Indigenous Olive Orchard Bacteria as Biocontrol Agents: An Integrated Culture-Dependent and Soil Microbiome Approach

TL;DR

This study explores bacteria in olive orchard soils that can fight harmful pathogens, suggesting a sustainable way to manage plant diseases.

Contribution

The study identifies phenology-related shifts in soil microbiomes and isolates bacteria with antagonistic potential against olive pathogens.

Findings

Higher cultivable bacterial diversity was observed during the fruit formation stage compared to the flowering stage.

Certain bacterial strains showed antagonistic activity against olive pathogens.

Soil microbiome analysis revealed taxa significantly enriched or depleted during olive fruit formation.

Abstract

Olive orchard soils are a source of microorganisms capable of inhibiting major olive pathogens. In this study, rhizobacteria were isolated and characterized based on plant growth-promoting traits, and soil 16 rRNA gene sequencing analysis was performed to analyze microbial communities at two key olive phenological stages (flowering and fruit formation). Using a culture-dependent approach, a total of 90 bacterial isolates representing distinct colony morphotypes were recovered from olive soils, with 35 during the flowering stage and 55 during the fruit formation stage, indicating a higher cultivable diversity during the latter period. We identified some bacterial strains with antagonistic activity and observed phenology-related shifts in the soil microbiome. Using differential abundance analysis, we identified bacterial taxa that were significantly enriched or depleted during olive fruit formation. Overall, this study demonstrates that olive-associated bacteria harbor antagonistic potential against olive pathogens. The use of bacteria adapted to olive agroecosystems represents a promising strategy for sustainable disease management.

Figures

Full text

1. Introduction

Mediterranean olive-growing regions, particularly Spain and Italy, face significant economic losses due to phytopathogenic diseases, with yield reductions of 10–20% reported in severely affected orchards, together with increased costs associated with labor and chemical treatments [1]. Among the most damaging diseases, olive knot disease and olive leaf spot represent major constraints to olive (Olea europaea L.) production.

Olive knot disease, caused by Pseudomonas savastanoi pv. savastanoi (Psv), is one of the most economically relevant bacterial diseases affecting olive trees. The pathogen induces hyperplastic and hypertrophic tumors (knots) on aerial organs, impairing vascular function, reducing tree vigor, and ultimately leading to yield losses [2]. Disease management has usually relied on preventive agronomic practices combined with copper-based bactericides. However, the long-term use of copper compounds has raised serious environmental concerns due to their accumulation in soils and toxicity to non-target organisms, including beneficial microbiota. In addition, the emergence of copper-tolerant Psv strains and recent regulatory restrictions, such as Commission Implementing Regulation (EU) 2018/1981 [3], have further limited the effectiveness and sustainability of copper-based control strategies [4]. In this context, biological control strategies based on beneficial microorganisms have gained increasing attention as environmentally friendly alternatives. Indigenous biological control agents (BCAs), particularly those adapted to olive agroecosystems, are of special interest due to their ability to suppress pathogens while enhancing plant health.

Several bacterial genera commonly associated with olive soils and rhizospheres, including Bacillus, Paenibacillus, and Pseudomonas, have demonstrated antagonistic activity against Psv through mechanisms such as antibiosis, competition for nutrients and ecological niches, and induction of systemic resistance [2,5]. Recent studies have shown that native Bacillus spp. strains can achieve up to 98% inhibition of tumor formation under in vivo conditions, highlighting the strong potential of indigenous microorganisms for sustainable olive disease management [6]. Beyond bacterial diseases, olive cultivation is also severely affected by fungal pathogens, particularly Venturia oleaginea, the causal agent of olive leaf spot (peacock spot disease). This pathogen causes leaf lesions, defoliation, and reduced photosynthetic capacity, leading to yield losses under favorable climatic conditions [7]. Although chemical fungicides, including copper formulations, remain the primary control option, several biological alternatives have shown promising results. Strains such as Bacillus subtilis QST 713, B. amyloliquefaciens D747, and Pseudomonas fluorescens Pf-5 have demonstrated significant reductions in disease severity under greenhouse and field conditions [8]. However, compared to bacterial diseases, the biological control of V. oleaginea remains less explored, underscoring the need for further research in this area.

