First Report of Ascochyta medicaginicola Causing Leaf Blight Disease of Pointed Gourd in India

Abstract

Pointed gourd (Trichosanthes dioica Roxb.) is a popular summer vegetable in India. During April 2017, severe leaf blight symptoms were observed in many fields located in the sub-Himalayan region in West Bengal state of India (21.14 to 21.30°N, 78.82 to 79.02°E). Lesions initially appeared as yellow spots (4 to 5 mm in diameter) on leaf margins that gradually expanded to cover almost the whole leaf between the veins. As lesions aged, they turned brown and dry, ultimately leading to defoliation. Tiny black pycnidia were scattered throughout the dried lesions. Symptomatic leaves were collected, cut into small square pieces approximately 5 mm², surface sterilized with 1% NaOCl, and transferred to potato dextrose agar. The agar plates were incubated at 28°C for 7 days. After 5 days of incubation, compressed, white velvety mycelia appeared on the plates, and the colonies gradually turned gray after sporulation. Conidia were hyaline, two to three septate, 4 to 8 μm long, 1.5 to 3 μm wide, thin and smooth walled, ellipsoidal to cylindrical conidia with both rounded ends. Fifteen isolates having similar morphology were recovered from diseased leaf tissue. Morphological characteristics matched those of Ascochyta medicaginicola var. macrospora Q. Chen & L. Cai (Chen et al. 2015). To confirm identity, DNA was extracted from the mycelia of the representative isolate PGALD2. The internal transcribed spacer (ITS) region of ribosomal RNA (rRNA) and the D1/D2 region of the 28S rRNA large subunit were amplified using primers ITS1/ITS4 (White et al. 1990) and NL1/NL4 (O’Donnell 1993), respectively. The sequences of the resulting polymerase chain reaction products were submitted to GenBank. BLAST analysis of the ITS region (accession no. MF447846) and D1/D2 region (accession no. MF447845) showed 99 and 100% nucleotide identity with ITS and D1/D2 regions of A. medicaginicola var. macrospora strain CBS 112.53 (ITS, GU237749; and D1/D2, GU238101), respectively. For pathogenicity testing, a conidial suspension (10⁶ spores/ml) of isolate PGALD2 was sprayed onto both surfaces of the leaves of 10 healthy 2-week-old potted pointed gourd plants until run-off. A set of 10 control plants was sprayed with sterile distilled water and maintained as a control. The plants were kept in a transparent and perforated polythene chamber for maintaining high humidity and aeration in the greenhouse for 15 days. Humidity was maintained by spraying sterile distilled water at 1-day intervals. The experiment was conducted three times. Three days after inoculation, yellow spots were visible on the inoculated leaves, which gradually expanded to cover a major part of the leaf. The controls remained symptomless. A. medicaginicola was successfully reisolated from symptomatic tissues. To our knowledge, this is the first report of A. medicaginicola var. macrospora causing leaf blight of pointed gourd. A. medicaginicola can cause severe yield losses of pointed gourd in India.