First report of Fusarium incarnatum-equiseti species complex as the causal agent of pod rot of black gram (Vigna mungo L.) in India.

Black gram (Vigna mungo L.) is important pulse crop and is the third-most cultivated pulse in India (Swaminathan et al. 2023). In August 2022, pod rot symptoms were observed on a black gram crop with 80 to 92% disease incidence in the Crop Research Center, Govind Ballabh Pant University of Agriculture & Technology, Pantnagar (29.0222° N, 79.4908° E), Uttarakhand, India. Disease symptoms included white to salmon pink fungal-like growth over the pods. The symptoms were more severe initially at the tip of the pods, which expanded at later stages to cover the entire pod. Seeds present in the symptomatic pods were severely shrivelled and non-viable. Ten plants from the field were sampled to identify the causal agent. Symptomatic pods were cut into pieces, surface-disinfested with 70% ethanol for 1 min to reduce contaminants, rinsed with sterilized water three times, dried on sterilized filter paper, and aseptically placed on potato dextrose agar (PDA) amended with 30 mg/liter streptomycin sulphate. After 7 days of incubation at 25°C, three Fusarium-like (FUSEQ1, FUSEQ2, and FUSEQ3) isolates were purified by single spore transfer and subcultured on PDA. Fungal colonies on PDA were initially white to light pink, aerial, and floccose, and later on, colonies turned ochre yellowish to buff brown. After transfer to carnation leaf agar (Choi et al. 2014), isolates formed hyaline, 3 to 5 septate macroconidia, 20.4 to 55.6 × 3.0 to 5.0 µm (n = 50) with tapered, elongated apical cells and prominent foot-shaped basal cells. Chlamydospores were thick, intercalary, globose, and abundant in chains. No microconidia were observed. Based on morphological characters, the isolates were identified as belonging to the Fusarium incarnatum-equiseti species complex (FIESC) (Leslie and Summerell 2006). For molecular identification of the three isolates, total genomic DNA was extracted using the PureLink® Plant Total DNA Purification Kit (Invitrogen™, ThermoFisher Scientific, Waltham, Massachusetts, United States) and used for amplification and sequencing of a portion of the internal transcribed spacer (ITS) region, the translation elongation factor-1 alpha (EF-1α) gene, and the second largest subunit of RNA polymerase (RPB2) gene (White et al. 1990; O'Donnell 2000). The sequences were deposited in the GenBank database (ITS: OP784766, OP784777, and OP785092; EF-1α: OP802797, OP802798, and OP802799; RPB2: OP799667, OP799668, and OP799669). Polyphasic identification carried out in fusarium.org indicated 98.72 % and 100% similarity of FUSEQ1 and FUSEQ2 respectively to F. clavum and FUSEQ3 had 98.72 % similarity to F. ipomoeae. Both of the identified species are members of the FIESC (Xia et al. 2019). Pathogenicity tests were carried out on 45-day-old potted plants of V. mungo with seed pods, which were kept in a greenhouse. Plants were sprayed with 10 ml of a conidial suspension of each isolate (107 conidia/ml). 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 25 ± 2°C. Within ten days, all the inoculated plants showed symptoms similar to those observed in the field, whereas control plants were symptomless. The pathogenicity test was repeated twice. Fungi consistently reisolated from the symptomatic pods were confirmed by morphological characterization and molecular assays as described above to belong to the FIESC, whereas no fungus was isolated from control pods. Fusarium spp. causing pod rot in green gram (V. radiata L.) has also been reported from India (Buttar et al. 2022). To our knowledge, this is the first report of FIESC as a causal agent of pod rot of V. mungo in India. The pathogen has the potential to cause considerable economic and production losses in black gram, and for that reason, disease management strategies should be implemented.

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

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), ß-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.