Cooperative Interactions Between Bacillus and Lysobacter Enhance Consortium Stability and Fusarium Wilt Suppression in Cucumber

Cooperative Interactions Between Bacillus and Lysobacter Enhance Consortium Stability and Fusarium Wilt Suppression in Cucumber | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cooperative Interactions Between Bacillus and Lysobacter Enhance Consortium Stability and Fusarium Wilt Suppression in Cucumber Xinli Sun, Riyan Xia, Jiyu Xie, Kun Duan, Weibing Xun, Nan Zhang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6904063/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Aug, 2025 Read the published version in Microbial Ecology → Version 1 posted 12 You are reading this latest preprint version Abstract The rhizosphere microbiome plays a pivotal role in plant health by mediating interactions between hosts, beneficial microbes, and pathogens. However, the ecological mechanisms underlying microbial consortia that suppress soil-borne diseases remain largely unexplored. In this study, we investigated how the biocontrol bacterium Bacillus velezensis SQR9 influences the assembly of the cucumber rhizosphere bacterial community in the presence of the pathogenic fungus Fusarium oxysporum FOC. Inoculation with B. velezensis SQR9 significantly enriched the genus Lysobacter , a group of known biocontrol bacteria potentially contributing to disease suppression. A meta-analysis of publicly available datasets revealed a positive correlation between Bacillus and Lysobacter abundances in healthy plant rhizospheres—a relationship absent in Fusarium wilt diseased soils—suggesting a conserved ecological association linked to disease suppression. Further mechanistic assays demonstrated that Lysobacter enzymogenes XL8, an antifungal bacterium isolated from the cucumber rhizosphere, formed synergistic biofilms with B. velezensis SQR9. Cross-feeding assays indicated that strain SQR9 facilitated the growth of L. enzymogenes XL8 through metabolic interactions, highlighting a cooperative mechanism that may stabilize the rhizosphere bacterial consortium. Greenhouse trials confirmed that this dual-species consortium outperformed single-species inoculations in suppressing Fusarium wilt, as evidenced by reduced pathogen abundance and enhanced plant growth. Together, our findings underscore the importance of microbial metabolic cooperation and biofilm-mediated coexistence in shaping rhizosphere community assembly and function, providing ecological insights for the development of synthetic microbial consortia aimed at sustainable plant disease management. microbial interaction SynCom biocontrol biofilm cross-feeding Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Plant disease management remains a major challenge in agriculture, particularly given the environmental concerns associated with chemical pesticides. The Fusarium oxysporum species complex is notorious for causing vascular wilt diseases in many crops, leading to severe yield losses worldwide [ 1 ]. The root microbiome, often referred to as the plant’s “second genome,” plays a crucial role in maintaining plant health [ 2 ]. Upon pathogen infection, plant roots can recruit beneficial microbes that inhibit pathogen invasion through direct antibiosis [ 3 , 4 ] or by inducing systemic resistance [ 5 , 6 ]. For instance, Arabidopsis thaliana selectively recruits protective rhizosphere bacteria following foliar infection by the downy mildew pathogen Hyaloperonospora arabidopsidis , enhancing disease resistance and growth [ 7 ]. Similarly, F. oxysporum modulates the tomato rhizosphere differently depending on host genotype, increasing the relative abundance of Lysobacter , Sphingobium , and Sphingomonas species in the resistant cultivar [ 8 ]. A recent meta-analysis revealed that the root microbiome of Fusarium wilt-infected plants is enriched in Xanthomonadaceae , Bacillaceae , Gibberella , and F. oxysporum , whereas healthy plants harbor greater abundances of Streptomyces , Mirabilis , Bradyrhizobiaceae , Comamonadaceae , and Mortierella [ 9 ]. Synthetic microbial communities (SynComs) have emerged as a promising tool for enhancing plant health [ 10 – 12 ]. One notable advantage of SynComs is their ability to perform complementary biocontrol functions, which offer a multifaceted defense against pathogens. Native root-associated bacterial consortia have been reported to protect plants from sudden wilt diseases through a suite of biocontrol traits [ 13 ]. Similarly, SynComs have been demonstrated to improve nutrient efficiency and yield in crops such as soybean and tomato and confer resistance to Fusarium wilt disease [ 14 – 16 ]. These communities mimic natural microbial interactions, providing novel mechanistic insights into microbiome stability, which is important for plant health [ 17 ]. Beyond disease suppression, the use of SynComs extends to increase plant growth by increasing nutrient acquisition efficiency [ 14 ], increasing resilience to salt and drought stress [ 18 , 19 ], and increasing resistance to aluminum toxicity [ 20 ]. Bacillus velezensis SQR9 is a well-characterized biocontrol bacterium that suppresses Fusarium wilt and promotes plant growth through various mechanisms [ 21 ]. It synthesizes various secondary metabolites, including bacillomycin D, fengycin, bacillaene, bacillibactin, difficidin, surfactin, macrolactin, and bacilycin [ 22 ]. It can also stimulate the resident rhizosphere species Pseudomonas stutzeri through metabolic interactions, which collectively improve plant growth and salt tolerance [ 23 ]. However, how B. velezensis SQR9 reshapes the rhizosphere microbiome under pathogen invasion remains unclear. In this study, we investigated the impact of B. velezensis SQR9 on cucumber rhizosphere bacterial diversity and structure during infection by pathogen F. oxysporum f. sp. cucumerinum FOC and explored its interaction with indeginous benefical bacteria. Our results revealed that Lysobacter spp., known antifungal biocontrol agents [ 24 , 25 ], were enriched by B. velezensis SQR9. A meta-analysis further revealed a positive correlation between Lysobacter and Bacillus abundances in healthy plant rhizospheres. We then isolated L. enzymogenes XL8 from the cucumber rhizosphere and constructed a dual-species consortium with SQR9. By assessing biofilm formation, cross-feeding interactions, and antifungal compound production, we demonstrated that this consortium enhances plant disease resistance and growth. These findings provide new insights into bacterial interactions in the rhizosphere and support the development of microbial consortia for sustainable plant disease management. Materials and Methods Strains and media B. velezensis SQR9 (CGMCC No.5808) and L. enzymogenes XL8 (GDMCC accession No.64920) were stored at − 80°C in Tryptone Soy Broth (TSB) supplemented with 25% glycerol. Lysobacter spp. were isolated previously from the rhizosphere of cucumber plants inoculated with B. velezensis SQR9 [ 23 ] and stored at − 80°C in Reasoner’s 2B broth (R2B) or TSB supplemented with 25% glycerol. Spores of F. oxysporum f. sp. cucumerinum J. H. Owen (FOC) were stored at − 80°C in potato dextrose broth (PDB) with 25% glycerol. The media were solidified with 1.5% agar when necessary. Strains were grown in TSB, R2B, or PDA as appropriate. Overnight cultures were harvested, washed, and resuspended in 0.9% NaCl to an OD 600 of 1 for inoculations. Pot experiments Soil was collected from a field in Taizhou City, Jiangsu Province, China (32.4555° N, 119.9229° E), in 2018. Surface sterilized Cucumber seeds (Jinchun 4) were pre-germinated before transplantation into pots. In the first pot experiment, treatments include: CTL (control group, no inoculation); B (inoculation with B. velezensis SQR9 suspension at a final concentration of 10 6 cells/g soil); F (inoculation with F. oxysporum FOC spore suspensions at a final concentration of 10 6 spores/g soil; and BF (inoculation with both B. velezensis SQR9 (10 6 cells/g soil) and F. oxysporum FOC (10 6 spores/g soil)). Each treatment group consisted of six replicates. The plants were grown in a greenhouse at 30°C with a 16:8 h light/dark cycle. After 16 days, rhizosphere soil was collected as described by Bai et al. [ 26 ]. In the second pot experiment, in addition to the CTL, B, F, and BF treatment groups, two additional treatment groups were included: LF (inoculation with both L. enzymogenes XL8 (10 6 cells/g soil) and F. oxysporum FOC (10 6 spores/g soil)), and BLF (inoculation with B. velezensis SQR9 (10 6 cells/g soil), L. enzymogenes XL8 (10 6 cells/g soil) and F. oxysporum FOC (10 6 spores/g soil)). Each treatment had ten replicates. After 30 days, photos were taken of the plants. Shoot height and fresh weight were recorded. The disease index was calculated as the number of diseased plants divided by the total number of all plants. Moreover, the rhizosphere soil from six replicates was collected for DNA extraction. The abundance of F. oxysporum in the rhizosphere soil was quantified via qPCR [ 27 ]. Amplicon sequencing and analysis Rhizosphere DNA was extracted using the DNeasy PowerSoil Pro Kit (QIAGEN cat no. 47014). Bacterial universal primers (338F/806R) targeting the V3 − V4 regions of the 16S rRNA gene were employed to construct the DNA library for sequencing. Paired-end sequencing of bacterial amplicons was performed on an Illumina MiSeq 300 platform (Genewiz, Suzhou, China). The reads were processed using the UPARSE pipeline (version 8.1) [ 32 ]. Specifically, the paired-end reads were merged with the “fastq_mergepairs” command. Following this, high-quality sequences were selected using the “fastq_filter” command, and dereplication was performed with the “derep_fulllength” command. Singletons were eliminated using USEARCH-unoise3 algorithm, while chimeric sequences were removed with the “uchime_ref” command. The remaining sequences were clustered into operational taxonomic units (OTUs) at a 97% similarity threshold using the “cluster_otus” command. Taxonomic assignment of the OTUs was performed using the Ribosomal Database Project classifier (version 11). Additionally, Bray − Curtis distance-based principal coordinates analysis (PCoA), and permutational multivariate analysis of variance (PERMANOVA) were conducted based on the OTU table using the vegan R package. Welch’s t-test was used to assess the significance of differences between the two treatments using STAMP [ 33 ]. Only OTUs with a relative abundance exceeding 0.05% across all samples were included in this analysis. Meta-analysis of amplicon sequencing data from Fusarium wilt disease studies Yuan et al. [ 9 ] collected amplicon sequencing data from several studies investigating wilt diseases that affect various plant species. We obtained the OTU table directly from the authors. Pearson correlation analysis was conducted using