Microbiological quality analysis of inoculants based on Bradyrhizobium spp. and Azospirillum brasilense produced "on farm" reveals high contamination with non-target microorganisms.

The use of inoculants carrying diazotrophic and other plant growth-promoting bacteria plays an essential role in the Brazilian agriculture, with a growing use of microorganism-based bioproducts. However, in the last few years, some farmers have multiplied microorganisms in the farm, known as "on farm" production, including inoculants of Bradyrhizobium spp. for soybean (Glycine max L. Merrill.) and Azospirillum brasilense for corn (Zea mays L.) or co-inoculation in soybean. The objective was to assess the microbiological quality of such inoculants concerning the target microorganisms and contaminants. In the laboratory, 18 samples taken in five states were serial diluted and spread on culture media for obtaining pure and morphologically distinct colonies of bacteria, totaling 85 isolates. Molecular analysis based on partial sequencing of the 16S rRNA gene revealed 25 genera of which 44% harbor species potentially pathogenic to humans; only one of the isolates was identified as Azospirillum brasilense, whereas no isolate was identified as Bradyrhizobium. Among 34 isolates belonging to genera harboring species potentially pathogenic to humans, 12 had no resistance to antibiotics, six presented intrinsic resistance, and 18 presented non-intrinsic resistance to at least one antibiotic. One of the samples analyzed with a shotgun-based metagenomics approach to check for the microbial diversity showed several genera of microorganisms, mainly Acetobacter (~ 32% of sequences) but not the target microorganism. The samples of inoculants produced on farm were highly contaminated with non-target microorganisms, some of them carrying multiple resistances to antibiotics.

Impact of the ahas transgene for herbicides resistance on biological nitrogen fixation and yield of soybean.

Studies on the effects of transgenes in soybean [Glycine max (L.) Merr.] and the associated use of specific herbicides on biological nitrogen fixation (BNF) are still few, although it is important to ensure minimal impacts on benefits provided by the root-nodule symbiosis. Cultivance CV127 transgenic soybean is a cultivar containing the ahas gene, which confers resistance to herbicides of the imidazolinone group. The aim of this study was to assess the effects of the ahas transgene and of imidazolinone herbicide on BNF parameters and soybean yield. A large-scale set of field experiments was conducted, for three cropping seasons, at nine sites in Brazil, with a total of 20 trials. The experiment was designed as a completely randomized block with four replicates and the following treatments: (T1) near isogenic transgenic soybean (Cultivance CV127) + herbicide of the imidazolinone group (imazapyr); (T2) near isogenic transgenic soybean + conventional herbicides; and (T3) parental conventional soybean (Conquista) + conventional herbicides; in addition, two commercial cultivars were included, Monsoy 8001 (M-SOY 8001) (T4), and Coodetec 217 (CD 217) (T5). At the R2 growth stage, plants were collected and BNF parameters evaluated. In general, there were no effects on BNF parameters due to the transgenic trait or associated with the specific herbicide. Similarly, at the final harvest, no grain-yield effects were detected related to the ahas gene or to the specific herbicide. However, clear effects on BNF and grain yield were attributed to location and cropping season.

Genomic basis of broad host range and environmental adaptability of Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 which are used in inoculants for common bean (Phaseolus vulgaris L.).

BACKGROUND: Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 are α-Proteobacteria that establish nitrogen-fixing symbioses with a range of legume hosts. These strains are broadly used in commercial inoculants for application to common bean (Phaseolus vulgaris) in South America and Africa. Both strains display intrinsic resistance to several abiotic stressful conditions such as low soil pH and high temperatures, which are common in tropical environments, and to several antimicrobials, including pesticides. The genetic determinants of these interesting characteristics remain largely unknown. RESULTS: Genome sequencing revealed that CIAT 899 and PRF 81 share a highly-conserved symbiotic plasmid (pSym) that is present also in Rhizobium leucaenae CFN 299, a rhizobium displaying a similar host range. This pSym seems to have arisen by a co-integration event between two replicons. Remarkably, three distinct nodA genes were found in the pSym, a characteristic that may contribute to the broad host range of these rhizobia. Genes for biosynthesis and modulation of plant-hormone levels were also identified in the pSym. Analysis of genes involved in stress response showed that CIAT 899 and PRF 81 are well equipped to cope with low pH, high temperatures and also with oxidative and osmotic stresses. Interestingly, the genomes of CIAT 899 and PRF 81 had large numbers of genes encoding drug-efflux systems, which may explain their high resistance to antimicrobials. Genome analysis also revealed a wide array of traits that may allow these strains to be successful rhizosphere colonizers, including surface polysaccharides, uptake transporters and catabolic enzymes for nutrients, diverse iron-acquisition systems, cell wall-degrading enzymes, type I and IV pili, and novel T1SS and T5SS secreted adhesins. CONCLUSIONS: Availability of the complete genome sequences of CIAT 899 and PRF 81 may be exploited in further efforts to understand the interaction of tropical rhizobia with common bean and other legume hosts.