ABSTRACT JOHNSON, KENDALL A. Characterization and Fungicide Efficacy of North Carolina Colletotrichum Populations Causing Glomerella Leaf Spot and Fruit Rot on Apple. (Under the direction of Dr. Sara Villani). Glomerella leaf spot (GLS) and fruit rot (GFR) is historically caused by members of the Colletotrichum gloeosporioides species complex. Although the disease was first reported in the 1970s in the United States, the disease has reemerged on apples in North Carolina (NC) causing economically devastating losses. These pathogens cause necrotic leaf lesions and rots of fruit that will ultimately lead to premature defoliation, a decrease in yield, and unsaleable fruit. According to previous research, ‘Red Delicious’ and ‘Fuji’ have shown partial or complete resistance to GLS and GFR. Cultivars from the ‘Golden Delicious’ lineage are the most susceptible which includes cvs. ‘Golden Delicious’, ‘Gala’, and ‘Pink Lady’ which are also amongst consumer favorites and most commonly grown in NC. Currently there is a paucity of information regarding the management of the disease, but fungicides, cultural practices, and the planting of resistant cultivars are primarily used to reduce disease within an orchard. Fungicide use is limited due to the risk of resistance, but has found to be the most effective form of control for this disease. The objectives of this research were to 1) characterize Colletotrichum species based on morphological and molecular techniques and evaluate in vitro QoI fungicide sensitivity shifts in Colletotrichum populations and 2) determine the most efficacious fungicide and timing for the management of GLS and GFR. Three hundred ninety-six isolates were collected from symptomatic apple leaves and fruit from a total of 17 orchards in North Carolina and Georgia in 2016. Three hundred and seventy- three of those recovered isolates were grown on potato dextrose agar (PDA) at 25°C under constant light for 10 d. After 10 d, isolates were characterized based on colony color, growth pattern and conidium shape and size. Six morphotypes were determined through morphological characterization in addition to multilocus sequence analysis identifying two Colletotrichum species, C. fructicola and C. fioriniae as causal agents of GLS and GFR. Mycelial growth inhibition and conidial germination inhibition assays were conducted to determine QoI fungicide sensitivity shifts in Colletototrichum populations. This study determined C. fioriniae isolates were more sensitive to pyraclostrobin and trifloxystrobin compared to C. fructicola isolates. All isolates were found to remain sensitive to pyraclostrobin in mycelial growth and conidial germination inhibition experiments. Through conidial germination assays, five orchards were found to have reduced sensitivity to trifloxystrobin. A field trial experiment was conducted in 2017 and 2018 to determine the most efficacious fungicide. A non-rotational fungicide program using single-site and multi-site fungicides was evaluated for the control of GLS and GFR. In a separate field experiment, applications for pyraclostrobin plus fluxapyroxad (Merivon) were evaluated to determine a critical time for application. In the non-rotational field experiment, pyraclostrobin plus fluxapyroxad and captan were observed to be the most efficacious treatments. Applications of pyraclostrobin and fluxapyroxad at eighth and ninth cover spray had significantly less disease in 2017. In 2018, no significant differences were found between pyraclostrobin plus fluxapyroxad treatments when applied at different timings. © Copyright 2019 by Kendall Anderson Johnson All Rights Reserved Characterization and Fungicide Efficacy of North Carolina Colletotrichum Populations Causing Glomerella Leaf Spot and Fruit Rot on Apple. by Kendall A. Johnson A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science Plant Pathology Raleigh, North Carolina 2019 APPROVED BY: _______________________________ _______________________________ Dr. Sara Villani Dr. James Kerns Committee Chair _______________________________ Dr. Marc Cubeta DEDICATION To my wonderful family and friends who have encouraged me throughout this journey. ii BIOGRAPHY Kendall Anderson Johnson was born on March 10, 1993 in Pensacola, Florida. In 1996, her family moved to Hamilton, OH, but then five years later moved to Raleigh, NC. This is considered home to her because she spent the majority of her life growing up and developing as a well-rounded individual. Throughout high school, Kendall was very involved in academic and extracurricular activities, holding leadership roles in Academy of Finance and Key Club; serving as captain for two years on the soccer team while playing on a travel soccer team, taking piano