阅读说明：本技术 一种多菌复合增效液体灭蚊菌剂及其制备方法与应用 (Multi-bacterium composite synergistic liquid mosquito-killing bacterium agent and preparation method and application thereof ) 是由 刘�文 韩莉华 刘海龙 于 2020-04-08 设计创作，主要内容包括：本发明提供一种多菌复合增效液体灭蚊菌剂及其制备方法与应用。所述液体灭蚊菌剂包括可侵染杀灭蚊子的五种好氧菌组成的A菌液和可抑制菌群拮抗的四种厌氧菌组成的B菌液；A菌液由苏云金芽孢杆菌以色列亚种,灭蚊链霉菌,球孢白僵菌,金龟子绿僵菌和玫烟色棒束孢组成；B菌液由保加利亚乳杆菌,嗜酸乳杆菌,植物乳杆菌和德氏乳杆菌组成；是通过将分别发酵培养的五种液体好氧菌A菌液,缓慢注入到,先分别再混合发酵培养的四种液体厌氧菌B菌液中,静置均质后得到的。作为灭蚊菌剂安全无害可直接大规模喷洒灭蚊；其弱酸气味利于诱蚊,可配制成多种诱杀灭蚊菌剂广泛应用；特别是多菌复合互不拮抗的方法,使灭蚊菌活力持久专长得以增效发挥。(The invention provides a multi-bacterium composite synergistic liquid mosquito-killing bacterium agent and a preparation method and application thereof. The liquid mosquito killing microbial inoculum comprises a bacterial solution A consisting of five aerobic bacteria capable of infecting and killing mosquitoes and a bacterial solution B consisting of four anaerobic bacteria capable of inhibiting flora antagonism; the A bacterial liquid consists of Bacillus thuringiensis Israeli subspecies, Streptomyces mosquitocidicus, Beauveria bassiana, Metarhizium anisopliae and Isaria fumosorosea; the B bacterial liquid consists of lactobacillus bulgaricus, lactobacillus acidophilus, lactobacillus plantarum and lactobacillus delbrueckii; the method is characterized in that five liquid aerobic bacteria A bacterial liquids which are respectively fermented and cultured are slowly injected into four liquid anaerobic bacteria B bacterial liquids which are respectively mixed, fermented and cultured, and then the mixture is stood and homogenized to obtain the aerobic bacteria B. The product is safe and harmless as a mosquito killing microbial inoculum and can be directly sprayed to kill mosquitoes in a large scale; the weak acid smell is beneficial to attracting mosquitoes, and can be prepared into various mosquito trapping and killing bactericides for wide application; in particular to a method for compounding multiple bacteria without mutual antagonism, which ensures that the activity of the mosquito-killing bacteria is lasting and the speciality can be enhanced and exerted.)

1. A multi-bacterium composite synergistic liquid mosquito-killing microbial inoculum is characterized in that: the mosquito killing bacteria comprise a bacteria liquid A consisting of five aerobic bacteria for killing mosquitoes and a bacteria liquid B consisting of four antagonistic anaerobic bacteria; mixing 50-70% of A bacterial liquid and 50-30% of B bacterial liquid;

the A bacterial liquid consists of the following five aerobic bacteria:

bacillus thuringiensis subspecies israelensis,

the mosquito-killing streptomyces is used for killing the mosquito,

the strain of the beauveria bassiana (balsamo) Vuillemin (Beauveria bassiana (balsamo) Vuillemin (Vuillemin) Vuillemin (,

the green muscardine fungus of the chafer,

isaria fumosorosea;

the B bacterial liquid consists of the following four anaerobic bacteria:

the bacterium Lactobacillus bulgaricus is a bacterium of the species Lactobacillus bulgaricus,

the bacteria of the lactobacillus acidophilus are selected,

the lactobacillus plantarum strain is a strain of lactobacillus plantarum,

lactobacillus delbrueckii.

2. The method for preparing the multi-bacterium composite synergistic liquid mosquito-killing bacterium agent as claimed in claim 1, wherein the method comprises the following steps: the five aerobic bacteria in the A bacterial liquid account for 10-30% respectively; and the four anaerobic bacteria in the B bacterial liquid respectively account for 25 percent.

