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Parasitism of Barley by Ustilago Hordei (Pers.) Lagerh. : some quantitative aspects of Disease expression Groth, James Vernon 1974

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P A R A S I T I S M O F B A R L E Y BY U S T I L A G O H O R D E I ( P E R S . ) L A G E R H • t S O M E Q U A N T I T A T I V E A S P E C T S O F D I S E A S E E X P R E S S I O N b y J A M E S V E R N O N GROTH B « S c « , U n i v e r s i t y o f M i n n e s o t a , 1 9 6 ? M . S c i , U n i v e r s i t y o f M i n n e s o t a , 1 9 6 9 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S FOR T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y i n t h e D e p a r t m e n t o f B o t a n y We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A O c t o b e r , 1 9 7 ^ In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at 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 , I ag ree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r ag ree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f 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 Vancouve r 8, Canada i A B S T R A C T Q u a n t i t a t i v e s t u d i e s w e r e m a d e o f d i s e a s e r e a c t i o n s i n t h e b a r l e y - U s t i l a g o h o r d e i s y s t e m . I n o c u l a t i o n s t u d i e s i n -v o l v i n g a c o m p a t i b l e c o m b i n a t i o n o f h o s t a n d p a r a s i t e , i n w h i c h o n e o f t h e m a t i n g - t y p e s w a s g r e a t l y d i l u t e d i n r e l a t i o n t o t h e o t h e r , i n d i c a t e d t h a t t h e p a r t i a l - v a c u u m i n o c u l a t i o n m e t h o d r e s u l t s i n a t l e a s t 100 s p o r i d i a p e r s e e d b e i n g a v a i l a b l e t o f o r m d i k a r y o n s w h i c h s m u t t h e p l a n t . R e s i s t a n c e , w h i c h c a n b e i n f l u e n c e d b y e n v i r o n e m n t , a c c o u n t s f o r t h e i n o c u l a t e d p l a n t s w h i c h a r e n o n s m u t t e d . Two a s p e c t s o f d i s e a s e e x i s t t w i t h i n - p l a n t r e a c t i o n a n d b e t w e e n - p l a n t r e a c t i o n . B o t h b e i n g g e n e t i c a l l y d e t e r m i n e d , t h e t w o w e r e c l o s e l y c o r r e l a t e d i n a l l d i s e a s e - p r o d u c i n g c o m b i n a t i o n s o f 12 b a r l e y c u l t i v a r s a n d 21 U . h o r d e i d i k a r y o n s . W i t h i n - c u l t -i v a r a n d w i t h i n - d i k a r y o n c o r r e l a t i o n s w e r e a l s o f o u n d . I n o v e r 500 p l a n t s , u s i n g a v i r u l e n t p a t h o g e n d i k a r y o n , s t u d i e s w e r e m a d e o f w i t h i n - p l a n t s m u t t i n g p a t t e r n s a n d t h e e f f e c t s o n p l a n t g r o w t h o f i n o c u l a t i o n a n d s m u t t i n g . D i s t r i b u t i o n o f s m u t t e d h e a d s w i t h i n s m u t t e d p l a n t s w a s n o t r a n d o m . N o d a l t i l l e r f a m i l i e s t e n d e d t o b e e i t h e r e n t i r e l y s m u t t e d o r e n t i r e l y h e a l t h y . W h e n f a m i l i e s w e r e d i f f e r e n t i a l l y s m u t t e d , t h e o l d e r m e m b e r s m o s t o f t e n w e r e n o n s m u t t e d . E n t i r e o l d e r f a m i l i e s m o s t o f t e n r e m a i n e d n o n s m u t t e d . T h e p r i n c i p a l c u l m d i d n o t f i t t h i s a g e - f r e q u e n c y p a t t e r n . R e g a r d l e s s o f t h e o c c u r r e n c e o f s m u t , i n o c u l a t i o n o f p l a n t s c a u s e d r e d u c t i o n o f t i l l e r i n g a n d c h a n g e d t h e p a t t e r n o f t i l l e r i n g s o t h a t i n o c u l a t e d p l a n t s p r o d u c e d l a t e r a l t i l l e r s a t h i g h e r n o d e s o f t h e p r i n c i p a l c u l i v — \ i i T A B L E O F C O N T E N T S P A G E G E N E R A L I N T R O D U C T I O N .' 1 L I T E R A T U R E R E V I E W 6 A . E a r l y D e v e l o p m e n t W i t h i n t h e S u s c e p t i b l e H o s t . . . 6 B . T h e E x p r e s s i o n o f R e s i s t a n c e • 1 1 C . T h e E f f e c t o f E n v i r o n m e n t o n B a r l e y C o v e r e d S m u t . 1 8 T H E E F F I C I E N C Y O F P A R T I A L - V A C U U M I N O C U L A T I O N O F * H A N N C H E N * B A R L E Y W I T H U S T I L A G O HORDE I 2 2 A . I n t r o d u c t i o n 2 2 B . M a t e r i a l s a n d M e t h o d s C . R e s u l t s 29 D . D i s c u s s i o n 3 1 T H E R E L A T I O N S H I P B E T W E E N W O A S P E C T S O F D I S E A S E E X P R E S S I O N 3 9 A . I n t r o d u c t i o n 3 9 B . M a t e r i a l s a n d M e t h o d s hS C s i S . B e f c . a L t s * . . . . 5 0 -D. D i s c u s s i o n * 5 9 T H E P A T T E R N O F S M U T T I N G A N D E F F E C T O N P L A N T GROWTH , B Y U S T I L A G O HORDE I I N ' H A N N C H E N ' B A R L E Y ° 7 A . I n t r o d u c t i o n 67 B . M a t e r i a l s a n d M e t h o d s 6$ E w s R e s u i t s • ? 1 * 1 . T h e D i s t r i b u t i o n o f S m u t t e d a n d H e a l t h y H e a d s i n S m u t t e d P l a n t s 7 5 2 . T h e E f f e c t o f I n o c u l a t i o n a n d S m u t t i n g o n P l a n t G r o w t h 8 3 D . D i s c u s s i o n 8 8 1 . T h e D i s t r i b u t i o n o f S m u t t e d a n d H e a l t h y H e a d s i n S m u t t e d P l a n t s 8 9 2 . T h e E f f e c t o f I n o c u l a t i o n a n d S m u t t i n g o n P l a n t G r o w t h 9 5 i i i T A B L E OF CONTENTS ( c o n t i n u e d ) PAGE GENERAL CONCLUSIONS 1 0 0 BIBLIOGRAPHY: APPENDIX A 1 1 3 APPENDIX B 1 1 5 i v L I S T O F T A B L E S T A B L E P A G E l . i T h r e e s e p a r a t e e s t i m a t e s o f t h e e f f e c t i v e s p o r i d i a l n u m b e r b a s e d o n t h r e e s p o r i d i a l r a t i o s a n d t h e i r r e s u l t i n g l e v e l s o f s m u t t i n g . ••••••«••« 3 2 1 . 2 . T h e n u m b e r o f m i n o r i t y o f m i n o r i t y s p o r -i d i a o v e r t o t a l c o l o n i e s , a s s h o w n b y t h e i r m a t i n g r e a c t i o n s , o f t w o c u l t u r e s w i t h u n e q u a l s p o r i d i a l r a t i o s s a m p l e d a t f o u r d i f f e r e n t t i m e s a f t e r m i x i n g . • 32 2 . 1 T h e b a s i s o f m e a s u r e m e n t o f s m u t r e a c t i o n s u s e d b y m a n y p a t h o l o g i s t s a n d b r e e d e r s w o r k i n g w i t h s m u t s o f c e r e a l s . . . • 4 1 2 . 2 C o m b i n a t i o n s o f h o s t c u l t i v a r a n d p a t h o g e n d i k a r y o n , a n d d i s e a s e r e a c t i o n o b s e r v e d . . 0 . 51 2 . 3 S o m e w i t h i n - c u l t i v a r a n d w i t h i n - d i k a r y o n . c o r r e l a t i o n a n a l y s e s i n v o l v i n g l o w -t i l l e r i n g p l a n t i n g s o n l y . • • • • • • • * • • • • • • • » • » 58 3 . 1 O b s e r v e d a n d e x p e c t e d n u m b e r s o f v a r i o u s l y s m u t t e d t w o . , t h r . e e a n d , f o u r o r m o r e , t i l l e r f a m i l i e s , a n d t h e a p p l i c a t i o n o f a C h i -s q u a r e t e s t f o r r a n d o m n e s s o f s m u t t i n g a m o n g f a m i l i e s , u s i n g a b i n o m i a l e x p a n s i o n t o c a l c u l a t e e x p e c t e d v a l u e s . • • • • • • • • • • • • • • 7 7 „ 3 . 2 T e s t f o r r a n d o m n e s s o f p o s i t i o n o f n o n -s m u t t e d t i l l e r ( s ) w i t h i n s m u t t e d t i l l e r f a m i l i e s , i n t e r m s o f a g e ( o r d e r o f a p p e a r a n c e ) o f t h e t i l l e r s . . . . . . . . . . . . . . . . . 78 3 . 3 T e s t f o r r a n d o m n e s s o f p o s i t i o n o f n o n -s m u t t e d t i l l e r f a m i l i e s w i t h i n t h e s m u t t e d b a r l e y p l a n t , i n t e r m s o f a g e ( o r d e r o f a p p e a r a n c e ) o f t h e f a m i l i e s . . . 9 . . . . . . . . . . . . 8 0 3 . 4 - T h e p r o d u c t i o n "by s m u t t e d p l a n t s a n d f a t e s o f t i l l e r f a m i l i e s , a n a l y z e d "by n o d a l p o s i t i o n . . . . . . o . . . . . » » . . * * . o . . . » • • « • • < > 8 1 3 . 5 M e a n n u m b e r s o f c u l m s i n i t i a t e d p e r p l a n t , a n d t h e i r f a t e s ( h e a d e d , s m u t t e d o r d e a d ) , w h e n p l a n t s w e r e u n i n o c u l a t e d , i n o c u l a t e d b u t f a i l e d t o b e c o m e s m u t t e d , o r i n o c u l a t e d a n d s m u t t e d . • . . . 0 • • 8 4 V TABLE PAGE 3 „ 6 C o m p a r i s o n s o f g r o w t h p a t t e r n s ( p e r c e n t o f p l a n t s a t t e m p t i n g l a t e r a l t i l l e r p r o d u c t i o n f r o m d i f f e r e n t n o d e s o f t h e p r i n c i p a l c u l m ) o f 2 0 9 s m u t t e d , 1 9 4 n o n s m u t t e d i n o c u l a t e d , a n d 8 3 n o n i n o c u l a t e d " H a n n c h e n 8 b a r l e y p l a n t s • » » » « » o o « o o » o 9 » » o « 9 » » » » » » e » » » » « » « » » « » » j 8 6 L I S T O F F I G U R E S v i FIGURE PAGE 1 , 1 D i s e a s e l e v e l , a s m e a s u r e d "by t h e p r o p o r - -t i o n o f m a x i m u m p o s s i b l e p e r c e n t o f p l a n t s s m u t t e d b y U s t i l a g o h o r d e i ( a m o n g t h e c o n t r o l p l a n t s — t h o s e i n o c u l a t e d w i t h a 1 « 1 s p o r i d i a l m a t i n g - t y p e r a t i o ) , i n r e l a t i o n t o m a t i n g - t y p e r a t i o o f t h e i n o c u l u m • * . . . . . . . . . . . . . . . . . » • 3 0 1 . 2 T h e o r e t i c a l r e l a t i o n s h i p b e t w e e n e f f e c t i v e n u m b e r s a n d m a t i n g - t y p e r a t i o s n e c e s s a r y t o r e a l i z e 0,95. 0.50^and 0.05 o f t o t a l p o s s i b l e p l a n t s s m u t t e d , i n c l u d i n g p l o t t i n g o f t h r e e o b s e r v e d v a l u e s . . . . . . . . . . 3 5 2 , 1 . C o r r e l a t i o n s c a t t e r d i a g r a m f o r 8 2 b a r l e y -U s t i l a g o h o r d e i g e n o t y p i c c o m b i n a t i o n s p l a n t e d u n d e r l o w - t i l l e r i n g c o n d i t i o n s . . . . . $6 2 . 2 C o r r e l a t i o n s c a t t e r d i a g r a m f o r 2 9 b a r l e y -U s t i l a g o h o r d e i g e n o t y p i c c o m b i n a t i o n s p l a n t e d u n d e r h i g h - t i l l e r i n g c o n d i t i o n s . . • • 5 7 3 . 1 I l l u s t r a t i o n o f t h e p a t t e r n o f e a r l y t i l l e r p r o l i f e r a t i o n i n a o n e - m o n t h o l d p l a n t o f ' H a n n c h e n * b a r l e y . . . « • • • • • • • . . . . . • » • • • . • • . . . 7 3 3 . 2 F r e q u e n c y o f c o v e r e d s m u t o c c u r r e n c e i n I r l t i l l e r f a m i l i e s o f b a r l e y p l a n t s i n r e l a t i o n t o r e l a t i v e a g e o f t h e t i l l e r f a m -i l y ( a s i n d i c a t e d b y p o s i t i o n ) • • » • • • • • * • • • » 8 2 3 » 3 T h e p e r c e n t o f s m u t t e d , n o n s m u t t e d i n o c u - a t s ^ l a t e d a n d n o n i n o c u l a t e d b a r l e y p l a n t s i n i t i a t i n g l a t e r a l t i l l e r p r o d u c t i o n a c c o r d * ? : i n g t o n o d a l p o s i t i o n o n t h e p r i n c i p a l c u l m 8 7 v i i A C K N O W L E D G E M E N T S I w o u l d e s p e c i a l l y l i k e t o t h a n k P r o f e s s o r C l a y t o n P e r s o n w h o , a s a n a d v i s o r a n d f r i e n d , h a s a l w a y s b e e n r e a d i l y a v a i l a b l e f o r g u i d a n c e a n d s u p p o r t . T h a n k s a l s o t o D r . T o n y G r i f f i t h s f o r h i s s o u n d a n d l o g i c a l a d v i c e ; t o T a d e s s a E b b a f o r p r o v i d i n g c u l t u r e s a n d f r e q u e n t i n f o r m a t i o n ; t o R o l a n d o R o b i l l o w h o d r e w t h e f i g u r e s a n d p r o v i d e d t e c h n i c a l h e l p ; t o D r . R . B a n d o n l f o r m y c o l o g i c a l a d v i c e ; a n d t o D r . R . K i e s l l n g , o f N o r t h D a k o t a S t a t e U n i v e r s i t y , f o r s a m p l e s o f t h e r a c e s u s e d . F i n a l l y , I s i n c e r e l y t h a n k m y w i f e , J o A n n , w h o , d e s p i t e t w o n e v e r - e n d i n g d i s t r a c t i o n s , h a s r e m a i n e d p a t i e n t a n d u n d e r s t a n d i n g . 1 G E N E R A L I N T R O D U C T I O N T h e c o v e r e d s m u t d i s e a s e o f c u l t i v a t e d b a r l e y ( H o r d e u r n  v u l g a r e L . ) I s c a u s e d b y a f u n g u s , U s t l l a g o h o r d e i ( P e r s . ) L a g e r h . . w h i c h I s o b l l g a t e l y p a r a s i t i c ( i . e . , t o t a l l y d e p e n d e n t o n g r o w t h w i t h i n t h e l i v i n g h o s t f o r c o m p l e t i o n o f i t s l i f e c y c l e ) . T h e p r i n c i p a l m a n i f e s t a t i o n o f t h e d i s e a s e i s s h o w n b y m a t u r e b a r l e y p l a n t s w h o s e f r u i t s ( c a r y o p s e s ) a r e r e p l a c e d b y m a s s e s o f t e l i o s p o r e s ( s o r i ) w h i c h h a v e b e e n p r o d u c e d b y t h e f u n g u s . T h i s p a r t i c u l a r h o s t - p a r a s i t e s y s t e m h a s b e e n t h e s u b j e c t o f r e c e n t g e n e t i c i n v e s t i g a t i o n w h o s e i n t e n t h a s b e e n t o l e a r n m o r e a b o u t 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 i n g e n e r a l t h r o u g h i n t e n s i v e l y s t u d y i n g a s i n g l e s y s t e m . T h u s f a r , t h e s t u d i e s h a v e c e n t e r e d o n t h e e f f e c t s o f s i n g l e g e n e s , e i t h e r i n t h e h o s t o r i n t h e p a r a s i t e , w h i c h h a v e r e l a t i v e l y l a r g e e f f e c t s o n d i s e a s e e x p r e s s i o n . H o w e v e r , a t t h i s t i m e a g r e a t e r u n d e r -s t a n d i n g o f r e s i s t a n c e o t h e r t h a n t h a t w h i c h a l l o w s n o s m u t t i n g w h a t s o e v e r I s e s p e c i a l l y d e s i r a b l e , s i n c e t h e u s e o f " p a r t i a l " r a t h e r t h a n " c o m p l e t e " r e s i s t a n c e m a y r e s u l t i n m o r e e f f e c t i v e l o n g - t e r m c o n t r o l o f t h e d i s e a s e b y a v o i d i n g t h e i n t e n s e s e l e c t i o n p r e s s u r e f a v o r i n g v i r u l e n t p a t h o g e n g e n o t y p e s ( P e r s o n , 1 9 6 7 ) 0 M o r e o v e r , f o r s e v e r a l r e a s o n s , t h i s w o u l d a p p e a r t o b e a u s e f u l h o s t - p s r a s i t e s y s t e m f o r t h e i n i t i a t i o n o f p o p u l a t i o n g e n e t i o s s t u d i e s a i m e d a t , a m o n g o t h e r t h i n g s , a g r e a t e r u n d e r s t a n d i n g o f h o s t - p a r a s i t e c o e v o l u t i o n . M u c h o f w h a t i s d e a l t w i t h i n t h i s t h e s i s c o n c e r n i n g i n t e r m e d i a t e 2 d i s e a s e e x p r e s s i o n w i l l b e e s s e n t i a l p r e l i m i n a r y I n f o r m a t i o n i f s u c h s t u d i e s a r e t o b e u n d e r t a k e n . W h e n c o n s i d e r i n g a n y h o s t - p a r a s i t e s y s t e m i n t e r m s o f p o p u l a t i o n g e n e t i c s , i t i s c o n v e n i e n t t o t h i n k i n t e r m s o f t h e r e l a t i v e r e p r o d u c t i v i t l e s o f t h e t w o i n t e r a c t i n g o r g a n i s m s * ' I t i s n e c e s s a r y t o e x a m i n e t h e e f f e c t s o f e x t r e m e s o f r e p r o -d u c t i v e s u c c e s s o f t h e h o s t a n d p a t h o g e n o n t h e s u c c e s s o f t h e s y s t e m , r a t h e r t h a n o n t h e s u c c e s s o f t h e c o m p o n e n t o r g a n i s m s . L o g i c a l l y , e a c h o r g a n i s m w i l l c h a n g e s o a s t o i n c r e a s e i t s r e p r o d u c t i v i t y . W i t h a n o b l i g a t e p a r a s i t e , h o w e v e r , h o s t c h a n g e s w h i c h a f f e c t p a r a s i t i c c o m p a t i b i l i t y m u s t b e c o n -s i d e r e d a l o n g w i t h c h a n g e s I n t h e p a r a s i t e i t s e l f . I n t h e c a s e w h e r e t h e p a t h o g e n i s a m a j o r , s e l e c t i v e , f o r c e , f o r t h e , h o s t , s t u d i e s o f h o s t r e p r o d u c t i v i t y m u s t i n v o l v e t h e p a t h o g e n . R e g a r d l e s s o f w h e t h e r o r n o t t h e p a t h o g e n i s l i m i t i n g , s t u d i e s o f t h i s k i n d a r e v a l i d l y o f h o s t - p a r a s i t e s y s t e m s a s m u c h a s t h e y a r e o f t h e c o m p o n e n t o r g a n i s m s . I n o r d e r f o r a h o s t -p a r a s i t e s y s t e m t o s u r v i v e , a n e q u i l i b r i u m o f h o s t a n d p a t h o g e n r e p r o d u o t l v l t l e s m u s t b e r e a c h e d , s u c h t h a t n e i t h e r c a n b e c o m e c o m p l e t e l y s u c c e s s f u l a n d c a u s e t h e e l i m i n a t i o n . o f t h e o t h e r . F o r t h e s y s t e m t o s u c c e e d , e a c h c o m p o n e n t o r g a n i s m m u s t s u f f e r a r e p r o d u c t i v e d e c r e a s e I n o r d e r t h a t t h e o t h e r m i g h t r e p r o d u c e a s w e l l . T h a t s u c h e q u i l i b r i a r e p r e s e n t t h e e n d r e s u l t o f h o s t - p a t h o g e n c o e v o l u t l o n h a s b e e n d i s c u s s e d m o r e c o m p l e t e l y b y P e r s o n ( I 9 6 7 ) . 3 T h e c o n c e p t o f r e l a t i v e r e p r o d u c t i v l t y a p p l i e s e s p e c i a l l y w e l l t o t h e b a r l e y - U . h o r d e i s y s t e m b e c a u s e ( 1 ) t h e p a t h o g e n i s t o t a l l y d e p e n d e n t o n t h e h o s t : o n l y b y c a u s i n g d i s e a s e c a n i t r e p r o d u c e s e x u a l l y ; a n d (2) t h e r e l a t i v e r e p r o d u c t i v l t i e s o f t h e h o s t a n d p a t h o g e n a r e c l e a r l y r e c i p r o c a l l y r e l a t e d : t e l l o -s p o r e p r o d u c t i o n o n l y o c c u r s a t t h e e x p e n s e o f s e e d p r o d u c t i o n a n d v i c e - v e r s a . A l t h o u g h t h e s e f e a t u r e s a r e a l s o s h o w n b y m a n y o t h e r h o s t - p a r a s i t e s y s t e m s i n v o l v i n g o b l i g a t e p a t h o g e n s , i t i s w i t h t h e h e a d - i n f e c t i n g s m u t s , w h e r e t h e g e n e r a t i o n t i m e o f t h e p a r a s i t e e x a c t l y m a t c h e s t h a t o f t h e h o s t , t h a t t h e f e a t u r e s a r e s h o w n m o s t c l e a r l y . I t s h o u l d b e n o t e d h e r e t h a t t h e e x i s t e n c e o f t h e b a r l e y -U . h o r d e i s y s t e m i s p r o b a b l y m o r e , o r , l e s s d e p e n d e n t o n , m a n , , a n d h i s a g r i c u l t u r a l p r a c t i c e s . T h e b a r l e y - U . h o r d e 1 s y s t e m i s n o t a " n a t u r a l " s y s t e m o f p a r a s i t i s m , a n d t h i s p o i n t m u s t b e k e p t i n m i n d w h e n d i s c u s s i n g i t i n t e r m s o f e v o l u t i o n a r y c o n c e p t s -s u c h a s f i t n e s s ( r e l a t i v e r e p r o d u c t i v l t y ) , i n o r d e r t o a v o i d d r a w i n g o v e r s i m p l i f i e d c o n c l u s i o n s . I n t h e p a s t , w h e n t h e n a t u r a l p r o g e n i t o r o f t h e b a r l e y -U . h o r d e i s y s t e m e x i s t e d , m e c h a n i s m s m u s t h a v e e v o l v e d w h e r e b y t h e p r o d u c t i o n o f b o t h s o m e s e e d a n d s o m e t e l l o s p o r e s w a s a s s u r e d . I t i s w i t h t h i s a s p e c t o f t h e s y s t e m ( o r t h e r e m n a n t s o f i t ) t h a t t h e t h e s i s i s c o n c e r n e d . F r o m t h e s t a n d p o i n t o f t h e b a r l e y h o s t , t h e r e a r e s e v e r a l w a y s i n w h i c h t h e p l a n t s c a n a v o i d b e i n g t o t a l l y s m u t t e d . F i r s t , a l t h o u g h s u s c e p t i b l e , 4 a p l a n t c a n s i m p l y e s c a p e b e c o m i n g i n f e c t e d . T h i s I s u n -d o u b t e d l y a n i m p o r t a n t f e a t u r e o f n a t u r a l h o s t - p a r a s i t e s y s t e m s j I t w i l l n o t b e d e a l t w i t h f u r t h e r , e x c e p t a s a p o s s i b l e a l t e r n a t i v e I n t h e f i r s t s e c t i o n o f t h e t h e s i s . S e c o n d , a p l a n t c a n e x p r e s s s o m e s o r t o f r e s i s t a n c e t o t h e d i s e a s e . T h i s r e s i s t a n c e c a n t a k e m o r e t h a n o n e f o r m . O n o n e h a n d , i t c a n b e e x p r e s s e d I n t e r m s o f a c e r t a i n p r o p o r t i o n of p l a n t s f a l l i n g t o d e v e l o p s m u t t e d s p i k e s . T h e e x t r e m e c a s e o f t h i s i s t h e m o n o g e n i c t o t a l r e s i s t a n c e , e x p r e s s e d b y s o m e b a r l e y c u l t l v s r s a g a i n s t c e r t a i n s m u t g e n o t y p e s , w h e r e s m u t t e d s p i k e s a r e n o t f o u n d . T h e p e r c e n t a g e o f p l a n t s f a l l i n g to d e v e l o p s m u t r a n g e s f r o m 1 0 0 ^ f o r t h e " t o t a l l y " I n c o m p a t i b l e c o m b i n a t i o n s d o w n t o a b o u t . 30% a n d s o m e t i m e s a s l o w a s \ $ % f o r t h e m o s t c o m p a t i b l e o n e s ( E b b a , 1 9 7 4 ; S i d h u , 1 9 7 2 ) . A n o t h e r w a y i n w h i c h r e s i s t a n c e c a n b e e x p r e s s e d i s t h r o u g h p r o d u c t i o n of h e a l t h y t i l l e r s o n s m u t t e d p l a n t s . L i t t l e I s k n o w n a b o u t t h i s f o r m of r e s i s t a n c e . I t m i g h t b e c o n s i d e r e d s o m e w h a t a n a l o g o u s t o t h e h o r i z o n t a l r e s i s t a n c e o r " t o l e r a n c e " f o u n d i n o t h e r h o s t - p a x a s i t e s y s t e m s ( R o b i n s o n , 1 9 7 3 ) . T h i s t y p e of r e s i s t a n c e ( p a r t i a l - s m u t t i n g o f d i s e a s e d p l a n t s ) r e s u l t s i n I n t e r m e d i a t e d i s e a s e l e v e l s w h i c h a r e n e i t h e r d e v a s t a t i n g to t h e h o s t n o r c r i t i c a l l y d e t r i m e n t a l t o t h e p a t h o g e n . F o r c o n v e n i e n c e , t h e t w o t y p e s o f r e s i s t a n c e w i l l h e r e a f t e r b e r e f e r r e d to a s b e t w e e n - p l a n t a n d w i t h i n - p l a n t r e s i s t a n c e , r e s p e c t i v e l y . 5 T h e p u r p o s e o f t h e s e s t u d i e s w a s t o f u r t h e r o u r u n d e r s t a n d i n g o f t h e d i s e a s e e x p r e s s i o n o f r e s i s t a n c e I n t h e b a r l e y - U . h o r d e i s y s t e m . T h i s i n c l u d e s t 1 ) e x a m i n i n g t h e a r t i f i c i a l i n o c u l a t i o n p h e n o m e n o n t o d e t e r m i n e t h e b a s i s o f n o n - e x p r e s s i o n o f d i s e a s e i n p l a n t s o f a n i n o c u l a t e d , s u s c e p t i b l e c u l t i v a r ; 2 ) d e t e r m i n i n g w h e t h e r o r n o t w l t h i n - p l a n t r e s i s t a n c e i s c o r r e l a t e d w i t h b e t w e e n - p l a n t r e s i s t a n c e ; a n d 3 ) e s t a b l i s h i n g t h e p a t t e r n o f s m u t t i n g w i t h i n t h e b a r l e y p l a n t , a s w e l l a s s o m e o f t h e e f f e c t s o f s m u t p r e s e n c e o n p l a n t g r o w t h . 