Silica Nanoparticles Suppress Fungal Pathogenic Allies to Alleviate Astragalus Root Rot

Silica Nanoparticles Suppress Fungal Pathogenic Allies to Alleviate Astragalus Root Rot | 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 Silica Nanoparticles Suppress Fungal Pathogenic Allies to Alleviate Astragalus Root Rot Jiamin Ai, Leilei Xu, Hao Ding, Zijing Dang, Liru Jian, Gehong Wei, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6006553/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Oct, 2025 Read the published version in Microbiome → Version 1 posted 8 You are reading this latest preprint version Abstract Background: Biological control mechanisms involve the inhibitory effect of antagonistic bacteria on pathogenic fungal growth. However, research on controlling crop diseases by inhibiting allies of pathogenic agents is relatively scarce. Results: In this study, the application of SiO 2 NPs resulted in an increase in the alpha diversity of the microbial communities in the rhizosphere caused by Astragalus , as well as an increase in the complexity of the co-occurrence network. SiO 2 NPs reduced the abundance of Pseudomonas and Microbacterium in the rhizosphere of Astragalus . Co-inoculated Fusarium with P. aeruginosa and M. oxydans could exacerbate the root rot of disease in Astragalus . In addition, M. oxydans SCK-308 and P. aeruginosa XS-134-7 promoted the growth of F. oxysporum and inhibited the growth of certain beneficial rhizosphere microorganisms, thereby facilitating the occurrence of the disease. Metabolomic analyses revealed that salicylic acid, indole-3-acetic acid, brassinosteroid, and palmitic acid were significantly enriched in the rhizosphere of Astragalus treated with SiO 2 NPs. Exogenous supplementation with these metabolites significantly inhibited the growth of P. aeruginosa and M. oxydans , thereby alleviating root rot in plants during coinfection with two bacteria and F. oxysporum . These results indicate that the metabolites enhance disease control efficacy through targeted inhibition of pathogen helpers. Additionally, SiO 2 NPs enhanced the enzymatic activities of ascorbate peroxidase, catalase, and peroxidase in Astragalus plants. Conclusions: Our findings suggest that SiO 2 NPs alter the composition of the rhizosphere microbial community and reduce the population of allies of F. oxysporum , activating salicylic acid-dependent systemic acquired resistance (SAR) in Astragalus and thereby decreasing the incidence of Fusarium root rot. These results suggest that SiO 2 NPs can serve as a sustainable agricultural practice. Silica nanoparticles (SiO2 NPs) Root rot disease Root exudates Microbial communities Metabolomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Background Soil-borne fungal pathogens pose substantial challenges to global agriculture, adversely affecting crop yield and sustainability, with potential yield losses of up to 75% for major crops [ 1 ]. Fusarium oxysporum is a prevalent soil-borne pathogen that can infect more than 150 different crops, leading to severe Fusarium root rot in species such as soybean [ 2 ], maize [ 3 ] and Astragalus [ 4 ]. The management of Fusarium root rot is particularly difficult due to persistence of pathogenic agents in the soil, substantial genetic diversity, and broad host range [ 5 ]. Historically, chemical methods have been employed to mitigate outbreaks of soil-borne diseases; however, these approaches have increasingly been phased out due to serious environmental concerns [ 6 ]. Microbiota transplantation has emerged as a promising strategy for the treatment of soil-borne diseases. Nevertheless, the efficacy of this process is contingent upon various factors, including the composition of the microbial community, the method of transplantation, the soil environment, and the physiological state of the plant host [ 7 ]. For large-scale implementation in agricultural production, further advancements are necessary. In the pursuit of safer agrochemicals, silica nanoparticles (SiO 2 NPs) have recently been proposed as novel tools to mitigate pathogen damage, including that caused by F. oxysporum [ 8 ]. In addition, several studies have shown that the application of SiO 2 NPs improves the resistance of various crops, including peanuts [ 9 ], rice [ 10 ], Arabidopsis [ 11 ], and potato [ 12 ], to biotic stress. The properties and chemical inertness of silicon contribute to its potential applicability in agricultural practices, as it is expected to have minimal negative impacts on environmental health and crop food safety [ 13 ]. In addition, SiO 2 NPs have emerged as an alternative and complementary approach to conventional methods of chemical control, biological control, and cultural practices, owing to their advantages of better efficacy, reduced input, and lower ecotoxicity [ 10 , 14 ]. It was initially proposed that silicon deposition creates a physical barrier beneath the leaf cuticle to prevent pathogen invasion [ 15 ]. However, subsequent research has indicated that the application of SiO 2 NPs inhibits the growth of pathogenic agents. For example, Ralstonia is prevalent in wilt-diseased soil, and the application of silicon has been found to reduce the incidence of bacterial wilt in peanuts by diminishing the relative abundance of Ralstonia in the rhizosphere [ 16 ]. Additionally, studies have shown that some SiO₂ NPs do not directly inhibit pathogens but instead enhance plant disease resistance by modulating the composition of the rhizosphere microbial community [ 17 ]. However, whether these altered microbes confer disease resistance or promote pathogenesis has not been well validated. For example, SiO₂ NPs promote disease resistance by increasing the relative abundance of microbial taxa with plant-beneficial potential. The relationships among microorganisms include neutral, positive, and negative interactions, thus in addition to antagonist, there are also some bacteria that can promote the growth of pathogen. This is in line with the recent finding where a considerable proportion of root-associated microorganisms can promote pathogen growth and pathogenicity, underscoring that such facilitation may be a key determinant of successful pathogen infection [ 18 – 20 ]. For example, Microbacterium paraoxydans and Pseudomonas syringae serve as bacterial helpers for Ralstonia solanacearum and Fusarium , respectively, exacerbating disease severity [ 21 , 22 ]. In addition, alterations in the levels or composition of root exudates can influence plant health by regulating pathogen populations and inducing plant resistance [ 23 ]. For example, Si-induced accumulation of defensive compounds such as phenols, flavonoids, lignin, and dopamine in the sclerenchyma and vascular tissues of roots may increase resistance to F. oxysporum in banana [ 24 ]. SiO 2 NPs can also promote resistance to pathogens in plants by mediating the production of endogenous phytohormones, including salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) [ 25 ]. Research has shown that peanuts irrigated with SiO 2 NPs exhibit a significant increase in resistance to bacterial wilt, attributed to SA-dependent plant immune responses that trigger systemic acquired resistance (SAR) [ 17 ]. The phenomenon of silicon-induced SAR in plants has also been corroborated in tomato, rice, and other crops [ 26 , 27 ]. Plant root exudates serve as vital carbon sources for soil microbes and play a crucial role in coordinating the composition of the rhizosphere microbiome. Conversely, microbes act as modifiers and limiters of root exudates, which are essential for soil health and significantly influence plant defence against soil-borne pathogens [ 28 ]. The interaction between root exudates and soil microbes enhances plant immunity and overall health during pathogen invasion. Nevertheless, reports addressing the effects of Si-mediated interactions between root exudates and soil microbial communities on plant disease resistance are scarce. This knowledge gap hinders our comprehensive understanding of how silicon modulates plant‒microbiome interactions to increase host plant defences. Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao (hereafter, Astragalus ) is an important perennial herbaceous leguminous plant recognized for its medicinal properties. This species is predominantly distributed in the provinces of Gansu, Shanxi, and Inner Mongolia, where it serves as a key economic crop [ 29 ]. A major threat to the yield and quality of Astragalus is the high incidence of root rot which is exacerbated by long-term monoculture. However, the extent to which SiO 2 NPs can increase the resistance of Astragalus to root rot, and the underlying mechanisms remain poorly understood. In this study, we investigated the effects of the addition of SiO 2 NPs through soil application and foliar spraying on the resistance of Astragalus to F. oxysporum . Our research focused on: (1) the impact of different concentrations of SiO 2 NPs and application methods on the resistance of Astragalus to root rot; (2) whether various methods of SiO 2 NPs application can alter the microbial community in the Astragalus rhizosphere and enhance root rot resistance; and (3) the relationship between SiO₂NP-modified microbes and resistance to root rot disease. Our comprehensive approach aims to examine the potential of SiO 2 NPs as an innovative strategy for managing soil-borne diseases in Astragalus , thereby promoting a transition to more sustainable agricultural practices. 2. Materials and methods 2.1. Effect of SiO 2 NPs on Fusarium oxysp or um growth The SiO 2 NPs (CAS: 7631-86-9, SKU: S490064, with a diameter of 17 nm) used in this study were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The morphology of the SiO 2 NPs was characterized using transmission electron microscopy (TEM, HT7800, Hitachi High-Tech, OR). The zeta potential of the SiO 2 NPs was measured using a Zetasizer Nano ZS instrument (Malvern Instruments Ltd., U.K.). The chemical composition and functional groups of SiO 2 NPs were determined by Thermo Scientific X-ray photoelectron spectroscopy (XPS) (ESCALAB 250Xi, Thermo, USA) and Fourier transform infrared spectroscopy (FTIR) (Frontier, PerkinElmer, USA). In addition, 1500 mg/L SiO 2 NPs suspension was used to observe its stability within 3 days of storage. The fungal pathogen Fusarium oxysporum used in this study was originally isolated from field-grown Astragalus plants exhibiting root rot symptoms [4].The toxicity of SiO 2 NPs to F. oxysporum was tested in solid and liquid media. PDA medium containing 200, 400, and 800 mg/L SiO 2 NPs was prepared respectively, and fungal plugs (6-mm diameter) were transferred to the PDA medium preparations and placed in a biochemical incubator at 28 °C for 7 days, after which fungal colony diameters were measured. Additionally, PDB medium (25 mL) containing the same concentrations of SiO 2 NPs was prepared, and 1 mL of F. oxysporum (~10 5 spores/mL) was added and cultured at 28 °C, and 220 rpm for 72 h. The culture medium was filtered with gauze, the spores in the filtrate were counted using a hemocytometer, and the filter residue was dried and weighed using analytical balance to obtain the weight of the fungal hyphae. Each treatment had six replicates. 2.2. Plant growth conditions and plant treatments The soils used in this study were collected from medicinal plant fields in Tanchang County, Gansu Province, China (N34° 15.123’; E104° 09.727’). The soil physical and chemical properties were as follows: pH, 8.17; soil organic matter, 27.66 g/kg; total nitrogen, 1.75 g/kg; total phosphorus, 1.31 g/kg; total potassium concentration, 10.86 g/kg, and available K, 223.77 mg/kg. SiO 2 NPs was added to the substrate (soil: vermiculite = 3: 1, v/v) to make its final concentrations 200, 400, and 800 mg/kg respectively. Then 500 g of substrate containing SiO 2 NPs was filled into planting bag. In addition, commercial carbendazim (50% active ingredient, Lanfeng Biol-Chemical Ltd. China) was added to the substrate to a final concentration of 100 mg/kg as a positive control. The Astragalus seeds were surface sterilized with 75% ethanol for 30 s and 1% sodium hypochlorite solution for 1 min and then rinsed six times with ddH 2 O water and soaked for 48 h. The surface-disinfected seeds were placed on sterile, moist filter paper to germinate. Ten germinated seedlings were sown in planting bags. After seven days, the seedlings in the planting bags were thinned to five uniform plants. The foliage-spraying SiO 2 NP treatment (Si_L) was performed as follows: SiO 2 NP suspensions were prepared by sonicating for 15 min in DI water, at concentrations of 200, 400, and 800 mg/L. After the true leaves of Astragalus seedlings had fully expanded, spraying was performed every seven days for a total of 5 times [30]. The soil surface was covered with paper towels to prevent soil contamination when SiO 2 NPs were sprayed. The other groups were sprayed with the same amount of DI water. The pathogen was cultured in PDB medium for 4-5 days, and the culture suspension was filtered using four layers of sterile gauze to obtain a spore suspension. The spore suspension was then serially diluted to 10 - 3 , the spores were counted with hemacytometers (Thoma, XB-K-25, 25×16), and the spore concentration was adjusted to approximately 10 5 spores/mL. One week after the first foliar spraying of SiO 2 NPs, 2 mL (per plant) of a spore suspension of F. oxysporum (approximately 10 5 spores per millilitre) was added to the roots of Astragalus plants. All the plants were grown in a greenhouse at 25 °C (16 h light/8 h darkness). The disease index and control effect were calculated after 21 days of inoculation with F. oxysporum . The diseased plants were divided into 4 levels based on their disease severity. Level 0 indicated root health without disease lesions. For Level 1, 1–2 black sunken lesions appeared in the roots. For Level 2, 3–5 sunken black lesions appeared in the roots. For Level 3, 5–8 sunken black lesions appeared in the roots. For Level 4, the root lesions had connected to form a network of vertical columns and rough epidermis. The disease index and control effect were calculated using the following formulas: disease index = [(∑disease grades × number of infected plants)/(total number of checked plants × 4)] ×100%, prevention effect = (control - treatment disease index)/control disease index × 100% [4]. After 21 days of pathogen inoculation, the indicators of plant growth status, i.e., plant height, root length, shoot fresh weight, shoot dry weight, root fresh weight, and root dry weight, were determined. The chlorophyll content in the leaves was determined with a hand-held chlorophyll meter. The malondialdehyde (MDA), superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and peroxidase (POD) contents were measured according to the instructions of relevant commercial assay kits from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). The amorphous Si contents of the aboveground parts and roots of Astragalus were determined using a plant silicon content test kit (Shanghai Yaji Biotechnology Co., Ltd., China) according to the manufacturer's protocol. 2.3. Rhizosphere soil collection and high-throughput sequencing Following the pot experiments, the roots were carefully transferred into enzyme-free centrifuge tubes with 25 mL of 1 × PBS and then vortexed for 5 minutes to collect the rhizosphere soil. After the roots were removed, the tubes were centrifuged at 8, 000 rpm for 7 minutes to pellet the soil particles. In total, 15 samples were obtained (3 treatments × 5 repetitions). All the samples were stored at −80 °C before DNA extraction. Microbial DNA was extracted from 0.5 g of rhizosphere soil using a FastDNA SPIN Kit for Soil (MP Biomedicals, USA) according to the manufacturer's protocol. The quality of the extracted DNA was assessed using 1% agarose gel electrophoresis, and its concentration and purity were determined with a NanoDrop 2000 spectrophotometer. The bacterial 16S rRNA gene (V4-V5 hypervariable regions) was amplified using the primers 515F (5'-GTGCCAGCMGCCGCGG-3') and 907R (5'- CCGTCAATTCMTTTRAGTTT-3'). The fungal hypervariable internal transcribed spacer (ITS) region was amplified using the primers ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS1R (5'-GCTGCGTTCTTCATCGATGC-3'). The library was subsequently constructed using a TruSeq DNA PCR-Free Sample Preparation Kit, quantified through qPCR, and then sequenced on an Illumina MiSeq platform. QIIME 2.0 [31] was used to filter the original tags and species annotations, and sequence alignment of bacteria and fungi was performed using the Silva and Unite databases, respectively. All reads assigned to plant plastids (chloroplasts and mitochondria) were discarded from the dataset, and the remaining effective sequences were clustered into amplicon sequence variants (ASVs) at 97% similarity [32]. The relative abundance of the ASVs was calculated in the R environment (version: V4.4.1, http://www.r-project.org/). The bacterial 16S rRNA and fungal ITS sequencing data for all the samples in this study were submitted to the NCBI Sequence Read Archive database under BioProject accession numbers PRJNA1192509 and PRJNA1192506. The 16S rRNA sequence of isolates were uploaded to NCBI GenBank accession numbers PV505490-PV505497. 2.4. Isolation and reinoculation of core microorganisms The bacteria were isolated from the rhizosphere soil of Astragalus using the method outlined by Oppenheimer-Shaanan [33] with minor modifications. Fresh rhizosphere soil was suspended in PBS and shaken at 180 rpm for 30 minutes. The dilutions were plated on LB medium solidified with 1.5% agar and then incubated at 30 °C for 2‒7 days [34]. All the purified isolates were stored in 30% glycerol (v/v) at −80 °C. To obtain the key bacteria, the 16S rRNA gene of the isolates were amplified using universal primer set (27F and 1492R) [35]. The obtained sequences were aligned with the sequences of core ASV in the co-occurrence network. Phylogenetic trees were constructed with MEGA software (v5) using the neighbour-joining (NJ) method. Finally, eight core microorganisms in the co-occurrence network were successfully isolated from the rhizosphere soil. To confirm the presence of these bacterial isolates in the rhizosphere soil, similarity analysis was performed based on the 16S rRNA sequences of the bacterial isolates and the gene sequences of ASVs (the same genus as the isolates). Isolates with the V4–V5 region matching with more than 97% identity were considered the same strain [36]. The core microorganisms were cultured in LB medium until they reached an optical density of 0.8. A 2-mL bacterial suspension (per plant) was subsequently added to the roots of 7-day-old Astragalus seedlings. One week after bacterial inoculation, a 2-mL (per plant) spore suspension of F. oxysporum (approximately 10 5 spores per millilitre) was generated. The disease index and control effect were calculated after 21 days of inoculation with F. oxysporum . 2.5. Metabolite extraction and LC‒MS/MS analysis The root exudates of Astragalus with and without SiO 2 NPs were collected for metabolomic analysis. The seedlings were removed from the soil, and the roots were washed with DI water and soaked for 2 h. Then, the seedlings were placed in sterile DI water and cultured in the dark for 24 h. The seedling exudates were collected and filtered with a 0.22- μm filter and then prefrozen at −80 °C, after which they were removed for lyophilization [37]. The dried samples were redissolved in 100 µL of methanol:water (v/v = 1:1). The dissolved samples were vortexed and centrifuged at 15,000 × g for 15 min (4 °C), and the resulting supernatants were used for LC‒MS/MS analysis [38]. Each treatment was performed in five independent biological replicates. A Vanquish UHPLC system (Thermo Fisher) and an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher) were used for untargeted Astragalus root exudate metabolomics analysis. The chromatographic conditions were as follows Hypesil Gold column (C18), 40 °C column temperature, 0.2 mL/min flow rate. In positive mode, mobile phase A was 0.1% formic acid, and mobile phase B was methanol; in negative mode, mobile phase A was 5 mM ammonium acetate (pH 9.0), and mobile phase B was methanol. The mass spectrometer scan range was 100–1500 m/z, and the ESI source settings were as follows: spray voltage, 3.5 kV; capillary temperature, 320 °C; sheath gas flow rate, 35 psi; and auxiliary gas flow rate, 10 arb. Positive/negative polarity operation was employed. The raw data generated by UHPLC-MS/MS were processed in CD 3.1 search software and compared with the mzCloud (https://www.mzcloud.org/), mzVault, and Masslist databases. Finally, the identification and relative quantification results of the metabolites were obtained. Metabolites were annotated in the KEGG, HMDB, and LIPIDMAPS databases. Differentially abundant metabolites were screened according to a fold change > 2 and p < 0.05, and pathway enrichment analysis was performed using the KEGG database. MetOrigin (http://metorigin.met-bioinformatics.cn/) was used to discriminate the sources of the metabolites, and the rhizosphere microbiome and metabolome were subjected to integrative analysis [39]. 