Abstract The current study investigates the potential of carbon dots (CDs) as an eco-friendly “plant vaccine” for controlling rice blast disease caused by Magnaporthe oryzae. These CDs offer a promising alternative to commercial fungicides that threaten environmental and human health. Foliar application of CDs (at 100 and 200 ​mg/L) at the tillering stage enhanced rice blast resistance across the entire life cycle. CDs significantly reduced the leaf blast disease index, with infection rates of 30.8%–49.5%, outperforming the commercial fungicide isoprothiolane (57.3%). CDs significantly increased grain yield (186%–198%), starch content in grains (27.0%–27.5%), and protein content in grains (25.4%–36.1%) relative to infected controls. Moreover, CDs demonstrated lower toxicity to soil organisms (Eisenia fetida and Caenorhabditis elegans) than isoprothiolane. Mechanistically, CDs stabilized chloroplast homeostasis, amplified photosynthesis, and enhanced carbohydrate allocation, thereby synchronously activating systemic resistance through indole acetic and jasmonic acid signaling. These dual agricultural and environmental benefits position CDs as a sustainable crop protection strategy, reconciling food security with ecological safety. Keywords: Carbon quantum dots, Magnaporthe oryzae, Green prevention, Nutritional quality, Disease resistance Graphical abstract [41]Image 1 [42]Open in a new tab Highlights * • The 100 and 200 ​mg/L carbon dots (CDs) have the potential as a “plant vaccine”. * • CDs activate the disease-resistant immune system to enhance rice blast resistance. * • CDs significantly increase the grain yield and quality at the mature stage. * • CDs (at 100 ​mg/L) are greener and safer than commercial fungicides. 1. Introduction Overusing chemical pesticides threatens environmental integrity and human health [[43][1], [44][2], [45][3]]. Rice blast, caused by Magnaporthe oryzae, ranks among the most devastating diseases of rice (Oryza sativa L.), resulting in approximately 10% annual global yield losses and exacerbating food insecurity [[46]4,[47]5]. Conventional control relies on isoprothiolane (IPT), which is applied prophylactically during tillering to suppress outbreaks and secure yields [[48]6,[49]7]. However, IPT exhibits critical limitations: high application doses, suboptimal efficiency, and pervasive environmental contamination [[50][8], [51][9], [52][10]]. Its persistence poses risks to non-target organisms and may accelerate pathogen resistance, thereby undermining long-term efficacy [[53]8]. Escalating ecological and antimicrobial resistance crises demand the development of sustainable, high-performance alternatives to balance crop protection with planetary health. The emergence of agricultural nanotechnology has introduced innovative strategies for protecting crops [[54]11,[55]12]. Nano-fungicides show a 30% increase in disease inhibition efficiency and an approximately 40% reduction in environmental risks compared to traditional agents [[56]13]. Although nanoscale and traditional fungicides share mechanisms such as cellular membrane disruption and organelle destabilization in pathogens, their transient bioactivity and single-target modes limit sustained efficacy [[57]14]. Nano-regulators, in contrast, activate host immunity and confer durable defense by reprogramming plant–pathogen interactions [[58]14]. For instance, lanthanum nanoparticles (La NPs) enhance antioxidant systems to combat oxidative stress induced by Fusarium oxysporum [[59]15]. At the same time, iron-, zinc-, or selenium-based nanomaterials stimulate the biosynthesis of salicylic acid (SA) and jasmonic acid (JA) to prime systemic acquired resistance (SAR) [[60]16,[61]17]. However, metal-based nanomaterials face scalability challenges due to high synthesis costs (2–5 times higher than those of carbon-based counterparts) and potential nano-bioaccumulation and risks, which complicates regulatory approvals [[62]18,[63]19]. Carbon dots (CDs) are carbon-based nanomaterials (<10 ​nm) characterized by multifunctional surface groups and tunable fluorescence properties [[64]20]. Their synthesis from renewable precursors—such as agricultural waste or citric acid—offers a 50%–70% cost reduction compared to metal-NPs, eliminating reliance on rare-earth elements or energy-intensive fabrication [[65]21]. Foliar-applied CDs have been demonstrated to enhance photosynthetic efficiency, carbohydrate assimilation, and crop yields [[66]22,[67]23]. CDs also modulate root architecture, improve nutrient uptake (e.g., N, P, and Fe), alter root exudate profiles, and activate phytohormone signaling pathways, collectively enhancing crop quality and disease resilience [[68]24]. The bioactivity of CDs in plants is strongly related to their physicochemical characteristics. For example, smaller CDs (e.g., 5 ​nm) demonstrate enhanced cellular uptake and subcellular organelle targeting (e.g., chloroplasts and mitochondria), thereby enabling more precise immune activation in specific locations