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Master of Science / Department of Entomology / David C. Margolies / James R. Nechols / A two-year greenhouse experiment was conducted to compare the relative effectiveness of biological control versus chemical control for western flower thrips, Frankliniella occidentalis, as a means of reducing the spread of Tomato spotted wilt virus (TSWV) on tomatoes. To compare efficacy of different thrips management tactics for reducing TSWV incidence, tomatoes were subjected to one of three treatments: 1) biological control based on weekly releases of the predatory mite, Amblyseius cucumeris, at a commercially-recommended rate, 2) a single chemical treatment with Conserve®, a spinosad formulation, or 3) no treatment. TSWV was introduced into the greenhouse either by starting with 20% of the crop already infected and releasing non-viruliferous thrips, or by making a single release of viruliferous thrips. Analyses were done among thrips management tactics for each virus introduction method to examine the cumulative number of weeks plants were infected, the weekly proportion of infected plants, and total marketable yield. The effects of different virus introduction methods were also compared. A comparison of virus introduction methods showed that, among all plants, the average number of weeks they were infected by TSWV was significantly lower when virus was introduced through infected plants than by infected thrips. In addition, when virus was introduced by infected thrips, a significantly greater proportion of plants were infected in any given week than when virus was introduced on infected plants. Finally, crop yields were significantly lower when virus was introduced via infected thrips than on infected plants. Among thrips management methods, plants were infected for significantly less time, and the proportion infected was lower in any given week, when biological or chemical control was applied compared to no thrips management. Tomato yields were not affected by thrips management tactic. There was no significant difference between biological and chemical control in the length of time that plants showed symptoms. However, the proportion of infected plants was marginally greater with biological control in weeks 4 and 5 than with chemical control; differences were not significant thereafter. My findings suggest that inundative releases of biological control may provide as adequate a level of protection from TSWV as chemical control in commercial greenhouse tomato crops.

Doctor of Philosophy / Department of Plant Pathology / Bikram S. Gill / The wild relatives of crop plants are sources of useful genes, but such genes when transferred to agricultural crops are often associated with deleterious traits. Because most of the recombination and the disease resistance genes are localized towards the ends of wheat chromosomes, cryptic terminal alien segments, carrying rust resistance genes, were transferred from Aegilops geniculata (UgMg) and Ae. triuncialis (UtCt) into common wheat without the usual linkage drag. The alien segment with the leaf rust and stripe rust resistance genes Lr57 and Yr40 in translocation T5DL•5DS-5MgS(0.95) was found to be less than 3.3 cM in genetic length and spans less than four overlapping BAC/PAC clones of the syntenic rice chromosome arm 12L. The alien segment with leaf rust resistance gene Lr58, transferred from Ae. triuncialis, was found to be less than 5% of the chromosome arm 2BL of wheat in T2BS•2BL-2tL(0.95), further suggesting that it is feasible to transfer small alien segments with disease resistance genes. Resistance genes Lr57, Yr40 and Lr58 were transferred to Kansas hard red winter wheat cultivars by backcrossing and marker assisted selection. Tillering, a key component of grain yield, and seed color which influences seed dormancy and pre-harvest sprouting in wheat, are agronomically important domestication traits in wheat. A tiller inhibition mutant with monoculm phenotype was isolated and the mutated gene (tin3) was mapped on the distal region of chromosome arm 3AmL of T. monococcum. As a first step towards isolating candidate gene(s), the tin3 and the seed color gene (R-A1) of chromosome 3A were mapped in relation to physically mapped ESTs and STS markers developed based on synteny with rice. Physically mapped wheat ESTs provided a useful framework to identify closely related rice sequences and to establish the most likely syntenous region in rice for the wheat tin3 and R-A1 region. Comparative genomic analysis of the tin3 and R-A1 genomic regions with the corresponding region in rice localized the tin3 gene to a 324 kb region spanned by two overlapping BACs and the R-A1 gene was mapped to a single BAC of the colinear rice chromosome arm 1L.

