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. 2023 Mar 20;12(6):1374.
doi: 10.3390/plants12061374.

Broad-Spectrum Antifungal, Biosurfactants and Bioemulsifier Activity of Bacillus subtilis subsp. spizizenii-A Potential Biocontrol and Bioremediation Agent in Agriculture

Karina Guillén-Navarro  1 , Tomás López-Gutiérrez  2 , Verónica García-Fajardo  1 , Sergio Gómez-Cornelio  3   4 , Eugenia Zarza  1   5 , Susana De la Rosa-García  6 , Manuel Chan-Bacab  7
Affiliations

Broad-Spectrum Antifungal, Biosurfactants and Bioemulsifier Activity of Bacillus subtilis subsp. spizizenii-A Potential Biocontrol and Bioremediation Agent in Agriculture

Karina Guillén-Navarro et al. Plants (Basel). .

Abstract

In this study, the antifungal, biosurfactant and bioemulsifying activity of the lipopeptides produced by the marine bacterium Bacillus subtilis subsp. spizizenii MC6B-22 is presented. The kinetics showed that at 84 h, the highest yield of lipopeptides (556 mg/mL) with antifungal, biosurfactant, bioemulsifying and hemolytic activity was detected, finding a relationship with the sporulation of the bacteria. Based on the hemolytic activity, bio-guided purification methods were used to obtain the lipopeptide. By TLC, HPLC and MALDI-TOF, the mycosubtilin was identified as the main lipopeptide, and it was further confirmed by NRPS gene clusters prediction based on the strain's genome sequence, in addition to other genes related to antimicrobial activity. The lipopeptide showed a broad-spectrum activity against ten phytopathogens of tropical crops at a minimum inhibitory concentration of 400 to 25 μg/mL and with a fungicidal mode of action. In addition, it exhibited that biosurfactant and bioemulsifying activities remain stable over a wide range of salinity and pH and it can emulsify different hydrophobic substrates. These results demonstrate the potential of the MC6B-22 strain as a biocontrol agent for agriculture and its application in bioremediation and other biotechnological fields.

Keywords: antifungal activity; bioemulsifier; biosurfactants; lipopeptide.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Maximum Likelihood Phylogenetic trees. Diagrams were obtained using 16S rRNA (a) and (b) Gyrase B Subunit gene sequences. The bootstrap consensus trees inferred from 1000 replicates were taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed; the percentage of replicate trees is shown next to the branches.
Figure 2
Figure 2
Kinetic of (a) antifungal (against C. gloeosporioides ATCC 42374) and (b) hemolytic activities over a period 132 h in LBMs of B. subtilis subsp. spizizenii MC6B-22.
Figure 3
Figure 3
Retention factor in thin layer chromatography (TLC) analysis of the crude lipopeptide extract CL84. (a) revealed with ninhydrin and cupric sulfate. (b) Bioautography against C. gloeosporioides growth in an agar overlay and dyed with an aqueous solution of 2,3,5 triphenyltetrazolium chloride and (c) in blood agar overlay.
Figure 4
Figure 4
Characterization of lipopeptide for (a) HPLC standard mycosubtilin, (b) 84 h purified lipopeptide analysis (PL84); MALDI–TOF-MS analysis of (c) iturin A6, A7 and (d) mycosubtilin.
Figure 5
Figure 5
Antifungal activity of CL84 by agar well diffusion showing broad spectrum against different phytopathogens (a) Aschochyta sp., (b) Colletorichum acutatum, (c) Colletotrichum gloeosporioides ATCC 42374, (d) Colletotrichum gloeosporioides, (e) Colletotrichum capsici, (f) Curvularia clavata, (g) Fusarium nivale, (h) Fusarium solani, (i) Moniliophthora roreri, (j) Pestalotiopsis maculans.
Figure 6
Figure 6
Emulsifying index obtained with different hydrocarbons using CL84 produced by Bacillus subtilis subsp. spizizenii MC6B-22. (a) Burnt motor oil, (b) Toluene, (c) Xylene, (d) Diesel, (e) n-Hexadecane, (f) Motor oil, (g) Crude oil (Petroleum), (h) n-Hexane, (i) Olive oil.
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