Micro-capsule suspended seed coating agent for preventing and treating clubroot of rape as well as preparation method and application of micro-capsule suspended seed coating agent

Abstract

The invention discloses a micro-capsule suspended seed coating for preventing and treating clubroot of rape and a preparation method and application thereof, the micro-capsule suspended seed coating for preventing and treating clubroot of rape is formed by coating fluazinam active compound through reaction of-NCO group of diisocyanamide ester and-NH2 group of organic amine on an oil-in-water interface. The preparation method of the microcapsule suspended seed coating for preventing and treating clubroot of rape comprises the following steps: fully and uniformly mixing fluazinam active compound, diisocyanate, fatty alcohol-polyoxyethylene ether and an organic solvent to prepare an organic solution, dripping the organic solution into water, and stirring for 25-30 minutes at the rotating speed of 1800-2200rpm to form a stable O / W emulsion; the preparation method comprises the following steps: adding the fluazinam microcapsule suspending agent into a reaction kettle, adjusting the rotating speed to 300-500rpm, dropwise adding an aqueous solution of organic amine within 8-10min, then reacting for 0.5-1h at the speed of 300-500rpm and the temperature of 50 + / -2 DEG C to obtain the fluazinam microcapsule suspending agent, adding sodium carboxymethyl cellulose and a coloring agent, and fully and uniformly mixing to prepare the fluazinam microcapsule suspending seed coating agent. The microcapsule suspended seed coating for preventing and treating clubroot of rape is high in encapsulation efficiency and good in slow release effect; the dosage is small, and the lasting time is long.

Description

Technical Field

[0001] The present invention relates to a microcapsule suspension seed coating agent for preventing and controlling clubroot of rape, a preparation method thereof, and an application thereof, belonging to the technical field of clubroot prevention and control of rape. Background Art

[0002] Rape is the largest oil and grain crop in China. The healthy development of the rape industry is related to China's oil and grain security. In recent years, with the increase in the rape sowing area, the spread range of the soil-borne disease clubroot has been expanding year by year, and it has become one of the main diseases restricting the development of the rape industry in China. Clubroot of rape is a soil-borne disease caused by the infection of Plasmodiophora brassica, commonly known as "rape cancer", which often causes the plant to grow slowly and even die due to root rot. With the improvement of the mechanization level, its spread range is showing a significant upward trend. In recent years, more and more main rape-producing areas in China have been invaded by clubroot, resulting in a rape yield loss between 5% and 60%. In severely affected areas, rape even fails to harvest. The resting spores of Plasmodiophora brassica can survive in the soil for more than ten years. Under suitable temperature and humidity, they can germinate and release primary zoospores to infect the root hairs of rape. Rape can be infected at both the seedling stage and the maturity stage, mainly at the seedling stage. Diseased plants do not show symptoms in the initial stage. In the later stage, the plants are short, the leaf color is light green and yellowish, and they often show wilting symptoms on sunny days. After infection, root nodules can usually be observed on the main root and lateral roots. With the growth of the plant and the deterioration of the disease, the tumors crack and rot in the later stage of infection, and the resting spores scatter in the soil, resulting in the wilting and death of diseased plants. Clubroot of rape occurs early and causes great harm, which has a significant impact on the plant height, number of branches, number of pods, and 1000-grain weight of the later-stage plants.

[0003] At present, for the prevention and control of clubroot of rape, the following main means are available:

[0004] 1. Disease-resistant breeding

[0005] There are physiological race differentiations in Plasmodiophora brassica, and the pathogenicities of different races are not the same. Identifying the physiological race category and pathogenicity is the basis of disease-resistant breeding. Breeders have constructed different identification systems according to the cultivation habits of cruciferous crops and the characteristics and laws of physiological races in different countries and regions. Hiroaki Yoshikawa (1989) identified the strains in the Kanto region of Japan through the Williams identification system and found that physiological races 1, 2, 3, 4, 8, and 9 appeared in this region, and the main physiological races were 1 and 3. Zhang Jing et al. (2019) used the single-spore isolation technique to identify the physiological races in 9 main Chinese cabbage-producing areas in China and found that the field bacterial sources included different physiological races, and physiological race 4 was the dominant race of Plasmodiophora brassica in China. Through the identification of different physiological races, a solid foundation has been laid for molecular breeding.

[0006] Cruciferous crops are all hosts of Plasmodiophora brassicae. Therefore, when breeding Brassica napus varieties resistant to clubroot, disease-resistant genes from other cruciferous crops are often introduced into Brassica napus. Foreign research on disease-resistant breeding started earlier. As early as 2006, researchers obtained a new strain of Brassica napus resources resistant to clubroot through interspecific hybridization of turnip ECD004 and cabbage ECD15.

[0007] Clubroot was introduced into China relatively late, but significant progress has also been made. Jia Ru (2019) used traditional breeding techniques combined with molecular marker-assisted breeding techniques to transfer the dominant disease-resistant gene CRd from the Chinese cabbage inbred line '85-74' into Brassica napus to cultivate new strains 'W3R' and 'Zhongshuang 11R'. Huazhong Agricultural University selected the first batch of conventional new varieties Huashuang 5R and hybrid new variety Huayouza 62R resistant to clubroot in China by crossing excellent parents of Brassica napus with disease-resistant turnip ECD04 and disease-resistant Chinese cabbage respectively.

[0008] Due to the race variability of Plasmodiophora brassicae, the resistance of disease-resistant varieties may decline after several years of continuous planting, and it takes a long time to cultivate a new Brassica napus variety resistant to disease. Therefore, cultivating disease-resistant varieties to control clubroot in Brassica napus is a long-term task.

