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First Report of Fruit Rot on Tomato (Lycopersicum esculentum) Caused by Phytophthora cactorum in Pakistan

Hussain, M., Zouhar, M., Ryšánek, P.
Plant disease 2019 v.103 no.4 pp. 776
DNA, DNA primers, Phytophthora cactorum, Solanum lycopersicum var. lycopersicum, agar, air drying, ampicillin, apples, chlamydospores, databases, ethanol, fruits, genes, hyphae, industry, internal transcribed spacers, mixing, mycelium, oospores, pathogen identification, pathogens, pears, peptide elongation factors, plant rots, polymerase chain reaction, rifampicin, risk, sodium hypochlorite, sporangia, surveys, texture, tomatoes, tubulin, virulence, zoospores, Pakistan
During April 2018, black-colored lesions were observed in 18% of tomato fruits (Lycopersicum esculentum L.) during a survey of fields near Kasur (31.656° N, 74.2648° E), Pakistan. Symptoms typical of fruit rot including black, water-soaked, necrotic lesions with concentric rings appeared on tomato fruits. Lesions enlarged and dark zonate bands were formed in the affected area. Diseased fruit turned spongy, ultimately covered with a leathery texture, and dropped off. Tissue from each diseased fruit was surface sterilized with 2% NaOCl solution for 1 min and rinsed twice in sterile distilled water. The lesions were cut from the fruit and plated on P5ARPH medium (Jeffers and Martin 1986). Plates were placed at 23 ± 2°C for 6 to 10 days in darkness, and white colonies appeared after 72 h. Colonies were subcultured by hyphal tips to V8 agar amended with rifampicin (10 mg/liter), ampicillin (250 mg/liter), and pimaricin (0.2% wt/vol). All isolates were homothallic and produced papillate, obpyriform sporangia. The size of sporangia ranged from 34.2 to 44.5 μm wide and 25.9 to 31.3 μm long. Sporangia were formed on long pedicels ranging from 1.2 to 3.1 μm in diameter. Chlamydospores were produced terminally, ranging from 31.5 to 41.2 μm in diameter, and oospores were plerotic (27.0 to 32.0 μm in diameter) with paragynous antheridia. On the basis of the morphological characteristics, the pathogen was identified as Phytophthora cactorum ([Lebert & Cohn] J. Schrot.) (Erwin and Ribeiro 1996). The internal transcribed spacer region (ITS), translation elongation factor 1 alpha (TEF1-α), and β-tubulin genes of three isolates were amplified with the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Btub2F/Btub4R, respectively. The amplified polymerase chain reaction products were purified, sequenced, and compared with the NCBI database using BLASTn analysis (Altschul et al. 1990). The resulting sequences of ITS (LS999935), β-tubulin (LS999936), and TEF1-α gene (LS999937) showed 99% similarity to EU109568, EU109572, and EU080286 sequenced P. cactorum isolates already available on the database after BLAST analysis. Pathogenicity tests were carried out on tomato fruits to confirm the virulence of sequenced P. cactorum isolates. Tomato fruits were surface sterilized with 95% ethanol and air dried. Mycelium was grown for 7 days on V8 medium, and a suspension was prepared by blending mycelia in 200 ml of sterile water. A zoospore suspension of 0.1 ml (20,000 zoospores/ml) of P. cactorum was applied onto the surface of the tomato fruit. Control tomato fruits were sprayed with sterile water. The inoculated fruits were incubated for 7 days in a moist chamber at 23 ± 2°C in darkness. Twenty-five tomato fruits were used as replicates, and the experiment was repeated thrice. After 7 days of incubation, lesions appeared on all inoculated fruits. Lesion size varied on tomato fruit from 35 to 48 mm and covered 20 to 25% of the total fruit surface. Control fruits remained healthy, and no pathogen was isolated from these fruits. The pathogen was reisolated from all inoculated fruits, and they showed black-colored lesions similar to those observed previously (Kao and Leu 1979). The isolated pathogen was identified as P. cactorum based on morphological features and DNA analysis. To the best of the authors’ knowledge, this is the first report of P. cactorum causing fruit rot on tomato in Pakistan. Fruit rots caused by P. cactorum have already been reported on pears and apples in different parts of the world (Waterhouse and Waterston 1966). The presence of P. cactorum on tomato fruit represents a new and major risk to the tomato industry in Pakistan.