Pantoea trifolii sp. nov., a novel bacterium isolated from Trifolium rubens root nodules.

A novel bacterium, designated strain MMK2T, 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 MMK2T 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 MMK2T 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 MMK2T 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 MMK2T (= DSM 115063T = LMG 33049T).

Assessment of Phenanthrene Degradation Potential by Plant-Growth-Promoting Endophytic Strain Pseudomonas chlororaphis 23aP Isolated from Chamaecytisus albus (Hacq.) Rothm.

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.

Cadmium-resistant Chryseobacterium sp. DEMBc1 strain: characteristics and potential to assist phytoremediation and promote plant growth.

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 Ca3(PO4)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.

Genetic and physiological diversity of white Spanish broom (Chamaecytisus albus) endophytes.

Chamaecytisus albus (Spanish broom) is a legume shrub that can be found in only one natural locality in Poland. This specimen is critically endangered; therefore, different actions focusing on protection of this plant in the natural habitat are undertaken, and one of them involves studies of the population of Chamaecytisus albus bacterial endophytes, which in the future could be used as bioprotectants and/or biofertilizers. A collection of 94 isolates was obtained from Spanish broom nodules, and the physiological and genetic diversity of these strains was studied. A few potentially beneficial traits were detected, i.e. secretion of cellulases (66 isolates), production of siderophores (60 isolates), phosphate solubilization (25 isolates), and production of IAA (58 isolates), indole (16 isolates), or HCN (3 isolates). Twenty-nine of the 94 tested isolates were able to induce the development of root nodules in plants grown in vitro and can therefore be assumed as Chamaecytisus albus symbionts. Genome fingerprinting by BOX-PCR, as well as gyrB and nodZ gene sequencing revealed a great genetic diversity of specimens in the collection. The symbiotic isolates were classified in different clades, suggesting they could belong to different species, however, most of them revealed sequence similarity to Bradyrhizobium genus.

Host-dependent symbiotic efficiency of Rhizobium leguminosarum bv. trifolii strains isolated from nodules of Trifolium rubens.

Trifolium rubens L., commonly known as the red feather clover, is capable of symbiotic interactions with rhizobia. Up to now, no specific symbionts of T. rubens and their symbiotic compatibility with Trifolium spp. have been described. We characterized the genomic diversity of T. rubens symbionts by analyses of plasmid profiles and BOX-PCR. The phylogeny of T. rubens isolates was inferred based on the nucleotide sequences of 16S rRNA and two core genes (atpD, recA). The nodC phylogeny allowed classification of rhizobia nodulating T. rubens as Rhizobium leguminosarum symbiovar trifolii (Rlt). The symbiotic efficiency of the Rlt isolates was determined on four clover species: T. rubens, T. pratense, T. repens and T. resupinatum. We determined that Rlt strains formed mostly inefficient symbiosis with their native host plant T. rubens and weakly effective (sub-optimal) symbiosis with T. repens and T. pratense. The same Rlt strains were fully compatible in the symbiosis with T. resupinatum. T. rubens did not exhibit strict selectivity in regard to the symbionts and rhizobia closely related to Rhizobium grahamii, Rhizobium galegae and Agrobacterium radiobacter, which did not nodulate Trifolium spp., were found amongst T. rubens nodule isolates.

Diversity and plant growth promoting properties of rhizobia isolated from root nodules of Ononis arvensis.

