UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Somatic recombination in Ustilago hordei during the parasitic phase on barly Megginson, Fiona Gertrude Ariel 1973

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

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1973_A6_7 M43_4.pdf [ 3.24MB ]
Metadata
JSON: 831-1.0101389.json
JSON-LD: 831-1.0101389-ld.json
RDF/XML (Pretty): 831-1.0101389-rdf.xml
RDF/JSON: 831-1.0101389-rdf.json
Turtle: 831-1.0101389-turtle.txt
N-Triples: 831-1.0101389-rdf-ntriples.txt
Original Record: 831-1.0101389-source.json
Full Text
831-1.0101389-fulltext.txt
Citation
831-1.0101389.ris

Full Text

SOMATIC RECOMBINATION IN USTILAGO HORDEI DURING THE PARASITIC PHASE ON BARLEY FIONA G.A. MEGGINSON B.Sc. Honours, U n i v e r s i t y of Edinburgh, 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Genetics We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1973 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 of the requirements 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 Columbia, 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 study. 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 copying 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 granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f C\ £"A/ ETICS The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada i ABSTRACT The question investigated i n t h i s study was whether or not somatic recombination can occur i n Ustilago  hordei whilst i t i s i n the p a r a s i t i c phase on barley. The inv e s t i g a t i o n was carried out i n two parts. In the f i r s t and major part, barley seeds were inoculated a r t i f i c i a l l y with mixtures of n u t r i t i o n a l l y d e f i c i e n t mutants of U. hordei. This was done i n such a way that i n f e c t i o n of the mature plants could only occur i f at l e a s t one recombination event had taken place asexually, between at l e a s t two i n f e c t i v e dikaryons, p r i o r to teliospore formation. One smutted plant was found. Detailed analysis of the teliospores from t h i s i n f e c t i o n was carried out. The second part of the study was designed to demonstrate that more than one i n f e c t i v e dikaryon can simultaneously occupy the host. Again, mixtures of n u t r i t i o n a l l y d e f i c i e n t mutants were used to inoculate barley seeds but t h i s time so that the o r i g i n of teliospores from any i n f e c t i o n could be traced back to the parental types i n the i n f e c t i v e dikaryon. I t was concluded that somatic recombination can i n f a c t occur whilst the fungus occupies the host tissue. i i TABLE OF CONTENTS Page LITERATURE REVIEW 1 INTRODUCTION 8 MATERIALS AND METHODS 11 A. Strains 11 1. Ustilago hordei... 11 2, Hordeum valgare .11 B. Culture of the fungus .12 1. Media 12 2. Growth of the fungus .1^ 3. Techniques 14 a. Mating type tests .14-b. Random spore analysis and replica plating .14-c. Tetrad analysis 15 C. Host inoculation and planting.. .16 1. Techniques 16 a. Basic method of seed inoculationJ.6 b. Experimental plan 17 2. Rationale .22 RESULTS 25 A. Somatic recombination experiment .25 1. Harvesting... .25 2. Random spore analysis ..........25 3. Tetrad analysis .29 B. Multiple infection experiment. * ^0 DISCUSSION .32 BIBLIOGRAPHY 51 i i i LIST OF TABLES Table Page No. 1. Mutants of y_. hordei used i n t h i s study............. 13 2. Plan f o r seed inoculation i n somatic recombination experiment • , 18 3 . Plan for seed inoculation i n multiple i n f e c t i o n experiment. 20 4. Preliminary random spore analysis of smut from head 501.. 26 5. Second random spore analysis of smut from head 501 ..2? 6. Tetrad analysis of 501 teliospores a f t e r micromanipulation. 29 7. Random spore analysis made from the multiple i n f e c t i o n experiment 31 8. Possible ways i n which somatic recombination could have occurred to give r i s e to teliospores 501 37 9. P o s s i b i l i t i e s for 501 teliospore formation.. 39 10. Table showing the meiotic segregations expected from germination of teliospore types kl i v LIST OF FIGURES Page No. Figure 1. Steps i n the parasexual cycle as they may occur i n U. hordei (after Pontecorvo) 4 V ACKNOWLEDGEMENTS I would l i k e to express my thanks to Dr. Clayton Person both for suggesting the problem and for helpful discussion. I am also g r a t e f u l to him for the use of his laboratory f a c i l i t i e s . Thanks are due, i n addition, to Dr. Tony G r i f f i t h s fo r help and encouragement during the early stages of the project when Dr. Person was away i n A u s t r a l i a . The author was enabled to carry out t h i s work by a scholarship awarded under the Commonwealth Scholarship and Fellowship Plan. 1. LITERATURE REVIEW The extreme v a r i a b i l i t y amongst plant pathogenic fungi has been known for a long time. Even where normal sexual reproduction i s impossible because the alternate host i s absent or mating types are incompatible, the pathogenic organisms often continue to show extensive v a r i a b i l i t y . This i s e s p e c i a l l y true of the smuts, which may be more variable than any other group of plant patho-genic fungi (Holton,1968j Cherewick ,1958). Here v a r i a t i o n abounds i n both morphology and physiology. In many cases where sexual reproduction i s absent, the d i v e r s i t y of progeny types cannot be explained on the basis of gene mutation alone. For t h i s reason, para-sexuality (Pontecorvo, 1956), otherwise known as somatic recombination, has been suggested as the mechanism by which these pathogenic fungi maintain t h e i r v a r i a b i l i t y . Parasexuality may be thought of as genetic recombination^ where no fi n e coordination between recombination, segrega-t i o n and reduction e x i s t s . In f a c t , the operation of parasexuality has been implicated i n a number of plant pathogens, including several rusts (Bartos et al . , 1 9 6 9 ; Bridgmon,1959? Ellingboe , 1 9 6 l j Sharma and Prasada,1969J V a k i l i and Caldwell,19575 Waterhouse,1952i Watson,19575 Watson and Luig , 1958, 1959• 1962;), smuts (Rowell,1955t Kozar ,1969; Day and Anagnostakis ,1971) and V e r t i c i l l i u m (Ingram,1968j Hastie , 1962), Cephalosporium (Tuveson and 2. Coy,1961), Phytophthora (Wilde,1961; Leach and Rich,1969) and Fusarium (Buxton,1956) species. I m p l i c i t and perhaps more important than v a r i a -b i l i t y i n a discussion of plant pathogenic fungi i s the means by which new races are originated. There are many cases i n the rusts, for example, where new races have been observed to a r i s e asexually (Bartos et a l . , 1 9 6 9 ; Bridgmon, 1959J Bridgmon and Wilcoxson,1959; Bugbee et a l . , 1 9 6 8 | Ellingboe , 1 9 6 l } Flor , 1 9 5 7 » I960; L i t t l e and Manners,19675 Nelson et al.,1955; Nelson,1966? Sharma and Prasada, 1969; V a k i l i and Caldwell, 1957; Waterhouse, 1952; Watson, 1957; Watson and Luig, 1958, 1959, 1962) . I t has been known for many years that new races and biotypes of p a r a s i t i c fungi a r i s e through hybridisation, mutation, and hetero-karyosis but i t seems not unreasonable to suspect that somatic recombination i s also responsible i n large part f o r the o r i g i n of new races (Person, 1958) . The parasexual cycle has been shown to operate i n a v a r i e t y of fungi including Aspergillus nidulans, (Pontecorvo and Raper, 1952), A. niger, and P e n i c i l l i u m  chrysogenum, (Buxton, 1956) , Coprinus species and Schizo-phyllum commune (Casselton, 1965)1 as well as i n the phytopathogenic fungi mentioned above. I t appears therefore, legitimate to conclude that the parasexual cycle i s not a rare oddity but that i t s occurrence i s f a i r l y widespread 3 . i n nature and that i t may well play a part i n the va r i a t i o n of host-parasite r e l a t i o n s h i p s . The precise extent of the operation of the parasexual cycle i n nature cannot be gauged since many species i n which i t might occur have so f a r not been suitable for experimentation of thi s kind. A major d i f f i c u l t y i n assessing the occurrence of parasexuality i n nature i s due to the lack of c r i t e r i a for i t s detection. Nevertheless, s u f f i c i e n t evidence i s availab l e to make a study of the process of somatic recombination i n a pathogen such as Ustilago hordei, a smut, worthwhile. Before describing the present study, a b r i e f outline of the steps i n the parasexual cycle as v i s u a l i s e d by Pontecorvo (1956) w i l l be given and the evidence for them considered. Three steps appear to be es s e n t i a l : (a) Heterokaryosis, (b) formation of heterozygous d i p l o i d n u c l e i and t h e i r m u l t i p l i c a t i o n and (c) occassional segregation and recombination at mitosis i n conjunction with haploidisation of d i p l o i d n u c l e i . A more detailed explanation of the parasexual cycle i s given i n Figure 1. FIGURE 1 STEPS IN THE PARASEXUAL CYCLE AS THEY MAY OCCUR IN U. HORDEI. (AFTER PONTECORVO). 13b^Haploid homokaryotic strain^-(abc d) 13D Haploid homokaryotic s t r a i n (ABC D)^ 3 32X10~ 6 unlike nuclei fuse Hyphal anastomosis haplo-diploid heterokaryotic myceliumi Haploid heterokaryotic mycelium (abc d) + (ABC DT (abc d) + (ABC D)+ [Jjg =5! 5a Heterozygous d i p l o i d nuclei _2 5b 10 heterozygous or ? d i p l o i d nuclei undergo mitotic crossing over 11 Haploid nuclei sort themselves out 8 12 Heterozygous d i p l o i d (abc d) homokaryotic mycelium (ABC D; (abc d) (ABc D) (aBC d) (ABC D) ^10" 3 d i p l o i d " n u c l e i undergo haploidisation Haploid homokarytic strains, some of which are recombinant 13a 13b 10 Haploid nu c l e i some of which are recombinants (abc :£.) 