Guidelines for the description of rhizobial symbiovars.

Rhizobia are bacteria that form nitrogen-fixing nodules in legume plants. The sets of genes responsible for both nodulation and nitrogen fixation are carried in plasmids or genomic islands that are often mobile. Different strains within a species sometimes have different host specificities, while very similar symbiosis genes may be found in strains of different species. These specificity variants are known as symbiovars, and many of them have been given names, but there are no established guidelines for defining or naming them. Here, we discuss the requirements for guidelines to describe symbiovars, propose a set of guidelines, provide a list of all symbiovars for which descriptions have been published so far, and offer a mechanism to maintain a list in the future.

Reclassification of type strains of Rhizobium indigoferae and Sinorhizobium kummerowiae into the species Rhizobium leguminosarum and Sinorhizobium meliloti, respectively.

The species Rhizobium indigoferae and Sinorhizobium kummerowiae were isolated from legume nodules and the 16S rRNA sequences of their respective type strains, CCBAU 71042T and CCBAU 71714T, were highly divergent from those of the other species of the genera Rhizobium and Sinorhizobium, respectively. However, the 16S rRNA gene sequences obtained for strains CCBAU 71042T and CCBAU 71714T several years after description, were different from the original ones, showing 100 % similarity to the type strains of Rhizobium leguminosarum and Sinorhizobium meliloti, respectively. Phylogenetic analyses of two housekeeping genes, recA and atpD, confirmed the high phylogenetic closeness of strains CCBAU 71042T and CCBAU 71714T to the respective type strains of R. leguminosarum and S. meliloti. In the present work, we compared the genomes of the type strains of R. indigoferae and S. kummerowiae available in several culture collections with those of the respective type strains of R. leguminosarum and S. meliloti, some of them obtained in this study. The calculated average nucleotide identity-blast and digital DNA-DNA hybridization values in both cases were higher than those recommended for species differentiation, supporting the proposal for the reclassification of the type strains of R. indigoferae and S. kummerowiae into the species R. leguminosarum and S. meliloti, respectively.

Definition of the symbiovar viciae in the species Rhizobium azibense and biogeographic implications.

Vicia faba L. (faba bean) is a legume cultivated worldwide which commonly establishes effective symbiosis with the symbiovar viciae of species from the Rhizobium leguminosarum phylogenetic group. However, on the basis of the rrs, recA, and atpD gene phylogenies, in this work we identified a strain named EFBRI 42 nodulating V. faba as Rhizobium azibense. This is the first report on the nodulation of Vicia by R. azibense which commonly nodulates P. vulgaris and to date encompasses strains harboring the nodC genes typical of the symbiovars gallicum and phaseoli. However, the strain EFBRI 42 carries a nodC gene typical of the symbiovar viciae for which we report here by the first time this symbiovar in R. azibense. This finding showed the existence of symbiotic genes horizontal transfer events during the coevolution of R. azibense with P. vulgaris and V. faba in their respective distribution centers of Mesoamerica and the Middle East.

Agrobacterium leguminum sp. nov., isolated from nodules of Phaseolus vulgaris in Spain.

Two endophytic strains, coded MOVP5T and MOPV6, were isolated from nodules of Phaseolus vulgaris plants grown on agricultural soil in Southeastern Spain, and were characterized through a polyphasic taxonomy approach. Their 16S rRNA gene sequences showed 99.3 and 99.4 %, 98.9 and 99.6 %, and 99.0 and 98.7% similarity to 'A. deltaense' YIC 4121T, A. radiobacter LGM 140T, and A. pusense NRCPB10T, respectively. Multilocus sequence analysis based on sequences of recA and atpD genes suggested that these two strains could represent a new Agrobacterium species with less than 96.5 % similarity to their closest relatives. PCR amplification of the telA gene, involved in synthesis of protelomerase, confirmed the affiliation of strains MOPV5T and MOPV6 to the genus Agrobacterium. Whole genome average nucleotide identity and digital DNA-DNA hybridization average values were less than 95.1 and 66.7 %, respectively, with respect to its closest related species. Major fatty acids in strain MOPV5T were C18 : 1 ω7c/C18 : 1 ω6c in summed feature 8, C19 : 0 cyclo ω8c, C16 : 0 and C16 : 0 3-OH. Colonies were small to medium, pearl-white coloured on YMA at 28 °C and growth was observed at 10-42 °C, pH 5.0-10.0 and with 0.0-0.5 % (w/v) NaCl. The DNA G+C content was 59.9 mol%. These two strains differ from all other genomovars of Agrobacterium found so far, including those that have not yet given a Latin name. The combined genotypic, phenotypic and chemotaxonomic data support the classification of strain MOPV5T as representing a novel species of Agrobacterium, for which the name Agrobacterium leguminum sp. nov. is proposed. The type strain is MOPV5T (=CECT 30096T=LMG 31779T).

