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The taxonomic status of two bacterial strains, KW56T and 2063T, isolated from root nodules of Chamaecytisus albus (Spanish broom), was investigated using a polyphasic approach. Both isolates belong to the genus Phyllobacterium, yet exhibit significant genotypic and phenotypic differences from all currently described species. Whole-genome comparisons revealed that strain KW56T is most closely related to Phyllobacterium trifolii PETP 02T, while strain 2063T is related to Phyllobacterium brassicacearum strains STM 196T and 29-15. However, digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values between the new isolates and their closest relatives were below established species delineation thresholds, supporting their recognition as novel species. Phenotypic analyses confirmed morphological and growth characteristics typical for Phyllobacterium, while also revealing several discriminatory physiological traits. Fatty acid profiling showed that cyclopropyl 19:0 was the major fatty acid in both strains, though each exhibited a unique fatty acid composition. Chemotaxonomic markers included 3-OH 14:0, a19:1, and 21:0 for strain KW56T, and 3-OH 14:0, 3-OH 17:0, and 3-OH 18:0 for strain 2063T. Based on the genomic, phenotypic, and chemotaxonomic data, we propose that strains KW56T and 2063T represent two novel species, for which the names Phyllobacterium chamaecytisi sp. nov. (DSM 113831) and Phyllobacterium lublinensis sp. nov. (DSM 113830) are proposed.

Fungal phytopathogens represent a large and economically significant challenge to food production worldwide. Thus, the application of biocontrol agents can be an alternative. In the present study, we carried out biological, metabolomic, and genetic analyses of three endophytic isolates from nodules of Chamaecytisus albus, classified as Pseudomonas chlororaphis acting as antifungal agents. The efficiency of production of their diffusible and volatile antifungal compounds (VOCs) was verified in antagonistic assays with the use of soil-borne phytopathogens: B. cinerea, F. oxysporum, and S. sclerotiorum. Diffusible metabolites were identified using chromatographic and spectrometric analyses (HPTLC, GC-MS, and LC-MS/MS). The phzF, phzO, and prnC genes in the genomes of bacterial strains were confirmed by PCR. In turn, the plant growth promotion (PGP) properties (production of HCN, auxins, siderophores, and hydrolytic enzymes, phosphate solubilization) of pseudomonads were bioassayed. The data analysis showed that all tested strains have broad-range antifungal activity with varying degrees of antagonism. The most abundant bioactive compounds were phenazine derivatives: phenazine-1-carboxylic acid (PCA), 2-hydroxy-phenazine, and diketopiperazine derivatives as well as ortho-dialkyl-aromatic acids, pyrrolnitrin, siderophores, and HCN. The results indicate that the tested P. chlororaphis isolates exhibit characteristics of biocontrol organisms; therefore, they have potential to be used in sustainable agriculture and as commercial postharvest fungicides to be used in fruits and vegetables.

Polycyclic aromatic hydrocarbons (PAHs) are common xenobiotics that are detrimental to the environment and human health. Bacterial endophytes, having the capacity to degrade PAHs, and plant growth promotion (PGP) may facilitate their biodegradation. In this study, phenanthrene (PHE) utilization of a newly isolated PGP endophytic strain of Pseudomonas chlororaphis 23aP and factors affecting the process were evaluated. The data obtained showed that strain 23aP utilized PHE in a wide range of concentrations (6–100 ppm). Ethyl-acetate-extractable metabolites obtained from the PHE-enriched cultures were analyzed by gas chromatography–mass spectrometry (GC-MS) and thin-layer chromatography (HPTLC). The analysis identified phthalic acid, 3-(1-naphthyl)allyl alcohol, 2-hydroxybenzalpyruvic acid, α-naphthol, and 2-phenylbenzaldehyde, and allowed us to propose that the PHE degradation pathway of strain 23aP is initiated at the 1,2-, 3,4-carbon positions, while the 9,10-C pathway starts with non-enzymatic oxidation and is continued by the downstream phthalic pathway. Moreover, the production of the biosurfactants, mono- (Rha-C8-C8, Rha-C10-C8:1, Rha-C12:2-C10, and Rha-C12:1-C12:1) and dirhamnolipids (Rha-Rha-C8-C10), was confirmed using direct injection–electrospray ionization–mass spectrometry (DI-ESI-MS) technique. Changes in the bacterial surface cell properties in the presence of PHE of increased hydrophobicity were assessed with the microbial adhesion to hydrocarbons (MATH) assay. Altogether, this suggests the strain 23aP might be used in bioaugmentation—a biological method supporting the removal of pollutants from contaminated environments.

