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Host : parasite interactions of rusts and axenic culture of Melampsora species Lane, William D. 1974

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HOST:PARASITE INTERACTIONS OF RUSTS AND AXENIC'CULTURE OF MELAMPSORA SPECIES by. WILLIAM DAVID LANE B.Sc.(Agr.), U n i v e r s i t y of B r i t i s h Columb: M.Sc. ,.• U n i v e r s i t y of B r i t i s h Columbia A Thesis Submitted i n P a r t i a l F u l f i l m e n t of the Requirements j f o r the Degree of DOCTOR OF PHILOSOPHY in' the Department of ,Plant Science i We accept t h i s t h e s i s as conforming to requ i r e d s/tandard THE UNIVERSITY OF BRITISH,COLUMBIA December, 1 9 7 4 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 of the r e -quirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree th a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Depart-ment or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed w i t h o u t my w r i t t e n permission. Department of Plant Science . U n i v e r s i t y of B r i t i s h Columbia Vancouver, B r i t i s h Columbia V 6 T 1W5 Date J e w l ; W > ABSTRACT The t h e s i s i s composed of two s e c t i o n s . The f i r s t sec-t i o n comprising Chapters I and I I i s designed to examine p o s s i b l e i n t e r a c t i o n s between i s o l a t e d f l a x p r o t o p l a s t s and germinating r u s t uredio'spores, or a x e n i c a l l y grown r u s t myce-lium in.an attempt to o b t a i n evidence f o r or against the theory that t o x i n s p l a y a r o l e i n r e s i s t a n c e and s u s c e p t i -b i l i t y i n the f l a x r f l a x r u s t system. A number of p r e v i o u s l y published observations form the b a s i s of the t o x i n theory of r e s i s t a n c e . These can be summarized as f o l l o w s : n e c r o t i c c e l l s are found surrounding the f l a x r u s t i n f e c t i o n s ; the cytoplasm of the r u s t and the host never come i n t o d i r e c t ' contact; plant c e l l s some distance removed from the s i t e of i n f e c t i o n become n e c r o t i c ; the n e c r o t i c area around an i n f e c -t i o n can be. extended away from the s i t e of i n f e c t i o n when an e l e c t r i c current i s passed through the l e a f , the n e c r o s i s extending i n the d i r e c t i o n of the p o s i t i v e pole; and the c h a r a c t e r i s t i c s of the n e c r o t i c area vary depending on the v i r u l e n c e and r e s i s t a n c e genes i n v o l v e d i n the i n t e r a c t i o n . ' Chapter I describes the i s o l a t i o n of f l a x p r o t o p l a s t s , and t h e i r growth and. development i n v i t r o . The medium used and other c u l t u r a l c o n d i t i o n s were, found to i n f l u e n c e both the type of development and the l o n g e v i t y of c u l t u r e d proto-p l a s t s . When c u l t u r a l c o n d i t i o n s were o p t i m a l , some proto-p l a s t s d i v i d e d m i t o t i c a l l y , and many remained a l i v e f o r a i v month or longer. Information obtained from these e x p e r i -ments formed the b a s i s of the experimental procedures em-ployed i n the work described in.Chapter I I . I n Chapter I I the t o x i n theory of r e s i s t a n c e and suscep-t i b i l i t y was examined u s i n g a p r o t o p l a s t b u r s t i n g bioassay to detect any fung a l t o x i n s , i f . t h e y were present i n the t e s t s o l u t i o n . P r o t o p l a s t s , are s u i t a b l e f o r a bioassay of t h i s kind because they' are f r a g i l e and s e n s i t i v e and burst when exposed to t o x i c substances. The b u r s t i n g response i s easy to detect and to q u a n t i f y . Because axenic c u l t u r e s of f l a x r u s t are now a v a i l a b l e a search f o r evidence of e x t r a c e l l u l a r products w i t h t o x i c p r o p e r t i e s i s t h e r e f o r e p o s s i b l e . Spore germination medium i s al s o a p o s s i b l e source of fung a l t o x i n s . I f the p r o t o p l a s t s i s o l a t e d from e i t h e r r e s i s t a n t or sus-c e p t i b l e v a r i e t i e s , or both, were burst when incubated w i t h exudate from axenic c u l t u r e s or spore germination medium t h i s would c o n s t i t u t e evidence i n support of.the t o x i n theory. The search f o r t o x i c substances i n both a x e n i c . c u l t u r e exudate and spore germination medium y i e l d e d negative r e -s u l t s . These, together with.other p r e v i o u s l y published, r e -s u l t s , argue against the involvement of t o x i n s i n the f l a x : f l a x r u s t system. An a l t e r n a t i v e explanation,..consistent w i t h the observations which o r i g i n a l l y suggested the t o x i n theory, i s t h a t , when the fungus invades the i n t a c t l e a f , t o x i n s are produced by the host p l a n t . This proposal i s discussed. . v The second s e c t i o n of the t h e s i s , comprising Chapters I I I and IV, describes techniques f o r growing r u s t s axenic-a l l y . I n Chapter I I I a technique i s described f o r the i s o l a -t i o n of c o l o n i e s of f l a x r u s t from i n f e c t e d cotyledons. The technique depends on the d i g e s t i o n of host c e l l w a l l s w i t h h y d r o l y t i c enzymes and washing the l i b e r a t e d c o l o n i e s f r e e from adhering f l a x p r o t o p l a s t s . Using t h i s method i t i s p o s s i b l e to c o l l e c t l a r g e numbers of f l a x r u s t c o l o n i e s w i t h o n l y a few host c e l l s adhering to them. The i s o l a t e d c o l o n i e s can be used as a source of uncontaminated f u n g a l t i s s u e . Axenic c u l t u r e s were e s t a b l i s h e d u s i n g c o l o n i e s i s o l a t e d i n t h i s way as inoculum. The r e s u l t s i n Chapter IV are an extension and m o d i f i c a -t i o n of those i n Chapter I I I and describe the axenic c u l t u r e of poplar r u s t f o r the f i r s t time. Surface s t e r i l i z e d l e a f pieces of black cottonwood leaves centered on u r e d i a l i n f e c -t i o n s of poplar r u s t (Melampsora o c c i d e n t a l i s ) were placed pustule s i d e up on a completely defined agar base medium. A l l the pustules grew f o r the f i r s t two weeks and a f t e r 1+ months about 30% of them became e s t a b l i s h e d as v e g e t a t i v e axenic c u l t u r e c o l o n i e s on the agar medium. The l e a f t i s s u e d i d not grow. The axenic c u l t u r e s were s u c c e s s f u l l y subcultured to f r e s h medium. Some produced spores i n c u l t u r e . The c o l o n i e s were capable of r e i n f e c t i n g excised leaves of the host i n v i t r o . The new axenic c u l t u r e techniques described i n Chapter I I I and IV are simpler than previous methods and have s e v e r a l other ad-vantages . v i TABLE OF CONTENTS Page ABSTRACT i i i TABLE: OF CONTENTS v i LIST OF.TABLES v i l i LIST OF FIGURES. . . i x ACKNOWLEDGEMENTS x GENERAL INTRODUCTION 1 H i s t o r i c a l Aspects of Rust 1 . . ' S u s c e p t i b i l i t y and Resistance 4 Axenic Culture 9 References 1 9 SECTION I . EXPERIMENTS USING PROTOPLASTS TO STUDY HOST:PARASITE INTERACTIONS 23 Chapter I . Development and D i v i s i o n of . P r o t o p l a s t s from F l a x Cotyledons 2 4 I n t r o d u c t i o n 21+ M a t e r i a l s and Methods . 2 5 Results and D i s c u s s i o n 26 References . 3 3 Chapter I I . Experiments u s i n g a P r o t o p l a s t Bioassay to Study F l a x : F l a x Rust I n t e r a c t i o n 35: I n t r o d u c t i o n , 3 5 M a t e r i a l s and Methods. 4 0 Results . 4 1 Discussion. 50 References 58. Appendix to Chapter I I 6 0 SECTION I I . AXENIC CULTURE 6 4 Chapter I I I . Axenic Culture of. F l a x Rust I s o l a t e d from Cotyledons- by C e l l W a l l D i -ge s t i o n 65 I n t r o d u c t i o n 6 5 M a t e r i a l s and Methods . 6 6 Results and Discussion. 6 8 References . 7 2 v i i Page Chapter IV. I s o l a t i o n and Axenic.Culture of Poplar Rust • 73 I n t r o d u c t i o n . . 7 3 M a t e r i a l s and Methods 7 4 R e s u l t s , and Discussion- 7 4 References BO SUMMARY 81 v i i i LIST OF TABLES .. Table . " Page I E f f e c t s of Spore Germination Medium Plus .Spores on P r o t o p l a s t s 43 I I E f f e c t s of Spore Germination Medium Plus Spores on P r o t o p l a s t s 4 5 I I I E f f e c t of 37°C Incubation:on P r o t o p l a s t s 46 IV E f f e c t of Spore Germination Medium on P r o t o p l a s t s 4 8 V E f f e c t s of Spore Germination Medium on Pr o t o p l a s t s 4 9 VI E f f e c t s of Axenic Exudate on P r o t o p l a s t s VII E f f e c t s of Axenic. Exudate on P r o t o p l a s t s 51 52 LIST OF FIGURES Figure Page . Chapter I . Development and D i v i s i o n of P r o t o p l a s t s from F l a x Cotyledons. 1-1 F r e s h l y i s o l a t e d p r o t o p l a s t s . 29 1-2 D i v i d e d p r o t o p l a s t . 29 .1-3 Budding p r o t o p l a s t . 29 1-4 • E x t r u s i o n of cytoplasm - type I . 29 • 1-5 • E x t r u s i o n of•.cytoplasm - type I I . 29 1-6 Divided giant c e l l . 29 1-7 B i n u c l e a t e giant c e l l . , 29 1-8 T y p i c a l giant c e l l . 29 Chapter I I I . Experiments u s i n g a P r o t o p l a s t Bioassay to Study Flax:F l a x . Rust I n t e r a c t i o n . . . I I I - l A colony i s o l a t e d a f t e r overnight i n c u b a t i o n 7 1 i n the enzyme s o l u t i o n . I I I - 2 Enlargement of a colony s i m i l a r to that 7 1 i n I I I - l . I I I - 3 I n t a c t h a u s t o r i a i n c l u d i n g h a u s t o r i a l 7 1 neck attached to i s o l a t e d colony. I I I - 4 Urediospores i n the orange spheres which. developed on i s o l a t e d c o l o n i e s at the beginning of c u l t u r e . . 7 1 I I I - 5 An axenic c u l t u r e which grew from an i s o -l a t e d colony. 7 1 Chapter IV. I s o l a t i o n and Axenic Culture of Poplar Rust. I'V-1 Poplar r u s t axenic c u l t u r e . • 7 9 IV-2 T y p i c a l mycelium obtained from an axenic 79 c u l t u r e . IV-3 R e i n f e c t i o n of black cottonwood l e a f . 79 IV-4 Spores produced by a x e n i c a l l y c u l t u r e d 79 poplar r u s t . X ACKNOWLEDGEMENTS As I near the end of my d o c t o r a l s t u d i e s I f e e l p a r t i c u l a r l y g r a t e f u l and f o r t u n a t e . t o have had Dr. Michael Shaw as my s u p e r v i s o r . He has provided.encouragement and help and h i s example has been an i n s p i r a t i o n . I would a l s o l i k e to thank the members of my committee f o r prudent ad-v i c e . They were: R.J. Copeman, G.W. Eaton, P.A. J o l l i f f e , V.C. Runeckles, G.H.N. Towers, and P.M. Townsley. Gayle Smith typed the t h e s i s and helped i n other ways; I thank h e r . . . I was supported f o r the l a s t three years w i t h a Leonard S. K l i n c k F e l l o w s h i p awarded by the U n i v e r s i t y of B r i t i s h Columbia. Chapters I I I and.IV have been p r e v i o u s l y published i n the Canadian J o u r n a l of Botany. 1 GENERAL INTRODUCTION The r u s t diseases are perhaps the most d e s t r u c t i v e and important diseases of man's crop plants.. Two important char-a c t e r i s t i c s of pathogenesis by r u s t f u n g i , which c o n t r i b u t e to making.rust diseases so important, are the s p e c i f i c i t y w i t h which races of r u s t s attack v a r i e t i e s of crop, p l a n t s and the h i g h l y evolved o b l i g a t e l y pathogenic nature of the r u s t f u n g i . Rust researchers have made e f f o r t s to under-stand the s p e c i f i c i t y of r u s t by attempting.to e x p l a i n the mechanism of r e s i s t a n c e and s u s c e p t i b i l i t y of host p l a n t s to r u s t s . The question of the o b l i g a t e l y pathogenic nature of r u s t s has been approached by c u l t u r i n g rusts, a x e n i c a l l y apart from c e l l s of the host plant which the r u s t normally . infects... . H i s t o r i c a l Aspects of Rust Chester (6*) began h i s book by summarizing e a r l y accounts of r u s t ; s e v e r a l of these are described here. The o r i g i n of the r u s t fungus, was documented in. Roman f o l k l o r e by Ovid ( 4 3 BC - 1 7 AD). According to the s t o r y . a poor farm couple i n the L a t i n s t a t e of C a r s e o l i had a w i l l -f u l son who caught a fox plu n d e r i n g . h i s chicken yard. The boy wrapped the f o x w i t h straw and hay, set t h i s on f i r e , and turned the f o x l o o s e . As punishment f o r t h i s . s i n , the gods v i s i t e d mankind w i t h r u s t . The Romans prayed and made s a c r i f i c e s to two r u s t gods, Robigus and Robigo. . Rust and 2 r u s t epidemics are a l s o mentioned i n many places i n the B i b l e . I n more modern times Shakespeare r e f e r s to i t i n "King Lear" . and as science and botany were developed many people s t u d i e d and wrote about i t . Chester ( 8 ) quotes Fontana ( 1 ? 6 7 ) as f o l l o w s : " I f the s t a l k and leaves are. attacked by t h i s t e r r i b l e malady, the best set (of k e r n e l s ) , pro-m i s i n g a heavy y i e l d , i s reduced to nothing or almost nothing because such great numbers of the greedy and gluttonous p l a n t s ( r u s t ) absorb n e a r l y a l l the n u t r i t i v e humor of the g r a i n , causing i t to become wasted and consumed because of the l o s s of the n o u r i s h i n g c h y l e . " The h i s t o r i c a l awareness of and concern w i t h r u s t t e s t i f y to the de s t r u c t i v e n e s s and importance of t h i s phyto-pathogen. Time has not lessened the dubious honor long held by r u s t of being the most d e s t r u c t i v e pathogen of man's food p l a n t s . Recent epidemics, of stem r u s t occurred i n 1 9 5 4 i - n North America and i n 1 9 6 4 i n A u s t r a l i a . Severe, outbreaks, of l e a f r u s t are s t i l l common i n the winter wheat growing areas of the United States and other c o u n t r i e s . At the present time coffee r u s t i s epidemic i n L a t i n America causing the d e s t r u c t i o n of many p l a n t a t i o n s . Most of the u s e f u l s c i e n t i f i c research on r u s t s has • been c a r r i e d out i n the l a s t s e v e n t y - f i v e years. E a r l y i n - ' v e s t i g a t i o n s described important aspects of r u s t b i o l o g y , the understanding of which are of paramount importance- i n understanding the h o s t : p a r a s i t e r e l a t i o n s of the r u s t s . The best example of useful' r e s u l t s a c c r u i n g from s t u d i e s 3 of h o s t : p a r a s i t e r e l a t i o n s was the r e c o g n i t i o n of genes f o r r u s t r e s i s t a n c e i n crop p l a n t s . Plant breeders have e f f e c -t i v e l y used t h i s i n f o r m a t i o n to breed r e s i s t a n t v a r i e t i e s . This has r e s u l t e d i n r e d u c t i o n of crop l o s s e s due to r u s t . A d d i t i o n a l i n f o r m a t i o n w i l l c o n t r i b u t e to reducing l o s s e s s t i l l further.. There are two outstanding o b j e c t i v e s of p h y s i o l o g i c a l s t u d i e s on r u s t pathogenesis of host p l a n t s . The f i r s t i s the e l u c i d a t i o n of the biochemical b a s i s of the.genetic spec- -i f i c i t y of r e s i s t a n c e and s u s c e p t i b i l i t y . The second i s to determine the n u t r i t i o n a l requirements f o r r u s t growth. S a t i s f a c t o r y answers to both of these questions are important to the understanding of two f a c t o r s which are par-t i c u l a r l y r e l e v a n t to the d e s t r u c t i v e n e s s of r u s t . These . are the extreme a d a p t a b i l i t y of r u s t s and t h e i r a b i l i t y t o p a r a s i t i z e and destroy l a r g e acreages of crops by. spreading i n an e xplosive manner when the c l i m a t i c c o n d i t i o n s are favourable. Measures used to c o n t r o l and prevent the des-t r u c t i o n by r u s t s i n the f u t u r e w i l l be based on the compre-hension of these f a c t o r s . Genetic s p e c i f i c i t y . o f r u s t p a r a s i t i s m has been shown i n a number of cases to. be extreme. Often one gene i n the fungus determines a v i r u l e n c e or v i r u l e n c e and the c o r r e s - . ponding gene i n the host determines r e s i s t a n c e and suscep-t i b i l i t y . The i n t e r a c t i o n of these genetic f a c t o r s determines whether s u c c e s s f u l establishment of the rust. 4 and pathogenesis of the host occurs or whether the i n t e r a c -t i o n r e s u l t s i n a r e s i s t a n t r e a c t i o n . Although, l i t t l e i s known at the present time about the biochemical- b a s i s of thes genetic i n t e r a c t i o n s , the knowledge of i t i n genetic terms has been instrumental i n the development of r u s t r e s i s t a n t v a r i e -t i e s of a number of crops and i n the p r e d i c t i o n of which v a r i e t i e s are p o t e n t i a l l y v u l n e r a b l e to new races of r u s t . S u s c e p t i b i l i t y and Resistance A number of hypotheses have been advanced to e x p l a i n the mechanism of p l a n t . r e s i s t a n c e and s u s c e p t i b i l i t y to patho gens. These i n c l u d e the n u t r i t i o n a l and the phytoal.exih t h e o r i e s and the h y p e r s e n s i t i v i t y defence r e a c t i o n . Two. others, which are d i f f e r e n t explanations of the s p e c i f i c gene product theory, have a l s o been proposed. They are Stahmann'-s hy b r i d enzyme' theory ( 3 7 ) and Albersheim's theory ( 1 ) . t h a t c e l l w a l l d i g e s t i n g enzymes produced by the fungus are regu-l a t e d by the s p e c i f i c composition and s t r u c t u r e of carbohy-drate polymers making up the host c e l l w a l l . Others ( 3 5 ) have suggested that the " s p e c i f i c gene product" may be RNA or DNA which could be exchanged i n one or both d i r e c t i o n s . • The b a s i s of the n u t r i t i o n a l theory of s u s c e p t i b i l i t y and r e s i s t a n c e i s derived from the basic f a c t that the para-s i t i z e d l e a f i s the substrate upon which the fungus depends f o r n u t r i t i o n . I t i s a simple step to suggest that a sus-c e p t i b l e h o s t : p a r a s i t e combination can e s t a b l i s h a s u i t a b l e metabolic and n u t r i t i o n a l environment wh i l e the r e s i s t a n t combination can not. The a b i l i t y to grow some r u s t s and other c l a s s i c a l l y o b l i g a t e p a r a s i t e s i n axenic c u l t u r e has been argued by some as evidence against t h i s theory. The f a c t s t i l l remains t h a t the i n t e r a c t i o n s of.a fungus w i t h . i t s host may r e g u l a t e f u n g a l metabolism and n u t r i t i o n i n such a way as.to be i n s t r u m e n t a l i n determining s u s c e p t i b i l i t y and • r e s i s t a n c e . The p h y t o a l e x i n theory of r e s i s t a n c e and s u s c e p t i b i l i t y became of considerable i n t e r e s t soon a f t e r the d i s c o v e r y of phytoalexins ( 1 . 