STUDIES ON SECONDARY METABOLITES ASSOCIATED WITH WITCHES’ BROOM DISEASE, FLORAL BIOLOGY, AND SEED FERMENTATION IN CACAO by Fábio Clasen Chaves A dissertation submitted to the Graduate School-New Brunswick Rutgers, The State University of New Jersey in partial fulfillment of the requirements for the degree of Doctor of Philosophy Graduate Program in Plant Biology written under the direction of Professor Thomas J. Gianfagna and approved by ________________________ ________________________ ________________________ ________________________ New Brunswick, New Jersey May, 2008 ABSTRACT OF THE DISSERTATION STUDIES ON SECONDARY METABOLITES ASSOCIATED WITH WITCHES’ BROOM DISEASE, FLORAL BIOLOGY AND SEED FERMENTATION IN CACAO by Fábio Clasen Chaves Dissertation Director: Professor Thomas J. Gianfagna Theobroma cacao L., a tree native to the Amazon, is cultivated in the tropics throughout the world for its seeds, used primarily for chocolate production. Cacao production is limited by several problems. Cocoa pod borer, an insect that burrows through pods, damages seeds, allowing contamination by toxigenic fungal species. Many fungal diseases infect cacao. Among them, Moniliophthora perniciosa, the causal agent of witches’ broom disease, severely affects plantations throughout South America and the Caribbean. Cacao yields are further limited by the naturally low rates of fruit set. Moreover, disease tolerant varieties are usually self-incompatible low producers and do not give a superior chocolate flavor compared to some disease susceptible and self- compatible genotypes with highly valued aroma compounds. During this project, problems associated with three main aspects of cacao were investigated: disease, production and processing. Studies on plant-endophyte-pathogen interactions allowed for the identification of new possible mechanisms of disease control; studies on cacao flower physiology indicated ways to improve pollination and therefore increase fruit set and crop yield; and investigations of the fermentation step of cacao processing permitted discovery of a method for maintaining higher levels of compounds valued by cacao manufacturers. ii Flavan-3-ol monomers and oligomers, purine alkaloids and salicylic acid, volatile organic compounds, polyketides and other phenolic compounds were among the determined bioactive compounds found in cacao, pathogens and endophytes, with influence on disease, production, and processing. iii ACKNOWLEDGMENTS I would like to thank: my advisor, Thomas J. Gianfagna, for his assistance, encouragement, guidance and support. His exemplar work ethic, sober advice, and personable demeanor have and will continue to influence my future career; the members of my graduate committee, Bingru Huang, James White Jr., Mark Kelm, and Prakash Hebbar, for their solicitude and keen guidance throughout my dissertation work; friends, colleagues and collaborators, Alan Pomella, Ajay Singh, Debora Esposito, Elisabeth Ng, Fernando Vega, Francisco Posada, Jeanne Peters, Jinpeng Xing, John Munafo, Logan Logendra, Luciana Ambrozevicius, Luis Mejia, Madhu Aneja, Marshall Bergen, Monica Torres, Patricia Alvarez, Patrick McCullough, Paulo Ribeiro, Richard Merrit, Rod Sharp, Shimon Rachmilevitch, Ulisses Hernandez, Yan Xu, and others that I may have inadvertently left out, whose friendship, partnership and shared experience positively contributed to my stay at Rutgers; all faculty members of Rutgers University, especially those from the Plant Biology and Pathology Department whose knowledgeable and informative lectures and seminars provided the basis for the development of my Ph.D. studies at Rutgers; CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for providing me with a graduate fellowship; Laura Cortese, my sweetheart, who has helped me avoid the use of ‘Portuguenglish’ during the writing of this dissertation; my family, Daniel, Morenei, Tania, and Vitor, for their constant support, love and care. iv You may travel the world, meet a million people and still if you do not know what you are looking for, you will never be satisfied with what you find. An experience may be very pleasant, but if you do not feel you are building something you won’t be happy. I may have heard them or may have read them; I sure did learn and believe them. v TABLE OF CONTENTS ABSTRACT OF THE DISSERTATION ................................................................................................... ii ACKNOWLEDGMENTS............................................................................................................................iv TABLE OF CONTENTS.............................................................................................................................vi LIST OF TABLES ........................................................................................................................................x LIST OF FIGURES ................................................................................................................................... xii 1. INTRODUCTION.....................................................................................................................................1 2. PLANT-ENDOPHYTE-PATHOGEN INTERACTIONS .....................................................................6 2.1. PLANT ....................................................................................................................................................6 2.1.1. INTRODUCTION...............................................................................................................................6 2.1.2. MATERIALS AND METHODS......................................................................................................12 2.1.2.1. CHEMICAL ELICITORS ............................................................................................................12 2.1.2.2. PATHOGEN INOCULATION .....................................................................................................12 2.1.2.3. LEAF SIZE EXPERIMENT .........................................................................................................13 2.1.2.4. SA AND SAG ANALYSIS.............................................................................................................15 2.1.2.5. PURINE ALKALOIDS AND PROCYANIDIN MONOMER ANALYSIS ..............................16 2.1.2.6. PROCYANIDIN OLIGOMER ANALYSIS ................................................................................17 2.1.2.7. STATISTICAL ANALYSIS..........................................................................................................17 2.1.3. RESULTS...........................................................................................................................................18 2.1.3.1. ENDOGENOUS LEVELS OF PURINE ALKALOIDS AND PROCYANIDIN MONOMERS IN FLUSH LEAVES OF GREENHOUSE GROWN TREES.................................................................18 2.1.3.2. LEVELS OF PURINE ALKALOIDS AND PROCYANIDIN MONOMERS IN FLUSH LEAVES OF GREENHOUSE GROWN TREES AFTER APPLICATION OF CHEMICAL ELICITORS.................................................................................................................................................19 2.1.3.3. ENDOGENOUS LEVELS OF SA AND SAG IN LEAVES OF GREENHOUSE-GROWN TREES..........................................................................................................................................................21 2.1.3.4. LEVELS OF SA AND SAG IN LEAVES OF GREENHOUSE-GROWN TREES AFTER APPLICATION OF CHEMICAL ELICITORS ......................................................................................22 2.1.3.5. LEVELS OF SA AND SAG IN LEAVES OF FIELD-GROWN TREES AFTER PATHOGEN INOCULATION..........................................................................................................................................25 2.1.3.6. CHANGES IN CACAO PROCYANIDIN CONTENT THROUGHOUT LEAF DEVELOPMENT........................................................................................................................................28 2.1.3.7. EFFECT OF PATHOGEN INOCULATION ON CACAO LEAF PROCYANIDINS ............32 2.1.3.8. PCA ANALYSIS COMPARING CAFFEINE, THEOBROMINE, (+)-CATECHIN, (-)- EPICATECHIN, SALICYLIC ACID, AND SALICYLIC ACID GLYCOSIDE LEVELS OF FLUSH LEAVES TREATED WITH ELICITORS ...............................................................................................36 2.1.4. DISCUSSION ....................................................................................................................................41 2.1.4.1. EFFECT OF CHEMICAL ELICITORS ON PURINE ALKALOIDS AND PROCYANIDIN MONOMERS ..............................................................................................................................................41 vi 2.1.4.2. EFFECT OF CHEMICAL ELICITORS AND PATHOGEN INOCULATION IN FREE SA AND SAG OF CACAO LEAVES..............................................................................................................44 2.1.4.3. LEAF SIZE EXPERIMENT .........................................................................................................46 2.1.4.4. EFFECT OF PATHOGEN INOCULATION IN PROCYANIDIN LEVELS ..........................48 2.1.5. CONCLUSIONS................................................................................................................................50 2.2. ENDOPHYTES AND THEIR METABOLITES...............................................................................53