Arbuscular mycorrhizal symbiosis suppresses tomato bacterial wilt by coordinating plant systemic resistance with microbiome antagonism

Arbuscular mycorrhizal symbiosis suppresses tomato bacterial wilt by coordinating plant systemic resistance with microbiome antagonism | 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 Arbuscular mycorrhizal symbiosis suppresses tomato bacterial wilt by coordinating plant systemic resistance with microbiome antagonism Dechao Zeng, Lingxiao Wang, Yijia Gong, Wenxuan Zhou, Wenbin Wang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8605735/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Tomato bacterial wilt, caused by Ralstonia solanacearum , is a globally devastating soil-borne disease that poses a serious threat to the sustainable development of tomato production. Arbuscular mycorrhizal fungi (AMF) are well-recognized beneficial soil microorganisms that significantly promote plant growth, enhance nutrient uptake, and bolster resistance to various biotic and abiotic stresses. However, evidence regarding the potential of AMF to suppress tomato bacterial wilt remains limited. In this study, we demonstrate that AMF inoculation remarkably reduces the disease index of bacterial wilt in tomato plants, upregulates the expression of pathogenesis-related (PR) genes, and enhances antioxidant enzyme activities, collectively strengthening systemic disease resistance. High-throughput 16S rRNA gene sequencing revealed that AMF colonization drives a substantial reassembly of the rhizosphere microbiome. Notably, AMF colonization promoted the recruitment of beneficial bacterial genera, including Bacillus and Brevibacillus , while significantly suppressing the abundance of Ralstonia bacterial genera. Furthermore, we isolated two Brevibacillus strains, named AQC211 and AQC296, from the mycorrhizosphere of healthy tomato plants, both of which exhibited antagonistic activity against R. solanacearum in vitro. Pot experiments confirmed that inoculation with the AQC211 strain significantly reduced the incidence and severity of bacterial wilt. These findings indicate that AMF can not only directly prime plant systemic resistance but also indirectly enhance protection against bacterial wilt by shaping a disease-suppressive rhizosphere microbiome. Arbuscular mycorrhizal fungi Ralstonia solanacearum tomato bacterial wilt Rhizosphere microbiome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Tomato ( Solanum lycopersicum ) is a globally important economic crop, yet its production is severely threatened by soil-borne diseases. Bacterial wilt, caused by Ralstonia solanacearum , is a devastating disease that can lead to systemic wilting and plant death, resulting in billions of dollars in economic losses annually (Sun et al. 2023; Yang et al. 2025). This pathogen secretes effector proteins (such as RipV2 and RipAC) through the Type III Secretion System (T3SS), which target core components of the plant immune system, thereby suppressing host defense responses (Sakata et al. 2023). Although traditional chemical control methods and the breeding of resistant cultivars have achieved somewhat success, the rapid evolution of R. solanacearum (e.g., phylotype II strain ES5-1) enables it to break plant resistance. Additionally, the overuse of chemical agents has further led to environmental pollution and issues of antimicrobial resistance (Del Rosario Villavicencio-Gutiérrez et al. 2024). Therefore, the development of green and environmentally friendly biological control strategies, especially those based on plant-microorganism interactions and systemic defense mechanisms, has become a central focus of current research (Jiang et al. 2025). Arbuscular mycorrhizal fungi (AMF), as the most widely distributed symbiotic microorganisms in soil, form mutualistic associations with over 80% of land plants and are vital components of the soil-plant micro-ecosystem. In addition to forming symbiotic structures with plant roots and extending the absorptive capacity of host roots, AMF can also activate plant systemic resistance via the jasmonic acid/ethylene signaling pathways and modulate the rhizosphere microbial network, thereby enhancing the adaptations and resilience of host plants to diverse environmental stresses, including drought, salinity, ultraviolet and heavy metal damages (Hartvig et al. 2024; Zhou et al. 2020; Deng et al. 2020; Ding et al. 2020). Additionally, growing evidence also points to the enhanced resistance of mycorrhizal plants to a wide range of soil-borne diseases caused by bacterial or fungal pathogens (Abarca et al. 2024). Earlier studies have shown that inoculation of Glomus sp. Zac-19 could reduce the incidence of onion white rot disease ( Sclerotium cepivorum Berk. ) from 92 to 28.7% at natural infestation and from 52.8 to 11.9% at artificial infestation (Lee et al. 2018). Recent studies has further revealed that AMF could induce changes in bacterial community composition, favoring the development of beneficial microbes for the host plant and pathogen antagonists (Lioussanne et al. 2010; Abarca et al. 2024). For instance, tomato plants colonized by F. mosseae and R. intraradices showed increased abundance of beneficial bacteria of the genera Pseudomonas , Herbaspirilium and Acidobacterium in the rhizosphere (Lioussanne et al. 2010). Inoculation of tomato with R. intraradices in field was shown to be associated with greater enrichment of Penicilium spp. and Thricoderma spp., two genera which include species with established antagonistic function against the pathogens R. solani and F. oxysporum (Patkowska et al. 2019; Jamiołkowska et al. 2019). These AMF-driven microbiome changes were closely related to alterations in root exudates, thereby creating a micro-environment that facilitates the suppression of pathogens (Umer et al. 2025). Recent studies have shown that beneficial microorganisms, such as Bacillus , enhance plant disease resistance through multiple mechanisms. On one hand, they activate the salicylic acid (SA)/jasmonic acid (JA) signaling pathways to induce systemic resistance in plants. On the other hand, they secrete lipopeptide metabolites, such as surfactin, to modulate the rhizosphere microbiome (Khatri et al. 2023; Song et al. 2021). For example, Bacillus subtilis inoculation could increase the diversity of the potato rhizosphere microbial community and suppress the colonization of pathogens by over 70% through antagonistic effects (Song et al. 2021). Application of a Bacillus velezensis strain SQR9 in organic fertilizers has been demonstrated to have positive effects in controlling and preventing banana Fusarium wilt, effectively curbing disease occurrence while also enhancing the diversity and richness of banana rhizosphere microorganisms (Lee et al. 2017; Sun et al. 2025). These findings highlight the great potential of microbiome engineering in disease control. While AMF have been repeatedly documented to exert a biological control effect on different crop diseases, the results obtained from different studies vary widely relying on the AMF strains used, the pathogenic agents, the plant species and the environmental conditions (Smith et al. 2009; Gianinazzi et al. 2008). This study aims to explore the potential of AMF in controlling tomato bacterial wilt and investigate its underlying mechanism. Through pot inoculation experiments, we demonstrate that AMF could indeed reduce the disease index of tomato bacterial wilt. Further physiological analyses combined with high-throughput sequencing revealed that AMF inoculation could not only enhance the expression of pathogenesis-related protein genes ( PRs ) and the activity of antioxidant enzymes in tomato roots but also enrich the abundance of beneficial bacteria such as Bacillus and Brevibacillus in mycorrhizosphere. Our findings suggest that AMF can alleviate bacterial wilt occurrence in host plants by operating two regulatory mechanisms, specifically the activation of plant systemic resistance and the reconstruction of rhizosphere bacterial community diversity. Materials and Methods Plant material, microbe strains and cultivation conditions Tomato ( Solanum lycopersicum L. cv. Dongshenghong ) seeds were surface-sterilized with 75% ethanol for 1 min and 10% sodium hypochlorite for 5 min, followed by six rinses with sterile water, and then germinated on sterile Petri dishes until cotyledons emerged fully. The seedlings were then transfer to sterilized quartz-sand and cultivated in a growth chamber for 10 days irrigated with a half-strength nutrient solution containing the following: 1 mM NH 4 + , 4 mM NO 3 − , 2 mM K + , 1 mM Pi, 0.75 mM Ca 2+ , 0.5 mM Mg 2+ , 0.25 mM Cl − , 0.5 mM SO 4 2− , 20 µM Fe 2+ , 9 µM Mn 2+ , 46 µM BO 3 3− , 8 µM Zn 2+ , 3 µM Cu 2+ , and 0.03 µM MoO 4 2− . The plantlets with the similar growth performance were then used for either sand-based or soil-based pot inoculation experiments. The three AMF strains Rhizophagus intraradices ( R. intraradices ), Funneliformis mosseae ( F. mosseae ) and Claroideoglomus etunicatum ( C. etunicatum ) used in this study were purchased from the Institute of Plant Nutrition and Resources of Beijing Academy of Agriculture and Forestry Sciences. The propagation of the AMF spores were conducted by co-cultivating the individual AMF strain with sorghum plants. The AMF spores were surface-sterilized by a mixed solution (containing 5% Chloramine-T, 0.04༅streptomycin and 1% Tween 20) for 15 min before used for inoculation. The Ralstonia solanacearum ( R. solanacearum ) strain QL-Rs1115 was provided by Prof. Zhong Wei from Nanjing Agricultural University. The preparation of the R. solanacearum inoculum was conducted according to the methods as described previously (Ling et al. 2020). For sand-based pot culture experiment, tomato plants were transplanted into 0.6-L pots filled with sterilized sand. A total of six treatments were designed, including CK (mock-inoculated control), Rs (inoculation with R. solanacearum alone), AMF (inoculation with AMF alone), Fm + Rs (dual inoculation with F. mosseae and R. solanacearum ), Ri + Rs (dual inoculation with R. intraradices and R. Solanacearum ), Ce + Rs (dual inoculation with C. etunicatum and R. Solanacearum ). Each plant was cultivated in an independent pot and each treatment included 9 biological replicates. For the AMF treatment, each plant was inoculated with approximately 300 spores around the roots. For the Rs treatment, each plant was irrigated with 50 ml R. solanacearum suspension with OD600 at 1.0. For the dual inoculation treatments, tomato plants were first inoculated with F. mosseae, R. intraradices , or C. etunicatum for four weeks, and then challenged with R. solanacearum for another two weeks. The CK control plants were obtained by