Identification of the Causal Agent of Downy Mildew of Plasmopara viticola Grapes by Quantitative PCR

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Abstract

A new method is proposed for the early diagnosis of the causal agent of grapes downy mildew, Plasmopara viticola, based on the method of quantitative real-time PCR (qPCR RT) using SYBR Green I fluorescence. Six pairs primers were developed for the diagnosis of P. viticola, among the designed primers, PvITS1_2-real-s/a demonstrated the highest effectiveness for early detection of grapevine downy mildew with a strong positive correlation with the metagenomic data of P. viticola distribution in Far Eastern grape species and varieties, where a linear dependence was found (R2 = 0.86). Thus, qPCR RT of PvITS1_2 can be used for early detection and monitoring of asymptomatic P. viticola infections. The developed method can be used as a basis for predicting epidemics of downy mildew of grapes and for its control in vineyards.

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About the authors

N. N. Nityagovsky

Federal Scientific Center of the Biodiversity, Far Eastern Branch of the Russian Academy of Sciences, Laboratory of Biotechnology

Email: aleynova@biosoil.ru
Russian Federation, Vladivostok, 690022

A. A. Dneprovskaya

Federal Scientific Center of the Biodiversity, Far Eastern Branch of the Russian Academy of Sciences, Laboratory of Biotechnology; Far Eastern Federal University, Institute of the World Ocean

Email: aleynova@biosoil.ru
Russian Federation, Vladivostok, 690022; Vladivostok, 690922

A. A. Ananev

Federal Scientific Center of the Biodiversity, Far Eastern Branch of the Russian Academy of Sciences, Laboratory of Biotechnology

Email: aleynova@biosoil.ru
Russian Federation, Vladivostok, 690022

K. V. Kiselev

Federal Scientific Center of the Biodiversity, Far Eastern Branch of the Russian Academy of Sciences, Laboratory of Biotechnology

Email: aleynova@biosoil.ru
Russian Federation, Vladivostok, 690022

О. А. Aleynova

Federal Scientific Center of the Biodiversity, Far Eastern Branch of the Russian Academy of Sciences, Laboratory of Biotechnology

Author for correspondence.
Email: aleynova@biosoil.ru
Russian Federation, Vladivostok, 690022

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2. Fig. 1. Quantitative determination of amplification of PvITS1_1, PvITS1_2 and PvCox1_1 sequence sites in grape DNA samples performed by PB PCR (a); relative abundance of P. viticola in NGS samples (b). The origin of all samples is indicated in Table 1. Nc is the PB PCR reaction without grape DNA. NM — it was not measured. The data is presented as an average value ± SE (combined data from samples of leaves and stems of the same plant). The average values for each digit followed by the same letter did not differ when using one-way analysis of variance (ANOVA) followed by the Tukey multiple comparison test.

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3. Fig. 2. Determination of the Pearson correlation coefficient of the relationship between estimates of the relative level of amplification according to RV PCR data (rel. units) and the relative representation of P. viticola ITS1 amplicons in NGS samples: 1, 2. 3 are linear regression lines for PvITS1_1, PvITS1_2 and PvCox1_1 amplicons, respectively.

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