2.6. Effects of metabolites on the resistance of Astragalus to root rot By comparing the relative abundance of root exudates from control and Si_S-treated plants, differentially abundant metabolites were identified. Standard materials of palmitic acid (CAS: 57-10-3, molecular weight: 256.42, AR), salicylic acid (CAS: 69-72-7, molecular weight: 138.12, AR), indole-3-acetic acid (CAS: 87-51-4, molecular weight: 175.18, AR), and brassinolide (CAS: 72962-43-7, molecular weight: 480.69, AR) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Each of the four compounds was dissolved in methanol to prepare a stock solution with a concentration of 10 mM. The effects of these metabolites on the growth of P. aeruginosa XS-134-7, M. oxydans SCK-308, and F. oxysporum were evaluated. The metabolite stock solutions were added to 24-well plates (Costar, Corning Incorporated, USA) containing LB medium or PDB medium to achieve final metabolite concentrations of 25, 50, 100, and 200 μM. The total volume in each well was 1 mL. Subsequently, 1 μL of bacterial suspension (OD 600 = 0.1) or fungal spore solution (approximately ~10 5 spores/mL) was transferred into the wells of a 24-well plate. The plates were incubated at 28 °C and shaken at 170 rpm for 24 hours. Bacterial growth was assessed by measuring the absorbance at OD 600 . The effects of metabolites on fungal spores were evaluated by counting spores with a haemocytometer. In addition, PDA medium was prepared for the four metabolites at final concentrations of 25, 50, 100, and 200 μM using the same method as described above. The 6-mm fungal plugs were subsequently transferred into PDA medium. The plates were then placed in a biochemical incubator at 28 °C for 7 days, after which the fungal colony diameter was measured. The control group (solvent control) consisted of medium supplemented with 200 μM methanol. Compared with no methanol addition, the additional concentration did not affect the growth of bacteria and fungi. Astragalus was infected on bi-compartmented Petri plates (90×15 mm) to explore the effects of metabolites on the resistance of Astragalus root rot. Briefly, 15 mL of water agar medium was added to the first compartment and 10 mL of Hoagland solution was added to the second compartment. After the true leaves of Astragalus seedlings were fully expanded, 100 μL of F. oxysporum (approximately 10 5 spores per millilitre), a bacterial suspension (OD 600 = 0.8), and metabolites were added to the side of the Hoagland solution. All the plates were placed in an artificial climate incubator at 25 °C (16 h light/8 h darkness). 2.7. Statistical analysis Data analysis and visualization were performed mainly in the R environment (V4.4.1). The 'vegan' package was used to calculate the alpha and β diversities. Principal coordinate analysis (PCoA) was conducted to assess β diversity. Redundancy analysis (RDA) was performed using the 'vegan' package in R. Spearman correlations were calculated using the 'ggcor' package in R. Cytoscape 3.4.0 was used to visualize the network. PICRUSt software was used to predict KEGG orthologue functional profiles of bacterial communities in rhizospheres [40]. The Sloan neutral model was applied to evaluate the effects of random dispersal and ecological drift on the assembly of microbial communities [41]. Phylogenetic bin-based null model analysis (iCAMP) was used to infer community assembly mechanisms [42]. To evaluate the influence of stochastic processes on community assembly, we employed a neutral community model (NCM) to predict the relationship between OTU detection frequency and relative abundance within a broader metacommunity [43]. This model, an adaptation of neutral theory, posits that taxa with high abundance in the metacommunity are more likely to disperse across sites, whereas rare taxa are more susceptible to loss due to ecological drift (i.e., stochastic fluctuations in population dynamics) [44]. The dispersal between communities is quantified by the parameter Nm, where N denotes the metacommunity size and m represents the immigration rate. The goodness-of-fit to the neutral model was assessed using R², and 95% confidence intervals for the model parameters were derived through bootstrapping with 1000 replicates. 3. Results 3.1. SiO 2 NPs increase pathogen resistance and promote plant growth The TEM images of the SiO 2 NPs showed that it exhibited a spherical shape, with an average size of 17 nm (Fig. S1a). The zeta potential of the SiO 2 NPs was found to be -23.47 mV. The results of FTIR spectra indicated that the peaks at 3438, 1660, 1170~1047 and 962 cm -1 were attributed to the -OH, H-O-H, Si-O-Si, and Si-OH vibration stretching, respectively (Fig. S1b). To further characterize the surface functional groups of the SiO 2 NPs, XPS spectroscopy was conducted. The results of XPS suggested that the full spectrum displays the existence of Si, C, and O on the surface of SiO 2 NPs (Fig. S1c). Furthermore, experiments on the stabilities of SiO 2 NPs were carried out (Fig. S1d). The results showed that the solution of SiO 2 NPs did not show the formation of aggregation between 0-12 hours. This indicated that the solution of SiO 2 NPs showed good nanoparticle dispersibility. To explore the potential of SiO 2 NPs in enhancing the disease resistance of Astragalus , the plants were subjected to various methods of SiO 2 NP application, including soil supplementation and foliar spraying, at concentrations of 200, 400, and 800 mg/kg, with the fungicide carbendazim used as a positive control. The findings demonstrated that carbendazim, soil application and foliar spraying of SiO 2 NPs significantly improved plant resistance to F. oxysporum infection, as indicated by ANOVA ( p < 0.05). Although the difference between the two application methods was not substantial, the disease index of Astragalus cultivated in the soil supplemented with SiO 2 NPs was lower than that of Astragalus cultivated with foliar spraying. Additionally, the efficacies of both treatments increased with increasing SiO 2 NP concentration (Fig. 1a). Because the application of SiO 2 NPs could significantly reduce the disease index, the efficacy of SiO 2 NPs in inhibiting pathogenic fungi was evaluated. However, the results indicated that SiO 2 NPs did not significantly influence the growth or sporulation of F. oxysporum in either solid or liquid medium (Fig. 1b). Therefore, the activities of antioxidant enzymes in plants treated with SiO 2 NPs were assessed. Both foliar spraying and soil supplementation with SiO 2 NPs resulted in significant increases in the activities of ascorbate peroxidase (APX), catalase (CAT), and peroxidase (POD) (ANOVA; p < 0.05). Regardless of the method of silicon application, the highest activities of the antioxidant enzymes were observed at a treatment concentration of 400 mg/kg SiO 2 NPs, with concentrations exceeding 400 mg/kg not yielding further increases in APX, CAT, and POD activities (Fig. 1c, d). This finding aligns with the observed accumulation of silicon in A stragalus , which peaked when SiO 2 NPs were applied at a concentration of 400 mg/kg (Fig. 1e). Soil supplemented with SiO 2 NPs at varying concentrations did not significantly affect on plant height or root length. However, the application of 400 mg/kg SiO 2 NPs resulted in notable increases in the relative chlorophyll content (measured by SPAD) and overall plant biomass (Fig. S2a). Increasing the concentration above 400 mg/kg did not lead to further improvements in these growth parameters; in fact, at a concentration of 800 mg/kg, decreases in both belowground and aboveground biomass were observed. These findings suggest that the increase in growth attributed to SiO 2 NPs is dose-dependent. Conversely, the foliar application of SiO 2 NPs at various concentrations did not significantly affect the height, root length, fresh root weight, or dry root weight of A stragalus (Fig. S2b). Notably, when SiO 2 NPs were applied at a concentration of 400 mg/L, there were significant increases in the chlorophyll content and biomass of the aboveground parts. Given that the application of SiO 2 NPs can influence plant metabolism, we further investigated whether SiO 2 NPs modulate the bacterial community structure in the rhizosphere and roots by altering the composition of root exudates. 3.2. SiO 2 NPs increased the stability and resilience of the microbial communities According to the results obtained from 16S and ITS amplicon sequencing, a total of 2,108,882 high-quality bacterial sequences and 1,815,835 fungal sequences were generated from 15 samples. Following the removal of low-quality and plant-derived reads, the remaining sequences were clustered into 1,731 bacterial and 978 fungal ASVs at a sequence identity threshold of ≥ 97%. The application of SiO 2 NPs to the soil (Si_S) significantly increased the alpha diversity of rhizosphere bacteria, whereas foliar spraying of SiO 2 NPs (Si_L) resulted in a significant increase in the alpha diversity of fungi (Fig. 2a). Principal coordinate analysis (PCoA) revealed significant differences in the composition of the rhizosphere bacterial (R 2 = 0.20; p = 0.001) and fungal communities (R 2 = 0.84; p = 0.001) of A stragalus under SiO 2 NP application (Fig. 2b and 2c). Compared with the control group, the Si_S-treated group presented significant increases in the relative abundance of two bacterial phyla (Acidobacteriota and Crenarchaeota) and two fungal phyla (Mortierellomycota and Basidiomycota) but a decrease in the abundance of Ascomycota (Fig. 2d and Fig. S3). The Si_L treatment significantly decreased the relative abundance of Actinobacteriota and increased the abundance of Crenarchaeota (ANOVA; p < 0.05). At the genus level, the application of SiO 2 NPs resulted in a greater number of significant changes in bacterial relative abundance (59 genera) than in fungal relative abundance (25 genera) (Fig. S4-S7). Notably, the relative abundance of Fusarium , the average disease index, and the prevention effect were significantly correlated with these 59 bacterial genera (Fig. 2e). Furthermore, the relative abundance of Fusarium in the rhizosphere significantly decreased with soil supplementation and foliar application of SiO 2 NPs. In both the bacterial and fungal communities, beta diversity was significantly positively correlated with the average disease index and negatively correlated with alpha diversity (Fig. 2f). The alpha diversity of bacteria was significantly positively correlated with the silicon contents in both the roots and above-ground parts of A stragalus , whereas fungi were positively correlated with only the silicon content in the roots. These findings suggest that F. oxysporum infection contributes to an increase in beta diversity and a decrease in alpha diversity within the rhizosphere, leading to significant alterations in the microbial community between diseased and healthy plants. The introduction of SiO 2 NPs mitigated these changes, increasing the diversity of the rhizosphere microbial community and reducing the incidence of disease. Co-occurrence network analysis was employed to investigate the alterations in the rhizosphere soil microbial co-occurrence patterns induced by the application of SiO 2 NPs (Fig. 3a). Compared with the control, the introduction of SiO 2 NPs resulted in a decrease in the density of microbial networks and the average clustering coefficient, while simultaneously leading to increases in the average degree and the numbers of communities, nodes, and links (Fig. 3b). Additionally, in the bacterial network, parameters such as network diameter, average path length, modularity, and negative correlation decreased, whereas these parameters increased in the fungal network. In conclusion, the introduction of SiO 2 NPs increased the complexity and interconnections within the network, thereby contributing positively to the stability and resilience of the microbial communities against disturbances. 3.3. Impact of SiO 2 NPs on the functional traits and assembly mechanisms of microbial communities To assess whether treatment with SiO 2 NPs alters the functional profiles of bacterial communities, we utilized PICRUSt software to predict community functions. The findings revealed that the functional profiles of the rhizosphere bacterial community were distinct between the SiO 2 NP-treated groups and the control group (Fig. 3c). Notably, amino acid metabolism and the biosynthesis of other secondary metabolites were enriched in the soils supplemented with SiO 2 NPs. Further analysis of the mechanisms underlying microbial community assembly across different treatments revealed that stochastic processes, including drift and other factors, predominantly influenced bacterial community assembly in the control group. In contrast, there was a shift towards dispersal limitation in the SiO 2 NP-treated groups, with contributions of 50.59% for soil supplementation and 39.09% for foliar spraying of SiO 2 NPs (Fig. 3d). The neutral community model (NCM) effectively captured a substantial portion of the relationship between the occurrence frequency of ASVs and their relative abundance variations (Fig. 3d). Specifically, the model explained 62.0%, 66.4%, and 65.6% of the community variance for the control, Si_S, and Si_L groups, respectively. The Nm- value for bacteria was greater in the Si_S treatment (Nm=4230) than in the Si_L treatment (Nm=3732), with the m value estimated at 0.09 for Si_S and 0.08 for Si_L, indicating greater dispersal of bacterial species in the Si_S group. In contrast, fungal community assembly was influenced primarily by deterministic processes, particularly homogeneous selection, across all the experimental groups (control: 55.58%; Si_S: 99.34%; Si_L: 64.01%), which resulted in a poor fit with the NCM. In conclusion, the application of SiO₂ NPs did not significantly influence the primary ecological processes of fungal community assembly. However, it did lead to a shift in the bacterial community assembly process, changing it from homogeneous selection to dispersal limitation. These findings suggest that SiO₂ NP treatment applied selective pressure on the survival and reproduction of bacterial communities, which in turn facilitated shifts in community composition and species diversity. 3.4. Effects of reinoculation with pathogen-associated bacteria on the root rot and growth of Astragalus The nodes in the bacterial co-occurrence networks of the Si_S- and Si_L-treated samples were ranked based on betweenness centrality, and the top 30 core ASVs were visualized (Fig. S8). Eight core ASVs were successfully isolated from rhizosphere soil, with greater than 97% sequence similarity between the isolated strains and the ASVs (Fig. 4a, Fig. S9). Among the four core ASVs in the Si_S-treated co-occurrence network, the relative abundances of ASV_794 (98.79% similarity to Microbacterium oxydans SCK-308) and ASV_349 (100.00% similarity to Pseudomonas aeruginosa XS-134-7) significantly decreased in the silicon treatments (ANOVA; p < 0.05) (Fig. 4b). Further analysis of the genera to which these ASVs belong revealed that the relative abundances of Microbacterium and Pseudomonas also significantly decreased with silicon treatment (Fig. 4c). In the Si_L-treated co-occurrence network, among the four core ASVs, the relative abundance of ASV_1492 ( Streptomyces ) significantly increased in the silicon treatment group. However, at the genus level, the relative abundance of Streptomyces did not change significantly, whereas that of Pseudarthrobacter significantly decreased. Therefore, we selected ASV_794 ( M. oxydans ), ASV_349 ( Pseudomonas aeruginosa ), and ASV_15 ( Pseudarthrobacter psychrotolerans ), which presented significantly reduced relative abundances at the genus level, for the pot experiments. The results of the pot experiments revealed that P. aeruginosa and M. oxydans promoted the infection of Astragalus by pathogenic fungi (Fig. 4d). Thus, we tested whether these two strains are also pathogens of Astragalus and can infect plants. The results revealed that neither P. aeruginosa nor M. oxydans could independently infect Astragalus and induce root rot (Fig. S10). Therefore, these two bacteria may serve as helpers in fungal pathogen infection of plants. Further experiments show that M. oxydans SCK-308 and P. aeruginosa XS-134-7 promoted the growth of F. oxysporum and inhibited the growth of certain beneficial rhizosphere microorganisms, thereby facilitating the occurrence of the disease (Fig. S11). There were no significant differences in growth indicators of plants inoculated with these bacteria. Therefore, the application of SiO 2 NPs to the soil reduced the abundance of Microbacterium and Pseudomonas in the rhizosphere, thereby reducing their incidence. 3.5. SiO 2 NP-induced resistance of Astragalus to F. oxysporum depends on salicylic acid Compared with foliar spraying, soil application of SiO 2 NPs had a better protective effect and significantly increased the diversity of rhizosphere bacteria. Therefore, we conducted an untargeted metabolomic analysis of the root exudates of soil SiO 2 NP-treated Astragalus . PCA and PLS-DA analysis revealed that the application of SiO 2 NPs significantly altered the root exudates of Astragalus (Fig. 5a and b). A total of 2043 metabolites were identified and initially classified into three groups: 209 host (plant) metabolites, 423 microbiota metabolites, and 2443 others (drug food and environment) (Fig. 5c). Based on the criteria fold change > 2 and p < 0.05, a total of 196 differentially abundant metabolites were identified, of which 96 increased and 100 decreased (Fig. 5d). The significantly upregulated metabolites included mainly lipids and lipid-like molecules, phenylpropanoids and polyketides, and organic acids and their derivatives (Fig. S12). The significantly downregulated metabolites included mainly lipids and lipid-like molecules, benzenoids, and organoheterocyclic compounds. The KEGG database was used to annotate the metabolic pathways of the differentially abundant metabolites in the Si_S-treated group, and the top 7 metabolic pathways were analysed (Fig. 5e). These pathways included five upregulated differentially abundant metabolites, and one was downregulated in the Si_S-treated group (Fig. 5f). Notably, the plant hormone signal transduction pathway was significantly enriched in the Si_S-treated plants and included three differentially abundant metabolites (auxin, brassinosteroid, and salicylic acid). Therefore, we analysed the pathways and metabolites related to the plant hormone signal transduction (Fig. 5g). These results indicate that tryptophan metabolism and the biosynthesis of phenylalanine, tyrosine, and tryptophan provide precursors for the synthesis of auxin. The biosynthesis of phenylalanine, tyrosine, tryptophan and phenylalanine provides precursor substances for the synthesis of salicylic acid (SA). Therefore, the untargeted metabolomic analysis of the root exudates of Astragalus revealed a significant impact of SiO 2 NPs on the modulation of metabolic profiles under F. oxysporum inoculation, with the involvement of SA playing a crucial role in enhancing root rot resistance. 3.6. Associations between the metabolome and microbiome Because of the interaction between root exudates and microbial communities, we assessed the relationships between the metabolites of Si_S-treated plants and the relative abundances of genera (all bacterial genera and Fusarium ) using Spearman's correlation coefficient. There were 572, 1055, 3844, 5451, and 12,680 closely associated bacteria‒metabolite pairs at the phylum, class, order, family, and genus levels, respectively ( p < 0.05) (Supplementary material 2). We further visualized the correlations between the four primary differentially abundant metabolites (salicylic acid, indole-3-acetic acid, brassinolide, and palmitic acid) and the differential bacterial genera (Fig. 6a). Notably, the four differentially abundant metabolites were significantly negatively correlated with Microbacterium and Pseudomonas , whose relative abundances were significantly reduced in the Si_S-treated group. Through the combined analysis of microbiota and metabolites, we identified two metabolic pathways related to the plant hormone signal transduction pathway: tryptophan metabolism and the biosynthesis of siderophore group nonribosomal peptide pathways. Three metabolites (L-tryptophan, indole-3-acetamide, and indole) are involved in this pathway for tryptophan metabolism. They are all precursors for the synthesis of indole-3-acetic acid and are engaged in two metabolic reactions in this pathway (R00679 and R00673) (Fig. 6b, c). For the biosynthesis of the siderophore group nonribosomal peptide pathway, the differentially abundant metabolite salicylic acid was involved in the R06602 metabolic reaction of this pathway (Fig. 6d). For each metabolic reaction, the biological and statistical correlations between the microbiota and metabolites were explored using the Bio-Sankey and STA-Sankey networks. In the Bio-Sankey and STA-Sankey networks, Pseudomonas and Microbacterium were identified as major genera closely associated with the metabolic reactions R00679 and R00673 and positively correlated with L-tryptophan, indole-3-acetamide, and indole (Fig. 6 and S13). In addition, Pseudomonas was identified as a significant genus closely associated with the metabolic reaction R06602 and negatively correlated with salicylic acid. The relative abundance of Pseudomonas was significantly reduced in the Si_S-treated group, and its reinoculation significantly increased the Astragalus root rot disease index. These findings indicate that the abundance of Pseudomonas is closely related to the occurrence of Astragalus root rot. In the fatty acid elongation pathway, palmitic acid and Fusarium are recognized as metabolites, and a fungal taxon involved in the R01274 metabolic reaction of this pathway has a negative correlation (Fig. 6e). Palmitic acid generates very long-chain fatty acids (VLCFAs) via the fatty acid synthesis pathway, which furthers the synthesis of the cuticle. The cuticle not only affects the ability of fungal hyphae to attach to the cuticular surface but also plays an essential role in systemic acquired resistance (SAR) by activating defence responses, such as regulating the active transport of salicylic acid across the apoplast, which is necessary for the induction of SAR. A comprehensive analysis of the microbiota and metabolites revealed that the addition of SiO 2 NPs to the soil significantly reduced the relative abundances of Pseudomonas and Fusarium and increased the synthesis of salicylic acid. This activated SAR and ultimately improved root rot resistance. In addition, soil-applied SiO 2 NPs promoted cuticle synthesis, forming a physical barrier against pathogen invasion. 3.7. Metabolites alleviate root rot by inhibiting the growth of pathogenic fungi and their "helpers" Salicylic acid, indole-3-acetic acid, brassinolide, and palmitic acid were significantly negatively correlated with the resistance of pathogenic fungi, and we further investigated the effects of these four differentially abundant metabolites on the resistance of Astragalus to F. oxysporum . The results demonstrated that when the concentrations of the four differentially abundant metabolites exceeded 100 μM, they significantly inhibited the growth of P. aeruginosa XS-134-7 (Fig. 6f). When palmitic acid was added at concentrations greater than 25 μM, it inhibited the growth of M. oxydans SCK-308. Salicylic acid, at a concentration of 200 μM, significantly inhibited the growth of M. oxydans SCK-308, whereas indole-3-acetic acid and brassinolide had no effect on the growth of M. oxydans SCK-308. The minimum concentrations of the metabolites required to inhibit bacterial growth were selected for the infection test. Compared with the control group ( P. aeruginosa XS-134-7 + F. oxysporum ), the addition of the four metabolites (100 μM) significantly reduced the disease index of Astragalus root rot (Fig. 6g). Similarly, compared with the control group ( M. oxydans SCK-308 + F. oxysporum ), the addition of the differentially abundant metabolites palmitic acid (25 μM) and salicylic acid (200 μM) also reduced the disease index of Astragalus root rot. Furthermore, we evaluated the impact of four differentially abundant metabolites on the growth and spore formation of F. oxysporum in both solid and liquid media. The results indicated that when the concentrations of these metabolites exceeded 100 μM, they significantly affected the growth of F. oxysporum (Fig. S14). However, brassinolide did not influence the spore germination of F. oxysporum . These findings suggest that the differentially abundant metabolites alleviate Astragalus root rot by inhibiting the growth of pathogenic fungal "helpers". 4. Discussion 4.1 SiO 2 NPs reduced the disease index of root rot Si-induced improvement in disease resistance is partly manifested by the reinforcement of cell walls, which prevents pathogen ingress. Moreover, the activation of defence-related enzymes, stimulation of antimicrobial compound production, and regulation of the complex network of signalling pathways are thought to be the key mechanisms by which Si-induced chemical defences against fungal pathogens transcend physical barriers [45] (Fig. 7). When investigating the effects of exogenous Si on plant diseases, many studies ignore the direct antifungal properties of Si, potentially overestimating the beneficial effect of Si on disease control [46]. Therefore, to better assess the role of Si in plant disease control and its potential mechanisms, selecting a Si concentration and an application method that does not directly affect pathogen growth but can activate plant defence responses would be more meaningful. Therefore, to examine the effects of SiO 2 NP application on F. oxysporum root rot in Astragalus , the effects of different concentrations of SiO 2 NPs on the disease index under different application methods were tested in pot experiments. In this study, different concentrations of SiO 2 NPs did not significantly promote or inhibit the growth of F. oxysporum (Fig. 1b). Both soil application and foliar spraying of SiO 2 NPs improved resistance to root rot. Nevertheless, soil applications have a lower disease index and better prevention ability than foliar spraying does. Furthermore, compared with foliar spraying of SiO₂ NPs, root treatment confers greater resistance to rice blast through the systemic acquired resistance (SAR) response [10]. Therefore, root treatment is a safer and more effective method for applying SiO₂ NPs. Compared with SiO₂ NP treatment, carbendazim treatment significantly inhibited the progression of root rot, achieving a disease index control rate of 71.54% (Fig. 1a), demonstrating superior disease prevention efficacy (57.70%). However, persistent carbendazim residues in soil pose bioaccumulation risks and potential groundwater contamination [47]. In this study, the application of SiO 2 NP did not have a negative impact on plant growth, and Kumari [48] also found that SiO 2 NP could improve soil fertility and Z. may s growth, suggesting the low toxicity and potential benefits for sustainable agriculture. This is similar to what others have discovered that SiO 2 NP have minimal negative impacts on environmental health and crop food safety due to its chemical inertness [49]. Therefore, although SiO₂ NPs exhibit marginally lower immediate efficacy than chemical pesticides do, their multidimensional advantages in ensuring medicinal material safety and maintaining soil health render them more suitable for the integrated management of medicinal plant diseases. However, SiO 2 NP can also have negative consequences depending on the conditions such as soil physical and chemical properties, nanoparticle concentration in the soil, therefore the environmental impact of SiO 2 NP needs to be further explored before its application in the field. Plants take up silicon from the soil and subsequently transport it from roots to shoots either passively (transpirational stream) or actively (specific transporter proteins) [46, 50]. Therefore, compared with the control, the soil-applicant SiO 2 NPs also significantly increased the Si content of the shoot (Fig. S2b). In addition, studies have shown that foliar application of SiO 2 NPs significantly increased the Si contents in leaves and stems, indicating the uptake of SiO 2 NPs by plant leaves and their translocation to stems, but no translocation from stems to roots was observed [51]. Interestingly, in our study, foliar application of SiO 2 NPs also significantly increased the content of Si in roots, but the content was lower than that in the soil treatment at the same concentration. The Si content in roots was significantly positively correlated with the prevention effect and negatively correlated with the disease index. We used SiO₂ NPs with a size of 17 nm, which is consistent with previous studies on the transmission threshold of approximately 15–40 nm into the central column or xylem of the root system [52]. Only a small amount of foliar-applied SiO₂ NPs was transported to the roots through the stomata, resulting in a relatively weak impact on the rhizosphere microbiome. In contrast, Astragalus root rot, caused by Fusarium , is a soil-borne disease. The application of SiO₂ NPs to the soil, plants accumulate more silicon through their root absorbtion, which has a greater impact on plant metabolism. Moreover, SiO₂ NPs applied to the soil may affect the growth of other microorganisms in the soil, resulting in a greater change in microbial community structure. All these reasons could lead to more effective disease control. Although foliar spraying of SiO 2 NPs is an effective method for controlling soil-borne diseases, soil application of SiO 2 NPs is typically more effective in improving plant disease resistance because of the transport and accumulation mechanism of silicon in plants. 4.2 SiO 2 NPs increased the diversity of rhizosphere microorganisms and reduced the abundance of pathogen-associated microorganisms The diversity and composition of the rhizosphere microbial community significantly influence soil health and are significant drivers of plant defence against soil-borne diseases [53, 54]. Previous study showed that the resistance to pathogenic colonization greatly increased with community diversity [55]. Our results also showed that SiO₂ NPs application significantly increased the Shannon α-diversity of bacterial community in the rhizosphere of Astragalus . Therefore, there is more overlap in nutrient requirements between rhizosphere microbial community and pathogen, and these rhizosphere microorganisms inhibit the growth of pathogen through nutrient competition. Active participation of Si in plant-microbe interactions has been demonstrated in several studies [56, 57]. Silicon can improve plant disease resistance through three principal mechanisms. The first silicon can directly inhibit the growth of pathogens to protect plants [58, 59]. Second, Si can modify soil microbial habitats by altering soil physicochemical properties and enzymes. For example, Si application can correct the decreases in soil pH and NH 4 + -N content caused by ginseng black spot disease, which affects soil microbial structure [23]. Third, Si can reshape the structure of the rhizosphere microbial community by altering root exudates, e.g., increasing the relative abundance of beneficial microbial taxa or reducing the relative abundance of microbial taxa of potential plant pathogens, which exerts the positive feedback effects on plant growth and resistance [60-62]. The SiO 2 NPs used in this study had no direct toxicity to F. oxysporum (Fig. 1b). Therefore, the improvement of its resistance to root rot was due to the change of the rhizosphere microbial community structure. The application of SiO 2 NPs significantly increased the α diversity of rhizosphere bacteria and fungi, and the index was positively correlated with the Si content of Astragalus roots (Fig. 2a, f). In addition, the application of SiO 2 NPs also increased the relative abundances of several microorganisms that promote plant growth, such as Lysobacter [63] and Sphingobium [64], which are highly important for plant growth and soil health. In addition to PGPR, some bacteria can promote the growth of pathogens in the plant rhizosphere, acting as helper of soil-borne pathogens during rhizosphere colonization. In this study, the relative abundances of microbial taxa associated with plant pathogens, such as Pseudomonas and Microbacterium [65], were significantly reduced (Fig. 4b). Crucially, both genera presented significant negative correlations with protection rates, whereas Microbacterium presented a markedly positive correlation with disease indices (Fig. 2e). As evidenced by multiple reports, P . aeruginosa has been identified as a natural soil inhabitant and a potential plant pathogen [66, 67]. Therefore, the coinoculation of P. aeruginosa and F. oxysporum may cause coinfection of Astragalus , thereby increasing the severity of root rot. In addition, a study showed that Microbacterium paraoxydans acts as a bacterial helpers for Ralstonia solanacearum , significantly enhancing its colonization in the tomato rhizosphere and increasing the severity of bacterial wilt disease [21]. Subsequent pot experiments revealed that coinoculation of F . oxysporum with either Pseudomonas or Microbacterium consistently increased root rot disease severity, suggesting a synergistic pathogenic interaction between the pathogen and its microbial allies during host colonization. Exogenous application of differentially abundant metabolites exhibiting antagonistic effects against the phytopathogen F . oxysporum and its "helpers" ( Pseudomonas and Microbacterium ) significantly ameliorated root rot severity under coinfection conditions (Fig. 6g). Numerous studies have shown that the increase in the antagonistic bacteria was found to enhance plant resistance to pathogen [68]. However, our results showed that M. oxydans SCK-308 and P. aeruginosa XS-134-7 promoted the growth of F. oxysporum and inhibited the growth of certain beneficial rhizosphere microorganisms (Fig. S11). This indicates that the propagation of pathogenic allies reduces the density of antagonistic bacteria and thus alleviates the inhibition of pathogen. Furthermore, interaction between pathogenic allies and F. oxysporum could have increased the niche space in root surface, which is conducive to the adsorption and infection of pathogenic fungi. These results elucidate a SiO 2 NP-mediated suppression strategy, in which targeted inhibition of pathogen-associated allies enhances disease control efficacy through disruption of cross-kingdom pathogenic synergies. Changes in the relative abundance and composition of microorganisms within a community can lead to alterations in their overall functional capacities. In this study, the addition of SiO 2 NPs increased amino acid metabolism in the soil. Research has shown that the resident microbial keystone taxa from disease-suppressive rice panicles subvert pathogen infection by manipulating BCAA flux in the host panicle [69]. Furthermore, a study indicated that the rhizosphere bacteria JR48 can produce phenylpyruvate to increase the accumulation of phenylalanine in plants, thereby promoting lignification and disease resistance dependent on phenylalanine metabolism [70]. Based on these findings, we speculate that the observed increase in amino acid metabolism in this study may be associated with increased disease resistance. In addition, the introduction of SiO 2 NPs increased the complexity and relevance of the microbial community network, increasing its stability and resistance (Fig. 3a, b). The application of SiO 2 NPs enhanced stochastic processes (dispersal limitation) during bacterial community construction and deterministic processes in fungal communities (Fig. 3d). Dispersal limitation usually refers to a low diffusion rate coupled with drift or weak selection, which may increase community variation or turnover [71]. These findings highlight the potential of SiO₂ NPs to influence microbial community structures, particularly bacteria, by altering ecological processes such as dispersal and selection, and suggest that such nanoparticles could influence microbial diversity in environments exposed to them. These findings also confirmed that the application of SiO 2 NPs changed the composition of the rhizosphere microbial community, thereby improving root rot resistance. 4.3 SiO 2 NP‑induced Astragalus resistance to Fusarium depends on salicylic acid Many previous studies on the impact of SiO 2 NPs on plants focused on the plant itself and did not consider its effects on root exudates. Plant roots absorb mineral nutrients and release organic exudates, such as fatty acids [72], plant hormones [73], and antimicrobial compounds [74]. These substances not only affect the physicochemical properties of the soil but also influence plant‒microbe interactions and help to build rhizosphere microbial communities. Therefore, changes in the level or composition of root exudates in soil–root–microbe interactions are essential for plants to protect themselves from soil-borne diseases. The application of SiO 2 NPs in this study significantly altered the root exudates of Astragalus , increasing the accumulation of lipids, lipid-like molecules, organic acids and their derivatives. This change increases bioavailable carbon storage in the soil, thereby improving the quality of the soil and affecting plant health. In addition, research has shown that organic acids can increase the anti-infection ability of plant cell walls, and certain organic acid derivatives have a direct fungicidal effects as part of the chemical strategy of plant defence against pathogens [75]. Notably, the application of SiO 2 NPs increased the accumulation of phenylpropanoids and polyketide substances in our research (Fig. S15). Secondary metabolites derived through the phenylpropanoid pathway, such as phenols and flavonoids, are well-known for their defensive roles against fungal pathogens [76]. For example, the silica-induced increased accumulation of phenolics, flavonoids, lignans, and dopamine in the scale and vascular tissues of roots may contribute to the enhancement of banana resistance to Fusarium oxysporum f. sp. cubense in banana [24]. Our results showed that applying SiO 2 NPs significantly increased the accumulation of the antimicrobial compounds naringenin [77], sinapyl alcohol, and gallocatechin gallate [78]. Among these, sinapyl alcohol is a critical monomer in the lignin biosynthetic pathway, and plays a role in cell wall lignification [79]. Lignification of the cell wall increases its mechanical strength and provides plants with a physical barrier against pathogen invasion. Thus, the accumulation of these substances helps improve the resistance of Astragalus root rot. Phytohormones are essential for plants to cope with biotic and abiotic stresses. Phytohormones can effectively regulate plant defence responses through complex signal networks and interactions [45]. Therefore, the use of silicon as a regulator by altering phytohormone homeostasis as well as a network of defence signalling components could be potential mechanisms for Si-triggered resistance responses. For example, Si-regulated SA synthesis and metabolism mediate resistance to peanut bacterial wilt [17]. In this study, salicylic acid, auxin, and brassinolide were significantly enriched in plant hormone signal transduction pathways as differential substances (Fig. 5). SA is a critical phytohormone that regulates numerous aspects of plant development and the activation of defenses against biotic stress. SA