CN108041102B - Method for preparing plutella xylostella killing biological agent by fermenting traditional Chinese medicine composite raw materials - Google Patents
Method for preparing plutella xylostella killing biological agent by fermenting traditional Chinese medicine composite raw materials Download PDFInfo
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Abstract
The invention relates to a method for preparing a plutella xylostella killing biological agent by fermenting traditional Chinese medicine composite raw materials, and belongs to the technical field of biological fermentation. In order to solve the problem of poor pertinence, the method for preparing the plutella xylostella killing biological agent by fermenting the traditional Chinese medicine composite raw materials comprises the steps of selecting the traditional Chinese medicine raw materials of chinaberry, radix sophorae flavescentis and radix stemonae, cleaning, crushing, adding into a fermentation tank, and adding water to dilute into a fermentation substrate; after sterilization, the activated Bacillus licheniformis bacteria was according to 108cfu/mL～109Inoculating cfu/mL of the fermentation substrate after sterilization, and performing fermentation treatment to obtain fermentation liquor; filtering to remove precipitate to obtain the diamondback moth killing biological agent. The biological preparation obtained by the invention can simultaneously contain toosendanin, matrine, stemonine and isocstemonine, thereby realizing the improvement of the insecticidal function on diamond back moths and having the effect of promoting the growth of vegetable plants.
Description
Technical Field
The invention relates to a method for preparing a plutella xylostella killing biological agent by fermenting traditional Chinese medicine composite raw materials, and belongs to the technical field of biological fermentation.
Background
Plutella xylostella (Plutel la xylostella la) is a lepidoptera plutella xylostella insect, which is named as cabbage worm, two-headed tip, diamond back moth, diease worm and the like, and belongs to the worldwide migratory flying pest. Diamondback moth has the characteristics of multiple generations, strong reproductive capacity, wide host range, high drug resistance level, difficult control and the like. Mainly contains brassicaceous plants such as harmful cabbage, purple cabbage, broccoli, cabbage, mustard, cauliflower, Chinese cabbage, rape, radish and the like, and the cost for preventing and controlling diamond back moths is up to 10 hundred million dollars every year all over the world. At present, chemical pesticides are mainly used for preventing and treating the plutella xylostella which is harmful to the plutella xylostella, so that the environment is polluted, plant phytotoxicity is easily caused, and poisoning of people and livestock is easily caused. Therefore, the development of biological plutella xylostella insecticides has been receiving attention.
Generally, chemical pesticides are adopted for killing insects, which is a socially accepted mode of killing insects, but the chemical pesticides all have certain toxicity, and the problem of long-term toxic residue exists when the pesticide is sprayed on plants. In the process of vegetable planting, the use of the pesticide is quite common, and various side effects caused by the adoption of chemical pesticides are caused, so that the pesticide is not beneficial to the health of human bodies. With the increasing awareness of the living environment and the natural ecology of people, the pesticide with high pesticide residue is limited. Therefore, the reduction of pesticide usage and the replacement of chemical pesticides with biological pesticides have become the current trend of pesticides.
At present, the discovered plant materials such as the melia azedarach, the sophora flavescens, the radix stemonae and the like have better insecticidal effect, the melia azedarach with insecticidal effect is developed according to the existing research situation, wherein the toosendanin has strong stomach toxicity effect on cabbage caterpillars and the like, can damage midgut tissues and block nerve conduction to cause paralysis, coma and death of pests. The sophora flavescens contains a plurality of secondary metabolites, and the sophora flavescens ether extract and the alcohol extract have strong bacteriostatic action on staphylococcus aureus; the soaking agent has inhibitory effect on Trichophyton violaceum, Trichophyton concentricum, Trichophyton schoenleinii, and Trichophyton ozi. Matrine can paralyze nerve center, solidify polypide protein, seal polypide pore, and suffocate pest; the stemona root contains various secondary metabolites such as stemonine, isobutramine, protostemonine, stemonine, hodorine, stemonine and the like, has killing effects on mosquito larvae, head lice, pubic louse, clothes lice, bed bugs and the like, and the stemona root with high concentration has the effect of killing rat enterobiasis in vitro. Therefore, there are studies to date which show that biological agents are prepared by fermentation. For example, Chinese patent application (publication No. CN106867947A) discloses a method for preparing insecticide by microbial fermentation of Melia azedarach L.which comprises inoculating Bacillus amyloliquefaciens, Bacillus pumilus and Bacillus thuringiensis to seed culture medium respectively, culturing to obtain mixed bacteria, treating Melia azedarach L.with ethanol and water, pulverizing, adding water, sterilizing, placing into a fermentation tank, inoculating the mixed bacteria, culturing at 37 deg.C for 3-12 days to obtain corresponding fermentation liquid, and filtering to obtain fermentation liquid as insecticide. Although the problems of inactivation and organic solvent pollution can be avoided to some extent by means of biological fermentation, the fermentation is carried out by means of coexistence of multiple biological bacteria, the cost is high, the conditions for coexistence of multiple bacteria are complex, and the like, and the method is only aimed at fermentation culture of a single plant.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method for preparing a plutella xylostella killing biological agent by fermenting a traditional Chinese medicine composite raw material, and solves the problem of how to realize the plutella xylostella killing performance which can improve the growth of plants and has high activity.
