No abstract

Brazilian sugarcane has been shown to obtain part of its nitrogen via biological nitrogen fixation (BNF). Recent reports, based on the culture independent sequencing of bacterial nifH complementary DNA (cDNA) from sugarcane tissues, have suggested that members of the Bradyrhizobium genus could play a role in sugarcane-associated BNF. Here we report on the isolation of Bradyrhizobium spp. isolates and a few other species from roots of sugarcane cultivar RB867515 by two cultivation strategies: direct isolation on culture media and capture of Bradyrhizobium spp. using the promiscuous legume Vigna unguiculata as trap-plant. Both strategies permitted the isolation of genetically diverse Bradyrhizobium spp. isolates, as concluded from enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR) fingerprinting and 16S ribosomal RNA, nifH and nodC sequence analyses. Several isolates presented nifH phylotypes highly similar to nifH cDNA phylotypes detected in field-grown sugarcane by a culture-independent approach. Four isolates obtained by direct plate cultivation were unable to nodulate V. unguiculata and, based on PCR analysis, lacked a nodC gene homologue. Acetylene reduction assay showed in vitro nitrogenase activity for some Bradyrhizobium spp. isolates, suggesting that these bacteria do not require a nodule environment for BNF. Therefore, this study brings further evidence that Bradyrhizobium spp. may play a role in sugarcane-associated BNF under field conditions.

The diversity of nitrogen-fixing bacteria was assessed in the rhizospheres of two cultivars of sorghum (IS 5322-C and IPA 1011) sown in Cerrado soil amended with two levels of nitrogen fertilizer (12 and 120 kg ha(-1)). The nifH gene was amplified directly from DNA extracted from the rhizospheres, and the PCR products cloned and sequenced. Four clone libraries were generated from the nifH fragments and 245 sequences were obtained. Most of the clones (57%) were closely related to nifH genes of uncultured bacteria. NifH clones affiliated with Azohydromonas spp., Ideonella sp., Rhizobium etli and Bradyrhizobium sp. were found in all libraries. Sequences affiliated with Delftia tsuruhatensis were found in the rhizosphere of both cultivars sown with high levels of nitrogen, while clones affiliated with Methylocystis sp. were detected only in plants sown under low levels of nitrogen. Moreover, clones affiliated with Paenibacillus durus could be found in libraries from the cultivar IS 5322-C sown either in high or low amounts of fertilizer. This study showed that the amount of nitrogen used for fertilization is the overriding determinative factor that influenced the nitrogen-fixing community structures in sorghum rhizospheres cultivated in Cerrado soil.

Forty-two strains representing the eight recognized nitrogen-fixing Paenibacillus species and 12 non-identified strains were examined by restriction fragment length polymorphism (RFLP) analysis of part of 16S and 23S rRNA genes amplified by polymerase chain reaction (PCR). Eleven different 16S rDNA genotypes were obtained from the combined data of RFLP analysis with four endonucleases and they were in agreement with the established taxonomic classification. Only one group of unclassified strains (Group I) was assigned in a separate genotype, suggesting they belong to a new species. Using the 23S PCR^RFLP method only six genotypes were detected, showing that this method is less discriminative than the 16S PCR^RFLP. Using the multilocus enzyme electrophoresis (MLEE) assay, the 48 strains tested could be classified into 35 zymovars. The seven enzymatic loci tested were polymorphic and the different profiles obtained among strains allowed the grouping of strains into 10 clusters. The PCR^RFLP methods together with the MLEE assay provide a rapid tool for the characterization and the establishment of the taxonomic position of isolates belonging to this nitrogen-fixing group, which shows a great potentiality in promoting plant growth.

