<p><p>Plants and their pathogens coevolve, with pathogen infection and host resistance acting in evolutionary antagonism of each other. Plant-pathogen coevolution has been shown to effect genetic divergence between populations and species, resulting in localized or specialized interactions between hosts and pathogens. Because most of the studies to date investigating plant-pathogen coevolution have been carried out in managed systems and have focused on pairwise interactions, we know little about three aspects of plant pathosystems in natural settings: 1) the role in nature of the gene-for-gene paradigm for genetic determination of resistance; 2) the relationship of host community diversity and structure, and host-pathogen interaction structure, to the coevolutionary process; and 3) the factors which underlie and drive local adaptation and specialization of interactions.</p><p><p>This dissertation constitutes the results of research in which I have begun addressing these aspects in a natural plant-fungus pathosystem comprising three <i>Ipomoea</i> host species and a single rust pathogen, <i>Coleosporium ipomoeae</i>. I have expanded previous characterization of the genetics of plant resistance in one constituent host species in the system by genetic crosses to characterize the basis of resistance in two additional species, finding support for the expectation that the gene-for-gene paradigm of interaction is important in natural systems. I conducted a cross-inoculation experiment designed to assess host and pathogen variation in infectivity and resistance, to investigate patterns of community interaction structure, and the role that antagonistic coevolution may play in structuring the communities which compose pathosystems. In these experiments I found that the coevolutionary interaction in this system leads to genetic divergence and the substantial amount of host and pathogen variation I discovered, but that it tends to preserve one pattern of community interaction structure across communities. I expanded my cross-inoculation experimental design to facilitate analysis of quantitative aspects of pathogenesis by measuring the intensity of infections, to test existing hypotheses concerning local adaptation and specialization in pathosystems. In this analysis I found strong host local adaptation and pathogen local maladaptation for the qualitative interaction trait of infectivity, and I found weak host local maladaptation and pathogen local adaptation for the quantitative interaction trait of aggressiveness. I also found host specialization among pathogens, and specialized resistance among hosts, to be common in this system. In light of these results, I hypothesize that the geographic scale of host-pathogen coevolution in this system is that of the local community, and that differences between host species result in persistent but incomplete host specialization in pathogen races.</p> / Dissertation

The impact of three pre-plant herbicides, pendimethalin, prometryn and trifluralin on the incidence and the development of Rhizoctonia solani-induced cotton seedling damping-off, on biocontrol activity of Burkholderia cepacia against R. solani, and on the efficacy of commonly used fungicides (metalaxyl, triadimenol, thiram) against R. solani-induced cotton seedling damping-off was investigated in the microcosm and in the field. In the microcosm experiments, pendimethalin, prometryn and trifluralin were applied to the soil at 2.4, 3.6 and 1.8 μg a.i. g⁻¹ soil, respectively (equivalent to the respective recommended field concentrations of 0.9, 1.3 and 0.7 kg a.i. ha⁻¹ In the microcosm experiments application of prometryn, to the soil caused a significant (P < 0.05) increase in pre-emergence damping-off incidence. Post emergence damping-off increased significantly (P < 0.05) in the presence of pendimethalin and prometryn. Biocontrol activity of B. cepacia (isolate D1) in controlling cotton seedling damping-off was significantly (P < 0.05) reduced by pendimethalin and prometryn in the microcosm experiments. In a field experiment conducted in Safford, Arizona, pre-plant application of pendimethalin or prometryn caused significant (P < 0.05) increases in disease incidence. In another field experiment in Tucson, Arizona, significant (P < 0.05) increase in disease incidence was observed in plots treated with prometryn. In both microcosm and field experiments, application of herbicides affected disease development as judged by the slope of disease progress curves. Biocontrol activity of an indigenous isolate (D1) of B. cepacia also was reduced significantly by pendimethalin and prometryn in both field experiments. Commercially available isolate (Deny®) of B. cepacia as a soil drench and as a seed treatment failed to protect cotton seedlings against R. solani-induced damping-off in both field experiments. The efficacy of commonly used fungicides (metalaxyl, triadimenol, thiram) against R. solani in the field was not significantly affected by herbicides in two field experiments at final assessment time. Pendimethalin and prometryn mediated changes in cotton seedling damping-off incidence and biocontrol activity of B. cepacia seem to be due to the changes in cotton seedlings susceptibility and changes in populations and structure of antagonistic bacteria in the plant rhizosphere.