lessons and finding time to work at a local restaurant. Kendall attended East Carolina University as a biology major in August 2011. Although she had a love for science beginning at a young age, she didn’t discover her love of plants until her junior year. She sought out every class she could involving plants such as plant biotechnology, plant physiology, and plant biology. She participated in a terrestrial field ecology and research problems in biology summer course with the Smithsonian Tropical Research Institute in Gamboa, Panama. Throughout this course, she had to design an experiment and assess data to draw conclusions. This established an interest in doing her own research. After graduation, Kendall accepted a job as Supply Chain Administration at Arysta LifeScience, an agrichemical company. She was immersed in the agricultural industry learning about crop protection products and various aspects of the business. She found her interest in plant pathology after interacting with several plant pathologists in the office and going on site visits for product efficacy evaluations. She also volunteered at Goodwill Community Foundation Farm. There, she carried out daily greenhouse operations as well as assisting with seeding, planting, and harvesting. Through these experiences, Kendall discovered her love for agriculture and crop protection. iii In 2016, Kendall began her graduate career in the research program under Dr. Sara M. Villani working with the characterization of causal agents of Glomerella leaf spot and fruit rot on apple, Colletotrichum spp. and fungicide efficacy for various FRAC groups and modes of action for her Master of Science degree. Her time spent in Dr. Villani’s lab allowed her to develop as a scientist as well as gain experience running lab experiments and field trials. Kendall hopes to continue her education in plant pathology through a PhD program at University of Georgia. iv ACKNOWLEDGMENTS I would like to thank my committee members, Drs. Sara M. Villani, James P. Kerns, and Marc A. Cubeta for their constant guidance and support. I would especially like to thank Dr. Sara Villani for taking a chance on me as her first student. I know this was a big risk, but I could never thank you enough for taking the time to be an amazing mentor and teaching me everything I know up to this point. Thank you for encouraging/forcing me to speak at grower and annual meetings even though you knew I was terrified of speaking in front of people, participate in educational outreach events and attend professional meetings. These experiences allowed me to develop professionally, build confidence while presenting to a wide range of audiences, as well as deepen my knowledge of plant pathology that will be beneficial throughout my career. I would like to acknowledge the funding sources for this work, NC Specialty Crops Block Grant and the various agrichemical companies. This project would not have been possible without their support. I would like to recognize everyone who has been a part of the Villani lab over the past couple of years including Rachel Kreis, Harris Kopp, Cody Justus, Charlie Mackenzie, Rebecca Littauer, and Alejandro Llanos. To Rachel, thank you so much for your help in the lab and field. You had answers for every lab related question I threw at you and made sure I was always using the correct techniques. Thank you for also becoming a great friend who is always up for concerts and Taylor Swift jam sessions. To Harris, thank you for being the best undergraduate student worker I could have ever asked for. You are one of the hardest working people I know and it was truly an honor to teach you about my project and establish new laboratory skills. To Cody, Charlie, Rebecca, and Alejandro, thank you for helping set up my field trial and rate disease. This was always a daunting task every week during the summer and without you it would have taken days! v I could not have done this without my friends and family. I am grateful for the constant support of my friends and the new friendships I have made along the way. Finally, thank you to my supportive parents Annetta Johnson and Paul Johnson and step parents, Steve Eyman and Karen Johnson, and my brother Kyle. Without you guys I would not be where I am today. vi TABLE OF CONTENTS LIST OF TABLES ........................................................................................................................ ix LIST OF FIGURES ...................................................................................................................... xi Chapter I: Literature Review ..................................................................................................... 1 Apple Cultivation and Production