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UBC Theses and Dissertations

Genetic studies of the host-parasite relationship between Ustilago hordei and Hordeum vulgare Ebba, Tadessa 1974

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C '. GENETIC STUDIES OF THE HOST-PARASITE RELATIONSHIP BETWEEN USTILAGO HORDEI AND HORDEUN VULGARE by TADESSA EBBA B .Sc . H a i l e S e l l a s s i e I U n i v e r s i t y , E t h i o p i a M .Sc . G e n e t i c s . U n i v e r s i t y o f W i s c o n s i n , M a d i s o n , U.S A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Depa r tmen t o f Botany 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 to the req u i red s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA J a n u a r y , 1974 In presenting th i s thesis in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f ree l y ava i l ab le for reference and study. I further agree that permission for extensive copying of this thesis for scholar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i cat ion of th is thesis for f inanc ia l gain sha l l not be allowed without my written permission. Department of The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada Date Sgjn. £ I, /f7</ ABSTRACT G e n e t i c s t u d i e s were c a r r i e d out on the f u n g a l p a r a s i t e Ustilago hordei ( P e r s . ) L a g e r h . and on i t s h o s t , Hordeum vulgave L . ( c u l t i v a t e d b a r l e y ) . In t h e s e s t u d i e s o f the h o s t - p a r a s i t e r e l a t i o n s h i p , s p e c i a l emphasis was p l a c e d on the g e n e t i c i n v e s t i g a t i o n o f the p a t h o g e n i c i t y . The t h e s i s i s d i v i d e d i n t o f o u r p a r t s . P a r t I d e a l s w i t h m u l t i a l 1 e l i s m of genes f o r v i r u l e n c e ( v - g e n e s ) i n the p a r a s i t e , and d e m o n s t r a t e d t h a t f o u r d i f f e r e n t l e v e l s of v i r u l e n c e ( o b t a i n e d on the b a r l e y c u l t i v a r T r e b i ) a r e c o n t r o l l e d by a l t e r n a t i v e a l l e l e s a t a s i n g l e g e n e t i c l o c u s i n the p a r a s i t e . T h i s i s t h e f i r s t d e m o n s t r a t e d example of a s e r i e s o f m u l t i p l e a l l e l e s d e t e r m i n i n g d i f f e r e n t l e v e l s of v i r u l e n c e . P a r t I I c o n c e r n s the i d e n t i f i c a t i o n and c h a r a c t e r i -z a t i o n of v -genes i n U. hordei and of r e s i s t a n c e genes ( R - g e n e s ) i n H. vulgave. Three v - g e n e s (two o f them new, one of them p r e v i o u s l y known) were i d e n t i f i e d . I t was shown t h a t the p r e v i o u s l y i d e n t i f i e d gene was e x p r e s s e d e i t h e r as a dominant or a r e c e s s i v e , d e p e n d i n g o o n the i i c o n d i t i o n s under w h i c h i t was t e s t e d , and t h a t the n e w l y -i d e n t i f i e d genes were both r e c e s s i v e . C u l t u r e s p o s s e s s i n g the n e w l y - d i s c o v e r e d v - g e n e s were used i n i d e n t i f y i n g two new R-genes i n the b a r l e y h o s t . A s t u d y of i n t e r a c t i o n s i n v o l v i n g the newly d i s c o v e r e d v - and R-genes l e d to the c o n c l u s i o n t h a t t h e s e i n t e r a c t i o n s have t h e i r b a s i s i n g e n e - f o r - g e n e r e l a t i o n s h i p s . P a r t I I I d e a l s w i t h the s y n t h e s i s of a complex b i o t y p e o f u. hordei p o s s e s s i n g v -genes a t two g e n e t i c l o c i . D i s e a s e r e a c t i o n o b t a i n e d w i t h t h i s complex b i o t y p e were compared b o t h q u a l i t a t i v e l y and q u a n t i t a t i v e l y w i t h t h o s e o b t a i n e d w i t h the s i m p l e r , p a r e n t a l b i o t y p e s . In t e s t s on c e r t a i n c u l t i v a r s the complex b i o t y p e p r o d u c e d e i t h e r the same or h i g h e r l e v e l s of d i s e a s e r e a c t i o n . Because the new b i o t y p e has ah e x t e n d e d h o s t range i t i s c o n s i d e r e d t h a t under c e r t a i n c o n d i t i o n s i t wou ld be c o m p a r a t i v e l y more f i t t h a n e i t h e r of the p a r e n t a l b i o t y p e s f rom w h i c h i t was d e r i v e d . P a r t IV of the t h e s i s c o n c e r n s the e f f e c t s o f n u t r i t i o n a l d e f i c i e n c y on the a c t i o n o f v - g e n e s . D i k a r y o n s w h i c h were homozygous f o r a r g , ad or met were i n a l l cases n o n - p a t h o g e n i c ; f o r t h o s e w h i c h were homozygous f o r p d x , p a t h o g e n i c i t y was u n a f f e c t e d . For d i k a r y o n s w h i c h were i i i heterozygous f o r one or more n u t r i t i o n a l def i c i ences , p a t h o -g e n i c i t y was e i t h e r u n i m p a i r e d o r r e d u c e d , d e p e n d i n g on t h e c o m b i n a t i o n ( d e f i c i e n c y : v i r u l e n c e gene : h o s t c u l t i v a r ) w h i c h was t e s t e d . I t was c o n c l u d e d t h a t the s p e c i f i c i t y o f pathogen b i o t y p e s was not d e t e r m i n e d by the a v a i l a b i l i t y or n o n - a v a i l a b i l i t y o f s p e c i f i c n u t r i t i o n a l f a c t o r s . However , the e f f e c t s were not e n t i r e l y n o n - s p e c i f i c , s i n c e changes i n l e v e l s o f v i r u l e n c e were shown o n l y i n c e r t a i n t e s t s . i v TABLE OF CONTENTS Page A b s t r a c t i i L i s t o f T a b l e s v i i L i s t o f F i g u r e s x i Acknowledgements x i i INTRODUCTION 1 LITERATURE REVIEW 6 PART I : MULTIPLE ALLELISM OF GENES CONTROLLING DIFFERENT LEVELS OF VIRULENCE IN USTILAGO HORDEI 21 I n t r o d u c t i o n 21 M a t e r i a l and Methods 21 R e s u l t s 27 D i s c u s s i o n 38 PART I I : USTILAGO HORDEI3 HORDEUM VULGARE AND THE GENE-FOR-GENE RELATIONSHIP 48 I n t r o d u c t i o n 48 M a t e r i a l and Methods 50 R e s u l t s 52 D i s c u s s i o n 69 v Page PART I I I : SYNTHESIS OF A COMPLEX USTILAGO HORDEI BIOTYPE AND ITS FITNESS RELATIVE TO SIMPLE BIOTYPES. 79 I n t r o d u c t i on 79 M a t e r i a l and M e t h o d s . 80 R e s u l t s 84 D i s c u s s i o n 90 PART I V : THE EFFECTS OF AUXOTROPHIC MUTATIONS ON PATHOGENICITY 95 I n t r o d u c t i o n . 95 M a t e r i a l and M e t h o d s . 97 R e s u l t s 102 D i s c u s s i o n . 112 GENERAL DISCUSSION. 122 BIBBIOGRAPHY. 125 APPENDICES A CULTURE MEDIUM 138 B SCHEMATIC REPRESENTATION OF THE L I F E CYCLE OF USTILAGO HORDEI . . . . • 140' v i LIST OF TABLES T a b ! e Page 1 P a r a s i t i c Systems f o r w h i c h t h e G e n e - F o r -Gene R e l a t i o n s h i p has been e i t h e r D e m o n s t r a t e d or P o s t u l a t e d 17 1-1 D i s e a s e R e a c t i o n s O b t a i n e d on the B a r l e y C u l t i v a r T r e b i by S e l f i n g and C r o s s i n g o f S p o r i d i a l L i n e s f rom Four s e t s o f U. hordei C u l t u r e s 29 1-2 D i s e a s e R e a c t i o n s O b t a i n e d on B a r l e y C u l t i v a r T r e b i F o l l o w i n g B a c k c r o s s i n g F i C u l t u r e s ( t e t r a d s e t s ) to the Two P a r e n t a l Se t s T l and T2 32 1-3 D i s e a s e R e a c t i o n s O b t a i n e d on B a r l e y C u l t i v a r T r e b i F o l l o w i n g B a c k c r o s s i n g If i C u l t u r e s ( t e t r a d s e t s ) to the Two P a r e n t a l S e t s , T l and T3 33 1-4 D i s e a s e R e a c t i o n s O b t a i n e d on B a r l e y C u l t i v a r T r e b i F o l l o w i n g B a c k c r o s s i n g F i C u l t u r e s ( t e t r a d s e t s ) t o t h e Two P a r e n t a l S e t s , T l and T4 34 1-5 D i s e a s e R e a c t i o n s O b t a i n e d on B a r l e y C u l t i v a r T r e b i F o l l o w i n g B a c k c r o s s i n g F i C u l t u r e s ( t e t r a d s e t s ) to the Two P a r e n t a l S e t s , T4 and T2 36 1-6 D i s e a s e R e a c t i o n s O b t a i n e d on B a r l e y C u l t i v a r T r e b i F o l l o w i n g B a c k c r o s s i n g F i C u l t u r e s ( t e t r a d s e t s ) to the two P a r e n t a l S e t s , T3 and T4 37 v i i T a b l e Page 11-1 The P o s t u l a t e d Genotypes of S p o r i d i a l C u l t u r e s o f S e t s D and G and of the D i k a r y o n s P r o d u c e d by S e l f i n g , T o g e t h e r w i t h the D i s e a s e R e a c t i o n s O b t a i n e d w i t h C u l t i v a r s E x c e l s i o r ( E ) , H i m a l a y a ( H i ) and K e y s t o n e (X) 54 11- 2 D i s e a s e R e a c t i o n O b t a i n e d on Three B a r l e y C u l t i v a r s F o l l o w i n g I n o c u l a t i o n w i t h D i k a r y o n s Formed by S e l f i n g and C r o s s i n g C u l t u r e Se t s D and G , and the Genotypes of the D i k a r y o n s . . . 57 11-3 D i s e a s e R e a c t i o n O b t a i n e d on Three B a r l e y C u l t i v a r s F o l l o w i n g I n o c u l a t i o n w i t h D i k a r y o n s Formed by B a c k c r o s s i n g Fx S p o r i d i a l L i n e s f rom D x G C r o s s to P a r e n t a l C u l t u r e Set D. 58 11-4 D i s e a s e R e a c t i o n O b t a i n e d on Three B a r l e y C u l t i v a r s F o l l o w i n g I n o c u l a t i o n w i t h D i k a r y o n s Formed by B a c k c r o s s i n g F i S p o r i d i a l L i n e s f rom D x G C r o s s to P a r e n t a l C u l t u r e Set G . . 60 11-5 D i s e a s e R e a c t i o n , i n P e r c e n t a g e s , Induced on E x c e l s i o r and K e y s t o n F o l l o w i n g I n o c u -l a t i o n w i t h D i k a r y o n s V 2 V 2 V i f V i t and V 2 V 2 V i t V i f o f U. hordei 63 11-6 F 3 L i n e s f rom E x c e l s i o r and K e y s t o n e C r o s s S e p a r a t e d i n t o Four C l a s s e s A c c o r d i n g to t h e i r I n t e r a c t i o n s w i t h Two D i k a r y o n s , V 2 V 2 V 4 V H . and V 2 V 2 V 4 V 4 , o f U. hordei and t h e Number o f L i n e s i n Each C u l t u r e 65 11- 7 D i s e a s e R e a c t i o n ( e x p r e s s e d i n p e r c e n t a g e ) Induced i n F 3 L i n e s D e r i v e d f rom the K x E C r o s s F o l l o w i n g I n o c u l a t i o n w i t h V 2 V 2 V 4 V 4 and V 2 V 2 V i t V i f D i k a r y o n s 66 v i i i T a b l e Page 11-8 T h e o r e t i c a l l y E x p e c t e d P a r e n t a l , F i and F 2 Genotypes of B a r l e y C u l t i v a r s E x c e l s i o r and K e y s t o n e and t h e i r R e a c t i o n s to v 2 v 2 V i tV it and V2Vzv>*vn1 Di k a r y o n s Based on F l o r ' s G e n e - F o r - G e n e R e l a t i o n s h i p 75 11-9 E x p e c t e d S e g r e g a t i o n of F 3 L i n e s o f B a r l e y D e r i v e d f rom E x c e l s i o r x K e y s t o n e C r o s s F o l l o w i n g I n o c u l a t i o n w i t h v 2 v ^ and V 2 V 2Vi tVi t Di k a r y o n s of U. hovdei (based on F l o r ' s g e n e - f o r - g e n e r e l a t i o n s h i p ) 76 I I I - l The r e a c t i o n of E l e v e n B a r l e y C u l t i v a r s to S e t s o f u. hovdei D i k a r y o n s w h i c h were D e r i v e d from S i n g l e T e l i o s p o r e s of Two P a r e n t a l D i k a r y o n s and t h e i r F i H y b r i d s 86 111 - 2 C r o s s e s o f F ( v i v 2 ) T e t r a d S e t s w i t h P a r e n t a l ( v i V 2 and V i v 2 ) S e t s and G , and t h e i r D i s e a s e R e a c t i o n on E x c e l s i o r and Vantage 87 111-3 C r o s s e s I n v o l v i n g Two T e t r a d Se ts (Ha and Hb) D e r i v e d f rom F i T e l i o s p o r e s of the V i V 2 x V i V 2 C r o s s and Three D i k a r y o n s ( v i V i V 2 v 2 , V i V i V 2 v 2 and V i V i V 2 V 2 ) , T o g e t h e r w i t h the D i s e a s e R e a c t i o n s (%) O b t a i n e d on E x c e l s i o r and Vantage . . . . . . . . 88 IV-1 D i s e a s e R e a c t i o n s , i n P e r c e n t a g e s , O b t a i n e d by D i f f e r e n t D i k a r y o n s ( v i V i V 2 V 2 , V i V i V 2 v 2 , o r v x V i V 2 v 2 ) o f U. hovdei oh Seven B a r l e y C u l t i v a r s 105 I V - 2 Summary o f S t a t i s t i c a l A n a l y s i s of D i f f e r e n c e s Between V i r u l e n c e L e v e l s Induced by D i f f e r e n t D i k a r y o n s on F i v e C u l t i v a r s as P r e s e n t e d i n T a b l e IV-1 . 107 1 x T a b l e Page I V - 3 D i s e a s e R e a c t i o n s , i n P e r c e n t a g e s , P r o d u c e d by D i f f e r e n t D i k a r y o n s ( V 1 V 1 v 2 v 2 > V ! V i V 2 V 2 , o r V 1 V 1 V 2 V 2 ) o f U. ho'vdei on Seven B a r l e y C u l t i v a r s 109 IV -4 Summary of S t a t i s t i c a l A n a l y s i s o f D i f f e r e n c e s Between V i r u l e n c e L e v e l s Induced by 15 D i f f e r e n t D i k a r y o n s on F i v e C u l t i v a r s as P r e s e n t e d i n T a b l e I V - 3 . . . . I l l x LIST OF FIGURES F i g u r e Page 1 A d i a g r a m to i l l u s t r a t e the g e n e r a l p l a n o f the e x p e r i m e n t 24 x i ACKNOWLEDGEMENTS I w i s h to e x p r e s s my d e e p e s t a p p r e c i a t i o n and g r a t i t u d e to my s u p e r v i s o r , P r o f e s s o r C l a y t o n P e r s o n f o r h i s g u i d a n c e and s u g g e s t i o n s i n academic w o r k , r e s e a r c h and i n the p r e p a r a t i o n of t h i s t h e s i s . I w o u l d l i k e to thank the members of my t h e s i s commit tee f o r v a l u a b l e d i s c u s s i o n s c o n c e r n i n g the p r e p a r a t i o n o f t h i s t h e s i s . I am i n d e b t e d to the C a n a d i a n Agency f o r I n t e r n a t i o n a l Development f o r the s c h o l a r s h i p a w a r d . Thanks to D r . Tony G r i f f i t h s f o r v a l u a b l e s u g g e s t i o n s and d i s c u s s i o n . Thanks are due to D r . B e l a S i v a k and M r . J i m G r o t h and the r e s t of the "Ustilago g r o u p " ( p a s t and p r e s e n t ) f o r t h e i r v a l u a b l e d i s c u s s i o n and moral s u p p o r t . I would l i k e to e x p r e s s my a p p r e c i a t i o n to M r . Ro lando R o b i l l i o f o r h i s h e l p i n d a t a c o l l e c t i o n i n the f i e l d and i n the l a b o r a t o r y w o r k . I w o u l d l i k e to acknowledge D r . A l f r e d o G a r c i a of the W o r l d Seeds I n c . f o r p r o v i d i n g l a n d and o t h e r f a c i l i t i e s i n C a l i f o r n i a , where p a r t of the work of t h i s t h e s i s was c a r r i e d o u t . And I w i s h to thank a l l my f r i e n d s who i n one way o r a n o t h e r made my s t a y i n Canada p i e a s a n t , a n d d w o r t h w h i 1 e . x i i INTRODUCTION The " s m u t s " i n c l u d e a l a r g e group o f f u n g a l s p e c i e s ( f a m i l y U s t i 1 a g i n a l e s ) , a l l o f w h i c h are p a r a s i t i c d u r i n g a t l e a s t p a r t o f t h e i r l i f e c y c l e . They cause e x t e n s i v e c r o p l o s s e s a n d , a c c o r d i n g to F i s h e r and H o l t o n ( 1 9 5 7 ) , they have done so s i n c e the b e g i n n i n g of a g r i c u l t u r e . O n l y a few of the many smut s p e c i e s have been used e x t e n s i v e l y i n g e n e t i c s t u d i e s o f m u t a t i o n , r e c o m b i n a t i o n , c o m p l e m e n t a t i o n , or i n s t u d i e s of h o s t - p a r a s i t e r e l a t i o n -s h i p s . VThese i n c l u d e Ustilago maydis ( H o l l i d a y , 1961 ; 1 9 6 4 ) , U. hordei (Thomas and P e r s o n , 1965 ; H o o d , 1966 ; K o z a r , 1969 ; D i n o o r and P e r s o n , 1969; Robb, 1971 ; S i d h u and P e r s o n , 1971 , 1 9 7 2 ) , U. Violacea (Day and J o n e s , 1969) and u. avenae ( H a l i s k y , 1 9 6 5 ) . C o n s i d e r i n g the amount o f work t h a t has been done i n d e t e r m i n i n g p h y s i o l o g i c r a c e s and m o r p h o l o g i c a l c h a r a c -t e r i s t i c s , s u r p r i s i n g l y l i t t l e i s known of the g e n e t i c s o f p a t h o g e n i c i t y , even though t h i s i s a f i e l d o f g r e a t t h e o r e t i c a l i n t e r e s t as w e l l as one w h i c h i s o f p r a c t i c a l ' u s e f u l n e s s to a g r i c u l t u r e . In the p a s t i t was common p r a c t i c e to breed c r o p p l a n t s f o r r e s i s t a n c e a g a i n s t d i s e a s e w i t h o u t c o n s i d e r i n g 1 2 the g e n e t i c p o t e n t i a l o f the p a r a s i t e . T h i s t r e n d i s c h a n g -i n g g r a d u a l l y and due emphasis i s b e i n g g i v e n to the s t u d y o f the g e n e t i c s o f p a t h o g e n i c i t y i n p a r a s i t e s ( f u n g i , i n s e c t s e t c . ) . In f a c t , a t p r e s e n t , the g e n e t i c s of p a t h o g e n i c i t y and the g e n e t i c s o f r e s i s t a n c e a r e s t u d i e d h a n d - i n - h a n d w h e r e v e r p o s s i b l e , i n o r d e r to e x p l o r e more f u l l y the g e n e t i c b a s i s o f the i n t e r a c t i o n s i n w h i c h both hos t and p a r a s i t e a re i n v o l v e d . A t the o u t s e t , i t i s i m p o r t a n t to d e f i n e some of the terms t h a t a re to be used i n t h i s t h e s i s . Some of them have been used i n a s l i g h t l y d i f f e r e n t c o n t e x t by o t h e r a u t h o r s ( M i l e s , 1955; D a y , 1960 ; Van der P l a n k , 1968; R o b i n s o n , 1969 ; N e l s o n , 1970 ; A i n s w o r t h , 1 9 6 1 ) . The d e f i n i t i o n s f o r the terms p a t h o g e n i c i t y and v i r u l e n c e a r e a d a p t e d f rom M i l e s (1 9 5 5 ) . P a t h b g e n i c i t y r e f e r s to the g e n e r a l c a p a c i t y or p r o p e r t y of one o r g a n i s m to cause d i s e a s e i n a n o t h e r o r g a n i s m . The o r g a n i s m t h a t p o s s e s s e s such a u p r o p e r t y i s a p a r a s i t e and i s r e f e r r e d to as a pathogen ( o t h e r w i s e as a n o n - p a t h o g e n ) . The p a t h o g e n i c i t y o f a pathogen i s o f t e n l i m i t e d to one o r , a t m o s t , a few hos t s p e c i e s . V i r u l e n c e r e f e r s to the r e l a t i v e p r o p e r t y or a b i l i t y o f a pathogen to cause d i s e a s e among s p e c i f i c members o f the h o s t s p e c i e s . Thus a pathogen can be v i r u l e n t on o n e , a f e w , or on most members o f a h o s t s p e c i e s and a v i r u 1 e n t on one or a few o t h e r members o f the h o s t s p e c i e s . The s p e c i f i c i t y shown by 3 a pathogen i n r e l a t i o n to a few members ( c u l t i v a r s ) o f a h o s t s p e c i e s forms the b a s i s f o r i d e n t i f y i n g p h y s i o l o g i c r a c e s . P a t h o g e n i c i t y i s a g e n e r a l p r o p e r t y of a p a t h o g e n ; v i r u l e n c e and a v i r u l e n c e a r e s p e c i f i c p r o p e r t i e s w i t h i n the g e n e r a l c o n t e x t o f p a t h o g e n i c i t y . In smut f u n g i v i r u l e n c e i s u s u a l l y measured i n terms o f the p e r c e n t a g e of i n o c u l a t e d p l a n t s w h i c h show the d i s e a s e . The degree of s e v e r i t y o f the d i s e a s e i s o f t e n d e s c r i b e d i n terms o f l e v e l s o f v i r u l e n c e o r o f r e s i s t a n c e ( e . g . l o w , i n t e r m e d i a t e , and h i g h ) . When the d i s e a s e i s f u l l y d e v e l o p e d the h o s t i s j u d g e d to be s u s c e p t i b l e and the p a r a s i t e v i r u l e n t . When the d i s e a s e d e v e l o p s p o o r l y the h o s t i s j u d g e d to be r e s i s t a n t and the p a r a s i t e a v i r u 1 e n t . G e n e t i c s t u d i e s have shown t h a t , f o l l o w i n g i n f e c t i o n , the e x t e n t o f d i s e a s e d e v e l o p m e n t i s c o n t r o l l e d t h r o u g h i n t e r a c t i o n o f g e n e t i c f a c t o r s i n both the h o s t and p a r a s i t e . The n a t u r e of t h e s e g e n e t i c i n t e r -a c t i o n s w i l l be d e s c r i b e d l a t e r . U. hordei ( P e r s . ) L a g e r h . , an o b l i g a t e p a r a s i t e " on b a r l e y , was used i n t h i s s tudy, , The g e n e t i c s o f p a t h o g e n i c i t y can o n l y be s t u d i e d i f the pathogen can go t h r o u g h a s e x u a l c y c l e . U. hordei s a t i s f i e s t h i s c o n d i t i o n ( F i s h e r and H o l t o n , 1 9 5 7 ) . The p a r a s i t i c s t a g e i n the l i f e c y c l e o f t h i s smut i s t h e d i k a r y o n , w h i c h i s formed upon the f u s i o n of h a p l o i d c e l l s ( s p o r i d i a ) o f o p p o s i t e m a t i n g t y p e s , o f t e n d e s i g n a t e d 4 A and a . The d i k a r y o n i n f e c t s and grows w i t h i n the h o s t . The smut s o r i , when t h e s e are f o r m e d , c o n t a i n l a r g e r numbers o f t e l i o s p o r e s w h i c h are d i p l o i d . They may appear as a d a r k mass on the l e a f b l a d e s and s h e a t h e s , and on the c u l m s , but m a i n l y t h e y r e p l a c e the seeds o f the b a r l e y p l a n t . Upon g e r m i n a t i o n o f a t e l i o s p o r e the d i p l o i d n u c l e u s undergoes m e i o s i s and forms a f o u r - c e l l e d p r o m y c e l i u m . The p r i m a r y s p o r i d i a p r o d u c e d by t h e s e f o u r l i n e a r l y - a r r a n g e d c e l l s c o m p r i s e an o r d e r e d t e t r a d . The f o u r p r i m a r y s - p o r i d i a can be s e p a r a t e d f rom the p r o m y c e l i u m by means o f m i c r o m a n i p u l a -t i o n . Each s p o r i d i u m can g i v e r i s e to a c o l o n y t h r o u g h y e a s t - l i k e b u d d i n g and g r o w t h on s y n t h e t i c medium. The m a t i n g sys tem i s d e t e r m i n e d a t a s i n g l e g e n e t i c l o c u s and i s b i p o l a r . Two of the f o u r s p o r i d i a a re d e s i g n a t e d A and two are a ( A p p e n d i x B ) . The c o m b i n a t i o n of s p o r i d i a l l i n e s d e r i v e d f rom a s i n g l e m e i o t i c e v e n t c o n s t i t u t e s " s e l f i n g , " w h i l e the com-b i n a t i o n o f two s p o r i d i a l l i n e s each f rom a d i f f e r e n t t e l i o s p o r e c o n s t i t u t e s " c r o s s i n g . " The h o s t , Hordeum vulgave L . ( c u l t i v a t e d b a r l e y ) , i s s e l f - p o l l i n a t i n g . T h u s , near i s o g e n i c and homozygous l i n e s can be d e v e l o p e d . I t can be c r o s s e d w i t h r e l a t i v e ease to s t u d y the i n h e r i t a n c e of r e s i s t a n c e and s u s c e p t i b i l i t y to d i s e a s e . I t can e a s i l y be grown i n p o t s or f l a t s i n the g r e e n -house under w e l l l i g h t e d c o n d i t i o n s as w e l l as i n the f i e l d . The main p u r p o s e s o f the work p r e s e n t e d i n t h i s t h e s i s were to o b t a i n i n f o r m a t i o n o n : I n t e r a c t i o n o f d i f f e r e n t v i r u l e n c e g e n e s h a v i n g q u a n t i t a t i v e l y d i f f e r e n t e f f e c t s . I d e n t i f i c a t i o n o f new v i r u l e n c e g e n e s i n U. hovdei and o f t h e i r c o m p l e m e n t a r y r e s i s t a n c e g e n e s i n H. vulgave. P o s s i b l e i n t e r a c t i o n b e t w e e n d i f f e r e n t v i r u l e n c e g e n e s (tl h v i and U h v 2 ) o f U. hovdei. E f f e c t s o f n u t r i t i o n a l d e f i c i e n c y on t h e a c t i o n ;Ga.s m e a s u r e d b o t h q u a l i -t a t i v e l y and q u a n t i t a t i v e l y ) o f v i r u -l e n c e g e n e s . LITERATURE REVIEW GENETICS OF PATHOGENICITY The d i s c o v e r y o f p a t h o g e n i c s p e c i a l i z a t i o n and l i f e c y c l e i n the p a t h o g e n i c f u n g i was f u n d a m e n t a l to the s t u d y of the g e n e t i c s o f p a t h o g e n i c i t y . E r i k s s o n , i n 1894, r e c o g -n i z e d forma speaiales w i t h i n Suoain-ia gvaminis3 a s p e c i e s o f r u s t p a t h o g e n , t h e r e b y s u b d i v i d i n g the s p e c i e s . A few y e a r s l a t e r B a r r u s ( 1 9 1 1 ) , w i t h the bean a n t h r a c n o s e f u n g u s , C o l l e t o t r i o h u m lindemuthianum, and Stakman ( 1 9 1 4 ) , w i t h wheat stem r u s t , P u o o i n i a g r a m i n i s f . s p . t r i t i a i 3 made f u r t h e r s u b d i v i s i o n s i n t o s t r a i n s , o r " p h y s i o l o g i c r a c e s , " based on the r e l a t i v e a b i l i t i e s o f d i f f e r e n t c u l t u r e to a t t a c k d i f f e r e n t c u l t i v a r s o f a p a r t i c u l a r c r o p s p e c i e s . The a p p r o a c h e s o f E r i k s s o n , B a r r u s and Stakman were f o l l o w e d by o t h e r w o r k e r s u s i n g o t h e r p a t h o g e n i c f u n g i as w e l l as by t h o s e u s i n g n o n f u n g a l p a r a s i t e s such as v i r u s e s and b a c t e r i a . I t was the d i s c o v e r y o f sex i n smut by K n i e p (1919) and l a t e r i n r u s t by C r a i g i e (1927) w h i c h f a c i l i t a t e d the s e l f i n g and c r o s s i n g o f p h y s i o l o g i c a l r a c e s , t h u s o p e n i n g the way f o r the g e n e t i c s t u d i e s t h a t f o l l o w e d . T h i s r e v i e w d e a l s o n l y w i t h the g e n e t i c s o f p a t h o g e n i c i t y i n smut f u n g i . 6 7 K n i e p (1919) was the f i r s t to d e m o n s t r a t e t h a t the s p o r i d i a o f u. v i o l a o e a f u n c t i o n as g a m e t e s , t h e r e b y d i s c o v e r i n g sex i n the smut f u n g i , and making p o s s i b l e the s u b s e q u e n t s t u d i e s by h y b r i d i z a t i o n between s p e c i e s and r a c e s o f the smut f u n g i . G o l d s c h m i d t (1928) was the f i r s t to s t u d y the g e n e t i c s o f p a t h o g e n i c i t y o f a smut s p e c i e s . He worked w i t h s i x r a c e s o f u. v i o l a o e a and t h e i r h y b r i d s , and f o u n d t h a t v i r u l e n c e i n two of t h e s e r a c e s was c o n t r o l l e d by a s i n g l e gene w h i c h was i n c o m p l e t e l y d o m i n a n t . T h i s was f o l l o w e d by C h r i s t e n s e n 1 s (1929) work i n u. maydis. He o b s e r v e d t h a t d i f f e r e n t c o m b i n a t i o n s o f m o n o s p o r i d i a l l i n e s showed d i f f e r e n t degrees o f i n f e c t i o n , r a n g i n g from l i g h t to h e a v y . He c o n c l u d e d t h a t the d i f f e r e n c e i n degrees of v i r u l e n c e had a m u l t i f a c t o r i a l b a s i s . In a s i m i l a r s t u d y Stakman et a l . (1933) o b t a i n e d s i m i l a r r e s u l t s , but c o n c l u d e d t h a t v i r u l e n c e was g o v e r n e d by s p e c i f i c g e n e s . On o f the o u t s t a n d i n g works i n t h i s f i e l d was t h a t o f N i c o l a i s e n (1934) w i t h U. avenae. He s e l f e d the t e t r a d p r o d u c t s o f a s i n g l e t e l i o s p o r e and o b t a i n e d s i m p l e s e g r e g a t i o n s f o r v i r u l e n c e and a v i r u l e n c e . He a l s o c r o s s e d d i f f e r e n t r a c e s and d e t e r m i n e d t h a t v i r u l e n c e genes c o u l d be d o m i n a n t , i n c o m p l e t e l y d o m i n a n t , o r r e c e s s i v e . Some o f the h y b r i d s e x h i b i t e d h i g h e r l e v e l s o f v i r u l e n c e than d i d the p a r e n t a l r a c e s . 