First report of Colletotrichum truncatum causing anthracnose of ashwagandha (Withania somnifera) in India.

Abstract

Ashwagandha (Withania somnifera), enriched in alkaloids, steroidal lactones and saponins, is a valuable herb in Indian Ayurvedic medicine. During December 2023, Va-1 (Vallabh Ashwagandha-1) plants at ICAR -Central Tobacco Research Institute, Vedasandur, Tamil Nadu (10.53717ºN, 77.9507°E) were noticed with anthracnose-like disease symptoms, characterized by irregular necrotic lesions on stem, leaves and pods. Infected plants dried entirely in the advanced stage and had pin-head-shaped fruiting bodies. In the assessment of 185 plants, 42 were symptomatic, while five were dried, and the disease severity varied between 17 to 22%. Pathogen was isolated from infected leaf and stem tissues (3 to 5 mm2) using a potato dextrose agar (PDA) medium amended with streptomycin sulphate (20 mg/l). Infected tissues were surface sterilized using 1% sodium hypochlorite, and the PDA plates were incubated at 27°C for three days. The pathogen was purified, and two cultures, AVCT01 and AVCT02, were used for the downstream characterization. Colonies on the PDA medium were initially pale white and later turned to dark grey, with an average growth of 42.6 mm (diameter) on the 7th day after inoculation (DAI). Conidia were smooth, hyaline, aseptate and curved, with a tapering end and truncate base. The conidial dimensions (n=30) were 22.8 ± 1.32 × 3.4 ± 0.29 µm (20.6 to 25.8 × 2.9 to 4.2). Acervuli were scattered, containing dark brown conidiomata with septate, pointed setae and measured 119 ± 16.6 × 4.2 ± 0.5 µm (81 to 148 × 3.1 to 5.3) in dimension. Morphological and cultural characteristics were identical to Colletotrichum truncatum described by Damm et al. (2009). Further, the molecular characterization was executed with primers targeting ITS (ITS1/ITS4) and three housekeeping genes; act (ACT-512F/ACT-783R), tub2 (Bt2a/Bt2b) and gapdh (GDF1/GDFR1) (Kim et al. 2020). The amplified products were sequenced bidirectionally and in BLASTn analysis, sequences revealed 100% identity with C. truncatum [(NCBI GenBank OM327672 (ITS), OR147336 (act), MG214130 (tub2) and MK675260 (gapdh)]. In the maximum likelihood phylogeny of concatenated alignments, these cultures clustered with CBS119189. All sequences were deposited in NCBI GenBank (ITS: PP919605 and PP919606; act: PP921525 and PP921526; tub2: PP921527 and PP921528; gapdh: PP921529 and PP921530). Pathogenicity was done by spraying conidial suspension of AVCT01 (1 × 106/ml) on leaves of three plants (two months old). Non-injury technique of Gupta et al. (2020) was conducted for stem inoculation. The inoculated and control plants (sprayed with sterile distilled water) were maintained separately at 25°C with 90% relative humidity. Necrotic lesions were observed on leaves and stems of inoculated plants on 8th DAI confirming Koch's postulates and the pathogen re-isolation was also done. Disease severity was assessed using the scale of Yang and Hartman, 2015. Previously, leaf spots caused by C. gloeosporioides and C. dematium were reported in ashwagandha from West Bengal (Sarkar and Dasgupta, 2017) and Uttar Pradesh (Wijeskara et al. 2005) states of India. However, our study marks the first report of C. truncatum in ashwagandha, alerting the adaptation of multiple Colletotrichum species to this highly valued medicinal herb. Elucidating the etiology of newly adapted pathogen species paves the way for rapid disease diagnostics to implement effective disease management strategies.