Bacillus velezensis S44 mediated chitosan nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses bacillus velezensis S44 mediated chitosan nano-particles as well as a preparation method and application of the bacillus velezensis S44 mediated chitosan nano-particles. The preparation method of the chitosan nanoparticles comprises the following steps: inoculating bacillus velezensis S44 into an LB culture medium, culturing for 36-60 hours, and filtering to remove cells to obtain a filtered supernatant; uniformly mixing the filtered supernatant and a chitosan solution, and incubating for 36 to 60 hours, so as to obtain chitosan nanoparticles; the bacillus velezensis S44 is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No: 66680. The preparation method is convenient and environmentally friendly, the obtained chitosan nanoparticles (S44-CNPs) are small in particle size (1t, 100 nm) and excellent in antibacterial activity, are equivalent to common pesticide azoxystrobin and are higher than common TPP-CNPs nanoparticles, and the huge potential of marine microorganism natural products for green synthesis of novel nano pesticides is proved.

Description

Technical Field

[0001] This invention relates to the field of biological control technology for plant diseases, and in particular to chitosan nanoparticles mediated by Bacillus belyssus S44, their preparation methods, and applications. Background Technology

[0002] strawberry( Fragaria × ananassa Strawberries are an important global economic crop, rich in antioxidants, polyphenols, flavonoids, anthocyanins, vitamins, and amino acids, thus possessing high economic value. Although strawberries have an enticing aroma and flavor, they are highly susceptible to spoilage after harvest due to physical damage, physiological changes, and microbial infection. Anthracnose (a bacterium) is a particularly dangerous pathogen. Colletotrichum gloeosporioides Penz. ), Anthrax bacillus ( C. acutatum Simmonds ) and strawberry anthracnose ( C. fragariae Brooks Anthracnose, caused by *Colletotrichum candida*, is particularly severe, leading to an annual yield loss of 25-30% and significantly reducing fruit quality and shelf life. *Colletotrichum candida*, as the most pathogenic species, poses a threat to strawberries both before and after harvest. Overall, post-harvest losses due to high respiration rates, dehydration, and fungal decay can reach 30%, reducing shelf life to only 1-2 days at room temperature and 3-5 days under refrigeration. Solving these problems requires a thorough understanding of the mechanisms leading to spoilage and the implementation of targeted post-harvest disease control strategies.

[0003] Traditional chemical fungicides (such as benomyl, mancozeb, carbendazim, tebuconazole, and prochloraz) have long been used to control postharvest fungal diseases. However, their widespread use has led to environmental and health problems, including increased pathogen resistance, disruption of beneficial microbial communities, and pesticide residues and accumulation in fruits. Therefore, developing environmentally friendly alternatives is urgently needed. Biocontrol agents can effectively and specifically inhibit pathogens with lower environmental and health risks, thus showing great promise. Furthermore, postharvest biocontrol technologies that can delay fruit ripening and inhibit microbial growth can further extend shelf life. Given the limited number of currently approved chemical agents and the increasing resistance of pathogens, developing sustainable disease control strategies has become a key issue in agriculture.

[0004] Therefore, existing technologies still need improvement and development. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide Bacillus vesiculosus S44-mediated chitosan nanoparticles, their preparation method and application, aiming to develop eco-friendly antibacterial agents and provide a comprehensive solution for field and post-harvest control of strawberry microbial diseases, quality maintenance and reduction of dependence on traditional fungicides.

[0006] The technical solution of the present invention is as follows: Firstly, it provides Bacillus vesiculosus (BVV) Bacillus velezensis The preparation method of S44-mediated chitosan nanoparticles includes the following steps: (1) Inoculate Bacillus belye S44 into LB medium and culture for 36-60 hours. Filter to remove cells and obtain the supernatant. (2) Mix the filtered supernatant and chitosan solution evenly and incubate for 36-60 hours to obtain chitosan nanoparticles; The Bacillus belyssus S44 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66680 and deposit date of July 14, 2025.

[0007] Secondly, Bacillus belye S44-mediated chitosan nanoparticles are provided, prepared using the chitosan nanoparticle preparation method described in the first aspect.

[0008] Thirdly, the application of chitosan nanoparticles as described in the second aspect in the preparation of products that inhibit plant pathogens is provided.

[0009] Fourthly, the application of chitosan nanoparticles as described in the second aspect in the prevention and control of anthracnose in strawberries after harvest is provided.

[0010] The main advantages of this invention are as follows: (1) S44-CNPs were synthesized by biosynthesis using the supernatant of Bacillus belye from mangroves and chitosan. The method is convenient and environmentally friendly. The obtained nanoparticles S44-CNPs have a small particle size (<100nm) and excellent antibacterial activity, which is comparable to that of the commonly used pesticide pyraclostrobin and significantly higher than that of common TPP-CNPs nanoparticles. This confirms the great potential of marine microbial natural products for the green synthesis of new nanopesticides. At present, there is very little research on this type of microorganism in the agricultural field, especially in the research and development of nanopesticides.

[0011] (2) This invention systematically evaluated the antibacterial activity spectrum of S44-CNPs against a variety of harmful pathogenic fungi and bacteria, as well as their ecotoxicity to beneficial biocontrol bacteria and indicator environmental organisms. The study is relatively systematic and comprehensive, providing necessary data support for clarifying whether S44-CNPs is an eco-friendly nano-pesticide.

[0012] (3) The inhibitory effect of S44-CNPs on anthracnose was verified in strawberry leaves and fruits, and the effects on fruit quality and storage period under cold storage conditions were verified. Furthermore, the effects and mechanisms of S44-CNPs on maintaining postharvest fruit metabolic homeostasis and improving fruit quality were clarified through quantitative analysis of 19 sugar substances and 146 central carbon compounds. Attached Figure Description

[0013] Figure 1 This refers to the biosynthesis of chitosan nanoparticles mediated by Bacillus belyssus S44; where (A) is the colony morphology of Bacillus belyssus S44 on MA plates; (B) is the appearance change of the reaction solution during the synthesis process; (C) is the characteristic absorption peak of the ultraviolet-visible spectrum of chitosan nanoparticles in the wavelength range of 200-600nm, Cs refers to chitosan, CFCs refers to the supernatant of bacterial filtration (cultural free supernatants), and S44-CNPs refers to the chitosan nanoparticles mediated by Bacillus belyssus S44.

[0014] Figure 2 The physicochemical properties of S44-CNPs are characterized as follows: (A) is a SEM micrograph of S44-CNPs; (B) is a FTIR spectral analysis of S44-CNPs; (C) is an XRD diffraction pattern of S44-CNPs; (D) is a statistical analysis of the particle size distribution of S44-CNPs based on the SEM image; and (E) is an EDS energy dispersive spectroscopy analysis of S44-CNPs.

[0015] Figure 3 S44-CNPs are against Colloidal anthracnose ( Colletotrichum gloeosporioides Results related to the inhibitory activity of CG growth were presented; (A) represents colony growth; (B) represents the colony diameter of CG on PDA plates; and (C) represents the inhibition rate of CG growth by different treatment groups. The experiment used PDA medium supplemented with 100 µg / mL chitosan (Cs) and azoxystrobin, as well as different concentrations of TTP-CNPs and S44-CNPs. The data represent independent experimental replicates, with three replicates per group (n=9). Different lowercase letters in the bar chart indicate significant differences between groups as determined by Tukey's HSD test (p value ≤ 0.05).