6 L I T E R A T U R E R E V I E W A . E A R L Y D E V E L O P M E N T W I T H I N T H E S U S C E P T I B L E HOST A n u m b e r o f s t u d i e s h a v e b e e n m a d e o f t h e d e v e l o p m e n t o f v a r i o u s c e r e a l s m u t s w i t h i n t h e i r h o s t s . S e e d l i n g , a s o p p o s e d t o f l o r a l , i n f e c t i n g s m u t s a r e m o r e l i k e l y t o s h e d l i g h t o n e v e n t s o c c u r r i n g w i t h U . h o r d e i I n b a r l e y , a l t h o u g h t h e l a t t e r a r e p r o b a b l y n o t s o d i f f e r e n t e x c e p t i n t h e i r m e a n s o f i n i t i a l e n t r y a n d i n t h e t i m i n g o f t h e e a r l y e v e n t s o f a n d f o l l o w i n g i n f e c t i o n . I n t e n s i v e s t u d i e s h a v e b e e n m a d e o n t h e d e v e l o p m e n t o f l o o s e a n d c o v e r e d s m u t s o f o a t s , U s t l l a g o  a v e n a e ( P e r s . ) R o s t r . a n d U . k o l l e r l W i l l i e , r e s p e c t i v e l y . B o t h , l i k e U . h o r d e 1 . a r e c o n s i d e r e d - t o b e s e e d l i n g I n f e c t i n g s m u t s . U . a v e n a e I s , h o w e v e r , a l s o c a p a b l e o f s o m e f l o r a l I n f e c t i o n a n d I s , t h e r e f o r e , c o n s i d e r e d t o b e a n i n t e r m e d i a t e t y p e f o r w h i c h I n f e c t i o n o f e m b r y o s a t t h e t i m e o f s e e d g e r m i n a -t i o n I s d e p e n d e n t o n m y c e l i u m r a t h e r t h a n o n t e l i o s p o r e s ( F i s h e r a n d H o l t o n , 1 9 5 7 ) • T h e m y c e l i u m o f U . a v e n a e p r o l i f e r a t e s e i t h e r i n t h e I n n e r h u l l o f t h e d o r m a n t s e e d o r i n t h e o u t e r p e r i c a r p o f t h e c a r y o p s l s . A n a t t e m p t b y T a p k e ( 1 9 4 8 ) t o m o r e a c c u r a t e l y l o c a t e t h e f u n g u s w a s a f a i l u r e . I n a n y c a s e , t h e l o c a t i o n I s r a t h e r s i m i l a r t o t h e l o c a t i o n o f t e l i o s p o r e s w h i c h a r e t h e s o u r c e o f i n f e c t i o n f o r t r u e s e e d l i n g i n f e c t o r s s u c h a s U . k o l l e r l . T h i s d i f f e r e n c e b e t w e e n t h e t w o s m u t s h a s b e e n m i n i m i z e d I n e x p e r i m e n t a l w o r k b y u s i n g I d e n t i c a l i n o c u -l a t i o n t e c h n i q u e s . K o l k ( 1 9 3 0 ) I n o c u l a t e d d e h u l l e d s e e d s o f a 7 s u s c e p t i b l e o a t c u l t l v a r b y d u s t i n g t h e m w i t h d r y t e l l o s p o r e s o f U . a v e n a e . T h e s e e d s w e r e e x a m i n e d f o r t h e p r e s e n c e o f s m u t m y c e l i u m a t v a r i o u s s t a g e s a f t e r p l a n t i n g . S h e f o u n d t h a t t h e f u n g u s d i d n o t r e a c h t h e c o l e o p t i l e u n t i l t h r e e d a y s a f t e r p l a n t i n g , a n d i t d i d n o t r e a c h e i t h e r t h e f i r s t v e g e -t a t i v e l e a f o r t h e m e s o c o t y l u n t i l f o u r d a y s a f t e r p l a n t i n g . G a g e ( 1 9 2 7 ) , u s i n g t h e s a m e f u n g u s a n d h o s t , f o u n d n o m y c e l i u m i n s e e d l i n g s w h i c h w e r e l e s s t h a n s e v e n d a y s o l d . T h e d i f f e -r e n c e b e t w e e n t h e s e o b s e r v a t i o n s c o u l d b e d u e t o t h e d i f f e r e n t m a t e r i a l s a n d t e c h n i q u e s u s e d i n t h e t w o s t u d i e s . I n a 1 3 - d a y -o l d s e e d l i n g , K o l k o b s e r v e d m y c e l i u m i n a l l p a r t s o f t h e c o l e o p t i l e , t h e m e s o c o t y l , t h e c o l e o p t i l e n o d e , a l l l e a f a x i l s a n d t h e c o l e o p t i l e a x i l l a r y b u d . N o n e w a s s e e n i n t h e m a i n t i l l e r g r o w i n g p o i n t . W o r k i n g w i t h t h e s a m e s y s t e m , L u t m a n ( 1 9 1 0 ) , h o w e v e r , d i d f i n d t h e m a i n t i l l e r g r o w i n g p o i n t i n v a d e d i n a 1 3 - d a y - o l d s e e d l i n g w h i c h h e b x a m l n e d . B e c a u s e s h e h a d o b s e r v e d s o m e m y c e l i u m i n t h e s p a c e b e t w e e n t h e c o l e o p t i l e a n d t h e f i r s t l e a f s h e a t h , K o l k f e l t t h a t s h e a t h - t o - s h e a t h I n w a r d p e n e t r a t i o n w a s a l i k e l y p a t h w a y . I n s e e d l i n g s , 13 t o 3 0 d a y s o l d , s h e o b s e r v e d g e n e r a l i n v a s i o n o f t h e c o n e o f t h e g r o w i n g p o i n t w i t h a p p a r e n t l y r a n d o m l a t e r a l d i s t r i b u t i o n o f t h e h y p h a e . T h e m y c e l i a l c o n c e n t r a t i o n a t t h e t i p o f t h e c o n e w a s s o m e w h a t l o w e r t h a n a t t h e b a s e . S a m p s o n ( 1 9 3 3 ) f o u n d a s i m i l a r m y c e l i a l d i s t r i b u t i o n a n d c h r o n o l o g y o f p e n e t r a t i o n w h e n s t u d y i n g U . k o l l e r l o n a s u s c e p t i b l e o a t c u l t l v a r . O n t h e b a s i s o f 8 n e g a t i v e e v i d e n c e , s h e f e l t t h a t p a s s a g e f r o m c o l e o p t i l e t o f i r s t v e g e t a t i v e l e a f m u s t b e I n d i r e c t , v i a t h e n o d e s , r a t h e r t h a n d i r e c t . M i l l s ( 1 9 6 6 ) o b s e r v e d t h a t a t t h e s e c o n d l e a f s t a g e ( 1 4 t o 17 d a y s a f t e r p l a n t i n g ) t h e m y c e l i u m o f U . a v e n a e w a s p r e s e n t i n t h e g r o w i n g p o i n t o f s u s c e p t i b l e o a t s e e d l i n g s , a n d t h a t t i l l e r p r i m o r d i a w e r e I n v a d e d b y m y c e l i u m f r o m t h e s u b t e n d i n g l e a f s h e a t h . T i l l e r b u d s I n t h e a x i l s o f t h e c o l e o p t l l e s , f i r s t l e a f , a n d s e c o n d l e a f w e r e i n f e c t e d a f t e r 1 4 , I ? , a n d 2 1 d a y s , r e s p e c t i v e l y . W h e n M i l l s e x a m i n e d p l a n t s a t t h e f o u r t h , f i f t h a n d s i x t h l e a f s t a g e s , h e f o u n d t h a t m o s t a p i c a l g r o w i n g p o i n t s h a d b e e n i n v a d e d . W e s t e r n ( 1 9 3 6 ) f o u n d m y c e l i u m o f U . a v e n a e I n s u s c e p t i b l e o a t a p i c a l g r o w i n g p o i n t s w i t h i n 2 1 d a y s . S o m e u s e f u l h i s t o l o g i c a l s t u d i e s h a v e b e e n m a d e u s i n g c o m m o n b u n t ( T l l l e t l a c a r l e s ( D e C . ) T u l . ) a n d w h e a t . W o o l m a n ( 1 9 3 0 ) d e t e r m i n e d t h a t I n a s u s c e p t i b l e c u l t l v a r , c o l e o p t i l e a n d l e a f s h e a t h I n v a s i o n h a d o c c u r r e d I n s o m e p l a n t s b y s e v e n d a y s a n d p r i m o r d i a l i n v a s i o n b y t w e l v e d a y s . H a n s e n ( 1 9 5 8 ) s a w i n t e r c e l l u l a r b u n t h y p h a e I n c o l e o p t l l e s o f 8 - d a y - o l d s e e d l i n g s o f a s u s c e p t i b l e w h e a t c u l t l v a r . He f a i l e d , h o w e v e r , t o f i n d h y p h a e I n t h e f i r s t l e a f s h e a t h u n t i l t h e s e e d l i n g s w e r e 3 0 t o 5 0 d a y s o l d . G e n e r a l I n v a s i o n o f a l l g r o w i n g p o i n t s d i d n o t o c c u r u n t i l s e e d l i n g s w e r e 50 d a y s o l d . I n a t h o r o u g h s t u d y o f b u n t d e v e l o p m e n t i n t w o d i f f e r e n t s u s c e p t i b l e c u l -t i v a r s , S w i n b u r n e ( I 9 6 3 ) f o u n d t h a t c r o w n n o d e s d i d n o t b e c o m e I n v a d e d u n t i l 3 1 d a y s a f t e r p l a n t i n g ( f i f t h l e a f f s t a g e ) . H i s 9 d i a g r a m s i n d i c a t e d t h a t , e v e n a t t h i s l a t e d a t e , l i t t l e t i l l e r b u d i n v a s i o n h a d o c c u r r e d . L o c a t i o n o f t h e m y c e l i u m w i t h i n t h e p l u m u l e a p p e a r s t o b e r a n d o m . O t h e r d e t a i l s o f d e v e l o p m e n t a r e e s s e n t i a l l y t h e s a m e a s t h o s e f o u n d b y K o l k ( 1 9 3 0 ) f o r t h e o a t s m u t s . S w i n b u r n e t h o u g h t t h a t t h e l e a f s h e a t h s w e r e i n v a d e d b y m y c e l i u m f r o m t h e n e x t o l d e s t l e a f , a n d t h a t i n h i s c u l t l v a r s t h e g r o w i n g p o i n t w a s f i r s t i n v a d e d b y m y c e l i u m f r o m t h e f o u r t h l e a f s h e a t h . H i s w o r k i s s o m e w h a t a t y p i c a l s i n c e t h e p l a n t s h a d n o t y e t b e g u n t o t i l l e r a t 5 0 d a y s b e c a u s e o f t h e g r o w t h c o n d i t i o n s e m p l o y e d . F o r t h e f l o r a l - i n f e c t i n g s m u t s t h e m y c e l i u m i s m u c h m o r e d e e p l y e m b e d d e d i n t h e s e e d a t p l a n t i n g t i m e . B a t t s ( 1 9 5 5 ) s h o w e d t h a t m y c e l i u m o f w h e a t l o o s e s m u t (Us . t . 1 1 a g o t r . l t l e i ( P e r s . ) B o s t r . ) i n s u s c e p t i b l e w h e a t s e e d s i s a b u n d a n t i n t h e s c u t e l l u m , t h e l o w e r e m b r y o , a n d t h e c o l e o r h i z a . I t i s a l s o b e g i n n i n g t o p e r m e a t e y o u n g n o d e s a n d i n t e r n o d e s , b u t i s n o t f o u n d i n t h e p l u m u l e , g r o w i n g p o i n t , o r r a d i c l e . A c c o r d i n g t o B a t t s a n d J e a t e r ( 1 9 5 8 ) , t h e m y c e l i u m i s i n s u c h a p o s i t i o n i n d o r m a n t s e e d s t h a t t h e r e i s l i t t l e p r o b a b i l i t y o f i t s b e i n g " l e f t b e h i n d " d u r i n g t h e p e r i o d o f r a p i d s t e m e l o n g a t i o n . T h e y s h o w e d t h a t a s t h e c r o w n n o d e d e v e l o p s t o f o r m t h e c r o w n r e g i o n c o n s i s t i n g o f t i l l e r b r a n c h e s a n d c o r r e s p o n d i n g n o d e s , I n t e r n o d e s , a n d t i l l e r c r o w n s , t h e m y c e l i u m g e n e r a l l y i s a b l e t o g r o w i n t o a l l p a r t s o f t h e c r o w n . T h e y a l s o n o t e d t w o o t h e r p o i n t s : ( 1 ) t h a t e l o n g a t i o n o f i n t e r n o d e s p r o c e e d s f r o m t h e 10 b a s e u p w a r d ( t h e l o w e r m o s t b e i n g f i r s t t o e l o n g a t e ) , a n d ( 2 ) t h a t n o m y c e l i u m w a s p r e s e n t i n i n t e r n o d e s . R u t t l e (193*0 o b s e r v e d a g e n e r a l i n v a s i o n o f a n I n o c u l a t e d s u s c e p t i b l e w h e a t s e e d l i n g 2 2 d a y s o l d , w i t h t h e s u r p r i s i n g e x c e p t i o n t h a t n o m y c e l i u m c o u l d b e f o u n d i n t h e p l u m u l e . M a l i k a n d B a t t s ( i 9 6 0 ) e s t a b l i s h e d t h a t t h e l o c a t i o n o f m y c e l i u m o f b a r l e y l o o s e s m u t ( U s t l l a g o n u d a ( J e n s . ) R o s t r . ) i n I n f e c t e d s u s c e p t i b l e b a r l e y s e e d s w a s n o t d i f f e r e n t f r o m t h e -l o c a t l o n o f w h e a t l o o s e - s m u t m y c e l i u m I n w h e a t s e e d s . No h i s t o l o g i c a l s t u d i e s o f p o s t - g e r m l n a t l o n l o o s e - s m u t d e v e l o p m e n t i n b a r l e y h a v e b e e n m a d e . . . I n f o r m a t i o n o n t h e d e v e l o p m e n t o f U . h o r d e i I n b a r l e y s e e d l i n g s I s s c a n t . K o z a r ( I 9 6 7 ) o b s e r v e d i n s u s c e p t i b l e O d e s s a b a r l e y , w h i c h h a d b e e n I n o c u l a t e d w i t h t e l i o s p o r e s , t h a t p e r i c a r p p e n e t r a t i o n o c c u r r e d a s e a r l y a s 4 8 h o u r s a f t e r g e r m i n a t i o n . I n v a s i o n o f t h e e m b r y o s a n d , s p e c i f i c a l l y , o f t h e c o l e o p t l l e s a n d n o d e p r i m o r d i a w a s s e e n a f t e r 1 0 0 h o u r s . L o n g , s l e n d e r , r e l a t i v e l y u n b r a n c h e d h y p h a e w e r e f o u n d I n t h e l n t e r n o d a l r e g i o n s o f a n e l o n g a t e d t i l l e r . A s w i t h a l l o t h e r s m u t s , t h e h i g h e s t c o n c e n t r a t i o n s o f m y c e l i u m I n o l d e r p l a n t s o c c u r r e d I n t h e s u b a p l c a l a r e a s o f t h e s p i k e a n d I n t h e c r o w n r e g i o n a t t h e b a s e o f t h e p l a n t . I n s u m m a r y , b a s e d o n r e s u l t s w h i c h a r e a d m i t t e d l y f a r f r o m c o m p l e t e , o t h e r t h a n f o r d i f f e r e n c e s i n t i m i n g , n o 1 1 I m p o r t a n t d i f f e r e n c e s i n e a r l y p o s t - g e r m i n a t i o n d e v e l o p m e n t o f t h e v a r i o u s c e r e a l s m u t s w i t h i n t h e i r h o s t s c a n b e s h o w n . T h i s s e e m s t o b e t r u e r e g a r d l e s s o f t h e l a r g e d i f f e r e n c e i n m o d e o f i n f e c t i o n b e t w e e n s e e d l i n g i n f e c t i n g s m u t s a n d f l o r a l I n f e c t i n g s m u t s . B . T H E E X P R E S S I O N O F R E S I S T A N C E I n t h e p r e v i o u s s e c t i o n , a g e n e r a l v i e w w a s p r o v i d e d o f t h e p a t h w a y o f i n f e c t i o n i n s u s c e p t i b l e s e e d l i n g s f o r s e v e r a l c e r e a l s m u t s . S t u d i e s h a v e a l s o b e e n m a d e c o m p a r i n g e v e n t s i n r e s i s t a n t ( s h o w i n g n o s m u t ) a n d s u s c e p t i b l e c u l -t l v a r s . H i s t o l o g i c a l s t u d i e s o f d i f f e r e n t c e r e a l s m u t s w i l l b e r e v i e w e d f i r s t , f o l l o w e d b y m a c r o s c o p i c s t u d i e s . W i t h b o t h t h e c o v e r e d a n d l o o s e s m u t s I n o a t s , r e s i s t a n t c u l t l v a r s u s u a l l y p r e s e n t n o b a r r i e r t o t h e i n i t i a l i n v a s i o n o f s e e d s a n d y o u n g s e e d l i n g s b y t h e f u n g u s . K o l k ( 1 9 3 0 ) a n d Z a d e ( 1 9 3 1 ) w e r e t h e f i r s t t o n o t i c e t h e i n v a s i o n o f s e e d l i n g s o f r e s i s t a n t c u l t i v a r s b y U . a v e n a e . S a m p s o n ( 1 9 3 3 ) o b s e r v e d n o r m a l ( s u s c e p t i b l e ) c o l e o p t i l e I n v a s i o n b y U . k o l l e r l I n f i v e d a y o l d o a t s e e d l i n g s o f a r e s i s t a n t c u l t l v a r , a f t e r w h i c h t h e f u n g u s w a s c h e c k e d I n I t s d e v e l o p m e n t . W e s t e r n ( 1 9 3 6 ) c o n f i r m e d t h i s , b u t a l s o f o u n d t h a t f o r s i n g l e r a c e s o f b o t h s m u t s t h e r e s i s t a n t c u l t l v a r • M a r k t o n ' d i d n o t a l l o w a n y p e n e t r a t i o n w h a t s o e v e r . T h e f u n g i w e r e s t o p p e d a t t h e c u t i c l e o f t h e e p i d e r m i s . A l l o t h e r r e s i s t a n t c o m b i n a t i o n s , i n c l u d i n g s e v e r a l o t h e r r a c e s t e s t e d o n ' K a r k t o n 1 , a l l o w e d 12 i n v a s i o n o f c o l e o p t i l e s ( o r e v e n d e e p e r t i s s u e s ) . T h e s e c o m b i n a t i o n s d i f f e r e d i n t h e l e n g t h o f t i m e d u r i n g w h i c h t h e m y c e l i u m c o u l d p e r s i s t i n a s t a t e w h i c h w a s i n d i s t i n -g u i s h a b l e f r o m t h a t o f a s u s c e p t i b l e c o m b i n a t i o n . I n m o s t c a s e s , p a t h o g e n a r r e s t w a s n o t a c c o m p a n i e d b y a n y t y p e o f h o s t t i s s u e a l t e r a t i o n . B r a n d w e l n ( 1 9 3 7 ) t w o r k i n g w i t h s e v e r a l c u l t i v a r s o f r e s i s t a n t o a t s , o b s e r v e d c o l e o p t i l e i n v a s i o n b y b o t h s m u t s t o b e g e n e r a l . T h i s i n d i c a t e s t h a t t h e c h o i c e o f m a t e r i a l c a n d e t e r m i n e w h a t w i l l b e o b s e r v e d . I n f e c t i o n b y T . c a r l e s i n r e s i s t a n t w h e a t c u l t i v a r s p r e s e n t e d a s i m i l a r p i c t u r e . G a i n e s ( 1 9 2 3 ) a n d C r e p i n e t a l . ( 1 9 3 7 ) r e p o r t e d t h a t s e e d l i n g s o f r e s i s t a n t a n d s u s c e p -t i b l e w h e a t c u l t i v a r s . s h o w e d , n o . d i f f e r e n c e i n e x t e n t , o f h y p h a l i n v a s i o n f o r t h e f i r s t t w o w e e k s o f g r o w t h . C r e p i n e t a l . o b s e r v e d p e n e t r a t i o n i n t o t h e s e c o n d l e a f s h e a t h i n b o t h c a s e s . W o o l m a n ( 1 9 3 0 ) d i s c e r n e d t h r e e " d i s t i n c t p h a s e s o f p a t h o g e n e s i s t ( 1 ) e n t r a n c e i n t o a n d d e v e l o p m e n t w i t h i n e p i d e r m a l c e l l s o f t h e c o l e o p t i l e ; ( 2 ) d e v e l o p m e n t w i t h i n d e e p e r c o l e o p t i l e t i s s u e s a n d l e a f s h e a t h s ; a n d ( 3 ) e n t r a n c e i n t o n o d e s , i n t e r n o d e s a n d g r o w i n g p o i n t s o f t h e e m b r y o . He f o u n d t h a t s t a g e ( 1 ) w a s a l w a y s a t t a i n e d b u t t h a t , i n r e -s i s t a n t m a t e r i a l , s t a g e ( 2 ) w a s a t t a i n e d o n l y i n s o m e p l a n t s ( w i t h n o d e l a y ) a n d s t a g e ( 3 ) w a s n e v e r a t t a i n e d . G r i f f i t h e t a l . ( 1 9 5 5 ) c o n f i r m e d t h i s o b s e r v a t i o n u s i n g d i f f e r e n t c u l t i v a r s a n d s m u t r a c e s . 1 3 W i t h U s t l l a g o n i g r a T a p k e , t h e s e m i - l o o s e s m u t o f b a r l e y , T i s d a l e a n d T a p k e ( 1 9 2 4 ) f o u n d t h a t p r i o r t o p e n e -t r a t i o n o f t h e c o l e o p t i l e a n d t h e f i r s t l e a f b a s e t h e r e a c t i o n s o f r e s i s t a n t a n d s u s c e p t i b l e c u l t i v a r s w e r e i n d i s -t i n g u i s h a b l e . T h e r e s i s t a n c e s h o w n t o w a r d f l o r a l i n f e c t i n g s m u t s , a l t h o u g h s i m i l a r , m u s t b e v i e w e d i n a d i f f e r e n t c o n t e x t . E m b r y o i n v a s i o n o f t h e d o r m a n t s e e d i s t h e n o r m a l c o n d i t i o n l e a d i n g t o a s u s c e p t i b l e d i s e a s e r e a c t i o n . R u t t l e ( 1 9 3 * 0 > B u b e n t z o v ( 1 9 4 1 ) a n d V a n d e r w a l l e ( 1 9 4 6 ) shoi<red t h a t i n s o m e r e s i s t a n t w h e a t a n d b a r l e y c u l t i v a r s , t h e m y c e l i u m w a s n o t p r e s e n t i n t h e e m b r y o s , a l t h o u g h i t w a s o f t e n f o u n d e l s e -w h e r e - i n t h e s e e d s . B u b e n t z o v f o u n d o n e r e s i s t a n t , w h e a t c u l t l v a r i n w h i c h a m i n o r i t y o f t h e e m b r y o s w e r e i n f e c t e d , h o w e v e r . V a n d e r w a l l e ( 1 9 4 2 ) a n d B a t t s a n d J e a t e r ( 1 9 5 8 ) a l s o f o u n d t e s i s t a n t w h e a t c u l t i v a r s I n w h i c h t h e e m b r y o s w e r e a s f r e q u e n t l y I n v a d e d a s t h o s e o f s u s c e p t i b l e c u l t i v a r s . A t h o r o u g h i n v e s t i g a t i o n w a s c a r r i e d o u t b y P o p p ( 1 9 5 1 a n d 1 9 5 9 ) w h o c o m p a r e d l e v e l s o f e m b r y o I n f e c t i o n , s e e d l i n g I n f e c t i o n a n d a d u l t p l a n t I n f e c t i o n I n b o t h w h e a t a n d b a r l e y . He f o u n d t h a t f o r w h e a t , m o s t c u l t i v a r s s h o w e d a h i g h p e r c e n t a g e o f e m b r y o i n f e c t i o n ( n o t g e n e r a l l y I n v o l -v i n g t h e p l u m u l a r a p e x ) , a n i n t e r m e d i a t e p e r c e n t a g e o f s e e d l i n g i n f e c t i o n , a n d ( I f t h e y p o s s e s s e d h i g h r e s i s t a n c e ) a l o w p e r c e n t a g e o f a d u l t p l a n t i n f e c t i o n . T h u s r e s i s t a n c e 1 4 a p p e a r s t o b e e x p r e s s e d t h r o u g h o u t t h e l i f e o f t h e p l a n t . O t h e r w h e a t c u l t i v a r s ( o r t h e s a m e c u l t i v a r s I n o c u l a t e d w i t h o t h e r r a c e s o f t h e p a t h o g e n ) , a n d a l l o f t h e s e v e r a l b a r l e y c u l t i v a r s e x a m i n e d s h o w e d a g o o d c o r r e l a t i o n b e t w e e n p e r c e n t e m b r y o a n d p e r c e n t a d u l t p l a n t I n f e c t i o n b y U . t r l t l c l a n d U . n u d a , r e s p e c t i v e l y . T h i s i m p l i e s t h a t e m b r y o e x c l u s i o n m a y b e a r e l a t i v e l y m o r e i m p o r t a n t c o n t r i b u t o r t o r e s i s t a n c e i n b a r l e y , a s c o m p a r e d w i t h w h e a t . Ohms a n d B e v e r ( 1 9 5 ^ a n d 1 9 5 5 ) f o u n d t h a t e m b r y o s o f r e s i s t a n t c u l t i v a r s w e r e a s f r e q u e n t l y I n v a d e d a s t h o s e o f s u s c e p t i b l e w h e n t w o w i n t e r w h e a t c u l t i v a r s w e r e i n o c u l a t e d w i t h t h r e e r a c e s o f U . t r l t l c l . G a s k l n a n d S c h a f e r ( I 9 6 2 ) h i s t o l o g i c a l l y e x a m i n e d f i v e r e s i s t a n t w h e a t c u l t i v a r s f o r t h e p r e s e n c e o f U . t r l t l c l m y c e l i u m a t v a r i o u s i n t e r v a l s f o l l o w i n g i n o c u l a t i o n . I n f o u r o f t h e m , r e s i s t a n c e o c c u r r e d a f t e r t h e e m b r y o , a n d , o c c a -s i o n a l l y , e v e n a f t e r t h e p l u m u l a r a p e x , h a d b e e n i n v a d e d . I n t h e f i f t h c u l t l v a r , e m b r y o I n f e c t i o n d i d n o t o c c u r . C o n v i n c i n g m a c r o s c o p i c e v i d e n c e I s a l s o a v a i l a b l e s h o w i n g t h a t s m u t m y c e l i u m I s o f t e n p r e s e n t i n r e s i s t a n t p l a n t s . H u b b a r d a n d S t a n t o n ( 1 9 3 4 ) , a n d S t e v e n s ( 1 9 3 6 ) f o u n d t h a t w h e n t w o o a t c u l t i v a r s w e r e I n o c u l a t e d w i t h U . k o l l e r l . r e d u c e d s t a n d s ( t h e p r o p o r t i o n o f p l a n t s e m e r g i n g ) r e s u l t e d e v e n t h o u g h t h e p l a n t s w e r e a p p a r e n t l y h e a l t h y . B r a n d w e l n ( 1 9 3 7 ) d i d n o t o b s e r v e t h i s e f f e c t i n g r e e n h o u s e s t u d i e s i n v o l v i n g t h e s e s a m e t w o c u l t i v a r s . He c o u l d , 15 however, produce the e f f e c t by p l a n t i n g loose and covered smut Inoculated seeds of r e s i s t a n t and s u s c e p t i b l e c u l t i v a r s i n s o i l which was afterward s e v e r e l y compacted (Brandweln, 1 9 3 8 ) . He concluded t h a t an i n t e r a c t i o n between i n f e c t i o n and de-h u l l i n g (which was commonly done t o o b t a i n higher l e v e l s of smutting) can s i g n i f i c a n t l y weaken the c o l e o p t i l e s , r e s u l t i n g i n reduced emergence. Zade ( 1 9 3 D » Welsh ( 1 9 3 2 ) and Hubbard and Stanton ( 1 9 3 * 0 a l l observed r e s i s t a n t oat c u l t i v a r s which e x h i b i t e d reduced h e i g h t , reduced y i e l d and delayed heading when Inoculated w i t h U. k o l l e r l and/or U„ avenae. S i m i l a r l y , w i t h f l a g smut of wheat, U r o c y s t l s t r l t i c l K o r n i k e , Churchward ( 1 9 3 7 - 8 ) observed s t u n t i n g and c h l o r o s i s o f i n o c u l a t e d p l a n t s which remained unsmutted, and were c l a s s i f i e d as r e s i s t a n t . B l a s t i n g (= s t e r i l i t y ) of heads of r e s i s t a n t oat c u l t i v a r s has been observed a f t e r i n o c u l a t i o n w i t h both U . k o l l e r l and U. avenae (Reed and Stanton, 1 9 3 8 ) and w i t h U. avenae -( H a l i s k y , 1 9 5 & ; Holton, 1 9 6 6 ) . Reed and Stanton a l s o found a few t e l i o s p o r e s i n some of the b l a s t e d heads* On occ a s i o n , workers were a b l e t o modify r e s i s t a n c e through c u l t u r a l methods. Some smutting was obtained I n otherwise " t o t a l l y r e s i s t a n t " 'Markton* oats when Smith and Bressman ( 1 9 3 1 ) cut back t he p l a n t s a t s i x t o seven w e e k s a f t e r p l a n t i n g . Drought c o n d i t i o n s produced the sane e f f e c t the f o l l o w i n g year. However, Woodward and Tingey ( 1 9 ^ 1 ) c u t back b a r l e y p l a n t s a t the boot stage and saw no e f f e c t 16 on smutting by U. h o r d e i , Stevens ( I 9 3 6 ) and Brandweln ( 1 9 3 7 ) could f i n d no e f f e c t on y i e l d , h e i g h t , date of m a t u r i t y , or amount of t i l l e r i n g when they i n o c u l a t e d s e v e r a l t o t a l l y r e s i s t a n t oat c u l t i v a r s w i t h U. k o l l e r l and U. avenae, G r i f f i t h et a l . ( 1 9 5 5 ) observed an e f f e c t on e a r l y growth of both r e s i s t a n t and s u s c e p t i b l e wheat 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 T. c a r l e s . The p l a n t s were g e n e r a l l y s m a l l e r than the uninoculated c o n t r o l s . The d i f f e r e n c e soon disappeared i n the r e s i s t a n t c u l t l v a r . They observed a few t e l i o s p o r e s i n the centre of seeds of some l a t e t i l l e r s of p l a n t s of the r e s i s t a n t c u l t l v a r , however, so i t would appear t h a t the fungus.was s t i l l present even a t m a t u r i t y . C r e p i n et a l . ( 1 9 3 7 ) a l s o saw s i m i l a r e f f e c t s on p l a n t growth i n o t h e r . r e s i s t a n t wheat c u l t i v a r s . U. n i g r a a l s o caused d i s t o r t i o n of seedlings developed from de h u l l e d seed of both s u s c e p t i b l e and r e s i s t a n t b a r l e y c u l t i v a r s a c c o r d i n g to T i s d a l e and Tapke (192'J-). L i t t l e has been recorded concerning the e f f e c t s of f l o r a l I n f e c t i n g smuts on r e s i s t a n t c u l t i v a r s of the host, Tingey and Tolman ( 1 9 3 * 0 c o u l d f i n d no c o r r e l a t i o n between percent of wheat stand ( s e e d l i n g s u r v i v a l ) and loose smut s u s c e p t i b i l i t y , i n d i c a t i n g t h a t i f the mycelium were present i n non-smutted p l a n t s i t s e f f e c t s were too s m a l l t o d e t e c t . 