The invention aims to realize the following technical scheme, and provides a method for preparing a plutella xylostella killing biological agent by fermenting traditional Chinese medicine composite raw materials, which is characterized by comprising the following steps:
A. selecting chinaberry, radix sophorae flavescentis and radix stemonae as traditional Chinese medicine raw materials, cleaning, crushing, adding into a fermentation tank, and adding water to dilute into a fermentation substrate;
B. sterilizing the obtained fermentation substrate, and then treating the activated Bacillus licheniformis according to the proportion of 108cfu/mL～109Inoculating cfu/mL of the fermentation substrate after sterilization, and performing fermentation treatment at the fermentation temperature of 25-35 ℃ to obtain fermentation liquor;
C. and filtering the obtained fermentation liquor to remove precipitates to obtain the diamondback moth killing biological agent.
The single bacillus licheniformis is used as a microorganism, so that the mixed raw materials of three traditional Chinese medicines of chinaberry, radix sophorae flavescentis and radix stemonae can be simultaneously used as substrates for fermentation culture, the problem of mutual competition caused by coexistence of multiple dominant microbial strains can be effectively avoided, the single bacillus licheniformis is used for fermentation culture, nutrient substances of the fermentation substrates can be better utilized, active ingredients in the chinaberry, the radix sophorae flavescentis and the radix stemonae can be effectively released, the obtained fermentation liquor can effectively kill plutella xylostella when being used as a biological insecticide, the bacillus licheniformis has a growth promoting effect, the biological insecticide can have a growth promoting effect on plants such as vegetables when being used, and the growth capacity of the plants is improved; meanwhile, the activity of active substances in the fermentation liquor can be ensured by a fermentation mode, and the function of killing the plutella xylostella is increased.
In the method for preparing the plutella xylostella-killing biological agent by fermenting the traditional Chinese medicine composite raw materials, preferably, the chinaberry fruit in the step A: flavescent sophora root: stemona root: the mass ratio of water is 1: 0.5-0.7: 0.7-0.9: 10 to 15. In order to make the active ingredients in the fermentation liquor obtained after fermentation have better insecticidal capacity on diamond back moths, the toosendanin obtained from chinaberry and the matrine obtained from radix sophorae flavescentis have higher proportion, and the effect of better blocking nerves and paralyzing nerves on diamond back moths is improved.
In the method for preparing the plutella xylostella-killing biological agent by fermenting the traditional Chinese medicine composite raw materials, preferably, ice powder is added in the crushing treatment in the step A, and the adding amount of the ice powder is 2.0-5.0% of the mass of the chinaberry. The ice powder has certain hardness and edges and corners, so that the cell walls of plant tissues can be damaged in the crushing process, and active ingredients in cells can be better released; meanwhile, the plant tissues can be more thoroughly crushed, the effective utilization of bacillus licheniformis to the plant tissues is improved, and the adopted ice powder is dissolved to directly form water fermentation liquor raw materials without generating side effects on the raw materials. More preferably, the temperature of the ice powder is-30 ℃ to-10 ℃. The hardness of the ice powder is improved, so that the ice powder has better puncture capability.
