Wheat stem rot prevention and control preparation based on plant source carbon nanodots and application

Abstract

The invention relates to a wheat stem rot prevention and control preparation based on plant source carbon nanodots and application, and aims to solve the problems of pathogenic bacterium drug resistance enhancement, pesticide residue and environmental risk caused by existing chemical prevention and control of wheat stem rot, complex fungus structure and lack of green prevention and control technology. According to the preparation method, a technical means of taking natural turmeric as a carbon source and hydrothermally synthesized carbon nanodots (CDs) as a photodynamic antibacterial agent is adopted, high reactive oxygen species (ROS) are excited under an illumination condition to efficiently kill fusarium pseudograminearum spores, and mycelial growth and spore germination of fusarium pseudograminearum spores are inhibited; therefore, the green prevention and control technical effects of no chemical residue, difficulty in resistance induction, environment friendliness and remarkable potted plant prevention effect (for example, the inactivation rate is 100% after 0.3 g / L CDs are illuminated for 30 minutes) are achieved.

Description

Technical Field

[0001] This invention relates to the field of crop disease control technology, and in particular to a control agent for wheat stem base rot based on plant-derived carbon nanodots and its application. Background Technology

[0002] Wheat stem rot ( Fusarium crown rot FCR is produced by Fusarium pseudograss (FCR). Fusarium pseudograminearum This disease, caused by a soil-borne fungus, is widespread in major wheat-producing areas worldwide, seriously threatening wheat yield and quality. Currently, control measures for this disease mainly rely on the following methods, but all have significant shortcomings: 1. Chemical control: Seed treatment or root drenching with chemical fungicides such as tebuconazole and difenoconazole. However, long-term use has led to increasing resistance in pathogens, excessive pesticide residues in the soil, disruption of the farmland's ecological balance, and potential food safety risks.

[0003] 2. Agricultural control: Measures such as crop rotation and deep plowing are adopted. However, the promotion and application of these measures are greatly limited due to the scarcity of arable land resources.

[0004] 3. Disease-resistant variety breeding: Due to the scarcity of wheat-available genes for resistance to stem rot, and the long breeding cycle and relatively slow progress, it is difficult to meet the current urgent production needs.

[0005] Therefore, developing new control strategies that are efficient, green, safe, and do not easily induce pathogen resistance is a major problem that urgently needs to be solved.

[0006] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0007] This invention addresses the shortcomings of existing chemical control methods for wheat stem rot, which lead to increased pathogen resistance, pesticide residues, and environmental risks, as well as the lack of green control technologies due to the complexity of fungal structures. It employs carbon nanodots (CDs) synthesized hydrothermally using natural turmeric as a carbon source, as a photodynamic antibacterial agent. Under light conditions, this agent activates highly reactive oxygen species (ROS) to efficiently kill Fusarium spores, inhibit mycelial growth, and suppress spore germination. This results in a green control technology that leaves no chemical residues, is less likely to induce resistance, is environmentally friendly, and exhibits significant potted plant efficacy (e.g., 100% inactivation rate with 0.3 g / L CDs after 30 minutes of light exposure).

[0008] According to one aspect of this disclosure, plant-derived carbon nanodots or nanoformations thereof are applied to at least one of the following (1) to (5): (1) Controlling wheat stem base rot or preparing agents for controlling wheat stem base rot; (2) Inactivating Fusarium spores or agents for preparing inactivating Fusarium spores; (3) Inhibit the germination of Fusarium spores or prepare a formulation to inhibit the germination of Fusarium spores; (4) Inhibit the elongation of germ tubes of Fusarium graminearum or prepare a formulation to inhibit the elongation of germ tubes of Fusarium graminearum; (5) Block the growth of Fusarium graminearum mycelium or prepare agents to block the growth of Fusarium graminearum mycelium.

