Sensitivity of Rhizoctonia Solani and Aphanomyces Cochlioides To
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SENSITIVITY OF RHIZOCTONIA SOLANI AND APHANOMYCES COCHLIOIDES TO FUNGICIDES, AND FITNESS OF TETRACONAZOLE-RESISTANT ISOLATES OF CERCOSPORA BETICOLA AFTER EXPOSURE TO DIFFERENT TEMPERATURE REGIMES A Dissertation Submitted to the Graduate Faculty Of the North Dakota State University of Agriculture and Applied Sciences By Sahar Ibrahim Arabiat In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY Major Department: Plant Pathology November 2014 Fargo, North Dakota North Dakota State University Graduate School Title SENSITIVITY OF RHIZOCTONIA SOLANI AND APHANOMYCES COCHLIOIDES TO FUNGICIDES, AND FITNESS OF TETRACONAZOLE-RESISTANT ISOLATES OF CERCOSPORA BETICOLA AFTER EXPOSURE TO DIFFERENT TEMPERATURE REGIMES By Sahar Ibrahim Arabiat The Supervisory Committee certifies that this disquisition complies with North Dakota State University’s regulations and meets the accepted standards for the degree of DOCTOR OF PHILOSOPHY SUPERVISORY COMMITTEE: Mohamed Khan Chair Gary Secor Melvin Bolton Marisol Berti Approved: December 4, 2015 Jack Rasmussen Date Department Chair ABSTRACT North Dakota and Minnesota produce 55% of USA sugarbeet production. Diseases caused by Rhizoctonia solani, Aphanomyces cochlioides, and Cercospora beticola are the major diseases affecting sugarbeet production in North Dakota and Minnesota. Growers mainly use partial resistant varieties and fungicides to manage diseases of sugarbeet. Sensitivity of R. solani and A. cochlioides to fungicides were evaluated in vitro using mycelium radial growth assay and by evaluating disease severity on inoculated plants treated with fungicides in the greenhouse. Phenotypic stability of tetraconazole-resistant isolates of C. beticola after exposure to different temperature regimes was evaluated. For R. solani, mean EC50 values for baseline isolates were 49.7, 97.1, 0.3, 0.2, and 0.9 µg ml-1 and for non-baseline isolates were 296.1, 341.7, 0.9, 0.2, and 0.6 µg ml-1 for azoxystrobin, trifloxystrobin, pyraclostrobin, penthiopyrad, and prothioconazole, respectively. The mean EC50 values of azoxystrobin, trifloxystrobin, and pyraclostrobin increased with a change factor of 6.0, 3.5, and 2.7, respectively. All fungicides at labeled rates effectively controlled R. solani in vivo. For A. cochlioides, tetraconazole, prothioconazole, and pyraclostrobin reduced mycelium radial growth in vitro with mean EC50 values of 3.5, 2.4, and 0.8 µg ml-1, respectively. However, these fungicides were not effective at controlling A. cochlioides in vivo. Sugarbeet plants up to three weeks old were found susceptible to A. cochlioides. Resistant isolates of C. beticola had no fitness penalty as measured by spore production, spore germination, mycelium radial growth, and disease severity after exposure to different temperature regimes. However, isolate 09-347, resistant to tetraconazole, reverted to a moderate resistance level after exposure to -20ºC, and -20ºC to 4ºC to -20ºC to 4ºC with a factor of change of 38.6 and 32.8, respectively. This research indicated that R. solani sensitivity to the evaluated QoIs had decreased, but they were