First report of <i>Curvularia dactylocteniicola</i> causing leaf spot disease of <i>Vigna unguiculata</i> in India

Vigna unguiculata (cowpea) is a leguminous crop in the family Fabaceae (Alemu et al., 2019). The seeds are highly nutritious and play a crucial role in commercial agriculture across Africa and Asia. In November 2023, dark-brown spot symptoms were observed on leaves of V. unguiculata in cultivated fields near Gauhati University (26°9'5.2“N, 91°39'17.5”E), Guwahati, Assam, India. In the early stages of infection, small, circular, brown spots appeared on the leaf blades. As the disease progressed, these spots expanded, developing a yellow-halo border around a tan centre. Eventually, the spots enlarged and the entire width of the leaf blade became blighted (Fig. 1). Fifteen diseased leaves were collected randomly from three diseased plants and washed under running tap water to remove soil and debris. The samples were surface sterilised by immersing in 70% ethanol for one minute, followed by 0.2% sodium hypochlorite solution for one minute, and then rinsed three times with distilled water (Omar et al., 2018). The leaves were cut into small fragments (5 × 5 mm) using a sterile scalpel. Each fragment was placed on a potato dextrose agar (PDA) plate and incubated at 28 ±2°C for seven days. Hyphal tips emerging from the fragments were subcultured onto freshly prepared PDA plates to obtain pure cultures. Altogether, 10 fungal isolates with similar morphological characteristics were obtained. The causal organism was isolated and a representative isolate was designated as ‘KR-CU1’. The colony of isolate KR-CU1 on PDA medium was circular, raised with an entire edge, rough, sparse to dense, and grey to whitish grey, whereas the reverse side was dark grey to blackish and did not produce pigment in artificial media (Fig. 2). The hyphae were septate, smooth, or asperulate. Conidiophores micronematous, septate, straight or flexuous, 63.5–93.5 × 2.8–4.7 µm in size. Conidia were asymmetrical to more or less curved at the third cell from the base, 2–3 septate, dark brown, smooth-walled, and measured 14.5–28.5 × 7.5–12.5 µm (n = 20). Microconidiation present, 1-celled, pale brown, thicker wall, globose or subglobose, 2–5 µm wide (Fig. 3). Morphological characteristics of isolate KR-CU1 were consistent with Curvularia dactylocteniicola as described by Marin-Felix et al. (2017). Genomic DNA was extracted using the CTAB method, and the ITS region was amplified and sequenced using the ITS1/ITS4 primers (White et al., 1990). The consensus sequence was submitted to GenBank (Accession No. PP794705). BLASTn analysis revealed that the sequence had 100% identity with the type sequence of Curvularia dactylocteniicola strain CPC 28810 (MF490815.1). A phylogenetic tree was constructed using the maximum likelihood method and the Kimura 2-parameter model with MEGA11 software (Tamura et al., 2021). The phylogenetic analysis showed that isolate KR-CU1 clustered closely with C. dactylocteniicola (Fig. 4). Three one-month-old plants of V. unguiculata cv. Kashi Kanchan cultivated in pots were used for a pathogenicity assay. One healthy leaf per plant was surface sterilised with 70% ethanol, washed with sterile distilled water three times and dried with sterile filter paper. Wounds were made using a sterile scalpel and 20 µL of a conidial suspension (1×107 conidia/ml) obtained from a culture of isolate KR-CU1 was inoculated using a micropipette; sterile distilled water served as a control. The inoculated area was covered with a punched transparent plastic bag and moistened with distilled water. Both control and inoculated plants were maintained in a greenhouse at 28 ±2°C with a 12-hour photoperiod and 80% relative humidity. After seven days, the leaves inoculated with the conidial suspension showed similar symptoms to those observed in the field, while the control plants exhibited no signs of disease symptoms (Fig. 5). The pathogen was re-isolated from symptomatic parts of the inoculated leaves and exhibited similar morphological characteristics to those described above, fulfilling Koch's postulates. Species of Curvularia are destructive pathogens causing necrotic and leaf spot diseases in various agricultural crops and Curvularia dactylocteniicola causes leaf spot disease in maize and sorghum in Sri Lanka (Ferdinandez et al., 2024). Curvularia caryopsidum, a closely related species to C. dactylocteniicola, has been reported to cause disease on V. unguiculata (Manamgoda et al., 2011). However, to the best of our knowledge, this is the first report of C. dactylocteniicola causing leaf spot disease on V. unguiculata in India and globally. This report will enable the development of proper management strategies to control leaf spot disease of cowpea and reduce potential damage caused by this pathogen. The authors are grateful to the Head of the Department of Botany, Gauhati University for providing the necessary facilities to conduct this work.