基于高通量测序鉴定慈姑黄化病病原

Identification of the pathogens associated with arrowhead yellows by high-throughput sequencing

【目的】明确引致广西平乐县慈姑大面积黄化的主要病原物,为该病害的防治提供参考。【方法】从广西平乐县采集自然表现褪绿黄化症状的慈姑植株,分别提取其叶片和茎组织的总RNA,利用RNA-Seq高通量测序技术对其转录组进行测序,并对序列组装获得的重叠群(contigs)进行BLAST注释,筛选出注释为植原体16S rRNA序列的contigs,根据其序列设计引物Phy-F和Phy-R,以采集的褪绿黄化症状慈姑植株和无症状植株的叶片样本总DNA为模板,进行PCR鉴定。【结果】对扩增获得的长度为1.5 kb的DNA片段进行序列测定,从分子水平证实慈姑黄化病的病原为植原体(strain:AY-China)。将测序获得的植原体16S rRNA序列与GenBank收录的15个植原体分组代表菌株的16S rRNA序列进行比对分析并构建系统发育进化树,发现AY-China与翠菊黄化植原体16SrI组(Aster yellows group:16SrI)聚类在同一分支,且各组代表菌株的16S rRNA序列相似性在88.3%～96.0%。进一步将AY-China与16SrI不同亚组代表菌株的16S rRNA序列进行多重比对并构建系统发育进化树,发现AY-China与翠菊黄化植原体16SrI-B亚组聚类到在一分支,且与16SrI-B亚组代表菌株的16S rRNA序列相似性高达99.5%,说明AY-China应属于16SrI-B亚组。【结论】广西平乐县慈姑黄化病病原为植原体,其隶属于翠菊黄化植原体16SrI-B亚组。

【Objective】The pathogen that caused arrowhead yellowing disease in Pingle,Guangxi was identified to provide reference for the prevention and control of the disease.【Method】Yellowing arrowhead samples with chlorisis and yellowing symptom were collected from Pingle,Guangxi.Total RNA in leaf and stem tissues were extracted,and transcriptome was sequenced by RNA-Seq high-throughput sequencing technology,and the contigs obtained by sequence assembly were annotated by BLAST.The contigs which were annotated as 16S rRNA sequence of phytoplasma were picked out,and the primers Phy-F and Phy-R were designed according to its sequence.The total DNA of leaf samples from collected yellowing arrowhead and non-yellowing arrowhead were as templet,PCR was used to identified them.【Result】 DNA fragments with 1.5 kb were amplified,and sequencing was conducted on it.It was confirmed that the pathogen of arrowhead yellow was phytoplasma(strain:AY-China)at molecular level.The 16S rRNA sequences of AY-China and the representative strains of 15 phytoplasma groups which downloaded from GenBank were compared by multiple sequence alignment,and the phylogenetic tree was constructed.It was found that AY-China and Aster yellows phytoplasma 16SrI group(Aster yellows group:16SrI)were clustered to the same branch,and the 16S rRNA sequences identities among phytoplasma groups were 88.3%-96.0%.The 16S rRNA sequences of AY-China and 16SrI subgroups representative strains were further compared with each other and the phylogenetic tree was constructed.It was found that AY-China and the Aster yellows phytoplasma 16SrI-B subgroup(Aster yellows subgroup:16SrI-B)was clustered to the same branch,and shared identities of more than 99.5% with other members in 16SrI group.These results indicated that AY-China was a member of phytoplasma 16SrI-B subgroup.【Conclusion】The pathogen of arrowhead yellowing disease found in Pingle County of Guangxi is identified as phytoplasma,belonging to Aster yellows subgroup 16SrI-B.