基于宏病毒组测序技术的苹果病毒病鉴定与分析

Identification and Analysis of Apple Viruses Diseases Based on Virome Sequencing Technology

采用宏病毒组测序技术(Virome Sequencing Technology)对河南地区染病苹果树进行病毒检测与分析,从建库测序的苹果叶片中共检测出13种病毒,其中有5种苹果病毒:苹果茎沟病毒(apple stem grooving virus,ASGV)、苹果茎痘病毒(apple stem pitting virus,ASPV)、苹果褪绿叶斑病毒(apple chlorotic leafspotvirus,ACLSV)、苹果坏死花叶病毒(applenecroticmosaicvirus,Ap NMV)和苹果绿皱缩相关病毒(apple green crinkle associated virus,AGCa V)。对病毒检测结果中的5种苹果病毒进行RT-PCR验证,扩增后均获得目的条带,与宏病毒组测序检测结果一致,表明宏病毒组测序检测结果精准可靠。根据测序结果设计特异性引物对ASPV全长进行扩增,得到1条长度为9246 nt的ASPV-HN分离物基因组,与NCBI数据库中已报道的19个ASPV分离物的基因组核苷酸序列一致性为75.48%~79.85%;宏病毒组测序拼接所得的两条长度均为6475 nt的ASGV基因组全长基因ASGV-HN1和ASGV-HN2,与NCBI数据库中已报道的18个ASGV分离物的基因组核苷酸序列一致性为79.98%~98.08%。对53份河南不同产区的苹果叶片样品进行苹果花叶病毒(apple mosaic virus,Ap MV)和Ap NMV的检测,结果显示花叶病症样品中均检测出Ap NMV,未检测到Ap MV,表明Ap NMV可能是引起河南地区苹果花叶病的主要病原。

Virome Sequencing Technology was used for virus detection and analysis of virus-infected apple trees in Henan Province,a total of 13 viruses were detected from apple leaves in the sequenced database,including five apple viruses:apple stem grooving virus(ASGV),apple stem pitting virus(ASPV),apple chlorotic leaf spot virus(ACLSV),apple necrotic mosaic virus(Ap NMV)and apple green crinkle associated virus(AGCa V).RT-PCR was used to verify the detection results of five apple viruses,and objective bands were obtained after amplification,it was consistent with virome sequencing results,indicating that the virome sequencing detection results were accurate and reliable.Based on the sequencing results,specific primers were designed to amplify the full length of ASPV,a length of 9246 nt ASPV-HN isolate genome was obtained and shared sequence identities 75.48%to 79.85%with complete genome of 19 ASPV isolates published in NCBI database;the two assembly sequences of ASGV genomes ASGV-HN1 and ASGV-HN2 based on virome sequencing results were both 6475 nt in length and shared sequence identities 79.98%to 98.08%with complete genome of 18 ASGV isolates published in NCBI database.Detection of apple mosaic virus(Ap MV)and Ap NMVwere carried out in 53 apple leaf samples collected from different apple producing areas in Henan Province.Ap NMV was detected in all samples with mosaic disease,while Ap MV was not detected in 53 samples.It indicated that Ap NMV may be the main pathogen which can cause apple mosaic disease in Henan Province.