3 ) . These compounds ( u s u a l l y phenolics) are i n h i b i t o r y to fungus growth and are o f t e n found i n increased concentrations s h o r t l y a f t e r f u n g a l i n v a s i o n . They are a l -ways found i n a s s o c i a t i o n w i t h n e c r o s i s . The p h y t o a l e x i n theory proposes t h a t during, r e s i s t a n t i n t e r a c t i o n s these compounds i n h i b i t , f u n g a l growth e v e n t u a l l y r e s u l t i n g i n f u n g a l death. I n the s u s c e p t i b l e i n t e r a c t i o n they are degraded i n t o harmless d e r i v a t i v e s . The most important observation argu-i n g against a primary r o l e of phytoalexins i n r e s i s t a n c e i s that t h e i r production i s n o n - s p e c i f i c . The importance of h y p e r s e n s i t i v i t y i n disease r e s i s t a n c e ' and s u s c e p t i b i l i t y has been debated s i n c e Stakman. f i r s t des-. c r i b e d . i t i n 1 9 1 5 . ^Although h y p e r s e n s i t i v i t y i s an i l l -d efined term, i t i s g e n e r a l l y considered to be the r a p i d n e c r o s i s . o f a l i m i t e d number of c e l l s around the s i t e of i n f e c t i o n . The theory proposes that r a p i d host c e l l death 6 i s o l a t e s the i n v a d i n g pathogen and causes i t s death. Several observations have lessened the s i g n i f i c a n c e of t h i s theory. F i r s t , h y p e r s e n s i t i v i t y i s not always associated w i t h r e s i s -tance, and second, i n c i p i e n t i n f e c t i o n s surrounded by necro-s i s f o r as long as twenty days are known to s t i l l be a l i v e . K i r a l y ( 2 6 ) i n a recent a r t i c l e has argued against t h i s , theory. When i t was discovered t h a t there was a complemen-t a r i t y of genes i n the fungus and host which determined r e s i s -tance and s u s c e p t i b i l i t y , one gene determining v i r u l e n c e i n the pathogen and one gene determining r e s i s t a n c e i n the corresponding host ( 2 0 ) , t h e o r i e s proposing s i n g l e gene pro-ducts of .both the fungus and i t s host become important. Stahmann ( 3 7 ) suggested that complementary genes i n the host and pathogen c a r r y i n f o r m a t i o n determining subunit s t r u c t u r e of the same enzyme i n both the host and the pathogen and t h a t these play a primary r o l e i n r e g u l a t i n g metabolism during h o s t : p a r a s i t e i n t e r a c t i o n . Exchange of subunits i n one or . both d i r e c t i o n s would make p o s s i b l e a h y b r i d enzyme w i t h p r o p e r t i e s d i f f e r e n t , from the corresponding enzyme i n e i t h e r the host or p a r a s i t e . Such i n t e r a c t i o n s could occur f o r a number of enzymes at l e a s t as great as the number of major genes c o n t r o l l i n g v i r u l e n c e and r e s i s t a n c e . During the s u s c e p t i b l e i n t e r a c t i o n a l l the h y b r i d en-zymes would c o n t r i b u t e to the r e g u l a t i o n of host metabolism i n a manner advantageous to the fungus. I n the incompatible r e a c t i o n one or more of the hybrid enzymes would cause 7 conditions, u n s u i t a b l e f o r f u n g a l growth, and pathogenesis, thus r e s u l t i n g i n f u n g a l death and hence .resistance. S i m i l a r t h e o r i e s have been proposed i n which i t i s pos-t u l a t e d that DNA or RNA may be t r a n s f e r r e d from the fungus to the host or v i c e versa ( 3 1 , 3 5 ) . Albersheim ( 1 ) has proposed a theory which i n v o l v e s host c e l l w a l l s p e c i f i c i t y . He proposes t h a t the composition and s t r u c t u r e of the host c e l l w a l l carbohydrates determine the pathogen's a b i l i t y to produce enzymes capable of degrading i t . He discusses evidence demonstrating the extreme s p e c i -f i c i t y ' of carbohydrate polymers as antigens as w e l l as c e l l w a l l polymers as s u b s t r a t e s f o r degradative enzymes. The r e g u l a t i o n of the degradative enzymes i s a l s o very s p e c i f i c and s p e c i f i c products -of c e l l w a l l degradation such as d i s -saccharides could e a s i l y be s p e c i f i c e f f e c t o r s and repressors of the f u n g a l degradative enzymes. Albersheim's theory s a t i s f i e s the t h e o r e t i c a l requirements f o r s p e c i f i c gene products from both the fungus and the host, the degradative enzymes of the pathogen and the composition or s t r u c t u r e of the plant c e l l w a l l being the components c o n f e r r i n g the s p e c i f i c i t y . I n Chapter I I experiments undertaken to i n v e s t i g a t e the f u n g a l t o x i n mechanism of s u s c e p t i b i l i t y and r e s i s t a n c e are presented. Scott ( 3 2 ) described the appeal of the f u n g a l t o x i n theory s u c c i n c t l y : " . . . i t i s d i f f i c u l t to escape the c o n c l u s i o n t h a t , compounds are released by the i n v a d i n g pathogen during the e a r l y stages of i n f e c t i o n , e s p e c i a l l y 8 since i t i s w e l l e s t a b l i s h e d that f u n g a l cytoplasm does not come i n t o d i r e c t contact w i t h host cyto-plasm. Such compounds would have to be r e a d i l y d i f f u s i b l e as c e l l s removed from the i n v a d i n g pathogen are a f f e c t e d . Yet these compounds would have to be l a r g e enough to c a r r y s p e c i f i c i t y . . . Now that some r u s t fungi.have been grown a x e n i c a l l y , i t should be p o s s i b l e to search t h e i r c u l t u r e media f o r compounds which w i l l r e a ct s p e c i f i c a l l y w i t h e i t h e r a s u s c e p t i b l e or r e s i s t a n t host." As no s e r i o u s attempts have been made to v e r i f y , or r e j e c t t h i s theory w i t h respect to the r u s t f u n g i I undertook 'to i n v e s t i g a t e t h i s problem. Chapter I describes the i s o l a t i o n of f l a x p r o t o p l a s t s from cotyledons and- t h e i r l o n g e v i t y and development i n c u l t u r e . The i n f o r m a t i o n and experience obtained from these s t u d i e s was used i n planning the experiments c a r r i e d out- i n Chapter I I . Experiments u t i l i z i n g p r o t o p l a s t s to study the t o x i n theory of r e s i s t a n c e and s u s c e p t i b i l i t y are described i n Chapter I I . When t e s t i n g t h e o r i e s designed to e x p l a i n the . biochemical b a s i s of.the genetic, s p e c i f i c i t y of r e s i s t a n c e and s u s c e p t i b i l i t y t e c h n i c a l d i f f i c u l t i e s . a r e encountered. These a r i s e because, at the time when primary events deter-mining s u s c e p t i b i l i t y and r e s i s t a n c e occur, the mass, of f u n g a l t i s s u e i s small compared to the mass of the host. Proto-p l a s t s were used,, i n an i n v i t r o system, so that a l a r g e population.of p r o t o p l a s t s would i n t e r a c t simultaneously w i t h the r u s t fungus., thus p e r m i t t i n g examination of the i n i t i a l responses of the p r o t o p l a s t s to the r u s t . 9 Axenic Culture Scott ( 3 2 ) , Scott and Maclean ( 3 3 ) and Yarwood ( 4 5 ) have reviewed axenic c u l t u r e of r u s t s and other o b l i g a t e phyto-pathogens. Much of the i n f o r m a t i o n i n the f i r s t part of t h i s . i n t r o d u c t i o n was drawn from these sources. Rust f u n g i are among the most advanced f u n g a l pathogens. They are o b l i g a t e p a r a s i t e s and i n nature only grow'on l i v i n g -host p l a n t s although r e c e n t l y s e v e r a l species have been suc-c e s s f u l l y grown In c u l t u r e . Most species of r u s t are only able to p a r a s i t i z e one, or at most, a few c l o s e l y r e l a t e d plant s p e c i e s . They seldom k i l l i n f e c t e d p l a n t s o u t r i g h t . Rusts are able to o b t a i n n u t r i e n t s from the host and produce large•• numbers of urediospores i n a c y c l i c manner w i t h a p e r i o d of only about ten days. This makes a v a i l a b l e a l a r g e inoculum pool and i s i n s t r u m e n t a l i n the r a p i d spread and the 'destruc-t i v e n e s s of r u s t epidemics. S t r i c t l y o b l i g a t e phytopathogenic f u n g i are those species which grow and reproduce i n nature only i n a s s o c i a t i o n w i t h l i v i n g host p l a n t s and cannot be c u l t u r e d a x e n i c a l l y ( 3 2 ) . Yarwood ( 4 5 ) c a l c u l a t e d t h a t f u l l y one quarter of the known plant pathogens are o b l i g a t e p a r a s i t e s and t h a t over 5.000 species of f u n g i are o b l i g a t e l y phytopathogenic .out of a t o t a l of approximately 3 7 , 0 0 0 known species of f u n g i . I n .the.Phycomycetes 1 4 0 o b l i g a t e pathogenic species are found i n the f a m i l i e s Peronosporaceae (downy mildews) and Albuginaceae (white r u s t s ) . Ascomycetes of the Order 1 0 E r y s i p h a l e s (powdery mildews), c o n s i s t i n g of about. 9 0 s p e c i e s , are a l s o o b l i g a t e p a r a s i t e s . The l a r g e s t number i s i n the Order Uredinales ( r u s t s ) which,, c l a s s i c a l l y , was considered to be composed.entirely of o b l i g a t e s . This Order i n the Class . Basidiomycetes i n c l u d e s 4 5 0 0 known s p e c i e s . Because few o b l i g a t e l y phytopathogenic f u n g i can be grown a x e n i c a l l y (growth of organisms of a s i n g l e species i n the absence of l i v i n g organisms or l i v i n g c e l l s of any other species ( 1 8 ) ) , d i f f i c u l t i e s are encountered i n studying host:, p a r a s i t e i n t e r a c t i o n s and mechanisms of r e s i s t a n c e and sus-c e p t i b i l i t y . I f o b l i g a t e f u n g i could be s t u d i e d other than i n the diseased p l a n t , d i f f e r e n t and p o t e n t i a l l y u s e f u l approaches could be taken. Three approaches have been considered to overcome, t h i s problem; f i r s t , studying germinating spores; second, in d u c i n g the fungus to grow out of the diseased p l a n t or i s o l a t i n g i t d i r e c t l y ; t h i r d , development of axenic c u l t u r e techniques using e i t h e r i n f e c t e d host c a l l u s c u l t u r e s or urediospores as inoculum. . The physiology of urediospores has. been reviewed ( 2 , 3 , .9, 1 0 , 2 8 , 3 4 , 3 6 ) and w i l l not be considered i n d e t a i l here. Urediospores, and to a l i m i t e d extent other spore forms ( 3 2 ) , have been used i n s t u d i e s of the development of the fungus, and as a source of f u n g a l t i s s u e separated from the host, f o r metabolic and other s t u d i e s ( 3 4 ) . . 1 1 1 Turel'and Ledingham ( 3 9 ) developed a method;for i n d u c i n g a e r i a l mycelium to grow from i n f e c t e d leaves maintained i n c u l t u r e . -Excised a e r i a l mycelium was used as a source of fungal t i s s u e i n metabolic s t u d i e s ( 2 9 , 4 0 , 4 3 ) . I n t h i s work d i f f i c u l t i e s i n i n t e r p r e t a t i o n of r e s u l t s arose because of the unknown e f f e c t s of e x c i s i o n on f u n g a l metabolism ( 4 3 ) . Another technique f o r . o b t a i n i n g f u n g a l t i s s u e separate from that of the pl a n t was used by Dekhuijzen et a l . (18, 1 9 ) . I n f e c t e d leaves were ground, f i l t e r e d to remove l e a f fragments and the hyphae segments concentrated by s e t t l i n g of the f i l -t r a t e i n sucrose g r a d i e n t s . A major disadvantage of t h i s method, i s the l o s s of c e l l contents from the broken hyphae and the a l t e r a t i o n of metabolism caused by g r i n d i n g . . Chapter I I I describes a method f o r the i s o l a t i o n of i n t a c t f u n g a l c o l o n i e s from i n f e c t e d l e a v e s . This source o f . fung a l m a t e r i a l has d i s t i n c t advantages when compared.to germinated spores., a e r i a l mycelium or. fragments I s o l a t e d by g r i n d i n g . The technique takes advantage of a property of the r u s t not p r e v i o u s l y e x p l o i t e d , the d i f f e r e n t composition of the r u s t c e l l w a l l compared to that of the host. . Enzymes were used which s e l e c t i v e l y d igest the host c e l l w a l l s ; the r e s u l t i n g p r o t o p l a s t s e i t h e r burst or f l o a t away- from the fung a l c o l o n i e s . l e a v i n g them i n t a c t . The technique i s gentle and few host c e l l s remain as contaminants. I t i s p o s s i b l e to o b t a i n s u f f i c i e n t f u n g a l m a t e r i a l f o r many experiments, 1 2 e a s i l y and i n a short time ( 2 gms f r e s h weight of f l a x r u s t fungus r e q u i r e s 1.5 h work). Perhaps the greatest advantage, i s t h a t greater than 9 5 % of the co l o n i e s are v i a b l e a f t e r i s o l a t i o n and washing. This, was demonstrated by us i n g them as.inoculum f o r e s t a b l i s h i n g axenic c u l t u r e s . The disadvantage of the method i s tha t the p o s s i b i l i t y of a l t e r e d metabolism due to the i s o l a t i o n remains. Twenty-seven years ago Hotson and Cutter (23) reported the f i r s t growth i n axenic c u l t u r e of a r u s t . This break-through made axenic c u l t u r e s a p o t e n t i a l source of uncontamin-ated r u s t t i s s u e . Development of techniques subsequent to Hotson and Cutter's work has made t h i s p o t e n t i a l a r e a l i t y f o r a small number of r u s t s p e c i e s . I n comparison w i t h the sources of r u s t m a t e r i a l described above axenic c u l t u r e s have s e v e r a l advantages.. Greater amounts of undisturbed t i s s u e are a v a i l -a b l e . A l s o , s t u d i e s of development past the germling stage are p o s s i b l e . Yarwood ( 4 5 ) i n 1 9 5 6 summed up the " s t a t e of the a r t " s a y i n g , "The Peronosporaceae, the Erysiphaceae, the Ur e d i n a l e s , R h i z e l l a . and Polystigma have s t i l l not been c u l t u r e d but some l i c h e n f u n g i many of the C h y t r i d i a l e s and at l e a s t one species of Rhytisma have si n c e been c u l t u r e d " . Other reviews on axenic c u l t u r e of r u s t s have been w r i t t e n since then by B r i a n ( 6 ) , Scott and Maclean (.33), Scott..