Areca palm (Areca catechu) is a major traditional medicinal plant in China. Areca palm leaf spot diseases which were different from the common morphology of leaf spot on areca palm were found on the locally adapted areca palm varieties in Dongsheng State Farm, Qionghai City, Hainan Province of China（19.34°N, 110.34°E） during the period of 2021 to 2022. Our survey indicates that the disease occurs throughout the year, with peak incidence during the rainy season. In the early stage, round or nearly round brown spots were distributed on the leaves. In the later stage, the spots became larger and turned dark brown as a whole (Fig. 1). The incidence rate of the disease in the sampling areas was about 10%. Infected trees exhibit overall weakened growth and, in severe cases, may die, thereby causing direct economic losses for growers. The pathogens causing the new type leaf spot disease in areca palm were identified employing the conventional tissue isolation methods (Fang. 1998). Leaves were collected from five plants for pathogen isolation. A total of five isolates were obtained, all of which exhibited similar cultural morphology. Healthy leaves from two-year-old plants of a locally adapted areca palm varieties collected and surface-sterilized. Ten small holes were pricked at the diagonal positions of the leaves with a sterilized needle. Subsequently, mycelial blocks about 3mm×3mm were cut and inoculated on the pricked and non-wound parts of the leaves. The inoculated leaves were placed in a sterilized petri dish with sterile filter paper and sterilized water, and incubated at 28°C under moist conditions. The Koch's postulates (Fang, 1998) showed that the same symptoms were found in the inoculated leaves of areca palm (Fig. 2, Fig. 3 A). Under microscopy, the hyphae of the pathogen were brown and septate, and the conidia were dark brown, with a darker color in the middle and lighter colors at both ends. Most of conidia were upright, a few were slightly curved, cylindrical or slightly swollen in the middle and tapering at both ends, with a truncated base (Fig. 3 B). Some conidia had obvious scars, with 4 - 9 false septa. The spore sizes were (34.86-115.94) μm×(11.18-17.71) μm, with an average size of 71.98μm×14.05μm. Based on PCR amplification using primers GPD l/GPD 2 (Berbee et al., 1999), ITS4/ITS5 (White et al., 1990) and EF1-983F/EF1-2218R (Rehner et al., 2005), the sequences of GPD, ITS and TEF of the pathogen strain Q-1-2 were confirmed and uploaded to GenBank (PV872889, PV798842, and PV872888 respectively). Sequence BLAST indicated that the GPD, ITS and TEF sequence of Q-1-2 showed the 100%, 100% and 99.9% similarity to that of Bipolaris bicolor strain YB450101 (MK372988.1, MH201400.1 and MK372987.1), as indicated in Table 1, Table 2 and Table 3. Phylogenetic analysis based on the combined sequences of GPD-ITS-TEF employing the method of Neighbor-Joining using MEGA-X (Kumar et al., 2018) with 1000 replicates bootstrap values (Felsenstein, 1985) demonstrated that the strain Q-1-2 and the strains of B. bicolor were clustered into one subclade with 99% bootstrap value (Fig.4). To our knowledge, this is the first report that areca palm leaf spot disease caused by the pathogen B. bicolor. The findings of this study will contribute to the epidemic monitoring and prevention and control of the diseases of the areca palm.
Yang et al. (Mon,) studied this question.