the R package “psych” to examine the correlation between the genera present in the rhizosphere. A t-test was performed to compare the relative abundance of genera in healthy and diseased plant rhizospheres. All the statistical analyses were performed via R software, with significance defined at a threshold of p < 0.05. Biofilm formation and quantification To examine the formation of pellicle biofilms, 20 µL of the starting inoculum was cultivated in 2 mL of TSB in a 24-well microtiter plate, and incubated statically for 48 h at 30°C. For coculture, the starting inoculum was prepared by mixing equal volumes of isolates. Photographs were taken to observe pellicle formation. Biofilm quantification was performed via a modified crystal violet (CV) staining method [ 28 ]. In brief, the growth medium and nonadherent cells were removed via a sterile syringe, and the wells were then rinsed with distilled water. The biofilm-containing cells were stained with 2 mL of 1% CV for 30 min at room temperature. Unbound CV was removed, and the wells were washed twice with distilled water. The biofilm-bound CV was solubilized with 2 mL of ethanol: acetone (4:1 v/v) for 20 min. Biofilm formation was quantified by measuring the OD 570 via a multifunctional plate reader. Each treatment had four biological replicates. Antagonism test of Lysobacter isolates against F. oxysporum In vitro, antagonism tests were performed on ½ potato dextrose agar (PDA) mixed with ½ R2A agar in 9-cm Petri dishes. F. oxysporum FOC spores from glycerol stocks (2 µL) were inoculated at the center of the plate and incubated at 28°C for 2 days. Then, 2 µL of bacterial suspension (OD 600 = 1) was inoculated 3 cm away from the center of the fungal colony. The plates were incubated at 28°C for another 4 days. Photographs were taken to assess the antagonistic activity. BIOLOG assay Carbon utilization profiles of B. velezensis SQR9 and L. enzymogenes XL8 were assessed using GEN III MicroPlate (Biolog cat no. 1030) contains 71 carbon source use assays and 23 chemical sensitivity tests. Overnight cultures were harvested, washed, and resuspended in inoculating fluid A (Biolog, cat no. 72401). Cell suspension (100 µL) was added per well and incubated at 30°C in an OmniLog instrument for 48 h. Color development, indicating metabolic activity, was automatically recorded. Each strain was tested in triplicate. Metabolic facilitation assays and metabolome analyses B. velezensis SQR9 was cultured in M9 glucose medium (0.2% glucose) at 30°C with shaking (180 rpm) until glucose was depleted, as confirmed by a Glucose GO Assay Kit (Sigma-Aldrich, GAGO20). Spent medium was collected by centrifugation and filter sterilized. L. enzymogenes XL8 was inoculated into the spent medium (1% v/v) and incubated under the same conditions for 5 days. Growth was measured by OD 600 (n = 6). Extracellular metabolites were collected and analyzed via ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Data were processed using Compound Discoverer 3.0 for peak alignment, detection, and quantification. Metabolites were annotated by matching spectra against mzCloud and ChemSpider databases. Each treatment included six replicates. Expression analysis of secondary metabolite biosynthetic genes B. velezensis SQR9 and L. enzymogenes XL8 were incubated in M9 glucose media at 30°C, 180 rpm for 48h. Supernatants were collected, filter-sterilized, and used (20% v/v) to supplement fresh cultures of the partner strain; M9-glucose medium alone served as control. After 48 h, cells were harvested for total RNA extraction (E.Z.N.A Bacterial RNA Kit), cDNA synthesis (Hiscript II Q RT SuperMix), and RT-qPCR (ChamQ Universal SYBR qPCR Master Mix). Biosynthetic gene clusters (BGCs) in the L. enzymogenes XL8 genome were predicted via antiSMASH 7.0 [ 29 ]. The transcription levels of genes involved in the biosynthesis of bacillomycin D, fengycin, bacillaene, surfactin, difficidin, bacillibactin, macrolactin, and bacilysin in B. velezensis SQR9, as well as WAP-8294A2, HSAF, and Le-pyrrolopyrazines in L. enzymogenes XL8, were quantified via RT‒qPCR. Primer sequences are listed in the Supplementary Table. The recA gene was used as the reference gene for both strains. RT‒qPCR was performed on an ABI 7500 system (ABI, USA) with the following thermal conditions: initial denaturation at 95°C for 30 s; 40 cycles of 95°C for 10 s and 60°C for 30 s; and standard melting curve analysis. The 2 −∆∆Ct method was used to calculate the relative expression changes [ 30 ]. This experiment had six biological replicates. Results Bacillus spp. and Lysobacter spp. are positively correlated in healthy plant rhizosphere The experimental design is presented in Fig. 1 A. Rhizosphere soil samples were for bacterial community profiling via amplicon sequencing. Compared to the non-inoculated control, inoculation with either B. velezensis or F. oxysporum did not affect Chao1 diversity (Fig S1 A) but significantly increased the community richness (Welch’s test, p < 0.05) (Fig S1 B). Principal coordinate analysis (PCoA) based on Bray‒Curtis distance matrices revealed significant differences in rhizosphere bacterial communities across the four treatment groups (PERMANOVA, R 2 = 0.74, p < 0.001) (Fig S1 C). Inoculated groups were clearly separated from the control along the first coordinate (PCoA1), whereas the dual inoculation (BF) treatment was distinguished from the single-inoculations along the second coordinate (PCoA2). In particular, the relative abundances of the genera Chitinophaga , Chryseobacterium , and Lysobacter were significantly greater in the BF group compared to the F group, whereas those of Cellvibrio , Erwinia , Flavobacterium , and Pseudoxanthomonas were significantly reduced (Welch’s test, p < 0.05) (Fig. 1 B). In a recent study, Yuan et al. [ 9 ] gathered amplicon sequencing data from rhizosphere samples of both healthy plants and plants affected by Fusarium wilt, across various plant species. From this dataset, we extracted the relative abundance of OTUs identified as Bacillus , Chitinophaga , Chryseobacterium , and Lysobacter . The results indicated that the abundance of Chryseobacterium spp. was below the detection limits. In the the rhizosphere of healthy plants, Bacillus and Lysobacter showed a significant positive correlation (R = 0.26, p < 0.01), whereas no such correlation was observed in diseased plants ( p = 0.1) (Fig. 1 C). Additionally, no significant correlation was detected between Bacillus and Chitinophaga in healthy plants, while a weak negative correlation emerged in diseased plants (R = -0.18, p = 0.032) (Fig. 1 D), which differs from our data. Both Bacillus and Chitinophaga presented significantly greater relative abundances in healthy compared to diseased plant rhizospheres (Fig. 1 E). Although Lysobacter was more abundant in the rhizosphere of healthy plants than in the rhizosphere of diseased plants, this difference was not statistically significant. In the present study, inoculation with either B. velezensis or F oxysporum enriched Lysobacter , with B. velezensis exerting a stronger effect (Fig. 1 F). Previous research showed that biofertilizers containing Bacillus amyloliquefaciens W19 also increases the relative abundance of Lysobacter in the rhizosphere [ 31 ]. Rather than acting through direct pathogen suppression by Bacillus spp., these enriched Lysobacter may serve as a keytone taxa in controlling F. oxysporum . Consistent with this, in the present study, the relative abundance of Bacillus itself did not significantly increase in the B or BF groups compared to the uninoculated control (Fig. 1 F). In addition, F. oxysporum has been reported to elevate Lysobacter abundance in the rhizosphere of disease-resistant tomato cultivars and decreases its abundance in susceptible cultivars, suggesting a link between the abundance of Lysobacter and the suppression of Fusarium pathogens [ 8 ]. In light of these findings, we sought to examine the interaction between Bacillus and Lysobacter . Biofilm formation and antifungal activity of Lysobacter spp. To investigate the interaction between B. velezensis SQR9 and Lysobacter spp., 15 Lysobacter isolates were selected from our strain collection (Fig. S2 ), which were isolated from the cucumber rhizosphere pre-treated with B. velezensis SQR9 [ 23 ]. On the root surface, rhizobacteria form biofilms that protect them from environmental stress and enable them to interact positively with the host plant [ 32 ]. Previous studies have shown that plant-beneficial bacteria can form biofilms synergistically within consortia [ 7 , 13 , 19 ]. To assees this, an in vitro biofilm formation assay was conducted. As shown in Fig. 2 A, none of the Lysobacter isolates formed visible biofilms individually. However, co-culturing with B. velezensis SQR9 enhanced biofilm wrinkling in isolates XL169, XL170, XL171, and XL8 compared to the strain SQR9 monoculture. Biofilm quantification assays further comfirmed that co-cultures produced significantly greater biofilm biomass than individual cultures (t-test, p < 0.05) (Fig. 2 B). Next, we assessed the antagonistic activity of these Lysobacter isolates against F. oxysporum in vitro. Only strains XL166 and XL8 inhibited the growth of F. oxysporum mycelia (Fig. 2 C). Given the superior antagonistic activity of L. enzymogenes XL8, we tested the additive effect of its culture supernatants with extracts of B. velezensis SQR9. The dual application produced the largest inhibition zone against F. oxysporum FOC (Fig. 2 D, 2 E). Based on it strong biofilm-promoting and antifungal activity, L. enzymogenes XL8 was selected for further study. Bacillus-Lysobacter consortium protects plants against wilt disease The protective effect of the dual-species consortium comprising B. velezensis SQR9 and L. enzymogenes XL8 against F. oxysporum FOC infection was evaluated in a greenhouse experiment. As shown in Fig. 3 A, plants inoculated with FOC alone (F group) exhibited severe disease symptoms, including wilting and leaf yellowing. Both single biocontrol bacterium treatments (BF and LF groups) provided partial protection, but the dual-species consortium (BLF group) was significantly more effective. Plants in the BLF group displayed the highest biomass (Fig. 3 B, 3 C) and the lowest disease index (Fig. 3 D), with differences among treatments being statistically significant. Moreover, the abundance of F. oxysporum FOC was also lowest in the BLF treatment group (Fig. 3 E), indicating superior pathogen suppresion. Taken together, these results demonstrate that the combined inoculation of B. velezensis SQR9 and L. enzymogenes XL8 exerts a synergistic effect, enhancing both plant growth and disease resistance. Bacillus velezensis SQR9 facilitates the growth of Lysobacter enzymogenes XL8 through cross-feeding To elucidate the mechanism underlying the synergistic interaction