1 7 An unusual type of r e s i s t a n c e i n v o l v i n g an apparent h y p e r s e n s i t i v e r e a c t i o n has been reported i n wheat again s t JJ. t r l t l c l by Oort ( 1 9 ^ 7 ) and by Ohms and Bever ( 1 9 5 5 ) , and i n oats against both oat smuts by Western ( 1 9 3 6 ) . Ohms and Bever observed t h a t , because of Infe c t e d seeds and s e e d l i n g s , stands were reduced. E a r l i e r mentioned stand r e d u c t i o n s may have been caused by t h i s form of r e s i s t a n c e , w i t h d e h u l l l n g being a necessary pre-r e q u i s i t e , however. With f l o r a l l n f e c t o r s , d e h u l l l n g was not p a r t of the i n o c u l a t i o n procedure. Oort ( 1 9 4 7 ) most f u l l y d e s c r i b e d the h y p e r s e n s i t i v i t y which he observed. I t r e s u l t e d In death of e i t h e r e n t i r e p l a n t s or of culms. Thus when heading occurred, a strong s e l e c t i o n f o r healthy p l a n t s or p l a n t p a r t s had taken p l a c e . D e t a i l s are l a c k i n g i n the case d e s c r i b e d by Western ( I 9 3 6 ) . Death of e n t i r e i n f e c t e d p l a n t s i s not mentioned, but i n f e c t e d culms were seen t o d i e . P r a c t i c a l l y nothing Is known about the presence or absence of U. hordei mycelium In i n o c u l a t e d p l a n t s of h i g h l y r e s i s t a n t b a r l e y c u l t i v a r s . No h i s t o l o g i c a l s t u d i e s of such m a t e r i a l have been made. F a r i s ( 1 9 2 4 a ) s t a t e d that p l a n t s which were i n f e c t e d w i t h the fungus were not d i s -t i n g u i s h a b l e from healthy p l a n t s u n t i l near matttSi^y* Johnston ( 1 9 3 * 0 observed reduced emergence of Glabron, a t o t a l l y smut-free c u l t l v a r , when both h u l l e d and deh u l l e d 18 seeds were Inoculated. The dehu l l e d seeds seemed to be more s e v e r e l y a f f e c t e d . Reduced stands were a l s o noted In two r e s i s t a n t b a r l e y c u l t i v a r s by Schafer et a l . ( 1 9 6 2 a ) . In t h i s case, reduced emergence occurred only when seeds were d e h u l l e d and pla n t e d deeply. I t Is p o s s i b l e t h a t In t h i s and other cases of stand r e d u c t i o n reviewed, s o i l pathogens were i n v o l v e d . C . THE EFFECTS OF ENVTRONMEOT ON BARLEY COVERED SMUT . Most of the p u b l i s h e d i n f o r m a t i o n on the e f f e c t s of environment on c e r e a l smuts has been reviewed by Tapke ( 1 9 4 8 ) . The purpose of t h i s s e c t i o n Is not to give a com-prehensive p i c t u r e o f what,is known, but r a t h e r to present i n f o r m a t i o n on environmental e f f e c t s which may r e l a t e to the r e s u l t s of t h i s t h e s i s . As Ba t t s and Jeater ( 1 9 5 8 ) s t a t e , environmental e f f e c t s on one s p e c i f i c smut disease cannot be g e n e r a l i z e d t o Include other smuts. One e s p e c i a l l y cannot apply what Is learne d from f l o r a l - i n f e c t i n g to s e e d l i n g -I n f e c t i n g smuts, and v i c e - v e r s a . Here f l o r a l i n f e c t o r s w i l l not be considered and, except f o r U. h o r d e i . the s e e d l i n g i n f e c t o r s w i l l be mentioned only b r i e f l y . F a r l s (1924a) demonstrated t h a t , f o r s u s c e p t i b l e genotypic combinations, u n i f o r m l y h i g h i n f e c t i o n l e v e l s c o u l d be obtained In the greenhouse over wide ranges of temperature, s o l i a c i d i t y and s o l i moisture. He concluded 1 9 that f i e l d and greenhouse c o n d i t i o n s n e a r l y always f a l l i n t o the f a v o r a b l e ranges of these v a r i a b l e s . His method of i n o c u l a t i o n was t o dust seeds w i t h dry t e l l o s p o r e s . Aamodt and Johnston ( 1 9 3 5 ) determined t h a t s o i l heterogeneity had l i t t l e e f f e c t on l e v e l of smut i n 1 3 8 b a r l e y c u l t i v a r s . Other s t u d i e s i n d i c a t e , however, that environment can be important. Briggs ( 1 9 2 7 ) was unable to o b t a i n high smut l e v e l s using the dry t e l i o s p o r e i n o c u l a t i o n technique f o l l o w e d by p l a n t i n g i n the f i e l d . Tapke ( 1 9 3 8 ) c l e a r l y determined t h a t the post-emergence environment could be important. He germinated seeds i n the greenhouse and, at v a r i o u s times a f t e r germination, t r a n s f e r r e d p l a n t s to the f i e l d . When seeds were dry dusted w i t h t e l l o s p o r e s , the-amount of smut was p r o p o r t i o n a l to the l e n g t h of time the seeds remained i n the greenhouse. When, however, i n o c u l a -t i o n was w i t h an aqueous suspension of t e l l o s p o r e s (which-was allowed to remain on the seeds f o r 2k hours before d r y i n g ) , no v a r i a t i o n i n smutting occurred among the d i f f e r e n t treatments. The study emphasized the importance of i n o c u l a t i o n technique on the degree of environmental s e n s i t i v i t y of i n o c u l a t e d s e e d l i n g s . The e f f e c t of de p t h of p l a n t i n g on smut l e v e l has been i n v e s t i g a t e d by Taylor and Zehner ( 1 9 3 1 ) and by Jones and S e l f El-Nasr ( 1 9 ^ 0 ) . They found t h a t , In g e n e r a l , the deeper the seeds were planted (up to 1 2 cm) the higher was the percent of p l a n t s 20 or heads smutted. The r e l a t i o n s h i p was not a l i n e a r one, so t h a t the most notable e f f e c t occurred at the deepest p l a n t i n g s . Both groups found that of a l l the smuts they s t u d i e d , U. hordei was l e a s t a f f e c t e d by depth of p l a n t i n g . Woodward and Tlngey (194-1) f a i l e d to f i n d any e f f e c t whatso-ever when p l a n t i n g s 1 . 5 and 3 . 0 inches deep were compared. Perhaps the most extreme case of an environmental e f f e c t on disease l e v e l s of covered smut of b a r l e y was r e p o r t e d by Ebba ( 1 9 7 3 ) . - He observed an i n t e r a c t i o n between smut genotype and environment when i d e n t i c a l p l a n t i n g s were made i n B r i t i s h Columbia and C a l i f o r n i a . On one b a r l e y c u l t l v a r a smut dlk a r y o n which caused some smut i n B.C. was a v t r u l e h S i n C a l i f o r n i a , w h i l e another dlkaryon,which caused a r a t h e r low l e v e l of smutting on another c u l t l v a r i n B.C., gave a much higher l e v e l i n C a l i f o r n i a . I t i s impossible t o say which environmental components are r e s p o n s i b l e . From the l a r g e number of other host and pathogen genotypes combined and s t u d i e d at these two l o c a t i o n s , i t would probably be safe to say t h a t t h i s o b s e r vation was e x c e p t i o n a l . The only other r e p o r t of such an i n t e r a c t i o n concerned T. c a r l e s and wheat. E e i c h e r t ( 1 9 3 0 ) found a s i t u a t i o n almost i d e n t i c a l to the one d e s c r i b e d above, w i t h the two l o c a t i o n s being Washington State and Germany. I t was of i n t e r e s t to note t h a t , i n each case, the l o c a l bunt genotype was the more s u c c e s s f u l o f the two. R e l c h e r t a l s o i n d i c a t e s t h a t t h i s i n t e r a c t i o n 2 1 w a s , i n h i s e x p e r i e n c e , a n e x c e p t i o n a l s i t u a t i o n . T h a t e n v i r o n m e n t a f f e c t s t h e d e g r e e o f r e a c t i o n b e t w e e n b a r l e y a n d c o v e r e d s m u t t h e r e c a n b e n o d o u b t . T a p k e ( 1 9 5 2 ) s u b d i v i d e d t h e e f f e c t s o f t h e e n v i r o n m e n t i n t o t h r e e c o n v e n i e n t i n t e r v a l s : ( 1 ) t h e p e r i o d o f i n o c u -l a t i o n — t h e m e t h o d o f a r t i f i c i a l i n o c u l a t i o n c h o s e n b e i n g i m p o r t a n t ; ( 2 ) t h e p r e - e m e r g e n c e e n v i r o n m e n t , a n d ( 3 ) t h e p o s t - e m e r g e n c e e n v i r o n m e n t . S c h a f e r e t a l . ( I 9 6 2 ) a n d T h o m a s ( I 9 6 5 ) s u m m a r i z e i t I n r o u g h l y t h e s a m e w a y , n o t , h o w e v e r , d i s t i n g u i s h i n g b e t w e e n t h e p r e - a n d p o s t - e m e r g e n c e e n v i r o n m e n t s . T h i s i s p r o b a b l y b e c a u s e n o t e n o u g h w o r k h a s b e e n d o n e t o i n d i c a t e a n y q u a l i t a t i v e d i f f e r e n c e b e t w e e n t h e m I n t e r m s o f e f f e c t s . . 2 2 PART I THE EFFICIENCY OF PARTIAL-VACUUM INOCULATION OF HANNCHEN BARLEY WITH USTILAGO HORDEI A. INTRODUCTION As s t a t e d i n the i n t r o d u c t i o n the smut d i s e a s e , when i t i s expressed i n i n d i v i d u a l p l a n t s , may be shown by one or more, and f r e q u e n t l y by a l l , spikes of the I n f e c t e d p l a n t . With one or more diseased spikes taken as the c r i t e r i o n f o r r e c o g n i z i n g the presence of disease In I n d i v i d u a l p l a n t s , the highest disease l e v e l s u s u a l l y r e s u l t i n about 5 0 to 6 0 per cent of the p l a n t s showing the d i s e a s e . Although Thomas ( I 9 6 5 ) and Sidhu ( 1 9 7 2 ) occa-s i o n a l l y observed disease l e v e l s as high as 80 to 8 5 per cent, Ebba ( 1 9 7 4 ) , who worked w i t h a range of smut genotypes and b a r l e y c u l t i v a r s I n t e r a c t i n g under wi d e l y v a r i e d environ-mental c o n d i t i o n s , d i d not r e p o r t disease l e v e l s higher than 6 0 per cent. For any p l a n t which shows a t l e a s t one smutted spike I t Is evident t h a t the i n f e c t i o n process has been success-f u l . A question a r i s e s concerning those p l a n t s which, f o l l o w i n g i n o c u l a t i o n , show no e x t e r n a l evidence of smutting. T h i s could represent the extreme case i n which, f o l l o w i n g s u c c e s s f u l i n f e c t i o n , none of the spikes became smutted. I t could a l s o represent those cases i n which i n f e c t i o n had not been accomplished. 2 3 This study was, t h e r e f o r e , d i r e c t e d to the question of whether, i n s u s c e p t i b l e combinations, the f a i l u r e t o express the disease f o l l o w i n g I n o c u l a t i o n was caused by the f a i l u r e of the i n f e c t i o n process, perhaps because of an i n e f f i c i e n t i n o c u l a t i o n technique, or t o other causes. The approach chosen t o answer t h i s question was q u i t e simple. The standard method of i n o c u l a t i o n has, i n the pa s t , i n v o l v e d inoculum made up of s p o r i d i a of the two necessary mating-types, A and a, which were mixed and shaken together f o r a t l e a s t 24 hours In l i q u i d b r o t h (Thomas 1 9 6 5 ) . More r e c e n t l y , Ebba (p e r s o n a l communication) found t h a t Inoculum which has been mixed and. allowed to stand f o r one or two days i s no;: more e f f e c t i v e than Inoculum which has been mixed Just p r i o r to i n o c u l a t i o n . From t h i s , I t mey be I n f e r r e d t h a t the dikaryons,which u l t i m a t e l y r e s u l t In I n f e c t i o n and smutting, can be formed a f t e r the seed has been i n o c u l a t e d ; they need not e x i s t p r i o r to the time of i n o c u l a t i o n . I t was thought t h a t by c a r e f u l l y a d j u s t i n g the r e l a t i v e amounts of A and a s p o r i d i a and observing the e f f e c t on l e v e l of smutting, an i n d i c a t i o n of the e f f i c i e n c y of the i n o c u l a t i o n procedure would be obtained. Moreover, the best expression of t h i s e f f i c i e n c y would be i n terms of an " e f f e c t i v e number" o f s p o r i d i a . This would represent the average number o f s p o r i d i a which, f o r each i n o c u l a t e d seed, would be poten-t i a l l y capable of t a k i n g p a r t In a " s u c c e s s f u l " I n f e c t i o n 24 ( I . e . , In an I n f e c t i o n which a c t u a l l y r e s u l t s In disease e x p r e s s i o n ) . The e f f e c t i v e number would thus r e l a t e to expression of the disease by i n d i v i d u a l p l a n t s , and would be a measure of the number of s p o r i d i a p o t e n t i a l l y capable of t a k i n g p a r t i n the i n f e c t i o n process. I t would, of course, give no i n d i c a t i o n of the a c t u a l p h y s i c a l l o c a t i o n of these s p o r i d i a . The above approach i s only one p o s s i -b i l i t y . For i n s t a n c e , d i l u t i o n of normal 1 : 1 mating-type inoculum would a l s o undoubtedly work. I t was f e l t t hat the mathematics and l o g i c of the approach chosen were s i m p l e r , however. The purpose of t h i s study was to determine the e f f e c t i v e number for. a r e p r e s e n t a t i v e h o s t - p a r a s i t e geno-type combination and, In so. doing, to assess the e f f i c i e n c y of the i n o c u l a t i o n procedure i n e s t a b l i s h i n g e f f e c t i v e host-pathogen contact. A short study was a l s o made to determine whether known s p o r i d i a l mixtures remain constant over time, or whether the i n i t i a l r a t i o s are brought c l o s e r to u n i t y by f a s t e r growth of the m i n o r i t y type component of the mixture. B. MATERIALS AND METHODS A s i n g l e smut d l k a r y o n and a s i n g l e b a r l e y c u l t l v a r were used i n t h i s study. The two smut haplolds which made up the dlkaryon were o r i g i n a l l y i s o l a t e d by Thomas ( I 9 6 5 ) 2 5 from separate smutted b a r l e y p l a n t s found near Winnipeg, Manitoba. They have been s i n c e used e x t e n s i v e l y as the standard h a p l o l d c u l t u r e s i n the l a b o r a t o r y of Dr. Person, and are designated E 3 a and JAA. The two-rowed b a r l e y c u l t l v a r 'Hannchen' was chosen f o r two reasons: ( 1 ) under normal c o n d i t i o n s a high percentage of p l a n t s show i n f e c -t i o n when 'Hannchen* seeds are i n o c u l a t e d w i t h the standard d l k a r y o n mentioned above; and ( 2 ) i n the greenhouse, where mainly t h i s study was c a r r i e d out, more p l a n t s of t h i s c u l t l v a r can be brought to m a t u r i t y i n a given amount of space than when other c u l t i v a r s are used. Seeds were t r e a t e d w i t h a d i l u t e s o l u t i o n of f o r m a l i n (one part, formalin^ to*.3.2,0V p a r t s water), p r i o r to i n o c u l a t i o n , i n order to k i l l any contaminating smuts and to loosen the seed h u l l s . They were soaked i n the s o l u t i o n f o r one hour^ r i n s e d f o r 3 0 minutes i n running tap water, and thoroughly d r i e d before i n o c u l a t i o n . , Seeds were Inoculated using the partial-vacuum technique d e s c r i b e d by Tapke and Bever ( 1 9 ^ 2 ) . Smut c u l t u r e s were maintained f o r up t o three weeks on modified Vogel's ( 1 9 5 6 ) complete agar medium i n p e t r i d i s h e s . These were st o r e d e i t h e r i n an Incubator a t 22°C o r , more commonly, i n a r e f r i g e r a t o r at 4°C. S p o r i d i a from each c u l t u r e were t r a n s f e r r e d , s e p a r a t e l y , to 1 2 5 tnl Erlenmeyer f l a s k s con-t a i n i n g 5 0 ml of complete b r o t h . A drop or two of aqueous 2 6 achromycin suspension (10 mg/ml) was added to i n h i b i t p o s s i b l e b a c t e r i a l growth, and the f l a s k s were shaken i n a 22°C incubator f o r 3 to 4 days, by which time s p o r i d i a l suspensions of maximum d e n s i t y were obtained. About 100 seeds were put Into a dram v i a l . For normal i n o c u l a t i o n , equal q u a n t i t i e s of the two haplolds (of opposite mating-type) were mixed and the mixture was p i p e t t e d onto the seed a t a r a t e of 8 to 10 ml of Inoculum per v i a l . The v i a l s were then placed i n a vacuum d e s s i c a t o r , without d e s s i c a n t , and sub-j e c t e d to a p a r t i a l vacuum ( r e s u l t i n g i n b o i l i n g ) f o r 30 minutes. Upon r a p i d r e l e a s e of the vacuum, s p o r i d i a are drawn under the seed h u l l s . The excess l i q u i d was then poured o f f , and the seeds were put Into s m a l l c o i n envelopes. Seeds were allowed to dry f o r a t l e a s t 2k hours i n the opened envelopes^ w e l l spaced on metal racks over paper towels. P l a n t i n g took p l a c e w i t h i n three days a f t e r I n o c u l a t i o n . In order t o manipulate the r a t i o s of the two smut h a p l o l d s , the absolute concentrations of the 3 to 4 day o l d s p o r i d i a l suspensions were f i r s t determined by d i l u t i n g each : 1:100 w i t h s t e r i l e water and counting the s p o r i d i a l concen-t r a t i o n s w i t h the a i d of a hemocytometer. Where necessary, the more concentrated suspension was adjusted by adding s t e r i l e water to o b t a i n equal concentrations of s p o r i d i a f o r the two c u l t u r e s . The s p o r i d i a l r a t i o s were then r e a l i z e d by mixing unequal amounts of the standardized c u l t u r e s i n 2 7 the r a t i o s d e s i r e d to gi v e a t o t a l of 8 to 10 ml of inoculum per v i a l . R a t i o s of 1 : 1 , 1 0 0 : 1 , 5 0 0 : 1 , 1 , 0 0 0 : 1 , and 1 0 , 0 0 0 : 1 (and t h e i r r e c i p r o c a l s ) were used. The extreme 1 0 , 0 0 0 : 1 and 1 : 1 0 , 0 0 0 r a t i o s were n e c e s s a r i l y obtained by using a 1 : 1 0 0 d i l u t i o n of the m i n o r i t y c u l t u r e . This r e s u l t e d i n a very s l i g h t decrease i n the s p o r i d i a l d e n s i t y of the f i n a l inoculum. Seeds were planted i n s o i l benches i n the greenhouse. About 5 0 seeds were sown i n each f o u r f o o t row. Rows were randomized. The p l a n t s were given 16 hours per day supplemental f l u o r e s c e n t l i g h t i n g . In the f i e l d , where some treatments were a l s o p l a n t e d , seeds were sown at a r a t e of 1 2 5 seeds per 1 5 -f o o t row. Disease l e v e l s , assessed i n terms of the percentage of p l a n t s showing a t l e a s t one smutted head, were recorded about three months a f t e r seeds were sown, when the pl a n t s had reached m a t u r i t y . Readings were made without knowledge of treatment. The e f f e c t i v e number of s p o r i d i a was determined by usin g the f o l l o w i n g r e l a t i o n s h i p (see d i s c u s s i o n ) : S- = 1 - P n where S = the disease l e v e l taken as a p r o p o r t i o n of the c o n t r o l p o p u l a t i o n (which had been i n o c u l a t e d w i t h a 1 : 1 m i x t u r e ) ! P = the p r o p o r t i o n of majority, s p o r i d i a i n the inoculum mixture; n = the e f f e c t i v e number of s p o r i d i a . 28 In l o g a r i t h m i c form the r e l a t i o n s h i p becomes! l o g ( 1 - S ) = n l o g (P) or H = l o g (1-5) l o g (P) which I s much more e a s i l y solved f o r n. Standard d i l u t i o n p l a t i n g techniques were used t o o b t a i n samples, as s i n g l e s p o r i d i a l c o l o n i e s , from 1 0 0 : 1 and 1 : 1 0 0 r a t i o mixtures of the two haplolds i n complete b r o t h shake c u l t u r e . From the d i l u t i o n p l a t e s , f i v e day o l d c o l o n i e s were t r a n s f e r r e d to f r e s h p e t r i p l a t e s of complete medium to form 2 5-colony master plates.. These were r e p l i c a -p l a t e d , according t o the method of Dinoor and Person ( I 9 6 9 ) , to complete agar p l a t e s containing: s p o r i d i a l lawns of E 3 a and I4A, the two standard mating-type t e s t e r s . Samples were taken at 0 , 7 , 24 and 72 hours a f t e r mixing. In a d d i t i o n , a t 24 hour s , , 5 0 ml of f r e s h medium was added to the c u l t u r e s t o i n i t i a t e another b u r s t of growth. Colony mating-types, based on the presence or absence of i n f e c t i o n hyphae or Msuchfaden M (Bauch, 1 9 3 2 ) , were recorded two days a f t e r t r a n s f e r t o the t e s t e r lawns. Determination of s i g n i f i c a n c e was c a r r i e d out, using a s t a t i s t i c a l technique which deals w i t h the confidence i n t e r v a l about the d i f f e r e n c e between two proportions (see appendix). The 1 : 1 c o n t r o l was the standard against which a l l other treatments were compared. 29 C. RESULTS Figure 1 . 1 shows the r e l a t i o n s h i p between d i f f e r i n g A:a r a t i o s i n the inoculum and the r e s u l t i n g l e v e l s of d i s e a s e . The h i g h e s t disease l e v e l , 30 per cent, was obtained w i t h the 1 : 1 mixture of A and a s p o r i d i a . In Figure 1 . 1 t h i s l e v e l i s set a r b i t r a r i l y at 1 . 0 , and other l e v e l s are shown as p r o p o r t i o n s of t h i s maximal l e v e l . Except f o r the 1 0 , 0 0 0 : 1 and 1 : 1 0 , 0 0 0 r a t i o s , which are based on samples of ca. 1 0 0 p l a n t s , each point on the curve Is based on a sample of at l e a s t 1 5 0 p l a n t s , and on 2 5 0 or more f o r the 1 : 1 , 1 0 0 : 1 and 1 : 1 0 0 r a t i o s . These numbers represent p l a n t s from a t l e a s t t h r e e , and i n some cases from f o u r , separate experiments. For the 1.1,1.,, 1 0 0 : 1 . . and. 1 : 1 0 0 r a t i o s one of the experiments was c a r r i e d out i n the f i e l d . R e s u l t s from d i f f e r e n t experiments were c o n s i s t e n t . The curve shown In Figure 1 . 1 i s not symmetrical. The skewness becomes evident when the 1 0 0 : 1 and 1 : 1 0 0 r a t i o s of A:a s p o r i d i a are compared w i t h each other; when the 1 0 0 : 1 r a t i o f a v o r s mating type a, the disease l e v e l i s s i g n i f i -c a n t l y lower than t h a t of the c o n t r o l s , and when i t favors mating type A the d i f f e r e n c e i n disease l e v e l s i s not s i g n i -f i c a n t . The skewness i s a l s o made evident when the 5 0 0 : 1 and 1 : 5 0 0 r a t i o s and the 1 , 0 0 0 : 1 and 1 : 1 , 0 0 0 r a t i o s are compared. In a l l cases i t Is the r a t i o which favors mating type a t h a t a s s o c i a t e s w i t h the lower disease l e v e l . FIGURE 1.1 A A to a sporidial ratio FIGURE 1.1. DISEASE LEVEL, AS MEASURED BY THE PROPORTION OF MAXIMUM POSSIBLE PERCENT OF BARLEY PLANTS SMUTTED BY USTILAGO HORDEI (AMONG THE CONTROL PLANTS— THOSE INOCULATED WITH A 1:1 SPORIDIAL MATING-TYPE RATIO) IN RELATION TO MATING-TYPE RATIO OF THE INOCULUM ' 3 1 Although the p o i n t s i n t h i s f i g u r e represent averages of. three or more experiments, the skewness was shown con-s i s t e n t l y i n a l l the separate experiments. At the extremes of the curve, where A:a r a t i o s of 1 0 , 0 0 0 : 1 and 1 : 1 0 , 0 0 0 were t e s t e d , only one diseased p l a n t was found. In accordance w i t h the skewness, t h i s s i n g l e diseased p l a n t r e s u l t e d from i n o c u l a t i o n w i t h the 1 0 , 0 0 0 : 1 i n mixture of A to a. The number of c o l o n i e s (over the t o t a l examined) which showed the mating-type of the m i n o r i t y c u l t u r e when 1 0 0 : 1 and 1 : 1 0 0 mating-type mixtures were made and sampled at v a r i o u s times- i s presented" i n Table 1 . 2 . These data show t h a t when s p o r i d i a are mixed together unequally and maintained JLn v i t r o , the r a t i o s do not change a p p r e c i a b l y toward l e s s d i s p a r i t y over a 3-day p e r i o d . D. DISCUSSION In q u a l i t a t i v e terms, one would expect t h a t i f the e f f e c t i v e number of s p o r i d i a i s l a r g e , so t h a t r e l a t i v e l y many s p o r i d i a have the p o t e n t i a l of forming dikaryons and of t a k i n g p a r t i n a s u c c e s s f u l i n f e c t i o n which r e s u l t s In smutting, then a r e d u c t i o n i n the percentage of diseased p l a n t s should not be r e a l i z e d u n t i l r a t h e r d i s p a r a t e r a t i o s of the two mating types are used. L i k e w i s e , I f the e f f e c t i v e number i s s m a l l , the number of p l a n t s showing 3 2 TABLE 1 . 1 THREE SEPARATE ESTIMATES OP THE EFFECTIVE SPORIDIAL NUMBER BASED ON THREE SPORIDIAL RATIOS AND THEIR RESULTING LEVELS OF INFECTION S p o r l d i a l I n f e c t i o n L e v e l C a l c u l a t e d r a t i o used* ( p r o p o r t i o n of E f f e c t i v e Number c o n t r o l ) . 100:1 0.643 103 500:1 0.331 201 1,000:1 0.175 195 *A combination of the r e c i p r o c a l r a t i o s of A and a s p o r i d i a TABLE 1 . 2 THE NUMBER OF MINORITY SPORIDIA OVER TOTAL COLONIES, AS SHOWN BY THEIR MATING-REACTIONS, OF TWO CULTURES WITH UNEQUAL SPORIDIAL RATIOS SAMPLED AT FOUR DIFFERENT TIMES AFTER M I X I N G Time of S p o r l d i a l Ratio Sampling 1 E3a:100 14A 1 I4A:100 E3a (hrs.)  0 . 1/54 1/55 7 0/54 0/54 2 4 2/54 0/55 7 2 * 0/55 . 0/54 •Sampled 48 hrs. a f t e r 50 ml of f r e s h medium was added to each c u l t u r e . 3 3 showing smut should drop o f f r e l a t i v e l y q u i c k l y as the r a t i o s i n c r e a s e . This i s because some of the seeds, which would otherwise become smutted p l a n t s i f Inoculated w i t h the usual 1*1 mating-type mixture, w i l l , at high r a t i o s , f a i l to r e c e i v e s p o r i d i a of the m i n o r i t y mating-type. Since a s i g n i f i c a n t decrease i n the percent of smutted pla n t s does not occur u n t i l the r a t i o reaches 1 0 0 IkA to 1 E 3 a s p o r i d i a on the one hand, and 5 0 0 E 3 a to 1 IkA on the other, i t would seem t h a t the e f f e c t i v e number must be r a t h e r l a r g e . The r e l a t i o n s h i p used i n i t s c a l c u l a t i o n i s an i n t u i t i v e one. From the percentage of smutted p l a n t s shown by the m a t e r i a l i n o c u l a t e d w i t h the 1 : 1 mating-type mixture, one s u b t r a c t s the p r o b a b i l i t y per seed (on the average) of miss i n g the m i n o r i t y mating type, t h i s p r o b a b i l i t y being f i r s t taken to a power equal to the e f f e c t i v e number. One would expect, then, t h a t i f the number of a v a i l a b l e s p o r i d i a i s l a r g e r , the p r o b a b i l i t y of not i n c l u d i n g the m i n o r i t y type w i l l be smaller f o r a given s p o r i d i a l r a t i o , and v i c e v e r s a . The use of average pro-b a b i l i t y i s v a l i d because l a r g e numbers of p l a n t s are-i n v o l v e d i n each case. An a l t e r n a t e way to i l l u s t r a t e the expectation Involved Is seen i n Figure 1 . 2 , which d i s p l a y s the r e l a -t i o n s h i p between s p o r i d i a l r a t i o and e f f e c t i v e number when three separate expectations ere considered. When a l o g - l o g 34 p l o t Is used, the s i z e of the e f f e c t i v e number necessary to o b t a i n each of three proportions of maximum smut; 0 . 9 5 , 0 . 5 and 0 . 0 5 , Increases i n a l i n e a r f a s h i o n as the s p o r i d i a l r a t i o becomes more d i s p a r a t e . The three observed estimates of the e f f e c t i v e number are a l s o p l o t t e d according t o s p o r i d i a l r a t i o and p r o p o r t i o n of t o t a l p o s s i b l e p l a n t s smutted. The e f f e c t i v e number estimates are then obtainable v i s u a l l y by observing the a b s c i s s a l values of the three p o i n t s . While I t i s p o s s i b l e t h a t other more complex approaches to the determination of the e f f e c t i v e number might y i e l d more^ p r e c i s e estimates, f o r the purpose of t h i s study the p r e c i s i o n a f f o r d e d by t h i s r a t h e r simple r e l a t i o n s h i p Is considered to be adequate. One can only speculate about the cause of the asymmetry of decrease i n smutting f o r corresponding r e c i p r o c a l r a t i o s . . At roughly maximum asymmetry,, a s p o r i d i a l r a t i o of around 2 5 0 E 3 a t o 1 I 4 A produces the same disease r e d u c t i o n as a r a t i o of 1 0 0 I 4 A to 1 E 3 a . The simples t explanation f o r t h i s Is t h a t , under p l a n t i n g c o n d i t i o n s , seedborne E 3 a s p o r i d i a do not s u r v i v e as w e l l as I 4 A s p o r i d i a . There Is no reason to con-clude t h a t t h i s Is mating-type r e l a t e d , s i n c e the two haplolds are known t o be g e n e t i c a l l y d i f f e r e n t . Another p o s s i b i l i t y Is t h a t the two mating-types may not be p h y s i o l o g i c a l l y M O a 3 PROPORTION OF MINORITY MATING-TYPE (1-P) M D t» > 8 * 3 S Q I o o 2 8 2 * ^ i 11 miii i 11 miii iiniwr i i n i i i i r i i i n i n r i in M o w 36 e q u i v a l e n t . I f , f o r i n s t a n c e , t h e A m a t i n g - t y p e i s t h e i n i t i a t o r o f d i k a r y o s i s , t h i s c o u l d d e t e r m i n e t h e s k e w n e s s . T h e r e i s a n o t h e r r e a s o n w h y t h e e f f e c t i v e n u m b e r e s t i -m a t e m a y b e l o w . A n a s s u m p t i o n v f h l c h i s m a d e I n t h i s w o r k i s t h a t a m i n o r i t y s p o r i d l u m a t v e r y d i s p a r a t e m a t i n g - t y p e r a t i o s w i l l a s f r e q u e n t l y p a r t i c i p a t e i n c o n j u g a t i o n a s a m i n o r i t y s p o r i d l u m w h i c h o c c u r s i n a l e s s u n e q u a l r a t i o . W h i l e n o t h i n g i s a c t u a l l y k n o w n f o r t h i s s p e c i e s , w o r k w i t h o n e o f t h e T r e m e l l a l e s , a g r o u p o f r e l a t e d f u n g i , s u g g e s t s t h a t t h e a m o u n t o f c o n j u g a t i o n - i n d u c i n g s u b s t a n c e ( s ) p r o d u c e d b y t h e m i n o r i t y m a t i n g - t y p e c o u l d b e i n s u f f i c i e n t w h e n t h e s e m i n o r i t y s p o r i d i a a r e v e r y i n f r e q u e n t ( B a n d o n i , I 9 6 5 ) . T h i s w o u l d m e a n t h a t s o m e o f t h e s e s p o r i d i a w o u l d f a l l t o t r i g g e r t h e n e a r b y m a j o r i t y s p o r i d i a t o c o n j u g a t e . T h e e f f e c t w o u l d b e t o m a k e t h e c a l c u l a t e d e f f e c t i v e n u m b e r a n u n d e r e s t i m a t e o f t h e a c t u a l n u m b e r o f s p o r i d i a a v a i l a b l e f o r d l k a r y o n f o r m a t i o n a n d i n f e c t i o n . A s w e l l , i f r e c i p r o c a l c o n j u g a t i o n - i n d u c i n g s u b s t a n c e s a r e p r o d u c e d b y t h e t w o m a t i n g - t y p e s , t h e s k e w n e s s o f t h e c u r v e c o u l d b e e x p l a i n e d b y u n e q u a l p r o d u c t i o n o r a c t i v i t i e s o f , o r s e n s i t i v i t y t o , s u c h s u b s t a n c e s . I s o g e n i c A a n d a l i n e s w i l l e l i m i n a t e s o m e o f t h e a b o v e p o s s i b i l i t i e s r e g a r d i n g s k e w n e s s . I n o r d e r t o m a k e u s e o f a s m a n y o b s e r v a t i o n s a s p o s s i b l e , t h e c o r r e s p o n d i n g r e c i p r o c a l r a t i o s w e r e c o m b i n e d t o c a l c u l a t e t h e e f f e c t i v e n u m b e r . 3 7 The two e f f e c t i v e number estimates from the 5 0 0 : 1 and 1 , 0 0 0 : 1 r a t i o s are i n good agreement, but the one from the 1 0 0 : 1 r a t i o s i s somewhat lower. A weighted average of the three gives an o v e r a l l estimate of 1 5 5 s p o r i d i a . From t h i s i t can be s a f e l y concluded t h a t the e f f e c t i v e number i s a t l e a s t 1 0 0 . Sampling over time from the two shake c u l t u r e s of r e c i p r o c a l 1 0 0 : 1 s p o r i d i a l r a t i o s shows c l e a r l y t h a t the r a t i o s do not decrease i n three days time. I f one assumes t h a t something s i m i l a r occurs In Inoculated and planted seeds, then the r a t i o s must remain unchanged duri n g the f i r s t three days of germination. This i s the c r i t i c a l p e r i o d f o r i n f e c -tIon- by U s t l l a g o hordei (Appel and Gassner, 1 9 0 7 ) . Results a l s o i n d i c a t e t h a t t h i s i s t r u e even i f , a f t e r I n o c u l a t i o n , the s p o r i d i a are able to d i v i d e s e v e r a l times more. These data support the v a l i d i t y of the concepts here employed to estimate the e f f e c t i v e number, f The c o n c l u s i o n to be drawn from these experiments i s t h a t v i r t u a l l y a l l seeds are making e f f e c t i v e contact w i t h the fungus when the inoculum i s composed of the normal 1 : 1 mating-type r a t i o . I f i t Is c o n s e r v a t i v e l y estimated that 1 0 0 s p o r i d i a are p o t e n t i a l l y capable of t a k i n g p a r t i n d i k a r y o n formation and i n f e c t i o n ( r e s u l t i n g i n disease expression) of a s i n g l e b a r l e y seed, and a l l o w i n g f o r 3 8 v a r i a t i o n d u e t o s a m p l i n g e r r o r ( a c c i d e n t a l d e v i a t i o n f r o m t h e 1 1 1 r a t i o i n t h e i n o c u l u m ) , t h e p r o b a b i l i t y o f o n e o f t h e t w o m a t i n g t y p e s b e i n g t o t a l l y a b s e n t i s e x p e c t e d t o b e e x t r e m e l y s m a l l . I n p r a c t i c e t h e n u m b e r s o f p l a n t s w h i c h f a i l t o s h o w t h e d i s e a s e a r e n e a r l y a l w a y s q u i t e a p p r e -c i a b l e . T h i s m u s t m e a n t h a t t h e p o t e n t i a l f o r d i s e a s e e x p r e s s i o n i s n o t e n t i r e l y d e t e r m i n e d b y t h e c a p a c i t y o f t h e i n o c u l u m t o p r o d u c e i n f e c t i v e d i k a r y o n s , a n d p o i n t s t o t h e c o n c l u s i o n t h a t h o s t r e s i s t a n c e i s a l s o a f a c t o r . How a n d w h e n s u c h h o s t r e s i s t a n c e m i g h t o p e r a t e i s n o t i n d i c a t e d b y t h e r e s u l t s o f t h i s s t u d y . S u c h r e s i s t a n c e a p p e a r s t o b e u n i v e r s a l I n b a r l e y , h o w e v e r , a n d n o t h a v i n g b e e n b r e d e i t h e r i n t o o r o u t o f t h e c r o p , i t m a y r e p r e s e n t a r e m n a n t o f t h e " n a t u r a l w r e s i s t a n c e o f w i l d b a r l e y a n c e s t o r s t o U. h o r d e i . 3 9 P A R T I I T H E RELATIONSHIP BETWEEN TWO ASPECTS O P DISEASE EXPRESSION. A . INTRODUCTION A b a s i o t e n e t o f t h e d i s e a s e p r o c e s s o f c e r e a l s m u t s h a s b e e n f r e q u e n t l y I g n o r e d o r a t l e a s t c o n s i d e r e d o f m i n o r i m p o r t a n c e . I t i s t h a t t w o a s p e c t s o f r e s i s t a n c e c a n b e d i s c e r n e d . O n e t y p e i s m a n i f e s t e d b y e n t i r e p l a n t s a n d i s m e a s u r e d a s t h e p e r c e n t a g e o f p l a n t s s h o w i n g s m u t i n a t l e a s t o n e c u l m . I t c a n b e c a l l e d b e t w e e n - p l a n t d i s e a s e s e v e r i t y a n d i s n o t h i n g m o r e t h a n t h e p r o p o r t i o n o f p l a n t s i n w h i c h s p o r u l a t i o n b y t h e p a t h o g e n i s p r e v e n t e d . A s p o i n t e d o u t i n t h e l i t e r a t u r e r e v i e w , i n " t o t a l l y r e s i s t a n t " c o m b i n a t i o n s o f h o s t a n d s m u t , t h e m y c e l i u m i s n o t n e c e s -s a r i l y p r e v e n t e d e i t h e r f r o m . e n t e r i n g t h e s e e d l i n g s o r f r o m g r o w i n g w i t h i n t h e i n f e c t e d p l a n t . Y i e l d l o s s m a y r e s u l t , e v e n t h o u g h t h e f u n g u s d o e s n o t s p o r u l a t e ; b u t f r o m t h e s t a n d p o i n t o f p a t h o g e n r e p r o d u c t i v l t y , t h i s i s a n e f f e c t i v e f o r m o f r e s i s t a n c e i n d e e d . T h e s e c o n d t y p e o f r e s i s t a n c e i n v o l v e s c u r t a i l m e n t o f s m u t t i n g w i t h i n p l a n t s k n o w n t o b e i n f e c t e d a t m a t u r i t y . I t c a n b e c a l l e d w i t h i n - p l a n t d i s e a s e s e v e r i t y . I t i s m e a s u r e d i n t e r m s o f p e r c e n t o f c u l m s s h o w i n g s m u t o n s m u t t e d p l a n t s ( p l a n t s w i t h a t l e a s t o n e s m u t t e d s p i k e ) . 4 0 In the pas t , breeders and p a t h o l o g i s t s have d i f f e r e d In the measure they chose t o employ In d e s c r i b i n g disease s e v e r i t y . Table 2 . 1 presents a summary of the b a s i s of measure used by many workers. The l i s t Is not exhaustive, but i t i s probably r e p r e s e n t a t i v e . The pl a n t b a s i s , as they used I t , i s a d i r e c t measure of the f i r s t type of r e s i s t a n c e discussed above. The head b a s i s r e f e r r e d to i n the t a b l e Is not simply a measure of the second type, however. Measured as smutted heads over t o t a l heads on a l l p l a n t s , i t i s r e a l l y a compounding of both types of r e s i s t a n c e . Few workers have attempted to l i m i t t h e i r measure of percent of smutted heads to p l a n t s which were known to be smutted. Some went so f a r as t o count both percent of p l a n t s and percent of heads smutted. One cannot s a f e l y assume, however, that simple d i v i s i o n of the percentage based on heads by the percentage based on p l a n t s w i l l g i v e an unequivocal measure of w i t h i n -p l a n t s e v e r i t y . This would only work i f i t were a c c u r a t e l y known t h a t smutted p l a n t s produce as many culms as non-smutted p l a n t s . As P a r t I I I w i l l show, t h i s was u s u a l l y not the case. C o r r e l a t i o n s between percent of p l a n t s and percent of heads smutted have been shown. C l a r k et a l . ( 1 9 3 3 ) obtained a high c o r r e l a t i o n ( r = 0 . 7 ^ 1 ) between the per-centage of p l a n t s and of heads which were bunted, f o l l o w i n g lnooula.tlonof wheat w i t h T. c a r l e s . They concluded t h a t , 4 1 TABLE 2 . 1 THE BASIS OF MEASUREMENT OF SMUT REACTIONS USED BY MANY PATHOLOGISTS AND BREEDERS WORKING WITH CEREAL SMUTS P l a n t Head R e f e r e n c e B a s i s a B a s i s ' 3 B o t h Aamodt and Johnson ( 1 9 3 5 ) x B a t t s ( 1 9 5 5 b ) x B a t t s and J e a t e r ( 1 9 5 8 ) x Brandweln ( 1 9 4 0 ) x B r l g g s ( 1 9 2 6 , 1 9 2 7 ) x C h e r e w l c k ( 1 9 5 8 ) x Churchward ( 1 9 3 7 - 8 ) x c x° C l a r k e t a l . ( 1 9 3 3 ) x Ebba (1974) x F a r i s ( 1 9 2 4 a , 1 9 2 4 b ) x F a r i s ( 1 9 3 4 ) x Ga i n e s ( 1 9 2 3 ) x G a s k i n and S c h a f e r ( 1 9 6 2 ) x G r i f f i t h e t a l . ( 1 9 5 5 ) x H a l l s k y ( 1 9 5 6 ) x H e a l d (.19.21.). x, Heyne and Han s i n g ( 1 9 5 5 ) x H o l t o n ( 1 9 6 4 ) x H o l t o n and H a l i s k y ( i 9 6 0 ) x° x c H o l t o n and R o d e n h i s e r ( 1 9 4 2 ) x Hubbard and S t a n t o n ( 1 9 3 4 ) x J o h n s t o n ( 1 9 3 4 ) x Jones e t a l . ( 1 9 4 0 ) x K o l k ( 1 9 3 07 x L e u k e l ( 1 9 3 6 ) x Mather and Han s i n g ( i 9 6 0 ) x M e t c a l f ( 1 9 6 2 ) x M e t c a l f and Helgason ( 1 9 6 2 ) x Middleman and Chapman ( 1 9 4 1 ) x Oor t ( 1 9 3 9 ) x O o r t ( 1 9 4 7 ) x P e r s o n and C h e r e w l c k ( 1 9 6 4 ) x Poehlman ( 1 9 4 9 ) x Poehlman and C l o n l n g e r ( 1 9 5 5 ) x Popp ( 1 9 5 D x Reed ( 1 9 2 9 , 1 9 3 8 ) x Reed and F a r i s ( 1 9 2 4 ) x Reed and S t a n t o n ( 1 9 3 8 ) x R e i c h e r t ( 1 9 3 0 ) x R o d e n h i s e r and H o l t o n ( 1 9 4 2 ) x Ross e t a l . ( 1 9 4 8 ) x 42 T A B L E 2 . 1 ( c o n t i n u e d ) R e f e r e n c e P l a n t B a s i s Head B a s i s B o t h R u t t l e ( 1 9 3 4 ) S a m p s o n e t a l . ( 1 9 2 7 ) S c h a f e r e t a l . ( 1 9 6 2 b ) S c h a l l e r e t a l . ( I 9 6 0 ) S h a n d s ( 1 9 5 6 ) x X S l d h u a n d P e r s o n ( 1 9 7 2 ) X X S m i t h ( 1 9 3 2 a ) S m i t h a n d B r e s s m a n ( 1 9 3 1 ) S w i n b u r n e ( I 9 6 3 ) T a p k e ( 1 9 2 9 , 1 9 3 1 ) T a p k e ( 1 9 3 7 , 1 9 4 5 , 1 9 5 2 , 1 9 5 5 ) T a p k e ( 1 9 3 8 ) T a p k e a n d B e v e r ( 1 9 4 2 ) T a y l o r a n d Z e h n e r ( 1 9 3 1 ) T e r v e t ( 1 9 4 0 , 1 9 4 1 , 1 9 4 4 ) T h o m a s a n d P e r s o n ( I 9 6 5 ) W e l l s ( 1 9 5 8 ) x W e l s h ( 1 9 3 2 ) x W e s t e r n ( 1 9 3 6 ) * W i l l i a m s a n d V e r m a ( 1 9 5 4 ) x W o o d w a r d a n d T l n g e y ( 1 9 4 1 ) ; • a P e r c e n t o f p l a n t s s h o w i n g o n e o r m o r e s m u t t e d h e a d . ^ P e r c e n t o f h e a d s s h o w i n g s m u t . © D i f f e r e n t m e a s u r e s w e r e u s e d i n d i f f e r e n t s e c t i o n s o f w o r k . T i n g e y e n d T o l m a n ( 1 9 3 4 ) T i s d a l e a n d T a p k e ( 1 9 2 4 ) W a u d a n d M e t z g e r ( 1 9 7 0 ) x x x X as a s e l e c t i o n c r i t e r i o n In p l a n t breeding, head counts would be n e a r l y as r e l i a b l e as p l a n t counts, In s p i t e of the f a c t t h a t the former was c o n s i s t e n t l y lower than the l a t t e r In I d e n t i c a l m a t e r i a l . E u t t l e (19 3*0 a l s o found a s i m i l a r c o r r e l a t i o n In a s i n g l e barley-U. hordei combina-t i o n . Tapke ( I 9 2 9 , 1931) recorded both head and p l a n t smut percentages f o r both f o u r wheat c u l t i v a r s Inoculated w i t h U. t r l t l c l and two b a r l e y c u l t i v a r s Inoculated w i t h U. nuda. The data show a s t r o n g c o r r e l a t i o n between percent of p l a n t s and percent of heads smutted. Tapke d i d not d i s c u s s t h i s f i n d i n g , however, In h i s paper. Gaines ( 1923) decided t h a t percent of smutted heads was the p r e f e r a b l e measure because, among other reasons, I t gave a more r e a l i s t i c estimate of the Impact which smut has on a crop. As w i l l be mentioned l a t e r , he was aware of the c o r r e l a t i o n as w e l l . B rlggs ( 1 9 2 6 ) and Churchward (1932-3) argued that f o r g e n e t i c s t u d i e s the p l a n t , not the culm, must be maintained as the u n i t . In t h e i r g e n e t i c s t u d i e s , even though they, too, were aware of the c o r r e l a t i o n , they used the p l a n t b a s i s . Bather than count both head and p l a n t smutting percentages, some workers t r i e d to get an I n d i c a t o r of w l t h l n - p l a n t disease s e v e r i t y i n another way: by r e c o r d i n g p l a n t s as e i t h e r d i s e a s e d , p a r t i a l l y d i s e a s e d , or healthy. Gaines ( I 9 2 3 ) and Churchward d i d t h i s w i t h wheat-T. c a r l e s . With oats and both smuts, Welsh ( 1 9 3 2 ) used t h i s method t o evaluate t h e o v e r a l l r e s i s t a n c e o f s i x c u l t i v a r s . I n n o c a s e I n t h e a b o v e w o r k s c o u l d a c o r r e l a t i o n b e s e e n b e t w e e n p e r c e n t o f d i s e a s e d p l a n t s a n d f r e q u e n c y o f p a r t i a l l y d i s e a s e d p l a n t s . S i n c e t h e d a t a w e r e n o t p a r t i c u l a r l y a b u n d a n t f o r t h i s p u r p o s e i n a n y o f t h e s t u d i e s , h o w e v e r , t h e r e s u l t s s h o u l d b e J u d g e d I n c o n c l u s i v e r a t h e r t h a n n e g a t i v e . E e e d ( 1 9 3 8 ) u s e d t h e s a m e s o r t o f s c o r i n g i n a n e x t e n s i v e s t u d y I n v o l v i n g e i g h t o a t c u l t i v a r s a n d a t o t a l o f f i v e r a c e s ; o f b o t h o a t s m u t s . He p r e s e n t e d e v i d e n c e o f a n e g a t i v e c o r r e -l a t i o n b e t w e e n p e r c e n t o f p l a n t s s m u t t e d a n d t h e n u m b e r o f p a r t i a l l y s m u t t e d p l a n t s . A t b e s t , t h i s t y p e o f s c o r i n g o f s m u t s e v e r i t y g i v e s a n o n p a r a m e t r i c o r q u a l i t a t i v e m e a s u r e o f w i t h i n - p l a n t d i s e a s e s e v e r i t y , u n l e s s l a r g e n u m b e r s o f o b s e r v a t i o n s a r e i n v o l v e d . I t I s n o t r e a l l y v a l i d t o e s t a b l i s h a c o r r e l a t i o n b e t w e e n p e r c e n t o f s m u t t e d p l a n t s a n d p e r c e n t o f s m u t t e d h e a d s ( o n a l l p l a n t s ) , b e c a u s e t h e s e t w o t h i n g s a r e l o g i c a l l y n o t I n d e p e n d e n t ( S t e e l a n d T o r r i e , i 9 6 0 ) . T h e y c a n n o t b e e x p e c t e d t o b e I n d e p e n d e n t b e c a u s e t h e h i g h e r t h e f r e q u e n c y o f s m u t t e d p l a n t s , t h e h i g h e r s h o u l d b e t h e f r e q u e n c y o f s m u t t e d c u l m s o n t h o s e p l a n t s , e v e n i f w i t h i n - p l a n t d i s e a s e s e v e r i t y d o e s n o t v a r y . B a r r i n g a n e g a t i v e c o r r e l a t i o n b e t w e e n w i t h i n - a n d b e t w e e n - p l a n t d i s e a s e s e v e r i t y , w h i c h I s u n l i k e l y , I t w o u l d b e s u r p r i s i n g i f a p o s i t i v e c o r r e l a -t i o n b e t w e e n t h e n o n - i n d e p e n d e n t v a r i a b l e s , t h a t i s , p e r c e n t ^ p l a n t s s m u t t e d a n d p e r c e n t c u l m s s m u t t e d , w a s n o t f o u n d . T h e r e I s n o a p r i o r i r e a s o n , o n t h e o t h e r h a n d , w h y t h e p e r c e n t o f s m u t t e d c u l m s o n o b v i o u s l y d i s e a s e d p l a n t s s h o u l d n o t b e I n d e p e n d e n t o f p e r c e n t o f p l a n t s s m u t t e d . H e n c e , t h e e m p i r i c a l I n v e s t i g a t i o n o f t h e r e l a t i o n s h i p b e t w e e n t h e s e t w o v a r i a b l e s I s v a l i d . S o m e w o r k e r s c a l l e d a t t e n t i o n t o t h e p o s s i b l e c o r r e -l a t i o n o f w i t h i n - a n d b e t w e e n - p l a n t d i s e a s e s e v e r i t y a n d v a l i d l y p r e s e n t e d e v i d e n c e I n f a v o r o f I t . A l t h o u g h t h e y p r e s e n t e d n o d a t e I n s u p p o r t o f i t , B a t t s ( 1 9 5 5 b ) a n d B a t t s a n d J e a t e r ( 1 9 5 8 ) c o n c l u d e d f r o m e a r l i e r l i t e r a t u r e a n d , p r e s u m a b l y , f r o m fchelr o w n o b s e r v a t i o n s , t h a t i t e x i s t 3 ^ I n o n l y t w o I n s t a n c e s w a s t h e r e l a t i o n s h i p p r o p e r l y i n v e s t i -g a t e d . G a i n e s ( 1 9 2 3 ) d e t e r m i n e d t h e p r o p o r t i o n o f t o t a l l y b u n t e d w h e a t p l a n t s , w h i c h h e c a l l e d c , t h e p r o p o r t i o n o f p a r t i a l l y b u n t e d p l a n t s , b , a n d a v a l u e , a , w h i c h w a s t h e p r o p o r t i o n o f b u n t e d h e a d s o n p a r t i a l l y b u n t e d p l a n t s . H e t h e n d e t e r m i n e d t h e o v e r a l l p e r c e n t o f b u n t e d h e a d s , d , b y t h e r e l a t i o n s h i p ! d = a b + c T h e I m p o r t a n t t h i n g i s t h a t h e n o t e d t h a t t h e v a l u e o f a w a s l o w e r o n t h e f o u r r e s i s t a n t c u l t i v a r s t h a n I t w a s o n t h e f o u r s u s c e p t i b l e c u l t i v a r s , ( O . 6 7 v s . O . 2 5 ) . Q u a l i t a t i v e l y , t h i s i s g o o d e v i d e n c e t h a t a p o s i t i v e c o r r e l a t i o n e x i s t e d i n h i s m a t e r i a l . O o r t ( 1 9 4 7 ) , w o r k i n g w i t h w h e a t a n d U , t r l t l c l , s h o w e d w h a t a p p e a r e d t o b e a v e r y c l o s e c o r r e l a t i o n o f w i t h i n - a n d b e t w e e n - p l a n t d i s e a s e s e v e r i t y i n a s t u d y w h i c h I n v o l v e d m a n y w h e a t c u l t i v a r s . H i s s a m p l e s i z e s a r e r a t h e r s m a l l i n m o s t c a s e s a n d h e I n c l u d e d n o s t a t i s t i c a l a n a l y s i s . H i s w a s t h e o n l y s t u d y f o u n d i n t h e l i t e r a t u r e i n w h i c h t h e c o r r e l a t i o n w a s i n v e s t i g a t e d a s t h e m a i n o b j e c t i v e a n d i n a m a n n e r s i m i l a r t o t h a t u n d e r t a k e n i n t h i s t h e s i s . T h e p u r p o s e o f t h i s s t u d y w a s t o d e t e r m i n e , u s i n g a s b r o a d a s a m p l i n g o f b a r l e y a n d U . h o r d e i g e n o t y p e s a s p o s s i b l e , w h e t h e r , i n t h i s h o s t - p a r a s i t e s y s t e m , t h e s e v e r i t y o f d i s e a s e r e a c t i o n w i t h i n p l a n t s i s ( 1 ) g e n e t i -c a l l y v a r i a b l e , a n d ( 2 ) c o r r e l a t e d w i t h s e v e r i t y b e t w e e n p l a n t s . B . M A T E R I A L S A N D M E T H O D S T w e l v e b a r l e y c u l t i v a r s a n d 2 1 U . h o r d e i d i k a r y o n s w e r e u s e d i n t h i s s t u d y . O n l y t h o s e g e n o t y p i c c o m b i n a t i o n s w h i c h s h o w e d s m u t i n a t l e a s t f i v e p e r c e n t o f i n o c u l a t e d p l a n t s w e r e u s e d . T h e s m u t d i k a r y o n s I n c l u d e d r e p r e s e n t a t i v e s o f t h e o r i g i n a l 1 3 p h y s i o l o g i c a l r a c e s d e s c r i b e d b y T a p k e ( 1 9 4 2 ) . T h e s e w e r e o b t a i n e d a s t e l i o s p o r e s a m p l e s f r o m N o r t h D a k o t a S t a t e U n i v e r s i t y , w h e r e t h e y h a v e b e e n m a i n -t a i n e d . T h e o t h e r e i g h t c u l t u r e s r e p r e s e n t a s w i d e a r a n g e o f s m u t b i o t y p e s a s i t w a s p o s s i b l e r e a d i l y t o o b t a i n . A l l a r e f r o m N o r t h A m e r i c a e x c e p t t h e t h r e e p r e f i x e d n E t " , w h i c h 4? a r e f r o m t h r e e s e p a r a t e c o l l e c t i o n s m a d e I n E t h i o p i a . O f t h e 21 d i k a r y o n s h e r e u s e d , o n e w a s d e r i v e d f r o m t h e h a p l o l d s E 3 a a n d I 4 A w h i c h were u s e d i n s e c t i o n I . F o r t h e o t h e r 20 d i k a r y o n s , t h e c o n s t i t u e n t h a p l o l d s w e r e b o t h d e r i v e d f r o m a s i n g l e t e l l b s p o r e . T h e s e 20 d i k a r y o n s w e r e t h u s p r o d u c e d t h r o u g h " s e l f l n g " o f t w o o f t h e f o u r p r o d u c t s o f 20 t e t r a d s d e r i v e d f r o m 20 d i f f e r e n t -t e l i o s p o r e s . T h e t e t r a d s w e r e d e r i v e d i n t h e f o l l o w i n g w a y i A p e t r i d i s h w a s p o u r e d w i t h a t h i n l a y e r o f c o m p l e t e a g a r . 2 2 B l o c k s a b o u t 15 mm w e r e c u t a n d e a c h w a s p l a c e d o n a 22 mm s t e r i l e c o v e r s l i p . F i v e m l a q u e o u s s u s p e n s i o n s o f t e l i o -s p o r e s w e r e p r e p a r e d I n t e s t t u b e s . A f t e r a d d i n g t o t h e s e s u s p e n s i o n s a d r o p o f a c h r o m y c i n s u s p e n s i o n (10 m g a c h r o m y c i n p e r m l H J J O ) , a s m a l l d r o p o f t h e t e l l o s p o r e s u s p e n s i o n w a s t r a n s f e r r e d t o t h e c e n t r e o f e a c h a g a r b l o c k w i t h a Pas toTas* p i p e t t e . D e p e n d i n g o n t h e g e r m i n a t i o n r a t e o f t h e t e l i o s p o r e s u s e d , t h e c o n c e n t r a t i o n w a s a d j u s t e d s o t h a t f r o m 10-100 t e l i o s p o r e s w e r e i n c l u d e d I n e a c h d r o p t r a n s f e r r e d . T h e a g a r b l o c k s w i t h t h e i r t e l i o s p o r e s w e r e t h e n I n c u b a t e d a t 22°C f o r a b o u t 12 h o u r s ( s o m e w h a t l o n g e r f o r o l d e r s a m p l e s ) , b y w h i c h t i m e m o s t o f t h e 5-10 v i a b l e t e l i o s p o r e s h a d g e r -m i n a t e d . O n e a c h b l o c k a s u i t a b l y g e r m i n a t e d t e l l o s p o r e , w i t h a l l f o u r s p o r i d i a l p r o d u c t s a c c e s s i b l e f o r m i c r o d i s -s e c t i o n , w a s s e l e c t e d . U s i n g a d e F o n b r u n e m i c r o m a n i p u l a t o r a n d a n u p w a r d - b e n t f i n e g l a s s n e e d l e , t h e f o u r p r i m a r y 4 8 s p o r i d i a w e r e d r a w n a w a y f r o m t h e p r o m y c e l i u m , o n e t o e a c h o f t h e f o u r s i d e s o f t h e b l o c k . T h e i r p o s i t i o n o n t h e p r o -m y c e l i u m w a s r e c o r d e d d i r e c t l y o n t h e c o v e r s l i p , n e a r t h e e d g e . I n 3-k d a y s , v i s i b l e c o l o n i e s h a d f o r m e d f r o m t h e f o u r s p o r i d i a . T h e y w e r e t r a n s f e r r e d s i n g l y t o f r e s h p l a t e s a n d , a f t e r s u f f i c i e n t g r o w t h , w e r e t e s t e d f o r m a t i n g - t y p e a s d e s c r i b e d I n P a r t I . S m a l l a m o u n t s o f s p o r i d i a w e r e p u t I n t o s c r e w - c a p t u b e s c o n t a i n i n g f i n e - t e x t u r e d , v e r y d r y s i l i c a g e l f o r l o n g - t e r m s t o r a g e . T w o o f t h e f o u r c u l t u r e s o f e a c h t e t r a d w e r e s e l e c t e d a t r a n d o m t o f o r m t h e d i k a r y o n s w h i c h w e r e u s e d t h r o u g h o u t t h e s t u d y . S e e d t r e a t m e n t a n d I n o c u l a t i o n w e r e a s d e s c r i b e d i n s e c t i o n I. C o n s i d e r i n g t h e s i z e o f t h e o v e r a l l e x p e r i m e n t i n r e l a t i o n t o t h e a m o u n t o f f i e l d s p a c e a n d r e c o r d i n g t i m e a v a i l a b l e , t h e m i n i m u m n u m b e r o f s m u t t e d p l a n t s w h i c h w o u l d b e e x a m i n e d f o r w i t h i n - p l a n t s e v e r i t y i n e a c h g e n o t y p l o c o m b i n a t i o n w a s s e t a t 30. O n l y p l a n t s w i t h t h r e e o r m o r e c u l m s w e r e I n c l u d e d . I n o r d e r t o b e 99 p e r c e n t c e r t a i n o f o b t a i n i n g 30 p l a n t s , f o r t h o s e c o m b i n a t i o n s w h e r e i n f o r m a t i o n w a s a v a i l a b l e c o n c e r n i n g t h e a m o u n t o f s m u t t o b e e x p e c t e d , t h e n u m b e r o f i n o c u l a t e d s e e d s n e e d e d f o r p l a n t i n g w a s d e t e r m i n e d b y u s i n g t h e r e l a t i o n s h i p ! (1 - P ) n = 0.99 w h e r e P = t h e p e r c e n t a g e o f p l a n t s s h o w i n g s m u t ; n = t h e n u m b e r o f p l a n t s n e e d e d t o b e 99 p e r c e n t c e r t a i n o f o b t a i n i n g a t l e a s t o n e s m u t t e d p l a n t . 