EVALUATION OF THE ENTOMOPATHOGENIC FUNGUS BEAUVERIA BASSIANA ON THE RED IMPORTED FIRE ANT, SOLENOPSIS INVICTA By ROBERTO MANOEL PEREIRA DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OFTHE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1991 A Eduardo, Ricardo, Angela, Alberto, e Rodrigo, que construiram a base de tudo. AJulia, Carolina, Marcel, Denise, e outros por vir, uma nova geragao de esperanqas. ACKNOWLEDGMENTS I thank Dr. Jerry L. Stimac for his guidance and friendship during the course of my studies at the University of Florida, and Dr. Sergio B. Alves for the support and teachings in the last 10 years. I am also very grateful for the help offered by Drs. Drion G. Boucias, Clayton W. McCoy, and James W. Kimbrough, and for the support from the faculty and staff of the Department of Entomology and Nematology of the University of Florida My gratitude is extended to Lois Wood for her ever-present helping hand, and for entertaining stories. I would also like to acknowledge the help of people who in many ways contributed to the completion of this work, namely: Lisa Huey, Robin Register, Reginald Coler, Hugh Smith, Dr. Tomas Zoebisch, and Dr. David Oi. I am grateful for the patience and understanding of my wife Bete, and for the loving and nurturing home she creates for our daughters and me. in TABLE OF CONTENTS page ACKNOWLEDGEMENTS iii LIST OF TABLES vii LIST OF FIGURES ix KEY TO ABBREVIATIONS xi ABSTRACT xii CHAPTERS INTRODUCTION 1 1 Geographic Distribution and Introduction of Fire Ants into the United States 1 Importance 2 Present Control Methods 4 Natural Enemies 5 Microbial Agents 7 Use of Beauveria bassiana in Insect Control 10 Beauveria bassiana as a Microbial Control for Fire Ants 11 Objectives 14 2 DOSE-RESPONSE OF THE RED IMPORTED FIRE ANTS, SOLENOPSIS INVICTA, WORKERS TO BEAUVERIA BASSIANA CONIDIA 17 Introduction 17 Materials and Methods 19 Fungus 19 Ants and Soil 20 IV Spray Tower and Spraying Procedure 21 Dose-Response Experiments 22 Mortality Assessment and Analysis of Results .... 24 Results 25 Direct Spray Experiments 25 Soil Incorporation Experiments 30 Surface Exposure Experiments 38 Discussion 42 Effect of Fungus in Direct Spray Experiments 42 Effect of Conidia Mixed into Soil 44 Infection of Ants by Conidia in Soil and on Plate Surface 46 TRANSMISSION OF BEAUVERIA BASSIANA 3 WITHIN ARTIFICIAL FIRE ANT NESTS IN THE LABORATORY 48 Introduction 48 Materials and Methods 51 Fungus and Inoculation of Ants 51 Preparation of Artificial Colonies 52 Experimental Procedures 52 Estimation of Number of Conidia in Cadaver Pile 54 . . Experiments with Conidia Formed on Ants 54 Assessment of Survival and Analysis of Results ... 55 Results 57 Experiments with Live Infected Ants in Plaster-Bottom Cups 57 Experiments with Live Infected Ants in Soil 61 Experiments with Dead Infected Ants 64 Conidia per Ant 70 Experiments with Conidia Formed on Ants 70 Discussion 74 4 GROWTH OF BEAUVERIA BASSIANA IN FIRE ANT NEST SOIL WITH AMENDMENTS 79 Introduction 79 Materials and Methods 81 Fungus 81 Amendments 82 Preparation of Soil Plates and Inoculation 83 Observations, Measurements and Analysis of Results 85 Results 86 Mycelial Inoculum 86 Conidial Inoculum 88 Ant Inoculum 93 Discussion 94 Effects of Inoculum Types 94 Effects of Amendments 99 5 CONCLUSIONS AND IMPLICATIONS OF RESULTS ON THE MICROBIAL CONTROL OF SOLENOPSIS INVICTA WITH THE ENTOMOPATHOGENIC FUNGUS BEAUVERIA BASSIANA 102 APPENDICES A EFFICIENCY OF WASHING PROCEDURE IN ESTIMATION OF NUMBER OF CONIDIA PER ANT AFTER APPLICATION OF CONIDIAL SUSPENSIONS 112 B EVALUATION OF EFFECT OF SOIL ANTAGONISM ON BEAUVERIA BASSIANA INFECTION OF FIRE ANT WORKERS 114 LITERATURE CITED 117 BIOGRAPHICAL SKETCH 135 VI LIST OF TABLES table page 1-1 Effective dose of B. bassiana against insect pests under several experimental conditions as reported by different authors 12 2-1 Lethal concentrations (conidia / ml of suspension) of B. bassiana against S. invicta, when suspensions are directly sprayed on the ants which are maintained in clean plastic cups for 15 days 28 2-2 Lethal times (days) for B. bassiana against S. invicta, when suspensions are directly sprayed on the ants, which are maintained in clean plastic cups for 15 days 29 . . . , 2-3 Lethal concentrations (conidia / ml of suspension) of B. bassiana against S. invicta, when suspensions are directly sprayed on the ants which are maintained in plastic cups containing either sterile or nonsterile fire ant nest soil for 15 days 33 2-4 Lethal concentrations (conidia / g of soil) of B. bassiana against S. invicta, when suspensions are applied to either