First Report of Curvularia lunata Causing Leaf Spots on Sorghum bicolor from Pakistan

In October 2012, reddish brown, oblong lesions with chlorotic centers were observed on the leaves of Sorghum bicolor in Punjab Province, Pakistan. Early symptoms appeared as reddish brown circular spots on the leaves. These spots increased in size and coalesced to form oblong lesions. Entire fields were severely affected by the disease. Pathogen isolations were made on malt extract agar (MEA) media. Symptomatic leaf samples were cut into 4 to 6 mm² pieces, surface sterilized (10% bleach for 1 min, 90% ethanol for 30 sec) and rinsed in sterilized water several times, followed by air drying. These samples were plated onto 2% MEA media, supplemented with 10 mg/liter chloramphenicol, and incubated at 25°C for 6 days in the dark. A mitosporic fungus of dark brown colony, bearing large stroma, appeared on the media. Conidiophores were brown, septate, geniculate, simple or unbranched, with dark brown scar. Conidia were brown, straight to pyriform, with 3 to 4 cells, with large and curved central cells, smooth walled, ranging in size from 7.3 to 21.26 μm, and produced apically in a sympodial manner. Based on morphological characteristics, the pathogen was identified as Curvularia lunata (Wakk.) Boedijn. (1,2). Morphological identification was also confirmed by the First Fungal Culture Bank of Pakistan (FCBP), Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan, and samples were submitted to FCBP (Accession No. 1201). The fungus was further identified by amplifying internal transcribed spacer region sequences (ITS1, rDNA, ITS2) by using ITS4 and ITS5 primers (4). The resulting 584-bp sequence was submitted to GenBank with Accession No. HG326308. This sequence showed 99% homology with C. lunata strain pingxiang (GenBank Accession No. JQ701897), causing leaf spots of lotus in China. Pathogenicity assay was conducted on 20-day-old seedlings of S. bicolor variety Indian Gold, grown from surface sterilized seeds. Fifteen replicate plants were sprayed with a spore suspension of 1 × 10⁶ spore/ml in distilled sterilized water, prepared from 1-week-old fungal culture, grown in the dark on 2% MEA media. Five replicate plants were sprayed with distilled sterilized water as control. Plants were covered with transparent polyethylene bags to retain moisture and enhance disease development, and kept in a greenhouse at ~30°C. Bags were removed after 5 days of incubation. Inoculated plants developed lesions similar to those observed on naturally infected plants. No symptoms were observed on control plants. The pathogen was re-isolated from infected leaves, and the morphology features were again studied, matching those of the pathogen isolated from field samples. Curvularia leaf spot diseases, caused by different Curvularia species, have been previously found on many grass species worldwide (3). To our knowledge, this is the first report of C. lunata leaf spots on S. bicolor in Pakistan. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, Surrey, England, 1971. (2) F. B. Rocha et al. Austral. Plant Pathol. 33:601, 2004. (3) J. D. Smith et al. Fungal diseases of amenity turf grasses. E & F.N. Spon., New York, 1989. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.