Development of conventional and quantitative real-time PCR assays for the detection and identification of Rhizoctonia solani AG-3 in potato and soil
Development of conventional and quantitative real-time PCR assays for the detection and identification of Rhizoctonia solani AG-3 in potato and soil
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DOI：
10.1046/j.1365-3059.2002.00712.x
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发表时间：
2002-06-01
期刊：
影响因子：
2.7
通讯作者：
Nicolson, MJ
中科院分区：
文献类型：
--
作者：
Lees, AK;Cullen, DW;Nicolson, MJ
建立了一种特异、敏感的PCR检测方法，用于马铃薯茎溃疡病和黑星病的主要病原菌Rhizoctonia solani AG-3的检测和鉴定。根据核糖体内转录间隔区（ITS1和ITS2）设计了一对常规引物（Rs1F2和Rs2R1）。索拉尼PCR扩增后，使用引物Rs1F2和Rs2R1从所有AG-3分离株的DNA扩增0.5-kb产物。当来自一系列其他R. Solani吻合组或来自其他马铃薯病原体的选择进行了测试，证实了引物仅对AG-3的特异性。在不同黑星病严重程度的马铃薯组织和接种了立枯丝核菌菌核的土壤中也检测到立枯丝核菌AG-3。solani的最低检测水平为5 × 10（-4）g菌核/g土壤。此外，设计特异性引物RsTqF1（基于Rs1F2序列）和RsTqR1以及TaqMan（TM）荧光探针RQP 1，以进行实时定量（TaqMan）PCR。传统的PCR和实时荧光PCR检测进行了比较，并结合直接从土壤中提取DNA和种子诱饵法，以确定最可靠的方法来检测和定量的AG-3在人工接种的田间土壤和自然侵染的土壤。结果表明，直接从土壤中提取DNA可能是有问题的，虽然AG-3是检测使用这种方法结合实时PCR检测。与直接从土壤中提取DNA相比，通过种子诱饵扩增立枯丝核菌提高了检测的灵敏度，并且当种子诱饵与常规PCR或实时PCR检测相结合时，在人工接种和自然侵染的土壤中均可检测到AG-3。潜在的使用这些快速和定量AG-3特异性检测，以解决流行病学问题，并在疾病管理的决策工具进行了讨论。
A specific and sensitive PCR assay was developed for the detection and identification of Rhizoctonia solani AG-3, the main causal pathogen of stem canker and black scurf of potato. A conventional primer set (Rs1F2 and Rs2R1) was designed from the nuclear ribosomal internal transcribed spacer (ITS1 and ITS2) regions of R. solani. Following PCR amplification, a 0.5-kb product was amplified from DNA of all isolates of AG-3 using primers Rs1F2 and Rs2R1. No product was amplified when DNA from isolates belonging to a range of other R. Solani anastomosis groups or from a selection of other potato pathogens was tested, confirming the specificity of the primers for AG-3 only. Rhizoctonia solani AG-3 was also detected in potato tissue with varying black scurf severity, and in soil inoculated with sclerotia of R. solani to a minimum detection level of 5 X 10(-4) g sclerotia/g soil. In addition, specific primers RsTqF1 (based on the Rs1F2 sequence) and RsTqR1, and a TaqMan(TM) fluorogenic probe RQP1, were designed to perform real-time quantitative (TaqMan) PCR. The conventional PCR and real-time PCR assays were compared and combined with direct DNA extraction from soil and a seed-baiting method to determine the most reliable method for the detection and quantification of AG-3 in both artificially inoculated field soil and naturally infested soils. It was shown that direct DNA extractions from soil could be problematic, although AG-3 was detectable using this method combined with the real-time PCR assay. The amplification of Rhizoctonia solani by seed baiting increased the sensitivity of the assay compared with direct extraction of DNA from the soil, and AG-3 was detectable in artificially inoculated and naturally infested soils when seed baiting was combined with either the conventional PCR or the real-time PCR assay. The potential for using these rapid and quantitative AG-3-specific assays to address epidemiological questions and as tools for decision-making in disease management is discussed.