Efforts to Characterize the Genome Organization and Gene Expression Patterns of the Cotton Root Rot Fungus, Phymatotrichopsis Omnivora

Several isolates of the fungus Phymatotrichopsis omnivora from cotton and alfalfa were used in attempts to study the fungus' genome structure and expression profiles of twenty-six genes during different growth stages. Pulsed field gel electrophoresis (PFGE), telomere fingerprinting, nucleic acid staining, and real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were employed to investigate the study's objectives. Efforts to characterize the genome of P. omnivora using PFGE and telomere fingerprinting were unsuccessful. However, chromosomal standards were separated by PFGE in two experiments. We confirmed the multinucleate nature of our P. omnivora isolates using fluorescent nucleic acid staining and microscopy, which showed younger hyphae have fewer nuclei than older hyphae and newly branching hyphal cells contained up to 24 nuclei. The gene ontology (GO) categorization and life stage expression patterns of twenty-six different genes were followed using qRT-PCR. However, because cDNAs were prepared using two different kits, sample bias prevented comparisons of gene expression across treatments. Of four housekeeping genes examined, expression of ITS-1 rRNA was most consistent across treatments and was used as an internal control to normalize expression of all other genes. Gene expression levels differed among the life stages of P. omnivora investigated. In vegetative mycelia, two virulence-related genes appeared repressed, while individual genes in all categories were highly expressed. In mycelia challenged with host (Medicago truncatula) or nonhost (Sorghum bicolor) root exudates, some genes involved in secondary metabolism, virulence and glycogen synthesis were expressed higher with nonhost root exudates. In sclerotia, genes involved in glycogen biosynthesis and secondary metabolism were expressed higher in 4 week-old sclerotia than 8 week-old sclerotia. Unfortunately, due to the high level of error, all gene expression data can only be considered preliminary. However, these findings point to interesting new directions for future gene expression and genomics studies comparing P. omnivora with other fungal pathogens and related fungi in the Pezizomycetes.Department of Entomology and Plant Patholog