4 9 T h e n e t r e s u l t w a s t h a t m o r e s e e d s w e r e p l a n t e d w h e n , f o r a p a r t i c u l a r g e n o t y p i c c o m b i n a t i o n , i t w a s e x p e c t e d t h a t t h e s m u t t i n g p e r c e n t a g e w o u l d b e l o w . I n o c u l a t e d s e e d s w e r e p l a n t e d i n t h e f i e l d i n 1 5 - f t . r o w s . T h e r a t e o f s e e d i n g v a r i e d f r o m 90-150 s e e d s p e r r o w , d e p e n d i n g o n p e r c e n t v i a b i l i t y o f t h e s e e d 8 n d o n t h e a m o u n t o f t i l l e r i n g e x h i b i t e d b y t h e c u l t l v a r a t a g i v e n s e e d i n g r a t e . U s u a l l y a p a r t i c u l a r g e n o t y p i c c o m b i n a t i o n w a s p l a n t e d i n 1-2 b l o c k s o f s e v e r a l r o w s e a c h . I n C a l i -f o r n i a , w h e r e a p l a n t i n g w a s m a d e d u r i n g t h e w i n t e r m o n t h s , t h e p r o c e d u r e s w e r e i d e n t i c a l e x c e p t t h a t t h e r o w s w e r e 20 f e e t l o n g . D a t a w e r e r e c o r d e d w h e n a l l s p i k e s h a d e m e r g e d . G e n e r a l l y , t h e p e r c e n t o f p l a n t s s m u t t e d w a s b a s e d o n s a m p l e s o f a t l e a s t 300 p l a n t s . I n t h e f e w c a s e s w h e r e f e w e r p l a n t s s e r e a v a i l a b l e , t h e p e r c e n t a g e o f p l a n t s s m u t t e d w a s h i g h e n o u g h s o t h a t s a m p l e s i z e s w e r e s t a t i s t i -c a l l y a d e q u a t e n o n e t h e l e s s ( S t e e l a n d T b r r l e , i 9 6 0 ) . S m u t t e d p l a n t s w h i c h w e r e t o b e e x a m i n e d o n a t i l l e r b a s i s w e r e c a r e f u l l y p u l l e d . W h e n t i m e w a s n o t l i m i t i n g a n d t h e p l a n t s w e r e a v a i l a b l e , 4 0 - 5 0 p l a n t s w e r e e x a m i n e d . P l a n t s w i t h o n e a n d t w o t i l l e r s w e r e a l s o r e c o r d e d , b u t f o r r e a s o n s t o b e g i v e n t h e y w e r e n o t u s e d i n d e t e r m i n i n g t h e d e g r e e o f w l t h i n - p l a n t s e v e r i t y . A l l p l a n t s i n a r o w w e r e p u l l e d 50 a n d r e c o r d e d b e f o r e g o i n g o n t o t h e n e x t r o w , i n o r d e r t o a v o i d b i a s e d s a m p l i n g . I n c o m p l e t e s m u t t i n g o f s p i k e s w a s a l s o r e c o r d e d o n a s i n g l e p l a n t b a s i s . W h e n s m u t t e d h e a d a n d s m u t t e d p l a n t p e r c e n t a g e s h a d b e e n c a l c u l a t e d , t h e c o r r e l a t i o n a n a l y s e s w e r e d o n e b y c o m p u t e r . C . R E S U L T S T a b l e 2.2 i s a m a t r i x . o f t h e 12 b a r l e y c u l t i v a r s a n d 21 s m u t d i k a r y o n s , s h o w i n g t h e d i s e a s e r e a c t i o n s w h i c h w e r e o b s e r v e d . T h e w o r k o f T a p k e (19^5) w ^ s c o n s u l t e d t o d e t e r -m i n e w h i c h c o m b i n a t i o n s o f h o s t a n d p a r a s i t e g e n o t y p e s t o I n v e s t i g a t e . A s , s h o w n b y h i s d a t a , a n y c o m b i n a t i o n w h i o h g a v e l e s s t h a n 1.0 p e r c e n t s m u t ( h e u s e d a h e a d p e r r o w b a s i s ) w a s n o t i n c l u d e d i n t h i s s t u d y . T h u s , m a n y o f t h e n e g a t i v e a n d l o w r e a d i n g s i n t h e t a b l e f o r t h e 13 r a c e s a n d e i g h t o f t h e b a r l e y c u l t i v a r s ( h i s w o r k d i d n o t i n c l u d e , ' C o n q u e s t , ' ' G a t e w a y J * K e y s t o n e ? o r ' V a n t a g e * ) a r e t h e f i n d i n g s o f T a p k e . A l s o , w h i l e t h e p r a c t i c a l l o w e r l i m i t i n t h e p r e s e n t w o r k w a s t a k e n a s 5 P e r c e n t p l a n t s s m u t t e d , I n t h r e e c a s e s w h e r e t h e r e w a s l e s s t h a n . 5 p e r c e n t p l a n t s s m u t t e d , s u f f i -c i e n t d a t a w e r e n o n e t h e l e s s o b t a i n e d s o t h a t t h e y c o u l d b e i n c l u d e d . I n f o r m a t i o n o n e x p e c t e d s m u t t i n g f o r s o m e o f t h e o t h e r c o m b i n a t i o n s w a s o b t a i n e d t h r o u g h p e r s o n a l c o m m u n i c a -t i o n w i t h E b b a . I f n o I n f o r m a t i o n w a s a v a i l a b l e a b o u t a 51 c o m b i n a t i o n , 5-8 r o w s ( a n d m o r e l a t e r I f n e c e s s a r y ) w e r e p l a n t e d . I n t h e t a b l e , t h e r e a r e t h r e e p o i n t s w h i c h a r e n o t p a r t o f t h e m a t r i x . W h i l e t h e y r e p r e s e n t c r o s s e s o f d i f f e r e n t r a c e s o r c o l l e c t i o n s , t h e y a r e n o t c r o s s e s o f t h e s a m e h a p l o l d p r o d u c t s a s s h o w n e l s e w h e r e i n t h e t a b l e . T h u s t h e i r p o s s i b l e g e n e t i c s i g n i f i c a n c e i s n o t d i s c u s s e d . T A B L E 2 . 2 C O M B I N A T I O N S O P H O S T C U L T I V A R A N D P A T H O G E N D I K A R Y O N , A N D D I S E A S E R E A C T I O N S O B S E R V E D G a t e w a y B a r l e y U s t i l a g o h o r d e i d l k a r y o n 3 C u l t l v a r H I R 2 B 3 R 4 R 5 R o R 7 C o n q u e s t - - 2 . 0 ° „ 1 9 . 0 E x c e l s i o r - L 50 .6 L - -8 7 . 4 H a n n c h e n 5 3 . 2 L — 2 0 . 7 - L -8 3 . 3 6 6 . 3 H i m a l a y a - - 4 5 . 8 L 8 9 . 8 K e y s t o n e L - 3 . 9 b - - L ;..... '.1 2 2 . 1 L i o n - 2 7 . 0 34.6 - 1 2 . 6 1 8 . 9 b -. t . 7 1 . 2 8 0 . 8 6 7 . 5 5 3 . 7 N e p a l - 5 5 . 5 5 7 . 5 5 6 . 2 - - -9 5 . 5 9 6 . 7 9 1 . 9 O d e s s a 3 1 . 5 b 4 7 . 1 5 9 . 5 4 2 . 3 3 7 . 2 2 2 . 9 b 2 5 . 0 b 64 . 4 7 2 . 6 9 4 . 6 6 9 . 0 7 5 . 2 7 0 . 4 5 2 . 8 P a n n i e r - L 3 9 . 9 - - L 7 9 . 0 T r e b i 7.6 L L 6 . 8 1 2 . 1 L 30 .6 3 1 . 0 4 4 . 7 V a n t a g e - - - - - -5 2 TABLE 2 . 2 ( c o n t i n u e d ) B a r l e y U s t l l e g o h o r d e i d i k a r y o n C u l t l v a r w R 9 R10 R l l HI 2 R13 E t l C o n q u e s t - L 6 . 8 b 1 8 . 1 L L L 2 7 . 6 71.** E x c e l s i o r - 2 6 . 6 7 0 . 8 - - L 4 6 . 8 8 3 . 6 -G a t e w a y - - - - - L -H a n n c h e n • - 2 8 . 2 7 8 . 3 4 8 . 4 8 2 . 5 5 8 . 6 8 1 . 0 3 2 . 3 7 1 . 0 3 5 . 2 6 9 . 9 3 7 . 6 8 5 . 9 H i m a l a y a 2 7 . 0 8 6 . 1 - 3 2 . 3 7 2 . 3 9 . 0 6 4 . 4 K e y s t o n e 6 . 0 4 2 . 1 5 . 3 b 2 4 . 2 - - 1 0 . 3 2 4 . 8 5 3 . 0 7 0 . 5 L i o n - 4 . 6 6 5 . 3 2 4 . 1 7 3 . 1 2 6 . 7 7 1 . ^ - 1 8 . 1 7 6 . 0 5 . 9 7 0 . 0 N e p a l L 3 0 . 7 5 6 . 0 4 9 . 3 8 3 . 2 6 1 . 5 93.*» 5^.1" a s . 4 4 0 . 7 8 4 . 1 O d e s s a 2 3 . 6 b 53.** 5 9 . 1 9 6 . 6 5 3 . 5 7 1 . 9 5*U8 7 7 . 9 4 4 . 6 8 6 . 6 5 5 . 8 8 0 . 7 I 7 . ^ b 3 5 . 7 P a n n i e r - L 2 9 . 4 9 5 . 7 - L — — T r e b i - - 7 . 6 4 0 . 2 4 4 . 3 6 9 . 5 L 6 . 8 b 1 3 . 8 2 . 8 3 2 . 2 V a n t a g e - 2 4 . 0 6 0 . 0 4 6 . 1 6 0 . 5 5 8 . 7 6 6 . 8 - 4 2 . 1 8 1 . 0 4 4 . 6 77.** 53 T A B L E 2 . 2 ( c o n t i n u e d ) B a r l e y U s t i l a . a r o h o r d e i d i k a r ? •on A d d i -C u l t l v a r E t 2 E t 3 Uh6 U h l 2 v l v 2 v 3 E X I t i o n a l C o n q u e s t - - - - 2 3 . 0 1 2 . 6 b 55.0 33.9 -E x c e l s i o r — — 56 .3 85.6 mm ( V 3 X U h - 6 ) 1 3 . 4 b 7 6 . 4 G a t e w a y - - - - L L H a n n c h e n 7.9 72.1 - - 67. l b 8 1 . 4 3 2 . 0 1 4 . 7 b 85 .7 4 6 . 0 2 4 . 0 b 4 6 . 2 H i m a l a y a - -• - 2 4 . 0 1 8 * 6 b 91.0 76.8 -K e y s t o n e L i o n 2 . 7 b 28.6 L 7 .4 55.1 2 0 . 6 83.2 19.8 7 5 . 2 1 0 . 3 8 . 5 5 3 .5 52.1 3 6 . 0 1 1 . l b 7 6 . 2 46 .6 : 8 . 0 b 4 2 . 4 ( U h - 6 x U h - 1 2 ) 6 1 . 7 b 67 .7 N e p a l L 26.8 53.9 4 7 . 3 95.2 - • -O d e s s a 30.3 8 1 . 9 19.7 b 4 8 . 2 4 0 . 3 8 5 . 7 62.2 9 5 . 6 4 5 . 5 1 6 . 0 b 8 6 . 9 5 3 . 4 3 2 . 8 b 4 8 . 1 P a n n i e r T r e b l 5.9 55.6 -3 0 . 1 77.5 1 1 . 2 5 3 . 9 23.2 55.6 2 9 . 7 -85.3 3 6 . 5 1 6 . 5 b 6 1 . 6 4 6 . 9 1 0 . 8 b 5 0 . 2 ( R 5 x R l l ) 4 8 . l b 8 5 . 1 V a n t a g e 14 .8 53.0 - - 31.5 b 6 6 . 1 27 .8 -72 .6 I 6 . 6 b 4 2 . 8 a N u m e r l c a l v a l u e s i n d i c a t e s u f f i c i e n t d a t a f o r a n a l y s i s . U p p e r v a l u e i s p e r c e n t o f p l a n t s s m u t t e d ; l o w e r i s p e r c e n t o f s m u t t e d c u l m s o n d i s e a s e d p l a n t s . - = n o s m u t o b s e r v e d . L = l e s s t h a n 5% p l a n t s s m u t t e d ; i n s u f f i c i e n t d a t a . b H i g h t i l l e r i n g p l a n t s 5* B e f o r e t h e c o r r e l a t i o n a n a l y s i s c o u l d b e p r o p e r l y d o n e , a c o m p l i c a t i o n i n v o l v i n g v a r i a t i o n I n p l a n t t i l l e r i n g h a d t o b e f i r s t d e a l t w i t h . A n I n v e r s e r e l a t i o n s h i p b e t w e e n t h e a v e r a g e n u m b e r o f c u l m s p e r p l a n t a n d t h e p e r c e n t o f s m u t t e d c u l m s o n s m u t t e d p l a n t s w a s d i s c o v e r e d . F o r e x a m p l e , N e p a l i n o c u l a t e d w i t h Uh6 w a s p l a n t e d b o t h a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a a n d i n C a l i f o r n i a . T h e l a t t e r p l a n t i n g w a s o n h i g h l y f e r t i l i z e d l a n d a n d o t h e r c o n d i t i o n s t h e r e w e r e a l s o c o n d u c i v e t o e x t r e m e l y h i g h t i l l e r i n g . A t B r i t i s h C o l u m b i a , w h e r e s m u t t e d p l a n t s p r o d u c e d a n a v e r a g e o f 4 . 2 6 c u l m s , 9 5 . 2 p e r c e n t o f t h e c u l m s w e r e s m u t t e d . I n C a l i f o r n i a , w h e r e s m u t t e d p l a n t s p r o d u c e d a n a v e r a g e o f 1 2 . 8 c u l m s , a s i g n i f i c a n t l y l o w e r 7 1 . 1 p e r c e n t o f t h e c u l m s w e r e s m u t t e d . T h e r e w a s n o s i g n i f i c a n t d i f f e r e n c e I n p e r c e n t o f p l a n t s s m u t t e d a t t h e t w o l o c a t i o n s . A s i m i l a r f i n d w a s m a d e w h e n L i o n a n d Uh6 w e r e p l a n t e d a t b o t h l o c a t i o n s ; o n l y h e r e t h e p e r c e n t a g e o f p l a n t s s m u t t e d w a s a c t u a l l y s i g n i f i c a n t l y h i g h e r I n C a l i f o r n i a . W h e t h e r t h e d a t a f o r a c o m b i n a t i o n w e r e o b t a i n e d a t B r i t i s h C o l u m b i a o r i n C a l i f o r n i a w a s l a r g e l y r a n d o m . Y e t t h e m e a n p e r c e n t o f s m u t t e d c u l m s o n d i s e a s e d p l a n t s f o r t h e 29 c o m b i n a t i o n s u n d e r h i g h - t l l l e r l n g c o n -d i t i o n s w a s 4 4 . 3 , w h i l e t h a t o f t h e 8 2 l o w - t l l l e r l n g c o m -b i n a t i o n s w a s 5 9 * ^ p e r c e n t . A n a l y s i s o f a l l 111 p o i n t s r e -v e a l e d a s i g n i f i c a n t n e g a t i v e r e g r e s s i o n o f f r e q u e n c y o f c u l m s o n d i s e a s e d p l a n t s o n a v e r a g e t i l l e r p r o d u c t i o n . W h e n 55 l o w - a n d h i g h - t l l l e r l n g p l a n t i n g s w e r e a n a l y z e d s e p a r a t e l y , h o w e v e r , t h e r e w a s n o s i g n i f i c a n t r e g r e s s i o n i n e i t h e r g r o u p i n g , ( a n a v e r a g e o f n i n e o r m o r e c u l m s p e r p l a n t b e i n g c o n s i d e r e d h i g h - t l l l e r i n g ) . D i v i s i o n o f d a t a i n t o t w o s e p a r a t e a n a l y s e s r e d u c e d t h e v a r i a t i o n o f a v e r a g e c u l m s p e r p l a n t w i t h e a c h d a t a s e t , t h u s m i n i m i z i n g t h e e f f e c t o f t h i s v a r i a b l e o n w i t h i n - p l a n t d i s e a s e s e v e r i t y . T h i s w a s f e l t t o b e t h e s i m p l e s t ' a n d m o s t e f f e c t i v e w a y t o a v o i d t h i s c o m p l i c a t i o n . F o r t h e l a r g e n u m b e r o f l o w - t i l l e r i n g p l a n -t i n g s , t h e v a r i a t i o n i n a v e r a g e n u m b e r o f c u l m s w a s l o w b e c a u s e o n l y p l a n t s w i t h t h r e e o r m o r e c u l m s w e r e c o n s i d e r e d . T h e m e a n w a s o f t e n n o t m u c h h i g h e r t h a n t h r e e c u l m s p e r p l a n t . T h e v a r i a t i o n I n a v e r a g e a m o u n t o f t i l l e r i n g w a s g r e a t e r f o r t h e h i g h - t l l l e r i n g p l a n t i n g s , b u t a r e g r e s s i o n w a s , n o n e t h e -l e s s , n o t f o u n d . F i g u r e s 2 . 1 a n d 2 . 2 a r e t h e w i t h i n - a n d b e t w e e n - p l a n t d i s e a s e s e v e r i t y s c a t t e r d i a g r a m s f o r l o w - a n d h i g h - t l l l e r i n g p l a n t i n g s , r e s p e c t i v e l y . I n d e t e r m i n i n g t h e r v a l u e s , t h e a r c s i n © t r a n s f o r m a t i o n w a s u s e d ( S t e e l a n d T o r r i e , i 9 6 0 ) . I n b o t h a n a l y s e s , t h e v a r i a n c e s o f x a n d y w e r e v e r y c l o s e ( e x p r e s s e d a s p e r c e n t a g e s ' . ) . H e n c e s c a t t e r d i a g r a m s i n v o l v i n g u n i t s o f s t a n d a r d d e v i a t i o n w e r e t h o u g h t u n n e c e s s a r y . A s s h a l l b e b r o u g h t o u t i n t h e d i s c u s s i o n , a s t r o n g g e n e t i c c o m p o n e n t I n t h e v a r i a n c e o f w i t h i n - p l a n t d i s e a s e s e v e r i t y I s i n d i c a t e d . 56 FIGURE 2.1 3 U to 2 S z^gj P©re©nt of Plates S l u t t e d 615 FIGURE 2.1. CORRELATION SCATTER DIAGRAM FOR 82 BARLEY-USTUAGO HORDEI GENOTYPIC COMBINATIONS PLANTED UNDER LOW-TILLERING CONDITIONS 57 FIGURE 2.2 90 J2 ?0 s <3 s © o 5 0 3 O i E 10 r = o.?6? 20 W Percent of plants smutted 6 0 FIGURE 2.2. CORRELATION SCATTER DIAGRAM FOR 29 BARLEY-USTILAGO HORDEI GENOTYPIC COMBINATIONS PLANTED UNDER HIGH-TILLERING CONDITIONS 58 Separate w i t h i n - c u l t l v a r and w i t h i n - d i k a r y o n c o r r e l a -t i o n s were made on l o w - t l l l e r l n g p l a n t i n g s of f i v e c u l t i v a r s and three d i k a r y o n s . Upon examination, i t appeared t h a t the other combinations d i d not possess s u f f i c i e n t i n f o r m a t i o n ( p o i n t s ) or s u f f i c i e n t v a r i a t i o n of one or both v a r i a b l e s t o warrant an a n a l y s i s . The r e s u l t s are summarized i n Table 2 . 3 . TABLE 2 . 3 SOME WITHTN-CULTIVAR AND WITHIN-DIKARYON CORRELATION ANALYSES INVOLVING LOW-TILLERING PLANTINGS ONLY Common C u l t l v a r .. or Dlkaryon Number of P o i n t s Value of r Hannchen 1 0 0 . 5 8 0 L i o n 1 2 0 . 6 6 9 * Nepal 1 0 0 * 9 1 1 * Odessa 1 3 0 . 4 5 0 T r e b l 1 0 0 . 8 1 5 * Race 9 7 0 . 7 5 8 * Race 1 3 8 0 . 7 6 2 * v l v 2 9 0 . 5 0 0 * S i g n l f l e a n t c o r r e l a t i o n a t the 5% l e v e l . 59 F i v e o f t h e e i g h t c o r r e l a t i o n s w e r e s i g n i f i c a n t . O f t h e t h r e e w h i c h w e r e n o t , v l v 2 a n d O d e s s a c a n b e e x p l a i n e d by-s m a l l s a m p l e s i z e a n d l a c k o r v a r i a n c e , r e s p e c t i v e l y . I t i s l i k e l y t h a t , h a d s u f f i c i e n t i n f o r m a t i o n b e e n a v a i l a b l e , t h e c o r r e l a t i o n w o u l d h a v e b e e n f o u n d i n a l l w i t h i n - c u l t l v a r a n d w i t h i n - d i k a r y o n g r o u p i n g s . I n c o n c l u s i o n , t h e r e i s a s t r o n g c o r r e l a t i o n b e t w e e n d i s e a s e s e v e r i t y w i t h i n a b a r l e y p l a n t a n d a m o n g b a r l e y p l a n t s , a s m e a s u r e d b y a m o u n t o f s m u t t i n g . D . D I S C U S S I O N I n c o m p a r i n g t h e p r e s e n t d a t a f o r t h e 1 3 o r i g i n a l r a c e s I n t e r a c t i n g w i t h t h e e i g h t c u l t i v a r s s t u d i e d b y T a p k e ( 1 9 ^ 5 ) w i t h h i s r e s u l t s , a f a i r l y g o o d q u a l i t a t i v e a g r e e m e n t w a s o b t a i n e d . B e c a u s e t h e p r e s e n t r e s u l t s w e r e r e c o r d e d o n a p l a n t b a s i s w h i l e t h o s e o f T a p k e w e r e o n a h e a d b a s i s , t h e p r e s e n t p e r c e n t a g e s w e r e n e a r l y a l w a y s m u c h h i g h e r . T h e r e w e r e s o m e e x c e p t i o n s , h o w e v e r . A l l o f t h e c o m b i n a t i o n s I n -v o l v i n g T r e b i g a v e p e r c e n t a g e s l o w e r t h a n t h o s e o b t a i n e d b y T a p k e . T h i s w o u l d l e a d o n e t o s u s p e c t t h a t t h e t w o s e e d l o t s w e r e d i f f e r e n t . A s w e l l , t h i s s t u d y r e v e a l e d t h a t s o m e o f t h e r a c e s a l s o h a d c h a n g e d . A l l d e t e c t e d c h a n g e s w e r e i n t e r m s o f r e d u c t i o n s o r l o s s e s I n v i r u l e n c e . G a i n s i n v i r u l e n c e , I f t h e y o c c u r r e d , w e n t u n d e t e c t e d s i m p l y b e c a u s e c o m b i n a t i o n s w h i c h T a p k e f o u n d g a v e l i t t l e e r n o s m u t 60 w e r e n o t i n c l u d e d i n t h i s s t u d y . L o s s e s o r r e d u c t i o n s i n v i r u l e n c e o c c u r r e d i n s e v e n d i f f e r e n t c o m b i n a t i o n s i n v o l v i n g e i t h e r r a c e 6 ( t w o c u l t i v a r s ) , r a c e 7 ( t h r e e c u l t i v a r s ) , o r r a c e 1 1 ( t w o c u l t i v a r s ) . A l l o t h e r r a c e s w e r e , q u a l i t a t i v e l y a t l e a s t , u n c h a n g e d . B e c a u s e o f p o o r s m u t t i n g i n t h e f i r s t t e s t , c o m b i n a t i o n s i n v o l v i n g r a c e s 6 a n d 7 w e r e g e n e r a l l y t e s t e d a s e c o n d t i m e , u s i n g p r o d u c t s f r o m d i f f e r e n t t e l l o -s p o r e s i n e a c h t e s t . S o m e q u a n t i t a t i v e d i f f e r e n c e s i n v i r u -l e n c e e x i s t e d b e t w e e n t h e t w o s e t s o f t e l i o s p o r e p r o d u c t s u s e d i n t h e t w o t e s t s ; t h e s e c o u l d h a v e b e e n d u e t o e n v i r o n m e n t . G r o s s r e d u c t i o n s o r l o s s e s o f v i r u l e n c e w e r e c o n s i s t e n t l y f o u n d i n b o t h d i k a r y o n s o f t h e t w o r a c e s . B a s e d o n t h i s a d m i t t e d l y s m a l l s a m p l i n g , i t m a y b e s u g g e s t e d t h a t t h e r a c e s a m p l e s a r e q u a l i t a t i v e l y u n i f o r m . O n e c a n a l s o c o n c l u d e t h a t , i n v i e w o f t h e n o t o r i o u s h e t e r o g e n e i t y e n d i n s t a b i l i t y o f s m u t r a c e s ( C h e r e w l c k , 1 9 5 8 ) a n d t h e d i f f i c u l t y o f m a i n t a i n i n g s u c h r a c e s o v e r a l o n g p e r i o d o f t i m e , t h e r a c e s a m p l e s o b t a i n e d f r o m N o r t h D a k o t a w e r e r e m a r k a b l y s i m i l a r t o t h o s e i d e n t i f i e d a n d c o l l e c t e d b y T a p k e i n t h e e a r l y 1 9 4 0 s . D e t a i l e d c o m p a r i s o n s o f q u a n t i t a t i v e r e s u l t s w e r e n o t m a d e , p r i m a r i l y b e c a u s e o f t h e d i f f e r e n t m e a s u r e s o f s m u t t i n g u s e d I n t h e t w o s t u d i e s . E v e n u n d e r c a s u a l e x a m i n a t i o n , t h e e x p e c t e d " c o r r e l a t i o n " b e t w e e n p e r c e n t o f p l a n t s s m u t t e d , a s m e a s u r e d I n t h e p r e s e n t s t u d y , a n d t h e p e r c e n t o f a l l h e a d s s m u t t e d , a s m e a s u r e d b y T a p k e , w a s q u i t e e v i d e n t , h o w e v e r . 6 1 T h e I n v e r s e r e l a t i o n s h i p b e t w e e n w i t h i n - p l a n t d i s e a s e s e v e r i t y a n d t h e a v e r a g e n u m b e r o f c u l m s p r o d u c e d b y t h e p l a n t s h a s b e e n i n d i r e c t l y a n d d i r e c t l y m e n t i o n e d b y o t h e r s . W o o d w a r d a n d T i n g e y ( 1 9 4 1 ) o b s e r v e d t h a t h i g h e r l e v e l s o f s m u t t i n g w e r e o b t a i n e d w i t h b a r l e y - U , h o r d e i o n l e s s f e r t i l e s o i l t h a n w e r e o b t a i n e d o n m o r e f e r t i l e s o i l . S i n c e t h i s m e a s u r e m e n t o f t h e d i s e a s e w a s b a s e d o n s m u t t e d h e a d s , i t c a n b e a s s u m e d t h a t t h e y w e r e p r o b a b l y o b s e r v i n g t h e i n c r e a s e d w i t h i n - p l a n t d i s e a s e s e v e r i t y w h i c h a c c o m p a n i e d t h e d e c r e a s e i n a v e r a g e t i l l e r p r o d u c t i o n o n p o o r e r s o i l , M i l a n ( 1 9 3 9 ) o b s e r v e d t h a t t h e r a t e o f s o w i n g , w h i c h a l s o c a n b e e x p e c t e d t o i n f l u e n c e t h e t i l l e r i n g o f p l a n t s , d i d n o t a f f e c t t h e p e r c e n t o f i n o c u l a t e d w h e a t p l a n t s s m u t t e d b y - U . t r l t l c l b u t d i d d i r e c t l y a f f e c t t h e p e r c e n t a g e o f c u l m s s m u t t e d , A p o s s i b l e e x p l a n a t i o n b y B a t t s a n d J e a t e r ( 1 9 5 8 ) i s t h a t e a c h i n f e c t e d p l a n t c o n t a i n s a l i m i t e d a m o u n t o f m y c e l i u m . T h u s , i f m a n y t i l l e r s a r e p r o d u c e d b y t h e p l a n t , a s m a l l e r p r o p o r -t i o n o f t h e m w i l l b e c o m e s m u t t e d . T h e y s u g g e s t e d a l s o t h a t t h e a m o u n t o f m y c e l i u m v a r i e s f r o m e m b r y o t o e m b r y o , w i t h t h e r e s u l t t h a t s o m e p l a n t s w i l l s h o w a h i g h e r f r e q u e n c y o f s m u t t e d h e a d s t h a n o t h e r s , e v e n i f o t h e r c o n d i t i o n s a r e e q u a l . U n f o r t u n a t e l y , t h e h i s t o l o g i c a l w o r k p r e s e n t e d b y B a t t s a n d J e a t e r d o e s n o t r e a l l y s u p p o r t t h e i r h y p o t h e s i s c o n c e r n i n g b o t h l i m i t e d a n d v a r i a b l e a m o u n t s o f m y c e l i u m I n e m b r y o s . N o n e t h e l e s s , t h e i r h y p o t h e s i s p r o v i d e s a s i m p l e 6 2 and p l a u s i b l e e x p l a n a t i o n f o r both Milan's and the present data ( d i f f e r e n c e s In mode of i n f e c t i o n of the two smuts n o t w i t h s t a n d i n g ) . I f Batt s had been able to study r e s i s -tance i n U. t r l t l c l t as was h i s i n t e n t i o n (Batts and J e a t e r , 1 9 5 8 ) he would undoubtedly have provided much a d d i t i o n a l evidence e i t h e r f o r or ag a i n s t h i s hypothesis. Regression a n a l y s i s f o r the e f f e c t of average t i l l e r p r o d u c t i o n on w i t h i n - p l a n t disease s e v e r i t y was used i n t h i s work only as a means to determine whether the data would e f f e c t i v e l y e l i m i n a t e v a r i a t i o n i n average t i l l e r i n g as a c o m p l i c a t i n g f a c t o r i n the between- and w i t h i n - p l a n t c o r r e l a -t i o n a n a l y s i s . Some problems which d e t r a c t from the r e -g r e s s i o n a n a l y s i s w i l l be di s c u s s e d b r i e f l y . For a r e -g r e s s i o n a n a l y s i s , one of the important i n i t i a l assumptions i s t h a t the independent v a r i a b l e i s normally d i s t r i b u t e d ( S t e e l and T o r r l e , i 9 6 0 ) . The d i s t r i b u t i o n of average t i l l e r numbers was de c i d e d l y bimodal In t h i s r e g r e s s i o n due to the two very d i f f e r e n t p l a n t i n g environments. The p o s s i -b i l i t y can be suggested t h a t some other environmental f a c t o r could be c o n t r i b u t i n g to the r e g r e s s i o n . However, from the e a r l i e r work d i s c u s s e d above, which supports the inverse cause and e f f e c t r e l a t i o n s h i p between the two v a r i a b l e s , In s i t u a t i o n s where gross environmental d i f f e r e n c e s are not prese n t , i t Is considered l i k e l y t h a t the a n a l y s i s i s v a l i d as presented. The second problem i s tha t s i n c e at l e a s t one 6 3 c u l m o f e a c h s m u t t e d p l a n t h a s t o b e s m u t t e d , o n e c u l m I s n o t f r e e t o v a r y . B y I n c l u d i n g t h e s e c u l m s i n t h e n u m e r a t o r s a n d d e n o m i n a t o r s , o n e o b t a i n s a s l i g h t l y h i g h e r w i t h i n - p l a n t d i s e a s e p e r c e n t a g e t h a n w o u l d b e o b t a i n e d i f t h e y w e r e e x -c l u d e d . A s t h e d a t a e x i s t , e x c l u s i o n w o u l d t e n d t o d e c r e a s e t h e s l o p e o f t h e r e g r e s s i o n , b u t t h e d e c r e a s e w o u l d b e s l i g h t . T h e s i g n i f i c a n c e o f t h e r e g r e s s i o n i s s u f f i c i e n t l y l a r g e ( p r o b a b i l i t y o f t h e s l o p e b e i n g 0 = l e s s t h a n 0 . 