Sessile plants rely on individual cells to perceive pathogens and coordinate defense. Guard cells (GCs), best known for regulating stomatal aperture, have poorly understood intrinsic immune roles. Here, we integrate single-cell and spatial analyses of Arabidopsis thaliana leaves infected with diverse phytopathogens, combining live and fixed imaging of transgenic immune reporters with single cell transcriptomic data. Powdery mildew infection triggers strong salicylic acid (SA) biosynthesis and transport in pavement cells, spreading to neighboring uninfected cells. In contrast, GCs fail to activate SA biosynthesis or SA-responsive genes. These cell-type-specific immune programs distinguishing GC and pavement cells are conserved across infections by the hemibiotrophic fungus Colletotrichum higginsianum and the bacterial pathogen Pseudomonas syringae. Despite impaired SA signaling and response, GCs display rapid calcium influx and pronounced reactive oxygen species bursts, transmitting immune signals to adjacent pavement cells via the apoplast. Notably, GCs are incompatible with adapted fungal pathogens and underwent hypersensitive cell death. Together, these findings uncover distinct immune programs among epidermal cell types, highlight GC-autonomous defense mechanisms, and provide a framework for understanding spatial immune coordination in plants.

Early detection of pre-symptomatic downy mildew (Pseudoperonospora cubensis) infection in cucumbers by using deep learning tools based on hyperspectral imagery

Botanical gardens host diverse plant assemblages that provide valuable opportunities to study fungal biodiversity and plant–fungal interactions. Powdery mildews ( Erysiphaceae ) are common pathogens in these settings, yet little is known about how they co‐occur with culturable fungi present on infected leaves. To document fungi recovered from infected tissue, we surveyed powdery mildew infections at the JC Raulston Arboretum, which is part of North Carolina State University. We identified the powdery mildew species present and the fungi that could be cultured from the infected phyllosphere. Eighteen powdery mildew species representing three genera were documented and confirmed through morphological and molecular methods. Using a dilution‐to‐extinction culturing approach, we recovered 147 isolates corresponding to 57 fungal species across 36 genera. Several genera including Aspergillus and Cladosporium were repeatedly recovered from powdery mildew‐infected leaves, and six isolates represent potentially novel lineages. Several of these genera have previously been shown to possess biological control potential against powdery mildew. These findings demonstrate that botanical gardens harbor rich, underexplored fungal diversity and that powdery mildew–infected tissues provide access to a broad community of co‐occurring fungi. Our results highlight the potential of culture‐based surveys in botanical gardens to uncover novel fungal taxa and identify candidate biological control agents for powdery mildew management.

Protein kinases (PKs) are key regulators of plant development and responses to biotic and abiotic stresses. However, a comprehensive characterization of this superfamily remains lacking in cucumber (Cucumis sativus), particularly regarding its role in powdery mildew resistance caused by Podosphaera xanthii. Here, we identified and characterized the cucumber kinome using Hidden Markov Models (HMMs), revealing 835 PKs distributed across seven chromosomes and classified into 20 groups and 123 families, with RLKs being the most abundant. Gene structure, domain composition, and subcellular localization analyses highlighted substantial structural and functional diversity. Tandem and whole-genome duplication events contributed significantly to PK expansion. Transcriptomic analyses using RNA-seq data from resistant and susceptible cultivars inoculated with P. xanthii revealed between 8 and 105 differentially expressed PK genes (DEGs), depending on the comparison. A greater number of PK DEGs were detected in susceptible genotypes, many of which are putatively associated with signaling pathways and cell cycle regulation. The expression of specific RLKs, MAPKs, CAMKs, and cyclin-dependent kinases suggests their role in defense responses. These findings enhance our understanding of the cucumber kinome and signal the potential of specific PKs in plant-pathogen interactions, providing a foundation for future functional studies and resistance-focused breeding strategies.

