Browsing by Author "Ralph A. Dean, Committee Member"

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Classical and Modern Genetic Approaches Reveal New Gene Associations with Aflatoxin Biosynthesis in Aspergillus parasiticus and A. flavus.
(2006-04-11) Price, Michael Scott; Ross W. Whetten, Committee Member; Ralph A. Dean, Committee Member; Gary A. Payne, Committee Chair; Margaret E. Daub, Committee Member
Production of aflatoxin (AF) in Aspergillus species is a highly regulated process involving transcriptional and post-transcriptional controls. Most of the regulation of AF production is focused through the pathway-specific transcriptional regulator aflR. While much is understood about the steps involved in biosynthesis, less is known about the regulatory circuits controlling AF production. A targeted cDNA microarray consisting of 768 genes was developed to investigate the effect of nitrogen source, carbon source, culture temperature and culture pH on AF production in A. parasiticus. Seventeen genes were identified as consistently differentially expressed with respect to AF, including three of the AF pathway structural genes. One of these genes, CA747470 was consistently downregulated with AF and was shown to repress AF production when overexpressed in A. flavus. Using an expanded cDNA microarray consisting of 5002 genes from an EST sequencing project (USDA-ARS, SRRC), we investigated the impact of aflR deletion on the transcriptome of A. parasiticus. In addition to the AF pathway genes, five additional genes were found to be regulated by aflR: niiA, hlyC, hypA, nadA, and hypB. These additional genes all possess putative consensus binding sites for AflR. The expression data from this study was also compared to the previous targeted array study by looking at expression of 324 genes shared by both microarrays. Expression profiles for the AF genes present on both arrays were consistent between experiments. CA747470 was shown to be highly expressed in all conditions. Overexpression of CA747470 resulted in increased radial growth and decreased AF production. Finally, a putative Rho-GDP dissociation inhibitor (Afrdi1) was deleted in A. flavus that was found to share a transcription profile with aflR with respect to AF. The Afrdi1 deletion strain exhibited repressed AF production, as well as a severe growth defect on minimal medium. The deletion mutant was phenotypically similar to the bem4 deletion strain of S. cerevisiae. The implication of this gene in AF regulation provides a direct link between vegetative growth and secondary metabolism in A. flavus. This work provides insight into the regulatory networks responsible for regulation of AF production in Aspergillus species, and indicates where future investigations are needed to understand the biology of this important mycotoxin.
Inheritance of Fruit Characteristics and Disease Resistance in Watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai]
(2005-03-24) Gusmini, Gabriele; Todd C. Wehner, Committee Chair; Gerald J. Holmes, Committee Member; Janet F. Spears, Committee Member; Ralph A. Dean, Committee Member
The watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus] is a major vegetable crop in the United States. The genetics of this crop have been widely studied and several genes reported. Nevertheless, further investigation was needed for genes determining the appearance of the fruit (rind and flesh colors), the weight of single fruit, and resistance to gummy stem blight, a severe disease of watermelon caused by Didymella bryoniae (Auersw.) Rehm. In this work the inheritance of novel rind phenotypes was measured and the genetics of flesh color verified. Three new genes were identified: Dr for the deep-red flesh color of 'Dixielee' and 'Red-N-Sweet', Yb for the yellow belly of 'Black Diamond Yellow Belly', and is for the intermittent stripes of 'Navajo Sweet'. The spotted phenotype from 'Moon and Stars' was transferred to light green and gray cultivar for the development of novel varieties with distinctive rind patterns. Yield of 80 diverse cultivars was evaluated in replicated experiments. Some of the new, elite hybrid cultivars were in the top yielding group, however old, inbred cultivars appeared in the top group as well. Consistent and significant differences among the 80 cultivars tested suggests genetic variation for yield. Since most watermelon consumers are interested in smaller fruit, six adapted cultivars bearing the largest and smallest fruit were crossed in a half-diallel, producing F1, F2, and backcross generations. Genotypic variances, heritability, and gain from selection were estimated. High environmental variance and low narrow- and broad-sense heritability were recorded. Finally, the inheritance of resistance to gummy stem blight, previously attributed to the db gene, was verified. A genetic system more complex than a single gene seems to regulate the transmission of resistance from resistant to susceptible germplasm. Due to the complexity of phenotypic testing for this disease in watermelon, a new project for the development of molecular markers linked to resistance was initiated. Nevertheless, the use of F3 phenotypic data and F2 genotypic markers and the apparent complexity of the trait did not allow the identification of a tightly linked marker.
