UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Effects of selection on populations of Ustilago hordei Christ, Barbara Jane 1984

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1984_A1 C47.pdf [ 7.91MB ]
Metadata
JSON: 831-1.0096396.json
JSON-LD: 831-1.0096396-ld.json
RDF/XML (Pretty): 831-1.0096396-rdf.xml
RDF/JSON: 831-1.0096396-rdf.json
Turtle: 831-1.0096396-turtle.txt
N-Triples: 831-1.0096396-rdf-ntriples.txt
Original Record: 831-1.0096396-source.json
Full Text
831-1.0096396-fulltext.txt
Citation
831-1.0096396.ris

Full Text

EFFECTS OF SELECTION ON POPULATIONS OF USTILAGO HORDEI By BARBARA JANE CHRIST B.Sc, The Pennsylvania State University, 1977 M.Sc, University of Minnesota, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE FACULTY OF GRADUATE STUDIES (DEPARTMENT OF BOTANY) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA Ap r i l 1984 © Barbara Jane Christ, 1984 In 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 the r equ i r ements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s tudy . I f u r t h e r agree 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 copy ing o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g ran ted by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s unde r s tood t h a t copy ing 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 no t be a l l owed w i thou t my w r i t t e n p e r m i s s i o n . The U n i v e r s i t y o f B r i t i s h Co lumbia 1956 Main Mall Vancouve r , Canada V6T 1Y3 Department o f A b s t r a c t The b a r l e y - U s t i l a g o h o r d e i system was examined i n p o p u l a t i o n g e n e t i c s t u d i e s . Three p o p u l a t i o n s of U s t i l a g o  h o r d e i were formed using t e l i o s p o r e s of f i v e d i f f e r e n t d i k a r y o n s f o r each p o p u l a t i o n . Each d i k a r y o n was heterozygous f o r a known v i r u l e n c e gene governing v i r u l e n c e t o the b a r l e y c u l t i v a r T r e b i . Each p o p u l a t i o n was s u b d i v i d e d and s i m u l t a n e o u s l y s e l e c t e d on T r e b i and Odessa, a r e s i s t a n t and s u s c e p t i b l e c u l t i v a r , r e s p e c t i v e l y . Changes i n the v i r u l e n c e a l l e l e frequency were monitored over the g e n e r a t i o n s of s e l e c t i o n . Increases i n frequency of the v i r u l e n t a l l e l e phenotype were observed f o r a l l p o p u l a t i o n s on both c u l t i v a r s . One p o p u l a t i o n was observed to d e c l i n e i n percent smutted p l a n t s w i t h i n the f i r s t g e n e r a t i o n of s e l e c t i o n whereas the o t h e r two p o p u l a t i o n s i n c r e a s e d . I t was observed t h a t the t e l i o s p o r e s of t h i s p o p u l a t i o n d i d not have the t y p i c a l g e r m i n a t i o n p a t t e r n f o r U s t i l a g o h o r d e i which was present i n the o t h e r two p o p u l a t i o n s . No apparent environmental f a c t o r s such as temperature or exogenous n u t r i e n t s were r e s p o n s i b l e f o r the abnormal germination. Abnormal germination was found to be passed on from the parents to the o f f s p r i n g and the next g e n e r a t i o n , suggesting t h a t i t was i n h e r i t e d . The r e s u l t s o f the g e r m i n a t i o n s t u d i e s d i r e c t l y c o r r e l a t e d abnormal ge r m i n a t i o n with reduced percent smutted p l a n t s . Tetrad a n a l y s i s of the i n d i v i d u a l d i k a r y o n s w i t h i n the p o p u l a t i o n s and the o r i g i n a l F-^  d i k a r y o n s r e v e a l e d t h a t the changes i n frequency of the v i r u l e n c e a l l e l e c o u l d have been produced by e i t h e r the i n c r e a s e of the v i r u l e n c e a l l e l e or by i n c r e a s e of the a p p r o p r i a t e combination o f polygenes which modify v i r u l e n c e . The s e g r e g a t i o n p a t t e r n s were s i m i l a r on both T r e b i and Odessa which may i n d i c a t e t h a t there i s a r e s i s t a n c e gene common to both c u l t i v a r s or t h a t the polygenes are n o n s p e c i f i c producing s i m i l a r e f f e c t s on both c u l t i v a r s . T h i s would e x p l a i n the r e s u l t s from the s e l e c t i o n experiments where there were i n c r e a s e s i n frequency of the v i r u l e n t phenotype r e g a r d l e s s of c u l t i v a r . i i i TABLE OF CONTENTS Page ABSTRACT i LIST OF TABLES V LIST OF FIGURES v i i i ACKNOWLEDGEMENTS X DEDICATION x i CHAPTER 1 INTRODUCTION 1 CHAPTER 2 POPULATION GENETICS OF HOST PARASITE SYSTEMS 10 Genetic Determination of Resistance and Virulence 10 Strategies for Disease Resistance 25 S t a b i l i z i n g Selection 40 CHAPTER 3 BARLEY: COVERED SMUT -BIOLOGY AND GENETIC ASPECTS 53 CHAPTER 4 GENERAL MATERIALS AND METHODS 68 Barley 68 Covered Smut 69 Inoculation and Planting 72 Selection Scheme 74 CHAPTER 5 SELECTION EXPERIMENTS WITH POPULATIONS OF USTILAGO HORDEI 7 6 Introduction 76 Materials and Methods 7 7 Results 80 Discussion 91 CHAPTER 6 LOWER DISEASE LEVELS CORRELATED WITH ABNORMAL GERMINATION OF USTILAGO HORDEI TELIOSPORES 97 Introduction 97 Materials and Methods 97 Results 99 Discussion I l l i v Page CHAPTER 7 FURTHER ANALYSIS OF POLYGENIC INHERITANCE OF VIRULENCE IN USTILAGO HORDEI 116 I n t r o d u c t i o n 116 M a t e r i a l s and Methods 117 R e s u l t s 118 D i s c u s s i o n 130 CHAPTER 8 GENERAL DISCUSSION AND CONCLUSIONS 137 LITERATURE CITED 152 APPENDICES A. Percent smut on b a r l e y c u l t i v a r T r e b i from s e l f i n g 10 U s t i l a g o Horde i t e l i o s p o r e s 173 B. Percent smut on b a r l e y c u l t i v a r T r e b i from d i a l l e l c r o s s o f s p o r i d i a o f F-^  U s t i l a g o h o r d e i t e l i o s p o r e s showing m o d i f i c a t i o n o f major v i r u l e n c e gene by po lygenes 17 4 C. Sc reen ing b a r l e y c u l t i v a r s to two U s t i l a g o h o r d e i r a c e s 17 5 D. E f f e c t o f seed ing r a t e on pe r cen t smutted p l a n t s 178 E. Pe rcent smut produced by F 2 d i k a r y o n s o f U s t i l a g o avenae on oa t c u l t i v a r Monarch 181 F. Percentages o f smut- in f ec t ed p l a n t s i n twelve v a r i e t i e s o f oa t s (Avena s a t i v a ) du r i ng e i g h t g e n e r a t i o n s o f s e l e c t i o n o f the pathogen ( U s t i l a g o avenae) on the oa t v a r i e t y Monarch 18 2 V LIST OF TABLES Page Tab le 2.1 H o s t - p a r a s i t e systems where e x c e p t i o n s to the t y p i c a l i n h e r i t a n c e p a t t e r n s o f r e s i s t a n c e i n gene-for-gene r e l a t i o n s have been r e p o r t e d . . 12 Tab l e 2.2 H o s t - p a r a s i t e systems where p o l y g e n i c a l l y determined r e s i s t a n c e has been demonstrated . . . . 13 Tab le 2.3 H o s t - p a r a s i t e systems where e x c e p t i o n s to the t y p i c a l i n h e r i t a n c e p a t t e r n s o f v i r u l e n c e i n gene-for-gene r e l a t i o n s have been r e p o r t e d 14 Tab le 2.4 H o s t - p a r a s i t e systems where p o l y g e n i c a l l y determined p a t h o g e n i c i t y has been demonstrated 22 Tab l e 2.5 Comparison o f s p e c i f i c terms o f t e n used i n p l a ce o f 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 28 Tab l e 3.1 Resu l t s o f " s e l f i n g " and b a c k c r o s s i n g a t e l i o s p o r e o f U s t i l a g o h o r d e i 6 3 Tab le 5.1 Comparison o f pe r cen t smutted p l a n t s o f b a r l e y c u l t i v a r T r e b i produced by us ing f o u r c o n c e n t r a t i o n s o f t e l i o s p o r e suspens ion o f U s t i l a g o h o r d e i 81 Tab le 5.2 Comparison o f pe r c en t smutted p l a n t s o f 15 U s t i l a g o h o r d e i d i k a r y o n s produced on b a r l e y c u l t i v a r s T r e b i and Odessa 81 Tab l e 5.3 Comparison o f pe r cen t smutted p l a n t s produced when th ree heterogeneous U s t i l a g o h o r d e i p o p u l a t i o n s and th ree c o n t r o l s were s e l e c t e d on two b a r l e y c u l t i v a r s , T r e b i and Odessa 8 3 Tab l e 5.4 Comparison o f pe r cen t smutted p l a n t s produced when th ree heterogeneous U s t i l a g o h o r d e i p o p u l a t i o n s were s e l e c t e d on one c u l t i v a r but i n o c u l a t e d onto another c u l t i v a r o f b a r l e y 84 Tab le 5.5 Summary o f a n a l y s i s o f v a r i a n c e o f seed weight and number o f b a r l e y c u l t i v a r T r e b i i n o c u l a t e d w i th th ree heterogenous U s t i l a g o h o r d e i p o p u l a t i o n s and one c o n t r o l 86 v i Page Tab le 5.6 Summary o f a n a l y s i s o f v a r i a n c e o f seed weight and number o f b a r l e y c u l t i v a r Odessa i n o c u l a t e d wi th th ree heterogeneous U s t i l a g o h o r d e i p o p u l a t i o n s and one c o n t r o l 86 Tab le 5.7 Comparison o f v i r u l e n c e a l l e l e phenotype f requency f o r the th ree heterogenous and two c o n t r o l U s t i l a g o h o r d e i p o p u l a t i o n s on b a r l e y c u l t i v a r T r e b i and Odessa 89 Tab le 5.8 Comparison o f v i r u l e n c e a l l e l e f r equency f o r the th ree heterogenous and two c o n t r o l U s t i l a g o  h o r d e i p o p u l a t i o n s on the c u l t i v a r s T r e b i and Odessa t e s t e d i n two yea r s 90 Tab le 6.1 Comparison o f the pe r cen t smutted p l a n t s produced by 15 U s t i l a g o h o r d e i d i k a r y o n s us ing s p o r i d i a l i n o c u l a t i o n v e r sus t e l i o s p o r e i n o c u l a t i o n on b a r l e y c u l t i v a r s T r e b i and Odessa 108 Tab l e 6.2 Pe rcen t smutted p l a n t s produced on b a r l e y ' c u l t i v a r s T r e b i and Odessa by f o u r U s t i l a g o  h o r d e i genotypes 110 Tab le 7.1 Pe rcen t smut on two b a r l e y c u l t i v a r s , T r e b i and Odessa , d e r i v e d from s e l f i n g 13 he te rozygous U s t i l a g o h o r d e i F 2 d i k a r y o n s 119 Tab le 7.2 Pe rcen t smut on two b a r l e y c u l t i v a r s , T r e b i and Odessa , d e r i v e d from s e l f i n g th ree homozygous U s t i l a g o h o r d e i d i k a r y o n s 122 Tab le 7.3 Pe rcent smut on two b a r l e y c u l t i v a r s , T r e b i and Odessa , d e r i v e d from s e l f i n g 10 F^  U s t i l a g o h o r d e i d i k a r y o n s 123 Tab l e 7 . 4 Pe rcent smut on two b a r l e y c u l t i v a r s , T r e b i and Odessa , d e r i v e d from s e l f i n g two p a r e n t a l U s t i l a g o h o r d e i d i k a r y o n s 125 Tab l e 7.5 Pe rcent smut on b a r l e y c u l t i v a r T r e b i d e r i v e d from b a c k c r o s s i n g th ree homozygous U s t i l a g o  h o r d e i d i k a r y o n s to p a r e n t a l d i k a r y o n s T^ and T„ 126 v i i P a g e Tab le 7.6 Pe rcent smut on b a r l e y c u l t i v a r T r e b i d e r i v e d from t e s t c r o s s i n g n ine he te rozygous to th ree homozygous U s t i l a g o h o r d e i d i k a r y o n s . . 127 Tab l e 8.1 P r o b a b i l i t y o f d i s e a s e l e v e l s when m u t l i p l e i n f e c t i o n s o c cu r w i th a heterogenous smut p o p u l a t i o n 143 Tab le C . l Pe rcen t smutted p l a n t s i n e i g h t b a r l e y c u l t i v a r s a f t e r i n o c u l a t i o n by two U s t i l a g o h o r d e i d i k a r y o n s 177 T a b l e D . l Summary o f a n a l y s i s o f v a r i a n c e o f U s t i l a g o  h o r d e i pe rcentage on b a r l e y c u l t i v a r Odessa a t f o u r seed ing r a t e s 180 Tab le D.2 Summary o f a n a l y s i s o f v a r i a n c e o f U s t i l a g o  h o r d e i percentage on b a r l e y c u l t i v a r T r e b i a t f ou r seed ing r a t e s 180 Tab l e D.3 Comparison o f average pe r cen t smut produced by a U s t i l a g o h o r d e i d i k a r y o n on b a r l e y c u l t i v a r T r e b i a t four, seed ing r a t e s 180 v i i i LIST OF FIGURES Page F igure 1.1 Termino logy o f d i s e a s e r e a c t i o n s , hos t and pathogen phenotypes i n host-pathogen i n t e r a c t i o n s 4 F i g u r e 1.2 The d i s e a s e r e a c t i o n s observed from the i n t e r a c t i o n s o f a r e s i s t a n c e gene and i t s a l l e l e s i n a hos t w i th the co r r e spond ing v i r u l e n c e gene and i t s a l l e l e s i n the pathogen 6 F igu re 1.3 The e v o l u t i o n o f gene-for-gene r e l a t i o n s by changes i n a l l e l e s i n hos t and pathogen as diagrammed w i t h i n the q u a d r a t i c check f o l l o w i n g the arrows s t a r t i n g a t 1 8 F i gu re 2.1 Models f o r the i n h i b i t o r s i n Melampsora l i n i ( la ) and Uromyces p h a s e o l i ( lb) 19 F i gu re 2.2 Comparison o f d i f f e r e n t i a l i n t e r a c t i o n s f o r v e r t i c a l r e s i s t a n c e (2.2a) to the cons t an t r ank ing observed f o r h o r i z o n t a l r e s i s t a n c e (2.2b) 27 F i gu re 3.1 L i f e c y c l e o f U s t i l a g o h o r d e i 55 F i g u r e 3.2 I l l u s t r a t i o n o f the t ypes o f t e t r a d s produced from a t e l i o s p o r e o f U s t i l a g o h o r d e i he te rozygous f o r a v i r u l e n c e gene and the r a t i o o f v i r u l e n t to a v i r u l e n t d i k a r y o n s produced from " s e l f i n g " 57 F i gu re 5.1 Comparison o f the f r equency o f number o f smutted t i l l e r s per p l a n t when i n o c u l a t e d w i th p o p u l a t i o n I o f U s t i l a g o h o r d e i between two b a r l e y c u l t i v a r s Odessa and T r e b i 87 F i gu re 5.2 Comparison o f the f r equency o f number o f smutted t i l l e r s per p l a n t when i n o c u l a t e d w i th p o p u l a t i o n I I I o f U s t i l a g o h o r d e i between two b a r l e y c u l t i v a r s Odessa and T e b i . 88 F i gu re 6.1 Pe rcen t spore g e r m i n a t i o n o f P o p u l a t i o n s I, I I , and I I I o f U s t i l a g o h o r d e i ove r th ree temperature reg imes 100 i x Page F igu re 6.2 Pe rcen t spore g e r m i n a t i o n o f P o p u l a t i o n s I, I I , and I I I o f U s t i l a g o h o r d e i on V o g e l ' s complete and min imal med ia , Po ta to dex t ro se agar (PDA), and water agar 102 F igu re 6.3 Pe rcen t spore g e r m i n a t i o n o f P o p u l a t i o n s I, I I , and I I I o f U s t i l a g o h o r d e i on Pota to dex t ro se agar (PDA) and seed e x t r a c t agar w i th e x t r a c t from b a r l e y c u l t i v a r T r e b i and Odessa 103 F i g u r e 6.4 Percent spore g e r m i n a t i o n o f P o p u l a t i o n s I, I I , and I I I o f U s t i l a g o h o r d e i a f t e r undergo ing one g e n e r a t i o n o f s e l e c t i o n on b a r l e y c u l t i v a r T r e b i 104 F i g u r e 6.5 Comparison o f pe r c en t spore g e r m i n a t i o n o f P o p u l a t i o n s I, C± and C 2 o f U s t i l a g o  h o r d e i 105 F i g u r e 6.6 Percent spore g e r m i n a t i o n o f the f i v e i n d i v i d u a l d i k a r y o n s i n each o f P o p u l a t i o n s I, II and I I I o f U s t i l a g o  h o r d e i 107 F i g u r e 6.7 Comparison o f pe r cen t spore ge rm ina t i on o f f o u r d i k a r y o n s o f U s t i l a g o h o r d e i 109 F i g u r e 8.1 Percent smutted p l a n t s expected f o r v a r y i n g numbers o f i n f e c t i o n s pe r seed by U s t i l a g o avenae 144 X ACKNOWLEDGEMENTS At v a r i o u s times throughout t h i s work, Dr. Person has f u n c t i o n e d as an a d v i s o r , teacher, c o l l e a g u e , f r i e n d and f a t h e r . He gave me many c h a l l e n g e s which have g i v e n me the c o n f i d e n c e needed as a s c i e n t i s t and educator. T h e r e f o r e , he deserves a s p e c i a l thanks. I thank Dr. Webster f o r the o p p o r t u n i t y to do p a r t of t h i s r e s e a r c h a t the U n i v e r s i t y o f C a l i f o r n i a , Davis and f o r h i s a d v i c e . He and the members of h i s l a b gave me tremendous support. Rolando R o b b i l l o deserves an award f o r h i s unending t e c h n i c a l and moral support. I a p p r e c i a t e the a d v i c e and e d i t o r i a l h e l p provided by my r e s e a r c h committee. I a l s o thank Dr. Groth f o r reading my t h e s i s and p r o v i d i n g u s e f u l comments. DEDICATION T h i s t h e s i s i s de d i c a t e d to two s p e c i a l people. F i r s t , to my a d v i s o r , Dr. Person, a s c i e n t i s t and n a t u r a l i s t who has a p p r e c i a t e d and questioned c o n s t a n t l y the i n t r i c a s i e s of t h i s world. Second, to my b r o t h e r , M a r t i n , whose handicap has never allowed him to understand or q u e s t i o n any wonder whether i t be g r e a t or s m a l l . Dr. Person and M a r t i n , each i n t h e i r own way gave me the i n i t i a t i v e , d r i v e and perseverance to undertake t h i s work, as w e l l as f u t u r e endeavors. CHAPTER 1 INTRODUCTION 1 Plant diseases were recognized and described as early as the t h i r d century B.C. by Theophrastus of Greece (Carefoot & Sprott, 1967). During the f i r s t century B.C., i t was a common Roman b e l i e f that plant diseases came from the gods. It was not u n t i l the seventeenth century a f t e r the invention of the microscope that some of the causal agents of plant diseases were discovered (Large, 1950). Theophrastus described how barley was more l i a b l e to mildew than wheat and within the barleys some mildewed more than others. Pliny, a Roman n a t u r a l i s t , noted that Roman wheat was more susceptible than barley to rust (Carefoot & Sprott, 1967). We know both of these observations to be quite accurate but of more importance i s the fact that these two n a t u r a l i s t s gave the f i r s t description of v a r i a t i o n i n disease resistance. Although differences i n disease l e v e l s were noted, no detailed analysis of these differences were made u n t i l the mid nineteenth century. As an example. Knight (1799) noted differences among wheat v a r i e t i e s i n rust resistance. Berkeley (1851) pointed out that white bulb onions were not r e s i s t a n t but colored bulb onions were r e s i s t a n t to a disease c a l l e d smudge. Goodridge published a paper in 1848 on resistance of potatoes to b l i g h t ( A l l a r d , 1960). It was not u n t i l the rediscovery of Mendel's work that the f i r s t genetic analysis of v a r i a t i o n i n disease resistance was performed. In 1905, Biffen crossed two wheat v a r i e t i e s and discovered that resistance to yellow rust was governed by a single recessive gene. Biffen's work demonstrated that high l e v e l s of resistance were inherited following simple Mendelian segrega-ti o n patterns. After B i f f e n , many papers covering a wide array of crops and t h e i r respective pathogens were published demon-str a t i n g single gene inheritance of resistance (Person & Sidhu 1971) . Resistance genes were incorporated into the common v a r i e t i e s of these crops and the v a r i e t i e s were widely grown. Plant breeders and pathologists believed at f i r s t that r e s i s t -ant c u l t i v a r s would provide permanent freedom from diseases and perhaps even eliminate the pathogen. This was not the case and in many cases the pathogen quickly adapted and gained virulence to overcome the resistance genes. The search for and incorporation of new resistance genes began an endless cycle that s t i l l continues. While others were observing differences i n disease r e s i s t -ance, Eriksson (1894) observed v a r i a t i o n for host species preference within Puccinia graminis. Using d i f f e r e n t host species he subdivided J P ^ graminis into formae s p e c i a l i s . The next step in pathogen v a r i a b i l i t y was observed by Barrus (1911). He discovered that within the species Colletotrichum  1indemuthianum, the anthracnose pathogen of beans, there existed variants or biotypes (now ca l l e d p hysiological races) which d i f f e r e d i n pathogenicity on d i f f e r e n t bean c u l t i v a r s . 3 Shortly a f t e r Barrus, Stakman (1914) showed that Eriksson's subspecies were not pathogenically homogeneous but also included many physiological races. These findings were subsequently extended to many other pathogenic fungi and other pathogens. It was soon recognized that through s h i f t s i n frequency of certa i n races, pathogens r e a d i l y overcame newly incorporated resistance genes. The discovery of sex in smut by Kniep (1919) and l a t e r in rust by Craigie (1927) made i t possible to examine the i n h e r i -tance of pathogenicity. Goldschmidt (1928) was the f i r s t to study the genetics of pathogenicity. Using races of Ustilago  violacea and t h e i r hybrids, he discovered that virulence (the degree of pathogenicity) was controlled by a single gene. Subsequently, several other pathogens were examined and again virulence was found to be under simple Mendelian segregation. Segregation patterns for both resistance in the host and virulence in the pathogen are based on disease reactions ( F i g . 1.1). It was not u n t i l the 1940's that Flor (1946, 1947, 1955) working with flax and flax rust (Melampsora l i n i ) , examined the genetic interactions that take place within a host-pathogen system. This was done by p a r a l l e l crossing experiments performed on the host and pathogen. For every resistance gene that he observed segregating in an F 2 host population, he observed segregation for a virulence gene i n the pathogen. From his re s u l t s he concluded that resistance was only expressed when matched to the corresponding avirulent 4 Hos t : Pathogen I n t e r a c t i o n D isease Reac t i on Compat ib le o r Incompat ib le ( + ) (-:) y Phenotypes y^ S u s c e p t i b l e R e s i s t a n t (Host) V i r u l e n t A v i r u l e n t (Pathogen) F i gu re 1.1 Termino logy o f d i s e a s e r e a c t i o n s , hos t and pathogen phenotypes i n host-pathogen i n t e r a c t i o n s . 5 (phenotype of "frustrated" pathogen producing no disease or very low levels) a l l e l e and avirulence was only expressed when matched with the corresponding resistance a l l e l e ( F i g . 1.2). Flor's work led him to the gene-for-gene concept, where for each gene conditioning a rust reaction i n the host, there i s a corresponding gene for virulence in the pathogen. Since then gene-for-gene r e l a t i o n s were demonstrated i n many host-parasite systems (Sidhu 1980). Person's (1959) t h e o r e t i c a l contribution of gene-for-gene interactions not only brought recognition to Flor's work but was the f i r s t to extrapolate the gene-for-gene in t e r a c t i o n to interactions between the host and pathogen populations and to apply the concepts of population genetics to host-parasite systems. He also proposed how gene-for-gene r e l a t i o n s possibly evolved by examining the interaction of a s p e c i f i c virulence and resistance gene and t h e i r changes at the population l e v e l (1968, 1974). Person suggested (Fig. 1.3) that i f a host population i s exploited by a pathogen so that there are high l e v e l s of disease, the presence of the pathogen exerts selection pressure on the host population for the procurement of a resistance gene. Because of the benefits of the resistance gene gained by the host, t h i s gene would probably spread r a p i d l y through the host population ( i n a g r i c u l t u r a l systems, the plant breeder incorporates the resistance genes in the common host v a r i e t i e s which are then widely grown). The resistance gene provides low 6 Host Pathogen RR/Rr r r AA/Aa aa R/r = resistance/susceptible a l le les A/a = avirulence/virulence a l le les = no disease or low levels (incompatible reaction) + = high levels of disease (compatible reaction) Figure 1.2 The disease reactions observed from the interactions of a resistance gene and i t s a l le les in a host with the corresponding virulence gene and i ts a l le les in the pathogen. This is the quadratic check from Ellingboe 1976. l e v e l s of disease or no disease - the incompatible reaction which i s known in gene-for-gene r e l a t i o n s to be attributed to the interaction of avirulent and resistance a l l e l e s . At t h i s time, only a genetic change has occurred i n the host, so the i n i t i a l i n teraction must have started i n #1 of Fig. 1.3 and has shif t e d to #2. Now the presence of the resistance gene i n the host population exerts pressure on the pathogen population so that the pathogen gains a virulence gene acting s p e c i f i c a l l y on that resistance gene to overcome resistance. This change moves the interaction of the host and pathogen to the compatible reaction of #3, Fig. 1.3. As soon as t h i s change occurs, the host population no longer requires the resistance a l l e l e and the removal of that a l l e l e w i l l make room for other resistance a l l e l e s . This change s h i f t s the interaction to #4 and now allows for the pathogen to lose the virulence a l l e l e by reverting to the avirulence a l l e l e of that locus. When the change i n the pathogen occurs, the interaction i s back to #1. This demonstration was for one p a r t i c u l a r gene i n the host and pathogen; but o v e r a l l s e l e c t i o n w i l l favor mutations i n the host that place the pathogen at a disadvantage and likewise, s e l e c t i o n w i l l favor mutations i n the pathogen that increases i t s reproductive c a p a c i t i e s . Those papers and others by Person (1966, 1967) gave insight on the rapid changes observed i n pathogen populations. These papers also supported Johnson's (1961) viewpoint that the plant breeder and pathologist are guiding the evolution of a Host Pathogen R/Rr rr AA/Aa aa R/r = resistance/susceptible a l le le A/a = avirulence/virulence a l le le -/+ = incompatible/compatible reaction ure 1.3 The evolution of gene-for-gene relations by changes in a l le les in host and pathogen as diagrammed within the quadratic check following the arrows starting at 1. See text for explanation. 9 pathogen. Therefore i t i s e s s e n t i a l t h a t the g e n e t i c s t r u c t u r e and dynamics of the host and pathogen p o p u l a t i o n s are understood. 10 CHAPTER 2 POPULATION GENETICS OF HOST-PARASITE SYSTEMS Genetic Determination of Resistance and Virulence* Since Flor's p a r a l l e l crossing experiments with flax and flax rust (1946, 1947, 1955), many host-parasite systems have been shown to have gene-for-gene interactions (Sidhu 1980). Based on t h e o r e t i c a l considerations, Person (1959) proposed that gene-for-gene interactions would be found to occur widely in p a r a s i t i c systems, and t h i s i s now known to be the case. These interactions have been found not only in plant-fungi interactions but also i n plant interactions with viruses, bacteria, nematodes and insects (Sidhu 1980). Certain simi-l a r i t i e s between gene-for-gene and antigen-antibody int e r a c t -ions have been recently discussed by Person and Christ (1983) . Several c h a r a c t e r i s t i c s are common to a l l plant-parasite systems where gene-for-gene r e l a t i o n s have been demonstrated. F i r s t resistance i s usually dominant and controlled by a single gene which may be linked, unlinked or i n a multiple a l l e l i c s e r i e s . Second, virulence i s usually recessive and inherited by single independent genes. Third, single combinations for incompatibility are e p i s t a t i c to a l l other interactions. Fourth, resistance and avirulence a l l e l e s function as condit-ional genes. Deviations have never been observed i n the l a s t * Part of t h i s section was used for a 1983 B r i t i s h Plant Pathology Symposium and w i l l be published i n 1984 as: The genetic determination of v a r i a t i o n i n pathogenicity. In: Populations of plant pathogens: Their dymanics and genetics. M.S. Wolfe and C E . Caten (eds.), Blackwell S c i . Pub., Oxford. 11 two c h a r a c t e r i s t i c s . I t i s now g e n e r a l l y agreed t h a t the s p e c i f i c t y o f gene-for-gene i n t e r a c t i o n s stems from the s p e c i f i c t y of i n t e r a c t i o n between the c o n d i t i o n a l r e s i s t a n c e and a v i r u l e n c e a l l e l e s . E x c e p t i o n s to the t y p i c a l c h a r a c t e r i s t i c s o f the i n h e r i t a n c e of r e s i s t a n c e i n c l u d e s : 1) r e s i s t a n c e genes t h a t are r e c e s s i v e ( r a t i o s are based on phenotypes which are the r e s u l t o f two i n t e r a c t i n g organisms), 2) two genes c o n t r o l l i n g r e s i s t a n c e to a p a r t i c u l a r pathogen genotype ( i t i s not always c l e a r whether two genes i n the host are i n t e r a c t i n g with o n l y one gene i n the pathogen), 3) m o d i f i e r s which can enhance the e f f e c t s of a major r e s i s t a n c e gene (there i s s p e c u l a t i o n t h a t "matched" r e s i s t a n c e genes may pro v i d e some r e s i d u a l r e s i s t a n c e and may a c t as m o d i f i e r s ) , 4) suppressors o f r e s i s t a n c e genes which are o c c a s i o n a l l y found e s p e c i a l l y when genes from a d i s t a n t r e l a t i v e of the host s p e c i e s are i n c o r p o r a t e d i n t o the host (Table 2.1). The major e x c e p t i o n t o gene-for-gene charac-t e r i s t i c s f o r r e s i s t a n c e i s r e s i s t a n c e c o n t r o l l e d by s e v e r a l genes t h a t have s m a l l a d d i t i v e a f f e c t s (Table 2.2). T h i s type o f r e s i s t a n c e i s con s i d e r e d as completely separate from major gene r e s i s t a n c e because the gene a c t i o n appears t o be non-s p e c i f i c as compared to the type of r e s i s t a n c e i n gene-for-gene systems. There are a l s o e x c e p t i o n s to a l l the c h a r a c t e r i s t i c i n h e r i t a n c e p a t t e r n s observed f o r v i r u l e n c e i n gene-for-gene systems. The f i r s t e x c e p t i o n i s t h a t dominant v i r u l e n c e has Tab l e 2.1 H o s t - p a r a s i t e systems where e x c e p t i o n s to the t y p i c a l i n h e r i t a n c e p a t t e r n s o f r e s i s t a n c e i n gene-for-gene r e l a t i o n s have been r e p o r t e d Systems r e c e s s i v e i n h e r i t a n c e : T r i t i c u m - P u c c i n i a s t r i i f o r m i s T r i t i c u m - P. g r a m i n i s t r i t i c i T r i t i c u m - P. r e c o n d i t a Avena - P. g r a m i n i s avenae Zea - P. s o r g h i Hordeum - U s t i l a g o h o r d e i c o n t r o l by 2 genes : Hordeum - U s t i l a g o nuda  T r i t i c u m - P. g r a m i n i s t r i t i c i T r i t i c u m - P. r e c o n d i t a  Avena - P. g r a m i n i s avenae Avena - P. co rona t a Reference B i f f e n 1905 Lupton & Macer 196 2 Knot t & Anderson 1956 Sawhney e_t a_l. 1981 Dyck & Samborski 1968 Bar tos e t al. 1969 McKenzie & Green 1965 McKenzie & Martens 1968 Malm & Hooker 196 2 Hooker & Saxena 1967 We l l s 1958 Konzak 1953 Knot t & Anderson 1956 Knot t 1957 Dyck & Samborski 198 2 Martens et a l . 1981 Baker 196 6 m o d i f i e r s : T r i t i c u m - P. r e c o n d i t a Dyck e t a_l. 1966 Samborski & Dyck 198 2 Avena - P. g r a m i n i s avenae McKenzie & Martens 1968 s u p p r e s s o r s : T r i t i c u m - P. g r a m i n i s t r i t i c i Kerber & Green 1980 Tab le 2.2 H o s t - p a r a s i t e systems where p o l y g e n i c a l l y determined r e s i s t a n c e has been demonstrated Systems T r i t i c u m - P u c c i n i a s t r i i f o r m i s T r i t i c u m - P. g r am in i s t r i t i c i  T r i t i c u m - P. r e c o n d i t a Avena - p. co rona t a  Hordeum - P. h o r d e i Solanum - Phytophthora i n f e s t a n s  Zea - C o c h l i o b u l u s carbonum  B r a s s i c a - L ep to sphae r i a maculans Reference Lewe l len & Sharp 1968 Lupton & Johnson 1970 Skovmand e_t a l . 1978 G a v i n l e r t v a t a n a & W i l coxson 1978 Luke e t a l . 1975 P a r l e v l i e t 1978 Johnson & W i l coxson 1979 Thurs ton 19 71 Hamid et_ al. 1982 Cargeeg & T h u r l i n g 1980 14 Tab l e 2.3 H o s t - p a r a s i t e systems where e x c e p t i o n s to the t y p i c a l i n h e r i t a n c e p a t t e r n s o f v i r u l e n c e i n gene-for-gene r e l a t i o n s have been r e p o r t e d Systems Reference dominant i n h e r i t a n c e : P u c c i n i a g r a m i n i s t r i t i c i T r l t i c u m P. g r am in i s avenae - Avena P. c o r o n a t a - Avena  Melampsora l i n i - Linum  Uromyces p h a s e o l i - Phaseo lus  U s t i l a g o h o r d e i - Hordeum Johnson 1954 Lu ig & Watson 1961 Green 1964, 1966 W i l l i ams eji al^. 1966 Kao & Knot t 1969 Green 1965 Green & McKenzie 1967 Haggag ejt al_. 197 3 S t a t l e r 1977, 1982 S t a t l e r & Jones 1981 Zimmer et: aJU 1965 F l o r 1946 C h r i s t & Gro th 1982 Ebba & Person (unpub l . ) c o n t r o l by 2 genes : P. g r a m i n i s t r i t i c i - T r i t i c u m P. r e c o n d i t a - T r i t i cu rn P. g r a m i n i s avena M. 1 i n i - Linum U. p h a s e o l i - Phaseo lus U. h o r d e i - Hordeum Kao & Knot t 1969 S t a t l e r 1977 ,1979a,1982 S t a t l e r & Jones 1981 Green 1965 S t a t l e r & Zimmer 1976 S t a t l e r 1979b Lawrence jet aJ., 1981 C h r i s t & Groth 1982 Ebba & Person 1975 Tab l e 2.3 ( cont inued) Systems l i n k a g e : P. r e c o n d i t a - T r i c t i c u m M. l i n i - Linum U. p h a s e o l i - Phaseo lus U. h o r d e i - Hordeum m o d i f i e r s : P. g r a m i n i s t r i t i c i - T r i t i c u m P. r e c o n d i t a - T r i t i c u m U. h o r d e i - Hordeum " i n h i b i t o r s " o r i n t e r a c t i o n o f 2 genes P. r e c o n d i t a - T r i t i c u m P. g r a m i n i s t r i t i c i - T r i t i c u m P. g r a m i n i s avenae - Avena U. p h a s e o l i - Phaseo lus M. l i n i - Linum Bremia l a c t u c a e - Lac tuca 15 Reference Samborski & Dyck 1976 S t a t l e r & Jones 1981 S t a l t e r 1982 S t a t l e r 1982 Lawrence e_t a J . 1981 C h r i s t & Gro th 1982 Pedersen & K i e s l i n g 1979 Green 1965 Green & McKenzie 1967 Samborski & Dyck 1968 Dyck & Samborski 1974 Pedersen & K i e s l i n g 1979 Samborski & Dyck 1968 Haggag e t a_l. 197 3 Kao & Knot t 1969 Green & McKenzie 1967 C h r i s t & Groth 1982 Lawrence e_t aJL. 1981 I.R. C r u t e , p e r s o n a l communicat ion 16 been r e p o r t e d f o r a number o f pathogens (Tab le 2 .3 ) . Person and Mayo (1974) p o i n t e d out t ha t the o v e r a l l p a t t e r n o f i n t e r -a c t i o n o f a hos t and p a r a s i t e i s not a l t e r e d by changes i n dominance r e l a t i o n s o f a hos t and/or p a r a s i t e . I t i s g e n e r a l l y accepted tha t a t the b i o c h e m i c a l l e v e l , dominance i s a s s o c i a t e d w i th a f u n c t i o n a l gene p roduc t and t h e r e f o r e , one would t h i n k t h a t the re i s a p roduc t from both the r e s i s t a n c e a l l e l e and a v i r u l e n c e a l l e l e t h a t may be needed t o form a h y b r i d mo lecu le t ha t a c t s as a " s t o p s i g n a l " . Because the g e n e t i c i n t e r p r e t a t i o n i n h o s t - p a r a s i t e systems i s based on the d i s e a s e r e a c t i o n which i s the phenotype expressed by two i n t e r a c t i n g o rgan i sms , i t i s d i f f i c u l t to d i s t i n g u i s h the u n d e r l y i n g g e n e t i c o r b i o c h e m i c a l mechanisms. I t i s p o s s i b l e t h a t the a v i r u l e n c e a l l e l e may p r o v i d e some s o r t o f r e c o g n i t i o n f o r the hos t which then may tu rn on c e r t a i n subsequent e v e n t s . /An example o f the complex na tu re o f dominance r e l a t i o n s i s i n P u c c i n i a g r a m i n i s t r i t i c i where W i l l i ams j3t a_l. (1966) r e p o r t e d an a v i r u l e n c e gene to be r e c e s s i v e on one c u l t i v a r but dominant on another c u l t i v a r . Kao and Knot t (1969) found a case where the re were two a l l e l e s f o r v i r u l e n c e , one which was dominant and the o the r r e c e s s i v e . The second e x c e p t i o n t o the g e n e r a l v i r u l e n c e c h a r a c t e r -i s t i c s i s the case o f two v i r u l e n c e genes matched t o one r e s i s t a n c e gene (Tab le 2 . 3 ) . There are two p o s s i b i l i t i e s f o r the e v o l u t i o n o f two v i r u l e n c e genes matched t o one r e s i s t a n c e 17 gene. One p o s s i b i l i t y i s a s tepwise g a i n i n v i r u l e n c e where the re i s a s i n g l e r e s i s t a n c e gene i n the hos t f o r which v i r u -l ence i n the pathogen p r o g r e s s i v e l y i n c r e a s e s by changes a t two l o c i such tha t both v i r u l e n c e genes are now necessa r y f o r p a t h o g e n i c i t y . The o the r p o s s i b i l i t y i s where two pathogen p o p u l a t i o n s have s e p a r a t e l y ga ined a v i r u l e n c e gene f o r a s p e c i f i c r e s i s t a n c e gene and , when these are brought t oge the r i n a s i n g l e genotype , e i t h e r o f these genes w i l l now p r o v i d e v i r u l e n c e . Haggag e_t a^L. (1973) had ev idence f o r two genes gove rn ing v i r u l e n c e where the second gene was p o s s i b l y a c t i n g as a supp re s so r o f the o the r a v i r u l e n t a l l e l e . C h r i s t and Groth (198 2) found two genes i n the pathogen matched to one r e s i s t -ance gene. The s e g r e g a t i o n p a t t e r n s o f the one gene showed tha t a v i r u l e n c e was dominant . The second gene was o n l y d i s c o v e r e d when a p o s t u l a t e d he te rozygous i s o l a t e t ha t when s e l f e d d i d not seg rega te 3 a v i r u l e n t r l v i r u l e n t but gave a l l a v i r u l e n t p rogeny . A d d i t i o n a l e v idence was p rov ided by a homozygous i s o l a t e tha t was s e l f e d which d i d not g i v e a l l v i r u l e n t progeny but segrega ted 3 v i r u l e n t : l a v i r u l e n t i n d i c a t i n g v i r u l e n c e was dominant f o r the second gene. Because o f the i n t e r a c t i o n o f these two genes they a l l u d e d to the p o s s i b i l i t y o f the second gene be ing an i n h i b i t o r gene . In Melampsora l i n i , Lawrence e_t a_l. (1981a) d i s c o v e r e d t h a t s e v e r a l v i r u l e n c e genes were a s s o c i a t e d w i th i n h i b i t o r genes . From the e x t e n s i v e a n a l y s i s w i th M. l i n i , a model can be proposed where the v i r u l e n c e gene and i n h i b i t o r gene are 18 matched to one r e s i s t a n c e gene , w i th the dominant i n h i b i t o r a l l e l e supp re s s i ng the e x p r e s s i o n o f the dominant a v i r u l e n t a l l e l e ( F i g . 2 .1a ) . The r e s u l t s from Uromyces p h a s e o l i ( C h r i s t & Groth 1982) do not f i t the Lawrence model ( F i g . 2 .1b ) . In U.  p h a s e o l i i t appears t ha t the e x p r e s s i o n o f the v i r u l e n c e a l l e l e i s i n h i b i t e d . Lawrence 's (1981a) use o f the term " i n h i b i t o r " was not in tended to imply any p a r t i c u l a r b i o c h e m i c a l mode o f a c t i o n f o r these genes , but mere ly to d e s c r i b e the phenotype observed when two genes i n t e r a c t . I n t e r a c t i o n s have been observed f o r s e v e r a l r u s t f u n g i (Tab le 2.3) and i n downy mildew o f l e t t u c e ( I .R. Crute p e r s o n a l communica t ion ) . The e x i s t e n c e o f these i n t e r a c t i o n s sugges t t ha t c a u t i o n shou ld be e x e r c i s e d i n i n f e r r i n g the genotype o f the pathogen from i t s phenotype . Other e x c e p t i o n s to the t y p i c a l v i r u l e n c e c h a r a c t e r i s t i c s are l i n k a g e o f v i r u l e n c e genes and m o d i f i e r s o f v i r u l e n c e genes (Tab le 2 .3 . ) L inkage has o n l y been r epo r t ed r e c e n t l y , p robab l y because o f the p a u c i t y o f d e t a i l e d g e n e t i c a n a l y s i s o f pa thogens . M o d i f i e r s o f v i r u l e n c e genes w i l l be d i s c u s s e d i n f u r t h e r d e t a i l i n a l a t e r s e c t i o n . So f a r these e x c e p t i o n s do not a l t e r the o v e r a l l p a t t e r n o f d i s e a s e r e c t i o n s observed w i t h i n the " q u a d r a t i c c h e c k " . Person and C h r i s t (1983) compared the d i s e a s e p a t t e r n o f the " q u a d r a t i c check" w i th the d i s e a s e mat r i x from a n t i g e n -an t i body i n t e r a c t i o n s and observed tha t the s p e c i f i c i n t e r -a c t i o n i n both i s the f a i l u r e o f d i s e a s e deve lopment . For both gene-for-gene i n t e r a c t i o n s o f p l a n t - p a r a s i t e systems and the a n t i g e n - a n t i b o d y i n t e r a c t i o n s , the phenotype h i d e s the 19 l a . M. l i n i model based on Lawrence et . a±. 1981 Parents F 2 gentotype1 a a l l x AAii Aali 9 A_I_:3 A _ i i : 3 aal_ :1 a a i i reaction 2 + + + - + + l b . U. phaseoli model based on Christ & Groth 1982 Parents F^ genotype a a l l x Aaii 1 Aali : 1 aal i reaction + - - + F± (selfed) F 2 genotype aa l i 3 aal_ : 1 aai i reaction + + -1. A/a = avirulence/virulence a l le les I / i = a l le les to inhibitor gene 2. +/- = compatible/incompatible reaction Figure 2.1 Models for the inhibitors in Melampsora l i n i (la) and Uromyces phaseoli (lb). 20 p o t e n t i a l complex i n t e r a c t i o n s t ha t a re i n v o l v e d . The complex b a s i s u n d e r l y i n g the a n t i b o d i e s was rev iewed by Leder (1982) . For a n t i g e n - a n t i b o d y i n t e r a c t i o n s the u n d e r l y i n g g e n e t i c s can be c o m p l i c a t e d . To take one example, A f r i c a n trypanosomes evade the hos t immune response by v a r y i n g t h e i r s u r f a c e a n t i -g ens . The a n t i g e n p r o p e r t i e s are due to a s i n g l e g l y c o p r o t e i n . A s i n g l e trypanosome can make more than 10 0 types o f g l y c o p r o t e i n s t ha t are so d i f f e r e n t t h a t the hos t a n t i b o d i e s a g a i n s t one g l y c o p r o t e i n are not e f f e c t i v e a g a i n s t o t h e r t y p e s . Recent s t u d i e s , rev iewed i n Bo r s t and Cross (1982) , show tha t each trypanosome seems to c o n t a i n the e n t i r e s e t o f a n t i g e n genes and tha t the a c t i v a t i o n o f some o f these genes i n v o l v e s t h e i r t r a n s p o s i t i o n i n t o an e x p r e s s i o n s i t e . The a n t i b o d i e s o f the hos t need o n l y e x e r t a s e l e c t i v e e f f e c t i n a heterogeneous trypanosome p o p u l a t i o n because a low degree o f s w i t c h i n g o f the a n t i g e n type i s an i n t r i n s i c g e n e t i c p r o p e r t y . For p l a n t - p a r a s i t e systems i t appears from ( F l o r ' s work) t ha t each r e s i s t a n c e gene o f an a l l e l i c s e r i e s i n t e r a c t s w i th a sepa ra te and independent g e n e t i c l o c u s o f the pathogen t h a t i s capab le o f p r o v i d i n g the "ma t ch ing " v i r u l e n c e a l l e l e . T r a n s -p o s i t i o n o f v i r u l e n c e from one s i t e to another i s not known. For both systems the hos t e x e r t s s e l e c t i o n f o r a s p e c i f i c v i r u l e n c e t ha t i s advantageous to the p a r a s i t e , r e g a r d l e s s o f the mechanism by which the v i r u l e n c e i s e x p r e s s e d . Leav ing the a n t i g e n - a n t i b o d y ana logy o f gene-for-gene r e l a t i o n s , the re i s one major d e v i a t i o n from the gene-for-gene c h a r a c t e r i s e s o f v i r u l e n c e . Th i s d e v i a t i o n , l i k e the one f o r r e s i s t a n c e genes , i s where v i r u l e n c e i s c o n t r o l l e d o r m o d i f i e d by s e v e r a l genes . Zimmer e_t ja_l. (1965) i d e n t i f i e d a major v i r u l e n c e gene i n P. c o r o n a t a . Some v a r i a t i o n was observed w i t h i n the proposed d i s c r e t e c l a s s e s . They suggested t h a t minor genes may be mod i f y i ng the e f f e c t s o f the v i r u l e n c e gene and tha t the minor genes would be hard t o d i s t i n g u i s h because t h e i r e f f e c t s cou ld be o f the same magnitude as env i ronmenta l a f f e c t s . The f i r s t r e p o r t on the c o n t r o l o f p a t h o g e n e c i t y by po lygenes was by Emara (1972) working w i th U s t i l a g o h o r d e i . S ince then a few o t h e r pathogens have been demonstrated to have p o l y g e n i c a l l y determined p a t h o g e n i c i t y (Tab le 2 . 4 ) . The major q u e s t i o n conce rn i ng po lygenes i n h o s t - p a r a s i t i c systems i s whether the gene i n t e r a c t i o n i s s p e c i f i c o r non -s p e c i f i c . In o r d e r to answer t h i s q u e s t i o n , more knowledge o f the gene a c t i o n i s needed. Most o f the arguments f o r gene a c t i o n i s p rov ided by p o l y g e n i c a l l y determined r e s i s t a n c e . One argument i s t ha t p o l y g e n i c r e s i s t a n c e i s r e a l l y r e s i s t a n t genes (of gene-for-gene i n t e r a c t i o n s ) t ha t have been matched by v i r u l e n c e genes . I t i s i n f e r r e d t ha t matched r e s i s t a n c e genes p r o v i d e some r e s i d u a l r e s i s t a n c e and t h a t when s e v e r a l are combined i n the hos t a q u a n t i t a t i v e e f f e c t o c c u r s on the r e s i s t a n t phenotype . Based on t h i s i n f e r e n c e i t i s then s a i d t h a t the re i s no such t h i n g as po lygenes a f f e c t i n g r e s i s t a n c e . Another argument i s t ha t p o l y g e n i c r e s i s t a n c e does e x i s t and can modi fy major gene r e s i s t a n c e . b l e 2.4 H o s t - p a r a s i t e systems where p o l y g e n i c a l l y determined p a t h o g e n i c i t y has been demonstrated Pathogen  U s t i l a g o h o r d e i U s t i l a g o maydis  C e r a t o c y s t i s u lmi Gaeumannomyces g r a m i n i s v a r . t r i t i c i Reference Emara 197 2 Emara & S idhu 1974 Person e_t a l . 198 2 Person e t a_l. 1983 Ba s s i & Bu rne t t 1980 B r a s i e r & G ibbs 1976 B r a s i e r 1977 B lanch e t a l . 1981 23 These same arguments can be a p p l i e d to v i r u l e n c e genes . For the f i r s t argument we cou ld say tha t p o l y g e n i c a l l y determined v i r u l e n c e are p a t h o g e n e c i t y genes f o r which the re are no matching r e s i s t a n c e genes . These genes would a c t i n an a d d i t i v e manner to modi fy v i r u l e n c e . T h i s argument can be extended to imply t ha t the re are no such t h i n g s as p o l y g e n e s . The o the r v i ewpo in t i s t ha t po lygenes a f f e c t i n g v i r u l e n c e do e x i s t , and tha t they e x e r t a n o n s p e c i f i c mod i f y i ng r o l e i n gene-for-gene i n t e r a c t i o n s . The h o s t - p a r a s i t e system p r o v i d i n g the most i n f o r m a t i o n a t t h i s t ime i s U s t i l a g o h o r d e i which causes the covered smut d i s e a s e o f b a r l e y . Emara (1972) used b i o m e t r i c a l a n a l y s i s to examine i n h e r i t a n c e p a t t e r n s o f U. h o r d e i . More r e c e n t l y a new expe r imen ta l approach was taken to r e c o g n i z e the e f f e c t s o f both major v i r u l e n c e genes and po lygenes (Person e_t a_l. 1982, 1983) . T h i s approach took advantage o f the f a c t t ha t w i th U. h o r d e i , the h a p l o i d p roduc t s o f m e i o s i s ( s p o r i d i a ) can be i s o l a t e d , grown i n c u l t u r e , s t o r ed f o r a long t ime and can be used i n t e t r a d a n a l y s i s . The use o f t e t r a d a n a l y s i s t o i d e n t i t y p o l y g e n i c i n h e r i t a n c e has been a p p l i e d to Neurospora  c r a s s a (Pateman 1955, Pateman & Lee 1960) and i s not unique to U. h o r d e i . Person and Ebba (unpubl i shed) observed 3:1 s e g r e g a t i o n p a t t e r n s i n s i n g l e p r o g e n i e s from a number o f F-^  t e l i o s p o r e s o f U. h o r d e i which meant a major gene was s e g r e g a t i n g . A compar ison o f s e l f e d p rogen i e s o f many g e n e t i c a l l y i d e n t i c a l t e l i o s p o r e s showed tha t the l e v e l s o f v i r u l e n c e were not c o n s i s t e n t but v a r i e d f o r d i f f e r e n t t e l i o s p o r e s . The v a r i a t i o n was not accounted f o r by another major gene and i t was h ypo thes i z ed tha t po lygenes were enhanc ing the e f f e c t s o f the major v i r u l e n c e gene . I t was expected tha t s p o r i d i a from a s i n g l e m e i o s i s may have a v a r i a b l e po lygene con ten t and t h e r e -f o r e , the d i k a r y o n s produced from the p roduc t s o f a s i n g l e m e i o s i s would va r y i n v i r u l e n c e l e v e l s . To t e s t the h y p o t h e s i s o f v a r y i n g po lygene c o n t e n t , the p r e d i c t i o n was made tha t s p o r i d i a cou ld be ranked on the b a s i s o f t h e i r per formance from s e l f i n g , and tha t when s p o r i d i a o f o p p o s i t e mat ing types were both ranked from h igh to low such a d i a l l e l c r o s s cou ld be pe r fo rmed , r ank ing o f the gametes shou ld be e x h i b i t e d . T h i s was the f i r s t time the concept o f cons t an t r ank ing was a p p l i e d t o p a t h o g e n i c i t y . The r e s u l t s o f a l a r g e exper iment (Person e_t a_l. 1982, 1983 ) , d i d show cons t an t r ank ing o f gametes both from a v i s u a l i n s p e c t i o n o f the r e s u l t s and from mathemat ica l a n a l y s i s . Pope (1982) working w i th some o f the same F-^  t e l i o s p o r e s , s e t up a d i a l l e l c r o s s us ing random s p o r i d i a and performed a b i o m e t r i c a l a n a l y s i s on the r e s u l t s . He demonstrated tha t the re was a major gene f o r v i r u l e n c e and po lygenes which enhanced v i r u l e n c e . The a n a l y s i s o f v a r i a n c e showed tha t the re were s i g n i f i c a n t n o n a d d i t i v e e f f e c t s , genotype x env i ronment i n t e r a c t i o n , and i n t e r a c t i o n s between the major gene and the p o l y g e n e s . Both o f these exper iments w i th U. h o r d e i gave 25 ev idence o f po lygenes mod i f y i ng gene-for-gene i n t e r a c t i o n s . At t h i s t ime , i t appears t h a t gene-for-gene systems are q u i t e complex i n i n h e r i t a n c e p a t t e r n s and po lygenes seem to p l a y a mod i f y i ng r o l e and can h ide the e f f e c t s o f a major gene. The v a r i a b i l i t y p rov ided by both gene-for-gene i n t e r a c t i o n s and po l ygenes p l a y an impor tan t r o l e i n d i s e a s e v a r i a b i l i t y . Much may be ga ined i n unders tand ing the u n d e r l y i n g mechanisms o f d i s e a s e v a r i a b i l i t y by comparing the an t i gen-an t i body system to p l a n t - p a r a s i t e sys tems . More r e s e a r c h i s needed to unders tand the comp l ex i t y o f d i s e a s e v a r i a b i l i t y . S t r a t e g i e s f o r D isease Res i s t ance D r i v e r (1962a, 1962b) d e f i n e d two types o f r e s i s t a n c e : h y p e r s e n s i t i v i t y , immunity to v a r i o u s r a c e s , and f i e l d r e s i s t a n c e , a g e n e r a l r e s i s t a n c e to a l l r a c e s . He s t a t e d tha t h y p e r s e n s i t i v i t y i s a f a l s e r e s i s t a n c e because i t i s r e a d i l y "b roken down" by the appearance o f new r aces which a r i s e by s e g r e g a t i o n o r m u t a t i o n ; whereas, f i e l d r e s i s t a n c e i s most l i k e l y p o l y g e n i c a l l y c o n t r o l l e d and t h e r e f o r e more s t a b l e because the pathogen would need to make s e v e r a l gene changes to overcome the r e s i s t a n c e . He a l s o r e cogn i zed tha t f i e l d r e s i s t a n c e i s based on the c a p a c i t y o f the p l a n t to l i m i t i n f e c t i o n and reduce the r a t e o f growth and development o f the pa thogen . H i s example o f f i e l d r e s i s t a n c e was non-immune v a r i e t i e s to Phytophthora i n f e s t a n s where these v a r i e t i e s can 26 be ranked i n the same o r d e r r e g a r d l e s s o f y e a r , env i ronment , o r i s o l a t e o f the fungus . D r i v e r ' s c o n c l u s i o n was t ha t b reeders shou ld t r y t o combine the two types o f r e s i s t a n c e . Vanderp lank (1963, 1968) a l s o i d e n t i f i e d two types o f r e s i s t a n c e , 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 . V e r t i c a l r e s i s t a n c e , c h a r a c t e r i z e d by hav ing d i f f e r e n t i a l i n t e r a c t i o n s (as i n gene-for-gene s y s t e m s ) , and has s i n g l e gene i n h e r i t a n c e . E p i d e m i o l o g i c a l l y , t h i s r e s i s t a n c e reduces the i n i t i a l inoculum o f the pathogen by e l i m i n a t i n g r a ces tha t cannot i n f e c t . H o r i z o n t a l r e s i s t a n c e , i s c h a r a c t e r i z e d by c o n f e r i n g some r e s i s t a n c e a g a i n s t a l l r a c e s w i th no 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 s p o l y g e n i c a l l y i n h e r i t e d . E p i d e m i o l o g i c a l l y , t h i s r e s i s t a n c e reduces the average r a t e o f i n f e c t i o n . V a n d e r p l a n k ' s method f o r t e s t i n g f o r 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 s to l ook a t the r ank ing o f a s e t o f v a r i e t i e s to a s e t o f pathogen r a ces ( F i gu re 2.2) and when r ank ing i s independent o f hos t o r pa thogen , r e s i s t a n c e i s h o r i z o n t a l . Vanderp lank d i d not i n t end to imply t h a t a l l r e s i s t a n c e f i t s i n t o e i t h e r v e r t i c a l o r h o r i z o n t a l r e s i s t a n c e . He d i d s t a t e t ha t p robab l y v e r t i c a l r e s i s t a n c e never o c c u r s unaccompanied by h o r i z o n t a l r e s i s t a n c e (1968) which was a l s o p o s t u l a t e d by D r i v e r (1962b) . U n f o r t u n a t e l y , many r e s e a r c h e r s ' t r y to d i s t i n g u i s h the two types as sepa ra te e n t i t i e s and t h e r e f o r e , have found l i t t l e use f o r V a n d e r p l a n k ' s te rms . Many r e s e a r c h e r s have d e f i n e d t h e i r own terms (Tab le 2 . 5 ) . 27 2.2a V e r t i c a l r e s i s t a n c e Host c u l t i v a r s A B 2.2b H o r i z o n t a l r e s i s t a n c e Host c u l t i v a r s in A B C +/- = c o m p a t i b l e / i n c o m p a t i b l e 1-5 = low-high l e v e l s of d i s e a s e F i g u r e 2.2 Comparison of d i f f e r e n t i a l i n t e r a c t i o n s f o r v e r t i c a l r e s i s t a n c e (2.2a) to the cons tan t r ank ing observed f o r h o r i z o n t a l r e s i s t a n c e (2 .2b) , b le 2.5 Comparison o f s p e c i f i c terms o f t e n used i n p l a c e o f 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 V e r t i c a l h y p e r s e n s i t i v e r a c e - s p e c i f i c major gene o l i o g e n i c , monogenic q u a l i t a t i v e s e e d l i n g d i f f e r e n t i a l d i s c r i m i n a t i n g H o r i z o n t a l f i e l d , g e n e r a l r ace n o n - s p e c i f i c minor gene p o l y g e n i c q u a n t i t a t i v e a d u l t p l a n t un i fo rm d i l a t o r y du rab l e s l ow-rus t i ng/mi ldew ing The terms v e r t i c a l and h o r i z o n t a l are u s e f u l i f used as a b s t r a c t terms d e s c r i b i n g a concept r a t h e r than hav ing a s p e c i f i c d e f i n i t i o n (Robinson 1976) . A l l o f the terms i n Tab le 2.5 have s p e c i f i c meanings e i t h e r g e n e t i c a l l y , p h e n o t y p i c a l l y o r e p i d e m i o l o g i c a l l y . For example s l o w - r u s t i n g g i v e s i n f o r m a t i o n about the phenotype but t e l l s no th ing about the i n h e r i t a n c e o f t h a t r e s i s t a n c e . D i s c r i m i n a t o r y and d i l a t o r y have e p i d e m i o l o g i c a l meanings but aga in r e a l l y do not t e l l us any th ing about the mode o f i n h e r i t a n c e o r gene a c t i o n . Major gene-minor gene r e s i s t a n c e g i v e s i n f o r m a t i o n on i n h e r i t a n c e p a t t e r n but s t a t e s no th ing o f the e p i d e m i o l o g i c a l consequences . Durab le r e s i s t a n c e means the r e s i s t a n c e has w i ths tood f i e l d c o n d i t i o n s f o r a number o f yea rs but a l s o g i v e s no i n f o r m a t i o n on i n h e r i t a n c e . A l s o , not a l l major r e s i s t a n c e genes p rov i de a h y p e r s e n s i t i v e response but may a l l o w some pathogen s p o r u l a t i o n . Many o f the terms do have a p r e c i s e meaning which can be used f o r s p e c i f i c c a s e s . I t becomes apparent t ha t the e p i d e m i o l o g i c a l consequences shou ld not be c o r r e l a t e d w i th one g e n e t i c term f o r r e s i s t a n c e because ep idemio logy o n l y c o n s i d e r s the consequences o f the e x p r e s s i o n o f genes which are a r e f l e c t i o n not o n l y o f r e s i s t a n c e genes but o f genes i n the h o s t - p a r a s i t e i n t e r a c t i o n (Een ink 1976) . Robinson (1969, 1973) r e t a i n e d V a n d e r p l a n k ' s terms but p o i n t e d out t ha t they c o u l d be d e f i n e d th ree ways: g e n e t i c a l l y , e p i d e m i o l o g i c a l l y o r m e c h a n i s t i c a l l y . S ince the terms are m u l t i d i s c i p l i n a r y , Robinson (1976) p r e f e r r e d to use the terms to d e s c r i b e subsystems o f a pa thosys tem. I f h o r i z o n t a l 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 c o n t r o l l e d then i t i s expected to be a more s t a b l e r e s i s t a n c e ( D r i v e r 1962 a,b) U n f o r t u n a t e l y , the e x p r e s s i o n o f h o r i z o n t a l r e s i s t a n c e i s more d i f f i c u l t to e v a l u a t e and i f i t i s p o l y g e n i c a l y c o n t r o l l e d , i t i s more d i f f i c u l t to i n c o r p o r a t e i n t o v a r i e t i e s (Simons 1972) . T h i s e x p l a i n s why the sea r ch f o r d i s e a s e r e s i s t a n c e has mos t l y been f o r s i n g l e major genes c o n f e r r i n g h i g h l e v e l s o f r e s i s t a n c e . Because o f the cons t an t b a t t l e to keep ahead o f the pathogen changes i n match ing the major r e s i s t a n c e gene, s e v e r a l s t r a t e g i e s u t i l i z i n g these r e s i s t a n c e genes have been proposed i n o rde r to curb the pathogen p o p u l a t i o n s h i f t s . These s t r a t e g i e s a r e : m u l t i l i n e s , gene deployment and gene p y r a m i d i n g . Jensen (1952) and Bor laug (1959) were the f i r s t to propose us ing m u l t i l i n e v a r i e t i e s . These v a r i e t i e s would be composed o f l i n e s w i th d i f f e r e n t r e s i s t a n c e genes . M u l t i l i n e s would be accompl i shed by b a c k c r o s s i n g i n d i v i d u a l r e s i s t a n c e genes i n t o sepa ra te pure l i n e s and then m ix ing the seed o f the s e r i e s o f pure l i n e s . /Another method would be to mix a s e r i e s o f compa t ib l e pure l i n e v a r i e t i e s and r e l e a s e them under a s i n g l e v a r i e t y name. The d i v e r s i f i c a t i o n o f r e s i s t a n t t ypes would be a method o f s t a b i l i z i n g the pathogen p o p u l a t i o n by r educ ing the p r o b a b i l i t y o f a s u c c e s s f u l i n f e c t i o n . Reducing the p r o b a b i l i t y o f a s u c c e s s f u l i n f e c t i o n o r r educ ing the amount o f d i s p e r s i n g inoculum i s b e l i e v e d to be the main reason f o r reduced d i s e a s e r a t e s on m u l t i l i n e s . Burdon (1978) i d e n t i f i e s s e v e r a l o t h e r mechanisms: l e s s s u s c e p t i b l e t i s s u e a v a i l a b l e f o r subsequent d i s p e r s a l o f i nocu lum, d e c l i n e i n d e n s i t y o f s u i t a b l e hos t s and t h e r e f o r e an i n c r e a s e i n d i s t a n c e inoculum must t r a v e l , and c r o s s p r o t e c t i o n ( the phenomenom i n which p l a n t t i s s u e s i n f e c t e d w i th one race are p r o t e c t e d from i n f e c t i o n by o t h e r r a c e s ) . I t i s apparent t h a t each o f these components i s i n t e r r e l a t e d and tha t i t would be d i f f i c u l t to sepa ra te the degree each component c o n t r i b u t e s . Leonard ( 1 9 6 9 a , b , c , ) was the f i r s t to e x p e r i m e n t a l l y e v a l ua t e the concept o f m u l t i l i n e s and p r o v i d e a mode l . From h i s expe r imen t s , Leonard d i s c o v e r e d t h a t the re can be c o m p e t i t i v e i n h i b i t i o n among pathogen r a ces on the same c u l t i v a r and tha t the r e p r o d u c t i o n per pathogen g e n e r a t i o n i s p r o p o r t i o n a l to the p r o p o r t i o n o f s u s c e p t i b l e p l a n t s i n the hos t m i x t u r e . H i s t h e o r e t i c a l work showed t h a t a h igh p r o p o r t -i o n o f s u s c e p t i b l e p l a n t s must be i n c l u d e d i n the mix ture i n o r d e r to pe rmi t the s u r v i v a l o f the pathogen r a ces w i th few v i r u l e n c e genes ( s imp le r a c e s ) . He a l s o conc luded t h a t m u l t i -l i n e s cannot p reven t o c cu r r ence o f a complex race o r " s u p e r r a c e " (many v i r u l e n c e genes , the race tha t can overcome a l l r e s i s t a n c e genes p resen t ) but can p revent the b u i l d up o f these r a ces to h igh f r e q u e n c i e s . Because o f some ev idence o f pathogen a d a p t a t i o n to hos t c u l t i v a r s on which they are c o n t i n u o u s l y c u l t u r e d , Leonard suggested us ing a s e r i e s o f compa t ib l e v a r i e t i e s w i th d i f f e r e n t g e n e t i c backgrounds r a t h e r 32 than a s e r i e s o f back c r o s s l i n e s . S e v e r a l models have been deve loped to e v a l u a t e m u l t i l i n e s and the e f f e c t s o f s t a b i l i z i n g s e l e c t i o n ( B a r r e t t 1978, B a r r e t t & Wolfe 1978, F leming & Person 1978, Groth 1976, 1978, Groth & Person 1977, Kiyosawa 1976, Kiyosawa & Shiyomi 1972, M a r s h a l l & P ryo r 1978, 1979, Os te rgaa rd 1983) . Leonard and Czochor (1980) have rev iewed models proposed f o r m u l t i l i n e s . One o f the models concerned w i th the long term f a t e o f m u l t i l i n e s i s t ha t o f Groth (1976) . Groth assumed tha t s t a b i l i z i n g s e l e c t i o n (sensu Vander P lank) f o r v i r u l e n c e genes does e x i s t and would be the same f o r a l l v i r u l e n c e genes . He does s t a t e t ha t t h i s i s p robab l y the most u n r e a l i s t i c assumpt ion o f the model b u t , a t t h i s t i m e , no th ing i s known about f i t n e s s c o n f e r r e d from v i r u l e n c e genes . Two v e r s i o n s were deve loped where f o r one the f i t n e s s , due to v i r u l e n c e genes , combines a d d i t i v e l y , and f o r the o t h e r , m u l t i p l i c a t i v e l y . Both v e r s i o n s l e d to the same p r e d i c t i o n tha t the re w i l l be a l i m i t to the number o f v i r u l e n c e genes accumulated through s e l e c t i o n . T h i s means t h a t s e l e c t i o n f o r the " supe r r a c e " need not o c c u r . B a r r e t t (1978) and B a r r e t t & Wolfe (1978) expanded on G r o t h ' s model and c l a r i f i e d a few p o i n t s . F i r s t , complete l e t h a l i t y o f n o n v i r u l e n t genotypes on r e s i s t a n c e hos t s i s a s p e c i a l case and does not a lways o c c u r . Second, the s e l e c t i o n c o e f f i c i e n t w i l l be d i f f e r e n t f o r each pathogen and a t d i f f e r e n t t imes d u r i n g deve lopment . They conc luded tha t more a c cu r a t e measurements o f the r a t e o f growth o f pathogens and o f the s e l e c t i o n f o r c e s 33 a c t i n g on the pathogens are needed to p r e d i c t w i th c e r t a i n t y whether m u l t i n e s w i l l s e l e c t complex r a c e s . The next s t r a t e g y i s gene deployment f i r s t proposed by Jensen (1952) . T h i s s t r a t e g y p r o v i d e s d i v e r s i t y on a n a t i o n a l o r r e g i o n a l b a s i s where o n l y s p e c i f i c r e s i s t a n c e genes would be u t i l i z e d i n a g i v e n r e g i o n . Johnson (1958) suppor ted the idea o f r e g i o n a l d i s t r i b u t i o n o f genes f o r r u s t r e s i s t a n c e . Browning e_t a_l (1969) a p p l i e d the concept to the oat-crown r u s t system and were the f i r s t to e x p l o r e the i m p l i c a t i o n s o f gene dep loyment . Knot t (1972) l a t e r extended the concept to wheat stem r u s t . Browning and coworke r ' s (1969) r eason ing f o r gene deployment as a s u i t a b l e s t r a t e g y f o r the oat-crown r u s t system i s based on the zones t ha t seem to o c cu r i n the " P u c c i n i a pathway. " The " P u c c i n i a pathway" i s a s i n g l e e p i d e m i o l o g i c a l u n i t f o r c e r e a l r u s t s and extends i n the Midwestern Nor th Amer ica from Mexico to Canada. Three major zones were e n v i s i o n e d . Zone 1 r e p r e s e n t s the over- w i n t e r i n g a rea o f the r u s t s (Mexico and the Southern Un i t ed S t a t e s ) . Zone 2 i s an i n t e r m e d i a t e zone ( C e n t r a l Un i t ed S ta tes ) and Zone 3 i s where the l a t e s p r i n g c e r e a l s are grown (Nor thern Un i t ed S ta tes and Canada) . The p r o p o s a l was to a s s i g n d i f f e r e n t r e s i s t a n c e genes to each zone and the reby break the u n i t y o f t h i s pathway. Gene development would s t a b i l i z e the pathogen p o p u l a t i o n by p roduc ing inoculum i n one zone t h a t would not be i n f e c t i v e i n the next zone. T h i s s t r a t e g y cou ld be the bes t s t r a t e g y i n 34 s i t u a t i o n s s i m i l a r to the " P u c c i n i a pathway" , where a pathogen i s widespread over a l a r g e g e o g r a p h i c a l a rea and moves i n a s p e c i f i c o r d e r l y manner ove r t ha t geog raph i c a rea by long d i s t a n c e spore d i s p e r s a l . T h i s s t r a t e g y would be hard to implement because o f the c o o p e r a t i o n p o s s i b l y needed among n a t i o n a l governments , as w e l l as g rowers . The t h i r d s t r a t e g y i s gene pyramid ing f i r s t proposed by Ne l son (1972) . Gene py ramid ing i s the i n c o r p o r a t i o n o f s e v e r a l r e s i s t a n c e genes o r as many as p o s s i b l e i n t o a s i n g l e g e n e t i c background. Ne lson f i r s t proposed t h i s s t r a t e g y based on ev idence from the Nor thern co rn l e a f b l i g h t - c o r n system (Ne lson e t a l . 1970) . Ne lson b e l i e v e s t h a t matched s p e c i f i c r e s i s t a n c e genes p r o v i d e some r e s i d u a l r e s i s t a n c e and t h e r e f o r e when combined i n a s i n g l e g e n e t i c background these genes would p r o v i d e a h o r i z o n t a l type o f r e s i s t a n c e (Ne lson 1978) . As d i s c u s s e d i n the p r e v i o u s s e c t i o n , the re appears to be two types o f r e s i s t a n c e , t ha t i n h e r i t e d i n s imp le Mendel ian manner as i n gene-for-gene systems and r e s i s t a n c e i n h e r i t e d by s e v e r a l genes a d d i t i v e i n e f f e c t . Day (1978) p o i n t s out t ha t wh i l e we rega rd these two types as sepa ra te e n t i t i e s , most l i k e l y , i n na ture both t ypes c o e x i s t and merge i n a spectrum o f g e n e t i c e f f e c t s . I f both types opera te s i m u l t a n e o u s l y , i t would be d i f f i c u l t to sepa ra te out the e f f e c t s c o n f e r r e d by the i n d i v i d u a l . The Nor the rn co rn l e a f b l i g h t - c o r n system has both types o f r e s i s t a n c e i n o p e r a t i o n . J enk ins and Robert (1952) had 35 ev idence o f r e s i s t a n c e c o n t r o l by s e v e r a l genes ; whereas, Hooker (1963 a,b) i d e n t i f i e d s i n g l e major r e s i s t a n c e genes . The co rn i n b r e d s used by Ne lson e t a_l. (1970) had r e s i s t a n c e i d e n t i f i e d by J enk ins and Robert (1961) through t r a n s l o c a t i o n s t u d i e s . The t e s t s i n d i c a t e d t ha t r e s i s t a n c e was l o c a t e d on s p e c i f i c chromosome arms and t h a t r e s i s t a n c e appeared to be major gene r e s i s t a n c e . Ne lson e_t a_l. (1965, 1970) assumed r e s i s t a n c e was i n h e r i t e d as major genes r a t h e r than i n a p o l y g e n i c manner. Ne l son e t a l . (1965, 1970) ob ta ined 750 m o n o s p o r i d i a l progeny from 10 c r o s s e s o f He lminthospor ium tu r c i cum i s o l a t e s . These 750 i s o l a t e s were t e s t e d on f o u r i nb red co rn l i n e s o f v a r y i n g number o f chromosome arms c a r r y i n g r e s i s t a n c e . They conc luded tha t i nb reds w i th g r e a t e r numbers o f chromosome arms w i th genes f o r r e s i s t a n c e tend to r e a c t more u n i f o r m l y to the i s o l a t e s than i nb r eds w i th fewer chromosome arms w i th genes f o r r e s i s t a n c e . T h e i r i m p l i c a t i o n from these r e s u l t s was t h a t genes f u n c t i o n v e r t i c a l l y when sepa ra te and the same genes f u n c t i o n h o r i z o n t a l l y when they are t o g e t h e r . Other ev idence s u p p o r t i n g gene pyramid ing i s tha t o f Ma r t i n and E l l i n g b o e (1976) and E l l i n g b o e (1976a) working w i th powdery mildew on wheat. T h e i r approach was to compare the th ree compa t ib l e i n t e r a c t i o n s from w i t h i n the q u a d r a t i c check (see Chapter 1) f o r a s p e c i f i c r e s i s t a n c e gene and i t s two a l l e l e s i n the hos t and the c o r r e s p o n d i n g major v i r u l e n c e gene and i t s two a l l e l e s i n the pa thogen . The th ree compat ib l e 36 r e a c t i o n s were observed to produce the same i n f e c t i o n t y p e , as e x p e c t e d , but the re were d i f f e r e n c e s i n the r a t e o f d e v e l o p -ment. A s lower r a t e o f growth was observed when the v i r u l e n c e a l l e l e was matched to the r e s i s t a n c e a l l e l e , as compared to the r a t e observed when the v i r u l e n c e / a v i r u l e n c e a l l e l e was matched to the s u s c e p t i b i l i t y a l l e l e . From these r e s u l t s they conc luded tha t overcome r e s i s t a n c e genes have a r e s i d u a l e f f e c t . They extended t h i s i dea to propose t ha t slow-mi ldew ing , which i s u s u a l l y a s s o c i a t e d w i th h o r i z o n t a l r e s i s t a n c e , to be the r e s u l t o f accumulated matched r e s i s t a n c e genes . Nas et_ _al_. (1981) a l s o worked w i th powdery mildew on wheat. He compared the e f f e c t s o f th ree matched r e s i s t a n c e genes and made s i m i l a r c o n c l u s i o n s . At t h i s t ime , there i s no i n f o r m a t i o n on the e f f e c t s gene py ramid ing would have on a pathogen p o p u l a t i o n o r whether many accumulated r e s i s t a n c e genes p r o v i d e a g e n e r a l r e s i s t a n c e . Without t h i s knowledge i t would be dangerous to i n c o r p o r a t e a l l known r e s i s t a n c e genes i n t o a s i n g l e g e n e t i c background. I t would appear t ha t a pathogen p o p u l a t i o n may g a i n the necessa r y v i r u l e n c e genes to overcome the r e s i s t a n c e genes . Most p l a n t b reede rs a re e s s e n t i a l l y py ramid ing r e s i s t a n c e genes . Breeders f i n d i t much e a s i e r j u s t to i n c o r p o r a t e new r e s i s t a n c e genes i n t o the w e l l e s t a b l i s h e d v a r i e t i e s w i thout concern f o r the overcome r e s i s t a n c e genes . " H o r i z o n t a l " r e s i s t a n c e can a l s o be c o n s i d e r e d a s t r a t e g y . As s t a t e d be fo r e h o r i z o n t a l r e s i s t a n c e i s c h a r a c t e r i z e d by e x h i b i t i n g no 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 e x h i b i t i n g cons tan t r a n k i n g . G e n e r a l l y i t i s thought t ha t t h i s type o f 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 c o n t r o l l e d . As s t a t e d i n the p r e v i o u s s e c t i o n , the re are s e v e r a l v i ewpo in t s on t h i s type o f r e s i s t a n c e . F i r s t the re i s Ne lson (1978) who d e f i n e s h o r i z o n t a l r e s i s t a n c e as a r e s i s t a n c e tha t reduces the apparent i n f e c t i o n r a t e and tha t i t can be i n h e r i t e d by s i n g l e genes o r by s e v e r a l . From h i s work on gene p y r a m i d i n g , he b e l i e v e s tha t r e s i s t a n c e genes (of gene-for-gene r e l a t i o n s ) when t oge the r g i v e a h o r i z o n t a l e f f e c t . One can i n f e r from t h i s t ha t the re i s no d i f f e r e n c e between major genes and po l ygenes f o r r e s i s t a n c e . E l l i n g b o e (1976a) a l s o b e l i e v e s major genes can have r e s i d u a l e f f e c t s and c l a ims tha t a l l d i s e a s e v a r i a b i l i t y f o l l o w s gene-for-gene i n t e r a c t i o n s . Those h o s t - p a r a s i t e systems where no gene-for-gene r e l a t i o n s are demonstrated may have i n t e r a c t i o n s t ha t are so weak tha t v e r y p r e c i s e c o n t r o l o f v a r i a b l e s i s necessa r y to demonstrate s p e c i f i c i n t e r a c t i o n s ( E l l i n g b o e 1976b) . He suppor t s t h i s w i th a compar ison o f s e g r e g a t i o n p a t t e r n s w i t h i n an i n o c u l a t e d F 2 hos t p o p u l a t i o n reco rded i n the f i e l d w i th r e s u l t s from c o n t r o l l e d chambers. In the f i e l d , r e s i s t a n c e appeared c o n t i n u o u s , but a 3:1 r e s i s t a n t to s u s c e p t i b l e r a t i o i s observed under c o n t r o l l e d c o n d i t i o n s . H i s c o n c l u s i o n i s t h a t h o r i z o n t a l r e s i s t a n c e i s r e s i s t a n c e which has not ye t been shown to be s p e c i f i e d (1976a, 1981) . 