Strawberry powdery mildew, caused by Podosphaera aphanis, severely impacts the yield and quality of strawberry fruit and necessitates sustainable control strategies. Here, we assessed the growth and hormone content of strawberry seedlings (Fragaria × ananassa ‘Hongyan’) to evaluate the abilities of four plant growth-promoting bacteria (PGPB), Azospirillum brasilense, Phytobacter diazotrophicus, Variovorax soli, and Microbacterium phyllosphaerae, in a 42-day consecutive culture. Among all treatments, our results showed that the strains V. soli and M. phyllosphaerae increased the seedling biomass by 25.6–25.8% and decreased the disease index by 30–32.5% compared to the control. Moreover, the strain M. phyllosphaerae could produce 22.32 μg·mg−1 indole-3-acetic acid (IAA) after 24 h in a consecutive culture system and still maintain the IAA at 8.12 ng·mg−1 in seedlings. In addition, the salicylic acid (SA) and jasmonic acid (JA) hormones of the seedlings increased by 293% and 35.3% after inoculating with V. soli and M. phyllosphaerae, respectively. Further analysis of the coordination between plant hormones and plant growth showed that the accumulation of SA might have had a positive relation with biomass, whereas JA was negative under powdery mildew infection. These findings highlight that the V. soli and M. phyllosphaerae strains could offer the dual benefits of growth promotion and different defense strategies against powdery mildew for sustainable strawberry production.

Maintaining grapevine health and resilience under climate change demands an understanding of how multiple stresses interact. Climate change has led to more frequent and intense droughts and heat waves, which often occur together and may affect disease pressure. Here, we explored a complex multi-stress framework by combining drought and heat stress with downy mildew infection in grapevine, determining their possible interactions (synergistic or antagonistic) on pathogenesis, and detailing their effects on grapevine leaf physiology. We subjected grapevines (Vitis vinifera L.) to drought, heat stress or a combination of both, subsequently inoculating leaves with Plasmopara viticola (Berk. & Curt. Ex de Bary). In these same leaves we quantified their physiological responses and changes in defense-related metabolite (H2O2 and phenols) concentrations. Our results revealed common and distinct responses to the multi-stress interactions. Drought and drought–heat treatments led to low stomatal conductance (a reduction in stomatal aperture), which acted as a biophysical mechanism restricting P. viticola entry and resulting in a lower P. viticola sporulation rate. Defense-related metabolites were differentially regulated according to the stress combination, suggesting a role in the biochemical mechanism against the P. viticola infection. Stilbene concentration increased by 36, 57 and 113% in response to drought, heat and drought–heat, respectively. Flavonoids and phenolic acids exhibited distinct patterns in response to different stress treatments. These results shed light on the mechanisms that drive complex interactions between abiotic and biotic stressors, enhancing our understanding of multi-stress resilience and allowing us to better predict how grapevines will respond to the changing climate.

Functional characterization of squalene epoxidase genes in Rosa chinensis upregulated by infection with Podosphaera pannosa

Powdery mildew, caused by the fungus Golovinomyces ambrosiae, is a common disease of greenhouse-grown hemp ( Cannabis sativa L.). Symptoms include white, powdery patches on the adaxial leaf surface that spread to other plant tissues including stems and inflorescences, impacting the plant's photosynthetic capacity and marketability. The purpose of this study was to evaluate the efficacy of organic fungicides commonly used to manage hemp powdery mildew in New York. The field trial was conducted in 2025 in Geneva, NY, on ‘White CBG’, a hemp cultivar with known susceptibility to G. ambrosiae. This report provides useful information for hemp growers who aim to reduce losses from powdery mildew infections and to develop disease management programs.

Volatile organic compounds (VOCs) produced by Medicinal Aromatic Plants (MAPs) are bioactive signaling molecules that play key roles in plant defense, acting against pathogens and triggering resistance responses. Intercropping with VOC-emitting MAPs can therefore enhance disease resistance. This study investigated VOCs emitted by sage (Salvia officinalis) as potential resistance inducers in grapevine (Vitis vinifera) against Plasmopara viticola, the causal agent of downy mildew, under consociated growth conditions. Sage and grapevine plants were co-grown in an airtight box system for 24 or 48 h, after which grape leaves were inoculated with P. viticola. Disease assessments were integrated with grapevine leaf metabolic profiling to evaluate responses to VOC exposure and pathogen infection. Untargeted and targeted metabolomic analysis revealed that sage VOCs consistently reprogrammed grapevine secondary metabolism, without substantial differences between 24 and 48 h exposures. Lipids, phenylpropanoids, and terpenoids were markedly accumulated following VOC exposure and persisted following inoculation. Correspondingly, leaves pre-exposed to sage VOCs exhibited a significant reduction in disease susceptibility. Overall, our results suggest that exposure to sage VOCs induces signaling and metabolic reprogramming in grapevine. Further research should elucidate how grapevines perceive and integrate these signals, as well as the broader processes underlying MAP VOC-induced defense, and evaluate their translation into sustainable viticultural practices.

Phytosanitary monitoring of strawberry cultivars and selected forms from the collection of the Federal Horticultural Center for Breeding, Agrotechnology and Nursery was conducted to identify differences in the pathogen complex between open and protected field conditions, with species identification clarified under laboratory conditions. Research was conducted in 2024–2025. We established that the pathogen complexes of micromycetes in strawberry plantings in Moscow and Bryansk oblasts include: Botrytis cinerea, Coniothyrium fuckelii, Cylindrocarpon spp., Dactylonectria macrodidyma, Fusarium solani, Marssonina potentillae var. fragariae, Pestalotia truncata, Phoma spp., Phomopsis spp., Pythium spp., and Rhizoctonia solani. Comparative analysis of the micromycete complexes revealed differences between the regions. For instance, Phytophthora nicotianae oomycete was not detected on plants from Bryansk oblast, while Coniothyrium fragariae leaf spot pathogen was not found in Moscow oblast. The lowest frequency of R. solani occurrence was noted in the Tsaritsa cultivar, with an average percentage of colonization by the fungus observed in Nashe Podmoskovye and Vostorg cultivars. At the same time, a lower incidence of phytophthora wilt was noted in Vityaz and Vostorg cultivars. In Moscow oblast under open field conditions during the growing season, the infection score for leaves by the Botrytis cinerea fungus across cultivars was: Bereginya – 1.5, Vostorg – 1.5, Nashe Podmoskovye – 1.0, Tsaritsa – 2.0. Powdery mildew infection was recorded within a range of 0–1 point on the studied cultivars. Under protected conditions, resistance to gray rot was noted in the Tsaritsa cultivar (0.5 points), while powdery mildew infection was observed in Vostorg and Bereginya cultivars at a level of 0.2–0.3 points. In the conditions of open field in Bryansk oblast, the Tsaritsa cultivar and selected forms Nos. 3-5- 1, 3-831-18, 3-434-5, 3-731-3 were distinguished by their resistance to powdery mildew (0 points). Selected form 3-395-3 showed notable resistance to gray mold.

The research was conducted on the resistance of garden strawberry cultivars with various ripening periods. The cultivars were grown in a climate chamber under controlled conditions simulating the spring–summer weather of the European North-East (Komi Republic). This approach was combined with the creation of an infectious background to study plants affected by powdery mildew. The cultivar samples were assessed for their resistance both to extreme growing conditions (with set parameters of humidity, light, and temperature in programmed regimes) and to infection of their aerial parts by powdery mildew. The study identified sharp diurnal and nocturnal temperature fluctuations (avg. 13±1 °C) as the primary factor limiting garden strawberry performance and the progression of powdery mildew infection. Under the simulated extreme conditions, cultivar samples were classified as showing low resistance or as being weakly or moderately susceptible to powdery mildew infection. The early-ripening Melodiya and the mid-season Feerverk cultivars exhibited the most infection of aerial parts among planted seedlings. Moderate susceptibility to the phytopathogen was observed in the Izbrannitsa and Urozhaynaya TsGL cultivars, while weak susceptibility was noted in the Festivalnaya and Torpeda cultivars.

Geranium carolinianum (commonly known as Carolina crane's-bill, Carolina geranium, and wild Geranium) is an annual herbaceous plant native to North America and widely naturalized in temperate regions worldwide. In Korea, this plant is considered an invasive species due to its rapid spread and competitive displacement of native flora (Secretariat, 2025; Oh et al., 2021). In May 2025, symptoms of downy mildew were observed on G. carolinianum populations growing in Buan, Korea, with a high disease incidence of 50–80%. Infected leaves exhibited chlorotic lesions with angular margins and dense whitish sporulation on the abaxial surfaces, typical of downy mildew infections. As the disease progressed, necrosis and defoliation occurred on severely infected plants. Two representative symptomatic specimens were deposited in the Kunsan National University Herbarium (KSNUH 2478, 2486). Sporangiophores (n=50) were slender, straight or slightly curved, monopodially branched in 2-5 orders, and measured 163–378 (av. 214 ± 49) × 4–8 (av. 6 ± 1) μm. Ultimate branchlets were straight to substraight, 6.7–21.8 (av. 12.1 ± 3.4) μm long, with a cup-like or swollen tip. Sporangia were subglobose to broadly ellipsoidal, rarely ovoidal or almost oblong, and measured 18.6–28.6 (av. 25.0 ± 2.5) × 14.9–22.8 (av. 19.4 ± 1.8) μm, with a length/width ratio of 1.15–1.43 (av. 1.3 ± 0.1). The morphological characteristics match a previous description of Plasmopara wilsonii (Voglmayr et al. 2006). To confirm the pathogen identity, total DNA was extracted from infected tissues. The internal transcribed spacer (ITS) region and the large subunit (LSU) gene of rDNA were amplified using the primer sets ITS1-O/LR0 and LR0R/LR6-O, respectively (Bachofer 2004; Moncalvo et al. 1995; Riethmüller et al. 2002). The obtained sequences were deposited in GenBank (Accession No. PV988127–8 for ITS and PV972918–9 for LSU). A BLASTn search revealed that the ITS sequences shared 99.0% identity with Plasmopara wilsonii sequence from the United States (DQ131919.1), and the LSU sequences showed 100% identity with those from Germany (KR349458.1, KR349457.1). In phylogenetic analyses of ITS and LSU sequences, the Korean isolates formed a well-supported group with P. wilsonii sequences, confirming its identity. For a pathogenicity test, 10 mL of a sporangial suspension (4.0 × 105 conidia/ml) was sprayed on the adaxial surfaces of all leaves of five healthy G. carolinianum plants. Five control plants were treated with sterile water. All plants were kept in darkness for 24 h, followed by incubation in a growth chamber at 90% relative humidity and 22℃, with a 10 h light/14 h dark cycle. After 10–14 days, typical downy mildew symptoms developed on the inoculated leaves, from which the pathogen was confirmed as P. wilsonii via morphological and molecular analyses. Control plants remained symptomless, fulfilling Koch’s postulates. Plasmopara wilsonii has previously been reported on Geranium carolinianum in the United Kingdom and the United States (Farr & Rossman 2025), but to our knowledge, this is the first report of downy mildew disease caused by P. wilsonii on G. carolinianum in Korea. Given that this plant is considered invasive in Korea, the emergence of this downy mildew may have potential implications for both plant health and management.

