Isolates of Bean pod mottle virus (BPMV), a member of the genus Comovirus, collected from soybean fields in Kentucky, Virginia, Arkansas, and Iowa were classified into two distinct subgroups, I and II, based on nucleic acid hybridization analysis using cloned cDNA probes to RNA-1 from BPMV strains K-G7 and K-Ha1. Slot blot hybridization analysis using cloned cDNA probes to RNA-2 from the same two strains (K-G7 and K-Ha1), however, revealed that some of the isolates, initially classified as belonging to subgroup I after analysis with RNA-1 probes, are in fact natural reassortants between the two strain subgroups. This was corroborated by nucleotide sequence analysis of full-length cDNA clones of both RNA-1 and RNA-2 from a putative reassortant strain (K-Ho1). These results indicate that BPMV strain diversity is more complex than initially anticipated, and that the use of cloned probes to both genomic RNAs during nucleic acid hybridization analysis is required to unravel the extent of such diversity. In a field plot experiment, BPMV isolates that belong to distinct strain subgroups induced symptoms that varied in severity and in the level of yield losses. In this regard, the reassortant strain K-Ho1 caused the most serious damage compared with four other BPMV isolates tested. Furthermore, the soybean alleles Rsv(1) and Rsv(4), known to confer resistance against Soybean mosaic virus, a member of the genus Potyvirus, did not provide any protection against BPMV. Additionally, we developed a reverse transcription-polymerase chain reaction procedure based on the sequence of a highly conserved region in the capsid polyprotein coding sequence that provides efficient and highly sensitive detection of all BPMV isolates tested, regardless of their strain classification.

A broad-spectrum anti-fungal protein of approximately 10 kDa, designated victoriocin, was purified from culture filtrates of a virus-infected isolate of the plant-pathogenic fungus Helminthosporium victoriae (teleomorph: Cochliobolus victoriae) by a multistep procedure involving ultrafiltration and reverse-phase high-performance liquid chromatography (RP-HPLC). Amino acid sequences, obtained by automated Edman degradation sequencing of RP-HPLC-purified victoriocin-derived peptides, were used to design primers for degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) amplification from H. victoriae DNA and cDNA templates. An open reading frame coding for a victoriocin precursor of 183 amino acids with calculated molecular mass of approximately 20 kDa was amplified by PCR from H. victoriae genomic DNA but not from the control fungus Penicillium chrysogenum. Southern hybridization analysis confirmed the presence of the victoriocin gene in all H. victoriae strains tested. Sequence analysis indicated that victoriocin has a sequence motif similar to that found in scorpion short toxin/charybdotoxin and a consensus sequence similar to that found in defensins. Victoriocin, like some other antifungal proteins, including the totivirus-encoded killer proteins, is predicted to be expressed in vivo as a preprotoxin precursor consisting of a hydrophobic N-terminal secretion signal followed by a pro-region and terminating in a classical Kex2p endopeptidase cleavage site that generates the N terminus of the mature victoriocin. A putative cell wall protein of approximately 30 kDa (P30) co-purified with victoriocin from cultural filtrates. The potential role of P30 in the antifungal activity of H. victoriae culture filtrates is discussed.

