Carbon quantum dots for plant oomycete diseases and preparation method and application thereof

Abstract

The application discloses carbon quantum dots for plant oomycete diseases and a preparation method and application thereof, and belongs to the technical field of nanometer material preparation. The preparation method comprises the following steps: dissolving salicylic acid, urea and copper chloride dihydrate in purified water, adjusting the pH value to 10-12, stirring to completely dissolve, and obtaining a mixed solution; transferring to a hydrothermal reaction kettle for reaction, cooling to room temperature, and obtaining carbon dots; centrifuging, collecting supernatant, filtering through a 0.22 mu m microporous filter membrane, dialyzing for 10-14 hours through a 1000 Da dialysis bag, and vacuum freeze-drying to obtain carbon quantum dot powder. The application discloses carbon quantum dots for plant oomycete diseases and a preparation method and application thereof, copper chloride is used as a dopant to dope heteroatoms to the carbon dots, so that a synergistic effect is generated between copper and the carbon dots, the antibacterial ability of the composite material is significantly enhanced, and the carbon quantum dots have high efficient antibacterial effect and excellent drug resistance.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanomaterial preparation, and particularly relates to a carbon quantum dot for plant oomycete diseases and a preparation method and application thereof. BACKGROUND

[0002] Plant pathogens, especially oomycetes, have caused huge economic losses to global agriculture. Oomycetes are a class of eukaryotic microorganisms similar to fungi, evolutionarily close to brown algae, but significantly different from fungi in biochemistry, physiology and genetics. For example, the cell wall of oomycetes is mainly composed of cellulose and glucan, while the cell wall of fungi is mainly composed of chitin. Therefore, fungicides targeting chitin are ineffective against oomycetes.

[0003] In addition, oomycete diseases such as downy mildew and pythium disease are extremely difficult to control due to their rapid growth and reproduction ability, as well as the ability to produce oospores that can survive in the environment for a long time. Traditional chemical fungicides have played an important role in controlling oomycete diseases, but long-term use has led to the emergence of drug resistance problems. For example, systemic fungicides are prone to drug resistance due to a single action site. In addition, many effective fungicides contain persistent chemicals such as tin and copper, which pose a potential threat to the ecosystem and human health.

[0004] In recent years, nanomaterials have gradually attracted attention in the field of antibiosis. New types of nanomaterials such as metal nanoparticles, borides, nanopolymers and carbon quantum dots have been used to kill bacteria or fungi. Carbon quantum dots (CQDs) are a kind of zero-dimensional carbon nanostructure material, which has the advantages of ultra-small size, easily modified structure, low cytotoxicity, high quantum yield and low cost. These characteristics make CQDs have broad application prospects in drug delivery and treatment of pathogenic bacterial infections. However, the current research on the antibacterial mechanism of CQDs is still insufficient, especially in the exploration of its specific antibacterial mechanism. SUMMARY

[0005] The application aims to provide a carbon quantum dot for plant oomycete diseases and a preparation method and application thereof, which has high antibacterial effect and excellent drug resistance.

[0006] To solve the above technical problems, the technical scheme adopted by the application is as follows:

[0007] A preparation method of a carbon quantum dot for plant oomycete diseases, comprising the following steps:

[0008] S1, dissolving salicylic acid, urea and copper chloride dihydrate in purified water, adjusting the pH value to 10-12, stirring to completely dissolve, and obtaining a mixed solution;

[0009] S2, transferring the mixed solution obtained in step S1 to a hydrothermal reaction kettle for reaction, and cooling to room temperature to obtain carbon dots;

[0010] S3, centrifuging the carbon dots obtained in step S1, collecting the supernatant, filtering through a 0.22 mu m microporous filter membrane, dialyzing for 10-14 h through a 1000 Da dialysis bag, and vacuum freeze-drying to obtain carbon quantum dots.

[0011] Preferably, in step S2, the reaction temperature is 160-200 DEG C, and the reaction time is 8-12 h.

[0012] Preferably, in step S3, the centrifugal speed is 7000-8000 rpm, and the centrifugal time is 15-25 min.