[0009] 2 Biological control

[0010] Biological control is highly favored in the control of plant diseases due to its wide source, environmental friendliness and safety. Research on the biological control of clubroot at home and abroad has become a hot topic. Existing research has shown that Bacillus subtilis and Bacillus cereus have good control effects on clubroot in Brassica napus. Liu Youzhou et al. reported that Bacillus amyloliquefaciens and Paenibacillus polymyxa can secrete a variety of antibacterial substances, including antibiotics, lipopeptides, antibacterial proteins, etc., thus inhibiting the germination of resting spores of Plasmodiophora brassicae. Lin Zihan et al. found that Bacillus velezensis can inactivate resting spores, and the field control effect on clubroot can reach 63.8%. Although biological control can achieve a certain control effect, its effect is not stable and requires more in-depth research.

[0011] 3 Chemical control

[0012] Chemical control is the most efficient measure for pest and disease control in agricultural production, with the characteristics of rapidity and high efficiency. In the chemical control of clubroot, the dosage forms are all suspension concentrates, with a short drug efficacy period, a large amount of drug use, mainly used for root irrigation, a high control cost and a large environmental residue.

[0013] Due to the lack of highly effective disease-resistant varieties and biocontrol agents, chemical control is still the fastest and most effective means of preventing and controlling clubroot. Currently, the main method of application is root irrigation with pesticides. Although this can effectively prevent and control clubroot, the short duration of effectiveness and large application amounts of the pesticides lead to waste of labor costs and environmental pollution. Summary of the invention

[0014] The present invention provides a microcapsule suspension seed coating agent for preventing and treating rapeseed clubroot, a preparation method and an application thereof. In this study, fluazinam original drug is used as a research material, and the microcapsule preparation process is optimized and screened by an interfacial polymerization method, which simplifies the operation, improves the efficacy, and provides a new solution for the prevention and control of clubroot.

[0015] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0016] A microcapsule suspension seed coating agent for preventing and controlling rapeseed clubroot is formed by reacting the -NCO group of diisocyanamide with the -NH2 group of organic amine at an oil-in-water (O / W) interface to coat the fluazinam original drug.

[0017] In order to improve the prevention and control effect, the raw materials used in the above-mentioned microcapsule suspension seed coating agent for preventing and controlling rapeseed clubroot disease include: fluazinam technical, diisocyanate, fatty alcohol polyoxyethylene ether and organic amine, among which the mass ratio of fluazinam technical, diisocyanate, fatty alcohol polyoxyethylene ether and organic amine is: (2.5-3): (2.25-2.75): (2.75-3.25): (0.1-0.4).

[0018] In the microcapsule suspension seed coating agent for preventing and controlling rapeseed clubroot, the mass content of fluazinam is 5-6%.

[0019] The microcapsule suspension seed coating agent for preventing and controlling rapeseed clubroot disease contains an organic solvent and water in a mass ratio of 1: (3.5-4).

[0020] In order to improve the stability and control effect of the microcapsule suspension seed coating agent for controlling rape clubroot, the organic solvent is cyclohexanone or ethyl acetate. The organic solvent is more preferably cyclohexanone.

[0021] In order to further improve the control effect, the organic amine is diethylenetriamine; the diisocyanate is 4,4'-diphenylmethane diisocyanate.

[0022] A method for preparing a microcapsule suspension seed coating agent for preventing and treating rape clubroot disease comprises the following steps:

[0023] 1) fully mixing fluazinam technical, diisocyanate (MDI), fatty alcohol polyoxyethylene ether (AEO-9) and an organic solvent to prepare an organic solution;

[0024] 2) Drop the organic solution obtained in step 1) into water and stir on a magnetic stirrer at 1800 - 2200 rpm for 25 - 30 min to form a stable O / W emulsion;

[0025] 3) Adjust the rotation speed to 300 - 500 rpm, dropwise add an aqueous solution of organic amine, complete the addition within 8 - 10 min, and then react at 300 - 500 rpm and 50 ± 2 °C for 0.5 - 1 h to obtain a fluazinam microcapsule suspending agent;

[0026] 4) Centrifuge the fluazinam microcapsule suspending agent prepared in step 3), and dry the obtained precipitate to obtain fluazinam microcapsule powder;

[0027] 5) Add sodium carboxymethylcellulose (CMC - Na) and a colorant to the fluazinam microcapsule suspending agent prepared in step 3), and mix well to prepare a fluazinam microcapsule suspending seed coating agent.

[0028] Adding sodium carboxymethylcellulose in the above step 5) can assist in film formation.

[0029] In the above step 5), the addition amount of sodium carboxymethylcellulose is 0.5 - 2% of the mass of the fluazinam microcapsule suspending agent. The addition amount of the colorant is 0.25 - 1% of the mass of the fluazinam microcapsule suspending agent.

[0030] The above microcapsule suspending seed coating agent for controlling clubroot of rape is used to control clubroot of rape; when in use, dilute the microcapsule suspending seed coating agent for controlling clubroot of rape with water and mix it evenly with the seeds. The mass ratio of the microcapsule suspending seed coating agent for controlling clubroot of rape to the mass of the seeds is 1:(10 - 30), and the mass content of the fluazinam technical in the diluted microcapsule suspending seed coating agent is 1.5 - 2.5%.

[0031] When in use, the addition order of the microcapsule suspending seed coating agent, seeds, and water is not limited, as long as the three are mixed evenly.

[0032] Unless otherwise specified in this application, all are mass percentages.

[0033] Further preferably, the mass ratio of the microcapsule suspending seed coating agent for controlling clubroot of rape to the mass of the seeds is 1:25. When diluting, the volume of water is 2.5 times the volume of the microcapsule suspending seed coating agent for controlling clubroot of rape. After dilution, the actual content of the fluazinam technical is 2.2%.

[0034] This application develops a sustained - release formulation of fluazinam technical and develops it into a suspending seed coating agent to achieve simultaneous application of seeds and pesticides in the field. It can not only greatly extend the pesticide persistence period, reduce the dosage, and lower the application cost, but also greatly improve its control effect on clubroot.

[0035] Technologies not mentioned in the present invention refer to the prior art.

