Mucuna is the source of L-Dopa (L 3,4 dihydroxyphenylalanine), a precursor of a dopamine used to treat Parkinson's disease. Leaf blight symptoms were observed on Mucuna pruriens plants in October to November 2010 in a field at the Indian Council of Agricultural Research Complex for the Northeastern Hill Region, Umiam, Meghalaya, India. Symptoms included black necrotic areas on leaves, collapsed leaf tissue and, occasionally, fungal growth visible on the leaf. In advanced infections, dead leaves were attached to the stem, followed by defoliation with only infected pods still attached. Approximately 10% of plants were infected in ~0.5 ha surveyed. Symptomatic leaf pieces were washed with sterile water, surface-sterilized using 4% NaOCl for 30 s, washed again, blotted dry, and plated on PDA amended with streptomycin (100 ppm). Characteristics of three fungal isolates were typical of Rhizoctonia solani J.G. Kühn [teleomorph = Thanatephorus cucumeris (A. B. Frank) Donk], i.e., hyphal branching at 90° angles, basal constriction at the hyphal branching point with a septum close to the lateral hyphum (3), and presence of multinucleate hyphal cells confirmed using DAPI (2-(4-amidinophenyl)-1H-indole-6-carboxamidine) staining (1). A culture was deposited at the Agharkar Research Institute, Pune, India (NFCCI No. 2602). The ITS1-5.8S-ITS2 region of nuclear rDNA of one isolate was sequenced after amplification with primers ITS1 and ITS4 (4), (GenBank Accession No. JQ675536). A BLAST search revealed 99% similarity of the sequence with that of 2 R. solani AG 1-IB isolates (AB122137 and AB000039). Sequences were aligned using MAFT Version 6. Maximum parsimony analysis using MEGA 5 placed the test isolate in AG 1-IB with 99% bootstrap support. PCR assays with primers for R. solani AG 1-IB produced a DNA band of ~300 bp (2). Koch's postulates were completed by inoculating 5-mm colonized plugs of PDA at the soil line of each of 5 potted, 40-day-old plants of M. pruriens, and covering the base of each plant with moistened cheesecloth. In addition, 3 plants were inoculated with colonized plugs at the junction of the lamina and petiole of 9 leaves/plant, spraying the plants with sterilized water, and covering the plants with polythene for 3 days. In addition, 10 detached leaves were inoculated with colonized PDA plugs and incubated in a moist chamber. Three non-inoculated plants served as a control treatment for the first 2 methods, and 10 leaves as a control treatment for the third method with sterilized PDA plugs. Symptoms of leaf blight (necrosis from base to leaf tip, with abundant fungal growth) developed in 6 to 7 days on plants inoculated at the soil line, 4 days on leaves inoculated at the junction of the lamina and petiole, and 2 to 3 days on detached leaves. Control plants and leaves remained asymptomatic for all 3 methods. R. solani was reisolated from inoculated plants as described above, and confirmed to be AG 1-IB. The fungus was not reisolated from control plants or leaves. To our knowledge, this is the first record of R. solani AG 1-IB causing leaf blight on M. pruriens in India.
References: (1) M. M. Kulik and P. D. Dery. Biotech. Histochem. 70:95, 1995. (2) M. Matsumoto. Mycoscience 43:185, 2002. (3) B. Sneh et al. Identification of Rhizoctonia Species. The American Phytopathological Society Press, St Paul, MN, 1991. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.