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 of
   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 Pseudomonas aeruginosa and Microbacterium 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
   Fusarium 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.
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Supplementary information

   The online version contains supplementary material available at
   10.1186/s40168-025-02183-x.

   Keywords: Silica nanoparticles (SiO[2] NPs), Root rot disease, Root
   exudates, Microbial communities, Metabolomics

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 [[41]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 [[42]2], maize [[43]3], and Astragalus [[44]4]. The
   constraints in early diagnosis, persistence of pathogenic agents in the
   soil, substantial genetic diversity, and broad host range make
   soil-borne pathogens management cumbersome and difficult [[45]5].
   Contemporary intensive farming systems using high levels of chemical
   fertilizers have disrupted the balance of soil microbial ecosystems,
   leading to disrupt the beneficial microbial populations and aggravate
   the prevalence of crop diseases [[46]6, [47]7]. Therefore, scientists
   have tried to transplant protective microbiomes from resistant plants
   to susceptible plants that regulate the rhizosphere microbiota and
   promote plant health. However, due to unstable colonization, soil type,
   plant development stage, genotype, etc., the success rate of
   rhizosphere microbiota transplantation (RMT) is about 10–20% [[48]8,
   [49]9]. Although the development of disease-resistant crop varieties
   can substantially mitigate these issues, the initial investment
   requirements are typically substantial and take a long time [[50]10,
   [51]11]. Nanotechnology provides a suitable alternative to overcome
   these challenges [[52]12]. 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 [[53]13]. In addition, silica
   nanoparticles (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 [[54]14,
   [55]15]. However, the exploration of SiO[2] NPs for biological control
   of soil-borne diseases is still limited, and the mechanism of its
   disease prevention needs further study to enhance our understanding.

   It was initially proposed that silicon deposition creates a physical
   barrier beneath the leaf cuticle to prevent pathogen invasion [[56]16].
   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 [[57]17]. 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 [[58]18]. 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 antagonists, some bacteria
   can promote the growth of pathogens. This is in line with the recent
   finding that 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 [[59]19–[60]21]. For example,
   Microbacterium paraoxydans and Pseudomonas syringae serve as bacterial
   helpers for Ralstonia solanacearum and Fusarium, respectively,
   exacerbating disease severity [[61]22, [62]23].

   In addition, alterations in the levels or composition of root exudates
   can influence plant health by regulating pathogen populations and
   inducing plant resistance [[63]24]. 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 [[64]25]. 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) [[65]26]. 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) [[66]18]. The
   phenomenon of silicon-induced SAR in plants has also been corroborated
   in tomato, rice, and other crops [[67]27, [68]28]. 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
   defense against soil-borne pathogens [[69]29]. 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
   defenses.

   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 [[70]30]. 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 SiO₂ NP-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.

Materials and methods

Effect of SiO[2] NPs on Fusarium oxysporum 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., UK). 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,
   according to the method described by Feng [[71]31], the dispersion
   stability of SiO[2] NPs in water was determined by observing the
   homogeneous of 1500 mg/L SiO[2] NPs suspension within 2 days. The
   solution was considered to be stable if the SiO[2] NPs solution did not
   precipitate. The fungal pathogen Fusarium oxysporum used in this study
   was originally isolated from field-grown Astragalus plants exhibiting
   root rot symptoms [[72]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.

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 were added to the substrate
   (soil: vermiculite = 3: 1, v/v) to make their final concentrations 200,
   400, and 800 mg/kg respectively. Then 500 g of substrate containing
   SiO[2] NPs was filled into a 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
   7 days, the seedlings in the planting bags were thinned to five uniform
   plants. The foliage-spraying SiO[2] NPs treatment (Si_L) was performed
   as follows: SiO[2] NPs 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 7 days for a total of 5 times [[73]32]. 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 milliliter) 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% [[74]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.

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 min to collect the rhizosphere soil. After the roots
   were removed, the tubes were centrifuged at 8000 rpm for 7 min 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 515 F (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
   [[75]33] 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 [[76]34]. The
   relative abundance of the ASVs was calculated in the R environment
   (version: V4.4.1, [77]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 was uploaded to NCBI GenBank accession
   numbers [78]PV505490-PV505497.

