First Report of Nigrospora sphaerica associated with Leaf Spot Disease of Cowpea (Vigna unguiculata) from India.

Cowpea (Vigna unguiculata (L.) Walp) is one of the main legume crops grown in arid and semi-arid regions in the world. Brazil, Haiti, Myanmar, Nigeria, Sri Lanka, United States, and India contributes to the substantial production of cowpea at the global level (Mahadevakumar and Janardhana, 2012, 2014). Field surveys conducted during 2017-19 (August-September) in major cowpea growing regions of southern Karnataka revealed the occurrence of characteristic leaf spot disease of unknown etiology with an incidence ranging from 6 to 8%. Initially, the symptoms developed as small specks (1.5 to 3.5 mm), characterized by circular or irregular shape. These lesions began to develop from the leaf margin and regularly extended and coalesced to form larger lesions. After the successful manifestation of the symptoms on leaves, the associated fungal pathogen was isolated. In brief, the infected leaves were surface sterilized with 2% NaOCl for 2 min, rinsed thrice in sterile distilled water (SDW) and blotter dried. The leaf sections were placed on potato dextrose agar (PDA) in Petri plates and incubated at room temperature (27 ± 2°C) for 10 to 12 days. Mycelia developed from infected tissues were transferred to fresh PDA plates and pure cultures were obtained. Mycelia were initially white and eventually turned into gray. The conidia were black, single-celled, smooth, spherical to subspherical, 10 to 22 μm in diameter (n=30), and borne singly on a hyaline vesicle at the tip of each conidiophore. Based on the cultural features and conidial morphology, the fungus was identified as Nigrospora sp. Further, to identify the pathogen to the species level, the ITS region of the ribosomal RNA gene was amplified using primers ITS1 and ITS4 (White et al. 1990). The amplified PCR products were purified and sequenced. The nBLAST analysis showed 100% similarity with reference sequences from the GenBank database Nigrospora sphaerica (MT225783.1; MN795578.1), and the sequences were deposited in GenBank (Accession No. MT305812.1, MT305813.1, MT305814.1). Based on the cultural, morphological, microscopic and molecular characteristics, the associated fungal pathogen was identified as N. sphaerica (Sacc.) Mason (Chen et al. 2018; Wang et al. 2017) and a voucher specimen was deposited at University of Mysore Herbarium with accession No. UOM20-NS1. Further, pathogenicity tests were conducted on healthy cowpea plants grown under greenhouse conditions. Inoculations were made with conidial suspension (105 conidia/ml) prepared in SDW and healthy plants sprayed with SDW served as a standard control. All the plants were covered with polyethylene bags for 24-48 hr and observations were made at regular intervals. Typical necrotic lesions developed after 12 days of inoculation and no such symptoms were observed on the standard control set. The associated pathogen was re-isolated from diseased leaves and its identity confirmed based on morphology and cultural characteristics. Leaf spots are becoming a major problem in cowpea growing areas in recent years (Dactuliophora sp., Pestalotiopsis leaf spot, Alternaria leaf spot, and many others) (Mahadevakumar and Janardhana 2012, 2014). Recently, Aplosporella hesperidica causing collar rot on cowpea has been reported from the same region (Deepika et al. 2020). The seed borne occurrence N. sphaerica on cowpea is reported from Brazil (Rodrigues and Menezes 2002), there are no previous reports available on the occurrence of N. sphaerica on cowpea leaf spots, the present investigation is the first report of N. sphaerica causing leaf spot disease on cowpea from India.