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Possible biochemical mechanisms of pathogenicity in Phytophthora sojae

Rivera-Vargas, Lydia Ivette, Ph.D.

The Ohio State University, 1994

UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106

POSSIBLE BIOCHEMICAL MECHANISMS OF

PATHOGENICITY IN Phytophthora aojae

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

by

Lydia I. Rivera Vargas, B.S., M.S.

The Ohio State University 1994

Dissertation Committee: Approved by Dr. T .L. Graham

Dr. I. Deep J JL Adviser Dr. A.F. Schmitthenner Dr. S. St. Martin Department of Plant Pathology To my niece, Adriana

ii ACKNOWLEDGMENTS

I would like to express my gratitude to my adviser. Dr. T.L. Graham for his guidance throughout the research. X am also very thankful to the other members of my committee. Dr. I. Deep, Dr. A.F. Schmitthenner and Dr. S. St. Martin for their suggestions and comments. Specials thanks to Dr. M.Y. Graham for her guidance with the electrophoretic work.

Also, X would like to express my gratitude to The Ohio State University, specially the Fellowship Office and the Dept, of Plant Pathology for their support throughout my studieB. X extend my thanks to Mr. J. Diaz, technician at the electron microscope lab for his advice during that part of the research.

X express my sincere appreciation to my friends for their encouragement and support throughout my studies. To my family, all my love for their continuous faith in me.

Columbus, Ohio Lydia I. Rivera-Vargas May, 1994

iii VITA

May 12, 1959 ...... Born - May ague z, Puerto Rico

EDUCATION 1977 - 1981 ...... B.S., University of Puerto Rico Mayaguez Campus 1981 - 1985 ...... M.S., University of Puerto Rico Mayaguez Campus 1985 -.1989 ...... Research Assistant, Plant Pathology Lab., Department of Crop Protection, University of Puerto Rico, Mayaguez Campus 1989 -.1992 ...... Patricia Robert Harris Fellow, Fellowship Office, The Ohio State University, Columbus, Ohio 1992 - 1994 ...... Teaching Assistant, Department of Plant Pathology, The Ohio State University, Columbus, Ohio

PUBLICATIONS

1. Rivera-Vargas, L.I. and Hepperly, P.R. 1987. Assessment of Chinese straw mushroom (Volvariella volvacea) fungal competitors on sugarcane bagasse. In: Cultivating Edible Fungi. Elsevier Publishing Co., The Netherlands, pp. 341 - 349. 2. Rivera-Vargas, L.I. and Hepperly, P.R. 1987. Internal mycoflora of Chinese straw mushroom basidiocarps - "in vitro" effects on mushroom growth. J. Agric. Univ. P.R. 71(2): 159-164. 3. Rivera-Vargas, L.I. and Hepperly, P.R. 1987. Fungicides to control fungal competitors in Chinese straw mushroom. J. Agric. Univ. P.R. 71(2): 165-175. 4. Rivera-Vargas, L.I., Schmitthenner, A.F. and Graham, T.L. 1993. effects on and metabolism by Phytophthora sojae. Phytochemistry. 32(4): 851-857. 5. Rivera-Vargas, L.I. and Graham, T.L. 1993. A Possible Role for Pectin Lyase in Phytophthora aojae Pathogenicity. (Abstr.) Phytopathology

iv FIELD OF STUDY

Major Field: Plant Pathology

Biochemical studies of host-pathogen interactions under the guidance of Dr. Terrence L. Graham.

v TABLE OF CONTENTS

DEDICATION ...... ii ACKNOWLEDGEMENTS ...... iii VITA ...... iv LIST OF TABLES ...... viii LIST OF FIGURES ...... xiii LIST OF PLATES ...... xvii

GENERAL INTRODUCTION ...... 1 CHAPTER PAGE I. SOYBEAN FLAVONOID EFFECTS ON AND METABOLISM BY Phytophthora sojae ...... 10 Introduction...... 10 Materials and Methods ...... 18 Results and Discussion ...... 25 Conclusions ...... 74

II. Phytophthora sojae CATALASE ...... 76 Introduction...... 76 Materials and Methods ...... 86 Results ...... 91 Discussion ...... 96 Conclusions ...... 102

III. Phytophthora sojae PECTOLYTIC ENZYMES ...... 104 Introduction...... 104 Materials and Methods ...... 114 Results ...... 129 Discussion ...... 176 Conclusions ...... 183

GENERAL CONCLUSION ...... 184 LIST OF REFERENCES ...... 185

vi APPENDICES ...... 198 A. Data Relative to Chapter I ...... 198 B. Data Relative to Chapter III ...... 231

vii LIST OF TABLES

TABLE PAGE 1. Effects of different plant natural compounds on P. sojae growth "in culture ...... 46 2. Microscopic affects of different plant natural compounds on P. sojae ...... 52 3. Possible mechanisms suggested in the chemical conversion of plant natural compounds by P. sojae ...... 73 4. Major functions of catalase ...... 79 5. Effect of different peroxide concentrations on P. sojae catalase activity ...... 91 6. Constitutive levels of catalase in different P. sojae isolates ...... 92

7. Constitutive levels of catalase in P. sojae isolate of race 3 over a period of 9 days of growth ...... 93 8. Induced catalase activity in P. sojae isolate of race 3 using peroxide and plant phenolic compounds ...... 95 9. Detection of different phenolic compounds used to induced catalase activity in 3 different P. sojae fractions using HPLC analysis ...... 95 10. Cotyledon lesion Bize of soybean cultivars W and W79 infected with P. sojae races 3 and 4 ...... 131 11. Histological data from soybean cultivar W7S infected with P. sojae races 3 (Incompatible) and 4 (Compatible) at different time intervals ...... 132 12. PG specific activity (U/mg of protein) in P. sojae race 4 mycelial portions ...... 151 13. PG specific activity (U/mg of protein) in P. sojae race 4 filtrate portions ...... 151 14. PNL specific activity in P. sojae race 3 induced using different soybean fractions ...... 159

viii TABLE PAGE

15. PL specific activity (U/mg of protein) from cotyledons and hypocotyls 36 h after inoculation with P. sojae (races 3 and 4) ...... 169 16. PL specific activity (U/mg of protein) from cotyledons and hypocotyls 42 h after inoculation with P. sojae (races 3 and 4) ...... 169 17. PG specific activity (U/mg of protein) from cotyledons and hypocotyls 36 h after inoculation with P. sojae (races 3 and 4) ...... 170 18. PG specific activity (U/mg of protein) from cotyledons and hypocotyls 42 h after inoculation with P. sojae (races 3 and 4) ...... 170 19. PME specific activity (U/mg of protein) from cotyledons and hypocotyls 36 h after inoculation with P. sojae (races 3 and 4} ...... 172 20. PME specific activity (U/mg of protein) from cotyledons and hypocotyls 42 h after inoculation with P. sojae (races 3 and 4) ...... 172 21. PME specific activity (U/mg of protein) from cotyledons and hypocotyls 36 h after inoculation with P. sojae (races 3 and 4) .... 173 22. PME specific activity (U/mg of protein) from cotyledons and hypocotyls 42 h after inoculation with P. sojae (races 3 and 4) ...... 173 23. P. sojae (race 1) growth on lima bean medium amended with various concentrations of ...... 199 24. P. sojae (race 3) growth on lima bean medium amended with various concentrations of apigenin...... 199 25. P. sojae (race 12) growth on lima bean medium amended with various concentrations of apigenin...... 199 26. P. sojae (race 1) growth on lima bean medium amended with various concentrations of ...... 200 27. P. sojae (race 3) growth on lima bean medium amended with various concentrations of biochanin A ...... 200 28. P. sojae (race 12) growth on lima bean medium amended with various concentrations of biochanin A ...... 200 29. P. sojae (race 1) growth on lima bean medium amended with various concentrations of ...... 201 30. P. sojae (race 3} growth on lima bean medium amended with various concentrations of coumestrol ...... 201 31. P. sojae (race 12) growth on lima bean medium amended with various concentrations of coumestrol ...... 201

ix TABLE PAGE

32. P. sojae (race 1) growth on lima bean medium amended with various concentrations of chrysin...... 202 33. P. sojae (race 3) growth on lima bean medium amended with various concentrations of chrysin ...... 202 34. P. sojae (race 12) growth on lima bean medium amended with various concentrations of chrysin...... 202 35. P. sojae (race 1) growth on lima bean medium amended with various concentrations of ...... 203 36. P. sojae (race 3) growth on lima bean medium amended with various concentrations of formononetin...... 203 37. P. sojae (race 12) growth on lima bean medium amended with various concentrations of formononetin...... 203 38. P. sojae (race 1) growth on lima bean medium amended with various concentrations of ...... 204 39. P. sojae (race 3) growth on lima bean medium amended with various concentrations of genistein ...... 204 40. P. sojae (race 12) growth on lima bean medium amended with various concentrations of genistein ...... 204 41. P. sojae (race 1) growth on lima bean medium amended with various concentrations of isorhamnetin...... 2 05 42. P. sojae (race 3) growth on lima bean medium amended with various concentrations of isorhamnetin ...... 205 43. P. sojae (race 12) growth on lima bean medium amended with various concentrations of isorhamnetin...... 205 44. P. sojae (race 1) growth on lima bean medium amended with various concentrations of isoquercetrin...... 206 45. P. sojae (race 3) growth on lima bean medium amended with various concentrations of isoquercetrin...... 206 46. P. sojae (race 12) growth on lima bean medium amended with various concentrations of isoquercetrin...... 206 47. P. sojae (race 1) growth on lima bean medium amended with various concentrations of ...... 207 48. P. sojae (race 3) growth on lima bean medium amended with various concentrations of kaempferol ...... 207 49. P. sojae (race 12) growth on lima bean medium amended with various concentrations of kaempferol ...... 207

50. P. sojae (race 1) growth on lima bean medium amended with various concentrations of ...... 2 08 51. P. sojae (race 3) growth on lima bean medium amended with various concentrations of naringenin ...... 208

x TABLE PAGE

52. P. sojae (race 12) growth on lima bean medium amended with various concentrations of naringenin ...... 208 53. P. sojae (race 1) growth on lima bean medium amended with various concentrations of ...... 209 54. P. sojae (race 3) growth on lima bean medium amended with various concentrations of quercetin ...... 209 55. P. sojae (race 12) growth on lima bean medium amended with various concentrations of quercetin ...... 209 56. P. sojae (race 1) growth on lima bean medium amended with various concentrations of rutin ...... 210 57. P. sojae (race 3) growth on lima bean medium amended with various concentrations of rutin ...... 210 58. P. sojae (race 12) growth on lima bean medium amended with various concentrations of rutin ...... 210 59. Concentration of apigenin after HPLC analysis from plates inoculated with 3 different P. sojae races ...... 211 60. Concentration of biochanin A after HPLC analysis from plates inoculated with 3 different P. sojae races ...... 212 61. Concentration of coumestrol after HPLC analysis from plates inoculated with 3 different P. sojae races ...... 213 62. Concentration of chrysin after HPLC analysis from plates inoculated with 3 different P. sojae races ...... 214 63. Concentration of formononetin after HPLC analysis from plates inoculated with 3 different P. sojae races ...... 215 64. Concentration of genistein after HPLC analysis from plates inoculated with 3 different P. sojae races ...... 216 65. Concentration of isoquercetrin after HPLC analysis from plates inoculated with 3 different P. sojae races ...... 217 66. Concentration of isorhamnetin after HPLC analysis from plates inoculated with 3 different P. sojae races .... 218 67. Concentration of kaempferol after HPLC analysis from plates inoculated with 3 different P. sojae races ...... 219 68. Concentration of naringenin after HPLC analysis from plates inoculated with 3 different P. sojae races ...... 220 69. Concentration of quercetin after HPLC analysis from plates inoculated with 3 different P. sojae races ...... 221 70. Concentration of rutin after HPLC analysis from plates inoculated with 3 different P. sojae races ...... 222

xi TABLE PAGE

71. Concentration o£ genistein after HPLC analysis from plates inoculated with 3 different P. aojae races (second set of experiments) ...... 223 72. Concentration of MGG (480 /xM) after HPLC analysis from plates inoculated with 3 different P. aojae races ...... 224 73. Concentration of KGG (240 /xM) after HPLC analysis from plates inoculated with 3 different P. aojae races ...... 225 74. Concentration of HGG (120 /xM) after HPLC analysis from plates inoculated with 3 different P. Bojae races ...... 226 75. Concentration of (240 /xM) after HPLC analysis from plates inoculated with 3 different P. aojae races ... 227 76. Concentration of daidzein (120 nM) after HPLC analysis from plates inoculated with 3 different P. aojae races ... 228 77. Concentration of MGD (240 /xM) after HPLC analysis from plates inoculated with 3 different P. aojae races ... 229 78. Concentration of MGD (120 jiM) after HPLC analysis from plates inoculated with 3 different P. aojae races ... 230 79. PNL specific activity in P. aojae race 4, mycelial portions ...... 232 80. PNL specific activity in P. aojae race 4, filtrate portions ...... 233 81. PNL specific activity (U/mg of protein) in soybean cotyledons and hypocotyls 12 h after inoculation with P. aojae races 3 and 4 ...... 234 82. PNL specific activity (U/mg of protein) in soybean cotyledons and hypocotyls 24 h after inoculation with P. aojae races 3 and 4 ...... 234 83. PNL specific activity (U/mg of protein) in soybean cotyledons and hypocotyls 30 h after inoculation with P. aojae races 3 and 4 ...... 235 84. PNL specific activity (U/mg of protein) in soybean cotyledons and hypocotyls 36 h after inoculation with P. aojae races 3 and 4 ...... 235 85. PNL specific activity (U/mg of protein) in soybean cotyledons and hypocotyls 42 h after inoculation with P. aojae races 3 and 4 ...... 236 86. PNL specific activity (U/mg of protein) in soybean cotyledons and hypocotyls 48 h after inoculation with P. aojae races 3 and 4 ...... 236

xii LIST OF FIGURES

FIGURE PAGE 1. Secondary product pathways in ...... 4 2. Structures o£ and isoflavonoids tested for their effect on P. aojae growth and development and for their metabolism by the fungus ...... 19 3. Schematic presentation of translocation of isoflavones on agar dishes ...... 24 4. Growth curves of P. aojae races 1, 3 and 12 in the presence of methanol (control) at different concentrations ...... 26 5. Growth curves of P. aojae races 1, 3 and 12 in the presence of Tween 20 at different concentrations...... 27 6. Growth curves of P. aojae races 1, 3 and 12 in the presence of glycerol at different concentrations...... 28 7. Growth curves of P. aojae races 1, 3 and 12 in the presence of N,N-dimethyl formamide (DMF) at different concentrations...... 29 8. Growth curves of P. aojae races 1, 3 and 12 in the presence of dimethyl sulfoxide (DMSO) at different concentrations...... 30 9. Growth curves of P. aojae races 1, 3 and 12 in the presence of the isoflavonoid, genistein at different concentrations...... 33 10. Growth curves of P. aojae races 1, 3 and 12 in the presence of the isoflavonoid, biochanin A at different concentrations...... 34 11. Growth curves of P. aojae races 1, 3 and 12 in the presence of the isoflavonoid, formononetin at different concentrations...... 35 12. Growth curves of P. aojae races 1, 3 and 12 in the presence of the isoflavonoid, coumestrol at different concentrations...... 36 13. Growth curves of P. aojae races 1, 3 and 12 in the presence of the flavonoid, apigenin at different concentrations...... 37

xiii FIGURE PAGE

14. Growth curvaB of P. sojae races 1, 3 and 12 in the presence of the flavonoid, chrysin at different concentrations...... 38 15. Growth curves of P, sojae races 1, 3 and 12 in the presence of the flavonoid, naringenin at different concentrations...... 39

16. Growth curves of P. sojae races 1, 3 and 12 in the presence of the flavonoid, isorhamnetin at different concentrations...... 40 17. Growth curves of P. sojae races 1, 3 and 12 in the presence of the flavonoid, isoquercetin at different concentrations...... 41 18. Growth curves of P. sojae races 1, 3 and 12 in the presence of the flavonoid, quercetrin at different concentrations...... 42 19. Growth curves of P. sojae races 1, 3 and 12 in the presence of the flavonoid, Kaenq>ferol at different concentrations...... 43 20. Growth curves of P. sojae races 1, 3 and 12 in the presence of the flavonoid, rutin at different concentrations...... 44 21. Metabolism of the flavonoids; kaempferol (KAEMP), quercetin (QUER), isorhamnetin (ISORHAM), isoquercetrin (ISOQUER) and rutin by P. sojae race 1 over a period of 8 days of g r o w t h . 55 22. Metabolism of the flavonoids; kaempferol (KAEMP), quercetin (QUER), isorhamnetin (ISORHAM), isoquercetrin (ISOQUER) and rutin by P. sojae race 3 over a period of 8 days of growth ...... 56 23. Metabolism of the flavonoids; kaempferol (KAEMP), quercetin (QUER), isorhamnetin (ISORHAM), isoquercetrin (ISOQUER) and rutin by P. sojae race 12 over a period of 8 days of growth ...... 57 24. Metabolic fate of the flavonoid quercetin (from Harborne, 1980) ...... 58 25. Metabolism of the isoflavone conjugates: KGG and MGD, by P. sojae race 3 over a period of 8 days of growth .... 60 26. Metabolism of the isoflavone conjugates: MGD and , by P. sojae race 3 over a period of 8 days of growth .... 61 27. Metabolism of the isoflavone conjugates: MGG and , by P. sojae race 3 over a period of 8 days of growth .... 62 28. Metabolism of the isoflavonoids, genistein (GEN), coumestrol (COUM), biochanin A (BIO A) and formononetin (FORM) by P. sojae race 1 over a period of 8 days of growth ...... 65

xiv FIGURE PAGE

29. Metabolism of the isoflavonoids, genistein (GEN), coumestrol (COUM), biochanin A (BIO A) and formononetin (FORM) by P. aojae race 3 over a period of 8 days of growth ...... 66 30. Metabolism of the isoflavonoids, genistein (GEN), coumestrol (COUM), biochanin A (BIO A) and formononetin (FORM) by P. aojae race 12 over a period of 8 days of growth ...... 67 31. Metabolism of the flavonoids; apigenin (APIG), chrysin (CHRY) and naringenin (NARIN) by P. aojae race 1 over a period of 8 days of growth ...... 69 32. Metabolism of the flavonoids; apigenin (APIG), chrysin (CHRY) and naringenin (NARIN) by P. sojae race 3 over a period of 8 days of growth ...... 70 33. Metabolism of the flavonoids; apigenin (APIG), chrysin (CHRY) and naringenin (NARIN) by P. aojae race 12 over a period of 8 dayB of growth ...... 71 34. Site of action of different pectolytic enzymes; Pectin lyase (PNL), pectate lyase (PL), polygalacturonase (PG) and pectin methyl esterase (PME) ...... 107 35. Growth curves of P. aojae race 3 and 4 on synthetic media amended with different pectic fractions: pectin and polygalacturonic acid ...... 146 36. Lima bean broth amended with different pectic fractions: polygalacturonic acid, pectin and dry hypocotyls was used to induced P. aojae (race 4) pectolytic enzymes. PNL activity in the mycelial portions was measured over a period of 12 days ...... 148 37. Lima bean broth amended with different pectic fractions: polygalacturonic acid, pectin and dry hypocotyls was used to induced P. aojae (race 4) pectolytic enzymes. PNL activity in the filtrate portions was measured over a period of 12 days ...... 14 9 38. Lima bean broth amended with different pectic fractions: polygalacturonic acid, pectin and dry hypocotyls was used to induced P. aojae (race 4) pectolytic enzymes. PL activity in the mycelial and filtrate portions was measured over a period of 12 days ...... 150 39. Lima bean broth amended with different pectic fractions: polygalacturonic acid, pectin from orange anf from apple was used to induced pectolytic enzymes in P. sojae race 3. PNL specific activity in the mycelial portions was measured over a period of 12 days ...... 153 40. Lima bean broth amended with different pectic fractions: polygalacturonic acid, pectin from orange and apple was used to induced pectolytic enzymes in P. aojae race 4. PNL specific activity in the mycelial portions was measured over a period of 12 days...... 154

xv FIGURE PAGE

41. Lima bean broth amended with different pectic fractions: polygalacturonic acid, pectin from orange anf from apple was used to induced pectolytic enzymes in P. sojae race 3. PNL specific activity in the filtrate portions was measured over a period of 12 days ...... 155 42. Lima bean broth amended with different pectic fractions: polygalacturonic acid, pectin from orange and apple was used to induced pectolytic enzymes in P. sojae race 4. PNL specific activity in the filtrate portions was measured over a period of 12 days ...... 156 43. Schematic of the electrophoretic pattern obtained in polyacrylamide gels ...... 161 44. PNL specific activity was measured in infected cotyledon tissues of soybean cultivar W7, with P. sojae races 3 and 4 ...... 163 45. PNL specific activity was measured in infected cotyledon tissues of soybean cultivar N with P. aojae races 3 and 4 ...... 164 46. PNL specific activity was measured in infected hypocotyl tissues of soybean cultivar with P. sojae races 3 and 4 ...... 165 47. PNL specific activity was measured in infected hypocotyl tissues of soybean cultivar W with P. sojae races 3 and 4 ...... 166 48. Schematic of the electrophoretic pattern obtained with IFG ...... 175

xvi LIST OF PLATES

PLATE PAGE

I. Toxicity aBsayB ...... 21 II. Treatments containing genistein conjugate (GT-2) caused frequent mycelial branching giving to the colony a feather-like appearance ...... 47

III. Clearing of the agar just ahead of the mycelial front was evidence of the metabolism of several of the compounds ...... 48 IV. The effects of various flavonoids on P. sojae morphology ...... 51 V. Tissue smples were taken from infected hypocotyls and cotyledons at different periods of time after inoculation ...... 116 VI. Eleven days old soybean cotyledons from cultivars W and W79 were infected with P. sojae races 3 and 4 .... 130 VII. A fungal hypha is surronded by an electron dense material at the plant surface ...... 135 VIII. Much of P. sojae colonization is intercellular, leading to the tissue maceration typical of the disease ...... 136 IX. Ultrastructure examination of P. sojae intercellular growth shows the formation of projections extending between the digested ends of the plant cell and the fungal cell ...... 138 X. Large number of cytoplasmic vesicles or lomasoxnes concentrated around the site of attachment and at the periphery of the fungal cell wall ...... 139 XI. Apparently, P. sojae has the ability to enzymatically disolve the pectic substances and other polymers that comprise the plant cell wall ...... 140 XII. P. sojae haustoria grew and enlarged intracellularly to a final club shape ...... 141 XIII. Multiple fungal hyphae penetrate the plant cell in the compatible interactions ...... 142

xvii PLATE PAGE XVX. Some changes also occurred in the plant cell wall, plasmalemma withdrawn from the cell wall and plant secretory vesicles were observed close to the cell membrane ...... 143

xviii GENERAL INTRODUCTION

Phytophthora sojae (Kaufmann & Gerdemann) Hansen and Maxwell [previously designated as Phytophthora megasperma Drechs. f.sp. glycinea (Hildeb.) Kuan and Erwin] is the causal agent of Phytophthora root rot and seedling damping-off in soybeans IGlycine max (L.) Merr.]. Millions of acres of soybean are infested with this pathogen, which causes severe economic losses under conditions that favor the disease (Schmitthenner, 1985). Despite current control options which include breeding for resistance, systemic fungicides and cultural and biological practices, the pathogen continues to be a problem, partly through the development of new physiological races (Paxton, 1983). P. sojae produces over 30 races that interact differentially with various soybean cultivars (Schmitthenner, personal communication). P. sojae races have been defined based on their reactions to specific resistance genes (Rps) present in various soybean differentials. Resistance in soybeans to P. sojae is mediated by seven loci with multiple alleles at two loci (Athow et al. 1980; Buzzell et al. 1987; Ploper et al. 1985). This disease has been a model for biochemical studies on host- pathogen interactions, particularly those involving secondary metabolites. It has been shown that soybean plants produce an array of secondary metabolites from the phenylpropanoid pathway, (eg. , flavonoids and isoflavonoids), constitutively and in response to biotic and abiotic elicitors (Buttery and Buzzell, 1973; De Wit, 1986; T.L. Graham, 1991b; T.L. Graham et al. 1990; Hahn et al. 1985; Ingham, 1982; Naim et al. 1974;, Morris et al. 1991 and Stossel, 1983). Some but not all of these have been strongly implicated in plant defense.

1 2

So far, studies on the soybean - P. sojae interaction have provided some of the most convincing evidence for the development of the phytoalexin model for disease resistance. Phytoalexins are defined as low molecular weight antimicrobial compounds that are produced or accumulate after exposure of plants to microorganisms or abiotic elicitors (Paxton, 1981) implying a role in defense of plants against microbes. Therefore, phytoalexins accumulation constitute part of the biochemical defense induced by microbial contact. They are not detected in significant quantities in healthy tissues but accumulate in stressed plant tissues. Accumulation of phytoalexins is often associated with a plant resistance mechanism termed hypersensitivity. Hypersensitivity (HR) is a rapid host cell death following infection. This resistance response is also called an incompatible response and involves necrosis of those plant cells that come in contact with an invading pathogen. It is a cell sacrifice to prevent the further spread and development of the pathogen to other plant cells and tissues. In incompatible interactions, it is well documented that the soybean plant responds to infection through the accumulation of phytoalexins such as coumestrol (Ingham, 1982) and four isomers of the , (Hahn et al. 1985) . It has been shown that accumulation of the is associated with the cessation of growth of the pathogen in resistant plants (Yoshikawa et al. 1978; Keen and Yoshikawa, 1983; Stossel, 1983; Hahn, 1985; Hard, 1989) and correlates well with race- specific resistance to P. sojae (Yoshikawa et al., 1978; Keen and Yoshikawa, 1983). The mechanisms for the toxicity of phytoalexins to fungi and bacteria have not been resolved, but alterations of cell membrane functions are proposed. In the specific case of the soybean phytoalexin, glyceollin, the impairment of the plant plasmalemma and tonoplast ATPase activities has been implied (Giannini, 1988). Besides the phytoalexins, glyceollin and coumestrol, another group of compounds, the isoflavonoids, genistein and daidzein, are produced in soybean tissues in response to infection {T.L. Graham, 1989 and T.L. Graham et al. 1990). The soybean isoflavonoid, daidzein, is the first committed precursor in the formation of glyceollin. Thus, it has been suggested that the release of constitutive daidzein may play a role in the overall accumulation of glyceollin (T.L. Graham et al. 1990). In addition, a preliminary study suggested that another isoflavonoid, genistein, might be directly toxic to P. sojae (T.L. Graham, 1989) . Fig. 1 shows a general diagram that summarizes the secondary product pathway in soybean which may relate to defense. Recent studies performed with isoflavone conjugates have also added to our understanding of host-pathogen interactions in chickpea (Roster et al. 1983) and soybean (T.L. Graham et al. 1990). Recently, Graham and his coworkers (1989, 1990 and 1991b) have emphasized the importance of isoflavone conjugate compounds in disease containment. Conjugates of the isoflavones, daidzein and genistein, have been shown to be constitutively present in soybean seedling tissues (T.L. Graham et al. 1991b). These conjugates of daidzein and genistein also accumulate in response to the pathogen. They start to accumulate in elicited tissues at 8 h and the soybean phytoalexin, glyceollin, starts to accumulate at 12 h. All these compounds are associated with race specific resistance mentioned above. Incompatible responses, in which soybean tissues are resistant to infection lead to the orchestrated expression of some of these related phenylpropanoid compounds. Cells ahead of the infection front respond to infection through the formation of the antibiotic genistein from the preformed conjugated form and the phytoalexins glyceollin and coumestrol. In addition to the accumulation of these different phenylpropanoid compounds in the incompatible response, phenolic polymers accumulate in cells immediately ahead of the infection front. M.Y. Graham and T.L. PHENYLPROPANOID DERIVED POLYMERS (LIGNIN AND SUBERIN)

PHENYLALANINE COUMES7ROL

GENISTEIN DAIDZEIN t GLYCEOLLIN GENISTEIN CONJUGATES DAIDZEIN CONJUGATES

Pig- 1- Secondary product pathways in soybeans: Some phenolic compounds are well-known fungitoxic and antibacterial substances and have many proposed effects on host-pathogen interaction. It is well documented that soybean responds to infection through the accumulation of phytoalexins such as glyceollin and coumestrol as well as the accumulation of conjugates of the isoflavones genistein and daidzein. In addition, accumulation of phenolic polymers like lignin and suberin, occur in cells ahead of the infection front. 5

Graham, (1992) observed that P. sojae elicitors (cell wall glucan) induce a rapid and massive accumulation of phenolic polymers in soybean cotyledon cell walls proximal to the point of elicitation. The deposition of phenolic polymers in elicited tissues was over 10 times more than that in wounded controls within 4 h of treatment. The response reached its maximum by 24 h, whereas in controls, phenolic polymer accumulation has just began by that time. The deposition of wall bound phenolics in elicited soybean tissues is greater than the glyceollin and isoflavone responses. The responses also include the covalently linked phenolics like lignin and suberin (Fig. 1) as well as simple esterified coumaric and ferulic acid monomers that are also involved in the defense reaction (M.Y. Graham and T.L. Graham, 1992). In soybean cotyledons the deposition of phenolic polymers seems to correlate with the induction of a specific group of anionic peroxidases (M.Y. Graham and T.L. Graham, 1992). Peroxidases are involved in the polymerization of hydroxycinnamyl alcohols in presence of peroxide to produced lignin. Peroxidases appear to be directly involved in the last steps of phenolic polymer deposition in the plant cell wall at the expense of H202 that acts as an oxidizing agent

(Stich and Eberman, 1984). In addition to the wall phenolics, soybean cells also transiently accumulate red pigments which are immediate precursors of glyceollin (M.Y. Graham and T.L. Graham, unpublished). Their role seems to be the protection of soybean cells from light induced phytoalexin toxicity. To get a more complete picture of the P. sojae-soybean interaction, M.Y. Graham and T.L. Graham (1994) have demonstrated the presence of wound-associated factors (competency factors) which are required for the response of soybean cells to P. sojae wall glucan elicitors. Competency factors might affect the glyceollin response as well as plant cell sensitivity to P. sojae wall glucan elicitors. Apparently, these wound factor(s) might be released from dead or dying cells during the HR in resistant plants and might be involved in cell to cell signaling (M.Y. 6

Graham and T.L. Graham, 1994). However, in distal cells there is no hydrolysis of pre-£ormed isoflavone conjugates; instead these molecules accumulate, building up a defense response that later on will contain the progression of the infection through the release of genistein. All of these various responses in proximal as well as distal cells in the incompatible interaction are induced in a spatial and temporal fashion by P. sojae cell wall glucan elicitor. Although we are beginning to better understand the resistance mechanisms of the host and their coordination, very little attention has been paid to the pathogen side of the interaction. He know very little about what determines virulence and pathogenicity in P. sojae and how these traits affect the overall outcome of the interaction. Therefore more information is needed on the mechanism of the disease regarding specifically defined events in disease development. The major objective of the research described in this thesis was to begin to define and characterize some particular P. sojae traits in terms of their biochemistry and relation to pathogenicity. Since so little is known on the mechanisms of pathogenicity and virulence in P. sojae, our goal was not so much to study any given mechanism of virulence of pathogenicity in depth, but to establish a broader perspective of what these mechanisms may be, allowing a framework for future research. This goal was pursued through meticulous observations on the molecular, spatial and temporal interactions of P. sojae isolates with different soybean cultivars. Parallel morphological, cytological and biochemical studies of P. sojae isolates were performed in culture to have a more complete picture and understanding of this plant pathogen.

Some potential morphological and biochemical factors involved in pathogenicity can include:

1) chemotaxis or any other factor that can help bring the pathogen into contact with the host (eg. adhesive materials that appear to consist of proteins and/or glycoproteins or polysaccharides 7

produced by the pathogen)

2) specialized infectious structures involved in the effective penetration of plant tissues (eg. infection peg, appresoria) 3) production of cell wall degrading enzymes and/or enzymes that

enable the pathogen to invade plant cells and tissues (eg. pectolytic enzymes and/or cutinase, an extracellular enzyme produced by germinating spores of various fungi that has been shown to be responsible in the penetration of cuticular barriers in plants) 4) production of enzymes that degrade preformed toxins or induced antimicrobial compounds (eg. phytoalexins and secondary metabolites) 5) production of toxins involved in conditioning plant tissue for colonization

6) production of substances that mask recognition factors at parasitic interfaces and substances that suppress the expression of the hypersensitive response and other plant defenses

Thus, many genes might be involved in the successful development of a parasite in its host tisBueB and in establishing or conditioning its specific relationship with the host. However, although many fungal morphological and biochemical factors have been proposed as pathogenicity factors, very few have been evaluated rigorously for their role(s) in disease. Some examples of fungal morphological and biochemical factors for which evidence indicates a possible pathological significance include:

1) chemoattractants: recent results on phytoalexin studies suggested that flavonoid compounds are involved in chemoattraction of the soybean symbiont, Bradyrhizobium japonicum [previosly designated as Rhizobium japonicum (Kirchner) Buchanan] (Khan and Bauer,

1988) and the soybean pathogen, P. sojae (Morris and Ward, 1992). 2) some biotrophic fungi are able to produce a puncture in the plant cell wall through special structures. For example the stachel, a rod-like structure present in the encysted zoospore of Plasmodiophora brasaicae Woronichin is produced to penetrate the root hair of its host (Ingram et al. 1976; Hisaghi, 1982a). 2) the role of cutinase in the degradation of plant cuticle (Kolattukudy, 1989). 3) pathogens such as Sclerotlnia aclerotiorum (Lib.) de Bary and Sclerotitm rolfaii Sacc. rapidly solubilize host cell walls by secreting polygalacturonases (Bateman, 1976; Cooper, 1983a and 1983b) and oxalate, a strong chelator of divalent cations which disrupts the integrity of the plant cell wall as a consequence of acidifying the host tissues (Godoy et al. 1990) 4) the production of a specific enzyme, demethylase, by Nectria haematococca Berk, and Br. which allows this pathogen to detoxify the pea phytoalexin, pisatin (VanEtten, 1981) 5) the importance of toxins in pathogenicity have been studied for many years with pathogens like Fusarium spp. and Helminthoaporivm Link (Bipolaria Schoem.) spp. A number of such toxins have been isolated and identified from fungi and bacteria (Yoneyama and Anzai, 1993) 6) the recognition factors like fungal and plant elicitors that have the ability to stimulate the accumulation of phytoalexins in plant cells (Yoshikawa, 1983; De Wit,1988). Fungal cell wall glucans as well as pectic fragments from the host plant have been observed to be synergistic in the accumulation of soybean phytoalexins (Davis et al. 1986). Another recognition factor that has attracted great attention are lectins; some of which

agglutinate bacteria and fungal spores. It seems that lectins provide surface receptor sites for rhizobia infections (Bauer, 9

1981). However, more experimental evidence is needed to suggest a dominant role in the recognition of fungal parasites.

