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Detection and Characterization of Plant Pathogenic Viruses in Hawaii.

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Title:Detection and Characterization of Plant Pathogenic Viruses in Hawaii.
Authors:Green, James C.
Contributors:Tropical Plant Pathology (department)
Date Issued:Aug 2018
Publisher:University of Hawaiʻi at Mānoa
Abstract:Historically and currently virus diseases have caused significant loss to agricultural stakeholders in HI (HI) and worldwide. Pineapple was one of HI’s most important agricultural crops for many years though recently production has declined. Mealybug wilt of pineapple (MWP) is one of the most destructive diseases of pineapple worldwide and the causal agents are single-stranded RNA (ssRNA) viruses know as Pineapple mealybug wilt-associated viruses (PMWaVs) of the Ampelovirus genus of the Closteroviridae family. Though the disease etiology of MWP is not completely understood much of the genomes of the various PMWaVs have been characterized previously; this study furthers the characterization of several PMWaVs. To clarify the mysterious etiology of MWP it is important that the function of the genes of the various PMWaVs are well understood, underlying the importance of genome characterization.
The genomes of PMWaV-1, PMWaV-2 and PMWaV-3 were first characterized in HI prior to this study, but only PMWaV-1 has been completely characterized. Two putative PMWaV member species: PMWaV-4 and PMWaV-5 had been reported from HI and Australia respectively. The putative PMWaV-4 was first reported in 2005. The PMWaV-4 heat shock protein 70 (HSP70) was the only gene reported. Prior to 2011, the criteria for species demarcation in the Ampelovirus genus was based off a 10% divergence of the amino acid (aa) sequence of the HSP70, RNA dependent RNA polymerase (RdRp) and coat protein (CP) genes; in 2011, the criteria was changed to a 25% divergence of the same three genes. In this study we characterize the remainder of the PMWaV-4 genome. Total RNA was used as the template for cDNA library construction and subjected to high-throughput sequencing (HTS), on an Illumina TruSeq 500 platform, multiple assembly pipelines were utilized for contig assembly and a 13,150 nucleotide (nt) scaffold was assembled that shares a high level of sequence homology with the 13,070 nt PMWaV-1 reference genome (AF414119). The aa similarity of the three species demarcation genes of PMWaV-4 compared to PMWaV-1 is 89, 87 and 85% for the RdRp, HSP70 and CP respectively. Based off the sequence similarity to PMWaV-1 we have determined PMWaV-4 to be a strain of PMWaV-1 rather than a distinct species. Additionally, HTS assemblies of PMWaV-1, PMWaV-2 and PMWaV -3 genomes shared a 99, 99 and 97%, respectively, nt similarity to their originally published genomes and supplemented the preexisting PMWaV-2 and PMWaV-3 sequences with 166 and 641 nt, respectively, in the 5’ termini of the viruses.
The potyvirus Banana bract mosaic virus (BBrMV) and badnavirus Canna yellow mottle virus (CaYMV) were previously identified infecting Alpinia purpurata (flowering ginger) in HI. Recently, farmers growing flowering ginger have reported severe virus-like disease symptoms from multiple farms on Oahu, HI. Surveys were conducted in September 2016 and April 2018 molecular and serological techniques were used to assay symptomatic flowering ginger for the presence of the previously identified viruses. CaYMV was found to be widespread at all but one location however no BBrMV was detected in any samples from either survey. Characteristic symptoms of CaYMV and BBrMV were not observed, however streaking symptoms in CaYMV and BBrMV negative leaves, stems and bracts were observed. The lack of conclusive evidence linking CaYMV or BBrMV infection with the symptomatic flowering ginger assayed during this study may indicate the presence of another pathogen’s involvement. Further study is necessary to conclude the casual organism of the disease found on flowering ginger.
Honolulu, HI is home to many community gardens which function to provide many urban dwellers the privilege of access to land for growing of edible and horticultural plants. Unfortunately, these areas are often ‘hotbeds’ of disease and pathogen dispersal. Recently, we identified several different plants infected with viruses from diverse crop and horticultural species. Potyvirus-specific ELISA detected potyviruses in symptomatic Passiflora spp. (passionfruit) and Phaseolus lunatus (lima bean). RT-PCR assay using degenerate potyvirus-specific primers then confirmed the ELISA results. Amplicons from the RT-PCR assay were cloned and sequenced using Sanger sequencing. Sequencing results revealed the potyviruses to be Watermelon mosaic virus (WMV) and Bean common mosaic virus (BCMV) infecting and passionfruit and lima bean respectively. WMV had previously been reported infecting passionfruit in HI. This was the first report of BCMV in HI and the sequence homology showed a high degree, 92% nt and 100% aa similarity to a BCMV isolate from China. A BCMV-specific ELISA was used to reconfirm the sequencing results. The unusually diverse amount of plants grown in a community garden as well as the high volume of individuals with access to the area provide the ideal circumstances for dissemination of plant pathogens.
Description:M.S. Thesis. University of Hawaiʻi at Mānoa 2018.
URI:http://hdl.handle.net/10125/62818
Rights:All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
Appears in Collections: M.S. - Tropical Plant Pathology


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