Strain ATCC 4720T is the authentic type strain of Agrobacterium tumefaciens, which is not a later heterotypic synonym of Agrobacterium radiobacter.

The original type strains of Agrobacterium radiobacter and Agrobacterium tumefaciens recorded in the eighth edition of Bergey's Manual of Determinative Bacteriology published in 1974 were NCIB 9042T and ATCC 4720T, respectively. However, in the list of the valid names of bacteria compiled in 1980, both strains were changed, A. radiobacter NCIB 9042T to ATCC 19358T and A. tumefaciens ATCC 4720T to ATCC 23308T. These changes were unjustified, particularly in the case of A. tumefaciens whose type strain was replaced by another strain from the same collection, although the original type strain ATCC 4720T was never lost and it is currently available in several culture collections. Therefore, we request that the type strain of A. tumefaciens be corrected from ATCC 23308T to ATCC 4720T.

Agrobacterium cavarae sp. nov., isolated from maize (Zea mays L.) roots.

A bacterial strain designated as RZME10T was isolated from a Zea mays L. root collected in Spain. Results of analysis of the 16S rRNA gene sequence showed that this strain belongs to the genus Agrobacterium with Agrobacterium larrymoorei ATCC 51759T being the most closely related species with 99.9 % sequence similarity. The similarity values of the rpoB, recA, gyrB, atpD and glnII genes between strain RZME10T and A. larrymoorei ATCC 51759T were 93.5, 90.0, 88.7, 87.9 and 90.1 %, respectively. The estimated average nucleotide identity using blast and digital DNA-DNA hybridization values between these two strains were 80.4 and 30.2 %, respectively. The major fatty acids of strain RZME10T are those from summed feature 8 (C18 : 1 ω6c/C18 : 1 ω7c) and C16 : 0. Pathogenicity tests on tomato and carrot roots showed that strain RZME10T was not able to induce plant tumours. Based on the results of genomic, chemotaxonomic and phenotypic analyses, we propose that strain RZME10T represents a novel species named Agrobacterium cavarae sp. nov. (type strain RZME10T=CECT 9795T=LMG 31257T).

Pseudomonas edaphica sp. nov., isolated from rhizospheric soil of Cistus ladanifer L. in Spain.

During a study on biodiversity of bacteria inhabiting rhizospheric soil of rockrose (Cistus ladanifer L.), we isolated a strain coded RD25T in a soil from Northern Spain. The 16S rRNA gene sequence showed 99.5 % identity with respect to the closest related species Pseudomonas brenneri DSM15294T, and 99.4 % with respect to P. paralactis WS4672T. The following related Pseudomonas species showed 99.3 % or less identity, and therefore RD25T was classified within genus Pseudomonas. The phylogenetic analysis of 16S rRNA and the housekeeping genes rpoB, rpoD and gyrB suggested that this strain could be a novel species. The strain RD25T has several polar-subpolar flagella. It can grow at 36 °C, at 0-6 % NaCl concentration and a range of pH 5-9. Positive for arginine dihydrolase and urease production, and negative for reduction of nitrate. The strain is catalase and oxidase positive. Major fatty acids are C16 : 1 ω7c / C16 : 1 ω6c in summed feature 3, C16 : 0, and C18 : 1 ω7c / C18 : 1 ω6c in summed feature 8. The respiratory ubiquinone is Q9. The DNA G+C content was 59.9 mol%. The digital DNA-DNA hybridisation average values (dDDH) ranged between 30-61.2 % relatedness and the ANIb values ranged between 93.9-80.5 % with respect to the type strains of the closely related species. Therefore, the genotypic, genomic, phenotypic and chemotaxonomic data support the classification of strain RD25 as a novel species of genus Pseudomonas, for which the name P. edaphica sp. nov. is proposed. The type strain is RD25T (=LMG 30152T=CECT 9373T).