than larger nanoparticles [[69]25]. Furthermore, negatively charged CDs on the surface inhibit the adhesion of bacterial pathogens through electrostatic repulsion and act as bactericidal agents by destroying the bacterial cell wall [[70]26]. Surface functional groups (e.g., –COOH, –NH[2]) contribute further to antimicrobial activity by competitively inhibiting fungal enzymes critical for chitin biosynthesis or respiration, thereby suppressing pathogen growth [[71]27]. Unlike metallic NPs, which solely enhance antioxidant enzymatic activity against Fusarium oxysporum [[72]15], or nanosilver restricted to direct bactericidal effects [[73]28], CDs' surface chemistry ensures non-phytotoxic foliar absorption and apoplastic/systemic transport, contrasting sharply with the soil contamination risks posed by metallic NPs [[74]29]. Moreover, the composition of CDs (C, O, N) aligns with OECD biodegradability guidelines, and scalable synthesis from food-grade precursors could simplify regulatory approval pathways [[75]30]. Despite these advancements, several unresolved questions persist: Can CDs serve as “plant vaccines” that induce immunological memory while uniquely integrating systemic resistance activation, pathogen inhibition, and biosecurity—an unprecedented triad unavailable in conventional or nano-fungicides? Addressing this knowledge gap could lead to innovative and sustainable strategies for managing crop diseases. Thus, the present study systematically evaluated CDs as an eco-friendly alternative to conventional fungicides for controlling M. oryzae-induced rice blast. Our multi-omics approach integrated disease resistance physiology, metabolic profiling, and differential gene expression analysis in infected rice following CD treatments. Additionally, we quantified yield enhancement, grain quality parameters, and biosafety. The present study provides the first evidence of CDs functioning as a green “plant vaccine” against rice blasts, offering a transformative strategy for sustainable crop protection while ensuring food safety and agricultural productivity. 2. Methods and materials 2.1. Synthesis and characterization of CDs CDs were synthesized using citric acid as the carbon source through a hydrothermal method [[76]31]. A 3.1521 ​g mixture of citric acid and 1.005 ​mL of ethylenediamine was dissolved in 30 ​mL of deionized water. The resulting solution was placed in a Teflon-lined stainless-steel autoclave and heated to 200 ​°C for 8 ​h. After naturally cooling to room temperature, 3 ​mL of 10% polyacrylic acid was incorporated, and the mixture was then heated to 80 ​°C for 4 ​h to yield the CD solution. This solution underwent dialysis for 48 ​h in a dialysis bag (500 ​kDa), followed by concentration using a rotary evaporator, and was then dried for 24 ​h using a vacuum freeze-dryer to obtain CD powders. The morphology and particle size of the CDs were analyzed with a transmission electron microscope (TEM, JEM-2100F, Japan). Fourier-transform infrared spectroscopy (Bruker, Germany) was employed to examine the surface functional groups of the CDs. The Zeta potential of 100 ​mg/L CDs was measured using a Malvern Zeta sizer (ZEN3600, Malvern, UK). 2.2. Plant growth and CDs application Rice seeds (cv. Chuangliangyou 699, blast-sensitive) were acquired from Anhui Luyi Seed Industry Co., Ltd. The seeds underwent sterilization in a 5% sodium hypochlorite solution for 10 ​min, followed by three rinses with deionized water and an 8-h soak. The seeds were subsequently placed on moist filter paper for germination in the greenhouse at the School of Environment & Ecology, Jiangnan University (Wuxi, China). During this phase, they were kept in darkness and regularly watered. Until the seedlings reached approximately 5 ​cm in height, uniformly developed seedlings were chosen and transferred into pots filled with 5 ​kg of paddy soil, all within the same greenhouse. The soil pH was measured at 5.3. The total organic carbon in the soil was measured at 2300 ​mg/kg using the potassium dichromate oxidation-external heating method. The soil-available nitrogen, phosphorus, and potassium were also quantified at 125.7 ​mg/kg, 23.2 ​mg/kg, and 92.1 ​mg/kg, respectively, using standard analytical methods [[77]19]. By the tillering stage (20 days post-transplantation), uniformly grown rice plants were assigned to various experimental conditions treatments. The groups included a healthy control group (NCK) treated with deionized water; an infected control group (RCK) also receiving deionized water; an infected treatment group sprayed with 800 ​mg/L of isoprothiolane (IPT, as per recommended dosage); and additional infected treatment groups sprayed with CDs at concentrations of 100 ​mg/L (CDs100), 200 ​mg/L (CDs200), and 400 ​mg/L (CDs400). The CDs and IPT solutions were freshly prepared in deionized water and used immediately. Foliar spraying occurred between 9:00 and 10:00 AM, after sonication of all treatment solutions. The spraying ensured uniform coverage on both leaf surfaces without dripping. Each plant received a 15 ​mL solution bi-daily for a week before rice blast infection. 