sterile or nonsterile fire ant nest soil, and ants are maintained in soil for 15 days 37 2-5 Lethal concentrations (conidia / mn\2 of plastic Petri dish) of B. bassiana against S. invicta, when suspensions are directly sprayed on Petri dishes and ants are allowed to walk in the dishes for 24 hours before being transferred to clean plastic cups for 15 days 40 VII 2-6 Lethal times (days) for B. bassiana against S. invicta, when suspensions are sprayed on Petri dishes and ants are allowed to walk in the dishes for 24 hours before being transferred to clean plastic cups for 15 days 41 3-1 Mean survivals of ants and mean weights of surviving ants in colonies receiving different percentages of B. bassiana-iniected individuals and maintained in containers with no soil (Experiment 1) 59 3-2 Mean survivals of ants and mean weights of surviving ants in colonies receiving different percentages of B. bassiana-iniected individuals and maintained in containers with no soil (Experiment 2) 60 3-3 Mean survivals of ants and mean weights of surviving ants in colonies receiving different percentages of B. bassiana-iniected individuals and maintained in soil (Experiment 1) 63 3-4 Mean survivals of ants and mean weights of surviving ants in colonies receiving different percentages of B. bassiana-iniected individuals and maintained in soil (Experiment 2) 65 3-5 Mean survivals of ants and mean weights of surviving ants in colonies receiving different percentages of B. bassiana-iniected cadavers and maintained in containers with no soil 66 3-6 Mean survivals of ants and mean weights of surviving ants in colonies receiving different percentages of B. bassiana-iniected cadavers and maintained in containers with soil 67 A-l Results of the estimation of washing efficiency of procedure used in the estimation of dose of conidia per ant after spraying of conidial suspension 113 B-l Percent mortality and infection of fire ant workers (S. invicta) exposed to nest soil containing conidia of the fungus B. bassiana 116 VIII LIST OF FIGURES E^ figure 2-1 Mortality and infection of S. invicta treated with direct spray of B. bassiana conidial suspensions and maintained in clean plastic cups for 15 days 27 2-2 Mortality and infection of S. invicta treated with direct spray of B. bassiana conidial suspensions and maintained in plastic cups containing sterile fire ant nest soil for 15 days 31 2-3 Mortality and infection of S. invicta treated with direct spray of B. bassiana conidial suspensions and maintained in plastic cups containing nonsterile fire ant nest soil for 15 days 32 2-4 Mortality and infection of S. invicta maintained for 15 days in sterile fire ant nest soil treated with B. bassiana 34 conidia 2-5 Mortality and infection of S. invicta maintained for 15 days in nonsterile fire ant nest soil treated with B. 36 bassiana conidia 2-6 Mortality and infection of S. invicta exposed to Petri dish surface treated with direct spray of B. bassiana conidial suspensions, and maintained in in clean plastic cups for 15 days 39 IX 3-1 Number of conidia per cup and per gram of ants produced on S. invicta cadavers, in population either with 100% of B. bassiana-infected insects or with mixture of 50-50% of healthy and infected ants 71 3-2 Mortality of S. invicta treated with direct spray of B. bassiana conidial suspensions prepared from conidia produced either on SDAY plates or on ant cadavers from experiments with or without soil 72 4-1 Diameter of 15-day old B. bassiana colonies on sterile and nonsterile fire ant nest soil amended with different materials and inoculated with dry mycelium particle 87 4-2 Number of B. bassiana colony forming units (CFU's) produced after 15 days of incubation on either sterile or nonsterile fire ant nest soil amended with different materials and inoculated with dry mycelium particle 89 4-3 Number of B. bassiana colony forming units (CFU's) produced after 15 days of incubation on either sterile or nonsterile fire ant nest soil amended with different materials and inoculated with conidial suspension 91 4-4 Diameter of 10-day old B. bassiana colonies on sterile fire ant nest soil amended with different materials and inoculated with conidial suspension 92 4-5 Diameter of 15-day old B. bassiana colonies on sterile and nonsterile fire ant nest soil amended with different materials and inoculated with with fire ant (S. invicta) abdomens 95 4-6 Number of B. bassiana colony forming units (CFU's) produced after 15 days of incubation on either sterile or nonsterile fire ant nest soil amended with different materials and inoculated with fire ant (S. invicta) abdomens 96