Bacillus megaterium WL-3 Lipopeptides Collaborate Against Phytophthora infestans to Control Potato Late Blight and Promote Potato Plant Growth.
ABSTRACT: Oomycete Phytophthora infestans [(Mont.) de Bary] is the cause of potato late blight, a plant disease which poses a serious threat to our global food security and is responsible for huge economic losses worldwide. Lipopeptides produced by Bacillus species are known to be potent antibacterial compounds against many plant pathogens. In this study, we show that Bacillus megaterium WL-3 has an antagonistic effect against potato late blight. Electrospray ionization mass spectrometry (ESI-MS) revealed that lipopeptides derived from the WL-3 strain contained three subfamilies, surfactin (C13 - C15), Iturin A (C14 - C16), and Fengycin A (C15 - C19). The Iturin A and Fengycin A lipopeptide families were each confirmed to have anti-oomycete effects against P. infestans mycelium growth as well as obvious controlling effects against potato late blight in greenhouse experiments and field assays. Furthermore, Iturin A and Fengycin A were able to promote plant photosynthetic efficiency, plant growth, and potato yield. Most importantly, the combination of Iturin A and Fengycin A (I + F) was superior to individual lipopeptides in controlling potato late blight and in the promotion of plant growth. The results of this study indicate that B. megaterium WL-3 and its lipopeptides are potential candidates for the control of late blight and the promotion of potato plant growth.
Project description:Potato late blight, caused by Phytophthora infestans (Mont.) de Bary, represents a great food security threat worldwide and is difficult to control. Recently, Bacillus spp. have been considered biocontrol agents to control many plant diseases. Here, Bacillus subtilis WL-2 was selected as a potent strain against P. infestans mycelium growth, and its functional metabolite was identified as Iturin A via electrospray ionization mass spectrometry (ESI-MS). Analyses using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed that Iturin A caused cell membrane disruption and an irregular internal cell structure. In addition, Iturin A triggered oxidative stress reactions similarly to reactive oxygen species (ROS) in P. infestans cells and caused mitochondrial damage, including mitochondrial membrane potential (MMP), mitochondrial respiratory chain complex activity (MRCCA), and ATP production decline. These results highlight that the cell structure disruption, oxidative stress, and energy supply dysfunction induced by Iturin A play an important role in inhibiting P. infestans. Additionally, B. subtilis WL-2 and Iturin A have great potential for inhibiting P. infestans mycelium growth and controlling potato late blight in the future.
Project description:In the present study, a deep-sea bacterial strain designated Bacillus sp. strain wsm-1 was screened and found to exhibit strong antifungal activity against many plant-pathogenic fungi, and corresponding antifungal agents were thereby purified and determined by tandem mass spectrometry to be two cyclic lipopeptide homologs. These homologs, which were different from any previously reported lipopeptides, were identified to possess identical amino acid sequences of ?-amino fatty acid-Asn-Ser-Asn-Pro-Tyr-Asn-Gln and deduced as two novel lipopeptides designated C14 iturin W and C15 iturin W. Electron microscopy observation indicated that both iturin W homologs caused obvious morphological changes and serious disruption of plasma membrane toward fungal cells, while C15 iturin W exhibited more serious cell damages than C14 iturin W did, which was well consistent with the results of the antifungal activity assays. To improve the yield and antifungal activity of iturin W, the effects of different carbon and nitrogen sources and amino acids on production of C14 iturin W and C15 iturin W were investigated. The results indicated that supplements of most of the detected carbon and nitrogen sources could increase the yield of C14 iturin W, but inhibit the yield of C15 iturin W, while supplements of tryptone and most of the detected amino acids could increase the yield of both C14 iturin W and C15 iturin W.IMPORTANCE Plant disease caused by pathogenic fungi is one of the most devastating diseases, which affects the food safety of the whole world to a great extent. Biological control of plant diseases by microbial natural products is more desirable than traditional chemical control. In this study, we discovered a novel lipopeptide, iturin W, with promising prospects in biological control of plant diseases. Moreover, the effects of different carbon and nitrogen sources and amino acids on production of C14 iturin W and C15 iturin W would provide a reasonable basis for the optimization of the fermentation process of lipopeptides. Notably, the structure of iturin W was different from that of any previously reported lipopeptide, suggesting that deep-sea microorganisms might produce many novel natural products and have significant potential in the development of biological products in the future.
Project description:Lipopeptides have been reported to exhibit anti-obesity effects. In this study, we obtained a <i>Bacillus velezensis</i> strain FJAT-52631 that could coproduce iturins, fengycins, and surfactins. Results showed that the FJAT-52631 crude lipopeptide, purified fengycin, iturin, and surfactin standards exhibited strong inhibition activities against lipase with dose-dependence manners (half maximal inhibitory concentration (IC<sub>50</sub>)?=?0.011, 0.005, 0.056, and 0.005?mg/mL, respectively). Moreover, fengycin and surfactin had the comparable activities with orlistat, but iturin not. It was revealed that the inhibition mechanism and type of the lipopeptides were reversible and competitive. The quenching mechanism of lipase was static and only one binding site between lipase and lipopoeptide was inferred from the fluorescence analysis. The docking analysis displayed that fengycin and surfactin could directly interact with the active amino acid residues (Ser or Asp) of lipase, but not with iturin. Our work suggests that the <i>B. velezensis</i> lipopeptides would have great potential to act as lipase inhibitors.
Project description:Bacillus amyloliquefaciens strain RHNK22 isolated from groundnut rhizosphere showed direct and indirect plant growth-promoting traits along with biosurfactant activity and reduction in surface tension of water. Biosurfactants were identified as lipopeptides (surfactin, iturin, and fengycin) by molecular and biochemical analysis in our studies.
Project description:A potential antagonist, designated strain Bacillus subtilis MBCU5 was previously isolated from vermicompost-amended soils of Gandhinagar, Gujarat, India. Crude allelochemicals from strain MBCU5 displayed strong antifungal activity against Macrophomina phaseolina as well as Rhizoctonia solani. These crude allelochemicals were tentatively identified as iturin, fengycin and surfactin through TLC and HPTLC analysis. Lipopeptides produced by MBCU5 were identified by MALDI-TOF-MS and LC-ESI-MS/MS analysis showed that iturin homologues (m/z 1020-1120), surfactin (m/z 1008.7 and m/z 1022.7), fengycin A and fengycin B (m/z 1400-1550) types of allelochemicals which are responsible for antifungal activity against pathogens. PCR analysis showed presence of genes (i.e. Iturin A synthetase KJ531680 and Surfactin synthetase KJ601726) involved in the biosynthesis of allelochemicals. Many reports showed lipopeptides from Bacillus species; this is the first report executed of multifarious allelochemicals from vermicompost-amended soil due to the presence of predominant Bacillus species.
Project description:Application of Bacillus cyclic lipopeptides (CLPs); fengycin, iturin A and surfactin has shown a great potential in controlling the spread of green mold pathogen invasion (Penicillium digitatum) in wounded mandarin fruit during postharvest period. The limited defensive protein profiles followed specific expression of pivotal genes relating to plant hormone mediating signaling pathways of the CLPs' action on stimulating host plant resistance have been exhibited. The present study aimed to elucidate the specific effect of individual CLP obtained from Bacillus subtilis ABS-S14 as elicitor role on activation of plant defensive system at transcriptional and proteomic levels with and without P. digitatum co-application in mandarin fruit. Fengycin and iturin A elevated the gene expression of PAL, ACS1, ACO, CHI, and GLU while significantly stimulating plant POD transcription was only detected in the treatments of surfactin both with and without following P. digitatum. An increase of LOX and PR1 gene transcripts was determined in the treatments of individual CLP with fungal pathogen co-application. Fengycin activated production of unique defensive proteins such as protein involved in ubiquinone biosynthetic process in treated flavedo without P. digitatum infection. Proteins involved in the auxin modulating pathway were present in the iturin A and surfactin treatments. CLP-protein binding assay following proteome analysis reveals that iturin A attached to 12-oxophytodienoate reductase 2 involved in the oxylipin biosynthetic process required for jasmonic acid production which is implicated in induced systemic resistance (ISR). This study suggests specific elicitor action of individual CLP, particularly iturin A showed the most powerful in stimulating the ISR system in response to stresses in postharvest mandarins.
Project description:To elucidate interactions between the antifungal cyclic lipopeptides iturin A, fengycin, and surfactin produced by Bacillus bacteria and the microtubular protein ?-tubulin in plant pathogenic fungi (Fusarium oxysporum, Colletrotrichum gloeosporioides, Alternaria alternata, and Fusarium solani) in molecular docking and molecular dynamics simulations, we retrieved the structure of tubulin co-crystallized with taxol from the Protein Data Bank (PDB) (ID: 1JFF) and the structure of the cyclic lipopeptides from PubChem (Compound CID: 102287549, 100977820, 10129764). Similarity and homology analyses of the retrieved ?-tubulin structure with those of the fungi showed that the conserved domains shared 84% similarity, and the root mean square deviation (RMSD) was less than 2 Å. In the molecular docking studies, within the binding pocket, residues Pro274, Thr276, and Glu27 of ?-tubulin were responsible for the interaction with the cyclic lipopeptides. In the molecular dynamics analysis, two groups of ligands were formed based on the number of poses analyzed with respect to the RMSD. Group 1 was made up of 10, 100, and 500 poses with distances 0.080 to 0.092 nm and RMSDs of 0.10 to 0.15 nm. For group 2, consisting of 1000 poses, the initial and final distance was 0.1 nm and the RMSDs were in the range of 0.10 to 0.30 nm. These results suggest that iturin A and fengycin bind with higher affinity than surfactin to ?-tubulin. These two lipopeptides may be used as lead compounds to develop new antifungal agents or employed directly as biorational products to control plant pathogenic fungi.
Project description:In this research, a strain with broad-spectrum antimicrobial activities was isolated from the gastrointestinal tract of hairtail (Trichiurus haumela) and identified as Bacillus siamensis JFL15 through morphological, 16S rRNA, and average nucleotide identity analyses. The genome of B. siamensis JFL15 was sequenced, and three gene clusters involved in the biosynthesis of surfactin (srf), bacillibactin (dhb), and fengycin (fen) were predicted through antiSMASH analysis. The combined genomics-metabolics profiling of the strain revealed 20 active compounds, which belong to four main types of cyclic lipopeptides produced by Bacillus species: bacillibactin, iturin, fengycin, and surfactin. Among these lipopeptides, two high-purity antifungal components, namely, components b and c, were successfully identified as iturin A and bacillomycin F. The minimum inhibitory concentrations (MICs) of iturin A for Magnapothe grisea, Rhizoctorzia solani, and Colletotrichum gloeosporioides were 125.00, 62.50, and 125.00 ?g/ml, respectively, whereas the MICs of bacillomycin F for these three organisms were 62.50, 31.25, and 62.50 ?g/ml, respectively. The mechanism of bacillomycin F and iturin A against M. grisea was also investigated. Scanning electron microscopy (SEM) indicated that the surface of the hypha treated with iturin A or bacillomycin F became sunk, lumpy, and wrinkled. The diversity of the identified and predicted compounds from B. siamensis JFL15 suggested that this strain might be a promising biocontrol agent for an effective and environmentally friendly control of pathogenic microorganisms. To the best of our knowledge, this study is the first to describe cyclic lipopeptides purified and identified from B. siamensis.
Project description:Several products of industrial interest are produced by Bacillus, including enzymes, antibiotics, amino acids, insecticides, biosurfactants and bacteriocins. This study aimed to investigate the potential of two bacterial isolates (P5 and C3) from puba, a regional fermentation product from cassava, to produce multiple substances with antimicrobial and surface active properties. Phylogenetic analyses showed close relation of isolates P5 and C3 with Bacillus amyloliquefaciens and Bacillus thuringiensis, respectively. Notably, Bacillus sp. P5 showed antimicrobial activity against pathogens such as Listeria monocytogenes and Bacillus cereus, in addition to antifungal activity. The presence of genes encoding pre-subtilosin (sboA), malonyl CoA transacylase (ituD), and the putative transcriptional terminator of surfactin (sfp) were detected in Bacillus sp. P5, suggesting the production of the bacteriocin subtilosin A and the lipopeptides iturin A and surfactin by this strain. For Bacillus sp. C3 the presence of sboA and spas (subtilin) genes was observed by the first time in members of B. cereus cluster. Bacillus sp. P5 showed emulsifying capability on mineral oil, soybean biodiesel and toluene, while Bacillus sp. C3 showed emulsifying capability only on mineral oil. The reduction of the surface tension in culture medium was also observed for strain P5, confirming the production of surface-active compounds by this bacterium. Monoprotonated molecular species and adducts of sodium and potassium ions of surfactin, iturin, and fengycin were detected in the P5 culture medium. Comparative MS/MS spectra of the peak m/z 1030 (C14 surfactin A or C15 surfactin B [M+Na]+) and peak m/z 1079 (C15 iturin [M+Na]+) showed the same fragmentation profile of standards, confirming the molecular identification. In conclusion, Bacillus sp. P5 showed the best potential for the production of antifungal, antibacterial, and biosurfactant substances.
Project description:Fusarium head blight (FHB) caused by infection with Fusarium graminearum leads to enormous losses to crop growers, and may contaminate grains with a number of Fusarium mycotoxins that pose serious risks to human and animal health. Antagonistic bacteria that are used to prevent FHB offer attractive alternatives or supplements to synthetic fungicides for controlling FHB without the negative effects of chemical management. Out of 500 bacterial strains isolated from soil, Bacillus amyloliquefaciens JCK-12 showed strong antifungal activity and was considered a potential source for control strategies to reduce FHB. B. amyloliquefaciens JCK-12 produces several cyclic lipopeptides (CLPs) including iturin A, fengycin, and surfactin. Iturin A inhibits spore germination of F. graminearum. Fengycin or surfactin alone did not display any inhibitory activity against spore germination at concentrations less than 30 ?g/ml, but a mixture of iturin A, fengycin, and surfactin showed a remarkable synergistic inhibitory effect on F. graminearum spore germination. The fermentation broth and formulation of B. amyloliquefaciens JCK-12 strain reduced the disease incidence of FHB in wheat. Furthermore, co-application of B. amyloliquefaciens JCK-12 and chemical fungicides resulted in synergistic in vitro antifungal effects and significant disease control efficacy against FHB under greenhouse and field conditions, suggesting that B. amyloliquefaciens JCK-12 has a strong chemosensitizing effect. The synergistic antifungal effect of B. amyloliquefaciens JCK-12 and chemical fungicides in combination may result from the cell wall damage and altered cell membrane permeability in the phytopathogenic fungi caused by the CLP mixtures and subsequent increased sensitivity of F. graminearum to fungicides. In addition, B. amyloliquefaciens JCK-12 showed the potential to reduce trichothecenes mycotoxin production. The results of this study indicate that B. amyloliquefaciens JCK-12 could be used as an available biocontrol agent or as a chemosensitizer to chemical fungicides for controlling FHB disease and as a strategy for preventing the contamination of harvested crops with mycotoxins.