Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (9): 1529-1540.doi: 10.3864/j.issn.0578-1752.2019.09.005

• PLANT PROTECTION • Previous Articles     Next Articles

Establishment and Application of Real-Time PCR for Quantitatively Detecting Plasmopara viticola in Vitis vinifera

LI WenXue1,XIAO RuiGang2,LÜ MiaoMiao1,DING Ning1,SHI HuaRong2,GU PeiWen1()   

  1. 1 School of Agriculture, Ningxia University, Yinchuan 750021
    2 Wine School, Ningxia University, Yinchuan 750021
  • Received:2018-12-04 Accepted:2019-01-17 Online:2019-05-01 Published:2019-05-16
  • Contact: PeiWen GU E-mail:gupeiwen2013@126.com

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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

Table 1

Tested strains"

菌株编号
Number		 拉丁学名
Latin name		 寄主（品种）
Host (Variety)		 菌株编号
Number		 拉丁学名
Latin name		 寄主（品种）
Host (Variety)
P1 P. viticola 葡萄（黑比诺）
Vitis vinifera (Pinot Noir)		 6 S. fuliginea 西葫芦
Cucurbita pepo
P2 P. viticola 葡萄（霞多丽）
V. vinifera (Chardonnay)		 7 C. diplodiella 葡萄（威代尔）
V. vinifera (Vidal)
P3 P. viticola 葡萄（赤霞珠）
V. vinifera (Cabernet Sauvignon)		 8 B. cinerea 葡萄（红地球）
V. vinifera (Red Globe)
1 A. brassicae 白菜
Brassica pekinensis		 9 S. sclerotiorum 番茄
Lycopersicon esculentum
2 C. gloeosporioides 葡萄（红地球）
V. vinifera (Red Globe)		 10 F. equiseti 马铃薯
Solanum tuberosum
3 C. capsici 辣椒
Capsicum annuum		 11 F. oxysporum 西瓜
Citrullus lanatus
4 F. solani 甘草
Glycyrrhiza uralensis		 12 B. dothidea 葡萄（红地球）
V. vinifera (Red Globe)
5 U. necator 葡萄（赤霞珠）
V. vinifera (Cabernet Sauvignon)		 13 T. harzianum 马铃薯
S. tuberosum

Fig. 1

Specificity detection of P. viticola primers F-Pv/R-Pv (A) and F-cox-Pv/ R-Pv (B) by using genomic DNA with conventional PCR"

Fig. 2

Melting curves of real-time PCR amplification"

Fig. 3

Sensitivity detection of P. viticola primers F-Pv/R-Pv (A) and F-cox-Pv/R-Pv (B) by using series genomic DNA dilutions with conventional PCR"

Fig. 4

Sensitivity detection of P. viticola primers with real-time PCR"

Fig. 5

Optimization of Tm value of real-time PCR assays"

Fig. 6

Construction of recombinant plasmid"

Fig. 7

Establishment of real-time PCR standard curve"

Fig. 8

Detection of leaf latent period of P. viticola (A) and its dynamic simulation (B)"

Table 2

Quantitative determination of P. viticola in latent infected leaves by real-time PCR"

接种后时间
Time after inoculation (h)		 Ct值
Ct value		 检测结果
Detection result (copies/μL)
6 32.36±0.57 (5.68±2.76) ×104c
12 31.04±0.81 (2.12±1.17) ×105c
24 28.89±1.06 (1.26±0.72) ×106c
48 26.33±0.40 (5.95±1.52) ×106c
72 23.77±0.21 (2.62±0.37) ×107b
96 20.15±0.29 (1.30±0.20) ×108a
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