[0016] Figure 4 The antibacterial spectrum of S44-CNPs and its activity against Colloidal anthracnose ( Colletotrichum gloeosporioidesThe results showed the effects of different concentrations of S44-CNPs (25µg / mL, 50µg / mL, 100µg / mL) on the mycelial growth of four pathogenic fungi (Fusarium graminearum, Alternaria alternata, Sclerotinia sclerotiorum, and Bacillus oryzae). (A) represents the inhibitory effect of different concentrations of S44-CNPs (25µg / mL, 50µg / mL, 100µg / mL) on the mycelial growth of four pathogenic fungi (Fusarium graminearum, Alternaria alternata, Sclerotinia sclerotiorum, and Bacillus oryzae). (B) represents the colony diameter (cm) of different concentrations of S44-CNPs (25µg / mL, 50µg / mL, 100µg / mL) on the mycelial growth of four pathogenic fungi (Fusarium graminearum, Alternaria alternata, Sclerotinia sclerotiorum, and Bacillus oryzae). (C) represents the colony diameter (cm) of different concentrations of S44-CNPs (25µg / mL, 50µg / mL, 100µg / mL). The inhibition rate of S44-CNPs (100 μg / mL) on the mycelial growth of four pathogenic fungi (Fusarium graminearum, Alternaria alternata, Sclerotinia sclerotiorum, and Blastomyces oryzae) was measured by comparing optical microscopy (CM) and scanning electron microscopy (SEM) images. Colletotrichum gloeosporioides The influence of mycelial morphology (CG).

[0017] Figure 5 The results show the inhibitory effect of S44-CNPs on pathogenic bacteria; (A) represents the bacterial concentrations of MRSA and PAO1 after 24 h of treatment with different concentrations of S44-CNPs; (B) represents the inhibition rates of MRSA and PAO1 after 24 h of treatment with different concentrations of S44-CNPs; different lowercase letters indicate the statistically significant differences between groups as determined by Tukey's HSD test (p value < 0.05).

[0018] Figure 6 These are the ecotoxicity assessment results of S44-CNPs; among them, (A) shows the *Trichoderma harzianum* on PDA medium with different concentrations of S44-CNPs. T. harzianum ) and Paecilomyces lilacinus ( P. lilacinum (A) Colony morphology of Trichoderma harzianum on PDA medium with different concentrations of S44-CNPs; (B) Colony morphology of Trichoderma harzianum on PDA medium with different concentrations of S44-CNPs. T. harzianum ) and Paecilomyces lilacinus ( P. lilacinum Analysis of colony diameter (cm) of (C) is that of Artemia salina ( Artemia salina Survival rate analysis of S44-CNPs, TTP-CNPs and fungicide azoxystrobin; different lowercase letters indicate the statistical significance between groups as determined by Tukey's HSD test (p value < 0.05).

[0019] Figure 7 This study investigated the inhibitory effect of S44-CNPs on anthracnose in strawberry leaves and fruits. (A) shows the disease incidence on leaves in each treatment group 5 days after inoculation with the pathogen, with a scale bar of 1 cm. (B) shows the lesion area (cm²) on leaves in each treatment group 5 days after inoculation with the pathogen. 2(C) shows the inhibition rate of different concentrations of S44-CNPs on anthracnose in strawberry leaves; (D) shows the disease incidence of strawberry fruits inoculated with *Colletotrichum gloeosporioides* at different observation time points under different concentrations of S44-CNPs treatment; (E) shows the lesion area (cm²) of strawberry fruits inoculated with *Colletotrichum gloeosporioides* under different concentrations of S44-CNPs treatment. 2 (F) represents the inhibition rate of strawberry fruits inoculated with Colletotrichum gloeosporioides under different concentrations of S44-CNPs treatment; the data are the mean ± standard deviation (SD) of three independent experiments; different lowercase letters in the bar chart indicate statistically significant differences between treatment groups as determined by Tukey's HSD test (p<0.05).

[0020] Figure 8 This is a study on the effect of S44-CNPs on the preservation of strawberry fruit at 4℃. Among them, (A) is a diagram showing the changes in appearance of strawberries in different treatment groups during 15 days of cold storage: water control (WC), anthracnose inoculation group (CG), chitosan nanoparticles (TTP-CNPs) and 25 / 50 / 100μg / mL S44-CNPs treatment groups; (B) is the rot rate (%) of strawberries in each group on day 15; (C) is the fruit weight loss rate (%) during storage; and (D) is the soluble sugar content (μg / g fresh weight) of strawberries after treatment.

[0021] Figures 9 to 11 This is the sugar content analysis result of strawberry fruits after storage at 4℃ for 15 days. Before storage, the fruits underwent water treatment (WT), *Colletotrichum candida* treatment (CT), S44-CNPs treatment (ST), or TTP-CNPs treatment (TT). Figure 9 (A) is a biplot of principal component analysis; Figure 9 The middle (B) is a cluster heatmap of 19 sugar metabolites, with colors from blue to red representing the abundance level of core sugars; Figure 10 (A) is a classification of carbohydrate metabolites based on KEGG annotations; Figure 10 (B) is the correlation matrix between carbohydrates and superclasses. The red and blue circles represent positive and negative correlations, respectively, and the size of the circles reflects the strength of the correlation. Figure 11 Box plots quantify the representative flavor-related sugars or the six sugar-related metabolites with the most significant changes in each treatment group.

[0022] Figures 12 to 14 This is a multivariate and pathway level analysis of central carbon metabolism in strawberry fruits under different treatment groups under refrigeration conditions. Among them, Figure 12 The pie chart in (A) shows the percentage distribution of identified metabolites across the 13 major chemical categories; Figure 12(B) is a PCA analysis diagram of central carbon metabolites in the four treatment groups (WT: water treatment group; CT: Colloidal anthracnose treatment group; TT: TTP-CNPs treatment group; ST: S44-CNPs treatment group); Figure 12 The middle (C) is a cluster heatmap of the 20 most abundant central carbon metabolites in all samples, with the blue-red color gradient representing low-high abundance; Figure 13 The middle (A) is a chord diagram showing the correlation between various central carbon metabolites and major metabolic categories. Red lines indicate positive correlations, blue lines indicate negative correlations, and the thickness of the lines reflects the strength of the correlation. Figure 13 The middle (B) is a bubble chart of KEGG pathway enrichment related to central carbon metabolism. The X-axis represents the pathway influence value, the Y-axis represents the statistical significance (-ln P value), the bubble size corresponds to the pathway influence value, and the color intensity represents the significance level. Figure 14 The dendrogram highlights pathways with significant alterations, including alanine / aspartate / glutamate metabolism, tyrosine metabolism, glycine / serine / threonine metabolism, and glutathione metabolism. Dark red areas indicate higher statistical significance.

[0023] Figure 15 The results of quantitative analysis of representative amino acids and organic acids under four treatments are presented by box plots: water treatment (WT), Colletotrichum gloeosporioides treatment (CT), TTP-CNPs only treatment (TT), and S44-CNPs only treatment (ST); where (A) is the quantitative level of key amino acids (nmol / g); and (B) is the quantitative level of key organic acids (nmol / g).

[0024] Figure 16 The results are the enrichment analysis results of the central carbon metabolism pathway in strawberry fruit under different postharvest treatment conditions; (A) is a cluster heatmap, where red indicates upregulation, blue indicates downregulation, and white indicates no change; (B) is a bubble chart of KEGG pathway enrichment, showing the significantly changed metabolic pathways, with the color intensity (red gradient) corresponding to the statistical significance level. Detailed Implementation

[0025] This invention provides chitosan nanoparticles mediated by Bacillus belye S44, their preparation method, and applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below.

[0026] Colloidal anthrax ( Colletotrichum gloeosporioides Anthracnose is a devastating postharvest pathogen in strawberries, causing severe anthracnose and fruit rot. Faced with the increasingly serious problem of pathogen resistance, the development of sustainable nanotechnology alternatives is urgently needed.

[0027] Nanoparticles (NPs) have shown significant potential in postharvest disease control due to their high specific surface area and unique physicochemical properties. Both metallic nanoparticles (such as silver, zinc oxide, and gold) and non-metallic nanoparticles (such as silica and carbon-based materials) exhibit excellent antibacterial properties. However, their ecotoxicity, environmental stability, and accumulation in plants remain controversial. In contrast, bio-based nanoparticles offer advantages such as higher safety and greater compatibility with sustainable development. In particular, biosynthesized nanoparticles offer advantages such as ease of operation, low cost, and avoidance of the use of toxic chemicals. Among them, chitosan, a naturally abundant biopolymer and an approved food additive, stands out for its biodegradability, safety, biocompatibility, and potent antifungal properties. More importantly, chitosan can serve as a highly efficient carrier for the bioactive components of pesticides and fertilizers, improving the physicochemical properties of these agents. Chitosan nanoparticles (CNPs) significantly enhance antibacterial efficacy: compared to ordinary chitosan, CNPs exhibit superior dispersibility, higher bioavailability, and stronger interactions with microbial cells, thereby penetrating cell membranes more efficiently to inhibit pathogens. Due to their superior properties, chitosan-based nanomaterials are playing an increasingly prominent role in green post-harvest technologies that reduce reliance on chemical fungicides and are widely recognized as promising alternatives to synthetic fungicides.

