Doctor of Philosophy / Genetics Interdepartmental Program / Bikram S. Gill / Leaf rust, caused by fungus Puccinia triticina, is an important foliar disease of wheat worldwide. Breeding for race-nonspecific resistant cultivars is the best strategy to combat this disease. Aegilops tauschii, D genome donor of hexaploid wheat, has provided resistance to several pests and pathogens of wheat. To identify potentially new adult plant resistance (APR) genes, 371 geographically diverse Ae. tauschii accessions were evaluated in field with leaf rust (LR) composite culture of predominant races. Accessions from Afghanistan only displayed APR whereas both seedling resistance and APR were common in the Caspian Sea region. Seventeen accessions with high APR were selected for production of synthetic hexaploid wheat (SHW), using ‘TetraPrelude’ and/or ‘TetraThatcher’ as tetraploid parents. Six SHWs were produced and evaluated for APR to LR and resistance to tan spot at seedling stage. Genetic analysis and mapping of APR introgressed from accession TA2474 was investigated in recombinant inbred lines (RIL) population derived from cross between SHW, TA4161-L3 and spring wheat cultivar, ‘WL711’. Genotyping-by-sequencing approach was used to genotype the RILs. Maximum disease severity (MDS) for LR was significantly correlated among all experiments and APR to LR was highly heritable trait in this population. Nine genomic regions significantly associated with APR to LR were QLr.ksu-1AL, QLr.ksu-1BS, QLr.ksu-1BL.1, QLr.ksu-1BL.2, QLr.ksu-2DS, QLr.ksu-2DL, QLr.ksu-5AL, QLr.ksu-5DL and QLr.ksu-6BL. Association of QLr.ksu-1BL.1 with marker Xwmc44 indicated this locus could be slow-rusting APR gene, Lr46/Yr29. QTLs detected on 2DS, 2DL and 5DL were contributed by TA4161-L3 and are novel, along with QLr.ksu-5AL. Tan spot, caused by necrotrophic fungus, Pyrenophora tritici-repentis, has recently emerged as a damaging disease of wheat worldwide. To identify QTLs associated with resistance to Race 1 of P. tritici-repentis, F[subscript]2:3 population derived from cross between SHW, TA4161-L1 and winter wheat cultivar, ‘TAM105’ was used. Two major effect QTLs, QTs.ksu-1AS.1 and QTs.ksu-7AS were significantly associated with tan spot resistance and contributed by TA4161-L1. QTs.ksu-7AS is a novel QTL and explained 17% of the phenotypic variation. Novel QTLs for APR to LR and tan spot identified in SHWs add new variation for broadening the gene pool of wheat and providing resources for breeding of durable resistant cultivars.

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.

Master of Science / Department of Plant Pathology / Karen A. Garrett / Ari Jumpponen / Traditional fallow periods in the Bolivian highlands are being shortened in an effort to increase short-term crop yields, with potential long-term impacts on soil communities. Using 454-pyrosequencing, we characterized fungal and bacterial community responses to (1) the length of fallow period and (2) the presence of the plants Parasthrephia sp. or Baccharis sp. (both locally known as ‘thola’). Thola is widely considered by farmers as beneficial to soil health, although it is also frequently harvested as a source of fuel by farmers. Soils in one study area, Ancoraimes, had higher levels of organic matter, nitrogen and other macronutrients compared to the other study area, Umala. In our analyses, Ancoraimes soils supported more diverse fungal communities, whereas Umala had more diverse bacterial communities. Unexpectedly, the longer fallow periods were associated with lower fungal diversity in Umala and lower bacterial diversity in Ancoraimes. Fungi assigned to genera Verticillium, Didymella, and Alternaria, and bacteria assigned to genera Paenibacillus, Segetibacter, and Bacillariophyta decreased in abundance with longer fallow period. The presence of thola did not significantly affect overall soil fungal or bacterial diversity, but did increase the frequency of some genera such as Fusarium and Bradyrhizobium. Our results suggest that fallow period has a range of effects on microbial communities, and that the removal of thola from the fields impacts the dynamics of the soil microbial communities.