(32) and Wolfe ( 4 4 ) . To o b t a i n axenic c u l t u r e s of r u s t s or any other o b l i -gate p a r a s i t e at l e a s t three c o n d i t i o n s must be met: 13 1, s e p a r a t i o n of the r u s t from b a c t e r i a and other contaminants w i t h which they are normally i n clo s e a s s o c i a t i o n i n nature; 2, p r o v i s i o n of a s u i t a b l e and favourable n u t r i t i o n a l environ-ment ; 3 , p r o v i s i o n of a favourable p h y s i c a l environment. Several approaches have been taken to f u l f i l l these r e q u i r e -ments r e s u l t i n g i n s u c c e s s f u l establishment of s e v e r a l r u s t s i n axenic c u l t u r e . Morel ( 3 0 ) f i r s t suggested u s i n g 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 c a l l u s as inoculum to i n i t i a t e r u s t axenic . cultures.. This was the technique used by Hotson and Cutter ( 2 3 ) to grow Gymnosporangium j u n i p e r i - v i r g i n i a n a e . The method was tedious and the. success r a t e low. I n one s e r i e s of experiments, 8 , 8 4 0 t e l i a l g a l l s on j u n i p e r were c o l l e c t e d and 1 3 , 5 0 4 c a l l u s c u l t u r e s i n i t i a t e d from them. Of these 3 5 8 were found to be s y s t e m i c a l l y i n f e c t e d w i t h r u s t . A f t e r 4 t o 9 months one of the primary c u l t u r e s and 6 of the many subcultures became n e c r o t i c and r u s t grew out of t h e . c a l l u s onto the medium, subsequently developing i n t o an axenic c u l t u r e ( 1 5 ) . Hotson and Cutter used Gautheret's n u t r i e n t no. 4 plant t i s s u e c u l -t u r e medium modified by the a d d i t i o n of 3 % dextrose and 5 0 0 ppm a s c o r b i c a c i d . L a t e r u s i n g the same technique Hotson and Cutter obtained 5 c u l t u r e s of the ru s t Uromyces a r i -t r i p h y l l i ( 1 5 , 1 6 ) and one of P u c c i n i a malvacearum (reported i n a posthumous paper examined by Scott and Maclean). A l -though others have not been able to repeat Cutter's work, i t i s respected, and these three r u s t s are considered to be 1 4 the f i r s t which were c u l t u r e d a x e n i c a l l y . Cronartium fusiforme has a l s o been grown usi n g the i n -fe c t e d host t i s s u e c u l t u r e method ( 2 2 ) . To i n i t i a t e i n f e c t e d plant t i s s u e c u l t u r e s 2 0 0 g a l l segments,were used. Mycelium . grew out of one of these and the r u s t was subsequently es-t a b l i s h e d i n axenic c u l t u r e . A t o t a l of 2 4 weeks was r e q u i r e d from c u l t u r i n g the g a l l s u n t i l establishment of the axenic c u l t u r e s . Aeciospores were produced i n c u l t u r e . Harvey and Grasham. ( 2 1 ) have grown Cronartium r i b i c o l a i n axenic c u l t u r e u s i n g the i n f e c t e d t i s s u e c u l t u r e method.. This r u s t was grown both on a defined medium c o n t a i n i n g g l u t a t h i o n e and c y s t e i n e and a medium c o n t a i n i n g yeast e x t r a c t , peptone and bovine albumin. S p o r e - l i k e bodies formed r e g u l a r l y i n some c u l t u r e s and p a t h o g e n i c i t y of the c u l t u r e s was demonstrated. The approach, other than use of i n f e c t e d t i s s u e c u l -t u r e s , has been to use urediospores as inoculum. . The f i r s t , success w i t h t h i s method was reported i n 1 9 6 6 by W i l l i a m s et a l . ( 4 1 ) w i t h P u c c i n i a graminis t r i t i c i race ANZ 1 2 6 - 6 , 7 , an A u s t r a l i a n i s o l a t e of stem r u s t of wheat. I n t h i s paper, no spores were reported to be produced i n c u l t u r e . . Axenic growth was confined to c o l o n i e s a maximum of 1 mm i n diameter and no claims were made of being able to subculture the r u s t by s e r i a l t r a n s f e r . L a t e r ( 4 2 ) p a t h o g e n i c i t y of the c u l t u r e s was demonstrated and spores were produced i n c u l t u r e . The . f i r s t mention of successful, s e r i a l subculture of A u s t r a l i a n stem r u s t c u l t u r e s was. i n 1 9 6 9 ( 3 3 ) . The medium upon which 1 5 t h i s r u s t grew contained Czapek's m i n e r a l s , sucrose, 0.1% yeast e x t r a c t and 0.1% Evans' peptone. Subsequent work by Bushnell ( 7 ) confirmed the success of W i l l i a m s et a l . ( 4 1 , 4 2 ) . , I n a comparative study of c e r e a l rusts i n v o l v i n g P u c c i n i a  graminis t r i t i c i , P. graminis avenae, P. graminis s e c a l i s , P. s o r g h i , P. coronata avenae and P. r e c o n d i t a t r i t i c i a , Kuhl et_ a l . ( 2 7 ) found that only P. graminis avenae grew, apart from the A u s t r a l i a n stem r u s t i s o l a t e p r e v i o u s l y c u l t u r e d . Growth was sporadic and v a r i a b l e but some spores were produced a f t e r several, weeks. No attempts were made to s e r i a l l y subculture P u c c i n i a graminis avenae. Soon.after W i l l i a m s f i r s t r e p o r t , T u r e l (38) success- ' f u l l y . g r e w Melampsora l i n i . ( f l a x r u s t ) i n axenic c u l t u r e from urediospores. M y c e l i a l growth developed i n 20 to 30%.of the c u l t u r e tubes which were seeded. Several c u l t u r e s were r e -ported to have survived • successive t r a n s f e r s over a p e r i o d of 5. months. Although spores were produced i n c u l t u r e , many of them were i r r e g u l a r l y shaped. The medium used contained Knop's m i n e r a l s , B e r t h e l o t ' s . t r a c e elements, sucrose, and 0.1% yeast e x t r a c t . Turel's work was confirmed by Coffey, Bose and Shaw ( 1 1 ) . Uromyces d i a n t h i ( c a r n a t i o n r u s t ) was c u l t u r e d by Jones ( 2 4 ) from urediospores. About 6 5 % of the i n o c u l a t i o n s de-veloped i n t o m y c e l i a l growths. At f i r s t growth stopped a f t e r 1 6 2-3months, but l a t e r t h i s d i d not occur. . To date, pathogen-i c i t y of axenic c u l t u r e s of t h i s r u s t has not been demon-•strated nor were spores produced i n c u l t u r e . The medium used contained Czapek Dox Broth, 0.2%, peptone and 0.2% yeast ex- . t r a c t . S e r i a l subcultures of Uromyces d i a n t h i were used sub-sequently, i n a n u t r i t i o n a l study ( 2 4 ) . Coffey and A l l e n ( 1 2 ) used urediospores to i n i t i a t e axenic c u l t u r e s of P u c c i n i a h e l i a n t h i (snap dragon r u s t ) . When c o n d i t i o n s were o p t i m a l , axenic c u l t u r e s developed i n 80-100% of f l a s k s . Colonies, reached a diameter of 1 2 mm a f t e r 25 weeks and could be s e r i a l l y suhcultured a f t e r 7 to. 1 4 weeks. This o p e r a t i o n was more s u c c e s s f u l l y done as the c o l o n i e s be-came o l d e r , presumably because they became adapted to growth i n v i t r o . P a t h o g e n i c i t y of these c u l t u r e s has not been demon-s t r a t e d but some t h i c k w a l l e d t e l i o s p o r e s were produced i n v i t r o . Growth occurred i n a medium c o n t a i n i n g the same minerals used s u c c e s s f u l l y f o r f l a x r u s t ( 3 6 ) , plus sucrose, d e f a t t e d bovine serum albumin and e i t h e r Evans' peptone, tryptone or casamino a c i d s . The requirements f o r bovine serum albumin ( 1 . 5 - 2 . 0 % ) was absolute but two amino a c i d s , glutamic a c i d , a s p a r t i c a c i d or al a n i n e i n combination w i t h c y s t e i n e chould' be s u b s t i t u t e d f o r the peptone, tryptone or casamino a c i d s . Two recent advances of considerable importance to the f i e l d of axenic c u l t u r e are the development of two defined 17 media, one s u i t a b l e f o r the growth of f l a x rust, and the other f o r wheat r u s t ( 4 ) , and the a b i l i t y to induce s p o r u l a t i o n of f l a x r u s t (race 3 ) by i n c l u d i n g amino a c i d s ( a s p a r t i c or glutamic acid) plus calcium i n the medium ( 5 ) . Because ur e d i o -spores and t e l i o s p o r e s are both produced i n c u l t u r e , the technique i s . p a r t i c u l a r l y v a l u a b l e f o r studying r u s t g e n e t i c s . Section I I of t h i s t h e s i s describes.two new approaches f o r the i n i t i a t i o n of axenic c u l t u r e s which avoid some of. the d i f f i c u l t i e s and l i m i t a t i o n s encountered when usi n g i n f e c t e d host t i s s u e c u l t u r e s or uhcontaminated urediospores as i n o c u -lum. Kuhl et a l . (27) d i s c u s s the d i f f i c u l t i e s of i n d u c i n g urediospores to grow past the s p o r e l i n g stage and become vegetat i v e c o l o n i e s . Concerning t h i s . t h e y wrote: . "...between germ tube el o n g a t i o n and the forma- . . t i o n of saprophytic hyphae a change occurs i n the expression of the r u s t genome w i t h consequent r e o r i e n t a t i o n of the r u s t metabolism. Of spore-l i n g s that do i n i t i a t e saprophytic growth only a small number s u r v i v e to form vigorous macro-s c o p i c a l l y v i s i b l e c o l o n i e s . This could be due to the extent to which developing hyphae adapt their-metabolism to the needs imposed by the a r t i f i c i a l medium. We take the view that e r r a t i c growth encountered w i t h present methods i s p a r t l y , due to the l a c k of c o n t r o l over the t r a n s i t i o n process,. • Thus a random event determines whether the t r a n s i t i o n does or does not take.place". By o b t a i n i n g m y c e l i a l inoculum from n a t u r a l l y i n f e c t e d l e a v e s , the t r a n s i t i o n from s p o r e l i n g to v e g e t a t i v e growth could be by-passed. As a r e s u l t , v a r i a b i l i t y would be decreased and c u l t u r e s e s t a b l i s h e d more e a s i l y and i t would perhaps be p o s s i b l e to grow species of r u s t s which have not been 1 8 s u c c e s s f u l l y grown before. , The i n i t i a l o b j e c t i v e of the work described i n Chapter I I I . was to devise a method f o r se p a r a t i n g the r u s t fungus t i s s u e from the i n f e c t e d l e a f . As w e l l as being a source of uncontaminated fung a l t i s s u e , c o l o n i e s i s o l a t e d from c o t y l e -dons by s e l e c t i v e enzymatic h y d r o l y s i s of host c e l l w a l l s were used as inoculum f o r e s t a b l i s h i n g axenic c u l t u r e s of t h e . f l a x r u s t fungus Chapter IV describes a method of e s t a b l i s h i n g axenic c u l t u r e s which has the advantages o f , and i s based on, the method described i n Chapter I I I . I t does not, however, r e -quire the use of enzymatic d i g e s t i o n . This method was used to e s t a b l i s h axenic c u l t u r e s of poplar r u s t f o r the f i r s t time. 19 REFERENCES 1. Albersheim, P., T.M. Jones and P.D. E n g l i s h . 1969. B i o -chemistry of the c e l l w a l l i n r e l a t i o n to i n f e c t i o n pro-cesses. Ann. Rev. Phytopathology 7: 171-194. 2 . A l l e n , P.J. 1965. Metabolic aspects of spore germination i n f u n g i . Ann. Rev. 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M o b i l i z a t i o n • f a c t o r s i n urediospores and bean leaves i n f e c t e d w i t h bean r u s t fungus. C o n t r i b . Boyce Thompson Inst.. 2 4 : 3 9 - 5 2 . 2 0 . F l o r , H.H. 1 9 7 L Current s t a t u s of the gene-for-gene . concept. Ann. Rev. Phytopathology 9: 2 7 5 - 2 9 6 . 2 1 . Harvey, A.E., and J.L. Grasham. 1 9 7 4 . Axenic c u l t u r e of the. mononucleate stages.of Cronartium r i b i c o l a . Phyto-pathology 6 4 : 1 0 2 8 - 1 0 3 5 . 2 2 . H o l l i s , C.A., R.A. Schmidt, and J.W. Kimbrough. 1 9 7 2 . . Axenic c u l t u r e of Cronartium f u s i f o r m e . Phytopathology 6 2 : 1 4 1 7 - 1 4 1 9 . 2 3 . Hotson, H.H., and V.M. Cu t t e r , J r . 1.951... The i s o l a t i o n and c u l t u r e of Gymnosporangium j u n i p e r i - v i r g i n i a n a e Schw. ... Proc. Nat. Acad. S c i . U.S.A. 3 7 : 4 0 0 - 4 0 3 . 2 4 . Jones, D.R. 1 9 7 2 . I n v i t r o c u l t u r e of ca r n a t i o n r u s t Uromyces d i a n t h i . Trans. B r i t . Mycol. 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Conversion to s o l u b l e products. Can. J . Bot. 4 6 : 4 3 5 - 4 6 0 . 3 0 . M o r e l ? G. 1 9 4 8 . Recherches sur l a c u l t u r e associee de p a r a s i t e s o b l i g a t o i r e s et de t i s s u e s vegetaux. Ann. Ep i p h y t i e s ( I I ) 1 4 : 1 - 1 1 2 . 3 1 . Rohringer ? R., N.K. Howes, W.K. Kim and D.J. Samborski. 1 9 7 4 . Evidence f o r a g e n e - s p e c i f i c RNA determining r e -s i s t a n c e i n wheat to stem r u s t . Nature 2 4 9 : 5 8 5 - 5 8 8 . 3 2 . S c o t t , K.J. 1972. Ob 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. 4 7 : 5 3 7 - 5 7 2 . 3 3 . S c o t t , K.J., and D.J. Maclean. 1 9 6 9 . C u l t u r i n g of r u s t f u n g i . Ann. Rev. Phytopathology 6: 1 2 3 - 1 4 6 . 3 4 . Shaw, M. 1 9 6 4 . The physiology of r u s t urediospores. Phytopath. Z. 5 0 : 1 5 9 - 1 8 0 . 3 5 . Shaw, M. 1 9 6 7 . The physiology and host p a r a s i t e r e l a -t i o n s of the r u s t s . Ann. Rev. Phytopathology 1: 2 5 9 - 2 9 4 . 3 6 . S t a p l e s , R.C. and W.K. Wynn. 1 9 6 5 . The physiology of urediospores of the r u s t f u n g i . Bot. Rev. 31: 5 3 7 - 5 6 4 . 3 7 . Stahmann, M.A., W. Woodbury, L. Lovrekovich and V. Macko. 1 9 6 8 . The r o l e of enzymes i n the r e g u l a t i o n , of disease r e s i s t a n c e and host-pathogen s p e c i f i c i t y . I n Biochemical Regulation i n Diseased P l a n t s or I n j u r y , pp. 2 6 4 - 2 7 4 . T. H i r a i (Ed.). Phytopath. Soc. Japan, Tokyo. 3 8 . T u r e l , F.L.M. 1 9 6 9 . Saprophytic development of the f l a x r u s t Melampsora l i n i , race no. 3 . Can. J . Bot. 4 7 : 8 2 1 -8 2 3 . 22 3 9 . T u r e l , F.L.M. and G.A. Ledingham. 1 9 5 7 . Production of a e r i a l mycelium and urediospores by Melampsora l i n i (Pers.) Lev on f l a x leaves 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: 8 1 3 - 8 1 9 . 4 0 . T u r e l , F.L.M. and G.A. Ledingham. 1 9 5 9 . U t i l i z a t i o n of l a b e l l e d substrates by the mycelium and urediospores of the f l a x r u s t fungus. Can. J . M i c r o b i o l . 5: 5 3 7 - 5 4 5 . 4 1 . W i l l i a m s , P.G., K.J. Scott and J.L. Kuhl. 1 9 6 6 . Vegeta-t i v e growth of P u c c i n i a graminis f . sp. t r i t i c i i n v i t r o . Phytopathology 5 6 : 1 4 1 5 - 1 4 1 9 . 4 2 . W i l l i a m s , P.G., K.J. S c o t t , J.L. Kuhl and D.J. Maclean. 1 9 6 7 . 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 graminis f. sp. t r i t i c i grown on a r t i f i c i a l medium. Phytopathology 5 7 : 3 2 6 - 3 2 7 . 4 3 . W i l l i a m s , P.G., and M. Shaw. 1 9 6 8 . Metabolism of glucose-C"^, pyruvate-C"*"^, and mannitol-C"^ by Melampsora l i n i . I . Uptake. Can. J . Bot. 4 6 : 4 3 5 - 4 4 0 . 4 4 . Wolf, F.T. 1 9 7 4 . The c u l t i v a t i o n of r u s t f u n g i upon a r t i f i c i a l media. Can. J . Bot. 5 2 : 767-772. 4 5 . Yarwood, C.E. 1 9 5 6 . Obligate p a r a s i t i s m . - Ann. Rev. Plant Physiology 7: 1 1 5 - 1 4 2 . SECTION I EXPERIMENTS USING PROTOPLASTS TO STUDY HOST:PARASITE INTERACTIONS 2 4 Chapter I Development and D i v i s i o n of P r o t o p l a s t s from F l a x Cotyledons INTRODUCTION I s o l a t e d or naked p r o t o p l a s t s , as they are sometimes c a l l e d , are c e l l s from which the c e l l w a l l has been removed. U s u a l l y , removal i s by d i g e s t i o n w i t h enzymes although mech-a n i c a l methods can a l s o be used. Once the c o n s t r a i n t s of the c e l l w a l l disappear, the p r o t o p l a s t must be o s m o t i c a l l y stab-i l i z e d i n s o l u t i o n to prevent death r e s u l t i n g from e i t h e r burst-i n g or c o n t r a c t i o n . U n l i k e normal p l a n t ' c e l l s , p r o t o p l a s t s are s p h e r i c a l . Each p r o t o p l a s t i s independent of others and they are not connected by plasmadesmata. Although p r o t o p l a s t suspensions can be c e n t r i f u g e d at low speed and p i p e t t e d , they must be handled g e n t l y because they are extremely f r a g i l e and are. e a s i l y • b u r s t . .Plant p r o t o p l a s t s have been shown capable, of t a k i n g up macromolecules, organelles.and b a c t e r i a ( 1 , 8 , 1 5 , 1 7 , 1 $ , 1 9 , 3 0 ) , and have, been used to o b t a i n parasexual hybrids (5.) and disease r e s i s t a n t mutants ( 4 ) . Their i s o l a t i o n and develop-ment has been reviewed by Cocking ( 6 ) . Great i n t e r e s t has • been shown i n the use of plant p r o t o p l a s t s i n new techniques of crop improvement. Gamborg ( 1 1 ) has l i s t e d 9 species i n . 