In the method for preparing the plutella xylostella-killing biological agent by fermenting the traditional Chinese medicine composite raw materials, preferably, a carbon source is added into the fermentation substrate after the sterilization in the step B, and the addition amount of the carbon source is 5-10% of the addition amount of the chinaberry. By adding a small amount of carbon source in the early stage, the growth and the propagation of the bacillus licheniformis can be improved, and a large amount of the bacillus licheniformis which grows in a propagation mode can be utilized more fully and quickly by adding the chinaberry, the radix sophorae flavescentis and the radix stemonae. As a further optimization, the carbon source is selected from one or more of glucose, fructose and sucrose. Is beneficial to improving the utilization efficiency of the bacillus licheniformis.
In the method for preparing the plutella xylostella-killing biological agent by fermenting the traditional Chinese medicine composite raw materials, the fermentation treatment time in the step B is preferably 7-10 days. The aim is to fully utilize the nutrient components of the fermentation substrate and improve the release of active components in plant tissue cells.
In the method for preparing the plutella xylostella-killing biological agent by fermenting the traditional Chinese medicine composite raw materials, the pH value of the system during fermentation treatment in the step B is 6.5-7.5 as preferable. The fermentation culture environment is more beneficial to the growth of the bacillus licheniformis.
In the method for preparing the diamondback moth-killing biological agent by fermenting the traditional Chinese medicine composite raw materials, preferably, the diamondback moth-killing biological agent in the step C contains toosendanin, matrine, stemonine and isocstemonine. By adopting the bacillus licheniformis of the invention as a growth-promoting bacterium for fermentation culture, the effective active ingredients of toosendanin, matrine, stemonine and isocstemonine in the melia azedarach, sophora flavescens and stemona sessilifolia can be better released, so that the obtained fermentation liquor has better insecticidal capacity on diamond back moths and has strong pertinence effect.
In summary, compared with the prior art, the invention has the following advantages:
the invention uses single bacillus licheniformis as the only microorganism bacterium for fermentation, so that the chinaberry, the radix sophorae flavescentis and the radix stemonae can be simultaneously subjected to one-time fermentation culture, and the single bacillus licheniformis is effectively used as a nutrient component for growth, thereby being beneficial to the release of active components in the chinaberry, the matrine, the stemonine and the isocstemonine, and the biological preparation can simultaneously contain toosendanine, the matrine, the stemonine and the isocstemonine to realize the improvement of the insecticidal function on diamond back moths.
Detailed Description
The technical solution of the present invention is further specifically described below by way of specific examples, but the present invention is not limited to these examples.
Example 1
Selecting respective traditional Chinese medicine raw materials according to the dosage proportion of chinaberry traditional Chinese medicine raw materials, sophora flavescens traditional Chinese medicine raw materials and stemona traditional Chinese medicine raw materials, firstly selecting fresh chinaberry traditional Chinese medicine raw materials which grow healthily, enabling the dry weight of chinaberry to be 900g, cleaning the chinaberry with clear water, then crushing the chinaberry into chinaberry pulp by using a crusher, adding a small amount of water in the crushing process to avoid overheating and damage to active ingredients, and adopting the fresh chinaberry with a part of water to play a role in preventing overheating; selecting fresh radix Sophorae Flavescentis to make its dry weight be 700g, cleaning with clear waterThen, crushing the mixture by a crusher to obtain sophora flavescens slurry, finally selecting fresh stemona traditional Chinese medicine raw materials to ensure that the dry weight of the stemona is 630g, cleaning the mixture, crushing the mixture to obtain stemona slurry, adding the three slurries into a fermentation tank for mixing, and adding water to dilute the three slurries into a 10L-volume aqueous fermentation substrate; sterilizing the obtained fermentation substrate at 80 deg.C for 15 min, and sterilizing by 10% activated Bacillus licheniformis9The amount of cfu/mL is inoculated to the sterilized fermentation substrate, wherein the bacillus licheniformis is directly obtained by purchasing, the specific activity is obtained by adopting a conventional seed culture medium to carry out culture activation treatment, and the activated bacillus licheniformis-containing liquid is obtained, and the specific seed culture medium comprises the following components in parts by weight: peptone: 10 g/L; enzyme mother cream: 5 g/L; sodium chloride: 10g/L, and the balance of water, taking a certain amount of the obtained culture solution to ensure that the inoculation amount meets the requirement, adding the culture solution into the solution of the fermentation substrate, controlling the fermentation temperature of a fermentation system to be 25-30 ℃ after inoculation, controlling the pH value of the system to be 7.1-7.3, and carrying out fermentation treatment for 10 days to obtain corresponding fermentation liquor after fermentation is finished; and filtering the obtained fermentation liquor by using a filter element to remove precipitates to obtain the plutella xylostella killing biological agent. The obtained biological agent was analyzed, and the specific analysis results were as follows:
the biological preparation contains toosendanin, matrine, stemonine and isocstemonine with higher components, and the toosendanin: matrine: baidotidine base: the content ratio of the isocratine is 1: 0.5: 0.2: 0.4. the content can also be analyzed, wherein the extraction rate of toosendanin in the melia azedarach is up to more than 60%, the extraction rate of matrine in the sophora flavescens is up to more than 40%, and the total extraction rate of the stemonine and the isocoudine in the stemona is up to more than 50%.
