First report of pear chlorotic leaf spot-associated virus on Japanese and European pears in Japan and its detection from an eriophyid mite.
First report of pear chlorotic leaf spot-associated virus on Japanese and European pears in Japan and its detection from an eriophyid mite.
复制标题
日本和欧洲梨上梨褪绿叶斑病相关病毒的首次报道及其从红叶螨中的检测。
DOI：
10.1094/pdis-09-20-2035-pdn
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发表时间：
2020
期刊：
影响因子：
4.5
通讯作者：
F. Kadono
中科院分区：
文献类型：
--
作者：
K. Kubota;Y. Chiaki;H. Yanagisawa;S. Takeyama;R. Suzuki;M. Kohyama;T. Horikawa;S. Toda;F. Kadono
2018年5月，从日本梨树cv.在茨木筑波的种质圃中表现出典型的褪绿斑症状的“丰水”（补充图S1）。如Kubota et al.（2020）所述，使用快速CTAB法（Gambino et al. 2008）制备总RNA，用于高通量测序。简而言之，在去除核糖体RNA后，通过将RNA片段化、合成cDNA和聚合酶链式反应（PCR）扩增来构建文库。使用NovaSeq 6000测序仪（Illumina，San Diego，CA，U.S.A.）具有双端150 nt读段。使用CLC Genomics 11.0软件（Qiagen，希尔登，德国）进行从头组装，最小长度为500 bp。截至2019年1月，共获得了来自33，565，182个读段的36，017个重叠群，并对GenBank序列数据库进行了BLASTX搜索。核果中常见的病毒，即，检测到苹果茎痘病毒、苹果绿色皱缩相关病毒、杏潜伏病毒（黄斑病毒）和苹果茎沟病毒（一种毛细病毒）。此外，检测到与已知emaraviruses的P1-P4和High Plains小麦花叶病毒的P7具有氨基酸序列同源性的5个重叠群（Tatineni等人，2014），并将其命名为PEV-Jp。使用补充表S1中所示的引物，通过克隆逆转录（RT）-PCR扩增产物的桑格测序，确定PEV-Jp 5个片段的完整核苷酸（nt）序列;对5 '和3'末端序列进行RACE验证（Takara Bio，滋贺，日本）。两两比较，PEV-Jp（LC 554756 -760）与PCLSaV-CG 1（MK 602177 -181）的RNA 1 ~ RNA 5核苷酸序列同源性为90.7%~ 98.7%，表明PEV-Jp是PCLSaV的一个分离株。使用新设计的片段特异性引物（补充表S1），12个有症状的日本梨树品种。2020年从同一苗圃取样的“Kosui”经RT-PCR检测呈PCLSaV阳性，而12棵无冠树则呈病毒阴性。类似的褪绿斑点，有时伴随坏死点，观察欧洲梨（梨）品种。“勒勒克捷。“（图S1 F）;通过RT-PCR在症状树的叶组织样品中检测到PCLSaV（n = 3/3），但在无症状树中未检测到PCLSaV（n = 0/2）。尚未确定PCLSaV的载体（Liu等人，2020），但早春喷洒杀螨剂可有效预防梨园中褪绿斑的发生（Nakai等人，2018）。由于千叶瘿螨（Eriophyes chibaensis Kadono，一种经常在日本梨上观察到的瘿螨）（图S1 G至S1 I）（Kadono，1981）的侵染与褪绿斑的发生有关（Shimizu等人，2019），E.从PCLSaV阳性的日本梨cvs中收集千叶梨个体。“Akizuki”和“Kosui“和P. communis cv.“勒勒克捷。对于通过酚-氯仿提取的总核酸分离，随后进行定量RT-PCR（补充表S1）。从Akizuki（n = 6/12）、Kosui（n = 13/18）和Le Lectier（n = 6/8）采集的螨样品中检测到预期的RNA 1和RNA 5特异性150 bp产物。结果表明，E. Chibaensis可以摄取PCLSaV，并且可能是该病毒的潜在载体，尽管需要额外的实验来证明其载体能力。据我们所知，这是日本首例PCLSaV报告，也是首例在大肠杆菌中检测到PCLSaV的报告。千叶。
In May 2018, three leaf samples were collected from Japanese pear trees cv. "Hosui" that exhibited typical chlorotic spot symptoms (Supplementary Figure S1) in a germplasm nursery in Tsukuba, Ibaraki. Total RNA was prepared using the rapid CTAB method (Gambino et al. 2008) for high-throughput sequencing, as described by Kubota et al. (2020). In brief, after removing ribosomal RNAs, a library was constructed by fragmenting RNA, synthesizing cDNA, and polymerase chain reaction (PCR) amplification. Sequencing was performed using NovaSeq 6000 sequencer (Illumina, San Diego, CA, U.S.A.) with paired-end 150 nt reads. De novo assembly was performed using CLC Genomics Workbench 11.0 Software (Qiagen, Hilden, Germany), with a minimum length of 500 bp. A total of 36,017 contigs derived