[0009] According to a second aspect of this disclosure, a nano-formulation product in which the active ingredient is plant-derived carbon nanodots is provided, which has at least one of the following (1) to (5) functions or effects: (1) Controlling wheat stem base rot; (2) Inactivation of Fusarium spores; (3) Inhibits the germination of Fusarium pseudograss spores; (4) Inhibits the elongation of germ tubes of Fusarium pseudograss; (5) Block the growth of Fusarium pseudograss mycelium.

[0010] In some embodiments of this disclosure, the nanoformulation contains plant-derived carbon nanodots ≥ 0.01 g / L.

[0011] In some embodiments of this disclosure, the application or the nano-formulation product is performed under the following photocatalytic conditions: light intensity ≥ 2000 Lux, and light exposure time ≥ 10 min.

[0012] According to a third aspect of this disclosure, a green control method for wheat stem base rot is provided, comprising: Plant-derived carbon nanodots were prepared into a working solution of 0.05 ~ 0.3 g / L; the working solution was applied to the base of wheat stems and then kept under the following light conditions: light intensity ≥ 2000 Lux and light duration ≥ 10 min.

[0013] One or more technical solutions provided in the embodiments of this application have at least one of the following technical effects or advantages: 1. Green and environmentally friendly materials, simple and economical preparation: CDs are synthesized using natural and renewable turmeric as a carbon source via a hydrothermal method. This process has widely available raw materials, a simple preparation method, and low cost. The resulting CDs possess excellent photocatalytic performance, good chemical stability and biocompatibility, and low cytotoxicity, thus solving the environmental pollution and residue problems of chemical pesticides.

[0014] 2. Highly efficient and specific antifungal mechanism: Under natural light conditions, CDs can efficiently generate high concentrations of reactive oxygen species (ROS). Through multiple ROS and their synergistic effects, this material can rapidly and efficiently kill Fusarium spores (e.g., 0.3 g / L concentration can completely inactivate 1×10⁻⁶ spores after 30 min of light irradiation). 6 The photodynamic antibacterial mechanism, which targets multiple sites and is less likely to induce drug resistance in pathogens, overcomes the problem of increased resistance to chemical fungicides. It also significantly inhibits mycelial growth and conidial germination (showing concentration-dependent inhibition, with complete inhibition at 0.3 g / L).

[0015] 3. Significant Field Disease Control Effects: Pot experiments confirmed that CDs can effectively control the occurrence and development of wheat stem base rot. Specifically, after CDs treatment (30 min of light), the length of lesions at the base of wheat stems decreased significantly with increasing CDs concentration; at a concentration of 0.3 g / L, the average lesion length was only 0.17 cm, the average disease index score was as low as 1.7, and the relative expression level of the pathogen in wheat tissues was also significantly reduced to 0.024. This clearly verifies its good efficacy in actual disease control.

[0016] 4. High safety and no adverse effects on growth: It has no adverse effects on wheat plant growth during application, leaves no chemical pesticide residues, and is highly safe for human health and the environment, meeting the requirements for safe agricultural production.

[0017] 5. Broad application prospects: This invention provides a new strategy for the prevention and control of wheat stem base rot that is efficient, green, safe, and less prone to resistance development. It has important theoretical significance and broad agricultural promotion value, and is especially suitable for replacing or reducing the use of traditional chemical pesticides. Attached Figure Description

[0018] Figure 1 The following are images showing the bactericidal effect of CDs on Fusarium oxysporum: (A) Dark environment treatment; (B) Treatment under 6000 Lux light intensity.

[0019] Figure 2 The effect of CDs on the mycelial growth of Fusarium pseudograss: (A) Mycelial growth diagram; (B) Mycelial inhibition rate.

[0020] Figure 3 The effect of CDs on spore germination of Fusarium pseudograss: (A) Spore germination photographs after 30 min of light treatment with different concentrations of CDs and 6 h of incubation in PDB; (B) Spore germ tube length at 6 h; (C) Spore germination rate at 4 h and 6 h.

