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Occurrence of ‘Candidatus Phytoplasma asteris’-Related Strains Infecting Chinaberry (Melia azedarach) Showing Chlorotic Leaf Symptoms on Hainan Island of China

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    Authors and Affiliations
    • Shao-Shuai Yu1
    • De Zhou1 2
    • Wei-Wei Song1
    1. 1Coconut Research Institute, Chinese Academy of Tropical Agricultural Sciences, Wenchang 571339, China
    2. 2College of Tropical Crops, Yunnan Agricultural University, Pu’er, Yunnan 665099, China

    Chinaberry (Melia azedarach), belonging to the family Meliaceae, is an ornamental tree distributed across southern China. In the autumn of 2021, in an area of 400 acres located in Wanning city of Hainan Province, a tropical island in China, with coordinates of 19°2′9.96″N, 110°28′42.72″E, about 20% (100) of the chinaberry trees showed disease symptoms including chlorotic leaves. The disease symptoms were consistent with infections by a phloem-limited prokaryotic pathogen, phytoplasma. The samples of six symptomatic and three asymptomatic trees were collected for pathogen detection. To identify the pathogen, total nucleic acids were extracted from 0.10 g of fresh leaf tissues from the diseased and healthy plants using the CTAB DNA extraction method based on Doyle and Doyle (1990). The primer pairs R16mF2/R16mR1 (Lee et al. 1993), secAfor1/secArev3 (Hodgetts et al. 2008), and fTuf1/rTuf1 (Schneider and Gibb 1997) were used for specific identification of phytoplasma conserved gene fragments of 16S rRNA, secA, and tuf by PCR amplification. Target PCR bands were amplified from DNA of six diseased chinaberry samples, but not from DNA of the healthy samples. The amplified products were cloned and sequenced by Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China). The phytoplasma gene sequences of 16S rRNA, secA, and tuf were obtained, and all the sequences were identical with the length of 1,336, 710, and 955 bp, respectively. Representative sequence data for the strain MaCL-hn were deposited in GenBank under accession numbers OR438638 (16S rRNA), OR513089 (secA), and OR860415 (tuf). The phytoplasma strain identified in the study was described as chinaberry chlorotic leaf (MaCL) phytoplasma (MaCL-hn strain). A BLAST search based on 16S rRNA genes showed that 43 strains in 16SrI group ‘Candidatus Phytoplasma asteris’ showed 100% similarity with the 16S rRNA sequence of MaCL-hn. A BLAST search based on secA genes showed that nine strains in the phytoplasma group showed 100% similarity with the 16SRNA sequence of MaCL-hn. A BLAST search based on tuf genes showed that 21 strains in the phytoplasma group showed 100% similarity with the 16S rRNA sequence of MaCL-hn. Restriction fragment length polymorphism analysis based on iPhyClassifier (Zhao et al. 2009) indicated that the MaCL-hn strain was a member of 16SrI-B subgroup with a similarity coefficient of 1.00 to the reference phytoplasma strain (AP006628). A phylogenetic tree was constructed based on 16S rRNA by MEGA 11.0 using the neighbor-joining method with 1,000 bootstrap value (Felsenstein 1985; Tamura et al. 2021). The results showed that the MaCL-hn strains clustered into one clade with 16SrI group ‘Ca. P. asteris’-related strains with 99% bootstrap value. Multilocus sequence analysis based on the concatenated sequences with the length of 3,001 bp including the sequences of 16S rRNA, secA, and tuf showed that the MaCL-hn strains were clustered into one clade with the phytoplasma strains in the group with 100% bootstrap value. To our knowledge, this is the first report that chinaberry can be infected by ‘Ca. P. asteris’-related strains belonging to 16SrI-B subgroup on Hainan Island of China. This finding will contribute to the epidemic monitoring and the preventive management of the phytoplasmas and their related diseases.

    The author(s) declare no conflict of interest.

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    Funding: This work was supported by the Hainan Provincial Natural Science Foundation of China (no. 323RC524).

    The author(s) declare no conflict of interest.