between B. velezensis SQR9 and L. enzymogenes XL8, we investigated their metabolic interaction. Figure 4 A depicts the experimental setup for the cross-feeding assay. L. enzymogenes XL8 showed limited growth in M9 glucose media but was able to grow in the presence of the culture supernatant of B. velezensis SQR9 (Fig. 4 B). Consistent with this finding, strain XL8 was found to grow in the vicinity of B. velezensis SQR9 colonies on plates (Fig. 4 C), suggesting metabolic cross-feeding. A metabolomic analysis was conducted to identify the metabolites potentially involved in this growth facilitation. The heatmap in Fig. 4 D shows the relative abundance of metabolites in the supernatant of B. velezensis SQR9 and the supernatant of L. enzymogenes XL8 grown on SQR9-derived medium. Metabolites enriched in the strain SQR9 supernatant but depleted in the strain XL8 supernatant were identified as potential cross-feeding compounds. Four commercially available metabolites—deoxycholic acid, 6-dimethylamino purine, 5-hydroxyindole, and D-phenylalanine—were tested to determine whether they could support the growth of L. enzymogenes XL8 in M9 minimal medium when used as the sole carbon source. The results revealed that the deoxycholic acid secreted by B. velezensis SQR9 can be utilized by L. enzymogenes XL8 (Fig. S3 ). The carbon source useability of strains SQR9 and XL8 was measured via BIOLOG assays, and the results demonstrated that L. enzymogenes XL8 can use a wide range of carbon sources (Fig. S4 ). Enhanced antifungal metabolites production in the dual-species consortium To assess whether B. velezensis SQR9 and L. enzymogenes influence each other’s secondary metabolite production, we performed RT‒qPCR analysis. Gene expression associated with secondary metabolite biosynthesis was compared between M9 glucose medium and M9 supplemented with 20% bacterial spent medium. B. velezensis SQR9 is known to produce eight antibioticsWhen exposed to XL8 spent medium, SQR9 showed significantly increased expression of genes responsible for bacillomycin D and bacillaene synthesis, while genes related to surfactin production were downregulated compared to the M9 control (Fig. 5 ). Bacillomycin D has been demonstrated to exert antifungal activity against F. oxysporum [ 33 ]. Bacillaene is a polysaccharide that exhibits antimicrobial activity against various bacteria and fungi, including Fusarium spp. [ 34 ]. Surfactin, a lipopeptide produced by various Bacillus species, exhibits broad-spectrum antimicrobial activity [ 35 ]. The downregulation of surfactin production by SQR9 may be correlated with reduced competition with XL8. AntiSMASH analysis revealed that L. enzymogenes XL8 contains three biosynthetic gene clusters (BGCs) for secondary metabolite production with 100% similarity to known clusters: HSAF (an antifungal antibiotic heat-stable antifungal factor that inhibits the growth of fungal hypha and the conidial germination) [ 25 ], WAP-8294A2 (a cyclopeptide with activity against Gram-positive bacteria) [ 36 ], and Le-pyrrolopyrazines (function unknown) [ 37 ]. Our results revealed that L. enzymogenes XL8 significantly upregulated HSAF and WAP-8294A2 biosynthetic genes when cultured in SQR9 spent medium (Fig. 5 ). These findings suggest mutual enhancement of antifungal metabolite production between the two strains, which likely contributes to their improved biocontrol efficacy observed in greenhouse experiments. Conclusion and Discussion Our findings demonstrated that the B. velezensis SQR9 significantly increases the abundance of Lysobacter in the rhizosphere of cucumber plants infected with pathogenic F. oxysporum . A positive correlation between the abundance of Lysobacter and Bacillus is commonly observed in the rhizosphere of healthy plants, suggesting a conserved ecological association. The synergistic biofilm formation and metabolic cross-feeding interactions between the biocontrol bacterium B. velezensis SQR9 and the antifungal agent L. enzymogenes XL8 suggest mechanisms that improve their fitness in the rhizosphere. Additionally, the upregulated expression bacillomycin D and HSAF biosynthetic genes—both encoding antifungal compounds known to antagonize F. oxysporum —along with the expanded inhibition zone observed in vitro , further indicate the synergistic biocontrol ability of this bacterial consortium. These combined mechanisms contribute to the effective suppression of cucumber Fusarium wilt disease. Enhanced biofilm formation is a common characteristic of plant-beneficial bacterial communities. Our study revealed that B. velezensis SQR9 and L. enzymogenes XL8 form synergistic biofilm, which may improve their colonizatoin and adaptability in the rhizosphere. Similar interactions have been reported between B. velezensis SQR9 and P. stutzeri XL272, resulting in improved plant growth promotion and salt tolerance [ 23 ]. Biofilm formation is crucial for microbial survival and functionality in diverse environments. For example, the emergent properties of biofilm-forming communities can confer increased drought tolerance in Arabidopsis [ 19 ] and facilitate the establishment of keystone species, ultimately promoting overall plant growth [ 38 ]. Disease-induced assemblage plant-beneficial bacterial consortia have also been reported to protect host plants against pathogens by synergistically forming biofilms [ 7 ]. Likewise, complementary biocontrol traits and cooperative biofilm formation contribute to the suppression of sudden wilt diseases in root-associated microbial communities [ 13 ]. Collectively, these findings emphasize the importance of synergistic biofilm formation in fostering microbial resilience, function stability, and plant health under various stress conditions. However, the ecological significance of beneficial bacterial biofilm formation under natural rhizosphere conditions remains poorly understood. Bridging this knowledge gap is essential for a deeper understanding of how microbial biofilms contribute to plant health, disease resistance, and environmental stress tolerance in real-world agricultural systems. Metabolic interactions play crucial roles in regulating the assembly of SynComs [ 39 ]. Our results suggest that the B. velezensis strain SQR9 promotes the growth of the L. enzymogenes strain XL8 by cross-feeding. Similarly, a seven-species SynCom has been reported to inhibit the maize fungal pathogen F. verticillioides , with Enterobacter cloacae identified as the keystone species [ 16 ]. Each member of SynCom occupies complementary metabolic niches, and spent media derived from the prototrophic strains can support the growth of two auxotroph strains [ 17 ]. The keystone species E. cloacae serves as a metabolic donor, enhancing overall SynCom biomass and utilizing diverse carbon sources from root exudates to prevent the dominance of undesirable taxa [ 17 ]. Keystone species may either promote SynCom biomass through facilitation or suppress it via competitive exclusion, depending on their metabolic roles [ 40 ]. A two-tiered cross-feeding system in which one strain alleviates toxic byproduct accumulation and the other detoxifies environmental toxins, demonstrates how cooperative exchanges extend beyond nutrient sharing to modulate community structure and enhance competitive fitness [ 41 ]. Such costless metabolic exchanges act as drivers of interspecies interactions within microbial communities and can be leveraged to design SynCom with increased ecological fitness and stability [ 42 ]. Our findings also suggest that exposure to spent media from consortium partners upregulates antifungal biosynthetic genes in both B. velezensis SQR9 and L. enzymogenes XL8, thereby enhancing their biocontrol potential. This is consistent with previous reports where SynCom improves their antifungal against maize seed-borne Fusarium pathogens by stimulating antibiotic biosynthetic genes expression [ 43 ]. A similar underlies the mutualism between B. velezensis and arbuscular mycorrhizal fungi, where fungal signals modulate bacterial secondary metabolite production, stabilizing the coexistence of both partners in the hyphosphere [ 44 ]. Such interactions not only promote systemic resistance in the host plant but also increase the overall biocontrol ability of the bacterial‒fungal consortium. Future studies should investigate the mechanisms of chemical signaling between B. velezensis SQR9 and L. enzymogenes XL8 to identify the key compounds regulating secondary metabolite expression in each organism. Moreover, L. enzymogenes OH11 has been reported to utilize a type VI secretion system (T6SS) to secrete toxic effectors to pathogenic filamentous fungi in a contact-dependent manner, which inhibits the growth of conidia into hyphae [ 45 ]. It remains to be investigated whether a similar antifungal mechanism exists in L. enzymogenes XL8 and whether its interaction with B. velezensis SQR9 influences this process. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Funding This work was financially supported by the National Natural Science Foundation of China (42307173), the China Postdoctoral Science Foundation (2023M747140), and the Rural Revitalization Strategy Project Seed Industry Vitalization Action Project of Guangdong Province (2023WPY00002). XS was supported by the Postdoctoral Fellowship Program of CPSF (GZB20230309), and the Excellent Postdoctoral Program of Jiangsu Province (2023ZB250). Author Contribution Nan Zhang, Ruifu Zhang, Qirong Shen, Kai Wu, and Zhihui Xu contributed to the study conception and design. Material preparation, experiment conduction, and data collection were performed by Xinli Sun, Riyan Xia, Jiyu Xie, and Kun Duan. Data analysis was performed by Xini Sun, Weibing Xun, and Guidong Huang. The first draft of the manuscript was written by Xinli Sun and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data Availability The raw sequencing data have been deposited in the NCBI Sequence Read Archive under the BioProject accession number PRJNA1209616. Metabolome data was provided in Supplementary Table. References Gordon TR (2017) Fusarium oxysporum and the Fusarium Wilt Syndrome. Annu Rev Phytopathol 55:23–39. https://doi.org/10.1146/annurev-phyto-080615-095919 Berendsen RL, Pieterse CMJ, Bakker PAHM (2012) The rhizosphere microbiome and plant health. 