t e d by e i t h e r soybean mosaic v i r u s and bean pod m o t t l e v i r u s (Ross, 1963, 1968) o r by soybean mosaic v i r u s and t o b a c c o r i n g s p o t v i r u s ( S c h m i t t h e n n e r and Gordon, 1969) The most s t u d i e d e f f e c t o f d o u b l e v i r u s i n f e c t i o n s i s t h a t w h i c h r e s u l t s i n a change i n symptom s e v e r i t y o r t y p e . When p l a n t s a r e s i m u l -t a n e o u s l y o r s e q u e n t i a l l y i n o c u l a t e d w i t h a p a i r o f v i r u s e s t h e c o m p l e x i o n o f symptoms can be more s e v e r e than t h e symptoms induced by each o f t h e v i r u s e s s e p a r a t e l y . One o f the e a r l y s t u d i e s ( V a n t e r p o o l , 1926) i n mixed v i r u s i n f e c t i o n was t h e o b s e r v a t i o n t h a t t h e s o - c a l l e d s t r e a k d i s e a s e o f tomato was due t o s y n e r g i s t i c e f f e c t s o f TMV and p o t a t o v i r u s X (PVX). Subsequent s t u d i e s r e v e a l e d o t h e r s y n e r g i s t i c i n t e r a c t i o n s such as the d o u b l e i n f e c t i o n o f t o b a c c o by TMV and t o b a c c o e t c h v i r u s ( B e n n e t t , 1949) by TMV and cucumber mosaic v i r u s ( G a r c e s - O r e j u e l a and Pound, 1957). I n t e r a c t i o n o f PVX and PVY i n t o b a c c o i s one system t h a t has been e x t e n s i v e l y s t u d i e d by Ross and h i s s t u d e n t s a t C o r n e l l U n i v e r s i t y . Double i n f e c t i o n o f t o b a c c o by PVX and PVY i n s y s t e m i c a l l y i n f e c t e d l e a v e s r e s u l t s i n e x t e n s i v e s e v e r e v e i n a l n e c r o s i s (Rochow and Ross, 1955; S t o u f f e r and Ross, 196lb) and i n s e v e r e n e c r o s i s a l o n g t h e m i d v e i n a d j a c e n t t o PVY-inocu 1ated h a l f l e a v e s when l e a v e s a r e i n o c u l a t e d s e p a r a t e l y on o p p o s i t e h a l v e s w i t h t h e two v i r u s e s (Damirdagh and Ross, 1967)-U s i n g t h e same v i r u s p a i r and ho s t Thomson (1961) and C l o s e (1964) found i n c r e a s e d symptom s e v e r i t y f o l l o w i n g d o u b l e i n f e c t i o n o f t o b a c c o . 7 Double i n f e c t i o n of pea by a l f a l f a mosaic virus and bean yellow mosaic virus results in the most severe symptoms (Ford, 1 9 6 7 ) . Soybean mosaic virus in combination with bean pod mottle virus induced severe necrosis,, d i s t o r t i o n and mottling of soybean f o l i a g e (Ross, 1 9 6 8 ; Lee and Ross, 1 9 7 2 a ) . Citrus doubly infected by yellow vein virus and psorosis virus showed only psorosis virus symptoms and when doubly infected by yellow vein virus and vein enation virus more severe symptoms were expressed (Weathers, I 9 6 0 , 1 9 6 l ) than when singly infected by vein enation virus alone or yellow vein virus alone. Tobacco mosaic virus on i t s own infects barley but not a l l plants become systemically infected. However, when barley plants were doubly infected by barley s t r i p e mosaic virus (BSMV) and TMV, the l a t t e r was able to move systemically, but symptoms in such doubly infected plants were no d i f f e r e n t from those in plants singly infected by BSMV (Dodds, 1 9 7 2 ; Dodds and Hami1 ton, 1 9 7 2 ). However, barley plants doubly infected by TMV and bromegrass mosaic virus (BMV) developed c h l o r o t i c mottle in systemically invaded leaves in addition to the general mosaic pattern caused by BMV alone (Hamilton and Nichols, 1 9 7 7 ) . Double infection of barley by BMV and BSMV resulted in severe symptoms, even though the nucleoprotein y i e l d of either virus was lower than in single infections (Peterson and Brakke, 1 9 7 3 ) . Cowpea plants doubly inoculated simultaneously with CCMV and CP-SBMV developed systemic symptoms of both viruses on the inoculated 8 primary leaves. CCMV sometimes caused necrotic etchings, whether in single or double infe c t i o n s . However when cowpea, singly infected with CP-SBMV was challenged k to 6 days later with CCMV, necrosis induced by CCMV was i n t e n s i f i e d and leaf abscision occurred (Kuhn and Dawson, 1 9 7 3 ) . !t was also noticed that a l l seed produced on doubly infected plants had mottled seed coats. B. E f f e c t on concentration and d i s t r i b u t i o n of viruses. In addition to the e f f e c t s discussed above there may also be a change in the con-centration or d i s t r i b u t i o n of viruses in a mixed i n f e c t i o n . The change may be either enhancement or suppression of one or both of the : interacting viruses. Such a change in virus concentration or d i s t r i -bution may not always be correlated with severity of symptoms. An increase in the concentration of dodder latent mosaic virus was observed (Bennett, 1949) when tomato plants were infected by either dodder latent mosaic virus and TMV or dodder latent mosaic virus and tobacco etch v i r u s . The concentration of PVX, in tobacco, doubly infected by PVX and PVY or PVX and TMV, was 2 to 11 times as much as that in singly infected plants (Rochow and Ross, 1 9 5 4 , 1 9 5 5 ) . The presence of PVY or TMV in the inoculum containing PVX also caused an increase in the number of local lesions caused by PVX (Thomson, I 9 6 0 - Stouffer and Ross ( I 9&la) found that the r a t i o of PVX in doubly infected plants to that in singly infected ones was 11.5 when plants were growing at 32°C, a temperature not optimal for PVX synthesis in singly inoculated 9 plants. The concentration of PVX in double infections was twice that of PVX in singly infected plants at a temperature (19°C) optimal for PVX synthesis. Close (1964) also observed that movement of PVX out of the inoculated leaves was r e s t r i c t e d at a temperature above 30°C. More recently, in an attempt to gain a better understanding of the mechanism by which PVX may be enhanced in double infections Goodman and Ross (1974b) used the close-timing concept of Damirdagh and Ross (19&7) and suggested that PVX is enhanced most by PVY when leaves in which greatest interaction takes place are invaded by PVX a f t e r they have been invaded by PVY. They observed that when PVX invaded the leaves before PVY, enhancement was less than when PVY preceded PVX by 12 hours; enhancement was minimal when PVX arrived 60 hours later than PVY. Another s t r a i n of PVX, however, was enhanced most only i f i t preceded PVY by 6 hours. It would appear that PVY predisposes the c e l l s to infection by PVX. The nature of the mechanism responsible for the enhancement could be a t r a n s l o c a t i b l e factor(s) and may not necessarily need the physical presence of the helper virus in the same c e l l . In another study (Goodman and Ross, 197^a) i t was found that enhancement of PVX occurs in c e l l s doubly infected by PVX and PVY. The degree of interaction may be influenced by the host as shown by Lee and Ross (1972a) who observed an increase in the concentration of soybean mosaic virus in soybean leaves also infected by bean pod mottle v i r u s . However, in bean, bean pod mottle virus interfered with the pro-duction of local lesions by soybean mosaic virus (Lee and Ross, 1972b). 10 Apart from enhancement in the concentration of one of the viruses there also could be antagonistic e f f e c t s on the concentration of one or both of the interacting viruses, even when they are considered unrelated. M u l t i p l i c a t i o n of PVY or Hyocyamus virus 3 was prevented when either virus and severe etch virus were inoculated simultaneously to tobacco (Bawden and Kassanis, 1 9 4 5 ) . However, severe etch virus and PVY have since been found to be s e r o l o g i c a l l y related ( P u r c i f u l l and Gooding, 1 9 7 0 ) and this type of interaction is common between related viruses. A c y c l i c pattern of reduction and increase in the concentration of either virus was observed when cucumber mosaic virus and TMV were in double infections (Garces-Orejuela and Pound, 1 9 5 7 ) . In pea doubly inoculated with a l f a l f a mosaic virus and bean yellow mosaic virus the concentration of a l f a l f a mosaic virus was reduced (Ross, 1 9 6 7 ) , even though the l a t t e r usually masked symptoms induced by bean yellow mosaic viru s . In simu1taneousy doubly inoculated plants turnip mosaic virus and cauliflower mosaic virus replicated independently; however the concentra-tion of turnip mosaic virus was reduced i f i t was introduced 15 to kO . days after inoculation with cauliflower mosaic virus (Kamei et a l , 1 9 6 9 ) . Double infections of barley by BSMV and BMV (Peterson and Brakke, 1 9 7 3 ) and of cowpea by CCMV and CP-SBMV (Kuhn and Dawson, 1 9 7 3 ) resulted in sy n e r g i s t i c e f f e c t s on symptomology, but the concentrations of both BSMV and BMV and of CP-SBMV were reduced by double infections. The con-centration of CP-SBMV in cowpea simultaneously inoculated with CCMV was reduced by 50% of that in singly inoculated plants (Kuhn and Dawson, 1 9 7 3 ) -11 The concentration of CCMV following simultaneous inoculations was not affected. When plants supporting rapid m u l t i p l i c a t i o n of one virus were challenge-inoculated with the other, the concentration of the challenging virus was considerably reduced. However, when the challenging virus was inoculated a f t e r the f i r s t virus had passed the rapid synthesis phase, the synthesis of the challenging virus was the same as in healthy plants inocu-. • lated at the same time. When two viruses interact in the same host, one of the consequences of such interaction may be an e f f e c t on the d i s t r i b u t i o n of one of the viruses. In cotton plants s i n g l y inoculated with the B r a z i l i a n tobacco streak virus the virus could be recovered only from the inoculated leaves and not from non-inocu1ated leaves (Costa, 1 9 6 9 ) . However, when the plants were doubly inoculated with Anthocyanosis virus and the B r a z i l i a n tobacco streak v i r u s , the l a t t e r could be recovered from the non-inocu1ated leaves, thus suggesting Anthocyanosis virus conditioned systemic invasion of cotton plants by the B r a z i l i a n tobacco streak v i r u s . Infection of barley by TMV is si m i l a r to that of cotton plants by the B r a z i l i a n tobacco streak v i r u s , but not i d e n t i c a l . In sin g l y inoculated barley plants TMV replicated very i n e f f i c i e n t l y in both the inoculated and non-inocu1ated leaves. However, when TMV and BSMV (Hamilton and Dodds, 1970; Dodds, 1972; Dodds and Hamilton, 1972) or TMV and BMV (Hamilton and Nichols, 1977) were doubly inoculated to barley, the concentration of TMV in doubly inoculated plants was very much enhanced in the 3 r d and 4 t h leaves. In some cases TMV could be not be: recovered from non-inoculated leaves when alone (Hamilton and Nichols, 1977) or could only be detected i f extracts were concentrated more than 10 times (Hamilton and Dodds, 1970; Dodds, 1972; Dodds and Hamilton, 1 9 7 2 ) . 12 Interaction of TMV with BSMV or with BMV in baeley is s i m i l a r , but not identical to that between PVX and PVY in tobacco (Rochow and Ross, 1 9 5 4 , 1 9 5 5 ) in that i t results in the enhancement of one of the pa r t i c i p a t i n g viruses. However, the two types of i n t e r a c t i o n . d i f f e r because PVX is readily detected in extracts of singly infected tissue, whereas TMV could be detected in tissue of singly infected plants only after extracts had been concentrated more than 1 0-fold. Also systemic spread of TMV in barley seems to be temperature-dependent since a high temperature ( 3 0°C) seems to be necessary (Hamilton and Dodds, 1 9 7 0 ; Hamilton and Nichols, 1 9 7 7 ) . For this reason i t was suggested (Hamilton and Dodds, 1 9 7 0 ) that under temperature stress, barley may become susceptible to TMV. Therefore the enhancement of TMV by BSMV or by BMV seems to be much .higher than that of PVX by PVY. Di s t r i b u t i o n of virus may also be affected by interaction between other viruses and s t r a i n s . Yellow aucuba mosaic virus did not become systemic when i noculated alone, but did so.in.the presence of TMV (Benda, 1 9 5 7 ) . Strains of tomato spotted w i l t virus that did not normal invade the host systemically when in single infections did so in the presence of a s t r a i n of the same virus that infected the host systemi-c a l l y (Norris, 1 9 5 1 ; Finlay, 1 9 5 2 ) . However, these r e s u l t s should be interpreted with caution, because i t appears they were based on mixed infections that occurred in the f i e l d , where a vector could have transmitted the s t r a i n s . 13 Double infection by two viruses may also a l t e r immunity of the host to one of the interacting viruses. The c i t r u s species Ponci rus t r i f o l i a t a L. is considered immune to yellow vein virus when i t is alone (Weathers, I 9 6 0 ) . However, when scions taken from lime plants doubly infected by yellow vein virus and vein enation virus were grafted to healthy P_. t r i f ol iata plants both viruses infected the l a t t e r host systemically (Weathers, 1961). Although th i s type of interaction has been termed exceptional (Ross, 197^) i t seems other s i m i l a r instances are being reported. Another example is the interaction between P v i r u s , a tymovirus, and cucumber mosaic virus (Tochihara, 1 9 5 9 ) . It was found that on i t s own P virus did not infect Nicotiana glutinosa or N_. tabacum (cv. Bright Yel low) , but. i t did infect N_. glutinosa (Tochihara, 1959) and Bright Yellow (Pound et al , 1962) in the presence of cucumber mosaic v i r u s . More recently i t was reported ( G i l l a s p i e and Koike, 1973) that Johnsongrass (Sorghum halepense L. Pers.) which is believed to be immune to sugarcane mosaic virus when inoculated alone did become infected by t h i s virus when it was in a " s t a b i l i z e d " mixture with maize dwarf mosaic v i r u s . The influence of maize dwarf mosaic virus on i n f e c t i o n of Johnsongrass by sugarcane mosaic virus is an interesting phenomenon since the two viruses are believed to be strains of the same v i r u s . When two viruses r e p l i c a t e together in the same host they may be synthe- < sized in the same c e l l or in adjacent c e l l s . This is of interest because i t is one of the r e q u i s i t e s for structural interactions and also because of the recent observation . (Goodman and Ross, 197^a) that enhancement of PVX . in tobacco occurred in c e l l s doubly infected by PVX and PVY. 14 C r y s t a l l i n e i n c l u s i o n s o f t o b a c c o e t c h v i r u s and TMV have been found i n the same c e l l s o f d o u b l y i n f e c t e d p l a n t s (McWhorter and P r i c e , 1949; F u j i s a w a e t a l , 1967)• I t was a l s o s u g g e s t e d by McWhorter and P r i c e (1949) t h a t the v i r u s e s c o u l d have r e p l i c a t e d i n t h e same c e l l s . C a u l i -f l o w e r mosaic v i r u s , a DNA v i r u s , i s a b l e t o c o - i n f e c t t u r n i p p l a n t s w i t h t u r n i p m o saic v i r u s and X - b o d i e s t o g e t h e r w i t h c a u l i f l o w e r m o saic v i r u s p a r t i c l e s and p a r t i c l e s o f t u r n i p mosaic v i r u s c o u l d be seen i n t h e same c e l l (Kamei e t a l , 1969)- Soybean m o s a i c v i r u s and bean pod m o t t l e v i r u s o c c u r r e d t o g e t h e r i n t h e same c e l l s o f d o u b l y i n f e c t e d p l a n t s (Lee and Ross, 1972a). Double i n f e c t i o n o f t o b a c c o by PVX and PVY o r TMV (Goodman and Ross, 1974a) and o f b a r l e y by e i t h e r BSMV and TMV (Dodds, 1974) or BMV and TMV ( H a m i l t o n and N i c h o l s , 1977) r e s u l t e d i n d o u b l e i n f e c t i o n o f i n d i v i d u a l c e l l s . These v i r u s - h o s t systems a l s o r e s u l t i n the enhance-ment o f one o f t h e v i r u s e s i n t h e m i x t u r e and i t has been shown (Goodman and Ross, 1974a) t h a t PVX enhancement o c c u r s i n d o u b l y i n f e c t e d c e l l s . C. E f f e c t on t r a n s m i s s i o n o f v i r u s e s by v e c t o r s A l t e r a t i o n s i n t h e t r a n s m i s s i o n b e h a v i o u r o f a v i r u s by a v e c t o r o r t h r o u g h seed i s a n o t h e r consequence o f d o u b l e i n f e c t i o n o f a p l a n t by v i r u s e s . The b e s t example o f t h i s phenomenon i s t h a t between s e r o l o -g i c a l l y d i f f e r e n t i s o l a t e s o f b a r l e y y e l l o w dwarf v i r u s . I t had been no-t i c e d t h a t t h e MAV i s o l a t e , n o r m a l l y not t r a n s m i t t e d from b a r l e y by Rhopalosiphum p a d i L. i n a s i n g l e : i n f e c t i o n , ' was t r a n s m i t t e d when i t was i n a d o u b l e i n f e c t i o n w i t h t h e a p h i d - t r a n s m i t t e d RPV i s o l a t e (Rochow, 1 9 7 0 ) . The same o b s e r v a t i o n was a l s o made when MAV and RMV i s o l a t e s d o u b l y i n f e c t e d b a r l e y (Rochow, 1 9 7 5 ) . In t h i s i n s t a n c e MAV c o u l d be t r a n s m i t t e d by R_. ma i d i s o n l y when i t o c c u r r e d i n mixed i n f e c t i o n s w i t h 1 5 i s o l a t e RMV which is normally transmitted by R. ma i d i s. Rhopalos i phum-ma id i s, however, did not transmit RMV and MAV when i t was fed or injected with extracts made from doubly infected plants. The f a i l u r e of R_. maid is to transmit RMV under in v i tro conditions was attributed to i n s t a b i l i t y of t h i s i s o l a t e in v i t r o (Rochow, 1975). The dependence of the MAV i s o l a t e on either RPV or RMV has been explained on the basis of structural interactions (genomic masking) (Rochow, 1970, 1972, 1975) which w i l l be discussed l a t e r . There is yet another type of dependent transmission which results from mixed in f e c t i o n s , but does not seem to involve structural interac-tions. Potato virus C and potato aucuba mosaic virus were only acquired and transmitted by aphid vectors i f each virus occurred in mixed infe c t -ions with PVY or i f aphids fed f i r s t on plants infected singly by PVY and then on plants infected by either potato virus C or potato aucuba mosaic virus (Kassanis and Govier, 1971). Formation of aggregates between p a r t i c l e s of the helper and the dependent viruses as well as modification of surface structures or surface charge of the s t y l e t s by PVY are some of the mechanisms which have been suggested to explain this phenomenon (Kassanis and Govier, 1971). The frequency of transmission of a l f a l f a mosaic virus by the pea aphid was increased in mixed infe c t i o n with pea streak virus while that of the l a t t e r virus was decreased (Hampton and Sylvester, 1 9 6 9 ) • From t h i s observation i t was inferred that the concentration of a l f a l f a mosaic virus may be increased in mixed infections with pea streak v i r u s , thus the high frequency of i t s transmission by the pea 16 a p h i d (Hampton and S y l v e s t e r , 1969). | t a p p e a r s , however, t h i s e x p l a -n a t i o n i s not t e n a b l e and indeed i t has been s u g g e s t e d (Rochow, 1972) t h a t v i r u s c o n c e n t r a t i o n , per se , seems not t o p l a y any s i g n i f i c a n t r o l e i n dependent t r a n s m i s s i o n . D. S t r u c t u r a l i n t e r a c t i o n s between v i r u s e s When two v i r u s e s i n f e c t t h e same host i n w h i c h they r e p l i c a t e o r t h e i r assembly s i t e s c o i n c i d e i n t h e same c e l l , t h e r e may be an exchange o f s t r u c t u r a l components between t h e two v i r u s e s . However, s e v e r a l o t h e r p r e r e q u i s i t e s p r e c l u d e s t r u c t u r a l i n t e r a c t i o n s between two v i r u s e s and t h e r e f o r e s y n t h e s i s , per s e , i n t h e same c e l l does not g u a r a n t e e t h a t t h e r e w i l l be an exchange o f v i r u s s t r u c t u r a l components. Some o f t h e f a c t o r s w h i c h f a v o u r o r p r e v e n t s t r u c t u r a l i n t e r a c t i o n s have been r e v i e w e d (Dodds and H a m i l t o n , 1976). In t h i s t h e s i s t h e t e r m i n o l o g y o f Dodds and H a m i l t o n (1976) f o r s t r u c t u r a l i n t e r a c t i o n s w i l l f o l l o w e d . Thus,genomic masking w i l l be used t o mean e n t i r e e n c a p s i d a t i o n o f a genome o f one v i r u s by h e t e r o l o g o u s p r o t e i n c a p s i d o f the o t h e r v i r u s , as adopted from Yamamoto and Anderson (1961) by Dodds and H a m i l t o n (1976). The term " p h e n o t y p i c m i x i n g " s i m i l a r l y w i l l be used i n t h e c o n t e x t o f S t r e i s i n g e r (1956), and adopted by Dodds and H a m i l t o n (1976), t o mean a p r o t e i n c a p s i d made o f a m i x t u r e o f h e t e r o l o g o u s p r o t e i n s u b u n i t s from a t l e a s t two v i r u s e s s u r r o u n d i n g th e genome o f one o f the v i r u s e s . W h e r e both p a r e n t a l v i r u s e s have a phenotype ( c o a t p r o t e i n ) , t h e r e c o u l d a l s o be a v i r i o n made o f both 17 parental p r o t e i n s and/or t h e i r n u c l e i c a c i d s . Obviously where.one of the parents e x i s t s only as a fr e e n u c l e i c a c i d , as in some TMV protein-defec-t i v e mutants, no second parental v i r i o n w i l l be formed nor wi1 1 phenotypic mixing occur. 1 - In v i t r o s t r u c t u r a l i n t e r a c t i o n s Studies of s t r u c t u r a l i n t e r a c t i o n s between plant v i r u s e s were f i r s t done in v i t r o and only r e c e n t l y have attempts been made to detect the phenomenon of s t r u c t u r a l i n t e r a c t i o n s in v i v o . Perhaps stimulated by the pioneer work of Fraenkel-Conrat and Wil l i a m s ( 1 9 5 5 ) which demons-tr a t e d that TMV coat p r o t e i n could be reassembled in v ? t r o w i t h i t s RNA to form i n f e c t i o u s rods,several l a b o r a t o r i e s have si n c e reported the re-c o n s t i t u t i o n of a number of v i r u s e s (Matthews and Hardie, 1 9 6 6 ; Hiebert et a l , 1 9 6 8 ; Wagner and Bancroft, 1 9 6 8 ; Atabekov et a l , 1 9 7 0 a ; Breck and Gordon, 1 9 7 0 ; Kado and Knight, 1 9 7 0 ; Jonard et a l , 1 9 7 2 ; F r i t s c h et a l , 1 9 7 3 ) . An important observation of these i n v t t r o studies i s that there is a high degree of s p e c i f i c i t y between the n u c l e i c a c i d of a v i r u s and i t s homologous coat p r o t e i n . Despite the s p e c i f i c i t y of r e c o n s t i t u t i o n between homologous com-ponents success has been achieved in v i t r o in assembling v i r i o n s from heterologous components. Thus, p r o t e i n s of small s p h e r i c a l v i r u s e s were reassembled into v i r i o n s w i t h RNAs from heterologous v i r u s e s (Hiebert et a l , 1 9 6 8 ) . In t h i s study CCMV-RNA was encapsidated by coat p r o t e i n s of BMV, broadbean mottle v i r u s and BMV-RNA by the coat p r o t e i n s of :CCMV 18 or broadbean mottle v i r u s . In v i t r o assembly of turnip yellow mosaic virus (TYMV)-RNA with TMV protein resulted in a much higher proportion of short rods than normal TMV rods (Matthews and Hardie, 1966). Further, the i n f e c t i v i t y of the assembled product was very much reduced a f t e r ". incubation with ribonuclease (RNase); thus suggesting the i n s t a b i l i t y of such p a r t i c l e s . Assembly, of PVX-RNA with TMV protein resulted in a product that was highly res i s t a n t to RNase treatment (Breck and Gordon, 1970), but no in vivo structural interactions were observed between these viruses (Goodman and Ross, 1974c). Other heterologous assembly products include RNA of cucumber virus-4 with TMV coat protein (Kado and Knight, 1970) and BSMV-RNA with cucumber virus-4 protein, PVX-RNA with TMV coat protein and TMV-RNA with cucumber virus-4 coat protein (Atabekov et a l , 1970a). F r i t s c h et al (1973) observed that reassembly of TMV-RNA with homologous protein occurred in the presence of excess host RNA. However, in competitive experiments in which TMV-RNA and TYMV-RNA were both present TMV protein assembled with the two RNAs. Because both TMV-RNA and TYMV-RNA were equally recognized by TMV protein, under some pH conditions, i t was suggested ( F r i t s c h et a l , 1973) that TYMV-RNA may have special s i t e s , possibly at the 5' OH end, s i m i l a r to those on TMV-RNA, which are also recognized equally by heterologous protein. However, since the 5' OH end was assigned to be the i n i t i a t i o n s i t e in TMV reconstitut ion (Butler and Klug, 1971) recent evidence indicates that reassembly of TMV may be b i d i r e c t i o n a l (Otsuki et a l , 1977) s t a r t i n g internally.near the 3' OH end..In another study (Jonard et a l , 1972) TMV-RNA 19 or a l f a l f a mosaic v i r u s RNA did not form nucleoprotein complexes with TYMV protein capsids. This suggests that TYMV protein may have greater a f f i n i t y for i t s own RNA than for heterologous RNAs. Recently SBMV was successfully assembled with i t s homolgous RNA and sowbane mosaic virus RNA (Tremaine and Ronald, 1977). Both assembly products were infectious on Pinto and Chenopodium armaranticolor, respec-t i v e l y . The assembly products were also resistant to 1% SDS (sodium dodecyl sulphate) to which native SBMV v i r i o n s are re s i s t a n t . They were also as stable as and. identical to SBMV v i r i o n s , in respect to u l t r a v i o l e t absorbance patterns, electrophoretic mobility in agarose gels, electron microscopy and i n f e c t i v i t y . However, when sowbane mosaic virus protein was assembled with i t s homologous RNA or with heterologous SBMV-RNA the new products were also unstable in ]% SDS.. In some cases, the heterologously reconstituted product is infectious (Hiebert et a l , 1968; Atabekov et a l , 1970a; Breck and Gordon, 1970; Kado anf Knight, 1970; F r i t s c h et al , 1973) although i t s s p e c i f i c i n f e c t i v i t y is usually less than that of the homologous reassembly product. In other cases the i n f e c t i v i t y of the heterologous product was d r a s t i c a l l y reduced by RNase treatment (Matthews and Hardie, 1966). The above examples are of in v i t r o genomic masking. Phenotypic mixing has been demonstrated in v i t r o between related and unrelated viruses. Protein subunits of CCMV, BMV and broadbean mottle virus were reconstituted in v i tro to form a;protein containing 20 mixed subunits of these viruses (Wagner and Bancroft, 1 9 6 8 ) . The recons-t i t u t e d product containing a mixture of two or more d i f f e r e n t proteins was resistant to snake venom phosphodiesterase, as indicated by residual i n f e c t i v i t y of the new product. In v i t r o phenotypic mixing between TMV st r a i n s was demonstrated when proteins of the common and tomato strains were assembled with RNA of the common s t r a i n (Otsuki et a l , 1 9 7 7 ) . 