Tobacco cyst nematodes (TCN), Globodera tabacum solanacearum, are one of the most serious pests for Virginia flue-cured tobacco (Nicotiana tabacum L.) farmers. These nematodes continue to spread to new farms every year and are now reported in 11 Virginia counties, seven North Carolina counties, and one farm in Maryland. The objectives of this study were to determine the ability of different populations of TCN to reproduce upon both a susceptible (K326) and resistant (NC567) cultivar, to compare TCN reproduction in non-infested soils with currently TCN-infested soils, and to examine reproduction and pathogenicity of TCN on other solanaceous crops. Tobacco cyst nematode reproduction on the TCN-resistant cultivar NC567 was significantly reduced when compared to the TCN-susceptible cultivar K326. Although significant differences among the populations were detected on both cultivars, the differences were not consistent across experiments. Results indicate similar pathogenicity among TCN populations on resistant and susceptible flue-cured tobacco cultivars. Plant breeders may use one population of the tobacco cyst nematode to screen tobacco germplasms for TCN resistance. Significant differences were noted in TCN reproduction among the various soils used in this study. However, the differences were inconsistent, making correlations between TCN reproduction and soil edaphic characteristics unreliable. Sterilizing the soils did not increase TCN reproduction. Reproduction in non-infested soils indicates that TCN will continue to spread throughout tobacco producing regions. Although traditionally an exclusive pest of tobacco, TCN's spread may threaten production of other solanaceous crops. Eggplant (Solanum melongena L.), pepper (Capsicum spp. L.), and tomato (Lycopersicon spp. L.) were confirmed to be hosts of TCN under field conditions. Eggplant was highly susceptible to TCN reproduction and yield suppression, exhibiting a 64% yield reduction in plots not treated with a nematicide compared to fumigated plots. Tomato allowed high rates of TCN reproduction but did not suffer yield loss. Tobacco cyst nematode reproduction was suppressed on pepper compared to the other two crops. No crop loss was noted in nontreated pepper plots compared to plots treated with a nematicide. Results would indicate that tobacco rotations including tomato or eggplant would be unacceptable due to high TCN reproduction rates. The tobacco cyst nematode does not seem to jeopardize the production of tomato and pepper in the southeastern United States, but does threaten profitability of eggplant production in this area. / Master of Science

The tobacco cyst nematode (TCN), Globodera tabacum solanacearum [(Miller and Gray, 1972) Behrens 1975] Stone 1983, is one of the most economically important pests of flue-cured tobacco (Nicotiana tabacum L.) in Virginia. Although TCN has been reported from other countries, the geographical distribution of G. t. solanacearum within the United States is limited to Virginia, North Carolina, and one county in Maryland. Approximately 30% of the tobacco acreage in Virginia is infested; average yield reduction is 15%, but complete crop failure can occur. The objectives of this research were to examine intraspecific variability within G. t. solanacearum and to evaluate the relative adaptability of G. t. solanacearum on a resistant (NC567) and a susceptible (K326) flue-cured tobacco cultivar. Nineteen geographic isolates of G. t. solanacearum, one isolate each of G. t. virginiae and the Mexican cyst nematode (G. "mexicana"), two isolates of G. t. tabacum, and five Heterodera species were characterized by DNA fingerprinting using the RAPD-PCR technique. Reproducible differences in fragment patterns allowed similar differentiation of the isolates and species with each primer. Hierarchical cluster analysis was performed to illustrate the relatedness between nematode isolates and species. In contrast to reports in the literature, we found a Miller isolate of G. "mexicana" to cluster more closely with G. t. solanacearum than with G. t. tabacum or G. t. virginiae. Although no pathotype differences have been found within G. t. solanacearum, the average Jaccard's similarity index among isolates of G. t. solanacearum was 74%, representing greater variation than that observed across different pathotypes of the closely related potato cyst nematode, Globodera pallida. This result suggests that the emergence of resistance-breaking biotypes is more likely than previous research suggests. If a new pathotype is reported, a RAPD marker associated with virulence against a specific host resistance gene could prove to be a valuable tool in population diagnosis, resistance screening, and overall TCN management. One isolate of G. t. solanacearum was cultured on a resistant (NC567) and a susceptible (K326) flue-cured tobacco cultivar over five generations. Variable TCN reproduction was observed on both cultivars over each generation. This variability in reproduction could be attributed to differences among generations in the time interval between inoculation and cyst extraction, temperature, possible diapause effects, and/or daylength. Ninety-eight cysts were produced in the fifth and final generation compared to the 14 to 50 cysts produced during each of the previous four generations. Increased reproduction on the resistant variety suggests that increased virulence might be selected, but research involving additional generations would need to be carried out in order to conclude whether or not TCN virulence is being selected. / Master of Science