This is the first report describing isolates from root nodules of Ononis arvensis (field restharrow). The aim of this investigation was to describe the diversity, phylogeny, and plant growth promoting features of microsymbionts of O. arvensis, i.e., a legume plant growing in different places of the southern part of Poland. Twenty-nine bacterial isolates were characterized in terms of their phenotypic properties, genome fingerprinting, and comparative analysis of their 16S rRNA, nodC and acdS gene sequences. Based on the nodC and 16S rRNA gene phylogenies, the O. arvensis symbionts were grouped close to bacteria of the genera Rhizobium and Mesorhizobium, which formed monophyletic clusters. The acdS gene sequences of all the isolates tested exhibited the highest similarities to the corresponding gene sequences of genus Mesorhizobium strains. The presence of the acdS genes in the genomes of rhizobia specific for O. arvensis implies that these bacteria may promote the growth and development of their host plant in stress conditions. The isolated bacteria showed a high genomic diversity and, in the BOX-PCR reaction, all of them (except three) exhibited DNA fingerprints specific only for them. Our studies showed that restharrow isolates formed effective symbiotic interactions with their native host (O. arvensis) and Ononis spinosa but not with Trifolium repens and Medicago sativa belonging to the same tribe Trifolieae as Ononis species and not with Lotus corniculatus, representing the tribe Loteae.

Electrophoretic profiles of lipopolysaccharides from Rhizobium strains nodulating Pisum sativum do not reflect phylogenetic relationships between these strains.

Rhizobia that nodulate peas comprise a heterogeneous group of bacteria. The aim of this study was to investigate the relationship between phylogeny and electrophoretic and hydroxy fatty acid lipopolysaccharide (LPS) profiles of pea microsymbionts. Based on amplified fragment length polymorphism (AFLP) fingerprinting data, the pea microsymbionts were grouped into two clusters distinguished at 58% similarity level. Based on the concatenated 16S rRNA, recA, and atpD housekeeping gene data, the microsymbionts appeared to be most closely related to Rhizobium leguminosarum biovars viciae and trifolii. Applying cluster analysis to their LPS electrophoretic profiles, the strains were assigned to two major groups with different banding patterns. All hydroxy fatty acids common to R. leguminosarum and R. etli were detected in each examined strain. Differences in the proportions of 3- to ω-1 hydroxy fatty acids allowed us to distinguish two groups of strains. This classification did not overlap with one based on LPS electrophoretic profiles. No clear correlation was apparent between the genetic traits and LPS profiles of the pea nodule isolates.

Purification and characterization of laccase from Sinorhizobium meliloti and analysis of the lacc gene.

The soil native bacterial strains were screened for laccase activity. Bacterial strain L3.8 with high laccase activity was identified as Sinorhizobium meliloti. The crude intracellular L3.8 enzyme extract was able to oxidize typical diagnostic substrates of plant and fungal laccases. Laccase L3.8 was purified 81-fold with a yield of 19.5%. The molecular mass of the purified bacterial laccase was found to be 70.0kDa and its pI was 4.77. UV-vis spectrum showed that L3.8 protein is a multicopper oxidase. The carbohydrate content of the purified enzyme was estimated at 3.2%. Moreover, the laccase active fraction was characterized in terms of kinetics, temperature, and pH optima as well as the effect of various chemical compounds on the laccase activity, and antioxidant properties, which indicated that the L3.8 laccase had unique properties that might be important in biotechnological applications. The lacc gene encoding S. meliloti laccase was cloned and characterized. The full-length sequence of 1950bp encoded a protein of 649 aa preceded by a signal peptide consisting of 26aa. Laccase L3.8 shared significant structural features characteristic of other laccases, including the conserved regions of four histidine-rich copper-binding sites. Potential biotechnological importance of a newly identified laccase is discussed.

Nodulation competitiveness of Ensifer meliloti alfalfa nodule isolates and their potential for application as inoculants.

Alfalfa (Medicago sativa) is a widely cultivated legume, which enters into nitrogen-fixing symbiosis with Ensifer (Sinorhizobium) spp. In this study, an autochthonous rhizobial population of Ensifer sp. occupying alfalfa nodules grown in arable soil was used as the basis for selection of potential inoculants. Alfalfa nodule isolates were identified as Ensifer meliloti by partial 16S rDNA, recA, atpD and nodC nucleotide sequencing. The sampled isolates displayed different symbiotic performance and diversity in the number of plasmids and molecular weight. Isolates that were the most efficient in symbiotic nitrogen fixation were tagged with a constitutively expressed gusA gene carried by a stable plasmid vector pJBA21Tc and used in competition experiments in soil under greenhouse conditions. Two E. meliloti strains LU09 and LU12, which effectively competed with indigenous soil rhizobia, were selected. The metabolic profiles of these selected strains showed differences in the use of carbon and energy sources. In addition, the LU09 strain exhibited bacteriocin production and LU12 mineral phosphate solubilization, which are valuable traits for soil survival. These strains may be considered as potential biofertilizers for alfalfa cultivation.

