Harnessing an endophytic Bacillus strain closely related to Bacillus velezensis for biocontrol of twig blight in bayberry caused by Pestalotiopsis versicolor

Twig blight disease caused by Pestalotiopsis versicolor threatens bayberry ( Myrica rubra ) cultivation in China, prompting extensive reliance on chemical fungicides with limited long-term efficacy. This study investigated the potential of endophytic bacteria isolated from bayberry leaves as sustainable biocontrol agents against this pathogenic fungus. An endophytic bacterium designated strain Bay9 was isolated from healthy bayberry leaves and identified as a Bacillus sp. closely related to Bacillus velezensis based on 16 S rRNA gene sequence analysis. Bay9 exhibited strong antifungal activity against P. versicolor in vitro and showed disease-suppressive effects under semi-controlled greenhouse conditions. On potato dextrose agar (PDA) and in potato dextrose broth (PDB), inhibition rates reached 74.44% and 87.65%, respectively, accompanied by severe ultrastructural damage to fungal hyphae observed by electron microscopy. Cell-free supernatants (CFSs) of Bay9 exhibited concentration-dependent antifungal activity, with inhibition rates of 15.77%, 34.99%, 66.74%, and 98.82% at concentrations of 2.5%, 5%, 10%, and 20% (v/v), respectively. CFSs at EC 50 and EC 90 concentrations (7.30% and 17.43%, respectively) significantly reduced spore production and germination, induced leakage of intracellular nucleic acids and proteins consistent with membrane perturbation and leakage, but not direct evidence of a defined molecular mechanism and substantially reduced colony formation. In detached leaf assays, EC 50 and EC 90 treatments reduced lesion development by 65.31% and 94.58%, respectively. Under semi-controlled greenhouse conditions, Bay9 treatment significantly reduced disease incidence (from 97% to 49.33%) and disease severity index (from 82.19% to 38.69%) compared with pathogen-inoculated plants ( p < 0.05), without causing phytotoxic effects on bayberry saplings. Bay9 exhibited strong antifungal activity associated with multiple putative mechanisms, potentially involving siderophore production, hydrolytic enzyme activities (protease, amylase, and lipase), and lipopeptide-related metabolites, as suggested by biochemical assays and MALDI-TOF-MS-based tentative identification of surfactin-, iturin-, and fengycin-like compounds. Collectively, these findings suggest that Bay9 has potential as a biocontrol agent against P. versicolor under laboratory, detached-leaf, and semi-controlled greenhouse conditions. However, further studies are required to elucidate the underlying molecular mechanisms and to validate its efficacy under field conditions before practical application can be recommended.