38 A second v i ewpo in t i s t ha t o f P a r l e v l i e t (1981b) . His v iew i s based on r e s u l t s from P u c c i n i a h o r d e i on b a r l e y . He has found p a r t i a l r e s i s t a n c e to be c o n t r o l l e d by s e v e r a l genes w i th a d d i t i v e e f f e c t s which have the c h a r a c t e r i s t i c s o f h o r i z o n t a l r e s i s t a n c e ( P a r l e v l i e t 1976a ,b , 1978) . However, s t a t i s t i c a l a n a l y s i s o f h i s da t a showed i n t e r a c t i o n s between i s o l a t e s and v a r i o u s components o f r e s i s t a n c e . He proposed t h i s was ev idence f o r d i f f e r e n t i a l i n t e r a c t i o n s . He conc luded tha t the s t a t i s t i c a l i n t e r a c t i o n and the r ank ing method are u n s a t i s f a c t o r y i n de t e rm in ing i f p a r t i a l r e s i s t a n c e i s h o r i z o n t a l (1976b) . P a r l e v l i e t and Zadocks (1977) proposed two mode l s , one w i th a d d i t i v e e f f e c t s o f genes and the o the r c h a r a c t e r i z e d by gene-for-gene i n t e r a c t i o n s . These were the a d d i t i o n and i n t e r a c t i o n mode l s , r e s p e c t i v e l y . From these models and the da t a r e p o r t e d by P a r l e v l i e t , they conc luded tha t p o l y g e n i c r e s i s t a n c e cou ld a c t i n a gene-for-gene manner and t h a t the o n l y d i f f e r e n c e between h o r i z o n t a l and v e r t i c a l r e s i s t a n c e i s the mode o f i n h e r i t a n c e . P a r l e v l i e t (1978) b e l i e v e s a l l r e s i s t a n c e genes ope ra te on a gene-for-gene b a s i s w i th v i r u l e n c e genes . The t h i r d v i ewpo in t i s t ha t p o l y g e n i c a l l y determined r e s i s t a n c e ( h o r i z o n t a l r e s i s t a n c e ) i s n o n s p e c i f i c and m o d i f i e s gene-for-gene i n t e r a c t i o n s . T h i s v i ewpo in t i s suppor ted by Person et_ a_l. (1982, 1983) . Robinson (1979) a l s o argues t ha t h o r i z o n t a l r e s i s t a n c e i s n o n s p e c i f i c w i th no gene-for-gene i n t e r a c t i o n s , t h e r e f o r e , cons t an t r ank ing i s the bes t c r i t e r i o n 39 f o r de t e rm in ing h o r i z o n t a l r e s i s t a n c e . He s t a t e s t ha t the sma l l d i f f e r e n t i a l i n t e r a c t i o n s observed by P a r l e v l i e t r e s u l t from expe r imen ta l e r r o r , e s p e c i a l l y i n t e r p l o t i n t e r f e r e n c e . Caten (1974) p o i n t e d out tha t i t i s not s imp l y the presence o r absence o f d i f f e r e n t i a l i n t e r a c t i o n s t ha t mat te r but t h e i r magnitude r e l a t i v e to the d i f f e r e n c e i n r e s i s t a n c e . Johnson and T a y l o r (1976) demonstrated t ha t i t depends on what was used as measurements o f r e s i s t a n c e ; v i s u a l assessments o r spore p r o d u c t i o n . They a l s o found t h a t s t a t i s t i c a l i n t e r a c t i o n s c o u l d be e l i m i n a t e d by t r ans fo rm ing the d a t a . The most impor tant aspec t o f h o r i z o n t a l r e s i s t a n c e i s i t s s t a b i l i t y . I t i s g e n e r a l l y accepted t h a t h o r i z o n t a l r e s i s t a n c e i s s t a b l e and tha t the s t a b i l i t y a r i s e s from the f a c t t ha t i t i s n o n s p e c i f i c and p o l y g e n i c a l l y c o n t r o l l e d . Most o f the ev idence i s l a r g e l y e m p i r i c a l , so expe r i emen ta l e v idence i s g r e a t l y needed. B a s i c a l l y , a l l the d i s e a s e r e s i s t a n c e s t r a t e g i e s t r y to s t a b i l i z e the pathogen p o p u l a t i o n i n one way o r a n o t h e r . Kiyosawa (1982) s t a t e d t h a t the q u e s t i o n o f which i s the bes t s t r a t e g y f o r d i s e a s e c o n t r o l cannot be answered u n t i l i n f o r m a t i o n about the f a c t o r s i n f l u e n c i n g the e f f e c t o f each i s known. He suggested t h a t the measurement o f f i t n e s s o f each gene was the most impor tan t f a c t o r i n s t a b i l i z i n g the pathogen p o p u l a t i o n . Vanderp lank (1968) borrowed the term " s t a b i l i z i n g s e l e c t i o n " from p o p u l a t i o n g e n e t i c s to d e s c r i b e the s e l e c t i o n a g a i n s t unnecessary v i r u l e n c e genes on the s u s c e p t i b l e hos t 40 p l a n t . A l l o f these s t r a t e g i e s are based on the assumpt ion t ha t s t a b i l i z i n g s e l e c t i o n o c c u r s . S t a b i l i z i n g S e l e c t i o n V a n d e r p l a n k ' s concept o f s t a b i l i z i n g s e l e c t i o n (see Chapter 1) has aroused c o n s i d e r a b l e c o n t r o v e r s y . Op in ions i n the l i t e r a t u r e i n p l a n t pa tho logy seems to be d i v i d e d r e g a r d i n g t h i s . c o n c e p t . The importance o f the concept i s man i f e s t ed i n the sea r ch f o r ways to improve and u t i l i z e d i s e a s e r e s i s t a n c e t h a t i s i n c o r p o r a t e d i n t o our c r op v a r i e t i e s . On the b a s i s o f a n a l y s i s o f s e l e c t e d e m p i r i c a l d a t a , Vanderp lank (1963) s t a t e d t ha t i t i s ax i oma t i c t ha t the pathogen genotype most f i t t o s u r v i v e on a s imp le hos t ( possess i ng few r e s i s t a n c e g e n e s ) , was the one w i th few v i r u l e n c e genes . T h i s i s h i s concept o f s t a b i l i z i n g s e l e c t i o n . Rather than rev iew the e m p i r i c a l e v idence Vanderp lank used i n s u p p o r t i n g s t a b i l i z i n g s e l e c t i o n , some o f the expe r imen ta l e v idence and a n a l y t i c a l f i e l d e v a l u a t i o n s w i l l be r e v i ewed . Even be fo re the concept o f s t a b i l i z i n g s e l e c t i o n was known, Loeger ing (1951) looked a t the s u r v i v a l o f wheat stem r u s t r a ces i n m i x t u r e s . He conc luded tha t the f a c t o r s a f f e c t i n g the r e l a t i v e s u r v i v a l o f c e r t a i n r a ces i n m ix tu res was not c l e a r l y e v i d e n t and most l i k e l y a number o f minor f a c t o r s o p e r a t i n g t oge the r a f f e c t e d s u r v i v a l . He argued tha t v i r u l e n c e a l o n e , c l e a r l y was not s u f f i c i e n t to enab le a race to 41 become p r e v a l e n t i n n a t u r e . F l o r (1953) , hav ing a c o n s i d e r a b l e amount o f expe r i ence wi th f l a x r u s t , d i s c o v e r e d tha t ove r s e v e r a l yea rs the re was a dec rease i n the number o f i s o l a t e s v i r u l e n t on d i f f e r e n t i a l v a r i e t i e s c o n t a i n i n g r e s i s t a n c e genes o the r than those p resen t i n commerc ia l v a r i e t i e s . He knew tha t t h i s d i d not n e c e s s a r i l y i n d i c a t e t ha t these r a ces were d i s a p p e a r i n g , because i t was not p o s s i b l e to i d e n t i f y l a t e n t v i r u l e n c e genes i n r a c e s c a r r y i n g matched a v i r u l e n c e a l l e l e s . He conc luded tha t the commerc ia l v a r i e t i e s on l y s e l e c t e d v i r u l e n c e necessa r y to overcome t h e i r p a r t i c u l a r r e s i s t a n c e genes . Under f i e l d c o n d i t i o n s he found tha t the predominant r a ces had the fewest v i r u l e n c e genes . He conc luded t h i s meant t ha t the dominant a v i r u l e n c e a l l e l e had some s u r v i v a l v a l u e . In greenhouse t e s t s he observed t h a t the re was no r e l a t i o n between v i g o r and the number o f dominant a v i r u l e n c e a l l e l e s c a r r i e d by a r a c e . Thurs ton (1961) found tha t i n m ix tu res o f Phy top thora  i n f e s t a n s , the s i m p l e s t race p redomina ted ; but under f i e l d c o n d i t i o n s o the r r a ces spread more r a p i d l y . He conc luded t h a t mic roenv i ronment was p robab l y a more impor tant f a c t o r than v i r u l e n c e genes i n the spread o f the fungus . S c h e i f e l e e_t _al . (1968) working w i th He lminthospor ium  t u r c i cum exper imented wi th m ix tu res o f i s o l a t e s on a s u s c e p -t i b l e co rn i n b r e d . The number o f v i r u l e n c e genes was i d e n t i f i e d by l e s i o n s i z e . The l a r g e r the l e s i o n , the g r e a t e r 42 the number o f v i r u l e n c e genes . The f i r s t exper iment compared an i s o l a t e w i th no v i r u l e n c e genes to one w i th s e v e r a l . The r e s u l t s were t h a t , a f t e r th ree g e n e r a t i o n s , 96% o f the p o p u l a t i o n was compr ised o f the i s o l a t e w i th no v i r u l e n c e genes . The second exper iment compared two i s o l a t e s w i th d i f f e r e n t numbers o f v i r u l e n c e genes . In t h i s c a s e , the i s o l a t e wi th the most v i r u l e n c e genes p redomina ted . L a t e r work (Ne lson and S c h e i f e l e 1970) showed t h a t the most p r e v a l e n t r ace a f t e r o v e r w i n t e r i n g was the l e a s t p r e v a l e n t r ace from these two expe r imen t s . T h e r e f o r e , o v e r w i n t e r i n g i s another f a c t o r de t e rm in ing the p reva l ence o f a race i n n a t u r e . S c h e i f e l e and Ne l son (1970) took the th ree r a ces from these exper iments and compared them f o r s p o r u l a t i o n c a p a c i t y , p e r c en t g e r m i n a t i o n and i n c u b a t i o n t ime . The c o n c l u s i o n was tha t these f a c t o r s were the major r eason f o r the race wi th no v i r u l e n c e genes be ing the most p r e v a l e n t i n m i x t u r e s . T h e i r o v e r a l l c o n c l u s i o n s were tha t f a c t o r s gove rn ing r e l a t i v e f i t n e s s are independent o f v i r u l e n c e genes . Leonard (1969) i n h i s work on m u l t i l i n e s t r i e d t o determine s u r v i v a l a b i l i t y o f P. g r a m i n i s avenae i s o l a t e s i n heterogeneous p o p u l a t i o n s . V i r u l e n t r a c e s had s u r v i v a l v a l u e s 14-46% lower than those o f r a ces a v i r u l e n t on the same d i f f e r e n t i a l c u l t i v a r . S epa ra t i on o f t h i s heterogeneous p o p u l a t i o n i n t o two s u b p o p u l a t i o n s , each c u l t u r e d f o r seven g e n e r a t i o n s on d i f f e r e n t c u l t i v a r s , r e s u l t e d i n i n c r e a s e d a b i l i t y o f spores to i n f e c t t h e i r r e s p e c t i v e hos t v a r i e t y . 43 Brown and Sharp (1970) compared the r e l a t i o n s h i p between r e l a t i v e s u r v i v a l a b i l i t y and the number o f v i r u l e n c e genes i n P u c c i n i a s t r i i f o r m i s . They used a mutant i s o l a t e w i th an a l b i n o marker f o r easy d i s t i n c t i o n from the w i l d type i s o l a t e s . The f i r s t compar ison was between the a l b i n o mutant and the w i l d type i s o l a t e from which the mutant was d e r i v e d . The w i l d type had a h i ghe r g e r m i n a t i o n p o t e n t i a l than the a l b i n o , but they dec i ded tha t the a l b i n o c o u l d compete f a i r l y w e l l w i th the w i l d t y p e . The a l b i n o was then p l a ced i n m ix tu res w i th o t h e r i s o l a t e s which con t a i ned more v i r u l e n c e genes . The a l b i n o would always d i s a p p e a r by the end o f e i g h t g e n e r a t i o n s . The a l b i n o i s o l a t e made coun t i ng the r u s t p u s t u l e s e a s i e r but was p r o b a b l y not the bes t i s o l a t e to compare i n m i x t u r e s . I f the compar isons were made w i th the w i l d t y p e , o the r r e s u l t s from the m ix tu res may have been o b s e r v e d . Another problem w i th m ix tu re s t u d i e s i s when on l y one c o l l e c t i o n i s made per u r e d i a l g e n e r a t i o n . S p o r u l a t i o n p o t e n t i a l s o f two i s o l a t e s may be s i m i l a r but the two i s o l a t e s may d i f f e r i n t ime r e q u i r e d to reach the maximum l e v e l o f s p o r u l a t i o n . Ogle and Brown (1970) examined m ix tu re s o f P u c c i n i a  g r am in i s t r i t i c i . Comparisons were made us ing d i f f e r e n t p r o p o r t i o n s o f i s o l a t e s i n the i n i t i a l m i x t u r e , v a r y i n g l e n g t h o f g e n e r a t i o n t i m e , and the e f f e c t o f the v a r i e t y on which the m ix tu re was c u l t u r e d . They found t h a t i n a l l c a s e s , the race w i th the w ides t hos t range (most v i r u l e n c e genes) had the h i g h e s t s u r v i v a l a b i l i t y . 44 Martens (1973) compared s u r v i v a l a b i l i t y - o f P. g r am in i s  avenae i s o l a t e s i n m i x t u r e s , both i n the greenhouse and i n the f i e l d . In the greenhouse the r a ce w i th the fewest v i r u l e n c e genes always i n c r e a s e d i n f r equency ; but under f i e l d c o n d i t i o n s i t was always outper formed by the race w i th more v i r u l e n c e genes . ' H i s f i e l d da t a were i n agreement w i th the e v o l u t i o n o f r a ces ove r 50 yea r s i n Canada. The c o n c l u s i o n was t ha t the number o f v i r u l e n c e genes was not the key i n de t e rm in ing success i n n a t u r e . Osoro and Green (1976) d i s c o v e r e d t h a t i n f i v e out o f s i x d i f f e r e n t m ix tu re s o f P. g r am in i s t r i t i c i i s o l a t e s , the complex race p redomina ted . Comparisons were made under two d i f f e r e n t temperature r eg imes . Only f o r one p a r t i c u l a r m i x t u r e , the complex race predominated a t 25 °C and the s imp le a t 1 5 ° C . The c o m p e t i t i v e a b i l i t i e s o f r a ces i n the o the r m ix tu res were not d i f f e r e n t i a l l y i n f l u e n c e d by tempera ture . They conc luded tha t i n c u b a t i o n p e r i o d and spore p r o d u c t i o n were the two major f a c t o r s a f f e c t i n g pathogen a g g r e s s i v e n e s s . Aslam and Browder (1977) r e p o r t e d mix ture exper iments (o f As l am ' s ) f o r P. r e c o n d i t a t r i t i c i i s o l a t e s c u l t u r e d f o r e i g h t g e n e r a t i o n s under sepa ra te reg imes o f temperature/day l e n g t h . The r e s u l t s suppor t the v iew tha t genotypes w i th few v i r u l e n c e genes tend to predominate ove r i s o l a t e s w i th more v i r u l e n c e genes . The i n c r e a s e o f the s imp le i s o l a t e was independent o f the i n i t i a l f r equency o f e i t h e r genotype i n the m i x t u r e . The 45 s i m p l e s t r ace a l s o e x h i b i t e d g r e a t e r a d a p t a b i l i t y to d i f f e r e n t temperature r eg imes . M ix tu re s t u d i e s w i th P. h o r d e i ( F a l a h a t i - R a s t e g a r e t a l . 1981) a l s o showed tha t the complex race cou ld p e r s i s t and be as a g g r e s s i v e as l e s s v i r u l e n t r a c e s . Aga in e f f e c t s o f temperature and inoculum d e n s i t y were impor tant f a c t o r s i n c o m p e t i t i v e a b i l i t y o f the r a c e s s t u d i e d . Katsuya and Green (1967) examined v a r i o u s f a c t o r s such as i n c u b a t i o n p e r i o d and spore v i a b i l i t y , t ha t may have caused the i n c r e a s e i n r ace 56 and d e c l i n e o f race 15B o f P. g r a m i n i s  t r i t i c i i n Canada. T h e i r c o n c l u s i o n s were t h a t not o n l y was s e l e c t i o n p r e s s u r e o f the hos t impor tant i n the change , but the lower temperature which p r e v a i l e d d u r i n g the p e r i o d i s a l s o impor t an t . Green (1975) examined the changes i n v i r u l e n c e o f P. g r am in i c t r i t i c i over a 10 year p e r i o d i n Canada. He d i s c o v e r d t h a t v i r u l e n c e changes were random r a t h e r than a p r o g r e s s i v e s tepwise g a i n i n v i r u l e n c e . In A u s t r a l i a , Lu ig and Watson (1970) found p r o g r e s s i v e s tepwise i n c r e a s e s i n v i r u l e n c e f o r P. g r a m i n i s t r i t i c i . In A u s t r a l i a , the s e l e c t i o n p r e s s u r e imposed by the hos t seems to be the most impor tant f a c t o r i n pathogen changes ; whereas, i n the U.S. and Canada o t h e r f a c t o r s were impor tan t i n i nduc i ng changes . MacKey (1977) made a compar ison o f v i r u l e n c e a n a l y s i s o f P. g r a m i n i s avenae between the U.S. and Sweden. In the U . S . , s e l e c t i o n p r e s s u r e o f the hos t was impor tan t but i n Sweden, i t was n o t . In Sweden, a wide range 46 o f v i r u l e n c e s o c c u r r e d w i th s e v e r a l combina t ions o f complex r a c e s . Person (1968) rev iewed the work o f Cherewick . Che rew i ck ' s (1958) o b j e c t i v e was to o b t a i n homogeneous i s o l a t e s o f U s t i l a g o  avenae which gave r e p e a t a b l e r e s u l t s on the d i f f e r e n t i a l oa t v a r i e t i e s . He t r i e d by i n o c u l a t i n g the d i f f e r e n t i a l s , t a k i n g the inoculum f o r f u t u r e g e n e r a t i o n s from the c u l t i v a r Monarch, and r e p e a t i n g the i n o c u l a t i o n and s e l e c t i n g f o r n ine g e n e r a -t i o n s . The pe r cen t smutted p l a n t s o f Monarch i n c r e a s e d ove r the n ine g e n e r a t i o n s (Appendix F ) ; t h i s would be the r e s u l t o f d i r e c t i o n a l s e l e c t i o n f o r v i r u l e n c e o r a g g r e s s i v e n e s s . For f i v e c u l t i v a r s which o r i g i n a l l y were h i g h l y smut ted , the pe r cen t smutted p l a n t s was d r a s t i c a l l y reduced a f t e r one g e n e r a t i o n o f s e l e c t i o n on Monarch. The dec rease cou ld be e x p l a i n e d by t h e ' f a c t tha t s i n c e s e l e c t i o n o c c u r r e d on Monarch, the genotypes s u c c e s s f u l on those f i v e c u l t i v a r s were be ing s e l e c t e d a g a i n s t . The g r a d u a l i n c r e a s e cou ld be e x p l a i n e d by the c u l t i v a r s hav ing some r e s i s t a n c e genes i n common. T h i s may be ev idence f o r s t a b i l i z i n g s e l e c t i o n . Che rew i ck ' s c o n c l u s i o n was t ha t he cou ld not o b t a i n a homogeneous p o p u l a t i o n o f smut. Ho l ton (1967) p r o v i d e d ve r y s i m i l a r ev idence f o r U. avenae. I s o l a t e A - l l , which was s e l e c t e d f o r s i x g e n e r a t i o n s on Anthony , dec reased i n pe r cen t smutted p l a n t s o f C l i n t l a n d . I s o l a t e C-576, which was s e l e c t e d on C l i n t l a n d , dec reased i n pe r cen t smutted p l a n t s o f B l ack Diamond but i n c r e a s e d i n pe r cen t smutted p l a n t s o f C l i n t l a n d . 47 Changes i n race compos i t i on were examined i n C a l i f o r n i a p o p u l a t i o n s o f Rhynchosporium s e c a l i s by Jackson and Webster ( 1 9 7 6 , a , b ) . The most f r equen t i s o l a t e s t ha t they found i n t h e i r c o l l e c t i o n s were from o p p o s i t e ends o f the spectrum f o r numbers o f v i r u l e n c e genes . In c o n t r o l l e d s t u d i e s they observed a s h i f t from the more complex r a ces to the s i m p l e r r a c e s i n j u s t two g e n e r a t i o n s . O r i g i n a l i s o l a t e s d i f f e r e d i n s p o r u l a t i n g a b i l i t y but t h i s was not r e l a t e d to the number o f v i r u l e n c e genes each i s o l a t e c o n t a i n e d . Young and P r e s c o t t (1974) looked a t the degree o f h e t e r o z y g o s i t y i n c o l l e c t i o n s o f P. r e c o n d i t a from Oklahoma where hos t s e l e c t i o n was b e l i e v e d to be m i n i m a l . They conc luded tha t the re was a c o n s i d e r a b l e degree o f h e t e r o z y g o s i t y . T h e i r o n l y c o n c l u s i o n r e l a t i n g to s t a b i l i z i n g s e l e c t i o n was t ha t the most p r e v a l e n t r a ces i n the f i e l d had few v i r u l e n c e genes and t h e r e f o r e s t a b i l i z i n g s e l e c t i o n must be i n f l u e n c i n g the p o p u l a t i o n . More r e c e n t l y R o e l f s and Gro th (1980) and Gro th and R o e l f s (1982) have taken a d i f f e r e n t approach f o r c e r e a l r u s t pa thogens . They have made compar isons o f v i r u l e n c e phenotypes between p o p u l a t i o n s o f P. g r a m i n i s t r i t i c i t h a t were r e p r o d u c i n g s e x u a l l y and those r ep roduc ing a s e x u a l l y . They were i n t e r e s t e d i n see ing i f d i f f e r e n c e s i n p o p u l a t i o n s t r u c t u r e e x i s t e d between these two p o p u l a t i o n s . One o f the o b s e r v a t i o n s from the gene d i s t r i b u t i o n f o r the s exua l p o p u l a -t i o n was t ha t a l a r g e number o f unnecessary v i r u l e n c e genes 48 appeared a t a h i g h f r equency , which cou ld be taken as ev idence f o r s t a b i l i z i n g s e l e c t i o n . Oard and Simons (1983) a l s o have made compar isons o f t r a i t s between sexua l and a sexua l p o p u l a t i o n s o f P. c o r o n a t a . They found no s i g n i f i c a n t d i f f e r e n c e s i n g e n o t y p i c v a r i a t i o n between the p o p u l a t i o n s . V i r u l e n c e a n a l y s i s o f E r y s i phe g r am in i s h o r d e i p o p u l a t i o n s (K lug-Andersen 1980) d i d not d i s c l o s e any s i g n i f i c a n t dec rease i n f i t n e s s caused by unnecessary v i r u l e n c e genes . I t was i n t e r e s t i n g to note t ha t many o f the v i r u l e n c e genes i n the p o p u l a t i o n s were p a r t i a l l y needed f o r the m u l t i l i n e s from which the p o p u l a t i o n was i n i t i a l l y o b t a i n e d ; t h e r e f o r e , these genes were exposed to d i r e c t i o n a l s e l e c t i o n when on the m u l t i l i n e but when the s e l e c t i o n p r e s s u r e was removed, no change i n v i r u l e n c e gene f r e q u e n c i e s was obse r ved . Os te rga rd (1982) po in t ed out t ha t t h i s work was not n e c e s s a r i l y m o n i t o r i n g changes i n gene f r e q u e n c i e s , but r a t h e r showed the r e s u l t s o f c o m p e t i t i o n among c l o n e s o f which the genotypes were mere l y l a b e l s . The l i t e r a t u r e i s d i v i d e d as to p r o v i d i n g ev idence f o r s t a b i l i z i n g s e l e c t i o n sensu Vander P l ank ; even w i t h i n i n d i v i d u a l papers there are oppos ing r e s u l t s . From the l i t e r a -tu re r e v i ewed , i t i s apparent t ha t b e t t e r des igned exper iments are needed. P a r l e v l i e t (1981a) rev iewed the v a r i o u s methods used to t r y to e v a l u a t e the concept o f s t a b i l i z i n g s e l e c t i o n . He e x p l a i n e d tha t most o f the expe r imen ta l r e s u l t s shou ld be viewed c a r e f u l l y s i n c e f i t n e s s measured under expe r imen ta l c o n d i t i o n s w i l l be d i f f e r e n t under n a t u r a l c o n d i t i o n s . I t i s 49 v e r y d i f f i c u l t to o b t a i n i n f o r m a t i o n on gene f r e q u e n c i e s and even ha rde r to o b t a i n i n f o r m a t i o n o f r e l a t i v e f i t n e s s v a l u e s a s s o c i a t e d w i th p a r t i c u l a r v i r u l e n c e genes . A number o f models have been deve loped to examine gene f r e q u e n c i e s and f i t n e s s v a l u e s . Ogle eMb a_l. (1973) deve loped a model to p r e d i c t the r e l a t i v e a b i l i t y o f r a ces to s u r v i v e i n m i x t u r e s . The model p r e d i c t s the d i f f e r e n c e s i n r e l a t i v e s u r v i v a l a b i l i t y , i f the f a c t o r s c o n t r i b u t i n g to r e l a t i v e s u r v i v a l a b i l i t y have been q u a n t i t a t i v e l y d e f i n e d . From the p r e v i o u s l y rev iewed p a p e r s , i t i s known tha t many o f these f a c t o r s have not been d e f i n e d and tha t ve r y l i t t l e q u a n t i t a t i v e i n f o r m a t i o n e x i s t s . Person e_t a_l. (1976) looked a t d i r e c t i o n a l and s t a b i l i z i n g s e l e c t i o n from the v i ewpo in t o f p o p u l a t i o n g e n e t i c s . They have a s s i gned t h e o r e t i c a l l y f i t n e s s v a l u e s to the pathogen v i r u l e n c e and a v i r u l e n c e a l l e l e s on both r e s i s t a n t and s u s c e p t i b l e h o s t s . T h e i r a n a l y s i s i n v o l v e d the e s t i m a t i o n o f changes i n r e l a t i v e f i t n e s s and changes i n f r e q u e n c i e s when on a hos t p o p u l a t i o n c o n t a i n i n g v a r i o u s p r o p o r t i o n s o f r e s i s t a n t and s u s c e p t i b l e hos t i n d i v i d u a l s . They d i s c o v e r e d tha t the s e l e c t i o n p r e s s u r e o p e r a t i n g a g a i n s t the v i r u l e n c e a l l e l e w i l l be maximal where the match ing r e s i s t a n c e gene i s absent from the p o p u l a t i o n . Kiyosawa and Yabuki (1976) and Kiyosawa (1980) modeled r a ce f requency changes and d i d v i r u l e n c e a n a l y s i s o f r ace f r e q u e n c i e s based on i n f o r m a t i o n o f the r i c e b l a s t fungus . The s i m u l a t i o n s produced i n the f i r s t a n a l y s i s gave ev idence f o r 50 s t a b i l i z i n g s e l e c t i o n . In a l a t e r a n a l y s i s us ing the r i c e b l a s t d a t a , f requency changes cou ld not be e x p l a i n e d by s t a b i l i z i n g s e l e c t i o n o r d i r e c t i o n a l s e l e c t i o n . Os te rgaa rd (1982, 1983) deve loped models f o r p r e d i c t i n g the development o f an ep idemic on c u l t i v a r m ix tu res and f o r dens i t y-dependent r e g u l a t i o n o f a p a r a s i t e p o p u l a t i o n , w i th a t tempts to e s t ima te f i t n e s s o f v i r u l e n c e genes . One o f her c o n c l u s i o n s was t ha t the most e f f i c i e n t way to improve the unders tand ing o f s e l e c t i o n on v i r u l e n c e genes might be to use c o m p e t i t i o n exper iments between mother and mutant i s o l a t e s , o r to mon i to r a c t u a l changes i n n a t u r a l p o p u l a t i o n s . A number o f t h e o r e t i c a l a t tempts have been made to e s t ima te f i t n e s s (MacKenzie 1978, Groth and B a r r e t t 1980, S k y l a k a k i s 1980, B a r r e t t 1983) , as w e l l a s , e s t i m a t e s based on da ta (Grant & A rche r 1983, Leonard 1977) . MacKenzie (1978) and Gro th and B a r r e t t (1980) d e r i v e d e x p r e s s i o n s d e s c r i b i n g changes i n the r e l a t i v e f r e q u e n c i e s o f two a s e x u a l l y r ep roduc i ng pathogen r aces i n the absence o f c o m p e t i t i o n . S k y l a k a k i s (198 0) looked a t the r a t e o f rep lacement o f one race by a second race i n an a s e x u a l l y r ep roduc ing pathogen when the r a c e s are compet ing . B a r r e t t (198 3) p o i n t s out some o f the g e n e r a l problems which u n d e r l i e these models and which make i t d i f f i c u l t to app l y these models to r e a l d a t a . The assumpt ions f o r s imp le p o p u l a t i o n growth models are g e n e r a l l y i gnored and i t i s c l e a r t ha t most o f the assumpt ions c o u l d not be met f o r many p l a n t d i s e a s e s . The f i n a l c o n c l u s i o n s o f t h i s r e v i ew , are t ha t much more q u a n t i t a t i v e da t a i s needed i n o r d e r to unders tand r e l a t i v e f i t n e s s a s s o c i a t e d w i th v i r u l e n c e genes , as w e l l as the changes i n gene f r e q u e n c i e s tha t occu r i n pathogen p o p u l a t i o n s . Attempts to observe changes i n gene f r e q u e n c i e s i n heterogeneous pathogen p o p u l a t i o n s on r e s i s t a n t and s u s c e p t i b l e c u l t i v a r s i s n e c e s s a r y . These types o f o b s e r v a t i o n s would p r o v i d e an unders tand ing o f s t a b i l i z i n g s e l e c t i o n . S t a b i l i z i n g s e l e c t i o n can a l s o app l y t o h o s t - p a r a s i t e systems i n the t r a d i t i o n a l p o p u l a t i o n g e n e t i c s d e f i n i t i o n . I f a h o s t - p a r a s i t e system has p o l y g e n i c d e t e r m i n a t i o n o f r e s i s t a n c e , v i r u l e n c e , o r b o t h , the type o f s e l e c t i o n o p e r a t i n g would be the t r a d i t i o n a l usage o f s t a b i l i z i n g s e l e c t i o n . At t h i s t ime one model e x i s t s (F leming and Person 1982) which a t tempts to examine the s t a b i l i t y o f p o l y g e n i c a l l y determined sys tems . T h e i r model examined a system w i th both p o l y g e n i c a l l y determined r e s i s t a n c e and p a t h o g e n i c i t y . Acco rd ing t o t h e i r mode l , a t any one p o i n t i n t ime , accumu la t ion o r l o s s o f po l ygenes i n one p o p u l a t i o n (host/pathogen) would be matched by a ccumu la t i on o r l o s s o f po lygenes i n the o t h e r . The s e l e c t i v e f o r c e s f o r r e p r o d u c t i v i t y o f the two i n t e r a c t i n g p o p u l a t i o n s a re d i a m m e t r i c a l l y opposed and c o u l d p rov i de the s t a b i l i t y thought to e x i s t i n these types o f h o s t - p a r a s i t e sys tems . Jenns et a_l. (1982) have t r i e d to deve lop a model to examine the d u r a b i l i t y o f q u a n t i t a t i v e r e s i s t a n c e . The model i n v o l v e d both g e n e r a l and s p e c i f i c i n t e r a c t i o n s w i th the pathogen and was des igned to t e s t the a b i l i t i e s o f s t a t i s t i c a l ana l y se s to d i s c r i m i n a t e between hos t genotypes w i th d i f f e r e n t l e v e l s o f major gene and p o l y g e n i c a l l y determined r e s i s t a n c e . The model was ab l e to rank new v a r i e t i e s a g a i n s t o l d ones but cou ld not p r o v i d e an e s t ima te o f d u r a b i l i t y . More r e s e a r c h i s a l s o needed to examine the s t a b i l i t y o f p o l y g e n i c a l l y determined sys tems . , No expe r imen ta l e v idence e x i s t s f o r the na ture o f the s t a b i l i t y o f these sys tems . At t h i s t ime a l l o f our knowledge r e g a r d i n g p o l y g e n i c determined p a t h o g e n i c i t y o r r e s i s t a n c e i s l a r g e l y e m p i r i c a l . 53 CHAPTER 3 BARLEY: COVERED SMUT - BIOLOGY AND GENETIC ASPECTS Ba r l e y (Hordeum vu lga r e L. emend. Bowden) be longs to the g r a s s f a m i l y Gramineae, t r i b e Hordeae and i s a s m a l l - g r a i n c e r e a l tha t i s used f o r a v a r i e t y o f p u r p o s e s . The o r i g i n o f b a r l e y i s u n c e r t a i n , but modern b a r l e y i s now grown throughout the temperate r e g i o n . C u l t i v a t e d b a r l e y s have e i t h e r a w in t e r o r s p r i n g growth h a b i t and the l a t t e r do not r e q u i r e a c o l d dormant p e r i o d be fo r e they mature . No rma l l y , a s i n g l e p l a n t w i l l produce 3 t o 6 stems ( t i l l e r s ) which may v a r y i n l e n g t h from 10cm to 150 cm. The number o f t i l l e r s and t h e i r l e n g t h are i n f l u e n c e d by the g e n e t i c makeup o f the b a r l e y , the d e n s i t y o f the s t a n d , and o t h e r env i ronmenta l f a c t o r s . The head o r s p i k e a t the top o f the t i l l e r i s made up o f s p i k e l e t s a t t ached to the nodes o f a z i g z a g r a c h i s . Each s p i k e l e t i s a s i n g l e f l owe r and 3 s p i k e l e t s are a t t a ched a t each node. In two-rowed b a r l e y s , o n l y the c e n t r a l f l o r e t i s f e r t i l e ; a l l a re f e r t i l e i n the s ix-rowed b a r l e y s (Anonymous 1979, Mathre 1982) . Most o f the v i g o r o u s improvement o f b a r l e y c u l t i v a r s by b reede r s began i n the 1 9 0 0 ' s . B a r l e y i s p redominan t l y s e l f -p o l l i n a t i n g , t h e r e f o r e a l l g e n e t i c improvement has been done through a r t i f i c i a l c r o s s e s . Hundreds o f c u l t i v a r s have been made w i th major improvements f o r most o f the agronomic t r a i t s , i n c l u d i n g d i s e a s e r e s i s t a n c e . 54 Covered smut, a d i s e a s e o f b a r l e y , i s caused by the fungus U s t i l a g o h o r d e i ( Pe rs . ) L age rh . The s i g n t h a t a b a r l e y p l a n t has the covered smut d i s e a s e i s a s o f t powdery mass o f b l a c k t e l i o s p o r e s t h a t r e p l a c e s the k e r n a l and i s covered by a p e r s i s t e n t g ray membrane. I n f ec t ed p l a n t s are f r e q u e n t l y i n d i s t i n g u i s h a b l e from h e a l t h y ones u n t i l the heads emerge. U. h o r d e i can o n l y complete i t s l i f e c y c l e by i n f e c t i n g b a r l e y p l a n t s . The l i f e c y c l e c o n s i s t s o f two phases , a p a r a s i t i c phase and n o n - p a r a s i t i c phase ( F i g . 3 .1 ) . The non -p a r a s i t i c phase beg ins w i th the ge rm ina t i on o f a d i p l o i d t e l i o s p o r e which undergoes m e i o s i s and forms a 4 - c e l l e d promycel ium w i th each c e l l c o n t a i n i n g a p roduc t o f m e i o s i s . M i t o s i s o c cu r s and s p o r i d i a bud o f f from each o f the f o u r c e l l s . Us ing a m i c roman ipu l a t o r the s p o r i d i a can be i s o l a t e d and then grown as a y e a s t - l i k e v e g a t a t i v e c l one on a r t i f i c i a l med ia . The mat ing system o f U. h o r d e i r e q u i r e s the un ion o f o p p o s i t e mat ing types to form the d i k a r y o n . Each t e l i o s p o r e t h e r e f o r e produces 2 s p o r i d i a o f each mat ing t ype . The two a l l e l e s o f the mat ing l o c u s are de s i gna t ed as + and - o r A and a . Only the d i k a r y o t i c hyphae can i n f e c t the b a r l e y p l a n t and t h i s i s the beg inn ing o f the p a r a s i t i c phase . The d i k a r y o t i c hypha e n t e r s through the c o l e o p t i l e , advances through the hos t t i s s u e and becomes e s t a b l i s h e d i n the growing p o i n t (Kozar 1969) . E v e n t u a l l y t e l i o s p o r e s are produced i n s o r i r e p l a c i n g the k e r n a l i n the s p i k e l e t s o f the p l a n t . 55 Intercellular nycellum Figure 3.1 Life Cycle of Ustilago hordei 56 In n a t u r e , the t e l i o s p o r e s would remain on the seed o r i n the s o i l between the growing seasons o f b a r l e y . The t e l i o -spores would germinate a t the same t ime as the b a r l e y seed and mat ing o f s p o r i d i a would o c c u r randomly . In the l a b o r a t o r y , a t e s t can be performed to determine the mat ing type (Bauch 19 32) . The a p p r o p r i a t e h a p l o i d s can be used to i n o c u l a t e the seed (Thomas and Person 196 5) w i th l i t t l e chance o f d i s e a s e escape (Groth and Person 1976) . Most o f the o l d e r l i t e r a t u r e on U. h o r d e i r e p o r t s t e l i o s p o r e s used as inocu lum and i n o c u l a t e d by a number o f methods ( T i s d a l e 1923, Tapke 1935, 1940, Tapke and Bever 1942, Popp and Cherewick 1953) . When the f ou r s p o r i d i a are i s o l a t e d i n o r d e r o f 1-4 ( F i g . 3 .2 ) , t h i s i s an o rde red t e t r a d . Us ing two g e n e t i c markers such as a v i r u l e n c e gene and mat ing t y p e , two types o f t e t r a d s can be o b s e r v e d . F i r s t , d i t y p e s are composed o f two types o f s p o r i d i a . I f both s p o r i d i a l t ypes are g e n e t i c a l l y i d e n t i c a l t o the p a r e n t a l s p o r i d i a , t h i s i s a p a r e n t a l d i t y p e ; and i f both s p o r i d i a l t ypes are r e comb inan t s , i t i s a non-p a r e n t a l d i t y p e . The o t h e r t e t r a d i s a t e t r a t y p e and the re are f ou r g e n e t i c a l l y unique s p o r i d i a . The t e t r a t y p e o f t e t r a d p r o v i d e s the most i n f o r m a t i o n on the s t a t u s o f the v i r u l e n c e gene . A 3:1 r a t i o w i l l be observed when the p rope r mat ings are pe r f o rmed , i f the v i r u l e n c e gene i s h e t e r o z y g o u s . Combining o r c r o s s i n g f ou r s p o r i d i a l c u l t u r e s o f a s i n g l e t e l i o s p o r e c o n -s t i t u t e s s e l f i n g and f o u r d i k a r y o n s can be formed. When the f o u r s p o r i d i a o f two t e l i o s p o r e s are combined, t h i s c o n s t i t u t e s 57 Heterozygous (W) t e l i o s p o r e s - segregations produced via s e l f i n g S p o r i d l a l mating t e l i o s p o r e Sporidium 1 2 3 4 Tetrad S e l f i n g 1 x 3 1 x A 2 x 3 2 x 4 Ratio V+ V+ v -v -PD Genotypes V-V-v+ v+ NPD Vv Vv Vv Vv Vv Vv Vv Vv V+ v+ V-v -T W Vv Vv w 4:0 4:0 3:1 F igu re 3.2 I l l u s t r a t i o n of the types o f t e t r a d s produced from a t e l i o s p o r e o f U s t i l a g o h o r d e i he te rozygous f o r a v i r u l e n c e gene and the r a t i o o f v i r u l e n t to a v i r u l e n t d i k a r yons produced from " s e l f i n g " . 58 c r o s s i n g and e i g h t d i k a r y o n s can be formed (Thomas and Person 1967) . D i sease r e a c t i o n s o f covered smut are determined by coun t i ng the number o f smutted p l a n t s out o f t o t a l number o f p l a n t s per t r ea tment . A p l a n t i s c o n s i d e r e d smutted i f one o r more o f the t i l l e r s has a smutted head (Thomas and Person 1967) . Other d i s e a s e assessments are to count the number o f smutted t i l l e r s out o f t o t a l number o f t i l l e r s pe r t rea tment (Emara 1972) . Groth e t a l . (1976) compared the p r o p o r t i o n o f smutted p l a n t s and the number o f smutted t i l l e r s per p l a n t t o see i f the re was a c o r r e l a t i o n between these two measurements. S t rong c o r r e l a t i o n s were found between the two measurements on the b a s i s o f 12 b a r l e y c u l t i v a r s and 21 d i k a r y o n s . In many i n s t a n c e s , the main culm escapes i n f e c t i o n , but the f i r s t , second o r a l l o t h e r t i l l e r s w i l l be i n f e c t e d . Groth and Person (1978) found t h a t o l d e r t i l l e r s were most f r e q u e n t l y nonsmutted and o l d e r f a m i l i e s o f t i l l e r s con t a i ned a smutted t i l l e r l e s s f r equen t than d i d younger f a m i l i e s . The i n f e c t e d heads are e a s i l y v i s i b l e due to the b l a c k spore mass, and bad l y d i s e a s e d heads u s u a l l y have p a l e r c o l o r e d awns and heads ( F a r i s 1924 ) . In some c a s e s , smutted heads do not emerge from the boot as do normal heads , but b u r s t through the base o f the f l a g l e a f ; t h i s i s r e f e r r e d to as c l u b b i n g . At t imes the upper p o r t i o n o f the head remains h e a l t h y wh i l e the b a s a l p a r t i s i n f e c t e d ; t h i s i s r e f e r r e d to as b a s a l s m u t t i n g . Sometimes the head appears h e a l t h y but i s p a l e r i n c o l o r than normal heads ; 59 a t c l o s e r o b s e r v a t i o n , spore masses can be seen underneath the lemma and t h i s i s r e f e r r e d to as p a r t i a l s m u t t i n g . A p l a n t has severe p a r t i a l smutt ing when o n l y a speck o f spores i s found a t the base o f the k e r n a l s . The t y p i c a l smutted head , w i th l i t t l e v i s i b l e p l a n t m a t e r i a l and masses o f t e l i o s p o r e s , i s termed complete s m u t t i n g . W i th in a s i n g l e smut genotype a l l o f the smut t ing types can be o b s e r v e d ; t h i s p o s s i b l y i s i n f l u e n c e d by growing s tage o f the t i l l e r and env i ronmenta l c o n d i t i o n s . S ince U. h o r d e i invades the hos t du r i ng the s e e d l i n g s t a g e , the env i ronmenta l f a c t o r s a t t h i s s tage o f p l a n t development are p r o b a b l y the most impor tant i n de t e rm in ing the d i s e a s e e x p r e s s i o n . Smut i n f e c t i o n appeared to be optimum at s o i l temperatures between 10 and 20 °C w i th a d r a s t i c r e d u c t i o n i n smut t ing i f temperature was <5° o r >30°C ( F a r i s 1924b) . Tapke (1938) con f i rmed tha t v e r y low temperatures reduced smut t ing but he l a t e r found t h a t d i f f e r e n t c u l t i v a r s respond d i f f e r e n t l y (Tapke 1952) . A c i d i c s o i l gave more smutt ing than sandy s o i l ( F a r i s 1924a, Jones and Abd E l Ghani S e i f - E l - N a s r 1940) . Increased mo i s tu r e i n the s o i l gave h i g h e r l e v e l s o f smut ( F a r i s 1924b, Jones and Abd E l Ghani S e i f - E l - N a s r 1940) , as long as the re was p rope r s o i l a e r a t i o n (Tapke 1944) . - The depth o f p l a n t i n g i s a l s o impor tan t w i th an optimum depth o f 2" t o 3" depending on s o i l c o n d i t i o n s (Jones and Abd E l Ghani S e i f - E l - N a s r 1940, T a y l o r and Zehner 1931) . Many o f the smut i n o c u l a t i o n p rocedures i n v o l v e the removal o r l o o s e n i n g o f the seed h u l l s which appear to i n c r e a s e the smut l e v e l s ( F a r i s 60 1924a, T i s d a l e 1923 ) , but a l s o reduces s e e d l i n g emergence ( S cha fe r e t a l . 