Selective enrichment of Pseudomonas spp. in the rhizoplane of different plant species

En contraste con la simbiosis entre rizobios y leguminosas, la especificidad de las pseudomonas en la colonización radicular parece menos estricta. Sin embargo, estudios sobre la diversidad bacteriana del nicho rizosférico resaltan la influencia de la especie vegetal en la selección específica de ciertos microorganismos a partir de la flora residente del suelo. Para evaluar el efecto que los cultivos extensivos de nuestro país tienen sobre la estructura de las comunidades de pseudomonas, se realizaron experimentos con plantas trampa, partiendo de semillas de trigo, maíz y soja, desinfectadas superficialmente y sembradas en un mismo suelo prístino. A partir de las suspensiones representativas de la microflora del rizoplano, se realizaron recuentos en placa en medio selectivo para pseudomonas. El conjunto de colonias de cada muestra se utilizó como fuente de ADN para analizar la estructura de comunidad a través del perfil de restricción de amplicones de los genes oprF y gacA. El análisis comparativo de estos perfiles agrupó a las muestras por especie de planta y las distinguió del patrón obtenido a partir del suelo prístino. La secuenciación parcial del gen 16S ADNr de aislamientos bacterianos representativos confirmó la existencia de genotipos enriquecidos diferencialmente en cada especie vegetal. Estos resultados apoyan la hipótesis de la existencia de mecanismos de selección específica de estirpes de pseudomonas a partir de la flora nativa del suelo en la interacción cooperativa entre estas PGPR y las raíces de diferentes cultivos como trigo, soja y maíz.In contrast to rhizobia-legume symbiosis, the specificity for root colonization by pseudomonads seems to be less strict. However, several studies about bacterial diversity in the rhizosphere highlight the influence of plant species on the selective enrichment of certain microorganisms from the bulk soil community. In order to evaluate the effect that different crops have on the structure of pseudomonad community on the root surface, we performed plant trap experiments, using surface-disinfected maize, wheat or soybean seeds that were sown in pots containing the same pristine soil as substrate. Rhizoplane suspensions were plated on a selective medium for Pseudomonas, and pooled colonies served as DNA source to carry out PCR-RFLP community structure analysis of the pseudomonads-specific marker genes oprF and gacA. PCR-RFLP profiles were grouped by plant species, and were distinguished from those of bulk soil samples. Partial sequencing of 16S rDNA genes of some representative colonies of Pseudomonas confirmed the selective enrichment of distinctive genotypes in the rhizoplane of each plant species. These results support the idea that the root systems of agricultural crops such as soybean, maize and wheat, select differential sets of pseudomonads from the native microbial repertoire inhabiting the bulk soil.Fil: Marrero, Mariana. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Agaras, Betina Cecilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Wall, Luis Gabriel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Valverde, Claudio Fabián. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

A black-pigmented pseudomonad isolate with antibacterial activity against phyllospheric pathogens

Bacteria of the Pseudomonas genus have been widely studied due to their antagonistic potential against a diverse group of fungal and bacterial phytopathogens, and their competence to colonize different plant tissues. We have isolated a rhizospheric pseudomonad that produced a black pigment, which is not a widespread trait within this genus. We confirmed that the isolate belonged to the P. putida complex through a MLSA analysis. We observed that the pigment synthesis was enhanced under high C:N ratios (25:1) and it was dependent of the carbon source, being maximized when we added glucose to M9. Besides, the supplementation of M9 with tryptophan inhibited the pigment production under C:N ratios of 4:1, and the addition of kojic acid reduced notably the pigment under favorable conditions. Ps. black presented several traits associated with plant-growth promoting potential with classical in vitro assays. Through a Tn5 mutagenesis approach, we found 2 representative clones, PB1 and PB5, that were consistently unable to produce the pigment under several growth conditions and were not altered in their in vitro probiotic traits. When comparing with PB1 and PB5 performances, we observed that the pigment gives Ps. black a higher tolerance to oxidative stress and UV radiation exposure. When confronting Ps. black with different bacterial phytopathogens, we demonstrated that Ps. black could inhibit in vitro the growth of Xanthomonas vesicatoria Bv5-4a, Pseudomonas syringae pv. tomato DC3000, P. syringae pv. syringae B728a, P. savastanoi pv. glycinea B076 and Clavibacter michiganensis subsp. michiganensis Cm9. Except for Psg B076, this antagonism was lost for PB1 and PB5 and when performing the test for Ps. black with tryptophan supplementation. Thus, we suggest that the pigment should be involved in the bacterial antagonisms, and that Ps black contains more than one antibacterial mechanism.Fil: Sosa, María Fernanda. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Centro de Bioquímica y Microbiología de Suelos. Laboratorio de Fisiología, Genética de Bacterias para Plantas; ArgentinaFil: Sobrero, Patricio Martín. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Centro de Bioquímica y Microbiología de Suelos. Laboratorio de Fisiología, Genética de Bacterias para Plantas; ArgentinaFil: Valverde, Claudio Fabián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Centro de Bioquímica y Microbiología de Suelos. Laboratorio de Fisiología, Genética de Bacterias para Plantas; ArgentinaFil: Agaras, Betina Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; Argentin