4 11 (abc dl (ABc D) (ABc d) (aBC d) etc. D i p l o i d i s a t i o n p r i o r to teliospore formation 1. abc/ABC represent 3 linked genes and d/D another one unlinked 2. The genotypes of the various kinds of nucleus are enclosed i n parentheses. 3. The occurrence figures are those given f o r Aspergillus nidulans. 5. There i s adequate evidence documented for each of the major steps involved. Heterokaryosis e n t a i l s association, separation and subsequent regrouping of g e n e t i c a l l y unlike nuclei i n multinucleate c e l l s as a r e s u l t of hyphal fusions. In the Basidiomycetes, dikaryosis, which i s simply heterokaryosis involving two d i f f e r e n t n u c l e i , i s a step i n the sexual process and as such i s a prerequisite for i n f e c t i o n of barley by U. hordei. The formation of heterozygous d i p l o i d s i n vegeta-t i v e c e l l s , or d i p l o i d i s a t i o n , i s not so c l e a r l y demonstrable i n smuts although there i s conclusive evidence f o r i t occur-r i n g i n other fungi. In V e r t i c i l l i u m (Ingram, 1968; Tolmsoff, 1972) vegetative d i p l o i d s occur i n nature whilst a r t i f i c i a l , genetic methods have been devised f o r the s e l e c t i o n of spontaneously occurring heterozygous d i p l o i d s i n A s pergillus nidulans (Roper, 1952), Ustilago maydis, (Holliday, 196lb), and U. violaceae (Day and Jones, 1968). Cytol o g i c a l evidence f o r d i p l o i d i s a t i o n i s contradictory. E h r l i c h (1958), working with U. maydis, observed that the mycelium r e s u l t i n g from a cross of two haploid l i n e s i n the host was i n i t i a l l y uninucleate. There was a progressive increase, however, i n the percentage of dikaryotic c e l l s u n t i l eleven days a f t e r inoculation at which time the trend reversed and uninucleate c e l l s again predominated. These changes were interpreted as being due to dikaryotisation 6. followed by nuclear unions. The l a t t e r were inferred from measurement of nuclear s i z e , which corresponded well with that of chlamydospore n u c l e i . Thus i t was concluded that karyogamy occurred p r i o r to spore formation. Indirect evidence f o r d i p l o i d i s a t i o n was also obtained by Leach and Rich (i960) i n Phytophthora infestans where hyphal fusions were observed c y t o l o g i c a l l y and production of parental and recombinant types of zoospores was obtained. The actual presence of d i p l o i d n u c l e i , however, was never observed. Bakerspigel (1965)» on the other hand, obtained inconclusive r e s u l t s upon c y t o l o g i c a l investigation of 19 d i f f e r e n t species and lk m i l l i o n nuclei for evidence of nuclear fusions i n heterokaryotic mycelium. He concluded that such fusions possibly occur at a rate lower than 1 i n 1 0 . Thus the evidence for the occurrence of hetero-zygous d i p l o i d s i n vegetative c e l l s of filamentous fungi has been almost exclusively genetic. I t seems certain that d i p l o i d i s a t i o n i s a rare event. F i n a l l y , the evidence f o r segregation and recom-bination at mitosis and for haploidisation i s extensive. These phenomena have been followed i n d i p l o i d s t r a i n s during both the sexual and parasexual cycles (Pontecorvo, 1956). M i t o t i c haploidisation has been induced i n U. violaceae by p-fluorophenylalanine (Day and Jones, 1968) 7 . and the r e s u l t i n g haploid l i n e s show a wide range of genotypes, both recombinant and parental for markers i n the o r i g i n a l d i p l o i d . I t also occurs spontaneously but at a much lower frequency, i n d i c a t i n g that d i p l o i d n u c l e i are r e l a t i v e l y stable through many generations of somatic c e l l s . This obviously would enhance the opportunity for mitotic crossing-over and segregation to occur. The f i r s t demonstration of mitotic recombination i n fungi followed the observation of homozygous d i p l o i d segregants from heterozygous d i p l o i d s i n A . nidulans. This was followed by a s i m i l a r demonstration i n U. maydis, where auxotrophic segregants, s t i l l d i p l o i d , were obtained from prototrophic heterozygous d i p l o i d s (Holliday, 1961) and another i n U. violaceae (Day and Jones, 1968). I t seems certain then that somatic recombination, an i n t e g r a l part of the parasexual cycle, i s an important feature of the l i f e cycle of certain fungi. Indeed, Raper (1959) has implicated i t s significance i n a l l Basidiomycetes, which of course includes the smuts. 8. INTRODUCTION The knowledge that somatic recombination occurs i n culture both spontaneously and af t e r induction i n Ustilago maydis (Rowell, 1955? Holliday, 196lb, 1965) and U. violaceae (Day and Jones, 1968, 1969) has stimulated s i m i l a r investigations of the process i n another smut, namely Ustilago hordei. Dinoor and Person (1969) obtained growth of the dikaryotic phase of U. hordei i n culture. The authors indicate that haploid recombinant sporidia have been obtained from "forced dikaryons" and i n view of the known parasexuality i n the two species mentioned above, conclude that somatic recombination can occur i n v i t r o i n U. hordei also. Further evidence that the parasexual cycle, as well as the normal sexual cycle, occurs i n U, hordei was obtained by Kozar (196?, 1969a). Compatible combinations of mutant spo r i d i a were used to i n f e c t the barley host. A f t e r some time the parasite was recovered as sporidia or as teliospores. Evidence indicates that the nuclei within the i n f e c t i v e dikaryon undergo fusion and produce d i p l o i d s p o r i d i a characterised by an i n d e f i n i t e Bauch test (see Materials and Methods) and biochemical "wild type" appearance. I f these d i p l o i d i n f e c t i o n sporidia were to undergo haploid-i s a t i o n , thereby providing somatic recombination of parental 9 . biochemical markers, a l l the requirements of the parasexual cycle would have been f u l f i l l e d (see L i t e r a t u r e review). In f a c t recombinant types were observed amongst sporidia extracted from the host p r i o r to teliospore formation. The d i f f i c u l t y i n i n f e r r i n g somatic recombination occurring i n vivo, however, l i e s i n the fa c t that recombination could possibly have occurred i n the inoculum mixture p r i o r to penetration of the host. A l t e r n a t i v e l y , i t may have occurred i n the i n v i t r o grown cultures a f t e r they had been extracted from the host. In an e f f o r t to c l a r i f y the process, Dinoor (unpublished) u t i l i s e d a "double inoculation technique". In t h i s technique some cultures were used on the seed (e.g. ad-arg-leuc+ad~) and others were injected into the seedlings grown from these inoculated seed (e.g. ad~arg-leuc""+leuc~). Recovery of wild type mutants, which could only have arisen by at l e a s t two consecutive recombin-a t i o n a l events, one of which would necessarily be asexual, taking place i n the host p r i o r to teliospore formation, would demonstrate the operation of somatic recombination inside the plant. Preliminary r e s u l t s were encouraging but the work was discontinued owing to the lack of repeat-a b i l i t y i n the experiment. In addition, the numbers of plants used were u n r e a l i s t i c a l l y small f o r the detection of such a rare phenomenon (see Li t e r a t u r e review). 10. The objective of the present study was to provide an unequivocal demonstration of somatic recombina-t i o n , i f indeed i t does occur, i n operation during the development of U. hordei inside the host plant. As i n the two previously described studies, barley seeds were inoculated a r t i f i c i a l l y with mixtures of n u t r i t i o n a l l y d e f i c i e n t mutants. In t h i s study, however, the gametic matings were arranged i n such a way as to preclude the occurrence of a recombinational event p r i o r to penetration of the host. Furthermore, the p o s s i b i l i t y of a recombin-at i o n a l event occurring aft e r extraction of sporidia from the host was eliminated by the selection process employed i n t h i s study (see Methods). 1 1 . MATERIALS AND METHODS. A . STRAINS 1 , Ustilago hordei (Pers.) Lagerh. The mutants used i n t h i s study were induced by Hood ( 1 9 6 6 ) using UV i r r a d i a t i o n and were derived from the wild type st r a i n s I ^ + and . I t was assumed that the majority of these mutations were point mutations rather than chromosomal aberrations, since the former are known commonly to r e s u l t a f t e r UV i r r a d i a t i o n . The p a r t i c u l a r auxotrophic st r a i n s used were chosen ( 1 ) for ease of detection i . e . non-leakiness, ( 2 ) f o r the absence of any evidence of close linkage with other markers such as the mating type locus or other n u t r i t i o n a l mutations and ( 3 ) f o r t h e i r non-pathogenicity on Hannchen and Vantage barley c u l t i v a r s when present i n the homozygous state. Table 1 shows the strains chosen. I t should be noted that the double mutant stra i n s used i n each case were derived from the corresponding single mutants. 2 . Hordeum vulgare L, For t h i s study, two v a r i e t i e s of barley, Hannchen and Vantage, were chosen. These v a r i e t i e s are known to be highly susceptible to i n f e c t i o n from crosses between the wild type st r a i n s of the fungus from which the auxotrophic mutants (see Table 1 ) were derived (Thomas, 1 9 6 5 ) . 