WLIP, WLIPß, and WLIPγ Produced from Pseudomonas canadensis Q3-1 via Precursor-Directed Biosynthesis and Their Roles on Biocontrol of Phytophthora Blight in Peppers.

White line-inducing principle (WLIP, 1), together with two new cyclic lipopeptides (CLPs) WLIPß (2) and WLIPγ (3), were characterized from the supernatant of Pseudomonas canadensis Q3-1 via precursor-directed biosynthesis (PDB) in the current study. They were purified from the supernatant of P. canadensis Q3-1 by solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC), and their structures were mainly determined via bioinformatic analyses, spectrometric and spectroscopic techniques, as well as single crystal X-ray diffraction (XRD). These WLIPs share (R)-3-hydroxydecanoic acid (HDA), but they differ from each other in the composition of peptidic sequences. In addition, these CLPs showed biocontrol activities against Phytophthora blight (caused by Phytophthora capsici) in peppers. Collectively, this study has shown that PDB could be used for generating new CLPs in Pseudomonas spp. Moreover, we have confirmed that WLIP, WLIPß, and WLIPγ could be used as lead agrochemicals to control Phytophthora blight in peppers.

Characterization and Production of a Biosurfactant Viscosin from Pseudomonas sp. HN11 and its Application on Enhanced oil Recovery During oily Sludge Cleaning.

Biosurfactants are renewable resources with versatile applications on environmental bioremediation and industrial processes. Pseudomonas species are one of the promising biosurfactant producers. However, besides rhamnolipids, little is known about Pseudomonas-derived biosurfactants on solubilization of polycyclic aromatic hydrocarbons (PAHs) and oily sludge treatment. In this study, Pseudomonas sp. HN11-derived biosurfactant was purified by chromatographic methods and was characterized as viscosin via bioinformatic analysis, spectrometric and spectroscopic analyses, Marfey's method and (C-H)α NMR fingerprint matching approach. Viscosin is a potent biosurfactant with critical micelle concentration of 5.79 mg/L and is stable under various stresses. Moreover, viscosin was produced at 0.42 g/L at 48 h of liquid fermentation. Further data have shown that emulsifying agent viscosin is capable of promoting the solubilization of PAHs and displays enhanced oil recovery during oily sludge treatment. More specifically, viscosin has shown significantly enhanced solubilization on fluoranthene compared with control (0.04 mg/L), 2.21 mg/L and 1.27 mg/L fluoranthene was recovered from 100 mg/L and 200 mg/L viscosin treatment, respectively. However, only 200 mg/L viscosin has significantly enhanced the solubilization of phenanthrene (0.75 mg/L) and benzo[a]pyrene (0.51 mg/L) compared to each control (0.23 mg/L for phenanthrene and 0.09 mg/L for benzo[a]pyrene). Viscosin treatment of oily sludge (recovering of 0.58 g oil) has shown a significant oil recovery compared to that of control (recovering of 0.42 g oil). This study shows the great potential of viscosin-type biosurfactant on oily sludge treatment.

Pseudophomins A-D Produced from Pseudomonas sp. HN8-3 Using an OSMAC Approach and Their Roles in Biocontrol of Phytophthora capsici in Cucumbers.

In this study, two new cyclic lipopeptides (CLPs) pseudophomins C (3) and D (4) and two known CLPs pseudophomins A (1) and B (2) were produced and characterized from the bacterial supernatant of Pseudomonas sp. HN8-3 by an OSMAC (one strain-many compounds) approach. OSMAC is a strategy that involves feeding of a single microorganism with divergent substrates to stimulate the production of new secondary metabolites. These pseudophomins were purified and identified via chromatographic methods, droplet collapse assay, genome mining, spectroscopic and spectrometric analyses, and single-crystal X-ray diffraction (XRD). Moreover, bioactivity tests showed that pseudophomins could lyse the zoospores of Phytophthora capsici in vitro, and coapplication of pseudophomins with zoospores of P. capsici further reduced the incidence of P. capsici on cucumber leaves. Collectively, these results indicated that pseudophomins have the potential to be developed as biopesticides for controlling P. capsici in cucumber.

