Abstract
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 103 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.
Highlights
Fusarium wilt of banana (Panama disease), caused by the soilborne fungus Fusarium oxysporum f. sp. cubense (Foc), is a serious disease in banana (Musa spp.) production worldwide [1,2,3]
The ESE-Tube scanner equipped with temperature settings to amplify DNA isothermally and spectral device to detect amplified product using fluorescence was applied for the detection of Foc tropical race 4 (TR4) in soil samples
The colour of loop-mediated isothermal amplification (LAMP) products of Foc TR4 isolate changed from orange to green when detected with SYBR Green I, whereas the colour of the other samples remained originally orange (Fig. 2C)
Summary
Fusarium wilt of banana (Panama disease), caused by the soilborne fungus Fusarium oxysporum f. sp. cubense (Foc), is a serious disease in banana (Musa spp.) production worldwide [1,2,3]. Fusarium wilt of banana (Panama disease), caused by the soilborne fungus Fusarium oxysporum f. Cubense (Foc), is a serious disease in banana (Musa spp.) production worldwide [1,2,3]. The ST4 isolates cause disease in Cavendish bananas in the subtropics [6,7,8], and TR4 isolates are pathogenic both under tropical and subtropical conditions [9,10,11,12]. In South China, Fusarium wilt of Xiang Jiao (AAA, Cavendish bananas) was first reported in Guangdong Province in 2001 [13], which caused by TR4 [14]
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