inoculation with autoclaved AMF and R. solanacearum inocula. All the plants were supplied with full-strength nutrient solution (described above) except Pi, which was reduced to 20 mM to guarantee high mycorrhizal colonization. For soil-based pot inoculation experiment, tomato plants were transplanted into 0.9-L pots filled with unsterilized soil (the soil contains 22.94 g/kg organic matter, 0.37 g/kg total N, 3.56 mg/kg available P, 418 mg/kg available K, and pH 6.7). This experiment comprised four treatments: CK (mock-inoculated control), Rs (inoculation with R. solanacearum alone), AMF (inoculation with R. intraradices alone) and Ri + Rs (dual inoculation with R. intraradices and R. Solanacearum ). Each plant was cultivated in an independent pot and each treatment included eight biological replicates. The inoculation procedures for AMF and R. solanacearum were the same as those mentioned in the sand-based pot culture. All these pot culture experiments were carried out in the controlled growth chambers with the following conditions: 35/28°C day/night temperature, a 14/10-h day/night photoperiod, and 60–70% relative humidity. Detection of mycorrhizal fungal colonization For visualization of AM fungal structures, the root segments were immersed into 1.8 M KOH solution heated to 90°C for 1 h, and then treated with 1% HCl (v⁄v) solution for 5 min. The roots were then counterstained for 2 h at 90°C with 0.3% Trypan Blue (dissolved in lactic acid/glycerol/water, 1 : 1 : 1, v/v/v). The mycorrhizal colonization rate was quantified based on the grid line intersect method (Montiel et al. 2023) using a binocular microscope (Leica, Germany). Disease assessment Disease severity was scored according to the methods described previously by Kemboi et al. (2022) with a 0–4 scale, namely, 0: No symptoms, 1: 1–25% foliar wilting, 2: 26–50% wilting, 3: 51–75% wilting, and 4: 76–100% wilting. Disease index (DI) and biocontrol efficacy (BE) were quantified according to the methodology established by Chen et al. (2020). DI=[∑(n i* i)/n T *i max ] * 100%;BE=(DI ck -DI treat /DI ck ) * 100%; i = Disease severity grade (0 to 4), i max =Maximum disease severity grade, n i =Number of plants with disease grade, n T =Total number of plants assessed per treatment. DI ck =Disease index of the control group, DI treat =Disease index of the treatment group. RNA extraction and quantitative reverse transcription- polymerase chain reaction (qRT‑PCR) analysis Total RNA was isolated from 100 mg of tomato leaves and roots using the guanidine thiocyanate extraction method with Trizol reagent (Accurate Biology, China). For performing qRT-qPCR analysis, 2 mg of total RNA from each sample was used to synthesize cDNA using a reverse transcription kit (Accurate Biology, China). The synthesized cDNAs were then used as templates for the qRT-PCR analysis conducted on an Applied Biosystems PlusReal-Time PCR System using the SYBR Green Pro Taq kit (Accurate Biology, China). The specific primer pairs for each of the target genes are listed in Supplemental Table S1 . The relative transcript abundance of each target gene was standardized to the transcript level of a tomato constitutive Actin gene (Liu et al. 2020). Determination of antioxidant enzyme activity Superoxide dismutase (SOD) activity was measured using the nitrogen blue tetrazolium (NBT) photochemical reduction method (Duan et al. 2025). Peroxidase (POD) activity was assessed using the guaiacol method (Liang et al. 2021), and catalase (CAT) activity is determined through the ultraviolet absorption method (Yuan et al. 2017). 16S ribosomal RNA (rRNA) sequencing and data analysis Soil samples were collected from the rhizosphere of tomato plants mock-inoculated (CK) or inoculated with AMF ( R. intraradices ), R. solanacearum , or the both. Four biological replicates for each treatment were used for DNA extraction and 16S rRNA gene amplification (V3-V4 region). The sequencing of the amplicons was performed on an Illumina NovaSeq 6000. After trimming and eliminating low-quality reads, 267146, 262514, 264144 and 262112 clean reads were obtained for the four inoculated and control plants, respectively, which accounted for over 95% of the total sequences. DNA extraction, 16S rRNA gene sequencing and data analysis were commercially conducted by Genesky Biotechnologies Inc. (Shanghai, China). Statistical Analysis The data were analyzed by Microsoft Excel and GraphPad Prism 8.0 and the significant differences ( P < 0.05) were determined by Student’s t-test or one-way ANOVA with Duncan’s multiple range test. Results AMF colonization inhibits the occurrence of tomato wilt disease To assess the potential of AMF in suppressing bacterial wilt, we initially conducted a sand-based pot experiment, in which tomato plants were cultivated in sterile sands as the substrate and inoculated separately with three AMF strains: Rhizophagus intraradices (Ri), Funneliformis mosseae (Fm), and Claroideoglomus etunicatum (Ce). One set of plants inoculated with autoclaved inoculum was used as the control (CK). After four weeks of growth, part of the CK plants and the AMF-inoculated plants were further challenged with Ralstonia solanacearum (Rs). Thus this experiment contained six treatments: CK, Rs (inoculated only with R. solanacearum ), AM (inoculated only with the AMF, Fm), Ri + Rs (dual-inoculation with Ri and Rs), Fm + Rs (dual-inoculation with Fm and Rs), and Ce + Rs (dual-inoculation with Ce and Rs). Following an additional two-week incubation, almost all the plants inoculated exclusively with R. solanacearum (Rs) exhibited the typical bacterial wilt symptoms, with the disease index (DI) high to 82%, whereas less 50% of the plants under dual inoculation (+ Ri / Fm/Ce + Rs) showed the disease symptoms. Within the three AMF strains, R. intraradices provided the highest level of disease suppression, with a disease index as low as 0.33 (Fig. 1 ). Thus, in the following experiments, we only chose R. intraradices as the AM inoculum. To confirm the capacity of AMF in the biocontrol of bacterial wilt, we further carried out a soil-based pot experiment. Tomato plants inoculated or mock-inoculated with R. irregularis were grown in unsterilized soil for four weeks, and then challenged with R. solanacearum (Rs) for another two weeks. Thus this experiment contained four treatments: CK, Rs (inoculated only with R. solanacearum ), AM (inoculated only with the AMF, Ri), AM + Rs (dual-inoculation with Ri and Rs). As observed, R. irregularis inoculation observably improved plant growth, even in the presence of R. solanacearum . The shoot and root biomass of the AMF-inoculated plants (referred as AM plants) increased by 210% and 74%, respectively, compared to those of the mock-inoculated control plants (CK). The AM plants also showed an over 20% increase in chlorophyll content and increases by over 60% and 200% in shoot N and P content, respectively, relative to the control plants (Fig. 2 b-e). Consistent with that found in the sand-based pot culture, we also observed a distinct suppressive effect of R. irregularis on the occurrence of bacterial wilt in the soil-based pot culture. The plants inoculated exclusively with R. solanacearum (Rs) showed an over 70% disease index (DI), while this index was reduced to about 40% in the dual-inoculated plants (AM + Rs), suggesting that R. irregularis inoculation could achieve a control efficacy of tomato bacterial wilt disease by about 28% under our experiment conditions (Fig. 2 f-g). To determine whether the R. solanacearum infection may affect mycorrhizal colonization, we further assessed the mycorrhizal colonization levels for the AM plants and dual-inoculated (AM + Rs) plants. As observed, both the AM plants and the dual-inoculated plants with no distinct wilt symptoms showed high mycorrhizal colonization levels, with the total root length colonization level reached to over 60% (Fig. S1 ). No significant difference in colonization levels was observed between the AM plants and the healthy dual-inoculated plants, while those diseased plants showed a much lower mycorrhizal colonization levels compared to that of the AM plants (data not shown). The control plants and the plants challenged with only R. Solanacearum (Rs) showed no distinct AMF structure in their roots. We also checked the transcript level of an AM-marker gene, SlHA8 , encoding an AM-specific H + -ATPase, which showed a highly induced expression in the roots of both AM and AM + Rs plants, but barely expressed in the roots of CK and Rs-treated plants. These results suggest that the AMF inoculation should be the causative factor that alleviates tomato bacterial wilt. AMF colonization enhances the expression of PR genes and the activity of antioxidant enzymes in tomato To explore the potential mechanisms underlying the AMF-mediated suppression of bacterial wilt, we examined the expression levels of four pathogen-related (PR) genes in the plants inoculated with AMF and/or R. solanacearum by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Inoculation only with R. solanacearum had no remarkable influence on the expression of the PR genes examined, except for SlPR4 , whose transcript level was significantly upregulated by R. solanacearum inoculation, compared to that in the control plants, while inoculation with AMF could substantially induce the expression of all the four SlPR genes (Fig. 3 a-d). The transcript levels of SlPR1 (encoding an antibacterial protein), SlPR2 (encoding a β-1,3-glucanase) and SlPR3 (encoding a chitinase) in the AM plants increased by 6-fold, 17-fold and 20-fold, respectively, relative to those in the control plants. Notably, in the dual-inoculated plants (AM + Rs), the transcript levels of SlPR1, 2 and 3 decreased by 47%, 67%, and 91%, respectively, compared to those observed in the AM plants. This suggests that R. solanacearum infection may partially inhibit the resistance response induced by AM symbiosis. An approximately 2-fold upregulation in the expression of SlPR4 was observed in both the AM plants and Rs -challeged plants compared to that in the control plants, suggesting that SlPR4 has a non-specific response to the invasion of exogenous symbiotic or pathogenic microorganisms. To determine whether AMF colonization and/or Rs infection may affect the oxidative stress response in tomato, we quantitatively analyzed the activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) in these plants (Fig. 3 e-g). Inoculation with AMF, Rs , or the both significantly increased the activities of all the three antioxidant enzymes. Particularly, the activity of SOD showed a more pronounced enhancement, with increases by almost 100% in the Rs -treated plants and nearly 200% in the AMF-inoculated plants and dual-inoculated plants, compared to that