Doctor of Philosophy / Department of Plant Pathology / Barbara S. Valent / Rice blast is a major fungal disease on rice, caused by the hemibiotrophic filamentous ascomycete fungus, Magnaporthe oryzae. This disease accounts for 157 million tons of grain loss annually. The fungus produces a specialized cell called appressorium to penetrate the host surface barrier and enter inside. It produces intracellular Invasive Hyphae (IH) that grow form cell to cell to colonize the host. The mechanisms of appressorium formation and host penetration have been studied in detail but the host colonization strategies remain largely unknown. We applied live-cell imaging to characterize spatial and temporal development of IH and plant responses inside successively-invaded rice cells. Early loading experiments with the endocytotic tracker, FM4-64, showed dynamic plant membranes around IH. These hyphae showed remarkable plasticity and recruited plant cell components. IH exhibited pseudohyphal growth and were sealed in plant membrane, termed the Extra-Invasive Hyphal Membrane (EIHM). The fungus spent up to 12 hours in the first cell, often tightly packing it with IH. IH that moved into neighboring cells were biotrophic, although they were initially thinner and grew more rapidly. IH in neighboring cells were wrapped in EIHM with distinct membrane caps at the hyphal tips. Time-lapse imaging showed IH scanning plant cell walls before crossing them, and transmission electron microscopy showed crossing occurring at pit fields. This and additional evidence strongly suggest that IH co-opt plasmodesmata for cell-to-cell movement. Our studies have revealed insights into a novel hemibiotrophic strategy employed by the blast fungus. Few genes have been previously characterized that impact the biotrophic IH. To understand the molecular basis of the biotrophic infection strategy we employed Laser Microdissection (LM) technology to isolate and purify the IH at this early growth stage. We compared the gene expression of these samples with axenically-grown mycelium using M. oryzae whole genome microarrays. We identified several hundreds of infection specific genes. We have shown that LM technology can be used to isolate homogenous cells from the infected rice tissues to study the underlying molecular mechanisms of signaling during disease formation. These studies will be very critical to understand the host-pathogen interactions to eventually develop durable management strategies.

Doctor of Philosophy / Department of Human Nutrition / Weiqun Wang / Wheat lignans are phenylpropane dimers linked by β-β bonds with a 1, 4-diarylbutane structure. They are biosynthesized in the cell cytoplasm through action of enzymes of the phenylpropanoid pathway. Pinoresinol lariciresinol reductase (PLR) catalyzes the final steps of biosynthesis of wheat lignans. In epidemiological and clinical investigations, studies show that high plasma lignan amounts correlate with reduced risks of breast, colon, and prostate cancers. However, in some of the studies, the results are not consistent. More consistent results are observed when animal and cell culture models are used. Our previous studies in the Wang lab demonstrated that treatment of human colon cancer cells, SW480 with lignans results in a dose and time dependent inhibition of cancer cell growth. In the first paper, we investigated direct experimental cancer preventative characteristics of a wheat lignan, secoisolariciresinol diglucoside (SDG) vs. its metabolite enterolactone in human colon cancer SW480 cells. Treatment of cancer cells with 0-40 µM SDG or enterolactone resulted into inhibition of cancer cell growth as observed by reduction of cell numbers. The reduction appeared related to induction of S-phase cell cycle arrest rather than cytotoxic effect. Further analysis revealed that SDG was more stable in cell culture medium than enterolactone. HPLC-MS/ESI showed that enterolactone is the principle metabolite in cancer cells but undetectable SDG or its metabolites were in the cells treated with SDG. In the second paper, we investigated over expression of the PLR gene and enhancement of lignan levels in transgenic wheat. We transformed wheat cultivars (‘Bobwhite’, ‘Madison’, and ‘Fielder’ respectively) with the Forsythia intermedia PLR gene under the regulatory control of the maize ubiquitin promoter. Of the total 217 transgenic wheat lines, we successfully obtained 7 transformants with the inserted ubiquitin PLR gene as screened by PCR. Real-time PCR further indicated 109-117% PLR over expression over the transgenic control in 3 transformants of the 7 at T0 generation. In addition, the levels of SDG, as determined by HPLC was found to be significantly elevated in one of the 3 positive transgenic plants. To the best of our knowledge, this is the first study reported that genetically engineered wheat with over expressed PLR enzyme enhancing phytochemical lignan has been successfully achieved.

Master of Science / Department of Plant Pathology / Karen A. Garrett / Ecological clines often result in gradients of disease pressure in natural plant communities, imposing a gradient of selection on disease resistance genes. We describe the diversity of a resistance gene homolog in natural populations of the dominant tallgrass prairie grass, Andropogon gerardii, across a precipitation gradient ranging from 47.63 cm/year in western Kansas to 104.7 cm/year in central Missouri. Since moisture facilitates infection by foliar bacterial pathogens, plants along this precipitation gradient will tend to experience heavier bacterial disease pressure to the east. In maize, the gene Rxo1 confers resistance to the pathogenic bacterium Burkholderia andropogonis. Rxo1 homologs have been identified in A. gerardii and B. andropogonis is known to infect natural populations of A. gerardii. The spatial genetic structure of A. gerardii was assessed from central Missouri to western Kansas by genotyping with AFLP markers. Samples were also genotyped for Rxo1 homologs by amplifying an 810 base pair region of the leucine-rich repeat and digesting with restriction enzymes. We compared Rxo1 homolog diversity to AFLP diversity across different spatial scales. Genetic dissimilarity based on AFLP markers was lower than would have occurred by chance at distances up to 30 m, and different prairies were more dissimilar than would have occurred by chance, but there was not a longitudinal trend in within-prairie dissimilarity as measured by AFLP markers. Dissimilarity of the Rxo1 homologs was higher in the east suggesting the presence of diversifying selection in the more disease-conducive eastern environments.