nu Innuflm a “1:; . . Tr .mnofihmw . awn «a; 5 3915». WWW. $5? . .gu 1. «5.23; . x... .3... {MN}. . . '1 I’ll. 4." ll. 1:! r (I \y . l. . vlnfilfin. .. 55...... 1.. tr . ‘ Isn‘t-x ... .3. v.9 ”:an it): ivfi.hahxlunimltl. .. .. .l .52....) nvutrs... l: 9.1 tfififdtfifi. r55 l 7 Va 0 33!!!! ”.2 > LI'I - 1ft! 21“."..hnnfliifivVu nun umkzi.zn . argflllfi. S..3...£.,. . . Pincus £22»... . ‘ “I..." +3.. F.4- .. .3 .5 .u... . 7:11‘35... ”2 :v :0. i... :n. v THESIS W937) NIVERSITY Ll IBRARIES IIIII II II IIIIIIIIIIIIIIIIIIIIIIII 3 1293 01564 This is to certify that the thesis entitled Induced Alternative Oxidase Activity and Attenuation of Virulence in Respiratory Mutants of Fusarium oxstorum E; ap:NBasilicum and Colletotrichum coccodes. presented by Mursel Catal has been accepted towards fulfillment of the requirements for M.Sc. degree in Botany & Plant Pathology haw/I LWW MajorJ Jrofegt' Date £376; 3/[77C 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution LIBRARY. Michigan State Universlty PLACE N RETURN BOX to remove thle checkout from your record. TO AVOID FINES return on or before one due. DATE DUE DATE DUE DATE DUE MSU to An Afflrmetive ActionlEmel Opportmlty lnetltulon .Wm-OJ I *7 fl, ..4.._ -— t _—__—-—— INDUCED ALTERNATIVE OXIDASE ACTIVITY AND ATTENUATION OF VIRULENCE IN RESPIRATORY MUTANTS or FUSARIUM OXYSPORUM F. SP. BASILICUM AND COLLETOYRICHW COCCODES By Mursel Catal A THESIS Submitted to Michigan State University in partial fitlfilment of the requirement for the degree of MASTER OF SCIENCE Department of Botany and Plant Patholog 1996 Itil I ABSTRACT INDUCED ALTERNATIVE OXIDASE ACTIVITY AND ATTENUATION OF VIRULENCE IN RESPIRATORY MUTANTS OF F USARI UM OXYSPORUM F. SP. BASILICUM AND COLLETOTRICHUM COCCODES by Mursel Catal Mitochondrial hypovirulence associated with cyanide-resistant alternative oxidase activity in the chestnut blight fungus ofl‘ers a unique opportunity to study both mitochondrial contributions to pathogenicity and the biological control of an important disease causing agent. Recently, it has been shown that mitochondrial hypovirulence can be induced in the laboratory by the treatment of conidia with mutagens that target the mitochondria. In this way, laboratory induced hypovirulent strains can be generated for use in the biological control of virulent chestnut blight strains. I report on attempts to use mitochondrial hypovirulence in other fimgal pathogens including F usarium oxysporum f. sp. basilicum and Colletom'chum coccodes both serious pathogens of their respective hosts, basil and tomato. To this end, cyanide-resistant respiration was induced in both fungi. Over 15 mutants isolated from F. oxysporum f. sp. basilicum had high levels of cyanide-resistant respiration accounting for 45 to l00% of total respiration. Twelve of those mutants were significantly less virulent and grew slower than a wild type strain. Transfer of the respiratory phenotype to a benomyl-resistant virulent strain with wild type respiration did not successfully demonstrate the cytoplasmic nature of the respiratory phenotype. However, transmission tests whereby nitrate non-utilizing (nit) mutants were used to form heterokaryons showed that at least one strain (Pm33 nit3) could transfer the abnormal respiratory phenotype and hypovirulence to a wild type strain (Fob nitM). It appeared that resulting heterokaryon single-spore cultures likely carried mixed populations of mitochondria (heteroplasmons) as alternative oxidase levels were not as high as the parental mutant phenotype. Attempts at biological control using the respiratory mutants to control virulent strains showed little evidence of control. High levels of cyanide-resistant respiration was also induced in Colletotrichum coccodes. Cyanide-resistant respiration was correlated with reduced virulence, slow growth and abnormal colony morphology in at least one of the mutants isolated. Attempts at cytoplasmic transmission of the mutant phenotype by forcing heterokaryons between respiratory mutants (CpmS nit3 and Cpm29 nit3) and a virulent wild type strain did not demonstrate the cytoplasmic nature of the mutation as the respiratory phenotype did not transfer. This indicates that the respiratory phenotypes were caused by nuclear mutations that induced cyanide-resistant respiration or that mitochondrial defect responsible for high levels of alternative oxidase will not freely exchange cytoplasmically in this fimgus. To my beloved wife, Zebra for her