First Confirmed Report of Leaf Blight on Browntop Millet Caused by Bipolaris setariae in Southern Peninsular India

Browntop millet (Brachiaria ramosa (L.) Stapf), which is native to the United States, was recently introduced into India as one of the small millet crop. In September 2018, leaf blight symptoms were observed on cv. Dundu Korale on the adaxial side of the leaves in a field at Bangalore, India (13.0784oN, 77.5793oE). Initial lesions were brown with small yellow halo that ranged from 1 to 5 mm and eventually enlarged exhibiting light brown centers. Afterwards, spots coalesced and leaves were blighted. About 75% of the plants were infected in the field of 0.5 ha. Samples of symptomatic and asymptomatic leaves were collected, and nine isolates were recovered from culture on potato dextrose agar (PDA). Single conidial isolation was performed. Colonies were grey to olive green with regular margins at 7 days when cultured on PDA at 27 ± 1oC and 16 h light and 8 h dark cycles. Conidiophores were single or in clusters from 4.92 to 6.04 μm in width. Conidia were fusoid, cylindrical to slightly curved ranging from 38.50 to 130 μm in length and from 8.30 to 17 μm in width, with 4 to 10 distosepta (n =100). Hilum was flat to inconspicuous or slightly protruded. Based on the morphology, the pathogen was identified as a Bipolaris species according to the genus standard descriptions of Helminthosporium (Alcorn 1988). Misra and Prakash (1972) reported that Helminthosporium setariae causing leaf spot on browntop millet in India, but they did not provide any morphological or molecular evidence of identification. For molecular identification, the genomic DNA of isolate BTMH3 was extracted and the internal transcribed spacer region (ITS) and glyceraldehyde-3-phosphate dehydrogenase gene (GPD) were amplified using the pairs of primers ITS1/ITS4 (White et al. 1990) and GPD1/GPD2 (Berbee et al. 1999), respectively. BLAST analysis of ITS (MT750301; 562 matching base pairs) and GPD (MT896702; 562 matching base pairs) sequences showed 99.82% of identity with sequences of Bipolaris setariae (Sawada) Shoemaker type strain CBS 141.31 (ITS: EF452444 and GPD: EF513206). Pathogenicity was proved by spraying 10 healthy 20-day-old browntop millet plants with conidial suspension (106 conidia/ml). Control plants were sprayed with distilled water. Plants were covered with transparent polythene bags in a greenhouse at 28 ± 2oC and high relative humidity of 90%. Symptoms were observed at five days post inoculation. The pathogen was re-isolated from infected areas using the same protocol as before