Isolation and reinoculation of core microorganisms

   The bacteria were isolated from the rhizosphere soil of Astragalus
   using the method outlined by Oppenheimer-Shaanan [[79]35] with minor
   modifications. Fresh rhizosphere soil was suspended in PBS and shaken
   at 180 rpm for 30 min. The dilutions were plated on LB medium
   solidified with 1.5% agar and then incubated at 30 °C for 2‒7 days
   [[80]36]. 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)
   [[81]37]. The obtained sequences were aligned with the sequences of
   core ASVs in the co-occurrence network. Phylogenetic trees were
   constructed with MEGA software (v5) using the neighbor-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 [[82]38]. 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 milliliter) was
   generated. The disease index and control effect were calculated after
   21 days of inoculation with F. oxysporum.

Metabolite extraction and LC‒MS/MS analysis

   The Astragalus seedlings used to collect root exudates were derived
   from the pot experiment described in “[83]Plant growth conditions and
   plant treatments” section. After collecting the rhizosphere soil, the
   roots of Astragalus seedlings were washed with DI water and soaked for
   2 h to ensure that the impurities on the root surface are cleaned.
   Then, the seedlings were placed in sterile DI water in the dark for
   24 h to collect root exudates of the Astragalus plants [[84]39]. Five
   replicates were set for each treatment, each replicate containing ten
   seedlings. This ensures that the rhizosphere soil samples, and root
   exudates are derived from the same Astragalus plants. 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
   [[85]40]. 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 [[86]41]. 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 ([87]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
   ([88]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 [[89]42].

Effects of metabolites on the resistance of Astragalus to root rot

   Plant-soil-microbiota interactions mediated by root exudates drive the
   rhizosphere microbial assembly and ultimately affect plant health. By
   comparing the relative abundance of root exudates of control and
   Si_S-treated plants to identify differentially abundant metabolites.
   This comparison aimed to reveal how Si_S treatment influences the
   metabolic pathways of plant root exudates, thereby uncovering potential
   disease resistance mechanisms. 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:
   72,962–43-7, 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 h. 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 hemocytometer. 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
   milliliter), 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).

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
   [[90]43]. The Sloan neutral model was applied to evaluate the effects
   of random dispersal and ecological drift on the assembly of microbial
   communities [[91]44]. Phylogenetic bin-based null model analysis
   (iCAMP) was used to infer community assembly mechanisms [[92]45].

   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 [[93]46]. 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) [[94]47]. 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^2, and 95%
   confidence intervals for the model parameters were derived through
   bootstrapping with 1000 replicates.

Results

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. S[95]1a). 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. S[96]1b). 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. S[97]1c). Furthermore, experiments on the stabilities
   of SiO[2] NPs were carried out (Fig. S[98]1d). The results showed that
   the solution of SiO[2] NPs did not show the formation of aggregation
   between 0 and 12 h. Instead, after 30 h, the SiO[2] NPs solution forms
   obvious settlement. 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] NPs 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. [99]1a).

Fig. 1.

   [100]Fig. 1
   [101]Open in a new tab

   SiO[2] NPs enhance Astragalus resistance to Fusarium root rot. a
   Disease indices and prevention effects of Astragalus under distinct
   treatments at 21 days after inoculation. Si_S: Soil application of
   SiO[2] NPs; Si_L: Foliar spray of SiO[2] NPs. b Effects of SiO[2] NPs
   on growth and sporulation of F. oxysporum. c Effects of foliar spraying
   with different concentrations of SiO[2] NPs on enzyme activities of
   Astragalus. d Effects of soil application of various concentrations of
   SiO[2] NPs on the enzyme activities of Astragalus. e Si contents of
   above- and belowground Astragalus under various treatments. Lowercase
   letters above the bars indicate significant differences (p < 0.05)
   between the different treatments (one-way ANOVA)

   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. [102]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. [103]1c, d). This finding aligns with the observed
   accumulation of silicon in Astragalus, which peaked when SiO[2] NPs
   were applied at a concentration of 400 mg/kg (Fig. [104]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. S[105]2a). 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 Astragalus (Fig. S[106]2b).
   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.

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 1731 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. [107]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 Astragalus under
   SiO[2] NPs application (Fig. [108]2b and c). 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. [109]2d and Fig. S[110]3). The Si_L treatment significantly
   decreased the relative abundance of Actinobacteriota and increased the
   abundance of Crenarchaeota (ANOVA; p < 0.05).

Fig. 2.

   [111]Fig. 2
   [112]Open in a new tab

   Effects of SiO[2] NPs on the microbial community composition in the
   rhizosphere of Astragalus. a Changes in the α diversity indices of
   rhizosphere bacteria and fungi under different SiO[2] NPs application
   methods. Si_S: Soil application of SiO[2] NPs; Si_L: Foliar spray of
   SiO[2] 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 Fusarium, the
   disease index, and the prevention effect. f RDA of the bacterial and
   fungal microbial communities

   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) (Figs.
   S[113]4–S[114]7). Notably, the relative abundance of Fusarium, the
   average disease index, and the prevention effect were significantly
   correlated with these 59 bacterial genera (Fig. [115]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. [116]2f). The alpha
   diversity of bacteria was significantly positively correlated with the
   silicon contents in both the roots and above-ground parts of
   Astragalus, 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. [117]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. [118]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.