8 H a l i s k y (1956) worked w i t h F i , F 2 and F 3 p r o g e n i e s w h i c h had been d e v e l o p e d t h r o u g h s e l f i n g and c r o s s i n g of the s p o r i d i a l p r o d u c t s o f s i n g l e t e l i o s p o r e s o f t h r e e r a c e s of U. avenae. The p r o g e n i e s were t e s t e d f o r v i r u l e n c e on the o a t c u l t i v a r s , Monarch and Camas. The h y b r i d s o f a c r o s s between the a v i r u l e n t r a c e (0% i n f e c t i o n ) and the two v i r u l e n t r a c e s ( a v e r a g i n g 53 and 95% i n f e c t i o n ) showed a v e r y low l e v e l o f v i r u l e n c e (1-9% i n f e c t i o n ) . The F 2 p r o g e n i e s s e g r e -g a t e d 1:2:1 f o r h i g h v i r u l e n c e (53 or 95%) , low v i r u l e n c e ( 1 - 9 % ) , and a v i r u l e n c e . H a l i s k y p o s t u l a t e d t h a t the h i g h v i r u l e n c e c l a s s was homozygous f o r v i r u l e n c e , t h a t the low v i r u l e n c e c l a s s was h e t e r o z y g o u s , and t h a t the a v i r u l e n t c l a s s was homozygous f o r a v i r u l e n c e . He i n t e r p r e t e d the d a t a as showing monogenic i n h e r i t a n c e of v i r u l e n c e and c o n c l u d e d t h a t v i r u l e n c e was dominant to a v i r u l e n c e . H o l t o n (1959) and Johnson (1960) r e i n t e r p r e t e d H a l i s k y ' s d a t a and s u g g e s t e d t h a t s i n c e the 1-9% l e v e l o f v i r u l e n c e was v e r y c l o s e to the z e r o l e v e l i t would be j u s t as l o g i c a l to c o n c l u d e t h a t a v i r u l e n c e was the dominant c h a r a c t e r . T h i s s u g g e s t i o n was a c c e p t e d by H o l t o n and H a l i s k y (1 9 6 0 ) . However , H o l t o n (1 9 6 4 , 1 966) , ' j u s i ng d i f f e r e n t r a c e s and d i f f e r e n t i a l s , r e a c h e d the c o n c l u s i o n , s i m i l a r to t h a t drawn e a r l i e r by N i c o l a i s e n ( 1 9 3 4 ) , t h a t v i r u l e n c e can be i n h e r i t e d as aodomd nantF,nannd n;opmpT.e'tel^o.d6m rinantQroi? a r e c e s s i v e c h a r a c t e r . He a l s o o b s e r v e d t h a t a h i g h l e v e l of 9 v i r u l e n c e i s dominant o v e r a low l e v e l o f v i r u l e n c e . Some-what s i m i l a r r e s u l t s were o b t a i n e d by B e c k e r (1936) i n s t u d i e s o f two h y b r i d s o f T i l l e t i a c a r i e s . In one of the c r o s s e s v i r u l e n c e was r e c e s s . i v e y e i n the o t h e r i t was i m -c o m p l e t e l y d o m i n a n t . In u. maydis t h e r e a r e two i n c o m p a t a b i 1 i t y l o c i " a " and " b " ( R o w e l l , 1955 ; H o l l i d a y , 1 9 6 1 ) . The " a " l o c u s c o n t r o l s s p o r i d i a l f u s i o n whereas the " b " l o c u s appears to c o n t r o l a l a t e r s t a g e i n d e v e l o p m e n t w h i c h has not y e t been i d e n t i f i e d . S i n c e the c o m p l e t i o n o f the s e x u a l c y c l e i s i n s e p a r a b l e f rom d i s e a s e d e v e l o p m e n t the " b " l o c u s has been c o n s i d e r e d as a p a t h o g e n i c i t y l o c u s . I t i s e s t i m a t e d t h a t t h e r e are 25 a l l e l e s a t t h i s l o c u s ( P u h a l l a , 1970 ; Day et a l . 3 1 9 7 1 ) . G e n e t i c s t u d y o f p a t h o g e n i c i t y i n u. hordei was begun o n l y r e c e n t l y . Thomas and P e r s o n (1965) i n v e s t i g a t e d the g e n e t i c c o n t r o l o f low l e v e l s o f v i r u l e n c e . . They c r o s s e d c u l t u r e s w h i c h gave about 5% and 0% r e a c t i o n on two b a r l e y c u l t i v a r s , Gateway and O l l i . From the s e g r e g a t i o n s o b t a i n e d t h e y c o n c l u d e d t h a t the p a t h o g e n i c i t y i n t h e s e c u l t u r e s was c o n t r o l l e d by two a l l e l e s a t a s i n g l e l o c u s . The z e r o r e -a c t i o n ( a v i r u l e n c e ) i s dominant to the 5% r e a c t i o n • ( v i r u l e n c e ) . Lade 4l»^*9)waiTsodw-0rkedlA!n;t^^ t a n c e o f genes c o n t r o l l i n g i n t e r m e d i a t e l e v e l s o f v i r u l e n c e , i . e . 10 5% to 35% l e v e l s o f d i s e a s e . He f o u n d t h a t the 5% l e v e l o f d i s e a s e was dominant to the 35% l e v e l . S i d h u and P e r s o n (1972) i n v e s t i g a t e d the g e n e t i c c o n t r o l o f h i g h l e v e l s o f v i r u l e n c e i n two c u l t u r e s on t h r e e b a r l e y c u l t i v a r s . Two i n d e p e n d e n t v i r u l e n c e g e n e s , bo th o f w h i c h were r e c e s s i v e , were i d e n t i f i e d i n t h i s s t u d y . They d e s i g n a t e d t h e s e genes " U h " ( r e p r e s e n t i n g the p a r a s i t e , Ustilago hordei), f o l l o w e d by V o r v (a dominant o r r e c e s s i v e v i r u l e n c e g e n e ) , f o l l o w e d by a number ( showing the l o c u s c o n c e r n e d ) . The two genes were thus l a b e l l e d Uh'vi and U h v 2 . In t h i s s t u d y , U h v i i s v i r u l e n t on b a r l e y c u l t i v a r s Hannchen and V a n t a g e , and U h v 2 on E x c e l s i o r . A number o f i n t e r s p e c i f i c c r o s s e s have been made among the v a r i o u s smut f u n g i i n o r d e r to s t u d y the i n h e r i t a n c e of p a t h o g e n i c i t y . A l l i s o n (1937) and Bever (1945) c r o s s e d U. hordei and u. nigra but f a i l e d to o b t a i n d e f i n i t e segre= g a t i o n p a t t e r n s f o r p a t h o g e n i c i t y . C h e r e w i c k (1964) p e r -formed i n t r a - and i n t e r s p e c i f i c c r o s s e s w i t h u. avenae and U. k o l l e r i , and w i t h u. nigra and u. hordei3 and t e s t e d the progeny on o a t s and b a r l e y r e s p e c t i v e l y . Some of the h y b r i d s e x h i b i t e d v i r u l e n c e as a dominant c h a r a c t e r , o t h e r s as an i n c o m p l e t e l y d o m i n a n t , and s t i l l o t h e r s as a r e c e s s i v e c h a r a c t e r . In n i n e out o f 78 t e t r a d s e t s o f s p o r i d i a l l i n e s , i s o l a t e d from F x h y b r i d t e l i o s p o r e s o f o a t s m u t , he 11 o b t a i n e d 3:1 s e g r e g a t i o n s of v i r u l e n c e to a v i r u l e n c e . H y b r i d s f rom i n t e r s p e c i f i c c r o s s e s showed a w i d e r range of p a t h o g e n i c i t y t h a n d i d e i t h e r the i n t r a s p e c i f i c c r o s s e s or the p a r e n t s . L a t e r , C h e r e w i c k (1967) t e s t e d the F i h y b r i d s and 27 s e t s o f d i k a r y o n s o f F 2 p r o g e n i e s f rom the u. hordei and u. n i g r a c r o s s e s on f i v e b a r l e y c u l t i v a r s . F i v e i n d e -p e n d e n t l y s e g r e g a t i n g v i r u l e n c e genes were i d e n t i f i e d i n t h i s s t u d y . Four o f t h e s e genes were r e c e s s i v e and one was domi n a n t . R e s i s t a n t Genes Soon a f t e r M e n d e l ' s work was d i s c o v e r e d , B i f f e n (1905) f o u n d t h a t r e s i s t a n c e to y e l l o w r u s t { P u o c i n i a glumarum) i n wheat was c o n t r o l l e d by one p a i r o f r e c e s s i v e g e n e s . He concluded t h a t d i s e a s e r e s i s t a n c e may be i n h e r i t e d a c c o r d i n g to M e n d e l ' s l a w s . T h i s l e d to a s e a r c h f o r genes g o v e r n i n g d i s e a s e r e s i s t a n c e and to b r e e d i n g f o r d i s e a s e r e s i s t a n c e i n c r o p p l a n t s . The l i t e r a t u r e i n t h i s f i e l d i s immense and many books and r e v i e w p a p e r s have been w r i t t e n ( e . g . Ausemus, 1943 ; D i c k s o n , 1956; W a l k e r , 1965 ; H o o k e r , 1967; Hooker et a l . 3 1 9 7 1 ) . I t can be s t a t e d t h a t i n most i n s t a n c e s r e s i s t a n c e i s i n h e r i t e d as a dominant c h a r a c t e r i s t i c . L i n k a g e and a l l e l i s m of r e s i s t a n c e genes i s commonly e n -c o u n t e r e d , whereas t h i s i s not the case w i t h v i r u l e n c e g e n e s . 12 In t h i s r e v i e w , o n l y the l i t e r a t u r e p e r t a i n i n g to r e s i s t a n c e i n b a r l e y to c o v e r e d smut w i l l be c o n s i d e r e d . In most o f the work c a r r i e d out i n N o r t h A m e r i c a , r e s i s t a n c e to r a c e 6 o f u. hovdei ( T a p k e , 1945) has been s t u d i e d . J o h n s t o n (1934) c r o s s e d two b a r l e y c u l t i v a r s G l a b r o n x T r e b i . G l a b r o n was h i g h l y r e s i s t a n t and i l tKebi was m o d e r a t e l y s u s c e p t i b l e to the i s o l a t e u s e d . J o h n s t o n d i d not r e c o g n i z e any d e f i n i t e p a t t e r n of i n h e r i t a n c e o f r e s i s t a n c e and s u s c e p t i b i l i t y i n the F 2 p r o g e n i e s . P u g s l e y and V i n e s (1946) r e p o r t e d t h a t more than two dominant genes c o n d i t i o n e d r e s i s t a n c e to an A u s t r a l i a n c o l l e c t i o n o f u. hovdei. Shand (1956) made c r o s s e s between a r e s i s t a n t c u l t i v a r and two s u s c e p t i b l e c u l t i v a r s , and s t u d i e d the s e g r e g a t i o n s i n F 2 , F 3 and b a c k c r o s s g e n e r a t i o n s when t h e s e were t e s t e d a g a i n s t r a c e 6 o f u. hovdei. He f o u n d one dominant gene i n the r e s i s t a n t c u l t i v a r . C o t t i n g h a m ( 1 9 5 8 ) , i n a c r o s s between P a n n i e r and O d e s s a , f o u n d t h a t two p a i r s o f genes a re res p o n s i b l e for r e s i s t a n c e i n P a n n i e r . In the c r o s s P a n n i e r x J e t , he o b s e r v e d a m o d i f i e d two f a c t o r r a t i o . From F i , F 2 and F 3 g e n e r a t i o n s o f d i a l l e l c r o s s e s between s i x c u l t i v a r s , f i v e r e s i s t a n t and one s u s c e p t i b l e to r a c e 6 , W e l l s (1958) i d e n t i f i e d two dominant genes ( U h x and U h 2 ) and two r e c e s s i v e genes ( u h 3 and u f u ) f o r r e s i s t a n c e . In c r o s s e s among f o u r r e s i s t a n t c u l t i v a r s he f a i l e d to g e t s e g r e g a t i o n and s u g g e s t e d a l l e l i s m o r c l o s e l i n k a g e among the r e s i s t a n c e genes p o s s e s s e d 13 by t h e s e c u l t i v a r s . From c r o s s e s o f J e t x Vantage and J e t x P l u s h , M e t c a l f (1962) i d e n t i f i e d a r e c e s s i v e gene f o r r e s i s -t a n c e i n the c u l t i v a r J e t . C h e r e w i c k and Buchannon (1969) r e p o r t e d a s i n g l e dominant r e s i s t a n c e gene i n c u l t i v a r s P a n n i e r and E x c e l s i o r , e f f e c t i v e a g a i n s t bo th u. hordei and U. n i g r a . S i h d u and P e r s o n (1972) c r o s s e d E x c e l s i o r to Vantage and to Hannchen . E x c e l s i o r was r e s i s t a n t to the u. hordei s t r a i n homozygous f o r v i r u l e n c e gene U h v i and s u s c e p t i b l e to t h e s t r a i n w h i c h was homozygous f o r U h v 2 . On the o t h e r h a n d , Vantage and Hannchen were both s u s c e p t i b l e to t h e U h v i and r e s i s t a n t to the U h v 2 b i o t y p e s . The s e g r e g a t i o n o f F 3 p r o g e n i e s f rom t h e s e c r o s s e s showed t h a t E x c e l s i o r c a r r i e d one r e s i s t a n c e g e n e , d e s i g n a t e d U h R 2 , and Vantage and Hannchen b o t h c a r r i e d a n o t h e r r e s i s t a n c e g e n e , U h R i . H o s t - P a r a s i t e R e l a t i o n s h i p s R e s i s t a n t and s u s c e p t i b l e phenotypes o f the hos t a r e c o n t r o l l e d by genes i n the h o s t , whereas v i r u l e n t and a v i r u l e n t phenotypes o f the p a r a s i t e a re c o n t r o l l e d by genes i n the p a r a s i t e . Y e t the d i s e a s e can be e x p r e s s e d o n l y when the h o s t and the p a r a s i t e i n t e r a c t w i t h one a n o t h e r . I t f o l l o w s t h a t the i n t e r a c t i o n w h i c h l e a d s to d i s e a s e d e v e l o p -ment , o r l a c k o f i t , must a l s o i n v o l v e i n t e r a c t i o n o f g e n e s , 14 or gene p r o d u c t s , p r e s e n t i n both the hos t and the p a r a s i t e . I t was t h r o u g h s t u d y i n g the g e n e t i c s o f bo th the h o s t and the p a r a s i t e t h a t F l o r d e v e l o p e d h i s c o n c e p t o f " g e n e - f o r -gene" r e l a t i o n s h i p s . In h i s p i o n e e r i n g s t u d i e s F l o r o b t a i n e d a s i n g l e -f a c t o r r a t i o o f a v i r u l e n c e to v i r u l e n c e when F 2 c u l t u r e s o f the r u s t fungus Melampsora l l n i were i n o c u l a t e d to f l a x c u l t i v a r s h a v i n g a s i n g l e dominant gene f o r r e s i s t a n c e . S i m i l a r l y , where the f l a x c u l t i v a r had two (or t h r e e ) dominance genes f o r r e s i s -t a n c e , two (or t h r e e ) f a c t o r s e g r e g a t i o n s f o r v i r u l e n c e and a v i r u l e n c e were o b t a i n e d . In p a r a l l e l s t u d i e s he a l s o f o u n d t h a t w i t h F 2 h o s t p o p u l a t i o n s a s i n g l e - f a c t o r s e g r e g a t i o n was o b t a i n e d when t h e s e were t e s t e d a g a i n s t r u s t c u l t u r e s h a v i n g a s i n g l e gene f o r v i r u l e n c e ; two (and t h r e e ) f a c t o r s e g r e g a t i o n s were o b t a i n e d when the F 2 h o s t p o p u l a t i o n s were t e s t e d a g a i n s t r u s t c u l t u r e s p o s s e s s i n g two (and t h r e e ) genes f o r v i r u l e n c e . These o b s e r v a t i o n s o f " c o m p l e m e n t a r y " i n t e r a c t i o n l e d f l o r to f o r m u l a t e h i s h y p o t h e s i s o f g e n e - f o r § g e n e r e l a t i o n -s h i p s , i n w h i c h he s u g g e s t e d t h a t f o r each gene p a i r t h a t c o n -d i t i o n s r e s i s t a n c e or s u s c e p t i b i l i t y i n the f l a x h o s t t h e r e i s a c o r r e s p o n d i n g gene p a i r i n the r u s t fungus t h a t c o n d i t i o n s a v i r u l e n c e o r v i r u l e n c e . Such " c o m p l e m e n t a r i t y " does n o t , o f c o u r s e , i m p l y a l l e l i s m of the genes i n the two o r g a n i s m s . However , genes f o r r e s i s t a n c e i n the h o s t and genes f o r 1 5 a v i r u l e n c e i n the p a r a s i t e can o n l y be i d e n t i f i e d when t h e y a r e b r o u g h t i n t o i n t e r a c t i o n w i t h one a n o t h e r . From h i s e x t e n s i v e i n v e s t i g a t i o n s F l o r ( f o r r e v i e w see F l o r , 1971) i d e n t i f i e d 26 genes f o r r e s i s t a n c e , f a l l i n g i n t o f i v e l o c i i n the f l a x h o s t , and 26 c o r r e s p o n d i n g v i r u l e n c e g e n e s , each a p p a r e n t l y a t a s e p a r a t e l o c u s i n the f l a x r u s t . In a l l t h e cases s t u d i e d i n the f l a x - f l a x r u s t s y s t e m , r e s i s -t a n c e was d o m i n a n t t o s u s c e p t i b i l i t y , and a v i r u l e n c e was dominant to v i r u l e n c e . P e r s o n (1959) p r o v i d e d a t h e o r e t i c a l b a s i s f o r F l o r ' s g e n e - f o r - g e n e h y p o t h e s i s by d e m o n s t r a t i n g that F l o r ' s d a t a f i t t e d an " i d e a l " g e n e t i c model of h o s t - p a r a s i t e r e l a t i o n -s h i p s . P e r s o n ' s model makes i t p o s s i b l e to p r e d i c t or s u g g e s t a g e n e - f o r - g e n e r e l a t i o n on the b a s i s o f g e n e t i c d a t a o b t a i n e d f rom s t u d i e s o f e i t h e r the h o s t o r the p a r a s i t e , o r f rom the d a t a o f p h y s i o l o g i c a r a c e s u r v e y s . T h i s i s o f s p e c i a l a d v a n t a g e i f the l i f e c y c l e o f one or both o f the members o f the h o s t - p a r a s i t e c c o m p l e x i s not known. P e r s o n et a l . ( 1 9 6 2 ) , a f t e r c o n s i d e r i n g the p o s s i b l e s i t u a t i o n s under w h i c h the g e n e - f o r - g e n e c o n c e p t i s a p p l i c a b l e , p r o v i d e d the f o l l o w i n g q u a l i f y i n g d e f i n i t i o n f o r the gene-f o r - g e n e r e l a t i o n s h i p : A gene-for-gene r e l a t i o n s h i p e x i s t s when the presence of a gene in one population i s contingent on the continued presence of a gene in another population3 and where the i n t e r a c t i o n between the two genes 16 leads to a s i n g l e phenotypic expression by which the presence or absence of the relevant gene in either organism may be recognized. A l t h o u g h the o p e r a t i o n of g e n e - f o r - g e n e r e l a t i o n -s h i p s has been s u g g e s t e d f o r more than 20 h o s t - p a r a s i t e systems i t has been s e c u r e l y e s t a b l i s h e d f o r o n l y a few s y s t e m s . Most o f t h e s e i n v o l v e p l a n t s and p a r a s i t i c f u n g i ( P e r s o n and S i d h u , 1971 ; P e r s o n and E b b a , u u n p u b l i s h e d ; and c f . T a b l e 1 ) . However , i n one case a t l l e a s t , the p a r a s i t e i s an i n s e c t ( H a t c h e t t et a l . 3 1 9 7 0 ) . In bunt and smut f u n g i g e n e - f o r - g e n e r e l a t i o n -s h i p s have been s u g g e s t e d or d e m o n s t r a t e d f o r f i v e s y s t e m s . S c h a l l e r et a l . (1960) s t u d i e d r a c e T - l o f T i l l e t i a c a r i e s and r a c e L - 9 o f T. f o e t i d a on two c u l t i v a r s of w h e a t , M a r t i n and E l g i n . M a r t i n c a r r i e s two r e s i s t a n t g e n e s , M and M i , w h i c h a r e e f f e c t i v e a g a i n s t r a c e s T - l and L - 9 , r e s p e c t i v e l y . E l g i n i s s u s c e p t i b l e to both of t h e s e r a c e s . Because of the s p e c i f i c i n t e r a c t i o n s between the two r e s i s t a n c e genes and the two r a c e s , t h e y s u g g e s t e d g e n e - f o r - g e n e r e l a t i o n -s h i p s . A few y e a r s l a t e r M e t z g e r et a l . (1962) s u g g e s t e d a g e n e - f o r - g e n e r e l a t i o n s h i p s i n the Triticum-T. f o e t i d a sys tem f o r the second t i m e , u s i n g the i n t e r a c t i o n s of seven r e s i s t a n c e genes i n the v u l g a r e wheat h o s t and 19 p a t h o g e n i c r a c e s o f bunt fungus and f o l l o w i n g P e r s o n ' s c r i t e r i a . Out o f t h e s e 19 r a c e s , r a c e T-14 was e f f e c t i v e a g a i n s t the M g e n e , 17 T a b l e 1 P a r a s i t i c Systems f o r Which the G e n e - f o r - G e n e R e l a t i o n s h i p has been e i t h e r D e m o n s t r a t e d o r P o s t u l a t e d P a r a s i t i c System R e f e r e n c e R u s t s : Linum-Melampsora l i n i F l o r 1947 , 1955 lea-Puccinia sorghi F l a n g a s and D i c k s o n 1961 Triticum-Puccinia s t r i i f o r m i s Zadoks 1961 Triticum-Puccinia graminis t r i t i c i L u i g and Watson 1961 Eelianthus-Puccini a h e l i a n t h i S a c k s t o n 1962 Coffea-Eemileia v a s t a t r i x Noronha-Wagner et al.. 1967 Triticum-Puccinia recondita S a m b o r s k i and Dyck 1968 Avena-Puccinia graminis avenae M a r t e n s et a l . 1970 •Mi 1 dews : Triticum-Evysiphe graminis t v i t i c i Powers and Sando 1957 Hordeum-Erysiphe graminis hordei Moseman 1959 Smuts : Triticum-Ustilago t r i t i c i O o r t 1963 Avena-Ustilago avenae H a l i s k y 1965 Hordeum-Ustilago hordei S i d h u and P e r s o n 1972 ( C o n t i n u e d ) 18 T a b l e 1 ( C o n t i n u e d ) P a r a s i t i c System R e f e r e n c e B u n t s : T r i t i c u m - T i l l e t i a cavies S c h a l l e r et al. 1960 T r i t i o u m - T i l l e t i a foetida S c h a l l e r et al. 1960 O t h e r s : Lycopersiaum-Cladosporium fulvum Day 1956 Solanum-Phytophthora infestans Toxopeus 1956 Malus-Venturia inaequalis Boone and K e i t t 1957 Phaseolus-Colletotvichum lindemuthianum A l b e r s h e i m et al. 1959 Oryza-Pyrioulavia ovyzae Kiyosawa 1967 Solanum-Synchytrium endobioticum Howard 1968 O t h e r s : ( N o n ? i f u n g a l ) : Gossypium-Xanthomonas malvaoearum B r i n k e r h o f 1970 Tritioum-May e t i o l a destructor H a t c h e t et al. 1970 19 r a c e T - l a g a i n s t the M 2 gene and T-10 a g a i n s t the gene i n c u l t i v a r H o h e n h e i m e r . V i r u l e n c e o f the r e m a i n i n g 16 r a c e s was i n each case s u g g e s t e d to be c o n d i t i o n e d by two or more g e n e s . From t h e i n t e r a c t i o n o f n i n e c u l t i v a r s o f wheat and s i x r a c e s o f l o o s e s m u t , O o r t (1963) s u g g e s t e d a g e n e - f o r -gene r e l a t i o n s h i p f o r the T v i t i o u m - U s t i l a g o t v i t i c i s y s t e m . The i n t e r a c t i o n o f the c u l t i v a r s and r a c e s do f i t the c o n -d i t i o n s pur f o r w a r d by P e r s o n f o r g e n e - f o r - g e n e r e l a t i o n s h i p . Murphy and Coffman (1961) s t u d i e d the g e n e t i c s o f r e s i s t a n c e i n c e r t a i n o a t c u l t i v a r s i n i n t e r a c t i o n w i t h c e r t a i n r a c e s of l o o s e smut o f o a t s . H o l t o n and H a l i s k y (1960) s t u d i e d the g e n e t i c s o f p a t h o g e n i c i t y i n l o o s e smut o f o a t s . From t h e s e two s e p a r a t e s t u d i e s , H a l i s k y (1965) s u g g e s t e d gene-f o r - g e n e r e l a t i o n s h i p s f o r the A v e n a - U s t i l a g o avenae s y s t e m . R e c e n t l y , S i d h u and P e r s o n (1972) c o n d u c t e d a p a r a l l e l g e n e t i c s t u d y o f the i n h e r i t a n c e o f r e s i s t a n c e and s u s c e p t i b i l i t y i n t h r e e b a r l e y c u l t i v a r s and o f the v i r u l e n c e and a v i r u l e n c e i n two b i o t y p e s o f c o v e r e d - s m u t o f b a r l e y . The two r e s i s t a n c e g e n e s , UhRi and U h R 2 , were both d o m i n a n t . In t h e smut f u n g u s the two c o r r e s p o n d i n g genes f o r v i r u l e n c e U h v a and U h v 2 , were both r e c e s s i v e . Genes U h v i and U h v 2 o f the fungus were e f f e c t i v e a g a i n s t UhRi and UhR 2 i n the h o s t , r e s p e c t i v e l y . Such a c o r r e s p o n d e n o e e b e t w e e n v - g e n e s i n the fungus and R-genes i n the h o s t , l e d them to the c o n c l u s i o n 20 t h a t t h e i n t e r a c t i o n s between Hordeum vulgave and U s t i l a g o hordei had t h e i r b a s i s i n g e n e - f o r - g e n e r e l a t i o n s h i p s . P A R T I MULTIPLE ALLELISM OF GENES CONTROLLING DIFFERENT LEVELS OF VIRULENCE IN USTILAGO HORDEI I n t r o d u c t i o n For p a r a s i t e s i n g e n e r a l , r e p o r t s o f l i n k a g e and * p a r t i c u l a r l y o f a l l e l i s m o f v i r u l e n c e genes are l a c k i n g . On the o t h e r h a n d , t h e r e are s e v e r a l r e p o r t s o f bo th l i n k a g e and a l l e l i s m o f r e s i s t a n c e genes i n the h o s t ( F l o r , 1971 ; Hooker et a l . 3 1962; Saxena et a l . , 1968; Moseman, 1966; K i y o s a w , 1967; P e r s o n et a l . 3 1 9 7 1 ) . The d i s e a s e r e a c t i o n s of c e r t a i n h o s t d i f f e r e n t i a l s , a p p a r e n t l y monogenic f o r r e s i s t a n c e , range f rom h i g h s u s -t c e p t i b i l i t y t h r o u g h i n t e r m e d i a t e r e s i s t a n c e to v e r y h i g h r e s i s t a n c e , d e p e n d i n g on the b i o t y p e of the pathogen w i t h w h i c h t h e y i n t e r a c t . S i m i l a r l y , c e r t a i n b i o t y p e s of the pathogen have shown v a r y i n g l e v e l s o f v i r u l e n c e on d i f f e r e n t h o s t s . Such a wide range of d i s e a s e has been o b s e r v e d i n most o f the sys tems ( e . g . r u s t s , s m u t s , m i l d e w s ) t h a t have been i n t e n s i v e l y s t u d i e d . A l i s t o f the many p o s s i b l e * For a p o s s i b l e e x c e p t i o n see Lawrence et al, ( P r o c . X I I I n t r . C o n g r . G e n e r i c s , 1 9 7 3 ) . 