阅读说明：本技术 一种利用虫草培养物制备具有抑菌活性精多糖的方法 (Method for preparing refined polysaccharide with antibacterial activity by using cordyceps culture ) 是由 厉晓腊 柴一秋 金轶伟 方鸣 陈官菊 刘又高 于 2018-07-12 设计创作，主要内容包括：本发明涉及生物技术领域,具体地,涉及一种利用虫草培养物制备具有抑菌活性精多糖的方法及其应用。本发明以虫草培养物为原料,提取粗多糖,采用大孔树脂吸附法和Sevag法两种方法相结合的手段去除粗多糖中的蛋白质而得到精多糖。进而采用分步醇沉得到了对三种革兰氏阴性植物病原细菌具有抑制活性的95%乙醇醇沉虫草精多糖CP95。本发明的制备方法具有操作简单、性能稳定、成本低廉的特点,本发明对更好地开发利用虫草培养物,以及降低虫草废弃培养基的清理成本具有重要的经济意义。(The invention relates to the technical field of biology, in particular to a method for preparing refined polysaccharide with bacteriostatic activity by using cordyceps culture and application thereof. The invention takes cordyceps culture as raw material, extracts crude polysaccharide, and adopts a means of combining a macroporous resin adsorption method and a Sevag method to remove protein in the crude polysaccharide to obtain refined polysaccharide. And further carrying out step-by-step alcohol precipitation to obtain 95% ethanol-precipitated cordycepin polysaccharide CP95 with inhibitory activity on three gram-negative plant pathogenic bacteria. The preparation method has the characteristics of simple operation, stable performance and low cost, and has important economic significance for better developing and utilizing the cordyceps culture and reducing the cleaning cost of the waste cordyceps culture medium.)

1. A method for preparing refined polysaccharide with bacteriostatic activity by using cordyceps culture is characterized by comprising the following steps:

(1) extracting crude polysaccharide: drying a proper amount of cordyceps culture at 70 ℃, adding 10 times of 50% ethanol according to the mass ratio of the culture to the volume of the extracting solution, extracting twice at 80 ℃, stirring for 0.5h each time, preserving heat for 2h, and filtering to obtain 50% ethanol extracting solution; adding 10 times of water solution into the residue after twice extraction with 50% ethanol, extracting at 100 deg.C, stirring for 0.5 hr, keeping the temperature for 1 hr, and filtering to obtain water extract. And respectively concentrating the 50% ethanol extract and the water extract to obtain 50% ethanol concentrated solution and water concentrated solution. Adding 3 times of 95% ethanol into the two concentrated solutions, standing overnight, and precipitating polysaccharide to obtain 50% ethanol-extracted precipitated polysaccharide and water-extracted precipitated polysaccharide, respectively. Mixing 50% ethanol extraction precipitated polysaccharide and water extraction precipitated polysaccharide, and spray drying to obtain Cordyceps crude polysaccharide;

(2) deproteinizing the crude polysaccharide: dissolving the crude polysaccharide with deionized water, loading the prepared crude polysaccharide solution on macroporous resin, eluting with 3 times volume of deionized water, and collecting the eluate; uniformly mixing the collected eluent and Sevag reagent according to the volume ratio of 5:1, violently oscillating for 30min, centrifuging for 15min at 4000rpm, placing the mixture in a separating funnel for separating to remove an intermediate denatured protein layer and a lower organic solution layer, and repeating the operation for 2 times; concentrating the water phase layer to obtain refined polysaccharide CP;

(3) ethanol precipitation by steps: uniformly stirring the concentrated solution of the refined polysaccharide with ethanol with the final concentration of 50% in volume ratio, standing at 4 ℃ overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and freeze-drying the precipitate at low temperature to obtain CP 50; regulating the volume of ethanol in the supernatant to 75%, standing overnight at 4 deg.C, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 75; concentrating and spin-drying the supernatant, dissolving with 95% ethanol, standing at 4 deg.C overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 95.

