Carrier-free self-assembled nano-pesticide, preparation method and application thereof

Abstract

The application provides a carrier-free self-assembled nano pesticide and a preparation method and application thereof, relates to the technical field of agricultural pesticides, and discloses a one-step nano precipitation self-assembly method for forming a flutianil carrier-free nano pesticide. Natural material tannic acid is used as an amphiphilic building unit, hydrophobic interaction, hydrogen bonding and pi-pi stacking are used to realize green nano-ization without a carrier and a chemical reaction, flutianil carrier-free nano pesticide realizes a nanoscale particle size, and the utilization rate of flutianil is effectively improved. In the preparation process of the carrier-free nano pesticide, no metal ion precursor is introduced, tannic acid maintains the original chemical structure and is not oxidized, the operation is simple, the efficacy of flutianil can be effectively improved, the use amount of flutianil can be significantly reduced, and the prevention and treatment effects of soybean root rot, cowpea wilt and litchi blight caused by phytophthora sojae, phytophthora phaseoli and phytophthora citri are significantly improved.

Description

Technical Field

[0001] This invention relates to the field of agricultural pesticide technology, and in particular to a carrier-free self-assembled nanopesticide, its preparation method, and its application. Background Technology

[0002] Oomycetes are an important class of pathogens, including genera such as *Phytophthora*, *Pythium*, and *Pythium*. These pathogens have well-developed, septate mycelia and can spread and infect through asexual reproduction (producing zoosporangia) and sexual reproduction (producing oospores). They can damage multiple parts of vegetables, including roots, stems, leaves, flowers, and fruits, and are highly pathogenic. Among the many oomycete diseases, those caused by the following pathogens are particularly prominent and pose a serious threat to agricultural production: *Phytophthora sojae*, which causes devastating soybean root rot, severely impacting global soybean production; *Phytophthora cowpeae*, which can cause root and stem rot and blight in cowpeas; *Phytophthora downyensis*, the pathogen of blight in litchi, mainly affects the fruit but can also infect flower spikes and leaves, posing one of the biggest threats to litchi yield and quality in my country; and *Phytophthora taroe*, the main pathogen causing blight in taro, which can lead to concentric ring spots on leaves, petiole rot, and wet rot in corms, causing yield reductions of more than 50% in severe cases, and is widely distributed in southern my country and Southeast Asia.

[0003] Fluoxazolidinone, developed by DuPont in 2007, is a novel fungicide for oomycetes. Its novel site of action involves binding to oxidosterol-binding proteins to inhibit the cellular function of pathogens, thereby exerting preventative, curative, and sporulation-inhibiting effects. However, due to its single site of action, fluoxazolidinone carries a medium to high risk of resistance. Furthermore, it degrades readily under acidic and alkaline conditions, and its photodegradation rate decreases with increasing initial concentration or humic acid concentration. While fluoxazolidinone exhibits good control of oomycete diseases, its high price, chemical instability, and large application rates in the field can negatively impact the environment and agricultural production.

[0004] Therefore, developing a new type of nano-pesticide that is simple and environmentally friendly, combining the high efficacy of fluthiazopyrone with reduced dosage, is of great significance for the effective control of oomycete diseases. Summary of the Invention

[0005] In view of this, the present invention proposes a carrier-free self-assembled nanopesticide, its preparation method, and its application, thereby solving the above-mentioned problems. The technical solution of the present invention is implemented as follows: A method for preparing carrier-free self-assembled nanopesticides, the specific preparation steps of which include: S1. Fluthiazopyrone was added to methanol to obtain a 10 mg / mL fluthiazopyrone solution; S2. Add tannic acid to dimethyl sulfoxide to obtain a 10 mg / mL tannic acid solution; S3. Mix the fluthiazopyrone solution and the tannic acid solution, add the mixed solution system to water, stir and centrifuge to remove residual free drug, and obtain the target carrier-free self-assembled nanopesticide; The fluthiazopyridine solution and tannic acid solution are mixed according to a molar ratio of fluthiazopyridine to tannic acid of 1:4.

[0006] Furthermore, in step S3, the volume ratio of the mixed solution system to water is 1:1; the stirring is carried out at 25°C and 500 rpm for 10 hours; and the centrifugation is carried out at 13528 × g for 5 minutes.

[0007] A carrier-free self-assembled nanopesticide is prepared by any of the preparation methods described above.

