Striking blight infecting mungbean germplasm was observed and the intensity of disease was devastating which varied from 11.45 to 48.77 per cent during flowering to pod set. After the disease become more severe, 10 infected leaves exhibiting Ascochyta leaf spot like symptoms were collected for microscopic studies based on morphological descriptions. Scores of black to brown pycnidia (85–275µm) were observed within the lesions arranged in concentric rings. Initially small yellow-coloured spots appeared on leaves which then enlarged and form circular to oval necrotic lesions (1mm-22mm) with dark brown margins and whitish grey to grey centre. Some lesions also exhibited circular rings giving typical target board like appearance. Infected plants with abundant necrotic lesions were collected then cut into near 3-5mm2 pieces and disinfected for one minute with 0.1 per cent Hgcl2, rinsed three times with sterile distilled water and transferred to potato dextrose agar (PDA). Colonies were transferred to water agar after a 7-day incubation period at 25 ± 1 oc, in order to obtain single spore isolates as described by Leslie and Summerell (2006). After five days of incubation on water agar, raised whitish velvety mycelium emerged on plates and the mycelium turned light grey to dark grey after few days with numerous pycnidia.
Morphological characteristics in culture revealed pycnidia were oval to circular, light brown to tan in colour, 80–250 µm in diameter. Conidia were hyaline, predominantly single septate or aseptate, measured 4.01–7.77 x 2.16–1.80µm, thin, oval to cylindrical with round ends and mycelium are hyaline, 3.20–4.88µm in diameter. Morphological characteristics matched with that of Phoma species as described by Bardas et al. (2008).
To confirm this pathogen at molecular level, genomic DNA was extracted from mycelia of SK-W-IU isolate. Two specific primers ITS4 (ITS F:TCCTCCGCTTATTGATATGC) developed by White (1990) and BT2a (β2a F: GGTAACCAAATCGGTGCTGCTTTC ) developed by Chen et al. (2015) were used to amplify the internal transcribed spacer (ITS) region of ribosomal RNA (rRNA) and β-tubulin region, respectively. Positive test results were obtained for this pathogen and the resulting PCR product sequences were submitted in GenBank (accession no. OM992361.1) and submission id of other sequence (2591999). It was found that ITS and tubulin region exhibited 99.78% and 99.81% nucleotide identity with ITS and BT2 regions of Boeremia exigua isolate 263Ph6 and Boeremia exigua strain HTY2 (ITS: MF599109.1 and BT2a: MW273782.1) respectively, in BLAST analysis. MEGA7 was used to perform evolutionary analysis as described by (Kumar et al. 2016; Saitou and Nei 1987; Tamura et al. 2004). Using the maximum likelihood method, a phylogenetic tree based on concatenated ITS and β-tubulin sequences from the genus Boeremia exigua obtained from National Centre for Biotechnology Information (NCBI) was constructed, revealed that the observed isolates and Boeremia exigua formed a monophyletic group and Fusarium solani from a separate cluster (out-group), thus validating the analysis. The combination of morphological and molecular data revealed that the fungus found associated with leaf spot of mungbean was B. exigua. Pathogenicity test of an isolate was confirmed on three-week-old mungbean seedlings of susceptible cultivar grown singly in pot. Conidial suspension of 1×104 spores ml− 1 of isolated pathogen was sprayed to run off on to three week old mungbean plants and a spray of sterile distilled water on control plants. After inoculation the sprayed plants were covered with polythene covers. At one day interval, sterile distilled water was sprayed to maintain humidity. Light brown lesions appeared on leaves 12 days after inoculation, which later expanded to cover larger portion of leaves, displaying typical symptoms (brown necrotic lesions with dark brown margins and greyish centre). Foliar lesions were similar to those originally seen in the field, while as control plants remain unaffected. B. exigua was successfully re-isolated from symptomatic tissues. To our knowledge this is the first report of Boeremia exigua causing Ascochyta leaf spot of mungbean in India. The outbreak of Ascochyta leaf spot on mungbean is of concern because of its severity and seed born nature. The future prospectus will be to device the efficient management strategies to combat this disease and to study the variability of the pathogen.