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The axenic culture of wheat and flax rust fungi Bose, Amitava 1973

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THE AXKNIC CULTURE OF WHEAT AND FLAX RUST FUNGI by AMETAVA BOSE 3 . S c . ( A g r . ) , B i h a r A g r i c u l t u r a l C o l l e g e M . S c . ( A g r . ) , B h a g a l p u r U n i v e r s i t y M.Sc., 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 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 f o r t h e Degree o f DOCTOR OF PHILOSOPHY i n t h e Department o f P l a n t S c i e n c e We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA O c t o b e r , 1973 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e -q u i r e m e n t s f o r an a d v a n ced d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t -ment o r b y h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y -i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D epartment o f P l a n t S c i e n c e , - U n i v e r s i t y o f B r i t i s h C o l u m b i a , V a n c o u v e r 8, B r i t i s h C o l u m b i a . Date im-M-1^ ABSTRACT R u s t f u n g i b e l o n g i n g t o t h e o r d e r U r e d i n a l e s have u s u a l l y b e e n c o n s i d e r e d t h e c l a s s i c a l e x a mples o f o b l i g a t e p a r a s i t e s amongst p l a n t p a t h o g e n i c f u n g i . The d e v e l o p m e n t o f o u r k n o w l e d g e o f t h e m e t a b o l i s m , n u t r i t i o n and p h y s i o l o g y o f t h e r u s t f u n g i has been r e s t r i c t e d b e c a u s e o f o u r i n a b i l i t y t o grow t h e s e f u n g i i n a x e n i c c u l t u r e . I n t h e p a s t t h e p h y s i o l o g i c a l and b i o c h e m i c a l i n v e s t i g a t i o n s on r u s t f u n g i p e r se have been l i m i t e d t o s t u d i e s o f t h e g e r -m i n a t i o n and d e v e l o p m e n t o f germ t u b e s . The c u l t i v a t i o n i n v i t r o o f P u c c i n i a g r a m i n i s t r i t i c i ( E r i k s s . and .Henri. ) , A u s t r a l i a n r a c e ANZ 1 2 6 - 6 , 7 , by W i l l i a m s e t a l . ( 1966, 1 9 6 7 ) , t o g e t h e r w i t h t h e r e p o r t o f T u r e l (1969) on t h e a x e n i c c u l t u r e o f M e l a m p s o r a l i n i ( E h r e n b ) L e v , r a c e 3 p r o m o t e d r e s e a r c h on c u l t u r i n g r u s t f u n g i on a r t i f i c i a l medium. An a r t i f i c i a l medium c o n t a i n i n g 3% g l u c o s e , C z a p e k ' s m i n e r a l s a l t s , 0.1% E v a n ' s p e p t o n e , p l u s d e f a t t e d BSA s u p p o r t -ed g r o w t h and s p o r u l a t i o n o f P u c c i n i a g r a m i n i s t r i t i c i r a c e ANZ 1 2 6 - 6 , 7 . T y p i c a l p i g m e n t e d u r e d o s p o r e s and t e l i o s p o r e s were f o r m e d a f t e r 6-3 weeks g r o w t h . The u r e d o s p o r e s were c a p a b l e o f i n f e c t i n g t h e m e s o p h y l l o f wheat l e a v e s e x p o s e d by s t r i p p i n g b a c k t h e l o w e r e p i d e r m i s . S c a n n i n g e l e c t r o n m i c r o s c o p y r e v e a l e d t h e p r e s e n c e o f a c o a t i n g o f unknown chem-i c a l c o m p o s i t i o n a r o u n d t h e u r e d o s p o r e s d e v e l o p e d i n v i t r o and not o b s e r v e d on u r e d o s p o r e s grown on wheat l e a v e s . i v Two d i f f e r e n t s t r a i n s o f M e l a m p s o r a l i n i ( E h r e n b ) Le v were grown on s o l i d m e d i a c o n t a i n i n g I+% s u c r o s e , m o d i f i e d Knop's t i s s u e c u l t u r e m a c r o n u t r i e n t s , B e r t h e l o t ' s m i c r o n u t -r i e n t s , y e a s t e x t r a c t and p e p t o n e . The m y c e l i u m was g e n e r a l l y b i n u c l e a t e . S p o r e - l i k e s t r u c t u r e s were r e c o r d e d i n t h e s t r o m a w h i c h r e s e m b l e d u r e d o s p o r e s and t e l i o s p o r e s . A d d i t i o n o f 1% d e f a t t e d BSA t o t h e medium d e s c r i b e d above g r e a t l y i n c r e a s e d t h e f r e q u e n c y o f e s t a b l i s h m e n t o f f l a x r u s t c o l o n i e s . A de-f i n e d l i q u i d medium, c o n t a i n i n g C z a p e k ' s m i n e r a l s y Ca++, g l u -c o s e , a s p a r t i c a c i d , g l u t a t h i o n e and c y s t e i n e , and i n o c u l a t e d w i t h u n c o n t a m i n a t e d u r e d o s p o r e s , s u p p o r t e d good v e g e t a t i v e g r o w t h and s p o r u l a t i o n o f wheat stem r u s t ( P u c c i n i a g r a m i n i s f . s p . t r i t i c i r a c e ANZ 1 2 6 - 6 , 7 ) . Of s i x N o r t h A m e r i c a n r a c e s o f wheat stem r u s t f u n g u s t e s t e d , o n l y t h r e e grew v e g e t a t i v e l y on a r t i f i c i a l medium. F i n a l l y a c h e m i c a l l y d e f i n e d l i q u i d medium c o n t a i n -i n g s u c r o s e , Knop's m i n e r a l s a l t s , m i c r o n u t r i e n t s , a s p a r t i c ( o r g l u t a m i c ) a c i d and c y s t e i n e s u p p o r t e d t h e g r o w t h o f v e g e -t a t i v e c o l o n i e s o f M e l a m p s o r a l i n i r a c e 3 f r o m u n c o n t a m i n a t e d u r e d o s p o r e s I n a h i g h l y r e p r o d u c i b l e manner. The f o r m a t i o n o f u r e d o s p o r e s and t e l i o s p o r e s o f t h e s e c o l o n i e s i n t h e l i q u i d medium was c o n t r o l l e d b y t h e l e v e l o f Ca++ ( a s CatNO-^)^ and t h e number o f c o l o n i e s p e r f l a s k . W i t h 6 0 - 7 0 c o l o n i e s p e r f l a s k , u r e d o s p o r e f o r m a t i o n o c c u r r e d on 6 0 t o 7 0 % o f t h e c o l o n i e s a t a Ca++ l e v e l 8 . 5 mM. A d e c r e a s e i n t h e Ca++ l e v e l t o 4 . 5 mM o r c o l o n y f r e q u e n c y t o 1 0 p e r f l a s k r e s u l t e d V i n o n l y i n f r e q u e n t s p o r u l a t i o n . v i t r o i n f e c t e d i n t a c t c o t y l e d o n s r e s u l t w i t h f l a x r u s t r e p r e s e n t s a b i l i t y t o c o n t r o l t h e g r o w t h o f p a r a s i t e I n a x e n i c c u l t u r e . The u r e d o s p o r e s p r o d u c e d i n I n a n o r m a l manner. T h i s a s u b s t a n t i a l a d v a n c e i n o u r t h i s i m p o r t a n t ' o b l i g a t e * v i TABLE OF CONTENTS Page FRONTISPIECE Xiv ABSTRACT i i i • TABLE OF CONTENTS v i L I S T OF TABLES i x L I S T OF FIGURES x ACKNOWLEDGEMENTS x i i i GENERAL INTRODUCTION 1 REFERENCES 16 CHAPTER I - S p o r u i a t i o n and P a t h o g e n i c i t y o f an A u s t r a l i a n I s o l a t e of-Wheat R u s t grown i n v i t r o . ABSTRACT..; 20 INTRODUCTION 21 MATERIALS AND METHODS 22 RESULTS 24 1. B e h a v i o u r o f G e r m i n a t i n g U r e d o s p o r e s on S o l i d Medium 21+ 2. Development o f C o l o n i e s on L i q u i d Medium 24 3 . P a t h o g e n i c i t y o f U r e d o s p o r e s p r o d u c e d i n A x e n i c C u l t u r e 30 4. S c a n n i n g E l e c t r o n M i c r o s c o p y , . . . 30 Page .CHAPTER I I . I n v i t r o C u l t u r e of the F l a x Rust, Melampsora l i n i . ABSTRACT 3 6 INTRODUCTION 3 6 MATERIALS AND METHODS 3 7 1 . Melampsora l i n i , S t r a i n 1 3 7 2 . Melampsora l i n i , S t r a i n 2 . . . . .- 4 0 3 . C y t o l o g i c a l Examination of the C u l t u r e s 4 1 RESULTS 4 1 1 • Melampsora l i n i , S t r a i n 1 . . . 4 1 2 . Melampsora l i n i , S t r a i n 2 4 3 3 . C y t o l o g i c a l S t u d i e s w i t h Melampsora l i n i 1+6 DISCUSSION 51 REFERENCES.' 5 9 CHAPTER I I I . I n v i t r o Growth of Wheat and F l a x Rust Fungi on C h e m i c a l l y D e f i n e d Media. ABSTRACT 60 INTRODUCTION •. . . 6 1 MATERIALS AND METHODS 68 1 . P r o d u c t i o n of Uncontaminated Uredospores 6 8 2 . C u l t u r e Media.. ' 6 9 3 . Sources of Organic N i t r o g e n and Sulphur 7 0 4 . I n o c u l a t i o n of Media 7 1 5. Assessment of Growth.. 7 2 RESULTS 7 2 Wheat Rust 7 2 1 . T r i a l s w i t h S o l i d Media 7 2 V X l l Page 2 . T r i a l s w i t h L i q u i d M e d i a 73 A. Growth o f r a c e s 15B4, 3 # , 3 2 - 1 1 3 and ANZ 1 2 6 - 6 , 7 7 3 B. E f f e c t o f cAMP on g r o w t h o f ANZ 1 2 6 - 6 , 7 73 C. E f f e c t o f a s p a r t i c a c i d , c y s t e i n e , g l u t a -t h i o n e and c a l c i u m on t h e g r o w t h o f ANZ 1 2 6 - 6 , 7 7 4 F l a x R u s t 7 9 1 . T r i a l s w i t h S o l i d M e d i a . , 7 9 A. E f f e c t o f BSA 7 9 B. R e i n f e c t i o n o f h o s t u s i n g v e g e t a t i v e c o l o n i e s grown on s o l i d medium 80 2 . T r i a l s w i t h L i q u i d M e d i a 8 l A. P r e l i m i n a r y t r i a l s and e f f e c t o f BSA 81 B. E f f e c t s o f m i n e r a l n u t r i e n t s 8 5 C. E f f e c t s o f a s p a r t i c a c i d , c y s t e i n e , g l u t a -t h i o n e and c a l c i u m 8 8 D. - R e i n f e c t i o n o f f l a x u s i n g u r e d o s p o r e s grown i n v i t r o 9 1 TABLE L I S T OF TABLES PAGE I Growth of o b l i g a t e p a r a s i t e s i n h o s t t i s s u e 5 I I I n v i t r o d e v e l o p m e n t o f r u s t f u n g i f r o m g e r m i n a t i n g u r e d o s p o r e s 8 I I I - I E f f e c t o f c y c l i c AMP and t h e o p h y l l i n e on g r o w t h o f P u c c i n i a g r a m i n i s t r i t i c i ( r a c e ANZ 1 2 6 - 6 , 7 ) 77 I I I - I I E f f e c t o f amino a c i d , g l u t a t h i o n e and C a ( N 0 ^ ) 2 on t h e g r o w t h o f P u c c i n i a g r a m i n i s t r i t i c i ( r a c e ANZ 126-6,7) 78 I I I - I I I E f f e c t o f d i f f e r e n t o r g a n i c n i t r o g e n s o u r c e s on t h e g r o w t h o f M e l a m p s o r a l i n i ( r a c e 3 ) $k I I I - I V Growth and d e v e l o p m e n t o f M e l a m p s o r a l i n i ( r a c e 3) on c o m p l e t e l y d e f i n e d l i q u i d m e d i a . . . X FIGURE TiTS-T OF FIGURES PAGE 1-1 U r e d o s p o r e s w i t h b u r s t germ t u b e on b a s i c medium 26 1-2 Two g e r m i n a t i n g u r e d o s p o r e s and t h e i r germ t u b e s have c r o s s e d each o t h e r 26 1-3 Two n u c l e i i n c y t o p l a s m e x t r u d e d f r o m b u r s t germ tu b e 26 1-4 Germ t u b e s o r i g i n a t i n g f r o m two u r e d o s p o r e s showing a n a s t o m o s i s 26 1-5 V e g e t a t i v e j u v e n i l e c o l o n i e s and s p o r u l a -t i n g c o l o n i e s d e v e l o p e d on l i q u i d medium 26 1-6 D e v e l o p i n g u r e d o s p o r e s i n c o l o n y grown on l i q u i d medium 1-7 D e v e l o p i n g t e l i o s p o r e s and m a t u r i n g u r e d o -s p o r e s f r o m c o l o n y grown on l i q u i d medium 28 1-8 F u l l y d e v e l o p e d t w o - c e l l e d t e l i o s p o r e s . . . . , , . . . 28 1-9 T h i c k mat o f hyphae a t p e r i p h e r y o f c o l o n y 28 grown on l i q u i d medium, 28 1-10 P u s t u l e s d e v e l o p e d on p r i m a r y l e a f o f L i t t l e C l u b wheat as a r e s u l t o f i n o c u l a t i o n w i t h u r e d o s p o r e s grown i n v i t r o 28 1-11 S c a n n i n g e l e c t r o n m i c r o g r a p h s of mature u r e d o s p o r e s grown on i n t a c t wheat l e a v e s 32 1-12 S c a n n i n g e l e c t r o n m i c r o g r a p h of mature u r e d o s p o r e s grown i n v i t r o . . '. 32 1-13 S c a n n i n g e l e c t r o n m i c r o g r a p h o f s p i n e s on s u r f a c e o f u r e d o s p o r e s grown on i n t a c t wheat l e a f 32 1-14 S c a n n i n g e l e c t r o n m i c r o g r a p h o f s p i n e s on s u r f a c e o f u r e d o s p o r e s grown on b a s i c medium... 32 1-15 S c a n n i n g e l e c t r o n m i c r o g r a p h o f d e v e l o p i n g s p i n e s on s u r f a c e o f u r e d o s p o r e s grown on i n t a c t wheat l e a f 32 1-16 S c a n n i n g e l e c t r o n m i c r o g r a p h of a t e l i o s p o r e grown on b a s i c medium 32 FIGURE x i PAGE I I - l A 12-day-old c o l o n y o f s t r a i n 1 ( f o u r t h t r a n s f e r ) . . , , 45 I I - 2 Same c o l o n y v i e w e d f r o m below... 45 I I - 3 An 8-week-old c o l o n y o f s t r a i n 2 grown f r o m a s e p t i c u r e d o s p o r e s 4$ I I - 4 A n " 8 - w e e k - o l d c o l o n y o f s t r a i n . 2 o f s m a l l s i z e c o n s i s t i n g e n t i r e l y o f a e r i a l m y c e l i u m . . . 48" I I - 5 F u s i o n o f germ t u b e s ,.. 48" I I - 6 B i n u c l e a t e hyphae o f an i n i t i a l c o l o n y . . 4$ I I - 7 B i n u c l e a t e m y c e l i u m o f a t r a n s f e r r e d c o l o n y , 50 I I - 3 U n i n u c l e a t e and b i n u c l e a t e h y p h a l c e l l s f r o m an o l d c o l o n y 50 II-9 A n a s t o m o s i s o f a e r i a l hyphae o f an o l d c o l o n y ' 50 11-10 T h i n w a l l e d s p h e r i c a l c e l l s f r o m s t r o m a t a . o f an i n i t i a l c o l o n y 50 11-11 T h i n w a l l e d s p h e r i c a l c e l l s i n t e r s p e r s e d by s h o r t h y p h a l c e l l s 50 11-12 " M i c r o s o r u s " s t r u c t u r e f r o m an i n i t i a l c o l o n y 53 11-13 Non-pigment ed u r e d o s p o r e l i k e c e l l s 53 11-14 U r e d o s p o r e l i k e c e l l s j o i n e d by s h o r t h y p h a l c e l l s 53 11-15 Dense s p o r e c l u s t e r f r o m s t r o m a t a o f a t r a n s f e r r e d c u l t u r e . ;. . . 55 11-16 S p o r e l i k e c e l l s f o r m e d a t t e r m i n a l hyphae..... 55 I I - 17 S p o r e l i k e c e l l s f o r m e d i n c h a i n s . 55 I I I - l N o r t h A m e r i c a n i s o l a t e s o f wheat stem r u s t s h o w i n g v e g e t a t i v e growth on l i q u i d medium 76 I I I - 2 Growth and d e v e l o p m e n t o f an A u s t r a l i a n r a c e of wheat stem r u s t on l i q u i d medium 76 tern r u s t ANZ 126-6 ,7 d e v e l o p e d on a c h e m i c a l i v d e f i n e d medium 7o .'.'hsat s FIGURE I I I - 4 F l a x r u s t c o l o n y i n i t i a l s r a i s e d on s o l i d medium I I I - 5 C o l o n i e s of f l a x r u s t o r i g i n a t i n g as a r e -s u l t of g e l a t i n spore suspension s e e d i n g on s o l i d medium I I I - 6 E c t o p a r a s i t i c mycelium of f l a x r u s t de-veloped on e x c i s e d c o t y l e d o n I I I - 7 F l a x r u s t c o l o n i e s developed on l i q u i d medium I l l - 8 F l a x r u s t c o l o n i e s developed on l i q u i d media s u r f a c e . The medium c o n t a i n s e i t h e r peptone p l u s BSA or amino a c i d m i x t u r e p l u s BSA as an o r g a n i c n i t r o g e n source..... I l l - 9 F l a x r u s t development under submerged c o n d i t i o n . . I l l - 1 0 Growth and development of. f l a x r u s t on c h e m i c a l l y d e f i n e d media I l l - 1 1 In. v i t r o developed uredospores of f l a x r u s t . a 111-12 I n v i t r o developed t e l l o s p o r e s of f l a x r u s t . . I l l - 1 3 V e g e t a t i v e and s p o r u l a t i n g c o l o n i e s of f l a x r u s t developed on a c h e m i c a l l y d e f i n e d medium 111-14 A x e n i c a l l y developed uredospores i n f e c t i n g the c o t y l e d o n when p l a c e d on exposed meso-p h y l l , 111-1$ A x e n i c a l l y developed uredospores i n f e c t i n g i n t a c t c o t y l e d o n X 1 X X ACKNOWLEDGEMENTS I e x p r e s s my s i n c e r e a p p r e c i a t i o n t o D r . M i c h a e l Shaw f o r s u g g e s t i n g t h e p r o b l e m and f o r a d v i c e and encouragement d u r i n g t h e c o u r s e o f t h e r e s e a r c h and i n t h e p r e p a r a t i o n o f t h e t h e s i s . C h a p t e r I I has been p u b l i s h e d i n t h e - C a n a d i a n J o u r n a l o f B o t a n y w i t h D r . M. D. C o f f e y as t h e s e n i o r a u t h o r . I t h a n k Dr. C o f f e y f o r h i s a c t i v e c o - o p e r a t i o n and g r a t e f u l l y acknow-l e d g e h i s h e l p . I a l s o t h a n k D r . C . 0. P e r s o n f o r h e l p w i t h t h e p h o t o m i c r o g r a p h y ; M r s . G a y l e S m i t h f o r t y p i n g t h e manu-s c r i p t ; and many o t h e r p e o p l e who have h e l p e d me i n v a r i o u s ways. T h i s r e s e a r c h was s u p p o r t e d by g r a n t s t o D r . M. Shaw f r o m t h e N a t i o n a l R e s e a r c h C o u n c i l o f Canada and t h e Canada Department o f A g r i c u l t u r e . till FRONTISPIECE "There i s h a r d l y a q u e s t i o n i n p h y t o p a t h o l o g y so a b s o r b i n g y e t so e l u s i v e as t h e o b l i g a t e p a r a s i t i s m o f t h e r u s t f u n g i . Many i n v e s t i g a t o r s have s o u g h t i n v a i n t h e p h i l o s o p h e r ' s s t o n e u n d e r l y i n g t h e p r o b l e m : a means o f c u l t u r i n g r u s t s on n u t r i e n t m e d i a o t h e r t h a n t h e l i v i n g p l a n t s " . ( C h e s t e r 1 9 4 6 ) . " P l a n t p a t h o l o g i s t s , p l a n t p h y s i o l o g i s t s , and o t h e r s have t r i e d u n s u c c e s s f u l l y f o r about 100 y e a r s t o c u l t u r e v i r u s e s , downy m i l d e w s , powdery m i l d e w s and r u s t s on n o n l i v i n g m e d i a " . (Yarwood 1 9 5 6 ) . Growth o f f l a x r u s t M elampsora l i n i ( E h r e n b .) Le"v on chemi-c a l l y d e f i n e d l i q u i d medium c o n t a i n i n g i n o r g a n i c s a l t s , s u c -r o s e , a s p a r t i c a c i d and c y s t e i n e . A. C o l o n y i n i t i a t i o n f r o m g e r m i n a t i n g u r e d o s p o r e s . X 5 . B. F u l l y d e v e l o p e d v e g e t a t i v e c o l o n i e s . X 15 . C. F u l l y d e v e l o p e d c o l o n y w i t h mature u r e d o s p o r e s . X 15 . D. I n t a c t f l a x c o t y l e d o n s i n o c u l a t e d w i t h u r e d o s p o r e s t a k e n f r o m an a x e n i c c o l o n y l i k e t h a t i n C and sho w i n g t y p i c a l f l a x r u s t u r e d o p u s t u l e s . 1 GENERAL INTRODUCTION DeBary (1387) d e f i n e d o b l i g a t e p a r a s i t e s as " s p e c i e s t o w h i c h a p a r a s i t i c l i f e i s e s s e n t i a l f o r t h e a t t a i n m e n t o f • t h e i r f u l l d e v e l o p m e n t " . He r e c o g n i z e d t h a t t h i s c a t e g o r y s h o u l d be f u r t h e r d i v i d e d i n t o (1) s t r i c t l y o b l i g a t e p a r a -s i t e s , (2) s p e c i e s w h i c h as f a r as i s known l i v e o n l y as p a r a -s i t e s , and (3) f a c u l t a t i v e s a p r o p h y t e s w h i c h c a n , t h o u g h u s u a l l y t h e y do n o t , l i v e as s a p r o p h y t e s f o r a t l e a s t p a r t o f t h e i r l i f e c y c l e s . The r u s t f u n g i have l o n g been r e g a r d e d as s t r i c t o b l i g a t e p a r a s i t e s o f v a s c u l a r p l a n t s , I . e . t h e y c o u l d n o t be c u l t i v a t e d a x e n i c a l l y . S i n c e p r e c i s e s t u d i e s on t h e n u t r i t i o n and m e t a b o l i s m o f t h e s e o b l i g a t e p a r a s i t e s c a n h a r d l y be c a r r i e d o u t i n t h e p r e s e n c e o f h o s t p l a n t t i s s u e , r e p e a t e d a t t e m p t s were made b y v a r i o u s g r o u p s t o ad a p t them t o a s a p r o p h y t i c mode o f e x i s t e n c e . Most o f t h e s e a t t e m p t s have n o t been r e c o r d e d i n p u b l i c a t i o n and many r e -g a r d e d t h e t a s k as I m p o s s i b l e . F o r example, C h e s t e r i n 1946 w r o t e , "There i s h a r d l y a q u e s t i o n i n p h y t o p a t h o l o g y so ab-s o r b i n g y e t so e l u s i v e as t h e o b l i g a t e p a r a s i t i s m o f t h e r u s t f u n g i . Many i n v e s t i g a t o r s have s o u g h t I n v a i n t h e p h i l o s o -p h e r ' s s t o n e u n d e r l y i n g t h e p r o b l e m : a means o f c u l t u r i n g r u s t s on n u t r i e n t m e d i a o t h e r t h a n t h e l i v i n g p l a n t " . He p o i n t e d o ut t h a t i t was b e l i e v e d t h a t i n t h e n u t r i t i o n o f t h e r u s t f u n g u s , l i e s t h e s e c r e t o f many b a s i c p r o b l e m s : t h e o b l i g a t e p a r a s i t i s m o f t h e r u s t s , t h e i r a l t e r n a t i o n o f h o s t s , 2 t h e i r n a r r o w h o s t r a n g e s , t h e i r p h y s i o l o g i c s p e c i a l i z a t i o n , t h e i r dependence on a c t i v i t y and v i g o r o f t h e h o s t p l a n t , and t h e m e c h a n i s m s f o r r e s i s t a n c e o f h o s t t o r u s t . W r i t i n g t e n y e a r s a f t e r C h e s t e r , Yarwood (1956) was t h e f i r s t t o c o n s i d e r t h e f i e l d o f o b l i g a t e p a r a s i t i s m i n a b r o a d c o m p r e h e n s i v e r e v i e w . He s t a t e d t h a t , " s i n c e a l l t h e v i r u s e s , most o f t h e c h y t r i d s , most p a r a s i t i c h i g h e r p l a n t s , and many m i s c e l l a n e o u s f u n g i a r e a l s o o b l i g a t e p a r a s i t e s a c c o r d i n g t o t h e p r e s e n t k n o w l e d g e , i t seems l i k e l y t h a t a b o u t o n e - f o u r t h o f p l a n t p a r a s i t e s a r e o b l i g a t e p a r a s i t e s " . These o b l i g a t e p a r a s i t e s i n c l u d e t h e c a u s e s o f many o f o u r most i m -p o r t a n t p l a n t d i s e a s e s , e.g. r u s t s , m i l d e w s , e t c . M o s t f u n g i r e g a r d e d as o b l i g a t e p a r a s i t e s by DeBary a r e s t i ] 1 so r e g a r d -ed ( Y arwood 1956) . P r i o r t o t h e 1960's l i m i t e d s u c c e s s v/as a c h i e v e d i n c u l t u r i n g l i c h e n f u n g i , and a number o f c h y t r i d s p r e v i o u s l y known t o be o b l i g a t e s . T h i s s i t u a t i o n o b v i o u s l y i n d i c a t e d t h e f e a s i b i l i t y o f c u l t u r i n g r u s t f u n g i on non-l i v i n g s u b s t r a t a . Yarwood e x p r e s s e d t h e o p i n i o n t h a t i t w o u l d u l t i m a t e l y be p o s s i b l e t o grow t h e s e o r g a n i s m s i n a x e n i c c u l -t u r e b u t q u e s t i o n e d w h e t h e r o r n o t t h e s u c c e s s f u l a x e n i c c u l -t u r e o f o b l i g a t e p a r a s i t e s o f p l a n t s w o u l d a i d i n t h e i r c o n -t r o l . Ten y e a r s a f t e r Yarwood's r e v i e w , P.W. B r i a n e n -t i t l e d The Leeuwenhoek L e c t u r e f o r 1966 t o t h e R o y a l S o c i e t y o f London " O b l i g a t e p a r a s i t i s m i n f u n g i " ( B r i a n 1967). I n h i s l e c t u r e B r i a n a l s o s u g g e s t e d t h a t f u n g i c o n s i d e r e d t o be 3 o b l i g a t e p a t h o g e n s w o u l d e v e n t u a l l y be grown I n a x e n i c c u l -t u r e o n c e t h e i r m e t a b o l i c p e c u l i a r i t i e s v/ere u n d e r s t o o d ( B r i a n 1967). I n d e s c r i b i n g t h e p h y s i o l o g y o f o b l i g a t e p a r a -s i t i s m he m e n t i o n e d t h a t g e r m i n a t i n g u r e d o s p o r e s o f r u s t do n o t l a c k t h e c a p a c i t y t o s y n t h e s i z e p r o t e i n . A d a p t i v e , as w e l l as c o n s t i t u t i v e enzymes, a p p e a r d u r i n g g e r m i n a t i o n and exogenous amino a c i d s c a n be i n c o r p o r a t e d i n t o s u c h p r o t e i n s . He e m p h a s i z e d t h e p o i n t t h a t , as o p p o s e d t o s p o r e l i n g s o f n o n - o b l i g a t e p a r a s i t e s , no n e t p r o t e i n s y n t h e s i s t a k e s p l a c e d u r i n g u r e d o s p o r e g e r m i n a t i o n . New p r o t e i n m o l e c u l e s f o r m e d a r e m a i n l y t h e r e s u l t o f t u r n o v e r a t t h e expense o f e x i s t i n g p r o t e i n . RNA and DNA s y n t h e s i s a r e s i m i l a r l y r e s t r i c t e d . Two a p p r o a c h e s have been employed i n a t t e m p t s t o c u l t u r e t h e r u s t f u n g i i n v i t r o : (1) The use o f dual-membered c a l l u s c u l t u r e s i . e . i n v i t r o g r o w t h o f i n f e c t e d h o s t t i s s u e l e a d i n g t o t h e e s t a b l i s h m e n t o f s a p r o b i c f u n g a l c o l o n i e s on t h e t i s s u e c u l t u r e medium, (2) A x e n i c c u l t u r e o f t h e p a t h o g e n s d i r e c t l y f r o m u r e d o s p o r e s g e r m i n a t e d on n u t r i e n t m e d i a . Dual-membered c a l l u s c u l t u r e s p r o v i d e a s i m p l e s y s -tem f o r t h e s t u d y o f t h e b i o c h e m i c a l and u l t r a s t r u c t u r a l e v e n t s t h a t accompany g r o w t h o f