A novel bacterium, designated strain MMK2 T , was isolated from a surface-sterilised root nodule of a Trifolium rubens plant growing in south-eastern Poland. Cells were Gram negative, non-spore forming and rod shaped. The strain had the highest 16S rRNA gene sequence similarity with P. endophytica (99.4%), P. leporis (99.4%) P. rwandensis (98.8%) and P. rodasii (98.45%). Phylogenomic analysis clearly showed that strain MMK2 T and an additional strain, MMK3, should reside in the genus Pantoea and that they were most closely related to P. endophytica and P. leporis . Genome comparisons showed that the novel strain shared 82.96–93.50% average nucleotide identity and 26.2–53. 2% digital DNA:DNA hybridization with closely related species. Both strains produced siderophores and were able to solubilise phosphates. The MMK2 T strain was also able to produce indole-3-acetic acid. The tested strains differed in their antimicrobial activity, but both were able to inhibit the growth of Sclerotinia sclerotiorum 10Ss01. Based on the results of the phenotypic, phylogenomic, genomic and chemotaxonomic analyses, strains MMK2 T and MMK3 belong to a novel species in the genus Pantoea for which the name Pantoea trifolii sp. nov. is proposed with the type strain MMK2 T (= DSM 115063 T  = LMG 33049 T ).

Bacteria belonging to the genus Micromonospora are recognized as microorganisms with the potential to be used in biotechnology processes, given their beneficial influence on plant growth and the biocontrol of phytopathogens. In this study, nineteen Micromonospora isolates originating from the root nodules of white clover plants were taxonomically assigned based on the phylogenetic analysis of the 16S rRNA gene and four housekeeping genes. The antifungal properties of the bacteria against phytopathogenic Botrytis cinerea, Fusarium oxysporum, Fusarium equiseti, Sclerotinia sclerotiorum, and Verticillium albo-atrum were tested with the agar plug test and the dual culture test. The ability to produce various metallophores was determined with the agar plug diffusion test on modified chrome azurol S (CAS) agar medium. International Streptomyces Project-2 medium (ISP2) broth amended with 0.2% L-tryptophan was used to indicate the bacterial ability to produce auxins. The strains belonging to M. tulbaghiae, M. inaquosa, and M. violae showed in vitro potential as antimicrobial agents against the tested fungi. M. inaquosa strain 152, M. violae strain 126, M. violae strain 66, and M. violae strain 45 were recognized as the most efficient metallophore producers. M. alfalfae strain 55 and M. lupini strain 5052 were identified as the most promising auxin compound producers and, therefore, show potential as plant-growth-promoting bacteria.

The aim of this study was to analyse the genetic and phenotypic differentiation of bacteria isolated from root nodules of Trifolium rubens, a rare clover species. The symbiotic compatibility of selected isolates was investigated using two agronomically important plants, red clover (Trifolium pratense L.) and pea (Pisum sativum L.). A high genetic diversity of analysed strains was shown using the Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) method. Most of the strains showed a high similarity to R. leguminosarum in the 16S rRNA sequence. Two strains were identified as Agrobacterium spp. There was a strong positive correlation between fresh clover weight and the number of root nodules and a statistically significant increase in the number of the root nodules in selected strains compared to the negative control. Phenotypic tests and BIOLOG analysis showed the ability of the analysed strains to grow in different experimental conditions (pH = 6–10, 0.5–3% NaCl, different carbon and nitrogen sources), which makes them suitable for application as an agronomically important factor.

Background Soil bacteria from the genus Rhizobium are characterized by a complex genomic architecture comprising chromosome and large plasmids. Genes responsible for symbiotic interactions with legumes are usually located on one of the plasmids, named the symbiotic plasmid (pSym). The plasmids have a great impact not only on the metabolic potential of rhizobia but also underlie genome rearrangements and plasticity. Results Here, we analyzed the distribution and sequence variability of markers located on chromosomes and extrachromosomal replicons of Rhizobium leguminosarum bv. trifolii strains originating from nodules of clover grown in the same site in cultivated soil. First, on the basis of sequence similarity of repA and repC replication genes to the respective counterparts of chromids reported in R. leguminosarum bv. viciae 3841 and R. etli CFN42, chromid-like replicons were distinguished from the pool of plasmids of the nodule isolates studied. Next, variability of the gene content was analyzed in the different genome compartments, i.e., the chromosome, chromid-like and 'other plasmids'. The stable and unstable chromosomal and plasmid genes were detected on the basis of hybridization data. Displacement of a few unstable genes between the chromosome, chromid-like and 'other plasmids', as well as loss of some markers was observed in the sampled strains. Analyses of chosen gene sequences allowed estimation of the degree of their adaptation to the three genome compartments as well as to the host. Conclusions Our results showed that differences in distribution and sequence divergence of plasmid and chromosomal genes can be detected even within a small group of clover nodule isolates recovered from clovers grown at the same site. Substantial divergence of genome organization could be detected especially taking into account the content of extrachromosomal DNA. Despite the high variability concerning the number and size of plasmids among the studied strains, conservation of the location as well as dynamic distribution of the individual genes (especially replication genes) of a particular genome compartment were demonstrated. The sequence divergence of particular genes may be affected by their location in the given genome compartment. The 'other plasmid' genes are less adapted to the host genome than the chromosome and chromid-like genes.