Thus, both morphological and biochemical factors are important in the infection process and may relate to genes that control pathogenicity. To pursue the major objectives of this study, that is, to define and characterize some of the biochemical traits of pathogenicity in P. sojae, we concentrated our research efforts on three major subjects: a) primarily, to more thoroughly characterize the toxicity of various soybean constitutive and induced flavonoids and isoflavonoids on P. sojae, including effects on morphology and growth as well as direct toxicity. Secondly, to evaluate the ability of the pathogen to metabolize these plant natural compounds "in culture" b) to evaluate the ability of the pathogen to produce catalase, a specific enzyme that degrades peroxide into water and oxygen, and to determine its possible role in P. sojae virulence or pa thogeni c i ty.

c) to characterize the role of various pectolytic enzymes produced by P. sojae "in culture" and "in planta" and to evaluate their possible role in pathogenicity or virulence. Detailed microscopic observations of soybean cell wall degradation and maceration were used to correlate and complement pectolytic enzyme activity with tissue deunage.

Each objective is discussed in more detail with results and interpretations in three separate chapters. CHAPTER I

Soybean Flavonoid Effects on and Metabolism

by Phytophthora sojae

INTRODUCTION

Plants defend themselves against pathogens by different combinations of structural or morphological characteristics as well as biochemical mechanisms. Structural or morphological characteristics act as physical barriers and inhibit pathogens from entering and spreading through plant cells and tissues. Some examples of these are: stomata sizes and shapes, thick mats of hairs on leaveB, quantity of wax and cuticle, among others (Anderson, 1982). Although structural characteristics are important, the resistance of a plant against pathogen attack and invasion depends mainly on their biochemical defenses (Anderson, 1991; Kuc, 1990 and Paxton, 1991). These biochemical reactions occur within host cells and tissues and produce substances that can be either toxic to the pathogen or create unfavorable conditions for its growth and development. Biochemical defense mechanisms can be divided in two major groups, preformed toxins and phytoalexins. Preformed toxins or chemicals are present in healthy plant cells constitutively. This group includes a variety of substances like phenolics and cyanogenic glycosides, for example. Cyanogenic glycosides are found specifically in cassava and sorghum and are known to play a role in plant resistance to pathogenic organisms (Anderson, 1982). On the other hand, phenolic compounds are a more diverse group and are widespread among plants, playing different

10 11

roles. They have been implicated in allelopathic roles, influencing germination and growth of neighboring plants (Lynn and Chang, 1990). In addition, they are structural components in the plant cell wall (eg. lignin and suberin) (Legrand, 1983 and Ride, 1983) and also are involved in the chemical defense of plants against microbes and insects (Barz et al. 1990; Deverall, 1982; Priend, 1985; Harborne, 1980, 1985 and 1990; Kuc, 1990; Matern and Kneusel, 1988; Metraux and Raskin, 1993 and Swain, 1985). Phenolics like protocatechuic acid and catechol, present in red onions, are responsible for the resistance against Colletotrichum spp. Corda (Anderson, 1982; Mansfield, 1983). Other phenolics like malonyl glycosyl conjugates of the isoflavonoids, daidzein and genistein, present in soybean seedlings, have been shown to be effective against P. sojae (T.L. Graham et al, 1990). Phenolics like DIMBOA (2,4-dihydroxy-7-methoxy- benzoazinone) and related glycosylated compounds have been implicated in constitutive resistance of corn, rye, wheat and other grasses to insects and certain fungi and specially to the phytopathogenic bacteria Erwinia spp. (Anderson, 1982). It seems that pre-formed phenolics are generally present in conjugated forms which are non-toxic for the plant cell itself (Anderson, 1991) and are usually stored in the vacuoles (Hrazdina and Wagner, 1985). However, pathogens required sugars and produce glycosidases that are able to hydrolyze such complex molecules (Anderson, 1991), The cleavage of the sugar groups releases toxic chemicals that are quite harmful to pathogens. Another important group of biochemical defense molecules produced by plants against invading pathogens are phytoalexins. These are induced and have been extensively studied especially in legumes and solanaceous plants in relation to resistance of plants to pathogens (Anderson, 1991 and Darwill and Albersheim, 1984). In legumes, isoflavonoid phytoalexins like pterocarpans, coumarins and isoflavans are more important whereaB in solanaceous plants, sesquiterpenoid phytoalexins like rishitin and lubimin

prevail (Ingham, 1982). In the case of soybean, glyceollin accumulation 12 has been implicated in race-specific resistance to P. sojae, and the isoflavonoid genistein might be directly toxic to the pathogen (T.L. Graham, 1989). Toxic effects of these phytoalexins are not yet understood. There is some evidence indicating their role by uncoupling oxidative phosphorylation (Smith, 1982) . In the specific case of P. sojae, experiments done "in vitro" showed that glyceollin caused ultrastructural plasma membrane abnormalities (Stossel, 1983). Also, impairment of the ATPase activities in plant plasmalemma and tonoplast have been implied (Giannini, 1988) . Although some studies have made possible the assessment of fungitoxic structure-activity relationships of some of the flavonoids (Adesanya et al. 1986), the precise structural requirements for their toxic activity are unknown. Apparently a certain degree of lipophilicity in these compounds seems to be required to penetrate cell membranes (Laks and Pruner, 1989 and Ingham, 1982).

Numerous bioassays have been utilized to assess the toxicity of phytoalexins (Bailey et al 1976; Skipp and Bailey, 1977). The purpose of bioassays "in vitro" is to provide information which could help clarify any role that phytoalexins may have in plant disease resistance. Studies of the effects on fungal spore germination, germ tube growth and mycelial growth on agar plates or liquid media are common (Bailey et al 197 6; Skipp and Bailey; Smith, 1982). Obviously any bioassay provides limited information and it is necessary to utilize a variety of assays in assessing phytoalexin toxicity. Unfortunately, many studies have not incorporated these elements in a systematic manner. Timing of growth measurements is critical in determining phytoalexin toxicity. Measurements of growth after only one period of time can provide incomplete or misleading data. It is dangerous to assume that all effects of inhibitors can be illustrated by simple radial growth inhibition. Monitoring the changes which occur during the bioassay allows fuller evaluation of the results and more meaningful conclusions. Relatively few studies have examined more subtle effects of secondary metabolites on 13 fungal growth and development. The examination of macroscopic as well as microscopic aspects is an additional important factor in determining the toxic effects of plant metabolites on fungal growth. Another draw-back in most studies about plant secondary metabolites is that they do not address the effects of multiple compounds on pathogens at the same time and very few address the effects of mixtures of compounds on pathogen growth and development. In nature, pre-formed toxins, phytoalexins or secondary metabolites rarely accumulate individually in diseased plants. Some plants species like common bean and soybean can produce more than one phytoalexin and various other metabolites (Smith, 1982; Hahn, 1985; T.L. Graham et al. 1991b). It seems possible that these metabolites might act synergistically to contain fungal invasion. In natural situations, plants seem to use them in orchestrated efforts to stop pathogen invasion (Barz, 1970; Fett and Osman, 1982; Wage and Heidin, 1985) . There are few studies that address this Bubject. For example common bean produced kievitone and in response to Rhizoctonia aolani Kuhn infection (Smith, 1982) . Studies "in vitro" showed that their effects were additive rather than synergistic. Effects of mixtures of naturally occurring soybean metabolites with genistein were tested "in vitro" against P. sojae. Genistein is a strong P. sojae inhibitor and is constitutively present in conjugated form in all soybean organs (T.L. Graham, 1990). These studies also suggested that the effects were additive rather than synergistic (Rivera-Vargas et al. 1993) Another very important aspect that has been over looked, especially in the interaction between soybean and P. sojae, is the active role of the pathogens to circumvent plant defenses. Differences in sensitivity to phytoalexins and their relation to pathogenicity can be explained in terms of various possible mechanisms present in the pathogen that make it able to cause disease despite plant efforts to holt its invasion. Generally speaking, several mechanisms have been suggested in order to explain pathogenicity in the presence of host antibiotics: The pathogen may have a cell wall or a plasmalemma less permeable to the toxic compound that helps to prevent the effective penetration of the metabolite {Smith, 1982) . Another possible explanation might be that there is a weak affinity between the phytoalexin and the fungal cell wall or sites of action {Smith, 1982}.

Some pathogens might have a metabolic by-pass for the specific process inhibited by the phytoalexin so that the fungus will be insensitive to its action (Smith, 1982). Models have also been proposed to explain these differences in sensitivity based on the presence of suppressor molecules produced by pathogens which directly interact with host receptor molecules at the cell wall level. The outcome, ie. disease, will depend on the different interaction of these two (De Wit, 1986; Callow, 1987). Finally, the best understood and studied mechanism is the detoxification of a phytoalexin before or after entry into the pathogen cell. Various examples of phytoalexin detoxification include: a. The pea phytoalexin, pisatin is a well documented case of phytoalexin degradation. Specifically, demethylation of the phytoalexin is accomplished by the pea pathogen, N. haematococca (Van Etten, 1981; Van Etten et al. 1982; Van Etten et al, 1989; Mackintosh et al. 1989). The importance of pisatin demethylation and its implications in pathogenicity has been well studied by Van Etten and his group. b. Other examples are the studies on the interaction of Bofcrytis spp. Micheli ex Pers. and Vicia fabae L. with their host. These studies suggested that phytoalexin accumulation restricts the growth of the non-pathogenJ9. cinerea Pers. whereas the pathogen,

B. fabae Micheli ex Pers. was able to metabolize and detoxify the 15

inhibitors to which it is exposed, thus preventing the accumulation of fungitoxic concentrations around the invading hyphae (Rosall et al. 1980) . In addition, B. fabae is less sensitive to derivatives of the phytoalexin wyerone and is able to detoxify this phytoalexin by reducing it to less fungitoxic products. This interaction showed a balance of phytoalexin production by the plant and phytoalexin degradation by the fungus {Mansfield, 1983). c. The degradation of the pea phytoalexin by Ascochyta rabiei Lib. (Leningrand) and detoxification of the bean phytoalexin, phaseollidin by Fusarium solani f.sp. phaaeoli Link has been reported (Smith, 1976). d. Studies on the interactions between Phaseolus vulgaris L. and Colletotrichum lindemuthianum (Sacc. & Magn.) Briosi & Cav. suggest that the host phytoalexin, phaseollin, appears to have marked fungicidal activity against C. lindemuthianum at concentrations above 10 fig/ml (Skipp and Bailey, 197 6). However, fungal colonies develop at higher concentrations of the phytoalexin. The authors suggest that the apparent insensitivity of the pathogen mycelium was associated with a localized depletion of phaseollin caused by fungal metabolism. e. Other examples are the two major sesquiterpenoid phytoalexins

produced by potato: lubimin and rishitin, both of which can be degraded by Gibberella pulicaris (Fries) Sacc. (anamorph = Fusarium aeunbucinium Fuchel) (Desjardin et al. 1989).

Regarding metabolic conversions by oomycetes, very little iB known. Phytophthora spp. are in fact cited as examples in which no phytoalexin metabolism was detected (Yoshikawa, 1979) , with the exception of the conversion of lubimin to 15-dihydrolubimin by Phytophthora capsici Leonian

(Ward and Stossel, 1983). Yoshikawa et al. (1979) studied glyceollin 16 degradation by P. sojae. They reported that the fungus failed to degrade the phytoalexin during a 24 h period. This finding was corroborated by Stossel in 1983. Thus it is assumed that in compatible interactions, the fungus circumvents the antibiotic by suppressing its synthesis, by repressing its function or by avoiding the phytoalexin by spatial separation (rapid colonization) or other unknown mechanisms. Stossel also showed that the ability of the pathogen, P. sojae, to adapt to glyceollin varies between races, and tolerance to glyceollin was not specifically induced (1983) . He found that some tolerance may be induced by low phytoalexin concentrations. However he concluded that this pathogen does not require tolerance to glyceollin for pathogenicity. Therefore, phytoalexin metabolism is not the only way to circumvent a phytoalexin-based defense mechanism (Van Etten et al, 1982). In most instances in which pathogens have been shown to metabolize a phytoalexin, other non-pathogenic species on that host were found to be capable of performing the same reaction. This observation is of little significance regarding phytoalexin metabolism and pathogenicity, since it could be naive to assume that the ability to metabolize a phytoalexin is the only trait required for pathogenicity in any fungus (VanEtten, 1982). However, in many of the examples on host-pathogen interactions mentioned above, the ability to degrade phytoalexins seem to play a decisive role. In terms of P. sojae-soybean interactions very little is known about the metabolism of secondary products and related compounds as well as their effects on fungal growth and development. Only one compound, glyceollin, has been examined in detail of the various constitutive and induced secondary products produced by soybean tissues in response to infection. Thus, more detailed systematic and coordinate biochemical and microscopical studies designed to examine the role of phytoalexins and secondary metabolites in resistance are needed. Although, research has been focused on their biocidal or biostatic effects, very few studies have emphasized other more subtle effects on fungal growth, development and 17 morphology. The understanding of those effects could lead u b to understand the natural resistance mechanism for disease control in the soybean-P. sojae interaction. 18

MATERIALS AND METHODS

Toxicity of Plant Natural Compounds in P. sojae Fungal Cultures: Fungal cultures were obtained from Dr. A.F. Schmitthenner (OARDC, Wooster, OH.). Isolates of races 1, 3 and 12 were used in all experiments. Cultures were stored on lima bean agar slants at 11 °C. To prepare the media for the slants as well as for the petri plates, 25 g of frozen lima beans were autoclaved 20 min to soften the beans. The soft beans were then passed through a sieve and the resulting suspension was centrifuged at 3/4 speed for 10 min. (Centrifuge Model HN, International Equipment, Co.Needham Hts, Mass, USA). Fifteen grams of agar were added to the clarified lima bean extract per liter. Lima bean extract are particularly low in aromatic metabolites which is ideal for these types of studies. It contains only very low levels of secondary products and yet provides the other nutrients and cofactors required for excellent P. sojae growth.

Plant Natural Products: Structures of the plant natural products used in toxicity studies are shown in Fig. 2. Genistein, daidzein, formononetin, kaempferol, rutin, quercetin, isoquercetrin and isorhhamnetin were obtained from Atomergic Chemetals Corporation (Farmingdale, NJ, USA). Apigenin and chrysin were obtained from Sigma Chemicals (St. Louis, MO, USA). Biochanin A was obtained from Aldrich

Chemical Company (Milwaukee, WI, USA). Coumestrol was obtained from Eastman Kodak Company (Rochester, NY, USA) . The conjugates 6"-malonyl-7-0- S-D-glucosyl and 7-0-6-glucosyl, of genistein and daidzein were purified (95%) using HPLC (Graham et al., 1990). Tween 20 (Fisher Biotech, Fair Lawn, N.J.), glycerol (Sigma, St.

Louis,MO), N,N - dimethyl formamide (DMF) (Sigma), dimethyl sulfoxide HO

NARINGENIN (NARIN) PLAVONOIDS: APIGENIN (APIG) : RlBRa==H, R3=OH CHRYSIN (CHRY) : Rl=R3=R3=H KAEMPFEROL (KAEMP) RX*H, R,-Rj»OH QUERCETIN (QDER) : Rj-Rj-Rj-OH ISORHAMNETIN (ISORHAM) : R^OCH,, R,«R,.OH ISOQUERCETRIN (ISOQDER) : Rj«R,=OH, Ra«0-GLUC0SE RUTIN : RjbRjbOH, Ra»O-RUTIN0SE

OH HO

ISOPLAVONOIDS : DAIDZEIN (DZ) : R1«H, Ra*OH COUMESTROL (COUM) GENISTEIN (GEN) : R . h R j b O H PORMONONETIN (FOR) : R2-H, Ra-OCH, BIOCHANIN A (BIO A) R2««0H, R 3 » O C H j ISOFLAVONOIDS CONJUGATES : 7-O-GLYCOSYL CONJUGATES : DAIDZIN (DZ-1) : RasH, Rj=0-GLUCOSE GENISTIN (GT-I) : Ra=OH, Ra=0-GLUC0SE 6"-MALONYL-7 -O-GLYCOSYL CONJUGATES : MGD (DZ-2) : Rj=Hr Ra-0-6"- MALONYLGLUCOSE MGG (GT-2) : Ra«OH, Ra=0-6" -MALONYLGLUCOSE Pig. 2 Structures of flavonoids and isoflavonoids tested for their effect on P. sojae growth and development and for their metabolism by the fungus. 20

(DUSO) (Sigma) and methanol were tested as possible solvents for the plant natural products used in these experiments. Toxicity of these different solvents on P. sojae was tested. Methanol was the best solvent and less toxic to P. sojae than the other solvents tested.

A 5 mM stock solution of each plant natural compound was prepared in 100% methanol. Sonication and heat were used with some of them to completely dissolve the compounds at this concentration. The plant natural compounds tested were very stable to heat and sonication as confirmed by HPLC. Six different concentrations were used for each compound tested. Aliquots of 6.25, 12.5, 25, 50, 100 and 200 fil from the stock solution were added to 4 ml of liquid lima bean agar in tubes maintained at 60°C in a heating block. Pure methanol was used as a control. After adding the

test sample to the media in each tube, it was vortexed and immediately poured into a 60 x 15 mm petri plate. After hardening, the plates were incubated in an oven for 8 hrs at 60°C with loose covers to evaporate any residual methanol.

Bioassays 1. Toxicity : Four mm diameter mycelial plugs from actively growing P. sojae cultures were transferred to the plates amended with the different concentrations of the different plant natural compounds. Three plugs were transferred onto each petri plate (Plate I) . Two replicates were tested for each concentration of the compound except for the purified conjugates of daidzein and genistein. Limited amounts of these chemicals were available and experiments were performed once. For all the rest, a total of Bix tests were run for each concentration of each compound.

Inoculated plates were incubated in the dark at 27°C. Samples were collected uBing a cork borer (no. 1) for microscopic examination as well

as HPLC analysis. Plate I illustrates the arrangement of the mycelial plugs on the plate and the sections examined in this experiment. Four 21

Plate X Toxicity assays: The effects of the flavonoids on P. sojae growth and morphology and the metabolism of these compounds was determined using amended lima bean agar. Three agar plugs (4mm) of P. sojae (isolates of races 1, 3 and 12) were symmetrically arranged on each plate to provide 3 replicates per plate. Observations were made on a daily basis and every other day after inoculation, samples; A, B, C and D were collected for HPLC analysis. 22 different sections (A, B, C and D) were taken from each fungal colony pertreatment. Section A and B included the oldest and the youngest area of mycelial growth, respectively. Sections C and D included two areas outside the fungal growth. Section C was sampled from the area immediately ahead of mycelial growth and D was from the section at the farthest edge of the plate. Section D thus represented a control to confirm the exact concentration of the compound in the medium. Only two sections A and B were sampled with those compounds that did not inhibit fungal growth and the mycelium was able to colonize the plate after a period of time. To determine the fungitoxic or fungistatic abilities of the different compounds tested, 4 mm agar plugs of mycelia were taken from treated plates and transferred back to unamended lima bean agar. The reestablishment of mycelial growth on control media was monitored for several days. Colony reestablishment was used to assess a fungistatic effect of the compound on P. sojae, whereas failure to reestablish a new colony was used to assess a fungitoxic effect.

2. Macroscopic Observations: On a daily basis radial growth measurements were made. Colony characteristics like mycelial color, density, formation of aerial mycelia and sectoring were recorded. 3. Microscopic Observations: Samples were taken every other day after inoculation and examined microscopically. For microscope observations, slides were prepared with double distilled water. Random fields of intact agar plugs were examined under low magnification {x 100) to count the number of sporangia and oogonia produced, and observe changes in color. More detailed examination of structure and morphology were obtained under high magnification (x 400). Mycelial abnormalities like hyphal swelling, cytoplasmic granulation, vesiculation, septation and abnormal coloration were examined.

Metabolism of Different Plant Natural Compounds by P. sojae HPLC Analysis: The protocol used for HPLC analysis was described by Graham et al. in 1990. The system used a C18 reverse phase column. 23

Sections A to D, described above, were transferred to microfuge tubes containing 600 fil of methanol, ground, centrifuged at 14,926 g's (Biofuge; Rotor no. C-1710-20, r„..= 7.9 cm) for 4 min and analyzed. Samples were stored at -20°C until analysis. HPLC analysis was carried out for all samples collected from treatments containing concentrations of 125 and 200 fiM of the different compounds tested.

Ujptake and Translocation of Different Plant Natural Compounds by P. sojae Hyphae In order to ascertain if isoflavonoids penetrate the fungal cytoplasm and are translocated in the fungal hyphae, a double dish technique was used (Fig. 3). The technique allows separation of the lima bean plant metabolite amended media from plain lima bean media using a small size petri plate (60 mm x 15 mm) inside a standard size plate (100 mm x 15mm). This prevents the diffusion of the plant metabolite in the media. Therefore, the mycelium outside of the inner dish has no direct contact with the compound tested. Fungal mycelia was transfered to two different points in separate plate sets (Fig. 3). In set 1, the mycelial plug was placed outside of the small plate amended with the isoflavonoid. In the other set 2, the mycelial plug was placed inside of the small plate amended with the plant metabolite. In thiB manner, P. sojae mycelium was growing towards and away from the plant metabolite source in separate plate sets. Three replications were made per compound at a 200 fiM concentration. Samples A, B, C and D were taken. Section A was an agar plug taken immediately adjacent to the point of inoculation. Section B was taken at the edge of the plate closest to the inoculum. Section C contained mycelia immediately outside the edge of the plate. Section D contain the active growing mycelia (hyphal tip). HPLC was used to examine the translocation of compounds tested at these various sections A, B, C and D from two different sets of fungal inoculation. 24

J 2

Fig. 3. Schematic presentation of the direction of translocation (arrows) of isoflavonoids on agar dishes. In set 1, the mycelial plug (H) was placed outside of the small plate amended with the isoflavonoid. In another set 2, the mycelial plug was placed inside of the small plate amended with the plant compound. Samples A, B, C and D were taken. 25

RESULTS AMD DISCUSSION

Toxicity o£ solvents on P. sojae growth Five different chemicals were used to dissolve the different metabolites tested: Tween 20, DMF, DMSO, glycerol and methanol. Of all possible additives tested, methanol was the least inhibitory to the three races of P. sojae used in the experiment {Fig. 4}. Tween 20 inhibited P. sojae growth for all isolates at almost all concentrations tested (Fig. 5) . Glycerol, DMF and DMSO were moderately inhibitory (Figs. 6 to 8) . Methanol was inhibitory only at concentrations of 5 % or greater. We thus choose methanol as the solvent for our bioassays. As a safeguard, however, to remove residual methanol, plates were incubated at 60°C for 8 h before their use in the bioassays.

Toxicity of Plant Natural Compounds on P. sojae Some of the diverse group of flavonoids tested in these studies are either constitutive or induced soybean metabolites. For example, isoflavones, like genistein and daidzein, are the major metabolites present in roots, cotyledons, hypocotyls and primary leaf tissues of soybeans as conjugated forms like 6"-malonyl-7-O-B-D-glycosyl and 7-O-lJ- D-glycosyl isoflavones (Fig. 2) . Coumestrol, an isoflavone-derived pterocarpan, is a major metabolite induced after infection in various soybean organs. Its structure is biogenetically related to the flavonoids but with additional complexities. Flavonols like quercetin and kaempferol are present in older soybean leaves. Rutin (a quercetin glucoside) and isoquercetrin are esterified with sugars and their structures are analogous to the glycosylated conjugates of daidzein and genistein present in all soybean organs (T.L. Graham et al. 1991b; Robinson 1991). Some of the flavonoids examined in these studies were selected on the basis of their structural similarities to soybean metabolites, for example. 26

? 120 E W •E 1 a£

I 40 IE

0 2 4 6 B D ays

M ethanol 120

BO

40

0 0 2 4 6 B D ays

E £ 100 £ 5O Uw • Control ev -•— 5 ul 50 -»— 20 ul o 320 ul oe

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Fig. 4. Growth curves of P. sojae races 1, 3 and 12 in the presence of methanol (control) at different concentrations. 27

120

SO

4 0

0 0 2 4 6 8 Day*

120 E E, £ i O 8 0 aL. «

40

0 0 2 4 6 8 Day*

120 Control 8 ul 32 ul % 128 ul o 8 0 o 512 ul «

40 c

0 Y ■ i ■ i ■— 0 2 4 6 B Day*

Fig. 5. Growth curves of P. sojae races 1, 3 and 12 in the presence of Tween 20 at different concentrations. 28

Glycsrol 120

80

40

0 0 2 4 6 8 Day*

120 £ £

a a ao cc

0 2 4 6 8 Day*

Glycarol

120 Conlrol S ul 32 ul 128 ul 80

40

0 0 2 4 6 e Day*

Fig. 6. Growth curves of P. sojae races 1, 3 and 12 in the presence of glycerol at different concentrations. 29

120 E E BO a

«o a (E

0 2 4 6 e D ays

120 E E

£ i e (3 to a au E 40

0 2 4 6 8 Day*

DMF 120 Control 5 ul 20 ul 80 ul 80 320 01

40

0 0 2 4 6 B D ays

Fig. 7. Growth curves of P. sojae races 1, 3 and 12 in the presence of N,N-dimethyl formamide (DMF) at different concentrations. 30

OMSO 120

8 0

40

0 0 2 4 6 8

OMSO

120

80

40

0 0 2 4 86 Day* —J# DMSO 120

80 Conlrol 5 ul 2 0 ul 40 80 ul 320 ul

0 0 2 4 6 8 D ays

Fig. 8. Growth curves o£ P. aojae races 1, 3 and 12 in the presence of dimethyl sulfoxide (DMSO) at different concentrations. 31 biochanin A and £ormononetin are related to genistein and daidzein, respectively (Fig. 2) . Both metabolites are present in chickpea roots (Harborne, 1980). Others were selected based on their common occurrence in legumes, (isorhamnetin), or in many types of plants (apigenin, chrysin, quercetin and kaempferol). Although all compounds tested can be designated as flavonoids, we will differentiate flavonoids from isoflavonoids because of their importance in soybean tissues as defense molecules. The group of isoflavonoids examined included genistein, daidzein and their conjugated forms; formononetin, biochanin A and coumestrol.

The results of "in vitro" bioassays for phytoalexin antifungal activity depend on many factors, such as the specific bioassay used, media composition and pH, growth stage of the fungus, inoculum size and incubation temperature. Morris et al. (1991) used Vg juice to assess the potential role of isoflavones from soybean flour in age related resistance. V8 juice agar has a very wide range of secondary products from a number of different plant genera. For this reason, it is not a well defined medium for studies on phytoalexin toxicity. We choose lima bean agar as the basal medium to perform the toxicity assays, since it contains only a very low level of secondary products and yet provides the other nutrients and cofactors required for excellent growth of P. sojae (T.L. Graham, 1990).

Concentrations ranging from 15 fiM to 500 fiK were selected to test the different compounds. This range is well within the levels of these metabolites seen constitutively or in induced responses in soybean. Glyceollin, for example has an ED50 of 100-150 nmoles/ml to inhibit P. sojae growth "in vitro" (Yoshikawa et al. 1978, Bhattacharyya and Ward, 1985) even though it can accumulate to levels as high as 1400 nmoles/g of tissue (T.L. Graham et al. 1990). Daidzein is synthesized or released within 24 hr after incompatible infections. Total, genistein accumulates up to 3000 nmoles/g of tissue during the same conditions (T.L. Graham and 32

H.Y. Graham, 1990).

In another example, the accumulation of phaseollin, the main phytoalexin produced by French bean hypocotyls, was calculated to be around 3000 jxg per cm within dead cells. This concentration is almost 300 times the minimum concentration needed to restrict the germ tube growth of all races of Colletotrichum lindemuthianum in vitro (Bailey and Deverall, 1971). Thus the range of 15 to 500 nM used in our experiments falls well below that expected for accumulation of these compounds "in vivo" and yet encompasses the established ED50 values for accepted phytoalexins.

Effects on Mycelial Growth In these studies, differential effects of the plant natural compounds on P. sojae were observed. Figs. 9 to 20 show the effects of the different compounds studied on P. sojae (isolates of races 1, 3 and 12). However, no differences in sensitivity to the compounds were observed between isolates with the exception of quercetin on race 12 isolate. While moderately inhibitory to other isolates, quercetin did not inhibit the growth of race 12 isolate at all (Fig. 18). Three groups of compounds can be distinguished based on their level of toxicity . The most toxic compounds were highly inhibitory of P. sojae mycelial growth at concentrations higher than 125 /xM (eg. the isoflavonoids genistein, biochanin A, formononetin and coumestrol (Figs. 9 to 12); and the flavonoids apigenin, chrysin, naringenin, isorhamnetin and isoquercetrin (the quercetin 3-0-fi-D-glucoside) (Figs. 13 to 17) . A second group, includes quercetin which caused a short lag period, but was strongly inhibitory only at concentrations higher than 200 /xM (Fig. 18). The last group included the compounds like kaempferol (a flavonol) and rutin (a complex glucosyl conjugate of quercetin) which were not inhibitory to the different P. sojae isolates (Figs. 19 and 20). 33

8C GENISTEIN

60

40

20

0 0 2 4 6 8

8 0

GENISTEIN

60

40

20

0 0 2 4 6 8

80

GENISTEIN

60 * ok. 0 40 C4 15 uM 31.23 uM 62.5 uM 20 125 uM 200 UM 500 uM 0 0 2 4 6 8 Day*

Fig. 9, Growth curves of P. sojae races 1, 3 and 12 in the presence of the isoflavonoid, genistein at different concentrations. 34

BG BIOCHANIN A

60

40

20

0 0 2 4 6 8 Day*

60 BIOCHANIN A

60

40

20

0 0 2 4 6 B

80 BIOCHANIN A

60

i o 40 -■— 15 uM 31.23 uM 62.5 uM 20 125 uM 200 uM ■O— 500 uM 0 0 2 4 6 e Day*

Fig. 10. Growth curves of P. sojae races 1, 3 and 12 in the presence of the isoflavonoid, biochanin A at different concentrations. 35

80 FORMONONETIN

E E 60

<5

o ■ IE 20

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FORMONONETIN

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FORMONONETIN

£E 60

* g 15 uM -a— 31.23 uM 6 2 .5 uM 125 uM «a 2 00 uM a ■Q— 500 uM IE

0 2 4 6 8 Day*

Fig. 11. Growth curves of P. aojae races 1, 3 and 12 in the presence of the isoflavonoid, formononetin at different concentrations. 36

80 COUMESTROL

60

40

20

0 0 2 4 6 8

80 COUMESTROL

60

40

20

0 0 2 4 6 8 Day*

80 COUMESTROL

60 i o 15 UM 40 31.23 uM 62.5 uM 125 uM o 2 00 uM ■ 20 IE 500 uM

0 0 2 4 6 a Daya

Fig. 12. Growth curves of P. sojae races 1, 3 and 12 in the presence of the isoflavonoid, coumestrol at different concentrations. 37

80

APIGENIN

60 I S u 40

*u IE 20

0 0 2 4 6 e Day*

BO APIGENIN

E £ 60 -C I s (3

a ■o IE 20

0 0 2 4 6 8 Day*

80

APIGENIN

60 i o 40 15 uM 31.25 UM 62.5 uM 20 125 uM 250 uM 500 uM 0 0 2 4 6 8 Day*

Fig. 13. Growth curves of P. sojae races 1, 3 and 12 in the presence of the flavonoid, apigenin at different concentrations. 38

CHRYSIN BO

60

40

20

0 0 2 A 6 e Day*

8 0 CHRYSIN

"e E n o* O 40 a um IE

o 2 4 6 e Day*

80

15 uM 60 31.23 uM 62.5 uM 125 uM

40 2 00 uM 500 uM

20

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Fig. 14. Growth curves of P. sojae races 1, 3 and 12 in the presence of the flavonoid, chrysin at different concentrations. 39

100

8C

c. *O 60

40 u■ C 20

0 4 Days

100

NARINGENIN eE s. * U>o o n « mo CC

0 2 4 6 0 Day*

100

NARINGENIN 80

60 •m bo o 15 UM 31.23 uM 40 62.5 uM 125 uM 200 uM 20 -O- 500 uM

0 4 Day*

Fig. 15. Growth curves of P. sojae races 1, 3 and 12 in the presence of the flavonoid, naringenin at different concentrations. 40

BO ISORHAMNETIN

60

o

■o cr 20

0 0 2 4 6 8 Day*

8 0 ISORHAMNETIN

6 0

so 4C

20

0 0 2 4 6 8 D ays

80 ISORHAMNETIN

60 io )5uM O 40 31.23 uM 62.5 uM o 125 uM ■ 20 200 UM

0 0 2 4 6 8 Day*

Fig. 16. Growth curves of P. sojae races 1, 3 and 12 in the presence of the flavonoid, isorhamnetin at different concentrations. 41

BO ISOQUERCETRIN

E E c i ko <3 40 * ■u C

0 2 4 6 B D ays

ISOQUERCETRIN

E S. 6 0

O 4 0 n u0 cc■ ■a

JX. 0 2 4 6 B D ays

BO ISOQUERCETRIN

60

15 uM 40 31.23 uM 62.5 uM 125 uM 20 200 uM 500 uM

0 0 2 4 B 8 D ays

Fig. 17. Growth curves of P. sojae races 1, 3 and 12 in the presence of the flavonoid, isoquercetrin at different concentrations. 42

100 QUERCETIN

E 80 £ * 60 WO

r- O DC 20

0 2 4 6 8 Day*

100

QUERCETIN 80

60

20

0

Day*

100

QUERCETIN ? E

a -■ — 15 uM CM 31.25 uM 40 62.5 UM O• 125 uM m 250 uM a 20 -D-- 500 uM

0 2 4 6 e Days

Fig. 18. Growth curves of P. sojae races 1, 3 and 12 in the presence of the flavonoid, quercetin at different concentrations. 43

KAEMPFEROL ? E

6 0 r!> (9

■ ■o DC

0 2 4 6 8 Day*

KAEMPFEROL 100 ? 6

O m 40 o• a E 20

0 2 4 6 8 Day*

120

KAEMPFEROL 100

80

15 UM 60 31.23 uM 62.5 uM 40 125 uM 200 uM 20 500 uM

0 0 2 4 6 8 Day*

Fig. 19. Growth curves of P. aojae races 1, 3 and 12 in the presence of the flavonoid, kaenpferol at different concentrations. 44

100

RUTIN E E

6 0 O

*■ 40 u■ 20

0 2 4 6 8 Days

100

RUTIN E E £ » 2 a m u E■

0 2 4 6 e Day*

too RUTIN E E 80 W £ So 60 k 15 uM (5 31.25 UM 40 62.5 uM 125 uM 250 uM 20 500 uM

0 0 2 4 6 8 Day a

Fig. 20. Growth curves of P. sojae races 1, 3 and 12 in the presence of the flavonoid, rutin at different concentrations. 45

I n some cases the fungal growth was inhibited (eg. quercetin) at the beginning of its growth on the amended agar. However, after the compound was metabolized by the funguB (see below) the growth was aggressive and the pathogen was able to colonize the whole plate by the end of the experiment.