t h e p a t h o g e n w h i c h a v o i d s some o f t h e p h y s i o l o g i c a l c o m p l e x i t i e s o f t h e i n f e c t e d w h o l e p l a n t . S u c h s t u d i e s may be a b l e t o d e f i n e t h e e v e n t s i n dual-membered s y s t e m s w h i c h e n a b l e t h e p a r a s i t e t o d e v e l o p a s a p r o b i c mode o f e x i s t e n c e . A summary o f t h e s u c c e s s f u l a t t e m p t s t o e s t a b l i s h o b l i g a t e p a r a s i t e s i n a x e n i c c u l t u r e t h r o u g h t h e use o f d u a l -4 membered c a l l u s c u l t u r e s I s p r e s e n t e d i n T a b l e 1 . These e a r l i e r w o r k s g r e a t l y a i d e d i n u n d e r s t a n d i n g t h e b a s i c p r i n -c i p l e s i n v o l v e d I n c u l t u r i n g r u s t f u n g i . I t was r e c o g n i z e d by M o r e l (194$) t h a t t h e i n o c u l a -t i o n o f c a l l u s c u l t u r e s b y u r e d o s p o r e s i s d i f f i c u l t o w i n g t o t h e h i g h r a t e o f c o n t a m i n a t i o n . He a t t r i b u t e d h i s l a c k o f s u c c e s s i n a t t e m p t i n g t o c u l t u r e r u s t on u n d i f f e r e n t i a t e d h o s t t i s s u e t o an i n a d e q u a t e i n o c u l a t i o n t e c h n i q u e , and s u g g e s t e d t h a t s u c c e s s f u l c u l t u r e s o f r u s t s m i g h t b e s t be a c h i e v e d s t a r t i n g f r o m s y s t e m i c a l l y i n f e c t e d p l a n t t i s s u e , Which c o u l d be v i g o r o u s l y s u r f a c e - s t e r i l i z e d . T h i s was i n f a c t a c h i e v e d by H o t s o n and C u t t e r (1951). U n f o r t u n a t e l y o t h e r w o r k e r s have n o t been a b l e t o o b t a i n a x e n i c c u l t u r e s o f t h e r u s t s c u l t u r e d by C u t t e r and hence h i s work h a s n o t r e -c e i v e d t h e r e c o g n i t i o n i t i s now c o n s i d e r e d t o d e s e r v e . However, i t was r e a l i z e d t h a t m a i n t e n a n c e o f t h e c u l -t u r e and dev e l o p m e n t o f t h e p a r a s i t e depends on e s t a b l i s h i n g a p r o p e r b a l a n c e between t h e g r o w t h o f p a r a s i t e and t h e h o s t t i s s u e . F o r example, T u r e l and Ledingham (1957) r e p o r t e d , t h a t , i f i n t a c t r u s t i n f e c t e d c o t y l e d o n s a r e p l a c e d on s o l i d medium a g r e a t number o f u n c o n t a m i n a t e d u r e d o s p o r e s a r e p r o -duced but t h e h o s t t i s s u e f a i l s t o p r o l i f e r a t e . On t h e o t h e r hand i f t h e c o t y l e d o n s a r e c u t i n h a l v e s b e f o r e c u l t u r i n g , h o s t c a l l u s g r o w t h o c c u r s f i r s t , f o l l o w e d b y t h e d e v e l o p m e n t of masses o f p a r a s i t i c m y c e l i u m , w h i c h o v e r g r o w s t h e h o s t c a l l u s . The s i t u a t i o n was e x p l o i t e d by s u b s e q u e n t w o r k e r s t o p r o d u c e u n c o n t a m i n a t e d u r e d o s p o r e s f o r s t a r t i n g a x e n i c TABLE I . Growth o f o b l i g a t e p a r a s i t e s i n h o s t t i s s u e c u l t u r e s and a x e n i c c u l t u r e . AUTHOR M o r e l ( 1 9 4 6 : RUST FUNGUS P l a s m o p a r a v i t i c o l a H o t s o n & G u t t e r Gymnosporangium ( 1 9 5 1 ) . j u n i p e r i - v i r g i n i a n a e C u t t e r (1959, I960) Heim & G r i e s ( 1 9 5 3 ) Gymnosporangium . j u n i p e r i - v i r g i n i a n a e Uromyces a r i - t r i -p h y l l i E r y s i p h e c i c h o r a -cearum T u r e l & M e l a m p s o r a l i n i Ledingham ( 1 9 5 7 ) N o z z o l i l l o & P u c c i n i a h e l i a n t h i C r a i g i e (I960) M a h e s h w a r i , H i l - P u c c i n i a a n t i r r h i n i d e b r a n d t & A l l e n (1967) MEDIUM G a u t h e r e t ' s medium G a u t h e r e t ' s n u t r i e n t s o l u t i o n , y e a s t ex-t r a c t and a s c o r b i c a c i d G a u t h e r e t ' s n u t r i e n t s o l u t i o n , y e a s t ex-t r a c t and a s c o r b i c a c i d S u c r o s e , m i n e r a l s , a u x i n s , v i t a m i n s , a d -e n i n e s u l p h a t e and c o c o n u t m i l k Knop's t i s s u e c u l t u r e s o l u t i o n , B e r t h e l o t ' s m i c r o n u t r i e n t s and c o c o n u t m i l k . M o d i f i e d H e l l e r ' s o r W h i t e T s . m e d i u m , y e a s t e x t r a c t and pe p t o n e M o d i f i e d M u r a s h i g e and Skoog's medium REMARK S u c c e s s f u l e s t a b l i s h m e n t o f downy m i l d e w o f g r a p e i n c a l -l u s c u l t u r e . The f i r s t s u c c e s s f u l i s o l a t i o n o f r u s t f u n g u s i n a x e n i c c u l -t u r e . U n c o n f i r m e d . B o t h t h e r u s t s were i s o l a t e d and s u b c u l t u r e d . Fungus was n o t i s o l a t e d a x e n i c a l l y . Fungus f a i l e d t o d e v e l o p ax-e n i c a l l y . Fungus f a i l e d t o d e v e l o p a x e n i c a l l y . Fungus f a i l e d t o d e v e l o p a x e n i c a l l y . TABLE I . ( C o n t i n u e d ) . AUTHOR RUST FUNGUS T e w a r i & A r y a S c l e r o s p o r a g r a m i n i -(1969) c o l a H o l l i s e t a l . C r o n a r t i u m f u s i f o r m e (1972) Harvey & Grasham C r o n a r t i u m r i b i c o l a (1973) Unpub-l i s h e d MEDIUM REMARK W h i t e ' s b a s a l m i n e r - Fungus d e v e l o p e d a x e n i c a l l y , a l s , c a s e i n h y d r o l y - s u r v i v e d t r a n s f e r R e s u l t s s a t g a r e v e r y s i m i l a r t o C u t t e r ' s work. M o d i f i e d B a r n e s & Fungus d e v e l o p e d a x e n i c a l l y . N a y l o r ' s m i n e r a l s , V e g e t a t i v e g r o w t h was p r e d i c -s u c r o s e , y e a s t e x t r a c t , t a b l e b u t s p o r u l a t i o n was m-pe p t o n e and amino a c i d f r e q u e n t . m i x t u r e . M i n e r a l s a l t s , s u e - V e g e t a t i v e g r o w t h was h i g h l y r o s e , p e p t o n e , y e a s t r e p r o d u c i b l e o n l y a f t e r t h e e x t r a c t o r amino a c i d p r o l o n g e d a s s o c i a t i o n o f t h e m i x t u r e . f u n g u s w i t h i t s h o s t m a i n -t a i n e d i n t i s s u e c u l t u r e . C u l t u r e s p o r u i a t e d a f t e r a p r o l o n g e d s u b c u l t u r i n g . P a t h o g e n i c i t y was r e c o r d e d . 7 c u l t u r e s o f r u s t f u n g i . A c h r o n o l o g i c a l l i s t o f p u b l i s h e d a t t e m p t s t o c u l -t u r e r u s t f u n g i f r o m u r e d o s p o r e s i s g i v e n i n T a b l e I I . F o l l o w -i n g u n c o n f i r m e d c l a i m s b y Ray i n 1901 ( P h r a g m i d i u m ) and G r e t s c u s h n i k o f f i n 1936 ( P u c c i n i a s p p . ) , t h e s u c c e s s f u l e s -t a b l i s h m e n t o f a x e n i c c u l t u r e s o f P u c c i n i a g r a m i n i s t r i t i c i f r o m u r e d o s p o r e s was r e p o r t e d b y a g r o u p o f A u s t r a l i a n w o r k e r s i n 1966 and 1967 ( W i l l i a m s e t a l . 1966, 1967). They grew an A u s t r a l i a n r a c e o f wheat stem r u s t on a r e l a t i v e l y s i m p l e medium ( T a b l e I I ) . T h e i r r e p o r t was q u i c k l y c o n f i r m e d ( B u s h n e l l 1968, C o f f e y e t a l . 1969) and marked t h e b e g i n n i n g o f a new e r a i n t h e s t u d y o f o b l i g a t e p a r a s i t i s m . I n 1969 S c o t t and M a c l e a n p u b l i s h e d a c o m p r e h e n s i v e r e v i e w d e s c r i b i n g t h e work t h a t l e d t o t h e a x e n i c c u l t u r e o f Uromyces a r i - t r i p h y l l i , Gymnosporangium j u n i p e r i - v i r g i n i a n a ( C u t t e r 1959, I960) and P u c c i n i a g r a m i n i s t r i t i c i ( W i l l i a m s e t a l . 1966, 1967). They d i s c u s s e d t h e i m p l i c a t i o n s o f t h e new r e s u l t s i n r e l a t i o n t o t h e c o n c e p t o f o b l i g a t e p a r a s i t i s m , and p o i n t e d o u t t h a t a m a j o r p r o b l e m i n o b t a i n i n g i n v i t r o d e v e l o p m e n t o f r u s t c o l o n i e s f r o m u r e d o s p o r e s was t h e e r r a t i c b e h a v i o u r o f t h e s p o r e s f r o m one e x p e r i m e n t t o t h e n e x t u n d e r a p p a r e n t l y i d e n t i c a l c o n d i t i o n s . I n a s u b s e q u e n t r e v i e w p a p e r S c o t t (1972) c r i t i c a l l y a s s e s s e d t h e c u r r e n t s t a t u s o f t h e b i o c h e m i c a l a s p e c t s o f 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 o f t h e r u s t s and m i l d e w s and t h e n u t r i t i o n o f r u s t f u n g i grown i n a x e n i c c u l t u r e . TABLE I I . I n v i t r o d e velopment o f r u s t f u n g i f r o m g e r m i n a t i n g u r e d o s p o r e s , AUTHOR Ray (1901) RUST FUNGUS Phragmidium subcor-t i c i u m G r e t s c h u s h n i k o f f P u c c i n i a spp, (1936) W i l l i a m s e t a l . (1966, 1957) B u s h n e l l (1968; S i n g l e t o n & Young (1968) C o f f e y e t a l . (1969) T u r e l (1969' P u c c i n i a g r a m i n i s t r i t i c i , ANZ 126 - 6 , 7 P u c c i n i a g r a m i n i s t r i t i c i , ANZ 126-6,7 P u c c i n i a r e c o n d i t a P u c c i n i a g r a m i n i s t r i t i c i ANZ 126-6 , 7 M e l a m p s o r a l i n i Race'3 MEDIUM G e l a t i n medium p l u s p l a n t e x t r a c t C u l t u r e medium c o n -t a i n i n g s u b s t a n c e s w h i c h a d s o r b e d ammonia Czapek's m i n e r a l s , g l u c o s e , y e a s t ex-t r a c t C z a p e k ' s m i n e r a l s , g l u c o s e , y e a s t ex-t r a c t and p e p t o n e C z a p e k ' s m i n e r a l s , g l u c o s e , y e a s t ex-t r a c t , p e p t o n e . C z a p e k ' s m i n e r a l s , y e a s t e x t r a c t , pep-t o n e , g l u c o s e and BSA M o d i f i e d Knop's t i s -sue c u l t u r e medium, B e r t h e l o t ' s m i c r o -n u t r i e n t s , s u c r o s e , y e a s t e x t r a c t . REMARK U n c o n f i r m e d r e p o r t . C a s u a l c l a i m : n o t c o n f i r m e d . S i m p l e t e c h n i q u e , a l t h o u g h c o n s i d e r a b l e v a r i a b i l i t y and u n p r e d i c t a b i l i t y was r e p o r t e d . P a r t l y c o n f i r m e d . W i l l i a m s ' work. U n s u b s t a n t i a t e d r e s u l t . S y n c h r o n u s c o l o n y d e v e l o p m e n t was r e c o r d e d , b u t i n c r e a s e d l a g p h ase p r e c e d e d t h e d e v e l o p ment o f t h e c o l o n i e s . S p o r u -l a t i o n was a l t o g e t h e r a b s e n t . V e g e t a t i v e g r o w t h o c c u r r e d w i t h s t r o m a f o r m a t i o n . F r e -q u e n c y o f e s t a b l i s h e d c u l t u r e was l o w . TABLE I I . ( C o n t i n u e d ) . AUTHOR C o f f e y e t a l . (1969) RUST FUNGUS Melampsora l i n i Race 3 & Race 210 Wong <S (1970; W i l l e t s P u c c i n i a g r a m i n i s M a c l e a n & S c o t t (1970) T u r e l (197i: t r i t i c i , ANZ 126 - 6 , 7 P u c c i n i a g r a m i n i s t r i t i c i , ANZ 126^6,7 M e l a m p s o r a l i n i Race No. 3 MEDIUM M o d i f i e d Knop's t i s -sue c u l t u r e medium, B e r t h e l o t ' s m i c r o -n u t r i e n t s , s u c r o s e , y e a s t e x t r a c t , pep-t o n e REMARK C o n f i r m e d t h e above. Cyto-l o g i c a l s t u d i e s showed t h e hyphae t o be b i n u c l e a t e . C z a p e k 's m i n e r a l s , 7 V e g e t a t i v e g r o w t h o c c u r r e d g l u c o s e , y e a s t e x t r a c t , b u t s p o r u l a t i o n was i n f r e -p e p t o n e , q u e n t . C z a p ek's m i n e r a l s , U n i n u c l e a t e c u l t u r e s , e v i -e l u c o s e y e a s t e x t r a c t dence i s p r e s e n t e d t h a t h y p h a l and peptone M o d i f i e d Knop's t i s -sue c u l t u r e medium, B e r t h e l o t ' s m i c r o -n u t r i e n t s , s u c r o s e , v i t a m i n f r e e casamino a c i d c e l l s a r e d i p l o i d . P o o r l y documented c l a i m s r e m a i n u n c o n f i r m e d . E s t a b l i s h e d c o l o n i e s i n f e c t e d and o v e r g r e w t h e e x c i s e d f l a x c o t y l e d o n s . S p o r e p r o d u c e d f r o m t h e s e i n f e c t e d i n t a c t p l a n t ; Races a r e f o u n d t o be t h e same as i n o c u l u m u s e d t o r a i s e a x e n i c c u l t u r e s . TABLE I I . ( C o n t i n u e d ) AUTHOR M a c l e a n et a l . (1971) RUST FUNGUS P u c c i n i a g r a m i n i s t r i t i c i , ANZ 126-6,7 MEDIUM Czapek's m i n e r a l s , g l u c o s e , y e a s t ex-t r a c t , p e p t o n e Bose and Shaw P u c c i n i a g r a m i n i s C z a p e k ' s m i n e r a l s , (1971) t r i t i c i , ANZ 126-6,7 g l u c o s e , y e a s t ex-v 7 t r a c t , p e p t o n e and BSA B u s h n e l l and S t e w a r t (1971] K u h l e t a l . (1971T H a r t l e y & W i l l i a m s (1971 (a) and ( b ) ) P u c c i n i a g r a m i n i s t t r i t i c i , Race 3 8 , P u c c i n i a g r a m i n i s C zapek's m i n e r a l s , g l u c o s e , y e a s t ex-t r a c t and p e p t o n e Czapek's m i n e r a l s , t r i t i c i , ANZ 126-6,7 g l u c o s e , y e a s t ex t r a c t and p e p t o n e P u c c i n i a g r a m i n i s C z a p e k ' s m i n e r a l s , t r i t i c i , ANZ 126-6,7 g l u c o s e , y e a s t ex-t r a c t and p e p t o n e REMARK U n i n u c l e a t e c u l t u r e s d i d r e -i n f e c t t h e h o s t and s u b s e q u e n t s p o r u l a t i o n o c c u r r e d on t h e p l a n t and t h o s e s p o r e s were f o u n d t o be u n i n u c l e a t e . These a u t h o r s b e l i e v e d t h e c u l t u r e s r e m a i n e d h a p l o i d a t u n i n u c l e a t e c o n d i t i o n . Needs c o n f i r m a t i o n , a l t h o u g h an un-n a t u r a l b i o l o g i c a l phenomenon. Change o f p l o i d y more common-l y s e e n i n p l a n t and a n i m a l c e l l s grown i n t i s s u e c u l t u r e . Growth and s p o r u l a t i o n was r e -p o r t e d . S.E.M. r e v e a l e d an unknown c o a t i n g a r o u n d t h e i n v i t r o grown u r e d o s p o r e s . F u r t h e r d e t a i l s t u d i e s a r e n e e d e d . Growth was p o o r o f t h e s e N o r t h A m e r i c a n r a c e s . On d e f i n e d medium g r o w t h was p o o r f o r t h i s i s o l a t e . H o V a r i a t i o n was r e p o r t e d r e g a r d -i n g s a p r o p h y t i c a b i l i t y be-tween d i f f e r e n t s t r a i n s be-l o n g i n g t o t h e same r a c e . TABLE I I . (Continued). AUTHOR H a r t l e y and W i l l i a m s (Con-tinued) RUST FUNGUS Masin and Andreev (1971) P u c c i n i a graminis t x^xt X C X S t r a i n 21 (USSR) V a s s i l ' e v and Melampsora p i n i -S a p l i n a torqua Kuhl et a l . P u c c i n i a g r a m i n i s (Unpublished) avenae Coffey and Shaw Melampsora l i n i (1972) Race 3 Jones (1972) Uromyces d i a n t h i Foudin and Wynn P u c c i n i a graminis (1972) t r i t i c i , ANZ 126-6,7 MEDIUM REMARK Czapek-'s m i n e r a l s , g l u c o s e , yeast ex-t r a c t and peptone Czapek's m i n e r a l s , g l u c o s e , yeast ex-t r a c t . Czapek's m i n e r a l s M o d i f i e d Knop's t i s -sue c u l t u r e medium, B e r t h e l o t ' s m i c r o -nut r i e n t s , sucrose & amino a c i d m i x t u r e Czapek Dox B r o t h , pep tone, g l u c o s e , yeast e x t r a c t Czapek's m i n e r a l s , glucose and amino a c i d m i x t u r e A g i v e n r a c e had m i x t u r e of two s t r a i n s and one showed d i f f e r e n t c u l t u r e proper-t i e s when s t u d i e d on i n v i t r o . B r i e f r e p o r t on v e g e t a t i v e growth of a Russian i s o l a t e of wheat stem r u s t . Needs c o n f i r m a t i o n . I n the l i g h t of present con-cept d i f f i c u l t t o e v a l u a t e . Needs c o n f i r m a t i o n . Unconfirmed. Evidence was presented t h a t e s t a b l i s h e d c l o n e s can adapt to broad range of carbohy-d r a t e s and amino a c i d s . V e g e t a t i v e growth was o b t a i n -ed. S p o r u l a t i o n absent. P a t h o g e n i c i t y t r i a l s were not r e p o r t e d . A d e f i n e d s o l i d medium was used. S p o r u l a t i o n was i n -f r e q u e n t . P a t h o g e n i c i t y t r i a l s were not r e p o r t e d . TABLE I I . ( C o n t i n u e d ) . AUTHOR RUST FUNGUS MEDIUM Howes and S c o t t (1972) P u c c i n i a g r a m i n i s C z a p e k's m i n e r a l s , t r i t i c i , ANZ 12b-6,7 glucose., c i t r a t e , c y s t e i n e , g l u t a m m e C o f f e y and A l l e n (1973) Melampsora l i n i Race 3, 79 P u c c i n i a h e l i a n t h i Race 1 M o d i f i e d Knop's t i s -sue c u l t u r e medium, B e r t h e l o t ' s m i c r o -n u t r i e n t s , s u c r o s e , y e a s t e x t r a c t , BSA, peptone T u r e l (1973 Melampsora l i n i Race 3 , 210 M o d i f i e d Knop's t i s -sue c u l t u r e medium, B e r t h e l o t ' s m i c r o -n u t r i e n t s , s u c r o s e , a s p a r t i c a c i d , c y s -t e i n e , g l u t a t h i o n e REMARK E s t a b l i s h e d c l o n e s r a i s e d on me d i a c o n t a i n i n g - p e p t o n e e t c were u s e d t o s t u d y S n u t r i -t i o n . A r a n g e o f S amino a c i d s was s u i t a b l e f o r r u s t g r o w t h , b u t i n o r g a n i c S was n o t . Growth o f f l a x r u s t and s u n -f l o w e r r u s t was po o r i n chem i c a l l y d e f i n e d l i q u i d medium T y p i c a l s p o r u l a t i o n was ab-s e n t . V e g e t a t i v e g r o w t h o c c u r r e d , s p o r u l a t i o n a b s e n t i n c h e m i -c a l l y d e f i n e d s o l i d medium. 13-F o l l o w i n g t h e i n i t i a l r e p o r t b y W i l l i a m s e t a l . s e v e r a l l a b o r a t o r i e s have p u b l i s h e d t h e r e s u l t s o f s u c c e s s f u l a t t e m p t s t o grow r u s t f u n g i i n a x e n i c c u l t u r e . I n a d d i t i o n t o t h e work o f t h e A u s t r a l i a n g r o u p , t h e most e x t e n s i v e p r o -grams have been c o n d u c t e d by T u r e l a t S a s k a t o o n , B u s h n e l l a t M i n n e s o t a , by J o n e s a t K e e l e , by C o f f e y and - A l l e n a t W i s c o n -s i n and i n t h e De p a r t m e n t o f P l a n t S c i e n c e a t U.B.C. At U.B.C. work on t h e a x e n i c c u l t u r e o f wheat r u s t s was s t a r t e d by t h e a u t h o r i n D r . Shaw's l a b o r a t o r y i n 1967. The f i r s t new f i n d i n g a t U.B.C. was t h a t when \ % g e l a -t i n was u s e d t o su s p e n d t h e u r e d o s p o r e i n o c u l u m v e g e t a t i v e g r o w t h o f an i s o l a t e o f t h e A u s t r a l i a n r a c e ANZ 1 2 6 - 6 , 7 o f P u c c i n i a g r a m i n i s t r i t i c i o c c u r r e d w i t h g r e a t e r f r e q u e n c y and p r e d i c t a b i l i t y i n v i t r o t h a n had been r e p o r t e d b y t h e A u s t r a l -i a n group a t t h a t t i m e ( C o f f e y , Bose and Shaw 1969) . An i n -t e r e s t i n g f e a t u r e o f t h e r e s u l t s was t h a t g r o w t h o c c u r r e d w i t h a l o w d e n s i t y o f u r e d o s p o r e s (20-40 spores/mm ) when t h e u r e d o s p o r e s were s u s p e n d e d i n g e l a t i n b e f o r e t h e p l a t e s were s e e d e d . U s i n g d r y u r e d o s p o r e s B u s h n e l l (1968) had f o u n d e a r l i e r t h a t c o l o n y d evelopment o n l y - o c c u r r e d i n r e g i o n s o f p l a t e s seeded a t a h i g h d e n s i t y (100-200 spores/mm^). A l -t h o u g h t h e g e l a t i n t e c h n i q u e d e s c r i b e d by C o f f e y e t a l . (1969) l e d t o t h e s y n c h r o n o u s d e v e l o p m e n t o f a x e n i c c o l o n i e s o f wheat r u s t on s o l i d m e d i a , t h e c o l o n i e s f a i l e d t o s p o r u -l a t e. 14 I n s u b s e q u e n t t r i a l s w i t h P u c c i n i a g r a m i n i s t r i t i c i c o n d u c t e d u s i n g a l i q u i d medium, t y p i c a l u r e d o p u s t u l e s and t e l e u t o p u s t u l e s d e v e l o p e d on t h e a x e n i c c o l o n i e s . R e i n f e c -t i o n o f t h e h o s t was o b t a i n e d u s i n g u r e d o s p o r e s grown i n v i t r o . These r e s u l t s , w h i c h c o n f i r m e d and e x t e n d e d t h o s e r e p o r t e d e a r l i e r by W i l l i a m s e t a l . (1967), a r e d e s c r i b e d i n C h a p t e r I o f t h i s t h e s i s , w h i c h has a l r e a d y b e e n p u b l i s h e d (Bose and Shaw 197D . C h a p t e r I I d e s c r i b e s t h e a x e n i c c u l t u r e and c y t o l o g y o f two s t r a i n s o f M e l a m p s o r a l i n i , t h e f l a x r u s t f u n g u s . These r e s u l t s c o n f i r m e d T u r e l ' s o r i g i n a l r e p o r t o f t h e a x e n i c c u l t u r e o f t h i s a u t o e c i o u s r u s t oh a s o l i d medium c o n t a i n i n g s u c r o s e , Knop.'s m i n e r a l n u t r i e n t s and y e a s t e x t r a c t . C h a p t e r I I h a s a l s o a l r e a d y been p u b l i s h e d ( C o f f e y , Bose and Shaw 1970). F u r t h e r s t u d i e s on t h e c a r b o h y d r a t e and amino a c i d n u t r i t i o n o f t h e s e f l a x r u s t c u l t u r e s were p u b l i s h e d by C o f f e y and Shaw (1972). C o f f e y and Shaw's r e s u l t s and f u r t h e r s t u d i e s by T u r e l (1973) l a i d t h e groundwork f o r t h e e x p e r i -ments d e s c r i b e d i n C h a p t e r I I I . C h a p t e r I I I d e a l s w i t h t h e g r o w t h o f b o t h wheat stem r u s t and f l a x r u s t on c h e m i c a l l y d e f i n e d l i q u i d m e d i a c o n -t a i n i n g o n l y a s i m p l e c a r b o h y d r a t e , m i n e r a l s a l t s and 2 o r 3 amino a c i d s . T y p i c a l u r e d o s p o r e s a n d t e l e u t o s p o r e s were p r o -d u c ed by b o t h r u s t s on t h e media employed and i n t a c t f l a x c o t y l e d o n s were s u c c e s s f u l l y i n o c u l a t e d w i t h f l a x u r e d o s p o r e s r a i s e d i n v i t r o . 15 The t h r e e c h a p t e r s i n t h e t h e s i s t h u s r e c o r d o u r i n c r e a s i n g a b i l i t y t o o b t a i n r e p r o d u c i b l e v e g e t a t i v e and r e p r o d u c t i v e g r o w t h o f t h e wheat and f l a x r u s t f u n g i . The i l l u s t r a t i o n s o f a x e n i c c u l t u r e s o f t h e f l a x r u s t f u n g u s i n t h e f r o n t i s p i e c e a r e s t r i k i n g t e s t i m o n y t o t h e p r o g r e s s w h i c h has been made s i n c e t h e w r i t e r b e gan h i s e x p e r i m e n t s i n 1967. 16 REFERENCES Bose, A., and M. Shaw. 1971 . S p o r u l a t i o n and p a t h o g e n i c i t y of an A u s t r a l i a n i s o l a t e of wheat r u s t grown i n v i t r o . Can. J . Botany 49 : 1961-1964. B r i a n , P.W. 1967. O b l i g a t e p a r a s i t i s m i n f u n g i . Proc. Roy. Soc., Ser. B, B i o l . S c i . 1968: 101-118. B u s h n e l l , W.R. 1968. I n v i t r o development of an A u s t r a l i a n i s o l a t e of P u c c i n i a g r a m i n i s f . sp. t r i t i c i . Phyto-p a t h o l o g y 5 8 : 5 2 6 - 5 2 7 . B u s h n e l l , W.R., and D.M. Stewart. 1971 . Development of Amer-i c a n i s o l a t e s of P u c c i n i a g r a m i n i s f . sp. t r i t i c i on an a r t i f i c i a l medium. Phytopathology 6 1 : 376 - 3 7 9 . C h e s t e r , K.S. 1946. The Nature and P r e v e n t i o n of the C e r e a l Rusts as e x e m p l i f i e d i n the Leaf Rust of Wheat. Ch r o n i c a B o t a n i c a Co., Waltham, Mass., U.S.A. 269 P. C o f f e y , M.D., A. Bose, and M. Shaw'. 1969. I n v i t r o growth of g e l a t i n suspensions of uredospores of P u c c i n i a g r a m i n i s t r i t i c i . Can. J . Botany 4 7 : 1291-1293. C o f f e y , M.D., A. Bose, and M. Shaw. 1970. I n v i t r o c u l t u r e of the f l a x r u s t , Melampsora l i n i . Can. J . Botany 4 3 : 773-776. C o f f e y , M.D., and M. Shaw. 1972. N u t r i t i o n a l s t u d i e s w i t h axenic c u l t u r e s of the f l a x r u s t , Melampsora l i n i . P h y s i o l . P l a n t Pathology 2 : 3 7 - 4 7 . C o f f e y , M.D., and P.J. A l l e n . 