湖南省长沙市番茄黄化曲叶病毒的检测与系统发育分析
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摘要
【目的】对湖南省长沙市发生的疑似番茄黄化曲叶病毒(TYLCV)样本进行检测,明确TYLCV在湖南长沙的发生情况,为TYLCV的防控提供科学依据。【方法】2016年3和11月,在湖南长沙采集疑似TYLCV样本48份,采用已发表的TYLCV不同株系的全基因组序列,利用DNAMAN进行序列比对,根据保守序列设计特异性引物TY-T-F和TYT-R、TY-P-F和TY-P-R对番茄样本的DNA进行PCR、Southern印迹杂交(Sourthern blotting)检测和系统发育分析。对呈阳性条带的PCR产物进行切胶回收并送样测序,在NCBI上进行BLAST比对分析,利用系统发育分析TYLCV湖南长沙分离物与国内外株系的相似性。【结果】采集的48份样本中有42份样本能扩增出目的条带,阳性检出率为87.5%;将42个阳性克隆序列导入MEGA 7.0,得到27个分离物。对测序结果进行同源性分析、多重比对、聚类分析及进化分析,结果显示27个湖南长沙TYLCV分离物与我国其他省(市)分离物的相似性在98.5%~99.8%,与伊朗株系(AJ132711和GU076453)、墨西哥株系(FJ012358)、澳大利亚株系(GU178814)和葡萄牙株系(AF105975)的相似性分别介于90.7%~91.5%、95.4%~95.5%、98.6%~98.8%和99.3%~99.4%。【结论】TYLCV已在湖南长沙发生危害,需加强检疫工作,防止从疫区调运感病番茄,以控制病害扩散蔓延。
【Objective】In order to clarify the occurrence of Tomato yellow leaf curl virus(TYLCV)in Changsha,Hunan,the scientific basis for the prevention and control of TYLCV was provided,and the suspected TYLCV samples collected in Changsha,Hunan were tested.【Method】In March and November of 2016,48 samples of suspected TYLCV were collected in Changsha,Hunan. Sequences from the published whole genomes of different TYLCV strains were aligned by using DNAMAN,then the specific primers TY-T-F and TY-T-R,TY-P-F and TY-P-R were designed according to the conserved fragments,and the DNA of the tomato sample was detected by PCR,Southern bloting and phylogenetic analysis.The PCR products of some positive bands were excised for electrophorosis gel and sequenced. The sequencing result were performed by BLAST comparison analysis on NCBI, and the similarity among Changsha isolates,domestic and foreign strains were studied by phylogenetic analysis.【Result】Among the 48 samples collected,42 samples were dected the target band with a positive detection rate of 87.5%. The 42 positive clone sequences were introduced into MEGA7.0,and 27 isolates were obtained. Homology analysis,multiple alignment,cluster analysis and evolution analysis were performed on the sequencing results. The results showed that the similarity between 27 Changsha TYLCV isolates and isolates from other provinces(municipalities)in China was 98.5%-99.8%,and the similarities between Changsha TYLCV isolates and Iranian strains(AJ132711 and GU076453),Mexican strain(FJ012358),Australian strain(GU178814)and Portuguese strain(AF105975)were 90.7%-91.5%,95.4%-95.5%,98.6%-98.8% and 99.3%-99.4% respectively.【Conclusion】TYLCV has outbroken in Changsha,Hunan. Quarantine work needs to be executed to prevent the transfer of susceptible tomatoes from the affected provinces in order to control the epidemic of the disease.