2.3. Pathogen inoculation and disease assessment Blast fungus was acquired from Mingzhou Biotechnology Co., Ltd. (Ningbo, China). M. oryzae was cultivated on a sterile 90 ​mm Petri dish at 30 ​°C for 5 days, until the surface was completely covered with fungus, followed by subculturing. The culture of M. oryzae was maintained on Potato Dextrose Agar (PDA) medium. The negative control group (NCK) was kept as the non-inoculated control, while the RCK, IPT, CDs100, CDs200, and CDs400 treatment groups were inoculated with M. oryzae according to the standardized protocol. After one week, rice plants were inoculated with M. oryzae during the tillering stage. From 9:00 to 10:00 AM, 45 ​mL of fungal suspension was evenly sprayed on each plant, ensuring both leaves were coated without dripping. To create a humid, warm environment, black plastic bags were placed over the rice pots until disease spots became visible on the rice surface and their quantity increased. The disease index was assessed by determining the infection rate of rice blast disease after one week of infection with M. oryza. The plants in each treatment group were closely observed, noting the presence of rice blast symptoms on the leaves, particularly when the lesions had stabilized after five days of infection. Subsequently, the infection rate was calculated, typically as the ratio of infected leaves to the total number of sampled leaves. The growth phenotype, plant height, shoot biomass, and root biomass of the rice were recorded after one week of infection with M. oryzae. Simultaneously, leaf and root samples were collected and washed with deionized water at least three times. The roots of intact rice plants were spread on a root scanner (Expression 12000XL, Japan) to analyze root parameters (root length, number of root tips, and root volume). The plant efficiency analyzer (Pocket PEA, Hansatech Instruments Ltd., UK) and the chlorophyll meter (SPAD-502 plus, Konica Minolta Inc., Japan) were used to measure chlorophyll fluorescence kinetics parameters and chlorophyll-related content (SPAD), respectively. A portable gas exchange system (CIRAS-3, PP-Systems, USA) was used to determine photosynthesis parameters such as intercellular CO[2] concentration (Ci), transpiration rate (Tr), net photosynthetic rate (Pn), and photochemical efficiency (Fv/Fm). Rice leaves were immediately placed in liquid nitrogen for further analysis of oxidative stress levels, antioxidant enzyme content, metabolite abundance, and gene expression levels in rice leaves. 2.4. Analysis of oxidative defense parameters and relative expression level of disease-resistant genes in rice leaves The activities of MDA, H[2]O[2], CAT, POD, and SOD in fresh rice leaves were measured [[78]15]. Detailed experimental procedures are described in Text S1. The relative expression level of genes was assayed with quantitative Real-Time PCR (qRT-PCR), as described in Text S2. The primer sequences of disease-resistant genes are listed in [79]Table S1. 2.5. The analysis of metabolites and hormone contents The current study detected and identified the metabolites in rice leaves of different treatments to elucidate the underlying mechanisms of CDs in regulating rice disease resistance. The metabolite extract and detection protocols are provided in Text S3. Samples were analyzed in negative and positive ionization modes in various runs. Partial least-squares discriminant analysis was used to analyze the LC-MS/MS data through an online web application ([80]http://www.metaboanalyst.ca). The methods described by Luo et al. were used to determine the contents of plant disease resistance hormones (SA, ABA, JA, and IAA) in rice leaves [[81]15]. The protocols are provided in Text S4. 2.6. Analysis of rice grain yield and quality To elucidate the impact of prevention with CDs on M. oryzae disease and its effects on grain yield and quality at maturity, a 135-day complete life-cycle experiment was conducted. The protocols of rice cultivation, spraying treatment, and M. oryzae inoculation were consistent with Sections 2.2 and 2.3. The difference was that the rice plants were cultivated until maturity post-infection during the tillering stage. The 100-grain weight, grain yield per plant, and starch and protein content in the grains were recorded at this stage. The detection methods of starch and proteins in rice grains are outlined in Text S5. 2.7. Biosafety evaluation of CDs on earthworms and nematodes The biosafety of CDs, a novel nanomaterial, necessitates further investigation and assessment. Eisenia fetida (E. fetida) and Caenorhabditis elegans (C. elegans) were employed as model organisms to evaluate the effects of various concentrations of CDs and IPT on their lethality or locomotion behaviors (head thrashes and body bends) after exposure to 1, 3, 5, 7, 10, and 14 days, or 24, 48, 72, and 96 ​h. Detailed experiment protocols are provided in Text S6. 