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09:38:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6904063/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6904063/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00248-025-02592-3","type":"published","date":"2025-08-29T15:56:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85076212,"identity":"744a9bd1-b546-412f-a72d-afdbb8ceae4c","added_by":"auto","created_at":"2025-06-20 16:39:05","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":436641,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePositive correlation between \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBacillus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLysobacter\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in the rhizosphere of healthy plants\u003c/strong\u003e. \u003cstrong\u003e(A) \u003c/strong\u003ePot experimental design. CTL: control group, no inoculation; B: inoculation with \u003cem\u003eB. velezensis\u003c/em\u003e SQR9; F: inoculation with \u003cem\u003eF. oxysporum\u003c/em\u003e FOC; BF: inoculation with both \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 and \u003cem\u003eF. oxysporum\u003c/em\u003e FOC.\u003cstrong\u003e (B) \u003c/strong\u003eDifferences in the relative abundance of genera between \u003cem\u003eF. oxysporum \u003c/em\u003eFOC inoculation and Co-inoculation of \u003cem\u003eF. oxysporum \u003c/em\u003eFOC and \u003cem\u003eB. velezensis \u003c/em\u003eSQR9 rhizosphere samples (Welch’s t-test; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). n = 6. \u003cstrong\u003e(C) \u003c/strong\u003ePearson correlation of the relative abundance of \u003cem\u003eBacillus\u003c/em\u003espp. and \u003cem\u003eLysobacter\u003c/em\u003e spp. in the rhizosphere of diseased vs. healthy plants. \u003cstrong\u003e(D)\u003c/strong\u003e Pearson correlation of the relative abundance of \u003cem\u003eBacillus\u003c/em\u003espp. and \u003cem\u003eChinitophaga\u003c/em\u003e spp. in the rhizosphere of diseased vs. healthy plants. \u003cstrong\u003e(E)\u003c/strong\u003e Relative abundances of \u003cem\u003eBacillus\u003c/em\u003e spp., \u003cem\u003eChitinophaga\u003c/em\u003espp., and \u003cem\u003eLysobacter\u003c/em\u003e spp. in the rhizospheres of diseased and healthy plants. Each boxplot indicates the median, interquartile range, and outliers. Statistical significance between groups was assessed with a t-test. Data collected by Yuan et al. [9]. \u003cstrong\u003e(F)\u003c/strong\u003e Relative abundances of \u003cem\u003eBacillus\u003c/em\u003e spp. and \u003cem\u003eLysobacter \u003c/em\u003espp. in different treatment groups in this study. Each boxplot indicates the median and interquartile range. Different letters indicate significant differences according to Welch’s ANOVA and Dunnett’s post hoc test (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). n = 6.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6904063/v1/531906b17c081616457716e8.jpeg"},{"id":85076631,"identity":"42a51c35-0803-452f-b6e1-3e3e1b8b8692","added_by":"auto","created_at":"2025-06-20 16:47:05","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":356496,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiofilm formation and antifungal activity of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLysobacter\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e spp. and the consortium. (A) \u003c/strong\u003eBiofilm phenotype. \u003cstrong\u003e(B) \u003c/strong\u003eBiofilm quantification by crystal violet staining. “*” indicates a significant difference from SQR9 based on a t-test (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05). The data are shown as the means ± SDs (n = 4). \u003cstrong\u003e(C) \u003c/strong\u003eAntagonistic activity of the \u003cem\u003eLysobacter\u003c/em\u003e isolates against \u003cem\u003eF. oxysporum\u003c/em\u003e. \u003cstrong\u003e(D) \u003c/strong\u003eAntagonistic effects of SQR9 extracts and the XL8 supernatant. The Oxford cup contained extracts from SQR9 monocultures or/and supernatants from XL8 monocultures. \u003cstrong\u003e(E)\u003c/strong\u003e Inhibition zone of SQR9 extractions and XL8 supernatant against \u003cem\u003eF. oxysporum\u003c/em\u003e. The data are shown as the means ± SDs (n = 4). Different letters indicate significant differences according to ANOVA and Tukey’s post hoc test (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05).\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6904063/v1/02bdd49980ebae5daa2d6817.jpeg"},{"id":85076216,"identity":"57f26361-a4a5-4b47-af9d-399684aaa180","added_by":"auto","created_at":"2025-06-20 16:39:05","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":267656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe dual-species consortium protects plants against pathogen infection. (A) \u003c/strong\u003ePlant phenotype. n=10. \u003cstrong\u003e(B)\u003c/strong\u003eShoot height. The data are shown as the means ± SDs (n = 10). \u003cstrong\u003e(C)\u003c/strong\u003e Shoot fresh weight. The data are shown as the means ± SDs (n = 10). \u003cstrong\u003e(D)\u003c/strong\u003eDisease index. \u003cstrong\u003e(E) \u003c/strong\u003eAbundance of \u003cem\u003eF. oxysporum \u003c/em\u003ein the rhizosphere soil. The data are shown as the means ± SDs (n = 6). Different letters indicate significant differences according to ANOVA and Tukey’s post hoc test (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6904063/v1/df406be5a4775ea9c8d5616e.jpeg"},{"id":85076225,"identity":"5e820c5d-1cac-4094-9470-049f73fe0551","added_by":"auto","created_at":"2025-06-20 16:39:05","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":376324,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eB. velezensis SQR9 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003efacilitates the growth of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. enzymogenes \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eXL8 through cross-feeding. (A) \u003c/strong\u003eExperimental design of the metabolic facilitation assay. \u003cstrong\u003e(B) \u003c/strong\u003eGrowth of XL8 in the spent medium of SQR9 on the 5\u003csup\u003eth\u003c/sup\u003e day. The data are shown as the means ± SDs (n = 6). \u003cstrong\u003e(C) \u003c/strong\u003eMetabolic attraction of M9 glucose medium. \u003cstrong\u003e(D) \u003c/strong\u003eMetabolic profile of bacterial spent medium. The heatmap shows the 30 compounds with the most significant differences in abundance between treatments. S represents the spent medium of SQR9 grown in M9 glucose media, and S_XL8 represents the spent medium of XL8 grown in SQR9 filtrate. N = 6.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6904063/v1/c67672fc4bab9137ca39050c.jpeg"},{"id":85076637,"identity":"bb97297e-75b3-4861-a2ea-503e13d81c15","added_by":"auto","created_at":"2025-06-20 16:47:05","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":101034,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelative expression of biosynthetic genes of secondary metabolites in cultures supplemented with bacterial spent medium. \u003c/strong\u003eBGC represents the biosynthesis-related gene cluster. The control group was grown in M9 glucose media, whereas the treatment group was grown in M9 glucose media supplemented with 20% each other’s spent media. The data are shown as the means ± SDs (n = 6). Log\u003csub\u003e10\u003c/sub\u003e(2^\u003csup\u003e-ΔΔCт\u003c/sup\u003e) value greater than 0.5 or less than -0.5 was considered significant.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6904063/v1/72cb49abea13d4db1f296ee9.jpeg"},{"id":90344861,"identity":"7895f15c-28b6-466e-9a2c-98380325054b","added_by":"auto","created_at":"2025-09-01 16:06:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2698233,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6904063/v1/99aa6393-0631-417a-9211-1aa30e7d28dd.pdf"},{"id":85076630,"identity":"4443d9f6-7c8d-45a9-bb43-bf577d8f4788","added_by":"auto","created_at":"2025-06-20 16:47:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":182954,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6904063/v1/30381fab0f2bd4a6c5b99e8e.pdf"},{"id":85076214,"identity":"9e96ad70-b9b4-48fb-8d6c-e2577a558da4","added_by":"auto","created_at":"2025-06-20 16:39:05","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":159276,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6904063/v1/1eabdbe5338baaf2f340e3e2.pdf"},{"id":85076220,"identity":"15a41e03-d802-4d9c-a584-e76b75f40364","added_by":"auto","created_at":"2025-06-20 16:39:05","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":146349,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6904063/v1/9177c227ed20b00159aa9cad.pdf"},{"id":85077191,"identity":"4110a44a-4078-4036-91f4-cee77ee8a895","added_by":"auto","created_at":"2025-06-20 16:55:05","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":209960,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6904063/v1/48b0065f5ab6d575d7eea4ea.pdf"},{"id":85076229,"identity":"e7ff04fa-a100-4f1e-948b-bd5ac9ba686e","added_by":"auto","created_at":"2025-06-20 16:39:05","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":364892,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6904063/v1/bf4804e450e3de41f37da470.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cooperative Interactions Between Bacillus and Lysobacter Enhance Consortium Stability and Fusarium Wilt Suppression in Cucumber","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlant disease management remains a major challenge in agriculture, particularly given the environmental concerns associated with chemical pesticides. The \u003cem\u003eFusarium oxysporum\u003c/em\u003e species complex is notorious for causing vascular wilt diseases in many crops, leading to severe yield losses worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The root microbiome, often referred to as the plant\u0026rsquo;s \u0026ldquo;second genome,\u0026rdquo; plays a crucial role in maintaining plant health [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Upon pathogen infection, plant roots can recruit beneficial microbes that inhibit pathogen invasion through direct antibiosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] or by inducing systemic resistance [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. For instance, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e selectively recruits protective rhizosphere bacteria following foliar infection by the downy mildew pathogen \u003cem\u003eHyaloperonospora arabidopsidis\u003c/em\u003e, enhancing disease resistance and growth [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Similarly, \u003cem\u003eF. oxysporum\u003c/em\u003e modulates the tomato rhizosphere differently depending on host genotype, increasing the relative abundance of \u003cem\u003eLysobacter\u003c/em\u003e, \u003cem\u003eSphingobium\u003c/em\u003e, and \u003cem\u003eSphingomonas\u003c/em\u003e species in the resistant cultivar [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. A recent meta-analysis revealed that the root microbiome of \u003cem\u003eFusarium\u003c/em\u003e wilt-infected plants is enriched in \u003cem\u003eXanthomonadaceae\u003c/em\u003e, \u003cem\u003eBacillaceae\u003c/em\u003e, \u003cem\u003eGibberella\u003c/em\u003e, and \u003cem\u003eF. oxysporum\u003c/em\u003e, whereas healthy plants harbor greater abundances of \u003cem\u003eStreptomyces\u003c/em\u003e, \u003cem\u003eMirabilis\u003c/em\u003e, \u003cem\u003eBradyrhizobiaceae\u003c/em\u003e, \u003cem\u003eComamonadaceae\u003c/em\u003e, and \u003cem\u003eMortierella\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSynthetic microbial communities (SynComs) have emerged as a promising tool for enhancing plant health [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. One notable advantage of SynComs is their ability to perform complementary biocontrol functions, which offer a multifaceted defense against pathogens. Native root-associated bacterial consortia have been reported to protect plants from sudden wilt diseases through a suite of biocontrol traits [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Similarly, SynComs have been demonstrated to improve nutrient efficiency and yield in crops such as soybean and tomato and confer resistance to \u003cem\u003eFusarium\u003c/em\u003e wilt disease [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These communities mimic natural microbial interactions, providing novel mechanistic insights into microbiome stability, which is important for plant health [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Beyond disease suppression, the use of SynComs extends to increase plant growth by increasing nutrient acquisition efficiency [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], increasing resilience to salt and drought stress [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and increasing resistance to aluminum toxicity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eBacillus velezensis\u003c/em\u003e SQR9 is a well-characterized biocontrol bacterium that suppresses \u003cem\u003eFusarium\u003c/em\u003e wilt and promotes plant growth through various mechanisms [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It synthesizes various secondary metabolites, including bacillomycin D, fengycin, bacillaene, bacillibactin, difficidin, surfactin, macrolactin, and bacilycin [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. It can also stimulate the resident rhizosphere species \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e through metabolic interactions, which collectively improve plant growth and salt tolerance [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, how \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 reshapes the rhizosphere microbiome under pathogen invasion remains unclear.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the impact of \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 on cucumber rhizosphere bacterial diversity and structure during infection by pathogen \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003ecucumerinum\u003c/em\u003e FOC and explored its interaction with indeginous benefical bacteria. Our results revealed that \u003cem\u003eLysobacter\u003c/em\u003e spp., known antifungal biocontrol agents [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], were enriched by \u003cem\u003eB. velezensis\u003c/em\u003e SQR9. A meta-analysis further revealed a positive correlation between \u003cem\u003eLysobacter\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e abundances in healthy plant rhizospheres. We then isolated \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 from the cucumber rhizosphere and constructed a dual-species consortium with SQR9. By assessing biofilm formation, cross-feeding interactions, and antifungal compound production, we demonstrated that this consortium enhances plant disease resistance and growth. These findings provide new insights into bacterial interactions in the rhizosphere and support the development of microbial consortia for sustainable plant disease management.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains and media\u003c/h2\u003e \u003cp\u003e \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 (CGMCC No.5808) and \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 (GDMCC accession No.64920) were stored at \u0026minus;\u0026thinsp;80\u0026deg;C in Tryptone Soy Broth (TSB) supplemented with 25% glycerol. \u003cem\u003eLysobacter\u003c/em\u003e spp. were isolated previously from the rhizosphere of cucumber plants inoculated with \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and stored at \u0026minus;\u0026thinsp;80\u0026deg;C in Reasoner\u0026rsquo;s 2B broth (R2B) or TSB supplemented with 25% glycerol. Spores of \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003ecucumerinum\u003c/em\u003e J. H. Owen (FOC) were stored at \u0026minus;\u0026thinsp;80\u0026deg;C in potato dextrose broth (PDB) with 25% glycerol. The media were solidified with 1.5% agar when necessary. Strains were grown in TSB, R2B, or PDA as appropriate. Overnight cultures were harvested, washed, and resuspended in 0.9% NaCl to an OD\u003csub\u003e600\u003c/sub\u003e of 1 for inoculations.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePot experiments\u003c/h3\u003e\n\u003cp\u003eSoil was collected from a field in Taizhou City, Jiangsu Province, China (32.4555\u0026deg; N, 119.9229\u0026deg; E), in 2018. Surface sterilized Cucumber seeds (Jinchun 4) were pre-germinated before transplantation into pots.\u003c/p\u003e \u003cp\u003eIn the first pot experiment, treatments include: CTL (control group, no inoculation); B (inoculation with \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 suspension at a final concentration of 10\u003csup\u003e6\u003c/sup\u003e cells/g soil); F (inoculation with \u003cem\u003eF. oxysporum\u003c/em\u003e FOC spore suspensions at a final concentration of 10\u003csup\u003e6\u003c/sup\u003e spores/g soil; and BF (inoculation with both \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 (10\u003csup\u003e6\u003c/sup\u003e cells/g soil) and \u003cem\u003eF. oxysporum\u003c/em\u003e FOC (10\u003csup\u003e6\u003c/sup\u003e spores/g soil)). Each treatment group consisted of six replicates. The plants were grown in a greenhouse at 30\u0026deg;C with a 16:8 h light/dark cycle. After 16 days, rhizosphere soil was collected as described by Bai et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the second pot experiment, in addition to the CTL, B, F, and BF treatment groups, two additional treatment groups were included: LF (inoculation with both \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 (10\u003csup\u003e6\u003c/sup\u003e cells/g soil) and \u003cem\u003eF. oxysporum\u003c/em\u003e FOC (10\u003csup\u003e6\u003c/sup\u003e spores/g soil)), and BLF (inoculation with \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 (10\u003csup\u003e6\u003c/sup\u003e cells/g soil), \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 (10\u003csup\u003e6\u003c/sup\u003e cells/g soil) and \u003cem\u003eF. oxysporum\u003c/em\u003e FOC (10\u003csup\u003e6\u003c/sup\u003e spores/g soil)). Each treatment had ten replicates. After 30 days, photos were taken of the plants. Shoot height and fresh weight were recorded. The disease index was calculated as the number of diseased plants divided by the total number of all plants. Moreover, the rhizosphere soil from six replicates was collected for DNA extraction. The abundance of \u003cem\u003eF. oxysporum\u003c/em\u003e in the rhizosphere soil was quantified via qPCR [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eAmplicon sequencing and analysis\u003c/h3\u003e\n\u003cp\u003eRhizosphere DNA was extracted using the DNeasy PowerSoil Pro Kit (QIAGEN cat no. 47014). Bacterial universal primers (338F/806R) targeting the V3\u0026thinsp;\u0026minus;\u0026thinsp;V4 regions of the 16S rRNA gene were employed to construct the DNA library for sequencing. Paired-end sequencing of bacterial amplicons was performed on an Illumina MiSeq 300 platform (Genewiz, Suzhou, China). The reads were processed using the UPARSE pipeline (version 8.1) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Specifically, the paired-end reads were merged with the \u0026ldquo;fastq_mergepairs\u0026rdquo; command. Following this, high-quality sequences were selected using the \u0026ldquo;fastq_filter\u0026rdquo; command, and dereplication was performed with the \u0026ldquo;derep_fulllength\u0026rdquo; command. Singletons were eliminated using USEARCH-unoise3 algorithm, while chimeric sequences were removed with the \u0026ldquo;uchime_ref\u0026rdquo; command. The remaining sequences were clustered into operational taxonomic units (OTUs) at a 97% similarity threshold using the \u0026ldquo;cluster_otus\u0026rdquo; command. Taxonomic assignment of the OTUs was performed using the Ribosomal Database Project classifier (version 11). Additionally, Bray\u0026thinsp;\u0026minus;\u0026thinsp;Curtis distance-based principal coordinates analysis (PCoA), and permutational multivariate analysis of variance (PERMANOVA) were conducted based on the OTU table using the vegan R package. Welch\u0026rsquo;s t-test was used to assess the significance of differences between the two treatments using STAMP [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Only OTUs with a relative abundance exceeding 0.05% across all samples were included in this analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMeta-analysis of amplicon sequencing data from\u003c/b\u003e \u003cb\u003eFusarium\u003c/b\u003e \u003cb\u003ewilt disease studies\u003c/b\u003e\u003c/p\u003e \u003cp\u003eYuan et al. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] collected amplicon sequencing data from several studies investigating wilt diseases that affect various plant species. We obtained the OTU table directly from the authors. Pearson correlation analysis was conducted using the R package \u0026ldquo;psych\u0026rdquo; to examine the correlation between the genera present in the rhizosphere. A t-test was performed to compare the relative abundance of genera in healthy and diseased plant rhizospheres. All the statistical analyses were performed via R software, with significance defined at a threshold of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003ch3\u003eBiofilm formation and quantification\u003c/h3\u003e\n\u003cp\u003eTo examine the formation of pellicle biofilms, 20 \u0026micro;L of the starting inoculum was cultivated in 2 mL of TSB in a 24-well microtiter plate, and incubated statically for 48 h at 30\u0026deg;C. For coculture, the starting inoculum was prepared by mixing equal volumes of isolates. Photographs were taken to observe pellicle formation. Biofilm quantification was performed via a modified crystal violet (CV) staining method [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In brief, the growth medium and nonadherent cells were removed via a sterile syringe, and the wells were then rinsed with distilled water. The biofilm-containing cells were stained with 2 mL of 1% CV for 30 min at room temperature. Unbound CV was removed, and the wells were washed twice with distilled water. The biofilm-bound CV was solubilized with 2 mL of ethanol: acetone (4:1 v/v) for 20 min. Biofilm formation was quantified by measuring the OD\u003csub\u003e570\u003c/sub\u003e via a multifunctional plate reader. Each treatment had four biological replicates.