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published_or_final_version / Botany / Master / Master of Science

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Doctor of Philosophy / Department of Plant Pathology / Harold N. Trick / Bread wheat (Triticum aestivum L.) is an important staple crop for 35% of the world’s population. One economically important pathogen of wheat is Puccinia triticina, the causal agent of leaf rust, can cause up to 50% yield loss during epidemics. Despite the lack of an alternate host to complete the sexual stages, P. triticina still has variation within the population, which can make achieving durable resistance difficult. This study aims to gain a better understanding of the P. triticina-wheat interaction by identifying wheat genes that are induced by individual and multiple races. Six P. triticina races were evaluated on a susceptible variety of wheat at six days post inoculation. RNA was sequenced and 63 wheat genes were identified that showed varying expression in response to the six P. triticina races. Fifty-four wheat genes were characterized during the first seven days of infection using real-time PCR. Race specific gene expression was found in three wheat genes with race differences on Lr2A, Lr2C, and Lr17A. Wheat genes that had similar expression in response to all six races were also identified. Seven of the characterized genes were then silenced using RNAi hairpin constructs. The transgenic plants were molecularly characterized and inoculated with a virulent P. triticina race in the T₂ generation. However, the endogenous genes were not silenced and the transgenic plants maintained susceptibility. A mutation approach was also used to identify wheat genes involved in infection. A mutant population of 3780 wheat plants was created using EMS. Fifteen hundred mutants from the M1 population were screened for plants with a different infection phenotype compared to the non-mutated control and 570 were selected. After two additional generations of selection, eight resistant mutants were obtained. The gene expression of the seven previously identified genes were evaluated and one mutant showed reduced expression of an ER molecular chaperone gene. This research uses a forward and reverse genetics approach to identify and evaluate the function of wheat genes in the wheat-P. triticina interaction. Although RNAi could not determine the gene function, the knockout mutant shows that the identified genes may have a crucial role in infection.

Green rusts (GRs), a group of layered Fe(II)-Fe(III) hydroxide salts, have been observed to be effective reductants for degrading organic and inorganic contaminants under suboxic conditions. Furthermore, the addition of a transition metal to GRs can produce high-activity modified green rusts (HMGRs) that demonstrate higher degradation rates. Methods of modifying GRs to obtain high reactivity for degradation of PCE and nitrate were developed and reduction kinetics of PCE and nitrate by HMGRs were characterized in this study. First, the most promising HMGRs were developed through screening tests. GRs modified with Pt, Cu, Ag, or Pb were found to be effective in improving degradation rates of PCE. GR-F(Pt) and GR-F(Cu) were chosen because they showed high reactivity and produced non-chlorinated by-products. Pt and Cu showed the capability of improving reduction kinetics of nitrate by GRs. GR-F(Pt) and GR-F(Cu) were selected for further study. Second, degradation of PCE by GR-F(Cu) and GR-F(Pt) was characterized using a batch reactor system. The reaction kinetics of PCE degradation by GR-F(Cu) and GR-F(Pt) was strongly dependent on pH over the range of pH 7.5-11, with the fastest rate at pH 11. Increasing concentrations of Cu(II) over the range of 0 to 5 mM resulted in improving the reduction kinetics by a factor of more than 400, although the rate at 7.5 mM of Cu(II) was unexpectedly lower than that at 5 mM. Surface saturation behavior was observed in the rates of dechlorination of PCE by GR-F(Cu). Finally, nitrate reduction by GR-F(Cu) and GR-F(Pt) was further studied to determine the effects on degradation rates of pH, Cu(II) addition, and initial nitrate concentration. A reaction model with four sequential steps was proposed to describe the process of nitrate being reduced to ammonium and GR being oxidized to magnetite. The reaction rates of nitrate reduction by GR-F(Cu) and GR-F(Pt) was highest at pH 9. The reaction rates of GR-NO3 were improved by three orders of magnitude when Cu(II) was added in the range of 0 to 2.5 mM, while reaction rate decreased at concentrations above 2.5 mM. Saturation behavior was also observed in nitrate reduction by GR-F(Cu).

