Carbon quantum dot aiming at plant oomycetes disease as well as preparation method and application of carbon quantum dot

Abstract

The invention discloses a carbon quantum dot aiming at plant oomycete diseases as well as a preparation method and application thereof, and belongs to the technical field of nano 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, and stirring until the materials are completely dissolved to obtain a mixed solution; transferring into a hydrothermal reaction kettle for reaction, and cooling to room temperature to obtain carbon dots; centrifuging, collecting supernate, filtering with a 0.22 mu m microporous filter membrane, dialyzing with a 1000Da dialysis bag for 10-14 hours, and performing vacuum freeze drying to obtain carbon quantum dot powder. The invention discloses a carbon quantum dot aiming at plant oomycete diseases and a preparation method and application thereof, copper chloride is used as a doping agent to perform heteroatom doping on the carbon dot, so that a synergistic effect is generated between copper and the carbon dot, the antibacterial ability of a composite material is remarkably enhanced, and the carbon quantum dot has an efficient antibacterial effect and excellent drug resistance.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano material preparation, and in particular relates to carbon quantum dots for plant oomycete diseases, and a preparation method and application thereof. Background Art

[0002] Plant pathogens, especially oomycetes, cause huge economic losses to global agriculture. Oomycetes are a class of eukaryotic microorganisms similar to fungi and evolutionarily close to brown algae, but they differ significantly 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 phytophthora are extremely difficult to control due to their rapid growth and reproduction capabilities, as well as their ability to produce oospores that can survive in the environment for a long time. Traditional chemical fungicides play an important role in the prevention and control of oomycete diseases, but long-term use has led to the emergence of drug resistance problems. For example, systemic fungicides have a single site of action, and pathogens can easily develop resistance to them. In addition, many effective fungicides contain persistent chemicals such as tin and copper, which pose a potential threat to ecosystems and human health.

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

[0005] The present invention aims to provide a carbon quantum dot for plant oomycete diseases and a preparation method and application thereof, wherein the carbon quantum dot has a highly efficient antibacterial effect and excellent drug resistance.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing carbon quantum dots for plant oomycete diseases comprises the following steps:

[0008] S1. Dissolve salicylic acid, urea and cupric chloride dihydrate in purified water, adjust the pH value to 10-12, and stir to completely dissolve to obtain a mixed solution;

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

[0010] S3. Centrifuge the carbon dots obtained in step S1, collect the supernatant, filter with a 0.22 μm microporous filter membrane, dialyze with a 1000 Da dialysis bag for 10-14 h, and freeze-dry in vacuum to obtain carbon quantum dots.

[0011] Preferably, in step S2, the reaction temperature is 160-200° 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°C, and the freeze-drying time is 24 hours.

[0014] The present invention also provides carbon quantum dots prepared by the preparation method described above.

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

[0016] The present invention also provides the carbon quantum dots prepared by the preparation method or the use of the carbon quantum dots in preparing products for preventing and controlling plant pathogenic oomycetes.

[0017] The present invention also provides a growth inhibitor for controlling plant pathogenic oomycetes, including the carbon quantum dots prepared by the preparation method or the carbon quantum dots.

[0018] The present invention also provides the use of the growth inhibitor for controlling plant pathogenic oomycetes in controlling pepper phytophthora.

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

[0020] (1) The present invention discloses a carbon quantum dot for plant oomycete diseases, a preparation method and an application thereof. Copper chloride is used as a dopant to dope the carbon dots with heteroatoms, so that a synergistic effect is produced between copper and carbon dots, significantly enhancing the antibacterial ability of the composite material. Copper-based nanoparticles can produce active oxygen under light conditions, and the carbon dots themselves also have certain antibacterial activity. After the two are combined, the antibacterial effect far exceeds that of a single material, especially in the prevention and treatment of oomycetes. And the metal loading is reasonably controlled, which effectively reduces the amount of antibacterial agent used, while ensuring that the antibacterial efficiency is not affected.

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

[0022] (3) Compared with traditional antibacterial materials, the carbon quantum dots disclosed in the present invention have lower production costs. The raw materials for preparing carbon quantum dots are widely available, the preparation process is simple and easy, and the amount of copper used is effectively controlled, which reduces the cost of raw materials and process. At the same time, its high antibacterial performance means that the amount required in practical applications is relatively small, further reducing the cost of use and meeting the market demand for economical and practical antibacterial agents.