We have previously reported the isolation and characterization of the broad-spectrum antifungal protein, victoriocin, from culture filtrates of a virus-infected isolate of the plant-pathogenic fungus Helminthosporium (teleomorph: Cochliobolus) victoriae. We predicted that the 10-kDa mature victoriocin is derived in vivo from a preprotoxin precursor that is processed by a signal peptidase and kexin-like endopeptidase. We also presented evidence that the victoriocin precursor is encoded by a host gene, designated the victoriocin (vin) gene. In the present study, an H. victoriae genomic DNA library was constructed in the cosmid vector pMLF-2, and a cosmid clone carrying the vin gene and flanking sequences was isolated and used to generate constructs for transformation of virus-free and virus-infected H. victoriae isolates with the vin gene. Culture filtrates of the virus-free vin transformants exhibited high levels of antifungal activity compared with that revealed by the nontransformed virus-free wild-type strain, which exhibited little or no antifungal activity. Moreover, transformation of the wild-type virus-infected H. victoriae strain with the vin gene resulted in still higher production of victoriocin and higher antifungal activity in the culture filtrates of the vin transformants compared with the virus-infected wild-type strain. As previously predicted, the presence in the vin transformants of the preprovictoriocin and its post-translationally generated products, the provictoriocin and the mature victoriocin, was clearly demonstrated. Processing of the victoriocin preprotoxin requires eukaryotic host factors because no processing occurred in an in vitro translation system or in bacteria. It is of interest that some of the virus-free isolates transformed with the vin gene exhibited some features of the virus-induced disease phenotype, including moderate stunting and sectoring. Present data suggests that victoriocin may play an indirect role in disease development. Taken together, these results indicate that victoriocin is the primary protein responsible for the antifungal activity in culture filtrates of virus-infected H. victoriae isolates and that virus infection upregulates the expression of victoriocin. Overproduction of victoriocin may give the slower-growing virus-infected fungal strains some competitive advantage by inhibiting the growth of other fungi.

Abstract. Plant pathogens of the genus Phytophthora may pose a threat to trees on reclaimed mine lands. The presence of these pathogens in forest soils of Appalachia has been documented, but their presence and relative distribution in mine spoils is unknown. Soil samples from mine spoils at the Bent Mountain, KY reforestation site were tested for presence of Phytophthora spp., and, specifically, P. cinnamomi, using a soil-baiting method. Loose-graded mine spoils (brown sandstone, gray sandstone, mixed spoil, and shale) dumped in 2005 and 2007 at the Bent Mountain reforestation site were tested monthly from May to October 2007 for Phytophthora at the surface (top 0-10 cm) using a soil baiting method. Soils collected from two non-mined control sites within Robinson Forest and Berea Forest were also tested using the same method. Colonies obtained from the baiting method were isolated in PARP-H V8 Phytophthora-selective medium and incubated at 25°C. Colonies with Phytophthora-characteristic morphology were tested by PCR. PCR results, confirmed by DNA sequencing, indicate that P. cinnamomi was successfully isolated using the baiting method from forest soils. No Phytophthora spp. were detected by the baiting method from mine spoils. Future work will involve testing water that has infiltrated through mine spoils for presence of Phytophthora spp. Water collected from lysimeters will be filtered to capture mycelial fragments or reproductive structures of Phytophthora species through exclusion on the filter membrane and the membrane will be subjected to DNA extraction and PCR. The presence of Phytophthora spp. in spoils and water in comparison to media physiochemical characteristics, aqueous geochemistry of infiltrated waters, and tree growth with regards to reforestation efforts on different tailing media will be evaluated.Additional

Phytophthora ramorum, cause of sudden oak death of trees or ramorum blight of other plant species, has an ever-increasing host range. Some geographic regions are considered to be at high risk of becoming infested with the pathogen, possibly causing plant mortality such as seen in native habitats of California and Oregon. One such region is the Appalachian range of the eastern United States, where known susceptible plants occur and climatic characteristics appear favorable for infections by this pathogen. We collected foliage of a range of plant species native to Appalachia in Kentucky during two summer seasons, and the foliage was shipped to Oregon for inoculation with P. ramorum to determine relative susceptibility. Leaves were needle-wounded and inoculated with either mycelium agar plugs or sporangia of a North American (A2 mating type) or European (A1 mating type) isolate. After 14 days incubation at 20°C in moist boxes, lesions caused by either inoculum type or isolate generally were comparable using digital photos and ASSESS software. Some genera, species, and cultivars within species were highly susceptible, while others were moderately susceptible or not susceptible. These results provide a basis for regional surveyors to select target hosts and to generate survey and management practices for nursery and forest areas. Accepted for publication 24 April 2007. Published 17 September 2007.