Wheat (Triticum aestivum L. em. Thell, 2n=6x=42, AABBDD) is subjected to significant yield losses by the endemic leaf rust pathogen, Puccinia triticina (Roberge ex Desmaz. F. sp. tritici). Breeding for resistance to this disease is a more appropriate option both environmentally and economically over fungicidal application. More than 57 leaf rust resistance genes in wheat have been identified and many of the resistance genes have been successfully introgressed into resistant cultivars, yet the continuous shifting of predominant races of P. triticina continues to be a challenge to breeders. Pyramiding multiple resistance genes into a single resistant cultivar is one of the preferred strategies to develop superior disease resistant cultivars. Efficient pyramiding requires the utilization of markers closely linked to the resistance genes. The objectives of this study were to characterize a novel source of resistance to leaf rust introgressed into the breeding line WX93D180-R-8-1, to determine its inheritance, map position, and linkage with molecular markers suitable for marker assisted selection. According to the pedigree of WX93D180, TX86D1310*3/TTCC417, the resistance in this breeding line should be derived from TTCC417 (Turkey tritici cereal collection), which was thought to be Triticum monococcum, which is a diploid species made up of only the A genome. However, our marker analyzes results indicated the resistance gene is located in the D genome and has the same location as the cloned leaf rust resistance gene Lr21. We verified the result in our population using primers from Lr21 and found the same segregation pattern with the phenotypic data (disease response). Therefore the pedigree is incorrect, TTCC417 was misidentified, or the resistance was not from TTCC417.

Green rusts (GRs), a group of layered Fe(II)-Fe(III) hydroxide salts, have been observed to be effective reductants for degrading organic and inorganic contaminants under suboxic conditions. Furthermore, the addition of a transition metal to GRs can produce high-activity modified green rusts (HMGRs) that demonstrate higher degradation rates. Methods of modifying GRs to obtain high reactivity for degradation of PCE and nitrate were developed and reduction kinetics of PCE and nitrate by HMGRs were characterized in this study. First, the most promising HMGRs were developed through screening tests. GRs modified with Pt, Cu, Ag, or Pb were found to be effective in improving degradation rates of PCE. GR-F(Pt) and GR-F(Cu) were chosen because they showed high reactivity and produced non-chlorinated by-products. Pt and Cu showed the capability of improving reduction kinetics of nitrate by GRs. GR-F(Pt) and GR-F(Cu) were selected for further study. Second, degradation of PCE by GR-F(Cu) and GR-F(Pt) was characterized using a batch reactor system. The reaction kinetics of PCE degradation by GR-F(Cu) and GR-F(Pt) was strongly dependent on pH over the range of pH 7.5-11, with the fastest rate at pH 11. Increasing concentrations of Cu(II) over the range of 0 to 5 mM resulted in improving the reduction kinetics by a factor of more than 400, although the rate at 7.5 mM of Cu(II) was unexpectedly lower than that at 5 mM. Surface saturation behavior was observed in the rates of dechlorination of PCE by GR-F(Cu). Finally, nitrate reduction by GR-F(Cu) and GR-F(Pt) was further studied to determine the effects on degradation rates of pH, Cu(II) addition, and initial nitrate concentration. A reaction model with four sequential steps was proposed to describe the process of nitrate being reduced to ammonium and GR being oxidized to magnetite. The reaction rates of nitrate reduction by GR-F(Cu) and GR-F(Pt) was highest at pH 9. The reaction rates of GR-NO3 were improved by three orders of magnitude when Cu(II) was added in the range of 0 to 2.5 mM, while reaction rate decreased at concentrations above 2.5 mM. Saturation behavior was also observed in nitrate reduction by GR-F(Cu).

Wheat (Triticum aestivum L. em. Thell, 2n=6x=42, AABBDD) is subjected to significant yield losses by the endemic leaf rust pathogen, Puccinia triticina (Roberge ex Desmaz. F. sp. tritici). Breeding for resistance to this disease is a more appropriate option both environmentally and economically over fungicidal application. More than 57 leaf rust resistance genes in wheat have been identified and many of the resistance genes have been successfully introgressed into resistant cultivars, yet the continuous shifting of predominant races of P. triticina continues to be a challenge to breeders. Pyramiding multiple resistance genes into a single resistant cultivar is one of the preferred strategies to develop superior disease resistant cultivars. Efficient pyramiding requires the utilization of markers closely linked to the resistance genes. The objectives of this study were to characterize a novel source of resistance to leaf rust introgressed into the breeding line WX93D180-R-8-1, to determine its inheritance, map position, and linkage with molecular markers suitable for marker assisted selection. According to the pedigree of WX93D180, TX86D1310*3/TTCC417, the resistance in this breeding line should be derived from TTCC417 (Turkey tritici cereal collection), which was thought to be Triticum monococcum, which is a diploid species made up of only the A genome. However, our marker analyzes results indicated the resistance gene is located in the D genome and has the same location as the cloned leaf rust resistance gene Lr21. We verified the result in our population using primers from Lr21 and found the same segregation pattern with the phenotypic data (disease response). Therefore the pedigree is incorrect, TTCC417 was misidentified, or the resistance was not from TTCC417.

Thesis (M.S. in plant pathology)--Washington State University, December 2008. / Title from PDF title page (viewed on Sept. 23, 2008). "Department of Plant Pathology." Includes bibliographical references.