Fig. 3.

   [119]Fig. 3
   [120]Open in a new tab

   Impact of SiO[2]NPs on the functional and assembly mechanisms of the
   microbial community. a Co-occurrence network analysis. Si_S: Soil
   application of SiO[2] NPs; Si_L: Foliar spray of SiO[2] NPs. b
   Topological parameters of co-occurrence networks with different
   samples. c Responses of microbial community functions to different
   SiO[2] NPs application methods. A heatmap was generated after
   standardizing the prediction results from PICRUSt. d Effects of SiO[2]
   NPs on the assembly mechanisms of microbial communities

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. [121]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 toward 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. [122]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. [123]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 NPs treatment applied selective pressure on
   the survival and reproduction of bacterial communities, which in turn
   facilitated shifts in community composition and species diversity.

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. S[124]8). Eight core ASVs
   were successfully isolated from rhizosphere soil, with greater than 97%
   sequence similarity between the isolated strains and the ASVs
   (Fig. [125]4a, Fig. S[126]9). 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. [127]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. [128]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. [129]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. S[130]10).
   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. S[131]11).
   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.

Fig. 4.

   [132]Fig. 4
   [133]Open in a new tab

   Effects of core microorganisms on Astragalus root rot. 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[2] NPs; Si_L: Foliar spray of
   SiO[2] NPs. c Relative abundances of core ASVs at the genus level. d
   Influence of core microorganisms on the disease index and plant
   phenotype

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. [134]5 a
   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. [135]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. [136]5d). The significantly
   upregulated metabolites included mainly lipids and lipid-like
   molecules, phenylpropanoids and polyketides, and organic acids and
   their derivatives (Fig. S[137]12). 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 analyzed (Fig. [138]5e). These pathways included five upregulated
   differentially abundant metabolites, and one was downregulated in the
   Si_S-treated group (Fig. [139]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 analyzed the
   pathways and metabolites related to the plant hormone signal
   transduction (Fig. [140]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.

Fig. 5.

   [141]Fig. 5
   [142]Open in a new tab

   Effects of SiO[2] NPs on root exudates and KEGG enrichment pathway
   analysis of Astragalus. a, b PCA and partial least-squares discriminant
   analysis of root exudates. Si_S: Soil application of SiO[2] NPs. c
   Analysis of possible sources of metabolites. d Volcanic plot of
   differentially abundant metabolites in control and SiO[2] 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

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 [143]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. [144]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.

Fig. 6.

   [145]Fig. 6
   [146]Open in a new tab

   Associations between the metabolome and microbiome. a Mantel test
   showing the significant relationships (i.e., Mantel’s p based on 999
   permutations, p < 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 [147]R00679 and
   [148]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 F. oxysporum

   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 ([149]R00679 and [150]R00673)
   (Fig. [151]6b, c). For the biosynthesis of the siderophore group
   nonribosomal peptide pathway, the differentially abundant metabolite
   salicylic acid was involved in the [152]R06602 metabolic reaction of
   this pathway (Fig. [153]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 [154]R00679 and [155]R00673 and positively
   correlated with l-tryptophan, indole-3-acetamide, and indole
   (Fig. [156]6 and S[157]13). In addition, Pseudomonas was identified as
   a significant genus closely associated with the metabolic reaction
   [158]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
   [159]R01274 metabolic reaction of this pathway has a negative
   correlation (Fig. [160]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.

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. [161]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. [162]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. S[163]14). 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.”

Discussion

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 [[164]48] (Fig. [165]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 [[166]49]. 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] NPs 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. [167]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 [[168]14]. Therefore, root treatment
   is a safer and more effective method for applying SiO₂ NPs. Compared
   with SiO₂ NPs treatment, carbendazim treatment significantly inhibited
   the progression of root rot, achieving a disease index control rate of
   71.54% (Fig. [169]1a), demonstrating superior disease prevention
   efficacy (57.70%). However, persistent carbendazim residues in soil
   pose bioaccumulation risks and potential groundwater contamination
   [[170]50]. In this study, the application of SiO[2] NPs did not have a
   negative impact on plant growth, and Kumari [[171]51] also found that
   SiO[2] NPs could improve soil fertility and Zea mays growth, suggesting
   the low toxicity and potential benefits for sustainable agriculture.
   This is similar to what others have discovered that SiO[2] NPs have
   minimal negative impacts on environmental health and crop food safety
   due to its chemical inertness [[172]13]. 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] NPs
   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] NPs needs to be
   further explored before its application in the field.

