黄芪根腐致病尖孢镰刀菌实时荧光定量PCR检测体系的建立及应用
Establishment and Application of Real-time Quantitative PCR Detection System for the Pathogenic Fusarium oxysporum Causing Astragalus membranaceus var.mongholicus Root Rot
赵丽梅 1赵利 1田洪岭 2高芬3
作者信息
- 1. 山西大学中医药现代研究中心,太原 030006
- 2. 山西农业大学经济作物研究所,太原 030031
- 3. 山西大学应用化学研究所,太原 030006
- 折叠
摘要
尖孢镰刀菌(Fusarium oxysporum)是引起蒙古黄芪(Astragalus membranaceus var.mongholicus,AMM)根腐病的优势致病菌之一.为了快速准确地检测和定量黄芪植株及其种植地土壤中的尖孢镰刀菌,根据延长因子(elongation factor-1α,EF-1α)序列设计引物并验证其特异性,进而建立该病菌的实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)检测体系,并运用该体系对黄芪和土壤样本内的尖孢镰刀菌进行检测.结果表明,设计的引物FaeF2/FaeR2特异性良好;所建体系对黄芪中尖孢镰刀菌DNA的检测灵敏度为0.896 pg/μL,土壤中尖孢镰刀菌孢子量的检测限度为102个分生孢子/g;标准曲线的相关系数分别为0.998和0.973;重复性评价显示该方法可靠、稳定.利用qRT-PCR方法对黄芪和土壤样本进行检测,接种发病样本、疑似发病样本和土壤样本中,尖孢镰刀菌的总检出率分别为100%、66.7%和43.75%;尖孢镰刀菌在黄芪中的定殖量与病情指数正相关,且相同种植年限下,发病土壤中尖孢镰刀菌孢子数显著大于健康土壤.综上结果表明,所建方法适用于黄芪及土壤中尖孢镰刀菌的检测.本研究可为黄芪根腐病的快速诊断、准确评价及土壤预警提供技术支持.
Abstract
Fusarium oxysporum is one of the dominant pathogens causing Astragalus membranaceus var.mongholicus(AMM)root rot.In order to rapidly and accurately detect and quantify F.oxysporum in AMM plants and soil,primers were designed based on the elongation factor-1α(EF-1α)sequence and verified for their specificity.The real-time quantitative PCR(qRT-PCR)detection system was established for the pathogen.The constructed qRT-PCR system was used to detect F.oxysporumin AMM plants and soil from AMM planting sites.The results showed that primer pair FaeF2/FaeR2 was highly specific,and the sensitivities of qRT-PCR detection were 0.896 pg/μL for F.oxysporum genomic DNA in AMM and 102 conidia/g for F.oxysporum conidia in soil.The correlation coefficients of the constructed standard curves for F.oxysporum detection in AMM and soil were 0.998 and 0.973,respectively.Repeatability evaluation showed that the system was stable and reliable.The AMM plants and soil samples were detected by the qRT-PCR detection system.The detection rate of the pathogenic F.oxysporum was 100%in F.oxysporum-inoculated samples,66.7%in suspected diseased samples and 43.75%in soil samples;the pathogen colonization in AMM was positively related with the disease index and the number of conidia in diseased soil was significantly greater than that in healthy soil at the same AMM cultivation years.The qRT-PCR system established in this study was suitable for the detection of the pathogenic F.oxysporum causing AMM root rot,and will provide technical support for the rapid diagnosis,accurate evaluation and soil warning of AMM root rot.
关键词
蒙古黄芪/根腐病/尖孢镰刀菌/实时荧光定量PCR(qRT-PCR)/检测体系Key words
Astragalus membranaceus var.mongholicus/Root rot/Fusarium oxysporum/Real-time quantitative PCR(qRT-PCR)/Detection System引用本文复制引用
基金项目
山西省自然科学研究面上项目(202103021224029)
山西省回国留学人员科研资助项目(2022-023)
出版年
2024
国家科技期刊平台
NSTL
万方数据