新疆地区四种草莓病毒病原的检测
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摘要
【目的】研究新疆草莓病毒病病源种类及田间发生情况,建立主要病毒病原多重RT-PCR检测体系,为无病毒草莓生产提供理论和技术支持。【方法】利用已报道的4种草莓主要病毒病原4对特异性引物,分别对新疆主要草莓种植区采集表现疑似病毒病症状的草莓样品共450份,采用单一反转录PCR(RT-PCR)及多重PCR对采集的样品进行检测,分析新疆草莓病毒病的发生情况。【结果】感染新疆草莓有3种主要病毒,南北疆15个不同栽培区均能检测出这3种病毒,每个采集地的病毒检出率均在26%以上。所有样品中病毒总检出率为64. 22%;其中,草莓镶脉病毒(strawberry vein banding virus,SVBV)检出率最高,为51. 78%;草莓斑驳病毒(strawberry mottle virus,SMoV)检出率为23. 78%,草莓轻型黄边病毒(strawberry mild yellow edge virus,SMYEV)检出率为9. 33%,草莓皱缩病毒(Strawberry crinkle virus,SCV)没有检出。病毒复合侵染率达18. 45%,两种病毒病复合侵染检出率为16. 67%,三种病毒病复合侵染检出率为1. 78%;所有样品中对3种病毒的多重RT-PCR与单一RT-PCR检测结果一致。【结论】新疆草莓受SVBV、SMoV和SMYEV三种病毒危害,单一病毒病感染率以及两种以上病毒复合侵染侵染率都较高,在无病毒种苗的培育和生产中加强病毒检测。
【Objective】To investigate the origin and occurrence of strawberry virus disease in Xinjiang,and to establish a multiplex RT-PCR detection system for main virus pathogens in the hope of providing theoretical and technical support for the plant production of virus-free strawberry.【Method】According to the 4 pairs of reported specific primers of the 4 species viral disease,450 strawberry samples with suspected viral diseases symptoms were collected from the different main planting areas in Xinjiang,all the samples were tested by single reverse transcription polymerase chain reaction( RT-PCR) and multiplex RT-PCR to analyze the occurrence characteristics of strawberry virus diseases.【Result】The results showed that there were 3 main viruses of SVBV( Strawberry vein banding virus),SMoV( Strawberry mottle virus) and SMYEV( Strawberry mild yellow edge virus),which could be detected from the samples from 15 different cultivation areas in northern and southern Xinjiang and the virus detection rate was above 26% in each collection area. The total detection rate of the virus in all samples was 64. 22%,among which,the detection rate of SVBV was the highest( 51. 78),the detection rate of SmoV was 23. 78 and strawberry of SMYEV was 9. 33%. Strawberry crinkle virus( SCV) was not detected. The complex infection phenomenon showed that co-infections were up to 18.45%,the detection rate of combined infection of two viruses was 16. 67% and the detection rate of combined infection of three kinds of viruses was up to 1. 78%. The results of multiplex RT-PCR for three viruses in all samples are consistent with the results of single RT-PCR detection.【Conclusion】Currently the primary viral disease in strawberry growing areas in Xinjiang is caused by SVBV,SMoV and SMYEV and the infection rate of one single virus and two or more viruses are relatively hight. Therefore,so it is necessary to strengthen virus detection in the cultivation and production of virus-free seedlings.
【Objective】To investigate the origin and occurrence of strawberry virus disease in Xinjiang,and to establish a multiplex RT-PCR detection system for main virus pathogens in the hope of providing theoretical and technical support for the plant production of virus-free strawberry.【Method】According to the 4 pairs of reported specific primers of the 4 species viral disease,450 strawberry samples with suspected viral diseases symptoms were collected from the different main planting areas in Xinjiang,all the samples were tested by single reverse transcription polymerase chain reaction( RT-PCR) and multiplex RT-PCR to analyze the occurrence characteristics of strawberry virus diseases.【Result】The results showed that there were 3 main viruses of SVBV( Strawberry vein banding virus),SMoV( Strawberry mottle virus) and SMYEV( Strawberry mild yellow edge virus),which could be detected from the samples from 15 different cultivation areas in northern and southern Xinjiang and the virus detection rate was above 26% in each collection area. The total detection rate of the virus in all samples was 64. 22%,among which,the detection rate of SVBV was the highest( 51. 78),the detection rate of SmoV was 23. 78 and strawberry of SMYEV was 9. 33%. Strawberry crinkle virus( SCV) was not detected. The complex infection phenomenon showed that co-infections were up to 18.45%,the detection rate of combined infection of two viruses was 16. 67% and the detection rate of combined infection of three kinds of viruses was up to 1. 78%. The results of multiplex RT-PCR for three viruses in all samples are consistent with the results of single RT-PCR detection.【Conclusion】Currently the primary viral disease in strawberry growing areas in Xinjiang is caused by SVBV,SMoV and SMYEV and the infection rate of one single virus and two or more viruses are relatively hight. Therefore,so it is necessary to strengthen virus detection in the cultivation and production of virus-free seedlings.