[0028] Currently, most bio-based nanoparticles are prepared using plant, fungal, bacterial, and algal extracts as reducing stabilizers. These particles, mostly with diameters of 10-100 nanometers, can effectively inhibit major pathogens such as *Botrytis cinerea*, *Colletotrichum gloeosporioides*, and *Penicillium expansum*. While marine microorganisms contain a rich variety of natural products with diverse biological activities, their application in agriculture lags far behind that in the pharmaceutical field due to insufficient research and development investment. In particular, marine microorganisms such as *Bacillus* can produce novel secondary metabolites in extreme marine environments such as high pressure, high salinity, and nutrient deprivation, often exhibiting multiple biological activities such as antibacterial, antitumor, and anti-inflammatory properties, thus showing great promise for applications in agriculture. Therefore, marine microorganism-mediated nanoparticle synthesis, due to its environmental friendliness and multiple advantages, is becoming a research hotspot in green nanotechnology, yet related research remains scarce.

[0029] Based on this, embodiments of the present invention provide Bacillus belysinus ( Bacillus velezensis The preparation method of S44-mediated chitosan nanoparticles includes the following steps: (1) Inoculate Bacillus belye S44 into LB medium and culture for 36-60 hours. Filter to remove cells and obtain the supernatant. (2) Mix the filtered supernatant and chitosan solution evenly and incubate for 36-60 hours to obtain chitosan nanoparticles; The Bacillus belyssus S44 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66680 and deposit date of July 14, 2025.

[0030] This embodiment utilizes marine-derived Bacillus belesia spp. ( Bacillus velezensis The S44 strain was used to develop an eco-friendly antifungal agent—chitosan nanoparticles (S44-Carbon Nanoparticles, S44-CNPs). The supernatant from the S44 bacterial suspension showed excellent biosynthetic efficiency of chitosan nanoparticles, successfully producing spherical nanoparticles (96.4±32.2 nm). Both the Fourier Transform Infrared (FTIR) and X-ray Diffraction (XRD) spectra of these nanoparticles exhibited typical chitosan characteristic peaks. S44-CNPs possess broad-spectrum antifungal activity against bacteria including *Colletotrichum gloeosporioides* (…). Colletotrichum gloeosporioides EC 50 =14.85 micrograms / ml), Fusarium graminearum ( Fusarium graminearum ), rice blast fungus ( Pyriculariaoryzae ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ) and Alternaria ( Alternaria alternata Multiple pathogenic fungi, including methicillin-resistant Staphylococcus aureus (MRSA), and methicillin-resistant Staphylococcus aureus (MRSA). Staphylococcus aureus MRSA) and Pseudomonas aeruginosa Pseudomonas aeruginosa They all showed significant inhibitory activity; while they exhibited inhibitory activity against beneficial fungi such as Trichoderma harzianum ( Trichoderma harzianum ) and Paecilomyces lilacinus ( Purpureocilliumlilacinum The impact is minimal. More importantly, S44-CNPs exhibit significantly lower ecotoxicity than the traditional fungicide azoxystrobin: at a concentration of 100 μg / mL, artichokes (… Artemia salinaThe survival rate reached 66.6% (compared to only 28% in the pyraclostrobin group). Strawberries treated with 100 μg / mL S44-CNPs showed a significant reduction in anthracnose lesion area compared to the untreated control group: approximately 78% less on leaves and approximately 88% less on fruit. This treatment also effectively inhibited postharvest water loss (1.8% vs. 6.7% in the control group) and maintained fruit quality during a 15-day cold storage period by inhibiting spoilage, including maintaining total sugar content (approximately 660 μg / g), appearance, and extending shelf life. Targeted metabolite quantitative analysis showed that S44-CNPs treatment maintained fruit flavor characteristics and sugar homeostasis. The marine microbial nanobiotechnology strategy established in this embodiment simultaneously achieves the triple goals of effective control of strawberry anthracnose, environmental friendliness, and maintenance of strawberry fruit metabolic homeostasis, providing a comprehensive solution for field and postharvest control of strawberry fungal diseases, quality maintenance, and reducing reliance on traditional fungicides.

[0031] In some embodiments, the chitosan solution is prepared with water and comprises: chitosan with a mass concentration of 0.05-0.3% and acetic acid with a volume concentration of 0.5-3%; the pH of the chitosan solution is 3-5.

[0032] In some more specific embodiments, the method for preparing the chitosan solution includes the following steps: Chitosan is dissolved in an aqueous solution containing acetic acid and stirred at 200-400 rpm for 8-16 hours. The filtrate is collected by filtration, and the pH is adjusted to 3-5 to obtain a chitosan solution.

[0033] In some embodiments, the volume ratio of the filtration supernatant to the chitosan solution is 1:(0.5-3).

[0034] In some implementations, the cultivation conditions in step (1) include: a temperature of 25-30°C and a rotation speed of 100-200 rpm.

[0035] In some implementations, the incubation conditions in step (2) include a temperature of 25-30°C.

[0036] In some embodiments, the filtration described in this invention is performed using a filter membrane with a diameter of 0.22-0.45 μm.

[0037] This invention provides chitosan nanoparticles mediated by Bacillus belye S44, which are prepared using the chitosan nanoparticle preparation method described above.

[0038] This invention provides the application of chitosan nanoparticles as described above in the preparation of products that inhibit plant pathogens.

[0039] In some embodiments, the plant pathogen is selected from *Colletotrichum gloeosporioides* (…).Colletotrichum gloeosporioides Fusarium graminearum ( ), Fusarium graminearum ), rice blast fungus ( Pyriculariaoryzae ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum Alternaria ( Alternaria alternata Methicillin-resistant Staphylococcus aureus Staphylococcus aureus ) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa One or more of the following.

[0040] This invention provides the application of chitosan nanoparticles as described above in the prevention and control of anthracnose in strawberries after harvest.

[0041] The present invention will be further described below through specific embodiments.

[0042] Example 1 This embodiment provides the biosynthesis of chitosan nanoparticles mediated by Bacillus belyssus from mangroves, and studies their dual inhibition and metabolic homeostasis regulation in postharvest anthracnose of strawberries, as detailed below: (1) Reagents, strains and culture media used Chitosan (deacetylation degree ≥85%, industrial grade, derived from crab shells) was purchased from Sigma-Aldrich (China); sodium tripolyphosphate (TTP) and glacial acetic acid were purchased from Aladdin Industries (Shanghai). Strain S44 was isolated in August 2021 from mangrove soil in the Mai Po Nature Reserve, Hong Kong (114.05°E, 22.49°N) and cultured in 2216 marine agar medium (MA, BD Difco™). Molecular identification of the strain was performed by amplification of the 16S rRNA gene (SEQ ID NO.1). The sequence was determined to be closely related by NCBI BLASTn alignment and identified as *Bacillus belesiensis*. Bacillus velezensis The sample has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66680 and deposit date of July 14, 2025.

[0043] Pathogenic fungi include: Colloidal anthracnose ( Colletotrichum gloeosporioides ACCC36353, provided by the China Agricultural Microbial Culture Collection Center), rice blast fungus ( Pyriculariaoryzae P131, a gift from Professor Peng Youliang of China Agricultural University), Fusarium graminearum ( Fusarium graminearum PH-1), Alternaria ( Alternaria alternata ) and Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum (Gifted by Zhejiang University). Beneficial fungi include: Trichoderma harzianum ( Trichoderma harzianum Th46) and Paecilomyces lilacinus (Purpureocilliumlilacinum (Gifted by the Trichoderma research team of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences). The bacterial strain contains methicillin-resistant Staphylococcus aureus (MRSA). Staphylococcus aureus ATCC 43300, MRSA) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa PAO1 (preserved in our laboratory). Fungal culture was performed on potato dextrose agar (PDA), and bacterial culture was performed on LB (Luria-Bertani) medium. Colloidal anthracnose was cultured on oat tomato agar (OTA) and incubated at 28°C in a light incubator (Shanghai Lanpa Instruments) with a 12h light / 12h dark cycle.

[0044] The culture medium is prepared with water and has the following components: LB medium: Tryptone 10 g / L, Yeast extract 1 g / L, Sodium chloride (NaCl) 5 g / L.

[0045] Oat tomato agar (OTA): 35g / L oat flakes, 150mL / L tomato juice (from freshly crushed tomatoes), 0.7g / L calcium carbonate (CaCO3), 18g / L agar, pH 7.0.

[0046] Potato glucose agar (PDA) medium: potato extract powder 6 g / L, glucose 20 g / L, agar 15 g / L.

[0047] (2) Biosynthesis and characterization of chitosan nanoparticles S44-CNPs Strain S44 was activated from -80℃ on MA medium and cultured at room temperature for 24-48 hours. Figure 1 (A) Afterwards, several single colonies were picked and placed in LB medium, and cultured for 48 hours at 28±2℃ and 150 rpm with shaking. The culture was then centrifuged at 10000×g for 15 minutes, and the supernatant was filtered through a 0.22μm membrane filter to remove residual cells and obtain the filtration supernatant (cultural free supernatants, CFCs). Figure 1 (B)

[0048] To prepare the chitosan solution (CS), chitosan was first dissolved at a concentration of 0.2% (w / v) in a 1% (v / v) aqueous acetic acid solution, and then stirred overnight with a continuous magnetic stirrer at 300 rpm. Afterwards, the solution was filtered through a 0.22 μm PVDF syringe, and the pH was adjusted to 4.0. Figure 1 (B)

[0049] In the biosynthesis stage, equal volumes of CFCs and CS solutions were mixed dropwise under continuous magnetic stirring at 350 rpm, and then incubated at 28±2℃ for 48 hours to promote the formation of chitosan nanoparticles through a cross-linking reaction. Figure 1 As shown in Figure (B), the initial chitosan solution (CS) and bacterial supernatant (CFCs-44) were both colorless and transparent solutions. After the reaction, the solution turned into a milky white suspension, indicating the synthesis of chitosan nanoparticles. The generated S44-CNPs were collected by centrifugation at 10000×g for 15 minutes, followed by washing three times with double-distilled water to remove unbound substances. For subsequent analysis, the purified nanoparticles were lyophilized to prepare a dry powder.

[0050] The optical properties of biosynthesized S44-CNPs in the 200-600 nm wavelength range were analyzed using a BioTek Synergy LX microplate reader (Biotek Instruments, USA). The results showed that the S44-CNPs solution contained characteristic absorption peaks of nanoparticles that were significantly different from those in chitosan solution and bacterial supernatant. Figure 1 (C) The size and surface morphology of chitosan nanoparticles were observed using a scanning electron microscope (SEM; JEOL JSM-IT200), confirming that most nanoparticles were spherical, with a few being irregularly shaped. Figure 2 The average particle size of the middle (A) is 96.40 ± 32.19 nm. Figure 2 (D) further confirms the successful synthesis of chitosan nanoparticles; Fourier transform infrared spectroscopy (FTIR) analysis ( Figure 2 (B) shows: 3416cm -1 The characteristic peak at 2924 cm⁻¹ corresponds to the stretching vibrations of OH and NH, indicating the presence of hydroxyl and amino groups in the chitosan backbone; -1 The aliphatic stretching groups (CH and CH2) observed at this location are attributed to the CH stretching vibration of the methylene group. Notably, at 1648 cm⁻¹... -1 (C=O stretching, amide I band) and 1536cm -1 The characteristic peak at (NH bending and CN stretching, amide II band) suggests the possible presence of protein components derived from bacterial metabolites. Venkatesan et al. (2023) reported that in CNPs synthesized mediated by Plectranthus vettiveroides, the peak at 1614.83 cm⁻¹... -1 The FTIR absorption band at 1386 cm⁻ corresponds to the stretching vibration of the carboxylate group (–COO⁻), which is consistent with the ion interaction mechanism between the protonated amino group of chitosan and the phenolic acid from plant sources. -1 (-CH3 and -CH2 bending vibrations) and 1072 cm -1Additional peaks, such as those indicating CO stretching vibrations, further confirmed the integrity of the polysaccharide structure. These spectral features collectively validated the successful formation of chitosan nanoparticles and indicated that bacterial CFCs played a stabilizing role in the synthesis process. The trace peaks in the terminal region of the FTIR spectrum reflected the vibrational characteristics of the chitosan glycoskeleton. Furthermore, the peak at 1648 cm⁻¹... -1 Up to 1072cm -1 The presence of multiple strong peaks in the interval suggests the involvement of capping agents (naturally occurring organic molecules, such as proteins, amino acids, polysaccharides, polyphenols, or other metabolites, which bind to the surface of nanoparticles during their synthesis)—these substances act as stabilizers to prevent excessive growth and aggregation of nanoparticles during colloid formation.

[0051] X-ray diffraction (XRD) analysis of S44-CNPs confirmed their semi-crystalline properties, showing characteristic peaks at 2θ = 11.6°, 18.9°, and 23.8°. ​ (C)). These diffraction peaks are consistent with the standard diffraction pattern of chitosan nanoparticles.

[0052] Energy dispersive spectroscopy (EDS) analysis determined the elemental composition of chitosan nanoparticles, revealing in detail the types and relative abundances of their constituent elements. Characteristic energy peaks identified carbon (C), nitrogen (N), and oxygen (O) as the three main elements of S44-CNPs. Trace amounts of Na, P, Fe, and K were also detected, which may originate from the bacterial supernatant. ​ (E)

[0053] (3) Determination of antifungal activity of S44-CNPs S44-CNPs were prepared into a suspension with a concentration of 1 mg / mL and added to potato dextrose agar (PDA) medium before solidification, resulting in final concentrations of 25 μg / mL, 50 μg / mL, and 100 μg / mL. Mycelial discs with a diameter of 5 mm were obtained by punching holes at the edge of *Colletotrichum gloeosporioides* colonies cultured on PDA for 7 days and placed in the center of PDA plates containing different concentrations of S44-CNPs. As controls, PDA plates containing 100 μg / mL CS, azoxystrobin (control), and 25 μg / mL, 50 μg / mL, and 100 μg / mL TTP-CNPs were added, respectively. PDA plates without any added substances served as negative controls. The synthesis of TPP-CNPs nanoparticles employed an ionogel method, with the following specific steps: Chitosan (2 mg / mL) was dissolved in a 1% (v / v) acetic acid solution and stirred overnight at room temperature. The pH was then adjusted to 4.6 using 1M NaOH. A 2 mg / mL sodium tripolyphosphate (TPP) solution was prepared with deionized water and filtered through a 0.22 µm needle filter. Under constant stirring at 400 rpm, 3 mL of TPP solution was added dropwise to 10 mL of chitosan solution to form nanoparticles. After the addition was complete, the reaction mixture was stirred for another hour to ensure complete nanoparticle formation. The resulting suspension was centrifuged at 10,000 rpm for 20 minutes at 4 °C. The precipitate was washed twice with deionized water to remove unreacted substances. The final nanoparticles could be immediately reconstituted in water for use or freeze-dried for storage.

[0054] All plates were incubated at 28±2℃ for 5 days. After incubation, fungal growth was assessed by colony diameter. The inhibition rate was calculated using the following formula: Inhibition rate (%) = (Rc - Rt) / Rc × 100 (Equation 1); Rc = diameter of fungal colonies in the control plate (cm); Rt = Diameter of fungal colonies in the treatment plate (cm); All experiments were performed in triplicate. The effective concentration (EC50) of all tested nanoparticles was calculated. 50 ).

