Pseudomonas chlororaphis metabolites as biocontrol promoters of plant health and improved crop yield.

The Pseudomonas fluorescens complex contains at least eight phylogenetic groups and each of these includes several bacterial species sharing ecological and physiological traits. Pseudomonas chlororaphis classified in a separate group is represented by three different subspecies that show distinctive traits exploitable for phytostimulation and biocontrol of phytopathogens. The high level of microbial competitiveness in soil as well as the effectiveness in controlling several plant pathogens and pests can be related to the P. chlororaphis ability to implement different stimulating and toxic mechanisms in its interaction with plants and the other micro- and macroorganisms. Pseudomonas chlororaphis strains produce antibiotics, such as phenazines, pyrrolnitrine, 2-hexyl, 5-propyl resorcinol and hydrogen cyanide, siderophores such as pyoverdine and achromobactine and a complex blend of volatile organic compounds (VOCs) that effectively contribute to the control of several plant pathogens, nematodes and insects. Phenazines and some VOCs are also involved in the induction of systemic resistance in plants. This complex set of beneficial strategies explains the high increasing interest in P. chlororaphis for commercial and biotechnological applications. The aim of this review is to highlight the role of the different mechanisms involved in the biocontrol activity of P. chlororaphis strains.

Modulation of Arabidopsis thaliana growth by volatile substances emitted by Pseudomonas and Serratia strains.

Many volatile compounds secreted by bacteria play an important role in the interactions of microorganisms, can inhibit the growth of phytopathogenic bacteria and fungi, can suppress or stimulate plant growth and serve as infochemicals presenting a new type of interspecies communication. In this work, we investigated the effect of total pools of volatile substances and individual volatile organic compounds (VOCs) synthesized by the rhizosphere bacteria Pseudomonas chlororaphis 449 and Serratia plymuthica IC1270, the soil-borne strain P. fluorescens B-4117 and the spoiled meat isolate S. proteamaculans 94 on Arabidopsis thaliana plants. We showed that total gas mixtures secreted by these strains during their growth on Luria-Bertani agar inhibited A. thaliana growth. Hydrogen cyanide synthesis was unnecessary for the growth suppression. A decrease in the inhibition level was observed for the strain P. chlororaphis 449 with a mutation in the gacS gene, while inactivation of the rpoS gene had no effect. Individual VOCs synthesized by these bacteria (1-indecene, ketones 2-nonanone, 2-heptanone, 2-undecanone, and dimethyl disulfide) inhibited the growth of plants or killed them. Older A. thaliana seedlings were more resistant to VOCs than younger seedlings. The results indicated that the ability of some volatiles emitted by the rhizosphere and soil bacteria to inhibit plant growth should be considered when assessing the potential of such bacteria for the biocontrol of plant diseases.

Pyoverdines Are Essential for the Antibacterial Activity of Pseudomonas chlororaphis YL-1 under Low-Iron Conditions.

Pseudomonas chlororaphis YL-1 has extensive antimicrobial activities against phytopathogens, and its genome harbors a pyoverdine (PVD) biosynthesis gene cluster. The alternative sigma factor PvdS in Pseudomonas aeruginosa PAO1 acts as a critical regulator in response to iron starvation. The assembly of the PVD backbone starts with peptide synthetase enzyme PvdL. PvdF catalyzes formylation of l-OH-Orn to produce l-N5-hydroxyornithine. Here, we describe the characterization of PVD production in YL-1 and its antimicrobial activity in comparison with that of its PVD-deficient ΔpvdS, ΔpvdF, and ΔpvdL mutants, which were obtained using a sacB-based site-specific mutagenesis strategy. Using in vitro methods, we examined the effect of exogenous iron under low-iron conditions and an iron-chelating agent under iron-sufficient conditions on PVD production, antibacterial activity, and the relative expression of the PVD transcription factor gene pvdS in YL-1. We found that strain YL-1, the ΔpvdF mutant, and the ΔpvdS(pUCP26-pvdS) complemented strain produced visible PVDs and demonstrated a wide range of inhibitory effects against Gram-negative and Gram-positive bacteria in vitro under low-iron conditions and that with the increase of iron, its PVD production and antibacterial activity were reduced. The antibacterial compounds produced by strain YL-1 under low-iron conditions were PVDs based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Moreover, the antibacterial activity observed in vitro was correlated with in vivo control efficacies of strain YL-1 against rice bacterial leaf blight (BLB) disease caused by Xanthomonas oryzae pv. oryzae. Collectively, PVDs are responsible for the antibacterial activities of strain YL-1 under both natural and induced low-iron conditions.IMPORTANCE The results demonstrated that PVDs are essential for the broad-spectrum antibacterial activities of strain YL-1 against both Gram-positive and Gram-negative bacteria under low-iron conditions. Our findings also highlight the effect of exogenous iron on the production of PVD and the importance of this bacterial product in bacterial interactions. As a biocontrol agent, PVDs can directly inhibit the proliferation of the tested bacteria in addition to participating in iron competition.