21 22 c o n t r i b u t i n g f a c t o r s would i n c l u d e a l l e l i s m , not o n l y f o r r e s i s t a n c e genes i n the h o s t but a l s o f o r v i r u l e n c e genes i n the p a t h o g e n . For f l a x and f l a x r u s t , F l o r (1970) o b s e r v e d t h a t a f l a x c u l t i v a r , monogenic f o r r e s i s t a n c e , gave t h r e e d i f f e r e n t l e v e l s o f d i s e a s e r e a c t i o n when i n o c u -l a t e d w i t h d i f f e r e n t c u l t u r e s o f f l a x r u s t . T h i s l e d him to p r o p o s e t h a t genes c o n t r o l l i n g v i r u l e n c e i n f l a x r u s t may o c c u r as m u l t i p l e a l l e l e s . The p u r p o s e o f the p r e s e n t s t u d y was to d e t e r m i n e w h e t h e r the genes i n U. hordei c o n t r o l l i n g d i f f e r e n t l e v e l s o f d i s e a s e r e a c t i o n on H. vulgar® a r e : ( i ) p o l y g e n e s ; ( i i ) a s e r i e s o f m u l t i p l e a l l e l e s ; or ( i i i ) s e p a r a t e major g e n e s . T h i s was done by s e l e c t i n g a p p r o p r i a t e t e s t c u l t u r e s o f u. hordei t h a t gave d i f f e r e n t l e v e l s o f d i s e a s e r e a c t i o n f o l l o w i n g i n o c u l a t i o n o f a s i n g l e b a r l e y c u l t i v a r , T r e b i . Se t s o f t e t r a d p r o d u c t s , d e v e l o p e d f rom s i n g l e t e l i o s p o r e s o f t h e s e c u l t u r e s , were s e l f e d , c r o s s e d , and b a c k c r o s s e d i n o r d e r to d e t e r m i n e the p a t t e r n of i n h e r i t a n c e of genes c o n -t r o l l i n g the v a r y i n g l e v e l s o f v i r u l e n c e . I t was d e m o n s t r a t e d t h a t t h e s e genes were members o f a m u l t i p l e a l l e l i c s e r i e s . M a t e r i a l s and Methods Samples o f t e l i o s p o r e s t a k e n from smut s o r i were o b t a i n e d f rom N. D a k o t a , U . S . A . The samples r e p r e s e n t e d the 23 13 r a c e s o f u. hordei d e s c r i b e d by Tapke ( 1 9 4 5 ) , w h i c h i n c i t e d i f f e r e n t l e v e l s o f d i s e a s e r e a c t i o n on v a r i o u s b a r l e y c u l t i v a r s . For the p r e s e n t s t u d y , f o u r s e t o f c u l t u r e s were e s t a b l i s h e d from s i n g l e t e l i o s p o r e s o f f o u r d i f f e r e n t r a c e s . These were chosen on the b a s i s o f the d i f f e r e n t l e v e l s o f v i r u l e n c e they e x h i b i t e d on a s i n g l e h o s t c u l t i v a r , C1-936 T r e b i . The f o u r t e l i o s p o r e s are d s i g n a t e d T l , T 2 , T 3 , and JA i n t h i s w o r k . The v a r i o u s l e v e l s o f d i s e a s e r e a c t i o n i n d u c e d by t h e s e c u l t u r e s a r e p r e s e n t e d i n T a b l e I - l and F i g u r e 1 . Se t s o f m e i o t i c p r o d u c t s ( t e t r a d s ) , t a k e n f rom a s i n g l e t e l i o s p o r e s were used to e s t a b l i s h the m o n o s p o r i d i a l c u l t u r e s . The t e t r a d s were i s o l a t e d by the use o f a de Fonbrune m i c r o m a n i p u l a t o r . A s i n g l e o r a few t e l i o s p o r e s were g e r m i n a t e d on an agar b l o c k p l a c e d on a c o v e r s l i p . A f t e r 8-12 hours o f i n c u b a t i o n a t 2 2 ° C , a c o m p l e t e s e t o f m e i o t i c p r o d u c t s a r r a n g e d i n a l i n e a r t e t r a d were then drawn away f rom the p r o m y c e l i u m , one a t a t i m e , w i t h t h e a i d . o f t h e m i c r o m a n i p u l a t o r . The p r o d u c t s were numbered 1 t h r o u g h 4 s t a r t i n g w i t h the s p o r i d i u m t a k e n f rom the apex of the p r o m y c e l i u m . The s p o r i d i a were l e f t f o r 4 -5 days a t 22°C on the agar b l o c k u n t i l the f o u r s m a l l coil-onies were j u s t v i s i b l e . The c o l o n i e s were then t r a n s f e r r e d to f r e s h c o m p l e t e medium i n a p e t r i - d i s h f o r f u r t h e r g r o w t h . A f t e r the c o l o n i e s grew to a s u i t a b l e s i z e , t e s t s f o r m a t i n g - t y p e were c a r r i e d o u t , u s i n g the Bauch (1932) 24a Figure 1. A diagram to i l l u s t r a t e the general plan of the experiment. Culture sets were derived from three te l iospores (Tl , T3 and T 4 ) ; these were used to develop three sets of Fi dikaryons through c r o s s i n g , as well as three sets of dikaryons through s e l f i n g ; they were also used to develop the six possible sets of backcross (BC) dikaryons. (For s i m p l i c i t y , a fourth set of parental cultures and other sets of Fi cul tures* aailso used in the experiment, are not i l l u s t r a t e d . ) 24 b v a v C (44%) 25 t e c h n i q u e . M a t i n g t y p e i n u. hordei i s b i p o l a r , i . e . two o f the s p o r i d i a a re ' A ' and the o t h e r two ' a ' . In B a u c h ' s t e c h n i q u e , the p r o d u c t i o n of S u c h f a d e n ( i n f e c t i o n hyphae) i s an i n d i c a t i o n t h a t the two mixed s p o r i d i a ! c u l t u r e s a r e o f o p p o s i t e m a t i n g t y p e s . The c u l t u r e s were m a i n t a i n e d on c o m p l e t e medium by t r a n s f e r e v e r y t h r e e w e e k s , or were s t o r e d on s i l i c a ge l f o r l o n g e r p e r i o d s of t i m e . In s e l f i n g the f o u r m o n o s p o r i d i a l l i n e s f r o m a t e l i o s p o r e can be p a i i t e d i n s i x d i f f e r e n t w a y s . However , o n l y f o u r o f the s i x c o m b i n a t i o n s are c o m p a t i b l e . A c o m p a t i b l e m a t i n g r e s u l t s i n the f o r m a t i o n of a d i k a r y o n , the o n l y s t a g e i n the l i f e c y c l e o f t h i s f u n g u s w h i c h i s p a r a s i t i c . In c r o s s i n g , w i t h e i g h t s p o r i d i a l l i n e s ( i . e . a s e t o f f o u r f rom each o f two t e l i o s p o r e s ) , 16 c o m b i n a -t i o n s a r e p o s s i b l e o f w h i c h o n l y 8 a re c o m p a t i b l e . For the b a c k c r o s s e s , to both p a r e n t s , two s e t s of t e t r a d s t a k e n from two F i t e l i o s p o r e s were used f o r each c r o s s , e x c e p t f o r the T l x T3 c r o s s , where f o u r s e t s o f t e t r a d s were u s e d . The s p o r i d i a l l i n e s were o b t a i n e d f rom F i t e l i o s p o r e s by t h e t e c h n i q u e d e s c r i b e d e a r l i e r f o r the p a r e n t a l t e l i o s p o r e s . As i n c r o s s i n g , t h e r e a r e 16 p o s s i b l e c o m b i n a t i o n s of the 26 s p o r i d i a l l i n e s i n a b a c k c r o s s , but o n l y 8 o f t h e s e m a t i n g s a r e c o m p a t i b l e . For i n o c u l a t i o n , the d i f f e r e n t s p o r i d i a l l i n e s were t r a n s f e r r e d to E r l e n m e y e r f l a s k s c o n t a i n i n g the d e s i r e d amount o f l i q u i d medium ( c f . A p p e n d i x A ) . The f l a s k s were shaken i n a 22°C i n c u b a t o r f o r 3-5 d a y s , d e p e n d i n g on the amount o f i n o c u l u m w h i c h was n e e d e d . B e f o r e i n o c u l a t i o n , seeds of the d i f f e r e n t i a l b a r l e y c u l t i v a r T r e b i were f r e e d f rom e x t e r n a l c o n t a m i n a t i o n . Seeds were soaked i n a d i l u t e s o l u t i o n of c o m m e r c i a l f o r m a l i n (1 p a r t f o r m a l i n to 320 p a r t s w a t e r ) f o r one hour and r i n s e d i n r u n n i n g w a t e r f o r about 30 m i n u t e s . The seeds were then s p r e a d o u t on paper t o w e l s to d r y . The p a r t i a l vacuum method d e s c r i b e d by Tapke and Bever (1943) was used to i n o c u l a t e the s e e d s . About 100-150 seeds were p l a c e d i n a dram v i a l . About 8 ml o f the s p o r i d i a l s u s p e n s i o n (enough to c o v e r the s e e d s ; 4 ml o f each m a t i n g t y p e ) , were poured on the s e e d s . ( s p o r i -d i a l l i n e s o f o p p o s i t e m a t i n g t y p e s can be grown e i t h e r s p e a r a t e l y o r m i x e d ) . The v i a l o f seeds and i n o c u l u m was then p l a c e d i n vacuum d e s i c c a t o r ( w i t h o u t d e s i c c a n t ) and s u b j e c t e d to p a r t i a l vacuum ( n e g a t i v e p r e s s u r e s o f ~ 20 i n c h e s ) f o r 20-30 m i n u t e s . The vacuum was r e l e a s e d a b r u p t l y to f a c i l i t a t e the p e n e t r a t i o n or adherance o f the i n o c u l u m 27 to t h e s e e d . A f t e r s e t t l i n g , the e x c e s s w a t e r was poured o f f and the seeds were put i n t o a paper c o i n e n v e l o p and s e t a s i d e , w i t h the tops o p e n , to d r y f o r a day or t w o . The d r i e d seeds were then p l a n t e d d i r e c t l y i n t t h e f i e l d o r i n pots i n the g r e e n h o u s e . However , i f the i n o c u l a t e d seeds were to be k e p t f o r more than t h r e e days b e f o r e p l a n t -i n g , they were s t o r e d i n a c o l d room ( 4 ° C ) u n t i l p l a n t i n g t i m e . In the f i e l d , about 100 o r 150 seeds were p l a n t e d to a 1 0 - f o o t and a 1 5 - f o o t r o w , r e s p e c i t v e l y . A f t e r the p l a n t s were h e a d e d , both s m u t t e d and h e a l t h y p l a n t s were c o u n t e d . D i s e a s e r e a c t i o n s were e x p r e s s e d as p e r c e n t a g e s o f smut ted p l a n t s out o f the t o t a l number of p l a n t s i n the r o w . In t h i s s t u d y , a p l a n t i s a u n i t made up of a s i n g l e o r s e v e r a l t i l l e r s a l l f rom the same s e e d . A p l a n t i s c o n s i d e r e d smut ted i f one or more o f the t i l l e r s a re e i t h e r p a r t i a l l y o r c o m p l e t e l y s m u t t e d . R e s u l t s S e l f i n g of the p r o d u c t s o f s i n g l e t e l i o s p o r e s gave r e s u l t s w h i c h i n d i c a t e d t h a t the t e l i o s p o r e s were homozygous f o r v i r u l e n c e . C u l t u r e s e t T l p o s s e s s e d v i r u l e n c e genes c o n t r o l l i n g the h i g h e s t l e v e l o f d i s e a s e r e a c t i o n ( a v . 44%) and c u l t u r e s e t T4 the l o w e s t l e v e l ( a v . 3%). In a l l the 28 c r o s s e s t h e h i g h e r l e v e l s of v i r u l e n c e were d o m i n a n t o v e r the l o w e r l e v e l s . In the f i r s t s e t o f d i a l l e l c r o s s e s , i n v o l v i n g c u l t u r e s e t s T l , T3 and T 4 , the h i g h v i r u l e n c e l e v e l o f c u l t u r e s e t T l was dominant i n a l l c r o s s e s . The v i r u l e n c e l e v e l o f c u l t u r e s e t T3 i s dominant to t h a t o f c u l t u r e s e t T 4 . In the second s e t o f d i a l l e l c r o s s e s among c u l t u r e s e t s T l , T2 and T 4 , the v i r u l e n c e l e v e l o f T l was dominant o v e r t h a t of T2 arid T 4 , whereas t h a t o f T2 was dominant o v e r t h a t o f T 4 . S e t s o f two or f o u r h y b r i d c u l t u r e s , t a k e n from F x t e l i o s p o r e s o f the i n t e r - c u l t u r e c r o s s e s , were used i n the b a c k c r o s s e s . In a l l the b a c k c r o s s e s s e g r e g a t i o n s f o r h i g h and low l e v e l s o f v i r u l e n c e were o b s e r v e d . B a c k c r o s s e s to t h e p a r e n t a l c u l t u r e s e t h a v i n g the h i g h - l e v e l o f v i r u l e n c e showed no s e g r e g a t i o n , w i t h a l l b a c k c r o s s d i k a r y o n s g i v i n g about the same l e v e l o f v i r u l e n c e as t h a t o f the p a r e n t h a v i n g the h i g h l e v e l o f v i r u l e n c e . However , the d i k a r y o n s f rom b a c k -c r o s s e s to the p a r e n t a l c u l t u r e w i t h the low l e v e l o f v i r u l e n c e s e g r e g a t e d to g i v e a 4 :4 r a t i o o f h i g h to low l e v e l s o f v i r u l e n c e . These o b s e r v a t i o n s are shown i n T a b l e 1 -2 . In the b a c k c r o s s e s i n v o l v i n g p a r e n t a l c u l t u r e s e t T4 ( T a b l e 1 - 4 ) , z e r o (-0%) v a l u e s a r e o f t e n o b s e r v e d . S i n c e the l e v e l o f i n f e c t i o n i s v e r y low i t i s p o s s i b l e t h a t a l l the p l a n t s i n a row may escape i n f e c t i o n . T h i s was o b s e r v e d when the p a r e n t a l c u l t u r e s e t T4 was s e l f e d , but t h i s i s not seen i n 29 T a b l e I - l D i s e a s e R e a c t i o n O b t a i n e d on the B a r l e y C u l t i v a r T r e b i by S e l f i n g and C r o s s i n g of S p o r i d i a l L i n e s f r o m Four S e t s of U. hordei C u l t u r e s D i k a r y o n s O b t a i n e d T h r o u g h : D i k a r y o t i c L i n e Di sease^^ R e a c t i o n (%) Genotype * S e l f i ng T l - 1 X T l -2 42 a a V V n X 4 49 n 3 X 2 44 n n X 4 43 T 2 - r X T2-2 15 b b V V II X 4 15 II 3 X 2 15 II n X 4 14 M T 3 - 1 " X T3-2 7 c c V V n X 3 8 II 4" X 2 9 II II X 3 7 II d d T 4 - T X IA-3 2 V V X 4 4 3 2" X 3 3 2 n X 4 4 3 ( C o n t i n u e d ) Tapke (1945) gave 43%, 23%, 13% and 5% f o r r a c e s f r o m w h i c h c u l t u r e s T l , T 2 , T3 and T 4 , r e s p e c t i v e l y , were o b t a i n e d . The d a t a shown are a v e r a g e s of two or more s e p a r a t e t e s t s . 30 T a b l e I - l ( C o n t i n u e d ) D i k a r y o n s O b t a i n e d T h r o u g h : D i k a r y o t i c L i n e D i s e a s e R e a c t i on (%) Genotype C r o s s i ng T l - 1 x T2-1 1 32 a b V V " x 3 2 37 II 2 x 2 3 40 n " x 4 4 50 n 3 x 1 5 40 II " x 3 6 53 n 4 x 2 7 36 II " x 4 8 40 T l - 1 x T3 -2 9 43 v a v c " x 3 10 46 2 x 1 11 48 •I " x 4 1 2 43 •I 3 x 2 13 46 n " x 3 14 35 II 4 x 1 1 5 41 n " * 4 16 47 II T l - T ' x T 4 - 3 17 37 a d V V " , x 4 18 49 n 2 x 1 19 44 n " x 2 20 48 n 3 x 3 21 49 II 4 x 4 22 47 II 4 x 1 23 43 II " x 2 24 43 II T2-1 x T4-1 25 11 b d V V " x 3 26 13 n 2 x 1 27 1 5 " x 3 28 14 n 3 x 2 29 9 " x 4 30 1 2 II 4 x 2 31 11 11 x 4 32 14 II 31 T a b l e 1-1 ( C o n t i n u e d ) D i k a r y o n s O b t a i n e d T h r o u g h : D i k a r y o t i c L i n e Di sease R e a c t i on {%) Genotype T3-1 x T 4 - 3 33 1 2 c d V V " x 4 34 10 2 x 1 35 9 " x 2 36 10 3 x 1 37 8 " x 2 38 10 4 x 3 39 8 4 40 7 Tab! e 1-2 ' lab'! e 1-2 D i s e a s e R e a c t i o n s O b t a i n e d on B a r l e y C u l t i v a r T r e b i F o l l o w i n g B a c k c r o s s i n g F i C u l t u r e s ( t e t r a d s e t s ) to the Two P a r e n t a l S e t s , T l and T2 R e s u l t s o f B a c k c r o s s e s to P a r e n t T l B a c k c r o s s e s o f S p o r i d i a l D e r i v e d From L i n e s R e s u l t s o f B a c k c r o s s e s P a r e n t T2 to Genotype A s s i g n e d Di sease R e a c t i o n * ^ ) P a r e n t T l X FI P a r e n t x T2 Di sease R e a c t i o n [%) Genotype Al ' s ' i g-ned a b V V 22 2 X ** 1 a - l x 1 15 b b v v I I 27 4 X X 3 13 n a a V V 33 1 X 2 X 2 16 d u V V I I 33 3 X n X 4 21 I I n 32 2 X 3 X 1 23 n I I a b V V 27 1:8 4 1 X X I I 4 X X 3 2 25 9 n b b V V 1! 21 3 X X 4 11 a b v " y a . 33 1 X l b - 1 X 2 16 b b V V - I I -•>'. I I 32 3 X I I X 4 13 n !l 28 2 X 2 X 1 15 n I I i 26 4 X n X 3 18 a v V 32 2 X 3 X 1 28 v " v u I I 41 4 X I I X 3 21 I I I I 24 1 X 4 X 2 22 I I I I 27 3 X n X 4 21 I I The d a t a shown i n Tables- r 1-2 t h r o u g h 1-6 a re a v e r a g e s o f two or t h r e e s e p a r a t e t e s t s . R e f e r s to the F i o f the T l x T2 c r o s s ; d i k a r y o t i c l i n e s a r i s i n g f rom t h i s c r o s s a re as numbered i n T a b l e 1 - 1 . For e x a m p l e , l a and l b a re d i f f e r e n t t e l i o s p o r e s o f the same c r o s s . T a b l e 1-3 D i s e a s e R e a c t i o n s O b t a i n e d on B a r l e y C u l t i v a r T r e b i F o l l o w i n g B a c k c r o s s i n g F i C u l t u r e s ( t e t r a d s e t s ) to the Two P a r e n t a l S e t s , T l and T3 R e s u l t s o f B a c k c r o s s e s to P a r e n t T l B a c k c r o s s e s o f S p o r i d i a l L i n e s D e r i v e d From R e s u l t s o f B a c k c r o s s e s P a r e n t T3 to Genotype T r D i s e a s e R e a c t i o n (%) P a r e n t T l FI P a r e n t :'[| T3T-' D i s e a s e R e a c t i o n (%) Genotype a c v v II a a v v II a c v v II a a v v v a v a a c v v 37 34 39 41 37 38 37 31 37 26 38 32 37 35 36 33 1 3 2 4 1 3 2 4 1 3 2 4 1 3 2 4 x x X X X X X X X X X X X X X X 1 5 b - l 1 X 1 II X 4 2 X 2 n X 3 3 X 1 II X 4 4 X 2 II X 3 1 X 1 II X 4 2 X 2 n X 3 3 X 1 II X 4 4 X 2 n X 3 1 2 9 39 41 7 4 33 37 41 39 42 35 9 5 9 3 c c V V II a c v v c c v v a c v v a c v v c c v v T a b l e 1-4 D i s e a s e R e a c t i o n s O b t a i n e d on B a r l e y C u l t i v a r T r e b i F o l l o w i n g B a c k c r o s s i n g F i C u l t u r e s ( t e t r a d s e t s ) to the Two P a r e n t a l S e t s , T l and T4 R e s u l t s o f B a c k c r o s s e s to P a r e n t T l B a c k c r o s s e s o f S p o r i d i a l L i n e s D e r i v e d From R e s u l t s o f B a c k c r o s s e s to Pare .nt tT4 Genotype D i s e a s e R e a c t i o n {%) P a r e n t T l P a r e n t T4 D i sease R e a c t i o n (%) Genotype a d v v I I a a v v a d v v II a d v v II a a v v I I I I 40 39 33 35 35 34 29 36 36 31 35 31 33 42 37 31 1 2 3 4 1 2 3 4 3 4 1 2 3 4 1 2 x x X X X X X X X X X X X X X X 2 3 a - l 2 3 b - l 1 X 1 I I X 3 2 X 2 n X 4 3 X 1 M X 3 4 X 2 II X 4 1 X 2 X 4 2 X 1 X 3 3 X 2 n X 4 4 X 1 n X 3 3 0 21 22 20 20 8 2 3 6 6 4 28 23 21 21 d d v v n a d v v I I d d v v d d v v II I I ( C o n t i nued) T a b l e 1-4 ( C o n t i n u e d ) R e s u l t s o f B a c k c r o s s e s P a r e n t T l to B a c k c r o s s e s o f S p o r i d i a l D e r i v e d From L i n e s R e s u l t s of B a c k c r o s s e s to P a r e n t T4 Genotype D i s e a s e R e a c t i o n P a r e n t T l X P a r e n t x T4 D i s e a s e R e a c t i o n (%) Genotype a a V V 33 1 X 2 3 c - l X 1 25 a d v V II a d V V 38 35 3 2 X X II 2 X X 2 3 25 0 d d V V 35 4 X II X 4 2 II a rl v a v a 33 1 X 3 X 1 23 Q U V V II a d V V 33 31 3 2 X X II 4 X X 2 3 26 0 II d d V V 31 4 X II X 4 0 II 34 2 X 24-1 X 3 3 d d V V II 32 4 X M X 4 2 ll a d a a V V 29 2 X 2 X 3 19 V V II a d V V 49 52 4 1 X < X II 3 X X 4 1 25 1 II d d V V n 39 3 X II X 2 4 II a d a d V V 53 1 X 4 X 1 23 v d v u 57 3 X M X 2 26 II GO T a b l e 1-5 D i s e a s e R e a c t i o n s O b t a i n e d on B a r l e y C u l t i v a r T r e b i F o l l o w i n g B a c k c r o s s i n g F i C u l t u r e s ( t e t r a d s e t s ) to the Two P a r e n t a l S e t s , T4 and T2 R e s u l t s o f B a c k c r o s s e s to P a r e n t T4 B a c k c r o s s e s o f S p o r i d i a l L i n e s D e r i v e d From R e s u l t s o f B a c k c r o s s e s to P a r e n t T2 Genotype D i s e a s e R e a c t i o n [%) P a r e n t P a r e n t T4 X X T2 3 X 2 5 a - l X 1 4 X II X 3 1 X 2 X 2 2 x ; II X 4 3 X 3 X 1 4 X X 3 1 X 4 X 2 2 X II X 4 1 X 2 5 b - l X 2 2 X H X 4 1 X 2 X 2 2 X II X 4 3 X 3 X 1 4 X II X 3 3 X 4 X 1 4 X II X 3 Di sease R e a c t i o n {%) Genotype d d v v n b d v v II n II d d v v II II II b d v v II II 1 0 2 0 19 19 20 16 1 2 4 3 25 26 24 20 9 10 12 9 33 29 32 23 11 13 26 28 37 41 47 47 b d v v M b b v v b d v v II n II b b v v M CO T a b l e 1-6 D i s e a s e R e a c t i o n s O b t a i n e d on B a r l e y C u l t i v a r T r e b i F o l l o w i n g B a c k c r o s s i n g F i C u l t u r e s ( t e t r a d s e t s ) to the Two P a r e n t a l S e t s , T3 and T4 R e s u l t s o f B a c k c r o s s e s to B a c k c r o s s e s of S p o r i d i a l L i n e s R e s u l t s o f B a c k c r o s s e s to P a r e n t T3 D e r i ved From P a r e n t T4 Genotype D i s e a s e R e a c t i o n (%) P a r e n t T3 X F i X P a r e n t T4 Di sease R e a c t i o n (%) Genotype c d " V V 7 2 X 3 3 a - l X 3 0 d d V V it 9 3 X X 4 3 n II 9 2 X 2 X 3 0 II II c c v v 10 8 3 1 X X n 3 X X 4 1 2 8 II c d v v II 6 4 X II X * 2 5 II II 5 1 X 4 X X 1 7 II 6 4 • X II X 2 9 II c c v v 5 2 X 3 3 b - l X 3 7 c d V V II c d V V 13 8 3 1 X X ' 2 X X 4 1 6 3 II d d V V II 6 4 X n X 2 0 H 7 2 X 3 X 3 0 c c V V 6 -14 3 1 X X n 4 X X 4 1 0 6 n c d V V II 6 4 X n X 2 8 38 the t a b l e because the v a l u e s i n the t a b l e r e p r e s e n t a v e r a g e s of t h r e e s e p a r a t e t e s t s i n v o l v i n g o v e r 200 p l a n t s per com-p a t i b l e c o m b i n a t i o n . In the case of F i c u l t u r e s t a k e n from the T l x T4 c r o s s and b a c k c r o s s e d to c u l t u r e s e t T 4 , t h e r e was s e g r e g a t i o n of h i g h and low l e v e l s o f v i r u l e n c e . However , the h i g h l e v e l s o f v i r u l e n c e were not as h i g h as t h o s e f o r p a r e n t T l . In b a c k c r o s s e s of h y b r i d c u l t u r e s f rom the T2 x T4 c r o s s to c u l t u r e s e t T 2 , a 4 : 4 s e g r e g a t i o n of hiigh and low v i r u l e n c e was o b s e r v e d . The low v i r u l e n c e c l a s s was as h i g h as the v i r u l e n c e l e v e l o f T 2 , whereas the h i g h v i r u l e n c e c l a s s was much h i g h e r than t h i s . Di s c u s s i on B i o t y p e s of p a t h o g e n i c f u n g i may d i f f e r i n t h e i r i n t e r a c t i o n w i t h h o s t c u l t i v a r s i n r e l a t i o n to w h e t h e r , on a s p e c i f i c c u l t i v a r , d i s e a s e w i l l o r w i l l not be e x p r e s s e d . These d i f f e r e n c e s i n s s p e c i f i c i t y a r e u s u a l l y e x p r e s s e d as v i r u l e n c e and a v i r u l e n c e . I t i s commonly a c c e p t e d t h a t such d i f f e r e n c e s a r e c o n t r o l l e d by one o r a few g e n e s , w h i c h , i n t h i s c o n t e x t , a r e r e f e r r e d to as " m a j o r " g e n e s . B i o t y p e s may a l s o f a i l to e x h i b i t s p e c i f i c i t y a n d , i n such c a s e s , two b i o t y p e s may d i f f e r i n the s e v e r i t y o f the d i s e a s e r e a c t i o n s w h i c h are e l i c i t e d f o l l o w i n g i n o c u l a t i o n to a number of 39 h o s t g e n o t y p e s or c u l t i v a r s . Such d i f f e r e n c e s a re r e f e r r e d to by Van der P l a n k as d i f f e r e n c e s i n " a g g r e s s i v e n e s s , " and i t i s h i s o p i n i o n t h a t d i f f e r e n c e s o f t h i s k i n d are c o n t r o l l e d by p o l y g e n e s (Van der P l a n k , 1968, 1 9 6 9 ) . The r e s u l t s o b t a i n e d i n t h i s s t u d y have shown t h a t d i f f e r i n g l e v e l s o f d i s e a s e r e a c t i o n , e l i c i t e d by d i f f e r e n t b i o t y p e s o f the pathogen on a s p e c i f i c h o s t c u l t i v a r can a l s o be c o n t r o l l e d by d i f f e r e n t a l l e l e s a t a s i n g l e v i r u l e n c e l o c u s of the p a t h o g e n . F l o r - $ 1 9 7 0 ) p r e s e n t e d i n d i r e c t e v i d e n c e f o r a l l e l i s m i n f l a x r u s t i In h i s s t u d y o f r e s i s t a n c e i n f l a x he noted t h a t a c u l t i v a r , C a s s , known to p o s s e s s a s i n g l e r e s i s t a n c e g e n e , gave t h r e e l e v e l s o f d i s e a s e r e a c t i o n ; immune ( i n f e c -t i o n t y p e 0 ) , i n t e r m e d i a t e ( i n f e c t i o n t y p e 1 ) , and h i g h l y s u s c e p t i b l e ( i n f e c t i o n t y p e s 3 - 4 ) , to d i f f e r e n t c u l t u r e s o f f l a x r u s t . From t h i s e v i d e n c e , w h i c h i s b o t h i n d i r e c t and g e n e t i c a l l y l i m i t e d . F l o r s u g g e s t e d t h a t v i r u l e n c e genes i n the f l a x r u s t may o c c u r as m u l t i p l e a l l e l e s . In the p r e s e n t s t u d y , the genes g o v e r n i n g d i f f e r e n t l e v e l s o f v i r u l e n c e showed p h e n o t y p i c i n t e r a c t i o n , a common f e a t u r e of a l l e l i c g e n e s . Dominance o f the h i g h o v e r the low l e v e l o f v i r u l e n c e i s e v i d e n t i n a l l the c r o s s e s . T h i s f a c t i s a l s o made e v i d e n t i n the b a c k c r o s s s t u d i e s . B a c k -c r o s s e s o f the h y b r i d s to the p a r e n t s w i t h the h i g h l e v e l o f v i r u l e n c e d i d not show any s e g r e g a t i o n . However , b a c k c r o s s e s 40 to the p a r e n t s w i t h low v i r u l e n c e gave 4 : 4 s e g r e g a t i o n s of h i g h and low v i r u l e n c e l e v e l s . T h i s i n d i c a t e s t h a t a s i n g l e g e n e t i c l o c u s i s i n v o l v e d i n d e t e r m i n i n g the two l e v e l s o f v i r u l e n c e . The a l l e l i c genes c o n t r o l l i n g f o u r v i r u l e n c e l e v e l s i n the f o u r c u l t u r e s were a s s i g n e d the f o l l o w i n g g e n o t y p e s . c u l t u r e s e t T l U h v a U h v a " T2 U h v b U h v b " T3 U h v c U h v c " T4 U h v d U h v d These a l l e l e s form a l i n e a r sequence i n r e l a t i o n to d o m i n a n c e , a f e a t u r e w h i c h i s c h a r a c t e r i s t i c o f o t h e r m u l t i p l e a l l e l i c s e r i e s . A l l e l e v a i s dominant to a l l t h e o t h e r a l l e l e s ; v b i s dominant to v c and v d ; and v c d b i s dominant to v . The a l l e l i c r e l a t i o n s h i p between v and v c was not d i r e c t l y d e m o n s t r a t e d i n t h i s s t u d y . However , t h i s r e l a t i o n s h i p can be deduced f rom what i s known of the r e m a i n i n g a l l e l e s o f the s e r i e s . T h u s , s i n c e v a i s a l l e l i c to v b , v c and v 0 * , and v b i s a l l e l i c to v ^ , then v b must a l s o be a l l e l i c to v c . In b a c k c r o s s e s to T4 o f F i c u l t u r e s f rom the T l x T:4 c r o s s , the h i g h v i r u l e n c e w h i c h s e g r e g a t e d was not as * For b r e v i t y when w r i t i n g g e n o t y p e s of the p a r a s i t e or o f the h o s t , the d e s i g n a t i o n Uh w i l l be o m i t t e d t h r o u g h o u t the r e m a i n d e r o f t h i s t h e s i i s ( e . g . UhvaUhva and UhRUhR w i l l be w r i t t e n v a v a and RR, r e s p e c t i v e ) . 41 h i g h as t h a t o f the h i g h p a r e n t , T l . However , the s e g r e -g a t i o n f o r h i g h and low v i r u l e n c e was u n a m b i g u o u s . H i g h v i r u l e n c e s i m i l a r to t h a t o f T l i s e x p e c t e d i f the v i r u l e n c e l e v e l o f T l i s dominant o v e r t h a t o f T 4 , as o b s e r v e d i n the F i . The r e d u c t i o n i n v i r u l e n c e l e v e l , i n t h i s c a s e , may have come about f o r two d i f f e r e n t r e a s o n s . F i r s t , e n v i r o n -ment c o u l d have had a pronounced e f f e c t on some c u l t u r e s . T h i s has been o b s e r v e d when p l a n t i n g d a t e s have d i f f e r e d and a l s o when t - p l a n t i h g s 9 were done i n d i f f e r e n t l o c a l i t i e s . In g e n e r a l , the l e v e l s o f d i s e a s e r e a c t i o n have been f o u n d to be s e n s i t i v e to e n v i r o n m e n t a l f a c t o r s ( c f . P a r t I I ) . S i n c e s i m i l a r r e s u l t s were o b s e r v e d a t d i f f e r e n t p l a n t i n g d a t e s and l o c a l i t i e s i n t h i s p a r t i c u l a r e x p e r i m e n t , the o b s e r v a t i o n c a n n o t be f u l l y e x p l a i n e d as due to e n v i r o n m e n t a l o n e . S e c o n d , the b a c k g r o u n d m i n o r genes p r e s e n t i n T4 may have a c t e d as m o d i f i e r s i n r e d u c i n g the v i r u l e n c e l e v e l s . The e f f e c t o f t h e s e m i n o r genes m i g h t have been overcome i n F i by the b a c k g r o u n d genes of T l . A n o t h e r o b s e r v a t i o n t h a t needs to be m e n t i o n e d c o n c e r n s the b a c k c r o s s to T2 of t e t r a d s f rom F i ' s o f the T2 x T4 c r o s s . The F i h y b r i d s o f the T2 x T4 c r o s s gave a v i r u l e n c e l e v e l c l o s e to t h a t o f T 2 . T h e r e f o r e , the h i g h v i r u l e n c e of T2 s h o u l d be d o m i n a n t , and b a c k c r o s s e s to T2 s h o u l d a l l g i v e the same v i r u l e n c e l e v e l as T 2 . However , t h e r e was a 4 : 4 s e g r e g a t i o n o f h i g h and low l e v e l s o f 42 v i r u l e n c e i n t h e s e b a c k c r o s s e s . The low v i r u l e n c e i s c l o s e to t h a t o f T 2 , but t h e r e i s a l a r g e d i f f e r e n c e between the v i r u l e n c e l e v e l o f T2 and the h i g h l e v e l o b s e r v e d i n t h i s w o r k . I t appears t h a t m i n o r genes are a l s o s e g r e g a t i n g i n the v b c u l t u r e s . Even though t h e r e a re few r e p o r t s o f l i n k a g e and of a l l e l i s m o f v i r u l e n c e genes i n the p a r a s i t e , t h e r e a re s e v e r a l w e l l documented cases of l i n k a g e and a l l e l i s m o f r e s i s t a n c e genes i n the h o s t ( F l o r , 1971 ; Hooker et a l . , 1962 ; .1971 ; Saxena et a l . , 1 968 ; Moseman, 1 966 ; P e r s o n et. a l . , 1971 ; K i y o s a w a , 1 9 6 7 ) . S i n c e the most e x t e n s i v e work has been done w i t h f l a x i t w i l l be c o n s i d e r e d h e r e . In f l a x , F l o r i d e n t i f i e d t w e n t y - s i x r e s i s t a n c e genes a t f i v e s e p a r a t e l o c i ( K , L , M , N and P , w i t h 1 , 12 , 6 , 3 and 4 a l l e l e s r e s p e c t i v e l y ) . The q u e s t i o n of w h e t h e r t h e s e genes are t r u e a l l e l e s o r c l o s e l y l i n k e d genes has been i n v e s t i g a t e d f o r two of the l o c i by Shepherd and Mayo ( 1 9 7 2 ) , u s i n g asmbdif :niedoci s - i t r a n s r t e s t v a d n u T : h e i » . e v i d e n c e a i i n d i c a t e s t h a t the L - l o c u s i s a complex l o c u s and t h a t the genes a t t h i s l o c u s are t r u e a l l e l e s . In the case of the M l o c u s t h e y were u n a b l e to d e c i d e whether the " a l l e l e s " a re t r u e or f u n c t i o n a l a l l e l e s , o r a s e r i e s o f c l o s e l y l i n k e d g e n e s . I t w i l l be r e c a l l e d t h a t F l o r a l s o i d e n t i f i e d the c o r r e s p o n d i n g v - g e n e s a t 26 s e p a r a t e l o