Oak powdery mildew, caused by Erysiphe alphitoides, is a widespread disease that affects oaks. We aimed to understand the complex interactions between extrinsic (environmental) and intrinsic (provenance) factors that influence the infection rate of E. alphitoides on Quercus robur seedlings. In a common garden controlled water experiment, we investigated the natural infection rate of E. alphitoides considering factors such as oak provenance, soil moisture availability, and the seedlings’ location in the microenvironment. We examined infection rates on first- and second-flush leaves, assessed oak gas exchange responses (net photosynthesis, transpiration, and stomatal conductance) to powdery mildew infections, and evaluated stomatal size and density as a potential pathway for pathogen entry. Our results showed that different factors influence first and second-flush leaf infection rates. First flush infections were milder, primarily depending on provenance (an intrinsic factor), with early leaf development correlating with lower infection rates. Second flush leaf infections in summer were more severe, driven by extrinsic factors such as the microenvironment and water availability, with spore dispersal influenced by prevailing wind direction. We observed a significant effect of E. alphitoides on photosynthesis, along with reduction in infection rates under drought. This suggests that stomatal closure—reflected in reduced stomatal conductance—under drought stress may prevent pathogen penetration. Moreover, our results suggest that stomatal closure itself, rather than the size of the stomatal pore, may play a more critical role in limiting pathogen entry. Further assessment of biochemical defenses in drought-stressed seedlings could help clarify if additional mechanisms are involved.

Abstract Physiological and molecular changes were investigated in barley hormone mutants after powdery mildew infection. Interactions between barley powdery mildew race A6 and barley cultivar Bowman, along with its various brassinosteroid hormone mutants (BW084, BW091, BW312, BW333, 828 and BW885) were all compatible. Fluorescence microscopy revealed strong autofluorescence signal of chlorophyll a at the early stage of the disease, which markedly decreased 10 days after inoculation. At 3 days post-inoculation (DPI), photosynthetic parameters Fv/Fm and qL significantly increased, while NPQ and qN decreased across all genotypes. Reflectance parameters Ctr1 (Carter index) significantly increased in Bowman and BW828 but decreased in BW 312. There was a significant decrease of Lic1 (Lichtenthaler index) in BW885 after infection with powdery mildew. Expression of the pathogenesis-related HvPR1-b gene increased markedly at 3 DPI, whereas the expression of the heat shock protein gene HvHsp90 increased significantly only at day 6 in powdery mildew-infected Bowman and its mutant lines. The 23kD jasmonate-induced protein gene HvJIP-23 was slightly down-regulated at both 3 and 6 DPI in each genotype. No significant difference was observed in expression of HvJIP-23 between Bowman and its near-isogenic mutants. The root abundant factor gene HvRAF was considerably down-regulated after powdery mildew infection at 3 DPI in each barley genotype as compared with the uninoculated control.

Grapevines are fruit crops that are of significant importance not only economically but also culturally to the world. Still, they are very vulnerable to diseases of mildew and other infections, which jeopardize the quality of fruits, health of plants, and the productivity of vineyards. These diseases can be effectively managed and cultivated by early and accurate disease detection. This review provides a summary of the major detection techniques that are in use such as traditional visual inspections,molecular diagnostic,sensor-based and the latest development in Artificial Intelligence.Deep Learning-based image recognition frameworks enable rapid and precise analysis of grapevine leaf and fruit images supporting early detection of disease symptoms and efficient vineyard monitoring. Regardless of these developments, there are still issues in tackling versatile interactions of pathogens, minimization of expenses, and applicability on the field. The future prospects would be the incorporation of the biological understanding with AI-driven detection systems to assist in early detection, better management of the diseases and long-term sustainability of the vineyards.