[0013] Preferably, in step S3, the freeze-drying temperature is-50 DEG C, and the freeze-drying time is 24 h.

[0014] The application also provides carbon quantum dots prepared by the preparation method.

[0015] The application also provides the application of carbon quantum dots prepared by the preparation method or the carbon quantum dots in preventing and treating plant pathogenic oomycete diseases.

[0016] The application also provides the application of carbon quantum dots prepared by the preparation method or the carbon quantum dots in preparing a product for preventing and treating plant pathogenic oomycete diseases.

[0017] The application also provides a growth inhibitor for preventing and treating plant pathogenic oomycete, comprising carbon quantum dots prepared by the preparation method or the carbon quantum dots.

[0018] The application also provides the application of the growth inhibitor for preventing and treating plant pathogenic oomycete in preventing and treating pepper Phytophthora capsici.

[0019] Compared with the prior art, the application has the following advantages and technical effects:

[0020] (1) The application discloses carbon quantum dots for plant oomycete diseases and a preparation method and application thereof, copper chloride is used as a dopant to dope heteroatoms to the carbon dots, so that a synergistic effect is generated between copper and the carbon dots, and the antibacterial capacity of the composite material is significantly enhanced. Copper-based nanoparticles can generate reactive oxygen under light conditions, and the carbon dots also have certain antibacterial activity, and the antibacterial effect of the combination of the two is far superior to that of a single material, especially in preventing and treating oomycetes. And reasonable control of the loading amount of the metal effectively reduces the amount of the antibacterial agent, while ensuring that the antibacterial efficiency is not affected.

[0021] (2) The carbon quantum dot material prepared by the application has excellent drug resistance, which can effectively cope with the increasingly severe problem of microbial drug resistance. This is because the antibacterial mechanism of the carbon quantum dots of the application is different from that of traditional antibacterial agents. The carbon quantum dots of the application destroy the cell structure and function of pathogens through multiple pathways, making it difficult for pathogens to develop effective drug resistance and prolonging the service life of the antibacterial material.

[0022] (3) Compared with traditional antibacterial materials, the production cost of the carbon quantum dots disclosed by the application is lower. The raw materials for preparing the carbon quantum dots are widely available, the preparation process is simple and easy to implement, and the amount of copper is effectively controlled, thereby reducing the cost of raw materials and process. At the same time, the efficient antibacterial performance makes the required amount in actual application less, further reducing the use cost and meeting the market demand for economic and practical antibacterial agents.

[0023] The technical solutions of the application will be further described in detail below with the aid of the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The morphology characterization results of Cu-CDs and CDs provided for Example 1 and Comparative Example 1 are as follows, wherein, Figure 1 (a) in the above figure is a transmission electron micrograph of Cu-CDs, the scale is 10 nm, Figure 1 (b) in the above figure is a crystal lattice fringe of Cu-CDs, the scale is 2 nm, Figure 1 (c) in the above figure is the potential results of Cu-CDs and CDs, Figure 1 (d) in the above figure is the particle size distribution graph of Cu-CDs;

[0025] Figure 2 is a Fourier infrared spectrum, wherein, Figure 2 (a) in the above figure is a Fourier infrared spectrum of Cu-CDs and CDs provided for Example 1 and Comparative Example 1, Figure 2 (b) in the above figure is a Fourier infrared spectrum of the carbon dot synthesis precursor salicylic acid and urea;

[0026] Figure 3 is an X-ray powder diffraction pattern of Cu-CDs and CDs provided for Example 1 and Comparative Example 1;

[0027] Figure 4 is an X-ray photoelectron spectrogram of Cu-CDs and CDs provided for Example 1 and Comparative Example 1, wherein, Figure 4 (a) in the above figure is an element full spectrum of Cu-CDs, Figure 4 (b) in the above figure is a C1s spectrum, Figure 4 (c) in the above figure is a N1s spectrum, Figure 4 (d) in the above figure is an O1s spectrum, Figure 4(f) is a Cu LM2 spectrum in (c), Figure 4 (f) is a Cu LM2 spectrum in (c),

[0028] Figure 5 A plate diagram of the mycelium growth inhibition ability of the carbon dot synthesis precursor SA, CDs and Cu-CDs provided by the present application on Phytophthora capsici;

[0029] Figure 6 A plate diagram of the mycelium growth inhibition ability of the copper chloride dihydrate with the same copper element content as the Cu-CDs provided by the present application on Phytophthora capsici;

[0030] Figure 7 Optical microscope pictures of the influence of SA, CDs and Cu-CDs on mycelium morphology, with a scale of 50 μm;

[0031] Figure 8 Optical microscope pictures of the influence of SA, CDs and Cu-CDs on the sporangium production of Phytophthora capsici, with a scale of 200 μm;

[0032] Figure 9 Optical microscope pictures of the influence of SA, CDs and Cu-CDs on the release of zoospores from the sporangium of Phytophthora capsici, with a scale of 200 μm;

[0033] Figure 10 Optical microscope pictures of the influence of SA, CDs and Cu-CDs on the morphology of zoospores, with a scale of 20 μm. DETAILED DESCRIPTION

[0034] The technical solutions of the present application are further described below through the drawings and examples.

[0035] Unless otherwise defined, the technical terms or scientific terms used in the present application shall have the usual meanings understood by those with ordinary skills in the art to which the present application belongs.

[0036] In the present application, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art, which can be purchased through commercial channels.

[0037] Example 1 The present example provides a preparation method of carbon quantum dots for plant oomycete diseases, comprising the following steps:

[0038] S1, 1.5 g of salicylic acid, 1.5 g of urea and 0.25 g of copper chloride dihydrate are dissolved in 40 mL of purified water, the pH value is adjusted to 11, and stirring is performed until complete dissolution to obtain a mixed solution;

[0039] S2, transferring the mixed solution obtained in step S1 into a hydrothermal reactor, reacting at 180℃ for 10h, cooling to room temperature to obtain carbon dots;

[0040] S3, centrifuging the carbon dots obtained in step S1 at 7800rpm for 20min, collecting the supernatant, filtering through a 0.22μm microporous filter membrane, dialyzing in a 1000Da dialysis bag for 12h, vacuum freeze-drying at-50℃ for 24h to obtain carbon quantum dot powder Cu-CDs.

[0041] Comparative Example 1 This comparative example provides a preparation method of carbon quantum dots for plant oomycete diseases, comprising the following steps:

[0042] S1, dissolving 1.5g salicylic acid and 1.5g urea in 40mL purified water, adjusting the pH value to 11, stirring to completely dissolve to obtain a mixed solution;

[0043] S2, transferring the mixed solution obtained in step S1 into a hydrothermal reactor, reacting at 180℃ for 10h, cooling to room temperature to obtain a carbon dot solution;

[0044] S3, centrifuging the carbon dot solution obtained in step S1 at 7800rpm for 20min, collecting the supernatant, filtering through a 0.22μm microporous filter membrane, dialyzing in a 1000Da dialysis bag for 12h, vacuum freeze-drying at-50℃ for 24h to obtain carbon dot powder CDs.

[0045] The carbon quantum dots Cu-CDs provided in Example 1 and the carbon dot powder CDs provided in Comparative Example 1 were verified for effect through the following tests:

[0046] 1. The morphology of Cu-CDs / CDs was observed by high-resolution transmission electron microscopy (HRTEM, Talos F200X G2, Thermo Fisher, America). At the same time, the potential of CDs and Cu-CDs was measured by a nanoparticle size and Zeta potential analyzer (Malvern, UK), and the size of Cu-CDs was counted by ImageJ, and the results are as follows Figure 1 .

[0047] From (a) in Figure 1 and (b) in Figure 1 , it can be seen that the morphology of the finally prepared Cu-CDs is spherical, and the lattice spacing is 0.21nm. From (c) in Figure 1 , it can be seen that the CDs provided in Comparative Example 1 have a negative charge (-5.21±0.37mV), while the charge of Cu-CDs provided in Example 1 is further reduced to (-14.4±0.5mV). From (d) in Figure 1 , it can be seen that the average size is 2.14±0.28nm.