12. In addition to the laboratory stock of Vantage seed, some Vantage was obtained from Buckerfields Ltd., New Westminster. Care was taken to di s t i n g u i s h between these two types of Vantage since t h e i r genetic ancestry was l i k e l y to be d i f f e r e n t . B. CULTURE OF THE FUNGUS. 1. Media. A modified Vogel's (1956) medium was used. Complete medium contained: 5g« Difco yeast extract, 5gt s a l t - f r e e casein hydrolysate (N.B. Co.), lOg. dextrose, 50mg. tryptophane, 20ml, Vogel's s a l t solution ( i b i d ) , 10ml. vitamin solution (Holliday, 1961), 1 l i t r e d i s t i l l e d water. For s o l i d medium 2% Difco bacto agar was added to make agar plates and 2.5$ to make slants. Minimal medium contained 20ml. Vogel's s a l t s olution and lOg. dextrose per l i t r e of d i s t i l l e d water. Supplemented minimal medium was prepared according to Holliday (1961). The i n d i v i d u a l growth factors were added to the minimal medium as required: amino acids lOOmg., purines and pyrimidines lOmg., and vitamins 1 mg. per l i t r e . A l l media were s t e r i l i s e d by autoclaving for 15 minutes at 151b. pressure and 121 degrees Centigrade before use. 13. TABLE 1 Mutants of U. h o r d e i used i n t h i s study. Experiment Strains Genotype Mating type N u t r i t i o n a l requirement Somatic Recombination AdU40 A d " N i a + a Adenine NiaX95 Ad^Nia" a N i a c i n 2-15 Ad~Nia~ A Adenine + N i a c i n MetV364 Met~Ser + a Methionine SerV138 Met s e r " a S e r i n e 2-34 Met"Ser" A . .Methionine + S e r i n e LeuV4-17 Leu"Arg* a Leucine ArgV240 Leu_Arg~ a A r g i n i n e 2-53 Leu"Arg" A Leucine + A r g i n i n e ArgV242 Arg~Leu* a A r g i n i n e LeuV4-17 Arg_Leu~ a Leucine 2-55 Arg~Leu~ A A r g i n i n e + Leucine M u l t i p l e I n f e c t i o n I 4 + A E/C * + a -ArgV35 Arg" A & a Arginine ProV324 Pro A & a Proline PanV271 Pan" A & a Pantothenic Acid PdxV26 Pdx" A & a Pyridoxine MetV375 Met" A & a Methionine LeuV4 Leu" A & a Leucine IlvU26 I l v " A & a Isoleucine and V a l i n e * Denotes wild type. Each culture was routinely tested f o r i t s n u t r i t i o n a l requirements and i t s mating type p r i o r to use. 14 2. Growth of the fungus. Shake c u l t u r e s were maintained i n Delong c u l t u r e f l a s k s f i t t e d w i t h Morton s t a i n l e s s s t e e l c l o s u r e s p l a c e d i n a New Brunswick Psychrotherm i n c u b a t o r - s h a k e r . These f l a s k s c o n t a i n e d l i q u i d medium to approximately 40$ of t h e i r t o t a l c a p a c i t y . A l l i n c u b a t i o n of c u l t u r e s , l i q u i d o r s o l i d , was made a t 22 degrees Centigrade (-2degrees). 3. Techniques. a. Mating type t e s t s . T h i s fungus e x h i b i t s b i p o l a r i n c o m p a t i b i l i t y and t h e r e f o r e i t i s important to e s t a b l i s h the mating types of a l l c u l t u r e s i n v o l v e d . C o m p a t i b i l i t y o f the s p o r i d i a l l i n e s was determined by the Bauch t e s t r e f e r r e d to by F i s c h e r and Ho i t o n (1957). Here, however, the t e s t s had to be c a r r i e d out on complete medium owing to the r e l u c t a n c e of s t r a i n s c a r r y i n g more than one auxotrophic marker to produce suchfaden ( i n f e c t i o n hyphae) on minimal medium. b. Random spore a n a l y s i s and r e p l i c a p l a t i n g . S p o r i d i a r e c o v e r e d from smutted heads of b a r l e y were taken from the shake c u l t u r e s when the c o n c e n t r a t i o n o f s p o r i d i a ft 7 approached 10 -10' per ml. One or two drops of achromycin (lOmg/ml.) were added to each shake c u l t u r e to prevent b a c t e r i a l p r o l i f e r a t i o n . Counts of the s p o r i d i a were made u s i n g a Spencer haemocytometer w i t h improved Neubauer r u l i n g 15. and appropriate d i l u t i o n s were made to g i v e approximately 100 c o l o n i e s per p l a t e . S t e r i l e c o n d i t i o n s were ensured throughout. When these c o l o n i e s became v i s i b l e (about 3-4 days), c o l o n i e s were picked o f f w i t h s t e r i l e t o o t h p i c k s and planted to "master" p l a t e s of complete medium on g r i d s o f 25 s e c t i o n s . For each a n a l y s i s made, approximately 200 c o l o n i e s were t e s t e d . A f t e r 3-4 days i n c u b a t i o n , the c o l o n i e s were ready f o r r e p l i c a t i o n . R e p l i c a p l a t i n g was performed according to Lederberg and Lederberg's technique ( 1 9 5 2 ) , otherwise known as the "Velvet Pad Method". Colonies were r e p l i c a t e d to minimal medium, to each of the r e q u i r e d supplemented media and to lawns of and on complete medium i n order to determine the mating type (see B3a. above). A f t e r 4-5 days the p l a t e s were examined to determine the biochemical c h a r a c t e r i s t i c s of the c o l o n i e s . c. Tetrad a n a l y s i s . I s o l a t i o n of the f o u r products of meiosis from the budding promycelium and t h e i r subsequent t r a n s f e r to new media to set up separate c u l t u r e s was done by standard procedure (D i c k i n s o n , 1926) using a De Fonbrune micromanipulator and microforge. A s l i g h t m o d i f i c a t i o n of the method was employed to e l i m i n a t e the d i f f i c u l t and hazardous process of s i n g l e t e l i o s p o r e i s o l a t i o n . Instead, a small drop of 16. the t e l i o s p o r e suspension was p l a c e d on the agar b l o c k , having f i r s t g r e a t l y d i l u t e d the suspension. In t h i s way 5-10 t e l i o s p o r e s were p l a n t e d t o each agar block and a f t e r germination, the most s u i t a b l e one could be chosen f o r micromanipulation, l e a v i n g the r e s t untouched i n the c e n t r e of the b l o c k . The t e t r a d s c o u l d then be analysed f o r t h e i r requirements on supplemented minimal media. C. HOST INOCULATION AND PLANTING. 1. Techniques. a. B a s i c method o f seed i n o c u l a t i o n . The b a r l e y seeds were i n o c u l a t e d w i t h the fungus a c c o r d i n g to the method o u t l i n e d by Tapke (1942). A l l seeds were t r e a t e d w i t h 0.12$ f o r m a l i n ( i . e . 40$ formaldehyde d i l u t e d 1 :320 w i t h water) f o r one hour i n order to s t e r i l i s e the seed s u r f a c e and l o o s e n h u l l s , which i s thought to a i d i n f e c t i o n . The seeds were then washed i n r u n n i n g tap water f o r 30 minutes. The wet seeds were spread t h i n l y on paper towels to a i r dry f o r 24-48 hours. S p o r i d i a l c u l t u r e s were i n o c u l a t e d to f r e s h agar s l a n t s and allowed to grow f o r 3-4 days. F r e s h inoculum from s l a n t s was p l a c e d i n the a p p r o p r i a t e q u a n t i t y o f l i q u i d complete medium and m u l t i p l i e d i n shake c u l t u r e f o r 48-60 hours u n t i l the c u l t u r e s appeared t h i c k . 1 7 . The barley seeds to be inoculated were counted into batches of about 100 and placed i n flat-bottomed #7 snap-cap c r y s t a l i t e v i a l s . S p o r i d i a l inoculum was poured over the seeds using equal quantities of the two mating types i n each v i a l . The v i a l s were placed uncovered i n a dessicator jar (without dessicant), and evacuated for 20 minutes at 20 l b . vacuum, i n order to draw the inoculum under the seed h u l l s . Excess inoculum was poured o f f and the seeds dumped into l a b e l l e d #2 Manilla coin envelopes. These envelopes were l e f t unsealed so that the seeds could a i r dry at room temperature. After a minimum of three days, the seeds were sown. b. Experimental plan. The f i r s t and main experiment was designed to detect somatic recombination occurring i n the fungus during i t s develop-ment inside the host. To t h i s end, seeds were inoculated i n the manner shown i n Table 2 . The seeds were then sown i n the f i e l d i n May, 1 9 7 2 . Each row was 10 feet long and contained about 100 seeds. The rows were spaced one foot apart. A f t e r about three months the barley plants were examined c a r e f u l l y f o r the presence of smut and any smutted heads c o l l e c t e d . The second experiment was carried out as a supportive study to the f i r s t experiment. I t was designed to demonstrate that multiple i n f e c t i o n , a prerequisite f o r 18. TABLE 2 P l a n f o r seed i n o c u l a t i o n i n somatic recombination experiment. T e s t C r o s s 3 , Number number Combination A a V a r i e t y o f rows 1 2-15 AdU4o + NiaX95 H 10 > 6 4 2 C 2-15 AdU4o H 10 l e V. y b 6 4 3° 2-15 NiaX95 H 10 4 C AdU40 d NiaX95 > 6 4 10 10 1 2-34 MetV364 + SerV138 H 20 v h 10 V b 10 2 2-34 MetV364 H 20 2 > 10 10 3 2-34 SerV138 H v h 20 10 V b 10 4 MetV364 a SerV138 H V b 20 10 10 1 2-53 LeuV4l7+ ArgV240 H 20 > 10 10 2 2-53 LeuV4l7 H 20 10 3 v b 10 3 2-53 ArgV240 H 20 V h 10 V b 10 4 LeuV4l7 ArgV240 H 20 8 12 19. TABLE 2 (CONT.) C r o s s a T e s t Combination A a V a r i e t y Number of rows 1 2-56 ArgV242+ LeuV4l7 H 20 V. 10 V b 10 2 2-56 ArgV242 H 20 4 V 10 V D 10 3 2-56 LeuV4l7 H 20 Vrb 1 0 ArgV242 a LeuV4l7 H 20 v * 8 V D 12 H = Hannchen V = Vantage a. As s t a t e d above, equal q u a n t i t i e s o f the two op p o s i t e mating types were used i n a l l combinations i . e . when two c u l t u r e s o f the same mating type, a^ . and a 2» are used i n one c r o s s , Volume of A = Volume a 1 + a 2 andV rolume a^ * Volume a, b. The Vantage seed d e s i g n a t e d V was obtained from B u c k e r f i e l d ' s L t d . (see A2 above). c. Combinations 2, 3 and 4 i n each t e s t c o