First report of rhizogenic strains of agrobacterium radiobacter biovar 1 causing root mat of cucumber and Tomato in Russia

Since 2013, many hydroponic cucumber (Cucumis sativus) and tomato (Solanum lycopersicum) crops in the Russian Federation have been affected by a root disorder known as “root mat” or “crazy roots.” The symptoms include extensive root proliferation within rockwool propagation cubes. Crazy roots has been reported in United Kingdom, France (Weller et al. 2000), and Japan (Sawada and Azegami 2014), where it caused significant reductions in marketable yield. The causal agent of this disease was shown to be wild-type Agrobacterium radiobacter biovar 1 strains harboring a Ri-plasmid (Weller et al. 2000). In the Russian Federation, crazy roots was found for the first time in winter 2013-2014 in the Novgorod Region on cucumber and tomato plants grown hydroponically in rockwool. The disease incidence in these outbreaks ranged from 50 to 100%. Rhizogenic strains of A. radiobacterbiovar 1 were isolated from affected roots, cucumber vines, tomato stems, internal tissue of fruits and seeds, rockwool, and irrigation water pumped from a local river during cultivation. Plant samples were surface-sterilized, homogenized, and 10-fold serial dilutions of the resulting suspensions were plated onto RS medium (Moore et al. 2001). Liquid from rockwool and water samples were used for serial dilutions and isolation of bacteria on RS medium as well. Isolation plates were incubated for 5 days at 28°C until bacterial colonies developed. Colonies consistent with the expected morphology ofAgrobacterium spp. on RS medium were subcultured on yeast dextrose calcium carbonate agar (YDC). Putative Agrobacterium isolates were confirmed by real-time-PCR assay as described previously (Weller and Stead 2002). In 2014, cucumber and tomato plants with similar symptoms were obtained from glasshouses in five regions of the Russian Federation. In Rostov and Novosibirsk regions, virulent Agrobacterium radiobacter bv1 was also isolated from irrigation water during cultivation. The identity of all recovered isolates (n = 36) was confirmed by biochemical tests as previously described (Moore et al. 2001) and real-time-PCR assay (Weller and Stead 2002). Suspensions of 10 isolates of the bacterium from 6 locations, deposited in the Russian Collection of Plant Pathogenic Microorganisms (Russian Institute of Phytopathology) as Ag2701 to Ag2710 were inoculated into seedlings of susceptible varieties ‘Marfinskii’ (cucumber) and ‘Gavrosh’ (tomato), as described by Weller et al. (2000). Symptoms were seen on all inoculated plants four to five weeks after inoculation, as increased root production across the propagation rockwool cube surface. Rhizogenic Agrobacterium strains were reisolated from all the slabs with symptomatic plants. No symptoms developed and no rhizogenicAgrobacterium radiobacter strains were isolated from control plants. Sequence typing of the housekeeping genes atpD, glnA, and recA was done according to the protocol and primers described by Puławska et al. (2012) for strains Ag2701-Ag2706 from six regions in Russia. Sequences were deposited in GenBank (atpD, glnA, and recA sequences as Accession Nos. KT831395 to KT831400, KT831401 to KT831406, and KT831407 to KT831412, respectively). BLAST analysis revealed that the sequenced genes for the recently isolated Agrobacterium spp. had 98 to 100% homology to those of A. radiobacter strain NCPPB 2659. This is the first record of rhizogenic Agrobacterium radiobacter causing root mat symptoms in cucumber and tomato in the Russian Federation. © 2016 The American Phytopathological Society