Polycyclic aromatic hydrocarbons (PAHs) are common xenobiotics that are detrimental to the environment and human health. Bacterial endophytes, having the capacity to degrade PAHs, and plant growth promotion (PGP) may facilitate their biodegradation. In this study, phenanthrene (PHE) utilization of a newly isolated PGP endophytic strain of Pseudomonas chlororaphis 23aP and factors affecting the process were evaluated. The data obtained showed that strain 23aP utilized PHE in a wide range of concentrations (6–100 ppm). Ethyl-acetate-extractable metabolites obtained from the PHE-enriched cultures were analyzed by gas chromatography–mass spectrometry (GC-MS) and thin-layer chromatography (HPTLC). The analysis identified phthalic acid, 3-(1-naphthyl)allyl alcohol, 2-hydroxybenzalpyruvic acid, α-naphthol, and 2-phenylbenzaldehyde, and allowed us to propose that the PHE degradation pathway of strain 23aP is initiated at the 1,2-, 3,4-carbon positions, while the 9,10-C pathway starts with non-enzymatic oxidation and is continued by the downstream phthalic pathway. Moreover, the production of the biosurfactants, mono- (Rha-C[sub.8]-C[sub.8], Rha-C[sub.10]-C[sub.8:1], Rha-C[sub.12:2]-C[sub.10], and Rha-C[sub.12:1]-C[sub.12:1]) and dirhamnolipids (Rha-Rha-C[sub.8]-C[sub.10]), was confirmed using direct injection–electrospray ionization–mass spectrometry (DI-ESI-MS) technique. Changes in the bacterial surface cell properties in the presence of PHE of increased hydrophobicity were assessed with the microbial adhesion to hydrocarbons (MATH) assay. Altogether, this suggests the strain 23aP might be used in bioaugmentation—a biological method supporting the removal of pollutants from contaminated environments.

The effectiveness of phytoremediation is closely related to the various interactions between pollutants, soil particles, rhizosphere microorganisms, and plants. Therefore, the object of current study was a cadmium-tolerant bacterium isolated from the rye rhizosphere, with a high degree of genetic similarity to the genus Chryseobacterium . Chryseobacterium sp. DEMBc1 was able to grow with 36 different BiologGN2 carbon sources and show the adaptation to stress factors such as Cd (100 μg ml −1 ), low temperature (8 °C), and salinity (2% NaCl). Furthermore, it was shown that DEMBc1 had the characteristics of plant growth–promoting microorganisms: it was able to produce ammonia, indole acetic acid, 1-aminocyclopropane-1-carboxylic acid deaminase, and siderophores, as well as solubilize Ca 3 (PO 4 ) 3 . After inoculation with DEMBc1, a significant decrease in the concentration of Cd was observed in the roots of Festuca ovina grown in Cd-polluted soil, compared to the non-inoculated Cd-polluted soil. It was also noticed that DEMBc1 produced a large amount of extracellular polymeric substances that were significantly higher than the cellular biomass. These polymers can form a barrier to reduce the translocation of Cd from the growth medium to the plant roots. According to the current study, DEMBc1 has a stabilizing potential and can decrease the mobility of Cd in the F. ovina rhizosphere, bioaccumulate metals in plant tissues, and effectively improve the bioavailability of nutrients, especially Fe, N, and P in a polluted environments. Graphical abstract

The competition potential of 14 Rhizobium leguminosarum bv. viciae isolates originating from nodules of Pisum sativum was estimated. Genotypic analyses of the isolates revealed a high level of chromosomal and plasmid content diversity. The isolates tagged with a plasmid-bearing constitutively expressed gusA gene were used to inoculate vetch (Vicia villosa) in competition experiments carried out under laboratory conditions. Soil extract containing autochthonous rhizobial population was used as competitor for gus-tagged strains, and the competition was studied by: (i) estimation of Gus⁺ root nodules on whole root systems, (ii) the pattern of individual nodule colonization by Gus⁺/Gus⁻ rhizobia, and (iii) the number of Gus⁺/Gus⁻ bacteria recovered from individual nodules. Several patterns of nodule colonization by Gus⁺/Gus⁻ bacteria were found. Some nodules identified as Gus⁺ contained gus-tagged bacteria only in the young and saprophytic zones, while the symbiotic zone was occupied by unmarked soil rhizobia. In other Gus⁺ nodules, despite the visible colonization of the entire nodule by gus-marked bacteria, a high number of Gus⁻ soil-derived rhizobia were recovered. The results suggest that rhizobial strains compete with each other also in the late stage of nodule development. Therefore, they may use different strategies to reach the late saprophytic zone of the nodule, which serves as an optimal environment for massive proliferation.