Genome mining and chemical characterization of a new cyclic lipopeptide associated with MDN-0066 from Pseudomonas moraviensis HN2 cultured in a valine-rich medium.

A new cyclic lipopeptide (CLP) MDN-0066-ß (1) and MDN-0066 (2) were isolated and characterized from the bacterial cultures of P. moraviensis HN2 in this study. The CLPs were purified by solid-phase extraction (SPE) and reversed-phase high performance liquid chromatography (RP-HPLC). Moreover, chemical structures of two CLPs were characterized by genome mining and analysis, nuclear magnetic resonance (NMR), high-resolution mass spectrometry (HR-MS), Marfey's method and (C-H)α NMR fingerprint matching approach. MDN-0066 (2) has an amino acid sequence of L-Leu1, D-Glu2, D-allo-Thr3, D-Leu4, D-Leu5, D-Ser6, L-Leu7, L-Ile8 linked to a saturated C10 ß-hydroxyl fatty acid moiety (R-configuration for 3-OH). The new CLP MDN-0066-ß (1) differs MDN-0066 (2) in the 8th position of L-valine in its peptide moiety, this variation in structure could be attributed to the supplement of L-valine in the cultural medium during liquid fermentation. Further antimicrobial tests showed that the two CLPs display moderate antagonistic activity against Staphylococcus aureus and Escherichia coli.

Analysis of the complete genome sequence of a rhizosphere-derived Pseudomonas sp. HN3-2 leads to the characterization of a cyclic lipopeptide-type antibiotic bananamide C.

A fluorescence and biosurfactant-producing strain HN3-2 was isolated from a rhizosphere soil sample of wheat plants and the chromosome of the strain HN3-2 was sequenced and was analyzed by multiple bioinformatics tools in this study. The genome size of the strain HN3-2 is 6,441,476 bp, with a GC content of 60.54%. 16Sr RNA-based phylogeny analysis showed that the strain HN3-2 belongs to Pseudomonas koreensis subgroup in Pseudomonas species. Preliminary data from genome mining have showed that the strain Pseudomonas sp. HN3-2 is capable of producing a peptide-type metabolite. Solid-phase extraction, reversed-phase high performance liquid chromatography (RP-HPLC) together with liquid chromatography-mass spectrometry, high-resolution mass spectrometry and tandem mass spectrometry analysis have led to the purification and identification of a cyclic lipopeptide (CLP) bananamide C (1) from the fermentative broth of the strain Pseudomonas sp. HN3-2. Moreover, the biological activity tests showed that banananmide 3 displays moderate antagonistic activity against Staphylococcus aureus and Escherichia coli. Collectively, these results provide the possibility of developing the CLP bananamide C as a drug leads for medical applications.

Isolation and characterization of a new cyclic lipopeptide surfactin from a marine-derived Bacillus velezensis SH-B74.

A marine-sediment-derived bacterium Bacillus velezensis SH-B74 can produce cyclic lipopeptides (CLPs). This study presented the isolation, characterization, and activity evaluation of a new CLP from the bacterial cultures of the strain SH-B74. Multiple chromatographic methods (solid-phase extraction and reversed-phase high-performance liquid chromatography) were applied to the purifying procedure of CLP, and the structural characterization of the new CLP was conducted by various spectroscopy (1D and 2D nuclear magnetic resonance together with Fourier transform infrared spectroscopy) and spectrometry (liquid chromatography-mass spectrometry, high-resolution mass spectrometry and tandem mass spectrometry) techniques as well as Marfey's method. The results displayed that the new CLP (anteiso-C15 Ile2,7 surfactin, 1) consists of a peptidic backbone of L-Glu1, L-Ile2, D-Leu3, L-Val4, L-Asp5, D-Leu6, L-Ile7, and an anteiso-C15 type saturated fatty acid chain. Further activity assay showed that the new CLP displays activity on the inhibition of the appressoria formation of rice blast causal pathogen Magnaporthe oryzae. To sum up, the results presented the perspective of potential application of the new CLP as a green agrichemical to control M. oryzae.

