CN114644996A - Microbial compound microbial inoculum for preventing and treating stem borers as well as preparation method and application thereof - Google Patents
Microbial compound microbial inoculum for preventing and treating stem borers as well as preparation method and application thereof Download PDFInfo
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- CN114644996A CN114644996A CN202210164577.0A CN202210164577A CN114644996A CN 114644996 A CN114644996 A CN 114644996A CN 202210164577 A CN202210164577 A CN 202210164577A CN 114644996 A CN114644996 A CN 114644996A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
- A01N63/23—B. thuringiensis
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention discloses a microbial compound microbial inoculum for preventing and treating borers, which is prepared by completely dissolving 4-7 parts of Empedobacter brevis fermentation liquor, 7-9 parts of Metarrhizium anisopliae fermentation liquor and 5-10 parts of Bacillus thuringiensis fermentation liquor in 40-60 parts of soybean oil, then adding-18 parts of palmityl alcohol and 10-20 parts of calcium alkyl benzene sulfonate, stirring to completely dissolve and mix the materials uniformly to obtain the microbial compound microbial inoculum, wherein the bacteria of the microbial compound microbial inoculum have synergistic effect in the prevention and treatment of the borers, and can be quickly spread and diffused into leafs when being sprayed on mulberry leaves, so that the prevention and treatment rate of the borers is improved, the generation overlapping is reduced, the prevention and treatment action time is prolonged, and the waste caused by the falling of the bacterial liquid is avoided.
Description
Technical Field
The invention relates to the technical field of microbial inoculant, and particularly relates to a microbial composite inoculant for preventing and treating borer as well as a preparation method and application thereof.
Background
At present, chemical pesticides are generally adopted in a method for preventing and treating stem borers. The prevention and treatment mode can kill natural enemies of the borers, pollute the environment, cause ecological damage and be not beneficial to ecological sustainable development; the sugarcane product also has pesticide residue which is harmful to human health; and the investment labor is large, the cost is high, and the effect of preventing and treating the borer is not high.
The mulberry stem borer is one of the important pests which harm mulberry trees, the mulberry stem borer eats and harms mulberry leaves, the mulberry leaves are raw materials for silkworm rearing by silkworm farmers, and once the mulberry stem borer causes serious adverse effects on the production of the mulberry.
Although the existing pesticide spraying is quick in effect, the prevention and the treatment are not thorough, and the mulberry crop safety can be influenced in the mulberry crop period, so that the problem can be effectively avoided by adopting a microbial agent for preventing and treating borers, for example, a method for preventing and treating borers disclosed in patent No. CN201810114554.2 releases a mixture of trichogramma eggs and beauveria bassiana spore powder to a target prevention and treatment area to prevent and treat target borers; the method has good biological control effect on corn borer and rice stem borer, and patent number CN201711292841.4 discloses a microbial composite microbial inoculum for killing rice overwintering borer, and the microbial inoculum has good control effect on rice overwintering borer. However, the mulworms have poor control effect on mulworms due to the phenomenon of generation overlapping and leaf rolling habit. Therefore, a microbial compound microbial inoculum is required to be researched for preventing and controlling the mulberry stem borer.
Disclosure of Invention
In view of the above situation, the invention aims to provide a microbial compound inoculant for preventing and treating borer and a preparation method thereof, and the microbial compound inoculant is applied to the prevention and treatment of the mulberry borer.
The technical scheme for solving the problem is as follows:
a microbial compound microbial inoculum for preventing and treating stem borers comprises the following components in parts by weight: 4-7 parts of Empedobacter brevis fermentation liquor, 7-9 parts of Metarrhizium anisopliae fermentation liquor, 5-10 parts of Bacillus thuringiensis fermentation liquor, 5-18 parts of palm alcohol, 10-20 parts of calcium alkyl benzene sulfonate and 40-60 parts of soybean oil.
Further, the Empedobacter brevis fermentation liquor is prepared by the following method: inoculating the activated seed liquid of the Empedobacter brevis into an LB slant culture medium, culturing for 28h at the temperature of 25 ℃, and then transferring to a fermentation culture medium for fermentation culture for 72h to obtain the Empedobacter brevis fermentation liquid.
Furthermore, the number of viable bacteria in the Empedobacter brevis fermentation liquor is not less than 1 multiplied by 109cfu/g, wherein the culture medium consists of 5% of sterilized soybean meal, 2.5% of shrimp meal, 1.2% of calcium carbonate, 0.6% of salt and the balance of water.
Further, the Metarrhizium anisopliae fermentation liquor is prepared by the following method: inoculating the activated seed liquid of the metarhizium anisopliae into a PDB liquid culture medium, culturing for 36h at the temperature of 30 ℃, and then transferring to a fermentation culture medium for fermentation culture for 60h to obtain the metarhizium anisopliae fermentation liquid.
