The mode of action of plant-associated Burkholderia against grey mould disease in grapevine revealed through traits and genomic analyses

Grapevine (Vitis vinifera L.) is a major crop globally and is highly susceptible to grey mould caused by Botrytis cinerea, which reduces yield and wine quality and leads to significant economic losses. Chemical control is widely used but has drawbacks including cost, limited efficacy across pathogens, ecological issues, and emergence of fungicide resistance. Biocontrol using plant growth-promoting bacteria (PGPB) offers an environmentally friendly alternative by enhancing plant resistance via mechanisms such as rhizosphere colonization, antimicrobial production, and induction of plant immunity (PTI), including ROS accumulation, callose deposition, and defense gene activation. Members of the Burkholderia genus (including Bcc and Paraburkholderia) have shown biocontrol and biofertilizer potential in various crops, yet their role against grapevine pathogens is underexplored. This study investigates two maize-rhizosphere-isolated Burkholderia strains (BE17 and BE24) for their ability to control grey mould in grapevine and to induce systemic resistance, and examines genomic traits underlying their biocontrol and plant growth-promotion potential.

The genus Burkholderia encompasses diverse species from multiple niches and has been reclassified into several genera (Paraburkholderia, Robbsia, Pararobbsia, Mycetohabitans, Trinickia, Caballeronia, and Burkholderia sensu stricto). Plant-associated Burkholderia and Bcc members have been reported as effective biocontrol agents and biofertilizers through secondary metabolites, volatiles, and induction of plant tolerance to stresses. Known antifungal compounds from Burkholderia include pyrrolnitrin, burkholdines, siderophores, xylocandin, cepacidin, and cepacin. PGPB-induced systemic resistance involves PTI-related responses such as ROS, callose deposition, and defense gene activation, often via SA or JA pathways. Despite the promise, the dual beneficial–pathogenic nature of Bcc (including opportunistic human pathogens) necessitates careful evaluation. The study builds on prior evidence of Burkholderia-mediated biocontrol in crops like tomato, pea, citrus, and soybean, and seeks to fill the gap for grapevine and B. cinerea.

- Strain isolation and culture: BE17 and BE24 were isolated in 2016 from maize rhizosphere in northeast France by serial dilution plating on LB. Colonies were purified and stored in LB with 20% glycerol at −80°C. Routine culture on LB at 30°C. - Phenotypic characterization: Morphology, motility, salt (0.5–1% NaCl), and pH (5–8) tolerance; BIOLOG GENIII biochemical profiling. - Molecular identification and phylogeny: 16S rRNA gene amplified (primers FD2/RP1), sequenced (>1420 nt), BLASTed. Phylogenetic tree built with MEGA X using neighbor-joining and Kimura 2-parameter; bootstrap (1000). Draft genomes analyzed for ANI via MiGA and digital DDH via TYGS to assess species relatedness. - In vitro antifungal assays: Dual culture on PDA to assess mycelial growth inhibition; spore germination assay in 96-well plates (PDB), mixing bacterial suspensions with B. cinerea conidia (final 5000 spores per well). Bacterial inocula typically at 10^8 CFU/mL for setup; spore germination inhibition reported at 10^6 CFU/mL. Incubation 24 h at 25°C (dark). Germination assessed microscopically; mycelial inhibition zones measured after 5–7 days at 25°C. - Plant material and bacterization: In vitro grapevine plantlets (Vitis vinifera L. cv. Chardonnay clone 7535) grown on MS medium at 25°C with 16/8 h light/dark (200 µmol m^-2 s^-1). For bacterization, 200 µL of BE17 or BE24 suspension (10^8 CFU/mL in PBS) applied to root zone. Controls received PBS. One week later, plantlets transferred to soil (120 g/magenta box) and acclimated 10 days. - Pathogen inoculation and disease assessment: B. cinerea maintained on PDA; conidial suspensions adjusted to 10^5 conidia/mL in PBS. Whole plantlets sprayed and incubated at 25°C (16/8 h). Detached leaf assay: leaves placed on 0.5% agar and inoculated with 5 µL of conidial suspension. Disease severity evaluated as necrosis diameter at 72 hpi. - Fungal growth quantification in planta: qRT-PCR of B. cinerea actin (Bc-Actin) transcripts at 0, 24, 48, 72 hpi. - ROS and callose assays: Histochemical detection at 8 hpi using DAB (H2O2) and NBT (O2−) staining; leaves destained in boiling ethanol and imaged. H2O2 quantification performed relative to standard curve (µmol g^-1 DW). Callose visualized at 24 hpi via aniline blue staining (0.01% in 150 mM K2HPO4) and fluorescence microscopy (DAPI filter). - Microscopy: 3D microscopy (Keyence) to visualize B. cinerea mycelium at 24, 48, 72 hpi. - Defense gene expression: qRT-PCR at 24 hpi for SA-pathway markers (GST1, PR5, PR10), JA markers (Chit4C, JAZ1). Normalization with EF1-α and 60S RPL18. Three independent experiments with technical replicates. - Genomics: Whole-genome sequencing (Illumina MiSeq/HiSeq 2×250 bp), de novo assembly (SPAdes), annotation (MicroScope, NCBI). Secondary metabolite BGC prediction using antiSMASH. Accession numbers: BE17 WHNU00000000 (WHNU01000000), BE24 WHNT00000000 (WHNT01000000). 16S accessions: MT912593 (BE17), MT912594 (BE24). - Statistics: One-way ANOVA with Tukey test (α=0.05) using GraphPad Prism; three independent experiments (each in triplicate).