3. A method for preparing a multi-bacterium composite synergistic liquid mosquito-killing bacterium agent as claimed in any one of claims 1 or 2, which is characterized in that: the method comprises the following steps:

the first step is as follows: respectively fermenting and culturing five aerobic bacteria in the bacteria liquid A into five liquid aerobic bacteria liquid,

(1) respectively preparing five liquid culture media of bacillus thuringiensis, streptomyces mosquitocidus, beauveria bassiana, metarhizium anisopliae and isaria fumosorosea, and inoculating corresponding strains into the five culture media; then carrying out primary amplification culture on the strains under the conditions of 25-37 ℃ and aerobic fermentation at the temperature of 120-;

(2) then respectively taking five aerobic bacteria liquid of primary fermentation, inoculating corresponding bacteria liquid into five liquid aerobic bacteria culture media, and carrying out secondary expansion culture on the bacteria liquid under the same conditions to obtain five secondary expansion aerobic fermentation bacteria liquid, wherein the inoculation amount of each primary bacteria liquid is 2-10% of the mass of the culture medium;

(3) respectively taking five aerobic bacteria liquid of secondary fermentation, inoculating corresponding bacteria liquid into five liquid aerobic bacteria culture media, and carrying out three-stage expanded culture on the bacteria liquid under the same conditions to obtain five bacteria liquid of three-stage expanded aerobic fermentation, wherein the inoculation amount of each secondary bacteria liquid is 2-10% of the mass of the culture medium;

(4) mixing the five bacterial liquids subjected to the three-stage culture according to the proportion of 10-30% of each bacterial liquid, standing and homogenizing for 24 hours to obtain a bacterial liquid A;

the second step is that: fermenting and culturing the four anaerobic bacteria in the B bacteria liquid into anaerobic bacteria liquid mixed with four liquids,

(1) respectively preparing liquid anaerobic bacteria culture media of lactobacillus bulgaricus, lactobacillus acidophilus, lactobacillus plantarum and lactobacillus delbrueckii, respectively inoculating respective corresponding strains to the four liquid anaerobic bacteria culture media, standing and fermenting hermetically for 10-30 days at 25-37 ℃, and performing primary amplification culture on the strains to obtain four primary anaerobic bacteria liquid;

(2) mixing the four anaerobic bacteria liquids of primary culture according to the proportion of 25 percent, sealing and standing for 24 hours to obtain anaerobic mixed bacteria liquid of primary fermentation;

(3) preparing a mixed fermentation liquid anaerobic bacteria culture medium, inoculating the anaerobic mixed bacteria liquid subjected to primary fermentation into the mixed fermentation liquid anaerobic bacteria culture medium, wherein the inoculation amount is 5-10% of the mass of the culture medium, standing the culture medium at the temperature of 30-37 ℃, performing closed anaerobic fermentation for 10-30 days, and performing secondary expansion culture on the strains to obtain a mixed fermentation secondary anaerobic bacteria liquid;

(4) repeating the step (3) to obtain a third-level anaerobic bacterial liquid of mixed fermentation, and obtaining a mixed bacterial liquid B after measuring the pH value to be less than or equal to 5;

the third step: proportioning: according to the proportion of 50-70% of the bacterial liquid A and 50-30% of the bacterial liquid B, slowly injecting the five bacterial liquids A into the mixed bacterial liquid B, uniformly stirring, standing for 24 hours, homogenizing and stabilizing to obtain the multi-bacterium composite synergistic liquid mosquito-killing microbial inoculum.

4. A method for preparing a multi-bacterium composite synergistic liquid mosquito-killing bacterium agent as claimed in claim 3, which is characterized in that:

the formula of the fermentation medium of five bacterial liquids which form the bacterial liquid A is as follows:

(a1) the formula of the bacillus thuringiensis culture medium comprises the following components: 20g of sucrose, 5g of peptone, 5g of yeast extract, 0.3g of dipotassium phosphate, 1g of disodium phosphate, 1g of magnesium sulfate, 0.02g of ferrous sulfate, 0.02g of calcium chloride and the balance of sterile water to 1L; the culture conditions are as follows: the initial pH value is 7.2, the temperature is 25-37 ℃, the temperature is 120-;

(a2) the formula of the mosquito-killing streptomyces culture medium comprises the following components: 20g of soluble starch, 1g of potassium nitrate, 0.5g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate, 0.5g of sodium chloride, 0.01g of ferrous sulfate and the balance of sterile water to 1L; the culture conditions are as follows: the initial pH value is 7.0, the light is shielded, the temperature is 25-37 ℃, the temperature is 120-;

