芒果畸形病病原菌Fusarium mangiferae的快速分子检测
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摘要
芒果畸形病是芒果上的重要病害之一,由镰孢菌侵染引起,其中以Fusarium mangiferae为主要致病菌。该病害诊断困难,且难于有效控制,因此,一旦发生则对芒果生产造成严重威胁。研究基于ISSR分子标记技术,从50条已知引物中筛选得到一条目的引物UBC 888,该引物可稳定扩增出大小为479bp的F.mangiferae特异性条带(Gen Bank Accession No.KJ526382)。根据获得的特异性片段序列设计引物,成功地将ISSR标记转化为SCAR标记,并获得一对SCAR特异性引物(W342,W1772)和一段大小为1 376bp的特异性扩增片段(Gen Bank Accession No.KJ526383)。通过优化特异性引物扩增条件,获得最适退火温度,构建芒果畸形病病原菌F.mangiferae的快速分子检测技术。此技术操作简单,特异性强,可检测真菌DNA的含量最低为10pg,适用于F.mangiferae和田间带菌芒果组织高灵敏度快速检测,为芒果畸形病的早期诊断和及时预防提供可靠理论依据和技术方法。
Mango malformation disease(MMD),caused by Fusarium,is one of the most destructive diseases on mango,imposing continuous threat on future mango industries.The disease is difficult to diagnose and short of effective control methods.Based on ISSR molecular marker,a purpose primer(UBC 888) was selected from 50 primers,and a stable and specific band of 479bp(Gen Bank Accession KJ526382) was generated.According to the specific sequence,the authors designed a pair of SCAR primers(W342,W1772) and got a specific gene fragment of 1 376bp(Gen Bank Accession KJ526383),and the ISSRmolecular markers were successfully transformed to SCAR ones.An effective detection technique of mango malformation disease was established based on this pair of primers.The established technique is simple,of high specificity and sensitivity to the pathogen both in vitro and in vivo,with the lowest detecting limit of 10 pg fungal DNA.This assay provided a theoretical basis and technical method for early diagnosis and proper prevention of mango malformation disease.
Mango malformation disease(MMD),caused by Fusarium,is one of the most destructive diseases on mango,imposing continuous threat on future mango industries.The disease is difficult to diagnose and short of effective control methods.Based on ISSR molecular marker,a purpose primer(UBC 888) was selected from 50 primers,and a stable and specific band of 479bp(Gen Bank Accession KJ526382) was generated.According to the specific sequence,the authors designed a pair of SCAR primers(W342,W1772) and got a specific gene fragment of 1 376bp(Gen Bank Accession KJ526383),and the ISSRmolecular markers were successfully transformed to SCAR ones.An effective detection technique of mango malformation disease was established based on this pair of primers.The established technique is simple,of high specificity and sensitivity to the pathogen both in vitro and in vivo,with the lowest detecting limit of 10 pg fungal DNA.This assay provided a theoretical basis and technical method for early diagnosis and proper prevention of mango malformation disease.
引文
Chang YH,Zhang L,Liu YZ,Chen ZY,2012.Advances of PCRdetection technology in plant pathogenic fungi.Jiangsu Agricultural Science,40(2):89-93(in Chinese)
Chen JS,Zheng FC,2007.Application of ITS sequences in classification and identification.Journal of Anhui Agricultural Sciences,35(13):3786-3792(in Chinese)
Flis B,Hennig J,Strzelczyk-Zyta D,Gebhardt C,Marczewski W,2005.The Ry-fsto gene from Solanum stoloniferum for extreme resistant to potato virus Y maps to potato chromosome XII and is diagnosed by PCR marker GP122718 in PVY resistant potato cultivars.Molecular Breeding,15(1):95-101
