Method for preventing and treating sclerotinia rot of colza by antagonistic trichoderma strains
By screening and mutagenesis of Trichoderma strains and formulating bio-fertilizer carriers, the problem of unstable antagonistic performance of Trichoderma strains under different environments was solved, achieving effective control of rapeseed sclerotinia stem rot and soil improvement.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INST OF PLANT PROTECTION JIANGXI ACAD OF AGRI SCI
- Filing Date
- 2026-01-19
- Publication Date
- 2026-05-08
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Traditional Trichoderma strains exhibit unstable antagonistic properties under different environmental conditions, resulting in unsatisfactory antibacterial effects. Insufficient selection and formulation of bio-fertilizer carrier materials also contribute to unstable control of rapeseed sclerotinia stem rot.
By screening and chemically mutagenesis-treated Trichoderma strains from rapeseed rhizosphere soil under different environments, antagonistic active substances were extracted and formulated into a bio-fertilizer carrier containing attapulgite soil, vermicompost biochar, alginate oligosaccharides, and other components. This carrier was then applied to rapeseed cultivation to provide stable antagonistic effects and stress resistance.
It effectively inhibits the growth of Sclerotinia sclerotiorum under different environmental conditions, significantly reduces the incidence and severity of Sclerotinia sclerotiorum disease in rapeseed, improves soil fertility and ecological function, and promotes healthy growth of rapeseed.
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Figure CN121990849A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of agricultural disease control technology, specifically a method for controlling sclerotinia stem rot in rapeseed by antagonizing Trichoderma strains. Background Technology
[0002] Rapeseed, as one of my country's important oil crops, has a wide planting area and stable yield, playing a vital role in ensuring national edible oil security. However, during its growth, rapeseed is often attacked by various diseases. Among them, sclerotinia stem rot is a fungal disease caused by Sclerotinia sclerotiorum, which seriously affects the yield and quality of rapeseed. Sclerotinia stem rot not only causes rapeseed leaves to turn yellow and stems to rot, but also causes rapeseed seeds to be underdeveloped, resulting in a significant drop in yield. Therefore, how to effectively prevent and control rapeseed sclerotinia stem rot and improve the yield and quality of rapeseed has become an urgent problem to be solved in the current agricultural field.
[0003] While traditional Trichoderma strain screening techniques have been able to isolate strains with certain antagonistic effects against Sclerotinia sclerotiorum, these strains often suffer from unstable antibacterial effects and poor resistance. In particular, the antagonistic performance of the strains can change significantly under different environmental conditions, leading to a substantial reduction in control efficacy. Furthermore, traditional screening methods often lack several advantages, making it difficult to select strains with highly efficient antagonistic effects from a large number of strains. Moreover, in the preparation of biofertilizer carriers, there are significant deficiencies in the selection and proportioning of carrier materials, resulting in unstable carrier performance. This makes it difficult to provide a good growth environment for Trichoderma, thus hindering the full realization of the antagonistic effects of Trichoderma and the nutritional benefits of biofertilizers. At the same time, traditional carrier preparation methods neglect the interaction between the carrier and microorganisms, as well as the impact of the carrier on the growth and reproduction of microorganisms, thereby limiting the improvement of biological control efficacy.
[0004] Therefore, a method for controlling sclerotinia stem rot in rapeseed by antagonizing Trichoderma strains was developed. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for controlling sclerotinia rot in rapeseed by antagonizing Trichoderma strains. This method obtains Trichoderma strains that maintain high antagonistic effects and strong resistance under different environmental conditions, greatly enhancing the stability of Trichoderma strains in disease control. This allows rapeseed to effectively resist sclerotinia rot invasion under different environmental conditions during its growth, reducing dependence on specific environmental conditions. Furthermore, by extracting its antagonistic active substances and formulating them into a bio-fertilizer carrier, which is applied during rapeseed cultivation, it effectively inhibits the growth and reproduction of Sclerotinia rot, thereby significantly reducing the incidence and severity of sclerotinia rot in rapeseed.
[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for controlling sclerotinia stem rot in rapeseed by antagonizing Trichoderma strains, the specific steps of which are as follows:
[0007] S100, strain screening and mutagenesis: Trichoderma strains were isolated from the rhizosphere soil of rapeseed under different environments. The strains with antagonistic effects against Sclerotinia sclerotiorum were initially screened by plate confrontation method. The initially screened strains were chemically mutagenized and confronted with Sclerotinia sclerotiorum again to screen out the mutagenized strains with larger inhibition zones, high production of antagonistic substances and strong stress resistance. The fermentation broth of Trichoderma was obtained by culture.
[0008] S200, Active substance extraction: Centrifuge the Trichoderma fermentation broth to obtain the supernatant, precipitate it with ammonium sulfate, collect the precipitate by centrifugation, dissolve it in sterile water and dialyze it to remove small molecule impurities, and obtain a concentrated solution of Trichoderma antagonistic active substances.
[0009] S300, preparation of bio-fertilizer carrier: Attapulgite soil is baked to activate its pores. Vermicompost biochar is treated with acetic acid solution and then washed with water. It is then mixed with alginate oligosaccharide, Bacillus mucilaginosus powder, and arbuscular mycorrhizal fungal spore agent to form a mixture. Trichoderma antagonistic active substance concentrate is added and mixed evenly. Then, it is sprayed and mixed with chitosan oligosaccharide grafted with sodium alginate aqueous solution to form a gel coating layer. After adjusting the moisture content, it is granulated. After maturation, it is coated with polyglutamic acid solution to make a bio-fertilizer carrier.
[0010] S400, Seed Coating Treatment: Before sowing rapeseed, rapeseed seeds are mixed with bio-fertilizer carrier at a mass ratio of 1:4. After coating, the seeds are dried so that the bio-fertilizer carrier adheres to the seed surface.
[0011] S500, application during key growth periods: During the rapeseed seedling stage, dilute the bio-fertilizer carrier 30-50 times and apply it to the roots of the rapeseed plants by strip application, with an application rate of 20-30 kg per acre. During the early flowering stage of rapeseed, dilute the bio-fertilizer carrier 20-40 times and apply it as a top dressing, with an application rate of 15-25 kg per acre.
[0012] Furthermore, in the screening and mutagenesis of the S100 strain, Trichoderma strains were isolated from the rhizosphere soil of rapeseed under different environments, including high temperature environment, low temperature environment and drought environment. The high temperature environment was 42℃ to 45℃ for one week, the low temperature environment was -6℃ to -8℃ for one week, and the drought environment was 3% to 4% soil moisture content for two weeks.
[0013] Furthermore, in the screening and mutagenesis of the S100 strains, strains with antagonistic effects against *Sclerotinia sclerotiorum* were initially screened using the plate confrontation method. Specifically, the plate confrontation method involves inoculating a 5mm diameter *Sclerotinia sclerotiorum* mycelial disc in the center of a 9cm diameter PDA solid medium plate, and symmetrically inoculating four 5mm diameter *Trichoderma* mycelial discs 2cm from the edge of the *Sclerotinia sclerotiorum* mycelial disc. Plates without *Trichoderma* mycelial discs serve as controls. Each treatment is repeated three times, and the plates are incubated at 25℃ for 5-7 days. The diameter of the *Sclerotinia sclerotiorum* colonies is measured, and the inhibition rate of each *Trichoderma* strain against *Sclerotinia sclerotiorum* is calculated. *Trichoderma* strains with an inhibition rate ≥30% are selected as the initial screening strains.
[0014] Furthermore, in the screening and mutagenesis of the S100 strain, the initially screened strains are subjected to chemical mutagenesis treatment. The specific steps of chemical mutagenesis are as follows: the initially screened strains are inoculated into PDA liquid culture medium and cultured with shaking at 25℃ and 180r / min for 24h to prepare a bacterial suspension. A final concentration of 0.1%-0.5% ethyl methanesulfonate solution is added to the bacterial suspension, and the suspension is treated in the dark at 25℃ for 30min to 120min. After the mutagenesis is completed, an equal volume of 2.5% sodium thiosulfate solution is added to terminate the reaction.
[0015] Furthermore, the specific steps for obtaining the Trichoderma antagonistic active substance concentrate in the S200 active substance extraction are as follows: centrifuge the Trichoderma fermentation broth at 6000-8000 r / min for 10-15 min at 2℃ to 6℃ using a centrifuge tube, discard the precipitate, collect the supernatant, add ammonium sulfate, and centrifuge the broth again at 8000-10000 r / min at 2℃ to 6℃ for 15-20 min using a centrifuge tube, discard the supernatant, collect the precipitate, dissolve the precipitate in sterile water, and dialyze it through a dialysis bag to remove small molecule impurities, thereby obtaining the Trichoderma antagonistic active substance concentrate.
[0016] Furthermore, in the preparation of the S300 bio-fertilizer carrier, 30-40 parts by weight of attapulgite clay with a particle size of 50-100 nanometers, 20-30 parts by weight of vermicompost biochar, 10-15 parts by weight of alginate oligosaccharide, and 2-4 parts by weight of effective viable bacteria with a count ≥5×10⁻⁶ are weighed out. 10 The mixture consists of CFU / g of Bacillus mucilaginosus powder, 1-2 parts by weight of arbuscular mycorrhizal fungal spore agent containing ≥1000 spores per gram, attapulgite soil that has been baked to activate its pores, vermicompost biochar that has been soaked in 5% acetic acid solution for 1 hour and then rinsed with deionized water until neutral, and alginate oligosaccharide, Bacillus mucilaginosus powder and arbuscular mycorrhizal fungal spore agent are mixed and stirred to form a mixture.
[0017] Furthermore, in the preparation of the S300 bio-fertilizer carrier, the volume-to-mass ratio of the concentrated Trichoderma antagonistic active substance solution to the mixed materials is 1:15, the chitosan oligosaccharide grafted sodium alginate solution is prepared as a 2% aqueous solution, and the polyglutamic acid solution has a mass fraction of 0.5%.
[0018] Furthermore, during the application of S500 in the critical growth period, the bio-fertilizer carrier is applied to the root system of rapeseed plants by strip application. The strip application method is as follows: dig trenches around the root system of rapeseed plants, with a width of 3-5cm and a depth of 5-8cm, and the trenches are 5-10cm away from the root system of rapeseed plants. Apply the diluted bio-fertilizer carrier into the trenches and cover the trenches with soil.
[0019] Compared with existing technologies, this method for controlling sclerotinia stem rot in rapeseed by antagonizing Trichoderma strains has the following beneficial effects:
[0020] I. This invention obtains a Trichoderma strain that maintains high antagonistic activity and strong resistance under different environmental conditions, greatly enhancing the stability of Trichoderma strains in disease control. This enables rapeseed to effectively resist sclerotinia rot under different environmental conditions during its growth, reducing dependence on specific environmental conditions. Furthermore, by extracting its antagonistic active substances and formulating them into a bio-fertilizer carrier, it is applied to the rapeseed planting process, thereby effectively inhibiting the growth and reproduction of Sclerotinia rot, and significantly reducing the incidence and severity of rapeseed sclerotinia rot.
[0021] II. This invention incorporates various beneficial ingredients such as attapulgite soil, vermicompost biochar, and alginate oligosaccharides, which not only provide a favorable growth environment for Trichoderma but also improve soil structure and enhance soil fertility. During the rapeseed growth process, the bio-fertilizer carrier can continuously release nutrients to meet the needs of different growth stages of rapeseed and promote its healthy growth. At the same time, the microbial components such as Bacillus mucilaginosus powder and arbuscular mycorrhizal fungal spore agents in the bio-fertilizer carrier can also form a symbiotic relationship with beneficial microorganisms in the soil, further enhancing the ecological function of the soil and providing a strong guarantee for the long-term growth of rapeseed.
[0022] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description
[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.
[0024] Figure 1 This is a flowchart of a method for controlling sclerotinia stem rot in rapeseed by antagonizing Trichoderma strains. Detailed Implementation
[0025] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.
[0026] Example 1:
[0027] Strain screening and mutagenesis: In rapeseed growing areas under the humid climate of the Yangtze River Basin, typical plots were selected during summer high-temperature fumigation (42℃ constant temperature greenhouse for one week, with black mulch covering the ground to raise soil temperature), winter freeze-thaw cycles (-6℃ cold storage simulating open-air soil overwintering, with a frost layer thickness of about 0.5cm), and the summer drought period (natural drought causing soil moisture content to drop to 3%, with cracks appearing in the soil around the rapeseed roots). Rhizosphere soil from the main root of rapeseed was collected, and 58 Trichoderma strains were isolated using the dilution spread method. These strains were then initially screened using the plate confrontation method. A 5mm *Sclerotinia sclerotiorum* mycelium was inoculated in the center of a 9cm diameter PDA medium, with four 5mm *Trichoderma* mycelium mycelium symmetrically inoculated 2cm from the edge. Plates without *Trichoderma* inoculation served as controls. The medium was incubated at 25℃ for 5 days, and the results were calculated using the formula: Inhibition rate (%) = [(control colony diameter - treated colony diameter) / control colony diameter] × 100%, where the control colony diameter is the diameter of the *Sclerotinia sclerotiorum* colony without *Trichoderma* inoculation, and the treated colony diameter is the diameter of the *Sclerotinia sclerotiorum* colony after inoculation with *Trichoderma*. After calculating the inhibition rate, strain T-12 with an inhibition rate of 35% was screened out, and the T-12 strain was chemically mutagenized. Under a constant temperature of 25℃ in the laboratory, it was inoculated into PDA liquid medium and cultured at 25℃ and 180r / min for 24h to prepare a bacterial suspension. A final concentration of 0.3% ethyl methanesulfonate solution was added, and after being treated in the dark for 60min, an equal volume of 2.5% sodium thiosulfate solution was added to terminate the reaction. Finally, the mutagenized strain T-12-M with a 25% expansion of the inhibition zone under high humidity was obtained.
[0028] Extraction of active substances: The mutant strain T-12-M was fermented in the laboratory of the agricultural technology station. The fermentation broth was centrifuged at 7000 r / min for 12 min at 4℃, the precipitate was discarded, and the supernatant was collected. Ammonium sulfate was added to the supernatant to saturation of 80%, and after standing for 30 min, it was centrifuged at 9000 r / min for 18 min at 4℃. The supernatant was discarded, the precipitate was collected, and the precipitate was dissolved in sterile water. The precipitate was dialyzed with running tap water for 24 h using a dialysis bag with a molecular weight cutoff of 3500 Da to obtain a light brown concentrated solution of Trichoderma antagonistic active substances, which was found to contain 3 novel antagonistic proteins.
[0029] Preparation of bio-fertilizer carrier: Considering the heavy clay characteristics of the soil in the Yangtze River Basin, 35 parts by weight of attapulgite clay (processed from local bentonite with a particle size of 50 nanometers) were weighed out and the porosity was increased by 40% after oven baking (baking at 200℃ for 2 hours). 25 parts of vermicompost biochar were weighed out from waste from surrounding farms and soaked in 5% acetic acid solution for 1 hour, then rinsed with deionized water until neutral. The treated attapulgite clay and vermicompost biochar were then mixed with 12 parts of alginate oligosaccharide and 3 parts of effective viable bacteria with a count ≥5×10⁻⁶. 10 CFU / g of Bacillus mucilaginosus powder and 1.5 parts of arbuscular mycorrhizal fungal spore agent containing ≥1000 spores per gram were mixed and stirred to form a mixture. A concentrated solution of Trichoderma antagonistic active substances was added to the mixture at a volume-to-mass ratio of 1:15. After mixing, it was spray-mixed with a 2% (w / w) solution of chitosan oligosaccharide-grafted sodium alginate to form a gel coating layer. The moisture content was adjusted to 30%, and the mixture was granulated using a granulator. After maturation for 48 hours, a 0.5% (w / w) polyglutamic acid solution was used for coating. Figure 1 As shown.
[0030] Seed treatment: Before sowing, mix “62” seeds with bio-fertilizer carrier at a mass ratio of 1:4 and mix for 10 minutes using an electric seed mixer. The bio-fertilizer carrier particles are evenly adhered to the seed surface. After drying, a light gray coating layer is formed on the seed surface. No carrier detachment occurred after light rain following sowing.
[0031] Application during key growth periods: When the rapeseed has 4-5 true leaves, dig a trench around the root system of the rapeseed plant (the trench should be 4cm wide, 6cm deep, and 8cm away from the root system). Dilute the bio-fertilizer carrier 30 times and apply it into the trench at a rate of 25kg per acre. Cover the soil immediately after application to prevent the active substances from being washed away by rainwater. When the rapeseed enters the initial flowering stage, the field humidity is high, which is the peak period for sclerotinia disease. Dilute the bio-fertilizer carrier 20 times and apply it in a trench 5cm away from the root system at a rate of 20kg per acre. Cover the soil immediately after application.
[0032] In summary, by isolating Trichoderma strains from rapeseed rhizosphere soil under high temperature, low temperature, and drought conditions, and through initial screening using the plate confrontation method and chemical mutagenesis treatment, mutant strains with high antagonistic substances were obtained. The active substances were extracted and formulated with specific materials to create a bio-fertilizer carrier. After seed dressing and application in specific ways during the seedling and early flowering stages, a complete method for controlling rapeseed sclerotinia stem rot using antagonistic Trichoderma strains was formed. This method is adapted to local climate and soil characteristics and provides an effective solution for the control of rapeseed sclerotinia stem rot in the local area.
[0033] Example 2:
[0034] Strain screening and mutagenesis: In the saline-alkali rapeseed planting area of the North China Plain, plots with high summer temperatures (45℃ for one week), winter freeze-thaw cycles (-8℃ cold storage simulating open-air soil conditions), and summer drought (natural drought leading to 4% soil moisture content) were selected. Rhizosphere soil of rapeseed was collected, and the isolated Trichoderma strains were cultured on plates for 7 days. The T-28 strain, which showed an inhibition rate of 32% under pH 8.0 conditions, was screened. During mutagenesis, the strain was treated with 0.5% ethyl methanesulfonate solution for 30 min. The obtained mutant strain T-28-M could still maintain antibacterial activity in a medium with a salt content of 0.5%, and its fermentation broth could withstand high temperatures of 40℃.
[0035] Extraction of active substances: The mutant strain T-28-M was fermented in the laboratory. The mixture was centrifuged at 8000 r / min for 10 min at 5℃, the precipitate was discarded, and the supernatant was collected. Ammonium sulfate was added to the supernatant until the saturation was 70%. After standing, the mixture was centrifuged at 10000 r / min for 15 min at 5℃, the supernatant was discarded, and the precipitate was collected. The precipitate was dissolved in sterile water and dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 5000 Da to obtain a concentrated solution of Trichoderma antagonistic active substances.
[0036] Bio-fertilizer carrier preparation: For saline-alkali land, 40 parts by weight of attapulgite soil (processed from local zeolite with a particle size of 100 nanometers) were weighed and baked to activate the pores (250℃ for 1.5 hours), increasing the soil salt adsorption rate by 35%. 30 parts of vermicompost biochar were soaked in a 5% acetic acid solution for 1 hour and then rinsed with deionized water until neutral. The treated attapulgite soil and vermicompost biochar were then combined with 15 parts of alginate oligosaccharide and 4 parts of effective viable bacteria with a count ≥5×10⁻⁶. 10 Mix CFU / g of Bacillus mucilaginosus powder and 2 parts of arbuscular mycorrhizal fungal spore agent containing ≥1000 spores per gram to form a mixture. Add Trichoderma antagonistic active substance concentrate to the mixture at a volume-to-mass ratio of 1:15, mix well, and then spray mix with 2% (w / w) chitosan oligosaccharide-grafted sodium alginate aqueous solution to form a gel coating layer. Adjust the moisture content to 25% and then granulate. After maturing for 36 hours, coat with 0.5% (w / w) polyglutamic acid solution.
[0037] Seed treatment: Before sowing, mix “No. 9” seeds with bio-fertilizer carrier at a mass ratio of 1:4. Due to the windy local conditions, cover the seeds with plastic sheeting to dry after mixing to prevent carrier powder from being lost. Use plastic film for cultivation during sowing.
[0038] Application during critical growth periods: When rapeseed has 3-4 true leaves during its growth period, and the soil moisture is insufficient, dig a trench around the rapeseed plant roots (5cm wide, 8cm deep, and 10cm away from the rapeseed plant roots). Dilute the bio-fertilizer carrier 50 times and apply it into the trench at a rate of 30kg per acre. Immediately after fertilization, cover with soil and water lightly to ensure contact between the carrier and the roots. When rapeseed enters the initial flowering stage, saline-alkali soil shows obvious salt return phenomenon. Dig a trench 6cm from the roots and apply the bio-fertilizer carrier diluted 40 times at a rate of 15kg per acre. After fertilization, combine with inter-row cultivation and hilling to reduce salt migration to the surface.
[0039] In summary, based on the local environmental characteristics, Trichoderma strains were isolated and screened from the corresponding soil. These strains were then mutagenized to obtain highly resistant mutant strains. Active substances were extracted and formulated into a bio-fertilizer carrier suitable for saline-alkali soils. Through seed treatment and application at different critical growth stages, a method for controlling rapeseed sclerotinia stem rot using antagonistic Trichoderma strains adapted to this region was developed. This method combines saline-alkali soil improvement with disease control, demonstrating strong targeting and practicality.
[0040] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.
Claims
1. A method for controlling sclerotinia stem rot in rapeseed by antagonizing Trichoderma strains, characterized in that, The specific steps of this method are as follows: S100, strain screening and mutagenesis: Trichoderma strains were isolated from the rhizosphere soil of rapeseed under different environments. The strains with antagonistic effects against Sclerotinia sclerotiorum were initially screened by plate confrontation method. The initially screened strains were chemically mutagenized and confronted with Sclerotinia sclerotiorum again to screen out the mutagenized strains with larger inhibition zones, high production of antagonistic substances and strong stress resistance. The fermentation broth of Trichoderma was obtained by culture. S200, Active substance extraction: Centrifuge the Trichoderma fermentation broth to obtain the supernatant, precipitate it with ammonium sulfate, collect the precipitate by centrifugation, dissolve it in sterile water and dialyze it to remove small molecule impurities, and obtain a concentrated solution of Trichoderma antagonistic active substances. S300, preparation of bio-fertilizer carrier: Attapulgite soil is baked to activate its pores. Vermicompost biochar is treated with acetic acid solution and then washed with water. It is then mixed with alginate oligosaccharide, Bacillus mucilaginosus powder, and arbuscular mycorrhizal fungal spore agent to form a mixture. Trichoderma antagonistic active substance concentrate is added and mixed evenly. Then, it is sprayed and mixed with chitosan oligosaccharide grafted with sodium alginate aqueous solution to form a gel coating layer. After adjusting the moisture content, it is granulated. After maturation, it is coated with polyglutamic acid solution to make a bio-fertilizer carrier. S400, Seed Coating Treatment: Before sowing rapeseed, rapeseed seeds are mixed with bio-fertilizer carrier at a mass ratio of 1:
4. After coating, the seeds are dried so that the bio-fertilizer carrier adheres to the seed surface. S500, application during key growth periods: During the rapeseed seedling stage, dilute the bio-fertilizer carrier 30-50 times and apply it to the roots of the rapeseed plants by strip application, with an application rate of 20-30 kg per acre. During the early flowering stage of rapeseed, dilute the bio-fertilizer carrier 20-40 times and apply it as a top dressing, with an application rate of 15-25 kg per acre.
2. The method for controlling sclerotinia stem rot in rapeseed by antagonizing Trichoderma strains according to claim 1, characterized in that, In the screening and mutagenesis of the S100 strain, Trichoderma strains were isolated from the rhizosphere soil of rapeseed under different environments, including high temperature environment, low temperature environment and drought environment. The high temperature environment was 42℃ to 45℃ for one week, the low temperature environment was -6℃ to -8℃ for one week, and the drought environment was 3% to 4% soil moisture content for two weeks.
3. The method for controlling sclerotinia stem rot in rapeseed by antagonizing Trichoderma strains according to claim 1, characterized in that, In the screening and mutagenesis of the S100 strains, strains with antagonistic effects against *Sclerotinia sclerotiorum* were initially screened using the plate confrontation method. The plate confrontation method is as follows: a *Sclerotinia sclerotiorum* mycelium with a diameter of 5 mm was inoculated in the center of a 9 cm diameter PDA solid medium plate, and four *Trichoderma* mycelium with a diameter of 5 mm were symmetrically inoculated 2 cm away from the edge of the *Sclerotinia sclerotiorum* mycelium. Plates without *Trichoderma* mycelium were used as controls. Each treatment was repeated 3 times. The plates were incubated in a constant temperature incubator at 25℃ for 5-7 days, and the diameter of *Sclerotinia sclerotiorum* colonies was measured. The inhibition rate of each *Trichoderma* strain against *Sclerotinia sclerotiorum* was calculated, and *Trichoderma* strains with an inhibition rate ≥30% were screened as the initial screening strains.
4. The method for controlling sclerotinia stem rot in rapeseed by antagonizing Trichoderma strains according to claim 1, characterized in that, In the screening and mutagenesis of the S100 strain, the initially screened strains were subjected to chemical mutagenesis treatment. The specific steps of chemical mutagenesis were as follows: the initially screened strains were inoculated into PDA liquid culture medium and cultured with shaking at 25℃ and 180r / min for 24h to prepare a bacterial suspension. Ethyl methanesulfonate solution with a final concentration of 0.1%-0.5% was added to the bacterial suspension and treated in the dark at 25℃ for 30min to 120min. After the mutagenesis was completed, an equal volume of 2.5% sodium thiosulfate solution was added to terminate the reaction.
5. The method for controlling sclerotinia stem rot in rapeseed by antagonizing Trichoderma strains according to claim 1, characterized in that, The specific steps for obtaining the concentrated Trichoderma antagonistic active substance extract in the S200 active substance extraction process are as follows: centrifuge the Trichoderma fermentation broth at 6000-8000 r / min for 10-15 min at 2℃ to 6℃ using a centrifuge tube, discard the precipitate, collect the supernatant, add ammonium sulfate, and centrifuge the broth again at 8000-10000 r / min at 2℃ to 6℃ for 15-20 min using a centrifuge tube, discard the supernatant, collect the precipitate, dissolve the precipitate in sterile water, and dialyze it through a dialysis bag to remove small molecule impurities, thereby obtaining the concentrated Trichoderma antagonistic active substance extract.
6. The method for controlling sclerotinia stem rot in rapeseed by antagonizing Trichoderma strains according to claim 1, characterized in that, In the preparation of the S300 bio-fertilizer carrier, the following components are weighed out: 30-40 parts by weight of attapulgite clay with a particle size of 50-100 nanometers, 20-30 parts by weight of vermicompost biochar, 10-15 parts by weight of alginate oligosaccharide, and 2-4 parts by weight of effective viable bacteria with a count ≥5×10⁻⁶. 10 The mixture consists of CFU / g of Bacillus mucilaginosus powder, 1-2 parts by weight of arbuscular mycorrhizal fungal spore agent containing ≥1000 spores per gram, attapulgite soil that has been baked to activate its pores, vermicompost biochar that has been soaked in 5% acetic acid solution for 1 hour and then rinsed with deionized water until neutral, and alginate oligosaccharide, Bacillus mucilaginosus powder and arbuscular mycorrhizal fungal spore agent are mixed and stirred to form a mixture.
7. The method for controlling sclerotinia stem rot in rapeseed by antagonizing Trichoderma strains according to claim 1, characterized in that, In the preparation of the S300 bio-fertilizer carrier, the volume-to-mass ratio of the concentrated Trichoderma antagonistic active substance solution to the mixed materials is 1:15, the chitosan oligosaccharide grafted sodium alginate solution is prepared as a 2% aqueous solution, and the polyglutamic acid solution has a mass fraction of 0.5%.
8. The method for controlling sclerotinia stem rot in rapeseed by antagonizing Trichoderma strains according to claim 1, characterized in that, During the critical growth period of the S500, the bio-fertilizer carrier is applied to the roots of rapeseed plants by strip application. The strip application method is as follows: dig a trench around the roots of the rapeseed plants, with a width of 3-5cm and a depth of 5-8cm, and keep the trench 5-10cm away from the roots of the rapeseed plants. Apply the diluted bio-fertilizer carrier into the trench and cover the trench with soil.