JIANG Dongshuai,XI Nana,YANG Junyu,et al.Effect of Sampling and Sending Conditions on RT-PCR Detection of Apple Virus Sample[J].Northern Horticulture,2019,43(19):13-20.[doi:10.11937/bfyy.20184618]
样本采集及寄送条件对苹果病毒RT-PCR检测效果的影响
- Title:
- Effect of Sampling and Sending Conditions on RT-PCR Detection of Apple Virus Sample
- Keywords:
- apple virus; RT-PCR detection; collection; conservation; sending condition
- 文献标志码:
- A
- 摘要:
- 为了解决采样季节、采样部位及寄送条件不规范影响苹果病毒样本检测可靠性的问题，以携带苹果锈果类病毒（Apple skin scar viroid，ASSVd）、苹果褪绿叶斑病毒（Apple chlorotic leafspot virus，ACLSV）、苹果花叶病毒（Apple mosaic virus,ApMV）、苹果茎痘病毒（Apple stem pitting virus,ASPV）、苹果茎沟病毒（Apple stem grooving virus,ASGV）的苹果植株为试材，采用RT-PCR检测技术，研究了在不同月份采集果树不同组织部位，带毒样本在不同温度下保存不同时间，比较RT-PCR检测效果,以期为规范苹果病毒检测样本的采集、保存与寄送方式提供依据。结果表明：10月采样进行ASSVd、ACLSV、ApMV、ASPV RT-PCR检测，与其它时期相比检出率相对较高，以冠层下部1年生枝皮顶梢为材料对4种病毒进行RT-PCR检测，与其它组织部位相比检出率相对较高。4月采样，进行ASGV RT-PCR检测，检出率相对较高，冠层上部1年生枝皮顶梢较其它组织部位检出率高。1年生枝皮样本在4、25 ℃保存对RT-PCR检测影响相对较小，保存1 d和3 d，3棵树5种病毒的检测结果均为阳性，4 ℃条件下保存5 d，除ApMV，其它4种病毒3棵树检测结果均为阳性，保存7 d及以上多数病毒在测试温度下（0、4、25、37 ℃）检测结果为阴性。
- Abstract:
- In order to standardize sample collecting and sending for the detection of apple virus.Apple trees infected with (Apple skin scar viroid,ASSVd)，（Apple chlorotic leafspot virus，ACLSV)，（Apple mosaic virus，ApMV)，（Apple stem pitting virus，ASPV)，（Apple stem grooving virus，ASGV) were used as test materials.Using RT-PCR detection technology，the different tissue parts of fruit trees were collected in different months，and the virus samples were stored at different temperatures for different time.The results provide the basis for standardizing the collection，preservation and sending of apple virus detection samples.The results showed that the detection rate of ASSVd，ACLSV，ApMV，ASPV by RT-PCR in October was higher than that in other periods.The detection rate of four viruses by RT-PCR was higher than that of other tissues by using the annual branch skin of the lower part of the canopy as the material.In April，the detection rate of ASGV RT-PCR was higher than that of other tissues，and the detection rate of the upper crown was higher than that of other tissues.When the annual branch skin samples were stored at 4，25 ℃ for 1 day and 3 days，the results of 5 kinds of viruses in three trees were all positive，and the results of three trees except ApMV，were all positive when stored at 4 ℃ for 5 days.Most of the viruses stored for 7 days or more were negative at any test temperature.
