榆树黄化病植原体的分子检测与鉴定

Molecular detection and identification of the phytoplasma associated with Elm yellows in China

利用植原体16S rRNA基因的通用引物R16mF2/R16mR1和R16F2n/R16R2对山东泰山上发生的榆树(Ulmus par-vifolia)黄化病感病植株总DNA进行巢式PCR扩增,得到了约1.2 kb的特异性片段,从分子水平证实了榆树黄化病的病原(EY-China)为植原体。将扩增到的片段测序,并进行一致性和系统进化树分析。结果表明,该分离物属于植原体榆树黄化组(CandidatusPhytoplasma ulmi),与该组成员16S rRNA序列的一致性均在98.2%以上,其中与16SrⅤ-B亚组中的纸桑丛枝(Paper mulberry witches’-broom)和枣疯病(Jujube witches’-broom)植原体一致性最高,达到99.4%,在系统进化树中与该亚组成员聚类到同一个分支,说明该分离物属于植原体16SrⅤ-B亚组。本研究首次对在中国引致榆树黄化病的植原体进行了分子检测,并通过核酸序列分析将其鉴定到亚组水平。

By use of nested-PCR, the 16S rRNA gene of phytoplasma associated with Elm yellows in China （EY-china） was amplified with universal primer pairs R16mF2/R16mRl and R16F2n/R16R2. DNA fragments of ca. 1.2 kb were obtained from the total DNA of diseased elm samples. The fragment was sequenced and subjected to RFLP and phylogenetic analyses. The results indicated that the phytoplasma EY-China was a member of Candidatus Phytoplasma ulmi （16SrV）, and shared identities of more than 98.2% with other members in this group. It had the highest identity of 99.4%, and formed the same branch in phylogentic tree with phytoplasmas associated with paper mulberry witches＇ -broom and jujube witches＇ -broom of subgroup 16SrV-B. These results indicate that EY-China belong to subgroup 16SrV-B. This is the first report of phytoplasma associated with elm yellows disease identified to subgroup level according to molecular evidences in China.