Detection and analysis of mycovirus-related RNA viruses from grape powdery mildew fungus Erysiphe necator.

The fungus, Erysiphe necator Schw., is an important plant pathogen causing powdery mildew disease in grapevines worldwide. In this study, high-throughput sequencing of double-stranded RNA extracted from the fungal tissue combined with bioinformatics was used to examine mycovirus-related sequences associated with E. necator. The results showed the presence of eight mycovirus-related sequences. Five of these sequences representing three new mycoviruses showed alignment with sequences of viruses classified in the genus Alphapartitivirus in the family Partitiviridae. Another three sequences representing three new mycoviruses showed similarity to classifiable members of the genus Mitovirus in the family Narnaviridae. These mycovirus isolates were named Erysiphe necator partitivirus 1, 2, and 3 (EnPV 1-3) and Erysiphe necator mitovirus 1, 2, and 3 (EnMV 1-3) reflecting their E. necator origin and their phylogenetic affiliation with other mycoviruses.

Powdery Mildew Outbreaks caused by Podosphaera macularis on Hop Cultivars Possessing the Resistance Gene R6 in the Pacific Northwestern United States.

Resistant cultivars of hop (Humulus lupulus) have been grown, with the aim of helping to manage powdery mildew in the Pacific Northwest since the first report of the disease in the field in 1997 (4). A major objective of many breeding programs is development of resistance to powdery mildew, and this has generally been achieved by single resistance genes (qualitative resistance). One such gene, R6 (3), has been utilized extensively in new cultivars and has prevented epidemics of the disease in those cultivars across the Pacific Northwestern United States for approximately 15 years. In 2011, a grower in Washington State reported outbreaks of powdery mildew on cv. Apollo, which is thought to possess powdery mildew resistance derived from R6. Fungicides and cultural control measures were applied, and the grower reported no substantial crop damage from the disease. During the winter of 2012, the same grower planted rhizomes of cv. Apollo in a greenhouse in the Yakima Valley of Washington State and later found the plants to be affected by powdery mildew. Affected leaves from plants of cvs. Apollo, Newport, and Nugget (all reported [3] or assumed to possess R6 based on pedigree) grown in the same greenhouse were later provided to the authors. Conidia obtained from each affected plants were transferred to plants of the highly susceptible cv. Symphony, which is not known to contain any resistance genes. After 10 to 14 days of incubation, resultant conidia from each cultivar above (total of three isolates) were transferred to greenhouse grown plants of cvs. Nugget and Symphony and incubated at 18°C. Within 7 days, all three isolates produced powdery mildew colonies characteristic of P. macularis (2) on both cultivars. Cleistothecia did not develop in any colonies. In addition, Nugget and Symphony plants were inoculated with a field population of P. macularis originating from cultivars lacking R6 in Oregon. These inoculations on Nugget did not develop powdery mildew whereas Symphony plants did. Non-inoculated controls remained free of powdery mildew. Results were identical in two additional experiments. The sequence of the mating type idiomorph, MAT1-1, was obtained to confirm identity of the pathogen as P. macularis as described previously (1). The sequences were identical among the three isolates obtained from the greenhouse in Washington and isolates of P. macularis obtained previously from Oregon and Washington. MAT1-2 idiomorph was not detected in the isolates collected. While R6-virulent strains have been detected previously in race characterization experiments, these strains have not caused widespread epidemics of powdery mildew. The increasing prevalence of virulent strains of P. macularis and outbreaks of powdery mildew on formerly resistant cultivars necessitates changes in breeding strategies and disease management efforts to minimize damage resulting from the disease. The distribution of virulent strains of the pathogen and susceptibility of formerly resistance cultivars to powdery mildew are currently under investigation. References: (1) B. Asalfet et al. Phytopathology 103:717, 2013. (2) R. Bélanger et al. The Powdery Mildews: a Comprehensive Treatise. APS Press, St. Paul, MN, 2002. (3) P. Darby. Brew Hist. 121:94, 2005. (4) C. Ocamb et al. Plant Dis. 83:1072, 1999.

Occurrence and Identification of Phytophthora spp. Pathogenic to Pear Fruit in Irrigation Water in the Wenatchee River Valley of Washington State.