Fig. 7.

   [173]Fig. 7
   [174]Open in a new tab

   Possible mechanisms of SiO[2] NP-induced enhanced resistance to root
   diseases caused by soilborne fungi. Application of SiO[2] NPs to the
   soil alters the root exudates of Astragalus, increasing the secretion
   of plant hormones, thereby activating salicylic acid-dependent systemic
   acquired resistance (SAR). This also enhances the activity of
   antioxidant enzymes, reducing cellular damage and improving plant
   resistance. Furthermore, the altered root exudates reduce the relative
   abundance of Fusarium and its “helpers” (Pseudomonas and
   Microbacterium), thus enhancing the resistance of Astragalus to root
   rot

   Plants take up silicon from the soil and subsequently transport it from
   roots to shoots either passively (transpirational stream) or actively
   (specific transporter proteins) [[175]49, [176]52]. Therefore, compared
   with the control, the soil-applicant SiO[2] NPs also significantly
   increased the Si content of the shoot (Fig. S[177]2b). 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 [[178]53].
   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 [[179]54]. 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 caused
   plants to accumulate more silicon through their root absorption, 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.

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 [[180]55, [181]56]. Previous
   study showed that the resistance to pathogenic colonization greatly
   increased with community diversity [[182]57]. 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 [[183]58, [184]59]. Silicon
   can improve plant disease resistance through three principal
   mechanisms. The first silicon can directly inhibit the growth of
   pathogens to protect plants [[185]60]. 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 [[186]24]. 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 [[187]61–[188]63]. The SiO[2] NPs used
   in this study had no direct toxicity to F. oxysporum (Fig. [189]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. [190]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 [[191]64] and Sphingobium [[192]65], 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
   [[193]66], were significantly reduced (Fig. [194]4b). Crucially, both
   genera presented significant negative correlations with protection
   rates, whereas Microbacterium presented a markedly positive correlation
   with disease indices (Fig. [195]2e). As evidenced by multiple reports,
   P. aeruginosa has been identified as a natural soil inhabitant and a
   potential plant pathogen [[196]67, [197]68]. 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 [[198]22]. 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. [199]6g). Numerous
   studies have shown that the increase in the antagonistic bacteria was
   found to enhance plant resistance to pathogen [[200]69]. 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
   [[201]22, [202]70–[203]72]. 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 [[204]22]. 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
   [[205]73]. 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 [[206]74]. 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. [207]3a, b). The application of
   SiO[2] NPs enhanced stochastic processes (dispersal limitation) during
   bacterial community construction and deterministic processes in fungal
   communities (Fig. [208]3d). Dispersal limitation usually refers to a
   low diffusion rate coupled with drift or weak selection, which may
   increase community variation or turnover [[209]75]. 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.

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 [[210]76], plant hormones [[211]77], and antimicrobial
   compounds [[212]78]. 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
   [[213]79]. Notably, the application of SiO[2] NPs increased the
   accumulation of phenylpropanoids and polyketide substances in our
   research (Fig. S[214]15). Secondary metabolites derived through the
   phenylpropanoid pathway, such as phenols and flavonoids, are well-known
   for their defensive roles against fungal pathogens [[215]80]. 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 [[216]25]. Our results
   showed that applying SiO[2] NPs significantly increased the
   accumulation of the antimicrobial compounds naringenin [[217]81],
   sinapyl alcohol, and gallocatechin gallate [[218]82]. Among these,
   sinapyl alcohol is a critical monomer in the lignin biosynthetic
   pathway, and plays a role in cell wall lignification [[219]83].
   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 [[220]48].
   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 [[221]18]. In this study, salicylic acid, auxin,
   and brassinolide were significantly enriched in plant hormone signal
   transduction pathways as differential substances (Fig. [222]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 [[223]84]. 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 [[224]85]. 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
   [[225]86]. 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 [[226]87]. 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 assisting in the detoxification of ROS in plants
   [[227]13, [228]88]. This function has also been confirmed in the
   prevention of Fusarium wilt in cucumber by Si [[229]89]. 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
   [[230]90].

   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.

Supplementary Information

   [231]Supplementary Material 1.^ (15.4MB, docx)
   [232]Supplementary Material 2.^ (605.5KB, xlsx)

Acknowledgements