Soil constitutes a major reservoir of microbial diversity and a key source of microorganisms with potential biocontrol activity. In perennial crops such as olive, soil microbial communities are influenced by plant developmental stage, root exudation patterns, and agricultural management practices. Phenological stages such as flowering and fruit formation are associated with shifts in plant physiology that may affect microbial recruitment and functional potential in the rhizosphere. Understanding these dynamics is essential for identifying microbial taxa and functions associated with disease suppression. The selection of microbial BCAs has increasingly focused on strains exhibiting plant growth-promoting (PGP) traits, including phosphate solubilization, indole-3-acetic acid (IAA) production, and siderophore secretion [9,10]. These traits contribute to plant nutrition and stress tolerance while also enhancing pathogen suppression through nutrient competition and indirect antagonism [11,12]. Strains combining multiple PGP traits may exert synergistic effects, reinforcing both plant growth promotion and disease control [13]. While culture-dependent approaches remain essential for isolating and functionally characterizing microbial antagonists, they capture only a fraction of the microbial diversity present in soil. Therefore, high-throughput 16S rRNA gene amplicon sequencing has become a standard tool for profiling bacterial community composition and structure across environmental conditions and phenological stages [14,15].

We have combined culture-dependent isolation and functional characterization of bacterial strains with 16S rRNA gene amplicon sequencing to investigate olive orchard soils as a source of microorganisms with antagonistic activity against Pseudomonas savastanoi pv. savastanoi and Venturia oleaginea. Specifically, we aimed to (i) isolate and screen native bacterial strains based on PGP traits and in vitro antagonistic activity, and (ii) assess changes in soil bacterial community composition between flowering and fruit formation stages. This integrative approach seeks to identify indigenous microbial candidates and support the development of sustainable, bio-based strategies for olive disease management.

2. Materials and Methods

2.1. Microbial Control Strains

Strains used as controls in the different assay procedures are described in Table 1.

2.2. Sampling and Isolation

Soil samples were collected from an olive orchard located in Almedíjar (Eastern Spain; 39.875638733590115, −0.434148993319731) during the 2024 growing season, covering the flowering and fruit formation stages from May to September. We established ten equidistant sampling points across the field and collected samples in a zigzag transect, avoiding areas near margins and hydrants. Samples were not collected directly from plant species (Figure 1).

The field was dry at the time of sampling, with no recent history of irrigation or fertilization. Soil samples were collected from a depth of 20 cm beneath the tree canopy, at a point away from the main trunk [16,17]. Following collection, samples were placed in labeled self-sealing bags and kept cold for transport to the laboratory. A composite sampling strategy was implemented to capture the high spatial heterogeneity of soil microbial communities and ensure a representative characterization of the orchard [18]. Sampling was conducted during two key phenological stages: flowering and fruit formation. At each site, ten random subsamples were collected within the same plot and thoroughly homogenized in the laboratory to obtain a single composite sample per plot. All samples were taken from a depth of 0–20 cm, targeting the upper soil layer where root density, organic matter, and microbial activity are highest, thereby maximizing the likelihood of isolating functionally diverse PGPR strains [19,20]. This approach provides a robust foundation for both culture-dependent isolation of antagonistic bacteria and subsequent 16S rRNA gene amplicon sequencing analysis.

Aliquots of 50 g of soil were sieved to remove stones and mixed with 50 mL of saline solution (0.85% NaCl) in three separated sterilized Erlenmeyer flasks. After 1 h of agitation at 180 rpm at room temperature, the supernatant was recovered and serial dilutions were prepared. From each dilution, 100 μL were spreaded in 10% Tryptic Soy Agar (TSA) supplemented with 0.01% (w/v) cycloheximide (ThermoFisher Scientific, Waltham, MA, USA). The plates were incubated at 30 $[eqn]$ C for five days. Culturable bacteria were isolated using a culture-dependent approach and purified on TSA. A total of 90 bacterial isolates were grown in TSB and stored at −80 $[eqn]$ C for further uses. To evaluate the properties of the isolates as PGP, some specific assays were carried out.

2.3. Isolates Characterization and Selection

2.3.1. Phosphate Solubilization

Phosphate solubilization was quantitatively assayed by measuring halos at 2, 5, and 7 days of incubation at 30 $[eqn]$ C as described by Nautiyal [21]. Control strain shown in Table 1.

