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Variation in resistance and virulence in the disease interaction between Melampsora rust (M. occidentalis).. 1984

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VARIATION IN RESISTANCE AND VIRULENCE IN THE DISEASE INTERACTION BETWEEN MELAMPSORA RUST (KL OCCIDENTAL!S) AND BLACK COTTONWOOD (POPULUS TRICHOCARPA) By TOM HSIANG B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of f o r e s t r y ) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1984 © Tom Hsiang, 1984 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s m a y b e g r a n t e d b y t h e H e a d o f m y D e p a r t m e n t o r b y h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t m y w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f F o r e s t r y T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 2 0 7 5 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V 6 T 1 W 5 D a t e : JUNE 22, 1984 V a r i a t i o n i n r e s i s t a n c e and v i r u l e n c e i n the dis e a s e i n t e r a c t i o n between Melampsora ru s t (M. o c c i d e n t a l i s ) and black cottonwood (Populus t r i c h o c a r p a ) . - ABSTRACT Disease s e v e r i t y , as expressed by spore p r o d u c t i o n r a t e was compared i n a t e s t of fourteen c l o n e s of black cottonwood (Populus t r ichocarpa) by ten i s o l a t e s of Melampsora ru s t (M. o c c i d e n t a l i s ) , a l l c o l l e c t e d from t h e i r n a t u r a l pathosystem. Spore pr o d u c t i o n r a t e was measured by average d a i l y p r o d u c t i o n on l e a f d i s k s d u r i n g twice the l a t e n t p e r i o d in days. The o v e r a l l average uredospore pr o d u c t i o n d u r i n g the time from i n o c u l a t i o n to twice the l a t e n t p e r i o d was 650 spores/disk/day. Latent p e r i o d ranged from 6 to 12 days with a median at 8 days. Clones as w e l l as i s o l a t e s d i f f e r e d s i g n i f i c a n t l y i n t h e i r c o n t r i b u t i o n s to spore p r o d u c t i o n r a t e s , while there was no i n d i c a t i o n of s p e c i f i c d i f f e r e n t i a l i n t e r a c t i o n . The lack of q u a l i t a t i v e r e s i s t a n c e and v i r u l e n c e i n d i c a t e s that q u a l i t a t i v e i n t e r a c t i o n s do not pl a y a major r o l e i n d i s e a s e i n t h i s n a t u r a l pathosystem. T h i s f i n d i n g holds the promise that black cottonwood r e s i s t a n c e w i l l not be d e v a s t a t i n g l y overcome when used i n p l a n t a t i o n s . TABLE OF CONTENTS INTRODUCTION h o s t r p a r a s i t e g e n e t i c s 4 LITERATURE REVIEW q u a l i t a t i v e r e s i s t a n c e 9 q u a n t i t a t i v e r e s i s t a n c e 10 METHODS sample s i z e 11 host s e l e c t i o n and t e l i a c o l l e c t i o n 12 uredospore inoculum p r e p a r a t i o n 14 v i r u l e n c e and r e s i s t a n c e t r i a l s 16 r a t i n g system 21 RESULTS AND DISCUSSION a n a l y s i s of v a r i a n c e 25 p h y s i o l o g i c a l s p e c i a l i z a t i o n 28 e f f e c t s of di s e a s e 30 n a t u r a l pathosystems 32 CONCLUSIONS 36 LITERATURE CITED 37 APPENDIX A: terms used to c l a s s i f y r e s i s t a n c e 43 APPENDIX B: l i s t i n g of a l l c l o n e s and i s o l a t e s with i s o l a t e c o l l e c t i o n and spore formation dates 44 APPENDIX C: l e a f age s u s c e p t i b i l i t y study 45 APPENDIX D: graph: mean average d a i l y spore p r o d u c t i o n of each p e t r i d i s h vs. inoculum c o n c e n t r a t i o n a p p l i e d to each p e t r i d i s h 46 APPENDIX E: graph of haemocytometer spore count v s . l i g h t absorbance of the same spore suspension 47 APPENDIX F: a n a l y s i s of v a r i a n c e f o r the dis e a s e s e v e r i t y parameters: average d a i l y spore p r o d u c t i o n , average d a i l y u r e d i a p r o d u c t i o n , t o t a l spores, and l a t e n t p e r i o d 48 APPENDIX G: a n a l y s i s of v a r i a n c e f o r s i g n i f i c a n t i n t e r a c t i o n . . 4 9 APPENDIX H: c a l c u l a t i o n s f o r the components of v a r i a n c e 50 i v LIST OF FIGURES FIGURE 1: l i f e c y c l e of Melampsora o c c i d e n t a l i s 3 FIGURE 2: a s i n g l e Melampsora r u s t i s o l a t e was a p p l i e d to each of the four t e e n c l o n e s of black cottonwood represented by 17 mm l e a f d i s k s i n each d i s h 17 V LIST OF TABLES TABLE I: a n a l y s i s of v a r i a n c e f o r average spore production d u r i n g twice the l a t e n t p e r i o d 26 TABLE I I : average d a i l y spore p r o d u c t i o n d u r i n g twice the l a t e n t p e r i o d f o r a l l i s o l a t e s on a l l c l o n e s 28 TABLE I I I : components of v a r i a n c e f o r the sources: i s o l a t e s , c l o n e s , b l o c k s , i n t e r a c t i o n , and e r r o r 30 ACKNOWLEDGEMENTS I s i n c e r e l y thank Dr. Bart van der Kamp f o r h i s guidance and unending p a t i e n c e . Thanks a l s o go to Dr. Jeanette Leach and Dr. Raoul Robinson f o r t h e i r c r i t i c a l examination of t h i s t h e s i s , and to Dr. Clayton Person f o r the d i s c u s s i o n s on h o s t r p a r a s i t e g e n e t i c s . T h i s r e s e a r c h was conducted while I was under an NSERC f e l l o w s h i p . The photos of a e c i a and pyc n i a on page 3 were taken by Richard McBride. 1 INTRODUCTION As a r u l e , a p a r a s i t e s p e c i e s has a very l i m i t e d range of host s p e c i e s on which i t can s u r v i v e ; but sometimes a p a r a s i t e s p e c i e s i s so s p e c i a l i z e d on a host s p e c i e s that only c e r t a i n p a r a s i t e races can p a r a s i t i z e a p a r t i c u l a r host v a r i e t y . Knowledge of t h i s s p e c i f i c i t y or p h y s i o l o g i c a l s p e c i a l i z a t i o n may be very important to the p l a n t breeder who seeks to produce v a r i e t i e s with r e s i s t a n c e to d i s e a s e , and to the p l a n t p a t h o l o g i s t who wishes to understand the source of d i s e a s e s t a b i l i t y . In a g r i c u l t u r a l c r o p s , one commonly observed phenomenon i s that r e s i s t a n c e i n c o r p o r a t e d i n t o c u l t i v a r s i s overcome w i t h i n a few seasons through p h y s i o l o g i c a l s p e c i a l i z a t i o n by the pathogen. But, there are cases i n which r e s i s t a n c e has not been l o s t or even decreased over decades. In such cases, the l o s s of r e s i s t a n c e through p h y s i o l o g i c a l s p e c i a l i z a t i o n of the p a r a s i t e a p p a r e n t l y cannot or does not occur. Thus, the r e c o g n i t i o n of the type of r e s i s t a n c e (Appendix A) i n c e r t a i n v a r i e t i e s stems from the h i s t o r y of t h e i r behaviour through p e r i o d s of d i s e a s e . With p o p l a r s and f o r e s t t r e e s i n g e n e r a l , such records are mostly n o n - e x i s t e n t . T h i s s o r t of data may be most q u i c k l y gathered i n the l a b o r a t o r y through a r t i f i c i a l i n o c u l a t i o n s i n order to assess the type of d i s e a s e i n t e r a c t i o n i n v o l v e d . The purpose of t h i s study i s to determine the type and extent of v a r i a t i o n i n r e s i s t a n c e of Populus t r i c h o c a r p a T o r r . & Gray, and i n v i r u l e n c e of Melampsora o c c i d e n t a l i s Jacks, toward each other, when sampled from t h e i r n a t u r a l pathosystem. Answers to two q u e s t i o n s are sought: f i r s t l y , i s there 2 p h y s i o l o g i c a l s p e c i a l i z a t i o n ( v a r i a t i o n i n v i r u l e n c e or r e s i s t a n c e s p e c i f i c to p a r t i c u l a r host c l o n e s or pathogen i s o l a t e s ) such that r u s t i s o l a t e s can be d i s t i n g u i s h e d by s p e c i f i c d i f f e r e n t i a l r e a c t i o n s on a s e r i e s of host c l o n e s , and v i c e versa? Secondly, does t h i s degree of p h y s i o l o g i c a l s p e c i a l i z a t i o n play a major r o l e i n t h i s w i l d pathosystem? I s e l e c t e d t h i s pathosystem f o r two reasons. F i r s t l y , I wanted to study h o s t r p a r a s i t e i n t e r a c t i o n s i n a n a t u r a l system where the host had not been under c u l t i v a t i o n and put i n t o p l a n t a t i o n s . N a t u r a l l y growing black cottonwood ( P. t r i c h o c a r p a ) was e a s i l y a c c e s s i b l e and had the h i g h l y v i s i b l e Melampsora l e a f r u s t . Secondly, I b e l i e v e that black cottonwood w i l l become an important commercial s p e c i e s i n western North America i n the near f u t u r e . P o p l a r s are e x c e l l e n t as short r o t a t i o n i n t e n s i v e c u l t u r e p l a n t a t i o n t r e e s (Hansen et a l . 1983, McNabb et a l . 1983), and black cottonwood i s the l a r g e s t of the North American p o p l a r s (Fowells 1965). Black Cottonwood belongs to the poplar s e c t i o n Tacamahaca (FAO/IPC 1980) and i s e a s i l y propagated both s e x u a l l y and a s e x u a l l y (Muhle-Larsen 1970). I t s n a t u r a l range extends along the P a c i f i c coast from Alaska to northern C a l i f o r n i a and east to Montana. I t i s mostly found on bottom land, r i v e r bars, and f o r e s t meadows and streambanks, but i t occurs throughout the p l a t e a u lands i n n o r t h - c e n t r a l B r i t i s h Columbia (Fowells 1965). Melampsora o c c i d e n t a l i s (Melampsora r u s t ) i s a h e t e r o e c i o u s e u c y c l i c r u s t . I t s l i f e c y c l e ( f i g u r e 1) i s as f o l l o w s : In s p r i n g , d i k a r y o t i c t e l i o s p o r e s , which overwintered on dead 3 PYCNIAL DROPLETS exuded by mo n o k a r y o t i c m y c e l i a from b a s i d i o s p o r e I n f e c t i o n s c o n t a i n p y c n i o s p o r e s t h a t a r e t r a n s f e r r e d between p y c n i a of d i f f e r e n t m ating types i n s p r i n g BASIDIOSPORES (X 750) i n s p r i n g I n f e c t c o n i f e r n e e d l e s TELIAL CRUSTS o v e r w i n t e r on f a l l e n l e a v e s . T e l i o s p o r e s g e r m i n a t e i n s p r i n g to g i v e b a s i d i a which germinate to g i v e m o n o k a r y o t i c b a s i d i o s p o r e * TELIA formed by d i k a r y o t i c m y c e l i a i n e a r l y f a l l A EC IA WITH LOOSE AECIOSPORES d i k a r y o t i c a e c i o s p o r e s formed from m y c e l i a a f t e r p y c n i a l t r a n s f e r i n f e c t b l a c k cottonwood l e a v e s i n l a t e s p r i n g UREDIA of h e a v i l y i n f e c t e d l e a f 1n l a t e summe"- UREDIA formed by d i k a r y o t i c m y c e l i a from a e c i o s p o r e i n f e c t i o n s or l a t e r from u r e d o s p o r e r e - 1 n f e c t 1 o n s produce u r e d o s p o r e s throughout summer UREDOSPORE (X 200) BESIDE ST0MATES w i l l form germ tube to p e n e t r a t e l e a f t h r o u g h open stomate on a b a x i a l s u r f a c e FIGURE 1 : L i f e - c y c l e of o c c i d e n t a l i s (read c o u nter-clockwise) 4 leaves of black cottonwood, undergo karyogamy and meiosis, and then germinate to produce b a s i d i a . These b a s i d i a produce h a p l o i d b a s i d i o s p o r e s which can i n f e c t the needles of Douglas- f i r ( Pseudotsuga m e n z i e s i i (Mirb.) Franco) and other c o n i f e r s to grow monokaryotic mycelia which form p y c n i a . A f t e r f e r t i l i z a t i o n by p y c n i a l t r a n s f e r , d i k a r y o t i c a e c i a develop on the same needles, and a e c i o s p o r e s are produced. These d i k a r y o t i c a e c i o s p o r e s i n f e c t black cottonwood leaves beginning i n l a t e s p r i n g . The i n f e c t i o n s g i v e r i s e to d i k a r y o t i c mycelia which produce u r e d i a and uredospores to r e i n f e c t black cottonwood l e a v e s . In l a t e f a l l , the d i k a r y o t i c mycelia form d i k a r y o t i c t e l i a which overwinter on the f a l l e n black cottonwood l e a v e s . T h i s r u s t s p e c i e s can be manipulated s e x u a l l y ( p y c n i a l t r a n s f e r ) and a s e x u a l l y ( r e p e a t i n g uredospores). ( Z i l l e r 1974). H o s t : P a r a s i t e G e n e t i c s The terms used i n t h i s paper to d e s c r i b e h o s t : p a r a s i t e r e l a t i o n s h i p s are d e f i n e d here. R e s i s t a n c e i s the host component of d i s e a s e i n t e r a c t i o n ; i t r e p r e s e n t s the a b i l i t y of a host to hinder i t s p a r a s i t e . V i r u l e n c e i s the p a r a s i t e component of d i s e a s e i n t e r a c t i o n ; i t denotes the a b i l i t y of a p a r a s i t e to reproduce on i t s host. R e s i s t a n c e , v i r u l e n c e , and the d i s e a s e i n t e r a c t i o n may appear to vary q u a l i t a t i v e l y or q u a n t i t a t i v e l y , depending on t h e i r g e n e t i c b a s i s and the ease with which they may be observed ( S c o t t et a l . 