Powdery mildew poses a significant threat to watermelon production. The development of disease-resistant varieties through gene editing represents a major focus in current breeding research. In this study, we identified an MLO family gene in watermelon, denoted by ClMLO5b, which is phylogenetically closely related to cucumber CsaMLO8 and melon CmMLO5. Homology modeling revealed high conservation of the three-dimensional protein structures among these orthologs. Expression analysis demonstrated that ClMLO5b is significantly up-regulated upon powdery mildew infection, and the protein localizes to the plasma membrane. To validate its function, we first employed an Agrobacterium rhizogenes-mediated hairy root transformation system to rapidly verify the editing efficiency of two CRISPR/Cas9 targets designed for ClMLO5b. Subsequently, stable transgenic watermelon plants were generated via Agrobacterium tumefaciens-mediated transformation, and a mutant line with homozygous substitutions at target site 2 was obtained. Disease resistance assays showed that, compared to wild-type plants, the Clmlo5b exhibited strongly inhibited mycelial growth, significantly reduced disease severity, and a substantial decrease in spore production after inoculation with powdery mildew. Our findings confirm that ClMLO5b is a key susceptibility gene in watermelon and provide both a promising genetic target and valuable breeding material for developing powdery mildew-resistant watermelon varieties.

Kentucky bluegrass powdery mildew, caused by the fungus Blumeria graminis f. sp. poae, is a destructive disease affecting Poa pratensis L. In this study, endophytic bacteria were isolated from the resistant Kentucky bluegrass cultivar ‘Taihang’. Employing a combination of conidia germination inhibition assays and control efficacy tests, the biocontrol endophytic bacterial strains were screened. The impact of inoculation with the powdery mildew pathogen and biocontrol endophytic bacteria on the difference in endophytic bacterial community in the leaves of Kentucky bluegrass were studied via Illumina Miseq high-throughput 16S ribosomal RNA gene sequencing technology. A total of 18 endophytic bacterial isolates were obtained from ‘Taihang’, belonging to 3 phyla: Proteobacteria (3 isolates), Actinobacteria (6 isolates), and Firmicutes (9 isolates). The conidia germination assay revealed that isolates 6213 (Bacillus sp.) and 718 (Neobacillus sp.) exhibited the strongest inhibitory against Blumeria graminis f. sp. poae, with inhibition rate exceeding 80%. Isolate 718 exhibited superior control efficacy over strain 6213. A concentration of 109 colony-forming units per milliliter (CFU/mL) was the most effective in suppressing powdery mildew on Kentucky bluegrass. The abundance of Proteobacteria on Kentucky bluegrass after the application of isolate 718 may enhance the resistance of Kentucky bluegrass to powdery mildew, and the dominant endophytic bacterial communities were Burkholderiales, Burkholderiaceae and Cupriavidus, indicating that the application of isolate 718 modulated the plant’s response to powdery mildew infection. These results demonstrate that isolate 718 enhanced the resistance of Kentucky bluegrass against powdery mildew by reshaping the endophytic bacterial community within the leaves. These findings provide molecular insights into plant−pathogen−endophytic bacteria interactions and support the development of sustainable strategies, eco-friendly strategies for plant diseases management.

The interactions between powdery mildews (Ascomycota, Erysiphaceae), obligate biotrophic pathogens of many plants, and pycnidial fungi belonging to the genus Ampelomyces, are classic examples of specific mycoparasitic relationships. These interactions are common and finely tuned tritrophic relationships amongst host plants, powdery mildews, and Ampelomyces mycoparasites wherever these organisms co-occur in the field. Selected Ampelomyces strains have already been developed as biocontrol agents of powdery mildew infections of some crops. In Australia, their study has received little attention so far. Only a single Ampelomyces strain, included in a whole-genome sequencing (WGS) project, was known from this continent. Here, we report the isolation of 20 more Ampelomyces strains from eight powdery mildew species in Australia. Multi-locus phylogenetic network analyses of all the 21 Australian Ampelomyces strains carried out in combination with 32 reference strains from overseas revealed that the Australian strains belonged to four molecular taxonomic units (MOTUs). All those MOTUs were delimited earlier based on Ampelomyces strains isolated in Europe, North America, and elsewhere. Based on the phylogenetic analyses, two Australian strains belonging to different MOTUs were selected for WGS. Long-read (MinION) and short-read (Illumina) technologies were used to provide genome assemblies with high completeness. Both assemblies have a bipartite structure, i.e., consisted of AT-rich, gene-sparse regions interspersed with GC-balanced, gene-rich regions. These new high-quality assemblies and evidence-based annotations are important resources for future analyses of mycoparasitic interactions to disentangle molecular mechanisms underlying mycoparasitism, possible new biocontrol applications, and naturally occurring tritrophic relationships.