undergoes glucosylation, methylation-demethylation, hydroxylation, or sulfonation to change its active/inactive forms. The activated SA subsequently induces SAR, reinforces cell walls, and activates pathogenesis-related (PR) proteins to resist pathogen [80]. In this study, the application of SiO₂ NPs significantly increased the salicylic acid content, suggesting that the enhanced resilience of Astragalus to Fusarium root rot may be mediated by salicylic acid-induced SAR. Plants synthesize salicylic acid through the phenylalanine and isochorismate pathways [81]. These two metabolic pathways were also annotated in the joint analysis of Astragalus rhizosphere microorganisms and root exudates. Disease-promoting Pseudomonas was negatively correlated with the isochorismate pathway. Indole-3-acetic acid (IAA) is an important plant hormone that regulates various biological processes, known as promoting plant growth. Recent studies indicated that IAA could also enhance the expression of genes related pathogenesis, thereby increasing disease resistance of plants [82]. Brassinolides and palmitic acid could inhibit the growth of some bacteria including the fungal pathogenic "helpers". This leads to the disorder of the microbial community in the rhizosphere of healthy plants, resulting in a decrease in their disease resistance. Additionally, palmitic acid has been identified as a differentially abundant metabolite involved in the cutin, suberine, and wax biosynthesis and fatty acid elongation pathways, as well as the fatty acid elongation pathway. The metabolism of cutin and waxes can influence plant disease resistance by modulating the permeability of the cuticle layer [83]. These findings indicated that SiO₂ NPs altered the community composition of Astragalus and its root exudates, triggering an SA-dependent SAR response through interaction, rather than directly inhibiting Fusarium growth to prevent root rot. Additionally, SA has been reported to induce the expression of antioxidant enzymes and increase the production of nonenzymatic antioxidants, thereby gassisting in the detoxification of ROS in plants [49, 84].This function has also been confirmed in the prevention of Fusarium wilt in cucumber by Si [85]. In this study, SiO₂ NPs significantly increased the activities of APX, CAT, and POD, while decreasing the MDA content at a concentration of 400 mg/kg. Therefore, exogenous silicon reduces the MDA content and promotes ROS scavenging by increasing antioxidant enzyme activity, thereby reducing cellular damage and improving the resistance of infected plants [86]. Overall, our findings suggest that the application of SiO 2 NPs increased the activity of defence-related enzymes and changed the diversity, composition, and functional spectrum of the rhizosphere microbial communities of Astragalus . The application of SiO 2 NPs induced the accumulation of defensive compounds and salicylic acid, thereby conferring resistance to root rot in Astragalus . Although the controlled pot experiments in this study allowed for precise measurement of the effects of SiO 2 NPs, they inevitably simplified the natural complexity of the rhizosphere environment. Field conditions, however, encompass additional variables such as microbial competition, soil heterogeneity, and climatic fluctuations, all of which could alter the observed plant‒microbe interaction patterns. Therefore, field experiments should be conducted under various seasonal conditions to assess the effects of SiO 2 NPs on plant resistance to soil-borne pathogens in the future. Conclusion In conclusion, the application of exogenous SiO 2 NPs increased the resistance of Astragalus to root rot, confirming that root treatment is an effective and safe application method. High-throughput sequencing and LC‒MS metabolomics techniques revealed the response of soil microbial communities and root exudates to SiO 2 NPs in Astragalus . SiO 2 NPs influence the structure of the rhizosphere microbial community by increasing its diversity and reducing the relative abundances of F. oxysporum and pathogenic allies ( Pseudomonas and Microbacterium ). Some plant root exudates alleviate Astragalus root rot by inhibiting the growth of pathogenic fungal helpers. The complex signalling network and interactions of phytohormones effectively regulate plant defence responses. In particular, SAR was activated through the accumulation of salicylic acid. In conclusion, this study demonstrates a promising approach for the use of SiO 2 NP-based plant elicitors for the effective management of Astragalus root rot. Abbreviations Si_S The soil-applied SiO 2 NPs treatment Si_L The foliage-spraying SiO 2 NPs treatment MDA Malondialdehyde SOD Superoxide dismutase APX Ascorbate peroxidase CAT Catalase POD Peroxidase SA Salicylic acid SAR Systemic acquired resistance VLCFAs Very long-chain fatty acids Declarations Acknowledgements We thank for the High-Performance Computing Center of Northwest A&F University for providing computing resources. Author contributions J.A. wrote and edited the text; L.X., H.D., Z.D., and L.J. conduct the experiments; G.W., and Z.L. made conception and design of the study; Z.L. reviewed and edited the final text. All authors read and approved the version of the manuscript. Funding This work was supported by the National Natural Science Foundation of China (42277317). National Key Research and Development Program of China (2021YFD1900702), and Special funds for the Major Science and Technology of Shaanxi Province (2020zdzx03–02-01). The funders played no role in the design of the study, analysis, and interpretation of data or in writing the manuscript. Competing interests The authors declare no conflict of interest. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Author details State Key Laboratory for Crop Stress Resistance and High-Efficiency Production, Shaanxi Key Laboratory of Agricultural and Environmental Microbiology, College of Life Science, Northwest A&F University, Yangling 712100, Shaanxi, People’s Republic of China. References Dutta P, Kumari A, Mahanta M, Upamanya Gunadhya K, Heisnam P et al. Nanotechnological approaches for management of soil-borne plant pathogens. 2023;14. https://doi.org/10.3389/fpls.2023.1136233. Jang Y, Yi H, Maharjan R, Jeong M, Yoon Y. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6006553","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":449837407,"identity":"c3260e50-7f4c-4e7f-8200-01558b5fe0ad","order_by":0,"name":"Jiamin Ai","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Jiamin","middleName":"","lastName":"Ai","suffix":""},{"id":449837408,"identity":"f8b3c712-acfe-4a7e-a086-941aa6cd12ce","order_by":1,"name":"Leilei Xu","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Leilei","middleName":"","lastName":"Xu","suffix":""},{"id":449837409,"identity":"aea59212-7143-4ed9-a7dd-8935eaf8db5a","order_by":2,"name":"Hao Ding","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Ding","suffix":""},{"id":449837410,"identity":"4cf92a51-664b-45b3-8585-f36ae7f507f0","order_by":3,"name":"Zijing Dang","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Zijing","middleName":"","lastName":"Dang","suffix":""},{"id":449837412,"identity":"dab7e69c-c89c-4b9f-a5d0-ee27865ed3ee","order_by":4,"name":"Liru Jian","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Liru","middleName":"","lastName":"Jian","suffix":""},{"id":449837414,"identity":"6c40eb94-f8cd-4e88-bd88-a8018899f129","order_by":5,"name":"Gehong Wei","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Gehong","middleName":"","lastName":"Wei","suffix":""},{"id":449837416,"identity":"d588a28f-2c4a-4a44-a240-652b808e2f28","order_by":6,"name":"Zhefei Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYBACPmYGNgYGGwYefhCvAkQcIKCFDawljYFHsgHIO0OUFgaIFgaDA0RrYWd/9uBDgo2M8e0eww8H2xjk+G4kMH4uwO+wdMMZCWk8ZnfOGEsAtRhL3khglp6BX8sxad4fh3nMbuRukP7YxpC44UYCGzMPXi2MbdJ/Ev7zGM/I3fwDaEs9EVqY2aQZEg7wGEjkbgM5LMGAsBY2NsmehGQeiRv53ywOnJMwnHnmYbM0Pi38/MefSfxIsLPnn5GWfONAmY083/Hkg5/xaUEHEkDM2ECChlEwCkbBKBgF2AAApQhFzKIvja4AAAAASUVORK5CYII=","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":true,"prefix":"","firstName":"Zhefei","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-02-11 10:53:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6006553/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6006553/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40168-025-02183-x","type":"published","date":"2025-10-21T16:16:28+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81719751,"identity":"99143c06-b1c7-4f89-a2c5-28674dff392b","added_by":"auto","created_at":"2025-04-30 15:58:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2996785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e NPs enhance \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAstragalus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e resistance to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFusarium\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e root rot.\u003c/strong\u003e (a) Disease indices and prevention effects of \u003cem\u003eAstragalus\u003c/em\u003e under distinct treatments at 21 days after inoculation. Si_S: Soil application of SiO\u003csub\u003e2\u003c/sub\u003e NPs; Si_L: Foliar spray of SiO\u003csub\u003e2\u003c/sub\u003e NPs. (b) Effects of SiO\u003csub\u003e2\u003c/sub\u003e NPs on growth and sporulation of \u003cem\u003eF\u003c/em\u003e.\u003cem\u003e oxysporium\u003c/em\u003e. (c) Effects of foliar spraying with different concentrations of SiO\u003csub\u003e2\u003c/sub\u003e NPs on enzyme activities of \u003cem\u003eAstragalus\u003c/em\u003e. (d) Effects of soil application of various concentrations of SiO\u003csub\u003e2\u003c/sub\u003e NPs on the enzyme activities of \u003cem\u003eAstragalus\u003c/em\u003e. (e) Si contents of above- and belowground \u003cem\u003eAstragalus\u003c/em\u003e under various treatments. Lowercase letters above the bars indicate significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) between the different treatments (one-way ANOVA).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6006553/v1/949db8f2128a2286fcd36dc7.png"},{"id":81719752,"identity":"6bcc517a-fcc7-4a40-9c8c-e1714c12a8a8","added_by":"auto","created_at":"2025-04-30 15:58:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5829273,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of SiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e NPs on the microbial community composition in the rhizosphere of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAstragalus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (a) Changes in the α diversity indices of rhizosphere bacteria and fungi under different SiO\u003csub\u003e2\u003c/sub\u003e NP application methods. Si_S: Soil application of SiO\u003csub\u003e2\u003c/sub\u003e NPs; Si_L: Foliar spray of SiO\u003csub\u003e2\u003c/sub\u003e NPs. (b-c) PCoA of the bacterial and fungal microbial communities. (d) Comparison of the major bacterial and fungal compositions at the phylum level. (e) Correlation analysis of bacterial differential genera with the relative abundance of \u003cem\u003eFusarium\u003c/em\u003e, the disease index, and the prevention effect. (f) RDA of the bacterial and fungal microbial communities.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6006553/v1/09d3344f034f2e2dd93b6ab8.png"},{"id":81720065,"identity":"66ad2509-25e5-42ec-8112-fd02a3a21215","added_by":"auto","created_at":"2025-04-30 16:06:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7072550,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of SiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e NPs on the functional and assembly mechanisms of the microbial community.\u003c/strong\u003e (a) Co-occurrence network analysis. Si_S: Soil application of SiO\u003csub\u003e2\u003c/sub\u003e NPs; Si_L: Foliar spray of SiO\u003csub\u003e2\u003c/sub\u003e NPs. (b) Topological parameters of co-occurrence networks with different samples. A heatmap was generated after standardizing the prediction results from PICRUSt. (c) Responses of microbial community functions to different SiO\u003csub\u003e2\u003c/sub\u003e NP application methods. (d) Effects of SiO\u003csub\u003e2\u003c/sub\u003e NPs on the assembly mechanisms of microbial communities.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6006553/v1/41d7985041a6598ff6368846.png"},{"id":81718739,"identity":"6e41485f-6c2d-4f2e-8f1a-e7f350052bf1","added_by":"auto","created_at":"2025-04-30 15:50:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4259712,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of core microorganisms on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAstragalus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e root rot. \u003c/strong\u003e(a) Potting experiment process for core microbial reinoculation. (b) Relative abundances of core ASVs in different treatments and matching to isolates. Si_S: Soil application of SiO\u003csub\u003e2\u003c/sub\u003e NPs; Si_L: Foliar spray of SiO\u003csub\u003e2\u003c/sub\u003e NPs. (c) Relative abundances of core ASVs at the genus level. (d) Influence of core microorganisms on the disease index and plant phenotype.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6006553/v1/5000b36f8e448e021a8d9a73.png"},{"id":81718742,"identity":"4b099509-216e-4ce9-967d-2df76a08b4f9","added_by":"auto","created_at":"2025-04-30 15:50:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4526875,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of SiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e NPs on root exudates and KEGG enrichment pathway analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAstragalus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(a-b) PCA and partial least-squares discriminant analysis of root exudates. Si_S: Soil application of SiO2 NPs. (c) Analysis of possible sources of metabolites. (d) Volcanic plot of differentially abundant metabolites in control and SiO\u003csub\u003e2\u003c/sub\u003e NP-treated plants. (e) KEGG enrichment pathway analysis. (f) Relative abundance of differentially abundant metabolites in different treatment groups. (g) Analysis of the plant hormone signal transduction pathway and its related metabolic pathways.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6006553/v1/77daa73b33d2f8e84fbbd947.png"},{"id":81720066,"identity":"9f317530-3232-448e-8f2a-51867954b8b6","added_by":"auto","created_at":"2025-04-30 16:06:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6473352,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssociations between the metabolome and microbiome.\u003c/strong\u003e (a) Mantel test showing the significant relationships (i.e., Mantel’s p based on 999 permutations, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05) of differential genera with differentially abundant metabolites. Mantel's r statistic is represented by line width, and the colour of the line indicates positive (red) and negative (blue) correlations. (b-c) BIO‐Sankey network for R00679 and R00673 metabolic reactions in tryptophan metabolism. (d) The correlation between salicylic acid and the growth of Pseudomonas in the BIO‐Sankey Network. (e) The correlation between palmitic acid and the growth of Fusarium in the BIO‐Sankey Network. (f) Effects of differential metabolites on the growth of fungal pathogenic allies. (g) Exogenous supplementation with differential metabolites alleviated root rot in plants during coinfection with two bacteria and \u003cem\u003eF. oxysporum\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6006553/v1/e40851251bab43d64c38baf8.png"},{"id":94490473,"identity":"6f559c4a-3fc7-403e-ae40-949926d870f0","added_by":"auto","created_at":"2025-10-27 17:10:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":32945842,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6006553/v1/9d963458-2c96-43cb-97bf-6824eb59407e.pdf"},{"id":81720859,"identity":"be9c01dd-79e9-4fce-86b0-3119de2f3abd","added_by":"auto","created_at":"2025-04-30 16:14:28","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":619997,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6006553/v1/36d8b31337858bf2383842f8.xlsx"},{"id":81718765,"identity":"bcd71c49-1b96-4f20-a721-85b13bbdeb51","added_by":"auto","created_at":"2025-04-30 15:50:29","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16190852,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterialrevised.docx","url":"https://assets-eu.researchsquare.com/files/rs-6006553/v1/0e3efd3fdda60b6a37ab61c1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Silica Nanoparticles Suppress Fungal Pathogenic Allies to Alleviate Astragalus Root Rot","fulltext":[{"header":"1. Background","content":"\u003cp\u003eSoil-borne fungal pathogens pose substantial challenges to global agriculture, adversely affecting crop yield and sustainability, with potential yield losses of up to 75% for major crops [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. \u003cem\u003eFusarium oxysporum\u003c/em\u003e is a prevalent soil-borne pathogen that can infect more than 150 different crops, leading to severe \u003cem\u003eFusarium\u003c/em\u003e root rot in species such as soybean [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], maize [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and \u003cem\u003eAstragalus\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The management of \u003cem\u003eFusarium\u003c/em\u003e root rot is particularly difficult due to persistence of pathogenic agents in the soil, substantial genetic diversity, and broad host range [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Historically, chemical methods have been employed to mitigate outbreaks of soil-borne diseases; however, these approaches have increasingly been phased out due to serious environmental concerns [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Microbiota transplantation has emerged as a promising strategy for the treatment of soil-borne diseases. Nevertheless, the efficacy of this process is contingent upon various factors, including the composition of the microbial community, the method of transplantation, the soil environment, and the physiological state of the plant host [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. For large-scale implementation in agricultural production, further advancements are necessary. In the pursuit of safer agrochemicals, silica nanoparticles (SiO\u003csub\u003e2\u003c/sub\u003e NPs) have recently been proposed as novel tools to mitigate pathogen damage, including that caused by \u003cem\u003eF. oxysporum\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In addition, several studies have shown that the application of SiO\u003csub\u003e2\u003c/sub\u003e NPs improves the resistance of various crops, including peanuts [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], rice [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and potato [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], to biotic stress. The properties and chemical inertness of silicon contribute to its potential applicability in agricultural practices, as it is expected to have minimal negative impacts on environmental health and crop food safety [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In addition, SiO\u003csub\u003e2\u003c/sub\u003e NPs have emerged as an alternative and complementary approach to conventional methods of chemical control, biological control, and cultural practices, owing to their advantages of better efficacy, reduced input, and lower ecotoxicity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt was initially proposed that silicon deposition creates a physical barrier beneath the leaf cuticle to prevent pathogen invasion [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, subsequent research has indicated that the application of SiO\u003csub\u003e2\u003c/sub\u003e NPs inhibits the growth of pathogenic agents. For example, \u003cem\u003eRalstonia\u003c/em\u003e is prevalent in wilt-diseased soil, and the application of silicon has been found to reduce the incidence of bacterial wilt in peanuts by diminishing the relative abundance of \u003cem\u003eRalstonia\u003c/em\u003e in the rhizosphere [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Additionally, studies have shown that some SiO₂ NPs do not directly inhibit pathogens but instead enhance plant disease