Sonia Fischer

Biocontrol and PGPR Features in Native Strains Isolated from Saline Soils of Argentina

A bacterial collection of approximately one thousand native strains, isolated from saline soils of Cordoba province (Argentina), was established. From this collection, a screening to identify those strains showing plant growth promotion and biocontrol activities, as well as salt tolerance, was performed. Eight native strains tolerant to 1 M NaCl and displaying plant growth promotion and/or biocontrol features were selected for further characterization. Strains MEP2 18, MRP2 26, MEP2 11a, MEP3 1, and MEP3 3b significantly increased the growth of maize seedlings under normal and saline conditions, whereas isolates ARP2 3, AEP1 5, and ARP2 6 were able to increase the root dry weight of agropyre under saline conditions. On the other hand, strains MEP2 18 and ARP2 3 showed antagonistic activity against phytopathogenic fungi belonging to Sclerotinia and Fusarium genus. Antifungal activity was found in cell-free supernatants, and it was heat and protease resistant. Strains MEP218 and ARP23 were identified as Bacillus sp. and strains MEP211a and MEP33b as Ochrobactrum sp. according to the sequence analysis of 16S rRNA gene.

Effect of root exudates on the exopolysaccharide composition and the lipopolysaccharide profile of Azospirillum brasilense Cd under saline stress

The effect of wheat root exudates on the exopolysaccharide (EPS) composition and the lipopolysaccharide (LPS) profile of Azospirillum brasilense Cd under saline stress was studied. EPS of A. brasilense Cd was composed of glucose (47%), mannose (3%), xylose (4%), fucose (28%), rhamnose (6%), arabinose (1%) and galactose (11%). Under saline stress, A. brasilense produced a totally different EPS, composed mainly of galactose. Root exudates induced changes in A. brasilense EPS composition only under normal conditions, consisting of higher amounts of arabinose and xylose compared with EPS of bacteria grown without root exudates. No changes were induced by root exudates when A. brasilense was grown under saline stress. Additionally, root exudates induced changes in the LPS profile, both under normal and stress conditions.

Characterization of a phage-like pyocin from the plant growth-promoting rhizobacterium Pseudomonas fluorescens SF4c

R-type and F-type pyocins are high-molecular-mass bacteriocins produced by Pseudomonas aeruginosa that resemble bacteriophage tails. They contain no head structures and no DNA, and are used as defence systems. In this report, we show that Pseudomonas fluorescens SF4c, a strain isolated from the wheat rhizosphere, produces a high-molecular-mass bacteriocin which inhibits the growth of closely related bacteria. A mutant deficient in production of this antimicrobial compound was obtained by transposon mutagenesis. Sequence analysis revealed that the transposon had disrupted a gene that we have named ptm, since it is homologous to that encoding phage tape-measure protein in P. fluorescens Pf0-1, a gene belonging to a prophage similar to phage-like pyocin from P. aeruginosa PAO1. In addition, we have identified genes from the SF4c pyocin cluster that encode a lytic system and regulatory genes. We constructed a non-polar ptm mutant of P. fluorescens SF4c. Heterologous complementation of this mutation restored the production of bacteriocin. Real-time PCR was used to analyse the expression of pyocin under different stress conditions. Bacteriocin was upregulated by mitomycin C, UV light and hydrogen peroxide, and was downregulated by saline stress. This report constitutes, to our knowledge, the first genetic characterization of a phage tail-like bacteriocin in a rhizosphere Pseudomonas strain.

