Marc Ongena

New linear lipopeptides produced by Pseudomonas cichorii SF1-54 are involved in virulence, swarming motility, and biofilm formation

Pseudomonas cichorii is the causal agent of lettuce midrib rot, characterized by a dark-brown to green-black discoloration of the midrib. Formation of necrotic lesions by several plant-pathogenic pseudomonads is associated with production of phytotoxic lipopeptides, which contribute to virulence. Therefore, the ability of P. cichorii SF1-54 to produce lipopeptides was investigated. A cell-free culture filtrate of SF1-54 showed surfactant, antimicrobial, and phytotoxic activities which are typical for lipopeptides. High-performance liquid chromatography analysis of P. cichorii SF1-54 culture filtrate revealed the presence of seven compounds with lipopeptide characteristics. Two related lipopeptides, named cichofactin A and B, were studied in more detail: they are linear lipopeptides with a decanoic and dodecanoic lipid chain, respectively, connected to the N-terminus of an eight-amino-acid peptide moiety. Both cichofactins are new members of the syringafactin lipopeptide family. Furthermore, two nonribosomal peptide synthethase-encoding genes, cifA and cifB, were identified as responsible for cichofactin biosynthesis. A cifAB deletion mutant no longer produced cichofactins and was impaired in swarming motility but showed enhanced biofilm formation. Upon spray inoculation on lettuce, the cichofactin-deficient mutant caused significantly less rotten midribs than the wild type, indicating that cichofactins are involved in pathogenicity of P. cichorii SF1-54. Further analysis revealed that P. cichorii isolates vary greatly in swarming motility and cichofactin production.

To settle or to move? The interplay between two classes of cyclic lipopeptides in the biocontrol strain Pseudomonas CMR12a

Pseudomonas CMR12a is a biocontrol strain that produces phenazine antibiotics and as yet uncharacterized cyclic lipopeptides (CLPs). The CLPs of CMR12a were studied by chemical structure analysis and in silico analysis of the gene clusters encoding the non-ribosomal peptide synthetases responsible for CLP biosynthesis. CMR12a produces two different classes of CLPs: orfamides B, D and E, whereby the latter two represent new derivatives of the orfamide family, and sessilins A-C. The orfamides are made up of a 10 amino acid peptide coupled to a β-hydroxydodecanoyl or β-hydroxytetradecanoyl fatty acid moiety, and are related to orfamides produced by biocontrol strain Pseudomonas protegens Pf-5. The sessilins consist of an 18-amino acid peptide linked to a β-hydroxyoctanoyl fatty acid and differ in one amino acid from tolaasins, toxins produced by the mushroom pathogen Pseudomonas tolaasii. CLP biosynthesis mutants were constructed and tested for biofilm formation and swarming motility. Orfamides appeared indispensable for swarming while sessilin mutants showed reduced biofilm formation, but enhanced swarming motility. The interplay between the two classes of CLPs fine tunes these processes. The presence of sessilins in wild type CMR12a interferes with swarming by hampering the release of orfamides and by co-precipitating orfamides to form a white line in agar.

Biosynthesis, Chemical Structure, and Structure-Activity Relationship of Orfamide Lipopeptides Produced by Pseudomonas protegens and Related Species

Orfamide-type cyclic lipopeptides (CLPs) are biosurfactants produced by Pseudomonas and involved in lysis of oomycete zoospores, biocontrol of Rhizoctonia and insecticidal activity against aphids. In this study, we compared the biosynthesis, structural diversity, in vitro and in planta activities of orfamides produced by rhizosphere-derived Pseudomonas protegens and related Pseudomonas species. Genetic characterization together with chemical identification revealed that the main orfamide compound produced by the P. protegens group is orfamide A, while the related strains Pseudomonas sp. CMR5c and CMR12a produce orfamide B. Comparison of orfamide fingerprints led to the discovery of two new orfamide homologs (orfamide F and orfamide G) in Pseudomonas sp. CMR5c. The structures of these two CLPs were determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis. Mutagenesis and complementation showed that orfamides determine the swarming motility of parental Pseudomonas sp. strain CMR5c and their production was regulated by luxR type regulators. Orfamide A and orfamide B differ only in the identity of a single amino acid, while orfamide B and orfamide G share the same amino acid sequence but differ in length of the fatty acid part. The biological activities of orfamide A, orfamide B, and orfamide G were compared in further bioassays. The three compounds were equally active against Magnaporthe oryzae on rice, against Rhizoctonia solani AG 4-HGI in in vitro assays, and caused zoospore lysis of Phytophthora and Pythium. Furthermore, we could show that orfamides decrease blast severity in rice plants by blocking appressorium formation in M. oryzae. Taken all together, our study shows that orfamides produced by P. protegens and related species have potential in biological control of a broad spectrum of fungal plant pathogens.