resistance by modulating the composition of the rhizosphere microbial community [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, whether these altered microbes confer disease resistance or promote pathogenesis has not been well validated. For example, SiO₂ NPs promote disease resistance by increasing the relative abundance of microbial taxa with plant-beneficial potential. The relationships among microorganisms include neutral, positive, and negative interactions, thus in addition to antagonist, there are also some bacteria that can promote the growth of pathogen. This is in line with the recent finding where a considerable proportion of root-associated microorganisms can promote pathogen growth and pathogenicity, underscoring that such facilitation may be a key determinant of successful pathogen infection [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. For example, \u003cem\u003eMicrobacterium paraoxydans\u003c/em\u003e and \u003cem\u003ePseudomonas syringae\u003c/em\u003e serve as bacterial helpers for \u003cem\u003eRalstonia solanacearum\u003c/em\u003e and \u003cem\u003eFusarium\u003c/em\u003e, respectively, exacerbating disease severity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, alterations in the levels or composition of root exudates can influence plant health by regulating pathogen populations and inducing plant resistance [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For example, Si-induced accumulation of defensive compounds such as phenols, flavonoids, lignin, and dopamine in the sclerenchyma and vascular tissues of roots may increase resistance to \u003cem\u003eF. oxysporum\u003c/em\u003e in banana [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. SiO\u003csub\u003e2\u003c/sub\u003e NPs can also promote resistance to pathogens in plants by mediating the production of endogenous phytohormones, including salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Research has shown that peanuts irrigated with SiO\u003csub\u003e2\u003c/sub\u003e NPs exhibit a significant increase in resistance to bacterial wilt, attributed to SA-dependent plant immune responses that trigger systemic acquired resistance (SAR) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The phenomenon of silicon-induced SAR in plants has also been corroborated in tomato, rice, and other crops [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Plant root exudates serve as vital carbon sources for soil microbes and play a crucial role in coordinating the composition of the rhizosphere microbiome. Conversely, microbes act as modifiers and limiters of root exudates, which are essential for soil health and significantly influence plant defence against soil-borne pathogens [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The interaction between root exudates and soil microbes enhances plant immunity and overall health during pathogen invasion. Nevertheless, reports addressing the effects of Si-mediated interactions between root exudates and soil microbial communities on plant disease resistance are scarce. This knowledge gap hinders our comprehensive understanding of how silicon modulates plant‒microbiome interactions to increase host plant defences.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAstragalus membranaceus\u003c/em\u003e Bge. var. \u003cem\u003emongholicus\u003c/em\u003e (Bge.) Hsiao (hereafter, \u003cem\u003eAstragalus\u003c/em\u003e) is an important perennial herbaceous leguminous plant recognized for its medicinal properties. This species is predominantly distributed in the provinces of Gansu, Shanxi, and Inner Mongolia, where it serves as a key economic crop [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. A major threat to the yield and quality of \u003cem\u003eAstragalus\u003c/em\u003e is the high incidence of root rot which is exacerbated by long-term monoculture. However, the extent to which SiO\u003csub\u003e2\u003c/sub\u003e NPs can increase the resistance of \u003cem\u003eAstragalus\u003c/em\u003e to root rot, and the underlying mechanisms remain poorly understood. In this study, we investigated the effects of the addition of SiO\u003csub\u003e2\u003c/sub\u003e NPs through soil application and foliar spraying on the resistance of \u003cem\u003eAstragalus\u003c/em\u003e to \u003cem\u003eF. oxysporum\u003c/em\u003e. Our research focused on: (1) the impact of different concentrations of SiO\u003csub\u003e2\u003c/sub\u003e NPs and application methods on the resistance of \u003cem\u003eAstragalus\u003c/em\u003e to root rot; (2) whether various methods of SiO\u003csub\u003e2\u003c/sub\u003e NPs application can alter the microbial community in the \u003cem\u003eAstragalus\u003c/em\u003e rhizosphere and enhance root rot resistance; and (3) the relationship between SiO₂NP-modified microbes and resistance to root rot disease. Our comprehensive approach aims to examine the potential of SiO\u003csub\u003e2\u003c/sub\u003e NPs as an innovative strategy for managing soil-borne diseases in \u003cem\u003eAstragalus\u003c/em\u003e, thereby promoting a transition to more sustainable agricultural practices.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e2.1. Effect of SiO\u003csub\u003e2\u003c/sub\u003e NPs on \u003cem\u003eFusarium\u0026nbsp;\u003c/em\u003e\u003cem\u003eoxysp\u003c/em\u003e\u003cem\u003eor\u003c/em\u003e\u003cem\u003eum\u0026nbsp;\u003c/em\u003egrowth\u003c/p\u003e\n\u003cp\u003eThe SiO\u003csub\u003e2\u003c/sub\u003e NPs (CAS: 7631-86-9, SKU: S490064, with a diameter of 17 nm) used in this study were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The morphology of the SiO\u003csub\u003e2\u003c/sub\u003e NPs was characterized using transmission electron microscopy (TEM, HT7800, Hitachi High-Tech, OR). The zeta potential of the SiO\u003csub\u003e2\u003c/sub\u003e NPs was measured using a Zetasizer Nano ZS instrument (Malvern Instruments Ltd., U.K.). The chemical composition and functional groups of SiO\u003csub\u003e2\u003c/sub\u003e NPs were determined by Thermo Scientific X-ray photoelectron spectroscopy (XPS) (ESCALAB 250Xi, Thermo, USA) and Fourier transform infrared spectroscopy (FTIR) (Frontier, PerkinElmer, USA). In addition, 1500 mg/L SiO\u003csub\u003e2\u003c/sub\u003e NPs suspension was used to observe its stability within 3 days of storage. The fungal pathogen \u003cem\u003eFusarium oxysporum\u003c/em\u003e used in this study was originally isolated from field-grown \u003cem\u003eAstragalus\u003c/em\u003e plants exhibiting root rot symptoms\u0026nbsp;[4].The toxicity of SiO\u003csub\u003e2\u003c/sub\u003e NPs to \u003cem\u003eF. oxysporum\u003c/em\u003e was tested in solid and liquid media. PDA medium containing 200, 400, and 800 mg/L SiO\u003csub\u003e2\u003c/sub\u003e NPs was prepared respectively, and fungal plugs (6-mm diameter) were transferred to the\u0026nbsp;PDA medium preparations\u0026nbsp;and placed in a biochemical incubator at 28 \u0026deg;C for 7 days, after which fungal\u0026nbsp;colony diameters were measured. Additionally, PDB medium (25 mL) containing the same concentrations of SiO\u003csub\u003e2\u003c/sub\u003e NPs was prepared, and 1\u0026nbsp;mL\u0026nbsp;of \u003cem\u003eF. oxysporum\u003c/em\u003e (~10\u003csup\u003e5\u003c/sup\u003e spores/mL) was added and cultured at 28 \u0026deg;C, and\u0026nbsp;220 rpm for 72 h. The culture medium was filtered with gauze, the spores in the\u0026nbsp;filtrate were counted using a hemocytometer, and the filter residue was dried and\u0026nbsp;weighed using analytical balance to obtain the weight of the\u0026nbsp;fungal hyphae. Each treatment had six replicates.\u003c/p\u003e\n\u003cp\u003e2.2. Plant growth conditions and plant treatments\u003c/p\u003e\n\u003cp\u003eThe soils used in this study were collected from medicinal plant fields in Tanchang County, Gansu Province, China (N34\u0026deg; 15.123\u0026rsquo;; E104\u0026deg; 09.727\u0026rsquo;). The soil physical and chemical properties were as follows: pH, 8.17; soil organic matter, 27.66 g/kg; total nitrogen, 1.75 g/kg; total phosphorus, 1.31 g/kg; total potassium concentration, 10.86 g/kg, and available K, 223.77 mg/kg.\u0026nbsp;SiO\u003csub\u003e2\u003c/sub\u003e NPs was added to the substrate (soil: vermiculite = 3: 1, v/v) to make its final concentrations 200, 400, and 800 mg/kg respectively. Then 500 g of substrate containing SiO\u003csub\u003e2\u003c/sub\u003e NPs was filled into planting bag. In addition, commercial carbendazim (50% active ingredient, Lanfeng Biol-Chemical Ltd. China) was added to the substrate to a final concentration of 100 mg/kg as a positive control. The \u003cem\u003eAstragalus\u003c/em\u003e seeds were surface sterilized with 75% ethanol for 30 s and 1% sodium hypochlorite solution for 1 min and then rinsed six\u0026nbsp;times with ddH\u003csub\u003e2\u003c/sub\u003eO water and soaked for 48 h. The surface-disinfected seeds were placed on sterile, moist filter paper to germinate. Ten germinated seedlings were sown in planting bags. After seven days, the seedlings in the planting bags were thinned to five uniform plants. The foliage-spraying SiO\u003csub\u003e2\u003c/sub\u003e NP treatment (Si_L)\u0026nbsp;was performed as follows: SiO\u003csub\u003e2\u003c/sub\u003e NP suspensions were prepared by sonicating for 15 min in DI water, at concentrations of 200, 400,\u0026nbsp;and\u0026nbsp;800 mg/L. After the true leaves of \u003cem\u003eAstragalus\u003c/em\u003e seedlings had fully expanded, spraying was performed every seven days for a total of 5 times [30]. The soil surface was covered with paper towels to prevent soil contamination when SiO\u003csub\u003e2\u003c/sub\u003e NPs\u0026nbsp;were sprayed. The other\u0026nbsp;groups were sprayed with the same amount of DI water.\u003c/p\u003e\n\u003cp\u003eThe pathogen was cultured in PDB medium for 4-5 days, and the culture suspension was filtered using four layers of sterile gauze to obtain a spore suspension. The spore suspension was then serially diluted to 10\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e3\u003c/sup\u003e, the spores were counted with hemacytometers (Thoma, XB-K-25, 25\u0026times;16), and the spore concentration was adjusted to approximately 10\u003csup\u003e5\u003c/sup\u003e spores/mL.\u0026nbsp;One week after the first foliar spraying of SiO\u003csub\u003e2\u003c/sub\u003e NPs, 2\u0026nbsp;mL (per plant) of a\u0026nbsp;spore suspension of \u003cem\u003eF. oxysporum\u003c/em\u003e (approximately 10\u003csup\u003e5\u003c/sup\u003e spores per millilitre) was added to the roots of \u003cem\u003eAstragalus\u003c/em\u003e plants. All the plants were grown in a greenhouse at 25 \u0026deg;C (16 h light/8 h darkness). The disease index and control effect were calculated after 21 days of inoculation with \u003cem\u003eF. oxysporum\u003c/em\u003e. The diseased plants were divided into 4 levels based on their disease severity. Level 0 indicated root health without disease lesions. For Level 1, 1\u0026ndash;2 black sunken lesions appeared in the roots. For Level 2, 3\u0026ndash;5 sunken black lesions appeared in the roots. For Level 3, 5\u0026ndash;8 sunken black lesions appeared in the roots. For Level 4, the root lesions had connected to form a network of vertical columns and rough epidermis. The disease index and control effect were calculated using the following formulas: disease index = [(\u0026sum;disease grades \u0026times; number of infected plants)/(total number of checked plants \u0026times; 4)] \u0026times;100%, prevention effect = (control - treatment disease index)/control disease index \u0026times; 100% [4].\u003c/p\u003e\n\u003cp\u003eAfter 21 days of pathogen inoculation, the indicators of plant growth status, i.e., plant height, root length, shoot fresh weight, shoot dry weight, root fresh weight, and root dry weight, were determined. The chlorophyll content in the leaves was determined with a hand-held chlorophyll meter. The malondialdehyde (MDA), superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and peroxidase (POD) contents were measured according to the instructions of relevant commercial assay kits from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). The amorphous Si contents of the aboveground parts and roots of \u003cem\u003eAstragalus\u003c/em\u003e were determined using a plant silicon content test kit (Shanghai Yaji Biotechnology Co., Ltd., China) according to the manufacturer\u0026apos;s protocol.\u003c/p\u003e\n\u003cp\u003e2.3. Rhizosphere soil collection and high-throughput sequencing\u003c/p\u003e\n\u003cp\u003eFollowing the pot experiments, the roots were carefully transferred into enzyme-free centrifuge tubes with 25 mL of 1 \u0026times; PBS\u0026nbsp;and then vortexed for 5 minutes to collect the rhizosphere soil. After the roots\u0026nbsp;were removed, the tubes were centrifuged at 8, 000 rpm for 7 minutes to pellet the soil particles. In total, 15 samples were obtained (3 treatments \u0026times; 5 repetitions). All the samples were stored at \u0026minus;80 \u0026deg;C before DNA extraction. Microbial DNA was extracted from 0.5 g of rhizosphere soil using a FastDNA SPIN Kit for Soil (MP Biomedicals, USA) according to the manufacturer\u0026apos;s protocol. The quality of the extracted DNA was assessed using 1% agarose gel electrophoresis, and its concentration and purity were determined with a NanoDrop 2000 spectrophotometer. The bacterial 16S rRNA gene (V4-V5 hypervariable regions) was amplified using the primers 515F (5\u0026apos;-GTGCCAGCMGCCGCGG-3\u0026apos;) and 907R (5\u0026apos;- CCGTCAATTCMTTTRAGTTT-3\u0026apos;). The fungal hypervariable internal transcribed spacer (ITS) region was amplified using the primers ITS1F (5\u0026apos;-CTTGGTCATTTAGAGGAAGTAA-3\u0026apos;) and ITS1R (5\u0026apos;-GCTGCGTTCTTCATCGATGC-3\u0026apos;). The library was subsequently\u0026nbsp;constructed using a TruSeq DNA PCR-Free Sample Preparation Kit, quantified through qPCR, and then sequenced on an Illumina MiSeq platform. QIIME 2.0 [31] was used to filter the original tags and species annotations, and sequence alignment of bacteria and fungi was performed using the Silva and Unite databases, respectively. All reads assigned to plant plastids (chloroplasts and mitochondria) were discarded from the dataset, and the remaining effective sequences were clustered into amplicon sequence variants (ASVs) at 97% similarity [32]. The relative abundance of the\u0026nbsp;ASVs was calculated in the R environment (version: V4.4.1, http://www.r-project.org/). The bacterial 16S rRNA and fungal ITS sequencing data for all the\u0026nbsp;samples in this study were submitted to the NCBI Sequence Read Archive database under BioProject accession numbers PRJNA1192509 and PRJNA1192506.\u0026nbsp;The 16S rRNA sequence of isolates were uploaded to NCBI GenBank\u0026nbsp;accession numbers PV505490-PV505497.\u003c/p\u003e\n\u003cp\u003e2.4. Isolation and reinoculation of core microorganisms\u003c/p\u003e\n\u003cp\u003eThe bacteria were isolated from the rhizosphere soil of \u003cem\u003eAstragalus\u003c/em\u003e using the method outlined by Oppenheimer-Shaanan [33] with minor modifications. Fresh rhizosphere soil was suspended in PBS and shaken at 180 rpm for 30 minutes. The dilutions were plated on LB medium solidified with 1.5% agar\u0026nbsp;and then incubated at 30 \u0026deg;C for 2‒7 days [34]. All the purified isolates were stored in 30% glycerol (v/v) at \u0026minus;80 \u0026deg;C. To obtain the key bacteria, the 16S rRNA gene of the isolates were amplified using universal primer set (27F and 1492R) [35]. The obtained sequences were aligned with the sequences of core ASV in the co-occurrence network. Phylogenetic trees were constructed with MEGA software (v5) using the neighbour-joining (NJ) method. Finally, eight core microorganisms in the co-occurrence network were successfully isolated from the rhizosphere soil. To confirm the presence of these bacterial isolates in the rhizosphere soil, similarity analysis was performed based on the 16S rRNA sequences of the bacterial isolates and the\u0026nbsp;gene sequences of ASVs (the\u0026nbsp;same genus as the\u0026nbsp;isolates). Isolates with the V4\u0026ndash;V5 region matching with more than 97% identity were considered the same strain [36]. The core microorganisms were cultured in LB medium until they reached an optical density of 0.8. A 2-mL bacterial suspension (per plant) was subsequently added to the roots of 7-day-old \u003cem\u003eAstragalus\u003c/em\u003e seedlings. One week after bacterial inoculation, a 2-mL (per plant) spore suspension of \u003cem\u003eF. oxysporum\u003c/em\u003e (approximately 10\u003csup\u003e5\u003c/sup\u003e spores per millilitre) was generated. The disease index and control effect were calculated after 21 days of inoculation with \u003cem\u003eF. oxysporum\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e2.5. Metabolite extraction and LC‒MS/MS analysis\u003c/p\u003e\n\u003cp\u003eThe root exudates of \u003cem\u003eAstragalus\u003c/em\u003e with and without SiO\u003csub\u003e2\u003c/sub\u003e NPs were collected for metabolomic analysis. The seedlings were removed from the soil, and the roots were washed with DI water and soaked for 2 h. Then, the seedlings were placed in sterile DI water and cultured in the dark for 24 h. The seedling exudates were collected and filtered with a 0.22-\u0026thinsp;\u0026mu;m filter and then prefrozen at \u0026minus;80 \u0026deg;C, after which they were removed for lyophilization [37]. The dried samples were redissolved in 100 \u0026micro;L of methanol:water (v/v = 1:1). The dissolved samples were vortexed and centrifuged at 15,000 \u0026times; g for 15 min (4 \u0026deg;C), and the resulting supernatants were used for LC‒MS/MS analysis\u0026nbsp;[38]. Each treatment was performed in five independent biological replicates.\u003c/p\u003e\n\u003cp\u003eA Vanquish UHPLC system (Thermo Fisher) and an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher) were used for untargeted \u003cem\u003eAstragalus\u003c/em\u003e root exudate metabolomics analysis. The chromatographic conditions were as follows Hypesil Gold column (C18), 40 \u0026deg;C column temperature, 0.2 mL/min flow rate.\u0026nbsp;In positive mode, mobile phase A was 0.1% formic acid, and mobile phase B was methanol; in negative mode, mobile phase A was 5 mM ammonium acetate (pH 9.0), and mobile phase B was methanol. The mass spectrometer scan range was 100\u0026ndash;1500 m/z, and the ESI source settings were as follows: spray voltage, 3.5 kV; capillary temperature, 320 \u0026deg;C; sheath gas flow rate, 35 psi; and auxiliary gas flow rate, 10 arb. Positive/negative polarity operation was employed. The raw data generated by UHPLC-MS/MS were processed in CD 3.1 search software and compared with the mzCloud (https://www.mzcloud.org/), mzVault, and Masslist databases. Finally, the identification and relative quantification results of the metabolites were obtained. Metabolites were annotated in the KEGG, HMDB, and LIPIDMAPS databases. Differentially abundant metabolites were screened according to a fold change \u0026gt; 2 and \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, and pathway enrichment analysis was performed using the KEGG database. MetOrigin (http://metorigin.met-bioinformatics.cn/) was used to discriminate the sources of the metabolites, and the rhizosphere microbiome and metabolome were subjected to integrative analysis [39].\u003c/p\u003e\n\u003cp\u003e2.6. Effects of metabolites on the resistance of \u003cem\u003eAstragalus\u003c/em\u003e to\u0026nbsp;root rot\u003c/p\u003e\n\u003cp\u003eBy comparing the relative abundance of root exudates from control and Si_S-treated plants, differentially abundant metabolites were identified.\u0026nbsp;Standard materials of palmitic acid (CAS: 57-10-3, molecular weight: 256.42, AR), salicylic acid (CAS: 69-72-7, molecular weight: 138.12, AR), indole-3-acetic acid (CAS: 87-51-4, molecular weight: 175.18, AR), and brassinolide (CAS: 72962-43-7, molecular weight: 480.69, AR) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Each of the four compounds was dissolved in methanol to prepare a stock solution with a concentration of 10 mM.