25 which p r o t o p l a s t s prepared from.cultured c e l l s have been . shown to-be capable of sustained c e l l d i v i s i o n . 'Thus f a r , proto p l a s t s , derived from i n t a c t . p l a n t s of three species have been shown to be capable of sustained d i v i s i o n , tobacco ( 1 9 , 2 7 ) , petunia ( 2 , 1 0 ) , and pea ( 7 ) ; but only w i t h tobacco have .complete p l a n t s been grown from p r o t o p l a s t s i s o -l a t e d d i r e c t l y from le a v e s . Carlson ( 3 ) has pointed out that the use of p r o t o p l a s t s derived d i r e c t l y from i n t a c t p l a n t s avoids the problem of genetic v a r i a b i l i t y inherent i n the use of p r o t o p l a s t preparations derived from c e l l c u l -t u r e s . I t i s t h e r e f o r e important to attempt to increase the number.of species from which p r o t o p l a s t s derived d i r e c t l y from i n t a c t p l a n t s can be induced to d i v i d e i n c u l t u r e and u l t i m a t e l y to grow i n t o complete p l a n t s . This chapter des-c r i b e s attempts to grow p r o t o p l a s t s derived d i r e c t l y from f u l l y expanded f l a x cotyledons. MATERIALS AND METHODS F u l l y expanded cotyledons were excised from f l a x seed-lings. (Linum u s i t a t i s s i m u m L., v a r i e t i e s Bison and Bombay) a f t e r 2 to 4 leaves had developed (17°C, 2 5 6 0 l u x ) . They ' were washed i n d i s t i l l e d water f o r 2 0 minutes, i n 7 0 % ethanol f o r 1 minute, 1% sodium h y p o c h l o r i t e f o r 2 0 minutes and then i n s t e r i l e d i s t i l l e d water u n t i l used (maximum 2 0 minutes). The a b a x i a l epidermis was removed and about 26 15 cotyledons incubated i n 2 ml of enzyme s o l u t i o n f o r 18 h at 22 °C. The enzyme s o l u t i o n contained. B5 medium ( 1 2 ) minus sucrose and hormones, 0.05 M glucose, 0.3 M s o r b i t o l plus 2 . 5 % c e l l u l a s e (Onozuka P - 5 0 0 0 ) , 2 . 5 % mascerozyme (both from A l l Japan B i o c h e m i c a l s ) , . 0 . 5 % pectinase (Sigma) and 1 0 0 u n i t s of s t r e p t o m y c i n / p e n i c i l l i n mixture. A f t e r d i g e s t i o n , v e s s e l elements and epidermis were removed and the proto-p l a s t s washed f r e e of enzyme by f i l t r a t i o n ( 1 3 ) . The wash medium contained .B5 medium minus sucrose,. 0.05 M glucose, 0.4 M s o r b i t o l , plus auxin or c y t o k i n i n (see r e s u l t s ) de-pending on the treatment. The washed p r o t o p l a s t s ( 5 - 1 0 ml) were then e i t h e r mixed w i t h an equal volume.of wash medium plus agar ( f i n a l agar concentration 0 . 5 % ) and spread i n a t h i n f i l m ( 0 . 5 mm) i n a p e t r i d i s h ( 3 5 n™) or w i t h an equal volume of wash medium without agar and incubated, as 0.1 ml d r o p l e t s i n p e t r i d i s h e s . The f i n a l c o n c e n t r a t i o n of proto-p l a s t s was 1 0 ^ / m l . i n both treatments. The p e t r i . d i s h e s were then sealed w i t h P a r a f i l m and kept at high, humidity i n a p l a s t i c box at 22°C. RESULTS AND DISCUSSION D i g e s t i o n at 3.7 °C decreased the time r e q u i r e d f o r proto-p l a s t i s o l a t i o n to about eight hours b u t . r e s u l t e d i n a smaller proportion.of l i v i n g p r o t o p l a s t s ( 4 0 % ) than d i g e s t i o n at 22°C (75-80%).. S a t i s f a c t o r y p r o t o p l a s t production occurred i n ' 27 d i g e s t i o n media ranging i n con c e n t r a t i o n from 0.15 M to.0.7 M ( s o r b i t o l plus glucose).. Adjustment of the "concentration of . the wash medium to 0.1 M greater than that of t h e . d i g e s t i o n medium increased the percentage of l i v i n g p r o t o p l a s t s a f t e r . washing. Although there are r e p o r t s of d e l e t e r i o u s e f f e c t s from prolonged exposure of p r o t o p l a s t s to the d i g e s t i v e en-zymes we d i d not observe t h i s e f f e c t . Even.after one month, p r o t o p l a s t s which had been l e f t i n the d i g e s t i o n medium were streaming v i g o r o u s l y . In b o t h . l i q u i d and agar treatments 75 to 80% of the p r o t o p l a s t s were i n t a c t at the beginning of i n c u b a t i o n . Immediately a f t e r i s o l a t i o n the c h l o r o p l a s t s were oft e n con-centrated i n the v i c i n i t y of the nucleus but were always at the periphery of the s p h e r i c a l p r o t o p l a s t s ( F i g . 1-1) . Some of the l a r g e r , chloro.plast-free p r o t o p l a s t s presumably o r i g i n a t e d from epidermal c e l l s (see r e f . 9). By day two, 15% of the p r o t o p l a s t s had d i e d . The remainder doubled i n s i z e , t h e i r c h l o r o p l a s t s becoming evenly d i s t r i b u t e d about, the. periphery of each c e l l and cytoplasmic strands and streaming becoming v i s i b l e . By day four many p r o t o p l a s t s had become e l l i p t i c a l or egg-shaped and "budding" ( F i g . 1-3) and d i v i s i o n ( F i g . 1-2) were f i r s t observed. The percentage of d i v i d e d c e l l s increased to a maximum frequency of 0.4%. on day 20. Both the c e l l s r e s u l t i n g from d i v i s i o n s con-t a i n e d c h l o r o p l a s t s , which o f t e n became concentrated near the cross" w a l l . Although c y c l o s i s was observed i n b o t h ' c e l l s 28 Figure I--1. F r e s h l y i s o l a t e d p r o t o p l a s t s . Day 1. X 260. Figure I--2. Divided p r o t o p l a s t . Day 5. X500. Figure I: -3 . Budding p r o t o p l a s t . Day 7. x.500. Figure. I -4. E x t r u s i o n of cytoplasm, type I . . Day 15 . X600. Figure I -5.' E x t r u s i o n of cytoplasm, type I I . Day 15 . X525. Figure I. -6.. Divided giant c e l l . Day 28. X430. Figure I -7. B i n u c l e a t e giant c e l l . Day 28. X260. Figure I -8. . T y p i c a l g i a n t c e l l . Day 28. X260. 29 30 a f t e r d i v i s i o n no m u l t i c e l l u l a r aggregates were observed. Budding was observed w i t h a frequency of'1% i n agar c u l t u r e s and 15% i n l i q u i d c u l t u r e s . E x t r u s i o n of p r o t o p l a s t conents, a process a k i n to budding, was common i n agar c u l t u r e s . Two types of e x t r u s i o n occurred ( F i g s . .1-4 & 1-5), the f i r s t , type occurred about day 5. The extruded m a t e r i a l was enclosed by a membrane and sometimes contained c h l o r o p l a s t s . I n the second type, which g r a d u a l l y increased i n frequency beginning on day 5, the extruded m a t e r i a l was l e s s organized and. o f t e n o r i g i n a t e d from more than one area on the p r o t o p l a s t s u r f a c e . About 5%.of the p r o t o p l a s t s began a second i n c r e a s e i n size, about day 20, by which time the c h l o r o p l a s t s had degen-erated. Many of these, p r o t o p l a s t s grew to ten times the diameter of f r e s h l y i s o l a t e d p r o t o p l a s t s , contained enlarged n u c l e i and e x h i b i t e d vigorous cytoplasmic streaming. D i v i s i o n of these c e l l s was al s o observed ( F i g . 1-6). Death of the c e l l s o r i g i n a t i n g from p r o t o p l a s t s occurred g r a d u a l l y from day .10 onwards and by day 40 n e a r l y a l l were dead A number of c o n d i t i o n s found to be s u i t a b l e f o r develop-ment and d i v i s i o n of p r o t o p l a s t s from other species were un-s u i t a b l e f o r f l a x . I ncubation i n the l i g h t caused r a p i d death as did a medium s u c c e s s f u l l y used w i t h tobacco (14). No d i f f e r e n c e s were observed between media which contained sucrose, glucose or r i b o s e alone or i n combination. Combin-at i o n s of 2,4-dichlorophenoxyacetic a c i d , naphthalene a c e t i c 31 a c i d , k i n e t i n or benzyl amino purine ranging i n concent r a t i o n from 0.1 to 10 mg/l had no n o t i c e a b l e e f f e c t s . V a r i a t i o n i n the conc e n t r a t i o n of B 5 medium from 0.1 to f u l l s t r e n g t h , use of cotyledons of d i f f e r e n t ages, use of mannitol e i t h e r . alone o r - i n combination w i t h s o r b i t o l , and v a r i a t i o n of the m o l a r i t y of the media a l s o had no n o t i c e a b l e e f f e c t on proto-p l a s t behaviour.. The medium s u c c e s s f u l l y used f o r h a p l o i d petunia p r o t o p l a s t s by Binding (2) promoted the growth of increased numbers of giant c e l l s (Figs.'. 1-7 j 1 -8) but d i d not increase the percentage of p r o t o p l a s t s which d i v i d e d . I f the c o n c e n t r a t i o n of p r o t o p l a s t s was l e s s than I x 10^/ml • they f a i l e d to develop and died w i t h i n a week; i f greater than 1 x 10^/ml, budding i n l i q u i d c u l t u r e s and e x t r u s i o n , of c e l l contents were increased s e v e r a l f o l d . . The responses of the two v a r i e t i e s Bison and Bombay to the var i o u s t r e a t -ments was i d e n t i c a l . Although i t was not p o s s i b l e to induce m u l t i p l e c e l l d i v i s i o n s and the formation of c a l l u s from c u l t u r e d f l a x p r o t o p l a s t s , 0.1+% d i v i d e d once. This r a t e of d i v i s i o n i s comparable to. that obtained by Gonstabel et a l . (7) w i t h p r o t o p l a s t s i s o l a t e d from pea stem t i p s . Both w i t h f l a x and pea.protoplasts the r a t e of d i v i s i o n i s much lower than has been achieved w i t h tobacco p r o t o p l a s t s . Takebe et a l . (21) were able t o induce d i v i s i o n i n 50% of t h e i r tobacco p r o t o p l a s t s . 32 Flax; cotyledons were -found to be a good t i s s u e from which to i s o l a t e p r o t o p l a s t s . Nearly 100% of the cotyledon c e l l s were converted to p r o t o p l a s t s and they were e a s i l y washed and separated from undigested d e b r i s . Some proto-p l a s t s were k i l l e d d u r i n g the i s o l a t i o n and washing proce- -dure, however, preparations c o n t a i n i n g 75% to 80% l i v i n g and healthy p r o t o p l a s t s were r o u t i n e l y prepared. 33 REFERENCES 1. Aoki, S., and I . Takebe. 1969. I n f e c t i o n of tobacco mesophyll p r o t o p l a s t s by tobacco mosaic v i r u s r i b o -n u c l e i c a c i d . V i r o l o g y 39: 439-449. 2. Binding, H. 1974.. C e l l c l u s t e r formation by l e a f • pr o t o p l a s t s from axenic c u l t u r e s of ha p l o i d Petunia  hybrida L. Plant Sciences L e t t e r s Z: 3. Carlson, P.S. 1973. The use of p r o t o p l a s t s f o r genetic research. Proc. Nat. Acad. S c i . (U.S.A.) 70: 598-602. 4. . Carlson, P.S. 1973. Methionine s u l f o x i m i n e - r e s i s t a n t mutants of tobacco. Science 180: 1366-1368. 5. Carlson,. P.S.-, H.H. Smith and R.D. Dearing. 1972. Para-sexual i n t e r s p e c i f i c plant h y b r i d i z a t i o n . Proc. Nat. Acad. S c i . (U.S.A.) 69: 2292-2294. 6. Cocking, E.C. 1972. Plant c e l l p r o t o p l a s t s - i s o l a t i o n and development. Ann. Rev. Plant Physiology 23: 24-50. 7. Constabel, F.f J.¥. K i r k p a t r i c k and O.L. Gamborg. 1973. C a l l u s formation from mesophyll p r o t o p l a s t s of Pisum  sativum. Can. J.. Bot. 51: 2105-2106. 8. Davey, M.R.,and E.C. Cocking. 1972. Uptake of b a c t e r i a by i s o l a t e d higher plant p r o t o p l a s t s . Nature 239: 455-456.. 9. Davey, M.R., E.M. Frearson, L.A. Withers and J..B. Powers. 1973 . Observations., on the morphology, u l t r a s t r u c t u r e and regeneration of tobacco l e a f epidermal- p r o t o p l a s t s . Plant Science L e t t e r s 2: 23-27. 10.. Durant, J . , I . Potrykus and G. Donn. .1973. Plantes i s s u e s de pr o t o p l a s t e s de Petunia. Z. P f l a n z e n p h y s i o l . 69: 26-34. 11. Gamborg, O.L. and R.A. M i l l e r . 1973... I s o l a t i o n , c u l t u r e and uses of plant protoplasts'. Can. J . Bot. 51: 1795-1799 12. Gamborg, O.L., R.A. M i l l e r and K. Ojima. 1968. N u t r i e n t • requirements of suspension, c u l t u r e s of soybean root c e l l s . Expt.- C e l l Res. .50: 151^158. 13. .Kao K.N. 'O.L. Gamborg, R.A. M i l l e r and. W.A. K e l l e r . 1971... C e l l d i v i s i o n , i n c e l l s regenerated from p r o t o p l a s t s of soybean and Haplopappus g r a c i l i s . Nature 232: 124. 34 14. Nagata, T., and I . Takebe. 1971. . P l a t i n g of i s o l a t e d .. mesophyll p r o t o p l a s t s on agar medium. Pla n t a 99: 12-20. 15. Ohyama, K., O.L. Gamborg and R. A. M i l l e r . 1972. Uptake of exogenous DNA by plan t p r o t o p l a s t s . Can. J . Bot. 50: 2077-2080. • . • •16. Ohyama, K., and J.P. N i t s c h . 1972. Flowering h a p l o i d . p l a n t s obtained from p r o t o p l a s t s of tobacco l e a v e s . Plant and C e l l P h y s i o l . 13: 229-236. 17. O t s u k i , Y. and I . Takebe. 1973. I n f e c t i o n of tobacco mesophyll p r o t o p l a s t s by cucumber mosaic v i r u s . V i r o l o g y 52.: 433-438. 18. O t s u k i , Y., T. Shimomma, and L. Takebe.: 1972. Tobacco mosaic v i r u s m u l t i p l i c a t i o n and expression of the N gene i n n e c r o t i c responding tobacco v a r i e t i e s . V i r o l o g y 50: 45-50. .19. . Potrykus, I . 1973. T r a n s p l a n t a t i o n of c h l o r o p l a s t s i n t o p r o t o p l a s t s of petunia. Z. f u r P f l a n z e n p h y s l o l o g i e 70: . 364-366. 20. Potrykus, I . and F. Hoffmann. 1973. T r a n s p l a n t a t i o n of n u c l e i c acids i n t o p r o t o p l a s t s of higher p l a n t s , Z. Pfl a n z e n p h y s i o l o g i e 69: 287-289. 21. Takebe, I . , G. Labib, and G. Melchers. 1971. Regenera-'. t i o n of whole p l a n t s from i s o l a t e d mesophyll p r o t o p l a s t s of tobacco. Naturwissenschaften 5$: 318-3.20,. 3 5 Chapter I I Experiments u s i n g a P r o t o p l a s t Bioassay . to Study Flax:Flax-Rust I n t e r a c t i o n INTRODUCTION The extremely d e l i c a t e nature of p l a n t c e l l p r o t o p l a s t s renders them p a r t i c u l a r l y s e n s i t i v e to chemical s t i m u l i or changes i n t h e i r environment. The absence of a c e l l w a l l allows r e a d i l y v i s i b l e expansion and b u r s t i n g of the proto-p l a s t t o occur when p r o t o p l a s t i n t e g r i t y i s d i s r u p t e d by l o s s of f u n c t i o n or s t r u c t u r e of the plasmalemma, by incr e a s e s i n c e l l volume, or by d i s r u p t i o n s w i t h i n the c e l l such as d i s -i n t e g r a t i o n of vacuoles. Thus t o x i n s or other compounds which i n f l u e n c e p r o t o p l a s t i n t e g r i t y and f u n c t i o n can be detected by a bioassay u s i n g p r o t o p l a s t s . The b u r s t i n g response of p r o t o p l a s t s has been used as the b a s i s of bioassays before. Rueslnk (1.9, 2 0 ) incubated p r o t o p l a s t s derived from Avena c o l e o p t i l e s . w i t h a number of compounds i n an i n v e s t i g a t i o n of plasmalemma s t r u c t u r e . Avena p r o t o p l a s t s burst when incubated i n s o l u t i o n s c o n t a i n -i n g a n i o n i c detergents (sodium dodecyl s u l f a t e , and t a u r o -c h o l i c acid) a c a t i o n i c detergent (hexadecyl t r i m e t h y l ammonium bromide).and a non-ionic detergent ( T r i t o n . X - 1 0 0 • but not Tween 8 0 ) . The basic p r o t e i n s , RNase and cytochrome 36 C a l s o caused b u r s t i n g b u t - t r y p s i n d i d not. P r o t e o l y t i c and. l i p o l y t i c enzymes as w e l l as peroxidase and polypheny! o x i -dase also f a i l e d to cause b u r s t i n g . Ruesink concluded from, evidence obtained w i t h h i s p r o t o p l a s t bioassay that t y r o s i n e plays no important r o l e i n the Avena plasma membrane in. con-t r a s t to some animal c e l l membranes where i t does. Ruesink was unable to demonstrate a b u r s t i n g response of Avena p r o t o p l a s t s to I n d o l e a c e t i c a c i d (IAA) although IAA i s known to cause b u r s t i n g of p r o t o p l a s t s obtained from other species ( 2 ) . Cocking (5, 6, 7) used a tomato root p r o t o p l a s t bioassay to i n v e s t i g a t e compounds known to have e f f e c t s on plant growth. Indole a c e t i c a c i d , naphthaleneacetic a c i d (NAA), g i b b e r e l l i c a c i d , e t h y l e n e d i a m i n e t e t r a c e t i c a c i d , 8-hydroxyquinoline and b e n z y l p e n i . c i l l i n a l l increased vacuo- ' l a t i o n of p r o t o p l a s t s and caused them to b u r s t . No f i r m conclusions could be drawn from t h i s work. Boulware and Camper (3) studied the e f f e c t s of s e v e r a l h e r b i c i d e s on the b u r s t i n g response of p r o t o p l a s t s . Although paraquat caused b u r s t i n g , p r e f o r a n , fluometuran, c h l o r b r o -muron and t r i f l u r a l i n d i d not. The study was undertaken to c o n t r i b u t e to the understanding of. the mode of a c t i o n of these p e s t i c i d e s . I n c o n t r a s t to the r e s u l t s of Ruesink and Thimann (20), Boulware and Camper. (3) found,Tween 80 to be t o x i c to tomato f r u i t p r o t o p l a s t s . P r o t o p l a s t s and c e l l s i s o l a t e d from leaves have been 37 used i n s e v e r a l s t u d i e s i n v e s t i g a t i n g h o s t : p a r a s i t e i n t e r a c -t i o n s . S t r o b e l and Hess ( 2 2 ) , studying the f u n g a l t o x i n Helminthosporicide' produced.by the sugarcane pathogen Helmin-thosporium s a c c h a r i , incubated p r o t o p l a s t s obtained from sus-c e p t i b l e and r e s i s t a n t v a r i e t i e s i n a s o l u t i o n c o n t a i n i n g the t o x i n . The hypothesis that the t o x i n binds to a s p e c i f i c p r o t e i n located.on the plasmalemma was supported by the f a c t that only p r o t o p l a s t s from the s u s c e p t i b l e v a r i e t y b u r s t . Four hours of i n c u b a t i o n were required to demonstrate the e f f e c t . S t r o b e l and Hess ( 2 2 ) found t h a t 5.0 mM t o x i n was required to burst i s o l a t e d p r o t o p l a s t s , whereas only 0 . 