1978 p.29). The g e n e t i c and e p i d e m i o l o g i c a l a t t r i b u t e s of q u a l i t a t i v e and q u a n t i t a t i v e i n t e r a c t i o n s a l s o vary g r e a t l y by the a u t h o r i t y (Appendix A). 5 Q u a l i t a t i v e i n t e r a c t i o n s occur i n systems where di s e a s e i n t e r a c t i o n s f a l l i n t o d i s t i n c t non-overlapping c l a s s e s . These are commonly termed compatible or incompatible, and may represent 100% or 0% d i s e a s e c l a s s e s r e s p e c t i v e l y ; or i n cases where the d i s e a s e i n t e r a c t i o n i s not an a l l or nothing s i t u a t i o n , the r e a c t i o n can s t i l l be grouped i n t o two c l a s s e s , for example above 60% and below 40% d i s e a s e . In any case, the d i f f e r e n t d i s e a s e s e v e r i t i e s should be rep e a t a b l y and s i g n i f i c a n t l y d e t e c t a b l e . T h i s i s simply i l l u s t r a t e d by a t a b l e : HOST CLONE + compatible i n t e r a c t i o n A B C (d i s e a s e occurs) - incompatible i n t e r a c t i o n a + - - ( l i t t l e or no d i s e a s e present) The d i f f e r e n t i a l i n t e r a c t i o n occurs PATHOGEN b - + - here s i n c e the cl o n e s are d i f f e r e n - ISOLATE t i a l l y r e s i s t a n t and the i s o l a t e s c - - + are d i f f e r e n t i a l l y v i r u l e n t . The q u a l i t a t i v e i n t e r a c t i o n forms the b a s i s of r e c o g n i t i o n of p h y s i o l o g i c a l s p e c i a l i z a t i o n i n pathogen races and host v a r i e t i e s . Races are thus d i s t i n g u i s h e d by d i f f e r e n t i a l r e a c t i o n s on host v a r i e t i e s as i n the preceding t a b l e . Q u a l i t a t i v e i n t e r a c t i o n s have been c o n c l u s i v e l y demonstrated to a r i s e from gene-for-gene i n t e r a c t i o n s i n a few pathosystems (Sidhu 1980). Accor d i n g to the gene-for-gene r e l a t i o n s h i p between a pathogen race and a host v a r i e t y as p o s t u l a t e d by F l o r (1955), a race w i l l be pathogenic to a v a r i e t y i f i t possesses v i r u l e n c e genes which correspond to a l l the r e s i s t a n c e genes i n the v a r i e t y . A race w i l l be non-pathogenic or a v i r u l e n t on a 6 v a r i e t y i f the v a r i e t y possesses at l e a s t one r e s i s t a n c e gene a g a i n s t which the race has no corresponding v i r u l e n c e gene. Thus a t e s t i n g of v a r i o u s s p e c i f i e d races a g a i n s t a host v a r i e t y w i l l g i v e i n f o r m a t i o n on the e x i s t e n c e of p a r t i c u l a r genes f o r r e s i s t a n c e i n the v a r i e t y . The other major i n t e r a c t i o n type i s q u a n t i t a t i v e . Disease s e v e r i t y must be r a t e d or q u a n t i t a t i v e l y measured as i t does not f a l l i n t o d i s t i n c t c l a s s e s . The commonly a s c r i b e d f e a t u r e s of t h i s q u a n t i t a t i v e i n t e r a c t i o n a r e : polygenes, constant ranking, n o n - s p e c i f i c i t y , and s t a b i l i t y i n the i n t e r a c t i o n (Fleming & Person 1982). By constant ranking, a s e r i e s of pathogenic races can be ranked f o r v i r u l e n c e , and t h i s ranking remains c o n s i s t e n t on any host v a r i e t y ; as w e l l , host v a r i e t i e s can be ranked and w i l l maintain the same ranking when t e s t e d a g a i n s t any pathogen race. I t i s q u i t e p o s s i b l e to f i n d minor q u a l i t a t i v e e f f e c t s super-imposed over a b a s i c q u a n t i t a t i v e system. T h i s i s w e l l p resented i n a t a b l e by Sc o t t et a l . (1978 p.33): VARIETY ISOLATES WYR 69/10 WYR 71/2 Hybrid 46 130 111 Joss Cambier 44 172 Q u a n t i t a t i v e i n t e r a c t i o n with reversed ranking, mg spores per 100 square cm l e a f produced on s e e d l i n g s of two wheat v a r i e t i e s by two i s o l a t e s of P u c c i n i a l e a f r u s t . However the q u a l i t a t i v e d i f f e r e n t i a l i n t e r a c t i o n may not always be s t a t i s t i c a l l y s i g n i f i c a n t , such as was found i n the example above (Sco t t et a l . 1978); and even when s t a t i s t i c a l l y s i g n i f i c a n t , the d i f f e r e n t i a l i n t e r a c t i o n may have l i t t l e 7 e p i d e m i o l o g i c a l impact, as h o s t - s p e c i f i c i t y here g i v e s only a s l i g h t advantage to the adapted race. The r e l a t i v e importance of q u a l i t a t i v e vs. q u a n t i t a t i v e e f f e c t s can be assessed by a n a l y z i n g the components of v a r i a n c e . I t i s easy to imagine e i t h e r p o s s i b i l i t y , q u a l i t a t i v e or q u a n t i t a t i v e o c c u r i n g i n t h i s Melampsora-Populus pathosystem. If a q u a l i t a t i v e system e x i s t s , such that cottonwood c o n s i s t s of a number of d i s t i n c t host v a r i e t i e s which can be d e f i n e d by t h e i r d i f f e r e n t i a l r e s i s t a n c e to a s e r i e s of pathogen races, then there would be i n e f f e c t a n a t u r a l m u l t i l i n e ; each host tr e e or c l o n a l group would be s u s c e p t i b l e to some races but r e s i s t a n t to o t h e r s . S e l e c t i o n and t e s t i n g f o r r e s i s t a n c e c o u l d be d i f f i c u l t s i n c e c e r t a i n c l o n e s might appear r e s i s t a n t simply because the t e s t d i d not expose them to p a r a s i t e races to which they were h i g h l y s u s c e p t i b l e . I f i t were not known that q u a l i t a t i v e i n t e r a c t i o n s c o u l d occur i n t h i s pathosystem, and i f these a p p a r e n t l y r e s i s t a n t c l o n e s were grown in p l a n t a t i o n s , then v i r u l e n t races would l i k e l y appear sooner or l a t e r and the q u a l i t a t i v e r e s i s t a n c e would be l o s t , p o s s i b l y before the p l a n t a t i o n reached m a t u r i t y . (Kiyosawa 1982 p. 1 1 2 ) . On the other hand, the system might be q u a n t i t a t i v e with t y p i c a l constant ranking. Experience with a g r i c u l t u r a l pathosystems has shown that such r e s i s t a n c e , though incomplete, i s q u i t e s t a b l e and l o n g - l a s t i n g . (Vanderplank 1982 p.77, Fry 1982 p. 200) . 8 For t h i s study, black cottonwood host c l o n e s w i l l be t e s t e d a g a i n s t Melampsora pathogen i s o l a t e s , and a n a l y s i s of v a r i a n c e p l u s a n a l y s i s of the components of v a r i a n c e should show whether the d i s e a s e i n t e r a c t i o n s d i f f e r s i g n i f i c a n t l y and whether there are s p e c i f i c d i f f e r e n t i a l i n t e r a c t i o n s . But no matter the outcome, i t i s not p o s s i b l e to conclude that there are no q u a l i t a t i v e genes for r e s i s t a n c e and v i r u l e n c e i n t h i s pathosystem. P o s s i b l y only a few t r e e s c a r r y s p e c i f i c powerful r e s i s t a n c e genes and only a few pathogen races the corresponding v i r u l e n c e genes. However, i f an a n a l y s i s of v a r i a n c e shows no s i g n i f i c a n t i n t e r a c t i o n , or i f the components of v a r i a n c e show that the i s o l a t e - c l o n e i n t e r a c t i o n term i s small r e l a t i v e to the main e f f e c t s , i s o l a t e s and c l o n e s , then i t may be concluded that such genes do not p l a y a major r o l e i n t h i s n a t u r a l pathosystem. The n u l l h y p o t h e s i s can then be s t a t e d as f o l l o w s : q u a l i t a t i v e d i f f e r e n t i a l i n t e r a c t i o n s p l a y a major r o l e i n t h i s n a t u r a l M;_ o c c i d e n t a l i s - P^ t r ichocarpa pathosystem. LITERATURE REVIEW Many i n v e s t i g a t o r s have shown the v a r i a b i l i t y of the Populus-Melampsora complex. The s e v e r i t y of Melampsora r u s t i s a f u n c t i o n of a complex of g e n e t i c (Heather & Sharma 1977), ontogenetic (Sharma, Heather & Winer 1980), and v a r i o u s environmental f a c t o r s such as temperature and l i g h t i n t e n s i t y (Heather & Chandrashekar 1982). These A u s t r a l i a n s t u d i e s were 9 done with l a r i c i - p o p u l i n a on h y b r i d p o p l a r s . Shain (1976) with M̂_ medusae on p l a n t a t i o n d e l t o i d e s i n eastern North America, and S p i e r s (1978) with M^ medusae and M_;_ l a r i c i - p o p u l i n a on h y b r i d p o p l a r s i n New Zealand reached s i m i l a r c o n c l u s i o n s . However, no e x t e n s i v e work has been done with the M. o c c i d e n t a l i s - P_j_ t r ichocarpa pathosystem. J o k e l a (1966), Wilcox & Farmer (1967), Farmer & Wilcox (1968), and Farmer (1970) report that the h e r i t a b i l i t y of r u s t r e s i s t a n c e i n the n a t i v e Populus d e l t o i d e s Marsh, p o p u l a t i o n toward Melampsora medusae Thuem. i s h i g h . However T h i e l g e s & Adams (1975) r i g h t l y p o i n t out that h e r i t a b i l i t y e stimates are of no value where dominance between genes occurs. There are r e p o r t s of both q u a l i t a t i v e and q u a n t i t a t i v e r e s i s t a n c e i n the Populus-Melampsora complex. Q u a l i t a t i v e R e s i s t a n c e P h y s i o l o g i c races i n Melampsora l a r i c i - p o p u l i n a Klebahn. were f i r s t r e p o r t e d by van V l o t e n (1949). Then Muhle-Larsen (1963) r e p o r t e d that progeny r e s u l t i n g from many c r o s s e s of r e s i s t a n t and s u s c e p t i b l e phenotypes of d e l t o i d e s and P. n i g r a L. showed Mendelian se g r e g a t i o n with respect to r e s i s t a n c e toward Melampsora l e a f r u s t . Chandrashekar & Heather (1980) demonstrate r a c i a l s p e c i a l i z a t i o n through the r e a c t i o n of s e v e r a l c l o n e s of P. d e l t o i d e s to mono-uredospore i s o l a t e s of M_̂  l a r i c i - p o p u l i n a . S e v e r a l pathogen races c o u l d be r e c o g n i z e d by q u a l i t a t i v e d i s t i n c t r e a c t i o n s on s e v e r a l c l o n e s of d e l t o i d e s . For Melampsora populnea (Pers.) K a r s t . , Gremmen (1980) 10 r e p o r t s t h a t d i f f e r e n t i a l r e a c t i o n s o c c u r f o r a e c i o s p o r e s f r o m d i f f e r e n t a e c i a l h o s t s p e c i e s , s o t h a t t h e r e i s a t l e a s t s p e c i e s s p e c i f i c i t y i f n o t r a c e s p e c i f i c i t y . F i n a l l y , a 1 9 8 0 F A O / I P C r e p o r t s t a t e s t h a t t h e r e s u l t s o f c r o s s i n g d e l t o i d e s w i t h P ^ n i g r a a n d t r i c h o c a r p a a f e w g e n e r a t i o n s r e m o v e d f r o m t h e i r w i l d a n c e s t o r s i n d i c a t e t h a t r e s i s t a n c e t o M e l a m p s o r a r u s t i s a d o m i n a n t c h a r a c t e r w h i c