Identification of synthetic consortia from a set of plant-beneficial bacteria.

The use of microbial inoculants in agriculture as biofertilisers and/or biopesticides is an appealing alternative to replace or reduce the practice of agrochemicals. Plant microbiota studies are revealing the different bacterial groups which are populating plant microbiomes re-energising the plant probiotic bacteria (PPB) translational research sector. Some single-microbial strain bioinoculants have proven valid in agriculture (e.g., based on Trichoderma, mycorrhiza or rhizobia); however, it is now recommended to consider multistrain consortia since plant-beneficial effects are often a result of community-level interactions in plant microbiomes. A limiting step is the selection of a fitting combination of microbial strains in order to accomplish the best beneficial effect upon plant inoculation. In this study, we have used a subset of 23 previously identified and characterised rice-beneficial bacterial colonisers to design and test a series of associated experiments aimed to identify potential PPB consortia which are able to co-colonise and induce plant growth promotion. Bacterial strains were co-inoculated in vitro and in planta using several different methods and their co-colonisation and co-persistence monitored. Results include the identification of two 5-strain and one 2-strain consortia which displayed plant growth-promoting features. Future practical applications of microbiome research must include experiments aimed at identifying consortia of bacteria which can be most effective as crop amendments.

Paraburkholderia atlantica sp. nov. and Paraburkholderia franconis sp. nov., two new nitrogen-fixing nodulating species isolated from Atlantic forest soils in Brazil.

A polyphasic study was conducted with 11 strains trapped by Mimosa pudica and Phaseolus vulgaris grown in soils of the Brazilian Atlantic Forest. In the phylogenetic analysis of the 16S rRNA gene, one clade of strains (Psp1) showed higher similarity with Paraburkholderia piptadeniae STM7183T (99.6%), whereas the second (Psp6) was closely related to Paraburkholderia tuberum STM678T (99%). An MLSA (multilocus sequence analysis) with four (recA, gyrB, trpB and gltB) housekeeping genes placed both Psp1 and Psp6 strains in new clades, and BOX-PCR profiles indicated high intraspecific genetic diversity within each clade. Values of digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) of the whole genome sequences were of 56.9 and 94.4% between the Psp1 strain CNPSo 3157T and P. piptadeniae; and of 49.7% and 92.7% between the Psp6 strain CNPSo 3155T and P. tuberum, below the threshold for species delimitation. In the nodC analysis, Psp1 strains clustered together with P. piptadeniae, while Psp6 did not group with any symbiotic Paraburkholderia. Other phenotypic, genotypic and symbiotic properties were evaluated. The polyphasic analysis supports that the strains represent two novel species, for which the names Paraburkholderia franconis sp. nov. with type strain CNPSo 3157T (= ABIP 241, = LMG 31644) and Paraburkholderia atlantica sp. nov. with type strain CNPSo 3155T (= ABIP 236, = LMG 31643) are proposed.

Paraburkholderia guartelaensis sp. nov., a nitrogen-fixing species isolated from nodules of Mimosa gymnas in an ecotone considered as a hotspot of biodiversity in Brazil.

A polyphasic approach was used to infer the phylogenetic position of six nitrogen-fixing symbiotic bacteria isolated from Mimosa gymnas nodules grown in an ecotone between the Brazilian biomes of Atlantic Forest and Cerrado, considered as a hotspot of biodiversity. The 16S rRNA gene phylogeny indicated the highest similarity with Paraburkholderia oxyphila (98.7-98.9%), but similar values were found with other Paraburkholderia species. The multilocus sequence analysis (MLSA) of five (recA, gyrB, trpB, gltB, and atpD) housekeeping genes indicated that the CNPSo strains represent a novel lineage, sharing less than 95.7% of nucleotide identity (NI) with other Paraburkholderia species, being more closely related to P. nodosa. Genome parameters were analyzed for strain CNPSo 3008T, and DNA-DNA hybridization revealed a maximum of 55.9% of DNA-DNA relatedness with P. nodosa, while average nucleotide identity with the two closest species was of 93.84% with P. nodosa and of 87.93% with P. mimosarum, both parameters confirming that the strain represents a new species. In the analysis of the nodulation nodC gene, all CNPSo strains showed the highest similarity with P. nodosa, and nodulation tests indicated host specificity with Mimosa. Other phylogenetic, physiological, and chemotaxonomic properties were evaluated. All data obtained support the description of the novel species Paraburkholderia guartelaensis sp. nov., with CNPSo 3008T (= U13000T = G29.01T) indicated as the type strain.

Mesorhizobium atlanticum sp. nov., a new nitrogen-fixing species from soils of the Brazilian Atlantic Forest biome.