Pseudomonas spp. isolates with high phosphate-mobilizing potential and root colonization properties from agricultural bulk soils under no-till management

Seven phosphate-mobilizing pseudomonads were isolated, identified, and characterized in terms of their biofertilizer potential and root-colonizing properties. Pseudomonas protegens (ex-fluorescens) CHA0 was used for comparative purposes. Four isolates (LF-MB1, LF-P1, LF-P2, and LF-P3) clustered with members of the "Pseudomonas fluorescens complex," whereas the other three (LF-MB2, LF-V1, and LF-V2) clustered with members of the "Pseudomonas putida/Pseudomonas aeruginosa complex." Assays in buffered liquid growth medium supplemented with tricalcium phosphate enabled the separation of the isolates into two groups: group A (LF-P1, LF-P2, LF-P3, and LF-V1) solubilized P from 151 up to 182 μg mL -1, and group B (LF-MB1, LF-MB2, and LF-V2) solubilized less than 150 μg P mL -1. All isolates displayed acid and alkaline phosphatase activities. With the exception of LF-MB2, all isolates were able to degrade phospholipids from lecithin. Additionally, all isolates exhibited extracellular protease activity, and four isolates produced hydrogen cyanide, two traits that are related to biocontrol of phytopathogens. To study root colonization in non-sterile soil, isolates were doubly tagged with gfp and a tetracycline resistance cassette. After 15 days of competition with the indigenous bacterial flora, all tagged isolates colonized soybean roots at counts ranging from 7. 6 × 10 5 to 1. 7 × 10 7 CFU g -1. The results indicate that there are already efficient phosphate-mobilizing pseudomonads adapted to agricultural bulk soils under no-till management in Argentina and thus having excellent potential for use as biofertilizers.Fil: Fernandez, Leticia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Sur. Departamento de Agronomía; ArgentinaFil: Agaras, Betina Cecilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Zalba, Pablo. Universidad Nacional del Sur. Departamento de Agronomía; ArgentinaFil: Wall, Luis Gabriel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Valverde, Claudio Fabián. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

7-hydroxytropolone is the main metabolite responsible for the fungal antagonism of Pseudomonas donghuensis strain SVBP6

