Jeffrey K. Stone

We report the first secondary metabolite, 8,8′-bijuglone, obtained from pure cultures of the slow growing Douglas fir- (Pseudotsuga menziesii var. menziesii) foliage-associated fungus Zasmidium pseudotsugae. The quinone was characterized using extensive LC/MS and NMR-based spectroscopic methods. 8,8′-Bijuglone exhibited moderate antibiotic activity against Gram-positive pathogens and weak cytotoxic activity in the NCI-60 cell line panel and in our in-house human colon carcinoma (HCT-116) cell line. An analysis of the fungal genome sequence to assess its metabolic potential was implemented using the bioinformatic tool antiSMASH. In total, 36 putative biosynthetic gene clusters were found with a majority encoding for polyketides (17), followed by non-ribosomal peptides (14), terpenes (2), ribosomal peptides (1), and compounds with mixed biosynthetic origin (2). This study demonstrates that foliage associated fungi of conifers produce antimicrobial metabolites and suggests this guild of fungi may present a rich source of novel molecules.

Timber durability is often assessed using small wood stakes exposed in direct soil contact, and the assessment generally emphasizes effects on wood rather than organisms involved. Understanding fungal colonization patterns can help identify key decay agents under varying conditions and use these patterns to improve wood protection strategies. Fungal colonization of red alder (Alnus rubra), Douglas-fir (Pseudotsuga menziesii) heartwood/sapwood, and western redcedar (Thuja plicata) field stakes was assessed over 2 y in western Oregon. Spatiotemporal fungal community variations were identified via culturing and DNA sequencing, where 814 isolates were identified from 84 stakes. Forty-six ascomycete genera were identified, with Phialophora, Trichoderma, and Epicoccum species occurring most frequently. Twenty-three basidiomycete genera were identified, with Trametes and Phanerochaete being the most common. Douglas-fir and western redcedar stakes contained the highest and lowest diversity levels, respectively, reflecting natural durability differences of these species. Fungal species abundance was higher below ground than in the above ground and groundline zones, likely reflecting more stable moisture regimes, proximity to soil-based fungi, and potential nutrient migration into wood beneath the soil surface. Ascomycetes were proportionally more abundant early in the exposure period, but basidiomycetes were also observed early in the process, and there appeared to be no consistent colonization pattern.

In August 2012, wilted hop bines were observed in a yard near Seneca Castle, New York, affecting 10 to 20% of the plants. Affected bines had a dark stem discoloration and wilted leaves, which remained attached after bines were killed. Dark brown to black erumpent pycnidia were aggregated in the cortical tissue on portions of affected stems. Isolations were made from surface-sterilized stem pieces onto potato dextrose agar (PDA), and hyphal tips were isolated and transferred to1% water agar amended with shredded sterilized hop stem pieces. A pycnidial fungus was consistently isolated that produced dark-brown to black colonies within two weeks. Conidia measuring (19.5–) 24.4 (–31.7) µm × (9.7–) 11 (–12.2) µm were dark brown when mature, thick-walled, aseptate, and ovoid, with a truncate to rounded base. These characteristics suggested Diplodia seriata (Phillips et al. 2007). Conidial measurements and other morphological characters were consistent with those of an ex-type specimen of D. seriata (CBS 112555). Elongation factor 1-α, beta-tubulin and ITS gene sequences were obtained from two isolates obtained from hop plants with standard primers (Úrbez-Torres et al. 2008; White et al. 1990) and deposited in GenBank (accession numbers KT460188 & KT460189, KT460190 & KT460191, KT447248 & KT447249). The sequences for each isolate and locus were 99%, 100%, and 100% homologous to corresponding sequences of Diplodia seriata isolate CBS 112555 (GenBank accessions AY573220, DQ458856, and NR_111151, respectively). Pathogenicity assays were conducted with D. seriata isolates DS-01 and DS-02 utilizing a toothpick inoculation procedure (Putnam 2004) to wound and deliver inoculum into the lower portions of stems near the soil. Hop plants of cv. Fuggle were pruned to two main stems, each trained separately onto bamboo stakes. On each of 7 to 8 replicate plants per isolate, one stem was inoculated by piercing with a PDA-coated toothpick colonized by the fungus; the other stem was not inoculated. Eight control plants were pierced with a PDA-coated toothpick without the fungus. Plants were kept in a mist chamber for 10 days and then maintained in a greenhouse at 24o C with a 14-hour light cycle for 9 months. Twenty-nine percent of plants inoculated with isolate DS-01 and 38% of plants inoculated with DS-02 had stunted growth and wilting, and developed diffuse lesions radiating outward from the inoculation site. The fungus was re-isolated from all symptomatic stems and confirmed as D. seriata based on morphological characters and resequencing of the three loci. The pathogen was not recovered from asymptomatic plants or non-inoculated controls. To our knowledge, this is the first report of infection of hop by D. seriata in the U.S. At present the disease appears to be only a minor issue on hop in New York State. D. seriata is a common and broadly distributed pathogen on grapevine and other woody hosts. The long incubation period of this fungus may limit symptom development on the annually produced shoots of hop. References: Phillips, A. J., et al. 2007. Fungal Diversity. 25:141-155. Putnam, M. L. 2004. Plant Pathol. 53:252. Úrbez-Torres, J. R., et al. 2008. Plant Dis. 92:519-529. White, T. J. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Innis, M. A., et al., eds. Academic Press, San Diego.

Swiss needle cast (SNC) is a foliar disease of Douglas-fir (Pseudotsuga menziesii) caused by Phaeocryptopus gaeumannii (Rohde) Petrak. This fungus is endemic to western North America, where it has historically had little impact in native forests. However, increasing disease severity in western Oregon since the 1990s has prompted renewed interest in P. gaeumannii and SNC. For this study, we analyze multilocus microsatellite genotypes from 482 single-spore isolates from 68 trees across 14 sites in the western Coast Range of Oregon and southwestern Washington. This study assesses genotypic variation and genetic structure at several levels of population hierarchy. Despite the observation that most of the genetic variation occurred within subpopulations, our analyses detected significant differentiation at all hierarchical levels. Clustering among the 482 isolates based on genetic distance clearly supports the existence of two previously described cryptic lineages of P. gaeumannii in the western United States. The two lineages occur in varying proportions along latitudinal and longitudinal gradients in western Oregon and Washington, suggesting a relationship between climate and phylogeography. Sites near Tillamook, Oregon, where SNC is most severe, consist of sympatric subpopulations in which the two lineages comprise roughly equal proportions.

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Germinating ascospores of Phaeocryptopus gaeumannii produce suprastomatal appressoria from which penetration pegs enter needles. Initial infection occurs between late May and early Jul and coincide...

Phaeocryptopus gaeumannii, the causal agent of Swiss needle cast disease, is widely distributed throughout New Zealand, where the disease

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