First Report of Alternaria alternata Causing Leaf Spot on Sweet Cherry ( Prunus avium L.) in the Western Himalayan Region of India

Sweet cherry (Prunus avium L.) is an economically important stone fruit cultivated in the Shimla district of Himachal Pradesh (H.P.), India (31.1050°N, 77.1640°E), which accounts for over 90% of India's cherry production (~3,000–3,500 ha). During surveys in June–July 2023 and 2024, leaf spot disease was observed at seven localities (Narkanda, Kotgarh, Baghi, Matiana, Theog, Kumarsain, and Thanadhar). Disease incidence ranged from 48.0 to 92.9% and severity from 21.7 to 62.9%, affecting all major commercial varieties. Symptoms consisted of small, irregular dark-brown spots with concentric rings on both leaf surfaces, coalescing to cause necrosis and defoliation. Tissue pieces (2–4 mm) from necrotic leaf margins (20 leaves/orchard; 3–5 orchards/locality; 7 localities) were surface-sterilized with 1% NaOCl (1 min), rinsed in sterile water, and plated on PDA at 25°C for 7 days. The pathogen was recovered from 17 of 20 sampled leaves per orchard (isolation frequency 85%). Ten morphologically consistent isolates were obtained; three representative isolates (UHF-CHERRY-1, -2, -3) were selected based on morphology and sporulation, and deposited at MTCC, CSIR-IMTECH, Chandigarh (MTCC 14201, 14202, 14203, respectively). Colonies turned dark olive-green; diameter 52–58 mm after 7 days. Conidia were obclavate, light to dark brown, 23.37–25.64 × 9.06–11.35 µm (n = 50, sampled equally from all three isolates), with 1–4 longitudinal and 1–5 transverse septa, consistent with Alternaria alternata (Simmons 2007) . DNA was extracted (CTAB method) from all three isolates. ITS was amplified with ITS1/ITS4 (White et al. 1990); gpd and Alt-a1 with GPD1/GPD2 (Berbee et al. 1999) and AltaF1/AltaR1 (Asturias and Arilla 1995). GenBank: ITS — PV628437, OR230257, OR230237; gpd — PZ243383, PZ243385, PV648928; Alt-a1 — PZ243382, PZ243384, PV648929 (UHF-CHERRY-1, -2, -3). BLASTN showed ≥99% ITS and ≥98% gpd and Alt-a1 identity with A. alternata CBS 916.96 (type strain). Sequences were concatenated in BioEdit, aligned with MUSCLE, and a maximum likelihood tree constructed in MEGA 11 (1,000 replicates) with A. alternata CBS 916.96 (type strain), A. arborescens CBS 110.48, A. tenuissima CBS 117.44, A. infectoria CBS 232.96 (Woudenberg et al. 2015), and Stemphylium lycopersici CBS 122803 as outgroup. All three cherry isolates formed a strongly supported clade (bootstrap 100%), which was sister to A. alternata CBS 916.96 with 87% bootstrap support, distinct from A. arborescens, A. tenuissima, and A. infectoria clades . Pathogenicity was assessed for each isolate separately on five potted cherry seedlings (var. Black Heart) per isolate (15 inoculated; five controls with 0.01% Tween-20). Spore suspensions (5×105 conidia/ml) were sprayed on leaf surfaces; plants held at >90% RH, 25±1°C for 24 h then moved to a greenhouse at 25°C. The experiment was repeated twice with similar results. Leaf spots appeared 5–7 days post-inoculation; controls were asymptomatic and isolation from control plants yielded no growth. The fungus was re-isolated and confirmed morphologically and by ITS sequencing, fulfilling Koch's postulates. To our knowledge, this is the first report of A. alternata causing leaf spot on P. avium in India, a finding of significance given the high disease pressure recorded and the importance of cherry production in H.P. Accurate pathogen identification is essential for developing targeted management strategies in this region.