关键词: 葡萄霜霉病菌, cox2, 实时荧光定量PCR, 潜伏侵染, 定量检测

Abstract:

【Objective】Grape downy mildew caused by Plasmopara viticola is one of the most important monoetic diseases on Vitis vinifera. The objective of this study is to establish a real-time PCR detection system of P. viticola based on the pathogen sequence information, and to provide a scientific basis for early diagnosis and prediction of grape downy mildew.【Method】According to the cox2 gene sequence of P. viticola in GenBank, a pair of specific primers, F-cox-Pv/R-Pv, was designed to establish and optimize the conventional PCR and real-time PCR reaction system. The mycelium DNA of 14 species of grape and other crop pathogens and antagonistic fungus (including P. viticola, Colletotrichum gloeosporioides, Uncinula necator, Botrytis cinerea, Coniella diplodiella, Botryosphaeria dothidea, Alternaria brassicae, C. capsica, Fusarium solani, Sphaerotheca fuliginea, Sclerotinia sclerotiorum, F. equiseti, F. oxysporum, Trichoderma harzianum) were used to detect the specificity of conventional PCR and real-time PCR. The sensitivity and repeatability of the system were also evaluated. The pathogen DNA in the grape leaves during the latent infection period of artificial inoculation with P. viticola was quantitatively detected by the established real-time PCR system. The relationship between inoculation time and latent infection amount of P. viticola in the grape leaves was analyzed by SPSS 19.0 software. 【Result】 The primers designed in this study had high specificity. A 139 bp fragment of genome DNA was only amplified from P. viticola by conventional PCR. The detection results of real-time PCR assays showed that the primers had only one absorption peak for P. viticola, and no product absorption peak was detected for other test strains. The sensitivity of conventional PCR was 10 pg·μL ^-1 genomic DNA, while the sensitivity of real-time PCR was 0.1 pg·μL ^-1, which was 100 times higher than that of the conventional PCR. The linear relationship between real-time PCR cycle threshold (Ct) and template concentration was constructed by using recombinant plasmid containing cox2 gene fragment as standard material. The standard curve was y=42.27-3.36x, with the correlation coefficient of 0.997 and amplification efficiency of 98.50%. The linear range was up to 7 orders of magnitude, showing a good linear relationship at 2.4×10 ³-2.4×10 ⁹ copies/μL. The real-time PCR system was used to detect the pathogen DNA in the grape leaves during the latent infection period of artificial inoculation with P. viticola. The results showed that the latent infection of P. viticola in the grape leaves increased exponentially with the change of inoculation time, the curve equation was y=6.34×10 ⁴·e ^{0.084 x}, with the correlation coefficient of 0.936. The real-time PCR system could detect the P. viticola DNA 6 h after inoculation, and the DNA content was 5.68×10 ⁴copies/μL.【Conclusion】The sensitivity of the established real-time PCR system for detection of grape downy mildew is much higher than that of conventional PCR, and the specificity and reproducibility of this real-time PCR system are good. There was a good linear relationship between Ct value and template concentration, and the amplification efficiency is high. This method can be used to quantitatively detect the latent infection of P. viticola.

Key words: Plasmopara viticola, cox2, real-time PCR, latent infection, quantitative detection