芸薹根肿菌及油菜根肿病分子检测与早期诊断
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摘要
近年来芸薹根肿菌引起的根肿病已成为我国油菜的主要病害,严重影响油菜及其它十字花科作物的产量及品质。为建立一套在土壤、植物组织和种子中精准检测根肿菌的高效分子方法,利用根肿菌ITS(internal transcribed spacer)序列,设计一对特异性引物(Pb ITS1),通过PCR反应优化体系进行根肿菌的分子检测及病害评估。结果表明,该引物及方法能够特异性检测根肿菌DNA,不受土传病原真菌、细菌、线虫及寄主植物内生菌的DNA干扰。灵敏性检测表明,模板DNA最低浓度达1pg·μL-1,土壤带菌量最低为1×103个孢子/g土,种子带菌量最低1×105个孢子/g种子。此外,该体系能够用于检测油菜及其它十字花科寄主植物(如组织、种子)和土壤类型。同时,可用于田间油菜不同生长阶段油菜根部及周围土壤的根肿菌检测。本研究建立的检测体系操作简便、适用范围广、灵敏度高、特异性强,可检测样品种类丰富,可为油菜及其它十字花科根肿病的早期诊断、流行监测与预警、综合防控等提供科学依据。
Clubroot has recently become one of the main diseases of oilseed rape and cruciferous crops in China,which seriously affected the yield of cruciferous crops.Therefore,an efficient method with a high efficiency for molecular detection of P.brassicae was developed for accurate diagnosis in various samples using a pair of specific primers(PbITS1) designed on ITS(internal transcribed spacer) sequence,and the molecular detection of P.brassicae and disease assessment were carried out by PCR procedure.The results indicated that PCR system could specifically detect P.brassicae without any amplification product from representative soil-borne pathogenic fungi,bacteria,nematodes and endophytes.Sensitivity assay showed that the minimum concentration of template DNA was 1× 10-3 ng/μL,the minimum number of spores in the contaminated soil was 1×103 spores/g soil,and the minimum level of spore with seeds was 1 × 105 spores/g seeds.Moreover,rape and other cruciferous plants(e.g.,tissues and seeds) and soils could be applied in this detection system,meanwhile roots and surrounding soils collected from the different growth stages could be used for disease assessment.The benefit of this detection system was convenient,sensitive and specific with a wide range of detectable samples,which provided insights on detection of P.brassicae as well as the diagnosis,monitoring epidemic pattern,and integrated management of clubroot.
Clubroot has recently become one of the main diseases of oilseed rape and cruciferous crops in China,which seriously affected the yield of cruciferous crops.Therefore,an efficient method with a high efficiency for molecular detection of P.brassicae was developed for accurate diagnosis in various samples using a pair of specific primers(PbITS1) designed on ITS(internal transcribed spacer) sequence,and the molecular detection of P.brassicae and disease assessment were carried out by PCR procedure.The results indicated that PCR system could specifically detect P.brassicae without any amplification product from representative soil-borne pathogenic fungi,bacteria,nematodes and endophytes.Sensitivity assay showed that the minimum concentration of template DNA was 1× 10-3 ng/μL,the minimum number of spores in the contaminated soil was 1×103 spores/g soil,and the minimum level of spore with seeds was 1 × 105 spores/g seeds.Moreover,rape and other cruciferous plants(e.g.,tissues and seeds) and soils could be applied in this detection system,meanwhile roots and surrounding soils collected from the different growth stages could be used for disease assessment.The benefit of this detection system was convenient,sensitive and specific with a wide range of detectable samples,which provided insights on detection of P.brassicae as well as the diagnosis,monitoring epidemic pattern,and integrated management of clubroot.
引文
[1] Dixon G R. The occurrence and economic impact of Plasmodiophora brassicae and clubroot disease[J]. J Plant Growth Regul,2009,28(3):194-202.
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[3] Cavalier-Smith T,Chao E Y. Phylogeny and classification of phylum cercozoa(Protozoa)[J]. Protist,2003,153(3/4):341-358.
[4] Dixon G R. Plasmodiophora brassicae in its environment[J]. J Plant Growth Regul,2009,28(3):212-228.
[5] Karling J S. The Plasmodiophorales[J]. Science,1968,99(2572):299-301.
[6] Dixon G R. The biology of Plasmodiophora brassicae Wor.-a review of recent advances[J]. Acta Hortic,2006(706):271-282.
[7] Faggian R,Strelkov S E. Detection and measurement of Plasmodiophora brassicae[J]. J Plant Growth Regul,2009,28(3):282-288.
[8] Buhariwalla H,Greaves S,Magrath R,et al. Development of specific PCR primers for the amplification of polymorphic DNA from the obligate root pathogen Plasmodiophora brassicae[J]. Physiol Mol Plant Pathol,1995,47(2):83-94.
[9] Cao T S,Tewari J,Strelkov S E. Molecular detection of Plasmodiophora brassicae,causal agent of clubroot of crucifers,in plant and soil[J]. Plant Dis,2007,91(1):80-87.