in the control plants. These results indicate that AMF colonization or Rs infection could significantly enhance the activities of these antioxidant enzymes, thereby potentially improving the plants' tolerance to adverse conditions. AMF colonization induces a reassembly of the rhizosphere microbial community To gain more insight into the AMF-mediated suppression of tomato bacterial wilt, we further employed the 16S rRNA sequencing technology to analyze the microbiota in the rhizospheric soil of the plants grown under four different treatments (CK, Rs, AM, AM + Rs). A total of 1,055,916 high-quality reads were obtained from 16 samples (four samples per treatment). The analysis led to the identification of 29 phyla, 57 classes, 72 orders, 161 families, 395 genera, and 462 species of microorganisms within these samples. Alpha diversity analysis indicated that the Chao1 index (species richness) and Shannon index (species diversity) in the three inoculation treatments (Rs, AM, AM + Rs) increased by 17.1%-19.6% and 7.9%-10.9%, respectively, compared to the control (CK); however, these differences were not statistically significant ( P > 0.05; Fig. S2). Moreover, no significant differences in either diversity or richness were observed among the three inoculation treatments ( P > 0.05). Permutational multivariate analysis of variance (PERMANOVA) (R² = 0.45, P = 0.0001), combined with unconstrained principal coordinates analysis (PCoA), revealed a significant separation in bacterial communities among the four treatments (Fig. 4 a). Specifically, the AM treatment showed the greatest divergence from the control group along PCoA axis 1, which explained 27.97% of the variation. In contrast, the presence or absence of R. solanacearum inoculation was the primary factor driving separation along PCoA axis 2, accounting for 17.39% of the variation. These results indicate that AMF colonization and/or R. solanacearum inoculation may induce a reassembly of the tomato rhizosphere microbial community. In the term of bacterial community composition, Proteobacteria , Acidobacteria , Actinobacteria , Chloroflexi , and Bacteroidetes were shown to be the dominant phyla across all the four treatments (Fig. 4 b). Inoculation with AMF significantly increased the relative abundances of Firmicutes and Planctomycetes (Fig. S3). At the genus level, Gemmatimonas , Streptomyces , and Bacillus exhibited relatively high abundances in all treatments (Fig. 4 c). Notably, the relative abundances of Bacillus , Brevibacillus , Nitrososphaera , and Nocardiopsis were significantly enhanced in the AMF-treated samples compared to those in the control samples, whereas those of Lysobacter , Streptomyces , and Dactylosporangium were significantly reduced following AMF inoculation (Fig. 4 d-e, Fig. S4). Additionally, AMF inoculation significantly decreased the relative abundance of Ralstonia bacteria (Fig. 4 f). Isolation of two beneficial Brevibacillus strains from tomato rhizosphere To further elucidate whether AMF-mediated systemic disease resistance involves the recruitment of beneficial rhizosphere bacteria, we screened the culturable bacteria from rhizospheric soils of the dual-inoculated (AM + Rs) plants that did not develop wilt disease. We successfully isolated two strains of Brevibacillus sp., named AQC211 and AQC296, both of which showed a distinct inhibitory capacity against R. solanacearum in our plate antagonism assays (Fig. 5 a). To further evaluate their biocontrol potential, a pot inoculation experiment was conducted. The results showed that inoculation with Brevibacillus sp. AQC211 could significantly reduce the severity of bacterial wilt and lower the disease index from 0.92 (in the control) to 0.70, corresponding to a disease control efficacy of 24%. The another strain, AQC296, showed a moderate level of biocontrol activity, reducing the disease index to 0.81, with a biocontrol efficacy of 12%. These findings collectively suggest that AMF-induced restructuring of the rhizosphere bacterial community may involve specific beneficial strains with biocontrol functions, such as Brevibacillus sp. AQC211, which may play a synergistic role with AMF in enhancing tomato resistance to bacterial wilt. Discussion Bacterial wilt, a prevalent soil-borne disease of solanaceous crops, have emerged as a major constraint to global solanaceous crop production, particularly in regions with intensive agricultural practices (Kashyap et al. 2021). Due to the high pathogenicity, strong environmental adaptability and broad host range of its causative pathogen, R. solanacearum , the conventional control strategies, such as chemical fumigation and crop rotation, are often largely ineffective (Wang et al. 2020). Recent studies in multiple plant species have shown a distinct advantage of using biological control strategies to suppress different soil-borne diseases, including the fungal and bacterial wilt (Fernández-González et al. 2020). While AMF, the beneficial symbionts of most land plants, have been repeatedly documented to be able to enhance host plant’s resistance and resilience against various biotic and abiotic stresses, the benefits of using AMF as a major biocontrol agent to suppress bacterial wilt are still lack of adequate and compelling proofs. In this study, we demonstrate that AM colonization could effectively alleviate tomato bacterial wilt, as indicated by the significantly lower disease index in those plants inoculated with the three AMF strains, R. intraradices , F. mosseae , or C. etunicatum , compared to that in the control plants under either sterile sand or unsterilised soil culture conditions (Fig. 1 a, Fig. 2 a). Previous studies have suggested that AMF may contribute to resistance to root and foliar pathogens via inducing plant systemic resistance, a phenomenon termed mycorrhiza-induced resistance (MIR). In this study, we indeed observed significantly up-regulated expression of four PR genes ( SlPR1 to 4 ) and enhanced activity of the three antioxidant enzymes (CAT, POD and SOD) in the leaves of AM plants, relative to that in control plants (Fig. 3 ), confirming that AMF colonization can induce the systemic resistance in plants. Notably, it seemed that R. solanacearum infection might be able to weaken the plant’s resistance induced by AMF, as infered from that the dual-inoculated (AM + Rs) plants showed significantly reduced expression levels of the three PR genes, SlPR1 , SlPR2 and SlPR3 , compared to the AM plants (Fig. 3 a-c). Consistent with that observed in the AM plants, the activity of CAT, POD and SOD in the Rs -challenged plants or dual-inoculated plants was also substantially enhanced compared to that in the control plants, suggesting that the mycorrhiza-induced resistance shares partially similar features with the systemic acquired resistance induced by pathogen infection. It is worth emphasizing that AMF colonization could not only enhance the systemic resistance, but also promote plant growth, chlorophyll accumulation and the uptake of Pi and N in tomato plants (Fig. 2 b-e). Previous studies reported that tomato plants colonized by AM fungi could substantially decrease root infection and disease severity caused by different pathogens, resulting in increased plant fresh weight (up to 198%) and fruit yield (14.3%) as compared to those non-mycorrhizal pathogen-infected plants (Berta et al. 2005; Utkhede 2006; Gianinazzi et al. 2008). The findings suggest that the improved growth and nutritional status of AM plants may impart them an advantage in compensating the damage caused the pathogenic infection. Increasing studies point to that the diversity of soil bacteria and their community structure are crucial for maintaining soil health and controlling soil-borne diseases (Jayaraman et al. 2021; Zhang et al. 2023). In this study, we found that the Rs- challenged tomato plants grown in unsterilized soil exhibited a lower disease index than those grown in sterile sand, even in the absence of AMF inoculation (Fig. 1 b, Fig. 2 f), suggesting that there might exist certain beneficial microorganisms that could antagonize R. solanacearum in the rhizospheric soil of tomato plants. Earlier studies reported that inoculation of mycorrhizal fungi could increase both the richness and diversity of rhizosphere microorganisms associated with host plants, thereby improving plant stress resistance (Toju et al. 2018; González Guzmán et al. 2022). While our 16S RNA gene sequencing showed no significant differences in Chao1 index (richness ) and Shannon index (diversity) between the control plants and those plants inoculated with AMF, R. solanacearum , or the both, we did observe a remarkable difference in the composition of the bacterial microbiota among the control group and the three inoculated treatments (Rs, AM, AM + Rs), as shown by the distinct separation in the unconstrained principal coordinate analysis (Fig. 4 a). These results strongly suggest that AMF colonization can reshape the rhizobacterial community of host plants. Our findings could gain well support from a recent study demonstrating that AMF colonization could recruit rhizobia to accumulate in the rhizosphere of M. truncatula (Cangioli et al. 2022). Increasing evidence points to that AMF may cooperate with certain beneficial rhizospheric microorganisms to synergistically enhance nutrient uptake and/or repel harmful microorganisms (Boyno et al. 2025). Bacillus and Brevibacillus species are well-documented beneficial microorganisms that can suppress the population growth and infection process of soil-borne pathogens through multiple approaches, such as resource competition, niche exclusion, secretion of antibiotic compounds and induction of plant systemic resistance (Ghani et al. 2019; Koza et al. 2022; Pan et al. 2025; Wang et al. 2021). In this study, we found that AMF inoculation led to a significant increase in the abundance of Bacillus and Brevibacillus , coupled with a remarkable decrease in Ralstonia bacteria, within the tomato rhizosphere. The successful isolation of two strains of Brevibacillus , both exhibiting a distinct antagonistic activity against R. solanacearum , strongly suggests that AMF can recruit beneficial microorganisms, such as Bacillus spp., to synergistically defend plants against infection by soil-borne pathogens like R. solanacearum . In summary, our present study demonstrates that AMF colonization can effectively alleviate tomato bacterial wilt by activating a series of physiological mechanisms, including improving plant growth and nutritional status, inducing plant systemic resistance, reshaping the rhizobacterial community and recruiting beneficial genera like Brevibacillus (Fig. 6 ). These findings highlight AMF as a sustainable, eco-friendly strategy for controlling bacterial wilt, offering significant potential to reduce reliance on chemical inputs and enhance both crop resilience and soil health in agricultural systems. Declarations Acknowledgements This work was supported by the National Natural Science Foundation of China (32472831), Zhongshan Biological Breeding Laboratory (ZSBBL-KY2023-03), Jiangsu Provincial Key Laboratory of Coastal Saline Soil Resources Utilization and Ecological Conservation and Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization. We thank Prof. Zhong Wei from Nanjing Agricultural University for the providing of R. solanacearum strain QL-Rs1115. Author contributions D.Z. and A.C. designed research and analyzed the data; D.Z., L.W., Y.G., W.Z., W.W. conducted the experiments; A.C., S.W. and G.X. supervised the project; A.C. and D.Z. wrote the manuscript. 