[0048] 2. CDs and Cu-CDs were analyzed by Fourier transform infrared spectroscopy (FTIR, Frontier, PerkinElmer, America), and the results are as follows: Figure 2 .

[0049] Depend on Figure 2 As shown in (a) of the spectrum, the CDs exhibit broad peaks for the stretching vibrations of OH and NH, as well as stretching vibration peaks for C=O and CN, and also include the in-plane bending vibration peak of NH in secondary amines. These features indicate successful nitrogen doping. Cu-CDs show peaks at 960 cm⁻¹. -1 The appearance of a new characteristic weak peak at this location, identified as a Cu-N group, indicates the successful formation of a Cu coordination compound. Figure 2 As shown in (b), the spectral peaks of salicylic acid include: a broad OH stretching vibration peak generated by the combined carboxyl and phenolic hydroxyl groups, a C=O stretching vibration peak of the carboxyl group, and a CO stretching vibration peak of the carboxyl group and phenolic compounds. The spectral peaks of urea include an NH stretching vibration peak of the amino group (primary amide doublet) and stretching vibration peaks of the C=O and CN groups of the amide group.

[0050] 3. X-ray powder diffraction (XRD, Rigaku D / MAX2500VL / PC, Japan) analysis, results are as follows: Figure 3 .

[0051] Depend on Figure 3 It can be seen that in the 2θ range of 10-60, CDs and Cu-CDs show only a single broad diffraction peak (002) centered at 2θ = 23°, which is a typical feature of polymerized carbon, indicating that sp 2 and sp 3 The combined characteristics of amorphous carbon. This result is consistent with... Figure 1 The lattice fringe analysis results in (b) are consistent with those in the XRD pattern, further confirming the amorphous nature of CDs. No other diffraction features were observed in the XRD pattern, indicating that there are no copper metal or copper chloride crystalline particles in the material.

[0052] 4. X-ray photoelectron spectroscopy (XPS, ESCALAB250Xi, Thermo Fisher, America) was performed on Cu-CDs, and the results are as follows: Figure 4 .

[0053] Depend on Figure 4It can be seen that the contents of C, N, O and Cu elements in Cu-CDs are 72.35%, 2.91%, 23.24% and 1.5% respectively, which confirms the successful doping of nitrogen element and copper element, and the content of copper atom is extremely low, which further reduces its potential harm. From the Cu 2p peak, it can be observed that there is a characteristic satellite peak of divalent copper ions in the range of 940-950eV, indicating that the copper atoms in Cu-CDs mainly exist in the form of divalent. Through the analysis of the Auger peak of copper, it is known that the copper atoms in Cu-CDs are a mixture of monovalent and divalent copper, mainly divalent copper.

[0054] In this experimental example, the inhibitory effect of a kind of salicylic acid-based CDs and Cu-CDs on oomycetes was investigated. The specific steps are as follows:

[0055] (1) Cultivation of P. capsici and stimulation of sporangium production and spore release:

[0056] a. The P. capsici isolate was inoculated into V8 juice agar medium, and the composition of the medium was 10 mL V8 juice, 0.02 g CaCO3 and 1.6 g agar per 100 mL. The culture temperature was set to 25°C, and the culture was carried out in the dark for 5 days.

[0057] b. Five agar blocks with a diameter of 8 mm were cut from the edge of the freshly cultured P. capsici colony and placed in a sterile culture dish, and 15 mL of 10% V8 liquid medium was added. The culture was incubated at 25°C in the dark for 3 days to form a mycelial mat. Then the V8 medium was removed, and 15 mL of sterile soil extract was added to completely immerse the mycelial mat. Continue to culture for 2 days until the motile sporangia are generated.

[0058] c. The sporangia were placed in a 4°C refrigerator for 30 min, then transferred to a 25°C environment for 30 min, and the cold and hot were alternated to stimulate the release of motile spores.

[0059] (2) The mycelial disc with a diameter of 8 mm was cut from the edge of the colony cultured for 5 days, and placed on V8 agar medium containing different concentrations (0, 25, 50, 100 and 200 μg mL -1 ) SA / CDs / Cu-CDs, and incubated at 25°C in the dark for 5 days until the mycelial diameter of the control group reached about 80 mm. Finally, the inhibition of mycelial growth of SA / CDs / Cu-CDs treatment group compared with the control group was evaluated by measuring the mycelial diameter. The inhibition rate (%) is calculated as follows:

[0060]

[0061] In the formula, D1 is the mycelial diameter of the control group, and D2 is the mycelial diameter of the treatment group. The results are as follows Figure 5 .

[0062] Depend on Figure 5 This indicates that the carbon dots synthesized from salicylic acid not only retain their active pharmaceutical components but also enhance their antibacterial effect to a certain extent. Furthermore, the experiment also showed that copper doping significantly improved the inhibitory performance of the carbon dots against oomycetes. When the concentration reached 200 μg / mL... -1 At that time, mycelial growth was completely inhibited.

[0063] To determine whether copper plays a dominant role in the sterilization of Cu-CDs, X-ray photoelectron spectroscopy was used to analyze the copper atomic content, which was found to be 1.5%, and the corresponding concentration of copper chloride dihydrate was calculated. 0.5, 1.0, 1.5, 2.0, and 3.0 μg mL of the solution were then analyzed. -1 Copper chloride dihydrate and Cu-CDs with an equivalent copper content were mixed with V8 agar medium and inoculated with mycelial cakes. The mixture was incubated in the dark at 25°C for 5 days. The antibacterial effect of copper chloride dihydrate was evaluated by measuring the mycelial diameter. The results are as follows: Figure 6 .

[0064] Depend on Figure 6 It can be seen that, at the same concentration, the antibacterial efficiency of copper ions is significantly lower than that of Cu-CDs, indicating that the extremely low concentration of copper atoms contained in Cu-CDs does not play a dominant role in the bactericidal effect.

[0065] The microscopic morphology of the treated hyphae was observed using an optical microscope to evaluate the antibacterial effect. The specific steps were as follows: First, five 8mm diameter agar plates containing *Phytophthora capsici* were cut and placed in a 25℃ incubator, then placed in petri dishes containing V8 liquid culture medium. 200 μg mL of the medium was added to each dish. -1 SA, CDs, and Cu-CDs were observed. After 4 days of cultivation, the morphology of the hyphae was observed using an optical microscope, and the results are as follows. Figure 7 .

[0066] Depend on Figure 7 It can be seen that the hyphal morphology of *Phytophthora capsici* changed significantly after treatment: the hyphae in the control group extended normally and had a regular shape; while the hyphae in the treatment group showed varying degrees of increased branching, distortion, swelling and other deformities. Among them, the Cu-CDs treatment group showed the most obvious hyphal distortion.

[0067] The antibacterial effects of CDs and Cu-CDs were evaluated using an in vitro sporangia formation inhibition assay. The specific steps were as follows: First, five 8 mm diameter agar plates containing *Phytophthora capsici* were selected and placed in petri dishes containing V8 liquid medium at 25°C for 3 days. Subsequently, the mycelia in the petri dishes were gently washed with sterile distilled water and then immersed in a solution containing SA, CDs, or Cu-CDs (200 μg / mL). -1The samples were cultured in sterile soil extract at 25°C in the dark for 3 days. Finally, the number of sporangia was counted using a hemocytometer under an optical microscope, and the sporangia formation rate was calculated. The results are as follows: Figure 8 .

[0068] Depend on Figure 8 It was found that different treatments significantly affected the formation of zoosporangia in *Phytophthora capsici*. In the SA and CDs treatment groups, the number of zoosporangia was slightly reduced compared to the control group; however, in the Cu-CDs treatment group, the number of zoosporangia decreased significantly, with only a very small number of zoosporangia forming on the hyphae, and zoosporangia formation was almost completely inhibited. These results indicate that Cu-CDs exhibit a significant advantage in inhibiting sporangia formation in *Phytophthora capsici*.