1973. N u t r i t i o n of Melampsora l i n i and P u c c i n i a h e l i a n t h i . Trans. B r i t . M y c o l . Soc. 60: 245-260. C u t t e r , V.M. 1 9 5 9 ( 1 9 6 1 ) . S t u d i e s on the i s o l a t i o n and growth of p l a n t r u s t s i n host t i s s u e c u l t u r e s and upon syn-t h e t i c media. I . Gymno sporangium. M y c o l o g i a 52: 248-2 9 5 . C u t t e r , V.M. 1960(1961) . S t u d i e s on the i s o l a t i o n and growth of p l a n t r u s t s i n host t i s s u e c u l t u r e s and upon syn-t h e t i c media. I I . Uromyces a r i - t r i p h y l l i . M y c o l o g i a $ 2 : 7 2 6 - 7 4 2 . DeBary, A. 1887. Comparative Morphology and B i o l o g y of the F u n g i , Mycetozoa and B a c t e r i a . (Eng. t r a n s , by Garnsey, H.E.F.; r e v . B a l f o u r , I . B . ) . Clarendon P r e s s , Oxford, 525p. 17 F o u d i n , A.S., and W.K. Wynn. 1972. Growth o f P u c c i n i a gram-i n i s f . s p . t r i t i c i on a d e f i n e d medium. P h y t o -p a t h o l o g y 62: 1032-1040. G r e t s e h u s h n i k o f f , A . I . • 1936. T o x i n s o f r u s t ( P u c c i n i a ) . Rev. A p p l . M y c o l . 15: 710 ( A b s t r . ) . H a r t l e y , M.J., and P.G. W i l l i a m s . 1971. M o r p h o l o g i c a l and c u l t u r a l d i f f e r e n c e s b e t ween r a c e s o f P u c c i n i a gram-i n i s 1. s p , t r i t i c i i n a x e n i c c u l t u r e . T r a n s . B r i t . M y c o l . S o c . 57: 137-144. Heim, J.M., and G.A. G r i e s . 1953. The c u l t u r e o f E r y s i p h e c i c h o r a c e a r u r . on s u n f l o w e r tumor t i s s u e . P h y t o -p a t h o l o g y 43: 343-344. Howes, N.K., and K . J . S c o t t . 1972. S u l p h u r n u t r i t i o n o f P u c c i n i a g r a m i n i s f . s p . t r i t i c i i n a x e n i c c u l t u r e . Can. J . B o t a n y 50: 1165-1170. H o t s o n , H.H., and V.M. C u t t e r . 1951. The i s o l a t i o n and c u l -t u r e o f Gymnosporangium j u n i p e r i - v i r g i n i a n a e Schw. upon a r t i f i c a l m e d i a . P r o c . N a t . A c a d . S c i . (U.S.A.) 37: 400-403. H o l l i s , C.A.,. R.A. S c h m i d t , and J.W. Ki m b r o u g h . 1972. A x e n i c c u l t u r e o f C r o n a r t i u m f u s i f o r m e . P h y t o p a t h o l o g y 6 2 : 1417-1419. J o n e s , D.R. 1972. I n v i t r o c u l t u r e o f c a r n a t i o n r u s t , U r o -myces d i a n t h i . T r a n s . B r i t . M y c o l . S oc. 58: 29-36. M a c l e a n , D.J., and K . J . S c o t t . 1970. V a r i a n t f o r m s o f s a p r o -p h y t i c m y c e l i u m grown f r o m u r e d o s p o r e s o f P u c c i n i a g r a m i n i s f . s p . t r i t i c i . J . Gen. M i c r o b i o l . 64 : 1 9 - 2 7 . M a c l e a n , D.J., K . J . S c o t t , and I.C. Tommerup. 1971. A u n i -n u c l e a t e w h e a t - i n f e c t i n g s t r a i n o f t h e stem r u s t f u n g u s i s o l a t e d f r o m a x e n i c c u l t u r e . J . Gen. M i c r o b i o l . 64 : 339-342. M a h e s h w a r i , R., A.C. H i l d e b r a n d t , and P . J . A l l e n . 1967. F a c t o r s a f f e c t i n g t h e g r o w t h o f r u s t f u n g i on h o s t t i s s u e c u l t u r e s . B o t a n . Gaz. 128: 153-159. M a s i n , V.V., and L.N. A n d r e e v . 1971. V e g e t a t i v e g r o w t h o f t h e p a t h o g e n o f wheat stem r u s t i n v i t r o . M i k o l o -g i y a i F i t o p a t h o l o g i y a 5 (2) : 197-200. M o r e l , G. 1948. R e c h e r c h e s s u r l a c u l t u r e a s s o c i e e de p a r a -s i t e s o b l i g a t i r i e s e t de t i s s u e vege'taux. Ann. E p i p h y t . ( P a r i s ) 14: 1-112. 18 N o z z o l i l l o , C., and J.H. C r a i g i e . I960. Growth of the r u s t fungus P u c c i n i a h e l i a n t h i on t i s s u e c u l t u r e s of I t s h o s t . Can. J . Botany 33: 227-233. Ray, J . 1901, C u l t u r e s et formes attenue'es des maladies cryptogamique des ve\getaux. C.R. Hebd. Seanc. Acad. S c i . , P a r i s 133: 307-309. S c o t t , K . J. 1972. O b l i g a t e p a r a s i t i s m by phytopathogenic f u n g i . B i o l . Rev. 47: 537-572. S c o t t , K . J . , and D.J. Maclean. 1969. C u l t u r i n g of r u s t f u n g i . Ann. Rev. Phytopathology 6: 123-146. S i n g l e t o n , L.L., and Young, H.C. 1968. The i n v i t r o c u l t u r e of P u c c i n i a r e c o n d i t a f . sp. t r i t i c i . P hytopathology 58: 1 0 6 8 (Abstr.1T Tewari, M.M., and A r y a , H.C. 1969. S c l e r o s p o r a g r a m i n i c o l a axenic c u l t u r e . Science (Washington) 163: 291-293. T u r e l , F.L.M. 1969. S a p r o p h y t i c development of the f l a x r u s t , Melampsora l i n i r a c e no. 3 . Can. J . Botany 47: 821-T u r e l , F.L.M. 1971. P a t h o g e n i c i t y and development d i f f e r -ences of t h r e e s a p r o p h y t i c a l l y growing i s o l a t e s of f l a x r u s t fungus Melampsora l i n i , race 3 . Can. J . Botany 49: 1993-1997. T u r e l , F.L.M. 1973. Growth of the f l a x r u s t Melampsora l i n i on c h e m i c a l l y d e f i n e d media. Can. J . Botany 51: 131-134. T u r e l , F.L.M., and G.A. Ledingham. 1957. P r o d u c t i o n of a e r i a l mycelium and uredospores by Melampsora l i n i ( Pers.) Lev. on f l a x l e a v e s I n t i s s u e c u l t u r e . Can. J . M i c r o b i o l . 3: 813-819. ' V a s s i l ' e v , O.A. and V . I . S a p l i n a . 1971. C u l t u r e of the f u n -gus Melampsora p i n i t o r q u a (A.BR.) R o s t r . - the pathogen of the stem r u s t of p i n e . M y c o l o g i y a i F i t o p a t h o l o g i y a 5 (2) : 182-183. W i l l i a m s , P.G., K.J. S c o t t , and J.L. K u h l . 1966. V e g e t a t i v e growth of P u c c i n i a g r a m i n i s f . sp. t r i t i c i n v i t r o . Phytopathology 56? 1418-1419. 19 W i l l i a m s , P.G., K . J . S c o t t , J . L . K u h l , and D.J. M a c l e a n . 1967. S p o r u l a t i o n and p a t h o g e n i c i t y o f P u c c i n i a g r a m i n i s f . s p . t r i t i c i grown on an a r t i f i c i a l medium. P h y t o -p a t h o l o g y 57: 326-327. Wong, A.L., and H.J. W i l l e t t s . 1970. O b s e r v a t i o n s on g r o w t h o f s e l e c t e d A u s t r a l i a n r a c e s o f wheat stem r u s t i n a x e n i c c u l t u r e . T r a n s . B r i t . M y c o l . S o c . 55: 231-23&\ Yarwood C.E. 1956. O b l i g a t e p a r a s i t i s m . Ann. Rev. P l a n t P h y s i o l . 7: 115-142. 20 CHAPTER I S p o r u l a t i o n and P a t h o g e n i c i t y o f an A u s t r a l i a n I s o l a t e o f Wheat R u s t grown i n v i t r o ABSTRACT C o l o n i e s o f P u c c i n i a g r a m i n i s P e r s . f . s p . t r i t i c i E r i k s s . & Henn., r a c e ANZ 1 2 6 - 6 , 7 were grown f r o m u r e d o s p o r e s on Czapek's m i n e r a l s a l t s , 3% g l u c o s e , 0.1% E v a n s ' p e p t o n e , p l u s d e f a t t e d b o v i n e serum albumen. D i k a r y o t i c v e g e t a t i v e hyphae a p p a r e n t l y d e v e l o p e d f r o m c e n t e r s o f germ t u b e a n a s t o -m o s i s , w i t h o u t t h e f o r m a t i o n o f t y p i c a l i n f e c t i o n s t r u c t u r e s . T y p i c a l p i g m e n t e d u r e d o s p o r e s and t e l i o s p o r e s were f o r m e d a f t e r 6 t o 8 weeks g r o w t h . B o t h s p o r e f o r m s were c o a t e d w i t h a l a y e r o f m a t e r i a l w h i c h was v i s i b l e u n d e r t h e s c a n n i n g e l e c t r o n m i c r o s c o p e and was n o t o b s e r v e d on u r e d o s p o r e s grown on i n t a c t wheat l e a v e s . The u r e d o s p o r e s were c a p a b l e o f i n f e c -t i n g t h e m e s o p h y l l o f wheat l e a v e s exposed by s t r i p p i n g b a c k t h e l o w e r e p i d e r m i s . The p o s s i b i l i t y i s c o n s i d e r e d t h a t t h e s u r f a c e c o a t i n g o f u r e d o s p o r e s grown i n v i t r o i s r e l a t e d t o t h e i r i n a b i l i t y t o i n f e c t i n t a c t l e a v e s v i a t h e s t o m a t a . 21 INTRODUCTION W i l l i a m s e t a l . (1966, 1967) and B u s h n e l l (1968) have d e s c r i b e d t h e p r o d u c t i o n o f u r e d o s p o r e s by P. g r a m i n i s P e r s . f . s p . t r i t i c i E r i k s s . & Henn., r a c e ANZ 126-6,7 i n v i t r o . W i l l i a m s ' g r oup (1967) f o u n d t h a t s u c c e s s f u l r e i n f e c -t i o n o f wheat l e a v e s by u r e d o s p o r e s grown i n v i t r o o n l y o c c u r r e d when t h e s p o r e s were p l a c e d on e x p o s e d m e s o p h y l l o f L i t t l e C l u b wheat. They c o n s i d e r e d t h a t t h e i n a b i l i t y o f s u c h u r e d o s p o r e s t o I n f e c t v i a s t o m a t a was due t o a l o w p o t e n t i a l f o r I n d e p e n d e n t g r o w t h d u r i n g t h e i n i t i a l s t a g e s o f i n f e c t i o n , r a t h e r t h a n t o a l a c k o f p a t h o g e n i c i t y p e r s e . I n f u r t h e r - ex-p e r i m e n t s w i t h an i s o l a t e o f t h e A u s t r a l i a n r u s t o b t a i n e d f r o m D r . P.G. W i l l i a m s , C o f f e y e t a l . (1969) o b t a i n e d b e s t g r o w t h o f v e g e t a t i v e c o l o n i e s b y s e e d i n g t h e u r e d o s p o r e s o n t o t h e medium i n s u s p e n s i o n o f 15% g e l a t i n . These c o l o n i e s e v e n t u a l l y p r o d u c e d n o n - p i g m e n t e d u r e d o s p o r e - l i k e s t r u c t u r e s . We have r e p e a t e d o u r e x p e r i m e n t s w i t h t h e A u s t r a l i a n i s o l a t e and have o b t a i n e d t h e p r o d u c t i o n o f t y p i c a l , p i g m e n t e d u r e d o -s p o r e s c a p a b l e o f r e i n f e c t i n g t h e h o s t p l a n t , as w e l l as t y p i c a l p i g m e n t e d t e l i o s p o r e s . S i n c e o b s e r v a t i o n s on t h e a x e n i c c u l t u r e o f r u s t f u n g i s t i l l c a n n o t be d e s c r i b e d as r o u t i n e , o u r new r e s u l t s a r e r e p o r t e d h e r e . 22 MATERIALS AND METHODS L i t t l e C l u b wheat was grown i n a c o n t r o l l e d e n v i r o n -ment c a b i n e t s e t a t 500 f t - c and 20°C f o r 16 h f o l l o w e d by 8 h i n d a r k n e s s a t 15°C. The f i r s t s e e d l i n g l e a v e s o f p l a n t s 1 week o l d were i n o c u l a t e d w i t h u r e d o s p o r e s o f P u c c i n i a g r a m i n i s P e r s . f . s p . t r i t i c i E r i k s s . & Henn., r a c e ANZ 126-6,7 s u s p e n d -ed i n w a t e r . The p l a n t s were s p r a y e d w i t h w a t e r b o t h b e f o r e and a f t e r i n o c u l a t i o n , c o v e r e d w i t h p l a s t i c bags t o e n s u r e h i g h h u m i d i t y , and i n c u b a t e d f o r 20-24 h i n d a r k n e s s a t 15°C. The bags were t h e n removed and t h e p l a n t s r e t u r n e d t o t h e g r o w t h c a b i n e t . A f t e r v i s i b l e i n f e c t i o n f l e c k s d e v e l o p e d , l e a v e s were s u r f a c e - s t e r i l i z e d , t h e i r c u t ends p l a n t e d i n a g a r i n c u l t u r e t u b e s , and i n c u b a t e d as d e s c r i b e d e a r l i e r ( C o f f e y e t a l . 1969). A f t e r 10 d a y s , d r y , c o n t a m i n a n t - f r e e u r e d o -s p o r e s were h a r v e s t e d and u s e d t o i n o c u l a t e s o l i d o r l i q u i d m e d i a . The b a s i c medium c o n t a i n e d C z a p e k ' s m i n e r a l s a l t s , 3% g l u c o s e , and 0.1% E v a n s ' p e p t o n e (Ev a n s M e d i c a l L t d . ) ( B u s h n e l l 1968), p l u s 1% d e f a t t e d b o v i n e serum albumen (BSA; f r a c t i o n V, Armour P h a r m a c e u t i c a l Co..), added a f t e r a u t o c l a v i n g , as d e s c r i b e d e a r l i e r ( C o f f e y e t a l . 1969). Medium s o l i d i f i e d w i t h 1.5% D i f c o B a c t o Agar i n p l a s t i c p e t r i d i s h e s was i n -o c u l a t e d w i t h a g e l a t i n s u s p e n s i o n of u r e d o s p o r e s ( C o f f e y e t al. 1969). The d i s h e s were t h e n c o v e r e d and s e a l e d w i t h 2 3 m a s k i n g t a p e . I n e x p e r i m e n t s w i t h l i q u i d medium, 2 0 m l a l i -q u o t s were p i p e t t e d i n t o 5 0 m l c o n i c a l , p y r e x f l a s k s . The pH was a d j u s t e d t o 6 . 4 b e f o r e a u t o c l a v i n g and a d d i t i o n o f BSA. The pH o f t h e medium d i d n o t change s i g n i f i c a n t l y a f t e r a u t o -c l a v i n g . U r e d o s p o r e s were c a r e f u l l y p l a c e d on t h e s u r f a c e o f t h e l i q u i d u s i n g a f l a t t e n e d n e e d l e and t h e f l a s k s were c l o s e d w i t h p o l y p r o p y l e n e s t o p p e r s . A g a r p i e c e s ( 1 cm ) b e a r i n g v e g e t a t i v e c o l o n i e s o r s p o r u l a t i n g c o l o n i e s grown on l i q u i d medium were s t a i n e d on c l e a r m i c r o s c o p e s l i d e s w i t h p h l o x i n e - K O H o r on u n d i l u t e d Giemsa r e a g e n t f o r 3 t o 4 m i n ( G i a t g o n g and F r e d e r i c k s e n 1 9 6 9 ) P h o t o m i c r o g r a p h s were t a k e n u n d e r b r i g h t - f i e l d o r p h a s e c o n -t r a s t , u s i n g a 3 5 mm camera. F o r e x a m i n a t i o n i n t h e s c a n n i n g e l e c t r o n m i c r o s c o p e (Cambridge I I S t e r e o s c a n ) , u r e d o s p o r e s grown i n v i t r o were f i r s t t e a s e d o n t o a d r y m i c r o s c o p e s l i d e and k e p t a t room temp e r a t u r e f o r 2 4 h. U r e d o s p o r e s h a r v e s t e d f r o m i n t a c t p l a n t s and t h o s e grown i n v i t r o were cemented t o s p e c i m e n s t u b s w i t h s i l v e r p a i n t and c o a t e d f i r s t w i t h c a r b o n and t h e n w i t h g o l d a t an a n g l e o f - 4 0 t o + 5 0 ° on a c o n t i n u o u s r o t a t i n g s t a g e , u s i n g a shadow c a s t e r . D i f f e r e n c e s i n t r e a t m e n t were e l i m i n -a t e d by p r o c e s s i n g b o t h s a m p l e s o f u r e d o s p o r e s a t t h e same t i m e . 24 RESULTS 1 . B e h a v i o u r of G e r m i n a t i n g U r e d o s p o r e s on S o l i d Medium The g e r m i n a t i o n and development o f u r e d o s p o r e s were examined 8 days a f t e r t h e p l a t e s were s e e d e d . I t was found t h a t t h e germ t u b e s of s i n g l e i s o l a t e d g e r m l i n g s had f r e q u e n t l y b u r s t , l i b e r a t i n g t h e c y t o p l a s m . T h i s was o b s e r v e d r e p e a t e d l y . F o r example i n one t r i a l a t l e a s t two b u r s t germ t u b e s were o b -s e r v e d i n e a c h . o f 10 c o n s e c u t i v e m i c r o s c o p e f i e l d s (10 t o 20 s p o r e s per f i e l d ; m a g n i f i c a t i o n X 100) a f t e r s t a i n i n g w i t h p h l o x i n e - K O H ( F i g s . I - l and 1-2). I n a few I n s t a n c e s two n u c l e i were seen i n t h e e x t r u d e d c y t o p l a s m a f t e r s t a i n i n g w i t h Giemsa r e a g e n t ( F i g . 1 - 3 ) . No t y p i c a l i n f e c t i o n s t r u c t u r e s were s e e n , but s w e l l i n g s o r v e s i c l e s were observed i n some i n s t a n c e s . I n a few i n s t a n c e s i n c i p i e n t b r a n c h i n g was o b s e r v e d i n s i n g l e i s o l a t e d germ t u b e s , but t h e s e d i d n o t d e v e l o p s e p t a . B u r s t -i n g was n o t o b s e r v e d when two o r more germ t u b e s had undergone a n a s t o m o s i s . Anastomosed t u b e s f r e q u e n t l y formed s e p t a ( F i g . 1 - 4 ) . I n f e c t i o n s t r u c t u r e s d i d not d e v e l o p a f t e r a n a s t o m o s i s but t h e r e were a number of v e s i c u l a r s w e l l i n g s a s s o c i a t e d w i t h each ' c e n t e r ' of a n a s t o m o s i s ( F i g . 1-4). A l l t h e hyphae examined were d i k a r y o t i c and t h e r e was no e v i d e n c e f o r the o c c u r r e n c e of the m o n o k a r y o t i c hyphae d e s c r i b e d by W i l l i a m s and H a r t l e y (1971). 2 . Development of C o l o n i e s on L i q u i d Medium Growth was s t r o n g l y i n f l u e n c e d by t e m p e r a t u r e . A f t e r i n c u b a t i o n f o r 4 weeks , growth was n e g l i g i b l e at 11 ° C , s low at F i g u r e 1-1. U r e d o s p o r e v ; i t h b u r s t germ t u b e ( a r r o w ) on b a s i c medium p l u s BSA. S t a i n e d w i t h p h l o x i n e - K O H . X 3 2 0 . F i g u r e 1 -2 . Two g e r m i n a t i n g u r e d o s p o r e s . The germ t u b e s have c r o s s e d e a c h o t h e r , b u t have n o t a n a s -t o m o s e d , and h a v e b u r s t ( a r r o w ) . Medium and s t a i n as f o r F i g u r e 1-1. X 634 . F i g u r e 1 -3 . Two n u c l e i ( a r r o w s ) i n c y t o p l a s m e x t r u d e d f r o m b u r s t germ t u b e . B a s i c medium. S t a i n e d w i t h G i e m s a - H C l . X 6 4 0 . F i g u r e 1-4. Germ t u b e s o r i g i n a t i n g f r o m two u r e d o s p o r e s s h o w i n g a n a s t o m o s i s ( u p p e r a r r o w ) and t h e d e v e l o p m e n t o f a v e g e t a t i v e hypha w i t h septum ( b o t t o m a r r o w ) . A t h i r d u r e d o s p o r e ( u g ) , c o n t a i n i n g two n u c l e i , has n o t g e r -m i n a t e d . X 3 4 1 . F i g u r e 1 -5 . C o l o n y d e v e l o p m e n t on l i q u i d medium. A, s p o r u l a t i n g c o l o n i e s w i t h c o l l a p s i n g m y c e l i u m , g r o w n - a t 17°C. B, j u v e n i l e c o l o n i e s i n v e g e t a t i v e s t a g e grown a t 14°C. P h o t o g r a p h e d a f t e r 6 weeks g r o w t h . 1t 2 6 27 14°C, and e x c e l l e n t a t 17°C. There was o n l y a l i t t l e growth a t 21°C, w h i c h caused a c o n s p i c u o u s c o l l a p s e o f t h e mycelium. At 11°C s m a l l c o l o n i e s d e v e l o p e d a f t e r 8 weeks and a few o f t h e s e s p o r u l a t e d a f t e r 10 weeks. S p o r u l a t i o n and t h e f o r m a -t i o n , o f a brown pigment o c c u r r e d a f t e r 8 weeks a t 14°C. F i g u r e I - 5 A shows s p o r u l a t i n g c o l o n i e s grown on l i q u i d medium f o r 6 weeks a t 17°C and F i g u r e I - 5 B shows v e g e t a t i v e c o l o n i e s of t h e same age grown a t 1 4 ° C At 17°C, 25 t o 30 f l u f f y w h i t e c o l o n i e s , w h i c h t e n d e d t o c o a l e s c e w i t h each o t h e r , d e v e l o p e d i n each f l a s k (15 r e p l i c a t e s ) . The r e s u l t s a t t h i s t e m p e r a t u r e were h i g h l y r e p r o d u c i b l e . A f t e r 6 weeks t h e c o l o n i e s had t u r n e d brown, some brown pigment had d i f f u s e d i n t o t h e medium, and l a r g e numbers o f u r e d o s p o r e s had d e v e l o p e d i n about 60% o f t h e f l a s k s ( F i g . I - 5 A ) . The d e v e l o p i n g u r e d o s p o r e s were .formed t e r m i n a l l y on s p o r o p h o r e s ( F i g . 1 - 6 ) , and, when mature, were r e l e a s e d by g e n t l e t e a s i n g w i t h a n e e d l e on a m i c r o s c o p e s l i d e . The u r e d o s p o r e s were s i m i l a r i n shape and s i z e t o t h o s e formed n o r m a l l y i n i n f e c t e d h o s t t i s s u e ( F i g . 1-7). T h i c k -w a l l e d , t w o - c e l l e d t e l i o s p o r e s d e v e l o p e d i n c l u s t e r s on s p o r o -phores ( F i g s . 1-7 and 1-8) about 2 weeks a f t e r t h e f o r m a t i o n o f u r e d o s p o r e s , and t h e c o l o n i e s t u r n e d dark brown i n c o l o r . C o f f e y et a l . (1969) r e p o r t e d t h e o c c u r r e n c e o f groups o f i r r e g u l a r l y shaped non-pigmented b o d i e s (5-10 u i n diam) i n t h e medium at t h e p e r i p h e r y of c o l o n i e s t h a t d e v e l o p e d when a g e l a t i n s u s p e n s i o n of u r e d o s p o r e s was seeded onto s o l i d medium. No such b o d i e s were o b s e r v e d i n l i q u i d medium but 2 3 F i g u r e 1 -6 . D e v e l o p i n g u r e d o s p o r e s i n c o l o n y g r o w n f o r 5 t o 6 w e e k s o n l i q u i d m e d i u m . P h l o x i n e / K O H . X 793. F i g u r e 1-7. D e v e l o p i n g t e l i o s p o r e ( a r r o w ) a n d m a t u r i n g u r e d o s p o r e s f r o m c o l o n y g r o w n o n l i q u i d m e d i u m f o r a b o u t 8 w e e k s . P h l o x i n e / K O H . X 1587. F i g u r e 1 - 8 . F u l l y d e v e l o p e d t w o - c e l l e d t e l i o s p o r e s . P h l o x i n e / K O H . X 6 3 4 . F i g u r e 1-9. T h i c k m a t o f h y p h a e a t p e r i p h e r y o f c o l o n y g r o w n o n l i q u i d m e d i u m f o r 6 w e e k s . X 3 0 7 . F i g u r e 1-10. P u s t u l e s d e v e l o p e d o n p r i m a r y l e a f o f L i t t l e C l u b w h e a t a b o u t 10 d a y s a f t e r i n -o c u l a t i o n w i t h u r e d o s p o r e s g r o w n i n v i t r o . A , t h e u p p e r s u r f a c e ; B , t h e l o w e r s u r -f a c e , o f u n i n f e c t e d l e a f . U r e d o s p o r e s w e r e a p p l i e d t o t h e l o w e r s u r f a c e o f t h e l e a f a f t e r t h e e p i d e r m i s h a d b e e n s t r i p p e d b a c k . T h e c e l l o p h a n e s t r i p u s e d t o c o v e r t h e i n o c u l u m i s v i s i b l e i n B . 29 30 t h e p e r i p h e r a l hyphae v/ere t h i c k and compact ( F i g . 1 -9) . A c t i v e l y g r o w i n g v e g e t a t i v e c o l o n i e s ( d i a m 2-4 mm) were t r a n s f e r r e d f r o m l i q u i d t o s o l i d medium ( p l u s BSA) i n t e s t t u b e s . About 5% o f t h e s e t r a n s f e r s v/ere s u c c e s s f u l , r e -newed v e g e t a t i v e g r o w t h b e i n g f o l l o w e d b y s p o r u l a t i o n . 3, P a t h o g e n i c i t y o f U r e d o s p o r e s P r o d u c e d i n A x e n i c C u l t u r e U r e d o s p o r e s grov/n i n v i t r o were a p p a r e n t l y u n a b l e t o i n f e c t i n t a c t v/heat l e a v e s . When s u c h u r e d o s p o r e s were p l a c e d d i r e c t l y on m e s o p h y l l t i s s u e , , w h i c h h a d been expo s e d b y s t r i p -p i n g o f f t h e e p i d e r m i s and c o v e r e d w i t h c e l l o t a p e , n o r m a l u r e d i a l p u s t u l e s d e v e l o p e d i n about 10 d a y s ( F i g . 1-10). E i g h t y p e r c e n t o f t h e s e t r i a l s were s u c c e s s f u l . The u r e d o -s p o r e s p r o d u c e d i n s u c h p u s t u l e s v/ere t h e m s e l v e s a b l e t o i n f e c t -n o r m a l , i n t a c t wheat l e a v e s . U r e d o s p o r e s f r o m p u s t u l e s f o r m e d u n d e r c e l l o t a p e were a l s o f o u n d t o be f r e e o f b a c t e r i a l c o n -t a m i n a n t s . When s e e d e d d i r e c t l y o n t o s o l i d o r l i q u i d m e d i a a t 17°C t h e y p r o d u c e d f l u f f y w h i t e c o l o n i e s w h i c h l a t e r s p o r u l a t e d . 4. S c a n n i n g E l e c t r o n M i c r o s c o p y On e x a m i n a t i o n by s c a n n i n g e l e c t r o n m i c r o s c o p y u r e d o -• s p o r e s grown i n v i t r o were f o u n d t o be c o v e r e d w i t h a l a y e r o f m a t e r i a l o f unknown c o m p o s i t i o n ( F i g . 1-12), w h i c h was n o t se e n on u r e d o s p o r e s t a k e n f r o m i n t a c t , i n f e c t e d p l a n t s ( F i g . I -11). The s p i n e s on u r e d o s p o r e s f r o m b o t h s o u r c e s were s i m i l a r i n s h a p e , s i z e , and d i s t r i b u t i o n ( F i g s / 1-13, 1-14, and 1-15). The. m o r p h o l o g y o f t h e u r e d o s p o r e s u r f a c e v/as s i m i l a r t o t h a t r e p o r t e d r e c e n t l y for ' P u c c i n i a c o r o n a t a ( C o r l e t t 1970). 31 F i g u r e s 1-11 - 1-16. S c a n n i n g e l e c t r o n m i c r o g r a p h s . F i g u r e 1-11. M a t u r e u r e d o s p o r e s grown on i n t a c t wheat l e a v e s . X 21,805. F i g u r e 1-12. M a t u r e u r e d o s p o r e s grown i n v i t r o . Note v / r i n k l e d s u r f a c e c o a t i n g n o t p r e s e n t i n ' F i g u r e I - 1 1 . X 23,291. F i g u r e I - 1 3. S p i n e s on s u r f a c e o f u r e d o s p o r e grown on i n t a c t wheat l e a f . X 1 0 . " ' " F i g u r e 1-14. S p i n e s on s u r f a c e o f u r e d o s p o r e grown on b a s i c medium. X 25,875. F i g u r e 1-15. D e v e l o p i n g s p i n e s on s u r f a c e o f u r e d o -s p o r e grown on i n t a c t wheat l e a f . X 10,797. F i g u r e I - l 6 , V / r i n k l e d s u r f a c e c o v e r i n g o f t e l i o s p o r e grown on b a s i c medium. X 2274. 