[0021] Figure 4The effects of CDs-treated Fusarium spores on wheat pathogenicity: (A) Photographs of wheat infected by spores treated with different concentrations of CDs under light irradiation for 30 min; (B) Length of lesions at the base of wheat stems; (C) Disease index score of wheat; (D) Relative expression level of Fusarium spores in wheat. Detailed Implementation

[0022] To better understand the technical solution of this application, the above technical solution will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0023] The turmeric carbon dots (CDs) involved in the following examples are prepared by hydrothermal method using natural turmeric as the carbon source (bulk, powder, or residue). Their surface is rich in functional groups such as hydroxyl, carboxyl, and pyridine nitrogen, exhibiting good water solubility and environmental friendliness. For the specific preparation method, please refer to patent document CN117158440A.

[0024] Example 1: Verification of the killing effect of CDs on Fusarium oxysporum spores (I) Experimental Methods 1. Preparation of pathogenic fungal spore suspension: ① Fusarium pseudograss, frozen at -80℃ ( Fusarium pseudograminearum Inoculate with fresh potato dextrose agar (PDA) plates and incubate at 28 °C in the dark for 3 days to activate.

[0025] ② Take the activated colony edge pieces and transfer them to 100 mL of sodium carboxymethyl cellulose (CMC) liquid culture medium.

[0026] ③ Incubate for 5 days at 28 ℃ and 180 rpm with shaking.

[0027] ④ The culture is filtered through sterile filter cloth, and the filtrate is collected.

[0028] ⑤ Centrifuge the filtrate at 6000 rpm for 10 min and discard the supernatant.

[0029] ⑥ The collected spore precipitate was washed twice with sterile water.

[0030] ⑦ Finally, resuspend the spores in sterile water and adjust the concentration for later use.

[0031] 2. CDs treatment and light exposure experiment: ① Dilute the turmeric CDs stock solution with sterile water to prepare CDs working solutions with concentrations of 0 g / L (Control), 0.01 g / L, 0.05 g / L, 0.1 g / L, 0.2 g / L, and 0.3 g / L, respectively.

[0032] ② Take 1.5 mL of CDs working solution of each concentration and dispense it into 1.5 mL centrifuge tubes.

[0033] ③ Add an appropriate amount of the spore suspension prepared in step 1 to each tube, and use a hemocytometer to precisely adjust the final spore concentration to 1×10⁻⁶. 6 CFU / mL.

[0034] ④ Set two sets of processing conditions: Dark treatment group: Place centrifuge tubes in a completely dark environment.

[0035] Illumination group: The centrifuge tubes were placed in a simulated sunlight environment (illuminance of 6000 Lux).

[0036] Samples were taken from both groups at 0 min, 10 min, 20 min, and 30 min after the start of treatment.

[0037] 3. Sterilization efficacy test (plate count method): Take 100 μL of spore suspension from each time point and perform serial dilutions with sterile water. Take 100 μL of each dilution and spread it evenly on a PDA plate. Incubate the PDA plates at 28 ℃ in the dark for 36 h. After incubation, count the colony-forming units (CFU) on each plate. Set up 3 replicates for each dilution gradient, and independently repeat the entire experiment 3 times.

[0038] (II) Experimental Results 1. Results of the dark processing group: such as Figure 1 As shown in Figure A, under completely dark conditions, treatment with different concentrations of CDs (0.01 ~ 0.3 g / L) for 30 min had no significant effect on the survival rate of Fusarium spores, indicating that CDs do not have direct sporicidal ability in the absence of light.

[0039] 2. Results of the light group: such as Figure 1 As shown in B, under a light intensity of 6000 Lux: CDs exhibited significant concentration- and time-dependent sporicidal effects; within a 30-minute treatment time, higher CDs concentrations resulted in higher spore inactivation rates; a 0.3 g / L CDs concentration treated for 30 minutes completely inactivated spores at an initial concentration of 1×10⁻⁶. 6 CFU / mL Fusarium spore suspension (100% inactivation rate).

