First Report of Pestalotiopsis Species Causing Leaf Spot of Cowpea (Vigna unguiculata) in India
Cowpea (Vigna unguiculata (L.) Walp) is an important legume crop cultivated in arid and semi-arid regions in underdeveloped and developing countries. India is a leading cowpea producer. In addition to India, Nigeria and Niger are the predominant producers of cowpea in the world. Brazil, Haiti, Myanmar, Sri Lanka, and the United States are also significant producers of cowpea. This is a drought-tolerant annual crop that thrives in warm weather (3), and is more well-adapted to the drier regions of the tropics than any other legume. Cowpea fields (190 ha) surveyed in Mysore district (Karnataka State) from 2010 to 2012 were found affected by a new leaf spot disease. Over 60% of surveyed fields were affected by this disease, with individual fields ranging from 30 to 75% disease incidence. Individual fields experienced an estimated 10 to 15% yield loss. Initially, leaf spot symptoms appeared as small, dark, necrotic lesions that increased to a diameter of 0.5 to 1.0 cm. These spots later enlarged to form brown, circular, elliptical, and irregular spots with halo margins. Symptoms persisted throughout the cropping season. Under severe infection, defoliation occurred. Black, sessile, discoid conidiomata were observed in lesions and exuded a pink spore mass that later turned brown. The fungus was isolated from affected leaf tissues that were surface sterilized with 2% NaOCl 2 solution, washed thrice with sterile water, blotter dried, and inoculated onto potato dextrose agar (PDA). White mycelia produced black globular acervuli with conidia on PDA after 7 days of incubation at 28 ± 2°C with a 12-h alternate light and dark period. Conidia had 5-celled (21.37 to 24.89 × 6.3 to 6.9 μm) segmentation with darker median cells and hyaline end cells. The apical cell typically had three appendages (sometimes 2 to 4) measuring 22.0 to 27.3 μm long and the basal appendage was 3.47 to 6.2 μm long. Based on these morphological features, the fungal pathogen was identified as Pestalotiopsis species. The isolated fungus was tested for pathogenecity on 30-day-old healthy cowpea plants grown under greenhouse conditions. A conidial suspension was prepared from 7-day-old culture by flooding with 2 to 4 ml of sterile distilled water. Spores were collected with a sterile micropipette and spore concentration was adjusted to 3 × 10 6 conidia/ml and applied as foliar spray onto 15 plants each in three replicates. Non-inoculated control plants were sprayed with sterile water. The plants were kept under high humidity (80%) for 5 days and at ambient temperature (28 ± 2°C). After 10 to 12 days post-inoculation, leaf spot symptoms appeared on inoculated plants, and the fungal pathogen was re-isolated and no such symptoms were found on control plants. The pathogen was confirmed by micro-morphological features. The ITS region of the ribosomal RNA gene was amplified using primers ITS1 and ITS4 (2). The amplified PCR product was purified and sequenced. nBLAST search comparison of sequences revealed 99% homology to Pestalotiopsis photiniae (AY682946.1). A representative sequence was deposited in GenBank (KC568288.1). Pestalotiopsis is an important pathogen on many crop plants and has been recorded on a wide variety of hosts, primarily on leaves, fruits, and in the rhizosphere. In recent times, cowpea is susceptible to a wide range of fungal pathogens causing severe yield loss at all stages of growth and development (1). Leaf spot caused by Pestalotiopsis species are becoming a major constraint for cowpea production in India. No previous reports are available on Pestalotiopsis species causing leaf spot of cowpea in India. References: (1) S. Mahadevakumar and G. R. Janardhana. New Dis. Rep. 25:17, 2012. (2) T. J. White et al. PCR Protocols: A Guide to Methods and Applications, page 315. Academic Press, San Diego, 1990. (3) A. A. Zohri et al. Korean Mycol. 20:252, 1992.