Furthermore, the viable count of the Metarrhizium anisopliae fermentation liquor is not less than 1 multiplied by 109cfu/g, wherein the culture medium consists of 7% of sterilized soybean meal, 3% of glucose, 1% of corn starch, 0.25% of potato powder, 0.01% of disodium hydrogen phosphate and the balance of water.
Further, the bacillus thuringiensis fermentation liquor is prepared by the following method: inoculating the activated seed liquid of the bacillus thuringiensis into an LB liquid culture medium, culturing for 20h at the temperature of 27 ℃, and then transferring to a fermentation culture medium for fermentation culture for 42h to obtain the bacillus thuringiensis fermentation liquid.
Furthermore, the number of viable bacteria in the bacillus thuringiensis fermentation liquor is not less than 1 × 109cfu/g, the culture medium is prepared from sterilized soybean powder 5%, flour 6%, dipotassium hydrogen phosphate 0.02%, magnesium sulfate0.01 percent of zinc sulfate, 0.01 percent of calcium powder, 1 percent of yeast powder, 0.5 percent of glucose, 0.5 percent of sodium chloride, 0.01 percent of manganese sulfate and the balance of water.
Further, the microbial compound inoculant is prepared by the following steps: and (2) completely dissolving the fermentation liquor of Empedobacter brevis, the fermentation liquor of Metarhizium anisopliae and the fermentation liquor of Bacillus thuringiensis in soybean oil according to a proportion, then adding the palmityl alcohol and the calcium alkyl benzene sulfonate, and stirring to completely dissolve and uniformly mix the materials to obtain the microbial composite inoculant.
Further, the microbial compound inoculant is used for preventing and treating the mulberry stem borers, and the application method is top-covering spraying.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the following advantages:
in the microbial composite inoculant prepared by the invention, Empedobacter brevis fermentation liquor, Metarrhizium anisopliae fermentation liquor and Bacillus thuringiensis fermentation liquor have synergistic effect in the prevention and treatment of the mulberry borers, so that the mulberry borers can be infected and have higher prevention and treatment effect; on the other hand, the microbial compound inoculant can secrete and synthesize various metabolites for eating by subsequently bred silkworms, and provides rich nutrient substances for the silkworms.
The palm alcohol, the calcium alkyl benzene sulfonate and the soybean oil added into the microbial compound inoculant can provide nutrient substances for the compound inoculant, improve the growth cycle of flora and promote the long-term effect of the flora, and can be mixed with the microbial strain fermentation liquor by taking the palm alcohol, the calcium alkyl benzene sulfonate and the soybean oil as solvents to form an oil spreading agent to be sprayed into mulberry leaves, so that the oil spreading agent can be quickly spread and diffused into the curled leaves, the prevention and treatment rate of the mulberry stem borers is improved, the generation overlapping is reduced, the prevention and treatment action time is prolonged, and the waste caused by the falling of the bacterial liquid is avoided.
Detailed Description
The technical solution of the present invention is described in detail with reference to the specific embodiments, but it should be emphasized that the present invention is not limited to the specific embodiments; the microbial species used in the examples below can be isolated by themselves or obtained by purchase; the starting materials described in the examples below are, without specific indication, either prepared by conventional methods or commercially available.
Example 1
(1) The preparation method of the Empedobacter brevis fermentation liquor comprises the following steps:
inoculating the activated seed solution of Empedobacter brevis in an LB slant culture medium, wherein the inoculation amount is 2%, culturing for 28h at the temperature of 25 ℃, and then transferring to a fermentation culture medium for fermentation culture for 72h to obtain Empedobacter brevis fermentation liquor; the culture medium consists of 5% of sterilized soybean meal, 2.5% of shrimp meal, 1.2% of calcium carbonate, 0.6% of salt and the balance of water; the number of viable bacteria in the Empedobacter brevis fermentation liquor is not less than 1 multiplied by 109cfu/g。
(2) The preparation method of the Metarrhizium anisopliae fermentation liquor comprises the following steps:
inoculating the activated seed liquid of the metarhizium anisopliae into a PDB liquid culture medium, wherein the inoculation amount is 2%, culturing for 36h at the temperature of 30 ℃, and then transferring to a fermentation culture medium for fermentation culture for 60h to obtain a metarhizium anisopliae fermentation liquid; the culture medium consists of 7% of sterilized soybean meal, 3% of glucose, 1% of corn starch, 0.25% of potato powder, 0.01% of disodium hydrogen phosphate and the balance of water; the viable count in the Metarrhizium anisopliae fermentation liquor is not less than 1 multiplied by 109cfu/g。
(3) The preparation method of the bacillus thuringiensis fermentation liquor comprises the following steps:
inoculating the activated seed liquid of the bacillus thuringiensis into an LB liquid culture medium, wherein the inoculation amount is 2%, culturing for 20h at the temperature of 27 ℃, and then transferring to a fermentation culture medium for fermentation culture for 42h to obtain bacillus thuringiensis fermentation liquid; the culture medium is composed of 5% of sterilized soybean meal, 6% of flour, 0.02% of dipotassium phosphate, 0.01% of magnesium sulfate, 0.01% of zinc sulfate, 0.01% of calcium powder, 1% of yeast powder, 0.5% of glucose, 0.5% of sodium chloride, 0.01% of manganese sulfate and the balance of water; the number of viable bacteria in the Bacillus thuringiensis fermentation liquor is not less than 1 × 109cfu/g。
Example 2
A microbial compound microbial inoculum for preventing and treating stem borers comprises the following components in parts by weight: 4 parts of Empedobacter brevis fermentation liquor, 7 parts of Metarrhizium anisopliae fermentation liquor, 5 parts of Bacillus thuringiensis fermentation liquor, 5 parts of palmityl alcohol, 10 parts of calcium alkyl benzene sulfonate and 40 parts of soybean oil.