Resistance to Globodera tabacum solanacearum in flue-cured tobacco was characterized in vitro, in the greenhouse, and field. The objectives of this study included evaluation of hatching of G. t. solanacearum as stimulated by root exudate, penetration and development of G. t. solanacearum on roots, and the effects of G. t. solanacearum on growth, yield, and quality of flue-cured tobacco. Root exudate from resistant (NC 567) and susceptible (K 326) flue cured tobacco cultivars had similar effects on hatching of G. t. solanacearum. Dilution of root exudate reduced hatching and hatching appeared to increase with increased temperature. More swollen and flask shaped nematodes developed in roots of K 326 than those of NC 567. Resistance reduced development of vermiform juveniles to swollen nematodes in NC 567. However, development of swollen nematodes to flask shaped nematodes was similar for both cultivars. Resistance to G. t. solanacearum was effective at 17, 22, 27, and 31°C. Infection by G. t. solanacearum suppressed number of leaves, plant height, fresh weight of leaves, and feeder roots. Reduction in fresh leaf weight at 11 weeks after transplanting was greater for the susceptible cultivar, K 326, than for the resistant cultivar, NC 567. However reduction in fresh feeder root weight and increases in the ratio of leaf weight to feeder root weight at 11 weeks after transplanting between both cultivars. Yield and grade index of cured leaves were negatively correlated with area under curves of total nematode population densities per gram of feeder root. The rate parameters of regression models suggested that the two cultivars responded similarly to infection by G. t. solanacearum in yield and quality reduction. However, K 326 had a higher yield potential in the absence of nematodes. / Ph. D.

Active disease resistance in plants is induced during the pathogen infection process that triggers multiple defense-related genes to establish broad-spectrum resistance. Several biotic and abiotic agents can mimic natural induced resistance (IR), categorized as systemic acquired (SAR) or induced systemic resistance (ISR). IR, triggered by acibenzolar-S-methyl (ASM) or plant growth-promoting rhizobacteria (PGPR), was evaluated on two-to-three types of tobacco in greenhouse and field studies. Tobacco mosaic virus (TMV) local lesion assays monitored induction and maintenance of ASM-induced SAR over a 21 day period via proportional reduction in the number of TMV local lesions between an untreated control and ASM-treated plants. Intraspecific variation in SAR was found among tobacco types; burley and flue-cured tobaccos responded by day 3, while oriental tobacco responded between day 3 and 6. The SAR signal was greatest between 6 and 15 days following ASM application, but IR was slightly evident even at 21 days after ASM application in all three tobacco types. Bottom and middle leaves responded similarly on all sample dates, but top leaves showed the weakest SAR response. Tobacco cyst nematode (TCN; Globodera tabacum solanacearum) is one of the most destructive pathogens of tobacco in Virginia. Among four PGPR combinations tested, a mixture of Bacillus amyloliquefaciens IN937a (GB99) and B. subtilis A13 (GB122) most consistently suppressed TCN reproduction in flue-cured and oriental tobacco. Application of ASM similarly reduced final numbers of TCN cysts, but also resulted in chlorosis, stunting, and lower plant fresh weight. GB99+GB122 also suppressed TCN development and reproduction in susceptible and resistant flue-cured cultivars, but reductions by ASM were less consistent. In a split-root trial, soil amendment with GB99+GB122 in one half of an oriental tobacco root system lowered final numbers of TCN more than did ASM. ASM exhibited undesirable effects in phytotoxicity trials in flue-cured and oriental tobacco, but GB99+GB122 was not phytotoxic. When oriental tobacco seedlings were grown in a GB99+GB122-treated soil-less media, a single application of 200 mg ASM/L one week after transplanting significantly suppressed TCN reproduction in the field without phytotoxicity. Further field research is needed to confirm this effect in flue-cured tobacco. / Ph. D.

Developing a flue-cured tobacco cultivar with high resistance to tobacco cyst nematode (TCN) is an important initiative in the Southern Piedmont of Virginia, where this pathogen causes severe yield losses. One hundred twenty eight lines representing a diverse geographic array of tobacco accessions, including cultivars from several types of tobacco, flue-cured-type tobacco introductions, and wild Nicotiana species were evaluated for TCN resistance under greenhouse conditions. Inheritance of TCN resistance has been reported to be closely linked or pleiotropic to inheritance of wildfire resistance. Consequently, accessions were also screened for wildfire resistance under greenhouse conditions to evaluate this relationship among a diverse group of tobacco accessions. Twenty-one accessions were identified with resistance to TCN. Response to the two pathogens was highly correlated. However, there was no relationship between resistance to the two pathogens for several accessions. 'KY 190', a fire-cured cultivar, possessed the N. longiflora source of wildfire resistance, but was found susceptible to TCN. This result seems to rule out pleiotropy and is consistent with the assertion that the two resistance genes are closely linked. six TCN resistant lines and two susceptible lines were selected and a diallel study was conducted to determine the inheritance of resistance to TCN. Additive gene action contributed significantly to inheritance of TCN resistance. Three accessions, 'Burley 64', 'Kutsaga 110', and 'Tl 1597', were determined to be the most promising parents for use in a breeding program designed to develop a flue-cured cultivar with a high level of TCN resistance. / Master of Science