Biological nitrogen fixation performed by diazotrophic bacteria is a vital process for agricultural and environmental sustainability. In recent years, bacterial classification has been based on genomic data, accelerating our understanding about the diversity, and resulting in the description of several new species. In this study, four strains (CNPSo 3140T, CNPSo 3235, CNPSo 3236 and CNPSo 3237) trapped by Phaseolus vulgaris and Mimosa pudica from soil samples of the Brazilian Atlantic Forest biome (Mata Atlântica) were submitted to polyphasic analysis to investigate their proper classification within the genus Mesorhizobium. The 16S rRNA gene phylogram showed that the strains present sequences identical to those of Mesorhizobium acaciaeand Mesorhizobium plurifarium, not allowing a clear taxonomic classification; however, when using multilocus sequence analysis methodology, the strains were grouped into a well-supported distinct clade, with <94.5 % nucleotide identity with the other species of the genus. The average nucleotide identity of CNPSo 3140T genome showed values below the threshold in relation to the closest species, of 89.75 % with Mesorhizobium plurifariumand of 88.83 % with Mesorhizobium hawassense; the digital DNA-DNA hybridization values were 39 and 37.70 % with the same species, respectively. Nodulation gene (nodC) phylogeny positioned the strains in an isolated cluster, showing greater similarity to Mesorhizobiumshonense. All data obtained in this study support the description of the novel species Mesorhizobiumatlanticum sp. nov. The type strain is CNPSo 3140T (=ABIP 206T=LMG 30305T=U1602T), isolated from a nodule of Phaseolus vulgaris.

Phylogenetic diversity of rhizobia nodulating native Mimosa gymnas grown in a South Brazilian ecotone.

Floristic surveys performed in "Campos Gerais" (Paraná, Brazil), an ecotone of Mata Atlântica and Cerrado biomes, highlights the richness and relative abundance of the family Fabaceae and point out the diversity and endemism of Mimosa spp. Our study reports the genetic diversity of rhizobia isolated from root nodules of native/endemic Mimosa gymnas Barneby in three areas of Guartelá State Park, an important conservation unit of "Campos Gerais". Soils of the sample areas were characterized as sandy, acid, poor in nutrients and organic matter. The genetic variability among the isolates was revealed by BOX-PCR genomic fingerprinting. Phylogeny based on 16S rRNA gene grouped the strains in a large cluster including Paraburkholderia nodosa and P. bannensis, while recA-gyrB phylogeny separated the strains in two groups: one including P. nodosa and the other without any described Paraburkholderia species. MLSA confirmed the separate position of this second group of strains within the genus Paraburkholderia and the nucleotide identity of the five concatened housekeeping genes was 95.9% in relation to P. nodosa BR 3437T. Phylogram based on symbiosis-essential nodC gene was in agreement with 16S rRNA analysis. Our molecular phylogenetic analysis support that Paraburkholderia are the main symbionts of native Mimosa in specific edaphic conditions found in South America and reveal the importance of endemic/native leguminous plants as reservoirs of novel rhizobial species.

Genetic diversity of symbiotic Paraburkholderia species isolated from nodules of Mimosa pudica (L.) and Phaseolus vulgaris (L.) grown in soils of the Brazilian Atlantic Forest (Mata Atlântica).

Some species of the genus Paraburkholderia that are able to nodulate and fix nitrogen in symbiosis with legumes are called ß-rhizobia and represent a group of ecological and biotechnological importance. We used Mimosa pudica and Phaseolus vulgaris to trap 427 rhizobial isolates from rhizospheric soil of Mimoseae trees in the Brazilian Atlantic Forest. Eighty-four representative strains were selected according to the 16S rRNA haplotypes and taxonomically characterized using a concatenated 16S rRNA-recA phylogeny. Most strains were assembled in the genus Paraburkholderia, including Paraburkholderia sabiae and Pa. nodosa. Mesorhizobium (α-rhizobia) and Cupriavidus (ß-rhizobia) were also isolated, but in smaller proportions. Multilocus sequence analysis and BOX-PCR analyses indicated that six clusters of Paraburkholderia represent potential new species. In the phylogenetic analysis of the nodC gene, the majority of the strains were positioned in the same groups as in the 16S rRNA-recA tree, indicative of stability and vertical inheritance, but we also identified horizontal transfer of nodC in Pa. sabiae. All α- and ß-rhizobial species were trapped by both legumes, although preferences of the host plants for specific rhizobial species have been observed.

Genome Sequence of Paraburkholderia nodosa Strain CNPSo 1341, a N2-Fixing Symbiont of the Promiscuous Legume Phaseolus vulgaris.