moral support iv ACKNOWLEDGENIENTS I would like to thank my adviser professor, Dr. Dennis W. Fulbright, for his guidance, financial support and friendship during my study. I would like to express my gratitude to all members of my guidance committee; Dr. Helmut Bertrand and Dr. Pat Hart for their guidance and help. I would like to thank all the members of Dr. Fulbright’s lab for their help and knowledge they provided to me. I would like to thank Julia Bell for her help in every dimculty I have had during my work. My appreciation goes to David Huber for providing me usefirl information and fungal strains that I used in my study. My endless thanks goes to my wife, Zehra, for her patience and support in every possible way. TABLE OF CONTENTS LIST OF TABLES .............................................................................................. viii LIST OF FIGURES .............................................................................................. xi CHAPTER 1. GENERAL INTRODUCTIONAND LITERATURE REVIEW ...... 1 Discovery of Hypovirulence ....................................................................... 1 Description of Hypovirulence ..................................................................... 2 Hypovirulence and Vegetative Incompatibility ............................................ 3 dsRNA-Associated Hypovirulence ............................................................. 4 Hypovirulence in North America ................................................................ 5 Genetic Characterization of dsRNA in Hypovirulent Strains ....................... 6 The origin of dsRNA .................................................................................. 8 Engineering Hypovirulence in other pathogens ........................................... 8 Role of dsRNA in Hypovirulence ............................................................... 9 The link between dsRNA and Hypovirulence in other firngi ........................ 9 Hypovirulence in dsRNA-Free Strains ........................................................ 10 Alternative Oxidase Activity ....................................................................... 11 Mitochondrial Hypovirulence ..................................................................... l3 Senescence and Alternative Oxidase .......................................................... 14 Mitochondrial Movement and Interactions ................................................. 16 Induction of Mitochondrial Hypovirulence ................................................. 17 CHAPTER 2. HYPOVIRULENCE ASSOCIATED WITH ELEVATED ALTERNATIVE OXIDASE ACTIVITY IN THE LABORATORY INDUCED MUTANT S OF FUSARIUM OXYSPORUM F. SP. BASILICUM Inroduction ..................................................................................................... 19 Material and Methods .................................................................................... 21 Fungal strains and growth conditions ......................................................... 21 Mutagenesis and selection of respiratory mutants ....................................... 21 Respiration ................................................................................................ 23 Pathogenicity assays .................................................................................. 24 Recovery of a benomyl-resistant mutant ..................................................... 25 Transmission using PBM2 .......................................................................... 25 Transmission during heterokaryon tests ...................................................... 26 Genetic transmission in planta and potential for biocontrol ......................... 27 Results ............................................................................................................. 28 Induction of alternative oxidase and isolation of possible mutants ............... 28 Pathogenicity of the respiratory mutants ..................................................... 33 Benomyl-resistant strains ........................................................................... 33 Transmission of respiratory phenotypes to a benomyl-resistant strain .......... 36 Heterokaryon formation and segregation of respiratory mutants ................. 42 Biological control ...................................................................................... 50 Discussion ..................................................................................................... 