0 1 |iM was required to cause symptoms i n an 8 cm l e a f p i e c e . The l a r g e d i f f e r e n c e . i n concentration between that r e q u i r e d to burst p r o t o p l a s t s compared to t h a t which caused l e a f symptoms suggests that something other than l o s s of plasmalemma func-t i o n i s involved i n symptom development. Samaddar and,Scheffer ( 2 1 ) studied the .effects of H e l -minthosporium v i c t o r i a e t o x i n on p r o t o p l a s t s prepared from c o l e o p t i l e s of both r e s i s t a n t and s u s c e p t i b l e oat v a r i e t i e s as w e l l as from corn and sorghum.. The. p r o t o p l a s t s were i n -cubated w i t h t o x i n at a c o n c e n t r a t i o n of 0 . 1 6 ug/ml. Complete i n h i b i t i o n of root growth of s u s c e p t i b l e oat v a r i e t i e s occurs at a t o x i n concentration, of 0 . 0 0 1 6 ug/ml. A f t e r 1 h incuba-t i o n i n the t o x i n s o l u t i o n , 1 0 0 % of the p r o t o p l a s t s from the s u s c e p t i b l e oat v a r i e t y had l y s e d . No b u r s t i n g of. e i t h e r the r e s i s t a n t oat p r o t o p l a s t s or those prepared from corn 38 or sorghum occurred. I f the t o x i n s o l u t i o n was d i l u t e d , the p r o t o p l a s t s from the s u s c e p t i b l e oat v a r i e t y took longer to b u r s t . Samaddar and Scheff e r (21) concluded from these r e s u l t s that membrane e f f e c t s are very important i n the ac-t i o n of t h i s t o x i n and that the a c t i o n of Helminthosporium  v i c t o r i a e t o x i n on s i n g l e p r o t o p l a s t s could be observed. . P r o t o p l a s t s have a l s o been used i n a study by Otsuki et_ a l . (16) i n which the n e c r o t i c response of tobacco to tobacco mosaic v i r u s was s t u d i e d . They found that although TMV m u l t i p l i e d i n p r o t o p l a s t s of both n e c r o t i c and systemic r e s -ponding v a r i e t i e s , no d i f f e r e n c e s between the two types of p r o t o p l a s t s were observable. From t h i s observation they con-cluded that n e c r o s i s i s a m a n i f e s t a t i o n r e q u i r i n g organized t i s s u e as i n an i n t a c t l e a f . The r e s u l t s of S t r o b e l and Hess (22) and Otsuki et a l . (16) both suggest that the d i s e a s e ' r e a c t i o n of i s o l a t e d p r o t o p l a s t s i s d i f f e r e n t from that of the i n t a c t p l a n t . P r o t o p l a s t s have not p r e v i o u s l y been used to study h o s t : r u s t i n t e r a c t i o n s . Although I- recognized that the r e -s u l t s might not be d i r e c t l y comparable w i t h the e f f e c t s of the fungus on an i n t a c t t i s s u e , which, i s composed of many contiguous c e l l s , I considered that i t would be valuable.' to examine the e a r l y i n t e r a c t i o n s between i s o l a t e d host p r o t o p l a s t s and the r u s t fungus. The. e a r l y i n t e r a c t i o n between o b l i g a t e fungal, p a r a s i t e s and t h e i r hosts i s an area of i n v e s t i g a t i o n of obvious importance, but has been 39 l a r g e l y neglected. The c h i e f reason f o r t h i s neglect i s the d i f f i c u l t y of i n v e s t i g a t i n g a stage of i n f e c t i o n In which the number of host c e l l s a f f e c t e d i s a very small p r o p o r t i o n of the t o t a l number i n the i n f e c t e d leaf;'- An important ad-, vantage.of the p r o t o p l a s t bioassay i s that any e f f e c t s of the fungus on- i t s products would be exerted i n a synchronized manner on the p r o t o p l a s t population i n the bioassay medium. I f a b u r s t i n g response occurred i t would be a s e n s i t i v e and e a s i l y observed measure of the i n t e r a c t i o n , p a r t i c u l a r l y i f a t o x i n were i n v o l v e d . Several observations suggested that t h i s might be the case. C a l d w e l l and Stone (1936) ob-served that guard c e l l s are k i l l e d when a germinating r u s t spore, grows through them. L i t t l e f i e l d (13) has pointed out that the n e c r o s i s of. c e l l s immediately adjacent to a n . i n c i -pient r u s t i n f e c t i o n i n f l a x , v a r i e s i n extent and character depending on the genotype of both the host and r u s t . Necro-t i c areas around wheat r u s t pustules .can be increased i n s i z e and extended away from the i n f e c t e d area of t h e . l e a f by the manipulation of an e l e c t r i c a l f i e l d (15). . The n e c r o t i c area extends towards the p o s i t i v e pole when a current of 2-4 microampere.s was a p p l i e d to the i n f e c t e d l e a f . The f o l l o w i n g experiments were t h e r e f o r e designed to i n v e s t i g a t e i n t e r a c t i o n ' between f l a x r u s t exudate and f l a x p r o t o p l a s t s prepared from both r e s i s t a n t and s u s c e p t i b l e v a r i e t i e s . B u r s t i n g of p r o t o p l a s t s when Incubated With r u s t 40 exudate would'indicate the presence of (a) f u n g a l t o x i n ( s ) and would support the t o x i n theory of r e s i s t a n c e and suscep-t i b i l i t y . . MATERIALS AND METHODS The i s o l a t i o n and washing of p r o t o p l a s t s i s described i n Chapter I . The p r o t o p l a s t s used i n these experiments were prepared i n an. identical.manner. The p r o t o p l a s t s were used w i t h i n 2 h a f t e r washing. Two v a r i e t i e s of f l a x were used; Bison ( s u s c e p t i b l e ) and Bombay ( r e s i s t a n t ) . Spore germination, medium was prepared by.germinating 20 mg of f l a x r u s t spores. (Melampsora l i n i , race 3) on the surface: of 1 ml of 0.35M s o r b i t o l spread out i n t o a t h i n l a y e r i n a p e t r i d i s h . A f t e r overnight germination (the per-cent germination was greater than 50%) the spores and l i q u i d were t r a n s f e r r e d to a tapered c e n t r i f u g e tube. They were then shaken i n a Vortex mixer, allowed to s i t f o r 0.5 h,, then shaken again and c e n t r i f u g e d t o sediment the spores. '••. The supernatant was the "spore germination medium". Exudate from a x e n i c a l l y c u l t u r e d f l a x r u s t (race 3)'was prepared i n . a s i m i l a r manner except t h a t 1 gm. ( f r e s h weight), of ru s t was incubated overnight i n 2 ml of 0.35 M s o r b i t o l . This i s r e f e r r e d to as "axenic exudate". To assay the percentage of l i v i n g p r o t o p l a s t s a suspen-s i o n of 250 or more p r o t o p l a s t s i n 0.1 ml of 0.35.M s o r b i t o l was placed on a depression s l i d e . The number of both l i v i n g 41 and dead p r o t o p l a s t s were counted u s i n g a microscope w i t h phase contrast o p t i c s . The t e s t or c o n t r o l s o l u t i o n was added and the percentage of l i v i n g p r o t o p l a s t s c a l c u l a t e d by counting the p r o t o p l a s t s on each s l i d e . This took about 10 minutes. Consecutive counts i n d i c a t e d that the v a r i a t i o n between counts was 10%. V i a b i l i t y of p r o t o p l a s t s was e a s i l y determined. A smooth o u t l i n e of the p r o t o p l a s t s , streaming, the c e n t r a l p o s i t i o n of the vacuole and evenly d i s t r i b u t e d c h l o r o p l a s t s i n the cytoplasm were a l l i n d i c a t i v e of healthy, p r o t o p l a s t s . RESULTS D i f f e r e n t preparations of p r o t o p l a s t s were c o n s i s t e n t i n appearance. P r o t o p l a s t s were prepared from the two v a r i e t i e s Bison and Bombay. In. each case 60 to 80% were a l i v e and i n an apparently h e a l t h y . s t a t e a f t e r washing and at the beginning of experiments. There was some morphological v a r i a t i o n amongst the v i a b l e p r o t o p l a s t s . For example, i n some, the c h l o r o p l a s t s were evenly spaced around the periphery of the c e l l but i n others they were concentrated on one side u s u a l l y i n c l u s t e r s around the nucleus. In attempting t o make the p r o t o p l a s t b u r s t i n g bioassay as s e n s i t i v e and c o n s i s t e n t as p o s s i b l e , a number of procedural v a r i a t i o n s were i n v e s t i g a t e d and d i f f e r e n t sources of fungal m a t e r i a l i n c l u d i n g germinating spores and axenic c u l t u r e s were used. Embedding the p r o t o p l a s t s i n a t h i n l a y e r of agar (0.5 mm) and ap p l y i n g the t e s t s o l u t i o n by f l o o d i n g the surface had 42 the advantage of i m m o b i l i z i n g the p r o t o p l a s t s . This method was, however, discarded because the presence of agar decreased, the v i s i b i l i t y and made i t more d i f f i c u l t to determine v i a b i l i t y . The agar a l s o caused a marked increased i n the r a t e of death of both t r e a t e d and c o n t r o l p r o t o p l a s t s during the f i r s t hours of the experiment. Varying the c o n c e n t r a t i o n of p r o t o p l a s t s , although i t had an e f f e c t on the growth and development' of p r o t o p l a s t s incubated f o r periods of up to a month (see Chapter I ) , d id not have an observable e f f e c t during the i n c u -b a t i o n periods of up to 20 h used i n these experiments. About 300 p r o t o p l a s t s i n 0.2 ml of s o l u t i o n could be counted e a s i l y and q u i c k l y (10 minutes). S e a l i n g the p r o t o p l a s t s i n a micro-i n c u b a t i o n chamber made of c o v e r s l i p s prevented evaporation . during the 10 minutes r e q u i r e d f o r counting. I t was so cum-bersome and time-consuming, however, t h a t i t was abandoned. Evaporation from depression s l i d e s was not a problem i f the s l i d e s were kept i n a p e t r i d i s h c o n t a i n i n g moist f i l t e r paper except during the counting procedure. Table I summarizes the r e s u l t s of an experiment i n which spore germination medium plus spores was used as.the t e s t s o l u t i o n . I n the c o n t r o l s o l u t i o n the p r o t o p l a s t s were i n c u -bated i n an equal volume of 0.35 M s o r b i t o l . Each f i g u r e i n Table I i s an average, of 2 separate counts.. The r e s u l t s show that i n t h i s experiment treatment w i t h the spore germin-a t i o n medium plus spores d i d not cause p r o t o p l a s t b u r s t i n g ; i f anything, a p r o t e c t i v e e f f e c t i s suggested, the decrease TABLE I . E f f e c t s of Spore Germination Medium Plus Spores on Prot o p l a s t s . . CHANGE IN PROTOPLAST . LIVING PROTOPLASTS[%) VIABILITY{%) HOURS^ 0 0.5 1.0 1.5 2.0, 17.0 A 1 B^ CONTROL^ Bison 68.7 - .66.0 5.7.5 63.4 5.2 Bombay 70.3 - 61.6 .57.4 54.3 16.0. TREATED^ Bison 6 1 . 9 53.7 63.4 • 58.4 56.4 . 57.3 4.6 +0.6 Bombay 67.3 59.7 52.7 48.8 53.6 59.5 7.8 +8.2 "'"Observed ^Observed decrease. decrease corrected f o r control. decrease of the same v a r i e t y . r^Hours from s t a r t of experiment.. ^ P r o t o p l a s t s (1 part) plus 0.35 M s o r b i t o l (9 p a r t s ) . ^ P r o t o p l a s t s (1 part) spore germination medium plus spores (9 p a r t s ) . 44 i n the percentage of l i v i n g p r o t o p l a s t s being 8 . 2 % l e s s i n the . t r e a t e d than i n the corresponding c o n t r o l . The.protec-t i v e e f f e c t of the treatment on Bombay p r o t o p l a s t s i s pro-bably not r e a l , because the percentage of l i v i n g p r o t o p l a s t s at 1 . 5 h was 11% l e s s than at 17 h, an i m p o s s i b i l i t y and a r e f l e c t i o n of the v a r i a b i l i t y i n counting. The r e s u l t s of a s i m i l a r experiment are presented i n Table I I . I n t h i s , case the spores were germinated overnight r a t h e r than f o r 2 h. The r e s u l t s . s u g g e s t a p o s s i b l e e f f e c t of the treatment i n i n c r e a s i n g b u r s t i n g , but there i s no d i f f e r e n c e between the s u s c e p t i b l e v a r i e t y Bison and the r e -s i s t a n t Bombay. There i s l i t t l e , i f any, evidence of incon-s i s t e n c y i n counting apparent i n t h i s experiment.. I n the experiment summarized i n Table I I I both proto-p l a s t i s o l a t i o n and the bioassay were conducted at 3 7 ° C f o r 8 h, i n s t e a d of at 2 2 ° C overnight as was the usual procedure. The b u r s t i n g response was measured at 3 7°C because t h i s temp-erature was known to be higher than the optimal temperature f o r f l a x p r o t o p l a s t growth and development. The p o s s i b i l i t y was considered t h a t the p r o t o p l a s t s would be more s e n s i t i v e at the higher temperature i f the k i n e t i c s of t o x i n a c t i o n (assuming a t o x i n was present) were c h a r a c t e r i z e d by a.high 0^ 10. The treatment c o n s i s t e d of germinated spore medium ad-justed to 0 . 3 5 M w i t h respect to s o r b i t o l and d i d not contain spores. The spores had been germinated overnight and then processed as described i n the methods s e c t i o n . TABLE I I . E f f e c t s of Spore Germination Medium Plus Spores on P r o t o p l a s t s CHANGE IN PROTOPLAST •~ LIVING PROTOPLASTS{%) • VIABILITY^) HOURSJ 0 1 . 0 1 . 5 3 . 0 . 1 8 . 0 A 1 B^ CQNTROlA Bison 6 3 . 7 . 6 3 . 3 4 8 . 7 5 6 . 1 5 7 . 7 6 . 0 Bombay 6 7 . 7 6 4 . 8 6 5 . 9 6 5 . 4 • 5 8 . 4 9 . 3 TREATED5 . Bison 6 1 . 5 " 5 6 . 6 5 6 .o- 4 4 . 8 4 5 . 2 1 6 . 3 - 1 0 . 3 .. Bombay 5 9 . 3 5 . 0 . 0 4 8 . 2 4 6 . 2 4 3 . 7 1 6 . 6 - 7 . 3 . Observed, decrease. 'Observed decrease c o r r e c t e d f o r the c o n t r o l decrease of the same v a r i e t y . Hours from s t a r t of experiment 'Protoplasts ( 1 part) plus 0 . 3 5 M s o r b i t o l ( 9 parts).-P r o t o p l a s t s ( 1 part) plus spore germination medium plus spores ( 9 parts) . . . .•••'. TABLE I I I . E f f e c t . o f 3 7 ° C Incubation on P r o t o p l a s t s 4 6 HOURS 3 LIVING , PROTOPLASTS\%) 0 1 . 5 CHANGE IN PROTOPLAST VIABILITY(%). . _ A 1 B ' CONTROL^ Bison 5 2 . 7 3 4 . 9 1 7 . 8 Bombay 4 9 . 9 3 3 . 2 1 6 . 7 TREATMENT l 5 . Bison 4 9 . 6 2 6 . 9 . 2 2 . 7 - 4 . 9 Bombay 5 2 . 2 2 7 . 2 2 5 . 0 - 8 . 3 TREATMENT 2 6 Bison 4 2 . 8 . . 1 6 . 3 . 2 6 . 5 - 8 . 7 Bombay 5 1 . 4 2 9 . 0 2 2 . 9 ^Observed decrease.. ^Observed decrease c o r r e c t e d f o r the c o n t r o l decrease of the same v a r i e t y . • . ^Hours from s t a r t of experiment. ^ P r o t o p l a s t s ( 1 part) plus 0 . 3 5 M s o r b i t o l ( 1 0 p a r t s ) . ^ P r o t o p l a s t s ( 1 part) plus spore germination medium , ( 1 0 parts) . .^Protoplasts ( 1 part) plus spore germination medium, ( 1 part) plus 0 . 3 5 M s o r b i t o l ( 9 p a r t s ) . 4 7 As can be seen from the r e s u l t s (Table' I I I ) , the per-, centage of l i v i n g p r o t o p l a s t s a f t e r washing was reduced by r a i s i n g the,temperature during i s o l a t i o n . The r a t e of death . of both the c o n t r o l and the tr e a t e d p r o t o p l a s t s was a l s o i n - , creased during the experiment. The increase in-temperature was not e f f e c t i v e i n I n c r e a s i n g the dif f e r e n c e , between the c o n t r o l s and the two treatments. . " Tables IV and V are two experiments, i n which spore ger-mination medium was tested.. . The two experiments were i d e n t i c a l i n a l l r e s p e c t s , except that they were not c a r r i e d out on the same day. They sum up the d i f f i c u l t i e s which were encountered i n demonstrating c o n s i s t e n t and r e l i a b l e evidence f o r the e x i s -tence of a t o x i n of f u n g a l o r i g i n . Although there was an . apparent treatment e f f e c t i n both experiments t h i s i s seen to be i n c o n s i s t e n t . For example, i n Table IV, treatment 1 (high c o n c e n t r a t i o n of spore germination medium), the decrease i n . p r o t o p l a s t v i a b i l i t y i s the same as i n the c o n t r o l . I n Table V, treatment 1 , the decrease i n p r o t o p l a s t v i a b i l i t y was 1 0 - . 