[0036] The microcapsule suspension seed coating agent for controlling clubroot of rape in the present invention has an encapsulation rate of over 98%, small microcapsule particle size, good morphology, no adhesion, normal temperature stability of more than 2 years, good thermal stability, and good slow-release effect. After coating seeds with it, compared with the non-coated treatment (CK), the seed germination potential and vigor index are increased by 18.96% and 14.32% respectively, the disease control effect at 10 weeks reaches 98.55% (the control is 0), and the yield per plant is increased by 36.15%. Compared with the non-slow-release fluazinam suspension seed coating agent treatment (SC), the seed germination potential and vigor index are increased by 31.12% and 224.47% respectively, the disease control effect is increased by 396.97%, and the yield per plant is increased by 279.72%. Moreover, the dosage is small and the duration of efficacy is long. Description of the Drawings

[0037] Figure 1 It is the preparation flow chart of the microcapsule suspension seed coating agent for controlling clubroot of rape in the present invention;

[0038] Figure 2 It is the microscopic observation diagram of emulsions obtained with different emulsifiers under a 1000-fold microscope; among them, a corresponds to agricultural emulsifier 600-3, b corresponds to Tween-80, and c corresponds to AEO-9;

[0039] Figure 3 It is the influence of different reaction temperatures on the encapsulation rate of microcapsules;

[0040] Figure 4 It is the scanning electron microscope diagram of the microcapsule suspension seed coating agent for controlling clubroot of rape in the present invention; among them, the solvent used in A is ethyl acetate, and the solvent used in B is cyclohexanone;

[0041] Figure 5 It is the particle size distribution diagram of the microcapsule suspension seed coating agent for controlling clubroot of rape in the present invention;

[0042] Figure 6 It is the infrared spectrum diagram of fluazinam technical, fluazinam microcapsules and isocyanate; among them, a is fluazinam technical, b is fluazinam microcapsules, and c is isocyanate;

[0043] Figure 7 It is the thermogravimetric analysis curve diagram of fluazinam technical, empty microcapsules and drug-loaded microcapsules; among them, a is the thermogravimetric analysis curve of fluazinam technical, b is the thermogravimetric analysis curve of empty microcapsules, and c is the thermogravimetric analysis curve of drug-loaded microcapsules;

[0044] Figure 8 It is the high performance liquid chromatography standard curve of fluazinam;

[0045] Figure 9 It is the slow-release curve of fluazinam microcapsules in the present invention;

[0046] Figure 10Scanning electron microscopy image of rapeseed seeds coated with fluazinam microcapsule suspension seed coating agent Detailed implementation mode

[0047] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with embodiments. However, the content of the present invention is not limited to the following embodiments only.

[0048] Unless otherwise specified in this application, all operations are carried out at room temperature (21 - 25 °C).

[0049] Example 1

[0050] Preparation of microcapsule suspension seed coating agent for controlling clubroot of rapeseed:

[0051] As Figure 1 shown, first, 2.75 g of fluazinam technical (purity 97%, purchased from Changshu Hengyao New Materials Co., Ltd.), 2.5 g of 4,4`-diphenylmethane diisocyanate (MDI), 3 g of fatty alcohol polyoxyethylene ether (AEO-9) and 8.5 g of cyclohexanone were fully mixed to prepare an organic solution; then this organic solution was dropped into 32.45 g of the aqueous phase at a rate of 60 drops / min and stirred on a magnetic stirrer at 2000 rpm for 30 min to form a stable O / W emulsion; then, the rotation speed was adjusted to 400 rpm, and an aqueous solution of 0.8 g of diethylenetriamine (mass concentration 30%) was dropped, and the dropping was completed within 10 min; the above solution was transferred to a magnetic stirrer water bath at a rotation speed of 400 rpm, the temperature was 50 °C, and MDI and the amine reacted at the oil-water interface for 1 h to obtain a 5.5% fluazinam microcapsule suspension. Further, 1 g of sodium carboxymethyl cellulose (CMC-Na) and 0.5 g of a colorant were added to the microcapsule suspension and mixed well to prepare a 5.5% fluazinam microcapsule suspension seed coating agent.

[0052] Comparative example 1

[0053] Different solvents were used to replace cyclohexanone in Example 1, and the rest were all referred to Example 1. The results of the fluazinam microcapsule suspension seed coating agents obtained with different solvents are shown in Table 1.

[0054] Table 1 Influence of different solvents on the emulsification results

[0055]

[0056]

[0057] Comparative example 2

[0058] Different emulsifiers were used to replace fatty alcohol polyoxyethylene ether (AEO-9) in Example 1, and the rest were all referred to Example 1. The results of the fluazinam microcapsule suspension seed coating agents obtained with different emulsifiers are shown in Table 2.

[0059] Table 2 Influence of Different Emulsifiers on Emulsification Results

[0060]

[0061] The experimental results show that when agricultural emulsifier 500 and Span - 80 are used, the emulsifying ability of the two emulsifiers is poor, and the stability of the formed oil - in - water emulsion is poor. When agricultural emulsifier 600 - 3 is used, the formed microcapsule has a larger particle size and is more adhesive( Figure 2 a). When Tween - 80 is used, although the formed microcapsules are not adhesive, the particle size is relatively large( Figure 2 b). When AEO - 9 is used, the particle size of the prepared microcapsules is significantly reduced, and the microcapsules are evenly distributed in water without aggregation( Figure 2 c).

[0062] Comparative Example 3

[0063] In Example 1, the reaction temperature is 50°C and the time is 1 h. In this example, the temperature is replaced with 40°C or 60°C respectively, and the rest refers to Example 1. The results are shown in Table 3

[0064] Table 3 Influence of Different Reaction Temperatures on the Surface Morphology of Microcapsules

[0065]

[0066] In Table 3, drying means that after preparing the 5.5% fluazinam microcapsule suspension, without adding sodium carboxymethylcellulose (CMC - Na) and colorants, directly centrifuging and drying the 5.5% fluazinam microcapsule suspension (drying to constant weight at 50°C).

