Article(id=1276598102840709946, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276597973173801322, articleNumber=null, orderNo=null, doi=10.3969/j.issn.1000-2561.2024.07.020, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1684425600000, receivedDateStr=2023-05-19, revisedDate=1690819200000, revisedDateStr=2023-08-01, acceptedDate=null, acceptedDateStr=null, onlineDate=1782294305762, onlineDateStr=2026-06-24, pubDate=1721836800000, pubDateStr=2024-07-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1782294305762, onlineIssueDateStr=2026-06-24, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1782294305762, creator=13701087609, updateTime=1782294305762, updator=13701087609, issue=Issue{id=1276597973173801322, tenantId=1146029695717560320, journalId=1235980609244409860, year='2024', volume='45', issue='7', pageStart='1303', pageEnd='1520', issueExtLink='null', onlineDate='null', pubDate='1721836800000', pubDateStr='2024-07-25', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1782294274847, creator='13701087609', updateTime=1782294274847, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext=null, issueFiles=null, downloadFileDto=null}, startPage=1478, endPage=1488, ext={EN=ArticleExt(id=1276598103230780220, articleId=1276598102840709946, tenantId=1146029695717560320, journalId=1235980609244409860, language=EN, title=Detection of Virus Species from Main Plantation Areas of Passion Fruit (Passiflora edulis) in Guizhou, columnId=1236292524264968282, journalTitle=Chinese Journal of Tropical Crops, columnName=Plant Protection & Bio-safety, runingTitle=null, highlight=null, articleAbstract=

Passion fruit (Passiflora edulis) is a characteristic and advantageous fruit in Guizhou, which is of great significance in poverty relief consolidation and the rural revitalization. However, in recent years, the viral diseases of passion fruit have become increasingly severe, leading to a sharp decline in yield and quality, which thereby seriously constrain industry development of passion fruit. In order to determine the virus species, distribution and compound infestation in the main plantation areas of passion fruit in Guizhou province, a total of 153 samples suspectedly infected by virus were collected from five main plantation orchards of passion fruit in five counties, i.e. Luodian, Congjiang, Rongjiang, Zhenning and Zhenfeng, and the virus detection was carried out by RT-PCR. The results showed that seven viruses were detected from the tested samples, namely East Asian Passiflora virus (EAPV), Telosma mosaic virus (TeMV), Cucumber mosaic virus (CMV), Turnip mosaic virus (TuMV), Passiflora mottle virus (PaMV), Passiflora latene carlavirus (PLV), and Potato virus Y (PVY), among which PVY infection was firstly reported from passion fruit. EAPV was the most prevalent one with infection rate as high as 96.08%, followed consecutively by TeMV (81.70%), CMV (78.43%), TuMV (44.44%), PaMV (26.80%), PVY (24.84%) and PLV (15.03%). In the present case, the distribution of passion fruit viruses in Guizhou province and the diversity of virus sequences were analyzed. Seven viruses were detected in Zhenning, six in Zhenfeng, five in Rongjiang and Congjiang, and three in Luodian. There’s a close identity between the nucleic acid of the seven detected viruses and the corresponding reference sequences (above 95%). Phylogenetic analysis revealed that EAPV, TeMV, PaMV, PLV and TuMV somewhat geographical clustered with passion fruit virus isolated from other provinces in China. CMV and PVY were most closely related to Cucumis melo CMV isolated from Spain and Capsicum annuum PVY isolated from Rwanda respectively, suggesting that the virus sequences diversely varies among the origins in China. A total of 24 types of mixed infection were detected in the main growing areas of passion fruit in Guizhou, and the complex infection rate was as high as 94.77%. The complex infection with 3-5 types predominated in the tested samples, and the combination of EAPV+TeMV+CMV was most frequent among the complex infections among which, EAPV, TeMV and CMV detected ranged from 78.43% to 96.08% in the five counties, somehow reflecting that they were the major viruses to prevent in passion fruit industry of Guizhou province. In addition, a multiplex PCR assay system for EAPV, TeMV and CMV was established in this study, which provides technical support for the rapid detection of related viruses.

, authors=null, authorsList=Yixin YAN, Guang QIAO, Kui ZHOU, Manying ZHANG, Ruirui LI, Xiaopeng WEN, authorCompany=null, correspAuthors=Xiaopeng WEN, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1276598106472977230, articleId=1276598102840709946, tenantId=1146029695717560320, journalId=1235980609244409860, language=CN, title=贵州百香果主要种植区病毒种类的检测, columnId=1236292524520820846, journalTitle=热带作物学报, columnName=植物保护与生物安全, runingTitle=null, highlight=null, articleAbstract=