2. In vivo structural interactions. In vivo structural interactions are one of the consequences of mixed infect i o n s . In order for genomic masking or phenotypic mixing to occur the viruses must have common or adjacent assembly s i t e s , and homologous s p e c i f i c i t y must be broken down. In addition, other subtle barriers must be removed. Structural interactions between plant vi ruses in vivo are not as common as those between animal viruses and bacteriophages (Dodds and Hamilton, 1 9 7 6 ) . In vivo structural interactions between bacteriophages were suspected (Delbruck and Bailey, 19^6) when i t was found that mixed infections with T2, Tk and T6 phages gave new types, Novick and S z i l a r d (1951) observed that when bacteriophages T2 and Jk were mixedly inoculated to th e i r bacterial host, the DNA of T2 was encapsidated in the coat protein of Th to form a genomically masked p a r t i c l e . In another study (Streisinger, 1956) i t was found that a mixed infe c t i o n of T2 and Tk resulted in the formation of both phenotypi-c a l l y mixed and genomically masked p a r t i c l e s . Reciprocal genomic masking was observed (Yamamoto and Anderson, 1961) in vivo between P221 and P22h 21 phages. Among the many examples between animal viruses, the inter-action between the genome of foot-and-mouth disease virus and the coat protein of bovine enterovirus (Trautman and Sutmoller, 1971) is of interest because of the economic importance of foot-and-mouth disease as a,lethal disease of c a t t l e in the southern hemisphere. Apparently detection of in vivo structural interactions between plant viruses was f i r s t observed between related and morphologically s i m i l a r viruses (Sarkar, 1969; Atabekov et a l , 1970b; Kassanis and Bastow, 1970a, 1970b; Kassanis and Conti, 1971; Atabekova et a l , 1975). The in vivo st r u c t u r a l interactions observed in these studies involved encapsidation of the RNAs of temperature-sensitive coat protein or protein-defective mutants of strains of TMV by the coat protein of the protein-competent s t r a i n s . The TMV mutant Ni 118, in mixed infections with the temperature-resistant vulgare s t r a i n of TMV, induced more lesions than when alone. From these results genomic masking of Ni 118-RNA by the coat protein of vulgare was inferred (Sarkar, 1969). These results were confirmed by Atabekov et al (1970b) who reported that the RNAs of Ni 118 and flavum mutants were genomically masked in the coat proteins of TMV stra i n s of vulgare, A14 and dolichos enation mosaic viru s . The CP-TMV protein also encapsidated the genomes of Ni 118 and PM2 mutants (Kassanis and Bastow, 1970a, 1970b) and of the Turin s t r a i n (Kassanis and Conti, 1971) of TMV. In other studies (Atabekov et a l , 1975) no stru c t u r a l interactions occurred between coat proteins and RNAs of the Ni 118 and i f they had access to the i r homologous components; however, encapsidated the RNA of Ni 118 at 22 a temperature which was non-permissive for the production of Ni 118 coat prote i n. Genomic masking can also occur in vivo between two s t r u c t u r a l l y similar, but unrelated viruses. Isolates of barley yellow dwarf virus are s t r u c t u r a l l y s i m i l a r but s e r o l o g i c a l l y unrelated and they are transmitted by d i f f e r e n t aphid species. Heterologous encapsidation of the RNA genome of a non-aphid transmitted s t r a i n by the coat protein of an aphid trans-mitting s t r a i n resulted in the aphid transmission of the former from doubly infected plants (Rochow, 1970, 1972, 1975). Recently in vivo structural interactions have been detected between s t r u c t u r a l l y d i s s i m i l a r viruses. In tissue extracted from barley doubly infected by BSMV and TMV the genome of TMV was found encapsidated in the coat protein of BSMV (Dodds and Hamilton, 1971; Dodds, 1972, 1974; Dodds and Hamilton, 197*0. When extracts prepared from doubly infected plants were treated with antiserum to TMV^not a l l TMV i n f e c t i v i t y was abolished whereas the i n f e c t i v i t y was removed by antiserum treatment of a mixture of extracts from plants singly infected by each v i r u s . Extracts., of doubly infected plants lost BSMV i n f e c t i v i t y a f t e r treatment with i t s homologous antiserum. The association of TMV-RNA with BSMV protein was about 8* of the RNA within BSMV v i r i o n s (Dodds, 1974). This genomic masking was detected only in leaves that synthesized more TMV than BSMV. In such doubly infected leaves p a r t i c l e s of both viruses appeared in the same c e l l . Genomic masking of BSMV-RNA by BMV protein was suspected 23 in doubly infected barley (Peterson and Brakke, 1973), but unfortunately this interaction was not confirmed by antibody n e u t r a l i z a t i o n of i n f e c t i -v i t y t est. Genomic masking of BMV-RNA by BSMV coat protein was not det-ected in another study (Morris, 1970) nor was i t detected between TMV . and BMV (Hamilton and Nichols, 1977). Although phenotypic mixing is limited by geometric barriers to st r u c t u r a l l y s i m i l a r viruses, i t was not u n t i l recently that i t s occurr-ence in vivo was reported between plant viruses. The phenomenon was reported to occur between protein subunits of sunh-hemp mosaic virus (= CP-TMV) and U 2 strains of TMV (Skotnicki et a l , 1977). Protein sub-units of aucuba and st r a i n s of TMV form aggregates in v i t r o and when these st r a i n s were doubly inoculated to tobacco plants, phenotypic mixing of t h e i r protein subunits was observed (Atabekova et a l , 1975; Taliansky et a l , 1977)- Mixed coat protein was also observed between protein sub-units of the common and tomato str a i n s of TMV when TMV was isolated from protoplasts that had been doubly inoculated with the two strai n s (Otsuki and Takebe, 1978). The results of structural interactions, discussed above, seem to indicate that conditions optimai for in v i t r o may not always be the same for in vivo interactions. The general approach in v i t ro reconstitution studies is to search for optimal conditions, be they buffer m o l a r i t i e s , pH gradients or the use of additives, that may favour interactions between v i r a l RNA and heterologous coat protein. Such optimal conditions in v i t r o ^ may not be those which are suitable for interaction in the host. 24 Although the success of in v i t r o reconstitut ion seems ; to surpass that found in vivo, the new product is either lower in y i e l d or less i n f e c t i v e than a product derived from homologous reassembly. In some experiements there seems to be a c o r r e l a t i o n between the success of in v i t r o assembly and In vivo structural interactions. Thus in vivo phenotypic mixing occurred between the protein subunits of and aucuba str a i n s of TMV which also formed protein aggregates in v i t r o (Taliansky et a l , 1 9 7 7 ) . Genomic masking of Ni 118-RNA by U2 TMV protein also occurred both in  vivo and in v i t r o (Atabekova et a l , 1 9 7 5 ) - Although ?n v i t r o assembly of PVX-RNA with TMV protein was achieved (Atabekov et a l , 1 9 7 0 a ; Breck and Gordon, 1 9 7 0 ) i t was not detected in vivo (Goodman and Rossj. 1 9 7 4 c ) . Where in vivo s t r u c t u r a l interactions have occurred between unrelated viruses (Dodds, 1 9 7 2 , 1 9 7 4 ; Dodds and Hamilton 197* * ; Peterson and Brakke, 1 9 7 3 ) two explanations have been given. It has been specula-ted (Dodds and Hamilton, 1 9 7 4 ) that the encapsidation of TMV-RNA by BSMV protein occurs because synthesis of large amounts of TMV-RNA in doubly infected tissue leads to excess RNA which becomes a v a i l a b l e to BSMV protein. Interaction between TMV and BSMV leads to great enhancement of TMV in leaves 3 and 4 of barley. Interaction between BMV and BSMV in doubly infected bar 1 eyrresii 1 ts in decreased y i e l d of either virus compared to thei r y i e l d in singly infected plants and i t has been suggested (Peterson and Brakke, 1 9 7 3 ) that encapsidation of BSMV-RNA by BMV protein could possibly be due to a great excess of BMV protein over that of BSMV protein. 25 The results obtained with TMV and-BMV in barley (Hamilton and Nichols, 1977) where no structural interactions were observed between the two viruses, suggest that one has to consider other reasons than excess of either RNA or coat protein of one of the viruses, because TMV was also enhanced in leaves 3 and 4 a lthough BMV was suppressed in these leaves. Indeed excess of either components may play a very minors role in leading to stru c t u r a l interactions unless homologous s p e c i f i c i t y can be mo-d i f i e d . S p e c i f i c i t y of RNA-protein and protein-protein interaction was, Indeed, found to be very high between TMV strains (Atabekova et a l , 1975). I I Synchronous systems for plant virus r e p l i c a t i o n . The study of plant virus r e p l i c a t i o n is limited to a great extent by the lack of a suitable system which affords synchronous inf e c t i o n and re p l i c a t i o n of virus in the c e l l s . However the advent of the proto-plast system in plant virology (Takebe and Otsuki, 1969) introduced a new approach to the study of plant viruses. Inoculation of protoplasts with TMV results in thei r rapid i n f e c t i o n and an accelarated synthesis of TMV. Between 8 and 22 hours a f t e r inoculation of protoplasts with TMV, TMV synthesis increased rapidly (Takebe and Otsuki, 1969) and continued to increase l i n e a r l y u n t i l 72 hours (Takebe et a l , 1971, c i t e d in Dawson et al , 1975). The usefulness of the protoplast system in the study of plant virus i n f e c t i o n and r e p l i c a t ion has recently been reviewed (Takebe 1975). The main l i m i t a t i o n s of the protoplast system are that the 26 isolated mesophyll c e l l s cannot usually be maintained for long times aft e r i n f e c t i o n . At about the same time as the development of the protoplast technique a nearly synchronous system in intact c e l l s was u t i l i z e d in the study of TMV synthesis in tobacco (Ni1sson-Ti11gren et a l , 1969). This system took advantage of rapidly developing young leaves which were in a nearly synchronous stage of infection k or 5 days after- inoculation with TMV. The obvious disadvantage with t h i s system is that the exact time at which infe c t i o n s t a r t s is not known. A system that afforded both synchronous inf e c t i o n and preservation of the c e l l s in th e i r native state was developed by Dawson and Schlegel (1973). This system has since proven to afford synchronous inf e c t i o n of c e l l s and r e p l i c a t i o n of viruses (Dawson and Schlegel, 1973; 1976a 1976b, 1976b, 1976c; Dawson et a l , 1975) s i m i l a r to that achieved in the protoplast system. The advantages of the system are: a large amount of tissue can be accomodated; c e l l s are synchronous with respect to t h e i r developmental stage and stage of i n f e c t i o n ; and i t is easy and inexpensive to build and use. Its main disadvantage is that, unlike protoplasts, there is spread of virus between intact c e l l s . This system was used as a working model for the interaction of CP-TMV and CP-SBM in some sections of this thesis and thus i t w i l l be discussed in d e t a i l s . The d i f f e r e n t i a l temperature synchronous system of Dawson and Schlegel (1973) is based on manipulation of the d i f f e r e n t parts of the plant a f t e r inoculation of the lower leaves with a v i r u s . E s s e n t i a l l y the lower 27 (leaves ace', i n o c u l a t e d i n a c o n v e n t i o n a l manner a f t e r w h i c h t h e p l a n t i s p l a c e d i n a p o l y s t y r o f o a m chamber m a i n t a i n e d a t a p e r m i s s i v e t e m p e r a t u r e ( h i g h ) f o r r a p i d v i r u s s y n t h e s i s . At t h e same ti m e t h e upper n o n - i n o c u -l a t e d l e a v e s a r e a l l o w e d t o p r o t r u d e o u t s i d e t h e s t y r o f o a m chamber where they a r e m a i n t a i n e d a t a n o n - p e r m i s s i v e t e m p e r a t u r e (low) f o r v i r u s s y n t h e -s i s but not f o r t r a n s l o c a t i o n . The n o n - p e r m i s s i v e t e m p e r a t u r e i s /. a c h i e v e d . b y p l a c i n g t h e e n t i r e s t y r o f o a m chamber'. i n a c o n t r o l l e d '. : • growth chamber m a i n t a i n e d a t t h e d e s i r e d low t e m p e r a t u r e . A f t e r a s u i t a b l e p e r i o d of v i r u s s y n t h e s i s i n the i n o c u l a t e d l e a v e s t h e e n t i r e p l a n t i s moved t o a growth chamber m a i n t a i n e d a t a t e m p e r a t u r e p e r m i s s i v e f o r v i r u s s y n t h e s i s i n t h e e n t i r e p l a n t . S i n c e i t s development the d i f f e r e n t i a l t e m p e r a t u r e synchronous system has proven a p p l i c a b l e t o t h e s t u d y o f v a r i o u s a s p e c t s o f t h e r e p l i c a t i o n o f p l a n t v i r u s e s . A r a p i d v i r u s s y n t h e s i s was a c h i e v e d w i t h CCMV and TMV (Dawson and S c h l e g e l , 1973, 1976b, 1976c; Dawson e t a l , 1975) whereby t h e degree o f s y n c h r o n y , as i n d i c a t e d by i n f e c t i v i t y and n u c l e o -p r o t e i n c u r v e s o f TMV, i s s i m i l a r t o t h a t f o r TMV i n p r o t o p l a s t s (Dawson e t a l , 1975). T h i s system has a l s o been used t o s t u d y t h e k i n e t i c s o f s y n t h e s i s o f TMV ss-RNA and i t s a t t e n d a n t d o u b l e - s t r a n d e d s t r u c t u r e s (Dawson and S c h l e g e l , 1976b) w h i c h a r e b e l i e v e d t o be i n t e r m e d i a t e s between p a r e n t a l v i r a l RNA and progeny v i r u s i n p l a n t ( J a c k s o n e t a l , 1972; K i 1 l a n d - B r a n d t and N i 1 s s o n - T i 1 1 g r e n , 1973; H a m i l t o n , 1974) and a n i m a l and b a c t e r i a l ( B i s h o p and L e v i n t o w , 1971) v i r u s e s . The system has a l s o been a p p l i e d i n t h e s t u d y o f i n h i b i t o r s w h i c h b l o c k d i f f e r e n t s t e p s i n t h e 28 r e p l i c a t i o n of CCMV a n d TMV (Dawson and Schlegel, 1 9 7 6 a , 1 9 7 6 c ) . In studying plant viruses in mixed infections i t is desirable to know i f the i n t e r a c t i o n , be i t synergism or antagonism, is affected by the order of a r r i v a l of the viruses in the tissue in which the int e r -action is being studied. Goodman and R o s s (1974b) showed evidence that for PVX to be maximally enhanced by PVY in doubly infected tissue there must be a coincidence of c r i t i c a l stages in the r e p l i c a t i o n cycle of the viruses. A synchronous system o f f e r s an opportunity to investigate i n t r a c e l l u l a r interactions of two d i f f e r e n t viruses with respect to t h e i r r e p l i c a t i o n in a mixed Infection. The protoplast system has already been used In the study of mixed virus infections (Otsuki and Takebe, 1 9 7 6 ; Barker and Harrison, 1 9 7 7 a , 1 9 7 7 b ) . Cucumber mosaic virus and TMV in doubly inoculated protoplasts multiplied independently of each other, that i s , an equal number of protoplasts in double and in s i n g l e inoculations was infected by either virus (Otsuki and Takebe, 1 9 7 6 ) . Also the concentration of TMV was the same in singly or doubly inoculated protoplasts but that of cucumber mosaic virus in doubly inoculated protoplasts was c o n s i s t e n t l y lower than that in singly inoculated protoplasts. In the same study proto-plasts were also doubly inoculated with PVX and TMV, a combination which others (Goodman and R o s s , 1 9 7 4 b ) found to result in the enhance-ment of PVX, but no mention was made whether the concentration of PVX was enhanced or otherwise affected under conditions of synchronous in f e c t i o n . 29 When protoplasts were doubly Inoculated with raspberfy ringspot virus and the CAM st r a i n of tobacco r a t t l e virus numerous aggregates of raspberry', ringspot virus were found throughout the cytoplasm of doubly inoculated protoplasts but not in singly inoculated protoplasts (Barker and Harrison, 1977a)- It was later found (Barker and Harrison, 1977b) that raspberry ringspot virus aggregates also occurred in doubly infec-ted leaf c e l l s and that t h e i r formation could be induced in v i tro by mixing the two viruses. Double infection did not a f f e c t the con-centration of either v i r u s . No structural interactions were detected in the protoplasts (Barker and Harrison, 1977b). I I I Seed transmission of plant viruses. Mixed v i r u s infections may also a l t e r seed transmission characteris-t i c s of the seed transmissible virus in the mixture. Thus seed trans-mission of soybean mosaic virus was reduced in double infections with bean pod mottle virus (Ross, 1963, 1968). On the other hand CP-SBMV was more readily transmitted in seed derived from cowpea plants doubly infected by CCMV and CP-SBMV than in singly inoculated plants (Kuhn and Dawson, 1973). In studies where structural interactions between viruses in double infections were reported (Dodds and Hamilton, 1974) and suspected (Peterson and Brakke, 1973) no seed transmission of TMV (Dodds and Hamilton, 1974) or of BSMV (Peterson and Brakke, 1973) occurred in plants inoculated with inoculum that contained the RNA of eithe r of these viruses encapsidated in the coat protein of heterologous 30 v i r u s . It is interesting that BSMV was not seed transmitted when i t s RNA was presumed to be encapsidated in BMV protein (Peterson and Brakke, 1973) because BSMV is known to be seed transmitted ( E s l i c k and Afanasiev, 1955; McKinney and Greeley, 1965). The mechanism of seed transmission i s , however, not f u l l y understood even where the seed-borne virus is in s ingle infections. Investigations on seed transmission in mixed virus infections must, of necessity, be preceded by an understanding of the d i s t r i b u t i o n and c h a r a c t e r i s t i c s of each of the viruses in the seed when i t is in a •.; single i n f e c t i o n . Where these have been resolved then one can study the seed transmission of the viruses in mixed infec t i o n s . With t h i s in mind, the l i t e r a t u r e dealing with seed transmission of viruses in single infections w i l l be reviewed with special emphasis on the CP-SBMV and CP-TMV alleged seed transmission. The phenomenon of seed transmission is puzzling in that r e l a t i v e l y few viruses are transmitted through seed and a majority have not been reported to be so transmitted. The bulk of evidence indicates that for a virus to be transmitted to the next progeny i t must be able to Infect the embryo in the early stages of Its development and there<-aft e r p e r s i s t in the embryo u n t i l seed germination. This type of seed transmission has been c a l l e d embryonic transmission (Bennett, 1969). Even in th i s case some viruses are able to infect embryos of some host plants and not of others. Why some viruses v i r t u a l l y f a i l to infect embryos of any host species is not c l e a r l y understood. Inactivation of the virus in the embryo has been suggested by some (Duggar, 1930; 31 Cheo, 1955)- The lack of d i r e c t vascular connections between the plant and the embryo has been implicated as a reason for the f a i l u r e of seed transmission in the case of viruses that are confined only to the vascular systems (Bennett and Esau, 1 9 3 6 ) . Immunity of the gametophytic generation (Bennett, 1940) is given as another reason for the lack of seed transmission by some viruses. The f a i l u r e of some viruses to invade the embryo has been explained on the basis of i n s u f f i c i e n t high-energy phosphorylated materials required for virus synthesis in the embryo (Caldwell, 1 9 6 2 ) . However, what appears clear is that seed transmi-ssion of virus through embryo infection depends on an interaction between host and virus (Shepherd, 1 9 7 2 ) . This interaction could be dependent on the composition of s p e c i f i c host and virus proteins. Indeed, glycoproteins were found in two seed transmitted viruses, BSMV and cowpea mosaic v i r u s , but not in three other viruses that were not seed transmitted (Partridge et a l , 1 9 7 4 ) . From th i s c o r r e l a t i o n i t was suggested by these workers that covalently-1inked carbohydrate residues on the v i r a l coat protein may act as recognition s i t e s for the attachment of the virus to the gametophytic c e l l surfaces. However, in nepoviruses i t has recenly been reported (Hanadarand Harrison, 1977) that seed t r a n s m i s s i b i 1 i t y was influenced more by RNA-1 than by RNA-2. RNA-2 of these viruses determines coat protein and serological s p e c i f i c i t y . As far as i t is known TMV is not seed transmissible through the 32 embryo except for the report of Gilmer and Wilks (1967). In spite of the fact that embryonic transmission of TMV is not known, e a r l i e r reports were c o n f l i c t i n g and these have been thoroughly reviewed (Taylor et a l , 1961; Bennett, 1969). Seed transmission of TMV has been reported in tomato seed, which is contained inside a fleshy or pulpy f r u i t , thus affording easy surface contamination by the v i r u s . Milbrath (1937) reported seed transmission of TMV in tomato seedlings of Indiana Canner which had not been handled. Crowley (1957) could find no evidence of TMV in the endosperm or embryo of tomato and pungent pepper af t e r decontamination of seed parts. Despite this he found that TMV was transmitted to 15 to 301 of pungent pepper seedlings derived from intact seed. He (Crowley, 1957) attributed this transmission to contamination of the germinating embryos from virus in the seed coats. Indeed, he found that no transmission of TMV occurred when embryos were removed from the seed coats and planted. Later studies (Taylor et a l , 1961; Broadbent, 1965) c l e a r l y showed that TMV is not seed transmitted in tomato i f seedlings are not handled ( i . e . transplanted) or when seed had been surface-decontaminated before planting and thus i t was u n l i k e l y that TMV in the endosperm would become absorbed into the germinating embryo. The embryonic transmission of TMV in seed of pome f r u i t trees (Gilmer and Wilks, 1967) is puzzling and exceptional for t h i s v i r u s . In t h i s study TMV was recovered from 16% of inner seed coat-endosperm tissues of Malus platycarpa, but not from corresponding 33 seeds whose inner seed coat-endosperm tissues had assayed p o s i t i v e l y for TMV. From this i t would appear the virus was associated with seed coats and endosperm. However, TMV was transmitted to 16% of seed-lings derived from planted seeds whose inner seed coat-endosperm tissue had been removed. The cowpea c h l o r o t i c spot i s o l a t e of sunn-hemp mosaic virus (=CP-TMV; Kassanis and Varma, 1 9 7 5 ) was seed-borne in k to 20% of seed of Vigna  sinensis cv. Pusa Dophasli (Kassanis and Varma, 1 9 7 5 ) while the type s t r a i n was not seed-borne (Capoor, 1 9 6 2 ) . Perhaps the seed transmission of the cowpea c h l o r o t i c spot i s o l a t e was a consequence of surface contamination s i m i l a r to that in pungent pepper (Crowley, 1 9 5 7 ) in which seedlings were not handled and, as suggested (Shepherd, 1972), is a rare example. Tobacco mosaic virus is very stable and infectious and i t may be that where seed transmission has been reported t h i s resulted from surface contamination by the v i r u s , e s p e c i a l l y i f decontamination procedures were not employed. Evidence so far a v a i l a b l e suggests that embryonic transmission of TMV. does not occur. The bean s t r a i n of southern bean mosaic virus (SBMV) was isolated for the f i r s t time from Fr.ench.'bean (Phaseol us vu Igar 1 s L.) and was found to be seed transmitted to 5% of seeds that had been stored for 7 months (Zaumeyer and Harter, 19^3). This virus is also very stable and infectious. Southern bean mosaic virus could be recovered from a l l 3h bean seed parts u n t i l maturity, by which time only seed coats contained infectious virus.(Cheo, 1955)- The absence of i n f e c t i v e SBMV in mature embryos was attributed by Cheo to ina c t i v a t i o n of the virus in mature seed. Although Cheo (1955) obtained 2 to 5% seed transmission of SBMV with 3 v a r i e t i e s of bean, he concluded, on the basis of 50 other planted mature seeds that f a i l e d to transmit the v i r u s , that the virus is not seed transmitted. Because he f a i l e d to recover the virus from buffer extracts of embryos of mature seed that were assayed d i r e c t l y on a local lesion host, a method others (Schippers, 1963) found less s e n s i t i v e than seedling assay, Cheo attributed the 2 to 5% seed transmission to accidental inclusion of "not-well matured" seed in the mature seed l o t . The apparent disappearance of SBMV in the embryos of mature seed was also reported by Crowley (1959)- It is s i g n i f i c a n t - t h a t Crowley (1959) could not recover SBMV from the embryos of two soybean v a r i e t i e s at any stage of development, but the virus was recovered from seed coats and perisperm tissues of more than 100 seeds tested; however, there was no mention of attempts to recover SBMV from planted soybean seed. McDonald (1971) and McDonald and Hamilton (1972) failed--.to recover infectious virus from either immature or mature bean embryos that had been decontaminated by running tap water wash for 30 minutes,although infectious virus could be readily recovered from corresponding seed coats treated like-wise. This evidence led these workers (McDonald 35 and Hamilton, 1972) to postulate-that the mechanism of SBMV seed trans-mission, where this has been reported (Zaumeyer and Harter, 1 9 4 3 ; Cheo, 1 9 5 5 ) , was due to surface contamination of the embryo at germination with virus from the seed coat in a sim i l a r manner to that reported for TMV in tomato seed (Taylor et a l , 1 9 6 1 ) . Thus the results of Cheo (1955) which indicated that SBMV could be recovered from immature embryos was attributed (McDonald, 1 9 7 1 ; McDonald and Hamilton, 1972) to the f a i l u r e of the e a r l i e r workers to decontaminate the embryos, a procedure that has been found to give r e l i a b l e results (Taylor et al , 1 9 6 1 ) . The Ghana s t r a i n of SBMV was transmitted through seed of cowpea (Lamptey, 1 9 7 2 ; Lamptey and Hamilton, 1 9 7 4 ) ; the rate of seed transmission in the seed l o t was 1 . 3 % . The Ghana s t r a i n could be recovered from a l l f l o r a l parts, some embryos and seed coats of immature and "maturing" . cowpea seeds which had been previously decontaminated with detergent plus a 30 minute running tap water wash. However the vir u s could not be recovered from embryos of dry mature seed even though i t was transmitted through planted seed. This agrees with the results of Cheo ( 1 9 5 5 ) who could not recover the bean s t r a i n of SBMV from embryos of mature seed. Although the 1 .3% seed transmission of the Ghana s t r a i n through cowpea seed was attributed (Lamptey and Hami1 ton, 1974) to possible contamination of the emerging seedlings by virus from the seed coat,no further explanation was given for the a c t i v i t y of the virus recovered, from extracts of decontaminated immature embryos. If seed transmission 36 of this virus through planted seeH was :due to contamination of the seed-lings with virus from the seed coat, then decontamination should have abolished the i n f e c t i v i t y from a l l embryos. During the course of these studies a manuscript, submitted for publication presenting evidence for embryonic transmission of the bean s t r a i n of SBMV in bean seed (Uyemoto and Grogan, 1 9 7 7 ) , was kindly made av a i l a b l e to t h i s laboratory. In t h i s study (Uyemoto and Grogan, 1 9 7 7 ) extracts of decontaminated immature embryos of three bean v a r i e t i e s were i n f e c t i v e . The virus was transmitted at a higher rate when decon-taminated immature embryos were planted than in s i m i l a r l y treated mature embryos. The equivocal evidence for embryonic transmission of SBMV is confusing. Other factors may have been responsible for the discrepancies reported in the recent studies (McDonald, 1 9 7 1 ; McDonald and Hamilton, 1 9 7 2 ; Lamptey, 1 9 7 2 ; Lamptey and Hamilton, 1 9 7 4 ; Uyemoto and Grogan, 1 9 7 7 ) since in a l l of these cases decontamination procedures were followed. It is not clear i f the differences in the results are due to a high r a t i o of buffer:embryo in the extraction of virus or whether some workers used many more seeds than others. Indeed absence of seed transmission in a few seeds does not mean i t does not occur at a l l (Shepherd, 1 9 7 2 ) . It is also l i k e l y that the negative results of some workers could be due to the presence of i n h i b i t o r s in mature embryo extracts which could have interfered with the i n f e c t i v i t y of the virus which may have been in the 3 7 embryo. Such i n h i b i t o r s have been reported (Cheo, 1 9 5 5 ; Crowley, 1 9 5 5 ) , although i t was thought (Cheo, 1 9 5 5 ) u n l i k e l y they would have been res-ponsible for the lack of transmission of SBMV In mature seed because the i n h i b i t o r y substance was present in embryos of both immature and mature bean seed. Perhaps because of i n h i b i t o r s of i n f e c t i o n , d i r e c t assay of embryo extracts on indicator plants has been found to be less s e n s i t i v e than seedling assay (Schippers, 1 9 6 3 ) . As far as CP-SBMV is concerned there seems to be consistency in its seed-borne nature since i t s f i r s t report (Shepherd and Fulton, 1 9 6 2 ) . These workers reported 3 to k% seed transmission of CP-SBMV in seedlings derived from seed produced on infected cowpea plants. However, since t h i s report of CP-SBMV being seed-borne very l i t t l e work has been done to study the d i s t r i b u t i o n of the virus in the seed. However, the l i t t l e information that is av a i l a b l e indicates that CP-SBMV can be consistently seed-borne (Gay, 1 9 7 3 ; Kuhn and Dawson, 1973). Although the source of infect i o n has not been thoroughly investigated, Gay ( 1 9 7 3 ) demonstrated that CP-SBMV could be recovered from a l l f l o r a l and seed parts of infec-ted plants of each of two cowpea v a r i e t i e s . Apparently Gay did not attempt recovery."