Bacillus amyloliquefaciens YN201732 Produces Lipopeptides With Promising Biocontrol Activity Against Fungal Pathogen Erysiphe cichoracearum

Bacillus amyloliquefaciens YN201732 is an endophytic bacteria with high biocontrol efficiency and broad-spectrum antimicrobial activities. In order to clarify the main active ingredients and their antifungal mechanisms against powdery mildew of tobacco, this study is focused on lipopeptide obtained through acid precipitation and organic solvent extraction. HPLC and LCMS-IT-TOF were used to separate and identify antimicrobial lipopeptides. Findings revealed that bacillomycin D plays an important role against surrogate fungal pathogen Fusarium solani. Synthetic pathways of sfp, bacillomycin D, and fengycin were separately disrupted. The sfp gene knockout mutant B. amyloliquefaciens YN201732M1 only showed minor antagonistic activity against F. solani. While Erysiphe cichoracearum spore germination was inhibited and pot experiments displayed a significant decrease in tobacco powdery mildew. The spore inhibition rate of YN201732M1 was only 30.29%, and the pot experiment control effect was less than 37.39%, which was significantly lower than that of the wild type. The inhibitory effect of mutant YN201732M2 (deficient in the production of bacillomycin D) and mutant YN201732M3 (deficient in the production of fengycin) on the spore germination of E. cichoracearum were 50.22% and 53.06%, respectively, suggesting that both fengycin and bacillomycin D had potential effects on spore germination of powdery mildew. Interestingly, in a greenhouse assay, both B. amyloliquefaciens YN201732M2 and YN201732M3 mutants displayed less of a control effect on tobacco powdery mildew than wild type. The results from in vitro, spore germination, and greenhouse-pot studies demonstrated that antimicrobial lipopeptides especially bacillomycin D and fengycin may contribute to the prevention and control of tobacco powdery mildew. In addition, gene mutation related to lipopeptide synthesis can also affect the biofilm formation of strains.

The ComQXPA Quorum Sensing System May Play an Important Role in the Synthesis of Bacillomycin D in Bacillus Amyloliquefaciens Q-426

Background: Bacillus amyloliquefaciens Q-426 can secrete numerous cyclic lipopeptides that have antifungal and antitumor activities. ComQXPA is a common quorum sensing (QS) system in Bacillus species. Most B. amyloliquefaciens strains are encoding the QS gene cluster comQXPA, however, the biological function of the ComQXPA system in B. amyloliquefaciens has not been well studied. In this study, we identified the comQXPA gene locus and the chemical structure of ComXQ-426 in B. amyloliquefaciens Q-426, and explored the function of ComXQ-426 in regulating lipopeptide production.Results: We identified and analyzed the comQXPA locus in Q-426. The full length of the comQXPA gene cluster was 4,014 bp, including 912 bp of comQ, 165 bp of comX, 2292 bp of comP, and 645 bp of comA. The comQXPA locus belongs to group B, as comQ and comX overlap by only one base pair. ComXQ-426 consists of six amino acids (GGDWKY) that contain a modified tryptophan residue. The antifungal activity of Q426ΔcomX was significantly affected, and almost no antifungal activity was observed, while the antifungal activity of strain Q426ΔcomX /comQX was restored to the same level as that of the wild-type strain. When the ComXQ-426 was added to the culture medium at a final concentration of 8 μg/L at the early stage of the log-phase, the antifungal activity of the wild-type strain Q-426 was significantly improved. Knocking out the comX gene did not affect the growth of the bacteria, however, the strain Q426ΔcomX lost its swimming ability, was unable to form colonies when spread on a solid surface, and could not form biofilms on the interface between the gas and liquid medium.Conclusions: Disruption of the ComPA signaling pathway in the Q-426 strain resulted in significant effects on bacillomycin D production, morphology, and motility.

Synergistic Inhibitory Activity of Bacillomycin D, Surfactin and Nisin against Thermoascus crustaceus, Neosartorya hiratsukae and Bacillus subtilis, Responsible for Cardboard Spoilage

Corrugated cardboard boxes are one of the largest paper-based packaging forms used for shipping and handling of wide variety of products in different end-use industries due to low cost, low weight and recyclability. Due to its organic composition, they are highly susceptible to spoilage from heat-resistant microbial spores, leading to economic losses and health risks. In this study, the efficacy of lipopeptides produced from Bacillus amyloliquefaciens MTCC 10456 against thermotolerant Thermoascus crustaceus, Neosartorya hiratsukae and Bacillus subtilis, isolated from spoiled cardboard boxes, was investigated. Lipopeptides were isolated by salt-precipitation of fermentation broth and activity-guided Reverse Phase-High Performance Liquid Chromatography (RP-HPLC). Inhibitory fractions consisted of bacillomycin D and surfactin, which were identified using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS/MS) analysis. Mixture of lipopeptides with nisin (3:2 w/w) asserted significant synergistic effect on the tested pathogens which reduced the minimum inhibitory concentrations (MIC) values and increased their inhibition spectra. Preservative coating containing lipopeptides and nisin was applied on the corrugated cardboard surfaces by mixing with starch-based additive by spread-coating method. It demonstrated biopreservative efficacy against the targeted microorganisms at during the observational period of 180 days. Reduction in microbial count of 4 log cycles was observed in 20 days and showed controlled release of coated peptides which indicate its suitability for packaging purposes. Findings from this study suggests an effective and scalable strategy to prevent microbial spoilage thereby extending the storage period of cardboard boxes.

Exploration of production of C14 and C15 bacillomycin D homologues with enzymatic hydrolysis from maize straws using fed-batch fermentation by Bacillus subtilis NS-174

A strain with strong antifungal activity, Bacillus subtilis NS-174, was identified and the antifungal compounds were purified and structurally analyzed by high performance liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS).