Master of Science / Department of Plant Pathology / William Bockus / Robert Bowden / Stem rust, caused by the fungus Puccinia graminis f. sp. tritici, has historically been one of the most important diseases of wheat. Although losses have been much reduced in the last fifty years, new highly virulent races of the pathogen have recently emerged in East Africa. These new races are virulent on nearly all of the currently deployed resistance genes and therefore pose a serious threat to global wheat production. The spring wheat variety ‘Kingbird’ is thought to contain multiple quantitative trait loci (QTLs) that provide durable, adult-plant resistance against wheat stem rust. Stem rust-susceptible Kansas winter wheat line ‘KS05HW14’ was backcrossed to Kingbird and 379 recombinant lines were advanced to BC₁F₅ and then increased for testing. The lines were screened for stem rust resistance in the greenhouse and field in Kansas and in the field in Kenya over multiple years. We identified 16,237 single nucleotide polymorphisms (SNPs) with the Wheat 90K iSelect SNP Chip assay. After filtering for marker quality, linkage maps were constructed for each wheat chromosome. Composite interval mapping and multiple-QTL mapping identified seven QTLs on chromosome arms 2BL, 2DS, 3BS, 3BSc, 5DL, 7BL, and 7DS. Six QTLs were inherited from Kingbird and one QTL on 7BL was inherited from KS05HW14. The location of the QTL on 2BL is approximately at locus Sr9, 3BS is at Sr2, 3BSc is at Sr12, and 7DS is at Lr34/Yr18/Sr57. Although no QTL was found on 1BL, the presence of resistance gene Lr46/Yr29/Sr58 on 1BL in both parents was indicated by the gene-specific marker csLV46. QTLs on 2DS and 5DL may be related to photoperiod or vernalization genes. Pairwise interactions were only observed with race QFCSC, most notably occurring with QTLs 2BL and 3BSc. These results confirm that there are multiple QTLs present in Kingbird. Ultimately, the identification of the QTLs that make Kingbird resistant will aid in the understanding of durable, non-race-specific resistance to stem rust of wheat.

Master of Science / Department of Agronomy / Guihua Bai / Stem rust, caused by Puccinia graminis f. sp. tritici, has successfully prevented rust epidemics by Deployment of resistant cultivars in the past several decades. Unfortunately, race TTKS (termed Ug99) has defeated most stem rust resistance genes existing in commercial cultivars. Sr40, a stem rust resistance gene from Triticum timopheevii ssp. araraticum, was transferred to wheat and provides effective levels of seedling and adult plant resistance against Ug99. To characterize Sr40 in wheat, two mapping populations were developed from the crosses RL6088 / Lakin and RL6088 / 2174. RL6088 is an Ug99-resistant parent with Sr40. Since race TTKS is a quarantined pathogen, a US stem rust isolate RKQQ that is avirulent to Sr40 was used to evaluate the rust resistance in the F[subscript]2 and F[subscript]2:3 populations at the seedling stage. A total of 83 simple sequence repeats (SSR) primers on chromosome 2B were used to screen the parents for polymorphism. Each F[subscript]2 population was analyzed with the markers polymorphic between two parents. Marker Xwmc344 was the most closely linked to Sr40, at 0.7 cM proximal, in the linkage map constructed from the population RL6088 / Lakin, while Xwmc474 and Xgwm374 were also tightly linked. Xwmc474 was mapped 2.5 cM proximal to Sr40 in the RL6088 / 2174 population. Xwmc474 and Xwmc661 were flanking markers for Sr40 in both populations. Markers linked to Sr40 will be useful for marker-assisted integration of Sr40 into elite wheat breeding lines. In addition, a unknown stem rust resistance gene from another source, OK01307, a breeding line from Oklahoma State University shows partial resistance to Ug99, and was characterized using SSRs in this study. Two mapping populations were developed from cross OK01307 / Chinese Spring and OK01307 / LMPG-6. A total of 1300 SSR primers were screened for polymorphism between OK01307 and Chinese spring, and 1000 SSR primers were screened for polymorphism between OK01307 and LMPG-6. Polymorphic primers between parents and between bulks were used to screen the corresponding population. One Sr gene in OK01307 was mapped on chromosome 1BS of the both populations, which was closely linked to Sr24. Whether the gene is Sr24 per se or a new Sr gene that closely linked to Sr24 needs further investigation.