The obtained organisms are adopted to carry out a diamondback moth killing experiment, and the biological preparation (the obtained liquid biological preparation is directly diluted to 100 times by water) is adopted to spray vegetable plants damaged by the diamondback moths for insecticidal treatment. After the biological agent is sprayed, the diamondback moth can be basically and completely killed within two days, the killing rate reaches over 95 percent, the diamondback moth can be basically and completely killed within more than five days, and the biological agent has a good effect of killing the diamondback moth; after the vegetable is sprayed, the growth condition of vegetables is better, and the vegetables with green leaves such as green vegetables are picked and selected by the vegetable tool, so that a certain growth promoting effect is achieved.
Example 2
Selecting respective traditional Chinese medicine raw materials according to the dosage proportion of the traditional Chinese medicine raw materials of chinaberry, radix sophorae flavescentis and radix stemonae, firstly selecting fresh healthy chinaberry raw materials, cleaning the raw materials with clear water, cutting chinaberry into small particles, and then crushing the particles into chinaberry pulp by a crusher; selecting fresh radix sophorae flavescentis as a traditional Chinese medicine raw material, cleaning the raw material with clear water, then crushing the raw material into radix sophorae flavescentis slurry by using a crusher, finally selecting fresh radix stemonae as a traditional Chinese medicine raw material, cleaning the raw material, then crushing the raw material into radix stemonae slurry, adding the obtained three slurries into a fermentation tank for mixing, and adding water to dilute the mixed slurry into a water-containing fermentation substrate; the addition amount of each component is as follows: flavescent sophora root: stemona root: the mass ratio of water is 1: 0.5: 0.7: 10, converting the mass of the chinaberry, the radix sophorae flavescentis and the radix stemonae according to dry weight, sterilizing the obtained fermentation substrate at 85 ℃ for 10 minutes, and then sterilizing the activated bacillus licheniformis according to the weight ratio of 108Inoculating cfu/mL to a sterilized fermentation substrate, wherein bacillus licheniformis is obtained directly by purchase, and the specific activity is obtained by adopting a conventional seed culture medium for culture activation treatment to obtain activated bacillus licheniformis liquid, the activation of the bacillus licheniformis is the same as that of the bacillus licheniformis liquid in the embodiment 1, after the inoculation is finished, the fermentation temperature of a fermentation system is controlled to be 30-35 ℃, the pH value of the system is controlled to be 6.5-7.0, and fermentation treatment is carried out for 7 days, and after the fermentation is finished, a corresponding fermentation liquid is obtained; coarse filtering to remove large solid residue in the fermentation liquid, and fine filtering to obtain the diamondback moth killing biological agent.
The biological preparation contains toosendanin, matrine, stemonine and isocstemonine with higher components, and the toosendanin: matrine: baidotidine base: the content ratio of the isocratine is 1: 0.4: 0.3: 0.5. the content can also be analyzed, wherein the extraction rate of toosendanin in the melia azedarach is up to more than 60%, the extraction rate of matrine in the sophora flavescens is up to more than 40%, and the total extraction rate of the stemonine and the isocoudine in the stemona is up to more than 50%.