百香果(Passiflora edulis)作为贵州特色优势水果,在巩固脱贫攻坚成果和深入实施乡村振兴战略中具有重要意义。但近年来百香果病毒病危害日趋严重,造成产量和品质急剧下降,严重影响产业发展。为确定贵州百香果主要种植区病毒种类、分布及复合侵染情况,本研究从贵州省罗甸县、从江县、榕江县、镇宁县和贞丰县5个百香果主要种植区采集疑似感病叶片153份,采用RT-PCR技术对其进行检测。结果表明:样本共检测到7种病毒,分别是东亚西番莲病毒(East Asian Passiflora virus,EAPV)、夜来香花叶病毒(Telosma mosaic virus,TeMV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、芜菁花叶病毒(Turnip mosaic virus,TuMV)、西番莲斑驳病毒(Passiflora mottle virus,PaMV)、西番莲潜隐病毒(Passiflora latene carlavirus,PLV)和马铃薯Y病毒(Potato virus Y,PVY)。EAPV检出率最高,达96.08%,以下依次为TeMV(81.70%)、CMV(78.43%)、TuMV(44.44%)、PaMV(26.80%)、PVY(24.84%)和PLV(15.03%);值得重视的是,PVY主要为害马铃薯,而侵染百香果为首次报道。本次检测到的7种病毒的测序结果与相应参考序列的核酸一致性均在95%以上,经构建进化树发现,EAPV、TeMV、PaMV、PLV、TuMV分别与中国各省份的百香果病毒分离物存在一定的地理聚集性,而CMV和PVY分别与西班牙CMV甜瓜分离物和卢旺达PVY辣椒分离物亲缘关系最近,这说明这些病毒来源及感染途径复杂,未在国内大面积传播。本次共检测到24种复合侵染类型,以3~5种复合侵染类型为主,复合侵染率达94.77%,其中EAPV+TeMV+CMV组合是复合侵染中出现最多的类型;EAPV、TeMV和CMV的检出率高达78.43%~96.08%,在5个百香果主要种植区均有检出,且在多种复合侵染类型中普遍存在,因此这3种病毒为侵染贵州百香果主要种植区的优势病毒。此外,本研究建立了EAPV、TeMV和CMV的多重PCR检测体系,为相关病毒的快速检测提供技术支持。

, authors=

闫艺心(1997—),女,硕士研究生,研究方向:生物技术与工程。

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* 文晓鹏(WEN Xiaopeng),E-mail:
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闫艺心(1997—),女,硕士研究生,研究方向:生物技术与工程。

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闫艺心(1997—),女,硕士研究生,研究方向:生物技术与工程。

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A: Curling; B: Crinkled, deformed; C: Flowering leaves, yellowing, yellow spots; D: Fruit, skin mottling.

, figureFileSmall=9yQfUo/1E4i29WGQV/54tw==, figureFileBig=lNDGkcRZeH1uc57jRsmE+Q==, tableContent=null), ArticleFig(id=1276598409251390047, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276598102840709946, language=CN, label=图1, caption=百香果病毒为害植株的田间症状表现

A:卷曲;B:皱缩、畸形;C:花叶、黄化、黄斑;D:果皮斑纹。

, figureFileSmall=9yQfUo/1E4i29WGQV/54tw==, figureFileBig=lNDGkcRZeH1uc57jRsmE+Q==, tableContent=null), ArticleFig(id=1276598409360441952, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276598102840709946, language=EN, label=Fig. 2, caption=RT-PCR amplification results of P. edulis sample

A: EAPV; B: TeMV; C: CMV; D: TuMV; E: PaMV; F: PLV; G: PVY; M: DL2000 DNA marker; 1–36: Samples from Luodian county; 37–72: Samples from Congjiang county; 73-93: Samples from Rongjiang county; 94-129: Samples from Zhenning county; 130–153: Samples from Zhenfeng county; 154: CK+; 155: CK.

, figureFileSmall=oshkm/xSnBvIS+pWqMp8UQ==, figureFileBig=z9gLA81mFYzjeSl18S5t/w==, tableContent=null), ArticleFig(id=1276598409431745121, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276598102840709946, language=CN, label=图2, caption=百香果样品病毒的RT-PCR扩增结果

A:EAPV;B:TeMV;C:CMV;D:TuMV;E:PaMV;F:PLV;G:PVY;M:DL2000 DNA marker;1~36:罗甸县样品;37~72:从江县样品;73~93:榕江县样品;94~129:镇宁县样品;130~153:贞丰县样品;154:阳性对照;155:阴性对照。

, figureFileSmall=oshkm/xSnBvIS+pWqMp8UQ==, figureFileBig=z9gLA81mFYzjeSl18S5t/w==, tableContent=null), ArticleFig(id=1276598409490465378, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276598102840709946, language=EN, label=Fig. 3, caption=Phylogenetic tree constructed from virus sequences of Guizhou P. edulis with GenBank-related virus sequences, figureFileSmall=N/QiwMzljjcG7px7QUWbcg==, figureFileBig=8oykZAW9nT2jLVr9mx23gg==, tableContent=null), ArticleFig(id=1276598409549185635, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276598102840709946, language=CN, label=图3, caption=基于贵州百香果病毒分离物与GenBank相关序列的系统进化树

A: EAPV; B: TeMV; C: CMV; D: TuMV; E: PaMV; F: PLV; G: PVY.

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M: DL2000 DNA marker.

, figureFileSmall=7R8bgKRx+1OnikaMeh7wfw==, figureFileBig=9iBEy5/iv21nbWrF5m7AXg==, tableContent=null), ArticleFig(id=1276598411579228776, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276598102840709946, language=EN, label=Fig. 6, caption=Amplification of EAPV, TeMV and CMV by multiplex RT-PCR with different annealing temperatures, figureFileSmall=o4ts2zlRgXPgyEEz4vUQFg==, figureFileBig=PDupQ3T7KwT9l44VubBM2w==, tableContent=null), ArticleFig(id=1276598411646337641, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276598102840709946, language=CN, label=图6, caption=不同退火温度下EAPV、TeMV和CMV的多重RT-PCR扩增

M: DL2000 DNA marker.