\u003c/p\u003e\n\u003cp\u003eThe effects of these metabolites on the growth of \u003cem\u003eP. aeruginosa\u003c/em\u003e XS-134-7, \u003cem\u003eM. oxydans\u003c/em\u003e SCK-308, and \u003cem\u003eF. oxysporum\u003c/em\u003e were evaluated. The metabolite stock solutions were added to 24-well plates (Costar, Corning Incorporated, USA) containing LB medium or PDB medium to achieve final metabolite concentrations of 25, 50, 100, and 200 \u0026mu;M. The total volume in each well was 1 mL. Subsequently, 1 \u0026mu;L of bacterial suspension (OD\u003csub\u003e600\u003c/sub\u003e = 0.1) or fungal spore solution (approximately ~10\u003csup\u003e5\u003c/sup\u003e spores/mL) was transferred into the wells of a 24-well plate. The plates were incubated at 28 \u0026deg;C and shaken at 170 rpm for 24 hours. Bacterial growth was assessed by measuring the absorbance at OD\u003csub\u003e600\u003c/sub\u003e. The effects of metabolites on fungal spores were evaluated by counting spores with a haemocytometer. In addition, PDA medium was prepared for the four metabolites at final concentrations of 25, 50, 100, and 200 \u0026mu;M using the same method as described above. The 6-mm fungal plugs were subsequently transferred into PDA medium. The plates were then placed in a biochemical incubator at 28 \u0026deg;C for 7 days, after which the fungal colony diameter was measured. The control group (solvent control) consisted of medium supplemented with 200 \u0026mu;M methanol. Compared with no methanol addition, the additional concentration did not affect the growth of bacteria and fungi.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAstragalus\u003c/em\u003e was infected on bi-compartmented Petri plates (90\u0026times;15 mm) to explore the effects of metabolites on the resistance of \u003cem\u003eAstragalus\u003c/em\u003e root rot. Briefly, 15 mL of water agar medium was added to the first compartment and 10 mL of Hoagland solution was added to the second compartment. After the true leaves of \u003cem\u003eAstragalus\u003c/em\u003e seedlings were fully expanded, 100 \u0026mu;L of \u003cem\u003eF. oxysporum\u003c/em\u003e (approximately 10\u003csup\u003e5\u003c/sup\u003e spores per millilitre), a bacterial suspension (OD\u003csub\u003e600\u003c/sub\u003e= 0.8), and metabolites were added to the side of the Hoagland solution. All the plates were placed in an artificial climate incubator at 25 \u0026deg;C (16 h light/8 h darkness).\u003c/p\u003e\n\u003cp\u003e2.7. Statistical analysis\u003c/p\u003e\n\u003cp\u003eData analysis and visualization were performed mainly in the R environment (V4.4.1). The \u0026apos;vegan\u0026apos; package was used to calculate the alpha and \u0026beta; diversities. Principal coordinate analysis (PCoA) was conducted to assess \u0026beta; diversity. Redundancy analysis (RDA) was performed using the \u0026apos;vegan\u0026apos; package in R. Spearman correlations were calculated using the \u0026apos;ggcor\u0026apos; package in R. Cytoscape 3.4.0 was used to visualize the network. PICRUSt software was used to predict KEGG orthologue functional profiles of bacterial communities in rhizospheres [40]. The Sloan neutral model was applied to evaluate the effects of random dispersal and ecological drift on the assembly of microbial communities\u0026nbsp;[41]. Phylogenetic bin-based null model analysis (iCAMP) was used to infer community assembly mechanisms [42].\u003c/p\u003e\n\u003cp\u003eTo evaluate the influence of stochastic processes on community assembly, we employed a neutral community model (NCM) to predict the relationship between OTU detection frequency and relative abundance within a broader metacommunity [43]. This model, an adaptation of neutral theory, posits that taxa with high abundance in the metacommunity are more likely to disperse across sites, whereas rare taxa are more susceptible to loss due to ecological drift (i.e., stochastic fluctuations in population dynamics) [44]. The dispersal between communities is quantified by the parameter Nm, where N denotes the metacommunity size and m represents the immigration rate. The goodness-of-fit to the neutral model was assessed using R\u0026sup2;, and 95% confidence intervals for the model parameters were derived through bootstrapping with 1000 replicates.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1. SiO\u003csub\u003e2\u003c/sub\u003e NPs increase pathogen resistance and promote plant growth\u003c/p\u003e\n\u003cp\u003eThe TEM images of the SiO\u003csub\u003e2\u003c/sub\u003e NPs showed that it exhibited a spherical shape, with an average size of 17 nm (Fig. S1a). The zeta potential of the SiO\u003csub\u003e2\u003c/sub\u003e NPs was found to be -23.47 mV. The results of FTIR spectra indicated that the peaks at 3438, 1660, 1170~1047 and 962 cm\u003csup\u003e-1\u003c/sup\u003e were attributed to the -OH, H-O-H, Si-O-Si, and Si-OH vibration stretching, respectively (Fig. S1b). To further characterize the surface functional groups of the SiO\u003csub\u003e2\u003c/sub\u003e NPs, XPS spectroscopy was conducted. The results of XPS suggested that the full spectrum displays the existence of Si, C, and O on the surface of SiO\u003csub\u003e2\u003c/sub\u003e NPs (Fig. S1c). Furthermore, experiments on the stabilities of SiO\u003csub\u003e2\u003c/sub\u003e NPs were carried out (Fig. S1d). The results showed that the solution of SiO\u003csub\u003e2\u003c/sub\u003e NPs did not show the formation of aggregation between 0-12 hours. This indicated that the solution of SiO\u003csub\u003e2\u003c/sub\u003e NPs showed good nanoparticle dispersibility.\u003c/p\u003e\n\u003cp\u003eTo explore the potential of SiO\u003csub\u003e2\u003c/sub\u003e NPs in enhancing the disease resistance of\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eAstragalus\u003c/em\u003e, the plants were subjected to various methods\u0026nbsp;of SiO\u003csub\u003e2\u003c/sub\u003e NP application, including soil supplementation and foliar spraying, at concentrations of 200, 400, and 800 mg/kg, with the fungicide carbendazim used as a positive control. The findings demonstrated that carbendazim, soil application and foliar spraying of SiO\u003csub\u003e2\u003c/sub\u003e NPs significantly improved plant resistance to \u003cem\u003eF. oxysporum\u003c/em\u003e infection, as indicated by ANOVA (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u0026nbsp;Although the difference between the two application methods was not substantial, the disease index of \u003cem\u003eAstragalus\u003c/em\u003e cultivated in\u0026nbsp;the soil\u0026nbsp;supplemented\u0026nbsp;with SiO\u003csub\u003e2\u003c/sub\u003e NPs was lower than that of \u003cem\u003eAstragalus\u003c/em\u003e cultivated with foliar spraying. Additionally, the efficacies of both treatments increased with increasing SiO\u003csub\u003e2\u003c/sub\u003e NP concentration\u0026nbsp;(Fig. 1a).\u003c/p\u003e\n\u003cp\u003eBecause the application of SiO\u003csub\u003e2\u003c/sub\u003e NPs\u0026nbsp;could significantly reduce the disease index, the efficacy of SiO\u003csub\u003e2\u003c/sub\u003e NPs in inhibiting pathogenic fungi was evaluated. However, the results indicated that SiO\u003csub\u003e2\u003c/sub\u003e NPs did not significantly influence the growth or sporulation of \u003cem\u003eF. oxysporum\u003c/em\u003e in either solid or liquid medium (Fig.\u0026nbsp;1b). Therefore, the activities of antioxidant enzymes in plants treated with SiO\u003csub\u003e2\u003c/sub\u003e NPs were assessed. Both foliar spraying and soil supplementation with SiO\u003csub\u003e2\u003c/sub\u003e NPs resulted in significant increases in the activities of ascorbate peroxidase (APX), catalase (CAT), and peroxidase (POD) (ANOVA; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Regardless of the method of silicon application, the highest activities of the antioxidant enzymes were observed at a treatment concentration of 400 mg/kg SiO\u003csub\u003e2\u003c/sub\u003e NPs, with concentrations exceeding 400 mg/kg not yielding further increases in APX, CAT, and POD activities (Fig.\u0026nbsp;1c, d). This finding aligns with the observed accumulation of silicon in \u003cem\u003eA\u003c/em\u003e\u003cem\u003estragalus\u003c/em\u003e, which peaked when SiO\u003csub\u003e2\u003c/sub\u003e NPs were applied at a concentration of 400 mg/kg (Fig.\u0026nbsp;1e).\u003c/p\u003e\n\u003cp\u003eSoil supplemented with SiO\u003csub\u003e2\u003c/sub\u003e NPs at varying concentrations did not significantly affect on plant height or root length. However, the application of 400 mg/kg SiO\u003csub\u003e2\u003c/sub\u003e NPs resulted in notable increases in the relative chlorophyll content (measured by SPAD) and overall plant biomass (Fig. S2a). Increasing the concentration above 400 mg/kg did not lead to further improvements in these growth parameters; in fact, at a concentration of 800 mg/kg, decreases in both belowground and aboveground biomass were observed. These findings suggest that the increase in growth attributed to SiO\u003csub\u003e2\u003c/sub\u003e NPs is dose-dependent. Conversely, the foliar application of SiO\u003csub\u003e2\u003c/sub\u003e NPs at various concentrations did not significantly affect the height, root length, fresh root weight, or dry root weight of \u003cem\u003eA\u003c/em\u003e\u003cem\u003estragalus\u003c/em\u003e (Fig. S2b). Notably, when SiO\u003csub\u003e2\u003c/sub\u003e NPs were applied at a concentration of 400 mg/L, there were significant increases in the chlorophyll content and biomass of the aboveground parts. Given that the application of SiO\u003csub\u003e2\u003c/sub\u003e NPs can influence plant metabolism, we further investigated whether SiO\u003csub\u003e2\u003c/sub\u003e NPs modulate the bacterial community structure in the rhizosphere and roots by altering the composition of root exudates.\u003c/p\u003e\n\u003cp\u003e3.2. SiO\u003csub\u003e2\u003c/sub\u003e NPs increased the stability and resilience of the microbial communities\u003c/p\u003e\n\u003cp\u003eAccording to the results obtained from 16S and ITS amplicon sequencing, a total of 2,108,882 high-quality bacterial sequences and 1,815,835 fungal sequences were generated from 15 samples. Following the removal of low-quality and plant-derived reads, the remaining sequences were clustered into 1,731 bacterial and 978 fungal ASVs at a sequence identity threshold of \u0026ge; 97%. The application of SiO\u003csub\u003e2\u003c/sub\u003e NPs to the soil (Si_S) significantly increased the alpha diversity of rhizosphere bacteria, whereas foliar spraying of SiO\u003csub\u003e2\u003c/sub\u003e NPs (Si_L) resulted in a significant increase in the alpha diversity of fungi (Fig.\u0026nbsp;2a). Principal coordinate analysis (PCoA) revealed significant differences in the composition of the rhizosphere bacterial (R\u003csup\u003e2\u003c/sup\u003e = 0.20; \u003cem\u003ep\u003c/em\u003e = 0.001) and fungal communities (R\u003csup\u003e2\u003c/sup\u003e = 0.84; \u003cem\u003ep\u003c/em\u003e = 0.001) of \u003cem\u003eA\u003c/em\u003e\u003cem\u003estragalus\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eunder SiO\u003csub\u003e2\u003c/sub\u003e NP application (Fig.\u0026nbsp;2b and\u0026nbsp;2c). Compared with the control group, the Si_S-treated group presented significant increases in the relative abundance of two bacterial phyla (Acidobacteriota and Crenarchaeota) and two fungal phyla (Mortierellomycota and Basidiomycota) but a decrease in the abundance of Ascomycota (Fig.\u0026nbsp;2d and Fig. S3). The Si_L treatment significantly decreased the relative abundance of Actinobacteriota and increased the abundance of Crenarchaeota (ANOVA; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eAt the genus level, the application of SiO\u003csub\u003e2\u003c/sub\u003e NPs resulted in a greater number of significant changes in bacterial relative abundance (59 genera) than in fungal relative abundance (25 genera) (Fig. S4-S7). Notably, the relative abundance of \u003cem\u003eFusarium\u003c/em\u003e, the average disease index, and the prevention effect were significantly correlated with these 59 bacterial genera (Fig. 2e). Furthermore, the relative abundance of \u003cem\u003eFusarium\u003c/em\u003e in the rhizosphere significantly decreased with soil supplementation and foliar application of SiO\u003csub\u003e2\u003c/sub\u003e NPs. In both the bacterial and fungal communities, beta diversity was significantly positively correlated with the average disease index and negatively correlated with alpha diversity (Fig. 2f). The alpha diversity of bacteria was significantly positively correlated with the silicon contents in both the roots and above-ground parts of \u003cem\u003eA\u003c/em\u003e\u003cem\u003estragalus\u003c/em\u003e, whereas fungi were positively correlated with only the silicon content in the roots. These findings suggest that \u003cem\u003eF. oxysporum\u0026nbsp;\u003c/em\u003einfection contributes to an increase in beta diversity and a decrease in alpha diversity within the rhizosphere, leading to significant alterations in the microbial community between diseased and healthy plants. The introduction of SiO\u003csub\u003e2\u003c/sub\u003e NPs mitigated these changes, increasing the diversity of the rhizosphere microbial community and reducing the incidence of disease.\u003c/p\u003e\n\u003cp\u003eCo-occurrence network analysis was employed to investigate the alterations in the rhizosphere soil microbial co-occurrence patterns induced by the application of SiO\u003csub\u003e2\u003c/sub\u003e NPs (Fig.\u0026nbsp;3a). Compared with the control, the introduction of SiO\u003csub\u003e2\u003c/sub\u003e NPs resulted in a decrease in the density of microbial networks and the average clustering coefficient, while simultaneously leading to increases in the average degree and the numbers of communities, nodes, and links (Fig.\u0026nbsp;3b). Additionally, in the bacterial network, parameters such as network diameter, average path length, modularity, and negative correlation decreased, whereas these parameters increased in the fungal network. In conclusion, the introduction of SiO\u003csub\u003e2\u003c/sub\u003e NPs increased the complexity and interconnections within the network, thereby contributing positively to the stability and resilience of the microbial communities against disturbances.\u003c/p\u003e\n\u003cp\u003e3.3. Impact of SiO\u003csub\u003e2\u003c/sub\u003e NPs on the functional traits and assembly mechanisms of microbial communities\u003c/p\u003e\n\u003cp\u003eTo assess whether treatment with SiO\u003csub\u003e2\u003c/sub\u003e NPs alters the functional profiles of bacterial communities, we utilized PICRUSt software to predict community functions. The findings revealed that the functional profiles of the rhizosphere bacterial community were distinct between the SiO\u003csub\u003e2\u003c/sub\u003e NP-treated groups and the control group (Fig. 3c). Notably, amino acid metabolism and the biosynthesis of other secondary metabolites were enriched in the soils supplemented with SiO\u003csub\u003e2\u003c/sub\u003e NPs. Further analysis of the mechanisms underlying microbial community assembly across different treatments revealed that stochastic processes, including drift and other factors, predominantly influenced bacterial community assembly in the control group. In contrast, there was a shift towards dispersal limitation in the SiO\u003csub\u003e2\u003c/sub\u003e NP-treated groups, with contributions of 50.59% for soil supplementation and 39.09% for foliar spraying of SiO\u003csub\u003e2\u003c/sub\u003e NPs (Fig. 3d).\u003c/p\u003e\n\u003cp\u003eThe neutral community model (NCM) effectively captured a substantial portion of the relationship between the occurrence frequency of ASVs and their relative abundance variations (Fig.\u0026nbsp;3d). Specifically, the model explained 62.0%, 66.4%, and 65.6% of the community variance for the control, Si_S, and Si_L groups, respectively.\u0026nbsp;The Nm-\u0026nbsp;value for bacteria was greater in the Si_S treatment (Nm=4230) than in the Si_L treatment (Nm=3732), with the \u003cem\u003em\u003c/em\u003e value estimated at 0.09 for Si_S and 0.08 for Si_L, indicating greater dispersal of bacterial species in the Si_S group. In contrast, fungal community assembly was influenced primarily by deterministic processes, particularly homogeneous selection, across all the experimental groups (control: 55.58%; Si_S: 99.34%; Si_L: 64.01%), which resulted in a poor fit with the NCM. In conclusion, the application of SiO₂ NPs did not significantly influence the primary ecological processes of fungal community assembly. However, it did lead to a shift in the bacterial community assembly process, changing it from homogeneous selection to dispersal limitation. These findings suggest that SiO₂ NP treatment applied selective pressure on the survival and reproduction of bacterial communities, which in turn facilitated shifts in community composition and species diversity.\u003c/p\u003e\n\u003cp\u003e3.4. Effects of reinoculation with pathogen-associated bacteria on the root rot and growth of \u003cem\u003eAstragalus\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe nodes in the bacterial co-occurrence networks of the Si_S- and Si_L-treated samples were ranked based on betweenness centrality, and the top 30 core ASVs were visualized (Fig. S8). Eight core ASVs were successfully isolated from rhizosphere soil, with greater than 97% sequence similarity between the isolated strains and the ASVs (Fig. 4a, Fig. S9). Among the four core ASVs in the Si_S-treated co-occurrence network, the relative abundances of ASV_794 (98.79% similarity to \u003cem\u003eMicrobacterium oxydans\u003c/em\u003e SCK-308) and ASV_349 (100.00% similarity to \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e XS-134-7) significantly decreased in the silicon treatments (ANOVA; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) (Fig. 4b). Further analysis of the genera to which these ASVs belong revealed that the relative abundances of \u003cem\u003eMicrobacterium\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e also significantly decreased with silicon treatment (Fig. 4c). In the Si_L-treated co-occurrence network, among the four core ASVs, the relative abundance of ASV_1492 (\u003cem\u003eStreptomyces\u003c/em\u003e) significantly increased in the silicon treatment group. However, at the genus level, the relative abundance of \u003cem\u003eStreptomyces\u003c/em\u003e did not change significantly, whereas that of \u003cem\u003ePseudarthrobacter\u003c/em\u003e significantly decreased. Therefore, we selected ASV_794 (\u003cem\u003eM. oxydans\u003c/em\u003e),\u0026nbsp;ASV_349\u0026nbsp;(\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e), and\u0026nbsp;ASV_15\u0026nbsp;(\u003cem\u003ePseudarthrobacter psychrotolerans\u003c/em\u003e), which presented significantly reduced relative abundances at the genus level, for the pot experiments. The results of the pot experiments revealed that \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eM. oxydans\u003c/em\u003e promoted the infection of \u003cem\u003eAstragalus\u003c/em\u003e by pathogenic fungi (Fig. 4d). Thus, we tested whether these two strains are also pathogens of \u003cem\u003eAstragalus\u0026nbsp;\u003c/em\u003eand can infect plants. The results revealed that neither \u003cem\u003eP. aeruginosa\u003c/em\u003e nor \u003cem\u003eM. oxydans\u003c/em\u003e could independently infect \u003cem\u003eAstragalus\u003c/em\u003e and induce root rot (Fig. S10). Therefore, these two bacteria may serve as helpers in fungal pathogen infection of plants. Further experiments show that \u003cem\u003eM. oxydans\u003c/em\u003e SCK-308 and \u003cem\u003eP. aeruginosa\u003c/em\u003e XS-134-7 promoted the growth of \u003cem\u003eF. oxysporum\u003c/em\u003e and inhibited the growth of certain beneficial rhizosphere microorganisms, thereby facilitating the occurrence of the disease (Fig. S11). There were no significant differences in growth indicators of plants inoculated with these bacteria. Therefore, the application of SiO\u003csub\u003e2\u003c/sub\u003e NPs to the soil reduced the abundance of \u003cem\u003eMicrobacterium\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e in the rhizosphere, thereby reducing their incidence.