Search

The plant-beneficial bacterium Pseudomonas brassicacearum forms phenotypic variants in vitro as well as in planta during root colonization under natural conditions. Transcriptome analysis of typical phenotypic variants using microarrays containing coding as well as noncoding DNA fragments showed differential expression of several genes relevant to secondary metabolism and of the small RNA (sRNA) genes rsmX , rsmY , and rsmZ . Naturally occurring mutations in the gacS-gacA system accounted for phenotypic switching, which was characterized by downregulation of antifungal secondary metabolites (2,4-diacetylphloroglucinol and cyanide), indoleacetate, exoenzymes (lipase and protease), and three different N -acyl-homoserine lactone molecules. Moreover, in addition to abrogating these biocontrol traits, gacS and gacA mutations resulted in reduced expression of the type VI secretion machinery, alginate biosynthesis, and biofilm formation. In a gacA mutant, the expression of rsmX was completely abolished, unlike that of rsmY and rsmZ . Overexpression of any of the three sRNAs in the gacA mutant overruled the pleiotropic changes and restored the wild-type phenotypes, suggesting functional redundancy of these sRNAs. In conclusion, our data show that phenotypic switching in P. brassicacearum results from mutations in the gacS-gacA system.

To shed light on the genetic equipment of the beneficial plant-associated bacterium Pseudomonas brassicacearum , we sequenced the whole genome of the strain NFM421. Its genome consists of one chromosome equipped with a repertoire of factors beneficial for plant growth. In addition, a complete type III secretion system and two complete type VI secretion systems were identified. We report here the first genome sequence of this species.

International audience; Pseudomonas brassicacearum forms phenotypic variants in vitro as well as in planta during root colonization under natural conditions, leading to subpopulations (phase I and II cells) that differ in colony morphology and production of exoenzymes/secondary metabolites. The maximal concentration of cadmium allowing both variants growth was 25 muM; however, phase II cells accumulated fivefold higher Cd than phase I cells, even though both variants showed the same growth rate and kinetics, comprising a long stasis period (50 h). The whole transcriptome analysis of both variants in response to Cd was investigated using the home-made DNA microarrays. This analysis revealed completely different adaptation mechanisms developed by each variant to withstand and grow in the presence of the toxic. A re-organization of the cell wall to limit Cd entrance was noticed for phase I cells, as genes encoding levan exopolymers were downregulated at the expense of an upregulation of genes encoding alginate, and an upregulation of transporters such as cadA, and a downregulation of copper transporters. Phase II cells were unable to prevent Cd entrance and recruited genes under the control of oxyR and soxR regulation to face osmotic and oxidant stresses generated by Cd. Putrescine and spermidine metabolism appeared to play a central role in Cd tolerance. Microarray data were validated by biological analyses such as motility, oxidative stress assay, metabolite profiling with ICR-FT/MS and UPLC, capillary electrophoresis analysis of biogenic amines.

Plant Growth Promoting Rhizobacteria (PGPR) is a group of bacteria that has the ability to enhance plant growth and yield via various plant growth promoting substances as well as biofertilizers. Ten bacterial isolates screened from rhizosphere of maize growing at Indo-Gangetic plain of eastern Uttar Pradesh, India showed potential nutrients solubilization and plant growth promoting activities. Rhizobacterial were able to produce phytohormone auxin (IAA) in the tryptophan supplemented medium which showed maximum production by strain BHU8. Phosphate solubilization activity was highly observed in isolate BHU8 followed by BHU3, BHU9 and BHU10. Plant cell wall hydrolyzing enzymes (Cellulase and Pectinase) were expressed in BHU3, BHU8, BHU9 and BHU10 and among ten rhizobacterial strains only six strains (BHU1, BHU2, BHU3, BHU5, BHU8 and BHU10) produced siderophore. Based on 16S rDNA gene sequence strains BHU1, BHU2, BHU3, BHU8, BHU10 showed 99.6%, 99.8%, 99.8%, 99.7% and 99.9% sequence alignment respectively with Pseudomonas brassicacearum, Pseudomonas taiwanensis Pseudomonas aeruginosa, Pseudomonas saponiphila and Pseudomonas oryzihabitans, respectively whereas strains BHU4 and BHU5 showed 99.7% similarity with Rhizobium radiobacter and strains BHU6 and BHU9 showed 99.9% sequence aligned with Bacillus oceanisediminis and Bacillus flexus, respectively. Strain BHU7 exhibited 99.9% similarity with Enterobacter asburiae.