-• of CP-SBMV from dry mature seed,and although asceptic methods were employed to separate seed parts, post-separation deconta-mination procedure is not mentioned, thus making his results meaningless. However, CP-SBMV was transmitted to an average of 2 3 and 2 6 % when seeds of the two v a r i e t i e s were planted (Gay, 1 9 7 3 ) . In mixed virus i n f e c t i o n 38 studies CP-SBMV was transmitted through planted seed to an average of 12.5% of seedlings derived from seeds from plants singly infected by CP-SBMV and to 20% of seedlings derived from seeds from plants doubly infected by CP-SBMV. and CCMV (Kuhn and Dawson, 1973)• The few studies (Gay, 1973; Kuhn and Dawson, 1973; Lamptey and Hamilton, 1974) on seed transmission of CP-SBMV through seeds indicated that t h i s s t r a i n of the virus can be transmitted through planted mature seed of cowpea. Also there seems to be no c o n f l i c t with respect to the frequency of seed transmission of CP-SBMV, except ofcourse, in studies (Lamptey and Hamilton, 1974) where care was taken to avoid spurious results by surface decontamination, no virus was recovered from embryos of dry mature seed when th e i r extracts were d i r e c t l y indexed on an in-dicator host even though the virus was transmitted through planted seed. It is not clear i f the negative results obtained with d i r e c t assay of embryos of dry seed (Lamptey and Hamilton, 1974) are due to i n h i b i t o r s of i n f e c t i o n , reported by others (Cheo, 1955; Crowley, 1955) to be present in the seed extracts, or due to inact i v a t i o n (Cheo, 1955). Apparently ina c t i v a t i o n was not responsible since the Ghana s t r a i n of SBMV was transmitted to 1.3% of the seedlings raised from planted mature seed. The p o s s i b i l i t y of in vivo i n h i b i t o r s to manual inoculation in the seed has been implicated (Powell and Schlegel, 1970) in the trans-mission of squash mosaic virus in seed of cantaloupe. In conclusion, a point that needs to be emphasized is that a number 39 of viruses are present in both t h e s e e d coat and endosperm, but these viruses are not seed transmitted. It has been stressed (Shepherd, 1972) that seed tran s m i s s i b i 1 i t y of viruses is a property of embryo infection and does not result from infection of other seed parts. Indeed i f virus in the seed coats or endosperm of some seeds was transmitted through seed as frequently as that in the embryo there would be abundant seed trans-mission of many more viruses. ko MATERIALS AND METHODS I. The viruses. The cowpea str a i n s of southern bean mosaic virus (CP-SBMV) and of tobacco mosaic virus (CP-TMV) used exclu s i v e l y in these studies were obtained from Dr. R.I. Hamilton, Ag r i c u l t u r e Canada Research Station, Vancouver, Canada (henceforth c a l l e d V.R.S. = Vancouver Research Station). The viruses had been maintained in lypholized t i s s u e . The CP-SBMV is the same as that used by Weintraub and Ragetli (1970) o r i g i n a l l y obtained from Dr. R.J. Shepherd, University of C a l i f o r n i a at Davis (Dr. J.H. Tremaine, personal communication). The CP-TMV is the same as that used by Morris (1974) brought from the University of C a l i f o r n i a at Riverside (Dr. Hamilton, personal communication). The viruses were increased separately in C a l i f o r n i a blackeye cowpea. Lypholized tissue was t r i t u r a t e d in 0.01 M potassium phosphate buffer pH 7.1 (henceforth c a l l e d phosphate buffer) in the presence of \% c e l i t e (diamatoceous earth). Purity, that i s , freedom from contamination,, was ascertained by extracting virus from systemically infected t r i f o l i a t e leaves of C a l i f o r n i a blackeye cowpea,diluting i t to 1/10,000 (w/v) in buffer containing ]% c e l i t e and inoculating the d i l u t i o n to local lesion hosts (Georgia 21 cowpea for CP-SBMV and Nicotiana glutinosa for CP-TMV). Single local lesions were then passaged s e r i a l l y through 5 transfers and then to C a l i f o r n i a blackeye cowpea. As a further check, sap from plants singly infected by either CP-SBMV or CP-TMV was back-inocu1ated to the local lesion hosts. Absence of local lesions of one virus in a preparation containing the other virus was taken as evidence of free-dom from contamination of one virus by the other. This check was r o u t i -nely done since working with mixed viruses provided an opportunity for cross-contarnination. Later in these studies the Cornell i s o l a t e of CP-TMV, kindly provi-ded by Dr. Milton Z a i t l i n , Cornell University, Ithaca, New York was used in some comparison studies on French bean (Phaseolus vul g a r i s L.). I I Hosts and the i r propagation. The hosts used in th i s study were C a l i f o r n i a blackeye cowpea (Vigna  ungu i cu1ata L. Walp, cv. Early Ramshorn), three Botswana local cowpea v a r i e t i e s (V. unguiculata, cvs. Botswana blackeye, V26-Bots and V45 _Bots), CP-SBMV hypersensitive cowpea (V_. unguiculata) variety Georgia 21 (GA 2 1 ) , French bean (Phaseolus vulgaris L. cvs. Pinto and Bountiful), N i cot iana  g 1 ut i nosa and N_. tabacum cv. Xanthi. The C a l i f o r n i a blackeye cowpea was used extensively in the studies for virus m u l t i p l i c a t i o n and seed trans-mission. It was produced by Bunton Seed Co. L o u i s v i l l e , Kentucky. The three Botswana cowpea v a r i e t i e s (or lines) were used as a comparison only in seed transmission. Only V45~Bots was used in virus synthesis in mixed infection studies, for reasons given l a t e r . The seeds of the Botswana v a r i e t i e s were kindly sent by Mr. N. Mahlatjie, Seed M u l t i p l i c a t i o n Unit, Department of Agricu 1trura 1 Research, Gaborone, Botswana. Georgia 21 42 cowpea was kindly provided by Drs. B.B. Brantley and CW. Kuhn, University of Georgia, Athens, Georgia. Pinto seed was purchased from Canada Safeway Stores and was routinely used by the greenhouse s t a f f at V.R.S. . Bountiful bean seed was produced by Dominion Seed House, Georgetown, Ontario. N_. glutinosa and Xanthi were from seeds produced and used routinely at V.R.S. Cowpea plants for propagation of virus for p u r i f i c a t i o n and seed transmission were raised in a steam-sterilized s o i l . Cowpea plants, C a l i f o r n i a blackeye.and V45~Bots, for- v irus nucleoprotein studies were raised in the same steam-sterilized s o i l during the early part of this project; however, this s o i l was often not sui t a b l e for cowpea growth and therefore most of the experiments were performed with plants raised in Hoagland's solution medium (Hoagland and Arnon, 1950) as modified by Johnson et al (1957)- When Hoagland's solution was used, seed was sown in p l a s t i c f l a t s containing a seeding mix, made up of : one part f i n e sand, one part peat moss and one part vermiculite. Six to 7 days after seeding the seedlings were transplanted to steam-sterilized r i v e r sand in 10-cm pots. The pots were put in small bowls, f i l l e d with Hoagland's solution and the plants were maintained hydroponica11y for the duration of the experiment in a controlled environment growth room, provided with two banks of Sylvania flourescent tubes to give a l i g h t intensity of 7,000 lux at the primary leaf surface and a temperature of 24 to 27°C, 18 hour photoperiod. A l l bean plants and indicator hosts raised in the greenhouse were maintained at a temperature of 20 to 43 25°C and 14 hour" photoperiod. A l l greenhouse compartments were insect-proof and were provided with heaters and coolers controlled thermostati-c a l l y . Pinto and Bountiful bean and GA 21 cowpea plants were used 8 to 10 days af t e r sowing and N_. g 1 ut i nosa and Xanthi were used when 6 to 7 weeks old. Inoculated cowpea plants for virus p u r i f i c a t i o n , seed transmission-and interaction of viruses, when raised in the greenhouse, were maintained at a temperature of 24 to 32°C and supplemented with fluorescent l i g h t to provide a photoperiod of 18 hours. When cowpea plants for mixed virus infections were raised in Hoagland's solution the plants were raised in the growth room as described above. Indicator hosts, a f t e r inoculation, were raised in a greenhouse maintained at 22 to 25°C or in a growth chamber with a temperature of 22 to 23°C, photoperiod of 16 hours and 13,000 lux at the primary leaf surface. III.Inoculation of hosts for virus p u r i f i c a t i o n , determination of virus  concentration and antisera preparation. A. Propagation and inoculation of hosts. The C a l i f o r n i a blackeye cowpea was used e x c l u s i v e l y for maintanence and propagation of viruses for p u r i f i c a t i o n . Seed was sown in 10-cm pots, 3 plants per pot. The primary leaves were inoculated with crude sap from infected source plants 8 to 10 days after sowing. Crude sap was obtained by grinding infected leaves in a mortar with a pestle in 0.01 M potassium kk phosphate buffer pH 7.1 plus \% c e l i t e (unless otherwise stated a l l ino-culations throughout the experiments reported herein included ]% c e l i t e ) . The primary leaves were inoculated using autoclaved gauze soaked in the inoculum. Immediately a f t e r inoculation the inoculated leaves were rinsed with running tap water. The inoculated plants were maintained in a green-house at temperatures of 2k to 32°C. Extra care was taken to keep plants apart so that fortuitous contamination did not occur. B. P u r i f i c a t i o n of viruses. Fifteen to 20 days a f t e r inoculation t r i f o l i a t e leaves were harvested from each lot of plants inoculated with either v i r u s . The leaves were stored at -20°C u n t i l needed for p u r i f i c a t i o n . Harvesting and p u r i f i c a -tion for the two viruses were never done on the same day in order to reduce the opportunity of contamination. At the end of the p u r i f i c a t i o n procedure for each virus the purity of each virus preparation was checked by back-inoculating i t to local lesion indicator hosts e.g. i f p u r i f i e d virus suspension was CP-SBMV i t was inoculated to N_. glut!nosa to check for the presence of CP-TMV and s i m i l a r l y a CP-TMV p u r i f i e d suspension was cross-checked on GA 21 for the presence of CP-SBMV. 1 . P u r i f i c a t i o n of CP-SBMV. The procedure for p u r i f i c a t i o n of CP-SBMV was that of Tremaine e t j a l (1976) since i t appeared to be the most su i t a b l e . (i) Tissue was crushed while frozen and sprinkled with D!;ECA (sodium diethyldithiocarbamate; 0.004 g/g of tissue) ( i i ) The tissue was homogenized in a Waring blender in 0.1 M sodium hS acetate (NaAc) buffer pH 5-0 containing 0.2% (v/v) mercaptoethanol (1 g tissue/2 ml of buffer), ( i i i ) The homogenate was squeezed through h layers of cheesecloth and the pH of the f i l t r a t e was adjusted to pH 5 with g l a c i a l a c e t i c acid. (iv) The f i l t r a t e was allowed to stand overnight at h°C. (v) This f i l t r a t e was then centrifuged foi* 20 minutes at 10,000 rpm in a Sorvall SS-34 rotor. The supernatant was saved, (vi) The supernatant was made 0. 1 M with respect to NaCl and 10% with respect to polyethylene glycol (PEG), M.W. 6,000 and s t i r r e d overnight to p r e c i p i t a t e the virus, ( v i i ) The p r e c i p i t a t e was pelleted by centrifugation at 10,000 rpm (low speed centrifugation) for 20 minutes, ( v i i i ) The p e l l e t was resuspended overnight in 0.1 M NaAc pH 5-0 buffer at h°C a f t e r which i t was c l a r i f i e d by low speed centrifugation for 10 minutes. (ix) The supernatant was centrifuged in a Spinco no. 30 rotor for 3 hours at 28,000 rpm. (x) The p e l l e t was resuspended overnight in 0.1 M sodium phosphate buffer pH 7-0 (this buffer allowed the virus p e l l e t to go into suspension r e a d i l y ) , (xi) The virus suspension was c l a r i f i e d by low speed centrifugation and the supernatant given another high speed centrifugation as in (ix) above. 46 ( x i i ) The f i n a l p e l l e t was extracted overnight in 0.01 M potassium phosphate buffer pH 7-1, given low speed centrifugation and the virus suspension was stored in this buffer containing ca one or 2 grains of chloro-butanol (as antimicrobial preservative) at 4°C. 2. P u r i f i c a t i o n of CP-TMV. (i) Frozen tissue was pulverized by hand and homogenized with a Waring blender in 0.1M potassium phosphate buffer pH 7-5 containing 0.1% (v/v) mercaptoethanol and 5-0 mM EDTA (ethyle-nediaminetetraacetic acid) (1 g tissue/2 ml of buffer), ( i i ) The homogenate was strained through 4 layers of cheesecloth or 2 layers of ,Wiracloth. ( i i i ) The f i l t r a t e was centrifuged for 30 minutes at 10,000 rpm in a Sorvall SS-34 rotor and the supernatant was saved, (iv) The supernatant was made 2% with respect to NaCl and 4% with res-pect to PEG and s t i r r e d for 4 hours or overnight at 4°C. (v) The extract was then c l a r i f i e d by centrifuging for 30 minutes at 10,000 rpm in a Sorval SS-34 rotor, (vi) The p e l l e t was extracted in 0.01 M potassium phosphate buffer pH 7.1 overn i ght. ( v i i ) The virus suspension was c l a r i f i e d by low speed centrifugation and supernatant saved, ( v i i i ) The supernatant was then centrifuged in a Spinco No. 30 rotor for hi 3 hours at 28,000 rpm. (ix) The p e l l e t was taken up in the potassium phosphate buffer (in 1/20 of o r i g i n a l sap volume) overnight, (x) The virus suspension was c l a r i f i e d by low speed centrifugation and when a very pure preparation was not necessary the p u r i f i -cation procedure was stopped at this stage. However when a very clean and pure preparation was required such as that used for preparation of antiserum the virus suspension was further cleaned by sucrose cushioning as follows: (xi) Four to 6 ml of a virus preparation (10 mg/ml) were floated on top of h to 6 ml of kS% ribonuclease-free sucrose in Beckman c e l l u l o s e n i t r a t e SW 27 tubes which were then f i l l e d with mineral o i l to prevent collapsing, ( x i i ) The virus was pelleted through sucrose by centrifuging in a Beckman SW: 27 rotor for 5 hours at 25,000 rpm.-( x i i i ) At the end of the high speed centrifugation the aqueous phase and mineral o i l were discarded and the clean p e l l e t was extracted overnight in 0.01 M potassium phosphate buffer pH 7.1. (xiv) The virus suspension was dialysed against several changes of d i s t i l l e d water to remove sucrose and f i n a l l y against 0.01 M potassium phosphate pH 7.1 buffer. (xv) The virus preparation was stored at h°C preserved with one or two grains of chloro-butanol. A l l c e ntrifugation procedures were performed at 5°C. 48 C. Determination of virus concentration. The concentrations of virus in p u r i f i e d preparations were determined by u l t r a v i o l e t spectrophotometry. The concentrations of virus in c l a r i f i e d extracts were estimated, a f t e r sucrose density gradient centrifugation, by comparing the area under absorbance pro-f i l e s with the area of known virus concentrations. 1. Determination of p u r i f i e d virus concentration. The concen-t r a t i o n of each p u r i f i e d v irus was determined in a Beckman Model DU u l t r a v i o l e t spectrophotometer at an absorbance of 0 U260" ^ e c o n c e n ~ t r a t i o n of CP-SBMV was determined by multiplying the OD^g'read i ng by the d i l u t i o n factor and then d i v i d i n g by the ex t i n c t i o n c o e f f i c i e n t (E 2£ Q) of 5-85 (E 2£n = f ° r concentration of 1 mg/ml, l i g h t path of 1 cm) (Shepherd, 1971); the concentration of CP-TMV was~simi1ar1y determined by using an assumed E^ Q^ of 3-2 ( E ^ g = 3.2 for concentration of 1 mg/ml, l i g h t path of 1 cm) (Kassanis and Varma, 1975). The r e l a t i v e purity of each virus preparation was determined by comparing the 0 D26r/^ D280 r a t ' ° °f p u r i f i e d v irus with published values for each virus (Table 1) 2. Estimation of concentration of p u r i f i e d virus by sucrose  density gradient centrifugation. Since p a r t i a l l y c l a r i f i e d extracts were used extensively in th i s thesis to estimate the concentra-tion of e i t h e r CP-SBMV or CP-TMV in both si n g l e and double infecti o n s , a rapid method for doing so was desirable. Because CP-SBMV and CP-TMV d i f f e r appreciably in sedimentation properties (Table!I), the technique of sucrose density gradient centrifugation (Brakke, 1963, 1967) would kS r e a d i l y s e p a r a t e the v i r u s e s and g i v e t h e i r c o n c e n t r a t i o n s i n a m i x t u r e . The s t a n d a r d c u r v e , f o r r a p i d d e t e r m i n a t i o n o f v i r u s c o n c e n t r a -t i o n s i n c l a r i f i e d e x t r a c t s , was p r e p a r e d from the s e d i m e n t a t i o n c h a r a c t e r i s t i c s o f known c o n c e n t r a t i o n s o f each p u r i f i e d v i r u s . P u r i f i e d v i r u s , o b t a i n e d as o u t l i n e d above, was made i n t o a s e r i e s o f d i l u t i o n s i n 0.02 M T r i s - H C l ( t r i s h y d r o x y m e t h y 1 - a m i n o m e t h a n e -h y d r o c h l o r i d e ) b u f f e r pH 6.5 f o r CP-SBMV and i n 0.02 M T r i s - H C l b u f f e r pH 6 .5 c o n t a i n i n g 0.1% Igepon T -73 (sodium-methy 1-N-oleoy 1taurate) f o r CP-TMV; t h e d e t e r g e n t was used t o p r e v e n t a g g r e g a t i o n o f CP-TMV. CP-SBMV was d i l u t e d t o c o n c e n t r a t i o n s o f 0 . 5 , 0 .25 , 0.1 and 0.05 mg/ml and CP-TMV was d i l u t e d t o c o n c e n t r a t i o n s o f 1.0, 0 . 5 , 0.25 and 0.125 mg/ml. S u c r o s e g r a d i e n t columns were p r e p a r e d m e c h a n i c a l l y w i t h a g r a d i e n t maker, Model 603, S c i e n t i f i c I n d u s t r i e s , I n c . , by m i x i n g kO ml o f 10% (w/v) s u c r o s e w i t h kO ml of k0% s u c r o s e so as t o pro-^ duce a l i n e a r g r a d i e n t c o n c e n t r a t i o n o f 10 mg/ml t o kO mg/ml, from t o p ; to bottom o f an SW k\ tube. The s u c r o s e s o l u t i o n was made by d i s s o l -v i n g Schwarz/Mann u t r a pure r i b o n u c l e a s e - f r e e s u c r o s e , o b t a i n e d from B e c t o n , D i c k s o n Co., Canada L t d , M i s s i s s a u g a , i n 0.01 M p o t a s s i u m phosphate b u f f e r pH 7-1 f o r CP-SBMV g r a d i e n t s and i n t h e same b u f f e r c o n t a i n i n g 0.02% Igepon T -73 f o r CP-TMV. G r a d i e n t columns made i n t h i s way were always l e f t o v e r n i g h t b e f o r e use. G r a d i e n t columns were l a y e r e d w i t h 0.2 ml o f t h e a p p r o p r i a t e v i r u s c o n c e n t r a t i o n and c e n t r i f u g e d a t 5°C f o r 105 minutes i n an SW k\ r o t o r 50 at 39,000 rpm. After centrifugation the gradient columns were analysed in an ISCO Model 640 density gradient f r a c t i o n a t o r coupled with an ISCO Model UA-5 u l t r a v i o l e t absorbance monitor, and chart recorder. The sucrose gradient columns were scanned at 254 nm and the absorbance of each virus was recorded as a peak (curve) on the chart. The area under the peak was calculated by counting small squares (each square is equivalent to appro-2 ximately 3 mm ) under the peak. The number of squares under each curve can be related to a known virus concentration. In this way, a standard curve between area under the curve and each virus concentration was plotted. This standard curve was used in a l l experiments to estimate the concentration of each virus in p a r t i a l l y c l a r i f i e d extracts. D. Preparation of CP-SBMV and CP-TMV antisera. Antisera were produced by injecting two young white rabbits. Before i n j e c t i o n with either virus antigen, each of the rabbits was appropriately tagged as no. 1 and no. 2. Normal serum (preimmune serum) was bled from each rabbit. Each of the viruses p u r i f i e d as described above (Section III. B. 1 and 2) was dialysed for 24 hours against several changes of 0.01 M potassium phosphate, 0.14, NaCl pH 7-0 (phosphate buffered-sa1ine, PBS) before i n j e c t i o n into the rabbits. One rabbit (no. 1) was injected with CP-SBMV only and the other rabbit (no. 2) with CP-TMV only. One ml (2 mg/ml) of each dialysed virus was mixed with an equal amount of Freund's complete adjuvant and emulsified in a 2-ml syringe u n t i l the mixture had thickened. One-half ml of the emulsion was then injected intramuscularly with a 22 gauge (1 inch) needle into each hind thigh. Injection with the same virus concentration was repeated for each rabbit two more times at weekly i n t e r v a l s . 51 Three weeks aft e r the last i n j e c t i o n each rabbit was bled a l t e r n a t e l y from each ear at monthly intervals u n t i l enough serum was c o l l e c t e d . The c o l l e c -ted blood at each bleeding was allowed to stand at 37°C for one hour and overnight at 4°C to allow c l o t t i n g . The serum was decanted and centrifuged at 5,000 rpm for 10 minutes. The supernatant was mixed with an equal volume of glycerine and stored at -20°C. The t i t r e of the antiserum for each virus was estimated by the mi c r o p r e c i p i t i n method in P e t r i dishes (van Slogteren, 1955) with ant i body^-ant i gen mixtures co-vered with mineral o i l . The highest antiserum d i l u t i o n that reacted with each homologous virus (0.1 mg/ml) antigen was taken as the t i t r e of the antiserum. The t i t r e of the antiserum against CP-SBMV was 1/512 and that against CP-TMV was 1/1024. Neither normal serum nor PBS reacted with any of the virus antigens. IV. Mixed infections of CP-SBMV and CP-TMV in cowpea. A. Interactions in C a l i f o r n i a blackeye and Botswana local v a r i e t y , V45~Bots. cowpeas. 1. Inocu1 at ions. C a l i f o r n i a blackeye cowpea or V45~Bots. cowpea seedlings were selected for uniformity 8 to 10 days af t e r sowing. Plants were either inoculated at that time or covered with black p l a s t i c for 24 hours at 28 to 30°C to enhance s u s c e p t i b i l i t y to virus i n f e c t i o n . In a l l experiments 0.05 mg/ml ( f i n a l concentration) of each p u r i f i e d virus was used in single and double infe c t i o n s . A l l inocula contained \% c e l i t e and were applied with cotton swabs (Q-tips). The inoculated plants were kept in the greenhouse at 24 to 32°C or in the growth room at 24 to 27°C. 52 (a) Simultaneous inoculation. When simultaneous inoculations were made one group of seedlings was mock-inocu1ated with phosphate buffer (control), another group was inoculated singly with either CP-SBMV or CP-TMV separately and the last group with a mixture of CP-SBMV and CP-TMV. Only the primary leaves were inoculated and they were rinsed immediately aft e r inoculation with running tap water. (b) Sequential inoculations. When sequential inoculations were made plants were not previously pre-conditioned in the dark. Plants were selected for uniformity and Inoculated as follows: the f i r s t lot was mock-inocu1ated with phosphate buffer only; the second one was singly inoculated with either CP-SBMV or CP-TMV; the t h i r d lot was doubly inoculated with a mixture of the two viruses, and the fourth l o t , which was to be sequentia 11y inocu1ated, was inoculated singly with either CP-SBMV or CP-TMV. A l l the inoculated leaves were rinsed with tap water and plants moved to the greenhouse or to the growth room. At various intervals a f t e r i n i t i a l inoculation plants of the fourth lot were sequentially inoculated with either CP-SBMV or CP-TMV. In se-que n t i a l l y inoculated plants the second virus was applied d i r e c t l y on the previously inoculated leaf and i f th i s was done less than 12 hours after, the previous inoculation c e l i t e was excluded from the second inoculum. When tissue was harvested a l l primary leaves were washed overnight in running tap water to remove residual inoculum. A l l harvested tissue was stored at-20°C u n t i l needed. 53 2. Assay. Assay of nucleoprotein was determined by a n a l y t i c a l sucrose density gradient centrifugation and by electron microscopy. (a) Sucrose density gradient centrifugation assay. When nucleo-protein content of CP-SBMV or CP-TMV in singly infected tissue was compared to that in doubly infected tissue the tissue was p a r t i a l l y c l a r i f i e d as fol1ows: (i) Frozen tissue was ground with a mortar and pestle in 0.2 M Tris-HCl buffer pH 6.5 (lg/0,5 - 2.0 ml of buffer depending on the stage of leaf a n a l y s i s ) , ( i i ) The homogenate was strained through k layers of cheesecloth and c l a r i f i e d immediately or l e f t to stand overnight at 4°C. ( i i i ) The f i l t r a t e was mixed with an equal volume of chloroform and emulsified by s t i r r i n g for 3 to 5 minutes at 4°C. (iv) The emulsion was centrifuged (SS-34 rotor, 10,000 rpm, 20 minutes, 5°C) and the supernatant saved, (v) The clear supernatant was used in estimation of virus concentra-tion. This method gave reasonably clean extracts with l i t t l e or no aggre-gation of CP-TMV, compared to other methods (Hamilton and Dodds, 1970). To determine the concentration of each virus in single and double infections, c l a r i f i e d sap (0.2 or 0.4 ml) was layered on sucrose density gradient columns and centrifuged as previously described (Section III.C.2). The centrifuged sucrose density gradient columns were scanned with u l t r a -5k v i o l e t absorbance monitor at 25k nm (Section I I I . C. 2) and the virus concentration of each sample was determined by counting squares under the peak and p l o t t i n g the number of squares under each absorbance peak on the prepared standard curve (Section I II.C.2). P r o f i l e s of gradients layered with extracts from healthy tissue were used to project base l i n e s . (b) Electron microscopy assay. Electron microscopy was used to determine the presence of CP-SBMV and CP-TMV in the same c e l l s of simul-taneously doubly infected and of each virus in c e l l s of singly infected leaves of C a l i f o r n i a blackeye cowpea. Plants used in this study were inoculated 10 days af t e r sowing. Sampling was done 12 days a f t e r inocu-l a t i o n in one series of experiments and in another 20 days af t e r inocu-l a t i o n . Small pieces of inoculated and systemically infected leaves were fixed in 5% g1utara1dehyde in 0.1 M potassium phosphate buffer pH 7-2 for 90 minutes, rinsed in phosphate buffer and then post-fixed in ]% osmium tetroxide in Palade's buffer for 60 minutes. The pieces were dehydrated in graded series of ethanol (50, 70, 35% and absolute) and then in propylene oxide. They were then i n f i l t r a t e d in propylene oxide; Epon 812 (1:1, v/v) a f t e r which they were embedded in Epon 812. Thin sections were cut on an LKB Ultratome I I I with a diamond knife. Thin sections were mounted on copper grids with a collodion carbon super f i l m and stained with 5% uranyl acetate (in 50% methanol) and then with Reynolds' lead c i t r a t e (Reynolds, 1963)• The sections were examined in a P h i l i p s EM 300 transmission microscope at a voltage output of 60 KV. 5 5 B. Movement of CP-SBMV and CP-TMV from inoculated primary leaves into preformed 3rd t r i f o l i a t e leaves of C a l i f o r n i a blackeye cowpea. The experiments in this section were aimed at studying interactions of CP-SBMV and CP-TMV in the 3rd t r i f o l i a t e leaves based on the sequence of a r r i v a l of each virus in these leaves. Therefore the f i r s t and 2nd t r i f o l i a t e leaves were trimmed before inoculation. 1. Trimming. When the f i r s t and 2nd t r i f o l i a t e leaves were 1.5 to 3-0 cm long they were trimmed o f f with a flamed razor blade or a pair of s c i s s o r s . The 3rd t r i f o l i a t e leaves were then allowed to grow to a length of 0.5 to 1 cm before inoculation; however i f by the time the 1st and 2nd t r i f o l i a t e leaves were trimmed the 3rd t r i f o l i a t e leaves had reached this s i z e inoculation was done the following day. 2. Inocu1 at ions. Inoculations were usually done at 14 days af t e r sowing. Group (i) plants were mock-inocu1ated on the primary leaves with buffer alone (control); group ( i i ) plants were inoculated on the right hand half leaf with 0.05 mg/ml CP-SBMV and on the l e f t hand half leaf with buffer; group ( i i i ) plants were inoculated with 0.05 mg/ml of CP-TMV on the l e f t hand half leaf and on the right hand half leaf with buffer; group (iv) plants were simultaneously inoculated on se-parate half leaves with CP-SBMV and CP-TMV as follows: the right hand half leaf was inoculated with 0.05 mg/ml of CP-SBMV and the l e f t hand half was immediately inoculated with 0.05 mg/ml of CP-TMV. A l l inocula-ted leaves were rinsed with water and the plants were put in the 24 ;to 27°C growth room for the duration of the experiment. At various 56 intervals a f t e r inoculation the 3rd t r i f o l i a t e leaves were removed with a flamed razor blade from three to four plants per treatment per i n t e r v a l . The removed leaves were floated on half-strength Hoagland 1s solution in Petri dishes and incubated at 2k to 27°C for 48 hours af t e r which they were stored at -20°C u n t i l a l l samples could be assayed together. In-f e c t i v i t y assay of the leaves was done by grinding leaves of each harvest in a small amount of phosphate buffer and inoculating i t to the local lesion indicator hosts. C. E f f e c t of sequence of a r r i v a l of each virus in the preformed  3rd t r i f o l i a t e leaf on the concentration of the other. 1. InoculatIons. Plants of the same age trimmed as above (Section IV.B.1) were inoculated e s s e n t i a l l y as above (Section IV.B.2) except that a f i f t h group (v) was added and inoculated as follows: one half of the plants were inoculated on the right hand half leaf with 0.05 mg/ml of CP-SBMV and the other half leaf was l e f t uninocu1ated; s i m i l a r l y , the other half of the plants were inoculated on the l e f t hand half leaf with 0.05 mg/ml of CP-TMV and the other half leaf was l e f t uninocu1ated. At various intervals a f t e r the f i r s t inoculation,plants of this f i f t h group were cha11enge-inocu1ated with either CP-TMV (0.05 mg/ml) on the uninoculated l e f t hand half leaf or CP-SBMV (0.05 mg/ml) on the uninoculated right hand half leaf. The plants were l e f t in the 2k to 27°C growth room for the duration of the experiment. 2. Assay. Nucleoprotein assay of singly and doubly infected tissue 57 was by a n a l y t i c a l sucrose density~~gradient centrifugation (Section IV.A.2 D. Interact ions of CP~SBMV and CP-TMV in C a l i f o r n i a blackeye cowpea  under synchronous conditions. 1. Trimming. C a l i f o r n i a blackeye cowpea plants raised in Hoagland's solution as previously described were trimmed to leave only the primary and 3rd t r i f o l i a t e leaves. The 3rd t r i f o l i a t e leaves at the time df trimming were 0.5 to 1.5 cm long. 2. Inocu1 at ions. Usually plants were trimmed the day before ino-c u l a t i o n . Plants selected for uniformity were pre-conditioned by dark treatment. One group was inoculated with CP-SBMV on the right hand half leaf of each primary leaf and with buffer on the l e f t hand half leaf; the other group was inoculated with CP-TMV on the l e f t hand half leaf and with buffer on the right hand half leaf; and the last group was simulta-neously inoculated with CP-SBMV on the right hand half leaf and with CP-TMV on the l e f t hand half leaf. Control plants were inoculated with buffer only. A l l inoculated leaves were rinsed with water. The inoculated plants were held for 42 hours (based on movement of viruses into 3rd t r i f o l i a t e leaves) (Section IV.B.) at 25 to 27°C in a growth chamber with a photoperiod of 18 hours and 13,000 lux. After incubation at 2 5 to 27°C for 42 hours some plants from each inoculation treatment were kept aside and the rest were moved to a polystyrofoam cham ber. From plants kept aside a small piece of tissue from the 3rd t r i f o -l i a t e leaves was cut with a flamed razor blade and the leaf pieces were incubated in Petri dishes for 48 hours. After this period the tissue 58 was homogenized in a spot plate and assayed on the respective local lesion indicator hosts for CP-SBMV and CP-TMV. The rest of the plants were put in the styrofoam chamber (Dawson and Schlegel 1973) with th e i r noninocu-lated 3 r d t r i f o l i a t e leaves extending out of the chamber. The styrofoam chamber was then placed in a plant growth chamber at 10°C in the dark. The inoculated primary leaves which were inside the styrofoam chamber were incubated at 27 to 28°C with heat furnished by two 15 W l i g h t bulbs controlled by a rheostat. The plants were subjected to a d i f f e r e n t i a l temperature treatment for 5 days a f t e r which they were moved to the 2k to 27°C growth room with r e l a t i v e humidity (R.H.) of 75 to 80%. The R.H. was maintained by heating a large water bath in the growth room. This was necessary because preliminary observations showed that i f treated plants were moved to a dry atmosphere some of the 3 r d t r i f o l i a t e leaves and primary leaves became necrotic and abscised. Due to space l i m i t a t i o n in the styrofoam chamber healthy control plants were raised in the 2k to 27°C growth room throughout. 3 . Assay. Accumulation of virus in the 3 r d t r i f o l a t e leaves was deter-mined by i n f e c t i v i t y of discs and by a n a l y t i c a l sucrose density gradient centrifugation. Only the 3 r d t r i f o l i a t e leaves were used for assay. (a) I n f e c t i v i t y assay. Third t r i f o l i a t e leaves were harvested beginning at the time plants were sh i f t e d to the 2k to 27°C growth room (zero time) and at various intervals u n t i l \kk hours since the s h i f t from the styrofoam chamber. Samples for i n f e c t i v i t y assay were removed 59 from leaves with a no. 3 cork borer (7 mm diameter) to give 8 discs for each sampling. The discs were stored at -20°C u n t i l a l l samples could be assayed together. Frozen samples were ground in phosphate buffer and were further dilu t e d to give 1/10 to 1/10,000 d i l u t i o n s (w/v). I n f e c t i v i t y was assayed by the h a l f - l e a f method based on a randomized block design for plants with two leaves (KIeczkowski, 1950) for 8 d i f f e r e n t virus concentrations extended to su i t 10 virus concentrations, replicated 18 times. Tissues from healthy control and zero time treatments were assayed separately. I n f e c t i v i t y of CP-SBMV and CP-TMV was assayed on primary leaves of GA 21 and N^. g 1 ut i nosa, trimmed to leave two middle leaves, respectively. (b) Nucleoprotein assay. Tissue which remained from 3rd t r i f o l i a t e leaves a f t e r removal of discs was stored at -20°C and was lat e r assayed for nucleoprotein y i e l d by a n a l y t i c a l sucrose density gradient c e n t r i f u -gation (Section IV.A.2.a). E. Analysis for stru c t u r a l interactions in C a l i f o r n i a blackeye cowpea  doubly infected by CP-SBMV and CP-TMV. 1. Inoculations. Cowpea plants were inoculated singly and doubly (simultaneously) with CP-SBMV and CP-TMV as previously described (Section IV.A.I.a). 2. Assay. Twenty days af t e r inoculation the 3rd t r i f o l i a t e leaves were harvested and stored at -20°C u n t i l needed. (a) Pur i f i cat ion. (i) Frozen tissue was pulverized and homogenized in 0.2 M Tris-HCl. buffer pH 6.5 with a Waring blender. 60 ( i i ) The homogenate was strained through k layers of cheesecloth, ( i i i ) The f i l t r a t e was emulsified with chloroform (1:1 v/v) by s t i r r i n g for 2 to 5 minutes at k°C. (iv) The emulsion was broken by centrifuging at 10,000 rpm for 20 minutes in a Sorvall SS-34 rotor, (v) The supernatant was centrifuged at 28,000 rpm in a Spinco no. 30 rotor for 3 hours, (vi) The p e l l e t was extracted overnight in 0.02 M Tris-HCl buffer pH 6.5 containing 0.1% Igepon T - 73. ( v i i ) The virus suspension was c l a r i f i e d at 10,000: rpm for. 1 0-minutes. The concentration of each virus in the extracts was estimated by a n a l y t i c a l sucrose density gradient centrifugation (Section IV.A.2.a) and the concentration of the viruses in the extracts from doubly infected plants (natural mixture) was adjusted to 0.25 and 0.5 mg/ml for CP-SBMV and CP-TMV, respectively. An a r t i f i c i a l mixture of the same concentra-tion as the natural one was made by mixing appropriate volumes of the extracts from singly infected plants. The mixtures were then analysed for genomic masking by i n f e c t i v i t y n e u t r a l i z a t i o n assay and also by i n f e c t i v i t y d i s t r i b u t i o n In sucrose gradients. (b) Serum n e u t r a l i z a t i o n of i n f e c t i v i t y . I n f e c t i v i t y n e u t r a l i z a -tion was performed following the method of Dodds (1972). A l l sera were dil u t e d 1/8 in PBS before use. Virus antigens p a r t i a l l y p u r i f i e d and prepared as above (Section IV. D.2.a) were neutralized as follows: 61 one mi 11i1itre-aliquots of natural (in vivo mixture) and a r t i f i c i a l (in v i t r o mixture) mixtures, as well as of each virus separately, were each incubated in 0.2 ml of CP-SBMV antibody, CP-TMV antibody, normal serum and PBS at room temperature (25°C) for one hour on a Fisher c l i n i -cal rotator and then at 4°C for three hours. The antigen-antibody mix-tures were then centrifuged at 10,000 rpm for 10 minutes. The super-natants were col l e c t e d from each mixture, combined with 1/10 volumes of serum, or buffer and incubated overnight. The antigen-antibody comp-lexes were then p r e c i p i t a t e d by low speed centrifugation as before, and at this time no further p r e c i p i t a t i o n was observed. The neutralized antigens were then used for i n f e c t i v i t y by the opposite half leaf method on GA 21 and N_. glutinosa for CP-SBMV and CP-TMV, respectively. C a l i f o r -nia blackeye cowpea was also inoculated separately by each of the prepa-rations in case the heterologous 1y encapsidated RNA could not recognize i t s s p e c i f i c local lesion host. (c) Sucrose density gradient centrifugation. A r t i f i c i a l and natural mixtures of the two viruses and preparations of CP-SBMV and of CP-TMV, p a r t i a l l y p u r i f i e d as above (Section IV.D.2.a) were layered on sucrose gradient columns and centrifuged at 39,000 rpm for 105 minutes at 5°C in a Beckman SW 41 rotor. Fractions were col l e c t e d with a syringe by puncturing on the side of the tube with a needle (Peterson and Brakke, 1973) at positions corresponding to the li g h t scattering zones of CP-SBMV and CP-TMV and to the zones between CP-SBMV and CP-TMV. The co l l e c t e d 62 f r a c t i o n s were dialysed overnight in phosphate buffer and inoculated to GA 21 and N_. glutinosa by the opposite half leaf method. C a l i f o r -nia blackeye cowpea and Pinto bean were also inoculated but separa-tely by each of the fra c t i o n s because both viruses infect C a l i f o r n i a blackeye cowpea systemically and CP-TMV infects Pinto systemically. V. Interactions of CP-SBMV and CP-TMV in Pinto bean. When fractio n s c o l l e c t e d a f t e r sucrose density gradient c e n t r i -fugation were assayed on Pinto as described above (Section IV.D .2.c), i t was inadvertently observed that f r a c t i o n s c o l l e c t e d between l i g h t scattering zones of CP-SBMV and CP-TMV in both natural and a r t i f i c i a l mixtures induced necrotic reddish local lesions on primary leaves of Pinto, s i m i l a r to those induced by the bean s t r a i n of SBMV on Pinto. These symptoms were always reproducible when Pinto primary leaves were doubly inoculated with either p u r i f i e d or crude sap extracts of a mix-ture of CP-SBMV and CP-TMV but not by CP-SBMV inoculated alone. A. Inoculations. 1. Inoculations with intact CP-SBMV and intact CP-TMV. In one series of experiments the primary leaves of Pinto were singly ino-culated with known concentrations of either p u r i f i e d CP-SBMV or CP-TMV or phosphate buffer alone. The inoculated leaves were thoroughly rinsed with running tap water to remove residual inoculum, and challenge-inoculated with varying concentrations of eit h e r CP-SBMV, CP-TMV or buffer 72 or 96 hours a f t e r i n i t i a l inoculation. In another series of experiments, time-course synthesis of CP-SBMV was studied in Pinto primary leaves doubly inoculated simultaneously with CP-SBMV 63 and CP-TMV and singly inoculated with the former v i r u s . The primary leaves of one group of plants were singly inoculated with CP-SBMV and those of another group were doubly inoculated simultaneously with 0.05 mg/ml of CP-SBMV and 0.05 mg/ml of CP-TMV. Control plants were inoculated with phosphate buffer only. At intervals a f t e r inoculation, the primary leaves were harvested and soaked in a detergent solution for 10 minutes and rinsed with tap water for 24 hours a f t e r which discs were cut with a no. 10 cork borer and stored at -20°C u n t i l a l l could be assayed together. 2. Inoculations with CP-SBMV-RNA and intact CP-TMV. Pinto primary leaves were singly inoculated with CP-SBMV-RNA prepared from either p u r i f i e d virus or from sap of infected leaves. (Section V.B.a and b), or they were doubly inoculated with CP-SBMV-RNA and intact CP-TMV. When simultaneous inoculations were made, a one ml aliquot of CP-SBMV-RNA was mixed with one ml (0.1 mg/ml) of intact CP-TMV and applied to Pinto primary leaves. For single inoculations the aliquot of CP-SBMV-RNA was dilute d with an equal volume of phosphate buffer and applied to the primary leaves. In sequential inoculations Pinto primary leaves were inoculated with the di l u t e d CP-SBMV-RNA or 0.05 mg/ml of intact CP-TMV or buffer only and later cha11enge-inocu1ated at various intervals : with either 0.05 mg/ml of intact CP-TMV or the d i l u t e d CP-SBMV-RNA. When the time-course synthesis of CP-SBMV was studied, the primary leaves were either doubly inoculated simultaneously with CP-SBMV-RNA and intact CP-TMV or singly with CR-SBMV-RNA. A l l inoculated leaves were rinsed with water. At 6k intervals a f t e r inoculation the primary leaves were harvested and rinsed in running water for 8 hours or overnight, blotted,and dfses were cut with a no.10 cork borer and stored at -20°C u n t i l a l l samples could be assayed together. 3. Assay. Assays for CP-SBMV on Pinto primary leaf t i s s u e , inocu-lated as mentioned above, were made by i n f e c t i v i t y , serology and electron microscopy. (a) I n f e c t i v i t y assay. The i n f e c t i v i t y assay for CP-SBMV was made on GA 21 whole leaves (replicated 20 times) rather than on half leaves in order to avoid contamination of the opposite h a l f - l e a f by one of the inocula. Tissues from discs stored as mentioned above (Section V.A.I and 2) were ground in a mortar with a pestle with a small amount of sand and dil u t e d 1:2 (w/v) in phosphate buffer and applied to GA 21 primary leaves. (b) Serological assay. The double d i f f u s i o n method of immunodiffu-sion (Ouchterlony, 1958) was used to detect antigens in tiss u e extracts. The d i f f u s i o n medium consisted of 10 ml of 0.75% Noble agar, 0.02% NaN^ in PBS in p l a s t i c Petri dishes (8.5 cm diameter). Several discs (total wt, 2.0 - 2.5 g) containing coalescing lesions were cut with a no. 5 cork borer from Pinto primary leaves that had been washed with 10% Lux detergent for 10 minutes and rinsed for 2k hours with running tap water. They were ground in PBS (1 g/0.2 ml) in a mortar with a pestle in the presence of p u r i f i e d sand (The B r i t i s h Drug Houses Ltd., Poole, England). The homogenate was then squeezed through a double layer of Miracloth, 65 heated at kO C for 60 minutes, and then c l a r i f i e d by centrifuging at 10,000 rpm for 10 minutes. Aliquots (0.02 ml) of supernatant were deposited in wells, 5 mm from a central well, containing either CP-SBMV or CP-TMV antiserum d i l u t e d to 1:128. The gels were incubated at room temperature (25°C) and examined for :timmunoprecipi tates 2k hours later u n t i l no more p r e c i p i t i n formed. (c) Electron microscopy assay. Seven to 10 days af t e r Inoculation primary leaves showing local lesions were prepared for EM or p u r i f i c a -t i o n . Sections for EM were prepared as before (Section IV.A.2.b) and so examined. Virus p u r i f i e d as for CP-SBMV (Section I M.B.I) was examined by EM but because of the preponderance of CP-TMV even af t e r an attempt to separate the two viruses by sucrose density gradient cent r i f u g a t i o n , i t was necessary to separate CP-SBMV by p r e c i p i t a -ting i t with homologous antiserum. The f r a c t i o n believed to be CP-SBMV was reacted with i t s homologous antiserum in agar gel double d i f f u s i o n . After CP-SBMV-antibody bands had formed the whole Petri dish was sub-merged in a beaker of 0.05 M NaCl overnight and rinsed in d i s t i l l e d water (Dr. J.H. Tremaine, personal communication). The bands were s l i c e d out with a razor blade and ground in a drop of 2% uracyl acetate negative s t a i n . The homogenate was mounted on grids and rinsed 5 times with 2% uracyl acetate negative s t a i n . The grids were viewed with a P h i l i p s EM 200 or 300. B. Extraction of ribonucleic acid. Infectious RNA was extracted from either p u r i f i e d virus preparation 66 or crude sap of infected C a l i f o r n i a blackeye cowpea. 1. Extraction of RNA from p u r i f i e d virus preparation. The infectious RNA was extracted as follows ( S a n t a l l i et a l , 1961): virus (2 ml, 3 mg/ml) was added to 6 ml of 0.05 M Kh^PO^, 0.13 M NaCl (adjusted to pH 7-0 with NaOH) which had been heated to 96.5 £ 0.5°C and the mixture was maintained at this temperature for one minute. The solution was immediately placed in ice and cooled for 5 minutes. After c h i l l i n g the mixture was c e n t r i -fuged at 6,000 rpm for 10 minutes. The RNA in the supernatant was p r e c i -pitated by adding two volumes of absolute ethanol containing a few drops of 3 M NaAc and allowed to stand on ice for 30 minutes. The RNA was c o l l e c -ted by centrifuging for 30 minutes at 9.000 rpm. The p e l l e t was extracted in 2.5 ml of 0.01 M potassium phosphate buffer pH 7.1 containing bento-nite (100 ^jg/ml) and the suspension was then used immediately for ino-culat ion. 2. Extraction of RNA from crude sap (Dawson and Kuhn, 1972). Twenty grams of fr e s h l y harvested leaf tissue of C a l i f o r n i a blackeye cowpea previously inoculated with CP-SBMV was blended in a Waring blender in an extraction medium containing 20 ml of water - saturated phenol, \% SDS (sodium dodecyl sulphate), \% bentonite 0.01 M EDTA, as modified by Kuhn and Adams (1976). The emulsion was centrifuged at 6,500 rpm for 10 minutes. The supernatant aqueous phase was mixed three times with petroleum ether to remove phenol. The f i n a l aqueous phase was preci p i t a t e d with 2 volumes of 35% ice-cold ethanol and centrifuged for 10 minutes at 6,500 rpm. The 67 RNA p e l l e t was extracted in 0.01 "M'potassium phosphate buffer pH 7-1 containing bentonite (100^ig/ml). The resuspended RNA was divided into 1 ml-aliquots and stored at -20°C u n t i l used. The RNA extracted in th i s way was used in most experiments. VI. Seed transmission of CP-SBMV and CP-TMV A. Propagation hosts for CP-SBMV and CP-TMV The C a l i f o r n i a blackeye cowpea and three Botswana local v a r i e t i e s , Botswana blackeye, V26-Bots (khaki colour) and V45-Bots (dark brown colour) cowpeas were grown in 21-cm pots, inoculated singly or doubly and raised in the greenhouse as described before (Section III), B. D i s t r i b u t i o n of CP-SBMV and CP-TMV in seed parts of C a l i f o r n i a  blackeye cowpea and 3 Botswana cowpeas. 1. Harvesting. The d i s t r i b u t i o n of CP-SBMV or CP-TMV in seeds de-rived from plants singly or doubly infected by CP-SBMV and CP-TMV was determined at various stages of seed maturity. This was done by..tagging the peduncle of the flower with a date tag as soon as a small pod was formed. The pods were then harvested when 15 to 20, 30 to 35 and 45 to 55 days old; these stages represented green immature, dough stage immature and vine-dry mature, respectively. 2. Decontamination procedures. Seeds were removed from the pods and separated into seed coats and embryos. In immature seed the seed coats were e a s i l y peeled o f f from the embryos (cotyledons and radicle-plumule shoot) without soaking. However, mature seed were soaked overnight 68 in d i s t i l l e d water before seed coats could be removed. The separated seed parts were separately decontaminated by either a tap water wash for 2k hours or they were soaked in 5% trisodium phosphate (Na^PO^) for 10 minutes and then rinsed overnight or at least for 12 hours in running tap water. Controls included healthy seed parts. The healthy embryos were a r t i f i c i a l l y contaminated with virus by soaking them in virus suspension (10 ug/ml) for 10 to 15 minutes. Half of the population of a r t i f i c i a l l y contaminated embryos was then decontaminated by tap water wash or by soaking them in 5% Na^PO^. The remainder was l e f t undeco-taminated. When cotyledons and radicle-plumule shoots were assayed separately, the whole embryo was decontaminated before d i s s e c t i o n . A plumule shoots which were then rinsed for 30 minutes in water. 3- Assay of embryos and seedlings derived from seeds produced on  plants infected with CP-SBMV and CP-TMV. (a) Direct assay of seed parts on indicator hosts. Embryos, radicle-plumules and seed coats were homogenized singly in 0.2, 0.05 and 0.25 ml, respectively, in phosphate buffer. Occasional samples containing f i v e pooled radicle-plumules or seed coats were homo-genized in 0.1 and 0.25 ml, respectively. The homogenates were inocula-ted to primary leaves of GA 21 and to middle leaves of N_. g 1 ut i nosa or Xanthi and the leaves were rinsed immediately with water. (b) Seedling assay. Undecontaminated embryos,, decontaminated embryos of mature seeds and flamed sharp forceps was used to dissect the cotyledons and r a d i c l e -69 intact mature seed were sown in steam-sterilized s o i l and allowed to germinate. In early experiments, tissue samples of 2nd and 3rd t r i f o l i a t e leaves of a l l seedlings were assayed i n d i v i d u a l l y on the respective i n d i -cator hosts. However, i t was lat e r realized that visual symptoms for CP-SBMV were as r e l i a b l e as the i n f e c t i v i t y assay so in subsequent expe-riments the c r i t e r i o n for infec t i o n by CP-SBMV in seedlings from seed of singly infected plants was based on visual symptoms. However, a l l seed-lings derived from seed produced by plants singly infected by CP-TMV or doubly infected by CP-SBMV were always assayed on the respective i n d i -cator hosts for each virus in case symptoms of one virus were masked by those of the other. Primary leaves were never assayed. Seedlings were never handled except when assayed. C. Ef f e c t of germination ori CP-SBMV and CP-TMV in seed coats. 1. Germination of seed. Seeds from plants previously infected singly by CP-SBMV or CP-TMV were sown in steam-sterilized r i v e r sand. Two to four days af t e r sowing seeds were removed and embryos and seed coats separated. One half of the population of the seed coats was de-contaminated overnight or for 2k hours in running tap water; the remainder was l e f t undecontaminated. 2. I n f e c t i v i t y assay. I n f e c t i v i t y of the seed coats obtained from germinated seed was compared with that of ungerminated seed coats. The seed coats were each ground in 0.25 ml of buffer and assayed by the half-leaf method on GA 21 for CP-SBMV and N. glutinosa for CP-TMV. The scheme of inoculation was as follows: (i) extracts of washed seed coats (from 70 germinated or ungerminated seeds) were inoculated to opposite halves of Pinto primary leaves; and ( i i ) those from unwashed seed coats were s i m i l a r l y inoculated. D. Effect of healthy mature seed extracts on i n f e c t i v i t y of  CP-SBMV and CP-TMV. 1. Extract ion. Healthy mature seeds were soaked overnight in d i s t i l l e d water and seed coats were removed. The embryos were ground in a Waring blender with 0.01 M potassium phosphate buffer pH 7-1 (one embryo/ 1 ml )• The homogenate was strained through a double layer of Miracloth. The f i l t r a t e was dilute d 1/10 and 1/100 (v/v). Virus was added to the f i l t r a t e and i t s d i l u t i o n made to a f i n a l concentration of 10jjg/rn]; buffer containing the same virus concentration was used as a control. 2. I n f e c t i v i t y assay. The virus preparations made as above were inoculated to GA 21 and N_. glutinosa for CP-SBMV and CP-TMV i n f e c t i v i t y tests, respectively , in a.completely randomized design by the half leaf method, replicated 8 times. VII. Production of seed for y i e l d analysis. The C a l i f o r n i a blackeye cowpea was used excl u s i v e l y for seed pro-duction. Seeds were sown in 21-cm pots and aft e r germination seedlings were thinned to 2 plants per pot. The treatments were arranged in a completely randomized design on a greenhouse bench. Because cowpea bears i n d e f i n i t e l y , peduncles of flowers were tagged and pods produced on these' ; flowers were the only ones harvested for y i e l d analysis. Pods were harvested when vine-dry at 55 to 65 days af t e r flowering. 71 RESULTS I . Estimation of concentration of p u r i f i e d virus by an a l y t i c a l  sucrose density gradient centrifugation. Absorbance p r o f i l e s of p u r i f i e d CP-SBMV and of p u r i f i e d CP-TMV are shown in Figure 1 and Figure 2, respectively and when the area under the peaks was calculated and plotted against virus concentration (mg/ml), a linear r e l ationship was obtained (Figures 3 and h). The shoulder on the l e f t hand side of the CP-TMV absorbance profi1e,probably, resulted from aggregation of the vi r u s . This is the best p r o f i l e pattern one could get with this s t r a i n of TMV, which seems prone to aggregation (Rees and Short, 1965) and i t did not seem to a f f e c t the r e p r o d u c i b i l i t y of the linear r e l a t i o n s h i p . The absorbance area values, in a r b i t r a r y units, are given in Table II and these were used to plot the standard curves (Figures 3 and 4). The standard curves were derived from data averaged from two ex-periments for each v i r u s . I I. Effect of single and double infections on C a l i f o r n i a  blackeye cowpea and Botswana cowpea v a r i e t i e s . A. Comparison of symptoms. 1. Symptoms in C a l i f o r n i a blackeye cowpea. When seedlings were s i n -gly inoculated with CP-SBMV, d i f f u s e c h l o r o t i c spots developed on the ino-culated primary leaves 4 to 5 days af t e r inoculation. The c h l o r o t i c spots developed also on primary leaves that were doubly inoculated with CP-SBMV 72 F i g u r e : l . Absorbance p r o f i l e s following centrifugation of 0.2 ml of p u r i f i e d CP-SBMV through sucrose density gradient columns in a Beckman SW 41 rotor, at 39,000 rpm at 5 C for 105 minutes and scanned at 254 nm (0.5 absorbance range). Direction of sed'imenitation is to the l e f t . A. CP-SBMV at 0.50 mg/ml before centrifugation. B. CP-SBMV at 0.25 mg/ml before ce n t r i f u g a t i o n . C. CP-SBMV at 0.10 mg/ml before centrifugation. D. CP-SBMV at 0.05 mg/ml before centrifugation. 0.16 0.14 0.12 0.10 E LTV CN 0.08 o c ro XI i_ o in < 0.06 0.04 0.02 0.0 0.04 0.02 0.0 7k Figure 2. Absorbance p r o f i l e s following centrifugation of 0.2 ml of pu r i f i e d CP-TMV through sucrose density gradient columns in a Beckman SW k] rotor, at 39,000 rpm at 5°C for 105 minutes and scanned at 254 nm (0.5 absorbance range). Direction of sedimentation is to the l e f t . A. CP--TMV at 1.00 mg/ml before ce n t r i f u g a t i o n . B. CP-•TMV at 0.50 mg/ml before ce n t r i f u g a t i o n . C. CP--TMV at 0.25 mg/ml before centrifugation. D. CP--TMV at 0.125 mg/ml before centrifugation 75 76 Figure 3- Relationship between CP-SBMV concentration (mg/ml) and absor-bance area of CP-SBMV in sucrose density gradient columns aft e r centrifugation in Beckman SW 41 rotor, at 39,000 rpm at 5 C for 105 minutes. The amount of virus in a suspension layered on sucrose density gradient columns was 0.2 ml for each concentration and the columns were scanned at 254 nm (0.5 absorbance range). The concentration of virus in the gradient columns is expressed in a r b i t r a r y absorbance area un i t s . 78 F i g u r e k. R e l a t i o n s h i p between CP-TMV c o n c e n t r a t i o n (mg/ml) and a b s o r -bance a r e a o f CP-TMV i n s u c r o s e d e n s i t y g r a d i e n t columns a f t e r c e n t r i f u g a t i o n i n Beckman SW 41 r o t o r , a t 3 9,000 rpm at 5 C f o r 105 m i n u t e s . The amount o f v i r u s i n a s u s p e n s i o n l a y e r e d on s u c r o s e d e n s i t y g r a d i e n t columns was 0.2 ml f o r each c o n c e n t r a t i o n and the columns were scanned a t 25b nm (0.5 a b s o r b a n c e r a n g e ) . The c o n c e n t r a t i o n o f v i r u s i n the g r a d i e n t columns i s e x p r e s s e d i n a r b i t r a r y a b s o r b a n c e a r e a un i t s . 80 Table II. Relationship between CP-SBMV and CP-TMV concent rations arid t h e i r absorbance areas (arbitrary units) a f t e r centrifuga-tion of 0.2 ml of each virus through sucrose density gradient columns at various concentrations Virus concentration (mg/ml) Absorbance area units CP-SBMV CP-TMV CP-SBMV CP-TMV 0.50 1.000 139 87 0.25 0.500 68 kl 0.10 0.250 27 22 0.05 0.125 13 11 Before centrifugation. 