First Report of Pythium myriotylum D., Causing Damping Off and Root Rot in Chili Pepper (Capsicum annum L.) from Punjab, Pakistan

Chili pepper (Capsicum annuum L.) has significant importance as a cash crop, like other vegetables, and occupies 20% of the total vegetable production area in Pakistan. Pythium spp. are notorious fungal-like agents that may result in 90% plant death as pre- and/or postemergence damping off. During surveys of nine major chili growing areas of Punjab, Pakistan, from September 2015 to November 2016, disease incidences ranging from 13.8 to 45.4% were recorded. The symptoms consisted of damping off, and on older seedlings (15 to 30 days posttransplanting) reduced growth, wilting, water soaking, brown discoloration, and root rot. Infected roots were cut into small pieces and surface sterilized using 1% sodium hypochlorite for 3 to 5 min, followed by a rinse with sterile distilled water. Root saps were serially diluted to appropriate dilutions, streaked onto corn meal agar medium supplemented with ampicillin (250 mg/liter), rifampicin (10 mg/liter), and pimaricin (10 mg/liter) (Jeffers and Martin 1986), and incubated at 28 ± 2°C (Koehler et al. 2017). A total of 13 hyphal tipped isolates, obtained from the infected chili roots of 30 plants, were characterized morphologically. Based on morphological characteristics—production of coenocytic hyphae with lobate sporangia (7 to 15 µm wide), knob-like appressorium, vesicles (43 to 52 µm in diameter) having 29 to 45 zoospores/vesicle, encysted zoospores (10 to 12 µm in diameter), terminal oogonia (30 to 38 µm in diameter), crooked necked antheridia (four to seven antheridia per oogonium), and aplerotic oospores (25 to 31 µm in diameter) (Drechsler 1943)—the pathogen was identified as Pythium myriotylum. DNA was extracted from two isolates using a cetyl trimethylammonium bromide method, and the internal transcribed spacer regions (ITS1 and ITS2) were amplified by polymerase chain reaction (White et al. 1990). The amplicons were purified and sequenced in both directions (GenBank accession nos. MF143429 and MF143430). BLAST analysis of approximately 700-bp fragments revealed that the sequences of the isolates had 99% identity with accession HQ643704.1 of P. myriotylum (Robideau et al. 2011). Koch’s postulates were fulfilled by adding 100 ml of a zoospore suspension (1 × 10⁶ zoospores/ml) from a 7-day-old culture to soil contained in 1.5 liter plastic pots just before seed sowing. Zoospores were produced as described by Rahimian and Banihashemi (1979). Twenty chili seeds (cultivar Sanam) were sown per pot, and pots were kept in a greenhouse at 30°C. The experiment was repeated twice with 10 replications. After 15 days, the inoculated plants exhibited symptoms of damping off and root rot, whereas control plants remained symptomless. Reisolation of the pathogen from symptomatic roots confirmed the association of P. myriotylum with the disease. This is the first report of P. myriotylum causing damping off in chili pepper crops from Pakistan. Damping-off and root rot symptoms on chili were also reported from the Sindh province, which contributes 89% of the total chili production in Pakistan, indicating the widespread occurrence of this pathogen. Therefore, the pathogen can have a great negative economic impact on chili production, and management options must thus be investigated.