The obtained organisms are adopted to carry out a diamondback moth killing experiment, and the biological preparation (the obtained liquid biological preparation is directly diluted to 200 times by water) is adopted to spray the vegetable plants damaged by the diamondback moths for insecticidal treatment. After the biological agent is sprayed, the diamondback moth can be basically and completely killed within two days, the killing rate reaches more than 93 percent, the diamondback moth can be basically and completely killed within five days, the effect of killing the diamondback moth is good, the growth condition of the vegetable is better after the biological agent is sprayed, and the vegetable with green leaves such as green vegetables can be selected, so that a certain growth promoting effect is achieved.
Example 3
Selecting respective traditional Chinese medicine raw materials according to the dosage proportion of the traditional Chinese medicine raw materials of chinaberry, radix sophorae flavescentis and radix stemonae, firstly selecting fresh healthy chinaberry raw materials, cleaning the raw materials with clear water, cutting chinaberry into small particles, and then crushing the particles into chinaberry pulp by a crusher; selecting fresh radix sophorae flavescentis as a traditional Chinese medicine raw material, cleaning the raw material with clear water, then crushing the raw material into radix sophorae flavescentis slurry by using a crusher, finally selecting fresh radix stemonae as a traditional Chinese medicine raw material, cleaning the raw material, then crushing the raw material into radix stemonae slurry, adding the obtained three slurries into a fermentation tank for mixing, and adding water to dilute the mixed slurry into a water-containing fermentation substrate; the addition amount of each component is as follows: flavescent sophora root: stemona root: the mass ratio of water is 1: 0.7: 0.9: 15, the mass of the chinaberry, the radix sophorae flavescentis and the radix stemonae is converted according to the dry weight, the obtained fermentation substrate is sterilized at 80 ℃ for 15 minutes, sucrose is added after the sterilization treatment, the addition amount of the sucrose is 5 percent of the addition amount of the chinaberry, preferably the dry weight of the chinaberry, and the activated lichen is convertedBacillus according to 1.5x 108Inoculating cfu/mL to a sterilized fermentation substrate, wherein bacillus licheniformis is directly obtained by purchasing, after inoculation is finished, controlling the fermentation temperature of a fermentation system to be 28-32 ℃, controlling the pH value of the system to be 7.1-7.5, and carrying out fermentation treatment for 8 days to obtain corresponding fermentation liquid after fermentation is finished; coarse filtering to remove large solid residue in the fermentation liquid, and fine filtering to obtain the diamondback moth killing biological agent.
The biological preparation contains toosendanin, matrine, stemonine and isocstemonine with higher components, and the toosendanin: matrine: baidotidine base: the content ratio of the isocratine is 1: 0.4: 0.3: 0.5. the content can also be analyzed, wherein the extraction rate of toosendanin in the melia azedarach is up to more than 60%, the extraction rate of matrine in the sophora flavescens is up to more than 40%, and the total extraction rate of the stemonine and the isocoudine in the stemona is up to more than 50%.
The obtained organisms are adopted to carry out a diamondback moth killing experiment, and the biological preparation (the obtained liquid biological preparation is directly diluted to 200 times by water) is adopted to spray the vegetable plants damaged by the diamondback moths for insecticidal treatment. After the biological agent is sprayed, the diamondback moth can be basically and completely killed within two days, the killing rate reaches more than 93 percent, the diamondback moth can be basically and completely killed within five days, the effect of killing the diamondback moth is good, the growth condition of the vegetable is better after the biological agent is sprayed, and the vegetable with green leaves such as green vegetables can be selected, so that a certain growth promoting effect is achieved.