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntagonism test of\u003c/b\u003e \u003cb\u003eLysobacter\u003c/b\u003e \u003cb\u003eisolates against\u003c/b\u003e \u003cb\u003eF. oxysporum\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn vitro, antagonism tests were performed on \u0026frac12; potato dextrose agar (PDA) mixed with \u0026frac12; R2A agar in 9-cm Petri dishes. \u003cem\u003eF. oxysporum\u003c/em\u003e FOC spores from glycerol stocks (2 \u0026micro;L) were inoculated at the center of the plate and incubated at 28\u0026deg;C for 2 days. Then, 2 \u0026micro;L of bacterial suspension (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1) was inoculated 3 cm away from the center of the fungal colony. The plates were incubated at 28\u0026deg;C for another 4 days. Photographs were taken to assess the antagonistic activity.\u003c/p\u003e\n\u003ch3\u003eBIOLOG assay\u003c/h3\u003e\n\u003cp\u003eCarbon utilization profiles of \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 and \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 were assessed using GEN III MicroPlate (Biolog cat no. 1030) contains 71 carbon source use assays and 23 chemical sensitivity tests. Overnight cultures were harvested, washed, and resuspended in inoculating fluid A (Biolog, cat no. 72401). Cell suspension (100 \u0026micro;L) was added per well and incubated at 30\u0026deg;C in an OmniLog instrument for 48 h. Color development, indicating metabolic activity, was automatically recorded. Each strain was tested in triplicate.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMetabolic facilitation assays and metabolome analyses\u003c/h2\u003e \u003cp\u003eB. velezensis SQR9 was cultured in M9 glucose medium (0.2% glucose) at 30\u0026deg;C with shaking (180 rpm) until glucose was depleted, as confirmed by a Glucose GO Assay Kit (Sigma-Aldrich, GAGO20). Spent medium was collected by centrifugation and filter sterilized. \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 was inoculated into the spent medium (1% v/v) and incubated under the same conditions for 5 days. Growth was measured by OD\u003csub\u003e600\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;6). Extracellular metabolites were collected and analyzed via ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Data were processed using Compound Discoverer 3.0 for peak alignment, detection, and quantification. Metabolites were annotated by matching spectra against mzCloud and ChemSpider databases. Each treatment included six replicates.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExpression analysis of secondary metabolite biosynthetic genes\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 and \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 were incubated in M9 glucose media at 30\u0026deg;C, 180 rpm for 48h. Supernatants were collected, filter-sterilized, and used (20% v/v) to supplement fresh cultures of the partner strain; M9-glucose medium alone served as control. After 48 h, cells were harvested for total RNA extraction (E.Z.N.A Bacterial RNA Kit), cDNA synthesis (Hiscript II Q RT SuperMix), and RT-qPCR (ChamQ Universal SYBR qPCR Master Mix). Biosynthetic gene clusters (BGCs) in the \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 genome were predicted via antiSMASH 7.0 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The transcription levels of genes involved in the biosynthesis of bacillomycin D, fengycin, bacillaene, surfactin, difficidin, bacillibactin, macrolactin, and bacilysin in \u003cem\u003eB. velezensis\u003c/em\u003e SQR9, as well as WAP-8294A2, HSAF, and Le-pyrrolopyrazines in \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8, were quantified via RT‒qPCR. Primer sequences are listed in the Supplementary Table. The \u003cem\u003erecA\u003c/em\u003e gene was used as the reference gene for both strains. RT‒qPCR was performed on an ABI 7500 system (ABI, USA) with the following thermal conditions: initial denaturation at 95\u0026deg;C for 30 s; 40 cycles of 95\u0026deg;C for 10 s and 60\u0026deg;C for 30 s; and standard melting curve analysis. The 2\u003csup\u003e\u0026minus;∆∆Ct\u003c/sup\u003e method was used to calculate the relative expression changes [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This experiment had six biological replicates.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eBacillus\u003c/b\u003e \u003cb\u003espp. and\u003c/b\u003e \u003cb\u003eLysobacter\u003c/b\u003e \u003cb\u003espp. are positively correlated in healthy plant rhizosphere\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe experimental design is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. Rhizosphere soil samples were for bacterial community profiling via amplicon sequencing. Compared to the non-inoculated control, inoculation with either \u003cem\u003eB. velezensis\u003c/em\u003e or \u003cem\u003eF. oxysporum\u003c/em\u003e did not affect Chao1 diversity (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA) but significantly increased the community richness (Welch\u0026rsquo;s test, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Principal coordinate analysis (PCoA) based on Bray‒Curtis distance matrices revealed significant differences in rhizosphere bacterial communities across the four treatment groups (PERMANOVA, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.74, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). Inoculated groups were clearly separated from the control along the first coordinate (PCoA1), whereas the dual inoculation (BF) treatment was distinguished from the single-inoculations along the second coordinate (PCoA2). In particular, the relative abundances of the genera \u003cem\u003eChitinophaga\u003c/em\u003e, \u003cem\u003eChryseobacterium\u003c/em\u003e, and \u003cem\u003eLysobacter\u003c/em\u003e were significantly greater in the BF group compared to the F group, whereas those of \u003cem\u003eCellvibrio\u003c/em\u003e, \u003cem\u003eErwinia\u003c/em\u003e, \u003cem\u003eFlavobacterium\u003c/em\u003e, and \u003cem\u003ePseudoxanthomonas\u003c/em\u003e were significantly reduced (Welch\u0026rsquo;s test, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn a recent study, Yuan et al. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] gathered amplicon sequencing data from rhizosphere samples of both healthy plants and plants affected by \u003cem\u003eFusarium\u003c/em\u003e wilt, across various plant species. From this dataset, we extracted the relative abundance of OTUs identified as \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eChitinophaga\u003c/em\u003e, \u003cem\u003eChryseobacterium\u003c/em\u003e, and \u003cem\u003eLysobacter\u003c/em\u003e. The results indicated that the abundance of \u003cem\u003eChryseobacterium\u003c/em\u003e spp. was below the detection limits. In the the rhizosphere of healthy plants, \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eLysobacter\u003c/em\u003e showed a significant positive correlation (R\u0026thinsp;=\u0026thinsp;0.26, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), whereas no such correlation was observed in diseased plants (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Additionally, no significant correlation was detected between \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eChitinophaga\u003c/em\u003e in healthy plants, while a weak negative correlation emerged in diseased plants (R = -0.18, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.032) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), which differs from our data. Both \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eChitinophaga\u003c/em\u003e presented significantly greater relative abundances in healthy compared to diseased plant rhizospheres (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Although \u003cem\u003eLysobacter\u003c/em\u003e was more abundant in the rhizosphere of healthy plants than in the rhizosphere of diseased plants, this difference was not statistically significant.\u003c/p\u003e \u003cp\u003eIn the present study, inoculation with either \u003cem\u003eB. velezensis\u003c/em\u003e or \u003cem\u003eF oxysporum\u003c/em\u003e enriched \u003cem\u003eLysobacter\u003c/em\u003e, with \u003cem\u003eB. velezensis\u003c/em\u003e exerting a stronger effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Previous research showed that biofertilizers containing \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e W19 also increases the relative abundance of \u003cem\u003eLysobacter\u003c/em\u003e in the rhizosphere [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Rather than acting through direct pathogen suppression by \u003cem\u003eBacillus\u003c/em\u003e spp., these enriched \u003cem\u003eLysobacter\u003c/em\u003e may serve as a keytone taxa in controlling \u003cem\u003eF. oxysporum\u003c/em\u003e. Consistent with this, in the present study, the relative abundance of \u003cem\u003eBacillus\u003c/em\u003e itself did not significantly increase in the B or BF groups compared to the uninoculated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). In addition, \u003cem\u003eF. oxysporum\u003c/em\u003e has been reported to elevate \u003cem\u003eLysobacter\u003c/em\u003e abundance in the rhizosphere of disease-resistant tomato cultivars and decreases its abundance in susceptible cultivars, suggesting a link between the abundance of \u003cem\u003eLysobacter\u003c/em\u003e and the suppression of \u003cem\u003eFusarium\u003c/em\u003e pathogens [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In light of these findings, we sought to examine the interaction between \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eLysobacter\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBiofilm formation and antifungal activity of\u003c/b\u003e \u003cb\u003eLysobacter\u003c/b\u003e \u003cb\u003espp.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the interaction between \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 and \u003cem\u003eLysobacter\u003c/em\u003e spp., 15 \u003cem\u003eLysobacter\u003c/em\u003e isolates were selected from our strain collection (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), which were isolated from the cucumber rhizosphere pre-treated with \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. On the root surface, rhizobacteria form biofilms that protect them from environmental stress and enable them to interact positively with the host plant [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Previous studies have shown that plant-beneficial bacteria can form biofilms synergistically within consortia [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. To assees this, an in vitro biofilm formation assay was conducted. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, none of the \u003cem\u003eLysobacter\u003c/em\u003e isolates formed visible biofilms individually. However, co-culturing with \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 enhanced biofilm wrinkling in isolates XL169, XL170, XL171, and XL8 compared to the strain SQR9 monoculture. Biofilm quantification assays further comfirmed that co-cultures produced significantly greater biofilm biomass than individual cultures (t-test, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eNext, we assessed the antagonistic activity of these \u003cem\u003eLysobacter\u003c/em\u003e isolates against \u003cem\u003eF. oxysporum\u003c/em\u003e in vitro. Only strains XL166 and XL8 inhibited the growth of \u003cem\u003eF. oxysporum\u003c/em\u003e mycelia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Given the superior antagonistic activity of \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8, we tested the additive effect of its culture supernatants with extracts of \u003cem\u003eB. velezensis\u003c/em\u003e SQR9. The dual application produced the largest inhibition zone against \u003cem\u003eF. oxysporum\u003c/em\u003e FOC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Based on it strong biofilm-promoting and antifungal activity, \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 was selected for further study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBacillus-Lysobacter\u003c/b\u003e \u003cb\u003econsortium protects plants against wilt disease\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe protective effect of the dual-species consortium comprising \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 and \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 against F. oxysporum FOC infection was evaluated in a greenhouse experiment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, plants inoculated with FOC alone (F group) exhibited severe disease symptoms, including wilting and leaf yellowing. Both single biocontrol bacterium treatments (BF and LF groups) provided partial protection, but the dual-species consortium (BLF group) was significantly more effective. Plants in the BLF group displayed the highest biomass (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) and the lowest disease index (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), with differences among treatments being statistically significant. Moreover, the abundance of \u003cem\u003eF. oxysporum\u003c/em\u003e FOC was also lowest in the BLF treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), indicating superior pathogen suppresion. Taken together, these results demonstrate that the combined inoculation of \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 and \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 exerts a synergistic effect, enhancing both plant growth and disease resistance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBacillus velezensis\u003c/b\u003e \u003cb\u003eSQR9 facilitates the growth of\u003c/b\u003e \u003cb\u003eLysobacter enzymogenes\u003c/b\u003e \u003cb\u003eXL8 through cross-feeding\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo elucidate the mechanism underlying the synergistic interaction between \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 and \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8, we investigated their metabolic interaction. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA depicts the experimental setup for the cross-feeding assay. \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 showed limited growth in M9 glucose media but was able to grow in the presence of the culture supernatant of \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Consistent with this finding, strain XL8 was found to grow in the vicinity of \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 colonies on plates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), suggesting metabolic cross-feeding.\u003c/p\u003e \u003cp\u003eA metabolomic analysis was conducted to identify the metabolites potentially involved in this growth facilitation. The heatmap in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD shows the relative abundance of metabolites in the supernatant of \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 and the supernatant of \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 grown on SQR9-derived medium. Metabolites enriched in the strain SQR9 supernatant but depleted in the strain XL8 supernatant were identified as potential cross-feeding compounds. Four commercially available metabolites\u0026mdash;deoxycholic acid, 6-dimethylamino purine, 5-hydroxyindole, and D-phenylalanine\u0026mdash;were tested to determine whether they could support the growth of \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 in M9 minimal medium when used as the sole carbon source. The results revealed that the deoxycholic acid secreted by \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 can be utilized by \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). The carbon source useability of strains SQR9 and XL8 was measured via BIOLOG assays, and the results demonstrated that \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 can use a wide range of carbon sources (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEnhanced antifungal metabolites production in the dual-species consortium\u003c/h2\u003e \u003cp\u003eTo assess whether \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 and \u003cem\u003eL. enzymogenes\u003c/em\u003e influence each other\u0026rsquo;s secondary metabolite production, we performed RT‒qPCR analysis. Gene expression associated with secondary metabolite biosynthesis was compared between M9 glucose medium and M9 supplemented with 20% bacterial spent medium. \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 is known to produce eight antibioticsWhen exposed to XL8 spent medium, SQR9 showed significantly increased expression of genes responsible for bacillomycin D and bacillaene synthesis, while genes related to surfactin production were downregulated compared to the M9 control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Bacillomycin D has been demonstrated to exert antifungal activity against \u003cem\u003eF. oxysporum\u003c/em\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Bacillaene is a polysaccharide that exhibits antimicrobial activity against various bacteria and fungi, including \u003cem\u003eFusarium\u003c/em\u003e spp. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Surfactin, a lipopeptide produced by various \u003cem\u003eBacillus\u003c/em\u003e species, exhibits broad-spectrum antimicrobial activity [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The downregulation of surfactin production by SQR9 may be correlated with reduced competition with XL8.\u003c/p\u003e \u003cp\u003eAntiSMASH analysis revealed that \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 contains three biosynthetic gene clusters (BGCs) for secondary metabolite production with 100% similarity to known clusters: HSAF (an antifungal antibiotic heat-stable antifungal factor that inhibits the growth of fungal hypha and the conidial germination) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], WAP-8294A2 (a cyclopeptide with activity against Gram-positive bacteria) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], and Le-pyrrolopyrazines (function unknown) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Our results revealed that \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 significantly upregulated HSAF and WAP-8294A2 biosynthetic genes when cultured in SQR9 spent medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These findings suggest mutual enhancement of antifungal metabolite production between the two strains, which likely contributes to their improved biocontrol efficacy observed in greenhouse experiments.\u003c/p\u003e\u003c/div\u003e "},{"header":"Conclusion and Discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003cp\u003eOur findings demonstrated that the \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 significantly increases the abundance of \u003cem\u003eLysobacter\u003c/em\u003e in the rhizosphere of cucumber plants infected with pathogenic \u003cem\u003eF. oxysporum\u003c/em\u003e. A positive correlation between the abundance of \u003cem\u003eLysobacter\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e is commonly observed in the rhizosphere of healthy plants, suggesting a conserved ecological association. The synergistic biofilm formation and metabolic cross-feeding interactions between the biocontrol bacterium \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 and the antifungal agent \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 suggest mechanisms that improve their fitness in the rhizosphere. Additionally, the upregulated expression bacillomycin D and HSAF biosynthetic genes\u0026mdash;both encoding antifungal compounds known to antagonize \u003cem\u003eF. oxysporum\u003c/em\u003e\u0026mdash;along with the expanded inhibition zone observed \u003cem\u003ein vitro\u003c/em\u003e, further indicate the synergistic biocontrol ability of this bacterial consortium. These combined mechanisms contribute to the effective suppression of cucumber \u003cem\u003eFusarium\u003c/em\u003e wilt disease.\u003c/p\u003e \u003cp\u003eEnhanced biofilm formation is a common characteristic of plant-beneficial bacterial communities. Our study revealed that \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 and \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 form synergistic biofilm, which may improve their colonizatoin and adaptability in the rhizosphere. Similar interactions have been reported between \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 and \u003cem\u003eP. stutzeri\u003c/em\u003e XL272, resulting in improved plant growth promotion and salt tolerance [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Biofilm formation is crucial for microbial survival and functionality in diverse environments. For example, the emergent properties of biofilm-forming communities can confer increased drought tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and facilitate the establishment of keystone species, ultimately promoting overall plant growth [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Disease-induced assemblage plant-beneficial bacterial consortia have also been reported to protect host plants against pathogens by synergistically forming biofilms [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Likewise, complementary biocontrol traits and cooperative biofilm formation contribute to the suppression of sudden wilt diseases in root-associated microbial communities [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Collectively, these findings emphasize the importance of synergistic biofilm formation in fostering microbial resilience, function stability, and plant health under various stress conditions. However, the ecological significance of beneficial bacterial biofilm formation under natural rhizosphere conditions remains poorly understood. Bridging this knowledge gap is essential for a deeper understanding of how microbial biofilms contribute to plant health, disease resistance, and environmental stress tolerance in real-world agricultural systems.