2.8. Quality control For oxidative defense and rice quality parameters, we included appropriate negative and positive controls, performed technical triplicates, and validated standard curves for each assay. The gene expression analyses were conducted under RNase-free conditions. We verified the A260/A280 and A260/A230 ratios with a NanoDrop 2000 and normalized the data using validated reference genes. For metabolomic studies, we included Quality Control samples (pooled from all biological samples) that were analyzed every 10 samples to monitor system stability, incorporated internal standards for retention time alignment, and performed blank runs to identify potential contamination. All experimental procedures were conducted with appropriate technical replicates and statistical validation. 2.9. Statistics All data from the treatment groups are presented as means and standard deviations. Data analysis was conducted using GraphPad Prism 10.1.2 and SPSS 22.0. A one-way ANOVA was used to assess the significance of differences between group means. The LSD test (p ​< ​0.05) further delineated these differences within the ANOVA framework. Different letters above the graphs visually indicate significant differences between group means. Each treatment group consisted of at least three data replicates. Metabolic pathway analysis was performed using the MetaboAnalyst 5.0 online tool. PCA (principal component analysis), volcano plots, Venn diagrams, and heat maps were generated using Origin 2022. 3. Results and discussion 3.1. Characteristics of CDs CDs are spherical and well-dispersed with noticeable inter-particle spacing and a crystal lattice spacing of 0.21 ​nm ([82]Fig. S1A and B). The particle size predominantly ranges around 2.5 ​nm ([83]Fig. S1C). The small size of CDs allows for easier infiltration into plant tissues, which influences gene expression and metabolic activities, ultimately impacting plant growth [[84]32]. [85]Fig. S1D illustrates distinct peaks: at 3410 ​cm^−1 for N–H/–OH stretching vibrations, at 2930 ​cm^−1 for C–H stretching vibrations, at 1660 ​cm^−1 for C Created by potrace 1.16, written by Peter Selinger 2001-2019 O stretching vibrations, at 1550 ​cm^−1 for C Created by potrace 1.16, written by Peter Selinger 2001-2019 C stretching vibrations, at 1390 ​cm^−1 for C–O–H stretching vibrations, and at 1220 ​cm^−1 for N–H stretching band. The strong and narrow peak at 857 ​cm^−1 likely relates to out-of-plane bending vibrations of C–H bonds in aromatic rings. The absorption peak at 599 ​cm^−1 shows a typically weak intensity. It may overlap with another vibrational mode [[86][33], [87][34], [88][35]]. [89]Fig. S1E shows a surface charge of −16.0 ​mV, indicating a significant negative charge. The findings confirmed that nitrogen-doped CDs (N-CDs) were produced with a small size and were abundant in –OH, –COOH, and –NH groups, resulting in a negative surface charge. Related studies have shown that N-CDs exhibit improved permeability, water solubility, and adsorption characteristics, rendering them highly effective for foliar application [[90]31]. 3.2. Integrated analysis of different concentrations of CDs-mediated disease resistance in M. oryzae-infected rice This study systematically evaluated the physiological and biochemical functions by which CDs (100, 200, and 400 ​mg/L) confer resistance to M. oryzae in rice leaves, integrating pathogen suppression, photosynthetic enhancement, and oxidative stress mitigation. The rice phenotype and leaf fluorescence intensity under various treatments in M. oryzae-infected rice are shown ([91]Fig. 1A and B). Compared to NCK, RCK-treated rice leaves showed pronounced blast lesions after fungal infection. In contrast, rice treated with IPT, CDs100, CDs200, and CDs400 exhibited ameliorated blast symptoms. Foliar application of CDs (100 ​mg/L) restores the fluorescence intensity to levels similar to those of NCK. Infected rice (RCK) exhibited severe blast lesions (disease index: 74.5%) compared to non-infected controls (NCK). CDs100, CDs200, and CDs400 reduced disease indices to 30.8%, 39.0%, and 49.5%, outperforming the fungicide IPT (57.3%) ([92]Fig. 1C). Fig. 1. [93]Fig. 1 [94]Open in a new tab The phenotype of rice (A) and leaf fluorescence intensity (B) in Magnaporthe oryzae-infected rice after treatment with carbon dots (CDs) at different concentrations and IPT. (C) Effect of foliar-applied CDs100, CDs200, CDs400, and IPT on disease index, plant height, fresh biomass, total sugar content, starch content, chlorophyll relative content (SPAD), net photosynthetic rate (Pn), photochemical efficiency (Fv/Fm), intercellular carbon dioxide content (Ci), and transpiration rate (Tr) of M. oryzae-infected rice leaves. (D) The heatmap of chloroplast homeostasis gene expression levels treated with CDs100, CDs200, and IPT on the M. oryzae-infected rice leaves. The significant difference among treatments is marked with different letters (p ​< ​0.05, n ​= ​3). NCK, the healthy