Genetic diversity and antifungal activity of native Pseudomonas isolated from maize plants grown in a central region of Argentina

Pseudomonas strains producing antimicrobial secondary metabolites play an important role in the biocontrol of phytopathogenic fungi. In this study, native Pseudomonas spp. isolates were obtained from the rhizosphere, endorhizosphere and bulk soil of maize fields in Córdoba (Argentina) during both the vegetative and reproductive stages of plant growth. However, the diversity based on repetitive-element PCR (rep-PCR) and amplified ribosomal DNA restriction analysis (ARDRA) fingerprinting was not associated with the stage of plant growth. Moreover, the antagonistic activity of the native isolates against phytopathogenic fungi was evaluated in vitro. Several strains inhibited members of the genera Fusarium, Sclerotinia or Sclerotium and this antagonism was related to their ability to produce secondary metabolites. A phylogenetic analysis based on rpoB or 16S rRNA gene sequences confirmed that the isolates DGR22, MGR4 and MGR39 with high biocontrol potential belonged to the genus Pseudomonas. Some native strains of Pseudomonas were also able to synthesise indole acetic acid and to solubilise phosphate, thus possessing potential plant growth-promoting (PGPR) traits, in addition to their antifungal activity. It was possible to establish a relationship between PGPR or biocontrol activity and the phylogeny of the strains. The study allowed the creation of a local collection of indigenous Pseudomonas which could be applied in agriculture to minimise the utilisation of chemical pesticides and fertilisers.

Colonization of wheat by Azospirillum brasilense Cd is impaired by saline stress.

The effect of saline stress on the colonization of wheat was analyzed by using Azospirillum brasilense Cd carrying the fusion of the reporter gene lacZ (β-galactosidase) with the N2 fixation gene promoter nifA. Colonization was also studied by inducing para-nodules on wheat roots using 2,4-D, establishing that these structures acted as bacterium protected niches. Bacteria grown under standard conditions were distributed along the whole root system, except the elongation zone, and colonized the para-nodules. Bacteria experiencing saline stress were mainly localized at the root tips and the lateral roots. In 2,4-D treated plants, most of the bacteria were present around the basal surface of the modified lateral root structures. Using the MPN method, there were not statistical differences between the numbers of control and stressed bacteria. As this method estimates endophytic colonization in contrast with the one using X-gal, which emphasizes colonization on the root surface, both procedures demonstrated to be necessary, concluding that salt treatment reduced surface colonization (X-gal) but not colonization inside the root. The bacterial counts made on inoculated wheat roots indicated higher numbers of both control and stressed bacteria in roots treated with 2,4-D compared with untreated roots.

A ptsP deficiency in PGPR Pseudomonas fluorescens SF39a affects bacteriocin production and bacterial fitness in the wheat rhizosphere

Pseudomonas fluorescens SF39a is a plant-growth-promoting bacterium isolated from wheat rhizosphere. In this report, we demonstrate that this native strain secretes bacteriocins that inhibit growth of phytopathogenic strains of the genera Pseudomonas and Xanthomonas. An S-type pyocin gene was detected in the genome of strain SF39a and named pys. A non-polar pys::Km mutant was constructed. The bacteriocin production was impaired in this mutant. To identify genes involved in bacteriocin regulation, random transposon mutagenesis was carried out. A miniTn5Km1 mutant, called P. fluorescens SF39a-451, showed strongly reduced bacteriocin production. This phenotype was caused by inactivation of the ptsP gene which encodes a phosphoenolpyruvate phosphotransferase (EI(Ntr)) of the nitrogen-related phosphotransferase system (PTS(Ntr)). In addition, this mutant showed a decrease in biofilm formation and protease production, and an increase in surface motility and pyoverdine production compared with the wild-type strain. Moreover, we investigated the ability of strain SF39a-451 to colonize the wheat rhizosphere under greenhouse conditions. Interestingly, the mutant was less competitive than the wild-type strain in the rhizosphere. To our knowledge, this study provides the first evidence of both the relevance of the ptsP gene in bacteriocin production and functional characterization of a pyocin S in P. fluorescens.

Inhibition of the phytopathogenic fungus Fusarium proliferatum by volatile compounds produced by Pseudomonas

The Fusarium head blight of grain cereals is a significant disease worldwide. In Argentina, high levels of contamination with Fusariumproliferatum have been found in crops. Many strains of the Pseudomonas genus antagonize the growth of fungi by different mechanisms, such as the production of antibiotics, siderophores, volatiles, and extracellular enzymes. In this work, we have designed a new system for studying the growth inhibition of F.proliferatum—namely by volatile compounds produced by Pseudomonas fluorescens MGR12. In both rich and minimal media, the bacterium released volatiles that negatively affected the mycelial growth of that phytopathogenic fungus. These bacterial compounds were analyzed by gas chromatography–mass spectrometry, but only a few could be identified by comparing their mass spectra with the libraries of the National Institutes of Standards and Technology MS search.

Fighting Plant Diseases Through the Application of Bacillus and Pseudomonas Strains

Plant growth-promoting bacteria (PGPBs) are capable of colonizing plants and influencing their growth by direct or indirect mechanisms. The direct mode of action occurs when metabolites or compounds synthesized by microorganisms are provided to the plant—for example, phytohormones—or when the bacteria facilitate the plant’s uptake of certain nutrients from the environment. In the indirect form of promotion, bacteria protect the plant against phytopathogenic organisms through the induction of systemic resistance and/or by the synthesis of antimicrobial compounds. The use of beneficial microorganisms as biopesticides offers a promising alternative to the use of chemical pesticides and an environmentally friendly strategy for agriculture. The PGPBs most studied and exploited as biocontrol agents are the species of Bacillus and fluorescent Pseudomonas. These strains produce a wide variety of metabolites involved in the biologic control of phytopathogenic fungi—for example, extracellular enzymes, siderophores, antibiotics, hydrogen cyanide, and volatile organic compounds, among others. Antibiosis is one mechanism of biologic control that is well characterized in Bacillus and Pseudomonas strains both genetically and biochemically. Among antibiotics identified in these two genera include the cyclic lipopeptides such surfactin, iturin, and fengycin in the bacilli and phenazines, 2,4-diacetylphloroglucinol, pyoluteorin, and pyrrolnitrin in the pseudomonads.

Mutation in a d-alanine–d-alanine ligase of Azospirillum brasilense Cd results in an overproduction of exopolysaccharides and a decreased tolerance to saline stress

Bacteria of the genus Azospirillum are free-living nitrogen-fixing, rhizobacteria that are found in close association with plant roots, where they exert beneficial effects on plant growth and yield in many crops of agronomic importance. Unlike other bacteria, little is known about the genetics and biochemistry of exopolysaccharides in Azospirillum brasilense. In an attempt to characterize genes associated with exopolysaccharides production, we generated an A. brasilense Cd Tn5 mutant that showed exopolysaccharides overproduction, decreased tolerance to saline conditions, altered cell morphology, and increased sensitivity to detergents. Genetic characterization showed that the Tn5 was inserted within a ddlB gene encoding for a d-alanine-d-alanine ligase, and located upstream of the ftsQAZ gene cluster responsible for cell division in different bacteria. Heterologous complementation of the ddlB Tn5 mutant restored the exopolysaccharides production to wild-type levels and the ability to grow in the presence of detergents, but not the morphology and growth characteristics of the wild-type bacteria, suggesting a polar effect of Tn5 on the fts genes. This result and the construction of a nonpolar ddlB mutant provide solid evidence of the presence of transcriptional coupling between a gene associated with peptidoglycan biosynthesis and the fts genes required to control cell division.

Draft Genome Sequences of Pseudomonas fluorescens Strains SF39a and SF4c, Potential Plant Growth Promotion and Biocontrol Agents

ABSTRACT Pseudomonas fluorescens SF4c and SF39a, strains isolated from wheat rhizosphere, have potential applications in plant growth promotion and biocontrol of fungal diseases of crop plants. We report the draft genome sequences of SF4c and SF39a with estimated sizes of 6.5 Mb and 5.9 Mb, respectively.