Browsing by Author "David Shew, Committee Co-Chair"

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    Biology and Management of Pythium Root Dysfunction in North Carolina
    (2008-04-24) Kerns, James Patrick; Lane Tredway, Committee Co-Chair; David Shew, Committee Co-Chair; Mike Benson, Committee Member; Tom Rufty, Committee Member
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    Evolutionary History and Population Dynamics of the M2 Double-stranded RNA of Rhizoctonia solani
    (2008-04-26) Charlton, Nikki De-Roi; Marc Cubeta, Committee Chair; David Shew, Committee Co-Chair; Mike Benson, Committee Member; Gary Payne, Committee Member
    M2 is a 3.57 kb double-stranded RNA (dsRNA) mycovirus first identified and sequenced from the soil fungus Rhizoctonia solani anastomosis group 3 (AG-3). This dsRNA has been hypothesized to regulate metabolic pathways associated with the parasitic and saprobic activity of the fungus. Isolates from a well-characterized field population of R. solani AG- 3 obtained from potato were examined for the M2 dsRNA. The detection frequency was 51.3%. The M2 dsRNA was isolated from 59 isolates and reverse-transcription PCR products were obtained for three genomic regions of the M2 dsRNA to examine genetic diversity, reconstruct their evolutionary history, and identify distinct evolutionary lineages. Site compatibility analysis performed using SNAP Workbench for clades in the M2 dsRNA strict consensus tree identified recombination events among clades. Recombination limited the genealogical analyses to specific haplotypes with non-recombining partitions in each data set. The reconstructed genealogies indicated differential selection pressures among the genome. Analysis of the ratio of nonsynonymous substitutions to synonymous substitutions concordantly revealed that regions within the genome are undergoing differential selection. Isolates from closely related fungi in the Rhizoctonia species complex were examined for the M2 dsRNA. M2 was detected in representative isolates belonging to three AG of R. solani (AG-1-IA, AG-4, and AG-6; teleomorph=Thanatephorus) and four AG of binucleate Rhizoctonia (AG-A, AG-F, AG-R, and AG-U; teleomorph=Ceratobasidium) using RT-PCR. Amplified PCR products from a representative sample of 12 isolates from eight different AG were sequenced and subjected to phylogenetic analysis and coalescent simulations to infer the ancestral lineage and determine the genetic relationship among these dsRNAs. Phylogenetic analysis of M2 dsRNA sequence data resulted in seven inferred haplotypes and there was no unique association with AG to support co-evolution of the M2 dsRNA haplotype within the fungal host. Based on coalescent analyses and the inferred genealogy, the ancestral M2 dsRNA haplotype likely evolved in R. solani AG-1-IA and has recently been acquired by isolates of Ceratobasidium. To our knowledge, this is the first report of a dsRNA occurring in isolates of binucleate Rhizoctonia that are also present in isolates of R. solani. Horizontal transmission of the M2 dsRNA between mycelia of somatically incompatible isolates of R. solani AG-3 was investigated. Eight donor isolates of R. solani AG-3 containing the M2 dsRNA were paired on potato dextrose agar with each of three different recipient isolates where the M2 dsRNA was absent. RT-PCR was used to detect horizontal transmission of the M2 dsRNA via hyphal anastomosis from donor to recipient isolates by examining hyphal explants taken 3-cm from the hyphal interaction zone. PCR-RFLP genetic-based markers of two nuclear loci and one mitochondrial locus were used to confirm identity and transmission between donor and recipient isolates of R. solani AG-3. The frequency of transmission observed between 72 pairings of the eight donor and three recipient isolates ranged from 0-33% and differences in the phenotype of the recipient isolates after acquisition of the M2 dsRNA via horizontal transmission were observed. To our knowledge, this represents the first demonstration of transmission of dsRNA between genetically different individuals of R. solani confirmed with nuclear and mitochondrial markers. These results suggest that transmission can occur between somatically incompatible isolates of R. solani AG-3, but that maintenance of the dsRNA in the recipient isolates was not stable following repeated subculturing on nutrient medium.