n s t i t u t e the nece s s a r y c o n t r o l s f o r t h i s experiment i . e . the p a i r - w i s e combinations o f each o f the c u l t u r e s used i n one t e s t . d. Although the c u l t u r e s used i n Combination 4 of each t e s t were of the one mating type and t h e r e f o r e i n c o m b a t i b l e , i t was s t i l l n e c essary to make t h i s c o n t r o l (see C2 below). e. Each t e s t was d i v i d e d i n t o two i d e n t i c a l p a r t s i n order to p r o v i d e r e p l i c a t e s . R e p l i c a t e s were t r e a t e d s e p a r a t e l y i n d i f f e r e n t batches and sown i n d i f f e r e n t p a r t s o f the f i e l d . TABLE 3 Plan for seed inoculations i n multiple i n f e c t i o n experiment Test Combination A a a a a a a a a 1^ E~ ArgV35 ProV324 PanV271 Pdxv26 MetV375 LeuV4 IlvU26 1 + + 2 + + 3 + + + 4- + + + + 5 + + + + + 6 + + + + + + 7 + + + + + + + 8 + + + + + + + + TABLE 3 (CONT.) Test Combination A A A A A A A a ArgV35 ProV32i+PanV271 PdxV26 MetV375 LueV4 IlvU26 - - ~ 2 + + + 3 + + + + B 4 + + + + + 5 + + + + . + + 6 + + + + + + + 7 + + + + + + + + Test B formed a reci p r o c a l (with respect to mating types) r e p l i c a t i o n of Test A. Once again, equal quantities of the two mating types were used i n each combination as explained i n Table 2, 2 2 . the success of the f i r s t experiment, can a c t u a l l y occur. Crosses were made according to the schedule shown i n Table 3. A l l the inoculations were made to the Hannchen c u l t i v a r and i n each case one row of 50 seeds was planted. Each row was 4-g- feet long and the rows were spaced 6 inches apart. This experiment was performed i n the greenhouse i n October, 1972. Seeds were grown at 75 degrees Fahrenheit (-5 degrees) using a 15 hour day as suggested by Schafer et a l . (1962). During i t s growth, the crop had to be treated with i n s e c t i c i d e for aphids and spider mites and with sulphur dust for powdery mildew. These problems and the crowding of the seeds i n the bench caused a number of plants to die but s u f f i c i e n t data was s t i l l obtained owing to the provision of a r e p l i c a t e being made. Smutted heads were co l l e c t e d a f t e r about 4 months. 2. Rationale. The somatic recombination experiment was designed to detect the occurrence of t h i s phenomenon i n the fungus within the infected host plant p r i o r to t e l i o -spore formation. The method depends on the occurrence of double i n f e c t i o n . The mutant strains chosen f o r each te s t were known not to permit sporulation when brought to homozygosity. For example, i n one t e s t , the markers were arranged i n the following way: 23. S t r a i n 1 : A Leu"Arg" St r a i n 2 » a Leu~Arg + S t r a i n 3 '> a Leu +Arg~ where A and a are the two mating types. When these strains are mixed together and poured over the susceptible barley seeds fo r inoculation, two matings are expected, both equally l i k e l y (providing that there i s no d i f f e r e n t i a l i n tendency to mate and that the two 'a' s t r a i n s were present i n equal quantities and the quantity of 'A' mating type equalled that of 'a* mating type)t #1 X #2 to give dikaryon Leu~Arg" + Leu~Arg + and #1 X #3 to give dikaryon Leu Arg + Leu Arg . Thus, those plants which have been infected singly, or carry a single i n f e c t i n g genotype, should show no smut at maturity, since neither dikaryon i s capable of causing i n f e c t i o n alone; both dikaryons are homozygous fo r one or the other of the two n u t r i t i o n a l markers. Consequently, any plant which does have smut would be of i n t e r e s t and investigation of the teliospores would be informative. Controls were necessary i n t h i s experiment to ensure that the i n d i v i d u a l pair-wise matings ( i . e . #1X#2, #1X#3, #2X#3) on the susceptible host gave no r e s u l t . These controls would also provide a check for mutation of the mutant cultures back to wild type. Otherwise the experiment consisted simply of inoculating with the mixtures of #1, #2 and #3, looking for smutted heads and checking these 24. f o r markers pres e n t i n s i n g l e t e l i o s p o r e s . The method d i c t a t e d by t h i s r a t i o n a l e cannot e n t i r e l y exclude the p o s s i b i l i t y o f an asexual recombination event o c c u r r i n g d u r i n g i n o c u l a t i o n , w h i l s t the s p o r i d i a are r e s t i n g on the s u r f a c e of the seed p r i o r to p e n e t r a t i o n . The time span i n v o l v e d , however, i s so b r i e f t h a t , f o r the purposes of t h i s study, such a l i k e l i h o o d may be s u b o r d i n -ated to the g r e a t e r p o s s i b i l i t y o f the event o c c u r r i n g w h i l s t the fungus i s a c t u a l l y i n s i d e the p l a n t . S i n c e the success of the somatic recombination experiment wholly depended upon the occurrence of double i n f e c t i o n , i t was c o n s i d e r e d necessary to show by e x p e r i -ment t h a t more than one i n f e c t i o n c o u l d i n f a c t occur. The experiment was s e t up i n a s i m i l a r manner to t h a t d e s c r i b e d by Person and Cherewick (1964). The mutants were known to produce i n f e c t i o n on Hannchen when crossed with e i t h e r 1^ or (depending on t h e i r mating t y p e ) . The s p o r i d i a l matings were arranged i n such a way t h a t up to seven d i f f e r e n t genotypes, each capable of b e i n g d i s t i n -guished from the others by v i r t u e of t h e i r n u t r i t i o n a l requirements, were prese n t together i n the inoculum mixture. In t h i s way, i t can be deduced, by random spore a n a l y s i s , whether any smut on a p l a n t has been produced by e i t h e r one, two, t h r e e , f o u r , f i v e , s i x or seven i n f e c t i o n s . 2 5 . RESULTS A. SOMATIC RECOMBINATION EXPERIMENT. 1. Harvesting. TEST 1. (see Table 2) I t was noticed that many of the 8000 plants i n th i s test were smutted, which was not expected. Therefore, the o r i g i n a l cultures used were retested and i t was found that the Ad U4-0 culture had reverted to wild type. This test was consequently disregarded. TEST 2 . Out of 16000 plants, none were smutted. TEST J3. One plant out of 16000 was smutted. I t occurred i n combina-t i o n 1 of the test and was therefore of great i n t e r e s t . I t had two smutted t i l l e r s and was designated 501 since t h i s was the number of the row i n which i t was found. TEST 4 . Out of 16000 plants, none were smutted. N.B. None of the control combinations i n tests 2 , 3 or 4 showed smut. 2 . Random Spore Analysis. Table 4 shows the r e s u l t s of the f i r s t random spore analysis that was made. 26. TABLE 4 P r e l i m i n a r y random spore a n a l y s i s o f smut from head 5 0 1 . 4 - 4 - 4. — — _ _ Leu A r g Leu Arg Leu A r g L e u Arg Unknown T o t a l Number of „ c o l o n i e s i 30 24 25 26 21 126 % T o t a l 1 2 3 . 8 19.1 1 9 . 9 20.6 16.6 100 I t can be seen from the t a b l e t h a t 17?° of the c o l o n i e s recovered d i d not have any of the expected requirements i . e . they d i d not grow on minimal medium or on medium supplemented w i t h a r g i n i n e , l e u c i n e or l e u c i n e + a r g i n i n e . These unknown c o l o n i e s were i n v e s t i g a t e d by a pr o c e s s of e l i m i n a t i o n a f t e r the manner d e s c r i b e d by H o l l i d a y (1961). They were s u c c e s s i v e l y t e s t e d on media l a c k i n g f i r s t amino a c i d s , then bases, then v i t a m i n s . Once i t was e s t a b l i s h e d t h a t the unknown requirement was f o r a v i t a m i n , i t was p o s s i b l e to narrow the i n v e s t i g a t i o n down s t i l l f u r t h e r , by a s i m i l a r p r o c e s s , to n i c o t i n i c a c i d ( n i a c i n ) as b e ing the m i s s i n g n u t r i e n t . I t was assumed t h a t t h i s was the r e s u l t of a spontaneous mutation s i n c e the o r i g i n a l c u l t u r e s , when r e t e s t e d , showed no t r a c e o f a n i a c i n requirement. With t h i s knowledge, a second random spore 2 7 . a n a l y s i s was c a r r i e d out, the r e s u l t s o f which are shown i n Table 5 . TABLE j> Second random spore a n a l y s i s o f smut from head $ 0 1 . Genotype as deduced from auxonography Mating type A a T o t a l number % of t o t a l c o l o n i e s L e u + A r g + N i a + 23 18 41 2 0 . 2 Leu~Arg~Nia~ 7 5 12 5 . 9 L e u ~ A r g + N i a ~ 9 6 15 7 . 4 L e u + A r g ~ N i a + 18 19 37 18 . 2 Leu A rg N i a 22 8 30 14 . 8 L e u + A r g ~ N i a " 6 11 17 8 . 4 L e u + A r g + N i a ~ 7 7 14 6 . 9 L e u ~ A r g ~ N i a + 17 20 37 1 8 . 2 T o t a l s 109 94 203 1 0 0 . 0 In t h i s case, the requirements of a l l the c o l o n i e s were d i s c o v e r e d and f i t t e d w i t h the e x p e c t a t i o n s i f there had been a mutation to N i a c i n d e f i c i e n c y i n one of the p a r e n t a l c u l t u r e s . I t was observed, however, t h a t the d i f f e r e n t genotypes d i d not occur i n equal f r e q u e n c i e s , as would be expected on the b a s i s o f random gene assortment a t m e i o s i s . 