Rhizobium aouanii sp. nov., efficient nodulating rhizobia isolated from Acacia saligna roots in Tunisia.

Three bacterial strains, 1AS14IT, 1AS12I and 6AS6, isolated from root nodules of Acacia saligna, were characterized using a polyphasic approach. Phylogenetic analysis based on rrs sequences placed all three strains within the Rhizobium leguminosarum complex. Further phylogeny, based on 1 756 bp sequences of four concatenated housekeeping genes (recA, atpD, glnII and gyrB), revealed their distinction from known rhizobia species of the R. leguminosarum complex (Rlc), forming a distinct clade. The closest related species, identified as Rhizobium laguerreae, with a sequence identity of 96.4% based on concatenated recA-atpD-glnII-gyrB sequences. The type strain, 1AS14IT, showed average nucleotide identity (ANI) values of 94.9, 94.3 and 94.1% and DNA-DNA hybridization values of 56.1, 57.4 and 60.0% with the type strains of closest known species: R. laguerreae, Rhizobium acaciae and 'Rhizobium indicum', respectively. Phylogenomic analyses using 81 up-to-date bacteria core genes and the Type (Strain) Genome Server pipeline further supported the uniqueness of strains 1AS14IT, 1AS12I and 6AS6. The relatedness of the novel strains to NCBI unclassified Rhizobium sp. (396 genomes) and metagenome-derived genomes showed ANI values from 76.7 to 94.8% with a species-level cut-off of 96%, suggesting that strains 1AS14I, 1AS12I and 6AS6 are a distinct lineage. Additionally, differentiation of strains 1AS14IT, 1AS12I and 6AS6 from their closest phylogenetic neighbours was achieved using phenotypic, physiological and fatty acid content analyses. Based on the genomic, phenotypic and biochemical data, we propose the establishment of a novel rhizobial species, Rhizobium aouanii sp. nov., with strain 1AS14IT designated as the type strain (=DSM 113914T=LMG 33206T). This study contributes to the understanding of microbial diversity in nitrogen-fixing symbioses, specifically within Acacia saligna ecosystems in Tunisia.

Rhizobium acaciae sp. nov., a new nitrogen-fixing symbiovar isolated from root nodules of Acacia saligna in Tunisia.

Three bacterial strains, 1AS11T, 1AS12 and 1AS13, members of the new symbiovar salignae and isolated from root nodules of Acacia saligna grown in Tunisia, were characterized using a polyphasic approach. All three strains were assigned to the Rhizobium leguminosarum complex on the basis of rrs gene analysis. Phylogenetic analysis based on 1734 nucleotides of four concatenated housekeeping genes (recA, atpD, glnII and gyrB) showed that the three strains were distinct from known rhizobia species of the R. leguminosarum complex and clustered as a separate clade within this complex. Phylogenomic analysis of 92 up-to-date bacterial core genes confirmed the unique clade. The digital DNA-DNA hybridization and blast-based average nucleotide identity values for the three strains and phylogenetically related Rhizobium species ranged from 35.9 to 60.0% and 87.16 to 94.58 %, which were lower than the 70 and 96% species delineation thresholds, respectively. The G+C contents of the strains were 60.82-60.92 mol% and the major fatty acids (>4 %) were summed feature 8 (57.81 %; C18 : 1 ω7c) and C18 : 1 ω7c 11-methyl (13.24%). Strains 1AS11T, 1AS12 and 1AS13 could also be differentiated from their closest described species (Rhizobium indicum, Rhizobium laguerreae and Rhizobium changzhiense) by phenotypic and physiological properties as well as fatty acid content. Based on the phylogenetic, genomic, physiological, genotypic and chemotaxonomic data presented in this study, strains 1AS11T, 1AS12 and 1AS13 represent a new species within the genus Rhizobium and we propose the name Rhizobium acaciae sp. nov. The type strain is 1AS11T (=DSM 113913T=ACCC 62388T).

Correction to: Salt stress mitigation in Lathyrus cicera by combining different microbial inocula.

[This corrects the article DOI: 10.1007/s12298-022-01205-4.].

Salt stress mitigation in Lathyrus cicera by combining different microbial inocula.

Arid and semi-arid areas are considered vulnerable to various environmental constraints which are further fortified by climate change. Salinity is one of the most serious abiotic factors affecting crop yield and soil fertility. Till now, no information is available on the effect of salinity on development and symbiotic nitrogen (N2) fixation in the legume species Lathyrus cicera. Here, we evaluated the effect of different microbial inocula including nitrogen-fixing Rhizobium laguerreae, arbuscular mycorrhizal fungus (AMF) Rhizophagus irregularis, a complex mixed inoculum of AMF isolated from rhizospheric soil in "Al Aitha", and various plant growth-promoting bacteria (PGPB) including Bacillus subtilus, Bacillus simplex and Bacillus megaterium combined with Rhizobium, the AMF consortium, or R. irregularis on alleviating salt stress in this legume. A pot trial was conducted to evaluate the ability of different microbial inocula to mitigate adverse effects of salinity on L. cicera plants. The results showed that salinity (100 mM NaCl) significantly reduced L. cicera plant growth. However, inoculation with different inocula enhanced plant growth and markedly promoted various biochemical traits. Moreover, the combined use of PGPB and AMF was found to be the most effective treatment in mitigating deleterious effects of salinity stress on L. cicera. In addition, this co-inoculation upregulated the expression of two marker genes (LcHKT1 and LcNHX7) related to salinity tolerance. Our findings suggest that the AMF/PGPB formulation has a great potential to be used as a biofertilizer to improve L. cicera plant growth and productivity under saline conditions. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01205-4.

