Browsing by Author "James W. Moyer, Committee Member"

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Factors affecting susceptibility to - and management of - postharvest soft rot of sweetpotatoes caused by Rhizopus stolonifer.
(2008-11-25) Edmunds, Brooke Aurora; James W. Moyer, Committee Member; D. Michael Benson, Committee Member; Gerald J. Holmes, Committee Chair; Lee-Ann Jaykus, Committee Member
Studies were undertaken to explore the relationship of R. stolonifer susceptibility with preharvest growing conditions and postharvest handling of sweetpotatoes. Additional studies were also completed to identify effective decay control products. A three-year study investigated the effect of preharvest conditions on R. stolonifer and Erwinia chrysanthemi susceptibility. Roots were harvested from 75 sweetpotato fields and information collected including soil samples, weather during the growing season, weed density, and insect injury (153 predictors). Roots were inoculated after 100 days in storage. Mean R. stolonifer incidence was 34.9% (standard deviation=31.7%) and mean E. chrysanthemi incidence was 51.0% (standard deviation=30.5%). Predictive models were developed using forward stepwise regression to identify predictors of interest, followed by mixed model analysis (p-value<0.05) to produce a final model. R. stolonifer susceptibility is best predicted by soil calcium (% CEC), plant-available soil phosphorus, soil humic matter (%), mean air temperature, mean volumetric soil moisture at 40 cm, and mean soil temperature at 2 cm (all over the growing season). E. chrysanthemi susceptibility is best predicted by soil pH and days that soil temperature exceeds 32 ÂºC (14 days pre- harvest). Studies were also conducted to define the relationship between postharvest handling and susceptibility to R. stolonifer. Experiments designed to simulate packingline handling found root ends are more susceptible that mid-sections and that increasing the number of time a root is dropped as well as increasing the impact force resulted in increased decay susceptibility. â€˜Hernandezâ€™ roots were significantly more susceptible than â€˜Beauregardâ€™ in all experiments. To confirm the relationship of impacts and disease development, Beauregard roots were sampled from locations along commercial packinglines. High decay in inoculated as compared to non-inoculated roots indicates that wounding is occurring that could result in disease if the pathogen was present at higher levels. Evaluations of reduced-risk fungicides, bio-fungicides and generally recognized as safe products for efficacy against R. stolonifer found that reduced-risk chemistries boscalid+pyraclostrobin and fludioxonil significantly reduced R. stolonifer development and performed similarly to dicloran. Pseudomonas syringae based products were moderately effective although results were extremely variable among tests. Generally recognized as safe treatments were ineffective by testing methods used.
Identification and Characterization of the Red Clover Necrotic Mosaic Virus Origin of Assembly
(2005-06-15) Basnayake, Veronica Roshani; James W. Moyer, Committee Member; Eric L. Davis, Committee Member; Steven A. Lommel, Committee Chair; Cynthia L. Hemenway, Committee Member
Red clover necrotic mosaic virus (RCNMV) is a single-stranded positive-sense RNA plant virus of the Dianthovirus genus, family Tombusviridae. Each RCNMV virion contains 180 subunits of the 37 kDa capsid protein (CP) forming a non-enveloped, isometric particle of T=3 quasi symmetry, 30-35 nm in diameter. The RCNMV genome consists of two RNAs, RNA-1 and RNA-2. RNA-1 codes for three proteins: i) p88, the RNA-dependent RNA polymerase, ii) p27, the replicase related protein and iii) the CP. RNA-2 is monocistronic and codes for the movement protein (MP). Currently, the RNA complement within RCNMV virions has not been determined. Density gradient centrifugation data has suggested the probability of a single type of particle, whereas the viral RNA profile from virions suggests otherwise. My thesis research has determined the RNA content within RCNMV virions after exposure to either heat or UV-irradiation. Both treatments result in the formation of a stable RNA-1: RNA-2 heterodimer. This leads to the conclusion that RCNMV virions co-package RNA-1 and RNA-2. Upon either treatment, RNA-2 multimers are also observed, suggesting the presence of particles packaging RNA-2 exclusively. These observations suggest that the RCNMV virion population consists of two distinct types of particles having similar densities. Based on the above RNA complement findings, I proceeded to delineate the specific viral sequences that determine the RNA content of an RCNMV virion. During virion assembly, the RCNMV CP must be able to distinguish and package both RNA-1 and RNA-2 while excluding heterologous host RNAs present in the cell. Viral CP subunits recognize specific cognate genomic sequences and/or structures that are unique to each viral genome. These are designated as origin of assembly sequences or packaging signals. To elucidate the assembly mechanism of RCNMV, I searched for the presence of distinct packaging signals on each of the genomic RNAs. Various constructs of RNA-1 and RNA-2 were tested for their assembly efficiencies in vivo using a plant assay system. While it has been previously demonstrated that RNA-1: RNA-2 base pairing directs the synthesis of the CP subgenomic RNA (sgRNA) from RNA-1 via the trans-activating (TA) element on RNA-2, it was not determined whether this interaction had a role in assembly. I have found that RNA-1 does not have the ability to package by itself given sufficient amounts of CP. Also, the CP sgRNA is not encapsidated into RCNMV virions. RNA-2 appears to play the major role in directing RCNMV assembly. My results indicate that a 209 nt sequence within the MP open reading frame on RNA-2 (containing the TA element) directs RCNMV assembly. Deletion mutagenesis of RNA-2 to delimit the packaging signal proved that the 34-nucleotide TA element was the origin of assembly. As further proof, expression of the TA element from a Tomato bushy stunt virus vector directed the co-packaging of RCNMV RNA-1 into virions proving it to be essential for RCNMV assembly. Deletion mutagenesis also revealed that RCNMV has an RNA packaging size requirement for production of stable virions. Based on all of the above observations, I propose the following model for the RCNMV packaging mechanism: the base pairing interaction of the TA element with RNA-1 initiates CP production and enables the formation of an RNA-1: RNA-2 heterodimer . This dimer formation allows the co-packaging of both RNAs into a single virion via the packaging signal on RNA-2. The discreet packaging signal on RNA-2 also allows the formation of some virions containing solely RNA-2.