Particular bacterial strains in certain natural environments prevent infectious diseases of plant roots. How these bacteria achieve this protection from pathogenic fungi has been analysed in detail in biocontrol strains of fluorescent pseudomonads. During root colonization, these bacteria produce antifungal antibiotics, elicit induced systemic resistance in the host plant or interfere specifically with fungal pathogenicity factors. Before engaging in these activities, biocontrol bacteria go through several regulatory processes at the transcriptional and post-transcriptional levels.

International audience; Pseudomonas brassicacearum was isolated as a major root-colonizing population from Arabidopsis thaliana. The strain NFM421 of P. brassicacearum undergoes phenotypic variation during A. thaliana and Brassica napus root colonization in vitro as well as in soil, resulting in different colony appearance on agar surfaces. Bacteria forming translucent colonies (phase II cells) essentially were localized at the surface of young roots and root tips, whereas wild-type cells (phase I cells) were localized at the basal part of roots. The ability of phase II cells to spread and colonize new sites on root surface correlates with over-production of flagellin as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of surface proteins and microsequencing. Moreover, phase II cells showed a higher ability to swim and to swarm on semisolid agar medium. Phase I and phase II cells of P. brassicacearum NFM421 were tagged genetically with green fluorescent protein and red fluorescent protein. Confocal scanning laser microscopy was used to localize phase II cells on secondary roots and root tips of A. thaliana, whereas phase I cells essentially were localized at the basal part of roots. These experiments were conducted in vitro and in soil. Phenotypic variation on plant roots is likely to be a colonization strategy that may explain the high colonization power of P. brassicacearum.

A total of 301 strains of fluorescent pseudomonads previously characterized by conventional phenotypic and/or genomic taxonomic methods were analyzed through siderotyping, i.e., by the isoelectrophoretic characterization of their main siderophores and pyoverdines and determination of the pyoverdine-mediated iron uptake specificity of the strains. As a general rule, strains within a well-circumscribed taxonomic group, namely the species Pseudomonas brassicacearum , Pseudomonas fuscovaginae , Pseudomonas jessenii , Pseudomonas mandelii , Pseudomonas monteilii , “ Pseudomonas mosselii ,” “ Pseudomonas palleronii ,” Pseudomonas rhodesiae , “ Pseudomonas salomonii ,” Pseudomonas syringae , Pseudomonas thivervalensis , Pseudomonas tolaasii , and Pseudomonas veronii and the genomospecies FP1, FP2, and FP3 produced an identical pyoverdine which, in addition, was characteristic of the group, since it was structurally different from the pyoverdines produced by the other groups. In contrast, 28 strains belonging to the notoriously heterogeneous Pseudomonas fluorescens species were characterized by great heterogeneity at the pyoverdine level. The study of 23 partially characterized phenotypic clusters demonstrated that siderotyping is very useful in suggesting correlations between clusters and well-defined species and in detecting misclassified individual strains, as verified by DNA-DNA hybridization. The usefulness of siderotyping as a determinative tool was extended to the nonfluorescent species Pseudomonas corrugata , Pseudomonas frederiksbergensis , Pseudomonas graminis , and Pseudomonas plecoglossicida , which were seen to have an identical species-specific siderophore system and thus were easily differentiated from one another. Thus, the fast, accurate, and easy-to-perform siderotyping method compares favorably with the usual phenotypic and genomic methods presently necessary for accurate identification of pseudomonads at the species level.