引文
[1]农业部.2015年全国各地蔬菜、西瓜、甜瓜、草莓、马铃薯播种面积和产量[J].中国蔬菜,2017,1(1):.Ministry of Agriculture,P.R.China(2017).Planting area and yield of vegetables,watermelons,melons,strawberries and potatoes throughout the country in 2015[J].China Vegetables,1(1):.(in Chinese)
[2]赵宝龙,郁松林,刘怀锋,等.新疆地区草莓生产历史、现状及发展建议[J].新疆农垦科技,2014,(1):15-17.ZHAO Bao-long,YU Song-ling,LIU Hai-feng,et al.(2014).History,present situation and Development of Strawberry production in Xinjiang[J].Xinjiang Farm Research of Science and Technology,(1):15-17.(in Chinese)
[3]张志宏,杨洪一,代红艳,等.应用多重RT-PCR检测草莓斑驳病毒和草莓轻型黄边病毒[J].园艺学报,2006,33(3):507-510.ZHANG Zhi-hong,YANG Hong-yi,DAI Hong-yang,et al.(2006).Detection of Strawberry mottle virus and Strawberry mild yellow edge virus by Multiplex RT-PCR[J].Acta Horticulturae Sinica,33(3):507-510.(in Chinese)
[4]盛宏亚,万继花,徐川,等.利用玻璃化超低温技术脱除草莓斑驳病毒(SMo V)的初步研究[J].中国农业大学学报,2016,21(3):53-57.SHENG Hong-ya,WAN Ji-hua,XU Chuan,et al.(2016).Preliminary study on elimination of Strawberry mottle virus(SMoV)from shoot tips by vitrification-cryopreservation treatment[J].Journal of China Agricultural University,21(3):53-57.(in Chinese)
[5]Martin,L.W.,&Converse,R.H.(1977).Influence of recent and chronic virus infections on strawberry growth and yield.Phytopathology,77(5):573-575.
[6]郑巧兮,黄剑清,沈菊英,等.上海地区草莓病毒病病原研究初报[J].中国病毒学,1988,(1):84-88.ZHENG Qiao-xi,HUANG Jian-qin,SHENG Ju-ying,et al.(1988).Preliminary Studies on Pathogen of Strawberry Virus Disease[J].Virologica Sinica,(1):84-88.(in Chinese)
[7]黄富琴,洪建源.田间草莓植株病毒病的鉴定和发病情况初报[J].中国果树,1989,(2):40.HUANG Fu-qin,HONG Jian-yuan.(1989).Identification and incidence of Strawberry Plant virus Disease in the Field[J].China Fruits,(2):40.(in Chinese)
[8]王国平,刘福昌,薛光荣,等.草莓病毒种类鉴定及培育无病毒种苗的技术研究[J].中国农业科学,1990,23(4):43-49.WANG Guo-ping,LIU Fu-chang,XUE Guang-rong,et al.(1990).Research on identification of strawberry viruses in China and techniques of obtaining virus-free strawberries[J].Scientia Agricultura Sinica,23(4):43-49.(in Chinese)
[9]王国平,刘福昌,国际翔.我国草莓主栽区病毒种类的鉴定[J].植物病理学报,1991,21(1):9-14.WANG Guo-ping,LIU Fu-chang,GUO Ji-xiang.(1991).Dentification on strawberry viruses in China[J].Acta Phytopathologica Sinica,21(1):9-14.(in Chinese)
[10]高庆玉,李光裕,周恩.关于草莓脱毒技术研究[J].东北农业大学学报,1993,(3):231-236.GAO Qin-yu,LI Guang-yu,ZHOU En.(1993).A study on virus-elimination techniques for strawberry[J].Journal of Northeast Agricultural College,(3):231-236.(in Chinese)
[11]韦石泉,吴元华.我国草莓斑驳病毒研究鉴定[J].植物病理学报,1994,24(4):293-298.WEI Shi-quan,WU Yuan-hua.(1994).The identification on strawberry mottle virus in China[J].Acta Phytopathologica Sinica,24(4):293-298.(in Chinese)
[12]吴元华,韦石泉,韩桂珍.草莓镶脉病发生调查、病毒鉴定及传播试验[J].植物保护,1994,20(5):27-28.WU Yuan-hua,WEI Shi-quan,HAN Gui-zhen.(1994).Investigation,virus Identification and Transmission Test of Strawberry vein setting Disease[J].Plant Protection,20(5):27-28.(in Chinese)
