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First Report of ‘Candidatus Phytoplasma asteris’ Causing Witches’-Broom Disease on Chinese Sumac (Rhus chinensis) in China

    Authors and Affiliations
    • S. Cai
    • J. Y. Chen , Institute of Forest Protection, Hubei Academy of Forestry, Hubei 430075, P. R. China
    • D. Y. Song , Wufeng County Forestry Beaurea, Hubei 443400, P. R. China
    • C. H. Hong
    • Y. P. Zha
    • Z. Y. Zhang
    • Y. X. Wang , Institute of Forest Protection, Hubei Academy of Forestry, Hubei 430075, P. R. China
    • N. Hong , College of Plant Science and Technology, Huazhong Agricultral University, Hubei 430070, P. R. China.

      Chinese sumac (Rhus chinensis: Anacardiaceae) has long been used as traditional plant medicine in Asia and its galls on leaves, called Galla chinensis, are widely used as the raw materials of industry. In August 2015, individual trees of Chinese sumac showing apical witches’-broom, shortened internodes, little leaf, flower phyllody, and virescence were found in the marginal lands of Wufeng county (30°10′N, 110°41′E), Hubei province. A survey conducted on 22 ha of 4-year-old R. chinensis in October 2015 indicated that disease incidence ranged from 20 to 37.5% in the different field plots and the diseased trees totaled 11,550. A total of 26 branch samples containing 20 symptomatic and six asymptomatic samples were collected for the next experiment. Total DNA was extracted from all twigs by CTAB method (Namba et al. 1993). DNA amplification was conducted from all samples, using the two primer pairs SN910601/SN910502 and SN920203/SN910502 (Namba et al. 1993). DNA samples from paulownia witches’-broom phytoplasma were used as positive control and that from asymptomatic plants as negative control. Direct PCR was performed using universal primers SN910601/SN910502 for amplification of the ribosomal 16S rDNA fragment of Mollicutes. The PCR product (1,370 bp) was diluted 1:10 with sterile distilled water and 1 µl of diluted DNA was used as a template for nested PCR with primers SN920203/SN910502. The 16SrI-specific primers (SN920203/SN910502) amplified the approximate 1,300 bp band from 15 samples out of 20 symptomatic samples. No PCR products were obtained for asymptomatic samples. The amplified products from three samples out of 15 positive samples were purified and sequenced by capillary electrophoresis (ABI 3730xl DNA Analyzer). In order to obtain the longest sequence of the ribosomal 16S rDNA, the 750 bp products amplified with the phytoplama-specific primers (SN910601/SN920204) from the PCR products (1,370 bp) of the three partial-sequenced plant samples were also purified and sequenced. The sequences were then assembled by DNAMAN 5.2.2 and the representative sequence was deposited in GenBank (KU529759). In silico restriction enzyme digestions and virtual gel plotting of 16S rDNA (Wei et al. 2007) indicated KU529759 had closest similarity with the reference strain OAY (M30790) with similarity coefficient of 1, which belonged to 16SrI group, subgroup B (‘Candidatus Phytoplasma asteris’). Additionally, the band (1,400 bp) of the partial groEL gene sequence were obtained by amplification with the primers (AYgroelF/AYgroelR) (Mitrović et al. 2011) from the above sequenced three plant samples. After purification, sequencing, and assembling, the representative sequence was deposited in GenBank (KX668147). Multiple alignment analysis indicated that KX668147 was 99% identical (100% query cover and E value = 0) to the corresponding sequence of the reference strain AY-W (AB559691), which also demonstrated that the phytoplasma associated with Chinese sumac witches’-broom belongs to the phytoplasma subgroup 16SrI-B (Mitrović et al. 2011). KU529759 and KX668147 were respectively 100% (query cover 97% and E value = 0) and 99% (query cover 100% and E value = 0) identical to the corresponding sequences AB738740 and AB738741 of the phytoplasma causing yellowing disease on hydrangea in Japan. To our best knowledge, this is the first report of ‘Ca. Phytoplasma asteris’ infecting R. chinensis in China.