Browsing by Author "Marc A. Cubeta, Committee Member"

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Alternaria Alternata Mannitol Metabolism in Plant-pathogen Interactions
(2006-12-30) Velez, Heriberto; Marc A. Cubeta, Committee Member; Gary A. Payne, Committee Member; Margaret E. Daub, Committee Chair; Greg G. Upchurch, Committee Member
Mannitol is purported to have role in fungi as a storage carbohydrate and has been shown to quench reactive oxygen species (ROS) both in vitro and in vivo. Mannitol metabolism in fungi is thought to occur through the mannitol cycle, which was proposed in the late 1970's from studies of cell free extracts of the fungus Alternaria alternata. In this cycle, mannitol 1-phosphate 5-dehydrogenase (MPDH; EC 1.1.1.17) reduces fructose 6-phosphate into mannitol 1-phosphate, which is dephosphorylated by a mannitol 1-phosphatase (EC 3.1.3.22) resulting in mannitol and inorganic phosphate. Mannitol also can be made through the enzyme mannitol dehydrogenase (MtDH; EC 1.1.1.138), which reduces fructose to mannitol. Here we report confirmation of these enzymes in the fungus A. alternata, the isolation of the genes, and the generation of strains mutated in MPDH, MtDH, or both genes. PCR confirmed gene replacement and enzyme assays using these mutants showed no activity for MtDH or MPDH. GC-MS analysis showed that double mutants did not produce mannitol, while single mutants had reduced mannitol production. Mannitol, as a quencher of ROS, may also have a role in host-pathogen interactions, by allowing the fungus to suppress ROS-mediated plant defense responses. To assess the contribution of mannitol in plant-pathogen interactions, wild type, single and double mutants were used in pathogenicity assays on tobacco plants. Severity of lesions caused by the MtDH disruptant was not significantly different from that of the wild type. By contrast, the MPDH disruptant and the double mutant caused significantly less disease. Microscopy analysis and histochemical staining for H2O2 showed that both the wild type strain and the double mutant were able to germinate, produced appressoria, and elicited a defense response from the host. Quantitative PCR studies showed that genes for both enzymes were upregulated in the presence of tobacco extracts, with MPDH having a stronger response. We conclude that mannitol biosynthesis is required for pathogenesis of A. alternata on tobacco, but is not required for normal spore germination either in vitro or in planta or for initial infection.
Biology and Management of Fairy Rings on Golf Putting Greens
(2010-04-23) Miller, Gerald Leo Jr; Lane P. Tredway, Committee Co-Chair; Larry F. Grand, Committee Co-Chair; Marc A. Cubeta, Committee Member; Michael A. Fidanza, Committee Member; D. Michael Benson, Committee Member
Fairy ring is a severe disease problem on golf putting greens in North Carolina and throughout the United States. Pathogen identification is difficult and very little is known about the etiology and management of this disease. Basidiocarps and soil samples were collected from 15 bermudagrass and 30 bentgrass greens exhibiting fairy ring symptoms in CA, FL, HI, IL, OK, NC, SC and WI. Genomic DNA was extracted from 122 total samples. Extractions were made from mycelium isolated from puffball or mushroom context tissue, from mycelium isolated from a soil block, or through direct DNA extraction from infested soil. DNA was also extracted from 16 type isolates for comparison to unknowns. The internal transcribed spacer (ITS) region of ribosomal (r)DNA was amplified and sequenced using the basidiomycete-specific primer sets ITS1f/ITS4b and Basid0001/2R. Most ITS sequences grouped into one of three clades corresponding to species within the Family Lycoperdaceae: Arachnion album, Bovista dermoxantha, and Vascellum curtisii. This is the first report of Arachnion album in association with fairy ring formation. Molecular identification was confirmed with morphological characterization of mature basidiocarps. Although many basidiomycete fungi have been associated with fairy ring on turfgrass, only 5 fairy ring forming species were detected in this survey of golf putting greens. Soil DNA extraction and a PCR detection assay was also specifically evaluated for diagnosis and identification of fairy ring pathogens. Genomic DNA was extracted from five soil samples from putting greens infested with fairy ring. The ITS region was amplified and directly sequenced from soil DNA extracts, yielding a consensus ITS sequence for four of the five samples. Four to eight cloned PCR fragments were sequenced per sample. Clones showed high sequence similarity (99%) to directly sequenced ITS fragments, indicating homogeneity in the resultant amplicon sequence. Target rDNA sequences were amplified successfully using primers specific to Vascellum curtisii and Bovista dermoxantha from both extracted soil DNA and DNA from known isolates. However, several false positive amplifications also occurred. Due to this variability in PCR detection assays, sequencing of extracted soil DNA may be a more viable alternative for detecting and identifying fairy ring pathogens. Field experiments were conducted to evaluate the impact of application rate, application timing, fungicide + surfactant tank-mix, and irrigation timing on the efficacy of preventive DMI fungicide applications for fairy ring control. No statistical difference was observed between the level of control afforded by single applications of low and high rates of triadimefon and tebuconazole. Single applications suppressed fairy ring early in the season, but did not provide season long control. No statistical difference was detected between application timing events based on average 5-day soil temperature thresholds, but a data trend showed lower disease severity in plots treated with fungicide at 13Â°C and 16Â°C. Two spring applications of triadimefon or triticonazole provided less residual control when tank-mixed with a surfactant than when applied alone. Irrigation timing treatments did not affect preventive fungicide control. An in vitro mycelial growth assay was used to determine the sensitivity [% 50 effective concentration (EC50)] of 16 isolates representing major fairy ring species to the fungicides flutolanil, propiconazole, tebuconazole, triadimefon, and triticonazole. No significant differences in fungicide sensitivity were detected among fairy ring species. Differences in in vitro sensitivity of isolates were detected among fungicides. Isolates were most sensitive to tebuconazole (EC50 = 0.149) and triticonazole (EC50 = 0.263), moderately sensitive to propiconazole (EC50 = 0.371) and flutolanil (EC50 = 0.546) and least sensitive to triadimefon (EC50 = 0.821).
Structural, Functional and Evolutionary Analyses of the Rice Blast fungal Genome
(2007-03-11) Deng, Jixin; Ralph A. Dean, Committee Chair; Gary A. Payne, Committee Member; Marc A. Cubeta, Committee Member; Jeffrey L. Thorne, Committee Co-Chair
Disease caused by rice blast results in a dramatic loss of rice production worldwide every year. The rice blast fungus, Magnaporthe oryzae, has long been the subject of intense investigation and has been adopted as a model system to elucidate molecular host-pathogen interactions. In recent years, a major initiative to decode the genome of this fungus was launched, which has greatly shaped current biological studies. This dissertation contains three projects developed to help with genome sequencing as well as analyses to explore genome function and evolution. The first project involved functional characterization of ACI1. This gene was identified in a yeast two-hybrid experiment to interact with MAC1, a key regulator of the infection process. However, deletion of ACI1 had no obvious effect on pathogenesis. The second project validated and refined the previously constructed physical map of chromosome 7 in preparation for a map based sequencing project. One hundred and forty out of the 155 non-repetitive DNA-containing ESTs were confirmed to be chromosome 7 anchored. Furthermore, 5 previously unanchored FPC contigs were anchored to chromosome 7. Following the release of the whole genome sequence and other related fungi, an evolutionary analysis of the cytochrome P450 superfamily was undertaken as a third project. Cytochrome P450s form a large protein superfamily, representing ˜ 1% of predicted ORFs and are present in many organisms from bacteria to human. The functions of P450 enzymes are very varied. In fungi, they are involved in detoxifying plant defense compounds as well as being required for biosynthesis of pathogenesis related toxins. Two plant pathogenic fungi Fusarium graminearum and M. oryzea with two saprophytic fungi Aspergillus nidulans and Neurospora crassa were chosen to compare their P450omes. Due to considerable sequence divergence that hampered application of standard phylogenetic approaches, a novel approach was developed to reconstruct their evolution. The results suggested that the ancestral genome of the four fungi contained similar number of P450s as found in current filamentous ascomycetes. As a general trend, during evolution, new lineage specific P450 have appeared with a corresponding loss of genes from ancestral clans. M. oryzae exhibited a larger expansion with less contraction compared to the other three fungi. In particular genes were expanded or maintained in two P450 clans that may be involved in the alkane and fatty acid assimilation.