First Report of <i>Colletotrichum siamense</i> Causing Fruit Rot of Sweet Orange ( <i>Citrus sinensis</i> ) in India

Citrus sinensis (sweet orange) is an economically important horticultural commodity in global fruit markets owing to its excellent organoleptic qualities, coupled with a rich nutritional profile that includes essential vitamins, minerals, and antioxidants. While conducting a field survey (350 hectares) during the kharif season of 2023 in Maharashtra state, India, fruit rot disease was observed with an incidence of 25-30% on sweet orange fruits. Infected fruits exhibited large, circular, sunken lesions on the surface, surrounded by a slightly raised or discoloured margin. In advanced stages, fruits exhibited extensive rotting and premature fruit drop. From the diseased fruit samples collected during the survey, four fungal isolates were obtained. These isolates were designated as CS vk_mpkv_101 (Amravati-21°14′4.26″N, 78°1′14.31″E), CS vk_mpkv_102 (Nagpur-21°15′38.2″N, 78°39′35.4″E), CS vk_mpkv_103 (Chhatrapati Sambhajinagar-19°51′19.6″N, 75°28′18.8″E), and CS vk_mpkv_104 (Jalna-19°51′11.50″N, 75°40′40.21″E). Isolation of the fungus was performed on PDA medium using the standard tissue isolation method, followed by purification using the single spore isolation method (Johnston and Booth 1983). Microscopic examination revealed that all four isolates of the pathogen produced septate mycelium and conidia. The conidia were unicellular, hyaline, cylindrical to fusiform, with a round apex and were guttulate. The size of conidia of all four isolates ranged from 11.5-15.9 × 2.9-5.5 μm. Cultural studies of all the isolates of fungi revealed that the colony colour varied from cream to greyish with whitish to slight yellowish pigmentation. Texture was cottony and fluffy with raised mycelial growth. Pathogenicity of all four isolates was confirmed on fresh sweet orange fruits by using the pin-prick method, satisfying Koch’s postulates. The characteristic rotting symptoms as observed on naturally infected fruits were reproduced during the pathogenicity test. All the isolates of the pathogen exhibited similar morphological and cultural characteristics. Based on these morphological and cultural characteristics, all four isolates were identified as Colletotrichum siamense (Fayyaz et al. 2020; Kaur et al. 2024). Among the four isolates, Colletotrichum siamense isolate vk_mpkv_101 was the most virulent, inducing severe rotting symptoms within the shortest incubation period (5 days), compared to other isolates. Consequently, the isolate vk_mpkv_101 was selected for molecular identification using ITS, β-tubulin, Actin, and GAPDH gene sequences, which were amplified with standard primers (White et al., 1990; Templeton et al., 1992; Glass and Donaldson, 1995). Blast analysis of the sequences generated from all four gene regions showed the highest sequence similarity with Colletotrichum siamense, thereby confirming the molecular identity of the isolate as C. siamense. Sequences were deposited in NCBI GenBank, and accession numbers received are PX248333 (ITS), PX113340 (Actin), PX117002 (β-tubulin), and PX113339 (GAPDH). The pure fungal culture was submitted to the National Fungal Culture Collection of India, and the accession number received for the culture is NFCCI-5995. Earlier, this fungus was reported as a causal agent of fruit rot on Kinnow fruit by Fayyaz et al. (2020) in Pakistan, on citrus by Wang et al. (2021) in Australia and on Kinnow fruit by Kaur et al. (2024) in India. To our knowledge, this is the first report of Colletotrichum siamense causing fruit rot in sweet orange (Citrus sinensis) in India. Identification of the pathogen provides a foundation for future research on effective management strategies, as it poses a significant threat to Citrus sinensis production in India.