苹果花脸和锈果症状伴随的苹果锈果类病毒分离物的分子特性分析

Molecular characteristics analysis of apple scar skin viroid isolates associated with dapple and scar symptoms of apple fruits

苹果锈果类病毒(apple scar skin viroid, ASSVd)侵染引起苹果花脸病和锈果病,近年来对我国苹果产业造成了严重损失,但造成两种症状差异的分子特性尚不明确。本研究前期从我国山东和山西收集表现花脸和锈果症状的苹果果实样品,进行了克隆和测序,获得56个ASSVd分子变种的全长核苷酸序列,长度在330~333 nt之间。通过对本研究获得的ASSVd分子变种比对分析及与报道的症状相关分子变种间的比较,表明花脸和锈果症状的ASSVd分子变种之间的一致率为86.9%~100%,变异率为0.0%~13.3%,均有4个主要变异区域(含21个变异位点),位于致病(P)区、P区与左端区(TL)邻接区域及P区与中央保守(C)区的邻接区域,二级结构呈紧凑的棍棒状。选取我国症状相关及国内外其他代表性ASSVd分子变种进行进化分析,显示我国ASSVd分离物分为7个簇(Ⅰ~Ⅶ),且两种不同症状相关分子变种聚在相同簇(Ⅰ、Ⅲ和Ⅵ)。其中,ASSVd簇Ⅰ为优势组群,含有我国新疆及其他地区和希腊及其他国家来源的分离物,推测为原始组群;簇Ⅱ、Ⅳ、V和Ⅶ表现出明显的我国地域专化性,推测为我国分离物的特有进化组群。对比本研究和国内外已报道两种症状相关ASSVd分子变种的核苷酸序列的长度、变异规律和进化关系,显示两种来源分子变种没有明显差异性,表明苹果两种症状之间的差异与ASSVd的分子变异没有相关性,而与苹果品种有关。研究结果为深入认识苹果花脸病和锈果病的病原分子特性及采用现代分子技术防治该病害提供了重要信息。

Apple scar skin viroid(ASSVd)causes dapple and scar symptoms on apple fruits,resulting in serious losses to apple industry in recent years in China,but the molecular characteristics responsible for the symptomatic diversion remain unclear.In the early stage of this study,apple fruits showing dapple and scar symptoms were collected from Shandong and Shanxi provinces in China,and subjected to cloning and sequencing.A total of 56 full-length nucleotide sequences in size of 330-333 nt were obtained.The variants obtained in this study were analyzed and compared with other reported variants related to these symptoms.The results showed that the ASSVd variants related to the dapple and scar symptoms have 86.9%-100%nucleotide sequence identity and 0.0%to 13.3%divergence among them,and all have four major diversity regions containing 21 diversity sites,which locate in the pathogenicity(P)region and the neighboring domains between P and terminal left regions and between P and central conserved regions,and fold into a compact rod-like secondary structure.The ASSVd variants related to the dapple and scar symptoms together with the representative variants isolated from China and abroad were subjected to phylogenetic analysis.It revealed that the Chinese ASSVd isolates were divided into seven clusters(I to VII),and the variants related to dapple and scar symptoms could be clustered together(in cluster I,III and IV).Among these,cluster I is the predominate group and harbors the variants collected worldwide,which is most likely the original group;cluster II,IV,V and VII showed a geographic specificity of our country,and are likely the specific clusters of Chinese isolates.Analyses of the nucleotide sequences of the two-symptom-related ASSVd variants obtained in this study and the others isolated from China and abroad revealed no obvious difference between variants of the two groups,indicating that the apple symptoms have no correlation with the molecular variation of ASSVd,but are related to apple varieties.This study provides important information for a better understanding of the molecular characteristics of ASSVd and developing modern molecular strategies for the disease control.