55 CHAPTER 3. CYANIDE-RESISTANT RESPIRATION AND REDUCED VIRULENCE IN COLLETOTRICHUM COCCODES Introduction .................................................................................................. 59 Material and methods ..................................................................................... 61 Fungal isolates and media used ................................................................. 61 Mutagenesis and selection of respiratory mutants ..................................... 62 Respiration ............................................................................................... 62 Heterokaryon tests for transmission .......................................................... 63 Segregation of heterokaryons ................................................................... 64 Pathogenicity tests on tomatoes ............................................................... 64 Results ........................................................................................................... 65 Nit mutant genotype determination ........................................................... 65 Heterokaryon tests .................................................................................... 65 Some characters of C. coccodes nit mutants ............................................... 69 Induction of alternative oxidase and mutagenesis ....................................... 69 Pathogenicity of respiratory mutants ......................................................... 71 Transmission of respiratory defects in heterokaryon tests ........................... 74 Alternative oxidase activity of heterokaryon-derived single-spore cultures. 80 Discussion ....................................................................................................... 82 CONCLUSION ..................................................................................................... 85 REFERENCES ..................................................................................................... 90 vii LIST OF TABLES CHAPTER 2 TABLE PAGE 2.1. F usarium oxysporum f. sp. basilicum strains used in this study ................................ 22 2.2. The induction of the alternative oxidase pathway in the presence of chlorarnphenicol for F usarium oxysporumf 5p. basilicum and Cryphonectria parasitica ................. 29 2.3. Characteristics of cyanide-resistant respiratory mutants of F usw'ium oxysporum f sp. basilicum ........................................................................................................ 32 2.4. Disease ratings of basil plants inoculated with F usarium oxysporumf 5p. basilicum wild type and respiratory mutants in greenhouse pathogenecity tests ......................... 34 2.5. Disease ratings of basil seedlings inoculated with F. oxysporumf 5p. basilicum wild type and respiratory mutants in a petri dish assay in the laboratory .................. 35 2.6.The growth rate and pathogenicity of selected benomyl-resistant isolates recovered from F usarium axysporum f sp. basilicum ............................................................... 3 8 2.7. The alternative oxidase (AO) activity of plugs from pairings between a benomyl- resistant strain (PBM2) and respiratory mutants ...................................................... 40 2.8. Alternative oxidase (AO) activity of single-spore cultures recovered from pairings between the benomyl-resistant strain (PBM2) and respiratory mutants ..................... 41 2.9. Heterokaryon formation between nitrogen nonutilizing mutants from F usarium oxysporumjfi 5p. basilicum wild type strain and respiratory mutants ........................ 43 2.10.The segregation of single spores on minimal medium amended with different nitrogen sources from heterokaryons between Fob nitM, a virulent strain and Pm33 nit3, a hypovirulent respiratory mutant ......................................................... 44 2.11. The segregation of single spores on minimal medium amended with different nitrogen sourcesfrom heterokaryon between Fob nitM and Fob nit3, two virulent strains ............................................................................................... 46 viii 2.12. Alternative oxidase (AO) activity of single-spore cultures from heterokaryons between Pm33 nir3 and Fob nitM .......................................................................... 