The genome sequence of hairy root Rhizobium rhizogenes strain LBA9402. Bioinformatics analysis suggests the presence of a new opine system in the agropine Ri plasmid

We report here the complete genome sequence of the Rhizobium rhizogenes (formerly Agrobacterium rhizogenes) strain LBA9402 (NCPPB1855rifR), a pathogenic strain causing hairy root disease. In order to assemble a complete genome we obtained short-reads from Illumina sequencing as well as long-reads from Oxford Nanopore Technology sequencing. The genome consists of a 3,958,212 bp chromosome, a 2,005,144 bp chromid (secondary chromosome) and a 252,168 bp Ri plasmid (pRi1855), respectively. The primary chromosome was very similar to that of the avirulent biocontrol strain K84, but the chromid showed a 724 kbp deletion accompanied by a large 1.8 Mbp inversion revealing the dynamic nature of these secondary chromosomes. The sequence of the agropine Ri plasmid was compared to other types of Ri and Ti plasmids. Thus we identified the genes responsible for agropine catabolism, but also a unique segment adjacent to the TL-region that has the signature of a new opine catabolic gene cluster including the three genes that together encode an opine dehydrogenase. Our sequence analysis also revealed a novel gene at the very right end of the TL-DNA, which is unique for the agropine Ri plasmid. The protein encoded by this gene was most related to the succinamopine synthases of chrysopine and agropine Ti plasmids and thus may be involved in synthesis of the unknown opine that can be degraded by the adjacent catabolic cluster. The available sequence will facilitate the use of R. rhizogenes and especially LBA9402 in both the laboratory and for biotechnological purposes.

Agrobacterium tumefaciens - associative microsymbionts of grain crop

The installed absence of Ti-plasmids in the genome structure of strains of diazotrophic bacteria Agrobacterium tumefaciens 32 and A. tumefaciens P3 was found, which characterizes the strains as non-pathogenic. Bacterization of grain seeds by associative bacterial strains stimulates root development by up to 40%.

A Novel Group of Rhizobium tumorigenes-Like Agrobacteria Associated with Crown Gall Disease of Rhododendron and Blueberry

Crown gall is an economically important and widespread plant disease caused by tumorigenic bacteria that are commonly affiliated within the genera Agrobacterium, Allorhizobium, and Rhizobium. Although crown gall disease was reported to occur on rhododendron, literature data regarding this disease are limited. In this study, an atypical group of tumorigenic agrobacteria belonging to the genus Rhizobium was identified as a causative agent of crown gall on rhododendron. Genome analysis suggested that tumorigenic bacteria isolated from rhododendron tumors are most closely related to Rhizobium tumorigenes, a new tumorigenic bacterium discovered recently on blackberry in Serbia. However, R. tumorigenes and novel rhododendron strains belong to separate species and form a homogenous clade within the genus Rhizobium, which we named the “tumorigenes” clade. Moreover, tumorigenic bacteria isolated from rhododendron seem to carry a distinct tumor-inducing (Ti) plasmid, compared with those carried by R. tumorigenes strains and Ti plasmids described thus far. To facilitate rapid identification of bacteria belonging to the “tumorigenes” clade, regardless of whether they are pathogenic or not, a conventional PCR method targeting putative chromosomal gene-encoding flagellin protein FlaA was developed in this study. Finally, our results suggested that this novel group of tumorigenic agrobacteria occurs on blueberry but it cannot be excluded that it is distributed more widely.

Complete Sequence of Succinamopine Ti-Plasmid pTiEU6 Reveals Its Evolutionary Relatedness with Nopaline-Type Ti-Plasmids

Agrobacterium tumefaciens is the etiological agent of plant crown gall disease, which is induced by the delivery of a set of oncogenic genes into plant cells from its tumor-inducing (Ti) plasmid. Here we present the first complete sequence of a succinamopine-type Ti-plasmid. Plasmid pTiEU6 is comprised of 176,375 bp with an overall GC content of 56.1% and 195 putative protein-coding sequences could be identified. This Ti-plasmid is most closely related to nopaline-type Ti-plasmids. It contains a single T-region which is somewhat smaller than that of the nopaline-type Ti-plasmids and in which the gene for nopaline synthesis is replaced by a gene (sus) for succinamopine synthesis. Also in pTiEU6 the nopaline catabolic genes are replaced by genes for succinamopine catabolism. In order to trace the evolutionary origin of pTiEU6, we sequenced six nopaline Ti-plasmids to enlarge the scope for comparison to this class of plasmids. Average nucleotide identity analysis revealed that pTiEU6 was most closely related to nopaline Ti-plasmids pTiT37 and pTiSAKURA. In line with this traces of several transposable elements were present in all the nopaline Ti plasmids and in pTiEU6, but one specific transposable element insertion, that of a copy of IS1182, was present at the same site only in pTiEU6, pTiT37, and pTiSAKURA, but not in the other Ti plasmids. This suggests that pTiEU6 evolved after diversification of nopaline Ti-plasmids by DNA recombination between a pTiT37-like nopaline Ti-plasmid and another plasmid, thus introducing amongst others new catabolic genes matching a new opine synthase gene for succinamopine synthesis.

Galling in Actinidia spp. seedlings

Several Actinidia spp. genotypes exhibit crown gall-like symptoms in both roots and canes. Galls form on roots and pruning wounds of canes. Investigations were undertaken to determine if an Agrobacterium species was responsible for gall formation in the Actinidia spp. material. Macerated galls were plated onto King’s B and various selective Agrobacterium agars, e.g. 1A and Roy & Sasser media. Bacterial isolates were tested by PCR for the presence of tumour-inducing (Ti) plasmids. Isolates that tested positive for the Ti plasmid were subsequently tested for pathogenicity by inoculation onto cut carrot discs, Nicotiana glutinosa and Solanum lycopersicum, and assessed for gall formation. Bacterial isolates that tested positive by PCR for the Ti plasmid were an orange tan colour on selective medium 1A, and clear with a red centre on the Roy & Sasser medium. Galls formed on cut carrots, S. lycopersicum and N. glutinosa after inoculation of Ti-positive bacterial isolates. Initial results indicate that an Agrobacterium species is associated with the formation of galls in Actinidia seedlings. However, biochemical characterisation and confirmation of Koch’s postulates using Actinidia species are needed for verification of this result.