2.3.2. Siderophore Production

Siderophore production was evaluated using Chromium azurol S (CAS) medium as described by Louden [22], with a modification in which 1:5 diluted TSA was used to reduce nutrient concentration and minimize potential toxicity of medium components [23]. Halos were measured at 2, 5, and 7 days of incubation at 30 $[eqn]$ C in the dark. The control strain is shown in Table 1.

2.3.3. Indole-3-Acetic Acid Production

Indole-3-acetic acid (IAA) production was determined according to Eckmann and Dessaux [24], with isolates grown in TSB at 30 $[eqn]$ C and 170 rpm. Absorbance was measured at 24, 48, and 120 h, and IAA concentration was calculated using a calibration curve. The control strain is shown in Table 1.

2.3.4. Criteria for Isolate Selection

Isolates were selected based on the consistent expression of at least one PGP trait, with particular emphasis on siderophore production. Priority was given to isolates from olive orchard soils, as indigenous microorganisms are expected to be better adapted to the olive agroecosystem. In addition, two reference strains, Bacillus amyloliquefaciens [25] and Priestia megaterium [6], isolated in our laboratory from citrus and potato soils, respectively, were also used.

Bibliographic evidence supports their use as biocontrol microorganisms.

2.4. Microbial Identification

2.4.1. 16S rDNA Partial Sequence

Microbial identification, including genomic DNA extraction, PCR amplification, and downstream analyses, was carried out following the protocol described by Arahal et al. [26]. The bacterial 16S rRNA gene was amplified using the universal primers SWI-F ( $[eqn]$ -AGAGTTTGATCCTGGCTCAG- $[eqn]$ ) and SWI-R ( $[eqn]$ -GGTTACCTTGTTACGACTT- $[eqn]$ ) [27] in a DLAB TC-1000G thermocycler (DLAB, Beijing, China). The resulting PCR amplicons were resolved by electrophoresis on a 1% (w/v) agarose gel, visualized under UV illumination, and subsequently purified using the High Pure PCR Product Purification Kit (Boehringer, Mannheim, Germany). Purified products were sequenced using the ABI Prism BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Stafford, TX, USA) at the SCSIE facility (Universitat de València, Burjassot, Spain). The obtained sequences were edited and aligned, and taxonomic identification was achieved by comparison with EMBL 16S rDNA reference sequences using the BLAST v2.17.0 algorithm [28].

2.4.2. MALDI-TOF

Bacterial strain identification was performed according to the standard Bruker Daltonics protocol using the extended direct transfer method. Freshly grown bacterial cultures were subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis with a Microflex L20 instrument (Bruker Daltonics, Billerica, MA, USA) equipped with a nitrogen (N_2_) laser. Mass spectra were acquired in positive linear ion mode at an acceleration voltage of 20 kV, accumulating 240 laser shots per target over a mass-to-charge (m/z) range of 2000–20,000 Da. For each strain, three independent spectra were generated using the MALDI Biotyper Realtime Classification (RTC) workflow to ensure reproducibility. Species-level identification was assigned based on the highest log score obtained by comparison with reference spectra contained in the MBT 7854 and MBT 7311-RUO databases [29].