3 2 33 T e l i o s p o r e s formed i n v i t r o a l s o appeared t o have a s u r f a c e c o a t i n g l i k e t h a t of the uredospores grown i n v i t r o , but t e l i o -spores grown on i n t a c t l e a v e s were not examined. The w r i n k l e d appearance of t h i s c o a t i n g i s c l e a r l y seen i n F i g u r e 1-16. DISCUSSION Our r e s u l t s suggest, t h a t i n the I s o l a t e made a v a i l -a b l e t o us by Dr. W i l l i a m s , anastomosis of germ tubes i s a n e c e s s a r y p r e r e q u i s i t e f o r the f o r m a t i o n of v e g e t a t i v e c o l o n i e s but we have not been a b l e t o o b t a i n c o n c l u s i v e evidence f o r t h i s . N e v e r t h e l e s s a l l attempts t o produce c o l o n i e s from s i n g l e spore i n o c u l a t i o n s of t h e media used have g i v e n n e g a t i v e r e -s u l t s . Our o b s e r v a t i o n s p r o v i d e no evidence to support W i l l i a m s ' (1971) r e c e n t demonstration t h a t d i k a r y o t i c m y c e l i a develop o n l y from uredospores which f i r s t develop c l e a r l y d i f f e r e n t i a t e d i n f e c t i o n s t r u c t u r e s . F u r t h e r work i s needed to r e s o l v e t h i s p o i n t . The uredospores and t e l i o s p o r e s t h a t develop from the d i k a r y o t i c mycelium appear to be e n t i r e l y nor-mal, except f o r the e x i s t e n c e of the c o a t i n g m a t e r i a l , which i s here r e p o r t e d f o r the f i r s t t i m e . Under the microscope, p i g -m entation of both uredospores and t e l i o s p o r e s was s i m i l a r to t h a t of spores produced on i n t a c t p l a n t s . I n our e a r l i e r ex-periments ( C o f f e y et al. 1969) u r e d o s p o r e - l i k e s t r u c t u r e s were found but were v i r t u a l l y c o l o r l e s s . I t i s p o s s i b l e t h a t a f a l l i n the water content of the medium prevented s p o r u l a t i o n i n our e a r l i e r attempts ( C o f f e y et_ a l . 1969); t h i s would not 34 o c c u r w i t h t h e l i q u i d medium u s e d i n t h e c u r r e n t e x p e r i m e n t s . The p a t h o g e n i c i t y o f t h e u r e d o s p o r e s p r o d u c e d on l i q u i d me-dium a l s o a p p e a r s t o be n o r m a l , e x c e p t f o r t h e i m p o r t a n t f a c t t h a t i n f e c t i o n o c c u r s o n l y on e x p o s e d m e s o p h y l l t i s s u e and n o t v i a t h e s t o m a t a on i n t a c t l e a v e s . I n t h i s r e s p e c t o u r o b s e r -v a t i o n s c o n f i r m t h o s e o f W i l l i a m s e t a l . ( 1 9 6 7 ) . I t i s temp-t i n g t o s u g g e s t t h e p o s s i b i l i t y t h a t t h e i n a b i l i t y o f u r e d o -s p o r e s grown i n v i t r o t o i n f e c t I n t a c t l e a v e s i s r e l a t e d t o t h e p r e s e n c e o f t h e unknown m a t e r i a l w i t h w h i c h s u c h s p o r e s a r e c o a t e d . W h i l e t h i s m a t e r i a l does n o t i n h i b i t g e r m i n a t i o n i n v i t r o o r on t h e i n t a c t l e a f , i t may w e l l a f f e c t c o n t a c t w i t h t h e i n t a c t l e a f and t h e d e v e l o p m e n t o f n o r m a l i n f e c t i o n s t r u c -t u r e s . We a r e o f t h e o p i n i o n t h a t t h e m a t e r i a l I s s e c r e t e d by t h e s p o r e s d u r i n g t h e i r d e v e l o p m e n t i n v i t r o . The p o s s i b i l i t y s h o u l d be e n v i s a g e d t h a t i t I s a t t a c k e d by enzymes r e l e a s e d f r o m t h e h o s t c e l l s when t h e e p i d e r m i s o f t h e l e a f i s s t r i p p e d o f f . These o b s e r v a t i o n s open a new a r e a i n t h e I n v e s t i g a t i o n o f t h e ' p r o p e r t i e s o f u r e d o s p o r e s p r o d u c e d i n v i t r o . 35 REFERENCES B u s h n e l l , W.R. 1968. I n v i t r o d e v e l o p m e n t o f an A u s t r a l i a n i s o l a t e o f P u c c i n i a g r a m i n i s f . s p . t r i t i c i . P h y t o -p a t h o l o g y 58: 526-527. C o f f e y , M.D., A. B o s e , and M. Shaw, 1969. I n v i t r o g r o w t h o f g e l a t i n s u s p e n s i o n s o f u r e d o s p o r e s o f P u c c i n i a g r a m i n i s f . s p . t r i t i c i . Can. J . B o t a n y 47: 1291-1293 . C o r l e t t , M. 1970. S u r f a c e s t r u c t u r e o f u r e d i n i o s p o r e s o f P u c c i n i a c o r o n a t a f . s p . a v e n a e . Can. J . B o t a n y 48: 2159-2161. G i a t g o n g , P., and R.A. F r e d e r i c k s e n . 1969. P a t h o g e n i c v a r i a -b i l i t y and c y t o l o g y o f m o n o c o n i d i a l s u b c u l t u r e s o f P i r i c u l a r i a o r y z a e . P h y t o p a t h o l o g y 59: 1152-1157. W i l l i a m s , P.G. 1971. A new p e r s p e c t i v e o f t h e a x e n i c c u l t u r e o f P u c c i n i a g r a m i n i s f . s p . t r i t i c i f r o m u r e d o s p o r e s . P h y t o p a t h o l o g y 6TT"994-1002. W i l l i a m s , P.G., K . J . S c o t t , and J . L . K u h l . 1966. V e g e t a t i v e g r o w t h o f P u c c i n i a g r a m i n i s f . s p . t r i t i c i i n v i t r o . P h y t o p a t h o l o g y 56: 1418-1419. W i l l i a m s , P.G., K . J . S c o t t , J . L . K u h l , and D.J. M a c l e a n . 1967. S p o r u l a t i o n and p a t h o g e n i c i t y o f P u c c i n i a g r a m i n i s f . sp . t r i t i c i grown on an a r t i f i c a l medium. P h y t o -p a t h o l o g y 57: 326-327. W i l l i a m s , P.G., and M.J. H a r t l e y . 1971. O c c u r r e n c e o f d i p l o i d l i n e s o f P u c c i n i a g r a m i n i s t r i t i c i i n a x e n i c c u l t u r e . N a t u r e New B i o l . 229: 181-182. 3 6 CHAPTER I I I n v i t r o C u l t u r e o f t h e F l a x R u s t , M e l a m p s o r a l i n i ABSTRACT Two d i f f e r e n t s t r a i n s o f M e l a m p s o r a l i n i ( P e r s . ) L e v . were grown on m e d i a s i m i l a r t o t h o s e d e s c r i b e d b y T u r e l ( 1 9 6 9 a ) . A l t e r a t i o n s i n o r g a n i c and i n o r g a n i c c o n s t i t u e n t s d i d n o t , i n g e n e r a l , p r e v e n t g r o w t h . P e p t o n e and y e a s t ex-t r a c t , s i n g l y o r i n c o m b i n a t i o n , s u p p o r t e d g r o w t h , b u t c o c o -n u t m i l k d i d n o t . M a i n t e n a n c e o f t h e a m b i e n t t e m p e r a t u r e b e l o w 17°C d u r i n g t r a n s f e r p r o c e d u r e s I n c r e a s e d t h e number o f c o l o n i e s w h i c h grew. The m y c e l i u m was g e n e r a l l y b i n u c -l e a t e . S p o r e - l i k e c e l l s o f v a r y i n g s h a p e s were f o u n d , some o f w h i c h r e s e m b l e d u r e d o s p o r e s and t e l i o s p o r e s . INTRODUCTION The g r o w t h i n a x e n i c c u l t u r e , t h a t i s , i n i s o l a t i o n f r o m a l l o t h e r l i v i n g o r g a n i s m s , o f m y c e l i u m f r o m g e r m i n a t e d u r e d o s p o r e s o f f l a x r u s t was r e p o r t e d b y T u r e l ( 1 9 6 9 a ) . She grew u r e d o s p o r e s o f r a c e 3 , M e l a m p s o r a l i n i ( P e r s . ) L e v . on a medium c o n t a i n i n g D i f c o y e a s t e x t r a c t , s u c r o s e , and I n o r g a n i c s a l t s . The i n a b i l i t y o f t h e c u l t u r e s t o e s t a b l i s h on a s i m i l a r medium i n w h i c h C z a p e k ' s m i n e r a l s were i n c l u d e d a s a s o u r c e o f i n o r g a n i c c o n s t i t u e n t s l e d T u r e l t o assume t h a t t h e b a l a n c e o f i n o r g a n i c s a l t s p l a y e d an i m p o r t a n t p a r t i n 37 d e t e r m i n i n g g r o w t h o f t h i s f u n g u s . I n a d d i t i o n , she f o u n d t h a t r a i s i n g t h e t e m p e r a t u r e above 17°C l e d t o t h e e v e n t u a l d e a t h o f t h e c u l t u r e s ( T u r e l 1969b). Her c u l t u r e s were c a p a b l e o f t r a n s f e r u n d e r c e r t a i n d e f i n e d c o n d i t i o n s and i n a g e i n g p a r t s o f t h e s t r o m a p r o d u c e d a r a n g e o f s p o r e - l i k e c e l l s , i n c l u d i n g some w h i c h r e s e m b l e d o n e - c e l l e d t e l i o s p o r e s ( T u r e l 1969a). An a t t e m p t was t h e r e f o r e made t o c o n f i r m T u r e l ' s work u s i n g two d i f f e r e n t s t r a i n s o f M. l i n i ' 1 ' . The i n f l u e n c e o f i n o r g a n i c , and more p a r t i c u l a r l y t h a t o f o r g a n i c c o n s t i t u e n t s , o f t h e c u l t u r e medium on g r o w t h was e x a m i n e d . N u c l e a r c o n d i t i o n o f t h e m y c e l i u m and o f s p o r e - l i k e c e l l s f o r m e d i n c u l t u r e was d e t e r m i n e d where p o s s i b l e and t h e mor-p h o l o g y o f t h e s p o r e - l i k e c e l l s compared w i t h t y p i c a l s p o r e t y p e s . MATERIALS AND METHODS 1. M e l a m p s o r a l i n i , S t r a i n 1 S e v e n - d a y - o l d B i s o n f l a x c o t y l e d o n s were i n o c u l a t e d w i t h an aqueous s u s p e n s i o n o f u r e d o s p o r e s o f M e l a m p s o r a l i n i . The i n o c u l a t e d s e e d l i n g s were k e p t i n t h e d a r k u n d e r h i g h h u m i d i t y f o r 21+ h and t h e n grown un d e r f l u o r e s c e n t l i g h t i n g ''"The m a t e r i a l u s e d i n t h e s e e x p e r i m e n t s was t h o u g h t t o be r a c e 3 ( s t r a i n 1) and r a c e 210 ( s t r a i n 2). Hov/ever, sub-s e q u e n t t e s t i n g o f t h i s m a t e r i a l on h o s t d i f f e r e n t i a l s h as r e v e a l e d t h a t t h e r a c e s a r e n o t p u r e . 33 (1000 f t - c f o r 16 h ) . C o t y l e d o n s were removed a f t e r 6 d a y s , when f l e c k i n g had o c c u r r e d , and p l a c e d i n a s o l u t i o n o f G r a m i c i d i n ( N u t r i t i o n a l B i o c h e m i c a l s ; 10 mg p e r l i t e r ) f o r 2-3 h. They were t h e n removed, s u r f a c e s t e r i l i z e d f o r 10 m i n w i t h 10% J a v e x (5.25% a v a i l a b l e c h l o r i n e ) and a d r o p o f Tween 80, and washed t h r e e t i m e s i n s t e r i l e g l a s s - d i s t i l l e d w a t e r . The c o t y l e d o n s were t h e n p l a c e d i n c u l t u r e t u b e s on a g a r s l a n t s w i t h t h e i r a d a x i a l s u r f a c e s f a c i n g u p w a r d s . Each t u b e c o n t a i n e d 6 m l o f t h e t i s s u e c u l t u r e medium u s e d by T u r e l (1969a). C u l t u r e s w e re p l a c e d u n d e r c o n t i n u o u s f l u o r e s c e n t l i g h t i n g (500 f t - c ) a t 17°C. A f t e r 13 d a y s , c o n t a m i n a n t f r e e c o t y l e d o n c u l t u r e s "were u s e d as a s o u r c e o f a s e p t i c u r e d o s p o r e s . - S m a l l numbers o f f l a x r u s t s p o r e s were p l a c e d d i r e c t l y on t h e s u r f a c e o f media i n p y r e x c u l t u r e t u b e s (125 x 16 mm- w i t h ' B a c t i C a p a l l ' p o l y p r o p y l e n e c l o s u r e s ) . F o u r d i f f e r e n t m e d i a were u s e d , a l l b a s e d on T u r e l ' s medium I I (1969a), b u t w i t h d i f f e r e n t o r g a n i c c o n s t i t u e n t s : ( i ) w i t h 0.1% D i f c o y e a s t e x t r a c t , ( i i ) w i t h 0.1% E v a n s ' p e p t o n e , ( i i i ) w i t h 0.1% D i f c o y e a s t e x t r a c t and 0 . 1 % 'Evans' p e p t o n e , ( i v ) w i t h 10% c o c o n u t m i l k . 39 M i c r o n u t r i e n t s ' ' ^ v/ere s u p p l i e d t o a l l f o u r m e d i a a t a l e v e l o f 0.8 m l p e r l i t e r . S i x m l o f medium were p l a c e d i n each t u b e ; 100 t u b e s v/ere u s e d f o r each medium. The pH a f t e r a u t o c l a v i n g v/as i n t h e r a n g e 4.9 t o $.1. A l l c u l t u r e t u b e s C o n t a i n i n g m e d i a and a l l i n s t r u m e n t s u s e d t o p l a c e t h e u r e d o -s p o r e s were c h i l l e d a t 3°C b e f o r e u s e . C u l t u r e t u b e s v/ere i n c u b a t e d a t 17°C i n t h e d a r k . At p e r i o d s b e t w e e n 4 and 6 weeks a f t e r c u l t u r i n g h ad been i n i t i a t e d , c o l o n i e s v/ere t r a n s f e r r e d t o t h e medium w i t h 0.1% p e p t o n e . A f t e r 3 weeks c o l o n i e s were a g a i n t r a n s f e r r e d t o a medium w i t h p e p t o n e p l u s m o d i f i e d i n o r g a n i c s c o n s i s t i n g o f K N 0 3 , 1.0 g; M g S 0 4 ' 7 H 2 0 , 0 .25 g; K H 2 P 0 ^ , 0 .25 g; NH^N0 3, 0.04 g; p e r l i t e r o f g l a s s - d i s t i l l e d w a t e r (pH a d j u s t e d t o 6.5 w i t h 0.1 N NaOH b e f o r e a u t o c l a v i n g ) . M i c r o n u t r i e n t s were added e i t h e r a t 0 . 8 o r 0 .5 m l i n l a t e r t r a n s f e r s . A f u r t h e r f i v e t r a n s f e r s were made t o m e d i a c o n t a i n i n g t h e m o d i f i e d i n -o r g a n i c s and e i t h e r p e p t o n e , o r b o t h p e p t o n e and y e a s t e x t r a c t , a t I n t e r v a l s o f 3 t o 6 weeks. ' ' " M i c r o n u t r i e n t s were S e q u e s t r e n e , 13% NaFe ( G e i g y ) , 10 .g; MnS0 4 - 7 H 20, 447 mg; K I , 10 mg; N i C l 2 - 6 H 2 0 , 18* mg; G o C l 2 - 6 H 2 0 , 18 mg; T i ( S 0 4 ) 2 ' 9 H 2 0 , 42 mg; ZnSO^, 35 rag; C u S O ^ H ^ O , 15 mg; BeSO^, 20 mg; H^PO^, 10 mg; H 2 S 0 ^ c o n c e n t r a t e d , 1/5 m l ; g l a s s - d i s t i l l e d w a t e r , 200 m l . M i c r o n u t r i e n t s added e i t h e r a t 0 .5 o r 0 . 8 ml p e r l i t e r o f medium. 40 To d e t e r m i n e i f c o l o n i e s c o u l d be i n i t i a t e d on t h e medium w i t h m o d i f i e d i n o r g a n i c s , an e x p e r i m e n t was c a r r i e d out w i t h M e l a m p s o r a l i n i u s i n g a l i q u i d c u l t u r e s y s t e m . S m a l l amounts o f a s e p t i c u r e d o s p o r e s were p l a c e d i n 10 m l o f t h e c u l t u r e medium ( m o d i f i e d i n o r g a n i c s w i t h m i c r o n u t r i e n t s , 4% s u c r o s e , 0.1% y e a s t e x t r a c t , and 0.1% p e p t o n e ) i n 50 m l c u l t u r e f l a s k s . The f l a s k s were i n c u b a t e d a t 17°C i n t h e d a r k on a r e c i p r o c a t i n g s h a k e r ( a b o u t 100 c y c l e s p e r m i n ) . S h a k i n g was s t o p p e d a f t e r 2 weeks and t h e c u l t u r e s t h e n grown u n d e r s t i l l c o n d i t i o n s . 2. M e l a m p s o r a l i n i , S t r a i n 2 A s e p t i c u r e d o s p o r e s were p r o d u c e d by methods a l r e a d y d e s c r i b e d i n t h e p r e v i o u s s e c t i o n . I n o c u l a t i o n p r o c e d u r e s were s i m i l a r t o t h o s e u s e d f o r s t r a i n 1, m e d i a and I n s t r u -ments b e i n g k e p t b e l o w 17°C and s p o r e s p l a c e d i n s m a l l quan-t i t i e s d i r e c t l y on t h e a g a r s u r f a c e . A m o d i f i e d v e r s i o n o f T u r e l ' s medium I (1969a) was u s e d w i t h 0.1% E v a n s ' p e p t o n e and 0.1% D i f c o casamino a c i d s as a d d i t i o n a l i n g r e d i e n t s . Tubes were examined a f t e r 6 weeks and a g a i n a t 8 weeks. A t 8 weeks c o l o n i e s were t r a n s f e r r e d t o f i v e d i f f e r e n t m e d i a : ( i ) T u r e l ' s I i n o r g a n i c s w i t h 0.1% p e p t o n e , ( i i ) T u r e l ' s I i n o r g a n i c s w i t h 0.1% y e a s t e x t r a c t , ( i i i ) m o d i f i e d i n o r g a n i c s ( s e e page 39) w i t h 0.1% p e p t o n e , 41 '" ( i v ) m o d i f i e d i n o r g a n i c s w i t h 0.1% y e a s t e x t r a c t , (v) m o d i f i e d I n o r g a n i c s w i t h 0.1% c a s a m i n o a c i d s . I n a l l f i v e m e d i a m i c r o n u t r i e n t s were added a t 0 . 8 -m l p e r l i t e r . 3. C y t o l o g i c a l E x a m i n a t i o n o f t h e C u l t u r e s Tease-mount p r e p a r a t i o n s o f i n i t i a l and t r a n s f e r r e d c o l o n i e s o f b o t h s t r a i n s were s t a i n e d w i t h p h l o x i n e / K O H o r h a e m a t o x y l i n f o r e x a m i n a t i o n o f c e l l s i n c u l t u r e s o f d i f f e r e n t a g e s , and o f t h e i r n u c l e a r c o n d i t i o n . RESULTS 1. M e l a m p s o r a l i n i , S t r a i n 1 T r e a t m e n t w i t h G r a m i c i d i n b e f o r e c o n v e n t i o n a l s u r -f a c e s t e r i l i z a t i o n m a r k e d l y r e d u c e d b a c t e r i a l c o n t a m i n a t i o n i n t h e c o t y l e d o n c u l t u r e s . I n a d d i t i o n , no a d v e r s e e f f e c t on t h e s u b s e q u e n t g r o w t h o f t h e c u l t u r e s was o b s e r v e d . D u r i n g t h e f i r s t 3 days i n c u l t u r e no e v i d e n c e o f b a c t e r i a l contam-i n a t i o n o f t h e c o t y l e d o n s c o u l d be f o u n d . A f t e r 13 days c o n -t a m i n a t i o n was o n l y f o u n d i n 3S out o f 126 t u b e s . I n e a r l i e r e x p e r i m e n t s i n w h i c h t h e a n t i b i o t i c was n o t u s e d , c o n t a m i n a -t i o n was sometimes as h i g h as 75% even a t 3 d a y s . O n l y v e r y s m a l l q u a n t i t i e s o f u r e d o s p o r e s were p l a c e d a t any p a r t i c u l a r p o i n t on t h e a g a r s u r f a c e . L a r g e q u a n t i t i e s o f u r e d o s p o r e s o f t h e o r d e r r e q u i r e d t o p r o d u c e m y c e l i a l g r o w t h o f P u c c i n i a g r a m i n i s ( B u s h n e l l 1968, W i l l i a m s et a l . 1966, 1967) r e s u l t e d i n no g r o w t h w i t h M e lampsora l i n i 42 b e c a u s e g e r m i n a t i o n was c o m p l e t e l y i n h i b i t e d . E s t i m a t i o n s o f t h e number o f e s t a b l i s h e d c o l o n i e s o f M e l a m p s o r a l i n i a f t e r 4 weeks i n c u l t u r e r e v e a l e d 8 0 % s u c c e s s on m e d i a w i t h y e a s t e x t r a c t o r p e p t o n e , o r b o t h t o g e t h e r . No d i s t i n c t i o n c o u l d be made i n g r o w t h on t h e t h r e e d i f f e r e n t m e d i a . Of t h e t o t a l , a b o u t 5% showed v e r y good g r o w t h a t t h i s s t a g e ( c o l o n i e s up t o 1 cm i n diam) w i t h p r o f u s e w h i t e a e r i a l m y c e l i u m . W i t h c o c o n u t m i l k , h o w e v e r , o n l y germ t u b e s were p r o d u c e d as a l o o s e mat on t h e a g a r s u r f a c e . At t h i s s t a g e , t r a n s f e r o f a l l t h e s m a l l c o l o n i e s ( 1 - 2 mm i n diam) was a t t e m p t e d . The f a s t e r g r o w i n g c o l o n i e s v/ere l e f t a f u r t h e r 2 weeks, d u r i n g w h i c h t i m e t h e y p r o d u c e d more a e r i a l m y c e l i u m . W i t h t h e s m a l l e r c o l o n i e s , s u r v i v a l on t r a n s f e r was a b o u t 0.5%; w i t h t h e f a s t e r g r o w i n g c o l o n i e s I t was a b o u t 30%. I n s u b s e q u e n t t r a n s f e r s o f t h e l a t t e r an a v e r a g e o f 50% s u r v i v a l was ob-t a i n e d . E n t i r e c o l o n i e s c o u l d e a s i l y be l i f t e d f r o m t h e a g a r s u r f a c e and I t was a p p a r e n t t h a t o n l y t h e s t r o m a t a and n o t t h e m y c e l i u m w ere i n c o n t a c t w i t h t h e medium. I n p r e p a r i n g T u r e l ' s medium I I i t was n o t e d t h a t p r e c i p i t a t i o n o f c a l c i u m p h o s p h a t e o c c u r r e d , a s a r e s u l t o f t h e r e a c t i o n o f t h e d i b a s i c p o t a s s i u m p h o s p h a t e w i t h c a l -c i u m n i t r a t e . A medium w i t h m o d i f i e d i n o r g a n i c s ( s e e page 39) was t h e r e f o r e u s e d f o r t h e t r a n s f e r r e d c o l o n i e s . Good g r o w t h was o b t a i n e d on t h i s m o d i f i e d medium ( F i g . I I - l ) and t h i s s h o u l d p r o v e u s e f u l I n f u t u r e n u t r i t i o n a l s t u d i e s w i t h d i f f e r e n t r a c e s o f M e l a m p s o r a l i n i . I n t h e i n i t i a l c o l o n y an o r a n g e s t r o m a f o r m e d be-n e a t h t h e m y c e l i u m a f t e r $ t o 8 weeks. I n t r a n s f e r r e d c o l o n i e s t h i s o r i g i n a l s t r o m a t u r n e d brown and a new o r a n g e s t r o m a l r e g i o n d e v e l o p e d a r o u n d i t ( F i g . I I - 2 ) . The i n i t i a -t i o n o f f r e s h m y c e l i a l g r o w t h was a l w a y s a s s o c i a t e d w i t h t h i s l a t t e r r e g i o n . S u c c e s s f u l t r a n s f e r o f t h e r u s t was o n l y a c -h i e v e d when l a r g e s t r o m a l segments ( f r o m 5 t o 10 mm i n diam) were i n c l u d e d i n t h e f u n g a l m a t e r i a l . W i t h t h e l i q u i d c u l t u r e s y s t e m s h a k i n g was s t o p p e d a f t e r 2 weeks as i t was f o u n d t h a t t h e l o o s e mat o f germ t u b e s t e n d e d t o become a t t a c h e d t o t h e w a l l s o f t h e c u l t u r e f l a s k s and removed f r o m t h e medium. A f t e r 5 weeks (3 weeks i n s t i l l c u l t u r e ) s m a l l amounts o f m y c e l i u m a p p e a r e d i n 23 o u t o f 24 f l a s k s . I n a b o u t 2$% o f t h e s e c o l o n i e s d e v e l o p m e n t was good (3-6 mm d i a m ) . C o l o n i e s grew on t h e s u r f a c e o f t h e medium and had a s i m i l a r a p p e a r a n c e t o t h o s e f o r m e d on s o l i d m e d i a , h a v i n g o r a n g e s t r o m a t a and p r o f u s e a e r i a l m y c e l i u m . A few f l a s k s had t h r e e o r f o u r i n d i v i d u a l c o l o n i e s . I t i s e v i d e n t , t h e r e f o r e , t h a t b o t h i n i t i a l and t r a n s f e r r e d c o l -o n i e s c a n be grown on t h e medium w i t h m o d i f i e d i n o r g a n i c s a l t s . 2. M e l a m p s o r a l i n i , S t r a i n 2 Growth o f s t r a i n 2 was a c h i e v e d on t h e medium d e s -c r i b e d ( s e e M a t e r i a l s ar.d M e t hods) . A f t e r 6 weeks about 60% o i t h e t u b e s had m y c e l i a l g r o w t h ; 15% o f t h e c u l t u r e s were l a r g e r (3-5 mm) and had orange s t r o m a t a . By 8 weeks about 44 F i g u r e I I - l . A 1 2 - d a y - o l d c o l o n y o f s t r a i n 1 ( f o u r t h t r a n s f e r ) g r o w i n g on a medium w i t h i n -o r g a n i c s , s u c r o s e , y e a s t e x t r a c t , and p e p t o n e . "White a e r i a l m y c e l i u m c o v e r s a c e n t r a l s t r o m a t a l r e g i o n , t h e l a t t e r p r o j e c t i n g t h r o u g h t h e c o l o n y a t one p o i n t . X 9 . F i g u r e I I - 2 . Same c o l o n y v i e w e d f r o m b e l o w . A r r o w s d e n o t e a v i s i b l e d e m a r c a t i o n between t h e o l d - c e n t r a l brown s t r o m a t a l r e g i o n ( o r i -g i n a l i n o c u l u m ) and an o r a n g e zone w h i c h f o r m e d s u b s e q u e n t t o t r a n s f e r . X 6. 46 30% o f t h e c o l o n i e s had o r a n g e s t r o m a t a and e x t e n s i v e a e r i a l m y c e l i u m ( F i g . I I - 3 ) . I n t h e o t h e r 50% o f e s t a b l i s h e d c u l -t u r e s , c o l o n i e s were w h i t e and f l u f f y (1-2 mm) bu t w i t h o u t s t r o m a t a ( F i g . I I - 4 ) ; 20% o f t h e t u b e s c o n t a i n e d n o t h i n g be-yond a n e t w o r k o f germ t u b e s . An i n t e r e s t i n g o b s e r v a t i o n made on t h e s e germ t u b e s was t h a t a n a s t o m o s i s had o c c u r r e d w h e r e v e r t h e y were i n c o n t a c t w i t h e a c h o t h e r ( F i g . I I - 5 ) . A f t e r 10 weeks a l a r g e p r o p o r t i o n o f t h e s m a l l c o l o n i e s h a d grown and d e v e l o p e d o r a n g e s t r o m a t a . A t t h i s t i m e h a l f t h e c u l t u r e s w h i c h showed no g r o w t h a t 8 weeks had now p r o d u c e d a e r i a l m y c e l i u m . C o l o n i e s t r a n s f e r r e d a t '8 weeks grew on a l l t h e m e d i a t e s t e d . I t was n o t p o s s i b l e t o d e t e r m i n e c o n s i s t e n t d i f f e r e n c e s i n g r o w t h b e tween m e d i a b e c a u s e t h e v a r i a b i l i t y i n c o l o n y s i z e was t o o h i g h , 3 . C y t o l o g i c a l S t u d i e s w i t h M e l a m p s o r a l i n i S t a i n i n g w i t h h a e m a t o x y l i n r e v e a l e d b i n u c l e a t e , s e p t a t e m y c e l i u m i n b o t h i n i t i a l and t r a n s f e r r e d c o l o n i e s o f both r a c e s ( F i g s . I I - 6 and I I - 7 ) . I n ab o u t 20% o f t h e c e l l s o n l y one n u c l e u s was n o t e d . T h i s c o u l d have been due t o m i g r a t i o n o f one o f t h e n u c l e i ( F i g . I I - 8 ) . A n a s t o m o s i s o f h y p h a l c e l l s was o b s e r v e d i n a f e w c a s e s ( F i g . I I - 9 ) . E x a m i n a t i o n o f t h e o r a n g e s t r o m a t a o f t h e i n i t i a l c o l o n i e s r e v e a l e d l a r g e numbers o f t h i n - w a l l e d r o u n d e d b o d i e s ( F i g . 