control; RCK, the infected control; CDs100, 100 ​mg/L CDs; CDs200, 200 ​mg/L CDs; CDs400, 400 ​mg/L CDs; IPT, 800 ​mg/L isoprothiolane. The height of RCK-treated rice decreased to 0.86 times that of NCK ([95]Fig. 1C). Rice treated with CDs100, CDs200, and CDs400 exhibited a notable increase in height, ranging from 21.1% to 22.9%, while the height of IPT-treated rice was similar to that of RCK. Compared to NCK, RCK treatment significantly decreased the shoot and root fresh biomass by 25.9% and 36.5%, respectively. The shoot fresh biomass in CDs100, CDs200, and CDs400 was significantly higher than that in RCK. Compared to RCK, the IPT and CDs400 treatments showed no significant difference in root biomass; the CDs100 and CDs200 treatments significantly increased root biomass by 50.9%–57.6%, indicating root recovery relative to the NCK. A healthy rice root system is crucial for plant growth and consistently high yields [[96]36,[97]37]. A similar study indicated that foliar application of CDs markedly enhances root growth, improves nutrient uptake and root biomass accumulation, and augments stress resistance [[98]24]. The present study demonstrated that rice roots in diseased plants treated with CDs100 and CDs200 recovered remarkably to healthy levels, facilitating improved water and nutrient absorption from the soil and thereby enhancing resistance to leaf blast disease ([99]Fig. S2). Moreover, in vitro, IPT inhibited the growth of M. oryzae by 97.3% over 24 ​h, which is 1.11–1.21 times the inhibition achieved by CDs100, CDs200, and CDs400 ([100]Fig. S2C). Lanthanum-based nanomaterials (1000 ​mg/L) exposed for 24 ​h effectively enhance plant disease resistance [[101]38]. During pathogen infection, the carbohydrate metabolism of crops is disrupted, resulting in nutrient depletion and gradual wilting [[102]39,[103]40]. Sugars and starches are products of photosynthesis. Compared to NCK, the total sugar and starch content in leaves in RCK decreased by 33.1% and 20.1%, respectively ([104]Fig. 1C). Conversely, the foliar application of CDs (100 or 200 ​mg/L) or IPT significantly enhanced the total sugar and starch content compared to RCK. These results demonstrated that foliar application of CDs (100 and 200 ​mg/L) promoted considerable carbohydrate supply in infected rice. A similar study indicated that the foliar application of CDs markedly enhances plant growth, improves nutrient uptake and biomass accumulation, and augments stress resistance [[105]24]. Compared to NCK, infection (RCK treatment) reduced photosynthetic efficiency ([106]Fig. 1C). Specifically, Ci, Tr, Pn, SPAD, and Fv/Fm were decreased by 32.1%, 43.1%, 49.1%, 32.6%, and 28.2%, respectively. Compared to RCK, the Ci, Tr, Pn, SPAD, and Fv/Fm of the IPT and CDs treatments increased by 22.8%–55.1%, 30.3%–71.8%, 48.8%–189%, 15.1%–60.5%, and 11.1%–42.9%, respectively. Ci, Tr, Pn, SPAD, and Fv/Fm levels in CDs100 and CDs200 treatments are higher than in the IPT treatment. This study restored the Pn, Fv/Fm, and SPAD of infected rice leaves treated with CDs (100 and 200 ​mg/L) to healthy levels. CDs promote better photosynthesis, ensuring a sufficient supply of carbohydrates in infected rice leaves and enhancing rice disease resistance. The relative gene expression levels of OsRBCL, OsRBCS, OsRCA, OsPSBA, OsPSBC, OsPETD, OsPSAC, OsNDHD, OsNDHE, and OsATPA in CDs100 were significantly higher than those in the RCK and IPT treatments ([107]Fig. 1D). OsRBCL, OsRBCS, and OsRCA encode RuBisCO enzyme activity [[108]41]. RuBisCO is crucial for maintaining chloroplast homeostasis, facilitating photosynthesis, and defending against biotic stress [[109]42]. PS II and PS I serve as the reaction centers of photosynthesis, and OsPSBA, OsPSBC, OsPETD, and OsPSAC encode PS II and PS I functions. These four genes are crucial for maintaining chloroplast homeostasis and improving plant stress resistance. The NDH and ATPase complexes affect chloroplast homeostasis and photosynthetic efficiency, with OsNDHD, OsNDHE, and OsATPA being genes that encode components of the NDH and ATPase complexes [[110]43]. It has been demonstrated that CDs can enhance electron transfer rates in PS I and PS II and increase RuBisCO activity, thereby boosting crop photosynthesis [[111]44,[112]45]. The results above indicate that foliar application of CDs (especially at 100 ​mg/L) significantly enhances chloroplast homeostasis and improves photosynthesis in M. oryzae-infected rice leaves. Compared to NCK, the levels of H[2]O[2] and MDA in rice leaves treated with RCK significantly increased by 2.73 and 2.86 times, respectively. The activities of SOD, CAT, and POD in RCK-treated leaves showed no significant difference from those with NCK treatment ([113]Fig. 2). This indicates that infection (RCK treatment) induced high levels of oxidative stress in rice leaves, leading to oxidative damage to cell membranes. Compared to RCK, treatments with IPT, CDs100, CDs200, and CDs400 significantly reduced the levels of H[2]O[2] and MDA by 21.1%–44.5% and 