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Development of Rapid in Vitro Assays and Current Status Of DEVELOPMENT OF RAPID IN VITRO ASSAYS AND CURRENT STATUS OF FUNGICIDE SENSITIVITY IN THE PECAN SCAB PATHOGEN FUSICLADIUM EFFUSUM by MURAT SEYRAN (Under the supervision of KATHERINE STEVENSON and TIMOTHY BRENNEMAN) ABSTRACT Isolates of F. effusum from three baseline orchards in Georgia were tested for sensitivity using a mycelial growth assay in microtiter plates. The 50% effective dose (EC50) values were estimated for azoxystrobin, dodine, fentin hydroxide, and propiconazole. The role of alternative oxidase inhibitors salicylhydroxamic acid and propyl gallate in quinone outside inhibitor assays also was evaluated. Both chemicals were found to be toxic to F. effusum and therefore were not used in the assays. A more rapid assay was developed based on conidial germination and micro- colony growth. Isolates of F. effusum from 35 commercial orchards were profiled using the new assay and in vitro resistance to fentin hydroxide, dodine, thiophanate-methyl, propiconazole, and azoxystrobin was detected in 20, 4, 6, 21, and 19 of the 35 orchards respectively. Taxonomy of F. effusum in the family Venturiaceae was supported based on the sequenced cytochrome b gene. INDEX WORDS: alternative oxidase inhibitors, benzimidazoles, cytochrome b gene, disease management, fungal taxonomy, fungicide sensitivity, fungicide resistance, fungicide resistance monitoring, guanidines, organotins, scab management, sterol inhibitors DEVELOPMENT OF RAPID IN VITRO ASSAYS AND CURRENT STATUS OF FUNGICIDE SENSITIVITY IN THE PECAN SCAB PATHOGEN FUSICLADIUM EFFUSUM by MURAT SEYRAN B.S., Ege University Izmir, Turkey, 2004 A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2008 © 2008 Murat Seyran All Rights Reserved DEVELOPMENT OF RAPID IN VITRO ASSAYS AND CURRENT STATUS OF FUNGICIDE SENSITIVITY IN THE PECAN SCAB PATHOGEN FUSICLADIUM EFFUSUM by MURAT SEYRAN Major Professors: Katherine L. Stevenson Timothy B. Brenneman Committee: Patrick J. Conner Ronald D. Gitaitis Albert K. Culbreath Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia December 2008 iv DEDICATION Pro prosum plebes For good people v ACKNOWLEDGEMENTS I would like to thank my advisors Dr. Stevenson and Dr. Brenneman for providing this assistantship oppurtunity, committee members Dr. Gitaitis, Dr. Conner and Dr. Culbreath; Dr. Ann Umbach, for critical review; Bonnie Evans, Kippy Lewis, Angenette Planter, Andrew Moore, Dr. Douda Kone for technical assistance and Sidney Law and Tim Varnadore for assistance in locating orchards. Financial support provided by E. I. Du Pont de Nemours and Company, Inc., United Phosphorus Inc., and the Georgia Agricultural Commodity Commission for Pecans is also appreciated. I would not reach to this point in life without the support my mother, fathter and sister, Ayse Nurla, Mustafa and Esra. I remember with respect, my elementary school teacher, Mrs. Gülsen Koçel, and my middle school teacher Mrs. Gülbetül Güven. And, I salute Mustafa Kemal and his soldiers. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .............................................................................................................v CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW......................................................1 Pecan Production and Pathology...............................................................................1 Disease cycle and Epidemiology of Pecan Scab Disease..........................................2 Pecan Scab Management...........................................................................................3 Mode of Action of Pecan Scab Fungicides ...............................................................4 Fungicide Resistance.................................................................................................9 Fungicide Resistance Management and the Current Status of Fungicide Resistance in F. effusum..........................................................................................22 Research Objectives ................................................................................................23 Literature Cited........................................................................................................24 2 BASELINE SENSITIVITY PROFILE OF FUSICLADIUM EFFUSUM TO DODINE, FENTIN HYDROXIDE, THIOPHANATE-METHYL AND PROPICONAZOLE ................................................................................................37 Abstract ...................................................................................................................38 Introduction.............................................................................................................39 Materials and Methods ............................................................................................40 Results .....................................................................................................................42 vii Discussion ...............................................................................................................44 Literature Cited........................................................................................................47 3 IN VITRO TOXICITY OF ALTERNATIVE OXIDASE INHIBITORS TO FUSICLADIUM EFFUSUM AND BASELINE SENSITIVITY TO AZOXYSTROBIN ..................................................................................................58 Abstract ...................................................................................................................59 Introduction.............................................................................................................60 Materials and Methods ............................................................................................61 Results .....................................................................................................................65 Discussion ...............................................................................................................66 Literature Cited........................................................................................................69 4 A RAPID METHOD TO DETECT FUNGICIDE RESISTANCE IN FUSICLADIUM EFFUSUM...............................................................................................................84 Abstract ...................................................................................................................85 Introduction.............................................................................................................86 Materials and Methods ............................................................................................88 Results .....................................................................................................................93 Discussion ...............................................................................................................96 Literature Cited......................................................................................................105 5 HYLOGENY OF THE PECAN SCAB FUNGUS FUSICLADIUM EFFUSUM (WINTER) BASED ON CYTOCHROME B GENE SEQUENCE......................129 Abstract .................................................................................................................130 Introduction...........................................................................................................131 viii Materials and Methods ..........................................................................................131 Results and Discussion..........................................................................................133 Literature Cited......................................................................................................135 6 SUMMARY ...............................................................................................................138 Literature Cited......................................................................................................139 APPENDIX..................................................................................................................................141 A DISCUSSION BASED ON THE INTRON STATUS OF MITOCHONDRIAL CYTOHROME B GENE SEQUENCE .............................141 Literature Cited......................................................................................................143 1 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW Pecan Production and Pathology Pecan (Carya illinoinensis (Wangenheim) K. Koch.), is the only native nut tree crop cultivated commercially in the United States (3). In 2005, U.S. growers produced 280 million pounds and the production in Georgia was 70 million pounds (2). Mexico is the second largest pecan producer in the world following the United States. Relatively small amounts of pecans are produced by Australia, Brazil, Israel, Peru, South Africa and Thailand (3,111). The utilized pecan production in the U.S. generally ranges from 85-150 million pounds and is fairly stable (111). However, the grower price of shelled pecans in the U.S. has gradually increased from $1.68 in 1980 to $3.32 per lb. in 2005 (111). The major economic constraints of pecan production in the southeastern U.S. are drought stress, insect pests and diseases. Pecan scab caused by the fungus Fusicladium effusum (Winter) is the most devastating disease of the pecan plant and is the major target for
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