11-10) w i t h two n u c l e i n o t v i s i b l e i n t h e f i g u r e . I n a s m a l l number of c a s e s these same t y p e s o f 47 F i g u r e I T - 3 . An 8 - w e e k - o l d c o l o n y o f s t r a i n 2 grown f r o m a s e p t i c u r e d o s p o r e s . X 13. F i g u r e I I - 4 . An 8-week-old c o l o n y o f s t r a i n 2 o f s m a l l s i z e (1-2 mm) c o n s i s t i n g e n t i r e l y o f a e r i a l m y c e l i u m . X 14. F i g u r e I I - $ , F u s i o n o f germ t u b e s o f u r e d o s p o r e s o f s t r a i n 2 . A r r o w i n d i c a t e s a r e g i o n o f germ t u b e f u s i o n . P h l o x i n e / K O H . X 768. F i g u r e I I - 6 . B i n u c l e a t e h yphae o f i n i t i a l c o l o n i e s o f s t r a i n 2. H y p h a l s w e l l i n g s a r e p r o -m i n e n t . H a e m a t o x y l i n . X 768. 49 F i g u r e 11-7. F i g u r e I 1 - 8 . F i g u r e I I - 9 . F i g u r e 11-10. F i g u r e 11-11. B i n u c l e a t e m y celium o f a t r a n s f e r r e d c o l o n y o f s t r a i n 1. A m i t o t i c f i g u r e (anaphase) i s v i s i b l e , i n d i c a t e d by an arrow. H a e m a t o x y l i n . X 2307. H y p h a l c e l l s f r o m an 8-week-old c o l o n y o f s t r a i n 2. One c e l l has two n u c l e i , o t h e r s have one. The arrow d e n o t e s a c e l l i n w h i c h t h e two n u c l e i a r e s e p a r -a t e d . H a e m a t o x y l i n . X 1920. H y p h a l a n a s t o m o s i s . An 8-week-old c o l o n y o f s t r a i n 2. H a e m a t o x y l i n . X 480. T h i n - w a l l e d s p h e r i c a l s p o r e c e l l s f r o m s t r o m a t a o f an I n i t i a l c o l o n y o f s t r a i n i n o c u l u m . P h l o x i n e / K O H . X 234. T h i n - w a l l e d c e l l s i n t e r s p e r s e d by s h o r t h y p h a l c e l l s . I n i t i a l c o l o n y o f s t r a i n 1 (8 weeks o l d ) . P h l o x i n e / K O H . X 560. 50 5 1 s t r u c t u r e s were s e e n i n t e r s p e r s e d b y s h o r t h y p h a l c e l l s ( F i g . 1 1 - 1 1 ) . These c e l l s were sometimes f o r m e d i n c h a i n s o r g a n -i z e d i n t o a ' m i c r o s o r u s ' - l i k e s t r u c t u r e ( F i g . 1 1 - 1 2 ) . T h e r e were a l s o s m a l l numbers o f t h i c k - w a l l e d , n o n - p i g m e n t e d c e l l s w i t h v e r r u c o s e w a l l s ( F i g . 1 1 - 1 3 ) , and w i t h d i m e n s i o n s s i m i l a r t o t h o s e o f t y p i c a l u r e d o s p o r e s ( 1 8 . 6 t o 3 1 . 0 by 2 4 . 0 t o 3 7 . 2 m i c r o n s ) , t h o u g h no germ p o r e s were o b s e r v e d . Some o f t h e s e were c o n n e c t e d by h y p h a l s t r u c t u r e s ( F i g . 1 1 - 1 4 ) . I n t r a n s -f e r r e d c o l o n i e s i n w h i c h t h e o r i g i n a l u r e d o s p o r e s were no l o n g e r p r e s e n t , t h i n - w a l l e d s p h e r i c a l c e l l s and t h i c k - w a l l e d c e l l s w i t h e l o n g a t e d and v a r i a b l e s h a p e s v/ere l o c a t e d i n t h e brown s t r o m a t a ( F i g . 1 1 - 1 5 ) . A t t e m p t s t o s t a i n t h e l a t t e r w i t h h a e m a t o x y l i n t o d i f f e r e n t i a t e t h e i r n u c l e a r c o n d i t i o n u s u a l l y f a i l e d , b u t i n a few p r e p a r a t i o n s two n u c l e i were ob-s e r v e d . Some o f t h e s e s p o r e - l i k e c e l l s f o r m e d t e r m i n a l l y and o t h e r s i n c h a i n s ( F i g s . I I - 1 6 , 1 1 - 1 7 , and I I - 1 8 ) . T h e i r d i m e n s i o n s were s i m i l a r t o t h o s e o f t y p i c a l t e l i o s p o r e s ( 2 4 . 8 t o 31.0 by 4 3 . 4 t o 60.0 m i c r o n s ) . No c y t o l o g i c a l d i f f e r e n c e s were n o t e d between t h e two s t r a i n s . DISCUSSION Growth o f b o t h s t r a i n s on media b a s e d upon t h o s e used by T u r e l (1969a) c o n f i r m s h e r f i n d i n g s . F u r t h e r m o r e , t h e u s e o f l o w t e m p e r a t u r e s (3-I7°C) d u r i n g t h e i n o c u l a t i o n and t r a n s f e r p r o c e d u r e s i m p r o v e d t h e c o n s i s t e n c y o f g r o w t h 52 F i g u r e 11-12. ' M i c r o s o r u s * s t r u c t u r e f r o m an i n i t i a l c o l o n y o f s t r a i n 1 (7 weeks o l d ) . P h l o x i n e / K O H . X 768. F i g u r e 11-13 . N o n - p i g m e n t e d , t h i c k - w a l l e d c e l l s . No germ p o r e s v i s i b l e . V e r r u c o s e . From i n i t i a l c o l o n y o f s t r a i n 2 . Haematoxy-l i n . X 1200. F i g u r e 11-14. Same, s h o w i n g two o f t h e s e c e l l s j o i n e d b y s h o r t h y p h a l c e l l s . H a e m a t o x y l i n . X 4 8 0 . 53 54 F i g u r e 11-15. Dense s p o r e c l u s t e r f r o m s t r o m a o f a t r a n s -f e r r e d c u l t u r e o f s t r a i n 1. A r r o w s d e n o t e t h i c k - w a l l e d c e l l s w i t h v a r i a b l e s h a p e s . P h l o x i n e / K O H . X 3 0 7 . F i g u r e s I I - 1 6 and 11-17. S p o r e - l i k e c e l l s f o r m e d t e r -m i n a l l y and i n c h a i n s . Some r e s e m b l e t e l i o -s p o r e s , o t h e r a r e a t y p i c a l . From a t r a n s -f e r r e d c o l o n y o f s t r a i n 1. P h l o x i n e / K O H . x 3 0 7 . F i g u r e 11-18. A c e l l w h i c h p r o b a b l y most c l o s e l y r e s e m b l e s a t y p i c a l t e l i o s p o r e . P h l o x i n e / K O H . X 7 6 8 . 55 56 ( c f . 80-90% g r o w t h w i t h 20% g r o w t h o b t a i n e d i n e a r l i e r w o r k ) . T h i s f i n d i n g a l s o s u p p o r t s T u r e l ' s (1969b) w o r k . However, t h e r e was s t i l l a g r e a t d e a l o f v a r i a b i l i t y i n g r o w t h w i t h some c u l t u r e s e x h i b i t i n g a l a g p e r i o d as l o n g as 8 weeks be-f o r e t h e i n i t i a t i o n o f m y c e l i a l g r o w t h f r o m t h e o r i g i n a l s p o r e i n o c u l u m . T h i s c o n t r a s t s w i t h t h e h i g h c o n s i s t e n c y and more r a p i d g r o w t h o b t a i n e d w i t h P u c c i n i a g r a m i n i s f . s p . t r i t i c i , A N Z 126-6,7 u s i n g g e l a t i n s u s p e n s i o n s o f u r e d o s p o r e s ( C o f f e y e t a l . 1969) . The a b i l i t y o f b o t h s t r a i n s t o grow on m e d i a w i t h d i f f e r e n t o r g a n i c c o n s t i t u e n t s and some changes i n i n o r g a n i c s s u g g e s t s t h a t t h e i r n u t r i t i o n a l r e q u i r e m e n t s may be r e l a t i v e l y n o n - s p e c i f i c . . The p r e c i p i t a t i o n w h i c h o c c u r r e d d u r i n g p r e p a r a -t i o n o f one o f t h e m e d i a i s a w a r n i n g t h a t c a r e must be e x e r -c i s e d i n I n t e r p r e t i n g t h e n u t r i t i o n o f m i c r o o r g a n i s m s i n t e r m s o f t h e c o n t e n t s of t h e c u l t u r e medium. A m o d i f i e d medium p r e p a r e d w i t h o u t t h e p r e c i p i t a t i o n o f i n o r g a n i c s a l t s s u p p o r t e d g r o w t h o f M e l a m p s o r a l i n i and w i l l be u s e d i n f u t u r e w o r k . The f a c t t h a t c a s a m i n o a c i d s s u p p o r t e d g r o w t h o f t r a n s f e r r e d c o l o n i e s o f s t r a i n 2 s u g g e s t s t h a t amino a c i d s may be t h e a c t i v e component o f t h e o r g a n i c c o m p l e x e s u s e d . However, t r a n s f e r r e d c o l o n i e s a r e n o t s u i t a b l e m a t e r i a l f o r n u t r i t i o n a l s t u d i e s s i n c e t h e r e c o u l d be a l a r g e c a r r y - o v e r o f n u t r i e n t s f r o m t h e p r e v i o u s medium. I f t h e r e were any d i f f e r e n c e s i n g r o w t h r a t e s between t h e two s t r a i n s , t h e s e were n o t a p p a r e n t b e c a u s e o f t h e h i g h i n h e r e n t v a r i a b i l i t y i n t h e c u l t u r e s o b t a i n e d . T h i s v a r i a b i l i t y i s i n p a r t due t o v a r i a t i o n i n 5 7 t h e l e n g t h o f t h e l a g p h a s e . G e n e t i c o r p h y s i o l o g i c a l f a c t o r s o r b o t h may be i n v o l v e d h e r e . I t i s l i k e l y t h a t t h e s t r a i n s u s e d c o n t a i n a h e t e r o g e n o u s m i x t u r e o f s p o r e s ( F i n c h a m and Day 1963), p o s s i b l y w i t h a w i d e r a n g e o f a b i l i t i e s f o r s a p r o -p h y t i s m . C y t o l o g i c a l e v i d e n c e showed t h a t t h e m y c e l i u m i s g e n e r a l l y b i n u c l e a t e and s e p t a t e . S i m i l a r s p o r e - l i k e c e l l s were f o u n d i n b o t h I n i t i a l and t r a n s f e r r e d m a t e r i a l o f t h e two s t r a i n s . Many o f t h e s e c e l l s were t h i n - w a l l e d and s p h e r i c a l b u t c e l l s w i t h s hapes and d i m e n s i o n s s i m i l a r t o u r e d o s p o r e s and t e l i o s p o r e s were r e c o r d e d . They were c e r t a i n l y n o t t y p i c a l s p o r e s , and t h e i r f o r m a t i o n i n a t o u g h l e a t h e r y zone r e n d e r e d them u n a v a i l a b l e f o r g e r m i n a t i o n s t u d i e s . I t i s p r o b a b l e t h a t t h e p h y s i c a l and c h e m i c a l c o n d i t i o n s n e c e s s a r y f o r n o r m a l s p o r u l a t i o n p r o -c e s s e s were n o t a c h i e v e d . I d e a l l y s p o r e f o r m a t i o n s h o u l d o c c u r s u p e r f i c i a l l y on t h e s u r f a c e o f t h e m y c e l i a l n e t w o r k as i t d i d w i t h P u c c i n i a g r a m i n i s ( W i l l i a m s e t al. 1967) b u t even w i t h t h i s s p e c i e s i t was n o t a l w a y s f o u n d ( B u s h n e l l 1968, C o f f e y e t a l . 1969). E a r l y work o f H o t s o n and C u t t e r (1959, I960, 1951) r e p o r t e d t h e g r o w t h o f s t r a i n s o f Gymno.sporangium j u n i p e r ! -v i r g i n i a n a e Schw. and Uromyces a r i - t r i p h y l l i Schw. on a r t i f i c i a l m e d i a . More r e c e n t l y , t h e g r o w t h o f r e p r e s e n t a t i v e s f r o m two d i f f e r e n t r u s t f a m i l i e s , M e l a m p s o r a c e a e and P u c c i n i a c e a e , and o f d i f f e r e n t p h y s i o l o g i c a l s t r a i n s w i t h i n a s i n g l e s p e c i e s , M e l a m p s o r a l i n i ( P e r s . ) L e v . , s u g g e s t s t h a t i n t i m e a l l r u s t f u n g i w i l l be grown i n a x e n i c c u l t u r e . The s e n s i -53 t i v i t y t o t e m p e r a t u r e and o t h e r , as y e t u n d e t e r m i n e d , e n v i r o n m e n t a l f a c t o r s has o b v i o u s l y been a c o n t r i b u t o r y f a c t o r i n t h e many p r e v i o u s a b o r t i v e a t t e m p t s a t c u l t u r i n g t h e U r e d i n a l e s . 5 9 REFERENCES B u s h n e l l , W.R. 1968. I n v i t r o d e v e l o p m e n t o f an A u s t r a l i a n i s o l a t e o f P u c c i n i a g r a m i n i s f . s p . t r i t i c i . P h y t o -p a t h o l o g y 5W: 526-527 . C o f f e y , M.D., A. B o s e , and M. Shaw. 1969. I n v i t r o g r o w t h o f g e l a t i n s u s p e n s i o n s o f u r e d o s p o r e s o f P u c c i n i a g r a m i n i s f . s p . t r i t i c i . C a n . J . B o t a n y 47: 1291-1293. C u t t e r , V.M., J r . 1 9 5 9 ( 1 9 6 1 ) . S t u d i e s on t h e i s o l a t i o n and g r o w t h o f p l a n t r u s t s i n h o s t t i s s u e c u l t u r e s and upon s y n t h e t i c m e d i a . I . Gymnosporangium. M y c o i o g i a 51: 2 4 8 - 2 9 5 . C u t t e r , V.M., J r . 1 9 6 0 ( 1 9 6 1 ) . S t u d i e s on t h e i s o l a t i o n and g r o w t h o f p l a n t r u s t s i n h o s t t i s s u e c u l t u r e s and upon s y n t h e t i c m e d i a . I I . Uromyces a r i - t r y p h y l l i . M y c o i o g i a 52: 7 2 6 - 7 4 2 . F i n c h a m , J . R . S . , and P.R. Day. 19-63. F u n g a l g e n e t i c s . I n B o t a n i c a l M o n o g r a p h s . V o l . 4 . B l a c k w e l l S c i e n t i f i c P u b l i c a t i o n s , O x f o r d , p. 254. H o t s o n , H.H. , 'and V.M. C u t t e r , J r . 1 9 5 L The i s o l a t i o n and c u l t u r e o f Gymnosporangium i u n i p e r i - v i r g i n i a n a e Schw. P r o c . N a t . A c a d . S c i . U.S. 37: 4 0 0 - 4 0 3 . S c o t t , K . J . 1968, The i n i t i a t i o n o f s a p r o p h y t i c g r o w t h o f P u c c i n i a g r a m i n i s . A b s t r a c t o f p a p e r s . F i r s t I n t e r n a t i o n a l C o n g r e s s o f P l a n t P a t h o l o g y , L o n d o n . T u r e l , F.L.M. 1969a. S a p r o p h y t i c d e v e l o p m e n t o f t h e f l a x r u s t M e l a m p s o r a l i n i , r a c e No. 3 . Can. J . B o t a n y 47: 8 2 1 - 8 2 3 . T u r e l , F.L.M. 1969b. Low t e m p e r a t u r e r e q u i r e m e n t f o r s a p r o -p h y t i c f l a x r u s t c u l t u r e s . C a n . J . B o t a n y 47: 163 7 - 1 6 3 8 . W i l l i a m s , F.G., K . J . S c o t t , and J . L . K u h l . 1966. V e g e t a t i v e g r o w t h of P u c c i n i a g r a m i n i s f . s p . t r i t i c i i n v i t r o . P h y t o p a t h o l o g y 56: 1418^1419. W i l l i a m s , P.G., K . J . S c o t t , J . L . K u h l , and D . J . M a c l e a n . 1967. S p o r u l a t i o n and p a t h o g e n i c i t y o f P u c c i n i a g r a m i n i s f , s p . t r i t i c i grown on an a r t i f i c i a l medium. P h y t o -p a t h o l o g y 57: 3 2 6 - 3 2 7 . 60 CHAPTER I I I I n v i t r o Grov/th o f Wheat and F l a x R u s t F u n g i on C h e m i c a l l y D e f i n e d M e d i a ABSTRACT A c h e m i c a l l y d e f i n e d , l i q u i d medium c o n t a i n i n g C z a p e k ' s m i n e r a l s , Ca++, g l u c o s e , a s p a r t i c a c i d , g l u t a t h i o n e and c y s t e i n e and se e d e d w i t h u n c o n t a m i n a t e d u r e d o s p o r e s , " s u p p o r t e d good v e g e t a t i v e g r o w t h and s p o r u l a t i o n o f an Aus-t r a l i a n i s o l a t e o f wheat stem r u s t ( P u c c i n i a g r a m i n i s P e r s . f . s p . t r i t i c i ( E r i k s s . & Henn.), r a c e ANZ 1 2 6 - 6 , 7 ) . O n l y one of s i x N o r t h A m e r i c a n i s o l a t e s t e s t e d (Race 38) f o r m e d c o l o n -i e s w h i c h a p p r o a c h e d t h o s e o f r a c e ANZ 1 2 6 - 6 , 7 i n f i n a l s i z e and g e n e r a l m o r p h o l o g y . 5'AMP had no e f f e c t on g r o w t h , b u t c y c l i c AMP i n h i b i t e d g rowth a f t e r u r e d o s p o r e g e r m i n a t i o n . H i g h l y r e p r o d u c i b l e g r o w t h o f f l a x r u s t ( M e l a m p s o r a l i n i ( E h r e n b , ) L e v . , Race 3) was o b t a i n e d r o u t i n e l y by s e e d -i n g u n c o n t a m i n a t e d u r e d o s p o r e s on a s o l i d medium c o n t a i n i n g D i f c o b a c t o - a g a r , s u c r o s e , Knop's m i n e r a l s a l t s and m i c r o -n u t r i e n t s p l u s y e a s t e x t r a c t , p e p t o n e and b o v i n e serum a l -bumen ( B S A ) . BSA was shown t o overcome t h e i n i t i a l i n h i b i -t i o n o f g r o w t h and t o promote t h e f o r m a t i o n o f u r e d o s p o r e s . I t c o u l d n o t be r e p l a c e d by p u t r e s c i n e . V e g e t a t i v e c u l t u r e s c o u l d be m a i n t a i n e d i n d e f i n i t e l y by s u b d i v i s i o n a t t h e 61 v e g e t a t i v e s t a g e and t r a n s f e r t o t h e same medium minus BSA. S u r f a c e s t e r i l i z e d f l a x c o t y l e d o n s became i n f e c t e d when p l a c e d w i t h t h e i r c u t ends i n c o n t a c t w i t h t h e s e c o l o n i e s . A c h e m i c a l l y d e f i n e d l i q u i d medium c o n t a i n i n g s u c -r o s e , Knopts m i n e r a l s a l t s , m i c r o n u t r i e n t s , a s p a r t i c ( o r g l u -t a m i c ) a c i d and c y s t e i n e s u p p o r t e d t h e growth o f v e g e t a t i v e c o l o n i e s o f Melampsora l i n i f r o m u n c o n t a m i n a t e d u r e d o s p o r e s i n a h i g h l y r e p r o d u c i b l e manner. The f o r m a t i o n o f u r e d o s p o r e s and t e l i o s p o r e s by t h e s e c o l o n i e s was c o n t r o l l e d by (a) t h e l e v e l o f Ca++ (as Ca^O-^^) a n d (b) t h e number o f c o l o n i e s p e r f l a s k . W i t h 40 t o 60 c o l o n i e s p e r f l a s k , u r e d o s p o r e f o r m a t i o n o c c u r r e d i n 60 t o 70% o f t h e c o l o n i e s a t a Ca++ l e v e l of 8 . 5 mM ( 2 . 0 g / l G a ( N 0 Q ) 0 ) . A d e c r e a s e i n t h e Ca++ l e v e l t o 4.25 mM or i n t h e c o l o n y f r e q u e n c y t o 10 per f l a s k r e s u l t e d I n o n l y i n f r e q u e n t s p o r u l a t i o n . The u r e d o s p o r e s produced i n v i t r o i n f e c t e d i n t a c t , week-old f l a x c o t y l e d o n s i n a normal manner. INTRODUCTION The s u c c e s s f u l growth of an i s o l a t e of P u c c i n i a g r a m i n i s P e r s . f . sp. t r i t i c i ( E r i k s s . & Henn.), Race ANZ 126-6,7 by W i l l i a m s e t a l . (1966, 1967) from u r e d o s p o r e s seeded on a s i m p l e medium i n d i c a t e d t h e f e a s i b i l i t y of c u l t u r i n g some of t h e r u s t f u n g i t r a d i t i o n a l l y known as o b l i g a t e p a r a s i t e s and s t i m u l a t e d r e s e a r c h i n t h i s f i e l d i n s e v e r a l l a b o r a t o r i e s . A number of h e t e r o e c i o u s and a u t o e c i o u s r u s t s b e l o n g i n g t o t h e P u c c i n i a c e a e and Melampsoraceae have s u b s e q u e n t l y been 62 grown i n c u l t u r e f r e e f r o m t h e i r h o s t s ( B u s h n e l l 1 9 6 8 , C o f f e y et a l . 1969, T u r e l 1969, 1971, M a c l e a n and S c o t t 1970, Wong and W i l l e t s 1970, K u h l e t a l . 1971 , M a s i n and A n d r e e v 1971, Bose and Shaw 1971 , J o n e s 1 9 7 2 ) . R e c e n t l y V a s s i l ' e v and • S a p l i n a (1971) v/ere a b l e t o e s t a b l i s h a x e n i c c u l t u r e o f M e l -ampsora p i n i t o r q u a (A. BR) R o s t r . , t h e c a u s a l o r g a n i s m o f stem r u s t o f p i n e , s t a r t i n g f r o m b a s i d i o s p o r e s , b u t t h e i r a c c o u n t i s t o o b r i e f t o a l l o w a c r i t i c a l a s s e s s m e n t o f t h e i r r e s u l t s , w h i c h r e q u i r e c o n f i r m a t i o n . The r e q u i r e m e n t s f o r a x e n i c g r o w t h o f t h e s e f u n g i I n c l u d e a s u i t a b l e c a r b o h y d r a t e s o u r c e , m i n e r a l n u t r i e n t s I n t h e ' c o r r e c t ' b a l a n c e ( T u r e l 1969, C o f f e y and Shaw 1972, C o f f e y and A l l e n 1973) and an o r g a n i c n i t r o g e n s o u r c e s u c h as p e p t o n e , b o v i n e serum albumen and y e a s t e x t r a c t . The c a l c i u m l e v e l a p p e a r s t o be p a r t i c u l a r l y i m p o r t a n t ( C o f f e y and A l l e n 1 9 7 3 ) . S a p r o b i c g r o w t h o f t h e r u s t s i s c h a r a c t e r i z e d by a l a g phase f o l l o w e d by v e g e t a t i v e g r o w t h and t h e f o r m a t i o n o f a s t r o m a . The f o r m a t i o n o f u r e d o s p o r e s and t e l i o s p o r e s i s i r r e g u l a r . I n d e e d , u n d e r c e r t a i n c u l t u r a l c o n d i t i o n s , some s t r a i n s r e m a i n p e r s i s t e n t l y v e g e t a t i v e ( M a c l e a n and S c o t t 1970, Howes and S c o t t 1 9 7 2 ) . S u c c e s s f u l r e - i n f e c t i o n o f t h e h o s t w i t h m y c e l i u m has been a c h i e v e d by s e v e r a l w o r k e r s ( W i l l i a m s e t a l . 1967, B u s h n e l l 1968, H a r t l e y and W i l l i a m s 1971, T u r e l 1 9 7 1 ) . R e - I n f e c t i o n by u r e d o s p o r e s p r o d u c e d i n a x e n i c c u l t u r e h as o n l y been r e p o r t e d by two gr o u p s ( W i l l i a m s e t a l . 1967, Bose and Shaw 1971) . 63 C u r r e n t l y , a t t e m p t s a r e b e i n g made i n s e v e r a l l a b o r a -t o r i e s t o d e v e l o p d e f i n e d m e d i a f o r t h e a x e n i c g r o w t h o f r u s t f u n g i by s u b s t i t u t i n g m i x t u r e s o f amino a c i d s f o r more c o m p l e x s o u r c e s o f o r g a n i c n i t r o g e n . Thus K u h l e t a l . (1971) r e p o r t e d t h a t P u c c i n i a g r a m i n i s t r i t i c i Race ANZ 1 2 6 - 6 , 7 showed some g r o w t h on a medium i n w h i c h y e a s t e x t r a c t and p e p t o n e were r e -p l a c e d by a s u l p h u r amino a c i d s u c h as c y s t e i n e . As measured by t h e t o t a l n i t r o g e n c o n t e n t o f t h e m y c e l i u m g r o w t h on t h i s medium v/as abo u t h a l f t h a t on y e a s t e x t r a c t o r p e p t o n e , and t h e a u t h o r s c o n c l u d e d t h a t no medium o r method o f i n o c u l a t i o n -was c o m p l e t e l y r e l i a b l e . F o u d l n and Wynn (1972) a c h i e v e d a g r e a t e r d e g r e e o f s u c c e s s w i t h t h e same s t r a i n o f wheat r u s t on a d e f i n e d medium c o n s i s t i n g o f 1% a g a r , 3% g l u c o s e , C z a p e k ' s m i n e r a l s , B u r k h o l d e r and N i c k e l l ' s t r a c e e l e m e n t s and a m i x -t u r e o f 16 amino a c i d s i n t h e p r o p o r t i o n s f o u n d i n p u r i f i e d c a s e i n h y d r o l y s a t e . Growth on t h i s medium was e q u a l t o t h a t i n t h e p r e s e n c e o f c a s e i n h y d r o l y s a t e and s u p e r i o r t o t h a t on E v a n s ' p e p t o n e - a b r a n d o f p e p t o n e w h i c h W i l l i a m s e t a l . (1967) had f o u n d t o be p a r t i c u l a r l y s u i t a b l e f o r t h e g r o w t h o f t h i s s t r a i n . B o t h u r e d o s p o r e s and t e l i o s p o r e s were f o r m e d on . F o u d i n and Wynn's (1972) medium, but. t h e r e i s o n l y a p a s s i n g r e f e r e n c e t o s p o r u l a t i o n i n t h e i r p a p e r and p a t h o g e n i c i t y t r i a l s w i t h s p o r e s p r o d u c e d i n a x e n i c c u l t u r e were n o t r e -p o r t e d . C o f f e y and Shaw (1972) r e p o r t e d t h a t a m i x t u r e o f e l e v e n 1-amino a c i d s ( f o u n d by a n a l y s i s t o be common t o b o t h E v a n s ' p e p t o n e and D i f c o y e a s t e x t r a c t and e q u i v a l e n t i n 64 amounts t o those s u p p l i e d by 0.1% peptone and 0.1% ye a s t ex-t r a c t ) , p l u s sucrose and m i n e r a l n u t r i e n t s , supported the s u s t a i n e d growth of an e s t a b l i s h e d i s o l a t e of t h e i r s t r a i n 1 of Melampsora l i n i (Ehrenb.) Le/V. U n f o r t u n a t e l y the percen-t a g e of s u c c e s s f u l t r a n s f e r s t o the amino a c i d medium was o n l y 25% compared w i t h 80% i n t r a n s f e r s t o the b a s i c medium p l u s peptone and yeast e x t r a c t . I n a d d i t i o n the " f i n a l s i z e of the c o l o n i e s was o n l y about h a l f t h a t o b t a i n e d w i t h peptone and y e a s t e x t r a c t . F u r t h e r t r i a l s i n d i c a t e d t h a t o n l y 1 - a l a n i n e and 1-methionine were e s s e n t i a l f o r the growth of t r a n s f e r r e d c u l t u r e s , b u t , once a g a i n , the r e s u l t s were i r r e g u l a r and growth was poor. I n subsequent work at W i s c o n s i n , u s i n g C o f f e y and Shaw's s t r a i n ' 1 and race 79 of Melampsora l i n i , C o f f e y and A l l e n (1973) r e p o r t e d t h a t the requirements of primary ( i . e . d e r i v e d d i r e c t l y from uncontaminated uredospores) and estab-l i s h e d ( i . e . t r a n s f e r r e d ) c u l t u r e s were d i f f e r e n t . The de-velopment of m y c e l i a l i n i t i a l s l e a d i n g t o the f o r m a t i o n of primary c u l t u r e s d i d not occur on as wide a range of carbo-hydrates as would support the growth of e s t a b l i s h e d c u l t u r e s and a combination of glutamic a c i d and c y s t i n e was the best s u b s t i t u t e f o r peptone or casamino a c i d s . M e t h i o n i n e d i d not support the growth of primary c u l t u r e s at the l e v e l t e s t e d . E s