1 5 % . Examination of the r e s u l t s f o r t r e a t m e n t s i n Tables IV and V ( d i l u t e spore germination medium) shows an e f f e c t which i s the reverse of tha t seen f o r treatment 1 . I n Table IV the d i l u t e spore germination medium decreased p r o t o p l a s t v i a b i l i t y 7 to 1 2 % but i n Table V i t was the same as In the c o n t r o l . The.results presented i n Tables VI and VII are from two i d e n t i c a l experiments which were c a r r i e d out on d i f f e r e n t TABLE TV. E f f e c t of Spore Germination Medium on Pr o t o p l a s t s CHANGE IN PROTOPLAST ~ LIVING. PROTOPLASTS (%) -, . VIABILITY(%) ' ' HOURS^ • 0 1.0 2.5 16 A X B CONTROL4 Bison 77, .6 75 .7 73.1 . 69 .2 .8 .4 Bombay 79, .5 81 .5 • 72.6 71 .2 8. .3 TREATMENT 1^  Bison 71, .3 68 .4 • 64.8 " 61 .1 10 .2 - 1.8 Bombay 74, .9 72 .1 . 65.7 69 .7 5 .2 + 3.1 TREATMENT 2 6 B i son 70, •2 68 .1 65.8 59 .7 20 .5 ; -12.1 Bombay 76. ,6 72 .7. 67.0 61 .4 15 .2 .. - 6.9 Observed decrease. 2 Observed decrease corrected f o r the decrease of the c o n t r o l o the same.variety. ^Hours from s t a r t of experiment. ' ^ P r o t o p l a s t s (1 part) plus 0.35 M s o r b i t o l . ^ P r o t o p l a s t s (1 part) plus spore germination medium (lO.parts) ^ P r o t o p l a s t s (1 part) plus spore germination medium (1 part) plus 0.35 M s o r b i t o l (9 p a r t s ) . TABLE V. E f f e c t s of Spore Germination Medium on P r o t o p l a s t s • . •' CHANGE IN PROTOPLAST '.. '• . LIVING PROTOPLASTS (%) \ VIABILITY(%) ? . HOURS3 . 0 0 . 5 2 . 0 3 . 0 2 0 A . B CONTROL^ . Bison 8 1 . 6 7 8 . 1 7 7 . 0 7 1 . 7 7 4 , . 6 7 . 0 . Bombay 6 2 . 7 6 7 . 6 6 1 . 3 5 7 . 9 5 5 : . 7 7 . 0 TREATMENT l 5 Bison 6 8 . 7 6 7 . 8 6 2 . 4 6 4 . 0 . 4 3 . . 1 1 7 . 2 - 1 0 , . 2 Bombay 6 6 . 1 7 4 . 0 7 1 . 4 6 8 . 5 5 1 . . 5 2 3 . 0 - 1 6 . . 0 TREATMENT 2 6 Bison 7 1 . 7 7 0 . 6 6 8 . 8 6 4 . 4 6 6 , . 8 . A . 9 . + 2 , . 1 Bombay 7 3 . 0 7 3 - . 4 7 0 . 3 6 8 . 6 6 6 , . 2 • 6 . 2 - o, . 8 "'"Observed decrease. . ^Observed decrease corre c t e d f o r the decrease of the c o n t r o l 'of the same v a r i e t y . 3Hours from s t a r t of experiment. -^ P r o t o p l a s t s ( 1 part) plus 0 . 3 5 M s o r b i t o l ( 1 0 p a r t s ) . ^ P r o t o p l a s t s ( 1 part) plus spore germination medium ( 1 0 p a r t s ) . ^ P r o t o p l a s t s ( 1 part) plus spore germination medium ( 1 part) plus 0 . 3 5 M s o r b i t o l ( 9 p a r t s ) . 50 days. The p r o t o p l a s t suspensions were incubated w i t h axenic exudate at two d i f f e r e n t concentrations (treatment 1 .and ' 2 ) . Although the r e s u l t s i n Table VI suggest t h a t the exudate obtained from axenic c u l t u r e s causes more b u r s t i n g than the exudate from germinating spores the e f f e c t s of the treatment (Table VT , treatment- 1 ) i s s t i l l not c o n c l u s i v e . Thus i n the experiment shown i n Table VII the e f f e c t of treatment 1 on p r o t o p l a s t s from the two d i f f e r e n t v a r i e t i e s of f l a x i s not as pronounced as i n the experiments i n Table VT. Moreover, p r o t o p l a s t s from Bombay f l a x which were the most s u s c e p t i b l e to b u r s t i n g i n Table VI, were h i g h l y r e s i s t a n t , to the t r e a t -ment i n the experiment i n Table VTI. F i n a l l y , there was i n these experiments (Tables.VI and VTI) no c o n s i s t e n t d i f f e r e n c e i n the response of the two v a r i e t i e s , Bison and Bombay. DISCUSSION In the preceding experiments which are described i n Tables I t o VII the p r o t o p l a s t bioassay was shown t o be u s e f u l and the counting of l i v i n g p r o t o p l a s t s to be c o n s i s t e n t . Pre-l i m i n a r y experiments designed to t e s t the consistency of . counting the percentage of l i v i n g p r o t o p l a s t s showed that when consecutive counts of the same s l i d e were made the v a r i a -b i l i t y i n the percentage of l i v i n g p r o t o p l a s t s was 1 0 % . I n the experiments described i n Tables I to VTI, the v a r i a b i l i t y of counting l i v i n g p r o t o p l a s t was a l s o u s u a l l y TABLE VI. E f f e c t s of Axenic. Exudate on P r o t o p l a s t s CHANGE IN PROTOPLAST . LIVING PROTOPLASTS[%). . ' VIABILITY^) • . HOURS^ 0 1 . 0 2 . 5 2 1 A B^ . CONTROL4 Bison 8 7 , . 1 8 4 . 1 8 1 . 3 7 4 , . 5 1 2 . 6 Bombay 7 3 , . 5 7 2 . 2 6 5 . 7 6 4 , . 3 8 . 7 TREATMENT l 5 Bison '80, . 0 7 6 . 0 7 4 . 4 . 5 1 ; . 4 2 8 . 6 - 1 6 . . 0 Bombay 7 5 . . 8 7 3 . 3 6 9 . 7 . 4 7 . . 7 2 8 . 1 - 1 9 , . 4 TREATMENT 2 6 • Bison 8 2 . . 9 7 8 . 7 7 6 . 9 7 2 . , 7 . 1 0 . 2 . - 2 . . 4 Bombay 8 2 . . 5 . 7 5 . 5 7 2 . 6 , 7 2 . , 6 9 . 9 . . + 1 , . 2 Observed decrease. Observed decrease c o r r e c t e d f o r the decrease of the c o n t r o l of the same v a r i e t y . Hours from s t a r t of experiment. P r o t o p l a s t s ( 1 part) plus 0 . 3 5 M s o r b i t o l ( 1 0 p a r t s ) . P r o t o p l a s t s ( 1 part) plus axenic exudate ( 1 0 p a r t s ) . P r o t o p l a s t s ( 1 p a r t ) plux.axenic exudate ( 1 part) plus 0 . 3 5 M s o r b i t o l ( 9 . p a r t s ) . • TABLE V I I . E f f e c t s of'Axenic Exudate on P r o t o p l a s t s CHANGE IN PROTOPLAST ' LIVING PROTOPLASTS (%) VIABILITY(%) ••• HOURS-3. 0 1.0 1.5 2.5 17 A 1 B^ CONTROL4 Bison 84 74 76 74 70 14 Bombay 83 79 80 70 68 15 TREATMENT5 •. Bison 1 83 68 63 68 .58' 25 -11 Bison 2 . 83 . 80 69 72 64 19 - 5 • Bombay 1 84 66 68 65 66 18 -.2.5 Bombay 2 83 69 71 68 68 • 15 • • .+. 0.5 ^Observed decrease. ^Observed decrease c o r r e c t e d f o r the decrease of the same con-t r o l v a r i e t y . •^Hours from s t a r t of experiment. ^ P r o t o p l a s t s (1 part) plus 0.35 M s o r b i t o l '(10 p a r t s ) . ^ P r o t o p l a s t s (1 part) plus axenic exudate (10 parts).' 1 and 2 are r e p l i c a t e s of the same experiment. 53 w i t h i n t h i s range. I f i n c o n s i s t e n c y of counting had occurred . the percentage of l i v i n g . p r o t o p l a s t s would.sometimes i n c r e a s e , r a t h e r than decrease or remain the same, as consecutive counts of the s l i d e were made durin g the experimental i n c u b a t i o n p e r i o d . The greatest i n c o n s i s t e n c y of t h i s k i n d was.in the v a r i e t y Bombay, Table I , treatment 1. Here, the percentage l i v i n g p r o t o p l a s t s was 48.8% a f t e r 1.5 h but at 17. h was 59.5%, an i n c o n s i s t e n c y of 10.7%.. Another measure of the consistency of counting percentage l i v i n g p r o t o p l a s t s i s a comparison of p r o t o p l a s t s of the same v a r i e t y , i n the same experiment at O h . Since a l l the proto-p l a s t s from each v a r i e t y were from the same stock,, suspension these counts should not vary more than 10%. Examination of Tables I-VTI shows, that a v a r i a t i o n of greater than 10% only occurred i n Table V. At 0 h Bison p r o t o p l a s t s ranged, i n per-centage l i v i n g p r o t o p l a s t s from 81.6% to 68.7%, a d i f f e r e n c e of 12.9% and Bombay p r o t o p l a s t s ranged from 62.7% to 73.0%, a. d i f f e r e n c e of 11.0%. In n e a r l y a l l the experiments (Tables I to VTI), the p r o t o p l a s t s from Bison and Bombay f l a x v a r i e t i e s when incuba-ted w i t h e i t h e r spore germination medium or axenic exudate, burst to a greater extent than those i n the corresponding c o n t r o l s o l u t i o n . The decrease, when corrected f o r the con-, t r o l , was u s u a l l y l e s s than 10%. I n the experiments presented i n Tables V and VT the treatment decrease a f t e r c o r r e c t i o n f o r the c o n t r o l decrease was greater than 10%. As has been 54 argued above, t h i s apparent e f f e c t of spore germination medium (Table V) and axenic exudate (Table VI) i s not r e a l . I t could not be demonstrated i n i d e n t i c a l experiments c a r r i e d out on d i f f e r e n t days.. There i s even l e s s evidence t h a t the two v a r i e t i e s . B i s o n and Bombay, responded d i f f e r e n t l y to any of the treatment s o l u t i o n s . An a d d i t i o n a l a n a l y s i s of the r e s u l t s , u sing s t a t i s t i c a l techniques i s presented i n the appendix at the end of t h i s chapter. From the discussion,. t h e r e f o r e , i t can be concluded.that although the bioassay technique i s r e l i a b l e , no t o x i c f a c t o r e i t h e r from germinated spores or axenic c u l t u r e s was detected. At l e a s t two p o s s i b i l i t i e s e x i s t . E i t h e r a t o x i c substance, was present at low c o n c e n t r a t i o n , but was not detected because of the 10% v a r i a b i l i t y i n c o u n t i n g . v i a b l e p r o t o p l a s t s o r , there was no t o x i c substance present. What then i s the explanation of the observations which prompted the search f o r t o x i c f a c t o r s produced by the fungus? I t seems apparent that the observations which suggested the i n -volvement of a t o x i c substance' r e s u l t from processes that only occur when the r u s t i n t e r a c t s w i t h the i n t a c t p l a n t . Otsuki et al. . (16) concluded from t h e i r experiments w i t h v i r u s i n f e c t e d p r o t o p l a s t s from n e c r o t i c and s u s c e p t i b l e responding tobacco th a t o r g a n i z a t i o n of c e l l s , as i n the i n t a c t l e a f , i s necessary f o r the n e c r o t i c response. This, a l s o seems to be the case w i t h f l a x r u s t and f l a x . Since the spores and axenic c u l t u r e s 55 used i n experiments are known to be pathogenic but d i d not cause p r o t o p l a s t s to b u r s t , the explanation of n e c r o s i s during disease may be. that the t o x i c f a c t o r s causing n e c r o s i s o r i g i n a t e from pl a n t c e l l s r a t h e r than from the fungus. A good p o s s i b i l i t y i s that, these t o x i c f a c t o r s of. plant o r i g i n are phytoalexins or compounds which act i n a s i m i l a r manner. Phytoalexins are produced by plant c e l l s and are r a r e l y found except i n a s s o c i a t i o n w i t h n e c r o s i s ( 1 4 ) . Their production can be induced by a wide range of pathogens as w e l l as by wounding ( 1 7 ) . Both t h e i r production and n e c r o s i s are non-s p e c i f i c responses i n c o n t r a s t to s u s c e p t i b i l i t y and r e s i s -tance. In the f l a x : f l a x r u s t system r e s i s t a n c e and s u s c e p t i -b i l i t y are dependent on one gene i n the fungus and one i n the host. ( 9 ) . The r e s u l t s support the Idea that r e s i s t a n c e i s . the r e s u l t of a process which i s d i s t i n c t and d i f f e r e n t from those causing the n e c r o t i c r e a c t i o n . This is. a new concept and one which i s important to the understanding of r e s i s t a n c e and s u s c e p t i b i l i t y of p l a n t s . t o pathogens. A very s i m i l a r proposal has r e c e n t l y been advanced by K i r a l y et a l . ( 1 1 ) . These authors suggest t h a t h y p e r s e n s i t i v i t y i s the r e s u l t and not.the cause of r e s i s t a n c e and have presented evidence t h a t r e s i s t a n c e can be expressed without the occurrence of n e c r o s i s (.10) . No compounds w i t h p h y t o a l e x i n - l i k e p r o p e r t i e s have been described from i n f e c t e d f l a x although many phenolic compounds are known to occur i n f l a x . I n the wheat:wheat r u s t system 56 .' i . ' . • although phytbalexiris- .per se are not thought to be produced other t o x i c phenolic compounds which may have many s i m i l a r p r o p e r t i e s have been i d e n t i f i e d ( 8 ). The phenolic g l y c o s i d e of 2 ,4-dihydroxy-7-methoxy-l ,4-benzoxazin-3-one' (.12) i s present i n wheat p l a n t s and i s known to break down i n t o the aglycone and benzoxazoline upon i n f e c t i o n of wheat by an a v i r u l e n t race of r u s t . Both of these breakdown products are known to have considerable a n t i f u n g a l a c t i v i t y . L i k e p h y t o a l e x i n produc-t i o n , breakdown of the g l y c o s i d e i s not gene s p e c i f i c . I f p h y t o a l e x i n production and the a s s o c i a t e d n e c r o s i s i s the r e s u l t of r e s i s t a n c e , what i s the nature of the s p e c i f i c i n t e r a c t i o n between the pathogen and the pla n t which r e s u l t s i n s u s c e p t i b i l i t y or r e s i s t a n c e ? There i s very l i t t l e e x peri-. mental i n f o r m a t i o n on t h i s subject but s e v e r a l proposals have been made. Albersheim et a l . (1) put forward an i n t e r e s t i n g hypothesis along w i t h a convincing d i s c u s s i o n and expla n a t i o n . They consider "That molecular i n t e r a c t i o n s between the carbo-hydrate c o n s t i t u e n t s of a host and the poly-saccharide degrading enzymes produced by a pathogen account f o r the inherent r e s i s t a n c e of p l a n t s to most microorganisms and that these i n t e r a c t i o n s account e q u a l l y w e l l f o r the r a r e instances i n which a microorganism s u c c e s s f u l l y i n f e c t s a p l a n t . " I f t h i s hypothesis i s e s s e n t i a l l y , c o r r e c t , then s u s c e p t i b i l i t y and r e s i s t a n c e would not be expected to occur or be observed i n a system i n which pathogens i n t e r a c t w i t h p r o t o p l a s t s be-cause p r o t o p l a s t s do not have c e l l w a l l s . The r e s u l t s of the 57 experiments :presented i n t h i s report are th e r e f o r e c o n s i s t e n t w i t h Albersheim's hypothesis but i t would nevertheless be d i f f i c u l t to argue th a t they provide any degree of proof of i t . A second hypothesis, i s that a s p e c i f i c gene product i s . ; produced by the fungus which when i n i n t e r a c t i o n w i t h plant c e l l s determines s u s c e p t i b i l i t y or r e s i s t a n c e . I n a recent report Rohringer et a l . (18) cla i m to have i s o l a t e d a gene-s p e c i f i c RNA d i r e c t l y i n v o l v e d i n r e s i s t a n c e of wheat to.wheat, r u s t from heavily, i n f e c t e d and n e c r o t i c wheat l e a v e s . Their bioassay i n v o l v e s i n j e c t i n g the RNA f r a c t i o n i n t o s u s c e p t i b l e or r e s i s t a n t wheat leaves infected, w i t h r u s t and counting the numbers of n e c r o t i c c e l l s i n each case a f t e r 11 h. The RNA i s a c t i v e only i n i n f e c t e d leaves c o n t a i n i n g h a u s t o r i a . This f i n d i n g and the conclusions drawn from the work remain to be confirmed. No evidence which supports the s p e c i f i c gene product, theory was obtained from the p r o t o p l a s t experiments reported -here. I f t h i s theory i s t r u e , the spore and axenic c u l t u r e e x t r a c t could c o n t a i n the "gene product" s i n c e the spores and axenic c u l t u r e s are known to.be pathogenic. The r e c o g n i t i o n of r e s i s t a n c e or s u s c e p t i b i l i t y would occur when the gene, product interacted, w i t h the p r o t o p l a s t s . P r o t o p l a s t b u r s t i n g would -not n e c e s s a r i l y . b e expected to occur because, as d i s -cussed above, the n e c r o t i c response may only occur when there i s an i n t e r a c t i o n of i n f e c t e d c e l l s w i t h surrounding t i s s u e s i n an i n t a c t l e a f . i l l ' : - 1 ; REFERENCES . 1 . Albersheim, P..-, ?.M. Jones and P.D.. E n g l i s h . . 1 9 6 9 . B i o -chemistry of the c e l l w a l l i n r e l a t i o n to i n f e c t i o n pro-cesses. Ann. Rev. Phytopathology 7 : . 1 7 1 - 1 . 9 4 . 2 . Boyer, M.H. 1 9 7 3 . Response of N i c o t i a n a mesophyll p r o t o -p l a s t s of normai and tumorous o r i g i n t o i n d o l e a c e t i c a c i d i n v i t r o . P l a n t . P h y s i o l o g y 5 1 : 8 9 8 - 9 0 1 . . 3 . Boulware, M.A. and N.D. Camper. 1 9 7 2 . E f f e c t s of s e l e c -ted h e r b i c i d e s on p l a n t p r o t o p l a s t s . P h y s i o l . Plantarum 2 6 : 3 1 3 - 3 1 7 . 