47 2.13. Alternative oxidase (AO) activity, pathogenicity and growth rate of single-spore cultures from individual isolates and from heterokaryons between Fob nitM and Pm33 nit3 mutants .......................................................................... 48 2.14. Disease ratings of basil plants inoculated with single-spore cultures fro heterokaryon between Fob nitM and Pm33 nit3 in the greenhouse pathogenicity assays .............................................................................................. 51 2.15. Ratings of pathogenicity tests performed in the greenhouse for biological control of F usarium oxysporumf 5p. basilicum with respiratory mutants ............... 52 2.16. Ratings of pathogenecity tests performed on petri dish-grown seedling in the laboratory ............................................................................................................ 54 CHAPTER 3 3.1. Grth of Colletotrichum coccodes nit mutants on different nitrogen sources ......... 66 3 .2. Heterokaryon formation between different nit mutants of Colletotrichum coccodes. 67 3 .3. The alternative oxidase activity, pathogenicity and growth of C. coccodes nit mutants inVogel medium ......................................................................................... 7O 3 .4. The number of single-spore cultures from mutagenesis screened for slow growt, colony morphology and, alternative oxidase activity and pathogenicity .................... 72 3.5. Some characteristics of respiratory mutants recovered from Colletotrichum coccodes strain Cc2 nit 3 ......................................................................................... 73 3.6. The pathogenicity and growth rate of hypovirulent strains determined in the preliminary studies .................................................................................................. 75 3.7. Segregation of single spores fi'om heterokaryons formed between virulent strain Cc3 nit2 and hypovirulent respiratory mutants CpmS MB and Cpm29 nir3 .............. 78 3.8. Alternative oxidase activity of single spores isolated from heterokaryons between Colletotrichum coccodes nit3 and respiratory mutants CpmS nit3and Cpm29 nit3....81 ix LIST OF FIGURES CHAPTER 2 FIGURE PAGE 2.1. The number of F usarium oxysporumf 5p. basilicum conidia surviving after treatment with UV-Light and ethidium bromide mutagens (Killing curve) ................ 30 2.2. The number of Fusarium oxymorum f. sp. basilicum single- spore colonies growing on PDA amended with difl‘erent concentrations of benomyl ....................... 37 CHAPTER 3 3.1. Heterokaryon formation between Cc3 nit2 and C02 nit3 isolates on minimal media amended with nitrate as sole nitrogen source ........................................................... 68 3.2. Antrachnose lesions formed by respiratory mutant Cpm29 nit3 and Cc3 nit2 and Colletotrichum coccodes on Roma tomatoes ..................................................... 76 3.3.Segregation of single-spore isolates on minimal medium amended with hypoxanthine as nitrogen source from heterokaryons formed between virulent Cc3 nitZ and hypovirulent respiratory mutant Cpm5 nit3 .......................................... 79 CHAPTER 1 GENERAL INTRODUCTION AND LITERATURE REVIEW GENERAL INTRODUCTION AND LITERATURE REVIEW Chestnut blight, caused by the filamentous firngus Cryphonectria (=Endothia) parasitica (Murr.) has eliminated the American chestnut (Castanea dentate (Mars) Birch) tree as a dominant or codominant species throughout its natural range in North America. The first infected trees were reported in 1904 in the Bronx zoo (Anagnostakis, 1982). Sprouts of the American chestnut tree have continued to grow from stumps, but before reaching maturity, they are infected, girdled and killed by the pathogen. The pathogen is still present and producing its abundant spores on the sprouts. European chestnut (Castanea sativa Mill.) trees are similar to the American trees and susceptible to blight (Anagnostakis, 1987 and 1988). Blight was transported to Europe and was first observed in 1938 near Genoa, Italy, where an epidemic ensued similar to the epidemic in North America (Anagnostakis, 1987). However, by the 1960's it was obvious that chestnut blight in Europe was less devastating to the European chestnut tree population than it was to the American chestnut tree population in North America. Discovery of Hypovirulence Hope for control of this canker disease came in 1965 with the discovery of a variant