Master of Science / Department of Plant Pathology / Christopher R. Little / Christopher R. Little / Charcoal rot, caused by Macrophomina phaseolina, is the most important soybean disease in Kansas. Several strategies have been recommended to control this disease including crop rotation, lower plant densities, biological control, plant resistance and tolerance, and fungicide application. However, those techniques have not been completely effective and the information concerning soil texture, irrigation and micronutrient fertility (particularly manganese) upon charcoal rot disease severity and the pathogen population is limited. The objective of this study was to determine key factors that affect the biology of M. phaseolina and charcoal rot processes under laboratory, greenhouse and field conditions. M. phaseolina microsclerotia were produced from PDA pure isolate and infested Japanese millet in the laboratory and characterized by different techniques such as serial dilutions in semi selective media with the aim to produce quality inoculum to reliably infect soybean seedling roots under greenhouse conditions; production of inoculum by infesting Japanese millet was the most efficient method. Root colonization and root infection of soybean seedlings was assessed through the use of M. phaseolina inoculum under controlled conditions in the greenhouse. Root infection by M. phaseolina and microsclerotia longevity in soil is determined by environmental factors such as soil moisture content, soil texture and source of inoculum. The objective of the greenhouse study was to determine the impact of these variables on seedling root infection at the V1 and V2 development stages. Artificial soils with different textures were infested; M. phaseolina microsclerotia and soybean seedlings were exposed to different soil moisture contents including pot saturation, pot (field) capacity, and permanent wilting point. Soil populations and levels of root colonization for the stages were assessed by estimating CFUs and root length. Results indicate that soil texture has a significant impact upon root morphology and root length. Root populations of M. phaseolina were significantly higher in sandy soil textures and lower in the fine-textured soils, suggesting an impact of soil water holding capacity in the root infection process. The effect of water stress on seedling root colonization by M. phaseolina indicates that early infection may be more important than previously thought. A field study was also conducted to determine the effect of the aforementioned variables in a 2-year field experiment conducted at two Kansas locations. Pathogen colonization was iii assessed by measuring colony-forming units (CFUs) from ground root tissue at R2-R4 (post-flowering/early pod development) and R8 (maturity) stages. Soil populations (pre-planting and post-harvest) of M. phaseolina, yield parameters, and plant characteristics were obtained. Results indicated that there are complex relationships between soil physiochemical properties (pH, NPK content, exchangeable cations, and organic matter) and soil texture (sand, soil, and clay composition), which may mitigate disease severity and pathogen levels in host tissue. Results also indicated that in natural M. phaseolina-infested soils, cropping history and soil texture play an important role in charcoal rot processes and influence the levels of pathogen soil populations, root colonization at maturity and, more importantly, soybean yield.

Some Arabidopsis lesion mimic mutants (LMM) show alterations in abiotic stress responses as well as pathogen resistance. cpr22 is a LMM which has a mutation in cyclic nucleotide-gated ion channels, is a typical LMM exhibiting elevated levels of salicylic acid (SA), spontaneous cell death, constitutive expression of defense genes, and enhanced resistance to various pathogens in an SA dependant manner. cpr22 defense responses are suppressed in high humidity and enhanced by low humidity. To investigate environmental effects, microarray analyses were conducted. Expression of several genes related to abscisic acid (ABA) signaling was altered and ABA levels increased in cpr22 after humidity shift. Furthermore, significant alterations in ABA-related phenotypes were observed. Double mutant analysis with nahG plants indicated that alterations in ABA signaling were attributable to elevated SA levels. These results suggest a negative effect of SA on ABA signaling/abiotic stress responses during the activation of defense responses.

The pathogenicity of plant pathogen Pseudomonas syringae depends on the type III secretion system which translocates effector proteins into host cells. In response, plants have evolved resistance proteins to detect presence of specific effectors and activate defense responses. The constant host surveillance imposes a strong selective pressure on effector proteins to evolve rapidly in order to evade detection. The P. syringae HopZ1 effector has evolved into three allelic forms as a result of diversifying selection. In this thesis, I aimed to investigate how sequence divergence contributes to the distinct allelic specificities of HopZ1. Mutational analysis of HopZ1a identified three amino acid residues that were potentially involved in dampening host defense responses, and two HopZ1a mutants partially lost the ability to trigger defense responses yet did not lose their virulence functions. These results suggested that distinct host targets could be involved in the defense-eliciting activity and virulence function of HopZ1a.

