First Report of Curvularia lunata Causing Leaf Spot on Lotus in China

Lotus (Nelumbo nucifera Gaertn.) is a flowering aquatic plant, and is widely planted as a vegetable and ornamental plant in China. In June 2011, a leaf spot was observed on lotus in Pingxiang City of Jiangxi Province, causing approximately 60% of leaves to die and leading to 10 to 15% yield loss. Initial symptoms were purple-brown spots emerging on the leaf surfaces with diameters ranging from 0.5 to 3 cm, which later developed grayish white centers and a black-brown banding pattern on the edges. Lesions often merged to form large necrotic areas, covering more than 70% of the leaf surface, which may have contributed to plant death. Small pieces (5 mm²) of symptomatic leaves were excised from the junction of diseased and healthy tissue, surface sterilized in 70% ethanol solution for 1 min and 0.1% mercuric chloride solution for 5 min, washed in three changes of sterile distilled water, and transferred to potato dextrose agar plates. Cultures were maintained in an incubator at 25°C for 5 to 7 days. After 7 days, six black-brown colonies were isolated, which developed dark brown septate conidiophores. Conidia were 20 to 25 × 9 to 13 μm, with three-horizontal septa, and curved at the third cell from the base that was longer and darker than the others. Cells at each end were subhyaline and intermediate cells were medium brown. These characteristics were consistent with Curvularia lunata (Wakker) Boedijng (1,2,4). Molecular characterization was based on rDNA sequence. For two isolates, DNA was extracted using a CTAB protocol with 0.8% mercaptoethanol, then the ITS1-5.8S-ITS2 region was amplified with primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') (3). PCR products were cloned and sequencing reactions were run on an AB3730 Stretch DNA sequencing system. On the basis of a comparison of 598 base pairs, both isolates had the same sequence (GenBank Accession No. JQ701798), which differed by one base pair from Cochliobolus lunatus NBRC 100173 (GenBank Accession No. JN943426) (conidial state: Curvularia lunata). Pathogenicity experiments were conducted by inoculating a conidial suspension (10⁶ CFU/ml) on five newly matured leaves of healthy lotus. Plants inoculated with sterile water served as the noninoculated controls. Plants were incubated in the greenhouse at 20 to 25°C. All the inoculated leaves started showing disease symptoms (purple flecks) after 7 days and the noninoculated control plants remained asymptomatic. C. lunata was consistently recovered from all inoculated plants, except the control, thus fulfilling Koch's postulates. To our knowledge, this is the first report of leaf spot caused by C. lunata on lotus in China. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, Surrey, England, 1971. (2) M. M. Hawa, et al. Plant Dis. 93: 971, 2009. (3) K. J. Martin and P. T. Rygiewicz. BMC Microbiol. 5:28, 2005. (4) F. B. Rocha et al. Austral. Plant Pathol. 33: 601, 2004.