Three hundred ninety one (391) spring wheat introductions from Asian, Middle Eastern and Mediteranean areas were screened for resistance to races C17, C20, C25, C49, C53 and C57 of Puccinia qraminis tritici; races 1, 5, 9, 15 and bulks 1, 4 and l0 of Puccinia recondita; races T2, T10, T13 and T39 of Ustilago tritici and to 6 isolates of Drechslera tritici-repentis prevalent in western Canada. Of the 34 introductions resistant to P. graminis tritici, 15 were genetically studied using F2 segregation data derived from the progeny of the crosses involving resistant introductions, their corresponding near isogenic lines and stem rust universal suscepts. Eleven of these were found to carry single Sr genes for resistance, notably, Sr30, Sr13 and Sr15. Of the 70 introductions resistant to P. recondita, 28 were studied and 9 were found to carry known Lr genes for resistance, notably Lr10 and the genes present in RL6057 and RL6061. Twenty two introductions are believed to carry either 1 or 2 unidentified dominant, recessive, partially dominant and/or complementary genes for resistance to either stem or leaf rust. Five introductions were immune and 6 highly resistant to the 4 races of U. tritici. Sixty-nine introductions were resistant to D. tritici-repentis . These were arbitrarily classified into 10 'phenotypic classes' according to their reactions to the 6 isolates used.

Three hundred ninety one (391) spring wheat introductions from Asian, Middle Eastern and Mediteranean areas were screened for resistance to races C17, C20, C25, C49, C53 and C57 of Puccinia qraminis tritici; races 1, 5, 9, 15 and bulks 1, 4 and l0 of Puccinia recondita; races T2, T10, T13 and T39 of Ustilago tritici and to 6 isolates of Drechslera tritici-repentis prevalent in western Canada. Of the 34 introductions resistant to P. graminis tritici, 15 were genetically studied using F2 segregation data derived from the progeny of the crosses involving resistant introductions, their corresponding near isogenic lines and stem rust universal suscepts. Eleven of these were found to carry single Sr genes for resistance, notably, Sr30, Sr13 and Sr15. Of the 70 introductions resistant to P. recondita, 28 were studied and 9 were found to carry known Lr genes for resistance, notably Lr10 and the genes present in RL6057 and RL6061. Twenty two introductions are believed to carry either 1 or 2 unidentified dominant, recessive, partially dominant and/or complementary genes for resistance to either stem or leaf rust. Five introductions were immune and 6 highly resistant to the 4 races of U. tritici. Sixty-nine introductions were resistant to D. tritici-repentis . These were arbitrarily classified into 10 'phenotypic classes' according to their reactions to the 6 isolates used.

Doctor of Philosophy / Department of Plant Pathology / Eduard Akhunov / The fungus Puccinia graminis f. sp. tritici (Pgt) is the causal agent of the wheat stem rust disease. Wheat stem rust has attracted a lot of attention after the emergence of the Ug99 race group, which at the time of its origin was virulent on most of the wheat varieties cultivated around the world. The evolution and spread of the Pgt isolates from the Ug99 race group posed a serious threat to worldwide wheat production. To mitigate the potential impact of new rust epidemics in major wheat production areas, it remains critical to identify new strategies for breeding durable resistance traits. A detailed understanding of the plant-pathogen interaction mechanisms in the wheat-Pgt pathosystem should be the foundation of these strategies. The interaction between the matching pair of resistance (R) and avirulence (Avr) genes, an important element of the plant-pathogen interactions, is described by the broadly documented gene-for-gene model. The cloning of the Sr35 gene, which confers near immunity against all isolates from the Ug99 race group provided a unique opportunity to investigate the molecular mechanisms of resistance to stem rust in wheat. The goals of the present study were: (1) to determine whether the Sr35 gene alone is sufficient for conferring resistance against Ug99, (2) to assess the Sr35 transcript levels during the time course of infection, and (3) to identify and validate the corresponding Avr gene interacting with Sr35. The cloning of Avr genes from the biotrophic fungi represents a substantial challenge due to the variability, redundant nature, the lack of similarity to known proteins, and lack of adequate functional tools to validate them. To overcome these limitations, we performed a comparative genomic analysis using multiple Sr35-avirulent and Sr35-virulent races, including 15 chemically mutagenized Pgt strains that acquired virulence on the Sr35 gene. Whole genome shotgun sequencing of the Pgt mutants identified a single candidate gene, which carried strong effect mutations in each mutant strain. The Avr gene candidate (AvrSr35) was expressed at early stages of infection and had a signal peptide indicating that the gene product is secreted. Comparative microscopic analysis of the infected tissues at different time points after infection indicated that AvrSr35 secretion occurs before haustoria formation. The re-sequencing of the AvrSr35 candidate gene in a panel of Sr35-virulent and Sr35-avirulent isolates including isolates from the Ug99 race group, revealed the presence of a mobile DNA element inserted into the coding sequence of virulent isolates. This insertion resulted in a premature termination codon and explains the origin of Pgt field isolates virulent in the presence of the Sr35 gene. Co-expression of AvrSr35 with the Sr35 in N. benthamiana leaves induced a specific hypersensitive response confirming the avirulence function of the candidate effector gene. Subcellular localization, bi-molecular fluorescence complementation, and co-immunoprecipitation assays in N. benthamiana leaves revealed that the AvrSr35 and Sr35 proteins interact and are likely associated with the endoplasmic reticulum and plasma membrane. Thus, this study identified and functionally characterized the first matching pair of Avr/R genes for cereal rusts.

Doctor of Philosophy / Genetics Interdepartmental Program - Plant Pathology / Bikram Gill / The diploid D genome species, Aegilops tauschii Coss. (2n=2x=14,DD) has provided numerous genes for resistance to diseases and insect pests that attack bread wheat (Triticum aestivum L. (2n=6x=42, AABBDD). Wheat production is currently threatened by broadly virulent races of the ‘Ug99’ lineage of wheat stem rust caused by the fungus, Puccinia graminis f.sp. tritici Pers. & Eriks. Screening of a large set of Ae. tauschii germplasm for resistance to TTKSK (Ug99) identified potentially novel sources of resistance. To expedite utilization of TTKSK resistance from Ae. tauschii, a direct hybridization approach was established that integrates gene transfer, mapping and introgression into one process. Direct crossing of Ae. tauschii accessions with an elite wheat breeding was used to initiate transfer of resistance. Genetic mapping of resistance was accomplished during gene transfer through development of BC[subscript]2 mapping populations. Bulked segregant analysis of BC[subscript]2F[subscript]1 genotypes at 70 SSR loci across the D genome identified the chromosome locations of stem rust resistance genes and facilitated genetic mapping. Using this approach, TTKSK resistance from CDL4424 and TA1662 was mapped on chromosome arm 1DS flanked distally by Xwmc432 and proximally by Xwmc222 at 4.4 cM, TA10187 on 6DS linked to Xcfd49 at 1.9 cM and TA10171 on 7DS linked Xwmc827 at 0.9 cM. TTKSK resistance from additional Ae. tauschii accessions CDL4366, TA1615, TA1642, TA1693 and TA1718 has been recovered in segregating populations but await mapping. Altogether, TTKSK resistance from eight Ae. tauschii accessions has been introgressed to a hard winter wheat genetic background. Three new stem resistance genes have been tagged with molecular markers for marker assisted breeding and will provide valuable material for stem rust resistance breeding and gene pyramids for effective control of stem rust.

Wheat diseases such as rusts and powdery mildews are among the most important and ancient diseases that affect wheat cultivation worldwide. The pathogen race specific resistance genes cannot maintain long lasting resistance. On the other hand, the presence of genes confers the non-race specific resistance last much longer. The durable resistance phenotypes in wheat against various rust and powdery mildew diseases were reported as Lr34, Yr18, and Pm38 separately / nevertheless, they were known to locate very close to each other based on linkage analysis. Recently, it was shown that all of these resistance phenotypes are indeed conferred by the same gene, encoding Adenosine triphosphate&ndash / binding cassette transporter (ABC-transporter) type protein. The way with which this transporter is functioning to maintain the durable resistance against different types of pathogens is still unknown. v The presence of LR34 (or ABC-transporter) gene resistant allele can be detected by specially designed markers. In spite of the few DNA sequence differences between the resistant and the susceptible alleles, the easily applicable PCR based markers allow the detection of the presence of this durable resistant allele on any given variety or cultivar. In our study, 62 different Turkish bread wheat varieties were screened by the gene specific molecular markers, developed from those LR34 gene mutation sites. The 11 cultivars determined to contain the resistant allele. This study is the first screening of Turkish cultivars for the presence of LR34 gene.

