应用荧光定量PCR 技术分析普通菜豆品种中尖镰孢菜豆专化型定殖量

尖镰孢菜豆专化型(Fusarium oxysporum f. sp. phaseoli)引起的菜豆枯萎病是菜豆生产中最严重的维管束类病害之一, 防治该病害有效方法是利用抗病品种。因此, 一种能够从菜豆受侵染组织中准确鉴定并定量检测枯萎病原菌含量的方法将有助于筛选抗性品种, 应用于普通菜豆枯萎病抗病育种。本研究基于荧光定量PCR技术开发出一种能够对定殖于菜豆组织中的枯萎病原菌准确定量的新方法。该技术对根、茎组织中病原菌DNA的最低检测量为1 pg, 能在接种病原菌6 d后明显区分抗病性不同的品种, 可在菜豆植株表现出明显发病症状前准确鉴定不同品种抗性水平的差异。经验证参试的感病品种BRB-130和A0640-1根、茎组织中定殖的病原菌DNA量显著高于抗病品种260205和黑芸豆, 与表型鉴定的结果完全符合。利用荧光定量PCR技术能够在病原菌侵染早期快速、准确、高效定量菜豆组织中定殖的病原菌, 这对指导菜豆抗病育种和植物病害传播的研究都具有非常重要价值。

Quantification of Fusarium oxysporum f.sp.phaseoli Detected by Real-time Quantitative PCR in Different Common Beans Cultivars

Fusarium wilt,caused by Fusarium oxysporum f.sp.phaseoli,is one of the most devastating vascular diseases of common bean.A efficient way to prevent and control the disease is to use resistant cultivars,so it is necessary to develop a rapid accurate method for detecting the pathogens.In this study,we developed a real-time quantitative polymerase chain reaction(qRT-PCR)protocol that quantified F.oxysporum f.sp.phaseoli DNA to a minimum of 1 pg in the plant roots and stems.Moreover,the qRT-PCR protocol asymptomatically distinguished resistant level of different bean cultivars challenged by the wilt pathogen FOP-DM01 at 6 d post inoculation.The result indicated FOP-DM01 DNA quantifications in the roots and stems of susceptible BRB-130 and A0640-1 were significantly higher than those in resistant 260205 and Heiyundou,which absolutely matched with the phenotypic identification.The use of this protocol for fast,reliable,and cost-effective quantification of F.oxysporum f.sp.phaseoli in asymptomatic tissues at early stages of the infection process is of great value for common bean breeding and studies of phytopathology and epidemiology.