0 0 0 0 1 ) , h o w e v e r , t h a t t h e c h a n g e w o u l d b e n e g l i g i b l e . H e n c e , t h e s i m p l e a p p r o a c h o f u s i n g u n a l t e r e d p e r c e n t a g e s i s j u s t a s r e l i a b l e a s t h e m o r e c o m p l e x a p p r o a c h w o u l d b e . T h i s w a s a l s o t h e c a s e i n t h e c o r r e l a t i o n a n a l y s i s . A l t h o u g h p r e v i o u s w o r k e r s , m e n t i o n e d i n t h e i n t r o d u c -t i o n , m o s t l y c h o s e m e a n s o f m e a s u r i n g w i t h i n - p l a n t d i s e a s e s e v e r i t y w h i c h d i f f e r e d f r o m t h o s e u s e d i n t h i s s t u d y , a n d a l t h o u g h m o s t o f t h e e a r l i e r s t u d i e s w e r e r e l a t i v e l y s m a l l , t h e c o n c l u s i o n s o f t h i s a n d t h e e a r l i e r s t u d i e s a r e i n g e n e r a l a g r e e m e n t . B u t t l e ( 1 9 3 * 0 m e n t i o n e d o n e b a r l e y - U . h o r d e i c o m b i n a t i o n i n w h i c h 30 p e r c e n t o f p l a n t s w e r e s m u t t e d b u t i n w h i c h o n l y 1 7 p e r c e n t o f a l l h e a d s w e r e s m u t t e d . A t t h i s i n t e r m e d i a t e p e r c e n t a g e o f s m u t t i n g ( o f p l a n t s ) s u c h a r e s u l t w o u l d b e I n a c c o r d a n c e w i t h t h e c o r r e l a t i o n d e r i v e d I n t h i s s t u d y . O n d i s e a s e d p l a n t s a p o r t i o n o f t h e c u l m s w o u l d b e h e a l t h y . I n d e e d , t h i s w a s f o u n d b y B u t t l e . A l s o c o n t r i b u t i n g t o t h e d i s p a r i t y b e t w e e n t h e t w o m e a s u r e s w a s 6 4 t h e f a c t t h a t s m u t t e d p l a n t s p r o d u c e d f e w e r t i l l e r s . O o r t ( 1 9 3 9 ) p r e s e n t e d s o m e e v i d e n c e t h a t f o r t h e l o o s e s m u t s o f b a r l e y a n d w h e a t p a r t i a l s m u t t i n g I s p e r h a p s l e s s f r e q u e n t t h a n f o r c o v e r e d s m u t o f b a r l e y . I n O o r t ' s s t u d y t h e i n o c u -l a t e d a n d d i s e a s e d w h e a t a n d b a r l e y p l a n t s w e r e n e a r l y a l w a y s t o t a l l y s m u t t e d . O n l y 3 - 9 p e r c e n t o f d i s e a s e d w h e a t p l a n t s a n d 4 . 0 p e r c e n t o f d i s e a s e d b a r l e y p l a n t s h a d h e a l t h y c u l m s , a n d t h e s e w e r e n e a r l y a l w a y s i n t h e m i n o r i t y . E v e n t h o u g h t h e p e r c e n t a g e o f s m u t t e d p l a n t s w a s h i g h ( o v e r 70 p e r c e n t ) t h i s w o u l d , i n c o m p a r i s o n w i t h h i g h d i s e a s e c o m b i n a t i o n s i n t h e p r e s e n t s t u d y , h a v e t o b e c o n s i d e r e d a s m a l l d e g r e e o f p a r t i a l s m u t t i n g , C u l t i v a r s > l i k e - O d e s s a , w h i c h p o s s e s s e d ? l i t t l e b e t w e e n - -o r w i t h l n - p l a n t r e s i s t a n c e t o a n y o f t h e d i k a r y o n s , a n d K e y s t o n e , w h i c h i n g e n e r a l p o s s e s s e d a h i g h l e v e l o f b o t h t y p e s o f r e s i s t a n c e , u n d o u b t e d l y c o n t r i b u t e d g r e a t l y t o t h e c o r r e l a t i o n . T h e w i t h i n - c u l t l v a r a n a l y s e s , h o w e v e r , r e v e a l e d t h a t t h e c o r r e l a t i o n i s n o t l i m i t e d t o b e t w e e n - c u l t l v a r c o m -p a r i s o n s . W h i l e c u l t i v a r s w h i c h p o s s e s s a g e n e r a l l y h i g h o r l o w l e v e l o f t h e t w o t y p e s o f r e s i s t a n c e c a n e a s i l y b e p i c k e d o u t , d i k a r y o n s d o n o t f o r m s u c h d i s t i n c t g r o u p s . A d l k a r y o n w a s n o t f o u n d , f o r i n s t a n c e , w h i c h p o s s e s s e d a r e l a t i v e l y h i g h b e t w e e n - a n d w l t h i n - p l a n t d i s e a s e - p r o d u c i n g a b i l i t y o n a l l o r m o s t c u l t i v a r s . T h i s l e a d s t o t h e c o n c l u s i o n t h a t , I n i t i a l l y a t l e a s t , s t u d i e s o n t h e i n h e r i t a n c e o f h o s t r e -s i s t a n c e w o u l d b e m o r e p r o d u c t i v e . 65 T h e v a r i a b i l i t y s h o w n f o r 1 1 1 c o m b i n a t i o n s i n w i t h i n -p l a n t d i s e a s e r e a c t i o n i s , t o a l a r g e e x t e n t , g e n e t i c a l l y d e t e r m i n e d . S a m p l i n g e r r o r i s o f t h e m a g n i t u d e o f - 5 p e r c e n t . E n v i r o n m e n t a l e f f e c t s c a n n o t b e i g n o r e d , b u t t h e y a r e a l m o s t c e r t a i n l y o f l e s s I m p o r t a n c e t h a n g e n e t i c d e t e r m i n a n t s . E a r l i e r s t u d i e s h a v e e s t a b l i s h e d t h a t b e t w e e n - p l a n t d i s e a s e r e a c t i o n s a r e l a r g e l y g e n e t i c a l l y d e t e r m i n e d , s o t h a t t h e c l o s e c o r r e l a t i o n o f b e t w e e n - a n d w l t h l n - p l a n t r e a c t i o n s c a n I t s e l f b e t a k e n a s e v i d e n c e f o r t h e g e n e t i c d e t e r m i n a t i o n o f w i t h i n - p l a n t d i s e a s e r e a c t i o n . T h e e x p e r i m e n t a l r e s u l t s a l s o a t t e s t t o t h e m i n o r r o l e p l a y e d b y e n v i r o n m e n t . O d e s s a , w h i c h a t B r i t i s h C o l u m b i a w a s c o n s i s t e n t l y h e a v i l y s m u t t e d , w a s p l a n t e d i n b l o c k s w h i c h w e r e m o r e o r l e s s r a n d o m l y l o c a t e d o v e r a t w o y e a r p e r i o d . S o m e b l o c k s w e r e p l a n t e d i n v e r y p o o r s o i l ; y e t s m u t t i n g w a s a l w a y s h i g h o n b o t h a w i t h i n - a n d b e t w e e n - p l a n t b a s i s . B l o c k s o f v a r i o u s c u l t i v a r s a n d d i k a r y o n s w e r e f r e q u e n t l y s p l i t i n t o t w o o r m o r e s u b - b l o c k s . S o m e t i m e s t h e s e w e r e p l a n t e d I n t w o d i f f e r e n t y e a r s . Y e t , a t U B C , e n v i r o n m e n t a l e f f e c t s o n d i s e a s e w e r e a l w a y s m i n i m a l ( u p t o a b o u t *5 p e r c e n t ) w h e n s u b - b l o c k r e s u l t s w e r e c o m p a r e d . B e c a u s e t h e c o r r e l a t i o n w a s r a t h e r c l o s e , I t I s n o t l i k e l y t h a t c o n t r a s t i n g c o m b i n a t i o n s f o r g e n e t i c s t u d i e s w i l l b e f o u n d e a c h o f w h i c h p o s s e s s e s o n l y o n e o f t h e h i g h d i s e a s e r e a c t i o n t y p e s . I n p a r t i c u l a r , t h e r e s e e m s t o b e a l a c k o f c o m b i n a t i o n s w h i c h g i v e h i g h b e t w e e n - p l a n t r e a c t i o n s a n d l o w 6 6 w i t h i n - p l a n t r e a c t i o n s . T w o p o s s i b i l i t i e s a r e l e f t f o r f u r t h e r s t u d y : v a r i e t i e s w i t h h i g h - w i t h l n a n d ' h l g h - b e t w e e n r e a c t i o n s c a n b e c r o s s e d w i t h t h o s e w i t h l o w - w i t h i n a n d l o w -b e t w e e n r e a c t i o n s ; o r c r o s s e s i n v o l v i n g v a r i e t i e s w h i c h d i f f e r o n l y i n t h e i r w i t h i n - p l a n t r e a c t i o n s c a n b e s t u d i e d . F o r e x a m p l e , t o s t u d y t h e i n h e r i t a n c e o f r e s i s t a n c e , T r e b l c o u l d b e c r o s s e d w i t h N e p a l a n d t h e p r o g e n y t e s t e d w i t h e i t h e r R 4 o r E t l , t o w h i c h g e n o t y p e s t h e p a r e n t c u l t i v a r s g i v e c o n t r a s t i n g r e a c t i o n s f o r b o t h r e s i s t a n c e t y p e s . G o o d e x a m p l e s o f p o t e n t i a l p a r e n t c u l t i v a r s w h i c h s e e m t o d i f f e r I n t h e i r w i t h i n - p l a n t r e a c t i o n s o n l y a r e K e y s t o n e a n d L i o n w i t h R 9 , a n d T r e b l a n d L i o n w i t h R 6 . T o s t u d y t h e i n h e r i t a n c e o f v i r u l e n c e I n t h e p a t h o g e n , R 4 c o u l d b e c r o s s e d w i t h R l l a n d t h e p r o g e n y t e s t e d o n T r e b l , t o w h i c h t h e p a r e n t s g i v e c o n t r a s t i n g b e t w e e n - p l a n t a n d w l t h i n - p l a n t d i s e a s e r e a c t i o n s . R l a n d E t 2 , w h i c h d i f f e r i n t h e i r w i t h i n - p l a n t v i r u l e n c e o n l y , c o u l d b e c r o s s e d a n d t h e p r o g e n y t e s t e d o n T r e b l . C o m b i n a t i o n s w h i c h d i f f e r o n l y i n t h e i r w i t h i n - p l a n t r e a c t i o n s a r e n o t e w o r t h y b e c a u s e t h e y m a y i n d i c a t e p o s s i b l e n o n - i d e n t i t y o f g e n e s g o v e r n i n g t h e t w o t y p e s o f r e s i s t a n c e . A l t e r n a t i v e l y , d i f f e r e n c e s i n e n v i r o n m e n t a l s e n s i t i v i t y o f c u l t i v a r s o r d i k a r y o n s c o u l d b e a n i m p o r t a n t , a l t h o u g h l e s s l i k e l y , e x p l a n a t i o n . B a c k g r o u n d p o l y g e n e s m i g h t a l s o b e I m p o r t a n t . A p r o p e r e x p e r i m e n t a l d e s i g n w o u l d d i s t i n g u i s h b e t w e e n t h e s e p o s s i b i l i t i e s I n c r o s s i n g s t u d i e s . 6 7 PART I I I THE PATTERN OF SMUTTING AND EFFECT ON PLANT GROWTH BY USTILAGO HORDEI IN 'HANNCHEN' BARLEY A . INTRODUCTION F a r l s ( 1 9 2 4 ) and Johnston ( 1 9 3 4 ) , In t h e i r s t u d i e s of the b a r l e y - U. hordei system, both reported t h a t , f o l l o w i n g I n o c u l a t i o n of b a r l e y w i t h U. h o r d e i , the f i r s t heads to appear i n a group of p l a n t s were u s u a l l y h e a l t h y , and t h a t diseased heads u s u a l l y appear at a l a t e r date. Johnston concluded t h a t diseased culms were slower t o mature than healthy r e g a r d l e s s of whether the l a t t e r were from healt h y or smutted p l a n t s . F a r l s r e p o r t e d t h a t i t was culms from healthy p l a n t s which one observed f i r s t . In n e i t h e r case was the e x p l a n a t i o n supported by o b s e r v a t i o n a l d a t a . An a l t e r n a t e , or at l e a s t c o n t r i b u t o r y , p o s s i b i l i t y i s t h a t o l d e r culms of smutted p l a n t s more f r e q u e n t l y remain healthy. In p a r t , t h i s work was undertaken to t e s t t h i s p o s s i b i l i t y — t h a t i s whether, w i t h i n a diseased b a r l e y p l a n t , the diseased and healthy t i l l e r s are produced randomly, or i n an age - r e l a t e d p a t t e r n . The work a l s o concerns the development of b a r l e y p l a n t s and how t h i s may be a f f e c t e d by U. h o r d e i . Since p a r t i a l r e s i s t a n c e probably i s , as p a r t s I and I I would i n d i c a t e , an Important c o n t r i b u t o r t o h o s t - p a r a s i t e accord, i t was f e l t t h a t s t u d i e s f u r t h e r i n g 68 our understanding of the d e t a i l s of such r e s i s t a n c e would be most worthwhile. Very l i t t l e i n f o r m a t i o n i s a v a i l a b l e concerning the exact l o c a t i o n of healthy and diseased culms i n p a r t i a l l y smutted or bunted c e r e a l s and p l a n t s . Churchward ( 1 9 3 7 - 3 8 ) found t h a t the r e s i s t a n t wheat c u l t l v a r Hope showed a low percentage of l a t e t i l l e r bunting when i n o c u l a t e d w i t h T. c a r l e s . U s u a l l y the f i f t h or s i x t h t i l l e r was diseased. Beed ( I 9 3 8 ) found t h a t the oat species Avena b r e v l s , when i n o c u l a t e d w i t h race 1 of U. k o l l e r l . showed smutted l a t e r a l t i l l e r s and healthy spikes of p r i n c i p a l culms i n nea r l y a l l of the 2 0 to 3 0 per cent of diseased p l a n t s . Neither of these workers paid a t t e n t i o n to exact t i l l e r r e l a t i o n s h i p s . At best they c a s u a l l y d i s t i n g u i s h e d the p r i n c i p a l culm from the sma l l e r l a t e r a l culms. No other work was found which i n any way f u r t h e r showed smutting p a t t e r n s i n c e r e a l s . The p a t t e r n of b a r l e y growth i s s i m i l a r to th a t of the more thoroughly s t u d i e d wheat. According to Hector ( 1 9 3 6 ) , the two are so s i m i l a r that the informat i o n on growth pa t t e r n s i n wheat a l s o applias to barley. A briaf account of the p a t t e r n of growth i n b a r l e y , given by - S a r v e l l a et a l . ( 1 9 6 2 ) , compares c l o s e l y w i t h the d e s c r i p -t i o n f o r wheat given by P e r c l v a l ( 1 9 2 1 ) . In g e n e r a l , the 6 9 p a t t e r n of t i l l e r i n g can be c a l l e d monopodial, w i t h a c e n t r a l unbranched culm (the p r i n c i p a l or a p i c a l culm) from s e v e r a l of whose lower nodes l a t e r a l culms (primary l a t e r a l s ) a r i s e . Each of the primary l a t e r a l culms i s i t s e l f capable of producing (secondary) l a t e r a l s . Except f o r the l i m i t e d number of nodes on each culm, the growth p a t t e r n i s , t h e o r e t i c a l l y , Indeterminate. P l a n t s can produce 1 0 0 culms i f the environment i s unusually f a v o r a b l e . In r e a l i t y , however, p l a n t s u s u a l l y do not p r o l i f e r a t e t o t h i s extent. A more d e t a i l e d d e s c r i p t i o n of growth patterns w i l l be presented i n the r e s u l t s s e c t i o n . B. MATERIALS AND METHODS: This work was c a r r i e d out e n t i r e l y i n the greenhouse. The b a r l e y c u l t l v a r *Hannchen' was chosen because of I t s h i g h t i l l e r i n g c a p a c i t y under greenhouse c o n d i t i o n s . The s i n g l e smut dl k a r y o n composed of haplolds E3a and Ik A was used throughout. Seed treatment and I n o c u l a t i o n were as d e s c r i b e d i n part I . Uninoculated seeds were t r e a t e d -e x a c t l y as those i n o c u l a t e d , except that s t e r i l e complete b r o t h ( f o l l o w e d by a d i s t i l l e d water r i n s e ) was s u b s t i t u t e d f o r inoculum. During the 3 to k months of growing time, pots and f l a t s were r o t a t e d and rearranged r e g u l a r l y . 7 0 When 2 to 3 culms had appeared on the p l a n t s , they were tagged In order to e s t a b l i s h a record of the t i l l e r r e l a t i o n s h i p s . They were again tagged at a l a t e r date, when more culms had appeared. Without such tagging, I t was n e a r l y Impossible to determine, w i t h c e r t a i n t y , the t i l l e r i n g p a t terns of mature p l a n t s . One by 1 . 5 Inch white marking tags w i t h s t r i n g s were used. To prevent tags from d e t e r i o r a t i n g , a f t e r the i n f o r m a t i o n on t i l l e r nodal o r i g i n and sequence of appearance had been recorded on them i n p e n c i l , they were dipped i n p a r a f f i n . I t was found t h a t even i f a tagged culm d i e d , i t s exact p o s i t i o n could s t i l l be determined. When a l l culms had headed, t h e i r p o s i t i o n , sequence and f a t e (headed, smutted or dead) were recorded. Informa-t i o n r e l a t i n g to s i n g l e p l a n t s was i n each case t r a n s f e r r e d to a schematic p l a n t diagram to a i d In subsequent a n a l y s i s . P o t s , which were found to be more s u i t a b l e , were used e x c l u s i v e l y i n the next three p l a n t i n g s . Three p l a n t s were grown i n each pot. One of the three p l a n t s i n each pot was uninoculated i n two of the p l a n t i n g s . These p l a n t s served as c o n t r o l s when observing p o s s i b l e e f f e c t s of l a t e n t I n f e c t i o n i n nonsmutted but i n o c u l a t e d p l a n t s . To o b t a i n p r o f u s e l y t i l l e r i n g p l a n t s , i n the l a s t p l a n t i n g , i n o c u l a t e d seeds were planted s i n g l y i n f o r t y pots and h i g h l y f e r t i l i z e d . 7 1 S t a t i s t i c a l t e c h n i q u e s u s e d t o a n a l y z e t h e s e d a t a I n c l u d e d t h e c o n f i d e n c e i n t e r v a l a b o u t t h e d i f f e r e n c e o f p r o p o r t i o n s , t h e s i m p l e t t e s t , t h e p a i r e d t t e s t a n d t h e C h l - s q u a r e t e s t . C . R E S U L T S I n t h e s e s t u d i e s , a t o t a l o f 528 b a r l e y p l a n t s w e r e l a b e l l e d a n d r e c o r d e d . N o n l n o c u l a t e d p l a n t s t o t a l l e d 8 3 . O f t h e 425 i n o c u l a t e d p l a n t s , 2 0 9 , o r 4-7.0 p e r c e n t b e c a m e s m u t t e d . T h e 209 s m u t t e d p l a n t s p r o d u c e d a t o t a l o f 1 ,198 c u l m s , f o r a m e a n o f 5 . 7 3 c u l m s p e r p l a n t . O f t h e s e , 9 2 7 , o r 7 7 . 4 p e r c e n t , w e r e s m u t t e d . O n e o f t h e p r i m a r y o b j e c t i v e s o f t h e s t u d y w a s t o d e t e r m i n e h o w t h e n o n s m u t t e d c u l m s w e r e d i s t r i b u t e d i n s m u t t e d p l a n t s . T o d o t h i s , a t h o r o u g h u n d e r s t a n d i n g w a s n e e d e d o f h o w a b a r l e y p l a n t g r o w s . F i g u r e 3 . 1 s h o w s a p h o t o g r a p h o f a r a t h e r t y p i c a l m o n t h - o l d b a r l e y p l a n t w i t h s i x y o u n g c u l m s . B e l o w i t t h e r e i s a l i n e d r a w i n g o f t h e p l a n t , n a m i n g t h e n o r m a l a n d m o d i f i e d l e a v e s w h i c h s u b t e n d t h e t i l l e r s . T h e t e r m i n o l o g y i s a l s o i n t r o d u c e d i n t h e d i a g r a m . A d i a g r a m m a t i c s k e t c h o f a h y p o t h e t i c a l p l a n t , I n w h i c h t h e i n t e r n o d e s h a v e b e e n g r e a t l y l e n g t h e n e d a n d a l l l e a v e s a r e e x c l u d e d , c a n b e s e e n I n t h e a p p e n d i x . T h e s e d i a g r a m s w e r e u s e f u l i n a n a l y z i n g t h e d a t a . I n r e a l i t y , t h e 7 2 r e g i o n of t i l l e r i n g , commonly c a l l e d the crown r e g i o n , i s g r e a t l y compressed, as the photograph would I n d i c a t e . Some q u a l i t a t i v e statements can be made about the ge n e r a l p a t t e r n of growth of 'Hannchen' b a r l e y under the experimental c o n d i t i o n s t h a t were employed. P l a n t s i n v a r i a b l y produced the p r i n c i p a l culm (IS) as the f i r s t and o l d e s t culm. I f , f o r some reason, I t d i e d very e a r l y , the next o l d e s t culm assumed I t s r o l e . I f a l a t e r a l culm was separated e a r l y from a p l a n t , I t a l s o acted as a p r i n c i p a l culm, t h a t Is to say, the l a t e r a l ' s lower nodes produced l a t e r a l culms to a much g r e a t e r extent than i t would have done had I t remained attached to the o r i g i n a l p l a n t . Only one primary l a t e r a l was produced from each node i n t h i s c u l t l v a r . With r a r e exception the l a t e r a l culm a r i s i n g i n the a x i l of a lower l e a f was o l d e r than one a r i s i n g from the a x i l of a higher l e a f , so t h a t , f o r example, the 2 L culm appeared before the JL culm In almost a l l p l a n t s . T h i s can be seen i n the photograph (Figure 3 * 1 ) » where the 1 L , 2 L and 3L l a t e r a l s are c l e a r l y i n order of descending age. In. l a b e l l i n g , the numerical s u b s c r i p t i n d i -cated the order of appearance of primary l a t e r a l culms (a primary l a t e r a l being d e f i n e d as the f i r s t l a t e r a l produced from a node of the p r i n c i p a l culm). The exceptions to t h i s order were o f t e n i n the form of l a t e primary l a t e r a l s from lower l e a v e s , u s u a l l y the CN or 1 L nodes. These n e a r l y 73 FIGURE 3.1 FIGURE 3 . 1 . ILLUSTRATION OF THE PATTERN OF EARLY T ILLER PROLIFERATION IN A ONE-MONTH OLD PLANT OF 'HANNCHEN• BARLEY FIGURE 3 . 1 coleoptile ( bud dormant in this plant) FIGURE 3 . 1 . CONTINUED 75 always d i e d before heeding. In only one case was an excep-t i o n a l order of appearance of primary l a t e r a l culms seen on a p a r t i a l l y smutted (and hence important) p l a n t . Because t h i s may not r e f l e c t r e l a t i v e t i l l e r bud age i n the very young s e e d l i n g , t h i s p l a n t was not included i n the analyses. P l a n t s produced l a t e r a l s from the c o l e o p t i l e node up to as hi g h as the e i g h t h v e g e t a t i v e l e a f node. In g e n e r a l , however, about 3 to 4 nodes were Involved. The nodes most commonly employed were 1L through 5L. Throughout the r e s t of t h i s t h e s i s a primary l a t e r a l together w i t h a l l of i t s l a t e r a l culms w i l l be r e f e r r e d t o as a t i l l e r f a m i l y . A t t e n t i o n was p a i d to the order of appearance of culms w i t h i n a t i l l e r f a m i l y , where the sequence d i d not vary from what would be expected (e.g. 2L f o l l o w e d by 2L-CN f o l l o w e d by 2L-1L). No attempt was made to compare r e l a t i v e ages of secondary l a t e r a l s a r i s i n g from d i f f e r e n t primary l a t e r a l s ( f o r instance 1L-CN w i t h 2L-CN). At a l l l e v e l s of t i l l e r p roduction there was o c c a s i o n a l absence of expected t i l l e r s (e.g. the 2L l a t e r a l d i d not appear). In many cases t h i s was probably due to I n j u r y t o the young bud. Q u a n t i t a t i v e data r e l a t i n g to l a t e r a l t i l l e r production w i l l be presented l a t e r f o r i n o c u l a t e d and noninoculated p l a n t s . 1. The D i s t r i b u t i o n of Smutted and Healthy Heads In Smutted P l a n t s Some simple s t a t i s t i c a l t e s t s were devised i n order to a l l o w d e f i n i t i v e statements to be made regarding the 7 6 r a n d o m n e s s o r n o n r a n d o m n e s s o f t h e l o c a t i o n o f h e a l t h y h e a d s i n s m u t t e d p l a n t s . T h e f i r s t o f t h e s e t e s t e d w h e t h e r o r n o t t h e d i s t r i b u t i o n o f s m u t t e d a n d n o n s m u t t e d h e a d s a m o n g t i l l e r f a m i l i e s w a s r a n d o m . T a b l e 3 . 1 p r e s e n t s t h e a n a l y s e s , s e p a r a t e l y , f o r t w o , t h r e e , a n d f o u r o r m o r e ( b u l k e d ) t i l l e r f a m i l i e s f r o m a l l s m u t t e d p l a n t s . T h e p r o c e d u r e w a s t o c l a s s i f y e a c h f a m i l y a c c o r d i n g t o w h e t h e r a l l c u l m s w e r e s m u t t e d , o r a l l w e r e h e a l t h y , o r w h e t h e r s o m e w e r e s m u t t e d a n d s o m e h e a l t h y ( m i x e d ) . F r o m t h e s e r e s u l t s t h e v a l u e s o f p , t h e p r o b a b i l i t y o f a c u l m i n t h a t f a m i l y g r o u p b e i n g s m u t t e d , a n d q , t h e p r o b a b i l i t y o f a c u l m r e m a i n i n g h e a l t h y , w e r e o b t a i n e d f o r e a c h f a m i l y s i z e . I f r a n d o m n e s s o f s m u t d i s t r i b u t i o n w e r e t h e c a s e , t h e p r o p o r -t i o n s o f f a m i l i e s s h o w i n g - - t h e v a r i o u s d e g r e e s o f s m u t t i n g s h o u l d f i t t h e e x p a n d e d b i n o m i a l ( p + q ) n , w h e r e n i s t h e n u m b e r o f c u l m s i n t h e f a m i l y . T h u s f o r t w o - t i l l e r f a m i l i e s , t h e e x p e c t e d f r e q u e n c y o f t h o s e w i t h b o t h h e a d s s m u t t e d i s p 2 , t h a t o f m i x e d i s 2 p q , a n d t h a t o f t h o s e w i t h b o t h h e a d s h e a l t h y i s q . F o r t i l l e r f a m i l i e s i n w h i c h t h e n u m b e r o f c u l m s w a s t h r e e o r m o r e , a t t e n t i o n w a s n o t p a i d t o t h e c o m p o s i t i o n o f t h e m i x e d f a m i l i e s o r t o t h e r e l a t i o n s h i p o f c u l m s w i t h i n t h e f a m i l i e s . B y a p p l y i n g a C h l - s q u a r e t e s t f o r g o o d n e s s - o f - f i t , i t c a n b e r e a d i l y s e e n t h a t t h e o b s e r v e d n u m b e r s d o n o t f i t t h e e x p e c t e d . I n e a c h c a s e t h e o b s e r v e d f r e q u e n c y o f m i x e d f a m i l i e s i s t o o l o w w h i l e t h a t o f t h e o t h e r t w o c l a s s e s I s t o o h i g h . H e n c e t h e r e i s a 77 TABLE 3 . 1 OBSERVED AND EXPECTED NUMBERS OF VARIOUSLY SMUTTED TWO, THREE AND FOUR OR MORE TILLER FAMILIES, AND THE APPLICATION OF A CHI-SQUARE TEST FOR RANDOMNESS OF SMUTTING AMONG FAMILIES, USING BINOMIAL EXPANSIONS TO CALCULATE EXPECTED VALUES A l l A l l G r o u p i n g S m u t t e d M i x e d H e a l t h y T o t a l , T w o t i l l e r f a m i l i e s O b s e r v e d 8 1 9 25 115 C a l c u l a t e d v a l u e s o f t a p = 0 . 