2.5. 16S rRNA Gene Sequencing and Bioinformatics Analyses

Once in the laboratory, and following the same rationale applied during sample preparation for isolations, the ten soil samples collected at each phenological stage were thoroughly homogenized to obtain a single composite sample representative of each stage, to enhance overall homogeneity and minimize the inherent spatial variability characteristic of agricultural samples. Three independent subsamples were collected from each composite sample and used as biological replicates for subsequent analyses. Amplicone-based microbiome analysis analyses were performed using these soil samples. Total DNA was extracted from each subsample, followed by amplification of the V3–V4 regions of the 16S rRNA gene according to the Illumina Amplicone-based Microbiome Analysis Sequencing Library Preparation protocol (Cod. 15044223 Rev. A), using primers with Illumina overhang adapters, as described by Klindworth et al. [30]. After amplification, samples were indexed using the Nextera XT Kit, and the expected amplicon size ( $[eqn]$ 50 bp) was confirmed with a Fragment Analyzer 5200 (Agilent, Santa Clara, CA, USA). Libraries were sequenced on a MiSeq platform using a 2 × 301 bp paired-end configuration (MiSeq Reagent Kit v3). Demultiplexed FASTQ files were processed using QIIME2 v2024.10. Quality filtering, denoising, chimera removal, and amplicon sequence variant (ASV) inference were performed using the DADA2 pipeline [31]. Taxonomic assignment was performed using the SILVA v138 reference database, and singleton ASVs were removed prior to downstream analyses. Alpha-diversity indices (Shannon, Pielou’s evenness, Faith’s phylogenetic diversity, and observed OTUs) and beta-diversity metrics (Bray–Curtis, Jaccard, unweighted UniFrac, and weighted UniFrac) were calculated after rarefying all samples to the minimum sequencing depth. Differences in alpha-diversity between phenological stages were assessed using the Kruskal–Wallis test with false discovery rate. To identify microbial features that were differentially abundant between stages, the Analysis of Compositions of Microbiomes with Bias Correction (ANCOM-BC) approach was applied, following the methodology described by Lin and Peddada [32].

2.6. Inhibition Assays

Inhibition assays were performed to evaluate the ability of the selected microorganisms to suppress olive pathogens, based on their key PGP activity profiles. All experiments were conducted in at least by triplicate to ensure representativeness. The incubation temperatures used corresponded to the optimal growth conditions for each pathogen, as determined from both the literature and prior experiences.

2.6.1. Inhibition Assay Against Pseudomonas savastanoi

In vitro antagonism against Pseudomonas savastanoi pv. savastanoi was evaluated using the disc diffusion method on TSA plates [33]. Both pathogen and antagonistic bacterial strains were standardized to $[eqn]$ nm = 0.5 and inoculated simultaneously, with 20 µL applied to sterile paper discs. Plates were incubated at 28 ± 2 °C, a temperature commonly reported as optimal for phytopathogenic Pseudomonas spp. [6]. The inhibitory effect was measured after 2, 5, and 7 days, as these intervals allowed detection of differences relative to the negative control and revealed variations in inhibition dynamics among isolates. These conditions were selected based on internal laboratory experience with in vitro inhibition assays.

2.6.2. Inhibition Assay Against Venturia oleaginea

Antagonistic activity against Venturia oleaginea was assessed by placing a fungal agar plug at the center of PDA plates containing a bacterial lawn of $[eqn]$ nm = 0.5. Control plates contained no bacterial inoculum. Plates were incubated at 21 °C, within the optimal growth range of this slow-growing pathogen [7,34]. Fungal growth was evaluated at 21 and 28 days, as extended incubation is required to allow sufficient mycelial development and reliable detection of inhibition. Incubation conditions were selected based on both literature reports and internal laboratory experience with fungal antagonism assays.

3. Results

3.1. Bacterial Abundance and Alpha/Beta Diversity Measures

Using a culture-dependent approach, a total of 90 bacterial isolates representing distinct colony morphotypes were recovered from olive orchard soils. We obtained 35 morphotypes from the flowering stage and 55 from the fruit formation stage; a higher cultivable diversity was detected in the latter period. From this collection, nine isolates were selected for further characterization based on their expression of key plant growth-promoting traits, including phosphate solubilization, siderophore production, and IAA synthesis.

In parallel, a culture-independent approach based on Illumina MiSeq sequencing was applied to soil samples, yielding a total of 23,303 high-quality reads. 7398 amplicon sequence variants (ASVs) were identified. The overwhelming majority of ASVs (99.86%) were assigned to the domain Bacteria, with a minor proportion of Archaea. Following rarefaction and the removal of singleton sequences, 48 operational taxonomic units (OTUs) were retained and used for subsequent diversity and community structure analyses.

Alpha-diversity metrics, including Shannon diversity, Pielou’s evenness, Faith’s phylogenetic diversity, and observed OTU richness, exhibited comparable values between the flowering and fruit formation stages, with no statistically significant differences (Figure 2). Nonetheless, variation in richness and phylogenetic diversity indices suggested the occurrence of subtle shifts in community composition and evolutionary breadth between the two phenological stages. Similarly, beta-diversity analyses based on Bray–Curtis, Jaccard, unweighted UniFrac, and weighted UniFrac distance metrics did not reveal statistically significant differences between the flowering and fruit formation stages (p > 0.05 for all metrics). Despite the lack of significance, ordination patterns consistently indicated a tendency toward partial separation of microbial communities, pointing to phenology-related restructuring that may not be fully captured by global distance-based comparisons.

With respect to the Adonis tests applied across the four distance-based analyses, the results indicate that there is insufficient statistical evidence to assert a significant difference between the two phenological states confidently. Nevertheless, a consistent trend toward community differentiation is apparent. Given the limited sample size, however, any further interpretation should be approached with caution, as the statistical power may be inadequate to support more definitive conclusions. A comparative analysis was also conducted between the two phenological states using selected metadata variables.

3.2. Soil Microbiome Dynamics During Fruit Formation in Olive Cultivation

Differential abundance analysis using ANCOM-BC identified several bacterial taxa that were significantly enriched or depleted in soil samples collected during the fruit formation stage (O2) relative to the reference phenological stage (Figure 3). During fruit formation, a significant enrichment of multiple taxa was detected, predominantly the phylum Actinobacteriota. The highest positive log fold change (LFC) was observed for Saccharothrix, Phytomonospora and Micromonosporaceae. Additional enriched taxa included Parachlamydiaceae, Terrabacter and Kribella.

In contrast, several taxa exhibited a significant decrease in relative abundance during the fruit formation stage. These depleted taxa were the phyla Firmicutes, Bacteroidota, Planctomycetota, and Patescibacteria. The most pronounced negative LFC values were observed for Paenarthrobacter, Planctomicrobium, Geobacteraceae and Candidatus Nomurabacteria. Additional reductions were detected for Clostridium sensu stricto, Tepidimicrobium, Fonticella, and members of the order Chitinophagales. The analysis revealed phenology-dependent changes in the relative abundance of specific bacterial taxa, despite the absence of significant differences in global diversity metrics.

3.3. Characterization and Selection of PGP Isolates

Based on the results of the PGP, an initial screening and selection of bacterial isolates was performed by prioritizing those that exhibited clear and consistent siderophore production. In addition, isolates that did not display strong siderophore-producing ability but showed particularly strong performance in other assessed traits—such as phosphate solubilization or IAA production—were also retained, as these functional attributes can independently contribute to improved plant nutrition and growth. Following this selection process, nine representative strains were chosen and taxonomically identified by 16S rRNA gene sequencing. A comprehensive description of these selected isolates, including their taxonomic affiliation and PGP traits, is presented in Table 2.

3.4. Inhibition of Olive-Tree Associated Pathogens

After a series of in vitro antagonistic assays, only two of the nine initial isolates— Bacillus mojavensis OliA 54 and Bacillus amyloliquefaciens CtA 59B1—exhibited measurable inhibitory activity against Pseudomonas savastanoi (Table 3).

Both strains produced inhibition halos that expanded progressively throughout incubation, with B. amyloliquefaciens showing stronger early activity and B. mojavensis increasing inhibition at later time points, ultimately reaching comparable levels. In subsequent assays, the antagonists were established as a bacterial lawn, and P. savastanoi was inoculated onto their surface. In all cases, no growth of P. savastanoi was observed on plates containing either antagonist, while normal growth occurred in control plates lacking antagonistic bacteria. These results confirm that both Bacillus isolates exert robust and time-dependent antagonistic effects, effectively suppressing P. savastanoi under experimental conditions. B. amyloliquefaciens displayed a stronger inhibitory effect at early evaluation points, whereas B. mojavensis increased its activity later and reached comparable levels by the final assessment. This pattern indicates that both isolates maintain robust and time-dependent antagonistic activity against P. savastanoi.

Interestingly, these two microorganisms also showed inhibitory activity against V. oleaginea, along with three other strains from our collection, suggesting that some strains possess broad-spectrum antagonistic potential against multiple olive pathogens.

