First Report of Leaf Spot Disease in Coconut Seedling Caused by Bipolaris setariae in China.

Coconut (Cocos nucifera L.), an important oilseed as well as a multipurpose perennial plantation crop, is distributed and planted in humid tropical areas. In October 2012, a new leaf spot disease was observed on 3-year-old coconut seedlings in Wenchang, Hainan Province, China. The symptom first appeared as spindly or elliptical and brown flecks with water-soaked lesions that became yellow with the progress of the disease. In the later stage of the disease, the lesions merged together, gradually expanding to the leaf apex. In recent years, the disease has been prevalent in all the nursery gardens surveyed. Once young leaves got infected and nearly all the leaves of the tree showed diseased symptoms, the coconut eventually became defoliated. The pathogen was isolated from the lesion margin, surface sterilized with 75% ethanol and 0.1% mercury bichloride, washed by sterile distilled water, and then placed excising pieces of leaves from the leision margin onto potato dextrose agar (PDA). Plates were incubated at 25°C for 4 days. After 7 days, the colony was grayish black and produced black pigment in the medium. Aerial mycelium was fluffy, septate, and branched, the conidiophores were slightly flexuous or straight, 5 to 11 μm thick, and produced curved, spindle-shaped, or fusiform, septate conidia with 4 to 10 septa, measuring 39 to 86 × 9 to 16 μm, with a slightly protuberant hilum, truncated. Based on the symptoms and mycelial and conidial characters above, the fungus was identified as Bipolaris setariae (1). The pathogenicity was established and repeated for six times by following Koch's postulates. Two 1-year-old coconut seedlings were washed with sterilized water and six leaves were wounded with a sterile needle and then inoculated by spraying them with a suspension of conidia of the isolate. The seedlings were kept in two incubators at 25°C for 12 days. Inoculated leaves showed typical symptoms similar to those described above. The pathogen was re-isolated from inoculated leaves. Morphological characteristics were identical to the original isolated fungus. In contrast, the control leaves did not show any symptoms. The genomic DNA of this fungus was extracted, amplification of the internal transcribed spacer (ITS) region was performed with primer ITS1 and ITS4, and the purified PCR product was sequenced (GenBank Accession No. KJ605157). BLASTn analysis revealed 99% sequence similarity with four B. setariae isolates (HE792936.1, JX462256, GU073108.1, and FJ606786.1). Morphologic characters and sequence analysis of the ITS rDNA confirmed that the pathogen was B. setariae. Bipolaris incurvata has been reported causing disease on coconut (2), but B. setariae was not previously reported on coconut. So far, this is the first report of B. setariae caused coconut seedling leaf spot disease in Hainan, China. References: (1) K. C. da Cunha et al. J. Clin. Microbiol. 50:4061, 2012. (2) A. Kamalakannan et al. New Dis. Rep. 12:18, 2005.