still effective at labeled rates under greenhouse iii conditions, and rotation of different fungicide classes could be a useful strategy to manage fungicide resistance. No fitness penalty was found after exposure of C. beticola isolates to cold treatments. However, C. beticola isolates resistant to tetraconazole became more sensitive to this fungicide after exposure to cold treatments. iv ACKNOWLEDGEMENTS I would like to thank my advisor Dr. Mohamed Khan for his guidance, encouragement, support, and patience throughout my research. Also I extend my thanks to the graduate committee Drs. Gary Secor, Melvin Bolton, and Marisol Berti for their help, support and valuable comments and suggestion to improve my research. Additionally, I would like to thank all faculty members, staff, and graduate students of the Plant Pathology Department for their kindness and support. I am thankful for all people helped me throughout my research especially Yangxi Liu and Beter Hack. Also I am thankfull for Jawahar Jyoti and James Hammond for their help in data analysis. I would also like to thank my mother, brothers, sisters, nephews and nieces for their love, prayers, and support. Special thanks go to my husband who helped me pursue my goal. I would like to extend my thanks to all my friends especially Dr. Kholoud Alananbeh. v TABLE OF CONTENTS ABSTRACT……………………………………………………………………………………...iii ACKNOWLEDGMENTS………….…………………………………………………………......v LIST OF TABLES…………………………………………..……………………………………ix LIST OF FIGURES………………………………………………………………………………xi LIST OF APPENDIX TABLES………………………………………………..……...……..….xii LIST OF APPENDIX FIGURES……………………………………………………...………..xvi CHAPTER ONE. LITERATURE REVIEW…………………...……………………...………….1 CHAPTER TWO. SENSITIVITY OF RHIZOCTONIA SOLANI TO AZOXYSTROBIN, TRIFLOXYSTROBIN, PYRACLOSTROBIN, PENTHIOPYRAD, AND PROTHIOCONAZOLE…...…………………………………………………………………….26 CHAPTER THREE. SENSITIVITY OF APHANOMYCES COCHLIOIDES TO TETRACONAZOLE, PROTHIOCONAZOLE, PYRACLOSTROBIN, AND HYMEXAZOL.…...………...…………………………………………………………………...52 CHAPTER FOUR. FITNESS OF TETRACONAZOLE-RESISTANT ISOLATES OF CERCOSPORA BETICOLA AFTER EXPOSURE TO DIFFERENT TEMPERATURE REGIMES…..………………………...……………………………………..…………………...66 APPENDIX A. RHIZOCTONIA SOLANI ISOLATES USED FOR SENSITIVITY TO FUNGICIDES IN VITRO.……………………………………………………………………....87 APPENDIX B. STATISTICAL ANALYSIS FOR DETERMINATION OF RHIZOCTONIA SOLANI AG-2-2 SUBGROUPS.…………………...……………………………………………91 APPENDIX C. DETERMINATION OF RHIZOCTONIA SOLANI AG-2-2 SUBGROUPS..………………………………....………………………………………………..92 APPENDIX D. STATISTICAL ANALYSIS SUMMARY FOR SENSITIVITY OF RHIZOCTONIA SOLANI TO FUNGICIDES.…………………………………………………...95 APPENDIX E. STATISTICAL ANALYSIS SUMMARY FOR SENSITIVITY OF RHIZOCTONIA SOLANI TO AZOXYSTROBIN WITH AND WITHOUT SALICYLHYDROXAMIC ACID.…………...……………………………………..…………..97 APPENDIX F. STATISTICAL ANALYSIS SUMMARY FOR EFFICACY OF FUNGICIDES AT CONTROLLING RHIZOCTONIA SOLANI IN VIVO.……………...……..98 vi APPENDIX G. STATISTICAL ANALYSIS SUMMARY FOR EFFICACY OF FUNGICIDES AT CONTROLLING RHIZOCTONIA SOLANI IN VIVO….………………….99 APPENDIX H. STATISTICAL ANALYSIS SUMMARY FOR RATE OF MYCELIUM RADIAL GROWTH OF RHIZOCTONIA SOLANI.….