4. Discussion

By integrating culture-dependent isolation with soil amplicone based microbiome analysis, we identified native bacterial strains with biocontrol activity and explored phenology-associated shifts in soil bacterial community composition. This combined approach provides complementary information on the functional potential of cultivable microorganisms and their ecological context within the olive agroecosystem. This integrative approach is particularly relevant in olive agroecosystems, where studies often focus either on the isolation of antagonistic microorganisms or on descriptive analyzes of soil microbial communities. By combining both strategies within the same experimental framework, our study allows functional screening of culturable bacteria to be interpreted in the context of the surrounding soil microbiome. This reduces the risk of selecting highly active isolates lacking ecological relevance and, conversely, of inferring potential functions from community-level data without experimental validation.

The selection of isolates was based on PGP traits, particularly phosphate solubilization, siderophore production, and indole-3-acetic acid (IAA) synthesis, traits frequently associated with both plant biostimulation and pathogen suppression [35].

The screened activities not only improve the nutritional status of the plant, promoting more vigorous growth and enhanced crop productivity, but also increase its capacity to withstand attacks by phytopathogens. In addition, some of the traits examined exert a direct effect on pathogen suppression. This is the case for siderophores, which, besides enhancing plant nutrition by making soil iron more accessible, also compete directly with fungi for this essential nutrient, thereby limiting their development. Most bacterial isolates obtained in this study belonged to genera commonly reported in soil and rhizospheric environments, including Bacillus, Priestia, Lysinibacillus, Micrococcus, and Pseudomonas [20,36]. These taxa are well known for their involvement in nutrient cycling, plant growth promotion, and biological control [37].

The antagonistic activity displayed by several strains against Pseudomonas savastanoi is consistent with previous reports highlighting the efficacy of Bacillus and Pseudomonas spp. as biocontrol agents through the production of antimicrobial compounds and lytic enzymes [20,38]. Among the tested strains, Bacillus amyloliquefaciens exhibited a particularly strong inhibitory effect, in agreement with studies reporting its ability to reduce disease severity without negatively affecting plant development [39]. These antagonistic assays were performed in vitro, and the observed inhibitory effects have not yet been validated under greenhouse or field conditions. Therefore, while these results suggest a promising biocontrol potential, their application in practical disease management should be considered preliminary. Culture-independent microbial community profiling analysis provided an ecological framework for these observations. Although alpha- and beta-diversity metrics did not reveal statistically significant differences between flowering and fruit formation stages, differential abundance analysis identified phenology-dependent shifts in specific bacterial taxa. Notably, fruit formation was associated with a significant enrichment of Actinobacteria, a phylum characterized by high metabolic versatility and the capacity to produce bioactive secondary metabolites with antimicrobial activity [40,41].

The enrichment of actinobacterial taxa such as Saccharothrix, Phytomonospora, and members of the Micromonosporaceae during fruit formation suggests a functional reorganization of the soil microbiome linked to increased metabolic activity and changes in root exudation patterns. Actinobacteria are known to contribute to the degradation of complex organic matter, nutrient cycling, and rhizosphere modulation [40], and their increased abundance may enhance disease-suppressive functions during this phenological stage. Conversely, a reduction in fermentative and anaerobic taxa, including Clostridium, Geobacteraceae, and Planctomicrobium, likely reflects changes in substrate availability, soil aeration, and microbial competition during fruit development. Although our study does not directly assess suppressiveness at the ecosystem level, the enrichment of Actinobacteria—frequently linked to antagonistic interactions and secondary metabolite production—supports the hypothesis that subtle changes in community composition may translate into functionally relevant outcomes.

Despite the overall stability of the soil microbiome structure, the detection of differentially abundant taxa through ANCOM-BC indicates that subtle, yet functionally relevant, microbial shifts occur in response to phenological changes [32], while several taxa detected by 16S rRNA gene sequencing analysis were also recovered through cultivation, others remained uncultured, underscoring both the complementarity of the two approaches and the need to integrate culture-dependent and culture-independent strategies to capture functional and taxonomic diversity due to the inherent limitations of culture-dependent methods [42].