[10]杨佩文,李家瑞,杨勤忠,等.根肿病菌核糖体基因ITS区段的克隆测序及其在检测中的应用[J].云南农业大学学报,2003,18(3):228-233.
[11]尹全,宋君,刘勇,等.土壤根肿病菌休眠孢子PCR快速检测方法的建立[J].西南农业学报,2010,23(2):390-392.
[12]王江岭,张建成,顾建锋.单条线虫DNA提取方法[J].植物检疫,2011,25(2):32-35.
[13] Rennie D C,Manolii V P,Cao T,et al. Direct evidence of surface infestation of seeds and tubers by Plasmodiophora brassicae and quantification of spore loads[J]. Plant Pathol,2011,60(5):811-819.
[14]杨佩文,杨勤忠,王群,等.十字花科蔬菜根肿病菌的PCR检测[J].云南农业大学学报,2002,17(2):137-139,157.
[15] Chee H Y,Kim W G,Cho W D,et al. Detection of Plasmodiophora brassicae by using polymerase chain reaction[J]. Kor J Plant Pathol,1998,14:589-593.
[16]杨华.湖南十字花科蔬菜品种抗根肿病鉴定、病菌小种及q PCR土壤病菌检测技术研究[D].长沙:湖南农业大学,2014.
[17]杜艳,刘邮洲,李建斌,等.十字花科根肿病研究现状及展望[J].江苏农业科学,2014,42(10):122-126.
[18]刘勇,罗一帆,黄小琴,等.芸薹根肿菌生理小种鉴别方法研究进展[J].中国油料作物学报,2011,33(4):420-426.
[19] Hwang S F,Strelkov S E,Feng J,et al. Plasmodiophora brassicae:a review of an emerging pathogen of the Canadian canola(Brassica napus)crop[J]. Mol Plant Pathol,2012,13(2):105-113.
[2] Chai A L,Xie X W,Shi Y X,et al. Research status of clubroot(Plasmodiophora brassicae)on cruciferous crops in China[J]. Can J Plant Pathol,2014,36(sup1):142-153.
[3] Cavalier-Smith T,Chao E Y. Phylogeny and classification of phylum cercozoa(Protozoa)[J]. Protist,2003,153(3/4):341-358.
[4] Dixon G R. Plasmodiophora brassicae in its environment[J]. J Plant Growth Regul,2009,28(3):212-228.
[5] Karling J S. The Plasmodiophorales[J]. Science,1968,99(2572):299-301.
[6] Dixon G R. The biology of Plasmodiophora brassicae Wor.-a review of recent advances[J]. Acta Hortic,2006(706):271-282.
[7] Faggian R,Strelkov S E. Detection and measurement of Plasmodiophora brassicae[J]. J Plant Growth Regul,2009,28(3):282-288.
[8] Buhariwalla H,Greaves S,Magrath R,et al. Development of specific PCR primers for the amplification of polymorphic DNA from the obligate root pathogen Plasmodiophora brassicae[J]. Physiol Mol Plant Pathol,1995,47(2):83-94.
[9] Cao T S,Tewari J,Strelkov S E. Molecular detection of Plasmodiophora brassicae,causal agent of clubroot of crucifers,in plant and soil[J]. Plant Dis,2007,91(1):80-87.
[10]杨佩文,李家瑞,杨勤忠,等.根肿病菌核糖体基因ITS区段的克隆测序及其在检测中的应用[J].云南农业大学学报,2003,18(3):228-233.
[11]尹全,宋君,刘勇,等.土壤根肿病菌休眠孢子PCR快速检测方法的建立[J].西南农业学报,2010,23(2):390-392.
[12]王江岭,张建成,顾建锋.单条线虫DNA提取方法[J].植物检疫,2011,25(2):32-35.
[13] Rennie D C,Manolii V P,Cao T,et al. Direct evidence of surface infestation of seeds and tubers by Plasmodiophora brassicae and quantification of spore loads[J]. Plant Pathol,2011,60(5):811-819.
[14]杨佩文,杨勤忠,王群,等.十字花科蔬菜根肿病菌的PCR检测[J].云南农业大学学报,2002,17(2):137-139,157.
[15] Chee H Y,Kim W G,Cho W D,et al. Detection of Plasmodiophora brassicae by using polymerase chain reaction[J]. Kor J Plant Pathol,1998,14:589-593.
[16]杨华.湖南十字花科蔬菜品种抗根肿病鉴定、病菌小种及q PCR土壤病菌检测技术研究[D].长沙:湖南农业大学,2014.
[17]杜艳,刘邮洲,李建斌,等.十字花科根肿病研究现状及展望[J].江苏农业科学,2014,42(10):122-126.
[18]刘勇,罗一帆,黄小琴,等.芸薹根肿菌生理小种鉴别方法研究进展[J].中国油料作物学报,2011,33(4):420-426.
[19] Hwang S F,Strelkov S E,Feng J,et al. Plasmodiophora brassicae:a review of an emerging pathogen of the Canadian canola(Brassica napus)crop[J]. Mol Plant Pathol,2012,13(2):105-113.