INSIGHTS INTO THE GENETIC AND ENVIRONMENTAL BASES OF MYCOTOXIN CONTAMINATION IN KENYAN MAIZE A Dissertation Presented To the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Samuel Kilonzo Mutiga January 2015 © 2015 Samuel Kilonzo Mutiga ii INSIGHTS INTO THE GENETIC AND ENVIRONMENTAL BASES OF MYCOTOXIN CONTAMINATION IN KENYAN MAIZE Samuel Kilonzo Mutiga, Ph.D. Cornell University, 2015 Mycotoxins are toxic fungal secondary metabolites that contaminate an estimated 25% of foods globally. Aflatoxin and fumonisin are major mycotoxins that contaminate maize in tropical countries. Kenya’s frequent aflatoxicosis outbreaks and the associated human fatality rates have received global attention. The objective of this dissertation was to investigate the extent and the drivers for mycotoxin contamination in Kenyan maize. Between May 2009 and March 2010, surveys were conducted in three provinces (Rift Valley, Western and Nyanza) of western Kenya, the country’s grain basket and a region where mycotoxin outbreak had not been recognized. Aflatoxin contamination above the regulatory limit of 10 ppb was observed in 15% of the flour samples that had been collected from the patrons of local mills. Drought and monocropping were identified as drivers for increased aflatoxin contamination. A longitudinal survey in farmers’ storage sheds and at local mills in Western Province revealed vulnerability of the most popular varieties to mycotoxins. Surveys were conducted in 10 districts of Eastern Province during an aflatoxin outbreak in 2010. Aflatoxin contamination above 10 ppb was observed in 39% of the flour samples from patrons (n=1500) of local mills, while 37% were above the 1 ppm regulatory limit for fumonisin. Reduced aflatoxin accumulation was associated with intercropping, larger farms and high grain yield. Visual grain sorting reduced fumonisin but not aflatoxin levels. Analysis of aflatoxin in naturally-infected diverse maize germplasm grown in eastern Kenya showed higher contamination under low nitrogen (N) than under optimal N. Early-maturing maize had reduced aflatoxin accumulation under low N, possibly because of faster utilization of the limiting and diminishing soil N. A mature kernel screening assay of diverse maize inbred lines showed that colonization and aflatoxin accumulation were influenced by the ear environment (where maize were grown). High sulfur content in grain was associated with reduced kernel colonization and aflatoxin accumulation. Management strategies should include: 1. surveillance across all maize-producing regions, 2. breeding for early maturing maize, and 3. spectral grain sorting. Experiments should be conducted to identify the intercrop species and the mechanism for aflatoxin control, and to elucidate the maize ionome - mycotoxin relationship. iii BIOGRAPHICAL SKETCH Samuel was born and brought up in eastern Kenya, where his parents were peasant farmers. While at high school, he had a great interest in Chemistry and Biology, and had a desire to pursue a career leading to the application of the knowledge. During his childhood, Samuel did not witness his parents buy certified seed or apply pesticides in their farming practices. Samuel has experienced the effect of food shortage and vividly recalls incidences of drought and famines that forced their family members to live on famine relief maize, locally termed as “kathĩĩka”, and moldy maize. Samuel did not know that moldy maize contained the mycotoxins which have been the major focus for his PhD research. Samuel’s career in agriculture begun when he joined Moi University (Kenya) for a B.Sc. degree in Horticulture in 2000. Despite his desire to be a Chemistry major, compelling advice from Dr. M. E. Omunyin, the then Head of Horticulture department, made him keep within. Samuel was undecided of his career until about his graduation with the B.Sc. in 2004, when he was compelled to be a crop protectionist by the larger grain borer, Prostephanus truncatus, which ground his nine 90-kg bags of maize grain that he had stored at his cousin’s store into unpalatable flour. Samuel earned a Master of Philosophy in Plant Protection from Moi University in 2008, under mentorship of Dr. L. S. Gohole and Prof. E. O. Auma. Samuel’s desire to advance his career in crop protection got a boost after his acceptance at Cornell University’s department of Plant Pathology & Plant-Microbe Biology in 2008. His research interests matched and were further shaped by Dr. Rebecca Nelson, who put enormous effort in developing research proposals to support Samuel’s research. Rebecca collaborated with Drs. Michael Milgroom, Charles Nicholson and Vivian Hoffmann in a research proposal on understanding aflatoxin accumulation in Kenyan maize, which was funded by the Cornell Center for Sustainable Future (CCSF, now ACSF) in 2008. During her 6-month (Jan-June 2009) sabbatical at Biosciences eastern and central Africa (BecA) – International livestock Research Institute (ILRI), Nairobi, Dr. Nelson discussed the aflatoxin project proposal with the host scientists