First Report of Leaf Spot Caused by Corynespora cassiicola on Acanthus ilicifolius in China.

Abstract

Acanthus ilicifolius, also referred to as holly mangrove, usually grows in the coastal areas in southern China. It is not only a pioneer species of mangroves but also an important medicinal mangrove plant. In June 2019, leaf spots on A. ilicifolius with disease incidence of about 30 to 40% were observed in about 150 plants in a field located in Zhanjiang City, Guangdong Province, China. The disease mainly occurred on the leaf margins or tips. Initial symptoms of the disease were dark brown spots (5 to 9 mm) surrounded by a yellow halo, that expanded irregularly or semicircularly, until finally the leaves turned chlorotic and defoliated. Six samples of symptomatic leaves were excised, surface sterilized with 75% ethanol 30 s, 0.1% HgCl2 30 s, and rinsed three times with sterile water, and cut into small pieces (5 mm × 5 mm), placed on potato dextrose agar (PDA) at 25°C and darkness, and two isolates with different morphological characteristics were obtained after 3 days. For pathogenicity tests of the isolates, wounded and non-wounded leaves were inoculated. Fresh wounds were made with a sterile needle. Mycelial plugs from 8-day-old cultures of two isolates, ALY1 and ALY2, on PDA were inoculated on adaxial surface of wounded and non-wounded healthy young leaves of the A. ilicifolius seedlings (2 years old, 4 leaves per plant, 2 replicate plants per isolate), covered with wet cotton and kept in moist chambers at 25 ± 1°C, 80% relative humidity. Leaves inoculated with sterile PDA plugs were used as the control. After 3-5 days, the wounded and non-wounded leaves inoculated with ALY1 showed symptoms of leaf spots, similar to those observed in the field, while leaves inoculated with ALY2 and the control leaves remained symptomless. The pathogenicity tests were repeated three times under the same conditions and similar results were observed. ALY1 was confirmed as the pathogen causing the leaf spot. On PDA, ALY1 produced a gray to grayish brown colony. The conidia were obclavate to cylindrical, straight to slightly curved, pale brown, with 6 to 18 pseudosepta, 62.5 to 225.0×3.8 to 14.8 μm (n=17). According to the morphological characteristics, ALY1 was preliminarily identified as Corynespora cassiicola (Zhang et al.2018). For molecular identification, the internal transcribed spacer (ITS), the actin gene (ACT), the β-tubulin gene (TUB2) and translation elongation factor 1-alpha gene (TEF1-α) of the three single-spore isolates from ALY1 (ALY1-1 to ALY1-3) were amplified and sequenced with primers ITS4/ITS5 (White et al.1990), ACT512F/ACT783R(Carbone and Kohn 1999), T1/T2 (Glass and Donaldson 1995), and EF1-728F/EF1-986R (O'Donnell et al.1998) respectively. The ITS region of ALY1 (-1, -2, -3) (GenBank accession number MN860006, -07, -08) was 99-100% identical (579/585, 586/586, 581/584 base pairs) to a corresponding ITS (MH255527) of C. cassiicola. Similarly, the ACT (MN887504, -05, -06), TUB2 (MN887507, -08, -09) and TEF1-α (MN887501, -02, -03) were 100% (342/342, 342/342, 342/342 base pairs), 100% (664/664, 662/662, 664/664 base pairs) and 100% (290/290, 290/290, 290/290 base pairs) to corresponding genes FJ853033, MH763700, and MK589886 sequences of C. cassiicola in GenBank, respectively. Phylogenetic tree generated with MEGA7 by the neighbor-joining analysis revealed that the isolate's ITS, ACT, TUB2 and TEF1-α sequences were grouped in the same clade to C. cassiicola. Based on both the morphological characteristics and sequences analyses, the pathogen causing leaf spot of A. ilicifolius was identified as C. cassiicola. This species had been reported on the A. ilicifolius in Guam America and Hong Kong China (Lu et al. 2000; Zhuang 2001; Dixon et al. 2009; Sumabat et al. 2018). The best of our knowledge, it is the first report of C. cassiicola causing leaf spot of A. ilicifolius in China. This disease may a potential threat to A. ilicifolius along the coastal regions of southern China.