ENDOPHYTIC ASSOCIATIONS OF SPECIES IN THE ASPERGILLUS SECTION NIGRI WITH MAIZE (ZEA MAYS) AND PEANUT (ARACHIS HYPOGEA) HOSTS, AND THEIR MYCOTOXINS by EDWIN RENE PALENCIA (Under the Direction of Charles W. Bacon) ABSTRACT Filamentous fungi in the Aspergillus section Nigri, the black aspergilli, are associated with several crops including maize, peanut, and grape where their infections can cause maize kernel rot, peanut blight, and grape rot, respectively. New evidence suggests black aspergilli can colonize plant tissue as endophytes. We developed a system to identify black aspergilli from peanut and maize in the southeastern United States. A survey indicated that 86.7 % (n= 150) of the isolates characterized by a rep-PCR system were A. niger, suggesting that this species complex is predominant in maize and peanut fields. To gain a better understanding of the plant- fungi interactions, we developed a genetic transformation system to generate fluorescent black Aspergillus strains. Microscopy showed that both A. niger and A. carbonarius fluorescent transformants colonized root tissue as intercellular hyphae, which is characteristic of endophytes. Plant experiments were carried out to determine the potential benefits of crop plants harboring endophytic fungi. Eleven black Aspergillus species were used to inoculate maize seeds, but only A. carbonarius and A. niger were able to systemically colonize seedlings; none of the isolates significantly promoted plant growth on two maize cultivars. In greenhouse studies in peanut, A. niger and its fluorescent transformants systemically colonized above and below-ground peanut tissue, but infections with both species had neutral effects on plant growth in the Florida 07 cultivar. We also tested the capacity of 150 isolates to produce fumonisins, carcinogenic secondary metabolites. We found that 27.8% (n=54) of the isolates produced FB1, 59.2% FB2, and 44.5% FB3. In conclusion, we report here that A. carbonarius and A. niger strains form endophytic associations with maize and peanut hosts. We also documented that some black Aspergillus isolates are capable of producing fumonisins. INDEX WORDS: Aspergillus, endophyte, mycotoxin, maize, corn, peanut ENDOPHYTIC ASSOCIATIONS OF SPECIES IN THE ASPERGILLUS SECTION NIGRI WITH MAIZE (ZEA MAYS) AND PEANUT (ARACHIS HYPOGEA) HOSTS, AND THEIR MYCOTOXINS by EDWIN RENE PALENCIA FORB, Univeresidad del Valle de Guatemala, Guatemala, 2000 M.S., University of Georgia, 2006 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2012 © 2012 Edwin Rene Palencia All Rights Reserved ENDOPHYTIC ASSOCIATIONS OF SPECIES IN THE ASPERGILLUS SECTION NIGRI WITH MAIZE (ZEA MAYS) AND PEANUT (ARACHIS HYPOGEA) HOSTS, AND THEIR MYCOTOXINS by EDWIN RENE PALENCIA Major Professor: Charles W. Bacon Committee: Harald Scherm Anthony E. Glenn Scott E. Gold Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2012 DEDICATION To my parents Eduarda del Rosario and Jose Victor Palencia for making me dream the fascinating game of life. To my beautiful wife, Kimberley, for filling my life with joy and happiness. The world is out there waiting for us. iv ACKNOWLEDGEMENTS First and foremost, I am grateful to my major advisor and mentor Dr. Charles Bacon for his patience, positive influence, guidance and continuous support through the years in the PhD program. He has shown me, by example, how to pursue excellence not only in science but in life in general. For the years to come, I will make sure to apply everything I have learned from him in any event of my life. Thanks Dr. Bacon, I deeply appreciate everything what you have done for me. I owe you much. I would also like to thank my advisory committee: Drs. Scott Gold, Anthony Glenn, and Harald Scherm for their invaluable encouragement, support, and for providing scientific guidance during the years of graduate school. I am thankful to Dr. Ronald Riley, for his encouragement, science, and mainly for his kind support throughout the years. My sincere thanks go to Dorothy M. Hinton for being instrumental in many aspects of this doctoral research, for her kindness, but especially for her friendship. Most of the experiments described in this doctoral dissertation would not have been possible without her assistance. Thanks Dot. I would also like to thank Dr. Maurice Snook for his help in the chemical analysis. I am thankful to Trevor Mitchell for devoting part of his time to the chemical analysis and for his friendship. I want to thank Johnna Garrish for being a great help in several experiments of this research, but especially for always helping me out with a great smile. Thanks to Britton Ormiston for her support, especially in my early years of graduate school. v I also want thank my friend Ada Bacetty for the great moments we have spent throughout all these years. It has been great to see how far you have gone, and you keep it coming. I am proud to be considered your friend. I thank my ARS-USDA-TMRU family who have helped me to discover this wonderful world of science. To all of you, past and present members of my USDA family, I will take you in my heart wherever I go. Last but not least, I want to thank Latoya Wiggins, Susan Brooks, Takiyah Ball, Rhonda Gantt, Calvin Williams, Nathaniel Upshaw (R.I.P), Susan Mize, and Joyce, for their invaluable friendship. You provided me with family love, especially in those days when a hug and hand shake were essential to pass through difficult times. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .............................................................................................................v LIST OF TABLES ...........................................................................................................................x LIST OF FIGURES ....................................................................................................................... xi CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW .....................................................1 BACKGROUND .....................................................................................................1 PROJECT OBJECTIVES ........................................................................................5 LITERATURE CITED ............................................................................................7 2 THE BLACK ASPERGILLUS SPECIES OF MAIZE AND PEANUT AND THEIR POTENTIAL FOR MYCOTOXIN PRODUCTION...................................................11 ABSTRACT ...........................................................................................................12 INTRODUCTION .................................................................................................12 SUMMARY ...........................................................................................................25 LITERATURE CITED ..........................................................................................27 3 USE OF A REP-PCR SYSTEM TO PREDICT SPECIES IN THE ASPERGILLUS SECTION NIGRI .........................................................................................................45 ABSTRACT ...........................................................................................................46 INTRODUCTION .................................................................................................47 MATERIALS AND METHODS ...........................................................................49 vii RESULTS AND DISCUSSION ............................................................................52 LITERATURE CITED ..........................................................................................57 4 GENETIC TRANSFORMATION OF THE FUNGI ASPERGILLUS NIGER VAR NIGER AND ASPERGILLUS CARBONARIUS USING FLUORESCENT MARKERS ..................................................................................................................68 ABSTRACT ...........................................................................................................69 INTRODUCTION .................................................................................................70 MATERIALS AND METHODS ...........................................................................72 RESULTS ..............................................................................................................79 DISCUSSION ........................................................................................................84 LITERATURE CITED ..........................................................................................89 5 ENDOPHYTIC ASSOCIATIONS BETWEEN BLACK ASPERGILLI AND MAIZE/PEANUT AS PLANT HOSTS ....................................................................102 ABSTRACT .........................................................................................................103 INTRODUCTION ...............................................................................................103 MATERIALS AND METHODS .........................................................................106 RESULTS ............................................................................................................110 DISCUSSION ......................................................................................................114 LITERATURE CITED ........................................................................................117 6 PRODUCTION