, figureFileSmall=o4ts2zlRgXPgyEEz4vUQFg==, figureFileBig=PDupQ3T7KwT9l44VubBM2w==, tableContent=null), ArticleFig(id=1276598411717640810, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276598102840709946, language=EN, label=Fig. 7, caption=Amplification of EAPV, TeMV and CMV by multiplex RT-PCR with different cycle index, figureFileSmall=mQEy8M2m5vH9eZpSHpEMmg==, figureFileBig=y5+hTG9SG8MfvD7grSvA0w==, tableContent=null), ArticleFig(id=1276598411793138283, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276598102840709946, language=CN, label=图7, caption=不同循环次数下EAPV、TeMV和CMV的多重RT-PCR扩增

M: DL2000 DNA marker.

, figureFileSmall=mQEy8M2m5vH9eZpSHpEMmg==, figureFileBig=y5+hTG9SG8MfvD7grSvA0w==, tableContent=null), ArticleFig(id=1276598411877024364, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276598102840709946, language=EN, label=Fig. 8, caption=Multiplex PT-PCR (EAPV, TeMV and CMV) virus detection on selected P. edulis samples, figureFileSmall=GaunVevS68YWvcTyZdyV0g==, figureFileBig=vWvevd7PvhtiIB9YTI0mgA==, tableContent=null), ArticleFig(id=1276598411948327533, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276598102840709946, language=CN, label=图8, caption=部分百香果样品的EAPV、TeMV和CMV多重PT-PCR病毒检测

M: DL2000 DNA marker; 1-12: P. edulis samples.

, figureFileSmall=GaunVevS68YWvcTyZdyV0g==, figureFileBig=vWvevd7PvhtiIB9YTI0mgA==, tableContent=null), ArticleFig(id=1276598412019630702, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276598102840709946, language=EN, label=Tab. 1, caption=

Field samples of P. edulis collected from main plantation areas in Guizhou province

, figureFileSmall=null, figureFileBig=null, tableContent=
品种Breed采样数Number of samples
罗甸县Luodian county从江县Congjiang county榕江县Rongjiang county镇宁县Zhenning county贞丰县Zhenfeng county
台农一号242791212
钦密九号9129
黄金果1212
黔香一号15
), ArticleFig(id=1276598412082545263, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276598102840709946, language=CN, label=表1, caption=

贵州百香果主要种植区田间样品

, figureFileSmall=null, figureFileBig=null, tableContent=
品种Breed采样数Number of samples
罗甸县Luodian county从江县Congjiang county榕江县Rongjiang county镇宁县Zhenning county贞丰县Zhenfeng county
台农一号242791212
钦密九号9129
黄金果1212
黔香一号15
), ArticleFig(id=1276598412145459824, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276598102840709946, language=EN, label=Tab. 2, caption=

Primers and reaction conditions used for P. edulis virus detection

, figureFileSmall=null, figureFileBig=null, tableContent=
引物名称Primer name引物序列(5′-3′)Primer sequence (5′-3′)退火温度Annealing temperature/℃PCR产物大小PCR product size/bp参考文献Reference
EAPV-FATGTGGGTAACACGCTTTC53286本研究
EAPV-RTGTGGCACCATCCTATCA
TeMV-FTGATAGTTCATTGACCCCTT54395本研究
TeMV-RCGCTGAAGTCATAATTTAACCC
CMV-FTTACATTAGAGACCGTAGAGC55690本研究
CMV-RTTGTCATTGCACCTACGTT
TuMV-FACGAGAGTATTTCCAACCGAT54306本研究
TuMV-RTCTTCTTTCATCTCGGGTGT
PaMV-FCATGTCACACAGAGCTATTGAG50840[14]
PaMV-RGCTTCGCATTTTCAACCATAGG
PLV-FAGTGTAGAGTTTGAGGGTGGTG55261[13]
PLV-RTGCGGTAGGTCTCCTGATT
PVY-FGGCATACGGACATAGGAGAAACT57447[15]
PVY-RCTCTTTGTGTTGTCCTCTTGTGT
CABMV-FTTCATACAATGTTGCACGAGA56352本研究
CABMV-RGAACTGCTCTACCTTATTCG
PWV-FAAGTACAAGAGTTGACATCCGTA56327本研究
PWV-RAATTGACAGACCAACGTCGAG
), ArticleFig(id=1276598412229345905, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276598102840709946, language=CN, label=表2, caption=

百香果病毒检测所用引物及反应条件

, figureFileSmall=null, figureFileBig=null, tableContent=
引物名称Primer name引物序列(5′-3′)Primer sequence (5′-3′)退火温度Annealing temperature/℃PCR产物大小PCR product size/bp参考文献Reference
EAPV-FATGTGGGTAACACGCTTTC53286本研究
EAPV-RTGTGGCACCATCCTATCA
TeMV-FTGATAGTTCATTGACCCCTT54395本研究
TeMV-RCGCTGAAGTCATAATTTAACCC
CMV-FTTACATTAGAGACCGTAGAGC55690本研究
CMV-RTTGTCATTGCACCTACGTT
TuMV-FACGAGAGTATTTCCAACCGAT54306本研究
TuMV-RTCTTCTTTCATCTCGGGTGT
PaMV-FCATGTCACACAGAGCTATTGAG50840[14]
PaMV-RGCTTCGCATTTTCAACCATAGG
PLV-FAGTGTAGAGTTTGAGGGTGGTG55261[13]
PLV-RTGCGGTAGGTCTCCTGATT
PVY-FGGCATACGGACATAGGAGAAACT57447[15]
PVY-RCTCTTTGTGTTGTCCTCTTGTGT
CABMV-FTTCATACAATGTTGCACGAGA56352本研究
CABMV-RGAACTGCTCTACCTTATTCG
PWV-FAAGTACAAGAGTTGACATCCGTA56327本研究
PWV-RAATTGACAGACCAACGTCGAG
), ArticleFig(id=1276598412309037682, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276598102840709946, language=EN, label=Tab. 3, caption=