of the pathogen showing reduced aggressiveness. Braghi, in Italy, observed European chestnut trees in which disease was in remission (Van Alfen et al.1975). The variant, later 2 found in France and the Pyrenes, was isolated and described as hypovirulent by Grente (Heiniger and Rigling, 1994; Nuss and Koltin, 1990). Reduced sporulation, white pigmentation in culture (compared to orange pigmentation of normal pathogenic isolates) and an abnormal grth rate were other phenotypic characteristics of the hypovirulent isolates (Heiniger and Rigling, 1994; Nuss, 1992). Furthermore, these hypovirulent isolates slowed or stopped canker development induced by virulent strains when they were inoculated around the cankers. It was suggested that the inoculation of the trees by hypovirulent isolates resulted in the conversion of resident virulent strains to the hypovirulent phenotype (Nuss, 1992; Heiniger and Rigling, 1994). Once a canker had been successfully cured by treatment with a hypovinrlent strain, much of the fungal mycelium in the original virulent infection appeared to be converted to the hypovirulence phenotype. Description of Hypovirulence Grente and Sauret (1960) described the behavior of hypovirulent strains in culture (Anagnostakis, 1987). Hypovirulent strains segregated, yielding normal looking strains; however, normal, virulent strains never segregated to hypovirulent strains. Grente and Sauret (1969) suggested that the hyphae of the virulent strain anastomosed with hyphae of the introduced hypovinrlent strain and a genetic determinant in the cytoplasm of the hypovirulent strain was transferred to the virulent strain. The cytoplasmic mode of hypovirulence transfer was genetically demonstrated by pairing auxotrophic strains of C. parasitica. A hypovirulent lysine auxotroph and a virulent methionine auxotroph were paired by inoculating the strains side-by-side in chestnut stems. Ninety days later, 3 methionine auxotrophs from the canker were recovered and found to be hypovirulent, indicating the hypovirulent phenotype was transferred from the lysine auxotroph. Additional evidence for the cytoplasmic nature of hypovirulence was provided when hypovirulent methionine and virulent arginine auxotrophic strains were paired to form a heterokaryon. Single-conidia] isolates from the heterokaryon required either methionine or arginine and all were hypovirulent (Grente and Sauret, 1969; Anagnostakis, 1982). The first hypovirulent strains of C. parasitica in North America were isolated from abnormal cankers from a chestnut grove near Rockford, Michigan in 1976 (Elliston et a1. 1977). Fulbright ct al. (1983) confirmed the presence of hypovirulent strains in difl‘erent locations throughout Michigan and provided evidence for ongoing biological control. Although it was first associated with C. parasitic hypovirulence has been found and studied in other fungal systems such as Ophiostoma ulmi (Brasier, 1983), Sclerotr‘nia sclerotiorum (Boland, 1992) and Helminthosporum victoriae (Lindberg, 1960). In these systems, hypovirulence appears similar to hypovirulence in C. parasitic in that it was characterized by slow growth and abnormal colony morphology. Also, a transmissible cytoplasmic element is thought to be a factor in reduced virulence. Hypovirulence and Vegetative Incompatibility Though it was clear that virulent strains were converted to hypovirulent, biological control by introduced transmissible hypovirulence has been less successfiil in North America than in Italy and France. Vegetatively incompatible strains of the pathogen might explain the failure of some cankers to be controlled if it blocks the transfer of the cytoplasmic determinants of hypovirulence. It is thought that vegetative incompatibility 4 results in the failure of hyphal anastomosis between incompatible strains. Anagnostakis estimated that between five and seven nuclear genes determine vegetative incompatibility. Two vegetatively compatible strains fi'eely undergo anastomosis if they have the same alleles at each vegetative incompatible locus (Nuss, 1992 and Anagnostakis, 1987 and 1977) It has been thought that the ability to form anastomosis increases as the number of heterogenic alleles decreases (Anagnostakis and Waggoner, 1981). However, vegetative incompatibility may be more complex than first reported. Huber and Fulbright (1992) reported that individual vegetative incompatibility genes in C. parasitica may have specific efl‘ects upon the