Detection of grapevine leaf roll-associated virus of grapes and the spread to vineyards of south Ukraine

Abstract

Introduction. Diseases of grapevines caused by viruses are often chronic, therefore, as a result of the use of infected bushes in the production of planting material, the further spread of these diseases [1, p.1-165]. In addition to planting material of domestic production, many seedlings of foreign producers are imported into the territory of Ukraine, namely from Austria, Serbia, France, Italy, including patients with viral and bacterial diseases. 

Analysis of recent researches and publications. The Grapevine Leaf Roll-Associated Virus (GLRaV) is a malicious virus of grape plants. The 9th serotypes of this virus have been established (GLRaV1-9). The most common are physiologically related 1st and 3rd serotypes. The spread of the disease, the causative agent of which is the leaf curling virus, occurs with infected planting material. Therefore, timely accurate diagnosis of the disease and identification of its pathogen is necessary. Grapple grafts are affected by the leaf twisting virus in a latent form and therefore it promotes the spread of viruses in the zone of inoculum viticulture. The visual inspection of industrial vineyards, as well as the testing of the vine showed that the viral disease - the twisting of leaves of grapes is the most common illness

The purpose to identify of Grapevine Leaf Roll-Associated Virus and its serotypes. To study the spread of Grapevine Leaf Roll-Associated Virus in the vineyards of southern Ukraine.

Methods. A phytosanitary survey of vineyards and sampling of external symptoms of viral diseases was carried out in the Odessa, Nikolaev and Kherson regions. A survey of vineyards was carried out from May to October.

To identify viruses of grapes used ELISA method. Diagnosis of viruses was performed according to the instructions for ELISA kits (Agritest, Italy). Preparation of solutions of immunoglobulins, conjugate and substrate was carried out in accordance with the instructions to the sets. N-nitrophenyl phosphate was used as a substrate. Incubation of the reaction mixture in microplates was carried out in a thermostat - shaker "Dynatec" (USA) for 2 hours at t = 37 ° C. The composition of the buffers was prepared according to the manufacturer's instructions for test kits Agritest, Italy.

Samples for carrying out PCR in real time were prepared by the method of the authors [8, p. 41-48]. Real-time PCR was performed using primer pairs and TaqMan® RT-PCR, according to the authors [9, p. 292-299]. To control the release of RNA, an internal control synthesized for amplifying mRNA from grape mitochondria, as well as a negative control from test systems for carrying out ELISA (Agritest), was used. Reverse transcription and amplification included the following cycles: at 45 ° C for 35 min., 95 ° C for 10 min. and 40 cycles of 95 ° C for 15 sec. and 60 ° C for 1 min. Amplification was carried out in a Rotor-Gene 6000 programmable thermocycler (Corbett Research, Australia).

Results. As a result of the survey of grape plantations of technical varieties in the Odesa, Mykolayiv and Kherson regions, the bushes of the grape varieties Cabernet Sauvignon, Merlot, Odesa Black, Pinot Noir, Chardonnay, Sukholimansky white with symptoms of Grapevine Leaf Roll-Associated Virus. The phytosanitary survey showed that the greatest percentage with the symptoms of Grapevine Leaf Roll-Associated Virus was in the Odessa region, this is probably due, first of all, to the planting material, as well as to the agro-climatic conditions of cultivation. It was established that the Grapevine Leaf Roll-Associated Virus was detected by the method of classical RT-PCR and RT-PCR in real-time in all symptomatic grape plants, while in the Sukholimansky and Chardonnay plants this virus was not identified by the ELISA method. It has been established that grapevines with symptoms of twisting leaves have not always been affected by this virus, the causative agent of Grapevine Leaf Roll-Associated Virus of grapes and not always could be identified by the ELISA method. Only a comprehensive study can guarantee the exact result of the identification of viruses of grapevine plants.

Discussion. The phytosanitary survey of grape plantations in Kherson, Nikolaev and Odessa regions showed that the most infected viruses of Grapevine Leaf Roll-Associated Virus grapes were found in the vineyards of the Odessa region, and probably the reason was the high percentage of import of seedlings of imported production. ELISA, RT-PCR with electrophoretic detection and RT-PCR in real-time fluorescence detection confirmed that the vineyards of southern Ukraine widespread viral disease of grape plants - Grapevine Leaf Roll-Associated Virus, caused by Grapevine Leaf Roll-Associated Virus 1st and 3rd serotypes. It is established that the symptoms of Grapevine Leaf Roll-Associated Virus do not always correspond to the presence of the virus in these plants. Leaf twisting virus was found in grape varieties of varieties: Cabernet Sauvignon, Merlot, Pinot Noir, Odessa Black, Sukholiman White, Chardonnay. It is shown that the ELISA method can not always identify the viruses, pathogens of viral diseases of grapes. So in the plants of grapes Sukholimansky white and Chardonnay by the ELISA method, the Grapevine Leaf Roll-Associated Virus of grapes, which was detected by the RT-PCR method, was not identified. For more accurate diagnostics it is necessary to carry out a comprehensive study of grape material in the production of certified seedlings, starting with phytosanitary examination and with obligatory identification by ELISA and PCR methods.