The pathogenicity of plant pathogen Pseudomonas syringae depends on the type III secretion system which translocates effector proteins into host cells. In response, plants have evolved resistance proteins to detect presence of specific effectors and activate defense responses. The constant host surveillance imposes a strong selective pressure on effector proteins to evolve rapidly in order to evade detection. The P. syringae HopZ1 effector has evolved into three allelic forms as a result of diversifying selection. In this thesis, I aimed to investigate how sequence divergence contributes to the distinct allelic specificities of HopZ1. Mutational analysis of HopZ1a identified three amino acid residues that were potentially involved in dampening host defense responses, and two HopZ1a mutants partially lost the ability to trigger defense responses yet did not lose their virulence functions. These results suggested that distinct host targets could be involved in the defense-eliciting activity and virulence function of HopZ1a.

Some Arabidopsis lesion mimic mutants (LMM) show alterations in abiotic stress responses as well as pathogen resistance. cpr22 is a LMM which has a mutation in cyclic nucleotide-gated ion channels, is a typical LMM exhibiting elevated levels of salicylic acid (SA), spontaneous cell death, constitutive expression of defense genes, and enhanced resistance to various pathogens in an SA dependant manner. cpr22 defense responses are suppressed in high humidity and enhanced by low humidity. To investigate environmental effects, microarray analyses were conducted. Expression of several genes related to abscisic acid (ABA) signaling was altered and ABA levels increased in cpr22 after humidity shift. Furthermore, significant alterations in ABA-related phenotypes were observed. Double mutant analysis with nahG plants indicated that alterations in ABA signaling were attributable to elevated SA levels. These results suggest a negative effect of SA on ABA signaling/abiotic stress responses during the activation of defense responses.

Successful pathogens like Pseudomonas syringae translocate type III effector proteins (T3SE) into host cells. Plant hosts react by specifically recognizing these effectors via R proteins that trigger defense responses. The T3SE family HopZ1 has evolved into three allelic forms as a result of diversifying selection. In this thesis, I investigated how virulence and immune-eliciting functions are determined in HopZ1a and HopZ1b in Arabidopsis. Mutational analysis of HopZ1a identified ten residues important for immune elicitation and at least three are involved in virulence functions. These results suggest that distinct key amino acid residues in HopZ1a mediate the two activities. The closely related HopZ1b T3SE elicits an inconsistent immune response in Arabidopsis. We found that HopZ1b-triggered immune response involves a TIR-type R protein and plastid-derived SA. Together, these results highlight an uncharacterized ETI response to the HopZ1 family of T3SEs.

Diss.--Evangelisch-theologische Fakultät--Tübingen, 1970. / Bibliogr. p. 275-292.

Doctor of Philosophy / Department of Plant Pathology / Jan E. Leach / Because it is predicted to be durable and broad spectrum, quantitative trait loci (QTL)-based resistance is an important option for rice disease control. However, manipulation of this type of resistance requires knowledge of the contributing genes. This study demonstrates the contribution of two of three defense response (DR) genes to QTL-governed resistance, and identifies a third gene that negatively regulates resistance. The contribution to QTL-governed resistance of one of nine rice OsPAL genes, which encode phenylalanine ammonia-lyase, was determined using reverse genetics. Mutant ospal4 contains a 750 bp deletion in OsPAL4 and was identified using a PCR-pooling strategy. OsPAL4 underlies a QTL on chromosome 2, and is located in cluster with three other OsPAL members. Rice lines mutated in OsPAL4 are more susceptible to a virulent strain of Xanthomonas oryzae pv. oryzae (Xoo) than lines with the wild type allele. RNAi suppression was used to evaluate the contributions of genes encoding oxalate oxidase-like proteins (OsOXL) and a 14-3-3 protein (GF14-e) to disease resistance. Silencing of 12 OsOXL genes clustered on chromosome 8, varied from suppression of a few gene members to silencing of all expressed family members. Screening of transgenic lines by challenge with Magnaporthe grisea (Mg), the rice blast pathogen, revealed that the more chromosome 8 OsOXL genes suppressed, the more susceptible the plants were to Mg. GF14-e co-localizes with a disease resistance QTL on chromosome 2. Specific suppression of GF14-e by RNAi silencing did not result in enhanced susceptibility to Mg. Instead, the lines exhibited spontaneous HR-type lesions. The presence of this lesion mimic phenotype correlated with enhanced resistance to a virulent strain of Xoo, suggesting that the GF14-e encoded 14-3-3 protein functions as a negative regulator of plant cell death and bacterial resistance in rice. This study supports the hypothesis that DR genes, such as OsOXL and OsPAL4 contribute to disease resistance governed by QTL. The role of GF14-e is less clear, however its down regulation may contribute to QTL-governed resistance. Thus, incorporation of regions harboring the effective DR gene alleles into rice will enhance broad spectrum and durable resistance.