[0040] As shown above, the CDs exhibit highly efficient and rapid killing effects on *Fusarium graminearum* spores under light conditions, with the sporicidal effect being concentration- and time-dependent. A concentration of 0.3 g / L of CDs completely inactivated spores within 30 minutes under natural light (6000 Lux). This fully demonstrates the excellent photoelectron conversion performance and photocatalytic antibacterial mechanism of turmeric CDs, providing important in vitro experimental evidence and theoretical basis for its application in the control of wheat stem rot in the field. The lack of effect under dark treatment further confirms that its action depends on photoexcitation.

[0041] Example 2: Verification of the inhibitory effect of turmeric carbon nanodots (CDs) on hyphal growth and spore germination of Fusarium pseudograss. (I) Experimental Methods: 1. Mycelial growth inhibition test: (1) Spore treatment: Take the *Fusarium graminearum* spore suspension prepared according to the method of Example 1 and treated with different concentrations of CDs (0 g / L Control, 0.01 g / L, 0.05 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L) under natural light (6000 Lux) for 30 minutes.

[0042] (2) Inoculation and culture: Take 5 μL of the treated spore suspension and add it to the center of the PDA solid medium. Three replicates are set for each concentration. After incubation in the dark at 28 ℃ for 72 h, the colony diameter is measured using the cross-sectional method. d c As a control group, d t (For processing groups).

[0043] (3) Inhibition rate calculation: Calculate the mycelial growth inhibition rate according to the formula: Inhibition rate (%) = (1 - d t / d c ) × 100.

[0044] 2. Spore germination inhibition test: (1) Spore recovery: Centrifuge the treated spore suspension at 6000 rpm for 10 min, resuspend the precipitate in sterile water and transfer it to 10 mL of PDB liquid culture medium.

[0045] (2) Germination observation: The culture was shaken at 28 ℃ and 180 rpm, and samples were taken at 4 h and 6 h respectively: 10 μL of culture suspension was taken and placed on a microscope slide; the germination of at least 200 spores was observed and counted under an optical microscope (germination standard was taken as germ tube length ≥ spore diameter), and the spore germination rate was calculated; at the same time, the germ tube length of the germinating spores was measured and recorded ( Figure 3 B).

[0046] (II) Experimental Results 1. Inhibition effect on mycelial growth ( Figure 2 ① CDs treatment significantly inhibited the growth of Fusarium pseudograss mycelium, and the inhibitory effect showed a clear concentration-dependent effect. Figure 2 A). ② As the concentration of CDs increased, the colony diameter decreased significantly. ③ The 0.3 g / L CDs treatment group completely inhibited the normal growth of mycelium, and the colony expansion was almost non-existent. Figure 2 A). ④ The mycelial growth inhibition rate increased significantly with increasing CDs concentration ( Figure 2 B), the inhibition rate of the 0.3 g / L treatment group was close to 100%.

[0047] 2. Spore germination inhibition effect ( Figure 3 ): (1) CDs treatment significantly inhibited the germination of Fusarium pseudograss spores, and the inhibitory effect was also concentration-dependent.

[0048] (2) Germination rate ( Figure 3 C): ① After 4 hours of culture, the spore germination rate of the control group was 79.86%; the spore germination rate of the 0.3 g / L CDs treatment group was 0% (completely non-germinating); ② After 6 hours of culture, the spore germination rate of the control group reached 100%; the spore germination rate of the 0.3 g / L CDs treatment group was still 0% (completely non-germinating).

[0049] (3) Germ tube growth ( Figure 3 B): In germinating spores, the germ tube length of spores in the CDs-treated group was significantly shorter than that in the control group, and the higher the CDs concentration, the more severe the inhibition of germ tube growth. Figure 3 A, 3B).

[0050] Therefore, it can be seen that the turmeric CDs effectively inhibited the mycelial growth and spore germination of *Fusarium graminearum* under light conditions (6000 Lux, 30 min), and the inhibitory effect was significantly concentration-dependent. A concentration of 0.3 g / L of CDs completely prevented mycelial growth (inhibition rate ≈ 100%) and completely inhibited spore germination (germination rate = 0%). This further confirms the potent inhibitory effect of turmeric CDs on *Fusarium graminearum* at key stages of pathogen growth and development, providing important experimental evidence for explaining its mechanism of control of wheat stem rot (such as blocking pathogen infection and spread).

[0051] Example 3: Evaluation of the control effect of turmeric carbon nanodots (CDs) on wheat stem base rot (I) Experimental Methods 1. Pot inoculation experiment design (1) Spore preparation: Take 1×10⁻⁶ Fusarium spore suspension (1×10⁻⁶) from the strain propagated in Example 1. 6 Add CDs solution according to the concentrations described in Table 1 to make the final concentrations 0 (Control), 0.01, 0.05, 0.1, 0.2, and 0.3 g / L. Also set up a sterile water treatment group (Mock).

[0052] (2) Light treatment: Place the mixture under natural light (6000 Lux) for 30 min.

[0053] (3) Inoculation procedure: Select wheat plants that have grown to the 3-leaf stage, make a slight wound at the base of the stem with a sterile blade, inject 200 μL of the treated spore suspension into each plant, and set 15 plants in each concentration group (3 replicates, 5 plants each time).

[0054] (4) Culture conditions: After inoculation, the plants were placed in a greenhouse (25 ± 2℃, humidity 70%) and exposed to light for 14 hours per day.

[0055] 2. Disease assessment: Disease surveys were conducted on the 7th day after inoculation (or when obvious disease was observed in the control group).

[0056] (1) Observation and measurement of lesions: Carefully peel off the tissue at the base of the wheat stem. Take photos to record the typical lesions at the base of the wheat stem in each treatment group. Figure 4 A). Use calipers or similar tools to accurately measure the length (cm) of the lesion at the base of each wheat stem (corresponding to...). Figure 4 B), and calculate the average lesion length for each group.

[0057] (2) Disease Severity Index Scoring: Based on pre-set disease grading standards (e.g., 0 = no symptoms; 1 = lesion length < 1 / 4 of stem circumference; 2 = lesion length 1 / 4 - 1 / 2 of stem circumference; 3 = lesion length 1 / 2 - 3 / 4 of stem circumference; 4 = lesion length > 3 / 4 of stem circumference or plant death), the severity of disease was graded for each wheat plant. The average disease index for each group was calculated using the following formula ( Figure 4 C): Disease index = [Σ(number of diseased plants at each level × corresponding level) / (total number of plants investigated × highest level)] × 100.

[0058] (3) Detection of relative expression levels of pathogens: Stem base tissue from the inoculation site was collected, and the specific genes of *Fusarium graminearum* (e.g., *Fusarium graminearum*) were detected using methods such as real-time quantitative PCR (qRT-PCR). FpTEF 1) Relative expression level in wheat tissues ( Figure 4D). Uninoculated healthy wheat or control wheat was used as a reference.

[0059] (II) Experimental Results Treatment with turmeric CDs significantly reduced the incidence of wheat stem rot, and the control effect showed a clear concentration-dependent effect. 1. Lesion phenotype ( Figure 4 A): The Control group showed large brown lesions at the base of the stem (3.7 ± 0.4 cm), while the lesion length in the CDs - 0.3 group was only 0.3 ± 0.1 cm, which was not significantly different from the Mock group (sterile water treatment) (p>0.05).

[0060] 2. Lesion length ( Figure 4 B): Lesion length decreases exponentially with increasing CDs concentration (R) 2 = 0.983), the lesion inhibition rate reached 81.1% after treatment with 0.1 g / L CDs. The average lesion length in the 0.3 g / L CDs treatment group was only 0.17 cm, which was much lower than that in the control group. CDs treatment significantly inhibited the spread of pathogens at the base of wheat stems.

[0061] 3. Disease index: Figure 4 The box plot showed that the median disease index in the Control group was 7 (range 5–9), while the median in the CDs-0.3 group was 1 (range 0–2), with an inhibition rate of 85.7%. 0.05 g / L CDs was sufficient to reduce the disease index to below 5, and it tended to stabilize after 0.2 g / L. The average disease index score in the 0.3 g / L CDs treatment group was as low as 1.7 (assuming a maximum score of 100), indicating extremely mild disease incidence.

[0062] 4. Relative expression level of pathogens: Figure 4 The bar chart (D-axis) showed that the relative fungal biomass in the Control group was 1.0, while that in the CDs-0.3 group it decreased to 0.1, with an inhibition rate of 90%. Fungal biomass was significantly positively correlated with lesion length and disease index (r = 0.976, p < 0.001). The relative expression level of pathogens in the 0.3 g / L CDs treatment group was only 0.024 (set to 1 relative to the control group), further confirming the potent inhibitory effect of CDs on pathogen invasion and spread.

[0063] As shown above, the CDs described in this invention, after pretreatment of pathogenic spores with light, can effectively control wheat stem rot caused by *Fusarium graminearum* infection. Its control efficacy exhibits a significant concentration dependence, with a concentration of 0.3 g / L CDs showing the best effect, controlling the average lesion length to 0.17 cm, reducing the disease index to 1.7, and significantly decreasing the relative expression level of the pathogen in the host (0.024). This directly verifies the actual control effect of turmeric CDs on wheat stem rot under simulated field conditions, and its excellent control efficacy is highly consistent with its strong in vitro bactericidal and bacteriostatic abilities (spore killing, mycelial growth inhibition, and spore germination inhibition) demonstrated in Examples 1 and 2, providing solid in vivo experimental evidence for the application of CDs in the field for green control of this disease.

[0064] Although some preferred embodiments of this application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this application.

[0065] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of its inventive concept. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. Application of plant-derived carbon nanodots or a nano-preparation thereof in at least one of the following (1)~(5): (1) preventing and treating wheat basal stem rot or preparing a medicament for preventing and treating wheat basal stem rot; (2) inactivating Fusarium pseudograminearum spores or preparing a medicament for inactivating Fusarium pseudograminearum spores; (3) inhibiting Fusarium pseudograminearum spore germination or preparing a preparation for inhibiting Fusarium pseudograminearum spore germination; (4) inhibiting Fusarium pseudograminearum spore germ tube elongation or preparing a preparation for inhibiting Fusarium pseudograminearum spore germ tube elongation; (5) blocking Fusarium pseudograminearum mycelium growth or preparing a medicament for blocking Fusarium pseudograminearum mycelium growth.

2. Use according to claim 1, characterized in that, The plant-derived carbon nanodots are curcumin carbon nanodots.

3. A nanoforumulation product, characterized in that, The effective component is plant-derived carbon nanodots, which has at least one of the following (1)~(5) functions or effects: (1) preventing and treating wheat basal stem rot; (2) inactivating Fusarium pseudograminearum spores; (3) inhibiting Fusarium pseudograminearum spore germination; (4) inhibiting Fusarium pseudograminearum spore germ tube elongation; (5) blocking Fusarium pseudograminearum mycelium growth.

4. The use of claim 1 or the nanoformulation product of claim 3, wherein, The nano-preparation contains plant-derived carbon nanodots ≥ 0.01 g / L.

5. The use of claim 1 or the nanoformulation product of claim 3, wherein, The application or the nano-preparation product is applied under the following photocatalytic conditions: light intensity ≥ 2000 Lux, light time ≥ 10 min.

6. A method for green control of wheat take-all disease, characterized by, It comprises: preparing plant-derived carbon nanodots into a working solution of 0.05 ~ 0.3 g / L; applying the working solution to the base of wheat stems.

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