Crown Gall of Grapevine Caused by Agrobacterium in Ukraine
DOI:
https://doi.org/10.15407/Keywords:
grapevine, polymerase chain reaction, Ti plasmids, opines, virulence genes, oncogenes, crown gall, Agrobacterium tumefaciens, Allorhizobium vitisAbstract
Crown gall of grapes is among the diseases affecting grape plants. This is the most dangerous chronic infection of the grapevine, caused by strains of tumorigenic bacteria Allorhizobium vitis (All. Vitis) (formerly Agrobacterium vitis) and tumorigenic Agrobacterium tumefaciens (A. tumefaciens), which are able to remain in the plant for a certain time in a latent state until they appear in favorable conditions for the manifestation of symptoms of this disease. The disease is not included in the European certification system, and this is due to the fact that in such large wine-growing countries as Italy, France, Germany, and Austria, which export a significant number of seedlings to Ukraine, they often have a latent form, and this leads to its spread (Milkus et al., 2012). The disease is systemic and vegetative propagation of affected bushes leads to the production of infected planting material, thereby contributing to its spread (Lehoczky, 1971; Tarbah & Goodman, 1986). The relevance of the study is due to the spread of crown gall grapevine in the vineyards of the Odesa and Mykolaiv regions of southern Ukraine, and for this purpose, the need for timely detection and identification of its pathogen, sequencing the genome of selected virulent isolates to determine the type of opines and their oncogenicity to prevent the further spread of the disease. Aim. To conduct a large-scale phytosanitary survey of grape plantations in the south of Ukraine (Odesa and Mykolaiv regions) to determine the presence of grape plants affected by crown gall. To isolate the causative agent of crown gall and its identification (All. vitis) by the PCR method in samples of symptomatic and asymptomatic material. To study the diversity of agrobacteria by the type of Ti-plasmid by means of genome sequencing of selected virulent bacterial isolates, and to determine their oncogenicity. Methods. The research was first carried out by visual inspection of grape plantations to determine the symptoms of the disease, and microbiological and molecular genetic research methods were used to identify the causative agent. Results. To identify symptoms, namely tumors, vineyards in the Odesa and Mykolaiv regions, with a total area of 2,660 hectares, were examined from 2012 to 2020. As a result of a visual inspection of the vineyards, grapevine plants showing symptoms of crown gall were found on the varieties: Cabernet Sauvignon, Merlot, and Chardonnay, on average, up to 25% of the total number of tested varieties affected in the Odesa region and up to 10% in the Mykolaiv region. In 130 grape samples out of 450 selected from vineyards with a total area of 320 hectares with symptoms of the disease, pathogenic strains of All. vitis and A. tumefaciens were detected and identified using the primers VCF3/VCR3. 300 asymptomatic samples were taken, and the latent form of the pathogen was detected in 20. Grape vine (Vitis vinifera L.) and tumors that were found on different parts of the grape plant were used to diagnose the disease. Identification of selected bacterial isolates by virulence properties was carried out using the polymerase chain reaction (PCR) method with A.tumefaciens-specific primers. Determination of the type of Ti-plasmids was carried out by means of genome sequencing of selected virulent bacterial isolates. It was established that almost all of them belong to biovar 1 (BV1 A. tumefaciens). When studying the diversity of agrobacteria according to the type of Ti-plasmid, it was found that they had the oncogenic Ti-plasmid, mainly of the octopine type, and only some (Od) Ti-plasmid of the nopaline type. Based on the obtained data, it was concluded that in the vineyards of the Odesa region, crown gall of grapes is caused by agrobacteria A. tumefaciens, mainly of the octopine type, and are oncogenic.
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