Doctor of Philosophy / Department of Plant Pathology / Xiaoyan Tang / P. syringae is a group of bacterial phytopathogens that can infect a wide variety of plants. These bacteria rely on the type III secretion system (TTSS) to deliver effectors into plant cells for infection. The TTSS genes, that encode the TTSS apparatus and the effectors, are repressed when bacteria grow in nutrient rich media but are strongly induced in the plants and in minimal medium (MM). Plant cutin monomers appear to negatively regulate the P. syringae TTSS genes. It is poorly understood how bacteria sense the environmental signals to regulate the TTSS genes. By genetic screen, four sets of transposon insertion mutants displaying aberrant TTSS gene expression were isolated: KB and fin mutants derepress the TTSS genes in rich medium KB and in the presence of a cutin monomer precursor in MM, respectively; min and pin mutants are defective in induction of TTSS genes in MM and in plants, respectively. A putative two-component sensor histidine kinase, RohS, is identified to be required for the induction of avrPto-LUC in MM and in plants. The rohS gene is in an operon containing a two-component response regulator gene rohR. Mutation of rohS in P. s. phaseolicola and P. s. tomato reduced the bacterial pathogenicity on hosts and HR-inducing activity on non-hosts. Our results suggested that RohS acts upstream of HrpR/HrpS. The phosphorylated RohR represses TTSS genes. It is likely that RohS acts as phosphatase of RohR in the TTSS-inducing conditions, and subsequently derepresses TTSS genes. Simple sugars such as glucose, sucrose and fructose are known to be inducers of the TTSS genes. Isolation of four min mutants defective in fructose-uptake enabled us to study if sugars serve as extracellular signals or as essential nutrients. Our results suggest that fructose acts as an essential nutrient for the activation of type III genes. These mutants slightly compromised induction of avrPto promoter in Arabidopsis and pathogenicity on the host bean plant, but displayed normal HR elicitation on non-host plant tobacco. The reduced pathogenicity suggested that exploitation of fructose from the host tissue is an important means for pathogenesis of P. s. phaseolicola.

Doctor of Philosophy / Department of Plant Pathology / Scot H. Hulbert / The rice cultivar Oryzica Llanos 5 (OL5) possesses a high level of resistance to the fungus Magnaporthe grisea. The number and chromosomal location of quantitative trait loci (QTL) conferring resistance against eight isolates of the blast fungus were tested in two different populations of recombinant inbred lines from the cross Fanny x OL5. Twenty one QTL were detected and associated with the resistance traits, disease leaf area and lesion type, on 9 rice chromosomes. Eight of these 21 resistance loci had significant resistance effects in both experiments, while the others had effects that were only statistically significant in one experiment. Most, but not all, of the QTL occurred in the same genomic regions as either genes with major race-specific effects or other resistance QTL that had been described in previous experiments. Most of the QTL appeared to be race-specific in their effects but it is possible some of the QTL with smaller effects were nonspecific. One of the blast isolates used was FL440, which causes limited disease on OL5 and was probably virulent on most or all of the major genes from OL5. Three QTL affected resistance to FL440 in both experiments, one of which mapped to a region on chromosome 9 where no blast resistance genes have yet been mapped. An advanced backcross strategy with marker-assisted selection for OL5 alleles in QTL regions was used to generate five BC2F3 populations carrying five different target regions associated with partial resistance to rice blast disease. Three of five of these populations were analyzed for segregation for resistance to the M. grisea isolate FL440. One QTL designated qrbr-11.3 near the bottom of rice chromosome 11 was found to be significantly associated with partial blast resistance in 120 lines of a BC2F3 population (P< 0.01). This QTL accounted for 12.4% and 8.0% of the phenotypic variation in diseased leaf area and lesion type observed under greenhouse inoculation. Examination of the genomic sequence at the qrbr-11.3 locus showed that twenty-nine candidate resistance genes are present at that locus (~1.8 Mb), twenty-seven of which are predicted NBS-LRR genes. Ultimately, the information from this study can be integrated into the development of improved lines with OL5-derived QTL for resistance.