Example 3
A microbial compound microbial inoculum for preventing and treating stem borers comprises the following components in parts by weight: 5 parts of Empedobacter brevis fermentation liquor, 8 parts of Metarrhizium anisopliae fermentation liquor, 7 parts of Bacillus thuringiensis fermentation liquor, 11 parts of palmityl alcohol, 15 parts of calcium alkyl benzene sulfonate and 50 parts of soybean oil.
Example 4
A microbial compound microbial inoculum for preventing and treating stem borers comprises the following components in parts by weight: 7 parts of Empedobacter brevis fermentation liquor, 9 parts of Metarrhizium anisopliae fermentation liquor, 10 parts of Bacillus thuringiensis fermentation liquor, 18 parts of palmityl alcohol, 20 parts of calcium alkyl benzene sulfonate and 60 parts of soybean oil.
Example 5
A microbial compound microbial inoculum for preventing and treating stem borers is prepared by the following steps: and (3) completely dissolving Empedobacter brevis fermentation liquor, Metarrhizium anisopliae fermentation liquor and Bacillus thuringiensis fermentation liquor in soybean oil according to the proportion described in the embodiment 2-4, then adding the palmityl alcohol and the calcium alkyl benzene sulfonate, and stirring to completely dissolve and uniformly mix the materials to obtain the microbial composite inoculant.
Comparative example 1
The same ratio as that described in example 3, except that the same amount of metarhizium anisopliae fermentation broth was substituted for the Empedobacter brevis fermentation broth, and the microbial composite inoculum was prepared according to the preparation method described in example 5.
Comparative example 2
The same ratio as that described in example 3, except that the same amount of Bacillus thuringiensis fermentation broth was used instead of the Metarrhizium anisopliae fermentation broth, and the microbial composite inoculum was prepared according to the preparation method described in example 5.
Comparative example 3
The same ratio as that described in example 3, except that the same amount of Empedobacter brevis fermentation broth was used instead of the Bacillus thuringiensis fermentation broth, and the microbial composite inoculum was prepared according to the preparation method described in example 5.
Comparative example 4
The microbial composite inoculum is prepared by directly and uniformly mixing the Empedobacter brevis fermentation broth, the Metarrhizium anisopliae fermentation broth and the Bacillus thuringiensis fermentation broth in the embodiment 3.
Test example 1
Evaluating the control effect of the microbial compound inoculant on the multocida:
the test object is mulberry which is attacked by mulberry borer, and the test product is the microbial compound inoculum prepared in the embodiments 2-4 and the comparative examples 1-4 of the invention;
the specific test method comprises the following steps: the mulberry field suffered from the mulberry borer insect pest is equally divided into eight areas, each area is not interfered with each other, wherein seven areas correspond to the experiment group which is sprayed with the same amount of the microbial compound fungicide prepared in the embodiment 2-4 and the comparative example 1-4 on the top, the rest area is sprayed with the same amount of clear water on the top as the control group, the normal fertilization management is carried out during the spraying period, and the spraying time is as follows: the first spraying time is before the 3 th instar of the first generation of mulberry borer larvae, spraying again every 15 days after spraying for the first time, spraying for 6 times totally, after spraying for 15 days, randomly picking 200 mulberry leaves from eight areas, detecting the number of mulberry borers suffered from insect pests, observing the distribution condition of the borers, calculating the prevention and treatment rate (the prevention and treatment rate/%, which is the number of mulberry borers suffered from insect pests in a clear water group-the number of mulberry borers suffered from insect pests in an experimental group)/the number of mulberry borers suffered from insect pests in a clear water group is multiplied by 100 percent), and calculating the integrity of the mulberry leaves according to the outline area of the mulberry leaves, wherein the results are shown in table 1:
as can be seen from table 1, compared with comparative examples 1 to 3, the composite microbial inoculum prepared by combining the fermentation broth of Empedobacter brevis, the fermentation broth of Metarhizium anisopliae and the fermentation broth of Bacillus thuringiensis in the microbial composite microbial inoculum has better prevention and treatment effects on the mulberry borers, and compared with comparative example 4, the composite microbial inoculum prepared by combining the fermentation broth of Empedobacter brevis, the fermentation broth of Metarhizium anisopliae and the fermentation broth of Bacillus thuringiensis with the palmityl alcohol, the calcium alkyl benzene sulfonate and the soybean oil has longer prevention and treatment time and better prevention and treatment effects on the mulberry borers.