Fifteen bacterial strains containing 1-aminocyclopropane-1-carboxylate (ACC) deaminase were isolated from the rhizoplane of pea (Pisum sativum L.) and Indian mustard (Brassica juncea L.) grown in different soils and a long-standing sewage sludge contaminated with heavy metals. The isolated strains were characterized and assigned to various genera and species, such as Pseudomonas brassicacearum, Pseudomonas marginalis, Pseudomonas oryzihabitans, Pseudomonas putida, Pseudomonas sp., Alcaligenes xylosoxidans, Alcaligenes sp., Variovorax paradoxus, Bacillus pumilus, and Rhodococcus sp. by determination of 16S rRNA gene sequences. The root elongation of Indian mustard and rape (Brassica napus var. oleifera L.) germinating seedlings was stimulated by inoculation with 8 and 13 isolated strains, respectively. The bacteria were tolerant to cadmium toxicity and stimulated root elongation of rape seedlings in the presence of 300 µM CdCl2 in the nutrient solution. The effect of ACC-utilising bacteria on root elongation correlated with the impact of aminoethoxyvinylglycine and silver ions, chemical inhibitors of ethylene biosynthesis. A significant improvement in the growth of rape caused by inoculation with certain selected strains was also observed in pot experiments, when the plants were cultivated in cadmium-supplemented soil. The biomass of pea cv. Sparkle and its ethylene sensitive mutant E2 (sym5), in particular, was increased through inoculation with certain strains of ACC-utilising bacteria in pot experiments in quartz sand culture. The beneficial effect of the bacteria on plant growth varied significantly depending on individual bacterial strains, plant genotype, and growth conditions. The results suggest that plant growth promoting rhizobacteria containing ACC deaminase are present in various soils and offer promise as a bacterial inoculum for improvement of plant growth, particularly under unfavourable environmental conditions.Key words: ACC deaminase, cadmium, ethylene, Indian mustard, pea, phytoremediation, rape, rhizobacteria.

Rhizobacteria closely related to two recently described species of pseudomonads, Pseudomonas brassicacearum and Pseudomonas thivervalensis , were isolated from two geographically distinct wheat field soils in South Australia. Isolation was undertaken by either selective plating or immunotrapping utilizing a polyclonal antibody raised against P. brassicacearum . A subset of 42 isolates were characterized by amplified 16S ribosomal DNA restriction analysis (ARDRA), BIOLOG analysis, and gas chromatography-fatty acid methyl ester (GC-FAME) analysis and separated into closely related phenetic groups. More than 75% of isolates tested by ARDRA were found to have >95% similarity to either Pseudomonas corrugata or P. brassicacearum-P. thivervalensis type strains, and all isolates had >90% similarity to either type strain. BIOLOG and GC-FAME clustering showed a >70% match to ARDRA profiles. Strains representing different ARDRA groups were tested in two soil types for biological control activity against the soilborne plant pathogen Gaeumannomyces graminis var. tritici , the causative agent of take-all of wheat and barley. Three isolates out of 11 significantly reduced take-all-induced root lesions on wheat plants grown in a red-brown earth soil. Only one strain, K208, was consistent in reducing disease symptoms in both the acidic red-brown earth and a calcareous sandy loam. Results from this study indicate that P. brassicacearum and P. thivervalensis are present in Australian soils and that a level of genetic diversity exists within these two novel species but that this diversity does not appear to be related to geographic distribution. The result of the glasshouse pot trial suggests that some isolates of these species may have potential as biological control agents for plant disease.

Bacteria isolates phenotypically related to Pseudomonas corrugata have frequently been isolated from the rhizosphere of Arabidopsis thaliana and Brassica napus grown on different soils. 16S rDNA (rrs) gene sequencing, DNA-DNA hybridization, biochemical characterization and siderophore typing showed that these isolates belong to two different species that are distinct from other species of the genus Pseudomonas, including P. corrugata. A description of properties of these two new species is given based on the study of 16 isolates. Proposed names are Pseudomonas brassicacearum (10 strains studied) and Pseudomonas thivervalensis (6 strains studied). The type strain of Pseudomonas brassicacearum is CFBP 11706T and that of Pseudomonas thivervalensis is CFBP 11261T.