2. A method for preparing refined polysaccharide with bacteriostatic activity by using cordyceps culture is characterized by comprising the following steps:

(1) extracting crude polysaccharide: drying a proper amount of cordyceps culture at 70 ℃, adding 10 times of 50% ethanol according to the mass ratio of the culture to the volume of the extracting solution, extracting twice at 80 ℃, stirring for 0.5h each time, preserving heat for 2h, and filtering to obtain 50% ethanol extracting solution; adding 10 times of water solution into the residue after twice extraction with 50% ethanol, extracting at 100 deg.C, stirring for 0.5 hr, keeping the temperature for 1 hr, and filtering to obtain water extract. And respectively concentrating the 50% ethanol extract and the water extract to obtain 50% ethanol concentrated solution and water concentrated solution. Adding 3 times of 95% ethanol into the two concentrated solutions, standing overnight, and precipitating polysaccharide to obtain 50% ethanol-extracted precipitated polysaccharide and water-extracted precipitated polysaccharide, respectively. Mixing 50% ethanol extraction precipitated polysaccharide and water extraction precipitated polysaccharide, and spray drying to obtain Cordyceps crude polysaccharide;

(2) deproteinizing the crude polysaccharide: dissolving the crude polysaccharide powder by using deionized water, uniformly mixing the prepared crude polysaccharide solution and a Sevag reagent according to the volume ratio of 5:1, violently oscillating for 30min, centrifuging for 15min at 4000rpm, placing in a separating funnel for separating to remove an intermediate denatured protein layer and a lower organic solution layer, and repeating the operation for 3 times; loading the aqueous phase layer treated by the Sevag method to macroporous resin, eluting by using deionized water with the volume of 3 times, and concentrating the aqueous phase to obtain refined polysaccharide CP;

(3) ethanol precipitation by steps: uniformly stirring the concentrated solution of the refined polysaccharide with ethanol with the final concentration of 50% in volume ratio, standing at 4 ℃ overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and freeze-drying the precipitate at low temperature to obtain CP 50; regulating the volume of ethanol in the supernatant to 75%, standing overnight at 4 deg.C, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 75; concentrating and spin-drying the supernatant, dissolving with 95% ethanol, standing at 4 deg.C overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 95.

3. The method according to claim 1 or 2, wherein CP95 is used for the preparation of a medicament against gram-negative plant pathogenic bacteria.

4. The method of claim 1 or 2, wherein the cordyceps culture comprises cordyceps culture such as cordyceps cicadae miq, cordyceps sinensis or cordyceps militaris.

5. The method of claim 1 or 2, wherein the cordyceps culture comprises a culture residue, a fruit body, a liquid fermentation mycelium, or a liquid fermentation mycelium filtrate.

Technical Field

The invention relates to the technical field of biology, in particular to a method for preparing refined polysaccharide with bacteriostatic activity by using cordyceps culture and application thereof.

Background

Cordyceps (Cordyceps) is a complex entomomycete complex, including Cordyceps sobolifera, Cordyceps sinensis, Cordyceps militaris and the like, contains various bioactive components, and has important health-care and medicinal values. Cordyceps cicadae (Ophiocerdyceps Sobolifer a, Isaria cicadae, asexual generation) is an entomomycete complex formed by infecting fungus of Clavipitaceae with nymphaea cicadae, and asexual fruiting body (named as peduncle) formed by head hypha is similar to flower bud, so that the Cordyceps cicadae is named as Cordyceps cicadae and is a high-quality Cordyceps sinensis with a long medicinal history in traditional Chinese medicine. Researches show that the Cordyceps sobolifera contains various bioactive substances, the main components of the Cordyceps sobolifera comprise Cordyceps Polysaccharide (CP), cordycepin, cordycepic acid, sterol, uracil, vitamins, alkaloids, inorganic salt mineral elements and the like, and the Cordyceps sobolifera has multiple effects of regulating immunity, resisting fatigue, protecting kidney, improving sleep, resisting tumor, protecting liver, resisting radiation, improving eyesight and the like. Cordyceps (Ophiocerdyceps sinensis) is a complex of fungal stroma developed after the Cordyceps fungi parasitize on the larva body of the hepialus armoricanus and dead larva filled with hyphae, contains cordycepin, Cordyceps polysaccharide, sterol, amino acids, etc., and has effects of immunoregulation, anticancer, endocrine regulation, antibacterial, hematopoiesis promotion, antivirus, liver protection, sedation and anticonvulsion. Cordyceps militaris (Cordyceps militaris L Link) also called Cordyceps militaris and Cordyceps militaris has similar active ingredients and pharmacological action to Cordyceps militaris.

Natural cordyceps is very rare, and in recent years, the development of artificial culture realizes large-scale industrial cultivation of cordyceps. The cordyceps culture medium is prepared by taking grain crops such as rice, wheat and the like as main raw materials, inoculating cordyceps strains, and performing solid fermentation culture to obtain a cordyceps culture, wherein the culture comprises two parts of asexual sporocarp at the upper part and culture residue at the bottom part, and the culture residue consists of transformed culture material, mycelium and conidiospore. In practical application, the asexual fruiting bodies of cordyceps sinensis are generally picked and developed as top quality, so that a large number of culture residues are generated. Detection shows that the culture residual gene after the fruiting body is harvested contains a large amount of mycelia and spores, is rich in adenosine, crude fat, crude protein, crude fiber, polysaccharide, amino acid, calcium, magnesium, potassium, linoleic acid and other substances, and also has a certain application value.

Disclosure of Invention

The invention discloses a method for preparing refined polysaccharide with bacteriostatic activity by using cordyceps culture and application thereof. The invention takes cordyceps culture as raw material, extracts crude polysaccharide, and adopts a means of combining a macroporous resin adsorption method and a Sevag method to remove protein in the crude polysaccharide to obtain refined polysaccharide. Further adopts fractional alcohol precipitation purification, and discloses the application of the 95 percent ethanol precipitation cordycepin polysaccharide extracted from the cordyceps culture in the aspect of inhibiting gram-negative plant pathogenic bacteria.

One of the purposes of the invention is to provide a method for preparing refined polysaccharide with bacteriostatic activity by using cordyceps culture.

Preferably, the method comprises the following steps:

(1) extracting crude polysaccharide: drying a proper amount of cordyceps culture at 70 ℃, adding 10 times of 50% ethanol according to the mass ratio of the culture to the volume of the extracting solution, extracting twice at 80 ℃, stirring for 0.5h each time, preserving heat for 2h, and filtering to obtain 50% ethanol extracting solution; adding 10 times of water solution into the residue after twice extraction with 50% ethanol, extracting at 100 deg.C, stirring for 0.5 hr, keeping the temperature for 1 hr, and filtering to obtain water extract. And respectively concentrating the 50% ethanol extract and the water extract to obtain 50% ethanol concentrated solution and water concentrated solution. Adding 3 times of 95% ethanol into the two concentrated solutions, standing overnight, and precipitating polysaccharide to obtain 50% ethanol-extracted precipitated polysaccharide and water-extracted precipitated polysaccharide, respectively. Mixing 50% ethanol extraction precipitated polysaccharide and water extraction precipitated polysaccharide, and spray drying to obtain Cordyceps crude polysaccharide;

(2) deproteinizing the crude polysaccharide: dissolving the crude polysaccharide with deionized water, loading the prepared crude polysaccharide solution on macroporous resin, eluting with 3 times volume of deionized water, and collecting the eluate; uniformly mixing the collected eluent and Sevag reagent according to the volume ratio of 5:1, violently oscillating for 30min, centrifuging for 15min at 4000rpm, placing the mixture in a separating funnel for separating to remove an intermediate denatured protein layer and a lower organic solution layer, and repeating the operation for 2 times; concentrating the water phase layer to obtain refined polysaccharide CP;

(3) ethanol precipitation by steps: uniformly stirring the concentrated solution of the refined polysaccharide with ethanol with the final concentration of 50% in volume ratio, standing at 4 ℃ overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and freeze-drying the precipitate at low temperature to obtain CP 50; regulating the volume of ethanol in the supernatant to 75%, standing overnight at 4 deg.C, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 75; concentrating and spin-drying the supernatant, dissolving with 95% ethanol, standing at 4 deg.C overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 95.

Preferably, the method comprises the following steps:

(1) extracting crude polysaccharide: drying a proper amount of cordyceps culture at 70 ℃, adding 10 times of 50% ethanol according to the mass ratio of the culture to the volume of the extracting solution, extracting twice at 80 ℃, stirring for 0.5h each time, preserving heat for 2h, and filtering to obtain 50% ethanol extracting solution; adding 10 times of water solution into the residue after twice extraction with 50% ethanol, extracting at 100 deg.C, stirring for 0.5 hr, keeping the temperature for 1 hr, and filtering to obtain water extract. And respectively concentrating the 50% ethanol extract and the water extract to obtain 50% ethanol concentrated solution and water concentrated solution. Adding 3 times of 95% ethanol into the two concentrated solutions, standing overnight, and precipitating polysaccharide to obtain 50% ethanol-extracted precipitated polysaccharide and water-extracted precipitated polysaccharide, respectively. Mixing 50% ethanol extraction precipitated polysaccharide and water extraction precipitated polysaccharide, and spray drying to obtain Cordyceps crude polysaccharide;

(2) deproteinizing the crude polysaccharide: dissolving the crude polysaccharide powder by using deionized water, uniformly mixing the prepared crude polysaccharide solution and a Sevag reagent according to the volume ratio of 5:1, violently oscillating for 30min, centrifuging for 15min at 4000rpm, placing in a separating funnel for separating to remove an intermediate denatured protein layer and a lower organic solution layer, and repeating the operation for 3 times; loading the aqueous phase layer treated by the Sevag method to macroporous resin, eluting by using deionized water with the volume of 3 times, and concentrating the aqueous phase to obtain refined polysaccharide CP;

(3) ethanol precipitation by steps: uniformly stirring the concentrated solution of the refined polysaccharide with ethanol with the final concentration of 50% in volume ratio, standing at 4 ℃ overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and freeze-drying the precipitate at low temperature to obtain CP 50; regulating the volume of ethanol in the supernatant to 75%, standing overnight at 4 deg.C, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 75; concentrating and spin-drying the supernatant, dissolving with 95% ethanol, standing at 4 deg.C overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 95.

Preferably, the CP95 has use in the preparation of a medicament for combating gram-negative phytopathogenic bacteria.

Preferably, the gram-negative phytopathogen is a rice bacterial brown streak RS-1 strain, a tomato bacterial wilt MYA-1 strain or a sweet potato stem rot Dd-1 strain.

Preferably, the cordyceps culture comprises cordyceps cicadae miq, cordyceps sinensis or cordyceps militaris and the like.

Preferably, the cordyceps culture comprises a culture residue, a fruit body, a liquid fermentation mycelium or a liquid fermentation mycelium filtrate.

The invention also aims to provide application of the 95% ethanol precipitated cordycepin polysaccharide CP95 in preparing a medicament for resisting gram-negative plant pathogenic bacteria.

Preferably, the CP95 is prepared from Cordyceps culture.

Preferably, the preparation method of the CP95 comprises the following steps:

(1) extracting crude polysaccharide: drying a proper amount of cordyceps culture at 70 ℃, adding 10 times of 50% ethanol according to the mass ratio of the culture to the volume of the extracting solution, extracting twice at 80 ℃, stirring for 0.5h each time, preserving heat for 2h, and filtering to obtain 50% ethanol extracting solution; adding 10 times of water solution into the residue after twice extraction with 50% ethanol, extracting at 100 deg.C, stirring for 0.5 hr, keeping the temperature for 1 hr, and filtering to obtain water extract. And respectively concentrating the 50% ethanol extract and the water extract to obtain 50% ethanol concentrated solution and water concentrated solution. Adding 3 times of 95% ethanol into the two concentrated solutions, standing overnight, and precipitating polysaccharide to obtain 50% ethanol-extracted precipitated polysaccharide and water-extracted precipitated polysaccharide, respectively. Mixing 50% ethanol extraction precipitated polysaccharide and water extraction precipitated polysaccharide, and spray drying to obtain Cordyceps crude polysaccharide;

(2) deproteinizing the crude polysaccharide: dissolving the crude polysaccharide with deionized water, loading the prepared crude polysaccharide solution on macroporous resin, eluting with 3 times volume of deionized water, and collecting the eluate; uniformly mixing the collected eluent and Sevag reagent according to the volume ratio of 5:1, violently oscillating for 30min, centrifuging for 15min at 4000rpm, placing the mixture in a separating funnel for separating to remove an intermediate denatured protein layer and a lower organic solution layer, and repeating the operation for 2 times; concentrating the water phase layer to obtain refined polysaccharide CP;

(3) ethanol precipitation by steps: uniformly stirring the concentrated solution of the refined polysaccharide with ethanol with the final concentration of 50% in volume ratio, standing at 4 ℃ overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and freeze-drying the precipitate at low temperature to obtain CP 50; regulating the volume of ethanol in the supernatant to 75%, standing overnight at 4 deg.C, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 75; concentrating and spin-drying the supernatant, dissolving with 95% ethanol, standing at 4 deg.C overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 95.

Preferably, the preparation method of the CP95 comprises the following steps:

(1) extracting crude polysaccharide: drying a proper amount of cordyceps culture at 70 ℃, adding 10 times of 50% ethanol according to the mass ratio of the culture to the volume of the extracting solution, extracting twice at 80 ℃, stirring for 0.5h each time, preserving heat for 2h, and filtering to obtain 50% ethanol extracting solution; adding 10 times of water solution into the residue after twice extraction with 50% ethanol, extracting at 100 deg.C, stirring for 0.5 hr, keeping the temperature for 1 hr, and filtering to obtain water extract. And respectively concentrating the 50% ethanol extract and the water extract to obtain 50% ethanol concentrated solution and water concentrated solution. Adding 3 times of 95% ethanol into the two concentrated solutions, standing overnight, and precipitating polysaccharide to obtain 50% ethanol-extracted precipitated polysaccharide and water-extracted precipitated polysaccharide, respectively. Mixing 50% ethanol extraction precipitated polysaccharide and water extraction precipitated polysaccharide, and spray drying to obtain Cordyceps crude polysaccharide;

(2) deproteinizing the crude polysaccharide: dissolving the crude polysaccharide powder by using deionized water, uniformly mixing the prepared crude polysaccharide solution and a Sevag reagent according to the volume ratio of 5:1, violently oscillating for 30min, centrifuging for 15min at 4000rpm, placing in a separating funnel for separating to remove an intermediate denatured protein layer and a lower organic solution layer, and repeating the operation for 3 times; loading the aqueous phase layer treated by the Sevag method to macroporous resin, eluting by using deionized water with the volume of 3 times, and concentrating the aqueous phase to obtain refined polysaccharide CP;

(3) ethanol precipitation by steps: uniformly stirring the concentrated solution of the refined polysaccharide with ethanol with the final concentration of 50% in volume ratio, standing at 4 ℃ overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and freeze-drying the precipitate at low temperature to obtain CP 50; regulating the volume of ethanol in the supernatant to 75%, standing overnight at 4 deg.C, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 75; concentrating and spin-drying the supernatant, dissolving with 95% ethanol, standing at 4 deg.C overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 95.

Preferably, the gram-negative phytopathogen is a rice bacterial brown streak RS-1 strain, a tomato bacterial wilt MYA-1 strain or a sweet potato stem rot Dd-1 strain.

Preferably, the cordyceps culture comprises cordyceps cicadae miq, cordyceps sinensis or cordyceps militaris and the like.

Preferably, the cordyceps culture comprises a culture residue, a fruit body, a liquid fermentation mycelium or a liquid fermentation mycelium filtrate.

The invention provides a method for preparing refined polysaccharide with bacteriostatic activity by using cordyceps culture and application thereof. The preparation method has the characteristics of simple operation, stable performance and low cost, and the protein removal method combining the Sevag method and the macroporous resin adsorption method can reduce the protein removal times of the Sevag method by 3-4 times, thereby not only saving the use amount of the Sevag reagent and reducing the treatment cost, but also effectively removing the protein in the polysaccharide, and the protein removal rate reaches more than 96 percent, thereby laying a good foundation for further separation and purification of polysaccharide components and research on physiological functions. The cordyceps sinensis essential polysaccharide CP95 prepared by the invention has stronger inhibiting effect on three important gram-negative plant pathogenic bacteria which are harmful and difficult to prevent and treat in agricultural production, and the antibacterial activity of the cordyceps sinensis essential polysaccharide CP95 is obviously higher than 5% of amino-oligosaccharide and 5% of chitosan, so that the cordyceps sinensis essential polysaccharide has important economic significance for better developing and utilizing cordyceps sinensis cultures and reducing the cleaning cost of cordyceps sinensis waste culture media.

Drawings

FIG. 1 is a schematic diagram of the preparation process of refined polysaccharide with bacteriostatic activity by using Cordyceps culture.

FIG. 2 is a graph showing the effect of deproteinization of crude polysaccharide, in which (A) shows deproteinization by Sevag method, (B) shows deproteinization by macroporous resin adsorption method, (C) shows deproteinization by Sevag method after macroporous resin adsorption, and (D) shows deproteinization by Sevag method before macroporous resin adsorption.

FIG. 3 is the elution curve of macroporous resin in Cordyceps cicadae polysaccharide solution, wherein (A) and (B) show the change of polysaccharide content, and (C) and (D) show the change of protein content.

FIG. 4 is a graph of the bacteriostatic effect of cordyceps cicadae miq polysaccharide on rice bacterial brown streak fungus RS-1, wherein 1 represents 2000mg/mL cordyceps cicadae polysaccharide solution, 2 represents 1000mg/mL cordyceps cicadae polysaccharide solution, 3 represents 500mg/mL cordyceps cicadae polysaccharide solution, 4 represents 100mg/mL cordyceps cicadae polysaccharide solution, and 5 represents negative control sterilized water.

FIG. 5 is a graph showing the inhibitory effect of OSP95 on three gram-negative plant pathogens, wherein (A) is the inhibitory effect of OSP95 on rice bacterial brown streak RS-1 strain, (B) is the inhibitory effect of OSP95 on tomato bacterial wilt MYA-1 strain, (C) is the inhibitory effect of OSP95 on sweet potato stem rot Dd-1 strain, 1 represents 25mg/ml OSP95-A, 2 represents 80mg/ml OSP95-A, 3 represents 20mg/ml OSP95-B, 3' represents 40mg/ml 95-B, 4 represents 40mg/ml 5% amino-oligosaccharin, 5 represents sterilized distilled water, and 6 represents 20mg/ml 5% OSP.

FIG. 6 is a graph showing the effect of different concentrations of 5% amino-oligosaccharin and OSP95 on bacterial inhibition and growth of tomato bacterial wilt MYA-1 strain, wherein (A) is 5% amino-oligosaccharin with different concentrations, (B) is OSP95-A with different concentrations, and (C) is OSP95-B with different concentrations, and 0-6 respectively represent concentrations of 0, 0.625mg/ml, 1.25mg/ml, 2.5mg/ml, 5mg/ml, 10mg/ml and 20 mg/ml.

Detailed Description

The present invention will be described in more detail with reference to examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.

In the present invention, all the equipment, materials and the like are commercially available or commonly used in the industry, if not specified. Unless otherwise indicated, the examples employ methods that are within the ordinary skill in the art.

The cordyceps sobolifera culture medium used in the invention adopts wheat as a main material, and the strain adopted in the artificial cultivation of cordyceps sobolifera is cordyceps sobolifera (O.sobolifera)022007-9 strain, is obtained by separation of plant protection and entomogenous fungi research laboratories of subtropical crop research institute in Zhejiang province, and is stored in China general microbiological culture Collection center (CGMCC No: 8989).

The LB solid culture medium used in the invention consists of 5.0g of yeast powder, 10.0g of peptone, 5.0g of NaCl, 17.0g of agar powder, 1000mL of distilled water, pH 7.0-7.2, high-pressure steam sterilization (121 ℃, 20 min); the NA solid medium consists of 3.0g of beef extract, 1g of yeast extract, 5g of peptone, 10g of glucose (or sucrose), 15g of agar powder and 1000mL of distilled water, and the pH value is 6.8-7.0. The rice bacterial brown streak (Acidovorax avenae sub sp. avenae) RS-1 strain used in the invention is given by professor plum, a institute of biotechnology, Zhejiang university, sweet potato stem rot (Dickeya dadantii) Dd-1 strain is given by microorganisms of the institute of agricultural sciences, Zhejiang and a Cayan researchers of the institute of plant protection, and the tomato bacterial wilt (Ralstoniasolanacearum) MYA-1 strain is collected by a research laboratory of the institute of subtropical crops, Zhejiang and is separated from a tomato bacterial wilt plant disease strain in the Town of Thyama. The Sevag reagent is prepared by mixing chloroform and n-butanol at a volume ratio of 4:1, and macroporous adsorption resin AB-8 is purchased from Cangzhou Baoyin adsorption material science and technology Limited.