and charted Samuel’s role in the project and his placement at the institution. As a member of R.Nelson lab, Samuel conducted his Ph.D research at Cornell University and at BecA–ILRI, Nairobi. At BecA, Samuel was co-supervised by Dr. Jagger Harvey, and had an opportunity to participate in grant proposal that was funded by the Australian Agency for International Development (AusAID). Besides attendance of several workshops relating to mycotoxin management in Eastern Africa, Samuel participated in the establishment of a mycotoxin platform at BecA-ILRI, Nairobi. Samuel’s interests are in management of plant diseases and pests using conventional and molecular breeding. He plans to spend the rest of his future career in doing research that could boost crop productivity, and food safety and security. iv I dedicate my thesis to my cousin, Paul Muchee Kairiba v ACKNOWLEDGMENTS My first acknowledgement is to Cornell’s department of Plant Pathology and Plant-Microbe Biology (PPPMB) for having accepted me to be their graduate student. I am very grateful for the Graduate Research and Teaching fellowship awarded to me by the department in 2008 which enabled me to join the University. I would like to acknowledge the support that my thesis committee members have given to me since I joined Cornell University. Dr. Rebecca Nelson worked hard to ensure that I got admitted to Cornell University, and I joined her lab. Dr. Nelson has been a great mentor and supportive in many ways during my research. I cannot forget the many times she introduced me, and made arrangements for me to meet Kenyan researchers who were to be of importance in my work. Dr. Nelson taught me the importance of database in handling large datasets. Besides the research advisory role, Dr. Nelson always alerted me about the need to take care of my health. I would also like to acknowledge Dr. Michael Milgroom for playing a key to role to ensure that I got admitted to Cornell University when he was the director of graduate studies in the PPPMB. Besides his support for my research in Kenya, Dr. Milgroom has been a source of my inspiration to pursue the understanding of the biology of plant pathogens. I would like to acknowledge Dr. Margaret Smith. I am honored to have Dr. Smith in my thesis committee as a mentor in Plant Breeding and Genetics (PB&G). My great desire to practice the plant breeding methods in managing plant diseases rhyme with the effort that Dr. Smith has put in the research extension and as a professor at Cornell University. I am very grateful to all my committee members for the advice, logistical support and handling of the paperwork that facilitated my 3-year leave of absence from Cornell University while I was doing my research at BecA-ILRI, Nairobi. I would like to appreciate the accommodation at BecA. I thank Drs. Segenet Kelemu (former BecA director) and Jagger Harvey, BecA-ILRI, Nairobi, for having accepted me to work in the BecA laboratories. The thousands of maize samples that I collected and stored in five freezers during my placement shocked most researchers, but the hub accommodated me. The research assistance provided by Gordon Otieno, Vincent Were, Damaris Njeri, and Samuel Angwenyi is appreciated. I would like to acknowledge RNelson lab. members who contributed to my work. Drs. Santiago Mideros and Chialin Chung introduced me to the laboratory standard operating procedures. Santiago maintained frequent check of my research progress even when I was in Kenya. Dr. Judy Kolkman ensured that I got my laboratory supplies in time. Laura Morales has been of great help in not only discussing my data, but also participating in exploratory analysis. Nelson Chepkwony provided technical support for my experiments in the lab at Cornell University. My work would not have been successful without consultations and collaboration with different researchers. Dr. Vivian Hoffmann (University of Maryland) participated in planning and the execution of vi Kenyan surveys. Vivian was involved in in-depth discussions about the study designs, participated in the development questionnaire and in sample collection. Dr. James Gethi (KARI, Kenya, now CIMMYT, Harare, Zimbabwe) has been a mentor and was always willing to share ideas about breeding methods and germplasm for reduction of aflatoxin accumulation in Kenyan maize. Lastly, my PhD program could not have been successful without the funding that I received from different sources. My admission into Cornell University was made possible by a teaching and research assistantship that I was offered by the department of Plant Pathology and Plant-Microbe Biology from 2008-2009. The Kenyan surveys were funded by the Cornell Center for Sustainable Future (CCSF, now ACSF) from 2009-2010. Dr. Christopher Barrett, a faculty fellow in the ACSF, played a big role in facilitating additional 3-year financial support for the Kenyan surveys through Stimulating Agricultural and Rural Transformation (StART) initiative of the Cornell International Institute of Food, and Agriculture Development (CIIFAD).