Microbial Pretreatment of Cotton Stalks by Phanerochaete chrysosporium for Bioethanol Production
(2007-07-31) Shi, Jian; Mari S. Chinn, Committee Member; Ralph A. Dean, Committee Member; Michael D. Boyette, Committee Co-Chair; Ratna R. Sharma-Shivappa, Committee Chair
Lignocellulosic biomass has been recognized as a widespread, potentially low cost renewable source of mixed sugars for fermentation to fuel ethanol. Pretreatment, as the first step towards conversion of lignocellulose to ethanol, remains one of the main barriers to technical and commercial success of the processing technology. Existing pretreatment methods have largely been developed on the basis of physiochemical technologies which are considered relatively expensive and usually involve adverse environmental impacts. In this study, an environmentally benign alternative, microbial pretreatment using Phanerochaete chrysosporium, was explored to degrade lignin in cotton stalks and facilitate their conversion into ethanol. Two submerged liquid pretreatment techniques (SmC), shallow stationary and agitated cultivation, at three inorganic salt concentrations (no salts, modified salts without Mn2+, modified salts with Mn2+) were compared by evaluating their pretreatment efficiencies. Shallow stationary cultivation with no salt was superior to other pretreatment conditions and gave 20.7% lignin degradation along with 76.3% solids recovery and 29.0% carbohydrate availability over a 14 day period. The influence of substrate moisture content (65%, 75% and 80% M.C. wet-basis), inorganic salt concentration (no salts, modified salts without Mn2+, modified salts with Mn2+) and culture time (0-14 days) on pretreatment effectiveness in solid state (SSC) systems was also examined. It was shown that solid state cultivation at 75% M.C. without salts was the most preferable pretreatment resulting in 27.6% lignin degradation, 71.1% solids recovery and 41.6% carbohydrate availability over a period of 14 days. A study on hydrolysis and fermentation of cotton stalks treated microbially using the most promising SmC (shallow stationary, no salts) and SSC (75% moisture content, no salts) methods resulted in no increase in cellulose conversion with direct enzyme application (10.98% and 3.04% for SmC and SSC pretreated samples, respectively) compared with untreated cotton stalk samples (17.93%). Washing of pretreated cotton stalks alone caused no significant increase in cellulose conversion. However, a heat treatment (autoclaving) followed by washing remarkably improved (P<0.05) cellulose conversion to 14.94% and 17.81% for SmC and SSC pretreatment, respectively. Mathematical models describing holocellulose consumption, lignin degradation, cellulase and ligninolytic enzyme production, and oxygen uptake associated with the growth of P. chrysosporium during 14 days fungal pretreatment were developed. For SmC pretreatment, model parameters were estimated by nonlinear regression and validated using an independent set of experimental data. Models yielded sufficiently accurate predictions for holocellulose consumption (R2=0.9772 and 0.9837, 1d and 3d oxygen flushing, respectively), lignin degradation (R2=0.9879 and 0.8682) and ligninolytic enzyme production (R2=0. 8135 and 0.9693) under both 1 and 3 d oxygen flushing conditions. However, the prediction capabilities for fungal growth (1d and 3 d), cellulase production (3d) and oxygen uptake (3d) were limited. For SSC, the models were established in three phases (I: day 0-4, II: day 4-7, III: day 7-14). After validation it was shown that the developed models can yield sufficiently accurate predictions for fungal growth (R2 =0.9724), holocellulose consumption (R2 =0.9686), lignin degradation (R2=0.9309) and ligninolytic enzyme production (R2=0.9203); however predictions of cellulase production were fair (R2=0.6133). Although significant delignification occurred during fungal pretreatment indicating the presence of ligninolytic enzymes, common spectrophotometric enzyme assays failed to detect lignin peroxidase (LiP) and manganese peroxidase (MnP) activities in fungal pretreatment cultures. Efforts were made to overcome the drawbacks of standardized assays by performing protein gel electrophoresis and crude enzyme delignification studies. Results from this research are expected to be beneficial in the development of pretreatment technologies that are environment friendly and utilize naturally occurring microorganisms.