[0008] Application of a carrier-free self-assembled nanopesticide in the control of plant diseases caused by oomycetes, wherein the oomycetes include Phytophthora soybeani (… Phytophthora sojae Phytophthora indicum ( ) Phytophthora vignae ), Phytophthora indicum ( Phytophthora litchii ) and Phytophthora indicum (Phytophthora colocasiae) At least one of the following.

[0009] Furthermore, the plant diseases include at least one of soybean root rot, cowpea wilt, and litchi downy mildew.

[0010] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention employs a one-step nanoprecipitation self-assembly method to form carrier-free nanopesticides of fluthiazopyrone. Using natural tannic acid as an amphiphilic building block, the carrier-free and chemically-free nanopesticides are achieved through hydrophobic interactions, hydrogen bonds, and π-π stacking, thus enabling the carrier-free nanopesticides of fluthiazopyrone to achieve nanoscale particle size and effectively improve the utilization rate of fluthiazopyrone.

[0011] 2. In the preparation process of the carrier-free nano-pesticide fluthiazopyrone of the present invention, no metal ion precursors are introduced throughout the process, and the tannic acid retains its original chemical structure without being oxidized. The operation is simple, which can not only effectively improve the efficacy of fluthiazopyrone, but also significantly reduce the amount of fluthiazopyrone used, avoiding a series of agricultural problems caused by the large amount of application in the field in traditional application methods. Attached Figure Description

[0012] Figure 1 The physicochemical characterization diagram of the fluthiazopyrone nanopesticide (TO) prepared in the example is shown in the figure. Figure 1 In the diagram, 'a' represents the Tyndall effect. Figure 1 Image b in the middle is a transmission electron microscope image. Figure 1 In the middle, c represents the particle size distribution of DLS. Figure 1In the diagram, d represents the Zeta potential. Figure 1 The symbol 'e' represents the elemental analysis of carbon, nitrogen, sulfur, and fluorine.

[0013] Figure 2 The diagram shows the inhibitory effects of the fluthiazopyrone nanopesticide (TO), fluthiazopyrone technical (OXA), and tannic acid (TA) prepared for the example on Phytophthora soybeanae, Phytophthora litchii, Phytophthora cowpeaae, and Phytophthora taroae.

[0014] Figure 3 The protective effects of the fluthiazopyrone nanopesticide (TO) and fluthiazopyrone technical grade (OXA) prepared for the examples on plant leaves are shown in the figures. Figure 3 In the middle, 'a' represents a litchi leaf. Figure 3 In the middle, b represents a cowpea leaf. Figure 3 In the middle, c represents a soybean leaf.

[0015] Figure 4 The effect of the fluthiazopyrone nanopesticide (TO) prepared for the example on the seed germination of litchi seeds is shown in the figure. Detailed Implementation

[0016] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0017] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0018] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0019] Example A method for preparing carrier-free self-assembled nanopesticides, the specific preparation steps of which include: S1. Add 10 mg of fluthiazopyrone to 1 mL of methanol and sonicate at 40 Hz for 10 min to prepare a 10 mg / mL fluthiazopyrone solution. S2. Add 10 mg of tannic acid to 1 mL of dimethyl sulfoxide and sonicate at 40 Hz for 10 min to prepare a 10 mg / mL tannic acid solution. S3. Mix fluthiazopyrone solution and tannic acid solution at a molar ratio of fluthiazopyrone to tannic acid of 1:4, and sonicate at 40 Hz for 10 min to obtain a mixed solution system. Gradually add 2 mL of the mixed solution system dropwise to 2 mL of deionized water, stir at 25 ℃ and 500 rpm for 10 h, centrifuge at 13528×g for 5 min, remove the supernatant, and resuspend in 2.0 mL of deionized water. Repeat three times to remove residual free drug to obtain fluthiazopyrone nanopesticide.

[0020] The contents of tannic acid and fluoxetine in the above-mentioned fluoxetine nanopesticide were determined by high performance liquid chromatography.

[0021] Test Example 1 The fluthiazopyrone nanopesticide (TO) prepared in the examples was characterized and measured. Specifically, the process involved redispersing the fluthiazopyrone nanopesticide particles in water and then using a laser pointer to perform a Tyndall effect test. The particle size distribution and surface potential of the prepared fluthiazopyrone nanopesticide particles were characterized using dynamic light scattering (DLS). TEM images of fluoxetine nanoparticles were obtained using transmission electron microscopy. The elemental composition of fluoxetine nanoparticles was analyzed using energy-dispersive X-ray spectroscopy.

