Multi-Omics Techniques for Analysis Antifungal Mechanisms of Lipopeptides Produced by Bacillus velezensis GS-1 against Magnaporthe oryzae In Vitro

Abstract

Magnaporthe oryzae is a fungal pathogen that causes rice blast, a highly destructive disease. In the present study, the bacteria strain GS-1 was isolated from the rhizosphere soil of ginseng and identified as Bacillus velezensis through 16S rRNA gene sequencing, whole genome assembly, and average nucleotide identity analysis. B. velezensis strain GS-1 exhibited significant antagonistic activity to several plant fungal pathogens. Through whole genome sequencing, 92 Carbohydrate-Active Enzymes and 13 gene clusters that encoded for secondary metabolites were identified. In addition, strain GS-1 was able to produce the lipopeptide compounds, surfactin, fengycin, and plantazolicin. The inhibitory effects of lipopeptide compounds on M. oryzae were confirmed, and the antagonistic mechanism was explored using transcriptomics and metabolomics analysis. Differential expressed genes (DEGs) and differential accumulated metabolites (DAMs) revealed that the inhibition of M. oryzae by lipopeptide produced by GS-1 downregulated the expression of genes involved in amino acid metabolism, sugar metabolism, oxidative phosphorylation, and autophagy. These results may explain why GS-1 has antagonistic activity to fungal pathogens and revealed the mechanisms underlying the inhibitory effects of lipopeptides produced by GS-1 on fungal growth, which may provide a theoretical basis for the potential application of B. velezensis GS-1 in future plant protection.

Keywords: Bacillus velezensis; Magnaporthe oryzae; biocontrol; genome sequencing; lipopeptide; metabolomics; transcriptomics.

Figure 1

Antagonistic activity of B. velezensis GS-1 against plant pathogenic fungi. (A) Magnaporthe oryzae, (B) Rhizoctonia solani, (C) Botrytis cirerea, (D) Fusarium graminearum, (E) Sclerotinia sclerotiorum, (F) Cercospora zeae, (G) Setosphaeria turcica, (H) Cochiliobolus heterostrophus. Scale bar: 1 cm.

Figure 2

Circular genome map of B. velezensis GS-1. From the innermost to the outermost: ring 1 for GC content, ring 2 for GC skew, ring 3 for distribution of rRNAs (green) and tRNAs (brown), ring 4 for COG classifications of protein-coding genes on the forward strand and reverse strand, ring 5 for restriction modification system, and ring 6 for genome size (black line).

Figure 3

Phylogenetic tree of GS-1 and 15 other Bacillus species based on 16S rRNA sequence analysis. The red color indicated the strain GS-1 isolated in this study.

Figure 4

Heatmap of pairwise average nucleotide identity (ANI) values for whole genomes of GS-1 and 15 other Bacillus species.

Figure 5

CAZy genes classification and glycoside hydrolase (GH) distribution in strain GS-1. (A) CAZy gene classification in GS-1 genome; (B) GH genes distribution in GS-1 genome.

Figure 6

Antagonistic activity of crude lipopeptide extract from B. velezensis GS-1 against indicated plant pathogenic fungi. Scale bar: 1 cm.

Figure 7

Matrix-assisted laser desorption ionization time-off light (MALDI-TOF) mass spectrometry (MS) spectra of the crude lipopeptide extracts.

Figure 8

KEGG enrichment scatter plot. The ordinate represents the KEGG pathway, the abscissa represents the Rich factor. The larger the Rich factor, the greater the enrichment. The larger the point, the greater the number of differential genes enriched in the pathway. The redder the color of the dots, the more significant the enrichment.

Figure 9

(A) Heatmap of differentially accumulated metabolites. (B) Top 10 DAMs in control and treatment.

Figure 10

Differential accumulated metabolites KEGG enrichment. The size of the points in the graph represents the number of distinct significant metabolites enriched into the corresponding pathway. The more p-Value approaches to 0, the more significant the enrichment is.

Figure 11

KEGG pathways significantly enriched in DEGs and DAMs.