DIAGNOSTICS OF VIRUSES OF GRAPE LEAFROLL AND VINE FANLEAF IN THE PROCESS OF PRODUCTION OF CERTIFIED SPROUT OF GRAPES

Abstract

The most damaging viral of viruses of grape leaf roll (GLRaV) and fan leaf (GFLV), which are very common and all wine regions of the world and lead to significant losses of grapes and its quality deterioration. So curl virus causes a decrease in grape leaves sugar in the berries to 16% in crop losses make 10-50%. Often viral disease occurring in a latent form, without current symptoms. Visual phytosanitary control prevents recognize bushes with latent infection and prevent the harvesting of them for vine vegetative propagation of plants. Modern serological and molecular genetic methods can accurately identify the affected grape plants.

Materials and methods research. To test the bushes grape clones in August - September selected upper leaves of plants. With the onset of cold weather virus isolation were carried out in the lignified shoots. Samples were transported to the laboratory and examined on the same day or stored at - 20 ° C for several months.

The reaction mixture for reverse transcription and polymerase chain reaction (RT-PCR) volume 25 ml water containingu, 10 × PCR buffer (KCl 500 m, 100 m Tris-NCl, pH 9,0), sucrose (20%) and red krezolov 2 mM dezoksinukleozydtryfosfat (dNTP), 0,1 M ditiotryyetol (DTT), 10 pmol of each primer, 1,25 U Taq-polymerase ("AmpliSens", Russia), 8 U revertazy ("Amplisense", Russia), 1, 5 mM MgSO4 (for GLRaV-1 and GLRaV-3), 1.3 mM MgSO4 (for GFLV). In the reaction mixture was brought to 2 ml prepared sample.

Reverse transcription was performed at 52 ° C in an incubator for 30 minutes. Amplification consisted of 35 cycles (94 ° C - 30 seconds, 56 ° C - 45 seconds, 72 ° C - 60 seconds) and elongation time in the last cycle reached 7 minutes (Rowhani A., personal communication). For GLRaV-1 annealing temperature was lowered to 53 ° C, and for GFLV increased to 61 ° C.

The reaction is carried out in a programmable thermostat "Tertsyk" Company "DNA - Technology" (Russia). Electrophoresis was performed in 1.5 % agarose gels. Etidiy bromide for visualization of PCR products was part trysborat buffer for electrophoresis ("Amplisense", Russia). The gel was photographed using video "Samsung" UV - radiation (wavelength 312 nm). To estimate the molecular weight fragments amplified using marker 800 - 100 base pairs ("Amplisense", Russia). grapevine fanleaf virus (GFLV) and twisting vine leaves grapes carried diagnosed by RT-PCR-Rt in real time. We used primers oligoC1 / oligoV1 for grapevine fanleaf virus (GFLV) and CPV / CPC for grapevine leafroll associated virus GLRaV 1. Based on the nucleotide sequences of the genes were chosen primers and probes labeled with fluorescent markers FAM, JOE, that allow detection of fluorescence in real time. Amplification was performed according to the recommendations in termotsykleri Rotor Gene 6000 (Corbett, Australia).

Results. As a result of RT-PCR analysis of the data from the distribution of latent forms of viral diseases of grapes, such as of viruses of grape leaf roll (GLRaV) and fan leaf  (GFLV) 1st and 3rd serotypes.

Optimized multiplex quantitative real-time PCR for the simultaneous detection of different viruses grapes are the best alternative to conventional PCR, so the results are available within 4-5 hours of highly standardized format without processing PCR products, which reduces the risk of false positives that can occur with manipulation of amplicon. This technique makes it possible to define multiple pathogens simultaneously, extremely cost for a small number of samples. Another advantage of the technology is the possibility of quantifying the amplified products.

The Cabernet Sauvignon, Rhine Riesling, Merlot Rose, Chardonnay and assayed for virus infestation grapes. It was found that the bush Rhine Riesling, Chardonnay and Merlot Rose did not contain pathogens of viral diseases of grapes. Cabernet Sauvignon was grapevine leafroll associated virus GLRaV 1.

Diagnosis grapevine fanleaf virus by RT-PCR in real time gives a highest-quality and accurate results of PCR analysis replaces product PCR detection by electrophoresis. This does not make sense to open and allows amplification products that minimizes the risk of contamination of the laboratory PCR products. Automatically record results eliminates human error in interpreting the data.

The technology allows to detect differentially in the samples of various viruses grapes. The presence of the studied sample nucleic acid of the virus is determined by the growth of certain dye fluorescence signal in one of the reaction mixture. Primers and probes were synthesized in the company Sintol (Russia).

The method recommended for the implementation of sanitary control system for production of certified planting material in nurseries Ukraine.

Conclusions

1. Research the presence of viral diseases among clones varieties of grapes.
2. Found that the most common viruses grape of viruses of grape leaf roll (GLRaV) and fan leaf  (GFLV) grapes.
3. Developed multiplex real-time PCR for detection of viruses grapes is the best alternative to conventional RT-PCR for diagnosis because. Results are available in 4-5 hours Continue in highly standardized format without processing PCR products that reduce not reliable results that can occur when amplykonas manipulation.
4. The technique makes it possible to identify several pathogens at once, which is very economical for samples in small quantities. And enables quantitative report amplyfikonas product.
5. For the first time developed a method of virus detection fan leaf  (GFLV) grapes by RT-PCR detection of fluorescence in real time.