Application of carboxylated chitin or carboxylated chitosan in promoting pest control of insecticidal fungi

Abstract

The invention relates to an application of carboxylated chitin or carboxylated chitosan in promoting insect-killing fungi to prevent and treat diseases and insect pests. Research finds that carboxylated chitin or carboxylated chitosan can significantly promote spore germination and hypha growth of insecticidal fungi, also can significantly enhance stress resistance of the insecticidal fungi, significantly enhance toxicity of the insecticidal fungi to pests, improve ultraviolet resistance and high temperature resistance of the insecticidal fungi, and improve the insecticidal activity of the insecticidal fungi. The invention also finds that the carboxylated chitin or chitosan can effectively inhibit the growth of Xanthomonas oryzae pv. Oryzae. Therefore, the carboxylated chitin or the carboxylated chitosan can be used for preparing a product for enhancing the insecticidal fungus effect and a product for preventing and treating the rice bacterial leaf blight, and the prevention and treatment of plant diseases and insect pests are realized. In addition, carboxylated chitin and carboxylated chitosan are derived from natural polymer materials, have the characteristics of safety, no toxicity, biodegradability, environmental friendliness and the like, are environment-friendly and economical, and have wide application prospects and market values.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and more specifically relates to the application of carboxylated chitin or carboxylated chitosan in promoting the use of insecticidal fungi to control pests and diseases. Background Art

[0002] With the increasing demands placed on agricultural products in modern agricultural production, the prevention and control of crop diseases and insect pests has become a crucial component of ensuring food security and sustainable agricultural production. While traditional chemical pesticides can effectively control pests and diseases to a certain extent, the long-term and extensive use of chemical pesticides not only easily leads to the development of pest resistance, but also seriously pollutes the ecological environment and even threatens human health. Therefore, the development of environmentally friendly, efficient, and sustainable biological pest control technologies is of great significance to modern agricultural production.

[0003] Insecticidal fungi (such as Mycelia javanica and Metarhizium anisopliae) have attracted much attention due to their environmental friendliness, safety for humans and animals, and significant control effects on a variety of pests. These insecticidal fungi can be widely used in the control of important agricultural pests such as rice planthoppers, rice borers, diamondback moths, fall armyworms, whiteflies, and locusts, and have shown unique advantages in the control of drug-resistant pests. However, in practical applications, insecticidal fungi still have some urgent problems to be solved: first, the insecticidal effect is relatively slow, which makes it difficult to meet the needs of rapid prevention and control during sudden pest outbreaks; second, the insecticidal effect is greatly affected by environmental factors (such as strong ultraviolet rays, high temperatures, etc.), resulting in unstable field control effects; third, its toxicity and adaptability need to be further improved to achieve efficient and rapid insecticide control and expand the range of insecticide control.

[0004] Based on the above background, developing products that can improve the reproductive efficiency, stress resistance and virulence of insecticidal fungi is of great significance for achieving green and efficient integrated prevention and control of agricultural pests and diseases. Summary of the Invention

[0005] The present invention aims to overcome the defects and deficiencies in the above-mentioned prior art and provide a new application of carboxylated chitin and / or carboxylated chitosan in improving the reproductive efficiency, stress resistance and toxicity of insecticidal fungi.

[0006] The first object of the present invention is to provide the use of carboxylated chitin and / or carboxylated chitosan in promoting the growth of insecticidal fungi and improving the ability to resist ultraviolet rays and high temperature.

[0007] The second object of the present invention is to provide a composition and its use in preventing and controlling crop diseases and insect pests.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] Chitin, also known as chitin or chitin, is widely found in the shells of crustaceans such as shrimp, crabs, and insects. It is a natural, high-molecular-weight, renewable polysaccharide. Chitosan is the product of chitin through decalcification, deproteinization, and deacetylation. Chitosan is insoluble in water but soluble in some dilute acids. Chitin and chitosan are safe, non-toxic, biodegradable, environmentally friendly, biocompatible, and edible.

[0010] The present invention has found that carboxylated chitin and / or carboxylated chitosan can increase the germination rate of insecticidal fungal spores and promote mycelial growth, effectively improving the fungal insecticidal speed and control effect. In addition, the present invention has also found that carboxylated chitin and / or carboxylated chitosan can improve the anti-ultraviolet and high temperature resistance of insecticidal fungi. Therefore, this application claims protection for the following scheme:

[0011] Application of carboxylated polysaccharides in promoting the growth of insecticidal fungi.

[0012] Application of carboxylated polysaccharides in the preparation of products for promoting the growth of insecticidal fungi.

[0013] Specifically, the promoting the growth of insecticidal fungi includes promoting the germination of insecticidal fungal spores and promoting the growth of insecticidal fungal hyphae.

[0014] Application of carboxylated polysaccharides in improving the UV resistance of insecticidal fungi.

[0015] Application of carboxylated polysaccharides in the preparation of products for improving the anti-ultraviolet ability of insecticidal fungi.

[0016] Application of carboxylated polysaccharides in improving the temperature tolerance of insecticidal fungi.

[0017] Application of carboxylated polysaccharides in the preparation of products for improving the temperature tolerance of insecticidal fungi.

[0018] Application of carboxylated polysaccharides in improving the toxicity of insecticidal fungi to pests.

[0019] Application of carboxylated polysaccharides in the preparation of insecticidal fungi to improve their toxicity to pests.

[0020] Specifically, the pests include brown planthopper, rice borer, diamondback moth, fall armyworm, whitefly or locust. In the embodiments of the present invention, brown planthopper is used as the research object.

[0021] Application of carboxylated polysaccharides combined with insecticidal fungi in controlling crop diseases and pests.

[0022] Application of carboxylated polysaccharides in inhibiting the growth of rice bacterial blight pathogen.

[0023] Application of carboxylated polysaccharides in the preparation of products for inhibiting the growth of rice bacterial blight pathogen.

[0024] Application of carboxylated polysaccharides in controlling rice bacterial blight.

[0025] Application of carboxylated polysaccharides in the preparation of products for preventing and controlling rice bacterial blight.

[0026] Specifically, the carboxylated polysaccharide is carboxylated chitin or carboxylated chitosan, and the insecticidal fungus is Corynespora or Metarhizium anisopliae.

[0027] As an optional embodiment, the Mycelia spp. is Mycelia javanica, and the Metarhizium anisopliae is Metarhizium niger.

[0028] The present invention also provides an insecticide, comprising a carboxylated polysaccharide and an insecticidal fungus; the carboxylated polysaccharide is carboxylated chitin or carboxylated chitosan, and the insecticidal fungus is Corynespora or Metarhizium anisopliae.

[0029] As an optional embodiment, the carboxylated chitosan is carboxymethyl chitosan.

[0030] As an optional embodiment, the preparation method of the carboxylated chitosan is as follows: chitosan and sodium hydroxide are added with water and isopropanol to prepare solution A, solution A is stirred and reacted for 0.5-1.5 hours, and then chloroacetic acid is added to obtain solution B, solution B is further reacted for 3-5 hours, ethanol is added, and solid particles are recovered to obtain carboxylated chitosan.

[0031] As an optional embodiment, the chitosan concentration in the solution A is 50-80 mg / mL, the sodium hydroxide concentration is 120-150 mg / mL; and the chloroacetic acid concentration in the solution B is 80-100 mg / mL.

[0032] As an optional embodiment, the ethanol is added to solution B at a volume ratio of solution B:ethanol = (75-95):100, and the concentration of the ethanol solution is 60-80%.

[0033] As a kind of selectable embodiment, the preparation method of described carboxylated chitosan is: get 5.0g chitosan, 10.3g sodium hydroxide (NaOH), join in 250mL conical flask, in conical flask, add water 15mL, Virahol 60mL subsequently, then, conical flask is placed on 50 ℃ magnetic stirring apparatus and stir, after 1h, 10mL chloroacetic acid solution (7.5g chloroacetic acid is dissolved in 10mL Virahol) is at the uniform velocity splashed in above-mentioned reaction conical flask, after under agitation, continue reaction 4h, in conical flask, add 100mL 70% ethanol termination reaction.Finally, remove reaction solution, collect solid particle and transfer in glass culture dish, in 60 ℃ baking oven, dry and namely obtain carboxylated chitosan.

[0034] As an optional embodiment, the Mycelia spp. is Mycelia javanica, and the Metarhizium anisopliae is Metarhizium niger.

[0035] As an alternative embodiment, the invention comprises a carboxylated polysaccharide and an insecticidal fungal spore suspension, wherein the concentration of the carboxylated polysaccharide is not less than 5 mM.

[0036] As an alternative embodiment, the invention comprises a carboxylated polysaccharide and an insecticidal fungal spore suspension, wherein the concentration of the carboxylated polysaccharide is 5-50 mM.

[0037] As an alternative embodiment, the carboxylated polysaccharide and the insecticidal fungus spore suspension are included, and the concentration of the carboxylated polysaccharide is 25 mM.

[0038] As an optional embodiment, the concentration of the spore suspension is not less than 1×10 3 spores / mL.

[0039] The use of the above-mentioned insecticides in preventing and controlling crop pests, or in preventing and controlling plant diseases caused by crop pests, should also be within the scope of protection of the present invention.

[0040] The present invention has the following beneficial effects:

[0041] The present invention has found that carboxylated chitin or carboxylated chitosan can significantly promote the germination of insecticidal fungal spores and the growth of mycelium, thereby improving the insecticidal efficiency of the insecticidal fungus; it can also significantly enhance the stress resistance of the insecticidal fungus, significantly enhance the toxicity of the insecticidal fungus to pests (such as brown planthoppers), and shorten the half-lethal time (LT50 of the insecticidal fungus). 50 ) and median lethal concentration (LC50) 50 ). In addition, carboxylated chitin or carboxylated chitosan can also significantly inhibit the bacterial blight of rice.

[0042] Therefore, carboxylated chitin or carboxylated chitosan can be used to prepare products that enhance the fungicidal effect and prevent and control rice bacterial blight, thereby improving the control effect of diseases and pests. In addition, carboxylated chitin and carboxylated chitosan are derived from natural polymer materials, are safe, non-toxic, biodegradable, and environmentally friendly, and are both environmentally friendly and economical, with broad application prospects and market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The spore germination rates of the WT and UV strains of the javanica fungus in different treatment groups (Figure a shows the spore germination rate of the WT strain mixed with carboxylated chitosan solution; Figure b shows the spore germination rate of the UV strain mixed with carboxylated chitosan solution; Figure c shows the spore germination rate of the WT strain mixed with carboxylated chitin solution; Figure d shows the spore germination rate of the UV strain mixed with carboxylated chitin solution).

[0044] Figure 2 The spore germination rates of the WT and UV strains of Metarhizium anisopliae in different treatment groups (Figure a shows the spore germination rate of the WT strain mixed with carboxylated chitosan solution; Figure b shows the spore germination rate of the UV strain mixed with carboxylated chitosan solution; Figure c shows the spore germination rate of the WT strain mixed with carboxylated chitin solution; Figure d shows the spore germination rate of the UV strain mixed with carboxylated chitin solution).

[0045] Figure 3 Effects of different treatments on the mycelial growth of Mycelium javanica (Figure a is the colony diameter of the WT strain on carboxylated chitosan medium; Figure b is the colony diameter of the UV strain on carboxylated chitosan medium; Figure c is the colony diameter of the WT strain on carboxylated chitin medium; Figure d is the colony diameter of the UV strain on carboxylated chitin medium).

[0046] Figure 4 Effects of different treatments on the mycelial growth of Metarhizium anisopliae (Figure a is the colony diameter of the WT strain on carboxylated chitosan medium; Figure b is the colony diameter of the UV strain on carboxylated chitosan medium; Figure c is the colony diameter of the WT strain on carboxylated chitin medium; Figure d is the colony diameter of the UV strain on carboxylated chitin medium).

[0047] Figure 5 The spore germination rate of Mycosporium javanica in carboxylated chitosan solution or carboxylated chitin solution after ultraviolet irradiation (Figure a shows the spore germination rate in carboxylated chitosan solution; Figure b shows the spore germination rate in carboxylated chitin solution).

[0048] Figure 6The spore germination rate of Metarhizium anisopliae in carboxylated chitosan solution or carboxylated chitin solution after ultraviolet irradiation (Figure a shows the spore germination rate in carboxylated chitosan solution; Figure b shows the spore germination rate in carboxylated chitin solution).

[0049] Figure 7 The spore germination rate of Mycosporium javanica in carboxylated chitosan or chitin solution under high temperature (Figure a is the spore germination rate of WT strain in carboxylated chitosan solution; Figure b is the spore germination rate of UV strain in carboxylated chitosan solution; Figure c is the spore germination rate of WT strain in carboxylated chitin solution; Figure d is the spore germination rate of UV strain in carboxylated chitin solution).

[0050] Figure 8 The spore germination rate of Metarhizium anisopliae in carboxylated chitosan or chitin solution under high temperature (Figure a is the spore germination rate of WT strain in carboxylated chitosan solution; Figure b is the spore germination rate of UV strain in carboxylated chitosan solution; Figure c is the spore germination rate of WT strain in carboxylated chitin solution; Figure d is the spore germination rate of UV strain in carboxylated chitin solution).

[0051] Figure 9 LT of brown planthopper infected by carboxylated chitosan or carboxylated chitin mixed with Coryneformis javanica 50 .

[0052] Figure 10 LC of brown planthopper infected by carboxylated chitosan or carboxylated chitin mixed with Coryneformis javanica 50 (a) LC on the 4th day of mixed infection 50 Measurement results; Figure b shows the LC on the 7th day of mixed infection 50 measurement results).

[0053] Figure 11 LT of brown planthopper after carboxylated chitosan or carboxylated chitin mixed with Metarhizium anisopliae was infected 50 .

[0054] Figure 12 LC of brown planthopper infected by carboxylated chitosan or carboxylated chitin mixed with Metarhizium anisopliae 50 (a) LC on the 7th day of mixed infection 50 Measurement results; Figure b shows the LC on the 12th day of mixed infection 50 measurement results).

[0055] Figure 13Figure 2 is the growth of pathogens on culture media containing carboxylated chitosan or carboxylated chitin (Figure a is the culture result of Xanthoceras oryzae on BPM culture medium containing 0.5x carboxylated chitosan; Figure b is the culture result of Xanthoceras oryzae on BPM culture medium; Figure c is the culture result of Xanthoceras oryzae on BPM culture medium containing 0.5x carboxylated chitin; Figure d is the culture result of Xanthoceras oryzae on water agar culture medium containing 0.5x carboxylated chitosan Figure e shows the culture results of Xanthophylloxera: oryzae on water agar medium; Figure f shows the culture results of Xanthophylloxera: oryzae on water agar medium containing 0.5x carboxylated chitin; Figure g shows the culture results of bacterial leaf streak pathogen on NA medium containing 0.5x carboxylated chitosan; Figure h shows the culture results of bacterial leaf streak pathogen on NA medium; Figure i shows the culture results of bacterial leaf streak pathogen on NA medium containing 0.5x carboxylated chitin). DETAILED DESCRIPTION

[0056] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0057] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0058] Chitosan, CAS: 9012-76-4, manufacturer: McLean, product number: C804726.

[0059] Chitosan, CAS: 1398-61-4, manufacturer: McLean, product number: C804531.

[0060] In the following examples, the wild strain (WT) of Corynespora javanica used has a deposition number of CCTCC NO: M2017709, which has been disclosed in patent CN108102929B.

[0061] The deposit number of the mutagenic strain of Coryneformis javanica (UV) used is: CCTCC NO: M2019800, which has been disclosed in patent CN110804555B.

[0062] The deposit number of the induced strain (UV) of Metarhizium anisopliae used is: CCTCC NO: M2016250, which has been disclosed in patent CN106434362B. The wild strain of Metarhizium anisopliae has also been disclosed in patent CN106434362B.

[0063] In the following examples, the carboxylated chitosan used is manufactured by MacLean, with a product number of C804727, a CAS number of 83512-85-0, and a structural formula of:

[0064] In the following examples, the preparation method of used carboxylated chitosan is: take by weighing 5.0g chitosan, 10.3g sodium hydroxide (NaOH) respectively, join in the 250mL conical flask, in conical flask, add entry 15mL, Virahol 60mL subsequently, then, conical flask is placed on 50 ℃ magnetic stirring apparatus and stir, behind the 1h, chloroacetic acid solution (7.5g chloroacetic acid is dissolved in 10mL Virahol) is at the uniform velocity splashed in the above-mentioned reaction conical flask, after continuing reaction 4h, in conical flask, add 100mL 70% ethanol termination reaction.At last, remove reaction solution, collect solid particle and transfer in the glass culture dish, in 60 ℃ baking oven, dry and promptly obtain carboxylated chitosan.The solid particle after the oven dry is water-soluble, observes the particulate matter without floating in the aqueous solution, and does not have precipitation after centrifugal, shows that the solid particle of preparation is carboxylated chitosan.

[0065] Example 1 Effects of chitosan or chitin before and after carboxylation on spore germination of Mycosporium javanica

[0066] 1. Experimental Methods

[0067] (1) Solution preparation

[0068] A certain amount of carboxylated chitosan, carboxylated chitin, chitosan, and chitin were weighed and prepared into four different stock solutions (pH = 7) with a molar concentration of 50 mM (1.0x) using sterile 0.05% Tween aqueous solution as the solvent. By diluting the stock solutions, 25 mM (0.5x) and 5 mM (0.1x) carboxylated chitosan solutions, carboxylated chitin solutions, chitosan solutions, and chitin solutions were prepared, with the pH of the prepared solutions all being 7. All prepared solutions were sterilized at 121°C and autoclaved for 20 min.

[0069] The specific preparation method is as follows:

[0070] 1.0x carboxylated chitosan or chitin solution: add 0.01 mol of carboxylated chitosan or carboxylated chitin to 200 mL of sterilized 0.05% Tween aqueous solution, adjust the solution to pH = 7, filter, and sterilize at 121°C and high temperature and high pressure for 20 min.

[0071] 0.5x, 0.1x carboxylated chitosan or chitin solution: add 0.01 mol of carboxylated chitosan or carboxylated chitin to 200 mL of sterilized 0.05% Tween aqueous solution, adjust the solution to pH = 7 and filter. Draw out 100 mL and 20 mL of the solution and mix them with 100 mL and 180 mL of water respectively, and sterilize at 121°C and high temperature and high pressure for 20 min.

[0072] 1.0x chitosan or chitin solution: add 0.01 mol chitosan or chitin to 200 mL of sterilized 0.05% Tween aqueous solution, adjust the solution pH to 7, and sterilize at 121°C under high temperature and high pressure for 20 min.

[0073] 0.5x, 0.1x chitosan or chitin solution: add 0.01 mol of carboxylated chitosan or chitin to 200 mL of sterilized 0.05% Tween aqueous solution, adjust the solution pH to 7, extract 100 mL and 20 mL of the solution and mix them with 100 mL and 180 mL of water respectively, and sterilize at 121°C and high temperature and high pressure for 20 min.

[0074] CZB solution: 2.0 g sodium nitrate (NaNO3), 0.5 g potassium chloride (KCl), 0.5 g magnesium sulfate (MgSO4), 1.0 g potassium phosphate (K2HPO4), 5.0 g tryptone (Tryptone), 0.01 g ferrous sulfate (FeSO4), 30 g sucrose, add water to 1000 mL, autoclave at 121°C for 20 min and set aside.

[0075] 0.05% Tween (TW) aqueous solution: 0.5 ml of Tween solution was diluted with water to 1000 ml, and sterilized by high pressure at 121°C for 20 min before use.

[0076] (2) Group processing

[0077] WT+0.1x carboxylated chitosan: WT strain mixed with 5 mM carboxylated chitosan solution.

[0078] WT+0.5x chitosan: WT strain mixed with 25 mM chitosan solution.

[0079] WT+0.1x carboxychitin: WT strain mixed with 5 mM carboxychitin solution.

[0080] WT+0.5x chitin: WT strain mixed with 25 mM chitin solution.

[0081] WT+0.05% TW: WT strain was mixed with 0.05% Tween aqueous solution.

[0082] WT+CZB: WT strain and CZB Czapek medium were mixed.

[0083] UV+0.1x carboxylated chitosan: UV strain mixed with 5 mM carboxylated chitosan solution.

[0084] UV+0.5x chitosan: UV strain mixed with 25 mM chitosan solution.

[0085] UV + 0.1x carboxychitin: Mix the UV strain and 5 mM carboxylated chitin solution.

[0086] UV+0.5x chitin: UV strain mixed with 25 mM chitin solution.

[0087] UV+0.05% TW: UV strain was mixed with 0.05% Tween aqueous solution.

[0088] UV+CZB: UV strain and CZB Czapek's culture medium are mixed.

[0089] The specific method is:

[0090] Take 1mL of the solution with a concentration of 1×10 7 A spore suspension of Combretum javanica (WT strain, UV strain) at spores / mL was added to a sterile conical flask (volume 50 mL), and 1 mL of chitosan solution (1.0x), chitin solution (1.0x), carboxylated chitosan solution or carboxylated chitin solution (1.0x) was added respectively. If the volume was less than 10 mL, 0.05% Tween water was added to make the volume 10 mL;

[0091] Positive control: 1 mL of fungal spores was mixed with 9 mL of CZB solution.

[0092] Negative control: 1 mL of fungal spores was mixed with 9 mL of 0.05% Tween water.

[0093] Each treatment group was cultured at 28°C and 180 rpm, and the germination of fungal spores was observed. The number of spores germinated at 5, 6, 7, 8, 9, 10, 11, and 12 hours was recorded. The germination rate of the fungal spores in different culture media was calculated.

[0094] 2. Experimental Results

[0095] The spore germination rates of the WT and UV strains of the different treatment groups are as follows Figure 1 As shown, the results show that:

[0096] At the same time point, the spore germination rates of the WT and UV strains in 0.1x carboxylated chitosan solution were significantly higher than those in 0.5x chitosan solution and 0.05% Tween water (p < 0.05), and even higher than those in CZB culture medium commonly used in the laboratory. Among them, the spore germination rate of the UV strain was significantly higher than that of the WT strain (p < 0.05). At 10 hours, the spore germination rates of the WT / UV strains in 0.1x carboxylated chitosan solution, CZB solution, 0.5x chitosan solution, and 0.05% Tween water were 66.0% / 73.6%, 62.0% / 70.2%, 55.8% / 61.3%, and 5.3% / 6.2%, respectively.

[0097] At the same time point, the spore germination rates of the WT and UV strains in 0.1x carboxylated chitin solution were significantly higher than those in 0.5x chitin solution and 0.05% Tween water (p < 0.05). Among them, the spore germination rate of the UV strain was significantly higher than that of the WT strain (p < 0.05). At 10 hours, the spore germination rates of the WT / UV strains in 0.1x carboxylated chitin solution, CZB solution, 0.5x chitin solution, and 0.05% Tween water were 62.2% / 72.4%, 58.4% / 65.8%, 50.2% / 59.1%, and 5.3% / 6.2%, respectively.

[0098] Based on the above experimental results, it was found that the spore germination rates of the WT / UV strain of Combretum javanicum in carboxylated chitosan and carboxylated chitin solutions were respectively increased by 60.7% / 67.4% and 56.9% / 65.8% compared with those in 0.05% Tween water.

[0099] Example 2 Effects of chitosan or chitin before and after carboxylation on spore germination of Metarhizium anisopliae

[0100] 1. Experimental Methods

[0101] (1) Solution preparation

[0102] The preparation methods of carboxylated chitosan solution, carboxylated chitin solution, chitosan solution, chitin solution, CZB solution and 0.05% Tween aqueous solution are the same as those in Example 1.

[0103] (2) Group processing

[0104] The treatment groups were set up the same as in Example 1, and the specific method was as follows:

[0105] Take 1mL of the solution with a concentration of 1×10 7 A spore suspension of Metarhizium anisopliae (WT strain, UV strain) with a spore volume of 50 mL / mL was added into a sterile conical flask (volume of 50 mL), and 1 mL of chitosan solution, chitin solution, carboxylated chitosan solution, and carboxylated chitin solution were added respectively. If the volume was less than 10 mL, 0.05% Tween water was added to make the volume up to 10 mL.

[0106] Positive control: 1 mL of fungal spores was mixed with 9 mL of CZB solution.

[0107] Negative control: 1 mL of fungal spores was mixed with 9 mL of 0.05% Tween water.

[0108] Each treatment group was cultured at 28°C and 180 rpm, and the germination of fungal spores was observed. The number of spores germinated at 16, 18, 20, 22, 24, and 26 hours was recorded. The germination rate of M. anisopliae spores in different culture media was calculated.

[0109] 2. Experimental Results

[0110] The spore germination of WT and UV strains of Metarhizium anisopliae in different treatment groups is shown in Figure 2. Figure 2 As shown, the structure shows that:

[0111] At the same time point, the spore germination rates of the WT and UV strains in 0.1x carboxylated chitosan solution were significantly higher than those in 0.5x chitosan solution and 0.05% Tween water (p < 0.05), and even higher than those in CZB culture medium commonly used in the laboratory. Among them, the spore germination rate of the UV strain was significantly higher than that of the WT strain (p < 0.05). At 24 hours, the spore germination rates of the WT / UV strains in 0.1x carboxylated chitosan solution, CZB solution, 0.5x chitosan solution, and 0.05% Tween water were 60.7% / 72.4%, 57.8% / 71.3%, 42.9% / 55.3%, and 4.0% / 5.3%, respectively.

[0112] At the same time point, the spore germination rates of the WT and UV strains in 0.1x carboxylated chitin solution were significantly higher than those in 0.5x chitin solution and 0.05% Tween water (p < 0.05), among which the spore germination rate of the UV strain was significantly higher than that of the WT strain (p < 0.05). At 24 h, the spore germination rates of the WT / UV strains in 0.1x carboxylated chitin solution, CZB solution, 0.5x chitin solution and 0.05% Tween water were 62.0% / 73.6%, 56.0% / 71.3%, 44.9% / 57.8%, and 2.4% / 5.3%, respectively.

[0113] Based on the above experimental results, it can be found that the spore germination rate of the WT / UV strain of Metarhizium anisopliae in carboxylated chitosan solution and carboxylated chitin solution is 56.7% / 67.1% and 59.6% / 68.3% higher than that in 0.05% Tween water.

[0114] Example 3 Effect of carboxylated chitosan or carboxylated chitin on the growth of Coryneformis javanica

[0115] 1. Experimental Methods

[0116] Water agar culture media (pH=7) containing chitosan, chitin, carboxylated chitosan, and carboxylated chitin at different concentrations (50 mM (1.0x), 25 mM (0.5x), and 5 mM (0.1x)) were prepared respectively.

[0117] The specific preparation method is as follows:

[0118] 1.0x carboxylated chitosan or carboxylated chitin water agar medium: add 200 mL of water to 0.01 mol of carboxylated chitosan or carboxylated chitin, adjust the solution pH to 7, add 4 g of agar powder, and sterilize at 121°C and high temperature and high pressure for 20 min.

[0119] 0.5x, 0.1x carboxylated chitosan or carboxylated chitin water agar medium: Add 200 mL of water to 0.01 mol of carboxylated chitosan or carboxylated chitin, adjust the solution to pH 7, extract 100 mL and 20 mL of the solution and mix them again with 100 mL and 180 mL of water, respectively, add 4 g of agar powder, and sterilize at 121°C and autoclave for 20 min.

[0120] 1.0x Chitosan or Chitin Water Agar Medium: Add 0.01 mol chitosan or chitin to 200 mL of water, adjust the solution to pH 7, add 4 g of agar powder, and sterilize at 121°C and autoclave for 20 min.

[0121] 0.5x, 0.1x chitosan or chitin water agar medium: Add 200 mL of water to 0.01 mol of carboxylated chitosan or chitin, adjust the solution to pH 7, extract 100 mL and 20 mL of the solution and mix them again with 100 mL and 180 mL of water, respectively, add 4 g of agar powder, and sterilize at 121°C and autoclave for 20 min.

[0122] Set up the processing groups:

[0123] WT (UV) + 1x carboxylated chitosan: WT strain (UV strain) was cultured in water agar medium containing 50 mM (1.0x) carboxylated chitosan;

[0124] WT (UV) + 0.5x carboxy chitosan: WT strain (UV strain) was cultured in water agar medium containing 25 mM (0.5x) carboxylated chitosan;

[0125] WT (UV) + 0.1x carboxylated chitosan: WT strain (UV strain) was cultured in water agar medium containing 5 mM (0.1x) carboxylated chitosan;

[0126] WT (UV) + 1x chitosan: WT strain (UV strain) was cultured in water agar medium containing 50 mM (1.0x) chitosan;

[0127] WT (UV) + 0.1x chitosan: WT strain (UV strain) was cultured in water agar medium containing 5 mM (0.1x) chitosan;

[0128] WT (UV) + 1x carboxychitin: WT strain (UV strain) was cultured in water agar medium containing 50 mM (1.0x) carboxychitin;

[0129] WT (UV) + 0.5x carboxychitin: WT strain (UV strain) was cultured in water agar medium containing 25 mM (0.5x) carboxychitin;

[0130] WT (UV) + 0.1x carboxychitin: WT strain (UV strain) was cultured in water agar medium containing 5 mM (0.1x) carboxychitin;

[0131] WT (UV) + 1x chitin: WT strain (UV strain) was cultured in water agar medium containing 50 mM (1.0x) chitin;

[0132] WT (UV) + 0.1x chitin: WT strain (UV strain) was cultured in water agar medium containing 5 mM (0.1x) chitin;

[0133] WT (UV) + water: WT strain (UV strain) was cultured in water agar medium.

[0134] The specific experimental methods are:

[0135] The inoculation concentration was 5×10 6 3 μL of a spore suspension of Coryneformis javanica (WT strain, UV strain) at a concentration of spores / mL was placed in an incubator at 28°C and incubated upside down. The diameter of the colonies was measured and recorded every day.

[0136] 2. Experimental Results

[0137] The colony diameters of Mycosporium javanicum on 1x carboxylated chitosan, 0.5x carboxylated chitosan, 0.1x carboxylated chitosan, 1x chitosan, 0.1x chitosan medium and water agar medium are as follows: Figure 3 As shown in Figures a and b, the results show that the colony diameters of the WT and UV strains of C. javanica on the six different culture media were ranked from largest to smallest: 1x carboxylated chitosan > 0.5x carboxylated chitosan > 0.1x carboxylated chitosan > 1x chitosan > 0.1x chitosan > water agar. On day 12, the colony diameters of the WT / UV strains of C. javanica on 0.5x carboxylated chitosan medium were 3.9 cm / 4.0 cm, respectively. These diameters were not significantly different from those on 1x carboxylated chitosan medium, but were significantly larger than those on 0.1x carboxylated chitosan medium, 1x chitosan medium, and 0.1x chitosan medium, and were 43.7% / 44.8% larger than those on water agar.

[0138] The colony diameters of Mycosporium javanicum on 1x carboxylated chitin, 0.5x carboxylated chitin, 0.1x carboxylated chitin, 1x chitin, 0.1x chitin medium and water agar medium are as follows: Figure 3As shown in Figures c and d, the results show that the colony diameters of the WT and UV strains of C. javanica on the six different culture media were ranked from largest to smallest: 1x carboxylated chitin > 0.5x carboxylated chitin > 0.1x carboxylated chitin > 1x chitin > 0.1x chitin > water agar. On day 12, the colony diameters of the WT / UV strains of C. javanica on 0.5x carboxylated chitin medium were 3.8 cm / 3.9 cm, respectively. These were not significantly different from those on 1x carboxylated chitin medium, but were significantly larger than those on 0.1x carboxylated chitin, 1x chitin, and 0.1x chitin medium, and were 38.8% / 41.2% larger than those on water agar.

[0139] Based on the above analysis data, it can be concluded that there is no significant difference between 0.5x carboxylated chitosan or chitin medium and 1x carboxylated chitin medium in supporting the growth of C. javanica (p>0.05). Considering the cost and other aspects, we used 0.5x carboxylated chitosan or chitin medium for subsequent experiments.

[0140] Example 4 Effect of carboxylated chitosan or carboxylated chitin on the growth of Metarhizium anisopliae

[0141] 1. Experimental Methods

[0142] Water agar culture media (pH=7) containing chitosan, chitin, carboxylated chitosan, and carboxylated chitin at different concentrations (50 mM (1.0x), 25 mM (0.5x), and 5 mM (0.1x)) were prepared respectively. The preparation method was the same as that in Example 3.

[0143] Set up the processing groups:

[0144] WT (UV) + 1x carboxylated chitosan: WT strain (UV strain) was cultured in water agar medium containing 50 mM (1.0x) carboxylated chitosan;

[0145] WT (UV) + 0.5x carboxy chitosan: WT strain (UV strain) was cultured in water agar medium containing 25 mM (0.5x) carboxylated chitosan;

[0146] WT (UV) + 0.1x carboxylated chitosan: WT strain (UV strain) was cultured in water agar medium containing 5 mM (0.1x) carboxylated chitosan;

[0147] WT (UV) + 1x chitosan: WT strain (UV strain) was cultured in water agar medium containing 50 mM (1.0x) chitosan;

[0148] WT (UV) + 0.1x chitosan: WT strain (UV strain) was cultured in water agar medium containing 5 mM (0.1x) chitosan;

[0149] WT (UV) + 1x carboxychitin: WT strain (UV strain) was cultured in water agar medium containing 50 mM (1.0x) carboxychitin;

[0150] WT (UV) + 0.5x carboxychitin: WT strain (UV strain) was cultured in water agar medium containing 25 mM (0.5x) carboxychitin;

[0151] WT (UV) + 0.1x carboxychitin: WT strain (UV strain) was cultured in water agar medium containing 5 mM (0.1x) carboxychitin;

[0152] WT (UV) + 1x chitin: WT strain (UV strain) was cultured in water agar medium containing 50 mM (1.0x) chitin;

[0153] WT (UV) + 0.1x chitin: WT strain (UV strain) was cultured in water agar medium containing 5 mM (0.1x) chitin;

[0154] WT (UV) + water: WT strain (UV strain) was cultured in water agar medium.

[0155] The specific experimental methods are:

[0156] The inoculation concentration was 5×10 6 3 μL of a spore suspension of Metarhizium anisopliae (WT strain, UV strain) at a spore count / mL was placed in an incubator at 28°C and inverted for culture. The diameter of the colonies was measured and recorded daily.

[0157] 2. Experimental Results

[0158] The colony diameters of Metarhizium anisopliae on 1x carboxylated chitosan, 0.5x carboxylated chitosan, 0.1x carboxylated chitosan, 1x chitosan, 0.1x chitosan medium and water agar medium are as follows: Figure 4As shown in Figures a and b, the results show that on six different culture media, the colony diameters of the WT and UV strains of Metarhizium anisopliae are ranked from largest to smallest in the following order: 1x carboxylated chitosan > 0.5x carboxylated chitosan > 0.1x carboxylated chitosan > 1x chitosan > 0.1x chitosan > water agar medium. Among them, at day 12, the colony diameters of the WT / UV strains of Metarhizium anisopliae on 0.5x carboxylated chitosan medium were 3.4 / 3.4 cm, respectively. These were not significantly different from the colony diameters on 1x carboxylated chitosan medium, but were significantly higher than the colony diameters on 0.1x carboxylated chitosan medium, 1x chitosan medium, and 0.1x chitosan medium, and were 32.1% / 27.2% higher than the colony diameters on water agar medium.

[0159] The colony diameters of Metarhizium anisopliae on 1x carboxylated chitin, 0.5x carboxylated chitin, 0.1x carboxylated chitin, 1x chitin, 0.1x chitin medium and water agar medium are as follows: Figure 4 As shown in Figures c and d, the results show that on six different culture media, the colony diameters of the WT and UV strains of Metarhizium anisopliae were ranked from largest to smallest in the following order: 1x carboxylated chitin > 0.5x carboxylated chitin > 0.1x carboxylated chitin > 1x chitin > 0.1x chitin > water agar medium. Among them, at day 12, the colony diameters of the WT / UV strains of Metarhizium anisopliae on 0.5x carboxylated chitin medium were 3.5 / 3.5 cm, respectively. These were not significantly different from the colony diameters on 1x carboxylated chitin medium, but were significantly higher than the colony diameters on 0.1x carboxylated chitin medium, 1x chitin medium, and 0.1x chitin medium, and were 33.3% / 30.9% larger than the colony diameters on water agar medium.

[0160] Based on the above analysis data, it can be concluded that there is no significant difference between 0.5x carboxylated chitin medium and 1x carboxylated chitin medium in supporting the growth of Metarhizium anisopliae (p>0.05). Considering the cost and other aspects, we used 0.5x carboxylated chitin medium for subsequent experiments.

[0161] Example 5 Carboxylated chitosan or carboxylated chitin can improve the ability of the fungus to resist ultraviolet radiation

[0162] 1. Experimental Methods

[0163] (1) Solution preparation

[0164] Weigh a certain amount of carboxylated chitosan and carboxylated chitin, and use 0.05% Tween aqueous solution as the solvent to prepare a carboxylated chitosan solution and a carboxylated chitin solution with a molar concentration of 5 mM (0.1x). Sterilize the carboxylated chitosan solution, carboxylated chitin solution, and 0.05% Tween aqueous solution at 121°C and autoclave for 20 minutes before use.

[0165] (2) Group processing

[0166] WT (UV) + 0.1x carboxylated chitosan: WT strain (UV strain) was cultured with 5 mM (0.1x) carboxylated chitosan solution;

[0167] WT(UV)+0.05% TW: WT strain (UV strain) cultured with 0.05% Tween aqueous solution;

[0168] WT (UV) + 0.1x carboxychitin: WT strain (UV strain) and 5 mM (0.1x) carboxylated chitin solution were cultured.

[0169] The specific experimental methods are:

[0170] The concentration of 1 mL of the solution irradiated with ultraviolet light for 40 min was 1×10 7 A spore suspension of Combresporium javanica (WT strain, UV strain) with a spore volume of 50 mL / mL was added to a sterile conical flask (50 mL), and then 9 mL of 0.1x carboxylated chitosan solution, carboxylated chitin solution, and 0.05% Tween aqueous solution were added to the conical flask respectively.

[0171] Each treatment group was cultured at 28°C and 180 rpm, and spore germination was observed. The number of spores germinated was recorded at 5, 6, 7, 8, 9, 10, 11, and 12 hours. The effects of carboxylated chitosan or chitin solutions on the germination rate of spores of Mycosporium javanicum or Metarhizium anisopliae after ultraviolet irradiation were calculated.

[0172] 2. Experimental Results

[0173] The spore germination rates of the WT and UV strains of C. javanica after irradiation with ultraviolet light for 40 minutes are shown in Figure 2. Figure 5 As shown, the results show that:

[0174] At the same time point, the spore germination rates of WT strain and UV strain in carboxylated chitosan solution were significantly higher than those in 0.05% Tween water (p<0.05). At 10 h, the spore germination rates of WT / UV strains in carboxylated chitosan solution were 36.4% / 62.7%, and those in 0.05% Tween water were 1.6% / 7.8%, respectively; among them, the spore germination rate of UV strain was significantly higher than that of WT strain (p<0.05).

[0175] At the same time point, the spore germination rates of the WT and UV strains in carboxylated chitin solution were significantly higher than those in 0.05% Tween water (p < 0.05). At 10 hours, the spore germination rates of the WT / UV strains in carboxylated chitin solution were 36.7% / 62.2%, and in 0.05% Tween water, respectively, were 2.4% / 7.8%. Among them, the spore germination rate of the UV strain was significantly higher than that of the WT strain (p < 0.05).

[0176] In summary, the spore germination rates of the WT / UV strain in carboxylated chitosan and chitin solutions after 40 minutes of ultraviolet irradiation were 34.8% / 54.9% and 34.3% / 54.4% higher than those in 0.05% Tween water.

[0177] Example 6 Carboxylated chitosan or carboxylated chitin can improve the ability of Metarhizium to resist ultraviolet rays

[0178] 1. Experimental Methods

[0179] (1) Solution preparation

[0180] Weigh a certain amount of carboxylated chitosan and carboxylated chitin, and use 0.05% Tween aqueous solution as solvent (pH = 7) to prepare a carboxylated chitosan solution and a carboxylated chitin solution with a molar concentration of 5 mM (0.1x). Sterilize the carboxylated chitosan solution, carboxylated chitin solution, and 0.05% Tween aqueous solution at 121°C and autoclave for 20 minutes before use.

[0181] (2) Group processing

[0182] WT (UV) + 0.1x carboxylated chitosan: WT strain (UV strain) was cultured with 5 mM (0.1x) carboxylated chitosan solution;

[0183] WT(UV)+0.05% TW: WT strain (UV strain) cultured with 0.05% Tween aqueous solution;

[0184] WT (UV) + 0.1x carboxychitin: WT strain (UV strain) and 5 mM (0.1x) carboxylated chitin solution were cultured.

[0185] The specific experimental methods are:

[0186] The concentration of 1 mL of the solution irradiated with ultraviolet light for 40 min was 1×10 7A spore suspension of Metarhizium anisopliae (WT strain, UV strain) with a spore / mL was added into a sterile conical flask (volume: 50 mL), and then 9 mL of 0.1x carboxylated chitosan solution, 0.1x carboxylated chitin solution and 0.05% Tween water were respectively added into the above conical flask.

[0187] Each treatment group was cultured at 28°C and 180 rpm, and spore germination was observed. The number of spores germinated was recorded at 5, 6, 7, 8, 9, 10, 11, and 12 hours. The effects of carboxylated chitosan or chitin solutions on the germination rate of spores of Mycosporium javanicum or Metarhizium anisopliae after ultraviolet irradiation were calculated.

[0188] 2. Experimental Results

[0189] The spore germination rates of the WT and UV strains of Metarhizium anisopliae after 40 minutes of ultraviolet irradiation are as follows: Figure 6 As shown, the results show that:

[0190] At the same time point, the spore germination rates of the WT and UV strains in carboxylated chitosan solution were significantly higher than those in 0.05% Tween water (p < 0.05). At 24 hours, the spore germination rates of the WT / UV strains in carboxylated chitosan solution were 22.0% / 67.3%, and those in 0.05% Tween water were 1.6% / 5.1%, respectively. Among them, the spore germination rate of the UV strain was significantly higher than that of the WT strain (p < 0.05).

[0191] At the same time point, the spore germination rates of the WT and UV strains in carboxylated chitin solution were significantly higher than those in 0.05% Tween water (p < 0.05). At 24 hours, the spore germination rates of the WT / UV strains in carboxylated chitin solution were 23.1% / 68.2%, and those in 0.05% Tween water were 1.6% / 5.1%, respectively. Among them, the spore germination rate of the UV strain was significantly higher than that of the WT strain (p < 0.05).

[0192] In summary, the spore germination rates of the WT / UV strain in carboxylated chitosan and chitin solutions after 40 minutes of ultraviolet irradiation were 20.4% / 62.2% and 21.5% / 63.1% higher than those in 0.05% Tween water.

[0193] Example 7 Carboxylated chitosan or carboxylated chitin can improve the high temperature resistance of Coryneformis javanica

[0194] 1. Experimental Methods

[0195] (1) Solution preparation

[0196] Weigh a certain amount of carboxylated chitosan and carboxylated chitin, and use 0.05% Tween aqueous solution as solvent (pH = 7) to prepare a carboxylated chitosan solution and a carboxylated chitin solution with a molar concentration of 5 mM (0.1x). Sterilize the carboxylated chitosan solution, carboxylated chitin solution, and 0.05% Tween aqueous solution at 121°C and autoclave for 20 minutes before use.

[0197] (2) Group processing

[0198] WT (UV)-10% carboxylated chitosan-33°C: WT strain (UV strain) and 0.1x carboxylated chitosan solution were cultured at 33°C;

[0199] WT (UV)-10% carboxylated chitosan-35°C: WT strain (UV strain) and 0.1x carboxylated chitosan solution were cultured at 35°C;

[0200] WT (UV)-10% carboxymethyl-33°C: WT strain (UV strain) and 0.1x carboxylated chitin solution were cultured at 33°C;

[0201] WT (UV)-10% carboxymethyl-35°C: WT strain (UV strain) and 0.1x carboxylated chitin solution were cultured at 35°C;

[0202] WT(UV)-0.05% TW-33°C: WT strain (UV strain) and 0.05% Tween aqueous solution were cultured at 33°C;

[0203] WT (UV)-0.05% TW-35°C: WT strain (UV strain) and 0.05% Tween aqueous solution were cultured at 35°C.

[0204] The specific experimental methods are:

[0205] Take 1mL of the solution with a concentration of 1×10 7 A spore suspension of Combresporium javanica (WT strain, UV strain) with a spore volume of 50 mL / mL was added to a sterile conical flask (50 mL), and then 9 mL of 0.1x carboxylated chitosan solution, carboxylated chitin solution, and 0.05% Tween aqueous solution were added to the conical flask respectively.

[0206] Each treatment group was cultured at 33°C and 35°C, at 180 rpm, and spore germination was observed. The number of spores germinated was recorded at 5, 6, 7, 8, 9, 10, 11, and 12 hours. The effects of carboxylated chitosan or chitin solutions on the spore germination rate of Mycosporium javanicum or Metarhizium anisopliae after ultraviolet irradiation were calculated.

[0207] 2. Experimental Results

[0208] The spore germination rates of WT and UV strains of C. javanica at 33℃ and 35℃ were as follows: Figure 7 As shown, the results show that:

[0209] At the same time point, the spore germination rates of the WT and UV strains in carboxylated chitosan solution were significantly higher than those in 0.05% Tween water (p < 0.05). At 33°C and 10 hours, the spore germination rates of the WT / UV strains in carboxylated chitosan solution were 40.2% / 72.4%, and in 0.05% Tween water, respectively. At 35°C and 10 hours, the spore germination rates of the WT / UV strains in carboxylated chitosan solution were 1.6% / 18.0%, and in 0.05% Tween water, respectively, were 0.7% / 4.2%.

[0210] At the same time point, the spore germination rates of the WT and UV strains in carboxylated chitin solution were significantly higher than those in 0.05% Tween water (p < 0.05). At 33°C and 10 hours, the spore germination rates of the WT / UV strains in carboxylated chitin solution were 46.9% / 67.1%, and in 0.05% Tween water, respectively, were 4.2% / 6.7%. At 35°C and 10 hours, the spore germination rates of the WT / UV strains in carboxylated chitin solution were 1.1% / 14.7%, and in 0.05% Tween water, respectively, were 0.7% / 4.2%.

[0211] Based on the above analysis, it can be found that carboxylated chitosan or chitin can improve the spore germination rate of Mycospora javanica under ultraviolet irradiation and high temperature, and the spore germination rate of UV strain is higher under the same conditions.

[0212] Example 8 Carboxylated chitosan or carboxylated chitin can improve the ability of Metarhizium anisopliae to resist high temperatures

[0213] 1. Experimental Methods

[0214] (1) Solution preparation

[0215] Weigh a certain amount of carboxylated chitosan and carboxylated chitin, and use 0.05% Tween aqueous solution as solvent (pH = 7) to prepare a carboxylated chitosan solution and a carboxylated chitin solution with a molar concentration of 5 mM (0.1x). Sterilize the carboxylated chitosan solution, carboxylated chitin solution, and 0.05% Tween aqueous solution at 121°C and autoclave for 20 minutes before use.

[0216] (2) Group processing

[0217] WT (UV)-10% carboxylated chitosan-33°C: WT strain (UV strain) and 0.1x carboxylated chitosan solution were cultured at 33°C;

[0218] WT (UV)-10% carboxylated chitosan-35°C: WT strain (UV strain) and 0.1x carboxylated chitosan solution were cultured at 35°C;

[0219] WT (UV)-10% carboxymethyl-33°C: WT strain (UV strain) and 0.1x carboxylated chitin solution were cultured at 33°C;

[0220] WT (UV)-10% carboxymethyl-35°C: WT strain (UV strain) and 0.1x carboxylated chitin solution were cultured at 35°C;

[0221] WT(UV)-0.05% TW-33°C: WT strain (UV strain) and 0.05% Tween aqueous solution were cultured at 33°C;

[0222] WT (UV)-0.05% TW-35°C: WT strain (UV strain) and 0.05% Tween aqueous solution were cultured at 35°C.

[0223] The specific experimental methods are:

[0224] Take 1mL of the solution with a concentration of 1×10 7 A spore suspension of Metarhizium anisopliae (WT strain, UV strain) with a spore / mL was added into a sterile conical flask (volume: 50 mL), and then 9 mL of 0.1x carboxylated chitosan solution, carboxylated chitin solution, and 0.05% Tween aqueous solution were respectively added into the above conical flask.

[0225] Each treatment group was cultured at 33°C and 35°C, at 180 rpm, and spore germination was observed. The number of spores germinated was recorded at 16, 18, 20, 22, 24, and 26 hours. The effects of carboxylated chitosan or chitin solutions on the spore germination rate of Mycosporium javanicum or Metarhizium anisopliae after ultraviolet irradiation were calculated.

[0226] 2. Experimental Results

[0227] The spore germination rates of WT and UV strains of Metarhizium anisopliae at 33℃ and 35℃ were as follows: Figure 8 As shown, the results show that:

[0228] At the same time point, the spore germination rates of the WT and UV strains in carboxylated chitosan solution were significantly higher than those in 0.05% Tween water (p < 0.05). At 33°C and 24 hours, the spore germination rates of the WT / UV strains in carboxylated chitosan solution were 28.2% / 61.6%, and in 0.05% Tween water were 1.7% / 4.7%, respectively. At 35°C and 24 hours, the spore germination rates of the WT / UV strains in carboxylated chitosan solution were 6.9% / 35.6%, and in 0.05% Tween water were 1.1% / 3.1%, respectively.

[0229] At the same time point, the spore germination rates of the WT and UV strains in carboxylated chitin solution were significantly higher than those in 0.05% Tween water (p < 0.05). At 33°C and 24h, the spore germination rates of the WT / UV strains in carboxylated chitin solution were 29.3% / 64.2%, and in 0.05% Tween water were 1.1% / 3.1%, respectively. At 35°C and 24h, the spore germination rates of the WT / UV strains in carboxylated chitin solution were 6.7% / 35.3%, and in 0.05% Tween water were 1.1% / 3.1%, respectively.

[0230] Based on the above analysis, it can be found that carboxylated chitosan or chitin can improve the spore germination rate of Metarhizium anisopliae under ultraviolet irradiation and high temperature, and the spore germination rate of UV strain is higher under the same conditions.

[0231] Example 9 Carboxylated chitosan or carboxylated chitin can enhance the toxicity of Mycosporium javanicum and Metarhizium anisopliae

[0232] 1. Experimental Methods

[0233] The toxicity of carboxylated chitosan and carboxylated chitin to brown planthopper was tested after they were mixed with Corynespora javanica and Metarhizium anisopliae, respectively.

[0234] The spores of Mycelia javanica and Metarhizium anisopliae were prepared with 0.05% Tween water to a concentration of 0 and 1.0×10 3 , 1.0×10 4 , 1.0×10 5 , 1.0×10 6 , 1.0×10 7 To a spore suspension having a concentration of spores / mL, carboxylated chitosan or carboxylated chitin is added to form a mixed solution, wherein the concentration of the carboxylated chitosan or carboxylated chitin in the mixed solution is 25 mM.

[0235] Different concentrations of the mixture were sprayed onto rice plants infested with fourth-instar nymphs of the brown planthopper. The rice plants were potted rice seedlings (35 cm tall) grown in 60 × 60 × 100 cm insect cages and placed in an insectary at 28°C. The mortality of brown planthopper nymphs was observed and recorded daily for 15 days, and the median lethal concentration (LC50) of the strain against brown planthoppers was then calculated. 50 ) and shortening the median lethal time (LT 50 ).

[0236] 2. Experimental Results

[0237] Carboxylated chitosan or carboxylated chitin were mixed with different concentrations of spores of Coryneformis javanica and infected to different degrees of death of brown planthoppers. In the control area treated with 0.05% Tween water, the mortality rate of brown planthoppers on the 4th / 7th day was 6.7% / 12.5%; while the concentration of 1×10 6 The mortality rates of brown planthopper nymphs in the treatment areas of WT strain of Coryneformis javanica and its mixture with carboxylated chitosan or chitin at 4 / 7 days were 41.7% / 74.2%, 65.8% / 88.3%, and 63.3% / 85.8%, respectively; the concentration of 1×10 6 In the treatment areas with spores of the UV strain of javanica and its mixture with carboxylated chitosan or chitin, the mortality rates of brown planthopper nymphs on the 4th / 7th day were 60.8% / 85.8%, 82.5% / 95.0%, and 82.5% / 93.3%, respectively.

[0238] LC of carboxylated chitosan or carboxylated chitin mixed with Corynespora javanica or Metarhizium anisopliae against Nilaparvata lugens 50 and LT 50 like Figure 9 、 Figure 10 、 Figure 11 and Figure 12 As shown in the results, carboxylated chitosan or chitin can enhance the toxicity of Coryneformis javanica or Metarhizium anisopliae to brown planthoppers. Among them, the LT of Coryneformis javanica WT strain mixed with carboxylated chitosan to brown planthopper nymphs was significantly higher than that of 50 The LC value of the mixture was reduced by 1.6d compared with that before mixing. 50 The LT of the mixture of the UV strain of Coryneformis javanica and carboxylated chitin against the nymphs of brown planthopper was reduced by 98.3% / 68.9% compared with that before mixing. 50 The LC value of the mixture was reduced by 1.3d compared with that before mixing, and the ... 50 The LT of Metarhizium anisopliae WT strain mixed with carboxylated chitin against brown planthopper nymphs was reduced by 80.3% / 30.1% compared with that before mixing. 50 Compared with the pre-mixing, the LC at 7d / 12d was reduced by 2.2d. 50The LT of the mixed green anisopliae UV strain and carboxylated chitin against brown planthopper nymphs was reduced by 96.5% / 94.6% compared with that before mixing. 50 The LC value of the mixture was reduced by 1.0d compared with that before mixing. 50 The UV strain was more virulent to brown planthoppers than the WT strain, with a higher mortality rate.

[0239] Example 10 Effect of carboxylated chitosan or chitin on the growth of bacterial blight pathogen

[0240] 1. Experimental Methods

[0241] The bacterial blight pathogen was inoculated on water agar medium containing 0.5x carboxylated chitosan or carboxylated chitin, BPM medium containing 0.5x carboxylated chitosan or carboxylated chitin, BPM medium and water agar medium, and the growth of the pathogen was observed regularly.

[0242] Bacterial leaf streak pathogens were inoculated into NA medium, NA medium containing 0.5x carboxylated chitosan, and NA medium containing 0.5x carboxylated chitin, respectively, and the growth of the pathogens was observed regularly.

[0243] The preparation method of 0.5x carboxylated chitosan or carboxylated chitin water agar culture medium is the same as that in Example 3.

[0244] Beef extract peptone medium (BPM): 3g beef extract, 10g peptone, 5g sodium chloride (NaCl), 20g agar powder, adjust the pH to 7.4-7.6, add water to 1000mL, and sterilize at 121℃ for 20min.

[0245] 0.5x carboxylated chitosan or carboxylated chitin BPM medium: Add 200 mL of water to 0.01 mol of carboxylated chitosan or chitin, adjust the solution to pH 7, and filter. Draw out 100 mL of the solution and add 0.3 g of beef extract, 1.0 g of peptone, 0.5 g of sodium chloride (NaCl), and 4.0 g of agar powder. Finally, adjust the volume to 200 mL and sterilize at 121°C and autoclave for 20 min.

[0246] Nutrient agar medium (NA): 5 g peptone, 1 g yeast extract, 3 g beef extract, 10 g sucrose, 20 g agar powder. Adjust the pH to 6-6.5 and add water to 1000 mL. Sterilize at 121°C and autoclave for 20 min.

[0247] 0.5x NA medium with carboxylated chitosan or carboxylated chitin: Add 200 mL of water to 0.01 mol of carboxylated chitosan or carboxylated chitin, adjust the solution to pH 7, and filter. Draw out 100 mL of the solution and add 0.5 g of peptone, 0.1 g of yeast extract, 0.3 g of beef extract, 1.0 g of sucrose, and 4.0 g of agar powder. Finally, adjust the volume to 200 mL and sterilize at 121°C and autoclave for 20 min.

[0248] 2. Experimental Results

[0249] In order to clarify the effects of carboxylated chitosan or carboxylated chitin on rice bacterial blight, we evaluated the effects of carboxylated chitosan or carboxylated chitin on the growth of rice bacterial blight under solid culture conditions. Figure 13 The results show that the bacterial blight pathogen grows well on BPM solid medium, but cannot grow when the BPM solid medium contains 0.5x carboxylated chitosan or carboxylated chitin. The bacterial blight pathogen also cannot grow on water agar medium or on water agar medium containing 0.5x carboxylated chitosan or carboxylated chitin. This indicates that carboxylated chitosan or carboxylated chitin can effectively inhibit the growth of rice bacterial blight.

[0250] Bacterial leaf streak disease can grow normally on NA medium. When the NA medium contains 0.5x carboxylated chitosan or chitin, the pathogen can also grow. This shows that the inhibitory effect of carboxylated chitosan or chitin on the growth of rice bacterial leaf streak disease is not obvious.

[0251] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of a carboxylated polysaccharide in promoting the growth of insecticidal fungi, or in preparing a product that promotes the growth of insecticidal fungi, characterized in that: The carboxylated polysaccharide is carboxylated chitin or carboxylated chitosan, and the insecticidal fungus is Corynespora or Metarhizium anisopliae.

2. The use of carboxylated polysaccharides in improving the anti-ultraviolet ability of insecticidal fungi, or in preparing products that improve the anti-ultraviolet ability of insecticidal fungi, characterized in that: The carboxylated polysaccharide is carboxylated chitin or carboxylated chitosan, and the insecticidal fungus is Corynespora or Metarhizium anisopliae.

3. Use of carboxylated polysaccharides in improving the temperature tolerance of insecticidal fungi, or in preparing products for improving the temperature tolerance of insecticidal fungi, characterized in that: The carboxylated polysaccharide is carboxylated chitin or carboxylated chitosan, and the insecticidal fungus is Corynespora or Metarhizium anisopliae.

4. Use of a carboxylated polysaccharide in improving the toxicity of an insecticidal fungus to pests, or in preparing a method for improving the toxicity of an insecticidal fungus to pests, characterized in that: The carboxylated polysaccharide is carboxylated chitin or carboxylated chitosan, and the insecticidal fungus is Corynespora or Metarhizium anisopliae.

5. The application of carboxylated polysaccharide in combination with insecticidal fungi in preventing and controlling crop diseases and insect pests, characterized in that: The carboxylated polysaccharide is carboxylated chitin or carboxylated chitosan, and the insecticidal fungus is Corynespora or Metarhizium anisopliae.

6. Use of a carboxylated polysaccharide in inhibiting the growth of bacterial blight pathogen, or in preparing a product for inhibiting the growth of bacterial blight pathogen, characterized in that: The carboxylated polysaccharide is carboxylated chitin or carboxylated chitosan.

7. An insecticide, characterized in that: The invention comprises carboxylated polysaccharide and insecticidal fungus; the carboxylated polysaccharide is carboxylated chitin or carboxylated chitosan, and the insecticidal fungus is Coryneformis or Metarhizium anisopliae.

8. The insecticide according to claim 7, characterized in that: The invention comprises a carboxylated polysaccharide and an insecticidal fungus spore suspension, wherein the concentration of the carboxylated polysaccharide is not less than 5 mM.

9. The insecticide according to claim 7, characterized in that: The concentration of spore suspension is not less than 1×10 3 spores / mL.

10. Use of the insecticide according to any one of claims 7 to 9 in controlling crop pests, or in controlling plant diseases caused by crop pests.

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