Example 4
Selecting respective traditional Chinese medicine raw materials of chinaberry, sophora flavescens and stemona sessilifolia which are respectively cleaned by clear water according to the dosage proportion of the chinaberry, sophora flavescens and stemona sessilifolia, shearing the three traditional Chinese medicine raw materials into small particles, adding ice powder, wherein the temperature of the initially added ice powder is-30 ℃, so that the adding amount of the ice powder is 5.0 percent of the dry weight of the chinaberry, then carrying out strong stirring and crushing treatment, and controlling the stirring speed to be about 10000 r/min to obtain a mixed material after direct mixingAdding water to dilute the slurry into an aqueous fermentation substrate; the addition amount of each component is as follows: flavescent sophora root: stemona root: the mass ratio of water is 1: 0.6: 0.7: 12, converting the mass of the chinaberry, the radix sophorae flavescentis and the radix stemonae into dry weight, sterilizing the obtained fermentation substrate at 80 ℃ for 20 minutes, adding glucose into the fermentation substrate until the addition amount of the glucose is 10% of the weight of the chinaberry, preferably converting the mass of the chinaberry into the dry weight of the chinaberry, and activating the bacillus licheniformis according to 1.2x 108Inoculating cfu/mL to a sterilized fermentation substrate, wherein the bacillus licheniformis is directly obtained by purchasing and is generally about equal to 100mL of activated bacillus licheniformis culture solution, after inoculation is finished, controlling the fermentation temperature of a fermentation system at 30-33 ℃, controlling the pH value of the system within the range of 7.1-7.5, and carrying out fermentation treatment for 9 days to obtain corresponding fermentation liquid; coarse filtering to remove large solid residue in the fermentation liquid, and fine filtering to obtain the diamondback moth killing biological agent.
The biological preparation contains toosendanin, matrine, stemonine and isocstemonine with higher components, and the toosendanin: matrine: baidotidine base: the content ratio of the isocratine is 1: 0.6: 0.4: 0.6. the content can also be analyzed, wherein the extraction rate of toosendanin in the melia azedarach is up to more than 75%, the extraction rate of matrine in the sophora flavescens is up to more than 60%, and the total extraction rate of the stemonine and the isocoudine in the stemona is up to more than 60%.
The obtained organisms are adopted to carry out a diamondback moth killing experiment, and the biological preparation (the obtained liquid biological preparation is directly diluted to 200 times by water) is adopted to spray the vegetable plants damaged by the diamondback moths for insecticidal treatment. After the biological agent is sprayed, the diamondback moth can be basically and completely killed within two days, the killing rate reaches more than 93 percent, the diamondback moth can be basically and completely killed within five days, the effect of killing the diamondback moth is good, the growth condition of the vegetable is better after the biological agent is sprayed, and the vegetable with green leaves such as green vegetables can be selected, so that a certain growth promoting effect is achieved.
Example 5
Selecting respective traditional Chinese medicine raw materials of chinaberry, sophora flavescens and stemona sessilifolia which are respectively cleaned by clear water according to the dosage proportion of the chinaberry, sophora flavescens and stemona sessilifolia, shearing the three traditional Chinese medicine raw materials into small particles, adding ice powder, controlling the temperature of the initially added ice powder to be-10 ℃ so that the adding amount of the ice powder is 2.0 percent of the dry weight of the chinaberry, then carrying out strong stirring and crushing treatment, controlling the stirring speed to be about 8000 revolutions per minute, obtaining mixed slurry after direct mixing, and adding water to dilute into a water-containing fermentation substrate; the addition amount of each component is as follows: flavescent sophora root: stemona root: the mass ratio of water is 1: 0.6: 0.8: 13, converting the mass of the chinaberry, the radix sophorae flavescentis and the radix stemonae into dry weight, sterilizing the obtained fermentation substrate at 90 ℃ for 10 minutes, adding sucrose to make the addition of the sucrose be 8% of the weight of the chinaberry, preferably converting the dry weight of the chinaberry, and then converting the activated bacillus licheniformis into 3.0x 108Inoculating cfu/mL to a sterilized fermentation substrate, wherein the bacillus licheniformis is directly obtained by purchasing and is generally about equal to 100mL of activated bacillus licheniformis culture solution, after inoculation is finished, controlling the fermentation temperature of a fermentation system at 30-33 ℃, controlling the pH value of the system within the range of 7.1-7.5, and carrying out fermentation treatment for 9 days to obtain corresponding fermentation liquid; coarse filtering to remove large solid residue in the fermentation liquid, and fine filtering to obtain the diamondback moth killing biological agent.
The biological preparation contains toosendanin, matrine, stemonine and isocstemonine with higher components, and the toosendanin: matrine: baidotidine base: the content ratio of the isocratine is 1: 0.5: 0.4: 0.5. the content can also be analyzed, wherein the extraction rate of toosendanin in the melia azedarach is up to more than 75%, the extraction rate of matrine in the sophora flavescens is up to more than 60%, and the total extraction rate of the stemonine and the isocoudine in the stemona is up to more than 60%.
