CN110558337A - biocontrol preparation for preventing and treating rice blast and preparation method thereof - Google Patents
biocontrol preparation for preventing and treating rice blast and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological control, and particularly relates to a biological control preparation for controlling rice blast and a preparation method thereof. The biocontrol preparation comprises the following components in parts by weight: 20-40 parts of gingko endophytic fungi fusarium oxysporum fermentation liquor, 10-20 parts of gingko leaf extract and 5-15 parts of gingko testa extract; and (2) mixing fermentation liquor prepared by performing solid culture and liquid culture on fusarium oxysporum, a ginkgo leaf extract and a ginkgo episperm extract according to the parts by weight to obtain the biocontrol preparation. After the biocontrol preparation disclosed by the invention is applied, the morbidity and disease index of rice blast can be obviously reduced, the relative prevention effect exceeds that of the conventional chemical prevention and control, meanwhile, the toxicity and residue problems caused by chemical pesticides can be avoided, and the biocontrol preparation is green, environment-friendly, efficient, difficult to generate drug resistance and has a certain application prospect.
Description
Technical Field
The invention belongs to the technical field of biological control, and particularly relates to a biological control preparation for controlling rice blast and a preparation method thereof.
Background
rice is one of the important food crops in the world, and more than half of the global population takes the rice as food. The planting area of the rice accounts for about 20 percent of the total planting area of the grain in the world. The rice blast (the pathogen is Magnaporthe oryzae, and the asexual generation is Pyricularia oryzae) is one of the most serious rice diseases harming the world. The rice blast can not be avoided in rice planting countries, the rice blast can not only reduce the yield of rice, but also has great influence on the quality of rice. According to the estimation of plant disease experts, 300 million tons of rice are directly lost due to the attack of rice blast in China every year.
At present, the rice blast prevention and treatment measures mainly comprise disease-resistant variety breeding, medicament prevention and treatment and cultivation management. The breeding of disease-resistant varieties is an economic and environment-friendly mode, but due to the complex and variable genetic background of rice blast germs and the simplification of blast-resistant varieties and the genetic homogeneity of main cultivars, the disease-resistant varieties are difficult to keep up with the variation speed of physiological races of pathogenic rice blast germs, so that a new rice variety with blast resistance loses resistance after being planted in a large area for several years and falls into an infected variety. The cultivation management is difficult to form a systematic and normative technical system, so that ideal effects are difficult to achieve in actual production. In order to meet the production needs, the chemical control is a common means for preventing and controlling rice blast, but due to the unreasonable application of chemical agents, the drug resistance of rice blast fungi to several main bactericides (cyproconazole and iprobenfos) commonly used in production is gradually enhanced, the prevention effect is continuously reduced, and considerable serious harm is brought to the ecological environment and human health.
disclosure of Invention
In view of the above technical problems, it is an object of the present invention to provide a biocontrol agent for controlling rice blast and a method for preparing the same, which can inhibit the occurrence of rice blast and avoid the toxicity and residue problems caused by chemical pesticides, are green, environmentally friendly, highly efficient and are not likely to cause drug resistance.
The invention aims to provide a biocontrol preparation for preventing and treating rice blast, which comprises the following components in parts by weight: 20-40 parts of gingko endophytic fungi fusarium oxysporum fermentation liquor, 10-20 parts of gingko leaf extract and 5-15 parts of gingko testa extract; and (2) mixing fermentation liquor prepared by performing solid culture and liquid culture on fusarium oxysporum, a ginkgo leaf extract and a ginkgo episperm extract according to the parts by weight to obtain the biocontrol preparation.
Preferably, the biocontrol agent comprises the following components in parts by weight: 30 parts of gingko endophytic fungi fusarium oxysporum fermentation liquor, 10 parts of gingko leaf extract and 15 parts of gingko testa extract.
Another object of the present invention is to provide a method for producing a biocontrol agent for controlling rice blast, which comprises the steps of:
(1) Inoculating fusarium oxysporum on a PDA (potato dextrose agar) solid slant culture medium for culturing for 24-48h, selecting mycelia to inoculate in a PDB (PDB) liquid culture medium when a strain grows vigorously, and performing fermentation culture for 5-7d in a constant-temperature oscillation incubator to obtain fusarium oxysporum fermentation liquor;
(2) Drying folium Ginkgo, pulverizing, sieving with 40-60 mesh sieve, placing into an extraction tank, simultaneously adding 60-80% ethanol, extracting for 2-3 times (each time for 60-70 min), and mixing extractive solutions; distilling the extractive solution under reduced pressure to 1/5-1/10 of the original volume to obtain concentrated solution, adding purified water with the same volume into the concentrated solution, precipitating for 12-15 hr, and collecting supernatant; drying the supernatant under reduced pressure, eluting with ethanol containing acetic acid, collecting eluate, distilling the eluate under reduced pressure to obtain fluid extract, and drying the fluid extract under reduced pressure to obtain folium Ginkgo extract;
(3) Decocting the ginkgo biloba sarcotesta with hot water at 90-99 ℃ for 1-2h each time for 2-3 times, wherein the material-liquid ratio is 1:6-12(W/V), filtering the decoctions, combining the filtrates, concentrating under reduced pressure, transferring the concentrated solution to a container, placing the container on a base of a magnetic stirrer, slowly dripping ethanol into the concentrated solution to make the final concentration of the ethanol reach 60-80% (V/V), stirring by starting the magnetic stirrer while adding the ethanol, standing, washing the precipitate with 80-90% ethanol for 3-5 times, and vacuum drying to obtain the ginkgo biloba sarcotesta extract;
(4) And uniformly mixing the fusarium oxysporum fermentation liquid, the ginkgo leaf extract and the ginkgo episperm extract according to the weight part to obtain the biocontrol preparation.
Further, the gingko endophytic fungus fusarium oxysporum is purchased from the institute of biotechnology of Chuanglian union, Beijing.
Further, the temperature of the fermentation culture in the step (1) is 26-28 ℃, and the rotating speed is 180-200 r/min.
Further, the thallus concentration of the fusarium oxysporum in the fusarium oxysporum fermentation liquor obtained in the step (1) is more than or equal to 1 multiplied by 107cfu/ml。
Further, the dosage of the ethanol in the extraction tank in the step (2) is 4-8 times of the weight of the ginkgo leaves.
Further, the ethanol containing acetic acid in the step (2) is composed of ethanol with the volume concentration of 80-90% and acetic acid with the volume percentage of 0.1-0.5%.
Further, the stirring temperature of the magnetic stirrer in the step (3) is 20-30 ℃, and the rotating speed is 1400-1800 r/min.
Compared with the prior art, the invention has the following beneficial effects:
(1) The ginkgo episperm extract contains abundant ginkgolic acid and flavonoid compounds, wherein the ginkgolic acid has certain bacteriostatic ability, and the alkyl side chain of the phenolic acid plays an important role in bacteriostasis. The flavonoids compounds in the ginkgo leaf extract can inhibit the activity of DNA topoisomerase in bacteria cells, so that the metabolic activity of nucleic acid in bacteria is inhibited, and the expression of related proteins in the bacteria is hindered, thereby achieving the bacteriostatic and bactericidal effects.
(2) The gingko endophytic fungus fusarium oxysporum and its metabolite can activate the protective enzyme system of rice. After the preparation containing the gingko endophytic fungi fusarium oxysporum and metabolites thereof is sprayed, the activities of Peroxidase (POD) and Catalase (CAT) in stems, leaves and ear organs can be improved, and the content of proline can be increased. The POD activity is improved, so that the content of phenol oxide can be greatly increased, the accumulation of lignin is promoted, and the cell wall is thickened, so that the invasion and expansion of pathogens are resisted.
(3) the ginkgo endophytic fungi fusarium oxysporum fermentation liquor, the ginkgo leaf extract and the ginkgo episperm extract are used as single active ingredients, so that the control effect is unstable in practical application due to various complex factors (such as pH, temperature, soil microbial environment and the like), and the control effect on rice blast is not obvious. After the ginkgo biloba endophytic fungi fusarium oxysporum and the metabolites thereof are compounded and mixed according to a certain proportion, the action targets of the ginkgo biloba endophytic fungi fusarium oxysporum and the metabolites thereof are different from those of the ginkgo biloba extract and the ginkgo biloba exocarp extract, the ginkgo biloba endophytic fungi fusarium oxysporum and the metabolites thereof are used for activating a protection enzyme system of rice, the ginkgo biloba exocarp extract is used for directly killing rice blast germs through the strong oxidizing action of phenolic acid, and flavonoids compounds in the ginkgo biloba extract inhibit the growth of the germs through the activity of DNA topoisomerase in germ cells. The three components are compounded to enhance the control effect on the rice blast and reduce the resistance of the rice blast to the preparation.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The endophytic fungus Fusarium oxysporum of Ginkgo used in the following examples was purchased from the institute of Biotechnology, Chuangliana, Beijing, and the ginkgo biloba leaves and the ginkgo biloba testa were collected by themselves, and other used chemical instruments and medicines were purchased from conventional approaches.