Doctor of Philosophy / Plant Pathology / John P. Fellers / Harold N. Trick / Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) are two viruses affecting wheat in the Great Plains region of the United States. Genetic resistance is severely limited, requiring management methods focusing on the deployment of resistant varieties and various cultural practices. Evaluation of resistance is complicated by the lack of a standard rating scale. The objective of this work was to develop new avenues to mitigate these challenges. A standardized virus symptom rating scale was developed using historical Kansas rating scales, and validated using multiple wheat populations. Two independent RNA interference (RNAi) expression vectors targeting portions of viral coat protein (CP) of WSMV and TriMV were previously transformed into wheat. T₂ plants and beyond were evaluated using PCR, reverse transcription-PCR and bioassays in which plants were challenged with their respective virus. These lines were evaluated for resistance through the T₆ generation. Crosses were made with the susceptible winter wheat cultivars, ‘Overley’ and ‘Karl 92.’ Real-time PCR results show viral titer was up to 20-fold lower in the T₆ transgenic lines, the F₁, and the BC₁F₁ compared to control plants. This provides evidence that this RNAi silencing method is stable in wheat over multiple generations. WSMV and TriMV use host eukaryotic initiation factors (eIF) in order to facilitate replication of their genomes. Previously created RNAi expression vectors were derived from the sequences of the wheat genes eIF(iso)4E-2 and eIF4G. Evaluation of these lines began in the T₁ generation. Resistance has been demonstrated in three lines of eIF(iso)4E-2 and four lines of eIF4G, derived by single seed descent. T₆ progeny co-infected with WSMV and TriMV continue to be resistant. Crosses have been performed with the winter wheat ‘Karl 92’ and three Kansas elite lines, KS030887K-6, KS09H19-2-3, and KS10HW78-1-1. RNAi construct effectiveness was evaluated using real-time PCR. Results show up to 18-fold reduction in viral titer in the transgenic lines, the F₁, and the BC₁F₁ in comparison to control plants. This research provides the first evidence that a single host transgene can provide resistance to multiple viruses and has great potential benefits to both breeders and producers.

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.

Master of Science / Department of Plant Pathology / William Bockus / Barley yellow dwarf (BYD) is one of the most important wheat diseases in the state of Kansas. Despite the development of cultivars with improved levels of resistance to BYD, little is known about the impact that this resistance has on yield loss from the disease. The intent of this research was to estimate yield loss in winter wheat cultivars in Kansas due to BYD and quantify the reduction in losses associated with resistant cultivars. During seven years, BYD disease incidence was visually assessed on numerous winter wheat cultivars in replicated field nurseries. Cultivars were planted about three weeks early to promote disease. When grain yields were regressed against BYD incidence scores, negative linear relationships significantly fit the data for each year and for the combined dataset covering all seven years. The models showed that, depending upon the year, 19-48% (average 33%) of the yields was explained by BYD incidence. For the combined dataset, 29% of the relative yield was explained by BYD incidence. The models predicted that cultivars showing high disease incidence had 25-86% (average 49%) less yield than a hypothetical cultivar that showed zero incidence. Using the models, the moderate level of resistance in the cultivar Everest was calculated to reduce yield loss from BYD by about 73%. Therefore, utilizing visual BYD symptom evaluations in Kansas, coupled with grain yields, is useful to estimate yield loss from the disease. Furthermore, linear models that incorporate those parameters can be used to calculate the impact of improving cultivar resistance to BYD on yield losses.

Doctor of Philosophy / Department of Plant Pathology / Anna E. Whitfield / Tomato spotted wilt virus (TSWV) is an economically important plant virus dependent on insects (thrips) for transmission to plant hosts. Like many animal-infecting viruses, TSWV replicates in the cells of its insect vector. The virus is an emergent disease threatening food and fiber crops worldwide. The aim of this work was to develop novel control strategies against TSWV through a better understanding of the virus-vector interaction. Previously, the TSWV GN protein was shown to be the viral attachment protein, a molecule mediating attachment of virus particles to the midgut epithelial cells of vector thrips. The specific goals of my research were to further examine the utility of disrupting the virus-vector interaction for effective virus control by exploiting GN properties, and to track the route of TSWV in thrips using confocal microscopy. To achieve these goals, I expressed soluble and insoluble forms of GN fused to green fluorescent protein (GFP) transiently and transgenically and examined their cellular localization in planta. GN::GFP recombinant protein localized to Golgi stacks throughout the cells as indicated by a punctate pattern or co-localization to a Golgi marker. In contrast, the soluble form of GN, GN-S::GFP, localized to the ER and apparently also to the cytoplasm. Virus acquisition and transmission assays with GN-S::GFP transgenic tomato plants demonstrated that transmission of TSWV by F. occidentalis was reduced by 35 to 100%. These results indicated that transgenic expression of GN-S in tomato plants may have the potential to prevent secondary spread of the virus. Novel features of the morphology of principal (PSGs) and tubular salivary glands (TSGs) of the insect vector F. occidentalis and of their infection with TSWV were described. The virus colonized different cell types and regions within the PSGs with variable intensity and distribution; and accumulated at the lumen of individual cells. The TSGs of F. occidentalis are proposed as a route for TSWV infection into the PSGs. The transgenic plants and the new knowledge of the virus vector interaction are promising tools to control TSWV and a model approach for the control of other vector-borne viruses.