[0067] The inventors found that during the formation of microcapsules, the reaction temperature affects the strength of the microcapsule wall material formation, and the strength of the wall material directly affects the appearance morphology and encapsulation rate of the microcapsules. Insufficient strength of the wall material will cause the microcapsule wall to be easily broken, greatly reducing the sustained - release effect of the microcapsules, or even unable to form microcapsules, resulting in a decrease in the microcapsule encapsulation rate. While too high strength will lead to a thicker wall, and fluazinam cannot be released smoothly, thus affecting the drug efficacy

[0068] From Table 3 and Figure 3It can be seen that the appearance morphology of the microcapsules after reacting for 1 h at different reaction temperatures and the encapsulation efficiency at the corresponding temperatures. When the reaction temperature is 50 °C, the surface of the formed microcapsules is smooth and there is no adhesion phenomenon. At this time, the encapsulation efficiency of the prepared microcapsules is the highest, reaching 98.78%. When the temperature is 40 °C, partial solvent precipitation can be observed and the microcapsules are adhered to each other. This is because the degree of microcapsule reaction is relatively low, making them prone to breakage, and some oil-in-water emulsions are not encapsulated by the capsule wall, ultimately resulting in a poor encapsulation efficiency of only 72.26%. When the temperature is 60 °C, the polymerization rate of the wall material is relatively fast, causing severe depressions on the surface of the microcapsules, resulting in a decrease in the encapsulation efficiency of the microcapsules to 71.54%.

[0069] Comparative Example 4

[0070] The difference from Example 1 is that the dropping step of the diethylenetriamine aqueous solution is omitted, and the rest are all referred to Example 1, and microcapsules cannot be formed. The inventor believes that the reason is that diethylenetriamine and 4,4'-diphenylmethane diisocyanate are two raw materials used to chemically react to form the microcapsule wall material. Among them, 4,4'-diphenylmethane diisocyanate is dissolved in the oil phase, and diethylenetriamine is dissolved in the water phase. When the two meet, a polymer containing multiple urea bonds is formed, which is the wall material of the microcapsules. If one of the components is removed or replaced, microcapsules cannot be formed.

[0071] Comparative Example 5

[0072] The difference from Example 1 is that diethylenetriamine is replaced by ethylenediamine, and the rest are all referred to Example 1, and microcapsules cannot be formed.

[0073] Comparative Example 6

[0074] The difference from Example 1 is that 4,4'-diphenylmethane diisocyanate is replaced by isophorone diisocyanate, and the rest are all referred to Example 1, and microcapsules cannot be formed.

[0075] Example 2

[0076] The 5.5% fluazinam microcapsule suspending seed coating agent prepared in Example 1 was tested as follows:

[0077] (1) Scanning electron microscopy of microcapsules:

[0078] Figure 4 It is the morphology diagram of the microcapsules characterized by SME (A: ethyl acetate solvent, B: cyclohexanone solvent). It can be seen from the figure that the microcapsules prepared with ethyl acetate as the solvent have larger particle sizes, uneven distribution, rough surfaces with pores, and the microcapsules are adhered to each other. The microcapsules prepared with cyclohexanone as the solvent have small and uniform particle sizes, smooth surfaces, and no obvious adhesion. Therefore, cyclohexanone is more suitable as a solvent for preparing fluazinam microcapsules.

[0079] (2) Particle size analysis:

[0080] Figure 5 It is the particle size distribution diagram of the microcapsules prepared in Example 1. As can be seen from the figure, the particle size of the microcapsules conforms to the normal distribution, and the average particle size of the microcapsules is 0.516 μm.

[0081] (3) Infrared characterization of microcapsules:

[0082] Figure 6 (a-c) are the infrared spectra of the original fluazinam, fluazinam microcapsules, and isocyanate, respectively. In curve a, absorption peaks appear at 1502.96 cm-1, 1546.15 cm-1, and 1600.46 cm-1, which may be attributed to the skeletal vibration of the benzene ring. And the strong absorption peak at 3389.35 cm-1 is the stretching vibration peak of the N-H bond. In curve c, the characteristic absorption peak of isocyanate -NCO is observed at 2274 cm-1. In curve b, the characteristic absorption peak of the isocyanate group at 2274 cm-1 disappears, indicating that the modified isocyanate has completely reacted with the amine to form a polyurea shell. In addition, the characteristic absorption peak of the original fluazinam can be observed in curve b. Therefore, it shows that the above pesticides have been successfully encapsulated by the microcapsule shell.

[0083] (4) Thermogravimetric characterization of microcapsules:

[0084] Figure 7(a-c) are the thermogravimetric analysis curves of fluazinam technical (a), empty microcapsules (b, compared with Example 1, only the fluazinam technical was omitted, and the rest was referred to Example 1), and drug-loaded microcapsules (c, prepared in Example 1), respectively. It can be seen from the figure that the weight of each sample decreases with the increase of temperature. It can be seen from Figure a that fluazinam has a large weight loss in the range of 0-313.18 °C, accounting for about 93.53% of the total sample amount. This is due to the thermal decomposition of fluazinam technical, which also indicates that fluazinam is easily decomposed at high temperatures. It can be seen from Figure b that the cracking process of empty microcapsules can be divided into three stages. In the first stage, within 267.01 °C, the weight loss reaches 2.86%, which is speculated to be the moisture in the preparation. In the second stage, between 276.01-431.76 °C, the weight loss reaches 58.29%, which is speculated to be the rapid decomposition of the blank microcapsules. In the third stage, between 431.76-596.26 °C, the weight loss is 38.65%, which may be the continuous decomposition of the blank microcapsules and the further cracking process of other residues in the preparation. It can be seen from Figure c that the degradation process of drug-loaded microcapsules is similar to that of blank microcapsules, also in three stages. Below 156.92 °C, 1.34% of the total sample amount is lost, which may be the thermal evaporation of the moisture in the preparation. The second stage is between 156.92-471.42 °C, and the weight loss is 60.96%, which is caused by the decomposition of the parent drug and microcapsules. The 35.51% weight loss between 471.42-612.67 °C is also caused by the decomposition of the microcapsules and residues. The fluazinam microcapsule suspension prepared by the interfacial polymerization method has good thermal stability and has good thermal stability below 260 °C. At the same time, it is also verified that the fluazinam technical has been encapsulated in the microcapsules.