【Objective】In order to clarify the occurrence of Tomato yellow leaf curl virus(TYLCV)in Changsha,Hunan,the scientific basis for the prevention and control of TYLCV was provided,and the suspected TYLCV samples collected in Changsha,Hunan were tested.【Method】In March and November of 2016,48 samples of suspected TYLCV were collected in Changsha,Hunan. Sequences from the published whole genomes of different TYLCV strains were aligned by using DNAMAN,then the specific primers TY-T-F and TY-T-R,TY-P-F and TY-P-R were designed according to the conserved fragments,and the DNA of the tomato sample was detected by PCR,Southern bloting and phylogenetic analysis.The PCR products of some positive bands were excised for electrophorosis gel and sequenced. The sequencing result were performed by BLAST comparison analysis on NCBI, and the similarity among Changsha isolates,domestic and foreign strains were studied by phylogenetic analysis.【Result】Among the 48 samples collected,42 samples were dected the target band with a positive detection rate of 87.5%. The 42 positive clone sequences were introduced into MEGA7.0,and 27 isolates were obtained. Homology analysis,multiple alignment,cluster analysis and evolution analysis were performed on the sequencing results. The results showed that the similarity between 27 Changsha TYLCV isolates and isolates from other provinces(municipalities)in China was 98.5%-99.8%,and the similarities between Changsha TYLCV isolates and Iranian strains(AJ132711 and GU076453),Mexican strain(FJ012358),Australian strain(GU178814)and Portuguese strain(AF105975)were 90.7%-91.5%,95.4%-95.5%,98.6%-98.8% and 99.3%-99.4% respectively.【Conclusion】TYLCV has outbroken in Changsha,Hunan. Quarantine work needs to be executed to prevent the transfer of susceptible tomatoes from the affected provinces in order to control the epidemic of the disease.
引文
郝永娟,王万立,金凤媚,刘春燕,霍建飞,徐维红.2010.天津市番茄黄化曲叶病毒病的发生与防治[J].天津农业科学,16(2):48-50.[Hao Y J,Wang W L,Jin F M,Liu C Y,Huo J F,Xu W H.2010.Occurrence and control of Tomato yellow leaf curl virus(TYLCV)in Tianjin[J].Tianjin Agricultural Sciences,16(2):48-50.]
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金凤媚,薛俊,郏艳红,周祥明,王建江,刘仲齐.2011.天津地区番茄黄化曲叶病毒DNA-A的克隆和序列分析[J].华北农学报,26(1):58-62.[Jin F M,Xue J,Jia Y H,Zhou X M,Wang J J,Liu Z Q.2011.Cloning and sequence analysis of TYLCV DNA-A in Tianjin[J].Acta Agriculturae Boreali-Sinica,26(1):58-62.]
李志勇,谢夏青,李兴红,董志平.2011.邯郸、保定番茄黄化曲叶病毒检测及其DNA-A全序列分析[J].华北农学报,26(3):64-67.[Li Z Y,Xie X Q,Li X H,Dong ZP.2011.Molecular detection and analysis of the complete nucleotide sequence of TYLCV isolates from Handan and Baoding[J].Acta Agriculturae Boreali-Sinica,26(3):64-67.]
宋晰,师迎春,张世晨,梁彦,王廿,陈善义,周涛.2013.北京地区番茄黄化曲叶病病毒分离物测定及株系的初步鉴定[J].植物病理学报,43(2):113-119.[Song X,Shi Y C,Zhang S C,Liang Y,Wang N,Chen S Y,Zhou T.2013.Detection and preliminary strain identification of the pathogen induing Tomato yellow leaf curl disease in Beijing[J].Acta Phytopathologica Sinica,43(2):113-119.]
汤亚飞,何自福,杜振国,佘小漫,蓝国兵,罗方芳.2015.番茄黄化曲叶病毒在湖北首次报道[J].植物保护,41(5):233-236[.Tang Y F,He Z F,Du Z G,She X M,Lan G B,Luo F F.2015.First report of Tomato yellow leaf curl virus in Hubei Province of China[J].Plant Protection,41(5):233-236.]
王浩权,纠敏,汪伦记,邱智军,黄狮,张敏.2016.侵染河南省番茄的番茄黄化曲叶病毒及伴随卫星DNA分子的基因组特征[J].河南农业科学,45(10):71-75.[Wang H Q,Jiu M,Wang L J,Qiu Z J,Huang S,Zhang M.2016.Genomic characterization of DNA-A and associated satellite DNA molecule of Tomato yellow leaf curl virus infecting tomato in Henan[J].Journal of Henan Agricultural Sciences,45(10):71-75.]
