DETECTION OF VIRUSES OF THE RUGOSE WOOD COMPLEX ON VINEYARDS OF THE ODESSA REGION
Authors: Nikolaeva N. 
Published: October 2022

Introduction.

Among these viruses, the most harmful are viruses that are part of the wood furrow complex (Rugose wood complex), namely: wood furrow Rupestris - Rupestris stem pitting (RSPaV); Grapevine virus B (GVB); Grapevine virus A (GVA) - Grapevine virus A (GVA); pitted wood LN 33 - LN 33 stem grooving. These viruses are part of the European Community certification system and must be tested for the presence of these viruses in the production of seedlings. Grape viruses cause great damage to vineyards in the south of Ukraine, especially viruses of the wood complex of the furrow complex (Rugose wood complex) (eng. - RWC).

The purpose of the research was to identify the presence of symptoms of viruses of this complex on vineyards in Odessa region and their identification. Methods. To conduct these studies, phytosanitary examination for the presence of wood furrow complex viruses was used, and real-time polymerase chain reaction (RT- -PCR) was used to identify viruses.

Materials and methods research.

The work used phytosanitary survey of vineyards in Ovidiopol, Bolgrad and Izmail districts of Odessa region, with a total area of 500 hectares. Samples with and without symptoms of virus infection were taken for real-time PCR diagnosis with hybridization-fluorescence detection (Real time PCR).

To identify viruses of the RWC complex, the selection, storage and preparation of samples of grape plants was performed according to ISO 16578: 2013. Samples for RT-PCR were prepared according to the method of the authors, leaves or scrapes of woody shoots, in the amount of 100 mg, placed in a homogenizer (Tube-mill control, IKA, China) carefully ground, poured 2 ml of extraction GGB) buffer: Na2CO3 - 1.59 g / l, NaHCO3 - 2.93 g / l, 2% PVP-40, 0.2% BSA, 0.5 g / l Tween-20, 10 g / l Na2S2O5 ( pH 9.0) and incubated at 95 ºC for 10 minutes in the thermostat "Dry block" TDB-120 (Biosan, Latvia). After that, the samples were kept in the refrigerator for 3 hours at +4 ºC. Isolation of RNA viruses was performed according to the method, namely: 2 μl of the sample was added to 23 μl of the reaction mixture (H2O - 12.0 μl; 10 × PCR buffer - 2.5 μl; sucrose + cresol - 2.5 μl; 4 mm dNTP - 1.25 μl (1.76 mm -2.84 μl), DTT (dithiothreitol) - 1.24 μl, pr1 (10 pmol) - 1.25 μl, pr2 (10 pmol) - 1.25 μl, Taq polymerase (2.5 u / µl) (Pfu DNA, Fermentas, Lithuania) - 0.25 μl, revertase (200 u / µl) (RevertAidTM M-MuLV, Fermentas, Lithuania) - 0.04 μl, Mg2 + (50mM) - 0.75 μl, covered with a layer of oil for PCR and performed RT-PCR. RT-PCR in real time was performed using forward and reverse primers, fluorescently labeled DNA probes, the reaction mixture in the amount of 20 μl (H2O - 8.5 μl; 10 × PCR buffer - 2.5 μl; sucrose + cresol - 2.5 μl, 4 mm dNTP - 2.5 μl (1.76 mm - 2.84 μl), DTT - 1.24 μl, pr1 - 0.5 μm, pr2 - 0.5 μm, fluorescent probe - 0 , 1 μM, Taq polymerase (2.5 u / µl) (Pfu DNA, Fermentas, Lithuania) - 0.25 μl, revertase (200 u / µl) (RevertAidTM M-MuLV, Fermentas, Lithuania) - 0.04 μg; Mg2 + - 3.0 mm and 5 μl of NС, or PС, or internal control, or test sample (at the bottom of the tube). Concentrations of forward, reverse primers, fluorescent DNA probes were selected empirically. used a negative control sample (NCS) - 1xPLR buffer and a positive control sample (PCR) - biomaterial from the test system for ELISA (Agritest, Italy). NC from grape mitochondria. The following primers and labeled probes (Fermentas, Lithuania) were used to identify RT-PCR viruses in real time according to]: for grape wood furrow complex A (GVA) virus: GVA-77 f - CGACCGAAATATGTACCTGAATACTC - direct; GVA-192 r1 - TTTGCTAGCTTTAGGACCTACTATATCTACCT - reverse; GVA-192 r2 - CTTGCTAGCcTTAGGtCCTACTATATCTACCT - reverse; GVA-104 p - CTTCGGGTACATCGCCTTGGTCG - probe. To the virus B of the grape wood furrow complex (GVB): GVB-92 f1 - CTAGGAGTGCGGCTAAACGAA - direct; GVB-95 f2 - GGAGTGCGGCCAAACGA - direct; GVB-202 r1 - CCTTAACCTCGTCCTGTGATATGGT - reverse; GVB-119p2 - ACCGTTACGGCCGTTGTTACTGTTGTGGTAG - probe Reverse transcription and amplification included the following cycles: at 50 ° C for 2 minutes, 95 ° C for 15 minutes and 45 cycles of 95 ° C for 15 sec. and 57 ° C - 1 min. Amplification was performed in a programmable thermal cycler Rotor-Gene 6000 (Corbett Research Pty Ltd., Australia). Accounting for analysis results, calculation of threshold cycles was performed using Rotor-Gene 6000 Series Software 1.7. The sample was considered positive, in the analysis of which there is an increase in the fluorescent signal on one of the color channels of the amplifier.

Results.

As a result of optimizing the conditions of real-time RT-PCR, a successful concentration of MgCl2 was selected for the highest fluorescence signal intensity and it was found that the fluorescent signal curve was more optimal at MgCl2 concentration in the range 3.0 - 2.5 mm. As a result of the conducted researches only the virus B of a complex of furrowing of grapes was identified, other viruses were not revealed As a result of phytosanitary inspection of vineyards of Bolgrad, Izmiil and Ovidiopol districts of Odessa region, symptoms of viral damage to grape plants were revealed. For the first time, grape viruses were identified by a modified RT- PCR method, and diagnostic conditions were selected.

Conclusions and prospects.

As a result of phytosanitary inspection, grape viruses belonging to the furrow complex were found. The lesions of grape bushes by viruses of the wood furrow complex on the vineyards of the Bolgrad district of Odessa region were detected and identified. During the diagnosis, the PCR parameters were optimized, namely, the annealing temperature and magnesium concentration were tested. The obtained data will allow timely detection of viruses of the grape furrow complex, which can lead to a significant reduction in yield and prevent their spread.

As a result of phytosanitary inspection of vineyards of Ovidiopol, Bolgrad and Izmail districts of Odessa region, 2 grape bushes with symptoms of viral disease of the grape furrow complex were found. For the first time in Ukraine, the method of real-time polymerase chain reaction with hybridization-fluorescence detection was used to diagnose viral disease, and the reaction conditions were selected and optimized. As a result of identification of the causative agent of the wood furrow complex by the RT-RF-PCR method, it was established that the vines were affected by the B complex virus (GVB).