Doctor of Philosophy / Department of Plant Pathology / Jianmin Zhou / Plants activate innate immune responses or innate immunity upon pathogen infection. There are two types of plant innate immunity: PAMP-triggered innate immunity (PTI) and effector-triggered innate immunity (ETI). The molecular basis for ETI has been well documented. However, the study on PTI and its interplay with pathogen virulence is in its infancy. My research focuses on the interplay between PTI and bacterial virulence in Pseudomonas-Arabidopsis interactions. NHO1, a gene required for nonhost resistance to Pseudomonas syringae, encodes for the 3-glycerol kinase in Arabidopsis genome. NHO1 functions, at least in part, by depriving glycerol from nonhost bacteria cells. NHO1 is induced by a well-known bacteria PAMP flg22. The induction of NHO1 correlates well with the resistance against Pseudomonas syringae pv. tabaci because a mutant strain of P. s. pv. tabaci deficient in NHO1 induction gains partial virulence on Arabidopsis plants. P. s. pv. tomato strain DC3000 induces transient NHO1 expression that is suppressed in a type III secrection system-dependent manner. Using protoplast assay, nine DC3000 effectors that are able to suppress NHO1 were identified. One of them, HopAI1, induces leaf chlorosis and helps nonpathogenic bacterial growth when expressed in Arabidopsis plants, suggesting that HopAI1 has virulence activity in planta. To study AvrB virulence activity in Arabidopsis plants, one mutant compromised in AvrB-specific RAR2.6 induction has been characterized in detail. rrb3 is more susceptible to a nonhost bacteria P. s. pv. tabaci strain 6505, a virulent bacteria P. s. pv. tomato strain DC3000 and an avirulent bacteria strain DC3000 (avrB). The mutant allele rrb3 carries a point mutation at the end of RAR1 CHORD II domain. RRB3 (RAR1), together with NDR1, is involved in the type II nonhost resistance to P. s. pv. tabaci but not in the type I nonhost resistance to P. s. pv. phaseolicola. RAR1 participates in basal resistance against DC3000 by antagonizing COI1 activity. AvrB targets RAR1 to trigger AvrB-dependent leaf chlorosis and enhanced bacterial growth. The AvrB-dependent enhanced bacterial growth but not leaf chlorosis requires COI1, suggesting that AvrB targets JA signaling pathway to promote parasitism.

Master of Science / Department of Plant Pathology / Karen A. Garrett / While plants are typically exposed to multiple stressors in the field, studies of genome-wide gene expression and phytohormone responses in wild plant species exposed to multiple stressors are rare. Our objectives were to determine the effects of drought and rust stress on gene expression in Andropogon gerardii, the dominant grass in tallgrass prairie, and associated levels of phytohormone production. In a factorial design, plants experiencing drought or non-drought conditions were either inoculated with the rust pathogen Puccinia andropogonis or not inoculated. Gene expression was evaluated with maize microarrays. Drought-stressed plants significantly decreased expression of genes associated with photosynthesis and the hypersensitive response, while expression of genes associated with chaperones and heat-shock proteins increased. No significant differences in gene expression in response to the rust treatment were detected using a mixed model analysis of variance and false discovery rate protection, probably because of the low infection rate. Phytohormone production increased when both stresses were present. The rust treatment significantly increased benzoic acid (BA) production in the presence of drought, while the drought treatment alone significantly increased salicylic acid (SA) production. Leaf tips usually had higher levels of all phytohormones in all treatments and the leaf section evaluated had a larger effect on phytohormone level than did the treatments applied.

