Genomic identification and characterization of the elite strains Bradyrhizobium yuanmingense BR 3267 and Bradyrhizobium pachyrhizi BR 3262 recommended for cowpea inoculation in Brazil

ABSTRACT The leguminous inoculation with nodule-inducing bacteria that perform biological nitrogen fixation is a good example of an "eco-friendly agricultural practice". Bradyrhizobium strains BR 3267 and BR 3262 are recommended for cowpea (Vigna unguiculata) inoculation in Brazil and showed remarkable responses; nevertheless neither strain was characterized at species level, which is our goal in the present work using a polyphasic approach. The strains presented the typical phenotype of Bradyrhizobium with a slow growth and a white colony on yeast extract-mannitol medium. Strain BR 3267 was more versatile in its use of carbon sources compared to BR 3262. The fatty acid composition of BR 3267 was similar to the type strain of Bradyrhizobium yuanmingense; while BR 3262 was similar to Bradyrhizobium elkanii and Bradyrhizobium pachyrhizi. Phylogenetic analyses based on 16S rRNA and three housekeeping genes placed both strains within the genus Bradyrhizobium: strain BR 3267 was closest to B. yuanmingense and BR 3262 to B. pachyrhizi. Genome average nucleotide identity and DNA-DNA reassociation confirmed the genomic identification of B. yuanmingense BR 3267 and B. pachyrhizi BR 3262. The nodC and nifH gene analyses showed that strains BR 3267 and BR 3262 hold divergent symbiotic genes. In summary, the results indicate that cowpea can establish effective symbiosis with divergent bradyrhizobia isolated from Brazilian soils.

Antagonistic endophytic bacteria associated with nodules of soybean (Glycine max L. ) and plant growth-promoting properties

Abstract A total of 276 endophytic bacteria were isolated from the root nodules of soybean (Glycine max L.) grown in 14 sites in Henan Province, China. The inhibitory activity of these bacteria against pathogenic fungus Phytophthora sojae 01 was screened in vitro. Six strains with more than 63% inhibitory activities were further characterized through optical epifluorescence microscopic observation, sequencing, and phylogenetic analysis of 16S rRNA gene, potential plant growth-promoting properties analysis, and plant inoculation assay. On the basis of the phylogeny of 16S rRNA genes, the six endophytic antagonists were identified as belonging to five genera: Enterobacter, Acinetobacter, Pseudomonas, Ochrobactrum, and Bacillus. The strain Acinetobacter calcoaceticus DD161 had the strongest inhibitory activity (71.14%) against the P. sojae 01, which caused morphological abnormal changes of fungal mycelia; such changes include fracture, lysis, formation of a protoplast ball at the end of hyphae, and split ends. Except for Ochrobactrum haematophilum DD234, other antagonistic strains showed the capacity to produce siderophore, indole acetic acid, and nitrogen fixation activity. Regression analysis suggested a significant positive correlation between siderophore production and inhibition ratio against P. sojae 01. This study demonstrated that nodule endophytic bacteria are important resources for searching for inhibitors specific to the fungi and for promoting effects for soybean seedlings.

Draft genome sequence of Bradyrhizobium manausense strain BR 3351T, an effective symbiont isolated from Amazon rainforest

ABSTRACT The strain BR 3351T (Bradyrhizobium manausense) was obtained from nodules of cowpea (Vigna unguiculata L. Walp) growing in soil collected from Amazon rainforest. Furthermore, it was observed that the strain has high capacity to fix nitrogen symbiotically in symbioses with cowpea. We report here the draft genome sequence of strain BR 3351T. The information presented will be important for comparative analysis of nodulation and nitrogen fixation for diazotrophic bacteria. A draft genome with 9,145,311 bp and 62.9% of GC content was assembled in 127 scaffolds using 100 bp pair-end Illumina MiSeq system. The RAST annotation identified 8603 coding sequences, 51 RNAs genes, classified in 504 subsystems.

Genome sequence of Bradyrhizobium embrapense strain CNPSo 2833T, isolated from a root nodule of Desmodium heterocarpon

Abstract Bradyrhizobium embrapense CNPSo 2833T is a nitrogen-fixing symbiont of the legume pasture Desmodium. Its draft genome contains 8,267,832 bp and 7876 CDSs. The symbiotic island includes nodulation and nitrogen fixation genes resembling the operon organization of B. japonicum. Several CDSs related to secretion proteins and stress tolerance were also identified.