2 8 . A possible explanation for the discrepancy ( i . e . the severe deficiency of a l l Nia~ containing colonies) could have been d i f f e r e n t i a l rates of m u l t i p l i c a t i o n of the various geno-types a f t e r spore germination. In order to fi n d out i f t h i s was the case, a competition experiment was performed. In t h i s experiment, a Leu Arg Nia culture and a Leu~Arg~Nia~ culture were each inoculated to 5ml. of l i q u i d complete medium. These were allowed to multiply i n shake culture for 2k hours. The c e l l s were then counted so that the concentrations could be adjusted and equal numbers of c e l l s of each type added to 50 ml. of l i q u i d complete medium i n a DeLong culture f l a s k . The mixture was shaken at constant temperature for three hours after which time the c e l l s were plated out onto complete medium to a concentra-tion of about 100 colonies per plate. Master plates were made af t e r three days and these were l a t e r replicated to minimal medium. I t was found that twice as many of the colonies + + . + — — selected were of the Leu Arg Nia type;., as of the Leu Arg Nia type, confirming the suspicion of d i f f e r e n t i a l rates of m u l t i p l i c a t i o n for the two genotypes. Evidently then, the ^discrepancy was caused not by a deficiency of Nia~ carrying genotypes a f t e r spore germination but merely by th e i r slower rate of d i v i s i o n . 29. 3. Tetrad Analysis. Great d i f f i c u l t y was experienced i n obtaining complete tetrads a f t e r micromanipulation of the 501 t e l i o -spores. Out of at lea s t 100 attempts, only 10 complete tetrads were obtained, with the help of J.V. Groth. Table 6 shows the r e s u l t s of the tetrad analysis. TABLE 6 Tetrad analysis of 501 teliospores a f t e r micromanipulation. Tetrad Leu Arg Nia Number Tetrad Leu Arg Nia Number type obtained type obtained + + + + + + + + + + + + + + + + + + + + + Arg = Arginine; Leu = Leucine; Nia = Niacin. - indicates a requirement for the nutrient. + indicates no requirement for the nutrient. 30. B. MULTIPLE INFECTION EXPERIMENT. Smutted plants were not obtained from every combination i n both tests (see Table 3) but at lea s t one smutted head was obtained for each type of inoculation. Where more than one smutted head was obtained for a p a r t i -cular combination, each head was tested i n d i v i d u a l l y . The r e s u l t s of the analysis appear i n Table 7. I t can be seen that i n only one head out of the 20 examined was there any evidence of multiple i n f e c t i o n . Combination k i n Test B showed d e f i n i t e l y that two d i f f e r e n t i n f e c t i v e dikaryons had participated i n producing the smut i n f e c t i o n . TABLE 7 RANDOM SPORE ANALYSIS MADE FROM THE MULTIPLE INFECTION EXPERIMENT Test Combination Head 1 Mating type 2 Mating type 3 Mating type Number A a Number A a Number A a 1 1 WT 96 40 56 Arg" 90 57 33 2 1 WT 104 44 60 Arg" 95 50 45 -2 WT 104 54 50 Arg" 93 ^5 48 -3 1 WT 93 57 36 Arg" 105 51 54 -A 6 1 WT 109 57 52 Leu" 91 40 51 -2 WT 127 69 58 Leu" 72 25 47 -7 1 WT 175. 86 89 I l v " 18 8 10 -2 WT 175 98 77 I l v " 15 2 14 — 8 1 WT 180 96 84 1 1 WT 107 56 51 Arg^ 92 47 45 2 1 WT 90 38 52 Arg 110 53 57 -2 WT 110 53 57 Arg" 84 34 50 -4 1 WT 105 51 54 Arg" 22 10 12 Pan" 68 26 42 5 1 WT 100 51 49 Pan" 99 48 51 B 6 1 WT 136 63 73 - -2 WT 190 97 93 - -7 1 WT 198 103 95 - -2 WT 192 107 85 - -3 WT 195 94 101 - -4 WT 183 98 85 WT = wild type Refer to Table 3 for the s p o r i d i a l crosses made i n each combination. 3 2 . DISCUSSION The detection of a rare recombinational event i n a micro-organism requires conditions that f a c i l i t a t e recognition of a novel phenotype i n a vast population having the parental phenotype. The se l e c t i o n system employed i n t h i s study met t h i s requirement! out of 12000 plants i n which somatic recombination could have occurred, i t was easy to pick out the one plant i n which i t had taken place (subject to confirmation by the i s o l a t i o n of the o r i g i n a l markers). N u t r i t i o n a l l y d e f i c i e n t mutants were used i n t h i s study as markers for investigating the process of somatic recombination. Analysis for t h e i r presence, subsequent to the putative somatic recombination event, provided confirmation that t h i s one smutted plant was indeed produced through somatic recombination rather than by mutation. An addit i o n a l safeguard against the l a t t e r p o s s i b i l i t y was provided i n the controls of the experiment (see Results, C 2 ) . Even though two spontaneous mutations were observed during the course of the experiment* namely the adenine reversion to wild type and the mutation to n i a c i n requirement i n one of the parental cultures of 501, none were observed i n the pair-wise control combinations of Test 3» i n which 501 was found. In these there was no chance of smut being produced unless wild type recombinants had been formed as a r e s u l t of 3 3 . some back mutation o c c u r r i n g i n one of the p a r e n t a l c u l t u r e s . From the r e s u l t s , then, i t i s p o s s i b l e to say tha t somatic recombination must have occurred i n the i n f e c t i v e dikaryons of 501 w h i l s t they occupied host t i s s u e . I n d e a l i n g w i t h an o b l i g a t e p a r a s i t e such as U. h o r d e i , i t i s not easy to give the exact sequence of events as Pontecorvo (1956) has done f o r the Ascomycetes. An attempt, however, w i l l be made to e x p l a i n the p o s s i b l e mechanism by which these r e s u l t s may have been obtained. C e r t a i n deductions can immediately be made from the t e t r a d a n a l y s i s (see Table 6 ) . Since non-parental d i t y p e s are as numerous as p a r e n t a l d i t y p e s , no matter which two markers we consider and no matter what we assume the genotype of 501 t e l i o s p o r e s to be, i t can be concluded t h a t there i s no l i n k a g e between the markers concerned; i . e . the l o c i determining a r g i n i n e , l e u c i n e and n i a c i n requirements are e i t h e r a l l s i t u a t e d on d i f f e r e n t chromosomes or s u f f i c i e n t l y f a r apart on a s i n g l e chromosome th a t recombina-t i o n would appear to be random. This i s supported by the f a c t t h a t i n no t e t r a d are a l l three p a i r s of markers t e t r a -type. This means th a t there can have been no three-strand double cross-overs at meiosis, which i s h i g h l y u n l i k e l y i f the three genes were l i n k e d . As was in t i m a t e d i n the l i t e r a t u r e review (p. 5)» segregation of markers f o r which a d i p l o i d i s heterozygous 34. occurs during vegetative m u l t i p l i c a t i o n , not only as a consequence of mitotic crossing-over between linked genes but also as a consequence of a process of haploidisation, i n which whole chromosomes, not chromosome parts, reassort at random. Thus somatic "recombination" of unlinked genes, on d i f f e r e n t chromosomes, should s t i l l occur. This, then, i s the form of somatic recombination thought to be active i n t h i s study. D e t a i l s of the process of haploidisation are almost completely unknown. Haploidisation could be a consequence of an accidental breakdown at mitosis i n the separation of chromatids to the two poles t i n a proportion of cases one daughter nucleus arises with a single set of chromosomes (Pontecorvo, 1956). Breakdowns of t h i s kind are well-known i n higher organisms and r e s u l t i n aneuploids. A further deduction which can be made from the tetrad data i s that since a l l three markers are segregating i n a l l the tetrads, the 501 .teliospores must have been heterozygous for a l l three markers. The wild type sporidia obtained i n both the random spore analyses and the tetrad analysis bear testimony to t h i s since wild types can only be generated i f there i s at l e a s t one wild type a l l e l e at each locus i n the d i p l o i d teliospore. Furthermore, the fact that wild type sporidia were obtained at a l l indicates that there must have been two successive recombination events? 35. one taking place asexually in the plant (somatic recombina-tion) and the other in the teliospores (meiotic recombina-tion) . This can be inferred because one member of each possible dikaryon (the 'A' mating type) i s mutant at both the original marker l o c i . Since each of the 'a' mating type members i s mutant at one or other of these l o c i , i t i s impossible for a wild type offspring to be generated by a single meiotic event. The following diagrammatic represen-tation w i l l help to make this clearer* DIKARYON TELIOSPORES #lt Leu"~Arg~A + Leu~Arg+a..... .Segregation for Arg"only, not for Leu . #2% Leu~Arg"A + LeufArg~a. Segregation for Leu~only, not for Argr Thus, i f any teliospore segregates for both Leu" and Arg"" (thereby generating some wild type offspring) a l l three nuclei must have participated in at least two recombinational events. With the above information and allowing for the fact that a mutation to niacin requirement occurred (see Results A2) in one of the parental cultures of 501, an hypothesis may be put forward which can explain how the present results were obtained. Originally, three sporidial types were present in the inoculum mixture of 501t #1 8 Leu""Arg~Nia+ A #2 I Leu'Arg +Nia + a #3 : Leu +Arg"Nia + a 36. The p o s s i b l e ways i n w h i c h s o m a t i c r e c o m b i n a t i o n c o u l d h a v e o c c u r r e d b e t w e e n t h e s e t y p e s a r e shown i n T a b l e 8. Now, a s s u m i n g f r e e a s s o c i a t i o n o f a l l n u c l e a r t y p e s when i t comes t o t e l i o s p o r e f o r m a t i o n , s u b j e c t o n l y t o m a t i n g t y p e r e s t r i c t i o n s , i t i s p o s s i b l e t o d e t e r m i n e t h e v a r i o u s ways i n w h i c h k a r y o g a m y c o u l d o c c u r i n o r d e r t o p r o d u c e t e l i o s p o r e s h e t e r o z y g o u s a t a l l t h r e e l o c i . T h i