t a b l i s h e d c u l t u r e s grew i n the presence of any one of a l a -n i n e , g l y c i n e , s e r i n e , a s p a r t i c a c i d , a s p a r a g i n e , glutamic a c i d , glutamine or a r g i n i n e , I n combination w i t h e i t h e r 65 c y s t e i n e o r c y s t i n e . M e t h i o n i n e c o u l d o n l y be s u b s t i t u t e d f o r c y s t e i n e o r c y s t i n e i f e s t a b l i s h e d c o l o n i e s were f i r s t c o n d i t i o n e d on t h e 11-amino a c i d m i x t u r e used by C o f f e y and Shaw. C o f f e y and A l l e n (1973) a l s o r e p o r t e d t h e f i r s t s u c c e s s -f u l a x e n i c g r o w t h o f P u c c i n i a h e l i a n t h i Schw. on s u c r o s e , i n -o r g a n i c s a l t s , b o v i n e serum albumen (BSA) and p e p t o n e o r c a s -amino a c i d s o r t r y p t o n e . C y s t e i n e i n c o m b i n a t i o n w i t h e i t h e r a s p a r t i c o r g l u t a m i c a c i d s o r a l a n i n e c o u l d be s u b s t i t u t e d f o r p e p t o n e , b u t no s u b s t i t u t e f o r BSA was f o u n d . I t must be e m p h a s i z e d t h a t i n a l l t h e e x p e r i m e n t s o f C o f f e y and Shaw (1972) and C o f f e y and A l l e n ( 1 9 7 3 ) , t h e g r o w t h o f M e l a m p s o r a l i n i on d e f i n e d m e d i a s o l i d i f i e d w i t h D i f c o - B a c t o a g a r was n o t p r e d i c t a b l e . The number and f i n a l s i z e o f t h e c o l o n i e s w h i c h d e v e l o p e d f r o m m y c e l i a l i n i t i a l s v/ere much s m a l l e r t h a n i n t h e p r e s e n c e o f a complex o r g a n i c s u b s t a n c e s u c h as p e p t o n e . M o r e o v e r , t h e m o r p h o g e n e t i c p a t t e r n o f de v e l o p m e n t was f a r f r o m n o r m a l w i t h r e s p e c t t o t h e f o r m a -t i o n o f a s t r o m a and t h e d i f f e r e n t i a t i o n o f u r e d o s p o r e s and t e l i o s p o r e s . I n d e e d , s p o r u l a t i o n was r a r e , and t h e o n l y d i -r e c t r e f e r e n c e t o i t i n t h e s e two p a p e r s i s C o f f e y and A l l e n ' s (1973) s t a t e m e n t t h a t t h e s t r o m a o f some c u l t u r e s o f P u c c i n i a h e l i a n t h i c o n t a i n e d t y p i c a l , t h i c k - w a l l e d t e l i o s p o r e s . More r e c e n t l y T u r e l (1973) has a c h i e v e d good s a p r o -b i c g r o w t h o f b o t h p r i m a r y ( i . e . d e r i v e d d i r e c t l y f r o m s t e r i l e u r e d o s p o r e s ) and e s t a b l i s h e d ( i . e . t r a n s f e r r e d ) c u l t u r e s o f r a c e 3 of M e l a m p s o r a l i n i on d e f i n e d media c o n t a i n i n g D i f c o B a c t o a g a r , m o d i f i e d Knop's m i n e r a l s , m i c r o n u t r i e n t s , s u c r o s e 66 and a s p a r t i c a c i d p l u s e i t h e r c y s t e i n e o r g l u t a t h i o n e ( p r i -mary c u l t u r e s ) o r c y s t i n e ( e s t a b l i s h e d c u l t u r e s ) . A l a n i n e and m e t h i o n i n e s u p p o r t e d o n l y s l o w and s p o r a d i c g r o w t h o f p r i m a r y c u l t u r e s . E s t a b l i s h e d c u l t u r e s s t a r t e d i n t h e p r e -s e n c e o f y e a s t e x t r a c t d i d n o t s u r v i v e on t r a n s f e r t o t h e b a s i c medium w i t h a l a n i n e and m e t h i o n i n e , c o n f i r m i n g t h e ob-s e r v a t i o n s o f C o f f e y and A l l e n (1973). T u r e l (1973) r e p o r t e d g r e a t v a r i a t i o n i n t h e de v e l o p m e n t o f p r i m a r y c u l t u r e s on h e r d e f i n e d m e d i a , b o t h w i t h i n and between i s o l a t e s f r o m one s p o r e h a r v e s t t o a n o t h e r . She d i d n o t r e c o r d t h e de v e l o p m e n t o f u r e d o s p o r e s ; c o n s p i c u o u s c l u s t e r s o f t e l i o s p o r e s f o r m e d i n t h e o l d e r p a r t s o f e s t a b l i s h e d c u l t u r e s I n one e x p e r i m e n t , b u t n o t i n a n o t h e r . S e v e r a l y e a r s p r i o r t o W i l l i a m s s (1966, 1967) w o r k , C u t t e r (1959, I960) had d e m o n s t r a t e d b y t h e use o f r u s t -i n f e c t e d c a l l u s c u l t u r e s t h a t r u s t s c o u l d be i s o l a t e d a x e n i c -a l l y , f o l l o w i n g a p e r i o d o f a d a p t a t i o n on h o s t t i s s u e c u l t u r e s m a i n t a i n e d i n G a u t h e r e t ' s medium. He was, I n f a c t , a b l e t o i s o l a t e s e v e r a l s t r a i n s o f Gymnosporangium j u n i p e r i - v i r g i n i a n a e and Uromyces a r i - t r i p h y l l i . A l t h o u g h h i s t e c h n i q u e s u f f e r e d f r o m a l a c k o f r e p r o d u c i b i l i t y i n ter m s o f s u c c e s s f u l i s o l a -t i o n , a few o f t h e i s o l a t e s were m a i n t a i n e d and s u b c u l t u r e d f o r a number o f g e n e r a t i o n s on s o l i d and l i q u i d m e d i a . A c c o r d -i n g t o C u t t e r t h e a p p e a r a n c e o f t h e r u s t i n v i t r o was f a r f r o m n o r m a l and t h e i n c i d e n c e o f s p o r u l a t i o n was v e r y l o w . T h i s s i t u a t i o n l o o g u t t e r ~ - • Vc -c- t h ? t t y p i c a l s p o r u l a t i o n i s 67 a h o s t - i n d u c e d phenomenon ( H o t s o n and C u t t e r 1951). However, r e - i n f e c t i o n o f h o s t c a l l u s t i s s u e as w e l l as t h e i n t a c t h o s t was a c h i e v e d s u c c e s s f u l l y , even a f t e r p r o l o n g e d s u b c u l t u r i n g on a r t i f i c i a l medium. The v a l i d i t y o f C u t t e r ' s a p p r o a c h , u s i n g dual-mem-b e r e d c a l l u s c u l t u r e s v/as c o n f i r m e d r e c e n t l y by H o l l i s e t a l . (1972). S t a r t i n g w i t h r u s t - i n f e c t e d t i s s u e " c u l t u r e s t h e y r e -p o r t e d t h e s u s t a i n e d a x e n i c c u l t u r e o f C r o n a r t i u m f u s i f o r m e Hedge, and Hunt ex Cumm, t h e f u s i f o r m r u s t o f s l a s h p i n e , on a c o m p l e x d e f i n e d medium c o n t a i n i n g 22 amino a c i d s , g l u t a m i n e , " g l u t a t h i o n e , v i t a m i n s , g r o w t h s u b s t a n c e s , c i t r i c a c i d , g l u c o s e , and a g a r . One c u l t u r e p r o d u c e d an a e c i a l s o r u s w i t h t y p i c a l , m a t u r e a e c i o s p o r e s i n a medium c o n t a i n i n g y e a s t e x t r a c t and p e p t o n e . S i m i l a r l y , w o r k i n g i n o u r l a b o r a t o r y , H a r v e y ( u n -p u b l i s h e d ) was a b l e t o i s o l a t e C r o n a r t i u m r i b i c o l a , t h e w h i t e p i n e b l i s t e r r u s t f u n g u s , f r o m t i s s u e c u l t u r e s o f i t s h o s t . V e g e t a t i v e g r o w t h o c c u r r e d a f t e r i s o l a t i o n o f t h e f u n g u s on a b r o a d r a n g e o f s u b s t r a t e s . I t w i l l be e v i d e n t f r o m t h i s b r i e f r e v i e w t h a t a l -t h o u g h much p r o g r e s s has been made i n t h e a x e n i c c u l t u r e o f r u s t f u n g i , n e v e r t h e l e s s a l l t h e r e s u l t s d e s c r i b e d s u f f e r f r o m a s a t i s f a c t o r y d e g r e e o f r e p r o d u c i b i l i t y , p a r t i c u l a r l y i n r e l a -t i o n t o s p o r u l a t i o n . M o r e o v e r t h e e x p e r i m e n t s w i t h ' d e f i n e d ' m e d i a a r e m a r r e d by t h e i n c l u s i o n o f b a c t o a g a r , w h i c h may w e l l s e r v e as a s o u r c e o f u n i d e n t i f i e d m a t e r i a l s . The p r i m a r y ob-j e c t i v e o f t h e work t o be d e s c r i b e d i n t h i s p a p e r was t o de-v e l o p f u l l y d e f i n e d l i q u i d m edia f o r t h e wheat and f l a x 6 8 r u s t s - m e d i a w h i c h c o u l d be s t e r i l i z e d b y m i l l i p o r e f i l t r a -t i o n o n l y and w h i c h w o u l d s u p p o r t g r o w t h and t h e f o r m a t i o n o f u r e d o s p o r e s c a p a b l e o f r e i n f e c t i n g h o s t t i s s u e , MATERIALS AMD METHODS 1. P r o d u c t i o n o f U n c o n t a m i n a t e d U r e d o s p o r e s T r i a l s were c o n d u c t e d w i t h an i s o l a t e o f r a c e ANZ 1 2 6 - 6 , 7 o f P u c c i n i a g r a m i n i s P e r s . f , s p . t r i t i c i ( E r i k s s . & Henn.) o r i g i n a l l y s u p p l i e d by Dr. P.G. W i l l i a m s and w i t h 6 N o r t h A m e r i c a n i s o l a t e s , 15B4, 5 6 , 17 , 3 2 - 1 1 3 , 15B-1L and 38 s u p p l i e d by D r . Gordon Green. I n o c u l u m o f a l l t h e s e i s o l a t e s was r a i s e d on t h e f i r s t s e e d l i n g l e a f o f L i t t l e C l u b wheat grown i n a c o n t r o l l e d e n v i r o n m e n t c a b i n e t s e t a t 500 f t - c . and 20°C f o r 16 h f o l l o w e d by 8 h d a r k n e s s a t l 6°C. A l l hand-l i n g p r o c e d u r e s were c a r r i e d out i n g l o v e boxes t o p r e v e n t c r o s s - c o n t a m i n a t i o n between i s o l a t e s . U n c o n t a m i n a t e d u r e d o -s p o r e s were o b t a i n e d a s d e s c r i b e d b y Bose and Shaw ( 1971) . Race 3 o f Me l a m p s o r a l i n i ( E h r e n b . ) L e v . was p r o -p a g a t e d on B i s o n f l a x . U n c o n t a m i n a t e d u r e d o s p o r e s were r a i s e d on c o t y l e d o n s on Gamborg*s B5" medium w i t h o u t a u x i n (Gamborg e t a l . 1 9 6 8 ) , B e t t e r r e s u l t s were o b t a i n e d when t h e c o t y l e d o n s were d r i e d on a s t e r i l e f i l t e r p a p e r a f t e r s u r f a c e s t e r i l i z a t i o n b e f o r e t r a n s f e r t o Gamborg's medium. U s i n g t h i s t e c h n i q u e a t l e a s t two b a t c h e s o f u r e d o s p o r e s can be ob-t a i n e d f r o m each c o t y l e d o n . 6 9 2. C u l t u r e M e d i a The b a s i c components ( p e r l i t e r ) o f t h e f o u r m e d i a u s e d a r e g i v e n b e l o w . I n a d d i t i o n , e a c h c o n t a i n e d a s o u r c e o f o r g a n i c n i t r o g e n . A l l c h e m i c a l s u s e d were o f r e a g e n t g r a d e . MI. C z a p e k 1 s - g l u c o s e , f o r wheat r u s t s : G l u c o s e , 30 g; NaN0 3, 2.0 g; KC1, 0 . 5 g; M g S 0 4 - 7 H 2 0 , 0 . 5 g; K H 2 P 0 4 , 1.0 g; F e S 0 ^ - 7 H 2 0 , 10 mg. M i l . C z a p e k ' s - g l u c o s e p l u s m i c r o n u t r i e n t s , f o r wheat r u s t s : Components o f MI p l u s 0 .5 o r 0 . 8 m l m i c r o n u t r i e n t s o l u -t i o n c o n t a i n i n g : s e q u e s t r e n e , 13% NaFe ( G e i g y ) , 10 g; M n S 0 4 « 7 H 2 0 , 447 mg; K I , 10 mg; N i C l 2 ' 6 H 2 0 , 18 rng; CoCl 2«6H 20, 18 mg; T i ( S 0 ^ ) 2 ' 9 H 2 0 , 42 mg; ZnSO^, 35 mg; CuSO^«'5H 20, 15 mg; BeSO^, 20 mg; KB^PO^ c o n c e n t r a t e d . 0.2 m l ; g l a s s d i s t i l l e d w a t e r , 200 m l . ( M o d i f i e d B e r t h e l o t ' s s o l u t i o n ; T u r e l 1 9 6 9 ) . M i l l . K n o p ' s - s u c r o s e p l u s m i c r o n u t r i e n t s , f o r f l a x r u s t : s u c r o s e , 40 g; C a ( N 0 3 ) 2 , 2.0 t o 6.0 g; K N 0 3 , 0 .25 g; M g S 0 4 ' 7 H 2 0 , 0 . 25 g; K H 2 P 0 4 , 0 . 25 g; NH^JTC^ , 40 mg; K 2 H P 0 4 , 0 . 75 g; 0.B m l m i c r o n u t r i e n t s o l u t i o n as i n Medium I I . ( W i t h s o l i d m e d i a t h e C a ( N 0 3 ) 2 was added l a t e r , j u s t b e f o r e t h e p l a t e s were p o u r e d ) . MIV. F o r f l a x r u s t : as f o r Medium I I I e x c e p t s u c r o s e , 50 g; and NH,_N0^ , 20 mg. 70 When r e q u i r e d , m e d i a were s o l i d i f i e d w i t h 15 g / l D i f c o B a c t o a g a r , s t e r i l i z e d by a u t o c l a v i n g a t 15 p s i f o r 20 m i n u t e s , and 10 m l a l i q u o t s p o u r e d i n t o p l a s t i c p e t r i d i s h e s ( L a b t e k 60 x ' 2 0 mm). A f t e r i n o c u l a t i o n , p l a t e s were s e a l e d w i t h p a r a f i l m t o m i n i m i z e e v a p o r a t i o n . 3 . S o u r c e s o f O r g a n i c N i t r o g e n and S u l p h u r E v a n s ' p e p t o n e (0.1% w/v, Evans M e d i c a l L t d . , U . K . ) , D i f c o y e a s t e x t r a c t (YE 0.1% w / v ) , and b o v i n e serum albumen (BSA, 1% w/v; F r a c t i o n V; d e f a t t e d a c c o r d i n g t o Chen 1967) s e r v e d as s o u r c e s o f o r g a n i c n i t r o g e n i n u n d e f i n e d m e d i a . Y e a s t e x t r a c t and p e p t o n e were a l w a y s added b e f o r e f i n a l ad-j u s t m e n t o f t h e pH and b e f o r e a u t o c l a v i n g . BSA was s t e r i l i z e d b y m i l l i p o r e ' f i l t r a t i o n ( p o r e s i z e 0,22 jj,) and added a f t e r a u t o c l a v i n g and c o o l i n g t h e medium. I n d e f i n e d m e d i a e i t h e r t h e 11 1-amino a c i d m i x t u r e * o f C o f f e y and Shaw (1972) o r c o m b i n a t i o n s o f a s p a r t i c a c i d , c y s t e i n e and g l u t a t h i o n e were s u b s t i t u t e d f o r Y E , p e p t o n e and BSA. F u l l y d e f i n e d m e d i a c o n t a i n e d no a g a r and were n o t a u t o -c l a v e d . A l l amino a c i d s and g l u t a t h i o n e were o b t a i n e d f r o m Sigma C h e m i c a l Company. ''Composition o f amino a c i d m i x t u r e ( C o f f e y and Shaw 1972). 1 - g l u t a m i c a c i d , 770 m g / l ; 1 - l e u c i n e , 160 m g / l ; 1 - p h e n y l a l -a n i n e , 1 5 0 m g/l; 1 - a l a n i n e , 120 m g / l ; 1 - i s o l e u c i n e , SO m g / l ; 1 - a s p a r t i c a c i d , 80 m g / l ; 1 - v a l i n e , 60 m g / l ; 1 - m e t h i o n i n e , 60 m g / l ; t h r e o n i n e , 50 m g / l ; 1 - s e r i n e , 50 m g / l ; 1 - g l y c i n e , 40 m g / l . 71 L i q u i d m e d i a were s t e r i l i z e d e i t h e r b y a u t o c l a v i n g o r , when com p l e x o r g a n i c n i t r o g e n s o u r c e s (YE and p e p t o n e ) were r e p l a c e d w i t h amino a c i d s m i x t u r e s , b y f i l t r a t i o n t h r o u g h N a l g e n e M i l l i p o r e f i l t e r s ( p o r e s i z e 0,22 u.) . When u s e d , 1 M P , CAMP and t h e o p h y l l i n e were added t o t h e c o o l medium a f t e r m i l l i p o r e f i l t r a t i o n . U n l e s s o t h e r w i s e s t a t e d , 1 0 m l a l i q u o t s w ere d i s p e n s e d i n t o $0 m l p y r e x c u l t u r e f l a s k s ( 4 . 5 cm i n t e r n a l b a s e d i a m e t e r ) w h i c h were c l o s e d w i t h b a c t i c a p a l l p o l y p r o p y -l e n e c a p s . The pH o f a l l m e d i a v/as a d j u s t e d t o 6 t o 6 . 4 f o r wheat stem r u s t and 5 . 5 f o r f l a x r u s t , e x c e p t i n t r i a l s w i t h h i g h l e v e l s o f c a l c i u m , i n w h i c h t h e pH was k e p t s t r i c t l y a t 5.0 t o a v o i d p r e c i p i t a t i o n . 4. I n o c u l a b i o n o f M e d i a A l l i n o c u l a t i o n s v/ere c o n d u c t e d on an a i r f l o w b e n c h u n d e r a s e p t i c c o n d i t i o n s a t 19 + 2°C. N o r m a l l y u r e d o s p o r e s were d u s t e d on t o t h e s u r f a c e o f t h e medium a t a d e n s i t y c o r r e s p o n d i n g t o 1000 t o 2000 p e r mm . I n t h o s e e x p e r i m e n t s i n w h i c h f l a x r u s t u r e d o s p o r e s v/ere s u s p e n d e d i n g e l a t i n f o r i n o c u l a t i o n o f s o l i d medium t h e s p o r e d e n s i t y v/as 4 0 0 t o 9 0 0 p e r mm^. Ten r e p l i c a t e p l a t e s were .used w i t h s o l i d m e d i a and 5 r e p l i c a t e f l a s k s w i t h l i q u i d m e d i a . T r i a l s w i t h l i q u i d m e d i a were r e p e a t e d t w i c e . A l l p l a t e s and f l a s k s were i n -c u b a t e d a t 17°C i n d a r k n e s s . 72 5. Assessment of Growth Growth of both Melampsora l i n i and P u c c i n i a g r a m i n i s was r a t e d a c c o r d i n g t o one of the f o l l o w i n g f i v e t y p e s . I no growth beyond germ tubes. I I sparse m y c e l i a l development, stroma not developed. I l l s m a l l w h i t e t u f t e d c o l o n i e s ; y e l l o w i s h stroma on under s u r f a c e . IV s m a l l w h i t e 'button' c o l o n i e s ; orange stroma f o r f l a x r u s t ; brownish stroma f o r wheat r u s t . V • l a r g e w h i t e 'button' c o l o n i e s w i t h prominent stroma; under the proper c o n d i t i o n s these c o l o n i e s developed s p o r u l a t i n g p u s t u l e s on t h e i r upper s u r f a c e s . M i c r o s c o p i c o b s e r v a t i o n s were made u s i n g p h l o x i n e / KOH s t a i n and photographs were takren w i t h a C a r l Z e i s s photo-microscope u s i n g b r i g h t f i e l d i l l u m i n a t i o n and a 35 nun camera. RESULTS Wheat Rust 1. T r i a l s w i t h S o l i d Media P e t r i p l a t e s c o n t a i n i n g s o l i d medium MI w i t h 0 . 1 % YE, 0 , 1 % Evans' peptone and 1% w/v BSA were i n o c u l a t e d w i t h dry uredospores of each of races 1 5 B 4 , 5 5 , 1 7 , 3 2 - 1 1 3 , 1 5 B 1 L , 3 3 and ANZ 1 2 6 - 6 , 7 of P u c c i n i a graminis t r i t i c i . ANZ 1 2 6 - 6 , 7 was i n c l u d e d t o p r o v i d e a standard by which the growth of the North American r a c e s c o u l d be judged. Races 1 5 B - 1 L , 1 7 , and 5 6 t u r n e d p a l e brown and showed d i s t i n c t s i g n s of senescence a f t e r 1 week. Races 1 5 B 4 , 3 8 , 3 2 - 1 1 3 and ANZ 1 2 6 - 6 , 7 7 3 d e v e l o p e d c o l o n i e s composed o f s e p t a t e a e r i a l hyphae and a l e a t h e r y s t r o m a . A t t h i s s t a g e t h e c o l o n i e s o f r a c e 3 8 and ANZ 1 2 6 - 6 , 7 were o f t y p e 4. Growth o f r a c e 3 2 - 1 1 3 was l e s s a d v a n c e d a n d - t h e s t r o m a was much t h i n n e r . Race 15B4 r e m a i n e d v e g e t a t i v e f o r a b o u t 2 months and m i c r o s c o p i c e x a m i n a t i o n i n -d i c a t e d t h a t t h e hyphae were composed o f s m a l l c i r c u l a r c e l l s . T hese t r i a l s showed t h a t g r o w t h a p p r o a c h i n g t h a t c h a r a c t e r i s t i c o f r a c e ANZ 1 2 6 - 6 , 7 v/as o b t a i n e d w i t h r a c e 3 8 and t h e r e v/ere d i f f e r e n c e s i n t h e d e g r e e o f de v e l o p m e n t o f t h e d i f f e r e n t r a c e s on t h e same medium. 2. T r i a l s w i t h L i q u i d M e d i a A. Growth o f r a c e s 15B4, 38\ 3 2 - 1 1 3 and ANZ 1 2 6 - 6 , 7 On .the b a s i s o f t h e p r e l i m i n a r y t r i a l s w i t h s o l i d medium, l i q u i d M i l medium w i t h p e p t o n e and BSA p l u s 0 . 5 g / l Ca(NO ) was i n o c u l a t e d w i t h e a c h o f r a c e s 15B4, 3 8 , 32-113 and ANZ 1 2 6 - 6 , 7 . Race 1$B4 d e v e l o p e d f l u f f y w h i t e c o l o n i e s , b u t g r o w t h was v e r y s l o w and no s t r o m a d e v e l o p e d ( F i g . I I I - l ) . R ace 32-113 d e v e l o p e d p r o f u s e a e r i a l hyphae b u t d i d n o t f o r m a s t r o m a ( F i g . I I I - l ) . A f t e r 4 weeks r a c e 38 d e v e l o p e d a brown s t r o m a submerged i n t h e medium ( F i g . I I I - l ) and g r o w t h a p p e a r e d t o be e q u i v a l e n t t o t h a t o f r a c e ANZ 1 2 6 - 6 , 7 w i t h r e s p e c t t o b o t h t h e f i n a l s i z e and t h e g e n e r a l m o r p h o l o g y o f t h e c o l o n i e s w h i c h were o f t y p e 4. B. E f f e c t o f cAMP on g r o w t h o f ANZ 1 2 6 - 6 , 7 S i n c e c y c l i c AMP i s i m p l i c a t e d I n hormone a c t i o n and t h e c o n t r o l o f enzyme s y n t h e s i s and m o r p h o g e n e s i s i n m i c r o -74 o r g a n i s m s ( R o b i s o n _et a l . 1971) i t w a s c o n s i d e r e d w o r t h w h i l e t o t e s t i t s e f f e c t o n t h e g r o w t h o f t h e w h e a t r u s t f u n g u s . T h e r e s u l t s a r e p r e s e n t e d i n T a b l e I I I - I . L o w c o n c e n t r a t i o n s ( 0 . 1 mM a n d 1 mM) h a d n o a p p a r e n t e f f e c t , b u t 10 mM c A M P c o m -p l e t e l y i n h i b i t e d g r o w t h o f A N Z 1 2 6 - 6 , 7 . C o m p l e t e i n h i b i t i o n o f g r o w t h w a s a l s o o b s e r v e d ' w i t h 1 mM c A M P i n t h e p r e s e n c e o f 1 mM t h e o p h y l l i n e ( F i g . I I I - 2 ) . T h e l a t t e r i n h i b i t s p h o s p h o -d i e s t e r a s e a n d t h u s i n c r e a s e s t h e e f f e c t i v e l e v e l o f c A M P . 5'AMP a l o n e o r i n c o m b i n a t i o n w i t h t h e o p h y l l i n e h a d n o e f f e c t o n g r o w t h . C . E f f e c t o f a s p a r t i c a c i d , c y s t e i n e , g l u t a t h i o n e a n d c a l -c i u m o n t h e g r o w t h o f A N Z 1 2 6 - 6 , 7 I n ' o u r a t t e m p t s t o d e v e l o p a c o m p l e t e l y d e f i n e d m e d i u m f o r t h e g r o w t h o f P u c c i n i a g r a m i n i s u s i n g o n l y a c a r b o -h y d r a t e s o u r c e , a m i n o a c i d s a n d m i n e r a l n u t r i e n t s we f o u n d t h a t i t w a s e s s e n t i a l t o p r o v i d e a s o u r c e o f c a l c i u m . T h i s e l e m e n t i s n o t a c o n s t i t u e n t o f t h e s t a n d a r d C z a p e k ' s m i n e r a l n u t r i e n t s o l u t i o n u s e d i n e a r l i e r a t t e m p t s t o c u l t u r e t h e w h e a t r u s t f u n g u s . U s i n g l i q u i d m e d i u m M i l c o n t a i n i n g v a r i o u s c o m b i n a -t i o n s o f a s p a r t i c a c i d , c y s t e i n e a n d g l u t a t h i o n e p l u s c a l c i u m n i t r a t e , b e s t r e s u l t s w e r e o b t a i n e d w h e n b o t h a m i n o a c i d s a n d t h e p e p t i d e w e r e c o m b i n e d w i t h f r o m 2 g / l ( 8 . 1 mM) t o 6 g / l ( 2 5 . 4 mM) C a ( N 0 3 ) 2 , a s s h o w n b y t h e d a t a i n T a b l e I I I - I I . A t c o n c e n t r a t i o n s o f C a ( N 0 3 ) 2 l e s s t h a n 2 g / l ( 8 . 1 mM) t h e l e n g t h o f t h e l a g p h a s e w a s i n c r e a s e d a n d t h e r e p r o d u c i b i l i t y o f t h e r e s u l t s w a s m a r k e d l y d e c r e a s e d . I n o u r e x p e r i e n c e t h e g r o w t h 75 F i g u r e I I I - l . N o r t h A m e r i c a n i s o l a t e s of wheat stem r u s t showing v e g e t a t i v e growth on l i q u i d medium M i l . Photographed a f t e r 6 weeks. (A) Race 3 2 - 1 1 3 , (B) 1 5 B - 4 , (C) 3 8 . X 1 . 0 . F i g u r e I I I - 2 . Growth and development of wheat stem r u s t r a c e ANZ 1 2 6 - 6 , 7 on l i q u i d medium M I . (A) 1 mM cAMP, (B) 1 mM t h e o p h y l l i n e , ( C ) 1 mM cAMP p l u s 1 mM t h e o p h y l l i n e . N o t e : Ihe growth i n ( C ) i s t h e r e s u l t of u r e d o s p o r e g e r m i n a t i o n o n l y . X 1 . 2 . F i g u r e I I I - 3 . Wheat stem r u s t ANZ 1 2 6 - 6 , 7 d e v e l o p e d on a c h e m i c a l l y d e f i n e d medium M i l h a v i n g a s p a r t i c a c i d , c y s t e i n e and g l u t a t h i o n e . D a r k e r r e g i o n a t t h e t o p of t h e c o l o n i e s i n d i c a t e t h e p o s i t i o n o f d e v e l o p i n g u r e d o p u s t u l e . X 2 . 4 . 76 77 TABLE I I I - I . E f f e c t s o f c y c l i c M P and t h e o p h y l l i n e on g r o w t h o f P u c c i n i a g r a m i n i s ( r a c e ANZ 126-6,7). Type o f C o l o n y on Medium MI w i t h 0.1% p e p t o n e + 0.1% p e p t o n e 1% BSA C o n t r o l I V V cAMP 0.1 mM 1 I I I V cAMP 1.0 mM I I I V cAMP 10 mM I I I I T h e o p h y l l i n e 1 mM I I I I V T h e o p h y l l i n e 1 mM and cAMP 1 mM I I I I T h e o p h y l l i n e 5 tAMP 1 mM 1 mM and I I I V 5 T AMP I V I V I d e n t i c a l r e s u l t s were o b t a i n e d w i t h d i b u t y r y l cAMP. s TABLE I I I - I I . E f f e c t o f amino a c i d s , g l u t a t h i o n e and C a ( N 0 3 ) 2 on t h e g r o w t h o f P u c c i n i a g r a m i n i s t r i t i c i ( r a c e ANZ 126-6,7) . O r g a n i c N i t r o g e n S o u r c e L e v e l o f C a ( N 0 o ) 2 S/ Weeks t o Development Type -of o f C o l o n i e s S t a l i n g Growth Remarks 1 . A s p a r t i c + g l u t a t h i o n e 2. A s p a r t i c + c y s t e i n e 6 6 2-3 4 4 I I I I V No s p o r u -l a t i o n No s p o r u -l a t i o n 3 . A s p a r t i c + c y s t e i n e + g l u t a t h i o n e 6 6-7 V U r e d o s p o r e s f o r m e d a f t e r 8 weeks 4. A s p a r t i c + c y s t e i n e + g l u t a t h i o n e 6-7 V U r e d o s p o r e s f o r m e d a f t e r 8 weeks A l l t r e a t m e n t s c o n t a i n e d t h e b a s i c medium M i l ( s e e t e x t ) . A s p a r t i c a c i d 4.5 mM ( 600 mg/ 100 m l ) ; c y s t e i n e 4 . 