Abstract

   Fusarium wilt, caused by Fusarium oxysporum f. sp. niveum (Fon), poses
   a significant threat to watermelon production globally. Traditional
   control methods often rely on chemical fungicides, which pose
   environmental risks and limited long-term efficacy. This study
   introduces biogenically-synthesized manganese nanoparticles (MnNPs) as
   a potent antifungal agent for managing Fusarium wilt. MnNPs were
   synthesized extracellularly using the culture supernatant of
   Lysinibacillus sphaericus NOTE11, a Mn-resistant bacterial strain
   isolated and characterized in this study. Comprehensive physicochemical
   analyses confirmed their crystalline structure, spherical morphology,
   and elemental composition. MnNPs demonstrated potent antifungal
   activity, significantly inhibiting Fon growth, conidiation, and
   conidial germination in vitro, with 100 µg/mL MnNPs reducing hyphal
   growth by 21.97% and conidial germination by 80% compared to untreated
   controls. Disease assays further confirmed that MnNPs significantly
   reduced Fusarium wilt severity in watermelon (~ 84%) compared with
   Fon-infected controls, with MnNP-treated infected-plants exhibiting
   minimal symptoms and reduced invasive fungal biomass in within
   watermelon tissues. Transcriptomic analysis revealed that MnNPs
   downregulated genes in the fusaric acid biosynthesis pathway, a key
   determinant of Fon virulence, disrupting its ability to infect host
   plants. Additionally, MnNPs modulated rhizosphere metabolites,
   enriching defense-related compounds, including phenolics, flavonoids,
   and organic acids. These findings establish MnNPs as a robust and
   impactful strategy for managing Fusarium wilt. By integrating
   nanotechnology and plant-rhizopshere interactions, this study provides
   a novel approach to mitigating soilborne diseases, emphasizing the
   potential of nano-enabled disease management approaches to enhance crop
   protection and sustainability in agriculture.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12951-025-03492-x.

   Keywords: Fusaric acid, Fusarium wilt, Manganese nanoparticles,
   Rhizosphere metabolome, Watermelon

Introduction

   Nanotechnology offers innovative tools for addressing agricultural
   challenges, including the management of plant diseases [[50]1, [51]2,
   [52]3]. Although a robust literature is available on the
   disease-controlling potential of chemically-synthesized nanoparticles
   (NPs) in various crops such as tomato (Solanum lycopersicum), wheat
   (Triticum aestivum), and rice (Oryza sativa) [[53]4, [54]5, [55]6,
   [56]7]; however, earlier studies often reported biocompatibility and
   environmental safety concerns of conventional chemical-based
   nano-agrochemicals. For example, chemically-synthesized copper NPs
   (450 mg/L) reduced root elongation up to 28% through increasing cell
   wall rigidity in lettuce (Lactuca sativa L.) compared with untreated
   controls [[57]8]. Similarly, chemically-synthesized silver NPs (20 ppm)
   inihibited the root weight (34%), stem length (43%), and seedling
   height (40%) in cucumber plants (Cucumis sativus L.) than untreated
   control plants [[58]9]. Recently, biologically-synthesized NPs have
   garnered significant attention for their eco-friendly and sustainable
   applications [[59]10, [60]11]. Biological synthesis methods, especially
   using bacteria, offer greater functional benefits and biocompatibility
   to NPs, thus replacing their chemically-synthesized counterparts
   [[61]12, [62]13, [63]14]. Biogenic maganese (Mn) NPs, synthesized
   through microbial processes, stand out for their remarkable
   antimicrobial properties and minimal environmental impact. These NPs
   exhibit potent activity against a range of phytopathogens, including
   Fusarium oxysporum f. sp. niveum (Fon), watermelon (Citrullus lanatus
   L.) Fusarium wilt pathogen [[64]12, [65]15]. Their antifungal efficacy
   is attributed to mechanisms such as disruption of pathogen cell
   membranes, inhibition of cellular processes, and modulation of
   oxidative stress [[66]16]. For example, biogenic MnNPs inhibited the
   growth of bacterial leaf blight pathogen, Xanthomonas oryzae pv.
   oryzae, by inducing oxidative damage and deforming cell wall [[67]15].
   Unlike conventional fungicides, Mn-based nanoagrochemicals not only
   mitigate pathogen growth but also augment plant nutrient supply and
   physiology, improving its growth and development [[68]17]. For example,
   MnNPs (1 mg/plant) improved the chlorophyll content (~ 20%) and
   photosynthesis rate (12%), ehancing the biomass of cucumber plants by
   ~ 21% [[69]18]. Given facts suggest biogenic MnNPs as a promising tool
   for sustainable disease management in agriculture, offering an
   innovative solution to combat one of the most devastating diseases in
   watermelon cultivation.

   Fon is a significant pathogen of watermelon, causing Fusarium wilt,
   which results in severe yield losses in this crop globally (up to 100%)
   [[70]19]. The pathogenicity of this strain is strongly correlated to
   the production of secondary metabolites, including mycotoxins (such as
   fusaric acid), effector proteins (such as secreted in xylem), and
   enzymes (such as cellulases, xylanases, and pectinases) that aid in
   host tissue colonization and disease progression [[71]20, [72]21,
   [73]22, [74]23]. These metabolites play essential roles in the
   physiological processes and virulence of Fon by facilitating the
   breakdown of plant cell walls and enhancing its survival in hostile
   environments [[75]24, [76]25]. As watermelon cultivars with inherent
   resistance to Fon are limited, soil treatment with chemical fungicides
   remain the primary method for managing Fusarium wilt. However,
   overreliance on these chemical treatments has raised serious
   environement safety concerns due their off-target effects and excessive
   buildup in soil, which compromises their repeated application in
   agriculture fields and exacerbates the problem of mycotoxin
   accumulation in infected plants [[77]12, [78]26, [79]27]. Recent
   studies suggest that alternative control methods, including the use of
   multifarious engineered NPs, may provide promising solutions [[80]28,
   [81]29, [82]30]. For example, cerium oxide NPs (250 mg/L) significantly
   suppressed Fusarium wilt upto 53% by augmenting antioxidative defense
   machinery (catalase and polyphenol oxidase) in F. oxysporum f. sp.
   lycopersici-infected tomato plants [[83]31]. Similarly, 100 µg/mL
   biogenic magnesium NPs inhibited Fon hyphal growth (~ 40%) by directly
   disrupting pathogen cell envelope, conidiation, and conidial
   germination [[84]30]. However, the mechanisms by which they influence
   secondary metabolite production and fungal virulence remain poorly
   understood. Given these facts, furture studies should investigate the
   safety and efficacy of nanomaterials for disease management and to
   understand their impacts on fungal biology.

