First report of black pepper (Piper nigrum L.) anthracnose caused by Colletotrichum siamense in north-east India.

Abstract

Black pepper (Piper nigrum L.) has been commonly cultivated as a spice crop in northeast India. In August 2021, anthracnose leaf spot was observed on black pepper vines with 50 to 60% of disease incidence in Assam Agricultural University, Jorhat (26.7509° N, 94.2037° E), Assam, India. On average, 80% of the leaves per individual vine were affected by this disease. Foliar symptoms initially appeared as chlorotic circular spots, which then coalesced into larger irregular lesions. The centers of the spots were brown, papery in texture, and surrounded by a yellow halo. Numerous acervuli at the center of the spots were observed. Ten vines from the orchard were sampled to identify the causal agent. Symptomatic leaves along with some healthy portion were cut (3 to 4.5 mm2), surface-sterilized in 70% ethanol for 30 s, rinsed in sterile distilled water twice, dried on sterilized filter paper, aseptically plated on potato dextrose agar (PDA) amended with Streptomycin sulphate (30 mg/L), and then incubated at 25°C for four days. Two Colletotrichum isolates were recovered from infected tissues and purified by the hyphal tip method. Fungal colonies on PDA were cottony, dense, white to gray in color, and with salmon pink conidial masses. Conidia (n = 50) were 13.6 to 19.8 × 4.2 to 6.4 μm, cylindrical, hyaline, single-celled, smooth-walled, and with rounded ends. Conidiophores were aseptate, hyaline, short and branched. Based on morphological features, the isolates were identified in the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For accurate identification of two isolates, the DNA was extracted from pure culture. The internal transcribed spacer (ITS) region, actin (ACT), β-tubulin 2 (TUB2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified by polymerase chain reaction (Weir et al. 2012) and sequenced. The sequences were deposited in the GenBank database (ITS: OP297054 and OP296876; ACT: OP327082 and OP327081; TUB2; OP327086 and OP327085; GAPDH: OP327084 and OP327083). A BLAST analysis of ITS, ACT, TUB2 and GAPDH sequences revealed 99.5-100%, 99.9-100%, 99.9-100% and 99.8-100% similarity respectively to C. siamense for both isolates in NCBI database. The pathogenicity tests were carried out on potted four months old vine cuttings of P. nigrum L., which were kept in a greenhouse. Ten healthy plants were sprayed with 50 µl of conidial suspension of each isolate (107 conidia ml-1, 10 ml/plant). Five control plants were sprayed with sterile distilled water. The plants were covered with sterilized plastic bags after inoculation to maintain humidity and kept in a greenhouse at day/night temperatures of 25 ± 2°C and 17 ± 2°C (Zhang et al., 2021). Within eight days, all the inoculated plants showed symptoms similar to those observed in the field, whereas control plants were asymptomatic. The pathogenicity test was repeated twice. C. siamense was consistently reisolated from the lesions and was confirmed by morphological characterization and molecular assays as described above in this note, whereas no fungus was isolated from control leaves. To our knowledge this is the first report of C. siamense causing black pepper anthracnose in northeast India. The pathogen has significant potential for causing high losses in black pepper production. These data will help researchers to develop effective management strategies for this disease.