Abiotic stressors impact outer membrane vesicle composition in a beneficial rhizobacterium: Raman spectroscopy characterization.

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have roles in cell-to-cell signaling, biofilm formation, and stress responses. Here, the effects of abiotic stressors on OMV contents and composition from biofilm cells of the plant health-promoting bacterium Pseudomonas chlororaphis O6 (PcO6) are examined. Two stressors relevant to this root-colonizing bacterium were examined: CuO nanoparticles (NPs)-a potential fertilizer and fungicide- and H2O2-released from roots during plant stress responses. Atomic force microscopy revealed 40-300 nm diameter OMVs from control and stressed biofilm cells. Raman spectroscopy with linear discriminant analysis (LDA) was used to identify changes in chemical profiles of PcO6 cells and resultant OMVs according to the cellular stressor with 84.7% and 83.3% accuracies, respectively. All OMVs had higher relative concentrations of proteins, lipids, and nucleic acids than PcO6 cells. The nucleic acid concentration in OMVs exhibited a cellular stressor-dependent increase: CuO NP-induced OMVs > H2O2-induced OMVs > control OMVs. Biochemical assays confirmed the presence of lipopolysaccharides, nucleic acids, and protein in OMVs; however, these assays did not discriminate OMV composition according to the cellular stressor. These results demonstrate the sensitivity of Raman spectroscopy using LDA to characterize and distinguish cellular stress effects on OMVs composition and contents.

Impact of spontaneous mutations on physiological traits and biocontrol activity of Pseudomonas chlororaphis M71.

Three morphological mutants (M71a, M71b, M71c) of the antagonist Pseudomonas chlororaphis M71, naturally arose during a biocontrol trial against the phytopathogenic fungus Fusarium oxysporum f.sp. radicis-lycopersisci. In this study, the three mutants were investigated to elucidate their role in the biocontrol of plant pathogens. M71a and M71b phenotypes were generated by a mutation in the two-component system GacS/GacA. The mutation determined an increase in siderophore production and an impaired ability to release proteases, to swarm, to produce phenazine and AHLs and to colonize tomato roots. In vitro antagonistic activity against different plant pathogens was partially reduced in M71a, while M71b resulted effective only against Pythium ultimum. Biocontrol efficacy against Fusarium oxysporum f.sp. radicis-lycopersisci, was partially reduced in M71a and completely lost in M71b. M71c phenotype was impaired in swarming motility, did not produce biofilms and its antagonistic activity was similar to the parental M71 strain. M71c showed an enhanced ability to colonize tomato roots, on which its progeny in part reverted to the M71 parental phenotype. Volatile organic compounds (VOCs) emitted by all four strains, inhibited the growth of Clavibacter michiganensis subsp. michiganensis and Seiridium cardinale in vitro. Real-time screening of VOCs by PTR-MS combined with GC-MS analysis, showed that methanethiol was the main component of the blend produced by all four M71 strains. However, the emissions of hydrogen cyanide, dimethyl disulfide, 1,3-butadiene and acetone were significantly affected by the three different mutations. These findings highlight that the simultaneous presence of different M71 phenotypes may improve, through the integration of different mechanisms, the ecological fitness and biocontrol efficacy of P. chlororaphis M71.

