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First Report of Botrytis cinerea Causing Postharvest Fruit Rot on Stored Pomegranates in Pakistan

Alam, M. W., Rehman, A., Ahmad, S., Sarwar, M., Naseem, M. K., Chattha, M. B., Malik, A. U., Ali, S.
Plant disease 2019 v.103 no.2 pp. 374
Botrytis cinerea, DNA primers, Punica granatum, air drying, conidia, conidiophores, culture media, fruits, fungal growth, fungi, genes, glyceraldehyde-3-phosphate dehydrogenase, grapes, industry, internal transcribed spacers, mycelium, pathogen identification, pathogenicity, pathogens, plant rots, polymerase chain reaction, pomegranates, ribosomal DNA, sclerotia, sodium hypochlorite, storage temperature, storage time, surveys, Europe, Pakistan
Pomegranate (Punica granatum L.) is an emerging fruit industry in Pakistan, and the crop is currently cultivated on 11,200 ha. During November 2017, a survey was conducted in Multan (30°7′40.9296″ N, 71°23′11.1984″ E) and Muzaffargarh (30°4′27.7572″ N, 71°11′4.7544″ E), major pomegranate-producing regions in Punjab Province. Postharvest fruit rots were observed on ‘Kandhari’ pomegranates after 35 days of storage at 6°C. The symptoms consisted of irregular, small, grayish-brown lesions on fruit crown that later developed into expanded (46 to 53 mm in diameter), dark brown lesions. In severe cases, the whole fruit rotted and was covered with gray fungal growth. The disease was severe in 47% of stored fruit. Small tissue segments were cut from 20 symptomatic fruit, surface disinfected with 1% NaOCl for 2 min, rinsed three times in sterile distilled water, air dried, placed onto potato dextrose agar (PDA), and incubated at 25°C. Fungal colonies obtained were initially whitish, becoming dark gray after 72 h. Conidia produced on PDA were single-celled, ellipsoid or ovoid in shape, and 6.3 to 10.5 × 4.5 to 9.0 μm in size. Conidiophores 16 to 31 μm in length, arose directly from the mycelia, were more or less straight, septate, branched at the apex, bearing bunches of conidia resembling grape clusters. After 13 to 15 days, round or irregular, black sclerotia (1.3 to 4.2 × 1.7 to 2.9 μm) were produced on the margins of the Petri plates. On the basis of morphological characteristics, the pathogen was identified as Botrytis cinerea (Ellis 1971). To verify the fungal species level, DNA from a representative isolate (PD410) was extracted. The internal transcribed spacer region (ITS) of the rDNA was amplified with primers ITS1/ITS4. BLAST analysis (GenBank accession no. MH329278) revealed 99% identity with B. cinerea strain XT5-2 (KX721051). Additionally, the glyceraldehyde-3-phosphate dehydrogenase gene (Staats et al. 2005) was sequenced, and the sequence (GenBank accession no. MH329277) was 100% homologous with B. cinerea strain SMGM003 (KX443702). Pathogenicity testing was performed twice on 10 surface-sterilized ‘Kandhari’ fruit. Fruit were internally inoculated by injecting a conidial suspension (0.5 ml/fruit, 10⁵ conidia/ml), using a 23-gauge syringe. Ten fruit inoculated with sterile distilled water served as controls. After 19 days at 6°C, all fruit inoculated with the fungus showed grayish brown lesions followed by rotting. Control fruit remained symptomless. The pathogen was reisolated from inoculated fruit, and morphological characteristics were similar to the original isolates. The disease has been reported in other parts of the world, including Asia (Kanwar and Thakur 1972) and Europe (Pala et al. 2009; Thomidis 2014), but this is the first report of B. cinerea causing postharvest fruit rot of pomegranate in Pakistan. The disease could cause heavy losses to the emerging pomegranate industry in Punjab Province of Pakistan. Therefore, appropriate management strategies should be investigated and applied.