熊艳,杨帅,青玲,周常勇,孙现超,杨水英.2011.四川番茄黄化曲叶病病原分子鉴定及变异分析[J].中国农业科学,44(3):477-484.[Xiong Y,Yang S,Qing L,Zhou C Y,Sun X C,Yang S Y.2011.Molecular identification and variation analysis of the pathogen causing Tomato yellow leaf curl disease in Sichuan Province[J].Scientia Agriculture Sinica,44(3):477-484.]
余文贵,赵统敏,杨玛丽,赵丽萍,季英华,周益军.2009.山东、安徽两省栽培番茄烟粉虱传双生病毒的PCR检测及序列分析[J].江苏农业学报,25(4):747-751.[Yu WG,Zhao T M,Yang M L,Zhao L P,Ji Y H,Zhou Y J.2009.PCR detection and sequence analysis of whiteny-transmitted gemiilivirus in tomato from Anhui and Shandong Provinces[J].Jiangsu Journal of Agricultural Sciences,25(4):747-751.]
赵统敏,余文贵,周益军,季英华.2007.江苏省番茄黄化曲叶病毒病(TYLCD)的发生与诊断初报[J].江苏农业学报,23(6):654-655.[Zhao T M,Yu W G,Zhou Y J,Ji Y H.2007.The occurrence and diagnosis of Tomato yellow leaf curl disease(TYLCD)in Jiangsu Provinc[J].Jiangsu Journal of Agricultural Sciences,23(6):654-655.]
Ammara U E,Mansoor S,Saeed M,Amin I,Briddon R W,Al-Sadi A M.2015.RNA interference-based resistance in transgenic tomato plants against Tomato yellow leaf curl virus-Oman(TYLCV-OM)and its associated betasatellite[J].Virology Journal,12(1):38.
Kil E J,Kim S,Lee Y J,Byun H S,Park J,Seo H,Kim C S,Shim J K,Lee J H,Kim J K,Lee,Choi H S,Lee S.2016.Tomato yellow leaf curl virus(TYLCV-IL):A seedtransmissible geminivirus in tomatoes[J].Scientific Reports,6:19013.
Li M,Li X J,Su Y L.2017a.Flue-cured tobacco confirmed as a reservoir host plant for Tomato yellow leaf curl virus by agro-inoculation and Bemisia tabaci MED-mediated transmission[J].PLo S One,12(12):e0190013.
Li Y Z,Qin L,Zhao J J,Muhammad T,Cao H H,Li H L,Zhang Y,Liang Y.2017b.Sl MAPK3 enhances tolerance to Tomato yellow leaf curl virus(TYLCV)by regulating salicylic acid and jasmonic acid signaling in tomato(Solanum lycopersicum)[J].PLo S One,12(2):e0172466.
Mabvakure B,Martin D P,Kraberger S,Cloete L,Brunschot S V,Geering A D W,Thomas J E,Bananej K,Lett J M,Lefeuvre P,Varsani A,Harkins G W.2016.Ongoing geographical spread of Tomato yellow leaf curl virus[J].Virology,498:257-264.
Mangal M,Srivastava A,Sharma R,Kalia P.2017.Conservation and dispersion of genes conferring resistance to tomato begomoviruses between tomato and pepper genomes[J].Frontiers in Plant Science,doi:10.3389/fpls.2017.01803.
PicóB,Díez M J,Nuez F.1996.Viral diseases causing the greatest economic losses to the tomato crop.II.The Tomato yellow leaf curl virus-A review[J].Scientia Horticulturae,67(3):151-196.
Segbefia M M,Amoatey H M,Ahiakpa K,Ahiakpa J K,Quartey E K,Appiah A S,Nunoo J,Kusi-Adjei R.2018.Field evaluation of tomato varieties/breeding lines against Tomato yellow leaf curl virus disease(TYLCV)[J].Pertanika Journal of Tropical Agricultural Science,41(1):423-440.
