Comparative virulence of Pyrenophora graminea Ito et Kurib isolates and the inheritance of resistance to P. graminea in barley by Richard Lee Ruff A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Plant Pathology Montana State University © Copyright by Richard Lee Ruff (1984) Abstract: Studies were continued to identify genes for resistance in barley to barley leaf stripe disease (causal organism : Pyrenophora graminea Ito et Kurib.). Because of the variability of infection, inoculation techniques were studied. Eight media were developed to compare substrate effects upon infection of a barley cultivar's seedlings by a P. graminea isolate. Although infection mean differences of the cultures were not significant, cultures grown on diffusate from germinated barley seed had slightly increased infectivity. Virulence of 24 P. graminea isolates was tested to determine the spectrum of resistance in three barley cultivars. Artificial inoculation of seeds was by the layered mycelium method. Both of the highly susceptible cultivars, 'Lami' and 'Summit', were found to have a low level of resistance to some of these isolates. 'Betzes' seeds inoculated with 18 of the isolates resulted in plants without symptoms. However, up to 16% symptomatic seedlings were found after inoculation with six other isolates. Specific resistance in these cultivars to the isolates was inferred. The Turkish cultivars, Tokak and Yesilkoy, were each reciprocally crossed to Betzes to study the inheritance of resistance in barley to leaf stripe disease. Tokak and Betzes are highly resistant to infection, and Yesilkoy has a low level of resistance. Seeds of the crosses were produced through the F3 and BC F2 generations and inoculated by the layered mycelium method. Seedlings were grown for six weeks and divided into a class with disease symptoms or a class without symptoms. Seedlings' reactions from the F1 and F2 generations of the Betzes X Tokak crosses fit models indicating that a single dominant gene was inherited from each parent. The lack of fit of the data in the BC F2 families indicated a modification of the dominant genes models. Seedlings' reactions indicated that a recessive gene was inherited from Yesilkoy. Qualitative gene action with multiple genes or quantitative gene action was inferred from the results of seed inoculations. A method to calculate the effects of penetrance and expressivity on expected gene ratios was discussed. COMPARATIVE VIRULENCE OF PYRENOPHORA GRAMINEA ITO ET KURIB ISOLATES AND THE INHERITANCE OF RESISTANCE TO JL4. GRAMINEA IN BARLEY. by Richard Lee Ruff A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Plant Pathology MONTANA STATE UNIVERSITY Bozeman, Montana December 1984 APPROVAL of a thesis submitted by Richard Lee Ruff This thesis has been read by each member of the thesis committee and has been found to be satisfactory regarding content, English usage, format, citations, bibliographic style, and consistency, and is ready for submission to the College of Graduate Studies. Date Chairperson, Graduate Committee Approved for the Major Department Date Approved for the College of Graduate Studies Date Graduate Dean iii STATEMENT OF PERMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for a master’s degree at Montana State University, I agree that the Library shall make it available to borrowers under the rules of the Library. Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgement of source is made. Permission for extensive quotation from or reproduction of this thesis may be granted by my major professor, or in his absence, by the Director of Libraries when, in the opinion of either, the proposed use of the material is for scholarly purposes. Any copying or use of the material in this thesis for financial gain shall not be allowed without my written' permission. V ACKNOWLEDGMENTS I express sincere appreciation and gratitude to the following persons and agencies (not in any particular order) : Dr. A. L. Scharen, for his continual encouragement, confidence, and personal concern as well as material facilities; Dr's. E. H. Hockett, J. H. Riesselman, and E. L. Sharp for their assistance and advice during my graduate studies; the staff and fellow graduate students of the Department of Plant Pathology for their assistance, abbetment, and perseverance; my mother and father, family, and friends for emotional, moral, and physical support; the Ground of my being for life; US-AID and the USN for financial support. vi TABLE OF CONTENTS Page APPROVAL ........................................ ..... ii STATEMENT OF PERMISSION TO USE ..................... ill VITA ....... ^ ......... ........ .'.............. iv ACKNOWLEDGMENTS ..... ........................ ...... v TABLE OF CONTENTS ...... ...................... vi LIST OF TABLES ..................................... viii ABSTRACT ................................ .............. xi CHAPTER I; INTRODUCTION .................. I CHAPTER 2: MEDIA EFFECTS ON INFECTION STABILITY OF PYRENOPHORA GRAMINEA ISOLATES WHEN ARTIFICIALLY CULTURED ................ 8 Introduction ........................ 8 Materials and Methods ............... 10 Results and Discussion ........... ...... 13 CHAPTER 3: VIRULENCE OF PYRENOPHORA GRAMINEA ISOLATES ON SPECIFIC BARLEY CULTIVARS. 19 Introduction ................. 19 Materials and Methods ........ 21 Results and Discussion ....... 24 CHAPTER 4: INHERITANCE OF RESISTANCE IN THREE BARLEY CULTIVARS TO TWO PYRENOPHORA GRAMINEA ISOLATES. ................... 31 Introduction........................ 3 I Materials and Methods ......... 33 Results ...................... ........... 3 5 Betzes - Tokak Crosses ............... 37 Betzes - Yesilkoy Crosses ............ 47 Discussion ................ 57 . vii TABLE OF CONTEMTS-Continued Page CHAPTER 5: SUMMARY ...... .......................... 65 REFERENCES CITED.... ...................... ...... 68 APPENDIX ................... ..................... 74 viii LIST OF TABLES Tables J Page 1. Percent of barley seedlings infected as a result of inoculation with cultures of Pvrenophora graminea isolate R PB grown on different media ...... ....... I if 2. Barley plants infected by Pvrenophora graminea as a percentage of total plants emerged (average of three replications) ..... 25 3 • Barley plants infected by Pvrenoohora graminea as a percentage of total plants emerged (average of three replications) 26 4. Barley plants infected by Pvrenoohora ■finuajm-i.ne a as a percentage of total plants emerged (average of three replications) ..... 27 5. Number of barley seedlings derived from check cultivars, parents, and progeny of two crosses with and without symptoms after inoculation with two isolates of Pvrenophora graminea ................... .................. 3 8 6. Number of specific parent and F g barley seedlings with and without symptoms after inoculation" with Pvrenoohora graminea isolate MT6 and Chi-square(X^ ) values when fit to a 15:1 ratio ......................... 41 7. Number of specific parent and F2 barley seed­ lings with and without symptoms after inocu­ lation with Pvrenonhora graminea isolate Kaya and Chi-square(X^)values when fit to a 15:1 ratio ......................................... 43 ix LIST OF TABLES-Continued Tables Page 8. Number of barley seedlings from bulked parents and backcrossed F^) seeds with and without symptoms after inoculation with two Pvre noohora graminea isolates ............ 4 5 9. Number of backcrossed Fg families resistant and segregating after inoculation with two Pvrenophora graminea isolates ............... 46 10. Number of barley seedlings derived from check cultivars, parents, and progeny of two crosses with and without symptoms after inoculation with two isolates of Pvrenophora graminea .............................. ....... 4 8 11. Number of specific parent and Fg barley seedlings with and without symptoms after inoculation with two Pvrenophora graminea isolates and Chi-square values when fit to a 13:3 ratio ................................... 52 12. Number of barley seedlings from check cultivars, bulked parents, and backcrossed F(.j) seeds with and without symptoms after inoculation with two Pvrenophora graminea isolates ............................ 55 13. Number of backcrossed Fg families resistant and segregating after inoculation with two Pvrenophora graminea isolates ............ 57 14. Potato dextrose agar plus diffusate of germ­ inated, barley seed...................... 75 15. Potato dextrose agar plus extract of ground, germinated, barley seed ..... ......... 7 6 16. Two-factorial analysis of variance of cultured isolate experiments and comparison of culture means ... ................... 77 X LIST OF TABLES-Continued Tables Page 17 • Comparison of emergence percentages of barley seedlings from the parent and check cultivars ..... t . 78 18. Analysis of covariance with emergence percentages as the covariable and infection percentages as the dependent variable of seedlings from the parent and check cultivars ........... 78 19. Number of Fo f amilies resistant and segrega­ ting after inoculation with two Pvrenophora graminea isolates (30-80 seedlings were read in each family) .......................... 79 20. Calculations of genetic ratios with the effects of penetrance and expressivity .... 81 xi ABSTRACT Studies were continued to identify genes for resis­ tance in barley to barley leaf stripe