Scientia Agricultura Sinica ›› 2015, Vol. 48 ›› Issue (1): 55-62.doi: 10.3864/j.issn.0578-1752.2015.01.06

• PLANT PROTECTION • Previous Articles     Next Articles

Establishment of SYBR Green I Real-Time PCR for Quantitatively Detecting Rhizoctonia cerealis in Winter Wheat

SUN Bing-jian, CHEN Qing-qing, YUAN Hong-xia, SHI Yan, LI Hong-lian   

  1. College of Plant Protection, Henan Agricultural University/Collaborative Innovation Center of Henan Grain Crops/National Key Laboratory of Wheat and Maize Crop Science, Zhengzhou 450002
  • Received:2014-06-24 Online:2015-01-01 Published:2015-01-01

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Abstract: 【Objective】Wheat sharp eyespot (WSE) caused by Rhizoctonia cerealis is one of the most important soil-born diseases on wheat in China. Early accurate quantitative detection is a foundation of forecast and control. Traditional method of organization isolation and identification of pathogen is time consuming, complicated and can’t be accurately quantified. In order to implement the early and quick quantitative determination of wheat sharp eyespot, a SYBR Green I real-time PCR method of R. cerealis was established based on the pathogen sequence information.【Method】 Based on the β-tubilin of R. cerealis, a pair of specific primers was designed. The SYBR Green I real-time PCR reaction system was established and optimized. The sensitivity, specificity and repeatability of the system were also evaluated, and RBipolaris sorokiniana, Fusarium graminearum, F. pseudograminearum were used for control fungi. The indoor potted plants of wheat which infected by R. cerealis were detected with optimized reaction system after inoculated for 5, 10 and 60 days, respectively.【Result】The primers were of great specificity, the specific PCR fragment was amplified from the DNA of R. cerealis isolates, but not from the DNA of other fungal isolates by conventional PCR. The real-time PCR assays also did not amplify DNA from control fungi. The sensitivity of conventional PCR was 6.5×103 copies/μL plasmid, while the sensitivity of real-time PCR was 6.5×102 copies/μL. The standard curve established by recombinant plasmid showed a fine linear relationship between threshold cycle and template concentration. The melt curve was specific with the correlation coefficient of 0.997 and with high amplification efficiency (0.91). For the indoor potted experiments, the detection results of real-time PCR of infected wheat samples, were showed a significant positive correlation with disease index and inoculum, respectively. 【Conclusion】 The developed real-time PCR assay for R. cerealis is fast, highly specific, sensitive, and reproducible. This method can be used to detect R. cerealis in wheat, and guidance prediction and control of wheat sharp eyespot.. cerealis, R. solani, AG-A, AG-F, Gaeumannomyces graminis var. tritici,

Key words: Rhizoctonia cerealis, SYBR Green I, real-time PCR, early detection

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