[0055] The results showed that S44-CNPs chitosan nanoparticles exhibited dose-dependent inhibitory effects at concentrations of 25 μg / mL, 50 μg / mL, and 100 μg / mL. Compared with the control group, the colony diameter was significantly reduced (p<0.05), and the inhibition rate was significantly increased. ​ (A)-(B)). Among them, S44-CNPs at 100 μg / mL showed the strongest antifungal activity against Colletotrichum gloeosporioides (C. gloeosporioides). C. gloeosporioidesThe inhibition rate of mycelial growth reached 62.4%, significantly better than chitosan itself (CS, 18.2%) and common TTP-CNP nanoparticles (57.5%), and comparable to that of azoxystrobin at the same concentration. Further determination of the half-maximal effective concentration (EC50) of S44-CNPs... 50 ), confirming its EC 50 The value (14.85 µg / mL) was significantly lower than the EC50 of commonly used chitosan nanoparticles TTP-CNPs. 50 The value (135.15 µg / mL, Table 1) further confirms that S44-CNPs have strong antifungal activity.

[0056] Table 1. Half-maximal effective concentration (EC50) of nanoparticles in inhibiting fungal growth. 50 )

[0057] (4) Determination of the antibacterial spectrum of S44-CNPs and its effect on the hyphal morphology of Colletotrichum gloeosporioides. Using the method described in (3), the antimicrobial spectrum of S44-CNPs against various plant pathogenic fungi at different concentrations (25 μg / mL, 50 μg / mL and 100 μg / mL) was evaluated, including Fusarium graminearum (…). F. graminearum PH-1), Alternaria ( A. alternata ), Sclerotinia sclerotiorum ( S. sclerotiorum ) and rice blast fungus ( P. oryzae P131 The results showed that at a concentration of 100 μg / mL, S44-CNPs significantly inhibited the hyphal growth of all tested fungi (inhibition rates ranged from 42.22% to 65.86%). Figure 4 Among the various groups (A)-(C), the one showing the strongest inhibitory activity (65.86%) against *Sclerotinia sclerotiorum*, a major pathogen causing diseases of soybeans and rapeseed, and a moderate inhibitory effect (52.34%) against *Alternaria alternata*. Figure 4 (C) Although different fungi have varying sensitivities to S44-CNPs, this study confirms that the S44-CNP nanoparticles have the potential to be developed into a broad-spectrum fungicide for the control of various crop diseases.

[0058] To further investigate the mechanism of action of S44-CNPs in inhibiting fungi, this example further determined the effect of S44-CNPs on fungal hyphae morphology. Following the method described in (3), *Colletotrichum gloeosporioides* (100 μg / mL) treated with S44-CNPs was subjected to... C. gloeosporioidesElectron microscopy was performed on the mycelium. After 5 days of culture, the peripheral mycelial region was carefully collected from the colony edge and observed under a Zeiss composite microscope at 40x magnification. The control group consisted of untreated cultures of S44-CNPs. For scanning electron microscopy (SEM) observation, samples were first fixed overnight at 4°C with 2.5% glutaraldehyde, followed by fixation with 1% (w / v) osmium tetroxide (OsO4) at 28±2°C for 1 hour. Dehydration was performed using a gradient series of ethanol (30-100%), with each concentration treated for 15 minutes. Morphological distortion of *Colletotrichum gloeosporioides* was observed using SEM according to the method previously described by Hafeez et al. (2024). Optical microscopy (CM) and scanning electron microscopy (SEM) analysis showed that untreated *Colletotrichum gloeosporioides* (… C. gloeosporioides The hyphae were straight, smooth, and plump; in contrast, the hyphae treated with S44-CNPs showed obvious structural abnormalities, including shrinkage, collapse, and surface fragmentation, indicating that the nanoparticles induced severe damage. Figure 4 (D)

[0059] This embodiment also evaluated the antibacterial potential of S44-CNPs against two pathogenic bacteria. Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PAO1) were selected as experimental subjects because they represent the type strains of Gram-positive and Gram-negative bacteria, respectively, and are both listed as pathogens of concern by the World Health Organization (WHO). The experiment was conducted in 96-well microtiter plates with a culture time of 24 hours. The test strains were first cultured overnight in LB medium and then adjusted to OD. 600 After reaching a concentration of 1.0, the plates were treated with 25 μg / mL, 50 μg / mL, and 100 μg / mL of S44-CNPs, respectively. Each well contained a total volume of 200 μL, including bacterial suspension and treatment solution. The plates were incubated at 37°C with shaking at 200 rpm. The microplates were then read from a microplate reader at a wavelength of 600 nm (OD200). 600 Bacterial cell density was measured to assess the growth inhibition effect.

[0060] The results showed that in the MRSA control group, bacterial cell density (OD) was... 600 After 24 hours of incubation, the OD value reached approximately 1.0. However, after treatment with S44-CNPs, the OD value decreased to approximately 0.80 at 25 µg / mL, to 0.74 at 50 µg / mL, and significantly decreased to 0.48 at 100 µg / mL, corresponding to a maximum inhibition rate of 69.02%. A similar trend was observed with PAO1, where treatment with 100 µg / mL S44-CNPs resulted in a bacterial growth inhibition rate of 55.91%. Figure 5 (A) and (B)). These findings indicate that S44-CNPs possess broad-spectrum antibacterial effects and have potential applications in agriculture and medicine.

[0061] (5) Ecotoxicity assessment of S44-CNPs The ecological risks were assessed by measuring and comparing the acute toxicity of S44-CNPs and azoxystrobin to beneficial microorganisms (Trichoderma harzianum and Paecilomyces lilacinus) and the indicator environmental organism Artemia nauplii. The toxicity evaluation indicators included the microbial growth inhibition rate (method as in (3)) and the survival rate of Artemia. The specific operation of the Artemia experiment was as follows: (i) Artemia egg hatching: 1g of Artemia eggs were placed in a conical flask, 500mL of artificial seawater (17g of sea salt dissolved in distilled water) was added and hydrated for 1 hour, and cultured at 25℃ with continuous oxygenation and light; (ii) Larval collection: after 48 hours, the hatched larvae were collected from the light side using a 20mL pipette; (iii) Exposure experiment: 15-20 nauplii were placed in each well of a 24-well plate, and 1mL of artificial seawater solution containing a specific concentration of S44-CNPs or azoxystrobin was added to each well; (iv) Result determination: after 24 hours of culture at 25℃, those without signs of internal or external movement within 10 seconds under a stereomicroscope were determined to be dead. All experiments were performed in triplicate, with each replicate in three parallel runs.

[0062] The results showed that at concentrations as high as 100 µg / mL, S44-CNPs did not inhibit the beneficial fungus *Trichoderma harzianum* (…). T. harzianum ) and Paecilomyces lilacinus ( P. lilacinum The growth of the bacteria was significantly inhibited, and the colony diameter (approximately 5.5 cm) of both strains was comparable to that of the control group. Figure 6 (A) and (B)). In contrast, acute environmental toxicity tests on the environmental indicator organism Artemia showed that S44-CNPs were significantly less toxic than TTP-CNPs and the commercial fungicide azoxystrobin. While S44-CNPs showed a moderate dose-dependent decrease in Artemia survival (66.6-60% survival at concentrations of 25-100 µg / mL), azoxystrobin exhibited significantly higher toxicity, with survival decreasing to 28% at a concentration of 100 µg / mL. Figure 6 (C)). These findings confirm the selective antimicrobial activity of S44-CNPs, which effectively inhibit pathogens while having minimal impact on non-target beneficial microorganisms and aquatic organisms. Therefore, they hold promise as a sustainable alternative to chemical pesticides in plant disease management.