Results of virus disease detection in P. edulis samples from Guizhou

, figureFileSmall=null, figureFileBig=null, tableContent=
项目Istem样品总数Number of samplesEAPVTeMVCMVTuMVPaMVPVYPLV
检出数Detection number检出率Detection rate/%检出数Detection number检出率Detection rate/%检出数Detection number检出率Detection rate/%检出数Detection number检出率Detection rate/%检出数Detection number检出率Detection rate/%检出数Detection number检出率Detection rate/%检出数Detection number检出率Detection rate/%
罗甸县363288.892877.782363.8900.0000.0000.0000.00
从江县3636100.002980.562877.782363.8900.001747.2200.00
榕江县2121100.001990.481990.481152.3800.00838.1000.00
镇宁县3636100.002980.563186.112158.332158.331336.111027.78
贞丰县242291.672083.331979.171354.172083.3300.001354.17
总数15314712512068413823
总检出率/%96.0881.7078.4344.4426.8024.8415.03
), ArticleFig(id=1276598412392923763, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276598102840709946, language=CN, label=表3, caption=

贵州百香果样品的病毒病检测结果

, figureFileSmall=null, figureFileBig=null, tableContent=
项目Istem样品总数Number of samplesEAPVTeMVCMVTuMVPaMVPVYPLV
检出数Detection number检出率Detection rate/%检出数Detection number检出率Detection rate/%检出数Detection number检出率Detection rate/%检出数Detection number检出率Detection rate/%检出数Detection number检出率Detection rate/%检出数Detection number检出率Detection rate/%检出数Detection number检出率Detection rate/%
罗甸县363288.892877.782363.8900.0000.0000.0000.00
从江县3636100.002980.562877.782363.8900.001747.2200.00
榕江县2121100.001990.481990.481152.3800.00838.1000.00
镇宁县3636100.002980.563186.112158.332158.331336.111027.78
贞丰县242291.672083.331979.171354.172083.3300.001354.17
总数15314712512068413823
总检出率/%96.0881.7078.4344.4426.8024.8415.03
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贵州百香果主要种植区病毒种类的检测
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闫艺心 , 乔光 , 周奎 , 张曼莹 , 李蕊蕊 , 文晓鹏 *
热带作物学报 | 植物保护与生物安全 2024,45(7): 1478-1488
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热带作物学报 |植物保护与生物安全 2024 , 45 (7) : 1478 -1488
贵州百香果主要种植区病毒种类的检测
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闫艺心, 乔光, 周奎, 张曼莹, 李蕊蕊, 文晓鹏*
作者信息
  • 贵州大学生命科学学院/农业生物工程研究院/山地植物资源保护与种质创新教育部重点实验室,贵州贵阳 550025
通讯作者:
* 文晓鹏(WEN Xiaopeng),E-mail:
Detection of Virus Species from Main Plantation Areas of Passion Fruit (Passiflora edulis) in Guizhou
Yixin YAN, Guang QIAO, Kui ZHOU, Manying ZHANG, Ruirui LI, Xiaopeng WEN*
Affiliations
  • College of Life Sciences, Guizhou University / Institute of Agro-bioengineering / Key laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guiyang, Guizhou 550025, China
出版时间: 2024-07-25 doi: 10.3969/j.issn.1000-2561.2024.07.020
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百香果(Passiflora edulis)作为贵州特色优势水果,在巩固脱贫攻坚成果和深入实施乡村振兴战略中具有重要意义。但近年来百香果病毒病危害日趋严重,造成产量和品质急剧下降,严重影响产业发展。为确定贵州百香果主要种植区病毒种类、分布及复合侵染情况,本研究从贵州省罗甸县、从江县、榕江县、镇宁县和贞丰县5个百香果主要种植区采集疑似感病叶片153份,采用RT-PCR技术对其进行检测。结果表明:样本共检测到7种病毒,分别是东亚西番莲病毒(East Asian Passiflora virus,EAPV)、夜来香花叶病毒(Telosma mosaic virus,TeMV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、芜菁花叶病毒(Turnip mosaic virus,TuMV)、西番莲斑驳病毒(Passiflora mottle virus,PaMV)、西番莲潜隐病毒(Passiflora latene carlavirus,PLV)和马铃薯Y病毒(Potato virus Y,PVY)。EAPV检出率最高,达96.08%,以下依次为TeMV(81.70%)、CMV(78.43%)、TuMV(44.44%)、PaMV(26.80%)、PVY(24.84%)和PLV(15.03%);值得重视的是,PVY主要为害马铃薯,而侵染百香果为首次报道。本次检测到的7种病毒的测序结果与相应参考序列的核酸一致性均在95%以上,经构建进化树发现,EAPV、TeMV、PaMV、PLV、TuMV分别与中国各省份的百香果病毒分离物存在一定的地理聚集性,而CMV和PVY分别与西班牙CMV甜瓜分离物和卢旺达PVY辣椒分离物亲缘关系最近,这说明这些病毒来源及感染途径复杂,未在国内大面积传播。本次共检测到24种复合侵染类型,以3~5种复合侵染类型为主,复合侵染率达94.77%,其中EAPV+TeMV+CMV组合是复合侵染中出现最多的类型;EAPV、TeMV和CMV的检出率高达78.43%~96.08%,在5个百香果主要种植区均有检出,且在多种复合侵染类型中普遍存在,因此这3种病毒为侵染贵州百香果主要种植区的优势病毒。此外,本研究建立了EAPV、TeMV和CMV的多重PCR检测体系,为相关病毒的快速检测提供技术支持。