[0022] The results are as follows Figure 1 As shown.

[0023] Results: See Figure 1 As shown in the Tyndall effect diagram of a, the fluoxetine nanopesticide prepared in the example exhibits a significant Tyndall effect, indicating the formation of a stable colloidal dispersion system without macroscopic precipitation.

[0024] See Figure 1 The transmission electron microscope image in Figure b shows that the fluoxetine nanoparticles prepared in the example have a smooth surface and a typical core-shell structure.

[0025] See Figure 1 The DLS particle size distribution diagram in Figure c shows that the average particle size of the fluthiazopyrone nanopesticide prepared in the example is 307.3 nm, indicating that the prepared fluthiazopyrone nanopesticide achieves nanoscale particle size, which can effectively improve the utilization rate of fluthiazopyrone.

[0026] See Figure 1 The Zeta potential diagram of d shows that the Zeta potential of the fluthiazopyrone nanoparticles prepared in the example is -31.81 mV, indicating that the prepared fluthiazopyrone nanoparticles have good stability.

[0027] See Figure 1 In the figure, e is an elemental analysis diagram. It can be seen that the fluoxetine nanoparticles prepared in the examples show a uniform distribution of carbon, nitrogen, sulfur, and fluorine signals, and no metal signals. This indicates that the self-assembly of the fluoxetine nanoparticles in the examples of the present invention is based entirely on non-covalent physical interactions and does not involve redox processes.

[0028] Test Example 2 The antibacterial activity of the fluthiazopyrone nanopesticide (TO) prepared in the examples was determined by indoor plate assay. Test strain: Phytophthora soybeanii ( Phytophthora sojae Phytophthora indicum ( ) Phytophthora vignae ), Phytophthora indicum ( Phytophthora litchii ) and Phytophthora indicum (Phytophthora colocasiae) ).

[0029] Test reagents: Fluthiazopyrone nanopesticide (TO), fluthiazopyrone technical grade (OXA), and tannic acid (TA) prepared in the examples.

[0030] Experimental Methods: The test agent was added to sterile melted V8 medium to prepare plates with a final test concentration of 0.001 μg / mL. Sterile melted V8 medium supplemented with sterile deionized water was used as a blank control (CK). For each activated strain after 5 days of culture, 3 mm × 3 mm mycelial blocks of uniform growth were cut with a sterile knife and inoculated into the center of sterile melted V8 medium. Five replicates were set for each group. The plates were incubated at 25℃ for 7 days. When the mycelium reached the edge of the medium, the colony diameter of each treatment was measured using the cross-crossing method, and the inhibition rate was calculated.

[0031]

[0032] The results are shown in Table 1.

[0033] Table 1

[0034] Results: From Table 1 and Figure 2 It can be seen that, at the same concentration of 0.001 μg / mL, the fluthiazopyrone nanopesticide and fluthiazopyrone technical prepared in the embodiments of the present invention all showed good inhibitory activity against Phytophthora soybeani, Phytophthora cowpeai, Phytophthora litchii, and Phytophthora taroi, with significantly better inhibitory effects than the blank control. Tannic acid alone did not show any inhibitory effect on the above four pathogenic oomycetes, and the inhibitory effect of the fluthiazopyrone nanopesticide prepared in the embodiments of the present invention was significantly better than that of the fluthiazopyrone technical.

[0035] The above experimental results show that the antibacterial efficacy of the fluthiazopyrone nanopesticide prepared in the embodiments of the present invention is significantly better than that of the fluthiazopyrone technical, and this synergistic effect is unrelated to the antibacterial activity of tannic acid itself. The fluthiazopyrone nanopesticide prepared in the embodiments of the present invention achieves both significantly improved efficacy and reduced pesticide use.

[0036] Test Example 3 The study investigated the protective effects of the fluthiazopyrone nanopesticide (TO) prepared in the examples on plant leaves of Phytophthora litica, Phytophthora cowpea, and Phytophthora soybean.

[0037] Test materials: Lychee leaves, cowpea leaves and soybean leaves with no disease spots and uniform size were selected.

[0038] Test reagents: Fluthiazopyrone nanopesticide (TO) prepared in the examples, and fluthiazopyrone technical grade (OXA).