Turaki A A,Ahmad B,Magaji U F,Abdulrazak U K,Yusuf BA.2017.Optimised cetyltrimethylammonium bromide(CTAB)DNA extraction method of plant leaf with high polysaccharide and polyphenolic compounds for downstream reliable molecular analyses[J].African Journal of Biotechnology,16(24):1354-1365.
Zhao W H,Ji Y H,Wu S H,Ma X F,Li S,Sun F,Cheng ZB,Zhou Y J,Fan Y J.2018.Single amino acid in V2 encoded by TYLCV is responsible for its self-interaction,aggregates and pathogenicity[J].Scientific Reports,doi:10.1038/s41598-018-21446-2
姜静,王银磊,李亚茹,赵丽萍,周蓉,陈伟男,赵统敏,余文贵.2018.江苏省及其他地区番茄黄化曲叶病毒的分子鉴定及序列分析[J].江苏农业学报,34(1):238-240.[Jiang J,Wang Y L,Li Y R,Zhao L P,Zhou R,Chen WN,Zhao T M,Yu W G.2018.Molecular identification and sequence analysis of Tomato yellow leaf curl virus in Jiangsu Province and its peripheral area[J].Jiangsu Journal of Agricultural Sciences,34(1):238-240.]
金凤媚,薛俊,郏艳红,周祥明,王建江,刘仲齐.2011.天津地区番茄黄化曲叶病毒DNA-A的克隆和序列分析[J].华北农学报,26(1):58-62.[Jin F M,Xue J,Jia Y H,Zhou X M,Wang J J,Liu Z Q.2011.Cloning and sequence analysis of TYLCV DNA-A in Tianjin[J].Acta Agriculturae Boreali-Sinica,26(1):58-62.]
李志勇,谢夏青,李兴红,董志平.2011.邯郸、保定番茄黄化曲叶病毒检测及其DNA-A全序列分析[J].华北农学报,26(3):64-67.[Li Z Y,Xie X Q,Li X H,Dong ZP.2011.Molecular detection and analysis of the complete nucleotide sequence of TYLCV isolates from Handan and Baoding[J].Acta Agriculturae Boreali-Sinica,26(3):64-67.]
宋晰,师迎春,张世晨,梁彦,王廿,陈善义,周涛.2013.北京地区番茄黄化曲叶病病毒分离物测定及株系的初步鉴定[J].植物病理学报,43(2):113-119.[Song X,Shi Y C,Zhang S C,Liang Y,Wang N,Chen S Y,Zhou T.2013.Detection and preliminary strain identification of the pathogen induing Tomato yellow leaf curl disease in Beijing[J].Acta Phytopathologica Sinica,43(2):113-119.]
汤亚飞,何自福,杜振国,佘小漫,蓝国兵,罗方芳.2015.番茄黄化曲叶病毒在湖北首次报道[J].植物保护,41(5):233-236[.Tang Y F,He Z F,Du Z G,She X M,Lan G B,Luo F F.2015.First report of Tomato yellow leaf curl virus in Hubei Province of China[J].Plant Protection,41(5):233-236.]
王浩权,纠敏,汪伦记,邱智军,黄狮,张敏.2016.侵染河南省番茄的番茄黄化曲叶病毒及伴随卫星DNA分子的基因组特征[J].河南农业科学,45(10):71-75.[Wang H Q,Jiu M,Wang L J,Qiu Z J,Huang S,Zhang M.2016.Genomic characterization of DNA-A and associated satellite DNA molecule of Tomato yellow leaf curl virus infecting tomato in Henan[J].Journal of Henan Agricultural Sciences,45(10):71-75.]
熊艳,杨帅,青玲,周常勇,孙现超,杨水英.2011.四川番茄黄化曲叶病病原分子鉴定及变异分析[J].中国农业科学,44(3):477-484.[Xiong Y,Yang S,Qing L,Zhou C Y,Sun X C,Yang S Y.2011.Molecular identification and variation analysis of the pathogen causing Tomato yellow leaf curl disease in Sichuan Province[J].Scientia Agriculture Sinica,44(3):477-484.]
