Genomic Analysis and Secondary Metabolites Production of the Endophytic Bacillus velezensis Bvel1: A Biocontrol Agent against Botrytis cinerea Causing Bunch Rot in Post-Harvest Table Grapes

Abstract

Botrytis bunch rot caused by Botrytis cinerea is one of the most economically significant post-harvest diseases of grapes. In the present study, we showed that the bacterial strain Bvel1 is phylogenetically affiliated to Bacillus velezensis species. The strain Bvel1 and its secreted metabolites exerted an antifungal activity, under in vitro conditions, against B. cinerea. UHPLC-HRMS chemical analysis revealed that iturin A2, surfactin-C13 and -C15, oxydifficidin, bacillibactin, L-dihydroanticapsin, and azelaic acid were among the metabolites secreted by Bvel1. Treatment of wounded grape berries with Bacillus sp. Bvel1 cell culture was effective for controlling grey mold ingress and expansion in vivo. The effectiveness of this biological control agent was a function of the cell culture concentration of the antagonist applied, while preventive treatment proved to be more effective compared to curative. The strain Bvel1 exhibited an adequate colonization efficiency in wounded grapes. The whole-genome phylogeny, combined with ANI and dDDH analyses, provided compelling evidence that the strain Bvel1 should be taxonomically classified as Bacillus velezensis. Genome mining approaches showed that the strain Bvel1 harbors 13 antimicrobial biosynthetic gene clusters, including iturin A, fengycin, surfactin, bacilysin, difficidin, bacillaene, and bacillibactin. The results provide new insights into the understanding of the endophytic Bacillus velezensis Bvel1 biocontrol mechanism against post-harvest fungal pathogens, including bunch rot disease in grape berries.

Keywords: biological control; colonization; endophytic bacteria; genome sequence; induced systemic resistance; iturin A; metabolites; surfactin.

Figure 1

Whole-genome-based phylogenetic tree highlighting the position of Bacillus Bvel1 relative to other closely related Bacillus species. Tree inferred with FastME 2.1.6.1 [24] from genome-blast distance phylogeny (GBDP) distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above the branches are GBDP pseudo-bootstrap support values > 75% from 100 replications, with an average branch support of 84%. The tree was rooted at the midpoint [24,25]. Leaf labels with different colors indicate species and subspecies clusters.

Figure 2

Antifungal activity of Bvel1 culture and its extracted secreted compounds (ESC) against B. cinerea. ESC1 and ESC2 represent extracted secreted compounds of Bvel1 when grown singly and paired with B. cinerea, respectively. (A) Direct antifungal activity of Bacillus velezensis Bvel1 using a dual culture assay at 3d (top) and 5d (bottom) of the interaction. (B) Antifungal activity of ESC1, ESC2, and M (methanol) using a well-diffusion confrontation assay at 3 days (i, iii) and 5 days of interaction (ii, iv). (C) TLC- bioautography using ESC1 and ESC2.

Figure 3

Spectra from Orbitrap high-resolution mass spectrometry (UHPLC-HRMS) analysis of the compounds secreted by Bvel1. (A) Surfactin A -C13, (B) Surfactin A -C15, (C) Iturin A2 -C14, (D) Oxydifficidin, (E) L-dihydroanticapsin, (F) Bacillibactin, and (G) Azelaic acid. The m/z of the compounds mentioned are circled on the spectra.

Figure 4

The effect of preventive and curative treatment with different concentrations (10⁶ and 10⁸ CFU/mL) of endophytic bacterial strain Bvel1 against grey mold on berries. (A) Disease incidence (% infected berries) of the preventive bacterial treatment, (B) disease incidence (%) of the curative bacterial treatment, (C) disease severity (% berry area with lesions) of the preventive bacterial treatment, (D) disease severity (%) of the curative bacterial treatment. Data values represent the mean of 3 biological replicates ± SD after t-test analysis. Asterisks indicate statistically significant differences of each treatment when compared to the control (B. cinerea) at each incubation timepoint (p < 0.05).

Figure 5

Biocontrol activity of antagonistic Bvel1 against B. cinerea on wounded red globe grapes. Wounded grapes treated with different concentrations (10⁶ and 10⁸ CFU/mL) of the endophytic bacterial strain Bvel1, 1 day prior to artificial inoculation with B. cinerea. Pictures were taken 3 days and 6 days after artificial inoculation (DAAI) and incubation at 25 °C.

Figure 6

Time growth curve of Bvel1 (Log10 CFU/wound) in grape wounds (one wound in each grape) with and without the presence of the fungus B. cinerea. Data values represent the mean of 3 biological replicates ± SD after t-test analysis. Asterisks indicate the statistical difference between the two treatments (p < 0.05).