贵州  /  百香果  /  病毒检测  /  RT-PCR  /  复合侵染

Passion fruit (Passiflora edulis) is a characteristic and advantageous fruit in Guizhou, which is of great significance in poverty relief consolidation and the rural revitalization. However, in recent years, the viral diseases of passion fruit have become increasingly severe, leading to a sharp decline in yield and quality, which thereby seriously constrain industry development of passion fruit. In order to determine the virus species, distribution and compound infestation in the main plantation areas of passion fruit in Guizhou province, a total of 153 samples suspectedly infected by virus were collected from five main plantation orchards of passion fruit in five counties, i.e. Luodian, Congjiang, Rongjiang, Zhenning and Zhenfeng, and the virus detection was carried out by RT-PCR. The results showed that seven viruses were detected from the tested samples, namely East Asian Passiflora virus (EAPV), Telosma mosaic virus (TeMV), Cucumber mosaic virus (CMV), Turnip mosaic virus (TuMV), Passiflora mottle virus (PaMV), Passiflora latene carlavirus (PLV), and Potato virus Y (PVY), among which PVY infection was firstly reported from passion fruit. EAPV was the most prevalent one with infection rate as high as 96.08%, followed consecutively by TeMV (81.70%), CMV (78.43%), TuMV (44.44%), PaMV (26.80%), PVY (24.84%) and PLV (15.03%). In the present case, the distribution of passion fruit viruses in Guizhou province and the diversity of virus sequences were analyzed. Seven viruses were detected in Zhenning, six in Zhenfeng, five in Rongjiang and Congjiang, and three in Luodian. There’s a close identity between the nucleic acid of the seven detected viruses and the corresponding reference sequences (above 95%). Phylogenetic analysis revealed that EAPV, TeMV, PaMV, PLV and TuMV somewhat geographical clustered with passion fruit virus isolated from other provinces in China. CMV and PVY were most closely related to Cucumis melo CMV isolated from Spain and Capsicum annuum PVY isolated from Rwanda respectively, suggesting that the virus sequences diversely varies among the origins in China. A total of 24 types of mixed infection were detected in the main growing areas of passion fruit in Guizhou, and the complex infection rate was as high as 94.77%. The complex infection with 3-5 types predominated in the tested samples, and the combination of EAPV+TeMV+CMV was most frequent among the complex infections among which, EAPV, TeMV and CMV detected ranged from 78.43% to 96.08% in the five counties, somehow reflecting that they were the major viruses to prevent in passion fruit industry of Guizhou province. In addition, a multiplex PCR assay system for EAPV, TeMV and CMV was established in this study, which provides technical support for the rapid detection of related viruses.