example 1
(1) inoculating Fusarium oxysporum (Fusarium oxysporum) as endophytic fungus of semen Ginkgo on PDA solid slant culture medium, culturing for 24-48 hr, and selecting mycelium for grafting when strain grows vigorouslyinoculating to PDB liquid culture medium, and performing fermentation culture at 26-28 deg.C and 180r/min for 7d in constant temperature shaking incubator to obtain Fusarium oxysporum fermentation liquid with thallus concentration of 1 × 10 or more7cfu/ml；
(2) Drying folium Ginkgo, pulverizing, sieving with 40 mesh sieve, placing into an extraction tank, simultaneously adding 60% ethanol 4 times of folium Ginkgo weight, extracting for 70min for 2 times, and mixing extractive solutions; distilling the extractive solution under reduced pressure to 1/5 of the original volume to obtain concentrated solution, adding purified water with the same volume into the concentrated solution, precipitating for 12-15h, and collecting supernatant; drying the supernatant under reduced pressure, eluting with ethanol containing acetic acid, collecting eluate, distilling the eluate under reduced pressure to obtain fluid extract, and drying the fluid extract under reduced pressure to obtain folium Ginkgo extract; the ethanol containing acetic acid consists of ethanol with the volume concentration of 80% and acetic acid with the volume percentage of 0.1%;
(3) decocting the ginkgo biloba sarcotesta with hot water at 90-99 ℃ for 1h each time for 3 times, wherein the material-liquid ratio is 1:6(W/V), filtering the decoctions respectively, combining the filtrates, concentrating under reduced pressure, transferring the concentrated solution into a container, placing the container on a base of a magnetic stirrer, slowly dripping ethanol into the concentrated solution to make the final concentration of the ethanol reach 60% (V/V), stirring with the magnetic stirrer while adding the ethanol, stirring at 20 ℃, the rotation speed is 1800r/min, standing, washing the precipitate with 80% ethanol for 3-5 times, and vacuum drying to obtain the ginkgo biloba sarcotesta extract;
(4) and uniformly mixing 20 parts of ginkgo endophytic fungi fusarium oxysporum fermentation liquor, 20 parts of ginkgo leaf extract and 10 parts of ginkgo episperm extract to obtain the biocontrol preparation.
Example 2
(1) Inoculating gingko endophytic fungus Fusarium oxysporum on PDA solid slant culture medium for culturing for 24-48h, selecting mycelia to inoculate in PDB liquid culture medium when bacterial strain grows vigorously, and performing fermentation culture at 26-28 deg.C and 200r/min for 5d in constant temperature shaking culture box to obtain Fusarium oxysporum fermentation liquid with thallus concentration of 1 × 10 or more7cfu/ml；
(2) Drying folium Ginkgo, pulverizing, sieving with 50 mesh sieve, placing into an extraction tank, simultaneously adding ethanol 6 times the weight of folium Ginkgo and 80% volume concentration, extracting for 3 times, each time for 60min, and mixing extractive solutions; distilling the extractive solution under reduced pressure to 1/10 of the original volume to obtain concentrated solution, adding purified water with the same volume into the concentrated solution, precipitating for 12-15h, and collecting supernatant; drying the supernatant under reduced pressure, eluting with ethanol containing acetic acid, collecting eluate, distilling the eluate under reduced pressure to obtain fluid extract, and drying the fluid extract under reduced pressure to obtain folium Ginkgo extract; the ethanol containing acetic acid consists of 85% ethanol by volume concentration and 0.3% acetic acid by volume percentage;
(3) decocting the ginkgo biloba sarcotesta with 90-99 ℃ hot water for 2 hours each time for 2 times, wherein the material-liquid ratio is 1:8(W/V), filtering the decoction liquids respectively, combining the filtrates, concentrating under reduced pressure, transferring the concentrated solution into a container, placing the container on a base of a magnetic stirrer, slowly dripping ethanol into the concentrated solution to enable the final concentration of the ethanol to reach 70% (V/V), stirring by starting the magnetic stirrer while adding the ethanol, wherein the stirring temperature is 25 ℃, the rotating speed is 1600r/min, washing the precipitate with 90% ethanol for 3-5 times after standing, and then carrying out vacuum drying to obtain the ginkgo biloba sarcotesta extract;
(4) and uniformly mixing 30 parts of ginkgo endophytic fungi fusarium oxysporum fermentation liquor, 10 parts of ginkgo leaf extract and 15 parts of ginkgo episperm extract to obtain the biocontrol preparation.