Nitrogen-fixing bacteria and arbuscular mycorrhizal fungi in Piptadenia gonoacantha ( Mart. ) Macbr

Abstract The family Leguminosae comprises approximately 20,000 species that mostly form symbioses with arbuscular mycorrhizal fungi (AMF) and nitrogen-fixing bacteria (NFB). This study is aimed at investigating and confirming the dependence on nodulation and biological nitrogen fixation in the specie Piptadenia gonoacantha (Mart.) Macbr., which belongs to the Piptadenia group. Two consecutive experiments were performed in a greenhouse. The experiments were fully randomized with six replicates and a factorial scheme. For the treatments, the two AMF species and three NFB strains were combined to nodulate P. gonoacantha in addition to the control treatments. The results indicate this species’ capacity for nodulation without the AMF; however, the AMF + NFB combinations yielded a considerable gain in P. gonoacantha shoot weight compared with the treatments that only included inoculating with bacteria or AMF. The results also confirm that the treatment effects among the AMF + NFB combinations produced different shoot dry weight/root dry weight ratios. We conclude that AMF is not necessary for nodulation and that this dependence improves species development because plant growth increases upon co-inoculation.

Characterization of rhizobia isolates obtained from nodules of wild genotypes of common bean

Abstract This study aimed to evaluate the tolerance to salinity and temperature, the genetic diversity and the symbiotic efficiency of rhizobia isolates obtained from wild genotypes of common bean cultivated in soil samples from the States of Goiás, Minas Gerais and Paraná. The isolates were subjected to different NaCl concentrations (0%, 1%, 2%, 4% and 6%) at different temperatures (28 °C, 33 °C, 38 °C, 43 °C and 48 °C). Genotypic characterization was performed based on BOX-PCR, REP-PCR markers and 16S rRNA sequencing. An evaluation of symbiotic efficiency was carried out under greenhouse conditions in autoclaved Leonard jars. Among 98 isolates about 45% of them and Rhizobium freirei PRF81 showed a high tolerance to temperature, while 24 isolates and Rhizobium tropici CIAT899 were able to use all of the carbon sources studied. Clustering analysis based on the ability to use carbon sources and on the tolerance to salinity and temperature grouped 49 isolates, R. tropici CIAT899 and R. tropici H12 with a similarity level of 76%. Based on genotypic characterization, 65% of the isolates showed an approximately 66% similarity with R. tropici CIAT899 and R. tropici H12. About 20% of the isolates showed symbiotic efficiency similar to or better than the best Rhizobium reference strain (R. tropici CIAT899). Phylogenetic analysis of the 16S rRNA revealed that two efficient isolates (ALSG5A1 and JPrG6A8) belong to the group of strains used as commercial inoculant for common bean in Brazil and must be assayed in field experiments.

Initial pH of medium affects organic acids production but do not affect phosphate solubilization

The pH of the culture medium directly influences the growth of microorganisms and the chemical processes that they perform. The aim of this study was to assess the influence of the initial pH of the culture medium on the production of 11 low-molecular-weight organic acids and on the solubilization of calcium phosphate by bacteria in growth medium (NBRIP). The following strains isolated from cowpea nodules were studied: UFLA03-08 (Rhizobium tropici), UFLA03-09 (Acinetobacter sp.), UFLA03-10 (Paenibacillus kribbensis), UFLA03-106 (Paenibacillus kribbensis) and UFLA03-116 (Paenibacillus sp.). The strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 solubilized Ca3(PO4)2 in liquid medium regardless of the initial pH, although without a significant difference between the treatments. The production of organic acids by these strains was assessed for all of the initial pH values investigated, and differences between the treatments were observed. Strains UFLA03-09 and UFLA03-10 produced the same acids at different initial pH values in the culture medium. There was no correlation between phosphorus solubilized from Ca3(PO4)2 in NBRIP liquid medium and the concentration of total organic acids at the different initial pH values. Therefore, the initial pH of the culture medium influences the production of organic acids by the strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 but it does not affect calcium phosphate solubilization.

In vitro synthesis of Frankia and mycorrhiza with Casuarina equisetifolia and ultrastructure of root system.

Casuarina equisetifolia is one of the ecologically and economically important tropical coastal trees nodulated by nitrogen-fixing actinomycete Frankia and forming symbiotic associations with both ecto- and endomycorrhizal fungi. The present study aims at the ultrastructural study of interactions between C. equisetifolia, Frankia, and mycorrhiza. C. equisetifolia seeds were sterilised and germinated under in vitro condition. The seedlings were transferred to conical flasks containing vermiculite and saw dust with Hoagland's solution. After 30 days, the inoculum of AM fungus--Glomusfasciculatum (A), ectomycorrhizal fungus-Pisolithus tinctorius (E) and actinorhizal Frankia (F) were inoculated individually and in various combinations, (A+E), (A+F), E+F) and (A+E+F). After 90 days, the experimental plant roots and nodules were harvested for assessment of growth characters of mycorrhizal and actinorhizal association by light and scanning electron microscope methods. C. equisetifolia roots were infected with arbuscles and vesicles of G. fasciculatum; P. tinctorius formed fungal sheath but no Hartig net. Large number of cortical cells were seen infected with Frankia, hyphae of Frankia were frequently seen penetrating from cell to cell directly through cell walls and Frankia occupied majority of the cell volume.