Guizhou  /  Passiflora edulis  /  virus detection  /  RT-PCR  /  complex infestation
闫艺心, 乔光, 周奎, 张曼莹, 李蕊蕊, 文晓鹏. 贵州百香果主要种植区病毒种类的检测. 热带作物学报, 2024 , 45 (7) : 1478 -1488 . DOI: 10.3969/j.issn.1000-2561.2024.07.020
Yixin YAN, Guang QIAO, Kui ZHOU, Manying ZHANG, Ruirui LI, Xiaopeng WEN. Detection of Virus Species from Main Plantation Areas of Passion Fruit (Passiflora edulis) in Guizhou[J]. Chinese Journal of Tropical Crops, 2024 , 45 (7) : 1478 -1488 . DOI: 10.3969/j.issn.1000-2561.2024.07.020
百香果又称西番莲(Passiflora edulis),是西番莲科(Passifloraceae)西番莲属(Passiflora Linn.)多年生常绿藤本热带水果,原产于南美洲,我国引种栽培后主要分布在台湾、广西、贵州、福建、广东、海南和云南等亚热带及热带地区[1]。百香果作为“第三代新兴果树”[2],集果用、药用、油用和庭院观赏为一体[3],且当年种植当年挂果,经济见效快,我国不少地区将其列为“短、平、快”的农业项目[4]
在百香果种植过程中,病毒病是其效益发挥的重要制约因素[5]。百香果感染病毒后,植株出现叶片黄化、花叶、畸变、卷曲,果实小且畸形,严重影响经济效益[1]。由于百香果多采用营养繁殖,且长期连作,使病毒病蔓延迅速[6]。目前,已报道能侵染百香果的病毒高达40余种,包括东亚西番莲病毒(East Asian Passiflora virus,EAPV)[7]、夜来香花叶病毒(Telosma mosaic virus,TeMV)[8]、黄瓜花叶病毒(Cucumber mosaic virus,CMV)[9]、西番莲木质化病毒(Passion fruit woodiness virus,PWV)[10]和豇豆蚜传花叶病毒(Cowpea aphidborne mosaic virus,CABMV)[11]等。在贵州种植的百香果上,严佳文等[12]检测到CMV和TeMV,任羽羽等[13]检测到PLV,这些结果为当地百香果产业发展及病毒病防控提供了参考,但这些检测仅涉及个别种植园,尚缺乏全省主要种植区病毒病种类和分布的报道。本研究拟采用RT-PCR法,对贵州百香果的病毒种类及复合侵染情况进行鉴定分析,旨在为全省范围内百香果果园规划、种苗繁育和病毒病防控等产业问题提供参考。
2021—2022年,从贵州省罗甸县、从江县、榕江县、镇宁县和贞丰县百香果种植基地,采集百香果疑似感病叶片153份(表1),样品症状主要包括花叶、黄化、皱缩、卷曲等;另采集健康百香果种子萌发的实生苗叶片作为阴性对照。所有样品经液氮速冻后于–80 ℃超低温冰箱保存备用。
采用RNA提取试剂盒(Omega Bio-Tek)提取疑似病毒侵染叶片混合样品总RNA,具体方法参照试剂说明书。用酶标仪测定其浓度和纯度,琼脂糖凝胶电泳检测其质量。
根据GenBank上已登录的序列,利用Oligo 7软件设计EAPV、TeMV、CMV、TuMV、CABMV、PWV的特异性检测引物,PaMV、PLV、PVY的检测引物参考相关文献报道(表2),本研究所有引物均委托生工生物工程(上海)股份有限公司进行合成。
以疑似病毒侵染的百香果叶片RNA为模板,逆转录合成cDNA第一链,具体过程见逆转录试剂盒说明书(TaKaRa宝生物工程有限公司)。以cDNA为模板,进行RT-PCR扩增,扩增体系为50 μL:2× Canace Gold PCR buffer 25 μL,Hieff Canace Gold High-Fidelity DNA Polymerase 0.5 μL,10 μmol/L的上下游引物各2.5 μL,cDNA模板5 μL,无菌ddH2O补足后混合均匀。PCR具体反应程序为:98 ℃预变性6 min;98 ℃变性10 s,60 ℃退火20 s,72 ℃延伸30 s,循环35次;72 ℃延伸5 min,4 ℃保存。取PCR产物进行1%琼脂糖凝胶电泳,通过凝胶成像系统观察结果。PCR阴性对照为百香果无病毒侵染的实生苗。
采用DNA凝胶回收试剂盒(TaKaRa宝生物工程有限公司)将特异性目的片段回收纯化,连接PEASY-Blunt载体(全式金生物技术股份有限公司产),转化至大肠杆菌感受态中,将PCR阳性克隆产物送至生工生物工程(上海)股份有限公司进行测序。测序结果与NCBI数据库中对应序列进行Blast比对。采用MEGA-7的邻接法(Neighbor-Joining,NJ)进行1000次置信度自展分析,并构建进化树。
采用RT-PCR法,对采集的153份百香果疑似感病叶片进行病毒检测,统计病毒检出样本数、采集样本数、分布情况及贵州百香果病毒的复合侵染类型。病毒检出率=病毒检出样本数/采集样本数×100%。
以复合感染EAPV、TeMV和CMV病毒的百香果样品cDNA为模板,适量稀释后进行多重RT-PCR扩增。对多重RT-PCR体系的退火温度(50、52、54、56、58、60 ℃)、引物含量(0.5、1、1.5、2 μL)和循环次数(20、25、30、35、40次)进行梯度设置,以优化该反应体系。PCR扩增产物进行1%琼脂糖凝胶电泳,通过凝胶成像系统观察结果。利用优化建立后的多重RT-PCR扩增体系对采集的部分复合侵染样品进行检测,以验证该体系的准确性。
田间调查发现,在贵州省罗甸县、从江县、榕江县、镇宁县和贞丰县5个百香果主要种植区,均有病毒病发生,主要表现为叶片卷曲(图1A)、皱缩、畸形(图1B)、花叶、黄化、黄斑(图1C)、果皮斑驳(图1D)等症状。长期连作地区如贞丰、镇宁等,病毒病发生率明显高于新种植地区,新果园也有发生,说明苗木带毒。