s i s shown i n T a b l e 9. TABLE 8 Possible ways i n which somatic recombination could have occurred to give r i s e to teliospores 5 0 1 . '1 Mutation to Event Dikaryons Nia~ in» 1 2 Nuclear Nia Mating Nuclear Mating Type Leu Arg Type Type Leu Arg Nia Type Dip l o i d i s a t i o n * 1 — _ A 1 _ _ _ A 2 + + a 3 + + a Haploidisation _ a a #1 5 + + A 8 + + A 6 - + a 6 - - + a 7 + - A 9 + - A 2 + + a 6 — - + a 1 •* A 9 + A D i p l o i d i s a t i o n * 1 + A 1 + A 2 - + - a 3 + + a „„ Haploidisation 6 — a 6 — - + a 7 + A 8 + + A - - a 6 - - + a 5 + + A 8 + + A 2 + a 6 - + a 1 + A 8 + + A TABLE 8 (CONT.) Dikaryons Mutation to Event Nia" i m 1 2 Nuclear Mating Nuclear Mating Type Leu Arg Nia Type Type Leu Arg Nia Type D i p l o i d i s a t i o n * 1 + A 1 + A 2 , - + + a 3 + — — a Haploidisation 6 — + a 6 — + a #3 5 + + A 9 + - - A 6 - + a - - - a 5 + + A 8 + + A 2 + + a - — - a 1 + • A 8 + + A * This event was inferred 39. TABLE 2 P o s s i b i l i t i e s for 501 teliospore formation Mutation to Karyogamy between Teliospore genotype Teliospore Nia" i n : nuclear types: Leu Arg Nia Mating Type Type  #2 + #9 + + a A + - - A #3 + #7 + - + a B + - A #1 #2 #3 #3 + #7 + - + a B + - A #2 + #8 + - + A C (=B) + - a #2 + #9 + + a A + - - A #3 + #5 + - - a D (=A) + + A With respect to the n u t r i t i o n a l markers, i t can be seen that there are only two basic types of teliospore possible fo r 5 0 1 , A or B, regardless of where we postulate the mutation to Nia" to have occurred. Therefore, whilst we can say that the mutation to Nia" did occur, i t i s impossible to say yet exactly where. The meiotic segrega-t i o n patterns expected from teliospore types A and B off e r further evidence on t h i s point, as shown in Table 1 0 . When the tetrad types given in t h i s table are compared with those a c t u a l l y obtained i n t h i s study (see Table 6 ) , i t 4-0. w i l l be seen that a l l the possible types of tetrad were present i f both types of teliospore, A and B, had germinated from the 501 head. By inspection of Table 9, only one of the three p o s s i b i l i t i e s f or teliospore formation f u l f i l l s t h i s requirement, namely that where the mutation to Nia" has occurred i n s t r a i n 1, the double mutant. I t i s concluded, therefore, that a mutation to ni a c i n requirement occurred i n parental s t r a i n #1 and that t h i s was followed by the somatic and meiotic recombination events described above i n the series of tables 8, 9, and 10. I t must be r e a l i s e d that t h i s whole hypothesis i s based on the conventional idea of parasexuality involving d i p l o i d s (see Figure 1). This need not necessarily be the case, as Hartley and Williams (1971) have pointed out. They envisage a mechanism f o r generating genetic v a r i a t i o n by errors i n migration of chromosomes at dikaryotic mitosis. Thus an exchange of whole chromosomes between haploid c e l l s could occur without a l t e r i n g the normal chromosome complement of the two n u c l e i . Somatic recombination could therefore be explained without invoking the hypothetical process of d i p l o i d i s a t i o n . To prove that somatic recombination i s a mitotic process involving d i p l o i d s requires the i s o l a t i o n of stable d i p l o i d s i n which segregation can be studied. This has been done successfully i n U. maydis (Holliday, 196lb), as mentioned belowj but the event of d i p l o i d i s a t i o n 41. can only be inferred i n U. hordei so f a r . For the purposes of t h i s hypothesis, however, i t i s f e l t that t h i s inference i s j u s t i f i e d . TABLE 10 Table showing the meiotic segregations expected from germination of teliospore types. Teliospore Type Tetrad Type _ A Leu Arg_Nia_ Leu Arg~Nia" Tetrad Type + B - + Leu_Arg Nia_ Leu~Arg Nia~ — + + + + i - - - L + + -+ + + - - + + — — - + -_ + + + — + p - + - < + - -+ - + D - + + + - — - + — _ + + + — + 3 - - + 6 + + + + + — — — — + + At t h i s point, consideration should be given to the d i f f i c u l t i e s experienced i n micromanipulation of the 501 teliospores. I t has already been mentioned that the various s p o r i d i a l types are subject to d i f f e r e n t i a l rates of m u l t i p l i c a t i o n (see Results A2) depending on the n u t r i -t i o n a l requirements which they carry. This explains why the 4 2 . a l l e l e r a t i o s (the proportions of recovery of the two homo-logous strands) depart from l s l , bearing i n mind that the segregants are recovered many nuclear generations aft e r the event which produced them. I t i s suggested that t h i s slower rate of growth and consequent lowered v i a b i l i t y of some of the s p o r i d i a l types, notably of those carrying the n i a c i n requirement, may account for the widespread f a i l u r e to recover a l l four meiotic products a f t e r teliospore germination i n t h i s experiment. Germination type i s known to be affected by the nutrient content of the medium (Holton et a l . , 1 9 6 8 ) and variations i n spore germination amongst the smut fungi are extensive ( i b i d . ) . Wild type teliospores, the o f f s p r i n g from which have no n u t r i t i o n a l requirements, are sensitive enough to manipulation, so i t i s not unreason-able to conclude that teliospores carrying one or more n u t r i -t i o n a l markers would be extremely d i f f i c u l t to handle. The only 1 0 complete tetrads obtained (Table 6 ) , however, do present unequivocal evidence for somatic recombination occurring i n the fungus whilst i t resided within the infected host plant. Additional evidence that t h i s was indeed the case comes from the multiple i n f e c t i o n experiment. The r r e s u l t s shown i n Table 7 demonstrate c l e a r l y that at l e a s t two i n f e c t i v e dikaryons can occupy an infected host plant at the same time. Furthermore, Person and Cherewick ( 1 9 6 4 ) 43. showed that i n U. k o l l e r i and U. avenae, multiple infections do occur and that more than one genotype of the pathogen may be found within a single diseased plant. In fact, the majority of diseased plants had been infected at l e a s t twice. The dangers of extrapolation of r e s u l t s between organisms must be r e a l i s e d but i t seems f a i r l y c e r t a i n from these two pieces of evidence that multiple i n f e c t i o n can occur generally i n the smuts and therefore that somatic recombination may also be a general phenomenon i n t h i s group of fungi. This w i l l be discussed at greater length below. The actual event of somatic recombination i n U. hordei seems to be d i f f e r e n t from that described for U. maydis. So f a r as i s known, none of the cultures of U. hordei that have been tested i s unstable or reacts l i k e a bisexual culture, although Kozar (1969b) did obtain i n d i c a -tions that some di p l o i d s may have been recovered along with haploid s p o r i d i a . In other words, workers f a i l e d to f i n d any d e f i n i t e l y d i p l o i d s p o r i d i a l cultures l i k e those described fo r U. maydis (Holliday, 196lb). This could simply indicate that any d i p l o i d s that may be formed i n the dikaryons are very t r a n s i t o r y by v i r t u e of extreme i n s t a b i l i t y . Although i n most recorded instances the d i p l o i d s p e r s i s t as a clone, a rare d i p l o i d i s a t i o n event followed immediately by haploid-i s a t i o n could produce much of the genetic d i v e r s i t y i m p l i c i t i n parasexuality, with no d i r e c t evidence of the presence of 44. a fusion nucleus. So t h i s i s c e r t a i n l y not evidence against the occurrence of somatic recombination i n U. hordei. The outcome of t h i s study has important implica-t i o n s . Primarily, i t has shown that t h i s event i s not simply a laboratory a r t i f a c t . Secondly, i t i s the f i r s t report of somatic recombination occurring i n vivo. Since the organism used here i s a parasite, t h i s suggests that a new and previously unsuspected source of v a r i a b i l i t y i s p o t e n t i a l l y available for a l l economically damaging fungi. The para-sexual cycle i n fungi provides a complete ersatz for sexual recombination i n that i t provides for both recombination and sh e l t e r i n g of gene v a r i a t i o n (Pontecorvo, 1 9 5 8 ) . In the l a t t e r respect, i t i s more v e r s a t i l e than sexual reproduction because i t includes two ways of storing gene v a r i a t i o n (heterokaryosis and heterozygosis) instead of only one. For example, the formation of d i p l o i d s could aid i n the retention of favourable mutations or i n the elimination of unfavourable ones. Too much emphasis, however, should not be placed on the storage of v a r i a t i o n i n the Basidiomycetes, since a l l d i p l o i d s reduce to haploids i n each generation by meiosis. More important here perhaps, i s the r e a l i s a t i o n that the parasexual cycle operating i n plant pathogenic fungi could provide an additional vehicle for the reassort-ment of virulence genes. The use of n u t r i t i o n a l l y d e f i c i e n t mutants here i s a tool i n the inves t i g a t i o n of processes of importance i n the reassortment of genes f o r pathogenicity. 