81 and CP-TMV. No symptoms developed on the primary leaves of seedlings ino-culated, 8 to 10 days af t e r sowing, with CP-TMV. However, when older plants (ca \k days) were inoculated with CP-TMV alone, or doubly with CP-SBMV, irregular purple lesions developed on the inoculated primary leaves. In systemically infected t r i f o l i a t e leaves of plants inoculated with CP-SBMV alone, vein clearing was induced in the newly-formed leaves which was replaced later by mosaic with mottling and at times d i s t o r t i o n and ch l o r o t i c spots appeared (Figure 5B). Leaves that appeared later seemed to recover from severe symptoms. Systemically infected leaves of plants inoculated with CP-TMV only developed mosaic and mottling accompanied by b l i s t e r s (Figure 5 C ) and d i s t o r t i o n of the leaves. Double infection caused symptoms of CP-TMV in the newly-developed leaves and in later leaves, leaf clearing and b l i s t e r s developed (Figure SO). Although CP-SBMV symptoms were sometimes expressed in double i n f e c t i o n , CP-TMV symptoms usually dominated and masked those of CP-SBMV. Leaf s i z e was also reduced by double infection (Figure 5D). When primary leaves inoculated with CP-TMV were cha11enge-inocu1ated with CP-SBMV, the number of c h l o r o t i c spots which developed on the inocu-lated leaves decreased with increase,in the interval between the two inoculations. When CP-SBMV-inocu1ated primary leaves were chal1enge-ino-culated with CP-TMV 2k and 72 hours af t e r i n i t i a l inoculation, more severe systemic symptoms, accompanied by necrosis, developed on newly-developed t r i f o l i a t e leaves than on those of plants simultaneously inoculated with 82 Figure 5- Third t r i f o l i a t e leaves of C a l i f o r n i a blackeye cowpea singly and doubly infected by CP-SBMV and CP-TMV. Photographed 20 days af t e r inoculation (28 days af t e r sowing). A. Healthy t r i f o l i a t e leaf-- from plants mock-inocu1ated with phosphate buffer. B. CP-SBMV-infected t r i f o l i a t e leaf. C. CP-TMV-infected t r i f o l i a t e leaf. D. CP-SBMV/CP-TMV-(doubly)-infected t r i f o l i a t e leaf. 83 the two viruses. When CP-TMV infected leaves were cha1 1enge-inocu1ated with CP-SBMV at sim i l a r i n t e r v a l s , severe symptoms developed on the t r i -f o l i a t e leaves that were not formed at the time of inoculation. In a l l the sequences, CP-TMV symptoms dominated those induced by CP-SBMV in t r i -f o l i a t e 1 eaves. 2 . Symptoms in Botswna cowpea v a r i e t i e s . CP-SBMV in single inocu-lations induced the c h l o r o t i c spots on the inoculated primary leaves of Botswana blackeye and V26-Bots cowpea v a r i e t i e s , but CP-TMV did not cause any obvious symptoms on the inoculated leaves. CP-SBMV systemic infection caused symptoms sim i l a r to those i t induced in C a l i f o r n i a blackeye cowpea, except that they were less severe. CP-SBMV single inoculation did not cause symptoms on the inoculated primary leaves or t r i f o l i a t e leaves of V45 -Bots, although the virus could be detected in primary leaves by infec-t i v i t y assay. CP-TMV readily infected a l l the three Botswana cowpea v a r i e -t i e s , causing symptoms sim i l a r to those in the C a l i f o r n i a blackeye cowpea. Double inoculation resulted in systemic infection of the three v a r i e t i e s by both viruses. Doubly inoculated primary leaves,, of a l l va r i e -t i e s developed c h l o r o t i c spots c h a r a c t e r i s t i c of CP-SBMV. When V45-Bots was doubly inoculated with both viruses, systemically infected leaves showed c h l o r o t i c spots, mosaic and mottling symptoms c h a r a c t e r i s t i c of CP-SBMV, but these were not masked or dominated by CP-TMV symptoms, in contrast to symptoms in C a l i f o r n i a blackeye cowpea. B. Effect on plant growth. 1. Plant growth in C a l i f o r n i a blackeye cowpea. Primary leaves of plants, singly or doubly inoculated, weighed the same as those inoculated 84 with buffer only. CP-SBMV in singly infected plants caused mild growth reduction, compared to buffer-inocu1ated plants. For instance, CP-SBMV in single infections reduced fresh weight and height of plants by 6.8 and 3.7%, respectively (not s t a t i s t i c a l l y s i g n i f i c a n t ) , while CP-TMV caused s i g n i f i c a n t reductions of 28.4 and 45-6%, respectively (Figure 6 and Table III). Although double infection did not cause any s i g n i f i c a n t reduction in height over that already caused by CP-TMV alone, i t , however, reduced fresh weight s i g n i f i c a n t l y over that of plants singly infected by CP-TMV (Figure 6 and Table III). The number of t r i f o l i a t e leaves produced on doubly or sin g l y infected or buffer-inoculated plants was about the same (Table III). 2. Plant growth in V45~Bots cowpea var i e t y v > . Double infection reduced weight and height of V45~Bots cowpea, as did CP-TMV infection alone, whereas no apparent difference was observed between buffer-inocu1ated and CP-SBMV-singly inoculated plants (Table IV). C. Comparison of e f f e c t s of CP-SBMV and CP-TMV on y i e l d and seed  character i s t i cs. 1. Yield of C a l i f o r n i a blackeye cowpea. Double infe c t i o n s i g n i f i c a n t l y reduced number of pods and seeds per plant by bO.b and bb.3%, respectively, compared to y i e l d of healthy plants. CP-SBMV alone; caused only s l i g h t reductions of 6.6 and 8.3% in pods and seeds, respectively, whereas CP-TMV single infection s i g n i f i c a n t l y reduced pod and seed y i e l d by 36.8 and 30.3%, respectively (Table V). Figure 6. C a l i f o r n i a blackeye cowpea plants singly and doubly infected by CP-SBMV and CP-TMV. Photographed 20 days aft e r inoculation (28 days af t e r sowing). A. Healthy p l a n t — mock-inocu1ated with phosphate buffer. B. CP-SBMV-infected plant. C. CP-TMV-infected plant. D. CP-SBMV/CP-TMV-(doubly)-infected plant. 86 Table I I I . E f f e c t of s i n g l e (Sl) and simultaneous double i n f e c t i o n s (Dl) of CP-SBMV and CP-TMV on growth of C a l i f o r n i a blackeye cowpea Height Percent Fresh wt. Percent No. of leaves per plant reduction per plant reduction per plant Treatment (cm) (g) Buffer 92.75 C 0.0 16.2 ? 0. .0 8. .0 CP-SBMV-SI 83-75 C 9-7 15.1 C . 6. .8 9. 0 CP-TMV-SI 50.50 d 45.6 11.6 d 28. ,4 8. 0 Dl 42.75 d 53.9 9.8 ? 39. 5 8. 0 Readings made 24 days a f t e r i n o c u l a t i o n (32 days a f t e r p l anting) Only t r i f o l i a t e leaves were counted. Values in each column not followed by the same l e t t e r vary s i g n i f i -c a n t l y at P = 0.05 by Duncan's M u l t i p l e Range (DMR) t e s t . 87 Table IV. Effect of single and simultaneous double infections by CP-SBMV and CP-TMV on growth of Botswana var i e t y of V45-Bots cowpea Percent Fresh wt. Percent wt. Height per height per plant reduction Treatment plant (cm) reduct ion (g) Buffer 29.0 0.0 11.0 0.0 CP-SBMV 29.3 -1.0 10.7 2.7 CP-TMV 21.3 30.0 8.0 27.3 CP-SBMV + CP-TMV 23.3 20.0 8.9 20.0 Readings made 20 days af t e r inoculation (28 days a f t e r sowing) 88 T a b l e V. E f f e c t o f s i n g l e and s i m u l t a n e o u s d o u b l e i n f e c t i o n s o f CP-SBMV and CP-TMV on pod and seed y i e l d o f C a l i f o r n i a b l a c k e y e cowpea Number per p l a n t  Treatment Pods % R e d u c t i o n Seed % R e d u c t i o n B u f f e r 13.6 b 0.0 87. • 5 b 0.0 CP-SBMV 12.7 b 6.6 80. .3 b 8.3 CP-TMV 8.6 c 36.8 61. .0 c 30.3 CP-SBMV + CP-TMV 8. 1 c 4o.4 48. .8 c 44.3 a P o d s and seeds h a r v e s t e d 60 days a f t e r f l o w e r i n g . it V a l u e s i n each column not f o l l o w e d by the same l e t t e r v a r y s i g n i f i c a n t l y a t P = 0.05 by DMR t e s t . 8 9 2 . Seed c h a r a c t e r i s t i c s in C a l i f o r n i a blackeye cowpea. Seeds produced by plants that had been buffer-inoculated and by plants singly infected by CP-SBMV had a normal seed coat colour. However, seeds produced by plants singly infected by CP-TMV and by plants doubly infected by CP-SBMV and CP-TMV had mottled and discoloured seed coat, with some internal necrosis. Double i n f e c t i o n , however, caused more seed coat mottling than CP-TMV alone. 3. Seed c h a r a c t e r i s t i c s in Botswana cowpea v a r i e t i e s . CP-SBMV sing l e inf e c t i o n did not cause seed coat mottling in Botswana blackeye, but 50% of seeds of V26-Bots from plants singly infected by CP-SBMV had mottled seed coats. CP-TMV and double i n f e c t i o n caused more seed coat discoloura-t i o n . D. D i s t r i b u t i o n and concentration of CP-SBMV and CP-TMV nucleoproteins  in cowpea. An a l y t i c a l sucrose density gradient centrifugation proved to be suitable for separation of the viruses in a natural mixture of p a r t i a l l y c l a r i f i e d sap (Figures 7 and 8 ) . Further, the Tris-HCl buffer pH 6 . 5 also proved to be s u i t a b l e as i t caused minimal or no aggregation of CP-TMV (Figures 7 and 8 ) . 1. D i s t r i b u t i o n and concentration of CP-SBMV and CP-TMV in the  inoculated primary leaves of C a l i f o r n i a blackeye cowpea. (a) Synthesis a f t e r simultaneous inoculations. CP-SBMV could not be detected by a n a l y t i c a l sucrose density gradient centrifugation of par-t i a l l y c l a r i f i e d sap prepared from primary leaves when analysed 48 hours a f t e r inoculation. However, CP-SBMV was detected at 9 6 to 1 9 2 hours af t e r 90 Figure 7- Relative absorbance p r o f i l e s following c e n t r i f u g a t i o n , through sucrose density gradient columns, of 0.2 ml of c l a -r i f i e d extracts of primary leaves of C a l i f o r n i a blackeye cowpea inoculated singly with either CP-SBMV or CP-TMV or with CP-SBMV and CP-TMV and harvested 8 days a f t e r inocula-t i o n . An equal amount (20 g f r . wt.) of tissue from each inoculum treatment was used and di l u t e d 1/2 (w/v), in this p a r t i c u l a r experiment. The gradients were centrifuged in a Beckman SW :4l rotor, at 39,000 rpm at 5°C for 105 minutes and scanned at 254 nm (0.5 absorbance range) scale. Direc-tion of sedimentation is to the l e f t . A. P r o f i l e s of extracts of and CP-TMV. B. P r o f i l e of extracts of t C. P r o f i l e of extracts of t D. P r o f i l e of extracts of t buffer only. tissue inoculated with CP-SBMV issue inoculated with CP-SBMV. issue inoculated with CP-TMV. issue mock-inocu Iated with Relative absorbance at 254 nm 92 Figure 8. Relative absorbance p r o f i l e s following c e n t r i f u g a t i o n , through sucrose density gradient columns, of 0.2 ml of c l a r i f i e d extracts of 3rd t r i f o l i a t e leaves which developed a f t e r ino-culation of C a l i f o r n i a blackeye cowpea with either CP-SBMV or CP-TMV or with CP-SBMV and CP-TMV and harvested 20 days aft e r inoculation of the primary leaves. An equal amount (4 g f r . wt.) of tissue from each treatment inoculum was used and d i l u t e d 1/3 (w/v), in th i s p a r t i c u l a r experiment; The gradients were centrifuged in a Beckman SW 41 rotor, at 39,000 rpm at 5 C for 105 minutes and scanned at 254 nm (0.5 absorbance range). Direction of sedimentation is to the l e f t . A. P r o f i l e s of extracts of t r i f o l i a t e tissue infected sys-temically by CP-SBMV and CP-TMV. B. P r o f i l e of extracts of t r i f o l i a t e tissue infected sys-temically by CP-SBMV. C. P r o f i l e of extracts of t r i f o l i a t e tissue infected syste-mically by CP-TMV. D. P r o f i l e of extracts of t r i f o l i a t e tissue a f t e r primary leaves were mock-inocu1ated with buffer only. Relative absorbance at 254 nm 3h inoculation, and i t was always reduced in doubly infected t i s s u e . Double inoculation caused reductions of 56 to 68% in the amount of CP-SBMV compared to that in singly infected tissue (Figure 7 and Table VI). Although the amount of CP-TMV was also reduced in double inoculation by 24 to 27%, com-pared to that in single inoculation, i t could be detected as early as 48 hours a f t e r inoculation (Figure 7 and Table VI). (b) Synthesis a f t e r sequential inoculations. When primary leaves inoculated with CP-SBMV, were cha11enge-inocu1ated with CP-TMV at various intervals (0.0, 0.5, 6.0 and 24.0 hr) a f t e r i n i t i a l inoculation, CP-SBMV was synthesized to only about 59 to 65% of that synthesized in singly ino-culated primary leaves. In contrast the challenging CP-TMV was reduced by about 50% of i t s concentration in singly infected primary leaf tissue (Figure 9 ) - When the reverse sequence was made on the primary leaves, so that CP-TMV was inoculated f i r s t and then cha11enge-inocu1ated with CP-SBMV, the synthesis of CP-SBMV was only 29 to 59% of that in singly inoculated leaves. 2. D i s t r i b u t i o n and concentration in primary leaves of Botswana  cowpea variety V 4 5-Bots. During the seed transmission studies with seeds of cowpea v a r i e t i e s from Botswana, i t was noticed that the vari e t y V45 _Bots did not show symp-toms when inoculated with CP-SBMV alone and no virus could be detected in the t r i f o l i a t e leaves of such plants by i n f e c t i v i t y and serological tests. It was therefore reasoned that CP-TMV might condition t h i s cowpea v a r i e t y Table VI. Relative amounts of CP-SBMV and CP-TMV nuc1eoprotein in single (SI) and double (Dl) infections of primary leaves of C a l i f o r n i a blackeye cowpea harvested at various intervals a f t e r inoculation Inoculum Nucleoprotein content (mg/g f r . wt.) at various intervals (hr) a f t e r inoculation 48 96 144 196 Buffer 0.0 CP-SBMV-SI 0.0 CP-SBMV-DI 0.0 Percent of CP-SBMV-DI as of CP-SBMV-SI 0.0 CP-TMV-Sl" 0.08 CP-TMV-DI 0.06 Percent of CP-TMV-DI as of CP-TMV-SI 75-0 0.0 0.03 0.01 33.3 0.34 0.25 73-5 0.0 0.13 0.05 38.5 0.85 0.65 76.5 0.0 0.16 0.07 43.8 1.49 1.14 76.5 Mean of 4 experiments 96 Figure 9. Relative amounts of CP-SBMV and CP-TMV nucleoproteins (mg/g f r . wt.) in primary leaves of C a l i f o r n i a black-eye cowpea inoculated singly and sequentially with either virus at various i n t e r v a l s . Tissue was analysed 192 hours af t e r i n i t i a l inoculation. A. Yi e l d of CP-SBMV in leaves cha11enge-inocu1ated with CP-TMV (T/S) or aft e r super inoculation to leaves pre-inoculated with CP-TMV (S/T) at various intervals a f t e r i n t i a l inoculation and in single inoculation ( S l ) . B. Yield of CP-TMV in leaves challenge-inoculated .;; with CP-SBMV (S/T) or aft e r superinoculat ion to leaves pre-inoculated with CP-SBMV (T/S) at various intervals a f t e r i n i t i a l inoculation and in single inoculation ( S l ) . A 98 to become systemically susceptible to CP-SBMV. In extracts obtained from t r i f o l i a t e leaves of doubly inoculated V45 _Bots plants, both viruses could be detected by i n f e c t i v i t y and serological t e s t s . This result pro-vided an opportunity for further investigation of th i s type of interaction. (a) Synthesis a f t e r simultaneous inoculations. When V45 _Bots cowpea plants were singly inoculated with CP-SBMV, the virus could be detected in extracts of primary leaves by i n f e c t i v i t y assay but not by a n a l y t i c a l sucrose density gradient c e n t r i f u g a t i o n , even when extracts were concentra-ted 2-fold (w/v) by 10% PEG. However, CP-SBMV could readily be detected by i n f e c t i v i t y and sucrose density gradient centrifugation in unconcentra-ted extracts of primary leaves that were doubly inoculated with both viruses (Tables VII and VIM). A time-course synthesis of CP-SBMV in the inoculated leaves indicated that CP-SBMV mult i p l i e d in these leaves when doubly inoculated with CP-TMV. CP-SBMV recovery from singly inoculated primary leaves was e r r a t i c and therefore i t was d i f f i c u l t to draw any conclusion (Table VIII) about the rate of i t s synthesis in these leaves. CP-TMV y i e l d was greater in singly infected tissue of primary leaves than in doubly inoculated leaves. 3. D i s t r i b u t i o n and concentration of CP-SBMV and CP-TMV in t r i f o l i a t e  leaves of C a l i f o r n i a blackeye cowpea. (a) Synthesis in systemically infected 1st, 2nd, 3rd and 4th t r i f o l i a t e leaves a f t e r simultaneous inoculations of primary leaves. The synthesis of CP-SBMV in doubly infected t r i f o l i a t e leaves was greatly enhanced. 99 Table VII. Nucleoprotein y i e l d of CP-SBMV and CP-TMV in singly (SI) and doubly (Dl) inoculated (simultaneously) primary leaves and systemically infected 3rd and 4th t r i f o l i a t e leaves of Botswana cowpea vari e t y V45~Bots, analysed at various intervals by a n a l y t i c a l sucrose density gradient c e n t r i -fugation Nucleoprotein y i e l d (mg/g. fr. - wt) CP- SBMV CP-TMV Leaf type Leaf agg (days) SI Dl SI Dl Pr imary 20 0 0.24 1.39 1.04 3rd t r i f o l i a t e 20 o c 0.20 0.32 0.30 Pr imary 30 o c 0.20 _d 1.34 4th t r i f o l i a t e 30 o e 0. 12 - 0. 10 aMean o f two e x p e r i m e n t s ^Days s i n c e i n o c u l a t i o n . C o n c e n t r a t e d 2- t o 1 0 - f o l d and scanned a t 254 nm (0.05 a b s o r b a n c e r a n g e ) . dNot tested C o n c e n t r a t e d 2 0 - f o l d and scanned a t 254 nm (0.05 a b s o r b a n c e r a n g e ) . 1 0 0 Table VIII. I n f e c t i v i t y of CP-SBMV in extracts from primary leaves of cowpea var. V45 -Bots, at d i f f e r e n t intervals a f t e r ino-culation with CP-SBMV alone or with CP-TMV Mean number of local lesions per half leaf of GA 21 at various intervals (days) a f t e r inoculation  Inoculum 1 3 6 10 20 Expt. No. 1 Buffer-inoculated extracts CP-SBMV-inoculated extracts CP-SBMV + CP-TMV Expt. No. 2 Buffer-inoculated extracts 0 0 0 0 CP-SBMV-inoculated extracts 0 0 0 71 CP-SBMV + CP-TMV 0 k 22 130 o b 62 273 aNot tested ^No virus detected-101 by the presence of CP-TMV. About f i v e times as much CP-SBMV was obtained from doubly infected 3rd t r i f o l i a t e 1 eaves, compared to that from singly infected leaves (Figure 8 ) . Double infection did not seem to a f f e c t synthe-s i s of CP-TMV, as i t was synthesized to about the same extent in both singly and doubly infected t r i f o l i a t e leaves. Both viruses, in both singly and doubly infected tissues, seemed to be synthesized more in the 1st and 2nd t r i f o l i a t e leaves, which at the time of inoculation were elongating rapidly, than in the 3rd and 4th t r i f o l i a t e leaves which appeared about 7 and 10 days a f t e r inoculation, respectively. However, the influence of CP-TMV on the synthesis of CP-SBMV seemed to be even more pronounced as each virus declined in single infections (Figures 8 and 10). (b) Synthesis in systemically infected 3rd t r i f o l i a t e leaves a f t e r  sequential inoculation of primary leaves. The syntheses of CP-SBMV and CP-TMV a f t e r sequential inoculation of primary leaves were studied in the 3rd t r i f o l i a t e leaves, because i t was in these leaves that the amount of CP-SBMV in doubly infected leaves r e l a t i v e to that in singly infected leaves was the highest,(Figures 8 and 10). When CP-SBMV was inoculated to primary leaves,7 2 hours af t e r i n i t i a l inoculation with CP-TMV, there was greater synthesis of CP-SBMV in the 3rd t r i f o l i a t e leaves than in comparable leaves of plants simultaneously inocu-lated on the primary leaves. The rate of CP-SBMV increase declined as the interval between inoculation was reduced. On the other hand, when CP-SBMV preceded CP-TMV in the inoculated primary leaves by 24 or 72 hours, only 102 Figure 10. Relative amounts of CP-SBMV and CP-TMV nucleoproteins (mg/g f r . wt.) in the 1st, 2nd, 3rd and 4th t r i f o l i a t e , leaves of C a l i f o r n i a blackeye cowpea analysed 10, 15, 20 and 25 days, respectively, a f t e r single and simultaneous double inoculations of primary leaves. The numbers on top of the figure correspond to the f i r s t to fourth t r i f o -1iate leaves. A Dl. CP-•SBMV from doubly infected tissue. A SI. CP-•SBMV from singly infected tissue. B Dl. CP-•TMV from doubly infected tissue. B SI. CP-•TMV from singly infected tissue. 1st 2nd 3rd 4th 1st 2nd 3rd .4th I I I I I I I I | Days af t e r inoculation 104 about twice as much CP-SBMV was synthesized in doubly infected t i s s u e , as in singly infected tissue. More CP-SBMV was synthesized in the t r i f o l i a t e leaves a f t e r simultaneous inoculation with both viruses than when CP-SBMV preceded CP-TMV in the inoculated primary leaves (Table IX). On the other hand, CP-TMV synthesis in the 3 r d t r i f o l i a t e leaf did not seem to be appreciably affected by double inf e c t i o n or by the sequence of inoculation of the primary leaves. In a l l doubly infected 3 r d t r i f o l i a t e leaves CP-SBMV was less than 50% of the:total nucleoprotein produced in such tissue. (c) Electron miscroscopy of singly and simultaneously inoculated  primary leaves and systemically infected t r i f o l i a t e leaves. When thin sections were examined with an electron microscope, both CP-SBMV-like p a r t i c l e s and CP-TMV p a r t i c l e s were.observed in the same veinal and parenchyma c e l l s of simultaneously doubly inoculated primary leaves and of systemically infected t r i f o l i a t e leaves (Figure 1 1 ) . Occasionally, a few c e l l s of doubly inoculated primary leaves and systemically infected t r i f o l i a t e leaves contained only CP-SBMV-like p a r t i c l e s and CP-TMV p a r t i c l e s separately. The CP-SBMV c r y s t a l s reported to occur in cowpea c e l l s infected by this virus (Weintraub and Ragetli, 1 9 7 0 ) were, s u r p r i s i n g l y , not found in singly or doubly infected plants in the present study, even when a delibe-rate attempt was made to search for them at the same sampling time, a f t e r inoculation, as used by these workers. This is interesting because the Table IX. Concentration of CP-SBMV and of CP-TMV in 3rd t r i f o l i a t e leaves (undifferentiated at time of inoculation) of C a l i f o r n i a blackeye cowpea af t e r single and sequential inoculations of primary leaves Concentration (mg/g f r . wt) of CP-SBMV and CP-TMV in I nocu1um tissue harvested 20 days a f t e r inoculation.  Inital Challenge CP-SBMV-Slb CP-SBMV-DIC CP-TMV-Sl b CP-TMV-D|C 0. k 2.02 0 CP-TMV CP-SBMV 1 .Ok 1 .80 2k CP-TMV CP-SBMV 1.17 1.91 12 CP-TMV CP-SBMV 1.86 2.19 2k CP-SBMV CP-TMV 0.87 2.08 12 CP-SBMV CP-TMV 0.85 1.95 Mean of 5 experiments. From tissue singly infected by CP-SBMV or CP-TMV. From doubly infected tissue. Interva1 between inoculat ions (hr)  106 Figure 11. Electron micrographs of c e l l s of primary and t r i f o l i a t e leaves of C a l i f o r n i a blackeye cowpea doubly infected by CP-SBMV and CP-TMV. Tissue was sectioned 10 days aft e r inoculation. A. Vascular c e l l of primary leaf showing both CP-SBMV-like p a r t i c l e s (S) and CP-TMV rods (T). B. Parenchyma c e l l of t r i f o l i a t e leaf showing both CP-SBMV-l i k e p a r t i c l e s (S) and CP-TMV rods (T). C. Vascular c e l l of t r i f o l i a t e leaf showing both CP-SBMV-l i k e p a r t i c l e s (S) and CP-TMV rods (T). D. Parenchyma c e l l of healthy t r i f o l i a t e l e a f - c o n t r o l . 107 108 109 same CP-SBMV is o l a t e used by Weintraub and Ragetli (1970) was used in this study. The only major diffe r e n c e in the studies reported here is that Hoagland's solution was used instead of s o i l as the growth medium. k. D i s t r i b u t i o n and concentration of CP-SBMV and CP-TMV in t r i f o l i a t e  leaves of Botswana cowpea variety V45-Bots that were not formed  at the time of simultaneous double and single inoculations of the primary leaves. When t r i f o l i a t e leaves of V45 -Bots were tested at a l l stages u n t i l 30 days a f t e r inoculation, no CP-SBMV could be detected by serology, infec-t i v i t y and a n a l y t i c a l sucrose density gradient centrifugation in preparations made from t r i f o l i a t e leaves of plants that were singly inoculated with . CP-SBMV, even when such preparations were concentrated 20-fold by 10% PEG p r e c i p i t a t i o n . A total of 70 plants, singly inoculated with CP-SBMV, were checked by i n f e c t i v i t y and serology. At 10 days a f t e r simultaneous inocu-l a t i o n with CP-SBMV and CP-TMV, less than 25% of the plants were infected by CP-SBMV based on assays of the f i r s t and second t r i f o l i a t e s ; at 20 days a f t e r simultaneous inoculation, assays of the th i r d t r i f o l i a t e s indicated that 75 to 100% of the plants were infected by CP-SBMV. The lower percentage of infection obtained when 1st and 2nd t r i f o l i a t e leaves were tested could mean that CP-SBMV did not move readily into these leaves, but since the same leaves were not re-tested 20 days af t e r inoculation when the 3rd t r i -f o l i a t e leaves were tested, no conclusion can be reached about th e i r virus content. 110 More of each v i r u s , e s p e c i a l l y CP-TMV, was synthesized in the primary leaves of V45 _Bots cowpea than in t r i f o l i a t e leaves (Table V I I ) . The values obtained for CP-TMV in the 3rd and 4 t h t r i f o l i a t e leaves were inor-dinately low for th i s virus (Table VII). Aggregation was suspected during centrifugation through sucrose columns, even though extracts of primary and t r i f o l i a t e leaves were prepared with the same buffer at the same time. However, i n f e c t i v i t y tests with virus preparations that were not c e n t r i -fuged through sucrose columns-, indicated that the primary leaves contained more virus than the t r i f o l i a t e leaves. As in C a l i f o r n i a blackeye cowpea, CP-TMV seems to be synthesized equally in single and double infection of the t r i f o l i a t e leaves. D. D i s t r i b u t i o n and concentration of CP-SBMV and CP-TMV in preformed  3rd t r i f o l i a t e leaves of C a l i f o r n i a blackeye cowpea. 1. A r r i v a l of CP-SBMV and CP-TMV at the 3rd t r i f o l i a t e leaves a f t e r  inoculation of primary leaves. Movement of CP-SBMV and CP-TMV into the 3rd t r i f o l i a t e leaves was studied in several experiments, and i t was found that CP-TMV always arrived at these leaves before CP-SBMV. In two experiments in which the inter-val between inoculation of the primary leaves and removal of the 3rd t r i -f o l i a t e leaves were c l o s e l y timed, CP-TMV arrived at the 3rd t r i f o l i a t e leaves between 62 and 64 hours af t e r inoculation. Sixty two hours was taken as the time required by CP-TMV to reach the 3rd t r i f o l i a t e leaves a f t e r inoculation (Table X). On the other hand, CP-SBMV required 84 hours to 111 a r r i v e at the 3rd t r i f o l i a t e leaves (Table X). Each virus moved at its own rate to the 3rd t r i f o l i a t e leaves, whether in single or double infectio n s , as long as the viruses were not mixed but were simultaneously inoculated separately to opposite half leaves. Preliminary observations had indicated that CP-SBMV was delayed i f i t was simultaneously inoculated in a mixed inoculum containing CP-TMV. The time required for each virus to reach the 3rd t r i f o l i a t e leaves (Table X) was based on data obtained when each virus was separately and simultaneously inoculated to opposite half leaves. Thus CP-TMV arrived at the 3rd t r i f o l i a t e leaf 22 hours before CP-SBMV. 2. Time-course synthesis of CP-SBMV and CP-TMV in preformed 3rd tr i f o l i a t e 1 eaves. Both viruses could be detected by an a l y t i c a l sucrose density gradient centrifugation in both singly and doubly infec-ted tissue extracts 5 days af t e r inoculation. However, in some experi-ments CP-SBMV was too low to be detected 5 days af t e r inoculation. It would appear that both viruses, in single and double infecti o n s , reach the peak of synthesis 10 to 12 days a f t e r inoculation of the primary leaves. The peak synthesis period is then followed by a period of apparent decline (Figure 12). 3. Effect of sequence of a r r i v a l of each virus on the concentration  of the other in the preformed 3rd t r i f o l i a t e leaves a f t e r single and sequential inoculations of the primary leaves. It can be seen (Table XI) that CP-SBMV was synthesized most when CP-TMV preceded i t by 22 hours; the r a t i o of CP-SBMV concentration in doubly 112 Table X. Movement of CP-SBMV "and of CP-TMV into 3rd t r i f o l i a t e leaves of C a l i f o r n i a blackeye cowpea which were formed i ( 0 . 5 - l - 0 cm long) at the time of inoculation Interval between inocu- Number of 3rd t r i f o l i a t e leaves that became la t i o n of primary leaves infected with either CP-SBMV or CP-TMV over and t r i f o l i a t e removal (hr) number of leaves tested CP-SBMV-SI CP-SBMV-DI CP-TMV-SI CP-TMV-DI Experiment 1 48 0/3 -b 0/3 50 0/2 - 0/3 60 0/3 0/3 0/3 0/3 66 0/3 0 /5 2/3 3/3 72 0/3 0/3 2/3 3/3 78 0/3 0/3 - 3/3 84 3/3 3/3 3/3 3/3 86 3/3 3/3 3/3 3/3 88 3/3 3/3 3/3 3/3 Experiment 2 54 - - 0/4 -56 - - 0/4 60 - 0/4 1/4 0/4 62 - 0/4 3/4 3/4 64 - 0/4 4/4 4/4 66 - 0/4 4/4 4/4 68 - 0/4 4/4 4/4 70 - 0/4 4/4 4/4 72 0/4 0/4 - 4/4 74 0/4 0/4 - 4/4 78 0/4 0/4 - 4/4 80 0/4 0/4 - 4/4 82 1/4 2/4 - 4/4 113 Table X. Cont. : Interval between inocu- Number of 3rd t r i f o l i a t e leaves that became la t i o n of primary leaves infected with either CP-SBMV or CP-TMV over and t r i f o l i a t e removal (hr) number of leaves tested  CP-SBMVSI CP-SBMV-DI CP-TMV-SI CP-TMV-DI Experiment 2 8k k/k k/k - k/k 86 k/k k/k - k/k "Inocula in each experiment were applied separately, i.e. CP-SBMV on the right half leaf and CP-TMV on the l e f t half when double ino-culations were made or applied as above and the l e f t half (CP-SBMV) was inoculated with buffer when single inoculations were made. b Not tested. 