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15:01:05","extension":"html","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":94823,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8605735/v1/feb448fc3e5da6bf0c3e6c1b.html"},{"id":101204531,"identity":"822f807a-7995-4cb9-9ba0-71e46a5504e5","added_by":"auto","created_at":"2026-01-27 09:43:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":347063,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAMF colonization alleviates the incidence of tomato bacterial wilt under sand-based pot culture. \u003c/strong\u003eTomato plants were cultivated in sterile sands as the substrate and inoculated separately with three AMF strains: \u003cem\u003eR. intraradices\u003c/em\u003e (Ri), \u003cem\u003eF. mosseae \u003c/em\u003e(Fm), and \u003cem\u003eC. etunicatum\u003c/em\u003e (Ce). The mock-inoculated control (CK) plants were obtained by inoculation with autoclaved inoculum. After four weeks of growth, part of the CK plants and the AMF-inoculated plants were further challenged with \u003cem\u003eR. solanacearum \u003c/em\u003e(Rs) for two weeks, and then the growth performance (a), disease index (b) and biocontrol efficacy (c) of these plants with different treatments were assessed. Note: CK (mock-inoculated control), Rs (inoculation with \u003cem\u003eR. solanacearum \u003c/em\u003ealone), AM (inoculation with the AMF, \u003cem\u003eF. mosseae,\u003c/em\u003ealone), Fm+Rs (dual inoculation with \u003cem\u003eF. mosseae \u003c/em\u003eand\u003cem\u003e R. solanacearum\u003c/em\u003e), Ri+Rs (dual inoculation with \u003cem\u003eR. intraradices \u003c/em\u003eand\u003cem\u003e R. Solanacearum\u003c/em\u003e),\u003cem\u003e \u003c/em\u003eCe+Rs (dual inoculation with \u003cem\u003eC. etunicatum \u003c/em\u003eand\u003cem\u003e R. Solanacearum\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8605735/v1/5593b3b2ad9a1dfc39208801.png"},{"id":100993641,"identity":"61ed3ec5-1f8a-42cd-895c-13fa793072a8","added_by":"auto","created_at":"2026-01-23 15:01:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":226971,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAMF colonization alleviates the incidence of tomato bacterial wilt under soil-based pot culture.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTomato plants were cultivated in unsterilized soil and inoculated or mock-inoculated with the AMF strain \u003cem\u003eR. intraradices\u003c/em\u003e (Ri). The control (CK) plants were inoculated with autoclaved inoculum. After four weeks of growth, part of the CK plants and the AMF-inoculated plants were further challenged with \u003cem\u003eR. solanacearum \u003c/em\u003e(Rs). Following an additional two-week incubation, the growth performance (a), fresh weight (b), chlorophyll content (c), shoot N content (d) and P content (e), disease index (f) and biocontrol efficacy (g) of these plants with different treatments were assessed. CK (mock-inoculated control), Rs (inoculation with \u003cem\u003eR. solanacearum\u003c/em\u003e), AM (inoculation with the AMF, \u003cem\u003eR. intraradices\u003c/em\u003e), AM+Rs (dual inoculation with \u003cem\u003eR. intraradices \u003c/em\u003eand\u003cem\u003eR. solanacearum\u003c/em\u003e). Different letters indicate significant differences (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8605735/v1/a4f1bd0e0480990039e3304c.png"},{"id":100993662,"identity":"03e794ea-1820-45f7-a575-8474141c0b36","added_by":"auto","created_at":"2026-01-23 15:01:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":114334,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAMF inoculation upregulates pathogen-related genes and enhances the activity of antioxidant enzymes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReal-time qRT-PCR analysis of the relative transcript levels of four pathogen-related (PR) genes (a-d) and determination of the activity of three antioxidant enzymes (e-g) in leaves of the tomato plants inoculated with AMF and/or \u003cem\u003eR. solanacearum. \u003c/em\u003eCK (mock-inoculated control), Rs (inoculation with \u003cem\u003eR. solanacearum\u003c/em\u003e), AM (inoculation with the AMF, \u003cem\u003eR. intraradices\u003c/em\u003e), AM+Rs (dual inoculation with \u003cem\u003eR. intraradices \u003c/em\u003eand\u003cem\u003e R. solanacearum\u003c/em\u003e). Different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8605735/v1/a74ff83783f87e16c82c2e25.png"},{"id":100993648,"identity":"a7cb9812-7008-44cc-af1f-c8f5d14af7e2","added_by":"auto","created_at":"2026-01-23 15:01:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":140046,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInoculation of AMF and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eR. solanacearum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e affects the rhizosphere bacterial communities of tomato plants.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Unconstrained principal coordinates analysis (PCoA) with Bray-Curtis distance showing a significant separation in rhizosphere\u003cstrong\u003e \u003c/strong\u003ebacterial communities among the four treatments. (b, c) Bar charts showing the relative abundances of bacterial taxa at the (b) phylum and (c) genus levels. Only the top 10 taxa with a relative abundance greater than 1% are shown (the remaining taxa are grouped as “Others”). Data represent mean values based on 16S rRNA gene sequencing results clustered at a 97% similarity threshold. (d-f) A comparison of the relative abundances of several genus-level bacterial species across different treatments. CK (mock-inoculated control), Rs (inoculation with \u003cem\u003eR. solanacearum\u003c/em\u003e), AM (inoculation with the AMF, \u003cem\u003eR. intraradices\u003c/em\u003e), AM+Rs (dual inoculation with \u003cem\u003eR. intraradices \u003c/em\u003eand\u003cem\u003eR. solanacearum\u003c/em\u003e). Different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8605735/v1/b5561f9d445d5f6d11a8056e.png"},{"id":100993643,"identity":"1b5ee889-074b-4dc6-b217-4977c3fee0ff","added_by":"auto","created_at":"2026-01-23 15:01:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":94387,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTwo \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBrevibacillus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e strains showing biocontrol activity against \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eR. solanacearum.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(a)\u003cstrong\u003e \u003c/strong\u003eInhibitory effects of \u003cem\u003eBrevibacillus\u003c/em\u003e strains AQC211 and AQC296 against \u003cem\u003eR. solanacearum\u003c/em\u003e in plate assays. (b–c) Disease index and biocontrol efficacy of AQC211 and AQC296 against bacterial wilt under pot culture conditions.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8605735/v1/7e0085026fe8fc442e6a3d1d.png"},{"id":100993655,"identity":"d595b1d0-6aa4-4ff2-9b33-14721e372984","added_by":"auto","created_at":"2026-01-23 15:01:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":297239,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA schematic model illustrating the effects of AMF colonization on the suppression of tomato bacterial wilt.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe working model describes three strategies by which AMF colonization enhances tomato resistance to R. solanacearum: (1) improving plant growth and nutritional status; (2) inducing systemic resistance responses by upregulating PR genes and enhancing the activity of antioxidant enzymes (CAT, POD and SOD); (3) recruitment of beneficial microbes like \u003cem\u003eBrevibacillus \u003c/em\u003ein mycorrhizosphere.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8605735/v1/7581bbe25d653cb989680e79.png"},{"id":101207888,"identity":"4d9304b4-37a1-4a9b-a530-54c48b46b0a4","added_by":"auto","created_at":"2026-01-27 10:07:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2165386,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8605735/v1/1790f7b1-2f1d-4256-b13f-5bd040dbc66c.pdf"},{"id":100993646,"identity":"43bee1b7-472a-44e9-b4cf-0213d6ca3d46","added_by":"auto","created_at":"2026-01-23 15:01:04","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":992030,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformations.docx","url":"https://assets-eu.researchsquare.com/files/rs-8605735/v1/dba7ec5279b09f95773133e5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Arbuscular mycorrhizal symbiosis suppresses tomato bacterial wilt by coordinating plant systemic resistance with microbiome antagonism","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) is a globally important economic crop, yet its production is severely threatened by soil-borne diseases. Bacterial wilt, caused by \u003cem\u003eRalstonia solanacearum\u003c/em\u003e, is a devastating disease that can lead to systemic wilting and plant death, resulting in billions of dollars in economic losses annually (Sun et al. 2023; Yang et al. 2025). This pathogen secretes effector proteins (such as RipV2 and RipAC) through the Type III Secretion System (T3SS), which target core components of the plant immune system, thereby suppressing host defense responses (Sakata et al. 2023). Although traditional chemical control methods and the breeding of resistant cultivars have achieved somewhat success, the rapid evolution of \u003cem\u003eR. solanacearum\u003c/em\u003e (e.g., phylotype II strain ES5-1) enables it to break plant resistance. Additionally, the overuse of chemical agents has further led to environmental pollution and issues of antimicrobial resistance (Del Rosario Villavicencio-Guti\u0026eacute;rrez et al. 2024). Therefore, the development of green and environmentally friendly biological control strategies, especially those based on plant-microorganism interactions and systemic defense mechanisms, has become a central focus of current research (Jiang et al. 2025).