   Among secondary metabolites, fusaric acid is pivotal for regulating
   pathogenicity and biology of phytopathogenic fungi [[85]21]. This
   phytotoxic compound has been shown to contribute to the virulence of
   Fon by impairing biochemical activities in plants such as respiration
   and nutrient acquisition, thus facilitating fungal infection [[86]32].
   Notably, fusaric acid has been implicated in the inhibition of plant
   immune responses, allowing the fungus to establish and maintain
   infection within the host [[87]33]. Previous studies have revealed that
   fusaric acid production is critical for fungal growth and sporulation,
   and its presence in infected tissues correlates with disease severity.
   Importantly, fusaric acid has been shown to enhance the ability of
   Fusarium species to colonize plant vascular systems, contributing to
   the development of wilt diseases [[88]34, [89]35]. To date, a cluster
   of 12 fusaric acid biosynthetic (FUB) genes has been identified and
   reported to regulate various physiological and biochemical processes in
   phytopathogenic fungi [[90]36]. Studies have indicated that deletion of
   FcFUB1 in Fusarium circinatum reduced fusaric acid production and
   pathogenicity on Pinus patula [[91]35]. Similarly, in F. oxysporum f.
   sp. cubense, disruption of FocFub4 induced defects in virulence on
   banana (Musa spp.) plants [[92]33]. These findings underscore the
   pivotal role of fusaric acid in pathogenicity of fungal phytopathogens
   and highlight its potential as a target for disease control strategies.

   During infection, pathogens alter the composition and abundance of
   rhizosphere metabolites to favor the disease progression [[93]37]. On
   the other hand, plants release a variety of antimicrobial metabolites,
   including organic acids, phenolics, and phytohormones, to antagonize
   pathogen growth or to enhance disease resistance mechanisms. These
   metabolites, including phenolic compounds, organic acids, and volatile
   organic compounds, have been shown to either suppress or enhance
   pathogen growth [[94]38, [95]39, [96]40]. Understanding the intricate
   networks between rhizosphere metabolites and NP-plant-Fon triad is
   vital for developing sustainable crop protection strategies. Although
   antagonistic potential of metallic NPs against Fon has been previously
   described, the specific molecular mechanisms driving these effects
   remain largely unexplored. In this study, we biosynthesized MnNPs using
   cell-free filtrate of a locally-isolated bacterial strain
   Lysinibacillus sphaericus NOTE11, showing higher resistance to
   Mn-containing precursor salt than other bacterial isolates.
   Specifically, our study investigated the molecular inhibition mechanism
   of MnNPs on Fon, focusing on their impact on fungal growth and
   virulence. Transcriptomic and metabolomic studies revealed that MnNPs
   significantly reduced hyphal growth, conidiation, and conidial
   germination by dysregulating key FUB genes, as well as suppressed Fon
   pathogenicity on watermelon plants by precisely regulating
   disease-suppressive rhizosphere metabolites. Moreover, we identified
   that MnNPs induced cellular damage, disrupted cell wall integrity, and
   inhibited key molecular pathways involved in fungal stress responses.
   Taken together, our study elucidates the antifungal potential of MnNPs
   and provides new insights into their molecular mechanisms of action
   against Fon, suggesting a better and sustainable alternative for
   watermelon Fusarium wilt management.

Materials and methods

Isolation and molecular identification of Mn-resistant bacteria

   Bacterial strains resistant to Mn were isolated from the rhizosphere of
   watermelon plants using the serial dilution method. Briefly,
   rhizosphere soil samples were serially diluted in sterile saline
   solution (0.85% w/v) [[97]41]. Aliquots (100 µL) from two different
   dilutions (i.e., 10^− 3 and 10^− 6) were plated onto manganese chloride
   (MnCl[2].4H[2]O, 99% purity)-supplemented nutrient agar and incubated
   at 28 ± 2 °C for 72 h. Following incubation, grown bacterial colonies
   were purified and tested for their resistance to Mn using the minimum
   inhibitory concentration (MIC) method [[98]42]. To characterize the
   strains molecularly, 16 S rRNA, commonly used for bacterial phylogeny,
   and the RNA polymerase beta subunit (rpoB), which provides more
   specific resolution at the species level, were apmilfied from the
   extracted DNA with fD1/rD1 and rpoB-F/rpoB-R primer pairs, respectively
   (Table [99]S1) [[100]43, [101]44]. The resulting sequences were
   analyzed using NCBI BLASTn to identify evolutionarily-linked bacterial
   species. Phylogenetic relationships were then inferred using the
   maximum likelihood method with bootstrap algorithm (1000 replicates),
   and evolutionary trees were constructed using MEGA7 software to align
   the sequences with homologous counterparts.

MnNPs biosynthesis and characterization

   MnNPs were extracellularly biosynthesized using the cell-free filtrate
   of the Mn-resistant strain, as described previously [[102]12]. Briefly,
   the 2-d old broth culture of Mn-resistant strain was centrifuged
   (5000×g) to obtain the extracellular metabolite-rich supernatant for
   MnNP synthesis. Following centrifugation, the cell-free filtrate and an
   equal volume of a Mn precursor solution (0.01 M MnCl[2].4H[2]O) was
   co-incubated at 28 ± 2 °C for an additional 72 h, resulting in the
   formation of MnNPs. The purified MnNPs were freeze-dried, homogenized,
   and stored in a sealed container for further analysis.

   The biosynthesized MnNPs were characterized for their physicochemical
   properties using a range of analytical techniques, as described
   previously [[103]45]. Fourier-transform infrared (FTIR) spectroscopy
   (Thermo Scientific NICOLET iS50FT-IR) was utilized to detect functional
   groups responsible for stabilizing the MnNPs and their interactions
   with biomolecules from the culture supernatant, within the
   4000–500 cm − 1 spectral range. For analyzing crystallinity and purity
   of the MnNPs, X-ray diffraction (XRD) analysis was performed using an
   XPertPro diffractometer (Almelo, the Netherlands). To determine shape,
   size range, and elemental composition of the MnNPs, energy-dispersive
   X-ray spectroscopy (EDS; Oxford Instruments, High Wycombe, UK)-coupled
   scanning electron microscopy (SEM; TM1000, Hitachi, Tokyo, Japan) was
   employed. For further analyzing ultrastructure and morphology at higher
   resultion, transmission electron microscopy (TEM; 1230 JEOL, Akishima,
   Japan) was performed.