Macroscopic effects of plant metabolites on P. aojae colony growth Table 1 summarizes some of the effects on colony growth of the different plant natural compounds on P. sojae (isolates of races 1, 3 and 12). Cottony growth, aerial mycelia and branching were also very common at higher concentrations. Thin and scanty mycelial growth was observed in treatments containing naringenin and isoquercetrin at concentrations higher than 200 ftM and kaempferol at concentrations higher than 125 ftM. Sectoring was observed with all the isoflavonoids and with the flavonoids, chrysin, isorhamnetin, naringenin and quercetin. Clearing, evidenced by a translucent halo was present in cultures amended with isorhamnetin (200 ftM) whereas a brownish halo was observed for isoquercetrin, quercetin and rutin (Plate II) . In treatments containing genistein conjugates, the mycelium branched in a manner giving the colony a feather-like appearance (Plate III). The enzymes involved in the hydrolysis of conjugates appear to be present in the hyphal tips (discussed below). Genistein conjugates are less toxic than genistein itself. This fact makes us believe that the rapid release of genistein at the hyphal tips may produce the branching or feather-like appearance of the colony. Table 1. Effects of Different Plant Natural Compounds on P.sojae growth "in culture". Colony Plant Compound Color Description Presence of Halo Isoflavonoids Genistein hyaline Cottony growth, sectoring No Coumestrol hyaline fl If II No Biochanin A hyaline n n n No Formononetin hyaline n n n No Flavonoids Apigenin yellowish Cottony growth No Chrysin hyaline Cottony growth, sectoring No Isoquercetrin brownish Thin & scanty growth Yes Isorhamnetin yellowish Cottony growth, sectoring Yes Kaempferol hyaline Thin & scanty growth No Naringenin hyaline " " ", sectoring No Quercetin brownish Cottony growth, sectoring Yes Rutin brownish Cottony growth Yes Control hyaline Normal growth No 47

Plate II Clearing of the agar just ahead of the mycelial front was evidence of the metabolism of several compounds. Plate III Treatments containing MGG caused frequent mycelial branching giving to the colony a feather like appearance. 49

Were these compounds fungicidal or fungistatic?

Flavonoids and isoflavonoids both affect P. sojae growth and development and their effects are non-race specific. All the compounds tested were fungistatic. Some compounds inhibited mycelial growth for a short period of time, but after a lag period the mycelium was able to grow

(eg. quercetin). Another group of compounds apparently did not affect the mycelial growth on agar media but their effects on hyphal morphology as well as in sporangia and oogonia development were observed (eg. rutin). This response is quite similar to that of R. aolani when it is exposed to the bean phytoalexin, phaseollin. Despite the phytoalexin damage to the fungal cell membrane, the fungus can subsequently recover its growth or adapt easily to the antibiotic (Van Etten et al. 1982). In another study with the pea phytoalexin, maackiain and its pathogen, Stemphylium botryosum Wallr., the germ tube growth was inhibited for a few hours. However, its elongation later resumed, and growth rapidly reached the rate of the controls (Higgins, 197 8). The authors suggest that germ tube growth was inhibited by a transient effect on the hyphal tip. They concluded that the resumption of growth of S. botryosum germ tubes was not the result of conversion of maackiain to non-inhibitory products. The resumed fungal growth was not caused by metabolism of the phytoalexin but by another unknown mechanism . Their conclusions were based on two observations, 1} germinated spores exposed to small additions of maackiain to induce its possible degradation were equally inhibited by later additions of maackiain and 2) germ tubes of Helminthosporium carbonum Link., a fungus that cannot metabolize maackiain, were similarly affected by the phytoalexin.

Microscopic effects of plant metabolites on P. soj'ae Effects on hyphal, sporangial and oogonial morphology as well as

development were observed over a period of time. Cytoplasmic granulation. 50 cellular disintegration, hyphal swelling and twisting were among the common effects of plant metabolites on fungal growth (Plate IV). Also an increase or decrease in number of sporangia and oogonia was seen in some cases. Table 2 summarizes the effects of the different plant metabolites on sporangial and oogonial production as well as the pigmentation of oogonia and oospores for the various isolates examined. It was very interesting to observe some isolates differences in the effects of the different compounds on the pathogen reproductive structures. Some compounds like quercetin, isoquercetrin and chrysin have -like activity (Wong, 1975) and stimulated the production of oogonia. Some isoflavonoids (genistein), also appeared to stimulate oogonium formation (Table 2) . This is a very interesting observation because P. sojae has sterol requirements for optimal growth and completion of its sexual cycle. This might be a mechanism of survival of the pathogen in nature, especially in response to soybean defense metabolites. Other compounds caused brownish coloration on oospore walls (eg. rutin, quercetin and isoquercetrin). It was interesting to observe that just the female reproductive structure incorporated this compounds into the cell wall whereas the male structure, the antheridium, remained hyaline (Plate IV).

Sporangium formation was inhibited by 60% of the compounds tested at concentrations higher than 200 fM (Table 2). This was of interest because sporangium formation leads to the production of zoospores. Zoospores are the most important means of dissemination of P. sojae, especially under flooding conditions in soils (Carlile, 1983). Further studies are needed to clarify these observations. 51

Plate IV The various flavonoids and isoflavonoids had distinctive effects on P. sojae morphology. The most frequent observations made regarding microscopic abnormalities in P. sojae growth and development are shown. A. Cytoplasmic granulation (arrows), B. hyphal branching , C. hyphal twisting and swelling, D. differential pigmentation of the oogonium (oog) compared to the male structure, the antheridium (anth), which was hyaline. Table 2. Microscopic effects of different plant natural compounds on P. sojae.

Compound Oogonium1' Sporangium -/ Ri R12 color Ri Ri Rli color isoflavonoids Genistein 7 3 1 hyaline --- hyaline Coumestrol 3 6 1 yellowish - + + hyaline Biochanin A 4 3 4 hyaline - - - hyaline Formononetin 1 3 4 hyaline -- + hyaline Flavonoids Apigenin 3 9 1 yellowish + + - hyaline Chrysin 4 8 8 hyaline - -- hyaline Isoquercetrin 8 12 5 brownish - - - hyaline Isorhamnetin 1 1 5 yellowish -- - hyaline Kaempferol 1 4 3 hyaline - + - hyaline Naringenin 0 2 0 hyaline - -- hyaline Quercetin 7 8 1 brownish - + + hyaline Rutin 2 5 1 brownish - + + hyaline Control 4 5 5 hyaline + + + hyaline 1/ Oogonia number were obtained using the mean of the observations taken from 3 different samples. 2/ Formation of P. sojae sporangia on plates: + ; absence of sporangia on plates: 53

Metabolism o£ plant metabolites by P. sojae 1. C o l o n y growth: Due to the incorporation o£ the compounds tested some agar plates turned yellowish to brownish in color. Compounds

like quercetin, isoquercetrin and rutin gave the agar a brownish color. Apigenin and isorhamnetin turned it yellowish. The rest o£ the compounds tested did not cause any color changes in the agar plates. The first indication of metabolism of a particular compound by P. sojae was the appearance of a halo around the mycelial plug. This was an important observation in some compounds like quercetin, isoquercetrin, isorhamnetin and rutin. The clearing of the agar media ahead of the mycelial tip made us hypothesize that the compound was broken down (Plate II) . This suggests the induction of specific enzymes at the hyphal tips might be involved in the metabolism by P. sojae of the some of the compounds tested. This observation has precedence in pisatin metabolism by N. haematococca, in which a turbid agar medium containing pisatin showed a clearing zone in advance of the growing mycelium (Van Etten et al., 1982). 2. HPLC Analysis: Plant phenolics are part of a dynamic equilibrium where there is a continuous synthesis and degradation (turnover) of these compounds "in planta". Studies involving plant pathogenesis have to take into account the many factors relating to turnover of a given compound both by the host and the pathogen to clarify the whole picture of what is occurring at the infection front. Although much work has been done on turnover at the host infection front (T.L. Graham et al., 1990; T.L. Graham and M.7. Graham, 1991), little has been done on possible fungal metabolism of plant phenolics by P. sojae. The metabolism of plant antimicrobial compounds is well documented (VanEtten et al. 1982; Barz, 1970; Barz et al. 1970; Ingham, 1982).

Fungal pathogens are able to demethylate, reduce, hydrate and oxidize plant phytoalexins in order to establish themselves inside plant tissues

(Van Etten et al., 1982 and Butt, 1985). Also bacteria are able to 54 metabolize plant antimicrobial compounds (Barz, 1970; Barz et al. 1985; Hildebrand and Caesar, 1989}. The metabolism of phytoalexins usually yields products that are less toxic to the pathogen. However, this is not always the case. As an example, the release of genistein from its conjugated forms leads to greater toxicity (T.L. Graham and M.Y. Graham, 1990) .

Valuable evidence of metabolism of various compounds by P. sojae (isolates of races 1, 3, and 12) was shown by HPLC analyses. Some of the plant compounds tested were partially or completely metabolized by the fungus regardless of the P. sojae isolate (Figs. 21 to 33). In order to facilitate the discussion on the metabolism of the different compounds studied, two major groups of compounds were differentiated: glycosides and aglycones, based on the presence or absence of sugar molecules in their chemical structure. A. Metabolism of glycosides 1. Flavonoid glycosides: Flavonoids glycosides include isoquercetrin and rutin. It was interesting to observe that P. sojae was able to completely metabolize the flavonoid glycosides, rutin and isoquercetrin into non aromatic products (Figs. 21 to 23). Rutin was completely metabolized very rapidly (2 days after inoculation) by isolates of races 1 and 12. Isolate of race 3 was also able to metabolized this compound 4 days after inoculation. Isoquercetrin was completely metabolized 4 days after inoculation for all isolates examined. Based on these data we can suggest a possible metabolic fate for the flavonoid glycosides:

Rutin — * Quercetin (metabolized further)+ Rutinose Isoquercetrin — » Quercetin (metabolized further) + Glucose

Isoquercetrin and rutin are quercetin glycosides which are commonly present in plants (Robinson, 1991), they are harmful compounds in their free forms (Harborne, 1980; Waage and Heidin, 1985). Rutin was completely i. 1 Mtbls f h faood, amfrl KEP, urei (QtJER) (ISORHAM), quercetin (KAEMP), , isorhamnetin kaempferol flavonoids, the of Metabolism 21. Fig.

CONCENTRATION (NMOLES) CONCENTRATION (NMOLES) 200 • 0 0 4 ■ 0 0 6 ■ 0 0 8 0 0 6 200 400 • 0 0 8 isoquercetrin (ISOQUER) and rutin by P. by rutin and (ISOQUER) isoquercetrin ■ KAEMP C U m ISORHAM BOCUm BOCUm ISORHAM m U C KAEMP AM CUm IOHM OCUm RUTIN m U C SO ISORHAM m U C KAEMP DAY 6 RACE 1 RACE 6 DAY DAY 2 RACE 1 RACE 2 DAY d fc >- a FLAVONOIDS FLAVONOIDS d Id a RUTIN d t d >- 2 t sojae o o z o ui t—OC< z ui H O z z s O _i 200 race 1 over a period of 8 days of growth. of days 8 of period a over 1 race 6 00 - 00 6 BOO DAY 8 RACE 1 RACE 8 DAY KAEMP KAEMP DAY 4 RACE 1 RACE 4 DAY LR ISORHAM CLER n FLAVONOIDS FLAVONOIDS SRA BOQUER ISORHAM 3 LER S3Q

RUTIN RUTIN >- ui ui 1200 DAY 2 RACE 3 01 DAY 4 RACE 3 111 1200 - O a 800 eoo

cc< K< “j 4 00 - 400 oUi I ou KAEMP CUm ISORHAM SDQUER RUTH KAEMP CLm ISORHAM SDQUER RUTIN FLAVONOIDS FLAVONOIDS

1200 DAY 6 RACE 3 DAY 8 RACE 3 09 Ui O 3 Q z 800 oz p < QCH z 4 00 ■ > oUi z ou i ■ l r KAEMP CUm ISORHAM BOQUER RUTIN KAEMP ISORHAM RUTIN FLAVONOIDS FLAVONOIDS

Fig. 22, Metabolism of the flavonoids, kaempferol (KAEMP), quercetin (QtJER), isorhamnetin (ISORHAM), isoquercetrin (ISOQUER) and rutin by P. sojae race 3 over a period of 8 days of growth.

ui a\ ■A H- ia

to CONCENTRATION (NMOLES) w CONCENTRATION (NMOLES) 8 8 -L. _ j_ g COMRETRY METABOLIZED a 5 ? a Ma* c o m r e t r y metabolized

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COMaETRY METABOLIZED I

CONCENTRATION (NMOLES) CONCENTRATION (NMOLES)

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Fig. 24. Metabolic fate of the flavonoid, quercetin (from Harborne, 1980) 59 metabolized by all isolates of P. sojae studied (Figs. 21 to 23). The cleavage of the rutinose group is probably the first reaction that occurs, since a metabolite with a retention time very similar to quercetin was detected, (Appendix A). After the cleavage of the rutinose group it could be possible that quercetin is further metabolized as shown in Fig. 24. Quercetin is apparently broken down into phloroglucinol carboxylic acid and protocatechuic acid in fungi by a peroxidase known as a quercetinase (Harborne, 1980 and Barz et al. 1985). Later on these metabolites are broken down further to C02 that goes to the atmosphere. Plants also break down quercetin to protocatechuic acid and phloroglucinol (Harborne, 1980) . In addition, it is well known that rutin is very efficiently cleaved by gastrointestinal microflora to yield aglycones in mammals (Barz et al. 1985) .

2. Isoflavonoid glycosides: The glycosylated conjugates of the isoflavones genistein and daidzein include HGG and genistin, and MGD and daidzin, respectively (Fig. 2). Isoflavone conjugates were hydrolyzed to their respective isoflavoneB very rapidly by P. sojae isolate of race 3. Fig. 25 shows the accumulation of genistein and daidzein throughout the experiment. However, when we look at the data in more detail, very interesting results were observed with the isoflavonoid glycosides compared to the flavonoid glycosides (discussed above). When the glycosides of genistein and daidzein were analyzed for their metabolism by P. sojae, many major peaks were detected (Fig. 26 and 27; Appendix A) . For instance, P. sojae rapidly metabolized the isoflavonoid conjugate MGD after inoculation, with the subsequent accumulation of daidzin and daidzein. Daidzin was further broken down into daidzein at the end of the experiment (Fig. 26). MGG was also rapidly metabolized (Fig. 27). MGG hydrolysis lead to the accumulation of genistin, genistein and another Fig. 25. Metabolism of the isoflavone conjugates, MGG and MGD, by P. by MGD, and MGG conjugates, isoflavone the of Metabolism 25. Fig. CONCENTRATION (NMOLES) CONCENTRATION (NMOLES) 2000 000 - 0 0 30 1000 2000 - 0 0 0 3 1000 - - - - DAY 6 RACE 3 RACE 6 DAY DAY 2 RACE 3 RACE 2 DAY G GNSE MGD GENtSTEN MGG G EtTN MGD GENtSTEN MGG over a period of 8 days of growth. of days 8 of period a over 4 OJGATES CONJUG CONJUGATES ~r DAJOZEN DAIDZEN u o 1000 z g Z H E < 0 0 30 2 u o 1000 ui < h- 2 OJ 01

0 0 0 2 3000 2000 - - i — DAY 4 RACE 3 RACE 4 DAY DAY 8 RACE 3 RACE 8 DAY MGG k BJSQ MGD GBiJtSTQN BITN MGD GB4ISTQN CONJUGATES CONJUGATES sojae rc 3 race DAIDZEIN DAIDZEIN os o CONCENTRATION (NMOLES) CONCENTRATION (NMOLES) H- <0 8 § § § i K> — i— ■ • ■ O*

I . COMPLETELY METABOLIZED

U H- (P a H-n B 0 0 Pi mi i 0 rr § ' OOMPLETaY METABOLIZED Mi ET z oo ID H- aip. oa *«iffl MlH pi 0 < Ml uo IP ID

rr3-^. 3 * C IP pi CONCENTRATION (NMOLES) CONCENTRATION (NMOLES) (Drr

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0la Vj. Pi n M 0PI ID

T9 . 7 2 . g i F CONCENTRATION (NMOLES) CONCENTRATION (NMOLES) 200 300 200 400 600 - . L I. G G^SW GYIBE GENSTHN GLYCITBNE Ge^lSTW MGG G GJSM LCTN GENtSTEN GLYCTTSNE GBJtSTM MGG Metabolism of the isoflavone conjugates, MGG and genistin, by by genistin, and MGG growth. of days 8 conjugates, of period isoflavone a the over of Metabolism MGG METABOLISM MGG DAY 6 RACE 3 RACE 6 DAY MGG METABOLISM MGG DAY 2 RACE 3 RACE 2 DAY l

J o ut o u I — LU tn cc < 200 Z 2 O 300 to o - o to MGG MGG ■r 1 MGG METABOLISM MGG DAY 4 RACE 3 RACE 4 DAY &tTN LCTN GENtSTEtN GLYCTTENE G<STlN 01TN LCTN GBvltSTEN GLYCTTENE G0I1ST1N MGG METABOLISM MGG DAY 8 RACE 3 RACE 8 DAY P.

sojae race race sojae 3 to S3

related isoflavonoid tentatively identified as glyciteine (T.L. Graham, unpublished), 4 days after inoculation with P. sojae isolate of race 3 (Fig. 27) . These studies showed that all isoflavone glycosides were metabolized to their aglycones.

Based on these data we can suggest a possible metabolic fate for the isoflavonoid glycosides:

MGG — » Genistin + Malonate MGD — » Daidzin + Malonate Genistin — » Genistein + Glucose Daidzin — ► Daidzein + Glucose

The release of genistein from MGG and genistin leads to a toxic product that can be fatal to the pathogen. Examination of the effects of the isoflavonoids, daidzein and genistein, showed that daidzein is essentially non-toxic whereas genistein is a potent inhibitor of P. sojae growth (T.L. Graham et al, 1989). Genistein is very little metabolized by P. sojae (Fig. 28, 29 and 30) and its role seems to be complementary to the glyceollin as an antibiotic whereas daidzein may play a role as a direct glyceollin precursor (T.L. Graham and M.Y. Graham, 1990). The reason why P. sojae hydrolyzed the conjugates to produce daidzein and genistein is unknown. Incompatible responses result in the accumulation of higher concentrations of conjugated genistein that are hydrolyzed to their isoflavones at the mycelial front in avery shorttime to detain fungal invasion (T.L. Graham et al., 1990). We can hypothesize that the fungus has glycosidases that metabolize the conjugates by removing the glucose molecules. Glycosylated compounds offer an easily available carbon source for pathogens. The production of glycosidases may play a primary role in the metabolism of these sugar containing compounds. Glycosidases might be commonly produced by plant pathogens to metabolize phenolic compounds which rarely occur in their free state in plants. Usually they are present in conjugated forms not only with sugars but with other groups (Barz et al. 1985; Hrazdina and Wagner, 1985). 64

We suspect that the fungal hyphal tips might be directly involved in the metabolism of isoflavonoid conjugates. However, this attempt to metabolize these conjugates compounds by the pathogen ironically favors the plant. In incompatible infected soybean tissues, nearly complete hydrolysis and release of daidzein and genistein from their conjugates occurs very early, a process which may lead to pathogen containment. Our work suggests that be P. sojae might be involved in the hydrolysis of conjugates at the infection front. However, a composite response of pathogen and plant glycosidases might be responsible for the accumulation of the isoflavones genistein and daidzein. The host glycosidases are currently under investigation (M.C. Hsieh and T.L. Graham, personal communication). In the compatible interaction slow release of glyceollin as well as isoflavone conjugates at the infection front have been reported (Keen and Yoshikawa, 1983; T.L. Graham and M.Y. Graham, 1991). In this case the pathogen will be able to invade the plant tissues more easily due to the spatial and temporal evasion of these toxic molecules.

B. Metabolism of aglycones 1. Isoflavonoid aglycones: In terms of aglycones, isoflavonoid compounds like coumestrol, genistein, formononetin and biochanin A were very little metabolized (Figs. 28 to 30) . This same group of compounds showed the highest toxicity to P. sojae (Figs. 9 to 12). The toxicity of the most biologically active compounds shows a nearly perfect inverse relationship to the rapidity and degree of metabolism. The partial metabolism observed with biochanin A and formononetin suggest possible cleavage of the methyl group in these compounds due to the action of a demethylase. This enzyme might be involved in their partial degradation. HPLC data make us suspect that this is the case based on the presence of other metabolites in the chromatograms• For instance, peaks co-eluting with genistein appear after biochanin A partial metabolism and peaks co-eluting daidzein occurs formononetin partial metabolism 1500 1500 DAY 2 RACE 1 DAY 4 RACE 1 o s * T000 - 5 . iooo zo H < < c Kc Z UJ 500 - ui 500 o o zo o CJ

GEM BIO A FORM COUM G&J BIO A FORM COUM ISOFLAVONOIDS ISOFLAVONOIDS

1500 1 5 0 0 - DAY 6 RACE 1 CO to DAY 6 RACE 1 UJ Ui

1000 1000

< < s IE z 1- III 500 z 500 o oui o z o o

GBJ BIO A FORM COJM Geg BIO A FORM COJM ISOFLAVONOIDS ISOFLAVONOIDS

Fig * 28. Metabolism of the isoflavonoids, genistein (GEN), coumestrol (COUM), biochanin A (BIO A) and formononetin (FORM) by P. sojae race 1 over a period of 8 days of growth.

<3\ UI □ > < O m CONCENTRATION (NMOLES) CONCENTRATION (NMOLES) £ s o s o o> ID CONCENTRATION (NMOLES) CONCENTRATION (NMOLES)

Fig. 29. Metabolism of the isoflavonoids, genistein (GEN), coumestrol (COUM), biochanin A (BIO A) and formononetin (FORM) by P. sojae race 3 over a period of 8 days of growth. 99 i. 0 Mtbls f h iolvnis gnsen GN, omsrl CU) bohnnA (BIOA) A biochanin (COUM), coumestrol (GEN), genistein isoflavonoids, the of Metabolism 30. Fig.

CONCENTRATION (NMOLES) CONCENTRATION (NMOLES) 1000 1000 and formononetin (FORM) by by (FORM) formononetin and 0 BOA OM OM 0 BOA OM OOUM FOFM BIO A G0I COUM FORM BIO A 001 SFAOOD ISOFLAVONOIDS ISOFLAVONOIDS . sojae P. OCUM race 12 over a period of 8 days of growth. of days 8 of aperiod over 12 race 1500 ISOFLAVONOIDS OOUM

(Appendix A ) .

It was interesting to observe that coumestrol was completely degraded by P. sojae isolate of race 1; however, it was not metabolized and toxic to the other two races. This suggests some isolate specificity regarding the metabolism of this specific pterocarpenoid compound which is structurally related to the glyceollins. Further experiments are needed to confirm this observation. 2. Flavonoid aglycones: Once again the relative toxicity of the most biologically active compounds shows a nearly perfect inverse relationship to the rapidity and degree of metabolism. More toxic flavonoids like apigenin, chrysin, naringenin were only weakly or partially metabolized (Fig. 31 to 33} . Non-toxic flavonoids like quercetin, isorhamnetin and kaempferol were completely metabolized 6 days after inoculation by almost all P. sojae isolates tested (Figs. 21 to 23).

The metabolic fate of the flavonoid, quercetin haB been elucidated in various organisms and was discussed in detail above (Fig. 24) (Harborne, 1980, Barz et al., 1985 and Stafford, 1990). Data obtained from our HPLC analysis is consistent with such metabolism. Complete degradation of quercetin-related compounds like kaempferol, rutin, isorhamnetin and isoquercetrin was also observed. He can hypothesize that these compounds may follow the same fate. All of them are related to quercetin, with variations only in the occurrence of a sugar or a methyl group. In the case of rutin and isoquercetrin, quercetin can be produced after the cleavage of the sugar group by a glycosidase. Quercetin can be produced from isorhamnetin by removal of a methyl group. The HPLC data support this hypothesis since a peak corresponding to quercetin is an intermediate metabolite of isorhamnetin (Appendix A).

Table III summarizes the possible mechanisms involved in the metabolism of the different flavonoids and isoflavonoids. The combined action of oxidases (e.g. demethylases), glycosidases, esterases and Fig. 1 Mtbls f h faood, pgnn AI) crsn CR) n aignn MRN by (MARIN) naringenin and (CHRY) chrysin (APIG), apigenin flavonoids, the of Metabolism 31. CONCENTRATION (NMOLES) CONCENTRATION (NMOLES) 2000 1000 - 1500 1500 500 500 . sojae P. - PG CHRY APIG APIG race 1 over a period of 8 days of growth. of days 8 of aperiod 1over race DAY 2 RACE 1 RACE 2 DAY D A Y S RACE 1 RACE S Y A D FLAVONOIDS FLAVONOIDS CHRY ii m NARIN NARIN

\prr%- c H A ■ o □ a b t- CL o o u o s < O 2000 1000 1500 - 1500 1200 - APIG PG CHRY APIG DAY 8 RACE 1 RACE 8 DAY DAY 4 RACE 1 RACE 4 DAY FLAVONOIDS FLAVONOIDS CHRY NARIN NARIN u> CTl 2000 2000 DAY 2 RACE 3 DAY 4 RACE 3 9) ■ A uj a b O 1500 O 1500 - a c □ D

si 1000 -

500 - ou

APIG CHRY NARIN APIG CHRY NARIN FLAVONOIDS FLAVONOIDS

1500 DAY 8 RACE 3 DAY 8 RACE 3

APIG CHRY NARIN APIG CHRY NARIN FLAVONOIDS FLAVONOIDS

Fig. 32. Metabolism of the flavonoids, apigenin (APIG), chrysin (CHRY) and naringenin (NARIN) by P. sojae race 3 over a period of 8 days of growth.

■o o i. 3 Mtbls f h faood, pgnn AI) crsn CR) n aignn NRN by (NARIN) naringenin and (CHRY) chrysin (APIG), apigenin flavonoids, the of Metabolism 33, Fig.

CONCENTRATION (NMOLES) CONCENTRATION (NMOLES) 1500 1000 1200 500 P. sojae race 12 over a period of 8 days of growth. of days 8 of aperiod over 12 race sojae APIG PG CHRY APIG DAY 6 RACE 12 RACE 6 DAY DAY 2 RACE 12 RACE 2 DAY FLAVONOIDS FLAVONOIDS CHRY NARIN NAP NAP IN o z o uJ 2 Z 3 <0 (J < t- o O _l UJ (J o z ui (C < UJ to o 000 0 10 1500 0 - 500 - APIG APIG DAY 4 RACE 12 RACE 4 DAY DAY B R AC E 12 E AC R B DAY FLAVONOIDS FLAVONOIDS CHRY CHRY NARIN NARIN 72 peroxidases is suggested. Some compounds like isorhamnetin can be degraded by two different mechanisms. However, our data based on HPLC analysis support demethoxylation and subsequent degradation of kaempferol. It seems more likely to occur because kaempferol's peak and no quercetin appeared in the chromatograms of all isolates of P. sojae tested. Demethylation of aromatic esters can be catalyzed by monooxygenases. Kaempferol might be further metabolized by peroxidases. However, further studies are needed in order to confirm these mechanisms in the degradation of these different compounds by fungi.

Translocation of the Different Plant Natural compounds by P. sojae The appearance of plant compounds throughout fungal mycelium has been used as proof that the compound can be translocated by the fungus. In our work, the first clue that some compounds were taken up by the fungus was the brownish pigmentation of oogonium and oospores of P. sojae (Plate IV). The translocation assays used by others did not work well with P. sojae. The density of mycelial growth was not great enough to detect uptake at any concentrations of the metabolites teBted. Table 3. Possible mechanisms suggested in the chemical conversion of plant natural compounds by P. sojae. 1) Oxidases : Demethy1ation Biochanin A — » Genistein + Methyl Group Formononetin — » Daidzein + Methyl Group Isorhamnetin ► Quercetin + Methyl Group 2) Oxidases : Demethoxylation Isorhamnetin » Kaempferol+ Methoxy Group

3) Esterases : Kalonyl Group Cleavage MGG: 6"-malonyl-7-0-B-D-glycosyl genistein — *■ Genistin + Malonate MGD: 6"-malonyl-7-0-J5-D-glucosyl daidzein — » Daidzin + Malonate

4) Glycosidases : Glycosyl Group Cleavage Rutin — * Quercetin + Rutinose Isoquercetrin » Quercetin + Glucose Genistin » Genistein + Glucose Daidzin — * Daidzein + Glucose

5} Quercetinase (Peroxidase): ring cleavage Quercetin — * Protocatechuic acid + Phloroglucinol carboxylic acid 74

CONCLUSIONS

Resistance to P. sojae in soybeans correlates well with the accumulation of the glyceollins (Keen and Yoshikawa, 1983) and other flavonoid compounds like the phytoalexin, coumestrol, and conjugates of genistein and daidzein in infected tissues (Darvill and Albersheim, 1984; Bbel, 1986; Graham et al. 1990 and T.L. Graham and M. Y. Graham, 1990). However, very little information on the dynamic interaction of P. sojae with soybean flavonoids has been known. This is the first study that looks in detail at both sides of the interaction; 1) the effects on P. sojae of the multiple constitutive and induced compounds that occur during natural infection processes and that might contribute to pathogen containment in plants and 2) the active role that P. sojae might play in the metabolism of some of these compounds. Toxicity of Plant Natural Compounds: This study showed that flavonoids and isoflavonoids have various effects on P. sojae, ranging from direct toxicity, to growth inhibition, to more subtle effects on hyphal morphology and sporangial and oogonial development. All isoflavonoids tested and the flavonoids naringenin and isorhamnetin were inhibitory at concentrations higher than 125 fiM. Certain compounds, like chrysin and apigenin, had severe effects on older hyphae at a cellular level, but little effect on hyphal extension and therefore on hyphal growth. This suggests that the detrimental effects of these compounds takes time to express or that hyphal tips may be somewhat more resistant to certain toxic effects. Other compounds were not inhibitory at all (e.g. rutin, kaempferol) Of particular importance, we have demonstrated that genistein is toxic to P. sojae, and we suggest that it may play a complementary role to

that of the glyceollins. In addition, genistein possesses steroid like activity (Wong, 1975) which is particularly interesting because the fungus has sterol requirement for optimal growth and completion of the sexual 75

cycle. Further studies are needed in order to investigate the mechanisms of toxicity of genistein.

Thus, the accumulation of multiple compounds in infected tissues like the phytoalexins, coumestrol and glyceollin, as well as constitutive and induced isoflavonoids might work together to contribute to the containment of the pathogen in a very localized fashion. Metabolism of Plant Natural Compounds by P. sojae: The presence of an active resistance mechanism in P. sojae against certain of the flavonoids is suggested by their rapid metabolism at the mycelial front. The relative toxicity of the biologically active compounds shows an inverse relationship to the rapidity and degree of metabolism. The rapid and complete degradation of the various flavonoids and isoflavonoids tested suggests that P. sojae may possess very effective degradation pathways, possibly including the rapid and coordinate action of oxidases

(e.g. demethylases), esterases, glycosidases and peroxidases. The nature and specificity of the various enzymes remains to be studied. However, based on our observations we can speculate that the pathogen might possess them in strategic locations for defense, most likely at the hyphal tip. CHAPTER II

Phytophthora sojae Catalaee

INTRODUCTION

Phytopathogenic fungi produce different kinds of enzymes that enable them to colonize and invade plant tissues. In order to understand the interaction between plants and pathogens it is important to look at the battery of enzymes that are produced by pathogenic organisms. The production of some of these enzymes (e.g. pectolytic enzymes, catalase), can make a parasite strongly pathogenic or virulent and able to cause severe disease in a particular host or over a host range. Not only the occurrence, but the rapidity and the amount of enzymeB produced are important virulence traits critical in the survival and further colonization of plant tissues by pathogens. Phytophthora sojae, for example, consists of over 30 races that cause disease in its host plant, soybean (Schmitthenner, 1985). Very little is known on P. sojae pathogenic traits which are crucial in its survival during compatible interactions. The pathogen must face the plant defensive reaction, which includes the production of phytoalexins (coumestrol and glyceollin) and isoflavonoids (genistein and daidzein) in order to multiply and colonize plant tissue (Yoshikawa, 1978; Keen and Yoshikawa, 1983; T.L. Graham, 1989; T.L. Graham, 1991; T.L. Graham et al. 1990; T.L. Graham and M.Y. Graham, 1990). The role of the different flavonoids and isoflavonoids in plant defense has been discussed in detail in Chapter I.

76 77

In addition to the isoflavonoids and flavonoids, soybean reacts to P. sojae defense elicitor (cell wall glucan) through the induction of a rapid and massive accumulation of phenolic polymers in soybean cotyledon cell walls immediately proximal to the point of elicitation (M.Y. Graham and T.L. Graham, 1991). The deposition of phenolic polymers is over 10 times more than that in wounded controls within 4 h of elicitor treatment. It reaches its maximum by 24 h, whereas in controls accumulation of phenolic polymer has just begun by that time. It has been shown that isoflavone conjugates start to accumulate in elicited tissues at 8 h and glyceollin at 12 h (M.Y. Graham and T.L. Graham, 1991) . The deposition of wall bound phenolics in soybean tissues is greater compared to the glyceollin and isoflavones responses. The responses include covalently linked phenolics like lignin, suberin as well as simple esterified coumaric and ferulic acid monomers (M.Y. Graham and T.L. Graham, 1991) . In soybean cotyledons the deposition of phenolic polymers seems to correlate with the induction of a specific group of anionic peroxidases (M.Y. Graham and T.L. Graham, 1992) . It is known that lignin is a polymerization product of hydroxycinnamyl alcohols (Legrand, 1983; Ride, 1983). This reaction is initiated by a peroxidase and hydrogen peroxide. Peroxidases appear to be directly involved in the last steps of phenolic polymer deposition in the plant cell wall at the expense of H202 that acts as an oxidizing agent. The origin of the H202 needed for the polymerization remains unknown. Studies in this regard have been done by Stich and Ebermann (1984). In nature it appears that peroxide formation is determined by the nature and amounts of various cofactors present in the lignifying tissue (e.g. Mn*2).

