A control agent for sclerotinia sclerotiorum of rape and a preparation method thereof

Abstract

The present application relates to the field of biological control, in particular to a kind of rape sclerotinia rot prevention and control agent and preparation method thereof.The rape sclerotinia rot prevention and control agent provided by the present application includes enterobacter cloacae fermentation liquor, methylotrophic bacteria fermentation liquor, trichoderma viride fermentation liquor and leptosporella gigantea fermentation liquor.The rape sclerotinia rot prevention and control agent is sprayed on the surface of rape at seedling stage, which can effectively prevent and control rape sclerotinia rot, and the field control efficiency reaches 79%;And enterobacter cloacae, methylotrophic bacteria, trichoderma viride and leptosporella gigantea have obvious synergistic effect in the prevention and control of rape sclerotinia rot.The present application provides a new combined microbial agent for preventing and controlling rape sclerotinia rot, which has wide application prospect due to wide source and remarkable effect.

Description

Technical Field

[0001] This invention relates to the field of biological control, and in particular to a control agent for sclerotinia stem rot in rapeseed and its preparation method. Background Technology

[0002] Rapeseed is one of the world's most important oilseed crops and a significant source of edible oil in my country. Its seeds contain 33%-50% oil, providing 57% of my country's domestically produced vegetable oil. Rapeseed is susceptible to various diseases and pests during its growth and development, with sclerotinia stem rot being the most common. Sclerotinia stem rot, caused by *Sclerotinia sclerotiorum*, is a global disease that severely restricts high and stable rapeseed yields. In recent years, with changes in ecological conditions and the widespread adoption of low-erucic acid and low-erucic acid rapeseed, the incidence of sclerotinia stem rot in rapeseed has shown an increasing trend.

[0003] Sclerotinia sclerotinia disease is a significant disease affecting rapeseed in the main rapeseed-producing areas of the middle and lower reaches of the Yangtze River in my country, and is classified as a Class A crop disease. Depending on the production region and corresponding climatic conditions, the incidence rate of sclerotinia sclerotinia disease generally ranges from 10% to 80%, resulting in yield losses of 10% to 70%. Furthermore, sclerotinia sclerotinia disease is also a major biological stress affecting rapeseed oil quality. When low-erucic acid and low-carboxylic acid (LDA) rapeseed varieties are infected with sclerotinia sclerotinia, the levels of erucic acid and hexadecyltrienoic acid in the rapeseed oil increase, as does the protein content, leading to a decrease in rapeseed oil quality. Therefore, sclerotinia sclerotinia disease not only has a significant impact on rapeseed yield but also greatly affects the oil content and quality of rapeseed.

[0004] Sclerotinia stem rot is characterized by a large number of hosts, wide adaptability, and strong resistance to adverse conditions. Coupled with the complexity of its pathogenic mechanism, chemical pesticides are primarily used for control in agricultural production. However, chemical control is costly, its effectiveness is unsatisfactory, and it easily causes environmental pollution. Therefore, there is an urgent need to develop a green control method that can effectively prevent and control sclerotinia stem rot in rapeseed. Summary of the Invention

[0005] The purpose of this invention is to provide a control agent for rapeseed sclerotinia stem rot and its preparation method, thereby solving the problems existing in the prior art. The control agent for rapeseed sclerotinia stem rot provided by this invention can effectively control rapeseed sclerotinia stem rot, achieving a field control efficacy of 79%, and has the advantages of being green and pollution-free.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a control agent for sclerotinia stem rot in rapeseed, comprising fermentation broth of Leuconostoc mesenteroides, fermentation broth of Bacillus methylotrophicus, fermentation broth of Trichoderma viride, and fermentation broth of Gastrodinium grandis. The effective viable count of Leuconostoc mesenteroides in the fermentation broth was 1×10⁻⁶. 7 CFU / mL; The effective viable count of Bacillus methyltrophicus in the fermentation broth was 1×10⁻⁶. 8 CFU / mL; The effective viable count of *Trichoderma viride* in the *Trichoderma viride* fermentation broth was 1 × 10⁻⁶. 8 CFU / mL; The effective viable count of *Gymnospermum macrocephala* in the fermentation broth was 1×10⁻⁶. 7 CFU / mL.

[0007] Preferably, the volume ratio of the *Leuconostoc mesenteroides* fermentation broth, the *Bacillus methyltrophicus* fermentation broth, the *Trichoderma viride* fermentation broth, and the *Gnaphalium fasciatus* fermentation broth is 1:2:1:1.

[0008] Preferably, the method for preparing the Leuconostoc mesenteroides fermentation broth includes the step of fermenting and culturing activated Leuconostoc mesenteroides to obtain the Leuconostoc mesenteroides fermentation broth; The method for preparing the fermentation broth of the methyltrophic Bacillus includes the step of fermenting and culturing activated methyltrophic Bacillus to obtain the fermentation broth of the methyltrophic Bacillus. The preparation method of the green Trichoderma fermentation broth includes the step of fermenting and culturing activated green Trichoderma to obtain the green Trichoderma fermentation broth; The preparation method of the fermentation broth of *Gastrodinium macrocarpa* includes the step of fermenting and culturing activated *Gastrodinium macrocarpa* to obtain the fermentation broth of *Gastrodinium macrocarpa*.

[0009] Preferably, in preparing the *Leuconostoc mesenteroides* fermentation broth, the culture medium used for fermentation culture comprises the following components: 4g soybean meal powder, 3g glucose, 1g dry yeast, 0.5g sodium acetate, 0.2g triammonium citrate, 0.3g dipotassium hydrogen phosphate, 0.058g magnesium sulfate, 0.025g manganese sulfate, and 100mL water; the fermentation culture time is 72h, the temperature is 30℃, and the inoculum size is 5%. When preparing the methyltrophic Bacillus fermentation broth, the culture medium used for fermentation culture includes the following components: 35 wt.% soybean meal, 45 wt.% corn flour, 15 wt.% distillers' grains, 4.2 wt.% corn flour, and 0.8 wt.% sucrose; the fermentation time is 58 h, the temperature is 32 °C, and the inoculum size is 5%. When preparing the *Trichoderma viride* fermentation broth, the culture medium used for fermentation culture includes the following components: 35 wt.% wheat bran, 15 wt.% rice husk, and 50 wt.% water; the fermentation culture time is 36 h, the temperature is 28 °C, and the inoculum size is 5%. When preparing the fermentation broth of *Gnaphalium fasciatus*, the fermentation culture medium used is PDA medium; the fermentation culture time is 24 h, the temperature is 25 °C, and the inoculum size is 5%.

[0010] This invention provides a method for preparing the above-mentioned control agent for sclerotinia stem rot in rapeseed, comprising the following steps: The fermentation broth of Leuconostoc mesenteroides, the fermentation broth of Bacillus methyltrophicus, the fermentation broth of Trichoderma viride, and the fermentation broth of Gastrodinium gracilis are mixed to obtain the control agent for sclerotinia rot of rapeseed.

[0011] Preferably, the volume ratio of the *Leuconostoc mesenteroides* fermentation broth, the *Bacillus methyltrophicus* fermentation broth, the *Trichoderma viride* fermentation broth, and the *Gnaphalium fasciatus* fermentation broth is 1:2:1:1.

[0012] Preferably, the method for preparing the Leuconostoc mesenteroides fermentation broth includes the step of fermenting and culturing activated Leuconostoc mesenteroides to obtain the Leuconostoc mesenteroides fermentation broth; The method for preparing the fermentation broth of the methyltrophic Bacillus includes the step of fermenting and culturing activated methyltrophic Bacillus to obtain the fermentation broth of the methyltrophic Bacillus. The preparation method of the green Trichoderma fermentation broth includes the step of fermenting and culturing activated green Trichoderma to obtain the green Trichoderma fermentation broth; The preparation method of the fermentation broth of *Gastrodinium macrocarpa* includes the step of fermenting and culturing activated *Gastrodinium macrocarpa* to obtain the fermentation broth of *Gastrodinium macrocarpa*.

[0013] Preferably, in preparing the *Leuconostoc mesenteroides* fermentation broth, the culture medium used for fermentation culture comprises the following components: 4g soybean meal powder, 3g glucose, 1g dry yeast, 0.5g sodium acetate, 0.2g triammonium citrate, 0.3g dipotassium hydrogen phosphate, 0.058g magnesium sulfate, 0.025g manganese sulfate, and 100mL water; the fermentation culture time is 72h, the temperature is 30℃, and the inoculum size is 5%. When preparing the methyltrophic Bacillus fermentation broth, the culture medium used for fermentation culture includes the following components: 35 wt.% soybean meal, 45 wt.% corn flour, 15 wt.% distillers' grains, 4.2 wt.% corn flour, and 0.8 wt.% sucrose; the fermentation time is 58 h, the temperature is 32 °C, and the inoculum size is 5%. When preparing the *Trichoderma viride* fermentation broth, the culture medium used for fermentation culture includes the following components: 35 wt.% wheat bran, 15 wt.% rice husk, and 50 wt.% water; the fermentation culture time is 36 h, the temperature is 28 °C, and the inoculum size is 5%. When preparing the fermentation broth of *Gnaphalium fasciatus*, the fermentation culture medium used is PDA medium; the fermentation culture time is 24 h, the temperature is 25 °C, and the inoculum size is 5%.

[0014] This invention provides the application of the described control agent for rapeseed sclerotinia stem rot in the prevention and control of rapeseed sclerotinia stem rot.

[0015] This invention provides a method for preventing and controlling sclerotinia stem rot in rapeseed, comprising the following steps: After diluting the above-mentioned rapeseed sclerotinia disease control agent, spray it onto the surface of the rapeseed plants.

[0016] The present invention discloses the following technical effects: This invention provides a control agent for rapeseed sclerotinia stem rot, comprising fermentation broths of *Leuconostoc mesenteroides*, *Bacillus methyltrophicus*, *Trichoderma viride*, and *Paecilomyces macrocephala*. Spraying this control agent onto the surface of rapeseed seedlings during the seedling stage effectively controls sclerotinia stem rot, achieving a field control efficacy of 79%, comparable to commercially available sclerotinia control agents. Furthermore, *Leuconostoc mesenteroides*, *Bacillus methyltrophicus*, *Trichoderma viride*, and *Paecilomyces macrocephala* exhibit a significant synergistic effect in controlling rapeseed sclerotinia stem rot. This invention provides a novel combined microbial agent for controlling rapeseed sclerotinia stem rot, with widely available sources, significant effects, and broad application prospects. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0022] Preparation Example Leuconostoc mesentery was purchased from Wuhan Gray Algae Biotechnology Co., Ltd., product number HZB114697; Preparation of *Leuconostoc mesenteroides* fermentation broth: Activated *Leuconostoc mesenteroides* was inoculated into a fermentation medium (4g soybean meal, 3g glucose, 1g dry yeast, 0.5g sodium acetate, 0.2g triammonium citrate, 0.3g dipotassium hydrogen phosphate, 0.058g magnesium sulfate, 0.025g manganese sulfate, and 100mL water), and then fermented. The medium was subsequently diluted to obtain an effective viable count of 1×10⁻⁶. 7 The fermentation broth of Leuconostoc mesenteroides at a concentration of CFU / mL was prepared; the inoculum size was 5%, the fermentation time was 72 h, and the temperature was 30℃.

[0023] Methylotrophic Bacillus was purchased from Wuhan Gray Algae Biotechnology Co., Ltd., product number HZB115648; Preparation of fermentation broth for methyltrophic Bacillus: Activated methyltrophic Bacillus was inoculated into a fermentation medium (35 wt.% soybean meal, 45 wt.% corn flour, 15 wt.% distiller's grains, 4.2 wt.% corn flour, and 0.8 wt.% sucrose; water was added to maintain a moisture content of around 55%), and fermentation was carried out. The medium was then diluted to obtain an effective viable count of 1 × 10⁻⁶ cells / mL. 8 The fermentation broth of Bacillus methyltrophicus was prepared at a concentration of CFU / mL; the inoculum size was 5%, the fermentation time was 58 h, and the temperature was 32℃.

[0024] Trichoderma viride was purchased from Wuhan Gray Algae Biotechnology Co., Ltd., product number HZB120088; Preparation of *Trichoderma viride* fermentation broth: Activated *Trichoderma viride* was inoculated into a fermentation medium (35 wt.% wheat bran, 15 wt.% rice husk, and 50 wt.% water), and fermented. The broth was then diluted to obtain an effective viable count of 1 × 10⁻⁶ cells / mL. 8 The fermentation broth of *Trichoderma viride* with a concentration of CFU / mL was prepared; the inoculum size was 5%, the fermentation time was 36 h, and the temperature was 28℃.

[0025] The giant branch genus *Gyrodactylus* was purchased from Wuhan Gray Algae Biotechnology Co., Ltd., product number HZB123411; Preparation of fermentation broth for *Gnaphalium fasciatus*: Activated *Gnaphalium fasciatus* was inoculated onto PDA medium and fermented. The broth was then diluted to obtain an effective viable count of 1 × 10⁻⁶ cells / mL. 7 The fermentation broth of *Gnaphalium affine* was prepared at a concentration of CFU / mL; the inoculum size was 5%, the fermentation time was 24 h, and the temperature was 25℃.

[0026] Bacillus amyloliquefaciens was purchased from Wuhan Gray Algae Biotechnology Co., Ltd., product number HZB112931; Preparation of Bacillus amyloliquefaciens fermentation broth: Activated Bacillus amyloliquefaciens was inoculated into LB medium for fermentation, followed by dilution to obtain an effective viable count of 1.8 × 10⁻⁶ cells / mL. 7 The fermentation broth of Bacillus amyloliquefaciens at CFU / mL was prepared with an inoculum size of 10%, a fermentation time of 72 h, a temperature of 35 °C, and a rotation speed of 220 rpm.

[0027] Bacillus subtilis was purchased from Wuhan Gray Algae Biotechnology Co., Ltd., product number HZB112205; Preparation of Bacillus subtilis fermentation broth: Activated Bacillus subtilis was inoculated into LB medium for fermentation, followed by dilution to obtain an effective viable count of 1.8 × 10⁻⁶ cells / ml. 7 The fermentation broth of Bacillus subtilis was prepared at a concentration of CFU / mL; the inoculum size was 10%, the fermentation time was 72 h, the temperature was 35℃, and the rotation speed was 220 rpm.

[0028] The fermentation broths of each strain prepared in this preparatory example were used in subsequent experiments.

[0029] Example 1 1. Experimental Methods Add 2 mL of different rapeseed sclerotinia disease control agents to PDA medium (see Table 1 for details). Use an 8 mm punch to collect sclerotinia mycelial blocks and inoculate them into the aforementioned medium. Use PDA medium as a blank control. After incubating at 24℃ for 72 h, observe the growth of sclerotinia mycelia. Calculate the inhibition rate based on the diameter of the pathogen in the blank control and the rapeseed sclerotinia disease control agent treatments.

[0030] Table 1 Different control agents for Sclerotinia stem rot in rapeseed 2. Experimental Results The antibacterial rates of different treatments are shown in Table 2.

[0031] Table 2 Antibacterial rates of different treatments As shown in Table 2, the control agent for rapeseed sclerotinia stem rot, No. 1, has the best antibacterial effect, with an antibacterial rate of 83%.

[0032] Example 2 1. Experimental Methods Experiments were conducted on rapeseed fields severely affected by sclerotinia stem rot in 2023, using the rapeseed variety "Zheshuang 6". Areas of equal size, each 50m², were randomly selected. 2 Each rapeseed sclerotinia disease control agent (rapsum sclerotinia disease control agents numbered 1-8 in Table 1) was applied three times and recorded as the rapeseed sclerotinia disease control agent group. A water control group and a sclerotinia control agent group were also set up. The spraying details for each area are as follows: Sclerotinia stem rot control formulation group: During the seedling stage of rapeseed, the sclerotinia stem rot control formulations in Table 1 were diluted 800 times and sprayed on the surface of the rapeseed plants; the amount of the diluted suspension was 30L / mu, and it was sprayed once every 10 days for a total of 3 times. Isocarbendazim group: During the seedling stage of rapeseed, dilute 40% isocarbendazim 800 times and spray it evenly on the leaves. The dosage is 40 grams per mu. Spray once every 10 days for a total of 3 times. Water control group: During the rapeseed seedling stage, spray the same amount of water as the rapeseed sclerotinia disease control agent onto the surface of the rapeseed plants; the amount of water used is 30L / mu, sprayed once every 10 days, for a total of 3 times; Simultaneously, the disease control effect was recorded before and 45 days after application. Fifty plants were randomly selected from each experimental area, and the number of diseased plants at each level was recorded. The disease index and control effect were calculated. The formulas for calculating the disease index and control effect are shown below: Disease index = ∑ (number of diseased plants at each level × corresponding level value) / (total number of plants surveyed × 4) × 100; Prevention and control effect = (disease index in control area - disease index in treatment area) / disease index in control area × 100%.

[0033] 2. Experimental Results The efficacy of different treatments is shown in Table 3.

[0034] Table 3 Statistical results of the prevention efficacy of different treatments As shown in Table 3, the control agent for rapeseed sclerotinia stem rot, No. 1, exhibited the best antibacterial effect, achieving a field efficacy of 79%, consistent with the results of the indoor antibacterial experiments. Furthermore, this invention screened the strains used, finding that a combination of *Leuconostoc mesenteriae* fermentation broth, *Bacillus methyltrophicus* fermentation broth, *Trichoderma viride* fermentation broth, and *Gnaphalium affine* fermentation broth improved the control efficacy against rapeseed sclerotinia stem rot. Screening the proportions of the fermentation broths revealed that altering the relative amounts of the fermentation broths reduced the control efficacy against rapeseed sclerotinia stem rot. This invention also included a treatment omitting the fermentation broth of one strain, and its efficacy was investigated. Although the efficacy increased compared to the control group, it was still significantly lower than that of the control agent No. 1. Moreover, the control efficacy of the control agent No. 1 was comparable to that of commercially available sclerotinia control agents.

[0035] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A control agent for sclerotinia stem rot in rapeseed, characterized in that, This includes fermentation broths of Leuconostoc mesenteroides, Bacillus methylotrophicus, Trichoderma viride, and Gastrodinium grandis; The effective viable count of Leuconostoc mesenteroides in the fermentation broth was 1×10⁻⁶. 7 CFU / mL; The effective viable count of Bacillus methyltrophicus in the fermentation broth was 1×10⁻⁶. 8 CFU / mL; The effective viable count of *Trichoderma viride* in the *Trichoderma viride* fermentation broth was 1 × 10⁻⁶. 8 CFU / mL; The effective viable count of *Gymnospermum macrocephala* in the fermentation broth was 1×10⁻⁶. 7 CFU / mL.

2. The rapeseed sclerotinia stem rot control agent according to claim 1, characterized in that, The volume ratio of the fermentation broth of Leuconostoc mesenteroides, the fermentation broth of Bacillus methyltrophicus, the fermentation broth of Trichoderma viride, and the fermentation broth of Gastrodinium grandis is 1:2:1:

1.

3. The rapeseed sclerotinia stem rot control agent according to claim 1, characterized in that, The method for preparing the Leuconostoc mesenteroides fermentation broth includes the step of fermenting and culturing activated Leuconostoc mesenteroides to obtain the Leuconostoc mesenteroides fermentation broth. The method for preparing the fermentation broth of the methyltrophic Bacillus includes the step of fermenting and culturing activated methyltrophic Bacillus to obtain the fermentation broth of the methyltrophic Bacillus. The preparation method of the green Trichoderma fermentation broth includes the step of fermenting and culturing activated green Trichoderma to obtain the green Trichoderma fermentation broth; The preparation method of the fermentation broth of *Gastrodinium macrocarpa* includes the step of fermenting and culturing activated *Gastrodinium macrocarpa* to obtain the fermentation broth of *Gastrodinium macrocarpa*.

4. The rapeseed sclerotinia stem rot control agent according to claim 3, characterized in that, When preparing the *Leuconostoc mesenteroides* fermentation broth, the culture medium used for fermentation culture includes the following components: 4g soybean meal powder, 3g glucose, 1g dry yeast, 0.5g sodium acetate, 0.2g triammonium citrate, 0.3g dipotassium hydrogen phosphate, 0.058g magnesium sulfate, 0.025g manganese sulfate, and 100mL water; the fermentation culture time is 72h, the temperature is 30℃, and the inoculum size is 5%. When preparing the methyltrophic Bacillus fermentation broth, the culture medium used for fermentation culture includes the following components: 35 wt.% soybean meal, 45 wt.% corn flour, 15 wt.% distillers' grains, 4.2 wt.% corn flour, and 0.8 wt.% sucrose; the fermentation time is 58 h, the temperature is 32 °C, and the inoculum size is 5%. When preparing the *Trichoderma viride* fermentation broth, the culture medium used for fermentation culture includes the following components: 35 wt.% wheat bran, 15 wt.% rice husk, and 50 wt.% water; the fermentation culture time is 36 h, the temperature is 28 °C, and the inoculum size is 5%. When preparing the fermentation broth of *Gymnospermum macrocephala*, the fermentation culture medium used is PDA medium; the fermentation culture time is 24 h, the temperature is 25 °C, and the inoculum size is 5%.

5. The method for preparing the rapeseed sclerotinia stem rot control agent according to any one of claims 1-4, characterized in that, Includes the following steps: The fermentation broth of Leuconostoc mesenteroides, the fermentation broth of Bacillus methyltrophicus, the fermentation broth of Trichoderma viride, and the fermentation broth of Gastrodinium gracilis are mixed to obtain the control agent for sclerotinia rot of rapeseed.

6. The preparation method according to claim 5, characterized in that, The volume ratio of the fermentation broth of Leuconostoc mesenteroides, the fermentation broth of Bacillus methyltrophicus, the fermentation broth of Trichoderma viride, and the fermentation broth of Gastrodinium grandis is 1:2:1:

1.

7. The preparation method according to claim 5, characterized in that, The method for preparing the Leuconostoc mesenteroides fermentation broth includes the step of fermenting and culturing activated Leuconostoc mesenteroides to obtain the Leuconostoc mesenteroides fermentation broth. The method for preparing the fermentation broth of the methyltrophic Bacillus includes the step of fermenting and culturing activated methyltrophic Bacillus to obtain the fermentation broth of the methyltrophic Bacillus. The preparation method of the green Trichoderma fermentation broth includes the step of fermenting and culturing activated green Trichoderma to obtain the green Trichoderma fermentation broth; The preparation method of the fermentation broth of *Gastrodinium macrocarpa* includes the step of fermenting and culturing activated *Gastrodinium macrocarpa* to obtain the fermentation broth of *Gastrodinium macrocarpa*.

8. The preparation method according to claim 7, characterized in that, When preparing the *Leuconostoc mesenteroides* fermentation broth, the culture medium used for fermentation culture includes the following components: 4g soybean meal powder, 3g glucose, 1g dry yeast, 0.5g sodium acetate, 0.2g triammonium citrate, 0.3g dipotassium hydrogen phosphate, 0.058g magnesium sulfate, 0.025g manganese sulfate, and 100mL water; the fermentation culture time is 72h, the temperature is 30℃, and the inoculum size is 5%. When preparing the methyltrophic Bacillus fermentation broth, the culture medium used for fermentation culture includes the following components: 35 wt.% soybean meal, 45 wt.% corn flour, 15 wt.% distillers' grains, 4.2 wt.% corn flour, and 0.8 wt.% sucrose; the fermentation time is 58 h, the temperature is 32 °C, and the inoculum size is 5%. When preparing the *Trichoderma viride* fermentation broth, the culture medium used for fermentation culture includes the following components: 35 wt.% wheat bran, 15 wt.% rice husk, and 50 wt.% water; the fermentation culture time is 36 h, the temperature is 28 °C, and the inoculum size is 5%. When preparing the fermentation broth of *Gymnospermum macrocephala*, the fermentation culture medium used is PDA medium; the fermentation culture time is 24 h, the temperature is 25 °C, and the inoculum size is 5%.

9. The application of the rapeseed sclerotinia stem rot control agent according to any one of claims 1-4 in the prevention and control of rapeseed sclerotinia stem rot.

10. A method for controlling sclerotinia stem rot in rapeseed, characterized in that, Includes the following steps: The rapeseed sclerotinia disease control agent according to any one of claims 1-4 is diluted and sprayed onto the surface of rapeseed plants.

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