In vitro antifungal activity and morphological observations

   The antagonistic potential of MnNPs against Fon were assessed on solid
   potato dextrose agar (PDA) media (200 g potato extract, 20 g glucose
   and 15 g agar per liter of ddH[2]O), with varying concentrations of
   bio-MnNPs (0, 25, 50, 100 µg/mL) as described previously [[104]12]. The
   antifungal activity was evaluated by calculating the percentage of
   growth inhibition based on radial growth diameter on PDA at 9 d
   post-inoculation (dpi). SEM (TM1000, Hitacchi, Tokyo, Japan) and TEM
   (1230 JEOL, Akishema, Japan) were used to examine changes in Fon
   morphology after treatment with bio-MnNPs. For microscopic
   observations, fungal hyphae grown on PDA with or without MnNPs were
   collected, washed, and processed as described previously [[105]30].
   Samples from six individual repeats per treatment were analyzed using
   TEM/SEM, with at least five fields examined per sample. The effects of
   bio-MnNPs on Fon conidiation, conidial length, conidial morphology,
   septal number, and germination were also assessed, with control
   experiments using untreated and bulk Mn-treated Fon as described
   previously [[106]30].

In planta disease and fungal biomass assays

   Watermelon plants of a susceptible cultivar (Zaojia) were used for
   disease assays, as described previously [[107]12]. Briefly, watermelon
   plants (3-week-old) were root inoculated with the Fon inoculum
   (5 × 10^5 spores/mL) [[108]46, [109]47]. The inoculated seedlings were
   then replanted in fresh potting mix, followed by treatment with 50 mL
   of MnNPs or bulk Mn (99.8% purity, particle size 60–300 μm) suspensions
   (100 µg/mL), as described previously [[110]12, [111]26]. To maintain
   conditions conducive to disease development, the plants were covered
   with cling film. Untreated mock-inoculated and Fon-inoculated plants
   served as controls, and disease severity was evaluated at 21 dpi using
   a previously reported 4-point scale [[112]47].

   In planta fungal biomass was assessed following an established protocol
   [[113]48]. Briefly, watermelon seedlings (3-week-old) treated with or
   without MnNPs and bulk Mn were exposed to Fon inoculum (5 × 10^5
   spores/mL) and maintained at 26 °C under shaking conditions (95 rpm).
   Plant tissues (roots and stems) were periodically collected (3, 6, and
   9 dpi), and relative invasive fungal growth was estimated by
   quantifying the genomic DNA levels of the FonOpm12 (Fon)/ClRps10
   (watermelon) using qPCR with gene-specific primers (Table [114]S1).

Transcriptomic analysis

   The Fon cultures were grown in PD broth media with three replicates at
   26 ℃ and 250 rpm. At 36 h post-incubation, Fon cultures were supplied
   with MnNPs (100 µg/mL) and incubated for another 12 h. After
   incubation, untreated and MnNP-treated Fon cultures were harvested,
   frozen in liquid nitrogen, and stored at -80 ℃ for subsequent
   transcriptome analysis. Solexa sequencing (Illumina) and data analysis
   were performed by Novogene (Beijing, China). Clean reads without low
   quality and duplicate sequences were then mapped to the reference
   genome (PRJNA18813) using TopHat2 software (v2.0.12) [[115]49]. The
   FPKM (Fragments per Kilobase per Million mapped reads) values
   indicating gene expression levels were determined using RSEM program
   [[116]50]. Differentially expressed genes (DEGs), with a fold
   change ≥ or ≤ 2 and a p-value ≤ 0.05, were determined using the DESeq R
   package [[117]51]. Kyoto Encyclopedia of Genes and Genomes (KEGG) and
   Gene Ontology (GO) enrichment analyses of the DEGs were conducted using
   the KEGG database ([118]http://www.genome.jp/kegg/) and GOseq R
   package, respectively [[119]52].

Generation of targeted deletion mutants and comparative growth assays

   Gene replacement fragments for FonFUB5, FonFUB8, FonFUB11, and FonFUB12
   were constructed by amplifying their 5′ and 3′ flanking regions using
   specific primers (Table [120]S1) and fusing them with a
   hygromycin-resistant cassette (HPH) through overlap polymerase chain
   reaction (PCR) as described previously [[121]53]. The purified
   constructs were introduced into wild-type (WT) protoplasts following
   previously established protocols. Positive transformants were selected
   on PDA plates containing hygromycin B (100 µg/mL) and verified through
   PCR using primers listed in Table [122]S1.

   For comparative growth assays, WT and deletion mutants were grown on
   PDA plates supplied with MnNPs, bulk Mn, or sterile ddH[2]O (controls)
   and alterations in growth rates were measured at 9 dpi.

Metabolomic profiling of rhizosphere soil

   Non-targeted metabolomic analysis of healthy and Fon-infected
   rhizosphere soil samples supplied with or without MnNPs was conducted
   by Central Laboratory of Zhejiang Academy of Agricultural Sciences,
   State Key Laboratory for Managing Biotic and Chemical Threats to the
   Quality and Safety of Agro-products (Hangzhou, China) using gas
   chromatography-mass spectrometry (GC–MS) as described previously
   [[123]54]. To ensure accuracy, quality control (QC) samples were
   included every 10 samples, prepared by pooling equal amounts of extract
   from each sample. The analysis was performed on a 7890B-5977 A GC/MS
   instrument (Agilent Technologies Inc., Santa Clara, CA, USA). Raw data
   were converted to mzXML format using Proteo Wizard software (version
   3.0.8789), and batch effects were minimized by normalization with QC
   samples. Metabolites with a relative standard deviation exceeding 30%
   in the QC samples were excluded from further analysis.

   Data profiling was performed by Central Laboratory of Zhejiang Academy
   of Agricultural Sciences (Hangzhou, China). Molecular weights were
   determined using mass-to-charge ratios (m/z) of parent ions, with
   molecular formulas predicted based on parts per million (ppm) and
   adduct ions. These were compared against database entries for
   identification through MS analysis. Fragment ion matching from MS/MS
   data provided further confirmation. Differentially abundant metabolites
   (DAMs) were analyzed using Metabo Analyst, and metabolic pathways were
   visualized using the KEGG Mapper tool, referencing the National
   Institute of Standards and Technology (NIST) standard spectrum.

RNA extraction and expression analysis

   RNA extraction was carried out using TRIzol reagent (Vazyme Biotech,
   Nanjing, China), and complementary DNA (cDNA) was synthesized with the
   HiScript II QRT SuperMix kit (Vazyme Biotech, Nanjing, China).
   Quantitative PCR (qPCR) reactions were prepared using AceQ qPCR SYBR
   Green Master Mix (Vazyme Biotech, Nanjing, China) and performed on a
   7500 Fast Real-Time System (Applied Biosystems, Foster City, CA, USA).
   Gene expression levels were normalized against FonActin using the
   2^−ΔΔCT method to calculate the relative expression of target genes
   [[124]55]. The primers used for this analysis are listed in Table
   [125]S1.

Statistical analysis

   Data were using one-way analysis of variance (ANOVA) with Data
   Processing System (DPS) package. Statistical significance was evaluated
   at a p-value threshold of ≤ 0.05 using the Least Significant Difference
   (LSD) test [[126]56]. Results are presented as the mean ± standard
   deviation (SD) derived from three independent replicates.

Results

Mn-resistant strain isolation and molecular characterization

   Here, a highly Mn-resistant bacterial strain, designated NOTE11, was
   identified, exhibiting a high MIC of 12 mM against MnCl[2].4H[2]O
   (Table [127]S2). The sequences of   16S rRNA (Accession no.
   [128]PQ669194) and rpoB (Accession no. [129]PQ678978) taxonomically
   classified strain NOTE11 as Lysinibacillus sphaericus. Sequence
   analysis revealed 99.80% similarity in the 16S rRNA gene with L.
   sphaericus Mc E09 ([130]OQ293909) and 100% similarity in the rpoB gene
   with L. sphaericus BS-pTAND672-2 ([131]CP071738). Phylogenetic analysis
   of both taxonomic marker genes (16 S rRNA and rpoB) indicated a
   evolutionary correlation among strain NOTE11 and other L. sphaericus
   and Bacillus spp. (Fig. [132]1A, B). These findings establish the
   taxonomic identity of strain NOTE11 as L. sphaericus.

Fig. 1.

   [133]Fig. 1
   [134]Open in a new tab

   Phylogenetic classification of bacterial strains based on 16S rRNA and
   rpoB gene sequences. (A) Phylogenetic tree constructed using the 16S
   rRNA gene sequences, showing the evolutionary relationship of the
   bacterial isolate with closely related strains from the genus Bacillus
   and Lysinibacillus. The tree was constructed using the maximum
   likelihood (ML) method with bootstrap values (> 50) indicated at branch
   nodes (percentage of 1,000 replicates). The scale bar represents the
   number of nucleotide substitutions per site. (B) Phylogenetic tree
   constructed based on the rpoB gene sequences. showing the evolutionary
   relationship of the bacterial isolate with closely related strains from
   the genus Bacillus and Lysinibacillus. The tree was constructed using
   the ML method with bootstrap values (> 50) indicated at branch nodes
   (percentage of 1,000 replicates). The scale bar represents the number
   of nucleotide substitutions per site. Escherichia coli served as an
   outgroup in both trees

Morpho-physicochemical properties of MnNPs

   Here, cell-free filtrate from L. sphaericus NOTE11 successfully
   biosynthesized MnNPs. The MnNPs synthesis was visually confirmed by a
   distinct color change in the reaction mixture from pale yellow to dark
   brown, indicating the reduction of Mn ions and the subsequent formation
   of MnNPs. FTIR analysis identified characteristic absorption bands at
   789, 1143, 1624, 2351, 3423, and 3645 cm^− 1, indicating the presence
   of diverse functional groups over the MnNP surface, such as C-H
   bending, C-N stretching, C = C stretching, O = C = O stretching, O-H
   stretching (alcohol), and O-H stretching (water) (Fig. [135]2A). These
   findings suggest that biomolecules in the bacterial culture supernatant
   play a crucial role in the biosynthesis and stabilization of MnNPs. The
   crystalline strucutre of biosynthesized MnNPs was confirmed by XRD
   analysis, showing specific diffraction peaks at 2θ, including 24.16°,
   31.36°, 37.29°, 41.49°, and 56.17°, corresponding to the (101), (201),
   (210), (211), and (212) planes of crystalline Mn, respectively
   (Fig. [136]2B). SEM imaging revealed predominantly spherical MnNPs with
   an irregular morphology and an average particle size of approximately
   32.28 nm (Fig. [137]2C). TEM further verified the spherical shape and
   uniform dispersion of MnNPs, exhibiting a narrow size range
   (Fig. [138]2D). EDS analysis depicted chemical constituents of the
   MnNPs, showing distinct peaks for Mn (30.26%) and O (oxygen; 63.78%)
   (Fig. [139]2E), indicating that the MnNPs consist primarily of Mn and
   O. These comprehensive characterizations provide a detailed
   understanding of the physicochemical properties and structural features
   of MnNPs, which were biosynthesized using the cell-free filtrate of L.
   sphaericus NOTE11.

Fig. 2.

   [140]Fig. 2
   [141]Open in a new tab

   Characterization of biosynthesized MnNPs. (A) FTIR spectrum showing the
   functional groups involved in MnNP synthesis. (B) XRD pattern
   confirming the crystalline structure of MnNPs. Prominent diffraction
   peaks validate the formation of MnNPs crystals. (C) SEM image revealing
   the spherical morphology of MnNPs with uniform size distribution at the
   nanoscale. (D) TEM image showing well-dispersed MnNPs with aggregated
   clusters, further confirming nanoscale size. (E) EDS analysis (left)
   and elemental mapping (right) highlighting the elemental composition of
   MnNPs

MnNPs disrupt Fon fundamental biological functions

   In vitro antifungal assays demonstrated that varying levels of MnNPs
   (except 25 µg/mL) significantly disrupted hyphal growth than bulk
   Mn-treated and untreated Fon. Biogenic MnNPs (100 µg/mL) highly reduced
   Fon hyphal growth by 21.97% on PDA, in contrast to untreated controls
   (Fig. [142]3A-C). Morphological observations of Fon treated with MnNP
   (100 µg/mL) revealed noticeable alterations in hyphal structure and
   morphology, including abnormal branching, distorted surfaces, and
   compromised cellular structure, indicative of cellular damage and
   cytoplasmic leakage. In contrast, 100 µg/mL bulk Mn-treated and
   untreated Fon samples exhibited intact hyphal and cellular structures
   (Fig. [143]3D).

Fig. 3.