Quantification of viable microorganisms is an important step in microbiological research as well as in microbial product formulation to develop biological control products or probiotics. Often, the efficiency of the resulting product is dependent on the microbial cell density and their viability, which may decrease over time. Commonly, the number of viable cells is determined by serial dilution and plating techniques or flow cytometry. In 2017, we developed a mathematical model for isothermal microcalorimetry (IMC) data analysis and showed that the new method allows for a more rapid quantification of viable fresh and freeze-dried anaerobic Lactobacillus reuteri cells than traditional viable count methods. This study developed the new method further by applying it to well-known aerophilic plant-beneficial microbial species (Pseudomonas brassicacearum, Bacillus amyloliquefaciens subsp. plantarum and Clonostachys rosea) used in biological control products. We utilized IMC to quantify viable cells in microbial pure cultures as well as when coated onto wheat seeds. The results from this study confirmed that thermal viable count methods are more rapid and sensitive than traditional viable count techniques. Most interestingly, a thermal viable count method was able to quantify microbes coated on seeds despite the presence of the natural microbiota of the seeds. Our results also showed that, in contrast to plating techniques for which clustered cells skew the results, IMC does not require single cells for accurate viable counts. Thermal viable count methods are novel methods for the rapid quantification of divergent bacterial and fungal species and enhance the speed, sensitivity, and accuracy of routine viable counts of pure cultures and controlled microbiomes such as plant seed coatings.

While the definition of a nanomaterial (NM) is mainly based on size, it is known that decreasing size can induce structural modifications to compensate for increased surface energy. Nevertheless, the influence of these structural modifications on NM toxicity, and in particular structural defects, is poorly studied mainly because of the difficulty in varying the crystallinity of a NM without changing any other morphological parameters. In this study, we used a single-walled alumino-germanate nanotube (Ge-imogolite) as a model, for which this can be achieved. Differences in toxicity of well-crystallized Ge-imogolite vs. Ge-imogolite presenting vacant sites towards the soil bacteria Pseudomonas brassicacearum were studied. Well crystallized tubes led to moderate toxicity attributed to a direct contact with the bacteria and the generation of reactive oxygen species, whereas tubes presenting vacant sites caused more severe toxic effects without any direct contact nor ROS generation. The bacterial growth inhibition in the presence of lacunar tubes was attributed to indirect mechanisms as their higher solubility leads to Al or Ge toxic ion release and/or to the retention of essential nutrients on the vacancies. This study highlights the close correlation between structural defects of a nanomaterial and the modulation of its mechanisms of toxicity.

The genus Pseudomonas comprises many known plant-associated microbes with plant growth promotion and disease suppression properties. Genome-based studies allow the prediction of the underlying mechanisms using genome mining tools and the analysis of the genes unique for a strain by implementing comparative genomics. Here, we provide the genome sequence of the strain Pseudomonas brassicacearum 3Re2-7, formerly known as P. trivialis and P. reactans, elucidate its revised taxonomic classification, experimentally verify the gene predictions by transcriptome sequencing, describe its genetic biocontrol potential and contextualize it to other known Pseudomonas biocontrol agents. The P. brassicacearum 3Re2-7 genome comprises a circular chromosome with a size of 6,738,544 bp and a GC-content of 60.83%. 6267 genes were annotated, of which 6113 were shown to be transcribed in rich medium and/or in the presence of Rhizoctonia solani. Genome mining identified genes related to biocontrol traits such as secondary metabolite and siderophore biosynthesis, plant growth promotion, inorganic phosphate solubilization, biosynthesis of lipo- and exopolysaccharides, exoproteases, volatiles and detoxification. Core genome analysis revealed, that the 3Re2-7 genome exhibits a high collinearity with the representative genome for the species, P. brassicacearum subsp. brassicacearum NFM421. Comparative genomics allowed the identification of 105 specific genes and revealed gene clusters that might encode specialized biocontrol mechanisms of strain 3Re2-7. Moreover, we captured the transcriptome of P. brassicacearum 3Re2-7, confirming the transcription of the predicted biocontrol-related genes. The gene clusters coding for 2,4-diacetylphloroglucinol (phlABCDEFGH) and hydrogen cyanide (hcnABC) were shown to be highly transcribed. Further genes predicted to encode putative alginate production enzymes, a pyrroloquinoline quinone precursor peptide PqqA and a matrixin family metalloprotease were also found to be highly transcribed. With this study, we provide a basis to further characterize the mechanisms for biocontrol in Pseudomonas species, towards a sustainable and safe application of P. brassicacearum biocontrol agents.