Trehalose polymer, preparation method thereof and application of trehalose polymer in improvement of stress tolerance of metarhizium anisopliae

Abstract

The invention discloses a trehalose polymer as well as a preparation method and application thereof. The structural formula of the trehalose polymer is as follows: trithioester on a main chain of the trehalose polymer can absorb ultraviolet rays; the hydrophobic carbon chain on the main chain of the polymer and the hydrophobic chain dodecyl acrylate on the side chain of the polymer can be embedded on the surface of metarhizium robertii conidia through hydrophilic and hydrophobic interaction, the polymer has the capability of protecting the conidia against ultraviolet stress, and the hydrophilic chain trehalose can reduce the influence of heat stress on the conidia. In practical application of metarhizium anisopliae, the trehalose polymer material is attached to the surfaces of metarhizium anisopliae spores, and a protective shell is formed on the surfaces of pathogenic unit conidia of the metarhizium anisopliae spores, so that the heat resistance and ultraviolet resistance of the metarhizium anisopliae are improved, and the problem that the metarhizium anisopliae is limited by high temperature and ultraviolet stress during field pest control is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pest control, and particularly relates to a trehalose polymer, a preparation method thereof and application of the trehalose polymer in improving stress tolerance of Metarhizium. BACKGROUND

[0003] Biological control using entomopathogens such as fungi, bacteria, viruses and nematodes has been proven to be one of the most promising pest management due to its environmental friendliness and safety. For example, Metarhizium, an entomopathogenic fungus used as a biological pesticide for centuries, has become a classic model for studying the interaction between fungi and insects to achieve industrial application of fungi. Conidia are the infectious units of entomopathogenic fungi, and during the process of fungal reproduction in insects, conidia can inherently adhere to the surface of insects and then enter the insect cuticle to kill the host.

[0004] However, the extensive use of conidia in the wild has been greatly limited due to the damage caused by ultraviolet light and heat stress to the conidia. Therefore, improving the heat tolerance and ultraviolet tolerance of fungi is crucial for their survival in various environments. SUMMARY

[0005] The technical problem to be solved by the present application is how to improve the high-temperature tolerance and ultraviolet stress tolerance of Metarhizium.

[0006] The present application solves the above technical problems by the following technical means:

[0007] A trehalose polymer has the following structural formula:

[0008]

[0009] In the structural formula of the trehalose polymer, "stat" represents random copolymerization.

[0010] The present application further provides a preparation method of the trehalose polymer, comprising the following steps:

[0011] S1, reacting (2-hydroxyethyl) benzyl trithiocarbonate and tetradecanoic acid in the presence of 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to obtain 2-(((benzylthio)carbonothioyl)thio) tetradecanoic acid ethyl ester;

[0012] S2, using 2-(((benzylthio)carbonothioyl)thio) tetradecanoic acid ethyl ester as a chain transfer agent, and using trehalose propenoate propenoate and dodecyl acrylate as monomers, and after freeze-thaw cycles, performing a polymerization reaction under a nitrogen environment initiated by an initiator to obtain the trehalose polymer.

[0013] Preferably, in S1, the molar ratio of the benzyl (2-hydroxyethyl)trithiocarbonat, tetradecanoic acid is 1:1.5.

[0014] Preferably, in S1, the molar ratio of the benzyl (2-hydroxyethyl)trithiocarbonat, 4-dimethylaminopyridine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1:0.5:1.

[0015] Preferably, in S2, the molar ratio of the 2-(((benzylthio)carbonothioyl)thio)ethyl tetradecanoate, trehalose acrylate, dodecyl acrylate is 1:20:5.

[0016] Preferably, in S2, the molar ratio of the 2-(((benzylthio)carbonothioyl)thio)ethyl tetradecanoate, initiator is 1:0.1-0.2.

[0017] Preferably, in S1, the reaction is carried out under reflux condition for 12h; in S2, the temperature of the reaction is 60-70℃, and the time is 12-20h.

[0018] Preferably, the dodecyl acrylate is obtained by reacting dodecanol and acryloyl chloride.

[0019] Preferably, the dodecyl acrylate is obtained by mixing dodecanol, acryloyl chloride, triethylamine and dichloromethane and then reacting at room temperature.

[0020] Preferably, the molar ratio of the dodecanol, acryloyl chloride and triethylamine is 1:1-1.5:1-1.5.

[0021] Preferably, the dodecyl acrylate is obtained by mixing dodecanol, acryloyl chloride, triethylamine and dichloromethane and then reacting at room temperature. The molar ratio of the dodecanol, acryloyl chloride and triethylamine is 1:1-1.5:1-1.5. The triethylamine is used to provide alkaline environment and as catalyst. The acryloyl chloride is added into the dichloromethane solvent containing dodecanol and triethylamine under ice bath, and the reaction is carried out at room temperature for 12 hours. Then the solvent is distilled under reduced pressure, and the dodecyl acrylate monomer LA is obtained by column chromatography separation and purification.