7 4 q = 0 . 2 6 E x p e c t e d ( i f r a n d o m l y ( p 2 ) (2pq) ( q 2 ) d i s t r i b u t e d ) 63 4 4 8 115 P r o b a b i l i t y t h a t s m u t t e d h e a d s a r e r a n d o m l y d i s t r i b u t e d = < 0 . 0 0 1 T h r e e t i l l e r f a m i l i e s O b s e r v e d 37 5 1 4 56 C a l c u l a t e d v a l u e s o f t p = 0 . 7 1 q = 0 . 2 9 E x p e c t e d ( i f r a n d o m l y ( p 3 ) ( 3 P 2 q + 3 P Q . 2 ) U 3 ) d i s t r i b u t e d ) 21 3 4 1 56 P r o b a b i l i t y t h a t s m u t t e d h e a d s a r e r a n d o m l y d i s t r i b u t e d = < 0 . 0 0 1 F o u r o r m o r e t i l l e r f a m i l i e s ( a v e r a g e 6 . 2 t i l l e r s ) O b s e r v e d 33 5 9 47 C a l c u l a t e d v a l u e s o f t p = O . 6 5 q = 0 . 3 5 E x p e c t e d ( i f r a n d o m l y ( p 6 ) ( l - p 6 - q 6 ) ( q 6 ) d i s t r i b u t e d ) 4 43 0 47 P r o b a b i l i t y t h a t s m u t t e d h e a d s a r e r a n d o m l y d i s t r i b u t e d = < 0 , 0 0 1 a p = t h e p r o b a b i l i t y , b a s e d o n t h e o b s e r v e d d a t a , o f a s i n g l e c u l m b e i n g s m u t t e d , q = t h e p r o b a b i l i t y o f a s i n g l e c u l m r e m a i n i n g h e a l t h y . 7 8 s t r o n g tendency f o r t i l l e r f a m i l i e s to be composed e n t i r e l y of e i t h e r smutted or healthy s p i k e s . A t t e n t i o n was next pai d only to the 1 9 t i l l e r f a m i l i e s which were mixed. They were c l a s s i f i e d according t o whether the nonsmutted s p l k e ( s ) were the o l d e s t , youngest, or n e i t h e r . I f smutting i s randomly d i s t r i b u t e d i n r e l a t i o n to age, the r a t i o of o l d e s t healthy to youngest healthy t i l l e r s should be 1 : 1 , The t e s t i s shown i n Table 3 . 2 . Sample s i z e s were s m a l l , so the Yates c o r r e c t i o n f a c t o r f o r c o n t i n u i t y was a p p l i e d i n c a r r y i n g out the C h i -square t e s t ( S t a n s f i e l d , 1 9 6 9 ) . A s i g n i f i c a n t d i f f e r e n c e e x i s t s between the observed and the expected r a t i o s . The co n c l u s i o n i s t h a t there i s a c l e a r tendency f o r o l d e r s p i k e s w i t h i n mixed smutted f a m i l i e s to remain healthy. TABLE 3 . 2 TEST FOR RANDOMNESS OF POSITION OF NONSMUTTED TILLER(S) WITHIN SMUTTED TILLER FAMILIES, IN TERMS OF AGE (ORDER OF APPEARANCE) OF THE TILLERS P o s i t i o n of nonsmutted t i l l e r or t i l l e r s  ' Oldest Neither Yo ungest Observed 1 5 0 4 Expected ( i f randomly d i s t r i b u t e d ) 8 . 5 8 . 5 P r o b a b i l i t y t h a t oldest:youngest r a t i o f i t s 1 : 1 = 0 . 0 1 5 * ^ A p p l y i n g Yates s m a l l sample c o r r e c t i o n f o r c o n t i n u i t y . 7 9 A l s o i n v e s t i g a t e d w a s t h e r e l a t i o n s h i p , i f a n y , b e t w e e n a g e ( p o s i t i o n ) o f a t i l l e r f a m i l y a n d I t s p r o b a -b i l i t y o f b e i n g s m u t t e d . A t e s t , s i m i l a r t o t h e o n e e x p l a i n e d a b o v e , w a s a p p l i e d t o s m u t t i n g a m o n g f a m i l i e s . A t i l l e r f a m i l y w a s c o n s i d e r e d s m u t t e d i f a n y o f I t s s p i k e s w e r e s m u t t e d . N o n s m u t t e d f a m i l i e s o n a l l s m u t t e d p l a n t s w e r e c l a s s i f i e d a c c o r d i n g t o t h e i r a g e r e l a t i v e t o t h e s m u t t e d f a m i l i e s . A n o n s m u t t e d f a m i l y ( o r a s e r i e s o f t h e m ) c o u l d t h u s b e c l a s s i f i e d a s o l d e r o r y o u n g e r t h a n t h e f a m i l y o r f a m i l i e s s h o w i n g s m u t , o r o f i n t e r m e d i a t e a g e , o r n e i t h e r , d e p e n d i n g o n w h e n t h e p r i m a r y l a t e r a l s o f t h e f a m i l i e s h a d a p p e a r e d . T a b l e 3.3 g i v e s t h e d e t a i l s o f t h e C h i - s q u a r e t e s t s o f h o w w e l l t h e r a t i o o f o l d e s t t o y o u n g e s t n o n s m u t t e d f a m i l y p o s i t i o n f i t s t h e e x p e c t e d 1»1 r a t i o ( b a s e d o n t h e a s s u m p t i o n t h a t t h e r e i s n o r e l a t i o n s h i p b e t w e e n f a m i l y a g e a n d s m u t t i n g p r o b a b i l i t y ) . T w o d i f f e r e n t r e s u l t s a r e t e s t e d - - o n e i g n o r i n g t h e p r i n c i p a l c u l m , w h i c h c a n n o t b e r e g a r d e d a s a t i l l e r f a m i l y , e n d o n e i n c l u d i n g t h e I S a s t h e o l d e s t c u l m ( a n d h e n c e c o n s i d e r i n g o n l y p l a n t s i n w h i c h I S s u r v i v e d ) . W h e n o n l y l a t e r a l t i l l e r f a m i l i e s a r e c o u n t e d i t i s c l e a r t h a t t h e r e I s a h i g h l y s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e a c t u a l o l d e s t ! y o u n g e s t r a t i o o b t a i n e d a n d t h e 1 « 1 e x p e c t e d . T h u s t h e r e . i s a s t r o n g t e n d e n c y f o r o l d e r t i l l e r f a m i l e s t o r e m a i n h e a l t h y i n p a r t i a l l y s m u t t e d p l a n t s . W h e n t h e I S c u l m I s i n c l u d e d , h o w e v e r , t h e t e n d e n c y c a n n o l o n g e r b e s e e n u s i n g t h e s i m p l e t e s t , a s t h e s e c o n d p a r t o f T a b l e 3-3 s h o w s . A m o r e c o m p l e t e p i c t u r e c a n b e 8 0 TABLE 3.3 TEST FOR RANDOMNESS OF POSITION OF NONSMUTTED TILLER FAMILES WITHIN THE SMUTTED BARLEY PLANT, IN TERMS OF AGE (ORDER OF APPEARANCE) OF THE FAMILIES P o s i t i o n of nonsmutted f a m i l y or f a m i l i e s Oldest Intermediate Youngest L a t e r t i l l e r s only Observed 55 8 26 Expected ( i f randomly d i s t r i b u t e d ) 4 o . 5 40.5 P r o b a b i l i t y t h a t oldesttyoungest r a t i o f i t s l t l = 0.001 P r i n c i p a l t i l l e r i ncluded (as o l d e s t t i l l e r ) Observed 31 30 25 Expected ( i f randomly d i s t r i b u t e d ) 28 . 2 8 P r o b a b i l i t y t h a t oldesttyoungest r a t i o f i t s 1»1 = 0.45 seen i n Table 3.4. Here t i l l e r f a m i l i e s , along w i t h the p r i n -c i p a l culm 9 are presented by p o s i t i o n and f e t e . The data of t h i s Table are shown g r a p h i c a l l y i n Figure 3.2, where t i l l e r f a m i l i e s are c l a s s i f i e d and the percentage i n each c l a s s which showed smut are given. The 13 culm i s the only p l a n t p a r t which does not f i t the p a t t e r n of decreasing frequency of smutting w i t h 8 g e . Data f o r the IS culms and, to a l e s s e r e x t e n t , f o r the CN and 1L f a m i l i e s , are e f f e c t e d by the r e l a -t i v e l y high death r a t e s ; had these culms a l l survived to 8 1 heading, the percentages of smut may have been changed. The graph and t a b l e show that the CN and 1 L p o s i t i o n s (bulked) were smutted s i g n i f i c a n t l y l e s s f r e q u e n t l y than the 2 L p o s i t i o n , which was i n t u r n smutted s i g n i f i c a n t l y l e s s f r e q u e n t l y than the 3 L , 4 L or (bulked) 5 D and up p o s i t i o n s . The l a t t e r three were not s i g n i f i c a n t l y d i f f e r e n t . One other notable p o i n t emerges i n Table 3.4. The frequency of t i l l e r f a m i l y death seemed to be d i r e c t l y propor-t i o n a l t o t h e i r age. This f a c t was n o t i c e d c a s u a l l y during l a b e l l i n g and h a r v e s t i n g . L a t e r emerging culms seemed to be "favored" by the p l a n t . Not only were they more l i k e l y to s u r v i v e , but they grew f a s t e r . The very l a t e culms, sometimes seen, e s p e c i a l l y i n response t o l a t e heavy watering, were the f a s t e s t growing of a l l . Because these arose s p u r i o u s l y , they were not in c l u d e d i n the analyses. TABLE 3.4 THE PRODUCTION BY SMUTTED PLANTS AND FATES OF TILLER FAMILIES, ANALYZED BY NODAL POSITION T i l l e r f a m i l y f a t e (%) Percent of Sample Nodal Headed Smutted Dead S u r v i v i n g Size P o s i t i o n Fam. Smutted* IS 1 4 . 6 50.2 3 5 . 2 8 0 . 4 D C 2 1 3 CN and 1 L 2 9 . 0 4 6 . 0 25.0 6 1 . 3 a 1 2 4 2 L 1 7 . 6 6 1 . 7 2 0 . 7 7 7 . 8 ° 1 8 8 3 L 1 0 . 3 6 8 . 5 2 1 . 2 8 6 . 9 ° 1 8 4 4 L 1 4 . 8 7 4 . 7 1 0 . 5 83 .40 1 6 2 5 L and above 1 2 . 5 7 2 . 2 1 5 . 2 8 5 . 3 ° 1 8 4 •Values not s h a r i n g the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n t (p=0.05). S t a t i s t i c a l comparisons are made v e r t i c a l l y . 100 FIGURE 3.2 100 Nodal position of t i l ler family FIGURE 3.2. FREQUENCY OF COVERED SMUT OCCURRENCE IN TILLER FAMILIES OF BARLEY PLANTS IN RELATION TO RELATIVE AGE OF THE TILLER FAMILY (AS INDICATED BY POSITION) 00 8 3 2 . T h e E f f e c t o f I n o c u l a t i o n a n d  S m u t t i n g o n P l a n t G r o w t h T h e m a i n e f f e c t s o f I n o c u l a t i o n a n d s m u t t i n g o n p l a n t g r o w t h w e r e e x a m i n e d : s t u n t i n g , e f f e c t s o n t i l l e r i n g , a n d e f f e c t s o n t h e p a t t e r n o f p l a n t g r o w t h . T h r e e w e e k s a f t e r p l a n t i n g , s e e d l i n g s o f t h e 1973-4 g r e e n h o u s e e x p e r i m e n t s e a s o n w e r e e x a m i n e d f o r s t u n t i n g . O u t o f 206 i n o c u l a t e d s e e d l i n g s , 25 e x h i b i t e d s o m e s t u n t i n g ( p l a n t s s h o r t w i t h n a r r o w , d a r k g r e e n l e a v e s ) . N o n e o f 83 u n i n o c u l a t e d p l a n t s w a s s t u n t e d . O f t h e 25 s t u n t e d p l a n t s , 16 b e c a m e s m u t t e d a n d n i n e d i d n o t . T h i s s a m p l e i s t o o s m a l l f o r s t a t i s t i c a l t r e a t m e n t . O n e c a n , h o w e v e r , c o n c l u d e t h a t w h i l e s t u n t i n g i s p r o b a b l y s m u t -r e l a t e d , n o c l e a r r e l a t i o n s h i p b e t w e e n s t u n t i n g a n d s u b -s e q u e n t s m u t t i n g w a s s e e n ( i . e . , s t u n t e d p l a n t s w e r e n o t a l w a y s s m u t t e d ) . B e c a u s e o f r a t h e r l a r g e a m o n g - p o t v a r i a t i o n o b s e r v e d i n 1972-3, i t w a s t h o u g h t t h a t p l a n t s o f d i f f e r e n t t r e a t m e n t s s h o u l d b e p a i r e d , e a c h p a i r b e i n g f r o m t h e s a m e p o t , i n o r d e r t o m e a s u r e t h e e f f e c t s , I f a n y , o f i n o c u l a t i o n a n d s m u t t i n g o n t i l l e r i n g . T h e a n a l y s i s i s p r e s e n t e d I n T a b l e 3.5» W h e t h e r o r n o t p l a n t s s h o w e d s m u t , t h e r e w a s a s i g n i f i c a n t r e d u c t i o n i n t h e n u m b e r o f t i l l e r s p e r i n o c u l a t e d p l a n t , b o t h I n i t i a t e d a n d s u r v i v i n g ( t o m a t u r i t y ) , w h e n s u c h p l a n t s w e r e c o m p a r e d w i t h u n i n o c u l a t e d p l a n t s . T h e r e w a s n o 8 4 T A B L E 3.5 M E A N NUMBERS O F C U L M S I N I T I A T E D P E R P L A N T , A N D T H E I R F A T E S ( H E A D E D , S M U T T E D OR D E A D ) , WHEN P L A N T S WERE U N I N O C U L A T E D , I N O C U L A T E D B U T F A I L E D TO B E C O M E S M U T T E D , OR I N O C U L A T E D A N D S M U T T E D T r e a t m e n t N u m b e r o f p a i r e d M e a n n u m b e r o f c u l m s p e r p l a n t * C o m p a r i s o n s O b s e r v a t i o n s H e a d e d D e a d T o t a l N o n i n o c u l a t e d I n o c u l a t e d n o n -s m u t t e d 6 8 5 . 5 6 4.91 2 . 9 3 2 . 4 7 8 . 4 9 7 . 8 3 I n o c u l a t e d n o n -s m u t t e d S m u t t e d 63 4 . 9 7 a 4 . 6 3 a 2.13 2 . 8 1 7 . 1 0 D 7 . 4 4 b N o n i n o c u l a t e d S m u t t e d 4 8 6.33 4 . 1 0 2 . 8 7 2 . 3 5 9 . 2 1 6 . 4 6 * M e a n s n o t s h a r i n g t h e s a m e s u p e r s c r i p t a r e s i g n i f i c a n t l y d i f f e r e n t (P=0.05, p a i r e d t a n a l y s i s ) . S t a t i s t i c a l c o m p a r i s o n s i n v o l v e d p a i r e d m e a n s o n l y . s i g n i f i c a n t d i f f e r e n c e , i n t o t a l t i l l e r s i n i t i a t e d o r s u r -v i v i n g , b e t w e e n s m u t t e d a n d n o n s m u t t e d i n o c u l a t e d p l a n t s . T h e s i t u a t i o n w a s d i f f e r e n t f o r d e a d t i l l e r s . N o n i n o c u l a t e d p l a n t s h a d s i g n i f i c a n t l y m o r e d e a d t i l l e r s t h a n e i t h e r s m u t t e d o r n o n s m u t t e d i n o c u l a t e d p l a n t s . T h i s i s p r o b a b l y a r e f l e c t i o n o f t h e o v e r a l l h i g h e r t i l l e r p r o d u c t i o n o f n o n i n o c u l a t e d p l a n t s . S m u t t e d p l a n t s h a d s i g n i f i c a n t l y m o r e d e a d t i l l e r s t h a n d i d n o n s m u t t e d I n o c u l a t e d p l a n t s . 85 To examine the e f f e c t of I n o c u l a t i o n and smutting on the p a t t e r n of p l a n t growth, smutted, nonsmutted i n o c u l a t e d and noninoculated p l a n t s were analyzed w i t h regard to the percentages of p l a n t s attempting to produce t i l l e r f a m i l i e s from each node. The d a t a , along w i t h the s t a t i s t i c a l ana-l y s e s , are presented i n Table 3 . 6 . The date are p l o t t e d , to show the d i f f e r e n c e s more c l e a r l y , i n Figure 3.3. Smutted and nonsmutted i n o c u l a t e d p l a n t s d i f f e r e d In t h e i r growth pat t e r n s only i n s o f a r as smutted p l a n t s attempted t i l l e r f a m i l y production w i t h a s i g n i f i c a n t l y higher frequency at the CN a n d ' 6 L nodes. This may r e l a t e to the higher death r a t e of culms i n smutted p l a n t s , whereby dead culms a r e r e p l a c e d by new ones. A marked e f f e c t of smutting and inocu-l a t i o n on the p a t t e r n of plant- growth Is seen when the non-i n o c u l a t e d p l a n t s were compared w i t h the other two groups. In the f i g u r e , i t i s c l e a r t h a t the noninoculated p l a n t s produced l a t e r a l t i l l e r s from lower nodes than e i t h e r non-smutted i n o c u l a t e d or smutted p l a n t s . In other words, I n o c u l a t i o n had the e f f e c t of decreasing l a t e r a l t i l l e r p r o d u c t i o n a t the 1 L node and I n c r e a s i n g i t at the 5 L >and 6 L ^ nodes. The CN node d i d not conform to t h i s e f f e c t , although i t might have been expected to do so. A f i n a l o b s e r v ation should be noted here. Smutted p l a n t s e x h i b i t e d a marked delay i n emergence of heads. In each p l a n t i n g the healthy p l a n t s were the f i r s t to be 86 TABLE 3.6 COMPARISONS OF GROWTH PATTERNS (PERCENT OF LATERAL TILLER PRODUCTION FROM DIFFERENT NODES OF THE PRINCIPAL CULM) OF 209 SMUTTED, 194 NONSMUTTED INOCULATED, AND 83 NONINOCULATED * HANNCHEN' BARLEY PLANTS P e r c e n t of p l a n t s attemutins: t i l l e r i n g from node* N o d a l P o s i t i o n Smutted Nonsmutted I n o c u l a t e d N o n i n o c u l a t e d C N 2 5 . 8 a 13 .4 * * 2 1 . 7 a I L 35.9 b 4 4 . 8 b 90.4 2 L 9 1 . 9 * * 93.3** 9 8 . 8 * * 3 L 87.6** 93.3** 96 .4 * * 4 L 78.5° 8 4 . 0 ° 62.6 5 L 56.0 d 4 6 . 4 d 13.2 6 L 2 8 . 2 1 2 . 9 3 . 6 * * * S t a t i s t i c a l c o m p a r i s o n s a r e m a d e h o r i z o n t a l l y . V a l u e s n o t s h a r i n g t h e s a m e s u p e r s c r i p t l e t t e r a r e s i g n i f i c a n t l y d i f f e r e n t (p=0.05). * * S a m p l e s i z e t o o s m a l l f o r s t a t i s t i c a l a n a l y s i s . r e c o r d e d a n d h a r v e s t e d , w h i l e t h o s e w h i c h a p p e a r e d l a s t w e r e i n v a r i a b l y s m u t t e d p l a n t s . H e a d s o f n o n s m u t t e d I n o c u l a t e d p l a n t s d i d n o t e m e r g e n o t i c e a b l y l a t e r t h a n t h o s e o f n o n -i n o c u l a t e d p l a n t s . FIGURE 3.3 100 e E s « a n -a 1= „ & . J _ tt> —* C to ~~ 80 *0 20 / smutted nonsmutted, inoculated noninoculated \ \ \ \ Cf4 1 L ZL 3L position on principal culm FIGURE 3.3. THE PERCENT OF SMUTTED, NONSMUTTED INOCULATED AND NONINOCULATED BARLEY PLANTS INITIATING LATERAL TILLER PRODUCTION ACCORDING TO NODAL POSITION ON THE PRINCIPAL CULM 0 0 8 8 D. DISCUSSION Since very l i t t l e has been w r i t t e n concerning growth of the b a r l e y p l a n t , the Information on growth of •Hannchen' b a r l e y was learned almost e n t i r e l y through f i r s t - h a n d obser-v a t i o n . The p a u c i t y of i n f o r m a t i o n about b a r l e y may be due t o the b e l i e f t h a t growth of b a r l e y Is q u i t e s i m i l a r to t h a t of wheat or o a t s , two much more thoroughly s t u d i e d c e r e a l s . The assumption can a l s o be made that plumular development i n the dormant seed of b a r l e y Is s i m i l a r t o that of wheat, as Hector ( I 9 3 6 ) s t a t e s In h i s chapter on b a r l e y . In wheat, both P e r c l v a l (1921) and McCall (193*0 found that two bud prlmordia are present i n the dormant seed. One of these Is the a p i c a l bud, which w i l l give r i s e to the IS culm, and the other i s i n the a x i l of the c o l e o p t i l e and w i l l develop i n t o the CN culm. Bonnett (1935) very b r i e f l y d e s c r i b e d the same c o n d i t i o n In seeds of both 2- and 6-rowed b a r l e y s . P e r c l v a l found t h a t I t was not u n t i l 10 to 15 days a f t e r germination t h a t the bud prlmordia i n the a x i l s of the o l d e s t v e g e t a t i v e leaves could be detected, lowermost f i r s t . In c o n c l u s i o n , the above s t u d i e s i n d i c a t e t h a t there i s a c l e a r d i f f e r e n c e In development among bud prlmordia a t the time of I n i t i a t i o n of smut i n f e c t i o n . Whether t h i s p a t t e r n of e a r l y development v a r i e s a p p r e c i a b l y has, u n f o r t u n a t e l y , never been determined. H i s t o l o g i c a l s t u d i e s have n e c e s s a r i l y i n v o l v e d only a s m a l l number of embryos, so that the r a r e 8 9 case of a t y p i c a l development, i f i t e x i s t s , would have gone undetected. As P e r c l v a l has pointed out, however, one can expect normal b i o l o g i c a l v a r i a t i o n i n t h i s and, indeed, as the present s t u d i e s show, i n e l l aspects of b a r l e y growth. 1 . The D i s t r i b u t i o n of Smutted and  Healthy Heads i n Smutted P l a n t s Smutted heads d i d not occur randomly i n p a r t i a l l y smutted p l a n t s . Members of a t i l l e r f a m i l y tended to e i t h e r be a l l h e a l t h y or a l l smutted. F u r t h e r , at both the w i t h i n ^ f a m i l y and between-family l e v e l , o l d e r culms remained healthy more f r e q u e n t l y than d i d younger culms. I t should be emphasized t h a t these were s t a t i s t i c a l tendencies only. Exceptions were not r a r e . A phenomenon s i m i l a r t o the above occurred w i t h i n i n d i v i d u a l s p i k e s . O c c a s i o n a l l y , spikes were found which contained both smut t e l i o s p o r e s and healthy seeds. I n -v a r i a b l y , the h e a l t h y seeds were found on the upper part of the s p i k e and the smut on the lower p a r t , The same obser-v a t i o n was recorded by F a r i s ( 1 9 2 4 a ) . Bennett and F i n c h ( 1 9 7 1 ) determined t h a t meiosls i n b a r l e y begins at the top of the s p i k e and proceeds downward, so that upper seeds are s l i g h t l y older.. Thus the w i t h i n - s p i k e smutting p a t t e r n Is the same as that, seen a t the w i t h i n - and b e t w e e n - t i l l e r f a m i l y l e v e l . The o n l y other r e p o r t found of w l t h l n - s p l k e p a r t i a l d i s e a s e was by Holton and Rodenhlser ( 1 9 4 2 ) . They 9 0 o b s e r v e d p a r t i a l l y d i s e a s e d h e a d s o f w h e a t i n f e c t e d w i t h T . c a r l e s i n w h i c h s o u n d s e e d a n d b u n t b a l l s w e r e b o t h r a n d o m l y l o c a t e d . A s I n d i c a t e d i n t h e l i t e r a t u r e r e v i e w , t h e p a t h w a y w h i c h s m u t s t a k e i n s p r e a d i n g t o v a r i o u s p a r t s o f t h e c r o w n h a s n o t b e e n c o n c l u s i v e l y d e t e r m i n e d . H e r e , t o o , i t i s l i k e l y t h a t c o n s i d e r a b l e v a r i a b i l i t y e x i s t s , s o t h a t t h e m y c e l i u m d o e s n o t n e c e s s a r i l y h a v e t o m o v e p r e c i s e l y t h r o u g h t h e s a m e t i s s u e s i n e a c h s e e d l i n g . H i s t o l o g i c a l e v i d e n c e p o i n t i n g t o a n y s p e c i f i c o r f r e q u e n t r o u t e , t a k e n b y t h e f u n g u s i n s p r e a d i n g t o v a r i o u s p a r t s o f t h e c r o w n , i s w e a k . A l s o w e a k i s e v i d e n c e f o r a d i f f e r e n c e i n d i s t r i b u t i o n o f m y c e l i u m w i t h i n t h e c r o w n w h i c h w o u l d e x p l a i n t h e t e n d e n c y f o r o l d e r c u l m s t o r e m a i n h e a l t h y . I n b o t h c a s e s , t h e w e a k n e s s o f t h e e v i d e n c e s t e m s f r o m t h e f a c t t h a t o n l y s m a l l n u m b e r s o f i n f e c t e d e m b r y o s h a v e b e e n e x a m i n e d . F r o m t h e l i m i t e d e v i d e n c e w h i c h i s a v a i l a b l e I t a p p e a r s t h a t t h e s m u t h y p h a e e n t e r t h e c r o w n f r o m b e l o w , s o t h a t o l d e r l e a f s h e a t h s a n d n o d e s a r e i n f e c t e d f i r s t , a n d t h a t t h e m y c e l i u m i s m o r e a b u n d a n t i n t h e l o w e r p a r t o f t h e c r o w n t h a n i n t h e u p p e r . T h i s a v a i l a b l e e v i d e n c e d o e s n o t p r o v i d e a r e a d y e x p l a n a t i o n o f t h e o b s e r v e d d i s t r i b u t i o n o f s m u t t e d c u l m s . O n t h e c o n t r a r y , I f t h e l o c a t i o n o f t h e h y p h a e w i t h i n t h e c r o w n w e r e t h e d e t e r m i n i n g f a c t o r , o n e w o u l d e x p e c t t h e o l d e r c u l m s t o b e m o r e f r e q u e n t l y s m u t t e d . I n c o n c l u s i o n , h i s t o -l o g i c a l w o r k o n t h e p a t h w a y o f i n f e c t i o n a n d t h e d i s t r i b u t i o n 9 1 of smut mycelium does l i t t l e to e x p l a i n the p a t t e r n of smutting which was most f r e q u e n t l y observed. In the l e s s frequent cases, where younger culms were nonsmutted, the i n f e c t i o n pathway and/or the d i s t r i b u t i o n p a t t e r n of mycelium w i t h i n the crown i s a p o s s i b l e e x p l a n a t i o n . In a few p l a n t s , the o l d e s t and youngest primary l a t e r a l culms were smutted w h i l e one or more intermediate t i l l e r f a m i l i e s were healthy. This may be the r e s u l t of m u l t i p l e i n f e c t i o n s ( i n f e c t i o n of a seed by more than one d i k a r y o n ) . Such m u l t i p l e I n f e c t i o n s have been c o n c l u s i v e l y demonstrated i n oats i n f e c t e d w i t h U. k o l l e r l by Person and Cherewick (1964). Although not determining smutting patterns based on t i l l e r r e l a t i o n s h i p s , they showed that d i f f e r e n t d i k a r y o n s can occur i n d i f f e r e n t culms of the same p l a n t . The s i m i l a r i t y of U. k o l l e r l to U. hordei i s such th a t i t would seem q u i t e l i k e l y t h a t such m u l t i p l e i n f e c t i o n s occur i n the l a t t e r a l s o . In U. k o l l e r l genotypic mosaics i n d i c a t e t h a t v a r i a t i o n i s p o s s i b l e i n how and/or when smut hyphae spread throughout the crown r e g i o n . A thorough study of the m u l t i p l e i n f e c t i o n phenomenon i n barley-U. hordei would w e l l complement the present s t u d i e s . A p o s s i b l e b a s i s f o r the observed p a t t e r n of smutting has been mentioned by other workers. Sampson (1932) observed a b a r r i e r of l i g n i f i e d c e l l s between the nodal t i s s u e s 9 2 c o n t a i n i n g U. k o l l e r l mycelium and the a p i c a l growing p o i n t In r e s i s t a n t oat p l a n t s . She concluded th a t i f the p l a n t could produce t h i s b a r r i e r before the fungus reached the growing p o i n t , the culm or culms would not become smutted. She suggested t h a t s u s c e p t i b l e p l a n t s were unable to l a y down t h i s b a r r i e r i n time, because the fungus was growing r a p i d l y , and t h a t growth of the mycelium i n t o t a l l y r e -s i s t a n t c u l t i v a r s was s t r o n g l y r e t a r d e d . Regardless of whether or not l l g n l f I c a t I o n i s the c r i t i c a l event, the hypothesis t h a t degree of smutting depends on r e l a t i v e r a t e s of growth of the embryo and the smut mycelium Is promising and popular, p a r t i c u l a r l y i n e x p l a i n i n g t o t a l r e s i s t a n c e . Ohms and Bever (1955) found t h a t the mechanism of r e s i s t a n c e t o U. t r l t l c l i n one wheat c u l t l v a r seemed t o be simply that the s e e d l i n g s outgrew the smut mycelium. The same can be s a i d f o r other s t u d i e s , mentioned i n the l i t e r a t u r e review, where e a r l y embryo i n f e c t i o n of t o t a l l y r e s i s t a n t c u l t i v a r s was as high as t h a t of s u s c e p t i b l e c u l t i v a r s . Swinburne (I963) b e l i e v e d t h a t f o r wheat Infected by T. c a r l e s , once the hyphae reached a growing p o i n t , smut was always expressed i n t h a t culm. In other words, r e s i s t a n c e could occur any time before.the smut had reached the growing p o i n t , but not a f t e r . The c r i t i c a l p e r i o d , then, appears t o be tha t period of growth p r i o r to development of some b a r r i e r t o hyphal growth, such as l l g n l f i c a t l o n or elon g a t i o n of the Internodes 93 o f a c u l m . I f t h e m y c e l i u m d o e s n o t g e t b e y o n d t h e a r e a o f t h e b a r r i e r , i t w i l l b e l e f t b e h i n d . T h i s a p p e a r s t o b e t h e k e y t o t h e s m u t t i n g p a t t e r n m o s t f r e q u e n t l y o b s e r v e d . T h e b a r r i e r w o u l d b e e x p e c t e d t o b e p r o d u c e d f i r s t i n t h e o l d e r c u l m s , a l l o w i n g a s h o r t e r p e r i o d f r o m g e r m i n a t i o n d u r i n g w h i c h t h e s m u t m y c e l i u m m u s t r e a c h t h e g r o w i n g p o i n t , w h e n c o m p a r e d w i t h y o u n g e r c u l m s . T h e r a r i t y o f m i x e d t i l l e r f a m i l i e s m i g h t b e e x p l a i n e d b y a c o m b i n a t i o n o f r e l a t i v e s i m i l a r i t y o f a g e a n d c l o s e p r o x i m i t y o f p r i m o r d i a o f t i l l e r f a m i l y m e m b e r s . T h e p r i n c i p a l c u l m s d i d n o t f i t i n t o t h e s a m e a g e t s m u t f r e q u e n c y p a t t e r n a s t h e l a t e r a l c u l m s , a n d t h e a b o v e h y p o t h e s i s " c a n n o t a p p l y t o t h e m . W h i l e t h e r e c a n b e n o d o u b t t h a t t h e I S c u l m i s t h e o l d e s t c u l m o n t h e p l a n t , i t i s n o t t h e l e a s t f r e q u e n t l y s m u t t e d , b y a n y m e a n s . H e n c e , t h e o b s e r v a t i o n t h a t h e a l t h y c u l m s a p p e a r b e f o r e s m u t t e d i n a g r o u p o f i n o c u l a t e d p l a n t s i s p r o b a b l y n o t d u e t o t h e s m u t t i n g - a g e r e l a t i o n s h i p o f c u l m s . I t s e e m s t o o c c u p y a s p e c i a l p l a c e w i t h r e g a r d t o s m u t t i n g . T h e a p i c a l g r o w i n g p o i n t c o u l d p e r h a p s b e c o n s i d e r e d t h e " t a r g e t " o f s m u t h y p h a e s p r e a d i n g t h r o u g h o u t t h e c r o w n r e g i o n . T h e m o s t t h a t c a n b e s a i d h e r e i s t h a t i t o c c u p i e s a u n i q u e p o s i t i o n w i t h r e g a r d t o t h e a g e i s m u t t i n g f r e q u e n c y r e l a t i o n s h i p , w h e n c o m p a r e d w i t h l a t e r a l c u l m s . 