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Korir Umd 0117E 20494.Pdf (1.137Mb) ABSTRACT Title of Dissertation: FUSARIUM SPECIES OF CUCUMIS MELO IN THE MID-ATLANTIC REGION OF THE US AND THEIR IMPACT ON SALMONELLA ENTERICA NEWPORT SURVIVAL AND INTERNALIZATION ON VARIOUS MELON CULTIVARS Robert Cheruiyot Korir, Doctor of Philosophy, 2019 Dissertation directed by: Dr. Kathryne L. Everts and Dr. Shirley Ann Micallef, Department of Plant Science and Landscape Architecture Fruit rots caused by Fusarium spp. can lead to economic yield losses on melon (Cucumis melo). However, which Fusarium spp. are the most prevalent in Maryland and Delaware has not been documented. Several Salmonella enterica subsp. serovar Newport (S. Newport) outbreaks on melon have occurred over the past 25 years. Fusarium spp. infestation on melon have potential impact on survival and colonization of Salmonella. Our objectives were to identify Fusarium spp. infestations on melons within the Delmarva region, and evaluate their impact on survival and internalization of S. Newport on various melon cultivars. Fifty-six isolates were molecularly identified, according to Fusarium-ID online database, as Fusarium spp. (Fusarium fujikuroi-20, Fusarium proliferatum-18, Fusarium oxysporum-15, Fusarium graminearium-2, Fusarium verticilloides-1). Our findings revealed that most of the Fusarium isolates we collected were not pathogenic to melon fruit. We evaluated the impact of four Fusarium spp. (F. armeniacum, F. oxysporum, F. fujikuroi, and F. proliferatum) on S. Newport survival in five melon cultivars; ‘Arava’ (C. melo var. reticulatus, Galia), ‘Athena’ (var. reticulatus, muskmelon), ‘Dulce Nectar’ (var. inodorus, honeydew), ‘Jaune de Canaries’ (var. inodorus, Canary), and ‘Sivan’ (var. cantalupensis, Charentais). Impact of F. proliferatum on survival and internalization of S. Newport was evaluated on honeydew (smooth) and cantaloupe (netted) melons. Generally, Fusarium did not impact the survival of S. Newport, however greater survival of S. Newport was observed on the netted cultivars compared to the smooth surface melons. Fusarium fujikuroi significantly enhanced survival of Salmonella when inoculated on riper ‘Jaune de Canaries’ melons (above ¾ slip). However, when the experiments were replicated with less ripe (about ¾ slip) melon, F. fujikuroi did not significantly influence the growth of S. Newport. Salmonella Newport internalized in all treatments and the cantaloupe and honeydew melons, but variation in population levels were observed across the treatments. Overall, Fusarium proliferatum did not impact internalization of S. Newport on either melon type. This may be attributed to that Fusarium species used during this study were nonpathogenic. Salmonella Newport recovered gradually decreased with time. Fusarium species on melon, influence S. Newport colonization differently. Also, melon rind type affects the ability of S. Newport to survive and colonize differently. Keywords: Melon, Cultivars, Fusarium spp., Salmonella enterica Newport, Survival, Internalization. FUSARIUM SPECIES OF CUCUMIS MELO IN THE MID-ATLANTIC REGION OF THE US AND THEIR IMPACT ON SALMONELLA ENTERICA NEWPORT SURVIVAL AND INTERNALIZATION ON VARIOUS MELON CULTIVARS. by Robert Cheruiyot Korir Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2019 Advisory Committee: Professor Kathryne Everts, Chair Associate Professor Shirley Micallef, Co-Advisor Dr. Manan Sharma Associate Professor Abani Pradhan Assistant Professor Rohan Tikekar © Copyright by Robert Cheruiyot Korir 2019 Dedication This dissertation is dedicated to my daughter, Meredith Chebet Korir, my parents; John and Esther Segem, and all other family members in Kenya. ii Acknowledgements First and foremost, I would like to acknowledge and thank my advisors, Dr. Kathryne Everts and Dr. Shirley Micallef, who worked tirelessly along with me during my doctoral studies. Many thanks are due Dr. Manan Sharma, Dr. Rohan Tikekar, and Dr. Abani Pradhan for their suggestions. Also, I sincerely thank Yacintha Johnson and Anthony LaBarck for their great help and support in the Lab., and David Armentrout and Michael Newell for their assistance in the field and high tunnel melon production. Furthermore, I would like to appreciate all the help I received from my colleagues in the Pathology and Food Microbiology Safety Labs, Department of Plant Science. Lastly, NIFA 2014 511062090 for funding this project. iii Table of Contents Dedication ..................................................................................................................... ii Acknowledgements ...................................................................................................... iii Table of Contents ......................................................................................................... iv Chapter 1: General Introduction ................................................................................... 1 Introduction ............................................................................................................... 1 1.1. Rationale for this study. ............................................................................ 1 1.2. Melon consumption in the U.S. ................................................................ 3 1.3. Melon Production in the US. .................................................................... 3 1.4. Melon Production in Maryland. ................................................................ 4 1.6. Microbial contamination of produce. ........................................................ 6 1.7. Fusarium spp. and Cucurbits. ................................................................... 6 1.8. Objectives. ................................................................................................ 8 1.9. The goal of this project was to test four main hypotheses: ....................... 9 Chapter 2: Literature Review ...................................................................................... 10 2.1. Salmonella outbreaks on cucurbits. ............................................................. 10 2.2. Phytopathogens and Human Pathogens on Plants (HPOPs). ....................... 11 2.3. Internalization of human bacteria in plants/produce. ................................... 15 2.4. Fusarium species Infection. ......................................................................... 16 Chapter 3: Fusarium spp. of Cucumis melo in the Mid-Atlantic Region of the U.S. 20 Abstract ................................................................................................................... 20 Introduction......................................................................................................... 21 Materials and Methods ....................................................................................... 22 3.1. Sample Collection. ....................................................................................... 22 3.2. Isolation of Fusarium spp. ........................................................................... 23 3.3. Pathogenicity test of Fusarium isolates; inoculum preparation. .................. 24 3.4. Identification of Fusarium Isolates. ............................................................. 26 3.5. Molecular Identification of Fusarium Isolates. ........................................... 27 3.6. PCR Amplifications. .................................................................................... 27 3.7. DNA Sequencing, Alignment, and Phylogenetic Analysis. ........................ 28 3.8. Data Analysis. .............................................................................................. 29 Results and Discussion. ...................................................................................... 29 4.1. Morphological analysis. ............................................................................... 29 4.2. Macroscopic Characteristics. ....................................................................... 30 4.3. Isolation and Pathogenicity Test. ................................................................. 30 4.4. Molecular Identification............................................................................... 31 4.5. Repeat Pathogenicity Test............................................................................ 33 Conclusion. ............................................................................................................. 33 Chapter 4: Interactions between Salmonella enterica Newport, Fusarium spp. and Melon Cultivars. ......................................................................................................... 35 Abstract ............................................................................................................... 35 Introduction. ............................................................................................................ 36 5.1. Melon Consumption and Salmonella Contamination. ................................. 36 5.2. Salmonella enterica on/in Plants. ................................................................ 36 iv 5.3. Fusarium spp. and Melon ............................................................................ 38 Materials and Methods ....................................................................................... 39 6.1. Melon Samples Production. ......................................................................... 39 6.2. Microbial Material Fusarium
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