Ph.D. - Tropical Plant Pathology
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ItemScreening genes encoding transcription factors associated with pathogenesis in alternaria brassicicola([Honolulu] : [University of Hawaii at Manoa], [May 2013], 2013-05)The necrotrophic fungus Alternaria brassicicola causes black spot disease of brassicaceous plants, including green cabbage (Brassica oleracea) and the oil-producing B. napus. Pathogenesis is a multistep process that includes germination, penetration and colonization of host tissues, and survival structure formation. The de novo synthesis of various catabolic enzymes and secondary metabolites needed during each step in pathogenesis are under the regulation of transcription factors. Zinc finger DNA-binding domains containing transcription factor forms the largest family of transcription factors in eukaryotes. Based on the zinc binding motifs transcription factors are of three classes: Cys2-His2, Cys2-Cys2, and the fungal specific, binuclear Zn(II)2Cys6His6. In the present study we made knock out mutants of all 184 C2H2 Zinc finger transcription factor encoding genes by targeted gene disruption and 35 Zn(II)2Cys6His6 motif containing transcription factors through targeted gene deletion/replacement in Alternaria brassicicola. Our bioassays on detached leaves of green cabbage have identified twelve genes associated with early and late stages of plant infection. Among these twelve genes, two genes are pathogenicity factors as their mutants were nonpathogenic, while one showed increase in pathogenicity by almost 100%. Remaining nine genes were strong virulence factors whose mutants showed 50-90% reduction in disease symptoms compared to the wild type. We also discovered a unique gene whose mutants showed complete loss of conidia yet no effect on pathogenesis. We further report a gene encoding transcription factor associated with detoxification of phytoalexins, which is important for early plant colonization. All the discovered pathogenesis associated genes were novel pathogenicity or virulence factors and only one gene (PacC) was previously identified as a pathogenicity factor in other fungal species. The molecular mechanisms of pathogenesis and their regulation in necrotrophic fungi are in an early stage of research. This study sets the platform for discovery of downstream genes associated with pathogenesis and the characterization of their functions. Currently available data from this study indicate the importance of transcription factors as regulators of pathogenesis and as future targets for disease management.
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ItemStudy of koa wilt disease : characterization of acacia koa and fusarium oxysporum([Honolulu] : [University of Hawaii at Manoa], [December 2011], 2011-12)Koa (Acacia koa) is a highly valuable tree species in Hawaii. Over the past few decades however, the species has suffered from a severe wilt and dieback disease. The goal of this study is to manage the disease and improve koa plantation health. Fungal species were isolated from koa trees showing typical dieback symptoms, and species of these isolates were identified. Fusarium pseudocircinatum was recovered from the samples and its virulence to koa was confirmed with Koch's postulates. This is the first report of this fungal species in Hawaii. The population structure of F. pseudocircinatum and F. oxysporum collected from the samples were analyzed with pathogenicity tests, vegetative compatibility group (VCG) tests, and Amplified Fragment Length Polymorphism (AFLP) analyses. Forty-six isolates including F. oxysporum and F. pseudocircinatum were grouped into 16 VCGs. Among the highly virulent isolates, 86% belonged to the single VCG group, and these isolated clustered together in AFLP analyses. VCG and AFLP could be used for detection and identification of F. oxysporum strains in soils, and therefore would help koa growers to establish new koa plantations. VCG2 of F. oxysporum is a significant biological entity for which the name F. oxysporum f. sp. acaciae is proposed to reflect its virulence on koa. Pathogenicity tests conducted on 18 koa families revealed genetic variations in wilt tolerance among koa families. Mortality rates of the 18 families inoculated with a highly virulent F. oxysporum f. sp. acaciae isolate ranged from 0 % to 100 %. However, a strong interaction, P < 0.0001, was observed when host-pathogen interaction among five koa families and ten F. oxysporum f. sp. acaciae isolates were tested. This result indicates that each koa family and F. oxysporum isolate behave differently depending on their host family/pathogen combination. When koa mortality data from the field trials and pathogenicity tests were compared, no direct correlation was observed (correlation coefficient =-0.013). Understanding the host-pathogen relationship between koa and F. oxysporum and trends in koa mortality in the field trials will ultimately lead to the establishment of more effective disease screening protocols for koa silviculture in Hawaii.
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ItemEcologically-based nematode management : exploiting nematode survival strategies for developing novel cover cropping and soil solarization practices([Honolulu] : [University of Hawaii at Manoa], [August 2011], 2011-08)Improving the use of cover crops, such as Crotalaria juncea and Tagates patula, is needed for management of plant-parasitic and free-living nematodes. These cover crops might improve soil health conditions through a strip-till cover cropping practice. The integration of cover cropping of C. juncea and soil solarization could further improve ecological-based nematode management more than C. juncea alone. Additional improvement could be achieved if T. patula or C. juncea are found to suppress Meloidogyne incognita or Rotylenchulus reniformis more efficiently when these nematodes are in a vulnerable state. Strip-till cover cropping (STCC) of C. juncea suppressed M. incognita, but T. patula only suppressed M. incognita when planted immediately following after a susceptible host. In addition, STCC of C. juncea also enhanced population densities of bacterivorous and fungivorous nematodes, whereas STCC of T. patula enhanced these free-living nematodes inconsistently. Integration of C. juncea cover cropping and soil solarization did not reduce R. reniformis opulation densities, but reduced weed coverage as compared to C. juncea alone. The benefits of integrating C. juncea to soil solarization were only observed where C. juncea biomass was high (3.6 Mt/ha). Leachate of T. patula did not suppress egg hatch and J2 activity of M. incognita. However, planting of T. patula in a greenhouse pot experiment suppressed M. incognita J2 activity if the soil was conditioned with irrigation or cucumber leachate but not with dry treatment. On the other hand, planting of C. juncea and cowpea (Vigna unguiculata) reduced the number of R. reniformis in anhydrobiotic state. Amending soil with C. juncea suppressed R. reniformis more efficiently if fewer anhydrobiotic R. reniformis were present. In conclusion, C. juncea outperformed T. patula in a STCC system, whereas integration of C. juncea with soil solarization did not improve the benefit of a cover crop, C. juncea. Based on the greenhouse studies, the nematode suppressive effect of T. patula could be improved by planting T. patula immediately after a susceptible host. Benefits of C. juncea amendment were more obvious if used immediately after nematode susceptible (poor or good) hosts.