Ph.D. - Tropical Plant Pathology

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    ( 2021) Boluk, Gamze ; ARIF, MOHAMMAD ; Tropical Plant Pathology
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    ( 2021) Olmedo Velarde, Alejandro ; Melzer, Michael J. ; Tropical Plant Pathology
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    Screening genes encoding transcription factors associated with pathogenesis in alternaria brassicicola
    ([Honolulu] : [University of Hawaii at Manoa], [May 2013], 2013-05) Srivastava, Akhil
    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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    Study of koa wilt disease : characterization of acacia koa and fusarium oxysporum
    ([Honolulu] : [University of Hawaii at Manoa], [December 2011], 2011-12) Shiraishi, Ayami
    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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    Ecologically-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) Marahatta, Sharadchandra Parasar
    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.
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    Developing a qPCR-based molecular technique for nematode community analysis
    ([Honolulu] : [University of Hawaii at Manoa], [May 2012], 2012-05) Wang, I-Chin
    Nematodes are good indicators for soil health. However performing nematode community analysis is laborious and technically challenging. This research seeks to develop a qPCR-based molecular tool for nematode community analysis. qPCR detecting 18S rDNA offered one approach to identify and quantify free-living nematodes. Owing to too many unpredictable nematode genera across soil ecosystems, one strategy is to develop universal qPCR markers selective for key nematode guilds (Ba1, Ba2, F2, P4, Om4, Om5, and P5) that are most critical for nematode faunal analysis. Universal qPCR primers were successfully being identified for all of these guilds except for Ba1. Two primers were needed for F2; Om4, Om5, and P5 cannot be differentiated and were thus being combined as Om4/ Om5/ P5 by one primer. These primers were then verified by BLAST and then run through artificial nematode mixture sample composed with known nematode guilds. The results confirmed the validity of these universal primers. The next logical step was to run these qPCR to nematodes collected from four natural ecosystems: forest, organic, pineapple field, and beach sites. Visual nematode identification on these four systems was being conducted to compare results. Two qPCR standard curves (plasmid DNA and genomic DNA) were used to obtain nematode abundance of the four ecosystems. Since both DNA standard curves did not estimate nematode abundance comparable to the visual count, ranking of nematode community indices of the four ecosystems were compared between molecular and the visual methods. While the ranking calculated by the plasmid DNA standard curve of qPCR assay were not consistent with most of the nematode community indices calculated by visual method, 4 out of 8 nematode indices estimated by the gDNA standard curve were relatively consistent. This research provided universal nematode guild qPCR primer sets and initial protocol of qPCR-based molecular tool for soil nematode community analysis. Further research need to be conducted on better estimation of nematode abundance, richness and diversity. More universal primers selective for Ba1, Ba3, F3, P3, also Om4, Om5, and P5 individual primers are needed.
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    Characterization and management of different Fusarium species associated with orchids cultivated in Hawaiʻi.
    ([Honolulu] : [University of Hawaii at Manoa], [May 2014], 2014-05) Srivastava, Shikha
    Commercially grown orchids are among the most valuable ornamental crops produced in the United States. Hawaii is one of the three largest orchids-growing states in the nation, producing the greatest variety of tropical species and unique intergeneric hybrids. A decrease in orchid production has been observed in Hawaii in association with spot and blights of shoots and flowers, as well as root rot that result in rapid death of juveniles and slow decline of mature plants. Such decline is now commonly observed in nearly all nurseries across the Oahu and the Island of Hawaii. Fungal pathogens are speculated to be one of the important sources of diseases in orchids in Hawaii. In the present study 16 fungal genera were isolated from 60 plant samples collected from Oahu and the Island of Hawaii. Fusarium was the most prevalent genus, with 78% of the isolates and frequently associated with several orchid cultivars, including over 9 genera and hybrids. Eleven Fusarium species, namely F. proliferatum, F. oxysporum, F. solani, F. subglutinans, F. poae, F. begoniae, F. bulbicola, F. anthophilum, F. denticulatum, F. circinatum and F. semitectum were found. Fusarium oxysporum was identified and was the most commonly isolated species from all the samples followed by F. proliferatum and F. solani. All Fusarium species were tested for pathogenicity on four orchid genera. Among the 11 Fusarium species, 6 were found pathogenic on 4 orchid genera. Three species, F. proliferatum, F. oxysporum and F. solani, showed moderate to high virulence on Dendrobium, Cymbidium and Miltonia. Fusarium circinatum and F. poae were low to moderate virulent on Dendrobium and Cymbidium whereas F. begoniae caused very low virulence on Dendrobium and Miltonia. Cattleya was susceptible to F. oxysporum only. Pathogenicity assays of Fusarium isolates on different cultivars of orchids resulted in identification of pathogenic Fusarium species was followed by testing efficacy of three fungicides, Pyraclostrobin, a.i. at 25%, Azoxystrobin a.i. at 50% and Triticonazole, a.i. at 20% on mycelial inhibition on four most prevalent Fusarium species namely-F. proliferatum, F. oxysporum, F. solani and F. subglutinans. Triticonazole was the most effective fungicide in the in vitro tests and was further evaluated for its efficacy in on Dendrobium plants in vivo against the most aggressive species of Fusarium, F. proliferatum under greenhouse conditions. This dissertation provides a thorough study to identify pathogenic Fusarium species on orchids and provides information regarding pathogenicity on orchid genera other than Dendrobium, and viable chemical methods to manage Fusarium species on orchids in Hawaii.