39.2%–61.5%. SOD, POD, and CAT activities in leaves treated with CDs100 and CDs200 increased significantly compared to those with RCK treatment. In contrast, the activities in IPT and CDs400-treated leaves showed no significant differences from those with RCK ([114]Fig. 2). Pathogens directly induce excessive accumulation of ROS in chloroplasts, leading to chloroplast oxidative damage that results in yellowing and death of the plants [[115]46]. These results suggest that, compared to IPT treatment, CDs significantly enhanced the SOD-CAT antioxidant pathway, which contributes to mitigating pathogen-induced accumulation of H[2]O[2] and MDA in the chloroplast and improving disease resistance [[116]47]. Fig. 2. [117]Fig. 2 [118]Open in a new tab Effect of foliar-applied CDs100, CDs200, CDs400, and IPT on H[2]O[2] content, MDA content, and the activities of SOD, POD, and CAT in M. oryzae-infected rice leaves. The significant difference among treatments is marked with different letters (p ​< ​0.05, n ​= ​3). Foliar application of CDs at 100 and 200 ​mg/L enhanced rice resistance to M. oryzae through three synergistic pathways: (1) indirect pathogen suppression, (2) photosynthetic recovery via chloroplast regulation, and (3) ROS scavenging. In contrast, higher CDs concentration (400 ​mg/L) induced phytotoxic effects, mirroring observations in CDs (>100 ​mg/L), where excessive nanoparticle accumulation disrupts cellular homeostasis [[119]48]. At supraoptimal doses, CDs likely overwhelm plant detoxification systems, causing ROS imbalance and metabolic dysregulation, which paradoxically exacerbate stress and diminish disease resistance. This dose-dependent duality aligns with the “hormesis” phenomenon, where low concentrations stimulate beneficial responses, while high concentrations inhibit them. The results underscore CDs' potential as sustainable alternatives to conventional fungicides, leveraging host-mediated resistance over direct antimicrobial action. To further explore the mechanisms by which CDs induce rice disease resistance, we focus solely on NCK, RCK, IPT, CDs100, and CDs200 treatments for analysis and comparison. 3.3. Mechanisms of CDs regulating disease resistance in rice Enhanced photosynthesis in plants activates “retrograde molecules” such as proteins, pigments, and hormones in chloroplasts, which migrate to the nucleus or cytosol. This movement modifies nuclear gene expression, initiating the plant's defense mechanisms and strengthening stress resistance [[120]17]. To illustrate how CDs can activate the disease-resistant immune system in rice by regulating chloroplast homeostasis, we further explored the metabolites and hormone biosynthesis pathways in rice leaves. 3.3.1. Effects of CDs on the biosynthesis pathway of metabolites in diseased rice leaves This study conducted metabolomic analysis on rice leaves from different treatment groups to elucidate the underlying mechanisms by which CDs regulate rice disease resistance. PCA results showed that the first and second principal components, accounting for 44.0% and 20.5% of the total variance, significantly distinguished between the NCK, RCK, IPT, and CDs treatment groups (CDs100 and CDs200) ([121]Fig. S3). Volcano plots and Venn diagrams were used to identify metabolites that were differentially responsive to CD treatment. The volcano plots indicated that, compared to RCK, NCK, IPT, CDs100, and CDs200, respectively, had 11, 9, 9, and 9 metabolites significantly down-regulated, as well as 2, 9, 17, and 16 metabolites considerably up-regulated ([122]Fig. 3A–D). Venn diagrams illustrated those 17 metabolites, uniquely responsive to CDs and regulating resistance to M. oryzae, including 15 significantly up-regulated and 2 significantly down-regulated metabolites ([123]Fig. 3E and F). Furthermore, metabolic pathway enrichment analysis of these 17 critical differential metabolites encompassed pathways related to amino acid metabolism, flavonoid biosynthesis, and plant hormone synthesis ([124]Fig. 3G). Fig. 3. [125]Fig. 3 [126]Open in a new tab The metabolites volcano plots of NCK (A), IPT (B), CDs100 (C), and CDs200 (D) in M. oryzae-infected rice leaves. The Venn plots show the significantly up-regulated (E) and down-regulated (F) metabolites in NCK, IPT, CDs100, and CDs200 groups. (G) The metabolic pathway network of flavonoid biosynthesis, hormone synthesis, and amino acid metabolism is regulated by CDs in M. oryzae-infected rice leaves. Compared to RCK, rice leaves treated with CDs exhibited significant differences in 17 metabolites, with 15 metabolite contents significantly increased and two metabolites significantly decreased ([127]Fig. S4A). Alanine, nicotinic acid, pyridoxine, histidine, ascorbic acid, 4-guanidinobutyric acid, and pyroglutamic acid are related to amino acid metabolism ([128]Fig. 3G). Amino acid metabolism is essential for plant protein synthesis and participates in various biochemical processes, including the biosynthesis of other amino acids, nucleotide metabolism, the