侵染广西红宝石青柚的柑橘褪绿矮化相关病毒遗传进化分析

【目的】明确引起广西多地泰国红宝石青柚产生叶片变形、扭曲、皱缩、褪绿花叶和矮化症状的病原，为有效防治该病害提供理论依据。【方法】从广西多地采集疑似发病的红宝石青柚叶片样品，利用柑橘褪绿矮化相关病毒（Citrus chlorotic dwarf-associated virus，CCDaV）特异引物进行PCR检测，通过分段扩增、克隆等方法对样品进行鉴定并获取全长基因序列，运用RDP5和MEGA 7.0对所获得的全长基因序列进行重组及遗传多样性分析。【结果】PCR检测结果显示采集的红宝石青柚叶片样品可扩增出642 bp的目的条带，证实红宝石青柚受到CCDaV感染；采用分段克隆的方法获得LA-1、LA-2、WM-1、GG-1、GG-2、WZ-1和WZ-2等7个各地代表分离物的全长序列，序列长度分别为3641、3642、3642、3640、3642、3644和3644 bp；核苷酸相似性比对发现，7个分离物间的核苷酸相似性在98%以上，与GenBank已登录的CCDaV各分离物间的核苷酸相似性在99%以上，说明研究获得的病毒分离物为CCDaV的分离物；重组分析发现，7个分离物未发生重组。系统发育进化树分析结果显示，研究获得的WZ-1和WZ-2分离物分别与Tha1-19、Tha30处于同一个小分支，说明这2个分离物与Tha1-19和Tha30具有较近的亲缘关系；其余几个分离物则分处不同的小分支，说明CCDaV广西分离物间仍有一定的进化多样性。【结论】引起广西红宝石青柚叶片呈V字型、皱缩、卷曲、褪绿花叶和植株严重矮化等症状的病原为CCDaV，部分地区分离物存在地域多样性。

Genetic and evolutionary analysis of Citrus chlorotic dwarf-associated virus infecting Ruby green pomelo in Guangxi

【Objective】To clarify the pathogen of the Ruby green pomelo showing symptoms of leaf deformation, curling, shrinking, chlorotic and dwarfing, and provide a theoretical basis for effective prevention and treatment of the disease.【Method】Several leaf samples were collected from suspected Ruby green pomelo in some cities of Guangxi, and two pairs of specific primers of Citrus chlorotic dwarf-associated virus(CCDaV) were designed and used for detected the suspected samples, the full-length of genome sequence were obtained by segmented amplification and gene cloning. RDP5 and MEGA 7.0 softwares were applied for analyze the recombination and the genetic diversity of the full length genome.【Result】PCR with expected size of 642 bp were obtained from all of the collected Ruby green pomelo samples, which confirmed that all of the samples were infected by CCDaV. The full-length genome sequences of seven locations representing isolates LA-1, LA-2, WM-1, GG-1, GG-2, WZ-1 and WZ-2 were obtained by using segmented amplification and gene cloning, and the full-length sequences of the seven isolates were 3641, 3642, 3642, 3640, 3642, 3644 and 3644 bp, respectively. The nucleotide sequence identities between the seven isolates were over 98%, and the identities against other CCDaV isolates available in GenBank were over 99%, this results showed that the seven isolates obtained in this research were CCDaV isolates. Recombination analysis showed that no recombination occurred in the seven isolates. Phylogenetic analysis showed that two CCDaV Guangxi isolates(WZ-1 and WZ-2) clustered with Tha1-19 and Tha30 in a sub-clade, indicating their close relation. The other isolates of Guangxi were distributed in different sub-clade, indicating that there were evolutionary diversity among the CCDaV Guangxi isolates.【Conclusion】CCDaV is the pathogen that infects the Ruby green pomeloshowing symptoms of V shape, shrinking, curling, chlorotic and dwarfing, and there is regional diversity of the isolates among different regions.