Pseudomonas donghuensis strain SVBP6, an isolate from an agricultural plot in Argentina, displays a broad-spectrum and diffusible antifungal activity, which requires a functional gacS gene but could not be ascribed yet to known secondary metabolites typical of Pseudomonas biocontrol species. Here, we report that Tn5 mutagenesis allowed the identification of a gene cluster involved in both the fungal antagonism and the production of a soluble tropolonoid compound. The ethyl acetate extract from culture supernatant showed a dose-dependent inhibitory effect against the phytopathogenic fungus Macrophomina phaseolina. The main compound present in the organic extract was identified by spectroscopic and X-ray analyses as 7-hydroxytropolone (7HT). Its structure and tautomerism was confirmed by preparing the two key derivatives 2,3-dimethoxy- and 2,7-dimethoxy-tropone. 7HT, but not 2,3- or 2,7-dimethoxy-tropone, mimicked the fungal inhibitory activity of the ethyl acetate extract from culture supernatant. The activity of 7HT, as well as its production, was barely affected by the presence of up to 50 μM added iron (Fe+2). To summarize, P. donghuensis SVBP6 produces 7HT under the positive control of the Gac-Rsm cascade and is the main active metabolite responsible for the broad-spectrum inhibition of different phytopathogenic fungi.Fil: Muzio, Federico Matías. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnologia. Centro de Bioquimica y Microbiologia de Suelos. Laboratorio de Fisiologia, Genetica de Bacterias Para Plantas.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Agaras, Betina Cecilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnologia. Centro de Bioquimica y Microbiologia de Suelos. Laboratorio de Fisiologia, Genetica de Bacterias Para Plantas.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Masi, Marco. Università degli Studi di Napoli Federico II; ItaliaFil: Tuzi, Angela. Università degli Studi di Napoli Federico II; ItaliaFil: Evidente, Antonio. Università degli Studi di Napoli Federico II; ItaliaFil: Valverde, Claudio Fabián. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnologia. Centro de Bioquimica y Microbiologia de Suelos. Laboratorio de Fisiologia, Genetica de Bacterias Para Plantas.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

A mutation in the glta gene from a native isolate of the Pseudomonas chlororaphis subgroup induces a phenotypic change associated with phenazine production

Members of the Pseudomonas genus colonise the rhizosphere of different plant species and display plant-probiotic traits. Among our collection of 19 isolates of native pseudomonads, isolate SPAN5 was obtained from a bulk soil sample of grasslands. This isolate, related with the Pseudomonas chlororaphis subgroup, showed several plant-growth promoting activities in vitro. We carried out Tn5 mutagenesis for identifying genes related to the biocontrol. We here report that the mutant clone SPAN5-135, which lost its orange pigmentation, suffered the insertion of Tn5 into the gltA gene encoding a type II citrate synthase (CS). The CS enzyme activity was reduced significantly in SPAN-135, as well as its phenazine production in several media. Also, siderophore and exoprotease secretion were affected. SPAN-135 could not recover the complete inhibition potential of the wild type version against the phytopathogenic fungi tested. This is the first report of the requirement of gltA for phenazine and siderophores production.Fil: Agaras, Betina Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; ArgentinaFil: Valverde, Claudio Fabián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; Argentin

A novel oligonucleotide pair for genotyping members of the pseudomonas genus by single-round PCR amplification of the gyrb gene

Pseudomonas is a phylogenetically diverse bacterial genus which is broadly distributed in different ecological niches, and whose taxonomy is continuously under revision. For that purpose, gyrB is one of the housekeeping genes routinely used for multilocus sequence analysis (MLSA). As we noticed that there was not a single primer pair available in the literature suitable for direct sequencing of this gene, we decided to design a unique oligonucleotide pair and to set up a polymerase chain reaction (PCR) protocol to obtain a single amplicon for the entire Pseudomonas genus. Based on the available gyrB sequence from 148 Pseudomonas species, we identified highly conserved regions to design oligonucleotides without fully degenerate positions. We then set up cycling conditions for achieving high specificity and yield of the PCR protocol. Then, we showed that the amplicons produced with this procedure were appropriate for direct sequencing with both primers, obtaining more than 95% of amplicons coverage. Finally, we demonstrated that a PCR-RFLP (restriction fragment length polymorphism) approach served to differentiate among Pseudomonas species, and even between members of the same species.Fil: Agaras, Betina Cecilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Valverde, Claudio Fabián. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Genomic insights into the broad antifungal activity, plant-probiotic properties, and their regulation, in Pseudomonas donghuensis strain SVBP6.