[13]王运生,陈笑瑜,邢冬梅,等.我国部分地区草莓病毒种类调查及RT-PCR检测[C].中国植物病理学会2015年学术年会论文集.2015.WANG Yun-sheng,CHEN Xiao-yu,XIN Dong-mei,et al.(2015).Investigation and RT-PCR Detection of Strawberry virus in some regions of China[C].Proceedings of the 2015 Annual meeting of the Chinese Society of Plant Pathology.(in Chinese)
[14]Thompson,J.R.,Wetzel SKlerks,M.M.,Vaskova,D.,Schoen,C.D.,&Spak,J.(2003).Multiplex RT-PCR detection of four aphid-borne strawberry viruses in FRagaria spp.in combination with a plant mRNA specific internal control.Journal of Virological Methods,111(2):85-93.
[15]周厚成,李思源,何水涛,等.草莓镶脉病毒的PCR检测及特异片段的序列分析[J].果树学报,2005,22(3):286-288.ZHOU Hou-cheng,LI Si-yuan,HE Shui-tao,et al.(2005).Detection of strawberry vein banding virus by PCR and sequence analysis of specific fragment[J].Journal of Fruit Science,22(3):286-288.(in Chinese)
[16]史芳芳.利用内标为基础的RT-PCR技术检测草莓皱缩病毒(SCV)和草莓镶脉病毒(SVBV)[J].安徽农业科学,2015,43(28):20-21.SHI Fang-fang.(2015).Detection of SCV and SVBV with Multiplex RT-PCR Based on Internal Control[J].Journal of Anhui Agricultural Sciences,43(28):20-21.(in Chinese)
[17]周厚成,何水涛.草莓病毒病研究进展[J].果树学报,2003,20(5):421-426.ZHOU Hou-cheng,HE Shui-tao.(2003).Advances in Strawberry Virus Research[J].Journal of Fruit Science,20(5):421-426.(in Chinese)
[18]王红,王菁菁,张科立,等.利用内标为基础的多重RT-PCR技术检测草莓斑驳病毒和草莓轻型黄边病毒[J].浙江农业学报,2014,26(1):105-109.WANG Hong,WANG Jing-jing,ZHANG Ke-li,et al.(2014).Detection of strawberry mottle virus and strawberry mild yellow edge virus with multiplex RT-PCR based on internal control[J].Acta Agriculturae Zhejiangensis,26(1):105-109.(in Chinese)
[19]常琳琳,张志宏.草莓病毒的简单、快速PCR检测方法[C].全国草莓大会,2009.CAHNG Lin-lin,ZHANG Zhi-hong.(2009).A simple and rapid PCR method for detection of strawberry virus[C].Proceedings of the National Strawberry Conferences.(in Chinese)
[20]朱海生,花秀凤,陈敏氡,等.四种草莓病毒SMoV、SVBV、SCV、SMYEV多重RT-PCR检测[J].核农学报,2013,27(11):1 630-1 635.ZHU Hai-sheng,HUA Xiu-feng,CHEN Min-dong,et al.(2013).Detection of four viruses(SMo V、SVBV、SMYEV and SCV)infecting strawberry by multiplex RT-PCR[J].Journal of Nuclear Agricultural Sciences,27(11):1,630-1,635.(in Chinese)
[21]班爱江,蔚承祥.四季草莓赛娃在新疆伊犁地区的栽培表现[J].落叶果树,2003,35(5):33.BAN Ai-jiang,WEI Cheng-xiang.(2003).Perfomance of everbearing strawberry'Selva'in Yili,Xinjiang Autonomous Region[J].Deciduous Fruits,35(5):33.(in Chinese)
[22]陈玉山,李四维,吴明武,等.草莓优良品种戈雷拉的试种[J].新疆农业科学,1992,(5):232.CHEN Yu-shang,LI Si-wei,WU Ming-wu,et al.(1992).Trial planting of Strawberry Variety Gorela[J].Xinjiang Agriculutral Sciences,(5):232.(in Chinese)
[2]赵宝龙,郁松林,刘怀锋,等.新疆地区草莓生产历史、现状及发展建议[J].新疆农垦科技,2014,(1):15-17.ZHAO Bao-long,YU Song-ling,LIU Hai-feng,et al.(2014).History,present situation and Development of Strawberry production in Xinjiang[J].Xinjiang Farm Research of Science and Technology,(1):15-17.(in Chinese)
[3]张志宏,杨洪一,代红艳,等.应用多重RT-PCR检测草莓斑驳病毒和草莓轻型黄边病毒[J].园艺学报,2006,33(3):507-510.ZHANG Zhi-hong,YANG Hong-yi,DAI Hong-yang,et al.(2006).Detection of Strawberry mottle virus and Strawberry mild yellow edge virus by Multiplex RT-PCR[J].Acta Horticulturae Sinica,33(3):507-510.(in Chinese)
[4]盛宏亚,万继花,徐川,等.利用玻璃化超低温技术脱除草莓斑驳病毒(SMo V)的初步研究[J].中国农业大学学报,2016,21(3):53-57.SHENG Hong-ya,WAN Ji-hua,XU Chuan,et al.(2016).Preliminary study on elimination of Strawberry mottle virus(SMoV)from shoot tips by vitrification-cryopreservation treatment[J].Journal of China Agricultural University,21(3):53-57.(in Chinese)
[5]Martin,L.W.,&Converse,R.H.(1977).Influence of recent and chronic virus infections on strawberry growth and yield.Phytopathology,77(5):573-575.
[6]郑巧兮,黄剑清,沈菊英,等.上海地区草莓病毒病病原研究初报[J].中国病毒学,1988,(1):84-88.ZHENG Qiao-xi,HUANG Jian-qin,SHENG Ju-ying,et al.(1988).Preliminary Studies on Pathogen of Strawberry Virus Disease[J].Virologica Sinica,(1):84-88.(in Chinese)
[7]黄富琴,洪建源.田间草莓植株病毒病的鉴定和发病情况初报[J].中国果树,1989,(2):40.HUANG Fu-qin,HONG Jian-yuan.(1989).Identification and incidence of Strawberry Plant virus Disease in the Field[J].China Fruits,(2):40.(in Chinese)
[8]王国平,刘福昌,薛光荣,等.草莓病毒种类鉴定及培育无病毒种苗的技术研究[J].中国农业科学,1990,23(4):43-49.WANG Guo-ping,LIU Fu-chang,XUE Guang-rong,et al.(1990).Research on identification of strawberry viruses in China and techniques of obtaining virus-free strawberries[J].Scientia Agricultura Sinica,23(4):43-49.(in Chinese)
[9]王国平,刘福昌,国际翔.我国草莓主栽区病毒种类的鉴定[J].植物病理学报,1991,21(1):9-14.WANG Guo-ping,LIU Fu-chang,GUO Ji-xiang.(1991).Dentification on strawberry viruses in China[J].Acta Phytopathologica Sinica,21(1):9-14.(in Chinese)
[10]高庆玉,李光裕,周恩.关于草莓脱毒技术研究[J].东北农业大学学报,1993,(3):231-236.GAO Qin-yu,LI Guang-yu,ZHOU En.(1993).A study on virus-elimination techniques for strawberry[J].Journal of Northeast Agricultural College,(3):231-236.(in Chinese)
[11]韦石泉,吴元华.我国草莓斑驳病毒研究鉴定[J].植物病理学报,1994,24(4):293-298.WEI Shi-quan,WU Yuan-hua.(1994).The identification on strawberry mottle virus in China[J].Acta Phytopathologica Sinica,24(4):293-298.(in Chinese)
[12]吴元华,韦石泉,韩桂珍.草莓镶脉病发生调查、病毒鉴定及传播试验[J].植物保护,1994,20(5):27-28.WU Yuan-hua,WEI Shi-quan,HAN Gui-zhen.(1994).Investigation,virus Identification and Transmission Test of Strawberry vein setting Disease[J].Plant Protection,20(5):27-28.(in Chinese)
[13]王运生,陈笑瑜,邢冬梅,等.我国部分地区草莓病毒种类调查及RT-PCR检测[C].中国植物病理学会2015年学术年会论文集.2015.WANG Yun-sheng,CHEN Xiao-yu,XIN Dong-mei,et al.(2015).Investigation and RT-PCR Detection of Strawberry virus in some regions of China[C].Proceedings of the 2015 Annual meeting of the Chinese Society of Plant Pathology.(in Chinese)
[14]Thompson,J.R.,Wetzel SKlerks,M.M.,Vaskova,D.,Schoen,C.D.,&Spak,J.(2003).Multiplex RT-PCR detection of four aphid-borne strawberry viruses in FRagaria spp.in combination with a plant mRNA specific internal control.Journal of Virological Methods,111(2):85-93.