The obtained organisms are adopted to carry out a diamondback moth killing experiment, and the biological preparation (the obtained liquid biological preparation is directly diluted to 500 times by water) is adopted to spray vegetable plants damaged by the diamondback moths for insecticidal treatment. After the biological agent is sprayed, the diamondback moth can be basically and completely killed within two days, the killing rate reaches over 90 percent, the diamondback moth can be basically and completely killed within five days, the effect of killing the diamondback moth is good, the growth condition of the vegetable is better after the biological agent is sprayed, and the vegetable with green leaves such as green vegetables can be selected, so that a certain growth promoting effect is achieved.
Comparative example 1
In the comparative example, a single bacillus thuringiensis was selected as the microorganism strain to be specifically proportioned, and the bacillus thuringiensis was purchased commercially.
Selecting respective traditional Chinese medicine raw materials according to the dosage proportion of chinaberry traditional Chinese medicine raw materials, sophora flavescens traditional Chinese medicine raw materials and stemona traditional Chinese medicine raw materials, firstly selecting fresh chinaberry traditional Chinese medicine raw materials which grow healthily, enabling the dry weight of chinaberry to be 900g, cleaning the chinaberry with clear water, then crushing the chinaberry into chinaberry pulp by using a crusher, adding a small amount of water in the crushing process to avoid overheating and damage to active ingredients, and adopting the fresh chinaberry with a part of water to play a role in preventing overheating; selecting fresh radix sophorae flavescentis traditional Chinese medicine raw materials to enable the dry weight of radix sophorae flavescentis to be 700g, cleaning the radix sophorae flavescentis with clear water, then crushing the radix sophorae flavescentis into radix sophorae flavescentis slurry by using a crusher, finally selecting fresh radix stemonae traditional Chinese medicine raw materials to enable the dry weight of radix stemonae to be 630g, cleaning the radix stemonae, then crushing the radix stemonae slurry into radix stemonae slurry, then adding the three obtained slurries into a fermentation tank to mix, and adding water to dilute the mixture into a 10L-volume aqueous fermentation substrate; sterilizing the obtained fermentation substrate at 80 deg.C for 15 min, and activating Bacillus thuringiensis according to the ratio of 109The amount of cfu/mL is inoculated to the sterilized fermentation substrate, the specific activity is cultured and activated by adopting a conventional seed culture medium to obtain the bacillus licheniformis liquid containing the activation, and the specific formula of the seed culture medium comprises the following components: proteinPeptone: 10 g/L; enzyme mother cream: 5 g/L; sodium chloride: 10g/L, and the balance of water, taking a certain amount of the obtained culture solution to ensure that the inoculation amount meets the requirement, adding the culture solution into the solution of the fermentation substrate, controlling the fermentation temperature of a fermentation system to be 25-30 ℃ after inoculation, controlling the pH value of the system to be 7.1-7.3, and carrying out fermentation treatment for 10 days to obtain corresponding fermentation liquor after fermentation is finished; and filtering the obtained fermentation liquor by using a filter element to remove precipitates to obtain the plutella xylostella killing biological agent. The obtained biological agent was analyzed, and the specific analysis results were as follows:
the biological preparation contains toosendanin, matrine, stemonine and isocstemonine with higher components, and the toosendanin: matrine: baidotidine base: the content ratio of the isocratine is 1: 0.1: 0.05: 0.1, the content proportion is relatively large, and the content condition can be analyzed, wherein the extraction rate of the toosendanin in the melia azedarach is up to more than 40 percent, the extraction rate of the matrine in the sophora flavescens is up to more than 10 percent, and the total extraction rate of the stemonine and the isocorydine in the stemona is up to more than 15 percent.
The obtained organisms are adopted to carry out a diamondback moth killing experiment, and the biological preparation (the obtained liquid biological preparation is directly diluted to 100 times by water) is adopted to spray vegetable plants damaged by the diamondback moths for insecticidal treatment. After the biological agent is sprayed, the diamondback moth can be basically and completely killed within two days, the killing rate reaches over 70 percent, the killing rate can basically reach about 85 percent within over five days, and the effect of killing the diamondback moth is relatively poor; after the spraying, the growth condition of the vegetable is general, and the vegetable tool selects the vegetable using green leaves such as green vegetables and the like, and has no obvious effect of promoting the growth.