h d e p e n d s o n a r e l a t i v e l y s m a l l n u m b e r o f g e n e s ( F A O / I P C 1 9 8 0 ) . Q u a n t i t a t i v e R e s i s t a n c e T h i e l g e s & A d a m s ( 1 9 7 5 ) r e p o r t m u c h v a r i a t i o n i n r e s i s t a n c e t o M e l a m p s o r a r u s t a m o n g a n d b e t w e e n f a m i l i e s o f o p e n g r o w n h a l f - s i b s e e d l i n g s o f P_^ d e l t o i d e s c o l l e c t e d f r o m s e v e r a l e a s t e r n N o r t h A m e r i c a n s t a t e s . P r o n o u n c e d d i f f e r e n c e s i n r u s t d a m a g e a n d r a t e o f p r o g r e s s i o n w e r e r e l a t e d t o g e o g r a p h i c o r i g i n o f t h e s e e d p a r e n t , t h u s t h e r e w a s a g e n e r a l c o n s t a n t r a n k i n g b y t h e l o c a l w i l d r u s t p o p u l a t i o n , w h i c h i s i n d i c a t i v e o f q u a n t i t a t i v e r e s i s t a n c e . E l d r i d g e e t a l . ( 1 9 7 3 ) r e p o r t t h a t p o p l a r c u l t i v a r s i n A u s t r a l i a d i f f e r e d i n r e s i s t a n c e w i t h t h e g r e a t e s t v a r i a t i o n i n d i s e a s e s e v e r i t y b e t w e e n p r o v e n a n c e s a n d m u c h l e s s w i t h i n . A g a i n t h e r e i s a g e n e r a l c o n s t a n t r a n k i n g o f p r o v e n a n c e s . T h r o u g h t e s t i n g o f f o u r M ; _ l a r i c i - p o p u l i n a i s o l a t e s a g a i n s t f o u r P o p l a r c u l t i v a r s , H e a t h e r , S h a r m a & M i l l e r ( 1 9 8 0 ) s u g g e s t t h a t t h e r e s i s t a n c e i s p o l y g e n i c a l l y b a s e d a n d c a u s e d b y m i n o r g e n e s w h i c h i n t e r a c t w i t h g e n e s i n t h e p a t h o g e n , a l t h o u g h n o g e n e t i c s t u d i e s w e r e d o n e i n t h i s e x p e r i m e n t . 11 METHODS There have been no p r e v i o u s s t u d i e s with t h i s p a r t i c u l a r pathosystem to search f o r p h y s i o l o g i c a l s p e c i a l i z a t i o n . S e v e r a l such s t u d i e s , however, have been done i n other Melampsora- Populus pathosystems, but the d i f f e r e n c e s between t h i s pathosystem and other Melampsora-Populus pathosystems warranted s e v e r a l p r e l i m i n a r y s t u d i e s i n methodology. These methodological s t u d i e s : l e a f age e f f e c t s on s u s c e p t i b i l t y , k i n e t i n c o n c e n t r a t i o n e f f e c t s on l e a f d i s k senescence, and h y p e r p a r a s i t e e f f e c t s on uredospores, are presented i n t h i s methods s e c t i o n i n the p e r t i n e n t a r ea. Sample S i z e U n l i k e many other Host-Pathogen s t u d i e s , my specimens were to be drawn from w i l d p o p u l a t i o n s and t h e i r d i s e a s e i n t e r a c t i o n c h a r a c t e r i s t i c s were not known. Thus a proper sample s i z e was needed to ensure s u f f i c i e n t sampling: n = ( t 2 ) ( S 2 ) / E 2 S 2 = 1.2 ( i n a d i s e a s e r a t i n g system of 0 to 5, Farmer & Wilcox 1968. S i m i l a r value given i n J o k e l a , 1966) E = 1 (set to be s i g n i f i c a n t d i f f e r e n c e i n d i s e a s e s e v e r i t y which I expect to be able to d e t e c t ) t = 2.447 (95% l e v e l of s i g n i f i c a n c e , 6 degrees of freedom) n = (2.447) 2 (1.2) / (1) = 7 T h i s c a l c u l a t i o n was v a l i d f o r q u a n t i t a t i v e systems, but as can be seen by the f o l l o w i n g c a l c u l a t i o n s , the sample s i z e h e l d , 1 2 even c o n s e r v a t i v e l y , f o r q u a l i t a t i v e systems. For q u a l i t a t i v e systems, the sample s i z e i s chosen such that there i s a 95% chance of s e l e c t i n g at l e a s t two d i s t i n c t types (clones or i s o l a t e s ) . I f q u a l i t a t i v e e f f e c t s are important, the most common type should not comprise more than, f o r example, 50% of the p o p u l a t i o n . The p r o b a b i l i t y of s e l e c t i n g only one type i n 'n' t r e e s i s .5 to the nth power. Hence, what i s 'n' such that .5 to the nth i s equal to 0.05 ( i . e . 95% c o n f i d e n c e ) ? The sample s i z e 'n' i s then found to be 5. T h e r e f o r e , at l e a s t seven t r e e s and seven pathogen races were needed i n sampling. As t h i s was the minimum, i t was decided that more than seven t r e e s along with t h e i r Melampsora rust i s o l a t e s were to be sampled. Host s e l e c t i o n and T e l i a c o l l e c t i o n Eleven c l o n e s of black cottonwood were s e l e c t e d from around the Lower F r a s e r V a l l e y i n the autumn of 1982 and the s p r i n g of 1983. T e l i a of the autumn c o l l e c t e d c l o n e s were a l s o gathered at that time and s t o r e d o u t s i d e i n open-meshed bags. During the e a r l y p a r t of 1983, two clones and t h e i r i s o l a t e s were c o l l e c t e d from the B.C. I n t e r i o r and one from Calgary, A l b e r t a . L i s t i n g s of a l l c l o n e s and i s o l a t e s as w e l l as the c o l l e c t i o n and spore formation dates f o r a l l i s o l a t e s are given i n Appendix B. The black cottonwood t r e e s were chosen on the b a s i s of being r e l a t i v e l y i s o l a t e d from other cottonwoods, and having a t t a i n e d r e p r o d u c t i v e age. F a l l e n twigs from the t r e e were u s u a l l y c o l l e c t e d , as r e p r o d u c t i v e buds and most of the branches 1 3 were in the higher p o r t i o n s of the t r e e . These a b s c i s e d twigs due to c l a d o p t o s i s are known to root r e a d i l y i n greenhouse c o n d i t i o n s (Galloway & W o r r a l l 1979). Leaves with t e l i a were a l s o c o l l e c t e d from around the base of the t r e e to be used i n v i r u l e n c e t r i a l s . Both c o l l e c t i o n s r e q u i r e d that the t r e e be i s o l a t e d so that the i d e n t i t y of the twigs and leaves c o u l d be c e r t a i n . I s o l a t i o n was a l s o r e q u i r e d so that uredospore c y c l i n g should be on the same t r e e , presumably a l l o w i n g b u i l d - u p of a s i n g l e Melampsora i s o l a t e . As i s o l a t i o n was the major c r i t e r i o n f o r sampling, t r e e s with leaves that had no t e l i a were a l s o s e l e c t e d . The requirement that the t r e e s had a t t a i n e d r e p r o d u c t i v e age was added so that sexual c r o s s e s c o u l d be made i f the parents were to e x h i b i t q u a l i t a t i v e r e s i s t a n c e . Since black cottonwoods are mostly d i o e c i o u s ( S c h r e i n e r 1974), a l l c r o s s e s were not p o s s i b l e i n any case. F o l l o w i n g the method of Muhle- Larsen (1970) with twigs p o s s e s s i n g r e p r o d u c t i v e buds set i n f l a s k s of d i s t i l l e d water, and with a p a i n t brush to repe a t e d l y dust p o l l e n onto newly emerging female f l o w e r s , I d i d achieve one s u c c e s s s f u l c r o s s which y i e l d e d 13 progeny. However, the parents d i d not d i f f e r s i g n i f i c a n t l y i n o v e r a l l r e s i s t a n c e , so these progeny were not of i n t e r e s t . S e v e r a l t r e e s from o u t s i d e the Lower F r a s e r V a l l e y were sampled to i n c l u d e geographic v a r i a t i o n . S t a t i s t i c a l a n a l y ses were l a t e r done with and without these c l o n e s and i s o l a t e s . The f i r s t a n a l y s i s was to show what type of v a r i a t i o n i n v i r u l e n c e and r e s i s t a n c e may e x i s t i n the t r i c h o c a r p a - M. o c c i d e n t a l i s pathosystem at a s p e c i e s l e v e l . The second 14 a n a l y s i s was to show what type of v a r i a t i o n may occur i n the system at a p o p u l a t i o n l e v e l . A l l c o l l e c t e d leaves with t e l i a were kept o u t s i d e i n open- meshed bags u n t i l A p r i l . Presumably, most of the t e l i a on these leaves belonged to the pathogen race which was most s u c c e s s f u l on the t r e e d u r i n g the past season. These t e l i a were probably the r e s u l t of a u t o - i n f e c t i o n d u r i n g the i n f e c t i o n c y c l e s w i t h i n the t r e e . These t e l i a c o u l d not be induced to germinate in the f a l l , probably because they probably r e q u i r e e x t e r n a l o v e r w i n t e r i n g s t i m u l i to germinate i n the s p r i n g (Longo et a l . 1980, Von Weissenberg 1980). The twigs were po t t e d i n s t e r i l i z e d s o i l and grown on long days i n the greenhouse i n order to produce enough leaves f o r the r e s i s t a n c e t r i a l s . The daylength and average temperature i n the greenhouse were 16 hrs and 20 degrees C. Greenhouse grown leaves have a low chance of w i l d i n f e c t i o n , and none of my cl o n e s became i n f e c t e d i n the greenhouse. Uredospore Inoculum P r e p a r a t i o n In e a r l y A p r i l , leaves with t e l i a were brought i n t o the l a b o r a t o r y , washed i n c o l d , running, tap water f o r one hour, and set on top of moist f i l t e r paper i n p e t r i d i s h e s to incubate. Upon v i s i b l e germination of the black t e l i a g i v i n g a golden- yellow l a y e r of b a s i d i a above the black t e l i a , the leaves were suspended over pots of D o u g l a s - f i r s e e d l i n g s p l a n t e d i n March and A p r i l . T h i s procedure f o l l o w s that of Z i l l e r (1965). The assumption was made that a l l t e l i a on the le a v e s of a s i n g l e 15 cottonwood t r e e were c l o s e l y r e l a t e d . The i n o c u l a t i o n of D o u g l a s - f i r s e e d l i n g s by these b a s i d i o s p o r e s should y i e l d p ycnia on the upper s u r f a c e of the D o u g l a s - f i r needles, but, u n l i k e Z i l l e r (1965), I d i d not f i n d i n o c u l a t i o n of D o u g l a s - f i r s e e d l i n g s by b a s i d i o s p o r e s of M. o c c i d e n t a l i s e a s i l y achieved. My i n o c u l a t i o n s y i e l d e d pycnia f o r two i s o l a t e s , Imp and P i c (Appendix B). P o s s i b l y my t e l i a l i s o l a t e s were adapted to an a e c i a l host other than D o u g l a s - f i r and thus were not able to cause i n f e c t i o n . I n o c u l a t i o n was attempted with f r e s h l y washed t e l i a that had not been set to germinate i n p e t r i d i s h e s . These i n o c u l a t i o n s d i d not y i e l d any p y c n i a . Attempts were a l s o made to monitor s p o r e f a l l by sporecounts of v a s e l i n e - c o a t e d c o v e r s l i p s set i n pots of D o u g l a s - f i r f o r the d u r a t i o n of the 3 - day i n o c u l a t i o n ; but contamination and the unevenness of the tr a p p i n g media prevented accurate counts. The pots of Douglas- f i r were kept i n i n s e c t f r e e cages to a v o i d s p u r i o u s f e r t i l i z a t i o n of py c n i a . Z i l l e r (1965) has shown that M^ o c c i d e n t a l i s i s h e t e r o t h a l l i c and thus i t i s p o s s i b l e to make c o n t r o l l e d sexual c r o s s e s by p y c n i a l t r a n s f e r s ( Z i l l e r 1959). S i n g l e t r a n s f e r s should l e a d to the formation of a e c i o s p o r e s , h a l f of the time i f there are only two mating types. The plan i n i t i a l l y was to make only s e l f i n g c r o s s e s , and then, i f the p a r e n t a l i s o l a t e l i n e s proved to be of i n t e r e s t d u r i n g l a t e r experimentation, non- s e l f i n g c r o s s e s would be made. The t r a n s f e r of d r o p l e t s of p y c n i a l f l u i d between py c n i a d e r i v e d from the same t e l i a l source y i e l d e d s e l f e d progeny i n 1 6 the form of a e c i o s p o r e s . T h i s was s u c c e s s f u l f o r both i s o l a t e s with p y c n i a , Imp and P i c . From s e l f i n g c r o s s e s between pycnia of the same t e l i a l source, some spore l i n e s were i s o l a t e d . The ae c i o s