TCA cycle, glycolysis, and hormone synthesis [[129]49]. Nanomaterials enhance amino acid metabolism pathways and improve plant stress resistance [[130]50,[131]51]. Diosmetin, apigenin, naringenin, glyceric acid, and isotretinoin are associated with the flavonoid biosynthesis pathway. Flavonoids are crucial in plant–pathogen interactions by acting as antimicrobial agents that scavenge excess free radicals under stress conditions [[132]52]. Corchorifatty acid, palmitic acid, JA, 12-OPDA, and zeatin are associated with the plant hormone synthesis pathway. Endogenous hormones such as SA and JA are crucial in plant disease resistance signaling pathways [[133]53]. Nano-regulators or plant growth regulators improve stress or disease resistance by promoting JA accumulation. JA can activate physical responses in plants when attacked by pathogens, prompting the synthesis of alkaloids or flavonoids that create defensive structures to impede pathogen spread [[134]17,[135]54,[136]55]. In the current study, the concurrent up-regulation of flavonoid and JA biosynthesis pathways suggests a potential crosstalk between these defense mechanisms, possibly contributing to the enhanced disease resistance observed in CD-treated plants. 3.3.2. Effects of CDs on hormone and defense pathways in diseased rice leaves To elucidate how CDs mediate rice blast resistance, we analyzed hormone levels and the expression of genes involved in flavonoid and hormone biosynthesis. Plant hormones such as abscisic acid (ABA), JA, and SA cooperatively regulate disease resistance [[137]56]. Notably, CDs (100 and 200 ​mg/L) significantly elevated indole acetic acid (IAA) and JA contents compared to the infected control (RCK) and fungicide (IPT) groups. At the same time, ABA and SA levels remained unchanged ([138]Fig. 4A). This aligns with the upregulation of IAA biosynthesis genes (OsYUCCA1, OsYUCCA4, OsYUCCA6, OsOASA1, OsOASB2, OsAMT2, OsTDD1) [[139]57] and JA pathway genes (OsOPR3, OsAOC, OsLOX, OsAOS) in CDs-treated leaves ([140]Fig. 4B and C) [[141]58,[142]59]. Synergistic interactions between JA and IAA amplify defense responses during pathogen infection [[143]60], supporting our findings. Chloroplasts, the primary sites for IAA and JA synthesis, are vulnerable to oxidative damage from pathogen-induced ROS. Here, CDs counteracted this by activating flavonoid biosynthesis genes (OsCHS1, OsCHI, OsF3H, OsFNSI) ([144]Fig. S4B) [[145]61,[146]62]. The correlation analysis revealed a strong positive correlation between the levels of IAA and JA and the genes related to chloroplast homeostasis that work on photosynthesis ([147]Fig. S5). These results suggest that foliar application of CDs activates the flavonoid biosynthesis pathway in pathogen-infected rice leaves, removes pathogen-induced ROS, enhances chloroplast homeostasis, and stimulates the biosynthesis of disease-resistance signals (IAA and JA). Fig. 4. [148]Fig. 4 [149]Open in a new tab Effect of foliar-applied CDs100, CDs200, and IPT on hormone contents (A), the relative gene expression of indoleacetic acid (B), jasmonic acid (C) biosynthesis, and anti-diseased gene (D) in M. oryzae-infected rice leaves. The significant difference among treatments is marked with different letters (p ​< ​0.05, n ​= ​3). The genes related to PR proteins (such as OsPR1a, OsPR1b, and OsPR10a) are essential in the plant defense system [[150]63]. When pathogens infect plants, plants activate the disease resistance signal transduction pathway and produce PR proteins, producing SAR and enhancing plant immunity to pathogens [[151]64]. The relative gene expression levels of OsPR1a, OsPR1b, and OsPR10a in rice leaves treated with CDs (CDs100 or CDs200) were significantly higher than those in the NCK, RCK, and IPT groups ([152]Fig. 4D). Similar studies have shown that nano-regulators (such as nano-selenium, -lanthanum, and -silica) can induce the expression of related disease resistance genes (AOC, LOX, PR1, PR3, or PR10), promote plants to acquire SAR, and enhance plant disease resistance [[153]15,[154]54,[155]65]. As expected, defense-related genes (OsOPR3, OsAOC, OsAOS, and OsPR1a) showed higher expression in infected controls (RCK) compared to healthy plants (NCK) ([156]Fig. 4C and D). This may reflect the well-established activation of JA biosynthesis (via OsOPR3, OsAOC, and OsAOS) and PR genes during M. oryzae infection as part of rice's innate immunity [[157]66]. These results indicate that CDs enhance rice disease resistance by activating the JA and IAA disease resistance signal transduction pathways, thereby enabling rice leaves to acquire SAR. 3.4. Impact of CDs prevention M. oryzae on rice yield and quality The grain yield phenotype at maturity revealed that compared to NCK, rice treated with RCK exhibited significantly reduced grain weight and quality. In contrast, the foliar application of IPT, CDs100, and CDs200 restored grain yield, with CDs100 and CDs200 outperforming IPT ([158]Fig. 5A). Compared to NCK, RCK-treated rice at maturity showed significant reductions of 17.9% and 60.5% in 100-grain weight and yield per plant, respectively, along with declines of 22.0% and 17.8% in starch and protein content. Relative to RCK, treatments with IPT, CDs100, and CDs200 resulted in increases of 21.9%–28.9% in 100-grain weight, 133%–198% in yield per plant, 23.3%–27.5% in starch content, and 12.9%–36.1% in protein content of rice grains at maturity ([159]Fig. 5B). Similar studies have indicated that CDs can act as growth regulators when sprayed on healthy crops to enhance photosynthesis and root nutrient uptake, improving crop growth, yield, and nutritional content, thus elevating crop quality [[160]67]. These results suggest that preventing M. oryzae in leaves with CDs (100 and 200 ​mg/L) at the tillering stage can significantly curb leaf blast disease and ensure the yield and quality of rice at maturity, which could have significant implications for sustainable agriculture. Fig. 5. [161]Fig. 5 [162]Open in a new tab (A) The phenotype of rice gain treated with CDs100, CDs 200, and IPT in M. oryzae-infected rice. (B) Effect of foliar-applied CDs100, CDs200, and IPT on 100-grain weight, grain yield per plant, and starch and protein contents. The significant difference among treatments is marked with different letters (p ​< ​0.05, n ​= ​3). 3.5. Biosafety evaluation of CDs The present study evaluated the biosafety of CDs as alternatives to traditional fungicides using E. fetida and C. elegans—established ecotoxicological models [[163]68,[164]69]. After 14 days, earthworm survival stabilized at 88.3% in CDs100 and CDs200 groups, while IPT treatment caused 100% mortality by day 7 ([165]Fig. 6A). Similarly, C. elegans exhibited >94% survival after 96-h CD exposure, showing no significant difference from the control group. However, the IPT-treated survival rate of C. elegans was 77.3% after 96 ​h, significantly lower than that in the control, CDs100, and CDs200 groups ([166]Fig. 6B). Fig. 6. [167]Fig. 6 [168]Open in a new tab Biosafety evaluation of CDs as nano-regulators of crop disease resistance. (A) The survival rate of E. fetida exposed to IPT, CDs100, and CDs200. The survival rate after 96 ​h exposure (B), head thrashes (C), and body bends (D) of C. elegans exposed to IPT, CDs100, and CDs200. The frequency of head thrashes and body bends in C. elegans is an essential marker for evaluating the damage to the nervous system of C. elegans [[169]70]. After 24 ​h of exposure, the IPT-treated C. elegans exhibited a rapid decline in head thrashes and body bends. Specifically, IPT reduced these neurobehavioral markers within 96 ​h, including a 45.2% decrease in head thrashes and a 46.3% reduction in body bends ([170]Fig. 6C and D), demonstrating acute neurotoxicity in IPT treatments. Compared to the control group, CDs100 resulted in an 11.2% decrease in head thrashes and a 15.0% reduction in body bends, while CDs200 led to a 14.5% decrease in head thrashes and a 19.9% reduction in body bends. These findings indicate that CDs maintain higher environmental compatibility than conventional fungicides, aligning with reports of lower ecotoxicity of nano-regulator [[171]36,[172]47,[173]71,[174]72]. Therefore, CDs, as a nano-regulator for crop disease prevention, offer higher biosafety and eco-friendliness than traditional fungicides. While current results suggest that CDs are safe for E. fetida and C. elegans, extended multi-species assessments remain necessary to fully characterize their ecological impacts. 4. Conclusions The present study first evidenced that foliar-applied CDs (100 and 200 ​mg/L) function as an effective “plant vaccine” by (1) inhibiting M. oryzae while preserving rice yield and quality; (2) exhibiting superior environmental safety over conventional fungicides; (3) activating the SOD-CAT-flavonoid axis to scavenge oxidative stress; and (4) inducing SAR via synergistic IAA-JA signaling and chloroplast homeostasis regulation. CDs represent a sustainable nano-agrotechnology with dual benefits in crop protection and environmental health. However, field validation of their long-term efficacy, economic feasibility, and ecological biosafety remains essential. Future research should focus on: (1) optimizing scalable CD formulations, (2) assessing synergies with integrated pest management strategies, and (3) elucidating transgenerational impacts on agroecosystems. These efforts will advance the transition toward precision green agriculture. CRediT authorship contribution statement Shuhan Lei: Writing – original draft, Visualization, Investigation, Funding acquisition. Wanjing Liu: Writing – original draft, Visualization, Investigation, Funding acquisition. Baoshan Xing: Supervision. Jun Wang: Supervision. Jiake Xu: Visualization. Chaoqi Wang: Investigation. Cheng Zhang: Writing – review & editing, Supervision, Funding acquisition, Conceptualization. Peng Gao: Writing – review & editing. Jun Wang: Supervision. Lusheng Zhu: Supervision. Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments