通辽市塞外红苹果中3种苹果潜隐病毒的检测及变异分析  

Detection and variation analysis of three types of apple latent viruses in Malus pumila‘Saiwaihong’in Tongliao

作　　者：张悦东 孙平平 李小燕[1] 杨荣[2] 王宝侠 张磊[1] 李正男 ZHANG Yuedong;SUN Pingping;LI Xiaoyan;YANG Rong;WANG Baoxia;ZHANG Lei;LI Zhengnan(College of Horticulture and Plant Protection,Inner Mongolia Agricultural University,Hohhot 010018,Inner Mongolia,China;Acade-my of Forestry Sciences of Inner Mongolia Autonomous Region,Hohhot 010010,Inner Mongolia,China;Tongliao Institute of Forestry and Grassland Sciences,Tongliao 028000,Inner Mongolia,China)

机构地区：[1]内蒙古农业大学园艺与植物保护学院,呼和浩特010018 [2]内蒙古自治区林业科学研究院,呼和浩特010010 [3]通辽市林业和草原科学研究所,内蒙古通辽028000

出　　处：《果树学报》2023年第5期978-987,共10页Journal of Fruit Science

基　　金：国家自然科学基金项目(32160031);内蒙古自然科学基金项目(2019MS03021);内蒙古农业大学高层次人才引进科研启动项目(NDYB2018-3,NDYB2019-1)。

摘　　要：【目的】明确通辽地区塞外红苹果(Maluspumila‘Saiwaihong’)中苹果褪绿叶斑病毒(ACLSV)、苹果茎痘病毒(ASPV)和苹果茎沟病毒(ASGV)3种苹果潜隐病毒的普遍率和遗传多样性。【方法】从通辽市开鲁县8个乡镇随机采集了96份塞外红苹果枝条,采用RT-PCR法对样品进行3种苹果潜隐病毒检测。对检测为阳性的样品进行病毒基因克隆、测序,并进行序列一致性分析和系统发育分析。【结果】检测结果表明,被检测的塞外红苹果中3种苹果潜隐病毒均有不同程度的发生,并且存在2种或3种病毒的复合侵染。基于CP基因对病毒一致性和变异情况进行分析,结果表明,来源于塞外红苹果的29个ACLSV分离物的核苷酸序列一致性为78.1%~99.9%,与其他15个已知的ACLSV分离物的一致性为69.1%~93.6%;来源于塞外红苹果的12个ASPV分离物的核苷酸序列一致性为83.6%~99.7%,与其他15个已知的ASPV分离物的一致性为75.4%~91.5%;来源于塞外红苹果的21个ASGV分离物的核苷酸序列一致性为96.4%~100.0%,与其他已知的40个ASGV分离物的一致性为89.9%~98.9%。系统发育分析结果表明,44个ACLSV分离物聚集在7个主支,其中29个塞外红苹果的ACLSV分离物分别位于JB组、Balatonl组、B6组和一个独立分支;27个ASPV分离物聚集为6支,其中12个塞外红苹果的ASPV分离物分别位于Ⅰ、Ⅳ、Ⅴ组;61个ASGV分离物聚集为8个支,其中塞外红苹果的21个ASGV分离物聚集在一起,形成独立分支。【结论】ACLSV、ASGV、ASPV均能侵染塞外红苹果,并且发生率较高。与其他来源的病毒相比,塞外红苹果的3种病毒分离物,各种内各分离物彼此之间的序列一致性较好并且进化关系较近。研究结果为塞外红苹果产业绿色、健康、持续发展提供基本理论依据。【Objective】The aim of this study was to determine the incidence and genetic diversity of three types of latent apple viruses,including apple chlorotic leaf spot virus(ACLSV),apple stem pitting virus(ASPV)and apple stem grooving virus(ASGV)in Saiwaihong apple(Malus pumila)in Tongliao.【Methods】Fresh twigs of Saiwaihong were randomly collected from 96 trees in 8 different counties in Kailu.Total RNA was extracted from the leaves of the samples using TaKaRa MiniBEST Plant RNA Extraction Kit and served as templates for cDNA synthesis using PrimeScriptⅡ1st Strand cDNA Syn-thesis Kit.The genes of the three viruses were amplified by RT-PCR,in which the primers were de-signed based on the conserved parts of the gene sequences published in NCBI GenBank.The amplified fragments were purified and cloned onto pTOPO-TA cloning vectors,and E.coli JM109 competent cells were transformed.Positive clones were sequenced at Beijing Genomics Institute.The sequences were analyzed and assembled using Vector NTI Advance 11,and the nucleotide sequence identity was determined using BLASTn program at NCBI website.Phylogeny analysis was performed based on the sequences using MEGA-X software.【Results】The incidence of the viruses in Saiwaihong in Tongliao was 30.2%(29/96)for ACLSV,12.5%(12/96)for ASPV and 22.0%(21/96)for ASGV.The triple mixed infection was 4.2%.The double mixed infection was 4.2%for ACLSV and ASPV,13.5%for ACLSV and ASGV,and 3.1%for ASPV and ASGV.The nucleotide sequence identities among the 29 ACLSV isolates from Saiwaihong were 78.1%-99.9%,while the identities were 69.1%-93.6%with ad-ditional 15 isolates published in NCBI.ACLSV isolate Kailu7-10(GenBank accession No.ON001713)had the highest nucleotide sequence identity of 93.6%with isolate XC-HF(MF678819),and the lowest nucleotide sequence identity of 69.1%was seen between ACLSV Kailu1-5(ON001689)and the isolate Ta Tao 5 fromAmerican peach(EU223295).The nucleotide sequence identities of the 12ASPV isolates from Saiwaihong were 83.6%-99.7%,and the identities were

关 键 词：塞外红苹果 RT-PCR 苹果褪绿叶斑病毒 苹果茎痘病毒 苹果茎沟病毒 

分 类 号：S661.1[农业科学—果树学]