Role of phenazines and cyclic lipopeptides produced by pseudomonas sp CMR12a in induced systemic resistance on rice and bean

Pseudomonas sp. CMR12a produces two different classes of cyclic lipopeptides (CLPs) (orfamides and sessilins), which all play a role in direct antagonism against soilborne pathogens. Here we show that Pseudomonas sp. CMR12a is also able to induce systemic resistance to Magnaporthe oryzae on rice and to the web blight pathogen Rhizoctonia solani AG2-2 on bean. Plant assays with biosynthesis mutants of Pseudomonas sp. CMR12a impaired in the production of phenazines and/or CLPs and purified metabolites revealed that distinct bacterial determinants are responsible for inducing systemic resistance in these two pathosystems. In rice, mutants impaired in phenazine production completely lost their ability to induce systemic resistance, while a soil drench with pure phenazine-1-carboxamide (PCN) at a concentration of 0.1 or 1 μM was active in inducing resistance against M. oryzae. In bean, mutants that only produced phenazines, sessilins or orfamides were still able to induce systemic resistance against Rhizoctonia web blight, but a balanced production of these metabolites was needed. This study not only shows that Pseudomonas sp. CMR12a can protect rice to blast disease and bean to web blight disease, but also displays that the determinants involved in induced systemic resistance are plant, pathogen and concentration dependent.

Complete genome sequence of Bacillus amyloliquefaciens subsp. plantarum S499, a rhizobacterium that triggers plant defences and inhibits fungal phytopathogens

Bacillus amyloliquefaciens subsp. plantarum S499 is a plant beneficial rhizobacterium with a good antagonistic potential against phytopathogens through the release of active secondary metabolites. Moreover, it can induce systemic resistance in plants by producing considerable amounts of surfactins. The complete genome sequence of B. amyloliquefaciens subsp. plantarum S499 includes a circular chromosome of 3,927,922bp and a plasmid of 8,008bp. A remarkable abundance in genomic regions of putative horizontal origin emerged from the analysis. Furthermore, we highlighted the presence of genes involved in the establishment of interactions with the host plants at the root level and in the competition with other soil-borne microorganisms. More specifically, genes related to the synthesis of amylolysin, amylocyclicin, and butirosin were identified. These antimicrobials were not known before to be part of the antibiotic arsenal of the strain. The information embedded in the genome will support the upcoming studies regarding the application of B. amyloliquefaciens isolates as plant-growth promoters and biocontrol agents.

Comprehensive comparison of the chemical and structural characterization of landfill leachate and leonardite humic fractions

AbstractHumic substances (HS) are complex and heterogeneous mixtures of organic compounds that occur everywhere in the environment. They represent most of the dissolved organic matter in soils, sediments (fossil), water, and landfills. The exact structure of HS macromolecules has not yet been determined because of their complexity and heterogeneity. Various descriptions of HS are used depending on specific environments of origin and research interests. In order to improve the understanding of the structure of HS extracted from landfill leachate (LHS) and commercial HS from leonardite (HHS), this study sought to compare the composition and characterization of the structure of LHS and HHS using elemental composition, chromatographic (high-performance liquid chromatography (HPLC)), and spectroscopic techniques (UV–vis, FTIR, NMR, and MALDI-TOF). The results showed that LHS molecules have a lower molecular weight and less aromatic structure than HHS molecules. The characteristics of functional groups of both LHS and HHS, however, were basically similar, but there was some differences in absorbance intensity. There were also less aliphatic and acidic functional groups and more aromatic and polyphenolic compounds in the humic acid (HA) fraction than in the fulvic acid (FA) and other molecules (OM) fractions of both origins. The differences between LHS and HHS might be due to the time course of humification. Combining the results obtained from these analytical techniques cold improve our understanding of the structure of HS of different origins and thus enhance their potential use.n Graphical AbstractChemical and structural and characterization of landfill leachate and leonardite humic fractions

Characterization and Evaluation of the Potential of a Diesel-Degrading Bacterial Consortium Isolated from Fresh Mangrove Sediment

Hydrocarbons are ubiquitous and persistent organic pollutants in the environment. In wetlands and marine environments, particularly in mangrove ecosystems, their increase and significant accumulation result from human activities such as oil and gas exploration and exploitation operations. Remediation of these ecosystems requires the development of adequate and effective strategies. Natural attenuation, biostimulation, and bioaugmentation are all biological soil treatment techniques that can be adapted to mangroves. Our experiments were performed on samples of fresh mangrove sediments from the Cameroon estuary and mainly from the Wouri River in Cameroon. This study aims to assess the degradation potential of a bacterial consortium isolated from mangrove sediment. The principle of our bioremediation experiments is based on a series of tests designed to evaluate the potential of an active indigenous microflora and three exogenous pure strains, to degrade diesel with/without adding nutrients. The experiments were conducted in laboratory flasks and a greenhouse in microcosms. In one case, as in the other, the endogenous microflora showed that it was able to degrade diesel. Under stress of the pollutant, the endogenous microflora fits well enough in the middle to enable metabolism of the pollutant. However, the Rhodococcus strain was more effective over time. The degradation rate was 77 and 90xa0% in the vials containing the sterile sediments and non-sterile sediments, respectively. The results are comparable with those obtained in the microcosms in a greenhouse where only the endogenous microflora were used. The results of this study show that mangrove sediment contains an active microflora that can metabolize diesel. Indigenous and active microflora show an interesting potential for diesel degradation.