余文贵,赵统敏,杨玛丽,赵丽萍,季英华,周益军.2009.山东、安徽两省栽培番茄烟粉虱传双生病毒的PCR检测及序列分析[J].江苏农业学报,25(4):747-751.[Yu WG,Zhao T M,Yang M L,Zhao L P,Ji Y H,Zhou Y J.2009.PCR detection and sequence analysis of whiteny-transmitted gemiilivirus in tomato from Anhui and Shandong Provinces[J].Jiangsu Journal of Agricultural Sciences,25(4):747-751.]
赵统敏,余文贵,周益军,季英华.2007.江苏省番茄黄化曲叶病毒病(TYLCD)的发生与诊断初报[J].江苏农业学报,23(6):654-655.[Zhao T M,Yu W G,Zhou Y J,Ji Y H.2007.The occurrence and diagnosis of Tomato yellow leaf curl disease(TYLCD)in Jiangsu Provinc[J].Jiangsu Journal of Agricultural Sciences,23(6):654-655.]
Ammara U E,Mansoor S,Saeed M,Amin I,Briddon R W,Al-Sadi A M.2015.RNA interference-based resistance in transgenic tomato plants against Tomato yellow leaf curl virus-Oman(TYLCV-OM)and its associated betasatellite[J].Virology Journal,12(1):38.
Kil E J,Kim S,Lee Y J,Byun H S,Park J,Seo H,Kim C S,Shim J K,Lee J H,Kim J K,Lee,Choi H S,Lee S.2016.Tomato yellow leaf curl virus(TYLCV-IL):A seedtransmissible geminivirus in tomatoes[J].Scientific Reports,6:19013.
Li M,Li X J,Su Y L.2017a.Flue-cured tobacco confirmed as a reservoir host plant for Tomato yellow leaf curl virus by agro-inoculation and Bemisia tabaci MED-mediated transmission[J].PLo S One,12(12):e0190013.
Li Y Z,Qin L,Zhao J J,Muhammad T,Cao H H,Li H L,Zhang Y,Liang Y.2017b.Sl MAPK3 enhances tolerance to Tomato yellow leaf curl virus(TYLCV)by regulating salicylic acid and jasmonic acid signaling in tomato(Solanum lycopersicum)[J].PLo S One,12(2):e0172466.
Mabvakure B,Martin D P,Kraberger S,Cloete L,Brunschot S V,Geering A D W,Thomas J E,Bananej K,Lett J M,Lefeuvre P,Varsani A,Harkins G W.2016.Ongoing geographical spread of Tomato yellow leaf curl virus[J].Virology,498:257-264.
Mangal M,Srivastava A,Sharma R,Kalia P.2017.Conservation and dispersion of genes conferring resistance to tomato begomoviruses between tomato and pepper genomes[J].Frontiers in Plant Science,doi:10.3389/fpls.2017.01803.
PicóB,Díez M J,Nuez F.1996.Viral diseases causing the greatest economic losses to the tomato crop.II.The Tomato yellow leaf curl virus-A review[J].Scientia Horticulturae,67(3):151-196.
Segbefia M M,Amoatey H M,Ahiakpa K,Ahiakpa J K,Quartey E K,Appiah A S,Nunoo J,Kusi-Adjei R.2018.Field evaluation of tomato varieties/breeding lines against Tomato yellow leaf curl virus disease(TYLCV)[J].Pertanika Journal of Tropical Agricultural Science,41(1):423-440.
Turaki A A,Ahmad B,Magaji U F,Abdulrazak U K,Yusuf BA.2017.Optimised cetyltrimethylammonium bromide(CTAB)DNA extraction method of plant leaf with high polysaccharide and polyphenolic compounds for downstream reliable molecular analyses[J].African Journal of Biotechnology,16(24):1354-1365.
Zhao W H,Ji Y H,Wu S H,Ma X F,Li S,Sun F,Cheng ZB,Zhou Y J,Fan Y J.2018.Single amino acid in V2 encoded by TYLCV is responsible for its self-interaction,aggregates and pathogenicity[J].Scientific Reports,doi:10.1038/s41598-018-21446-2