Table 1.
Summary of identity, source, and cross-reactivity against food-associated fungal indicators of honey bacterial isolates.
Fig 1.
Deferred inhibition assay of purified products from Bacillus velezensis WRB-ZX-001 and WRB-ZX-002 against food-isolated Aspergillus fumigatus.
Precipitates of WRB-ZX-001 and WRB-ZX-002 from 60% ammonium sulfate were shown in A and B. Solid phase extraction eluates of 60% acetonitrile for WRB-ZX-001 and WRB-ZX-002 were shown in C and D. Two-fold serial dilution was performed for all samples to determine the antifungal activity units.
Fig 2.
Reversed-phase HPLC of purified products of Bacillus velezensis WRB-ZX-001 and WRB-ZX-002.
Purification process included ammonium sulfate precipitation, solid phase extraction, and HPLC fraction collection. Single peaks shown in A and C were from isolate WRB-ZX-001 and WRB-ZX-002, respectively, and both have shown inhibition against fungal indicator strain Aspergillus fumigatus as shown in B and D.
Fig 3.
Mass spectra for purified antifungal compounds produced by Bacillus velezensis WRB-ZX-001 and WRB-ZX-002.
A and B are LC-MS spectra for HPLC collected active fraction of WRB-ZX-001 and WRB-ZX-002. Ion with m/z value of 1057.57 was assigned to C15 iturin A [M+H]+. Ion with m/z value of 1079.55 was assigned to C15 iturin A [M+Na]+.
Table 2.
Antifungal activity of purification products of Bacillus velezensis isolates against food-isolated Aspergillus fumigatus.
Fig 4.
Core genome phylogeny of 43 Bacillus amyloliquefaciens group isolates.
Maximum likelihood tree was constructed with core genome SNPs identified by kSNP. 41 reference genomes of Bacillus amyloliquefaciens group isolates were obtained from NCBI genome database. The core genome of Bacillus subtilis 168 was used as outgroup. Phylogeny was inferred by RAxML under time-reversible model with gamma distributed substitution sites and 1000 bootstrap repetitions. Bar represents 0.2 substitution per site. Isolates from this study were labeled with red circle.
Fig 5.
Genome comparison of Bacillus velezensis WRB-ZX-001 and WRB-ZX-002 against closely related Bacillus type strains.
Bacillus velezensis FZB42 was used as the reference strain. The circular ring map was constructed by BLAST Ring Image Generator (BRIG, version 0.95). From inner to outer ring: 1) GC content; 2) Bacillus velezensis FZB42 nucleotide sequence; 3) GC Skew; 4) Bacillus velezensis WRB-ZX-001 nucleotide sequence; 5) Bacillus velezensis WRB-ZX-002 nucleotide sequence; 6) Bacillus velezensis CBMB205 nucleotide sequence; 7) Bacillus amyloliquefaciens DSM7 nucleotide sequence.
Table 3.
Potential secondary metabolite synthesis gene clusters identified in Bacillus velezensis WRB-ZX-001 and WRB-ZX-002 by antiSMASH 5.0.
Fig 6.
Growth curve and antifungal activity curve for Bacillus velezensis WRB-ZX-001 (A) and WRB-ZX-002 (B). Growth curve was plotted by measuring absorption at OD600nm every 30 min and a standard form of logistic equation was used to fit the absorption data (red line). Antifungal activity was measured by well diffusion overlay inhibition assay of serially diluted cell-free supernatant every two hours against fungal indicator strain Aspergillus fumigatus and data were shown in bar plots.
Table 4.
Antifungal activity of heat-treated and protease-treated purified products of Bacillus velezensis isolates against food-isolated Aspergillus fumigatus.