   [144]Fig. 3
   [145]Open in a new tab

   Antifungal potential of MnNPs against Fon. (A) Radial growth of Fon on
   PDA plates supplemented with different concentrations (25, 50, and
   100 µg/mL) of bulk Mn (B-Mn) and MnNPs. Control (CK) plates show normal
   fungal growth without any treatment. (B) Quantitative measurement of
   radial growth (mm) at varying concentrations of B-Mn and MnNPs.
   Different letters above bars represent significant differences
   (p-value ≤ 0.05). (C) Antifungal activity (%) of B-Mn and MnNPs. (D)
   Microscopic analysis of fungal structures treated with MnNPs
   (100 µg/mL). SEM images (upper panel) reveal damage to fungal hyphae,
   including irregular morphology and structural disruption (red arrows),
   compared to intact hyphae in CK and B-Mn-treated samples. TEM images
   (lower panel) highlight intracellular damage in MnNP-treated fungal
   cells, including vacuolization, disrupted cell membranes (red arrows),
   while CK and B-Mn treatments show intact cellular ultrastructure

   Moreover, MnNP treatments at different concentrations (25, 50, and
   100 µg/mL) significantly reduced conidial morphogenesis, germination
   rate, and septation than bulk Mn-treated and untreated Fon.
   Specifically, 100 µg/mL MnNP-treated Fon produced significantly less
   conidia (63.20%) at 24 h post-treatment compared to untreated Fon
   (Fig. [146]4A). The conidial germination rate was also notably
   decreased (10%) in Fon condial suspesion supplied with 100 µg/mL MnNP,
   whereby 100 µg/mL bulk Mn-treated and untreated controls showing a
   germination rate of around 65% and 90%, respectively (Fig. [147]4B, C).
   Calcofluor white staining and microscopic observation revealed that Fon
   cultures treated with varying MnNPs concentrations (50 and 100 µg/mL)
   produced macroconidia with no or 1 septum, whereas bulk Mn-treated and
   untreated Fon produced macroconidia with 1 to 3 septa (Fig. [148]4D,
   E). Additionally, majority of the macroconidia from 100 µg/mL
   MnNP-treated Fon were significantly shorter than untreated controls
   (Fig. [149]4F). These results underscore the promissing Fon-suppressing
   potential of biogenic MnNPs, deciphering their significant impact on
   key pathogen biological processes such as conidiation, spore
   germination, and conidial morphology.

Fig. 4.

   [150]Fig. 4
   [151]Open in a new tab

   Effect of MnNPs on fundamental biological processes in Fon. (A)
   Macroconidia production by Fon in MBL medium supplemented with or
   without MnNPs and bulk Mn (B-Mn). (B) Conidial germination rate and (C)
   germinated conidia micrograph [scale bar = 20 μm] of Fon in YEPD medium
   supplemented with or without MnNPs and B-Mn. Atleast 100 spores per
   field were observed for estimating conidial germination rates. (D)
   Septa number, (E) macroconidial morphology [scale bar = 20 μm], and (F)
   conidia length with or without MnNPs and B-Mn. Different letters above
   bars represent significant differences (p-value ≤ 0.05)

MnNPs suppressed Fusarium wilt and invasive fungal growth

   In this study, we assessed the effectiveness of biogenic MnNPs
   (100 µg/mL) and bulk Mn (100 µg/mL) in controlling watermelon Fusarium
   wilt. Disease assays demonstrated significantly reduced disease
   severity in MnNP-treated plants than bulk Mn-treated and untreated
   Fon-infected controls (Fig. [152]5A–C). Notably, the proportion of
   healthy plants with no or minor chlorosis symptoms was notably higher
   in the MnNP-treated group (~ 88.89%) than bulk Mn-treated (22.22%) and
   untreated Fon-infected controls (11.11%) (Fig. [153]5B). Specifically,
   MnNP-treated plants exhibited a significantly lower disease severity
   index (DSI = 14.36) with minimal or no disease appearance than bulk
   Mn-treated (DSI = 85.01) and untreated Fon-infected plants
   (DSI = 88.38) (Fig. [154]5C). These results demonstrate the promissing
   disease-suppressive potential of biogenic MnNPs, especially for
   watermelon Fusarium wilt management.

Fig. 5.

   [155]Fig. 5
   [156]Open in a new tab

   MnNPs-induced suppression of fungal biomass reduce Fon pathogenicity on
   watermelon. (A) Disease phenotype of plants supplied with or without
   MnNPs at 21 dpi. (B) Disease scores and (C) disease severity indexes of
   Fon-infected plants supplied with or without MnNPs at 21 dpi. Invasive
   fungal growth in (D) roots and (E) stems of Fon-infected plants
   supplied with or without MnNPs. Different letters above bars represent
   significant differences (p-value ≤ 0.05)

   We also examined the impact of MnNPs and bulk Mn at 100 µg/mL
   concentration on invasive pathogen growth within watermelon tissues.
   The results showed that MnNP treatment resulted in the most significant
   reduction in fungal biomass in both root and stem tissues, compared to
   bulk Mn treatment and untreated Fon-infected controls. Specifically, at
   6 dpi, MnNP-treated plants showed significantly reduced fungal biomass
   in roots and stems (41.57% and 68.89%, respectively), than untreated
   Fon-infected controls (Fig. [157]5D, E). In contrast to bulk Mn, MnNP
   treatment showed the highest reduction in fungal biomass at 9 dpi, with
   a 51.70% reduction in roots and 67.62% in stems (Fig. [158]5D, E).
   These data clealry depict the superior efficacy of MnNPs in controlling
   watermelon Fusarium wilt, precisely by inhibiting the accumulation of
   fungal biomass in plant tissues.

MnNPs induced transcriptomic alterations in Fon

   To investigate the potential inhibitory molecular mechanisms of MnNPs
   against Fon, transcriptomic changes in the MnNP-treated and untreated
   Fon were compared using the RNA-seq technique. A total of 649 DEGs,
   comprising 137 upregulated and 512 downregulated genes (with a fold
   change ≥ or ≤ 2 and a p-value ≤ 0.05), were identified in MnNP-treated
   Fon (Table [159]S3 and Table [160]S4; Fig. [161]S1).