45. Since multiple i n f e c t i o n can occur i n barley, there i s also the p o s s i b i l i t y of a synthesis i n the host, by para-sexual recombination, of biotypes possessing genes fo r virulence from more than two parental biotypes. As mentioned above (see L i t e r a t u r e Review), there are many reports of new races of rusts a r i s i n g asexually but i n the majority of cases the authors are reluctant to a t t r i b u t e t h e i r occurrence to parasexual recombination. For example, F l o r (1964) states c a t e g o r i c a l l y that "there was no i n d i c a t i o n that parasexual processes were involved i n the pathogenic v a r i a t i o n of Melampsora l i n i " even though he did obtain several asexual variants upon mixing uredio-spores of two rust races. Instead he explained these variants on the basis of nuclear exchange followed by single gene mutations or deletions. The importance of mutations i n the o r i g i n of new races has been mentioned by a number of other authors (Christensen, 1959; Day, i960, 1966? Fischer and Holton, 1957; Parmeter et a l , 1963? Toxopeus, 1956? Zimmer et a l . , 1963). Whilst i t i s r e a l i s e d that mutations are undoubtedly a factor i n the development of new races, i t i s f e l t that one cannot r u l e out somatic recombination as another p o s s i b i l i t y , as F l o r points out i n his 1971 paper. Other workers at various times have suggested somatic hybridisation, heterokaryosis, nuclear p a i r i n g regardless of sex and cytoplasmic influences as being singly or s e v e r a l l y responsible for the production of new asexual 46. variants i n the ru s t s . More recently, Bartos et a l . ( 1 9 6 9 ) » Hartley and Williams ( 1 9 7 1 ) . and Sharma and Prasada (1969) have explained si m i l a r r e s u l t s on the basis of parasexual recombination, as i t has been defined here. I t seems that neither nuclear exchange nor any of the other mechanisms suggested above can account for both the o r i g i n and d i v e r s i t y of new s t r a i n s among the progeny of mixtures of spores. Somatic recombination, on the other hand, can accomodate both of these factors and as such may be an important mode of o r i g i n of new str a i n s i n both rusts and smuts. I t i s i n t e r e s t i n g to note at t h i s point that i n biochemical studies with species of U s t i l a g i n a l e s , mitotic analysis could have considerable advantage over standard meiotic analysis (Halisky, 1 9 6 5 ). Mitotic recombination could become important i n the mapping of chromosomes of organisms l i k e U. hordei, which have so f a r frustrated a l l attempts at meiotic analysis, i n the same manner as i t has been employed i n chromosome studies of A. nidulans (Pontecorvo and Roper, 1952) and U. violaceae (Day and Jones, 1 9 6 9 ) . Furthermore, i n v i t r o production of somatic recombinants could be used as a means of circumventing passage through the host, barley i n t h i s case, thus obviating the necessity of a three month generation i n t e r v a l . Standard parasexuality, despite the many unknowns, may also prove to be a useful t o o l f o r the elucidation of genetic controls of and the biosynthetic processes involved i n virulence. In any case, well-marked 47. arms i n a l l the chromosomes are necessary before the true nature of "virulence" can be determined. There i s l i t t l e doubt that t h i s prerequisite could be achieved through somatic recombination. The r e s u l t s of t h i s study suggest cer t a i n l i n e s f o r further research into t h i s subject. With t h i s system we could study the extent of penetration and development of avirulent biotypes i n r e s i s t a n t plants, using the method of s p o r i d i a l extraction p r i o r to teliospore formation described by Kozar (1967). Further, by inoculating with various combinations of virulence genes, we could investigate (a) the p o s s i b i l i t y that inexpressible genes for virulence from an avirulent i s o l a t e w i l l be recovered by a biotype with other expressible genes and (b) the p o s s i b i l i t y that two phenotypically avirulent biotypes w i l l recombine asexually i n the host to produce a v i r u l e n t biotype, which w i l l thus be able to reproduce. This work also has implications for the study of fungi whose sexual phase i s not common but i n which there i s a considerable d i v e r s i t y of races. Since various mechanisms of v a r i a t i o n may be operational and since v a r i a t i o n i s complex and dynamic, the i d e n t i f i c a t i o n of any one mechanism, when a l l are i n t e r - r e l a t e d , i s problematic. Mutation, heterokaryosis and somatic recombination are a l l nuclear mechanisms thought to produce v e r s a t i l i t y i n Fungi Imperfecti, V e r s a t i l i t y 48. i s extremely important to the s u r v i v a l of a l l plant pathogens, since without i t , extension of host range to include new r e s i s t a n t v a r i e t i e s i s impossible. The f a c t that parasexual recombination can determine v a r i a t i o n i n the host range, at l e a s t i n the Fusaria, has been shown adequately by Buxton (1956). Therefore a wide f i e l d of research has been opened, both fundamental and applied, by work on somatic recombination. Nevertheless, i t i s only possible to speculate on the importance of parasexuality i n the s u r v i v a l and evolution of plant pathogens i n nature, since evidence of parasexuality i n natural populations i s wanting. I t seems ce r t a i n , however, that the phenomenon i s not confined to a p a r t i c u l a r taxonomic unit or to any s p e c i f i c group of pathogens because i t occurs i n fungi producing disease on a wide range of hosts (see L i t e r a t u r e Review). The group to which i t may have the greatest significance i s the Fungi Imperfecti, where i t may be a major evolutionary mechanism. More complete r e a l i s a t i o n of i t s p o t e n t i a l requires research not only into the genetics and cytology of recombination events per se but also into problems of anastomosis, incom-p a t i b i l i t y , heterokaryosis and extranuclear inheritance. There i s no doubt for example, that the number of haploid st r a i n s of fungi greatly outweighs the number of d i p l o i d s t r a i n s i n nature. In f a c t , there i s only one report of a n a t u r a l l y occurring stable d i p l o i d (Ingram, 1968). I f the 49. parasexual cycle provides such a good source of v a r i a b i l i t y , why are d i p l o i d s not more common i n nature? What genetic controls are imposed on parasexuality i n nature? On the whole, the parasexual cycle seems les s perfect but more f l e x i b l e than the sexual cycle. I t would be s u r p r i s i n g i f a system with p o t e n t i a l i t i e s as great as those of sexual reproduction were merely a laboratory c u r i o u s i t y . Indeed, the r e s u l t s of t h i s study would indicate that i t i s not. That parasexuality probably does play a relevant part i n natural populations i s demonstrated by the work of Luig and Watson (1971) i n A u s t r a l i a . Here sexual reproduction i n wheat rust i s v i r t u a l l y non-existent owing to the absence of the barberry host. Yet the rust has been able to attack successively a l l the r e s i s t a n t wheat v a r i e t i e s that have been introduced, causing great concern. There i s l i t t l e doubt that asexual recombinants and mutants of the fungus provide the necessary v a r i a b i l i t y . In conclusion, i t may be said that v a r i a t i o n i n smut fungi, i n a l l i t s ramifications, has i t s most funda-mental impact on pathogenicity. This study has shown that somatic recombination, a l b e i t a very rare event, may contribute to the production of new and more v i r u l e n t races of smut. Furthermore, i t has shown that somatic recombination can occur i n pathogenic fungi whilst they are occupying the host plant. The importance of t h i s f i n d i n g i s easy to perceive 50. when one considers that most of the fungi pathogenic to c u l t i v a t e d plants and, i n c i d e n t a l l y , most of those employed i n i n d u s t r i a l fermentation processes, are asexual. I t i s r e a l i s e d , however, that although currently recognised processes of parasexuality l a r g e l y conform to those described fo r A. nidulans (see Literature Review), other viable processes of recombination are not precluded. One thing i s certain: many more explorations, using parasexual recombination i n plant pathogenic fungi, into the modification of host range, inter-genic e f f e c t s on virulence and blocks i n pathogenicity appear warranted. 5 1 . BIBLIOGRAPHY 1 . B a k e r s p i g e l , A. 1 9 6 5 . C y t o l o g i c a l i n v e s t i g a t i o n s of the p a r a s e x u a l c y c l e i n f u n g i . 1 . Nuclear f u s i o n . Mycopathol. Mycol. A p p l . 2 6 1 2 3 3 - 2 4 0 . 2 . B a r t o s , P., Fleischmann, G., Samborski, D.J. and S h i p t o n , W. 1 9 6 9 . S t u d i e s on a s e x u a l v a r i a t i o n i n the v i r u l e n c e of oat crown r u s t , P u c c i n i a  coronata, and a wheat l e a f r u s t , P. r e c o n d i t a . Can. J . Botany 4 7 : 1 3 8 3 - 1 3 8 7 . 3 . B r a d l e y , S.G. 1 9 6 2 , P a r a s e x u a l phenomena i n micro-organisms. Ann. Rev. M i c r o b i o l . 1 6 : 3 5 - 5 2 . 4 . Bridgmon, G.H. 1 9 5 9 . P r o d u c t i o n of new r a c e s of P u c c i n i a graminis v a r . t r i t i c i by v e g e t a t i v e f u s i o n . P h y t o p a t h _ n : 9 : 3 8 6 - 3 8 8 . 5 . Bridgmon G.H. and Wilcoxson, R.D. 1 9 5 9 . New r a c e s from mixtures of u r e d i o s p o r e s of v a r i e t i e s o f P u c c i n i a g r a m i n i s . Phytopath. 49:428-429, 6 . Bugbee, W.M., L i n e , R.F. and Kernkamp, M.F. 1 9 6 8 . P a t h o g e n i c i t y of progenies from s e l f i n g Race 15B and 5 6 of P u c c i n i a graminis f . sp_. t r i t i c i . Phytopath. 