Doctor of Philosophy / Department of Plant Pathology / Megan Kennelly / Large patch, caused by the fungus Rhizoctonia solani anastomosis group (AG) 2-2 LP, is the most common and severe disease of zoysiagrass (Zoysia spp). Despite the importance of this disease, few studies have examined pathogen biology, cultivar susceptibility, cultural controls, and chemical controls. The objectives of this dissertation were: (1) Characterize large patch isolates based on anastomosis pairing, in-vitro mycelial growth rates, nuclear counts, virulence, PCR, and amplified fragment length polymorphism (AFLP); (2) Determine the effects of cultivation (aerification, verticutting, and sand topdressing) on disease severity; (3) Evaluate different fall and spring applications of the fungicides flutolanil, azoxystrobin, and triticonazole; (4) Evaluate the susceptibility of fifteen new zoysiagrass germplasm lines from parental crosses including Z. japonica, Z. matrella, and Z. pacifica. All the R. solani isolates from large patch-infected zoysiagrass from Kansas belonged to AG 2-2 LP. Variations were observed among the isolates in their average number of nuclei per cell, mycelial growth rates and virulence. There was also variation in the amplified fragment length polymorphism (AFLP) DNA fingerprints, suggesting possible underlying genetic differences of biological significance among members of AG 2-2 LP. Cultivation did not affect soil moisture or temperature. Cultivation also did not reduce patch sizes, nor influence turf recovery rate from large patch. From 2009 to 2011, spring and fall N fertility was consistently associated with lower percentages of diseased turf in both cultivated and non-cultivated plots at Manhattan and Haysville. In general, two fall applications of fungicide did not reduce disease compared to one fall application. Fungicides applied in the fall when thatch temperatures ranged from 17.8oC to 23.2oC reduced disease compared to untreated controls. Early spring applications reduced disease compared to later spring applications. In germplasm screening studies, all progeny had similar disease levels compared to Meyer in the growth chamber, but only 6 consistently had disease levels as low as Meyer in the field. Growth chamber results did not correlate to field results.

Master of Science / Department of Plant Pathology / Robert L. Bowden / John F. Leslie / Fusarium graminearum Schwabe sensu lato (teleomorph: Gibberella zeae (Schwein.) Petch), a homothallic ascomycete fungus, is the causal agent of Fusarium head blight (FHB) of wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), and other small grains. FHB occurs worldwide and serious outbreaks have been reported in North America, South America, Asia, and Europe. According to the phylogenetic species concept (PSC), F. graminearum is composed of at least 15 phylogenetic lineages known as the Fusarium graminearum species complex. Although F. graminearum is homothallic, some members of different phylogenetic lineages are known to intercross in the laboratory. It has been suggested that F. graminearum sensu lato fits the biological species concept (BSC). According to the BSC, “species are groups of actually or potentially interbreeding natural populations, which are reproductively isolated from other such groups”. Previous reports of intercrossing were qualitative, so the degree of reproductive isolation, if any, is not clear. Since intrinsic reproductive isolation is the key criterion to identify species by the BSC, more detailed quantitative information is needed. Chromosome rearrangements between fungal strains may reduce fertility in sexual crosses through the production of genetically inviable recombinant progeny. As such, rearrangements can be important postzygotic reproductive barriers between species. Following methods used in Neurospora crassa, ascospore tetrads were analyzed for patterns of ascospore viability. Crosses were made with three lineage 7 (F. graminearum sensu stricto according to PSC) strains as female. Each female was a MAT1-2 knockout mutant that rendered it obligately heterothallic. Males were several members of lineages 6 (F. asiaticum according to PSC) and lineage 7. Crosses with lineage 7 males formed complete asci with 8 ascospores indicating that their genomes are isosequential with the testers. Crosses with one strain from lineage 6 with two known inversions produced asci containing 8, 6, and 4 ascospores, consistent with it not being isosequential. However, three other strains of lineage 6 appeared to be isosequential with the testers. Therefore, chromosome rearrangements did not appear to be common to strains of lineage 6 and probably do not contribute significantly to reproductive isolation of lineage 6 and lineage 7. Interlineage fertility studies with the three lineage 7 tester strains were performed to quantify interlineage fertility parameters including the total number of ascospores produced, perithecial density, and perithecium internal development scores. All lineage 7 female testers successfully crossed to all 23 male strains from lineages 1 to 9. For total ascospore production, one female tester crossed equally well with all lineages and the other two testers showed statistically significant differences for a few lineages. For perithecial density, there was a significantly lower density with all three testers when crossed with lineage 6, but the other lineages were not statistically different from lineage 7. For perithecial development, there was large variation for every lineage. Therefore, in the crosses with reduced fertility, the reduction can be attributed to a postzygotic effect since mature perithecia and asci developed. All of the tested lineages of the Fusarium graminearum species complex can produce viable progeny with F. graminearum lineage 7, which was the taxonomic type of the original species before it was split into phylogenetic species. There are a few examples of reduced fertility with two lineage 7 testers, the remaining tester crossed equally well with all lineages. Therefore members of lineages 1-9 all should be considered members of Fusarium graminearum according to the BSC. The existing female testers could be used to identify members of the F. graminearum clade by performing test crosses in the laboratory. The PSC and BSC species concepts do not agree for this group of fungi. This disagreement indicates that the F. graminearum species complex is in the early stages of speciation. The lack of intrinsic reproductive barriers supports the hypothesis that these lineages have developed in geographic isolation. As the lineages have apparently been brought together through global trade, interlineage hybrids have been reported in the field. The discrepancy between PSC and BSC will eventually be resolved by whether the lineages fuse or remain separate in nature. Even if the lineages remain separate, this study demonstrates the potential for gene flow between lineage 7 and lineages 1 through 9.