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First Report of a New ‘Candidatus Phytoplasma asteris’ Subgroup of 16SrI Associated with Willow Phyllody in China

    Affiliations
    Authors and Affiliations
    • Y.-M. Luan
    • J.-W. Zhang
    • F.-T. Zhang , Department of Plant Pathology, College of Plant Protection, Shandong Agricultural University, Tai’an 271018, China
    • T.-S. Zhu , College of Plant Science, Tarim University, Alar, Xinjiang Province, 843300, China
    • X.-D. Li
    • Y.-P. Tian , Department of Plant Pathology, College of Plant Protection, Shandong Agricultural University, Tai’an 271018, China.

      Willow (Salix babylonica Linn.) is a common deciduous tree and widely planted in Asia and Europe. Willow plants displaying symptoms of phyllody, bushy and small leaves, were observed in southern Xinjiang in 2005. The incidence of willow phyllody was less than 10% in Bazhou (9/100, number of the symptomatic trees/total number of trees visited) and Kezhou districts (12/200) and was 48.0–64.3% in Kashi (185/400), Tumushuke (154/300), Akesu (193/300), Alar (308/600), and Hetian districts (336/700). In total, seven symptomatic and seven asymptomatic samples were collected from the above regions in 2007. Total DNA was extracted from the phloem tissue of the collected samples. The 16S rRNA and rp gene were amplified using universal primer pairs R16mF2/R16mR1 and R16F2/R16R2, and rpF1/rpR1 and rp(I)F1/rp(I)R1, respectively, via nested polymerase chain reaction (PCR) (Gundersen et al. 1996). PCR products of 1.25 kb (accession no. MG738364) and 0.83 kb (accession no. MG727563) in length were obtained from all seven collected symptomatic but not asymptomatic willow samples, respectively. Both results indicated that a phytoplasma was associated with the willow phyllody symptom. The associated phytoplasma was designated as willow phyllody phytoplasma (WPP). The 16S rRNA of WPP was compared with the representative isolates using Clustal X in BioEdit software, and it showed the highest sequence identity of 99.5% with that of onion yellows (AP006628), which is a representative phytoplasma strain of ‘Candidatus Phytoplasma asteris’ 16SrI-B. In the phylogenetic tree constructed using the neighbor-joining method based on 16S rRNA sequences, WPP formed a close branch with 16SrI group isolates. The virtual restriction fragment length polymorphism analysis using iPhyClassifier online server showed that WPP is most similar to the reference pattern of the 16Sr group I, subgroup B (AP006628), with a similarity coefficient of 0.94, which is less than 0.97 (Zhao et al. 2009). The rp gene of WPP shared the highest identity of 99.6% with that of rpI-D (AY264857) and 98.9% with that of rpI-B (AY183717). Taken together, above results suggested that WPP belongs to a new subgroup of 16SrI. The previously identified phytoplasmas associated with yellows, ball-like structures, and small leaves symptoms of willow in Spain belonged to the stolbur group (16SrXII; Alfaro-Fernendez et al. 2011), whereas those associated with willow yellows and willow proliferation in China belonged to 16SrI-C and 16SrVI, respectively (Wei et al. 2009; Zhang et al. 2012). To our knowledge, this is the first report that willow phyllody was associated with infection of phytoplasma related to ‘Ca. Phytoplasma asteris’ 16SrI group, subgroups B and D in China. The results presented here provide useful information for the precise control of willow phyllody disease.

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      Funding: Funding was provided by National Natural Science Foundation of China (grant nos. 31501612 and 31571984).