\u003c/p\u003e\n\u003cp\u003e3.5. SiO\u003csub\u003e2\u003c/sub\u003e NP-induced resistance of \u003cem\u003eAstragalus\u003c/em\u003e to \u003cem\u003eF. oxysporum\u003c/em\u003e depends on salicylic acid\u003c/p\u003e\n\u003cp\u003eCompared with foliar spraying, soil application of SiO\u003csub\u003e2\u003c/sub\u003e NPs had a better protective effect and significantly increased the diversity of rhizosphere bacteria. Therefore, we conducted an untargeted metabolomic analysis of the root exudates of soil SiO\u003csub\u003e2\u003c/sub\u003e NP-treated \u003cem\u003eAstragalus\u003c/em\u003e. PCA and PLS-DA analysis revealed that the application of SiO\u003csub\u003e2\u003c/sub\u003e NPs significantly altered the root exudates of \u003cem\u003eAstragalus\u003c/em\u003e (Fig. 5a and b). A total of 2043 metabolites were identified and initially classified into three groups: 209 host (plant) metabolites, 423 microbiota metabolites, and 2443 others (drug food and environment) (Fig. 5c). Based on the criteria fold change \u0026gt; 2 and \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, a total of 196 differentially abundant metabolites were identified, of which 96 increased and 100 decreased (Fig. 5d). The significantly upregulated metabolites included mainly lipids and lipid-like molecules, phenylpropanoids and polyketides, and organic acids and their derivatives (Fig. S12). The significantly downregulated metabolites included mainly lipids and lipid-like molecules, benzenoids, and organoheterocyclic compounds. The KEGG database was used to annotate the metabolic pathways of the differentially abundant metabolites in the Si_S-treated group, and the top 7 metabolic pathways were analysed (Fig. 5e). These pathways included five upregulated differentially abundant metabolites, and one was downregulated in the Si_S-treated group (Fig. 5f). Notably, the plant hormone signal transduction pathway was significantly enriched in the Si_S-treated plants and included three differentially abundant metabolites (auxin, brassinosteroid, and salicylic acid). Therefore, we analysed the pathways and metabolites related to the plant hormone signal transduction (Fig. 5g). These results indicate that tryptophan metabolism and the biosynthesis of phenylalanine, tyrosine, and tryptophan provide precursors for the synthesis of auxin. The biosynthesis of phenylalanine, tyrosine, tryptophan and phenylalanine provides precursor substances for the synthesis of salicylic acid (SA). Therefore, the untargeted metabolomic analysis of the root exudates of \u003cem\u003eAstragalus\u003c/em\u003e revealed a significant impact of SiO\u003csub\u003e2\u003c/sub\u003e NPs on the modulation of metabolic profiles under \u003cem\u003eF. oxysporum\u003c/em\u003e inoculation, with the involvement of SA playing a crucial role in enhancing root rot resistance.\u003c/p\u003e\n\u003cp\u003e3.6. Associations between the metabolome and microbiome\u003c/p\u003e\n\u003cp\u003eBecause of the interaction between root exudates and microbial communities, we assessed the relationships between the metabolites of Si_S-treated plants and the relative abundances of genera (all\u0026nbsp;bacterial genera and \u003cem\u003eFusarium\u003c/em\u003e) using Spearman\u0026apos;s correlation coefficient. There were 572, 1055, 3844, 5451, and 12,680 closely associated bacteria‒metabolite pairs at the phylum, class, order, family, and genus levels, respectively (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) (Supplementary material 2). We further visualized the correlations between the four primary differentially abundant metabolites (salicylic acid, indole-3-acetic acid, brassinolide, and palmitic acid) and the differential bacterial\u0026nbsp;genera (Fig. 6a). Notably, the four differentially abundant metabolites were significantly negatively correlated with \u003cem\u003eMicrobacterium\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e, whose relative abundances were significantly reduced in the Si_S-treated group. Through the combined analysis of microbiota and metabolites, we identified two metabolic pathways related to the plant hormone signal transduction pathway: tryptophan metabolism and the biosynthesis of siderophore group nonribosomal peptide pathways.\u003c/p\u003e\n\u003cp\u003eThree metabolites (L-tryptophan, indole-3-acetamide, and indole) are involved in this pathway for tryptophan metabolism. They are all precursors for the synthesis of indole-3-acetic acid and are engaged in two metabolic reactions in this pathway (R00679 and R00673) (Fig.\u0026nbsp;6b, c). For the biosynthesis of the siderophore group nonribosomal peptide pathway, the differentially abundant metabolite salicylic acid was involved in the R06602 metabolic reaction of this pathway (Fig. 6d). For each metabolic reaction, the biological and statistical correlations between the microbiota and metabolites were explored using the Bio-Sankey and STA-Sankey networks. In the Bio-Sankey and STA-Sankey networks, \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eMicrobacterium\u003c/em\u003e were identified as major genera closely associated with the metabolic reactions R00679 and R00673 and positively correlated with L-tryptophan, indole-3-acetamide, and indole (Fig. 6 and S13). In addition, \u003cem\u003ePseudomonas\u003c/em\u003e was identified as a significant genus closely associated with the metabolic reaction R06602 and negatively correlated with salicylic acid. The relative abundance of \u003cem\u003ePseudomonas\u003c/em\u003e was significantly reduced in the Si_S-treated group, and its reinoculation significantly increased the \u003cem\u003eAstragalus\u003c/em\u003e root rot disease index. These findings indicate that the abundance of \u003cem\u003ePseudomonas\u003c/em\u003e is closely related to the occurrence of \u003cem\u003eAstragalus\u003c/em\u003e root rot. In the fatty acid elongation pathway, palmitic acid and \u003cem\u003eFusarium\u003c/em\u003e are recognized as metabolites, and a fungal taxon involved in the R01274 metabolic reaction of this pathway has a negative correlation (Fig. 6e). Palmitic acid generates very long-chain fatty acids (VLCFAs) via the fatty acid synthesis pathway, which furthers the synthesis of the cuticle. The cuticle not only affects the ability of fungal hyphae to attach to the cuticular surface but also plays an essential role in systemic acquired resistance (SAR) by activating defence responses, such as regulating the active transport of salicylic acid across the apoplast, which is necessary for the induction of SAR.\u003c/p\u003e\n\u003cp\u003eA comprehensive analysis of the microbiota and metabolites revealed that the addition of SiO\u003csub\u003e2\u003c/sub\u003e NPs to the soil significantly reduced the relative abundances of \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eFusarium\u003c/em\u003e and increased the synthesis of salicylic acid. This activated SAR and ultimately improved root rot resistance. In addition, soil-applied SiO\u003csub\u003e2\u003c/sub\u003e NPs promoted cuticle synthesis, forming a physical barrier against pathogen invasion.\u003c/p\u003e\n\u003cp\u003e3.7.\u0026nbsp;Metabolites alleviate root rot by inhibiting the growth of pathogenic fungi and their \u0026quot;helpers\u0026quot;\u003c/p\u003e\n\u003cp\u003eSalicylic acid, indole-3-acetic acid, brassinolide, and palmitic acid were significantly negatively correlated with the resistance of pathogenic fungi, and we further investigated the effects of these four differentially abundant metabolites on the resistance of \u003cem\u003eAstragalus\u003c/em\u003e to \u003cem\u003eF. oxysporum\u003c/em\u003e. The results demonstrated that when the concentrations of the four differentially abundant metabolites exceeded 100 \u0026mu;M, they significantly inhibited the growth of \u003cem\u003eP. aeruginosa\u003c/em\u003e XS-134-7 (Fig. 6f). When palmitic acid was added at concentrations greater than 25 \u0026mu;M, it inhibited the growth of \u003cem\u003eM. oxydans\u003c/em\u003e SCK-308. Salicylic acid, at a concentration of 200 \u0026mu;M, significantly inhibited the growth of \u003cem\u003eM. oxydans\u003c/em\u003e SCK-308, whereas indole-3-acetic acid and brassinolide had no effect on the growth of \u003cem\u003eM. oxydans\u003c/em\u003e SCK-308. The minimum concentrations of the metabolites required to inhibit bacterial growth were selected for the infection test. Compared with the control group (\u003cem\u003eP. aeruginosa\u003c/em\u003e XS-134-7 + \u003cem\u003eF. oxysporum\u003c/em\u003e), the addition of the four metabolites (100 \u0026mu;M) significantly reduced the disease index of \u003cem\u003eAstragalus\u003c/em\u003e root rot (Fig. 6g). Similarly, compared with the control group (\u003cem\u003eM. oxydans\u003c/em\u003e SCK-308 + \u003cem\u003eF. oxysporum\u003c/em\u003e), the addition of the differentially abundant metabolites palmitic acid (25 \u0026mu;M) and salicylic acid (200 \u0026mu;M) also reduced the disease index of \u003cem\u003eAstragalus\u003c/em\u003e root rot. Furthermore, we evaluated the impact of four differentially abundant metabolites on the growth and spore formation of \u003cem\u003eF. oxysporum\u003c/em\u003e in both solid and liquid media. The results indicated that when the concentrations of these metabolites exceeded 100 \u0026mu;M, they significantly affected the growth of \u003cem\u003eF. oxysporum\u003c/em\u003e (Fig. S14). However, brassinolide did not influence the spore germination of \u003cem\u003eF. oxysporum\u003c/em\u003e. These findings suggest that the differentially abundant metabolites alleviate \u003cem\u003eAstragalus\u003c/em\u003e root rot by inhibiting the growth of pathogenic fungal \u0026quot;helpers\u0026quot;.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e4.1 SiO\u003csub\u003e2\u003c/sub\u003e NPs reduced the disease index of root rot\u003c/p\u003e\n\u003cp\u003eSi-induced improvement in disease resistance is partly manifested by the reinforcement of cell walls, which prevents pathogen ingress. Moreover, the activation of defence-related enzymes, stimulation of antimicrobial compound production, and regulation of the complex network of signalling pathways are thought to be the key mechanisms by which Si-induced chemical defences against fungal pathogens transcend physical barriers [45]\u0026nbsp;(Fig.\u0026nbsp;7). When investigating the effects of exogenous Si on plant diseases, many studies ignore the direct antifungal properties of Si, potentially overestimating the beneficial effect of Si on disease control [46]. Therefore, to better assess the role of Si in plant disease control and its potential mechanisms, selecting a Si concentration and an application method that does not directly affect pathogen growth but can activate plant defence responses would be more meaningful. Therefore, to examine the effects of SiO\u003csub\u003e2\u003c/sub\u003e NP application on \u003cem\u003eF. oxysporum\u003c/em\u003e root rot in \u003cem\u003eAstragalus\u003c/em\u003e, the effects of different concentrations of SiO\u003csub\u003e2\u003c/sub\u003e NPs on the disease index under different application methods were tested in pot experiments.\u0026nbsp;In this study, different concentrations of SiO\u003csub\u003e2\u003c/sub\u003e NPs did not significantly promote or inhibit the growth of \u003cem\u003eF. oxysporum\u003c/em\u003e (Fig. 1b). Both soil application and foliar spraying of SiO\u003csub\u003e2\u003c/sub\u003e NPs improved resistance to root rot. Nevertheless, soil applications have a lower disease index and better prevention ability than foliar spraying does. Furthermore, compared with foliar spraying of SiO₂ NPs, root treatment confers greater resistance to rice blast through the systemic acquired resistance (SAR) response\u0026nbsp;[10].\u0026nbsp;Therefore, root treatment is a safer and more effective method for applying SiO₂ NPs. Compared with SiO₂ NP treatment, carbendazim treatment significantly inhibited the progression of root rot, achieving a disease index control rate of 71.54% (Fig. 1a), demonstrating superior disease prevention efficacy (57.70%). However, persistent carbendazim residues in soil pose bioaccumulation risks and potential groundwater contamination [47]. In this study, the application of SiO\u003csub\u003e2\u003c/sub\u003e NP did not have a negative impact on plant growth, and Kumari [48] also found that SiO\u003csub\u003e2\u003c/sub\u003e NP could improve soil fertility and \u003cem\u003eZ. may\u003c/em\u003es growth, suggesting the low toxicity and potential benefits for sustainable agriculture. This is similar to what others have discovered that SiO\u003csub\u003e2\u003c/sub\u003e NP have minimal negative impacts on environmental health and crop food safety due to its chemical inertness [49]. Therefore, although SiO₂ NPs exhibit marginally lower immediate efficacy than chemical pesticides do, their multidimensional advantages in ensuring medicinal material safety and maintaining soil health render them more suitable for the integrated management of medicinal plant diseases. However, SiO\u003csub\u003e2\u003c/sub\u003e NP can also have negative consequences depending on the conditions such as soil physical and chemical properties, nanoparticle concentration in the soil, therefore the environmental impact of SiO\u003csub\u003e2\u003c/sub\u003e NP needs to be further explored before its application in the field.\u003c/p\u003e\n\u003cp\u003ePlants take\u0026nbsp;up silicon from the soil and subsequently transport it from roots to shoots either passively (transpirational stream) or actively (specific transporter proteins) [46, 50]. Therefore, compared with the control, the\u0026nbsp;soil-applicant SiO\u003csub\u003e2\u003c/sub\u003e NPs also significantly increased the Si content of the shoot\u0026nbsp;(Fig. S2b). In addition, studies have shown that foliar application of SiO\u003csub\u003e2\u003c/sub\u003e NPs significantly increased the\u0026nbsp;Si contents in leaves and stems, indicating the uptake of SiO\u003csub\u003e2\u003c/sub\u003e NPs by plant leaves and their\u0026nbsp;translocation to stems, but no translocation from stems to roots was observed [51]. Interestingly, in our study, foliar application of SiO\u003csub\u003e2\u003c/sub\u003e NPs also significantly increased the content of Si in roots, but the content was lower than that in\u0026nbsp;the soil treatment at the same concentration. The Si content in roots was significantly positively correlated with the prevention effect and negatively correlated with the disease index. We used SiO₂ NPs with a size of 17 nm, which is consistent with previous studies on the transmission threshold of approximately 15\u0026ndash;40 nm into the central column or xylem of the root system [52]. Only a small amount of foliar-applied SiO₂ NPs was transported to the roots through the stomata, resulting in a relatively weak impact on the rhizosphere microbiome. In contrast, \u003cem\u003eAstragalus\u003c/em\u003e root rot, caused by \u003cem\u003eFusarium\u003c/em\u003e, is a soil-borne disease. The application of SiO₂ NPs to the soil, plants accumulate more silicon through their root absorbtion, which has a greater impact on plant metabolism. Moreover, SiO₂ NPs applied to the soil may affect the growth of other microorganisms in the soil, resulting in a greater change in microbial community structure. All these reasons could lead to more effective disease control. Although foliar spraying of SiO\u003csub\u003e2\u003c/sub\u003e NPs is an effective method for controlling soil-borne diseases, soil application of SiO\u003csub\u003e2\u003c/sub\u003e NPs is typically more effective in improving plant disease resistance because of the transport and accumulation mechanism of silicon in plants.\u003c/p\u003e\n\u003cp\u003e4.2 SiO\u003csub\u003e2\u003c/sub\u003e NPs increased the diversity of rhizosphere microorganisms and reduced the abundance of pathogen-associated microorganisms\u003c/p\u003e\n\u003cp\u003eThe diversity and composition of the rhizosphere microbial community significantly influence soil health and are significant drivers of plant defence against soil-borne diseases [53, 54]. Previous study showed that the resistance to pathogenic colonization greatly increased with community diversity [55]. Our results also showed that SiO₂ NPs application significantly increased the Shannon \u0026alpha;-diversity of bacterial community in the rhizosphere of \u003cem\u003eAstragalus\u003c/em\u003e. Therefore, there is more overlap in nutrient requirements between rhizosphere microbial community and pathogen, and these rhizosphere microorganisms inhibit the growth of pathogen through nutrient competition.\u0026nbsp;Active participation of Si in plant-microbe interactions has been demonstrated in several studies [56, 57]. Silicon can improve plant disease resistance through three principal mechanisms. The first silicon can directly inhibit the growth of pathogens\u0026nbsp;to protect plants [58, 59]. Second, Si can modify soil microbial habitats by altering soil physicochemical properties and enzymes. For example, Si application can correct the decreases in soil pH and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N content caused by ginseng black spot disease, which affects soil microbial structure [23]. Third, Si can reshape the structure of the rhizosphere microbial community by altering root exudates, e.g., increasing the relative abundance of beneficial microbial taxa or reducing the relative abundance of microbial taxa of potential plant pathogens, which exerts the positive feedback effects on plant growth and resistance [60-62].\u0026nbsp;The SiO\u003csub\u003e2\u003c/sub\u003e NPs used in this study had no direct toxicity to \u003cem\u003eF. oxysporum\u003c/em\u003e (Fig. 1b). Therefore, the improvement of its resistance to root rot was due to the change of the rhizosphere microbial community structure. The application of SiO\u003csub\u003e2\u003c/sub\u003e NPs significantly increased the \u0026alpha;\u0026nbsp;diversity of rhizosphere bacteria and fungi, and the index was positively correlated with the Si content of \u003cem\u003eAstragalus\u003c/em\u003e roots (Fig. 2a, f).\u003c/p\u003e\n\u003cp\u003eIn addition, the application of SiO\u003csub\u003e2\u003c/sub\u003e NPs also increased the relative abundances of several microorganisms that promote plant growth, such as \u003cem\u003eLysobacter\u003c/em\u003e [63] and \u003cem\u003eSphingobium\u003c/em\u003e [64], which are highly important for plant growth and soil health.\u0026nbsp;In addition to PGPR, some bacteria can promote the growth of pathogens in the plant rhizosphere, acting as helper of soil-borne pathogens during rhizosphere colonization.\u0026nbsp;In this study, the relative abundances of microbial taxa associated with plant pathogens, such as \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eMicrobacterium\u003c/em\u003e [65], were significantly reduced (Fig. 4b).\u0026nbsp;Crucially, both genera presented significant negative correlations with protection rates, whereas \u003cem\u003eMicrobacterium\u003c/em\u003e presented a markedly positive correlation with disease indices (Fig. 2e). As evidenced by multiple reports, \u003cem\u003eP\u003c/em\u003e\u003cem\u003e. aeruginosa\u003c/em\u003e has been identified as a natural soil inhabitant and a potential plant pathogen [66, 67]. Therefore, the coinoculation of \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eF. oxysporum\u003c/em\u003e may cause coinfection of \u003cem\u003eAstragalus\u003c/em\u003e, thereby increasing the severity of root rot. In addition, a study showed that \u003cem\u003eMicrobacterium paraoxydans\u003c/em\u003e acts as a bacterial helpers for \u003cem\u003eRalstonia solanacearum\u003c/em\u003e, significantly enhancing its colonization in the tomato rhizosphere and increasing the severity of bacterial wilt disease [21]. Subsequent pot experiments revealed that coinoculation of \u003cem\u003eF\u003c/em\u003e\u003cem\u003e. oxysporum\u003c/em\u003e with either \u003cem\u003ePseudomonas\u003c/em\u003e or \u003cem\u003eMicrobacterium\u003c/em\u003e consistently increased root rot disease severity, suggesting a synergistic pathogenic interaction between the pathogen and its microbial allies during host colonization. Exogenous application of differentially abundant metabolites exhibiting antagonistic effects against the phytopathogen \u003cem\u003eF\u003c/em\u003e\u003cem\u003e. oxysporum\u003c/em\u003e and its \u0026quot;helpers\u0026quot; (\u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eMicrobacterium\u003c/em\u003e) significantly ameliorated root rot severity under coinfection conditions (Fig. 6g). Numerous studies have shown that the increase in the antagonistic bacteria was found to enhance plant resistance to pathogen [68]. However, our results showed that \u003cem\u003eM. oxydans\u003c/em\u003e SCK-308 and \u003cem\u003eP. aeruginosa\u003c/em\u003e XS-134-7 promoted the growth of \u003cem\u003eF. oxysporum\u003c/em\u003e and inhibited the growth of certain beneficial rhizosphere microorganisms (Fig. S11). This indicates that the propagation of pathogenic allies reduces the density of antagonistic bacteria and thus alleviates the inhibition of pathogen. Furthermore, interaction between pathogenic allies and \u003cem\u003eF. oxysporum\u003c/em\u003e could have increased the niche space in root surface, which is conducive to the adsorption and infection of pathogenic fungi. These results elucidate a SiO\u003csub\u003e2\u003c/sub\u003e NP-mediated suppression strategy, in which targeted inhibition of pathogen-associated allies enhances disease control efficacy through disruption of cross-kingdom pathogenic synergies.