c i i n f l a x r u s t . I f 43 i t i s assumed t h a t the R-genes i n f l a x , i d e n t i f i e d as e l l e l e s by F l o r , a re t r u e a l l e l e s and t h a t t h e r e i s no a l l e l i s m o f the r e l a t e d v - g e n e s , i t means t h a t the p a r a s i t e has 26 s e p a r a t e or i n d i v i d u a l l o c i w h i c h engage i n g e n e - f o r - g e n e i n t e r a c t i o n w i t h the f i v e l o c i and t h e i r m u l t i a l l e l e s i n the h o s t . S i n c e the f i v e l o c i i n the h o s t can accomodate a maximum o f f i v e p a i r s of homozygous r e s i s t a n c e genes a t one t i m e , or a maximum of 10 i f the r e s i s t a n c e genes are h e t e r o z y g o u s a t a l l l o c i , a minimum o f 16 and a maximum of 21 l o c i o f the p a r a s i t e w i l l r e m a i n unmatched by r e s i s t a n c e genes of the h o s t . In c o n s i d e r i n g the s t r u c t u r e o f the v i r u l e n c e genes i n t h i s s t u d y , i t i s u s e f u l to c o n s i d e r t h e i r p o s s i b l e o r i g i n . The f a c t t h a t e x t e n s i v e s e r i e s o f a l l e l e s a r e f o u n d i n n a t u r e , and i n h i g h e r o r g a n i s m s e s p e c i a l l y , s u g g e s t s t h e e x i s t e n c e o f e v o l u t i o n a r y mechanisms by w h i c h s e v e r a l d i f f e r e n t v a r i a n t s o f a gene a r e a l l f a v o u r e d by s e l e c t i o n . The most d i r e c t mechanism f o r the o r i g i n o f d i f -f e r e n t v a r i a n t s o f a gene i s m u t a t i o n . W i t h m u t a t i o n o c c u r r i n g a t d i f f e r e n t s i t e s a l o n g the gene r e s u l t s s h o u l d b b e t r u e , or f u n c t i o n a l a l l e l i s m . As an a l t e r n a t i v e mechanism i t i s p o s s i b l e t h a t t h e gene of an a l l e l i c group may a r i s e t h r o u g h unequal c r o s s i n g o v e r . B e g i n n i n g w i t h a s i n g l e pathogen 44 gene c o n t r o l l i n g one l e v e l o f v i r u l e n c e , r a r e and unequal c r o s s i n g o v e r would have p r o d u c e d a s e r i a l d u p l i c a t i o n of this gene a n d , i f the phenotype a s s o c i a t e d w i t h the new d u p l i c a t e d gene i s somewhat d i f f e r e n t , i t may be r e c o g n i z e d as an a l l e l e o f the o r i g i n a l g e n e . A l l e l e s p r o d u c e d by e i t h e r o f t h e s e two mechanisms s h o u l d show a f u n c t i o n a l , as w e l l as a s t r u c t u r a l , r e l a t e d n e s s . A t h i r d mechanism, p o s t u l a t e d many y e a r s ago by F i s h e r , i n v o k e s s e l e c t i o n f a v o u r i n g e v e r - c l o s e r l i n k a g e of genes w h i c h , though d i f f e r i n g i n f u n c t i o n , c o n f e r an added s e l e c t i v e a d v a n t a g e when i n h e r i t e d t o g e t h e r , as a u n i t . In such a case the " a l l e l e s " w h i c h are s e l e c t e d f o r wou ld not be f u n c t i o n a l l y a l l e l i c , and the c l o s e l y l i n k e d genes of the u n i t s h o u l d be s e p a r a b l e by c r o s s i n g o v e r . A l t h o u g h the m u l t i p l e a l l e l e s d i s c o v e r e d i n t h i s s t u d y c o u l d have a r i s e n by any o f t h e s e t h r e e p o s s i b l e mechanisms the f i r s t m e n t i o n e d , b e i n g s i m p l e s t , i s the h y p o t h e s i s w h i c h i s f a v o u r e d . When, f rom the v i e w p o i n t of p o p u l a t i o n g e n e t i c s , the f i t n e s s o f the d i f f e r e n t v - a l l e l e s on the c u l t i v a r T r e b i i s c o n s i d e r e d , the a l l e l e s t h a t c o n t r o l the h i g h l e v e l o f v i r u l e n c e would be c o n s i d e r e d f i t t e s t . T h i s wou ld be v a f o l l o w e d by v b , v c and v d . However , the o r d e r o f f i t n e s s c o u l d w e l l be d i f f e r e n t i f a d i f f e r e n t h o s t c u l t i v a r were g r o w n . For e x a m p l e , on c u l t i v a r Nepal ( T a p k e , 1945) the most f a v o u r e d a l l e l e , based on i t s l e v e l o f v i r u l e n c e , wou ld 45 c d ct b be v f o l l o w e d by v , v and v . The d i f f e r e n t a l l e l e s c o u l d thus a s s i s t i n a t t a i n i n g immedia te f i t n e s s on d i f f e r e n t h o s t c u l t i v a r s . As the p o p u l a t i o n ( a c r e a g e ) of the d i f f e r e n t c u l t i v a r s f l u c t u a t e s , the f r e q u e n c y of the d i f f e r e n t v - a l l e l e s w o u l d f l u c t u a t e w i t h i t . S i m i l a r l y , as a p l a n t b r e e d e r r e -p l a c e s d i f f e r e n t R - a l l e l e s i n h i s b r e e d i n g programme the c o r r e s p o n d i n g v - a l l e l e s w i l l d i m i n i s h i n f r e q u e n c y o n l y to i n c r e a s e when the f r e q u e n c y of the r e l a t e d R - a l l e l e i s i n c r e a s e d a g a i n . Mode (1958 , 1960 and 1961) and P e r s o n (1966) have c o n s i d e r e d the problems of c o - e v o l u t i o n o f h o s t and p a r a s i t e and the r o l e t h a t b a l a n c e d p o l y m o r p h i s m p l a y s i n i t . P e r s o n c o n s i d e r e d f i v e a l l e l e s a t a s i n g l e h o s t l o c u s and the f i v e r e l a t e d v - g e n e s a t f i v e d i f f e r e n t l o c i i n the p a r a s i t e . He d e m o n s t r a t e d , t h e o r e t i c a l l y , how the f i v e R - a l l e l e s and the'ir f i v e r e l a t e d v - l o c i may i n t e r a c t i n a system of b a l a n c e d p o l y m o r p h i s m . As the f r e q u e n c y of a s p e c i f i c v - a l l e l e r i s e s , t h a t o f the r e l a t e d R a l l e l e f a l l s a n d , when a new R a l l e l e i s b r o u g h t i n by s e l e c t i o n , the p r e v i o u s v - a l l e l e goes down and i s r e p l a c e d by a new v - a l l e l e . In the p r e s e n t s t u d y , t h e r e are f o u r v - a l l e l e s a t a s i n g l e l o c u s s i n the paras i te a n d , on the a s s u m p t i o n t h a t the f o u r r e l a t e d R-genes i n the h o s t a re a l s o a t a s i n g l e r e l a t e d l o c u s , the f l u c t u -a t i o n s i n f r e q u e n c i e s o f g e n o t y p e s i n both s p e c i e s wou ld be s i m i l a r to thatcoff P e r s o n ' s model of b a l a n c e d p o l y m o r p h i s m . 46 The k i n d o f r e s u l t o b t a i n e d i n t h i s s t u d y i s i n agreement w i t h t h e g e n e - f o r - g e n e c o n c e p t . I t shows t h a t v i r u l e n c e g e n e s , as w e l l as r e s i s t a n c e g e n e s , can o c c u r as m u l t i p l e a l l e l e s . I t i s c o n c e i v a b l e t h a t f o r e v e r y a l l e l e o f a m u l t i p l e a l l e l i c s e r i e s i n the smut , t h e r e i s a c o n r e s p o n d -i n g R - a l l e l e o f a h o s t . V e r y l i t t l e i s known about the g e n e t i c s o f d i f f e r -ences i n degree of e x p r e s s i o n amohgnv-genes a s - w e T U ^ as among R - g e n e s . A t p r e s e n t the g e n e t i c p r o c e d u r e s f o r c o n t r o l l i n g p l a n t d i s e a s e s caused by p a r a s i t i c f u n g i a re m a i n l y based on our knowledge of d i f f e r e n c e s i n k i n d among R-genes r a t h e r than d i f f e r e n c e s i n d e g r e e . The c h o i c e , and u s e , o f R-genes h a v i n g .1arge e f f e e t s o a p p e a r s " t o be aimed a t e l i m i n a t i n g p a r a s i t i c p o p u l a t i o n s r a t h e r than r e d u c i n g t h e i r numbers , a n d , so f a r , i t has f a i l e d more o f t e n t h a n , n o t . I t i s p o s s i b l e t h a t more e f f e c t i v e and l a s t i n g c o n t r o l can be a c h i e v e d by the m a n i p u l a t i o n o f the v a r i o u s degrees ( l e v e l s ) o f p a r a s i t i s m . B r e e d i n g a g a i n s t f u n g a l pathogens i n c r o p p l a n t s i s based on the a s s u m p t i o n t h a t a gene f o r v i r u l e n c e i s i n d i v i s i b l e and t h a t no more than one a l l e l e can o c c u r a t each l o c u s . The p r e s e n t s t u d y has p r o v i d e d e v i d e n c e f o r the e x i s t e n c e of a s e r i e s o f m u l t i p l e a l l e l e s o f v i r u l e n c e genes c o n t r o l l i n g f o u r l e v e l s o f d i s e a s e r e a c t i o n . I t would a l s o 47 be o f i n t e r e s t to i n v e s t i g a t e whether the R-genes of the h o s t , w i t h w h i c h t h e s e v - a l l e l e s i n t e r a c t , are a l s o a l l e l i c . PART II USTILAGO HORDEI, HORDEUM VULGARE AND THE GENE-FOR-GENE RELATIONSHIP I n t r o d u c t i o n In g e n e r a l , a v i r u l e n c e i n the p a r a s i t e and r e s i s -t a n c e i n the h o s t a re i n h e r i t e d as dominant c h a r a c t e r s , and v i r u l e n c e i n the p a r a s i t e and s u s c e p t i b i l i t y i n the h o s t as r e c e s s i v e . However , i n a m i n o r i t y of c a s e s , v i r u l e n c e i n the p a r a s i t e and s u s c e p t i b i l i t y i n the h o s t a r e dominant ( c f . P e r s o n et a l . , 1971 ; J o h n s o n , 1954; G r e e n , 1965 ; Haggag et a l . , 1973 ; N i c o l a i s e n , 1934; H o l t o n , 1 9 6 4 ) . S t u d i e s on the g e n e t i c s o f p a t h o g e n i c i t y i n the b a r l e y c o v e r e d s m u t , u. hovdei, have shown t h a t v i r u l e n c e i s r e c e s s i v e , t h a t a v i r u l e n c e i s d o m i n a n t , and t h a t i n r e l a t i o n to a s p e c i f i c h o s t c u l t i v a r t h e s e a l t e r n a t i v e s a r e d e t e r m i n e d by a s i n g l e g e n e t i c l o c u s . Thomas and P e r s o n (1965) i n v e s t i g a t e d the g e n e t i c c o n t r o l o f a low (5%) l e v e l s o f v i r u l e n c e i n t h i s s m u t . L a t e r , Lade (1969) s t u d i e d the g e n e t i c c o n t r o l o f a somewhat h i g h e r ( i n t e r m e d i a t e ) l e v e l o f v i r u l e n c e and r e c e n t l y , 48 49 S i h d u and P e r s o n (1971) i n v e s t i g a t e d the g e n e t i c c o n t r o l o f a h i g h v i r u l e n c e l e v e l . The l a t t e r a u t h o r s i d e n t i f i e d two v i r u l e n c e genes i n two smut c u l t u r e s : gene U h v i d e t e r m i n e d v i r u l e n c e on c u l t i v a r s Hannchen and V a n t a g e , and U h v 2 on E x c e l s i o r . S i m i l a r s t u d i e s have been c a r r i e d out w i t h the o a t s m u t , u. avenae ( N i c o l a i s e n , 1934; H a l i s k y , 1965; H o l t o n , 1964) and w i t h an i n t e r s p e c i f i c h y b r i d o f the b a r l e y s m u t s , U. n i g r a and U. hordei ( C h e r e w i c k , 1 9 6 7 ) . As f o r the h o s t , a number o f s t u d i e s have been done on the g e n e t i c s of r e s i s t a n c e i n b a r l e y to U. hordei ( c f . the l i t e r a t u r e r e v i e w s e c t i o n ) . Dominant as w e l l as r e c e s s i v e r e s i s t a n c e genes have been i d e n t i f i e d i n b a r l e y a g a i n s t U. hordei (Pugs 1 e y e e t a a l . 3 1946; S h a n d , 1956 ; C o t t i n g h a m , . 1958; W e l l s , 1958 ; M e t c a l f e , 1 962 ; Cherewte.kJ.et a l . 3 1 9 6 9 ) . More r e c e n t l y , S i h d u and p e r s o n (1972) s t u d i e d t h e g e n e t i c b a s i s f o r r e s i s t a n c e and s u s c e p t i b i l i t y i n t h r e e b a r l e y c u l t i v a r s . They i d e n t i f i e d two r e s i s t a n c e g e n e s , UhRi and U h R 2 ; t h e s e genes i n t e r a c t e d w i t h v i r u l e n c e genes U h v i and U h v 2 , r e s p e c t i v e l y . For f u r t h e r a n a l y s i s o f g e n e t i c phenomena ( i n c l u d i n g l i n k a g e , a l l e l i s m , and e p i s t a s i s t h a t may p r e v a i l among v i r u l e n c e genes as w e l l as among r e s i s t a n c e g e n e s ) , i t i s n e c e s s a r y t h a t more genes be i d e n t i f i e d i n both s y s t e m s . As B l a c k (1952) p o i n t e d o u t , the i d e n t i f i c a t i o n and d e t e r m i n a -t i o n o f the r e l a t i o n s h i p o f the genes f o r p a t h o g e n i c i t y i n 50 m i c r o o r g a n i s m s i s i m p o r t a n t not o n l y f rom the s t a n d p o i n t of t h e i r use i n a b r e e d i n g program but a l s o as an a i d i n d e t e r -m i n i n g b i o s y n t h e t i c pathways a n d , u l t i m a t e l y , i n d e v e l o p i n g an u n d e r s t a n d i n g o f the n a t u r e o f r e s i s t a n c e i n the h o s t . The p r e s e n t s t u d y c o n c e r n s U. hordei and H. v u l g a r e . I t s p u r p o s e was to d i s c o v e r new genes both f o r v i r u l e n c e i n U. hordei and f o r r e s i s t a n c e i n H. v u l g a r e , to d e t e r m i n e the p a t t e r n s of i n h e r i t a n c e of t h e s e g e n e s , and to i n v e s t i g a t e t h e i r mode of i n t e r a c t i o n . Two s e t s of smut c u l t u r e s , d e s i g n a t e d D and G , and t h r e e b a r l e y c u l t i v a r s ( E x c e l s i o r , H i m a l a y a and K e y s t o n e ) , were u s e d . M a t e r i a l s and Methods  The Pathogen Two s e t s o f u. hordei c u l t u r e s (D and G ) , . r e p r e s e n t -i n g t h e l i n e a r t e t r a d p r o d u c t s d e r i v e d f rom two s i n g l e t e l i o s p o r e s , and t h e i r i n t e r a c t i o n w i t h t h r e e b a r l e y c u l t i -v a r s , C I - 1 2 4 8 E x c e l s i o r , H i m a l a y a and 292 ( C . A . N . ) K e y s t o n e , were s t u d i e d . C u l t u r e s e t D , o b t a i n e d f rom D r . P e r s o n ' s l a b o r a t o r y a t UBC, had been s t u d i e d p r e v i o u s l y by S i h d u and P e r s o n ( 1 9 7 1 ) . The second s e t o f c u l t u r e s , w h i c h was o b t a i n e d f rom D r . P . Thomas o f the CDA R e s e a r c h L a b o r a t o r y a t W i n n i p e g , C a n a d a , had been d e r i v e d from a t e l i o s p o r e o b t a i n e d f rom the 51 A - l x A - 2 c r o s s d e s c r i b e d by Thomas ( 1 9 6 5 ) . The second c u l t u r e s e t i s d e s i g n a t e d G i n t h i s s t u d y . Methods o f c u l t u r i n g , s e l f i n g , c r o s s i n g , b a c k c r o s s -i n g and i n o c u l a t i o n are as d e s c r i b e d i n P a r t I . In the b a c k c r o s s e s , f i v e s e t s o f t e t r a d s , d e r i v e d f rom f i v e F i t e l i o s p o r e s , were u s e d . E x c e p t f o r t e l i o s p o r e s 13A and 13B, w h i c h came from the same c r o s s , the F i t e l i o s p o r e s were a l l d e r i v e d f rom d i f f e r e n t c r o s s e s of the p a r e n t a l c u l t u r e s e t s . The s t u d y w i t h c u l t i v a r E x c e l s i o r was c a r r i e d out t w i c e a t The 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 (UBC) and once i n C a l i f o r n i a ( I m p e r i a l V a l l e y ) ; t h a t w i t h H i m a l a y a t w i c e a t UBC, never i n C a l i f o r n i a ; and t h a t w i t h K e y s t o n e once a t UBC and once i n C a l i f o r n i a . The Hos t Two b a r l e y c u l t i v a r s , E x c e l s i o r and K e y s t o n e were c r o s s e d . Because E x c e l s i o r has a number of d i s t i n c t dominant m o r p h o l o g i c a l markers t h a t w o u l d be i d e n t i f i a b l e i n F i p l a n t s , i t was used as the p o l l e n p a r e n t . With. E x c e l s i o r used as the f e m a l e p a r e n t , the a d v a n t a g e of b e i n g a b l e to d i s t i n g u i s h h y b r i d f rom a c c i d e n t a l l y - s e l f e d progeny would not be r e a l i z e d . About 165 s e e d s t w e r e x o b t a i n e d b f r b ' m y . t h e n i x c e d i s . i . o r by K e y s t o n e c r o s s . Of t h e s e , 70 seeds were i n o c u l a t e d w i t h 52 the D - 2 A / D - 3 a d i k a r y o n and 80 seeds w i t h the G - l a / G - 3 a d i k a r y o n . Ten of the r e m a i n i n g seeds were grown i n t o h e a l t h y F i p l a n t s . S i n c e i t i s not p o s s i b l e , a t p r e s e n t , to t e s t a s i n g l e F 2 a g a i n s t two t e s t c u l t u r e s o f u. hordei, seeds c o l l e c t e d f r o m t h r e e F i p l a n t s were used to e s t a b l i s h the F 2 g e n e r a t i o n . Seeds were h a r v e s t e d f rom 81 s i n g l e F 2 p l a n t s ( o r i g i n a t e d from one F i p l a n t ) . T h i s number of F 2 p l a n t s i s s u f f i c i e n t to t e s t f o r s e g r e g a t i o n of two genes ( M a t h e r , 1938; H a n s o n , 1 9 5 9 ) . Seeds t a k e n f r o m each o f the 81 F 2 p l a n t s were d i v i d e d i n t o two l o t s o f about 100 seeds e a c h ; f o r each p a i r o f 100 -seed l o t s o b t a i n e d i n t h i s way , one l o t was i n o c u l a t e d w i t h the D - 2 A / D - 3 a d i k a r y o n and the o t h e r w i t h the G - l A / G - 3 a d i k a r y o n . The i n o c u l a t e d seed l o t s were p l a n t e d a t U l B C i n t o 15 f o o t r o w s . F 3 rows i n w h i c h t h e r e were s m u t t e d p l a n t s were c l a s s i f i e d as s u s c e p t i b l e and t h o s e w h i c h d i d not show any d i s e a s e were c l a s s i f i e d as r e s i s t a n t . R e s u l t s The V i r u l e n c e Genes C u l t u r e s e t D, when s e l f e d , was v i r u l e n t on E x c e l s i o r and a v i r u l e n t on H i m a l a y a and K e y s t o n e . On the o t h e r h a n d , c u l t u r e s e t G , on s e l f i n g , was a v i r u l e n t on E x c e l s i o r but s e g r e g a t e d f o r v i r u l e n c e on H i m a l a y a and K e y s t o n e i n a 2 :2 53 r a t i o of a v i r u l e n t to v i r u l e n t . These r e s u l t s were e x p l a i n e d by p o s t u l a t i n g v i r u l e n c e genes a t a s i n g l e g e n e t i c l o c u s i n c u l t u r e s e t D and a t two l o c i i n c u l t u r e s e t G (as shown i n T a b ! e 11 — 1 ) . In the c r o s s of the D and G c u l t u r e s e t s , the F z h y b r i d s were v i r u l e n t on E x c e l s i o r but a v i r u l e n t on H i m a l a y a and K e y s t o n e i n t e s t s c o n d u c t e d a t UBC. The l e v e l o f v i r u -l e n c e e x h i b i t e d by t h e F i c u l t u r e s on E x c e l s i o r was much l o w e r ( c a . 9%) than t h a t o b t a i n e d w i t h the s e l f e d c u l t u r e s o f s e t D ( c a . 40%). T h i s r e s u l t i n d i c a t e s t h a t v i r u l e n c e of c u l t u r e s e t D on E x c e l s i o r i s i n h e r i t e d as an i n c o m p l e t e l y dominant t r a i t , whereas the v i r u l e n c e of c u l t u r e s e t G on c u l t i v a r s H i m a l a y a and K e y s t o n e i s i n h e r i t e d as a r e c e s s i v e t r a i t . In s i m i l a r t e s t s c o n d u c t e d i n C a l i f o r n i a , . a l l F i c u l t u r e s were avdiriiilemt on a ] l t h r e e c u l t d i v a r s / Thus the v i r u l e n t phenotype t h a t was i n c o m p l e t e l y dominant a t UBC was r e c e s s i v e i n C a l i f o r n i a . B a c k c r o s s to P a r e n t a l C u l t u r e D F i v e s e t s o f t e t r a d p r o d u c t s , d e r i v e d f rom s i n g l e F i t e l i o s p o r e s , were b a c k c r o s s e d to t h e D - s e t i n o r d e r to d e t e r m i n e the mode of s e g r e g a t i o n of v i r u l e n c e . The b a c k -c r o s s e s p r o d u c e d d i k a r y o n s w h i c h were a v i r u l e n t on H i m a l a y a and K e y s t o n e but v i r u l e n t on E x c e l s i o r i n t e s t s c a r r i e d o u t T a b l e 11 -1 The P o s t u l a t e d Genotypes o f S p o r i d i a l C u l t u r e s o f Se t s D and G and of the D i k a r y o n s P r o d u c e d by S e l f i n g , T o g e t h e r w i t h the D i s e a s e R e a c t i o n s O b t a i n e d w i t h C u l t i v a r s E x c e l s i o r (E) , H i m a l a y a ( H i ) and K e y s t o n e (K) S p o r i d i a l M a t i n g S p o r i d i a l Compati b l e Di k a r y o n R p a r t i o n * n f L i n e Type Genotype C o m b i n a t i o n Genotype r\ c ci \+ it 1 v 11 E Hi K D - l a v 2 V i t V 5 D - l a x D-2A V 2 V 2 V 4 V 4 V 5 V 5 S R R 2 A II x D-4A n S R R 3 a II D-3a x D-2A S R R 4 A II x D-4A s R R G - l A V 2 V 4 V 5 G-IA x G-2a V 2 V 2 V i t V i t V 5 v 5 R R R 2 a V z V ^ V s x G-3a "• v ^ V g V s R S S 3 a V 2 V 4 V 5 G-4A x G-2a " V ^ V s V s R S S 4 A V 2 V 4 V 5 x G-3a •" V ^ V s V s R R R S = s u s c e p t i b l e ; R = r e s i s t a n c e . -p=> 55 a t UBC. Three l e v e l s o f v i r u l e n c e , l o w , i n t e r m e d i a t e and h i g h , were e x h i b i t e d by t h e s e c u l t u r e s when they were i n o c u -l a t e d to E x c e l s i o r . In some b a c k c r o s s e s the t h r e e l e v e l s o f v i r u l e n c e s e g r e g a t e d i n the r a t i o s 2 low : 2 i n t e r m e d i a t e : 4 h i g h . In o t h e r s the s e g r e g a t i o n was i n the r a t i o o f 4 i n t e r m e d i a t e : 4 h i g h . The h i g h v i r u l e n c e l e v e l a p p r o x i m a t e d the v i r u l e n c e l e v e l of the p a r e n t a l c u l t u r e D ( T a b l e 11- 3) . A r e c e s s i v e m o d i f y i n g gene (m) was p o s t u l a t e d to e x p l a i n the d i f f e r e n c e s i n . t h e . l e v e l s o f v i r u l e n c e shown by the h e t e r o z y g o u s c l a s s e s . When the same t e s t s were r e p e a t e d i n C a l i f o r n i a , r e s u l t s s i m i l a r to t h o s e o b s e r v e d a t UBC were o b t a i n e d on c u l t i v a r K e y s t o n e ( H i m a l a y a was not used i n C a l i f o r n i a ) . However , t e s t s on E x c e l s i o r gave r e s u l t s d i f f e r e n t f rom t h o s e o b t a i n e d a t UBC; C r o s s e s between D and G c u l t u r e s e t s were a v i r u l e n t on E x c e l s i o r , and t h i s r e s u l t was s u b s t a n t i a t e d by the b a c k c r o s s S i t i i d i i e s . In the b a c k c r o s s e s , a 4 : 4 s e g r e g a -t i o n r a t i o o f a v i r u l e n t t o v i r u l e n t was o b t a i n e d . That i s , v i r u l e n c e i s r e c e s s i v e i n C a l i f o r n i a , but i n c o m p l e t e l y dominant a t UBC. The F i s p o r i d i a l l i n e s w h i c h a t UBC p r o d u c e d h i g h v i r u l e n c e l e v e l s i n c r o s s e s to c u l t u r e s e t D a r e the same as t h o s e w h i c h were v i r u l e n t i n C a l i f o r n i a . In b a c k c r o s s e s the same d i k a r y o n s o f t e n p r o d u c e d h i g h e r v i r u l e n c e l e v e l s i n C a l i f o r n i a than a t UBC. The o v e r - a l l r e s u l t s i n d i c a t e t h a t 56 a s i n g l e p a i r o f v i r u l e n c e genes i s i n v o l v e d . The v i r u l e n c e gene i n c u l t u r e s e t D i s d e s i g n a t e d v 2 . The d i k a r y o n 1 3 B - 3 / D - 2 ( T a b l e 11-3) was e x p e c t e d to show a h i g h e r l e v e l o f v i r u l e n c e , t h a n t h a t o b t a i n e d i n t h i s s t u d y . P l a n t s w h i c h showed d i s e a s e r e a c t i o n s f o l l o w i n g i n o c u l a t i o n w i t h t h i s d i k a r y o n d i d not show normal d i s e a s e symptoms. R a t h e r , the s p i k e s were s t u n t e d and upon e x a m i n a -t i o n v e r y few t e l i o s p o r e s were f o u n d . H o l t o n (1966) has d e s c r i b e d what appears to be a s i m i l a r case i n u. avenae, w h i c h he r e f e r r e d to as " b l a s t . " B a c k c r o s s e s to P a r e n t a l C u l t u r e G The b a c k c r o s s e s to c u l t u r e G showed s e g r e g a t i o n on a l l t h r e e c u l t i v a r s when t e s t e d a t UBC. The a v i r u l e n t : v i r u l e n t s e g r e g a t i o n s on E x c e l s i o r were i n a 4 : 4 r a t i o f o r b a c k c r o s s e s i n v o l v i n g f o u r o f the F i c u l t u r e s e t s , and i n a 2 :6 r a t i o f o r the f i f t h s e t ( T a b l e 1 1 - 4 ) . The F i s p o r i d i a l l i n e s t h a t p r o d u c e d d i s e a s e r e a c t i o n on E x c e l s i o r when c r o s s e d to G were t h o s e w h i c h p r o d u c e d h i g h v i r u l e n c e l e v e l s i n the b a c k c r o s s to D. These s p o r i d i a l l i n e s p o s s e s s the a l l e l e U h v 2 , and t h e r e f o r e form a h e t e r o z y g o u s d i k a r y o n when c r o s s e d to G. The same b a c k c r o s s e s , when t e s t e d i n C a l i f o r n i a , were a l l a v i r u l e n t on E x c e l s i o r . S e g r e g a t i o n on H i m a l a y a was i n a r a t i o o f 2 :6 ( v i r u l e n t to a v i r u l e n t ) i n t h r e e of T a b l e 11-2 D i s e a s e R e a c t i o n O b t a i n e d on Three B a r l e y C u l t i v a r s F o l l o w i n g I n o c u l a t i o n w i t h D i k a r y o n s Formed by S e l f i n g and C r o s s i n g C u l t u r e Se ts D and G , and the Genotype of the D i k a r y o n s D i s e a s e R e a c t i o n s ( i n P e r c e n t a g e s ) P a r e n t a l C u l t u r e s S p o r i d i a l M a t i ngs Di k a r y o t i c L i nes A t UBC In C a l i f o r n i a Genotypes D x G D - l X D-2 1 II X 4 2 3 X 2 3 II X 4 4 G - l X G-2 5 n X 3 6 4 X 2 7 II X 3 8 D - l X G - l 9 II X 4 10 2 X 2 11 X 3 12 3 X 1 13 II X 4 14 4 X 2 1 5 II X 3 16 E x c e l s i o r 56 17 58 29 0 0 0 0 9 10 8 14 8 10 3 13 H i m a l a y a 0 0 0 Q0 0 21 20 0 0 0 0 0 0 0 0 0 K e y s t o n e E x c e l s i o r 0 0 0 0 0 1 5 13 0 0 0 0 0 0 0 0 0 56 27 38 43 0 0 0 0 0 0 0 0 0 0 0 0 K e y s t o n e 0 0 0 0 0 2 2 0 0 0 0 0 0 0 0 0 m m v 2 V 2 V i t V i t V 5 V 5 M M V 2 V 2 V i f V i ( V s V 5 " v ^ V s V s " V i ^ V s V s " V ^ V s V s M m V 2 v 2 V i t V i f V 5 V 5 " V ^ V . V s V s " V ^ V - V s V s " V i ^ V s V s " V ^ V s V s " V ^ V s V s " V ^ V s V s " V ^ v ^ V s V s T a b l e 11-3 D i s e a s e R e a c t i o n O b t a i n e d on Three B a r l e y C u l t i v a r s F o l l o w i n g I n o c u l a t i o n w i t h D i k a r y o n s Formed by B a c k c r o s s i n g F x S p o r i d i a l L i n e s f rom D x G C r o s s to P a r e n t a l C u l t u r e Se t D D i s e a s e R e a c t i o n ( i n P e r c e n t a g e s ) A t UBC In C a l i f o r n i a S p o r i d i a l M a t i ngs E x c e l s i or H i m a l a y a Keys tone E x c e l s i o r K e y s t o n e Genotype 9-1 n 2 n x x X X X X X X 11-1 x D-n 2 II 3 II 4 M •1 3 2 4 22 4 1 3 •2 4 1 3 2 4 1 3 15 15 39 33 37 34 6 24 5 6 41 37 22 29 53 45 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 75 58 60 70 0 0 0 0 32 24 0 0 74 70 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 mmV 2 v 2 Mmv 2 v 2 n n mmV 2v : ) vlMmV 2v 2 M II " v 2 v 2 II H mmV 2 v 2 " v 2 v 2 II II ** I n f e c t i o n was u n e x p e c t e d . A v a l u e of about 23% was e x p e c t e d ( C o n t i n u e d ) 00 T a b l e 11-3 ( C o n t i n u e d ) D i s e a s e R e a c t i o n ( i n P e r c e n t a g e s ) At UBC In C a l i f o r n i a S p o r i d i a l M a t i ngs E x c e l s i o r H i m a l a y a Keys tone E x c e l s i or K e y s t o n e Genotype 13A-1 x D-2 " x 4 2 x 1 " xx 3 3 x 1 " x 3 4 x 2 " x 4 13B-1 x D-1 " x 3 2 x 1 " x 3 3 x 2 " x 4 4 x 2 " x 4 15-1 x D-2 " x 4 2 x i " x 3 3 x 1 " x 3 4 x 2 " x 4 3 8 37 39 41 39 22 24 26 20 18 22 7 46 42 64 25 22 36 45 39 39 23 6* oooooooo oooooooo oooooooo oooooooo oooooooo oooooooo 0 6* 38 63 72 55 0 0 0 0 0 0 5 39 69 45 0 0 41 24 44 69 0 0 oooooooo oooooooo oooooooo MmV 2 v 2 I I I I " V 2 V 2 I I I I mm " 1 1 1 1 " V 2 v 2 n n mmV2v 2 n 11 11 1 1 n n Mmv 2 v 2 11 n n 11 n 11 mmV 2 v 2 n 11 Mmv 2v 2 11 1 1 n n 1 1 1 1 mmV2v 2 n 11 T a b l e 11-4 u ' " D i s e a s e R e a c t i o n O b t a i n e d on Three B a r l e y C u l t i v a r s F o l l o w i n g I n o c u l a t i o n w i t h D i k a r y o n s Formed by B a c k c r o s s i n g F i S p o r i d i a l L i n e s f rom D x G C r o s s to P a r e n t a l C u l t u r e G D i s e a s e R e a c t i o n ( i n P e r c e n t a g e s ) A t UBC In C a l i f o r n i a S p o r i d i a l M a t i ngs E x c e l s i or H i m a l a y a Keystone E x c e l s i or K e y s t o n e - G e n o t y p e 9-1 X G-2 0 0 II X 3 0 7 2 X 1 4 0 II X 4 2 0 3 X 1 6 0 n X 4 6 0 4 X 2 0 0 II X 3 0' 13 11-1 X G - l 0 0 II X 4 0 7 2 X 2 8 0 n X 3 5 0 3 X 1 2* 0 II X 4 8* 0 4 X 2 2 5 II X 3 8 0 0 3 0 0 0 0 0 6 0 2 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 0 0 0 0 0 7 0 1 0 0 0 0 2 0 V 2 V 2 V 4 V i t V 5 v 5 II V4V4V5V5 V 2 v 2 V - V „ v 5 v 5 II V ^ V s V s II v k \ l h \ l 5 \ l 5 V i ^ V s V s V 2 V 2 V i t V i t V 5 v 5 II V i t V i t V s V s V 2 V 2 V i t V i t V 5 V 5 II V it V t v 5V 5 V a v 2 V 4 V t V 5V5 V i t V t V s V s V a V 2 V i*v i t V 5 V 5 n V h\lkV 5V 5 V 2 v 2 V i t V ^ V 5 V 5 V nv i t V 5 V 5 ( C o n t i n u e d ; ** D i s e a s e was u n e x p e c t e d D i s e a s e was e x p e c t e d O T a b l e 11-4 ( C o n t i n u e d ) D i s e a s e R e a c t i o n ( i n P e r c e n t a g e s ) A t UBC S p o r i d i a l M a t i ngs E x c e l s i or H i m a l a y a Keys tone E x c e l s i or K e y s t o n e Genotype 13A.