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Blinova S.A., Shvartsev A.A., Syksin S.V., Bondarenko G.N., Bashkirova I.G., Gorislavets S.M., Risovannaya V.I., Stranishevskaya E.P., Volodin V.A., Alekseev Ya.I. Development of real time pcr kit for diagnostics of grapevine black wood causative agent Candidatus Phytoplasma solani.Sel'skokhozyaistvennaya Biologiya [Agricultural Biology], 2020, Vol. 55, № 1, p. 194-204.
(how to cite this article)

doi: 10.15389/agrobiology.2020.1.194eng

UDC: 634.8:632.08:577.2

Acknowledgements:
The studies were carried out as part of the VNNIIViV Magarach RAS State Task No. 0833-2015-0019.

 

DEVELOPMENT OF REAL TIME PCR KIT FOR DIAGNOSTICS
OF GRAPEVINE BLACK WOOD CAUSATIVE AGENT
Candidatus Phytoplasma solani

S.A. Blinova1, A.A. Shvartsev1, S.V. Syksin1, G.N. Bondarenko2, I.G. Bashkirova2, S.M. Gorislavets3, V.I. Risovannaya3, E.P. Stranishevskaya3, V.A. Volodin3, Ya.I. Alekseev1, 3, 4

1LLC Syntol, 42, Timiryazevskaya ul., Moscow, 127434 Russia, e-mail Sofya.blinova@yandex.ru (✉ corresponding author), alexey.sva@yandex.ru, stason_16@inbox.ru, jalex@syntol.ru;
2All-Russian Plant Quarantine Center, 32, ul. Pogranichnaya, pos. Bykovo, Ramenskii Region, Moscow Province, 140150 Russia, e-mail reseachergm@mail.ru, bashkirovaid@mail.ru;
3National Research Institute for Grape and Wine Magarach RAS, 31, ul. Kirova, Yalta, Republic of Crimea, 298600 Russia, e-mail mgr.magarach@gmail.com, stranishevskayaelena@gmail.com, vitaliivolodin1988@gmail.com;
4Institute for Analytical Instrumentation RAS, 31-33, ul. Ivana Chernyh, St. Petersburg, 198095 Russia, e-mail jalex@syntol.ru

ORCID:
Blinova S.A. orcid.org/0000-0001-6782-8353
Gorislavets S.M. orcid.org/0000-0002-6749-8048
Shvartsev A.A. orcid.org/0000-0002-2786-9860
Risovannaya V.I. orcid.org/0000-0003-2208-798X
Syksin S.V. orcid.org/0000-0002-2753-3857
Stranishevskaya E.P. orcid.org/0000-0002-2840-5638
Bondarenko G.N. orcid.org/0000-0002-3826-1009
Volodin V.A. orcid.org/0000-000-2842-6092
Bashkirova I.G. orcid.org/0000-0001-9014-4179
Alekseev Ya.I. orcid.org/0000-0002-1696-7684

Received October 16, 2019

 

Today, phytoplasmas are causative agents of about three hundred different plant diseases. The greatest damage in European vineyards is due to two types of phytoplasmas, Candidatus Phytoplasma vitis Marzorati et al. 2006, the pathogen of flavescence dorée of grapevine, and Candidatus Phytoplasma solani Qualino et al., 2013, the causative agent of black wood of grapevine. Phytoplasma damage of vineyards can lead to crop losses of up to 25-30 %, and when infected up to 70 %, the vineyards should be completely uprooted. Symptoms of various phytoplasma diseases in grape are similar with each other and with viral and bacterial diseases that makes their visual differentiation to species impossible. The wide spread and high damage by phytoplasma diseases require deeper research of phytoplasma epidemiology and relevant molecular genetic diagnostic methods for monitoring phytopathogen in planting material. We have developed the first Russian kit for detection these pathogens by real-time polymerase chain reaction (Real-Time PCR) which allows effective identification of Candidatus Phytoplasma solani. A comparison of the developed kit with the recommended primers and probes for Real-Time PCR has shown a higher sensitivity and specificity as compared to existing diagnostic PCR systems. The goal of this work was to develop and test a kit for the detection of Candidatus Phytoplasma solani by the real-time polymerase chain reaction (qPCR). Candidatus Phytoplasma solani DNA samples and infected grape vines, roots and leaves of Chardonnay, Pinot noir and Bastardo Magarachsky varieties with visual signs of infection collected in the autumn of 2018 from the vineyards of the South Coast region of the Crimean peninsula were tested. Phytoplasma DNA was extracted as recommended by EPPO, with modifications, as well as with Cytosorb reagent kit (Syntol LLC, Russia). A pair of primers, SolaSeq_F 5'-AACTTAACCTTTTAACTAGGGC-3' and SolaSeq_R 5'-CATCAAGGCATTTGCC-3', was designed for Candidatus Phytoplasma solani DNA sequencing. To estimate the analytical sensitivity of the test system, a vector construct based on the Pal2T plasmid (Evrogen, Russia) was created with the insertion of the Candidatus Phytoplasma solani target 119 bp fragment of SecY gene. The sequence of SecY gene is conservative, unlike other genes recommended for diagnosis. The designed primers allow identification of all Candidatus Phytoplasma solani strains which sequences we found in the GenBank NCBI Nucleotide database on January 16, 2019. The developed reagent kit was tested using various Real-Time PCR instruments. We have assessed the main characteristics of the reagent kit, i.e. sensitivity, specificity, and reproducibility. Analytical sensitivity of the developed test system isn’t less than 15 copies per PCR reaction. The analytical specificity was 100 % when tested with 37 closely related and accompanying microorganisms, as well as four samples of grapes suspected to be infected by Candidatus Phytoplasma solani. There were no false-positive results in the analysis of other types of phytoplasmas and related microorganisms. Also, in analyzing target organism DNA samples, false-negative results were not found. The developed kit was tested on 194 samples of grapes suspected of being infected by Candidatus Phytoplasma solani. The specificity of Candidatus Phytoplasma solani detection was confirmed in all cases by DNA sequencing of positive samples. The developed kit allows rapid, accurate and high sensitive DNA identification of Candidatus Phytoplasma solani in plants at all stages of their vegetative development, including planting material, and can also be used for full-scale screening studies.

Keywords: phytoplasma, Candidatus Phytoplasma solani, grapes, real-time PCR, diagnostics, qPCR test kit, specificity, sensitivity, reproducibility, repeatability.

 

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