While the invention has been described in further detail with reference to specific embodiments thereof, it is not intended that the invention be limited to the specific embodiments thereof; for those skilled in the art to which the present invention pertains and related technologies, the extension, operation method and data replacement should fall within the protection scope of the present invention based on the technical solution of the present invention.
Claims (9)
1. The microbial compound microbial inoculum for preventing and treating stem borers is characterized by comprising the following components in parts by weight: 4-7 parts of Empedobacter brevis fermentation liquid, 7-9 parts of Metarrhizium anisopliae fermentation liquid, 5-10 parts of Bacillus thuringiensis fermentation liquid, 5-18 parts of palm alcohol, 10-20 parts of calcium alkyl benzene sulfonate and 40-60 parts of soybean oil.
2. The microbial compound inoculant for preventing and treating borer according to claim 1, wherein the Empedobacter brevis fermentation liquor is prepared by the following method: inoculating the activated seed liquid of the Empedobacter brevis into an LB slant culture medium, culturing for 28h at the temperature of 25 ℃, and then transferring to a fermentation culture medium for fermentation culture for 72h to obtain the Empedobacter brevis fermentation liquid.
3. The microbial compound inoculant for preventing and treating borer according to claim 2, wherein the viable count of the Empedobacter brevis fermentation liquor is not less than 1 x 109cfu/g, wherein the culture medium consists of 5% of sterilized soybean meal, 2.5% of shrimp meal, 1.2% of calcium carbonate, 0.6% of salt and the balance of water.
4. The microbial compound inoculant for preventing and treating borer according to claim 1, wherein the Metarrhizium anisopliae fermentation broth is prepared by the following method: inoculating the activated seed liquid of the metarhizium anisopliae into a PDB liquid culture medium, culturing for 36h at the temperature of 30 ℃, and then transferring to a fermentation culture medium for fermentation culture for 60h to obtain the metarhizium anisopliae fermentation liquid.
5. The control of borer according to claim 4The microbial compound microbial inoculum of the insect is characterized in that the viable count in the metarrhizium anisopliae fermentation liquor is not less than 1 multiplied by 109cfu/g, wherein the culture medium consists of 7% of sterilized soybean meal, 3% of glucose, 1% of corn starch, 0.25% of potato powder, 0.01% of disodium hydrogen phosphate and the balance of water.
6. The microbial compound inoculant for preventing and treating borer according to claim 1, wherein the bacillus thuringiensis fermentation liquid is prepared by the following method: inoculating the activated seed liquid of the bacillus thuringiensis into an LB liquid culture medium, culturing for 20h at the temperature of 27 ℃, and then transferring to a fermentation culture medium for fermentation culture for 42h to obtain the bacillus thuringiensis fermentation liquid.
7. The microbial composite inoculant for preventing and treating borer according to claim 6, wherein the number of viable bacteria in the Bacillus thuringiensis fermentation broth is not less than 1 x 109cfu/g, the culture medium is composed of 5% of sterilized soybean meal, 6% of flour, 0.02% of dipotassium phosphate, 0.01% of magnesium sulfate, 0.01% of zinc sulfate, 0.01% of calcium powder, 1% of yeast powder, 0.5% of glucose, 0.5% of sodium chloride, 0.01% of manganese sulfate and the balance of water.
8. The microbial compound inoculant for preventing and treating borer according to any one of claims 1 to 7, which is prepared by the following steps: and (2) completely dissolving the fermentation liquor of Empedobacter brevis, the fermentation liquor of Metarhizium anisopliae and the fermentation liquor of Bacillus thuringiensis in soybean oil according to a proportion, then adding the palmityl alcohol and the calcium alkyl benzene sulfonate, and stirring to completely dissolve and uniformly mix the materials to obtain the microbial composite inoculant.
9. The microbial compound inoculant for preventing and treating borer according to any one of claims 1 to 7, wherein the microbial compound inoculant is used for preventing and treating mulberry borer by using a top-spraying method.
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