[0023] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The morphological characterization results of Cu-CDs and CDs provided in Example 1 and Comparative Example 1, wherein: Figure 1 (a) is a transmission electron microscopy image of Cu-CDs, with a scale of 10 nm. Figure 1 (b) shows the lattice fringes of Cu-CDs, with a scale of 2 nm. Figure 1 (c) in the figure is the potential result of Cu-CDs and CDs. Figure 1 (d) in the figure is the particle size distribution diagram of Cu-CDs;

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

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

[0027] Figure 4 X-ray photoelectron spectra of Cu-CDs and CDs provided in Example 1 and Comparative Example 1, wherein: Figure 4 (a) is the full spectrum of Cu-CDs elements. Figure 4 (b) is the C1s spectrum. Figure 4 (c) in the figure is the N1s spectrum. Figure 4 (d) in the figure is the O1s spectrum. Figure 4(e) in the figure is the Cu 2p spectrum. Figure 4 (f) in the figure is the CuLM2 spectrum;

[0028] Figure 5 A plate diagram showing the ability of the carbon dot synthesis precursors SA, CDs and Cu-CDs provided by the present invention to inhibit the mycelial growth of Phytophthora capsici;

[0029] Figure 6 A plate diagram showing the ability of cupric chloride dihydrate with a copper content equivalent to that in Cu-CDs provided by the present invention to inhibit the mycelial growth of Phytophthora capsici;

[0030] Figure 7 Optical microscope pictures of the effects of SA, CDs and Cu-CDs on hyphae morphology provided by the present invention, with a scale of 50 μm;

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

[0032] Fig. 9 Optical microscope pictures of the effects of SA, CDs and Cu-CDs provided by the present invention on the release of zoospores from sporangium of Phytophthora capsici, with a scale of 200 μm;

[0033] Fig.10 Optical microscope pictures showing the effects of SA, CDs and Cu-CDs provided by the present invention on the morphology of zoospores, scale: 20 μm. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0035] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

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

[0037] Example 1 This example provides a method for preparing carbon quantum dots for plant oomycete diseases, comprising the following steps:

[0038] S1. Dissolve 1.5 g of salicylic acid, 1.5 g of urea and 0.25 g of cupric chloride dihydrate in 40 mL of purified water, adjust the pH value to 11, and stir to completely dissolve to obtain a mixed solution;

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

[0040] S3. Centrifuge the carbon dots obtained in step S1 at 7800 rpm for 20 min, collect the supernatant, filter with a 0.22 μm microporous filter membrane, dialyze with a 1000 Da dialysis bag for 12 h, and freeze-dry at -50°C in a vacuum freeze-drying process for 24 h to obtain carbon quantum dot powder Cu-CDs.

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

[0042] S1. Dissolve 1.5 g of salicylic acid and 1.5 g of urea in 40 mL of purified water, adjust the pH to 11, and stir until completely dissolved to obtain a mixed solution;

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

[0044] S3. Centrifuge the carbon dot solution obtained in step S1 at 7800 rpm for 20 min, collect the supernatant, filter it with a 0.22 μm microporous filter membrane, dialyze it with a 1000 Da dialysis bag for 12 h, and freeze-dry it at -50°C in a vacuum freeze-drying process for 24 h to obtain carbon dot powder CDs.

[0045] The effects of the carbon quantum dots Cu-CDs provided in Example 1 and the carbon dot powder CDs provided in Comparative Example 1 were verified by the following experiments:

[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 nanoparticle size and Zeta potential analyzer (Malvern, UK), and the size of Cu-CDs was counted by ImageJ. The results are shown in Figure 1 .

[0047] Depend on Figure 1 (a) and Figure 1 As shown in (b), the morphology of the finally prepared Cu-CDs is that the carbon quantum dots are spherical and the lattice spacing is 0.21nm. Figure 1 As shown in (c), the CDs provided in Comparative Example 1 have a negative charge (-5.21±0.37 mV), while the charge of the Cu-CDs provided in Example 1 is further reduced to (-14.4±0.5 mV). Figure 1 From (d) in the figure, we can see 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). Figure 2 .

[0049] Depend on Figure 2 As shown in (a), CDs show broadband peaks of OH and NH stretching vibrations, as well as stretching vibration peaks of C=O and CN, and also include in-plane bending vibration peaks of NH in secondary amines. These features indicate the success of nitrogen doping. Cu-CDs at 960 cm -1 A new characteristic weak peak appears at , which is determined to be the Cu-N group, indicating the successful formation of the Cu coordination compound. Figure 2 As shown in (b), the spectral peaks of salicylic acid include: the OH stretching vibration broadband peak generated by the carboxyl group and the phenolic hydroxyl group, the C=O stretching vibration peak of the carboxyl group, and the CO stretching vibration peaks of the carboxyl group and phenolic compounds. The spectral peaks of urea include the NH stretching vibration peak of the amino group (primary amide doublet) and the stretching vibration peaks of C=O and CN of the amide.