[15]周厚成,李思源,何水涛,等.草莓镶脉病毒的PCR检测及特异片段的序列分析[J].果树学报,2005,22(3):286-288.ZHOU Hou-cheng,LI Si-yuan,HE Shui-tao,et al.(2005).Detection of strawberry vein banding virus by PCR and sequence analysis of specific fragment[J].Journal of Fruit Science,22(3):286-288.(in Chinese)
[16]史芳芳.利用内标为基础的RT-PCR技术检测草莓皱缩病毒(SCV)和草莓镶脉病毒(SVBV)[J].安徽农业科学,2015,43(28):20-21.SHI Fang-fang.(2015).Detection of SCV and SVBV with Multiplex RT-PCR Based on Internal Control[J].Journal of Anhui Agricultural Sciences,43(28):20-21.(in Chinese)
[17]周厚成,何水涛.草莓病毒病研究进展[J].果树学报,2003,20(5):421-426.ZHOU Hou-cheng,HE Shui-tao.(2003).Advances in Strawberry Virus Research[J].Journal of Fruit Science,20(5):421-426.(in Chinese)
[18]王红,王菁菁,张科立,等.利用内标为基础的多重RT-PCR技术检测草莓斑驳病毒和草莓轻型黄边病毒[J].浙江农业学报,2014,26(1):105-109.WANG Hong,WANG Jing-jing,ZHANG Ke-li,et al.(2014).Detection of strawberry mottle virus and strawberry mild yellow edge virus with multiplex RT-PCR based on internal control[J].Acta Agriculturae Zhejiangensis,26(1):105-109.(in Chinese)
[19]常琳琳,张志宏.草莓病毒的简单、快速PCR检测方法[C].全国草莓大会,2009.CAHNG Lin-lin,ZHANG Zhi-hong.(2009).A simple and rapid PCR method for detection of strawberry virus[C].Proceedings of the National Strawberry Conferences.(in Chinese)
[20]朱海生,花秀凤,陈敏氡,等.四种草莓病毒SMoV、SVBV、SCV、SMYEV多重RT-PCR检测[J].核农学报,2013,27(11):1 630-1 635.ZHU Hai-sheng,HUA Xiu-feng,CHEN Min-dong,et al.(2013).Detection of four viruses(SMo V、SVBV、SMYEV and SCV)infecting strawberry by multiplex RT-PCR[J].Journal of Nuclear Agricultural Sciences,27(11):1,630-1,635.(in Chinese)
[21]班爱江,蔚承祥.四季草莓赛娃在新疆伊犁地区的栽培表现[J].落叶果树,2003,35(5):33.BAN Ai-jiang,WEI Cheng-xiang.(2003).Perfomance of everbearing strawberry'Selva'in Yili,Xinjiang Autonomous Region[J].Deciduous Fruits,35(5):33.(in Chinese)
[22]陈玉山,李四维,吴明武,等.草莓优良品种戈雷拉的试种[J].新疆农业科学,1992,(5):232.CHEN Yu-shang,LI Si-wei,WU Ming-wu,et al.(1992).Trial planting of Strawberry Variety Gorela[J].Xinjiang Agriculutral Sciences,(5):232.(in Chinese)