Freeman S,2007.Epidemiology and control of mango malformation disease,submitted to the U.S.Agency for International Development,Bureau for Global Programme,Field Support and Research Centre for Economic Growth and Agriculture Development.Plant Pathology,56:39
Jia J,Pu JJ,Lv YC,Zhan W,Wang F,Xie YX,2012.Detection of mango malformation disease by nested-PCR.Guangdong Agricultural Sciences,22:162-164(in Chinese)
Khaskheli MI,Pathan MA,Jiskani MM,Wagan KH,Soomro MH,Poussio GB,2008.First record of Fusarium nivale(Fr.)Ces.associated with mango malformation disease(MMD)in Pakistan.Pakistan Journal of Botany,40(6):2641-2644
Kojima T,Nagaoka T,Noda K,Ogihara Y,1998.Genetic linkage map of ISSR and RAPD markers in Einkorn wheat in relation to that of RFLP markers.Theoretical and Applied Genetics,96:37-45
Liao F,Zhao L,Luo JF,Huang GM,Liu YT,Li MG,2009.Molecular detection of T illetia bromi on brome grasses.Mycosystema,28(6):776-782(in Chinese)
Liu F,Lu NH,Zhan RL,Wei JG,2012a.Advances in mango malformation disease.Chinese Journal of Tropical Crops,33(11):2104-2109(in Chinese)
Liu F,Wei JG,Zhan RL,Chang JM,2012b.Vegetative compatibility of Fusarium mangiferae strains from mango malformation disease.Journal of Fruit Science,29(6):1092-1096(in Chinese)
María D,Jiménez-Gasco M,Jiménez-Díaz RM,2003.Development of a specific polymerase chain reaction-based assay for the identification of Fusarium oxysporum f.sp.ciceris and its pathogenic races 0,1A,5,and 6.Phytopathology,2(93):200-209
Mohamed NMI,Salleh B,Leslie JF,2013.Fusarium species associated with mango malformation in peninsular Malaysia.Journal of Phytopathology,161:617-624
Newman Z,Freeman S,Biton I,Sa’ada D,Paz T,Maymon M,Lavi U,2012.Molecular diagnosis of mango malformation disease and phylogeny of Fusarium mangiferae.Phytoparasitica,40:287-297
Pu JJ,Xie YX,Zhang H,Zhang X,Qi YX,Peng J,2014.Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium mangiferae associated with mango malformation.Physiological and Molecular Plant Pathology,86:81-88
Ratnaparkhe MB,Santra DK,Tullu A,Muehlbauer FJ,1998.Inheritance of inter-simple-sequence-repeat polymorphisms and linkage with a fusarium wilt resistance gene in chickpeal.Theoretical and Applied Genetics,96:348-353
Rodriguez-Alvarado G,Fernández-Pavia SP,Ploetz RC,Valenzuela-Vázquez M,2008.A Fusarium sp.different from Fusarium oxysporum and F.mangiferae is associated with mango malformation in Michoacán,Mexico.Plant Pathology,57:781
Wang JB,Wang LX,Liu ZY,Du ZJ,Yi XP,Xv BY,2005.Comparison of genomic DNA extraction methods and optimization of ISSR-PCR reaction systems for mango(Mangifera indica L.).Biotechnology,15(5):37-41(in Chinese)
Wu JB,Zhan RL,Liu F,Li GP,Zhao YL,Chang JM,2013.Isolation,identification and diversity analysis of endophytic fungi in Mangifera indica.Journal of Fruit Science,30(6):1030-1039(in Chinese)
XüMY,Tang CH,Zhang JS,Tang QJ,Yang Y,Jia W,Pan YJ,2008.Development of SCAR markers based on SRAP and ISSR for rapid identification of Ganoderma strains.Mycosystema,27(5):707-717(in Chinese)
XüYM,Liu XM,Wang JM,2011.Applications of ISSR in the researches of the fungal plant pathogens.Chinese Agricultural Science Bulletin,27(9):358-361(in Chinese)