Genotypic and symbiotic diversity studies of rhizobia nodulating Acacia saligna in Tunisia reveal two novel symbiovars within the Rhizobium leguminosarum complex and Bradyrhizobium.

Acacia saligna is an invasive alien species that has the ability to establish symbiotic relationships with rhizobia. In the present study, genotypic and symbiotic diversity of native rhizobia associated with A. saligna in Tunisia were studied. A total of 100 bacterial strains were selected and three different ribotypes were identified based on rrs PCR-RFLP analysis. Sequence analyses of rrs and four housekeeping genes (recA, atpD, gyrB and glnII) assigned 30 isolates to four putative new lineages and a single strain to Sinorhizobium meliloti. Thirteen slow-growing isolates representing the most dominant IGS (intergenic spacer) profile clustered distinctly from known rhizobia species within Bradyrhizobium with the closest related species being Bradyrhizobium shewense and Bradyrhizobium niftali, which had 95.17% and 95.1% sequence identity, respectively. Two slow-growing isolates, 1AS28L and 5AS6L, had B. frederekii as their closest species with a sequence identity of 95.2%, an indication that these strains could constitute a new lineage. Strains 1AS14I, 1AS12I and 6AS6 clustered distinctly from known rhizobia species but within the Rhizobium leguminosarum complex (Rlc) with the most closely related species being Rhizobium indicum with 96.3% sequence identity. Similarly, the remaining 11 strains showed 96.9 % and 97.2% similarity values with R. changzhiense and R. indicum, respectively. Based on nodC and nodA phylogenies and cross inoculation tests, these 14 strains of Rlc species clearly diverged from strains of Sinorhizobium and Rlc symbiovars, and formed a new symbiovar for which the name sv. "salignae" is proposed. Bacterial strains isolated in this study that were taxonomically assigned to Bradyrhizobium harbored different symbiotic genes and the data suggested a new symbiovar, for which sv. "cyanophyllae" is proposed. Isolates formed effective nodules on A. saligna.

Phylogenetic study of rhizobia nodulating pea (Pisum sativum) isolated from different geographic locations in Tunisia.

Nodulated Pisum sativum plants showed the presence of native rhizobia in 16 out of 23 soil samples collected especially in northern and central Tunisia. A total of 130 bacterial strains were selected and three different ribotypes were revealed after PCR-RFLP analysis. Sequence analyses of rrs and four housekeeping genes (recA, atpD, dnaK and glnII) assigned 35 isolates to Rhizobium laguerreae, R. ruizarguesonis, Agrobacterium radiobacter, Ensifer meliloti and two putative genospecies. R. laguerreae was the most dominant species nodulating P. sativum with 63%. The isolates 21PS7 and 21PS15 were assigned to R. ruizarguesonis, and this is the first report of this species in Tunisia. Two putative new lineages were identified, since strains 25PS6, 10PS4 and 12PS15 clustered distinctly from known rhizobia species but within the R. leguminosarum complex (Rlc) with the most closely related species being R. indicum with 96.4% sequence identity. Similarly, strains 16PS2, 3PS9 and 3PS18 showed 97.4% and 97.6% similarity with R. sophorae and R. laguerreae, respectively. Based on 16S-23S intergenic spacer (IGS) fingerprinting, there was no clear association between the strains and their geographic locations. According to nodC and nodA phylogenies, strains of Rlc species and, interestingly, strain 8PS18 identified as E. meliloti, harbored the symbiotic genes of symbiovar viciae and clustered in two different clades showing heterogeneity within the symbiovar. All these strains nodulated and fixed nitrogen with pea plants. However, the strains belonging to A. radiobacter and the two remaining strains of E. meliloti were unable to nodulate P. sativum, suggesting that they were non-symbiotic strains. The results of this study further suggest that the Tunisian Rhizobium community is more diverse than previously reported.

Genotypic and symbiotic diversity of native rhizobia nodulating red pea (Lathyrus cicera L.) in Tunisia.

Nodulation and genetic diversity of native rhizobia nodulating Lathyrus cicera plants grown in 24 cultivated and marginal soils collected from northern and central Tunisia were studied. L. cicera plants were nodulated and showed the presence of native rhizobia in 21 soils. A total of 196 bacterial strains were selected and three different ribotypes were revealed after PCR-RFLP analysis. The sequence analysis of the rrs and two housekeeping genes (recA and thrC) from 36 representative isolates identified Rhizobium laguerreae as the dominant (53%) rhizobia nodulating L. cicera. To the best of our knowledge, this is the first time that this species has been reported among wild populations of the rhizobia-nodulating Lathyrus genus. Twenty-five percent of the isolates were identified as R. leguminosarum and isolates LS11.5, LS11.7 and LS8.8 clustered with Ensifer meliloti. Interestingly, five isolates (LS20.3, LS18.3, LS19.10, LS1.2 and LS21.20) were segregated from R. laguerreae and clustered as a separate clade. These isolates possibly belong to new species. According to nodC and nodA phylogeny, strains of R. laguerreae and R. leguminosarum harbored the symbiotic genes of symbiovar viciae and clustered in three different clades showing heterogeneity within the symbiovar. Strains of E. meliloti harbored symbiotic genes of Clade V and induced inefficient nodules.