[0050] 3. X-ray powder diffraction (XRD, Rigaku D / MAX2500VL / PC, Japan) analysis, the 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 only show a single broad diffraction peak (002) centered at 2θ = 23°, which is a typical feature of polymeric carbon, indicating that the sp 2 and sp 3 This result is consistent with the amorphous carbon characteristics of Figure 1 The lattice fringe analysis results in (b) are consistent, further confirming the amorphous nature of CDs. No other diffraction features were found in the XRD pattern, indicating that there are no crystalline particles of copper metal or copper chloride in the material.

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

[0053] Depend on Figure 4It can be seen that the contents of C, N, O, and Cu in Cu-CDs are 72.35%, 2.91%, 23.24%, and 1.5%, respectively, confirming the successful doping of nitrogen and copper elements. At the same time, the content of copper atoms is extremely low, further reducing its potential harmfulness. From the Cu 2p peak, it can be observed that there are characteristic satellite peaks of divalent copper ions in the range of 940-950eV, indicating that the copper atoms in Cu-CDs mainly exist in divalent form. 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, among which divalent copper is the main component.

[0054] Experimental Example This experimental example explores the inhibitory effect of CDs and Cu-CDs based on salicylic acid on oomycetes. The specific steps are as follows:

[0055] (1) Cultivation of pepper phytophthora and stimulation of sporangium production and spore release:

[0056] a. The isolate of Phytophthora capsici was inoculated into V8 juice agar medium, 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 at 25°C, and the culture was carried out in a dark environment for 5 days.

[0057] b. Cut 5 agar blocks with a diameter of 8 mm from the edge of the freshly cultured Phytophthora colony, place them in a sterile culture dish, add 15 mL of 10% V8 liquid culture medium, and culture them in a dark environment at 25°C for 3 days to allow the formation of mycelium. Then remove the V8 culture medium and add 15 mL of sterile soil extract to completely immerse the mycelium. Continue to culture for 2 days until zoosporangia are formed.

[0058] c. Place the sporangium in a 4°C refrigerator for 30 minutes, then transfer it to a 25°C environment for 30 minutes, alternating between hot and cold to stimulate the release of zoospores.

[0059] (2) Mycelial disks with a diameter of 8 mm were cut from the edge of the colony after 5 days of culture and placed in a tube containing different concentrations (0, 25, 50, 100, and 200 μg mL -1 )SA / CDs / Cu-CDs V8 agar medium, incubated in a dark environment at 25°C for 5 days, until the mycelial diameter of the control group reached about 80 mm. Finally, the mycelial diameter was measured to evaluate the degree of inhibition of mycelial growth in the SA / CDs / Cu-CDs treatment group compared with the control group. The inhibition rate (%) was calculated as follows:

[0060]

[0061] Where D1 is the mycelium diameter of the control group, and D2 is the mycelium diameter of the treatment group. Figure 5 .

[0062] Depend on Figure 5 It can be seen that the carbon dots synthesized with salicylic acid as raw material not only retain its active medicinal ingredients, but also enhance the antibacterial effect to a certain extent. In addition, the experiment also showed that the doping of copper atoms significantly improved the inhibitory performance of carbon dots against oomycetes. When the concentration reached 200μg mL -1 The mycelial growth was completely inhibited.

[0063] In order to determine whether the copper element in Cu-CDs plays a dominant role in sterilization, the copper atomic content obtained by X-ray photoelectron spectroscopy was 1.5%, and the corresponding concentration of cupric chloride dihydrate was calculated. -1 Copper chloride dihydrate and Cu-CDs with equivalent copper content were mixed with V8 agar medium and inoculated with bacterial cakes. The culture was carried out in the dark at 25°C for 5 days, and the antibacterial effect of copper chloride dihydrate was evaluated by measuring the diameter of mycelium. The results are shown in Table 1. 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 mycelium was observed by optical microscopy to evaluate the antibacterial effect. The specific steps were as follows: First, 5 8 mm diameter agar plates of Phytophthora capsici were cut and placed in a 25 °C incubator in a culture dish containing V8 liquid culture medium. 200 μg mL -1 SA, CDs and Cu-CDs. After 4 days of culture, the morphology of mycelium was observed using an optical microscope. Figure 7 .

