Graduate Thesis Or Dissertation
 

Biological and molecular characterization of wild type parental and nonpathogenic mutant strains of Pseudomonas syringae pathovars phaseolicola and syringae

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  • The in planta growth of three wild type strains of phytopathogenic bacteria and three nonpathogenic mutant strains was studied to determine if mutations eliminating the ability of the mutant strains to cause disease had affected their growth in leaf tissue. The mutant strains were obtained by Tn5 mutagenesis of Pseudomonas svringae pv. phaseolicola strain PP7010 and P. svringae pv. svringae strain PS9020, organisms which respectively cause halo blight of bean and bacterial brown spot of bean. Through cloning of Tn5-containing genomic DNA, bacterial conjugation, auxanography, and genetic marker exchange experiments, these studies demonstrated that the Tn5 mutation in strain PP7014 of P. svringae pv.. phaseolicola had created an auxotrophic requirement for uracil and had caused an inability to produce typical necrotic and chlorotic disease symptoms in bean leaves. A simple and reproducible bioassay was developed to monitor bacterial growth in leaf tissue. Using this bioassay, mutant PP7014 was shown to be incapable of growth in planta, presumably due to its inability to obtain uracil for growth in the leaf. However, a similar Tn5 arginine auxotroph, PP7510, was capable of multiplication in bean leaves, demonstrating that prototrophy of P. svringae pv. phaseolicola is not a prerequisite for pathogenicity. Growth studies with pathogenic wild type P. syringae pv. syringae strains J900 and PS9020 revealed a correlation between growth in planta and the severity of symptom expression. Altered growth patterns and symptom expression were shown for two nonpathogenic strains derived by Tn5. mutagenesis of PS9020. Mutant strain PS9021 was incapable of in planta growth and disease symptom expression. Mutant strain PS9024, although initially capable of rapid multiplication in bean leaves, was unable to maintain the high population levels and produce the characteristic symptoms of the parental wild type strain. Further growth studies were conducted to investigate the complementation of the Tn5 mutation in PS9021 by cosmids containing wild type PS9020 DNA homologous to the mutated region. Although stable throughout multiple rounds of bacterial replication, the cosmids could partially, but not completely, restore the wild type in planta growth and symptom expression pattern to PS9021.
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