114 Figure 12. Time-course r e p l i c a t i o n of CP-SBMV and CP-TMV as indicated by t h e i r respective nucleoprotein contents (mg/g. f r . wt.) in preformed 3rd t r i f o l i a t e leaves of C a l i f o r n i a blackeye cowpea af t e r single and simultaneous double inoculations of the primary leaves. A. CP-SBMV. B. CP-TMV. Third t r i f o l i a t e leaves doubly infected ( A A ) Third t r i f o l i a t e leaves singly infected ( O O ) Days a f t e r i nocu1 at ion 116 infected t i s s u e to that in singly infected tissue was 2 . 7 - When simulta-neous inoculations were made with the two viruses on the primary leaves, t h e o r e t i c a l l y , this should have resulted in the a r r i v a l of CP-SBMV 22 hours after CP-TMV had arrived at the 3 r d t r i f o l i a t e leaves. S i m i l a r l y when CP-SBMV was inoculated 22 hours before CP-TMV, t h i s should have resulted in both viruses a r r i v i n g simu1tanous1y at the 3 r d t r i f o l i a t e leaves. When CP-SBMV was inoculated so that i t arrived at the 3 r d t r i f o l i a t e leaves 72 hours before CP-TMV, enhancement was apparent, but to a lesser extent. On the other hand, when either virus arrived at the 3 r d t r i f o l i a t e leaves 72 hours af t e r the other, i t s synthesis was greatly retarded, compared to that reached in singly infected plants (Table XI). Although s l i g h t reductions in CP-TMV are apparent when i t was introduced a f t e r CP-SBMV, on the whole this virus was s l i g h t l y affected, i f i t reached the 3 r d t r i f o l i a t e leaves within 22 hours of CP-SBMV a r r i v a l at these leaves. E. Concentration of CP-SBMV and CP-TMV in the preformed 3 r d t r i f o l i a t e  leaves of C a l i f o r n i a blackeye cowpea synchronously infected by  d i f f e r e n t i a l temperature manipulation. The objective of the experiments reported in this section was to investigate the e f f e c t of synchronous infection system on the interaction between CP-SBMV and CP-TMV and contrast i t with the interaction obtained under asynchronous conditions. The hypothesis was, under synchronous in f e c t i o n , that synthesis of each virus would proceed independently. 1. Effect of synchronous infection on accumulation of virus in the  3 r d t r i f o l i a t e leaves. The i n f e c t i v i t y of CP-SBMV and CP-TMV from t r i f o l i a t e leaves of Table XI. Concentration of CP-SBMV when CP-TMV arrived before (+) and aft e r (-) CP-SBMV and of CP-TMV when CP-SBMV arrived before (+) and a f t e r (-) CP-TMV at the preformed 3rd t r i f o l i a t e leaves of C a l i f o r n i a blackeye cowpea Predicted interval between a r r i v a l of CP-SBMV and CP-TMV at 3rd t r i f o l i a t e s Concentration (mg/g f r . wt) in tissue harvested 15 days of CP-SBMV and afte r i n i t i a l CP-TMV i nocu1 at ion Ratio of DI/SI (hr) CP-SBMV-D|b CP-SBMV-SI CP-TMV-DIC CP-TMV-SI CP-SBMV CP-TMV +72 0.53 0 . 6 4 0 . 6 0.2 +22 2.30 2.50 2.7 0.8 + 12 1.70 2.78 2.0 0.9 0 1.57 0.85 3.00 3.1 1.9 1.0 -12 1.08 3.20 1.3 1.0 -22 1 . 1 4 3.00 1.3 1 . 1 -72 1 .02 3.10 1.2 1.0 Mean of 4 experiments. bConc. of CP-SBMV in relation to a r r i v a l of CP-TMV. CConc. of CP-TMV in relation to a r r i v a l of CP-SBMV. 118 plants that had been inocu1ated "and incubated for 42 hours before they were shifted to the styrofoam chamber was negative for either virus when extracts were assayed on their respective indicator hosts. Thus, incuba-tion of plants for 42 hours a f t e r ino'culation was r o u t i n e l y used, mainly because i t had been found in preliminary experiments that cowpea plants could not survive long periods of cold treatment (10°C). An incubation for 42 hours, before the s h i f t , allowed the viruses to be established in the inoculated primary leaves, long before they could move into the 3rd tr i f o l i ate 1 eaves. Plants were used unexcised, because excised plants (Dawson and Schlegel, 1976c),under the conditions of these experiments, performed poorly and usually did not grow for some time a f t e r excision. I n f e c t i v i t y of tissue harvested at zero time (5 days) was negative for either v i r u s , thus suggesting that the 3rd t r i f o l i a t e leaves were not infected at the time plants were moved from the styrofoam chamber. However, i n f e c t i v i t y of CP-TMV was routinely detected in tissue analysed 8 hours af t e r the s h i f t to the permissive temperature, but that of CP-SBMV was occasionally (in 3 experiments out of 7 successful ones) detected in doubly infected tissue and not at a l l in singly infected tissue (Figures 13 and 14). When CP-SBMV was detected in singly infected tissue at 24 hours, i t s infec-t i v i t y was always lower than that in extracts from doubly infected tissue. The rate of synthesis of each virus increased rapidly between 24 and 96 hours a f t e r the s h i f t to the permissive temperature. These results 119 Figure 13- Relative i n f e c t i v i t y of CP-SBMV in 3rd t r i f o l i a t e leaves of C a l i f o r n i a blackeye cowpea infected synchronously singly by CP-SBMV and doubly by CP-SBMV and CP-TMV at 10 C and shifted to 27 C. I n f e c t i v i t y is expressed as mean number of local lesions per half leaf of GA 21. Zero time is when plants were moved from the styrofoam chamber to the permissive temperature of 27 C. Third t r i f o l i a t e leaves doubly infected expressed as mean number of local lesions ( A A ) Third t r i f o l i a t e leaves singly infected expressed as mean number of local lesions ( O O ) . 120 121 Figure 14. Relative i n f e c t i v i t y of CP-TMV in 3rd t r i f o l i a t e leaves of C a l i f o r n i a blackeye cowpea infected synchronously singly by CP-TMV and doubly by CP-SBMV and CP-TMV at 10°C and shifted to 27 C. I n f e c t i v i t y is expressed as mean number of local lesions per half leaf of N_. glut!nosa. Zero time is when plants were moved from the styrofoam chamber to the permissive temperature of 27 C. Third t r i f o l i a t e leaves doubly infected expressed as mean number of local lesions ( A— ; A ). Third t r i f o l i a t e leaves singly infected expressed as mean number of local lesions ( O O ) . Mean no. of lesions/half leaf x di1. x 10 N> -t" ON CO O M 4=-o o o o o o o o ro 123 (Figures 13 and \k) indicate that both viruses in singly and doubly infec-ted 3rd t r i f o l i a t e tissue replicated synchronously. As before, under asynchronous conditions, CP-TMV was synthesized equally in singly and doubly synchronously infected tissue. Similary, CP-SBMV under synchronous conditions was enhanced in doubly infected t i s s u e , compared to that in singly infected tissue (Figures 13 and 15). It is interesting that under synchronous infe c t i o n more (up to 11 mg/g f r . wt) CP-TMV was synthesized in both singly and doubly infected tissue (Figure 15) than under asynchronous conditions (up to k mg/g f r . wt) (Figure 12). However, the level of synthesis of CP-SBMV did not appear to be altered s i g n i f i c a n t l y by synchronous infection (Figure 15). Neither did the high rate of synthesis of CP-TMV, under synchronous i n f e c t i o n , a f f e c t CP-SBMV synthesis in doubly infected tissue. The size of the t r i f o l i a t e leaf at the time of inoculation of the primary leaves affected the d i s t r i b u t i o n of CP-SBMV to the t r i f o l i a t e leaves. When t r i f o l i a t e leaves were 2.5 to 5-0 cm in length at the time of inoculation, no CP-SBMV could be detected in these leaves despite pro-longed incubation under d i f f e r e n t i a l temperature. However, CP-TMV was able to enter these leaves readily. The optimal length of the 3rd t r i f o -l i a t e leaves for entry of both viruses was 0.5 to 1.5 cm. I I I . Analysis for structural interactions between CP-SBMV and CP-TMV  in C a l i f o r n i a blackeye cowpea. 1. A n a l y t i c a l sucrose density gradient centrifugation. I n f e c t i v i t y of CP-SBMV was associated only with the top f r a c t i o n and 124 Figure 15- Nucleoprotein accumulation of CP-SBMV and CP-TMV in C a l i f o r n i a blackeye cowpea 3rd t r i f o l i a t e leaves synchronously infected by the two viruses together and separately at 10 C and sh i f t e d to 27 C. Zero time is when plants were moved from the styrofoam chamber to the permissive temperature of 27 C. Leaves were harvested at various intervals a f t e r s h i f t to 27 C. Third t r i f o l i a t e leaves doubly infected ( A A ) . Third t r i f o l i a t e leaves singly infected ( O- O ) . A. CP-SBMV nucleoprotein. B. CP-TMV nucleoprotein. 126 that of CP-TMV with only the bottom f r a c t i o n . Further, when fr a c t i o n s between CP-SBMV and CP-TMV bands from a r t i f i c i a l and natural mixtures were assayed on GA 21 and Xanthi, about 2 local lesions were induced on each of these hosts, thus indicating contamination of the middle f r a c t i o n s by CP-SBMV and CP-TMV. The same fra c t i o n s also caused systemic symptoms of CP-SBMV on C a l i f o r n i a blackeye cowpea and of CP-TMV on Bountiful bean and C a l i f o r n i a blackeye cowpea. Surpri s i n g l y , they also caused reddish necrotic local lesions on Pinto bean. This was unexpected since CP-TMV infects Pinto systemically without any symptoms on the inoculated primary leaves, and CP-SBMV alone is not known to infect this host. This unusual inte r -action of CP-SBMV and CP-TMV in Pinto is described in d e t a i l l a t e r . The an a l y t i c a l sucrose density gradient centrifugation technique was not used further in subsequent structural interaction experiments, because i t s re-l i a b i l i t y in separating i n f e c t i v i t y of these viruses from a mixture was doubtfu1. 2. I n f e c t i v i t y n e u t r a l i z a t i o n . The i n f e c t i v i t y of each virus in a r t i f i c i a l and natural mixtures was abolished by treatment with i t s homo-logous antiserum, but not by treatment with heterologous antibody, normal serum or buffer (PBS) (Table XII). This indicated that neither CP-SBMV-RNA nor CP-TMV-RNA was encapsidated in the coat protein of the other v i r u s . However, because of the remote p o s s i b i l i t y that a putative heterologous 1y encapsidated RNA could have lost recognition s i t e s for i t s s p e c i f i c host, C a l i f o r n i a blackeye cowpea, which is susceptible to both viruses, was i n -cluded in the test plants to exclude this p o s s i b i l i t y . However, when the 127 Table XII. Relative amounts of CP-SBMV and CP-TMV on GA 21 and N. glutinosa r e s p e c t i v l y recovered a f t e r n e u t r a l i z a -tion of i n f e c t i v i t y of a r t i f i c i a l and natural mixtures Inocu1um I n f e c t i v i t y of CP-SBMV and CP-TMV afte r n e u t r a l i z a t i o n Treatment C a l i f o r n ia blackeye CP-SBMV/ CP-TMV GA 21 CP-SBMV N. g1ut i nosa CP-TMV A. Natural m ixtures CP-SBMV/CP-TMV CP-SBMV/CP-TMV CP-SBMV/CP-TMV CP-SBMV/CP-TMV CP-TMV-antibody CP-SBMV-antibody Normal serum Bu f f e r d + + + + + + + + 82 c 60 139 6 7 113 B. A r t i f i c i a l mixtures CP-SBMV/CP-TMV CP-SBMV/CP-TMV CP-SBMV/CP-TMV CP-SBMV/CP-TMV CP-TMV-antibody + + CP-SBMV-antibody + + Normal serum + + Buffer + + 117 41 200 12 14 190 a Chlorotic spots on primary leaves for on t r i f o l i a t e leaves for CP-TMV ^ Average no. of local lesions per half •: No local lesions observed. . ^ PBS (phosphate buffered-sa1ine). CP-SBMV or mottling and b l i s t e r s leaf of each host. 128 t r i f o l i a t e leaves of C a l i f o r n i a blackeye cowpea plants were assayed a f t e r inoculation with either virus i n f e c t i v i t y could.only be associated with the inoculum that had been treated with a heterologous antiserum, normal serum or PBS. It can also be seen that CP-TMV was very much affected by normal serum and CP-SBMV antibody, whereas CP-SBMV i n f e c t i v i t y was s l i g h t l y affected by normal serum and CP-TMV antibody (Table XII). This is not surprising as i t has been observed (Rappaport and Siegel, 1955) that a non-specific reaction can occur between TMV and other sera. IV. Interaction of CP-SBMV and CP-TMV in Pinto bean. A r t i f i c i a l mixtures of CP-SBMV and CP-TMV (p u r i f i e d or crude prepara-tions) always induced necrotic local lesions when they were inoculated to primary leaves of Pi;nto. When the local lesions were isolated and ino-culated to Bountiful bean, the causal agent inducing lesions on Pinto could not be recovered from extracts of t r i f o l i a t e s of the inoculated Bountiful; however; CP-TMV could be recovered from such extracts. The f a i l u r e of the local lesion causal agent to go systemic in Bountiful suggests that i t is not the bean s t r a i n of SBMV. Investigations were therefore made to determine which virus was responsible for the induction of local lesions. A. Interaction of CP-SBMV and CP-TMV a f t e r sequential inoculation  of Pinto bean. 1. Inoculation with intact viruses. When local lesions were counted, 5 to 7 days af t e r the last inoculation, t h e i r numbers varied with the concen-r tr a c t i o n of CP-SBMV in the inoculum that was cha11enge-inocu1ated (Table XIII). 129 Table XIII. Relationship between number of local lesions and varying concentrations of either CP-SBMV or CP-TMV challenge-inoculated on Pinto primary leaves pre-inoculated with a constant amount of either virus 72 hours e a r l i e r I n i t i a l Inocu1um Concentrat ion (yg/ml) Cha11enge Concentrat ion (jjg/ml) Mean no. of local lesions per half l e a f 3 CP-TMV 50 CP-SBMV 50 75 CP-TMV 50 CP-SBMV 5 27 CP-TMV 50 CP-SBMV 0.5 16 Buffer CP-SBMV 50 0 CP-SBMV 50 CP-TMV 50 k CP-SBMV 50 CP-TMV 5 0 CP-SBMV 50 CP-TMV 0.5 0 Buffer CP-TMV 50 0 Mean of two experiments 130 The primary leaves, previously inoculated with a constant concentration of CP-SBMV, developed only a few or no local lesions when cha11enge-inocu-lated with varying concentrations of CP-TMV (Table XIII). No local lesions developed when CP-SBMV or CP-TMV-inocu1ated leaves were inoculated with phosphate buffer only. Figure 16A shows the type of local lesions induced on Pinto leaves simultaneously doubly inoculated with CP-SBMV and CP-TMV. Leaves singly inoculated by either virus alone did not develop local lesions (Figure 16B and C). These results strongly suggest that the necrotic local lesions were induced by CP-SBMV in the presence of CP-TMV. 2. Inoculation with CP-SBMV-RNA and intact CP-TMV. When Pinto primary leaves were preinocu1ated with a constant amount of CP-SBMV-RNA and then cha11enge-inocu1ated with CP-TMV at various i n t e r v a l s , the number of local lesions that formed decreased sharply as the interval from preino-culation increased (Table XIV). However, on leaves preinocu1ated with intact CP-TMV (0.05 mg/ml), when they were challenge-inoculated 2b hours later with CP-SBMV-RNA, the local lesions that developed were about the same in number as those induced when intact CP-TMV was inoculated simul-taneously (0 hr) with the same amount of CP-SBMV-RNA (Table XIV). No local lesions were induced a f t e r RNase treatment of an inoculum containing CP-SBMV-RNA and CP-TMV, or either virus alone (Table XIV). Inocula were treated with 50_jjg/ml of pancreatic RNase and incubated for at least, 30 minutes (room temperature) before inoculation. 3. Starch lesions. A p o s s i b i l i t y existed that CP-TMV could have induced micro-1esions i n v i s i b l e to the naked eye. To test this p o s s i b i l i t y , Figure 16. Primary leaves of Pinto singly and doubly inoculated with intact CP-SBMV and intact CP-TMV. A. Primary leaf inoculated with CP-SBMV and CP-TMV B. Primary leaf inoculated with CP-TMV C. Primary leaf inoculated with CP-SBMV. 132 Table XIV. Ef f e c t of pre-inocu1 at ion with CP-TMV on i n f e c t i v i t y of CP-SBMV-RNA (purified) on Pinto primary leaves inoculated simultaneously and sequent ial ly Mean no. of local lesions produced per half leaf of Pinto by CP-SBMV aft e r challen-I n i t i a l 1 noc. Cha11enge i noc. ge inoculation at various intervals (hr) 0.0 6.0 2k k8 CP-SBMV-RNA CP-TMVb 36.0 2.0 0.0 0.0 CP-SBMV-RNA buffer 0.0 0.0 0.0 0.0 CP-SBMV-RNA + intact CP-TMV + RNAse 0.0 0.0 0.0 0.0 CP-SBMV-RNA RNase 0.0 0.0 0.0 0.0 CP-SBMVb CP-TMV 68.5 NDC ND ND CP-TMV CP-SBMV-RNA ND ND 30.0 ND CP-TMV + buffer 0.0 0.0 0.0 0.0 Local lesions read 4 - 5 days af t e r the last challenge inoculation. 0.05 mg/ml of either intact CP-SBMV or intact CP-TMV used. Not done 1 3 3 Pinto primary leaves were simultaneously inoculated with intact CP-SBMV and intact CP-TMV or singly with either v i r u s . Six days af t e r inoculation the primary leaves were harvested and stored overnight at k°Z in the dark to allow removal of excess starch. The leaves were then tested for starch by bleaching them at 80°C in 70% ethanol. The starch lesions were developed by soaking the leaves in a potassium-1 a c t i c acid mixture (1:20) (Lindner et a l , 1959) fo r , at least, 15 minutes. Leaves were examined for starch lesions using a dissecting microscope with a low power objective. The only lesions that could be seen were those on the primary leaves that had been simultaneously inoculated wi:th both viruses. B. Time-course synthesis of CP-SBMV in tissue also inoculated with  CP-TMV. I n f e c t i v i t y of CP-SBMV, as indicated by the number of local lesions, increased with time from inoculation to ]bb hours a f t e r inoculation (Figure 17). Simi1arly,CP-SBMV synthesis increased with time when extracts prepared from Pinto primary leaves, previously doubly inoculated with CP-SBMV-RNA and intact CP-TMV, were assayed on GA 21 (Figure 18). No increase in CP-SBMV t i t r e was apparent when extracts prepared from Pinto primary leaves that had pre-viously been singly inoculated with either intact CP-SBMV or CP-SBMV-RNA were assayed on GA 21 (Figures 17 and 18). However, there was some infec-t i v i t y in extracts of tissue that had been singly inoculated with either intact CP-SBMV or i t s RNA, thus suggesting that t h i s virus could have infected Pinto subliminally. 134 Figure 17- Time-course r e p l i c a t i o n of CP-SBMV in Pinto primary leaves singly and doubly inoculated with intact CP-SBMV and intact CP-TMV, as shown by r e l a t i v e i n f e c t i v i t y of Pinto primary leaf extracts harvested at various intervals after inoculation and assayed on GA 21. Extracts of doubly inoculated leaves ( A-Extracts of singly inoculated leaves ( O O ). 136 Figure 18. Time-course r e p l i c a t i o n of CP-SBMV in Pinto primary leaves singly and doubly inoculated with CP-SBMV-RNA and intact CP-TMV, as shown by r e l a t i v e i n f e c t i v i t y of Pinto primary leaf extracts harvested at various intervals a f t e r inocula-tion and assayed on GA 21. Extracts of doubly inoculated leaves ( A-Extracts of singly inoculated leaves ( O •A ). O ). 2k 48 72 96 120 144 156 Hours after inoculation 138 C. Confirmation of CP-SBMV ,in local lesions of Pinto primary leaves. 1. Serological assay. Sap from doubly infected tissue reacted with CP-SBMV antiserum as well as with CP-TMV antiserum (Figure 19). P u r i f i e d CP-SBMV, but not sap from Pinto primary leaves singly inoculated with CP-SBMV, reacted with CP-SBMV antiserum. P u r i f i e d CP-TMV and sap from Pinto primary leaves singly inoculated with CP-TMV reacted with CP-TMV antiserum. Anti-serum against CP-SBMV or normal serum did not react with crude or p u r i f i e d preparations of CP-TMV. These results indicate that CP-SBMV was able to re p l i c a t e in the presence of CP-TMV in Pinto primary lea f , to a level detec-table by this method. Residual inoculum cannot account for the results because p r e c i p i t i n bands should have developed between the wells containing the a n t i -serum against CP-SBMV and those containing sap from primary leaves previously inoculated singly with this v i r u s . 2. Electron microscopy. (a) Thin sections. CP-SBMV v i r i o n s could not be detected by electron microscopy in sections of local lesions. This does not, however, invalidate the serological evidence and the time-course synthesis r e s u l t s , which show that CP-SBMV did multiply in Pinto. The f a i l u r e to detect CP-SBMV v i r i o n s by electron microscopy could be eit h e r due to the preponderance of CP-TMV p a r t i c l e s which were synthesized and thus masked the presence of the sphe-r i c a l CP-SBMV or to the inherent d i f f i c u l t i e s of proper f i x a t i o n in necro-t i c tissue. Indeed others (Weintraub and Ragetli, 1964) f a i l e d to detect TMV p a r t i c l e s in necrotic lesions on N. glutinosa. 139 Figure 19- Immunodiffusion reactions of crude sap from Pinto primary leaves singly and doubly inoculated with CP-SBMV and CP-TMV and of p u r i f i e d (control) CP-SBMV and CP-TMV. Gels were made with 0.75% Noble agar (w/v) in PBS plus 0.02% NaN . Pr e c i p i -t i n bands were photographed 72 hours a f t e r incu-bation at room temperature. Antisera wel1s. D i l u t i o n S-A — CP-SBMV 1/128 T-A -- CP-TMV 1/128 N-S -- Normal serum 1/128 Antigen, wells (outside). 1. Crude sap from primary leaves of Pinto singly inoculated with CP-SBMV. 2. Crude sap from primary leaves of Pinto doubly inoculated with CP-SBMV and CP-TMV. 3 . Crude sap from primary leaves of Pinto singly inoculated with CP-TMV. b. P u r i f i e d ( 0 . 2 5 mg/ml) CP-TMV -- con t r o l . 5 . P u r i f i e d ( 0 . 1 0 mg/ml) CP-SBMV -- control. 6. Crude sap from primary leaves of Pinto doubly inoculated with CP-SBMV and CP-TMV (same as 2 above). 141 (b) P u r i f i e d v i r u s . When the CP-SBMV-antibody p r e c i p i t i n band was cut and mounted on electron microscope grids and examined, a few p a r t i c l e s which resembled CP-SBMV were observed (Figure 20). D. Influence of Cornell i s o l a t e of CP-TMV on CP-SBMV infe c t i o n of Pinto. When CP-SBMV and CP-TMV (Cornell isolate) were simultaneously inocula-ted to Pinto primary leaves, necrotic local lesions, s i m i l a r to those obtained with a mixture of CP-SBMV and CP-TMV (V.R.S. i s o l a t e ) , developed. No local lesions developed on leaves singly inoculated with CP-SBMV or CP-TMV (Cornell isolate) (Figure 2 1 ) . Contrary to the report (Bruening et a l , 1976) that the Cornell i s o l a t e of CP-TMV induces local lesions on Pinto, no local lesions were observed in several t r i a l s by the writer when the l a t t e r was inoculated with CP-TMV (Cornell i s o l a t e ) . V. Seed transmission of CP-SBMV and CP-TMV in cowpea. A. Direct planting of seed produced on C a l i f o r n i a blackeye cowpea  plants singly and doubly infected by CP-SBMV and CP-TMV. 1. Freshly harvested vine-dry mature seed. CP-SBMV, but no CP-TMV, was transmitted to seedlings derived from planted seeds produced on C a l i f o r -nia blackeye cowpea plants that were infected singly by CP-SBMV or doubly by the two viruses. CP-SBMV was transmitted up to 38% (x = 13.5%) in seed-lings from singly infected plants and up to 35% (x = 7-6%) in seeds produced on doubly infected plants (Table XV). CP-TMV was never transmitted through planted seeds of cowpea. When seedlings were infected by CP-SBMV, symptoms 142 Figure 20. Electron micrographs of CP-SBMV-like p a r t i c l e s (arrows) p u r i f i e d from Pinto primary leaf lesions. The p u r i f i e d virus suspension was subjected to immuno-d i f f u s i o n and the p r e c i p i t i n band was crushed on an electron microscope grid for observation. 143 Figure 21. Pinto primary leaves inoculated singly with intact CP-TMV (Cornell isolate) and doubly with intact CP-SBMV and intact CP-TMV (Cornell i s o l a t e ) . A. Primary leaf inoculated with CP-TMV (Cornell isolate) B. Primary leaf inoculated with CP-SBMV and CP-TMV (Cornell i s o l a t e ) . 1 4 4 Table XV. Transmission of CP-SBMV and CP-TMV from seed produced by singly or doubly infected C a l i f o r n i a blackeye cowpea Total number of infected seed 1ings/tota 1 number of germi-nated seed 1 i ngs 3  CP-SBMV CP-TMV Seed source No. Percent range Mean Percent No. Percent range Mean Percent Healthy seeds 0 / 4 4 4 ' 0 0 0 / 4 4 4 0 0 Singly infected 7 1 / 5 2 8 0 - 3 8 1 3 . 5 0 / 4 8 0 0 0 Doubly infected 4 5 / 5 9 4 0 - 3 5 7 . 6 0 / 5 9 4 0 0 Total number of seedlings derived from d i f f e r e n t seed lots produced at d i f f e r e n t times. 145 o f v e i n c l e a r i n g always appeared f i r s t on the 1st and 2nd t r i f o l i a t e l e a v e s , but no symptoms were o b s e r v e d on t h e p r i m a r y l e a v e s . 2. E f f e c t o f seed s t o r a g e d u r a t i o n on v i r u s t r a n s m i s s i o n . Seeds were sown from t h e same seed l o t a f t e r s t o r a g e a t room t e m p e r a t u r e (25°) f o r v a r i o u s l e n g t h s o f time a f t e r h a r v e s t i n g . S t o r a g e up t o 18 months d i d not s i g n i f i c a n t l y a f f e c t seed t r a n s m i s s i b i 1 i t y o f CP-SBMV i n t h e s e e d l i n g s • ( T a b l e X V I ) . 3. E f f e c t o f seed m o t t l e on t r a n s m i s s i o n o f CP-SBMV and CP-TMV  th r o u g h p l a n t e d seed. When normal and m o t t l e d seeds were sown, CP-SBMV was t r a n s m i t t e d t o 4.5 and ~l,k% o f the s e e d l i n g s , r e s p e c t i v e l y ( T a b l e XVI I ) . A p p a r e n t l y , seed c o a t c o l o u r d i d not a f f e c t seed t r a n s m i s s i o n o f CP-SBMV, because i f t h i s was so one would have e x p e c t e d h i g h e r seed t r a n s m i s s i o n l e v e l s from seed d e r i v e d ;from d o u b l y i n f e c t e d p l a n t s than from t h o s e d e r i v e d from plants'. s i ngl:y i n f e c t e d by. CP-SBMV. B. D i r e c t p l a n t i n g o f seeds from s i n g l y and d o u b l y i n f e c t e d  Botswana cowpea v a r i e t i e s . N e i t h e r CP-SBMV nor CP-TMV was t r a n s m i t t e d t h r o u g h seeds o f t h e 3 Botswana v a r i e t i e s ( T a b l e XVII I) and n e i t h e r v i r u s was d e t e c t e d by i n f e c t i v i t y o r s e r o l o g i c a l a s s a y s o f l e a f e x t r a c t s made from s e e d l i n g s produced by p l a n t e d seeds d e r i v e d from i n o c u l a t e d p l a n t s . S i n c e fewer seeds were used i n t h e s e a s s a y s , compared w i t h the a s s a y s o f C a l i f o r n i a b l a c k -eye c o w p e a . i t i s l i k e l y t h a t CP-SBMV c o u l d be t r a n s m i t t e d i n Botswana b l a c k e y e and V26-Bots cowpea, but p r o b a b l y t o a l e s s e r e x t e n t than in t h e C a l i f o r n i a b l a c k e y e cowpea seed. 146 Table XVI. Effect of storage duration of seed on seed transmission of CP-SBMV to seedlings of C a l i f o r n i a blackeye cowpea 3 Number of infected Age of seed seedlings/out of Percent Seed source (months) number germinated 'Infect ion Healthy seeds 3 0/10 0 8 0/25 0 18 0/25 0 CP-SBMV-SIb 3 1/10 10 8 0/24 0 18 8/86 9-3 CP-SBMV-DIC 3 2/10 20 8 1/26 4 18 0/24 0 A l l seeds were from the same seed lot and were stored in p l a s t i c bags at laboratory room temperature. bSeed derived from plants singly infected with CP-SBMV. CSeed derived from plants doubly infected with CP-SBMV and CP-TMV. 147 T a b l e X V I I . E f f e c t o f seed m o t t l e on t r a n s m i s s i o n o f CP-SBMV and CP-TMV t h r o u g h p l a n t e d seeds of C a l i f o r n i a b l a c k e y e cowpea t h a t were s i n g l y (SI) and d o u b l y (Dl) i n f e c t e d P r o p o r t i o n o f s e e d l i n g s i n f e c t e d Seed c o a t No. I n f e c t e d / P e r c e n t Seed s o u r c e c o l o u r no. assayed t r a n s m i ss ion H e a l t h y seed Normal 0/90 0. .0 M o t t l e d a CP-SBMV-SI Norma 1 19/96 19. .8 M o t t l e d -CP-SBMV-DI Norma 1 3/67 4. • 5 M o t t l e d 10/135 7. ,4 CP-TMV-SI Normal 0/51 0. .0 M o t t l e d 0/59 0. , 0 CP-TMV-DI Norma 1 0/67 0. .0 M o t t l e d 0/135 0. .0 No seed was m o t t l e d 148 Table XVIII. Seed transmission of CP-SBMV and CP-TMV in planted seed of three Botswana local cowpea v a r i e t i e s Total number of seedlings infected/total of Seed source Variety seedlings germinated  CP-SBMV CP-TMV No. %' No. % Heal, thy Blackeye 0/20a 0 0 0 s . b Blackeye 0/47 0 c -D l d Blackeye 0/11 0 0/1 1 0 SI V26-Bots 0/116 0 - -Dl V26-Bots 0/38 0 0/38 0 SI V45-Bots - - 0/28 0 - a Al1 seedl ings were also tested by serology. Singly infected plants. Not tested - seed unavailable. Doubly infected plants. 149 C. D i s t r i b u t i o n of CP-SBMV and CP-TMV in seed parts. Evidence of seed-borne transmission of CP-SBMV made i t necessary that d i s t r i b u t i o n of both viruses be investigated in d i f f e r e n t seed parts. 