参考文献/References:
[1]何水涛，刘沛镇，俞宏.苹果病毒及其无病毒化技术概述[J].河南农业科学，1995(5)：23-27.[2]李小燕，蔺国菊，葛红霞，等.苹果病毒病发生及防治趋势[J].北方园艺，2002(3)：66-67.[3]王亚南，胡同乐，刘淑香，等.我国苹果病毒病的研究现状[J].安徽农业科学，2010,38(15):7933-7936.[4]刘福昌.苹果病毒病的发生和研究现状[J].植物检疫,1984(5):6-10.[5]刘福昌，王焕玉.苹果病毒病的研究和防治[J].中国果树,1983(2):31-33.[6]潘季淑，徐绍颖，孙文彬，等.苹果病毒病害调查初报[J].中国果树,1979(2)：44-51.[7]赵英,牛建新.苹果凹果类病毒(ADFVd)的检测与序列分析[J].果树学报，2008(5)：682-685.[8]赵英,牛建新.新疆苹果皱果类病毒(AFCVd)的检测与序列分析[J].农业生物技术学报,2009,17(1):164-169.[9]刘秀丽.苹果绿皱果病的发生与预防技术[J].山西果树,2011(3):22-23.[10]魏宁生.苹果花叶病试验初报[J].西北农林科技大学学报，1959(3)：41-74.[11]NODA H，YAMAGISHI N，YAEGASHI H，et al.Apple necrotic mosaic virus，a novel ilarvirus from mosaic-diseased apple trees in Japan and China[J].Journal of General Plant Pathology，2017，83：83-90.[12]张涛,吴云锋,曹瑛,等.李属坏死环斑病毒病研究进展[J].北方果树,2012(1):1-3.[13]杜国荣，杜俊杰.果树病毒病与脱毒技术综述[J].山西果树，2004(5)：36-37.[14]SHAMLOUL A M，FAGGIOLI F，KEITH J M，et al.A novel multiplex RT-PCR probe capture hybridization(RT-PCR-ELISA) for simultaneous detection of six viroids in four genera：Apscaviroid，Hostuviroid，Pelamoviroid，and Pospiviroid[J].Journal of Virological Methods，2002，105(1)：115.[15]WINKOWSKA L，GRIMOVA L，RYSANEK P.Quantitative detection of four pome fruit viruses in apple trees throughout the year[J].Phytopathologia Mediterranea,2016，55(2):207-224.[16]SVOBODA J，POLK J.Relative concentration of Apple mosaic virus coat protein in different parts of apple tree[J].Horticultural Science-UZEI (Czech Republic)，2010，37(1)：22-26.[17]吴然.苹果锈果类病毒的时空分布规律及对花青苷合成和相关基因的影响[D].保定：河北农业大学，2015.[18]冀志蕊.我国苹果主要病毒的检测及苹果褪绿叶斑病毒分子变异研究[D].保定：河北农业大学，2012.[19]PARK H，YOON J，KIM H，et al.Mutiplex RT-PCR assay for the detection of apple stem grooving virus and apple chlorotic leaf spot virus in infected Korean apple cultivars[J].Plant Pathology Journal，2006，22(2)：168-173.[20]杨金凤，吕运霞，唐兴敏，等.苹果锈果类病毒RT-PCR检测体系的建立[J].中国果树，2015(5)：62-67.[21]吕婵娟，周常勇，周彦，等.运用一步法RT-PCR检测柑橘鳞皮病毒及其在橘橙不同部位中的全年分布[J].植物保护，2007，33(3)：42-45.
相似文献/References:
[1]孙国珍,高利利,陆文利,等.河北省设施番茄褪绿病毒分子检测和鉴定研究[J].北方园艺,2015,39(09):95.[doi:10.11937/bfyy.201509026]
SUN Guo-zhen,GAO Li-li,LU Wen-li,et al.Molecular Detection and Identification of Tomato chlorosis virus Infecting Greenhouse-grown Tomato Plants in Hebei Province[J].Northern Horticulture,2015,39(19):95.[doi:10.11937/bfyy.201509026]
备注/Memo
第一作者简介：蒋东帅（1992-），男，内蒙古察右中旗人，硕士研究生，研究方向为分子植物病理学。E-mail：2419893153@qq.com.责任作者：王亚南（1979-），女，河北三河人，博士，教授，研究方向为果蔬病毒与寄主的互作及其综合防治。E-mail：wyn3215347@163.com.基金项目：国家重点研发计划资助项目（2016YFD0201100）；国家苹果现代产业技术体系资助项目（CARS-27）；河北省自然科学基金资助项目（C2019204327）。收稿日期：2019-03-26