Plant-growth promotion has been linked to the Pseudomonas genus since the beginning of this research field. In this work, we mined the genome of an Argentinean isolate of the recently described species P. donghuensis. Strain SVBP6, isolated from bulk soil of an agricultural plot, showed a broad antifungal activity and several other plant-probiotic activities. As this species has been recently described, and it seems like some plant-growth promoting (PGP) traits do not belong to the classical pseudomonads toolbox, we decide to explore the SVBP6 genome via an bioinformatic approach. Genome inspection confirmed our previous in vitro results about genes involved in several probiotic activities. Other genetic traits possibly involved in survival of SVBP6 in highly competitive environments, such as rhizospheres, were found. Tn5 mutagenesis revealed that the antifungal activity against the soil pathogen Macrophomina phaseolina was dependent on a functional gacS gene, from the regulatory cascade Gac-Rsm, but it was not due to volatile compounds. Altogether, our genomic analyses and in vitro tests allowed the phylogenetic assignment and provided the first insights into probiotic properties of the first P. donghuensis isolate from the Americas

A CsrA/RsmA translational regulator gene encoded in the replication region of a Sinorhizobium meliloti cryptic plasmid complements Pseudomonas fluorescens rsmA/E mutants

Members of the CsrA/RsmA family are global regulatory proteins that bind to mRNAs, usually at the ribosome-binding site, to control mRNA translation and stability. Their activity is counteracted by small non-coding RNAs (sRNAs), which offer several binding sites to compete with mRNA binding. The csrA/rsmA genes are widespread in prokaryotic chromosomes, although certain phylogenetic groups such as Alphaproteobacteria lack this type of global regulator. Interestingly, a csrA/rsmA-like sequence was identified in the replication region of plasmid pMBA19a from the alphaproteobacterium Sinorhizobium meliloti. This rsmA-like allele (rsmASm ) is 58 % identical to Xanthomonas axonopodis pv. citri chromosomal rsmA and bears an unusual C-terminal extension that may fold into an extra α-helix. Homology-based modelling of RsmA Sm suggests that all key mRNA-binding residues are conserved and correctly positioned in the RNA-binding pocket. In fact, a 1.6 kb fragment from pMBA19a encompassing the rsmASm locus restored rsmA/E-dependent phenotypes of rsmA/E gacS Pseudomonas fluorescens mutants. The functionality of RsmA Sm was confirmed by the gain of control over target aprA′–′lacZ and hcnA′–′lacZ translational fusions in the same mutant background. The RsmA Sm activity correlated with Western blot detection of the polypeptide. Phenotype and translational fusion data from rsmA/E P. fluorescens mutants expressing RsmX/Y/Z RNAs indicated that RsmA Sm is able to bind these antagonistic sRNAs. In agreement with the latter observation, it was also found that the sRNA RsmY was stabilized by RsmA Sm . Deletion of the C-terminal extra α-helix of RsmA Sm affected its cellular concentration, but increased its relative RNA-binding activity. This is believed to be the first report of the presence and characterization of a functional csrA/rsmA homologue in a mobile genetic element.Fil: Agaras, Betina Cecilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sobrero, Patricio Martín. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Valverde, Claudio Fabián. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Specific enumeration and analysis of the community structure of culturable pseudomonads in agricultural soils under no-till management in Argentina

Members of the Pseudomonas genus have been isolated and identified worldwide as plant probiotic microorganisms. Little is known about the dynamics of pseudomonads population in extensive agricultural systems under the influence of biotic and abiotic factors. As part of the national consortium BIOSPAS devoted to study soil biology and its relationship with productivity, our lab began to characterize pseudomonad populations in agricultural soils under no-till management. We here report the set up and application of a combination of selective plating and PCR-RFLP analysis of the genus specific gacA and oprF genes, to quantify and study the community structure of culturable pseudomonads in bulk soil and rhizosphere samples. The specificity of both methods has been verified by 16S rDNA and oprF sequencing. The application of selective plating and gacA/oprF PCR-RFLP analyses revealed differences in the amount and predominant type of culturable pseudomonads among geographical locations, among treatments, and between rhizospheric and bulk soil. The results indicate that these simple methods prove useful to systematically monitor pseudomonads structure in bulk and rhizospheric soils along sampling periods covering crop rotations and agricultural practices at different locations.Fil: Agaras, Betina Cecilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Wall, Luis Gabriel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Valverde, Claudio Fabián. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin