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Kong Hawii 0085O 10931.Pdf VIRAL DISEASES OF CROTALARIA IN HAWAII THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI‘I AT MĀNOA IIN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN TROPICAL PLANT PATHOLOGY MAY 2021 By Alexandra Kong Thesis Committee: Michael Melzer, Chairperson John Hu Koon-Hui Wang ACKNOWLEDGEMENTS I would like to start off by thanking my committee members Dr. Michael Melzer, Dr. John Hu, and Dr. Koon-hui Wang for their patience and mentorship during my time here at the University of Hawaii at Manoa. I would also like to thank my family and fellows in tropical plant pathology for their unwavering support during these past couple of years. I’m very thankful for the members of the Agrosecurity lab, past and present, for their kindness and putting up with me for the past 4 years. This includes Dr. Michael Melzer, Dr. Shizu Watanabe, Miriam Long, Tomie Vowell, Asoka de Silva, Alejandro Olmedo Velarde, Brandi-Leigh Adams, Jarin Loristo, Cheyenne Barela, Nelson Masang Jr., and Lisa Lowe. The biggest thanks to Dr. Michael Melzer for being an outstanding mentor and giving me the opportunity to work in his lab as both an undergraduate and graduate student; his kindness and leadership being a true inspiration during my years working in his lab. Thanks to Flora Samis and Dr. Koon-hui Wang for collecting the infected sunn hemp material from the Poamoho Research Station and to the extension agent Alton Aragaki for finding and collecting infected Crotalaria samples from the Big Island. Dr. John Hu, Dr. Wayne Borth, and the Spring 2019 PEPS602 class that helped with the purification of SHMoV virus particles that were later used in the transmission electron microscopy. Additional thanks to Miriam Long, a previous member of the Argosecurity Lab, whose work served as a foundation upon which this thesis was built. ii ABSTRACT Sunn hemp (Crotalaria juncea) is a leguminous cover crop valued for its ability to rapidly accumulate biomass and fix nitrogen. In October 2016, farmers in Maui County noticed symptoms of leaf mottle, reduced seed pod numbers, and a reduction in seed yield of their sunn hemp crop. Although these symptoms are indicative of Sunn hemp mosaic virus (SHMV) infection, next generation sequencing of a dsRNA-based library revealed the presence of two viruses not yet reported in Hawaii. The first, Tobacco streak virus (TSV) is Ilarvirus previously reported in the continental United States, but not in Hawaii. The second virus is a novel viral species with notable homology to members of the genus Tobamovirus and most similar in sequence identity to SHMV. In 2018 sunn hemp samples from Poamoho with symptoms of leaf chlorosis were also sampled. Next generation sequencing of a dsRNA-based library revealed the presence of another virus not yet reported in Hawaii, the Fabavirus Broad bean wilt virus 2. A weedy sunn hemp relative, Crotalaria micans was also sampled from the Big Island and was found to be infected with the potyvirus Bean common mosaic virus. The presence of these new viruses could cause potential problems not only to the sunn hemp industry in Hawaii, but may potentially impact local agriculture. The purpose of this study was to identify the pathogen(s) responsible for the observed symptoms on C. juncea and other Crotalaria species as well as better characterize this new tobamovirus. iii Table of Contents Acknowledgements………………………………………………………………………………..ii Abstract…………………………………………………………………………………………...iii List of Tables……………………………………...….………………………………………….vii List of Figures………………………………………………………………………...…………viii Preface……………………………………………………………………………………………..x Chapter 1: Literature Review…………………………………………………………………...…1 Crotalaria juncea…………………………..…………………………………………...…1 Tobamoviruses…………………………………………………………………………….5 Sunn-hemp mosaic virus…………………………………………………………………..5 Seed Transmission of Tobamoviruses……………………...……………………………..6 Chapter 2: Molecular and Physical Characterization of a Tobamovirus Infecting Sunn Hemp in Hawaii……………………………………………………………………………………………..8 Introduction………………………………………………………………………………..8 Materials and Methods…………………………………………………………………...10 Viral Source and Maintenance…………………………………………………...10 Sequencing and Phylogenetic Analyses………..………………………………...11 Molecular Detection of sunn-hemp mottle virus………………………………...12 Transmission Electron Microscopy……………………………………………...14 Results……………………………………………………………………………………15 Sequencing and Phylogenetic Analyses………..………………………………...15 Molecular Detection of sunn-hemp mottle virus……………………………...…26 Transmission Electron Microscopy……………………………………………...29 iv Discussion………………………………………………………………………………..32 Chapter 3: Biological Characterization of sunn-hemp mottle virus……………………………..34 Introduction………………………………………………………………………………34 Materials and Methods………………………………………………………...…………35 Virus Source and Maintenance……………………………………………..……36 Effects of sunn-hemp mottle virus Infection on Plant Development……….……36 Experimental Host Range……………………………………………………..…37 Seed Contamination and Transmission………………………………………..…37 Plant to Plant Transmission………………………...……………………………38 Results……………………………………………………………………………………41 Effects of sunn-hemp mottle virus Infection on Plant Development………….…41 Experimental Host Range……………………………………………………..…43 Seed Contamination and Transmission………………………………………..…47 Plant to Plant Transmission………………………...……………………………47 Discussion………………………………………………………………………………..52 Chapter 4: Characterization of Other Viruses Infecting Crotalaria………………………...……55 Introduction………………………………………………………………………………55 Materials and Methods…………………………………………………….……………..55 Tobacco streak virus in Crotalaria juncea ‘Tropic Sun’………………………...55 Big Island Crotalaria micans……………………...………….…………………56 Poamoho Crotalaria juncea…………………………………...…………………56 Results……………………………………………………………………………………60 Tobacco streak virus in Crotalaria juncea ‘Tropic Sun’……………….………..60 v Big Island Crotalaria micans………...……………………………….…………60 Poamoho Crotalaria juncea……………………………………..………………60 Discussion………………………………………………………………..………………61 Chapter 5: Conclusion and Future Studies……………………………………….………………63 Literature Cited……………………………………………………………………..……………65 vi List of Tables 1. Percent identity matrix of the tobamovirus genomes and proteomes……………………18 2. Primers used in this study……………………………………………………………..…31 3. Inoculation rates and symptoms observed on experimental host range for SHMoV……42 vii List of Figures 1. Immature sunn hemp (Crotalaria juncea) plants in the field……………………………..2 2. Foliar mottle on sunn hemp plants in Maui County in 2016 and on inoculated sunn hemp plants grown in the greenhouse……………………………………………………………9 3. Genome organization of sunn-hemp mottle virus (SHMoV), sunn-hemp mosaic virus (SHMV), and tobacco mosaic virus (TMV)……………………………………………..17 4. Neighbor joining and maximum likelihood trees generated from the amino acid sequence of the movement and coat proteins………………………………………………………21 5. Amplification plot and standard curve generated from serial 10-fold dilutions of cDNA made from the total nucleic acids extracted from symptomatic sunn hemp plants grown in a greenhouse inoculated with sunn-hemp mottle virus…………………………………..28 6. Transmission electron micrograph of purified sunn-hemp mottle virus particles extracted from Crotalaria juncea leaf and stem tissue……………………………………………..30 7. 98-cone setup used in transmission experiments………………………………………...39 8. Barplots of comparing sunn hemp grown for 90 days in greenhouse conditions measuring height (A), days till first flower (B), above-ground biomass (C), and below- ground biomass (D) …………………………………………………………………..….40 9. Symptoms of mottle and shoestringing observed on the leaves of an inoculated C. pallida plants grown in the greenhouse…………………………………………………………..43 10. Spatial pattern maps of SHMoV transmission in a greenhouse setting with no wind…...44 11. Spatial pattern maps of SHMoV transmission in a greenhouse setting with an average wind speed of 0.16kph and gusts averaging 0.48kph during the day…………………46 viii 12. Spatial pattern maps of SHMoV transmission in a greenhouse setting with an average wind speed of 1.28kph and gusts averaging 2.31kph during the day…………………47 13. Line graph showing the average and standard deviations of plants infected (%) over an 8 week period………………………………………………………………………………48 14. Left: Mosaic symptoms on C. micans collected from the Big Island. Right: Chlorosis seen on infected sunn hemp on Oahu……………………………………………………56 ix PREFACE Many species of Crotalaria are considered weeds; however, there is one species of significant agronomical importance, Crotalaria juncea, more commonly known as sunn hemp. Sunn hemp originates in India where it was commonly cultivated as a fiber crop. In the United States sunn hemp has gained popularity as a cover crop due to its ability to rapidly accumulate biomass and its ability to fix nitrogen as it grows. In 2016 a sunn hemp farmer noticed virus-like symptoms of leaf mottle and stunting on his sunn hemp crop. Though these symptoms are reminiscent of infection by SHMV initial sequencing data showed infection by a tobamovirus of about 80% nucleotide homology to SHMV. Since the cutoff for new tobamovirus species is less than 90% nucleotide homology these results suggest that the virus infecting these sunn hemp plants could be a new species of tobamovirus. Work was done to better characterize this new tobamovirus. Additionally, at Poamoho Research Station symptoms of leaf chlorosis were observed on cultivated sunn hemp. These plants were sampled and processed for high throughput sequencing to identify viruses
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