A loop-mediated isothermal amplification assay can rapidly diagnose soybean root-rot and damping-off diseases caused by Pythium spinosum
The oomycete pathogen causing root-rot and damping-off diseases in soybean (Glycine max) fields in the Huang-Huai region of China was identified as Pythium spinosum. To detect P. spinosum for disease diagnosis and control, we developed a loop-mediated isothermal amplification (LAMP) reaction with a primer set designed from the rDNA internal transcribed spacer 2 (ITS2) sequence of P. spinosum. The LAMP assay can efficiently amplify the target gene within 60 min at 63 °C. In specificity tests using 31 Pythium spp., 12 Phytophthora spp., 6 Phytopythium spp., and 9 other fungi strains, no cross-reactions were observed in the LAMP assay. The detection limit was 100 pg·μL−1 of genomic DNA per reaction. In cases of suspected disease, P. spinosum could be detected directly, using the LAMP assay, from soybean tissues and soil collected from fields in soybean production areas in the Huang-Huai region. This study provides a rapid method for diagnosing soybean root-rot and damping-off diseases caused by P. spinosum.
KeywordsPythium spinosum ITS2 LAMP assay Soybean root rot
We thank Xiaoli Wang and Yue Yang for maintaining the strains used in this study. This work was supported by the China Agriculture Research System (CARS-004-PS14), the Fundamental Research Funds for the Central Universities (KJQN201738), the National Natural Science Foundation of China (31601618) and the Special Fund for Agro-scientific Research in the Public Interest of China (201303018).
Conceived and designed the experiments: XBZ and YCW.
Performed the experiments: HF, JJC, ZY, and ZL.
Analyzed the data: HF, JJC, WWY, and XBZ.
Contributed reagents/materials/analysis tools: HF, JJC, ZY, ZL, WWY, and YCW.
Wrote the paper: HF, JJC, XBZ, and WWY.
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