Grapevine (*Vitis vinifera*) is a major fruit crop globally, particularly in France, but its productivity is threatened by grey mold disease, caused by *Botrytis cinerea*. Chemical pesticides are commonly used for control but are costly, less effective against a wide range of pathogens, and pose ecological risks such as the development of pesticide resistance. Biological control strategies, using plant growth-promoting bacteria (PGPB), offer a more sustainable solution. PGPB enhance plant resistance through mechanisms including root colonization, competition with other microbes, production of antimicrobial compounds, and induction of plant-mediated resistance responses. The genus *Burkholderia* contains various soil bacteria found in diverse ecological niches, some of which have been shown to have biocontrol properties in other plants. However, their role as biocontrol agents for grapevine pathogens remains understudied. This study investigated two *Burkholderia* strains, BE17 and BE24, isolated from the maize rhizosphere, to determine their biocontrol potential and mechanism of action against *B. cinerea* in grapevine.

Extensive research highlights the devastating impact of *Botrytis cinerea* on grapevine production, causing significant economic losses. Chemical control methods, while widely employed, present environmental drawbacks. The rise of pesticide resistance necessitates the exploration of alternative approaches. The use of PGPB, including *Burkholderia* species, has gained traction as a sustainable solution. Studies have demonstrated the ability of some *Burkholderia* strains to suppress fungal growth and enhance plant immunity in various species. However, the detailed mechanisms by which *Burkholderia* strains achieve biocontrol in grapevines remain poorly understood. This gap in knowledge underscores the importance of this study in evaluating the effectiveness and elucidating the mechanisms of *Burkholderia*-mediated resistance against *B. cinerea* in grapevines.

Two *Burkholderia* strains, BE17 and BE24, isolated from the maize rhizosphere, were characterized morphologically and biochemically using BIOLOG GENIII microtiter plates and 16S rRNA sequencing. Phylogenetic analysis and ANI/DDH calculations were used to determine their taxonomic classification. *In vitro* antifungal activity was assessed by measuring the inhibition of *B. cinerea* spore germination and mycelium growth. The biocontrol efficacy of the strains against *B. cinerea* was evaluated *in vivo* using detached leaves and *in vitro* grapevine plantlets. The plantlets were treated with bacterial suspensions and subsequently infected with *B. cinerea*. Disease severity was assessed by measuring the size of necrotic lesions. Reactive oxygen species (ROS) accumulation (H₂O₂ and O₂⁻) and callose deposition were determined using histochemical staining methods. Gene expression analysis using qRT-PCR was conducted to evaluate the expression of defense-related genes (PR5, PR10, GST, Chit4C, JAZ1) in control and bacterized plants after *B. cinerea* infection. Whole-genome sequencing of BE17 and BE24 was performed to identify genes involved in biocontrol and plant growth promotion. *In silico* analysis was conducted using the antiSMASH server to identify secondary metabolite gene clusters. Statistical analysis using one-way ANOVA with Tukey's test was performed to determine significant differences.

Both *Burkholderia* strains, BE17 and BE24, effectively inhibited *B. cinerea* spore germination and mycelium growth *in vitro*. *In vivo*, both strains significantly reduced grey mold disease severity in grapevine plantlets compared to the control. Bacterized plantlets showed increased ROS accumulation (H₂O₂) and callose deposition upon *B. cinerea* infection. Gene expression analysis revealed significantly upregulated expression of PR5 and PR10 genes in bacterized plants after pathogen challenge, indicating activation of the SA-signaling pathway. Genome sequencing and analysis revealed the presence of numerous genes associated with plant growth promotion (e.g., ACC deaminase, IAA production, phosphate solubilization) and biocontrol (e.g., secondary metabolite biosynthesis, cell wall degrading enzymes). Specifically, both strains possessed gene clusters involved in ornibactin biosynthesis, and BE17 contained an additional cluster predicted to produce a nonribosomal peptide. The ANI values between BE17 and BE24, and their closest relatives, were lower than 95%, and digital DDH values were less than 70%, suggesting that these strains represent novel species.

The findings demonstrate that *Burkholderia* strains BE17 and BE24 effectively control grey mold disease in grapevines through a combination of direct antagonism and induced systemic resistance (ISR). The *in vitro* and *in vivo* assays confirmed the antifungal activity of these strains. The increased ROS production and callose deposition observed in bacterized plants are consistent with the activation of plant defense mechanisms. The upregulation of PR5 and PR10 genes further supports the involvement of the SA-signaling pathway in ISR. The genomic analysis identified several genes related to plant growth promotion and biocontrol. These findings highlight the multi-faceted mechanisms of action employed by BE17 and BE24, enhancing their effectiveness in disease control. These strains thus represent a promising alternative strategy for sustainable agriculture. Further research could focus on field trials to assess the strains' effectiveness under diverse environmental conditions and to investigate the role of specific secondary metabolites in biocontrol.

This study demonstrated the significant biocontrol potential of two novel *Burkholderia* strains, BE17 and BE24, against grapevine grey mold. Both strains exhibited strong antifungal activity and induced systemic resistance in grapevine. Genomic analysis confirmed their capacity for plant growth promotion and revealed mechanisms for direct antagonism. These findings support the development of *Burkholderia*-based biopesticides for sustainable grapevine disease management. Further research is warranted to optimize application strategies and assess long-term efficacy and environmental impact.

The study was conducted under controlled laboratory and greenhouse conditions. Further research is needed to assess the efficacy of these strains under field conditions, where environmental factors can influence biocontrol effectiveness. The specific roles of the various secondary metabolites identified in the genomic analysis need further investigation to confirm their contribution to biocontrol activity. A larger sample size in field studies would also strengthen the generalizability of the findings.