First Report of Causing Brown Leaf Spot of Potato in South India.

Potato (Solanum tuberosum L.), as one of the major food crops, is cultivated in both temperate and subtropical climatic regions worldwide. During the field survey conducted in December 2023, black to brown colored spots with dark brown to black margins were observed in the leaves of the potato cultivar Kufri Jyothi, grown annually to monitor pest and disease incidence at the Indian Council of Agricultural Research (ICAR) - Central Potato Research Institute (CPRI) Research Station, Ooty, Tamil Nadu, India (Latitude - 11.370796 N, Longitude - 76.664094 E, Elevation - 2110 m above mean sea level. As the disease progressed, the spots were scattered throughout the leaves and coalesced to form necrotic lesions. The disease incidence reached 40 - 45%, attributed by airborne inoculum spread. Tissues collected from the margin of the infected leaf (5 × 5 mm) were surface disinfected with 1% NaOCl for 1 min, 70% ethanol for 30 sec and rinsed with sterilized water for three consecutive times before incubation at 25 ± 1°C on the potato dextrose agar (PDA) medium for pathogen culturing. Five cultures were obtained through single spore isolation and denoted as PTAA-01 to PTAA-05. The colony morphology of all five isolates was identical, with a greyish-brown and black appearance on the upper and undersides of the Petri dishes. The mycelia were septate, light grey and had a geniculate brown conidiophore with a short conidial chain. Conidia were brown, short-beaked, obclavate to obpyriform in shape, one to six and zero to two transverse and oblique septa. The dimensions of the conidia (n = 50) are 18 - 53 x 9 - 14 (length × width) µm. Morphological characters of the pathogen were consistent with Alternaria alternata (Fr.) Keissl, described earlier by Simmons (2007). Genomic DNA was extracted from the representative isolate PTAA-01 using CTAB method (Murray and Thompson 1980). PCR was performed with primers ITS1 / ITS4 targeting internal transcribed spacer (ITS) sequence (White et al. 1990), gpd1/gpd2 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), PG3/PG2b for endopolygalacturonase (EndoPG), Alt a1F/Alt a1R for Alternaria major allergen gene and EF1-728F/EF1-986R for translation elongation factor (TEF) 1-α (Woudenberg et al. 2015). Amplified products were sequenced and deposited in NCBI GenBank with accession numbers; PP864706 (ITS), PP943430 (GAPDH), PP968830 (EndoPG), PQ031066 (Alt a1) and PQ031067 (TEF1-α) and all shared > 99% identity with A. alternata (GenBank accession numbers: MN919390, KX226447, KP123997, OK040811, and MZ648042), respectively. A neighbour-joining phylogenetic tree was constructed based on concatenated sequences of ITS, GAPDH, EndoPG, Alt a1, and TEF1-α using MEGA 11, where PTAA-01 isolates formed a clade with the strain of CBS 916.96 of A. alternata. For pathogenicity test, the conidial suspension (106 spores/ml) prepared from 15 days old culture was spray inoculated on one-month-old potato cultivar Kufri Jyothi in triplicates and maintained under glasshouse condition with an ambience of 16 h photoperiod, 20 - 25°C temperature and > 70% relative humidity. Symptoms appeared on the 5th day after inoculation and were identical to the natural symptoms observed earlier in the field. The reisolated pathogen had morphological and molecular procedures identical to those of A. alternata, fulfilling Koch's postulate test. While Lingwal et al. (2022) reported brown leaf spots of potatoes in Eastern parts of India, our study marks the first report in Southern India alerting the potato growers of this specific region to the A. alternata outbreak.