Paraburkholderia nodosa CNPSo 1341 is a N2-fixing symbiont of Phaseolus vulgaris isolated from an undisturbed soil of the Brazilian Cerrado. Its draft genome contains 8,614,032 bp and 8,068 coding sequences (CDSs). Nodulation and N2-fixation genes were clustered in the genome that also contains several genes of secretion systems and quorum sensing.

Rhizobium paranaense sp. nov., an effective N2-fixing symbiont of common bean (Phaseolus vulgaris L.) with broad geographical distribution in Brazil.

Nitrogen (N), the nutrient most required for plant growth, is key for good yield of agriculturally important crops. Common bean (Phaseolus vulgaris L.) can benefit from bacteria collectively called rhizobia, which are capable of fixing atmospheric nitrogen (N2) in root nodules and supplying it to the plant. Common bean is amongst the most promiscuous legume hosts; several described species, in addition to putative novel ones have been reported as able to nodulate this legume, although not always effectively in terms of fixing N2. In this study, we present data indicating that Brazilian strains PRF 35(T), PRF 54, CPAO 1135 and H 52, currently classified as Rhizobium tropici, represent a novel species symbiont of common bean. Morphological, physiological and biochemical properties differentiate these strains from other species of the genus Rhizobium, as do BOX-PCR profiles (less than 60 % similarity), multilocus sequence analysis with recA, gyrB and rpoA (less than 96.4 % sequence similarity), DNA-DNA hybridization (less than 50 % DNA-DNA relatedness), and average nucleotide identity of whole genomes (less than 92.8.%). The novel species is effective in nodulating and fixing N2 with P. vulgaris, Leucaena leucocephala and Leucaena esculenta. We propose the name Rhizobium paranaense sp. nov. for this novel taxon, with strain PRF 35(T) ( = CNPSo 120(T) = LMG 27577(T) = IPR-Pv 1249(T)) as the type strain.

Rhizobium freirei sp. nov., a symbiont of Phaseolus vulgaris that is very effective at fixing nitrogen.

Common bean (Phaseolus vulgaris L.) can establish symbiotic associations with several Rhizobium species; however, the effectiveness of most strains at fixing nitrogen under field conditions is very low. PRF 81(T) is a very effective strain, usually referred to as Rhizobium tropici and used successfully in thousands of doses of commercial inoculants for the common bean crop in Brazil; it has shown high rates of nitrogen fixation in all areas representative of the crop in the country. Here, we present results that indicate that PRF 81(T), although it belongs to the 'R. tropici group', which includes 10 Rhizobium species, R. tropici, R. leucaenae, R. lusitanum, R. multihospitium, R. miluonense, R. hainanense, R. calliandrae, R. mayense, R. jaguaris and R. rhizogenes, represents a novel species. Several morpho-physiological traits differentiated PRF 81(T) from related species. Differences were also confirmed in the analysis of rep-PCR (sharing less than 45 % similarity with the other species), MLSA with recA, atpD and rpoB genes, and DNA-DNA hybridization. The novel species, for which we propose the name Rhizobium freirei sp. nov., is able to establish effective root nodule symbioses with Phaseolus vulgaris, Leucaena leucocephala, Leucaena esculenta, Crotalaria juncea and Macroptilium atropurpureum. The type strain is PRF 81(T) ( = CNPSo 122(T) = SEMIA 4080(T) = IPR-Pv81(T) = WDCM 440(T)).

Novel Rhizobium lineages isolated from root nodules of the common bean (Phaseolus vulgaris L.) in Andean and Mesoamerican areas.

The taxonomic affiliations of nineteen root-nodule bacteria isolated from the common bean (Phaseolus vulgaris L.) in Mexico, Ecuador and Brazil were investigated by analyses of 16S rRNA and of four protein-coding housekeeping genes. One strain from Mexico could be assigned to Rhizobium etli and two from Brazil to Rhizobium leucaenae, whereas another from Mexico corresponded to a recently described bean-nodulating species-level lineage related to R. etli and Rhizobium phaseoli. Ten strains isolated in Ecuador and Mexico corresponded to three novel Rhizobium lineages that fall into the R. phaseoli/R. etli/Rhizobium leguminosarum clade. One of those lineages, with representatives isolated mostly from Ecuador, seems to be dominant in beans from that Andean region. Only one of the Mexican strains clustered within the Rhizobium tropici clade, but as an independent lineage. Interestingly, four strains were affiliated with species within the Rhizobium radiobacter clade. The existence of yet non-described native Rhizobium lineages in both the Andean and Mesoamerican areas is discussed in relation to common-bean diversity and environmental conditions.