Rhizobium pisi sv. trifolii K3.22 harboring nod genes of the Rhizobium leguminosarum sv. trifolii cluster.

The taxonomic status of the Rhizobium sp. K3.22 clover nodule isolate was studied by multilocus sequence analysis (MLSA) of 16S rRNA and six housekeeping chromosomal genes, as well as by a subsequent phylogenic analysis. The results revealed full congruence with the Rhizobium pisi DSM 30132(T) core genes, thus supporting the same taxonomic position for both strains. However, the K3.22 plasmid symbiosis nod genes demonstrated high sequence similarity to Rhizobium leguminosarum sv. trifolii, whereas the R. pisi DSM 30132(T)nod genes were most similar to R. leguminosarum sv. viciae. The strains differed in the host range nodulation specificity, since strain K3.22 effectively nodulated red and white clover but not vetch, in contrast to R. pisi DSM 30132(T), which effectively nodulated vetch but was not able to nodulate clover. Both strains had the ability to form nodules on pea and bean but they differed in bean cultivar specificity. The R. pisi K3.22 and DSM 30132(T) strains might provide evidence for the transfer of R. leguminosarum sv. trifolii and sv. viciae symbiotic plasmids occurring in natural soil populations.

Competitiveness of Rhizobium leguminosarum bv. trifolii strains in mixed inoculation of clover (Trifolium pratense).

Rhizobium leguminosarum by. trifolii (Rlt) establishes beneficial root nodule symbiosis with clover. Twenty Rlt strains differentially marked with antibiotic-resistance markers were investigated in terms of their competitiveness and plant growth promotion in mixed inoculation of clover in laboratory experiments. The results showed that the studied strains essentially differed in competition ability. These differences seem not to be dependent on bacterial multiplication in the vicinity of roots, but rather on complex physiological traits that affect competitiveness. The most remarkable result of this study is that almost half of the total number of the sampled nodules was colonized by more than one strain. The data suggest that multi-strain model of nodule colonization is common in Rhizobium-legume symbiosis and reflects the diversity ofrhizobial population living in the rhizosphere.

Intragenomic diversity of Rhizobium leguminosarum bv. trifolii clover nodule isolates.

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.

Genetic and metabolic divergence within a Rhizobium leguminosarum bv. trifolii population recovered from clover nodules.

Rhizobia are able to establish symbiosis with leguminous plants and usually occupy highly complex soil habitats. The large size and complexity of their genomes are considered advantageous, possibly enhancing their metabolic and adaptive potential and, in consequence, their competitiveness. A population of Rhizobium leguminosarum bv. trifolii organisms recovered from nodules of several clover plants growing in each other's vicinity in the soil was examined regarding possible relationships between their metabolic-physiological properties and their prevalence in such a local population. Genetic and metabolic variability within the R. leguminosarum bv. trifolii strains occupying nodules of several plants was of special interest, and both types were found to be considerable. Moreover, a prevalence of metabolically versatile strains, i.e., those not specializing in utilization of any group of substrates, was observed by combining statistical analyses of Biolog test results with the frequency of occurrence of genetically distinct strains. Metabolic versatility with regard to nutritional requirements was not directly advantageous for effectiveness in the symbiotic interaction with clover: rhizobia with specialized metabolism were more effective in symbiosis but rarely occurred in the population. The significance of genetic and, especially, metabolic complexity of bacteria constituting a nodule population is discussed in the context of strategies employed by bacteria in competition.

Response to flavonoids as a factor influencing competitiveness and symbiotic activity of Rhizobium leguminosarum.

Flavonoids play a crucial role as signal molecules in promoting the formation of nodules by symbiotic bacteria commonly known as rhizobia. The early interaction between flavonoids and NodD regulatory protein activates nod gene transcription and the synthesis of Nod factor that initiates nodule primordium. In this study, we assessed response to flavonoids as factors influencing competitiveness of rhizobia and their symbiotic activity. Rhizobium leguminosarum nodule isolates belonging to three biovars, trifolii, viciae and phaseoli characterized earlier as competitive or uncompetitive relative to native rhizobia, were used. Investigating nodA promoter induction using plasmid lacZ fusion, we found that competitive strains more readily responded to a wide range of synthetic flavonoids and seed exudates in comparison to uncompetitive strains, albeit some exceptions were noticed. Of all the synthetic flavonoids and seed exudates studied, naringenin, hespertin and clover and vetch exudates were the most effective inducers of nodA promoter in competitive strains. Only one of the nine examined uncompetitive strains was highly induced by clover seed exudate. Subsequently, the effect of preinduction of R. leguminosarum bv. trifolii with clover exudate was assessed. Out of 18 pre-activated strains, nine strains (including competitive ones) increased clover wet mass of shoots and nodule number when used as inoculants. Our results demonstrate a plausible approach of isolating and characterizing flavonoid-responsive field isolates that could be further developed into relevant legume inoculants.

Pretreatment of clover seeds with nod factors improves growth and nodulation of Trifolium pratense.

The increase in legume production in sustainable agriculture depends not only on the effectiveness of the selected nitrogen-fixing inoculants but also on their competitiveness in a soil environment containing an indigenous rhizobial population. In this study, we investigated the effect of pretreatment of red clover seeds with specific Nod factor (LCOs) on germination, growth, and nodulation of clover growing under sterile conditions and in the soil. We demonstrated that, although the symbiotic ability and competitiveness of the inoculant strain RtKO17 was not improved under competitive soil conditions, LCOs treatment of clover seeds significantly enhanced clover nodulation and growth of plants.

Increased metabolic potential of Rhizobium spp. is associated with bacterial competitiveness.

Of 105 rhizobial isolates obtained from nodules of commonly cultivated legumes, we selected 19 strains on the basis of a high rate of symbiotic plant growth promotion. Individual strains within the species Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viciae, and Rhizobium etli displayed variation not only in plasmid sizes and numbers but also in the chromosomal 16S-23S internal transcribed spacer. The strains were tagged with gusA gene and their competitiveness was examined in relation to an indigenous population of rhizobia under greenhouse conditions. A group of 9 strains was thus isolated that were competitive in relation to native rhizobia in pot experiments. Nineteen selected competitive and uncompetitive strains were examined with respect to their ability to utilize various carbon and energy sources by means of commercial Biolog GN2 microplate test. The ability of the selected strains to metabolize a wide range of nutrients differed markedly and the competitive strains were able to utilize more carbon and energy sources than uncompetitive ones. A major difference concerned the utilization of amino and organic acids, which were metabolized by most of the competitive and only a few uncompetitive strains, whereas sugars and their derivatives were commonly utilized by both groups of strains. A statistically significant correlation between the ability to metabolize a broad range of substrates and nodulation competitiveness was found, indicating that metabolic properties may be an essential trait in determining the competitiveness of rhizobia.

The nadA gene of Pseudomonas fluorescens PGPR strain 267.1.

An insertion mutant of Pseudomonas fluorescens PGPR strain 267.1 was found to be auxotrophic for niacin (nicotinic acid) and could not synthesize quinolinic acid. The Tn5 interrupted gene was cloned and sequenced. The cloned fragment contained an open reading frame, nadA, capable of encoding a 359-amino-acid protein (39.0 kDa) with substantial identity to various bacterial quinolinate synthetases. The nadA gene complemented quinolinic acid synthesis deficiency and niacin auxotrophy of the P. fluorescens 106 P nadA mutant.