[0066] Depend on Figure 7 It can be seen that the hyphae morphology of pepper phytophthora has changed significantly after treatment: the hyphae of the control group stretched normally and had a regular shape, while the hyphae of the treated groups showed different degrees of increased branching, distortion, expansion and other distortion phenomena. Among them, the Cu-CDs treated group showed the most obvious hyphae distortion.

[0067] The in vitro inhibition of sporangium formation test was used to evaluate the antibacterial effect of CDs and Cu-CDs. The specific steps were as follows: First, five 8 mm diameter agar plates of Phytophthora capsici were selected and placed in a culture dish containing V8 liquid medium at 25 °C for 3 days. Subsequently, the mycelium in the culture dish was gently washed with sterile distilled water and immersed in a medium containing SA, CDs or Cu-CDs (200 μg mL -1) in sterile soil extract and continue to culture at 25°C in the dark for 3 days. Finally, the number of sporangia was counted by hemocytometer under an optical microscope, and the sporangium formation rate was calculated. The results are as follows: Figure 8 .

[0068] Depend on Figure 8 It can be seen that different material treatments had a significant effect on the formation of zoosporangium of pepper. In the SA and CDs treatment groups, the number of zoosporangium of pepper was slightly reduced compared with the control group; while in the Cu-CDs treatment group, the number of zoosporangium decreased significantly, only a very small number of zoosporangium was formed on the hyphae, and the production of zoosporangium was almost completely inhibited. These results show that Cu-CDs has a significant advantage in inhibiting the formation of sporangium of pepper.

[0069] The zoospore release experiment was used to evaluate the inhibitory performance of CDs and Cu-CDs against Phytophthora capsici. The specific steps were as follows: First, the same number of sporangia were added to the medium containing SA, CDs or Cu-CDs (200 μg mL -1 ) in sterile distilled water. Subsequently, the samples were placed in a 4°C refrigerator for stimulation for 30 min and incubated at 25°C for 30 min to induce the sporangium to release zoospores. Finally, the zoospores in all treatment groups were counted using a hemocytometer and a microscope. The results are shown in Fig. 9 .

[0070] Depend on Fig. 9 It can be seen that the zoospores in the control group were almost completely released, while after SA and CDs treatment, the zoospore release process was inhibited and some spores failed to be released. Especially in the Cu-CDs treatment group, the number of released zoospores was extremely limited, and the spore release rate was less than 20%. These results show that Cu-CDs has a significant effect in inhibiting the release of zoospores of pepper phytophthora.

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

[0072] Depend on Fig.10It can be seen that most zoospores in the control group showed normal morphology and maintained a uniform round shape; while in the SA-treated group, the spore morphology was basically intact. In contrast, in the CDs and Cu-CDs-treated groups, some spores lysed and released their contents. In addition, the number of lysed spores gradually increased with the extension of incubation time. These results indicate that CDs and Cu-CDs have a significant effect on the morphology of zoospores, suggesting that they may play an important role in the antibacterial process.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing carbon quantum dots for plant oomycete diseases, characterized in that: The following steps are involved: S1. Dissolve salicylic acid, urea and cupric chloride dihydrate in purified water, adjust the pH value to 10-12, and stir to completely dissolve to obtain a mixed solution; S2, transferring the mixed solution obtained in step S1 to a hydrothermal reactor for reaction, cooling to room temperature, and obtaining carbon dots; S3. Centrifuge the carbon dots obtained in step S1, collect the supernatant, filter with a 0.22 μm microporous filter membrane, dialyze with a 1000 Da dialysis bag for 10-14 h, and freeze-dry in vacuum to obtain carbon quantum dots.

2. The preparation method according to claim 1, characterized in that: In step S2, the reaction temperature is 160-200° C., and the reaction time is 8-12 h.

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

4. The preparation method according to claim 1, characterized in that: In step S3, the freeze-drying temperature is -50°C and the freeze-drying time is 24 hours.

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

6. Use of the carbon quantum dots prepared by the preparation method according to any one of claims 1 to 4 or the carbon quantum dots according to claim 5 in preventing and controlling plant pathogenic oomycete diseases.

7. Use of the carbon quantum dots prepared by the preparation method according to any one of claims 1 to 4 or the carbon quantum dots according to claim 5 in the preparation of products for controlling plant pathogenic oomycetes.

8. A growth inhibitor for controlling plant pathogenic oomycetes, characterized in that The carbon quantum dots prepared by the preparation method according to any one of claims 1 to 4 or the carbon quantum dots according to claim 5.

9. Use of the growth inhibitor for controlling plant pathogenic oomycetes as claimed in claim 8 in controlling Phytophthora capsici.

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