………………………………………..111 APPENDIX I. RATE OF MYCELIUM RADIAL GROWTH OF RHIZOCTONIA SOLANI ISOLATES WITH LOW AND HIGH EC50 VALUES..……………………………………….112 APPENDIX J. STATISTICAL ANALYSIS SUMMARY FOR SENSITIVITY OF APHANOMYCES COCHLIOIDES TO FUNGICIDES.………………………………………..113 APPENDIX K. ISOLATES USED FOR EVALUATING SENSITIVITY OF APHANOMYCES COCHLIOIDES TO FUNGICIDES.……………………………...………..114 APPENDIX L. STATISTICAL ANALYSIS SUMMARY FOR DETERMINING THE SUSCEPTIBLE STAGES OF SUGARBEET TO APHANOMYCES COCHLIOIDES AND EFFICACY OF FUNGICIDES IN VIVO.……………………………………………………..116 APPENDIX M. STATISTICAL ANALYSIS FOR SPORE PRODUCTION FOR CERCOSPORA BETCOLA ISOLATES FROM DIFFERENT TEMPERATURE REGIMES……..………………………………………………………………………………..118 APPENDIX N. STATISTICAL ANALYSIS FOR SPORE GERMINATION FOR CERCOSPORA BETCOLA ISOLATES FROM DIFFERENT TEMPERATURE REGIMES…..…………………………………………………………………………………..120 APPENDIX O. STATISTICAL ANALYSIS FOR RADIAL GROWTH OF CERCOSPORA BETCOLA ISOLATES FROM DIFFERENT TEMPERATURE REGIMES………..………..122 APPENDIX P. STATISTICAL ANALYSIS FOR SENSITIVITY OF CERCOSPORA BETCOLA ISOLATES FROM DIFFERENT TEMPERATURE REGIMES TO TETRACONAZOLE.…………………………………………………………………………..124 APPENDIX Q. STATISTICAL ANALYSIS SUMMARY FOR EFFECT OF DIFFERENT TEMPERATURE REGIMES ON TETRACONAZOLE-SENSITIVE AND -RESISTANT CERCOSPORA BETICOLA ISOLATES BASED ON DISEASE SEVERITY....……………..126 APPENDIX R. NON-PARAMETRIC ANALYSIS SUMMARY FOR EFFECT OF DIFFERENT TEMPERATURE REGIMES ON TETRACONAZOLE-SENSITIVE AND - RESISTANT CERCOSPORA BETICOLA ISOLATES BASED ON DISEASE SEVERITY……………………………………………………………………………………..127 vii APPENDIX S. EFFICACY OF DIFFERENT CONCENTRATIONS OF AZOXYSTROBIN, TRIFLOXYSTROBIN, PYRACLOSTROBIN, PENTHIOPYRAD, AND PROTHIOCONAZOLE AT CONTROLLING RHIZOCTONIA SOLANI AG-2-2 IIIB WITH HIGH AND LOW EC50………………………..…………………………131 APPENDIX T. MYCELIUM RADIAL GROWTH OF CERCOSPORA BETICOLA ISOLATES AT DIFFERENT TETRACONAZOLE CONCENTRATIONS.……………..…..133 viii LIST OF TABLES Table Page 2.1. Year of collection, state of origin, and number of Rhizoctonia solani isolates used for mycelium radial-growth assay and in greenhouse studies. ......................................... 31 2.2. Properties of the fungicides used to evaluate sensitivity of Rhizoctonia solani in vitro and in the greenhouse studies. .................................................................................. 32 2.3. Means and confidence intervals for the EC50 values of Rhizoctonia solani isolate, 393 used as the control. ..................................................................................................... 33 2.4. Subgroups, isolates, azoxystrobin and trifloxystrobin EC50 values for Rhizoctonia solani isolates that were used in the greenhouse study. .................................................... 34 2.5. Pearson correlation coefficient of 27 baseline isolates between EC50 values of azoxystrobin, trifloxystrobin, pyraclostrobin, penthiopyrad, and prothioconazole. Numbers in parentheses refer to p value. .......................................................................... 39 2.6. Pearson correlation coefficient of 78 non-baseline isolates between EC50 values of azoxystrobin, trifloxystrobin, pyraclostrobin, penthiopyrad, and prothioconazole. Numbers in parentheses