Yang SJ,Zhan RL,Liu F,Zhao YL,Chang JM,He YB,2011.The pathogen causing mango malformation in Jinsha River dry-hot valley,China.Mycosystema,30(1):27-31(in Chinese)
Zhan RL,2011.Diagnosis and treatment of the primary mango diseases and insect pests.China Agricultural Press,Beijing.5(in Chinese)
Zhang BJ,Li ZG,Zhang JR,Wang JM,2008.Comparative study on 4 kinds of extraction methods of genomic DNA from Fusarium spp.Journal of Anhui Agricultural Sciences,36(31):13559-13560(in Chinese)
Zietkiewicz E,Rafalski A,Labuda D,1994.Genome fingerprinting by simple sequence repeat(SSR)-anchored polymerase chain reaction amplification.Genomics,20:176-183
常有宏,张磊,刘邮洲,陈志谊,2012.植物病原真菌PCR检测技术研究进展.江苏农业科学,40(2):89-93
陈剑山,郑服丛,2007.ITS序列分析在真菌分类鉴定中的应用.安徽农业科学,35(13):3786-3792
柳凤,卢乃会,詹儒林,韦继光,2012a.芒果畸形病研究进展.热带作物学报,33(11):2104-2109
柳凤,韦继光,詹儒林,常金梅,2012b.芒果畸形病病原菌的营养体亲和性研究.果树学报,29(6):1092-1096
廖芳,赵磊,罗加凤,黄国明,刘跃庭,李明刚,2009.雀麦上雀麦腥黑粉菌Tilletia bromi的分子检测.菌物学报,28(6):776-782
贾静,蒲金基,吕延超,占魏,王芳,谢艺贤,2012.芒果畸形病的巢式PCR检测.广东农业科学,22:162-164
王家保,王令霞,刘志媛,杜中军,易小平,徐碧玉,2005.芒果DNA提取方法及ISSR反应体系的优化.生物技术,15(5):37-41
吴婧波,詹儒林,柳凤,李国平,赵艳龙,常金梅,2013.杧果内生真菌分离鉴定及多样性分析.果树学报,30(6):1030-1039
许美燕,唐传红,张劲松,唐庆九,杨焱,贾薇,潘迎捷,2008.利用SRAP和ISSR建立快速鉴定灵芝属菌株的SCAR标记.菌物学报,27(5):707-717
徐玉梅,刘小妹,王建明,2011.ISSR标记技术在植物病原真菌研究中的应用.中国农学通报,27(9):358-361
杨顺锦,詹儒林,柳凤,赵艳龙,常金梅,何衍彪,2011.金沙江干热河谷地区芒果畸形病的病原菌.菌物学报,30(1):27-31
詹儒林,2011.芒果主要病虫害诊断与防治原色图谱.北京:中国农业出版社.5
张宝俊,李志岗,张家榕,王建明,2008.4种镰刀菌基因组DNA提取方法的比较研究.安徽农业科学,36(31):13559-13560
Chen JS,Zheng FC,2007.Application of ITS sequences in classification and identification.Journal of Anhui Agricultural Sciences,35(13):3786-3792(in Chinese)
Flis B,Hennig J,Strzelczyk-Zyta D,Gebhardt C,Marczewski W,2005.The Ry-fsto gene from Solanum stoloniferum for extreme resistant to potato virus Y maps to potato chromosome XII and is diagnosed by PCR marker GP122718 in PVY resistant potato cultivars.Molecular Breeding,15(1):95-101
Freeman S,2007.Epidemiology and control of mango malformation disease,submitted to the U.S.Agency for International Development,Bureau for Global Programme,Field Support and Research Centre for Economic Growth and Agriculture Development.Plant Pathology,56:39
Jia J,Pu JJ,Lv YC,Zhan W,Wang F,Xie YX,2012.Detection of mango malformation disease by nested-PCR.Guangdong Agricultural Sciences,22:162-164(in Chinese)
Khaskheli MI,Pathan MA,Jiskani MM,Wagan KH,Soomro MH,Poussio GB,2008.First record of Fusarium nivale(Fr.)Ces.associated with mango malformation disease(MMD)in Pakistan.Pakistan Journal of Botany,40(6):2641-2644
Kojima T,Nagaoka T,Noda K,Ogihara Y,1998.Genetic linkage map of ISSR and RAPD markers in Einkorn wheat in relation to that of RFLP markers.Theoretical and Applied Genetics,96:37-45
Liao F,Zhao L,Luo JF,Huang GM,Liu YT,Li MG,2009.Molecular detection of T illetia bromi on brome grasses.Mycosystema,28(6):776-782(in Chinese)
Liu F,Lu NH,Zhan RL,Wei JG,2012a.Advances in mango malformation disease.Chinese Journal of Tropical Crops,33(11):2104-2109(in Chinese)
Liu F,Wei JG,Zhan RL,Chang JM,2012b.Vegetative compatibility of Fusarium mangiferae strains from mango malformation disease.Journal of Fruit Science,29(6):1092-1096(in Chinese)
María D,Jiménez-Gasco M,Jiménez-Díaz RM,2003.Development of a specific polymerase chain reaction-based assay for the identification of Fusarium oxysporum f.sp.ciceris and its pathogenic races 0,1A,5,and 6.Phytopathology,2(93):200-209