4 . C a l d w e l l , R.M., and .G.M. Stone. 1 9 3 6 . R e l a t i o n of stomatal f u n c t i o n of wheat t o . i n v a s i o n and i n f e c t i o n by l e a f r u s t P u c c i n i a t r i t i c i . J . Agr. Res. 5 2 : 9 1 7 - 9 3 2 . 5 . Cocking, E.C. 1 9 6 1 . P r o p e r t i e s of i s o l a t e d p r o t o p l a s t s . Nature 1 9 1 : 7 8 0 - 7 8 2 . 6 . Cocking, E.C. 1 9 6 2 . The a c t i o n of i n d o l y l - 3 - a c e t i c a c i d on i s o l a t e d p r o t o p l a s t s of tomato cotyledons. Biochem. J . 8 2 : 7 . . Cocking, E.C. 1 9 6 2 . Action, of growth substances, che-l a t i n g agents and a n t i b i o t i c s on i s o l a t e d root p r o t o p l a s t s . Nature 1 9 3 : 9 9 8 - 9 9 9 . .8. EINaghy, M.A. and M. Shaw. 1 9 6 6 . : C o r r e l a t i o n between r e s i s t a n c e to stem r u s t and the c o n c e n t r a t i o n of a gluco-s i d e i n wheat. Nature 2 1 0 . 4 . 1 7 - 4 1 8 . 9 . F l o r , H.H. 1 9 7 1 . Current s t a t u s of the gene-for-gene concept. Ann. Rev. Phytopathology 9 : 2 7 5 - 2 9 6 . 1 0 . K i r a l y , Z. 1 9 7 4 . Tissue n e c r o s i s and plant disease r e -s i s t a n c e . Am. Phytopath. Soc./Can. Phytopath. Soc. Ann. Meeting 1 9 7 4 , Abstr. I 6 5 . 1 1 . K i r a l y , Z., B. Barna and T. Ersek. . 1 9 7 2 . H y p e r s e n s i t i v i -t y as a consequence,. not the cause, of p l a n t r e s i s t a n c e to i n f e c t i o n . Nature 2 3 9 : 4 5 6 - 4 5 7 . 1 2 . Knott, D.R. and J . Kumar. 1 9 7 2 . Tests of the r e l a t i o n -s h i p between a s p e c i f i c phenolic glucoside and stem r u s t r e s i s t a n c e i n wheat. P h y s i o l . Plant Pathology 2 : 3 9 3 - 3 9 9 . 59 13. L i t t l e f i e l d , L . J . 1973. H i s t o l o g i c a l evidence f o r d i -verse mechanisms of r e s i s t a n c e to f l a x r u s t , Melampsora  l i n i (Ehrenb.) Lev. Physiol'. Plant Pathology. 3: 241-247. 14. M i i l l e r , K. and H. Berger. 1940. Experimentelle unter-suchungen uber d i e Phytophthora r e s i s t e n z der- k a r t o f f e l . . Arb. B i o l . Reichsanstat Land-U F o r s t w r i t s c h B e r l i n 23: 189-231. 15. O l i e n , C.R. 1957. E l e c t r o p h o r e t i c displacement of the n e c r o t i c area from the region of m y c e l i a l development i n Kh a p l i emmer i n f e c t e d w i t h race 56 of Puccinia.graminis var. t r i t i c i .,, Phytopathology 45 : 26 ( Abstr. ) . 16. O t s u k i , I . , T. Shimomura and I . Takebe. 1972.. Tobacco mosaic v i r u s m u l t i p l i c a t i o n and expression of the N gene i n n e c r o t i c responding tobacco v a r i e t i e s . V i r o l o g y 50: 45-50. 17. Rahe, J.E.. 1974. Accumulation of p h a s e o l l i n . i n hypo-c o t y l s of e t i o l a t e d Phaseolus v u l g a r i s i n response to mechanical i n j u r y . Am. Phytopath. Soc./Can. Phytopath.. Soc.'1974 Meeting. Abstr. 196. 18. Rohringer, R., N.K. Howes, W.K. Kim and D.J. Samborski. 1974. Evidence f o r a g e n e - s p e c i f i c RNA determining r e -s i s t a n c e i n wheat to stem r u s t . Nature 249: 585-588. 19. Ruesink, A.W. 1971. The plasma membrane, of Avena coleo-p t i l e p r o t o p l a s t s . Plant Physiology 47: 192-195. 20. Ruesink, A.W. and K.V. Thimann. 1965. P r o t o p l a s t s from the Avena c o l e o p t i l e . Proc. Nat. Acad. S c i . (U.S.A.) 54: 56-64. 21. Samaddar, K.R. and R.P. Sc h e f f e r . 1966. E f f e c t s of Helminthosporium v i c t o r i a e t o x i n on p r o t o p l a s t s from Avena c o l e o p t i l e s . Phytopathology 56: 898 ( A b s t r . ) . 22. S t r o b e l , G..A, and W.M. Hess. 1974. Evidence f o r the presence of the t o x i n - b i n d i n g p r o t e i n on the plasma mem-brane of sugar cane c e l l s . Proc. Nat. Acad. S c i . (U.S.A.) 71: I413-I417. 60 • i • APPENDIX TO CHAPTER I I . . . A more- d e t a i l e d a n a l y s i s of the r e s u l t s which are pre-sented i n Tables I-VTI'and discussed above i s p o s s i b l e using s t a t i s t i c a l techniques. I n p a r t i c u l a r , the p o s s i b l e e f f e c t on the b u r s t i n g of p r o t o p l a s t s caused by spore germination medium and axenic exudate can be q u a n t i f i e d i n more p r e c i s e terms and l i m i t s on these e f f e c t s s e t . Two analyses were c a r r i e d out, the f i r s t on those e x p e r i -ments which i n c l u d e d c o n t r o l p r o t o p l a s t s and one treatment (Tables. I , I I and VII) and the second which i n c l u d e d two t r e a t -ments plus the' c o n t r o l (Tables IV, V and V I ) . The experiment presented i n Table I I I was not included i n e i t h e r . a n a l y s i s because only two counts of the percentage l i v i n g p r o t o p l a s t s were made (at 0 and 1.5 h ) . In both the f i r s t and second analyses a randomized block design was used, the d i f f e r e n t experiments being designated as block s . . For the purposes of the a n a l y s i s only the counts taken at f o u r d i f f e r e n t times during the experiment were used. Thus i n the experiment i n Table I those counts made at, 0.5 and 1.5. h were not used. S i m i l a r i l y i n Table I I those at. 1.5 h, i n Table VII those at 1.5 h and in Table V those .at 2.0 h were not used. In.the experiment i n Table VII two s l i d e s f o r each, v a r i e t y were incubated w i t h the treatment s o l u t i o n . The two s l i d e s f o r each v a r i e t y were averaged to give one count f o r each v a r i e t y . These adjustments, mad-e the design of the. e x p e r i -ments s i m i l a r so that the a n a l y s i s could be done. 61 A n a l y s i s of Variances, Tables I , I I and VII Source df MS F Experiment Treatment '. . • V a r i e t y . . Time Time X V a r i e t y Treatment X Time V a r i e t y X Time Treatment X V a r i e t y X b-1 = 2 a -1 = 1 c -1 = 1 h -1 = 3 1236.67 446.52 5.88 380.56 .53NS .64NS ..88NS ( a - D ( c - l ) = 1 (a -1 )(h -1 ) = 3 ( c - l ) ( h - l ) = 3 8.71 9.75 13.42 Time Experimental E r r o r T o t a l ( a - l ) ( c - l ) ( h - l ) • = 3 ( b - l ) ( a c h - l ) = 30 bach -1 = 47 26.89 15.29 1.7.5NS h i g h l y s i g n i f i c a n t , PZ .0.01 NS not s i g n i f i c a n t , P y> 0.05 The r e s u l t s of t h e . a n a l y s i s of variance f o r the data i n Tables I , I I and VTI show that i n these experiments the t r e a t e d p r o t o p l a s t s burst to a s i g n i f i c a n t l y greater extent than those incubated i n the c o n t r o l s o l u t i o n . The mean percentage of l i v i n g p r o t o p l a s t s f o r the c o n t r o l was 66.60% and f o r the t r e a t e d was 60.50%. A 95% confidence i n t e r v a l about the d i f f e r e n c e can be constructed•as f o l l o w s : Numerically., the d i f f e r e n c e between the means i n c l u d i n g the 95% confidence i n t e r v a l c a l c u l a t e d u s i n g the above equation i s 6.10% + 1.13%. The conclusions t h a t can be drawn from the above a n a l y s i s are tha t . t h e decrease of percentage l i v i n g p r o t o p l a s t s f o r the t r e a t e d p r o t o p l a s t s over t h a t . o f the c o n t r o l p r o t o p l a s t s was s i g n i f i c a n t and that the d i f f e r e n c e (95% confidence i n t e r v a l ) i s between 4.18% and 8.01%. ( x x - x 2 )•+ t 3 0 d f ( 1-tailed) + EMS 62' Analysis: o£i Variance.', .'Tables IV, V and VI Source df MS F Experiment ; Treatment V a r i e t i e s Time Treatment X V a r i e t y Treatment X Time V a r i e t y X Time Treatment X V a r i e t y b-1 = 2. a-1 = 2 c-1 = 1 h-1 = 3 (a-1)(c-1) = 2 (a-1)(h-1) = 6 ( c - l ) ( h - l ) = 3 258.84 261.35 72.20 555.94 198.54 57.97 6.86 X Time Experimental E r r o r T o t a l • (a-1)(c-1)(h-1) =6 ( b - l ) ( a c h - l ) = 46 bach-1 = 71 5.19 27.49 0.19NS h i g h l y s i g n i f i c a n t , p<,0.01 NS not s i g n i f i c a n t , P^0.05 The r e s u l t s of the a n a l y s i s of variance f o r the data i n Tables IV, V and VI are that i n . these experiments, as i n those i n Tables I , I I and. V I I , the t r e a t e d p r o t o p l a s t s burst t o a s i g -n i f i c a n t l y greater extent than those incubated i n the c o n t r o l s o l u t i o n . I n t h i s a n a l y s i s although the v a r i e t i e s d i d not respond s i g n i f i c a n t l y d i f f e r e n t l y there was a v a r i e t y X t r e a t -ment i n t e r a c t i o n which shows that the nature of the responses of the two v a r i e t i e s was d i f f e r e n t . The same method as t h a t described above can,be used to c a l c u l a t e the magnitude of the d i f f e r e n c e s , w i t h 95% c o n f i -dence. The. two v a r i e t i e s are d e a l t w i t h s e p a r a t e l y because of the v a r i e t y X treatment i n t e r a c t i o n . Bison c o n t r o l (mean 77.3%) was l e s s than Bison, treatment 1, by the i n t e r v a l 7.87% to 15.06%. Bombay c o n t r o l (mean 69.0%) did not d i f f e r from Bombay, treatment 1 (-2.27%.to 4.92%). Bison c o n t r o l was l e s s than Bison, treatment 2, by the i n t e r v a l 3.08% to 63 10.27%. Bombay c o n t r o l did not d i f f e r from Bombay, treatment 2 (-6.69 t o 0.49). Although,the. r e s u l t s from the s t a t i s t i c a l a n a l y s i s c l e a r -l y show a greater decrease of percentage l i v i n g . p r o t o p l a s t s f o r the t r e a t e d than f o r the c o n t r o l p r o t o p l a s t s , the magni-tude of the d i f f e r e n c e i s s m a l l , u s u a l l y less, than 10%. Samaddar and Scheffer (21) f o r example, observed 100% b u r s t i n g of p r o t o p l a s t s a f t e r 1 h when they incubated p r o t o p l a s t s from a s u s c e p t i b l e oat v a r i e t y w i t h Helminthosporium v i c t o r i a e t o x i n . Even i f the t o x i c substance i n the treatment s o l u t i o n s was v e r y . d i l u t e i t . w o u l d be expected that the d i f f e r e n c e between the c o n t r o l and. t r e a t e d p r o t o p l a s t s would be greater than 10% a f t e r 20 h i n c u b a t i o n . The d i f f e r e n c e i n the d e c l i n e i n the percentage of l i v i n g p r o t o p l a s t s between treatment 1 and the d i l u t e treatment 2 would a l s o be. expected to be grea t e r . There i s at l e a s t one p o s s i b l e reason why a d i f f e r e n c e between t r e a t e d and c o n t r o l p r o t o p l a s t s could occur even i f no t o x i c substance was present, namely an unconscious psycho-l o g i c a l b i a s i n the counting. This p o s s i b i l i t y e x i s t s because the i d e n t i t y of the c o n t r o l and'treatment, s l i d e s was known to the c o u n t e r . . . 6 4 ! Ti l- ! . •: • SECTION IT AXENIC CULTURE 65 Chapter- I l l Axenic Culture of F l a x Rust I s o l a t e d from  Cotyledons by C e l l W a l l D i g e s t i o n INTRODUCTION Otsuki and Takebe (8) i s o l a t e d i n t a c t mesophyll c e l l s and t h e i r p r o t o p l a s t s from a number of. p l a n t species by u s i n g mixtures of h y d r o l y t i c enzymes to dig e s t the p l a n t c e l l walls.. They noted that, some species, were more r e s i s t a n t to the en-zymes than others. Maheshwari (7) used a s i m i l a r technique to i s o l a t e the epidermis of sunflower and snapdragon leaves but found i t more d i f f i c u l t to ob t a i n s u i t a b l e preparations from wheat and corn. A method i s reported here f o r the i s o l a t i o n of i n t a c t c o l o n i e s of the o b l i g a t e p a r a s i t e (Melampsora l i n i . (Pers.) Lev., ; Race No. 3) from f l a x by the use of h y d r o l y t i c enzymes which s e l e c t i v e l y d igest the c e l l w a l l s of the host p l a n t , without apparently damaging the f u n g a l c e l l wall.. Once i s o - . l a t e d , the colonies.may be u s e f u l f o r metabolic and other s t u d i e s of the. r u s t fungus i n the absence of host t i s s u e . Such c o l o n i e s can a l s o be used as inoculum for. the e s t a b l i s h -ment of axenic c u l t u r e s . "Published I n Canadian J o u r n a l of Botany V o l . 50:2601-2603, 1972.. 66 MATERIALS AND METHODS Growth a n d . i n o c u l a t i o n of f l a x seedlings ( v a r i e t y Bison) and surface s t e r i l i z a t i o n of the cotyledons were c a r r i e d out as described by Coffey et a l . (3). Cotyledons were harvested when i n f e c t i o n f l e c k s were well-developed but not yet orange i n c o l o u r . A f t e r surface s t e r i l i z a t i o n , the a b a x i a l epidermis was s t r i p p e d away as c a r e f u l l y as p o s s i b l e u s i n g f i n e forceps and a s p a t u l a . The cotyledons were then immersed i n the B-5 medium described by Gamborg et_ a l . (5), modified by the omission of 2,4-D and c o n t a i n i n g i n a d d i t i o n 2.5% macerozyme and 2.5% 0nozuka-P5000 c e l l u l a s e (both obtained from A l l Japan Biochem-i c a l s ) , 0.5% pectinase (Sigma) and Gramicidin D (6 ug/ml). I f a l a r g e y i e l d of c o l o n i e s was d e s i r e d , about s i x t y cotyledons were immersed i n 25 ml of the enzyme mixture i n a 50 ml E r l e n -myer f l a s k , i n f i l t r a t e d under vacuum u n t i l they sank, and i n -cubated f o r 90 minutes at room temperature w i t h gentle shaking. This was f o l l o w e d by a g i t a t i o n w i t h a m a g n e t i c . s t i r r e r f o r 30 minutes. The speed of the s t i r r e r was adjusted to avoid foam-i n g . The l i b e r a t e d c o l o n i e s were now suspended i n the medium and were c o l l e c t e d on a nylon screen (3&*5 (-0. They were then . washed w i t h modified B-5 medium ( i . e . minus 2,4-D), resuspend-ed i n modified B-5 medium and c e n t r i f u g e d at low speed to concentrate them i n a loose p e l l e t . This procedure i s r e f e r r e d to as procedure I . 67 Procedure I I ; was used to. obtain c o l o n i e s which were v i r t u a l l y f r e e of c h l o r o p l a s t s and f l a x c e l l s . About s i x cotyledons w i t h the abaxial"epidermis removed were incubated i n a p l a s t i c p e t r i d i s h (Falcon, 60 mm), c o n t a i n i n g 2 ml of enzyme s o l u t i o n , at room temperature overnight. The c o l o n i e s were then f r e e d from the epidermis by gentle shaking, t r a n s -f e r r e d t o about 25 ml of modified B-5 medium and a g i t a t e d w i t h a magnetic s t i r r e r f o r 10 minutes. This suspension was poured i n t o a p e t r i d i s h . Colonies were spotted through a d i s s e c t i n g microscope and picked up w i t h a Pasteur p i p e t t e . Axenic c u l -t u r e s were i n i t i a t e d by t r a n s f e r r i n g the. colonies,, suspended i n modified B-5 medium to the agar medium used f o r axenic c u l t u r e of the r u s t . The dr o p l e t of l i q u i d was then, removed, to ensure d i r e c t contact of the c o l o n i e s w i t h the agar s u r f a c e . Each l i t e r of the agar medium contained KNO^, 0.25 gm; Ca(NO^)-4H2'0, 2 gm; K HPO^,'-0.75 gm; KHgPC^, 0.25 gm; -NH^NCy 0.04 gm; the m i c r o n u t r i e n t s described by Coffey and Shaw (3); sucrose, 40 gm; agar, 16 gm; and bovine serum albumin.