Another part of the defensive strategy of plants to limit invading pathogens is the induction of oxidative stress by producing H202 and active oxygen species like: superoxide anion (02-), hydroxy radical (-OH) and singlet oxygen (0*) (Sutherland, 1991). Of all the biologically occurring radicals, *0H is by far the most reactive and the strongest oxidizing 7a agent known. H202, while not a radical, is stable and has been shown to exhibit toxicity.

H202 has been shown to be produced by animals as well as plant cells as part of their defense mechanisms. H202 at a very low concentration (26 /tM) prevented the germination of Peronospora tabacina Adam sporangiospores and Colletotrlchum lagenarium (Pass.) Ell. and Halst. and Cladoaporium cucumericum Ell. and Arth. conidia in vitro (Peng and Kuc, 1992) . With the phytopathogenic bacteria, like Pseudomonas ayringae van Hall, it is known that one of the initial responses of invaded plant tissue during compatible and incompatible interactions is the production of H202 at elevated amounts (Klotz and Hutcheson, 1992). Peroxide can penetrate easily through membranes and affect a variety of cellular processes. The levels of superoxide and hydroxyl radicals also increase during incompatible interactions in which disease fails to develop. It has been shown that the ability of P. ayringae to multiply in plant tissue can be enhanced by infiltration of the plant tissue with agents that reduce the free active oxygen radicals. Various enzymes and chemicals have been implicated in the reduction of oxygen radicals. Enzymes like catalase and superoxide dismutase and chemical agents like iron-chelating and hydroxyl radical-quenching agents are some examples.

As stated above, the capacity of phytopathogenic fungi and bacteria to multiply in plant tissue may be due in part to the ability of these organisms to detoxify peroxide. Catalase seems to be the most important peroxide-consuming enzyme at physiological peroxide concentrations . It was one of the first enzymes described and has been the object of intense research for nearly a century. Since then, a considerable amount of biochemical information has been accumulated (Deisseroth and Dounce, 197 0 and Schonbaum and Chance, 1976). It is a widely occurring enzyme present primarily in the intracellular space in mammalian and non-mammalian cells. Because it converts peroxide to water and 02 very rapidly it is also sometimes referred to as a hydroperoxidase. It also plays a role in the 79 oxidation of H+ donors like phenolic compounds. Table 4 summarizes the major reactions of catalase.

Catalase is able to catalyze the destruction of peroxide by forming a primary complex between peroxide and the iron of its hematin prosthetic group. The importance of the heme complex of the prosthetic group will be discussed further in regards to its relationship with virulence in some phytopathogenic bacteria.

Table 4. Major Functions of Catalase 1) Degrades peroxide to oxygen and water

■ —► 2E]0 + O] 2) Oxidation of H donors; eg. phenols ROOH + AH} --•* HjO + ROH + A

Catalase has been shown to play an important part in the survival of a variety of organisms (bacteria, yeast, fungi and plants) and has been regard as a virulent trait in pathogens. In animals, it is well known that in response to mammalian pathogens, leukocytes are able to produce peroxide and free radicals at higher levels (Baehner et al.1982). Many mammalian pathogenic bacteria are resistant to these compounds by decomposing peroxide through the action of catalases (Baehner et al. 1982). Various examples of the importance of catalase in bacterial organisms are: 1) protection from the toxic and mutagenic effects of peroxide by catalase induction has been reported in the bacteria Salmonella typhimurium (Loeffler) Castellani and ChalmerB (Winguist et al., 1984). 2) Escherichia coli (Migula) Castellani & Chalmers produces two different catalases. Catalase deficient mutants of E. coli are 50-60 fold more sensitive to killing by 1 mH H202 than their parental strains, confirming the role of catalase as a key protective enzyme against peroxide (Doewen, 1984) . 3) Multiple catalases have been reported in 80

Bacillus subtilis (Ehrenburg) Conn., by the same author. Significant increase in catalase activity was observed during the transition from

stationary phase to the onset of sporulation (Loewen and Switala, 1987). In fungi some work has been done, but to a lesser extent. Multiple catalases have been reported in Neurospora crassa (Turian & Bianchi) and Aspergillus niger Micheli ex LinK (Chary and Natvig, 1989 and Witteveen et al. 1992). In the specific case of A. niger, four distinct catalases were identified. Two are constitutively present, one intracellular (cat I) and the other one located in the cell wall (cat II). Another two are induced under conditions when glucose oxidase is formed (cat III and IV). Strong induction of these later catalases, which were localized at the site of peroxide formation, in the cell wall, makes it very likely that this enzyme playB a major role in the breakdown of peroxide formed by glucose oxidase.

Catalase has also been associated with senescence in plant and animal tissues. In plants the senescence of leaves is accompanied by an increase in peroxide concentration and decrease in peroxide destruction. Catalase activity seems to be a reliable indicator of wheat leaf senescence (Khan and Choudhuri, 1988). The enzyme activity gradually decreased in all leaves of the wheat plant during reproductive development. However peroxidase activity later increased up to the grain maturation stage and thereafter decreased. Catalases have been more thoroughly studied in some plant species. Three maize catalases that respond to cercosporin-containing fungal extracts have been recently reported (Williamson and Scandalios, 1993). The catalases respond differentially to applied Cercospora Fres. toxin (cercosporin), and the response varies with developmental stage. They concluded that catalase accumulates in response to the toxin but also to other fungal compounds present in all extracts. In tobacco leaves, multiple forms of catalase have been identified. They appear at specific stages of seedling development and undergo changes 81

in distribution during leaf maturation. These forms can be separated by elution at different pH values from chromatofocusing columns and possess differences in thermal stability (Havir and HcHale, 1987). In addition to catalyzing the direct decomposition of peroxide, some catalases can utilize peroxide for peroxidative oxidation of various substrates including short-chain alcohols such as methanol and ethanol (Deisseroth and Dounce, 1970) . All forms of catalase in tobacco show peroxidative oxidation of alcohols as well as peroxide degrading activities. Also, catalase has been used extensively as a general peroxisome marker. De Felipe et al. (1988), in studies with the plant, Lolium rigidum Gaud, found that catalase was mainly present in the fibrous component of microbodies and peroxidase was mainly localized in the cell walls, tonoplast and chloroplasts. When plants were treated with herbicide, an increase in C02 photorespiration of those plants was observed. This gave rise to the generation of peroxide and thereafter to catalase activity in the peroxisomes. Based on the evidence stated above it is well established that this particular enzyme is important in many physiological processes and seems to be present in all aerobic organisms. In terms of its importance as a pathogenicity and/or virulent trait, recently, very powerful evidence has been found that linked catalase with the Systemic Acquired Resistance (SAR) response in plants (Chen et al. 1993, Jones, 1994) . SAR is a defense response that is induced locally by pathogens but that spreads systemically to protect the entire plant. Chen and his coworkers (1993) have shown that salicylic acid might act as a natural signal molecule activating plant defense responses inducing systemic acquired resistance. Surprisingly, some members of the catalase family of enzymes share high sequence identity with a salicylic acid binding protein. This suggests that some member of the catalase family might be the salicylic acid binding protein receptor and it explains why salicylic acid can protect plants against pathogens that produce catalase to overcome the plant 82

resistant response. It has been shown that salicylic acid seems to bind to catalase inhibiting its enzyme activity in vitro by 80% (Chen et al. 1993). Consequently, the increase of reactive oxygen species, especially

H20j concentrations, might activate the expression of genes related to defense in the plants. Even though we are beginning to better understand plant resistance mechanisms recently, the importance of catalase as a pathogenicity and/or virulent trait has been extensively studied for a long time, especially with bacterial pathogens of the genus Pseudomonas. In experiments performed by Digat in 1972, with avirulent and virulent strains of Pseudomonas solanacearum (Smith) Smith, they proposed that catalase activity appeared to be involved in bacterial virulence in tomato plants. The higher the enzyme activity, the higher the virulence. Their results indicate that only the bacterial cells with high levels of catalase activity can establish themselves in the xylem of tomato plants and multiply to such an extent that they may damage the vascular system. "In vitro" experiments revealed that avirulent mutants were able to recover catalase activity when provided with preformed prosthetic heme (Fe) group. When avirulent mutants were grown on a heme-containing medium, they regained not only their catalase activity but partial virulence also (Goodman et al. 1986). Apparently the enzyme is part of a complex interaction that controls virulence in this pathogen. It appears that an increase in catalase activity engendered by virulent strains reduces the efficacy of plant natural defenses.

It has been known for many years that the bacterial pathogen, Pseudomonas syringae, and recently, the saprophytic bacteria, Pseudomonas putida (Trevisan) Migula, have catalase activity . It has been shown that catalase activity increases after contact with legume roots. P. putida catalase isoenzymes appear to play a role in the survival of this bacterium in the rhizosphere (Katsuwon and Anderson, 1990) . In the case of halo blight of bean, caused by Pseudomonas Byringae pv. phaseolicola 83

[(Burkholder) Young et al.] on Phaaeolus vulgaris L.(common bean), it was suggested that peroxidases were involved in the resistant response. Increased activities of peroxidases caused by infection were detected earlier in the resistant varieties and the activity was higher during the first days after infection compared to susceptible varieties. The appearance of a high activity of a bacterial catalase in infected tissues suggested that this enzyme might influence host peroxide metabolism. The significance of the enzyme could be that it might play a role in the critical first stage of the establishment of the pathogen by competing with peroxidaBe of the host for peroxide substrates (Rudolph and Stahmann, 1964). This suppression of peroxidase activity can be explained based on the antimicrobial phenolics produced by plant defense reactions. Reaction 1 shows an example of how specifically catalase may act in the destruction of the substrate for the peroxidase enzymes.

Reaction 1

catalase I------«■ Ha0 + O jt

AH2 + H2Oj ---- ► 2H20 + A (in its reduced form)

A may represent a phenolic compound in its non-reduced form.

Due to the action of catalase, phenolic compounds are maintained in their reduced, hence antimicrobially less active form rather than as antimicrobially active quinones . Catalase activity has also been studied with fungal pathogens but to a lesser extent. In studies with the rust, Uromyces phaseoli (Pers.) Wint. and its host P. vulgaris, a general increase in activity was stimulated 2-3 days after inoculation in susceptible and resistant cultivars. However its activity decreased 4-5 days after inoculation when necrotic lesions differentiate in the incompatible response but not in the compatible. In the compatible response (susceptible cultivar), a drop in catalase activity was observed after 9 days of inoculation and during 84 uredospores differentiation favoring the fungal infection (Hontalbini and Buonaurio, 1989) . The authors suggested that this might be related to cell collapse occurring during the later stages of infection. Recently, the specific activity of various enzyme associated with defense against toxic species was measured for various soil pathogens. Enzymes such as superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase were studied in fungi belonging to diverse taxonomic groups. Various levels of catalase activity were reported depending on the fungal species and age of culture (Cohem and Teomi, 1992).

Peroxide production in soybean tissues haB recently received some attention. Hydrogen peroxide metabolism in soybean embryonic axes at the onset of germination was studied by Puntarulo et al. (1988). They concluded that peroxide production was an early event and soybean catalase appears to be the predominant peroxide consuming activity in early seedlings tissues. In another study, it was shown that soybean cells produce hydrogen peroxide and 02- as an early response to P. sojae wall glucan elicitor (Lindner et al. 1988). Using the same system, Apostol et al. (1989) investigated the rapid stimulation of H202 production and its use by cell wall peroxidases to oxidize susceptible substrates. They also propose a role for the peroxidase and the peroxide burst in the overall disease resistance mechanism. Exogenous H202 stimulates formation of the soybean phytoalexin glyceollin, whereas inhibition of elicitor-stimulated phytoalexin production was observed after addition of catalase among other inhibitors. The occurrence of active oxygen species as well as peroxide at the infection front in P. sojae-soybean interactions is very likely, given the evidence reported. It is very probable that P. sojae will be exposed to the deleterious effects of these active oxygen species on soybean root surfaces because it is a primarily a root pathogen. We hypothesized that P. sojae must have mechanisms to overcome active oxygen Bpecies and their effects and that the production of catalase may be one of them. Catalase 85

has been reported in Phytophthora spp. In 1986, Powell and Bracker did a cytochemical study to determine the presence of catalase in Phytophthora palmivora (Butler) Butler zoospores. They showed that more than one organelle contained catalase activity. However to our knowledge no cytochemical or any other investigation on the role that P. aojae catalase may play in P. aojae virulence or pathogenicity has been made. The major objectives of these studies were to examine 1) first, the constitutive levels of catalase activity in P. aojae races (isolates) 2) second, the effects of peroxide and plant phenolics in the induction of P. sojae catalase activity and relate this to its importance in P. aojae pathogenicity. 86

MATERIALS AND METHODS

Response o£ P. sojae colonies to the addition o£ HjOj To estimate the hydrogen peroxide splitting activity (catalase activity), different H203 concentrations (from 0.1 mM - 100 mM) were prepared. Four day old P. sojae colonies, growing on lima bean agar, were used for the assay. The colonies were flooded and the effervescence upon the addition of peroxide was recorded. The results were taken immediately after flooding and 30 min after peroxide addition. The generation of such effervescence,due to 02 evolution, is a common preliminary assay for catalase (Digat, 1972).

Preparation of extracts from P. sojae Several isolates of P. sojae were evaluated for constitutive catalase levels. Four P. sojae isolates (races 1, 3, 4, and 12) were grown in 3 1 flasks containing 500 ml of lima bean broth. Flasks were inoculated with 10 mycelial plugs and mycelium was harvested 4 days after inoculation. To harvest the mycelium, a nylon filter was used and two portions were obtained; mycelial and filtrate portions. After harvesting, the mycelium was weighed and stored an -80°C. The filtrate was divided in two portions and stored at -20 °C. For enzyme extraction, ice cold 50 mM phosphate buffer (pH = 7.8, with 0.1 mM EDTA) was added to the mycelium in a 3:1 (v/w) ratio (w/v). A sonic cell disruptor (Sonicator Model 350, Branson Sonic Power, Inc.) was used to extract the enzymes. Using a microtip, ten pulses were applied (duty cycle = 45%, output controls 2) . This was repeated 5-6 times, cooling the extract on an ice bath between cycles. After sonication, samples were centrifuged at 15000 x g (Biofuge, Rotor no. C-1710-20, rmox=

7.9 cm) for 5 min. Supernatant of the mycelial extracts was kept at -80°C for enzyme assay. 87

One hundred ml of the filtrate was lyophilized (Lyophilizer Model Dura-dry Microprocessor from FTS Systems, Inc.) and 1 ml of extraction buffer was added to redissolve the powder. Samples were then centrifuged at 15000 x g (Biofuge, Rotor no. C-1710-20, r = 7.9 cm) for 2 min before enzyme assay. Enzyme activity was measured using a spectrophotometer (Shimadzu Model UV 360) as reported by Abei (1984) . The change in absorbance per unit time was the measure of the catalase activity. Culture filtrates and supernatants of mycelial extracts were assayed for enzyme activity. For each 1 ml of substrate, 20 fil of enzyme was used for the assay. Two repetitions per sample for each P. sojae isolate were done. Protein concentration was determined by the method of Lowry (1951) as modified by Ohnishi and Barr (1978) with bovine serum albumin as standard.

Constitutive levels of catalase activity in P. sojae

isolates during culture growth In order to determine the constitutive levels of catalase produced by P. sojae during different phases of culture growth, P. sojae isolate of race 3 was grown in 125 ml lima bean broth for 9 days. The top of ten 4mm agar plugs of P. sojae mycelium were used to inoculate 500 ml liquid media. Samples were taken every day from day 1 to 3, then every other day from days 5 to 9. Enzyme activity as well as mycelium dry weight were determined at each Btage of growth. Extracts were prepared as noted above. Three portions were examined for catalase activity: mycelial (supernatant and pellet), and filtrate. To extract enzymes from mycelia pellets, 1 ml of phosphate buffer was added. After vortexing the pellet was ground up with drill tip and then spun down again. All supernantant was removed from the pellet and enzyme activity was measured, pellet was discarded. To detect enzyme activity from filtrates 20 and 50 fil were examined from fresh cultures. Enzyme activity was determined aB stated above. 88

Induction of catalase activity in P. aojae isolate (race 3)

using H^Oj and plant phenolic compounds

Five different compounds were selected to detect induced enzyme activity in lima bean broth. These were peroxide, the soybean isoflavones, daidzein and genistein, and the chickpea isoflavones, formononetin and biochanin A. All compounds tested were used at concentrations below toxic levels. Stock solutions of peroxide at 10 mM and all isoflavones at 50 jiM were prepared. One 1 flasks containing 100 ml of lima bean broth were inoculated with 10 agar plugs (4 mm) of P. aojae isolate (race 3). Four days after inoculation, 2.5 ml of the compound stock solution was added per flask. Twenty four hrs after, the mycelium and filtrate were harvested and enzyme activity was determined following the protocol described above. Three replicates for each treatment were done. In order to monitor the toxicity of the compounds, fungal mycelia was examined microscopically. Also at each time point, extra samples from the supernatant from mycelial extracts and the culture filtrate were analyzed by HPLC to detect the levels and possible metabolism of the phenolic compounds in the samples.

Induction of catalase activity in P. aojae isolate (race 3)

using different H2Oj concentrations This set of experiments were performed in order to examine the peroxide dose response in the enzyme induction at two different time points with two P. aojae isolates (races, 3 and 12) . Four different peroxide concentrations were used (0.1, 1, 10 and 100 mM) and water was used as control. Four and 5 day old P. aojae lima bean agar cultures were covered with the different peroxide concentrations for 1 h. Four repetitions per treatment were done. The peroxide solution was then drained from the cultures and 12 h later, P. aojae hyphae were sampled. Four sections A, B, C and D (refer to the first chapter Plate I) were taken to detect enzyme activity at different ages of fungal growth. Ten 89 plugs per each section were taken and the top portions o£ the agar plugs {containing the mycelium) were then harvested. These sections of the plugs were transferred to Eppendorf tubes containing 200 n 1 of buffer and ground with a drill tip. The samples were then sonicated with a cell disruptor as described above. Enzyme activity was determined as stated above.

Visualization of catalase activity on polyacrylamide gels Polyacrylamide gels were run to detect catalase activity in P. sojae mycelial extracts as well as in the filtrates. Catalase activity was induced using H202 and the isoflavonoids, genistein, daidzein, formononetin and biochanin A (described in the section above) . To prepare the gels (pH = 8.5), stock solutions of the following were used: a) 30% acrylamide: 29 g acrylamide (Sigma, St. Louis,MO) + 1 g methylene bis acrylamide (Sigma) in 100 ml of deionized double distilled water (DDDW). b) lower buffer: 18.5 g Tris base (Trizma, Sigma, St. Louis,MO) in 100 ml of DDDW (pH = 8.8). c) upper buffer; 5.98 g Tris base in 100 ml of DDDW (pH = 6.8) d) sample buffer: upper buffer + 80% glycerol (Sigma) in a 1:1 ratio e) running buffer: 12 g Tris base + 57.6 g glycine (Sigma) in 1 1 of DDDW. f) 10% Ammonium persulfate (APS) (Sigma) solution was prepared daily.

A Biorad vertical gel apparatus was used for the electrophoresis. Upper gel (stacking gel) and lower gel at 5 and 7% concentrations were prepared using the following solutions: Lower gel 7.5% 5% Upper gel Lower buffer 12 ml 12 ml Upper buffer 1.2 ml 30% polyacrylamide 12 ml 8 ml 1.2 ml

DDDW 24 ml 28 ml 7.6 ml 10% APS 200 n 1 200 nl 50 pil

TEMED (Sigma) 48 48 pil 10 fil 90

Lower gel was aspirated under vacuumed for 10 min in order to remove air bubbles and then poured in the gel apparatus. Lower gel was 16 x 2 0 cm with a 4 cm stacking (total gel size was 20 x 20 cm). Two 1 of running buffer were prepared diluting the stock solution (refer to e above) 1:20

(v/v) with DDDW. Six or 12 /xl of sample buffer were added to 17 or 35 /il of the sample, respectively. One ^il of bromophenol blue (Sigma) was added to each sample as tracking dye (Sigma, St. Louis,HO). The electrophoresis was carried out at 4°C and the voltage applied was constant 80 V for the upper gel and constant 185 V (Power supplier, Hoefer Scientific Instrument, San Francisco, Ca) when the samples reached the lower gel. After the run, gels were stained for an hour with 0.1% of cooumassie brilliant blue prepared in a 40:10% solution of methanol and acetic acid, respectively. After that, the gels were left overnight in a 0.005% cooumasie brilliant blue and destained twice with methanol:acetic acid solution (40:10%) until protein bands appeared against a clear background. Gels were dried using a Slab Gel Dryer (Biorad, Model 438) for examination and interpretation. 91

RESULTS

Response of P. sojae colonies to the addition of Hj02 The activity of the various P. sojae isolates (races 3 and 12) was visually demonstrated by effervescence upon addition of peroxide solutions to the colonies. Table 5 shows the results of this preliminary test. Both P. sojae isolates tested showed no effervescence immediately after the addition of peroxide at the various concentrations used. Effervescence was recorded after 30 min of the peroxide addition with both P. sojae isolates. Strong effervescence was observed at 100 mM peroxide concentration at this time. Based on our visual test, P. sojae isolate of race 3 appeared to respond to slightly lower peroxide concentrations than isolate of race 12.

Table 5. Effect of different peroxide concentration on P. sojae catalase activity. Catalase Activity!7 Isolate H202 Concentration (mM) 0 min 30 min Race 3 0.0 0 0 0.1 0 0 1.0 0 + 10.0 0 + + 100.0 0 +++ 12 0.0 0 0 0.1 0 0 1.0 0 0 10.0 0 ++ 100.0 0 +++

1/ 0: no effervescence; +: weak effervescence; ++: effervescence; +++: strong effervescence. The catalase activity was estimated based on bubble formation after peroxide addition on P. sojae colonies grown on lima bean agar.

Constitutive levels of catalase in different P. sojae isolates Table 6 shows the results of the experiments on constitutive levels of catalase activity 4 days after inoculation for various P. sojae isolates. The higheBt constitutive level of catalase activity (55 U/mg of 92 protein) was obtained with the isolate of race 3 in the supernatant of mycelial extracts. The other three isolates showed much lower activities ranging from 8 to 12 U/mg of protein. No activity was obtained in any of the filtrates of the isolates examined.

Table 6. Constitutive levels of catalase in different P. sojae isolates.

Isolates Enzyme Activity -1 (Races) (U/mg of protein) 1 8.30 3 55.00 4 10.35 12 12.10 1/ 4 days after inoculation

Catalase activity in P. sojae isolate of race 3 during different

phases of culture growth Table 7 shows constitutive levels of catalase activity measured in P. sojae isolate of race 3 over a period of 9 dayB growth in liquid culture. The highest enzyme activity in mycelial extracts was observed the third day after inoculation (73.11 U/mg of protein). However, other than the low value at day 2, activity remained steady throughout the experiment, ranging from 58 to 73 U/mg of protein, suggesting that age of culture had little effect on activity. No activity was observed in the filtrates whereas some activity seems to occur in the pellets. However, these results are questionable due to the difficulties in suspending the pellet portions in phosphate buffer before the assay, and may be due to interference of particulate material with the spectrophotometric assay. 93

Table 7. Constitutive levels of catalase in P. aojae isolate of race 3 over a period of 9 days of growth.

Enzyme Activity {U/mg of protein) Mycelial Extraction Days Filtrate Supernatant Pellet 1 0.00 57.86 45.55 2 0.00 20.59 23.68 3 0.00 73.11 27.83 5 0.00 60.83 31.08 7 0.64 60.92 245.30 9 0.06 59.23 19.63

Induction of catalase activity in P. aojae isolate of race 3 1) Induction using H2Oj and legume isoflavonoids as inducers Catalase activity was examined 24 h after its induction with various phenolic compounds. The reason for this was to give the fungus a period of time to synthesize the enzyme so that we were able to measure its activity. In typical enzyme induction experiments an increase of many fold (10 to 100 times) in enzyme activity is to be expected. The results, shown in Table 8 suggest that, as seen above, the mycelial supernatants seem to have higher enzyme activity compared to the extremely low activity in the culture filtrates. The highest enzyme activity waB detected when the soybean isoflavonoid, daidzein, was the inducer. A slight increase in enzyme activity was observed with peroxide and the other phenolic compounds tested compared to the control. However, the enzyme activity was not dramatically increased over that of the controls suggesting that P. aojae catalase is not effectively induced under these conditions by any treatment. HPLC data is summarized in Table 9. Almost all phenolic compounds tested remained associated with the pellet fractions, with the exception of formononetin. We observed that particularly high concentrations of the compounds genistein and biochanin A appeared in this fraction whereas low 94 concentration of compounds were present in the supernatant of mycelial extracts and filtrate fractions. This is very interesting and may reflect binding of these compounds to the fungal cell wall. As noted above, this is the normal site of catalase accumulation. Why we see so little induction despite the surface binding of the inducers remains unclear. Microscopic observations of the hyphae and other fungal structures were taken to examine the toxicity of the different compounds after their addition. In summary, no major cytological changes were observed in the treated mycelium compared to the control. The only exception was that genistein seemed to promote the production of female reproductive structures, the oogonia.

2) Induction using different concentrations Due to the lack of induction of catalase by phenolics and the normally used levels of peroxide, various H202 concentrations ranging from 0.1 to 100 mM were evaluated as P. sojae catalase inducers. This time P. sojae (races 3 and 12) colonies were flooded with H20a for 1 h and Bamples were taken to evaluate enzyme activity. No enzyme activity appeared to be induced with the different peroxide concentrations used in this set of experiments with P. sojae isolates of races 3 and 12.

Polyacrylamide gels to detect catalase induction Polyacrylamide gels were run to detect any protein induced by peroxide and or any of the phenolic compounds from legumes tested. Even though 7 bands appeared clearly on gels, we were not able to distinguish any new bands in the gels of treated samples vs. controls. 95

Table 8. Induced catalase activity in P. sojae isolate o£ race 3 using peroxide and plant phenolic compounds. Enzyme Activity^ Compounds-7 Supernatant-7 Filtrates Control 21.40 0.45 Peroxide 32.84 0.59 Daidzein 41.97 0.36 Genistein 23.28 0.29 Biochanin A 28.06 0.32 Formononetin 31.89 0.37 1/ 4 Days After Inoculation 2/ Final compound concentrations per treatment: peroxide, 0.01 pM; genistein, daidzein, biochanin A and formononetin, 1.25 fiM. 3/ Supernatant from mycelial extracts

Table 9. Detection of different phenolic compounds used to induced catalase activity in 3 different P. sojae fraction using HPLC analysis.

INDUCERS-7

Race 3 Fractions CK h 2o 2 G D BF

Supernatant^7 - - +a' + + + Filtrate - - + + + + + Pellet-7 - - +++ + +++ + + + + - 1/ Inducers: G = genistein, D = daidzein, B = biochanin A, F = formononetin and CK = control. 2/ Supernatant of mycelial extracts; pellets from mycelial extracts 3/ - : no compound detected; + :< 100 nmoles; ++:< 1,000 nmoles; +++:< 10,000 nmoles and ++++:< 100,000 nmoles of phenolic compound detected. 96

DISCUSSION

Constitutive Levels of Catalase in P. sojae Isolates It is well established that biocontrol agents as well as pathogens need to protect themselves against defense mechanisms of plants. Plant roots can produce activated 03 species and superoxide anion (Katsuwon and Anderson, 1989). This can cause membrane damage, mutagenesis, altered metabolism and necrosis. Recently the importance of catalase has been stressed in the survival of pathogenic as well as saprophytic bacteria. CatalaBe activity in saprophytic fluorescent pseudomonads, like P. putida, has been detected using cell extracts and it seems to be dependent upon age culture and growth medium. Higher levels of enzyme activity (180 - 375 U/mg of protein) were obtained with media containing root wash components from 10 day old P. vulgaris plants. Similar findings were obtained with P. fluorescens Migula, where the enzyme activity ranged from 126 - 289 U/mg of protein in culture using root wash components (Katsuwon and Anderson, 1992). On the other hand, much higher catalase activity was reported from phytopathogenic bacteria. At stationary growth phases, P. syringae pv. glycinea (Coerper) catalase activity ranged from 593 - 1,141 U/mg of protein whereas for P. syringae pv. syringae it was about 681 U/mg of protein (Klotz and Hutcheson, 1992) . In the specific case of the P. syringae strains, a unique isozyme appeared to help overcome the stress caused by peroxide burst as well as from other different oxidative radicals (Katsuwon and Anderson, 1990). The importance of catalase activity in the survival of phytopathogenic as well as saprophytic bacteria is well established. However, very little information has been accumulated on the role of catalase in the establishment and development of phytopathogenic fungi. Hj02 at very low concentrations prevented the germination of sporangiospores of the fungal pathogens Peronospora tabacina and Colletotrichum lagenarium and Cladosporiurn cucumerinum conidia in vitro. 97

When catalase was added the Inhibitory effects on spore germination and disease development were abolished (Feng and Kuc, 1992).

Recently, the pattern of catalase activity of several soil pathogens was studied by Cohem and Teomi (1992) . The levels of enzyme activity were measured over a 7 day period. Enzyme activity did not change with age of the fungus culture. They divided the fungi in three different groups with low, intermediate and high catalase activity. Low catalase activity levels ranged from 12.3 to 25.9 U/mg of protein for Rhlzoctonia aolani and intermediate levels range from 52.3 to 84.3 U/mg of protein for Fusarium oxyaporum f.ap. melon!a Snyder and Hansen and Sclerotium rolfaii Sacc. High levels of catalase activity (up to 195 U/mg of protein) were reported for Rhizopus arrhizus A. Fisher and for an oomycete, Pythium aphanidermaturn (Edson) Fitzp. The highest activity reported was 340.3 U/mg of protein for Trichodeirma harzianum Rifai.

The first clue that P. aojae may have some catalase activity was the effervescence observed when H20, was added to P. aojae cultures (Table 5). That observation made us believe that catalase was constitutively present in this soybean pathogen. So we next decided to examine the constitutive levels of catalase activity in e different P. aojae isolates. When we compared our findings with Cohem and Teomi findings, we observed that P. aojae catalase activity in the isolates grown on lima bean broth for 4 days ranged from 8 to 55 U/mg of protein. Catalase activity in the mycelial extracts (Table 6) ranged from low in isolates of races 1, 4 and 12 (8 - 12 U/mg of protein) to intermediate in race 3 (55 U/mg of protein). Overall the constitutive levels of the enzyme seemed low compared with other phytopathogenic organisms. Since catalase is often dependent on growth phase, the next step was to collect data on constitutive levels of catalase activity during growth of P. sojae cultures. 98

Catalase activity in P. aojae isolate o£ race 3 during different phaseb of culture growth Enzyme production was evaluated in P. sojae isolate of race 3 over a period of 9 days of growth. Enzyme activity was measured in 3 different fractions: mycelial extracts (pellet and supernatant) and filtrates. As stated above, there was little overall effect of culture age of P. sojae on catalase activity. With the exception of a low level at 2 days (20 U/mg of protein) , it ranged from 58 - 73 U/mg of protein over the time of culture. This compares to the intermediate levels of activity observed by Cohem and Teomi (1992) in F. oxysporum Schlecht. and S. rolfsii. Since constitutive, enzyme activity was low and fairly constant through the growth curve (20 - 73 U/mg of protein), we next examined if higher enzyme activities might be induced by peroxidase or phenolic compounds.

Induction of catalase activity in P. sojae isolate of race 3 1) HjOj and legume isoflavonoids as inducers: Hydrogen peroxide as well as various phenolic compounds from the legumes soybean and chickpea were used as inducers of enzyme activity in P. sojae isolate of race 3. The various legume phenolics selected, including the soybean isoflavonoids genistein and daidzein, are produced in response to fungal invasion. They therefore represent compounds that the pathogen may encounter in plant tissues and might be direct inducers of catalase synthesis. Reaction 1 shows the importance that catalase might have in P. sojae pathogenicity and its role in the destruction of peroxidase substrates (therefore, maintaining various antimicrobial phenolic compounds in their reduced or less active forms). Although slight increases in catalase activity occurred upon addition of peroxide or soybean isoflavonoids to the lima bean media, there was no dramatic induction of enzyme activity. This is very unusual since catalase seems to be strongly inducible in many organisms including pathogens (Fridovich, 1976). 99

When we analyzed the different fungal fractions for presence of phenolic compounds, we observed that they appeared to be strongly associated with the pellet fractions. HPLC data is summarized in Table 9. We observed that especially the soybean isoflavones daidzein and genistein and biochanin A (a chickpea isoflavone structurally related to genistein) were present in these fractions. This HPLC data suddest to us that during culture the phenolic compounds became associated with the fungal cell wall. Since no appreciable induced activity was observed with supernatants of mycelial extracts and culture filtrates it seemed possible that catalase enzyme might be localized in the P. sojae cell wall. In A. niger very strong induction of catalase activity was observed with a specific catalase that seems to be localized at the site of H202 formation in the cell wall. The authors stated that this catalase probably plays a major role in the breakdown of peroxide formed by a glucose oxidase (Witteveen et al. 1992). Due to the difficulties we encountered in solubilizing catalase from P. sojae pellet portion of mycelial extracts and therefore detecting any catalase activity in this fraction, just speculations on this regard can be made and further studies are necessary to determine if catalase is present in the P. sojae cell wall.

2) Various HjOa concentrations as inducers: Due to the lack of induction by the usual levels of peroxide used, another Bet of experiments were performed using various peroxide concentrations (0.1 to 100 mM) with P. sojae isolates of races 3 and 12. This time samples from P. sojae colonies were taken 12 h after induction to be evaluate for catalase activity. Unfortunately, no significant enzyme activity was detected with any of the treatments. Stimulation of catalase synthesis upon H202 addition has been observed in some enteric bacteria. In experiments performed with Salmonella typhimurium, using H202 (80 fiM) as inducer of catalase, a lag of 10 to 15 min after H202 addition was observed before catalase synthesis 100 was detected (Finn and Condon, 1975) . However the concentrations of peroxide they used (0.1 (M to 10 mM) were lower than the concentrations we used (0.1 to 100 mM). The maximum stimulation of catalase synthesis was observed at H202 concentration of 80 pM whereas addition of 10 mM resulted in rapid lysis of the culture without increase in the specific activity of catalase.

Since under no conditions were appreciable catalase activities found in the P. sojae fractions examined, we suggest various hypothesis to explain our findings. To reconcile our observations, four hypothesis regarding this subject might be considered: 1) catalase might not play an important role in peroxide degradation in P. sojae, 2) catalase might be bound to the P. sojae cell wall and we failed to detect its activity, 3) the presence of both peroxide and inducing phenolic compounds are needed or 4) compounds other than those we examined (peroxide and the isoflavonoids) may be responsible for induction "in planta". The first hypothesis presented implies no role for catalase at all in P. aojae pathogenicity. This is entirely possible and implies that another enzyme or enzymes may be involved in decomposition of peroxide, for example a peroxidase (gluthatione peroxidase). The second hypothesis seems possible based on evidence found in the literature, specially with A. niger. The catalase enzyme is located in the cell wall of this fungus where it probably play a major role in peroxide breakdown. Its role seems to be protect glucose oxidase which is located in the cell wall from inactivation due to high concentration of peroxide.