From the initial collection of 90 bacterial isolates, nine strains were selected based on their PGP traits. Siderophore production was considered particularly relevant due to its established role in iron competition and pathogen suppression [43]. However, inhibition assays against P. savastanoi revealed that strains with lower siderophore production often exhibited stronger antagonistic effects. This suggests that iron competition was not the primary mechanism of inhibition in this case, and that alternative strategies such as antibiosis played a dominant role. Indeed, Bacillus spp. are known to produce antimicrobial lipopeptides (e.g., surfactins, iturins, and fengycins) and volatile organic compounds that can suppress bacterial pathogens independently of siderophore-mediated competition [38]. It is also noteworthy that strains exhibiting strong phosphate-solubilizing activity tended to show enhanced suppression of the bacterial pathogen. This pattern is consistent with previous studies demonstrating that organic acid production and local acidification—key processes in phosphate solubilization—can also inhibit bacterial growth and modify the rhizosphere environment in ways that reduce pathogen fitness [44,45]. Conversely, isolates with a higher siderophore-producing capacity displayed more pronounced antagonistic activity against the fungal pathogen. This aligns with the recognized role of siderophores in limiting iron availability, a critical factor for fungal development, and in mediating competitive interactions that suppress fungal pathogens [46]. Together, these findings reinforce the idea that different PGP traits contribute selectively to pathogen suppression and that the functional profile of each strain may determine its biocontrol potential against specific types of phytopathogens. In this context, the complementary inhibition assay in which the antagonistic strains were established as a bacterial lawn provides additional insight into the mechanisms underlying pathogen suppression. Observations, such as the complete absence of P. savastanoi growth under these conditions suggests a strong competitive exclusion effect, likely resulting from a combination of rapid surface colonization, niche occupation, and the accumulation of inhibitory metabolites. Such priority effects, whereby early colonizers prevent the establishment of later-arriving competitors, are considered ecologically relevant mechanisms in soil and rhizosphere environments. Together with the diffusion-based inhibition assays, these results indicate that antagonism against P. savastanoi is mediated not only by diffusible antimicrobial compounds but also by direct competitive interactions. The use of both experimental approaches, therefore, allows a more comprehensive assessment of biocontrol potential, capturing both antibiosis-driven inhibition and suppression through competitive dominance, which are likely to operate simultaneously under natural soil conditions [47].

Taken together, these results highlight that biocontrol efficacy is not associated with a single functional trait, but rather with specific combinations of mechanisms whose relevance may vary depending on the target pathogen. This functional specificity underscores the importance of tailoring microbial-based strategies to particular disease contexts, rather than relying on generalized biocontrol solutions [38]. Despite the robustness of the observed trends, this study has some limitations that should be acknowledged. In particular, the relatively limited sample size and the use of composite soil samples, although they reduce spatial variability and enable greater homogeneity among samples belonging to the same temporal interval, may have reduced the statistical power to detect subtle differences in global microbial diversity between phenological stages, especially in beta-diversity analyses, while no statistically significant separation was observed, consistent trends across multiple distance metrics suggest that moderate community restructuring may occur, but it remains under-detected. Increasing the number of biological replicates, avoiding sample pooling, and extending sampling across additional seasons or orchards would help to further validate these patterns and strengthen future ecological inferences.

5. Conclusions

We propose a combined strategy to address the management of two of the most relevant olive plant pathogens. The integration of culture-dependent and culture-independent approaches provides a comprehensive framework for identifying microbial taxa and functions associated with disease suppression in olive. This dual strategy enables both the discovery of key microbial groups and the experimental validation of their antagonistic or plant-growth-promoting (PGP) activities. We have demonstrated that olive orchard soils harbor diverse microbial communities with antagonistic potential against major olive pathogens. Although overall microbial diversity remained relatively stable between flowering and fruit formation, differential abundance analyses revealed taxon-specific shifts, with a marked enrichment of the phylum Actinobacteria during fruit formation, suggesting subtle functional rearrangements of the soil microbiome. Among the cultivable fraction, only a limited number of bacterial isolates exhibited consistent antagonistic activity, indicating that biocontrol potential is highly strain-dependent and associated with specific plant growth–promoting traits. The use of native microorganisms adapted to olive agroecosystems emerges as a promising strategy for sustainable and environmentally friendly disease management. Our results indicate that plant growth-promoting traits traditionally associated with nutritional enhancement may also contribute to pathogen suppression, depending on the biological characteristics of the target pathogen. Understanding these functional trade-offs is therefore essential for optimizing the exploitation of soil microbial resources. Future research should focus on validating the biocontrol efficacy of selected strains under both greenhouse and field conditions, as well as elucidating their interactions within complex soil microbial communities.