1. D i s t r i b u t i o n of CP-SBMV and CP-TMV in seed parts of d i f f e r e n t  developmental stages of seed of C a l i f r o n i a blackeye cowpea  d i r e c t l y assayed on the respective indicator hosts. When seed of C a l i f o r n i a blackeye cowpea was harvested at d i f f e r e n t developmental stages a f t e r flowering, CP-SBMV was recovered from extracts of pooled embryos of a l l stages of seed maturity produced on doubly infec-ted plants,but not from extracts of those produced on plants singly infec-ted by CP-SBMV. No CP-TMV was recovered from any of the embryos. Both viruses could be recovered from a l l seed coats of a l l stages (Table XIX). The results of Table XIX are based on one experiment which employed a total of only 5 seeds for each assay, so the data may not be representative of a larger population of seeds. More extensive experiments were performed with embryos of d i f f e r e n t stages of development, but instead of u t i l i z i n g pooled seed parts the seed parts were assayed i n d i v i d u a l l y . However, because in pooled embryo experi-ments CP-SBMV i n f e c t i v i t y could be recovered from embryos of doubly infec-ted plants, i t was necessary to test the e f f i c a c y of the tap water wash decontamination procedure. Healthy embryos were a r t i f i c i a l l y contaminated with 10 jjg/ml of CP-SBMV. The contaminated embryos were then decontamina-ted by washing in tap water for 30 minutes and 24 hours or by soaking in 150 Table XIX. D i s t r i b u t i o n of CP-SBMV and CP-TMV in pooled seed parts of C a l i f o r n i a blackeye cowpea of d i f f e r e n t development stages of maturity whose extracts were d i r e c t l y assayed on indicator hosts a f t e r decontamination by an 8-hour tap water wash I nocu1um Seed part° Age of Pgds (days) Mean number of local lesion per half leaf of:  GA 21 CP-SBMV N^. gl ut inosa CP-TMV SI SI Dl Dl embryo seed coat embryo seed coat 15-20 15-20 15-20 15-20 0 229 2 138 N/As N/A 0 72 SI SI Dl Dl embryo seed coat embryo seed coat 45-50 9 45-50 45-50 45-50 0 27 3 102 0 65 0 0 Healthy Heal thy Heal thy Heal thy embryo seed coat embryo seed coat 15-20 15-20 45-50 45-50 0 0 0 0 0 0 0 0 Each seed parts were pooled into either 5 embryos or 5 seed coats and ground together. 3Age in days since flowering. 'Seeds produced on plants singly infected by either CP-SBMV or CP-TMV. Green immature seed. Vine-dry mature seed. 'Not ava i1able. Seeds produced on plants doubly infected by CP-SBMV and CP-TMV 151 5% Na^PO^ for 10 minutes, followed by rinsing in tap water for 8 to 12 hours. Extracts from a r t i f i c i a l l y contaminated embryos that had been decontaminated by washing for 30 minutes were infecti o u s , but not those decontaminated for 2k hours or in StNa^PO^ (Table XX). I n i t i a l l y lO^Na^PO^ was used, but i t was abandoned af t e r i t was found that i t affected germination of embryos. Thus a l l experiments that follow used either 2k hour tap water wash or 5% Na^PO^. The radicle-plumule shoots were separated from cotyledons with flamed tweezers or sharp forceps. When extracts of individual whole embryos or radicle-plumule shoots and seed coats were assayed, CP-SBMV was transmitted to 10.3% of r a d i c l e -plumule shoots of green immature embryos (Table XXI), 6.0% of dough stage immature whole embryos (Table XXII) and 18.0% of radicle-plumule shoots of vine-dry mature embryos (Table XXIII). Neither CP-SBMV nor CP-TMV was transmitted by artificially-contaminated-decontaminated embryos. Further, CP-TMV was not detected in embryos of naturally infected seeds that were decontaminated. When embryos were shaken in d i s t i l l e d water before decon-tamination, i n f e c t i v i t y for either virus could be recovered from such washings when assayed,but not from washings made aft e r decontamination. I n f e c t i v i t y of either virus in the seed coat was not eliminated by any of the decontamination procedures, except that at times Na^PO^ reduced the i r i n f e c t i v i t y . When 10% Na^PO^ was used in one experiment ' (Tab!e XX I, experiment no.l) CP-SBMV i n f e c t i v i t y was not recovered. In a single experiment, when i n f e c t i v i t y of extracts was determined after cotyledons and radicle-plumule shoots were separated, i t was found 152 Table XX. E f f i c a c y of decontaminating CP-SBMV and CP-TMV from a r t i f i c i a l l y -contaminated embryos with tap water and 5% Na PO, a f t e r which the i r extracts were assayed d i r e c t l y on either GA 21 or N. glut inosa Decontamination Proportion of i n f e c t i v e embryos  Inoculum procedure No. infected/ Mean no. assayed lesions Percent Healthy embryos CP-SBMV-contaminated CP-SBMV-contami nated CP-SBMV-contaminated CP-SBMV-contam i nated CP-SBMV-contam i nated CP-SBMV standard (10 jug/ml) CP-TMV- b contam i nated CP-TMV-contam i nated CP-TMV standard (10 jig/ml) unwashed unwashed 30 min.-wash 2k hr.-wash 5% Na PO^ + 12 hr-wash untreated 5% Na PO^ + 12 hr-wash untreated 0/10 10/10 6/20 0/20 0/50 10/10 0/40 3/10 0 k 127 100 30 100 30 146 Mean number of local lesions per half leaf of either GA 21 or N. g1ut i nosa, 'Seed soaked in virus (lOjug/ml) Table XXI. D i s t r i b u t i o n of CP-SBMV and CP-TMV In 15~20 day o l d 3 i n f e c t e d and a r t i f i c i a l l y contaminated greeen immature seed p a r t s of s i n g l y i n o c u l a t e d C a l i f o r n i a b l a c k e y e cowpea which were d i r e c t l y assayed i n d i v i d u a l l y on i n d i c a t o r h o s t s a f t e r d econtamination w i t h 5% and \0% Na.PO. f o r 10 and 30 minutes, r e s p e c t i v e l y P r o p o r t ion h a l f l e a f of i n f e c t e d seeds and o f : number of l o c a l l e s i o n s per GA 21 N. g l u t i nosa N a t u r a l i n f e c t i o n (N) or a r t i f i c i a l c o n t a m i n a t i o n (A) CP-SBMV CP-TMV Exper iment number Seed part T r e a t e d (T) or u n t reated(U) Range** Mean c No. o f seeds i n f e c t e d / no. assayed Range Mean No. of seeds i n f e c t e d / no. assayed Expt. no. 1 With 10% Healthy N embryo 0' embryo T T 0 0 0 0 0/10 0/52 0 0 0 0 0/10 0/52 N embryo U 1-195 65 20/20 1-4 2 11/20 A embryo T 0 0 0/52 0 0 0/52 N Seed coat T 15-267 107 10/10 2-208 63 10/10 Expt. no. 2 With 5% Heal thy r a d i c l e - p l u m u l e T 0 0 0/10 0 0 0/10 N r a d i c l e - p l u m u l e T 1-2 2 6/57 0 0 0/52 N rad i c l e - p l u m u l e U 5-159 10 28/52 0-28 U 12/52 T a b l e XXI. c o n t i n u e d : Exper iment number N a t u r a l i n f e c t i o n (N) or a r t i f i c i a l contami nat ion P r o p o r t i o n of i n f e c t e d seeds and number of l o c a l l e s i o n s per h a l f l e a f o f : GA 21 CP-SBMV (A) Seed p a r t T r e a t e d (T) or t  u n t r e a t e d (U) Range Mean No. of seeds i n f e c t e d / no. assayed N^ . g 1 ut i nosa CP-TMV Range Mean No. of seeds i n f e c t e d / no. assayed Expt. no. 2 w i t h 5% A rad i c l e - p l u m u l e T 0 0 0/52 0 0 0/52 N seed coat T 23-2*i5 120 10/10 3^-101 93 10/10 N seed coat U 30-300 166 10/10 3-109 32 10/10 St a n d a r d (10 ug/ml) 33-198 10/10 136-168 156 10/10 S i n c e f l o w e r i n g . 'Local l e s i o n range. L o c a l l e s i o n mean, 'whole embryo Table XXII. Distribution of CP-SBMV and CP-TMV in 30-35 day- o l d 3 infected and a r t i f i c i a l l y contaminated immature (dough stage) seed parts of C a l i f o r n i a blackeye cowpea d i r e c t l y assayed i n d i v i d u a l l y on indicator hosts a f t e r decontamination with 5% Na-P0, for 10 minutes Proportion of infected seeds and number of local lesions Natural per half leaf of: .  infec t i o n (N) GA 21 R. -g 1 lit innsa  or a r t i f i - Treated,!. (T) CP-SBMV-SI CP-TMV-SIL ~ c i a l conta- or No. of seeds No. of seeds mination (A) Seed part Untreated (U) , infected/ no. infected/no. Range Mean assayed Range Mean assayed N embryo 6 T 1-2 2 3/5D 0 0 0/50 N embryo U 3-260 61 48/48 1-1 1 2/50 "A embryo T 0 0 0/50 0 0 0/40 A embryo U 1-11 4 10/10 0 0 0/40 N seed coat T 1-8 3 9/10 3-34 15 7/10 N seed coat U 24-402 182 10/10 5-116 46 10/10 N embryo washings U 82-170 119 19-59 44 Standa rd (10 ug/ml) ;• 67-216 127 42-274 146 Since flowering. Singly infected Local l e s i o n ; range Local lesion mean Whole embryo Washings obtained by shaking 5 embryos in 2.5 ml of phosphate buffer before decontamination. Table XXIII. Di s t r i b u t i o n of CP-SBMV and CP-TMV in naturally infected (N) and a r t i f i c i a l l y contaminated (A) seed parts of vine dry mature seed of C a l i f o r n i a blackeye cowpea whose seed parts extracts were d i r e c t l y assayed i n d i v i d u a l l y on indica-tor hosts a f t e r decontamination with 5% Na,P0^ for 10 minutes followed by 12 hour tap water wash Proportion of infected seed parts and mean number of local lesions per half leaf of:  GA 21 N. glutinosa Natural(N) Treated(T) CP-SBMV - s i b CP-TMV- s . b or or No. infected/ No. i nfected/ A r t i f i c i a l (A) Seed part untreated(U) Range 3 Mean no. assayed Range Mean noc assayed N R-P6 T 1-5 2 9/50 • 0 0 0/50 N R-P U 1-15 4 10/13 1-2 2 4/20 A R-P T 0 0 0/50 0 0 0/50 A R-P U 3-30 13 10/10 1-1 1 4/10 N s . c f T 2-8 5 3/10 3-182 86 10/10 N s . c U 2-60 14 10/10 5-471 120 10/10 Standard (lOjug/m 11) 76-189 129 1 12-248 180 CP-SBMV - D l 9 CP-TMV - D l 9 No. infected/ No. i nfected/ Range Mean no. assayed Range Mean no. assayed N E h T 1-11 4 2/64 0 0 0/64 N E U 1-1 1 2/64 0 0 0/64 N R-P T 1-1 1 2/40 0 0 0/40 N S.C T 1-1 1 1/10 1-10 3 8/10 Table XXI I I. Continued: Proportion of infected seed parts and mean number of local lesions per half leaf of: GA 21 N. glutinosa Natural (N) Treated CP-SBMV-DI9 CP-TMV-DI9 or or No. infected/ No. infected/ A r t i f i c i a l ( A ) Seed part untreated Range , Mean no. assayed Range Mean no. assayed N S.C U 2-22 9 9/10 3-23 13 10/10 Seeds harvested k5 to 55 days since flowering. 'Seeds produced on plants singly infected by CP-SBMV or CP-TMV. Range of local lesions per half leaf. 'Mean local lesions per half leaf. Rad icle-plumule. Seed coat. 'Doubly infected with CP-SBMV and CP-TMV 'whole embryo. 158 that CP-SBMV was transmitted equally by cotyledons as by radicle-plumule shoots and the virus was transmitted to about the same extent in planted seed (Table XXIV). Although the t i t r e of each virus was always higher in the seed coats than in the embryos, i t is not possible to state c a t e g o r i c a l l y which stage of seed coat development contained more v i r u s , because d i f f e r e n t assay plants were used at d i f f e r e n t times of assay. 2. D i s t r i b u t i o n of CP-SBMV and CP-TMV in seed parts of Botswana  cowpea v a r i e t i e s . When embryos of seeds produced on Botswana cowpea v a r i e t i e s , previously inoculated with CP-SBMV and CP-TMV, were assayed, neither virus could be recovered from the embryos of v a r i e t i e s tested. However, each virus was recovered from the seed coats of v a r i e t i e s tested, except that CP-SBMV could not be recovered from the seed coats of seed derived from doubly inoculated V45~Bots plants (Table XXV). 3. D i s t r i b u t i o n of CP-SBMV and CP-TMV in planted embryos of C a l i f o r n i a  blackeye cowpea. When embryos from seeds produced on singly or doubly infected plants were decontaminated by a 2k hour tap wateriwash or in 5% Na^PO^ and then planted, CP - S B M V was transmitted to the seedlings. No virus was transmitted to the seedlings derived from healthy embryos that had been a r t i f i c i a l l y contaminated and decontaminated. In embryos derived from natu r a l l y infected seeds and in those that had been a r t i f i c i a l l y contaminated and planted without decontamination, 1.€P'TS'BMV> .. was' , however, transmitted to the seedlings (Tables XXVI and XXVII). In each table seeds from the same seed lot were used and, for comparison with Table XXIV. D i s t r i b u t i o n of CP-SBMV and CP-TMV in seed parts of singly infected (SI) vine dry mature seed of C a l i f o r n i a blackeye cowpea whose extracts were d i r e c t l y assayed i n d i v i d u a l l y on indicator hosts a f t e r decontamination by a 24 ghour tap water wash and transmission by planted intact seed Seed part Proport ion induced by of infected seeds and mean number of local lesions seed part. ; extracts per half leaf of: Q/\ 21 glutinosa CP-SBMV-SI CP-TMV-SI" Meanc No. no. i nfected/ assayed Percent Meanc No. infected/ Percent no. assayed Coty d 1 1/26 4 0 0/22 0 R-P6 2 1/26 4 0 0/22 0 Planted i ntact seed 1/49 2 0 0/32 0 aSeeds and embryos from the same seed lot that had been stored in p l a s t i c bags for 2 weeks aft e r harvest. Singly infected. Mean number of local lesions per h a l f . l e a f . Cotyledons without radicle-plumule shoot. Radicle-plumule shoot without cotyledons. Table XXV. Distribution of CP-SBMV and CP-TMV in seed parts of Botswana cowpea v a r i e t i e s singly (SI) and doubly (Dl) inoculated with the two vi ruses Seed part Treated (T) Proportion of transmi ss ion V i rus Sou ree Var iety or untreated (U) No. infected/ no. assayed Percent CP-SBMV SI Blackeye s . c a U 10/10(100)B 100 CP-SBMV SI B1ackeye R-PC T d 0/20 ( 0) 0 CP-SBMV SI V26-Bots s . c . U 20/20( 5 3 ) 100 CP-SBMV SI V26-Bots R-P T 0/20( 0) 0 CP-TMV SI V26-Bots S.C U 10/10(150) 100 CP-SBMV D l 6 V45-Bots S.C U 0/20( 0) 0 CP-TMV Dl V45-Bots S.C u 10/20( 2) 50 CP-TMV Dl 4 5-Bots R-P T 0/20( 0) 0 a" Seed coat ^Number in parenthesis refers to number of local les ions per ha 1f leaf CRad i c l e -plumule shoot ^Treated Doubly with 5% NajPO^ inoculated plants not checked for double infection by both v i ruses. 161 Table XXVI. Transmission of CP-SBMV in embryos of naturally infected seeds and a r t i f i c i a l l y contaminated embryos of C a l i f o r n i a blackeye cowpea planted a f t e r decontamination with deter-gent followed by 24 hour tap water wash Treatment Natural i nfect ion(N) or a r t i f i c i a l contaminat ion(A) Washed (W) No. or unwashed(U) no. Proportion of seedlings infected i nfected/ assayed Percent Embryos Embryos Embryos Soaked intact seed Dry intact seed Embryo Dry natura 1 1y infected intact seed Heal thy A a Av N U U W U W 0/10 1/11 0/40 20/20 12/24 7/24 8 / 3 4 0 . 0 9 - 1 0 . 0 1 0 0 . 0 5 0 - 0 2 9 . 2 2 3 . 5 Embryos separated from seed coats and then soaked in 10^|ig/ml of virus for 1 0 - 1 5 minutes. Intact (dry) seed soaked d i r e c t l y in 1 0 ug/ml of virus for 8 hours and then planted. Intact (dry) seed soaked overnight in d i s t i l l e d water and then soaked intact in 1 0 lig/ml of virus and then planted. 162 Table XXVII. Transmission CP-SBMV and CP-TMV in embryos of natur a l l y infected seeds and in a r t i f i c i a l l y contaminated embryos of C a l i f o r n i a blackeye cowpea planted a f t e r decontamination in 5% Na_P0^ followed by rinsing in running tap water for 8 hours and the i r transmission in planted naturally infected intact seed Natural i nfect ion(N) or a r t i f i -Proportion of i nfected seed 1i ngs Treatment c i a l conta- Treated (T) No. infected/ V i rus Source mination (A) untreated (U) no. assayed Percent CP-SBMV s i a N T 7 / 5 0 1 4 , 0 CP-SBMV SI N U 8 / 4 8 1 6 . 7 CP-SBMV A b T 0 / 5 0 0 . 0 CP-SBMV A U 8 / 4 9 1 6 . 3 CP-TMV SI N T 0 / 5 0 0 . 0 CP-TMV SI N U 5 / 4 9 1 0 . 2 0 CP-TMV A T 0 / 4 5 0 . 0 CP-TMV A U 0 / 5 0 0 . 0 CP-SBMV DI C N T 3 / 4 0 7 . 5 CP-SBMV Dl N planted intact seedd 1 3 / 2 0 2 6 . 4 CP-TMV Dl N T 0 / 4 0 0 . 0 CP-TMV Heal thy embryo Dl N planted intact seed U 0 / 2 0 2 0 / 2 0 0 . 0 0 . 0 Seeds produced on singly infected plants ^Healthy embryos a r t i f i c i a l l y contaminated with either CP-SBMV or CP-TMV (10 >jg/ml) c d Seeds produced on doubly infected plants. Naturally infected seeds that were planted untreated. 163 transmission of decontaminated planted embryos, data on intact planted seeds are included in each table. There is a good agreement between trans-mission of CP-SBMV in seedlings derived from planted embryos and that in seedlings derived from planted intact seed (Tables XXVI and XXVIl). 4 . Effect of age of C a l i f o r n i a blackeye cowpea seedling on suscepti- b i l i t y to either CP-SBMV or CP-TMV. The fact that CP-SBMV, but not CP-TMV, was consistently seed-borne in C a l i f o r n i a blackeye cowpea did not rule out the p o s s i b i l i t y that seedlings could be refractory to CP-TMV infection at some stage of germination and early growth. Healthy embryos and seeds were sown in steam-sterilized sand. Embryos were then inoculated with either virus ( I 0 j j g / m l ; no c e l i t e ) on the cotyle-don or radicle-plumule by rubbing with Q-tips, just before planting or 5 days af t e r planting. Whether either virus was introduced before germination or 5 days a f t e r planting, both viruses readily infected the young seedlings (Table XXVIII). Therefore seedling age cannot explain the d i f f e r e n t i a l transmission of the viruses from seeds of infected plants. Because of the reports (Schneider and Worley, 1 9 5 9 ; Roberts and Price, 1 9 6 7 ) that SBMV can enter and infect apparently uninjured c e l l s of bean, it was necessary to test t h i s . Suspensions of CP-SBMV and CP-TMV (lOjjg/ml) were delivered by pipette on the primary leaves of 4 to 6 day-old seedlings without touching the seedlings. In one experiment in which the two viruses were separately applied to seedlings CP-SBMV infected one of 14 seedlings tested. In another experiment in which mixed inoculum of both viruses was 164 T a b l e X X V I I I . T r a n s m i s s i o n o f CP-SBMV and CP-TMV when i n o c u l a t e d t o h e a l t h y c o t y l e d o n s and o r r a d i c l e - p l u m u l e s h o o t s o f C a l i f o r n i a b l a c k e y e cowpea a t d i f f e r e n t s t a g e s a f t e r sowi ng Inocu1um Ages a t : i noc. (days) C o t y l e d o n s o r r a d i c l e - p l u m u l e P r o p o r t i o n i n f e c t e d by No. o f p l a n t s e i t h e r v i r u s . % CP-SBMV a 0 coty1edons 2/20 10 CP-SBMV 0 rad i cle-p1umu1e 4/20 20 CP-SBMV 5 b c o t y l e d o n s 1/10 10 CP-TMV 0 c o t y l e d o n s 4/20 20 CP-TMV 0 rad i cle-p1umu1e 3/20 15 CP-TMV 5 coty1edons 6/10 60 B u f f e r 0 coty1edons 0/20 0 3 1 n o c u 1 a t e d ^ F i v e days j u s t b e f o r e p l a n t i n g , a f t e r p l a n t i n g and 2 days a f t e r emergence. 165 applied on primary leaves, CP-SBMV infected 2 of 53 tested seedlings. CP-TMV did not infect any of the seedlings. The nature of symptoms produ-ced, however, was d i f f e r e n t from that produced by seedlings derived from natur a l l y infected seed. Naturally infected seedlings never showed symptoms on the primary leaf whereas seedlings inoculated by the drop method showed c h l o r o t i c spot symptoms on the primary leaves. In contrast to natural infect i o n s , where systemic symptoms are c l e a r l y obvious on the f i r s t t r i f o -l i a t e to form 10 to 12 days af t e r sowing, symptoms on seedlings inoculated by the drop method developed systemic symptoms of mosaic and mottling on the 1st and 2nd t r i f o l i a t e leaves 18 days af t e r inoculation. Clearly entry of CP-SBMV through apparently uninjured c e l l s does not seem to account for its seed-borne nature. 5. Effect of healthy seed extract on infection of CP-SBMV and CP-TMV on GA 21 and H_. glutinosa respectively. The low level of local lesions obtained when naturally infected and a r t i f i c i a l l y contaminated embryos were assayed for CP-SBMV or CP-TMV promp-ted the suspicion that embryo extracts may contain an i n h i b i t o r ( s ) . When this was investigated, i t was found that the greater the concentration of the embryo extract used to d i l u t e either virus to 10 ^ug/ml the greater the degree of i n h i b i t i o n . I n f e c t i v i t y of CP-SBMV was reduced by 45 and 25% when undiluted and 1/10 d i l u t i o n extracts were used, respectively, and CP-TMV i n f e c t i v i t y was reduced by 96 and 69%, respectively, by sim i l a r extracts (Table XX.I.X). 166 Table XXIX. Ef f e c t of an extract of healthy seed of C a l i f o r n i a blackeye cowpea on i n f e c t i v i t y of CP-SBMV and CP-TMV on GA 21 and N. g1ut i nosa, respectively Mean number of local lesions per half leaf of: GA 21 H_. glutinosa CP-SBMV CP-TMV Di l u t i o n Mean Percent Mean Percent treatment lesions reduction lesions reduction Standard 3 227 b 0.0 196 b 0.0 1/100 178 c 21.6 164 C 16.3 1/10 171 C 24.7 6 l d 68.9 und i1uted 124d 45.4 7 e 96.4 3Standard was made of 10^jg/ml of eit h e r virus in phosphate buffer, pH 7.1 bed G Values in each column not followed by the same l e t t e r vary s i g n i f i -cantly at P = 0.05 by DMR test. 167 D. Effect of germination o'n survival and i n f e c t i v i t y of virus on seed  of C a l i f o r n i a blackeye cowpea. This study was carried out because i t was suspected that perhaps CP-TMV is not seed-borne because of i t s inactivation during germination. When naturally infected seeds were sown in sand, i n f e c t i v i t y of CP-SBMV in germi-nated seed coats was reduced or eliminated compared to that of ungerminated seed coats (Figure 22). No s i g n i f i c a n t difference was apparent between CP-TMV extracts from germinated and ungerminated seed (Table XXX). The reason for the low i n f e c t i v i t y of CP-SBMV in seed coats of germi-nated seeds is not clear. 168 Figure 22. Relative i n f e c t i v i t y of CP-SBMV in seed coat extracts of germinated and ungerminated seeds derived from C a l i f o r n i a blackeye cowpea plants, singly infected by CP-SBMV. Seed coats used in th i s experiment were not washed before assay. I n f e c t i v i t y is indicated by local lesions on primary leaf of GA 21 cowpea. Each assay was made up of 5 pooled seed coats (one seed coat/0.25 ml of phosphate buffer pH 7.1). A. Inoculated with seed coat extracts of ungerminated seeds. B. Inoculated with seed coat extracts of germinated seeds. 1 6 9 Table XXX. Ef f e c t of germination on i n f e c t i v i t y and survival of CP-SBMV and CP-TMV in the seed coats of seed of C a l i f o r n i a blackeye cowpea Treatment Mean number of local lesions per half leaf of: N. glutinosa GA 21 CP-SBMV Mean 1es ions Percent reduct ion CP-TMV Mean 1es ions Percent reduct ion Ungerminated Germinated 51 0 . 0 9 2 . 2 klC 38C 0 . 0 9 - 5 A l l seed coats were washed overnight in running tap water af t e r separat ion. 'Percent reduction expressed r e l a t i v e to lesions induced by ungerminated seed coats. '^Values in each column not followed by the same l e t t e r vary s i g n i f i c a n t l y at P = 0 . 0 5 by Student-t-distribut ion. 170 DISCUSSION In the present studies evidence has been presented to show that the r e p l i c a t i o n of CP-SBMV is greatly enhanced by that of CP-TMV in two cowpea v a r i e t i e s . This influence of CP-TMV on CP-SBMV synthesis was e f f e c t i v e , in C a l i f o r n i a blackeye cowpea, under both asynchronous and synchronous condi-tions of in f e c t i o n . Evidence also indicates that CP-TMV infection conditions Pinto bean to become susceptible to CP-SBMV. Evidence also indicates that double infection and consequent enhancement of one virus in a mixture by the other does not necessarily result in interaction of the i r structural components, even i f the i r v i r i o n s are present in the same c e l l . These studies also present evidence, which suggests that seed-borne nature of CP-SBMV is very l i k e l y a consequence of embryo infe c t i o n rather than surface contamination of seedlings by the testae during germination. I. Interaction in cowpea. A. Effects on plant growth. The fact that double infection did not reduce plant growth in C a l i -f o r n i a blackeye cowpea any more than CP-TMV alone,and that the systemic symptoms, produced on t r i f o l i a t e leaves of such plants, resembled those of CP-TMV more than those of CP-SBMV alone, suggest that the e f f e c t s of double infection were additive. The results also suggest that virus concentration may not necessarily be correlated with severity of symptoms. Others (Dodds and Hamilton, 1972; Kuhn and Dawson, 1973) made si m i l a r observations. 171 The i s o l a t e of CP-SBMV used in these studies could have been a milder s t r a i n than the one used by Kuhn and Dawson (1973). This is '(indicated by the i n a b i l i t y of our i s o l a t e to cause any s i g n i f i c a n t reductions in plant growth and y i e l d components in C a l i f o r n i a blackeye cowpea, whereas that used by Kuhn and Dawson caused s i g n i f i c a n t plant growth reductions of 18 to 32%. B. D i s t r i b u t i o n and concentration of viruses. It is not clear why each of the viruses was synthesized less in doubly infected primary leaves than singly infected s i m i l a r leaves of C a l i f o r n i a blackeye cowpea and yet CP-SBMV was greatly enhanced in the t r i f o l i a t e leaves. It is u n l i k e l y that CP-SBMV and CP-TMV in the inoculated primary leaf of cowpea were competing for the same i n f e c t i b l e s i t e s (centres), because syn-thesis of each v i r u s , e s p e c i a l l y of CP-SBMV, was retarded more as the inter-val between pre-inocu1 at ion by one virus and cha11enge-inocu1 at ion by the other increased. In other studies where competition for i n f e c t i b l e s i t e s was implicated, i t was found that the number of soybean mosaic virus local lesions on bean was very much reduced when i t was inoculated simultaneously with bean pod mottle v i r u s , and reduction was greater as the interval between inoculation decreased (Lee and Ross, 1972b). It may be that in the inoculated leaves of C a l i f o r n i a blackeye cowpea, CP-SBMV and CP-TMV may be competing for some component, such as ribosomes, which may be needed for t r a n s l a t i o n of the messenger RNAs for their r e p l i -case or coat protein. Replicase may be one of the proteins synthesized early in infection (Hunter et a l , 1976; Siegel et a l , 1978). However, 172 unless age is also a factor, overloading of the t r a n s l a t i o n a l system is in-congruent with the fact that doubly infected t r i f o l i a t e leaves synthesized each of the viruses e f f i c i e n t l y and that the concentration of CP-SBMV in double inf e c t i o n was greatly enhanced. Another pl a u s i b l e theory could be that since the two viruses are introduced by mechanical (manual) inoculation, which is a very i n e f f i c i e n t way of introducing v i r u s , not many c e l l s are i n i t i a l l y infected and also mechanical injury may induce some sort of resistance of virus c e l l - t o - c e l l spread and synthesis. If one remembers that challenge-inoculation of primary leaves was superimposed d i r e c t l y on pre-inoculated areas of the same leaf , then extra wounding of the same leaf and p r o b a b i l i t y of excluding the second virus even becomes greater. However, simultaneously inoculated viruses were in one mixture. Sl i g h t increase in the y i e l d of PVX in tobacco leaves doubly inocu-lated with PVX and PVY have been observed (Rochow and Ross, 1955; Stouffer and Ross, 1961b; Close, 1964; Damirdagh and Ross, 1967) and these have been attributed partly to age (Stouffer and Ross, 196lb). Lack of enhancement of TMV in leaves 1 and 2 of barley, doubly inoculated with BSMV and TMV, was attributed to age of the leaves (Dodds 1972). In the primary leaves of V45 _Bots, which is apparently systemically resistant to CP-SBMV sing1e i n f e c t i o n , CP-SBMV t i t r e was greater in doubly inoculated leaves than in singly inoculated ones. CP-SBMV t i t r e in the ino-culated primary leaves was about the same as that in the systemically infected t r i f o l i a t e leaves. Although the concentration of CP-TMV was s l i g h t l y greater 173 in singly inoculated than doubly -inocu1ated primary leaves, i t was greater in the inoculated leaves than in the systemically infected t r i f o l i a t e leaves. However, the results of \A5-B0ts were based only on two experiments, and they may not j u s t i f y any conclusion about the r e p l i c a t i o n of CP-TMV. What is c l e a r , is that systemic invasion of V45~Bots by CP-SBMV is dependent on infection of th i s host by CP-TMV. For marked enhancement of PVX by PVY, i t has been suggested (Stouffer and Ross, 1961b) that PVX is enhanced more by PVY, only i f the l a t t e r enters the leaf through the vascular system. This explanation f i t s the CP-SBMV/ CP-TMV interaction in C a l i f o r n i a blackeye cowpea, but i t does not seem te-nable for the same pair of viruses in V45 -Bots. The syntheses of CP-SBMV and CP-TMV were greatest in rapidly elongating 1st and 2nd t r i f o l i a t e l e a f l e t s of C a l i f o r n i a blackeye cowpea. These results indicate that the synthesis of both viruses is greatest when leaves are in-vaded when elongating and expanding rapidly. Similar results were obtained for CP-SBMV and CCMV by Kuhn and Dawson ( 1 9 7 3 ) . The results of sequential inoculation suggest that for CP-SBMV to be greatly enhanced in the 3 r d t r i f o l i a t e leaves that were not formed at ino-c u l a t i o n , CP-TMV inoculation must precede CP-SBMV inoculation, but they do not indicate which virus a r r i v e s f i r s t at these 3 r d t r i f o l i a t e leaves. It can also be seen that for CP-SBMV to be enhanced, CP-TMV must either be inoculated simultaneously with CP-SBMV on the primary leaf, or CP-TMV must precede CP-SBMV into the preformed 3 r d t r i f o l i a t e leaf. It is therefore 174 suggested that for enhancement of CP-SBMV to occur in the t r i f o l i a t e 1 eaves, CP-TMV must be inoculated 12 to 2 2 hours before or 12 to 2 2 hours a f t e r CP-SBMV. Perhaps infection of CP-TMV, within a r e l a t i v e l y short space of time of infection by CP-SBMV, represses CP-SBMV-specific in h i b i t o r y subs-tance(s). Such an i n h i b i t o r of CP-SBMV r e p l i c a t i o n may be produced or activated as soon as the host c e l l s are infected by CP-SBMV. The i n h i b i -tor could be an inherent mechanism in the host genotype to protect i t against CP-SBMV infe c t i o n . The i n h i b i t o r may not necessarily be coded for by CP-SBMV. For the e f f e c t of CP-TMV, to be maximally e f f e c t i v e , i t must be introduced, shortly before or a f t e r , or simultaneously with CP-SBMV, so that the host system is no longer able to produce t h i s substance against CP-SBMV infection or synthesis. When CP-SBMV infection precedes that of CP-TMV by 7 2 hours, the influence of CP-TMV on counteracting such a subs-tance is reduced, and CP-SBMV synthesis is enhanced only s l i g h t l y . Simi-l a r l y when CP-TMV infection precedes that of CP-SBMV by a wide margin ( 7 2 hr), i t s e f f e c t in n u l l i f y i n g the i n h i b i t o r of CP-SBMV is no longer e f f e c t i v e , as the host, by that time, may have developed non-specific re-sistance against i n f e c t i o n . It is therefore suggested that for CP-TMV to influence r e p l i c a t i o n of CP-SBMV (i) the two viruses must be introduced into the leaf within short time of each other, so that CP-TMV can n u l l i f y the mechanism by which CP-SBMV synthesis is controlled and ( i i ) that enhancement is a result of predisposition of c e l l s by the helper virus or i t s by-product which allows c e l l - t o - c e l l movement and translocation of the dependent vir u s . 