\u003c/p\u003e \u003cp\u003eArbuscular mycorrhizal fungi (AMF), as the most widely distributed symbiotic microorganisms in soil, form mutualistic associations with over 80% of land plants and are vital components of the soil-plant micro-ecosystem. In addition to forming symbiotic structures with plant roots and extending the absorptive capacity of host roots, AMF can also activate plant systemic resistance via the jasmonic acid/ethylene signaling pathways and modulate the rhizosphere microbial network, thereby enhancing the adaptations and resilience of host plants to diverse environmental stresses, including drought, salinity, ultraviolet and heavy metal damages (Hartvig et al. 2024; Zhou et al. 2020; Deng et al. 2020; Ding et al. 2020). Additionally, growing evidence also points to the enhanced resistance of mycorrhizal plants to a wide range of soil-borne diseases caused by bacterial or fungal pathogens (Abarca et al. 2024). Earlier studies have shown that inoculation of \u003cem\u003eGlomus sp. Zac-19\u003c/em\u003e could reduce the incidence of onion white rot disease (\u003cem\u003eSclerotium cepivorum Berk.\u003c/em\u003e) from 92 to 28.7% at natural infestation and from 52.8 to 11.9% at artificial infestation (Lee et al. 2018). Recent studies has further revealed that AMF could induce changes in bacterial community composition, favoring the development of beneficial microbes for the host plant and pathogen antagonists (Lioussanne et al. 2010; Abarca et al. 2024). For instance, tomato plants colonized by \u003cem\u003eF. mosseae\u003c/em\u003e and \u003cem\u003eR. intraradices\u003c/em\u003e showed increased abundance of beneficial bacteria of the genera \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eHerbaspirilium\u003c/em\u003e and \u003cem\u003eAcidobacterium\u003c/em\u003e in the rhizosphere (Lioussanne et al. 2010). Inoculation of tomato with \u003cem\u003eR. intraradices\u003c/em\u003e in field was shown to be associated with greater enrichment of \u003cem\u003ePenicilium\u003c/em\u003e spp. and \u003cem\u003eThricoderma\u003c/em\u003e spp., two genera which include species with established antagonistic function against the pathogens \u003cem\u003eR. solani\u003c/em\u003e and \u003cem\u003eF. oxysporum\u003c/em\u003e (Patkowska et al. 2019; Jamiołkowska et al. 2019). These AMF-driven microbiome changes were closely related to alterations in root exudates, thereby creating a micro-environment that facilitates the suppression of pathogens (Umer et al. 2025).\u003c/p\u003e \u003cp\u003eRecent studies have shown that beneficial microorganisms, such as \u003cem\u003eBacillus\u003c/em\u003e, enhance plant disease resistance through multiple mechanisms. On one hand, they activate the salicylic acid (SA)/jasmonic acid (JA) signaling pathways to induce systemic resistance in plants. On the other hand, they secrete lipopeptide metabolites, such as surfactin, to modulate the rhizosphere microbiome (Khatri et al. 2023; Song et al. 2021). For example, \u003cem\u003eBacillus subtilis\u003c/em\u003e inoculation could increase the diversity of the potato rhizosphere microbial community and suppress the colonization of pathogens by over 70% through antagonistic effects (Song et al. 2021). Application of a \u003cem\u003eBacillus velezensis\u003c/em\u003e strain SQR9 in organic fertilizers has been demonstrated to have positive effects in controlling and preventing banana \u003cem\u003eFusarium\u003c/em\u003e wilt, effectively curbing disease occurrence while also enhancing the diversity and richness of banana rhizosphere microorganisms (Lee et al. 2017; Sun et al. 2025). These findings highlight the great potential of microbiome engineering in disease control. While AMF have been repeatedly documented to exert a biological control effect on different crop diseases, the results obtained from different studies vary widely relying on the AMF strains used, the pathogenic agents, the plant species and the environmental conditions (Smith et al. 2009; Gianinazzi et al. 2008).\u003c/p\u003e \u003cp\u003eThis study aims to explore the potential of AMF in controlling tomato bacterial wilt and investigate its underlying mechanism. Through pot inoculation experiments, we demonstrate that AMF could indeed reduce the disease index of tomato bacterial wilt. Further physiological analyses combined with high-throughput sequencing revealed that AMF inoculation could not only enhance the expression of pathogenesis-related protein genes (\u003cem\u003ePRs\u003c/em\u003e) and the activity of antioxidant enzymes in tomato roots but also enrich the abundance of beneficial bacteria such as \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eBrevibacillus\u003c/em\u003e in mycorrhizosphere. Our findings suggest that AMF can alleviate bacterial wilt occurrence in host plants by operating two regulatory mechanisms, specifically the activation of plant systemic resistance and the reconstruction of rhizosphere bacterial community diversity.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material, microbe strains and cultivation conditions\u003c/h2\u003e \u003cp\u003eTomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e L. cv. \u003cem\u003eDongshenghong\u003c/em\u003e) seeds were surface-sterilized with 75% ethanol for 1 min and 10% sodium hypochlorite for 5 min, followed by six rinses with sterile water, and then germinated on sterile Petri dishes until cotyledons emerged fully. The seedlings were then transfer to sterilized quartz-sand and cultivated in a growth chamber for 10 days irrigated with a half-strength nutrient solution containing the following: 1 mM NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, 4 mM NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, 2 mM K\u003csup\u003e+\u003c/sup\u003e, 1 mM Pi, 0.75 mM Ca\u003csup\u003e2+\u003c/sup\u003e, 0.5 mM Mg\u003csup\u003e2+\u003c/sup\u003e, 0.25 mM Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e, 0.5 mM SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e, 20 \u0026micro;M Fe\u003csup\u003e2+\u003c/sup\u003e, 9 \u0026micro;M Mn\u003csup\u003e2+\u003c/sup\u003e, 46 \u0026micro;M BO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e, 8 \u0026micro;M Zn\u003csup\u003e2+\u003c/sup\u003e, 3 \u0026micro;M Cu\u003csup\u003e2+\u003c/sup\u003e, and 0.03 \u0026micro;M MoO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e. The plantlets with the similar growth performance were then used for either sand-based or soil-based pot inoculation experiments.\u003c/p\u003e \u003cp\u003eThe three AMF strains \u003cem\u003eRhizophagus intraradices\u003c/em\u003e (\u003cem\u003eR. intraradices\u003c/em\u003e), \u003cem\u003eFunneliformis mosseae\u003c/em\u003e (\u003cem\u003eF. mosseae\u003c/em\u003e) and \u003cem\u003eClaroideoglomus etunicatum\u003c/em\u003e (\u003cem\u003eC. etunicatum\u003c/em\u003e) used in this study were purchased from the Institute of Plant Nutrition and Resources of Beijing Academy of Agriculture and Forestry Sciences. The propagation of the AMF spores were conducted by co-cultivating the individual AMF strain with sorghum plants. The AMF spores were surface-sterilized by a mixed solution (containing 5% Chloramine-T, 0.04༅streptomycin and 1% Tween 20) for 15 min before used for inoculation. The \u003cem\u003eRalstonia solanacearum\u003c/em\u003e (\u003cem\u003eR. solanacearum\u003c/em\u003e) strain QL-Rs1115 was provided by Prof. Zhong Wei from Nanjing Agricultural University. The preparation of the \u003cem\u003eR. solanacearum\u003c/em\u003e inoculum was conducted according to the methods as described previously (Ling et al. 2020).\u003c/p\u003e \u003cp\u003eFor sand-based pot culture experiment, tomato plants were transplanted into 0.6-L pots filled with sterilized sand. A total of six treatments were designed, including CK (mock-inoculated control), Rs (inoculation with \u003cem\u003eR. solanacearum\u003c/em\u003e alone), AMF (inoculation with AMF alone), Fm\u0026thinsp;+\u0026thinsp;Rs (dual inoculation with \u003cem\u003eF. mosseae\u003c/em\u003e and \u003cem\u003eR. solanacearum\u003c/em\u003e), Ri\u0026thinsp;+\u0026thinsp;Rs (dual inoculation with \u003cem\u003eR. intraradices\u003c/em\u003e and \u003cem\u003eR. Solanacearum\u003c/em\u003e), Ce\u0026thinsp;+\u0026thinsp;Rs (dual inoculation with \u003cem\u003eC. etunicatum\u003c/em\u003e and \u003cem\u003eR. Solanacearum\u003c/em\u003e). Each plant was cultivated in an independent pot and each treatment included 9 biological replicates. For the AMF treatment, each plant was inoculated with approximately 300 spores around the roots. For the Rs treatment, each plant was irrigated with 50 ml \u003cem\u003eR. solanacearum\u003c/em\u003e suspension with OD600 at 1.0. For the dual inoculation treatments, tomato plants were first inoculated with \u003cem\u003eF. mosseae, R. intraradices\u003c/em\u003e, or \u003cem\u003eC. etunicatum\u003c/em\u003e for four weeks, and then challenged with \u003cem\u003eR. solanacearum\u003c/em\u003e for another two weeks. The CK control plants were obtained by inoculation with autoclaved AMF and \u003cem\u003eR. solanacearum\u003c/em\u003e inocula. All the plants were supplied with full-strength nutrient solution (described above) except Pi, which was reduced to 20 mM to guarantee high mycorrhizal colonization.\u003c/p\u003e \u003cp\u003eFor soil-based pot inoculation experiment, tomato plants were transplanted into 0.9-L pots filled with unsterilized soil (the soil contains 22.94 g/kg organic matter, 0.37 g/kg total N, 3.56 mg/kg available P, 418 mg/kg available K, and pH 6.7). This experiment comprised four treatments: CK (mock-inoculated control), Rs (inoculation with \u003cem\u003eR. solanacearum\u003c/em\u003e alone), AMF (inoculation with \u003cem\u003eR. intraradices\u003c/em\u003e alone) and Ri\u0026thinsp;+\u0026thinsp;Rs (dual inoculation with \u003cem\u003eR. intraradices\u003c/em\u003e and \u003cem\u003eR. Solanacearum\u003c/em\u003e). Each plant was cultivated in an independent pot and each treatment included eight biological replicates. The inoculation procedures for AMF and \u003cem\u003eR. solanacearum\u003c/em\u003e were the same as those mentioned in the sand-based pot culture.\u003c/p\u003e \u003cp\u003eAll these pot culture experiments were carried out in the controlled growth chambers with the following conditions: 35/28\u0026deg;C day/night temperature, a 14/10-h day/night photoperiod, and 60\u0026ndash;70% relative humidity.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetection of mycorrhizal fungal colonization\u003c/h3\u003e\n\u003cp\u003eFor visualization of AM fungal structures, the root segments were immersed into 1.8 M KOH solution heated to 90\u0026deg;C for 1 h, and then treated with 1% HCl (v\u0026frasl;v) solution for 5 min. The roots were then counterstained for 2 h at 90\u0026deg;C with 0.3% Trypan Blue (dissolved in lactic acid/glycerol/water, 1 : 1 : 1, v/v/v). The mycorrhizal colonization rate was quantified based on the grid line intersect method (Montiel et al. 2023) using a binocular microscope (Leica, Germany).