Comparative example 2
In this comparative example, a single Bacillus pumilus was selected as the microorganism strain to be specifically proportioned, and the Bacillus pumilus was purchased on the market, and the specific embodiment was identical to that of comparative example 1 except that Bacillus pumilus was used instead of Bacillus thuringiensis. Obtaining the corresponding biological preparation.
The biological preparation contains toosendanin, matrine, stemonine and isocstemonine with higher components, and the toosendanin: matrine: baidotidine base: the content ratio of the isocratine is 1: 0.2: 0.1: 0.1, the content proportion is relatively large, and the content condition can be analyzed, wherein the extraction rate of the toosendanin in the melia azedarach is up to more than 45 percent, the extraction rate of the matrine in the sophora flavescens is up to more than 15 percent, and the total extraction rate of the stemonine and the isocorydine in the stemona is up to more than 15 percent.
The obtained organisms are adopted to carry out a diamondback moth killing experiment, and the biological preparation (the obtained liquid biological preparation is directly diluted to 100 times by water) is adopted to spray vegetable plants damaged by the diamondback moths for insecticidal treatment. After the biological agent is sprayed, the diamondback moth can be basically and completely killed within two days, the killing rate reaches over 75 percent, the killing rate can basically reach about 85 percent within over five days, and the effect of killing the diamondback moth is relatively poor; after the spraying, the growth condition of the vegetable is general, and the vegetable tool selects the vegetable using green leaves such as green vegetables and the like, and has no obvious effect of promoting the growth.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.
Claims (9)
1. A method for preparing a plutella xylostella killing biological agent by fermenting traditional Chinese medicine composite raw materials is characterized by comprising the following steps:
A. selecting chinaberry, radix sophorae flavescentis and radix stemonae as traditional Chinese medicine raw materials, cleaning, crushing, adding into a fermentation tank, and adding water to dilute into a fermentation substrate;
B. sterilizing the obtained fermentation substrate, and then treating the activated Bacillus licheniformis according to the proportion of 108cfu/mL～109Inoculating cfu/mL of the fermentation substrate after sterilization, and performing fermentation treatment at the fermentation temperature of 25-35 ℃ to obtain fermentation liquor;
C. and filtering the obtained fermentation liquor to remove precipitates to obtain the diamondback moth killing biological agent.
2. The method for preparing a plutella xylostella-killing biological agent by fermenting traditional Chinese medicine composite raw materials according to claim 1, wherein in the step A, the chinaberry fruit: flavescent sophora root: stemona root: the mass ratio of water is 1: 0.5-0.7: 0.7-0.9: 10 to 15.
3. The method for preparing a plutella xylostella killing biological agent by fermenting traditional Chinese medicine composite raw materials according to claim 1, wherein ice powder is added in the crushing treatment in the step A, and the adding amount of the ice powder is 2.0-5.0% of the mass of the chinaberry.
4. The method for preparing the diamondback moth killing biological agent by fermenting the traditional Chinese medicine composite raw material according to claim 3, wherein the temperature of the ice powder is-30 ℃ to-10 ℃.
5. The method for preparing a plutella xylostella-killing biological agent by fermenting traditional Chinese medicine composite raw materials according to claim 1, wherein a carbon source is added into a fermentation substrate after sterilization in the step B, and the addition amount of the carbon source is 5-10% of the addition amount of the chinaberry.
6. The method for preparing a plutella xylostella-killing biological agent by fermenting the traditional Chinese medicine composite raw materials according to claim 5, wherein the carbon source is selected from one or more of glucose, fructose and sucrose.
7. The method for preparing the plutella xylostella killing biological agent by fermenting the traditional Chinese medicine composite raw materials according to any one of claims 1 to 6, wherein the fermentation treatment time in the step B is 7 to 10 days.
8. The method for preparing a plutella xylostella-killing biological agent by fermenting the traditional Chinese medicine composite raw material according to any one of claims 1 to 6, wherein the pH value of the system during fermentation treatment in the step B is 6.5-7.5.
9. The method for preparing a diamondback moth-killing biological agent by fermenting traditional Chinese medicine composite raw materials according to any one of claims 1 to 6, wherein the diamondback moth-killing biological agent in the step C contains toosendanin, matrine, stemonine and isocstemonine.
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