   A total of 76 KEGG pathways (17 upregulated and 59 downregulated) were
   identified in the MnNP-treated Fon (Fig. [162]6A, B). Notably, the
   largest group of pathways included Starch and sucrose metabolism (3/3
   upregulated/downregulated DEGs), followed by Nitrogen metabolism (2/2
   upregulated/downregulated DEGs), ABC transporters (2/2
   upregulated/downregulated DEGs), Nucleocytoplasmic transport (2/2
   upregulated/downregulated DEGs), Meiosis-yeast (2/2
   upregulated/downregulated DEGs), and Biosynthesis of amino acids (2/2
   upregulated/downregulated DEGs) (Fig. [163]6A, B). In addition, GO
   analysis categorized the upregulated and downregulated DEGs into three
   main functional groups: molecular function, cellular component, and
   biological process (Fig. [164]6C, D). Both upregulated and
   downregulated DEGs were significantly enriched in regulation of binding
   of carbohydrate, cofactor and coenzyme, formation of intracellular
   organelles, and regulation of gene expression or stress responses at
   the molecular, cellular, and biological levels, respectively, compared
   to the untreated controls (Fig. [165]6C, D). Overall, the findings
   suggest that MnNPs induce significant transcriptomic changes in Fon,
   particularly in pathways related to growth, cell division, and
   virulence.

Fig. 6.

   [166]Fig. 6
   [167]Open in a new tab

   MnNPs-induced transcriptomic alterations in Fon suprress its growth,
   development, and pathogenicity. Highly enriched (A) upregulated and (B)
   downregulated KEGG pathways in MnNPs-treated Fon. The size of each
   circle represents gene number involved in the corresponding pathway.
   The gene ratio of a pathway was represented as the ratio of the number
   of DEGs (p-value ≤ 0.05) to the total number of background genes in the
   corresponding particular pathway. GO annotation for (C) upregulated and
   (D) donwregulated DEGs in MnNPs-treated Fon. In the plots, y-axis
   represents roles of the DEGs at different levels of biological
   activity, while x-axis represents number of genes involved in
   corresponding function

MnNPs dysregulated fusaric acid biosynthetic pathway to inhibit Fon growth
and pathogenicity

   Further analysis of the RNA-seq data from MnNP-treated Fon revealed
   numerous mycotoxin production-related DEGs involved in the fusaric acid
   biosynthesis pathway, such as FonFUB5 (FOXG_15243; downregulated),
   FonFUB8 (FOXG_17660; downregulated), FonFUB11 (FOXG_17181;
   downregulated) (Table [168]S4), and FonFUB12 (FOXG_15235; upregulated)
   (Table [169]S3), which are known to play key roles in pathogen biology
   and virulence on host plants.

   To investigate the regulatory roles of FonFUB genes in Fon, we analyzed
   the expression of FonFUB5, FonFUB8, FonFUB11, and FonFUB12 across
   different developmental stages and in infected watermelon roots. The
   results demonstrated that FonFUB5, FonFUB8, FonFUB11, and FonFUB12
   expression level was significantly higher in germinating conidia than
   that in mycelia and conidia at 12 h post-inoculation (hpi)
   (Fig. [170]7A–D). Additionally, a substantial upregulation of these
   genes was observed in infected watermelon roots at 3 dpi
   (Fig. [171]7A–D), suggesting a critical role of FonFUB genes at the
   early infection development stages, such as root penetration and
   colonization. These data indicate that FonFUBs function in regulating
   the development and pathogenicity of Fon in watermelon.

Fig. 7.

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

   MnNPs-mediated dysregulation of fusaric acid biosynthesis genes,
   FonFUB5, FonFUB8, FonFUB11, and FonFUB12, inhibit physiological and
   pathogenicity-related processes in Fon. Expression profiling of (A)
   FonFUB5, (B) FonFUB8, (C) FonFUB11, and (D) FonFUB12 in mycelia,
   conidia, germinating conidia, and infected watermelon roots. (E)
   Expression levels of FonFUB5, FonFUB8, FonFUB11, and FonFUB12 in Fon
   grown with or without MnNPs and bulk Mn (B-Mn). (F) Radial growth
   diameters and (G) colony phenotype of Fon strains (WT, ΔFonFub5,
   ΔFonFub8, ΔFonFub11, and ΔFonFub12) grown with or without MnNPs and
   B-Mn. Different letters above bars represent significant differences
   (p-value ≤ 0.05)

   Further, we verify the impact of MnNPs (100 µg/mL) on the expression of
   FonFUB5, FonFUB8, FonFUB11, and FonFUB12. The results showed a
   significant downregulation of FonFUB5, FonFUB8, and FonFUB11 and a
   notable upregulation of FonFUB12 following MnNP treatment than bulk
   Mn-treated and untreated Fon (Fig. [174]7E), which coincide with the
   RNA-seq data. This suggests that MnNPs may dysregulate these genes
   involved in fusaric acid biosynthesis, potentially affecting Fon
   pathogenicity.

FonFUB genes regulate MnNPs sensitivity in Fon

   To investigate the involvement of FonFUB genes in regulating the
   sensitivity response of Fon to MnNPs, we assessed the impact of MnNPs
   (100 µg/mL) on the radial growth of deletion mutants ΔFonFub5,
   ΔFonFub8, ΔFonFub11, and ΔFonFub12, which were confirmed using
   PCR-based detection (Fig. [175]S2). Notably, in the absence of MnNPs,
   the deletion mutants showed significantly less hyphal growth compared
   to the WT, confirming their critical role in Fon growth. Intriguingly,
   the deletion mutants showed greater sensitivity to MnNPs treatment with
   significantly less hyphal growth than bulk Mn-treated and untreated Fon
   strains (Fig. [176]7F, G). Specifically, hyphal growth of MnNP-treated
   ΔFonFub5, ΔFonFub8, ΔFonFub11, and ΔFonFub12 was significantly reduced
   by ~ 44%, ~ 45%, ~ 44%, and 41%, respectively, than untreated deletion
   mutant strains. These findings suggest that fusaric acid biosynthesis
   genes, including FonFUB5, FonFUB8, FonFUB11, and FonFUB12, are likely
   involved in the stress sensitivity response to MnNPs in Fon, indicating
   their dysregulation is one of the antifungal mechanisms of MnNPs in
   controlling Fusarium wilt in watermelon.

