Zhang Xin, Zhang He, Pu Jinji, Qi Yanxiang, Yu Qunfang, Xie Yixian, Peng Jun
Ministry of Agriculture Key Laboratory of Integrated Pest Management on Tropical Crops, Environmental and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, China.
Ministry of Agriculture Key Laboratory of Integrated Pest Management on Tropical Crops, Environmental and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, China ; State Key Laboratory of Agro-biotechnology and Ministry of Agriculture Key Laboratory for Plant Pathology, China Agricultural University, Beijing, China.
PLoS One. 2013 Dec 20;8(12):e82841. doi: 10.1371/journal.pone.0082841. eCollection 2013.
Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10(3) spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China.
尖孢镰刀菌古巴专化型（Foc）是香蕉枯萎病（巴拿马病）的病原菌，是香蕉（芭蕉属）最具毁灭性的病害之一。Foc热带4号小种（TR4）是目前全球香蕉生产中的主要关注点。在芭蕉属植物中尚未发现对Foc有效的抗性，且香蕉植株一旦感染Foc，就没有有效的防治措施。早期准确检测Foc TR4对于保护香蕉产业和指导香蕉种植至关重要。开发了一种实时荧光环介导等温扩增检测法（RealAmp），用于快速定量检测土壤中的Foc TR4。当与提取的土壤DNA混合或与每克人工接种土壤含10³个孢子混合时，RealAmp检测法的检测限约为0.4 pg/µl质粒DNA，且未观察到与其他相关病原菌的交叉反应。通过对人工接种和自然感染的样本进行检测，证实了用于定量TR4基因组DNA的RealAmp检测法。与经典实时荧光定量PCR相比，自然感染样本中Foc TR4土壤传播病原菌DNA的定量结果无显著差异（P>0.05）。此外，通过在扩增前向盖子内部添加SYBR Green I荧光染料，RealAmp检测法采用改进的闭管可视化检测系统实现了可视化，避免了染料对DNA扩增的抑制作用，使该检测法在现场更方便，因此可能成为一种简单、快速且有效的技术，有潜力作为田间检测和监测Foc TR4的替代工具，这将成为中国南方土壤传播病原菌基于DNA的常规检测服务。