example 3
(1) Inoculating gingko endophytic fungus Fusarium oxysporum on PDA solid slant culture medium for culturing for 24-48h, selecting mycelia to inoculate in PDB liquid culture medium when bacterial strain grows vigorously, and performing fermentation culture at 26-28 deg.C and 190r/min for 6d in constant temperature shaking culture box to obtain Fusarium oxysporum fermentation liquid, wherein the thallus concentration of Fusarium oxysporum in the fermentation liquid is more than or equal to 1 × 107cfu/ml；
(2) Drying folium Ginkgo, pulverizing, sieving with 60 mesh sieve, placing into an extraction tank, simultaneously adding 70% ethanol 8 times of folium Ginkgo weight, extracting for 2 times (each time for 70 min), and mixing extractive solutions; distilling the extractive solution under reduced pressure to 1/8 of the original volume to obtain concentrated solution, adding purified water with the same volume into the concentrated solution, precipitating for 12-15h, and collecting supernatant; drying the supernatant under reduced pressure, eluting with ethanol containing acetic acid, collecting eluate, distilling the eluate under reduced pressure to obtain fluid extract, and drying the fluid extract under reduced pressure to obtain folium Ginkgo extract; the ethanol containing acetic acid consists of ethanol with the volume concentration of 90% and acetic acid with the volume percentage of 0.5%;
(3) Decocting the ginkgo biloba sarcotesta with hot water at 90-99 ℃ for 1h each time for 3 times, wherein the material-liquid ratio is 1:10(W/V), filtering the decoctions respectively, combining the filtrates, concentrating under reduced pressure, transferring the concentrated solution into a container, placing the container on a base of a magnetic stirrer, slowly dripping ethanol into the concentrated solution to make the final concentration of the ethanol reach 80% (V/V), stirring with the magnetic stirrer while adding the ethanol, stirring at 30 ℃, rotating at 1400r/min, standing, washing the precipitate with ethanol with the volume concentration of 85% for 3-5 times, and vacuum drying to obtain the ginkgo biloba sarcotesta extract;
(4) And uniformly mixing 40 parts of ginkgo endophytic fungi fusarium oxysporum fermentation liquor, 15 parts of ginkgo leaf extract and 5 parts of ginkgo episperm extract to obtain the biocontrol preparation.
Example 4
(1) Inoculating gingko endophytic fungus Fusarium oxysporum on PDA solid slant culture medium for culturing for 24-48h, selecting mycelia to inoculate in PDB liquid culture medium when bacterial strain grows vigorously, and performing fermentation culture at 26-28 deg.C and 180r/min for 7d in constant temperature shaking culture box to obtain Fusarium oxysporum fermentation liquid, wherein the thallus concentration of Fusarium oxysporum in the fermentation liquid is more than or equal to 1 × 107cfu/ml；
(2) Drying folium Ginkgo, pulverizing, sieving with 40 mesh sieve, placing into an extraction tank, simultaneously adding 70% ethanol 6 times of folium Ginkgo weight, extracting for 3 times (60 min each time), and mixing extractive solutions; distilling the extractive solution under reduced pressure to 1/5 of the original volume to obtain concentrated solution, adding purified water with the same volume into the concentrated solution, precipitating for 12-15h, and collecting supernatant; drying the supernatant under reduced pressure, eluting with ethanol containing acetic acid, collecting eluate, distilling the eluate under reduced pressure to obtain fluid extract, and drying the fluid extract under reduced pressure to obtain folium Ginkgo extract; the ethanol containing acetic acid consists of ethanol with the volume concentration of 80% and acetic acid with the volume percentage of 0.3%;
(3) Decocting the ginkgo biloba sarcotesta with 90-99 ℃ hot water for 2 hours each time for 2 times, wherein the material-liquid ratio is 1:12(W/V), filtering the decoction liquids respectively, combining the filtrates, concentrating under reduced pressure, transferring the concentrated solution into a container, placing the container on a base of a magnetic stirrer, slowly dripping ethanol into the concentrated solution to enable the final concentration of the ethanol to reach 60% (V/V), stirring by starting the magnetic stirrer while adding the ethanol, wherein the stirring temperature is 20 ℃, the rotating speed is 1800r/min, washing the precipitate with 90% ethanol by volume concentration for 3-5 times after standing, and then carrying out vacuum drying to obtain the ginkgo biloba sarcotesta extract;
(4) Uniformly mixing 25 parts of ginkgo endophytic fungi fusarium oxysporum fermentation liquor, 10 parts of ginkgo leaf extract and 10 parts of ginkgo episperm extract to obtain the biocontrol preparation.