[0085] (5) Determination of encapsulation efficiency:

[0086] The effective content of fluazinam was determined by high performance liquid chromatography. After optimizing the liquid chromatography conditions, the chromatographic conditions for this experiment were obtained as follows: C18 stainless steel column (150 mm × 4.6 mm); mobile phase: 85% methanol: 15% water; flow rate: 1.0 ml / min; column temperature: 30 °C; detection wavelength: 240 nm; injection volume: 5 μl.

[0087] The high performance liquid chromatography standard curve of fluazinam is as Figure 8 shown. Its linear regression equation is y = 8.7307x - 69.667, and the correlation coefficient is 0.9993, showing good linear correlation and meeting the requirements of the next experiment.

[0088] The encapsulation efficiency of the microcapsules was determined by using a method of extracting external active ingredients with a solvent. After centrifuging the prepared fludioxonil microcapsule suspension seed coating agent, the volume of the supernatant was measured. 1 mL of the supernatant was passed through a 0.22-μm organic membrane for liquid phase analysis, with three replicates in total. The measured encapsulation efficiency was 98.78%. The encapsulation efficiency of the microcapsules prepared in Example 3 was determined by the same method to be 81.94%.

[0089] (6) Determination of sustained-release performance:

[0090] The 5.5% fludioxonil microcapsule suspension obtained in Example 1 was centrifuged at a speed of 8000 rpm for 10 min to obtain a precipitate. The supernatant was poured off, and the precipitate was placed in a vacuum dryer and dried at 50 °C to a constant weight to obtain fludioxonil microcapsule powder for the determination of sustained-release performance.

[0091] 1 g of the fludioxonil microcapsule powder was weighed and placed in a bag. During the rape sowing period, the bag was buried in the rape experimental field (simultaneously with the rape seeds), with a burial depth of 1 cm (the same as the sowing depth of the rape seeds). One dialysis bag was taken every 10 days (d), and sampling was completed after 70 d, with a total of 7 samplings. The fludioxonil content in the microcapsule samples in the bag was determined by liquid chromatography.

[0092] Figure 9 The left figure in the middle is the 5.5% fludioxonil microcapsule suspension seed coating agent prepared in Example 1, and the right figure is the sustained-release curve of the 5.5% fludioxonil microcapsule suspension seed coating agent prepared in Example 1. It can be seen from Figure 9 that: after 30 d of release, the cumulative release amount of the microcapsules reached 53.6%, and this period was a process of burst release; then, with the increase of time, it was slowly released. After 50 d of release, it was rapidly released for a period of time, and the cumulative release amount reached 72.3%; finally, after 70 d of release, the cumulative release amount reached 92.5%. The sustained-release performance of the microcapsules prepared in Example 3 was determined by the same method, and the sustained-release curve was as shown in Figure 9 , and the cumulative release amount reached 72.6% after 20 d of release and 96.89% after 40 d of release.

[0093] (7) Seed coating:

[0094] The seeds of the Brassica napus variety Qianyou 32 were first disinfected, and the prepared microcapsule suspension seed coating agent was used to coat the rape seeds. Coating was carried out at the drug-seed ratios of 1:10, 1:25, 1:50, and 1:100 (liquid volume mL: seed mass g) respectively. The coated seeds were naturally dried, with water as the blank control (CK). Specific coating method: Weigh 50 g of seeds and put them into a petri dish, then add the corresponding volume of the liquid medicine according to the drug-seed ratio. Finally, make up the total volume of the liquid to 5 mL with water, and then hold the petri dish with hand and rotate it clockwise until the medicine and the seeds are fully mixed evenly and the inner wall of the petri dish is clean without medicine, and dry it in a cool place.

[0095] Figure 10 The SEM images of rapeseed seeds coated with the fluazinam microcapsule suspension seed coating agent obtained in Example 1 at a drug-seed ratio of 1:25 are shown. The pictures from left to right are at different magnifications. It can be seen from the figure that many microcapsules containing the active ingredient of fluazinam are successfully coated on the seed surface.

[0096] Example 3

[0097] Ethyl acetate was used to replace cyclohexanone in Example 1, and the rest was the same as in Example 1, coded as YCS.

[0098] Comparative Example 7

[0099] A fluazinam suspension seed coating agent without microcapsule coating, that is, the addition of 4,4'-diphenylmethane diisocyanate and diethylenetriamine in Example 1 was omitted, and the rest was the same as in Example 1, coded as SC.

[0100] Comparative Example 8

[0101] A suspension seed coating agent was prepared with empty microcapsules without fluazinam coating, that is, the addition of fluazinam technical in Example 1 was omitted, and the rest was the same as in Example 1, with a fluazinam content of 0%, coded as KN.

[0102] Example 4

[0103] Application effect evaluation of the 5.5% fluazinam microcapsule suspension seed coating agent prepared in Example 1 on the vigor of rapeseed seeds, the control effect of clubroot disease and yield traits.