Characterization of Cichopeptins, New Phytotoxic Cyclic Lipodepsipeptides Produced by Pseudomonas cichorii SF1-54 and Their Role in Bacterial Midrib Rot Disease of Lettuce

The lettuce midrib rot pathogen Pseudomonas cichorii SF1-54 produces seven bioactive compounds with biosurfactant properties. Two compounds exhibited necrosis-inducing activity on chicory leaves. The structure of the two phytotoxic compounds, named cichopeptin A and B, was tentatively characterized. They are related cyclic lipopeptides composed of an unsaturated C12-fatty acid chain linked to the N-terminus of a 22-amino acid peptide moiety. Cichopeptin B differs from cichopeptin A only in the last C-terminal amino acid residue, which is probably Val instead of Leu/Ile. Based on peptide sequence similarity, cichopeptins are new cyclic lipopeptides related to corpeptin, produced by the tomato pathogen Pseudomonas corrugata. Production of cichopeptin is stimulated by glycine betaine but not by choline, an upstream precursor of glycine betaine. Furthermore, a gene cluster encoding cichopeptin synthethases, cipABCDEF, is responsible for cichopeptin biosynthesis. A cipA-deletion mutant exhibited significantly less virulence and rotten midribs than the parental strain upon spray inoculation on lettuce. However, the parental and mutant strains multiplied in lettuce leaves at a similar rate. These results demonstrate that cichopeptins contribute to virulence of P. cichorii SF1-54 on lettuce.

The cyclic lipopeptide orfamide induces systemic resistance in rice to Cochliobolus miyabeanus but not to Magnaporthe oryzae

Key messageThePseudomonas-derived cyclic lipopeptide orfamide can induce resistance toCochliobolus miyabeanus but not toMagnaporthe oryzae in rice. Abscisic acid signaling is involved in the induced systemic resistance response triggered by orfamide.AbstractDiverse natural products produced by beneficial Pseudomonas species have the potential to trigger induced systemic resistance (ISR) in plants, and thus may contribute to control of diseases in crops. Some beneficial Pseudomonas spp. can produce cyclic lipopeptides (CLPs), amphiphilic molecules composed of a fatty acid tail linked to an oligopeptide which is cyclized. CLPs can have versatile biological functions, but the capacity of Pseudomonas-derived CLPs in triggering ISR responses has barely been studied. Pseudomonas protegens and related species can produce orfamide-type CLPs. Here we show that in rice, orfamides can act as ISR elicitors against the necrotrophic fungus Cochliobolus miyabeanus, the causal agent of brown spot disease, but are not active against the blast fungus Magnaporthe oryzae. Orfamide A can trigger early defensive events and activate transcripts of defense-related genes in rice cell suspension cultures, but does not cause cell death. Further testing in rice cell suspension cultures and rice plants showed that abscisic acid signaling, the transcriptional activator OsWRKY4 and pathogenesis-related protein PR1b are triggered by orfamide A and may play a role in the ISR response against C. miyabeanus.

Change in ATP-binding cassette B1/19, glutamine synthetase and alcohol dehydrogenase gene expression during root elongation in Betula pendula Roth and Alnus glutinosa L. Gaertn in response to leachate and leonardite humic substances.

Humic substances (HS) are complex and heterogeneous compounds of humified organic matter resulting from the chemical and microbiological decomposition of organic residues. HS have a positive effect on plant growth and development by improving soil structure and fertility. They have long been recognized as plant growth-promoting substances, particularly with regard to influencing nutrient uptake, root growth and architecture. The biochemical and molecular mechanisms through which HS influence plant physiology are not well understood. This study evaluated the bioactivity of landfill leachate and leonardite HS on alder (Alnus glutinosa L. Gaertn) and birch (Betula pendula Roth) during root elongation inxa0vitro. Changes in root development were studied in relation to auxin, carbon and nitrogen metabolisms, as well as to the stress adaptive response. The cDNA fragments of putative genes encoding two ATP-binding cassette (ABC) transporters (ABCB1 and ABCB19) belonging to the B subfamily of plant ABC auxin transporters were cloned and sequenced. Molecular data indicate that HS and their humic acid (HA) fractions induce root growth by influencing polar auxin transport (PAT), as illustrated by the modulation of the ABCB transporter transcript levels (ABCB1 and ABCB19). There were also changes in alcohol dehydrogenase (ADH) and glutamine synthetase (GS) gene transcript levels in response to HS exposure. These findings confirmed that humic matter affects plant growth and development through various metabolic pathways, including hormonal, carbon and nitrogen metabolisms and stress response or signalization.