[0039] Experimental Methods: The fluthiazopyrone nanopesticide (TO) and fluthiazopyrone technical (OXA) prepared in the examples were formulated into three concentrations: 0.1 μg / mL, 0.2 μg / mL, and 0.5 μg / mL, respectively. Leaves were placed face down in 150 mm petri dishes lined with moistened filter paper, with the petioles wrapped in moistened filter paper to maintain humidity. After uniformly spraying each concentration of pesticide solution, the dishes were incubated in a 25°C dark incubator for 24 h. Subsequently, mycelial blocks of the corresponding pathogens (Phytophthora lychee, Phytophthora cowpea, and Phytophthora soybean) were inoculated onto the surface of leaves of various plants. After inoculation, the plants were continued to be incubated at 25°C, kept in darkness for the first 24 h, and then subjected to alternating light and dark conditions. Disease incidence was observed and photographed regularly. To clearly visualize the lesions, soybean and cowpea leaves were photographed using a UV gel electrophoresis apparatus. The lesion area of ​​all leaves was counted, and the inhibition rate against the three Phytophthora diseases was calculated.

[0040]

[0041] The results are shown in Table 2.

[0042] Table 2

[0043] Results: From Table 2 and Figure 3 It can be seen that the preventive effects of both the fluthiazopyrone nanopesticide and the fluthiazopyrone technical prepared in the examples on the leaves of the three Phytophthora species showed a concentration-dependent increasing trend. At a concentration of 0.2 μg / mL, the preventive effects of the fluthiazopyrone nanopesticide prepared in the examples of this invention against Phytophthora litchii, Phytophthora cowpea, and Phytophthora soybean were 87.14%, 100%, and 37.70%, respectively, all significantly better than the control effect of the fluthiazopyrone technical. This indicates that the nano-sizing of the fluthiazopyrone nanopesticide of this invention brings about a synergistic effect, which is not a simple improvement in physical structure, but rather a significant improvement in bioavailability. This can greatly reduce the effective dosage, providing an innovative technical solution for the efficient and reduced-dosage application of fluthiazopyrone.

[0044] Test Example 4 The effects of the fluthiazopyrone nanopesticide (TO) prepared in the examples on litchi seed germination were investigated.

[0045] Experimental Methods: Ten litchi seeds of similar size were selected, rinsed with clean water to remove surface impurities, and placed in a 60 mm petri dish. 15.0 mL of sterile deionized water was added and the seeds were soaked for 5 min. After soaking, the seeds were air-dried. A 5.0 μg / mL solution of the fluthiazopyrone nanopesticide prepared in the previous example was prepared, and 10.0 mL was placed in a clean container for soaking the litchi seeds. A control group (CK) was also established, using an equal volume of sterile deionized water to soak the litchi seeds. The soaking solution was changed every 24 h, and the germination status of the litchi seeds was recorded by photographing after 48 h.

[0046] Result: As Figure 4 As shown, litchi seeds treated with a 5.0 μg / mL fluoxetine nanopesticide solution were able to germinate normally, and their germination status was not significantly different from the blank control. The results indicate that the fluoxetine nanopesticide of this invention did not significantly affect the germination of the tested litchi seeds at this concentration.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing carrier-free self-assembled nanopesticides, characterized in that, The specific preparation steps include: S1. Fluthiazopyrone was added to methanol to obtain a 10 mg / mL fluthiazopyrone solution; S2. Add tannic acid to dimethyl sulfoxide to obtain a tannic acid solution with a concentration of 10 mg / mL; S3. Mix the fluthiazopyrone solution and the tannic acid solution, add the mixed solution system to water, stir and centrifuge to obtain the target carrier-free self-assembled nanopesticide; The fluthiazopyridine solution and tannic acid solution are mixed according to a molar ratio of fluthiazopyridine to tannic acid of 1:

4.

2. The method for preparing a carrier-free self-assembled nanopesticide as described in claim 1, characterized in that, In step S3, the volume ratio of the mixed solution system to water is 1:1; the stirring is carried out at 25°C and 500 rpm for 10 hours; and the centrifugation is carried out at 13528 × g for 5 minutes.

3. A carrier-free self-assembled nanopesticide, characterized in that, It is prepared by the preparation method described in claim 1 or 2.

4. The application of a carrier-free self-assembled nanopesticide as described in claim 3 in the prevention and control of plant diseases caused by oomycetes, wherein the oomycetes include at least one of Phytophthora soybeani, Phytophthora cowpeai, Phytophthora litchiensis, and Phytophthora taroi.

5. The application as described in claim 4, characterized in that, The plant diseases mentioned include at least one of soybean root rot, cowpea wilt, and litchi downy mildew.

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