Detection and quantification ofFusarium communein host tissue and infested soil using real-time PCR

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Abstract

Fusarium wilt caused by Fusarium commune is a major limiting factor for Chinese water chestnut (Eleocharis dulcis) production in China. A SYBR Green I real-time quantitative polymerase chain reaction (qPCR) assay was developed based on the mitochondrial small subunit rDNA of F. commune. Assay specificity of the FO1/FO2 primer set was tested on 41 fungal isolates, and only a single PCR band of c. 178 bp from F. commune was amplified. The detection limits of the assay were 1 fg μL−1 pure F. commune genomic DNA, 1 pg μL−1F. commune genomic DNA mixed with host plant genomic DNA (0·5 ng μL−1), and 1000 conidia/g soil (artificially inoculated). The amount of F. commune DNA in stem tissues detected by qPCR was significantly correlated with the disease severity (DS) ratings; however, the qPCR assay showed no significant positive correlation between spore densities in soil of different fusarium wilt DS groupings and the DS ratings. The qPCR assay was further applied to 76 soil samples collected from commercial fields of E. dulcis during the 2011 and 2012 growing seasons. The spore density of F. commune detected was positively correlated with disease index in the 2012 growing season but not in 2011. The qPCR method can be used for rapid and specific detection of F. commune in plant and soil samples, which will facilitate monitoring of the pathogen and improvement of disease management.

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