[0104] (1) Evaluation method

[0105] 1) Determination of seed vigor index:

[0106] A seed germination experiment was conducted on a total of 17 treatments, namely the control CK (clean water), Example 1 (including four ratios of four medicinal seeds: 1:10, 1:25, 1:50, 1:100), Example 3 (including four ratios of four medicinal seeds: 1:10, 1:25, 1:50, 1:100), Comparative Example 7 (including four ratios of four medicinal seeds: 1:10, 1:25, 1:50, 1:100), and Comparative Example 8 (including four ratios of four medicinal seeds: 1:10, 1:25, 1:50, 1:100), to evaluate seed vigor. Specific method: ① Evenly place rapeseed seeds in a culture dish (90 mm) lined with double-layer filter paper, and conduct moist cultivation in a constant temperature incubator at a temperature of 25 °C, a light time of 16 h / 8 h / d (light / dark / day), and a light intensity of 120 μmol / m2 / s; ② Set 3 replicates for each treatment, with 100 seeds in each replicate (observe carefully during the cultivation process, supplement distilled water in a timely manner to keep the filter paper moist), and define the radicle breaking through the seed coat and reaching a length of 1-2 mm (showing white) as the sign of seed germination; ③ After sowing, record data every 8 h until the seed germination situation no longer changes. On the 7th day after sowing, randomly select 5 germinated seeds from each replicate to record the root length.

[0107] Seed germination potential = number of germinated seeds at 24 h after sowing / number of tested seeds × 100%.

[0108] Seed germination rate = number of germinated seeds on the 7th day after sowing / number of tested seeds × 100%.

[0109] Germination index = ∑(Gt / Dt), where Dt is the number of days of germination and Gt is the number of germinated seeds on the current day.

[0110] Seed vigor index V = GI × S

[0111] Gt is the number of germinated seeds at time t, and Dt is the corresponding number of days of germination;

[0112] GI is the germination index, and S is the root length (mm).

[0113] 2) Determination of yield traits:

[0114] Sow a total of 17 treatments, namely the control CK, Example 1 (including four ratios of four medicinal seeds), Example 3 (including four ratios of four medicinal seeds), Comparative Example 7 (including four ratios of four medicinal seeds), and Comparative Example 8 (including four ratios of four medicinal seeds), in a field without clubroot disease occurrence, and measure the yield-related traits at the yellow ripening stage of the siliques. Set 3 replicates for each treatment, plant 1 plot for each replicate, with a total of 51 plots, and the area of each plot is 20 m 2(5 m in length and 4 m in width), a randomized block experimental design was adopted, and half-row protective rows were planted around the experimental field. One week before rapeseed harvest, in each plot, the five-point sampling method was used, and 5 plants were taken from each sampling point for the number of siliques per plant, the number of seeds per silique, 1000-grain weight, and the seed yield per plant. The specific measurement methods refer to the methods in "Analysis of Agronomic Trait Variation and Correlation in Brassica napus Germplasm Populations": ① The number of siliques per plant: the number of effective siliques on the whole plant, in units of "pcs"; ② The number of seeds per silique: randomly take 50 siliques from the upper, middle, and lower parts of the pod-bearing layer of the plant, count the number of seeds in each silique, and calculate the average value as the number of seeds per silique, in units of "pcs"; ③ 1000-grain weight: dry the seeds of the whole rapeseed plant to a constant weight, randomly weigh the weights of three portions of seeds using an electronic balance with a precision of one-thousandth, and count the number of seeds in each seed sample using a seed analyzer (Hengmei HM-KZ01), and calculate the 1000-grain weight.

[0115] 3) Determination of the control effect against clubroot

[0116] a. Preparation of the resting spore suspension of Plasmodiophora brassicae

[0117] Refer to the sucrose extraction method in "Morphology of Plasmodiophora brassicae and Biological Characteristics of Resting Spores" to prepare the resting spore suspension of Plasmodiophora brassicae, with slight modifications:

[0118] ① Weigh 10 g of clubroot tissue that has rotted for 1 week under natural conditions, add 50 ml of sterile water, and homogenize it with a tissue homogenizer (JJ-2B). After filtering with gauze, transfer the filtrate into a 50 mL sterilized centrifuge tube;

[0119] ② Centrifuge at 500 r / min for 5 min; take the supernatant and the upper gray solution;

[0120] ③ Centrifuge at 3100 r / min for 15 min; discard the supernatant;

[0121] ④ Re-suspend the precipitate with 30 mL of sterile water, centrifuge at 3100 r / min for 10 min, and discard the supernatant (this step is repeated 2 - 3 times);

[0122] ⑤ Suspend the precipitate with 5 mL of 50% sucrose solution, shake well, and centrifuge at 3100 r / min for 10 min;

[0123] ⑥ Transfer the supernatant into a sterilized centrifuge tube, add 30 mL of sterile water, centrifuge at 3100 r / min for 10 min, and discard the supernatant;

[0124] ⑦ Re-suspend the precipitate in 30 mL of sterile water, mix well, centrifuge at 3100 min for 10 min, and discard the supernatant (this step is repeated 1 - 2 times);

[0125] ⑧ Add distilled water to the prepared resting spore suspension, and use a hemocytometer to adjust the concentration of the resting spore suspension to 1x109 per mL of resting spores, to be prepared and used immediately.

[0126] b. Pot experiment with Plasmodiophora brassicae by mixing with soil

[0127] Weigh 2 kg of soil sample for each nutrient bowl, pour 2 mL of resting spore suspension with a concentration of 1x10 9 per mL evenly into the soil sample, and mix well to make the concentration of resting spores in the soil 1x10 6 per gram of soil sample (far greater than the spore concentration of 1.34×10 3 per gram of soil, which indicates a high risk of clubroot disease shown in "Establishment of a quantitative detection method for Plasmodiophora brassicae in soil and investigation of clubroot disease of rapeseed in Changde City"). Sow a total of 17 treatments including control CK, Example 1 (with four drug-seed ratios), Example 3 (with four drug-seed ratios), Comparative Example 7 (with four drug-seed ratios), and Comparative Example 9 (with four drug-seed ratios) into the pot soil. Each treatment has 3 replicates, and each replicate plants 30 plants. 70 days after sowing, count the disease incidence of rapeseed seedlings, and calculate the control effect of each treatment. The grading standard for clubroot disease at the seedling stage is slightly modified with reference to "Research on the control technology of clubroot disease of rapeseed in the Jianghan Plain area".