HPLC data gathered make us believe that this could be a reasonable explanation for the high concentrations of phenolic compounds present at the pellet fractions. If any phenolic compound were involved in catalase induction along with H202, probably the best location for P. sojae catalase will be the cell wall of the fungus. The pathogen would react faster in response to plant defenses because the enzyme will be in direct contact with the toxic compound, knocking out soybean defenses. However, more 101

exhaustive methods need to be developed to address this possibility. Based on. the very limited data examined on catalase activity,

indirect evidence suggests that HjOj itself is not the direct inducer of catalaBe synthesis. This is very unusual since peroxide is a nearly universal inducer of catalase in other organisms. It is quite possible that certain specific phenolics are needed along with peroxide in order to have catalase synthesis. It is important to point out that in all of our experiments we were

evaluating enzyme activities "in vitro" rather than "in planta". In the specific case of soybean-P. sojae interaction there might be a specific soybean compound or factor, or cell environment needed for catalase induction which we did not explore. Experiments "in planta" would be necessary to answer some of these questions. It is also important to consider, however, that catalases are produced by plants as well, in different tissues and organelles or cellular locations. For example, the distribution of catalase activity in three soybean organs (leaf, cotyledon and root nodule) are different (Stegink et al.,1987). These authors found catalase as a common enzyme in all tissue extracts, detecting the highest enzyme activity in the cotyledons. Therefore, specific methods would need to be developed in order to distinguish the enzymes produced by the plant from those produced by the pathogen. Only in this way would we will be able to get a better picture of what is going on in infected vs. healthy tissues "in planta". 102

CONCLUSIONS

Respiring cells produce toxic levels of H202. Because of this toxicity, peroxide must not be allowed to accumulate. Catalases and peroxidases prevent this fatal accumulation, serving a very important role. However there are respiring organisms that lack catalase or peroxidase. Commonly, these organisms live in mixed cultures with cells that contain these enzymes or in soil which contains inorganic catalysts for the decomposition of H202 (Fridovich, 1976) . The lack of catalase can also be partially compensated by another enzyme, glutathione peroxidase. This enzyme seems to be important in scavenging H202. This enzyme is widely distributed in mammalian cells and is effective at low concentrations of peroxide (Fridovich, 1976). Some contradiction regarding it presence in plants and microorganisms seems to exist. Plants and microorganisms are apparently deficient in this enzyme system. However, the enzyme has been detected in yeast and filamentous fungi. Moreover, it has been shown that cellular concentrations of reduced gluthatione are higher in various fungi (Cohen et al., 1986). More recently, Cohem and Teomi (1992) were not able to detect this enzyme in any of the six different fungi examined. So the presence of this enzyme in fungal tissues is still an enigma. Based on the limited data that we have analyzed, catalase is constitutively present at relatively low levels in all P. sojae isolates examined. Activity is associated with the mycelial fractions. Very little activity was associated with the filtrate portions analyzed suggesting that is not secreted and remains intracellular or wall bound. From the present investigation and the literature reviewed, several hypothesis were presented to explain our findings. In summary these are: 1. Catalase activity might not play an important role in P. sojae pathogenicity. The low catalase activity obtained upon induction suggests that catalase is not important in P. sojae

pathogenicity on soybean as it is for phytopathogenic bacteria like Pseudomonas,

Catalase might be bound to P. sojae cell walls and we failed to detect its activity based on the methods used. The presence of both H202 and inducing phenolic compound are needed. Compounds other than those we examined (H202 and the isoflavones) may be responsible for induction "in planta". Chapter III

P. aojae pectolytic enzymes

INTRODUCTION

The plant cell wall is a complex structure divided into three major functional and structural regions: the middle lamella, primary and secondary wall. It is primarily composed of 3 classes of polysaccharides: pectic substances, hemicelluloses and celluloses. In addition, other non­ polysaccharide components are common, like lignin, suberin, proteins such as hydroxyproline-rlch glycoproteins (HRGP's) and extensin, ions, small molecular weight conjugates and wall esterified hydroxycinnamic acids (Bateman and Basham, 1976) . Pectic polymers predominate in the middle lamella, acting as an intercellular cement. Hemicelluloses are predominant in primary and secondary walls. They link the pectic and cellulosic fractions. Cellulose 1b the major structural component of plant secondary walls. It makes up ca. 30% of primary walls and about 40% of secondary walls (Cooper, 1983a). Various functions are played by the cell walls: they 1) provide a rigid structure for plant support, 2) comprise the major component of water conducting vessels, 3) act as intercellular cement, especially the middle lamella, 4) counteract the osmotic pressure resulting from cell contents, and 5) play a pivotal role in plant pathogenesis (Cassab and Varner, 1988 , Ride, 1983). In terms of plant-pathogen interactions, the cell wall serves several roles. The most important role is that it acts as a physical barrier to infectious agents. The majority of phytopathogenic

104 105 microorganisms must degrade cell walls either to enter and colonize tissues or to release nutrients essential to their growth and development within intercellular spaces (Karr and Albersheim, 197 0). The ability to produce cell wall degrading enzymes is present not only in parasites but in saprophytes as well. However, saprophytes appear to lack genes for basic pathogenicity (Schafer et al., 1989). Another difference is that pathogen secretion of cell wall degrading enzymes, during penetration and colonization of plant tissues, appears to be under strict control. Many of them can produce a wide range of cell wall degrading enzymes that can act on different pectic fractions under different conditions of pH and temperature. In terms of pathogenicity or virulence, this contact between the plant cell wall and pathogen cell wall degrading enzymes is probably the first molecular interaction that occurs during infection.

Enzymes that are responsible for the degradation of pectic polymers are collectively called pectolytic enzymes and they are the subject of this research. Pectolytic enzymes can be classified according to substrate specificity and mechanism and position of cleavage. Generally speaking, these enzymes are usually low molecular weight, stable, extracellular glycoproteins and can be present in multiple forms or isoenzymes (Cooper, 1983a). They are subdivided on the basis of their specificity for pectic polymers. For example, some of them are specific for esterified (methoxylated) or non-esterified polymers, known as pectin or polygalacturonic acid, respectively. Another way to subdivide them is baBed on the position of cleavage: endo or exo. Endo-enzymes attack internal regions of the chain at random whereas exo-enzymes attack at the terminal region of the chain. Some pectolytic enzymes like: polygalacturonases (PG) and pectate lyases (PL) act on polygalacturonic acid, whereas others like pectin lyase (PNL) and pectin methyl esterase (PME) act upon pectin (Fig. 34). According to its mechanisms of cleavage they are divided in hydrolases and lyases. Hydrolases cleave by 106 hydrolysis like the polygalacturonases. Lyases, like pectate and pectin lyases cleave by lysis or transelimination, which leads to an unsaturated bond (Rexova-Benkova and Markovic, 1976). Other differences between lyases and hydrolases are that the pH optima for hydrolases is acidic whereas lyases have higher pH optima (alkaline) and require divalent cations like Ca2\

It is well known that plant pathogenic bacteria and fungi produce pectolytic enzymes in culture and on isolated plant cell walls (Collmer and Keen, 1986) . In terms of the role of pectolytic enzymes in disease, it is known that necrotrophic organisms cause extensive wall degradation. This condition is found in a wide range of diseases which include damping- off, soft rots, root rot and leaf and stem lesions. In contrast to necrotrophic organisms, biotrophic fungi pectolytic enzymes must be under Btrict control (Ingram et al., 1983). In the case of obligate parasites, tissue infection may involve minimal changes in host walls. Highly localized action of enzymes is suggested based on ultrastructure studies with Bremia lactucae Regel, a biotrophic fungi which causes a downy mildew in lettuce (Ingram et al., 1983). Taking this in consideration, pectolytic enzymes might be important factors in virulence and pathogenicity. For example, partially purified enzymes can recreate tissue maceration and release oligomers from plants cell wall (Darvill et al., 1984; Davis et al., 1986; Burns, 1991). These oligomers are elicitors that induce plant defense responses, including phytoalexin accumulation in soybean (Davis et al. 1986). This suggest that pectolytic enzymes have dual and apparently opposing functions; they macerate plant tissues and trigger defense responses. This subject will be discussed in more detail further. However, the role of such enzymes in pathogenesis has not been fully established regardless of the great amount of research done on this subject. Evidence against and in favor of the importance of some of these enzymes in pathogenesis has been collected and will be presented. Of all PG PL PME PNL ! COOH •COOCH

OH OH OH

OH OH OH OH

Fig. 34. Sit# of action of different pectolytic enzymes; Pectin lyase (PNL), pectate lyase (PL), polygalacturonase (PO) and pectin methyl esterase (PMX). 107 108 pectolytic enzymes, PL and PNL have been studied most extensively. A great deal of research has been dedicated to the PL produced by plant pathogenic bacteria (Collmer and Keen, 1986; Chatterjee et al. 1991; Van Gijsegem, 1986; Yang et al. 1992). Several PL forms are induced in culture by soft rotting bacteria belonging to Erwinia spp. Deletion of the genes coding for four endo-PL isoenzymes in E. chrysanthemi Burkholder, McFadden & Dimock dramatically reduced its pathogenicity (Collmer and Keen, 1986}. Transfer of these PL genes to E. coll, which is not a plant pathogen, confer plant pathogenic ability to a certain degree (Chatterjee et al. 1991). Yang et al. (1992) studied the expression of pectolytic enzymes in

Erwinia carotovora subsp. carotovora (Jones) Bergey et al., a soft rotting bacteria. They found sequential accumulation of mRNA for exo-PL, endo-PL and endo-PG, respectively. These suggest that exo-PL reaction products activate other pectolytic enzyme genes. They hypothesized that host responses are affected by pectolytic enzymes products, regulation of these enzyme levels is iportant because they may activate host defense genes. PLs have been studied in fungi to a lesser extent. Highly virulent races of Fusarium oxysporum Schlecht. Snyd. & Hans. f.sp. ciceri (Padwick) Snyd. & Hans, have been shown to produce two PL forms in culture, and on cell wall preparations. The least virulent race has been shown to produce only one form of PL (Perez-Artes and Tena, 1990) suggesting a role for PL in F. oxysporum f. ap. ciceri virulence. On the other hand, PNL has been extensively studied in plant pathogenic fungi (Cooper and Wood, 1980; Wijesundera et al. 1984 and 1989). PNL degrades highly esterified pectin independent of the action of other pectolytic enzymes. PNLs are mostly extracellular enzymes, secreted in response to inducing events (Collmer and Keen, 1986) . PNL has been found exclusively in fungi and bacteria. For example, two formB of PNL has been detected in Colletotrichum lindemuthianum grown in culture and lesions on hypocotyls of P. vulgaris inoculated with this pathogen (Wijesundera et al., 1984 and 1989). High endo-PNL activity was 109

associated with vascular tissues of susceptible tomato cuttings infected with Verticillium albo-atrum Reinke & Berth. (Cooper and Wood, 1980). Apparently, PNL seems to play an important role in the development of some diseases, especially those related to wilting and rotting. It is

interesting to point out that endo-PNL is the only PNL form known; no exo forms have been detected (Burns, 1991) . In adittion, PNLs have not been reported in higher plants, with the exception of a report in pea (Albersheim and Killias, 1962).

In the specific case of PG, its activity has been studied in many bacterial and fungal diseases. In the case of soft rotting bacteria a more clear picture of the importance of microbial PGs in addition to PLs in pathogenesis has been established (Collmer and Keen, 1986). In fungi, the detection of endo-PG in wheat root seedlings inoculated with Gaeumannomycea graminis (Sacc.) von Arx and Olivier var. tritici Walker prior to visual symptoms suggests a role in penetration and cell wall degradation (Dori et al. 1992). Development of PG and PNL deficient mutants from V. albo atrum showed less severe, late or no symptoms in tomato plants (Durrands and Cooper, 1988). However, evidence against the role of PG in pathogenesis has also been presented. Recently, a mutant of Cochliobolua carbonum Drechler, lacking endopolygalacturonase activity, produced the same symptoms in maize plants as the wild type, indicating that the PG enzyme was not required in pathogenicity (Scott-Craig et al. 1990). However, the role of PG in pathogenicity might be related to the fact that PG activity appears to be sequentially induced by the reaction products of earlier pectolytic enzymes like PLs or PNLs. Secondly its activity appears to be associated with PME in order to hydrolyze highly esterified pectins. Of all the enzymes discussed, PME's role in pathogenesis has received little attention. PME demethylates highly esterified pectins, in such a way that the pectic molecule is more susceptible to the action of other pectolytic enzymes like PG and PL. It is known that in some 110 organisms it is present in complex with PL (Rexova-Benkova and Markovic, 197 6). Overall, PME is widespread in higher plants and a great deal of information has come from studies in plants. Apparently, it is a key enzyme in plant cell growth (Sajjaanantakul and Pitifer, 1991). In terms of microbial PME, it appears to be extracellular and can be a constitutive or inducible enzyme (Forster and Rasched, 1985). The PMEs from plants and microbes often differ in pH optima and isoelectric point (pi). Plant PMEs have more alkaline requirements than fungal enzymeB. The pH optimum for PME of fungal origin is also lower than that of bacterial origin (Fogarty and Kelly, 1983). No evidence of a direct role in pathogenicity has been found to our knowledge. In summary, it appears that the importance of pectolytic enzymes importance in plant diseases varies regarding the disease examined. Some enzymes appear to be more important than others. The whole picture of the importance of pectolytic enzymes in pathogenicity or virulence is complicated by the fact that these enzymes are normally produced not only by fungi and bacteria, but by plants itselves (Bateman, 1976; Cooper, 1983b). The cell wall fulfills various structural roles and even though the cell wall is rigid, morphological changes that occur during the normal development of plants are accompanied by partial degradation of cell walls. Normal processes like elongation, abscission, germination and fruit ripening require cell wall degradation to some extent. Middle lamella disruption by plant pectolytic enzymes may resemble ultrastruetural changes induced during pathogenesis. The regulation of synthesis of microbial pectolytic enzymes in culture and in planta differs. This also complicates the picture because of the different enzymes that can act on a substrate in a particular environment and because of all the factors that can be involved in the regulation of these different enzymes: pH, temperature, cofactors, etc. Pectolytic enzymes produced in culture may be quite different from those associated with pathogenesis. Because of that, some studies in culture can Ill be misleading in attempts to explain the role of pectolytic enzymes in a disease. In culture, mono- or oligosaccharides can act as catabolite repressors of these enzymes. In a few cases, cell wall degrading enzymes are produced constitutively and are not subject to catabolite repression. An example of this is a polygalacturonase produced by Helminthosporium maydis Link and an extracellular PME produced by Phytophthora infestans (Mont.) de Bary (Cooper, 1983a; Forster and Rasched, 1985). P. sojae infection of soybean seedlings is accompanied by extense tissue maceration in compatible interactions. The symptoms that characterize this disease: damping-off, watery lesions, stem and root rot, led us to suspect that an array of pectolytic enzymes might be involved in this disease. Cells within infected tissues are separated from each other suggesting digestion of the middle lamella. Pectolytic enzymes are associated with middle lamella disruption and might play an important role in P. sojae pathogenicity and/or virulence. Another important consideration is that both induction of pectolytic enzyme synthesis in pathogens and elicitation of defense reactions in plants appear to be mediated by pectic components that can be released from the primary cell wall. Probably, such inducers are released after the action of pre-existing basal levels of these enzymes. This suggests that the success of pectolytic pathogens results from the interaction of a complex of factors that may differ from one disease to the next. In the P. sojae-soybean system, generation of host cell wall elicitor fragments (galacturonides), has been shown to enhance soybean phytoalexin, glyceollin, accumulation in response to fungal elicitors from P. sojae wall (glucans) (Nothnagel et al., 1983). These elicitors of soybean cell wall origin can also be generated by the exogenous application of pectolytic enzymes (Darvill and Albersheim, 1984, Darvill et al., 1984, Davis et al., 1986). T.L. Graham and M.Y. Graham (1991) reported several factors that seem to be involved in the accumulation of glyceollin in soybean tissues after treatment with P. sojae wall glucan in incompatible 112 interactions. These are: 1) higher concentrations of wall glucan, 2) increase in tissue age, 3) proximity of responding cells to the treated surface and 4) co-treatment with pectolytic enzymes. These results suggest an important role for pectolytic enzymes in plant response to pathogen attack in incompatible interactions. Another important aspect of the interaction of pectin polymers with Phytophthora spp. is the report that pectin accelerates the encystment and germination of P. palmivora zoospores (Grant et al., 1985) . This suggests that a pectin-like material on root surface may act as a recognition signal which results in the encystment of this species. Also zoospores encystment is accompanied by a rapid Ca2* excretion, suggesting that pectic substances apparently trigger a stimulus involving changes in cellular Ca2* that activates early events of encystment (Irving et al. 1984). Thus, it is important to characterize the array of pectolytic enzymes produced by P. sojae in infected tissues to understand the nature of this disease. The production of pectolytic enzymes by compatible, in contrast to, incompatible P. sojae races in tissues from different soybean cultivars deserves attention. To study the role of pectolytic enzymes in pathogenesis, we examined some of the various criteria enumerated by Cooper (1983a) that can be evaluated to implicate these enzymes in pathogenicity or virulence. These are: 1) ability to produce the enzymes in culture or "in vitro" 2) detection of these enzymes in infected tissue or "in vivo" 3) depletion of cell wall polysaccharides 4) correlation of enzyme production with pathogenicity 5) microscopic alterations in walls of infected tissue 6) reproduction of wall changes or disease symptoms with purified enzymes

7) inhibition of these enzymes during pathogenesis by application of specific antiserum to a particular enzyme. 113

The major objective of this investigation was to examine and characterize the various pectolytic enzymes produced in culture and in infected soybean tissueB by P. sojae. This was fulfilled through various approaches and quantitative assays performed under different conditions. 114

MATERIALS AMD METHODS

As we stated before, we based our research on some of the various criteria listed by Cooper (1983a) to determine the role of pectolytic enzymes in pathogenicity. Four major approaches were taken to study the production of pectolytic enzymes by P. sojae:

1) Microscopic alterations of cell wall in infected tissues 2) Ability to produce pectolytic enzymes in culture or in vitro 3) Detection of pectolytic enzymes in planta or in vivo 4) Correlation of enzyme production with pathogenicity

Microscopic techniques The first approach was to use basic microscopic techniques to observe any alteration in soybean cell wall following infection with P. sojae. Two different techniques were used: 1) histological sections of infected soybean cotyledons from cultivar Williams 79 (W79) with compatible and incompatible P. sojae races and 2} more detailed observations of cell ultrastructure using the electron microscope. 1. Histological Work on Soybean Cotyledons Infected with P. sojae During these studies we used the soybean cultivar Williams 7 9 (w79). It possess the Rpsl-c resistance gene for P. sojae race 3, but is susceptible to P. sojae race 4. In order to observe the damage caused by compatible and incompatible races of P. sojae during infection, 11 day old W,9 cotyledons were inoculated with P. sojae races 3 and 4. The abaxial surface of the cotyledons were disinfected with 95% ethanol. Five cotyledons were then placed abaxial side up in inverted petri plates containing moistened filter paper. Cotyledons were wounded through the pit with a sterile needle. After that, the cotyledons were infected with P. sojae by placing a mycelial agar plug (1 mm) on the wounded region. Control cotyledons were wounded and agar plugs containing no mycelium were placed on top of the wounded tissue. Cotyledons were incubated in moist 115 chambers at 100 fiEm^s1' for 24, 28, 32 and 36 h for each treatment. Observations on the damage caused by the P. sojae infection on cotyledon tissue were taken at each of these 4 time periods. Infected cotyledons were fixed in formalin-acetic acid-alcohol (FAA; 5ml 40% formaldehyde, 5ml glacial acetic acid and 90ml of 50% ethyl alcohol) overnight. After that, the tissue was subjected to ethyl alcohol dehydration and embedded in paraffin {Jensen, 1962, O'Brien and McCulley, 1981). Three replicates for each cotyledon at each time period were done. Serial sections of 10 fi thick for each treatment were prepared. Tissue was stained with safranine and fast green, in sequence (O'Brien and McCulley, 1981) . Data on tissue maceration as well as epidermal cell size and number were evaluated for each treatment.

2. Electron Microscope (EM) work on soybean cotyledons infected with P. sojae: Soybean cotyledons and hypocotyls from Williams (W) , the universal susceptible, and W79 were infected with P. sojae races 3 and 4. Cotyledons and hypocotyls were treated as stated above. This time tissue was infected for 30, 36, 42, 48 h. Two different sections, A and B, including the lesion and surrounding area were taken from infected hypocotyls and controls (Plate V) . For cotyledon tissue three different sections were sampled (A, B and C) for infected tissues and the controls (Plate V). Each tissue section was cut in four (0.5 mm) pieces and fixed in a solution containing potassium phosphate buffer at pH = 7.2, 2% glutaraldehyde and 1% paraformaldehyde for 3 h. All samples were post­ fixed in 1% 0s04 in the same buffer for 2 h. Later the samples were washed with buffer and dehydrated with a series of alcohols. Finally, tissues were embedded in an Epon 812 resin for 24 h, Ultrathin cotyledon and hypocotyl sections (6/0 were cut with a diamond knife in a ultramicrotome. Sections were poststained with 0.5% uranyl acetate and lead citrate and collected on coated nickel grids (Gluert, 1975). Samples were examined with the electron microscope (Model Zeiss 10). Plate V Tissue samples were taken from infected hypocotyls (sections A and B) and cotyledons (sections A, B and C) at different periods of time after inoculation. 117

Ability of P. sojae to produce pectolytic enzymes in culture Our second approach was to determine the ability of P. sojae races to produce pectolytic enzyme in culture media amended with different pectic fractions. The experiment was planned to determine if the fungus was able to grow in pectic fractions on solid media and second to study the different kinds of pectolytic enzymes induced in liquid culture that might be involved in P. sojae pathogenesis. 1. Growth of P. sojae races on different pectic fractions in solid media: P. sojae races 1, 3, 4, 7 and 12 agar plugs (4mm), were placed on plates containing synthetic media (SH) as basal media. SM contained 2.5g sucrose (EM Science, Cherry Hill, N.J.), 0.3 g asparagine monohydrate (Sigma), 0.15 g KH2P04 (Jenneile Chemical Co, Cinn, OH.), 0.15 g K2HP04 (J.T. Baker, Inc. Phillisburg, N. J) , 0.10 g MgS04 • 7H20, 0.08 g CaCl2 • 2H20 (Sigma), 0.01 g Ascorbic acid (Sigma), 0.002 g thiamine (Sigma), 0.001 g FeS04 • 7H20 (Sigma), 0.0044 g ZnS04 • 7H20, 1 ml stock solution MnCl2 ■ 4H20 (Em Science, Cherry Hill, N.J.), 2 ml of stock solution cholesterol, 20 g agar (Bacto agar, Difco) in 1 1 double distilled water (pH = 6.5). To prepared stock solution of MnCl2 • 4H20, 7.0 g were added to 100 ml of double distilled water. For stock solution of cholesterol, 0.005 g were added in 1 ml dimethyl formamide. SM was amended with two different pectic fractions: pectin or polygalacturonic acid (Pac) (both from citrus; Sigma). Carbon and nitrogen sources were removed from the agar media in order to obligate the pathogen to use the particular pectic fraction. Several treatments were made: 1) SM + pectin or Pac (0.5 %) (with sucrose and asparagine) 2) SM + 11 " (without sucrose, with asparagine) 3) SM + " " (without asparagine and sucrose but with ammonium sulfate)

The reason to use ammonium sulfate (0.26 g) as a substitute for asparagine was to reduce the carbon source, while at the same time supplying a nitrogen source. P. sojae radial growth was measured every other day for 118

10 days. Three replicates per treatment per race were included.

Methods used to detect pectolytic enzyme activity Induction of pectolytic enzyme activity in P. sojae cultures was detected using two major methods: polyacrylamide gel electrophoresis (PAGE) and spectrophotometric assays. Protein content was determined by the method of Bradford (1976).

1. PAGE: Cruickshank and Wade (1980) were able to detect various pectolytic enzyme activities by incorporating pectin in polyacrylamide gels with subsequent staining of unhydrolyzed pectin with ruthenium red. This method allows the simultaneous detection of isoenzymes of PNL, PME and PG. Because of the convenience of this method, we decided to modify and adapt it for a Biorad vertical slab gel apparatus. We used a 7% polyacrylamide gel (pH = 8.7), modified by adding 0.1% citrus pectin (Sigma). For detailed description on gel preparation refer to Chapter II (page 89). Electrophoresis was carried out at 4°C. Voltage applied for the upper and lower gels was 80 V and 185 V, respectively. Gel wells were filled with 17 or 35 p. 1 of fungal enzyme extracts, 6 or 12 /il of sample buffer, respectively and 1 /xl of 0.05% bromophenol blue. Gels were stained according to Cruickshank and Wade (1980). First, they were incubated for 90 min at 25°C in 100 ml 0.1 M Malic acid. This acid causes a gradual change in pH in the gels down to pH = 3 in 90 min. After that gels were rinsed with deionized double distilled water and stained for 1 h in 0.002% ruthenium red (Sigma). Then the gels were washed with several changes of double distilled water and left overnight. Gels were dried for examination and interpretation. 2. Spectrophotometric assays: Four pectolytic enzymes were studied in P. sojae races, PL, PNL, PG and PME. The assays used to measure enzyme activity were the following: 119

a) Pectate lyase (PL): PL activity was determined measuring the increase in absorbance at 232 nm of 4,5-unsaturated reaction products. Substrate solution was prepared with 60 mM Tris HCl buffer pH = 8.5, 0.6 mM CaClj and 24% (w/v) polygalacturonic acid (Sigma) . Reactions were monitored for 1 min at 232 nm in a spectrophotometer (Model Shimadzu) . The methodology was as described by Collmer et al. (1988) . b) Pectin lyase (PNL): A modification of the assay described by Collmer et al. (1988) for PL was used. To detect PNL activity, 0.25% citrus pectin was added as substrate instead of polygalacturonic acid. Increase in the absorbance at 232 nm was monitored for 1 min in order to calculate enzyme activity. Eight different assay protocols were tested in order to examine the best conditions for the PL and PNL spectrophotometric assays. Various factors were considered, including pH and the addition of CaJ*. These were:

1) 50 mM Acetate buffer + polygalacturonic acid (0.24% w/v), with or without 0.60 mM CaCl2, pH 5.2 2) 50 mM Acetate buffer + pectin (0.25% w/v), with or without 0.60 mM CaCl,, pH 5.2 3) 60 mM Tris-HCl + Polygalacturonic acid (0.24% w/v), with or without 0.60 mM CaCl2, pH 8.5 4) 60 mM Tris-HCl + Pectin (0.25% w/v), with or without 0.60 mM CaCl2, pH 8.5

Of the protocols investigated, 60 mM Tris-HCl with 0.6 mM CaCl2 (pH = 8.5) was selected as optimal for PNL and PL spectrophotometric assays.

Enzyme activity was calculated using the molar extinction coefficient (e = 4600 M'1 c m 1) for the unsaturated product at 232 nm. One unit of enzyme forms 1 /mol of 4,5-unsaturated product in 1 min under the conditions of the assay. 120

c) Polygalacturonase (PG): PG activity was measured using the assay described by Collmer et al. (1988). The Nelson & Somogyi reagent was used to measure the increase in reducing groupB (oligogalacturonic acid) released from polygalaturonic acid during 30 min. A D-galacturonic acid standard curve was developed to quantitate the oligogalacturonic acid produced in the reaction mixture. One unit of enzyme forms 1 junol of reducing sugar (oligalacturonic acid) in 1 min under the conditions of the assay.

d) Pectin methyl esterase (PHE): The assay for PME was based on the color change of a pH indicator, bromophenol blue, which detected the release of free uronic acid residues, following demethylation of substrate. This assay was performed at pH = 7.5. The color change was monitored by spectrophotometer at 620 nm for 3 min (Hagerman and Austin, 1986). This assay was originally developed for plant PME. We successfully modified the assay using another pH indicator, bromocresol purple, to detect changes in absorbance at a lower pH (6.5) in order to characterize fungal PME. The color change was monitored at 570 nm. One unit of enzyme forms 1 ftmol of acid in 1 min to react with the bromothymol blue or bromocresol purple under the conditions of the assay.

Pectolytic enzyme induction experiments in culture In order to select a suitable basal media to examine the induction of various pectolytic enzymes by pectic fragments in P. sojae, we tested 3 different broths: synthetic media, lima bean and soybean flour. 1. Synthetic media {SM} and lima bean broths: P. sojae races 3 and 4 were grown on 1 1 flasks for 4 days in 200 ml of lima bean broth or SM broth as basal media. The different media were amended with two different pectic fractions: pectin (0.1%) and polygalacturonic acid (0.1%). The pH in all treatments ranged from 7 to 7.3. At the time of harvest, fungal cultures were filtered through a polyester macrofilter (diameter 30 cm sq., mesh opening 10 pm and thickness 70 nm) . The mycelium was washed with 121

50 ml double distilled deionized water. After harvesting, mycelium was weighted and stored at -80°C. The fungal filtrate portions were collected and divided in two equal portions and stored at -20SC. For enzyme extraction from mycelial portions, acetate buffer pH = 5.2 (1:3 w/v) was used. Mycelium was sonicated with a cell disruptor (Sonifier Model 350, manufactured by Branson Sonic Power, Inc.). Ten pulses (45% duty cycle, output control 2) were applied 5-6 times with cooling between sonications. After sonication, samples were centrifuged at 15000 x g (Biofuge, Rotor no. C-1710-20, r...= 7.9 cm) for 5 min. Supernatant was kept at -80°C and used to run activity PAGE. Filtrates were used directly without further treatment. 2. Soybean flour: Soybean flour (1.3% w/v) from W, W7, and Williams 82 (W82) seeds was also used to grow P. sojae race 3 in some experiments. The reason to use soybean flour was to have in the media all of the different metabolites present in soybean that might act as inducers or coinducers of pectolytic enzymes. The various treatments used as pectolytic enzyme inducers were: a.) soybean flour from W + pectin (1%) or Pac (1%) b.) " " " W75 + " " c.) " " W82 + " " d.) controls, soybean flour from each soybean cultivar (no pectic fraction added)

The reason to select the different cultivars was to observe possible differential enzyme induction using susceptible and resistant soybean cultivars to P. sojae race 3.

Pectolytic enzyme induction in lima bean broth ("in vitro") Following the above evaluations, lima bean broth was selected as the most suitable media to grow P. sojae races. Lima bean provides the nutrients and cofactors required for excellent P. sojae growth. Two major sets of experiments were performed to study the 4 different pectolytic 122

enzymes induced in culture and examine their activity over a period of time (12 days).

Growth curves P. sojae race 4 was grown on lima bean broth amended with different pectin fractions. The various treatments were; 0.5% pectin, 1.0% Pac and 1.0% soybean dry hypocotyl powder from Williams cultivar. The dry hypocotyl fractions were obtained from 172.28 g of fresh hypocotyls (W). The fresh tissue was oven dried at 60°C and finely ground. Lima bean broth was used as control. One liter flasks containing 200 mis of Lima Bean Broth amended with the different pectic fractions were used in this experiment. A half petri plate of pure P. sojae cultures were used to inoculate each flask. A sterile syringe (20 cc, needle size 20 gauge) was used to break up the agar cultures for inoculation. A very thin layer from the top of a half plate of a P. sojae culture was transferred to the syringe and pressed through the needle. The advantage of this technique is that many foci of inocula were produced and subsequent fungal growth was homogenous and vigorous after two days of inoculation. Fungal cultures were aerated daily using very gentle circular movements. Flasks were incubated at room temperature for 12 days. Mycelia and filtrate were harvested every other day. Enzyme extraction protocol for the mycelial portion was as stated above, using 50 mM sodium acetate buffer, pH 5.2 (1:3 w/v). One hundred mis of filtrate portions were lyophilized (Lyophilizer Model Dura-dry Microprocessor from FTS Systems, Inc.). Filtrate portions were reconstituted for spectrophotometric assay with 1 ml double distilled deionized water. The same experiment was repeated a second time using pectin (0.5%)

from two different sources (orange and apple) and purified polygalacturonic acid (1.0%) (all from Sigma). Apple and citrus pectins have 7.1 and 8.0% methoxy and 80% and 7 8% galacturonic acid content 123

respectively. In this experiment, mycelium and filtrates were harvested every day for 8 days, then every other day until 12 days. Protocols for pectolytic enzyme extraction and detection were as stated above. Pectin extraction from soybean hypocotyls

To complement results with soybean hypocotyl powder and commercial pectins, we were interested in extracting pectin from soybean hypocotyls for pectolytic enzyme induction in culture. The methodology reported by Huang (1973) was followed. However, we were unable to test pectolytic enzyme induction on soybean pectin due to the very low yield of pectin extracted during these experiments.

Induction of pectolytic enzyme activity in P. sojae race 3 using plant phenolic compounds Since virulence genes, including pectolytic enzymes, are often induced by plant signal molecules such as flavonoids, we wished to determine if soybean isoflavonoids could induce pectic enzymes in P. sojae. Four different compounds were selected to induce pectolytic enzyme activity in lima bean broth. The soybean isoflavones, daidzein and genistein, and the chickpea isoflavones, formononetin and biochanin A were used as enzymes inducers. All compounds were tested at concentrations below toxic levels. Fifty /xM stock solutions of all isoflavones were prepared. Flasks containing 100 ml of lima bean broth were inoculated with 10 agar plugs (4 mm) of P. sojae race 3. Four days after inoculation, 2.5 ml of the stock solution for the appropriate compound was added per flask. Mycelial and filtrate enzymes were extracted as stated above and enzyme activity was determined using PAGE as described above.