- Taxonomy and novelty: 16S rRNA placed BE17 and BE24 within Burkholderia (close to B. ambifaria, B. metallica, B. seminalis, B. cepacia). ANI/DDH analyses indicated both are novel species within the B. cepacia complex (BE24 closest to B. cenocepacia at 94.27% ANI; BE17 closest to B. pyrrocinia 95.18% and B. stabilis 95.16%; DDH <70%). ANI between BE17 and BE24 = 91.91% (DDH 52.4%). - In vitro antifungal activity: Both strains inhibited B. cinerea mycelial growth in dual culture and reduced spore germination by >90% when mixed with 10^6 CFU/mL bacterial suspension. - Disease suppression in planta: Detached leaf assays showed significant reductions in grey mould lesions at 72 hpi. Necrosis diameter reduced by 57% (BE24) and 36% (BE17) vs. non-bacterized control (control necrosis length ~14 mm). Bc-Actin transcript levels were significantly lower in BE17/BE24-treated plants at 24–72 hpi, indicating reduced fungal growth. - Priming of defenses: Upon pathogen challenge, bacterized plants showed increased ROS accumulation at 8 hpi (H2O2 level ~1.5× in BE24-treated vs. control; BE17 measured at 6.97 µmol g^-1 DW) with stronger DAB/NBT staining than controls. Callose deposition at 24 hpi was markedly higher in BE17/BE24 plants (more pronounced with BE24). - Defense gene activation: At 24 hpi, PR5 and PR10 (SA-pathway markers) were significantly upregulated in BE17/BE24-bacterized plants upon B. cinerea challenge; GST, Chit4C, and JAZ1 were not significantly modulated compared to controls. - Genomes: BE17 genome 8,457,567 bp, GC 65.95%, 8034 genes (7958 coding; 7524 protein-coding; 11 rRNA; 61 tRNA; 4 ncRNA). BE24 genome 7,422,622 bp, GC 66.90%, 7090 genes (7001 coding; 6655 protein-coding; 19 rRNA; 66 tRNA; 4 ncRNA). - Secondary metabolism and PGP traits: antiSMASH revealed multiple BGCs (NRPS, bacteriocin, arylpolyene, Hserlactone, terpene, phosphonate, pyrrolnitrin). Both strains possess an ornibactin siderophore NRPS cluster. BE17 harbors an additional NRPS cluster with four NRPS genes (nine modules) predicted to produce a nonribosomal peptide of nine monomers. PGP-related genes detected: ACC deaminase; IAA biosynthesis; phosphate solubilization (Pepc, PQQ-dependent GDH) and phosphate uptake (pstA/B/C/S; Pho regulon). Motility/colonization genes abundant (flagella: BE17 74, BE24 48; signaling: BE17 41, BE24 59), cell wall–degrading enzymes, and multiple secretion systems (T2SS, T3SS, T4SS, T6SS). - Overall: BE17 and BE24 deploy combined direct antibiosis (antifungal metabolites) and indirect ISR (SA-pathway activation, ROS, callose) to suppress B. cinerea in grapevine.

The study addresses the need for sustainable management of grey mould in grapevine by evaluating Burkholderia strains as biocontrol agents. BE17 and BE24 directly inhibited B. cinerea growth and spore germination, likely via secondary metabolites (e.g., pyrrolnitrin, siderophores such as ornibactin, and other NRPS products) and potential nutrient competition. In planta, both strains primed grapevine defenses, as evidenced by elevated ROS production, increased callose deposition, and upregulation of SA-pathway PR genes (PR5, PR10) after pathogen challenge, consistent with induced systemic resistance. Genomic analyses corroborate these mechanisms, revealing rich BGC repertoires, PGP traits (ACC deaminase, IAA, phosphate solubilization/uptake), motility/colonization genes, and secretion systems that facilitate plant association and antagonism. The results demonstrate that BE17 and BE24 reduce disease severity and fungal proliferation, offering a dual mode of action (direct antibiosis + ISR). Given the placement within the Bcc and genomic distinctiveness suggesting novel species, these strains represent promising candidates for biocontrol in viticulture, with the caveat of safety assessments due to Bcc’s association with opportunistic infections. Their multifaceted traits align with the requirements for effective, durable biocontrol under sustainable agriculture paradigms.

Burkholderia sp. BE17 and BE24, two novel Bcc members isolated from maize rhizosphere, effectively control grey mould in grapevine via combined direct antifungal activity and ISR mediated through the SA signaling pathway. They significantly inhibit B. cinerea mycelial growth and spore germination, reduce lesion development, prime ROS and callose responses, and upregulate PR5 and PR10 upon infection. Genomes encode extensive secondary metabolite BGCs (including ornibactin and additional NRPS clusters) and PGP functions (ACC deaminase, IAA, phosphate solubilization/uptake), as well as traits for motility, colonization, and secretion. These features support their deployment as biocontrol agents for sustainable viticulture. Potential future research directions include: comprehensive safety and risk assessments given Bcc affiliations; detailed metabolomic characterization and purification of antifungal compounds; functional genetics to validate key BGCs and ISR determinants; optimization of formulation and delivery methods; evaluation under greenhouse and multi-site field conditions; and assessment of impacts on grapevine microbiomes and wine quality.

- Experiments were conducted on in vitro plantlets and controlled conditions; greenhouse and field validations were not reported. - While within-plant fungal load and lesions were measured, long-term persistence and colonization dynamics were not detailed. - The B. cepacia complex includes opportunistic human pathogens; safety, virulence factor profiling, and regulatory compliance were not addressed experimentally in this study. - Antifungal compounds responsible for activity were predicted genomically but not isolated/chemically validated here. - Host range, spectrum of activity against other grapevine pathogens, and potential non-target effects were not evaluated.