[0128] c. Disease investigation

[0129] ① The grading standard for clubroot disease is as follows:

[0130] Grade 0: Normal roots, without any tumors;

[0131] Grade 1: No tumor on the main root, tumors formed on the lateral roots;

[0132] Grade 2: Tumors begin to form on the main root, and the diameter of the tumor is less than 2 times the diameter of the stem base;

[0133] Grade 3: The main root is swollen, and the diameter of its tumor is 2 - 3 times the diameter of the stem base.

[0134] ② The calculation formulas for disease-related trait indicators are as follows:

[0135]

[0136]

[0137] (2) Evaluation results

[0138] 1) Influence on seed vigor

[0139] The fludioxonil microcapsule suspension seed coating agents prepared in Example 1 and Example 3 were used to coat rapeseed seeds at the drug-seed ratios of 1:10, 1:25, 1:50, and 1:100 (liquid volume ml: seed mass g), respectively, and the non-coated treatment (CK) was used as the control. The results showed that the germination potential of the control (CK) was 64.67%, the germination rate was 94.67%, the germination index was 5.83, the root length was 5.75 cm, and the vigor index was 33.52, showing a medium seed vigor level overall (Table a).

[0140] Example 1 (HCS1:25) showed the best performance among all treatments. The germination potential (76.93%) was increased by 18.96% compared with CK, the germination rate (98.16%) and the germination index (6.63) reached the highest values, the root length (5.78 cm) was close to that of CK, and the vigor index (38.32) was increased by 14.3% (Table a, b). Its germination potential, germination rate, and vigor index were significantly better than those of Example 3 and all comparative examples, demonstrating a comprehensive ability to promote seed germination and root growth.

[0141] The germination potential of Example 3 (YCS1:25) was 68.51%, and the germination rate was 92.21%, both of which were significantly lower than those of HCS1:25; the root length (3.91 cm) and the vigor index (22.01) were decreased by 47.83% and 74.10% respectively compared with HCS1:25 (Table a, b), indicating that its effect on improving seed vigor was inferior to that of Example 1.

[0142] Comparative Example 7 (SC 1:25) showed the worst seed vigor performance, with a root length of only 2.12 cm (decreased by 172.64% compared with HCS1:25), a vigor index of 11.81 (decreased by 224.47% compared with HCS1:25), and the germination potential (58.67%) and the germination index (5.57) were also significantly lower than those of Example 1 (Table a, b), indicating that this treatment had an obvious inhibitory effect on seed vigor.

[0143] The germination potential (44.67%) and the germination rate (89.33%) of Comparative Example 8 (KN 1:25) were the lowest among all treatments, and the vigor index was 25.72 (lower than 33.52 of CK and decreased by 48.99% compared with HCS1:25) (Table a, b), indicating that it not only could not improve seed vigor but might even inhibit the germination process.

[0144] In summary, Example 1 (HCS1:25) showed the best performance in all seed vigor indexes, and its germination potential, germination rate, and vigor index were significantly higher than those of the control (CK), Example 3, and the comparative examples (YCS, SC, KN). SC 1:25 and YCS 1:25 were significantly weaker than HCS1:25 in terms of root length and vigor index, and KN 1:25 had the worst seed vigor.

[0145] 2) Control effect on diseases

[0146] Coat seeds with the fluazinam microcapsule suspension seed coating agent of the present invention, and investigate the disease incidence of plants 70 days after sowing. The results show that the incidence rate and disease index of the control (CK) are both close to 100%, the disease control effect is 0%, and there is no disease resistance ability at all (Table a). The disease prevention and control effect of Example 1 (HCS1:25) is outstanding. The incidence rate (1.42%) and disease index (1.42%) are the lowest among all treatments, and the disease control effect is as high as 98.55%. Its control effect is significantly better than that of Example 3 (YCS1:25: 58.18%) and Comparative Example 7 (SC 1:25: 19.83%) (Table a), and completely avoids the high infection risk of the control.

[0147] For Example 3 (YCS1:25), the incidence rate is 41.03%, the disease index is 41.03%, and the disease control effect is only 58.18%. The prevention and control ability is reduced by 69.39% compared with HCS1:25 (Tables a, b), indicating that its disease resistance mechanism has limited effect.

[0148] The disease control effect of Comparative Example 7 (SC 1:25) is extremely low (19.83%), the incidence rate is 78.66%, and the disease index is 78.66% (Table a), indicating that this treatment can hardly inhibit the development of the disease and may even aggravate the disease condition.

[0149] For Comparative Example 8 (KN 1:25), the incidence rate and disease index are both close to the CK level (99.63% and 99.3%), and the disease control effect is only 0.03% (Table a), indicating that it has no disease resistance effect at all.

[0150] In summary, Example 1 (HCS1:25) shows an absolute advantage in disease prevention and control, with a control effect as high as 98.55%, which is significantly better than Example 3, Comparative Examples (YCS, SC) and the control (CK). The control effects of YCS1:25 and SC1:25 are less than 60% and 20% respectively, and KN 1:25 has no disease resistance ability at all.

[0151] 3) Influence on yield traits

[0152] Coat seeds with the fluazinam microcapsule suspension seed coating agent of the present invention, and in the test area without clubroot disease, it can significantly increase the number of siliques per plant, the number of grains per silique and the grain yield per plant (Tables a, b). The yield per plant of the control (CK) is 47.86 g, the number of siliques is 418.0, the number of grains per silique is 26.2, and the 1000-grain weight is 4.37 g, which is the benchmark yield level.

[0153] The comprehensive yield index of Example 1 (HCS1:25) performed the best. The yield per plant (65.16 g) was 36.15% higher than that of the CK. The number of siliques per plant (465.8) and the number of seeds per silique (33.2) were both higher than those of the control and comparative examples, and the 1000-seed weight (4.21 g) was slightly lower than that of the CK but significantly higher than that of the comparative example (Tables a, b). Its high-yield advantage mainly stems from the significant increase in the number of siliques and the number of seeds per silique.