\u003c/p\u003e\n\u003ch3\u003eDisease assessment\u003c/h3\u003e\n\u003cp\u003eDisease severity was scored according to the methods described previously by Kemboi et al. (2022) with a 0\u0026ndash;4 scale, namely, 0: No symptoms, 1: 1\u0026ndash;25% foliar wilting, 2: 26\u0026ndash;50% wilting, 3: 51\u0026ndash;75% wilting, and 4: 76\u0026ndash;100% wilting. Disease index (DI) and biocontrol efficacy (BE) were quantified according to the methodology established by Chen et al. (2020).\u003c/p\u003e \u003cp\u003eDI=[\u0026sum;(n\u003csub\u003ei*\u003c/sub\u003ei)/n\u003csub\u003eT\u003c/sub\u003e*i\u003csub\u003emax\u003c/sub\u003e]\u003csub\u003e*\u003c/sub\u003e100%;BE=(DI\u003csub\u003eck\u003c/sub\u003e -DI\u003csub\u003etreat\u003c/sub\u003e/DI\u003csub\u003eck\u003c/sub\u003e)\u003csub\u003e*\u003c/sub\u003e100%;\u003c/p\u003e \u003cp\u003ei\u0026thinsp;=\u0026thinsp;Disease severity grade (0 to 4), i\u003csub\u003emax\u003c/sub\u003e=Maximum disease severity grade, n\u003csub\u003ei\u003c/sub\u003e=Number of plants with disease grade, n\u003csub\u003eT\u003c/sub\u003e=Total number of plants assessed per treatment. DI\u003csub\u003eck\u003c/sub\u003e=Disease index of the control group, DI\u003csub\u003etreat\u003c/sub\u003e=Disease index of the treatment group.\u003c/p\u003e\n\u003ch3\u003eRNA extraction and quantitative reverse transcription- polymerase chain reaction (qRT‑PCR) analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated from 100 mg of tomato leaves and roots using the guanidine thiocyanate extraction method with Trizol reagent (Accurate Biology, China). For performing qRT-qPCR analysis, 2 mg of total RNA from each sample was used to synthesize cDNA using a reverse transcription kit (Accurate Biology, China). The synthesized cDNAs were then used as templates for the qRT-PCR analysis conducted on an Applied Biosystems PlusReal-Time PCR System using the SYBR Green Pro Taq kit (Accurate Biology, China). The specific primer pairs for each of the target genes are listed in Supplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The relative transcript abundance of each target gene was standardized to the transcript level of a tomato constitutive \u003cem\u003eActin\u003c/em\u003e gene (Liu et al. 2020).\u003c/p\u003e\n\u003ch3\u003eDetermination of antioxidant enzyme activity\u003c/h3\u003e\n\u003cp\u003eSuperoxide dismutase (SOD) activity was measured using the nitrogen blue tetrazolium (NBT) photochemical reduction method (Duan et al. 2025). Peroxidase (POD) activity was assessed using the guaiacol method (Liang et al. 2021), and catalase (CAT) activity is determined through the ultraviolet absorption method (Yuan et al. 2017).\u003c/p\u003e \u003cp\u003e \u003cb\u003e16S ribosomal RNA (rRNA) sequencing and data analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSoil samples were collected from the rhizosphere of tomato plants mock-inoculated (CK) or inoculated with AMF (\u003cem\u003eR. intraradices\u003c/em\u003e), \u003cem\u003eR. solanacearum\u003c/em\u003e, or the both. Four biological replicates for each treatment were used for DNA extraction and 16S rRNA gene amplification (V3-V4 region). The sequencing of the amplicons was performed on an Illumina NovaSeq 6000. After trimming and eliminating low-quality reads, 267146, 262514, 264144 and 262112 clean reads were obtained for the four inoculated and control plants, respectively, which accounted for over 95% of the total sequences. DNA extraction, 16S rRNA gene sequencing and data analysis were commercially conducted by Genesky Biotechnologies Inc. (Shanghai, China).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe data were analyzed by Microsoft Excel and GraphPad Prism 8.0 and the significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were determined by Student\u0026rsquo;s t-test or one-way ANOVA with Duncan\u0026rsquo;s multiple range test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAMF colonization inhibits the occurrence of tomato wilt disease\u003c/h2\u003e \u003cp\u003eTo assess the potential of AMF in suppressing bacterial wilt, we initially conducted a sand-based pot experiment, in which tomato plants were cultivated in sterile sands as the substrate and inoculated separately with three AMF strains: \u003cem\u003eRhizophagus intraradices\u003c/em\u003e (Ri), \u003cem\u003eFunneliformis mosseae\u003c/em\u003e (Fm), and \u003cem\u003eClaroideoglomus etunicatum\u003c/em\u003e (Ce). One set of plants inoculated with autoclaved inoculum was used as the control (CK). After four weeks of growth, part of the CK plants and the AMF-inoculated plants were further challenged with \u003cem\u003eRalstonia solanacearum\u003c/em\u003e (Rs). Thus this experiment contained six treatments: CK, Rs (inoculated only with \u003cem\u003eR. solanacearum\u003c/em\u003e), AM (inoculated only with the AMF, Fm), Ri\u0026thinsp;\u003cem\u003e+\u003c/em\u003e\u0026thinsp;Rs (dual-inoculation with Ri and Rs), Fm\u0026thinsp;+\u0026thinsp;Rs (dual-inoculation with Fm and Rs), and Ce\u0026thinsp;+\u0026thinsp;Rs (dual-inoculation with Ce and Rs). Following an additional two-week incubation, almost all the plants inoculated exclusively with \u003cem\u003eR. solanacearum\u003c/em\u003e (Rs) exhibited the typical bacterial wilt symptoms, with the disease index (DI) high to 82%, whereas less 50% of the plants under dual inoculation (+\u0026thinsp;\u003cem\u003eRi\u003c/em\u003e/\u003cem\u003eFm/Ce\u003c/em\u003e\u0026thinsp;+\u0026thinsp;Rs) showed the disease symptoms. Within the three AMF strains, \u003cem\u003eR. intraradices\u003c/em\u003e provided the highest level of disease suppression, with a disease index as low as 0.33 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Thus, in the following experiments, we only chose \u003cem\u003eR. intraradices\u003c/em\u003e as the AM inoculum.\u003c/p\u003e \u003cp\u003eTo confirm the capacity of AMF in the biocontrol of bacterial wilt, we further carried out a soil-based pot experiment. Tomato plants inoculated or mock-inoculated with \u003cem\u003eR. irregularis\u003c/em\u003e were grown in unsterilized soil for four weeks, and then challenged with \u003cem\u003eR. solanacearum\u003c/em\u003e (Rs) for another two weeks. Thus this experiment contained four treatments: CK, Rs (inoculated only with \u003cem\u003eR. solanacearum\u003c/em\u003e), AM (inoculated only with the AMF, Ri), AM\u0026thinsp;\u003cem\u003e+\u003c/em\u003e\u0026thinsp;Rs (dual-inoculation with Ri and Rs). As observed, \u003cem\u003eR. irregularis\u003c/em\u003e inoculation observably improved plant growth, even in the presence of \u003cem\u003eR. solanacearum\u003c/em\u003e. The shoot and root biomass of the AMF-inoculated plants (referred as AM plants) increased by 210% and 74%, respectively, compared to those of the mock-inoculated control plants (CK). The AM plants also showed an over 20% increase in chlorophyll content and increases by over 60% and 200% in shoot N and P content, respectively, relative to the control plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-e). Consistent with that found in the sand-based pot culture, we also observed a distinct suppressive effect of \u003cem\u003eR. irregularis\u003c/em\u003e on the occurrence of bacterial wilt in the soil-based pot culture. The plants inoculated exclusively with \u003cem\u003eR. solanacearum\u003c/em\u003e (Rs) showed an over 70% disease index (DI), while this index was reduced to about 40% in the dual-inoculated plants (AM\u0026thinsp;+\u0026thinsp;Rs), suggesting that \u003cem\u003eR. irregularis\u003c/em\u003e inoculation could achieve a control efficacy of tomato bacterial wilt disease by about 28% under our experiment conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef-g).\u003c/p\u003e \u003cp\u003eTo determine whether the \u003cem\u003eR. solanacearum\u003c/em\u003e infection may affect mycorrhizal colonization, we further assessed the mycorrhizal colonization levels for the AM plants and dual-inoculated (AM\u0026thinsp;+\u0026thinsp;Rs) plants. As observed, both the AM plants and the dual-inoculated plants with no distinct wilt symptoms showed high mycorrhizal colonization levels, with the total root length colonization level reached to over 60% (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). No significant difference in colonization levels was observed between the AM plants and the healthy dual-inoculated plants, while those diseased plants showed a much lower mycorrhizal colonization levels compared to that of the AM plants (data not shown). The control plants and the plants challenged with only \u003cem\u003eR. Solanacearum\u003c/em\u003e (Rs) showed no distinct AMF structure in their roots. We also checked the transcript level of an AM-marker gene, \u003cem\u003eSlHA8\u003c/em\u003e, encoding an AM-specific H\u003csup\u003e+\u003c/sup\u003e-ATPase, which showed a highly induced expression in the roots of both AM and AM\u0026thinsp;\u003cem\u003e+\u003c/em\u003e\u0026thinsp;Rs plants, but barely expressed in the roots of CK and Rs-treated plants. These results suggest that the AMF inoculation should be the causative factor that alleviates tomato bacterial wilt.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAMF colonization enhances the expression of PR genes and the activity of antioxidant enzymes in tomato\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore the potential mechanisms underlying the AMF-mediated suppression of bacterial wilt, we examined the expression levels of four pathogen-related (PR) genes in the plants inoculated with AMF and/or \u003cem\u003eR. solanacearum\u003c/em\u003e by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Inoculation only with \u003cem\u003eR. solanacearum\u003c/em\u003e had no remarkable influence on the expression of the PR genes examined, except for \u003cem\u003eSlPR4\u003c/em\u003e, whose transcript level was significantly upregulated by \u003cem\u003eR. solanacearum\u003c/em\u003e inoculation, compared to that in the control plants, while inoculation with AMF could substantially induce the expression of all the four \u003cem\u003eSlPR\u003c/em\u003e genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-d). The transcript levels of \u003cem\u003eSlPR1\u003c/em\u003e (encoding an antibacterial protein), \u003cem\u003eSlPR2\u003c/em\u003e (encoding a β-1,3-glucanase) and \u003cem\u003eSlPR3\u003c/em\u003e (encoding a chitinase) in the AM plants increased by 6-fold, 17-fold and 20-fold, respectively, relative to those in the control plants. Notably, in the dual-inoculated plants (AM\u0026thinsp;+\u0026thinsp;Rs), the transcript levels of \u003cem\u003eSlPR1, 2\u003c/em\u003e and \u003cem\u003e3\u003c/em\u003e decreased by 47%, 67%, and 91%, respectively, compared to those observed in the AM plants. This suggests that \u003cem\u003eR. solanacearum\u003c/em\u003e infection may partially inhibit the resistance response induced by AM symbiosis. An approximately 2-fold upregulation in the expression of \u003cem\u003eSlPR4\u003c/em\u003e was observed in both the AM plants and \u003cem\u003eRs\u003c/em\u003e-challeged plants compared to that in the control plants, suggesting that \u003cem\u003eSlPR4\u003c/em\u003e has a non-specific response to the invasion of exogenous symbiotic or pathogenic microorganisms.\u003c/p\u003e \u003cp\u003eTo determine whether AMF colonization and/or Rs infection may affect the oxidative stress response in tomato, we quantitatively analyzed the activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) in these plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-g). Inoculation with AMF, \u003cem\u003eRs\u003c/em\u003e, or the both significantly increased the activities of all the three antioxidant enzymes. Particularly, the activity of SOD showed a more pronounced enhancement, with increases by almost 100% in the \u003cem\u003eRs\u003c/em\u003e-treated plants and nearly 200% in the AMF-inoculated plants and dual-inoculated plants, compared to that in the control plants. These results indicate that AMF colonization or \u003cem\u003eRs\u003c/em\u003e infection could significantly enhance the activities of these antioxidant enzymes, thereby potentially improving the plants' tolerance to adverse conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAMF colonization induces a reassembly of the rhizosphere microbial community\u003c/h2\u003e \u003cp\u003eTo gain more insight into the AMF-mediated suppression of tomato bacterial wilt, we further employed the 16S rRNA sequencing technology to analyze the microbiota in the rhizospheric soil of the plants grown under four different treatments (CK, Rs, AM, AM\u0026thinsp;+\u0026thinsp;Rs). A total of 1,055,916 high-quality reads were obtained from 16 samples (four samples per treatment). The analysis led to the identification of 29 phyla, 57 classes, 72 orders, 161 families, 395 genera, and 462 species of microorganisms within these samples.\u003c/p\u003e \u003cp\u003eAlpha diversity analysis indicated that the Chao1 index (species richness) and Shannon index (species diversity) in the three inoculation treatments (Rs, AM, AM\u0026thinsp;+\u0026thinsp;Rs) increased by 17.1%-19.6% and 7.9%-10.9%, respectively, compared to the control (CK); however, these differences were not statistically significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig. S2). Moreover, no significant differences in either diversity or richness were observed among the three inoculation treatments (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Permutational multivariate analysis of variance (PERMANOVA) (R\u0026sup2; = 0.45, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001), combined with unconstrained principal coordinates analysis (PCoA), revealed a significant separation in bacterial communities among the four treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Specifically, the AM treatment showed the greatest divergence from the control group along PCoA axis 1, which explained 27.97% of the variation. In contrast, the presence or absence of \u003cem\u003eR. solanacearum\u003c/em\u003e inoculation was the primary factor driving separation along PCoA axis 2, accounting for 17.39% of the variation. These results indicate that AMF colonization and/or \u003cem\u003eR. solanacearum\u003c/em\u003e inoculation may induce a reassembly of the tomato rhizosphere microbial community.\u003c/p\u003e \u003cp\u003eIn the term of bacterial community composition, \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eAcidobacteria\u003c/em\u003e, \u003cem\u003eActinobacteria\u003c/em\u003e, \u003cem\u003eChloroflexi\u003c/em\u003e, and \u003cem\u003eBacteroidetes\u003c/em\u003e were shown to be the dominant phyla across all the four treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Inoculation with AMF significantly increased the relative abundances of \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003ePlanctomycetes\u003c/em\u003e (Fig. S3). At the genus level, \u003cem\u003eGemmatimonas\u003c/em\u003e, \u003cem\u003eStreptomyces\u003c/em\u003e, and \u003cem\u003eBacillus\u003c/em\u003e exhibited relatively high abundances in all treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Notably, the relative abundances of \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eBrevibacillus\u003c/em\u003e, \u003cem\u003eNitrososphaera\u003c/em\u003e, and \u003cem\u003eNocardiopsis\u003c/em\u003e were significantly enhanced in the AMF-treated samples compared to those in the control samples, whereas those of \u003cem\u003eLysobacter\u003c/em\u003e, \u003cem\u003eStreptomyces\u003c/em\u003e, and \u003cem\u003eDactylosporangium\u003c/em\u003e were significantly reduced following AMF inoculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-e, Fig. S4). Additionally, AMF inoculation significantly decreased the relative abundance of \u003cem\u003eRalstonia\u003c/em\u003e bacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIsolation of two beneficial\u003c/b\u003e \u003cb\u003eBrevibacillus\u003c/b\u003e \u003cb\u003estrains from tomato rhizosphere\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further elucidate whether AMF-mediated systemic disease resistance involves the recruitment of beneficial rhizosphere bacteria, we screened the culturable bacteria from rhizospheric soils of the dual-inoculated (AM\u0026thinsp;+\u0026thinsp;Rs) plants that did not develop wilt disease. We successfully isolated two strains of \u003cem\u003eBrevibacillus\u003c/em\u003e sp., named AQC211 and AQC296, both of which showed a distinct inhibitory capacity against \u003cem\u003eR. solanacearum\u003c/em\u003e in our plate antagonism assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eTo further evaluate their biocontrol potential, a pot inoculation experiment was conducted. The results showed that inoculation with \u003cem\u003eBrevibacillus\u003c/em\u003e sp. AQC211 could significantly reduce the severity of bacterial wilt and lower the disease index from 0.92 (in the control) to 0.70, corresponding to a disease control efficacy of 24%. The another strain, AQC296, showed a moderate level of biocontrol activity, reducing the disease index to 0.81, with a biocontrol efficacy of 12%. These findings collectively suggest that AMF-induced restructuring of the rhizosphere bacterial community may involve specific beneficial strains with biocontrol functions, such as \u003cem\u003eBrevibacillus\u003c/em\u003e sp. AQC211, which may play a synergistic role with AMF in enhancing tomato resistance to bacterial wilt.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBacterial wilt, a prevalent soil-borne disease of solanaceous crops, have emerged as a major constraint to global solanaceous crop production, particularly in regions with intensive agricultural practices (Kashyap et al. 2021). Due to the high pathogenicity, strong environmental adaptability and broad host range of its causative pathogen, \u003cem\u003eR. solanacearum\u003c/em\u003e, the conventional control strategies, such as chemical fumigation and crop rotation, are often largely ineffective (Wang et al. 2020). Recent studies in multiple plant species have shown a distinct advantage of using biological control strategies to suppress different soil-borne diseases, including the fungal and bacterial wilt (Fern\u0026aacute;ndez-Gonz\u0026aacute;lez et al. 2020). While AMF, the beneficial symbionts of most land plants, have been repeatedly documented to be able to enhance host plant\u0026rsquo;s resistance and resilience against various biotic and abiotic stresses, the benefits of using AMF as a major biocontrol agent to suppress bacterial wilt are still lack of adequate and compelling proofs.\u003c/p\u003e \u003cp\u003eIn this study, we demonstrate that AM colonization could effectively alleviate tomato bacterial wilt, as indicated by the significantly lower disease index in those plants inoculated with the three AMF strains, \u003cem\u003eR. intraradices\u003c/em\u003e, \u003cem\u003eF. mosseae\u003c/em\u003e, or \u003cem\u003eC. etunicatum\u003c/em\u003e, compared to that in the control plants under either sterile sand or unsterilised soil culture conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Previous studies have suggested that AMF may contribute to resistance to root and foliar pathogens via inducing plant systemic resistance, a phenomenon termed mycorrhiza-induced resistance (MIR). In this study, we indeed observed significantly up-regulated expression of four PR genes (\u003cem\u003eSlPR1\u003c/em\u003e to \u003cem\u003e4\u003c/em\u003e) and enhanced activity of the three antioxidant enzymes (CAT, POD and SOD) in the leaves of AM plants, relative to that in control plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), confirming that AMF colonization can induce the systemic resistance in plants. Notably, it seemed that \u003cem\u003eR. solanacearum\u003c/em\u003e infection might be able to weaken the plant\u0026rsquo;s resistance induced by AMF, as infered from that the dual-inoculated (AM\u0026thinsp;+\u0026thinsp;Rs) plants showed significantly reduced expression levels of the three PR genes, \u003cem\u003eSlPR1\u003c/em\u003e, \u003cem\u003eSlPR2\u003c/em\u003e and \u003cem\u003eSlPR3\u003c/em\u003e, compared to the AM plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c). Consistent with that observed in the AM plants, the activity of CAT, POD and SOD in the \u003cem\u003eRs\u003c/em\u003e-challenged plants or dual-inoculated plants was also substantially enhanced compared to that in the control plants, suggesting that the mycorrhiza-induced resistance shares partially similar features with the systemic acquired resistance induced by pathogen infection. It is worth emphasizing that AMF colonization could not only enhance the systemic resistance, but also promote plant growth, chlorophyll accumulation and the uptake of Pi and N in tomato plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-e). Previous studies reported that tomato plants colonized by AM fungi could substantially decrease root infection and disease severity caused by different pathogens, resulting in increased plant fresh weight (up to 198%) and fruit yield (14.3%) as compared to those non-mycorrhizal pathogen-infected plants (Berta et al. 2005; Utkhede 2006; Gianinazzi et al. 2008). The findings suggest that the improved growth and nutritional status of AM plants may impart them an advantage in compensating the damage caused the pathogenic infection.