Insights into plant-beneficial traits of probiotic Pseudomonas chlororaphis isolates.

Pseudomonas chlororaphis isolates have been studied intensively for their beneficial traits. P. chlororaphis species function as probiotics in plants and fish, offering plants protection against microbes, nematodes and insects. In this review, we discuss the classification of P. chlororaphis isolates within four subspecies; the shared traits include the production of coloured antimicrobial phenazines, high sequence identity between housekeeping genes and similar cellular fatty acid composition. The direct antimicrobial, insecticidal and nematocidal effects of P. chlororaphis isolates are correlated with known metabolites. Other metabolites prime the plants for stress tolerance and participate in microbial cell signalling events and biofilm formation among other things. Formulations of P. chlororaphis isolates and their metabolites are currently being commercialized for agricultural use.

Novel sodium alkyl-1,3-disulfates, anionic biosurfactants produced from microbial polyesters.

A sodium alkyl disulfate mixture (SADM) synthesised from microbially produced 3-hydroxy fatty acids methyl esters (HFAMEs), showed 13-fold surface tension decrease when compared with the reference surfactant sodium dodecyl sulfate (SDS). Polyhydroxyalkanoates, accumulated by bacteria intracellularly when supplied with a mixture of fatty acids derived from hydrolysed rapeseed oil, were isolated, depolymerised and methylated to produce HFAMEs in very high yield (90%). A sequential chemical reduction and sulfation of the HFAMEs produced the sodium alkyl disulfates in high yields (>65%). SADM performs also 1.3-times better than dodecyl (1,3) disulfate, in surface tension tests. SADM shows also the formation of a specific critical micelle concentration (CMC) at a concentration 21-fold lower than SDS. The wettability of the SADM mixture is similar to SDS but the foaming volume of SADM is 1.5-fold higher. The foam is also more stable with its volume decreasing 3 times slower over time compared to SDS at their respective CMC values. Established sulfation technologies in chemical manufacturing could use the 3-hydroxy fatty acids methyl esters moiety (3-HFAME) given its origin from rapeseed oil and the extra OH residue on 3-position in the molecule, which affords the opportunity to produce disulfate surfactants with a proven superior performance to monosulphated surfactants. Thus, not only addressing environmental issues by avoiding threats of deforestation and monocultivation associated with palm oil use but also achieve a higher performance with lower use of surfactants.

Volatile Organic Compounds Produced by Pseudomonas chlororaphis subsp. aureofaciens SPS-41 as Biological Fumigants To Control Ceratocystis fimbriata in Postharvest Sweet Potatoes.

The biocontrol activity and chemical composition of the volatile organic compounds (VOCs) produced by Pseudomonas chlororaphis subsp. aureofaciens SPS-41 were investigated. The VOCs inhibited mycelial growth and spore germination in Ceratocystis fimbriata, which causes black rot disease in sweet potato tuber roots (TRs) and showed wide-spectrum antifungal activity against several plant pathogenic fungi. A microscopic examination of C. fimbriata cells suggested morphological changes and a loss of cellular contents. Different inoculation strategies significantly affected the antifungal activity of the VOCs. In the volatile profile of SPS-41, the most abundant compound, 3-methyl-1-butanol, followed by phenylethyl alcohol and 2-methyl-1-butanol showed strong inhibition toward C. fimbriata. The weight loss rate and disease severity of the TRs were significantly reduced in response to the VOCs emitted by SPS-41. The results suggest that the VOCs produced by P. chlororaphis subsp. aureofaciens SPS-41 might constitute an attractive biological fumigant for controlling black rot disease in sweet potato TRs.

Demonstration of the adhesive properties of the medium-chain-length polyhydroxyalkanoate produced by Pseudomonas chlororaphis subsp. aurantiaca from glycerol.