The interactions between powdery mildews (Ascomycota, Erysiphaceae), obligate biotrophic pathogens of many plants, and pycnidial fungi belonging to the genus Ampelomyces, are classic examples of specific mycoparasitic relationships. These interactions are common and finely tuned tritrophic relationships amongst host plants, powdery mildews, and Ampelomyces mycoparasites wherever these organisms co-occur in the field. Selected Ampelomyces strains have already been developed as biocontrol agents of powdery mildew infections of some crops. In Australia, their study has received little attention so far. Only a single Ampelomyces strain, included in a whole-genome sequencing (WGS) project, was known from this continent. Here, we report the isolation of 20 more Ampelomyces strains from eight powdery mildew species in Australia. Multi-locus phylogenetic network analyses of all the 21 Australian Ampelomyces strains carried out in combination with 32 reference strains from overseas revealed that the Australian strains belonged to four molecular taxonomic units (MOTUs). All those MOTUs were delimited earlier based on Ampelomyces strains isolated in Europe, North America, and elsewhere. Based on the phylogenetic analyses, two Australian strains belonging to different MOTUs were selected for WGS. Long-read (MinION) and short-read (Illumina) technologies were used to provide genome assemblies with high completeness. Both assemblies have a bipartite structure, i.e., consisted of AT-rich, gene-sparse regions interspersed with GC-balanced, gene-rich regions. These new high-quality assemblies and evidence-based annotations are important resources for future analyses of mycoparasitic interactions to disentangle molecular mechanisms underlying mycoparasitism, possible new biocontrol applications, and naturally occurring tritrophic relationships.

BackgroundGrapevines are among the most economically important fruit crops, and the microbiome profoundly influences their health, yield, and quality. However, mechanistic insights into microbiome-orchestrated grapevine biology remain limited.ResultsHere, we conduct large-scale pan-metagenomic and pan-metatranscriptomic analyses of the phyllosphere microbiome from 107 grapevine accessions spanning 34 Vitis species. We show that the grapevine core microbiome is dominated by phyla Bacillota and Pseudomonadota. Leveraging PacBio sequencing, we assembled 19 high-quality metagenome-assembled genomes (MAGs) from the grapevine pan-microbiome, representing the first MAG reconstruction in plant-associated microbial communities using PacBio reads. These MAGs encode genes associated with antibiotic resistance, secondary metabolism, and carbohydrate-active enzymes (CAZymes), which could potentially influence grapevine biology. During downy mildew (DM) infection, DM-resistant grapevines exhibit significantly higher microbial network complexity than susceptible counterparts. Among the key taxa contributing to this complexity, Bacillota emerged as the dominant phylum, displaying strong abundance correlations with phylum Euglenozoa and Cyanobacteriota, and an isolated Bacillota species from the grapevine leaves, Bacillus cereus, demonstrated potent biocontrol activity against DM infection. Pan-metatranscriptomic analysis further revealed significant upregulation of eukaryotic microbial genes involved in primary and secondary metabolism.ConclusionsThis pan-metagenomic study offers unprecedented insights into the complex structure, diversity, and functional roles of the grapevine phyllosphere microbiome and presents valuable genomic and microbial resources for microbiome research and engineering to enhance viticulture productivity and quality.Video Supplementary InformationThe online version contains supplementary material available at 10.1186/s40168-025-02287-4.