Master of Science / Department of Horticulture, Forestry, and Recreation Resources / Edward E. Carey / With acceptance and utilization of chemical pesticides declining, some vegetable producers are turning to alternative methods to manage plant health issues. Compost tea (CT) has provided control of some foliar pathogens and may provide benefits beyond disease suppression. Despite an increasing body of popular and scientific literature focusing on CT as a biological control option for growers, information on the efficacy of CT is still lacking for many pathosystems. In this study, field trials were conducted to evaluate the efficacy of CT on Septoria lycopersici, causal agent of Septoria leaf spot on tomato, in Kansas, in 2006 and 2007. Previous research done at KSU with a similar CT showed adequate control of this pathogen in field and greenhouse studies conducted. Additional work to develop a rapid screening method for efficacy of CT formulations was carried out in the greenhouse at Manhattan, KS. CT sprayed weekly on tomato plants prior to and after disease onset led to no significant difference in control of the pathogen compared to untreated controls. A contact fungicide (chlorothalonil) provided significant control of the pathogen in 2007, but not in 2006. These results contrast with those obtained in previous K-State research. It is difficult to assess why such striking differences were obtained, but the variation in these results point to the need to identify optimal recipes of CT for this pathosystem. Preliminary investigations standardized plant age, inoculum concentration, incubation conditions, and incubation interval for measurable Septoria leaf spot disease development on young tomato plants in the greenhouse. Ingredients of the field-tested CT were used to make a variety of CTs to test using the greenhouse-screening assay. Further work on identifying effective CT recipes is needed to substantiate the validity of this screening protocol and to evaluate the correlation of this method with disease suppression in the field.

Doctor of Philosophy / Department of Plant Pathology / Robert L. Bowden / John F. Leslie / Gibberella zeae is an ascomycete filamentous fungus and the major cause of Fusarium head blight, also called scab, in small grains. This dissertation contains three related studies of G. zeae. In the first, the genetic map was aligned with the first assembly of the genomic sequence released by The Broad Institute (Cambridge, MA). Approximately 99% of the sequence was anchored to the genetic map, indicating the high quality of the sequence assembly and validity of the genetic map. The alignments grouped the linkage groups and supercontigs into four sets, which is consistent with the hypothesis that there are four chromosomes in this fungus. In the second, the sex pheromone precursor genes (ppg1 and ppg2) and the pheromone receptor genes (pre1 and pre2) were identified and characterized. Deletion of ppg1 or pre2 ([Delta]ppg1 or [Delta]pre2 strains) reduced the number of perithecia produced by self-fertilization, but did not completely block perithecial formation. The proportion of crosses resulting from outcrossing increased when the [Delta]ppg1 strains were used as the female in crosses with male strains containing an intact ppg1 gene. [Delta]ppg2 and [Delta]pre1 mutants had no discernable effect on morphological phenotype or self-fertilization. Thus, one of the pheromone/receptor pairs (ppg1/pre2) found in many Ascomycetes has a role in, but is not essential for, selfing or outcrossing in G. zeae, whereas the other pheromone/receptor pair (ppg2/pre1) no longer has a detectable function in sexual reproduction. In the third study, spore germination of G. zeae was tested in the presence of α- factor-like pheromone peptides of G. zeae or N. crassa. The pheromone peptide of N. crassa more efficiently inhibited spore germination than did the peptide from G. zeae. Arginine and lysine residues were the most important determinants in blocking spore germination. In conclusion, this research has validated the genetic map and the genomic assembly of G. zeae, characterized sex pheromone functions and characterized pheromone peptide ability to inhibit spore germination. The pheromone peptides of G. zeae and N. crassa may be useful as control agents for G. zeae and pheromone peptide efficacy might be further enhanced by judicious substitutions for some of the amino acids.

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