MnNPs altered rhizosphere metabolites to suppress Fon pathogenicity in
watermelon

   Metabolomic profiling revealed significant shifts in rhizosphere
   metabolites in response to MnNP treatment in both healthy and
   Fon-inoculated soils. A total of 215 metabolites were identified and
   categorized into 12 chemical classes, with fatty acyls (19.5%),
   glycerophospholipids (14.6%), and phenol lipids (14%) being the most
   prominent groups (Fig. [177]8A). PCA demonstrated clear clustering of
   CK, CK + NP, Fon, and Fon + NP groups along PC1 (45% variance
   explained), indicating distinct metabolic shifts induced by MnNPs and
   Fon infection. The distinction between Fon + NP and Fon along PC1 and
   PC2 suggests that MnNP treatment significantly alters the metabolic
   landscape in Fon-infected soils (Fig. [178]8B). Notably, MnNP treatment
   (Fon + NP) altered the metabolome of Fon-inoculated soils, as evidenced
   by a unique set of DAMs compared to untreated Fon (Fig. [179]8C, D).
   Volcano plots highlighted significant enrichment of defense-related
   metabolites, including spriamycin iii and rubromycin ca1, in
   MnNP-treated soils (Fig. [180]8D). Heatmap analysis further
   corroborated the differential abundance of key metabolites, such as
   alpha-elaterin 2-d-glucopyranoside acid and hexosylsphingosine, which
   were elevated in the Fon + NP group (Fig. [181]8E). Pathway enrichment
   analysis revealed that MnNPs upregulated key metabolic pathways,
   including glycerophospholipid metabolism, biosynthesis of unsaturated
   fatty acids, and phenylalanine, tyrosine, and tryptophan biosynthesis,
   all of which are integral to plant defense and stress signaling
   (Fig. [182]8F). Collectively, these findings indicate that MnNPs
   modulate rhizosphere metabolites to enhance soil health and suppress
   Fon pathogenicity in watermelon.

Fig. 8.

   [183]Fig. 8
   [184]Open in a new tab

   MnNP-induced shifts in rhizosphere metabolomics suppress Fon
   pathogenicity. (A) Classification of identified metabolites into major
   chemical classes. (B) PCA plot depicting distinct metabolic profiles
   among MnNP-treated or untreated heathy and Fon-infected groups. (C)
   Venn diagram showing overlap of DAMs across MnNP-treated or untreated
   heathy and Fon-infected groups. (D) Volcano plots of DAMs highlighting
   significant metabolites in MnNP-treated or untreated heathy and
   Fon-infected groups. (E) Heatmap showing the relative abundance of key
   metabolites across MnNP-treated or untreated heathy and Fon-infected
   groups. (F) KEGG pathway analysis indicating enrichment of key
   metabolic pathways in MnNP-treated or untreated heathy and Fon-infected
   groups

Discussion

   Fusarium wilt fungus, Fon, remains one of the most devastating
   soilborne pathogens affecting watermelon crops worldwide, necessitating
   innovative and sustainable control strategies. In this study, we
   explored the potential of MnNPs, derived from L. sphaericus NOTE11
   culture supernatant (Figs. [185]1 and [186]2), as a potent antifungal
   agent to combat Fon, demonstrating their superior efficacy in
   inhibiting fungal growth, suppressing Fusarium wilt, and modulating
   rhizosphere metabolites compared to bulk Mn. Previous studies have
   reported the antifungal activities of various metallic NPs, such as
   silver and magnesium NPs, against F. oxysporum (including Fon), largely
   attributing their effects to oxidative stress induction and disruption
   of fungal membrane integrity [[187]57, [188]58]. However, these studies
   primarily focused on in vitro effects without addressing the potential
   in planta applications or the underlying molecular mechanisms. In
   contrast, our study not only demonstrated significant inhibitory
   effects of MnNPs on Fon growth and conidiation but also revealed their
   ability to dysregulate key virulence pathways, including fusaric acid
   biosynthesis, and suppress fungal biomass accumulation in watermelon
   plants. Furthermore, the transcriptomic and metabolomic analyses
   highlighted the multifaceted impact of MnNPs on Fon physiology and soil
   health, underscoring their novelty in modulating host-pathogen
   interactions and rhizosphere dynamics.

   The comprehensive characterization of biosynthesized MnNPs provides
   valuable insights into their physicochemical and structural attributes,
   which are crucial for understanding their stability and functionality.
   The presence of diverse functional groups, as indicated by FTIR
   analysis, suggests the involvement of biomolecules from L. sphaericus
   NOTE11 in the reduction, capping, and stabilization of MnNPs,
   contributing to their dispersity and overall functionality [[189]59].
   The XRD, microscopic and EDS analyses revealed the crystalline nature
   of MnNPs, which were predominantly spherical with slight irregularities
   and showed Mn or O as their active ingredients, consistent with
   previous reports on biosynthesized metal NPs [[190]12, [191]45]. These
   findings collectively establish a strong foundation for understanding
   the structural and functional properties of MnNPs, supporting their
   role in plant disease suppression.

   The biogenic MnNPs exhibited significant antifungal activity by
   disrupting fundamental biological processes in Fon, including hyphal
   growth, conidiation, and conidial germination, all of which are
   critical for fungal development and pathogenicity. Fungal growth,
   conidiation, spore length, and germination rates were also notably
   suppressed in MnNP-treated Fon, with treated macroconidia exhibiting
   reduced septation (Figs. [192]3 and [193]4), suggesting impaired
   cellular division and spore viability. These inhibitory effects on
   basic fungal biological processes can directly be corelated to a
   weakened ability of Fon to establish infection, thereby reducing its
   pathogenicity in host plants (Fig. [194]5). Previous studies have
   highlighted the antifungal properties of metallic NPs, such as
   zinc-copper oxide NPs (1000 µg/mL) and chitosan-silver NPs (25 mg in 5
   mL acetic acid), against phytopathogenic F. oxysporum [[195]60,
   [196]61]. These NPs primarily exert their effects through mechanisms
   such as oxidative damage and membrane disruption [[197]62]. However,
   unlike the non-specific oxidative stress mechanisms reported in these
   studies, our findings demonstrate that MnNPs not only damage fungal
   cellular structures but also interfere with key developmental processes
   like conidiation and septation even at lower concentrations (i.e.
   100 µg/mL), as supported by our previous results [[198]12].
   Intriguingly, the proficient antifungal activity of MnNPs can be
   attributed to the biomolecule-assisted synthesis of MnNPs, which could
   introduce additional functional groups that amplify their antifungal
   properties [[199]30]. These findings provide deeper insights into the
   antifungal potential of MnNPs and highlight their primary mechanism of
   action against Fon.

   The transcriptomic analysis revealed that MnNPs dysregulated key
   pathways in Fon, particularly those associated with virulence and
   pathogenicity, such as the fusaric acid biosynthesis pathway
   (Fig. [200]6). Several key genes in this pathway, including FonFUB5,
   FonFUB8, and FonFUB11, were significantly downregulated following MnNP
   treatment, while FonFUB12 was upregulated. These genes are critical
   regulators of fusaric acid production, a mycotoxin known to play a
   central role in suppressing host defenses and promoting fungal
   colonization [[201]63, [202]64]. The observed suppression of FonFUB
   genes in MnNP-treated Fon suggests a direct impact on its ability to
   produce fusaric acid, thereby reducing its pathogenic potential
   (Fig. [203]7). Previous studies have established the role of fusaric
   acid in fungal virulence [[204]33, [205]34, [206]65], with research
   showing that disruption of fusaric acid biosynthesis genes, such as
   FUB1, FUB2, FUB3, FUB4, FUB5, and FUB10, reduces the aggressiveness of
   pathogens like F. oxysporum f. sp. cubense TR4 on banana plants
   [[207]66]. While earlier work has primarily focused on genetic
   knockouts or chemical inhibitors to suppress fusaric acid production,
   this study uniquely demonstrates that MnNPs can achieve similar
   outcomes by directly modulating the expression of fusaric acid
   biosynthesis genes (Fig. [208]7). Notably, our results also show that
   the deletion mutants ΔFonFubs exhibited heightened sensitivity to MnNPs
   (Fig. [209]7), further highlighting the importance of this pathway in
   regulating stress response and survival under MnNP treatment in Fon.
   Additionally, our findings extend beyond toxin suppression, as we show
   that MnNPs also alter the broader transcriptomic landscape of Fon,
   including pathways involved in stress responses, carbohydrate
   metabolism, and cell division (Fig. [210]6). Unlike chemical inhibitors
   that target specific biosynthetic pathways, MnNPs appear to have a more
   comprehensive impact, simultaneously disrupting multiple facets of
   fungal metabolism and virulence. This broader mode of action
   distinguishes MnNPs from traditional antifungal approaches, making them
   a more robust alternative for managing Fon infections.

   Previous studies have established that rhizosphere metabolites suppress
   plant diseases directly by inhibiting the growth or pathogenicity of
   phytopathogens, including fungi, and indirectly by enhancing plant
   immunity, thereby maintaining a healthy soil ecosystem for better plant
   growth and development [[211]40, [212]67, [213]68]. Previously, key
   metabolites involved in defense pathways, including ABC transporters,
   biosynthesis of antibiotics, and biosynthesis of phenylpropanoids, in
   tabacco (Nicotiana tabacum) against bacterial wilt pathogen, Ralstonia
   solanacearum, were found to be enriched in disease-suppressive
   rhizospheric soils [[214]69]. In this study, metabolomic analysis
   revealed that MnNPs significantly altered the metabolic profile of the
   watermelon rhizosphere, promoting the accumulation of specific
   secondary metabolites with antifungal properties (Fig. [215]8). Key
   metabolites, including phenolic compounds, flavonoids, and organic
   acids, were enriched in MnNP-treated rhizosphere samples (Fig. [216]8).
   These metabolites are known to strengthen plant defense mechanisms by
   directly inhibiting fungal growth or modulating plant-pathogen
   interactions [[217]70, [218]71, [219]72]. For instance, phenolics and
   flavonoids, which were elevated in the MnNP-treated rhizosphere, have
   been reported to inhibit the growth and the biosynthesis of the
   phytotoxins beauvericin and fusaric acid in banana wilt fungus F.
   oxysporum f. sp. cubense TR4 [[220]70]. The increased abundance of
   these metabolites likely contributed to the reduced pathogenicity of
   Fon observed in MnNP-treated plants. Moreover, the presence of these
   antifungal metabolites may create an unfavorable environment for fungal
   colonization, thereby indirectly supporting plant health. For example,
   biogenic selenium NPs (50 mg/kg) improved various metabolites,
   including citrate, isocitrate, succinate, malate, 2-oxo-glutarate, and
   khelmarin D, in lettuce plants to suppress F. oxysporum f. sp.
   Lactucae-induced wilt disease [[221]73]. Overall, the influence of
   multifaceted MnNPs on rhizosphere dynamics highlights their potential
   as an innovative and sustainable approach to managing Fusarium wilt and
   other soilborne plant diseases.

Conclusions

   This study presents a comprehensive exploration of the antifungal
   potential of biogenically-synthesized MnNPs against Fon, the causative
   agent of Fusarium wilt in watermelon. MnNPs demonstrated significant
   antifungal activity by disrupting fundamental biological processes,
   including hyphal growth, conidiation, and conidial germination,
   ultimately impairing Fon pathogenicity. Transcriptomic analysis
   revealed that MnNPs induced substantial alterations in gene expression,
   specifically on the fusaric acid biosynthesis pathway, which plays a
   critical role in Fon virulence. Furthermore, rhizosphere metabolomic
   profiling highlighted the ability of MnNPs to modulate the soil
   metabolome, enriching defense-related metabolites and creating an
   inhibitory environment for Fon colonization and infection
   (Fig. [222]9). The findings underscore the potential of MnNPs as a
   novel and effective tool for managing Fusarium wilt in watermelon,
   offering a sustainable alternative to conventional chemical fungicides.
   By integrating nanotechnology with agricultural disease management,
   this study advances our understanding of the molecular and ecological
   interactions between NPs, pathogens, and plants. Future research should
   focus on field-scale evaluations and the environmental safety of MnNPs
   to facilitate their practical application in sustainable agriculture.
   Furthermore, future studies should conduct field-scale evaluations to
   validate the efficacy of MnNPs under diverse environmental conditions
   and cropping systems. Investigating the mechanisms underlying
   plant-nanoparticle interactions and optimizing MnNP formulations for
   controlled release and targeted action will further enhance their
   applicability.

Fig. 9.

   [223]Fig. 9
   [224]Open in a new tab

   Proposed model of mechanism of action of MnNPs against Fon-induced
   Fusarium wilt in watermelon. MnNPs enter Fon cells by disintegrating
   membrane, followed by fusaric acid biosynthesis genes (FonFUB5,
   FonFUB8, FonFUB11, and FonFUB12) interruption and conidiation
   disruption, thereby reducing invasive fungal growth within plant
   tissues. These events collectively contribute to compromised Fon
   infection and pathogenicity on watermelon plants. Further, MnNPs
   modulate the rhizosphere metabolome, enriching phytobeneficial
   metabolites in the root-vicinal area that antagonize pathogen growth
   and promote plant health. This dual-action mechanism effectively
   controls Fon infection and enhances watermelon resistance to Fusarium
   wilt

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [225]Supplementary Material 1^ (244.8KB, docx)

Acknowledgements