Induction of pectolytic enzyme activity in P. sojae using plant fractions

Soybean cultivars: Soybean seeds from cultivars W and W79 were planted in flats (size 18 x 13.5 cm) of metro mix 360 (Grace and Sierra 124

Horticultural Products Co., Milpitas, Ca) and grown in a growth chamber at 23°C with 10,000 /xEinsteins of light on a 14 h photoperiod. These two cultivars were selected based on susceptibility (W) or resistance (W79) to P. sojae race 3, respectively. The soybean hypocotyls were harvested 11 days after planting. For W and W7, cultivars a total of 126.82 and 165g of fresh hypocotyls were harvested. Plant extracts: Soybean hypocotyl tissue was first homogenized in an extraction buffer. Potassium phosphate buffer 100 mM (pH = 7) at 4°C was used. This constituted the "aqueous fraction". The remaining plant residue was then subjected to serial extractions with various organic solvents starting from the most polar to the less polar. 1. Aqueous fraction: This fraction contains proteins, carbohydrates and some secondary metabolite conjugates. A ratio of 2:1 (buffer to tissue) was used for W and W7, cultivars. After homogenization the plant tissue was centrifuged (Centrifuge Model HN, International Equipment Co. , Needham Hts. Mass, USA) twice at 3/4 speed for 4 min. The pellet was kept and the supernatant was divided into 2 portions and frozen. The remaining pellet was homogenized again in 200 ml of phosphate buffer for 1 min and centrifuged again under the same conditions. This time the pellet was kept and the supernatant was discarded. 2. 80% Ethanol fraction: This fraction contains flavonoids, most free and conjugated secondary products, such as genistein and daidzein, and conjugated sterols. The pellet obtained at the first step of the aqueous fraction was resuspended in 200 ml of 80% ethanol. The suspension was stirred for 10 min and then centrifuged for 4 min at same speed stated before. The supernatant was divided in 2 portions and kept in the freezer. The pellets were resuspended in 200 ml of 80% ethanol and stirred for another 10 min. The suspension was centrifuged 6 min. The supernatant was discarded and the pellet was used in the next extraction. 3. Chloroform : methanol fraction: This fraction contains free sterols and lipids. A mixture of chloroform : methanol (2:1), was 125

prepared. Pellets were stirred in this solvent mixture for 10 min and then centrifuged for 4 min under the same centrifugation conditions explained above. The supernatant was kept in the freezer. Pellets were resuspended in chloroform : methanol (2:1). After 10 min of stirring the suspension was centrifuged again. The pellet was kept for the last extraction and the supernatant discarded.

4. Cell wall fraction: This fraction contains cell walls and phenolic polymers. The pellet obtained in the chloroform : methanol extraction was left in the hood on a glass plate to dry. This cell wall fraction was weighed and reduced to very fine powder for the experiments.

Enzyme induction with plant fractions Lima bean broth was used as basal media to grow P. sojae for two days before enzyme induction with the different plant fractions. A half petri plate of P. sojae race 3 was used to inoculate 200 ml lima bean broth using a syringe {as described above). Cultures were aerated daily with very gentle circular movements and incubated at room temperature. Two days after inoculation the cultures were ready for enzyme induction.

The aqueous fraction was sterilized with a membrane filter (4.5/4 and 115 ml capacity) after centrifugation (Sorvall RC-5B Refrigerated Superspeed Centrifuge, DuPont Instruments, Wilmington Delaware; rotor SS- 34) for 40 min at 12,000 x g. Two concentrations of the aqueous fraction (10 and 40% v/v equivalent to the weight of original tissue) from soybean cultivars W and W79 were used for induction. The 80% ethanol and chloroform : methanol fractions were evaporated using a rotary evaporator and then reconstituted with ethanol or methanol respectively for the induction experiments. The ethanol and chloroform : methanol fractions were reconstituted so that the amount in the treatment was equivalent to 10 and 40% fresh weight of the original tissue.

Cell wall fractions from soybean hypocotyls of W and W,, cultivars

were sterilized with ethanol (80%). Also 10 and 40% concentration 126

equivalent of the total cell wall preparation were added to the lima bean broth for the induction experiments.

Lima bean broth was used as control.

Enzyme extraction Fungal cultures were harvested as explained above. Acetate buffer 50 mM (pH = 5.2) was used for mycelial extraction and was added in proportion to mycelial weight (1:3 w/v). For mycelial extraction the buffer was kept

cold and the samples on ice at 4°C. Mycelium was sonicated to disrupt fungal cellB as described above. After sonication, samples were centrifuged at 14,926 g (Biofuge, Rotor no. C-1710-20 r„„= 7.9 cm) for 5 min. Supernatant, pellets and freeze dried filtrates (Lyophilizer Model Dura-dry Microprocessor from FTS Systems, Inc.) were kept at -20°C. Activity gels were used to detect pectolytic enzymes in P. sojae fractions. Supernatants from extracted mycelia were examined directly. Before electrophoresis, pellets from these extracts were soaked for 10 h in acetate buffer, 50 mM (pH = 5.2) at 10°C. The supernatant from the soaked pellets were used for electrophoresis. Lyophilized filtrate powder was reconstituted with 1 ml deionized double distilled water.

Pectolytic enzyme induction in planta Soybean cultivars W and W7, and P. sojae races 3 and 4 were used in these experiments. Eleven day old cotyledons and hypocotyls were infected with P. sojae races as stated before. Control cotyledons were wounded and agar plugs containing no mycelium were placed on top of the wounded tissue. Fifty cotyledons and hypocotyls were infected and incubated on shelves at room temperature with 100 fiEn^'s1" of light for 12, 24, 30, 36, 42 and 48h for each cultivar and P. sojae race. Samples from sections of infected cotyledon and hypocotyl tissues were taken as shown in Plate V. Pectolytic enzymes were extracted with 50 mM sodium acetate buffer (pH =

5.2) (1:3 w/v). Tissue was ground with buffer and centrifuged at 127

15000 x g as described above for 4 min. The supernatant was used for the spectrophotometric assay.

IFG for plant and fungal enzyme differentiation Isoelectric focusing gels (IFG) is able to separate the various forms or isoenzymes of a particular enzyme based on their isoelectric points (pi). This technique was used to separate pectolytic enzymes from host as well as from pathogen origin. Also we intend to compare P. sojae pectolytic enzymes produced in culture from those produced in infected tissues. Protocol for IFG: Isoelectric focusing was carried out in a Biorad vertical apparatus using tube gels (size 5 x 7 x 125 mm) according to Garfin, (1990). The gels were prepared using the following reagents: 1) Polyacrylamide monomer concentrate 5 ml 24.25 g acrylamide (Sigma) 0.75 g bisacrylamide (Sigma) 7 0.00 ml deionizd double distilled water 2) 25 % glycerol (Sigma) 4 ml 3) Carrier ampholites 40%; pH 3-10 (Biolite, Biorad, Ca.) 1 ml 4) 0.1% riboflavin 5' phosphate (Sigma) 100 /xl 5) 10% Ammonium persulfate (Sigma) 30 ^1 6) TEMED (Sigma) 6.67 jil 7) Deionized double destilled water 10 ml

First, the polyacylamide, glycerol and the carrier ampholites were mixed and aspirated under vacuum for 5 min. After that, riboflavin, ammonium persulfate and TEMED were added to chemically polymerize the gel. Two ml of the gel solution were poured per tube. Eighteen tube gels were placed under a fluorescent light in order to let the gel polymerize completely. Eighty percent glycerol was added to the samples of P. sojae enzyme extracts in a 0.5 : 1 (v/v) ratio, respectively. Samples from

infected tissues as well as filtrates from P. sojae pure culture were 128 used for the electrophoresis. The upper cathodic reservoir was filled with 0.01M NaOH and the lower anodic reservoir with 0.02 M H3P04. Electrophoresis was carried out for 12 h at 6°C, applying 200 V for the first 2 h and then raising the voltage to 300 V which waB maintained constant until the end of the run. After electrophoresis, the gels were placed in 0.02 H Tris-HCl buffer (pH = 8.3) for PNL isoenzyme determination according to Lisker and Retig (1974) . After 10 min, the buffer was replaced with 0.9% pectin from citrus (Sigma) in Tris-HCl buffer (pH = 8.3) containing 0.002 M CaCl2. The gels were incubated for 15 min at 30°C, washed with deionized double distilled water and immersed in 0.05% ruthenium red (Sigma) solution for staining. The gels were left overnight in the staining solution, after which the staining solution was replaced with deionized double distilled water. Commercial pectin lyase from Aspergillus niger (125 U/mg of protein; Sigma) and IF markers (Sigma) ranging from 3.55 to 9.3 were used as standards. To stain IF markers a solution of 0.25% cooumassie brilliant blue (CBB) was prepared in 10 and 40 % (v/v) of acetic acid and methanol respectively. IF markers were stained with 0.25% for half an hour. After that the staining solution was changed successively to lower concentrations (first to 0.025% (w/v) CBB and then to 0.0025% (w/v) CBB for another half hour for each concentration). Finally, the gels were left overnight in a methanol : acetic acid solution (40% ; 10% v/v). 129

RESULTS

Macroscopic observations: cotyledon and hypocotyl lesions Cotyledons and hypocotyls from soybean cultivars W and W7S were infected with P. sojae races 3 and 4. Table 10 summarizes the lesion areas sampled in cotyledon tissues for the different soybean cultivars and P. sojae interactions studied. No infected area was observed in cotyledons inoculated for 12 and 24 h. Water-soaked lesions, around agar plugs, were first observed in hypocotyls 24 h after inoculation in the compatible responses. Necrotic lesions were observed 30 h after inoculation in both organs with the incompatible response (soybean cultivar W79 and race 3) (Plate VI) . Water-soaked lesions were observed in all the compatible responses in both organs, 30 h after inoculation (Plate VI) . Hypocotyl and cotyledon tissues were completely rotten after 48 h in compatible responses. Thus, the major soft-rotting phase of growth occurred very rapidly between 30 and 48 hrs.

Microscopic observations Alterations in soybean tissues infected with P. sojae races were observed with both the light and electron microscope. Compatible and incompatible responses were examined over time and/or at various distances from the point of inoculation and compared.

1. Histological Work on Soybean Cotyledons Infected with P. sojae Soybean cultivar W7, was infected with P. sojae races 3 and 4 (incompatible and compatible respectively) and samples were taken at 24, 28, 32 and 36 h in order to monitor infection. Necrotic tissue was observed 36 h after inoculation in the resistant tissues. Compatible responses otherwise showed extremely macerated tissues 32 h after inoculation (Table 10). Cotyledons were infected at the pit area, which is a slightly concave structure on the abaxial side of the cotyledon consisting of enlarged cells. Although the pit has been studied in detail 130

Plate VI Eleven days old soybean cotyledons and hypocotyls from cultivars W and W7, were infected with P. sojae races 3 and 4. W is a universal susceptible cultivar whereas W19 is resistant to race 3 and susceptible to race 4. Table 10. Cotyledon lesion size of soybean cultivars W and W 79 infected with P. sojae races 3 and 4. Lesion size (cm) Soybean P. sojae Hours*7 cultivar Race 3 Racei 4 30 W 0.5Section A 0.5 Section iIV w79 0.4 n 0.5 n 36 w 1.4 Sections A+B 1.3 Sections A+B W7S 0.5 " A 0.7 n A+B 42 w 1.7 Sections A+B+C 1.5 Sections A+B+C w79 0.6 " A 1.3 n A+B 48 w 2.0 Sections A+B+C-7 2.0 Sections A+B+C W7S 0.6 " A 2 . 0 n A+B+C 1/ Hours after inoculation. 2/ Sections sampled (refer to Plate V). Whole cotyledon is infected after 48h in the susceptible responses. Table 11. Histological data from soybean cultivar W7S infected with P. sojae races 3 (Incompatible) and 4 (Compatible) at different time intervals.

Hours after inoculation 24 28 32 36 Pit Cells^7 R3 R4 R3 R4 R3 R4 R3 R4 CK Size (/un) 72 ± 4.1 85 ± 3.7 130 ± 5.0 75 ± 3.2 64 ± 2.5 -V 80 ± 5.0 - 66 ± 2.2 Number-'' 14 ± 7.9 13 ± 8.6 15 ± 8.8 11 ± 7.9 8 ± 7.2 N17 - 9 ± 3.1 Columns-7 2 ± 1.3 1 ± 1.0 1 ± 1.4 1 ± 1.7 2 ± 0.0 N- 1 ± 1.0 Infection-7 NI NI I I I I I I NA 1/ Gigantic epidermal cell at the pit area were evaluated in terms of size (/iM) , number of cells per row. Normal epidermal cell size was 25 ± 0.99 pm. 2/ Data on cell size or number was not taken 32 and 36 h after inoculation because of the severity of tissue maceration with P. sojae race 4. 3/ Number of gigantic cells in the epidermis 4/ N : necrotic tissue. 5/ Number of columns of gigantic cells 6/ No infected tissue: NI ; Infected tissue: I; do not apply: NA. 133 by Yaklich and his coworkers (1987 and 1989), its function in Glycine spp. is still unknown. We observed an increase in the number as well as in size of these enlarged epidermal cells in infected soybean cotyledon tissues from 24 to 28h (Table 11) . It was interesting to notice an increase in epidermal cell size up to 130 (jlM in the incompatible response 28 h after inoculation. Epidermal cell size in the compatible responses and controls range from 64 to 85 /tM. The increase in pit cell size in the incompatible response might imply drastic metabolic changes related to defense mechanisms that can be occurring in those epidermal cells. Hypersensitive cell death of these cells occurred in incompatible response by 36 h (Table 11) . It is known that the hypersensitive response involves very early membrane alterations (Goodman et al. 1986) and changes in ion transport (Misaghi, 1982b). Also in experiments performed by T.L. Graham and H.Y. Graham, 1990) glyceollin accumulation at the point of inoculation was detected within the first 24 h in the incompatible response and isoflavones conjugates were hydrolyzed in those cells to their aglycones very early (<12 h) after inoculation. It is intriguing to speculate that probably the increase in cell size in the pit cells might be due to these different kinds of metabolic processes that are occurring within those cells in a short period of time in the incompatible response. This would required further investigation.

2. Electron Microscope Nork on Soybean Cotyledons Infected with P. aojae Soybean cotyledons and hypocotyls from W and W79 were infected for 30, 36, 42, 48 h with P. aojae races 3 and 4. Sections from the lesion area and control tissues were taken as shown in Plate V and examined with the electron microscope. We were interested in plant cell wall damage caused by penetrating hyphae and in the disruption caused at the hyphae-plant cell wall interface. Plates VII to XIV show the electron micrographs of tissue damage caused by P. aojae in compatible and incompatible responses. We 134 observed a great degree of maceration in the compatible responses (W-P. sojae races 3 and 4 and W79-P. sojae race 4) 36 h after inoculation. Thus, data taken 30 h after inoculation will be discussed in more detail in the compatible responses due to extensive maceration of tissues 36 h after inoculation. However, in the incompatible response lesions are very well differentiated 36h after inoculation, therefore data taken 36h after inoculation will be discussed in more detail.

General observations (compatible and incompatible interactions) 1. Intercellular observations: In Borne interactions the first morphologically observable event between a pathogen and its host is the production of electron dense material at the plant cell surface. In some cases, this has been hypothesized to cause an adhesion of the pathogen to the host cell wall to allow later penetration of the host cell (Hohl, 1991). In others, it is viewed as a host defense response to immobilize the pathogen (Sequeira and Graham, 1977). Plate VII shows the production of a material surrounding a fungal cell that has make contact with a plant cell wall. In P. sojae-soybean interaction, a material that might consist of proteins and/or glycoproteins and even polysaccharides has been reported to be produced by the fungus (Hohl, 1991). Different types of molecules have been implicated in adhesion. Evidence to implicate a lectin-ligand type interaction in the adhesion of P. sojae germinated cysts to beads coated with galactosamine residues and host protoplast has been obtained (Hohl, 1991). However, it has not been established that adhesion is required for infection. In fact, much of the colonization cauBed by P. sojae is intercellular, leading to the tissue maceration typical of the disease. Consistent with this, EM observations revealed a predominance of intercellular fungal infection (Plate VIII). The middle lamella was 135

Plate VII

A fungal hypha (FH) is surrounded by an electron dense material (arrows) at the plant cell surface. 136

Plate VIII Much of P. aojae colonization is intercellular, leading to the tissue maceration typical of the disease. In these electron micrographs the middle lamella is disrupted and various fungal hyphae (FH) were present in the intercellular space in compatible interactions. (Plant cell; PC) 137 disrupted, and various fungal hyphae were present in the intercellular spaces. That suggested to us that intercellular hyphae could be responsible for middle lamella degradation. Ultrastructure examination of intercellular P. sojae growth also shows the formation of cellular infection pegs and projections extending between the digested ends of the host walls (Plate IX and XI) . Large numbers of cytoplasmic vesicles (lomasomes) concentrated around the site of attachment and at the periphery of the fungal cell membrane (Plate X). 2. Penetration and Intracellular observations: Based on observations at the ultrastructural level it appeared that P. sojae has the ability to enzymatically dissolve the pectic substances and other polymers that comprise the cell wall and thus grow in and colonize the intercellular spaces (Plate XI). Very localized cell wall disruption also occurred during cellular penetration and hauBtoria formation (Plate XI). Haustoria grew and enlarged intracellularly forming a club shape (Plate XII) . The haustoria possessed very dense cytoplasm and many organelles like mitochondria, ribosomes and endoplasmic reticulum. Apparently a great deal of cellular processes were triggered. The nature and function of the components of the interface between haustoria and host protoplast is unknown. Some changes also occurred in the host cell. It was interesting to observe that the plasmalemma formed large folds and projections extending toward the fungal cell (Plate IX).

Specific observations for compatible and incompatible interactions So far, we have discussed the results of what was occurring in infected cells in incompatible and compatible responses in general. However, some differences in the nature and magnitude of the responses were seen. Compatible responses were accompanied by multiple fungal hyphae

(Plate XIII) penetrating the same cell as well as various hyphae in the intercellular spaces 30 h after infection (Plate XI). The plant Plate IX

Ultrastructure examination of P. sojae intercellular growth shows the formation of projections (arrows) extending between the digested ends of the plant cell (PC) and the fungal cell (FC).

UJ CD Plate X

Large number of cytoplasmic vesicles or lomasomes (arrows) concentrated around the site of attachment and at the periphery of the fungal cell membrane (FCM). Plant cell: PC; Fungal cell; FC. Plate XI

A . Apparently P. sojae has the ability to enzymatically disolve the pectic substances and other polymers that comprise the plant cell wall (PCW). B. Localized cell disruption (arrows) also occurred during infection peg (IP) formation and cellular penetration. C. Close up of cell wall disruption at the host and fungal cell (FC) interface. Plant cell: PC; Intercellular space: IS.

PCW

O 141

Plate XII

P. sojae haustoria (HT) grew and enlarged intracellularly to a final club shape (PC: plant cell; IS: intercellular space) 142

Plate XIII

Multiple fungal hyphae penetrate the plant cell in the compatible interaction. Plant cell: PC; Fungal cell: FC; Intercellular space:IS. 143

Plate IVX

Some changes also occurred in the plant cell (PC), plasmalemma (PM) withdrawn from the cell wall and plant secretory vesicles (arrows) were observed close to the cell membrane

PC 144 plasmalemma was withdrawn from the cell wall. Plant secretory vesicles were observed close to the infection area (Plate IVX). Plant tissue was badly macerated, especially the middle lamella, 3 6 h after inoculation. Although the incompatible response also produced cell wall degradation, it was to a lesser extent, enabling us to look at more detail at the plant- pathogen interaction 36 h after inoculation. Contact areas between pathogen and host cell were noticed as well as deposition of secretory vesicles of fungal origin at the periphery of the cell (Plate XX and X). Penetration pegs as well as haustoria development were accompanied by cell wall degradation but to a lesser extent compared to the susceptible response. A notable feature of penetration is the narrowness of the zone of alterations of host walls around infection pegs in the incompatible and very early stages of the compatible response. This suggests that diffusible extracellular enzymes are produced under strict control (Plate XX) . This also suggests that they are not produced in large quantities like in the later stages of the compatible response.

Compatible and incompatible interactions are characterized by callose formation in soybean hypocotyls infected with P. sojae (Stossel et al. 1980 and 1981; Coffey and Wilson, 1983). However, no direct evidence of callose deposition was observed surrounding the site of entry in any of the interactions examined in this study.

P. aojae ability to produce pectolytic enzymes in culture To determine the ability of P. sojae races to grow on different pectic fractions and the different kinds of pectolytic enzymes that might be involved in P. sojae pathogenesis, various set of experiments were performed in culture.

Growth of P. sojae races on solid media amended with different pectic fractions: The first experiments were done to study the growth of different P. sojae races on two different pectic fractions as carbon 145 sources: pectin and polygalacturonic acid. A synthetic minimal media was chosen as the basal media for this set of experiments. Five different P. sojae isolates representing 5 different races were tested. Fig. 35 shows the growth of races 3 and 4 in pectin and polygalacturonic acid. Overall, pectin was a better carbon source than polygalacturonic acid for P. sojae. The same results were obtained with races 1, 7 and 12 (data not shown). Growth of P. sojae races on liquid media amended with different pectic fractions: In order to select a suitable basal media to examine the induction of various pectolytic enzymes produced by P. sojae, we tested various liquid media. Synthetic medium, soybean flour and lima bean broths were used as basal media to grow P. sojae races 3 and 4 for further studies on P. sojae pectolytic enzyme induction in culture. The different media were amended with two different pectic fractions: pectin and polygalacturonic acid.

Soybean flour from W, W7, and We2 seeds were used to supply in the media all the various metabolites present in soybean tissues that can act as inducers or coinducers of pectolytic enzymes. These cultivars were selected to compare data on enzyme induction using susceptible and resistant cultivars. Of the three media evaluated, lima bean broth was selected over the others. P. sojae growth in SM was very poor and the mycelium was too thin to get enough mycelium for enzyme extraction. In contrast soybean flour broth was too rich, especially in oils and produced a very dense broth which was very easily contaminated. Also, mycelial growth was poor.

Pectolytic enzyme induction in lima bean broth ("in vitro") 1. Growth curves: P. sojae race 4 was grown on lima bean broth amended with various pectin fractions : pectin, polygalacturonic acid and soybean dry hypocotyl powder. Unamended lima bean broth was used as control. 2000 2000 POLYGALACTURONIC ACID PECTM

EI 5Q 1500 PeC T N LLf PAC PEC (NO SUC) i a PAG (NO SUC) PEC (NO SUC. NO ASP, AMM) ii PAC (N O SU C . NO ASP. AMM) 5a O H SC 1000 CONTROL 1000 CONTROL «£ n X>- ui" >(A UI CD z 500 500 o

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0 2 4 6 8 10 DAYS

Fig. 35. Growth curves of P. sojae race 3 and 4 on synthetic media amended with different pectic fractions: pectin (PEC) and polygalacturonic acid (PAC). Carbon and nitrogen sources were removed from the agar media in order to obligate the pathogen to use a particular pectic fraction. (SUC: sucrose; ASP: asparagine; AMM: ammonium sulfate). i-* ■b

FigB. 36 to 42 and Table 12 and 13 show the results of pectolytic enzyme induction in P. sojae race 4 in culture. Enzyme activities were examined in both the mycelia as well as in the culture filtrate. Three different enzyme activities were detected: PNIi, PL and PG. Of all the enzymes tested PNL was the major enzyme produced in culture (Fig. 36 and 37) . In the mycelial fractions, P. aojae PNL developmental expression is very late in controls, the highest enzyme activity was observed 10 days after inoculation. Pectin and dry hypocotyl powder had more dramatic effects on enzyme induction. Pectin induces PNL activity somewhat earlier than controls, 8 days after inoculation (Fig. 36) . However, it was interesting to observe that soybean dry hypocotyl powder induces PNL activity even earlier than pectin, 2 days after inoculation. Polygalacturonic acid was a very poor inducer (see baseline. Fig. 36). In summary, PNL showed a transient response and seems to be under developmental control in the mycelial extracts. When we look at enzyme activity in filtrates, the highest induction was observed at very early Btages of fungal growth for treatments containing dry hypocotyl powder and pectin. These data also support the somewhat earlier and more dramatic induction of PNL by dry hypocotyl powder than pectin (Fig. 37). Polygalacturonic acid again, was a very poor inducer of enzyme activity. PNL appears to be present as an extracellular enzyme early in culture. Although most of the enzyme is detected extracellularly, a small proportion also appears to be associated with the mycelial fractions and not secreted. These various observations made us hypothesize that these enzymes may be produced at the hyphal tip in the fungus and are secreted to the external environment. PL and PG activities were very low compared with PNL activity in both fractions (Fig. 38 and Tables 11 and 12). In fact, if anything, PL activity was suppressed in the presence of pectic substrates (Fig. 38). PL activity was induced earlier in controls and showed the highest enzyme activity at the mycelial fractions suggesting that just a small MYCELIAL FRACTION 4000 PECTIN DRY CONTROL HYPOCOTYLS

DAYS

Fig. 36. Lima bean broth amended with different pectic fractions: polygalacturonic acid (PAC), pectin and dry hypocotyls was used to induce P. sojae (race 4) pectolytic enzymes. PNL activity in the nycelial portions was measured over a period of 12 days. 148 eoooo -r A 400 a FILTRATES ✓ \ FILTRATES 3 PECTIN / \ POLYGALACTURONIC ACID 60 0 0 0 ■ '' 3 0 0 - 1 / ' ' > u /' \ ' / / 1~— O4 4 0 0 0 0 - // V v' “ 200 LL tt

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Fig. 37. Lima bean broth amended with different pectic fractions: polygalacturonic acid (PAC), pectin and dry hypocotyls was used to induced P. sojae (race 4) pectolytic enzymes. P. sojae PNL activity in the filtrate portions was measured over a period of 12 days.

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Fig. 38. Lima bean broth amended with different pectic fractions: polygalacturonic acid (PAC), pectin and dry hypocotyls was used to induced P. sojae (race 4) pectolytic enzymes. P. sojae PL activity in the mycelial and filtrate portions was measured over a period of 12 days. 151

Table 12. PG specific activity (U/mg of protein) in P. sojae race 4 mycelial portions. Pectic Fractions Day Pacy Pectin Dry hypocotyls Control 0 0.00 0.00 0.00 0.00 2 0.25 0.64 0.13 0.09 4 0.33 0.24 0.50 0.46 6 0.28 0.06 0.06 0.07 8 0.16 0.07 0.13 0.08 10 0.19 0.13 0.13 0.11 12 0.23 0.14 0.13 0.07 y Pac = polygalacturonic acid

Table 13. PG specific activity (O/mg of protein) in P. sojae race 4 filtrate portions.

Pectic Fractions Day Pacy Pectin Dry hypocotyls Control 0 0.00 0.00 0.00 0.00 2 0.61 2.13 0.76 4 0.66 1.82 1.91 0.65 6 0.98 1.40 0.45 8 1.19 1.35 1.22 0.42 10 0.78 2.12 1.85 0.40 12 1.01 1.66 0.50 -7 Pac = polygalacturonic acid 152 level of the enzyme is secreted to the culture media. PG activity in the filtrate was higher than in the mycelial extract. However, the induction of PL and PG was not seen or was very weak. A second set of experiments were done to reconfirm our findings using P. aojae races 3 and 4. The mycelium as well as filtrate were harvested everyday for 8 days and then every other day until the 12th day. Two different pectin sources were used: orange and apple. The reason for this was to observe differences in pectolytic enzyme induction based on the degree of pectin methylation. Although we tried to extract pectin from soybean hypocotyls to get a more complete view of what is occurring in the plant we were unsuccessful. As an herbaceous plant the pectin content of soybean was too low to obtain the large quantities needed for incorporation into growth medium. The second set of experiments looked at PNL activity induction in culture in more detail and confirmed the results obtained in the first set of experiments. PNL showed the highest enzyme activity in the filtrate compared to mycelial fractions for both P. aojae isolates examined (Figs. 39 to 42). PNL again appeared to be secreted into the media to degrade the pectic fraction. Pectin from orange appeared to be the best PNL inducer and polygalacturonic acid the poorest. In fact, no enzyme activity was detected in filtrates using polygalacturonic acid as inducer in race 4 and enzyme activity was very low in race 3. It was interesting to notice that when enzyme activity was higher in the filtrate, low or no activity was present in the mycelial portion and vice versa. This suggests that although the enzyme is secreted, there is apparently some physiological control over this process. PNL activity was not steadily induced in any of the experiments performed. These findings may be related to the very low levels of enzyme measured. However, they confirmed that pectin is the stronger inducer of PNL and the vast majority of the enzyme activity is extracellular. It would be very interesting to investigate the factors controlling enzyme secretion in further work. In terms of isolates of A r <3 o MYCELIAL PORTION 2 MYCELIAL PORTION ' s BOO ■ / PECTIN (ORANGE) i 5 800 - PECTIN (APPLE) / / 4 >- n >- r> £ m i ' > o 600 ■ / £ 600 - / \ / w < / *“ s i \ 0 O OC / % / < • / < » V / s A « 400 ' ! 400 - o o' 1 a f - 4 . 5 * LL * u a 1 O CL /' V' \ > \ / U i _ 1 U J _ Q . Z a 5 200 ■ 200 - V / \ / ffl / \ i CO / * » . . \ / 1 • \ -J * 1 i » 1 z / Q. 0 - , ^ 4 , „ . . V . -p 0 - , T , , , , , F l ...... , , 10 6 6 10 12 DAYS DAYS

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Fig. 42. Lima bean broth amended with different pectic fractions: polygalacturonic acid (FAC) and pectin from orange and apple was used to induced pectolytic enzymes in P. sojae race 4. PNL specific activity from the filtrate portions was measure over a 12 days period. 157

P. sojae, some differences were observed. For example, PNL activity in race 4 was higher than in race 3 in the controls. Perhaps there is an

isolate specific effect on activity. However, PNL was the major extracellular enzyme activity detected regardless of the isolate. Also, the race 3 isolate showed higher enzyme induction in the filtrates than the race 4 isolate on the higher esterified pectin. Based on these results it appears that P. sojae races respond to changes in pectin composition. Methylation of the polygalacturonic acid structure is critical since polygalacturonic acid is a very weak inducer. Further studies are needed to determine what precise degree of esterification is optimal for induction. It was interesting to observe that some enzyme induction was seen in controls although at very low levels. Apparently lima bean broth possesses some kind of metabolites that induces pectolytic enzymes in P. sojae.

Induction of pectolytic enzyme activity in P. sojae race 3

using plant phenolic compounds Four different isoflavonoid compounds were selected to induce pectolytic enzyme activity: genistein, daidzein, formononetin and biochanin A. These flavonoids were examined for induction of pectolytic enzyme activity due to their predominant role in P. sojae -soybean interactions, their presence in the apoplast and the gene inducing activity of flavonoids in other host parasite systems (Peters and Verma, 1990) . Enzyme activity was determined in the mycelium and filtrate portions by activity staining of polyacrylamide electrophoretic gels. Unfortunately, the technique was not sensitive enough to detect any enzyme activity during this set of experiments or these compounds are not inducers of pectolytic enzyme activity in P. sojae. 158

Induction o£ pectolytic enzyme activity in P. sojae

using soybean fractions Various soybean fractions were also used to induce pectolytic enzymes in P. sojae cultures. Lima bean broth was used to grow P. sojae for two days before enzyme induction with the different plant fractions. Four different fractions were used as inducers: 1. Aqueous fraction: This fraction contains proteins, carbohydrates and

some secondary product conjugates. It may also contain cell wall polygalacturonic acids. 2. 80% Ethanol fraction: This fraction contains flavonoids, phenolics and most secondary product and sterol conjugates. 3. Chloroform : methanol fraction: This fraction contains free sterols and lipids. 4. Cell wall fraction: This fraction contains cell walls and most associated cell wall polymers, including the more highly esterified pectic polymers.

Spectrophotometric assays and PAGE were used to detect enzyme activity. Table 14 shows the specific activity of PNL induced with various plant fractions. The best pectolytic enzyme inducer of all plant fractions tested was the aqueous fraction. PNL activity was more highly induced by the aqueous fractions of the susceptible cultivar (H) compared to the resistant cultivar w79. Since pectic acid is soluble in hot water, it is possible that some of the less esterified pectic polymers are in this fraction. Surprisingly, very low activity was obtained using plant cell wall preparations where the more highly esterified pectic fractions should be. Modifications of pectins may occur during the extraction of cell walls. Preserving the integrity of the cell wall during isolation to ensure minimal modifications of structural constituents is important. Studies in 159

Table 14. PNL specific activity in P. sojae race 3 induced using different soybean fractions. Specific activity (U/mg of protein) induced with soybean fractions from W and W„

W w„ Treatments*7 PNL PNL Cell wall (10%) 27 161 254 " (40%) 27 ND 115

Aqueous (10%) 37 6 , 908 1,458 (40%) !7 6,910 1,970 Control 0.67 393 1/ Lima bean media was amended with different plant fractions, among them aqueous and cell wall preparations. 2/ Two treatments consisted of 10 and 40% equivalent of the total cell wall preparation were used. 3/ Two concentrations of aqueous fractions were used; 10 and 40% (v/v) equivalent to the weight of the fresh tissue. 160 the extraction of tomato pectins showed that deesterification of pectin presumably caused by PME occurs during the initial aqueous washes (Koch and Nevins, 1989). Possibly demethylation might occur in hypocotyl cell wall preparations during the initial stages of extraction. If this is the case, it might explain the low PNL specific activity obtained with the cell wall preparation from soybean. The chloroform : methanol and ethanol (80%) fractions did not show any enzyme inducing activity (data not shown). The 80 % ethanol fraction contains flavonoids and phenolics , these findings are consistent with the lack of induction of enzyme activity by the major flavonoids in soybean, daidzein and genistein. Electrophoresis was used to detect enzyme activity induced by different plant cell fractions. Based on color differences after staining with ruthenium red, we should be able to sort out the various pectolytic enzymes produced by P. sojae. Only a faint yellow band was observed in the gels with treatments containing cell wall preparations and aqueous portions from H and W7, cultivars. This indicates the possible induction of some PNL by those plant fractions. However, migration of the protein was constrained and it remained very close to the point where the sample was loaded (Fig. 43) . Thus all of our work in vitro would suggest that P. sojae does not produce a wide array of pectolytic enzymes. PNL seems to be the predominant enzyme produced

Pectolytic enzyme induction in planta Although in vitro studies are of great importance in defining the precise conditions for induction and the various isoenzymes that a microorganism produces, different conditions or signal molecules may stimulate additional enzymes in vivo. Thus, the third approach taken was to detect pectolytic enzyme production in infected tissues. It was shown in previous experiments that PNL was the major enzyme produced in culture by P. sojae. He decided to monitor enzyme activity at six different time intervals: 12, 24, 30, 36, 42 and 48 h post inoculation. PNL activity was 161

1 2

Fig. 43. Schematic of the electrophoretic pattern obtained with polyacrylamide gels. Enzyme activity was induced in P. sojae with soybean fractions. Only a yellow band was seen very close to the point where the sample were loaded, indicating the possible induction of PNL by 1) aqueous 2) cell wall preparation treatments. Treatments containing 10 and 40% equivalent of the total preparation from fresh tissues from W79, respectively are shown. 162 detected in two different soybean organs, hypocotyls and cotyledons infected with P. sojae races 3 and 4. Supernatants of mycelial extracts were used to detect the enzyme through spectrophotometric assays. Consistent with in vitro experiments PNL seems to be the most important pectolytic enzyme involved in soybean tissue maceration as well. The enzyme showed higher activity in cotyledons than in hypocotyls. PNL specific activity was higher in infected tissues than in controls (Figs. 44 to 47) . Very little PNL activity was detected 12 and 24 h after inoculation, this is consistent with the observation on lesion development. No infected area was observed during that period oftime in cotyledons. Although, PNL was detected early during infection, the highest PNL specific activity was detected between 30 and 36 h after inoculation in both organs, the same period during which rapid intercellular growth and tissue maceration occurred. Specifically, enzyme activity was highest in infected cotyledon tissues (section B, Fig. 43) and infected hypocotyl tissue (section B, Fig. 47). This implies that higher enzyme activity was detected in those sections just ahead of the infection front. Again this is consistent with a role in tisBue maceration. For example, at 30 h after inoculation, PNL specific activity was 15,000 U/mg of protein, in cotyledon section B of the compatible response, W7J with P. sojae race 4 and around 6,000 U/mg of protein, in cotyledon section C, 36 h after inoculation. In hypocotyl section B, enzyme activity was approximately 8,000 U/mg of protein in the compatible interaction , W and P. sojae race 4. It was interesting to observe PNL activity (3,000 u/mg of protein) in incompatible responses ahead of the infection front, (eg. cotyledon section B) at 36 h (Fig. 44). Very high activity (15,000 U/mg of protein) was associated with the compatible response at earlier time (3 0 h). This may imply a correlation between enzyme activity and virulencewith p. sojae race 4. However, more experiments are needed in order to clarify these observations.