from 33,565,182 reads were obtained and subjected to BLASTX search against the GenBank sequence database as of January 2019. Viruses commonly found in stone fruits, i.e., apple stem pitting virus, apple green crinkle-associated virus, apricot latent virus (foveaviruses), and apple stem grooving virus (a capillovirus), were detected. In addition, five contigs with amino acid sequence homologies to P1-P4 of known emaraviruses and the P7 of High Plains wheat mosaic virus (Tatineni et al. 2014) were detected and designated as PEV-Jp. The complete nucleotide (nt) sequences of the five segments of PEV-Jp were determined by Sanger sequencing of cloned reverse transcription (RT)-PCR amplification products using the primers shown in Supplementary Table S1; the 5'- and 3'-terminal sequences were RACE verified (Takara Bio, Shiga, Japan). In pairwise comparisons, the complete RNA1 to RNA5 of PEV-Jp (LC554756-760) shared 90.7% to 98.7% nt identities with those of PCLSaV-CG1 (MK602177-181), indicating that PEV-Jp is an isolate of PCLSaV. Using newly designed segment-specific primers (Supplementary Table S1), 12 symptomatic Japanese pear trees cv. "Kosui" sampled in 2020 from the same nursery tested positive for PCLSaV by RT-PCR while 12 symptomless trees were negative for the virus. Similar chlorotic spots, sometimes accompany necrotic spots, were observed on European pear (Pyrus communis) cv. "Le Lectier." (Fig. S1F) in Niigata in 2019; PCLSaV was detected by RT-PCR in leaf tissue samples from symptomatic trees (n = 3/3) but not in symptomless trees (n = 0/2). No vector for PCLSaV has been identified (Liu et al. 2020) but acaricide sprays in the early spring are effective for preventing occurrence of chlorotic spots in pear orchards (Nakai et al. 2018). Since the infestations of Eriophyes chibaensis Kadono, an eriophyid mite often observed on the Japanese pear (Fig. S1G to S1I) (Kadono, 1981), has been associated with occurrences of the chlorotic spots (Shimizu et al. 2019), samples of E. chibaensis individuals were collected from PCLSaV-positive Japanese pear cvs. "Akizuki" and "Kosui"and P. communis cv. "Le Lectier." for total nucleic acid isolations via phenol-chloroform extraction, followed by quantitative RT-PCR (Supplementary Table S1). The expected RNA1 and RNA5 specific 150 bp products were detected from mite samples collected from Akizuki (n = 6/12), Kosui (n = 13/18), and Le Lectier (n = 6/8). The results indicate that E. chibaensis can ingest PCLSaV and may be a potential vector for the virus, although additional experiments are needed to demonstrate its vector competency. To our knowledge, this is the first report of PCLSaV in Japan and the first report of its detection in E. chibaensis.