94 Some workers have t r i e d to r e l a t e s i z e of seed and, r a p i d i t y of germination to smut s u s c e p t i b i l i t y i n determining i f , from the standpoint of the p l a n t , the l e n g t h of the c r i t i c a l p e r i o d i s important. R e s u l t s vary. Tervet (1944) found t h a t r a p i d , v i g o r o u s l y germinating oat seeds were l e s s s u s c e p t i b l e to loose and covered smut than l e s s r a p i d l y ger-minating seeds of each of s e v e r a l c u l t i v a r s . Reed and F a r i s ( 1 9 2 4 ) had found t h i s not to be the case, however,using the same host and pathogens. Brandwein ( 1 9 3 3 ) could f i n d no c o r r e l a t i o n between covered smut s u c e p t i b i l l t y and the ger-m i n a t i o n and growth r a t e of s u s c e p t i b l e and r e s i s t a n t oat c u l t i v a r s . F a r i s ( 193*0 a c t u a l l y found bunting percentages to be higher on r a p i d l y germinating seeds of s e v e r a l wheat c u l t i v a r s i n o c u l a t e d w i t h T l l l e t l a FoetIda (Wall.) L i v o and T. c a r l e s ; but Smith ( 1 9 3 2 a ) could f i n d no r e l a t i o n between growth r a t e and bunt s u s c e p t i b i l i t y i n the wheat c u l t i v a r s he examined. These i n c o n s i s t e n c i e s might best be explained by d i f f e r e n c e s i n experimental procedures and by the f a c t t h a t v a r i a t i o n i n seed s i z e , v i g o r of germination or growth r a t e s are complex phenomena about which g e n e r a l i z a t i o n i s r i s k y , p a r t i c u l a r l y when one t r i e s to r e l a t e them to a concept e q u a l l y complex, such as smut s u s c e p t i b i l i t y . Increased smut s u s c e p t i b i l i t y caused by such t r e a t -ments as deep sowing and c o o l e r growing temperatures, which tend to slow germination and p l a n t growth r a t e , can a l s o be 95 explained on the b a s i s t h a t they a l l o w a longer c r i t i c a l p e r i o d . Swinburne (1963) expressed t h i s idea f o r wheat-T. c a r i e s , and Jones and S e i f - E l - N a s r (1939), w i t h regard to p l a n t i n g depth, f o r s e v e r a l c e r e a l smuts, i n c l u d i n g U . h o r d e i . 2 . The E f f e c t of I n o c u l a t i o n and  Smutting on P l a n t Growth Seeds which had been i n o c u l a t e d grew i n t o p l a n t s w i t h fewer s u r v i v i n g culms than those which had not, r e g a r d l e s s of whether or not these p l a n t s became smutted. This r e p r e s e n t s , as f a r as the author i s aware, the f i r s t r e p o r t of any e f f e c t of i n o c u l a t i o n on nonsmutted p l a n t s when a compatible host-pathogen combination Is invo l v e d . I t i s a l s o the f i r s t r e p o r t , of any k i n d , of such an e f f e c t i n the barley-U. hordei system. Reports of e f f e c t s on germination or growth of non-smutted i n o c u l a t e d p l a n t s when the host possessed t o t a l or near t o t a l r e s i s t a n c e to smutting were covered i n the l i t e r a t u r e review. Rather s u r p r i s i n g l y , the e f f e c t s observed i n t h i s work were much more s u b t l e than any of those r e p o r t e d . Neither apparent b l a s t i n g , s e e d l i n g stand r e d u c t i o n s , nor increased t i l l e r m o r t a l i t y were observed i n nonsmutted i n o c u l a t e d p l a n t s i n the present work. The e a r l y f i n d i n g t h a t nonsmutted "Immune" c u l t i v a r s were In r e a l i t y being r a t h e r s e v e r e l y a f f e c t e d by smut caused some 9 6 a l a r m . O f c o u r s e , I t w a s a l s o r e a l i z e d t h a t i f s u c h c u l t i v a r s w e r e w i d e l y p l a n t e d , t h e s m u t w o u l d , b a r r i n g l a r g e p o p u l a t i o n s h i f t s t o w a r d v i r u l e n c e o n t h e r e s i s t a n t c u l t i v a r s , s o o n b e e l i m i n a t e d b e c a u s e o f l o w r e p r o d u c t i v i t y . T h e o n l y o t h e r e f f e c t s o b s e r v e d o n n o n s m u t t e d i n o c u -l a t e d * H a n n c h e n 1 b a r l e y p l a n t s w e r e t h e i n f r e q u e n t s t u n t i n g o f s e e d l i n g s a n d t h e s h i f t i n t h e p a t t e r n o f t i l l e r i n g . A l l o f t h e s e e f f e c t s c o u l d b e c a u s e d b y s m u t m y c e l i u m b e i n g p r e s e n t I n t h e p l a n t o n l y a t t h e s e e d l i n g s t a g e . D u e t o s p a c e l i m i t a t i o n s , p l a n t s w e r e c o m p e t i n g f o r r o o t s p a c e a n d l i g h t , a n d d i f f e r e n c e s b e t w e e n t r e a t m e n t s w e r e p r o b a b l y m a g n i f i e d b y t h i s i n t e r - t r e a t m e n t p l a n t c o m p e t i t i o n . A l t e r n a t i v e l y , i f i t c o u l d b e s h o w n - t h a t n o n s m u t t e d i n o c u l a t e d p l a n t s w»r©, o v e r a l l , l e s s v i g o r o u s t h a n n o n i n o c u l a t e d p l a n t s , i t w o u l d s u p p o r t t h e p r e m i s e t h a t t h e m y c e l i u m i s p r e s e n t i n t h e p l a n t t i s s u e f o r a r e l a t i v e l y l o n g t i m e , p e r h a p s t o m a t u r i t y . O n e c a n s a f e l y c o n c l u d e t h a t i n t h i s p a r t i c u l a r g e n o t y p l c c o m b i n a t i o n , s m u t m y c e l i u m i s p r e s e n t f o r a t l e a s t a s h o r t p e r i o d i n i n o c u l a t e d s e e d l i n g s o f u l t i m a t e l y " h e a l t h y " p l a n t s . T h e s i t u a t i o n I s n o t a s h a d b e e n f o u n d i n s o m e r e s i s t a n t c o m b i n a t i o n s o f h o s t a n d s m u t ( d i s c u s s e d I n t h e l i t e r a t u r e r e v i e w ) w h e r e t h e p a t h o g e n w a s u n a b l e t o p e n e -t r a t e b e y o n d t h e p e r i c a r p . W h i l e e f f e c t s o n p l a n t s w e r e n o t i n v e s t i g a t e d i n t h e s e l a t t e r c a s e s , i t i s d o u b t f u l t h a t t h e p a t h o g e n c o u l d h a v e h a d a s i g n i f i c a n t e f f e c t o n p l a n t g r o w t h 9 7 w h e n e x c l u s i o n w a s s o e f f e c t i v e . O f c o u r s e , a s t h e s a m e w o r k e r s w h o d e m o n s t r a t e d s u c h r e s i s t a n c e s h o w e d , o n e c a n n o t g e n e r a l i z e a b o u t t h e d e t a i l s o f r e s i s t a n c e i n t h e h o s t - p a r a s i t e s y s t e m b a s e d o n t t f ha t w a s f o u n d i n a s i n g l e c o m b i n a t i o n , p a r t i c u l a r l y w h e n t h a t c o m b i n a t i o n e x h i b i t s a d i f f e r e n t ( i . e . , p a r t i a l ) s o r t o f r e s i s t a n c e t h a n t h a t w h i c h h a s b e e n c o m m o n l y s t u d i e d . R e s i s t a n c e t o U . h o r d e i b y n e a r - e x c l u s i o n f r o m t h e s e e d m a y o c c u r I n b a r l e y . I t h a s y e t t o b e d e m o n s t r a t e d , h o w e v e r . C o m p a r e d w i t h h i s t o l o g i c a l o b s e r v a t i o n o f s e e d l i n g s , t h e p r e s e n t w o r k i s a d m i t t e d l y a m o r e i n d i r e c t w a y o f d e m o n -s t r a t i n g t h e p r e s e n c e o f s m u t m y c e l i u m i n n o n s m u t t e d p l a n t s . I t has- t h e a d v a n t a g e t h a t " m u c h l a r g e r n u m b e r s o f p l a n t s c a n b e o b s e r v e d . D e t a i l e d h i s t o l o g i c a l s t u d i e s o f s m u t i n f e c t i o n a l l s u f f e r f r o m t h e m a j o r c r i t i c i s m t h a t c o n c l u s i o n s a r e b a s e d o n a s m a l l , o f t e n I n a d e q u a t e , n u m b e r o f o b s e r v a t i o n s . N e v e r t h e l e s s , h i s t o l o g i c a l s t u d i e s w o u l d c o m p l e m e n t t h e p r e s e n t s t u d i e s , a n d v i c e - v e r s a . S m u t t e d p l a n t s e x h i b i t e d o t h e r s y m p t o m s w h i c h n o n -s m u t t e d i n o c u l a t e d p l a n t s d i d n o t . I n a d d i t i o n t o r e d u c e d t i l l e r i n g a n d o c c a s i o n a l s t u n t i n g o f y o u n g s e e d l i n g s , s m u t t e d p l a n t s a l s o e x h i b i t e d s o m e c h l o r o t i c s t r e a k i n g a n d d i s t o r t i o n o f u p p e r l e a v e s o f s m u t t e d c u l m s ( b u t n e v e r o f h e a l t h y c u l m s ) . I n o n e o f t h e s p r i n g p l a n t i n g s , s t r i p e 98 smutting was common i n the upper 1 to 2 leaves of smutted culms. High temperature probably induced t h i s symptom (Schafer et a l . t 1962). That smutted p l a n t s should be a f f e c t e d i n ways other than the smutting of f l o r e t s i s w e l l documented. Here, too, g e n e r a l i z a t i o n i s impossible. Reports of e f f e c t s on t i l l e r i n g , f o r example, are found f o r n e a r l y a l l c e r e a l smuts. Some workers reported reductions i n t i l l e r i n g (Welsh, 1932; Mather and Hansing, i960; Gaunt and Manners, 1971). Sampson (I927) and Holton and Rodenhiser (19^2) found increased t i l l e r i n g w i t h wheat-T. c a r l e s . Although no supporting data were presented, R u t t l e (193*0 observed reduced t i l l e r i n g by smutted p l a n t s of one b a r l e y c u l t l v a r i n o c u l a t e d w i t h U. h o r d e i . I t must be remembered th a t i n a l l of these experiments, nonsmutted p l a n t s were a l s o i n o c u l a t e d . Whether they were Infected or not one cannot say, s i n c e i n o c u l a t i o n techniques v a r i e d . In the present work, however, there was no s i g n i f i c a n t d i f f e r e n c e i n number of t i l l e r s per p l a n t r e a c h i n g m a t u r i t y when nonsmutted i n o c u l a t e d end smutted p l a n t s were compared. This c o n t r a s t s w i t h R u t t l e ' s f i n d i n g s . Although smutted p l a n t s produced an unusually high number of culms which l a t e r d i e d before reaching m a t u r i t y , t h i s phenomenon i s not confined t o smutted, or even to inocu-l a t e d , p l a n t s . I t seems to be a normal c h a r a c t e r i s t i c of p l a n t growth. Even under the best of co n d i t i o n s i n the 9 9 f i e l d , such dead culms were commonly observed. P e r c l v a l (1921) noted them i n wheat and speculated t h a t they were probably the r e s u l t of I n s u f f i c i e n t a d v e n t i t i o u s r o o t s coupled w i t h dry growing c o n d i t i o n s . I t i s not c l e a r why smutting should have the e f f e c t of i n c r e a s i n g t h e i r frequency. P o s s i b l y i t represents a p h y s i o l o g i c change caused by the developing s o r l , which deprives some of the culms of water or n u t r i e n t s . One can only speculate on the reason f o r the change i n p a t t e r n of p l a n t development shown i n Figure 30» ' I t Is f u r t h e r evidence f o r the presence of smut mycelium i n non-smutted i n o c u l a t e d p l a n t s , and i t p o i n t s up how much more s i m i l a r smutted, and. nonsmutted inoculated, p l a n t s are when compared to noninoculated p l a n t s . I t appears t h a t the presence of the smut i n h i b i t e d t i l l e r production at the lowermost nodes, which then caused the p l a n t s t o produce t h e i r l a t e r a l t i l l e r s from higher nodes. This Is c l e a r d e s p i t e the f a c t t h a t Inoculated p l a n t s i n i t i a t e d fewer culms than noninoculated p l a n t s . Whether t h i s i n h i b i t i o n i s due to a c t u a l death of p r l m o r d i a or to some other (more s u b t l e ) f a c t o r i s not c l e a r . 1 0 0 GENERAL CONCLUSIONS A huge l i t e r a t u r e has developed over many years concerning r e s i s t a n c e In c e r e a l s to smuts and bunts. Because of the o v e r r i d i n g economic c o n s i d e r a t i o n s , t o t a l or n e a r - t o t a l r e s i s t a n c e has been d e a l t w i t h almost e x c l u -s i v e l y ; but from the standpoint of h o s t - p a r a s i t e homeo-s t a s i s , p a r t i a l or Incomplete r e s i s t a n c e , such as that w i t h which t h i s t h e s i s d e a l s , Is a t l e a s t as important and pro-b a b l y more so. One must be c a r e f u l i n t r y i n g to extend the c o n s i d e r a b l e knowledge about t o t a l r e s i s t a n c e to i n c l u d e p a r t i a l r e s i s t a n c e . This work has not revealed any great d i f f e r e n c e s , however. P a r t s I and I I P g i v e two separate l i n e s of evidence which show t h a t i n •Hannchen' b a r l e y Inoculated w i t h a d i k a r y o n to which I t I s , i n the standard t h i n k i n g , " s u s c e p t i b l e , " nonsmutted Inoculated p l a n t s have not escaped I n f e c t i o n but, r a t h e r , have remained unsmutted as the r e s u l t of a type of a c t i v e r e s i s t a n c e . This i s shown i n P a r t I , which demonstrates the extreme e f f i c i e n c y w i t h which the i n o c u l a t i o n procedure insures that smut w i l l be In c o n t a c t w i t h the germinating seed. P a r t I I I shows t h a t , as In the m a j o r i t y of t o t a l or n e a r - t o t a l types of r e s i s -tance which have been I n v e s t i g a t e d i n s e v e r a l host-smut systems, U. hordei a f f e c t s the growth of nonsmutted p l a n t s , i n d i c a t i n g t h a t the mycelium Is present i n a t l e a s t a 1 0 1 p r o p o r t i o n o f t h e s e e d l i n g s f o r a n u n d e t e r m i n e d l e n g t h o f t i m e . P a r t I I p r e s e n t s , f o r t h e f i r s t t i m e i n a n y o f t h e c e r e a l s m u t s , c l e a r s t a t i s t i c a l e v i d e n c e t h a t t h e r e a r e t w o s e p a r a t e c o m p o n e n t s t o s m u t r e s i s t a n c e w h i c h a r e c l o s e l y c o r r e l a t e d . T h i s k n o w l e d g e w i l l b e e s s e n t i a l i n p o p u l a t i o n g e n e t i c s s t u d i e s , w h e r e a l l I m p o r t a n t e f f e c t s w h i c h a d d t o o r s u b t r a c t f r o m a g e n e ' s f i t n e s s m u s t b e u n d e r s t o o d a n d a c c o u n t e d f o r . I n p a r t i c u l a r , t h i s I n f o r m a t i o n s h o w s t h a t i n f i t n e s s s t u d i e s t h e m e a s u r e m e n t b a s e d o n p e r c e n t a g e o f i n f e c t e d p l a n t s , w h i c h h a s i n t h e p a s t b e e n m o s t o f t e n e m p l o y e d , w i l l b e i n a d e q u a t e s i n c e i t I g n o r e s w l t h i n - p l a n t d i s e a s e r e a c t i o n s . I n P a r t I I , f o r r e a s o n s g i v e n t h e r e , I t w a s n e c e s s a r y t o e x c l u d e n o n s m u t t e d I n o c u l a t e d p l a n t s w h e n o b t a i n i n g a m e a s u r e o f w i t h l n - p l a n t d i s e a s e s e v e r i t y . A t t h i s p o i n t , i t w o u l d b e u s e f u l t o c o n s i d e r t h e m o n c e a g a i n , a l o n g w i t h t h e p a r t i a l l y a n d t o t a l l y s m u t t e d p l a n t s , b e c a u s e P a r t s I a n d I I I i n d i c a t e t h a t t h e s e n o n s m u t t e d p l a n t s , a s w e l l a s t h e p a r t i a l l y s m u t t e d p l a n t s , c o n t r i b u t e t o d i s e a s e c u r t a i l m e n t v i a h o s t r e s i s t a n c e . T h e r e s e e m t o b e t w o d i s t i n c t t h r e s h o l d s , o r s t a g e s , t h r o u g h w h i c h t h e f u n g u s m u s t p a s s i n o r d e r t o p r o d u c e t e l l o s p o r e s . I f i t g e t s p a s t t h e f i r s t I t w i l l b e a b l e t o c a u s e s o m e s m u t t i n g i n t h e p l a n t . T h i s e v e n t i s m e a s u r e d i n t e r m s o f b e t w e e n p l a n t 1 0 2 d i s e a s e s e v e r i t y . I f I t c r o s s e s t h e s e c o n d t h r e s h o l d t h e f u n g u s w i l l f r e q u e n t l y s m u t m a n y o r a l l c u l m s o f t h e p l a n t . O n e c a n b e s t t h i n k o f t h i s e v e n t a s t a k i n g p l a c e s e p a r a t e l y i n e a c h c u l m . F o r c o m b i n a t i o n s w h i c h d i s p l a y a h i g h d e g r e e o f r e s i s t a n c e t h e d i s t i n c t i o n b e t w e e n t h e s e t w o t h r e s h o l d s i s n o t d e a r . F o r t h e s e c o m b i n a t i o n s t h e n o n s m u t t e d p l a n t s o c c u r r e d i n t h e h i g h e s t f r e q u e n c y , f o l l o w e d b y p l a n t s w i t h o n l y a m i n o r i t y o f c u l m s s m u t t e d ; t h e m o s t i n f r e q u e n t c l a s s e s w e r e t h o s e w i t h p l a n t s h a v i n g m o s t o r a l l c u l m s s m u t t e d . T h i s c o n t i n u o u s a n d s m o o t h g r a d a t i o n i s e v i d e n c e f o r a s i m i -l a r i t y o f m e c h a n i s m s i n v o l v e d i n w i t h i n - a n d b e t w e e n - p l a n t r e s i s t a n c e . H o w e v e r , t h e g r a d a t i o n i s n o t s m o o t h f o r c o m -b i n a t i o n s i n w h i c h d i s e a s e s e v e r i t y i s h i g h . H e r e t h e m o s t f r e q u e n t c l a s s e s w e r e t h e t w o e x t r e m e s . P l a n t s w e r e u s u a l l y e i t h e r t o t a l l y n o n s m u t t e d o r t o t a l l y s m u t t e d . I f t h e f i n d i n g s w i t h * H a n n c h e n ' a n d E x I c a n b e a p p l i e d t o o t h e r c o m b i n a t i o n s , a n d t h e r e I s n o r e a s o n w h y t h e y c a n n o t , t h e n i t i s c l e a r t h a t t h e f u n g u s i s p r e s e n t f o r a t i m e I n a p r o p o r t i o n , p o s s i b l y I n a l l , o f t h e i n o c u l a t e d s e e d l i n g s . I n m a n y p l a n t s t h e f u n g u s d o e s n o t g e t b e y o n d t h e f i r s t t h r e s h o l d , I . e . , I t d o e s n o t s u c c e e d i n s p o r u l a t i n g . I n p l a n t s i n w h i c h t h e s m u t d o e s s u r m o u n t t h i s b a r r i e r , i t u s u a l l y a l s o s u c c e e d s i n s u r m o u n t i n g t h e s e c o n d b a r r i e r , s o t h a t a l l c u l m s a r e s m u t t e d ; b u t I n a m i n o r i t y o f c u l m s t h e f u n g u s I s s t o p p e d a t t h e s e c o n d t h r e s h o l d . A l l l e v e l s o f r e s i s t a n c e a r e f o u n d a n d , I n 103 i n t e r m e d i a t e c o m b i n a t i o n s , t h e s e c o n d t h r e s h o l d b e c o m e s r?. r e l a t i v e l y m o r e i m p o r t a n t . T h u s t h e h i g h i n c i d e n c e o f n o n s m u t t e d p l a n t s , e v e n i n h i g h d i s e a s e c o m b i n a t i o n s , s h o w s t h a t t w o s e p a r a t e e v e n t s o c c u r . 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A P P E N D I X A 113 S t a t i s t i c a l c o m p a r i s o n o f b i n o m i a l r a t i o s T w o b i n o m i a l r a t i o s c a n b e s t a t i s t i c a l l y c o m p a r e d u s i n g a c o n f i d e n c e i n t e r v a l a b o u t t h e d i f f e r e n c e o f t h e i r p o p u l a t i o n m e a n s ( L i , 1964), T h e s h o r t h a n d f o r m , u s i n g 1.96*, t h e z, v a l u e f o r 0,95 a l p h a , l i s : w h e r e 1 D = T h e d i f f e r e n c e b e t w e e n t h e o b s e r v e d r a t i o s w h i c h l a r e t o b e c o m p a r e d ( a n e s t i m a t e o f t h e i r p o p u l a t i o n d i f f e r e n c e ) , p-j_= T h e p r o p o r t i o n o f s a m p l e o n e o b s e r v a t i o n s o f a g i v e n o u t c o m e , P g = T h e p r o p o r t i o n o f s a m p l e t w o o b s e r v a t i o n s o f a g i v e n o u t c o m e , n i = T h e n u m b e r o f s a m p l e o n e o b s e r v a t i o n s , n £ = T h e n u m b e r o f s a m p l e t w o o b s e r v a t i o n s , I n a p p l y i n g t h i s t e s t , i f t h e d i f f e r e n c e b e t w e e n t h e s a m p l e p r o p o r t i o n s , D , i s g r e a t e r t h a n t h e c a l c u l a t e d c o n f i d e n c e i n t e r v a l ( i n o n e d i r e c t i o n ) , t h e r e i s a s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e t w o r a t i o s . I f n o d i f f e r e n c e e x i s t s , t h e i n t e r v a l a b o u t D , o f c o u r s e , c o n t a i n s z e r o • A c c o r d i n g t o S t e e l a n d T o r r i e ( i960), c e r t a i n m i n i m u m s a m p l e s i z e s m u s t b e o b s e r v e d i n o r d e r f o r t h e s t a t i s t i c s p ^ a n d p g t o a c c u r a t e l y e s t i m a t e t h e 1 1 4 corresponding p o p u l a t i o n parameters as f o r a normal d i s t r i b u t i o n . This i s shown i n t a b l e A . l . TABLE A . l BINOMIAL SAMPLE SIZES FOR NORMAL APPROXIMATION TO APPLY P Number observed i n ~~~ ' s m a l l e r c l a s s Sample s i z e _ . . (ml ; ( n ) 0.5 15 30 0 . 4 2 0 50 0 . 3 2 4 8 0 0 . 2 4 0 2 0 0 0 . 1 60 6 0 0 0.05 7 0 1 4 0 0 115 APPENDIX B i / * 3 / / / or / ^ -z. ( ")—^ / \J >s— ^ X -J y/ o •2 ^ , o> II - « o <^ > "ci W -<? o o •-. o V s: <=i E 2 . 3 - o =j s "d •<? <j II «? ^ •>> o S "ci ci 3 -"CJ Si « = ^ 3 cJ s; o ci S E -a <j -Ed S i £ ^> O - S ! 0 •o *e~cs ^ c^ J # 3 PUBLICATIONS Groth, J.V., V.E. Comstock and N.A. Anderson. 1968. Resistance of p a i r s of yellow-and brown-seeded is o g e n i c l i n e s of f l a x to Rhi z o c t on i a s e e d l i n g b l i g h t . Phyto-pathology 58:1052 (Abst.). Groth, J.V. and N.A. Anderson. 1969. Het-erokaryon i n c o m p a t i b i l i t y i n Rh i z o c t o n i a  s o l a n i . . Phytopathology 59:1028 (Abst.). Groth, J.V., V.E. Comstock.and N.A. Anderson. 1970. E f f e c t of seed color on t o l e r -- ance of f l a x to see d l i n g b l i g h t caused by Rh i z o c t o n i a s o l a n i . Phytopathology 60:379-380. Anderson, N.A., H.M. S t r e t t o n , J.V. Groth and N.T. F l e n t j e . 1972. Genetics of heterokaryosis i n Thanatephorus cu-c urne r i s. Phytopathology 62 : 105 7-1065. Groth, J.V. 1973. The e f f e c t of unequal mating-type r e p r e s e n t a t i o n on i n f e c -t i o n of ba r l e y by U s t i l a g o h o r d e i . Proceedings of the Second Interna-t i o n a l Congress of Pla n t Pathology #708 (Ab s t . ) . Person, CO. and J.V. Groth. 19 73. Sta-b i l i z i n g s e l e c t i o n i n polygenic par-a s i t i c systems. I n v i t e d paper pre-sented at the Second I n t e r n a t i o n a l Congress of Plant Pathology. Groth, J.V., and CO. person. 197A. The expression of r e s i s t a n c e i n barley to U s t i l a g o hordei. 66th Annual Meeting of the American Phytop a t h o l o g i c a l S o c i e t y , Vancouver (Abst.). AWARDS . 1963 Chapman Foundation Forestry Schol-arship . 1965 Member Xi Sigma P i Honorary Forestry F r a t e r n i t y . 1967. Chaney Award to Outstanding Forestry Senior. 

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