Population genetic analysis of Tomato spotted wilt virus (TSWV) on peanut in North Carolina and Virginia.
(2009-07-07) Kaye, Amanda Claire; George G. Kennedy, Committee Co-Chair; James W. Moyer, Committee Member; Marc A. Cubeta, Committee Co-Chair; Barbara B. Shew, Committee Member
Exploring the genetic diversity and evolutionary history of plant viruses is critical to understanding their ecology and epidemiology. Tomato spotted wilt virus (TSWV) is an ambisense RNA virus that infects over 1000 species of plants and has been shown to reassort its genome. To further investigate this virus, maximum-likelihood and population genetics-based methods were used to investigate the population structure, genetic diversity, and sources of genetic variation in field isolates of TSWV from peanut in North Carolina and Virginia. Selected regions of the nucleocapsid, movement, and RNA-dependent RNA polymerase genes were amplified and sequenced to identify haplotypes and infer genetic relationships between isolates of TSWV with heuristic methods. The haplotype structure of each locus consisted of one or two predominant haplotypes and more than 100 haplotypes represented by a single isolate. No specific haplotypes or clades were associated with geographic area, peanut cultivar or year. The population was panmictic at the regional level and high levels of genetic diversity were observed among isolates. There was evidence for negative selection acting upon each locus and maximum-likelihood analyses indicated that exponential growth was occurring in the population. The results of compatibility analyses and the persistence of specific gene sequences in isolates collected over three field seasons suggest that recombination was occurring in the population. Phylogenetic analysis supports that each locus has an independent evolutionary history. Also, high diversity in viruses has been attributed to the occurrence of recombination and mutation To investigate recombination in TSWV, matrix compatibility and ancestral recombination methods were used to detect, quantify, and reconstruct the history of recombination in three genes of TSWV in isolates collected from three cultivars of peanut (Gregory, NC12C, and Perry) sampled in multiple years. Site commonalities among the isolates collected from different cultivars were found in each gene where the majority of the recombination appeared to be occurring. These corresponded to sequence near the 3Ã¢â‚¬Â² terminus in the N and RdRP genes and to sequence near the 5Ã¢â‚¬Â² terminus in the NSm gene. Between isolates collected from different peanut cultivars, the Gregory isolates proportionally had the most recombination occurring in the N gene and the RdRP genes, while the Perry isolates had the most recombination occurring in the NSm. Recombination rates per site estimated for the N, NSm, and RdRP genes in the NC12C isolates were 0.00934, 0.01559, and 0.00495, respectively.
The Role of Temperature and Precipitation on Thrips Populations in Relation to the Epidemiology of Tomato Spotted Wilt Virus.
(2008-11-09) Morsello, Shannon Cathleen; Fred L. Gould, Committee Member; James F. Walgenbach, Committee Member; James W. Moyer, Committee Member; George G. Kennedy, Committee Chair
ABSTRACT MORSELLO, SHANNON CATHLEEN. The Role of Temperature and Precipitation on Thrips Populations in Relation to the Epidemiology of Tomato Spotted Wilt Virus. (Under the direction of George G. Kennedy.) The influence of temperature and precipitation on spread of Tomato spotted wilt virus (family Bunyaviridae, genus Tospovirus, TSWV) in chickweed (Stellaria media), population growth of its? primary vector, Frankliniella fusca (Hinds), on chickweed, and the timing and magnitude of F. fusca and Thrips tabaci Lindeman dispersal from winter hosts was characterized using population data collected during spring 2004, 2005 and 2006, and trapping data from North Carolina and Virginia from 1997 ? 2001 and 2004 ? 2007. Frankliniella fusca populations on chickweed were suppressed immediately following rain events, but precipitation occurring in early May delayed senescence of chickweed and ultimately resulted in higher populations by the end of May. Spread of TSWV within chickweed plots was directly related to the size of the immature F. fusca population in the plot. Regression analysis determined that temperature, total precipitation and number of days with precipitation during January through May explained 70% and 55% of the total variation in the numbers of F. fusca and 57% and 63% of T. tabaci, respectively captured from 1 April ? 10 May and 1 April ? 31 May. Further analysis determined that temperature measured throughout the overwintering period positively influenced the number of dispersing F. fusca throughout Spring, and precipitation that occurred during late March, late April, early May and late May influenced the dispersal of F. fusca by killing immature larvae, suppressing adult flight or delaying senescence of host plants. Additional models explained 60%, 74%, 68% and 69% of the variation in the number of dispersing F. fusca captured during 1 ? 15 April, 16 ? 30 April, 1 ? 15 May and 16 ? 31 May, respectively. Validation resulted in weak correlations of observed and expected values for each trapping interval, but similar trends over time. Future work developing risk models for thrips flights and TSWV on an area-wide basis must be species specific and include additional parameters to better capture late-season host plant dynamics.