Diversity Of Root Nodule Bacteria Research Articles

Phenotypic and genotypic diversity of root nodule bacteria from wild Lathyrus and Vicia species in Gaziantep, Turkey.

This study identified the phenotypic and genotypic characteristics of the bacteria that nodulate wild Lathyrus and Vicia species natural distribution in the Gaziantep province of Turkey. Principle component analysis of phenotypic features revealed that rhizobial isolates were highly resistant to stress factors such as high salt, pH and temperature. They were found to be highly sensitive to the concentrations (mg/mL) of the antibiotics neomycin 10, kanamycin, and tetracycline 5, as well as the heavy metals Ni 10, and Cu 10, and 5. As a result of REP-PCR analysis, it was determined that the rhizobial isolates were quite diverse, and 5 main groups and many subgroups being found. All of the isolates nodulating wild Vicia species were found to be related to Rhizobium sp., and these isolates were found to be in Clades II, III, IV, and V of the phylogenetic tree based on 16S rRNA. The isolates that nodulated wild Lathyrus species were in Clades I, II, IV, V, VI, VII, and VIII, and they were closely related to Rhizobium leguminasorum, Rhizobium sp., Phyllobacterium sp., Serratia sp., and Pseudomonas sp. According to the genetic analyses, the isolates could not be classified at the species level, the similarity ratio was low, they formed a distinct group that was supported by strong bootstrap values in the phylogenetic tree, and the differences discovered in the network analysis revealed the diversity among the isolates and gave important findings that these isolates may be new species.

Diversity of root nodule bacteria from leguminous crops

In the present study, a total of 353 nodule-associated bacteria were isolated from 220 legume plant samples belonging to Cicer arietinum (85), Glycine max (74), Vigna radiata (21) and Cajanus cajan (40). A total of 224 bacteria were identified as fast-growing Rhizobium spp. on the basis of differential staining (Gram staining and carbol fuchsin staining) and biochemical tests. All the isolates were tested for indole acetic acid production (IAA), phosphate solubilization and siderophore production on plate assay. To examine the effect of volatile organic metabolites (VOM) and water soluble soil components (WSSC) on nodule bacteria, culture conditions were optimized by observing the effects of various parameters such as pH, salt content and temperatures on the growth of bacteria. Selected rhizobia were subjected to random amplified polymorphic DNA (RAPD) and amplified ribosomal DNA restriction analysis (ARDRA) analysis to identify their species. On the basis of RAPD and ARDRA, 10 isolates were identified as Rhizobium meliloti. In this study, Rhizobium GO4, G16, G20, G77, S43, S81, M07, M37, A15 and A55 were observed as the best candidates among the tested bacteria and can be further used as potent bioinoculants.

Estimating Divergence Times and Substitution Rates in Rhizobia.

Accurate estimation of divergence times of soil bacteria that form nitrogen-fixing associations with most leguminous plants is challenging because of a limited fossil record and complexities associated with molecular clocks and phylogenetic diversity of root nodule bacteria, collectively called rhizobia. To overcome the lack of fossil record in bacteria, divergence times of host legumes were used to calibrate molecular clocks and perform phylogenetic analyses in rhizobia. The 16S rRNA gene and intergenic spacer region remain among the favored molecular markers to reconstruct the timescale of rhizobia. We evaluate the performance of the random local clock model and the classical uncorrelated lognormal relaxed clock model, in combination with four tree models (coalescent constant size, birth–death, birth–death incomplete sampling, and Yule processes) on rhizobial divergence time estimates. Bayes factor tests based on the marginal likelihoods estimated from the stepping-stone sampling analyses strongly favored the random local clock model in combination with Yule process. Our results on the divergence time estimation from 16S rRNA gene and intergenic spacer region sequences are compatible with age estimates based on the conserved core genes but significantly older than those obtained from symbiotic genes, such as nodIJ genes. This difference may be due to the accelerated evolutionary rates of symbiotic genes compared to those of other genomic regions not directly implicated in nodulation processes.

Ribosomal protein biomarkers provide root nodule bacterial identification by MALDI-TOF MS

Accurate identification of soil bacteria that form nitrogen-fixing associations with legume crops is challenging given the phylogenetic diversity of root nodule bacteria (RNB). The labor-intensive and time-consuming 16S ribosomal RNA (rRNA) sequencing and/or multilocus sequence analysis (MLSA) of conserved genes so far remain the favored molecular tools to characterize symbiotic bacteria. With the development of mass spectrometry (MS) as an alternative method to rapidly identify bacterial isolates, we recently showed that matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) can accurately characterize RNB found inside plant nodules or grown in cultures. Here, we report on the development of a MALDI-TOF RNB-specific spectral database built on whole cell MS fingerprints of 116 strains representing the major rhizobial genera. In addition to this RNB-specific module, which was successfully tested on unknown field isolates, a subset of 13 ribosomal proteins extracted from genome data was found to be sufficient for the reliable identification of nodule isolates to rhizobial species as shown in the putatively ascribed ribosomal protein masses (PARPM) database. These results reveal that data gathered from genome sequences can be used to expand spectral libraries to aid the accurate identification of bacterial species by MALDI-TOF MS.

Genetic diversity of root nodulating bacteria associated with Retama sphaerocarpa in sites with different soil and environmental conditions

The genetic diversity of root nodulating bacteria isolated from Retama sphaerocarpa was studied using BOX-A1R PCR and phylogenetic analysis of the 16S rRNA region, as well as the housekeeping genes atpD, glnII and recA. A total of 193 isolates were obtained from eight different sites with different soil and environmental conditions in the Iberian Peninsula. These isolates corresponded to 31 different strains that successfully nodulated R. sphaerocarpa seedlings in reinoculation trials. About one-third of the strains clustered with B. canariense or B. cytisi within Bradyrhizobium group I. The remaining strains clustered with B. elkanii/B. pachyrhizi within Bradyrhizobium group II or in separate clades that could represent new lineages. Based on the 16S rRNA and combined atpD+glnII+recA sequences, two to three lineages of root nodulating bacteria were found at each sampling site, except for Collado Garcia where five species were detected. B. canariense and B. elkanii/B. pachyrhizi were the most abundant species, whereas the least abundant were those related to B. retamae and a putative new lineage. B. canariense was found only in soils with neutral and acid pH, whereas B. retamae was the dominant species in alkaline soils.

Genetic diversity of root nodule bacteria nodulating Lotus corniculatus and Anthyllis vulneraria in Sweden

Very little is known about the genetic diversity and phylogeny of rhizobia nodulating Lotus species in northern temperate regions. We have therefore studied the genetic diversity among a total of 61 root nodule bacteria isolated from Lotus corniculatus and Anthyllis vulneraria from different geographic sites and habitats in Sweden by restriction fragment length polymorphism (RFLP) of the internal transcribed spacer between their 16S rRNA and 23S rRNA (IGS) region. A high diversity consisting of 26 IGS types from 54 L. corniculatus isolates and five IGS types from seven A. vulneraria isolates was found. The 16S rRNA sequences and phylogeny of representatives of the different IGS types showed four interesting exceptions from the majority of the isolates belonging to the genus Mesorhizobium: Two isolates were both found to be closely related to Rhodococcus spp., and two other isolates showed close relationship with Geobacillus spp. and Paenibacillus spp., respectively. The nodA sequences and phylogeny showed that all the isolates, including those not belonging to the traditional rhizobia genera, harbored nodA sequences which were typical of Mesorhizobium loti. Generally, the 16S rRNA and nodA phylogenetic trees were not congruent in that isolates with similar 16S rRNA sequences were associated with isolates harboring different nodA sequences. All the isolates were confirmed to nodulate L. corniculatus in an inoculation test. This is the first report of members of these non-rhizobia genera being able to nodulate legumes, and we suggest that they may have acquired their nodulating properties through lateral gene transfer.

Genetic Diversity of Rhizobia Associated with Acacia longifolia in Two Stages of Invasion of Coastal Sand Dunes

We examined the genetic diversity of root nodule bacteria associated with the Australian legume Acacia longifolia in two stages of invasion of a coastal sand dune system. All isolates belonged to the genus Bradyrhizobium. A higher diversity was found in the long-established trees. The results suggest the introduction of exotic bradyrhizobia with the plant.