First Report of Phytophthora pseudosyringae on Chestnut Nursery Stock in Spain.

Phytophthora pseudosyringae causes stem necrosis and collar rot of deciduous tree species (Quercus spp., Fagus silvatica, and Alnus glutinosa) in several European countries (1,2). In November 2006, we received diseased Castanea sativa seedlings from a nursery in Galicia (northwest Spain). These plants had tongue-shaped necroses of the inner bark and cambium. Reddish, sunken lesions occurred on the surface of the bark, either in the stem base or higher on the stem. Tissue from the leading edge of the lesions was transferred to a selective V8 agar medium (4) and incubated for 7 days at 20°C in the dark. A Phytophthora sp. was isolated, transferred to cornmeal agar (CMA) and V8 agar, and incubated in the dark. Colonies were appressed with stellate to rosaceous growth patterns on CMA and stellate, limited aerial mycelium on V8 agar. Growth on V8 occurred from 2 to 25°C with an optimum at 20°C and a radial growth rate of 4.5 mm per day at 20°C. Chains of inflated spherical to deltoid hyphal swellings with radiating hyphae were abundantly produced in water (2). Chlamydospores were not observed on agar media. The deciduous, sympodial, semipapillate, rarely bipapillate sporangia with pedicels had a length/breadth average ratio of 1.55. Oogonia, antheridia, and oospores were produced within a single culture. Oogonia were spherical and smooth walled, antheridia were predominantly paraginous, but some were amphyginous, and oospores were plerotic that turned golden yellow with age (2). Internal transcribed spacer (ITS)-rDNA and mitochondrial DNA (mtDNA) regions were amplified by nested-PCR and sequenced with DNA extracted from mycelium. The amplicon sizes obtained were similar to those reported for P. pseudosyringae (2,3). DNA sequences showed 99 to 100% homology with those previously identified as P. pseudosyringae and deposited in GenBank. Pathogenicity of the isolate was confirmed by inoculating 10 C. sativa seedlings, as well as three detached leaves from each of another 10 young plants growing in containers. For the seedlings, one shallow cut was made into the bark on the main stem. A colonized agar plug was inserted beneath the flap that was sealed with Parafilm. Unwounded and wounded detached leaves of C. sativa were dipped into a zoospore aqueous suspension (1 × 10 zoospores ml) for 10 s., seedlings and leaves were incubated at 20°C and 95% humidity for 60 and 7 days, respectively. After 7 days, foliar lesions that developed exceeded 25 mm, and the pathogen was consistently reisolated. Leaves inoculated with sterile water did not develop symptoms. On inoculated seedlings, the external surface of the bark was reddish and sunken. Stem lesions progressed bidirectionally from the wound. P. pseudosyringae was recovered from inoculated seedlings but not from controls. On the basis of its unique combination of morphological and physiological characters, pathogenicity, and ITS and mtDNA sequences, the Phytophthora isolated from chestnut was identified as P. pseudosyringae. To our knowledge, this is the first report of P. pseudosyringae on C. sativa in Spain. References: (1) EPPO Reporting Service. Online publication. No. 10 2005/162, 2005. (2) T. Jung et al. Mycol. Res. 107:772, 2003. (3) F. N. Martin et al. Phytopathology 94:621, 2004. (4) C. Pintos Varela et al. Plant. Dis. 87:1396, 2003.