\u003c/p\u003e \u003cp\u003eIncreasing studies point to that the diversity of soil bacteria and their community structure are crucial for maintaining soil health and controlling soil-borne diseases (Jayaraman et al. 2021; Zhang et al. 2023). In this study, we found that the \u003cem\u003eRs-\u003c/em\u003echallenged tomato plants grown in unsterilized soil exhibited a lower disease index than those grown in sterile sand, even in the absence of AMF inoculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef), suggesting that there might exist certain beneficial microorganisms that could antagonize \u003cem\u003eR. solanacearum\u003c/em\u003e in the rhizospheric soil of tomato plants. Earlier studies reported that inoculation of mycorrhizal fungi could increase both the richness and diversity of rhizosphere microorganisms associated with host plants, thereby improving plant stress resistance (Toju et al. 2018; Gonz\u0026aacute;lez Guzm\u0026aacute;n et al. 2022). While our 16S RNA gene sequencing showed no significant differences in Chao1 index (richness ) and Shannon index (diversity) between the control plants and those plants inoculated with AMF, \u003cem\u003eR. solanacearum\u003c/em\u003e, or the both, we did observe a remarkable difference in the composition of the bacterial microbiota among the control group and the three inoculated treatments (Rs, AM, AM\u0026thinsp;+\u0026thinsp;Rs), as shown by the distinct separation in the unconstrained principal coordinate analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). These results strongly suggest that AMF colonization can reshape the rhizobacterial community of host plants. Our findings could gain well support from a recent study demonstrating that AMF colonization could recruit rhizobia to accumulate in the rhizosphere of \u003cem\u003eM. truncatula\u003c/em\u003e (Cangioli et al. 2022).\u003c/p\u003e \u003cp\u003eIncreasing evidence points to that AMF may cooperate with certain beneficial rhizospheric microorganisms to synergistically enhance nutrient uptake and/or repel harmful microorganisms (Boyno et al. 2025). \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eBrevibacillus\u003c/em\u003e species are well-documented beneficial microorganisms that can suppress the population growth and infection process of soil-borne pathogens through multiple approaches, such as resource competition, niche exclusion, secretion of antibiotic compounds and induction of plant systemic resistance (Ghani et al. 2019; Koza et al. 2022; Pan et al. 2025; Wang et al. 2021). In this study, we found that AMF inoculation led to a significant increase in the abundance of \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eBrevibacillus\u003c/em\u003e, coupled with a remarkable decrease in \u003cem\u003eRalstonia\u003c/em\u003e bacteria, within the tomato rhizosphere. The successful isolation of two strains of \u003cem\u003eBrevibacillus\u003c/em\u003e, both exhibiting a distinct antagonistic activity against \u003cem\u003eR. solanacearum\u003c/em\u003e, strongly suggests that AMF can recruit beneficial microorganisms, such as Bacillus spp., to synergistically defend plants against infection by soil-borne pathogens like \u003cem\u003eR. solanacearum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn summary, our present study demonstrates that AMF colonization can effectively alleviate tomato bacterial wilt by activating a series of physiological mechanisms, including improving plant growth and nutritional status, inducing plant systemic resistance, reshaping the rhizobacterial community and recruiting beneficial genera like Brevibacillus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These findings highlight AMF as a sustainable, eco-friendly strategy for controlling bacterial wilt, offering significant potential to reduce reliance on chemical inputs and enhance both crop resilience and soil health in agricultural systems.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32472831), Zhongshan Biological Breeding Laboratory (ZSBBL-KY2023-03), Jiangsu Provincial Key Laboratory of Coastal Saline Soil Resources Utilization and Ecological Conservation and Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization. We thank Prof. Zhong Wei from Nanjing Agricultural University for the providing of\u0026nbsp;\u003cem\u003eR. solanacearum\u003c/em\u003e strain QL-Rs1115.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.Z. and A.C. designed research and analyzed the data; D.Z., L.W., Y.G., W.Z., W.W. conducted the experiments; A.C., S.W. and G.X. supervised the project; A.C. and D.Z. wrote the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that all data supporting the findings of this study are available within the article are available upon request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbarca C, Fernandez Bidondo L, Bompadre J, et al. 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RSC Adv. 2020;10(7):3978-3990.\u003c/li\u003e\n\u003cli\u003eWang S, Na X, Yang L, et al. Bacillus megaterium strain WW1211 promotes plant growth and lateral root initiation via regulation of auxin biosynthesis and redistribution. Plant Soil. 2021;466(1):491-504.\u003c/li\u003e\n\u003cli\u003eYang Y, Zhang X, Ma J, et al. Integrated analysis of transcriptome, sRNAome and degradome sequencing provides insights into bacterial wilt resistance in potato. BMC Plant Biol. 2025.\u003c/li\u003e\n\u003cli\u003eYuan XY, Zhang LG, Huang L, et al. Spraying Brassinolide improves Sigma Broad tolerance in foxtail millet (Setaria italica L.) through modulation of antioxidant activity and photosynthetic capacity. Sci Rep. 2017;7(1):11232.\u003c/li\u003e\n\u003cli\u003eZhang Z, Wei Y, Peng Z, et al. Exploration of microbiome diversity of stacked fermented grains by flow cytometry and cell sorting. Front Microbiol. 2023;14:1160552.\u003c/li\u003e\n\u003cli\u003eZhou J, Chai X, Zhang L, et al. Different Arbuscular Mycorrhizal Fungi Cocolonizing on a Single Plant Root System Recruit Distinct Microbiomes. mSystems. 2020;5(6).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"mycorrhiza","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcor","sideBox":"Learn more about [Mycorrhiza](http://link.springer.com/journal/572)","snPcode":"572","submissionUrl":"https://submission.nature.com/new-submission/572/3","title":"Mycorrhiza","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Arbuscular mycorrhizal fungi, Ralstonia solanacearum, tomato bacterial wilt, Rhizosphere microbiome","lastPublishedDoi":"10.21203/rs.3.rs-8605735/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8605735/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTomato bacterial wilt, caused by \u003cem\u003eRalstonia solanacearum\u003c/em\u003e, is a globally devastating soil-borne disease that poses a serious threat to the sustainable development of tomato production. Arbuscular mycorrhizal fungi (AMF) are well-recognized beneficial soil microorganisms that significantly promote plant growth, enhance nutrient uptake, and bolster resistance to various biotic and abiotic stresses. However, evidence regarding the potential of AMF to suppress tomato bacterial wilt remains limited. In this study, we demonstrate that AMF inoculation remarkably reduces the disease index of bacterial wilt in tomato plants, upregulates the expression of pathogenesis-related (PR) genes, and enhances antioxidant enzyme activities, collectively strengthening systemic disease resistance. High-throughput 16S rRNA gene sequencing revealed that AMF colonization drives a substantial reassembly of the rhizosphere microbiome. Notably, AMF colonization promoted the recruitment of beneficial bacterial genera, including \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eBrevibacillus\u003c/em\u003e, while significantly suppressing the abundance of \u003cem\u003eRalstonia\u003c/em\u003e bacterial genera. Furthermore, we isolated two \u003cem\u003eBrevibacillus\u003c/em\u003e strains, named AQC211 and AQC296, from the mycorrhizosphere of healthy tomato plants, both of which exhibited antagonistic activity against \u003cem\u003eR. solanacearum\u003c/em\u003e in vitro. Pot experiments confirmed that inoculation with the AQC211 strain significantly reduced the incidence and severity of bacterial wilt. These findings indicate that AMF can not only directly prime plant systemic resistance but also indirectly enhance protection against bacterial wilt by shaping a disease-suppressive rhizosphere microbiome.\u003c/p\u003e","manuscriptTitle":"Arbuscular mycorrhizal symbiosis suppresses tomato bacterial wilt by coordinating plant systemic resistance with microbiome antagonism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-23 15:00:59","doi":"10.21203/rs.3.rs-8605735/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-25T22:53:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-06T20:42:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-26T11:56:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-12T09:22:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"314718561464612149415779791505561677875","date":"2026-02-10T07:10:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"225778456016705893958807594864904684131","date":"2026-02-07T04:10:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"318470563114496773773221376829796353834","date":"2026-02-05T23:26:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"329030474775328740220465249555925877327","date":"2026-02-02T13:58:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-21T13:16:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-20T14:29:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-20T12:12:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Mycorrhiza","date":"2026-01-15T01:00:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"mycorrhiza","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcor","sideBox":"Learn more about [Mycorrhiza](http://link.springer.com/journal/572)","snPcode":"572","submissionUrl":"https://submission.nature.com/new-submission/572/3","title":"Mycorrhiza","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1ca5c700-31f6-4687-b292-79b73212c2e4","owner":[],"postedDate":"January 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-14T14:53:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-23 15:00:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8605735","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8605735","identity":"rs-8605735","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}