transmission of cytoplasmically carried genetic elements and that a two gene difference can be more permissive to the transmission than a particular one gene difi‘erence. Since the hypovirulence phenotype is transmitted only during hyphal anastomosis with related vegetatively compatible groups, the vegetative compatibility structure within the fungal population can afl‘ect the dissemination and persistence of introduced hypovirulence. It was reported that mixtures of different hypovirulent mycelia efi‘ectively overcome vegetative incompatibility and rapidly arrest canker development (Jaynes and Elliston, 1980). dsRNA-Associated Hypovirulence The answer to the question as to the nature of the cytoplasmic determinant in hypovirulent strains came partly in the late 1970's. Day et a1. (1977) showed that hypovirulent strains, carried double-stranded ribonucleic acid (dsRNA) in the cytoplasm and that this dsRNA could be transferred to virulent strains via anastomosis. 5 The role of dsRNA and its relationship to hypovirulence was based on correlative evidence until it was shown that transformation of virulent C. parasitica strains with a fill] length complementary DNA copy of a hypovirulence-associated viral dsRNA conferred the complete hypovirulence phenotype (Choi and Nuss, 1992). Cytoplasmic dsRNA was resurrected fiom the chromosomally integrated cDNA copy. These dsRNA molecules were capable of converting compatible virulent strains to hypovirulence, thus establishing dsRNA as the causal agent of hypovirulence in C. parasitica. There is wide variation in the expression of hypovirulence-associated phenotypes in dsRNA-containing strains of C. parasitica. Virulence expression of dsRNA-containing strains ranged from avirulent to almost normally virulent, indicating that difl‘erent levels of hypovirulence are determined by cytoplasmically transmitted dsRNA independently of the nuclear genetic background of the recipient vimlent strains ( Nuss and Koltin, 1990; Nuss, 1992). In addition to reduced virulence, hypovirulent strains can exhibit several different symptoms including altered colony morphology, suppressed conidiation, reduced oxalate accumulation, reduced laccase production, reduced pigment production, as well as noted changes in several other proteins and mRN A transcripts (Nuss and Koltin, 1990). Hypovirulence in North America Surveys in Michigan indicated that the chestnut blight pathogen isolated from abnormal cankers had abnormal colony morphology, reduced virulence and contained dsRNA (Fullbright et al. 1983). Pathogenicity tests showed that native Michigan hypovirulent isolates of C. parasitica may be responsible for recovering chestnut groves in Michigan and that hypovirulence is naturally spreading (Brewer, 1995) . The most obvious 6 morphologic difference between European and Michigan hypovirulent strains were sporulation and pigmentation differences. Michigan strains are pigmented whereas European strains generally lack pigmentation and sporulation was not as noticeably suppressed in Michigan hypovirulent strains as in European strains (Heiniger and Rigling, 1994) Elliston (1985) compared the cultural characteristics, pathogenicity and fruiting capacities of Italian and American dsRNA-containing strains relative to dsRNA-free strains to determine if any consistent cultural indicators of dsRNA or reduced virulence could be found. In his studies, he found that the dsRNA-containing strains all difi‘ered in culture fi'om dsRNA-flee strains and from one another. All dsRNA-containing strains were deficient in pathogenicity and fi'uiting capacity. He suggested that overall appearence in culture may be a usefirl criterion to select strains to be tested for dsRNA. Dunn and Boland (1993) reported that naturally occuring isolates of C. parasitica collected from different native stands of American chestnut in Ontario possessed dsRNA and were hypovirulent. Genetic Characterization of dsRNA in Hypovirulent Strains Double-stranded RNAs associated with difi‘erent strains vary considerably with respect to size, number of bands, concentration and sequence homology. Even isolates recovered from different cankers on the same tree can contain dsRNA molecules of varying size. Most hypovirulent isolates harbor one to three large dsRNA molecules and several small molecules (Fulbright, 1990). The recent analyses of dsRNAs associated with a European (EP713) hypovirulent strain and an North American hypovirulent strain have