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University of Florida Thesis Or Dissertation Formatting FUNGICIDE RESISTANCE PROFILE OF QUIESCENT Botrytis cinerea INFECTIONS ON STRAWBERRY TRANSPLANTS By MICHELLE SOUZA OLIVEIRA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2015 © 2015 Michelle Souza Oliveira To my family and my fiancé, Leandro ACKNOWLEDGMENTS First I would like to thank God, my creator, who is always with me and strengthen me in my most difficult times. “Fear not, I am with you; be not dismayed; I am your God. I will strengthen you, and help you, and uphold you with my right hand of justice” (Isaiah 41:10). I am sincere thankful to Dr. Natalia Peres, my advisor, for giving me the opportunity to develop such interesting and important work, believing in my work and for all her guidance and teachings in plant pathology, research and extension. I would like to gratefully thank my committee members, Dr. Nicholas Dufault and Dr. Philip F. Harmon, for their support with laboratory space while I was in Gainesville and for all their valuable suggestions to my work. I thank all the lab members and interns of the Strawberry Pathology lab at Gulf Coast Research and Education Center for their teamwork and the help with some evaluations. I thank my family that besides the physical distance are always present in my life achievements, and I gratefully thank my fiancé Leandro for his support and for all his encouragement, patience and unconditional love. And after all the difficulties I’m proud to say I always believed in my dreams and that’s what made me have my accomplishments. “It’s the possibility of having a dream come true that makes life interesting” (Paulo Coelho) 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 9 ABSTRACT ................................................................................................................... 11 CHAPTER 1 LITERATURE REVIEW .......................................................................................... 13 Strawberry Production ............................................................................................ 13 Nursery Transplants................................................................................................ 15 Botrytis cinerea ....................................................................................................... 18 Epidemiology and Life Cycle ............................................................................ 18 Symptoms and Signs ....................................................................................... 19 Disease Control ...................................................................................................... 20 Chemical Control .............................................................................................. 21 Fungicide Resistance ....................................................................................... 22 Objectives ............................................................................................................... 26 2 TRANSPLANTS AS A SOURCE OF INFECTION OF Botrytis cinerea TO STRAWBERRY FIELDS IN FLORIDA .................................................................... 30 Introduction ............................................................................................................. 30 Materials and Methods............................................................................................ 32 Fungal Isolates ................................................................................................. 32 Fungicide Sensitivity Tests ............................................................................... 33 Results .................................................................................................................... 35 Frequency of Infection ...................................................................................... 35 Fungicide Sensitivity Tests ............................................................................... 36 Discussion .............................................................................................................. 41 3 OPTIMIZATION OF A RESAZURIN-BASED ASSAY TO EVALUATE SENSITIVITY OF Botrytis cinerea TO RESPIRATION-INHIBITOR FUNGICIDES .......................................................................................................... 60 Introduction ............................................................................................................. 60 Materials and Methods............................................................................................ 62 Fungal Isolates ................................................................................................. 62 Pathogenicity Tests .......................................................................................... 63 Effect of Conidial Concentration on Resazurin Reduction ................................ 63 Evaluation of the Best Medium for the Reduction of Resazurin........................ 64 5 Time of Evaluation of the Microplates and its Effect on Resazurin Reduction .. 65 Effect of SHAM and DMSO on the Respiration of Botrytis cinerea ................... 65 Resazurin Reduction Calculations .................................................................... 66 Conidial Germination Test ................................................................................ 67 Results .................................................................................................................... 68 Pathogenicity Tests .......................................................................................... 68 Effect of Conidial Concentration on Resazurin Reduction ................................ 68 Evaluation of the Best Medium for the Reduction of Resazurin........................ 69 Time of Evaluation of the Microplates and its Effect on Resazurin Reduction .. 70 Effect of SHAM and DMSO on the Respiration of Botrytis cinerea ................... 70 Conidial Germination Test ................................................................................ 70 Discussion .............................................................................................................. 72 4 CONCLUSIONS ..................................................................................................... 88 LIST OF REFERENCES ............................................................................................... 90 BIOGRAPHICAL SKETCH .......................................................................................... 100 6 LIST OF TABLES Table page 2-1 Frequency of strawberry transplants from different nurseries infected with Botrytis cinerea in 2011, 2012 and 2013. ........................................................... 55 2-2 Analysis of variance of the frequency of strawberry transplants infected with Botrytis cinerea by nursery and region in 2011, 2012 and 2013. ........................ 55 2-3 F-statistics and P-values for the fungicide inhibition of the mycelial growth of Botrytis cinerea isolates collected from fourteen nurseries in 2011, 2012, and 2013. .................................................................................................................. 56 2-4 Frequency of resistance of Botrytis cinerea isolates from nurseries to seven different fungicides tested in the mycelial growth assay. .................................... 57 2-5 F-statistics and P-values for the fungicide inhibition of the conidial germination of Botrytis cinerea isolates collected from fourteen nurseries in 2011, 2012, and 2013. ........................................................................................ 58 2-6 Frequency of resistance to seven different fungicides of Botrytis cinerea isolates from nurseries tested in the conidial germination assay. ....................... 59 3-1 Botrytis cinerea isolates from strawberry nursery transplants and their sensitivity to boscalid and pyraclostrobin. ........................................................... 83 3-2 Molar extinction coefficient for Resazurin (Invitrogen 2009). .............................. 83 3-3 The EC50 (μg/ml) values of Botrytis cinerea isolates in a resazurin reduction assay and the effect of salicylhydroxamic acid (SHAM) and dimethyl sulfoxide (DMSO) on pyraclostrobin-amended medium (complete medium). ..... 83 3-4 The EC50 values (μg/ml) of sensitive Botrytis cinerea isolates in a resazurin- reduction assay and the effect of SHAM and DMSO on a pyraclostrobin- amended medium (HA at pH 6.5). ...................................................................... 84 3-5 The EC50 values (μg/ml) of Botrytis cinerea isolates on boscalid and pyraclostrobin-amended medium comparing a resazurin reduction assay and the conidial germination test. .............................................................................. 84 3-6 Analysis of variation of the EC50 values (μg/ml) of Botrytis cinerea sensitive isolates in a conidial germination test and a resazurin reduction assay with boscalid- and pyraclostrobin-amended medium. ................................................ 85 3-7 Correlation of the EC50 values (μg/ml) of Botrytis
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