175 This proposal does not take physical presence of CP-TMV or, for that matter, i t s rapid synthesis into account, except that perhaps CP-TMV must continue to repress production of such a substance. Indeed, the e f f e c t of CP-TMV may be translocated in the host without necessarily being accompanied by CP-TMV v i r i o n s . The results of synchronous infe c t i o n also support the theory that the magnitude of CP-TMV synthesis may not be a factor in the enhancement of CP-SBMV, for CP-TMV was greatly synthesized and yet CP-SBMV was replicated to about the same extent as under asynchronous conditions. Additional support for the thesis that physical presence of CP-TMV does not seem to be a factor in CP-SBMV enhancement is that both viruses occurred in the same c e l l s of doubly infected primary leaves and t r i f o l i a t e leaves. Both viruses could be found in the same c e l l of primary leaves in which CP-SBMV was reduced by the presence of CP-TMV. Hamilton and Nichols (1977) could find no proof that physical presence of BMV was reponsible for enhanced synthesis of TMV in barley. The main influence of CP-TMV, on CP-SBMV infection and i t s systemic invasion of t r i f o l i a t e leaves of V45 _Bots, could be predisposing the c e l l s by either removal of inhibitory substances or enabling increased CP-SBMV synthesis in the inoculated leaves. This increased synthesis may lead to movement of th i s virus from c e l l - t o - c e l l and eventually into the vascular system, wherefrom i t can be translocated into the t r i f o l i a t e leaves. The observation that i t was d i f f i c u l t to detect CP-SBMV in 1st and 2nd t r i f o -l i a t e leaves of doubly infected V45~Bots plants may indicate that move-ment of CP-SBMV may be partly dependent on metabolite translocation, which should have been p r e f e r e n t i a l l y routed to the newly expanding 3rd 176 and subsequent t r i f o l i a t e leaves.' This is perhaps analogous to BSMV/TMV interaction in barley (Dodds, 1972; Dodds and Hamilton, 1972). But to at t r i b u t e that to metabolite transport only, is not congruent with synthe-s i s of CP-SBMV and CP-TMV in the 1st and 2nd t r i f o l i a t e s of C a l i f o r n i a blackeye cowpea, where they were synthesized more than in the later-formed 1 eaves. The influence of CP-TMV on CP-SBMV systemic invasion of V45 -Bots is sim i l a r to the influence of BSMV or BMV on systemic spread and enhancement of TMV in barley (Hamilton and Dodds, 1970; Dodds, 1972; Dodds and Hamilton, 1972; Hamilton and Nichols, 1977). The two systems d i f f e r , however, in that in some cases (Hamilton and Dodds, 1970; Dodds, 1972; Dodds and Hamilton, 1972), TMV could be detected in concentrated extracts of barley singly ino-culated with TMV, whereas no CP-SBMV could be detected by i n f e c t i v i t y or an a l y t i c a l sucrose density gradient centrifugation in extracts of singly infected t r i f o l i a t e 1 eaves that were concentrated 20-fold. If CP-SBMV invades this host systemically when inoculated alone then, i t must be synthesized to a level below that which could be detected by the methods employed in this study. The two systems also d i f f e r in that whereas the influence of BSMV or BMV on TMV in barley was dependent on high temperature, that of CP-TMV on CP-SBMV in V45 _Bots was e f f i c i e n t under normal temperature regimes, suitable for plant growth. The influence of CP-TMV on systemic invasion of V45~Bots by CP-SBMV is si m i l a r to the e f f e c t of Anthocyanos i s virus on conditioning cotton plants to become systemically invaded by the Br a z i l i a n tobacco streak virus (Costa, 1969)-177 The present studies have u t i l i z e d the synchronous d i f f e r e n t i a l tempe-rature system (Dawson and Schlegel, 1973), for the f i r s t time, in studying mixed virus infections and i t s application is promising. The system can be extended to include sequential inoculations and there is no doubt i t can be used with less problems i f cold-tolerant plants, such as barley, can be used as virus hosts. Another synchronous system has recently been used to study mixed virus infections (Otsuki and Takebe, 1976, 1978; Barker and Harrison, 1977a, 1977b). C. In vivo structural interactions. The present studies f a i l e d to reveal any evidence of genomic masking between CP-TMV and CP-SBMV. However, this is not surprising when one con-siders that only two cases of in vivo genomic masking have been reported between s t r u c t u r a l l y d i s s i m i l a r plant viruses (Dodds and Hamilton, 1971, 1974; Dodds, 1974; Peterson and Brakke, 1973). Others (Morris, 1970; Goodman and Ross, 1974c; Hamilton and Nichols, 1977) could not detect such in vivo structural interactions. In the present studies, i t is not known why structural interactions between CP-SBMV and CP-TMV did not occur. One can only postulate, even then without much cer t a i n t y , since our knowledge of what happens in the mil l i e u of the infected c e l l is meagre. It was stated in the section dealing with the l i t e r a t u r e review that some of the conditions that must be met before interactions between pools of v i r a l components can occur are that the homologous s p e c i f i c i t y between v i r a l RNA and coat protein be broken down and that assembly s i t e s of the viruses more or less coincide. It is 178 also necessary that there be temporal coincidence in the l i f e cycles of the viruses for interaction to occur. Although the present studies show that both CP-SBMV and CP-TMV were present in the same c e l l , there is no proof that both replicated in that c e l l . Assembly s i t e s of viruses, including those of TMV, whose l i f e cycle has been extensively studied, are s t i l l in doubt. The nucleus (Langenberg and Schlegel, 1969), chloroplasts (Singer, 1972), mitochondria (Jackson et a l , 1971), and cytoplasmic membranes (Ni1sson-Ti11gren et a l , 1974) have a l l been implicated in the synthesis and assembly of TMV. However, chloroplasts have been discounted (Shalla et a l , 1975), as s i t e s for TMV synthesis, because the intrap 1astidia 1 p a r t i c l e s present in the these orga-nelles could have been previously mistaken for TMV v i r i o n s . The most plau-s i b l e s i t e s for TMV r e p l i c a t i o n , and perhaps assembly, are mitochondria (Jackson et a l , 1971) because the r e p l i c a t i v e forms, believed to be inter-mediates in the virus l i f e cycle, were found to sediment with these organelles. However, recent evidence (Ni1sson-Ti11gren et a l , 1974) discounts mitochon-dria and implicates cytoplasmic membranes as s i t e s of TMV synthesis, because they were associated with TMV ds-RNA. Studies on r e p l i c a t i o n and or s i t e s of assembly of SBMV are lacking. The only evidence a v a i l a b l e for SBMV is that in which v i r u s - l i k e p a r t i c l e s of the cowpea and bean str a i n s of SBMV were found in the nucleus and cytoplasm (Weintraub and Ragetli, 1970). In additon, accumulation of v i r u s - l i k e p a r t i c l e s of the bean s t r a i n were found in the phloem; i t was also implied that these may be s i t e s for r e p l i -cation. It is d i f f i c u l t to speculate whether CP-SBMV and CP-TMV assembled 179 at common s i t e s . If they did not assemble at common s i t e s , then this could be one reason why the i r s tructural components did not interact. It is l i k e l y that the viruses did not assemble at the same s i t e s . Electron micrographs show that in some c e l l s , CP-SBMV, was enclosed by membranes whose s i g n i f i -cance is not clea r . Further, they could have had common r e p l i c a t i o n and assembly s i t e s but the s p e c i f i c i t y of the RNA-protein interaction precluded such interaction. Indeed RNA-protein s p e c i f i c i t y was found to be high even between TMV st r a i n s (Atabekova et a l , 1 9 7 5 ) . Time-course synthesis studies of CP-SBMV and CP-TMV in primary and t r i f o l i a t e leaves of C a l i f o r n i a blackeye cowpea indicated that CP-TMV synthesis was always more rapid than that of CP-SBMV. T h e o r e t i c a l l y one would assume that i f the kinetics of r e p l i c a -tion for the two viruses, as measured by i n f e c t i v i t y and spectrophometry, represent what happens inside the c e l l , then the accelerated r e p l i c a t i o n of CP-TMV would have l e f t protein subunits in excess of that needed to en-capsidate homologous RNA, thus furnishing subunits that would encapsidate CP-SBMV-RNA. The r a p i d i t y at which BSMV may be synthesized in barley is given (Dodds, 1 9 7 4 ; Dodds and Hamilton, 1974) as one p o s s i b i l i t y that may have favoured TMV-RNA genomic masking by excess BSMV protein, in the absence of homologous s p e c i f i c i t y . In the present studies, i t is also possible that genomic masking of RNA of virus by the coat protein of the other occurred, but because of the i n s t a b i l i t y of the reassembly product, attack by c e l l u l a r RNase made their detection, by i n f e c t i v i t y , d i f f i c u l t . Also because of non-specific reactions 180 between v i r a l antigen and antibodies as well as normal serum, detection of a low percentage of heterologously encapsidated product would be d i f f i -c u l t . I I. Interaction in Pinto. The present studies also show that CP-SBMV can infect and multiply to a detectable level in the inoculated leaves of Pinto bean, a host otherwi regarded as immune (Shepherd and Fulton, 1962; Shepherd, 1971). It is not clear why Pinto is infected by CP-SBMV in the presence of CP-TMV. However, this type of s i t u a t i o n does not seem to be novel and unique; others (Tochiha 1959; Weathers, 196l;Pound et a l , 1962) reported incidents, where a virus not known to infect a host, did so in the presence of another. The extent of i n t e r a c t i o n , by these viruses was not characterized further. The simplest explanation of how CP-TMV influences CP-SBMV infection of Pinto could be that a f t e r the Pinto leaf c e l l s are infected by CP-TMV, they become pre-disposed to CP-SBMV infe c t i o n . As hypothesized for enhan-cement of CP-SBMV, in cowpea, i t could be that CP-TMV infection n u l l i f i e s or represses production of substance(s) that would normally act against infection by CP-SBMV. Another p o s s i b l i t y could be that CP-SBMV v i r i o n s or RNA molecules are adsorbed to CP-TMV p a r t i c l e s and enter the c e l l s in that manner during inoculation. P a r t i c l e s of raspberyy ringspot and tobacco r a t t l e viruses have been observed to form in vivo and in v i t r o aggregates (Barker and Harrison, 1977b). However, this does not seem tenable for two reasons: (i) mere physical a t t r a c t i o n at level of the v i r i o n would not 181 necessarily mean that after uncoating, the host genotype would recognize and accept CP-SBMV-RNA template and ( i i ) because CP-SBMV-RNA, challenge-inoculated 2k hours af t e r pre-inoculation with CP-TMV, s t i l l induced local lesions of the same magnitude as in a simultaneous inoculation with CP-TMV. When CP-TMV dissociated protein was mixed with intact CP-SBMV and inocula-ted to Pinto, no local lesions developed (Molefe, unpublished observation). A thi r d p o s s i b i l i t y , less favoured, could be that CP-TMV infection stimulates the host c e l l native RNA-dependent RNA polymerase which then becomes a v a i l a b l e to the CP-SBMV-RNA template. However, unless such stimulated replicase is determined by the host-virus genotype there would be an infection of hosts otherwise regarded as immune, so long as the "incompetent" virus is accompanied by a competent v i r u s . This is not the case. This hypothesis is a t t r a c t i v e , however, because of recent reports (Ikegami and Fraenkel-Conrat, 1978; Romaine and Z a i t l i n , 1978; White and Dawson, 1978) that soluble RNA polymerases are present in healthy c e l l s and are stimu-lated by in f e c t i o n . However, the strength of th i s hypothesis is weakened by the report (White and Dawson, 1978) that the a c t i v i t y of these poly-merases may be stimulated by mock-inocu1 at ion in cowpea and tobacco leaves, thus suggesting that the increase in enzyme a c t i v i t y is not exclu-s i v e l y v i r u s - s p e c i f i c . However, modification of the a c t i v i t y of such poly-merases may occur in the interaction of the host and virus genomes, that i s , the dependent virus must possess some genes with the potential to infect such a host. It may be that host enzyme, as suggested else-182 where (Romaine and Z a i t l i n , 1978), " i s the 'core' polymerase which lacks t r a n s c r i p t i o n a l c o n t r o l , and through i t s association with other virus-coded and host-coded proteins on the membrane i t forms the fully-competent r e p l i -case". The suggestion that a dependent virus might have the .potential to infect an immune host during a mixed infection is not u n l i k e l y , in the case of SBMV, because two of i t s r e l a t i v e s have been reported to infect bean, when isolated from cowpea (Lamptey, 1972) and.'infect., cowpea-.when i solated from bean (Yerkes and Patino, I960) in single i n f e c t i o n . Thus in the case of CP-SBMV one would assume that the potential to infect bean e x i s t s , but for some reason the genome of th i s v irus is unable to stimulate the v i r u s -s p e c i f i c replicase to a s u f f i c i e n t level to d i r e c t the synthesis of v i r a l ' RNA . III. Seed transmission. The present studies have also presented evidence which suggests that CP-SBMV and not CP-TMV is consistently seed-borne in cowpea. Transmission of CP-SBMV through planted seed of cowpea confirms previous results (Shepherd and Fulton, 1962; Gay, 1973; Kuhn and Dawson, 1973). It is doubtful that contamination of the embryo or developing seedling with virus from the seed coats would account for such consistency in the seed t r a n s m i s s i b i 1 i t y of CP-SBMV, and yet not result in CP-TMV seed transmission. There was a good agreement between seed transmission of CP-SBMV through planted seeds and planted embryos. The f a i l u r e to recover CP-SBMV consistently from embryo extracts, d i r e c t l y assayed on GA 21, could be attributed to the apparent 183 presence of i n h i b i t o r ( s ) of infection in the seed of cowpea that may int e r f e r e with virus at the infection s i t e s . The presence of this i n h i b i -tor (s) may have been the factor responsible for the f a i l u r e of others to recover infectious SBMV from either immature embryos (McDonald, 1971; McDonald and Hamilton, 1972) or embryos of dry mature seed (Cheo, 1955; McDonald and Hamilton, 1972) of bean infected with the bean s t r a i n of SBMV, and embryos of dry mature seed (Lamptey, 1972; Lamptey and Hamilton, 1974) of C a l i f o r n i a blackeye cowpea infected with the Ghana s t r a i n of SBMV. Indeed an i n h i b i t o r of infection which reduced a c t i v i t y of SBMV has been reported in bean seed extract (Cheo, 1955), and Crowley (1955) reported others which reduced i n f e c t i v i t y of cucumber mosaic virus and TMV in cucumber and tobacco seeds,respectively. The presence of i n h i b i t o r s in seed extracts has been implicated in lower seed transmission percentages obtained with embryo extracts assayed d i r e c t l y on indicator plants than with seedling assay of bean common mosaic virus (Schippers, 1963) and of squash mosaic (Powell and Schlegel, 1970). Negative results of d i r e c t assay of embryo extracts on indicator hosts could also be due to too low a concentration of the virus in the embryo. SBMV has been reported to be seed-borne (Zaumeyer and Harter, 19^3; Uyemoto and Grogan, 1977) and embryonica11y-transmitted (Uyemoto and Grogan, 1977) in bean. Recent results at V.R.S., using sero-log i c a l 1 y-spec i f i c electron microscopy (SSEM), indicate evidence of v i r u s - l i k e p a r t i c l e s in washed embryos of bean, var. Early G a l l a t i n , infected with SBMV, and i n f e c t i v i t y was n i l when p a r t i c l e s observed were few (Hamilton and Nichols, unpublished r e s u l t s ) . 184 The best evidence of CP-SBMV infection of embryos was the transmi-ssion of the virus to seedlings by decontaminated embryos. If surface contamination or fortuitous entrance of CP-SBMV into uininjured embryos was the cause of i t s seed-borne nature, then a r t i f i c i a l l y contaminated-decontaminated embryos should have transmitted the vi r u s ; they did not. Additional evidence for embryo infection by CP-SBMV comes from the results which indicate that i n f e c t i v i t y of CP-SBMV in the seed coats was reduced by germination of seed. These results suggest that, i f the source of CP-SBMV transmitted through planted seed is the seed coat, then the embryo or developing seedlin must be infected (contaminated) at or before germinat ion. It is not clear why CP-SBMV was not transmitted through planted seed or embryo extracts of two Botswana local cowpea v a r i e t i e s . That seed coat contamination is not a factor in seed transmission of CP-SBMV is also supported by the results of these v a r i e t i e s whose seed coats contained both viruses. CP-SBMV may be seed-transmitted in these v a r i e t i e s , but to a low level not detected by the number of seeds used in these studies. An- ii other p o s s i b i l i t y is that CP-SBMV may not be seed-transmitted in these v a r i e t i e s . It is not unusual that a virus may be transmitted through seed of one variety or c u l t i v a r of a plant and not so seed transmitted in another of the same species or genus. Thus, squash mosaic virus was seed-transmitted in some v a r i e t i e s of squash, but not in others (Grogan et a l , 1959) and the M2 is o l a t e of peanut mottle virus was transmitted in seed of some c u l t i v a r s of groundnut but not in others (Adams and Kuhn, 1977 ) . 185 It is therefore proposed that CP-SBMV is embryonica11y transmitted through seed of C a l i f o r n i a blackeye cowpea and that virus in the seed coat plays very l i t t l e , i f any, role in seed transmission of th i s virus or, for that matter, of CP-TMV. Some viruses occur in the seed coat of some hosts, but they are not seed-borne; thus curly top virus is not transmit-ted through planted seed of sugar beet (Bennett and Esau, 1936) and nor is CP-TMV in the present studies. Where i n h i b i t o r s of manual inoculation are suspected to interfere-; with the i n f e c t i v i t y assay of the virus a better way to circumvent t h i s problem would be to assay, for presence of virus in the seed, seedlings derived from decontaminated embryos. The use of techniques as s e n s i t i v e as emzyme-1inked immunosorbent assay (Clark and Adams, 1977) or se r o l o g i c a 1 1 y - s p e c i f i c elec-tron microscopy (Derrick and Brlansky, 1976), could also be employed to avoid i n h i b i t o r s ; however these methods may not indicate the v i a b i l i t y of the virus so scanned. The mechanism of seed transmission is poorly understood. In the present studies CP-SBMV was transmitted less in seeds from doubly infected plants than in seeds of singly infected plants. S i m i l a r l y , soybean mosaic virus was transmitted less in seeds of soybean doubly infected by soybean mosaic virus and bean pod mottle virus (Ross, 1963)- In contrast, CP-SBMV was transmitted more in seed of C a l i f o r n i a blackeye cowpea, doubly infected by CCMV and CP-SBMV (Kuhn and Dawson, 1973). It is tempting to speculate that, perhaps seed transmission of the seed-transmissible virus is reduced in seed of 186 doubly infected plants, because th e i r embryos possess an inherent protective mechanism against in f e c t i o n by the virus which is not normally embryo-borne. In doubly infected plants, infection by either CP-TMV or bean pod mottle virus would induce a non-specific i n h i b i t o r y substance(s), which would reduce the a b i l i t y of the embryo-borne virus to infect the embryo. This hypothesis is very simple and i t may not r e f l e c t what a c t u a l l y happens between host-virus genotypes. Bennett (1940) explained lack of embryo infec-tion by some viruses, by suggesting that the gametophytic generation may be immune to infection by non-embryo-borne viruses due to some physiological c h a r a c t e r i s t i c s inherent in the gametophytic make-up. The observation that double i n f e c t i o n , i n the present studies, pro-duced more mottled seed than single infection confirms the results of Kuhn and Dawson (1973). In neither study was there evidence to indicate that seed mottling affected seed transmission of CP-SBMV. By contrast, seed mottling in soybean, infected by soybean mosiac v i r u s , was correlated with higher seed transmission of this virus than was the case in normal seed (Ross, 1963; Kennedy and Cooper, 1967). It was also not possible to corr e l a t e seed transmission of CP-SBMV with i t s concentration in the leaves. This observation is sim i l a r to that made for soybean mosaic virus when i t was in double inf e c t i o n with bean pod mottle v i r u s . The l a t t e r interaction and that of CP-SBMV/CP-TMV, reported in the present studies, resulted in the enhancement of the seed-transmissi-ble v i r u s . However, in double infections CP-SBMV and soybean mosaic virus were transmitted less than in seed derived from singly infected 187 plants. In contrast, CCMV/CP-SBMV interaction, which leads to depressed CP-SBMV nucleoprotein y i e l d s , resulted in higher seed transmission rates of CP-SBMV than those found in seed derived from singly infected plants. Adams and Kuhn (1977) could not co r r e l a t e seed transmission of peanut mottle virus with the level of virus in the leaves and flowers of groundnut. The influence of CP-TMV on systemic infection of V45 _Bots and on in-fectio n of Pinto bean by CP-SBMV is not without s i g n i f i c a n c e in the epide-miology of diseases in the f i e l d . Although no vector of TMV is known, i t is very l i k e l y that the two viruses may be adsorbed to the claws of crawling insects and deposited by them on a host otherwise regarded as immune or re-sis t a n t to CP-SBMV; in the presence of CP-TMV the two viruses may i n i t i a t e i nfection through fo r t u i t o u s injury. Indeed, Scott and Fulton (1978) reported that beetles could acquire and deposit both CP-TMV and the bean s t r a i n of SBMV. Although only the l a t t e r was transmitted by the beetles, infectious CP-TMV could be recovered from leaf surfaces in the areas where the beetles had fed. The fact that both CP-SBMV and CP-TMV occur in seed coats of cowpea, means that these viruses can be transported in this manner to distant places. Their presence in the seed coat also means that the seed coat can provide source of inoculum for either v i r u s . Although cowpea is not normally trans-planted, but seeded d i r e c t l y , the viruses may enter the embryo during handling of wet seed. For instance in Botswana, where cowpea is grown extensively, under dry-land farming, i t is sometimes necessary to soak seed for a period of time before sowing, to supplement the low level of moisture a v a i l a b l e in 188 in the s o i l . After soaking the seed is broadcast or sown with a planter, both of which may injure the embryos. This could be an inadvertent way of introducing the viruses into embryos. The influence of CP-TMV on infe c t i o n of Pinto by CP-SBMV may also lend support to the evolution theory speculated for the o r i g i n of SBMV strains (Lamptey and Hamilton, 1974). It could be that, however, CP-SBMV evolved and lost the a b i l i t y to infect bean, i t and the host s t i l l possess the potential for an interaction ( i n f e c t i o n ) , under conditions not favourable to Pinto and in th i s case infection by CP-TMV would be unfavourable conditions. It would also be interesting to see what would happen i f CP-SBMV, in the local lesions of Pinto leaf, was continuously passaged in the presence of CP-TMV and a f t e r several passages, attempt to inoculate i t singly to Pinto as we11 as cowpea. 189 SUMMARY Double infection of C a l i f o r n i a blackeye cowpea by CP-SBMV and CP-TMV resulted in lower y i e l d s of each virus in the inoculated primary leaves and in the enhancement of CP-SBMV synthesis in the systemically infected t r i f o l i a t e leaves compared to yi e l d s in singly infected tissue. The influence of CP-TMV infection on enhanced synthesis of CP-SBMV was e f f e c t i v e under both asynchronous and synchronous conditions of in f e c t i o n . CP-TMV infection also conditioned V45-Bots cowpea to become systemically infected by CP-SBMV. Evidence is also presented which suggests that in the presence of CP-TMV infe c t i o n CP-SBMV infected and mul t i p l i e d in Pinto bean. Although double infection enhanced CP-SBMV synthesis and that both viruses were detected in the same c e l l s of doubly infected C a l i f o r n i a blackfeye cowpea, no genomic masking was detected between the two viruses. CP-SBMV, but not CP-TMV, was transmitted through planted seed and planted decontaminated embryos of C a l i f o r n i a blackeye cowpea. Double i n f e c t i o n ; caused CP-SBMV to be transmitted less than in seed from singly infected plants. It is theorized that CP-TMV infection predisposes the host p h y s i o l o g i c a l l y by allowing more c e l l s to be infected by CP-SBMV and thus increased synthesis of CP-SBMV. It is also concluded that CP-SBMV seed transmission is a result of embryo inf e c t i o n . The influence of CP-TMV on systemic infection of V45-Bots and on rendering Pinto bean to become susceptible to CP-SBMV emphasizes the f a c t that the importance of studies on mixed virus infections is not just a laboratory c u r i o s i t y , but can also have important epidemiological s i g n i f i c a n c e . Transmission of CP-SBMV through seed of cowpea should be considered in breeding programmes aimed at obtaining v i r u s - f r e e germ-plasm. 190 LITERATURE CITED Adams, D.B. a n d CW. Kuhn. 1 9 7 7 . Seed transmission of peanut mottle virus in peanuts. Phytopathology 6 7 : 1 1 2 6 - 1 1 2 9 . Atabekov, J.G., V.K. Novikov, V.K. Vishnichenko and V.G. Javakhia. 1 9 7 0 a . 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redpajama_set_name: RedPajamaCommonCrawl