p o r e s were i n o c u l a t e d onto t h e i r r e s p e c t i v e o r i g i n a l host c l o n e . A s e r i e s of uredospore t r a n s f e r s f o l l o w e d to y i e l d pure spore l i n e s . For a l l other i s o l a t e s which c o u l d not be s e l f e d due to la c k of py c n i a , u r e d i a was c o l l e c t e d i n l a t e summer from the o r i g i n a l t r e e s . (The c o l l e c t i o n dates are presented i n Appendix B). The uredospores of s e v e r a l u r e d i a of each l e a f were spread onto green-house grown leaves of t h e i r r e s p e c t i v e o r i g i n a l host c l o n e . S e v e r a l uredospore t r a n s f e r s f o l l o w e d before v i r u l e n c e t r i a l s . I thought that these uredospores of l a t e summer were l i k e l y those of the race which were w e l l adapted towards t h e i r p a r t i c u l a r black cottonwood c l o n e s . I f p h y s i o l o g i c a l s p e c i a l i z a t i o n were o c c u r r i n g i n t h i s n a t u r a l system, w e l l adapted spores of l a t e summer should be expected to e x h i b i t i t . V i r u l e n c e and Re s i s t a n c e T r i a l s The most recent f u l l y expanded l e a f of each c l o n e was c o l l e c t e d i n the greenhouse. A f t e r s u r f a c e s t e r i l i z a t i o n with .35% sodium h y p o c h l o r i t e f o r 1 minute, each l e a f was cut i n t o ten 17 mm d i s k s with a number 6 cork borer. D i s k s of one clone were p l a c e d i n t o ten d i f f e r e n t p e t r i d i s h e s on top of number 3 Whatman f i l t e r paper s a t u r a t e d with a 5 ppm s o l u t i o n of k i n e t i n . Each p e t r i d i s h ended up with 14 d i s k s r e p r e s e n t i n g the fourteen 17 c l o n e s ( f i g u r e 2); In one experimental block, there were ten p e t r i d i s h e s , one f o r each i s o l a t e . The block was r e p l i c a t e d at ten d i f f e r e n t times l e a d i n g to ten randomized complete blocks as the experimental d e s i g n . One block was d i s c a r d e d the e i g h t h day a f t e r i n o c u l a t i o n due to the unacceptably low i n f e c t i o n . The inoculum q u a l i t y f o r t h i s block was suspect. F i g u r e 2: A s i n g l e Melampsora r u s t i s o l a t e was a p p l i e d to each of the fourteen c l o n e s of black cottonwood represented by 17 mm l e a f d i s k s i n each p e t r i d i s h . To s t a n d a r d i z e the e f f e c t s of l e a f age and p o s i t i o n on s u s c e p t i b i l i t y to r u s t , the most recent f u l l y expanded l e a f was used. I t has been suggested that a r e c e n t l y f u l l y expanded l e a f e x h i b i t s maximum s u s c e p t i b i l i t y , and that subsequently s u s c e p t i b i l i t y decreases with age (Sharma & Heather & Winer 1980, L i n & Edwards 1974). As w e l l , the most r e c e n t l y expanded 18 l e a f may be expected to senesce l e s s q u i c k l y than o l d e r l e a v e s . A minor experiment was conducted to measure these age e f f e c t s . S e v e r a l cottonwood stems were s t r i p p e d e n t i r e l y of t h e i r leaves f o r use i n t h i s l e a f age s u s c e p t i b i l i t y study. Leaf halves were i n o c u l a t e d i n p e t r i d i s h e s and incubated f o r 25 days at which time the d i s e a s e s e v e r i t y was assessed by number of u r e d i a and t e l i a . The youngest leaves were the most r e s i s t a n t , and d i d not become d i s e a s e d . The most r e c e n t l y f u l l y expanded l e a f was found to be the youngest or the second youngest l e a f s u s c e p t i b l e to Melampsora r u s t . (Appendix C ) . The host clones were represented by l e a f d i s k s cut from leaves s u r f a c e s t e r i l i z e d with .35% sodium h y p o c h l o r i t e f o r 1 min (Waller 1981). These d i s k s were f l o a t e d on f i l t e r paper i n a 5 ppm k i n e t i n s o l u t i o n i n a p e t r i d i s h , as has been done in other s t u d i e s of the Melampsora-Populus complex (Shain & C o r n e l i u s 1979, Singh & Heather 1981) In a small study, the e f f e c t of v a r y i n g the c o n c e n t r a t i o n of k i n e t i n was t e s t e d on h a l v e s of r e c e n t l y matured l e a v e s . One h a l f of these l e a f halves were s u r f a c e s t e r i l i z e d . The r e s u l t s showed that while there were no s i g n i f i c a n t d i f f e r e n c e s between s t e r i l i z e d and n o n - s t e r i l i z e d l e a v e s , with the higher c o n c e n t r a t i o n s of k i n e t i n (10, 50, or 100 ppm), heavy senescence occurred up to four times f a s t e r than with water. There were no great d i f f e r e n c e s between water and up to 5 ppm k i n e t i n . Chandrashekar (1982) found s i m i l a r r e s u l t s . I n o c u l a t i o n s f o l l o w e d the method of Shain & C o r n e l i u s (1979). Uredospores were suspended i n a .1% agar s o l u t i o n . Three d r o p l e t s of suspension, each approximately .02 ml, were 19 i n o c u l a t e d with a m i c r o p i p e t t e onto each d i s k of one p e t r i d i s h . Shain and C o r n e l i u s (1979) s t a t e that the optimum i n o c u l a t i o n c o n c e n t r a t i o n i s between 1250 to 5000 sp o r e s / d i s k (1.7 cm diameter). Indeed, I obtained heavy i n f e c t i o n at 1300 spores/disk (1.7 cm diameter) i n e a r l i e r i n o c u l a t i o n t e s t s . Attempts were made to s t a n d a r d i z e inoculum c o n c e n t r a t i o n s to around 3000 sp o r e s / d i s k (three .02 ml drops of 50,000 spores/ml suspension) through a constant spore suspension absorbance reading ( .05 absorbance u n i t s or 90% tra n s m i t t a n c e ) on a spectrophotometer p r i o r to i n o c u l a t i o n ; but the achieved range was 1000 to 20000 with an average at 6000 s p o r e s / d i s k . These spore counts were made on a haemocytometer a f t e r the i n o c u l a t i o n s r a t h e r than before, which may have c o n t r i b u t e d to to t h i s great range i n inoculum c o n c e n t r a t i o n . The uredospore inoculum l o a d was purposely set higher than the c o n c e n t r a t i o n at which heavy i n f e c t i o n c o u l d occur on some c l o n e s . In i n o c u l a t i o n curves ( i n f e c t i o n l e v e l vs. spore c o n c e n t r a t i o n ) , i t i s thought that slope decreases e x p o n e n t i a l l y (concavely c u r v i l i n e a r ) and reaches a c o n c e n t r a t i o n s a t u r a t i o n l e v e l (slope goes to 0) where a g r e a t e r c o n c e n t r a t i o n of inoculum does not cause g r e a t e r i n f e c t i o n . My o b j e c t i v e was to ensure that inoculum c o n c e n t r a t i o n s onto a l l the clones exceeded t h e i r s a t u r a t i o n p o i n t so i n f e c t i o n l e v e l should be due p u r e l y to r e s i s t a n c e and v i r u l e n c e r a t h e r than escape e f f e c t s . As a f i n a l check on proper inoculum l o a d , a c o r r e l a t i o n was l a t e r made between inoculum c o n c e n t r a t i o n and s i n g l e p e t r i d i s h i n f e c t i o n averages. (A s i n g l e spore p r e p a r a t i o n was a p p l i e d to a s i n g l e p e t r i d i s h ) . The r e s u l t s (r= -.14) showed no 20 s i g n i f i c a n t c o r r e l a t i o n between spore c o n c e n t r a t i o n and i n f e c t i o n l e v e l s , which meant that my i n o c u l a t i o n s were, as proper, in the range of s a t u r a t i o n c o n c e n t r a t i o n s . As w e l l , a graph of the two showed no apparent trends even among the lowest c o n c e n t r a t i o n s . (Appendix D). A f t e r each p e t r i d i s h i n o c u l a t i o n , spores were p l a t e d onto 5% water agar to determine the percent germination. There was a imperfect fungus commonly a s s o c i a t e d with the uredospores which may have been i n h i b i t i n g uredospore germination i n the water agar p l a t e s . Readings of 0% germination were obtained, although i n f r e q u e n t l y , yet i n f e c t i o n of l e a f d i s k s d i d occur from these same spore suspensions. I t was suspected that a h y p e r p a r a s i t e which was e s p e c i a l l y promoted i n the water agar p e t r i p l a t e c o n d i t i o n s was r e s p o n s i b l e . An unpublished experiment was done in the same l a b to t e s t the e f f e c t s of t h i s presumed h y p e r p a r a s i t e . T h i s experiment was conducted by Elena K l e i n as a d i r e c t e d s t u d i e s i n the f a l l of 1983. She showed that although t h i s presumed h y p e r p a r a s i t e had a great e f f e c t i n r e d u c i n g i n f e c t i o n when i n o c u l a t e d onto the l e a f a week p r i o r to uredospore i n o c u l a t i o n , i t had no e f f e c t s i n a 24 hr p r i o r i n o c u l a t i o n . However, there was c o n s i d e r a b l e background v a r i a t i o n i n t h i s experiment. H y p e r p a r a s i t e s and a n t a g o n i s t s of Melampsora uredospores have been commonly repo r t e d ( B i e r 1965, McBride 1969, Omar & Heather 1979, Sharma & Heather 1981 & 1982), however t h e i r r o l e i n n a t u r a l pathosystems i s not known. These p u b l i s h e d s t u d i e s were a l l conducted i n l a b o r a t o r y c o n d i t i o n s , and while 21 a n t a g o n i s t s can be promoted i n l a b o r a t o r y c o n d i t i o n s , a n t a g o n i s t i c c o n c e n t r a t i o n s may be uncommon i n Nature. F i n a l l y , the p e t r i d i s h e s of the main experiment were a l l placed i n t o an i n c u b a t i o n chamber at a temperature of 18 degrees C with constant c o o l white f l u o r e s c e n t l i g h t and watered every 3 days with d i s t i l l e d water. Krzan (1980) shows that optimal a i r temperature f o r germination of l a r i c i - p o p u l i n a uredospores i s 16 degrees C, with higher temperatures more i n h i b i t i n g than lower temperatures. Toole (1967) holds that the temperature optima f o r the germination of uredospores of medusae and M. l a r i c i - p o p u l i n a i s 18 degrees C. Shain & C o r n e l i u s (1979) demonstrate that l e a f d i s k s of d e l t o i d e s i n o c u l a t e d with M. medusae w i l l have more severe i n f e c t i o n s at 18 degrees C than at 23 degrees C. Rating System Disease s e v e r i t y may be assessed by s e v e r a l parameters. In c l a s s i c a l s t u d i e s of host:pathogen r e l a t i o n s h i p s , i n f e c t i o n type with d e f i n e d c a t e g o r i e s of i n f e c t i o n has been the main s c r e e n i n g c r i t e r i o n , but many workers emphasize the importance of e p i d e m i o l o g i c a l c h a r a c t e r i s t i c s such as number of p u s t u l e s , i n c u b a t i o n p e r i o d , number of spores per p u s t u l e , and l o n g e v i t y of s p o r u l a t i o n . (Zadoks & Schein 1978 p.7, Sharma & Heather 1979a). Number of spores would be an a b s o l u t e measure of the v i r u l e n c e / r e s i s t a n c e i n the a e g r i c o r p u s (hostrpathogen a s s o c i a t i o n , Loegering 1978 p. 3 1 1 ) ; but, depending on spore p r o d u c t i o n r a t e s , the l a t e n t p e r i o d (time from i n o c u l a t i o n to 22 symptoms) c o u l d a l s o have important e p i d e m i o l o g i c a l consequences. Thus a combination of the two, such as #spores produced / l a t e n t p e r i o d , which i s spore p r o d u c t i o n r a t e , may give a b e t t e r assessment. Other s t u d i e s in the Melampsora-Populus complex have used d i v e r s e measures f o r d i s e a s e s e v e r i t y . In the multitude of p u b l i c a t i o n s by Heather and a s s o c i a t e s at the A u s t r a l i a n N a t i o n a l U n i v e r s i t y , the measures used are: I P F ( i n c u b a t i o n p e r i o d ) , ULD(uredia per d i s k ) , and USM(uredospores per mm2 ). Measurements are made at 15 days a f t e r i n o c u l a t i o n . Shain and C o r n e l i u