Mohamed NMI,Salleh B,Leslie JF,2013.Fusarium species associated with mango malformation in peninsular Malaysia.Journal of Phytopathology,161:617-624
Newman Z,Freeman S,Biton I,Sa’ada D,Paz T,Maymon M,Lavi U,2012.Molecular diagnosis of mango malformation disease and phylogeny of Fusarium mangiferae.Phytoparasitica,40:287-297
Pu JJ,Xie YX,Zhang H,Zhang X,Qi YX,Peng J,2014.Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium mangiferae associated with mango malformation.Physiological and Molecular Plant Pathology,86:81-88
Ratnaparkhe MB,Santra DK,Tullu A,Muehlbauer FJ,1998.Inheritance of inter-simple-sequence-repeat polymorphisms and linkage with a fusarium wilt resistance gene in chickpeal.Theoretical and Applied Genetics,96:348-353
Rodriguez-Alvarado G,Fernández-Pavia SP,Ploetz RC,Valenzuela-Vázquez M,2008.A Fusarium sp.different from Fusarium oxysporum and F.mangiferae is associated with mango malformation in Michoacán,Mexico.Plant Pathology,57:781
Wang JB,Wang LX,Liu ZY,Du ZJ,Yi XP,Xv BY,2005.Comparison of genomic DNA extraction methods and optimization of ISSR-PCR reaction systems for mango(Mangifera indica L.).Biotechnology,15(5):37-41(in Chinese)
Wu JB,Zhan RL,Liu F,Li GP,Zhao YL,Chang JM,2013.Isolation,identification and diversity analysis of endophytic fungi in Mangifera indica.Journal of Fruit Science,30(6):1030-1039(in Chinese)
XüMY,Tang CH,Zhang JS,Tang QJ,Yang Y,Jia W,Pan YJ,2008.Development of SCAR markers based on SRAP and ISSR for rapid identification of Ganoderma strains.Mycosystema,27(5):707-717(in Chinese)
XüYM,Liu XM,Wang JM,2011.Applications of ISSR in the researches of the fungal plant pathogens.Chinese Agricultural Science Bulletin,27(9):358-361(in Chinese)
Yang SJ,Zhan RL,Liu F,Zhao YL,Chang JM,He YB,2011.The pathogen causing mango malformation in Jinsha River dry-hot valley,China.Mycosystema,30(1):27-31(in Chinese)
Zhan RL,2011.Diagnosis and treatment of the primary mango diseases and insect pests.China Agricultural Press,Beijing.5(in Chinese)
Zhang BJ,Li ZG,Zhang JR,Wang JM,2008.Comparative study on 4 kinds of extraction methods of genomic DNA from Fusarium spp.Journal of Anhui Agricultural Sciences,36(31):13559-13560(in Chinese)
Zietkiewicz E,Rafalski A,Labuda D,1994.Genome fingerprinting by simple sequence repeat(SSR)-anchored polymerase chain reaction amplification.Genomics,20:176-183
常有宏,张磊,刘邮洲,陈志谊,2012.植物病原真菌PCR检测技术研究进展.江苏农业科学,40(2):89-93
陈剑山,郑服丛,2007.ITS序列分析在真菌分类鉴定中的应用.安徽农业科学,35(13):3786-3792
柳凤,卢乃会,詹儒林,韦继光,2012a.芒果畸形病研究进展.热带作物学报,33(11):2104-2109
柳凤,韦继光,詹儒林,常金梅,2012b.芒果畸形病病原菌的营养体亲和性研究.果树学报,29(6):1092-1096
廖芳,赵磊,罗加凤,黄国明,刘跃庭,李明刚,2009.雀麦上雀麦腥黑粉菌Tilletia bromi的分子检测.菌物学报,28(6):776-782
贾静,蒲金基,吕延超,占魏,王芳,谢艺贤,2012.芒果畸形病的巢式PCR检测.广东农业科学,22:162-164
王家保,王令霞,刘志媛,杜中军,易小平,徐碧玉,2005.芒果DNA提取方法及ISSR反应体系的优化.生物技术,15(5):37-41
吴婧波,詹儒林,柳凤,李国平,赵艳龙,常金梅,2013.杧果内生真菌分离鉴定及多样性分析.果树学报,30(6):1030-1039
许美燕,唐传红,张劲松,唐庆九,杨焱,贾薇,潘迎捷,2008.利用SRAP和ISSR建立快速鉴定灵芝属菌株的SCAR标记.菌物学报,27(5):707-717
徐玉梅,刘小妹,王建明,2011.ISSR标记技术在植物病原真菌研究中的应用.中国农学通报,27(9):358-361
杨顺锦,詹儒林,柳凤,赵艳龙,常金梅,何衍彪,2011.金沙江干热河谷地区芒果畸形病的病原菌.菌物学报,30(1):27-31
詹儒林,2011.芒果主要病虫害诊断与防治原色图谱.北京:中国农业出版社.5
张宝俊,李志岗,张家榕,王建明,2008.4种镰刀菌基因组DNA提取方法的比较研究.安徽农业科学,36(31):13559-13560