ABSTRACT Seven hundred forty-nine isolates of Phytophthora spp. were obtained from irrigation canals in eastern Washington State during the 1992 to 1995 and 1999 growing seasons. Isolates were retrieved using pear baiting techniques. All isolates were pathogenic to pear and were present in irrigation water beginning early in fruit development. Over the course of the 5 year study, 10 and 5% of isolates were identified as P. cactorum and P. citricola, respectively, using morphological criteria. The remaining isolates could not be identified using morphological criteria. Colony morphology of these isolates was characterized during all years of the study. In 1999, more detailed studies utilizing polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of entire internal transcribed spacer (ITS) regions (ITS1, 5.8S, and ITS2) of ribosomal DNA for 180 isolates, and sequence analysis of ITS2 for 50 isolates, were used to investigate genetic variation and phylogenetic relationships among isolates. Isolates were divided into 12 groups based on their growth type on corn meal agar. Restriction digestion of the entire ITS region with three enzymes revealed 11 restriction digestion patterns among 180 isolates. PCR-RFLP and sequence data were obtained for 12 reference Phytophthora spp. (two species in each of Waterhouse's six morphological groups). Phylogenetic analysis of ITS2 regions revealed nine clades, each with strong bootstrap support. Molecular analyses revealed 23 isolates that were in the P. gonapodyides clade, 9 in the P. parasitica clade, 1 in the P. cactorum clade, 7 in the P. citricola/capsici clade, and 4 in the P. cambivora/pseudotsugae clade. The three isolates comprising clade 5 were significantly distinct from all other Phytophthora spp. in the databases and may represent a new Phytophthora sp. Colony morphology was not consistently correlated to PCR-RFLP pattern or ITS2 phylogeny, suggesting that the former criterion is insufficient for species identification. The results of this study indicate that at least nine phylogenetically distinct taxa of Phytophthora pathogenic to pear are present in irrigation water in North Central Washington.

Perennation of Uncinula necator in Vineyards of Eastern Washington.

Studies on the mode of perennation of Uncinula necator in Eastern Washington were conducted over a 4-year period. Evidence of perennation of U. necator in infected dormant buds was not evident during vineyard surveys conducted over the period. Cleistothecia retrieved from bark fissures and senesced leaves contained viable ascospores at bud burst and later. The proportion of cleistothecia retrieved from bark that contained viable ascospores at bud burst ranged from 0.19 to 0.48, 0.09 to 0.72, 0.18 to 0.22, and 0.48 to 0.67 in 1998, 1999, 2000, and 2001, respectively. Viability of cleistothecia retrieved from senesced leaves in two vineyards at bud burst was 0.41 and 0.40 in 1998 and was 0.5 and 0.4 in 1999. Ascospore release in lab studies occurred from the late-dormant stage through the prebloom and (in some cases) the bloom stages. The initial ascosporic infection of Chardonnay leaves began at the late-dormant stage; colony numbers then declined through the prebloom and bloom stages. In vineyard studies, ascospores were trapped as late as 70 days after bud burst during rain events of 3.9 to 9.6 mm. Detection of ascospores in vineyard air preceded the initial occurrence of powdery mildew symptoms and signs and the occurrence of conidia in volumetric spore traps by several days. Cleistothecia are the only known source of primary inoculum in the grape-production regions of Eastern Washington.

First Report of Phytophthora syringae Causing Rot on Apples in Cold Storage in the United States.

A decay of 'Granny Smith' apples (Malus domestica Borkh.) was observed in 1988, 1990, and 1991 on fruit grown in the lower Hood River Valley of Oregon and stored at 0°C. Harvested fruit were drenched with thiabendazole and stored in October in all years. In mid-November, fruit were sized, drenched with sodium hypochlorite, and returned to cold storage. Decay was observed in January when fruit were removed from cold storage, sorted, and packed. Decayed areas were light brown and firm with a slightly indefinite margin. Losses were less than 1% of fruit packed. Diseased fruit were surface-disinfested with 95% ethanol, and tissue pieces were transferred aseptically to potato dextrose agar acidified with lactic acid and incubated at approximately 22°C. The fungus consistently isolated was identified as Phytophthora syringae (Kleb.) Kleb. based on morphological characters (3). Sporangia were persistent and averaged 60 µm long (range 59 to 69) × 40 µm wide (range 37 to 43). Antheridia were paragynous, and oospores averaged 37 µm (range 31 to 46). 'Golden Delicious', 'Granny Smith', and 'Gala' apples were inoculated with mycelial plugs from a 7-day-old culture of P. syringae and incubated 12 days at 5°C and 7 to 12 days at 22°C. Twenty fruit of each cultivar were used-ten were inoculated, and ten uninoculated fruit served as controls. Lesions developed on all inoculated fruit but not on uninoculated controls. Lesions were spherical, chocolate brown, and firm with no evidence of external mycelia. Lesion morphology was similar on all cultivars. P. syringae was reisolated from lesion margins of all infected fruit. This postharvest decay of apples has not been observed in the Hood River Valley since 1991. Fruit rot of apples caused by P. syringae is known in Canada (1) and is common in the United Kingdom (2), but has not been reported previously in the United States. To our knowledge, this is the first report of postharvest decay of apples by P. syringae in the United States. References: (1) R. G. Ross and C. O. Gourley. Can. Plant Dis. Surv. 49:33, 1969. (2) A. L. Snowdon. A Color Atlas of Postharvest Diseases. CRC Press, Inc., Boca Raton, FL, 1990. (3) G. M. Waterhouse. The Genus Phytophthora. Misc. Publ. 12. The Commonwealth Mycological Institute, Kew, Surrey, England, 1956.