Isolation and characterization of a new cyclic lipopeptide orfamide H from Pseudomonas protegens CHA0.

A new cyclic lipopeptide (CLP) orfamide H (1) was purified and identified from the cultural broth of the bacterial strain Pseudomonas protegens CHA0. The crude extract of the strain CHA0 was obtained by an acid-aided precipitation process, then the compound 1 was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). Subsequently, the chemical structure of orfamide H was determined by 1D and 2D nuclear magnetic resonance (NMR) and mass spectrometry (MS). Further biological assays indicate that the new CLP orfamide H shows the activity on inhibiting the appressoria formation of the fungus Magnaporthe oryzae, the causal agent of the blast disease in rice. Taken all together, these results indicated that the new CLP orfamide H has the capacity to be developed as an agrichemical to control blast disease in rice.

Complete genome sequence of a marine-sediment-derived bacterial strain Bacillus velezensis SH-B74, a cyclic lipopeptides producer and a biopesticide.

A marine-sediment sample-derived strain Bacillus velezensis SH-B74 has the capacity to produce cyclic lipopeptides (CLPs), and these CLPs secreted by the strain show biological activities against various pests under both in vitro and in planta conditions, such evidence has supported that the strain SH-B74 is a biopesticide. To get a better insight into the mechanisms on the control of the pesticides by the strain, a genome sequencing project has been applied to the genomic DNA of the strain SH-B74. The results show that the strain SH-B74 has a chromosome size of 4,042,190 bp, with a GC content of 46.5%, in addition, the strain contains a 61,634 bp plasmid pSH-B74, with a GC content of 40.8%. Data from bioinformatic analysis reveal that the strain SH-B74 has genes with the capacity to increase environmental adaptation, promote the rhizosphere fitnesses and secrete a spectrum of antibiotics, including nonribosomal peptide synthetases (NRPSs)-derived CLPs bacillopeptin, plipastatin, and surfactin. The presence of CLPs in the bacterial cultures of the strain SH-B74 was confirmed further by LC-MS analysis. Thus, genome sequencing and analyses together with chemical analysis reveal the promising perspectives of the strain SH-B74 that are of spectacular importance to its trait as a plant beneficial microbe to be used in agriculture practices.

Plipastatin A1 produced by a marine sediment-derived Bacillus amyloliquefaciens SH-B74 contributes to the control of gray mold disease in tomato.

Certain Bacillus species have the capacity to produce cyclic lipopeptides and these lipopeptides are promising determinants contributing to the biocontrol of plant diseases. In the current study, a cyclic lipopeptide plipastatin A1 was isolated from the fermentation broth of a marine sediment-derived Bacillus amyloliquefaciens SH-B74 by the combination of solid-phase extraction and reversed-phase high-performance liquid chromatography, and its structure was identified by tandem mass spectrometry, high-resolution electro-spray ionization mass spectrometry, and gas chromatography-mass spectrometry together with nuclear magnetic resonance analysis. Moreover, data from activity evaluation revealed that plipastatin A1 has excellent in vitro activity on the suppression of the conidia germination of B. cinerea, the causal agent of gray mold disease in tomato. Furthermore, plipastatin A1 can successfully decrease the incidence of gray mold disease on tomato leaves at 50 µM concentration. This study indicates that B. amyloliquefaciens SH-B74 appears to be a potentially sustainable pesticide to control gray mold disease in tomato plants, and its cyclic lipopeptide plipastatin A1 plays an important role in the in vitro and in planta biocontrol of B. cinerea.

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

KEY MESSAGE: The Pseudomonas- derived cyclic lipopeptide orfamide can induce resistance to Cochliobolus miyabeanus but not to Magnaporthe oryzae in rice. Abscisic acid signaling is involved in the induced systemic resistance response triggered by orfamide. Diverse 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.

Antimicrobial and Insecticidal: Cyclic Lipopeptides and Hydrogen Cyanide Produced by Plant-Beneficial Pseudomonas Strains CHA0, CMR12a, and PCL1391 Contribute to Insect Killing.

Particular groups of plant-beneficial fluorescent pseudomonads are not only root colonizers that provide plant disease suppression, but in addition are able to infect and kill insect larvae. The mechanisms by which the bacteria manage to infest this alternative host, to overcome its immune system, and to ultimately kill the insect are still largely unknown. However, the investigation of the few virulence factors discovered so far, points to a highly multifactorial nature of insecticidal activity. Antimicrobial compounds produced by fluorescent pseudomonads are effective weapons against a vast diversity of organisms such as fungi, oomycetes, nematodes, and protozoa. Here, we investigated whether these compounds also contribute to insecticidal activity. We tested mutants of the highly insecticidal strains Pseudomonas protegens CHA0, Pseudomonas chlororaphis PCL1391, and Pseudomonas sp. CMR12a, defective for individual or multiple antimicrobial compounds, for injectable and oral activity against lepidopteran insect larvae. Moreover, we studied expression of biosynthesis genes for these antimicrobial compounds for the first time in insects. Our survey revealed that hydrogen cyanide and different types of cyclic lipopeptides contribute to insecticidal activity. Hydrogen cyanide was essential to full virulence of CHA0 and PCL1391 directly injected into the hemolymph. The cyclic lipopeptide orfamide produced by CHA0 and CMR12a was mainly important in oral infections. Mutants of CMR12a and PCL1391 impaired in the production of the cyclic lipopeptides sessilin and clp1391, respectively, showed reduced virulence in injection and feeding experiments. Although virulence of mutants lacking one or several of the other antimicrobial compounds, i.e., 2,4-diacetylphloroglucinol, phenazines, pyrrolnitrin, or pyoluteorin, was not reduced, these metabolites might still play a role in an insect background since all investigated biosynthetic genes for antimicrobial compounds of strain CHA0 were expressed at some point during insect infection. In summary, our study identified new factors contributing to insecticidal activity and extends the diverse functions of antimicrobial compounds produced by fluorescent pseudomonads from the plant environment to the insect host.

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.

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.

Production and characterization of surfactin-type lipopeptides as bioemulsifiers produced by a Pinctada martensii-derived Bacillus mojavensis B0621A.

Bacillus mojavensis B0621A was isolated from the mantle of a pearl oyster Pinctada martensii collected from South China Sea. Semi-purified surfactins (225 mg L(-1)) were obtained by acid precipitation and vacuum flash chromatography. The component of the semi-purified surfactins was preliminarily analyzed by liquid chromatography mass spectrometer system, and the results showed that all these surfactins could be a group of homologues. Eight surfactin homologues were isolated and afforded by reversed phase high-performance liquid chromatography. Furthermore, their structure was characterized by mass spectrometry analysis combined with nuclear magnetic resonance spectroscopy techniques. These surfactins shared seven amino acids as peptide backbone and a saturated ß-hydroxy fatty acid chain residue (from C13 to C15), differed each other from peptide sequence in the position of Leu7 or Val7. All these surfactins had significant activity and stability of emulsification under various pH (from 7.0 to 12.0), temperature range (from 20 to 115 °C) and sodium chloride concentration (from 2.5 to 20.0 %, w/v). Taken all together, these results indicated that B. mojavensis B0621A have potential to be an alternative source as a biological-derived emulsifying agent.

Interplay between orfamides, sessilins and phenazines in the control of Rhizoctonia diseases by Pseudomonas sp. CMR12a.