Bacillus safensis subsp. osmophilus subsp. nov., isolated from condensed milk, and description of Bacillus safensis subsp. safensis subsp. nov.

A bacterial strain, designated BC09T, was isolated from a contaminated sample of condensed milk. Phylogenetic analyses based on 16S rRNA gene sequences placed strain BC09T into the genus Bacillus with its closest relatives being Bacillus safensis and Bacillus australimaris with 100 and 99.9 % similarity, respectively. Analysis of the gyrB gene confirmed the closeness of strain BC09T with respect to the species B. safensis since it presented 97.8 and 95.2 % similarity values, respectively, to the type strains of B. safensis and B. australimaris. DNA-DNA hybridization confirmed these results showing averages of 67 and 56 %, respectively, between strain BC09T and the type strains of B. safensis and B. australimaris. Average nucleotide identity blast values obtained for BC09T compared to the closest relative type strains were 95.7 and 67.6 %, respectively, and predicted DNA-DNA hybridization values were 93.1 and 51.9 %, respectively. However, strain BC09T differs from the type strains of its closest relatives in several phenotypic characteristics. MK-7 was the only menaquinone detected and iso-C15:0 and anteiso-C15:0 were the major fatty acids. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, two unidentified phospholipids, two unidentifed glycolipids, three unidentified lipids and one unidentifed phosphoglycolipid. Meso-diaminopimelic acid was detected in the peptidoglycan. The G+C content was 40.9 mol%. Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain BC09T represents a new subspecies of B. safensis, for which the name Bacillus safensis subsp. osmophilus subsp. nov. is proposed. The type strain is BC09T (=LMG 30124T, =CECT 9344T).

Minimal standards for the description of new genera and species of rhizobia and agrobacteria.

Herein the members of the Subcommittee on Taxonomy of Rhizobia and Agrobacteria of the International Committee on Systematics of Prokaryotes review recent developments in rhizobial and agrobacterial taxonomy and propose updated minimal standards for the description of new species (and genera) in these groups. The essential requirements (minimal standards) for description of a new species are (1) a genome sequence of at least the proposed type strain and (2) evidence for differentiation from other species based on genome sequence comparisons. It is also recommended that (3) genetic variation within the species is documented with sequence data from several clearly different strains and (4) phenotypic features are described, and their variation documented with data from a relevant set of representative strains. Furthermore, it is encouraged that information is provided on (5) nodulation or pathogenicity phenotypes, as appropriate, with relevant gene sequences. These guidelines supplement the current rules of general bacterial taxonomy, which require (6) a name that conforms to the International Code of Nomenclature of Prokaryotes, (7) validation of the name by publication either directly in the International Journal of Systematic and Evolutionary Microbiology or in a validation list when published elsewhere, and (8) deposition of the type strain in two international culture collections in separate countries.

Phyllobacterium salinisoli sp. nov., isolated from a Lotus lancerottensis root nodule in saline soil from Lanzarote.

A Gram-negative rod, designated strain LLAN61T, was isolated from a root nodule of Lotus lancerottensis growing in a saline soil sample from Lanzarote (Canary Islands). The strain grew optimally at 0.5 % (w/v) NaCl and tolerated up to 3.5 %. The 16S rRNA gene sequence analysis showed that strain LLAN61T belonged to genus Phyllobacterium and that Phyllobacteriumleguminum ORS 1419T and Phyllobacteriummyrsinacearum IAM 13584T are the closest related species with 97.93 and 97.86% similarity values, respectively. In the atpD phylogeny, P. leguminum ORS 1419T and P. myrsinacearum ATCC 43591T, sharing similarities of 87.6 and 85.8% respectively, were also the closest species to strain LLAN61T. DNA-DNA hybridization showed an average value of 21 % between strain LLAN61T and P. leguminum LMG 22833T, and 6 % with P. myrsinacearum ATCC 43590T. The predominant fatty acids were C19 : 0 cyclo ω8c and C18 : 1ω6c/C18 : 1ω7c (summed feature 8). The DNA G+C content was 58.0 mol%. Strain LLAN61T differed from its closest relatives in some culture conditions and in assimilation of several carbon sources. Based upon the results of phylogeny, DNA-DNA hybridization, phenotypic tests and fatty acid analysis, this strain should be classified as a novel species of Phyllobacterium for which the name Phyllobacterium salinisoli sp. nov. is proposed (type strain LLAN61T=LMG 30173T = CECT 9417T).