First Report of Phytophthora palmivora Causing Fruit Rot on Pomegranate in Greece

Pomegranate (Punica granatum) is a dynamically increasing alternative crop for Greece grown on more than 2,000 ha. In September 2016, a severe fruit rot disease of pomegranate cv. Wonderful was observed in fields of Lamia, Fthiotida, Greece. Symptoms appeared after heavy early-autumn rainfalls, on fruits that were mainly at the lower part of the trees. Initially, small, circular, light brown, water-soaked lesions covered by white Phytophthora-like spores appeared on the fruit surface. The lesions rapidly enlarged superficially and internally, causing partial or entire fruit rot, whereas no other part of the tree was affected. Disease incidence in orchards was estimated at 10 to 30%. A Phytophthora sp. was consistently and readily isolated from the edges of symptomatic fleshy mesocarp tissue, previously surface-sterilized with 95% ethanol, on potato dextrose agar (PDA). To obtain single hyphal isolates, hyphal tips were transferred into new PDA and the growth rate of the oomycete was 9.0 mm/day at 25°C in the dark. Microscopic observations revealed papillated, ovoid and ellipsoid sporangia measuring 27.6 to 72.0 × 23.0 to 36.8 μm (avg. 47.7 × 30.4 μm) with short pedicels (1.0 to 9.0 μm, avg. 3.4 μm) developing on sympodial sporangiophores after 10 days of growth on PDA. Terminal or intercalary chlamydospores were spherical, thick-walled, ranging from 18.5 to 45.9 μm (avg. 32.7 μm) in diameter. DNA from a representative single-hyphae isolate (code PH5ROD) was extracted and the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) was amplified using the universal primers ITS5 and ITS4 (White et al. 1990). The PCR product was sequenced and deposited in GenBank (accession no. KY242491). On the basis of morphological characteristics (Erwin and Ribeiro 1996) and a BLAST search with 100% identity to published ITS sequences of P. palmivora isolates in GenBank (KT148926, KT148928), the oomycete was identified as P. palmivora. For pathogenicity tests, 20 pomegranate fruits cv. Wonderful were artificially inoculated with the isolate PH5ROD by removing a 4.0 × 2.0 mm disc of the leathery fruit exocarp, inserting a 4.0 mm-diameter mycelial plug of a 10-day-old PDA culture, and covering the hole with the detached exocarp disc. Fruits were sprayed with sterilized distilled water, enclosed in plastic bags, and kept at 25°C with a 12-h photoperiod. Control fruits were inoculated with sterilized PDA plugs. Five days post inoculation, all inoculated fruits exhibited symptoms similar to those observed in orchards, whereas P. palmivora was consistently reisolated, thus confirming Koch’s postulates. Neither symptoms nor positive isolations were observed in control plants. Pathogenicity tests were repeated twice. P. palmivora has been reported as the causal agent of pomegranate fruit rot in India (Erwin and Ribeiro 1996) and crown and root rot on pomegranate trees in Turkey (Türkölmez et al. 2016). This is the first report of P. palmivora causing fruit rot on pomegranate in Greece. This disease could result in destructive epidemics after severe rainfalls and cause heavy losses to pomegranate production. Therefore, effective management practices should be investigated and applied. © 2017, American Phytopathological Society. All rights reserved.