[0022] Preferably, in S1, the benzyl (2-hydroxyethyl)trithiocarbonat, tetradecanoic acid, 4-dimethylaminopyridine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide are mixed with dichloromethane, and the reaction is carried out under reflux to obtain the 2-(((benzylthio)carbonothioyl)thio)ethyl tetradecanoate.

[0023] Preferably, the molar ratio of the benzyl (2-hydroxyethyl)trithiocarbonat, tetradecanoic acid, 4-dimethylaminopyridine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1:1-1.5:0.5-1:0.5-1.

[0024] Preferably, (2-hydroxyethyl) benzyl trithiocarbonate, tetradecanoic acid, 4-dimethylaminopyridine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide are weighed according to a molar ratio of 1:1-1.5:0.5-1:0.5-1, wherein 4-dimethylaminopyridine is used to activate the hydroxyl group and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is used to activate the carboxyl group. The reaction is carried out in dichloromethane at 50°C for 12 hours, then the solvent is distilled under reduced pressure, and then the product is separated and purified by column chromatography to obtain the prepared hydrophobic chain transfer agent 2-(((benzylthio)carbonothioyl)thio) tetradecanoic acid ethyl ester.

[0025] Preferably, in S2, trehalose acrylate, dodecyl acrylate, 2-(((benzylthio)carbonothioyl)thio) tetradecanoic acid ethyl ester, initiator, dimethyl sulfoxide are mixed uniformly, and after freeze-thaw cycles, the polymerization reaction is carried out at 60-70°C under a nitrogen atmosphere to obtain the trehalose polymer.

[0026] Preferably, in S2, after the polymerization reaction, a dialysis treatment is further included, the dialysis bag used for dialysis has a molecular weight cut-off of 3500-4000, the dialysis time is 2-5h, and at least five times of water change is carried out during the dialysis process.

[0027] Preferably, 2-(((benzylthio)carbonothioyl)thio) tetradecanoic acid ethyl ester is used as the chain transfer agent, trehalose acrylate TreA and dodecyl acrylate LA are used as monomers, the chain transfer agent, monomer TreA and monomer LA are dissolved in dimethyl sulfoxide, an initiator is added and mixed uniformly, freeze-thaw cycles are carried out, and the polymerization reaction is carried out at 60-70°C under a nitrogen atmosphere for 12-20h, then the polymerization reaction product is purified by dialysis with a dialysis bag to obtain the purified trehalose polymer PTLA.

[0028] Preferably, the initiator is azobisisobutyronitrile.

[0029] Preferably, the preparation method of the trehalose acrylate comprises the following steps:

[0030] a. 8 hydroxyl groups of trehalose are fully protected using trimethylchlorosilane to obtain T-1; trehalose and trimethylchlorosilane are weighed according to a molar ratio of 1:8-12, added to a pyridine solution and stirred, the reaction is carried out at room temperature for 12 hours, then diluted with ethyl acetate, extracted with pure water for three times, dried with anhydrous Na2SO4, filtered under suction and the solvent is distilled under reduced pressure to obtain the product T-1.

[0031] b. solid potassium carbonate is used as a catalyst to remove one protection of trehalose to obtain T-2; T-1 and solid K2CO3 are weighed according to a molar ratio of 1:0.8-1.2, added to methanol and stirred, the reaction is carried out at 0°C for 18min, then diluted with n-hexane, extracted with pure water for three times, dried with anhydrous Na2SO4, filtered under suction and the solvent is distilled under reduced pressure. The product T-2 is separated and purified by column chromatography.

[0032] c. Acryloyl chloride was added to the de-protected trehalose to obtain T-3. T-2, acryloyl chloride and triethylamine were weighed according to the molar ratio of 1:1-1.5:1-1.5, and added to dichloromethane, and reacted at room temperature for 12 hours. Then the solvent was distilled under reduced pressure, and the product T-3 was obtained by column chromatography separation and purification.

[0033] d. The remaining protection of trehalose was removed to obtain the prepared monomer TreA. T-3 and Amberlyst 15 ion exchange resin were weighed according to the molar ratio of 1:7-12, and added to a mixed solvent of methanol and water (volume ratio of 11:1), and reacted at room temperature for 1 hour. After suction filtration, the obtained filtrate was extracted once with n-hexane, and then the solvent was distilled under reduced pressure. After water membrane, the final product TreA was obtained by freeze-drying.

[0034] The application also provides a use of the trehalose polymer in improving the stress tolerance of Metarhizium.

[0035] Preferably, the stress tolerance is one or both of heat stress tolerance and ultraviolet stress tolerance.

[0036] Preferably, the use of the trehalose polymer in improving the stress tolerance of Metarhizium includes mixing Metarhizium spores with a solution of the trehalose polymer and incubating at room temperature.

[0037] Preferably, after incubation at room temperature, it further includes heating to 39-45℃ or treating with ultraviolet irradiation.

[0038] Preferably, the solution of the trehalose polymer contains a dissolution aid.

[0039] Preferably, the dissolution aid is at least one of Tween 20 and Tween 80.

