First report of Agrobacterium vitis causing crown galls of wine grape in Russia

Symptoms of crown gall were observed in 9 of 15 surveyed vineyards located along Black Sea coast of Russia. Sampled tumor tissue was placed in a mortar and pestle for maceration. Serial dilutions of the resulting suspension were plated onto RS medium described by Roy and Sasser (1983). Isolation plates were incubated for 5 days at 28°C until bacteria developed. Colonies were consistent with morphology expected of Agrobacterium spp. On RS medium (opaque red center, translucent margin, mucoid) were purified on yeast dextrose calcium carbonate agar (YDC). Sixty-nine putative Agrobacterium isolates were confirmed by PCR with consensus primers virD2A/2C from the virD2 gene (Haas et al. 1995). Isolates—identified by PCR and producing tumors on indicator plants (carrot, red beet, sunflower) and on grapevine plants in 30 days after needle prick inoculation—were subjected to additional biochemical and physiological tests for Agrobacterium spp. (Moore et al. 2001). The tests included evaluation of 3-ketolactose production, alkaline reaction in litmus milk, growth on 2% and 5% NaCl, growth at 36°C, acid production from erythritol and melezitose, and alkali production from malonic acid and l-tartaric acid. Bacteria reisolated from inoculated grapevine plants were similar to original isolates in PCR test and 3-ketolactose production. The PGF/PGR primers amplifying the chromosomal polygalacturonase gene pehA (Szegedi and Bottka 2002) were used to identify A. vitis isolates and differentiate them from A. tumefaciens. Based on PCR, 18 of 69 tested isolates belonged to A. vitis and showed results of biochemical tests consisted with this species. In addition, for nine isolates, DNA sequence analysis of the housekeeping genes dnaK and trpE confirmed the isolates as A. vitis (Aujoulat et al. 2011). Sequences were deposited in GenBank as Accessions Nos. KT831413 to KT831421 for the dnaK gene and KT831422 to KT831430 for the trpE gene. Sequences were compared with corresponding genes of sequenced strain Agrobacterium vitis S4 (Accession No. CP000633.1). BLAST analysis revealed 99% homology for dnaK and 100% homology for trpE gene. This is the first documented Russian record of Agrobacterium vitis. © 2016, American Phytopathological Society. All rights reserved