\u003c/p\u003e\n\u003cp\u003eChanges in the relative abundance and composition of microorganisms within a community can lead to alterations in their overall functional capacities. In this study, the addition of SiO\u003csub\u003e2\u003c/sub\u003e NPs increased amino acid metabolism in the soil. Research has shown that the resident microbial keystone taxa from disease-suppressive rice panicles subvert pathogen infection by manipulating BCAA flux in the host panicle [69]. Furthermore, a study indicated that the rhizosphere bacteria JR48 can produce phenylpyruvate to increase the accumulation of phenylalanine in plants, thereby promoting lignification and disease resistance dependent on phenylalanine metabolism [70]. Based on these findings, we speculate that the observed increase in amino acid metabolism in this study may be associated with increased disease resistance.\u0026nbsp;In addition, the introduction of SiO\u003csub\u003e2\u003c/sub\u003e NPs increased the complexity and relevance of the microbial community network, increasing its stability and resistance (Fig. 3a, b). The application of SiO\u003csub\u003e2\u003c/sub\u003e NPs enhanced stochastic processes (dispersal limitation) during bacterial community construction and deterministic processes in fungal communities (Fig. 3d). Dispersal limitation usually refers to a low diffusion rate coupled with drift or weak selection, which may increase community variation or turnover [71].\u0026nbsp;These findings highlight the potential of SiO₂ NPs to influence microbial community structures, particularly bacteria, by altering ecological processes such as dispersal and selection, and suggest that such nanoparticles could influence microbial diversity in environments exposed to them.\u0026nbsp;These findings also confirmed that the application of SiO\u003csub\u003e2\u003c/sub\u003e NPs changed the composition of the rhizosphere microbial community, thereby improving root rot resistance.\u003c/p\u003e\n\u003cp\u003e4.3 SiO\u003csub\u003e2\u003c/sub\u003e NP‑induced \u003cem\u003eAstragalus\u003c/em\u003e resistance to \u003cem\u003eFusarium\u003c/em\u003e depends on salicylic acid\u003c/p\u003e\n\u003cp\u003eMany previous studies on the impact of SiO\u003csub\u003e2\u003c/sub\u003e NPs on plants focused on the plant itself and did not consider its effects on root exudates. Plant roots absorb mineral nutrients and release organic exudates, such as fatty acids [72], plant hormones [73], and antimicrobial compounds [74]. These substances not only affect the physicochemical properties of the soil but also influence plant‒microbe interactions and help to build rhizosphere microbial communities. Therefore, changes in the level or composition of root exudates in soil\u0026ndash;root\u0026ndash;microbe interactions are essential for plants to protect themselves from soil-borne diseases. The application of SiO\u003csub\u003e2\u003c/sub\u003e NPs in this study significantly altered the root exudates of \u003cem\u003eAstragalus\u003c/em\u003e, increasing the accumulation of lipids, lipid-like molecules, organic acids and their\u0026nbsp;derivatives. This change increases bioavailable carbon storage in the soil, thereby improving the quality of the soil and affecting plant health. In addition, research has shown that organic acids can increase the anti-infection ability of plant cell walls, and certain organic acid derivatives have a direct fungicidal effects as part of the chemical strategy of plant defence against pathogens [75]. Notably, the application of SiO\u003csub\u003e2\u003c/sub\u003e NPs increased the accumulation of phenylpropanoids and polyketide substances in our research\u0026nbsp;(Fig. S15). Secondary metabolites derived through the phenylpropanoid pathway, such as phenols and flavonoids, are well-known for their defensive roles against fungal pathogens [76]. For example, the silica-induced increased accumulation of phenolics, flavonoids, lignans, and dopamine in the scale and vascular tissues of roots may contribute to the enhancement of banana resistance to \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. cubense in banana [24]. Our results showed that applying SiO\u003csub\u003e2\u003c/sub\u003e NPs significantly increased the accumulation of the antimicrobial compounds naringenin [77], sinapyl alcohol, and gallocatechin gallate [78]. Among these, sinapyl alcohol is a critical monomer in the lignin biosynthetic pathway, and plays a role in cell wall lignification [79]. Lignification of the cell wall increases its\u0026nbsp;mechanical strength and provides plants with\u0026nbsp;a physical barrier against pathogen invasion. Thus, the accumulation of these substances helps improve the resistance of \u003cem\u003eAstragalus\u003c/em\u003e root rot.\u003c/p\u003e\n\u003cp\u003ePhytohormones are essential for plants to cope with biotic and abiotic stresses. Phytohormones can effectively regulate plant defence responses through complex signal networks and interactions [45]. Therefore, the use of\u0026nbsp;silicon as a regulator by altering phytohormone homeostasis as well as a network of defence signalling components could be potential mechanisms for Si-triggered resistance responses.\u0026nbsp;For example, Si-regulated SA synthesis and metabolism mediate resistance to peanut bacterial wilt [17]. In this study, salicylic acid, auxin, and brassinolide were significantly enriched in plant hormone signal transduction pathways as differential substances (Fig. 5). SA is a critical phytohormone that regulates numerous aspects of plant development and the activation of defenses against biotic stress. SA undergoes glucosylation, methylation-demethylation, hydroxylation, or sulfonation to change its active/inactive forms. The activated SA subsequently induces SAR, reinforces cell walls, and activates pathogenesis-related (PR) proteins to resist pathogen [80]. In this study, the application of SiO₂ NPs significantly increased the salicylic acid content, suggesting that the enhanced resilience of \u003cem\u003eAstragalus\u003c/em\u003e to \u003cem\u003eFusarium\u003c/em\u003e root rot may be mediated by salicylic acid-induced SAR. Plants synthesize salicylic acid through the phenylalanine and isochorismate pathways [81]. These two metabolic pathways were also annotated in the joint analysis of \u003cem\u003eAstragalus\u003c/em\u003e rhizosphere microorganisms and root exudates. Disease-promoting \u003cem\u003ePseudomonas\u003c/em\u003e was negatively correlated with the isochorismate pathway. Indole-3-acetic acid (IAA) is an important plant hormone that regulates various biological processes, known as promoting plant growth. Recent studies indicated that IAA could also enhance the expression of genes related pathogenesis, thereby increasing disease resistance of plants [82]. Brassinolides and palmitic acid could inhibit the growth of some bacteria including the fungal pathogenic \u0026quot;helpers\u0026quot;. This leads to the disorder of the microbial community in the rhizosphere of healthy plants, resulting in a decrease in their disease resistance. Additionally, palmitic acid has been identified as a differentially abundant metabolite involved in the cutin, suberine, and wax biosynthesis and fatty acid elongation pathways, as well as the fatty acid elongation pathway. The metabolism of cutin and waxes can influence plant disease resistance by modulating the permeability of the cuticle layer [83].\u0026nbsp;These findings indicated that SiO₂ NPs altered the community composition of \u003cem\u003eAstragalus\u003c/em\u003e and its root exudates, triggering an SA-dependent SAR response through interaction, rather than directly inhibiting \u003cem\u003eFusarium\u003c/em\u003e growth to prevent root rot.\u0026nbsp;Additionally, SA has been reported to induce the expression of antioxidant enzymes and increase the production of nonenzymatic antioxidants, thereby gassisting in the detoxification of ROS in plants\u0026nbsp;[49, 84].This function has also been confirmed in the prevention of \u003cem\u003eFusarium\u003c/em\u003e wilt in cucumber by Si [85]. In this study,\u0026nbsp;SiO₂ NPs significantly increased the activities of APX, CAT, and POD, while decreasing the MDA content\u0026nbsp;at a concentration of 400 mg/kg. Therefore, exogenous silicon reduces the MDA content and promotes ROS scavenging by increasing antioxidant enzyme activity, thereby reducing cellular damage and improving the resistance of infected plants\u0026nbsp;[86].\u003c/p\u003e\n\u003cp\u003eOverall, our findings suggest that the application of SiO\u003csub\u003e2\u003c/sub\u003e NPs increased the activity of defence-related enzymes and changed the diversity, composition, and functional spectrum of the rhizosphere microbial communities of \u003cem\u003eAstragalus\u003c/em\u003e. The application of SiO\u003csub\u003e2\u003c/sub\u003e NPs induced the accumulation of defensive compounds and salicylic acid, thereby conferring resistance to root rot in \u003cem\u003eAstragalus\u003c/em\u003e.\u0026nbsp;Although the controlled pot experiments in this study allowed for precise measurement of the effects of\u0026nbsp;SiO\u003csub\u003e2\u003c/sub\u003e NPs, they inevitably simplified the natural complexity of the rhizosphere environment. Field conditions, however, encompass additional variables such as microbial competition, soil heterogeneity, and climatic fluctuations, all of which could alter the observed plant‒microbe interaction patterns. Therefore, field experiments should be conducted under various seasonal conditions to assess the effects of SiO\u003csub\u003e2\u003c/sub\u003e NPs on plant resistance to soil-borne pathogens in the future.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, the application of exogenous SiO\u003csub\u003e2\u003c/sub\u003e NPs increased the resistance of \u003cem\u003eAstragalus\u003c/em\u003e to root rot, confirming that root treatment is an effective and safe application method. High-throughput sequencing and LC‒MS metabolomics techniques revealed the response of soil microbial communities and root exudates to SiO\u003csub\u003e2\u003c/sub\u003e NPs in \u003cem\u003eAstragalus\u003c/em\u003e. SiO\u003csub\u003e2\u003c/sub\u003e NPs influence the structure of the rhizosphere microbial community by increasing its diversity and reducing the relative abundances of \u003cem\u003eF. oxysporum\u003c/em\u003e and pathogenic allies (\u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eMicrobacterium\u003c/em\u003e). Some plant root exudates alleviate \u003cem\u003eAstragalus\u003c/em\u003e root rot by inhibiting the growth of pathogenic fungal helpers. The complex signalling network and interactions of phytohormones effectively regulate plant defence responses. In particular, SAR was activated through the accumulation of salicylic acid. In conclusion, this study demonstrates a promising approach for the use of SiO\u003csub\u003e2\u003c/sub\u003e NP-based plant elicitors for the effective management of \u003cem\u003eAstragalus\u003c/em\u003e root rot.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSi_S\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThe soil-applied SiO\u003csub\u003e2\u003c/sub\u003e NPs treatment\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSi_L\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThe foliage-spraying SiO\u003csub\u003e2\u003c/sub\u003e NPs treatment\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMalondialdehyde\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSuperoxide dismutase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAPX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAscorbate peroxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCAT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCatalase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePeroxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSalicylic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSAR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSystemic acquired resistance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVLCFAs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVery long-chain fatty acids\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank for the High-Performance Computing Center of Northwest A\u0026amp;F University for providing computing resources.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.A. wrote and edited the text; L.X.,\u0026nbsp;H.D., Z.D., and L.J. conduct the experiments; G.W., and Z.L. made conception and design of the study; Z.L.\u0026nbsp;reviewed and edited the final text.\u0026nbsp;All authors read and approved the version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of\u0026nbsp;China (42277317). National Key Research and Development Program of China (2021YFD1900702), and\u0026nbsp;Special funds for the Major Science and Technology of Shaanxi Province (2020zdzx03–02-01). The funders played no role in the design of the study, analysis, and interpretation of data or in writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eState Key Laboratory for Crop Stress Resistance and High-Efficiency Production, Shaanxi Key Laboratory of Agricultural and Environmental Microbiology, College of Life Science, Northwest A\u0026amp;F University, Yangling 712100, Shaanxi, People’s Republic of China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eDutta P, Kumari A, Mahanta M, Upamanya Gunadhya K, Heisnam P et al. 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Silicon enhances plant resistance to \u003cem\u003eFusarium\u003c/em\u003e wilt by promoting antioxidant potential and photosynthetic capacity in cucumber (\u003cem\u003eCucumis sativus\u003c/em\u003e L.). Frontiers in plant science. 2022;13:1011859. https://doi.org/10.3389/fpls.2022.1011859.\u003c/li\u003e\n \u003cli\u003eCarneiro-Carvalho A, Pinto T, Ferreira H, Martins L, Pereira C et al. Effect of silicon fertilization on the tolerance of Castanea sativa Mill. seedlings against Cryphonectria parasitica Barr. J Plant Dis Prot. 2020;127(2):197-210. https://doi.org/10.1007/s41348-019-00283-z.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbio","sideBox":"Learn more about [Microbiome](http://microbiomejournal.biomedcentral.com/)","snPcode":"40168","submissionUrl":"https://submission.nature.com/new-submission/40168/3","title":"Microbiome","twitterHandle":"@MicrobiomeJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Silica nanoparticles (SiO2 NPs), Root rot disease, Root exudates, Microbial communities, Metabolomics","lastPublishedDoi":"10.21203/rs.3.rs-6006553/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6006553/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Biological control mechanisms involve the inhibitory effect of antagonistic bacteria on pathogenic fungal growth. However, research on controlling crop diseases by inhibiting allies of pathogenic agents is relatively scarce. \u003cstrong\u003eResults:\u003c/strong\u003e In this study, the application of SiO\u003csub\u003e2\u003c/sub\u003e NPs resulted in an increase in the alpha diversity of the microbial communities in the rhizosphere caused by \u003cem\u003eAstragalus\u003c/em\u003e, as well as an increase in the complexity of the co-occurrence network. SiO\u003csub\u003e2\u003c/sub\u003e NPs reduced the abundance of \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eMicrobacterium\u003c/em\u003e in the rhizosphere of\u003cem\u003e Astragalus\u003c/em\u003e. Co-inoculated \u003cem\u003eFusarium\u003c/em\u003e with \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eM. oxydans\u003c/em\u003e could exacerbate the root rot of disease in \u003cem\u003eAstragalus\u003c/em\u003e. In addition, \u003cem\u003eM. oxydans\u003c/em\u003e SCK-308 and \u003cem\u003eP. aeruginosa\u003c/em\u003e XS-134-7 promoted the growth of \u003cem\u003eF. oxysporum\u003c/em\u003e and inhibited the growth of certain beneficial rhizosphere microorganisms, thereby facilitating the occurrence of the disease. Metabolomic analyses revealed that salicylic acid, indole-3-acetic acid, brassinosteroid, and palmitic acid were significantly enriched in the rhizosphere of \u003cem\u003eAstragalus\u003c/em\u003e treated with SiO\u003csub\u003e2\u003c/sub\u003e NPs. Exogenous supplementation with these metabolites significantly inhibited the growth of \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eM. oxydans\u003c/em\u003e, thereby alleviating root rot in plants during coinfection with two bacteria and \u003cem\u003eF. oxysporum\u003c/em\u003e. These results indicate that the metabolites enhance disease control efficacy through targeted inhibition of pathogen helpers. Additionally, SiO\u003csub\u003e2\u003c/sub\u003e NPs enhanced the enzymatic activities of ascorbate peroxidase, catalase, and peroxidase in \u003cem\u003eAstragalus\u003c/em\u003e plants.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Our findings suggest that SiO\u003csub\u003e2\u003c/sub\u003e NPs alter the composition of the rhizosphere microbial community and reduce the population of allies of \u003cem\u003eF. oxysporum\u003c/em\u003e, activating salicylic acid-dependent systemic acquired resistance (SAR) in \u003cem\u003eAstragalus\u003c/em\u003e and thereby decreasing the incidence of \u003cem\u003eFusarium\u003c/em\u003e root rot. These results suggest that SiO\u003csub\u003e2\u003c/sub\u003e NPs can serve as a sustainable agricultural practice.\u003c/p\u003e","manuscriptTitle":"Silica Nanoparticles Suppress Fungal Pathogenic Allies to Alleviate Astragalus Root Rot","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-30 15:50:23","doi":"10.21203/rs.3.rs-6006553/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-25T08:46:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-17T23:38:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-01T07:42:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"336435769183449048949561259313889730765","date":"2025-04-29T17:39:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"67236894251721958167401947429769117860","date":"2025-04-29T15:31:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-29T07:18:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-28T12:28:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbiome","date":"2025-04-26T10:36:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbio","sideBox":"Learn more about [Microbiome](http://microbiomejournal.biomedcentral.com/)","snPcode":"40168","submissionUrl":"https://submission.nature.com/new-submission/40168/3","title":"Microbiome","twitterHandle":"@MicrobiomeJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7dc9150a-a5df-4cb7-bc3d-2fb72bf5e66f","owner":[],"postedDate":"April 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-27T16:30:13+00:00","versionOfRecord":{"articleIdentity":"rs-6006553","link":"https://doi.org/10.1186/s40168-025-02183-x","journal":{"identity":"microbiome","isVorOnly":false,"title":"Microbiome"},"publishedOn":"2025-10-21 16:16:28","publishedOnDateReadable":"October 21st, 2025"},"versionCreatedAt":"2025-04-30 15:50:23","video":"","vorDoi":"10.1186/s40168-025-02183-x","vorDoiUrl":"https://doi.org/10.1186/s40168-025-02183-x","workflowStages":[]},"version":"v1","identity":"rs-6006553","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6006553","identity":"rs-6006553","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}