-1 x G -1 0 17 7 0 11 V 2 V 2 V 4 V 4 V 5 V 5 II x 4 0 0 0 0 0 " V ^ V s V s 2 x 2 0 * * 0 0 0 0 V 2 v 2 V k\lu\l 5 v 5 II X 3 1 6 0 0 0 " V ^ v ^ V s V s 3 X 2 0** 6 6 0 0 " V ^ V s V s II X 3 1 0 6 0 0 " V ^ V s V s 4 X 1 0 17 H.8 0 3 V 2 V 2 V 4 V 4 V 5 V 5 II X 4 0 11* 11* 0 0 "• v ^ V s V s 13B-1 x G - 2 0 0 0 0 _ V 2 V 2 v i t V W 5 v 5 X 3 0 9 4 0 - " v ^ V s V s 2 X 2 0 0 0 0 - " V ^ V s V s n X 3 0 13 5 0 : - " V ^ V g V s 3 X 1 0 0 0 - V 2 v 2 V i » v 4V 5 V 5 n X 4 0* * 0 0 0 - " V ^ V i t V s V s 4 X 1 0* * 0 0 0 - " VnVkVsVs X 4 8 0 0 0 - " V . V ^ V s V s 15-1 x G- 1 0 0 0 0 0 V 2 V 2 V l f v l t v 5 V 5 n x 4 0 7 12 0 9 " V ^ V ^ V s V s 2 X 2 1 0 0 0 0 V 2 v 2 V i t V i f V 5 V 5 II ':. X 3 1 0 0 0 0 "• V . v ^ V s V s 3 X 2 2 11 25 0 3 " V . V ^ V s V s II X 3 3 19* 0 0 0 " V ^ v ^ V s V s 4 X 1 0 0 0 0 0 \l 2V 2\l »v ,V 5V 5 II X 4 0 0 0 0 0 " V ^ V ^ V s V s In C a l i f o r n i a CT> 62 the b a c k c r o s s e s , 3 :5 i n the r e m a i n d e r . On K e y s t o n e a 2 :6 r a t i o o f v i r u l e n c e to a v i r u l e n c e was o b t a i n e d w i t h a l l f o u r c u l t u r e s e t s when t h e s e were t e s t e d i n C a l i f o r n i a . In t e s t s on c u l t i v a r K e y s t o n e , c o n d u c t e d a t UBC, f o u r o f the F x b a c k c r o s s e s s e g r e g a t e d 2 :6 and one s e g r e g a t e d i n a 3 :5 r a t i o of v i r u l e n c e to a v i r u l e n c e . For the b a c k c r o s s to s e t G , thec idi k a r y o n s t h a t p r o d u c e d d i s e a s e on H i m a l a y a a l s o p r o d u c e d d i s e a s e on the c u l t i v a r K e y s t o n e . T h i s s u g g e s t s t h a t a s i n g l e g e n e t i c l o c u s d e t e r m i n e s a v i r u l e n t r e a c t i o n on both c u l t i v a r s . D u p l i c a t e v i r u l e n c e g e n e s , v 4 and v 5 , were p r o p o s e d to e x p l a i n the r e s u l t s o b t a i n e d on H i m a l a y a and K e y s t o n e . The R e s i s t a n c e Genes The v i r u l e n c e g e n e s , v 2 a a n d v^, w h i c h had a l r e a d y been i d e n t i f i e d , were used to d e t e c t or r e v e a l the i n h e r i t a n c e p a t t e r n o f the r e l a t e d r e s i s t a n c e genes i n the h o s t . E x c e l s i o r i s s u s c e p t i b l e and r e s i s t a n t to d i k a r y o n s D - 2 A / D-3a and G - l A / G - 3 a , r e s p e c t i v e l y . D i k a r y o n D - 2 A / D - 3 a i s homozygous f o r v i r u l e n c e gene v 2 and d i k a r y o n G - l a / G - 3 a i s homozygous f o r v ^ . K e y s t o n e and E x c e l s i o r showed c o n t r a s t i n g d i s e a s e r e a c t i o n s t o w a r d s t h e s e d i k a r y o n s . These i n t e r a c t i o n s a r e summarized i n T a b l e 11-1 . Because H i m a l a y a showed the same r e a c t i o n as K e y s t o n e to t h e s e d i k a r y o n s , i t was not used i n t h i s s t u d y . 63 T a b l e 11-5 D i s e a s e R e a c t i o n i n P e r c e n t a g e s , Induced on E x c e l s i o r and K e y s t o n e F o l l o w i n g I n o c u l a t i o n w i t h D i k a r y o n s v 2 v 2Vi tVi » ; - ; a n d V 2 y 2 V i t V i t o f v. hovdei T e s t C u l t u r e Y e a £ a n ^ P l a c e of T e s t i n g D i s e a s e R e a c t i o n (•%) o f : VaVaV^V^ 1 971 ,1 972,1 973,UBC 1972 C a l i f , a v e r a g e V a V z V ^ v ^ 1 971 ,1973 UBC • 1 972 C a l i f , a v e r a g e E x c e l s i o r K e y s t o n e 58 0 38 0 48 0 0 1 5 0 2 0 8 .5 Seeds o b t a i n e d from the K e y s t o n e by E x c e l s i o r c r o s s e s were i n o c u l a t e d w i t h D - 2 A / D - 3 a (70 s e e d s ) and G - I A / G-3a (80 s e e d s ) . No s m u t t e d s p i k e was d e t e c t e d i n t h e s e F i p l a n t s . Data o b t a i n e d f o l l o w i n g t h e i n o c u l a t i o n o f F 3 b a r l e y l i n e s w i t h both d i k a r y o n s , D - 2 A / D - 3 a , a r e p r e s e n t e d i n T a b l e 11- 7. The ddsease r e a c t i o n s of F 3 l i n e s o f E x c e l s i o r by K e y s t o n e c r o s s f e l l i n t o f o u r c l a s s e s : t h o s e w h i c h were s u s c e p t i b l e to both c u l t u r e s ( S - S ) ; t h o s e w h i c h were 64 s u s c e p t i b l e to the f i r s t c u l t u r e and r e s i s t a n t to the second ( S - R ) ; t h o s e r e s i s t a n t to the f i r s t and s u s c e p t i b l e to the second ( R - S ) ; and t h o s e w h i c h were r e s i s t a n t to b o t h c u l t u r e s ( R - R ) . The c o u n t o f the number of rows f a l l i n g i n t o each o f t h e s e c l a s s e s showed t h a t the d a t a f i t s a 9 : 3 : 3 : 1 ( S - S : S -R: R - S : R-R) r a t i o , t y p i c a l o f two i n d e p e n -d e n t l y s e g r e g a t i n g dominant c h a r a c t e r s . A c h i - s q u a r e t e s t for g o o d n e s s - o f - f i t showed t h a t t h i s i n t e r p r e t a t i o n i s i n agreement w i t h the o b s e r v a t i o n s ( T a b l e 11-6) . Had i t been p o s s i b l e to t e s t t h e s e rows i n the F 2 g e n e r a t i o n s a g a i n s t b o t h c u l t u r e s the r a t i o s h o u l d have been 1 : 3 : 3 : 9 ( S - S : S -R: R - S : R - R ) . The r e s i s t a n c e gene i n E x c e l s i o r i s r e v e a l e d by i n t e r a c t i o n w i t h c u l t u r e D w h i c h p o s s e s s e s v i r u l e n c e gene v 2 . The c o r r e s p o n d i n g r e s i s t a n c e gene i n E x c e l s i o r i s d e s i g n a t e d R 2 . S i m i l a r l y , t h e r e s i s t a n c e gene i n K e y s t o n e i s r e v e a l e d by i n t e r a c t i n g w i t h d i k a r y o n G - l A / G - 3 a , w h i c h i s homozygous f o r the v i r u l e n c e gene vh. The c o r r e s p o n d i n g r e s i s t a n c e gene i n K e y s t o n e i s d e s i g n a t e d R^. As shown by the F x and the s e g r e g a t i n g F 3 p r o g e n i e s , r e s i s t a n c e i s dominant i n both i n s t a n c e s . The g e n o t y p e s of E x c e l s i o r and K e y s t o n e , as f a r as t h e s e . r e s i s t a n c e genes a r e c o n c e r n e d , a re d e s i g n a t e d R 2 R 2 r i ( r i t and r 2 r 2 R i ( R i t . T a b l e 11-6 F 3 L i n e s f rom E x c e l s i o r and Keystone C r o s s S e p a r a t e d i n t o Four C l a s s e s A c c o r d i n g to T h e i r I n t e r a c t i o n s w i t h Two D i k a r y o n s , yzViMh^lh and V 2 V 2 V i + V 4 , o f u. hordei and the Number of L i n e s i n Each C u l t u r e R e a c t i o n to T e s t of F 3 L i n e C u l t u r e : Number of F Each C l a s s 3 b! i m e s r i n f F ( E E x c K ) C • a s s v 2 v 2 V ^ V 4 V 2 V 2 v 4 V 4 C l a s s O b s e r v e d E x p e c t e d f d 2 ) [E J S S S-S 41 4 5 . 6 ~ 0 .46 S R S-R 1 3 15 .2 0 .33 R S R-S 18 15 .2 0 .52 R R R-R 9 5.1 2 .98 TOTAL 81 81 .1 X 2 P = 3 .29 = 0 .35 tn 66 T a b l e 11-7 D i s e a s e R e a c t i o n ( e x p r e s s e d i n p e r c e n t a g e ) Induced i n F 3 - ns ls ines D e r i v e d f rom the K x E C r o s s F o l l o w i n g I n o c u l a t i o n s wi t h ' v 2 v 2 V i ( V i t and V 2 V 2 v nv4"Dik a r y o n s R e a c t i o n of F 3 L i n e s of the C r o s s K x E t o D i k a r y o n s F 3 L i n e v 2 v 2 V i l V i t (%• age) V 2 V 2 V t f V i t (% age) C l a s s Number 1 14 10 S-S 2 3 3 S-S 3 4 1 S-S 4 0 0 R-R 5 2 2 S-S 6 2 1 S-S 7 0 0 R-R 8 8 2 S-S 9 0 0 R-R 10 2 2 S-S 11 7 0 S-R 1 2 4 8 S-S 13 5 6 S-S 14 2 0 S-R 15 2 8 S-S 16 0 2 R-S 1 7 0 2 R-S 18 6 10 S-S 19 4 0 S-R 20 9 0 S-R 21 4 4 S-S 22 6 6 S-S 23 0 5 R-S 24 2 5 S-S 25 6 1 7 S-S 26 4 1 2 S-S 27 0 0 R-R 28 10 5 S-S 29 0 2 R-S 30 3 2 S-S ( C o n t i nued) 67 T a b l e 11-7 ( C o n t i n u e d ) R e a c t i o n of F 3 L i n e s of the C r o s s K x E to D i k a r y o n s F 3 L i n e v 2 v 2V1+ V it (%• age) V 2V 2VitVit (% age) C l a s s Number 31 4 3 S-S 32 0 2 R-S 33 0 0 R-R 34 0 0 R-R 35 3 9 SS-S 36 0 4 R-S 37 4 0 S-R 38 7 3 S-S 39 2 11 S-S 40 0 2 R-S 41 3 3 S-S 42 0 10 R-S 43 0 0 R-R 44 4 0 S-R 45 13 14 S-S 46 0 6 R-S 47 4 0 S-R 48 3 2 S-S 49 0 0 R-R 50 15 9 S-S 51 4 0 S-R 52 0 0 R-R 53 5 3 S-S 54 4 5 S-S 55 0 10 R-S 56 0 8 R-S 57.7 8 2 S-S 58 6 2 S-S 59 5 15 S-S 60 0 2 R-S 61 14 6 S-S 62 7 3 S-S 63 5 2 S-S 64 2 0 S-R 65 5 0 S-R 66 5 0 S-R ( C o n t i nued) 68 T a b l e 11-7 ( C o n t i n u e d ) R e a c t i on C r o s s K of F 3 L i n e s o f the x E to Di k a r y o n s F 3 L i n e V2V2V1+V1+ V. 3 V 2 V 4 V 4 Number (% age) •(? I- age) C l a s s 67 2 3 S -S 68 0 8 R -S 69 9 6 S -S 70 5 6 S -S 71 5 8 S -S 72 0 3 R -S 73 0 2 R -S 74 2 3 S -S 75 0 2 S -S 76 4 0 S -R 77 3 9 S -S 78 2 2 S -S 79 9 0 S -R 80 0 15 R -S 81 0 10 R -S 69 D i s c u s s i o n Three v i r u l e n c e genes U h v 2 , Uhvzt and U h v 5 were i d e n t i f i e d i n t h i s s t u d y . V i r u l e n c e gene U h v 2 i s the same as the v i r u l e n c e gene i d e n t i f i e d by S i h d u and p e r s o n ( 1 9 7 1 ) , s i n c e the same c u l t u r e s e t D, and d i f f e r e n t i a l c u l t i v a r , E x c e l s i o r , were used i n both s t u d i e s . The o t h e r two v i r u l e n c e genes a r e new and were i d e n t i f i e d i n c u l t u r e s e t G on c u l t i v a r s H i m a l a y a and K e y s t o n e . In t h i s s t u d y the b a c k c r o s s method was used to i d e n t i f y and d e t e r m i n e the mode of i n h e r i t a n c e of v i r u l e n c e genes p r e s e n t ( indtherD a r i d n G i s e t s ' . i n T h i s method b y p a s s e s the f a i l u r e to d e t e c t s e g r e g a t i o n t h a t may o c c u r e i t h e r as a consequence of m a t i n g i n c o m p a t i b i l i t y , or o f l i n k a g e o f v i r u l e n c e genes to t h e i r c e n t r o m e r e . For a h y b r i d t e l i o -s p o r e w h i c h i s h e t e r o z y g o u s f o r one p a i r o f a l l e l e s , the two a l l e l e s g i v e r i s e to a 2 :2 s e g r e g a t i o n among the f o u r s p o r i d i a l ( g a m e t i c ) p r o d u c t s w h i c h make up the t e t r a d . I f t h e t e t r a d i s e i t h e r p a r e n t a l d i t y p e (DP) or n o n - p a r e n t a l d i t y p e (NPD) f o r the m a t i n g t y p e l o c u s and the s e g r e g a t i n g a l l e l e s , no s e g r e g a t i o n w i l l be d e t e c t e d when the gametes a r e s e l f e d , a n d , as a c o n s e q u e n c e , no u s e f u l g e n e t i c i n f o r -m a t i o n w i l l be o b t a i n e d . ( F o r e x a m p l e , i f the f o u r s p o r i d i a l p r o d u c t s a re o f the f o l l o w i n g g e n o t y p e s , i n r e s p e c t to p a t h o g e n i c i t y l o c u s and m a t i n g t y p e l o c u s : V A , V A , va and v a , a l l c o m p a t i b l e A x a m a t i n g s w i l l be h e t e r o z y g o u s Vv 70 and t h e s e w i l l be e i t h e r v i r u l e n t or a v i r u l e n t d e p e n d i n g on w h e t h e r v i r u l e n c e i s dominant o r r e c e s s i v e . The o n l y t e t r a d t h a t can p r o v i d e u s e f u l g e n e t i c i n f o r m a t i o n , t h e r e -f o r e , i s t e t r a t y p e . ) The r e s u l t s o b t a i n e d a t UBC from s e l f i n g and c r o s s i n g the two c u l t u r e s e t s on E x c e l s i o r and t e s t i n g the d i k a r y o n s p e r m i t the p r e d i c t i o n o f the r e a c t i o n o b t a i n e d i n the b a c k c r o s s e s . B a c k c r o s s e s to c u l t u r e s e t D s h o u l d a l l r e s u l t i n v i r u l e n t progeny and b a c k c r o s s e s to c u l t u r e s e t G s h o u l d s e g r e g a t e the r a t i o 4 a v i r u l e n t :4 v i r u l e n t . S i n c e the v i r u l e n c e l e v e l s o f the F i d i k a r y o n s were l o w e r t h a n the v i r u l e n c e l e v e l s o f the d i k a r y o n s of c u l t u r e s e t D, b a c k c r o s s e s to c u l t u r e s e t D were e x p e c t e d to s e g r e g a t e f o r l e v e l o f v i r u l e n c e i n the r a t i o 4 h i g h :4 l o w . H e r e , the h i g h v i r u l e n c e l e v e l r e p r e s e n t s the homozygous g e n o t y p i c ( v 2 v 2 ) c l a s s and the low l e v e l r e p r e s e n t s the h e t e r o z y g o u s ( V 2 v 2 ) c l a s s . A l t h o u g h the t h e o r e t i c a l l y - e x p e c t e d s e g r e g a -t i o n r a t i o was o b t a i n e d , the v i r u l e n c e l e v e l o f the h e t e r o -zygous c l a s s was m o d i f i e d . In some of the b a c k c r o s s e s ( i n the c l a s s e x p e c t e d to show about the same v i r u l e n c e l e v e l as the F i ) , h i g h e r v i r u l e n c e l e v e l s ( u p . t o 29%), were o b t a i n e d . In o t h e r b a c k c r o s s e s two d i f f e r e n t l e v e l s o f v i r u l e n c e , one about the same as the F i l e v e l (3 -4%) , the o t h e r i n t e r m e d i a t e (15 -29%) , were o b s e r v e d . The p r e s e n c e of a m o d i f i e r gene i n c u l t u r e s e t D was p r o p o s e d to e x p l a i n t h i s u n e x p e c t e d 71 d e v i a t i o n . The r o l e o f the m o d i f i e r i s to enhance the v i r u -l e n c e of gene v 2 when i t i s homozygous. T h i s m o d i f i e r i s d e s i g n a t e d m and i s r e c e s s i v e . I t s dominant a l l e l e , M , i s p o s s e s s e d by c u l t u r e s e t G . The F i ( V 2 v 2 ) d i k a r y o n from the D x G c r o s s and the h e t e r o z y g o u s d i k a r y o n ( V 2 v 2 ) f rom b a c k c r o s s e s to D were b o t h a v i r u l e n t i n C a l i f o r n i a but v i r u l e n t a t UBC when i n o c u -l a t e d to E x c e l s i o r . A t p r e s e n t , t h i s can o n l y be e x p l a i n e d as due to e n v i r o n m e n t a l f a c t o r ( s ) . Gene v 2 must have been s e n s i t i v e to a s p e c i f i c e n v i r o n m e n t a l f a c t o r such as t e m p e r a -t u r e , s o i l t y p e , or m o i s t u r e . I t i s not p o s s i b l e , a t t h i s t i m e , to s i n g l e out the s p e c i f i c f a c t o r i n the e n v i r o n m e n t to w h i c h the v 2 gene i s s e n s i t i v e . S e n s i t i v i t y o f a pathogen a n d / o r i t s h o s t to e n v i r o n m e n t has been o b s e r v e d o n l y i n a few h o s t : p a r a s i t e s y s t e m s . Wheat r u s t ( r a c e 5 6 ) , c a r r y i n g v i r u l e n c e gene P 6 , w h i c h i s e f f e c t i v e a g a i n s t a r e s i s t a n t gene S r 6 i n wheat c u l t i v a r s , i s known to be t e m p e r a t u r e s e n s i t i v e ( K n o t t e t a l . 3 1956; L o e g e r i n g et a l . 3 1969 ; S e e v e r s et a l . 3 1970 ; D a l y , 1 9 7 2 ) . When a wheat c u l t i v a r c a r r y i n g S r 6 i s i n o c u l a t e d w i t h a c u l t u r e o f r a c e 56 at 20°C the p l a n t s e x h i b i t i n f e c t i o n t y p e 0 ( r e s i s t a n t ) , . b u t ' a t 2 4 - 2 5 ° C the d i s e a s e r e a c t i o n i s t y p e 4 ( s u s c e p t i b l e ) . T h i s c l e a r l y i n d i c a t e s t h a t the t y p e of d i s e a s e r e a c t i o n i s r e v e r s i b l e by e n v i r o n m e n t a l f a c t o r s . In the p r e s e n t work the dominance or r e c e s s i v e n e s s o f gene v 2 dependscon some 72 f a c t o r i n the e n v i r o n m e n t . The s p e c i f i c e n v i r o n m e n t a l f a c t o r r e s p o n s i b l e remains to be i d e n t i f i e d . S i n c e c u l t u r e s e t 6 p roduces a v i r u l e n t progeny on s e l f i n g , but v i r u l e n t progeny (on E x c e l s i o r ) when c r o s s e d to s e t D , the b a c k c r o s s o f the D x G h y b r i d progeny to G s h o u l d y i e l d a 4:4 s e g r e g a t i o n r a t i o o f a v i r u l e n c e to v i r u -l e n c e on the c u l t i v a r E x c e l s i o r . The v i r u l e n t c l a s s was e x p e c t e d to show about the same v i r u l e n c e l e v e l as the F i . H o w e v e r , the l e v e l o f v i r u l e n c e i n the b a c k c r o s s was f o u n d to be r e l a t i v e l y l o w . In f a c t , i n some of the b a c k c r o s s e s the c o m b i n a t i o n t h a t was e x p e c t e d to be v i r u l e n t p r o d u c e d no d i s e a s e . In t h e s e cases the h o s t c o u l d have escaped i n f e c t i o n . The same b a c k c r o s s e s , when compared to the p a r e n t a l c u l t u r e s e t G , p r o d u c e d r e l a t i v e l y low l e v e l s o f v i r u l e n c e on b o t h H i m a l a y a and K e y s t o n e . These r e s u l t s may be e x p l a i n e d as due to the l a c k of m i n o r g e n e s , p r e s e n t i n c u l t u r e s e t G , w h i c h enhanced the v i r u l e n c e l e v e l s o f the s e l f e d progeny of s e t G . B a c k c r o s s e s of the F r to c u l t u r e s e t G were e x p e c t e d to g i v e 2 : 6 v i r u l e n c e to a v i r u l e n c e r a t i o s on b o t h H i m a l a y a and K e y s t o n e . T h i s e x p e c t a t i o n was based on the r e s u l t s o f c u l t u r e s e t G when s e l f e d , and when c r o s s e d to s e t D and t e s t e d on t h e s e c u l t i v a r s . The g e n o t y p e s a s s i g n e d to the c u l t u r e s e t G s p o r i d i a l l i n e s were V 4 V 5 A , V i t V s a , V i t V s a and V 4 V 5 A ( see T a b l e I I - l ) . The F i (G x D ) t e l i o s p o r e s would 73 be e i t h e r V 4 v 4 V 5 V 5 o r V 4 V 5 v 5 d e p e n d i n g on the p a r e n t a l G s p o r i d i a l l i n e s w h i c h p a r t i c i p a t e d i n the c r o s s w i t h c u l t u r e D. For e x a m p l e , d i k a r y o n l i n e number 9 (see T a b l e 11- 2) , w o u l d be V k v h M 5 V 5 . T h i s would produce F i s p o r i d i a l l i n e s o f the f o l l o w i n g g e n o t y p e s : \ U V 5 , V 4 V 5 and V 4 V 5 . Among t h e s e the o n l y g e n o t y p e t h a t would g i v e any u s e f u l g e n e t i c i n f o r m a t i o n i n the b a c k c r o s s t o G i s v 4 V 5 . The two s p o r i d i a l l i n e s o f V 4 V 5 g e n o t y p e c o u l d be of the same or o f d i f f e r e n t m a t i n g t y p e s . These s p o r i d i a l l i n e s ( v ^ V s ) w o u l d p a r t i c i p a t e i n f o u r out o f the e i g h t p o s s i b l e c r o s s e s i n the b a c k c r o s s to G ( F i x G ) . However , the p a r e n t a l s p o r i d i a l l i n e s w i t h w h i c h t h e y show s e g r e g a t i o n a r e of d i f f e r e n t m a t i n g t y p e s , G-1A {vk\ll) or G-3a ( v ^ V s ) . I f bo th F ^ v ^ V s ) s p o r i d i a l l i n e s a re o f the same m a t i n g t y p e o n l y one of the p a r e n t a l s p o r i d i a l l i n e S j , G-1A or G - 3 a , c a n p a r t i c i p a t e i n the c r o s s , g i v i n g a 2 :6 r a t i o o f v i r u l e n c e to a v i r u l e n c e . On the o t h e r hand i f the F i ( v i t V 5 ) s p o r i d i a l l i n e s a re o f d i f f e r e n t m a t i n g t y p e s ( e . g . d i k a r y o n l i n e n o . 11 i n T a b l e 11-4) , t h e y can p a r t i c i p a t e i n m a t i n g w i t h two d i f f e r e n t p a r e n t a l s p o r i d i a l l i n e s . T h i s , a g a i n , would y i e l d a 2 :6 r a t i o of v i r u l e n c e to a v i r u l e n c e . The r e s u l t s o b t a i n e d i n C a l i f o r n i a on K e y s t o n e c o n f o r m to t h i s e x p e c t a t i o n . In t e s t s a t UBC, some of the F i s e g r e g a t i o n d i d not conform to t h e e x p e c t a t i o n . The o n l y e x p l a n a t i o n t h a t can be o f f e r e d a t t h i s t i m e i s t h a t t h i s 74 c o u l d be due t o . s e e d c o n t a m i n a t i o n s f rom a c u l t i v a r , such as O d e s s a , w h i c h i s s u s c e p t i b l e to a l l known b i o t y p e s of s m u t . U s i n g two of the v i r u l e n c e g e n e s , v 2 and V i + , a l r e a d y d e t e r m i n e d i n the p a t h o g e n , two r e s i s t a n c e genes were i d e n -t i f i e d f rom the c r o s s between c u l t i v a r s E x c e l s i o r and K e y s t o n e . R 2 was i d e n t i f i e d i n E x c e l s i o r and i s the same as the r e s i s t a n c e gene i d e n t i f i e d by S i h d u and P e r s o n ( 1 9 7 2 ) . was i d e n t i f i e d i n K e y s t o n e and was not p r e v i o u s l y known. S i n c e i t was not p r a c t i c a l t o t e s t the s e g r e g a t i o n f o r r e s i s t a n c e and s u s c e p t i b i l i t y i n the F 2 , the p r o g e n i e s were advanced to the F 3 g e n e r a t i o n and t e s t e d . The F 3 g e n e r a t i o n l i n e s s e g r e g a t e d i n t o a 3:1 r a t i o o f s u s c e p t i b i l i t y (S) to r e s i s t a n c e (R) to the i n d i v i d u a l c u l t u r e s . The 3:1 r a t i o o f S t o R o b t a i n e d i n the F 3 l i n e s i s e x p e c t e d i f the r e -s i s t a n c e i s e x p r e s s e d as a dominant c h a r a c t e r i s t i c on each o f the two c u l t i v a r s . T h i s w o u l d have g i v e n a 3:1 r a t i o o f R:S had i t been p o s s i b l e to t e s t i n the F 2 . When the r e a c t i o n s to the two t e s t c u l t u r e s were examined i n the F 3 g e n e r a t i o n , a 9 : 3 : 3 : 1 s e g r e g a t i o n r a t i o o f S - S ; S - R : R - S ; R - r was o b s e r v e d . T h i s would have g i v e n a 1 : 3 : 3 : 9 r a t i o o f S - S : S - R : R - S : R-R had i t been p o s s i b l e to t e s t f o r i t i n the F 2 g e n e r a t i o n . The b a s i s f o r t h i s e x p e c t a t i o n i s shown i n T a b l e s 11-8 and 9 . The c o n c l u s i o n t h a t r e s i s t a n c e 75 i s d o m i n a n t , d e t e r m i n e d on the b a s i s of F 3 d a t a , i s c o n s i s -t e n t w i t h the f a c t t h a t the F i o f the E x c e l s i o r x K e y s t o n e c r o s s were r e s i s t a n t to both c u l t u r e s . T a b l e 11-8 T h e o r e t i c a l l y E x p e c t e d P a r e n t a l , F x and F 2 g e n o t y p e s of B a r l e y C u l t i v a r s E x c e l s i o r and K e y s t o n e and t h e i r R e a c t i o n to v 2 v 2 V i f V i t and V 2 V 2Vi tVi t d i k a r y o n s , Based on F l o r ' s G e n e - f o r - G e n e R e l a t i o n s h i p Host Genotypes I n t e r a c t i o n s w i t h Di k a r y o n s * v 2 v 2 V ^ V it V 2 V 2 V i f V ^ E x c e l s i o r (E) R 2 R 2 r i f r i f S R K e y s t o n e (K) r 2 r 2 R i t R i t R S K x E ( F 2 ) R 2 r 2 R i t r i t R R 9 R 2 - R i » - R R F 2 -3 R 2 - r i t r i t -3 r 2 r 2 R i t -1 r 2 r 2 r ^ r i t R S S S R S R = R e s i s t a n c e ; S = S u s c e p t i b l e T a b l e 11-9 E x p e c t e d S e g r e g a t i o n of F 3 L i n e s o f B a r l e y D e r i v e d f rom E x c e l s i o r x K e y s t o n e C r o s s F o l l o w i n g I n o c u l a t i o n w i t h v 2 v 2 V < t V i t and V 2 V 2 v i+v i + D i k a r y o n s o f u. hordei (based on F l o r ' s G e n e - f o r - G e n e H y p o t h e s i s ) . G e n e t i c C o m p o s i t i o n of F o l l o w i n g I n o c u l a t i o n w i t h P h e n o t y p e o f F 3 C l a s s i f i c a t i o n o f the F 3 L i n e F r e q u e n c y F 2 F 3 v 2 v 2 V i t V i t V 2 V 2 V n V i + R-R 1/16 1/16 R z R a R ^ R ^ R 2 R 2 R i t R % R R R 2/16 R 2 R 2 R i t r 1+ 3/4 R 2 R 2 R i t -1/4 R 2 R 2 r , t r 4 R R R S R-S 3/16 1/16 R z R ^ t r i t R 2 R 2 r i>rn R S 2/16 R 2 r 2 R i t R i t 3/4 R 2 - R^R4 1/4 r 2 r 2 R l ( R 1 t R S R R S-R * \ 3/16 1/16 r z r z R t R ^ r 2 r 2 R i t R i t S R 4/16 R z r z R ^ 9/16 R 2 - R i t -3/16 R 2 - r i t r i t 3/16 r 2 r 2 R t -1/16 r 2 r 2 r i t r i t R R S S R S R S S-S 9/16 2/16 R 2 r 2 r i t r i t 3/4 R z - r i t T i t 1/4 r 2 r 2 r 4 r i t R S S S 2/16 r 2 r 2 R , t r i t 3/4 r 2 r 2 R , t -1/4 r 2 r 2 r 4 r i t S S R S 1/16 r 2 r 2 r ^ r i t r 2 r 2 r . t r i t S S 77 The d a t a showed c l e a r l y t h a t two r e s i s t a n c e g e n e s , one i n each p a r e n t a l c u l t i v a r , were i n v o l v e d and t h a t they s e g r e g a t e d i n d e p e n d e n t l y . In summary, the g e n e t i c s t u d y w i t h the p a r a s i t e r e v e a l e d t h r e e v i r u l e n c e g e n e s : one i d e n t i f i e d i n i n t e r a c t i o n w i t h E x c e l s i o r , the o t h e r two w i t h H i m a l a y a and K e y s t o n e j o i n t l y . The g e n e t i c s t u d y w i t h the h o s t r e v e a l e d two i n d e -p e n d e n t l y i n h e r i t e d r e s i s t a n c e g e n e s , one i n E x c e l s i o r , the o t h e r i n K e y s t o n e . These were i d e n t i f i e d w i t h the a i d of two smut d i k a r y o n s , v 2 v 2 V 4 V / 4 and V 2V 2V tv >*, r e s p e c t i v e l y . The g e n o t y p e of E x c e l s i o r i s d e s i g n a t e d Rz^z^^rn and t h a t o f K e y s t o n e , r 2 r 2 R < t R i t . The k i n d of g e n e - f ors-gene r e l a t i o n s h i p f i r s t demon-s t r a t e d by F l o r ( 1942 , 1955) i n f l a x and f l a x r u s t has been d e m o n s t r a t e d , r e c e n t l y , i n b a r l e y and c o n v e r e d - s m u t of b a r l e y , by S i h d u and Rerson ( 1 9 7 2 ) . The r e s u l t s o f the p r e s e n t s t u d y a l s o f i t the c r i t e r i a of the g e n e - f o r - g e n e r e l a t i o n s h i p as d e s c r i b e d by F l o r ( 1 9 5 5 ) , and by P e r s o n ( 1 9 5 9 ) , and s t r e n g t h e n the c o n c l u s i o n s r e a c h e d by S i h d u and P e r s o n (1972) c o n c e r n i n g the g e n e - f o r - g e n e r e l a t i o n s h i p i n t h i s s y s t e m . I n t e r a c t i o n s i n v o l v i n g r e s i s t a n c e gene R^ and v i r u l e n c e genes v ^ , r e v e a l e d i n the p r e s e n t s t u d y , a re a l ; l c i n c c o m p l e t e s a c c o K d ' w r t h FI or 1 s h y p o t h e s i s . A t the p r e s e n t , g e n e - f o r - g e n e r e l a t i o n s h i p s have been e i t h e r d e m o n s t r a t e d o r p o s t u l a t e d i n a b o u t 23 h o s t : p a r a s i t e 78 s y s t e m s , a l l o f w h i c h have some f e a t u r e s i n common: 1. R e s i s t a n c e i n t h e h o s t i s u s u a l l y d o m i n a n t w h e r e a s v i r u l e n c e i n t h e p a r a s i t e i s u s u a l l y r e c e s s i v e . 