Example 5
(1) inoculating gingko endophytic fungus Fusarium oxysporum on PDA solid slant culture medium for culturing for 24-48h, selecting mycelia to inoculate in PDB liquid culture medium when bacterial strain grows vigorously, and performing fermentation culture at 26-28 deg.C and 190r/min for 6d in constant temperature shaking culture box to obtain Fusarium oxysporum fermentation liquid, wherein the thallus concentration of Fusarium oxysporum in the fermentation liquid is more than or equal to 1 × 107cfu/ml；
(2) Drying folium Ginkgo, pulverizing, sieving with 50 mesh sieve, placing into an extraction tank, simultaneously adding ethanol 8 times the weight of folium Ginkgo and 80% volume concentration, extracting for 2 times, each time for 70min, and mixing extractive solutions; distilling the extractive solution under reduced pressure to 1/10 of the original volume to obtain concentrated solution, adding purified water with the same volume into the concentrated solution, precipitating for 12-15h, and collecting supernatant; drying the supernatant under reduced pressure, eluting with ethanol containing acetic acid, collecting eluate, distilling the eluate under reduced pressure to obtain fluid extract, and drying the fluid extract under reduced pressure to obtain folium Ginkgo extract; the ethanol containing acetic acid consists of 85% ethanol by volume concentration and 0.5% acetic acid by volume percentage;
(3) decocting the ginkgo biloba sarcotesta with hot water at 90-99 ℃ for 1h each time for 3 times, wherein the material-liquid ratio is 1:8(W/V), filtering the decoctions respectively, combining the filtrates, concentrating under reduced pressure, transferring the concentrated solution into a container, placing the container on a base of a magnetic stirrer, slowly dripping ethanol into the concentrated solution to make the final concentration of the ethanol reach 70% (V/V), stirring with the magnetic stirrer while adding the ethanol, stirring at 25 ℃ and 1600r/min, washing the precipitate with ethanol with the volume concentration of 85% for 3-5 times after standing, and vacuum drying to obtain the ginkgo biloba sarcotesta extract;
(4) And uniformly mixing 35 parts of ginkgo endophytic fungi fusarium oxysporum fermentation liquid, 15 parts of ginkgo leaf extract and 15 parts of ginkgo episperm extract to obtain the biocontrol preparation.
Example 6
(1) Inoculating gingko endophytic fungus Fusarium oxysporum on PDA solid slant culture medium for culturing for 24-48h, selecting mycelia to inoculate in PDB liquid culture medium when bacterial strain grows vigorously, and performing fermentation culture at 26-28 deg.C and 200r/min for 5d in constant temperature shaking culture box to obtain Fusarium oxysporum fermentation liquid with thallus concentration of 1 × 10 or more7cfu/ml；
(2) Drying folium Ginkgo, pulverizing, sieving with 60 mesh sieve, placing into an extraction tank, simultaneously adding 60% ethanol 4 times of folium Ginkgo weight, extracting for 3 times (60 min each time), and mixing extractive solutions; distilling the extractive solution under reduced pressure to 1/8 of the original volume to obtain concentrated solution, adding purified water with the same volume into the concentrated solution, precipitating for 12-15h, and collecting supernatant; drying the supernatant under reduced pressure, eluting with ethanol containing acetic acid, collecting eluate, distilling the eluate under reduced pressure to obtain fluid extract, and drying the fluid extract under reduced pressure to obtain folium Ginkgo extract; the ethanol containing acetic acid consists of ethanol with the volume concentration of 90% and acetic acid with the volume percentage of 0.1%;
(3) Decocting the ginkgo biloba sarcotesta with hot water at 90-99 ℃ for 2 hours each time for 2 times, wherein the material-liquid ratio is 1:10(W/V), filtering the decoction liquids respectively, combining the filtrates, concentrating under reduced pressure, transferring the concentrated solution into a container, placing the container on a base of a magnetic stirrer, slowly dripping ethanol into the concentrated solution to enable the final concentration of the ethanol to reach 80% (V/V), stirring by starting the magnetic stirrer while adding the ethanol, wherein the stirring temperature is 30 ℃, the rotating speed is 1400r/min, washing the precipitate with ethanol with the volume concentration of 80% for 3-5 times after standing, and then carrying out vacuum drying to obtain the ginkgo biloba sarcotesta extract;
(4) And uniformly mixing 30 parts of ginkgo endophytic fungi fusarium oxysporum fermentation liquor, 20 parts of ginkgo leaf extract and 5 parts of ginkgo episperm extract to obtain the biocontrol preparation.
Comparative example 1 is a blank control with application of clear water.
Comparative example 2 is a control to which a conventional chemical pesticide, isoprothiolane EC 40% (x 500-fold liquid) was applied.
Comparative example 3 differs from example 1 in that only the ginkgo endophytic fungus fusarium oxysporum fermentation broth is administered.
Comparative example 4 is different from example 1 in that only ginkgo biloba leaf extract is administered.
comparative example 5 differs from example 1 in that only the ginkgo biloba testa extract is administered.
Comparative example 6 differs from example 1 in that only the ginkgo endophytic fungi fusarium oxysporum fermentation broth and the ginkgo biloba leaf extract are administered.
Comparative example 7 differs from example 1 in that only the ginkgo endophytic fungus fusarium oxysporum fermentation broth and the ginkgo episperm extract are administered.
comparative example 8 differs from example 1 in that only ginkgo biloba leaf extract and ginkgo biloba testa extract are administered.
inoculating rice blast fungus conidium suspension (concentration 1 × 10) by in vivo inoculation method of rice5one/mL) induced rice blast, and the agents in the above 6 examples and 8 comparative examples were applied, respectively, 3 pots per treatment mode, 30 plants per pot. The incidence and disease index of the rice blast are investigated 10 days after inoculation, and the effect of preventing and treating the rice blast is evaluated.
TABLE 1 control of rice blast by different treatments
As can be seen from the data in the above table, compared with comparative example 1 in which only an equal amount of clear water is applied and comparative example 2 in which a conventional chemical pesticide is applied, the incidence and disease index of blast disease in examples 1 to 6 in which the biocontrol agent of the present invention is applied are significantly reduced, wherein the incidence of blast disease is reduced by 82.63% and 10.83% on average, the disease index is reduced by 91.32% and 54.69% on average, and the relative control effect is more than 90%, indicating that the biocontrol agent of the present invention has a good control effect on blast disease.
Compared with the example 1, the treatment of the comparative example 3 only applying the fermentation liquor of the gingko endophytic fungus fusarium oxysporum and the comparative example 5 only applying the gingko testa extract has higher morbidity and disease index, and the relative control effect is only 28.19 percent and 13.58 percent; comparative example 4, which applied only ginkgo biloba extract, had a certain control effect on rice blast, which was 55.14% but less than 40% isoprothiolane. In comparative examples 6-8 in which any two components are compounded, comparative example 6 in which the ginkgo endophytic fungi fusarium oxysporum fermentation liquid and the ginkgo biloba extract are applied and comparative example 8 in which the ginkgo biloba endophytic fungi fusarium oxysporum fermentation liquid and the ginkgo biloba exocarp extract are applied have similar effects, and comparative example 7 in which the ginkgo endophytic fungi fusarium oxysporum fermentation liquid and the ginkgo exocarp extract are applied has poorer control effects and only 21.38% of relative control effects, which indicates that the single or two components in the biocontrol preparation of the invention have certain rice blast control effects, but the effects are still lower than the conventional chemical control and are also lower than the biocontrol preparation of the invention.
Claims (8)
1. a biocontrol preparation for preventing and treating rice blast is characterized by comprising the following components in parts by weight: 20-40 parts of gingko endophytic fungi fusarium oxysporum fermentation liquor, 10-20 parts of gingko leaf extract and 5-15 parts of gingko testa extract; and (2) mixing fermentation liquor prepared by performing solid culture and liquid culture on fusarium oxysporum, a ginkgo leaf extract and a ginkgo episperm extract according to the parts by weight to obtain the biocontrol preparation.
2. The biocontrol formulation for controlling rice blast of claim 1, characterized in that it comprises the following ingredients in parts by weight: 30 parts of gingko endophytic fungi fusarium oxysporum fermentation liquor, 10 parts of gingko leaf extract and 15 parts of gingko testa extract.
3. The method for producing a biocontrol agent for controlling rice blast as claimed in claim 1 or 2, characterized by comprising the steps of:
(1) Inoculating fusarium oxysporum on a PDA (potato dextrose agar) solid slant culture medium for culturing for 24-48h, selecting mycelia to inoculate in a PDB (PDB) liquid culture medium when a strain grows vigorously, and performing fermentation culture for 5-7d in a constant-temperature oscillation incubator to obtain fusarium oxysporum fermentation liquor;
(2) drying folium Ginkgo, pulverizing, sieving with 40-60 mesh sieve, placing into an extraction tank, simultaneously adding 60-80% ethanol, extracting for 2-3 times (each time for 60-70 min), and mixing extractive solutions; distilling the extractive solution under reduced pressure to 1/5-1/10 of the original volume to obtain concentrated solution, adding purified water with the same volume into the concentrated solution, precipitating for 12-15 hr, and collecting supernatant; drying the supernatant under reduced pressure, eluting with ethanol containing acetic acid, collecting eluate, distilling the eluate under reduced pressure to obtain fluid extract, and drying the fluid extract under reduced pressure to obtain folium Ginkgo extract;
(3) Decocting the ginkgo biloba sarcotesta with hot water at 90-99 ℃ for 1-2h each time for 2-3 times, wherein the material-liquid ratio is 1:6-12(W/V), filtering the decoctions, combining the filtrates, concentrating under reduced pressure, transferring the concentrated solution to a container, placing the container on a base of a magnetic stirrer, slowly dripping ethanol into the concentrated solution to make the final concentration of the ethanol reach 60-80% (V/V), stirring by starting the magnetic stirrer while adding the ethanol, standing, washing the precipitate with 80-90% ethanol for 3-5 times, and vacuum drying to obtain the ginkgo biloba sarcotesta extract;
(4) and uniformly mixing the fusarium oxysporum fermentation liquid, the ginkgo leaf extract and the ginkgo episperm extract according to parts by weight to obtain the biocontrol preparation.
4. The method for preparing a biocontrol agent for controlling rice blast as claimed in claim 3, characterized in that the temperature during fermentation culture in step (1) is 26-28 ℃ and the rotation speed is 180-200 r/min.
5. The method for preparing a biocontrol agent for controlling rice blast of claim 3, wherein the cell concentration of Fusarium oxysporum in the Fusarium oxysporum fermentation broth obtained in step (1) is not less than 1X 107cfu/ml。
6. The method for preparing a biocontrol agent for controlling rice blast as claimed in claim 3, characterized in that the amount of ethanol in the extraction tank of step (2) is 4-8 times the weight of ginkgo leaves.
7. The method for preparing a biocontrol agent for controlling rice blast as claimed in claim 3, characterized in that said ethanol containing acetic acid in step (2) is composed of ethanol with a volume concentration of 80-90% and acetic acid with a volume percentage of 0.1-0.5%.
8. The method for preparing a biocontrol agent for controlling rice blast as claimed in claim 3, characterized in that the stirring temperature of the magnetic stirrer in the step (3) is 20-30 ℃ and the rotation speed is 1400-1800 r/min.
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| WO2022188162A1 (en) * | 2021-03-12 | 2022-09-15 | 德州学院 | Endophytic fungus in ginkgo biloba and anti-bacterial and antitumor application thereof |
| CN115449501A (en) * | 2022-07-25 | 2022-12-09 | 福建华闽晟业生物科技有限公司 | Application of Jujun grass juice as non-pathogenic fusarium oxysporum fermentation culture medium |
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