6 mM (56 mg /100 m l ) ; g l u t a t h i o n e 3 .3 mM ( 1 0 1 . 4 rng/lOO m l ) . 03-' • 79 o f ANZ 126-6,7 was b e t t e r and more e a s i l y r e p r o d u c i b l e on t h i s new d e f i n e d medium ( T r e a t m e n t s 3 & 4 , T a b l e I I I - I I ) t h a n on m e d i a c o n t a i n i n g p e p t o n e , BSA and YE ( F i g , I I I - 3 ) . I n p a r -t i c u l a r t h e c u l t u r e s I n a b o u t 50% o f t h e f l a s k s p r o d u c e d u r e d o -s p o r e s a f t e r 8 weeks. There was no r e l e a s e o f brown p i g m e n t i n t o t h e medium as i n o u r e a r l i e r e x p e r i m e n t s (Bose and Shaw 1971). T e l i o s p o r e f o r m a t i o n was i n f r e q u e n t . The m o r p h o l o g y o f t h e u r e d o s p o r e s v/as v e r y s i m i l a r t o t h a t o f t h o s e d e s c r i b e d e a r l i e r (Bose and Shaw 1971). These s p o r e s were c a p a b l e o f r e i n f e c t i n g wheat s e e d l i n g s when p l a c e d on expos e d m e s o p h y l l t i s s u e o f t h e f i r s t s e e d l i n g l e a f . F l a x R u s t 1. T r i a l s w i t h S o l i d M e d i a A. E f f e c t o f BSA Medium M i l l w i t h 2 g / l C a ( N 0 3 ) 2 p l u s 0.1% Evans' pep-t o n e , 0.1% YE and 1% d e f a t t e d BSA was u s e d r o u t i n e l y f o r t h e c u l t u r e o f f l a x r u s t . U r e d o s p o r e s were s e e d e d i n clumps (1000 t o 2000 p e r mm ) e i t h e r d i r e c t l y o n t o t h e s u r f a c e o f t h e medium o r o n t o p r e - s t e r i l i z e d M i l l i p o r e f i l t e r s l y i n g on t h e a g a r s u r f a c e ( Q u i c k and C r o s s 1971). Type 5 c o l o n i e s w i t h a l u x u r i o u s f l u f f y m y c e l i a l mat and an u n d e r l y i n g o r a n g e s t r o m a d e v e l o p e d , u s u a l l y i n abou t 5 weeks ( F i g . I I I - 4 ) . I n t h e p r e -s e n c e o f BSA, t h e a e r i a l p a r t s o f t h e s e c o l o n i e s o f t e n de-v e l o p e d i n t o u r e d o s o r i . S u b c u l t u r i n g was t h e r e f o r e done a t t h e v e g e t a t i v e s t a g e by d i v i s i o n and t r a n s f e r t o t h e same medium minus BSA. I n o u r e x p e r i e n c e c u l t u r e s o f t h i s t y p e can be s u b c u l t u r e d i n d e p e n d e n t l y . C o n t i n u e d i n c u b a t i o n o f 80 the p l a t e s r e s u l t e d i n the development of secondary c o l o n i e s from the r e s i d u a l germ tubes at the s i t e s of the o r i g i n a l p r i -mary c o l o n i e s which had been removed f o r t r a n s f e r . At the same time c o l o n i e s a l s o developed i n r e g i o n s of the p l a t e s which had o r i g i n a l l y been seeded at lower d e n s i t i e s of 4 0 0 -o 500 uredospores per mm . These o b s e r v a t i o n s suggested t h a t the M i l l medium p l u s BSA might be i n t e r a c t i n g i n some manner w i t h the i n h i b i -t o r s r e l e a s e d from uredospores which prevented g e r m i n a t i o n i n our e a r l i e r experiments ( C o f f e y et a l . 1 9 7 0 ) . I n order t o t e s t t h i s p o s s i b i l i t y uredospores were t h e r e f o r e suspended i n 15% w/v g e l a t i n and seeded onto s o l i d medium M i l l w i t h and w i t h o u t BSA. A f t e r e i g h t weeks l a r g e c o l o n i e s had developed over the i n o c u l a t e d zones on the p l a t e s w i t h BSA ( F i g . I I I - ? ) . These c o l o n i e s were about 50% l a r g e r i n diameter than the c o l o n i e s which developed on p l a t e s seeded d i r e c t l y w i t h dry uredospores. They remained v e g e t a t i v e and c o l l a p s e d a f t e r 12 weeks. N e g l i -g i b l e growth o c c u r r e d on the c o n t r o l p l a t e s l a c k i n g BSA. This r e s u l t c o n f i r m s the c o n c l u s i o n t h a t BSA overcomes the i n i t i a l i n h i b i t i o n of growth. B. R e i n f e c t i o n of host u s i n g v e g e t a t i v e c o l o n i e s grown on s o l i d medium Surface s t e r i l i z e d c o tyledons of B i s o n f l a x were p l a c e d w i t h t h e i r cut ends i n co n t a c t w i t h month o l d c o l o n i e s of f l a x r u s t on s o l i d M i l l medium p l u s peptone, BSA and YE. A f t e r 4 weeks r u s t mycelium developed e c t o p a r a s i t i c a l l y on 8 l t h e u p p e r s u r f a c e o f t h e c o t y l e d o n s , w h i c h became c o m p l e t e l y c o v e r e d by t h e f u n g u s . The e c t o p a r a s i t l c m y c e l i u m t h e n p r o -d u c e d - masses o f o r a n g e c o l o u r e d u r e d o s p o r e s ( F i g . I I I - 6 ) . N o r -m a l e r u m p e n t _ r u s t p u s t u l e s d e v e l o p e d on t h e l o w e r s u r f a c e s o f t h e c o t y l e d o n s . U r e d o s p o r e s f r o m t h e s e p u s t u l e s i n f e c t e d i n t a c t f l a x p l a n t s i n a n o r m a l manner. 2. T r i a l s w i t h L i q u i d M e d i a A. P r e l i m i n a r y t r i a l s and e f f e c t o f BSA W i t h l i q u i d m e d i a i t was f o u n d t h a t , a f t e r t h e de-v e l o p m e n t o f a s t r o m a , i s o l a t e d f l a x r u s t c o l o n i e s have a m a r k e d t e n d e n c y t o s i n k as a r e s u l t o f s l i g h t d i s t u r b a n c e s d u r i n g h a n d l i n g o f t h e c u l t u r e f l a s k s ( F i g . I I I - 7 ) . T h i s p r o -b l e m was n e v e r e n c o u n t e r e d w i t h wheat r u s t c o l o n i e s . I t was o v e rcome by s e e d i n g f l a x r u s t u r e d o s p o r e s as e v e n l y as p o s s i -b l e o v e r an a r e a a t l e a s t 3 cm i n d i a m e t e r . A n a s t o m o s i s and i n t e r l o c k i n g o f t h e germ t u b e s f o r m e d a mat w h i c h , p r e s u m a b l y due t o s u r f a c e t e n s i o n e f f e c t s , h e l d t h e d e v e l o p i n g c o l o n i e s a b o v e t h e l i q u i d s u r f a c e . The r e s u l t s i n T a b l e I I I - I I I show t h a t M i l l medium c o n t a i n i n g e i t h e r 0 .1% E v a n s ' p e p t o n e p l u s YE o r C o f f e y and Shaw's (1972) 11-amino a c i d m i x t u r e , b u t l a c k i n g BSA, s u p p o r t e d o n l y p o o r g r o w t h w i t h r e s p e c t t o b o t h t h e p e r c e n t a g e number o f f l a s k s i n w h i c h g r o w t h o c c u r r e d and t o t h e t y p e o f c o l o n y f o r m e d (Type 3 ) . A d d i t i o n o f 1% BSA m a r k e d l y i n c r e a s e d t h e p e r c e n t a g e o f f l a s k s s h o w i n g g r o w t h and r e s u l t e d i n t h e f o r m a t i o n o f t y p e 5 c o l o n i e s ( F i g . 8 ) . N o r m a l l y when 82 F i g u r e I I I - 4 . F l a x r u s t c o l o n y i n i t i a l s r a i s e d on medium M i l l h a v i n g YS, p e p t o n e and BSA. P h o t o g r a p h e d a f t e r 6 weeks. F i g u r e I I I - 5 . C o l o n i e s o f f l a x r u s t o r i g i n a t i n g as a r e s u l t o f g e l a t i n s p o r e s u s p e n s i o n s e e d -i n g on medium M i l l . (A) medium c o n t a i n -i n g p e p t o n e , n o t e v e r y l i t t l e v i s i b l e g r o w t h . (B) p e p t o n e p l u s BSA, p h o t o g r a p h e d a f t e r 8 weeks, F i g u r e I I I - 6 . E c t o p a r a s i t i c m y c e l i u m o f f l a x r u s t de-v e l o p e d on e x c i s e d c o t y l e d o n s . A r r o w s i n d i c a t e t h e r e g i o n o f u r e d o s p o r e s . P h o t o g r a p h e d a f t e r 6 weeks. X 1.2. x TABLE I I I - I I I . E f f e c t s o f d i f f e r e n t o r g a n i c n i t r o g e n s o u r c e s on t h e g r o w t h o f M e lampsora l i n i ( r a c e 3 ) . N i t r o g e n S o u r c e Weeks t o Develop-ment o f C o l o n i e s % o f F l a s k s show- Type o f i n g Growth C o l o n y 0.1% Evan's peptone 0.1% Evan's peptone + 1% BSA Amino a c i d s 1 Amino a c i d s 1 + 1% BSA Amino a c i d s 1 + 1 mg/l p u t r e s c i n e Amino a c i d s 1 + 10 mg/l p u t r e s c i n e 7 5- 6 8 6- 7 6-7 20 50 35 60 40 50 I I I V I I t o I I I V I I I I I I A l l t r i a l s w i t h l i q u i d medium M i l l c o n t a i n i n g 2 g / l Ca(N0^)2 1 Amino a c i d m i x t u r e o f C o f f e y and Shaw (1972^ 85 c o l o n i e s became submerged i n t h e medium, d e v e l o p m e n t s t o p p e d , b u t when, as i n a f e w i s o l a t e d c a s e s i n t h e p r e s e n c e o f BSA, submerged c o l o n i e s d i d c o n t i n u e t o grow, d e v e l o p m e n t was ab-n o r m a l and was c h a r a c t e r i z e d by f r a g m e n t a t i o n and t h e f o r m a -' t i o n o f s e v e r a l s u b c o l o n i e s w h i c h r e m a i n e d c o m p l e t e l y d e v o i d o f p i g m e n t a t i o n ( F i g . I I I - 9 ) . S i n c e i t i s p o s s i b l e t h a t one o f t h e e f f e c t s o f BSA may be due t o i t s p o l y c a t i o n i c p r o p e r t i e s , and s i n c e t h e p o l y -a m i nes 'and t h e c l o s e l y r e l a t e d d i a m i n e , p u t r e s c i n e , a r e now c o n s i d e r e d t o have a r e l a t i o n s h i p t o n u c l e i c a c i d m e t a b o l i s m ( S m i t h 1 9 7 2 ) an a t t e m p t was made t o r e p l a c e BSA by p u t r e s c i n e . The r e s u l t s o f t h e s e t r i a l s a r e g i v e n i n T a b l e I I I - I I I and i n -d i c a t e t h a t w h i l e p u t r e s c i n e does n o t i n h i b i t g r o w t h , i t does n o t s u b s t i t u t e f o r BSA. B. E f f e c t s o f m i n e r a l n u t r i e n t s F l a x r u s t showed o n l y v e r y p o o r g r o w t h on m e d i a c o n -t a i n i n g C z a p e k ' s m i n e r a l s a l t s and m i c r o n u t r i e n t s ( i . e . medium M i l , f o r wheat r u s t ) t o g e t h e r w i t h a s p a r t i c a c i d and c y s t e i n e i . e . when C z a p e k ' s were s u b s t i t u t e d f o r Knop's m a c r o n u t r i e n t s a l t s . One e f f e c t o f s u b s t i t u t i n g C z a p e k's f o r Knop's m i n e r a l s a l t s i s t h e e l i m i n a t i o n o f c a l c i u m . I n o t h e r e x p e r i m e n t s t h e g r o w t h o f f l a x r u s t on Knop's m i n e r a l s p l u s m i c r o n u t r i e n t s ( I . e . medium MIV) t o g e t h e r w i t h a s p a r t i c a c i d and c y s t e i n e was c o n -s p i c u o u s l y r e d u c e d when t h e c a l c i u m l e v e l was l e s s t h a n 1.5 gm/l C a ( N O ^ ) 9 ( T a b l e I I I - I V . These e f f e c t s o f v a r y i n g t h e l e v e l s o f c a l c i u m a r e d e s c r i b e d i n g r e a t e r d e t a i l i n S e c t i o n C. 86 F i g u r e I I I - 7 . F l a x r u s t c o l o n y d e v e l o p e d on l i q u i d medium M i l l c o n t a i n i n g p e p t o n e and BSA. (A) i n i t i a l f l o a t i n g c o l o n i e s , (B) i s o l a t e d s i n g l e c o l o n y i s p a r t i a l l y s u b m e r g e d . X 1.1. F i g u r e I I I - 8 . F l a x r u s t c o l o n i e s on l i q u i d medium M i l l (A) p e p t o n e p l u s BSA, (B) amino a c i d m i x t u r e p l u s BSA, p h o t o g r a p h e d a f t e r 7 weeks g r o w t h , X 1.6. F i g u r e I I I - 9 . F l a x r u s t d e v e l o p m e n t u n d e r submerged c o n d i t i o n . N o t e t h e f r a g m e n t e d c l u s t e r e d h y p h a e . X 1.2. F i g u r e 1 1 1 - 1 0 . G r o w t h and d e v e l o p m e n t o f f l a x r u s t on c h e m i c a l l y d e f i n e d medium MIV. (A) a s p a r t i c a c i d , c y s t e i n e p l u s g l u t a -t h i o n e , (B) a s p a r t i c a c i d p l u s c y s t e i n e , (C) a s p a r t i c a c i d p l u s g l u t a t h i o n e . X 1 . 1. 87 88 Growth was a l s o s t r o n g l y i n f l u e n c e d by t h e m i c r o n u t r i e n t s o l u -t i o n u s e d . Thus, no g r o w t h o c c u r r e d when a l l t h e components o f t h e m o d i f i e d B e r t h e l o t ' s m i c r o n u t r i e n t s o l u t i o n was o m i t t e d f r o m m e d i a M i l l and MIV. On t h e o t h e r h a n d , t h e s i m u l t a n e o u s e l i m i n a t i o n o f Ti(SO,)„, BeSO,, K I and H o P 0 , f r o m t h e m i c r o -4 2 4 J 4 n u t r i e n t s o l u t i o n had no e f f e c t on g r o w t h and d e v e l o p m e n t . These components were u s e d r o u t i n e l y b e c a u s e i t was o b s e r v e d t h a t when t h e y were i n c l u d e d t h e s t o c k s o l u t i o n r e m a i n e d s t e r i l e f o r l o n g p e r i o d s a t room t e m p e r a t u r e . When B e r t h e l o t ' s m i c r o -n u t r i e n t s were r e p l a c e d w i t h T u r e l ' s (1973) STE s o l u t i o n ( F e ( N 0 3 ) 2 - 9 H 2 0 , 139 mg; EDTA - 2 N a , 134 mg; MnSO^, 895 mg; a l l I n 8 0 ml d i s t i l l e d H 2 0 ) g r o w t h was h i g h l y u n p r e d i c t a b l e , w i t h o n l y 10% o f t h e c u l t u r e f l a s k s d e v e l o p i n g v e g e t a t i v e c o l o n i e s . C. E f f e c t s o f a s p a r t i c a c i d , c y s t e i n e , g l u t a t h i o n e and c a l c i u m The e f f e c t s o f a s p a r t i c a c i d , c y s t e i n e , g l u t a t h i o n e and c a l c i u m n i t r a t e on t h e gro w t h o f f l a x r u s t i n medium MIV a r e summarized i n T a b l e I I I - I V . At c o n c e n t r a t i o n s s i m i l a r t o t h o s e u s e d by T u r e l * ( 1 9 7 3 ) a s p a r t i c a c i d ( 2 2 . 5 mM), c y s t e i n e ( 2 . 3 mM), g l u t a t h i o n e ( 1 . 6 5 mM) and C a ( N 0 ^ ) 2 ( 2 . 0 g / l ) r e -s u l t e d i n t h e d e v e l o p m e n t o f v e g e t a t i v e t y p e 3 c o l o n i e s a f t e r 6 weeks i n 20 t o 50% o f t h e f l a s k s ( T r e a t m e n t s 1 t o 3, T a b l e I I I - I V ) . T h i s i s an u n s a t i s f a c t o r y l e v e l o f p r e d i c t a b i l i t y . * T u r e l u s e d 28 mg/100 ml c y s t e i n e - H C l , i . e . I . 6 5 mM c y s t e i n e . TABLE I I I - I V . Growth and development of Melampsora l i n i (race 3) on completely defined l i q u i d media. of Colony Developed Flasks T r i a l Aspartic Acid Cys-teine Gluta-thione Calcium Nitrate Within 4 After b Weeks Weeks showing Growth (g/D 1 2 3 4 5 6 7 8 + + + + + ++ ++ ++ + + + + ++ ++ ++ + + + 2 2 2 4 4 0 0.5 1 I I I I I I I I I I I I I I I I I I IV IV 50 30 20 50 40 20 50 80 9 ++ ++ 1.5 V 100 10 ++(G) •++ 2 V 100 11 ++ + • 2 V 100 12 13 + ++ ++ ++ 2 6 IV V 60 90 14 ++(G) ++ ++ 6 V 100 15 ++ ++ 6 V 90 Remarks No sporulation No sporulation No sporulation No sporulation No sporulation No sporulation No sporulation Uredospores i n f r e -quent. No t e l i o s p o r e s . Uredosporess& t e l i o -spores formed with high frequency. Uredospores & t e l i o -spores formed with high frequency. Uredospores & t e l i o -spores formed with high frequency. 1 Uredospores infrequent Uredospores & t e l i o -spores formed with high frequency. Uredospores & t e l i o -spores formed with high frequency. Uredospores & t e l i o -spores infrequent. A l l t r i a l s on l i q u i d medium MIV with Ca(N0Q) 2 l e v e l s as noted + denotes aspartic a c i d , 22.5 mM (300 mg/lOO ml); cysteine 2.3 mM (28 mg/100 ml); g l u t a -thione 1 65 mM (50.7 mg/100 ml); ++ denotes twice the foregoing concentrations, (a) denotes that i n otherwise s i m i l a r t r i a l s 48.5 mM (68.5 mM (684 mg/l00 ml) glutamic acid was substituted f o r aspartic acid with i d e n t i c a l r e s u l t s . 90 D o u b l i n g t h e l e v e l o f Ca(N0 )9 d i d n o t i m p r o v e g r o w t h o r l e a d 3 <~ t o t h e d e v e l o p m e n t o f s p o r e s ( T r e a t m e n t s 4 & 5 ) . I n t r i a l s 6 t o 10 t h e c o n c e n t r a t i o n s o f a s p a r t i c a c i d and c y s t e i n e were d o u b l e d t o 45 and 4 . 6 mM r e s p e c t i v e l y and t h e l e v e l o f G a ( N 0 ^ )2 was v a r i e d f r o m 0 t o 2 g / l . I n t h e a b s e n c e o f Ca(N0^)2 ( t r i a l 6) g r o w t h was t h e same a s i n t r e a t -ment 2 . As t h e l e v e l o f c a l c i u m n i t r a t e was i n c r e a s e d t o 2 g / l v e g e t a t i v e g r o w t h was i m p r o v e d and a c c e l e r a t e d and t h e p e r c e n t a g e number o f f l a s k s s h e w i n g g r o w t h i n c r e a s e d . Thus w i t h 1 g / l o f Ca(N0^)2> bype 4 c o l o n i e s d e v e l o p e d i n f o u r weeks i n 80% o f t h e f l a s k s b u t u r e d o s p o r e f o r m a t i o n o c c u r r e d o n l y i n f r e q u e n t l y ( T r i a l 8 ) . I n t r i a l s 9 and 10 , a t C a U O ^ ^ l e v e l s o f 1.5 and 2 . 0 g / l , t y p e 5 c o l o n i e s d e v e l o p e d i n f o u r weeks i n 100% o f t h e f l a s k s and u r e d o s p o r e s and t e l i o s p o r e s were f o r m e d by 60 t o 70% o f t h e c o l o n i e s i n each f l a s k . T r i a l s 11 and 12 show t h a t a h i g h l e v e l (45 mM) o f a s p a r t i c a c i d i s n e c e s s a r y f o r good g r o w t h and s p o r u l a t i o n w h i l e t h e c o n c e n t r a t i o n o f c y s t e i n e can be d e c r e a s e d t o 2 .3 mM w i t h o u t a d v e r s e e f f e c t . T r i a l s 13 t o 15 show f u r t h e r t h a t t h e l e v e l o f c a l c i u m n i t r a t e c a n be i n c r e a s e d t o 6 . 0 g / l and t h a t c y s -t e i n e c a n be c o m p l e t e l y r e p l a c e d b y - g l u t a t h i o n e w i t h o u t ad-v e r s e l y a f f e c t i n g v e g e t a t i v e g r o w t h . S p o r u l a t i o n was, how-e v e r , m a r k e d l y d e c r e a s e d i n t h e c o m p l e t e a b s e n c e o f c y s t e i n e . No g r o w t h o c c u r r e d when m e t h i o n i n e ( 4 . 6 mM) was s u b s t i t u t e d f o r c y s t e i n e I n o t h e r w i s e s i m i l a r t r i a l s . I n t h e m e d i a used i n t r i a l s 9 , 10 , 11 , 1 3 , 14 and 15 g r o w t h was h i g h l y r e p r o d u c i b l e . Type 5 c o l o n i e s d e v e l o p e d 91 i n v i r t u a l l y 100% o f t h e f l a s k s ( F i g . 111-10). A most i m p o r -t a n t f e a t u r e o f t h e s e r e s u l t s i s t h a t when 40 t o 60 c o l o n i e s grew i n a s y n c h r o n o u s manner i n a s i n g l e f l a s k o r a n g e u r e d o -s p o r e s d e v e l o p e d on t h e u p p e r s u r f a c e s o f t h e c o l o n i e s and p r o d u c e d masses o f d r y u r e d o s p o r e s a f t e r 5 weeks ( F i g . I I I - l l ) . One t o two weeks l a t e r d a r k brown t e l i o s o r i had a p p e a r e d and d e v e l o p e d a c r u s t y mass o f t e l i o s p o r e s ( F i g . 1 11 -12) . These r e s u l t s a r e i n s t r i k i n g c o n t r a s t w i t h t h e s i t u a t i o n t h a t de-v e l o p e d when t h e r e were l e s s t h a n 10 c o l o n i e s p e r f l a s k . Under t h e s e c o n d i t i o n s each c o l o n y d e v e l o p e d a l e a t h e r y s t r o m a on t h e u n d e r s u r f a c e and u r e d o p u s t u l e s n e v e r d e v e l o p e d ( F i g . I I I -1 3 ) . Such c o l o n i e s r e m a i n e d v e g e t a t i v e even a f t e r t h e o r i g i n a l medium was removed by p i p e t t e and r e p l a c e d w i t h f r e s h medium, a l t h o u g h t h e y d i d i n c r e a s e i n s i z e . When t h e o r i g i n a l medium was r e p l e n i s h e d i n f l a s k s w i t h 40 t o 60 c o l o n i e s , renewed v e g e t a t i v e g r o w t h o c c u r r e d and t h e m y c e l i u m o v e r g r e w t h e s p o r u l a t i o n p u s t u l e s . S u c c e s s i v e r e p l e n i s h m e n t s o f t h e medium d i d n o t r e s u l t i n r e - s p o r u l a t i o n . D. R e i n f e c t i o n o f f l a x u s i n g u r e d o s p o r e s grown i n v i t r o . U r e d o p u s t u l e s t a k e n f r o m c o l o n i e s grown r o u t i n e l y on t h e medium u s e d i n t r i a l 10 ( T a b l e I I I - I V ) were p l a c e d on t h e e x p o s e d m e s o p h y l l o f f l a x c o t y l e d o n s and c o v e r e d w i t h c e l l o t a p e . A f t e r 14 days a l a r g e p u s t u l e d e v e l o p e d on b o t h s i d e s o f each c o t y l e d o n and p r o d u c e d n o r m a l u r e d o s p o r e s ( F i g . 111-14) . S i n c e i t was p o s s i b l e t h a t r e i n f e c t i o n v/as accomp-l i s h e d by m y c e l i u m i n t h e u r e d o p u s t u l e s r a t h e r t h a n by g e r m i n -92 a t i n g u r e d o s p o r e s , t h e f o l l o w i n g p r o c e d u r e was a d o p t e d . Axen-i c a l l y grown c o l o n i e s b e a r i n g u r e d o p u s t u l e s were d r i e d a t room t e m p e r a t u r e (20 + 2°C) f o r 43 h and h e l d i n g e l a t i n c a p s u l e s i n t h e r e f r i g e r a t o r f o r about one week. The d r y u r e d o s p o r e s were t h e n d u s t e d o n t o s l i d e s u n d e r t h e d i s s e c t i n g m i c r o s c o p e and p l a c e d i n s m a l l clumps on exposed m e s o p h y l l t i s s u e w h i c h was c o v e r e d w i t h c e l l o t a p e as b e f o r e . Normal u r e d o p u s t u l e s d e v e l o p e d on t h e c o t y l e d o n s i n o c u l a t e d i n t h i s way i n 10 d a y s . I n o t h e r t r i a l s m a t u r e , d r y u r e d o s p o r e s were c a r e -f u l l y h a r v e s t e d d i r e c t l y f r o m t h e t o p s of u r e d o p u s t u l e s on c o l o n i e s grown i n v i t r o . When i n t a c t f l a x c o t y l e d o n s were I n o c u l a t e d d i r e c t l y w i t h w a t e r o r 1%, g e l a t i n s u s p e n s i o n s o f u r e d o s p o r e s h a r v e s t e d i n t h i s way, n o r m a l u r e d o p u s t u l e s de-v e l o p e d a f t e r ' t w o weeks ( F i g . 1 11-15) . I n some t r i a l s m a t u r e d r y u r e d o s p o r e s , t a k e n d i r e c -t l y f r o m t h e t o p s o f u r e d o p u s t u l e s on c o l o n i e s grown i n v i t r o , showed l e s s t h a n 10%0 g e r m i n a t i o n a f t e r 24 h on s o l i d M i l l medium p l u s E v a n s ' p e p t o n e , BSA and YE. I n o t h e r t r i a l s s u c h u r e d o s p o r e s showed up t o 60% g e r m i n a t i o n . A f t e r 2 months a few o f t h e seeded zones w h i c h had shown a h i g h p e r c e n t a g e o f g e r m i n a t i o n d e v e l o p e d i n t o t y p e 3 c o l o n i e s . T h i s r e s u l t shows t h a t i t i s p o s s i b l e t o e s t a b l i s h a x e n i c c u l t u r e s f r o m f l a x r u s t u r e d o s p o r e s w h i c h v/ere t h e m s e l v e s p r o d u c e d i n a x e n i c c u l -t u r e . U n f o r t u n a t e l y o n l y a few t r i a l s o f t h i s k i n d have been made t o d a t e and f u r t h e r e x p e r i m e n t s o f t h i s k i n d a r e u r g e n t l y r e q u i r e d . 93 F i g u r e I I I - 1 1 . M a t u r e d u r e d o s p o r e s i n c l u s t e r t e a s e mount p r e p a r a t i o n . P h l o x i n e / K O H . X 660. F i g u r e 111-12. Tease mount p r e p a r a t i o n o f t e l e u t o -s p o r e s . P h l o x i n e / K O H . X 660. F i g u r e 111-13. Growth and d e v e l o p m e n t o f f l a x r u s t on c h e m i c a l l y d e f i n e d medium MIV c o n -t a i n i n g a s p a r t i c a c i d and c y s t e i n e . (A) V e g e t a t i v e c o l o n i e s a f t e r s t r o m a f o r m a t i o n medium was r e p l e n i s h e d a f t e r f o u r weeks. (B) S p o r u l a t i n g c o l o n i e s w i t h p u s t u l e f o r m a t i o n . U i n d i c a t e s t h e p o s i t i o n o f u r e d o p u s t u l e , T d e n o t e s t e l e u t o s o r i . X 1 . 5 . F i g u r e I I I - 1 A . A x e n i c a l l y d e v e l o p e d u r e d o p u s t u l e i n -f e c t i n g t h e c o t y l e d o n , when p l a c e d on ex p o s e d m e s o p h y l l . Note t h e p r o d u c -t i o n o f u r e d o s p o r e s on b o t h s i d e s o f c o t y l e d o n . F i g u r e 111-15. A x e n i c a l l y d e v e l o p e d u r e d o s p o r e s i n -f e c t i n g i n t a c t c o t y l e d o n and t h e p r o -d u c t i o n o f n o r m a l u r e d o s p o r e s . P h o t o -g r a p h e d a f t e r two weeks. 94 95 DISCUSSION B u s h n e l l and S t e w a r t (1-971) r e p o r t e d t h a t 25 A m e r i -c a n i s o l a t e s o f P u c c i n i a g r a m i n i s t r i t i c i showed w i d e v a r i a -t i o n s i n g r o w t h i n a x e n i c c u l t u r e and t h a t t h e i r t e c h n i q u e s v/ere n o t a d e q u a t e f o r t h e l o n g t e r m c u l t u r e o f most o f t h e i s o l a t e s t e s t e d . Two i s o l a t e s ( r a c e s 56 and 23) d i d , h o w e v e r , s u r v i v e s u b c u l t u r i n g f o r n i n e months. Of t h e s i x r a c e s t e s t e d by u s t h r e e (15B4, 3 8 , and 32-113) d e v e l o p e d c o l o n i e s b u t o n l y one ( r a c e 38) f o r m e d t y p e 4 c o l o n i e s w i t h a s t r o m a a p p r o a c h i n g t h e f i n a l s i z e o f t h e c o l o n i e s o b t a i n e d r o u t i n e l y w i t h t h e i s o -l a t e o f t h e A u s t r a l i a n r a c e ANZ 1 2 6 - 6 , 7 a v a i l a b l e t o u s . 