2. A l l e l i s m has been shown t o e x i s t i n t h e h o s t b u t n o t i n t h e p a r a s i t e ( s e e P a r t I ) . There i s no r e p o r t , known to the a u t h o r , where a g e n e - f o r - g e n e r e l a t i o n s h i p has been e s t a b l i s h e d or p o s t u l a t e d i n w h i c h v i r u l e n c e i s d o m i n a n t . The f a c t t h a t v i r u l e n t was i n c o m -p l e t e l y dominant i n the p r e s e n t s t u d y ( i n t e s t s c o n d u c t e d a t U B C ) , does not a l t e r the g e n e - f o r - g e n e c o n c e p t . The gene-f o r - g e n e r e l a t i o n s h i p i s not a f f e c t e d by the dominance r e l a -t i o n s h i p s o f e i t h e r v i r u l e n c e o r r e s i s t a n c e genes ( P e r s o n and Mayo, 1 9 7 3 ) . T h e r e f o r e the g e n e - f o r - g e n e r e l a t i o n s h i p has been d e m o n s t r a t e d i n t h i s s y s t e m . Becausedoifietheasritmri i l iar i t y pf isreaetdionsashown^ by both H i m a l a y a and K e y s t o n e i n i n t e r a c t i o n w i t h d i k a r y o n s formed from c u l t u r e s e t G , i t would be r e a s o n a b l e to p o s t u l a t e t h a t the r e s i s t a n c e gene p o s s e s s e d by H i m a l a y a i s the same as the one i d e n t i f i e d i n K e y s t o n e . T h i s R - g e n e (R^ ) w o u l d i n t e r a c t s p e c i f i c a l l y w i t h the two d u p l i c a t e v i r u l e n c e g e n e s , v 4 and v 5 a n d , i n so d o i n g , i t w o u l d have a d u a l s p e c i f i c i t y . The geno-t y p e o f t h e s e two c u l t i v a r s would be r 2 r 2 R i t R i t . Based on the g e n e r a l c o n d i t i o n s put f o r w a r d by P e r s o n ( 1 9 5 9 ) , t h i s w o u l d be a s p e c i a l case o f the g e n e - f o r - g e n e r e l a t i o n s h i p . P A R T I I I SYNTHESIS OF A COMPLEX USTILAGO HORDEI BIOTYPE AND ITS FITNESS RELATIVE TO SIMPLE BIOTYPES I n t r o d u c t i on When two d i f f e r e n t genes are b r o u g h t t o g e t h e r i n a s i n g l e o r g a n i s m they may i n t e r a c t i n such a way as to a f f e c t the f i t n e s s o f the o r g a n i s m , whose r e p r o d u c t i o n may be enhanced or r e d u c e d . I t i s a l s o p o s s i b l e t h a t t h e f i t n e s s o f t h e o r g a n i s m w i l l r e m a i n u n c h a n g e d . Van der P l a n k ( 1 9 6 9 ) , f rom h i s s t u d y o f the p u b l i s h e d work on Phytophthora infestans, Fusavium oxysporum f . s p . lyoopevsioi, and on Puccinia gvaminis f . s p . t v i t i c i , c o n c l u d e d t h a t u n n e c e s s a r y v i r u l e n c e i n a b i o t y p e o f a pathogen w i l l r e d u c e i t s f i t n e s s to s u r v i v e as w e l l as i t s a g g r e s s i v e n e s s . Van der P l a n k ' s c o n c l u s i o n has been d i s -c u s s e d i n s e v e r a l r e c e n t p u b l i c a t i o n s ( G r e e n , 1971 ; M a r t e n s et a l . , 1973 ; MacKey , 1973) and c o n f l i c t i n g e v i d e n c e , bo th f o r and a g a i n s t the c o n c l u s i o n s , has been p r e s e n t e d . 79 80 T h i s s t u d y was u n d e r t a k e n , f i r s t to s y n t h e s i z e a b i o t y p e of 177 hordei, homozygous f o r two v i r u l e n c e genes ( V 1 V 1 V 2 V 2 ) . The two v i r u l e n c e g e n e s , V i and v 2 , i n the s i m p l e b i o t y p e s were i d e n t i f i e d by S i h d u and P e r s o n ( 1 9 7 1 ) . The o b j e c t i v e was to d e t e c t i n t e r a c t i o n t h a t may t a k e p l a c e between t h e s e genes i n the s y n t h e s i z e d c u l t u r e . The d i s e a s e r e a c t i o n s o f the p a r e n t a l c u l t u r e s ( h a v i n g s i n g l e g e n e s ) , and of the d e r i v e d c u l t u r e ( h a v i n g both g e n e s ) , were compared both q u a l i t a t i v e l y and q u a n t i t a t i v e l y . M a t e r i a l s and Methods Two h a p l o i d c u l t u r e s e t s d e s i g n a t e d D and F ( S i h d u , 1 9 7 2 ) , and of g e n o t y p e s V i V 2 and V i V 2 , r e s p e c t i v e l y , were mated and i n o c u l a t e d on the b a r l e y c u l t i v a r O d e s s a . The F i t e l i o s p o r e s p r o d u c e d by t h i s c r o s s w o u l d g e n e r a t e equa l numbers o f bo th m a t i n g t y p e s a n d , f o r s p o r i d i a o f each m a t i n g t y p e , f o u r p o s s i b l e g e n o t y p e s , as f o l l o w s : V i V 2 , V i V 2 , V i V 2 and V i V 2 . S i n c e the p u r p o s e of t h i s s t u d y was to s y n t h e s i z e a d i k a r y o n of g e n o t y p e V i V i V 2 v 2 , bo th m a t i n g t y p e s of o n l y one of the f o u r p o s s i b l e g e n o t y p e s , namely V i V 2 , were n e e d e d . There i s no e v i d e n c e to d a t e o f l i n k a g e i n v o l v i n g V i and v 2 , e i t h e r w i t h each o t h e r or w i t h the m a t i n g t y p e l o c u s . I t was e x p e c t e d , t h e r e f o r e , t h a t f o r s p o r i d i a o f 81 e i t h e r m a t i n g t y p e , one i n f o u r w o u l d be of g e n o t y p e V i V 2 . T h u s , the p r o b a b i l i t y o f not o b t a i n i n g t h i s genotype i s 3 / 4 , and the number (n) o f s p o r i d i a needed i n o r d e r to i n c l u d e a t l e a s t one s p o r i d i u m of the d e s i r e d g e n o t y p e s , w i t h a known p r o b a b i l i t y o f f a i l u r e (F) i s r e p r e s e n t e d by the f o l l o w -i n g e q u a t i o n : ( 3 / 4 ) n = F where n = number of s p o r i d i a , o f one m a t i n g t y p e , needed F = chance of f a i l u r e s e l e c t e d as s a t i s f a c t o r y . In t h i s c a s e , w i t h F s e t a t 1 5 / 1 0 0 0 , the minimum number of s p o r i d i a needed i s n l o g ( 3 / 4 ) = l o g ( 1 5 / 1 0 0 0 ) ; The minimum number o f s p o r i d i a o f one m a t i n g t y p e t h a t s h o u l d be i s o l a t e d i s 1 5 . By the same r e a s o n i n g the number of s p o r i d i a o f the o p p o s i t e m a t i n g t y p e t h a t i s a l s o needed i s 1 5 , making a t o t a l o f 30 s p o r i d i a l l i n e s . The 30 s p o r i d i a l l i n e s were c u l t u r e d and the 225 ( i . e . 15 x 15) c o m p a t i b l e m a t i n g s were made. These were used to i n o c u l a t e two b a r l e y c u l t i v a r s , E x c e l s i o r and V a n t a g e , s u s c e p t i b l e to d i k a r y o n s v 2 v 2 and V i V i , r e s p e c t i v e l y ( i . e . 225 i n o c u l a t i o n s of each c u l t i v a r were s t u d i e d ) . The 82 e x p e c t a t i o n was t h a t any V j . v 2 and V i V 2 s p o r d i a l c o m b i n a t i o n o f o p p o s i t e m a t i n g t y p e s w i l l produce d i s e a s e on both c u l t i v a r s s i m u l t a n e o u s l y . The methods of c u l t u r i n g , i n o c u l a t i n g , s e l f i n g and c r o s s i n g were the same as t h o s e d e s c r i b e d f o r P a r t I . Out o f the 225 i n o c u l a t i o n s to both E x c e l s i o r and V a n t a g e , o n l y one caused d i s e a s e i n both h o s t c u l t i v a r s . As a p r e l i m i n a r y t e s t , two random s p o r i d i a of o p p o s i t e m a t i n g t y p e were i s o l a t e d from t e l i o s p o r e s c o l l e c t e d f rom d i s e a s e d p l a n t s o f each o f the two b a r l e y c u l t i v a r s . The two s e t s o f random s p o r i d i a were used to i n o c u l a t e seeds of the two c u l t i v a r s to d e t e r m i n e w h e t h e r , i n f a c t , the V 1 V 1 V 2 V 2 d i k a r y o n has been o b t a i n e d . A c o m p l e t e t e t r a d s e t , o b t a i n e d from a s i n g l e t e l i o s p o r e f rom one of the smutted s p i k e s , was a l s o used i n an i n v e s t i g a t i o n to t e s t whether the new d i k a r y o n w a s , i n f a c t , V i V i V 2 v 2 . Two s e p a r a t e t e s t s were c o n d u c t e d . F i r s t , t e t r a d p r o d u c t s o f the new d i k a r y o n were mated w i t h the two p a r e n t a l c u l t u r e s e t s , V i and v 2 . I t was e x p e c t e d t h a t the V i V 2 gametes ( s p o r i d i a ) o f the new d i k a r y o n , when c r o s s e d w i t h v x V 2 would g i v e i n f e c t i o n on Vantage and not on E x c e l s i o r . S i m i l a r l y , a c r o s s between the new c u l t u r e s e t and V i V 2 w o u l d p r o d u c e i n f e c t i o n on E x c e l s i o r but not on V a n t a g e . In the second t e s t , t e t r a d s e t s o f V i V 2 ) 83 V i V 2 and V i V 2 c u l t u r e s were c r o s s e d to two s e t s o f t e t r a d p r o d u c t s i s o l a t e d f rom F i t e l i o s p o r e s o f the V i V 2 x V i v 2 c r o s s . T e t r a d p r o d u c t s o f the new d i k a r y o n were a l s o c r o s s e d to t e t r a d p r o d u c t s o f c u l t u r e G and i n o c u l a t e d onto E x c e l s i o r . C u l t u r e G was a v i r u l e n t on E x c e l s i o r , whereas c u l t u r e s D and T5 were v i r u l e n t on E x c e l s i o r . T e t r a d p r o d u c t s f rom a s i n g l e t e l i o s p o r e ( v i V i V 2 v were s e l f e d and i n o c u l a t e d to e l e v e n b a r l e y c u l t i v a r s . Out o f t h e s e e l e v e n c u l t i v a r s , seven were used i n a s t u d y to compare the l e v e l s o f v i r u l e n c e i n d u c e d by the V i V i V 2 v 2 , V i V i V 2 V 2 and V i V i V 2 v 2 d i k a r y o n s . The e l e v e n c u l t i v a r s and t h e i r r e a c t i o n to the d i k a r y o n s w h i c h were p r o d u c e d by s e l f i n g and c r o s s i n g the t h r e e c u l t u r e s e t s a re p r e s e n t e d i n T a b l e I I I - l and 1 1 1 - 2 . S i n c e the d i s e a s e r e a c t i o n s were r e c o r d e d i n terms of p e r c e n t a g e s o f smut ted p l a n t s , the d a t a were t r a n s formed to a r c s i n e to a l l o w f o r a v a l i d a n a l y s i s of v a r i a n c e One-way a n a l y s i s o f v a r i a n c e was u s e d , t o g e t h e r w i t h T u k e y ' t e s t f o r m u l t i p l e c o m p a r i s o n , to compare the l e v e l s o f d i s e a s e r e a c t i o n c o n t r o l l e d by the new c u l t u r e w i t h t h o s e of the p a r e n t a l c u l t u r e s . In the a n a l y s i s , the d i s e a s e r e -a c t i o n v a l u e s o b t a i n e d f rom the f o u r d i f f e r e n t c o m b i n a t i o n s o f a t e t r a d s e t were used as o b s e r v a t i o n s . 84 Results The newly synthesized dikaryon is designated T 5 in this study. That i t s genotype was V i V i V 2 v 2 was evident from the resul ts of three sets of experiments. 1. A l l t h e p r o g e n y p r o d u c e d by s e l f i n g t h e new d i k a r y o n d i s p l a y e d t h e v i r u l e n c e o f b o t h i n i t i a l d i k a r y o n s , and we re a b l e t o i n c i t e d i s e a s e on b o t h h o s t c u l t i v a r s . T h i s i n d i c a t e d t h a t t h e new d i k a r y o n was homozygous f o r b o t h v i and v 2 . 2. C r o s s e s b e t w e e n t e t r a d s e t s o b t a i n e d f r o m t h e new d i k a r y o n and V i V i V 2 V 2 we re u n i f o r m l y v i r u l e n t ? on V a n t a g e , a nd ra v m n ill I en t E© neExcie t s i ©h<;>s<bhosewbeiiiwee n s e t s d e r i v e d f r o m t h e new d i k a r y o n and V i V i v 2 v 2 we re u n i f o r m l y v i r u l e n t on E x c e l s i o r b u t n o t on V a n t a g e . T h e s e r e s u l t s showed t h a t t h e new d i k a r y o n was homozygous f o r b o t h v i and v 2 and was o f g e n o t y p e v i V i v 2 v 2 . 3. When t h e t e t r a d s e t d e r i v e d f r o m t h e new d i k a r y o n ( i . e . f r o m d i k a r y o n T5, o f p r e sumed g e n o t y p e v i V i v 2 v 2 , and a i l c u l t u r e s o f t h e s e t p r e s umed t o be v i v 2 ) was c r o s s e d w i t h two d i f f e r e n t t e t r a d s e t s o b t a i n e d f r o m F i d i k a r y o n s o f t h e D x F c r o s s ( F i g e n o t y p e V i V i V 2 v 2 ; c u l t u r e s o f t h e s e t s e g r e g a t i n g f o r b o t h V i : v i and V 2 . : v 2 ) t h e e x p e c t e d 4 : 4 s e g r e g a t i o n s we re o b t a i n e d on b o t h E x c e l s i o r and V a n t a g e 85 t h e s e two c u l t i v a r s r e v e a l t h e V i : v i and V 2 : v 2 s e g r e g a t i o n s , r e s p e c t i v e l y ) . The g e n o t y p e s p o s t u l a t e d , on t h e b a s i s o f t h e s e s e g r e g a t i o n s , f o r t h e f o u r s p o r i d i a l c u l t u r e s o f e a c h o f t h e two F i t e t r a d s e t s we re t h e n t e s t e d by b a c k c r o s s i n g t h e s e t s t o p a r e n t a l s p o r i d i a o f g e n o t y p e s V i V i and V 2 v 2 . When t h e b a c k c r o s s d i k a r y o n s were t e s t e d on c u l t i v a r s V a n t a g e and E x c e l s i o r , 4 : 4 s e g r e g a t i o n s we re o b t a i n e d w h i c h c o n f i r m e d t h e g e n o t y p i c a s s i g n m e n t s , made e a r l i e r , t o t h e f o u r c u l t u r e s o f e a c h o f t h e two s e t s . When s e l f e d , the new c u l t u r e p r o d u c e d the same l e v e l o f v i r u l e n c e as d i d the p a r e n t a l c u l t u r e , F , on c u l t i v a r s H a n n c h e n , V a n t a g e , O d e s s a , T r e b i and L i o n . A c r o s s between t e t r a d s e t s o f T5 and F showed a s i g n i f i c a n t l y h i g h e r l e v e l o f v i r u l e n c e than d i d e i t h e r T5 or F a l o n e . When s e l f e d c u l t u r e T5 showed a s i g n i f i c a n t l y l o w e r l e v e l o f v i r u l e n c e on c u l t i v a r s s u s c e p t i b l e o n l y to c u l t u r e D ( v i z . E x c e l s i o r , Nepal and P a n n i e r ) . C u l t u r e T5 gave a r e l a t i v e l y h i g h e r l e v e l o f v i r u l e n c e on c u l t i v a r s Odessa and T r e b i . A c r o s s between t e t r a d s e t s d e r i v e d f rom d i k a r y o n s T5 and D e x h i b i t e d the same l e v e l o f v i r u l e n c e as t h a t o f D. As shown i n T a b l e I I I - l , d i k a r y o n s D and F were a v i r u l e n t on K e y s t o n e , H i m a l a y a and C o n q u e s t . However , T5 was v i r u l e n t on t h e s e c u l t i v a r s . D i k a r y o n G was a v i r u l e n t and T5 was v i r u l e n t on E x c e l s i o r . However , a c r o s s between T a b l e I I I - l The R e a c t i o n of E l e v e n B a r l e y C u l t i v a r s , t o Se t s o f u. hordei D i k a r y o n s w h i c h were D e r i v e d f rom S i n g l e T e l i o s p o r e s of Two P a r e n t a l D i k a r y o n s - i d and t h e i r F i H y b r i d S p o r i d i a l M a t i n g E x c e l s i or Nepal P a n n i e r Hannchen Vantage Odessa T r e b i L i o n K e y s t o n e Hi C T5-1 x T5-2 6 16 14 27 31 40 32 24 23 41 31 " x 4 12 15 5 28 33 42 24 25 17 40 23 3 x 2 9 12 17 24 30 47 27 16 23 46 20 " x 4 15 15 13 20 25 42 35 10 12 42 17 D-1 x D-2 56 62 26 0 0 30 19 1 5 0 0 0 11 x 4 22 46 20 0 0 17 16 12 0 0 0 3 x 2 48 24 25 0 0 27 21 18 0 0 0 " x 4 36 38 21 0 0 38 18 11 0 0 0 F-1 x F -2 0 0 0 26 25 24 20 16 0 0 0 " x 3 0 0 0 31 27 27 31 14 0 0 0 4 x 2 0 0 0 22 31 37 1 5 13 0 0 0 " x 3 0 0 0 27 35 41 25 16 0 0 0 CO 87 T a b l e 111-2 C r o s s e s o f F i ( v i V 2 ) T e t r a d Se t w i t h P a r e n t a l ( v i V 2 and V i v 2 ) Se t s and G , and T h e i r D i s e a s e R e a c t i o n on E x c e l s i o r and Vantage S p o r i d i a l M a t i n g E x c e l s i o r Vantage Genotype of Pathogen T5-1 x D -2 38 0 V i v i v 2 v 2 II X 4 21 0 M 2 X 1 28 0 II X 3 33 0 II 3 X 2 33 0 II n X 4 51 0 n 4 X 1 46 0 n II X 3 38 0 n -T-5^ 1) x F -2 0 61 v i v iV 2 v z II X ~3 0 42 2 X 1 0 63 II X 4 0 51 n 3 X 2 0 67 M II X 3 0 59 n 4 X 1 0 61 II II X 4 0 42 n T5-1 x G -1 1 - V 2 V 2 n X 4 5 II 2 X 2 0 II n X 3 5 II 3 X 1 1 II II X X 4 4 II 4 X 2 1 II II X 3 2 II T a b l e 111 - 3 C r o s s e s I n v o l v i n g Two T e t r a d Se ts (Ha and Hb) D e r i v e d f rom F-i T e l i o s p o r e s o f the V i v 2 x V i V 2 c r o s s and Three D i k a r y o n s ( v i V i V 2 v 2 ; V i V i V 2 v 2 and V i V i V 2 V 2 ) , T o g e t h e r w i t h the D i s e a s e R e a c t i o n s (%) O b t a i n e d on E x c e l s i o r and Vantage S p o r i d i a l M a t i n g E x c e l s i o r Vantage S p o r d i a l M a t i n g E x c e l s i or Vantage Ha-1 x V i V 2 - l 0 0 Hb-1 X V i V 2 - 2 40 0 X y 3 0 0 n X 4 38 0 2 % x 2 8 21 2 X 1 0 0 n X 4 • 18 19 11 X 3 0 0 3 X 2 1 0 44 3 X 2 0 0 11 X 4 9 29 11 X 4 0 0 4 X X i ; 8 0 4 X 1 33 0 11 3 0 n X 3 44 0 Hb-1 X V i V 2 - 2 4 0 Ha-1 X V l V 2 - l 0 0 11 X 4 6 0 n X 4 0 0 2 X 1 0 22 2 X 2 0 45 n X 3 0 30 n X 3 0 36 3 X 2 0 0 3 X 2 0 58 n X 4 0 0 11 X 3 0 43 4 X 1 8 28 4 X 1 0 0 11 X 3 8 29 n X 4 0 0 ( C o n t i n u e d ) CO OO T a b l e 111-3 ( C o n t i n u e d ) S p o r i d i a l M a t i ng E x c e l s i or Vantage S p o r d i a l M a t i n g E x c e l s i or Vantage Ha-1 x V i v 2 - l 0 0 Hb-1 x V 1 V 2 - 2 0 0 " X 3 0 0 " x 3 0 0 2 x 2 41 0 2 x 1 0 50 11 X 4 35 0 " X 4 0 39 3 x 2 0 0 3 x 2 0 0 " X 4 0 0 " X 3 0 0 4 x 1 39 0 4 x 1 0 40 " X 3 43 0 " X 4 0 35 00 90 t e t r a d s of t h e s e d i k a r y o n s was v i r u l e n t on E x c e l s i o r , i n d i -c a t i n g v i r u l e n c e m i g h t be dominant on E x c e l s i o r ( c f . P a r t I I ) . D i s c u s s i o n The new u. hovdei b i o t y p e , v 1 v 1 v 2 v 2 j has a^.reduced l e v e l o f v i r u l e n c e on t h o s e c u l t i v a r s t h a t a r e s u s c e p t i b l e to the p a r e n t a l d i k a r y o n D ( V i V i V 2 v 2 ) . T h i s c o u l d be due t o : ( i ) the V i gene h a v i n g a n e g a t i v e e p i s t a t i c e f f e c t on v 2 , t h e r e b y s i g n i f i c a n t l y r e d u c i n g i t s l e v e l o f v i r u l e n c e on c u l t i v a r s E x c e l s i o r , Nepal and P a n n i e r ; ( i i ) m i n o r g e n e s , p o s s i b l y t r a n s f e r r e d w i t h V i g a m e t e s , w h i c h m o d i f y the v i r u -l e n c e l e v e l o f v 2 when a c t i n g i n c o m b i n a t i o n w i t h i t . The p r e s e n t work does not d i s c r i m i n a t e between t h e s e a l t e r n a t i v e s . On c u l t i v a r s s u s c e p t i b l e to both p a r e n t a l c u l t u r e s as w e l l as on t h o s e s u s c e p t i b l e to v x v i o n l y , the new c u l t u r e p r o d u c e d the same l e v e l o f d i s e a s e r e a c t i o n as d i d the p a r e n t a l c u l t u r e . The new b i o t y p e has a l s o e x t e n d e d i t s v i r u l e n c e range by b e i n g v i r u l e n t on c u l t i v a r s w h i c h were r e s i s t a n t to b o t h p a r e n t a l c u l t u r e s . The r e d u c e d d i s e a s e r e a c t i o n s , shown by c u l t u r e T5 on E x c e l s i o r , Nepal and P a n n i e r (as compared w i t h c u l t u r e D) , i r i i n d i c a t e s t h a t the new b i o t y p e i s l e s s f i t on t h e s e 91 c u l t i v a r s than the p a r e n t a l c u l t u r e s . T h i s d i f f e r e n c e w o u l d be i m p o r t a n t i f c u l t i v a r s o f g e n o t y p e r i r i R 2 R 2 had been s e l e c t e d f o r i n a g r i c u l t u r e and were w i d e l y g r o w n . On the o t h e r h a n d , i f c u l t i v a r s o f genotype R 1 R 1 R 2 R 2 , R i R i ^ 2 r 2 , F i r i R 2 R 2 and r i r i r 2 r 2 were a l l w i d e l y g r o w n , the d e c r e a s e i n v i r u l e n c e l e v e l on some c u l t i v a r s w ou ld be compensated f o r by the e x t e n d e d h o s t r a n g e . The new c u l t u r e c a r r i e s a t l e a s t one u n n e c e s s a r y gene f o r v i r u l e n c e on c u l t i v a r s O d e s s a , T r e b i and L i o n , s i n c e t h e s e c u l t i v a r s were s u s c e p t i b l e to both p a r e n t a l c u l t u r e s and p o s s e s s e d r e s i s t a n c e to n e i t h e r o f them. The new c u l t u r e p r o d u c e s the same l e v e l o f v i r u l e n c e as d i d the p a r e n t a l c u l t u r e F , and h i g h e r l e v e l s o f v i r u l e n c e than p a r e n t a l c u l t u r e D on Odessa and T r e b i . T h i s i n d i c a t e s t h a t the u n n e c e s s a r y gene f o r v i r u l e n c e on t h e s e two c u l t i v a r s , does not make i t l e s s f i t than the p a r e n t a l c u l t u r e s , w h i c h p o s s e s s o n l y one of the two v i r u l e n c e g e n e s . Van der P l a n k ( 1 9 6 8 , 1969) c o n c l u d e d t h a t u n n e c e s -s a r y v i r u l e n c e i n a r a c e r e d u c e s i t s f i t n e s s t o s u r v i v e as w e l l as i t s a g g r e s s i v e n e s s . L e o n a r d (1969) showed e x p e r i -m e n t a l l y t h a t o a t stem r u s t c o m p l i e s w i t h t h i s p r i n c i p l e . However , t h e r e a r e a number o f w o r k e r s who are z e a l o u s a d h e r e n t s o f the o p p o s i t e v i e w . Green ( 1 9 7 1 ) , u s i n g some of the same wheat stem r u s t d a t a as Van der P l a n k had a n a l y z e d , c o n c l u d e d t h a t t h e r e i s i n s u f f i c i e n t e v i d e n c e f o r 92 c o n c l u d i n g t h a t u n n e c e s s a r y v i r u l e n c e i s h a r m f u l o r t h a t s t a b i l i z i n g s e l e c t i o n i s o p e r a t i v e . Ogle and Brown (1970) i n A u s t r a l i a , f o u n d t h a t wheat stem r u s t s t r a i n s w i t h h i g h e r r e l a t i v e s u r v i v a l a b i l i t y a l s o had the w i d e r h o s t r a n g e . Mac Key ( 1 9 7 3 ) , i n Sweden, r e a c h e d the same c o n c l u s i o n as Green and Ogle et a l . f o r the same o r g a n i s m . Mar tens et a l . (1970) c o n c l u d e d t h a t the most s u c c e s s f u l r a c e s o f o a t stem r u s t i n Canada o v e r many y e a r s have c a r r i e d genes f o r v i r u -l e n c e i n excess o f t h o s e r e q u i r e d f o r s u c c e s s f u l p a r a s t i s m i n N o r t h A m e r i c a . S i m i l a r l y , Brown and Sharp (1970) f o u n d t h a t the s t r i p e r u s t (p. s t r i i f o r m i s ) b i o t y p e showing the w i d e s t h o s t range had a g r e a t e r s u r v i v a l a b i l i t y . Mac Key (1973) f o u n d t h a t i n wheat l e a f r u s t c e r t a i n c o m b i n a t i o n s o f v i r u l e n c e genes d e c r e a s e f i t n e s s w h i l e o t h e r s have no a f f e c t . In crown r u s t of o a t s (P. coronata f . s p . avenae), M a r t e n s et a l . (1973) r e a c h e d the c o n c l u s i o n t h a t complex r a c e s a r e n o t l e s s f i t . These s t u d i e s i n d i c a t e t h a t , f o r c e r e a l r u s t s , t h e r e i s good e v i d e n c e to s u p p o r t the g e n e r a l c o n c l u s i o n t h a t complex r a c e s a re common. T h i s would i n d i c a t e t h a t r a c e s w i t h l a r g e r number o f v i r u l e n c e genes a re b e t t e r f i t t h a n t h o s e h a v i n g a s i n g l e v i r u l e n c e gene a n d , a t the v e r y l e a s t , t h a t u n n e c e s s a r y v i r u l e n c e does not r e d u c e f i t n e s s . T h e r e f o r e , Van der P l a n k ' s i d e a o f s i m p l e r a c e s does not seem to h o l d i n c e r e a l r u s t s . 93 Mac Key (1973) i n d i c a t e d t h a t the d a t a f o r powdery m i l d e w and wheat a r e i n a c c o r d w i t h Van d e r P l a n k ' s i d e a t h a t complex r a c e s a r e l e s s f i t . W o l f e et a l . ( 1 9 7 3 ) , w o r k i n g w i t h powdery m i l d e w of b a r l e y , f o u n d t h a t s i m p l e r a c e s a r e b e t t e r c o m p e t i t o r s when c o m p e t i n g on a s i n g l e h o s t . B u t , of c o u r s e , complex r a c e s have a w i d e r h o s t r a n g e . The p r e s e n t d a t a , because of t h e i r l i m i t e d s c o p e , may not s u p p o r t a g e n e r a l c o n c l u s i o n on the d i f f e r e n t i a l f i t n e s s o f complex and s i m p l e b i o t y p e s o f V. hordei. N e i t h e r do t h e y s u p p o r t Van der P l a n k ' s h y p o t h e s i s t h a t complex b i o t y p e s a r e n e c e s s a r i l y l e s s f i t than s i m p l e o n e s . I t i s e v i d e n t f rom the r e s u l t s t h a t the new b i o t y p e w i t h two known v i r u l e n c e genes e x h i b i t e d , on c u l t i v a r s w i t h o u t R i and R 2 , the same or h i g h e r l e v e l o f v i r u l e n c e as the p a r e n t a l b i o t y p e s f rom w h i c h i t was s y n t h e s i z e d . A l s o , the new c u l t u r e , by i t s c a p a c i t y to i n f e c t c u l t i v a r s r e s i s t a n t ' t o both p a r e n t a l c u l t u r e s , has i n c r e a s e d i t s v i r u l e n c e r a n g e . O b v i o u s l y , the complex b i o t y p e has the a d v a n t a g e of a t t a c k -i n g more c u l t i v a r s ; t h e r e f o r e , as l o n g as s e l e c t i o n p r e s s u r e i s m a i n t a i n e d , and the c u l t u r e comes i n c o n t a c t w i t h both R i and R 2 , whether t h e s e o c c u r s i n g l y o r i n c o m b i n a t i o n , complex r a c e s a r e a t an a d v a n t a g e , even though the l e v e l o f v i r u l e n c e may be l o w e r on some c u l t i v a r s . 