5 8 : 1 2 9 1 - 1 2 9 3 . 7 . Buxton, E.W. 1 9 5 6 . H e t e r o k a r y o s i s and p a r a s e x u a l recombin-a t i o n i n pathogenic s t r a i n s of Fusarium oxysporum. J . Gen. M i c r o b i o l . 1 5 : 1 3 3 - 1 3 9 . 8 . C a s s e l t o n , L.A. 1 9 6 5 . Somatic recombination i n f u n g i . S c i . P r ogr. (Lond.) 5 3 : 1 0 7 - 1 1 5 . 9 . Cherewick, W.J. 1 9 5 8 . C e r e a l smut r a c e s and t h e i r v a r i a b i l i t y . Can. J . P I . S c i . 3 8 : 4 8 1 - 4 8 9 . 1 0 . C h r i s t e n s e n , J . J . 1 9 5 9 . Somatic v a r i a t i o n i n the r u s t f u n g i . I n t e r n . Bot. Cong. R e p o r t s . 9 : 5 7 1 - 5 7 4 . 1 1 . Day, A.W. and Jones, J.K. 1 9 6 8 . The p r o d u c t i o n and c h a r a c t e r i s t i c s of d i p l o i d s i n U s t i l a g o  v i o l a c e a e . Genet. Res. 1 1 : 6 3 - 8 1 . 52. 12. Day, A.W. and Jones, J.K. 1969. Sexual and p a r a s e x u a l a n a l y s i s of U s t i l a g o v i o l a c e a e . Genet. Res, 14:195-221. 13. Day, P.R. i960 V a r i a t i o n i n phytopathogenic f u n g i . Ann. Rev. M i c r o b i o l . 14:1-16. 14. . 1966. G e n e t i c s o f the h o s t - p a r a s i t e system. Ann. Rev. Phytopath. 4:245-268. 15. and An a g n o s t o s t a k i s , S.L. 1971, M e i o t i c p roducts from n a t u r a l i n f e c t i o n s of U s t i l a g o  maydis. Phytopath. 61:1020-1021. 16. D i c k i n s o n , S. 1926. A method o f i s o l a t i n g and h a n d l i n g i n d i v i d u a l spores and b a c t e r i a . Proc. Roy. Soc. Med. 19:1-4. 17. Dinoor, A. and Person, C.O. 1969. Genetic complementa-t i o n i n U s t i l a g o h o r d e i . Can. J . Botany 47:9-14. 18. E h r l i c h , H.G. 1958. Nuclear behaviour i n mycelium of a solopathogenic l i n e and i n a c r o s s of two h a p l o i d l i n e s of U s t i l a g o maydis. Mycologia 50:622-627. 19. E l l i n g b o e , A.H. 1961. Somatic recombination i n P u c c i n i a graminis v a r . t r i t i c i . Phytopath. 51:13-15. 20. F i s c h e r , G.W. 1951. The smut f u n g i . The Ronald Press Company, New York. 21. and Holton, C.S. 1957. B i o l o g y and c o n t r o l of the smut f u n g i . The Ronald P r e s s Company, New York, 22. F l o r , H.H. 1957. The v e g e t a t i v e o r i g i n of a new race of the f l a x r u s t fungus. Phytopath. 47:11. 23. . i960. Asexual v a r i a n t s o f Melampsora l i n i . Phytopath. 50:223-226. 24. . 1964. Genetics of somatic v a r i a t i o n f o r p a t h o g e n i c i t y i n Melampsora l i n i . Phytopath. 54:823-826. 25. . 1971. Current s t a t u s of the gene-for-gene concept. Ann. Rev. Phytopath. 9:275-296. 53. 2 6 . Halisky, P.M. 1965• Physiologic s p e c i a l i s a t i o n and genetics of the smut fungi, 3. Botan. Rev. 311114-150. 2 7 . Hartley, M.J. and Williams, P.G. 1971 Genotypic v a r i a t i o n within a phenotype as a possible basis f o r somatic hybridisation i n rust fungi. Can. J. Botany. 4 9 i 1 0 8 5 - 1 0 8 7 . 28. Hastie, A.C. 1 9 6 2 . Genetic recombination i n the Hop-wilt fungus, V e r t i c i l l i u m albo-atrum. J . Gen. Microbiol. 27:373-382. 2 9 . Holliday, R. 1 9 6 l a . The genetics of Ustilago maydis. Genet. Res. 2:204 - 2 3 0 . 3 0 . . 1 9 6 l b . Induced mitotic crossing-over i n Ustilago maydis. Genet. Res. 2:231-248. 31. . 1 9 6 5 . Induced mitotic crossing-over i n r e l a t i o n to genetic r e p l i c a t i o n i n synchronously d i v i d i n g c e l l s of Ustilago maydis. Genet. Res. 6:104 - 1 2 0 . 3 2 . Holton, C.S., Hoffmann, J.A. and Duran, R. 1 9 6 8 . V a r i a t i o n i n the smut fungi. Ann. Rev. Phytopath. 6 :213-242. 33. Hood, H.C. I 9 6 6 . UV i r r a d i a t i o n s e n s i t i v i t y and mutation production i n haploid sporidia of Ustilago  hordei. Ph. D. Di s s e r t a t i o n . Univ. of Alberta, Edmonton. 3 4 . Ingram, R. 1 9 6 8 . V e r t i c i l l i u m dahliae var. longisporum, a stable d i p l o i d . Trans. Br, Mycol. Soc. 51:339-341. 3 5 . Kozar, F. 1 9 6 7 . Studies on the development of Ustilago hordei. M. Sc. Thesis: Univ. of Alberta, Edmonton. 3 6 . _ _ . 1 9 6 9 a . M i t o t i c recombination i n biochemical mutants of Ustilago hordei. Can. J . Genet. Cytol. 111 9 6 1 - 9 6 6 . 3 7 . . 1 9 6 9 b . The pathway of i n f e c t i o n of Ustilago hordei. Can. J . Genet. Cytol. 11:977-986. 54. 38. Leach, S.S. and R i c h , A.E. 1969. The p o s s i b l e r o l e o f p a r a s e x u a l i t y and c y t o p l a s m i c v a r i a t i o n i n ra c e d i f f e r e n t i a t i o n i n Phytophthora i n f e s t a n s . Phytopath. 59:1360-1365. 39. Lederberg, J . and Lederberg, E.M. 1952. R e p l i c a p l a t i n g and i n d i r e c t s e l e c t i o n o f b a c t e r i a l mutants. J . B a c t e r i o l . 63>399-406. 40. L i t t l e , R. and Manners, J.G. 1967. P r o d u c t i o n o f new p h y s i o l o g i c r a c e s i n P u c c i n i a s t r i i f o r m i s (Yellow Rust) by h e t e r o k a r y o s i s . Nature 213s422. 41. L u i g , N.H. and Watson, I.A. 1971. 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 graminis f . sp_. t r i t i c i . P r oc. L i n n . Soc. N.S.W. 95«22-45. 42. Nelson, R.R. 1956. T r a n s m i s s i o n o f f a c t o r s f o r u r e d i o -spore c o l o u r 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 by means o f n u c l e a r exchange between vegeta-t i v e hyphae. Phytopath. 46t538-540. 43. , Wilcoxson, R.D. and C h r i s t e n s e n , J . J . 1955. H e t e r o k a r y o s i s as a b a s i s f o r v a r i a t i o n i n P u c c i n i a graminis v a r . t r i t i c i . Phytopath. 45«639-643. 44. Parmeter, J.R., Snyder, W.C. and R e i c h l e , R.E. 1963. H e t e r o k a r y o s i s and v a r i a b i l i t y i n p l a n t patho-genic f u n g i . Ann. Rev. Phytopath. 1:51-76. 45. Person, C O . 1958. Development of concepts i n r u s t g e n e t i c s . P r o c . Gen. Soc. Canada. 3«25-29. 46. , and Cherewick, W.J. 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. 47. Pontecorvo, G. and Roper, J.A. 1952. Genetic a n a l y s i s without s e x u a l r e p r o d u c t i o n by means of p o l y -p l o i d y i n A s p e r g i l l u s n i d u l a n s . J . Gen. M i c r o b i o l . 6 : v i i ( A b s t r . ) . 48. . 1956. The p a r a s e x u a l c y c l e i n f u n g i . Ann. Rev. M i c r o b i o l . 10:393-400. 55. 49. Pontecorvo, G. 1958. Trends i n genetic analysis. Columbia Univ. Press, New York. 50. Raper, J.R. 1959. Parasexual phenomena i n Basidiomycetes, Intern. Bot. Cong. Reports. 9:379-383. 51. Roper, J.A. 1952. Production of heterozygous d i p l o i d s i n filamentous fungi. Experientia 8s14 - 1 5 . 52. Rowell, J.B. 1955« Segregation of sex factors i n a d i p l o i d l i n e of Ustilago zeae induced by alpha r a d i a t i o n . Science 121s304-306. 53. Schafer, J.F., Dickson, J.G. and Shands, H.L. 1962. E f f e c t s of temperature on covered smut expression i n two barley v a r i e t i e s . Phytopath. 52:II6I-H63. 54. Sharma, S.K. and Prasada, R. 1969. Production of new races of Puccinia graminis var. t r i t i c i from mixtures of races on wheat seedlings. Aust. J . Agric. Res. 20s981-985. 55. Tapke, V.F. and Bever, W.M. 1942. E f f e c t i v e methods of inoculating seed barley with covered smut (Ustilago hordei). Phytopath. 32s1015-1021. 56. Thomas, P. 1965. Virulence i n Ustilago hordei (Pers.) Lagerh. M. Sc Thesiss Univ. of Alberta, Edmonton. 57. T i n l i n e , R.D. and Macneill, B.H. 1969. Parasexuality i n plant pathogenic fungi. Ann, Rev. Phytopath. 7*147-170. 58. Tolmsoff, W.J. 1972. D i p l o i d i s a t i o n and heritable gene repression-derepression as major sources for v a r i a b i l i t y i n morphology, metabolism and pathogenicity of V e r t i c i l l i u m species, Phytopath. 62:407-413. 59. Toxopeus, H.J. 1956. Reflections on the o r i g i n of new physiologic races i n Phytophthora infestans and the breeding for resistance i n potatoes. Euphytica 5:221-237. 60. Tuveson, R.W. and Coy, D.0. 1961. Heterokaryosis and somatic recombination i n Cephalosporium  mycophylum. Mycologia 53:244-253. 56. 61. V a k i l i , N.G. and Caldwell, R.M. 1957. Recombination of spore colour and pathogenicity between ur e d i a l clones of Puccinia recondita f . sp. t r i t i c i . Phytopath. 47:536 (Abstr.). 62. Waterhouse, W.L. 1952. Australian rust studies. 9. Physiologic race determinations and surveys of cereal rusts. Proc. Linn. Soc. N.S.W. 77:209-258. 6 3 . Watson, I.A. 1957. Further studies on the production of new races from mixtures of races of Puccinia  graminis var. t r i t i c i on wheat seedlings. Phytopath*7~4"7:510-512. 64. a n d L uig» N.H. 1958. Somatic hybridisation i n Puccinia graminis var. t r i t i c i . Proc, Linn. Soc. N.S.W. 83«190-195. 65. and 1 1959. Somatic hybridisation between Puccinia graminis var. t r i t i c i and P. graminis var. s e c a l i s . Proc. Linn. Soc. N.S.W. 84:207-20oT~ 6 6 . and 1962. Asexual intercrosses between somatic recombinants of Puccinia graminis. Proc. Linn. Soc. N.S.W. 87:99-104. 67. . 1970. Changes i n virulence and population s h i f t s i n plant pathogens. Ann. Rev. Phytopath. 8:209-230. 11 6 8 . Wilde, P. 1 9 6 1 . Ein Beitrag zur Kenntnis der V a r i a b i l i t a t von Phytophthora infestans (Mont.) de Bary. Arch. Mikrobiol. 40:163-195. 69. Zimmer, D.E., Schafer, J.F. and Patterson, F.L. 1963. Mutations for virulence i n Puccinia coronata. Phytopath. 53:171-176. 

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}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            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:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0101389/manifest

Comment

Related Items