[0154] The yield per plant (38.87 g) and the number of siliques per plant (396.3) of Example 3 (YCS1:25) were both significantly lower than those of HCS1:25 (decreased by 40.4% and 14.9% respectively), and the 1000-seed weight (3.83 g) was the lowest among all treatments (Tables a, b), indicating that this treatment had no promoting effect on yield improvement.

[0155] The yield performance of Comparative Example 7 (SC 1:25) was the worst. The yield per plant was only 17.16 g (279.72% lower than that of HCS1:25), the number of siliques per plant (257.4) and the number of seeds per silique (20.2) were extremely low, and the 1000-seed weight was 3.30 g (Tables a, b), showing that it severely inhibited the yield formation of the crop.

[0156] The yield per plant (49.53 g) of Comparative Example 8 (KN 1:25) was close to that of the CK. The number of siliques per plant (422.3) and the number of seeds per silique (26.9) were slightly higher than those of the CK but still lower than those of HCS1:25 (Tables a, b), indicating that its yield improvement ability was limited and it could not break through the control level.

[0157] In summary, Example 1 (HCS1:25) was significantly higher than the control and comparative examples in terms of the number of siliques per plant (465.8), the number of seeds per silique (33.2), and the yield per plant (65.16 g), and had the best comprehensive yield. The yield indexes of YCS1:25 and SC1:25 were significantly behind, and KN1:25 was only close to the CK level.

[0158] In conclusion, after coating the seeds of Brassica napus with the fludioxonil microcapsule suspension seed coating agent of the present invention, the seed vigor, disease control effect, and yield traits were all significantly improved compared with the control and comparative examples. Example 1 (HCS1:25) (fludioxonil content 2.2%) showed the best effects on these three traits. The germination potential, germination rate, and vigor index of the seeds were all improved comprehensively, promoting germination and root growth. The incidence rate (1.42%) and disease index (1.42%) were the lowest, and the control effect was close to 99%. The number of siliques per plant, the number of seeds per silique, and the yield per plant were all significantly higher than those of the control and all comparative examples. Therefore, HCS1:25 is the optimal treatment plan, with high vigor, strong disease resistance, and high-yield characteristics, and is suitable as the first choice for practical applications.

[0159]

[0160]

Claims

1. A microcapsule suspension seed coating agent for preventing and treating rape clubroot, characterized in that: The fluazinam original drug is coated by the reaction of the -NCO group of diisocyanamide and the -NH2 group of organic amine at the water-in-oil (O / W) interface.

2. The microcapsule suspension seed coating agent for preventing and treating rape clubroot according to claim 1, characterized in that: The raw materials used include: fluazinam technical, diisocyanate, fatty alcohol polyoxyethylene ether and organic amine, wherein the mass ratio of fluazinam technical, diisocyanate, fatty alcohol polyoxyethylene ether and organic amine is: (2.5-3): (2.25-2.75): (2.75-3.25): (0.1-0.4).

3. The microcapsule suspension seed coating agent for preventing and treating rape clubroot according to claim 1 or 2, characterized in that: The mass content of fluazinam is 5-6%.

4. The microcapsule suspension seed coating agent for preventing and treating rape clubroot according to claim 1 or 2, characterized in that: Contains organic solvent and water in a mass ratio of 1:(3.5-4).

5. The microcapsule suspension seed coating agent for preventing and treating rape clubroot according to claim 4, characterized in that: The organic solvent is cyclohexanone or ethyl acetate.

6. The microcapsule suspension seed coating agent for preventing and treating rape clubroot according to claim 5, characterized in that: The organic solvent is cyclohexanone.

7. The microcapsule suspension seed coating agent for preventing and treating rape clubroot according to claim 2, characterized in that: The organic amine is diethylenetriamine; and the diisocyanate is 4,4'-diphenylmethane diisocyanate.

8. A method for preparing the microcapsule suspension seed coating agent for preventing and treating rape clubroot according to any one of claims 1 to 7, characterized in that: The steps include: 1) fully mixing the fluazinam technical, diisocyanate, fatty alcohol polyoxyethylene ether and organic solvent to prepare an organic solution; 2) dropping the organic solution obtained in step 1) into water, and stirring on a magnetic stirrer at 1800-2200 rpm for 25-30 min to form a stable O / W emulsion; 3) adjusting the rotation speed to 300-500 rpm, adding the aqueous solution of the organic amine dropwise within 8-10 min, and then reacting at 300-500 rpm and 50±2° C. for 0.5-1 h to obtain a fluazinam microcapsule suspension; 4) centrifuging the fluazinam microcapsule suspension prepared in step 3), and drying the obtained precipitate to obtain fluazinam microcapsule powder; 5) adding sodium carboxymethyl cellulose and a colorant to the fluazinam microcapsule suspension prepared in step 3), and mixing thoroughly to prepare a fluazinam microcapsule suspension seed coating agent.

9. The method for preparing the microcapsule suspension seed coating agent for preventing and treating rape clubroot according to claim 8, characterized in that: In step 4), the amount of sodium carboxymethyl cellulose added is 0.5-2% of the mass of the microcapsule suspension.

10. Use of the microcapsule suspension seed coating agent for preventing and treating rape clubroot according to any one of claims 1 to 7, characterized in that: Used for preventing and controlling clubroot disease of rapeseed; when in use, the microcapsule suspension seed coating agent for preventing and controlling clubroot disease of rapeseed is diluted with water and mixed with seeds, the mass ratio of the microcapsule suspension seed coating agent for preventing and controlling clubroot disease of rapeseed to the mass ratio of seeds is 1:(10-30), and the mass content of fluazinam original drug in the microcapsule suspension seed coating agent after dilution is 1.5-2.5%.

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