The aim of this work is the accurate diagnosis of the Puccinia species of the selected turf grass species under field and laboratory conditions and evaluate influences on the intensity of occurrence. The laboratory tests will be recommended for the best stage of plants to artificial infection. The experiment was carried out in collaboration with SŠ Větrov. In the two-year follow-up (in 2011 and 2012) were at particular times of samples of infected plants, which were then evaluated microscopically. And at the same time experimental work was carried out in laboratory conditions (artificial infection) in plant material of the same origin and assessed degree assault with a precise diagnosis of the type of pathogen. Between 2011 and 2012, was higher occurence stem rust than crown rust.. From a total of 514 tests was stem rust in 379 cases, while crown rust in only 40 cases. In 95 samples was observed incidence of both simultaneously rust and mildew spores remaining plants were discovered. Greenhouse resistance tests were performed on younger plants (3 sheets) and older (6 sheets) in 16 variants, of which 4 were control. Infected leaves were spray uredispor with talc and injected with an aqueous suspension followed by looking drops and were then sealed under insulators at 23 ° C for 24 - 48 hours.

Stem rust and Yellow rust diseases are the two major wheat fungal diseases causing considerable yield losses in Turkey and all around the world. There are studies which are carried out to identify and utilize resistance sources in order to obtain resistant lines of wheat. However, virulent pathotypes are continuously being important threats to wheat production and yield. For that reason, new approaches for rapid identification are needed. The aim of this study was to investigate and to understand the structural and functional differences between the resistant and sensitive durum wheat cultivars to the plant fungal diseases of stem and yellow (stripe) rusts. To aim this, forty durum wheat recombinant inbred lines (RILs), which were previously determined to be resistant or sensitive to stem and yellow rust diseases, were investigated by the noninvasive Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy. Also, classification of the resistant and sensitive lines depending on the structural and functional differences has been attempted. The FTIR spectra for stem rust disease showed that, resistant durum wheat lines had a significant increase in the population of unsaturation in acyl chains of lipid molecules, an increase in lipid and in total protein content and also an increase in carboxylic acids and alcohols. For yellow rust disease, resistant lines had a significant increase in hydrogen bonding and they had also a more ordered membrane structure. In Principal Component Analysis for stem rust disease, according to 3700-650 cm-1 region, amide III band (1213-1273 cm-1 region) and C-H stretching region (3020- 2800 cm-1), the resistant and sensitive groups were separated successfully. For yellow rust disease, according to 3700-650 cm-1 region, Amide A and Amide III bands, the resistant and sensitive lines were grouped distinctly. FTIR spectroscopy provides a useful approach to determine the differences in molecular structure of durum wheat RILs regarding resistance of lines to fungal diseases. However, further research is still needed to ensure if the structural and functional differences in biomolecules of the samples could be used as molecular markers for discrimination of rust resistant materials from rust sensitive ones.

The aim of the thesis is evaluation of occurence of rusts on the selected types of grass, their specific diagnosis and valorization of their influence on the important agricultural qualities. The experiment was carried out in a cooperation with SS Větrov. In a two-year period (in 2009 and 2010) the samples of infested plants, which were subsequently evaluated microscopically, were taken. The ascertained results were processed through the Kruskal-Wallis test and the Independent Group T-Test. In 2009 and 2010 the occurrence of stem rust on the tested types of grass was significantly higher than occurence of crown rust. From the total number of 513 tests stem rust was found in 353 cases, whereas crown rust was found only in 103 cases. In 27 samples the occurrence of both types of rusts was found and there was no occurence found on the rest of the plants. The results show that in our climatic conditions stem rust becomes the most frequent pathogen which causes rustiness of grass. In 2010 the results of turf experiments proved that the selection of genotype of perenne reygrass with a lower infestation by stem rust had a positive impact on health conditions and better appearance of the offspring of the selected plans. Effectivenes of selection was confirmed. Among the tested materials there were chosen very predisposed genotypes which could be useful as the infectious material in the planned greenhouse tests for the resistance against rusts. Currently, we have also been abble to recognize the genotypes which are relatively resistant against particular rusts. If their resistance is confirmed in the following period, they might be used as donors of resistance in xenogamy and as immune standards in tests. The introduced results from the first stage of cooperation are only preliminary and it will be necessary to verify them at the following stages.