Prevention or cure of different illnesses through the use of plant latex is a worldwide known concept. The antifungal activity of Hancornia speciosa latex has been observed against Candida albicans. However, H. speciosa latex is not a sterile plant exudate and secondary metabolites produced by bacteria could be involved in fungal inhibition. In the present study, the bacterial communities of the latex from three H. speciosa trees were characterized using traditional plating and molecular methods. Twelve strains isolated from the latex samples were clustered into four groups by amplified ribosomal DNA restriction analysis (ARDRA). One representative of each group was sequenced and they were identified as belonging to the genera Bacillus, Klebsiella, Enterobacter and Escherichia. None of the 12 isolates showed antifungal activity against C. albicans. A lack of a microbial origin for the antifungal properties of latex was noted. DGGE profiles generated from each of the three latex samples showed unique patterns. Sequencing of the DGGE bands demonstrated the affiliation with the genera Klebsiella, Pantoea, Enterobacter and Burkholderia. In addition, clone libraries were generated and the phylogenetic distribution of the 50 analyzed clones was similar to that obtained using DGGE. The presence of some potential pathogens should be considered before using H. speciosa latex in folk medicine.

Molecular approaches [PCR-denaturing gradient gel electrophoresis (DGGE)] were used to determine whether three different vetiver (Chrysopogon zizanioides) genotypes, commercially used in Brazil and considered economically important over the world, select specific bacterial populations to coexist in their rhizospheres. DGGE profiles revealed that the predominant rhizospheric bacterial community hardly varies regarding the vetiver genotype. Moreover, using traditional cultivation methods, bacterial strains were isolated from the different rhizospheres. Colonies presenting different morphologies (83) were selected for determining their potential for plant growth promotion. More than half of the strains tested (57.8%) were amplified by PCR using nifH-based primers, specific for the enzyme nitrogenase reductase. The production of siderophores was observed in 88% of the strains, while the production of antimicrobial substances was detected in only 14.5% of the isolates when Micrococcus sp. was used as the indicator strain. Production of indole-3-acetic acid and the solubilization of phosphate were observed in 55.4% and 59% of the isolates, respectively. In total, 44 strains (53%) presented at least three characteristics of plant growth promotion and were submitted to amplified ribosomal DNA restriction analysis. Twenty-four genetic groups were formed at 100% similarity and one representative of each group was selected for their identification by partial 16S rRNA gene sequencing. They were affiliated with the genera Acinetobacter, Comamonas, Chryseobacterium, Klebsiella, Enterobacter, Pantoea, Dyella, Burkholderia, or Pseudomonas. These strains can be considered of great importance as possible biofertilizers in vetiver.

No abstract

The mung bean has a great potential under tropical conditions given its high content of grain protein. Additionally, its ability to benefit from biological nitrogen fixation (BNF) through association with native rhizobia inhabiting nodule microbiome provides most of the nitrogen independence on fertilizers. Soil microbial communities which are influenced by biogeographical factors and soil properties, represent a source of rhizobacteria capable of stimulating plant growth. The objective of this study is to support selection of beneficial bacteria that form positive interactions with mung bean plants cultivated in tropical soils, as part of a seed inoculation program for increasing grain yield based on the BNF and other mechanisms. Two mung bean genotypes (Camaleão and Esmeralda) were cultivated in 10 soil samples. Nodule microbiome was characterized by next-generation sequencing using Illumina MiSeq 16S rRNA. More than 99% of nodule sequences showed similarity with Bradyrhizobium genus, the only rhizobial present in nodules in our study. Higher bacterial diversity of soil samples collected in agribusiness areas (MW_MT-I, II or III) was associated with Esmeralda genotype, while an organic agroecosystem soil sample (SE_RJ-V) showed the highest bacterial diversity independent of genotype. Furthermore, OTUs close to Bradyrhizobium elkanii have dominated in all soil samples, except in the sample from the organic agroecosystem, where just B. japonicum was present. Bacterial community of mung bean nodules is mainly influenced by soil pH, K, Ca, and P. Besides a difference on nodule colonization by OTU sequences close to the Pseudomonas genus regarding the two genotypes was detected too. Although representing a small rate, around 0.1% of the total, Pseudomonas OTUs were only retrieved from nodules of Esmeralda genotype, suggesting a different trait regarding specificity between macro- and micro-symbionts. The microbiome analysis will guide the next steps in the development of an inoculant for mung bean aiming to promote plant growth and grain yield, composed either by an efficient Bradyrhizobium strain on its own or co-inoculated with a Pseudomonas strain. Considering the results achieved, the assessment of microbial ecology parameters is a potent coadjuvant capable to accelerate the inoculant development process and to improve the benefits to the crop by soil microorganisms.