94 C u l t i v a r s H i m a l a y a , K e y s t o n e and Conques t a re r e s i s t a n t to b o t h V i V i V 2 V 2 and V i V i V 2 v 2 d i k a r y o n s . However , t h e s e c u l t i v a r s a r e s u s c e p t i b l e to the new c u l t u r e w h i c h i s homozygous f o r b o t h V i and v 2 . The g e n e - f o r - g e n e c o n c e p t p r e d i c t s t h a t f o r e v e r y v - g e n e i n the p a r a s i t e t h e r e i s a r e l a t e d R-gene i n the h o s t ( F l o r , 1955 ; P e r s o n , 1 9 5 9 ) . T h i s l e a d s me to s u g g e s t t h a t t h e s e t h r e e c u l t i v a r s p o s s e s s two r e s i s t a n c e g e n e s , R i and R 2 , i n common. When the new c u l t u r e and c u l t u r e G , v i r u l e n t and a v i r u l e n t on E x c e l s i o r j . r e s p e c t i v e l y , were c r o s s e d and i n o c u -l a t e d to E x c e l s i o r , the h y b r i d d i k a r y o n s were v i r u l e n t . However , the v i r u l e n c e l e v e l o f the h y b r i d d i k a r y o n s was v e r y l o w . T h i s i n d i c a t e s t h a t the v 2 v i r u l e n c e gene i n the new c u l t u r e i s i n c o m p l e t e l y d o m i n a n t to i t s a v i r u l e n c e a l l e l e i n G . In P a r t I I o f t h i s t h e s i s , i t i s shown t h a t the v i r u l e n c e of c u l t u r e D i s dominant when c r o s s e d to c u l t u r e G on E x c e l s i o r . The same phenomenon appears to h o l d t r u e h e r e . P A R T I V THE EFFECTS OF AUXOTROPHIC MUTATIONS ON PATHOGENICITY I n t r o d u c t i on Because the i n t e r a c t i o n t h a t t a k e s p l a c e between the h o s t and the p a r a s i t e i s so p o o r l y u n d e r s t o o d , e s p e c i a l l y a t the b i o c h e m i c a l l e v e l , i t was t h o u g h t w o r t h w h i l e to e s t a b l i s h w h e t h e r the a b i 1 i t y to m e t a b o l i z e c e r t a i n n u t r i e n t s i s r e l a t e d to p a t h o g e n i c i t y . B i o c h e m i c a l m u t a t i o n s have been used e x t e n s i v e l y as g e n e t i c m a r k e r s as w e l l as f o r d e t e r m i n -i n g the s t e p w i s e p r o g r e s s of b i o c h e m i c a l reacti ons ^ An v v i v o . Perhaps the f i r s t p e r s o n to use i n d u c e d b i o c h e m i c a l mutants o f p a t h o g e n i c m i c r o o r g a n i s m s , h a v i n g known n u t r i t i o n a l d e f i c i e n c e s , i n the s t u d y of h o s t - p a r a s i t e r e l a t i o n s h i p s , was P e r k i n s ( 1 9 4 9 ) . H i s work w i t h the smut f u n g u s , U s t i l a g o maydis ( D C . ) C d a , was f o l l o w e d by the w o r k s s o f Bacon et a l . (1950 , 1951) w i t h Bacterium typhosum and of K e i t t and Boone ( 1 9 5 2 , 1954) w i t h V e n t u r i a i n a e q u a l i s . In the l a s t 20 y e a r s s e v e r a l i n v e s t i g a t i o n s have been c o n d u c t e d to d e t e r m i n e the 95 96 r o l e w h i c h n u t r i t i o n p l a y s i n p a t h o g e n i c i t y . The n u t r i t i o n a l h y p o t h e s i s o f p a r a s i t i s m of L e w i s (1953) and the n u t r i t i o n a l -i n h i b i t i o n h y p o t h e s i s o f p a t h o g e n i c i t y o f G a r b e r (1954 , 1956) 1960) have s t i m u l a t e d c o n t i n u e d r e s e a r c h and d e b a t e . W i t h U s t i l a g o hordei ( P e r s . ) L a q e r h . , Hodd (1966) i n d u c e d about 500 n u t r i t i o n a l mutants by uv i r r a d i a t i o n . R o b e r t s (1967) and P e r s o n (1968) s t u d i e d the e f f e c t o f the n u t r i t i o n a l m u t a t i o n s on p a t h o g e n i c i t y on some b a r l e y c u l -t i v a r s . They f o u n d t h a t a l l n u t r i t i o n a l d e f i c i e n c i e s behaved as r e c e s s i v e s d u r i n g the p a t h o g e n i c p h a s e . As f o r d i k a r y o n s , w h i c h were made homozygous f o r n u t r i t i o n a l d e f i c i e n c i e s , some were n o n - p a t h o g e n i c , o t h e r s were p a t h o g e n i c , and s t i l l o t h e r s were p a t h o g e n i c i n some t e s t s , n o n - p a t h o g e n i c i n o t h e r t e s t s . For t h o s e homozygotes w h i c h were n o n - p a t h o g e n i c , the d e f i c i e n c y was u s u a l l y f o r an amino a c i d o r a n i t r o g e n base a n d , l e s s f r e q u e n t l y , f o r a v i t a m i n . The p u r p o s e s o f the p r e s e n t s t u d y w e r e : 1. To t r a n s f e r g e n e s g o v e r n i n g n u t r i t i o n a l r e -q u i r e m e n t s f r o m a c u l t u r e h a v i n g a known v i r u l e n c e gene t o a n o t h e r c u l t u r e h a v i n g a d i f f e r e n t v i r u l e n c e g e n e . 2. To d e t e r m i n e t h e e f f e c t s o f d i f f e r e n t n u t r i -t i o n a l d e f i c i e n c i e s on t h e a c t i o n ©f v i r u l e n c e g e n e s , as m e a s u r e d q u a l i t a t i v e l y and q u a n t i t a t i v e l y on s e v e n b a r l e y c u l t i v a r s . 97 . 3 . To a t t e m p t t o s u p p l e m e n t t h e h o s t w i t h t h e n u t r i e n t r e q u i r e d by a m u t a n t c u l t u r e , i n t h e e x p e c t a t i o n t h a t t h e p a t h o g e n i c i t y o f t h e n o n - p a t h o g e n i c a u x o t r o p h i c h o m o k a r y o n s may be r e s t o r e d . M a t e r i a l s a n d M e t h o d s T r a n s f e r of genes f o r b i o c h e m i c a l m u t a t i o n s f rom the  V i to the v 2 c u l t u r e and t e s t f o r the e f f e c t o f b i o - c h e m i c a l m u t a t i o n s on v i r u l e n c e g e n e s . Four b i o c h e m i c a l l y - d e f i c i e n t m u t a t i o n s of u. hovdei ( P e r s . ) L a g e r h . were used i n t h i s s t u d y . The d e f i c i e n c i e s were f o r a d e n i n e ( a d ) , a r g i n i n e ( a r g ) , m e t h i o n i n e ( m e t ) , and p y r i d o x i n e ( p d x ) . These mutants were i n d u c e d by Hood ( 1 9 6 6 ) , t h r o u g h uv i r r a d i a t i o n of the w i l d t y p e h a p l o i d c u l t u r e , Ii* +, w h i c h had been i s o l a t e d by Thomas (1 9 6 5 ) . Hood d e s i g n a t e d the mutants V -201 , V - 2 4 0 , V - 2 5 and V - 2 1 0 , r e p r e s e n t i n g a d , a r g , met and p d x , r e s p e c t i v e l y . These mutants were o b t a i n e d i n both m a t i n g t y p e s by c r o s s i n g the muta ted K + to the w i l d t y p e c u l t u r e E 3 ~ a n d s e l e c t i n g mutant progeny of b o t h tiiamng t y p e s . The w i l d t y p e c u l t u r e s , Ih + and E 3 ~ p o s s e s s the v i gene ( E b b a , u n p u b l i s h e d ) . The p r o c e d u r e f o r t r a n s f e r r i n g s i n g l e genes f o r b i o c h e m i c a l d e f i c i e n c i e s f rom the V i monokaryon to the v 2 and i 98 V i V 2 monokaryons was the same i n a l l c a s e s . I t w i l l be d e s c r i b e d i n r e l a t i o n to o n l y one o f the d e f i c i e n c i e s , n a m e l y , the a r g i n i n e d e f i c i e n c y . In t h i s d e s c r i p t i o n the symbol " + " . r e f e r s to w i l d t y p e , and symbols A and a to the two m a t i n g t y p e s ( r e p l a c i n g "+" and r e s p e c t i v e l y ; e x c e p t f o r the o r i g i n a l w i l d t y p e s , E 3 ~ and Thus the f o r m u l a arg A/+a r e f e r s to a h e t e r o k a r y o n w h i c h i s h e t e r o z y g o u s f o r the a r g i n i n e d e f i c i ency . The c u l t u r i n g and i n o c u l a t i o n methods used i n t h i s s t u d y were the same as t h o s e d e s c r i b e d f o r P a r t I . For t r a n s f e r oft fche a r g i n i n e d e f i c i e n c y , monokaryon V i V 2 a r g a was c r o s s e d w i t h V i V 2 + A to produce a d i k a r y o n w h i c h was i n o c u l a t e d to the s u s c e p t i b l e c u l t i v a r O d e s s a . The V i V 2 a r g a / V i V 2 + A t e l i o s p o r e s were suspended i n 5 ml o f s t e r i l e w a t e r c o n t a i n i n g a drop of a c h r o m y c i n (TO mg o f a c h r o m y c i n d i s o l v e d i n 1 ml o f H 2 0 ) to c o n t r o l b a c t e r i a l g r o w t h . T h i s i s t h e n s p r e a d on c o m p l e t e medium (CM) to g e r m i n a t e and p r o d u c e h a p l o i d s p o r i d i a . F i f t y r a n d o m l y s e l e c t e d s p o r i d i a were t r a n s -f e r r e d w i t h s t e r i l e w i r e n e e d l e to CM, where t h e y were a l l o w e d grow f o r 3-4 days to produce c o l o n i e s . These c o l o n i e s were r e p l i c a p l a t e d ( H o o d , 1966) to m i n i m a l medium (MM; see A p p e n d i x and to MM s u p p l e m e n t e d w i t h a r g i n i n e . A r g i n i n e r e q u i r i n g c o l o m were s e l e c t e d and c l a s s i f i e d w i t h r e s p e c t to m a t i n g t y p e . The a r g i n i n e r e q u i r i n g monokaryons s e l e c t e d i n t h i s way a r e e x p e c t e d to be of f o u r g e n o t y p e s ( i n r e s p e c t to v i r u l e n c e ) as f o l l o w s : V X V 2 , V x v 2 , V i V 2 and V i V 2 . The 99 d e s i r e d g e n o t y p e s was ( a r g ) v 2 , and the number of c u l t u r e s needed i n o r d e r to i n c l u d e t h i s g e n o t y p e w i t h 99% c e r t a i n t y was 7 • (F = 1/100 = ( l / 2 ) n , and n = 6 . 6 , ( s e e page 8 1 ) . Seven m o n o k a r y o n s , f o u r i n A and t h r e e i n a m a t i n g t y p e s , were t h e r e f o r e c r o s s e d w i t h the a p p r o p r i a t e m a t i n g t y p e of V i V 2 + a n d , f o r each c r o s s , the d i k a r y o n was t e s t e d on the c u l t i v a r E x c e l s i o r (100 p l a n t s i n each t e s t ) . On t h i s c u l t i v a r any d i k a r y o n w h i c h i n c i t e s t h e d i s e a s e i s known to be homozygous v 2 v 2 as w e l l as h e t e r o z y g o u s f o r the a r g i n i n e r e q u i r e m e n t . T e l i o s p o r e s , t a k e n from d i s e a s e d p l a n t s o f the c u i l i t i v a r E x c e l s i o r , were then g e r m i n a t e d a n d , f rom the r e s u l t i n g r a n -domly chosen s p o r i d i a l c o l o n i e s , monokaryons o f g e n o t y p e v 2 a r g were s e l e c t e d . A s i m i l a r p r o c e d u r e y i e l d e d monokaryons i n w h i c h v 2 was combined w i t h each of the o t h e r two n u t r i t i o n a l d e f i c i e n c i e s (met and p d x ) . For t r a n s f e r o f n u t r i t i o n a l d e f i c i e n c i e s to V i V 2 monokaryons i t was n e c e s s a r y to t e s t the d i k a r y o n on the c u l t i v a r Vantage ( w h i c h s e l e c t s f o r v x V i ) as w e l l as on E x c e l s i o r ( w h i c h s e l e c t e d f o r v 2 v 2 ) . The e f f e c t o f n u t r i t i o n a l d e f i c i e n c y on p a t h o -g e n i c i t y was t e s t e d by c o m p a r i n g c u l t u r e s w h i c h were made e i t h e r h e t e r o z y g o u s or homozygous f o r one o r more d e f i c i e n c i e s ; they were compared w i t h each o t h e r and w i t h the w i l d t y p e . S i n c e the d i s e a s e r e a c t i o n s were e x p r e s s e d i n p e r c e n t a g e s , the d a t a were t r a n s f o r m e d to a r c s i n e to a l l o w a v a l i d a n a l y s i s 100. o f v a r i a n c e . One-way a n a l y s i s o f v a r i a n c e , t o g e t h e r w i t h T u k e y ' s t e s t f o r m u l t i p l e c o m p a r i s o n was used to d e t e r m i n e whether the d i f f e r e n c e s were s t a t i s t i c a l l y s i g n i f i c a n t . Three s e t s o f d a t a , two from t e s t s a t C a l i f o r n i a and the o t h e r f rom t e s t s a t UBC, were a n a l y z e d . The n u t r i t i o n a l mutants a s s o c i a t e d w i t h v i r u l e n c e gene V i were t e s t e d on seven b a r l e y c u l t i v a r s , f i v e o f w h i c h were s u s c e p t i b l e to the w i l d t y p e d i k a r y o n , V i V i V 2 V 2 . Those w i t h v 2 were t e s t e d on the same seven c u l t i v a r s , f i v e o f w h i c h were s u s c e p t i b l e to V i V i V 2 v 2 . T h r e e of t h e c u l t i v a r s were s u s c e p t i b l e to both c u l t u r e s . Mutant V i V 2 . a r g was t e s t e d on a l l seven c u l t i v a r s i n c o m b i n a t i o n w i t h the w i l d t y p e and the o t h e r m u t a n t s . The c u l t i v a r s used w e r e : CI -541 Hannchen ; CI -7324 V a n t a g e ; C l - O d e s s a ; C I - 9 3 6 T r e b i ; C I - 9 2 3 L i o n ; C I - 1 2 4 8 E x c e l s i o r ; and N e p a l . I t i s p o s s i b l e t h a t d u r i n g the g r o w t h o f the c u l t u r e i n v i t r o o r i n v i v o mutant genes m i g h t r e v e r t to w i l d t y p e w i t h the r e s u l t t h a t the c u l t u r e would be i n c o r r e c t l y a s s e s s e d . However , to guard a g a i n s t t h i s , samples t a k e n from s m u t t e d s p i k e s were t e s t e d f o r the p r e s e n c e of r e v e r t a n t s . J S u p p l e m e n t a t i o n o f the h o s t w i t h t h e n u t r i e n t r e q u i r e d  by the mutant p a r a s i t e . The h o s t was p r o v i d e d w i t h the s p e c i f i c n u t r i e n t f o r w h i c h the fungus was d e f i c i e n t . The n u t r i e n t s ( a r g i n i n e , 1 01 m e t h i o n i n e and a d e n i n e ) were p r o v i d e d i n w a t e r s o l u t i o n . Three methods were u s e d : 1. B a r l e y s e e d s o f c u l t i v a r s Hannchen and V a n t a g e we re we re i n o c u l a t e d w i t h d i k a r y o n s homozygous f o r a c e r t a i n n u t r i e n t r e q u i r e m e n t s . The i n o c u l a t e d s e e d s were go rwn f o r a week i n l a r g e p e t r i e d i s h e s on f i l t e r p a p e r s w h i c h had been s o a k e d w i t h a s o l u t i o n o f t h e r e q u i r e d n u t r i e n t . The s e e d l i n g we re t h e n t r a n s p l a n t e d t o f l a t s i n t h e g r e e n h o u s e whe re t h e p l a n t s weee i n j e c t e d e a c h day u n t i l t h e s p i k e s we re a b o u t t o emerge w i t h t h e r e q u i r e d n u t r i e n t . The n u t r i e n t s we re i n j e c t e d i n t o t h e s t e m s a t 2.-3 l o c a t i o n s , u n t i l t h e s o l u t i o n s t a r t e d t o emerge a l o n g t h e a x i l s o f t h e l e a v e s . T h r e e d i f f e r e n t c o n c e n t r a t i o n s were u sed f o r e a c h o f t h e n u t r i e n t s : f o r a r g i n i n e and m e t h i o n i n e 5, 10, o r 25 mg p e r 100 ml o f m a t e r ; f o r a d e n i n e , 0 . 5 , 1.0, o r 2 .5 mg p e r 100 ml o f w a t e r . The l o w e s t c o n c e n t r a t i o n i n e a c h c a s e was b a s e d on t h a t e m p l o y e d i n g r o w t h - r e s p o n s e s t u d i e s in v i t r o . The n u t r i e n t s o l u t i o n was a l s o i n j e c t e d i n t o t h e p l a n t s e a c h d a y , f r o m t h e day o f t r a n s p I a t i n g . A t h e a d i n g , t h e young s p i k e s we re i n s p e c t e d f o r s m u t . .F.i.f.t.y. p l a n t s o f e a c h c u l t i v a r ( H a n n c h e n and V a n t a g e ) we re u sed f o r e a c h t r e a t m e n t o f e a c h n u t r i e n t . 2 . S eed s were i n o c u l a t e d w i t h homozygous d i k a r y o n s f o r e a c h m u t a n t and p l a n t e d i n f l a t s i n t h e g r e e n h o u s e . 1 0 2 As s oon as t h e s e e d l i n g s emerged t h e y we re i n j e c t e d w i t h t h e s p e c i f i c n u t r i e n t r e q u i r e d by t h e f u n g u s . The method o f i n j e c t i o n , c o n c e n t r a t i o n s , and c u l t i v a r s and number o f p l a n t s u sed were t h e same as t h o s e d e s c r i b e d a b o v e . 3. U n i n o c u l a t e d s e e d s we re a l s o p l a n t e d i n f l a t s i n t h e g r e e n h o u s e . A t t h e two l e a f s t a g e , an i n o c u l u m , c a p a b l e o f p r o d u c i n g a d i k a r y o n homozygous f o r a n u t r i t i o n a l d e f i c i e n c y , was i n j e c t e d i n t o t h e s e e d l i n g a t two o r t h r e e l o c a t i o n s . T h i s was f o l l o w e d by i n j e c t i o n o f t h e r e q u i r e d n u t r i e n t e v e r y day u n t i l h e a d i n g . The me thod s o f i n j e c t i o n , c o n c e n t r a t i o n o f t h e n u t r i e n t s , c u l t i v a r s , and numbers o f p l a n t s u sed we re t h e same as t h o s e d e s c r i b e d f o r (I.) a b o v e . The c o n t r o l s f o r t h e s e e x p e r i m e n t s were the i n o c u -l a t i o n of s e t s o f 1 0 0 seeds o f each c u l t i v a r w i t h homozygous d i k a r y o n s of each mutant and no" h u t r i e h t h i nj ec i t i on ,-' as w e l l asKwi- thnthe w i l d - t y p e d i k a r y o n w i t h and w i t h o u t the i n j e c -t i o n of< n u t r i e n t s . R e s u l t s Genes c o n t r o l l i n g the a r g , met and pdx r e q u i r e m e n t s were t r a n s f e r r e d f rom a V i to a v 2 c u l t u r e . That the mutant genes were t r a n s f e r r e d was e v i d e n t f rom the r e s u l t s o f the c r o s s e s between the monokaryons w h i c h were presumed to c a r r y t h e v 2 gene and the w i l d t y p e c u l t u r e w h i c h was known to 103 have the v 2 g e n e . The r e s u l t i n g d i k a r y o n s were p a t h o g e n i c on E x c e l s i o r , a c u l t i v a r w h i c h was s u s c e p t i b l e to v 2 / v 2 d i k a r y o n s o n l y . A u x o t r o p h s i n the b a c k g r o u n d of . Vi g e n e : D i k a r y o n s homozygous f o r a r g , met and ad were n o n - p a t h o g e n i c on a l l c u l t i v a r s ( T a b l e IV-1 and I V - 3 ) . Homo-qygous pdx d i k a r y o n s were p a t h o g e n i c on a l l c u l t i v a r s . How-e v e r , the l e v e l o f v i r u l e n c e of t h e s e c u l t u r e s , when compared w i t h the w i l d t y p e , was s i g n i f i c a n t l y r e d u c e d on c u l t i v a r s H a n n c h e n , Vantage and O d e s s a . A l l d i k a r y o n s h e t e r o z y g o u s f o r genes c o n t r o l l i n g the d i f f e r e n t d e f i c i e n c i e s were p a t h o g e n i c . The l e v e l o f v i r u l e n c e e x h i b i t e d by such d i k a r y o n s was e i t h e r h i g h e r , l o w e r , o r equal to t h a t o f the p r o t o t r o p h d e p e n d i n g on the c u l t u r e and c u l t i v a r u s e d . A l l ad h e t e r o z y g o t e s showed s i g n i f i c a n t l y l o w e r l e v e l s of v i r u l e n c e on Hannchen when compared w i t h w i l d t y p e . A l s o , ad h e t e r o z y g o t e s e x h i b i t e d s i g n i f i c a n t l y l o w e r l e v e l s b f v i r u l e n c e on V a n t a g e , T r e b i and L i o n , and met h e t e r o z y g o t e s showed l o w e r v i r u l e n c e l e v e l s on L i o n and O d e s s a . The a r g - p d x d i k a r y o n s ( v x a r g +/vi + pdx) showed l o w e r v i r u l e n c e l e v e l s on c u l t i v a r s Hannchen and L i o n , T a b l e IV-1 * D i s e a s e R e a c t i o n s , i n P e r c e n t a g e s , Produced by D i f f e r e n t D i k a r y o n s ( v i V i V 2 V 2 , V i V i V 2 v 2 , or V i V i V 2 v 2 ) of u. hordei on Seven ** B a r l e y C u l t i v a r s Di k a r y o n No. Genotypes of the Pathogen H V 0 T L Homo z y g o t e s 1 V i V 2 a / V i V 2 A 41 43 36 1 2 10 2 V i V 2 a rg A / v i V 2 arg a 0 0 0 0 0 3 V i V 2 a rg A v i V 2 arg a 0 0 0 0 0 4 V i V 2 ad A / v i V 2 ad a 0 0 0 0 0 5 V i V 2 met A / v i V 2 met a 0 . 3 0 0 0 0 6 V i V 2 pdx A / v i V 2 pdx a 16 29 13 14 12 Heterozyg .o tes 7 V i V 2 a rg a / V i V 2 + A 44 42 54 19 1 5 8 V i v 2 arg a / " 50 36 59 18 28 9 V i V 2 ad a / 14 20 26 6 5 10 V i V 2 met a / " 51 30 34 13 17 11 V i V 2 pdx/ 58 41 36 20 14 ( C o n t i nued) The v a l u e s i n t h i s t a b l e are averages of t h r e e t e s t s . H = Hannchen ; V = V a n t a g e ; 0 = O d e s s a ; T = T r e b i and L = L i o n . o -Fa T a b l e I V - l ( C o n t i n u e d ) Di k a r y o n No. Genotypes o f the Pathogen H V 0 T L 12 V i V 2 a rg A / v i V 2 a rg a 0 0 0 0 0 13 " + / v i V 2 ad + a 11 42 22 16 10 14 " + / v i V 2 met + a 24 41 41 29 19 15 " + / V i V 2 pdx + a 15 39 26 23 4 16 V i V 2 + arg A / V i V 2 ad + a 23 38 29 23 11 17 II / v i V 2 met + a 37 49 47 37 24 18 / v i V 2 pdx + a 55 55 42 34 24 19 V i V 2 + card} A / v i V 2 me't++aa 1 2 27 22 16 4 20 n / V i V 2 pdx + a 12 28 39 21 14 21 V i V 2 + met A / v i V 2 pdx + a 41 27 36 26 11. T a b l e I V - 2 Summary o f S t a t i s t i c a l A n a l y s i s o f D i f f e r e n c e s Between V i r u l e n c e L e v e l s Induced by D i f f e r e n t D i k a r y o n s on F i v e C u l t i v a r s as P r e s e n t e d i n T a b l e IV-1 (Any mean not underscored by the same l i n e are s i g n i f i c a n t l y d i f f e r e n t at 5% l e v e l ) H A N N C H E N D i k a r y o n n o . 2 18 9 7 6 1 21 1 7 14 16 11 1 5 8 19 20 13 10 1 2 5 4 3 Mean d i s e a s e 58 55 51 50 44 41 41 37 24 23 16 1 5 14 1 2 1 2 11 0 , 3 .0 0 0 0 r e a c t i o n I n p e r c e n t a g e V A N T A G E D i k a r y o n n o . 18 14 17 1 6 13 2 1 5 16 7 9 1 1 20 1.9 21 8 1 2 10 5 4 3 Mean d i s e a s e 55 51 49 43 42 42 41 39 38 36 30 29 28 27 27 20 0 0 0 0 0 r e a c t i o n i n p e r c e n t a g e 0 D E S S A D i k a r y o n n o . 7 6 17 18 14 20 1 2 21 9 16 8 1 5 13 19 11 1 2 10 5 4 3 Mean d i s e a s e 59 54 47 42 41 39 36 36 36 34 29 26 26 22 22 1 3 0 0 0 0 0 r e a c t i o n i n p e r c e n t a g e T R E B I D i k a r y o n n o . 17 18 14 21 16 1 5 20 2 6 7 1 9 1 3 11 9 1 8 1 2 10 5 4 3 Mean d i s e a s e 37 34 29 26 23 23 21 20 19 18 16 16 1 4 13 1 2 6 0 0 0 0 0 r e a c t i o n i n p e r c e n t a g e L I 0 N D i k a r y o n n o . 7 17 18 14 9 6 2 20 11 1 6 21 1 1 3 8 1 5 1 9 1 2 10 5 4 3 Mean d i s e a s e 28 24 24 19 17 1 5 14 14 1 2 1 1 1 1 10 1 0 5 4 4 0 0 0 0 0 r e a c t i o n i n p e r c e n t a g e 107 whereas the V i + a r g / v i ad + h e t e r o k a r y o n e x h i b i t e d a l o w e r v i r u l e n c e l e v e l on Hannchen o n l y . The met h o m o k a r y o n s , V i V 2 m e t / v i V 2 met and V i v 2 m e t / V i V 2 met p r o d u c e d v e r y low d i s e a s e r e a c t i o n s on Hannchen ( 0 . 3 % ) , E x c e l s i o r (0.3%) and Nepal ( 0 . 6 % ) . When s p o r i d i a f rom t e l i o s p o r e s o f smut ted s p i k e s were t e s t e d , s e g r e g a t i o n f o r met and w i l d t y p e was d e t e c t e d . The low i n f e c t i o n c o u l d have o c c u r r e d f o r two r e a s o n s : ( i ) one of the mutant genes c o u l d have r e v e r t e d back to the w'iild t y p e , e i t h e r in v i t r o o r in vivo; o r ( i i ) the met c u l t u r e s c o u l d have been c o n t a m i -nated w i t h w i l d t y p e s p o r i d i a . The second p o s s i b i l i t y i s most u n l i k e l y , s i n c e o t h e r c u l t i v a r s i n o c u l a t e d w i t h the same c u l t u r e were not s m u t t e d . Thus t h e f i r s t p r o p o s a l i s the most p r o b a b l , e . A u x o t r o p h s i n the b a c k g r o u n d of the v 2 gene : Homokaryons f o r met and a r g were n o n - p a t h o g e n i c . Homokaryons f o r pdx showed a v e r y d i s t i n c t s p e c i f i c i t y i n a s s o c i a t i o n w i t h the v 2 g e n e . The l e v e l o f v i r u l e n c e was r e d u c e d to 0-1% on c u l t i v a r s E x c e l s i o r and Nepal w h i l e on the o t h e r c u l t i v a r s the v i r u l e n c e l e v e l was h i g h e r . The i n c r e a s e on the c u l t i v a r Odessa was s t a t i s t i c a l l y s i g n i f i -c a n t , whereas the i n c r e a s e on c u l t i v a r s T r e b i and L i o n was n o t . 1 08 The d i f f e r e n c e s i n the v i r u l e n c e l e v e l s shown by d i k a r y o n s w h i c h were h e t e r o z y g o u s f o r n u t r i t i o n a l d e f i c i -e n c i e s were not s i g n i f i c a n t . However , mutants h e t e r o z y g o u s f o r genes d e t e r m i n i n g two d i f f e r e n t r e q u i r e m e n t s , e . g . v 2 + a r g / v 2 met +, showed r e d u c e d v i r u l e n c e on E x c e l s i o r and Nepal but not on the o t h e r c e u l t i v a r s . The a u x o t r o p h i c m u t a n t , V i V 2 a r g : S e l f s o f v i v 2 a rg and c r o s s e s of V i v 2 a r g w i t h v i V 2 a rg and V i v 2 a rg gave d i k a r y o n s homozygous f o r the a r g i n i n e d e f i c i e n c y w h i c h were n o n - p a t h o g e n i c on a l l " c u l t i v a r s . C r o s s e s w h i c h p r o d u c e d d i k a r y o n s h e t e r o z y g o u s f o r the a r g i n i n e r e q u i r e m e n t showed v a r i a b l e l e v e l s of v i r u l e n c e , d e p e n d i n g on w h e t h e r they were homozygous f o r V i o r f o r v 2 , o r h e t e r o z y g o u s f o r d e f i c i e n c i e s o t h e r than a r g i n i n e ( c f . T a b l e s IV-1 and I - V T 3 ) . V I V 2 a r g , when c r o s s e d w i t h o t h e r n u t r i t i o n a l mutants c a r r y i n g the v x g e n e , showed h i g h e r v i r u l e n c e l e v e l s t h a n d i d the w i l d t y p e d i k a r y o n s . (The f . e x e e p t i o n : was the V i V 2 a r g + / v i V 2 + ad d i k a r y o n s , w h i c h gave low l e v e l s of d i s e a s e on c u l t i v a r s H a n n c h e n , Vantage and O d e s s a . ) C u l t u r e V i V 2 a r g , when c r o s s e d w i t h the w i l d t y p e s as w e l l as w i t h the o t h e r mutant m o n o k a r y o n s , showed s i g n i f i c a n t l y l o w e r v i r u l e n c e on E x c e l s i o r , and when c r o s s e d w i t h v 2 met or v 2 T a b l e I V - 3 * D i s e a s e R e a c t i o n s , i n P e r c e n t a g e s , Produced by D i f f e r e n t D i k a r y o n s (V1V1V2Y2, V 1 V 1 V 2 V 2 , o r V 1 V 1 V 2 V 2 ) o f U. hordei on Seven ** B a r l e y C u l t i v a r s Di k a r y o n No. Genotypes of the D i k a r y o n s E N 0 T L Homozy.gotes 1 V i V 2 + a / V i V 2 + a 46 45 37 21 20 2 V i V 2 a rg A/V1V2 a rg a 0 0 0 0 0 3 V i V 2 a r g A/V1V2 a r g a 0 0 0 . 0 0 4 V i v 2 met A/V1V2 met 0 . 3 0 .6 0 0 0 5 V i V 2 pdx A/V1V2 pdx 0 .3 0 . 3 48 25 31 H e t e r o z y g o t e s 6 V iv 2 a r g A / V iv 2 + a 32 61 53 17 27 7 V1V2 a r g A/ " 9 44 33 34 21 8 V i v 2 met A/ 28 47 30 30 13 9 V i v 2 pdx A / 31 66 48 30 23 ( C o n t i nued) The v a l u e s i n t h i s t a b l e a re averages o f t h r e e t e s t s . E = E x c e l s i o r ; N = N e p a l ; 0 = O d e s s a ; T = T r e b i and L = L i o n . • o cu 3 o o o o i cu -Q CO O >> s-_ ^ O. CU +J M -O CO cu O->) 4-> O sz cu CD sz o >> • s - o o . i — CM O CM CM OO CM O 00 o o I — OO I — CM OO o o o O 0! CO CM OO CM t^" o L O O CO CM CM o o o o O CM i — CO o fO (0 (O + + CD4-> X S- (DT) ra £ CL CM CM CN) > > > rH l-t I-H > > > + + CD S-r0 + + + -f-> X X CU " O - a £ a- Q. CM CM CM > > > i—1 rH rH > > < O l s - CU E + + CM = CM > . > rH i-H > > CM 0 O ^ -T a b l e I V - 4 Summary of S t a t i s t i c a l A n a l y s i s o f D i f f e r e n c e s Between V i r u l e n c e L e v e l s Induced by 15 D i f f e r e n t D i k a r y o n s on F i v e C u l t i v a r s as P r e s e n t e d i n T a b l e I V - 3 (Means-' not underscored by the same l i n e are s i g n i f i c a n t l y d i f f e r e n t at 5% l e v e l ) E X C E L S I O R Di Icaryon n o . 1 6 9 8 11 7 13 12 14 1 5 4 5 2 3 10 Mean d i s e a s e r e a c t i o n i n 46 32 31 28 1 2 9 4 1 1 .6 .3 .3 0 0 0 p e r c e n t a g e N E P A L D i k a r y o n n o . 9 6 . 8 1 7 11 12 15 a 5 14 2 3 10 1 3 Mean d i s e a s e r e a c t i o n i n '--.-66 61 57 45 44 26 2 1 0 .6 0 . 3 0 . 3 0 0 0 0 p e r c e n t a g e O D E S S A D i k a r y o n n o . 6 1 5 9 .5 14 1 12 7 8 11 13 2 3 4 10 Mean d i s e a s e r e a c t i o n i n 53 50 48 48 47 37 36 33 30 29 27 0 0 0 0 p e r c e n t a g e T R E B I D i k a r y o n n o . 7 . 12 14 8„ 9 1 5 13 5 1 11 6 2 3 .4 10 Mean d i s e a s e r e a c t i o n i n p e r c e n t a g e 34 38 31 30 30 30 27 25 21 18 17 0 0 0 0 L I O N D i k a r y o n n o . 