Generally speaking, all the data evaluated from these experiments 163

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Bhowed a strong correlation between PNL activity and infection, especially with the compatible responses. However, there was not a clear pattern regarding enzyme activity in susceptible vs. resistant cultivars suggesting a role in pathogenesis but not in virulence. Since activity falls off after the tissues became macerated, the data also support all previous observations that indicate that the expression of the enzyme is inducible and transient (ie. under very tight regulation). Lower PNL specific activity was obtained in samples from tissue 12, 24, 42 and 48 h after inoculation. This might be explained in terms of the infection process, since 12 and 24 h after inoculation, tissue maceration has not occurred (Figs. 44 to 47) . The activity observed at these stages might be due to basal levels of enzymes or the smaller amounts of fungal mycelia. By 42 h a decrease in enzyme activity is observed. Apparently, most of the intercellular growth of the pathogen has occurred and enzyme activity would no longer needed. In summary, PNL induction is tightly regulated and corresponds both spatially and temporally with maximal tissue maceration by the pathogen. Induction is somewhat earlier in P. sojae pathogenesis compared with other pathogens. PNL activity was detected at very high levels 30 and 36 h after inoculation of soybean cotyledons and hypocotylB. In P. vulgaris infected with C. 2indemuthianum, for example, PNL activity was detected in lesions 4 days after inoculation (Hijesundera et al. 1989) . Also, high endo-PNL activity was found in vessels of susceptible tomato cells infected with the wilting pathogen V. albo-atrum 3 days after infection (Cooper and Wood, 1980). In some diseases, PNL seems to be very important. For example, symptoms were reproduced in stems and leaves of beans with partially purified endo-PNL from C. lindemuthianum (Cooper and Wood, 1980). In other diseases, it might be a virulence factor. A correlation between the production of PNL in virulent, but not in hypovirulent isolates, of P. solani has been shown (Marcus et al. 1986). PNL is considered to be an 168 extracellular enzyme in many fungi and might be produced at the hyphal tip and secreted to the surrounding environment. Various Phytophthora spp. produce extracellular PNL on solid media (McIntyre and Hankin, 1978). This might help the pathogen in penetration and tissue colonization. Our work with P. sojae is consistent with this notion. Based on our results with PNL in the first set of experiments we decided to examine other pectolytic enzymes induced in infected tissues 36 and 42 h after inoculation to confirm and extend our observations. If PNL is the major early endo-pectolytic enzyme produced to break up polymers and allow intercellular growth, it seemed possible that additional enzymes might be induced at later times to further break up the polymers into monomeric sugars for P. sojae metabolism. Four different enzyme activities were studied in infected tissues: PNL, PL, PG and PME. Specific activity was examined in infected cotyledons and hypocotyls using the same soybean- P. sojae interactions described above. In summary, PNL again showed the highest activity in infected tissue compared to controls. Specific activities of PG, PL and PME compared to PNL in infected tissues were extremely low (Tables 15 to 22). PL activity in the controls waB very high compared to its activity in infected tissues at 36 h after inoculation (Table. 15). This suggested that the activity we detected was due mainly to PL of host origin. It is probable that highly esterified pectins are present in soybean cell walls and PL is not important in terms of its pathogenicity of P. sojae. PG specific activities were slightly higher at 36 than at 42 h after inoculation and were detected in control tissues as well (Tables 17 and 18) . It was interesting to observe that PG activity was higher in culture than in infected tissues. The lower PG activity in infected tissues might 169

Table 15. PL specific activity (U/mg of protein) from cotyledons and hypocotyls 36 h after inoculation with P. sojae (races 3 and 4).

Sample R3 R4 Control

W Cot A 00 00 1, 078 " B 56 00 79 " C 203 00 294 W Hyp A 00 00 00 " B 00 98 00

W7 9 Cot A 173 15 430 II B 00 30 132 II C 85 00 223 W79 Hyp A 00 66 00 n B 00 00 37

Table 16. PL specific activity (U/mg of protein) from cotyledons and hypocotyls 42 h after inoculation with P. sojae (races 3 and 4).

Sample R3 R4 Control

W Cot A 00 52 00 " B 00 66 00 " C 00 00 00 W Hyp A 00 100 00 " B 00 00 00

W79 Cot A 74 00 96 II B 82 89 00 II C 00 20 00 W79 Hyp A 00 00 00 II B 00 00 00 17 0

Table 17. PG specific activity (U/mg of protein) from cotyledonB and hypocotyls 36 h after inoculation with P. sojae.

Section R3 R4 Control

W Cot A 0.214 0.432 0.322 " B 0.239 0.211 0.187 " C 0.331 0.496 0.310 W Hyp A 0.225 0.461 0.248 " B 0.166 0.229 0.219 W79 Cot A 0.394 0.399 0.720 II B 0.373 0.308 0.133 II C 0.515 0.491 0.648 W79 Hyp A 0.140 0.158 0.212 ii B 0.427 0.255 0.287

Table 18. PG specific activity (U/mg of protein) from cotyledons and hypocotyls 42 h after inoculation with P. sojae.

Section R3 R4 Control

W Cot A 0.046 0.021 0.074 " B 0.025 0.012 0.438 " C 0.448 0.006 0.130 W Hyp A 0.009 0 . 0 0 0 0.161 " B 0.067 0.009 0.365 W7 9 Cot A 0.324 0.031 0.360 II B 0.035 0.005 0.118 It C 0.513 0.145 0.544 W79 Hyp A 0.048 0 . 0 0 0 0.526 ii B 0.052 0.005 0.113 171

be due to a endo-FG inhibitor £actor (PGIF) that has been shown to be produced by plant cells. PGIF has been isolated from cell walls of tomato Btems infected with Fusarium oxysporum f.sp. lycopersici (Jones et al. 1972) and from cell walls of P. vulgaris infected with C. lindemuthianum (Anderson and Albersheim, 1972). Also, an endo-pectin lyase inhibiting factor has been reported (Brown, 1984 and Brown and Adikaram, 1983) . Inhibitor factors can play a crucial role in infection process as well as in pathogenicity. These factors greatly influence the rate of rot development in plant tissues (Brown and Adikaram, 1983) . PG and PL seem to be much less effective than PNLs in degrading cell walls (Baldwin and Pressey, 1989). The relative ineffectiveness of these enzymes might be due to highly esterified pectin in some plant cell walls which would not be susceptible to their action. Probably, the prior action of a PME is needed in order for PL and PG to hydrolyze soybean pectic fractions. PME showed the lowest activity of all enzymes tested. Its activity was extremely low in infected tissues (Tables 19 to 22). It's specific activity was higher in hypocotyls ranging from 0.191 to 0.601 U/mg of protein than in cotyledons (range from 0.095 to 0.428), 42 h after inoculation. This result might be due in part to PME from plant origin. To try to separate plant from fungal PME we used a different pH indicator with lower pH to run the assay. Fungal PME's have lower pH optima than the plant enzymes. The results showed essentially no PME activity 36 and 42 h after inoculation (Tables 21 and 22}. This supports that the PME activity we were detecting was mainly from plant origin. In conclusion, PME might not play an important role in P. sojae pathogenicity.

In summary, PL, PG and PME do not show significant activity in planta based on the data analyzed. These findings confirm the results obtained with our experiments in culture and suggest that although P. sojae produces PNL, a very effective enzyme for dissolution of the middle lamella and thus intercellular colonization, little PME, PL or PG are 172

Table 19. PME specific activity (U/mg of protein) in soybean cotyledons and hypocotyls 36 h after inoculation with P. sojae (races 3 and 4)^.

Section R3 R4 Control

W Cot A 0.002 0.012 0.000 " B 0.012 0.027 0.000 " C 0.013 0.007 0.000 W Hyp A 0.034 0.291 0.000 " B 0.091 0.083 0.000

W79 Cot A 0.037 0.047 0.000 II B 0.000 0.000 0.004 II C 0.000 0.000 0.000 W79 Hyp A 0.035 0.017 0.160 n B 0.101 0.012 0.000 1/ Enzyme activity was detected using the pH indicator bromothymol blue at 620 nm. Assay was performed at pH = 7.5

Table 20. PME specific activity (U/mg of protein) in soybean cotyledons and hypocotyls 42 h after inoculation with P. sojae (races 3 and 4)^.

Section R3 R4 Control W Cot A 0.286 0.321 0.428 11 B 0.238 0.076 0.217 " C 0.114 0.241 0.084 W Hyp A 0.390 0.473 0.418 " B 0.601 0.452 0.191 W79 Cot A 0.463 0.405 0 . 1 1 1 II B 0.095 0.158 0.240 II C 0.095 0.258 0.291 W79 Hyp A 0.231 0.545 0.195 ii B 0.395 0.403 0.407 1/ Enzyme activity was detected using the pH indicator bromothymol blue at 620 nm. Assay was performed at pH = 7.5 173

Table 21. PME specific activity (TJ/mg of protein) in soybean cotyledons and hypocotyls 36 h after inoculation with P. sojae (races 3 and 4)

Section R3 R4 Control

W Cot A 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0

" B 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 " C 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 W Hyp A 0.055 0.032 0 . 0 0 0 " B 0.032 0.022 0 . 0 0 0

W79 Cot A 0.000 0.000 0.000 II B 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 It C 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 W79 Hyp A 0.012 0.013 0 . 0 0 0 n B 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 1/ Enzyme activity was detected using the pH indicator bromo cresol purple at 570 nm. Assay was performed at pH = 6.5

Table 22. PME specific activity (U/mg of protein) in soybean cotyledons and hypocotyls 42 h after inoculation with P. sojae (races 3 and 4)

Section R3 R4 Control

W Cot A 0 . 0 0 0 0.044 0 . 0 0 0 " B 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 ii c 0.000 0.000 0.000

W Hyp A 0.053 0 . 0 0 0 0 . 0 0 0 " B 0.048 0.044 0 . 0 0 0

W7 9 Cot A 0 . 0 0 0 0 . 0 0 0 0.029 It B 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 tl C 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 W79 Hyp A 0.018 0 . 0 0 0 0 . 0 0 0 n B 0.044 0 . 0 0 0 0.026

1/ Enzyme activity was detected using the pH indicator bromo cresol purple at 570 nm. Assay was performed at pH = 6.5 174 produced. Taken together with our studies on P. sojae utilization of pectic polymers as sole carbon source, these studies suggest that P. sojae may not effectively utilize pectic polymers as a source of monomeric sugars.

Plant and fungal enzymes separation

IF6 was used to separate pectolytic enzymes from host and pathogen origin. The various PNL isoenzymes that were separated based on their pi are shown in Fig. 48. Very complex enzyme patterns were observed in infected soybean tissue extracts especially in the compatible interaction

(W73 race 4 and W race 3). Around 12 to 13 bands can be differentiated in the compatible interactions. In P. sojae filtrate from pure culture seven major bands were observed at pi around 5 and 6 . The enzymes were induced in culture 5 days after inoculation on lima bean broth amended with pectin from citrus. When we compared the enzyme patterns between infected tissues and enzymes induced in pure culture, approximately 7 bands appeared to be from pathogen origin. These enzymes were consistently present in compatible infections but were not seen in control host tissues or incompatible infections. This is very interesting and may confirm the role of these enzymes in tissue maceration. Some activity was observed in the control tissue especially at pi (3.9 to 4.2). However, further experiments are needed to clearly differentiate host from pathogen enzymes. 175

Fig. 48. Schematic of the electrophoretic pattern obtained using IF (pH range 3-10) from soybean cotyledon tissues infected with P. aojae races 3 and 4. Fungal PNL isoenzymes pattern from pure culrure filtrate are also shown. 1) Sample from P. sojae race 3 pure culture (filtrate). 2) Extract from W7, control (no infected) . 3) Extract from W79 infected with P. aojae race 3 (Incompatible response). 4) Extract from W7, infected with race 4 (compatible response). 5) Extract from w control. 6) Extract from W infected with race 3. Solid lines : high enzyme activity; broken lines: weak activity. Gels were loaded with 200 fxl concentrated crude extract. 176

DISCUSSION In order to facilitate the discussion an overview of all our investigation will be presented to help us comprehend the results of our microscopic, in planta and in culture experiments . A model on the importance of P. sojae PNL in pathogenicity will be proposed based on our findings.

Light and electron microscope studies of the interactions of compatible and incompatible isolates of P. aojae with soybean cultivars Studies on the histology of P. sojae - soybean interactions in hypocotyl tissues have been done (Stossel et al. 1980 and 1981). However, soybean cell wall degradation caused by this pathogen has not been investigated until now. Based on our studies on soybean-P. sojae interactions, host cell wall degradation in the incompatible and compatible responses at earlier stages of infection, less than 30 h, reflect that these are basically the same. The real differences in wall degradation occur after 30 h, consistent with the rapid colonization and maceration of tissues in compatible responses at that time. The evidence presented support the idea that differences between susceptible and resistant responses are quantitative rather than qualitative and that cell wall degradation may play a role in pathogenicity rather than virulence. Electron micrographs (Plate VIII) showed that P. sojae growth is primarily intercellular regardless of the type of interaction. Highly localized cell wall degradation in cotyledon tissues was also associated with fungal infection pegs and haustorial development (Plate XI and XII). Haustoria appeared to grow and form within host cells. Regardless of the interaction, P. sojae seems to exhibit an initial period of biotrophic intercellular growth followed by limited penetration of cells without killing the infected cells. The electron micrographs suggest that the pathogen may produce very low levels of pectolytic enzymes during early colonization and penetration. These enzymes might be produced at the 177 hyphal tips and might be wall bound and/or selectively secreted. Following the slow initial stage of colonization and cellular penetration (0 - 30 hrs), the pathogen grows very rapidly in compatible but not in incompatible interactions, causing extense maceration of tissues. Thus, the microscope work suggests that the early stages of infection are similar in compatible and incompatible interactions, followed by an explosive intercellular growth of the fungus in compatible but not incompatible interactions. The determinative events leading to resistance or susceptibility seem to occur at about 30 h, consistent with the defense responses studied (T.L. Graham and H.Y. Graham, 1991). Even though such extensive maceration of tissues is part of P. sojae symptoms in susceptible cultivars, no attempts have been made to measure pectolytic enzyme activity in this fungus or during infection of soybean tissues until now.

In planta experiments The data analyzed in our in vivo experiments imply a role for a P. sojae PNL that appears to be produced at the hyphal tip and rapidly secreted to the extracellular environment. Although limited amounts of the enzyme are produced very early in infection, consistent with the early stages of infection noted from microscope observations, the majority of the enzyme activity is associated with tissue maceration ahead of the infection front. All of our investigations (microscopic, in culture and in plant) support this hypothesis. Although high PNL activity correlates to pathogenesis, it was interesting to detect its activity in control tissues. These findings suggest that enzymes from host origin may be involved in cell wall degradation during pathogenesis and that we were looking at a composite response. In studies on the interactions between

Pseudomonas syringae pv. phaseolicola and french beans, PG activity appeared to have two different origins, partly from the bacteria and the other from the host or as the result of the interaction (Longland et al., 178

1992). However, they were able to observe differences in PG activity between susceptible and resistant cultivars. Unfortunately, our data do not allow us to observe differences between susceptible or resiBtant cultivars and more detailed experiments are needed. The somewhat erratic data obtained in our in vivo experiments might be explained by the production of various isoenzymes that might be induced under different conditions from pathogen and host origin. Alternatively, it may be explained by the fact that the production and secretion of these enzymes seems to be under very tight regulation, occurring only in a narrow window of time. Some evidence on the complexity of fungal enzymes was obtained through IFG electrophoresis of extracts from fungal pure culture. Although we were unable to separate all PNL isoenzymes from host and pathogen origin clearly, it seems clear that several of the isoenzymes are mainly from pathogen origin, particularly those in compatible infections. The complexity observed might also be due to specificities reflected by polymer esterification and glycosidic linkages and anomeric configuration (a and S) among others. In terms of plant cell walls, the number of possible variations in plant cell wall polysaccharide structure is very large (Albersheim et al., 1969). Developing tissues undergo drastic changes in terms of their sugar composition during the first weeks of development. Cell walls of susceptible bean seedlings can be degraded by R. solani enzymes, but not cell walls from older plants (Bateman et al. 1969). Likewise, resistance and susceptibility in the soybean-P. sojae system occur in a short period of time during plant development. Very young seedling leaves are universally susceptible to P. sojae races, and later on they develop race specific resistance. Old plant leaves in contrast show universal resistance (Ward, 1989). Thus, changes that occur during development and maturation of soybean plants are crucial in P. sojae pathogenicity and might be cell wall related. For example, during the normal process of tissue maturation, plant pectins are converted to calcium pectate, changing the neutral sugar composition of cell walls 179

(Ride, 1983). In studies with Botrytis cinerea, PG and PNL were detected in infected swede bulb tissues and also in culture. Studies measuring the Ca*2 release during pathogenesis suggest that the pathogen has the potential to release Ca*2 bound to the pectic substances in the middle lamella during host tissue maceration. Ca*3 is responsible for cell wall shrinkage and swelling which occurs when it is detached from pectates present in the middle lamella. The swelling observed in the middle lamella in the presence of fungal hyphae might be due to the disorganization of the cell wall with the subsequent release of Ca*2 (Kaile et al. 1991) . Also deposition of lignin or related substances can be involved in the resistance of older tissues to enzymatic degradation (Ride, 1983). For example, a PG from Cladosporium cucvmericum elicits lignification in cucumber hypocotyls in susceptible and resistant cultivars (Robertsen, 1987) . Thus, many events occurring at the plant cell as well as at the pathogen cell level complicate the picture of resistance and susceptibility, and they need to be carefully taken into account to understand plant pathogenesis. Pectic-derived signals in the environment are recognized by both the plant and the pathogen serving different roles. In the pathogen they may act as signals involved in pathogenicity like the acceleration of the encystment and germination of zoospores and at the same time turn on geneB that allow the pathogen to invade host tissues (Grant, 1985). In the host those molecules can act as signals of pathogen invasion turning on defense genes. As we mentioned before, phytoalexin responses can be induced by the presence of plant cell wall oligomers and that response will depend upon oligomers size.

In culture experiments

The first experiments done using different pectic fractions as sole

carbon source showed that P. sojae races preferred pectins over polygalacturonate fractions for growth. Enzyme induction of P. sojae grown 180 on various commercial pectic fractions and soybean hypocotyls showed that the pathogen was able to produce various pectolytic enzymes to break down the pectic polymers. However, of all enzymes tested, PNL was the major enzyme produced by P. sojae in culture consistent with its preferential utilization of pectin as a carbon source. High PNL specific activity was detected in culture filtrates and mycelium at very early stages of growth especially when pectin or soybean dry hypocotyls were used as inducers. Unfortunately, we were not able to test enzyme induction using purified soybean pectin due to the large amounts needed and its low yield from this herbaceous plant. Instead, pectin from two different sources: apple and orange were used. When we tested commercial pectins varying in the degree of methylation we found that orange pectin was a stronger inducer of extracellular PNL than apple pectin. Orange pectin appeared to be a better PNL inducer for the isolate of race 3 examined than for the isolate of race 4. In summary, P. sojae produces limited and specialized enzymes and does not seem to use pectic polymers effectively as a carbon source.

A model on PNL role in P. sojae pathogenicity Taking all of our results into account, a model can be proposed to explain the importance of PNL in pathogenicity in the soybean-P. sojae system, as well as in the hypersensitive response. Pectate lyase has been shown to enhance soybean phytoalexin, glyceollin, accumulation along with fungal glucan elicitors from the P. sojae cell wall (Nothnagel et al., 1983). Elicitors from soybean cell wall origin can in fact, be generated by the exogenous application of pectolytic enzymes (Darvill and Albersheim, 1984, Darvill et al., 1984 and Davis et al., 1986). In addition, T.L. Graham and M.Y. Graham, (1991) reported several factors that seem to be involved in the accumulation of the soybean phytoalexin, glyceollin, in soybean tissues after treatment with P. sojae wall glucan. One factor was the cotreatment with pectate lyase. These support a possible role for PL in the elicitation of plant defense responses to 181 pathogen attack in incompatible interactions. On the other hand, no studies to actually measure PL in infected tissues have been carried out. Our studies suggest that P. sojae, at least, produces PNL and little PL. Moreover, it produces little if any PME, Thus, the oligomers released by P. sojae are likely to be pectin oligomers and not pectate oligomers. Pectin oligomers are very poor elicitor synergists (T.L. Graham, unpublished). Thus, P. sojae may minimize the induction of host defenses by effective wall dissolution and the lack of elicitor release. It has been further hypothesized that elicitation of plant defenses in soybean cells depends upon an optimal size of released pectic oligomers (Nothnagel et al., 1983). Host endo-PG inhibitor proteins have been suggested to play a crucial role in limiting the rate of digestion of pectic fractions by PL, leading to more effective elicitor molecules. In compatible interactions PNL might thus play a doubly important role in P. sojae pathogenicity. Importantly, this enzyme would not be sensitive to endo-PG inhibitor protein. Thus, P. sojae could act on pectin polymers without interference and reduce the oligomers to shorter molecules that show lower or no elicitation of phytoalexin in the invaded tissues even if they were consequently demethylated. Finally, PNL is the only enzyme known to be able to cleave pectin polymers without the prior action of other enzymes. This is very important in termB of pathogenesis. It can be an added advantage over PL and PG that needs the action of PME in order to hydrolyze pectin (Baldwin and Pressey, 1989).

Further studies are need to explain the role of P. sojae PNL in soybean infection. It will be interesting to purify P. sojae PNL and use it in immunocytochemistry studies to locate in situ the production of this enzyme in the pathogen and to follow its secretion during soybean infection. The method has been applied to detect pectic fractions in tomato tissues infected with F. oxysporum f.sp. radicis-lycopercisi

(Benhamou et al., 1990). They precisely detected the accumulation of 182 galacturonlc acid-rich molecules at cell wall appositions and intercellular spaces during pathogen invasion. Further studies trying to detect PNL activity in situ will be valuable in order to understand the mechanisms of P. sojae invasion of soybean tissues. It will also be interesting to actually follow the products of digestion of cell walls in planta by P. sojae and to evaluate their activity as elicitors or elicitor synergists. If our model is correct, we would expect that P. sojae infection will lead to minimal release of elicitor-active fragments. Another tool that can be used to determine the role of P. sojae PNL, would be to raise antibodies against the purified enzyme and block its activity in order to reduce cell wall disruption and pathogenesis. This technique was used by Crawford and Kolattukudy (1987) to inhibit the activity of a PL from Fusarium solani f.sp. pisi on pea stems. The ability of the pathogen to infect pea stems was reduced by 80%. 183

CONCLUSIONS

In conclusion, the results from our studies on pectolytic enzymes produced by P. sojae in soybean tissues suggest a possible role for a PNL in infection. In culture and in plant studies support that a PNL appears to be produced at the hyphal tips and to be rapidly secreted to the extracellular environment. Its detection in cotyledon and hypocotyl tissues 30 and 36h after inoculation also suggests that it is produced during the rapid colonization phase of infection. It seemB to be responsible for the tissue maceration ahead of the infection front. There was a strong correlation between high PNL activity and P. sojae pathogenicity. However, we were not able to demonstrate any race specificity or any PNL role in P. sojae virulence. Although we cannot rule out the possibility that a unique PNL isoenzyme is produced by the fungus or host in compatible interactions that allows rapid colonization by the fungus, the determinative phase of resistance seems to occur before 30 h, and it is likely that the fungus in incompatible interactions is simply severely inhibited or dead by this time. All other pectolytic enzymes studied (PG, PL and PME) did not show significant activity under the experimental conditions of these studies. Apparently they do not play an important role in P. sojae pathogenicity and/or virulence. In fact, as discussed above, the lack of these activities may serve as a distinct advantage for P. sojae in that it would limit the elicitation of the soybean phytoalexin, glyceollin. 184

GENERAL CONCLUSIONS

P. sojae has developed the ability to recognize the plant defense responses and to successfully survive them in the compatible interactions.

In incompatible interactions, the pathogen is also able to colonize the plant tissues but to a lesser extent. Various processes that are taking place in a very short time, at both sides of the interaction, will determine the final result. The P. so j ae - soybean system has shown to be an excellent model to study biochemistry of host-pathogen interactions, and a lot of information has been generated in terms of the biochemistry of the host. With all this in mind we were determined to study in a broader sense the biochemistry of the pathogen. First we looked at the array of plant natural compounds that might have some potential as plant defense molecules. Studies using plant natural compounds in vitro showed that P. sojae possesses the basic mechanisms to deal with many of the phenolic compounds present in plants. An array of different enzymes (eg. glycosidases, esterases, peroxidases) appear to be produced in order to metabolize partially or completely these phenolic compounds. Secondly, P. sojae's basic mechanisms for tissue penetration and colonization appear to be extremely efficient. P. sojae probably produces only one kind of pectolytic enzyme, PNL, minimizing the induction of host defense. It seems that even though the plant has evolved the ability to recognize the pathogen and react to its presence, the pathogen has evolved the ability to maximize its efforts to cause disease in the compatible interactions. Even though we were unable to clarify any role of these

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198 Table 23. P. sojae growth (race 1) on lima bean medium amended with various concentrations of apigenin. Race 1 Growth per day (mm)

Concentrations (jxM) 0 1 2 3 4 5 6 500 0 0.5 1.3 5 10 15 27 200 0 0.2 6.0 20 48 80 80 125 0 1.0 7.1 27 59 80 80 62.5 0 1.3 15.4 70 80 80 80 31.2 0 1.3 18.5 70 80 80 80 15 0 1.1 18.5 65 80 80 80

Table 24. P. sojae (race 3) growth on lima bean medium amended with various concentrations of apigenin Race 3 iGrowth per day (ran)

Concentrations (/xM) 0 1 2 3 4 5 6

500 0 0.03 0. 03 1 3 6 7 200 0 0.52 1 5 11 14 20 125 0 0.79 5 12 29 43 59 62.5 0 3.14 10 48 80 80 80 31.2 0 0.79 12 45 80 80 80 15 0 0.79 11 45 80 80 80

Table 25. P. sojae (race 12) growth on lima bean medium , amended with various concentrations of apigenin

Race 12 Growth per day (mm)

Concentrations (/xM) 0 1 2 3 4 5 6

500 0 0.0 00 00 00 00 00 200 0 0.4 5 18 25 38 43 125 0 4 26 62 80 59 80 62.5 0 5 45 80 80 80 80 31.2 0 6 50 80 80 80 80

15 0 5 43 80 80 80 80 199 Table 26. P. sojae (race 1) growth on lima bean medium amended with various concentrations of biochanin A. Race 1 Growth per day (mm)

Concentrations (fiM) 0 1 2 3 4 5 6

500 0 0.3 0.5 0.6 0.6 0.6 0.6 200 0 0.8 0.9 5 15 11 15 125 0 0.6 60 18 43 56 65 62.5 0 1.3 13 33 53 80 80 31.2 0 1.3 14 38 70 80 80 15 0 1.3 24 65 80 80 80

Table 27. P. sojae (race 3) growth on lima bean medium amended with various concentrations of biochanin A. Race 3 Growth per day (nan) Concentrations (^H) 0 1 2 3 4 5 6

500 0 0.00 0.03 0.0 00 00 0.0 200 0 0.00 0.94 6.9 11 14 5.1 125 0 0.03 6.0 16.9 36 36 80 62.5 0 0.15 11.3 31.4 62 80 80 31.2 0 0.31 14.0 40.4 65 80 80 15 0 0.05 9.0 27.4 53 80 80

Table 28. P. sojae (race 12) growth on lima bean medium amended with various concentrations of biochanin A. Race 12 Growth per day (mm)

Concentrations (y.M) 0 1 2 3 4 5 6

500 0 0.0 0.0 00 00 00 00 200 0 0.2 8.1 17 28 45 49 125 0 0.6 7.8 38 68 80 80 62 .5 0 0.5 25 65 80 80 80 31.2 0 0.5 31 80 80 80 80 15 0 0.5 22 56 80 80 80 200 Table 29. P. sojae (race 1} growth on lima bean medium amended with various concentrations of coumestrol. Race 1 Growth per day (mm)

Concentrations (fiM) 0 1 2 3 4 5 6 500 0 0.0 00 00 00 0.5 0.6 200 0 0.3 8 20 38 80 80 125 0 0.9 17 50 80 80 80 62.5 0 3.6 29 68 80 80 80 31.2 0 1.3 14 36 59 80 80 15 0 1.8 24 71 80 80 80

Table 30. P. sojae (race 3) growth on lima bean medium amended with various concentrations of coumestrol. Race 3 Growth per day (mm) Concentrations {/iM) 0 1 2 3 4 5 6 500 0 0.0 0.0 0.0 0.0 0.2 2.3 200 0 0.3 0.2 2.3 9.3 8 9.0 125 0 0.9 5 16 27 46 66 62.5 0 3.6 10 34 53 80 80 31.2 0 1.3 7 24 45 80 80 15 0 1.8 14 56 80 80 80

Table 31. P. sojae (race 12) growth on lima bean medium amended with various concentrations of coumestrol. Race 12 Growth per day (mm) Concentrations (nM) 0 1 2 3 4 5 6

500 0 0.0 00 00 00 00 00 200 0 0.4 8 19 26 56 56 125 0 1 17 38 53 80 80 62 .5 0 6 25 56 74 80 80 31.2 0 9 59 80 80 80 80 15 0 10 59 80 80 80 80 Table 32. P. sojae (race 1) growth on lima bean medium amended with various concentrations of chrysin. Race 1 Growth per day (mm) Concentrations (^iM) 0 1 2 3 4 5 6 500 0 0.000 0.3 1.8 3.6 4.5 9.4 200 0 0.002 18 23 27 40 74 125 0 0.000 22 42 56 80 80 62.5 0 0.001 11 45 77 80 80 31.2 0 0.107 2.5 18 28 65 80 15 0 0.31 6.0 14 30 19 80

Table 33. P. sojae (race 3) growth on lima bean medium amended with various concentrations of chrysin. Race 3 Growth per day (am)

Concentrations (ftM) 0 1 2 3 4 5 6 500 0 0.00 0.3 1.3 1.3 5.0 7.3 200 0 0.10 1.3 7.9 11 15 24 125 0 0.31 10 31 50 68 77 62.5 0 0.62 15 59 80 80 80 31.2 0 2.3 20 65 80 80 80 15 0 1.3 20 59 80 80 80

Table 34. P. sojae (race 12) growth on lima bean medium amended with various concentrations of chryi Race 12 Growth per day (am) Concentrations (|xM) 0 1 2 3 4 5 6

500 0 0.0 00 00 1 3 7 200 0 0.6 13 40 65 32 80 125 0 64 454 80 80 80 80 62 .5 0 69 452 80 80 80 80 31.2 0 69 530 80 80 80 80

15 0 90 530 80 80 80 80 202 Table 35. P. sojae (race 1) growth on lima bean medium amended with various concentrations of formonone tin. Race 1 Growth per day (mm)

Concentrations (/iM) 0 1 2 3 4 5 6 500 0 4 8 1.3 1.3 1.3 3.8 200 0 0.3 8 18 54 53 69 125 0 1.3 13 38 80 80 80 62.5 0 1.3 18 56 80 80 80 31.2 0 1.3 18 56 80 80 80 15 0 1.3 31 71 80 80 80

Table 36. P. sojae (race 3) growth on lima bean medium amended with various concentrati formonone tin. Race 3 Growth per day (mm) Concentrations (jxK) 0 1 2 3 4 5 6 500 0 0.0 0.0 0.0 0.0 0.0 1.3 200 0 0.0 0.9 6.2 7.8 11 17 125 0 0.79 6.0 18 40 80 80 62.5 0 2.36 11 34 56 80 80 31.2 0 1.57 13 38 71 80 80 15 0 0.79 13 48 80 80 80

Table 37. P. sojae (race 12) growth on lima bean medium amended with various concentrati formononetin. Race 12 Growth per day (mm) Concentrations (^M)

500 0 0.0 0.0 00 00 00 00 200 0 0.2 7.8 25 40 56 71 125 0 2.8 20 53 71 77 80 62.5 0 2.5 43 80 80 80 80 31.2 0 9.0 56 80 80 80 80 15 0 9.0 41 80 80 80 80 to o u> Table 38. P. sojae (race 1) growth on lima bean medium amended with various concentrations of genistein. Race 1 Growth per day (mn) Concentrations (fiM) 0 1 2 3 4 5 6 500 0 0.1 0.6 0.6 1.2 1.3 1.6 200 0 0.3 7.8 18 31 53 71 125 0 0.4 11 31 61 80 80 62.5 0 1.3 15 34 59 80 80 31.2 0 1.3 20 59 80 80 80 15 0 1.3 25 59 80 80 80

Table 39. P. sojae (race 3) growth on lima bean medium amended with various concentrations of genistein Race 3 Growth per day (mm) Concentrations (fiM) 0 1 2 3 4 5 6

500 0 2.2 1.3 0.0 0.0 0.2 1.4 200 0 0.0 0.3 0.3 5.0 53 10 125 0 0.08 7.8 25 48 80 80 62.5 0 0.23 11 20 53 80 80 31.2 0 0.08 11 31 68 80 80 15 0 0.16 16 38 68 80 80

Table 40. P. sojae (race 12) growth on lima bean medium amended with various concentrations genistein. Race 12 Growth per day (nan)