Doctor of Philosophy / Department of Plant Pathology / Barbara S. Valent / The hemibiotrophic rice blast fungus Magnaporthe oryzae undergoes complex morphological development throughout its infection cycle. From 8-20 hours after a fungal spore lands on a leaf surface, the fungus differentiates a complex appressorium that punctures the host cuticle. By ~24 hours post inoculation (hpi), the fungus grows inside an epidermal cell as a primary hypha, and by 36 hpi the fungus has differentiated specialized biotrophic invasive hyphae (IH) that are filling the first-invaded cell and moving into neighbor cells. Throughout its life cycle, IH invade living rice cells although invaded cells appear dead when the fungus moves into the next cell. Biotrophic invasion must be mediated by fungal effectors, proteins that pathogens secrete inside live host cells to control them. However, little is known about blast effectors, and the low fungal biomass in early infection stages complicates identification of effector genes, as well as identification of rice genes controlled by effectors. The characterized AVR-Pita effector gene is specifically expressed in planta, but it was not clear how its gene expression pattern changed in different infection stages. We found that AVR-Pita is first expressed around the time of penetration. AVR-Pita is highly expressed in IH developing in asymptomatic tissue from 36 hpi to as late as 7 days post inoculation when lesions are maturing. Using inoculated rice sheaths, we successfully enriched for infected tissue RNA that contained ~20% IH RNA at 36 hpi. We compared IH gene expression to expression in mycelium from pure culture using a whole-genome M. oryzae oligoarray, and we compared infected rice gene expression to expression in mock-inoculated tissue using a rice oligoarray. Rice genes that were induced >50-fold during infection were enriched for genes involved in transferring information from sensors to cellular responses. Fungal genes that were induced >50-fold in IH included known effectors and many IH-specific genes encoding hypothetical secreted proteins that are candidate effectors. Gene knock-out analyses of three putative effector genes failed to show major effects on pathogenicity. Details of the blast interaction transcriptome will provide insights on the mechanisms of biotrophic plant disease.

Master of Science / Department of Electrical and Computer Engineering / Karen A. Garrett / Caterina M. Scoglio / With rapid global movement of epidemics, research efforts to characterize dynamics of epidemics have gained much focus. Traditional epidemiological models have focused on only temporal components of epidemics. Development of spatio-temporal models proved to be a notable achievement in epidemiology. Network-based epidemiological models enable better handling of spatial and temporal components of an epidemic. Early network models considered a binary level of contact between infected entities, which is an idealistic approach. A realistic approach would use weighted edges which signify the level of interaction between the nodes where the edge-weights change over time as a function of environmental factors. Estimation of edge weights from observed time series is a relatively less explored area for network modeling. Dynamic networks make the problem more complicated as edge weights change over time. Estimation of parameters for models describing the edge weights as a function of variables that change in time has the potential to provide better general models. Soybean rust (caused by Phakopsora pachyrhizi) is an important disease globally and its occurrence in the US has been studied extensively since its introduction in 2004. Rust is a fungal disease which propagates as a result of the fungal spores being carried by the wind. In this thesis, a network network based model is proposed to predict the intensity of spread of the disease in space and time. This model uses the host abundance and wind data and the observed rust incidence time series to compute the edge-weights. Also, the edge-weights in the model change over time thus following a dynamic approach. In order to cut costs involved with the establishment and maintenance of infection monitoring sites, the effect of removal of monitoring nodes using various strategies has also been analyzed in this thesis. The model has been tested with observed soybean rust data from sentinel plot network from across the United States.