Pseudomonas chlororaphis subsp. aurantiaca (DSM 19603) was grown on crude glycerol from biodiesel production to produce a medium-chain-length polyhydroxyalkanoate (mcl-PHA), composed of 3-hydroxydodecanoate (43 ±â€¯1.8 mol%), 3-hydroxydecanoate (29 ±â€¯3.1 mol%), 3-hydroxytetradecanoate (12 ±â€¯0.4 mol%), 3-hydroxyoctanoate (10 ±â€¯1.5 mol%) and 3-hydroxyhexanoate (6 ±â€¯0.3 mol%). The biopolymer had an average molecular weight of 1.1 × 105 Da, with a polydispersity index of 1.5, and was semi-crystalline, as shown by its crystallinity index of 37 ±â€¯0.2%. It had low melting (44 °C) and glass transition (-48 °C) temperatures, and was thermally stable up to 285 °C. The biopolymer films were elastic and translucid, were hydrophobic and presented relatively high permeability to oxygen and carbon dioxide. The films demonstrated to have good adhesion properties towards porcine skin and human skin. The tension (61.1 ±â€¯20.6 kPa) and shear (12.7 ±â€¯2.14 kPa) bond strength of the mcl-PHA for porcine skin suggest its potential as a biomaterial for the development of novel natural adhesives for wound closure or wound dressings.

Complete Genome Sequence of Pseudomonas chlororaphis subsp. aurantiaca Reveals a Triplicate Quorum-Sensing Mechanism for Regulation of Phenazine Production.

Pseudomonas chlororaphis subsp. aurantiaca StFRB508 regulates phenazine production through N-acyl-l-homoserine lactone (AHL)-mediated quorum sensing. Two sets of AHL-synthase and AHL-receptor genes, phzI/phzR and aurI/aurR, have been identified from the incomplete draft genome of StFRB508. In the present study, the complete genome of StFRB508, comprising a single chromosome of 6,997,933 bp, was sequenced. The complete genome sequence revealed the presence of a third quorum-sensing gene set, designated as csaI/csaR. An LC-MS/MS analysis revealed that StFRB508 produced six types of AHLs, with the most important AHL being N-(3-hydroxyhexanoyl)-l-homoserine lactone (3-OH-C6-HSL). PhzI mainly catalyzed the biosynthesis of 3-OH-C6-HSL, while AurI and CsaI catalyzed that of N-hexanoyl-l-homoserine lactone and N-(3-oxohexanoyl)-l-homoserine lactone, respectively. A mutation in phzI decreased phenazine production, whereas that in aurI or csaI did not. A phzI aurI csaI triple mutant (508ΔPACI) did not produce phenazine. Phenazine production by 508ΔPACI was stimulated by exogenous AHLs and 3-OH-C6-HSL exerted the strongest effects on phenazine production at the lowest concentration tested (0.1 µM). The plant protection efficacy of 508ΔPACI against an oomycete pathogen was lower than that of wild-type StFRB508. These results demonstrate that the triplicate quorum-sensing system plays an important role in phenazine production by and the biocontrol activity of StFRB508.

Structure of the O-specific polysaccharides of Pseudomonas chlororaphis subsp. chlororaphis UCM B-106.

O-specific polysaccharide was obtained from the lipopolysaccharide of Pseudomonas chlororaphis subsp. chlororaphis UCM B-106 and studied by composition analysis along with 1D and 2D (1)H and (13)C NMR spectroscopy. The polysaccharide was found to contain a derivative of pseudaminic acid (Pse) and the following structure of the trisaccharide repeating unit was established: →4)-ß-Psep5Ac7Hb-(2 â†’ 6)-ß-d-Galf-(1 â†’ 3)-ß-d-Galp-(1→ where Pse5Ac7Hb indicates 5-acetamido-3,5,7,9-tetradeoxy-7-[(R)-3-hydroxybutanoylamino]-l-glycero-l-manno-non-2-ulosonic acid.