## 3 Discussion  
This study was developed with the goal of identifying and characterizing endophytic bacteria with potential utility as biocontrol agents capable of protecting against pear Valsa canker caused by _V. pyri_. Strain HF1 was ultimately identified as the endophyte with strong inhibitor activity. Based on the results of morphological and molecular characterization efforts, this strain was identified as _B. atrophaeus_. _B. atrophaeus_ has previously been shown to inhibit the growth of a range of fungal pathogens, including _B. atrophaea_ and _C. atrophaea_[10; 14]. Consistently, _B. atrophaeus_ was herein found to readily antagonize the growth of _V. pyri_ in vitro and in vivo in this study. As such, _B. atrophaeus_ strain HF1 offers potential value as a novel biocontrol agent that can protect against pear Valsa canker. Figure 9: Antifungal activity of pure compounds against _V. pyri_-induced pear Valsa canker. (**A**) Antifungal activity of three pure compounds against _V. pyri_ in vivo. Healthy twigs were inoculated with mycelial plugs with following fumigation with the indicated pure compounds (125 or 250 \(\mu\)L L\({}^{-1}\) headspace). Water was used as CK treatment. Bar: 1 cm. (**B**) Disease lesion size. Data are presented as the mean \(\pm\) SD of six biological replicates. Different letters represent significantly differences at \(p\) < 0.05. This experiment was performed independently three times with similar results. _B. atrophaeus_ strain HF1 culture filtrate also exhibited significant antagonistic activity, confirming the presence of antimicrobial compounds therein. Lipopeptides are among the main types of antimicrobial compounds produced by _Bacillus_ species [21] and include fenygcin, iturin, and surfactin, all of which reportedly exhibit robust antagonistic activity [22]. [PERSON] et al. [17] found that _B. atrophaeus_ strain CAB-1 was able to produce fengycin, while [PERSON] et al. [14] revealed that _B. atrophaeus_ strain J-1 harbored genes encoding fengycin, iturin A, and surfactin synthetases. Here, strain HF1 was similarly found to encode genes associated with fengycin, iturin, and surfactin biosynthesis. Endophyte-derived lytic enzymes can directly facilitate pathogen biocontrol. For example, Bacillus and Pseudomonas species can produce proteases and chitinases that can degrade the cell walls of fungal pathogens [23]. Here, strain HF1 secretions were found to exhibit robust protease and chitinase activity (Supplementary Table S1), potentially contributing to the observed damage to _V. pyri_ mycelial growth and morphological integrity. Moreover, SYTOX green staining confirmed that culture filtrate prepared from strain HF1 induced severe damage to _V. pyri_ hyphal membranes. These results clearly demonstrated the ability of strain HF1 culture filtrate to partially mediate the observed antifungal activity of this endophytic bacterial strain through the targeting of fungal cell walls and membranes, in line with antifungal mechanisms reported previously for other biocontrol agents [9; 24]. VOCs are secondary metabolites produced by endophytes that can serve as candidate biocontrol agents [25]. Here, strain HF1 was found to exert its antifungal activity against _V. pyri_ at least in part through the production of VOCs, consistent with what has been reported for other Bacillus species [18]. Strain HF1-derived VOCs were able to effectively inhibit _V. pyri_ mycelial growth while causing pronounced morphological and structural damage of the hyphae, indicating the antimicrobial properties of these VOCs. Further SPME-GC/MS-based characterization led to the identification of 27 specific VOCs present within headspace mixtures prepared for strain HF1. Of these, thirteen pure forms were tested for antimicrobial activity, of which hexanoic acid, bivervin, and 2-methylvaleraldehyde were able to effectively suppress _V. pyri_ growth and survival.