Jean Luiz Simões de Araújo

Bradyrhizobium stylosanthis sp. nov., comprising nitrogen-fixing symbionts isolated from nodules of the tropical forage legume Stylosanthes spp.

The introduction of legumes and nitrogen-fixing bacteria in tropical areas under pasture is a key factor for improvement of soil fertility. However, there are still very few studies concerning the symbionts of tropical forage legumes. We performed a polyphasic study with three strains representing the genus Bradyrhizobium (BR 446T, BR 510 and BR 511) isolated from the tropical perennial forage legume of the genus Stylosanthes. On the basis of 16S rRNA gene sequences, the three strains showed highest similarity with B. huanghuaihaiense, and in the analysis of the intergenic transcribed spacer (ITS) they showed less than 93.4 % similarity to all described species of the genus Bradyrhizobium. Multilocus sequence analysis (MLSA) with three, four or five (dnaK, glnII, gyrB, recA and rpoB) housekeeping genes confirmed that the BR strains belong to a distinct clade, with <96.5 % nucleotide identity with other members of the genus Bradyrhizobium. Average nucleotide identity (ANI) of genome sequences between strain BR 446T and B.huanghuaihaiense was below the threshold for species circumscription (90.7 %). DNA-DNA hybridization resulted in ΔTm values over 6.7 °C with the most closely related species. Similarities among the BR strains and differences from other species were confirmed by rep-PCR analysis. Interestingly, the BR strains were grouped in the analysis of nifH and nodC genes, but showed higher similarity with B. iriomotense and B. manausense than with B.huanghuaihaiense, indicating a different evolutionary history for nitrogen-fixation genes. Morpho-physiological, genotypic and genomic data supported that these BR strains represent a novel species for which the name Bradyrhizobium stylosanthis sp. nov. is suggested. The type strain is BR 446T (=CNPSo 2823T=HAMBI 3668T=H-8T), isolated from Stylosanthes guianensis.

Bradyrhizobium sacchari sp. nov., a legume nodulating bacterium isolated from sugarcane roots

Members of the genus Bradyrhizobium are well-known as nitrogen-fixing microsymbionts of a wide variety of leguminous species, but they have also been found in different environments, notably as endophytes in non-legumes such as sugarcane. This study presents a detailed polyphasic characterization of four Bradyrhizobium strains (type strain BR 10280T), previously isolated from roots of sugarcane in Brazil. 16S rRNA sequence analysis, multilocus sequence analysis (MLSA) and analysis of the 16S-23S rRNA internal transcribed spacer showed that these strains form a novel clade close to, but different from B. huanghuaihaiense strain CCBAU 23303T. Average nucleotide identity (ANI) analyses confirmed that BR 10280T represents a novel species. Phylogenetic analysis based on nodC gene sequences also placed the strains close to CCBAU 23303T, but different from this latter strain, the sugarcane strains did not nodulate soybean, although they effectively nodulated Vigna unguiculata, Cajanus cajan and Macroptilium atropurpureum. Physiological traits are in agreement with the placement of the strains in the genus Bradyrhizobium as a novel species for which the name Bradyrhizobium sacchari sp. nov. is proposed.

Tn5 insertion in the tonB gene promoter affects iron-related phenotypes and increases extracellular siderophore levels in Gluconacetobacter diazotrophicus

Abstract TonB-dependent receptors in concert with the TonB–ExbB–ExbD protein complex are responsible for the uptake of iron and substances such as vitamin B12 in several bacterial species. In this study, Tn5 mutagenesis of the sugarcane endophytic bacterium Gluconacetobacter diazotrophicus led to the isolation of a mutant with a single Tn5-insertion in the promoter region of a tonB gene ortholog. This mutant, named Gdiaa31, displayed a reduced growth rate and a lack of response to iron availability when compared to the wild-type strain PAL5T. Several efforts to generate null-mutants for the tonB gene by insertional mutagenesis were without success. RT-qPCR analysis demonstrated reduced transcription of tonB in Gdiaa31 when compared to PAL5T. tonB transcription was inhibited in the presence of Fe3+ ions both in PAL5T and in Gdiaa31. In comparison with PAL5T, Gdiaa31 also demonstrated decreased nitrogenase activity and biofilm formation capability, two iron-requiring physiological characteristics of G. diazotrophicus. Additionally, Gdiaa31 accumulated higher siderophore levels in culture supernatant. The genetic complementation of the Gdiaa31 strain with a plasmid that carried the tonB gene including its putative promoter region (pPtonB) restored nitrogenase activity and siderophore accumulation phenotypes. These results indicate that the TonB complex has a role in iron/siderophore transport and may be essential in the physiology of G. diazotrophicus.

PCR assay for direct specific detection of Bradyrhizobium elite strain BR 3262 in root nodule extracts of soil-grown cowpea

AimsSuccessful inoculation of legume crops with rhizobia depends on dominating nodule occupancy with highly efficient strains. The aim of this study was to develop a rapid and reliable conventional PCR methodology to specifically detect an elite Bradyrhizobium strain in root nodule extracts from soil-grown cowpea plants.MethodsThe draft genome sequence of Bradyrhizobium pachyrhizi BR 3262 was compared to the closely related strain PAC 48T. BR 3262-specific regions were selected to design specific primer pairs, which were tested with respect to PCR amplification specificity and efficiency on extracted DNA, bacterial cells and root nodules from cowpea plants grown under gnotobiotic conditions and in soil.ResultsEleven designed primer pairs were specific for BR 3262 amplification and two of them (pairs 2645 and 2736) were highly sensitive and selected for further analyses. Experiments with gnotobiotic and soil-grown plants showed that both primer pairs were suitable to reliably determine nodule occupancy and confirmed the competitiveness of strain BR 3262 in natural soil.ConclusionsPrimer pairs 2645 and 2736 are novel tools to accompany the fate of strain BR 3262 in inoculation experiments of cowpea in soil. This strategy should be applicable to other rhizobium/legume symbioses in the field.

COMUNIDADE DE FUNGOS MICORRÍZICOS ARBUSCULARES: DIVERSIDADE, COMPOSIÇÃO E GLOMALINA EM ÁREA REVEGETADA COM SESBÂNIA

The community composition and diversity of arbuscular mycorrhizal fungi (AMF) can be affected by several factors, including the climate, soil biota and host plants. This study aimed to evaluate the influence of Sesbania virgata (SV) revegetation in pure and intercropped with plantation Eucalyptus camaldulensis (EC) and Acacia mangium (AM), in the composition and diversity of arbuscular mycorrhizal fungi (AMF), and the amount glomalin-related soil protein (GRSP) in an area damaged by clay extraction. The experimental design was a randomized block with four treatments (planting pure SV - 100SV); consortium of SV + EC - 50SV: 50EC; consortium AM SV + - 50SV: 50AM) and degraded area with natural vegetation - ADVE) and three replications. Revegetation of mining digging clay with SV in pure or intercropped planting reduced the abundance of spores and increased species diversity of arbuscular mycorrhizal fungi. Furthermore, increased the amount of glomalin-related soil protein compared with the degraded area and spontaneous vegetation.