Bruguiera gymnorrhiza (L.) Savigny is an important mangrove tree species that grows in the intertidal regions of the tropical and subtropical coastlines. In a survey conducted in March 2014, a leaf spot disease on this plant was observed in Sea View Promenade in Zhanjiang, Guangdong Province, China. Symptoms on leaves initially appeared as small circular to irregular, dark brown, necrotic, sunken spots with an average diameter of 4 to 7 mm. The spots gradually enlarged in size, becoming irregular, or remained circular with concentric rings or zones. In the latter, the spots coalesced, and the leaves withered, dried, and fell from the plants. Leaf tissues (3 × 5 mm), cut from the margins of lesions, were surface-disinfected, placed on potato dextrose agar (PDA), and incubated at 28°C with a 12-h photoperiod. Five fungi (MLL1 to MLL5) with different morphological characteristics were obtained. To fulfill Koch's postulates, wounded and nonwounded leaves were inoculated. Fresh wounds were made with a sterile needle on 10 detached leaves and 10 leaves on five living plants for fungi MLL1 to MLL5 independently. Mycelial plugs of each fungus were applied to wounded and nonwounded leaves. For the control, 10 leaves on five living plants were inoculated with agar plugs in a similar manner, to both wounded and nonwounded leaves. All treatments were incubated in a humid chamber in the dark at 28°C. Leaf spots identical to those observed in the field were observed on the wounded leaves inoculated with fungus MLL3 after 3 to 4 days, while the other four fungi and the control remained symptomless. The 10 nonwounded leaves inoculated with fungus MLL3 were also infected after 5 days. The fungus, with the same colony and conidial morphology as MLL3, was re-isolated from the affected leaves. The pathogenic test was repeated three times under the same conditions. Hyphal tips of MLL3 were transferred to PDA for morphological observation. Colonies of white-to-dark-gray mycelia, black on the underside, formed on PDA. The colonies were further identified as Alternaria sp., based on the dark brown, obclavate to obpyriform catenulate conidia with longitudinal and transverse septa tapering to a prominent beak attached in chains on a simple and short conidiophore (3). Conidia varied from 22.5 × 40.26 to 3.95 × 5.79 μm and had three to eight transverse and zero to four longitudinal septa, with a beak length of 0 to 7.25 μm. For molecular identification, PCR was carried out using internal transcribed spacer (ITS) region primers ITS1/ITS4, partial sequences of the beta tubulin gene primers Bt1a-Bt1b (1), and A. alternata species-specific primers AAF2/AAR3 (2). The PCR products were subjected to direct sequencing. The resulting sequences were compared against the GenBank nucleotide database by using a BLAST alignment, which revealed that MLL3 had 99 to 100% identity with A. alternata for the ITS, Bt1a-Bt1b, and AAF2/AAR3 regions (GenBank Accession Nos. KF669893, GQ240308, and KJ716876, respectively). Sequences for MLL3 were deposited in GenBank under accession numbers KJ767515, KJ921779, and KJ921778. According to both morphological and sequence analyses, the pathogen of the leaf spot of B. gymnorrhiza was identified as A. alternata. To our knowledge, this is the first report of A. alternata on leaves of B. gymnorrhiza in China. This pathogen could cause serious foliar damage and threaten the survival, growth, and fitness of the local B. gymnorrhiza community.
References: (1) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (2) P. Konstantinova et al. Mycol. Res. 106:23, 2002. (3) E. G. Simmons. Alternaria: An identification Manual. CBS Fungal Biodiversity Center, Utrecht, Netherlands, 2007.