土壤中烟草根黑腐病菌的real-time PCR分子检测
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摘要
根据烟草根黑腐菌与其它近缘真菌在ITS序列上的差异,设计PCR检测引物TbF/TbR,并通过对各菌株基因组DNA扩增验证其特异性;以接种10倍浓度梯度的根黑腐菌孢子的土壤DNA为模板,建立土壤烟草根黑腐菌real-time PCR检测的标准曲线,得出每个反应体系中分生孢子数目的对数(x)和对应的Ct值(y)之间呈线性关系:y=-1.5373x+24.955,R2=0.9909(P<0.01)。通过对5块烟田土壤烟草根黑腐菌检测和烟田烟草根黑腐病的调查,证实上述方法可以准确检测土壤中烟草根黑腐菌孢子数量。
According to the difference in ITS sequence between black rot fungus of tobacco root and other relative fungi, a pair of specific primer TbF/TbR was designed, and its specificity was verified by genomic DNA amplification. The standard curve for real-time PCR detection of black rot fungi of tobacco root was established by using soil DNA inoculated with spores of black rot fungi(10 times in concentration gardient) as a template. It was found that there was a linear relationship between the logarithm(x) of number of conidia in each reaction system(x) and the corresponding Ct value(y): y=-1.5373 x+24.955, R~2=0.9909(P<0.01). Through detection of black rot fungi of tobacco root in five tobacco fields and the investigation of black rot disease of tobacco root in tobacco field, the above method was proved to be capable of accurately detecting the number of black rot fungi of tobacco root in soil.
According to the difference in ITS sequence between black rot fungus of tobacco root and other relative fungi, a pair of specific primer TbF/TbR was designed, and its specificity was verified by genomic DNA amplification. The standard curve for real-time PCR detection of black rot fungi of tobacco root was established by using soil DNA inoculated with spores of black rot fungi(10 times in concentration gardient) as a template. It was found that there was a linear relationship between the logarithm(x) of number of conidia in each reaction system(x) and the corresponding Ct value(y): y=-1.5373 x+24.955, R~2=0.9909(P<0.01). Through detection of black rot fungi of tobacco root in five tobacco fields and the investigation of black rot disease of tobacco root in tobacco field, the above method was proved to be capable of accurately detecting the number of black rot fungi of tobacco root in soil.
引文
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[7]窦彦霞,彭雄,余佳敏,等.中国烟草根黑腐病菌根串珠霉菌群及r DNA-ITS序列分析[J].菌物学报, 2012, 31(4):531-539.DOU Yanxia, PENG Xiong, YU Jiamin, et al. Grouping and rDNAITS sequence analysis of Thielaviopsis basicola on tobacco in China[J]. Mycosystema, 2012, 31(4):531-539.
[8]窦彦霞,徐林,马寇华,等.烟草根黑腐病菌ISSR标记的遗传多样性分析[J].烟草科技, 2017, 11(50):9-15.DOU Yanxia, XU Lin, MA Guanhua, et al. Genetic diversity analysis of Thielaviopsis basicola by ISSR method[J]. Tobacco Science&Technology, 2017, 11(50):9-15.
[9]Amy E. Paulin-Mahady, Thomas C. Harrington and Doug McNew.Phylogenetic and Taxonomic Evaluation of Chalara, Chalaropsis,and Thielaviopsis Anamorphs Associated with Ceratocystis[J].Mycologia,2002, 94(1):62-72.
[2]边传红,丁玥琪,赵世明,等.河南省烟草根黑腐病菌的分子鉴定及致病性分析[J].烟草科技, 2017, 50(3):8-14.BIAN Chuanhong, DING Yueqi, ZHAO Shimin, et al. Molecular identification and pathogenicity analysis of Thielaviopsis basicola in Henan Province[J]. Tobacco Science&Technology, 2017, 50(3):8-14.
[3]张顺.烟草“两黑病”的防治措施[J].植物保护,2017(4):25-89.ZHANG Shun. Prevention and control measures of"two black disease"in tobacco[J]. plant protection,2017(4):25-89.
[4]陆星星,刘伟阳,徐后娟.烟草三类根腐类病害病原真菌多重PCR检测方法的建立[J].山东农业大学学报(自然科学版),2016, 47(4):520-524.LU Xingxing,LIU Weiyang, XU Hou, et al. Establishment and Application of Multiplex PCR Method for Detection of Three Root Rotting Fungi on Tobacco[J]. Joumal ofShandongAgricultural University(Natural Science Edition), 2016, 47(4):520-524.
[5]赵永强,张成玲,张薇,等.烟草根黑腐病菌的PCR分子检测[J].植物病理学报, 2009, 39(1):23-29.ZHAO Yongqiang,ZHANG Chengling, ZHANG Wei, et al.Molecular detection of Thielaviopsis basicola by PCR assay[J].Acta Phytopathologica Sinica, 2009, 39(1):23-29.
[6]康振辉,黄俊丽.土壤中烟草根黑腐病菌的实时定量PCR检测技术研究[J].植物病理学报,2010, 40(2):210-213.KANG Zhenhui, HUANG Junli. Detection of Thielaviopsis basicolainsoilwithreal-timequantitativePCR[J]. Acta Phytopathologica Sinica, 2010, 40(2):210-213.
[7]窦彦霞,彭雄,余佳敏,等.中国烟草根黑腐病菌根串珠霉菌群及r DNA-ITS序列分析[J].菌物学报, 2012, 31(4):531-539.DOU Yanxia, PENG Xiong, YU Jiamin, et al. Grouping and rDNAITS sequence analysis of Thielaviopsis basicola on tobacco in China[J]. Mycosystema, 2012, 31(4):531-539.
[8]窦彦霞,徐林,马寇华,等.烟草根黑腐病菌ISSR标记的遗传多样性分析[J].烟草科技, 2017, 11(50):9-15.DOU Yanxia, XU Lin, MA Guanhua, et al. Genetic diversity analysis of Thielaviopsis basicola by ISSR method[J]. Tobacco Science&Technology, 2017, 11(50):9-15.
[9]Amy E. Paulin-Mahady, Thomas C. Harrington and Doug McNew.Phylogenetic and Taxonomic Evaluation of Chalara, Chalaropsis,and Thielaviopsis Anamorphs Associated with Ceratocystis[J].Mycologia,2002, 94(1):62-72.