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. 2019 Apr 2;19(1):71.
doi: 10.1186/s12866-019-1440-8.

Lipopeptide mediated biocontrol activity of endophytic Bacillus subtilis against fungal phytopathogens

Dibya Jyoti Hazarika  1 , Gunajit Goswami  1 , Trishnamoni Gautom  1 , Assma Parveen  1 , Pompi Das  1 , Madhumita Barooah  1 , Robin Chandra Boro  2
Affiliations

Lipopeptide mediated biocontrol activity of endophytic Bacillus subtilis against fungal phytopathogens

Dibya Jyoti Hazarika et al. BMC Microbiol. .

Abstract

Background: The use of chemical fungicides against fungal pathogens adversely affects soil and plant health thereby resulting in overall environmental hazards. Therefore, biological source for obtaining antifungal agents is considered as an environment-friendly alternative for controlling fungal pathogens.

Results: In this study, seven endophytic bacteria were isolated from sugarcane leaves and screened for its antifungal activity against 10 fungal isolates belonging to the genera Alternaria, Cochliobolus, Curvularia, Fusarium, Neodeightonia, Phomopsis and Saccharicola isolated from diseased leaves of sugarcane. Among the seven bacterial isolates, SCB-1 showed potent antagonistic activity against the tested fungi. Based on the phenotypic data, Fatty Acid Methyl Esters (FAME) and 16S rRNA gene sequence analysis, the isolate SCB-1 was identified as Bacillus subtilis. The bacterial isolate was screened negative for chitinase production; however, chloroform and methanol extracts of the bacterial culture caused significant inhibition in the growth of the fungal isolates on semisolid media. Volatile component assay showed highest inhibitory activity against Saccharicola bicolor (SC1.4). A PCR based study detected the presence of the genes involved in biosynthesis of surfactin, bacillaene, difficidin, macrolactins and fengycin. Mass spectrometric analysis of the bacterial extract detected the presence of antifungal lipopeptide surfactin, but other metabolites were not detected. The biocontrol activity of the bacterial isolate was established when bacterial pretreated mung bean seeds were able to resist Fusarium infection, however, the untreated seeds failed to germinate.

Conclusion: The antifungal potential of isolate Bacillus subtilis SCB-1 was established against taxonomically diverse fungal pathogens including the genera Saccharicola, Cochliobolus, Alternaria and Fusarium. The potent antifungal compound surfactin as well as volatiles produced by the bacterial isolate could be responsible for its bio-control activity against fungal infections.

Keywords: Antifungal activity; Bacillus subtilis; Biological control; FAME; LC-MS; Surfactin.

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Conflict of interest statement

Ethics approval and consent to participate

No animals, human subjects, human material, or human data are used in this study.

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Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
Antagonistic activity of isolate SCB-1 against different sugarcane phytopathogens. The dual culture represents inoculation of SCB-1 (on the right side of each dual culture plate) with the respective fungal strains
Fig. 2
Fig. 2
Antagonistic activity of the SCB-1 culture supernatant, crude extracts, and volatile organic components. a Antifungal activity of the culture supernatant against four test fungi (Cochliobolus hawaiiensis SC2.3, Curvularia senegalensis SC4.1, Alternaria alternata SC6.2 and Fusarium oxysporum SC7.1) on PDA plates. Photographs were taken after 72 h of inoculation. b and c Growth diameter of the fungal colonies challenged with 0 ppm (control), 250 ppm and 500 ppm of bacterial crude extract after 48 h and 72 h, respectively. Asterisk (*) and double Asterisk (**) indicates level of significance over control with p ≤ 0.05 and p ≤ 0.01, respectively (t-test, Microsoft Excel 2013). d Inhibitory activity of bacterial volatile organic components against the test fungi. Results are represented as the inhibition percentage of the colony diameter of the test fungi at different time interval. The different letters (a-e) above the bars represent significant differences (p ≤ 0.05) in the inhibition percentage for individual test fungi, otherwise non-significant
Fig. 3
Fig. 3
Detection of secondary metabolites produced by bacterial isolate SCB-1. a PCR amplified products of the eight secondary metabolite biosynthetic genes on 1.2% agarose gel. b UPLC chromatogram of the bacterial crude extract. c Mass spectra of surfactin corresponding fraction eluted at retention time of 12.45 min
Fig. 4
Fig. 4
Biocontrol potential of SCB-1 against three Fusarium isolates on germinating seeds mung bean. a Photographs of bacteria untreated and treated germinating seeds after 48 h of fungal inoculation. b Graph representing germination percentage of mung bean seeds after different treatments. c Graph representing the root and shoot lengths of germinating seeds after different treatments. The different letters (a-d) above the bar represent significant difference (p ≤ 0.05) in the germination percentage, root lengths and shoot lengths of germinating seeds, otherwise non-significant
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