[0040] Preferably, the concentration of the solution of the trehalose polymer is 0.1-2 mg / mL.

[0041] Preferably, the incubation time at room temperature is 30 min.

[0042] Preferably, the heating time is 10-60 min.

[0043] Preferably, the ultraviolet light is 6-12 mJ cm -2 of ultraviolet light.

[0044] Preferably, the use of the trehalose polymer in improving the stress tolerance of Metarhizium includes the following steps:

[0045] (1) The trehalose polymer is added into a dissolving aid solution, ultrasonic treatment is carried out for 10-20 min, and the solution is placed at room temperature for 10-20 min, and the ultrasonic treatment and the placing are repeated for three times, so that a PTLA solution with a concentration of 0.1-2 mg / mL is obtained;

[0046] (2) The Metarhizium spores are incubated in the PTLA solution with a concentration of 0.1-2 mg / mL at room temperature, and heated at 39-45 ℃ for 10-60 min; or, the Metarhizium spores are incubated in the PTLA solution with a concentration of 0.1-2 mg / mL at room temperature, and irradiated with ultraviolet light at 6-12 mJ cm -2 Ultraviolet light energy irradiation.

[0047] The application further provides a Metarhizium control agent containing Metarhizium and the trehalose polymer.

[0048] Preferably, the Metarhizium is Metarhizium spores.

[0049] Preferably, the trehalose polymer is attached to the surface of the Metarhizium spores.

[0050] The application has the following advantages:

[0051] (1) The application uses the reversible addition fragmentation chain transfer polymerization method, referred to as RAFT polymerization, ethyl 2-(((benzylthio)carbonothioyl)thio)tetradecanoate as a chain transfer agent, trehalose acrylate TreA and lauryl acrylate LA as monomers, and a polymer PTLA is obtained by initiating a polymerization reaction under a nitrogen environment after multiple freeze-thaw cycles. The trehalose polymer PTLA prepared by copolymerizing TreA, LA monomers and ethyl 2-(((benzylthio)carbonothioyl)thio)tetradecanoate can absorb ultraviolet on the trithioester of the polymer main chain, and the hydrophobic carbon chain on the polymer main chain and the hydrophobic side chain lauryl acrylate can be embedded on the surface of the Metarhizium anisopliae conidium by hydrophilic-hydrophobic interaction, so as to have the ability to protect the conidium from ultraviolet stress, and the hydrophilic chain trehalose can reduce the influence of heat stress on the conidium.

[0052] (2) In the actual application of Metarhizium, the trehalose polymer material of the application is attached to the surface of Metarhizium spores, and a protective shell is formed on the surface of the pathogenic unit conidium, so as to improve the heat resistance and ultraviolet resistance of Metarhizium, and solve the problem that Metarhizium is limited by high temperature and ultraviolet stress when used to control pests in the wild.

[0053] Trehalose can improve the tolerance of cells to extreme conditions, and has the ability to stabilize biological macromolecules under stress conditions. The present application uses reversible addition-fragmentation chain transfer polymerization to prepare trehalose polymers with trehalose side chains and hydrophobic main chains, and by using trehalose polymers to act on the pathogenic units of Metarhizium, a protective shell is formed on the surface of conidia, so as to improve the heat resistance and ultraviolet resistance of fungi to a greater extent, and provide a new way for the application of biocontrol fungi. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 The preparation route diagram of the trehalose polymer in Example 1 of the present application is shown in the figure;

[0055] Figure 2 The Fourier transform infrared spectrum of the monomer TreA in Example 1 of the present application is shown in the figure;

[0056] Figure 3 The nuclear magnetic hydrogen spectrum of the monomer T-1 in Example 1 of the present application is shown in the figure;

[0057] Figure 4 The nuclear magnetic hydrogen spectrum of the monomer T-2 in Example 1 of the present application is shown in the figure;

[0058] Figure 5 The nuclear magnetic hydrogen spectrum of the monomer T-3 in Example 1 of the present application is shown in the figure;

[0059] Figure 6 The nuclear magnetic hydrogen spectrum of the monomer TreA in Example 1 of the present application is shown in the figure;

[0060] Figure 7 The nuclear magnetic hydrogen spectrum of the monomer LA in Example 1 of the present application is shown in the figure;

[0061] Figure 8 The nuclear magnetic hydrogen spectrum of the hydrophobic chain transfer agent in Example 1 of the present application is shown in the figure;

[0062] Figure 9 The nuclear magnetic hydrogen spectrum of the polymer PTLA in Example 1 of the present application is shown in the figure;

[0063] Figure 10 The Fourier transform infrared spectrum of the polymer PTLA in Example 1 of the present application is shown in the figure;

[0064] Figure 11 The gel permeation chromatogram of the polymer PTLA in Example 1 of the present application is shown in the figure;

[0065] Figure 12 The germination rate of Metarhizium spores after heat treatment at different temperatures after mixing with the polymer PTLA solution in Test Example 1 of the present application is shown in the figure;

[0066] Figure 13The germination rate of the green muscardine spores after being mixed with different concentrations of polymer PTLA solution and free trehalose T solution and then heat treated in Test Example 2 of the present application;

[0067] Figure 14 The germination rate of the green muscardine spores after being mixed with different concentrations of polymer PTLA solution and free trehalose T solution and then ultraviolet treated in Test Example 2 of the present application;

[0068] Figure 15 The germination rate of the green muscardine spores after being treated for different heat treatment times in Test Example 3 of the present application;

[0069] Figure 16 The germination rate of the green muscardine spores after being treated for different ultraviolet energy in Test Example 4 of the present application. DETAILED DESCRIPTION

[0070] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described below in a clear and complete manner in combination with the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0071] The test materials and reagents used in the following examples, and the like, can be obtained from commercial channels unless otherwise specified.

[0072] The specific techniques or conditions not specified in the examples can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0073] Embodiment 1

[0074] The present embodiment provides a preparation method of a trehalose polymer, the preparation route of which is shown in Figure 1 and includes the following steps:

[0075] (1) Preparation of trehalose monomer

[0076] a. Preparation of T-1

[0077] Trehalose (7.5 g, 21.91 mmol) and trimethylchlorosilane (28.56 g, 262.92 mmol) were weighed according to a molar ratio of 1:12, and the trehalose was added to 80 mL of pyridine while stirring, and then trimethylchlorosilane was slowly added dropwise in an environment at 0°C. After the reaction was completed, the temperature was restored to room temperature, and the reaction was continued at 25°C for 12 hours. Then, 100 mL of ethyl acetate was added for dilution. The mixture was extracted with pure water three times, dried with anhydrous Na2SO4, and then the solvent was distilled under reduced pressure after suction filtration to obtain the product T-1.

[0078] b.Preparation of T-2

[0079] T-1 (4 g, 4.35 mmol), solid K2CO3 (0.55 g, 3.98 mmol) were weighed according to the molar ratio of 1 :0.9, added into 270 mL of methanol for stirring, diluted with 200 mL of n-hexane after reaction for 18 min at 0 °C. Extracted with pure water for three times, dried with anhydrous Na2SO4, and the solvent was distilled under reduced pressure after suction filtration. Separated by silica gel wet column chromatography, and the eluent was V 石油醚 :V 乙酸乙酯 = 50:1, and the product T-2 was obtained after purification.

[0080] c.Preparation of T-3

[0081] T-2 (3 g, 3.54 mmol), acryloyl chloride (0.48 g, 5.31 mmol), triethylamine (0.54 g, 5.34 mmol) were weighed according to the molar ratio of 1 :1.5:1.5, T-2 and triethylamine were added into 100 mL of dichloromethane, acryloyl chloride was diluted with 5 mL of dichloromethane, and then the diluted acryloyl chloride was added dropwise in an environment of 0 °C. After the completion of the addition of the reactant, the temperature was restored to room temperature, and the reaction was continued at 25 °C for 12 hours. Then the solvent was distilled under reduced pressure, and separated by silica gel wet column chromatography, and the eluent was V 石油醚 :V 乙酸乙酯 = 50:1, and the product T-3 was obtained after purification.

[0082] d.Preparation of TreA.

[0083] T-3 (2 g, 2.22 mmol), Amberlyst 15 ion exchange resin (7.67 g, 24.4 mmol) were weighed according to the molar ratio of 1 :11, added into 180 mL of a mixed solvent of methanol and water (volume ratio of 11:1), and reacted at room temperature for 1 hour. After suction filtration, the obtained filtrate was extracted once with n-hexane, and then the solvent was distilled under reduced pressure for about 15 mL. The final product TreA was obtained after water membrane and freeze-drying.

[0084] (2) Preparation of hydrophobic acryl monomer

[0085] Dodecanol (1 g, 5.37 mmol), acryloyl chloride (0.73 g, 8.07 mmol), triethylamine (0.81 g, 8.005 mmol) were weighed according to the molar ratio of 1 :1.5:1.5, dodecanol and triethylamine were added into 25 mL of dichloromethane, acryloyl chloride was diluted with 7 mL of dichloromethane, and then the diluted acryloyl chloride was added dropwise in an environment of 0 °C ice bath. After the completion of the addition of the reactant, the temperature was restored to room temperature, and the reaction was continued at 25 °C for 12 hours. Then the solvent was distilled under reduced pressure, and separated by silica gel wet column chromatography, and the eluent was V 石油醚:V 乙酸乙酯 = 50:1, the product dodecyl acrylate LA was purified.

[0086] (3) Preparation of hydrophobic chain transfer agent

[0087] (2-hydroxyethyl) benzyl trithiocarbonate (1 g, 4.09 mmol), myristic acid (1.4 g, 6.13 mmol), 4-dimethylaminopyridine (0.25 g, 2.05 mmol), 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide (0.63 g, 4.06 mmol) were weighed according to the molar ratio of 1:1.5:0.5:1 and added into 100 mL dichloromethane, and the reaction was condensed backflow at 50°C for 12 hours. Then the solvent was distilled under reduced pressure, and the product was separated by silica gel wet column chromatography, and the eluent was V 石 = 50:1, the product dodecyl acrylate LA was purified.

[0088] (4) Preparation of trehalose polymer

[0089] a. Acryl trehalose TreA (50 mg, 0.126 mmol), dodecyl acrylate LA (7.58 mg, 0.0315 mmol), 2-(((benzylthio)carbonothioyl)thio)tetradecanoic acid ethyl ester (2.87 mg, 0.0063 mmol) were added to an ampoule and dissolved in 605.5 μL of dimethyl sulfoxide.

[0090] Azobisisobutyronitrile (AIBN) was dissolved in dimethyl sulfoxide to form a 1 mg / mL solution, and 100 μL (0.1 mg, 0.00063 mmol) was added to the ampoule and mixed, and the ampoule was sealed.

[0091] b. The mixed solution sealed in the ampoule was subjected to three freeze-thaw cycles, and then polymerization was carried out at 70°C under nitrogen for 16 h, and then the polymerization product was dialyzed with a dialysis bag with a molecular weight cut-off of 3500 for 3 h, and the water was replaced five times during the dialysis, to obtain the purified polymerization product, namely PTLA

[0092] During the synthesis, the synthesis product of step (1) was characterized by infrared spectroscopy, and the obtained infrared spectrum is shown in Figure 2 It can be seen that the characteristic peak of hydroxyl group appears at 3325 cm -1 -1, and the characteristic peak of carbon-oxygen double bond in ester bond appears at 1729 cm -1 -1, proving the synthesis of the target product TreA. T-1, T-2, T-3 were characterized by nuclear magnetic resonance analysis, and the nuclear magnetic hydrogen spectrum is as follows: Figure 3 、 Figure 4 andFigure 5 As shown, due to the special structure of trehalose, Figure 4 The decrease of CH3 peak area in the nuclear magnetic hydrogen spectrum proves that one protection is successfully removed to obtain T-2, Figure 5 The peak area ratio of double bond again proves that one protection is removed. The nuclear magnetic characterization analysis of TreA is obtained, and the nuclear magnetic hydrogen spectrum is as shown in the figure Figure 6 As shown, further indicating that TreA is successfully synthesized. The nuclear magnetic characterization analysis of the synthesis product in step (2) is obtained, and the nuclear magnetic hydrogen spectrum is as shown in the figure Figure 7 As shown, indicating that the product LA is successfully synthesized. The nuclear magnetic characterization analysis of the synthesis product in step (3) is obtained, and the nuclear magnetic hydrogen spectrum is as shown in the figure Figure 8 As shown, indicating that the hydrophobic chain transfer agent is successfully synthesized. The nuclear magnetic resonance analysis and Fourier infrared spectrum analysis of the polymerization product prepared in step (4) are obtained, and the nuclear magnetic hydrogen spectrum is as shown in the figure Figure 9 As shown, proving the successful synthesis of the polymer PTLA. The Fourier transform infrared spectrum is as shown in the figure Figure 10 As shown, 1729cm-1 -1 The characteristic peak of carbon-oxygen double bond in ester bond appears, 3367cm-1 -1 The characteristic peak of hydroxyl group appears, further proving the synthesis of the target polymer.

[0093] The synthesis route of the trehalose polymer material is as follows:

[0094]

[0095] The gel permeation chromatography analysis of the prepared polymer PTLA is obtained, and the gel permeation chromatogram is as shown in the figure Figure 11 As shown, the number average molecular weight M n =9800Da, the polydispersity coefficient PDI=1.15, and the molecular weight distribution is narrow.

[0096] Example 2

[0097] The present embodiment provides an application of a trehalose polymer material in improving the stress tolerance of Metarhizium anisopliae, comprising the following steps:

[0098] (1) Dissolve Tween 20 in pure water to prepare a 0.55mg / mL Tween 20 solution. Add the Tween 20 solution to the polymer PTLA prepared in Example 1, ultrasonic treatment for 10min, and then place it in a 25℃ environment for 10min. Repeat the steps of ultrasonic treatment and placement for three times to obtain a PTLA solution with a concentration of 2mg / mL.

[0099] (2) Take 20μL of the PTLA solution with a concentration of 10 7 / mL of Metarhizium spore suspension was mixed with 200 μL of 2 mg / mL PTLA solution, incubated at room temperature for 30 min, and then heated in a water bath at 39°C for 30 min. After heating, 10 μL was dropped on the medium, and the liquid was allowed to evaporate.

[0100] Example 3

[0101] This example provides a use of trehalose polymer material in improving the stress tolerance of Metarhizium, which is different from Example 2 in that the heating treatment temperature after mixing the Metarhizium spore suspension with the PTLA solution is 42°C.

[0102] Example 4

[0103] This example provides a use of trehalose polymer material in improving the stress tolerance of Metarhizium, which is different from Example 2 in that the heating treatment temperature after mixing the Metarhizium spore suspension with the PTLA solution is 45°C.

[0104] Example 5

[0105] This example provides a use of trehalose polymer material in improving the stress tolerance of Metarhizium, which is different from Example 2 in that the heating treatment temperature after mixing the Metarhizium spore suspension with the PTLA solution is 48°C.

[0106] Example 6

[0107] This example provides a use of trehalose polymer material in improving the stress tolerance of Metarhizium, which includes the following steps:

[0108] (1) Dissolve Tween 20 in pure water to prepare a 0.55 mg / mL Tween 20 solution. Add the Tween 20 solution to the polymer PTLA prepared in Example 1, ultrasonic treatment for 10 min, and then place it in a 25°C environment for 10 min. Repeat the ultrasonic treatment and placement steps three times to obtain a 2 mg / mL PTLA solution. Similarly, add the Tween 20 solution to the free trehalose, ultrasonic treatment for 10 min, and then place it in a 25°C environment for 10 min. Repeat the ultrasonic treatment and placement steps three times to obtain a 2 mg / mL trehalose T solution.

[0109] (2) Mix 20 μL of 10 7 / mL Metarhizium spore suspension with 200 μL of 2 mg / mL PTLA solution and 2 mg / mL free trehalose T solution, respectively, incubate at room temperature for 30 min, and then heat in a water bath at 45°C for 30 min. After heating, 10 μL was dropped on the medium, and the liquid was allowed to evaporate.

[0110] (3) Mix 20 μL of 10 7 / mL of Metarhizium sporesuspension was mixed with 200 μL of 2 mg / mL PTLA solution and 2 mg / mL free trehalose T solution respectively, incubated at room temperature for 30 min, 10 μL was dropped on the medium, and after the liquid evaporated, the sample was irradiated under UV light at 8 mJ cm -2 under UV light.

[0111] Example 7

[0112] This example provides an application of trehalose polymer material in improving the stress tolerance of Metarhizium, which is different from Example 6 in that the concentration of PTLA solution and T solution is 1 mg / mL.

[0113] Example 8

[0114] This example provides an application of trehalose polymer material in improving the stress tolerance of Metarhizium, which is different from Example 6 in that the concentration of PTLA solution and T solution is 0.5 mg / mL.

[0115] Example 9

[0116] This example provides an application of trehalose polymer material in improving the stress tolerance of Metarhizium, which is different from Example 6 in that the concentration of PTLA solution and T solution is 0.25 mg / mL.

[0117] Example 10

[0118] This example provides an application of trehalose polymer material in improving the stress tolerance of Metarhizium, which is different from Example 6 in that the concentration of PTLA solution and T solution is 0.1 mg / mL.

[0119] Example 11

[0120] This example provides an application of trehalose polymer material in improving the stress tolerance of Metarhizium, which includes the following steps:

[0121] (1) Tween 20 was dissolved in pure water to prepare a 0.55 mg / mL Tween 20 solution. The Tween 20 solution was added to the polymer PTLA prepared in Example 1, ultrasonically treated for 10 min, and then placed at 25°C for 10 min. The steps of ultrasonic treatment and placement were repeated three times to obtain a PTLA solution with a concentration of 2 mg / mL.

[0122] (2) 20 μL of Metarhizium spore suspension with a concentration of 10 7 / mL was mixed with 200 μL of 2 mg / mL PTLA solution, incubated at room temperature for 30 min, and then heated in a water bath at 45°C for 10 min. After heating, 10 μL was dropped on the medium, and after the liquid evaporated.

[0123] Example 12

[0124] The present example provides a use of trehalose polymer material in improving the stress tolerance of Metarhizium anisopliae, which is different from example 11 in that the heating treatment time after mixing the Metarhizium anisopliae spore suspension with the PTLA solution is 20 min.

[0125] Example 13

[0126] The present example provides a use of trehalose polymer material in improving the stress tolerance of Metarhizium anisopliae, which is different from example 11 in that the heating treatment time after mixing the Metarhizium anisopliae spore suspension with the PTLA solution is 30 min.

[0127] Example 14

[0128] The present example provides a use of trehalose polymer material in improving the stress tolerance of Metarhizium anisopliae, which is different from example 11 in that the heating treatment time after mixing the Metarhizium anisopliae spore suspension with the PTLA solution is 40 min.

[0129] Example 15

[0130] The present example provides a use of trehalose polymer material in improving the stress tolerance of Metarhizium anisopliae, which is different from example 11 in that the heating treatment time after mixing the Metarhizium anisopliae spore suspension with the PTLA solution is 50 min.

[0131] Example 16

[0132] The present example provides a use of trehalose polymer material in improving the stress tolerance of Metarhizium anisopliae, which is different from example 11 in that the heating treatment time after mixing the Metarhizium anisopliae spore suspension with the PTLA solution is 60 min.

[0133] Example 17

[0134] The present example provides a use of trehalose polymer material in improving the stress tolerance of Metarhizium anisopliae, which includes the following steps:

[0135] (1) Dissolve Tween 20 in pure water to prepare a 0.55 mg / mL Tween 20 solution. Add the Tween 20 solution to the polymer PTLA prepared in example 1, ultrasonic treatment for 10 min, and then place it in a 25℃ environment for 10 min. Repeat the steps of ultrasonic treatment and placement for three times to obtain a PTLA solution with a concentration of 2 mg / mL.

[0136] (2) Mix 20 μL of the 10 7Example 2, 3, 4 and 5, respectively. The culture medium was placed in an incubator at 25°C and the germination rate was observed after 12-18h. The results are shown in Figure 1, wherein the figure shows the germination rate of the spores of Metarhizium anisopliae under different conditions. -2 Irradiation under UV light.

[0137] Example 18

[0138] This example provides a use of trehalose polymer material in improving the stress tolerance of Metarhizium anisopliae, which is different from Example 17 in that the spores of Metarhizium anisopliae are irradiated at 10mJ cm -2 Irradiation under UV light.

[0139] Example 19

[0140] This example provides a use of trehalose polymer material in improving the stress tolerance of Metarhizium anisopliae, which is different from Example 17 in that the spores of Metarhizium anisopliae are irradiated at 8mJ cm -2 Irradiation under UV light.

[0141] Example 20

[0142] This example provides a use of trehalose polymer material in improving the stress tolerance of Metarhizium anisopliae, which is different from Example 17 in that the spores of Metarhizium anisopliae are irradiated at 6mJ cm -2 Irradiation under UV light.

[0143] Test Example 1

[0144] According to the method of Example 2, four groups of Metarhizium anisopliae spore suspensions were mixed with Tween 20 solution without PTLA as a control group (WT), and incubated at room temperature for 30min, and then heat treated at temperatures of 39, 42, 45 and 48°C for 30min, respectively. After heating, 10μL was dropped on the culture medium, and the liquid was evaporated. The culture medium after heat treatment of Example 2, Example 3, Example 4, Example 5 and the control group was placed in an incubator at 25°C, and the germination rate was observed after 12-18h. The results are shown in Figure 1, wherein the figure shows the germination rate of the spores of Metarhizium anisopliae under different conditions. Figure 12

[0145] PTLA-39°C, PTLA-42°C, PTLA-45°C, PTLA-48°C represent the results of Examples 2, 3, 4 and 5, and WT-39°C, WT-42°C, WT-45°C, WT-48°C are the results of the control group.

[0146] ​It can be seen that, compared with the control group, the PTLA solution can improve the germination rate of Metarhizium spores after heat treatment at a temperature of 39-45°C. When the heat treatment temperature is 48°C, Metarhizium spores do not germinate. When the heat treatment temperature is 45°C, the germination rate of Metarhizium spores in the mixed PTLA solution is higher than that of the control group, and the improvement is greater than that at 39°C and 42°C.

[0147] Test Example 2

[0148] According to the method of Example 6, Tween 20 solution without PTLA was used as the control group (WT, concentration 0 mg / mL). Metarhizium spore suspension was mixed with Tween 20 solution without PTLA and incubated at room temperature for 30 min. The heating control group was heated in a water bath at 45°C for 30 min. After heating, 10 μL of the spore suspension was dropped on the medium and the liquid was allowed to evaporate. The ultraviolet control group was 10 μL dropped on the medium, and after the liquid evaporated, 8 mJ cm-2 of ultraviolet light was irradiated for 30 s. The medium was incubated in a 25°C incubator, and the germination rate after 18 h of growth was observed. The results are shown in Table 1. -2 Ultraviolet irradiation treatment. The medium after heat and ultraviolet treatment in Examples 6-10 and the control group was incubated in a 25°C incubator, and the germination rate after 18 h of growth was observed. The results are shown in Table 2. Figure 13 、 14 As shown in Table 2, the horizontal coordinate in the figure represents the concentration of PTLA solution and T solution.

[0149] It can be seen that, compared with the control group, PTLA solution with a concentration of 0.1-2 mg / mL can improve the germination rate of Metarhizium spores after heat treatment and ultraviolet irradiation. However, free trehalose solution can only improve the germination rate after heat treatment and has no effect on ultraviolet irradiation treatment. The germination rate of PTLA solution with a concentration of 2 mg / mL after heat treatment can reach 86.07%, which is significantly higher than that of the control group of 48.75%. Moreover, PTLA solution with a concentration of 0.1-2 mg / mL also has a significant improvement in the germination rate after heat treatment and ultraviolet irradiation compared with free trehalose solution.

[0150] Test Example 3

[0151] According to the method of Example 11, Tween 20 solution without PTLA was used as the control group (WT). Metarhizium spore suspension was mixed with Tween 20 solution without PTLA and incubated at room temperature for 30 min. Then, it was heated at 45°C for 10, 20, 30, 40, 50, and 60 min, respectively. 10 μL of spore suspension treated at different heating times was dropped on the medium, and the liquid was allowed to evaporate. The medium after heat treatment in Examples 11-16 and the control group was incubated in a 25°C incubator, and the germination rate after 18 h of growth was observed. The results are shown in Table 3. Figure 15

[0152] ​It can be seen that, compared with the control group, the PTLA solution can improve the germination rate of Metarhizium spores after different heat treatment times. When the heat treatment time is 30 min, the germination rate of Metarhizium spores is increased from 51.66% to 84.24%, an increase of 32.58%. The germination rate is better than that of 10, 20, 40, 50 and 60 min, which is increased by 15.09%, 21.21%, 18.13%, 13.83% and 15.7%, respectively.

[0153] Test Example 4

[0154] According to the method of Example 17, Tween 20 solution without PTLA was used as a control group (WT), Metarhizium spore suspension was mixed with Tween 20 solution without PTLA, incubated at room temperature for 30 min, 10 μL was dropped on the medium, and after the liquid volatilized, 12, 10, 8, 6 mJ cm -2 was irradiated under ultraviolet light. The medium after ultraviolet treatment in Examples 17-20 and the control group was cultured in a 25℃ incubator, and the germination rate after 18h of growth was observed, and the results are shown in Table 5. Figure 16

[0155] It can be seen that, compared with the control group, the PTLA solution can improve the germination rate of Metarhizium spores after different ultraviolet energy irradiation treatments. And under the ultraviolet energy irradiation of 8 mJ cm -2 , the PTLA solution can improve the germination rate of Metarhizium spores by 24.18%, which is the most significant compared with other ultraviolet energy irradiation.

[0156] During the test of the germination rate, there is a slight fluctuation in the test results of different batches, but the overall trend and data difference are not large.

[0157] The implementation principle of the present application is as follows: the present application uses the method of reversible addition fragmentation chain transfer polymerization to copolymerize TreA, LA polymer monomer and 2-(((benzylthio) carbonylthio) thio) tetradecanoic acid ethyl ester to prepare trehalose polymer PTLA. The trithioester on the polymer main chain can absorb ultraviolet, and the hydrophobic carbon chain on the polymer main chain and the hydrophobic chain dodecyl acrylate on the side chain can be embedded on the surface of Roberts Metarhizium conidia through hydrophilic and hydrophobic interaction, which has the ability to protect conidia from ultraviolet stress, and the hydrophilic chain trehalose can reduce the influence of heat stress on conidia.

[0158] In the practical application of Metarhizium, the trehalose copolymer material of the present application is attached to the surface of Metarhizium spores to form a protective shell on the surface of the pathogenic unit conidia, so as to improve the heat resistance and ultraviolet resistance of Metarhizium, and solve the problem that Metarhizium is limited by high temperature and ultraviolet stress when preventing and controlling pests in the wild.​

[0159] The above examples are only used to illustrate the technical solutions of the present application, but not to limit the present application; although the present application has been described in detail with reference to the foregoing examples, those ordinarily skilled in the art should understand: the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced equivalently; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A trehalose polymer, characterized by: The structural formula is as follows:

2. A method of preparing a trehalose polymer as claimed in claim 1, characterized by: The method comprises the following steps: S1, benzyl (2-hydroxyethyl) trithiocarbonate and tetradecanoic acid are used as raw materials, and the reaction is carried out in the presence of 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to obtain 2-(((benzylthio) carbonothioyl) thio) tetradecanoic acid ethyl ester; S2, ethyl 2-(((benzylthio)carbonothioyl)thio)tetradecanoate as chain transfer agent, trehalose propenoate The polymer of trehalose is obtained by polymerization of dodecyl acrylate as monomer under nitrogen atmosphere after freeze-thaw cycle, and the polymerization is initiated by initiator.

3. The method of claim 2, wherein the trehalose polymer is prepared by: In S1, the molar ratio of the benzyl (2-hydroxyethyl) trithiocarbonate and tetradecanoic acid is 1:1.

5.

4. The method for preparing the trehalose polymer according to claim 2, characterized in that: In S1, the molar ratio of the benzyl (2-hydroxyethyl) trithiocarbonate, 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1:0.5:

1.

5. The method for preparing the trehalose polymer according to claim 2, characterized in that: In S2, the molar ratio of the 2-(((benzylthio) carbonothioyl) thio) tetradecanoic acid ethyl ester, trehalose propenoate and dodecyl acrylate is 1:20:

5.

6. The method for preparing the trehalose polymer according to claim 2, characterized in that: In S2, the molar ratio of the 2-(((benzylthio) carbonothioyl) thio) tetradecanoic acid ethyl ester and the initiator is 1:0.1-0.

2.

7. The method of claim 2-6, wherein: In S1, the reaction is carried out under reflux conditions for 12 h; in S2, the reaction temperature is 60-70 DEG C, and the reaction time is 12-20 h.

8. Use of the trehalose polymer of claim 1 in improving the stress tolerance of Metarrhizium anisopliae.

9. The use of trehalose polymers according to claim 8 for increasing the stress tolerance of Metarhizium spp., characterized in that: The method comprises mixing Metarrhizium anisopliae spores with the trehalose polymer solution and incubating at room temperature.

10. A Metarhizium control agent, characterized by: Metarrhizium anisopliae and the trehalose polymer of claim 1.

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