[0063] (6) Detection of the inhibitory effect of S44-CNPs on anthracnose in detached strawberry leaves and fruits Leaf experiment: Healthy, fully expanded leaves of third-stage strawberry (variety: Miaoxiang No. 7) were selected and treated within 2 hours of collection, following the method of Miller-Butler et al. (2018). Before inoculation, the leaves were sequentially rinsed with tap water, surface-sterilized with 70% (v / v) ethanol for 30 seconds, and rinsed three times with sterile distilled water. Anthracnose spores were eluted from OTA plates cultured for 5 days using sterile ddH2O, collected by centrifugation, and resuspended to a final concentration of 1×10⁻⁶. 6 Conidia / mL. Using a micropipette, each wound was inoculated: the experimental group was inoculated with 10 μL of spore suspension containing different concentrations of S44-CNPs (25 μg / mL, 50 μg / mL, and 100 μg / mL), while the control group was inoculated with pathogenic spore suspension without nanoparticles or sterile deionized water. All leaves were placed in 9 cm petri dishes lined with moistened sterile filter paper, then placed in a sealed transparent plastic container. The containers were first incubated in the dark at 28°C and 100% relative humidity for 36 hours, then transferred to light conditions (light / dark cycle: 12 / 12 h) for 3 days.

[0064] The results showed that S44-CNPs effectively inhibited leaf anthracnose in a dose-dependent manner. Figure 7 (A)-(C)). The healthy control group showed no symptoms, while the leaves inoculated with the pathogen developed an average growth of 1.2 cm 5 days after inoculation. 2 Large, dark-colored lesions were observed. Treatment with 25 μg / mL, 50 μg / mL, and 100 μg / mL S44-CNPs significantly reduced the lesion area to 0.6 cm². 2 0.5cm 2 and 0.3cm 2 The corresponding inhibition rates were approximately 48%, 58%, and 78%. These results confirm the inhibitory effect of S44-CNPs on leaf diseases.

[0065] Fruit Experiment: Strawberry fruits were harvested from the field and transported to the laboratory. After surface sterilization with 1% (w / v) sodium hypochlorite, they were rinsed twice with ddH2O, dried, and then a wound approximately 1 mm long was made at the neck of the fruit using a sterile blade. The experiment included a pathogen-only inoculation group and a co-treatment group with different concentrations of S44-CNPs (25 μg / mL, 50 μg / mL, and 100 μg / mL). The spore solution used for inoculation was obtained using the same methods and concentrations as in the leaf experiment. Inoculated fruits were placed in sealed plastic trays and incubated at 28℃ and 70% relative humidity for 120 hours. The experiment employed a completely randomized design, with each treatment group containing 6 biological replicates, for a total of 3 replicates (n=18). The lesion diameter (LD) was measured using ImageJ software at 48, 72, 96, and 120 hours post-inoculation, with the average diameter of the damaged area used as the quantitative indicator. Following the standards of Li et al. (2024) and Liu et al. (2019), fruits with a LD exceeding 0.3 cm (initial wound size) are considered rotten.

[0066] The results showed that the fruit in the control group rotted rapidly, with lesions reaching an area of ​​approximately 2.0 cm² after 120 hours. 2 ( Figure 7 Fruits treated with S44-CNPs (especially the 100 µg / mL concentration group) maintained a good appearance throughout the culture period, with lesions barely spreading from the wounds. Quantitative analysis showed that 25 µg / mL, 50 µg / mL, and 100 µg / mL S44-CNPs achieved disease inhibition rates of approximately 58%, 78%, and 88%, respectively. It should be noted that the use of 100 µg / mL S44-CNPs alone did not cause significant negative effects on plant leaves and fruits, indicating that S44-CNPs have no significant phytotoxicity under the test conditions.

[0067] (7) Study on the preservation effect of S44-CNPs on strawberry fruit Based on the excellent antibacterial and disease-suppressing effects of S44-CNPs in previous studies, this example further evaluated the preservation effect of S44-CNPs on strawberries. Strawberry fruits of uniform maturity and without mechanical damage (15-20 grams per fruit, 80-90% surface coloration) were selected for the experiment. All samples were first washed three times with sterile distilled water, and then air-dried for 30 minutes in a sterile operating table at 25±1℃. The strawberries were randomly divided into four treatment groups (6 fruits per group), including different concentrations of S44-CNPs immersion treatment groups (25 μg / mL, 50 μg / mL, and 100 μg / mL), a nanomaterial positive control group (100 μg / mL TTP-CNPs), a sterile water negative control group (WC), and a pathogen control group (CG, 1×10⁻⁶). 6(1 conidia / mL). Treated strawberries were stored in plastic containers at 4°C using a completely randomized design to eliminate positional bias. Observations were recorded every 5 days during the 15-day storage period. The experiment was repeated three times, with the final results for each treatment group derived from a total of 18 fruits across the three rounds. Evaluation indicators included rot rate and weight loss (moisture loss). Rot rate was calculated according to the method of Bahmani et al. (2022) by counting the number of fruits exhibiting typical signs of decay such as mycelial growth, tissue rot, or discoloration. The formula was: Rot rate (%) = (Number of rotten fruits / Total number of fruits) × 100. Weight loss was calculated according to the methods of Sani et al. (2025) and Kavi et al. (2024), by weighing the fruits (accurate to 0.01 g) on ​​day 0 and day 15 of storage. The formula was: Weight loss (%) = (Initial weight - Final weight) / Final weight × 100.

[0068] The results showed that postharvest application of S44-CNPs significantly improved the quality and shelf life of strawberry fruits during cold storage. During the 15-day storage period, both the sterile water-negative control group (WC) and the pathogen control group (CG) exhibited rapid and severe decay, with the CG group showing the highest decay rate (approximately 65%). Although TTP-CNPs reduced the decay rate to approximately 20%, a concentration of 100 µg / mL of S44-CNPs showed the best protective effect, controlling the decay rate to only approximately 5%. Figure 8 (A) and (B)). This appearance preservation effect is consistent with a significantly reduced water loss rate (approximately 1.8% in the S44-CNPs treatment group, compared to 6.7-8.4% in the WC and CG groups). Figure 8 (C)). Furthermore, S44-CNPs helped maintain total sugar levels (approximately 660 µg / g), comparable to healthy fruit, while the total sugar content of the fruit in the pathogen control group was significantly lower (approximately 600 µg / g), indicating that S44-CNPs can maintain the stability of fruit metabolism. Figure 8 (D)

[0069] (8) Quantitative analysis of the effects of S44-CNPs on the content of 19 sugars in strawberry fruit Given that S44-CNPs increased the total sugar content in strawberry fruits during cold storage, this example further quantified 19 sugar components in fruits from different treatment groups to further elucidate its metabolic protection mechanism. The extraction and analysis method for sugar metabolites is as follows: Approximately 50 mg of the above-mentioned frozen strawberry tissue was mixed with 500 μL of 50% methanol-water solution (v / v) and two 2 mm zirconium beads. The sample was homogenized at 50 Hz for 20 seconds, with a 10-second interval, repeated for 9 cycles to ensure thorough tissue disruption. Subsequently, 500 μL of 50% methanol-water solution (v / v) was added, vortexed for 1 minute, and sonicated for 5 minutes. After centrifugation at 12000 rpm for 5 minutes, 400 μL of the supernatant was transferred to a clean 1.5 mL centrifuge tube and vacuum concentrated and dried. The dried residue was reconstituted with 80 μL of methoxypyridine solution (20 mg / mL), vortexed for 30 seconds, and incubated at 60 °C for 60 minutes. Then, 100 μL of BSTFA-TMCS (99:1, v / v) was added, vortexed, and incubated at 70 °C for 90 min. The final solution was centrifuged at 12000 rpm for 5 min, and 100 μL of the supernatant was transferred to an autosampler for GC-MS analysis (BIOTREE, Shanghai, China).

[0070] The standard preparation process is as follows: Each monosaccharide standard was prepared into a 1000 ppm stock solution using 50% methanol-water solution (v / v). 100 μL of each standard was mixed and diluted to 2 mL with 50% methanol-water solution (v / v) to prepare a 50 ppm mixed standard working solution. A series of standard solutions of 10 ppb, 20 ppb, 50 ppb, 100 ppb, 200 ppb, and 500 ppb were obtained through a methanol gradient dilution. 200 μL of each concentration was aliquoted into 1.5 mL centrifuge tubes, centrifuged overnight to remove water, and stored at -20°C. Derivatization procedure: 80 μL of MEOX (20 mg / mL) was added to each dried sample, and incubated at 60°C for 1 hour; then 100 μL of BSTFA was added, and incubated at 70°C for 1.5 hours before centrifugation. The final supernatant was transferred to a brown sample vial containing a glass inner tube, sealed, and stored at room temperature for analysis.

[0071] Principal component analysis (PCA) plot clearly shows ( Figure 9In the middle (A) group, the contents of 19 sugars showed significant separation in different treatment groups, highlighting the significant impact of pathogen infection and the unique protective effect of S44-CNPs on sugar homeostasis in refrigerated strawberry fruit. The first principal component (PC1) accounted for 40.3% of the total variation, and the second principal component (PC2) accounted for 35.7%, together explaining 76% of the variation in the data. Hierarchical clustering analysis of sugar metabolites heatmaps showed that the S44-CNPs treatment group (ST) exhibited the highest relative abundance (shown as dark red squares) among almost all targeted sugar metabolites, including D-(-)-fucose, L-rhamnose, D-sorbitol, L-arabinose, D-(+)-mannose, xylose, D-glucose-6-phosphate, D-glucuronic acid, trehalose-6-phosphate, sucrose, raffinose, D-galacturonic acid, D-(-)-fructose, and D-glucose, indicating the superior efficacy of S44-CNPs in maintaining fruit carbohydrate reserves. Figure 9 (B) KEGG pathway enrichment analysis revealed significant differences in metabolic reprogramming among the four treatment groups, including carbohydrate metabolism, membrane transport, signal transduction, and global metabolic pathways. Figure 10 (A)), while the correlation heatmap ( Figure 10 (B) comprehensively illustrates the pairwise relationships between targeted sugar metabolites in strawberry fruit, where the size and color of the circles represent the strength and direction of the Spearman correlation coefficients. A group of major sugars and sugar alcohols, including D-galactose, sucrose, inositol, maltose, lactose, D-glucose, and D-(+)-mannose, showed strong positive correlations, indicating that they are synergistically regulated in interconnected metabolic pathways, which is crucial for maintaining fruit quality and energy balance.

[0072] The flavor profile of strawberries is regulated by key sugars and sugar alcohols, including fructose and xylose, which affect sweetness; mannose-derived esters, which produce fruity aroma; and galacturonic acid, which affects texture. For example... Figure 11 As shown, the *Colletotrichum gloeosporioides* treatment group (CT group) resulted in severe depletion of key flavor metabolites, including: L-(-)-fucose for caramel flavor, L-rhamnose for tartness, D-(+)-mannose as a precursor to ethyl hexanoate, xylose for enhanced sweetness, D-sorbitol for a cooling sensation, and D-galacturonic acid for maintaining fruit texture. This phenomenon confirms pathogen-driven carbohydrate catabolism and pectinase-mediated fruit softening. Notably, under pathogen-free conditions, the S44-CNPs treatment group (ST group) not only maintained these carbohydrate levels compared to the untreated control group but also significantly increased D-sorbitol and D-mannose content, indicating that S44-CNPs induced metabolic preconditioning under cold stress.

[0073] (9) Analysis of the effects of S44-CNPs on central carbon metabolites in strawberry fruit Central carbon metabolism (CCM) analysis in strawberry fruit provides a method for systematically understanding the core dynamics of energy and mass flow during fruit development, ripening, storage, and stress response. Furthermore, the CCM response to stress conditions such as hypoxia or pathogen infection can reveal mechanisms of plant defense and adaptation. Therefore, to further clarify the influence of S44-CNPs on metabolic reprogramming in strawberry fruit, this embodiment further employed ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) to quantitatively analyze 146 central carbon metabolites in strawberry fruit. The specific method is as follows: Frozen strawberry fruit samples (25 mg) were extracted with 500 μL of methanol:water (3:1, v / v) solution containing an internal standard, and two 3.2 mm stainless steel beads were added. The samples were homogenized at 40 Hz for 4 minutes, sonicated on ice for 5 minutes (repeated 3 times), incubated at -40℃ for 1 hour, centrifuged (12000 rpm, 4℃, 15 minutes), and 400 μL of the supernatant was vacuum dried. Residues were redissolved in 200 μL of methanol:acetonitrile:water (1:1:2, v / v / v), filtered, and transferred to an autosampler vial. LC-MS / MS analysis was performed at BIOTREE (Shanghai, China) using an Agilent 1290 UPLC system equipped with a BEHC18 column (reversed-phase) or a BEH Z-HILIC column, connected to an AB Sciex Triple Quad 6500+ mass spectrometer. Injection volume was 1 μL, autosampler temperature was 6 °C, and mobile phase was configured according to reversed-phase or HILIC mode. Mass spectrometry parameters included: ion spray voltage (+5000 / -4500 V), curtain gas (35 psi), ion source temperature (400 °C), and gas source 1 / 2 (50 psi). Standard curves were prepared by serial dilution of a mixed standard solution containing an internal standard. Limits of detection (LOD) and limits of quantitation (LOQ) were determined based on signal-to-noise ratio (LOD: S / N = 3; LOQ: S / N = 10). Precision and accuracy were assessed using quality control replicates, and results are expressed as relative standard deviation (RSD%) and recovery (%). The processed dataset (including peak number, sample name, and normalized peak area) was analyzed using SIMCA 18.0.1 (Sartorius Stedim, Sweden), with logarithmic transformation and Pareto scaling to reduce variance and noise. Principal component analysis (PCA) was used to visualize sample distribution and identify outliers (95% confidence interval), while orthogonal partial least squares discriminant analysis (OPLS-DA) was used to assess intergroup separation and screen for differentially expressed metabolites. Model performance was validated using 7-fold cross-validation and 200 permutation tests, with R... 2 and Q 2 The VIP value reflects the model's goodness of fit and predictive ability. Metabolites with a VIP value > 1 and p < 0.05 (Student t-test) are considered significantly different. Pathway enrichment analysis was performed using the KEGG and MetaboAnalyst databases.

[0074] CCM analysis of refrigerated strawberries detected 146 carbon-based metabolites, with organic acids (22.66%), carbohydrates (21.09%), and amino acids (22.66%) being the most abundant. Other important categories included nucleotides (11.72%) and benzene derivatives (5.47%). Figure 12 (A)

[0075] PCA analysis revealed significant differences in metabolic profiles among the treatment groups. PC1 (23.3%) and PC2 (20.1%) showed clear separation, with the CT group (anthrax treatment group) separating from the healthy WT group (water treatment group), indicating that infection induced metabolic reprogramming. The TT group (TTP-CNPs treatment group) was located between CT and WT, reflecting partial protective effects, while the ST group (S44-CNPs treatment group) formed an independent cluster along PC2, demonstrating unique, pathogen-stress-independent metabolic regulation specific to nanoparticles. Figure 12 (B)

[0076] Heatmap analysis showed significant depletion of metabolites in the CT group, while S44-CNPs treatment upregulated specific amino acids in strawberries, including methionine (an ethylene / volatile precursor), threonine / serine (osmotic protectants), branched-chain amino acids (leucine / isoleucine; flavor ester precursors), glutamine (a nitrogen transporter), lysine (an antibacterial agent), cysteine ​​(a glutathione synthesis precursor), and histidine (a pH buffer). Figure 12 The study (C) revealed the metabolic reprogramming mechanism regulated by S44-CNPs. The role of the TT group was intermediate between that of the S44-CNPs and the CT group. Methionine-derived sulfur volatiles (such as methylthion) can retain fruit aroma, while threonine / serine, synthesized through glycine and betaine, can stabilize fruit membrane structure under cold stress. Branched-chain amino acids provide energy for the TCA cycle and produce fruit aroma esters, while cysteine / histidine enhances antioxidant capacity (GSH / SOD) and fruit disease resistance. The significant upregulation of these amino acid contents explains the dual role of S44-CNPs in maintaining flavor complexity and extending shelf life during refrigeration.