[0069] The inhibitory effects of CDs and Cu-CDs on *Phytophthora capsici* were evaluated using a zoospore release assay. The specific steps were as follows: First, the same number of sporangia were added to a solution containing SA, CDs, or Cu-CDs (200 μg / mL). -1 The samples were then placed in sterile distilled water. Subsequently, these samples were placed in a 4°C refrigerator for 30 min and incubated at 25°C for 30 min to induce the release of zoospores from the sporangia. Finally, the zoospores in all treatment groups were counted using a hemocytometer and microscope, and the results are shown below. Figure 9 .

[0070] Depend on Figure 9 It was found that almost all zoospores were released in the control group, while the release of zoospores was inhibited after treatment with SA and CDs, and some spores failed to be released. In particular, in the Cu-CDs treatment group, the number of released zoospores was extremely limited, with a spore release rate of less than 20%. These results indicate that Cu-CDs have a significant effect on inhibiting the release of zoospores from Phytophthora capsici.

[0071] The antibacterial properties of carbon dots were evaluated by observing the morphological changes of zoospores after co-incubation with CDs and Cu-CDs. The specific steps were as follows: First, zoospores were released from the sporangia using hot and cold stimulation as described above, and the zoospores were collected and their concentration adjusted to 10. 4 mL -1 Then, the zoospores were mixed with 200 μg mL -1 SA, CDs, and Cu-CDs were incubated for 30 min each, and then the morphological changes of the spores were observed under an optical microscope. The results are as follows: Figure 10 .

[0072] Depend on Figure 10It can be seen that most of the zoospores in the control group presented normal morphology, maintaining uniform round shape; while in the SA treatment group, the morphology of the spores was basically intact. In contrast, in the CDs and Cu-CDs treatment groups, part of the spores appeared to be lysed and released their contents. In addition, with the extension of incubation time, the number of lysed spores gradually increased. These results suggest that CDs and Cu-CDs have a significant impact on the morphology of zoospores, suggesting that they may play an important role in the process of inhibiting bacteria.

[0073] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, but not to limit it. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can still be modified or equivalently replaced, and these modifications or equivalent replacements should not make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A method for preparing carbon quantum dots against plant oomycete diseases, characterized by, The method comprises the following steps: S1, dissolving salicylic acid, urea and copper chloride dihydrate in purified water, adjusting the pH value to 10-12, stirring to completely dissolve, to obtain a mixed solution; S2, transferring the mixed solution obtained in step S1 to a hydrothermal reaction kettle for reaction, cooling to room temperature to obtain carbon dots; S3, centrifuging the carbon dots obtained in step S1, collecting the supernatant, filtering through a 0.22 μm microporous filter membrane, dialysis in a 1000 Da dialysis bag for 10-14 h, vacuum freeze-drying to obtain carbon quantum dots.

2. The production method according to claim 1, characterized by, In step S2, the reaction temperature is 160-200℃, and the reaction time is 8-12h.

3. The production method according to claim 1, characterized by, In step S3, the centrifugal speed is 7000-8000rpm, and the centrifugal time is 15-25min.

4. The method of claim 1, wherein, In step S3, the freeze-drying temperature is-50℃, and the freeze-drying time is 24h.

5. The carbon quantum dots prepared by the preparation method of any one of claims 1-4.

6. The carbon quantum dots prepared by the preparation method of any one of claims 1-4 or the carbon quantum dots of claim 5 for use in preventing and treating plant pathogenic oomycete diseases.

7. The carbon quantum dots prepared by the preparation method of any one of claims 1-4 or the carbon quantum dots of claim 5 for use in the preparation of a product for preventing and treating plant pathogenic oomycete diseases.

8. A growth inhibitor for controlling a plant pathogenic Oomycete, characterized by, The carbon quantum dots prepared by the preparation method of any one of claims 1-4 or the carbon quantum dots of claim 5.

9. The use of the growth inhibitor for preventing and treating plant pathogenic oomycetes of claim 8 in preventing and treating Phytophthora capsici.

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