First report of Agrobacterium rubi and Agrobacterium rhizogenes, causing crown and root gall and hairy root on blueberry in Argentina
From 2006 to 2009, crown gall and hairy root symptoms were observed on blueberry (Vaccinium corymbosumcvs. O'Neil, Millennia, and Misty) plants from six nurseries in Tucumán, Concordia, Pilar, Morón, and Baradero, Argentina. Bacteria were isolated from galls of all three cultivars and from hairy roots of Millenia and O'Neil onto D1 and D1M agar media at 27°C. TypicalAgrobacteriumcolonies developed in 5 days (2). Seven bacterial strains (five from galls and two from hairy roots) were studied further. All were gram negative, aerobic, and catalase positive with rod-shaped cells that synthesized β--galactosidase and metabolizedD-glucose,D-arabinose,n-acetyl-glucosamine, maltose, mannitol, and malonate. Strains were negative for lysine decarboxylase, H2S production, indole, and 3-ketolactose production. While gall strains were urease positive and citrate variable (mostly positive), hairy root strains were urease negative, citrate positive, had poly-β-hydroxybutyrate inclusion granules, and clarified acid on potato dextrose agar containing 0.5% CaCO3(2).Agrobacterium tumefaciensATCC 15955 and LBA 958 were included as controls. PCR with virA/C primers amplified a 338-bp product corresponding to thevirD2operon and confirmed that the strains harbored a pathogenic plasmid (1). Bacterial strains were assigned to biovars with a multiplex PCR assay targeting 23S rRNA sequences (3). Two strains produced PCR amplicons typical ofA. rhizogenesbv. 2. The other five strains produced PCR amplicons typical ofA. rubi, which were insensitive to agrocin in a bioassay withA. radiobacterstrain K1026. Identity was confirmed by sequencing the 16S rDNA of strains F 266 (GenBank No. GU580894) and F 289 (No. GU580895), which had 99% homology to 16sRNA sequences ofA. rubiICMP 11833 (AY626395.1) andA. rhizogenesATCC 11325 (AY945955.1), respectively. Pathogenicity of all seven strains was tested onV. corymbosumcv. Misty,Bryophyllum daigremontiana, tobacco cv. Xanthi, tomato cv. Presto, and pepper cv. California Wonder. Plants were inoculated by a needle stabbed into the stems with the appropriate cell suspension (108CFU/ml) of each strain or with sterile distilled water (control treatment). Two plants of each species were tested per strain. Plants were grown for at least 45 days at 23 ± 3°C and symptoms were recorded. Inoculations with the five strains isolated from galls caused development of spherical, white to flesh-colored, rough, spongy wart-like galls at the inoculation sites. Root strains induced root proliferation on all inoculated plants as well as in a carrot disk bioassay (4). On blueberry plants, galls were dark brown to black, rough, and woody 6 months after inoculation. No lesions were observed on control plants. Bacteria were reisolated from symptomatic tissues of inoculated plants. Enterobacterial repetitive intergeneric consensus-PCR confirmed that the DNA fingerprints of the reisolated strains were identical to those of the original strains. To our knowledge, this is the first report ofA. rubiandA. rhizogenescausing hairy root and crown gall on blueberry in Argentina.