(Miles, F r a c t i o n V), 0.5 gm. The calcium n i t r a t e was autoclaved s e p a r a t e l y and added to the medium when i t had cooled. The bovine serum albumin was d e f a t t e d (1) and. f i l t e r s t e r i l i z e d . This medium was known to support growth of axenic c u l t u r e s i n i t i a t e d from f l a x r u s t urediospores (unpublished r e s u l t s ) / Incubation was i n the dark at 17°C. 68 .RESULTS AND DISCUSSION This technique of i s o l a t i n g f u n g a l c o l o n i e s from i n -f e c t e d p l a n t t i s s u e depends "on the use of enzymes which di g e s t or c r i t i c a l l y weaken the host c e l l w a l l s but not those of the fungus. When i n f e c t e d f l a x cotyledons were incubated i n the enzyme mixture, plant p r o t o p l a s t s were produced but the fungus was l e f t i n t a c t . The epidermis and v e s s e l elements, however, were r e s i s t a n t to the a c t i o n of the enzymes and fragments of . . these o f t e n remained attached to i s o l a t e d c o l o n i e s . The pro-cedure can be s i m p l i f i e d by making up the enzyme mixture i n the medium used f o r axenic c u l t u r e of the r u s t . I f the r u s t was s p o r u l a t i n g at the time of i s o l a t i o n , the w a l l s . o f the host c e l l s adjacent to the f u n g a l c o l o n i e s were more d i f f i c u l t to d i g e s t i o n . The p e l l e t s of c o l o n i e s i s o l a t e d by procedure I were pale green because the three hour i n c u b a t i o n l e f t some apparently i n t a c t host c e l l s i n the center of the f u n g a l mass. When procedure IT was used many c o l o n i e s were v i r t u a l l y f r e e of contaminating host c e l l s (Figs.. I I I - l , I I I - 2 and I I I - 3 ) . A l l of the 82 c o l o n i e s p l a t e d on the agar medium grew, assumed a s p h e r i c a l shape and turned orange In c o l o u r . Microscopic- examination confirmed that urediospore development had occurred ( F i g . I l l - 4 ) . None of these spores were observed to. have germinated. Three c o l o n i e s continued to increase 'in s i z e , the orange spheres becoming about f o u r times the diameter of the o r i g i n a l l y p l a t e d colonies.. White 69 mycelium-began to develop from the base of these a f t e r about one month i n cul t u r e . a n d growth has continued a f t e r ' t r a n s f e r to f r e s h medium ( F i g . I I I - 5 ) . Small fragments of cotyledons, attached to the c o l o n i e s n e i t h e r i n h i b i t e d nor promoted fung a l growth. There i s l i t t l e chance that the axenic c u l t u r e s of. the f l a x r u s t fungus p r e s e n t l y a v a i l a b l e o r i g i n a t e from a s i n g l e spore, s i n c e s e v e r a l dozen spores i n c l o s e a s s o c i a t i o n are r e -quired to i n i t i a t e axenic growth (9) . I t has a l s o been suggested that anastomosis of germ tubes precedes the develop-ment of c o l o n i e s i n c u l t u r e (3). I n o c u l a t i n g cotyledons w i t h s i n g l e . s p o r e s as described by Fleischmann et a l . (4) and estab-l i s h i n g axenic c u l t u r e s from these i n f e c t i o n s using t h i s tech--nique would y i e l d c o l o n i e s derived from a s i n g l e urediospore. Kuhl et a l . (6) c u l t u r e d s e v e r a l races of P u c c i n i a graminis  t r i t i c i from urediospores but growth was i n c o n s i s t e n t . These authors suggested, that the t r a n s i t i o n from s p o r e l i n g t o vegeta-t i v e hyphae was the c r i t i c a l stage i n the establishment of c u l -t u res capable of continued growth. The procedure described here may prove u s e f u l f o r the establishment of axenic c u l t u r e s of other races of P. graminis as w e l l as other r u s t f u n g i which have not been grown apart, from t h e i r hosts, because i t . bypasses i n v i t r o s p o r e l i n g development and the i n i t i a t i o n of v e g e t a t i v e hyphae. 70 Figure I I I - l . . A c o l o n y . i s o l a t e d a f t e r , overnight i n c u b a t i o n i n the enzyme, s o l u t i o n . XU3 . Figure I I I - 2 . Enlargement of a colony s i m i l a r to t h a t i n Figure I I I - l . Note that most hyphal strands have not been severed during i s o l a t i o n . X 2 2 4 . Figure 111-3. I n t a c t h a u s t o r i a (H) i n c l u d i n g h a u s t o r i a l neck (Hn) attached to i s o l a t e d colony. X1828. Figure I I I - 4 . Urediospores i n the orange spheres which developed several- weeks a f t e r the i s o l a t e d c o l o n i e s were p l a t e d on axenic c u l t u r e medium. X224. Figure I I I - 5 . An axenic culture, which grew from an i s o -l a t e d colony. Photographed 2 weeks a f t e r the f i r s t t r a n s f e r and about 2 months '•' a f t e r the beginning of a x e n i c • c u l t u r e . X2.5. 72 REFERENCES 1. Chen, R.F. 1967." Removal of f a t t y a c i d s from serum albumin by charcoal treatment. J . B i o l . Chem. 242: 173-181.-. 2. Coffey, M.D., A. Boseand M. Shaw. 1970. I n v i t r o c u l -t u r e of f l a x r u s t , Melampsora l i n i . Can. J . Bot. 48: 773-776. • . 3. Coffey, 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 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: 37-46. 4. Fleischmann, G., J . Khair and A. Dinoor. .1966. Dry twi'st: a new system of c u l t u r i n g r u s t from s i n g l e spores. Can. J . Bot. 44: 1009-1013. 5. Gamborg, O.L., R.A. M i l l e r and K. Ojima. 1968. N u t r i e n t requirements of suspension c u l t u r e s of soybean root c e l l s Expt. C e l l . R e s . 50: I 5 I - I 5 8 . .6. Kuhl., J.L., D.J. Maclean, K.J. Scott and P.G. W i l l i a m s . 1971. The axenic c u l t u r e of P u c c i n i a species from uredio spores: experiments on i n h i b i t i o n and v a r i a t i o n . Can. J . Bot. 49? 2.01-209. 7. Maheshwari, R. 1966,. The physiology of p e n e t r a t i o n and i n f e c t i o n by. urediospores of r u s t f u n g i . Ph.D. Thesis,. . U n i v e r s i t y of Wisconsin, U n i v e r s i t y M i c r o f i l m s , L t d . , A Xerox,Co., Ann Arbor, Michigan. 8. O t s u k i , Y. and I . Takebe. 1969. I s o l a t i o n of i n t a c t mesophyll c e l l s and t h e i r p r o t o p l a s t s from higher p l a n t s . Plant & C e l l P h y s i o l . 10: 917-921. • . . 9. T u r e l , F.L.M. 1969. Saprophytic development of the f l a x r u s t Melampsora l i n i race no. 3 . Can. J . Bot. 47: 821-823 . 7 3 Chapter IV . I s o l a t i o n and Axenic Culture of Poplar Rust ;,; INTRODUCTION The d i f f i c u l t i e s of studying the h o s t : p a r a s i t e r e l a -t i o n s of the r u s t f u n g i are compounded because .they are ob-l i g a t e p a r a s i t e s and normally cannot grow apart from t h e i r host p l a n t s . Recently, s e v e r a l species of r u s t s have been s u c c e s s f u l l y grown by seeding uncontaminated urediospores onto appropriate media ( 1 , 5 ) . In t h i s report a technique i s described f o r the establishment, f o r the f i r s t time, of axenic c o l o n i e s of poplar r u s t from ur e d i o p u s t u l e s excised from i n f e c t e d leaves u s i n g a defined medium developed f o r f l a x r u s t ( 7 ) . L i k e the enzymatic technique f o r i s o l a t i o n and axenic growth of f l a x r u s t which we described e a r l i e r ( 3 ) the. ' e x c i s i o n ' technique does not r e q u i r e uncontaminated urediospores, and i s si m p l e r . .Poplar r u s t (Melampsora o c c i d e n t a l i s Pers.) i s a hetero-ecious rust, w i t h s e v e r a l a l t e r n a t e hosts ( 4 ) , i n which the u r e d i a l and t e l i a l stages occur on black cottonwood (Populus  t r i c h o c a r p a Torr. and Gray). 'Published i n Canadian J o u r n a l of Botany V o l . 5 2 : 2 2 2 8 - 2 2 3 0 , 1 9 7 4 . 7 4 MATERIALS AND METHODS Leaves of i n f e c t e d Cottonwood, growing on the.U.B.C. campus, were c o l l e c t e d ,at the beginning of October, when orange urediopustules were abundant, and s t e r i l i z e d by con-s e c u t i v e immersion i n 1 % sodium l a u r y l s u l p h a t e . ( 2 0 min), 7 0 % ethanol ( 2 min) and 1 % Na h y p o c h l o r i t e ( 5 min),, w i t h a thorough r i n s e i n d i s t i l l e d water a f t e r each treatment. S i n g l e r u s t p u s t u l e s , i n c l u d i n g . a surrounding zone of unin-f e c t e d t i s s u e 1 mm wide were excised and placed, s i x per p e t r i d i s h ( 6 0 mm diam.), on agar medium. The dishes were sealed w i t h p a r a f i l m and incubated a t l 7 ° C i n darkness. The medium (see legend, F i g . 1 ) was prepared at double concen-t r a t i o n , f i l t e r e d s t e r i l i z e d and mixed w i t h an equal volume of agar ( p u r i f i e d by 5 r i n s e s i n d i s t i l l e d water over 2 4 h, 1 r i n s e i n ethanol and 1 r i n s e i n chloroform). RESULTS AND DISCUSSION Pustules on the l e a f pieces doubled i n s i z e during the f i r s t week of i n c u b a t i o n , pale stromata developing t o pro-trude above the l e a f s u r f a c e . Three weeks l a t e r mycelium emerged from some pustules and a f t e r 4 months mycelium had grown onto the agar medium from 3 0 % . of the vpustules.. This mycelium developed from stromatic hyphae of the fungus,.not. from the germination of the urediospores o r i g i n a l l y present i n the excised pustule.. No growth of l e a f t i s s u e occurred. 75. Once e s t a b l i s h e d on the m e d i u m c o l o n i e s . were roughly semi-s p h e r i c a l i n shape ( F i g . IV-1) w i t h a compact stromatic center, i n which i r r e g u l a r l y shaped s p o r e - l i k e bodies were embedded ( F i g . IV-2).,.and w i t h a surface of f l u f f y white my-celium. Doubling i n s i z e occurred every three weeks during the f i r s t months of c u l t u r e , but subsequently took four or f i v e weeks. Colonies were t r a n s f e r r e d to f r e s h medium at .three-week i n t e r v a l s and b i s e c t e d when they reached 1.5 cm i n diameter. - Apparently normal urediospores were produced on the surfaces of some c o l o n i e s a f t e r three months i n c u b a t i o n ( F i g . IV-4). On exposure to continuous l i g h t (2570 l u x ) the my-, celium became orange i n colour.. To date, subcultures have. been maintained continuously f o r one year, w i t h l o s s e s o c c u r r i n g only when t r a n s f e r was i n f r e q u e n t . R e i n f e c t i o n of black cottonwood leaves by a x e n i c a l l y grown poplar r u s t was demonstrated as f o l l o w s . Newly emerged leaves 5 cm lo n g were surface s t e r i l i z e d and. a s t r i p (1 cm x 1 mm) cut from the edge of one lob e . Each l e a f was l a i d on the agar w i t h i t s cut edge i n contact w i t h a r u s t colony. The sealed dishes were then incubated i n the l i g h t (2570 l u x ) . The. fungus invaded the l e a f and a e r i a l mycelium grew from the cut surface a f t e r twenty days, then the colony which served as inoculum was removed ( F i g . IV-3).' Ten days l a t e r u r e d i o -pustules developed on the l e a f s u rfaces; urediospores c o l l e c t e d from these pustules germinated normally. 76 The technique was al s o t e s t e d using s e v e r a l other species of. r u s t . These were Hollyhock r u s t ( P u c c i n i a malva-cearum), snapdragon r u s t ( P u c c i n i a a n t i r r h i n i ) , mint r u s t ( P u c c i n i a mentha), t h i s t l e r u s t ( P u c c i n i a suaveolens), wheat r u s t ( P u c c i n i a graminis t r i t i c i ) races ANZ-126 and 15B, and bean r u s t (Uromyces phaseolus)' race PRE-2. S u i t a b l e pustules were processed as described above and p l a t e d onto the medium used w i t h the poplar r u s t and a defined medium p r e v i o u s l y used s u c c e s s f u l l y t o grow wheat r u s t (1). I t was not p o s s i b l e to e s t a b l i s h v e g e t a t i v e c o l o n i e s of any of these r u s t s u s i n g the new technique. However, the wheat r u s t pustules (both races) grew to.a l i m i t e d extent when used as inoculum and p l a t e d on the medium p r e v i o u s l y used to grow wheat r u s t a x e n i c a l l y u s i n g urediospores as i n -oculum. The growth was s i m i l a r to the i n i t i a l growth of f l a x r u s t (described i n Chapter I I I ) . Dark brown i r r e g u l a r l y shaped spheres grew from the inoculum i n c r e a s i n g i n s i z e to a maximum of'"0.5 cm. The spheres were composed of i r r e g u l a r s p o r e - l i k e bodies intermixed w i t h hyphae. The growth of the. wheat r u s t stopped about a month a f t e r the beginning of i n c u b a t i o n . The enzymatic (3) and e x c i s i o n techniques both by-pass s p o r e l i n g development and the i n i t i a t i o n of vegetative,hyphae i n v i t r o . They may t h e r e f o r e be u s e f u l i n e s t a b l i s h i n g axenic c u l t u r e s of species of r u s t f u n g i which are d i f f i c u l t 77 to grow from urediospores seeded d i r e c t l y onto the medium. Use. of e i t h e r technique, f o l l o w i n g s i n g l e spore i n o c u l a t i o n s . (2) of the host, would y i e l d axenic c u l t u r e s , and u l t i m a t e l y c l o n e s , each derived o r i g i n a l l y from a s i n g l e urediospore. Since t h i s cannot be accomplished by mass seeding of s u i t -able media with.urediospores, the procedure we describe should be u s e f u l i n s t u d i e s on the biochemical genetics of the r u s t f u n g i . The success of both the techniques described i n Chapters I I I and IV depends on the use of a s u i t a b l e medium which w i l l support the growth of the p a r t i c u l a r species of r u s t under i n v e s t i g a t i o n . 78 Figure IV-1. T y p i c a l colony of Melampsora o c c i d e n t a l i s three months a f t e r i s o l a t i o n . Grown on the f o l l o w i n g medium ( i n g / l ) : KNOy, 0.2$; MgS0 4-7H 20, 0 .25; KH 2P0 4 ,: 0.25 ; KgHPO/,., 0 .75; NH4NO3, 0.02; Cat NO3 ) 2 ' 4 ^ 0 , 2.0; sucrose, 50; a s p a r t i c a c i d , 5.99; c y s t e i n e , 0.558; plus 0.8 ml of m i c r o n u t r i e n t stock • s o l u t i o n c o n t a i n i n g i n mg/200 ml): 13% NaFe (Geigy), 10,000; MnSO^-HgO, 447; KI, 10; NiCl 2.-6H 20, 18; CoCl 2-6H 20, 18; Ti(S0 / t_) 2-9H 20, 42; ZnS0^-7H 20, 35; CuSO^-5H 20, 15; BeSO/,., 20; H3PO4 (85%), 10; H2S.O^ (cone .) , 0.2 ml. Figure IV-2. T y p i c a l mycelium obtained from axenic c u l t u r e s . S p o r e - l i k e bodies were o f t e n observed. X300. Figure TV-3. R e i n f e c t i o n of black cottonwood l e a v e s . S p o r u l a t i o n of the pustule i n f e c t i n g the . l e a f occurred one week a f t e r t h i s photo-graph was taken. X2. Figur e I V - 4 . Spores produced by axenic c u l t u r e s of poplar r u s t . X500. 80 REFERENCES 1 . -Bose, A, and .M. Shaw. 1 9 7 4 . I n v i t r o growth of wheat and f l a x r u s t f u n g i on complex and che m i c a l l y defined .media. Can.'J. Bot. 52:1118-1195 2 . Fleischmann, G., J . Khair and A. Dinoor. . 1 9 6 6 . Dry tw i s t : , a new system of c u l t u r i n g r u s t from s i n g l e spores. Can. J . Bot. 4 4 : 1 0 0 9 - 1 0 1 3 . 3 . Lane, W.D. and M. Shaw. 1 9 7 2 . Axenic c u l t u r e of f l a x r u s t i s o l a t e d from cotyledons by c e l l w a l l d i g e s t i o n . Can. J . Bot. 5 0 : 2 6 0 1 - 2 6 0 3 . . 4 . Molnar, A.C. and B. Sivak. 1 9 6 4 . Melampsora i n f e c t i o n of pine i n . B r i t i s h Columbia. Can. J . Bot. 4 2 : 1 4 5 - 1 5 8 . 5 . S c o t t , K.J. and D.J. Maclean. 1 9 6 9 . C u l t u r i n g of r u s t f u n g i . Ann. Rev. Phytopathology 6 : 1 2 3 - 1 4 6 . 81 SUMMARY . 1. F l a x p r o t o p l a s t s were i s o l a t e d from cotyledons. Optimum co n d i t i o n s of d i g e s t i o n , washing-and manipulation were, determined so t h a t preparations c o n s i s t i n g of 100% proto-p l a s t s w i t h an average of 75% a l i v e and healthy were c o n s i s t e n t l y obtained. 2. • A p r o t o p l a s t b u r s t i n g bioassay procedure, based on pre-v i o u s l y published work, was developed and the c o n d i t i o n s of I t s use determined so that c o n s i s t e n t and u s e f u l r e s u l t s were obtained from i t . 3... The theory of fungal t o x i n involvement i n the phytopath-ogenic sequence i n v o l v e d i n r u s t p a r a s i t i s m was t e s t e d u s i n g the p r o t o p l a s t b u r s t i n g bioassay and exudates from germinating spores and pathogenic axenic c u l t u r e s . 4. Evidence arguing .against the f u n g a l t o x i n theory was ob-, .tained and an a l t e r n a t e e x p l a n a t i o n , c o n s i s t e n t w i t h the observations which suggested t h i s theory, was proposed. 5. Other current t h e o r i e s of s u s c e p t i b i l i t y and r e s i s t a n c e to o b l i g a t e pathogens were discussed i n r e l a t i o n to the evidence obtained from, the p r o t o p l a s t b u r s t i n g bioassay. 6. Factors i n f l u e n c i n g f l a x p r o t o p l a s t s maintained i n c u l -. t.ure f o r as l o n g as f o r t y days were i n v e s t i g a t e d . 7. By v a r y i n g these f a c t o r s a d i v e r s i t y of types of develop-ment occurred i n c l u d i n g d i v i s i o n of c e l l s o r i g i n a t i n g from p r o t o p l a s t s i s o l a t e d from f l a x cotyledons. 82 8. A technique was developed f o r i s o l a t i n g i n t a c t . r u s t colonies, from p a r a s i t i z e d l e a v e s . The b a s i s of t h i s technique i s the s e l e c t i v e enzymatic d i g e s t i o n of host c e l l ' w a l l s . 9. E n z y m a t i c a l l y i s o l a t e d c o l o n i e s were a l i v e a f t e r i s o l a t i o n and could be used as inoculum to- e s t a b l i s h axenic c u l t u r e s of f l a x r u s t . 10. A. new method, simpler and w i t h s e v e r a l d i s t i n c t advan-tages over the two t r a d i t i o n a l l y used methods was developed, the b a s i s of which was the use of.pu s t u l e s growing i n p a r a s i t i z e d leaves as inoculum. 11. Poplar r u s t was grown.for the f i r s t time u s i n g t h i s method. 12. The poplar r u s t c u l t u r e s were maintained i n s e r i a l sub c u l t u r e f o r a year. The c o l o n i e s were shown to be. pathogenic and to produce spores i r r e g u l a r l y . 

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