'The f o r m a t i o n o f i r r e g u l a r t h i c k - w a l l e d c e l l s by c u l t u r e s o f r a c e 38 on b o t h l i q u i d and s o l i d m e d i a r e f l e c t s an u n f a v o u r a b l e p h y s i c o - c h e m i c a l e n v i r o n m e n t i n s p i t e o f t h e p r e s e n c e o f pep-t o n e and BSA. Our o b s e r v a t i o n s s u p p o r t t h e v i e w t h a t t h e a b i l i t y o f d i f f e r e n t i s o l a t e s t o d e v e l o p i n a x e n i c c u l t u r e depends on i n h e r e n t c h a r a c t e r i s t i c s o f t h e i s o l a t e s ( B u s h n e l l and S t e w a r t 1 9 7 1 ) . The g r o w t h and dev e l o p m e n t o f p r i m a r y c u l t u r e s o f r u s t f u n g i f r o m u r e d o s p o r e s i n v i t r o i s o f t e n v a r i a b l e and u n p r e d i c t a b l e ( S c o t t and M a c l e a n 1 9 6 9 ) . The a d d i t i o n o f BSA t o t h e medium has been shown t o i m p r o v e t h e f r e q u e n c y w i t h w h i c h c o l o n i e s o f wheat r u s t a r e e s t a b l i s h e d and t h e i r s u b s e q u e n t g r o w t h ( C o f f e y et a l . 1970, K u h l et a l . 1971) . The r e s u l t s o f o u r t r i a l s w i t h b o t h s o l i d and l i q u i d m edia show t h a t BSA a l s o i n c r e a s e s t h e f r e q u e n c y o f e s t a b l i s h m e n t o f f l a x r u s t 96 c o l o n i e s as w e l l as t h e i r d i f f e r e n t i a t i o n t o f o r m a s t r o m a . P o s s i b l y BSA i n h i b i t s o r compensates f o r t h e l o s s o f metabo-l i t e s by t h e g e r m i n a t i n g s p o r e s . Whether i t s e f f e c t I s r e l a -t e d t o i t s p r o p e r t i e s as a. p o l y c a t i o n i s not known but t h e r e s u l t s i n T a b l e I I I - I I I show t h a t i t cannot be r e p l a c e d by t h e d i a m i n e , p u t r e s c i n e , w h i c h s t i m u l a t e s growth i n a number o f m i c r o o r g a n i s m s ( S m i t h 1972). A d d i t i o n o f cAMP t o t h e medium i n h i b i t e d t h e growth o f r a c e ANZ 126-6,7 a f t e r g e r m i n a t i o n of u r e d o s p o r e s ( T a b l e I I I - I . I t might t h e r e f o r e be r e w a r d i n g t o i n v e s t i g a t e t h e l e v e l s o f cAMP i n r e s i s t a n t and s u s c e p t i b l e h o s t p l a n t s i n t h e l i g h t o f t h e c u r r e n t c o n c e p t t h a t t h e i n -c r e a s e d hormonal l e v e l c a uses a c o n c o m i t a n t i n c r e a s e i n cAMP ( P o l l a r d 1970). The r e s u l t s i n T a b l e s I I I - I I and I I I - I V show t h a t p r i m a r y c u l t u r e s o f b o t h wheat and f l a x r u s t grew and s p o r u -l a t e d on s i m p l e , c o m p l e t e l y d e f i n e d media. Czapek's m i n e r a l s have been used I n t h e c u l t u r e medium f o r wheat r u s t whereas Knop's m i n e r a l s p l u s m o d i f i e d B e r t h e l o f s m i c r o n u t r i e n t s have been used f o r f l a x r u s t . The r e l a t i v e c o n c e n t r a t i o n s and comp-o s i t i o n o f t h e m a c r o n u t r i e n t s a l t s r e q u i r e d a r e t h e r e f o r e a p p a r e n t l y q u i t e d i f f e r e n t f o r t h e two r u s t s . One i m p o r t a n t d i f f e r e n c e between Czapek's and Knop's m i n e r a l n u t r i e n t s o l u -t i o n s i s t h a t t h e l a t t e r i n c l u d e s C a ( N 0 ^ ) 2 . I n our e x p e r i -ments b e s t growth of wheat r u s t was o b t a i n e d on Czapek's m i n e r a l s supplemented by t h e a d d i t i o n of t h e same l e v e l s o f Ca(N0-3) 2 a-n<3 m i c r o n u t r i e n t s and c o n t a i n i n g t h e same amino a c i d s t h a t gave t h e b e s t growth o f f l a x r u s t ( T a b l e s I I I - I I 97 and I I I - I V ) . These o b s e r v a t i o n s s u g g e s t t h a t t h e n u t r i t i o n a l r e q u i r e m e n t s f o r a x e n i c g r o w t h o f p r i m a r y c u l t u r e s o f P u c c i n i a and M e l a m p s o r a a r e n o t d i s s i m i l a r , i n s e v e r a l i m p o r t a n t r e s -p e c t s . C a l c i u m has been i m p l i c a t e d as a m i c r o n u t r i e n t i n t h e n u t r i t i o n o f f u n g i by S t e i n b e r g ( 1 9 4 8 ) , b u t i t i s n o t r e a l l y known w h e t h e r t h e c a l c i u m r e q u i r e m e n t o f f u n g i i s s p e c i f i c o r n o t ( C o c h r a n e 1 9 5 8 ) . C a l c i u m e x e r t s a p r o t e c t i v e e f f e c t a g a i n s t a number o f a g e n t s ( L i n d e b e r g 1944, O l s e n 1972) and has a s t a b i l i z i n g e f f e c t on c e l l membranes ( B e n e d e t t i and Emmelot 1 9 6 8 ) . The r e l a t i v e l y h i g h l e v e l o f c a l c i u m f o u n d t o be n e c e s s a r y f o r t h e s p o r u l a t i o n o f f l a x r u s t ( T a b l e I I I - I V ) s u g g e s t s t h a t t h e e f f e c t s o f c a l c i u m on t h e g r o w t h o f r u s t f u n g i i n v i t r o may be r e l a t e d t o i t s p r o t e c t i v e and s t a b i l i z -i n g a c t i o n . A l t h o u g h g l u c o s e and s u c r o s e a r e t h e p r e f e r r e d c a r b o -h y d r a t e s o u r c e s f o r wheat and f l a x r u s t s , r e s p e c t i v e l y , b o t h r u s t s c a n u t i l i z e e i t h e r o f t h e s e two s u g a r s and a wi d e r a n g e o f o t h e r c a r b o h y d r a t e s ( W i l l i a m s e t a l . 1966, 1967, C o f f e y and Shaw 1 9 7 2 ) . The two r u s t s a l s o have s i m i l a r amino a c i d r e -q u i r e m e n t s . A s p a r t i c ( o r g l u t a m i c ) a c i d and c y s t e i n e i n c o m b i n a t i o n s u p p o r t e d b o t h v e g e t a t i v e g r o w t h and s p o r u l a t i o n o f f l a x r u s t . On t h e o t h e r hand, w h i l e wheat r u s t grew on a s p a r t i c a c i d p l u s c y s t e i n e , s p o r u l a t i o n was o n l y o b t a i n e d w i t h t h e f u r t h e r a d d i t i o n o f g l u t a t h i o n e . The new r e s u l t s o b t a i n e d w i t h f l a x r u s t ( T a b l e I I I -I V , T r i a l s 9 t o 15) show t h a t t h e use o f h i g h e r c o n c e n t r a t i o n s 98 o f amino a c i d s t h a n t h o s e employed b y e a r l i e r w o r k e r s ( e . g . T u r e l 1973) i m p r o v e s v e g e t a t i v e g r o w t h and p e r m i t s s p o r u l a t i o n . The r e s u l t s w i t h f l a x r u s t r e p r e s e n t a d i s t i n c t a dvance i n o u r a b i l i t y t o o b t a i n a p p a r e n t l y m o r p h o g e n e t i c a l l y ' n o r m a l 1 v e g e -t a t i v e d e v e l o p m e n t and s p o r u l a t i o n i n v i t r o w i t h a h i g h d e g r e e o f r e p r o d u c i b i l i t y . On t h e b a s i s o f a n i m a l c e l l c u l t u r e work . H a r r i s (1964) s u g g e s t e d t h a t t h e r e may be a g r e a t e r l o s s o f c e r t a i n m e t a b o l i t e s i n a c h e m i c a l l y d e f i n e d l i q u i d medium. T h i s l e a c h i n g e f f e c t c a n be c o u n t e r b a l a n c e d by i n c r e a s i n g t h e e x t e r n a l s u p p l y o f c e r t a i n e s s e n t i a l i n g r e d i e n t s o f t h e medium. I t seems p r o b a b l e t h a t s p o r u l a t i o n i s c o r r e l a t e d w i t h c hanges i n t h e c h e m i c a l c o m p o s i t i o n o f t h e medium b r o u g h t a b o u t by t h e f u n g u s i t s e l f , s i n c e renewed v e g e t a t i v e g r o w t h was i n d u c e d b y r e p l a c e m e n t o f t h e o r i g i n a l w i t h f r e s h medium. I n a d d i t i o n , t h e p o s s i b i l i t y s h o u l d n o t be o v e r l o o k e d t h a t i n t e r a c t i o n s b etween c o l o n i e s may a l s o be m e d i a t e d by v o l a t i l e m e t a b o l i c p r o d u c t s and t h a t t h e s e may a l s o p l a y a r o l e i n c o n t r o l l i n g d e v e l o p m e n t and s p o r u l a t i o n . The r e s u l t s w i t h wheat r u s t ( T a b l e I I I - I I ) do n o t r e f l e c t t h e same d e g r e e o f c o n t r o l and were l e s s p r e d i c t a b l e t h a n t h o s e f o r f l a x r u s t . We cannot,, e x p l a i n t h e d i f f e r e n c e i n p e r f o r m a n c e between t h e two r u s t s i n a x e n i c c u l t u r e a t p r e -s e n t , b u t i t i s p o s s i b l e t h a t f u r t h e r m o d i f i c a t i o n s i n t h e m e d i a u s e d may i m p r o v e t h e g r o w t h and dev e l o p m e n t o f t h e wheat r u s t . One f a c t o r w h i c h may have some b e a r i n g on t h e r e s u l t s i s t h e p r o c e d u r e u s e d t o r a i s e a s e p t i c u r e d o s p o r e s t o p r o v i d e t h e i n o c u l u m f o r t h e e x p e r i m e n t a l work. The p r o c e d u r e f o r 99 f l a x r u s t y i e l d s a f r i a b l e mass o f e a s i l y m a n i p u l a t e d u r e d o -s p o r e s b u t t h a t f o r wheat r u s t (Bose and Shaw 1971) does n o t . The wheat r u s t u r e d o s p o r e s have a s t r o n g t e n d e n c y t o clump t o g e t h e r and .are more d i f f i c u l t t o m a n i p u l a t e . "Whether t h e s e d i f f e r e n t c h a r a c t e r i s t i c s o f t h e u r e d o s p o r e s o f t h e two r u s t s have any e f f e c t on t h e i r s u b s e q u e n t g r o w t h i n a x e n i c c u l t u r e i s e n t i r e l y a m a t t e r o f c o n j e c t u r e a t p r e s e n t . The s u c c e s s f u l e s t a b l i s h m e n t o f t h e r u s t f u n g i i n a x e n i c c u l t u r e c l e a r l y i n v o l v e s a p e r i o d o f a d a p t a t i o n on t h e medium w h i c h s u p p o r t s g r o w t h . We a r e o f t h e o p i n i o n t h a t s u c h a d a p t a t i o n o c c u r s as a r e s u l t o f t h e c l o s e a s s o c i a t i o n and a n a s t o m o s i s o f germ t u b e s f o l l o w i n g u r e d o s p o r e g e r m i n a t i o n . I n p a r t i c u l a r a n a s t o m o s i s may r e s u l t i n a m u l t i n u c l e a t e s i t u a t i o n , a n a l o g o u s t o t h a t w h i c h o c c u r s i n t h e i n f e c t i o n s t r u c t u r e s n o r -m a l l y f o r m e d on t h e h o s t p l a n t . I n dual-membered ( i . e . i n -f e c t e d ) c a l l u s c u l t u r e s t h e n e c e s s a r y a d a p t a t i o n f o r s u b s e q u e n t s a p r o b i c g r o w t h p r e s u m a b l y o c c u r s d u r i n g p r o l o n g e d a s s o c i a t i o n o f t h e two o r g a n i s m s i n v i t r o . D e s p i t e o u r s u c c e s s i n o b t a i n i n g r e p r o d u c i b l e v ege-t a t i v e and r e p r o d u c t i v e g r o w t h , p a r t i c u l a r l y o f f l a x r u s t , u n d e r p r e c i s e l y d e f i n e d c o n d i t i o n s , t h e mechanism o r n a t u r e o f t h e a d a p t i v e changes t h a t p e r m i t s a p r o b i c g r o w t h a r e com-p l e t e l y unknown. At t h e g e n e t i c l e v e l a d a p t a t i o n may i n v o l v e t h e d e r e p r e s s i o n o f a p a r t i c u l a r segment o f t h e f u n g a l genome ( S c o t t and M a c l e a n 1969) o r t h e exchange of chromosome segments between a d j a c e n t f u n g a l n u c l e i ( H a r t l e y and W i l l i a m s 1971). The d i f f u s i o n o f c y t o p l a s m i c f a c t o r s o r even v o l a t i l e compon-100 e n t s b e tween g e r m l i n g s , i n t h e ease o f p r i m a r y c u l t u r e s de-r i v e d f r o m u r e d o s p o r e s , o r between h o s t and p a r a s i t e i n d u a l -membered c u l t u r e s , may a l s o be i n v o l v e d . I n d u c e d a d a p t a t i v e c h a n g e s i n v o l v i n g c y t o p l a s m i c r a t h e r t h a n n u c l e a r e l e m e n t s a r e known i n f u n g i and t h e i r t r a n s m i s s i b i l i t y has been demon-s t r a t e d ( J i n k s 1957, 1958). F i n a l l y , a d a p t a t i o n may I n v o l v e , t h e s e l e c t i v e r e m o v a l o r i n a c t i v a t i o n o f i n h i b i t o r s . . I n h i b i -t o r s o f u r e d o s p o r e g e r m i n a t i o n a r e w e l l known ( A l l e n and D u n k l e 1971) b u t t o o u r k n o w l e d g e n a t u r a l l y o c c u r r i n g i n h i b i -t o r s o f h y p h a l g r o w t h have n o t been r e p o r t e d . W h a t e v e r t h e mechanism o f a d a p t a t i o n t o s a p r o b i c g r o w t h , e s t a b l i s h e d a x e n i c c o l o n i e s have been shown t o u t i l i z e a b r o a d e r r a n g e o f sub-s t r a t e s t h a n t h a t w h i c h p e r m i t s t h e I n i t i a t i o n o f p r i m a r y c o l o n i e s from u r e d o s p o r e s o r f r o m i n f e c t e d c a l l u s ( C u t t e r 1959, I960, C o f f e y and Shaw 1972). 101 REFERENCES A l l e n , P.J., and L.D. Dunkle. 1971. Natural a c t i v a t o r s and i n h i b i t o r s of spore germination. In Morphological and Biochemical Events i n Plant Parasite I n t e r a c t i o n . S. Akai and S. Ouchi (Eds.). Mochizukl Pub. Co., Japan. 415 p. Benedetti, E.L., and P. Emmelot. 1968. Structure and f u n c t i o n of plasma membranes i s o l a t e d from l i v e r . In The Membranes. A.J. Dalton and F. Haguenau (Eds.). Academic Press, Nev/ York, 223 P . Bose, A., and M. Shaw. 1971. Sporulation and pathogenicity of an A u s t r a l i a n i s o l a t e of wheat rust grown i n v i t r o . Can. J . Botany 49: 1961-1964. . Bushnell, W.R. 1968. In v i t r o development of an A u s t r a l i a n i s o l a t e of Puccinia graminis f . sp. t r i t i c i , Phyto-pathology 5 8 : 3 2 6 - 3 2 7 . Bushnell, W.R., and D.M. Stewart. 1971. Development of Ameri-can i s o l a t e s of Puccinia graminis f. sp. t r i t i c i on an a r t i f i c i a l medium, phytopathology 61 : 3 7 6 - 3 7 9 . Cochrane, V.M. 195#. Physiology of Fungi. John Wiley & Sons, Inc., New York. 524 p. Coffey, M.D., and P.J. A l l e n . 1973. N u t r i t i o n of Melampsora l i n i and Puccinia h e l i a n t h i . Trans. B r i t . Mycol. Soc. 60: 2413-26707 Coffey, M.D., A. Bose and M. Shaw. 1969. In v i t r o growth of g e l a t i n suspensions of uredospores of Puccinia gramin-i s f . sp. t r i t i c i . Can. J , Botany 47: 1291-1293. Coffey, M.D., A. Bose and M. Shaw. 1970. In v i t r o culture of the f l a x r u s t , Melampsora l i n i . Can. J . Botany 4 8 : 773-776. Coffey, M.D., and M. Shaw. 1972. N u t r i t i o n a l studies with axenic cultures of the f l a x r u s t , Melampsora l i n i . P h y s i o l . Plant Pathology 2: 3 7 - 4 7 . Cutter, V.M., J r . 1959(1961) . Studies on the i s o l a t i o n and growth of plant rusts i n host t i s s u e cultures and upon synthetic media. I . Gymnosporangium. Mycologia 52: 248-295. Cutter V.M., J r . 1960(1961) . Studies on the i s o l a t i o n and growth of plant rusts i n host t i s s u e cultures and upon synthetic media. I I . Uromyces a r i - t r i p h y l l i . Mycologia 52: 7 2 6 - 7 4 2 . 102 F o u d i n , A.S., and ¥ . K . ¥ y n n . 1972. Growth o f P u c c i n i a g r a m i n -i s f . s p . t r i t i c i on a d e f i n e d mediura. P h y t o p a t h o l o g y h~2: 1032-lUlAT. Gamborg, O.L., R.A. M i l l e r , and K. O j i m a . 1968. N u t r i e n t r e -q u i r e m e n t s o f s u s p e n s i o n c u l t u r e s o f s o y b e a n r o o t c e l l s . E x p t . C e l l R e s . 50: I 5 I - I 5 8 . H a r r i s , M. 1964. C e l l C u l t u r e and S o m a t i c V a r i a t i o n . H o l t , R i n e h a r d & W i n s t o n , I n c . , New Y o r k , 547 p. H a r t l e y , M.J., and P.G. W i l l i a m s . 1971a. M o r p h o l o g i c a l and c u l t u r a l d i f f e r e n c e s between r a c e s o f P u c c i n i a g r a m i n -i s f . s p . t r i t i c i i n a x e n i c c u l t u r e . T r a n s . B r i t . M y c o l . S o c . 57: 137-144. H a r t l e y , M.J., and P . J . W i l l i a m s , 1971b. G e n o t y p i c v a r i a t i o n w i t h i n a p h e n o t y p e as a p o s s i b l e b a s i s f o r s o m a t i c h y b r i d i z a t i o n i n r u s t f u n g i . Can. J . B o t a n y 4 9 : 1 0 8 5 - 1 0 8 8 . H o l l i s , C.A., R.A. S c h m i d t , and J.W. K i m b r o u g h t . 1972. A x e n i c c u l t u r e o f C r o n a r t i u m f u s i f o r m e . P h y t o p a t h o l o g y 6 2 : 1417-1419. H o t s o n , H.H., and V.M. C u t t e r , J r . 1 9 5 1 . The i s o l a t i o n and c u l t u r e o f Gymnosporangium j u n i p e r i - v i r g i n i a n a e Schw. upon a r t i f i c i a l m e d i a . P r o c . N a t . A c a d . S c i . ( U . S . A . ) 37: 4 0 0 - 4 0 3 . Howes, N.K., and K . J . S c o t t . 1972. S u l p h u r n u t r i t i o n o f P u c c i n i a g r a m i n i s f . s p . t r i t i c i I n a x e n i c c u l t u r e . C a n . J . B o t a n y 50: 1165-1170. J i n k s J L . 1957. S e l e c t i o n f o r c y t o p l a s m i c d i f f e r e n c e s . ' P r o c . R o y . S o c . B. 146: 527-540. J i n k s J . L . 1958. C y t o p l a s m i c d i f f e r e n t i a t i o n I n f u n g i . ' P r o c . Roy. S o c . B. 11+8: 3 1 4 - 3 2 1 . J o n e s D.R. 1972. I n v i t r o c u l t u r e o f c a r n a t i o n r u s t , U r o -' 'myces d i a n t h i . T r a n s . B r i t . M y c o l . S o c . 58: 29' =J6". K u h l , J . L . , D . J . M a c l e a n , K . J . S c o t t , and P.G. W i l l i a m s . 1971. ' The a x e n i c c u l t u r e o f P u c c i n i a s p e c i e s f r o m u r e d o -s p o r e s - e x p e r i m e n t s on n u t r i t i o n and v a r i a t i o n . C a n . J . B o t a n y 59: 2 0 1 - 2 0 9 . L i n d e b e r g G. 1944. Uber d i e p h y s i o l o g i e l i g n i n a b l a u e n d e r bodenhymenomyzeten. Symb. B o t . U p s . 8 ( 2 ) : 1-183. 103 M a c l e a n , D.J.., and K . J . S c o t t . 1970. V a r i a n t f o r m s . o f s a p r o -p h y t i c m y c e l i u m grown f r o m u r e d o s p o r e s o f P u c c i n i a g r a m i n i s f . s p . t r i t i c i . J . Gen. M i c r o b i o l . 64: 1 9 - 2 7 . . M a s i n , V.V., and L.N. A n d r e e v . 1971. V e g e t a t i v e g r o w t h o f t h p a t h o g e n o f wheat stem r u s t i n v i t r o . M I k o l o g i y a i F i t o p a t h o l o g i y a 5 ( 2 ) : 1 9 7 - 2 0 0 . ' O l s e n , R.A. 1972. T r i t e r p e n e g l y c o s i d e s as i n h i b i t o r s o f f u n -g a l g r o w t h and m e t a b o l i s m . P h y s i o l . P l a n t a r u r n 27: 2 0 2 - 2 0 8 . P o l l a r d , C . J . 1970. I n f l u e n c e o f g i b b e r e l l i c a c i d on t h e i n -c o r p o r a t i o n o f 8-lA-c a d e n i n e i n t o a d e n o s i n e 3 S , 5 T -c y c l i c p h o s p h a t e i n b a r l e y a l e u r o n e l a y e r s . B i o c h i m . B i o p h y s . A c t a 2 0 1 : 511-512. Q u i c k , W.A., and S.L.C. C r o s s . 1971. The use o f m i l l i p o r e f i l t e r d i s c s i n a x e n i c c u l t u r e o f f l a x r u s t . Can. J . B o t a n y 4 9 : 187-188. R o b l s o n , G.A., R.W. B u t c h e r , and E.W. S u t h e r l a n d . 1971. C y c l AMP i n l o w e r o r g a n i s m s . I n C y c l i c AMP. R o b l s o n , G.A R.W. B u t c h e r and E.W. S u t h e r l a n d ( E d s . ) . Academic Press', New Y o r k , p. 422-453 . S c o t t , K . J . , and D.J. M a c l e a n . 1969. C u l t u r i n g o f r u s t f u n g i Ann. Rev. P h y t o p a t h o l o g y 6: 123-146. S m i t h , T.A. 1972. The p h y s i o l o g y o f p o l y a m i n e s and r e l a t e d compounds. E n d e a v o u r 3 1 ( 1 1 2 ) : 2 2 - 2 8 . S t e i n b e r g , R.A. 1948. E s s e n t i a l i t y o f c a l c i u m i n t h e n u t r i -t i o n o f f u n g i . S c i e n c e ( W a s h i n g t o n ) 107: 4 2 3 . T u r e l , F.L.M, 1969. S a p r o p h y t i c d e v e l o p m e n t o f t h e f l a x r u s t M e l a m p s o r a l i n i r a c e no. 3 . Can. J , B o t a n y 47: 821-823 . T u r e l , F.L.M. 1971. P a t h o g e n i c i t y and d e v e l o p m e n t a l d i f f e r -e n c e s o f t h r e e s a p r o p h y t i c a l l y g r o w i n g i s o l a t e s o f f l a x r u s t f u n g u s , M e l a m p s o r a l i n i , r a c e 3 . Can. J . B o t a n y 49 : 1993-1997. T u r e l , F.L.M. 1973. Growth o f t h e f l a x r u s t M e l ampsora l i n i on c h e m i c a l l y d e f i n e d m e d i a . Can. J . B o t a n y 51 : 131-134. V a s s i l ' e v , O.A., and V . I . S a p l i n a . 1971. C u l t u r e o f t h e f u n -gus M e l a m p s o r a p i n i t o r q u a (A. BR.) R o s t r . - t h e patho-gen o f t h e stem r u s t o f p i n e . M I k o l o g i y a i F i t o -p a t h o l o g i y a 5 ( 2 ) : 182-183. 104 W i l l i a m s , P.G., K . J . S c o t t , and J . L . K u h l . 1966. V e g e t a t i v e g r o w t h o f P u c c i n i a g r a m i n i s f . s p . t r i t i c i i n v i t r o . P h y t o p a t h o l o g y $67 1413 -1419. W i l l i a m s , P.O., K . J . S c o t t , J . L . K u h l , and D.J. M a c l e a n . 1967, S p o r u l a t i o n and p a t h o g e n i c i t y of P u c c i n i a g r a m i n i s f . s p . t r i t i c i grown on an a r t i f i c i a l medium. P h y t o -p a t h o l o g y 57 : 326-327. Wong, A.L., and H.J. W i l l e t t s . 1970. O b s e r v a t i o n s on g r o w t h o f s e l e c t e d A u s t r a l i a n r a c e s o f wheat stem r u s t i n a x e n i c c u l t u r e . T r a n s . B r i t . M y c o l . Soc. 55 : 231-238. 1 105 SUMMARY 1. L i q u i d mediurri c o n t a i n i n g C z a p e k ' s m i n e r a l s , 3% g l u c o s e , 0.1% E v a n s ' p e p t o n e and 1% d e f a t t e d b o v i n e serum a l b u m i n s u p p o r t e d t h e v e g e t a t i v e g r o w t h and s p o r u l a t i o n o f P u c c i n i a g r a m i n i s t r i t i c i r a c e ANZ 1 2 6 - 6 , 7 . 2 . T y p i c a l p i g m e n t e d u r e d o s p o r e s and t e l i o s p o r e s were f o r m e d a f t e r 6-8 weeks o f g r o w t h . 3. The u r e d o s p o r e s were c a p a b l e o f i n f e c t i n g t h e m e s o p h y l l o f wheat l e a v e s e x p o s e d by s t r i p p i n g back t h e l o w e r e p i -d e r m i s . 4. S o l i d medium c o n t a i n i n g D i f c o .bacto a g a r , s u c r o s e , Knop's m i n e r a l s a l t s and m i c r o n u t r i e n t s p l u s y e a s t e x t r a c t and p e p t o n e s u p p o r t e d t h e v e g e t a t i v e g r o w t h o f two d i f f e r e n t s t r a i n s o f f l a x r u s t M e l a m p s o r a l i n i ( E h r e n b . ) L e v . 5 . I n c i d e n c e o f e s t a b l i s h m e n t of t h e c o l o n i e s s h a r p l y i n -c r e a s e d when d e f a t t e d BSA was added t o t h e above m e d i a . 6. S u r f a c e s t e r i l i z e d f l a x c o t y l e d o n s become i n f e c t e d when p l a c e d w i t h t h e i r c u t ends i n c o n t a c t w i t h t h e s e c o l o n i e s . 7. Good v e g e t a t i v e g r o w t h was o b t a i n e d when t h r e e N o r t h A m e r i c a n r a c e s (32-113, 15B4 and 38) o f P u c c i n i a g r a m i n i s i t r i t i c i were t e s t e d on a l i q u i d medium c o n t a i n i n g C z a p e k ' s m i n e r a l s , 3% g l u c o s e , 0 .1% E v a n s ' p e p t o n e and i • . - • BSA p l u s 0 . 5 g / l C a ( N 0 3 ) 2 . ' ' ' ' 8. A c h e m i c a l l y d e f i n e d l i q u i d medium c o n t a i n i n g s u c r o s e , Knop's m i n e r a l s a l t s , B e r t h e l o t ' s m i c r o n u t r i e n t s , a s p a r t i c ( o r g l u t a m i c ) a c i d and c y s t e i n e s u p p o r t e d t h e gro w t h o f 106 v e g e t a t i v e c o l o n i e s o f M e l a m p s o r a l i n i r a c e 3 f r o m un-c o n t a m i n a t e d uredospores.. The f o r m a t i o n o f u r e d o s p o r e s and t e l i o s p o r e s o c c u r r e d a t a r e l a t i v e l y h i g h f r e q u e n c y and s p o r u l a t i o n seems t o be d e p e n d e n t upon t h e l e v e l o f Ca++ and number o f c o l o n i e s o r i g i n a t i n g p e r f l a s k . The u r e d o s p o r e s o f f l a x r u s t d e v e l o p e d i n a x e n i c c u l t u r e i n f e c t e d h e a l t h y week o l d f l a x c o t y l e d o n s i n a n o r m a l manner. 

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