## 2 Results
### Amplification of B. atrophaeus Strain HF1 Genes Associated with Lipopeptide Biosynthesis  
Lipopeptides such as iturin, surfactin, and fengycin are among the most common antimicrobial compounds produced by Bacillus species. To detect whether strain HF1 contained these lipopeptide biosynthesis-related genes, PCR amplification was used. Specific bands of the expected size, as well as _fortiuD_, _sff_, and _fen_ genes, were identified in strain HF1 (Supplementary Figure S1), and sequencing analyses further confirmed the presence of gene clusters associated with the biosynthesis of iturin, surfactin, and fengycin in strain HF1. Figure 3: _B. atrophaeus_ strain HF1 exerts antifungal activity against pear Valsa canker. (**A**) Biocontr of pear Valsa canker with strain HF1 cell suspension. Sterile water (CK) and Tebuconazoleide treatments were, respectively, used for negative and positive control treatments. Scale bar: 1 cm. (**B**) Disease lesion size. Data are means \(\pm\) SD from six biological replicates. Different letters correspond to significant differences at the \(p\) < 0.01 threshold. This experiment was independently repeated three times and yielded similar results. Figure 4: Effect of _B. atrophaeus_ strain HF1 culture filtrate on _V. pyri_ mycelial growth. (**A**) _V. pyri_ colony morphology at 6 dpi on PDA medium containing 2%, 5%, or 10% strain HF1 culture filtrate concentrations. The untreated PDA medium was served as CK. (**B**) _V. pyri_ colony diameters. (**C**) Inhibition rates. Data are means \(\pm\) SD from three biological replicates. Different letters correspond to significant differences at the \(p\) < 0.05 threshold. This experiment was independently repeated three times and yielded similar results. Figure 5: Effect of _B. atrophaeus_ strain HF1 culture filtrate on the membrane permeability of _V. pyri_ hyphae. The hyphae were stained with 1 \(\mu\)M SYOX green at 24 h after being treated with strain HF1 culture filtrate. NA liquid medium treatment was used as CK. Bar: 20 \(\mu\)m. Bright: bright-field; green fluorescence: green fluorescence field; merged: merged images of bright-field and green fluorescence-field. The Impact of B. atrophaeus Strain HF1-Derived VOCs on V. pyri Mycelial Growth and Membrane Permeability  
The impact of VOCs derived from strain HF1 on _V. pyri_ mycelial growth was evaluated. These analyses demonstrated that these VOCs were able to readily suppress the growth of _V. pyri_ mycelia in a dose-dependent fashion (Figure 6A,B). At a strain HF1 concentration of \(1\times 10^{8}\) CFU mL\({}^{-1}\), these VOCs exhibited an inhibition rate of 84.09% (Figure 6C). These VOCs were also able to readily inhibit a range of other pathogenic fungi that damage fruit plants, with respective mycelial growth inhibition rates for _V. mali_, _B. dothidea_, _C. gloeosporoides_, and _B. cinerea_ of 77.90%, 45.52%, 29.2%, and 76.62% (Supplementary Figure S2). Further analyses demonstrated that strain HF1 VOCs fumigation was sufficient to disrupt hyphal morphology, with treated hyphae appearing curved and thinner when examined via microscopy, whereas CK hyphae were smooth and appropriately shaped (Figure 6D). In addition, _V. pyri_ hyphae cell membrane permeabilization was destroyed by strain HF1 VOCs fumigation, since a strong GFP fluorescent signal was detected in strain HF1 VOCs-treated hyphae after SYTOX green staining. There was no GFP fluorescent signal in CK hyphae (Figure 7). These results provided strong evidence that VOCs derived from strain HF1 exhibit high levels of antifungal activity. Figure 6: The impact of _B. atrophaeus_ strain HF1-derived VOCs on _V. pyri_ mycelial growth. (**A**) _V. pyri_ colony morphology following fumigation with 100 \(\upmu\)L of different concentrations of strain HF1 cell suspension. Bacteria-free liquid NA medium fumigation served as CK. (**B**) _V. pyri_ colony diameters. (**C**) Inhibition rates. Data are means \(\pm\) SD from three biological replicates. Different letters correspond to significant differences at the \(p<0.05\) threshold.