14 5 6 11 1 5 9 7 1 13 11 8 2 3 4 10 Mean d i s e a s e r e a c t i o n i n 32 31 27 27 27 23 21 20 20 14 1 3 0 0 0 0 p e r c e n t a g e — 112 p d x , i t e x h i b i t e d s i g n i f i c a n t l y l o w e r v i r u l e n c e l e v e l s on the c u l t i v a r N e p a l . S u p p l e m e n t a t i o n w i t h the r e q u i r e d n u t r i e n t i n v i v o : A t t e m p t s to s u p p l y the needed n u t r i e n t by the methods d e s c r i b e d d i d not r e s t o r e p a t h o g e n i c i t y . A d e n i n e a p p e a r e d to be p h y t o t o x i c a t the h i g h e r c o n c e n t r a t i o n . The w i l d t y p e d i k a r y o n , w h i c h was i n j e c t e d i n t o t h e p l a n t a t the t w o - l e a f s t a g e , and w h i c h was used as a c o n t r o l , d i d ' p r o d u c e s m u t . However , the l e v e l s o f d i s e a s e r e a c t i o n on Hannchen and V a n t a g e , as shown by the number o f s m u t t e d p l a n t s (10% and 12% r e s p e c t i v e l y ) was v e r y low as compared w i t h t h o s e (45% and 50% r e s p e c t i v e l y ) p r o d u c e d i n the g r e e n -house by the s t a n d a r d method of i n o c u l a t i o n . D i s c u s s i o n In t h i s s t u d y the e f f e c t s o f n u t r i t i o n a l d e f i c i e n c y were s t u d i e d i n r e l a t i o n to v i r u l e n c e genes V i and v 2 . For s t u d i e s i n v o l v i n g v i , the v x v i d i k a r y o n s w h i c h were made homozygous f o r n u t r i t i o n a l d e f i c i e n c i e s f e l l i n t o two g r o u p s : ( i ) t h o s e w h i c h were n o n - p a t h o g e n i c ; and ( i i ) t h o s e w h i c h r e t a i n e d p a t h o g e n i c i t y s i m i l a r to t h a t o f the p a r e n t a l geno-t y p e f rom w h i c h the mutants had been d e r i v e d . The f i r s t i 113 group i n c l u d e d a r g , ad and met h o m o z y g o t e s . The second group was r e p r e s e n t e d by the pdx h o m o z y g o t e , w h i c h was p a t h o g e n i c . I t s h o u l d be noted t h a t homozygotes o f the f i r s t d group a r e n o n - p a t h o g e n i c (as d i s t i n g u i s h e d f rom a v i r u l e n t ) , s i n c e t h e y do not cause d i s e a s e on any o f t h e t e s t e d c u l t i v a r s . The l o s s o f p a t h o g e n i c i t y of b i o c h e m i c a l l y d e f i c i e n t d i k a r y o n s i s a p p a r e n t l y due to t h e i r i n a b i l i t y to s y n t h e s i z e the r e q u i r e d n u t r i e n t s . I t i s i n f e r r e d t h a t t h e y f a i l to p r o d u c e d i s e a s e symptoms on c u l t i v a r s w h i c h do n o t p r o v i d e the needed s u b s t a n c e . Assuming t h a t t h i s i n t e r p r e t a t i o n i s c o r r e c t , the h o s t w h i c h d o e s . n o t p r o v i d e the needed s u b s t a n c e i s the e q u i v a l e n t o f m i n i m a l medium. ( I t i s i n t e r e s t i n g i n t h i s c o n n e c t i o n to note t h a t , f o r t h e s e r e q u i r e m e n t s , com-p l e m e n t a t i o n p a t t e r n s shown by d e f i c i e n t m u t a n t s a r e the same whether c a r r i e d o u t on the h o s t o r on m i n i m a l medium; P e r s o n , u n p u b l i s h e d ) ) G a r b e r ( 1 9 5 6 ) , i n p r e s e n t i n g h i s n u t r i t i o n a l h y p o t h e s i s o f p a r a s i t i s m , emphas ized t h a t the h o s t must p r o v i d e an adequate n u t r i t i o n a l e n v i r o n m e n t i f the p a r a s i t e i s to s u c c e e d . From s t u d i e s o f b i o c h e m i c a l mutants o f Salmonella typhosa, Bacon et a l . (1951) a l s o c o n c l u d e d t h a t n o n - p a t h o g e n i c i t y i s r e l a t e d d i r e c t l y to the n u t r i t i o n a l d e f i c i e n c i e s o f t h e i r c u l t u r e s . T h i s i n d i c a t e s t h a t f o r S a l m o n e l l a , as w e l l as f o r U s t i l a g o 3 the e s s e n t i a l 114 c o n d i t i o n f o r p a t h o g e n i c i t y i s t h a t the p a r a s i t e be a b l e to m a i n t a i n i t s e l f on the h o s t . The second group o f homozygous a u x o t r o p h s were t h o s e w h i c h d i d not l o s e t h e i r p a t h o g e n i c i t y . The mutant f o r pdx f e l l i n t h i s c a t e g o r y . From p r e v i o u s work i t i s known t h a t v i t a m i n s , i n g e n e r a l , a r e not c r i t i c a l i n d e t e r m i n i n g l o s s of p a t h o g e n i c i t y ( D u t t a et a l . 3 1960 ; Boone , 1 9 7 1 ) . T h e r e f o r e , i t was not s u r p r i s i n g t h a t the pdx r e q u i r i n g d i k a r y o n s r e m a i n e d f u l l y p a t h o g e n i c i n t h i s s t u d y , a t l e a s t i n the V i V i d i k a r y o n . The p a t h o g e n i c i t y o f p d x / p d x d i k a r y o n s c o u l d be due to the p r e s e n c e i n the h o s t of q u a n t i t i e s o f pdx or pdx p r e c u r s o r s s u f f i c i e n t to meet the r e q u i r e m e n t of the p a t h o g e n . The l o s s o r l a c k of l o s s o f p a t h o g e n i c i t y by homo-zygous a u x o t r o p h s can be l o o k e d a t i n a n o t h e r way. The l o s s o f p a t h o g e n i c i t y by mutants homozygous f o r a r g , m e t , or ad can be v i e w e d as an example of e p i s t a s i s , w i t h the a c t i o n o f the mutant gene e p i s t a t i c o v e r t h a t o f the v i r u l e n c e g e n e . P a t h o g e n i c i t y was not l o s t i f a d i k a r y o n c a r r i e d a b i o c h e m i c a l m u t a t i o n i n a s i n g l e n u c l e u s ( i . e . i f i t was h e t e r o -zygous f o r the m u t a t i o n ) . S i m i l a r l y , when c u l t u r e s o f o p p o s i t e m a t i n g t y p e s c a r r y i n g d i f f e r e n t b i o c h e m i c a l m u t a t i o n s were i n o c u l a t e d , p a t h o g e n i c i t y was u n i m p a i r e d . The d i f f e r e n t n u c l e i o f the h e t e r o k a r y o n complement each o t h e r . The f a c t t h a t the c o m b i n a t i o n of w i l d 115 t y p e and mutant n u c l e i showed n o n - m u t a n t phenotype i n a l l cases i n d i c a t e d t h a t the mutant c o n d i t i o n i s r e c e s s i v e . H e t e r o z y g o u s mutants ( h e t e r o k a r y o n s ) showed v i r u l e n c e l e v e l s w h i c h were h i g h e r or l o w e r , or equal to t h o s e o f the w i l d t y p e , d e p e n d i n g on the p a r t i c u l a r d i k a r y o n and c u l t i v a r u s e d . S p e c i f i c d i f f e r e n c e s i n the l e v e l s o f v i r u l e n c e i n d i c a t e d t h a t the n u t r i t i o n a l mutants show s p e c i f i c i t y i n d e t e r m i n i n g l e v e l s o f v i r u l e n c e . For e x a m p l e , h e t e r o z y g o t e s f o r the ad r e q u i r e m e n t exh ib i ted a r e l a t i v e l y low l e v e l o f v i r u l e n c e (compared w i t h w i l d t y p e and w i t h c e r t a i n o t h e r h e t e r o z y g o t e s ) on the c u l t i v a r H a n n c h e n . S i m i l a r i n s t a n c e s o f s p e c i f i c i t y shown by a u x o t r o p h s have been r e p o r t e d by G a r b e r et a l . (1956) and G a r b e r (1954) i n E r w i n i a a v o i d e a e a a n d K K l e b s i e l l a pneumoniae. The e f f e c t o f n u t r i t i o n a l m u t a t i o n s on p a t h o g e n i c i t y o f homozygous v 2 v 2 d i k a r y o n s w i l l now be c o n s i d e r e d . I n d e -pendent genes d e t e r m i n i n g n u t r i t i o n a l r e q u i r e m e n t s , f o r a r g , met and pdx were t r a n s f e r r e d from the v x to the v 2 m o n o k a r y o n . D i k a r y o n s ( v 2 v 2 ) w h i c h were a l s o homozygous f o r e i t h e r a r g o r met were r e n d e r e d n o n - p a t h o g e n i c on a l l c u l t i v a r s . Homo-z y g o t e s f o r the pdx r e q u i r e m e n t were w e a k l y p a t h o g e n i c (1%) on c u l t i v a r s E x c e l s i o r and Nepal i n t e s t s t h a t were c o n d u c t e d a t UBC whereas they were n o n - p a t h o g e n i c i n the C a l i f o r n i a t e s t s . H o w e v e r , the same pdx homozygotes showed s i g n i f i c a n t l y h i g h e r v i r u l e n c e l e v e l s , as compared w i t h the w i l d t y p e , on 116 t h e o t h e r t h r e e c u l t i v a r s i n both 1 o c a l i t i e s . These r e s u l t s c o u l d be e x p l a i n e d as due t o : (1) the pdx homozygotes showing marked h o s t s p e c i f i c i t y i n the v 2 v 2 gene b a c k g r o u n d ; (2) Some m i n o r genes c o u l d have been t r a n s f e r r e d , a l o n g w i t h the mutant genes f rom the v i to t h e v 2 c u l t u r e , w h i c h l o w e r e d the l e v e l o f v i r u l e n c e of the v 2 v 2 d i k a r y o n on c u l t i v a r s E x c e l s i o r and N e p a l , and enhanced i t on the o t h e r c u l t i v a r s ; (3) C u l t i v a r s E x c e l s i o r and Nepal a p p a r e n t l y do not p r o v i d e s u f f i c i e n t f r e e pdx or s u b s t r a t e s f o r pdx s y n t h e s i s . Thus the pdx m u t a t i o n has the e f f e c t o f n u l l i f y i n g or r e d u c i n g the e x p r e s s i o n of the v 2 v 2 d i k a r y o n on t h e s e c u l t i v a r s . Even though i t i s d i f f i c u l t to d e t e r m i n e . w h i c h of the above a l t e r n a t i v e s i s more l i k e l y , the f i r s t a l t e r n a t i v e appears to p r o v i d e the s i m p l e s t e x p l a n a t i o n . However , f rom the s t u d y p r e s e n t e d i n P a r t I'DI o f t t h i s t h e s i s the second a l t e r n a t i v e c o u l d e x p l a i n the r e s u l t s as w e l l . I n j / P a r t I I I i t was shown t h a t the complex c u l t u r e homozygous f o r both Vj. and v 2 g e n e s , had r e d u c e d v i r u l e n c e l e v e l s on E x c e l s i o r and N e p a l . T h i s was e x p l a i n e d as d u e , p a r t l y , to g e n e t i c modi f i e r s . T h e r e are two main c l a s s e s of a u x o t r o p h i c h e t e r o -z y g o t e s . The f i r s t c l a s s i n c l u d e s t h o s e d i k a r y o n s h e t e r o -zygous f o r a gene c o n t r o l l i n g a n u t r i t i o n a l d e f i c i e n c y ( v 2 a r g / v 2 + ) . They a r e f u l l y p a t h o g e n i c , a l t h o u g h the l e v e l s o f v i r u l e n c e a r e v a r i a b l e . For e x a m p l e , v 2 a r g / v 2 + 117 shows a r e l a t i v e l y low l e v e l o f v i r u l e n c e on E x c e l s i o r but shows e i t h e r the same or h i g h e r l e v e l s o f v i r u l e n c e on the r e m a i n i n g c u l t i v a r s (as compared to the w i l d t y p e ) . In the second c l a s s a re t h o s e d i k a r y o n s h e t e r o -zygous f o r two genes t h a t c o n t r o l d i f f e r e n t n u t r i t i o n a l r e -q u i r e m e n t s ( e . g . v 2 + a r g / v 2 met +) . These h e t e r o z y g o t e s showed s i g n i f i c a n t l y r e d u c e d l e v e l s o f v i r u l e n c e on c u l t i v a r s E x c e l s i o r and N e p a l . However , the v i r u l e n c e l e v e l s were not changed s i g n i f i c a n t l y on the o t h e r t h r e e c u l t i v a r s ( i . e . i n ceWpTa'r'iisioim w i t h the w i l d t y p e ) . The h e t e r o z y g o t e v 2 + a r g / v 2 met + gave s i g n i f i c a n t l y h i g h e r l e v e l s o f v i r u l e n c e on E x c e l s i o r and Nepal when compared w i t h t h e r e m a i n i n g h e t e r o z y g o t e s , even though t h i s l e v e l was l o w e r than t h a t o f the w i l d t y p e . The d r a m a t i c r e d u c t i o n i n e f f i c i e n c y of c o m p l e m e n t a t i o n among d i f f e r e n t a u x o t r o p h s on E x c e l s i o r and Nepal ( e x c e p t f o r v 2 + a r g / v 2 met •+) was u n e x p e c t e d . Such a marked r e d u c t i o n n o r m a l l y w ou ld be e x p e c t e d o n l y i f the mutant genes d e t e r m i n e the same f u n c t i o n and a r e a l l e l e s o f the same l o c u s . The r e a c t i o n o f the o t h e r c u l t i v a r s to the same h e t e r o k a r y o n s c l e a r l y shows t h a t the genes d e t e r m i n i n g the v a r i o u s n u t r i t i o n a l r e q u i r e m e n t s i n t h i s s t u d y a r e a t d i f f e r e n t l o c i . I t c o u l d be t h a t the two c u l t i v a r s p r o v i d e d a l i m i t e d amount o f the needed n u t r i e n t or i t s p r e c u r s o r a n d , i n so d o i n g , p r o v i d e d an e n v i r o n m e n t i n w h i c h the " w i l d - t y p e " was i n c o m p l e t e l y d o m i n a n t . The 118 e x p l a n a t i o n s p r e s e n t e d above to a c c o u n t f o r the r e d u c e d v i r u l e n c e o f pdx homokaryons on E x c e l s i o r and Nepal c o u l d e x p l a i n the o b s e r v a t i o n s on t h e s e a u x o t r o p h i c h e t e r o k a r y o n s as w e l 1 . A number o f w o r k e r s have a t t e m p t e d to o f f e r e x p l a n a -t i o n s f o r the e f f e c t o f n u t r i t i o n on p a t h o g e n i c i t y . L e w i s ( 1 9 5 3 , 1957) p r o p o s e d the " b a l a n c e h y p o t h e s i s , " a c c o r d i n g to w h i c h the outcome of the h o s t - p a r a s i t e r e l a t i o n s h i p i s d e t e r m i n e d by i n t e r a c t i o n s between the p a r a s i t e and s u b s t a n c e s p r o v i d e d by the h o s t . T h i s v i e w was based on the g r o w t h and absence of growth of p a r a s i t e s i n c u l t u r e s c o n t a i n i n g d i f f e r e n t c o m b i n a t i o n s of n u t r i e n t s . G a r b e r (1954 , 1956) worked w i t h a number of b a c t e r i a l p l a n t p a t h o g e n s . He r e l a t e d the p a t h o g e n i c i t y , or l a c k of i t , shown by t h e s e b a c t e r i a on a c e r t a i n h o s t s p e c i e s to two e n v i r o n m e n t s i n the h o s t : the n u t r i t i o n a l e n v i r o n m e n t and the i n h i b i t o r y e n v i r o n m e n t . G a r b e r (1960) a l s o p r o p o s e d t h a t even i f t h e r e a r e adequate c o n c e n t r a t i o n s o f the r e q u i r e d n u t r i e n t s , s u b s t a n c e s c o u l d be p r e s e n t t h a t i n h i b i t t h e i r u p t a k e , or subs tances c o u l d be a b s e n t t h a t promote t h e i r u p t a k e , r e s u l t i n g i n l o s s of p a t h o g e n i c i t y . K e i t t , Boone and c o - w o r k e r s ( c f . Boone 1971) worked w i t h s e v e r a l b i o c h e m i c a l mutants o f V. i n a e q u a l i s . They c o n c l u d e d t h a t the n u t r i t i o n a l mutants when p a t h o g e n i c , or when made p a t h o g e n i c by p r o v i d i n g 119 t h e needed s u b s t a n c e , showed the same h o s t s p e c i f i c i t y as the w i l d t y p e c u l t u r e f rom w h i c h t h e y had been d e r i v e d . W i t h u. hordei, Hood (1966) t e s t e d d i k a r y o n s e i t h e r homozygous or h e t e r o z y g o u s f o r b i o c h e m i c a l d e f i c i e n c i e s on Hannchen and Vantage b a r l e y . D i k a r y o n s homozygous f o r a b i o -c h e m i c a l d e f i c i e n c y were a l l n o n - p a t h o g e n i c w h i l e d i k a r y o n s h e t e r -zygous f o r a b i o c h e m i c a l d e f i c i e n c y were p a t h o g e n i c . R o b e r t s ( 1 9 6 7 ) , u s i n g H o o d ' s m u t a n t s , a l s o i n v e s t i g a t e d the p a t h o g e n i c i t y o f homokaryons and h e t e r o k a r y o n s on v a r l e y c u l t i v a r s . He r e p o r t e d t h a t some of the homokaryons and a l l o f the h e t e r -i k a r y o n s were p a t h o g e n i c . He a l s o o b s e r v e d v a r i a t i o n i n l e v e l s o f v v i T K u i l i e n e e among the p a t h o g e n i c h e t e r o k a r y o n s . He e x p l a i n e d h i s r e s u l t s as due to the b a l a n c e o f n u t r i l i t e s s u p p l i e d by the h o s t , w h i c h he d e s c r i b e d by the term "gene e q u i p o i s e . " H i s i n t e r p r e t a t i o n was s i m i l a r to the " b a l a n c e h y p o t h e s i s " o f L e w i s ( 1 9 5 3 ) . R o b e r t s f o u n d t h a t pdx v-210 ( i . e . V i pdx) homokaryons were p a t h o g e n i c on Hannchen and Odessa but n o n -p a t h o g e n i c on L i o n and on two o t h e r c u l t i v a r s . In u n p u b l i s h e d work ( P e r s o n ) as w e l l as i n t h i s s t u d y , t h i s c u l t u r e has been p a t h o g e n i c on a l l c u l t i v a r s , i n c l u d i n g L i o n . In s e v e r a l p a t h o g e n i c o r g a n i s m s (Bacon et a l . 3 1950 , 1951 ; Burrows et a l . 3 1954; B u x t o n , 1956; Boone , 1971 ; H o l l i d a y , 1961 ; G a r b e r , 1954 , 1956) p a t h o g e n i c i t y was r e s t o r e d to n o n - p a t h o g e n i c a u x o t r o p h s by p r o v i d i n g the needed n u t r i l i t e d u r i n g the i n c u b a t i o n p e r i o d on the h o s t . 1 20 The r e q u i r e d s u b s t a n c e s were s u p p l i e d by s p r a y i n g on the f o l i a g e or by i n j e c t i o n i n t o stems or p e t i o l e s . The s u p p l e -mented n u t r i e n t may or may not r e s t o r e p a t h o g e n i c i t y d e p e n d i n g on the k i n d of n u t r i e n t and the o r g a n i s m s i n v o l v e d ( K e i t t et al., 1959 ; G a r b e r , 1 9 5 4 ) . R o b e r t s (1967) s p r a y e d b a r l e y p l a n t s w i t h the r e q u i r e d n u t r i e n t s but d i d not r e s t o r e p a t h o g e n i c i t y to the b i o c h e m i c a l mutants o f u. hordei. In the p r e s e n t s t u d y , p a t h o g e n i c i t y was not r e s t o r e d by the a p p l i c a t i o n o f the r e q u i r e d n u t r i e n t in vivo even though g r o w t h was r e s t o r e d when a p p l i e d in v i t r o . T h i s w o u l d i n d i c a t e t h a t t h e r e i s no d i r e c t r e l a t i o n s h i p between the c a p a c i t y f o r in y i t r o g r o w t h and p a t h o g e n i c i t y . T h i s v i e w i s a l s o s u p p o r t e d by the u n i m p a i r e d p a t h o g e n i c i t y o f a u x o -t r o p h i c pdx homokaryons . The r e s u l t s o f t h i s s t u d y and e v i d e n c e p r o v i d e d by d i f f e r e n t o r g a n i s m s i n o t h e r s t u d i e s , wou ld l e a d to the c o n c l u s i o n s t h a t the d i f f e r e n c e i n p a t h o g e n i c i t y shown by the . b i o t y p e s o f u. hordei i s not due to d i f f e r e n c e s i n n u t r i t i o n a l r e q u i r e m e n t s ( w h i c h a r e met by the d i f f e r e n t b a r l e y c u l t i v a r s ) , but r a t h e r to the s p e c i f i c i t y c o n t r o l l e d by v i r u l e n c e g e n e s . T h i s p r o p o s a l i s s u p p o r t e d by the f a c t t h a t t h e l o s s o f p a t h o g e n i c i t y due to b i o c h e m i c a l d e f i c i e n c i e s r e s u l t e d i n l o s s o f p a t h o g e n i c i t y on a l l c u l t i v a r s . E v i d e n c e f rom o t h e r s o u r c e s , where g r o w t h o f both v i r u l e n c e and a v i r u l e n t pathogens was promoted by t i s s u e e x t r a c t s and 121 homogenates , t a k e n f rom the h o s t , show t h a t i t i s not the n u t r i t i o n t h a t d e t e r m i n e s s p e c i f i c i t y ( K l i n e et a l . , 1957; Boone , 1971 ; G a r b e r , 1954, 1 9 5 6 ) . On the o t h e r h a n d , i t was c l e a r l y d e m o n s t r a t e d i n t h i s s t u d y t h a t the a u x o t r o p h s can show s p e c i f i c i t y * by m o d i f i n g the l e v e l s o f v i r u l e n c e shown by p a t h o g e n i c d i k a r y o n s i n i n t e r a c t i o n s w i t h s p e c i f i c h o s t c u l t i v a r s . The r e s u l t s o f t h i s s t u d y w o u l d l e a d to the s u g g e s -t i o n t h a t f o r an o r g a n i s m to be a pathogen two c o n d i t i o n s are e s s e n t i a l . (1) The p a r a s i t e must have a g e n e , or g e n e s , f o r v i r u l e n c e i n o r d e r to overcome the g e n e t i c r e s i s t a n c e t h a t the h o s t may p o s s e s s . (2) I t must be g e n e t i c a l l y c a p a b l e o f p r o v i d i n g , f o r i t s e l f , a l l m e t a b o l i t e s w h i c h are e s s e n t i a l d u r i n g the p a t h o g e n i c phase and w h i c h a r e not p r o v i d e d by the h o s t . GENERAL CONCLUSIONS To a r r i v e a t an u n d e r s t a n d i n g of the g e n e t i c i n t e r -a c t i o n s i n v o l v e d i n the h o s t - p a r a s i t e r e l a t i o n s h i p i t i s n e c e s s a r y f i r s t to d e t e r m i n e the g e n e t i c b a s i s o f p a r a s i t i c v i r u l e n c e and o f h o s t r e s i s t a n c e . Both the p a r a s i t e , u. hordei, and the h o s t , H.vulgare, o f f e r s p e c i a l a d v a n t a g e s f o r g e n e t i c a n a l y s i s o f t h i s k i n d w i t h the o n l y d i s a d v a n t a g e b e i n g t h e i r l o n g l i f e c y c l e . The v a r i o u s g e n e t i c phenomena, such as a l l e l i s m , d o m i n a n c e , e p i s t a s i s and m o d i f i e r s , d i s c o v e r e d i n the pathogen i n t h i s s t u d y r e v e a l t h a t a v a r i e t y of g e n e t i c mechanisms may be i n v o l v e d i n a h o s t - p a r a s i t e i n t e r a c t i o n . In P a r t I i t i s d e m o n s t r a t e d t h a t genes g o v e r n i n g f o u r d i f f e r e n t v i r u l e n c e l e v e l s form a s e r i e s o f m u l t i p l e a l l e l e s . More s t u d y i s r e q u i r e d to d e t e r m i n e whether the phenomenon o f a l l e l i s m among v -genes g o v e r n i n g v a r i o u s l e v e l s o f v i r u l e n c e i s common or w h e t h e r i t ceases to e x i s t a t some l e v e l . I t i s known t h a t h o s t c u l t i v a r s show d i f f e r e n t l e v e l s o f r e s i s t a n c e . C e r t a i n l y , one of the n e x t s t e p s i s t o a p p l y t e c h n i q u e s s i m i l a r to t h o s e used i n t h i s work to d e t e r m i n e w h e t h e r the genes i n the h o s t g o v e r n i n g the d i f f e r e n t r e s i s t a n c e l e v e l s a l s o form a s e r i e s o f m u l t i p l e a l l e l e s . 1 22 1 2 3 I t was shown i n P a r t I I t h a t v i r u l e n c e i n u. hordei can be e i t h e r dominant or r e c e s s i v e . For the v i r u l e n c e genes w h i c h have been i d e n t i f i e d , a knowledge o f t h e i r mode o f i n t e r a c t i o n w i t h r e s i s t a n c e genes o f t h e h o s t s h o u l d u l t i m a t e l y c o n t r i b u t e to a b e t t e r u n d e r s t a n d i n g of h o s t r e s i s t a n c e . I t i s now p o s s i b l e to combine t h e s e v i r u l e n c e genes w i t h o t h e r p r e v i o u s l y - i d e n t i f e d genes and to e x t e n d the a n a l y s i s to i n c l u d e o t h e r p o s s i b l e i n t e r a c t i o n s , both among v i r u l e n c e genes i n the p a r a s i t e and between t h e s e and the r e s i s t a n c e genes o f the h o s t . In one s t u d y i n t h i s t h e s i s i t was shown t h a t a complex b i o t y p e ( v x V i V 2 v 2 ) was f i t t e r t h a n the s i m p l e r b i o t y p e s ( v i V i o r v 2 v 2 ) f rom w h i c h i t was s y n t h e s i z e d . More s t u d i e s o f t h i s k i n d a r e n e e d e d . Complex b i o t y p e s of d i f f e r e n t l e v e l s of c o m p l e x i t y ( i . e . b i o t y p e s p o s s e s s i n g t w o , t h r e e , f o u r or more v - g e n e s ) s h o u l d be s y n t h e s i z e d and compared w i t h each o t h e r and w i t h s i m p l e r b i o t y p e s , f o r f i t n e s s on v a r i o u s h o s t c u l t i v a r s b e f o r e a more g e n e r a l c o n c l u s i o n i s a t t e m p t e d . T h i s t y p e o f s t u d y w o u l d be v a l u a b l e i n the u n d e r s t a n d i n g o f the c o - e v o l u t i o n o f the p a r a s i t e and i t s h o s t . S i m i l a r work of the k i n d s u g g e s t e d above f o r the p a r a s i t e a l s o needs to be done w i t h the h o s t . H o s t s c a r r y i n g t w o , t h r e e o r more R-genes s h o u l d be s y n t h e s i z e d , u s i n g the R-genes i d e n t i f i e d 124 i n t h i s and i n p r e v i o u s s t u d i e s , to i n v e s t i g a t e the e f f e c t s t h a t R-genes may have on each o t h e r and on v - g e n e s . The p r e s e n t work w i t h n u t r i t i o n a l mutants has shown t h a t the s p e c i f i c i t y o f pathogen b i o t y p e s i s not d e t e r -mined by the a v a i l a b i l i t y or n o n - a v a i l a b i l i t y o f n u t r i t i o n a l f a c t o r s . The a u x o t r o p h s e x h i b i t e d s p e c i f i c i t y i n t h e i r r e g u l a t i o n of v i r u l e n c e l e v e l s . One of the q u e s t i o n s r a i s e d i n t h i s work was whether the r e d u c e d v i r u l e n c e l e v e l o f the v 2 v 2 d i k a r y o n , when h e t e r o z y g o u s f o r a d e f i c i e n c y , s h o u l d be a t t r i b u t e d to the e f f e c t o f the d e f i c i e n c y o r to the e f f e c t o f m i n o r genes t r a n s f e r r e d from the V i c u l t u r e , a l o n g w i t h the d e f i c i e n c y g e n e s . More work s h o u l d be done a l o n g t h i s l i n e to e x p l o r e t h i s q u e s t i o n . One way of d i f f e r e n t i -a t i n g between t h e s e a l t e r n a t i v e s wou ld be to b a c k c r o s s the v 2 c u l t u r e s w i t h the v a r i o u s d e f i c i e n c y genes to the w i l d t y p e v 2 c u l t u r e i n an a t t e m p t to remove the presumed e f f e c t of the mi nor g e n e s . 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P I a n t e n z i e k t e n 6 7 : 6 9 - 2 5 6 . APPENDIX A CULTURE MEDIUM (A) COMPLETE BROTH V o g e l ' s s o l u t i o n ( d i l u t e ) 20 ml D i s t i l l e d w a t e r 1 1 T r y p t o p h a n e 50 mg C a s e i n h y d r o l y s a t e 5 gm Y e a s t e x t r a c t ( D i f c o ) 5 gm S u c r o s e 20 gm o r D e x t r o s e 1 0 gm V i t a m i n S o l u t i o n 1 0 ml N o t e : I. V i t a m i n s o l u t i o n t o be added a f t e r a u t o c l a v i n g . 2. To make c o m p l e t e p l a t e s add 20 gm B a c t o - a g a r b e f o r e a u t o c l a v i n g . (B) MINIMAL MEDIUM V o g e l ' s s o l u t i o n D i s t i l l e d w a t e r B a c t o r - a g a r D e x t r o s e (C) SUPPLEMENTED MINIMAL MEDIUM Amino a c i d s S t o c k S o l u t i o n : 500 mg i n 100 ml 50% e t h a n o l o r 100 ml d i s t i l l e d w a t e r . Use 2 ml o f the s t o c k s o l u t i o n per 100 ml medium (100 mg per 1 i t r e ) . 1 38 20 ml 1 1 20 gm 10 gm B a s e s : S t o c k S o l u t i o n : 50 mg i n 100 ml 50% e t h a n o l o r 100 ml d i s t i l l e d w a t e r . Use 2 ml o f the s t o c k s o l u t i o n per 100 ml medium (10 mg per 1 i t r e ) . V i tami n s : S t o c k S o l u t i o n : . 5 mg i n 100 ml 50% e t h a n o l o r 100 ml d i s t i l l e d w a t e r . Use 2 ml o f the s t o c k s o l u t i o n per 100 ml medium (1 mg per 1 i t r e ) . (D) VOGEL'S SOLUTION ( c o n c e n t r a t e ) N a 3 c i t r a t e • 2 H 2 0 1 23 gm K H 2 P O 4 a n h y d r o u s 250 gm N r U N 0 3 anhydrous 100 gm MgSOzt • 7 H 2 0 10 gm C a C l 2 • 2 H 2 0 5 gm T r a c e e lement s o l u t i o n 5 ml D i s t i l l e d w a t e r 750 ml C h i o r o f o r m 2 ml N o t e : I. Add c h e m i c a l s s u c c e s s i v e l y w i t h s t i r r i n g . 2. S t o r e a t room t e m p e r a t u r e . 3 . D i l u t e 5 0 - f o l d w i t h d i s t i l l e d w a t e r b e f o r e u s e . (E) VITAMIN SOLUTION T h i a m i n 100 mg R i b o f l a v i n 50 mg P y r i d o x i n 50 mg C a l c i u m p a n t o t h e n a t e 200 mg B e n z o i c a c i d 50 mg N i c o t i n i c a c i d < 200 mg C h o l i n e c h l o r i d e 200 mg I n o s i t o l 400 mg F o l i c a c i d 50 mg D i s t i l l e d w a t e r to a t o t a l o f 1 1 N o t e : I . S t S t © r e t a * e ? t ? C . 2 . Use 10 ml o f v i t a m i n s o l u t i o n p e r l i t r e o f s t e r i Ie med i um. APPENDIX B A S c h e m a t i c R e p r e s e n t a t i o n of the L i f e C y c l e o f U s t i l a g o Hovdei 

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