Aims: This work was conducted to identify the antifungal compounds produced by two previously isolated Bacillus sp. strains: ARP₂3 and MEP₂18. Both strains were subjected to further analysis to determine their taxonomic position and to identify the compounds responsible for their antifungal activity as well as to evaluate the efficiency of these strains to control sclerotinia stem rot in soybean. Methods and Results: The antifungal compounds were isolated by acid precipitation of cell-free supernatants, purified by RP-HPLC and then tested for antagonistic activity against Sclerotinia sclerotiorum. Mass spectra from RP-HPLC eluted fractions showed the presence of surfactin C₁₅, fengycins A (C₁₆–C₁₇) and B (C₁₆) isoforms in supernatants from strain ARP₂3 cultures, whereas the major lipopeptide produced by strain MEP₂18 was iturin A C₁₅. Alterations in mycelial morphology and sclerotial germination were observed in the presence of lipopeptides-containing supernatants from Bacillus strains cultures. Foliar application of Bacillus amyloliquefaciens strains on soybean plants prior to S. sclerotiorum infection resulted in significant protection against sclerotinia stem rot compared with noninoculated plants or plants inoculated with a nonlipopeptide-producing B. subtilis strain. Conclusions: Both strains, renamed as B. amyloliquefaciens ARP₂3 and MEP₂18, were able to produce antifungal compounds belonging to the cyclic lipopeptide family. Our data suggest that the foliar application of lipopeptide-producing B. amyloliquefaciens strains could be a promising strategy for the management of sclerotinia stem rot in soybean. Significance and Impact of the Study: Sclerotinia stem rot was ranked as one of the most severe soybean disease in Argentina and worldwide. The results of this study showed the potential of B. amyloliquefaciens strains ARP₂3 and MEP₂18 to control plant diseases caused by S. sclerotiorum.

Bacillus amyloliquefaciens MEP₂18 is an autochthonous bacterial isolate with antibacterial and antifungal activities against a wide range of phytopathogenic microorganisms. Cyclic lipopeptides (CLP), particularly fengycins, produced by this bacterium; are the main antimicrobial compounds responsible for the growth inhibition of phytopathogens. In this work, the CLP fraction containing fengycins with antibacterial activity was characterized by LC-ESI-MS/MS. In addition, the antibacterial activity of these fengycins was evaluated on the pathogens Xanthomonas axonopodis pv. vesicatoria (Xav), a plant pathogen causing the bacterial spot disease, and Pseudomonas aeruginosa PA01, an opportunistic human pathogen. In vitro inhibition assays showed bactericidal effects on Xav and PA01. Atomic force microscopy images revealed dramatic alterations in the bacterial surface topography in response to fengycins exposure. Cell damage was evidenced by a decrease in bacterial cell heights and the loss of intracellular content measured by potassium efflux assays. Furthermore, the viability of MRC-5 human normal lung fibroblasts was not affected by the treatment with fengycins. This study shows in vivo evidence on the less-known properties of fengycins as antibacterial molecules and leaves open the possibility of using this CLP as a novel antibiotic.

The interaction between Azospirillum brasilense and plants is not fully understood, although several bacterial surface components like exopolysaccharides (EPS), flagella, and capsular polysaccharides are required for attachment and colonization. While in other plant–bacteria associations (Rhizobium–legume, Pseudomonas–potato), lipopolysaccharides (LPS) play a key role in the establishment of an effective association, their role in the root colonization by Azospirillum had not been determined. In this study, we isolated a Tn5 mutant of A. brasilense Cd (EJ1) with an apparently modified LPS core structure, non-mucoid colony morphology, increased EPS production, and affected in maize root colonization. A 3790-bp region revealed the presence of three complete open reading frames designated rmlC, rmlB and rmlD. The beginning of a fourth open reading frame was found and designated rmlA. These genes are organized in a cluster which shows homology to the cluster involved in the synthesis of dTDP-rhamnose in other bacteria. Additionally, the analysis of the monosaccharide composition of LPSs showed a diminution of rhamnose compared to the wild-type strain.

Background: The bacterial Hfq protein is able to interact with diverse RNA molecules, including regulatory small non-coding RNAs (sRNAs), and thus it is recognized as a global post-transcriptional regulator of gene expression. Loss of Hfq has an extensive impact in bacterial physiology which in several animal pathogens influences virulence. Sinorhizobium meliloti is a model soil bacterium known for its ability to establish a beneficial nitrogen-fixing intracellular symbiosis with alfalfa. Despite the predicted general involvement of Hfq in the establishment of successful bacteria-eukaryote interactions, its function in S. meliloti has remained unexplored. Results: Two independent S. meliloti mutants, 2011-3.4 and 1021Δhfq, were obtained by disruption and deletion of the hfq gene in the wild-type strains 2011 and 1021, respectively, both exhibiting similar growth defects as free-living bacteria. Transcriptomic profiling of 1021Δhfq revealed a general down-regulation of genes of sugar transporters and some enzymes of the central carbon metabolism, whereas transcripts specifying the uptake and metabolism of nitrogen sources (mainly amino acids) were more abundant than in the wild-type strain. Proteomic analysis of the 2011-3.4 mutant independently confirmed these observations. Symbiotic tests showed that lack of Hfq led to a delayed nodulation, severely compromised bacterial competitiveness on alfalfa roots and impaired normal plant growth. Furthermore, a large proportion of nodules (55%-64%) elicited by the 1021Δhfq mutant were non-fixing, with scarce content in bacteroids and signs of premature senescence of endosymbiotic bacteria. RT-PCR experiments on RNA from bacteria grown under aerobic and microoxic conditions revealed that Hfq contributes to regulation of nifA and fixK1/K2, the genes controlling nitrogen fixation, although the Hfq-mediated regulation of fixK is only aerobiosis dependent. Finally, we found that some of the recently identified S. meliloti sRNAs co-inmunoprecipitate with a FLAG-epitope tagged Hfq protein. Conclusions: Our results support that the S. meliloti RNA chaperone Hfq contributes to the control of central metabolic pathways in free-living bacteria and influences rhizospheric competence, survival of the microsymbiont within the nodule cells and nitrogen fixation during the symbiotic interaction with its legume host alfalfa. The identified S. meliloti Hfq-binding sRNAs are predicted to participate in the Hfq regulatory network.