First report of Alternaria destruens causing leaf spot disease of Prunella vulgaris in India

Prunella vulgaris (self-healing plant, family Lamiaceae) is a perennial herbaceous plant which grows in temperate regions of the world, and is used in traditional medicine (Wang et al., 2019). Huge losses have been observed in P. vulgaris due to a leaf spot disease. In August 2023, fifty P. vulgaris leaves displaying symptoms of dark-brown to brown, circular to irregular necrotic patches were collected from three different areas of the Kashmir Valley, India (Fig. 1). The leaves were surface sterilised with 1% sodium hypochlorite, rinsed with distilled water and cut into small pieces, and placed on potato dextrose agar supplemented with chloramphenicol (50 µg/ml). The petri plates were incubated at 25 ±2°C. The fungal subcultures exhibited cotton-like, white and dark-brown to grey mycelium in the centre with circular edges (Fig. 2). Conidia were dark brown, measuring 14–53 × 6–18 µm, often obpyriform to obclavate or ellipsoid in shape, with a tiny conical beak at the tip. The conidia had one to two longitudinal and two to five transverse septa (Fig. 3). The morphological and microscopic characteristics resembled those of Alternaria sp. Identification was confirmed by molecular and phylogenetic analysis based on the Internal Transcribed Spacer (ITS) rDNA. Genomic DNA was extracted using a GeneJET Plant Genomic Mini Kit (ThermoFisher Scientific, USA) and the ITS region was amplified using the ITS1F/ITS4R universal primers (White et al., 1990) and sequenced. The sequence was deposited in GenBank (Accession No. PP180006) and had 99.82% identity with Alternaria destruens ATCC 204363 (NR137143). A phylogenetic tree was constructed using the maximum likelihood method and RAxML (Fig. 4). The fungus isolate matched the morphological and molecular characteristics of A. destruens (Simmons, 2007). Confirmation of the pathogenicity of the fungus was performed on healthy mature leaves of P vulgaris which were sprayed with 2 mL of a conidial suspension of A. destruens (107 conidia/mL). Plants were sprayed with sterile water as a control. The plants were grown in 15 × 15 cm pots containing a 3:1 (v/v) mixture of soil and manure for 25 days, in a greenhouse at 25 ±2°C with a relative humidity of over 85%. The inoculated leaves produced leaf spot symptoms while the control plants remained symptomless (Fig. 5). Alternaria destruens was re-isolated from the inoculated leaves and confirmed by microscopic examination, satisfying Koch's postulates, Alternaria destruens was reported to be pathogenic on Cuscuta gronovii (Simmons, 1998), aubergine, barley, mango and sunflower from Iran (Ershad, 2009) and on Ligustrum sinense from China (Yang et al., 2019). This is the first report of A. destruens causing leaf spot of P. vulgaris in Kashmir, India.