[0077] Chord diagram ( Figure 13The middle (A) section shows the correlation of metabolites, indicating a strong positive correlation among amino acids (such as leucine, lysine, methionine, threonine, and serine), which are crucial for protein synthesis and defense signal transduction. They are negatively correlated with stress-related compounds (oxalic acid, nicotinamide, and UDP-N-acetylglucosamine), especially in the CT group, indicating metabolic imbalance. S44-CNP treatment maintained amino acid abundance and reduced the accumulation of stress metabolites, suggesting that it alleviates infection-induced metabolic burden and maintains postharvest quality. KEGG pathway enrichment analysis showed that amino acid biosynthesis, carbon metabolism, and the alanine / aspartate / glutamate pathway significantly participated in the metabolic reprogramming of S44-CNPs. Figure 13 (B) and Figure 14 The CT group infected with *Colletotrichum gloeosporioides* showed persistent downregulation in amino acid and nucleotide biosynthesis, indicating severe metabolic inhibition; while ST treatment upregulated these pathways, showing active metabolic activity and carbon flux. Dendrograms and bubble diagrams further revealed changes in energy-related pathways such as tyrosine, glycine / serine / threonine, glutathione metabolism, TCA cycle, and glyoxylate metabolism. Overall, these results indicate that fungal infection leads to widespread metabolic inhibition in fruit, while S44-CNPs can effectively maintain central metabolic function and enhance postharvest fruit stress resistance at 4°C.

[0078] (10) Analysis of the effects of S44-CNPs on the amino acid and organic acid content in strawberry fruit Targeted analysis of amino acids and related metabolites in strawberry fruits showed significant differences compared to the water treatment group (WT) among the *Colletotrichum gloeosporioides* treatment group (CT), the TTP-CNPs treatment group (TT), and the S44-CNPs treatment group (ST). Figure 15Infection led to a significant accumulation of stress-related compounds, with proline and betaine increasing by approximately 3.5-fold and 1.7-fold, respectively, compared to the WT level. The TT group showed a similar but weaker trend, suggesting a partial protective effect. The accumulation of these protective substances is consistent with typical plant responses to biotic stress, contributing to the maintenance of osmotic balance and membrane stability under adverse conditions. Conversely, infection was accompanied by a reduction in several essential amino acids: threonine decreased by 50% in the CT group and 40% in the TT group; cysteine ​​decreased by 30% and 40%, respectively. This depletion may reflect the pathogen's utilization of the host's amino acid reserves to support its own proliferation. Notably, S44-CNPs treatment (ST) alleviated these changes: proline and betaine concentrations in the ST group were close to WT levels, indicating a weakened stress response; threonine content was comparable to WT, cysteine ​​increased twofold, and leucine slightly increased (1.2-fold). The increase in cysteine, as a precursor to glutathione biosynthesis (a core component of the plant's antioxidant defense system), is particularly important. Furthermore, the CT group showed increased nicotinamide levels (1.6-fold increase) reflecting oxidative stress and energy turnover, while this trend decreased by 50% in the ST group, further supporting its effect of stabilizing metabolic status and reducing disturbances. These results indicate that S44-CNPs provide effective metabolic protection during postharvest storage by maintaining amino acid balance, limiting oxidative stress, and preserving the nutritional integrity of fruits during cold storage.

[0079] (11) Central carbon metabolism analysis showed that S44 chitosan nanoparticles maintained the metabolic stability and homeostasis of strawberries during refrigeration. Compared to the water-treated group (WT), the S44-CNPs-treated group (ST) maintained higher amino acid abundance while reducing the levels of stress-related metabolites (oxalate, nicotinamide, and uridine diphosphate N-acetylglucosamine), indicating a more balanced metabolic profile. Figure 16 (A). This suggests that S44 chitosan nanoparticles help maintain metabolic homeostasis under refrigerated conditions, thereby improving postharvest fruit quality. Furthermore, KEGG heatmaps and pathway enrichment analysis showed significant activation of amino acid biosynthesis, carbon metabolism, and the alanine / aspartate / glutamate pathway. Figure 16 (B) Compared to the water-treated group (WT), the *Colletotrichum gloeosporioides* treatment group (CT) showed a sustained downregulation of amino acid and nucleotide biosynthetic pathways, indicating severe metabolic inhibition. Conversely, the ST treatment group upregulated these pathways, demonstrating its ability to maintain vigorous metabolic activity and efficient carbon transport under refrigerated conditions.

[0080] In summary, this invention confirms that the marine bacterium *Bacillus belesiensis* (… Bacillus velezensis S44 biosynthesized chitosan nanoparticles (S44-CNPs) are a multifunctional and eco-friendly solution for controlling postharvest anthracnose in strawberries. S44-CNPs exhibit multiple functional advantages: compared to other nanoparticle formulations, they are more effective against *Colletotrichum gloeosporioides* (…).C. gloeosporioides It has a superior inhibitory effect (EC). 50 = 14.85µg / mL). Crucially, S44-CNPs not only effectively inhibited disease development in detached leaves and fruits but also maintained fruit quality—reducing weight loss (approximately 1.5%) and maintaining sugar metabolic homeostasis during cold storage. Targeted metabolomics analysis revealed a novel mechanism: S44-CNPs alleviate metabolic disorders by stabilizing sugar content, central carbon metabolism pathways (such as glycolysis and the TCA cycle), and preventing amino acid depletion. Unlike synthetic fungicides, S44-CNPs exhibit selective toxicity, protecting beneficial microorganisms (Trichoderma harzianum). T. harzianum Paecilomyces lilacinus P. lilacinum Furthermore, it significantly reduces environmental risk (survival rate of Artemia salina: 66.6%, compared to 28% in the pyraclostrobin-treated group). This marine-derived nanobiotechnology provides a sustainable solution for post-harvest management, minimizing environmental impact while achieving disease control, quality maintenance, and shelf-life extension.

[0081] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. Bacillus belye ( Bacillus velezensis A method for preparing S44-mediated chitosan nanoparticles, characterized in that, Including the following steps: (1) Inoculate Bacillus belye S44 into LB medium and culture for 36-60 hours. Filter to remove cells and obtain the supernatant. (2) Mix the filtered supernatant and chitosan solution evenly and incubate for 36-60 hours to obtain chitosan nanoparticles; The Bacillus belyssus S44 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66680 and deposit date of July 14, 2025.

2. The method for preparing chitosan nanoparticles according to claim 1, characterized in that, The chitosan solution is prepared with water and includes: chitosan with a mass concentration of 0.05-0.3% and acetic acid with a volume concentration of 0.5-3%; the pH of the chitosan solution is 3-5.

3. The method for preparing chitosan nanoparticles according to claim 2, characterized in that, The method for preparing the chitosan solution includes the following steps: Chitosan is dissolved in an aqueous solution containing acetic acid and stirred at 200-400 rpm for 8-16 hours. The filtrate is collected by filtration, and the pH is adjusted to 3-5 to obtain a chitosan solution.

4. The method for preparing chitosan nanoparticles according to claim 1, characterized in that, The volume ratio of the filtration supernatant to the chitosan solution is 1:(0.5-3).

5. The method for preparing chitosan nanoparticles according to claim 1, characterized in that, In step (1), the cultivation conditions include: a temperature of 25-30℃ and a rotation speed of 100-200 rpm; The filtration is performed through a filter membrane with a diameter of 0.22-0.45 μm.

6. The method for preparing chitosan nanoparticles according to claim 1, characterized in that, In step (2), the incubation conditions include a temperature of 25-30℃.

7. Chitosan nanoparticles mediated by Bacillus belyssus S44, characterized in that, Chitosan nanoparticles were prepared using the preparation method described in any one of claims 1-6.

8. The application of chitosan nanoparticles as described in claim 7 in the preparation of products that inhibit plant pathogens.

9. The application according to claim 8, characterized in that, The plant pathogen is selected from *Colletotrichum gloeosporioides* (… Colletotrichum gloeosporioides Fusarium graminearum ( ), Fusarium graminearum ), rice blast fungus ( Pyriculariaoryzae ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum Alternaria ( Alternaria alternata Methicillin-resistant Staphylococcus aureus Staphylococcus aureus ) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa One or more of the following.

10. The application of chitosan nanoparticles as described in claim 7 in the prevention and control of anthracnose in strawberries after harvest.

Images