1962) . Other than these s t u d i e s r e l a t i v e l y l i t t l e i s known about the optimum c o n d i t i o n s f o r smut i n f e c t i o n . The f i r s t s tudy w i th b a r l e y on i n h e r i t a n c e o f r e s i s t a n c e to covered smut was done by Johnston (1934) , but u n f o r t u n a t e l y , the re were no c l e a r cu t s e g r e g a t i o n p a t t e r n s i n h i s F3 l i n e s d e r i v e d from r e c i p r o c a l c r o s s e s o f G labron ( r e s i s t a n t ) and T r e b i (moderate ly s u s c e p t i b l e ) . Woodward and T ingey (1941) t e s t e d F3 l i n e s from c r o s s e s at Winter C lub ( s u s c e p t i b l e ) to Union Bea rd l e ss and Co lo rado 3063 (both r e s i s t a n t ) ; however, the l e v e l s o f i n f e c t i o n were too low to make any c o n c l u s i v e i n t e r p r e t a t i o n s . Pugs ley and V ines (1946) t e s t e d F3 l i n e s from a c r o s s o f Cape ( s u s c e p t i b l e ) to Kwan ( r e s i s t a n t ) and o b t a i n e d a range o f 0-25% i n f e c t i o n ; whereas, no smut was observed i n the F 2 g e n e r a t i o n . Shands (1956) was the f i r s t to o b t a i n r e s u l t s t ha t cou ld be i n t e r p r e t e d g e n e t i c a l l y . He c ro s sed B r a c h y t i c ( r e s i s t a n t ) to Chevron and C o l s e s s (both s u s c e p t i b l e ) . He d i s c o v e r e d t h a t , f o r both c r o s s e s , r e s i s t a n c e was dominant and c o n d i t i o n e d by one gene. He worked w i th l a r g e numbers ,of p l a n t s and a l s o examined the r e l a t i o n s h i p o f i n h e r i t a n c e o f r e s i s t a n c e to s i x o t h e r p l a n t t r a i t s which were a l l independent o f smut r e s i s t a n c e . We l l s (1958) used d i a l l e l c r o s s e s among f i v e r e s i s t a n t and one s u s c e p t i b l e c u l t i v a r . I n t e r p r e t a t i o n s were based l a r g e l y 61 on F 3 d a t a w i th the b a s i s o f smut l e v e l o f the s u s c e p t i b l e pa ren t as a c o r r e c t i o n f o r e s capes . Three o f the v a r i e t i e s had a common dominant gene f o r r e s i s t a n c e and two o t h e r c u l t i v a r s each had a s i n g l e r e c e s s i v e gene f o r r e s i s t a n c e . Cott ingham (1958) s t u d i e d r e s i s t a n c e to race s i x o f covered smut and found two genes c o n d i t i o n i n g r e s i s t a n c e ; r e s i s t a n c e was r e c e s s i v e . M e t c a l f (1962) s t u d i e d the i n h e r i t -ance o f b a r l e y r e s i s t a n c e to l o o s e smut, f a l s e l o o s e smut and covered smut; he found a dominant gene f o r r e s i s t a n c e to l o o s e smut and p o s s i b l y one to covered and f a l s e l o o s e smut. He observed a c l o s e a s s o c i a t i o n between the r e a c t i o n to f a l s e l o o s e smut and covered smut. Cherewick and Buchannon (1969) examined the i n h e r i t a n c e o f r e s i s t a n c e i n two b a r l e y c r o s s e s and d i s c o v e r e d one dominant gene to each f a l s e l o o s e smut and covered smut, and aga in the r e a c t i o n s to the two smuts appeared to be c l o s e l y a s s o c i a t e d . K i e s l i n g (1970) found a case where th ree independent dominant genes governed r e s i s t a n c e to a smut race and another case where f o u r genes governed r e s i s t a n c e . S idhu and Person (1972) i n v e s t i g a t e d the g e n e t i c s o f r e s i s t a n c e i n th ree c u l t i v a r s by t e s t i n g w i th two smut geneo types . Aga in r e s i s t a n c e was dominant and c o n t r o l l e d by s i n g l e genes f o r each smut genotype . Cau t i on shou ld be taken on the i n t e r p r e t a t i o n s o f the e a r l y s t u d i e s on r e s i s t a n c e to covered smut. Some r e s e a r c h e r s may not have been t e s t i n g w i th homogeneous smut c u l t u r e s , o t h e r s had low smut i n f e c t i o n and h igh p l a n t m o r t a l i t y . A few 62 papers had skewed F 2 and d i s t r i b u t i o n s and r e a l l y were not c l e a r i n d i c a t i o n s o f s imp le i n h e r i t a n c e . Kn iep (1919) not on l y d i s c o v e r e d sex i n smuts but was a l s o the f i r s t to demonstrate 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 the smuts . F a r i s (1924) r e p o r t e d f i v e p h y s i o l o g i c r a c e s o f U. h o r d e i us ing f o u r d i f f e r e n t i a l c u l t i v a r s . Two more r a ces were r e p o r t e d by Aamodt and Johnson (1935) . Tapke (1937) d i s t i n g u i s h e d e i g h t r a c e s us ing e i g h t b a r l e y c u l t i v a r s as d i f f e r e n t i a l hos t s f o r 200 c o l l e c t i o n s o f covered smut. Tapke used th ree c l a s s e s o f d i s e a s e r e a c t i o n s i n a n a l y z i n g h i s d a t a . These were r e s i s t a n t : 0-5%, i n t e r m e d i a t e : 6-35%, and s u s c e p t i b l e : 36%. Tapke (1945) l a t e r r e p o r t e d f i v e a d d i t i o n a l r a ces from 244 new c o l l e c t i o n s o f smut. Us ing o n l y f o u r d i f f e r e n t i a l c u l t i v a r s , Yu and Fang (1945) r e p o r t e d n ine r a c e s i n C h i n a . Semeniuk (1940) r e p o r t e d on p h y s i o l o g i c r a c e s which appeared i n A l b e r t a ; however, h i s r e s u l t s were not c o n s i s t e n t from yea r to year and were p o s s i b l y changing due to changes i n r a ces o r changes i n env i ronment . Cherewick (1958) examined f i e l d c o l l e c t i o n s over n ine g e n e r a t i o n s o f s e l e c t i o n on a s i n g l e c u l t i v a r and conc luded tha t i t was not p o s s i b l e to form a homogeneous smut p o p u l a t i o n . Because U. h o r d e i forms an o rde red t e t r a d , i t i s easy t o do g e n e t i c s t u d i e s us ing t e t r a d a n a l y s i s . The genotype w i th rega rd to v i r u l e n c e can e a s i l y be i d e n t i f i e d f o r a s i n g l e s p o r i d i a l c u l t u r e . When we observe a 3:1 r a t i o w i t h i n the f ou r d i k a r y o n s r e s u l t i n g from s e l f i n g as i n Tab le 3 . 1 , where th ree Tab le 3.1 T e l i o s p o r e 17 B S e l f e d 1 x 2 1 x 4 3 x 2 3 x 4 63 Resu l t s o f " s e l f i n g " and b a c k c r o s s i n g a t e l i o s p o r e o f U s t i l a g o h o r d e i % smutted Backc ross % smutted p l a n t s 17 B x T 4 p l a n t s 39 1 1 25 ) ) 25 35 1 2 26 ) 16 2 1 2 ) ) 4 30 2 1 7 ) 3:1 3 3 1 ) ) 3 3 4 5 ) 4 3 19 ) ) 23 4 4 28 ) 2:2 64 d i k a r y o n s gave ove r 30% smutted p l a n t s and one gave o n l y 16%/ s e v e r a l c o n c l u s i o n s can be made. F i r s t , v i r u l e n c e i s dominant , the t e l i o s p o r e i s he te rozygous f o r v i r u l e n c e , spo r i d i um 2 and 3 c a r r y the r e c e s s i v e a l l e l e f o r a v i r u l e n c e , and l a s t , spor id ium 1 and 4 c a r r y the dominant a l l e l e f o r a v i r u l e n c e . These c o n -c l u s i o n s can e a s i l y be v e r i f i e d by pe r fo rming a t e s t c r o s s . With the p r e d i c t i o n t h a t spo r i d i um 2 and 3 w i l l c o n s i s t e n t l y y i e l d low l e v e l s o f smut wh i l e 1 and 4 w i l l g i v e h igh l e v e l s , a 2:2 gamet ic r a t i o shou ld be o b s e r v e d . The f i r s t g e n e t i c a n a l y s i s o f t h i s s o r t f o r v i r u l e n c e i n covered smut was performed by Thomas and Person (1965) . They s e l f e d and c ro s sed th ree t e l i o s p o r e s and s e p a r a t e l y i n o c u l a t e d the d i k a r y o n s onto th ree b a r l e y c u l t i v a r s . From the r e s u l t s i t was conc luded tha t a s i n g l e gene c o n t r o l l e d v i r u l e n c e even w i th low pe r cen t i n f e c t i o n . Cherewick (1967) i n t e r c r o s s e d U. h o r d e i and U. n i g r a s t r a i n s and found v i r u l e n c e i n h e r i t e d by a s i n g l e gene . In some cases v i r u l e n c e was dominant and o t h e r s r e c e s s i v e . F u l l e r t o n and N i e l s e n (1974) a l s o made i n t e r c r o s s e s o f these two s p e c i e s and s e l f e d each s p e c i e s . T h e i r r e s u l t s i n d i c a t e d t ha t v i r u l e n c e i n the two s p e c i e s may be the same gene o r the genes are ve ry c l o s e l y l i n k e d . Lade (1969) examined the i n h e r i t a n c e o f v i r u l e n c e i n r ace 6 and race 10 o f U. h o r d e i and i d e n t i f i e d a s i n g l e gene w i th a v i r u l e n c e dominant . He a l s o found tha t the re might be m o d i f i e r genes . U n f o r t u n a t e l y , L a d e ' s c o n c l u s i o n s r e l i e d on 65 da ta from ve ry few p l a n t s and t h e r e f o r e might not be tha t r e l i a b l e . The next major examina t ion o f the i n h e r i t a n c e o f v i r u l e n c e was tha t o f S idhu and Person (1971, 1972) . They examined the g e n e t i c d e t e r m i n a t i o n o f l a r g e r d i f f e r e n c e s i n the l e v e l s o f v i r u l e n c e as compared t o the p r e v i o u s work o f Thomas and Person (1965) . They i d e n t i f i e d two v i r u l e n c e genes , each matched to a r e s i s t a n c e gene i n the h o s t . T h i s was the f i r s t e x t e n s i v e g e n e t i c a n a l y s i s w i th a smut pathogen and i t s hos t showing a gene-for-gene r e l a t i o n s h i p . Ebba and Person (1975) a l s o found a gene-for-gene r e l a t i o n s h i p i n t h i s h o s t - p a r a s i t e system but t h i s t ime the re were two v i r u l e n c e genes matched to one r e s i s t a n c e gene. There were a f f e c t s o f m o d i f i e r genes and env i ronment on one v i r u l e n c e - r e s i s t a n c e gene i n t e r a c t i o n . Thomas (1976) p r o v i d e d r e s u l t s from h i s a n a l y s i s o f v i r u l e n c e t ha t d i d not c o i n c i d e w i th those o f Ebba and Person (197 5) because an i n d i v i d u a l v i r u l e n c e gene would have to a f f e c t more than one r e s i s t a n c e gene . Pedersen and K i e s l i n g (1979) examined the e f f e c t s o f i n b r e e d i n g race 8 o f U. h o r d e i which was known to c o n t a i n th ree r e c e s i v e v i r u l e n c e genes . They a l l u d e d to the p o s s i b i l i t y o f a r e g u l a t o r gene which may have been r e s p o n s i b l e f o r i n c r e a s e d v i r u l e n c e . These l a t t e r papers demonstrate the comp l ex i t y o f the i n t e r a c t i o n s i n U. h o r d e i -b a r l e y sys tem. Emara (1972) tu rned to us ing b i o m e t r i c a l ana l y ses to i n v e s t i g a t e U. h o r d e i . He showed tha t the re was a component o f 66 v i r u l e n c e ( agg ress i veness ) t h a t f i t s a con t i nuous d i s t r i b u t i o n . T h i s suggested t h a t a g g r e s s i v e n e s s was under p o l y g e n i c c o n t r o l . From the b i o m e t r i c a l a n a l y s i s , he conc luded tha t most o f the g e n e t i c c o n t r o l was caused by a d d i t i v e e f f e c t s r a t h e r than i n t e r a c t i o n s but h a l f o f the pheno typ i c v a r i a n c e was caused by env i ronmenta l f a c t o r s . Emara and S idhu (1974) went on to examine the v a r i a b i l i t y w i t h i n two t e l i o s p o r e s known to be homozygous r e c e s s i v e f o r a major v i r u l e n c e gene. The two t e l i o s p o r e s were s e l f e d and c r o s s e d . Agg re s s i v eness was found to be a con t i nuous c h a r a c t e r both w i t h i n and between t e l i o s p o r e s . D ika ryons r e s u l t i n g from c r o s s i n g were s i g n i f i c a n t l y more a g g r e s s i v e than those r e s u l t i n g from s e l f i n g ; sugges t i ng h e t e r o s i s . In t h i s s tudy o n l y 35% o f the pheno typ i c v a r i a b i l i t y was due to env i ronmenta l f a c t o r s . Emara and Freake (1981) t e s t e d 16 d i f f e r e n t d i k a r y o n s i n f i v e d i f f e r e n t env i ronments and found tha t the genotype-envi ronment i n t e r a c t i o n was not s i g n i f i c a n t . T h i s meant t h a t a f a v o r a b l e env i ronment would i n c r e a s e the l e v e l s o f smut f o r a l l genotypes and an un f a vo rab l e env i ronment would dec rease the l e v e l s o f smut f o r a l l geno t ypes . The re fo r e the r e l a t i v e d i f f e r e n c e s between genotypes remained cons t an t i n any e n v i r o n -ment. Ebba (1974) had p o s t u l a t e d t ha t v i r u l e n c e i n f o u r r a c e s o f smut were determined by f o u r d i f f e r e n t a l l e l e s a t a s i n g l e l o c u s . F u r t h e r a n a l y s i s o f F-, t e l i o s p o r e s i d e n t i f i e d a gene with two a l l e l e s s e g r e g a t i n g and v i r u l e n c e l e v e l s seemed to be m o d i f i e d by o the r genes . T h i s work and subsequent work was d i s c u s s e d i n Chapter 2. The impor tant p o i n t from t h i s work i s t ha t gene-for-gene r e l a t i o n s do e x i s t but can be m o d i f i e d by p o l y g e n e s . There i s s t i l l much to l e a r n about the U. h o r d e i - b a r l e y system both a t the g e n e t i c l e v e l and the p h y s i o l o g i c a l l e v e l . The g e n e t i c s o f b a r l e y can e a s i l y be e l u c i d a t e d . Because t e t r a d a n a l y s i s can be used w i th U. h o r d e i , some v e r y r e f i n e d g e n e t i c a n a l y s i s can be done. S ince l a r g e numbers o f t e l i o -spores o f a s i n g l e genotype can be produced and l a r g e p o p u l a t i o n s can be examined, U. h o r d e i l ends i t s e l f to be ing a good organism f o r p o p u l a t i o n g e n e t i c e x p e r i m e n t s . CHAPTER 4 68 GENERAL MATERIALS AND METHODS B a r l e y The b a r l e y c u l t i v a r s Odessa and T r e b i were used i n the f o l l o w i n g expe r imen t s . The seed o f each c u l t i v a r was d e r i v e d from a s i n g l e p l a n t and was i n c r e a s e d each year d u r i n g the summer i n the f i e l d p l o t s on south campus a t UBC and du r i ng the w in t e r a t e i t h e r D a v i s , CA o r Yuma, AZ. When the p l a n t s were mature the heads were cut o f f and t h r e s h e d . Seed was t r e a t e d f o r one hour w i th 0.12% f o r m a l i n s o l u t i o n (40% formaldehyde d i l u t e d one p a r t t o 320 p a r t s water) to s t e r i l i z e the seed s u r f a c e and to l oosen the h u l l s . The seeds were then r i n s e d v i g o r o u s l y f o r 30 minutes i n runn ing tap water and d r i e d tho rough l y f o r 48 hours b e f o r e i n o c u l a t i o n (Tapke and Bever 1942) . I f l o o s e smut were p r e sen t i n the f i e l d d u r i n g the growing season , the seed was a l s o sub j ec t ed to a hot water t rea tment (Tapke 1926) . Seed was p l a ced i n 48 °C water f o r one hour and 50 minutes to i n h i b i t l oose smut g rowth . A f t e r the t rea tment the seed was d r i e d t ho rough l y f o r 48 h o u r s . One hundred seeds o f each c u l t i v a r were p l a ced i n a t r a y c o n t a i n i n g mo is t v e r m i c u l i t e and were incuba ted a t room tempera tu re . A f t e r f i v e d a y s , the number o f s e e d l i n g s was coun t ed . The average o f th ree r e p l i c a t e s was used to determine the pe r cen t ge rm ina t i on o f each seed l o t . 69 Covered Smut I n d i v i d u a l s p o r i d i a l c u l t u r e s were i s o l a t e d w i th the use o f a de Fonbrune m i c roman ipu l a to r and an upward-bent f i n e g l a s s need le (Groth e t a l . 1976) . F i r s t , a t h i n l a y e r o f V o g e l ' s complete medium (Vogel 1956) was poured i n t o s t e r i l e p l a s t i c p l a t e s . B l o cks about 15 mm x 15 mm were c u t us ing a s t e r i l e s c a l p e l and were p l a ced on s t e r i l e 22 mm x 22 mm cove r s l i p s . F i v e cover s l i p s were p l a ced i n a p e t r i d i s h c o n t a i n i n g a t h i n l a y e r o f 2% water a g a r . A 5 ml aqueous t e l i o s p o r e suspens ion was made by p l a c i n g a p i n c h o f t e l i o s p o r e s i n a s t e r i l e t e s t t u b e , adding a few drops o f s t e r i l e d i s t i l l e d wa te r , t e a s i n g the t e l i o s p o r e s wi th a s t e r i l e g l a s s r o d , adding 5 ml o f s t e r i l e d i s t i l l e d water and s t i r r i n g w i th a Vor tex s t i r r e r . A s m a l l d rop o f t h i s suspens ion was p l a ced i n the c e n t e r o f each agar b l o c k w i th a s t e r i l e Pas teur p i p e t t e . The agar b l o c k s were then incuba ted a t 22 °C f o r 15 to 24 h o u r s . On each b l o c k a ge rm ina t i ng t e l i o s p o r e was s e l e c t e d . I f a t e t r a d was r e q u i r e d the chosen t e l i o s p o r e needed t o have a l l f o u r m e i o t i c p r o d u c t s . Each o f these p roduc t s would be drawn away from the promycel ium by the g l a s s n e e d l e , one to each o f the f o u r s i d e s o f the agar b l o c k . T h e i r p o s i t i o n on the promycel ium was r e co rded on the edge o f the cove r s l i p . I f random p roduc t s o f m e i o s i s were r e q u i r e d f o u r ge rm ina t i ng t e l i o s p o r e s were randomly chosen w i th o n l y one p roduc t o f m e i o s i s from each t e l i o s p o r e drawn to the edge o f the agar b l o c k . In 3-4 d a y s , v i s i b l e c o l o n i e s were formed where the i n d i v i d u a l s p o r i d i a had 70 been d e p o s i t e d . These were t r a n s f e r r e d s i n g l y to p e t r i p l a t e s o f V o g e l ' s complete medium (VCM) o r po t a to-dex t ro se agar (PDA) us ing s t e r i l e t o o t h p i c k s . Random p roduc t s o f m e i o s i s were a l s o ob t a i ned by another method where a t e l i o s p o r e suspens ion was made up as p r e v i o u s l y d e s c r i b e d ; but t h i s t ime f ou r d rops o f aqueous Achromycin suspens ion (10 mg/ml) were added to i n h i b i t b a c t e r i a l g rowth . The suspens ion was a g i t a t e d w i th a Vor tex mixer and a l lowed to s e t f o r 8 min . i n o r d e r f o r p l a n t d e b r i s and l a r g e r yeas t spo res to s e t t l e to the bottom o f the s u s p e n s i o n . Us ing a s t e r i l e p i p e t t e p o s i t i o n e d h a l f way down i n the s u s p e n s i o n , 0.1 ml o f suspens ion was taken and t r a n s f e r r e d to p e t r i p l a t e s c o n t a i n i n g VCM. With the a i d o f a sp reader the t e l i o s p o r e s were d i s t r i b u t e d even l y ove r the s u r f a c e o f the aga r . These p l a t e s were incuba ted f o r 24 hours a t 2 2 ° C , then 1.0 ml o f s t e r i l e d i s t i l l e d water was p l a ced on the s u r f a c e o f the agar and s w i r l e d to l oosen s p o r i d i a from the ge rm ina t i ng t e l i o s p o r e s . The s p o r i d i a l suspens ion was drawn o f f w i th a s t e r i l e Pas teur p i p e t t e and p l a ced i n a s t e r i l e t e s t t ube . Haemocytometer counts o f s p o r i d i a were taken so t ha t the suspens ion cou ld be d i l u t e d to o b t a i n app rox ima te l y 100 c o l o n i e s / p l a t e . One ml o f the d i l u t e d suspens ion was p l a ced i n p e t r i d i s h e s o f VCM. In 3-4 days v i s i b l e c o l o n i e s were i n d i v i d u a l l y t r a n s f e r r e d to PDA or VCM us ing s t e r i l e t o o t h p i c k s . A l l s p o r i d i a l c u l t u r e s were ma in ta ined on e i t h e r PDA o r VCM; and were incuba ted a t 22 °C f o r 2 o r 3 weeks and then s t o r e d f o r 2 weeks a t 4 °C to s low the growth u n t i l the t r a n s f e r t o f r e s h med ia . As soon as p o s s i b l e , c u l t u r e s were prepared f o r l ong term s to rage by t r a n s f e r r i n g to s l a n t s o f PDA o r VCM and a l lowed to grow f o r 3 days a t 2 2 ° C . Then 1 ml o f s t e r i l i z e d 7% skim m i l k suspens ion was added t o each s l a n t . Us ing a s t e r i l e i n v e r t e d 1 ml d i s p o s a b l e p i p e t t e the i n d i v i d u a l c o l o n i e s were teased apa r t and t r a n s f e r r e d t o a s t e r i l e screw cap (13 x 100 mm) t e s t tube c o n t a i n i n g s t e r i l e , oven-dr i ed s i l i c a g e l (6-16 mesh) . The emu ls ion and s i l i c a g e l were a g i t a t e d w i th a Vor tex mixer and immediate ly p l a ced i n an i c e bath w i th the caps t i g h t e n e d . The t e s t tubes were l a t e r p l a ced i n Tupperware c o n t a i n e r s c o n t a i n i n g i n d i c a t o r s i l i c a g e l and s t o r e d i n a r e f r i g e r a t o r a t 4 °C f o l l o w i n g the procedure o f P e r k i n s (1962) . The mat ing type o f each c u l t u r e was determined by making s p o r i d i a l suspens ions o f two s tandard t e s t e r s p o r i d i a , E ja and I^A, r esuspend ing the - and + mat ing t y p e , r e s p e c t i v e l y and s p o t t i n g these suspens ions s e p a r a t e l y on 1.5% PDA o r V o g e l ' s min ima l medium i n p e t r i p l a t e s to form s p o r i d i a l lawns. Then us ing s t e r i l e t o o t h p i c k s , the unknown s p o r i d i a were i n d i v i d u a l l y t r a n s f e r r e d to a spot o f each t e s t e r mat ing t y p e , i ncuba ted f o r 24 hours a t 22 °C and examined f o r the presence o f i n f e c t i o n hypae o r " s u c h f a d e n " (Bauch 1932) . 72 Smutted heads were s t o r e d a t room tempera tu re . Smutted heads whose t e l i o s p o r e s were to be used as inoculum were ground up w i th a Thomas-Wiley In te rmed ia te M i l l . The t e l i o s p o r e s were p l a ced i n a d e s i c c a t o r w i th d e s i c c a n t f o r s e v e r a l d a y s , then t r a n s f e r r e d t o a i r t i g h t c o n t a i n e r s and s t o r e d a t 4 ° C . I n o c u l a t i o n and P l a n t i n g S p o r i d i a used f o r inoculum were t r a n s f e r r e d from p e t r i p l a t e s w i th a s t e r i l e l o o p to t e s t tubes c o n t a i n i n g 5 mis o f V o g e l ' s complete b r o t h . A d rop o r two o f aqueous Achromycin suspens ion was added and the tubes were shaken i n a 22 °C i n c u b a t o r f o r 3-4 days to o b t a i n s ynch ronous l y growing c u l t u r e . A f t e r p rope r growth was ob t a i ned some o f the suspens ion o f each spo r id ium was t r a n s f e r r e d to Er lenmeyer f l a s k s c o n t a i n i n g the d e s i r e d amount o f V o g e l ' s complete b r o t h . Aqueous Achromycin was added to the b ro th and the f l a s k s were shaken i n a 22 °C i n c u b a t o r f o r 3-4 days by which t ime s p o r i d i a l suspens ions o f maximal d e n s i t y were o b t a i n e d . The amount o f s p o r i d i a l suspens ion t r a n s f e r r e d from t e s t tubes to the f l a s k s depended on the volume o f b ro th i n the f l a s k . Seeds were i n o c u l a t e d us ing the p a r t i a l vacuum method o f Tapke and Bever (1942) . Seed o f the a p p r o p r i a t e c u l t i v a r was p l a ced i n dram v i a l s , the amount v a r i e d depending on the pe r cen t ge rm ina t i on o f the seed . Equa l q u a n t i t i e s o f the a p p r o p r i a t e h a p l o i d s p o r i d i a l suspens ions were p i p p e t t e d onto the s e e d s , g e n e r a l l y 3 mis per h a p l o i d c u l t u r e . The v i a l s were 73 then p l a ced i n a vacuum d e s i c c a t o r , w i thout d e s i c c a n t , and sub j e c t ed to a p a r t i a l vacuum f o r 30 m inu t e s . The excess l i q u i d was poured o f f and the seeds were p l a ced i n t o c o i n enve lopes and a l lowed to d r y f o r 24 hours i n the open e n v e l o p e s . A f t e r the seeds were d r y , the enve lopes were o r g a n i z e d f o r p l a n t i n g and then s t o r e d a t 4 ° C u n t i l p l a n t i n g which was no more than 3-4 days l a t e r . When t e l i o s p o r e s were used as inocu lum, the smutted heads were ground up as p r e v i o u s l y d e s c r i b e d and the d e s i r e d amount o f t e l i o s p o r e s was weighed us ing a M e t t l e r b a l a n c e . The measured spores o f d i f f e r e n t t rea tments were s e p a r a t e l y p l a ced i n a s t e r i l e Wheaton-dounce t i s s u e g r i n d e r w i th 5 mis o f 0.1% water agar and were teased a p a r t . T h i s was then added to an Er lenmeyer f l a s k a l s o c o n t a i n i n g 0.1% water agar so t h a t the inoculum c o n c e n t r a t i o n was 1 g / 1 . F i ve mis o f t e l i o s p o r e suspens ion were p i p p e t t e d onto the seeds and sub j e c t ed to a p a r t i a l vacuum as b e f o r e . T h i s t i m e , a f t e r pou r ing o f f the excess inocu lum, the v i a l s wre p l a c e d i n an a i r t i g h t c o n t a i n e r and incuba ted f o r 24 hours at 1 5 ° C . Then the seed was p l a ced i n c o i n enve lopes and t r e a t e d i n the same manner as f o r s p o r i d i a l i n o c u l a t i o n s . For each exper iment a t UBC, each t rea tment was p l an t ed i n a 3 m row us ing a P l ane t J r . s i n g l e b e l t p l a n t e r . The rows were 0.3 m a p a r t and an a l l e y o f 0.9 m was kept between each b l o c k o f rows. There were th ree r e p l i c a t e s o f each exper iment and t rea tments were randomized . Weeding was done by hand; 74 f e r t i l i z e r was l i m i t e d and a s p r i n k l e r i r r i g a t i o n system was used i f n e c e s s a r y . P l a n t s were g e n e r a l l y mature a f t e r th ree months. At D a v i s , each t reatment was p l a n t e d by hand i n a 3 m row. Each row was 0.6 m apa r t w i th a 0.6 m a l l e y between b l o c k s o f rows. Each row was on a r a i s e d bed so tha t furrow i r r i g a t i o n c o u l d be used i f n e c e s s a r y . Weeding was done by a c u l t i v a t o r . F e r t i l i z e r l e v e l s were h igh (100 u n i t s o f ammonia n i t r a t e ) . P l a n t s were mature 6-7 months a f t e r s e e d i n g . S e l e c t i o n Scheme 1. D ikaryons would be o b t a i n e d from the exper iment r e p o r t e d by Person e t a l . 1982, 1983. The d i k a r y o n s would be chosen a long the d i a g o n a l l i n e s t a r t i n g i n the top r i g h t c o r n e r o f the g r i d (Appendix B) and moving to the bottom l e f t c o r n e r . These d i k a r y o n s would be he te rozygous f o r a major v i r u l e n c e gene and a l s o max ima l l y heterogeneous f o r the median number o f p o l y g e n e s . 2. Three p o p u l a t i o n s / c u l t i v a r would be c r e a t e d to r e p r e s e n t r e p l i c a t e s ove r the g e n e r a t i o n s o f s e l e c t i o n . For each o f the s i x p o p u l a t i o n s , two s u b p o p u l a t i o n s would be s e l e c t e d ; one o f which would be s e l e c t e d f o r h i g h l e v e l s o f smut and the o t h e r low l e v e l s o f smut. S e l e c t i o n would con t i nue f o r a number o f g e n e r a t i o n s such tha t i f the p o p u l a t i o n was s e l e c t e d on T r e b i , i t was always i n o c u l a t e d onto T r e b i f o r the next g e n e r a t i o n . 3. A f t e r eve ry g e n e r a t i o n random s p o r i d i a would be i s o l a t e d from the p o p u l a t i o n o f t e l i o s p o r e s , then mated and t e s t e d a g a i n s t an a v i r u l e n t s p o r i d i u m . T h i s would a l l o w the phenotypes o f the randomly i s o l a t e d s p o r i d i a to be apparent so t ha t a f requency o f the v i r u l e n t v s . a v i r u l e n t s p o r i d i a cou ld be o b t a i n e d . The changes i n f requency cou ld be observed from one g e n e r a t i o n to ano the r . 4 . T h e o r e t i c a l l y , the s u b p o p u l a t i o n s t ha t are f o r h i g h l e v e l s o f smut shou ld e v e n t u a l l y be homozygous v i r u l e n t and the sub-p o p u l a t i o n s f o r low l e v e l s o f smut shou ld be homozygous r e c e s s i v e . S p o r i d i a from the h igh subpopu l a t i ons would be c r o s s e d to s p o r i d i a from the low subpopu l a t i ons and t e s t e d on both c u l t i v a r s which shou ld sepa ra te out the e f f e c t s o f s e l e c t i o n on the p o l y g e n e s . CHAPTER 5 SELECTION EXPERIMENTS WITH POPULATIONS OF USTILAGO HORDEI I n t r o d u c t i o n Vanderp lank (1963, 1968) proposed t h a t unnecessary v i r u l e n c e genes o f a pathogen would be s e l e c t e d a g a i n s t when c u l t u r e d on a s u s c e p t i b l e h o s t . Many peop le have attempted to v a l i d a t e o r i n v a l i d a t e V a n d e r p l a n k ' s ax ium. The r e s u l t s r e p o r t e d i n the l i t e r a t u r e are d i v i d e d and o f t e n c o n f l i c t i n g ev idence e x i s t s i n an i n d i v i d u a l r e p o r t . S tud i e s which are g e n e r a l l y r e p o r t e d a r e : (1) a re m ix tu re s t u d i e s where d i f f e r e n t i s o l a t e s compete on a s i n g l e h o s t ; (2) a re r e p o r t s o f genotype f r e q u e n c i e s o r f r e q u e n c i e s o f v i r u l e n c e gene combina t ions which are found i n n a t u r e . Ev idence t ha t hos t c u l t i v a r s can have a major r o l e on the e f f e c t s o f s e l e c t i o n o f a pathogen p o p u l a t i o n was observed i n U s t i l a g o avenae (Cherewick 1958, Ho l ton 1967) . Both papers gave ev idence o f d i r e c t i o n a l s e l e c t i o n f o r v i r u l e n c e by hos t c u l t i v a r s and had d a t a t h a t cou ld be i n t e r p r e t e d as s t a b i l i z i n g s e l e c t i o n sensu Vanderp l ank . With a l l the g e n e t i c a n a l y s i s p r e v i o u s l y done on U. h o r d e i and the a b i l i t y to i s o l a t e p roduc t s o f m e i o s i s , i t was proposed tha t the U. h o r d e i -b a r l e y system cou ld be an e x c e l l e n t system f o r pe r fo rm ing s e l e c t i o n expe r imen t s . The o r i g i n a l o b j e c t i v e o f the exper iment (Chapter 4 -s e l e c t i o n scheme) was to form a U. h o r d e i p o p u l a t i o n w i th 77 v i r u l e n c e and a v i r u l e n c e a l l e l e s i n equa l f r equency w i th r e s p e c t to a p a r t i c u l a r v i r u l e n c e gene. T h i s p o p u l a t i o n would be sub-d i v i ded and undergo s e l e c t i o n on a r e s i s t a n c e and s u s c e p t i b l e b a r l e y c u l t i v a r , r e s p e c t i v e l y . The changes i n a l l e l e f r equency would be de t e rm ined . M a t e r i a l s and Mathods I n o c u l a t i o n T r i a l s Smutted heads o f b a r l e y , which were p r e v i o u s l y i n o c u l a t e d w i th a genotype o f smut homozygous v i r u l e n t on c u l t i v a r T r e b i , were ground up us ing a Thomas-Wiley In termedia te m i l l . The spores were weighed w i th a M e t t l e r b a l a n c e , were p l a ced i n t o s t e r i l e Wheaton-dounce t i s s u e g r i n d e r w i th 5 mis o f 0.1% wa te r . aga r and were teased a p a r t . Four c o n c e n t r a t i o n s o f t e l i o s p o r e suspens ions were p r e p a r e d : 0.5 g / 1 , 1.0 g / 1 , 1.5 g/1 and 2.0 g / 1 . Haemocytometer counts were made o f each s u s p e n s i o n . Seed o f T r e b i was t r e a t e d as d e s c r i b e d by Groth and Person (1976) . Seeds were i n o c u l a t e d f o l l o w i n g the pa r t i a l - va cuum techn ique d e s c r i b e d by Tapke and Bever (1942) . I nocu l a ted seed f o r each t rea tment was p l a n t e d i n the f i e l d i n rows 6 m long a t UBC. There were th ree r e p l i c a t e s i n a random b l o c k d e s i g n . Data were r eco rded when a l l s p i k e s had emerged. The percentage o f smutted p l a n t s was based on a t l e a s t 100 p l a n t s . Format ion o f P o p u l a t i o n s Hap lo id s p o r i d a were ob ta ined from s i l i c a g e l , c u l t u r e d and prepared f o r i n o c u l a t i o n as d e s c r i b e d by Groth and Person (1976) . The a p p r o p r i a t e h a p l o i d 78 c u l t u r e s were mixed i n a v i a l c o n t a i n i n g seed o f the c u l t i v a r T r e b i , a modera te l y r e s i s t a n t c u l t i v a r , and i n another v i a l c o n t a i n i n g seed o f Odessa , a s u s c e p t i b l e c u l t i v a r . The i n o c u l a t i o n procedure was the same as p r e v i o u s l y used . Seed f o r each t rea tment was p l an t ed i n the f i e l d i n rows 3 m long a t UBC. There were th ree r e p l i c a t e s i n a s p l i t p l o t d e s i g n . Data were recorded as b e f o r e . S e l e c t i o n Exper iments F i f t e e n d i k a r y o n s were formed on both T r e b i and Odessa from the above . The r e s u l t i n g smutted heads were ground up as be fo re and each kept s e p a r a t e l y i n a i r t i g h t v i a l s . The f i f t e e n d i k a r y o n s were randomly a s s i gned to one o f th ree p o p u l a t i o n s per c u l t i v a r w i th f i v e d i k a r y o n s per p o p u l a t i o n . The ass ignment w i t h i n each p o p u l a t i o n was the same f o r each c u l t i v a r . Three c o n t r o l s were used i n t h i s expe r imen t . Cj_ and C 2 were p a r e n t a l smut geno t ypes , homozygous f o r v i r u l e n c e and a v i r u l e n c e on T r e b i , r e s p e c t i v e l y . C3 was i n o c u l a t e d w i th no smut but w i th on l y the 0.1% water aga r . T e l i o s p o r e s were we ighed, suspended, as b e f o r e , i n a c o n c e n t r a t i o n o f 1 g/1, and i n o c u l a t e d on both c u l t i v a r s . Seed f o r each t rea tment was p l an t ed i n a 3 m row wi th th ree r e p l i c a t e s i n a s p l i t p l o t f i e l d d e s i g n a t D a v i s , CA. Data were r eco rded as b e f o r e . A l l smutted heads were c o l l e c t e d . Inoculum f o r each o f the s i x p o p u l a t i o n s was taken from the c u l t i v a r on which i t was undergo ing s e l e c t i o n and the p rocedure was repea ted f o r a t o t a l o f th ree g e n e r a t i o n s . When da ta were c o l l e c t e d f o r the f i r s t g e n e r a t i o n o f s e l e c t i o n , a l l non-smutted heads were c o l l e c t e d from a randomly chosen row o f each p o p u l a t i o n per r e p l i c a t e . The heads were t h r e s h e d , the seed was d r i e d and then weighed on a M e t t l e r b a l a n c e . The seed was a l s o counted on an e l e c t r o n i c seed coun t e r (Model 850-2 The Old M i l l C o . ) . The we ights o r t o t a l number o f seed was ad jus t ed f o r the number o f p l a n t s f o r t h a t p a r t i c u l a r row. T h e r e f o r e , a l l d a t a are based on the average weight o r number o f seeds per p l a n t . Comparisons were made among the p o p u l a t i o n s and the u n i n o c u l a t e d c o n t r o l . For one randomly chosen r e p l i c a t e o f the s e l e c t i o n exper iment o f g e n e r a t i o n 1, the t o t a l number o f t i l l e r s and the number o f smutted t i l l e r s were reco rded f o r each p l a n t w i t h i n the th ree p o p u l a t i o n s . T o t a l s o f 406, 490, 625 and 520 p l a n t s were examined f o r P o p u l a t i o n I on T r e b i , I on Odessa , I I I on T r e b i and I I I on Odessa , r e s p e c t i v e l y . Random s p o r i d i a were o b t a i n e d from the t e l i o s p o r e s t h a t were not used f o r i nocu lum. These s p o r i d i a were i s o l a t e d by one o f two methods: (1) random s p o r i d i a were i s o l a t e d us i ng a de Fonbrune m i c roman ipu l a t o r as the p rocedures d e s c r i b e d by Groth et_ j a l . (1976) ; (2) s p o r i d i a were i s o l a t e d by a p l a t i n g t e c h n i q u e . For the p l a t i n g t echn ique 0.1 ml a l i q u o t s o f the t e l i o s p o r e suspens ions were t r a n s f e r r e d to p e t r i p l a t e s c o n t a i n i n g V o g e l ' s Complete medium. With the a i d o f a s p r e a d e r , the t e l i o s p o r e s were e ven l y d i s t r i b u t e d ove r the s u r f a c e o f the aga r . These p l a t e s were i ncuba ted f o r 24 h r s . 80 a t 2 2 ° C . A f t e r i n c u b a t i o n , 1 ml o f s t e r i l e d i s t i l l e d water was p l a c e d on the s u r f a c e o f the agar and s w i r l e d to l oosen s p o r i d i a from the ge rm ina t i ng t e l i o s p o r e s . The s p o r i d i a l suspens ion was drawn o f f w i th a s t e r i l e Pas teur p i p e t t e and p l a ced i n a s t e r i l e t e s t t ube . The c o n c e n t r a t i o n o f the suspens ion was ad jus t ed to 1 x 1 0 3 s p o r i d i a pe r ml and 0.1 ml was p l a ced on V o g e l ' s complete agar p l a t e s . In 3 - 4 days v i s i b l e c o l o n i e s were obse r ved . I s o l a t e d c o l o n i e s were immedia te ly t r a n s f e r r e d to f r e s h agar p l a t e s us ing s t e r i l e t o o t h p i c k s . The mat ing t ypes o f the random s p o r i d i a were determined by the Bauch t e s t . F i f t e e n s p o r i d i a / m a t i n g t y p e / p o p u l a t i o n were then p a i r e d w i th the a p p r o p r i a t e t e s t c u l t u r e , V-A-2 + and e i t h e r V - A - l " o r V - A - 3 " , (see Chapter 7 ) , and used to i n o c u l a t e seed o f T r e b i as d e s c r i b e d p r e v i o u s l y . The seed per t rea tment was p l a n t e d i n 3 m rows w i th th ree r e p l i c a t e s i n a random b l o c k d e s i g n a t UBC. The da t a were r eco rded as be fo re and used i n de t e rm in ing the phenotype o f each random s p o r i d i a . R e s u l t s I n o c u l a t i o n T r i a l s The f o u r c o n c e n t r a t i o n s o f t e l i o s p o r e suspens ions y i e l d e d an average o f 32%, 34%, 41% and 41% f o r 2.0 g / 1 , 1.5 g / 1 , 1.0 g/1 and 0.5 g/1 , r e s p e c t i v e l y . A n a l y s i s o f v a r i a n c e i n d i c a t e d the re were no s i g n i f i c a n t d i f f e r e n c e s among the t rea tments o r w i t h i n the t rea tments (Tab le 5 .1 ) . The o r i g i n a l d i k a r y o n tha t produced the t e l i o s p o r e s used i n t h i s 81 Tab l e 5.1 Comparison o f pe r c en t smutted p l a n t s o f b a r l e y c u l t i v a r T r e b i produced by us ing 4 c o n c e n t r a t i o n s o f t e l i o s p o r e suspens ion o f U s t i l a g o h o r d e i Source o f V a r i a t i o n d f SS MS F-va lue a C o n c e n t r a t i o n s R e p l i c a t e s E r r o r 3 2 6 188.88 151.76 331.04 62.96 75.88 55.17 1.14 NS 1.38 NS T o t a l 11 671.68 a . NS ='not s i g n i f i c a n t Tab l e 5.2 Comparison o f pe r c en t smutted p l a n t s o f 15 U s t i l a g o h o r d e i d i k a r y o n s produced on b a r l e y c u l t i v a r s T r e b i and Odessa P o p u l a t i o n and % smutted p l a n t s d i k a r yon T r e b i Odessa I- A 20 22 B 12 14 C 15 48 D 32 37 E 15 49 x = 19 34 II- A 12 13 B 4 60 C 20 6 D 25 8 E 19 36 x = 16 25 III-A 19 14 B 34 38 C 35 32 D 41 51 E 29 44 x = 32 36 82 exper iment was r e p o r t e d to produce an average o f 40% smutted p l a n t s . Format ion o f P o p u l a t i o n s The averages o f the f i v e d i k a r y o n s f o r p o p u l a t i o n s I, II and I I I on T r e b i were 19%, 16% and 32%, r e s p e c t i v e l y . The average va lue f o r the i n d i v i d u a l d i k a r y o n s i s g i v e n i n Tab le 5 .2 . S e l e c t i o n Exper iments P o p u l a t i o n I and I I I showed i n c r e a s e s i n the percentage o f smutted p l a n t s whether on T r e b i o r Odessa a f t e r one g e n e r a t i o n o f s e l e c t i o n (Tab le 5 .3 ) . Over the next 2 g e n e r a t i o n s p o p u l a t i o n I s tayed cons t an t a t 28% when s e l e c t e d on T r e b i ; whereas , when s e l e c t e d on Odessa the re were random f l u c t u a t i o n s i n the l e v e l o f smut. A f t e r g e n e r a t i o n one , p o p u l a t i o n I I I a l s o dec reased i n l e v e l s o f smut on both c u l t i v a r s . P o p u l a t i o n II d e c l i n e d i n pe r cen t smutted p l a n t s u n t i l the re was no smut i n g e n e r a t i o n 3. C o n t r o l s C^ and C 2 gave f a i r l y c o n s i s t e n t l e v e l s o f smut. The u n i n o c u l a t e d c o n t r o l , C-j, never had any smut. P o p u l a t i o n I when s e l e c t e d on T r e b i (Tab le 5.3) gave s i m i l a r l e v e l s o f smut to the l e v e l s observed when the p o p u l a t i o n was s i m u l t a n e o u s l y i n o c u l a t e d on Odessa (Tab le 5 .4 ) . P o p u l a t i o n I s e l e c t e d on Odessa but s i m u l t a n e o u s l y i n o c u l a t e d on T r e b i (Tab le 5.4) gave lower l e v e l s o f smut than when s e l e c t e d on T r e b i (Tab le 5 .3 ) . P o p u l a t i o n II I s e l e c t e d on T r e b i and i n o c u l a t e d on T r e b i (Tab le 5.3) gave v e r y s i m i l a r l e v e l s o f smut when i n o c u l a t e d on Odessa (Tab le 5 .4 ) . P o p u l a t i o n II I s e l e c t e d on Odessa but i n o c u l a t e d on T r e b i Tab l e 5.3 Comparison o f pe r c en t smutted p l a n t s produced when th ree heterogeneous U s t i l a g o h o r d e i p o p u l a t i o n s and th ree c o n t r o l s were s e l e c t e d on P o p u l a t i o n two b a r l e y c u l t i v a r s C u l t i v a r s S e l e c t e d on , T r e b i and Odessa % smutted p l a n t s Gene ra t i ons 0 1 2 i n 3 I T r e b i 19 31 28 28 Odessa 34 42 29 38 II T r e b i 16 2 2 0 Odessa 25 2 1 0 II I T r e b i 32 66 23 20 Odessa 36 55 25 34 c l T r e b i 18 15 20 20 X Odessa 31 16 33 33 T r e b i 0 0 0 0 Odessa 6 9 — — C 3 T r e b i 0 0 0 0 Odessa 0 0 0 0 84 Tab l e 5.4 Comparison o f pe r c en t smutted p l a n t s produced when th ree heterogeneous U s t i l a g o h o r d e i p o p u l a t i o n s were s e l e c t e d on one c u l t i v a r but i n o c u l a t e d onto another c u l t i v a r o f b a r l e y % smutted p l a n t s C u l t i v a r s C u l t i v a r i n g e n e r a t i o n P o p u l a t i o n s e l e c t e d on i n o c u l a t e d 2 3 I T r e b i Odessa 29 29 I Odessa T r e b i 21 23 II T r e b i Odessa 1 0 II Odessa T r e b i 4 0 II I T r e b i Odessa 28 24 I I I Odessa T r e b i 26 20 85 (Tab le 5.4) gave s i m i l a r l e v e l s to the s u b p o p u l a t i o n s e l e c t e d on T r e b i (Tab le 5 .3 ) . Compar isons were made o f average seed p r o d u c t i o n pe r p l a n t produced w i t h i n the th ree heterogeneous p o p u l a t i o n s t o the c o n t r o l w i th no smut. The a n a l y s i s o f v a r i a n c e showed t h a t the re was no s i g n i f i c a n t d i f f e r e n c e f o r the average seed weight o r seed number per p l a n t among the f o u r t rea tments on the c u l t i v a r T r e b i o r Odessa (Tab les 5.5 and 5 .6 ) . For eve ry a n a l y s i s , r e p l i c a t e s were s i g n i f i c a n t . F i g u r e s 5.1 and 5.2 graph the f requency o f p l a n t s w i th which v a r y i n g l e v e l s o f i n f e c t e d t i l l e r s / p l a n t were observed f o r P o p u l a t i o n I on T r e b i and Odessa , and I I I on T r e b i and Odessa , r e s p e c t i v e l y . For both p o p u l a t i o n s , the major d i f f e r e n c e i n the f r equency d i s t r i b u t i o n was the d i f f e r e n c e i n non-smutted p l a n t s . For p o p u l a t i o n I I I on T r e b i , i t appears t ha t the p r o b a b i l i t y o f g e t t i n g one t i l l e r smutted i s equa l to any o t h e r comb ina t ion o f smutted t i l l e r s , but on Odessa , t he re i s a h i g h e r p r o b a b i l i t y o f g e t t i n g one o r two t i l l e r s smut ted . The a l l e l e f requency exper iments were per formed on the i n i t i a l p o p u l a t i o n s (Gen n ) and a f t e r the f i r s t g e n e r a t i o n o f s e l e c t i o n . The f r equency o f the v i r u l e n c e a l l e l e phenotype f o r a l l p o p u l a t i o n s on the 2 c u l t i v a r s i s g i v e n i n Tab le 5 .7 . For the 3 heterogeneous p o p u l a t i o n s the re were i n c r e a s e s i n the v i r u l e n c e phenotype on e i t h e r c u l t i v a r . The a l l e l e f r equency t e s t was repea ted f o r o n l y the i n i t i a l g e n e r a t i o n . Us ing the same means o f d i s t i n g u i s h i n g between v i r u l e n t and a v i r u l e n t 86 Tab l e 5.5 Summary o f a n a l y s i s o f v a r i a n c e o f seed weight and number o f b a r l e y c u l t i v a r T r e b i i n o c u l a t e d w i th th ree heterogeneous U s t i l a g o h o r d e i p o p u l a t i o n s and one c o n t r o l T o t a l Seed Weight Seed Number Source o f v a r i a t i o n d . f . Mean square a F-value Mean square F - va l ue a P o p u l a t i o n s 3 2.85 1.02 NS 5327.7 1.8 NS R e p l i c a t e s 2 20.92 7.47+ 22542.0 7.7 + E r r o r 6 2.80 2927.9 a NS = not s i g n i f i c a n t * = s i g n i f i c a n t , P = 0.05 Tab l e 5.6 Summary o f a n a l y s i s o f v a r i a n c e o f seed weight and number o f b a r l e y c u l t i v a r Odessa i n o c u l a t e d w i th th ree heterogeneous U s t i l a g o h o r d e i p o p u l a t i o n s and one c o n t r o l Seed Weight T o t a l Seed Number Source o f v a r i a t i o n d . f . Mean square a F-value Mean square F - v a l u e a P o p u l a t i o n s 3 0.06 1.6 NS 276.3 0.8 NS R e p l i c a t e s 2 2.09 50.1 * * 2333.8 7.1* E r r o r 6 0.042 329.5 a NS = not s i g n i f i c a n t * s i g n i f i c a n t , P = 0.05 * * = s i g n i f i c a n t , P = 0.01 87 F i g u r e 5.1 Comparison o f the f requency o f number o f smutted t i l l e r s per p l a n t when i n o c u l a t e d w i th P o p u l a t i o n I o f U s t i l a g o h o r d e i between two b a r l e y c u l t i v a r s Odessa and T r e b i . 88 0.801 0.60 Odessa Trebi o S g. 0.40 o 0.20 Number of infected tillers per plant F i g u r e 5.2 Comparison o f the f requency o f number o f smutted t i l l e r s per p l a n t when i n o c u l a t e d w i th P o p u l a t i o n I I I o f U s t i l a g o h o r d e i between two b a r l e y c u l t i v a r s Odessa and T r e b i . 89 Comparison o f v i r u l e n c e a l l e l e phenotype f requency f o r the th ree heterogeneous and two c o n t r o l U s t i l a g o h o r d e i p o p u l a t i o n s on b a r l e y c u l t i v a r T r e b i and Odessa V i r u l e n c e a l l e l e phenotype C u l t i v a r f r equency i n g e n e r a t i o n s e l e c t e d on 0 1 T r e b i 0.40 0.70 Odessa 0.47 0.85 T r e b i 0.46 0.42 Odessa 0.53 0.72 T r e b i 0.33 0.80 Odessa 0.24 0.84 T r e b i 1.00 Odessa 1.00 T r e b i Odessa 0.10 0.17 90 Tab l e 5.8 Comparison o f v i r u l e n c e a l l e l e f r equency f o r the th ree heterogeneous and two c o n t r o l U s t i l a g o  h o r d e i p o p u l a t i o n s on the c u l t i v a r s T r e b i and Odessa t e s t e d i n two yea r s V i r u l e n c e a l l e l e phenotype C u l t i v a r f r equency i n g e n e r a t i o n 0 P o p u l a t i o n s e l e c t e d on 1982 1983 I T r e b i 0.40 0.57 Odessa 0.47 0.60 II T r e b i 0.46 0.53 Odessa 0.53 0.70 II I T r e b i 0.33 0.53 Odessa 0.24 0.73 C-, T r e b i 1.00 1.0 Odessa 1.00 1.0 C T r e b i 0.10 0.36 Odessa 0.17 0.32 91 phenotypes as f o r the f i r s t t e s t , t he re was an i n c r e a s e i n the v i r u l e n t t ypes observed f o r a l l p o p u l a t i o n s (Tab le 5 .8 ) . D i s c u s s i o n Tapke (1935) r e p o r t e d us ing a t e l i o s p o r e suspens ion o f 1 g/1 . Because the i n o c u l a t i o n t r i a l produced 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 d i f f e r e n c e s among the f o u r c o n c e n t r a t i o n s t e s t e d , 1 g/1 was used as the s tandard c o n c e n t r a t i o n f o r a l l the f u t u r e t e l i o s p o r e i n o c u l a t i o n s . I t i s p o s s i b l e t ha t when a c e r t a i n inoculum c o n c e n t r a t i o n i s r e a c h e d , the re i s no room on the seed s u r f a c e f o r any more t e l i o s p o r e s to ge rm ina t e ; t h e r e f o r e add ing more t e l i o s p o r e s does not a l t e r the observed percentage o f smutted p l a n t s . The p r o b a b i l i t y o f s t a t i s t i c a l l y a l t e r i n g the l e v e l o f smut observed i s not a l t e r e d by i n c r e a s i n g the t e l i o s p o r e c o n c e n t r a t i o n beyond a c e r t a i n p o i n t and the c o n c e n t r a t i o n s t e s t e d may a l l have been over t h i s t h r e s h o l d . Three p o p u l a t i o n s were formed to r e p r e s e n t r e p l i c a t e s o f the F 2 p o p u l a t i o n over the g e n e r a t i o n s o f s e l e c t i o n . F i v e d i k a r y o n s were i n c o r p o r a t e d i n each p o p u l a t i o n t o g i v e enough t e l i o s p o r e s to be used f o r the f u t u r e i n o c u l a t i o n . There was c o n s i d e r a b l e v a r i a t i o n o f pe r c en t smutted p l a n t s produced from i n d i v i d u a l d i k a r y o n s w i t h i n each o f the p o p u l a t i o n s (Tab le 5 .2 ) . T h i s v a r i a b i l i t y shou ld a l l o w f o r change to o c cu r by s e l e c t i o n . S e ve r a l o f the d i k a r y o n s produced no s i g n i f i c a n t d i f f e r i n g percentage o f smutted p l a n t s on T r e b i o r Odessa , r e s i s t a n t and s u s c e p t i b l e c u l t i v a r s , r e s p e c t i v e l y . 92 Because the p o p u l a t i o n s were he te rozygous f o r a major v i r u l e n c e gene and were maximal l y heterogeneous f o r p o l y g e n e s , changes i n average l e v e l o f smut over the g e n e r a t i o n s were e x p e c t e d . These changes would be a consequence o f m e i o t i c r eassor tment and s e l e c t i o n o f the most s u c c e s s f u l d i k a r y o n s . I t was expected tha t the more s u c c e s s f u l d i k a r y o n s t ha t were s e l e c t e d may have i n c r e a s e d r e p r o d u c t i v i t y ove r the p a r e n t a l d i k a r y o n s . Random changes i n the l e v e l o f smut over the g e n e r a t i o n s cou ld be consequences o f v a r i n g geno t ypes , env i ronmenta l f a c t o r s , o r the i n t e r a c t i o n o f the two. Because o f the c o n t r o l geno t ypes , and C2» d i d not show any i n c r e a s e d o r major changes , env i ronmenta l d i f f e r e n c e s a lone were not the major f a c t o r r e s p o n s i b l e f o r the changes observed i n the th ree p o p u l a t i o n s . The average pe r cen t smut f o r p o p u l a t i o n I I I a f t e r one g e n e r a t i o n o f s e l e c t i o n was g r e a t e r than the amounts produced p r e v i o u s l y by the i n d i v i d u a l d i k a r y o n s composing p o p u l a t i o n I I I . The i n c r e a s e cou ld be the r e s u l t o f the a p p r o p r i a t e complement o f po lygenes a s s o c i a t e d w i th the v i r u l e n c e a l l e l e . P o p u l a t i o n II d e c l i n e d d r a s t i c a l l y i n smutted p l a n t s which was a t t r i b u t e d to abnormal g e r m i n a t i o n o f the t e l i o s p o r e s (Chapter 6 ) . An o b j e c t i v e o f t h i s exper iment was to s e l e c t d i v e r g i n g s u b p o p u l a t i o n s w i t h i n each p o p u l a t i o n , one w i th h igh l e v e l s o f smut and the o the r w i th low l e v e l s . T h i s was not ach ieved because each p o p u l a t i o n as ex t r eme l y heterogeneous i n both the l e v e l o f smut and the type o f smut t ing ( comple te , p a r t i a l o r b a s a l ) . A l a r g e amount o f v a r i a t i o n f o r the number o f t i l l e r s smutted and the type o f smut t ing e x i s t e d w i t h i n s i n g l e p l a n t s . In g e n e r a l more v a r i a t i o n was observed on T r e b i than on Odessa . Because o f the d i f f i c u l t y i n s o r t i n g out a l l o f these v a r i a b l e s and i n de t e rm in ing how the genotype o f the i n f e c t i n g d i k a r y o n c o n t r i b u t e s to each v a r i a b l e , o n l y t e l i o s p o r e s from h e a v i l y smutted p l a n t s were used f o r the f u t u r e g e n e r a t i o n ' s i nocu lum. These t e l i o s p o r e s were t o r e p r e s e n t the h igh s u b p o p u l a t i o n . S e l e c t i o n f o r the o t h e r s u b p o p u l a t i o n was abandoned. There are s e v e r a l r e p o r t s demons t ra t ing a d a p t a t i o n o f pathogen i s o l a t e s to a c u l t i v a r a f t e r s e v e r a l g e n e r a i t o n s o f c u l t u r i n g on tha t c u l t i v a r (Caten 1974, C l i f f o r d and C l o t h i e r 1974, Habgood 1976, James and Fry 1983, J e f f r e y e_t a l . 1962, J i n k s and G r i n d l e 1963) . The p o p u l a t i o n s s t u d i e d here cou ld p r o v i d e ev idence f o r hos t a d a p t a t i o n . P o p u l a t i o n I o r I I I when s e l e c t e d on T r e b i gave s i m i l a r l e v e l s o f smut when i n o c u l a t e d on e i t h e r T r e b i o r Odessa (Tab le 5.3 and 5 .4 ) . C o n t r a s t i n g to t h i s i s when p o p u l a t i o n I o r I I I were s e l e c t e d on Odessa ; then Odessa tended to have h i g h e r l e v e l s o f smut than T r e b i . I t appears t ha t genotypes s e l e c t e d on Odessa were adap t ing f o r a h i g h e r p a r a s i t i c a b i l i t i y s p e c i f i c to Odessa and t h e r e f o r e , tended to produce l e s s smut on T r e b i . P o p u l a t i o n I s e l e c t e d and i n o c u l a t e d on T r e b i gave s l i g h t l y h i g h e r l e v e l s o f smut compared to p o p u l a t i o n I s e l e c t e d on Odessa but i n o c u l a t e d on T r e b i . In o rde r to observe hos t a d a p t a t i o n these exper iments would need to con t i nue f o r s e v e r a l g e n e r a t i o n s . When d e a l i n g w i th a pathogen such as smut, which r e p l a c e s the k e r n a l w i th s p o r e s , i t i s expected t h a t the number o f h e a l t h y seeds i n a smutted hos t p o p u l a t i o n i s reduced compared to a non-smutted p o p u l a t i o n . Host r e p r o d u c t i v i t y shou ld be an impor tant measurement to determine the e f f e c t s o f the smut i n f e c t i o n . Comparisons were made between a smutted and non-smutted hos t p o p u l a t i o n f o r t o t a l seed weight and t o t a l seed number. No s i g n i f i c a n t d i f f e r e n c e was found . T h i s was unexpected because the re were h i g h l e v e l s o f smut t ing i n g e n e r a t i o n I f o r which the compar iosn was made. A p o s s i b l e e x p l a n a t i o n i s t ha t the smutted p l a n t s produced more t i l l e r s than the non-smutted p l a n t s . T h i s i s o p p o s i t e o f a p r e v i o u s r e p o r t f o r U. h o r d e i (Groth and Person 1978) . S t i m u l a t i o n o f the t i l l e r i n g p r o c e s s o f the hos t was r e p o r t e d f o r the T i l l e t i a s p e c i e s (Purdy e_t a_l, 186 3 ) . U n f o r t u n a t e l y , i n these exper iments the t i l l e r counts were not r ecorded f o r the u n i n o c u l a t e d c o n t r o l . Future exper iments are needed to c l a r i f y the p o i n t o f no d i f f e r e n c e s between the smutted and non-smutted p o p u l a t i o n s . In these e x p e r i m e n t s , r e p l i c a t e s were s i g n i f i c a n t l y d i f f e r e n t . I t was noted tha t one r e p l i c a t e was under ex t reme ly wet c o n d i t i o n s as compared t o the o the r two. T h i s cou ld account f o r the v a r i a b l i l i t y among r e p l i c a t e s . Another way o f o b s e r v i n g d i f f e r e n c e s o f d i s e a s e l e v e l between a r e s i s t a n t and s u s c e p t i b l e c u l t i v a r i s by comparing the f r equency d i s t r i b u t i o n o f number o f smutted t i l l e r s / p l a n t . Gro th e t . a l (1976) demonstrated t h a t the re was a s t r ong c o r r e -95 l a t i o n between the p r o p o r t i o n o f smutted p l a n t s and the p r o p o r -t i o n o f smutted heads on smutted p l a n t s . T h i s was based on examining smutted p l a n t s o n l y . He suggested t ha t the re were genes which c o n d i t i o n e d each o f these d i s e a s e r e a c t i o n s . Us ing da t a from o n l y the smutted p l a n t s f o r p o p u l a t i o n I and I I I , no c o r r e l a t i o n between the two d i s e a s e r e a c t i o n s was obse r ved . In o rde r to o b t a i n an a c cu r a t e account both d i s e a s e r e a c t i o n s are needed. Data o f those k inds c o u l d produce much more comprehen-s i v e i n f o r m a t i o n o f r e p r o d u c t i v i t y o f the pathogen and the v a r i a b i l i t y tha t might e x i s t w i t h i n the pathogen p o p u l a t i o n . F i f t e e n s p o r i d i a o f each mat ing type per p o p u l a t i o n were mated to an a p p r o p r i a t e s p o r i d i u m , i d e n t i f i e d as c a r r y i n g the r e c e s s i v e a l l e l e f o r a v i r u l e n c e , i n o c u l a t e d onto T r e b i and r eco rded as v i r u l e n t o r a v i r u l e n t . A v i r u l e n t phenotype was any th ing g r e a t e r than 10% smutted p l a n t s . S ince the i n i t i a l smut p o p u l a t i o n s were a l l from he te rozyous d i k a r y o n s , i t was expected t ha t the v i r u l e n t phenotype shou ld be expressed a t the f r equency o f 0 . 5 0 . D e v i a t i o n s from 0.50 f o r the th ree he te rozyous p o p u l a t i o n s were observed (Tab le 5.7) and can be e x p l a i n e d by sampl ing e r r o r . The p o p u l a t i o n was a p a r e n t a l p o p u l a t i o n t ha t was expected to be homozygous v i r u l e n t . A l l s p o r i d i a t e s t e d from C-^  were o f the v i r u l e n t phenotype . The C 2 p o p u l a t i o n was another p a r e n t a l p o p u l a t i o n t ha t was expected to be homozygous r e c e s s i v e but a few s p o r i d i a were c l a s s i f i e d as be ing v i r u l e n t . S ince i t was demonstrated t ha t the re were po lygenes which m o d i f i e d the e x p r e s s i o n o f the 96 major gene (Person e_t a_l. 1982, 1983) , the v i r u l e n t s p o r i d i a observed i n the C 2 p o p u l a t i o n c o u l d be the r e s u l t o f the po lygenes r a t h e r than the v i r u l e n c e a l l e l e o f the major gene. I f t h i s i s the c a s e , then i t would not be p o s s i b l e to a c c u r a t e l y i d e n t i f y the v i r u l e n c e a l l e l e . The f r e q u e n c i e s observed cou ld be a r e f l e c t i o n o f e i t h e r the major v i r u l e n c e a l l e l e o r the a p p r o p r i a t e complement o f p o l y g e n e s . Inc reases were observed i n the f requency o f the v i r u l e n t s p o r i d i a a f t e r 1 g e n e r a t i o n o f s e l e c t i o n r e g a r d l e s s o f which c u l t i v a r on which s e l e c t i o n took p l a c e . T h i s s e l e c t i o n exper iment was o r i g i n a l l y des igned to observe changes i n a l l e l e f r equency o f a major gene when s e l e c t i o n took p l a c e on a r e s i s t a n t v e r sus s u s c e p t i b l e c u l t i v a r . T h i s o b j e c t i v e was not r e a l i z e d because o f the c o m p l i c a t i o n o f the e f f e c t s o f the po lygenes which modi fy v i r u l e n c e . Be fo re o the r s e l e c t i o n exper iments are d e s i g n e d , the v a r i a b i l i t y i n smut l e v e l s and type o f smutt ing must be b e t t e r q u a n t i f i e d and r e l a t e d to the a c t i o n o f e i t h e r the po l ygenes o r the v i r u l e n c e gene . The number i n which the po l ygenes modi fy the e f f e c t s o f the major gene (such as number o r smutted t i l l e r s per p l a n t o r number o f t e l i o s p o r e s per p l a n t and o r sp i ke ) must be i d e n t i f i e d . The e f f e c t s o f smut i n f e c t i o n on the hos t r e p r o d u c t i v i t y must be s o r t e d o u t . 97 CHAPTER 6 LOWER DISEASE LEVELS CORRELATED WITH ABNORMAL GERMINATION OF USTILAGO HORDEI TELIOSPORES I n t r o d u c t i o n A l a r g e amount o f v a r i a t i o n f o r m o r p h o l o g i c a l and p h y s i o l o g i c a l a spec t s o f t e l i o s p o r e g e r m i n a t i o n e x i s t s w i t h i n the smuts (Ho l ton e t a l . 1968) . Spore g e r m i n a t i o n i s not on l y fundamental f o r c l a s s i f i c a t i o n but i s a p r e r e q u i s i t e to i n f e c t i o n and t h e r e f o r e , has i m p l i c a t i o n s conce rn ing the success as a pa thogen . Ge rmina t ion o f a p o p u l a t i o n o f U s t i l a g o h o r d e i t e l i o s p o r e s was observed to be a t y p i c a l and to have reduced s p o r i d i a l f o rma t i on from norma l . I t was s p e c u l a t e d t h a t the abnormal g e r m i n a t i o n might account f o r reduced l e v e l s o f smutted p l a n t s (observed i n Chapter 5 ) . Exper iments were s e t up to determine i f the ge rm ina t i on o f the one t e l i o s p o r e p o p u l a t i o n was d i f f e r e n t from o t h e r p o p u l a t i o n s , to p o s s i b l y f i n d an e x p l a n a t i o n f o r the a t y p i c a l g e r m i n a t i o n p a t t e r n , and t o observe the r e l a t i o n s h i p between ge rm ina t i on r a t e s and pe r cen t smutted p l a n t s . M a t e r i a l s and Methods The th ree t e l i o s p o r e p o p u l a t i o n s under examina t ion were those ment ioned i n Chapter 5. A " p i n c h " o f t e l i o s p o r e s from each o f the th ree p o p u l a t i o n s was p l a c e d i n a t e s t tube w i th a few drops o f s t e r i l e d i s t i l l e d water . The spores were teased apa r t w i th a s t e r i l e g l a s s r o d . Then 10 mis o f s t e r i l e 98 d i s t i l l e d water were added to each t u b e , which was the s t i r r e d w i th a Vor tex m ixe r . The c o n c e n t r a t i o n o f each suspens ion was a d j u s t e d to 1 x 10^ spores/ml and 0.1 ml o f each suspens ion was s e p a r a t e l y p l a ced on agar p l a t e s . There were th ree r e p l i c a t e s / t r e a t m e n t f o r eve ry 5 hour t ime i n t e r v a l s t a r t i n g a t 5 hours c o n t i n u i n g to 30 h o u r s . For each t r ea tmen t , the number o f t e l i o s p o r e s p roduc ing s p o r i d i a , p roduc ing promycel ium o n l y , and non-germinat ing t e l i o s p o r e s were recorded f o r 10 random mic roscope f i e l d s a t 400X. Genera l o b s e r v a t i o n s were a l s o r e c o r d e d . Pe rcen t g e r m i n a t i o n ( s p o r i d i a produced) was o b t a i n e d by ave rag ing the th ree r e p l i c a t e s . Pe rcent ge rm ina t i on was graphed over t ime . Temperature s t u d i e s T e l i o s p o r e s o f the th ree p o p u l a t i o n s were ob ta ined from the c u l t i v a r Odessa . Spore suspens ions were p l a t e d onto V o g e l ' s complete medium and incuba ted a t 4 ° , 1 5 ° , 22° and 35 °C f o r 25 h r s . N u t r i t i o n a l s t u d i e s T e l i o s p o r e s o f the th ree p o p u l a t i o n s were ob t a i ned from the c u l t i v a r T r e b i . Spore suspens ions were p l a t e d onto V o g e l ' s complete and m i n i m a l , Po ta to Dextrose Agar (PDA) and water aga r . Spore suspens ions ob t a i ned from Odessa were p l a t e d onto PDA, and water agar c o n t a i n i n g e x t r a c t from ge rm ina t i ng seeds o f c u l t i v a r T r e b i o r Odessa s eeds . Seeds were germinated by p l a c i n g 50 g o f seed mois tened w i th d i s t i l l e d water i n a weigh boat and i n c u b a t i n g a t 20 °C f o r 48 h o u r s . Germinated seeds were b lended i n an O s t e r i z e r w i th 100 mis o f d i s t i l l e d water , s t r a i n e d through cheese c l o t h and 99 f i l t e r e d through a m i l l i p o r e f i l t e r (.22 u ) . S i x t y mis o f e x t r a c t were added to 540 mis o f water and 20 g agar to produce the seed e x t r a c t aga r . A l l the p l a t e s were incuba ted a t 22 °C f o r 20 h r s . P o p u l a t i o n s t u d i e s T e l i o s p o r e s formed f o r the th ree p o p u l a t i o n s a f t e r a g e n e r a t i o n o f s e l e c t i o n on the c u l t i v a r T r e b i , t e l i o s p o r e s o f the f i v e i n d i v i d u a l d i k a r y o n s which formed each p o p u l a t i o n , and t e l i o p s o r e s o f and C 2 were p l a t e d onto PDA and incuba ted a t 2 2 ° C . T e l i o s p o r e s o f each d i k a r y o n were d i r e c t l y i n o c u l a t e d onto seed o f T r e b i and Odessa f o l l o w i n g the p rocedure i n Chapter 4. Pe rcent smutted p l a n t s was r eco rded as d e s c r i b e d i n Chapter 4. Phenotype s t u d i e s Smutted heads from a s i n g l e genotype were randomly s e l e c t e d from the p o p u l a t i o n s to r e p r e s e n t the phenotypes p a r t i a l l y o r comp l e t e l y smut ted . Two genotypes r ep r e sen t ed each phenotype. The t e l i o s p o r e s o f each were p l a t e d onto PDA and incuba ted a t 22 °C f o r 20 h r s . The p rocedure o f d i r e c t i n o c u l a t i o n was used to i n o c u l a t e seed o f T r e b i and Odessa f o r each o f the genotypes s e l e c t e d . Pe rcen t smutted p l a n t s was r eco rded as d e s c r i b e d i n Chapter 4. R e s u l t s Temperature s t u d i e s Each o f the p o p u l a t i o n s had i t s o p t i m a l pe r cen t g e r m i n a t i o n a t 22 °C ( F i g . 6 . 1 ) . Ge rmina t ion r a t e s a t 15° and 35 °C were s i m i l a r , p a r t i c u l a r l y f o r I and I I I , but were s lower than a t 2 2 ° C . The same average pe r cen t o f g e r m i n a t i o n was reached by 25 hours f o r I and I I I , r e g a r d l e s s 100 Time (hrs.) F i g u r e 6.1 Pe rcent spore g e r m i n a t i o n o f P o p u l a t i o n s I, I I , and I I I o f U s t i l a g o h o r d e i ove r th ree temperature r eg imes . 101 o f tempera ture . No ge rm ina t i on ( s p o r i d i a l p roduc t i on ) occu r r ed w i t h i n 48 hours a t 4 °C f o r any p o p u l a t i o n . P o p u l a t i o n II had ve r y l i t t l e g e r m i n a t i o n a t any tempera tu re , had fewer t e l i o -spores p roduc ing s p o r i d i a , and many t e l i o s p o r e s d i d not produce a l l f o u r s p o r i d i a . The s p o r i d i a were g e n e r a l l y l a r g e r than n o r m a l , and when i s o l a t e d , they were s low g row ing . The promycel ium o f many t e l i o s p o r e s was u s u a l l y l o n g e r than normal and o f t e n would d i s i n t e g r a t e . N u t r i t i o n a l s t u d i e s P o p u l a t i o n I and I I I had s i m i l a r g e r m i n a t i o n p a t t e r n s on V o g e l ' s comp le t e , min imal and PDA w i th I r e a c h i n g 73%, 75% and 70% r e s p e c t i v e l y , and I I I r e a c h i n g 66%, 68% and 68% r e s p e c t i v e l y ( F i g . 6 . 2 ) . II had v e r y l i t t l e g e r m i n a t i o n r e g a r d l e s s o f media . Water agar suppor ted ve r y l i t t l e s p o r i d i a l p r o d u c t i o n f o r a l l p o p u l a t i o n s . S i m i l a r g e r m i n a t i o n p a t t e r n s to PDA were observed when seed e x t r a c t agar was t e s t e d ( F i g . 6 . 3 ) . P o p u l a t i o n s t u d i e s The g e r m i n a t i o n p a t t e r n s observed f o r the t e l i o s p o r e s produced a f t e r one g e n e r a t i o n o f s e l e c t i o n on T r e b i gave ve r y s i m i l i a r p a t t e r n s to those p r e v i o u s l y observed ( F i g . 6 . 4 ) . P o p u l a t i o n II d i d have s l i g h t l y b e t t e r g e r m i n a t i o n . Ge rmina t i on p a t t e r n s o f the p a r e n t a l p o p u l a t i o n s , and C 2 » were obse r ved . Both C-^  and C 2 had l e s s than 40% g e r m i n a t i o n a f t e r 15 hours o f i n c u b a t i o n ( F i g . 6 . 5 ) . Germina -t i o n o f C, and C 0 was l e s s than the F- p o p u l a t i o n I. 102 Time (hrs.) Tims (hrs.) F i g u r e 6.2 Percent spore g e r m i n a t i o n o f P o p u l a t i o n s I, I I , and I I I o f U s t i l a g o h o r d e i on V o g e l ' s complete and min ima l med ia , Po ta to d e x t r o s e agar (PDA), and water aga r . 103 F i g u r e 6.3 Pe rcent spore g e r m i n a t i o n o f P o p u l a t i o n s I, I I , and I I I o f U s t i l a g o h o r d e i on Po ta to dex t rose agar (PDA) and seed e x t r a c t agar w i th e x t r a c t from b a r l e y c u l t i v a r T r e b i and Odessa . 104 Time (hrs.) F i g u r e 6 .4 Pe rcent spore g e r m i n a t i o n o f P o p u l a t i o n s I, I I , and I I I o f U s t i l a g o h o r d e i a f t e r undergo ing one g e n e r a t i o n o f s e l e c t i o n on b a r l e y c u l t i v a r T r e b i . 105 Time (hrs.) F i g u r e 6.5 Comparison o f pe r cen t spore ge rm ina t i on o f P o p u l a t i o n s I, C1 and C 2 o f U s t i l a g o h o r d e i . 106 T e l i o s p o r e s o f i n d i v i d u a l d i k a r y o n s w i t h i n each o f the p o p u l a t i o n s were observed to va r y i n g e r m i n a t i o n p a t t e r n s ( F i g . 6.6) . Each p o p u l a t i o n has a t l e a s t one d i k a r y o n whose t e l i o -spo res had l e s s than 30% g e r m i n a t i o n . A l l f i v e d i k a r y o n s o f II had ve r y l i t t l e g e r m i n a t i o n . The i n d i v i d u a l d i k a r y o n s f o r each p o p u l a t i o n and the r e s u l t i n g pe r cen t smutted p l a n t s o f T r e b i and Odessa are g i v e n i n Tab le 6 . 1 . In g e n e r a l the re were lower l e v e l s o f smut produced by i n o c u l a t i n g w i th the t e l i o s p o r e s compared to the l e v e l s o r i g i n a l l y produced by the s p o r i d i a . There i s a c o r r e l a t i o n between reduced g e r m i n a t i o n and the l e v e l o f smut produced when i n o c u l a t i n g w i th the t e l i o s p o r e s . Ge rmina t ion o f I, A and B were l e s s than the o t h e r th ree d i k a r y o n s ( F i g . 6.6) and they produced ve r y few smutted p l a n t s (Tab le 6 . 1 ) . Germina t ion o f I I I B and E were l e s s than the o t h e r th ree d i k a r y o n s and they a l s o produced ve r y few smutted p l a n t s . A l l d i k a r y o n s o f II produced v e r y l i t t l e smut. Phenotype s t u d i e s In t rea tment 1 and 2 the t e l i o s p o r e s were from comp l e t e l y smutted heads , and t rea tment 3 and 4 were t e l i o s p o r e s from p a r t i a l l y smutted heads . There was no r e l a t i o n s h i p between phenotype and g e r m i n a t i o n p a t t e r n s ( F i g . 6.7) . There were reduced l e v e l s o f smutted p l a n t s produced from t e l i o s p o r e l i n e s showing low l e v e l s o f g e r m i n a t i o n (Tab le 6 . 2 ) . Genotype 1, 2 and 4 produced comp le t e l y smutted heads r e g a r d l e s s o f c u l t i v a r . 107 F i g u r e 6.6 Percent spore ge rm ina t i on o f the f i v e i n d i v i d u a l d i k a r y o n s i n each o f P o p u l a t i o n s I, II and I I I o f U s t i l a g o h o r d e i . 108 T a b l e 6.1 Comparison o f the pe r cen t smutted p l a n t s produced by 15 U s t i l a g o h o r d e i d i k a r y o n s us i ng s p o r i d i a l i n o c u l a t o n ve r sus t e l i o s p o r e i n o c u l a t i o n on b a r l e y c u l t i v a r s T r e b i and Odessa P o p u l a t i o n & d i k a r y o n % smutted p l a n t s produced on c u l t i v a r T r e b i Odessa s p o r i d i a l t e l i o s p o r e s p o r i d i a l t e l i o s p o r e A B C D E 20 12 15 32 15 6 0 10 15 9 22 14 48 37 49 1 5 31 27 24 II A B C D E 12 4 20 25 19 0 1 0 0 0 13 60 6 8 36 0 0 0 1 1 I I I A B C D E 19 34 35 41 29 13 1 9 11 0 14 38 32 51 44 24 2 33 40 0 109 I Time (hrs.) F i g u r e 6.7 Comparison o f pe r c en t spore ge rm ina t i on o f f o u r d i k a r y o n s o f U s t i l a g o h o r d e i . 110 Tab l e 6.2 Pe rcent smutted p l a n t s produced on b a r l e y c u l t i v a r s T r e b i and Odessa by f ou r U s t i l a g o  h o r d e i genotypes % smutted p l a n t s on c u l t i v a r Genotype T r e b i Odessa 1 17 18 2 1 3 3 0 0 4 25 24 I l l D i s c u s s i o n Temperature d i d not appear to be a f a c t o r a f f e c t i n g the reduced ge rm ina t i on o f I I . S i m i l a r average pe r cen t g e r m i n a t i o n was reached f o r each p o p u l a t i o n ove r a l l the temperatures t e s t e d except f o r 4 ° C . U. avenae and U. maydis germinate w i t h i n the range o f 4-35°C and 8-38 °C , r e s p e c t i v e l y ( Jones 1923 a , b ) , t h e r e f o r e , i t i s not s u r p r i s i n g t o f i n d U. h o r d e i ge rm ina t i ng w i t h i n the same temperature range . The o p t i m a l temperature f o r U. h o r d e i appeared to be 2 2 ° C , t h e r e f o r e , a l l the subsequent exper iments were performed a t 2 2 ° C . Exogenous n u t r i e n t s d i d not seem to be a f a c t o r i n the reduced ge rm ina t i on o f I I . Very s i m i l a r g e r m i n a t i o n p a t t e r n s were observed on media c o n t a i n i n g some n u t r i e n t s . Seed e x t r a c t agar p r o v i d e s s i m i l a r l e v e l s o f n u t r i e n t s as PDA or V o g e l ' s comp le t e . S p o r i d i a l f o rma t i on was reduced but c o n j u g a t i o n was enhanced on water aga r . C a l t r i d e r and G o t t l i e b (1966) s t u d i e d the n u t r i t i o n a l r equ i rements o f ge rm ina t i ng U. maydis t e l i o s p o r e s and found marked d i f f e r e n c e s i n the promot ion o f ge rm ina t i on by s u g a r s . S a r t o r i s (1924) compared growth r a t e s o f U. h o r d e i h a p l o i d c u l t u r e s on v a r i o u s sugars and d i s c o v e r e d ma l tose to g i v e the bes t g rowth . He a l s o d i s c o v e r e d the a d d i t i o n o f po tass ium d ihyd rogen phosphate and magnesium s u l f a t e to ma l tose caused c o n j u g a t i o n o f s p o r i d i a . Bowman (1946) r e p o r t e d t h a t c o n j u g a t i o n o f U. maydis s p o r i d i a o c cu r r ed o n l y when c u l t u r e d on low n u t r i e n t s . T h i s e x p l a i n s why c o n j u g a t i o n was observed 112 on water agar and none o f the o the r t ypes o f med ia . There was no d i f f e r e n c e i n ge rm ina t i on p a t t e r n s on the two seed e x t r a c t med ia . I t would be i n t e r e s t i n g to examine r a t e o f growth o f the h a p l o i d c u l t u r e s on the two d i f f e r e n t e x t r a c t media to see i f obse r vab l e d i f f e r e n c e s o c c u r . T h i s type o f i n f o r m a t i o n cou ld shed l i g h t on the d i f f e r e n c e s between a r e s i s t a n t and s u s c e p t i b l e c u l t i v a r . T e l i o s p o r e s o f the th ree p o p u l a t i o n s c u l t u r e d on T r e b i o r Odessa showed no d i f f e r e n c e s i n g e r m i n a t i o n p a t t e r n s . Th i s can be observed by comparing F i g . 6.1 w i th F i g . 6.2 ( V o g e l ' s complete med i a ) . Some s p o r i d i a o f II were observed to be l a r g e . Jo rgensen (1962) i d e n t i f i e d a s i n g l e gene i n U. h o r d e i r e s p o n s i b l e f o r s p o r i d i a l s i z e . He found tha t s i z e o f s p o r i d i a d i d not appear to a f f e c t p a t h o g e n i c i t y o r ge rm ina t i on o f the t e l i o s p o r e s . He s p e c u l a t e d t h a t the l a r g e s p o r i d i a l s i z e may have o r i g i n a t e d by an i n t e r - s p e c i f i c h y b r i d i z a t i o n between U. nuda and U. h o r d e i . Many o t h e r r e s e a r c h e r s (Kernkamp & Pe t t y 1941, L a s k a r i s 1941, C h i l t o n 1940, 194 3) have r e p o r t e d abnormal g e r m i n a t i o n o f smut t e l i o s p o r e s where v e r y few s p o r i d i a are p roduced . Others have observed d i p l o i d s p o r i d i a among those produced ( C h i l t o n 1940, 1943, C h r i s t e n s e n 1931) . L a s k a r i s (1934 and 1941) and C h i l t o n (1943) have r e p o r t e d cases o f abnormal g e r m i n a t i o n where the promycel ium and s p o r i d i a are much l a r g e r than norma l . Many r e s e a r c h e r s ( C h i l t o n 1938, 1940, L a s k a r i s 1939, Stakman e t 113 a l . 1943) have reported disintegration or lys is of the promycelium either before or after sporidial formation and Chilton (1943) stated that lys is seemed to be confined to the time when meiosis occurs. Inheritance of abnormal germination has been postulated or demonstrated for many smut species (Chilton 1938, Laskaris 1941, Holton 1951, 1957, Holton & Dietz 1960, Fischer 1940); i t is most prevalent when there is inbreeding or interspecific hybridization (Christensen & Rodenhiser 1940, Nielsen 1968). Also associated with abnormal germination are sporidia that bud once or twice and then disintegrate (Fisher 1940, Grasso 1955) or sporidia that grow slowly (Chilton 1938). Most of the observations of abnormal germination for other smut species were observed during these experiments with U. hordei. It appears that abnormal germination of U. hordei is inherited and that there is a problem with meiosis. Most of the previous l i terature on abnormal germination in smut agrees with this speculation. Meiotic mutations (mutation that modifies the normal pattern of chromosome behavior during meiosis) have been found in many organisms including fungi, Drosophila, plants and man. A generalization made from studies of meiotic mutants is that there is abnormal disjunction and consequently some of the meiotic products are often inviable . Chilton (1943) and Popp & Hanna (1935) suggested that abnormal germination in smuts was associated with chromosome nondisjunction. A meiotic mutation 114 as the cause o f abnormal g e r m i n a t i o n i n smuts may be a r easonab l e s p e c u l a t i o n . I t was not s u r p r i s i n g t ha t a f t e r another g e n e r a t i o n o f c u l t u r i n g on the h o s t , i n c r e a s e s i n pe r cen t g e r m i n a t i o n were o b s e r v e d . S ince g e r m i n a t i o n a f f e c t s the success o f the smut, i t i s expected tha t the genotypes t ha t are b e t t e r adapted and produce more s p o r i d i a would be more s u c c e s s f u l , and t h e r e f o r e , would be s e l e c t e d through the c u l t u r i n g p r o c e s s . The o n l y o t h e r d i f f e r e n c e between the t e l i o s p o r e s from the i n i t i a l g e n e r a t i o n and a f t e r s e l e c t i o n i s the age d i f f e r e n c e . These exper iments were performed when the t e l i o s p o r e s o f the i n i t i a l g e n e r a t i o n were a year o l d ; whereas, the t e l i o s p o r e s o f the l a t e r g e n e r a t i o n were o n l y a few months o l d . G r ea t e r spore v i a b i l i t y c o u l d account f o r the i n c r e a s e d g e r m i n a t i o n observed i n the l a t t e r g e n e r a t i o n . The low g e r m i n a t i o n and the o b s e r v a t i o n s o f abnormal s p o r i d i a l f o rma t i on o f the p a r e n t a l t e l i o s p o r e s , and C 2 i n d i c a t e s t ha t the phenomenom o f abnormal ge rm ina t i on may be i n h e r i t e d . S ince I, II and I I I were F 2 p o p u l a t i o n s , v a r i a t i o n i n g e r m i n a t i o n can be expected to be g r e a t e r than t ha t observed i n the p a r e n t s . The F 2 ' s are d e r i v e d from s p o r i d i a which are c l o s e l y r e l a t e d and may account f o r the i n c r e a s e i n abnormal g e r m i n a t i o n which has been obse r ved . The amount o f v a r i a t i o n i n g e r m i n a t i o n observed among the f i v e d i k a r y o n s which made up each p o p u l a t i o n showed t h a t II was randomly composed o f abnormal ge rm ina to r s and t h a t I and I I I 115 a l s o had d i k a r y o n s t h a t have abnormal g e r m i n a t i o n . T e l i o -spores o f each o f the d i k a r y o n s were i n o c u l a t e d onto both T r e b i and Odessa . The d i k a r y o n s tha t had abnormal g e r m i n a t i o n b a r e l y produced any smutted p l a n t s wh i l e the o t h e r d i k a r y o n s produced much h i g h e r pe r cen tages o f smutted p l a n t s . T h i s then accounts f o r the d r a s t i c d e c l i n e i n l e v e l s o f smut produced by II (Chapter 5 ) . The r e s u l t s o f the exper iment i n v o l v i n g p a r t i a l l y smutted heads i n d i c a t e d tha t the re i s no c o r r e l a t i o n between p a r t i a l smut t ing and abnormal g e r m i n a t i o n . The phenotypes o f complete and p a r t i a l smut t ing were not s t a b l e and seemed to be e a s i l y i n f l u e n c e d by env i ronmenta l c o n d i t i o n s w i t h i n , as w e l l a s , o u t s i d e the h o s t . The c o r r e l a t i o n between d i s e a s e l e v e l s and g e r m i n a t i o n i s accounted f o r by the p r o d u c t i o n o f fewer s p o r i d i a which are then a v a i l a b l e f o r i n f e c t i o n . Fewer s p o r i d i a reduce the p r o b a -b i l i t y o f i n f e c t i o n and subsequen t l y y i e l d fewer smutted p l a n t s . The r e fo r e g e r m i n a t i o n i s one t r a i t t h a t a f f e c t s p a t h o g e n i c i t y o f the smut pa thogen . Pedersen et a_3-. (1977) have i d e n t i f i e d a gene t h a t c o n d i -t i o n s poor development o f i n f e c t i o n hyphae o f U. h o r d e i , which a l s o was c o r r e l a t e d w i th d i s e a s e l e v e l s . F a c t o r s t ha t a f f e c t growth r a t e s are l i k e l y t o be p a r t o f the f a c t o r s t h a t a re a s s o c i a t e d w i th a g g r e s s i v e n e s s o f the pa thogen . Agg res s i v eness i s most impor tant i n pathogens such as smut t h a t a re sys temic o r i n c l o s e a s s o c i a t i o n w i th the hos t f o r l ong p e r i o d s . 116 CHAPTER 7 FURTHER ANALYSIS OF POLYGENIC INHERITANCE OF VIRULENCE IN USTILAGO HORDEI I n t r o d u c t i o n Gene t i c a n a l y s i s o f v i r u l e n c e i n U s t i l a g o h o r d e i has shown tha t v i r u l e n c e i s i n h e r i t e d by s i n g l e genes tha t a re e i t h e r dominant or r e c e s s i v e (Thomas & Person 1965, Thomas 1976, S idhu & Person 1971, Ebba 1974, Ebba & Person 1975) . Gene-for-gene r e l a t i o n s have a l s o been demonstrated i n the U. h o r d e i - b a r l e y system (S idhu & Person 1972, Ebba 1974) . More r e cen t a n a l y s i s has shown tha t w i t h i n the gene-for-gene system the re i s v a r i a b i l i t y f o r a g g r e s s i v e n e s s and tha t v i r u l e n c e i s mod i f i ed by po lygenes (Emara 1972, Emara & S idhu 1974, Pope 1983) . Person e t a_l. (1982, 198 3) have d i s c u s s e d some r e cen t work i n v o l v i n g t e t r a d a n a l y s i s o f U. h o r d e i F^  t e l i o s p o r e s . In s e l f ing and b a c k c r o s s i n g w i th the s p o r i d i a o f the F-^  t e l i o s p o r e s , s e g r e g a t i o n p a t t e r n s r e v e a l e d tha t there was a major v i r u l e n c e gene and v i r u l e n c e was dominant on the c u l t i v a r T r e b i . There was a c o n s i d e r a b l e v a r i a t i o n i n percentage o f smut among t e l i o s p o r e s . The v a r i a t i o n cou ld not be accounted f o r by another major v i r u l e n c e gene so i t was proposed tha t po l ygenes were mod i f y i ng the major gene. They hypo thes i zed tha t the s p o r i d i a (gametes) c o u l d be ranked on t h e i r b a s i s o f per formance from the s e l f i n g and c r o s s i n g d a t a . A d i a l l e l c r o s s was per formed w i th 12 s p o r i d i a o f each mat ing type and 117 the r e s u l t s showed cons tan t r ank ing o f gametes and c o n s i d e r -ab l e v a r i a t i o n i n l e v e l s o f smut as p r e d i c t e d by t h e o r e t i c a l a n a l y s i s o f p o l y g e n i c determined t r a i t s . The f o l l o w i n g exper iments were performed to observe s e g r e g a t i o n p a t t e r n s f o r d i k a r y o n s d e r i v e d from the p r e v i o u s exper iment on both the c u l t i v a r T r e b i and Odessa , a modera te l y r e s i s t a n t and s u s c e p t i b l e c u l t i v a r , r e s p e c t i v e l y . M a t e r i a l s and Methods T e l i o s p o r e s o f U. h o r d e i were ob t a i ned from smutted heads produced i n 1977 a t U n i v e r s i t y o f B r i t i s h Co lumbia . The p a r e n t a l and F^ c u l t u r e s were ob t a i ned from s i l i c a g e l . T h i r t e e n he te rozygous and th ree homozygous d i k a r y o n s , t h a t were be ing s t u d i e d i n p o p u l a t i o n s t u d i e s (Chapter 5 ) , were chosen f o r f u r t h e r g e n e t i c a n a l y s i s . The m e i o t i c p roduc t s from s i n g l e t e l i o s p o r e s o f each d i k a r y o n were o b t a i n e d , c u l t u r e d and t e s t e d f o r mat ing t ypes as d e s c r i b e d by Groth et: a_l. (1976) . The seed t reatment and i n o c u l a t i o n procedure fo l l owed Groth and Person (1976) . Approx imate l y 100 seeds pe r t reatment were p l an t ed i n a 3 m row wi th th ree r e p l i c a t e s per t r ea tment . D isease r e a c t i o n s were r eco rded as the number o f smutted p l a n t s ( a t l e a s t one t i l l e r smutted) out o f a t o t a l number o f p l a n t s pe r t r ea tmen t . Pe rcent smutted p l a n t s was based on the average o f the th ree r e p l i c a t e s . The f o l l o w i n g exper iments were pe r fo rmed : 118 1. T h i r t e e n he te rozygous F 2 d i k a r y o n s , 3 homozygous F 2 d i k a r y o n s and p a r e n t a l s T± and T 4 were " s e l f e d " and t e s t e d on c u l t i v a r s T r e b i and Odessa . 2. Ten F-^  d i k a r y o n s were " s e l f e d " and t e s t e d on T r e b i and Odessa . 3. Three homozygous F 2 d i k a r y o n s were " b a c k c r o s s e d " to both p a r e n t s , T^ and T^ , and t e s t e d on T r e b i . 4. Nine o f the 13 he te rozygous F 2 d i k a r y o n s were " t e s t -c r o s s e d " to the three homozygous F 2 d i k a r y o n s and t e s t e d on T r e b i . R e s u l t s D ikaryon I-B had no c l e a r s e g r e g a t i o n on T r e b i but had a r a t i o o f 3 l o w : l h i gh on Odessa . D ikaryons I-C, I-D, I-E, I I-E, and IV-C had s e g r e g a t i o n p a t t e r n s f o r v i r u l e n c e t h a t f o l l owed a 3 l o w : l i n t e rmed i a t e o r h igh l e v e l o f smut on both T r e b i and Odessa as expected f o r one gene s e g r e g a t i n g (Tab le 7 . 1 ) . D ika ryons II-B, III-B, IV-A and IV-B showed no c l e a r s e g r e g a t i o n on T r e b i but a l l excep t II-B had s e g r e g a t i o n p a t t e r n s o f 2 low:2 i n t e rmed i a t e l e v e l s o f smut on Odessa . II- B had s e g r e g a t i o n r a t i o o f 3 l o w : l i n t e r m e d i a t e on Odessa . III- A had a 2:2 s e g r e g a t i o n r a t i o on both T r e b i and Odessa . III-C had a 2:2 on T r e b i but 1 low:2 i n t e r m e d i a t e : ! h igh on Odessa . III-D segrega ted 2 l o w : l i n t e r m e d i a t e : ! h i gh on T r e b i but 1 l o w : ! i n t e r m e d i a t e d h igh on Odessa . 119 Tab le 7.1 Pe rcen t smut on two b a r l e y c u l t i v a r s , T r e b i and Odessa , d e r i v e d from s e l f i n g 13 he te rozygous U s t i l a g o h o r d e i F 2 d i k a r y o n s s p o r i d i a l % smut on c u l t i v a r * d i k a r yon mat ing T r e b i Odessa I-B 1 x 2 11 42 1 x 3 7 10 4 x 2 10 5 4 x 3 2 7 I-C 1 x 2 0 5 1 x 4 0 0 3 x 2 8 34 3 x 4 2 15 I-D 1 x 3 0 2 1 x 4 1 5 2 x 3 24 34 2 x 4 12 17 I-E 1 x 2 12 29 1 x 4 4 3 3 x 2 0 3 3 x 4 0 0 II-B 1 x 3 0 2 1 x 4 0 11 2 x 3 0 0 2 x 4 0 1 II-E 1 x 2 6 16 1 x 4 4 16 3 x 2 18 21 3 x 4 4 13 II I A 1 x 2 0 0 1 x 4 0 7 3 x 2 22 32 3 x 4 21 35 III-B 1 x 2 0 0 1 x 3 0 7 4 x 2 3 9 4 x 3 0 0 Tab le 7.1 - c o n t ' d s p o r i d i a l % smut on c u l t i v a r d i k a r y o n mating T r e b i Odessa III-C 1 x 2 8 20 1 x 4 10 32 3 x 2 1 10 3 x 4 3 18 III-D 1 x 2 4 41 1 x 4 39 45 3 x 2 0 8 3 x 4 16 23 IV-A 1 x 3 0 2 1 x 4 2 12 2 x 3 0 0 2 x 4 7 14 IV-B 1 x 2 24 23 1 x 4 30 35 3 x 2 20 43 3 x 4 35 48 IV-C 1 x 2 0 0 1 x 3 17 19 4 x 2 3 25 4 x 3 5 11 Smut l e v e l s on Odessa are s i g n i f i c a n t l y h i g h e r than l e v e l s on T r e b i (W i l coxson s igned rank t e s t P = . 0 5 ) . Ranks o f smut l e v e l s on T r e b i a re c o r r e l a t e d w i th ranks on Odessa (Spearman's rank c o r r e l a t i o n r s = . 8 7 ) . 121 D ika ryon V-A had s e g r e g a t i o n p a t t e r n s o f 2 low: 2 i n t e r m e d i a t e on both T r e b i and Odessa (Tab le 7 . 2 ) . V-B had no s e g r e g a t i o n on Odessa but had a r a t i o o f 2 i n t e r m e d i a t e : 2 h igh on T r e b i . V-C segrega ted 3 i n t e r m e d i a t e : 1 h i g h on T r e b i but segregated 3 h i g h : l i n t e r m e d i a t e on Odessa . F^ d i k a r y o n 17B segregated 3 i n t e r m e d i a t e : ! low on T r e b i but segrega ted 3 i n t e r m e d i a t e : 1 h igh on Odessa . 18C had no c l e a r s e g r e g a t i o n on e i t h e r c u l t i v a r . 18D, 19B, 19D and 20D had s e g r e g a t i o n p a t t e r n s o f 2:2 on both c u l t i v a r s (Tab le 7 . 3 ) . 21E and 23C segrega ted 3 l o w : l i n t e r m e d i a t e o r h i g h on both c u l t i v a r s . 21C had no c l e a r s e g r e g a t i o n on T r e b i but had 3 l o w : l i n t e r m e d i a t e on Odessa . 22G segrega ted 2 l o w : l i n t e r -m e d i a t e : ! low on T r e b i and segrega ted 2:2 on Odessa . P a r e n t a l d i k a r y o n gave s e g r e g a t i o n p a t t e r n o f 3 l o w : l i n t e r m e d i a t e on both c u l t i v a r s (Tab le 7 . 4 ) . T^ gave no s e g r e g a t i o n on e i t h e r c u l t i v a r and g e n e r a l l y gave v e r y low l e v e l s o f smut. Backc ross o f V-A to T^ segrega ted 4 low:4 i n t e rmed i a t e on T r e b i but when V-A was backc rossed to T 4 , the re were no s e g r e g a t i o n p a t t e r n s (Tab le 7 . 5 ) . Backc ross V-B to Tj_ segrega ted 2 low:4 i n t e r m e d i a t e d h i g h but when V-B was back-c r o s s e d to T 4 , there was a 5 low:3 h igh s e g r e g a t i o n p a t t e r n . Backc ross o f V-C to T^ gave a 5 low:3 h igh s e g r e g a t i o n p a t t e r n but the backc ross to T 4 gave a 6 low:2 h igh s e g r e g a -t i o n p a t t e r n . I-C x V-A gave no c l e a r s e g r e g a t i o n f o r v i r u l e n c e . I-C x V-B gave s e g r e g a t i o n p a t t e r n s t h a t c o u l d e i t h e r be a 2:6 o r 3:5 Tab le 7.2 Percent smut on two b a r l e y c u l t i v a r s . T r e b i Odessa , d e r i v e d from s e l f i n g th ree homozygous U s t i l a g o h o r d e i d i k a r y o n s d i k a r y o n V-A V-B s p o r i d i a l mating 1 X 2 1 X 4 3 X 2 3 X 4 1 X 3 1 X 4 2 X 3 2 X 4 1 X 2 1 X 3 4 X 2 4 X 3 % smut on c u l t i v a r * T r e b i Odessa 6 9 3 12 12 20 10 19 34 34 17 35 45 34 24 30 8 29 27 32 13 16 17 27 Smut l e v e l s on Odessa are s i g n i f i c a n t l y h i ghe r than l e v e l s on T r e b i (W i l coxson s igned rank t e s t P = . 0 5 ) . 123 Tab le 7.3 Percent smut on two b a r l e y c u l t i v a r s , T r e b i and Odessa , d e r i v e d from s e l f i n g 10 F-^  U s t i l a g o  h o r d e i d i k a r y o n s s p o r i d i a l % smut on c u l t i v a r d i k a r y o n mating T r e b i Odessa 17B 1 x 2 7 17 1 x 4 1 11 3 x 2 8 40 3 x 4 10 18 18C 1 x 2 3 9 1 x 4 1 3 3 x 2 7 13 3 x 4 3 13 18D 1 x 3 0 12 1 x 4 10 27 2 x 3 0 12 2 x 4 14 27 19B 1 x 2 1 5 1 x 4 26 34 3 x 2 1 4 3 x 4 14 25 19D 1 x 2 5 14 1 x 3 5 32 4 x 2 0 1 4 x 3 1 3 20D 1 x 2 4 8 1 x 4 5 11 3 x 2 0 2 3 x 4 1 3 21C 1 x 2 0 0 1 x 4 8 18 3 x 2 0 5 3 x 4 3 5 21E 1 x 2 2 1 1 x 3 1 5 4 x 2 16 19 4 x 3 0 1 124 Tab le 7.3 - c o n t ' d . s p o r i d i a l % smut on c u l t i v a r * d i k a r y o n mat ing T r e b i Odessa 22G 1 x 3 2 6 1 x 4 12 11 2 x 3 7 30 2 x 4 21 22 23C 1 x 3 4 1 1 x 4 2 19 2 x 3 1 6 2 x 4 17 30 Smut l e v e l s on Odessa are s i g n i f i c a n t l y h i ghe r than on T r e b i (W i l coxson s igned rank t e s t P = . 0 5 ) . Ranks o f smut l e v e l s on T r e b i are c o r r e l a t e d wi th ranks on Odessa (Spearman's rank c o r r e l a t i o n r s = . 7 8 ) . 125 Tab le 7.4 Pe rcen t smut on two b a r l e y c u l t i v a r s . T r e b i and Odessa , d e r i v e d from s e l f i n g two p a r e n t a l U s t i l a g o  h o r d e i d i k a r y o n s d i k a r y o n T, s p o r i d i a l mating 1 x 2 1 x 4 3 x 2 3 x 4 % smut on c u l t i v a r T r e b i Odessa 3 0 10 5 4 0 17 7 1 x 3 1 x 4 2 x 3 2 x 4 1 0 0 0 2 3 3 4 126 Tab le 7.5 Pe rcen t smut on b a r l e y c u l t i v a r T r e b i d e r i v e d from b a c k c r o s s i n g th ree homozygous U s t i l a g o h o r d e i d i k a r y o n s to p a r e n t a l d i k a r y o n s , T^ and T^ Backcross o f d i k a r y o n s and s p o r i d i a l mat ings % smut % smut 1 1 12 18 9 3 5 17 2 4 1 3 2 4 1 3 V-A 1 1 2 2 3 3 4 4 3 4 1 2 3 4 1 2 1 1 0 0 0 5 0 0 16 4 12 6 13 26 13 23 2 4 2 4 1 3 1 3 V-B 1 1 2 2 3 3 4 4 3 4 3 4 1 2 1 2 23 20 19 11 4 4 2 1 10 20 8 0 25 2 6 17 1 3 2 4 2 4 1 3 V-C 1 1 2 2 3 3 4 4 1 2 3 4 3 4 1 2 0 0 3 4 34 35 1 3 127 Tab le 7.6 Pe rcen t smut on b a r l e y c u l t i v a r T r e b i d e r i v e d from t e s t c r o s s i n g n ine he te rozygous to th ree homozygous U s t i l a g o h o r d e i d i k a r y o n s he te rzygous Homozygous d i k a r y o n s  d i k a r y o n and V-A V-B V-C s p o r i d i a l s p o r i d i a % s p o r i d i a % s p o r i d i a % mat ing smut smut smut I-C 1 1 1 1 6 2 1 1 3 1 2 7 3 11 2 2 4 3 21 1 10 2 4 4 4 19 4 16 3 1 2 1 23 2 4 3 3 8 2 20 3 22 4 2 2 3 16 1 2 4 4 2 4 10 4 10 I-D 1 2 0 3 1 1 0 1 4 2 4 0 4 2 2 2 17 3 42 1 15 2 4 8 4 31 4 26 3 1 8 1 22 2 19 3 3 18 2 23 3 27 4 1 0 1 13 2 9 4 3 1 2 11 3 14 I-E 1 1 7 1 16 2 9 1 3 10 2 19 3 30 2 2 11 3 31 1 15 2 4 12 4 23 4 20 3 1 0 1 0 2 0 3 3 0 2 0 3 1 4 2 25 3 32 1 28 4 4 20 4 32 4 15 II-E 1 1 4 1 24 2 11 1 3 9 2 22 3 31 2 2 17 3 32 1 4 2 4 7 4 25 4 10 3 1 5 1 26 2 31 3 3 6 2 21 3 26 4 2 11 3 11 1 11 4 4 10 4 8 4 0 128 Tab le 7.6 - c o n t ' d . he te rzygous Homozygous d i k a r y o n s d i k a r y o n and V-A V-B V-C s p o r i d i a l s p o r i d i a % s p o r i d i a % s p o r i d i a % mat ing smut smut smut III-B 1 2 5 3 5 1 14 1 4 3 4 8 4 10 2 1 0 1 7 2 2 2 3 4 2 2 3 19 3 1 3 1 32 2 4 3 3 18 2 37 3 35 4 2 11 3 28 1 5 4 4 8 4 16 4 6 III-C 1 1 2 3 3 4 4 2 4 1 3 4 1 3 7 2 0 2 2 1 0 3 4 1 2 4 1 2 19 23 20 27 15 26 31 1 4 2 3 4 2 3 3 13 1 25 7 7 34 IV-A 1 1 2 2 3 3 4 4 1 3 1 3 2 4 2 4 1 7 0 3 4 3 22 27 1 2 1 2 3 4 3 4 21 1 8 7 29 17 39 29 2 3 2 3 1 4 1 4 1 28 0 16 2 13 36 19 IV-B 1 1 2 2 3 3 4 4 2 4 1 3 2 4 1 3 20 19 11 15 27 21 13 28 3 4 1 2 3 4 1 2 23 16 18 19 13 18 35 40 1 4 2 3 1 4 2 3 19 16 26 33 24 14 36 42 129 Tab le 7.6 - c o n t ' d . he te rzygous Homozygous d i k a r y o n s d i k a r y o n and V-A V-B V-C s p o r i d i a l s p o r i d i a % s p o r i d i a % s p o r i d i a % mat ing smut smut smut IV-CB 1 2 1 3 8 1 5 1 4 1 4 5 4 3 2 1 0 1 28 2 6 2 3 5 2 26 3 34 3 1 5 1 20 2 27 3 3 10 2 23 3 28 4 2 8 3 13 1 0 4 4 4 4 12 4 8 130 r a t i o . I-C x V-C segrega ted 3 low:5 i n t e r m e d i a t e . I-D gave s e g r e g a t i o n p a t t e r n s o f 4 low:2 i n t e r m e d i a t e d h i g h , 2 low:2 i n t e r m e d i a t e d h igh and 2 low:4 i n t e r m e d i a t e d h i g h when c r o s s e d to V-A, V-B and V-C , r e s p e c t i v e l y . IE x V-A and I-E x V-C gave s e g r e g a t i o n p a t t e r n s o f 2 low:4 i n t e r m e d i a t e d h i g h , whereas , I-E x V-B segregated 2 low:3 i n t e r m e d i a t e d h i g h . II- E gave s e g r e g a t i o n p a t t e r n s o f 4 low:4 i n t e r m e d i a t e , 2 i n t e r m e d i a t e : 6 h i g h , and 2 low:3 i n t e r m e d i a t e d h igh when c r o s s e d w i th V-A, V-B and V-C, r e s p e c t i v e l y . III-C x V-A and III- C x V-B gave no s e g r e g a t i o n f o r v i r u l e n c e but III-C x V-C gave 6 l o w d h i g h . IV-A x V-A gave 6 l o w d h i g h , whereas, IV-A x V-B and IV-A x V-C segregated 3 low:5 h i g h . IV-B x V-A gave no c l e a r s e g r e g a t i o n f o r v i r u l e n c e . IV-B segrega ted 6 i n t e r m e d i a t e d h igh and 3 i n t e r m e d i a t e d h i g h when c r o s s e d w i th V-B and V-C, r e s p e c t i v e l y . IV-C x V-A gave no c l e a r s e g r e g a t i o n but IV-C x V-B and IV-C x V-C segrega ted 4 i n t e r m e d i a t e d h i g h and 5 l o w d h i g h , r e s p e c t i v e l y . D i s c u s s i o n Person e t a l . (1982, 1983) p o i n t e d ou t tha t s e g r e g a t i o n f o r a v i r u l e n c e gene would o n l y be observed w i th a t e t r a t y p e t e t r a d and then a 3:1 r a t i o would be obse r ved . S ince the d i k a r y o n s (Tab le 7.1) were hypo thes i zed to be he te rozygous w i th v i r u l e n c e dominant , s e g r e g a t i o n f o r a v i r u l e n c e gene on T r e b i was expected upon " s e l f i n g " o f some o f them. There were p o t e n t i a l 3:1 s e g r e g a t i o n p a t t e r n s observed on both T r e b i and 131 Odessa but the d i f f e r e n c e i n v i r u l e n c e l e v e l s w i t h i n a s i n g l e m e i o s i s was much s m a l l e r than p r e v i o u s l y observed f o r the major v i r u l e n c e gene (Appendix A ) . In s e v e r a l c a s e s , such as I-E, the a v i r u l e n t : v i r u l e n t phenotype was i n a 3:1 r a t i o , which was the r e v e r s e o f p r e v i o u s r e s u l t s (Appendix A ) . Odessa was h i g h l y s u s c e p t i b l e t o both p a r e n t s , Tj_ and T 4 , t h e r e f o r e , Odessa would not c a r r y a r e s i s t a n c e gene match ing an a v i r u l e n c e a l l e l e i n the F 2 d i k a r y o n s . Seg rega t i on p a t t e r n s were observed on both c u l t i v a r s and i n some cases the o n l y d i f f e r e n c e was tha t Odessa g e n e r a l l y had h i g h e r l e v e l s o f smut. III-D had o p p o s i t e s e g r e g a t i o n p a t t e r n s on the two c u l t i v a r s . Wi thout any o f the p r e v i o u s c r o s s i n g e x p e r i m e n t s , one c o u l d assume tha t the observed s e g r e g a t i o n p a t t e r n s were f o r a major v i r u l e n c e gene. The 2:2 r a t i o tha t was observed f i t s the s e g r e g a t i o n p a t t e r n f o r two genes . S ince the p r e v i o u s work had i d e n t i f i e d a major gene t ha t was m o d i f i e d by s e v e r a l o t h e r genes wi th minor e f f e c t s , the s e g r e g a t i o n p a t t e r n s i n these exper iments i s bes t e x p l a i n e d by e f f e c t s o f the minor genes . I t i s p o s s i b l e tha t when the a p p r o p r i a t e complement o f minor genes o r po lygenes i n the h a p l o i d s p o r i d i a a re combined, the d i k a r y o n cou ld have a g r e a t l y enhanced v i r u l e n c e and produce much g r e a t e r number o f smutted p l a n t s . S ince po lygenes are thought to be n o n - s p e c i f i c i n t h e i r a c t i o n , these genes would a f f e c t the observed l e v e l s o f smut t ing on both c u l t i v a r s and 132 c o u l d e x p l a i n the s e g r e g a t i o n p a t t e r n s observed on both c u l t i v a r s . No s e g r e g a t i o n was expected f o r the homozygotes (Tab le 7.2) but y e t the re appeared to be sma l l increment d i f f e r e n c e s . D ikaryons V-B and V-C were expected t o be s i m i l a r t o V-A but the o r i g i n o f V-B and V-C i s now u n c e r t a i n , t h e r e -f o r e , the g e n e t i c i n f o r m a t i o n conce rn i ng them was not as impor tant as V-A. The F-^  d i k a r y o n s (Tab le 7.3) had p r e v i o u s l y been t e s t e d i n 1976 but o n l y on the c u l t i v a r T r e b i . They were r e t e s t e d to compare s e g r e g a t i o n p a t t e r n s on T r e b i w i th those on Odessa . No s e g r e g a t i o n was expected on Odessa but aga in the re were ve r y s i m i l a r p a t t e r n s on both c u l t i v a r s . These s e g r e g a t i o n p a t t e r n s on Odessa suggest t ha t maybe the re i s a r e s i s t a n c e gene i n Odessa tha t has not been p r e v i o u s l y i d e n t i f i e d . The l e v e l s o f smut and the s e g r e g a t i o n p a t t e r n s observed d i d not a lways agree w i th the 1976 d a t a . P a r e n t a l s T-^  and T 4 gave r e s u l t s q u i t e d i f f e r e n t from r e s u l t s ob t a i ned by Ebba (1974) . Ebba ob ta ined an average o f 44% and 2.5% smut on T r e b i w i th and T 4 , r e s p e c t i v e l y . The r e s u l t s observed were ten f o l d l e s s than p r e v i o u s l y o b s e r v e d . Ebba had conc luded t h a t T^ and T 4 were homozygous v i r u l e n t and a v i r u l e n t , r e s p e c t i v e l y . The minor d i f f e r e n c e s observed between the f o u r d i k a r y o n s o f each T-^  and T 4 would mean t h a t the re i s a 10% random v a r i a t i o n o r a t l e a s t a 5% d e v i a t i o n i n e i t h e r d i r e c t i o n o f the observed l e v e l 133 o f smut. In g e n e r a l most o f the observed l e v e l s o f smut over the 1981-1983 p e r i o d a t UBC were ten f o l d l e s s than p r e v i o u s l y o b s e r v e d . A l s o c u l t u r e s ob t a i ned from s i l i c a g e l were g i v i n g reduced l e v e l s ove r those p r e v i o u s l y obse r ved . T h i s can be seen f o r both the p a r e n t a l and F^ c u l t u r e s . The backc ros ses (Tab le 7.5) were performed wi th the i d e a t h a t TJL and T^ , as w e l l as the th ree F 2 d i k a r y o n s , were homozygous f o r the gene c o n d i t i o n i n g v i r u l e n c e to T r e b i . With t h i s assumpt ion i t was expected t h a t any observed s e g r e g a t i o n would r e s u l t from the minor genes . A 4:4 s e g r e g a t i o n can be e x p l a i n e d by one gene, i f one t e l i o s p o r e was homozygous r e c e s s i v e and the o t h e r he te rozygous such tha t the re was a 2:2 gamet ic s e g r e g a t i o n p a t t e r n f o r the h e t e r o z y g o t e . There was a 4:4 r a t i o observed wi th T-j_ x V-A, i f a g a i n , the s m a l l i n c r e -menta l d i f f e r e n c e s i n p e r c e n t smut i s impor t an t . The 6:2 and 5:3 s e g r e g a t i o n r a t i o s can be e x p l a i n e d by two genes , i f both t e l i o s p o r e s are he te rozygous and both formed a t e t r a t y p e t e t r a d a t m e i o s i s . The observed s e g r e g a t i o n p a t t e r n s are o n l y bes t e x p l a i n e d by the i n t e r a c t i o n o f the a p p r o p r i a t e complement o f minor genes and the major gene. Spor id ium V-C-3 combined w i th e i t h e r 3 o r 4 o f T^ r e s u l t s i n a g r e a t enhancement o f v i r u l e n c e . Spor id ium V-C-3 a l s o had the h i g h e s t average v a l ue o f V-C s p o r i d i a i n the s e l f i n g expe r imen t s . Other examples are V-B 3 o r 4 x T^-3 and V-B-l x T 4 ~ 3 o r 4. When V-B was s e l f e d spo r id ium th ree had the h i g h e s t average o f the f ou r s p o r i d i a . The enhanced v i r u l e n c e observed f o r s p e c i f i c combina t ions demonstra tes tha t the re are i n t e r a c t i o n s o c c u r r i n g , whether these i n t e r a c t i o n s r e s u l t from the minor genes i n t e r a c t i n g w i th the major gene o r from the a p p r o p r i a t e comb ina t ion o f minor genes i s unknown. The t e s t c r o s s e s a l s o have s e g r e g a t i o n p a t t e r n s t h a t cannot be e x p l a i n e d e a s i l y by the major gene t h a t was i d e n t i f i e d p r e v i o u s l y . Aga in some i n t e r e s t i n g i n t e r a c t i o n s between s p e c i f i c s p o r i d i a are g r e a t l y enhanc ing v i r u l e n c e . One example i s I-D-sporidium 2 and 3 g i v e h igh l e v e l s i n s e l f i n g and a l s o h igh l e v e l s i n a l l the t e s t c r o s s e s . Another example, d i k a r y o n 2 x 4 was the h i g h e s t i n s e l f i n g o f IV-A and when t e s t c r o s s e d , spor id ium IV-A-4 was always p roduc ing h i g h l e v e l s o f smut. An e x c e p t i o n i s I-E where spo r i d i um 4 was h i g h f o r a l l the t e s t c r o s s e s but was not n e c e s s a r i l y h igh when s e l f e d . Another e x c e p t i o n i s III-B where spo r i d i um 3 i s a s s o c i a t e d w i th the h igh v a l ues from the t e s t c r o s s e s but i s not h igh i n s e l f i n g . A concept t h a t has become impor tan t f o r p o l y g e n i c a l l y determined systems i s cons tan t r a n k i n g . T h i s concept was a p p l i e d to the U s t i l a g o - b a r l e y system p r e v i o u s l y (Person e t a l . 1982, 1983) and cons t an t r ank ing o f gametes was demons t ra ted . Cons tan t r ank ing o f gametes can be examined i n the da ta where the d i k a r y o n s from " s e l f i n g " were i n o c u l a t e d on both T r e b i and Odessa . In g e n e r a l the rank o f the d i k a r y o n s on T r e b i i s i d e n t i f i e d to the rank on Odessa . An example i s I-D where the o r d e r o f the d i k a r y o n s on both c u l t i v a r s i s 2 x 3, 2 x 4, 1 x 4 135 and 1 x 3 . Because o f l i m i t e d t ime and space , the c r o s s i n g exper iments were o n l y i n o c u l a t e d onto T r e b i . I t would have been i n t e r e s t i n g to be ab l e to make compar isons o f d i k a r yons on the two c u l t i v a r s . From p r e v i o u s t e t r a d a n a l y s i s (Person e_t a_l. 1982, 1983) and b i o m e t r i c a l a n a l y s i s (Pope 1983) o f t h i s p a r t i c u l a r hos t-p a r a s i t e sys tem, a dominant major v i r u l e n c e gene was i d e n t i f i e d w i th po lygenes mod i f y i ng the e f f e c t s o f the major gene . Most o f t h i s d e t a i l e d a n a l y s i s was o n l y performed on one c u l t i v a r . T h i s f u r t h e r a n a l y s i s was per formed to g i v e more g e n e t i c i n f o r m a t i o n when i n o c u l a t i o n s were on another c u l t i v a r . Not o n l y d i d the r e s u l t s g i v e more g e n e t i c i n f o r m a t i o n but they a l s o r e v e a l e d some areas where more i n f o r m a t i o n i s needed. When g e n e t i c a n a l y s i s o f both the hos t and p a r a s i t e can be per formed i t shou ld be pe r fo rmed . Because no g e n e t i c a n a l y s i s was per formed from the v i ewpo in t o f the h o s t , the number o f r e s i s t a n c e genes or the gene a c t i o n tha t i s i n v o l v e d i n the hos t i s not known. An unknown r e s i s t a n c e gene may have been i d e n t i f i e d i n Odessa , but a t t h i s t ime because o f the nature o f the gene a c t i o n i n the pa thogen , i t i s not c e r t a i n i f we are d e a l i n g w i th a r e s i s t a n c e gene o r the i n t e r a c t i o n s o f the h a p l o i d pathogen geno types . The compar ison o f r e s u l t s from these exper iments w i th those from p r e v i o u s yea rs exper iments demonstrated t h a t compar i sons can not be e a s i l y made between d i f f e r e n t yea r s o r env i ronmenta l c o n d i t i o n s . Env i ronmenta l m o d i f i c a t i o n s o f smut 136 l e v e l s from macro condit ions (Ebba & Person 197 5) and mic ro c o n d i t i o n s (Emara & Freake 1981) have p r e v i o u s l y been demons t ra ted . Much more work i s needed to understand the v a r i a b i l i t y observed w i t h i n a s i n g l e t rea tment (d ikaryon) on a s p e c i f i c c u l t i v a r and to b e t t e r e s t ima te d i s e a s e l e v e l s when t h i s v a r i a b i l i t y e x i s t s . C r o s s i n g exper iments o f T r e b i and Odessa are needed to unders tand the i n h e r i t a n c e o f r e s i s t a n c e to these smut d i k a r y o n s . Some o f the genotypes from these exper iments c o u l d be used to examine the e f f e c t s o f the po l ygenes independent o f the major gene . T h i s would a l l ow one to observe how the po lygenes modi fy the e f f e c t s o f the v i r u l e n c e gene , such a s , changes i n number o f smutted p l a n t s , a l t e r i n g the number o f smutted t i l l e r s per p l a n t o r the t o t a l number o f t e l i o s p o r e s per p l a n t . 137 CHAPTER 8 GENERAL DISCUSSION AND CONCLUSIONS The o r i g i n a l exper iment was des igned (Chapter 4) to shed l i g h t on V a n d e r p l a n k 1 s concept o f s t a b i l i z i n g s e l e c t i o n . The theo ry behind the concept o f s t a b i l i z i n g s e l e c t i o n appears to be s i m p l i s t i c and i n r e a l i t y the events i n v o l v e d i n p o p u l a t i o n changes f o r a pathogen o r a hos t are q u i t e complex. Because o f t h i s comp l ex i t y the o r i g i n a l o b j e c t i v e was not r e a l i z e d . One g o a l o f the exper iments was to o b t a i n v a l u e s f o r a l l e l e f r e q u e n c i e s and observe the pathogen p o p u l a t i o n s ove r s e v e r a l g e n e r a t i o n s o f s e l e c t i o n f o r any changes i n a l l e l e f r e q u e n c i e s o f a s p e c i f i c v i r u l e n c e gene. These r e s u l t s were confounded by the i n t e r a c t i o n o f po l ygenes w i th the v i r u l e n c e gene such tha t i t was unknown whether smut l e v e l s were a r e f l e c t i o n o f the v i r u l e n c e gene o r the enhancement o f v i r u l e n c e by the p o l y g e n e s . Inc rease i n number o f pathogen gametes p roduc ing a v i r u l e n t phenotype was observed over one g e n e r a t i o n and most l i k e l y g r e a t e r i n c r e a s e s would have been observed ove r s e v e r a l g e n e r a t i o n s . Because o f the l a c k o f knowledge o f the two hos t c u l t i v a r geno t ypes , a t rue compar ison o f the changes o c c u r r i n g on a r e s i s t a n t v s . s u s c e p t i b l e c u l t i v a r cou ld not be made. The c u l t i v a r s T r e b i and Odessa were chosen because o f some p r e v i o u s g e n e t i c i n f o r m a t i o n (Ebba 1974, Apendix A & B ) . Other c u l t i v a r s were t e s t e d w i th the p a r e n t a l c u l t u r e s but a l l proved to be comp le t e l y r e s i s t a n t 138 to both smut pa r en t s (Appendix C ) . I t was impor tant t ha t the r e s i s t a n t c u l t i v a r a l l ow some i n f e c t i o n t o take p l a c e so t ha t s e r i a l passages o f the smut p o p u l a t i o n s c o u l d be made. Changes i n a l l e l e f r equency o r the s h i f t i n g from a v i r u l e n t to a v i r u l e n t a l l e l e w i t h i n a pathogen p o p u l a t i o n on a s u s c e p t i b l e c u l t i v a r i s the b a s i s f o r s t a b i l i z i n g s e l e c t i o n . At t h i s t ime the re i s no i n f o r m a t i o n , o n l y t h e o r y , on v i r u l e n c e a l l e l e f r equency changes i n pathogen p o p u l a t i o n s and what s e l e c t i o n p r e s s u r e i s needed f o r these changes to o c c u r . T h i s type o f i n f o r m a t i o n i s needed to c o n t r o l pathogen p o p u l a t i o n l e v e l s us ing r e s i s t a n c e genes . A s s o c i a t e d w i th changes i n a l l e l e f r equency i s the concept o f f i t n e s s . Most o f the l i t e r a t u r e i n p l a n t pa tho logy at tempts to a s s o c i a t e f i t n e s s v a l u e s w i th v i r u l e n c e genes . F i t n e s s of- a c e r t a i n genotype i s not j u s t the f u n c t i o n o f a p a r t i c u l a r l o c u s but a complex f u n c t i o n o f many genes . Most au tho rs assume f i t n e s s o r s e l e c t i v e v a l u e s f o r a g i v e n genotype to be cons t an t ove r t ime but i n an a b s o l u t e sense are not (Koj ima 1971) . Maynard Smith (1962) p o i n t e d out t ha t a s t a b l e polymorphism can be ma in ta ined i f the f i t n e s s o f p a r t i c u l a r genotypes i n c r e a s e s as t h e i r f r equency i n the p o p u l a t i o n d e c r e a s e s . Mode (1958) and Person (1966) d i s c o v e r e d how a ba lanced polymorphism c o u l d deve l op i n h o s t - p a r a s i t e sys tems . They po in t ed out tha t a gene f o r r e s i s t a n c e has low s e l e c t i v e advantage as long as the match ing v i r u l e n c e gene i s common i n the p a r a s i t e p o p u l a t i o n but the v a l ue i s h i gh i f the co r r e spond ing v i r u l e n c e gene i s 139 r a r e . L i kew ise i n the pa thogen , the v i r u l e n c e gene has a h igh s e l e c t i v e advantage when the co r r e spond ing r e s i s t a n c e gene i s p r e sen t i n the hos t p o p u l a t i o n , but has a low va lue when the r e s i s t a n c e gene i s absen t . T h i s i s why Vanderp lank suggested t ha t unnecessary v i r u l e n c e genes a re somewhat d i s advan t ageous . At tempts were made w i t h i n these exper iments to o b t a i n measurements f o r both hos t and pathogen r e p r o d u c t i v e a b i l i t y . For most o f the g e n e r a t i o n s , e x t e r n a l c i r cums tances d i d not a l l o w f o r these measurements to be t a k e n . For the h o s t , e i t h e r seed number o r seed weight per p l a n t c o u l d be used . For the pathogen the bes t measurement would be number o f spores o r we ight o f s p o r e s . With the l i m i t e d da t a on spore weight a s s o c i a t e d w i th pe r cen t smutted p l a n t s from these e x p e r i m e n t s , the re was a p o s i t i v e c o r r e l a t i o n f o r the pathogen genotypes examined ( C h r i s t u n p u b l i s h e d ) . However, o b s e r v a t i o n s on the d i f f e r e n t phenotypes o f smut t ing per head would i n d i c a t e t h a t pe r cen t smutted p l a n t s does not have to c o r r e l a t e p o s i t i v e l y w i th number o f spores produced ( p e r s o n a l o b s e r v a t i o n s ) . In the s e l e c t i o n scheme (Chapter 4) i t was proposed tha t two s u b p o p u l a t i o n s would be s e l e c t e d , one p roduc ing h igh l e v e l s o f smut and the o the r p roduc ing low l e v e l s on each c u l t i v a r . These s u b p o p u l a t i o n s would r e p r e s e n t the end p o i n t s below and above the mean o f the p o p u l a t i o n . T h i s i s d i s r u p t i v e s e l e c t i o n U n f o r t u n a t e l y , because o f the i n a b i l i t y to determine these phenotypes w i t h i n each p o p u l a t i o n , t h i s type o f s e l e c t i o n was not a c h i e v e d . G e n e r a l l y , s e l e c t i o n was f o r the genotypes t ha t 140 shou ld g i v e h igh l e v e l s o f smut. Re su l t s i n d i c a t e d t ha t the re was enough v a r i a b i l i t y w i t h i n each p o p u l a t i o n so t h a t the p o p u l a t i o n s were s t a b i l i z i n g a t a p a r t i c u l a r l e v e l o f smut which i s what one would expec t a c c o r d i n g to the Hardy-Weinberg law. Cherewick (1958) a l s o conc luded tha t i t was not p o s s i b l e to o b t a i n a homogeneous p o p u l a t i o n o f smut t e l i o s p o r e s . N i c h o l a i s e n (1934) , a l s o working w i th U. avenae , made s i m i l a r o b s e r v a t i o n s to those o f Cherewick . He conc luded tha t v a r i e t i e s impose changes i n p a t h o g e n i c i t y o f the smut c u l t u r e s and w i t h i n a s i n g l e i n c r e a s e o f a heterogeneous p o p u l a t i o n the re i s a d e c l i n e i n the l e v e l o f smut. He specu l a t ed t ha t the d e c l i n e p o s s i b l y r e s u l t e d from the e x c l u s i o n o f some pathogen geno types . L e i t z k e (1937) a l s o observed changes i n p a t h o g e n i c i t y o f heterogeneous smut c u l t u r e s w i th passages through d i f f e r e n t hos t c u l t i v a r s . He a l s o observed a r e d u c t i o n i n the l e v e l o f smut w i th the heterogeneous p o p u l a t i o n s , whereas the i n d i v i d u a l components o f the p o p u l a t i o n con t i nued to produce h igh l e v e l s o f smut when on t h e i r own. L e i t z k e gave reasons f o r the changes observed w i th passag ing o f c u l t u r e s through v a r i o u s c u l t i v a r s . These i n c l u d e d : d i f f e r e n c e s o f spore g e r m i n a t i o n o f d i f f e r e n t smut geno t ypes , c o l o n y t ypes produced from a t e l i o s p o r e , and new geno types . For f u r t h e r p o p u l a t i o n s t u d i e s w i th U. h o r d e i , i t would be u s e f u l to form a p o p u l a t i o n s t a r t i n g from a s i n g l e he te rozygous d i k a r y o n and observe changes o f a v i r u l e n c e a l l e l e ove r g e n e r a t i o n s o f s e l e c t i o n on a r e s i s t a n t and s u s c e p t i b l e 141 c u l t i v a r . Some p o p u l a t i o n s o f t h i s type were formed, but s e l e c t i o n has not gone f u r t h e r than one g e n e r a t i o n . Aga in w i th these p o p u l a t i o n s , the re would be d i f f i c u l t y i n s o r t i n g out the e f f e c t s o f the major gene from the e f f e c t s o f the p o l y g e n e s . Whi le r e c o r d i n g r e s u l t s f o r the a l l e l e f r equency t e s t s , o b s e r v a t i o n s were made on the amount o f v a r i a b i l i t y o f degree o f smutt ing ( p a r t i a l , comp le t e , e t c . ) w i t h i n a s i n g l e geno type . T h i s v a r i a b i l i t y must stem from the v a r i o u s p h y s i o l o g i c a l i n t e r a c t i o n s o f the hos t and p a r a s i t e t ha t occu r du r i ng the growing p e r i o d o f the h o s t . To q u a n t i f y t h i s v a r i a b i l i t y , would r e q u i r e more knowledge o f these p h y s i o l o g i c e v e n t s . The v a r i a b i l i t y cou ld a l s o stem from m u l t i p l e i n f e c t i o n s o f a p a r t i c u l a r s e e d l i n g . M u l t i p l e i n f e c t i o n s by a s i n g l e smut s p e c i e s was c l e a r l y demonstrated by Person and Cherewick (1964) Us ing combina t ions o f a bu f f mutant and w i l d type cu l tu re ' s o f U. K o l l e r i , r e s u l t s i n d i c a t e d t h a t the m a j o r i t y o f d i s e a s e d p l a n t s had been i n f e c t e d tw ice and s e v e r a l p l a n t s may have had up to f o u r i n f e c t i o n s . I f a s i n g l e p l a n t can have s e v e r a l i n f e c t i o n s , then i n a heterogeneous smut p o p u l a t i o n i n c r e a s e d l e v e l s o f smut may be obse r ved . The i n c r ea sed l e v e l s would r e s u l t from c e r t a i n genotypes be ing b e t t e r ab l e to i n f e c t the s e e d l i n g than o the r geno t ypes , t h e r e f o r e , an i n c r e a s e i n the p r o b a b i l i t y o f o b s e r v i n g d i s e a s e pe r s e e d l i n g may be o b s e r v e d . An e x c e l l e n t example where t h i s phenomenon may be observed i s a r e i n t e r p r e t a t i o n o f da ta from H a l i s k y (1956) . H a l i s k y c rossed th ree r a ces o f U. avenae i n a l l combina t ions and t e s t ed s e v e r a l oat c u l t i v a r s . On the c u l t i v a r Monarch, the homozygous domin -ant smut genotype was a v i r u l e n t (0% smut ) , the he te rozygous genotype produced an average o f 8% smutted p l a n t s and the homozygous r e c e s s i v e produced an average o f 80% smutted p l a n t s . Each o f the i n d i v i d u a l F 2 d i k a r y o n s was i n c r ea sed by us ing the t e l i o s p o r e s f o r i n o c u l a t i o n . What i s i n t e r e s t i n g i s the i n c r e a s e d l e v e l s o f smutted p l a n t s observed d u r i n g the i n c r e a s e (Appendix E ) . T h i s i n c r e a s e i n smut l e v e l s can be e x p l a i n e d by m u l t i p l e i n f e c t i o n s . Even f o r the homozyous v i r u l e n t genotypes the re were i n c r e a s e s r ang ing from 3 to 19% r e s u l t i n g from an i n c r e a s e i n p r o b a b i l i t y o f g e t t i n g i n f e c t i o n . I n o c u l a t i n g w i th the he te rozygous F 2 d i k a r y o n s produces the p o s s i b i l i t y o f i n f e c t i o n o c c u r r i n g by any o f the th ree genotypes w i th random ma t i ng . The i n c r e a s e d l e v e l s observed i n the i n c r e a s e o f the he te rozygous F 2 ' s i s a comb ina t ion o f the he te rozygous and homozygous v i r u l e n t genotypes which can both be i n f e c t i n g the s e e d l i n g s . Us ing H a l i s k y ' s d a t a , p r e d i c t i o n s o f observed pe r cen t smut w i th a 1, 2, 3 o r 4 i n f e c t i o n s per s e e d l i n g can be made (Tab le 8.1 and F i gu re 8.1) and i n c r e a s e s by a p a r t i c u l a r pe rcentage can be expected i f m u l t i p l e i n f e c t i o n s o c c u r . Pe r cen t smut was not c a l c u l a t e d f o r over f o u r i n f e c t i o n s pe r seed because Person and Cherewick o n l y observed up to f o u r i n f e c t i o n s per p l a n t . In t h e o r y , however, the re can be many i n f e c t i o n s per seed and the curve ( F i g . 8.1) would approach 100 p e r c e n t . M u l t i p l e i n f e c t i o n s not o n l y can account f o r the v a r i a b i l i t y observed but can e x p l a i n the i n c r e a s e o f smutted 143 Tab l e 8.1 P r o b a b i l i t y o f d i s e a s e l e v e l s when m u l t i p l e i n f e c t i o n s o c cu r w i th a heterogenous smut p o p u l a t i o n 1 - % Prob. T o t a l % I n f e c t i o n / smutted h e a l t h y Prob Smut .Seed Genotype Freg p l a n t s p l a n t s (H) 1-H 1 AA .25 1.00 .25 Aa .50 .92 .46 .76 .24 aa .25 .20 .05 AA: AA .06 1.00x1.00 .06 AA: Aa .25 l.OOx .92 .23 AA:aa .13 l .OOx .20 .03 Aa: Aa .25 .92x .92 .21 Aa : aa .25 .92x .20 .05 aa :aa .06 .20x .20 0 Theory based on da ta o f H a l i s k y (1956) where on average AA, Aa , aa gave 0, 8 and 80% smutted p l a n t s , r e s p e c t i v e l y . Theory i n p a r t from C O . Person ( pe r sona l communica t ion ) . F i g u r e 8.1 Pe rcen t smutted p l a n t s expected f o r v a r y i n g numbers o f i n f e c t i o n s per seed by U s t i l a g o avenae p l a n t s observed d u r i n g the f i r s t g e n e r a t i o n o f s e l e c t i o n . Exper iments s i m i l a r to those o f Person and Cherewick were s e t up us ing bu f f mutants i n comb ina t ion w i th w i l d type U. h o r d e i . Mixed i n f e c t i o n s were o b s e r v e d , but q u a n t i t a t i v e da t a were d i f f i c u l t to o b t a i n ( C h r i s t u n p u b l i s h e d ) . S i m i l a r f l u c t u a t i o n s i n l e v e l s o f smut were observed on both c u l t i v a r s . Whether these s i m i l a r p a t t e r n s are e x p l a i n e d by the i n c r e a s i n g o f smut genotypes w i th the o p t i m a l po lygene con ten t o r by the two c u l t i v a r s hav ing some r e s i s t a n c e gene i n common i s not known. C r o s s i n g exper iments w i th the two c u l t i v a r s were s t a r t e d but the F 2 ' s have not ye t been t e s t e d . P r e v i ous c r o s s i n g exper iments o f b a r l e y which were t e s t e d w i th U. h o r d e i have demonstrated r e s i s t a n c e c o n t r o l l e d by s i n g l e genes (S idhu & Person 197 2 ) . B a r l e y i s known to have q u a n t i t a t i v e l y i n h e r i t e d r e s i s t a n c e to o the r pathogens ( P a r l e v l i e t 1978) and t h e r e f o r e , cou ld have q u a n t i t a t i v e l y i n h e r i t e d r e s i s t a n c e to U. h o r d e i . I t would be i n t e r e s t i n g to f i n d a h o s t - p a r a s i t e system w i th r e s i s t a n c e and v i r u l e n c e q u a n t i t a t i v e l y i n h e r i t e d and observe p o p u l a t i o n f l u c t u a t i o n s o f both the hos t and pa thogen . In these types o f s t u d i e s s t a b i l i z i n g s e l e c t i o n , as used by p o p u l a t i o n g e n e t i c i s t s , might be o b s e r v e d . R e c e n t l y , the re i s major i n t e r e s t i n q u a n t i t a t i v e l y i n h e r i t e d r e s i s t a n c e and the s t a b i l i t y o f i t . Now tha t the re i s ev idence f o r p o l y g e n i c a l l y determined a g g r e s s i v e n e s s i n U. h o r d e i , t h i s type o f i n h e r i t a n c e may be 146 found i n o t h e r smut s p e c i e s . P o l y g e n i c a l l y determined a g g r e s s i v e n e s s i n c u l t u r e s o f U. avenae used by Cherewick (1958) cou ld a l t e r the i n t e r p r e t a t i o n o f h i s s e l e c t i o n e x p e r i -ments . Changes i n f requency o f a l l e l e s o f a major v i r u l e n c e gene were proposed as a c coun t i ng f o r the changes i n pe r cen t smut observed ove r e i g h t g e n e r a t i o n s (Chapter 2 ) . These changes may have r e s u l t e d from i n c r e a s e s i n a g g r e s s i v e n e s s i n the smut p o p u l a t i o n s . That cou ld e a s i l y e x p l a i n why s i m i l a r i n c r e a s e s were observed on the s i x s u s c e p t i b l e c u l t i v a r s . I t i s i n t e r e s t i n g to note t h a t L e i t z k e (1937) c o n s i d e r e d t e l i o s p o r e ge rm ina t i on r a t e to be r e s p o n s i b l e f o r changes observed i n a smut p o p u l a t i o n and , i n the s e l e c t i o n exper iment o f t h i s r e s e a r c h , ge rm ina t i on was a major f a c t o r . L e i t z k e a l s o noted d i f f e r e n c e s i n mat ings between h a p l o i d c u l t u r e s depending on co l ony morphology such as m y c e l i a l o r s p o r i d i a l . Groth ( p e r s o n a l communication) has found tha t m y c e l i a l c o l o n i e s o f U. h o r d e i tend to produce low l e v e l s o f smut. I f Groth i s c o r r e c t , the co lony morphology would e x p l a i n the d r a s t i c r e d u c t i o n i n smut l e v e l s observed i n s e v e r a l o f these expe r imen t s . Kernkemp (1939, 1942) showed tha t the re were th ree types o f h a p l o i d c u l t u r e s , e n t i r e l y s p o r i d i a l , e n t i r e l y myce l ium, and a m i x t u r e . The f i r s t two never change i n morphology but the l a t t e r does under c e r t a i n env i ronmenta l c o n d i t i o n s . M y c e l i a l f o rma t i on was s t i m u l a t e d by env i ronmenta l c o n d i t i o n s t ha t are g e n e r a l l y un f a vo rab l e to the growth o f the o rgan i sm , e . g . , p o i s o n s added 147 to the media o r low n u t r i e n t c o n c e n t r a t i o n s . N i e l s e n (1966) d i s c o v e r e d a v o l a t i l e subs tance r e l e a s e d by the smut which a l s o a l t e r e d the co l ony morphology to a m y c e l i a l t y p e . Many o f the h a p l o i d c o l o n i e s taken from s i l i c a g e l were o f the m y c e l i a l type and r a r e l y r e v e r t e d to the s p o r i d i a l t y p e . For a p a r a s i t e such as smut, the h e a l t h and v i g o r o f the hos t i s v e r y impor t an t . A sma l l s tudy on seed ing r a t e was made to see i f space p l a n t i n g would a l t e r the l e v e l s o f smut (Appendix D) . The r e s u l t s i n d i c a t e d t h a t the re were d i f f e r e n c e s . These da t a can a l s o be used to examine hos t sample s i z e needed to d e t e c t a s p e c i f i c l e v e l o f smut. Where the number o f p l a n t s i s l e s s than 100 an a c cu r a t e i n d i c a t i o n o f the l e v e l o f smutt ing i s not o b t a i n e d . The i n i t i a l o b j e c t i v e o f t h i s exper iment was not ob t a i ned f o r the f o l l o w i n g r e a s o n s : 1. I t was not p o s s i b l e t o p r e d i c t which t e l i o s p o r e s from the s e l e c t i o n exper iment would r e p r e s e n t genotypes r e s u l t i n g i n h igh o r low l e v e l s o f smut. The r e fo r e t e l i o s p o r e s were randomly s e l e c t e d to r e p r e s e n t the h igh l e v e l s o f smut. 2. F i v e d i f f e r e n t F 2 t e l i o s p o r e l i n e s were used per p o p u l a t i o n so tha t the re would be enough t e l i o s p o r e s t o use f o r i n o c u l a t i o n o f the s e l e c t i o n exper iment and f o r t e s t i n g the a l l e l e f r equency . Th i s made the p o p u l a t i o n s ve r y heterogeneous and made i t d i f f i c u l t to q u a n t i f y the v a r i a b i l i t y . 148 3. There was no g e n e t i c i n f o r m a t i o n conce rn ing the r e s i s t a n c e genes o f T r e b i and Odessa . 4. T e l i o s p o r e l i n e s d i d not have equa l ge rm ina t i on r a t e s and p o s s i b l y had d i f f e r e n t l e v e l s o f i n f e c t i o n a b i l i t i e s . 5. The i n a b i l i t y to d i s t i n g u i s h between the e f f e c t s o f the v i r u l e n c e gene and the e f f e c t s o f the po l ygenes made i t i m p o s s i b l e to determine a l l e l e f requency o f the v i r u l e n c e gene. 6. There was c o n s i d e r a b l e v a r i a t i o n i n smut phenotypes ( p a r t i a l , comp le te , basa l ) and i n number o f smutted t i l l e r s per p l a n t . I t i s not known whether t h i s v a r i a t i o n i s caused by the a c t i o n o f the po l ygenes o r how the po lygenes modi fy the e f f e c t s o f the v i r u l e n c e gene. Because o f c o m p l i c a t i o n s , the s e l e c t i o n exper iment was o n l y c a r r i e d out f o r th ree g e n e r a t i o n s and the a l l e l e f r equency t e s t s were stopped a f t e r one g e n e r a t i o n . More g e n e r a t i o n s o f s e l e c t i o n are needed to make any s ta tement on s t a b i l i z i n g s e l e c t i o n o r on hos t a d a p t a t i o n . The a l l e l e f requency t e s t s a re p robab l y not a good r e f l e c t i o n o f the change i n the v i r u l e n c e a l l e l e . C o n c l u s i o n s from these s t u d i e s a r e : 1. T e l i o s p o r e s from s i n g l e d i k a r y o n s shou ld have been used f o r each p o p u l a t i o n i n the s e l e c t i o n exper iments to dec rease the h e t e r o g e n e i t y . 149 2. U. h o r d e i - b a r l e y may not be the bes t system f o r examining the concept o f s t a b i l i z i n g s e l e c t i o n because o f the p o i n t s l i s t e d above. 3. Abnormal g e r m i n a t i o n o f t e l i o s p o r e s i n t e l i o s p o r e i n o c u l a t i o n i s a s s o c i a t e d w i th reduced l e v e l s o f smut. 4. Exogenous n u t r i e n t s and temperature had no e f f e c t on the abnormal ge rm ina t i ng t e l i o s p o r e s . 5. There was some ev idence t ha t the abnormal g e r m i n a t i o n i s an i n t r i n s i c p r o p e r t y o f s p e c i f i c t e l i o s p o r e l i n e s . 6. The abnormal ge rm ina t i on o f s p e c i f i c t e l i o s p o r e l i n e s made i t i m p o s s i b l e to o b t a i n t e t r a d s o f those l i n e s f o r compar ison o f o the r t e l i o s p o r e l i n e s i n the g e n e t i c a n a l y s i s . 7. Because o f the n o n s p e c i f i c a c t i o n o f the p o l y g e n e s , the d i k a r y o n s o f i n d i v i d u a l t e l i o s p o r e s were s e g r e g a t i n g i n f a i r l y s i m i l i a r f a s h i o n on the two c u l t i v a r s . T h i s c o u l d e x p l a i n the s i m i l i a r response o f the two c u l t i v a r s i n the s e l e c t i o n and a l l e l e f requency expe r imen t s . Many q u e s t i o n s were genera ted by these s t u d i e s : 1. Why i s there so much v a r i a b i l i t y i n smut phenotype and the number o f smutted t i l l e r s pe r p l a n t w i t h i n a s i n g l e genotype o f smut? 2. Is t h i s v a r i a b i l i t y the r e s u l t o f p h y s i o l o g i c i n t e r a c t i o n s between the p l a n t and smut, p o s s i b l e c o m p e t i t i o n between d i f f e r e n t smut d i k a r y o n s w i t h i n the p l a n t , o r both? 150 3. O lde r l i t e r a t u r e r e p o r t s seed i n f e c t i n g smuts are not hos t s p e c i f i c i n i n i t i a l i n f e c t i o n , t h e r e f o r e , where and when does the v i r u l e n c e gene p l a y i t ' s p a r t i n de te rm in ing the f u t u r e course o f events w i t h i n the i n f e c t e d p l an t ? 4. Why d i d n ' t the smutted p o p u l a t i o n o f b a r l e y have fewer seeds than the c o n t r o l p o p u l a t i o n ? 5. Do U. h o r d e i i n f e c t i o n s s t i m u l a t e t i l l e r i n g ? 6. Is the r e s i s t a n c e o f T r e b i and Odessa matched to these smut c u l t u r e s i n h e r i t e d by s i n g l e genes o r s e v e r a l genes? 7. Does Odessa have a r e s i s t a n c e gene matched t o these smut c u l t u r e s ? 8. Is the a t y p i c a l g e r m i n a t i o n an i n h e r i t e d t r a i t ? 9. How do the po lygenes modi fy the e f f e c t s o f the v i r u l e n c e gene? 10. What env i ronmenta l f a c t o r s have the g r e a t e s t i n f l u e n c e on a l t e r i n g smut l e v e l s and can they be q u a n t i f i e d ? 11. I t i s known t h a t a s i n g l e p l a n t may be i n f e c t e d by s e v e r a l d i k a r y o n s . Is the re c o m p e t i t i o n among these d i k a r y o n s w i t h i n the p l a n t and are o n l y few o f these d i k a r y o n s s u c c e s s f u l i n p roduc ing t e l i o s p o r e s ? S e v e r a l g u i d e l i n e s a r i s e from these s t u d i e s : 1. It i s necessa r y to unders tand the b a s i c b i o l o g y o f the h o s t - p a r a s i t e system under exam ina t i on . 2. When p o s s i b l e , g e n e t i c a n a l y s i s o f the hos t and p a r a s i t e shou ld be done s i m u l t a n e o u s l y . Smuts have a c o n s i d e r a b l e amount o f v a r i a b i l i t y w i t h i n a p o p u l a t i o n . With more knowledge o f t h e i r p h y s i o l o g y and b e t t e r means o f q u a n t i f y i n g d i s e a s e l e v e l s , they cou ld be f a v o r a b l e pathogens to work w i t h . P o p u l a t i o n g e n e t i c s o f h o s t - p a r a s i t e sys tems , a t t h i s t ime , has no i n f o r m a t i o n on a l l e l e f r e q u e n c i e s , muta t ion r a t e s o r s e l e c t i o n c o e f f i c i e n t s . S ince we do not expec t s e l e c t i o n a g a i n s t v i r u l e n c e genes to be to the same d e g r e e , the re i s no reason to assume V a n d e r p l a n k 1 s s t a b i l i z i n g s e l e c t i o n shou ld app l y e q u a l l y f o r a l l pathogens and a l l v i r u l e n c e genes . 152 LITERATURE CITED Aamodt, O.S . and W.H. J ohns ton . 1935. Reac t ions o f b a r l e y v a r i e t i e s to i n f e c t i o n w i th covered smut ( U s t i l a g o h o r d e i ( Pe r s . ) K & S ) . Can. J o u r . Res. 12 :590-613. A l l a r d , R.W. 1960. P r i n c i p l e s o f P l a n t B r e e d i n g . John Wi ley & Sons , I n c . , New Yo rk , 485 pp . Anonymous. 1979. B a r l e y : O r i g i n , Botany , C u l t u r e , Winter H a r d i n e s s , G e n e t i c s , U t i l i z a t i o n , P e s t s . U .S .D .A . Ag . Handbook No. 338, 154 pp . As l am , M. and L . E . Browder. 1977. S t a b i l i z i n g s e l e c t i o n f o r p a t h o g e n i c i t y i n c e r e a l r u s t f u n g i . Pages 205-212. In : Gene t i c d i v e r s i t y i n p l a n t s . Eds . Muhammed, A . , R. A k s e l , and R.C. VonBo r s t e l Plenum P r e s s , New York . Baker , B.S. A . T . C . C a r p e n t e r , M.S. E s p o s i t o , R.E. E s p o s i t o , and L. S and l e r . 1976. The g e n e t i c c o n t r o l o f m e i o s i s . Ann. Rev. G e n e t i c s 10 :53-134. Baker , E.P. 1966. I s o l a t i o n o f complementary genes c o n d i t i o n i n g crown r u s t r e s i s t a n c e i n the oa t v a r i e t y Bond. Euphy t i c a 15:313-318. B a r r e t t , J . A . 1978. A model o f ep idemic development i n v a r i e t y m i x t u r e s . Pages 129-137. In : P l a n t D isease Ed idemio logy . e d s . S c o t t , P.R. and A. B a i n b r i d g e . B l a c k w e l l S c i . P u b l . , O x f o r d . B a r r e t t , J . A . 1983. E s t i m a t i n g r e l a t i v e f i t n e s s i n p l a n t p a r a s i t e s : Some g e n e r a l p rob lems . Phy topa tho logy 73:510-512. B a r r e t t , J . A . and M.S. Wo l fe . 1978. M u l t i l i n e s and supe r- races — a r e p l y . Phy topa tho logy 68:1535-1537. B a r r u s , M.F. 1911. V a r i a t i o n i n v a r i e t i e s o f beans i n t h e i r s u s c e p t i b i l i t y to a n t h r a c n o s e . Phy thopatho logy 1:190-195. B a r t o s , P., P .L . Dyck and D.J . Samborsk i . 1969. A d u l t - p l a n t l e a f r u s t r e s i s t a n c e i n Tha tcher and Marquis wheat: a g e n e t i c a n a l y s i s o f the h o s t - p a r a s i t e i n t e r a c t i o n . Can. J . Bo t . 47 :267-269. B a s s i , F . G . A . , J . H . B u r n e t t . 1980. The g e n e t i c a r c h i t e c t u r e o f a g g r e s s i v e n e s s i n U s t i l a g o mayd i s . Ann. A p p l . B i o l . 94 :281 . 153 Bauch, R. 1932. Die S e x u a l i t a t von U s t i l a g o Scorzonera and U s t i l a g o zeae . Phy topa th . Z e i t s . 5:315-321. B i f f e n , R.H. 1905. Mende l ' s law o f i n h e r i t a n c e and wheat b r e e d i n g . J ou r . A g r i c . S c i . 1:4-48. B l a n c h , P.A. M . J .C . Asher & J . H . B u r n e t t . 1981. I nhe r i t a nc e o f p a t h o g e n i c i t y and c u l t u r a l c h a r a c t e r s i n Gaeumannomyces  g r a m i n i s v a r . t r i t i c i . T r a n s . Br . M y c o l . Soc . 77 :319-399. B o r l a u g , N.E . 1959. The use o f m u l t i l i n e a l o r compos i te v a r i t i e s to c o n t r o l a i r b o r n e ep idemic d i s e a s e s o f s e l f - p o l l i n a t e d c rop p l a n t s . P roc . I n t e r n . Wheat Genet . Symp. 1:12-26. B o r s t , P. and G .A .M . C r o s s . 1982. M o l e c u l a r b a s i s f o r Trypanosome a n t i g e n i c v a r i a t i o n . C e l l 29:291-303. Bowman, D. 1946. S p o r i d i a l f u s i o n i n U s t i l a g o mayd i s . J ou r . A g r i . Res. 72 :233-243. B r a s i e r , C M . 1977. I nhe r i t a nc e o f p a t h o g e n i c i t y and c u l t u r a l c h a r a c t e r s i n C e r a t o c y s t i s u l m i ; h y b r i d i z a t i o n o f p r o t o p e r i c i t h e c i a l and non-aggress i ve s t r a i n s . T r a n s . B r . Mycol Soc . 68 :45-52 . B r a s i e r , C M . and J . N . G i b b s . 1976. I nhe r i t ance o f p a t h o g e n i c i t y and c u l t u r a l c h a r a c t e r s i n C e r a t o c y s t i s  u l m i : h y b r i d i z a t i o n o f a g g r e s s i v e and non-aggress i ve s t r a i n s . Ann. A p p l . B i o l . 83 :31-37 . Brown, J . F . and E .L . Sharp . 1970. The r e l a t i v e s u r v i v a l a b i l i t y o f pa thogen i c t ypes o f P u c c i n i a s t r i i f o r m i s i n m i x t u r e s . Phy topatho logy 60:529-533. Browning. J . R . , M.D. S imons, K. J . Frey and H.C. Murphy. 1969. Reg iona l deployment f o r c o n s e r v a t i o n o f oa t c rown-rust r e s i s t a n c e genes . Iowa A g r i c . Home Econ. Exp. S t a t . Spec. Rep. 64 :49-56 . Burdon. J . J . 1978. Mechanisms o f d i s e a s e c o n t r o l i n heterogeneous p l a n t p o p u l a t i o n s - an e c o l o g i s t ' s v i ew. Pages 193-200. In : P l an t D i sease Ep idemio logy e d s . P.R. S co t t and A. Ba i nb r i dge B l a c k w e l l S c i . P u b l . , O x f o r d . C a r e f o o t , G .L . and E.R. S p r o t t . 1967. Famine i n the Wind. Rand McNal ly & Co. 229 pp . 154 Cargeeg , L .A. and N. T h u r l i n g . 1980. Seed l i ng and a d u l t r e s i s t a n c e to B l a c k l e g ( L ep tosphae r i a maculans Desm. Ces . e t . de Not . ) i n Spr ing Rape (B rass i ca " napus L.) . A u s t . J . A g r i c . Res. 31 :37-46. C a t e n , C . E . 1974. I n t r a - r a c i a l v a r i a t i o n i n Phy tophthora i n f e s t a n s and a d a p t a t i o n to f i e l d r e s i s t a n c e f o r p o t a t o b l i g h t . Ann. A p p l . B i o l . 77 :259-270. C a t r i d e r , P.G. and D. G o t t l i e b . 1966. E f f e c t o f sugars on g e r m i n a t i o n and metabol ism o f t e l i o s p o r e s o f U s t i l a g o  mayd i s . Phy topa tho logy 56:479-484. Cherew ick , W.J. 1958. C e r e a l smut r a ces and t h e i r v a r i a b i l i t y . Can. J . P l an t S c i . 38 :481-489. Cherew ick , W.J. 1967. I nhe r i t a nc e o f v i r u l e n c e i n U s t i l a g o  h o r d e i and U s t i l a g o n i g r a . Can. J . Genet . C y t o l . 9 :141-146. Cherew ick , W.J. and K.W. Buchannon. 1969. I nhe r i t ance o f r e s i s t a n c e to covered and f a l s e l o o s e smut i n the b a r l e y v a r i e t i e s , Pann ie r and E x c e l s i o r . Can. J . Genet . C y t o l . 11 :250-253. Cherewick , W.J. and R . I .H . McKenz ie . 1969. I nhe r i t a nc e o f r e s i s t a n c e to l o o s e smut and covered smut i n the oa t v a r i e t i e s , B l ack Mesdag, Camas, and Rodney. Can. J . Genet . C y t o l . 11 :919-923. C h i l t o n , S t . John P. 1938. The occu r r ence o f l y s i s i n c e r t a i n c r o s s e s o f U s t i l a g o zeae ( A b s t . ) . Phy topa tho logy 2 8 : 5 . C h i l t o n , S t . John P. 1940. Delayed r e d u c t i o n o f the d i p l o i d nuc l eus i n p r o m y c e l i a o f U s t i l a g o zeae . Phy topa tho logy 30:622-623. C h i l t o n , S t . John P. 1943. A h e r i t a b l e abno rma l i t y i n the g e r m i n a t i o n o f ch lamydospores o f U s t i l a g o zeae . Phy topatho logy 33 :749-765. C h r i s t , B.J . and J . V . G r o t h . 1982. I nhe r i t ance o f v i r u l e n c e t o th ree bean c u l t i v a r s i n th ree i s o l a t e s o f the bean r u s t pa thogen . Phy topa tho logy 72 :767-770. C h r i s t e n s e n , J . J . 1931. S t u d i e s on the g e n e t i c s o f U s t i l a g o  zeae . Phy topa th . Z e i t s . 4 :129-188. C h r i s t e n s e n , J . J . and H.A. Rodenh ise r . 1940. 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 o f g e n e t i c s o f smut f u n g i . B o t a n i c a l Rev. 8 :389-425. 155 C l i f f o r d , B .C. and R.B. C l o t h i e r . 1974. 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 o f P u c c i n i a h o r d e i on b a r l e y hos t s w i th n o n - h y p e r s e n s i t i v e r e s i s t a n c e . T r a n s . Br . M y c o l . Soc . 63 :421-430. Co t t i ngham, C . 1958. I nhe r i t ance o f r e s i s t a n c e to covered smut i n s e v e r a l h y b r i d s o f b a r l e y . Ph .D. T h e s i s , M ich igan S t a t e U n i v e r s i t y . C r a i g i e , J . C . 1927. D i s cove r y o f the f u n c t i o n o f the p y c n i a o f the r u s t f u n g i . Nature 120:765-767. Day, P.R. 1978. The g e n e t i c b a s i s o f e p i d e m i c s . Pages 263-285. I n : P l a n t D i sease V o l . 2: How d i s e a s e deve lops i n p o p u l a t i o n s , e d s . J . G . H o r s f a l l and E.D. Cow l ing . Acad . P r e s s . , N.Y. D r i v e r , C M . 1962a. B reed ing f o r r e s i s t a n c e t o d i s e a s e s and p e s t s . Amer. Po ta to J o u r . 39 :57-62. D r i v e r , C M . 1962b. B reed ing f o r D i sease r e s i s t a n c e . S co t . P l an t Breed ing S t a t . B u l l . 1-11. Dyck, P.L. and D.J . Samborsk i . 1968. G e n e t i c s o f r e s i s t a n c e to l e a f r u s t i n the common wheat v a r i e t i e s . Webster , L o r o s , B r e v i t , C a r i n a , Malakof and C e n t e n a r i o . Can. J . Genet . C y t o l . 10 :7-17 . Dyck, P.L. and D. J . Samborsk i . 1974. I nhe r i t ance o f v i r u l e n c e i n P u c c i n i a r e c o n d i t a on a l l e l e s a t the Lr2 Locus f o r r e s i s t a n c e i n wheat. Can. J . Genet . C y t o l 16:323-332. Dyck, P.L. and D.J . Samborsk i . 1982. The i n h e r i t a n c e o f r e s i s t a n c e t o P u c c i n i a r e c o n d i t a i n a group o f common wheat c u l t i v a r s . Can. J . Genet . C y t o l . 24 :273-283. Dyck, P . L . , D. J . Samborsk i , and R.G. Anderson . 1966. I nhe r i t ance o f a d u l t - p l a n t l e a f r u s t r e s i s t a n c e d e r i v e d from the common wheat v a r i e t i e s Exchange and F ron t ana . Can. J . Genet . C y t o l . 8 :665-671. Ebba, T . 1974. Gene t i c S t u d i e s o f the h o s t - p a r a s i t e r e l a t i o s h i p between U s t i l a g o h o r d e i and Hordeum v u l g a r e . Ph .D . T h e s i s U n i v e r s i t y o f B r i t i s h Co lumbia . T40 pp . Ebba, T . and C. P e r son . 1975. Gene t i c c o n t r o l o f v i r u l e n c e i n U s t i l a g o h o r d e i . IV D u p l i c a t e genes f o r v i r u l e n c e and g e n e t i c and env i ronmenta l m o d i f i c a t i o n o f a gene-for-gene r e l a t i o n s h i p . Can. J . Genet . C y t o l . 17:631-636. 156 Een ink , A . H . 1976. G e n e t i c s o f h o s t - p a r a s i t e r e l a t i o s h i p and un i fo rm and d i f f e r e n t i a l r e s i s t a n c e . Ne the r l and J ou r . P l a n t . Pa th . 82 :133-145. E l l i n g b o e , A . H . 1976a. 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 o f Expe r imen ta l Procedure? . A u s t . P i . P a th . Soc . News le t t e r 4 :44-46. E l l i n g b o e , A . H . 1976b. G e n e t i c s o f h o s t - p a r a s i t e i n t e r a c t i o n s . Pages 761-778. In : P h y s i o l o g i c a l P l a n t Pa tho logy , e d s . H e i t e f u s , R. and P .H. W i l l i a m s . S p r i n g e r - V e r l a g , B e r l i n . E l l i n g b o e , A . H . 1981. Changing concep ts i n host-pathogen g e n e t i c s . Ann. Rev. Phy topatho logy 19:125-143. Emara, Y .A . 1972. Gene t i c c o n t r o l o f a g g r e s s i v e n e s s i n U s t i l a g o h o r d e i . I. N a t u r a l v a r i a b i l i t y among p h y s i o l o g i c a l r a c e s . Can. J . Genet . C y t o l . 14 :919-924. Emara, Y . A . and G.W. F reake . 1981. E f f e c t o f env i ronment and genotype and t h e i r i n t e r a c t i o n on p a t h o g e n i c i t y o f U s t i l a g o h o r d e i : 1. P a r a s i t e - env i ronment e f f e c t s . J o u r n a l o f H e r e d i t y 72 :261-263. Emara, Y . A . and G. S i d h u . 1974. P o l y g e n i c i n h e r i t a n c e o f a g g r e s s i v e n e s s i n U s t i l a g o h o r d e i . H e r e d i t y 32:219-224. E r i k s s o n , J . 1894. Uber d i e S p z i a l i s i e r u n g des P a r a s i t i s m a s b i e den G e t r e i d e r o s t p i l z e n . B e r i c h t e de r Deutschen Bo tan i s chen G e s e l l s c h a f t 12:292-331. F a l a h a t i - Ras tega r , M . , J . S . Manners, and J . Smar t t . 1981. E f f e c t s o f temperature and inocu la tum d e n s i t y on c o m p e t i t i o n between r a ces o f P u c c i n i a h o r d e i . Trans.- B r . Myco l . Soc. 77 :359-368. F a r i s , J . A . 1924a. P h y s i o l o g i c a l s p e c i l i z a t i o n o f U s t i l a g o  h o r d e i . Phy topa tho logy 14:537-557. F a r i s , J . A . 1924b. F a c t o r s i n f l u e n c i n g i n f e c t i o n o f Hordeum  s a t i v i u m by U s t i l a g o h o r d e i . Am. J o u r . Bot . 11 :189-214. F i s c h e r , G.W. 1940. Two cases o f h a p l o - l e t h a l d e f i c i e n c y i n U s t i l a g o b u l l a t a o p e r a t i v e a g a i n s t s ap rophy t i sm . M y c o l o g i a 23 :275-289. F l e m i n g , R.A. and C O . Pe r son . 1978. D i sease c o n t r o l through the use o f m u l t i l i n e s : A T h e o r e t i c a l c o n t r i b u t i o n . Phy topa tho logy 68:1230-1233. 157 F l e m i n g , R.A. and C O . Pe r son . 1982. Consequences o f p o l y g e n i c d e t e r m i n a t i o n o f 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 . Can. J . P l a n t . P a t h o l . 4 :89-96. F l o r , H.H. 1946. G e n e t i c s o f p a t h o g e n i c i t y i n Melampsora  l i n i . J ou r . Ag r . Res. 73 :335-357. F l o r , H.H. 1947. I nhe r i t ance o f r e a c t i o n to r u s t i n f l a x . J o u r . Ag r . Res. 74 :241-262. F l o r , H.H. 1953. Ep idemio logy o f f l a x r u s t i n the North C e n t r a l S t a t e s . Phy topa tho logy 43:624-628. F l o r , H.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 o the r i m p l i c a t i o n s . Phy topa tho logy 45:680-685. F u l l e r t o n , R.A. and J . N i e l s e n . 1974. I d e n t i c a l genes f o r v i r u l e n c e i n the smuts U s t i l a g o h o r d e i and U s t i l a g o n i g r a , and the i n h e r i t a n c e o f v i r u l e n c e on the b a r l e y c u l t i v a r s Keystone and Conquest . Can. J . P l a n t . S c i . 54 :253-257. G a v i n l e r t v a t a n a , S. and R.D. W i l coxson . 1978. I nhe r i t ance o f slow r u s t i n g o f s p r i n g wheat by P u c c i n i a r e c o n d i t a f . s p . t r i t i c i and 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 . T r a n s . Br . M y c o l . Soc . 71 :413-418. G o l d s c h m i d t , V. 1928. Verebungsversuche m i t den b i o l o g i s c h e r Ar ten des Antherenbrandes ( U s t i l a g o v i o l a c e a P e r s . ) E in B e i t r a g zur Frage der P a r a s i t a r e n S p e x i a l i s i e r u n g . Z e i t . f u r Bot . 21 :1-90 . G r a n t , M.W. and E.A. A r c h e r . 1983. C a l c u l a t i o n o f s e l e c t i o n c o e f f i c i e n t s a g a i n s t unnecessary genes f o r v i r u l e n c e from f i e l d d a t a . Phy topatho logy 73:547-551. G r a s s o , V. 1955. A h a p l o - l e t h a l d e f i c i e n c y i n U s t i l a g o  h o r d e i . Phy topatho logy 45:521-522. G reen , G . J . 1964. A c o l o r m u t a t i o n , i t s i n h e r i t a n c e , and the i n h e r i t a n c e o f p a t h o g e n i c i t y i n P u c c i n i a g r a m i n i s P e r s . Can. J . Bo t . 42:1653-1664. G reen , G . J . 1965. I nhe r i t ance o f v i r u l e n c e i n oa t stem r u s t on the v a r i e t i e s Sevnothree , R i ch l and and White Russ i an . Can. J . Genet . C y t o l . 7 :641-650. Green , G . J . 1966. S e l f i n g s t u d i e s w i th r a ces 10 and 11 o f wheat stem r u s t . Can. J . Bo t . 44:1255-1260. 158 G r e e n f G . J . 1975. V i r u l e n c e changes i n P u c c i n i a g r am in i s f . s p . t r i t i c i i n Canada. Can. J . Bo t . 53:1377-1386. G reen , G . J . and R . I .N . McKenzie . 1967. Mendel ian and extrachromosomal i n h e r i t a n c e o f v i r u l e n c e i n P u c c i n i a  g r a m i n i s f . s p . avenae. Can. J . Genet . C y t o l . 9 :785-793. G r o t h , J . V . 1974. P a r a s i t i s m o f b a r l e y by U s t i l a g o h o r d e i ( Pe r s . ) L a g e r h . : Some q u a n t a t i v e a spec t s o f d i s e a s e e x p r e s s i o n . Ph .D. T h e s i s U n i v e r s i t y o f B r i t i s h Columbia 115 pp . G r o t h , J . V . 1976. M u l t i l i n e s and "Super Races" a s imp le mode l . Phy topa tho logy 66:937-939. G r o t h , J . V . 1978. Rebu t t a l to " M u l t i l i n e s and super r a ces - a r e p l y . " Phy topa tho logy 68 :1538-1539. G r o t h , J . V . and J . A . B a r r e t t . 1980. E s t i m a t i n g p a r a s i t i c f i t n e s s : A r e p l y . Phy topa tho logy 70:840-842. G r o t h , J . V . and C O . Pe r son . 1976. E s t i m a t i n g the e f f i c i e n c y o f pa r t i a l - va cuum i n o c u l a t i o n o f b a r l e y wi th U s t i l a g o  h o r d e i . Phy topatho logy 66 :65-69 . G r o t h , J . V . and C O . P e r son . 1977. Gene t i c in te rdependence o f hos t and p a r a s i t e i n e p i d e m i c s . N.Y. Acad. S c i . 287:97-106. G r o t h , J . V . and C O . Pe r son . 1978. Smutt ing p a t t e r n s i n b a r l e y and some p l a n t growth e f f e c t s caused by U s t i l a g o  h o r d e i . Phy topatho logy 68:477-483. G r o t h , J . V . , C O . Person and T . Ebba. 1976. R e l a t i o n between two measures o f d i s e a s e e x p r e s s i o n i n b a r l e y - U s t i l a g o  h o r d e i i n t e r a c t i o n s . Phy topa tho logy 66:1324-1347. G r o t h , J . V . and A . P . R o e l f s . 1982. E f f e c t o f s exua l and asexua l r e p r o d u c t i o n on race abundance i n c e r e a l r u s t fungus p o p u l a t i o n s . Phy topatho logy 72:1503-1507. Habgood, R.M. 1976. D i f f e r e n t i a l a g g r e s s i v e n e s s o f Rhynchosporium s e c a l i s i s o l a t e s towards s p e c i f i e d b a r l e y geno t ypes . T r a n s . Br . M y c o l . Soc . 66:201-204. Haggag, M . E . A . , D.J . Samborski and P.J. Dyck. 1973. G e n e t i c s o f p a t h o g e n i c i t y i n th ree r a ces o f l e a f r u s t on f o u r wheat c u l t i v a r s . Can. J . Genet . C y t o l . 15 :73-82. H a l i s k y , P.M. 1965. I nhe r i t ance o f p a t h o g e n i c i t y i n U s t i l a g o avenae . Research S tud i e s S ta te C o l l . Wash. 24:348-386. 159 Hamid, A . H . , J . E . Ayers and R.R. H i l l , J r . 1982. The i n h e r i t a n c e o f r e s i s t a n c e i n co rn to C o c h l i o b o l u s carbonum Race 3. Phy topa tho logy 72:1173-1177. H o l t o n , C S . 1951. Methods and r e s u l t s o f s t u d i e s on h e t e r o t h a l l i s m and h y b r i d i z a t i o n i n T i l l e t i a c a r r i e s and T . f o e t i d a . Phy topatho logy 41:511-521. H o l t o n , C S . 1967. Race p o p u l a t i o n dynamics i n U s t i l a g o  avenae as r e l a t e d to the p e r s i s t a n c e o f l o o s e smut i n o a t s . P l a n t D isease Rep t r . 51:844-846. H o l t o n , C S . and S .M. D i e t z . 1957. Hap lo id l e t h a l f a c t o r s a s s o c i a t e d w i th two unique so rus t ypes i n the oa t smut f u n g i . (Abs t . ) Phy topatho logy 42:524-525. H o l t o n , C S . and S.M. D i e t z . 1960. An apparent a s s o c i a t i o n o f h a p l o i d l e t h a l f a c t o r s w i th two unique so rus types o f oa t smut. Phy topa tho logy 50:749-751. Hooker , A . L . 1963a. Monogenic r e s i s t a n c e i n Zea mays L. to He lminthospor ium t u r c i c u m . Crop S c i . 3:381-383. Hooker , A . L . 1963b. I nhe r i t ance o f c h l o r o t i c l e s i o n r e s i s t a n c e to He lminthospor ium tu r c i cum i n s e e d l i n g c o r n . Phy topatho logy 53:660-662. Hooker , A . L . and K.M.S. Saxena. 1967. Apparent r e v e r s a l o f dominance o f a gene i n co rn f o r r e s i s t a n c e to P u c c i n i a  s o r g h i . Phy topa tho logy 57:1372-1374. J a c k s o n , L . F . and R.K. Webster . 1976a. Race d i f f e r e n t i a t i o n , d i s t r i b u t i o n , and f r equency o f Rhynchosporiurn s e c a l i s i n C a l i f o r n i a . Phy topatho logy 66:719-725. J a c k s o n , L . F . and R.K. Webster . 1976b. The dynamics o f a c o n t r o l l e d p o p u l a t i o n o f Rhynchosporium s e c a l i s , changes i n race compos i t i on and f r e q u e n c i e s . Phy topa tho logy 66:726-728. James, R.V. and W.E. F r y . 1983. P o t e n t i a l f o r Phy tophthora  i n f e s t a n s P o p u l a t i o n s to adapt to po t a to c u l t i v a r s w i th r a t e - r e d u c i n g r e s i s t a n c e . Phy topatho logy 73:984-988. J e f f r e y , S . I . B . , J . L . J i n k s and M. G r i n d l e . 1962. I n t r a r a c i a l v a r i a t i o n i n Phy tophythora i n f e s t a n s and f i e l d r e s i s t a n c e to p o t a t o b l i g h t . G e n e t i c a 32:323-338. J e n k i n s , M.T. and A . L . Rober t . 1952. I nhe r i t ance o f r e s i s t a n c e to the l e a f b l i g h t o f co rn caused by He lminthospor ium t u r c i c u m . Agron . J . 44:136-140. 160 J e n k i n s , M.T. and A . L . Rober t . 1961. Fu r the r g e n e t i c s t u d i e s o f r e s i s t a n c e to He lminthospor ium tu rc i cum Pass , i n maize by means o f chromosomal t r a n s l o c a t i o n s . Crop S c i . 1:450-455. J enns , A . E . , K. J . Leonard and R.A. M o l l . 1982. S t a b i l i t y a n a l y s e s f o r e s t i m a t i n g r e l a t i v e d u r a b i l i t y o f q u a n t i t a t -i v e r e s i s t a n c e . Theor . A p p l . Genet . 63:183-192. J ensen , N.F. 1952. I n t r a - v a r i e t a l d i v e r s i f i c a t i o n i n oa t b r e e d i n g . Agron . J . 44 :30-34. J i n k s , J . L . and M. G r i n d l e . 1963. Changes induced by t r a i n i n g i n Phy tophthora i n f e s t a n s . H e r e d i t y 18:245-264. Johnson , D.A. and R.D. W i l coxson . 1979. I nhe r i t ance o f s low r u s t i n g o f b a r l e y i n f e c t e d w i th P u c c i n i a h o r d e i and s e l e c t i o n o f l a t e n t p e r i o d and number o f u r e d i a . Phy topatho logy 69:145-151. Johnson , R. and A . J . T a y l o r . 1976. Spore y i e l d o f pathogens i n i n v e s t i g a t i o n s o f the r a c e - s p e c i f i c i t y o f hos t r e s i s t a n c e . Ann. Rev. Phy topa tho logy 14 :97-119 . Johnson , T . 1954. S e l f i n g s t u d i e s w i th p h y s i o l o g i c r a c e s o f wheat stem r u s t , P u c c i n i a g r a m i n i s v a r . t r i t i c i . Can. J . Bo t . 32:506-522. J ohnson , T . 1958. Reg iona l d i s t r i b u t i o n o f genes f o r r u s t r e s i s t a n c e . Robigo 6 :16-17. Johnson , T . 1961. Man gu ided e v o l u t i o n i n p l a n t r u s t s . Sc i ence 133:357-362. J o h n s t o n , W.H. 1934. S t u d i e s on the d e h u l l i n g o f b a r l e y k e r n a l s w i th s u l p h u r i c a c i d and on the i n h e r i t a n c e o f r e a c t i o n to covered smut, U s t i l a g o h o r d e i (Pers) K & S i n f e c t i o n i n c r o s s e s between Glabrum and T r e b i b a r l e y s . Can. J . Res. 11 :458 :473 . J o n e s , E.S. 1923a. I n f l uence o f t empera tu re , mo i s tu re and oxygen on spore g e r m i n a t i o n o f U s t i l a g o avenae. J o u r . A g r i . Res. 24:577-591. J o n e s , E.S. 1923b. I n f l uence o f temperature on the spore g e r m i n a t i o n o f U s t i l a g o zeae . J o u r . A g r i . Res. 24 :593-597. J o n e s , G .H . and Abd E l Ghani S e i f - E l - N a s r . 1940. The i n f l u e n c e o f sowing depth and mo i s tu r e on smut d i s e a s e s , and the p r o s p e c t s o f a new method o f c o n t r o l . Ann. App. B i o l . 27 :35-57. 161 J o rgensen , J . 1962. V a r i a t i o n i n s i z e o f s p o r i d i a i n U s t i l a g o  h o r d e i ( Pe rs . ) Lage rh . and i t s g e n e t i c background. F r i e s i a 7:97-104. Kao, K.N. and D.R. K n o t t . 1969. The i n h e r i t a n c e o f patho-g e n e c i t y i n r a ces 111 and 29 o f wheat stem r u s t . Can. J . Genet . C y t o l . 11:266-274. K a t s u y a , K. and G . J . Green . 1967. Reproduc t i ve p o t e n t i a l s o f r a ces 15B and 56 o f wheat stem r u s t . Can. J . Bot . 45:1077-1091. Kernkamp, M.F. 1939. Gene t i c and env i ronmenta l f a c t o r s a f f e c t i n g growth types o f U s t i l a g o zeae . Phy topatho logy 29:473-484. Kernkamp, M.F. 1942. The r e l a t i v e e f f e c t o f env i ronmenta l and g e n e t i c f a c t o r s on growth types o f U s t i l a g o zeae . Phy topatho logy 32:554-567. Kernkamp, M.F. and M.A. P e t t y . 1941. V a r i a t i o n i n the g e r m i n a t i o n o f ch lamydospores o f U s t i l a g o zeae . Phy topa tho logy 31:333-340. K e r b e r , E.R. and G . J . Green . 1980. Suppress ion o f stem r u s t r e s i s t a n c e i n the h e x a p l o i d wheat c v . Canthatch by chromosome 7DL. Can. J . Bo t . 58:1347-1350. K i e s l i n g , R.L. 1970. The i n h e r i t a n c e o f r e s i s t a n c e to U s t i l a g o h o r d e i i n s p r i n g b a r l e y . Pages 500-507. In : B a r l e y Gene t i c s I I . Ed . R.A. W i l s o n . Wash. S ta te Un i v . P r e s s . K i yosawa , S. 1976. A Comparison by S i m u l a t i o n o f d i s e a s e d i s p e r s a l i n pure and mixed s t ands o f s u s c e p t i b l e and r e s i s t a n t p l a n t s . Japan J . B reed . 26:137-145. K iyosawa, S. 1980. On the v i r u l e n c e a n a l y s i s o f pathogen r ace f r e q u e n c i e s . Ann. Phy topa th . Soc. Japan 46:582-693. K iyosawa, S. 1982. Gene t i c s and e p i d e m i o l o g i c a l model ing o f breakdown o f p l a n t d i s e a s e r e s i s t a n c e . Ann. Rev. Phy topa tho logy . 20 :93-117. K iyosawa, S. and M. Sh i yomi . 1972. A t h e o r e t i c a l e v a l a u t i o n o f the e f f e c t o f m ix ing r e s i s t a n t v a r i e t y w i th s u s c e p t i b l e v a r i e t y f o r c o n t r o l l i n g p l a n t d i s e a s e s . Ann. Phy topa th . Soc . Japan 38 :41-51 . 162 K iyosawa, S. and S. Y a b u k i . 1976. Model ing on the race f r equency change i n a host-pathogen system wi th genes f o r r e s i s t a n c e and a v i r u l e n c e . Japan J . B reed . 26:237-246. K lug-Andersen , Susanne. 1980. S e l e c t i v e v a l u e s o f unnecessary v i r u l e n c e genes i n p o p u l a t i o n s o f b a r l e y powdery mildew ( E r y s i p h e g r a m i n i s o f s p . h o r d e i ) . Ph .D. T h e s i s Royal V e t e r i n a r y A g r i c u l t u r a l Un i v . Copenhagen, Denmark, 63 pp . K n i e p , H. 1919. Untersuchungan Uber den Anthernband U s t i l a g o  v i o l a c e a e . P e r s . E in B e i t r a g zum S e x u a l i t a t s p r o b l e m . Z e i t . f u r Bot . 11:275-284. K n o t t , D.R. 1957. The i n h e r i t a n c e o f r u s t r e s i s t a n c e I I . The i n h e r i t a n c e o f stem r u s t r e s i s t a n c e i n 6 a d d i t i o n a l v a r i e t i e s o f common wheat. Can. J . P l an t S c i . 37:177-192 K n o t t , D.R. 1972. Us ing r a c e - s p e c i f i c r e s i s t a n c e to manage the e v o l u t i o n o f p l a n t pa thogens . J . E n v i r o n . Q u a l i t y 1:227-231. K n o t t , D.R. and R.G. Anderson . 1956. The i n h e r i t a n c e o f r u s t r e s i s t a n c e . 1. The i n h e r i t a n c e o f stem r u s t r e s i s t a n c e i n ten v a r i e t i e s o f common wheat. Can. J . A g r i c . S c i . 36: 174-195. Ko j ima , Ken- I ch i . 1971. Is the re a cons t an t f i t n e s s va lue f o r a g i v e n gentoype? No! E v o l u t i o n 25:281-285. Konzak, C F . 1953. I nhe r i t ance o f r e s i s t a n c e i n b a r l e y to p h y s i o l o g i c r a ces o f U s t i l a g o nuda. Phy topa tho logy 43:369-375. K o z a r , F. 1969. The p a t h o g e n i c i t y o f i n f e c t i o n o f U s t i l a g o  h o r d e i . Can. J . Genet . C y t o l . 11 :977-986. Lade , D.H. 1969. I nhe r i t ance o f p a t h o g e n e c i t y o f U s t i l a g o  h o r d e i . Ph .D . T h e s i s . Nor th Dakota S ta te Un i v . 44 p p . L a r g e , E . C 1950. The advance o f the f u n g i . Johnathan Cape, London. 48 8 pp . L a s k a r i s , T . 1939. The o c cu r r ence o f l y s i s i n c e r t a i n c r o s s e s o f Sphace lo theca s o r g h i . (Abs t . ) Phy topa tho logy 2 9 : 1 4 . L a s k a r i s , T . 1941. A h e r i t a b l e l y s i s i n ge rm ina t i ng c l amytospores o f Space lo theca s o r g h i . phy topa tho logy 31:254-263. 163 Lawrence, G . J . , G .M.E . Mayo and K.W. Shepherd. 1981a. I n t e r a c t i o n s between genes c o n t r o l l i n g p a t h o g e n i c i t y i n the f l a x r u s t fungus . Phy topa tho logy 71 :12-19 . Lawrence, G . J . , K.W. Shepherd, and G .M.E . Mayo. 1981b. F ine s t r u c t u r e o f genes c o n t r o l l i n g pa tho genec i t y i n f l a x r u s t , Melampsora l i n i . H e r e d i t y 46:297-313. Lede r , P. 1982. The g e n e t i c s o f an t i body d i v e r s i t y . S c i . /American 246:102-115. L e i t z k e , B. 1937. I n f e c t i o n s v e r s u c h e m i t h a f e r f l u g b r a n d -sporen-germischen . Phy topa th . Z e i t s . 10 :504-553. Leona rd , K. J . 1969a. F a c t o r s a f f e c t i n g r a t e o f stem r u s t i n c r e a s e i n mixed p l a n t i n g s o f s u s c e p t i b l e and r e s i s t a n t oa t v a r i e t i e s . Phy topatho logy 59:1845-1850. Leona rd , K. J . 1969b. S e l e c t i o n i n heterogeneous p o p u l a t i o n s o f P u c c i n i a g r am in i s f . s p . avenae. Phy topatho logy 59:1851-1857. Leona rd , K. J . 1969c. Gene t i c e q u i l i b r i a i n host-pathogen sys tems . Phy topa tho logy 59:1858-1863. Leona rd , K. J . 1977. S e l e c t i o n p r e s s u r e s and p l a n t pa thogens . Ann. N.Y. Acad S c i . 287:207-222. Leona rd , K. J . and R.J. Czochor . 1980. Theory o f g e n e t i c i n t e r a c t i o n s among p o p u l a t i o n s o f p l a n t s and t h e i r pa thogens . Ann. Rev. Phy topatho logy 18:237-258. L e w e l l e n , R.T. and E .L . Sharp. 1968. I nhe r i t ance o f minor r e a c t i o n gene combina t ions i n wheat to P u c c i n i a  s t r i i f o r m i s a t two temperature p r o f i l e s . Can. J . Bo t . 46 :2155-2172. L o e g e r i n g , W.Q. 1951. S u r v i v a l o f r a ces o f wheat stem r u s t i n m i x t u r e s . Phy topa tho logy 41 :56-65. L u i g , N.H. and I.A. Watson. 1961. A s tudy o f i n h e r i t a n c e o f p a t h o g e n e c i t y i n P u c c i n i a g r a m i n i s v a r . t r i t i c i . P r o c . L i n . Soc. N.S.W. 86 :217-229. L u i g , N.H. and I.A. Watson. 1970. The e f f e c t o f complex g e n e t i c r e s i s t a n c e i n wheat on the v a r i a b i l i t y o f P u c c i n i a  g r a m i n i s f . s p . t r i t i c i . P r o c . L i n . Soc . N.S.W. 95 :22-45 . 164 Luke, H . H . , R.D. Ba rne t t and P.L. P f a h l e r . 1975. I nhe r i t ance o f h o r i z o n t a l r e s i s t a n c e t o crown r u s t s i n o a t s . Phy topa tho logy 65:631-632. Lup ton , F .G .H . and R. Johnson. 1970. B reed ing f o r mature p l a n t r e s i s t a n c e t o y e l l ow r u s t i n wheat. Ann. A p p l . B i o l . 66 :137-143. Lup ton , F .G .H . and R .C . F . Macer . 1962. I nhe r i t ance o f r e s i s t a n c e to y e l l ow r u s t ( P u c c i n i a glumarum E r i k e s and Henn.) i n seven v a r i e t i e s o f wheat. T r a n s . B r . Myco l . Soc . 45 :21-45 . MacKenz ie , D.R. 1978. E s t i m a t i n g p a r a s i t i c f i t n e s s . Phy topa tho logy 68 :9-13 , 1108 ( E r ra tum) . MacKey, J . 1977. S t r a t e g i e s o f r a c e - s p e c i f i c p h y t o p a r a s i t i s m and i t s c o n t r o l by p l a n t b r e e d i n g . Gene t i ka 9 :237-255. Malm, N.R. and A . L . Hooker. 1962. Res i s t ance t o r u s t , P u c c i n i a s o r g h i Schm., c o n d i t i o n e d by r e c e s s i v e genes i n two co rn i nb red l i n e s . C rop . S c i . 2 :145-147. M a r s h a l l , D.R. and A . J . P r y o r . 1978. M u l t i l i n e V a r i e t i e s and D i sease C o n t r o l I. The " D i r t y Crop" approach w i th each component c a r r y i n g a unique s i n g l e r e s i s t a n c e gene . Theor . A p p l . Genet . 51:177-184. M a r s h a l l , D.R. and A . J . P r y o r . 1979. M u l t i l i n e v a r i e t i e s and d i s e a s e c o n t r o l I I . The " D i r t y Crop" approach w i th components c a r r y i n g two or more genes f o r r e s i s t a n c e . E u p h y t i c a 28:145-159. Ma r t ens , J .W. 1973. Compe t i t i v e a b i l i t y o f oa t stem r u s t r a ces i n m i x t u r e s . Can. J . Bo t . 51:2233-2236. Mar tens , J . W . , P.G. Rothman, R . I .H . McKenzie and P.D. Brown. 1981. Ev idence f o r complementary gene a c t i o n c o n f e r r i n g r e s i s t a n c e to P u c c i n i a g r a m i n i s avenae i n Avena s a t i v a . Can. J . Genet . C y t o l . 23:591-595. M a r t i n , T . J . and A . H . E l l i n g b o e . 1976. D i f f e r e n c e s between compa t ib l e p a r a s i t e / h o s t genotypes i n v o l v i n g the Pm4 l o c u s o f wheat and the c o r r e s p o n d i n g genes i n E r y s i p h e g r a m i n i s f . s p . t r i t i c i . Phy topa tho logy 66:1435-1438 . Mathre , D.E . (Ed. ) 1982. Compendium o f B a r l e y D i s e a s e s . The Amer ican P h y t o p a t h o l o g i c a l S o c i e t y . S t . P a u l , MN. 78 pp . Maynard Smi th , J . 1962. D i s r u p t i v e s e l e c t i o n , polymorphism and s ympa t r i c s p e c i a t i o n . Nature 195:60-62. 165 McKenz ie , R . I .H . and G . J . Green . 1965. Stem r u s t r e s i s t a n c e i n o a t s . I. The i n h e r i t a n c e o f r e s i t a n c e to race 6AF i n 6 v a r i e t i e s o f o a t s . Can. J . Genet . C y t o l . 7:268-274. McKenz ie , R . I .H . and J .W. Mar tens . 1968. I nhe r i t ance i n the oa t s t r a i n C . I . 3034 o f a d u l t p l a n t r e s i s t a n c e to race C 10 o f stem r u s t . Crop S c i . 8 :625-627. M e t c a l f , D.R. 1962. I nhe r i t ance o f r e s i s t a n c e to l o o s e smut, covered smut and f a l s e l o o s e smut i n the b a r l e y v a r i e t y J e t . Can. J . P l an t S c i . 42 :176-189. Mode, C . J . 1958. A mathemat i ca l model f o r the c o - e v o l u t i o n o f o b l i g a t e p a r a s i t e s and t h e i r h o s t s . E v o l u t i o n 12:158-165. Nass , H . A . , W.L. Pedersen , D.R. Mackenzie and R.R. N e l s o n . 1981. The r e s i d u a l e f f e c t s o f some " d e f e a t e d " powdery mildew r e s i s t a n c e genes i n i s o l i n e s o f w in t e r wheat. Phy topa tho logy 71:1315-1318. N e l s o n , R.R. 1972. S t a b i l i z i n g r a c i a l p o p u l a t i o n s o f p l a n t pathogens by use o f r e s i s t a n c e genes . J . E n v i r o n . Q u a l i t y 1:220-227. N e l s o n , R.R. 1978. G e n e t i c s o f h o r i z o n t a l r e s i s t a n c e to p l a n t d i s e a s e s . Ann. Rev. Phy topa tho logy 16:359-378. N e l s o n , R.R., D.R. MacKenzie and G .L . S c h e i f l e . 1970. I n t e r a c t i o n o f genes f o r p a t h o g e n i c i t y and v i r u l e n c e i n T r i c h o m e t a s p h a e r i a t u r c i c a w i th d i f f e r e n t numbers o f genes f o r v e r t i c a l r e s i s t a n c e i n Zea mays. Phy topatho logy 60:1250-1254. N e l s o n , R.R., A . L . Robert and G . F . Sprague. 1965. E v a l u a t i n g g e n e t i c p o t e n t i a l s i n Helminthosporiurn t u r c i c u m . Phy topatho logy 55:418-420. N e l s o n , R.R. and G . L . S c h e i f e l e . 1970. F a c t o r s a f f e c t i n g the o v e r w i n t e r i n g o f T r i chome ta sphae r i a t u r c i c a on ma ize . Phy topa tho logy 60:369-370. N i c o l a i s e n , W. 1934. Die Grundlagen de r Immunitatszuchtung gegen U s t i l a g o avenae. ( Pe rs . ) J ens . Z e i t . f u r P f l a n z . 19 :1-56 . N i e l s e n , J . 1966. Changes i n g e r m i n a t i o n type i n some U s t i l a g o s p e c i e s caused by an u n i d e n t i f i e d v o l a t i l e s u b s t a n c e . Can. J . Bo t . 44:163-170. 166 N i e l s e n , J . 1968. Exper iments on v e g e t a t i v e d i s s o c i a t i o n o f the d i k a r y o n and on l y s i s o f h y b r i d s p o r i d i a o f the c r o s s U s t i l a g o avenae U. k o l l e r i . Can J . Bot . 46 :487-496. Oa rd , J . H . and M.D. S imons. 1983. A growth chamber compar ison o f t r a i t s o f a g g r e s s i v e n e s s i n s exua l and asexua l p o p u l a -t i o n s o f P u c c i n i a c o r o n a t a . Phy topatho logy 73:1226-1229. O g l e , H. J . and J . F . Brown. 1970. R e l a t i v e a b i l i t y o f two s t r a i n s o f P u c c i n i a g r a m i n i s t r i t i c i to s u r v i v e when mixed . Ann. A p p l . B i o l . 66 :273-279. O g l e , H . J . , N.W. T a y l o r , and J . F . Brown. 1973. A mathemat i ca l approach to the p r e d i c t i o n o f d i f f e r e n c e s i n the r e l a t i v e a b i l i t y o f r a ces o f P u c c i n i a g r a m i n i s t r i t i c i to s u r v i v e when mixed . A u s t . J . B i o l . S c i . 26:1137-1143. Oso ro , M.O. and G . J . Green. 1976. S t a b i l i z i n g s e l e c t i o n i n P u c c i n i a g r a m i n i s t r i t i c i i n Canada. Can. J . Bo t . 54:2204-2214. O s t e r g a a r d , H. 1982. P o p u l a t i o n s b i o l o g i s k e S t u d i e r o f P a r a s i t a e r a Svampe PA P l a n t e r , S p e c i e l t Bygmeldug. Ph .D . T h e s i s , Royal A g r i . & V e t . Un iv . Copenhagen, Denmark. O s t e r g a a s r d , H. 1983. P r e d i c t i n g development o f ep idemics on c u l t i v a r m i x t u r e s . Phy topatho logy 73:166-172. P a r l e v l i e t , J . E . 1976a. P a r t i a l r e s i s t a n c e o f b a r l e y to l e a f r u s t , P u c c i n i a h o r d e i . I I I . The i n h e r i t a n c e o f the hos t p l a n t e f f e c t on l a t e n t p e r i o d i n f o u r c u l t i v a r s . Euphy t i c a 25:241-248. P a r l e v l i e t , J . E . 1976b. E v a l u a t i o n o f the concept o f h o r i z o n t a l r e s i s t a n c e i n the b a r l e y / P u c c i n i a h o r d e i hos t-pathogen r e l a t i o n s h i p . Phy topatho logy 66:494-497. P a r l e v l i e t , J . E . 1978. Fu r the r ev idence o f p a r t i a l r e s i s t a n c e i n b a r l e y to l e a f r u s t , P u c c i n i a h o r d e i . Euphy t i c a 27 :369-379. P a r l e v l i e t , J . E . 1981a. S t a b i l i z i n g s e l e c t i o n i n c r o p pa thosys tems : An empty concept o r a r e a l i t y ? Euphy t i c a 30:259-269. P a r l e v l i e t , J . E . 1981b. R a c e - n o n - s p e c i f i c d i s e a s e r e s i s t a n c e . Pages 47-54. I n : S t r a t e g i e s f o r the c o n t r o l o f c e r e a l d i s e a s e s . Eds . J enkyn , J . F . and Plumb, R.T. B l a c k w e l l S c i . P u b l . O x f o r d . 167 P a r l e v l i e t . J . E . and J . C . Zadoks. 1977. The i n t e g r a t e d concept of d i s e a s e r e s i s t a n c e ; A new view i n c l u d i n g h o r i z o n t a l and v e r t i c a l r e s i s t a n c e i n p l a n t s . Euphy t i ca 26 :5-21 . Pateman, J . A . 1955. P o l y g e n i c i n h e r i t a n c e i n Neurospora . Nature 176:1274-1275. Pateman, J . A . and B .T .O . Lee. 1960. Seg rega t i on o f po lygenes i n o rde red t e t r a d s . H e r e d i t y 15 :351-361. Pede rsen , W., L. J ensen , R.L. K i e s l i n g and L. D a r l i n g t o n . 1977. I nhe r i t ance o f a m o r p h o l o g i c a l f a c t o r l i m i t i n g i n f e c t i o n by U s t i l a g o h o r d e i . Phy topatho logy 67:1397-1399. Pede rsen , W.L. and R.L. K i e s l i n g . 1979. E f f e c t o f i n b r e e d i n g on pa thogenec i t y i n r ace 8 o f U s t i l a g o h o r d e i . Phy topatho logy 69:1207-1212. P e r k i n s , D.D. 1962. P r e s e r v a t i o n o f Neurospora s t o ck c u l t u r e s w i th anhydrous s i l i c a g e l . Can. J . M i c r o b i o l . 8 :591-594. P e r s o n , C O . 1959. Gene-for-gene r e l a t i o n s i n h o s t : p a r a s i t e sys tems . Can. J . Bot 37:1101-1130. P e r s o n , C. 1966. Gene t i c polymorphism i n p a r a s i t i c s ys tems . Nature 212:266-267. P e r s o n , C 1967. Gene t i c a spec t s o f p a r a s i t i s m . Can. J . Bo t . 45:1193-1204. P e r s o n , C O . 1968. Gene t i c ad justment o f f u n g i to t h e i r env i ronment . Pages 395-415. The F u n g i : An Advanced T r e a t i s e V o l . I I I . Eds . A i n s w o r t h , G . C and Sussman, Academic P r e s s , New York . P e r s o n , C. and W.J. Cherewick . 1964. I n f e c t i o n m u l t i p l i c i t y i n U s t i l a g o . Can. J . Genet . C y t o l . 6 :12-18. P e r s o n , C O . and B.J. C h r i s t . 1983. P o p u l a t i o n g e n e t i c s and e v o l u t i o n o f h o s t - p a r a s i t e i n t e r a c t i o n s . Pages 379-386. In : C h a l l e n g i n g problems i n P l an t H e a l t h . T . Kommendahl and P. W i l l i a m s , Eds . Am. P h y t o p a t h o l o g i c a l Soc . S t . P a u l , MN. P e r s o n , C , R. F leming and L. Cargeeg . 1982. N o n - s p e c i f i c i n t e r a c t i o n based on p o l y g e n e s . Pages 318-325. In : R e s i s t a n c e to d i s e a s e s and pes t s i n f o r e s t t r e e s . H.M. Heybroek, B.R. Stephan and K. von We issenberg , e d s . Pudoc, Wageningen. 168 P e r s o n , C , R. F l em ing , L. Cargeeg and B. C h r i s t . 1983. P r esen t knowledge and t h e o r i e s conce rn ing du r ab l e r e s i s t a n c e . Pages 27-37. In : Durable Res i s t ance i n C r o p s . F. L a m b e r t i , J . M . Wa l l e r and N.A. Van de r G r a f f , e d s . Plenum P r e s s , New York . P e r s o n , C , J . V . Groth and O.M. My lyk . 1976. Gene t i c change i n h o s t - p a r a s i t e p o p u l a t i o n s . Ann. Rev. Phy topa tho logy 14:177-188. P e r s o n , C O . and G .M.E . Mayo. 1974. Gene t i c l i m i t a t i o n s o f s p e c i f i c i n t e r a c t i o n s between a hos t and a p a r a s i t e . Can. J . Bot . 52:1339-1347. P e r s o n , C. and G. S i d h u . 1971. G e n e t i c s o f h o s t - p a r a s i t e i n t e r r e l a t i o n s h i p s . Pages 31-38. In : Muta t ion Breed ing f o r D i sease Res i s t ance IAEA, V i e n n a . Poehlman, J . and C .K . C l o n i n g e r . 1955. Res i s t ance to t h r ee b a r l e y smut d i s e a s e s i n M i s s o u r i E a r l y B ea rd l e s s S t r a i n s . Agronomy J ou r . 47 :243-246. Popp, W. and Hanna, W.J. 1935. S t ud i e s on the p h y s i o l o g y o f the oa t smuts. S c i . A g r i . 15 :424-434. Pope, D.D. 1982. B i o m e t r i c a l a n a l y s i s o f P a t h o g e n i c i t y i n the U s t i l a g o h o r d e i - Hordeum vu lga r e h o s t - p a r a s i t e sys tem. M.Sc. T h e s i s , U n i v e r s i t y o f B r i t i s h Co lumbia . 156 pp . P u g s l e y , A . T . and A. V i n e s . 1946. Breeding A u s t r a l i a n b a r l e y s r e s i s t a n t to covered smut. J . A u s t r . I n s t . A g r i . S c i . 12 :44-47 . Pu rdy , L . H . , E .L . K e n d r i c k , J . A . Hoffman, C S . H o l t o n . 1963. Dwarf bunt o f wheat. Ann. Rev. M i c r o b i o l . 17 :199-222. Rob inson , R.A. 1969. D i sease r e s i s t a n c e t e r m i n o l o g y . Rev. App. M y c o l . 48:593-606. Rob inson , R.A. 1973. H o r i z o n t a l r e s i s t a n c e . Rev. P l a n t . P a th . 42 :483-501. Rob inson , R.A. 1976. P l a n t Pa thosys tems. S p r i n g e r - V e r l a g , New York . 184 pp . Rob inson , R.A. 1979. Permanent and impermanent r e s i s t a n c e to c rop p a r a s i t e s ; A r e-examina t i on o f the pathosystem concept w i th s p e c i a l r e f e r e n c e to r i c e b l a s t . Z. P f l anzenzuch tg 83 :1-39 . 169 R o e l f s , A . P . , and J . V . G r o t h . 1980. A compar ison o f v i r u l e n c e phenotypes i n wheat stem r u s t p o p u l a t i o n s r ep roduc ing s e x u a l l y and a s e x u a l l y . Phy topa tho logy 70:855-862. Samborks i , D. J . and P.L . Dyck. 1968. I nhe r i t ance o f v i r u l e n c e i n wheat l e a f r u s t on the s tandard d i f f e r e n t i a l wheat v a r i e t i e s . Can. J . Genet . C y t o l . 10 :24-32. Samborsk i , D. J . and P.L. Dyck. 1976. I nhe r i t ance o f v i r u l e n c e i n P u c c i n i a r e c o n d i t a on s i x backc ross l i n e s o f wheat w i th s i n g l e genes f o r r e s i s t a n c e to l e a f r u s t . Can. J . Bo t . 54:1666-1671. Samborsk i . D. J . and P.L . Dyck. 1982. Enchancement o f r e s i s t a n c e to P u c c i n i a r e c o n d i t a by i n t e r a c t i o n s o f r e s i s t a n c e genes i n wheat. Can. J . P l a n t . P a th . 4:152-156 S a r t o r i s , G. 1924. S tud i e s i n the l i f e h i s t o r y and p h y s i o l o g y o f c e r t a i n smuts . Am. J ou r . Bo t . 2:617-644. Sawhney, R .N . , V .R. Chopra and M.S. Swaminathan. 1981. An a n a l y s i s o f genes f o r r e s i s t a n c e a g a i n s t two Ind ian c u l t u r e s o f stem r u s t r a c e s o f two bread wheats. Theor . A p p l . G e n e t i c s 60:157-160. S c h a f e r , J . F . , J . S . D i ckson and H.L. Shands. 1962. E f f e c t s o f temperature on covered smut e x p r e s s i o n i n two b a r l e y v a r i e t i e s . Phy topatho logy 52:1161-1163. S c h e i f e l e , G .L . and R.R. N e l s o n . 1970. F a c to r s a f f e c t i n g the s u r v i v a l o f T r i c h o m e t a s p h a e r i a t u r c i c a (He lminthospor ium  tu rc i cum) on Zea mays. Can. J . Bo t . 48:1603-1607. S c h e i f e l e , G . L . , R.R. N e l s o n , and C . C . Werham. 1968. S t ud i e s on s t a b i l i z i n g s e l e c t i o n i n T r i chome tasphae r i a t u r c i c a (He lminthospor ium t u r c i c u m ) . P l an t D i sease Rep t r . 52:427-430. Semenick, W. 1940. P h y s i o l o g i c r a ces o f U s t i l a g o h o r d e i ( Pe r s . ) K & S. i n A l b e r t a . Can. J . Res. Sec . C 18 :76-78 . Shands, R.G. 1956. I nhe r i t ance o f covered smut r e s i s t a n c e i n two b a r l e y c r o s s e s . Agron . J . 48 :81-86 . S i d h u , G .S . 1980. Gene t i c a n a l y s i s o f p l a n t p a r a s i t i c sys tems . P r o c . XIV I n t . Cong. Genet . 1:391-408. S i d h u , G. and C. Pe r son . 1971. Gene t i c c o n t r o l o f v i r u l e n c e o f U s t i l a g o h o r d e i I I . S eg rega t i ons f o r h i g h e r l e v e l s o f v i r u l e n c e . Can. J . Genet . C y t o l . 13 :173-178. 170 S i d h u , G. and C. Pe r son . 1972. Gene t i c c o n t r o l o f v i r u l e n c e i n U s t i l a g o h o r d e i I I I . I d e n t i f i c a i t o n o f genes f o r hos t r e s i s t a n c e and demons t r a t i on o f gene-for-gene r e l a t i o n s . Can. J . Genet . C y t o l . 14:209-213. Simmons, M.D. 1972. P o l y g e n i c r e s i s t a n c e to p l a n t d i s e a s e s and i t s use i n b reed ing r e s i s t a n t c u l t i v a r s . J . E n v i r o n . Q u a l i t y 1:232-240. Skovmand, B., R.D. W i l coxson , B.L. Shea re r , and R.E. S t u cke r . 1978. I nhe r i t ance o f s low r u s t i n g to stem r u s t i n wheat. E u p h y t i c a 27 :95-107. S k y l a k a k i s , G. 1980. E s t i m a t i n g p a r a s i t i c f i t n e s s o f p l a n t pa thogen i c f u n g i : A t h e o r e t i c a l c o n t r i b u t i o n . Phy topa tho logy 70:696-698. Stakman, E .C . 1914. A s tudy i n c e r e a l r u s t s . P h y s i o l o g i c r a c e s . Minn. A g r i c . Exp. S t a t . B u l l . 138. Stakman, E . C , M.F. Kernkamp, T . H . K ing and W.J. M a r t i n . 1943. Gene t i c f a c t o r s f o r m u t a b i l i t y and mutant c h a r a c t e r s i n U s t i l a g o zeae . Am. J . Bo t . 30 :37-48. S t a t l e r , G.D. 1977. I nhe r i t ance o f v i r u l e n c e o f c u l t u r e 73-47 P u c c i n i a r e c o n d i t a . Phy topa tho logy 67:906-908. S t a t l e r , G.D. 1979a. I nhe r i t ance o f p a t h o g e n i c i t y o f c u l t u r e 70-1 , Race 1, o f P u c c i n i a r e c o n d i t a t r i t i c i . Phy topatho logy 69:661-663. S t a t l e r , G.D. 1979b. I nhe r i t ance o f v i r u l e n c e o f Melampsora  l i n i r ace 218. Phy topatho logy 69:257-259. S t a t l e r , G.D. 1982. I nhe r i t ance o f v i r u l e n c e i n P u c c i n i a  r e c o n d i t a f . s p . t r i t i c i on Durum and s p r i n g wheat c u l t i v a r s . Phy topa tho logy 72:210-213. S t a t l e r , G.D. and D.A. J ones . 1981. I nhe r i t ance o f v i r u l e n c e and u r e d i a l c o l o r and s i z e i n P u c c i n i a r e c o n d i t a t r i t i c i . Phy topa tho logy 71:652-655. S t a t l e r , G.D. and D.E . Zimmer. 1976. I nhe r i t ance o f v i r u l a n c e o f r a ce 370, Melampsora l i n i . Can. J . Bo t . 54 :73-75 . Tapke , V . F . 1926. S i n g l e hot-water and steam t rea tments o f seed wheat f o r c o n t r o l o f l o o s e smut. U.S. Dept . Ag r . B u l l . 1383. 171 Tapke , V . F . 1935. An e f f e c t i v e and e a s i l y a p p l i e d method o f i n o c u l a t i n g seed b a r l e y w i th covered smut. Phy topatho logy 25:1038-1039. Tapke , V . F . 1937. P h y s i o l o g i c r a c e s o f U s t i l a g o h o r d e i . J ou r . A g r i . Res. 55:683-692. Tapke , V . F . 1938. I n f l uence o f Env i ronment , a f t e r S e e d l i n g Emergence, on covered smut i n B a r l e y . Phy topa tho logy 28:370-371. Tapke , V . F . 1945. New p h y s i o l o g i c r a ces o f U s t i l a g o h o r d e i . . Phy topa tho logy 35:970-976. Tapke , V . F . 1948. Environment and the C e r e a l Smuts. B o t a n i c a l Rev. 14:359-412. Tapke , V . F . 1952. Fu r the r s t u d i e s on env i ronment a f t r s e e d l i n g emergence and o the r f a c t o r s t h a t i n f l u e n c e the i n c i d e n c e o f b a r l e y covered smut. Phy topatho logy 42:117-118. Tapke , V . F . and W.M. Bever . 1942. E f f e c t i v e methods o f i n c o u l a t i n g seed b a r l e y w i th covered smut ( U s t i l a g o  h o r d e i ) . Phy topatho logy 32:1015-1021. T a y l o r , J .W. and M.G. Z e l i n e r . 1931. E f f e c t o f depth o f s e e d l i n g s on the o c cu r r ence o f covered smut and l o o s e smut i n w in te r b a r l e y . J ou r . Am. Soc. Agron . 23 :132-141. Thomas, P.L. 1976. I n t e r a c t i o n o f v i r u l e n c e genes i n U s t i l a g o  h o r d e i . Can. J . Genet . C y t o l . 18 :141-149. Thomas, P.L. and C. Pe r son . 1965. Gene t i c c o n t r o l o f low v i r u l e n c e i n U s t i l a g o . Can. J . Genet . C y t o l . 7:583-588. T h u r s t o n , H.D. 1961. The r e l a t i v e s u r v i v a l a b i l i t y o f r a ces o f Phytophthora i n f e s t a n s i n m i x t u r e s . Phy topatho logy 51:748-755. T h u r s t o n , H.D. 1971. R e l a t i o n s h i p s o f g e n e r a l r e s i s t a n c e : l a t e b l i g h t o f p o t a t o . Phy topa tho logy 61:620-626. T i s d a l e , W.H. 1923. An e f f e c t i v e method o f i n o c u l a t i n g b a r l e y w i th covered smut. Phy topa tho logy 13:551-554. Vande rp l ank , J . E . 1963. P l an t D i s e a s e s : Ep idemics and C o n t r o l . Acad . P r e s s , New York . 349 pp . Vande rp l ank , J . E . 1968. D i sease r e s i s t a n c e i n p l a n t s . Acad . P r e s s . New York . 206 p p . 172 V o g e l , H. J . 1956. A conven ien t growth medium f o r Neurospora (Medium N ) . M i c r o b i a l G e n e t i c s B u l l . 13 :42-43 . W e l l s , S .A. 1958. I n h e r i t a n c e o f r e c t i o n to U s t i l a g o h o r d e i i n c u l t i v a t e d b a r l e y . Can. J . P l an t S c i . 38 :45-60. W i l l i a m s , N . D . , F . J . Gough and M.R. Rondon. 1966. I n t e r a c t i o n o f p a t h o g e n i c i t y genes i n P u c c i n i a g r a m i n i s f . s p . t r i t i c i and r e c t i o n o f genes i n T r i t i c u m aes t i vum s p . vu lga re •Marquis ' and ' R e l i a n c e ' . Crop S c i . 6 :245-248. Woodward, R.W. and D.C. T i ngey . 1941. I n o c u l a t i o n exper iments w i th covered smut o f b a r l e y . J . Am. Soc. Agron . 33:632-662. Yong, H.C. J r . , and J . M . P r e s c o t t . 1977. A s tudy o f r ace p o p u l a t i o n s o f P u c c i n i a r e c o n d i t a f . s p . t r i t i c i . Phy topatho logy 67:528-532. Y u , T . F . and C T . Fang. 1945. A p r e l i m i n a r y r e p o r t on f u r t h e r s t u d i e s o f 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 U s t i l a g o h o r d e i . Phy topatho logy 35:517-520. Zimmer, D . E . , J . F . S cha fe r and F . L . P a t t e r s o n . 1965. Nature o f f e r t i l i z a t i o n and i n h e r i t a n c e o f v i r u l e n c e i n P u c c i n i a  c o r o n a t a . Phy topa tho logy 55:1320-1324. 173 Appendix A pe r cen t smut on b a r l e y C u l t i v a r T r e b i from s e l f i n g 1 0 F 1 U s t i l a g o h o r d e i t e l i o s p o r e s U. h o r e d i d i k a r y o n 17-B 18-C 18-D 19-B 19-D 20-D 21-C 21-E 2 2-G 23-C i a l mat ing % smut 1 x 2 39 1 x 4 35 3 x 2 16 3 x 4 30 1 x 2 48 1 x 4 50 3 x 2 35 3 x 4 16 1 x 3 37 1 x 4 38 2 x 3 8 2 x 4 38 1 x 2 14 1 x 4 38 3 x 2 9 3 x 4 26 1 x 2 35 1 x 3 13 4 x 2 63 4 x 3 35 1 x 2 26 1 x 4 10 3 x 2 41 3 x 4 38 1 x 2 26 1 x 4 38 3 x 2 36 3 x 4 37 1 x 2 35 1 x 3 19 4 x 2 40 4 x 3 28 1 x 3 30 1 x 4 42 2 x 3 15 2 x 4 41 1 x 3 38 1 x 4 13 2 x 3 54 2 x 4 42 T h i s i s unpub l i shed da ta from a 1976 exper iment performed by Ebba, T . , & C. Person a t U . B .C . 174 Appendix B Pe rcen t smut on b a r l e y c u l t i v a r T r e b i from d i a l l e l c r o s s o f s p o r i d i a o f F, U s t i l a g o h o r d e i t e l i o s p o r e s showing m o d i f i c a t i o n o f major v i r u l e n c e gene by po lygenes Sporidia, mat] .ng type ja(-) Means Genotype: l I Cenotype: v Sporidial 17 A 21 C 19 B 20 A 21 E 20 C 21 C 21 B 24 C 24 A 20 C 20 A VV Vv vv Over-Lints 4 4 4 4 4 1 2 4 2 4 4 3 a l l 18 D 1 + 60 62 48 35 33 42 42 48 43 28 32 35 50 38 44 21 B 1 + 42 42 50 41 34 27 35 41 34 26 28 26 39 32 36 . S <i i 21 B 3 + 20 A 2 + SO 53 44 34 61 34 50 47 54 31 49 25 33 34 25 43 13 29 8 24 10 14 ' 5 20 51 37 16 27 34 32 I u 23 C 3 + 43 30 18 24 29 17 31 31 21 17 16 23 27 23 25 ting 21 C 1 + 50 48 50 56 45 38 16 16 11 4 10 3 48 10 29 a >i 19 A 3 + 55 47 55 60 48 32 14 0 4 6 5 9 50 6 28 * ffl a 22 G 3 + 47 35 27 32 27 23 12 17 18 14 8 8 32 13 22 •a ts 4J 0 20 A 1+ 48 46 36 36 1 7 13 2 0 1 4 0 29 3 16 Spoi J 24 D 2+ 24 A 3 + 45 38 31 38 33 21 25 16 13 8 19 15 5 5 2 2 3 0 1 2 2 1 2 1 28 23 3 2 15 12 22 P 2 + 24 16 24 6 10 2 8 4 4 1 3 1 14 4 9 vy 50 42 42 39 40 32 41 • £ Vv 44 37 35 33 22 19 35 38 28 21 20 22 30 vv 10 6 6 4 5 3 6 Overall 46 39 38 36 29 25 21 19 15 11 11 11 25 Data from Person e t a l . 1982, 1983. 175 Appendix C Sc reen ing b a r l e y c u l t i v a r s to two U s t i l a g o h o r d e i r a c e s I n t r o d u c t i o n Tapke (1945) r e p o r t e d the r e s u l t s o f i n f e c t i o n o f s e v e r a l U s t i l a g o h o r d e i r a ces on s e v e r a l c u l t i v a r s o f b a r l e y . C u l t i v a r s T r e b i and Odessa , were used by Tapke as d i f f e r e n t i a l v a r i e t i e s . The o b j e c t i v e o f t h i s exper iment was to o b t a i n i n f o r m a t i o n on whether s i x b a r l e y c u l t i v a r s were r e s i s t a n t o r s u s c e p t i b l e to two U. h o r d e i d i k a r y o n s , T^ and T 4 . M a t e r i a l s and Methods The c u l t i v a r s used were T r e b i , Odessa , Van tage , Keys tone , Conques t , Wo l fe , Gateway and O l l i . P r e p a r a t i o n o f the seed f o l l o w s the method d e s c r i b e d i n Chapter 4. The 4 h a p l o i d s p o r i d i a o f each o f the d i k a r y o n s , and T 4 , were ob t a i ned from s i l i c a g e l and prepared f o r i n o c u l a t i o n as s t a t e d i n Chapter 4. Each t reatment was p l a n t e d i n a 3 m row and the re were th ree r e p l i c a t e s f o r each t r ea tmen t . The t rea tments w i t h i n each o f the two U. h o r d e i r a ces were randomized i n a random b l o c k d e s i g n . The number o f smutted p l a n t s w i th a t l e a s t one smutted head out o f a t o t a l number o f p l a n t s was used as the b a s i s o f de t e rm in ing the pe r cen t smutted p l a n t s . 176 R e s u l t s and D i s c u s s i o n The c u l t i v a r s t e s t e d were r e s i s t a n t to the d i k a r y o n s , T-^  and T 4 ( Tab le C . l ) . T h i s was not s u r p r i s i n g because a l l o f the c u l t i v a r s , excep t T r e b i and Odessa , were not used by Tapke o r anyone e l s e as d i f f e r e n t i a l v a r i e t i e s f o r d i f f e r e n t i a t i n g the r a ces o f U. h o r d e i . What was unusua l i s t ha t Tapke had o b t a i n e d 43% and 5% smutted p l a n t s o f T r e b i when t e s t e d w i th T-^  and T^ , r e s p e c t i v e l y . He ob t a i ned 39% and 34% smutted p l a n t s o f Odessa when t e s t e d w i th T-^  and T 4 , r e s p e c t i v e l y . Ebba (1974) ob ta ined an average o f 44% and 2.5% smutted p l a n t s o f T r e b i when t e s t e d w i th T-^  and T 4 r e s p e c t i v e l y . The c u l t u r e s used i n t h i s exper iment were i d e n t i c a l to those used by Ebba, the on l y d i f f e r e n c e be ing tha t f o r t h i s expe r imen t , the c u l t u r e s had been s t o r e d i n s i l i c a g e l f o r s e v e r a l y e a r s . E i t h e r the c u l t u r e s have l o s t some o f t h e i r pa thogen i c a b i l i t y o r the i n o c u l a t i o n p rocedure was p o o r . There i s no reason to suspec t t ha t the l a t t e r i s t r u e . The o n l y p o t e n t i a l problem was tha t weather c o n d i t i o n s a f t e r p l a n t i n g a l lowed the seed to germina te q u i c k l y and p o s s i b l y the a p i c i a l mer is tem r e g i o n o f the p l a n t s was ahead o f the smut myce l ium. Groth (1974) had observed tha t Vantage , Keys tone , Conquest and Gateway were r e s i s t a n t to a number o f U. h o r d e i d i k a r y o n s . 177 Tab l e C . l Pe rcent smutted p l a n t s i n 8 b a r l e y c u l t i v a r s a f t e r i n o c u l a t i o n by 2 U s t i l a g o h o r d e i d i k a r y o n s Pe rcen t smutted p l a n t s produced C u l t i v a r by d i k a r y o n T r e b i 4 0 Odessa 7 2 Vantage 3 0 Keystone 0 0 Conquest 0 0 Wolfe 0 0 Gateway 0 0 O l l i 0 0 178 Appendix D E f f e c t o f seed ing r a t e on pe r cen t smutted p l a n t s I n t r o d u c t i o n I t has been p r e v i o u s l y r e p o r t e d tha t env i ronment can a f f e c t the l e v e l s o f smut observed (Ebba & Person 1975/ Emara & Freake 1981) . I t i s g e n e r a l l y b e l i e v e d tha t the c o n d i t i o n s o f growth o f the hos t and the v i g o r o f the hos t can g r e a t l y i n f l u e n c e the amount o f smut observed ( F a r i s 1924a & b ) . The e f f e c t o f seed ing r a t e on the l e v e l o f smut has not r e c e i v e d much a t t e n t i o n . Tapke (1948) gave a b r i e f rev iew o f the l i t e r a t u r e and found one r e p o r t where heavy seed ings produced more d i s e a s e d p l a n t s . Because seed ing c o n d i t i o n s seemed to be q u i t e v a r i a b l e , t h i s exper iment was per formed to compare seed ing r a t e s . M a t e r i a l s and Methods Two c u l t i v a r s , T r e b i and Odessa , were i n o c u l a t e d w i th s p o r i d i a 3 and 4 o f IV B. Seed t rea tment and i n o c u l a t i o n was performed as d e s c r i b e d i n Chapter 4. Four seed ing r a t e s were 50, 100, 150, and 200 seeds/3 m row. There were th ree r e p l i c a t e s f o r each t reatment i n a random b l o c k d e s i g n f o r each c u l t i v a r . The number o f smutted p l a n t s , w i th a t l e a s t one smutted head, out o f t o t a l number o f p l a n t s was r e c o r d e d . 179 Resu l t s and D i s c u s s i o n On the c u l t i v a r Odessa , there were no s i g n i f i c a n t ( p > 0 . 0 5 ) d i f f e r e n c e s i n l e v e l s o f smut ove r the f o u r seed ing r a t e s (Tab le D . l ) . There were s i g n i f i c a n t d i f f e r e n c e s (p = 0.05) o f l e v e l o f smut on the c u l t i v a r T r e b i (Tab le D.2 ) . Comparison o f the t rea tment means us ing L . S .D . i s shown i n Tab le D.3. The da t a from T r e b i compared to r e s u l t s from Chapter 7 f o r IV 3 x 4, show tha t seed ing o f 100 seeds/3 m g i v e s an o p t i m a l l e v e l o f smut. T h i s i s the seed ing r a t e t ha t i s used g e n e r a l l y f o r most expe r imen t s . I t i s not known why no d i f f e r e n c e s were observed on Odessa . The d i f f e r e n c e between the two c u l t i v a r s i s t ha t T r e b i i s c a r r y i n g a r e s i s t a n c e gene t ha t i s matched by the smut geno t ype , whereas, Odessa a p p a r e n t l y does not have t h a t p a r t i c u l a r r e s i s t a n c e gene. 180 T a b l e D.1 Summary o f a n a l y s i s o f v a r i a n c e o f U s t i l a g o  h o r d e i percentage on b a r l e y c u l t i v a r Odessa a t f o u r seed ing r a t e s Source o f v a r i a t i o n d . f . Mean square F-va lue a seed ing r a t e s r e p l i c a t e s e r r o r 3 2 6 28 22 143 .196 NS .154 NS NS = not s i g n i f i c a n t Tab l e D.2 Summary o f a n a l y s i s o f v a r i a n c e o f U s t i l a g o  h o r d e i pe rcentage on b a r l e y c u l t i v a r T r e b i a t f ou r seed ing r a t e s Source o f Mean v a r i a t i o n d . f . square F - v a l u e a seed ing r a t e 3 382 9.4 * r e p l i c a t e s 2 60 1.5 NS e r r o r 6 41 a NS = not s i g n i f i c a n t * s i g n i f i c a n t , P = 0.05 Tab le D.3 Comparison o f average pe r cen t smut produced by a U s t i l a g o h o r d e i d i k a r y o n on b a r l e y c u l t i v a r T r e b i a t f ou r seed ing r a t e s Seeding r a t e/3 m Ave % smut L . S .D . * 50 4 A 150 17 B 200 22 B 100 31 C * mean s e p a r a t i o n by Leas t S i g n i f i c a n t D i f f e r e n c e Appendix E Pe r cen t smut produced by F 2 d i k a r y o n s o f U s t i l a g o avenae on oa t c u l t i v a r Monarch 11 12 15 16 17 % smut F : it n. it * F± spore d i k a r y o n F 2 " £ 3 "  genotype 1 x 2 8 12 Mm 1 X 4 1 19 Mm 3 X 2 5 21 Mm 3 X 4 3 22 Mm 1 X 3 8 29 Mm 1 X 4 0 0 MM 2 X 3 93 80 mm 2 X 4 8 31 Mm 1 X 3 74 91 mm 1 X 4 2 26 Mm 2 X 3 19 25 Mm 2 X 4 3 0 MM 1 X 2 0 0 MM 1 X 3 9 42 Mm 4 X 2 8 48 Mm 4 X 3 98 79 mm 1 X 2 11 19 Mm 1 X 4 93 90 mm 3 X 2 0 0 MM 3 X 4 7 32 Mm 1 X 2 0 0 MM 1 X 4 9 26 Mm 3 X 2 12 31 Mm 3 X 4 99 89 mm F 3 i s i n c r e a s e o f F 2 d i k a r y o n s by t e l i o s p o r e i n o c u l a t i o n . Data o f H a l i s k y , P.M. 1965. 182 Appendix F Percentages o f smut- in fec ted p l a n t s i n twelve v a r i e t i e s o f o a t s (Avena s a t i v a ) d u r i n g e i g h t g e n e r a t i o n s o f s e l e c t i o n o f the pathogen ( U s t i l a g o avenae) on the oat v a r i e t y monarch Year and percentage infection Oat variety 1948" 1949 1950 1951 1952 1953 1954 1955 1956 Monarch 8 12 2 6 5S 60 68 - 60 99 9 2 Anthony 6 5 10 19 3 2 4 4 4 S 5 3 71 8 5 Mabel 64 10 14 58 73 57 72 8 3 8 2 Beacon 38 12 31 61 62 . 5 5 51 4 9 8 0 Black Diamond 10 3 e 21 3 5 26 15 38 74 Victory 14 2 10 28 19 2 6 33 3 9 5 0 Gothland 15 1 1 13 28 13 7 ' 2 7 2 5 Fulghum 1 0 1 • 3 3 8 0 10 9 Black Mesdag 0 0 0 1 0 0 0 0 1 Camas 0 0 0 0 0 0 3 4 0 Nicol 0 0 1 8 11 5 0 6 4 Atlantic 0 0 2 13 0 0 0 3 0 0 Data from Cherewick (1958). * The original field collection was tested in 194S; inoculum in all other years was produced in preceding year on variety Monarch. Taken from P e r s o n , C. 1966. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0096396/manifest

Comment

Related Items