\u003c/p\u003e \u003cp\u003eMetabolic interactions play crucial roles in regulating the assembly of SynComs [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Our results suggest that the \u003cem\u003eB. velezensis\u003c/em\u003e strain SQR9 promotes the growth of the \u003cem\u003eL. enzymogenes\u003c/em\u003e strain XL8 by cross-feeding. Similarly, a seven-species SynCom has been reported to inhibit the maize fungal pathogen \u003cem\u003eF. verticillioides\u003c/em\u003e, with \u003cem\u003eEnterobacter cloacae\u003c/em\u003e identified as the keystone species [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Each member of SynCom occupies complementary metabolic niches, and spent media derived from the prototrophic strains can support the growth of two auxotroph strains [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The keystone species \u003cem\u003eE. cloacae\u003c/em\u003e serves as a metabolic donor, enhancing overall SynCom biomass and utilizing diverse carbon sources from root exudates to prevent the dominance of undesirable taxa [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Keystone species may either promote SynCom biomass through facilitation or suppress it via competitive exclusion, depending on their metabolic roles [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. A two-tiered cross-feeding system in which one strain alleviates toxic byproduct accumulation and the other detoxifies environmental toxins, demonstrates how cooperative exchanges extend beyond nutrient sharing to modulate community structure and enhance competitive fitness [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Such costless metabolic exchanges act as drivers of interspecies interactions within microbial communities and can be leveraged to design SynCom with increased ecological fitness and stability [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur findings also suggest that exposure to spent media from consortium partners upregulates antifungal biosynthetic genes in both \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 and \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8, thereby enhancing their biocontrol potential. This is consistent with previous reports where SynCom improves their antifungal against maize seed-borne \u003cem\u003eFusarium\u003c/em\u003e pathogens by stimulating antibiotic biosynthetic genes expression [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. A similar underlies the mutualism between \u003cem\u003eB. velezensis\u003c/em\u003e and arbuscular mycorrhizal fungi, where fungal signals modulate bacterial secondary metabolite production, stabilizing the coexistence of both partners in the hyphosphere [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Such interactions not only promote systemic resistance in the host plant but also increase the overall biocontrol ability of the bacterial‒fungal consortium. Future studies should investigate the mechanisms of chemical signaling between \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 and \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 to identify the key compounds regulating secondary metabolite expression in each organism. Moreover, \u003cem\u003eL. enzymogenes\u003c/em\u003e OH11 has been reported to utilize a type VI secretion system (T6SS) to secrete toxic effectors to pathogenic filamentous fungi in a contact-dependent manner, which inhibits the growth of conidia into hyphae [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. It remains to be investigated whether a similar antifungal mechanism exists in \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 and whether its interaction with \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 influences this process.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was financially supported by the National Natural Science Foundation of China (42307173), the China Postdoctoral Science Foundation (2023M747140), and the Rural Revitalization Strategy Project Seed Industry Vitalization Action Project of Guangdong Province (2023WPY00002). XS was supported by the Postdoctoral Fellowship Program of CPSF (GZB20230309), and the Excellent Postdoctoral Program of Jiangsu Province (2023ZB250).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eNan Zhang, Ruifu Zhang, Qirong Shen, Kai Wu, and Zhihui Xu contributed to the study conception and design. Material preparation, experiment conduction, and data collection were performed by Xinli Sun, Riyan Xia, Jiyu Xie, and Kun Duan. Data analysis was performed by Xini Sun, Weibing Xun, and Guidong Huang. The first draft of the manuscript was written by Xinli Sun and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe raw sequencing data have been deposited in the NCBI Sequence Read Archive under the BioProject accession number PRJNA1209616. 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Proceedings of the National Academy of Sciences 122:e2418766122. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas\u003c/span\u003e\u003cspan address=\"10.1073/pnas\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbial-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"meco","sideBox":"Learn more about [Microbial Ecology](https://www.springer.com/journal/248)","snPcode":"248","submissionUrl":"https://submission.nature.com/new-submission/248/3","title":"Microbial Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"microbial interaction, SynCom, biocontrol, biofilm, cross-feeding","lastPublishedDoi":"10.21203/rs.3.rs-6904063/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6904063/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe rhizosphere microbiome plays a pivotal role in plant health by mediating interactions between hosts, beneficial microbes, and pathogens. However, the ecological mechanisms underlying microbial consortia that suppress soil-borne diseases remain largely unexplored. In this study, we investigated how the biocontrol bacterium \u003cem\u003eBacillus velezensis\u003c/em\u003e SQR9 influences the assembly of the cucumber rhizosphere bacterial community in the presence of the pathogenic fungus \u003cem\u003eFusarium oxysporum\u003c/em\u003e FOC. Inoculation with \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 significantly enriched the genus \u003cem\u003eLysobacter\u003c/em\u003e, a group of known biocontrol bacteria potentially contributing to disease suppression. A meta-analysis of publicly available datasets revealed a positive correlation between \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eLysobacter\u003c/em\u003e abundances in healthy plant rhizospheres\u0026mdash;a relationship absent in \u003cem\u003eFusarium\u003c/em\u003e wilt diseased soils\u0026mdash;suggesting a conserved ecological association linked to disease suppression. Further mechanistic assays demonstrated that \u003cem\u003eLysobacter enzymogenes\u003c/em\u003e XL8, an antifungal bacterium isolated from the cucumber rhizosphere, formed synergistic biofilms with \u003cem\u003eB. velezensis\u003c/em\u003e SQR9. Cross-feeding assays indicated that strain SQR9 facilitated the growth of \u003cem\u003eL. enzymogenes\u003c/em\u003e XL8 through metabolic interactions, highlighting a cooperative mechanism that may stabilize the rhizosphere bacterial consortium. Greenhouse trials confirmed that this dual-species consortium outperformed single-species inoculations in suppressing \u003cem\u003eFusarium\u003c/em\u003e wilt, as evidenced by reduced pathogen abundance and enhanced plant growth. Together, our findings underscore the importance of microbial metabolic cooperation and biofilm-mediated coexistence in shaping rhizosphere community assembly and function, providing ecological insights for the development of synthetic microbial consortia aimed at sustainable plant disease management.\u003c/p\u003e","manuscriptTitle":"Cooperative Interactions Between Bacillus and Lysobacter Enhance Consortium Stability and Fusarium Wilt Suppression in Cucumber","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-20 16:39:00","doi":"10.21203/rs.3.rs-6904063/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-21T18:19:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-21T09:18:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-20T09:01:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-10T06:13:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"157743492813434687622348948614135221351","date":"2025-07-06T06:45:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"306539489105637744547269853498217250964","date":"2025-07-05T12:36:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"322755047920962978485171207492089613825","date":"2025-06-21T03:48:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"221909351790835442207660261796371931655","date":"2025-06-20T14:12:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-18T12:38:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-17T04:03:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-17T04:02:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Ecology","date":"2025-06-16T09:29:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"meco","sideBox":"Learn more about [Microbial Ecology](https://www.springer.com/journal/248)","snPcode":"248","submissionUrl":"https://submission.nature.com/new-submission/248/3","title":"Microbial Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"87d72a61-6327-4e6f-92a4-c5e80460ac1f","owner":[],"postedDate":"June 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-01T16:00:02+00:00","versionOfRecord":{"articleIdentity":"rs-6904063","link":"https://doi.org/10.1007/s00248-025-02592-3","journal":{"identity":"microbial-ecology","isVorOnly":false,"title":"Microbial Ecology"},"publishedOn":"2025-08-29 15:56:56","publishedOnDateReadable":"August 29th, 2025"},"versionCreatedAt":"2025-06-20 16:39:00","video":"","vorDoi":"10.1007/s00248-025-02592-3","vorDoiUrl":"https://doi.org/10.1007/s00248-025-02592-3","workflowStages":[]},"version":"v1","identity":"rs-6904063","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6904063","identity":"rs-6904063","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}