s (1979) use t o t a l u r e d i a and t e l i a counts f o r di s e a s e s e v e r i t y , and measurements are made at 9 to 11 days a f t e r i n o c u l a t i o n . S p i e r s (1978) s i m i l a r l y made u r e d i a l counts 10 days a f t e r i n o c u l a t i o n . In a l l of these s t u d i e s , the p a r t i c u l a r reason f o r the date of measurement i s not g i v e n . For each of my d i s k s , which were observed d a i l y , measurements were made at a p e r i o d of twice the l a t e n t p e r i o d so that a l l d i s e a s e i n t e r a c t i o n s c o u l d be measured at r e l a t i v e l y the same stage of development. A l l u n i n f e c t e d d i s k s were observed up to a minimum of 22 days. The number of spores may be measured i n d i r e c t l y by measuring absorbance of a spore suspension with a spectrophotometer. (Both t r a n s m i t t a n c e and absorbance s c a l e s are given on a S p e c t r o n i c 20 spectrophotometer, but i t i s e a s i e r to read the l i n e a r t r a n s m i t t a n c e s c a l e because the absorbance s c a l e i s n o n - l i n e a r . A f t e r o b t a i n i n g t r a n s m i t t a n c e readings, one can e a s i l y convert them i n t o absorbance u n i t s , s i n c e absorbance i s the l o g of the i n v e r s e of t r a n s m i t t a n c e ) . Sharma 23 & Heather (1979b) have shown that Melampsora uredospore c o n c e n t r a t i o n as measured by a haemocytometer has a high c o r r e l a t i o n (r=.998) with l i g h t absorbance at 640 nm of the same spore suspension. Spore suspensions f o r readings on a Sp e c t r o n i c 20 were made with m o d i f i c a t i o n s of Sharma & Heather's (1979b) procedure. As l a t e n t p e r i o d has a strong r e l a t i o n s h i p with spore p r o d u c t i o n , a l l d i s k s were assessed upon reaching twice t h e i r l a t e n t p e r i o d . Disease assessment took two forms: the f i r s t i n v o l v e d m i c r o s c o p i c examination of the d i s k s to count the number of u r e d i a and t e l i a . U r edia were r a t e d i n t o one of three diameter c l a s s e s : <.2mm, .2mm to .5 mm, and >.5mm. The second method i n v o l v e d spore counts f o r d i s e a s e s e v e r i t y . The d i s k s were p l a c e d s i n g l y i n t o t e s t tubes with 1 ml of 5% Tween-20, and the tubes were v i g o r o u s l y a g i t a t e d f o r 1 hour one hour on a B u r r e l l w r i s t - a c t i o n shaker. One and a h a l f mis of water were then added to each tube to make the 2.5 mis needed f o r absorbance measurements on a spectrophotometer. The f i n a l 2% Tween-20 c o n c e n t r a t i o n d i d not d e t e c t a b l y decrease t r a n s m i t t a n c e i n a Tween-20 absorbance t e s t . Random checks were c a r r i e d out on one out of ten tubes with a haemocytometer to d e r i v e a c o r r e l a t i o n f o r absorbance ( c o l o r i m e t e r ) and spore count (haemocytometer). Spore suspensions f o r 154 d i s k s were measured f o r absorbance and spore count. The d e r i v e d equation was: SPORE COUNT = 1228300 * ABSORBANCE - 7294, which had a s i g n i f i c a n t r=.9l (Appendix E ) . T h i s negative y- i n t e r c e p t value i n d i c a t e s that there may have been d i s c o l o r a t i o n 24 of the spore suspensions by the d i s k s . Indeed, over 20 of these suspensions were made from d i s k s with no i n f e c t i o n , and these suspensions a l l showed a small degree of absorbance. Since spore suspensions cannot take t e l i a i n t o account, a value of 500 spores was a s s i g n e d to each t e l i a to give the f i n a l e q uation: SPORE COUNT = 1228300 * ABSORBANCE - 7294 + 500 * TELIA. T h i s value of 500 spores f o r each t e l i u m was decided upon a f t e r c a l c u l a t i n g that a medium s i z e uredium r e l e a s e d t h i s number of spores i n the d i s k shaking p r o c e s s . Sharma & Heather (1979b) d e r i v e d a s i m i l a r equation o f : UREDOSPORES = 1170000 * ABSORBANCE + 2800 f o r l a r i c i - p o p u l i n a on h y b r i d p o p l a r s . A n a l y s i s of v a r i a n c e was then performed on spore p r o d u c t i o n ra t e s as c a l c u l a t e d from average spore p r o d u c t i o n during twice the l a t e n t p e r i o d . A n a l y s i s of v a r i a n c e was a l s o c a r r i e d out on t o t a l p u s t u l e count over twice l a t e n t p e r i o d , on t o t a l spore count, and on l a t e n t p e r i o d i n days. Components of v a r i a n c e were then c a l c u l a t e d f o r the v a r i a n c e sources used i n the a n a l y s i s of v a r i a n c e on average d a i l y spore p r o d u c t i o n to determine the v a r i a n c e c o n t r i b u t i o n by each source. 25 RESULTS AND DISCUSSION The spore production r a t e s are presented i n Table I I . Latent p e r i o d v a r i e d from 6 to 12 days with the median at 8 days. Out of 1260 d i s k s , 235 d i d not become i n f e c t e d , although no s i n g l e c l o n e - i s o l a t e combination was without i n f e c t i o n throughout the nine b l o c k s . A n a l y s i s of Variance The analyses of v a r i a n c e f o r a l l specimens and f o r only Lower Fr a s e r V a l l e y specimens are presented in Table I. The f i r s t a n a l y s i s was done to show what type of v a r i a t i o n i n v i r u l e n c e and r e s i s t a n c e may e x i s c i n the P_;_ t r ichocarpa - M. o c c i d e n t a l i s pathosystem at a s p e c i e s l e v e l . The second a n a l y s i s was to show what type of v a r i a t i o n may occur i n the system at a p o p u l a t i o n l e v e l . Both analyses showed that there were h i g h l y s i g n i f i c a n t d i f f e r e n c e s between c l o n e s of black cottonwood and between i s o l a t e s of Melampsora r u s t , which meant that there were great d i f f e r e n c e s i n c l o n a l r e s i s t a n c e and i n i s o l a t e v i r u l e n c e at both the p o p u l a t i o n and s p e c i e s l e v e l s . The c l o n a l means in spore p r o d u c t i o n r a t e v a r i e d from 311 to 1008 spores/disk/day, and the i s o l a t e means v a r i e d from 283 to 1074 spores/disk/day (Table I I ) . These a n a l y s e s a l s o showed that there were no s i g n i f i c a n t i n t e r a c t i o n s between c l o n e s and i s o l a t e s . Thus there was no i n d i c a t i o n of d i f f e r e n t i a l i n t e r a c t i o n . The s i g n i f i c a n t F-value f o r blocks meant that s i g n i f i c a n t v a r i a t i o n was removed by b l o c k i n g . T h i s v a r i a t i o n c o n s i s t e d of random e f f e c t s , time e f f e c t s (as b l o c k s were r e p l i c a t e d at 26 TABLE I: A n a l y s i s of v a r i a n c e f o r average d a i l y spore p r o d u c t i o n d u r i n g twice the l a t e n t p e r i o d , and i s o l a t e and c l o n a l rankings (***). a. For a l l specimens SOURCE Block Treatment I s o l a t e s Clones I n t e r a c t i o n E r r o r T o t a l D. . F. 8 1 39 F-VALUE 6.942 2.357 5.318 14.442 .929 F-PROB ** 9 1 3 1 1 7 .0000 .0000 .0000 .0000 .6884 1112 1259 C l o n a l ranking: * * * Ken<Pond<Thun<Sal<Poco<Mar<Pic<Hebb<Rup<Imp<Gran<Cord<TrQ<Cal I s o l a t e ranking: * * * IMP <GRAN <KEN <THUN <PIC <PRG <HEBB <ALK <RUP <POCO b. For Lower F r a s e r V a l l e y Specimens SOURCE D. F. . F-VALUE F-PROB Block 8 5.003 .0000 Treatment 76 1 .885 .0001 I s o l a t e s 6 8.600 .0000 Clones 1 0 5.319 .0000 I n t e r a c t i o n 60 .870 .7457 E r r o r 608 T o t a l 692 C l o n a l r a n k i n g : Ken<Thun<Sal<Pond<Mar<Poco<Hebb<Rup<Imp<Cord<Gran I s o l a t e ranking: IMP <KEN <GRAN <THUN <HEBB <POCO <RUP * Specimens from o u t s i d e the Lower F r a s e r V a l l e y ** F-prob i s the p r o b a b i l i t y that the n u l l h y p o t h e s i s , which s t a t e s that there are no d i f f e r e n c e s , i s t r u e . *** The rankings r e s u l t from Duncans m u l t i p l e range t e s t s (95% c o n f i d e n c e ) , and a l l specimens underscored by the same l i n e are not s i g n i f i c a n t l y d i f f e r e n t . 27 d i f f e r e n t t i m e s ) , v a r i a t i o n between leaves of a s i n g l e c l o n e , and v a r i a t i o n from separate p r e p a r a t i o n s of the same i s o l a t e . The lack of s i g n i f i c a n t c l o n e - i s o l a t e i n t e r a c t i o n coupled with s i g n i f i c a n t d i f f e r e n c e s beteen c l o n e s and between i s o l a t e s means that there i s constant r a n k i n g . From the i s o l a t e and clone rankings (Table I ) , i t can be seen that although the absolute rankings d i f f e r s l i g h t l y between the Lower Fr a s e r V a l l e y rankings and the a l l - s p e c i m e n s rankings, there are no s t a t i s t i c a l l y s i g n i f i c a n t changes i n ranking. Within one un d e r l i n e d group (Table I ) , specimens may change order between a l l - s p e c i m e n s and F r a s e r V a l l e y specimens, but rankings do not change between groups. Analyses of v a r i a n c e on other parameters of dis e a s e s e v e r i t y (average p u s t u l e p r o d u c t i o n , t o t a l spore count, and l a t e n t p e r i o d ) showed the same r e s u l t s (Appendix F ) . Clones as w e l l as i s o l a t e s d i f f e r e d s i g n i f i c a n t l y , while there was no s i g n of d i f f e r e n t i a l i n t e r a c t i o n . These a n a l y s e s were done because, depending on the r e s i s t a n c e mechanism i n v o l v e d , i t would have been p o s s i b l e f o r one parameter to demonstrate d i f f e r e n t i a l i n t e r a c t i o n while another d i d n ' t . For example, i f the r e s i s t a n c e mechanism were i n v o l v e d i n impeding the p e n e t r a t i o n process, t h i s c o u l d show up most c l e a r l y i n l a t e n t p e r i o d d i f f e r e n c e s . Whereas i f the r e s i s t a n c e mechanism were i n v o l v e d in impeding p a r a s i t i c growth and s p o r u l a t i o n a f t e r p e n e t r a t i o n , t h i s would probably show up b e t t e r i n t o t a l spore count than i n l a t e n t p e r i o d d i f f e r e n c e s . 28 P h y s i o l o g i c a l S p e c i a l i z a t i o n Another p r e s e n t a t i o n of the l a c k of d e t e c t a b l e s p e c i a l i z a t i o n or adapta t i o n by the i s o l a t e s toward t h e i r o r i g i n a l host clones i s given i n Table I I . I t shows that i n no case d i d an i s o l a t e produce more spores on i t s o r i g i n a l host than on a l l other h o s t s . A t - t e s t done on the d i f f e r e n c e i n spore p r o d u c t i o n r a t e s between means of a l l o r i g i n a l h o s t - c l o n e combinations and of a l l experimental combinations showed no s i g n i f i c a n t d i f f e r e n c e between the two. Thus there was no i n d i c a t i o n of p h y s i o l o g i c a l s p e c i a l i z a t i o n by pathogen i s o l a t e s toward t h e i r o r i g i n a l host c l o n e s . TABLE I I : Average d a i l y spore p r o d u c t i o n d u r i n g twice the l a t e n t p e r i o d f o r a l l i s o l a t e s on a l l c l o n e s ( u n i t s are sp o r e s / d i s k / d a y ) . ( i s o l a t e s ) Alk Gran Hebb Imp Ken P i c Poco PrG Rup Thun MEAN (clones) CAL 2152 639 2964 1 020 1 346 1 740 3443 1 270 2700 3245 2052 CORD 1 333 813 1466 1 56 367 783 1 362 903 1110 1 669 997 GRAN 377 723* 348 392 1 125 1034 1 937 733 1 767 1089 953 HEBB 1 263 622 358* 316 631 623 672 885 817 470 666 IMP 955 1596 324 1 1 3* 547 560 583 671 1115 723 719 KEN 1 54 353 1 2 1 58 241 * 361 591 490 405 341 31 1 MAR 890 376 953 221 237 433 870 1093 1 1 43 31 1 653 PIC 645 261 804 509 473 1 1 06* 813 773 602 481 655 POCO 678 406 868 89 688 847 644* 779 914 264 618 POND 339 178 181 196 364 456 642 313 456 203 333 RUP 770 316 490 398 328 868 1037 885 1 091 * 657 684 SAL 714 337 524 80 835 292 556 403 528 164 443 THUN 338 37 266 76 425 233 565 747 806 289* 371 TRQ 986 1322 1068 313 1516 987 1323 798 1 159 606 1008 MEAN 828 570 765 283 652 737 1074 767 1 044 751 I 747 * O r i g i n a l c l o n e - i s o l a t e combinations 29 In my sampling of i s o l a t e d t r e e s , there was i n h e r e n t l y a b i a s toward sampling those cottonwoods which had s u r v i v e d s i n g l y i n t h e i r immediate area. They may have had some s p e c i a l c h a r a c t e r i s t i c s such as r e s i s t a n c e genes which allowed them to s u r v i v e . In a d d i t i o n , i s o l a t e s were taken from these same sampled t r e e s , so that i f p h y s i o l o g i c a l s p e c i a l i z a t i o n were o c c u r i n g , these i s o l a t e s should s u r e l y demonstrate i t . T h i s can be c o n t r a s t e d to t o t a l random s e l e c t i o n of both c l o n e s and i s o l a t e s where the t h e o r e t i c a l c orresponding genes may not have been sampled. The sampling procedure thus f u r t h e r strengthens the r e s u l t , that there are no sign s of p h y s i o l o g i c a l s p e c i a l i z a t i o n i n t h i s n a t u r a l pathosystem. Because I d i d not f i n d d i f f e r e n t i a l i n t e r a c t i o n s i n the samples need not mean that there are none. Quite p o s s i b l y d i f f e r e n t i a l i n t e r a c t i o n s do occur to a minor extent i n t h i s pathosystem. M a n i p u l a t i o n of the data by e l i m i n a t i n g s e v e r a l c l o n e s and i s o l a t e s l e a d to a s i g n i f i c a n t F-value f o r i s o l a t e - c lone i n t e r a c t i o n (Appendix G), but t h i s data manipulation may be s t a t i s t i c a l l y unsound, and a l s o t h i s i n t e r a c t i o n may have no b i o l o g i c a l importance. If d i f f e r e n t i a l i n t e r a c t i o n s o r i g i n a t i n g from p h y s i o l o g i c a l s p e c i a l i z a t i o n do occur i n t h i s pathosystem, they are very r a r e . M a thematically, the sample s i z e of 14 randomly s e l e c t e d c l o n e s allows me to say with 95% c o n f i d e n c e that t r e e s with q u a l i t a t i v e r e s i s t a n c e w i l l p l a y a pa r t i n 20% or l e s s of a l l d i s e a s e i n t e r a c t i o n s i n t h i s pathosystem ( c a l c u l a t i o n s : what i s x such that ( 1 - x ) 1 * = .05? x i s found to be . 2 0 ) . 30 T h i s i s f u r t h e r demonstrated by an a n a l y s i s of the components of v a r i a n c e : although t h i s experiment c o n t a i n s c o n s i d e r a b l e e r r o r , i t was s t i l l p o s s i b l e to see the s t r o n g l y s i g n i f i c a n t d i f f e r e n c e s between c l o n e s and between i s o l a t e s (Table I I I ) . TABLE I I I : Components of v a r i a n c e f o r the sources: i s o l a t e s , c l o n e s , b l o c k s , i n t e r a c t i o n , and e r r o r . i s o l a t e s c l o n e s b l o c k s i s o l a t e - c l o n e i n t e r a c t i o n e r r o r percent v a r i a n c e accounted f o r : a l l specimens Vancouver only 2.6% 5.7% 11.5% 4.6% 3.5% 5.0% 0.0% 0.0% 82.4% 84.7% If there had been no d e t e c t a b l e d i f f e r e n c e s between i s o l a t e s or between c l o n e s , then one c o u l d c l a i m that t h i s experiment was not s e n s i t i v e enough to d e t e c t d i f f e r e n t i a l c l o n e - i s o l a t e i n t e r a c t i o n e i t h e r . The r e l a t i v e v a r i a n c e c o n t r i b u t i o n s change somewhat f o r a l l sources except i s o l a t e - c l o n e i n t e r a c t i o n , which remained at 0% (Table I I I ) . C a l c u l a t i o n s f o r components of v a r i a n c e are given i n Appendix H. E f f e c t s of Disease Aside from the hypothesized g e n e t i c s of d i s e a s e i n t e r a c t i o n , t h i s d i s e a s e may have a profound e f f e c t on i t s h o s t . In an unpublished experiment conducted i n t h i s l a b by Susan Hruszowy on the e f f e c t s of M_;_ o c c i d e n t a l i s on p h o t o s y n t h e s i s i n black cottonwood, she found that 31 p h o t o s y n t h e s i s of d i s e a s e d leaves was reduced s i g n i f i c a n t l y by 31% as compared to d i s e a s e - f r e e leaves on the same p l a n t . T h i s experiment was done i n the greenhouse on leaves s t i l l a t tached to the p l a n t . S i m i l a r r e s u l t s have been r e p o r t e d i n p u b l i s h e d s t u d i e s . Uredopustules of some r u s t s a ct as si n k s to accumulate metabolic products i n competition with other p l a n t p a r t s (Wang 1961). Heavy i n f e c t i o n by Melampsora r u s t causes premature senescence of leaves and d e f o l i a t i o n (Schipper & Dawson 1974). Moderate r u s t i n f e c t i o n can cause a 46% growth r e d u c t i o n (Widin & Schipper 1976). D e f o l i a t i o n has a great e f f e c t on stem height and b a s a l stem diameter and consequently on the volume of wood produced by i n f e c t e d t r e e s ( J o l y 1959, Widin & Schipper 1976). Donnelly (1974) r e p o r t s that b a s a l leaves of p o p l a r s export photosynthate mainly to the b a s a l stem while higher leaves c o n t r i b u t e more toward stem h e i g h t ; thus as b a s a l leaves are most o f t e n f i r s t i n f e c t e d and a b s c i s e d , b a s a l stem diameter i s g r e a t l y a f f e c t e d . However, these f i g u r e s f o r growth r e d u c t i o n are extreme and i t may have been environmental i n t e r a c t i o n s which allowed f o r such great s e v e r i t y . Palmberg (1977) found that poplar d i s e a s e r e s i s t a n c e to Melampsora r u s t was not s i g n i f i c a n t l y d i f f e r e n t between d i f f e r e n t c l o n e s on the same s i t e , but was s i g n i f i c a n t l y d i f f e r e n t between s i t e s . He a l s o found very high genotype X environment i n t e r a c t i o n s . Heather & Chandrashekar (1982) c l a i m that environmental f a c t o r s are most important i n d i s e a s e s t a b i l i t y , and that l i g h t and temperature c o n t r i b u t e more to v a r i a t i o n than do c u l t i v a r s or ra c e s . 32 N a t u r a l Pathosystems There have been very few s t u d i e s done on n a t u r a l pathosystems. Robinson (1980) s t a t e s t h a t : " v i r t u a l l y no f a c t u a l s t u d i e s of a w i l d p l a n t pathosystem have ever been undertaken" (p. 204), but does mention that two r e s e a r c h e r s have been working on a w i l d p l a n t pathosystem i n the Netherlands. Other s t u d i e s of w i l d p l a n t pathosystems i n c l u d e P u c e i n i a spp. on Avena spp. i n I s r a e l (Wahl et a l . 1978, Dinoor 1977), two f o l i a r d i s e a s e s i n a T r i f o l i u m repens p o p u l a t i o n i n Wales (Burdon 1980), E r y s i p h e graminis h o r d e i on Hordeum sp. (Wahl et a l . 1978), a l e a f r u s t d i s e a s e on G l y c i n e sp. i n A u s t r a l i a (Burdon & M a r s h a l l 1981), Puce i n i a spp. on Avena spp. i n A u s t r a l i a (Burdon, Oates & M a r s h a l l 1983), and a r u s t d i s e a s e of w i l d sunflower s p e c i e s (Zimmer & Rehder 1976). The problem with s e v e r a l of these s t u d i e s i s that the w i l d p l a n t s grew near t h e i r c u l t i v a t e d r e l a t i v e s , so that the s e l e c t i o n pressure posed by the nearby uniform c u l t i v a r s c o u l d have a l t e r e d the pathogen p o p u l a t i o n such that these w i l d pathosystems may not have been n a t u r a l . I c o u l d f i n d no s t u d i e s i n the l i t e r a t u r e on n a t u r a l f o r e s t t r e e pathosystems i n t h i s area of pathosystem the o r y . Even my study i s not wholly n a t u r a l s i n c e most of the specimens were s e l e c t e d from near urban s e t t i n g s . However, n e i t h e r the host black cottonwood nor the pathogen Melampsora r u s t have been manipulated to any extent i n the area, so that the unnatural and powerful s e l e c t i o n f o r c e s a r i s i n g from homogenous p l a n t a t i o n s have not had an e f f e c t . 33 Robinson (1979 p.21) spe c u l a t e s that q u a l i t a t i v e r e s i s t a n c e can evolve i n n a t u r a l d i s c o n t i n u o u s pathosystems but i t need not do so. He a l s o suggests that d i s c o n t i n u i t y (which r e f e r s to the c o n t i n u i t y of the h o s t r p a r a s i t e i n t e r a c t i o n ) would be the f o r c e which c r e a t e s and maintains q u a l i t a t i v e r e s i s t a n c e , s i n c e continuous systems would have no use f o r q u a l i t a t i v e r e s i s t a n c e . However, unl e s s the pathogen poses a great s e l e c t i o n pressure on i t s host, q u a l i t a t i v e r e s i s t a n c e would not be necessary i n the host i n whichever system. The M^ occ i d e n t a l i s - P_j_ t r ichocarpa pathosystem can be co n s i d e r e d d i s c o n t i n u o u s , s i n c e the ru s t must c y c l e to the a l t e r n a t e host every year and there i s normally no i n t e r a c t i o n between the rus t and l i v e cottonwood leaves d u r i n g the winter. F o l i a g e emerges each s p r i n g f r e e of Melampsora r u s t . However, Melampsora r u s t does not appear to pose a continuous s e l e c t i o n pressure on i t s host. Some years, n e a r l y a l l t r e e s are h e a v i l y i n f e c t e d ; d u r i n g other years, many of the same t r e e s remain f r e e from i n f e c t i o n . Furthermore, even d u r i n g years of heavy i n f e c t i o n , major d e f o l i a t i o n i s not o f t e n observed and at any ra t e does not occur u n t i l l a t e i n the growing season. Major r e s i s t a n c e genes and v i r u l e n c e genes have been i s o l a t e d from n a t u r a l p o p u l a t i o n s , and from t h i s i t i s o f t e n i n f e r r e d t h at major genes do have a r o l e to play i n di s e a s e systems that are s t a b l e . However another i n t e r p r e t a t i o n c o u l d be that these genes have a f u n c t i o n other than i n di s e a s e r e s i stance. I t i s p o s s i b l e to imagine that major v i r u l e n c e genes can be maintained i n the pathogen p o p u l a t i o n i f there are powerful 34 s e l e c t i o n p r e s s u r e s which act to favor d i s t i n c t polymorphisms. One such pressure c o u l d be a p h y s i o l o g i c a l b a r r i e r such as l a c k of sexual r e p r o d u c t i o n . For example, in a m i c r o c y l i c r u s t without spermogonia, a mutation c o u l d give r i s e to a powerful v i r u l e n c e gene which all o w s i t s possessors to spread through the p o p u l a t i o n . Lack of m e i o t i c c r o s s i n g - o v e r would prevent gene combinations or a new g e n e t i c background f o r t h i s major v i r u l e n c e gene where i t s e f f e c t s would be m o d i f i e d , or where i t would be incompatible. Another s e l e c t i o n p r e s s s u r e c o u l d be the powerful a r t i f i c i a l s e l e c t i o n posed by monocultures of a c u l t i v a r with a s i n g l e set of strong r e s i s t a n c e genes. Powerful v i r u l e n c e genes would then be r e q u i r e d i n the pathogen p o p u l a t i o n f o r s u r v i v a l . Sidhu (1980 p. 396) l i s t s 27 pathosystems i n which gene-for- gene r e l a t i o n s h i p s have been i m p l i e d , suggested, or demonstrated. None of these systems are n a t u r a l l y w i l d . However the o c c i d e n t a l i s - P_;_ t r i c h o c a r p a pathosystem which I s t u d i e d i s w i l d , and not under strong a r t i f i c i a l s e l e c t i o n p r e s s u r e s to produce major r e s i s t a n c e or v i r u l e n c e genes. As w e l l both p a r t n e r s are q u i t e capable of o u t c r o s s i n g with other members of t h e i r s p e c i e s so no p h y s i o l o g i c a l pressure i s present to maintain d i s t i n c t d i s e a s e i n t e r a c t i o n polymorphisms. There i s a v a l u a b l e p r a c t i c a l i m p l i c a t i o n from these r e s u l t s . The l a c k of q u a l i t a t i v e r e s i s t a n c e and v i r u l e n c e i n t h i s system holds the promise that cottonwood r e s i s t a n c e w i l l not be d e v a s t a t i n g l y overcome when used in p l a n t a t i o n s . Cottonwoods reproduce very e a s i l y a s e x u a l l y by c l a d o p t o s i s (twig 3 5 d r o p ) , and so i t would not be uncommon to f i n d neighboring i n d i v i d u a l s of the same genotype. Thus one c o u l d i n f e r that even l i m i t e d monoculture c o u l d be p o s s i b l e f o r p l a n t a t i o n s of black cottonwood. However, uniform p l a n t a t i o n s might s t r o n g l y favor other d i s e a s e s and d i s o r d e r s of black cottonwood. There i s a l s o a t h e o r e t i c a l i m p l i c a t i o n of these r e s u l t s . Very l i t t l e work has been done on n a t u r a l pathosystems i n the area of h o s t : p a r a s i t e g e n e t i c s . The lack of q u a l i t a t i v e r e s i s t a n c e and v i r u l e n c e i n the M. o c c i d e n t a l i s - P. t r i c h o c a r p a pathosystem i n d i c a t e s that q u a l i t a t i v e i n t e r a c t i o n s do not p l a y a major r o l e i n d i s e a s e i n t h i s system. However, many more s t u d i e s of other n a t u r a l pathosystems are r e q u i r e d before making the ge n e r a l c o n c l u s i o n that q u a l i t a t i v e i n t e r a c t i o n s a r i s i n g from p h y s i o l o g i c a l s p e c i a l i z a t i o n due to gene-for-gene e f f e c t s do not occur i n n a t u r a l pathosystems. 36 CONCLUSION An a n a l y s i s of v a r i a n c e of average d a i l y spore p r o d u c t i o n by 10 i s o l a t e s of Melampsora o c c i d e n t a l i s on 14 clones of Populus t r i c h o c a r p a showed no i n d i c a t i o n s of p h y s i o l o g i c a l s p e c i a l i z a t i o n . C l o n a l r e s i s t a n c e as we l l as i s o l a t e v i r u l e n c e d i f f e r e d s i g n i f i c a n t l y . A n a l y s i s of v a r i a n c e of t o t a l spores, average d a i l y p u s t u l e p r o d u c t i o n , and l a t e n t p e r i o d a l l gave t h i s same r e s u l t . The o v e r a l l average spore p r o d u c t i o n d u r i n g the time from i n o c u l a t i o n to twice the l a t e n t p e r i o d was 650 spores/disk/day. Latent p e r i o d ranged from 6 to 12 days with a median at 8 days. The sampling technique was b i a s e d toward s e l e c t i n g specimens which had the o p p o r t u n i t y to be p h y s i o l o g i c a l l y s p e c i a l i z e d , s i n c e i s o l a t e s were c o l l e c t e d along with t h e i r hosts i n l a t e summer; yet t h i s s p e c i a l i z a t i o n was not found. The l a c k of q u a l i t a t i v e r e s i s t a n c e and v i r u l e n c e i n d i c a t e s that q u a l i t a t i v e i n t e r a c t i o n s do not pl a y a major r o l e i n dis e a s e i n t h i s system. T h i s f i n d i n g holds the promise that cottonwood r e s i s t a n c e w i l l be of a durable nature and not d e v a s t a t i n g l y overcome when used i n p l a n t a t i o n s . » 37 LITERATURE CITED B i e r , J . 1965. Some e f f e c t s of f o l i a g e saprophytes i n the c o n t r o l of Melampsora l e a f r u s t on Black Cottonwood. F o r e s t r y C h r o n i c l e 41:306-315. Browning, A., M. Simons & E. T o r r e s . 1977. Managing host genes: epi d e m i o l o g i c and g e n e t i c concepts. i n : Plant Disease, V o l . I. eds: J . H o r s f a l l & E. Cowling. Academic Press, New York. pp. 191-212. Burdon, J . 1980. V a r i a t i o n i n d i s e a s e - r e s i s t a n c e w i t h i n a po p u l a t i o n of T r i f o l i u m repens . J o u r n a l of Ecology 68:737-744. Burdon, J . & D. M a r s h a l l . 198.1. I n t e r - and I n t r a - s p e c i f i c d i v e r s i t y i n the disease-response of G l y c i n e s p e c i e s to the l e a f - r u s t fungus Phakopsora p a c h y r h i z i . J o u r n a l of Ecology 69:381-390 Burdon, J . , J . Oates & D. M a r s h a l l . 1983. I n t e r a c t i o n s between Avena and P u c c i n i a s p e c i e s : I. Wild h o s t s : Avena barabata r A_̂_ fatua , A_j_ l u d o v i c i a n a . J o u r n a l of A p p l i e d Ecology 20:571-584. Chandrashekar, M. 1982. E f f e c t s of some chemicals employed i n the detached l e a f c u l t u r e of Populus on the i n f e c t i o n of Melampsora l a r i c i - p o p u l i n a . Eur. J . For. Path. 12(6/7):30l-307. & W. Heather. 1980. Rea c t i o n s of poplar c l o n e s to p h y s i o l o g i c races of Melampsora l a r i c i - p o p u l i n a . Phytopathology 72:327-330. Dinoor. A. 1977. Genes f o r d i s e a s e r e s i s t a n c e from n a t i v e p o p u l a t i o n s , pp. 15-25 i n : Induced Mutations a g a i n s t Plant D i s e a s e s . I n t e r n a t i o n a l Atomic Energy Agency, Vienna, A u s t r i a . Donnelly, J . 1974. Seasonal changes i n photosynthate t r a n s p o r t w i t h i n e l o n g a t i n g shoots of Populus g r a n d i d e n t a t a . Can. J . Bot. 52:2547-2559. E l d r i d g e , K. & A. Matheson & W. S t a h l . 1973. Genetic V a r i a t i o n i n r e s i s t a n c e t o po p l a r l e a f r u s t . A u s t r a l . For. Res. 6:53-59. E l l i n g b o e , A. 1975. H o r i z o n t a l r e s i s t a n c e : An a r t i f a c t of experimental procedure? A u s t r a l i a n P l a n t Pathology S o c i e t y Newsletter 3:44-46. E l l i n g b o e , A. 1981. Changing concepts i n host:pathogen g e n e t i c s . Annual Review of Phytopathology 19:125-143. FAO/IPC. 1980. Poplars and Willows i n wood p r o d u c t i o n and land use. FAO f o r e s t r y s e r i e s #10, Rome. 328 pp. 38 Farmer, R. 1970. Genetic v a r i a t i o n among o p e n - p o l l i n a t e d progeny of eastern cottonwood. Ann. Appl. B i o l . 31:149— 151. Farmer, R. & J . Wilcox. 1968. P r e l i m i n a r y t e s t i n g of e a s t e r n cottonwood c l o n e s . T h e o r e t i c a l and A p p l i e d Genetics 38:197-201 . Fleming, R. & C. Person. 1982. Consequences of p o l y g e n i c d e t e r m i n a t i o n of r e s i s t a n c e and a g g r e s s i v e n e s s i n n o n s p e c i f i c h o s t - p a r a s i t e r e l a t i o n s h i p s . Canadian J o u r n a l of P l a n t Pathology 45:680-685. F l o r , H. 1955. H o s t - p a r a s i t e i n t e r a c t i o n i n f l a x r u s t — i t s g e n e t i c s and other i m p l i c a t i o n s . Phytopathology 45:680- 685. Fowells, H. 1965. S i l v i c s of F o r e s t Trees of the United S t a t e s . Black Cottonwood. pp. 508-518. USDA F o r e s t S e r v i c e A g r i c u l t u r e Handbook 271. 762 pp. Fry, W. 1982. P r i n c i p l e s of P l a n t D i s e a s e . Academic Press, Toronto. pp. Galloway, G. & J . W o r r a l l . 1979. C l a d o p t o s i s : a r e p r o d u c t i v e s t r a t e g y i n black cottonwood? Can. J . For. Res. 9:122- 125. Gremmen, J . 1980. Problems and p r o s p e c t s i n breeding Melampsora ru s t r e s i s t a n t p o p l a r s . F o l i a F o r e s t a l i a 422:5- 9. Hansen, E., L. Moore, D. Netzer, M. O s t r y & H. Phipps & J . Z a v i t k o v s k i . 1983. E s t a b l i s h i n g i n t e n s i v e l y c u l t u r e d h y b r i d poplar p l a n t a t i o n s f o r f u e l and f i b e r . USDA For. Serv. Gen. Tech. Rep. NC-78. 24 pp. Heather, W. & M. Chandrashekar. 1982. S t a b i l i t y of d i s e a s e r e s i s t a n c e i n n a t u r a l and agro-ecosystems. Trans. B r i t Mycol. Soc. 78(2) : 381-383 - & J . Sharma. 1977. Some aspects of poplar r u s t r e s e a r c h in A u s t r a l i a . A u s t r a l i a n F o r e s t r y 40(l):28-43. , & A. M i l l e r . 1980. P h y s i o l o g i c s p e c i a l i z a t i o n i n Melampsora l a r i c i - p o p u l i n a on c l o n e s of poplar demonstrating p a r t i a l r e s i s t a n c e to l e a f r u s t . A u s t r a l i a n F o r e s t r y Research 10:125-131. Heybroek, H., B. Stephan & K. von Weissenberg. 1982. 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E f f e c t of c l i m a t i c f a c t o r s on development of the d i s e a s e caused by Melampsora l a r i c i - p o p u l i n a . F o l i a F o r e s t a l i a 422:10-13. L i n , M. & H. Edwards. 1974. Primary p e n e t r a t i o n process i n powdery mildewed b a r l e y r e l a t e d to host c e l l age, c e l l type, and occurrence of b a s i c s t a i n i n g m a t e r i a l . New P h y t o l o g i s t 73:131-137. Loegering, W. 1978. Current concepts of i n t e r o r g a n i s m a l g e n e t i c s . Annual Review of Phytopathology 16:309-320. Longo, N., F. Moriondo S< B. Longo. 1980. Overwintering and g e r m i n a b i l i t y of t e l i o s p o r e s of Melampsora p i n i t o r q u a . F o l i a F o r e s t a l i a 422:19-24. McBride, R. 1969. A m i c r o b i o l o g i c a l c o n t r o l of Melampsora medusae . Can. J . Bot. 47:711-715. McNabb, H., R. H a l l & M. Os t r y . 1982. 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E f f e c t of sa p r o p h y t i c p h y l l o p l a n e f u n g i on germination and development of Melampsora l a r i c i - p o p u l i n a . Trans. B r i t . Mycol. Soc. 72:225-231. 40 Palmberg, C. 1977. S e l e c t i n g f o r r u s t r e s i s t a n c e i n p o p l a r s i n A u s t r a l i a . T h i r d World C o n s u l t a t i o n on F o r e s t Tree Breeding. Canberrra, 1977. pp. 220-230. Robinson, R. 1979. Permanent and impermanent r e s i s t a n c e to crop p a r a s i t e s ; A re-examination of the Pathosystem concept with s p e c i a l r e f e r e n c e to Rice B l a s t . A. Pflanzenzuchtung 83:1-39. Robinson, R. 1980. New concepts i n breeding f o r d i s e a s e r e s i s t a n c e . Annual Review of Phytopathology 18:189-210. Schipper, A.L. & D.H. Dawson. 1974. Poplar l e a f r u s t -- a problem i n maximum wood f i b e r p r o d u c t i o n . P l a n t Disease Reporter 58:721-723. S c h r e i n e r , J . 1974. Populus L . 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Genetic v a r i a t i o n and h e r i t a b i l i t y of Melampsora l e a f r u s t r e s i s t a n c e i n Eastern cottonwood. F o r e s t Science 21:278-282 T h i e l g e s , B. & S. Land. eds. 1976. Symposium on eastern cottonwood and r e l a t e d s p e c i e s . L o u i s i a n a State U n i v e r s i t y , Baton Rouge, L o u i s i a n a . Toole, R. 1967. Melampsora medusae causes cottonwood r u s t in the lower M i s s i s s i p p i v a l l e y . Phytopathology 57:1361-1363. Vanderplank, J . 1963. P l a n t d i s e a s e s , epidemics and c o n t r o l . Academic Press, New York. 349 pp. 1982. Host-pathogen I n t e r a c t i o n s i n P l a n t Disease. Academic Press, Toronto. 207 pp. V l o t e n , H. van. 1949. K r u i s i n g s p r o e v e n met rassen van Melampsora l a r i c i - p o p u l i n a (klebahn) . ( C r o s s i n g experiments with s t r a i n s of Melampsora l a r i c i - p o p u l i n a , summary i n e n g l i s h ) T i j d s c h r i f t over P l a n t e n z i e k t e n 55:196-209. Wahl, I., N. Eshed, A. Segal & Z. Sobel. 1978. S i g n i f i c a n c e of w i l d r e l a t i v e s of small g r a i n s and other w i l d grasses i n c e r e a l powdery mildews. i n : The Powdery Mildews. ed: D. Spencer. Academic Press, New York. pp. 83- 100 W a l l e r , J . 1981. Fungal pathogens i n the a i r and i n p l a n t shoots. i n : Methods in p l a n t pathology, ed: R. B u r c h i l l . P h y t o p a t h o l o g i c a l Papers 26:9-16. Wang, D. 1961. The nature of s t a r c h accumulation at the r u s t i n f e c t i o n s i t e s i n leaves of p i n t o bean p l a n t s . Can. J . Bot 39:1595-1604. Weissenberg, K. von. 1980. E f f e c t of temperature on dormancy, a c t i v a t i o n , and long-term storage of t e l i o s p o r e s of Melampsora p i n i t o r q u a . F o l i a F o r e s t a l i a 422:32-36. Widin, K. & A. Schipper. 1976. Epidemiology and impact of Melampsora medusae l e a f r u s t on h y b r i d p o p l a r s . i n : I n t e n s i v e p l a n t a t i o n c u l t u r e : f i v e years r e s e a r c h . USDA 42 For. Serv. Gen. Tech. Rep. NC-21, p. 63-74. Wilcox, J . & R. Farmer. 1967. V a r i a t i o n and i n h e r i t a n c e of j u v e n i l e c h a r a c t e r i s t i c s of e a s t e r n cottonwood. S i l v a e Genetica 16:162-165. Zadoks, J . 1961. Yellow r u s t on wheat, s t u d i e s i n epidemiology and p h y s i o l o g i c s p e c i a l i z a t i o n . T. P l z i e k t e n 67:69-256. (as quoted in Zadoks, 1972. pp 43-63 in B i o l o g y of Rust Re s i s t a n c e in F o r e s t T r e e s ) . Zadoks, J . & R. Schein. 1978. Epidemiology and p l a n t d i s e a s e management, the known and the needed. i n : Comparative Epidemiology. eds: J . P a l t r i & J . Kranz. T h i r d I n t e r n a t i o n a l Congress of P l a n t Pathology, pp. 1-17. Z i l l e r , W. 1959. S t u d i e s of western t r e e r u s t s IV: U r e d i n o p s i s h a s h i o k a i and tL p t e r i d i s c ausing p e r e n n i a l needle r u s t of f i r . Can. J . Bot. 43:217-230. Z i l l e r , W. 1965. S t u d i e s of western t r e e r u s t s VI: The a e c i a l host ranges of Melampsora a l b e r t e n s i s , M̂_ medusae , and M._ o c c i d e n t a l i s . Canadian J o u r n a l of Botany 43:217-230. 1974. The t r e e r u s t s of Western Canada. Canadian F o r e s t S e r v i c e Dept. of Environment P u b l i c a t i o n #1329. 272 pp. Zimmer, D. & D. Rehder. 1976. Rust r e s i s t a n c e of w i l d H e l i a n t h u s s p e c i e s of the North C e n t r a l U n i t e d S t a t e s . Phytopathology 66:208-211. 43 APPENDIX A: V a r i o u s l a b e l s have been a p p l i e d to the r e s i s t a n c e , v i r u l e n c e and d i s e a s e i n t e r a c t i o n . The f o l l o w i n g l i s t i s adapted from Browning, Simons & To r r e s (1977): VERTICAL PERPENDICULAR SPECIFIC MAJOR GENE MONOGENIC OLIGOGENIC MULTIPLE ALLELE QUALITATIVE HIGH SEEDLING HYPERSENSITIVE PROTOPLASMIC DISCRIMINATORY DIFFERENTIAL HORIZONTAL LATERAL NON-SPECIFIC MINOR GENE POLYGENIC MULTIGENIC MULTIPLE GENE QUANTITATIVE LOW,MODERATE ADULT NON-HYPERSENSITIVE NON-PROTOPLASMIC DILATORY UNIFORM FIELD DURABLE TOLERANCE Vanderplank 1963 Vanderplank 1963 Zadoks 1961 Browning et a l . 1977 Vanderplank 1963 Johnson 1979 These are but some of the terms which have been used to d e s c r i b e r e s i s t a n c e , v i r u l e n c e , and d i s e a s e i n t e r a c t i o n . Within each column the terms given are at l e a s t p a r t i a l l y synonymous. P u b l i c a t i o n s i n the f i e l d of h o s t - p a r a s i t e g e n e t i c s most commonly use the terms v e r t i c a l and h o r i z o n t a l r e s i s t a n c e . However, these terms have become burdened with both g e n e t i c and e p i d e m i o l o g i c a l d e f i n i t i o n s which, a c c o r d i n g to many people, are not n e c e s s a r i l y e q u i v a l e n t (Johnson 1979, E l l i n g b o e 1981). Furthermore, the two columns represent two d i s t i n c t c a t e g o r i e s to some authors (as above) while others do not see a d i s t i n c t i o n and view the corr e s p o n d i n g terms as r e p r e s e n t i n g two extremes of a continuum (Nelson, 1978; E l l i n g b o e , 1975). The terms used i n t h i s paper, q u a l i t a t i v e and q u a n t i t a t i v e , are not n e c e s s a r i l y synonymous with other terms i n the same column, and should be read s o l e l y with the d e f i n i t i o n s given them i n t h i s paper. APPENDIX B: 1. LISTING OF ALL CLONES AND ISOLATES Name I s o l a t e Clone L o c a t i o n ALK X A l l i s o n Lake, B.C. CAL X Calgary, A l b e r t a CORD X Corduroy T r a i l , U.B.C Endowment Lands GRAN X X Grandview & Nanaimo S t r , Vancouver HEBB X X Hebb S t r . (Renfrew & 10th), Vancouver IMP X X Imperial D r i v e & 16th, Vancouver KEN X X Boundary & Nelson S t r , Vancouver MAR X Fr a s e r Viewpoint, U.B.C. PIC X X P i c n i c P o i n t , Kamloops, B.C. POCO X X Port Coquitlam, B.C. POND X Ponderosa C a f e t e r i a , U.B.C. PRG X Pr i n c e George, B.C. RUP X X Rupert & Grandview, Vancouver SAL X S a l i s h T r a i l near B.C. Res., U.B.C. THUN X X Thunderbird Stadium, U.B.C. TRQ X T r a n q u i l l e Farm, Kamloops, B.C. These c l o n e s d i d not s u r v i v e i n the greenhouse: ALK and PRG. These i s o l a t e s were not presen t : Cal,Cord,Mar,Pond,Sal,TrQ. 2. ISOLATE COLLECTION AND FORMATION DATES COLLECTION FORMATION I s o l a t e T e l i a Uredia B a s i d i a Pycnia A e c i a Uredia Alk 10/83 Gran 10/82 8/83 7/83 Hebb 8/83 Imp 1 0/82 20/5/83 31/5/83 16/6/83 3/7/83 Ken 1 1/82 8/83 6/83 Pi c 1 0/82 18/6/83 27/6/83 15/7/83 27/7/83 Poco 8/83 PrG 8/83 Rup 8/83 Thun 10/82 8/83 7/83 APPENDIX C: Leaf age s u s c e p t i b i l i t y study Three stems were s t r i p p e d e n t i r e l y of t h e i r leaves and the lea v e s were cut i n t o h a l v e s f o r i n o c u l a t i o n . The le a v e s were i n o c u l a t e d with s e v e r a l d r o p l e t s of the spore suspensions of an i s o l a t e known to be v i r u l e n t on that c l o n e . The d r o p l e t s were then spread a l l over the l e a f h a l v e s with a p a i n t b r u s h . l e a f h e i g h t * l e n g t h width p u s t u l e s (cm) (cm) (cm) (number) 1-1 2.2 8.0 2.5 0 1-2 4.6 11.6 4.7 1 1-3 7.2 14 . 9 6.5 g ** 1-4 10.5 15.5 5.7 8 1-5 14.0 14.6 5.5 4 1-6 18.0 13.8 5.9 1 1-7 2 1 . 2 10.8 5.4 10 1-8 2 4 . 8 11 . 5 5.2 10 2-1 .2 5.2 1 .8 0 2-2 .5 5.5 1 .9 0 2-3 1 .2 4.9 2.0 1 o ** 2-4 2.4 4.6 2. 1 2 2-5 4.7 4.8 1 .9 5 2-6 6.5 5.4 1 .8 5 2-7 7.8 4.3 1 .7 10 2-8 9.0 5.0 1 .9 1 2-9 10.2 5.0 1 .9 12 2-10 12.0 6.0 2.3 8 3-1 1 .0 7.5 3.9 0 3-2 2.0 7.0 3.4 0 3-3 2.3 8.2 3.5 7 * * 3-4 4.5 9.2 4.3 8 3-5 6.0 6.2 3.5 1 3-6 7.2 8.1 3.8 25 3-7 8.2 7.5 3.5 2 3-8 9.8 8.0 4.0 5 3-9 11.0 9.2 4.0 7 3-10 13.2 7.2 3.4 8 * h e i g h t r e f e r s to d i s t a n c e from the top of the p l a n t , and the s m a l l e s t height means the youngest l e a f , (e.g. l e a f 1-1 was the youngest l e a f on stem 1 ) . ** T h i s was judged to be the most r e c e n t l y expanded l e a f based upon the l e n g t h and width of the l e a f . By p u s t u l e number, t h i s l e a f was a l s o found to be the youngest or second youngest l e a f s u s c e p t i b l e to the r u s t . APPENDIX D: Mean average d a i l y spore p r o d u c t i o n df each p e t r i d i s h The c o r r e l a t i o n i s r=-.14, which i s not s i g n i f i c a n t . vs. inoculum c o n c e n t r a t i o n a p p l i e d to i d i s h . MEAN SPORE PRODUCTION 23396. + 20276. 17156. 14037. 10917. 7796.8 + * 4676.9 + * * * * 1557.0 + + 1000.0 5111.1 9222.2 3055.6 7166.7 11278. 13333. 17444. 15389. INOCULUM CONCENTRATION 19500. ON O N APPENDIX E: Haemocytometer spore count vs. l i g h t absorbance of the same spore s u s p e n s i o n . T h i s r e l a t i o n s h i p was used to d e r i v e an e q u a t i o n f o r c a l c u l a t i n g s p o r e s from absorbance. The d e r i v e d e q u a t i o n : SPORES = 1,228,300 * ABSORBANCE - 7294 has an r=.91 .14000 +6+ HAEMOCYTOMETER SPORE COUNT + * .12000 +6+ + .10000 +6+ 80000. + 60000. 40000. 20000. * * * 2 22 * * *** * *2 + 3 * * 22*2*2* *2 * * 3 5*2*2*3 * * +*789*722*** 2 * + + + + + +- .86950 -3 .23426 -1 .12148 -1 .45982 -1 .68538 -1 .91095 -1 .34704 -1 .57260 -1 .79817 -1 LIGHT ABSORBANCE 10237 -P- 48 APPENDIX F: ANALYSIS OF VARIANCE 1. Average spore p r o d u c t i o n d u r i n g twice the l a t e n t p e r i o d f o r a l l specimens SOURCE Block Treatment I s o l a t e s Clones I n t e r a c t i o n E r r o r T o t a l i. F, 8 1 39 1112 1 259 9 1 3 1 1 7 F-VALUE 6.944 2.357 5.318 14.442 .923 F-PROB .0000 .0000 .0000 .0000 .6884 2. Average spore p r o d u c t i o n f o r Vancouver Region specimens SOURCE D. F. F-VALUE F-PROB Block 8 5.003 .0000 Treatment 76 1 .885 .0001 I s o l a t e s 6 8.600 .0000 Clones 1 0 5.319 .0000 I n t e r a c t i o n 60 .870 .7457 E r r o r 608 T o t a l 692 3. Pu s t u l e count over twice l a t e n t p e r i o d SOURCE Block Treatment I s o l a t e s Clones I n t e r a c t i o n E r r o r T o t a l i. F, 8 139 1112 1 259 9 1 3 1 17 F-VALUE 6.693 2.514 8. 160 17.293 .851 F-PROB .0000 .0000 .0000 .0000 .8673 4. Number of spores produced d u r i n g twice l a t e n t p e r i o d SOURCE Block Treatment I s o l a t e s Clones I n t e r a c t i o n E r r o r T o t a l D. F, 8 1 39 1112 1 259 1 1 F-VALUE 6.865 341 219 1 1 7 ,831 2, 7, 16, F-PROB .0000 .0000 .0000 .0000 .8989 5. Latent P e r i o d (time i n days from i n o c u l a t i o n to symptoms) SOURCE Block Treatment I s o l a t e s Clones I n t e r a c t i o n E r r o r T o t a l D. F, 8 1 39 1112 1259 1 1 F-VALUE 3.548 1 .825 7.714 8. 105 .869 F-PROB .0000 .0000 .0000 .0000 .8321 49 APPENDIX G: A n a l y s i s of v a r i a n c e f o r i s o l a t e - c l o n e i n t e r a c t i o n where lowly c o r r e l a t e d c l o n e s and i s o l a t e s have been subject to a n a l y s i s on U.B.C. MTS program *ANOVAR MODEL,SPORIN=A+B+C+AC+E LIMITS,2,9,2 FACTORIAL DESIGN A1=Hebb A2=Gran C1=CAL C2=IMP DATA,-DAT RANDOM,B RENAME,BLOCK=B,ISOLATES=A,CLONES=C,INTERACTION=AC RANGE,(SOURCE=ALL),(SIGREQD=NONE),(CORREL=YES) OPTIONS,PRNTEMS INPUT,A(5,2),B(2,2),C(8,2),SPORIN(55,8) ANALYSIS OF VARIANCE FOR AVERAGE SPORE PRODUCTION RATE SOURCE D.F. F-VALUE F PROB ISOLATES 1 0.9022 0 .3543 CLONES 1 3.1251 0 .0864 INTERACTION 1 10.8899 0 .0031 BLOCK 8 1.1114 0 .3905 ERROR 24 TOTAL 35 I SOL CLONE MEAN STD ERROR RANGE TEST 1. Hebb * CAL 2324.000 168.444 A 2. Hebb * IMP 454.889 40.725 B 3. Gran * CAL 756.556 26.846 B 4. Gran * IMP 1321.667 177.073 B A ** T h i s was the only s i g n i f i c a n t F-value ( < .05 ), so a Duncan's M u l t i p l e Range Test was performed on these means of i s o l a t e - c l o n e i n t e r a c t i o n . Means f o l l o w e d by the same l e t t e r (A or B) are not s i g n i f i c a n t l y d i f f e r e n t . The c o n c l u s i o n can be made that e l i m i n a t i o n of c l o s e l y c o r r e l a t e d c l o n e s and i s o l a t e s (with r e s p e c t to di s e a s e s e v e r i t y ) can le a d to a s i g n i f i c a n t i s o l a t e - c l o n e i n t e r a c t i o n but without the more important s i g n i f i c a n t d i f f e r e n c e s between clones or between i s o l a t e s . 50 APPENDIX H: CALCULATIONS FOR COMPONENTS OF VARIANCE Begin with an a n a l y s i s of v a r i a n c e with mean squares: Source degrees of freedom mean square blocks 8 9013334 i s o l a t e s 9 6415802 cl o n e s 13 17423792 i n t e r a c t i o n 117 1206473 e r r o r 1112 1298330 Then c a l c u l a t e the expected mean squares (EMS): EMS(blocks) = VAR(error) + N * A * C * VAR(blocks) E M S ( i s o l a t e s ) = VAR(error) + N * B * C * V A R ( i s o l a t e s ) + N * B * V A R ( i n t e r a c t i o n ) EMS(clones) = VAR(error) + N * A * B * VAR(clones) + N * B * V A R ( i n t e r a c t i o n ) E M S ( i n t e r a c t i o n ) = VAR(error) + N * B * V A R ( i n t e r a c t i o n ) EMS(error) = VAR(error) Let the EMS values equal t h e i r mean square c o u n t e r p a r t s , and from the a n a l y s i s of v a r i a n c e : N i s the number of r e p l i c a t i o n ' s 0 A i s the number of i s o l a t e s 10 B i s the number of bl o c k s 9 C i s the number of c l o n e s 14 Then s o l v e f o r the v a r i a n c e s (VAR): VAR(blocks) = ( MS(blocks) - MS(error) ) / N * B = 55107 V A R ( i s o l a t e s ) = ( M S ( i s o l a t e s ) - M S ( i n t e r a c t ) ) / N * B * C =41344 VAR(clones) = ( MS(clones) - M S ( i n t e r a c t ) ) / N * A * B = 180192 V A R ( i n t e r a c t ) = ( M S ( i n t e r a c t ) - MS(error) ) / N * B = 0 VAR(error) = MS(error) =1298330 VAR(total)= 1574973 (the sum of these v a r i a n c e s ) Now c a l c u l a t i n g a l l the v a r i a n c e s as a percentage of the t o t a l w i l l g i v e what i s found i n Table I I I .

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