We investigated the role of phenazines and cyclic lipopeptides (CLPs) (orfamides and sessilins), antagonistic metabolites produced by Pseudomonas sp. CMR12a, in the biological control of damping-off disease on Chinese cabbage (Brassica chinensis) caused by Rhizoctonia solani AG 2-1 and root rot disease on bean (Phaseolus vulgaris L.) caused by R. solani AG 4-HGI. A Pseudomonas mutant that only produced phenazines suppressed damping-off disease on Chinese cabbage to the same extent as CMR12a, while its efficacy to reduce root rot on bean was strongly impaired. In both pathosystems, the phenazine mutant that produced both CLPs was equally effective, but mutants that produced only one CLP lost biocontrol activity. In vitro microscopic assays revealed that mutants that only produced sessilins or orfamides inhibited mycelial growth of R. solani when applied together, while they were ineffective on their own. Phenazine-1-carboxamide suppressed mycelial growth of R. solani AG 2-1 but had no effect on AG 4-HGI. Orfamide B suppressed mycelial growth of both R. solani anastomosis groups in a dose-dependent way. Our results point to an additive interaction between both CLPs. Moreover, phenazines alone are sufficient to suppress Rhizoctonia disease on Chinese cabbage, while they need to work in tandem with the CLPs on bean.

Production and characterization of Iturinic lipopeptides as antifungal agents and biosurfactants produced by a marine pinctada martensii-derived Bacillus mojavensis B0621A.

Bacillus mojavensis B0621A was isolated from a pearl oyster Pinctada martensii collected from South China Sea. While screening for cyclic lipopeptides potentially useful as lead compounds for biological control against soil-bone fungal plant pathogens, three lipopeptides were isolated and purified from the fermentation broth of B. mojavensis B0621A via vacuum flash chromatography coupled with reversed-phase high performance liquid chromatography (RP-HPLC). The structural characterization and identification of these cyclic lipopeptides were performed by tandem mass spectrometry (MS/MS) combined with gas chromatography-mass spectrometry (GC-MS) analysis as well as chemical degradation. These lipopeptides were finally characterized as homologues of mojavensins, which contained identical amino acids back bones of asparagine1, tyrosine2, asparagine3, glutamine4, proline5, asparagine6, and asparagine7 and differed from each other by their saturated ß-amino fatty acid chain residues, namely, iso-C14 mojavensin, iso-C16 mojavensin, and anteiso-C17 mojavensin, respectively. All lipopeptide isomers, especially iso-C16 mojavensin and anteiso-C17 mojavensin, displayed moderate antagonism and dose-dependent activity against several formae speciales of Fusarium oxysporum and presented surface tension activities. These properties demonstrated that the lipopeptides produced by B. mojavensis B0621A may be useful as biological control agent to fungal plant pathogens.

Isolation and characterization of a new iturinic lipopeptide, mojavensin A produced by a marine-derived bacterium Bacillus mojavensis B0621A.

Three lipopeptides were isolated by bioactivity-guided fractionation from the fermentation broth of Bacillus mojavensis B0621A. A new iturinic lipopeptide, named mojavensin A, was tentatively characterized by 1D, 2D NMR and MS spectroscopy, Marfey's method containing a novel peptide backbone of L-Asn1, D-Tyr2, D-Asn3, L-Gln4, L-Pro5, D-Asn6, L-Asn7 and an anteiso-type of the saturated ß-fatty acid side chain. Compound 2 and 3 were tentatively identified as iso-C16 fengycin B and anteiso-C17 fengycin B, respectively. These lipopeptides displayed dose-dependent antifungal activity against a broad spectra of phytopathogens and were weakly antagonistic to Staphylococcus aureus. Moreover, they all revealed cytotoxic activities against the human leukemia (HL-60) cell line. Mojavensin A, iso-C16 fengycin B, and anteiso-C17 fengycin B inhibited the growth of HL-60 with IC50 of 100, 100 and 1.6 µM, respectively.