Concentrations (j*M) 0 1 2 3 4 5 6

500 0 0.000 0.0 0.0 00 00 00 200 0 0.002 1.5 9.0 20 36 45 125 0 0 .89 14 31 65 80 80 62.5 0 2.5 34 80 80 80 80 31.2 0 5.0 31 63 80 80 80 15 0 2.8 22 71 80 80 80 204 Table 41. P. sojae {race 1) growth on lima bean medium amended with various concentrations o£ isorhaametin, Race 1 Growth per day (mm) Concentrations (pM) 0 1 2 3 4 5 6

500 0 0.000 0.00 0.0 0.0 00 0.9 200 0 0 .001 0.31 2.3 4.7 11 13 125 0 ------62.5 0 0.83 4 34 41 42 46 31.2 0 0.16 16 36 62 65 80 15 0 0.44 17 30 53 62 80

Table 42. P. sojae (race 3) growth on lima bean medium amended with various concentrate isorhaametin. Race 3 Growth per day (mm) Concentrations (pK) 0 1 2 3 4 5 6

500 0 0.00 0.0 0.0 0.0 00 4.3 200 0 0.03 0.2 1.8 4.2 9 10 125 0 0.21 2.0 11 24 48 80 62.5 0 0.23 5.2 16 43 77 80 31.2 0 0.16 10 34 65 74 80 15 0 0.10 27 36 80 80 80

Table 43. P. sojae (race 12) growth on lima bean medium amended with various concentrate isorhaametin. Race 12 Growth per day (ana) Concentrations {pti) 0 1 2 3 4 5 6

500 0 0.0 00 00 0.0 00 1.2 200 0 0.0 00 0.1 0.5 2 2.5 125 0 0.6 13 46 74 80 80 62.5 0 0.5 17 68 80 80 80 31.2 0 1.8 34 77 80 80 80 15 0 2.2 36 68 80 80 80 too U1 Table 44. P. sojae (race 1) growth on lima bean medium amended with various concentrations o£ isoquercetrin. Race 1 Growth per day (mm)

Concentrations (jjM) 0 1 2 3 4 5 6 500 0 0.0 0.01 2 5 8 12 200 0 0.2 4 18 41 56 80 125 0 2.1 18 71 80 80 80 62.5 0 0.3 20 74 80 80 80 31.2 0 2 . 0 28 80 80 80 80 15 0 0.6 26 80 80 80 80

Table 45. P. sojae (race 3) growth on lima bean medium amended with various concentrations isoquercetrin. Race 3 Growth per day (mm) Concentratiisns (pili) 0 1 2 3 4 5 6 500 0 0.03 0.1 0.5 3.5 4.3 9 200 0 0.21 4 11 15 29 41 125 0 0.24 11 34 50 71 80 62.5 0 0.55 14 45 80 80 80 31.2 0 1.2 22 62 80 80 80 15 0 0.9 18 53 80 80 80

Table 46. P. sojae (race 12) growth on 1 ima bean medium amended with various concentrations i soquercetrin. Race 12 Growth per day (mm) Concentrations {fiM) 0 1 2 3 4 5 6 500 0 0.0 0.0 00 00 00 1.5 200 0 0.6 7 . 8 15 38 54 77 125 0 4.3 27 62 77 80 80 62 .5 0 5.2 36 68 80 80 80 31.2 0 4.5 41 80 80 80 80 15 0 5.2 51 80 80 80 80

CTl Table 47 . P. sojae (race 1) growth on lima bean medium amended with various concentrations of Kaempferol. Race 1 Growth per day (nan) Concentrations (fiM.) 0 1 2 3 4 5 6 500 0 3.9 21 45 77 87 83 200 0 2.8 27 62 81 95 98 125 0 5.3 33 71 84 81 91 62 .5 0 5.7 28 71 88 88 88 31.2 0 6.9 40 71 77 84 92 15 0 6.6 41 71 84 84 98

Table 48. P. sojae (race 3) growth on lima bean medium amended with various concentrati kaempferol. Race 3 Growth per day (nan) Concentrations (ftM) 0 1 2 3 4 5 6 500 0 2.1 19 43 77 81 91 200 0 5.0 29 59 84 77 91 125 0 5.0 26 53 92 85 102 62 .5 0 4.5 28 68 85 85 98 31.2 0 5.0 32 74 84 91 91 15 0 5.7 31 71 109 109 109

Table 49. P. sojae (race 12) growth on lima bean medium amended with various concentrati kaempferol. Race 12 Growth per day (am) Concentrations (piM)

500 0 11 53 91 98 98 98 200 0 6.6 48 98 106 106 113 125 0 8.8 41 87 94 94 94 62.5 0 7.7 40 91 92 102 102 31.2 0 3.5 31 91 91 91 91 15 0 5.6 41 87 106 106 106 to o Table 50. P. sojae (race 1) growth on lima bean medium amended with various concentrations of naringenin* Race 1 Growth per day (mm) Concentrations (fi M) 0 1 2 3 4 5 6 500 0 1.3 1.6 6 9 22 26 200 0 1.6 9 13 20 43 48 125 0 2.6 27 27 53 80 80 62.5 0 5.0 36 40 74 87 91 31.2 0 5.0 20 62 80 94 94 15 0 5.0 18 62 80 80 84

Table 51. P. sojae (race 3) growth on lima bean medium amended with various concentrati^ naringenin. Race 3 Growth per day (am) Concentrations (piM) 0 1 2 3 4 5 6 500 0 1.3 3 3 5 8,8 12 200 0 1.6 7 17 20 50 61 125 0 2.5 16 57 61 80 80 62.5 0 2.5 15 43 77 91 94 31.2 0 2.8 16 43 77 87 94 15 0 3.3 11 50 91 94 98

Table 52. P. sojae (race 12) growth on lima bean medium amended with various concentrati* nar xngenin. Race 12 Growth per day (mm) Concentrations (^M) 0 1 2 3 4 5 6 500 0 0.0 0.9 1.9 5.6 8 10 200 0 0.7 3.8 11 18 53 59 125 0 0.7 13 38 74 87 87 62.5 0 1.6 20 71 84 91 94 31.2 0 1.6 32 71 81 81 81 15 0 2.3 24 77 84 84 84 oto 00 Table 53. P. sojae (race 1) growth on lima bean medium amended with various concentrations o£ quercetin. Race 1 Growth per day (mm)

Concentrations (.fM) 0 1 2 3 4 5 6 500 0 3.1 13 22 48 62 84 200 0 5.7 35 71 81 92 92 125 0 1.0 15 24 65 85 98 62.5 0 2.5 71 84 91 94 94 31.2 0 5.2 32 61 106 110 110 15 0 6.6 41 71 84 84 98

Table 54. P. sojae (race 3) growth on lima bean medium amended with various concentrations o£ quercetin. Race 3 Growth per day (am) Concentrations (/M ) 0 1 2 3 4 5 6 500 0 1.7 10 15 25 61 84 200 0 2.9 11 19 30 53 69 125 0 2.6 13 21 50 63 91 62.5 0 3.1 56 45 84 91 91 31.2 0 2.1 20 45 84 85 98 15 0 3.7 29 56 84 94 98

Table 55. P. sojae (race 12) growth on lima bean medium amended with various concentrations of quercetin. Race 12 Growth per day (mm) Concentrations (fj.M) 0 1 2 3 4 5 6 500 0 4.6 48 91 91 94 94 200 0 6.1 48 91 91 91 91 125 0 4.8 38 74 77 87 91 62.5 0 9.4 51 91 91 91 91 31.2 0 4.8 45 84 88 94 84

15 0 5.6 11 50 91 91 94 209 Table 56. P. sojae (race 1) growth on lima bean medium amended with various concentrations of rutin. Race 1 Growth per day (mm) Concentrations (fiM) 0 1 2 3 4 5 6 500 0 3.8 20 56 87 87 87 200 0 2.9 22 56 81 91 91 125 0 3.8 20 68 91 91 91 62 .5 0 4.2 22 74 88 91 91 31.2 0 4.2 20 59 95 98 98 15 0 4.6 20 68 95 95 95

Table 57. P. sojae (race 3) growth on lima bean medium amended with various concentrations of rutin. Race 3 iGrowth per day (mm) Concentrations (fiM) 0 1 2 3 4 5 6 500 0 2.6 3 37 71 91 98 200 0 4.2 14 40 81 91 98 125 0 5.6 22 56 74 95 98 62.5 0 3.8 22 80 102 102 102 31.2 0 6.5 25 61 91 102 102 15 0 6.5 20 59 91 102 102

Table 58. P. sojae (race 12) growth on lima bean medium amended with various concentrations of rutin Race 12 Growth per day (nm) Concentrations (/xM) 0 1 2 3 4 5 6 500 0 1.6 15 38 61 87 102 200 0 2.2 24 56 87 94 102 125 0 1.6 25 80 91 102 102 62.5 0 1.3 22 77 87 98 102 31.2 0 2.6 26 68 91 98 102

15 0 2.3 27 74 91 98 102 210 211

Table 59. Concentration of apigenln after HPLC analysis from plates Inoculated with 3 different P. sojae races-'' Days 2 4

Race Peak-7 A B CD AB C D 1 21.57 1,223 1,223 1,340 1,968 938 1,448 2,026 2. 026 3 21.41 969 969 1,137 1,704 800 1,202 1,689 1,689

12 21.34 1,024 1,204 1,264 1,264 878 1,031 --

Days

6 8

Race Peak-7 A B C DABCD 1 21.57 865 1,442 - - 562 1,063 - - 3 21.41 834 1, 075 - - 848 1,262 - -

12 21.34 750 1, 168 - - 871 1,440 - -

-/ Samples were taken from plates containing 120 fiM of the compound tested per 8 days. -7 A specific peak for apigenin at 21.7 retention time was reported previously (Graham, 1991a) . However the peak was observed at its retention time with some small variation which depend on the sample. 212

Table 60. Concentration of biochanin A after HPLC analysis from plates inoculated with 3 different P. sojae races-7 Days 2 4 Race Peak-7 A BC D A B CD

1 21.50 0 0 6 6 6 0 0 0 25.88 535 535 705 766 161 628 573 1,106

3 21.50 6 6 0 0 0 0 12 12 26.02 572 572 915 538 718 711 892 892 12 21.59 5 6 5 8 0 0 5 5 26.06 434 451 628 655 555 666 756 756

Days

6 8

Race Peak-7 AB C D AB CD

1 21.50 5 23 _ _ 7 17 _- 25.88 524 715 - - 446 634 - -

3 21.50 0 0 0 0 0 0 _ _ 26.02 645 546 944 944 579 474 --

12 21.59 6 5 _ _ 0 0 _ 26.06 408 831 - - 307 506 -

- Samples were taken from plates containing 120 fiM of the compound tested per 8 days. -7 A specific peak for biochanin A at 27.7 retention time was reported previously (Graham, 1991a). However the peak was observed at its retention time with some small variation which depend on the sample. 213

Table 61. Concentration o£ coumestrol after HPLC analysis from plates inoculated with 3 different P. sojae races-7 Days 2 4

Race Peak-7 ABC D A B C D

1 21.56 912 985 1,155 1,155 562 1,162 _ 24.34 7 8 14 14 0 10 -

3 21.59 897 944 878 1,519 634 1,421 _ 24.39 11 10 11 22 0 19 -

12 21.53 825 884 __ 724 1,012 _ 24.39 8 11 - - 0 8 -

Dayo

6 8 Race Peak A B CDAB C D

1 21.56 546 857 _ 363 892 - 24.34 6 13 - - 0 0 -

3 21.59 615 1,226 __ 2 69 852 - 24.39 0 12 - 0 6 -

12 21.53 456 805 _ _ 505 634 _ 24.39 7 8 - - 16 14 - - Samples were taken from plates containing 120 (M of the compound tested per 8 days. -7 A specific peak for coumestrol at 21.9 retention time was reported previously (Graham, 1991a). However the peak was observed at its retention time with some small variation which depend on the sample. 214

Table 62. Concentration o£ chrysin after HPLC analysis from plates inoculated with 3 different P. sojae races^ Days 2 4

Race Peak-/ A B CD A B C D 1 25.60 353 353 427 617 335 346 351 436 3 25.56 242 242 242 389 258 313 307 307

12 25.50 211 236 348 348 164 254 _ _

Days

i 8 Race Peak-/ ABCDA BCD 1 25.60 337 414 - - 204 369 - - 3 25.56 306 488 - - 166 208 - - 12 25.50 123 256 - - 125 207 - - y Samples were taken from plates containing 120 fiM of the compound tested per 8 days. A specific peak for chrysin at 26.4 retention time was reported previously (Graham, 1991a). However the peak was observed at its retention time with some small variation which depend on the sample. 215

Table 63. Concentration of formononetin after HPLC analysis from plates inoculated with 3 different P. sojae races^

Days 2 4

Race Peak-/ A B CDABC D

1 18.44 117 117 0 10 0 22 _ _ 22.78 1,163 1,163 892 1,291 926 1,250 - - 3 22.75 1,091 1,092 1,356 1,615 1,226 1,606 - -

12 22.84 1,458 1,586 -_ 1,297 1,369 _-

Days

6 8 Race Peak-'' A B CD A B C D

1 18.44 26 86 _ _ _ _ _ 22.78 1,398 1,530 - - 1,002 1,368 - - 3 22.75 1,226 1,781 -- 1,096 1,678 -- 12 22.84 737 1,442 - - 939 1,268 - -

- Samples were taken from plateB containing 120 fM of the compound tested per 8 days. A specific peak for formononetin at 24.5 retention time (RT) was reported previously (Graham, 1991a). However the formononetin peak was observed at its retention time with some small variation which depend on the sample. Also a peak at 18.44 RT was observed with race 1 that might be due to the appearance of daidzein (rt = 18.9) in the sample. 216

Table 64. Concentration of genistein -/ after HPLC analysis from plates inoculated with 3 different P. sojae races.

Days 2 4 Race Peak!/ ABCDAB C D 1 21.05 84 84 107 1,267 102 758 - - 3 21.08 553 646 620 1,089 430 946 --

12 21.08 599 1,243 1,112 2,207 813 1,010 - -

Days

6 8 Race Peak-/ A B C D A B C D

1 21.05 410 889 527 701 - - 3 21.08 362 630 544 411 -- 12 21.08 33 576 36 481 - - 1/ Data based on the first set of experiments. ll Samples were taken from plates containing 120 fiM of the compound tested per 8 days. A specific peak for genistein at 21.6 retention time was reported previously (Graham, 1991a). However the peak was observed at its retention time with some small variation which depend on the sample. 217

Table 65. Concentration o£ isoquercetrin after HPLC analysis from plates inoculated with 3 different P. sojae races^

Days 2 4 Race Peak-' A B CDA B c D 1 15.33 86 86 440 823 0 0 265 265 3 15.26 37 37 37 618 0 0 __ 21.58 20 20 20 6 10 0 0 0

12 15.30 0 187 656 656 0 0 5 5

Days

6 8 Race Peak-' A B CD AB c D

1 15.33 0 0 -- 0 0 --

3 15.26 0 0 _ _ 0 11 - _ 21.58 0 0 - - 6 60 --

12 15.30 0 0 0 0 0 0 -- Samples were taken from plates containing 120 fiM of the compound tested per 8 days. 2J_ A specific peak for isoquercetrin at 15.20 retention time was reported previously (Graham, 1991a). However the isoquercetrin peak was observed at its retention time with some small variation which depend on the sample. Also a peak at 21.58 RT was observed with race 3 that might be due to the appearance of quercetin (rt) = 2 0 .0) in the sample. 218

Table 66. Concentration of isorhamnetin after HPLC analysis from plates inoculated with 3 different P. sojae races-7

Days 2 4 Race Peak-7 A B C D AB C D

1 6.50 0 0 0 0 5 8 _ 15.80 11 11 7 0 9 43 - - 21.96 396 396 629 822 12 712 - -

3 6.50 15 15 _ 20 __ __ 15.80 29 29 16 16 64 103 12 12 21.89 720 720 899 1,305 224 544 934 934

12 6.10 0 0 0 0 10 0 _ _ _ 15.80 17 0 10 26 6 32 - 21.93 444 668 861 1,926 11 533 - -

Days 6 8

Race Peak-7 ABCDABC D

1 6.50 13 5 5 0 22 - _ 15.80 0 24 13 13 0 13 - 21.96 0 588 929 929 0 0 --

3 6.50 6 0 __ 0 0 _ - 15.80 0 44 - - 0 0 - - 21.89 0 368 - - 0 0 - -

12 6.10 20 13 __ 0 13 __ 15.88 0 5 -- 0 6 - - 21.93 0 97 -- 0 42 - - Samples were taken from plates containing 120 jiM of the compound tested per 8 days. A specific peak for isorhamnetin at 22.50 retention time was reported previously (Graham, 1991a). However the isorhamnetin peak was observed at its retention time with some small variation which depend on the sample. Also two peaks at 6.50 and 15.80 (rt) were observed with all races that might be due to the appearance of phenylalanine (rt = 7.6) and an unknown compound in the sample, respectively. 219

Table 67. Concentration of kaempferol after HPLC analysis from plates inoculated with 3 different P. sojae races!''

Days 2 4

Race Peak2/ A B CD ABCD

1 10.55 123 59 41 41 31 59 _ _ 15.60 92 66 0 0 0 0 -- 17.22 97 131 31 31 129 190 - - 18.41 69 40 0 0 0 0 - - 21.09 81 78 166 0 0 0 - -

3 10.64 21 21 13 0 29 55 _ _ 15.55 17 17 0 14 0 47 - - 17.22 13 13 0 12 21 77 - - 19.45 25 25 27 95 0 0 -- 21.44 113 113 407 667 8 87 - -

12 10.86 0 0 0 0 18 50 _- 17.13 12 27 12 12 114 242 -- 21.70 0 64 80 80 0 0 - -

Days 6 8 Race Peak1' AB C DABC D

1 10.55 46 76 _ _ 57 110 - - 15.60 0 5 - - 7 0 - - 17.22 166 317 - - 120 213 - - 18.41 0 0 - - 0 0 - - 21.09 0 0 -- 0 0 - -

3 10.64 56 117 _ 60 89 __ 15.55 6 38 - - 9 13 -- 17.22 32 44 -- 28 47 -- 19.45 0 0 - - 0 0 - - 21.44 0 29 - - 0 0 --

12 10.86 23 82 _ _ 61 60 __ 17.13 99 199 - - 100 194 - - 21.70 0 0 - - 0 0 - y Samples were taken from plates containing 120 fiM of the compound tested for race 1 and 3; and 60 (M for race 12 during a 8 days period. y A specific peak for kaempferol at 21.80 retention time was reported previously (Graham, 1991a). However the kaempferol peak was observed at its retention time with some small variation which depend on the sample. Also various peaks were observed with all races that might be due to the appearance of several unknown metabolites. 220

Table 6 8 . Concentration o£ naringenin after HPLC analysis from plates inoculated with 3 different P. sojae races- Days 2 4

Race Peak-' A B C DAB CD 1 20.50 79 84 93 134 177 205 663 663 3 20.53 297 297 249 294 303 544 558 761 12 20.56 68 115 156 146 154 203 217 295

Days

6 8

Race Peak-7 A BCDABC D 1 20.50 466 664 - - 387 573 - - 3 20.53 365 514 759 759 341 523 - - 12 20.56 396 469 783 783 0 359 584 584 Samples were taken from plates containing 120 of the compound tested per 8 days. -7 A specific peak for naringenin at 21.1 retention time was reported previously (Graham, 1991a). However the peak was observed at its retention time with some small variation which depend on the sample. 221

Table 69. Concentration of quercetin after HPLC analysis from plates inoculated with 3 different P. sojae races1'' Days 2 4 Race Peak-/ A B C D A B C D

1 19.39 25 117 114 137 0 0 --

3 19.42 0 0 506 506 0 23 158 158

12 19.38 0 16 62 62 0 0 __

Days

6 8

Race Peak!7 A B C D A B c D

1 19.39 0 0 - 0 0 - - 3 19.42 0 0 - 0 0 - -

12 19.3 8 0 0 - 0 0 - - i/ Samples were taken from plateB containing 120 of the compound tested per 8 days. - A specific peak for quercetin at 20.0 retention time was reported previously {Graham, 1991a). However the peak was observed at its retention time with some small variation which depend on the sample. 222

Table 70. Concentration of rutin after HPLC analysis from plates inoculated with 3 different P. sojae races-''

Days 2 4 Race Peak-7 A B c DA BC D

1 14.84 17 55 195 259 0 0 _ _ 15.80 56 51 91 0 6 6 -- 17.53 28 16 67 0 0 0 -- 18.69 41 27 80 0 110 0 - -

3 14.84 0 21 147 323 0 18 _ _ 15.33 0 24 0 11 0 0 -- 19.45 15 25 19 19 0 0 - - 21.61 13 0 0 0 5 0 - -

12 14.90 0 0 83 83 0 0 __

Days 6 8 Race Peak^ A B c D A B c D

1 14.84 0 0 _- 0 0 - _ 15.80 0 0 -- 0 0 - - 17.53 0 0 - - 0 0 - - 18.69 0 0 - - 0 0 - -

3 14.84 0 0 _ 0 0 __ 15.33 0 0 - - 0 0 -- 19.45 0 0 -- 0 0 -- 21.61 0 0 - - 0 0 - “

12 14.90 0 0 - - 0 0 - - Samples were taken from plates containing 120 of the compound tested per 8 days. -/ A specific peak for rutin at 14.80 retention time was reported previously (Graham, 1991a) . However the rutin peak was observed at its retention time with some small variation which depend on the sample. Also various peaks were observed with races 1 and 3 that might be due to the appearance of quercetin (rt = 2 0 .0) and other unknown compounds seem to be present in the sample at the beginning of the experiments. All of them desappeared after 6 days. 223

Table 71. Concentration of genistein after HPLC analysis from plates inoculated with P. sojae race 3 Days 2 4 Race Peak2{ A B C DA B CD

3 13.82 51 51 46 _ _ 2,186 __ 15.74 56 56 70 - - 5,796 - 28 17.44 18 18 27 - - 50 -- 19.25 34 34 35 31 - 44 34 32 21.23 3, 981 3,981 3,970 5,294 3,121 4,102 4,581 6,054

Days

Race Peak3/ 13.82 15.74 - 16 17.44 - .... 19.25 35 35 21.23 2,409 4,318 - - 1,746 4,094 -! Data based on the second set of experiments. Samples were taken from plates containing 240 fiK of the compound tested per 8 days. A specific peak for genistein at 21.60 retention time was reported previously (Graham, 1991a). However the genistein peak was observed at its retention time with some small variation which depend on the sample. Also various peaks were observed that might be due to the presence of metabolites like daidzin (rt * 13.60), genistin (rt = 15.50) and glyciteine (rt = 19.30) in the sample at the beginning of the experiments. 224

Table 72. Concentration of MGG after HPLC analysis from plates inoculated with P. sojae race 3 Days 2 4 Race Peak-' A B C DAB CD

3 13.69 _ • _ _ - 892 __ 15.68 20 20 294 658 - 1,129 267 661 17.40 58 58 367 1,519 42 782 154 890 19.22 37 37 38 30 34 191 34 34 21.08 1,484 1,484 681 213 3,844 1,161 980 690

Days 6 8 Race Peak-'' A BCD AB CD

3 13.69 _ _ - _ _ _ _ _ 15.68 - 16 97 383 - -- - 17.40 11 20 26 209 --- - 19.22 35 40 38 37 - --

21.08 2,018 1,481 1,215 1,568 746 356 - - Samples were taken from plates containing 480 /.iM of the compound tested per 8 days. A specific peak for MGG at 17.30 retention time was reported previously (Graham, 1991a) . However the MGG peak was observed at its retention time with some small variation which depend on the sample. Also various peaks were observed that are due to the metabolism of the genistein conjugate. Metabolites like genistin (rt = 15.50), genistein (rt = 21.60) and glyciteine (rt = 19.30) seem to be present in the sample at the beginning of the experiments. Genistein accumulates at the end of the experiment. 225

Table 73. Concentration of MGG after HPLC analysis from plates inoculated with P. sojae race 3

Days 2 4

Race Peak-7 ABCD A B CD

3 13.91 83 83 . 58 ___ 15.85 103 103 169 295 88 - 160 289 17 .53 58 58 284 870 30 - 149 472 19.44 44 44 42 32 42 35 43 34 21.24 1, 810 1,810 1,563 2,510 2,448 1,541 1,462 1 , 880

Days 6 8

Race Peak2' ABC DA BC D

3 13 .91 _ _ __ _ 34 _ _ 15.85 34 -- - - 67 -- 17.53 - 11 - - - 19 - - 19.44 37 38 ------

21.24 1,714 1,185 - - 3,030 1,559 - -

Samples were taken from plates containing 240 fiM of the compound tested per 8 days. -/ A specific peak for MGG at 17.30 retention time was reported previously (Graham, 1991a). However the MGG peak was observed at its retention time with some smallvariation which depend on the sample. Also various peaks were observed that are due to the metabolism of the genistein conjugate. Metabolites like genistin (rt = 15.50), genistein (rt = 21.60) and glyciteine (rt = 19.30) seem to be present in the sample at the beginning of the experiments. Genistein accumulates at the end of the experiment. 226

Table 74. Concentration of MGG after HPLC analysis from plates inoculated with P. sojae race 3 XJ

Days 2 4

Race Peak-7 AB C D A B CD

3 13,99 _ _ 409 _ __ _ 15.81 - 10 75 449 10 - 53 53 17.52 - 14 211 607 -- 93 93 19.37 - 34 35 73 41 18 32 32 21.95 263 273 180 161 123 275 371 371

Days 6 8

Race Peak-7 A B C D A B CD

3 13.99 136 _ _ 61 __ 15.81 141 15 - 61 -- - 17.52 35 -- 12 - - - 19.37 62 31 - 33 - -- 21.95 17 98 - 33 33 - -

Samples were taken from plates containing 120 /jM of the compound tested per 8 days. A specific peak for MGG at 17.30 retention time was reported previously (Graham, 1991a). However the MGG peak was observed at its retention time with some small variation which depend on the sample. Also various peaks were observed that are due to the metabolism of the genistein conjugate. Metabolites like genistin (rt = 15.50), genistein (rt = 21.60) and glyciteine (rt = 19.30) seem to be present in the sample at the beginning of the experiments. Genistein accumulates at the end of the experiment. 227

Table 75. Concentration of daidzein after HPLC analysis from plates inoculated with P. sojae race 3 -f Days

Race Peak-7 AB C D A BC D

3 13.67 --_ _ 11 59 _ _ 15.67 - - - - - 65 - - 17.38 - -- -- 23 - - 18.44 4, 901 5,283 5, 843 7,332 4,464 6,347 7,436 7,436 22.63 17 16 21 21 11 10 15 15

Days 6 8 Race Peak-7 A BC D A B CD

3 13.67 _ - ______15.67 - 11 -- - 11 - - 17.38 ------18.44 4,096 7,337 -- 4,958 6, 653 -- 22.63 17 - - - 14 19 - - y Samples were taken from plates containing 240 of the compound tested per 8 days. i/ A specific peak for daidzein at 18.90 retention time was reported previously (Graham, 1991a) . However the daidzein peak was observed at its retention time with some small variation which depend on the sample. Also various peaks were observed that might be due to the presence of metabolites like daidzin (rt = 13.60), genistin (rt = 15.50) and glyciteine (rt = 19.30) in the sample at the beginning of the experiments. 228

Table 76. Concentration of daidzein after HPLC analysis from plates inoculated with P. sojae race 3 ff Days 2 4

Race Peak-7 AB C D A BC D

3 13.71 _ _ 55 __ 15.58 - -- - 42 -- 17 .39 - - - - 15 - - 18.45 2,389 2,389 103 3,474 2,310 3,008 3,511 3,511 22.67 16 16 21 - 13 10 10 Days 6 8 Race Peakff A B C D ABCD

3 13.71 _ _ - 50 __ 15.58 - 13 -- 59 - - 17.39 ------18.44 1,918 3,226 - 1,823 3,068 - - 22.67 15 14 - 13 26 - - Samples were taken from plates containing 120 of the compound tested per 8 days. -7 A specific peak for daidzein at 18.90 retention time was reported previously (Graham, 1991a) . However the daidzein peak was observed at its retention time with some small variation which depend on the sample. Also various peaks were observed that might be due to the presence of metabolites like daidzin (rt = 13.60), genistin (rt = 15.50) and genistein (rt = 21.60) in the sample at the beginning of the experiments. 229

Table 77. Concentration of MGD after HPLC analysis from plates inoculated with P. aojae race 3 -1 Days 2 4 Race Peak2/ ABCDA B C D

3 11.25 296 296 __ _ __ 13.70 1,511 1,511 529 1,258 95 10 290 290 15.71 1,494 1,494 796 2,415 93 73 521 521 17.39 608 608 12 - 24 17 22 22 18.46 2,917 2,917 1,623 929 3,295 3,335 3,671 3,671

Days 6 8 Race Peak-/ A B C D A B C D

3 11.25 _ ___- _ 13.70 43 29 ------15.71 29 23 ------17.39 ------18.46 2,820 2, 951 - 1,102 1,275 - - Samples were taken from plates containing 240 nM of the compound tested per 8 days. A specific peak for MGD at 15.70 retention time was reported previously (Graham, 1991a). However the HGDpeak was observed at its retention time with some small variation which depend on the sample. Also various peaks were observed that might be due to the presence of metabolites like daidzein (rt = 18.90) and daidzin (rt = 13.60) in the sample. Daidzein accumulated at the end the experiments. 230

Table 78. Concentration of MGD after HPLC analysis from plates inoculated with P. sojae race 3 Days 2 4 Race Peak^ A BC DA B CD

3 13.72 143 29 245 777 172 _ 686 686 15.73 130 38 482 1,363 252 12 866 866 17 .44 28 - -- 90 - 351 351 18.97 1,019 1,202 811 84 846 1,408 2,994 2,994 22 .71 - 14 13 10 18 14 148 148

Days 6 8

Race Peak^ A B C DA B CD

3 13.72 ______- 15.73 ------17.44 ------18.97 272 444 -- 120 183 - - 22.71 19 37 -- 16 20 - - -/ Samples were taken from plates containing 120 of the compound tested per 8 days. -1 A specific peak for MGD at 15.70 retention time was reported previously (Graham, 1991a). However the MGD peak was observed at its retention time with some small variation which depend on the sample. Also various peaks were observed that might be due to the presence of metabolites like daidzein (rt = 18.90) and daidzin (rt = 13.60) in the sample. Daidzein accumulated at the end the experiments. APPENDIX B.

231 Table 79. PNL specific activity in P. sojae race 4, mycelial portions.

Pectic Fractions^ Pacl/ Pectin (Orange) Pectin (Apple) Control Days R3iy R4 R3 R4 R3 R4 R3 R4 0 00 00 00 0 00 00 00 00 1 00 397 00 1,581 362 1,292 00 00 2 226 1,317 00 344 416 429 00 00 3 00 294 00 633 308 293 00 135 4 84 554 00 992 400 00 00 00 5 153 359 00 543 211 236 341 00 6 140 375 00 00 143 - 526 00 7 320 500 596 1,432 315 988 00 00 8 - 291 00 00 731 00 250 00 10 237 104 00 00 112 881 00 90 12 00 00 902 00 919 1,637 528 00 P. sojae was grown on lima bean media ammended with various pectic fractions. 2J Pac = polygalacturonic acid P. sojae race 3: R3 ; P. sojae race 4: R4. 232 Table 80. PNL specific activity in P. sojae race 4, filtrate portions.

Pectic Fractions Pac-7 Pectin (Orange) Pectin (Apple) Control Days R3-/ R4 R3 R4 R3 R4 R3 R4

0 00 00 00 00 000 00 000 00 1 00 00 87,617 94 116 690 000 00 2 00 00 59,170 00 000 373 000 1,672 3 828 00 00 805 000 633 652 00 4 00 00 114,085 494 19,857 11,321 000 16,304 5 3,126 00 120,970 1,337 22,433 00 000 00 6 00 00 71,537 1,627 000 1,449 000 - 7 00 00 00 1,863 000 543 000 2,249 8 00 00 23,056 3,623 27,173 1,224 000 1, 672 10 87 00 5,219 2,609 - 2,536 64 00 12 46 00 10,014 373 163 1,976 43 00 P. sojae was grown on lima bean media ammended with various pectic fractions. If Pac = polygalacturonic acid P. sojae race 3: R3 ; P. sojae race 4: R4. 234

Table 81 . PNL specific activity (U/mg) in soybean cotyledons and hypocotyls 12h after inoculation with P. sojae races 3 and 4.

Section R3 R4 Control w Cot A 00 00 00 II B 00 51 00 It C 453 86 24

Hyp A 00 62 1,058 n B 00 134 220

W7J Cot A 00 00 00 ii B 00 00 00 it C 185 00 242

Hyp A 00 101 37 n B 00 364 106

Table 82. PNL specific activity {U/mg) in soybean cotyledons and hypocotyls 24h after inoculation with P. sojae races 3 and 4 .

Section R3 R4 Control

W Cot A 424 830 00 II B 102 00 562 II C 148 312 113

Hyp A 00 1,542 00 II B 556 87 72

W„ Cot A 652 00 00 II B 247 00 284 tl C 99 1,277 1,743

Hyp A 00 00 00 n B 223 00 00 235

Table 83. PNL specific activity (U/mg) in soybean cotyledons and hypocotylB 3Oh after inoculation with P. sojae races 3 and 4.

Section R3 R4 Control W Cot A 145 3,135 665 II B 552 00 153 II C 00 437 00

Hyp A 572 755 00 II B 200 8,619 2 , 608 Cot A 734 295 206 ii B 1,377 14,900 597 ii C 913 3,274 662 Hyp A 393 2,149 00 it B 840 00 00

Table 84. PNL specific activity (U/mg) in soybean cotyledons and hypocotyls 36h after inoculation with P. sojae races 3 and 4.

Section R3 R4 Control

W Cot A 1,314 120 351 It B 1,291 484 182 II C 316 7 49 Hyp A 483 606 75 11 B 65 1,289 480

W„ COt A 268 729 111 II B 2,796 90 33 II C 444 7,308 266 Hyp A 1,500 185 906 II B 3,443 150 137 236

Table 85. PNL specific activity (U/mg) in soybean cotyledons and hypocotyls 42h after inoculation with P. sojae races 3 and 4.

Section R3 R4 Control

w Cot A 00 00 00 n B 134 00 00 II C 152 68 00 Hyp A 82 00 00 n B 112 00 00 W7S Cot A 409 22 00 ti B 602 67 00 ii C 00 290 70 Hyp A 78 00 00 If B 00 00 00

Table 86. PNL specific activity (U/mg) in soybean cotyledons and hypocotyls 48h after inoculation with P. sojae races 3 and 4.

Section R3 R4 Control W Cot A 2,299 45 153 11 B 67 145 275 II C 1,200 00 413

Hyp A 00 528 00 H B 331 00 00 Cot A 00 73 433 ii B 00 262 663 n C 156 00 770

Hyp A 00 245 00 II B 00 00 148