采用RT-PCR法对采集的153份样品进行9种病毒检测,结果显示,EAPV、TeMV、CMV、TuMV、PaMV、PLV和PVY共7对引物对可分别在286、395、690、306、261、840、447 bp处扩增出特异性条带,条带位置与预期大小吻合,清晰明亮(图2)。CABMV与PWV未扩增到预期大小的目的条带,表明样品中未检测出这2种病毒;百香果实生苗未扩增出条带,说明百香果实生苗不带病毒,可以作为阴性对照。
将上述检测到的7种病毒随机选取部分样品进行克隆测序,将获得的序列分别命名为EAPVGZ、TeMV-GZ、CMV-GZ、TuMV-GZ、PaMV-GZ、PLV-GZ和PVY-GZ,结果显示,这些序列与EAPV(KP114136)、TeMV(KJ789129)、CMV(KX-525733)、TuMV(KF246570)、PaMV(MK492286)、PLV(MK340756)和PVY(ON604844)核苷酸序列一致性非常高,分别为97.56%、98.41%、95.67%、98.37%、99.69%、99.23%和97.54%。
采用邻接法对7种病毒测序结果构建系统发育树,结果表明,EAPV-GZ、TeMV-GZ、PaMV-GZ、PLV-GZ和TuMV-GZ均与中国不同省份的百香果病毒分离物在同一个亚组,存在一定的地理聚集性。EAPV-GZ与台湾百香果EAPV分离物(KP-114136)有较近的亲缘关系(图3A);TeMV- GZ与广西百香果TeMV分离物(KJ789129)聚为一支(图3B);TuMV-GZ除了与福建百香果TuMV分离物(MK340758)亲缘关系较近外,还与福建厦门蝴蝶兰TuMV分离物(KF246570)、中国TuMV芥菜分离物(LC537522)同处于一个亚组(图3D);PaMV-GZ分离物与海南百香果PaMV分离物(MK492286)以较高的自展值(Bootstrap value=100)聚为同一分支(图3E);PLV-GZ分离物和福建百香果PLV分离物(MK340756)有较近的亲缘关系,而与福建厦门百香果PLV分离物(MK340757)亲缘关系较远(图3F)。
CMV-GZ和PVY-GZ则与不同物种的病毒分离物亲缘关系较近。CMV-GZ和西班牙甜瓜CMV分离物(KX525733)、西班牙番茄CMV分离物(AM183115)、意大利西葫芦CMV分离物(OM867855)聚为一支,有较近的亲缘关系,而与浙江半夏CMV分离物(EU723570)、中国山东矮牵牛CMV分离物(EU414799)亲缘关系较远(图3C);PVY-GZ和卢旺达辣椒PVY分离物(ON604844)亲缘关系较近,而与贵州烟草PVY分离物亲缘关系较远(图3G)。
在贵州省5个百香果主产区采集的153份样品中,全部检测出病毒。不同的病毒分布存在一定差异(表3),EAPV的总检出率最高,达96.08%,其余依次为TeMV(81.70%)、CMV(78.43%)、TuMV(44.44%)、PaMV(26.80%)、PVY(24.84%)和PLV(15.03%)。EAPV、TeMV和CMV分布最为广泛,在所有百香果种植基地均有检出,EAPV的检出率在88.89%~100%之间,TeMV的检出率在77.78%~90.48%之间,CMV的检出率在63.89%~90.48%之间,是贵州省侵染百香果的主要病毒。TuMV在罗甸县未检出,在其余四县的检出率达52.38%~63.89%;PaMV和PLV的分布仅涉及镇宁县和贞丰县;PVY在从江县的检出率最高。镇宁县检测到7种病毒,贞丰县检测到6种病毒,榕江县和从江县检测到5种病毒,罗甸县检测到3种病毒。
本研究采集的153份样品中,共有8份为单一病毒侵染,分别为罗甸县EAPV样品3份、TeMV样品2份、CMV样品1份和贞丰县PaMV样品2份,其余145份样品均为病毒复合侵染,复合侵染率为94.77%。共有24种复合侵染类型,其中3~5种复合侵染类型占比较大,仅在7份样品中检测到6种病毒复合侵染类型,均出现在镇宁县和贞丰县,未发现7种病毒复合侵染类型。EAPV+TeMV+CMV的复合侵染组合检出23份,是复合侵染出现最多的类型(图4)。
对侵染贵州百香果的主要病毒EAPV、TeMV和CMV建立多重RT-PCR扩增体系。设置不同引物浓度进行扩增,结果显示引物加入量为0.5~ 2 μL时,3种病毒均能扩增出目的条带,因此为节约成本,选取引物加入量为0.5 μL(反应终浓度为2.0 mmol/L)为最佳引物浓度(图5)。采用优化后的引物浓度对多重RT-PCR体系的退火温度进行筛选,结果显示当退火温度为54 ℃时,3种病毒扩增的目的条带清晰无杂带,因此选取54 ℃为多重RT-PCR体系的最优退火温度(图6)。采用优化后的引物浓度和退火温度对多重RT-PCR体系的循环次数进行筛选,结果显示当循环次数为35次和40次时,扩增出的条带较清晰,因此为节约循环时间,选取循环次数35次为最佳循环次数(图7)。
综上所述,多重RT-PCR的最佳反应体系为:2×Taq PCR MasterMix Ⅱ 10 μL,10 μmol/L的上下游引物各0.5 μL,cDNA模板0.5 μL,无菌ddH2O补足至20 μL。最佳反应条件为:94 ℃预变性3 min;94 ℃变性30 s,54 ℃退火30 s,72 ℃延伸50 s,循环35次;72 ℃延伸10 min,4 ℃保存。如图8所示,利用优化后的多重RT-PCR体系对12份EAPV、TeMV和CMV复合侵染的样品进行扩增,条带位置与预期大小吻合,检测结果与单一RT-PCR扩增结果一致,因此该体系适用于EAPV、TeMV和CMV的多重检测。
目前,国内外报道能侵染百香果的病毒高达44种[16],但对其复合侵染的研究较少。谢慧婷等[17]在广西百香果上检测出3种病毒,存在4种复合侵染类型,复合侵染率为34.30%;陈礼浪等[16]在海南百香果上检测出7种病毒,存在32种复合侵染类型,以2~4种病毒复合侵染为主,复合侵染率达78.91%。本研究对贵州百香果主要种植区进行检测,共检测到7种病毒及24种复合侵染类型,3~5种复合侵染类型占比较大,复合侵染率达94.77%。其中EAPV、TeMV和CMV的总检出率高达78.43%~96.08%,在5个百香果主要种植区都有检出,且在11种复合侵染类型中普遍存在,因此,这3种病毒为侵染贵州百香果主要种植区的优势病毒。
此外,本研究首次在百香果上检测出PVY。PVY最早发现于英国的马铃薯上[18],我国于1950年在烟草上首次发现[19],该病毒传播方式多样,主要以蚜虫为媒介进行传播,也可以通过汁液摩擦和嫁接等方式传播。PVY的寄主种类繁多,可侵染茄科、豆科和藜科34个属170余种植物,严重影响植物品质及产量[20]。PVY与EAPV、TeMV、PaMV等常侵染百香果的病毒均属于马铃薯Y病毒属(Potyvirus),本研究通过孙琦等[15]已建立的PVY检测体系,在百香果上首次检测出PVY,说明百香果是PVY的新宿主,这为百香果病毒病监测及防控提供了依据,为后续的相关研究提供技术支持。鉴于病毒发生和传播的特性,后续应加强对该病毒的监管与检测,防止其大面积传播。
多重RT-PCR法可同时检测多种病毒,显著提高了检测效率,但目前关于百香果的多重RT-PCR报道较少。本研究前期利用单一RT-PCR法检测出贵州百香果的主要优势病毒为EAPV、TeMV和CMV,这3种病毒在福建[10]、海南[16]、广西[17]等地也被广泛检出,是感染国内百香果的主要病毒。本研究以单一RT-PCR检测为基础,对引物浓度和退火温度等因素进行了筛选优化,建立了这3种病毒的多重RT-PCR体系,为百香果病毒的快速检测提供了技术支持。
病毒侵染百香果后会呈现不同的田间症状,植物的表型症状会随宿主种类、生长状况、生长年龄、感染阶段和外部条件不同而变化,在某些情况下,植物病毒病的症状与病虫害的发生、植物营养缺乏、逆境胁迫和除草剂损害的症状非常相似,难以辨别[21]。本研究中百香果病毒病复合侵染情况严重,存在“一毒多症”和“多毒一症”的情况,仅通过表型观察难以区分,因此在田间防治过程中,遇到疑似病毒感染植株应及时送检,以明确病毒种类,及时防治,从源头上控制该病害的发生及传播。值得注意的是,病毒病在新果园也有发生,说明苗木带病,为了贵州百香果产业的可持续性发展,培育本地脱毒苗并实行严格的种苗检验检疫措施刻不容缓。
序列分析对病毒溯源有一定参考价值,通过构建进化树发现,EAPV、TeMV、PaMV、PLV、TuMV均与中国各省份的百香果病毒分离物在同一亚组,存在一定的地理聚集性,这说明它们可能具有相同的进化来源,病毒是通过苗木的跨地区引种销售而实现的远距离传播。其中,EAPV-GZ分离物与台湾百香果分离物(KP114136)一致性最高,与大陆首次检测的百香果EAPV分离物(ON641738)亲缘关系较近,因此合理推测大陆地区发现的EAPV是从台湾引种时传入的,这与谢丽雪等[7]的研究结果一致。TeMV-GZ与广西百香果亲缘关系较近,在本次采样的5个县均有检出,严佳文等[12]和任羽羽等[13]也先后检测到,这说明TeMV-GZ在贵州百香果中已经广泛流行。
将测序结果与NCBI数据库进行比对时发现,CMV和PVY与相应参考序列的相似性较低,经构建进化树可以看出,CMV和PVY分别与西班牙甜瓜和卢旺达辣椒的病毒分离物亲缘关系较近,这说明病毒来源和感染途径复杂,合理推测这也是PVY在贵州被首次发现的原因:(1)病毒的引种携带。据余亚白等[22]报道,国内百香果品种多引自于台湾,而台湾百香果均引自于海外,其中不乏民间个人引进,由于病毒病潜伏期长,缺乏引种检疫意识,病毒可能通过国外引种时传入。(2)病毒的跨物种传播。CMV和PVY的自然寄主类型丰富,传播方式多样,蚜虫等媒介可从通过啃食该病毒的其他宿主将其传播至百香果中。因此在百香果的种植过程中,除了要增强引种检疫意识,还要加强对传播媒介的防治,把进化关系较近的物种在同一地区分开种植,以免造成病毒的大面积播散。
近年来,在农村产业发展的推动下,贵州百香果产业实现了高速发展,现种植规模已跃居全国第三。百香果作为贵州的特色优势水果,对巩固脱贫攻坚成果和深入实施乡村振兴战略具有深刻的意义。本研究对贵州百香果主要种植区的病毒侵染情况进行综合分析,对该产业的健康可持续发展具有一定的参考意义。
  • 中央引导地方科技发展资金项目(黔科合中引地[2023]-9)
  • 贵州省农业农村厅种业工程项目(黔农计财[2022]-9)
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2024年第45卷第7期
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doi: 10.3969/j.issn.1000-2561.2024.07.020
  • 接收时间:2023-05-19
  • 首发时间:2026-06-24
  • 出版时间:2024-07-25
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  • 收稿日期:2023-05-19
  • 修回日期:2023-08-01
基金
中央引导地方科技发展资金项目(黔科合中引地[2023]-9)
贵州省农业农村厅种业工程项目(黔农计财[2022]-9)
作者信息
    贵州大学生命科学学院/农业生物工程研究院/山地植物资源保护与种质创新教育部重点实验室,贵州贵阳 550025

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* 文晓鹏(WEN Xiaopeng),E-mail:
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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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