(a3) the formula of the beauveria bassiana culture medium comprises the following components: 50g of cane sugar, 1g of dipotassium hydrogen phosphate and 1 percent of corn leaching liquor, wherein 1L of the corn leaching liquor is complemented; the culture conditions are as follows: the initial pH value is 7.0, the light is shielded, the temperature is 25-37 ℃, the temperature is 120-; wherein, the 1% corn leaching liquor is prepared by adding water 1L into 10g of fresh corn kernels, boiling for 30 minutes, filtering to remove residues, and supplementing sterile water to 1L;

(a4) the formula of the metarhizium anisopliae culture medium comprises the following components: 50g of cane sugar, 1g of dipotassium hydrogen phosphate and 1 percent of corn leaching liquor, wherein 1L of the corn leaching liquor is complemented; the culture conditions are as follows: the initial pH value is 7.0, the light is shielded, the temperature is 25-37 ℃, the temperature is 120-; wherein, the 1% corn leaching liquor is prepared by adding water 1L into 10g of fresh corn kernels, boiling for 30 minutes, filtering to remove residues, and supplementing sterile water to 1L;

(a5) the formula of the Isaria fumosorosea culture medium comprises the following components: 1L of 20% potato extract and 20g of sucrose; the culture conditions are as follows: initial pH value is 7.0, shading is carried out at 25-37 ℃, temperature is 120-; wherein 20% potato extractive solution is prepared from peeled potato 200g, cutting into small pieces, adding water 1L, boiling for 30 min, filtering to remove residue, and supplementing sterile water to 1L;

and (II) the formula of the fermentation culture medium of four bacterial liquids which form the bacterial liquid B is as follows:

(b1) the formula of the lactobacillus bulgaricus culture medium comprises the following components: 20g of sucrose, 35g of peptone, 1.5g of calcium carbonate, 30g of yeast extract, 2g of dipotassium hydrogen phosphate, 1.5g of magnesium sulfate, 1g of calcium carbonate, 0.02g of manganese sulfate, 1.5g of ammonium hydrogen phosphate and the balance of sterile water to 1L; the culture conditions are as follows: initial pH value of 6.0, 30-37 deg.C, shading, sealing, standing for anaerobic culture for 10-30 days;

(b2) the lactobacillus acidophilus culture medium formula comprises: 20g of sucrose, 20g of peptone, 1g of sodium chloride, 0.01g of manganese sulfate, 0.02g of magnesium sulfate, 0.5g of sodium acetate and the balance of sterile water to 1L; the culture conditions are as follows: initial pH value of 6.0, 30-37 deg.C, shading, sealing, standing for anaerobic culture for 10-30 days;

(b3) the lactobacillus plantarum culture medium formula comprises the following components: 20g of sucrose, 25g of yeast extract, 2g of dipotassium phosphate and sterile water for complementing 1L; the culture conditions are as follows: initial pH value of 6.0, 30-37 deg.C, shading, sealing, standing for anaerobic culture for 10-30 days;

(b4) the formula of the lactobacillus delbrueckii culture medium is as follows: 20g of sucrose, 35g of peptone, 1.5g of calcium carbonate, 30g of yeast extract, 2g of dipotassium hydrogen phosphate, 1.5g of magnesium sulfate, 1g of calcium carbonate, 0.02g of manganese sulfate, 1.5g of ammonium hydrogen phosphate and the balance of sterile water to 1L; the culture conditions are as follows: initial pH value of 6.0, 30-37 deg.C, shading, sealing, standing for anaerobic culture for 10-30 days;

and (III) the formula of the mixed fermentation medium of the four bacterial liquids of the B bacterial liquid is as follows:

20g of sucrose, 35g of peptone, 1.5g of calcium carbonate, 30g of yeast extract, 2g of dipotassium hydrogen phosphate, 1.5g of magnesium sulfate, 1g of calcium carbonate, 0.02g of manganese sulfate, 1.5g of ammonium hydrogen phosphate and the balance of sterile water to 1L; the culture conditions are as follows: initial pH value of 6.0, 30-37 deg.C, shading, sealing, and standing for anaerobic culture for 10-30 days.

5. The application of the multi-bacterium composite synergistic liquid mosquito killing bactericide in mosquito trapping agents is characterized in that:

(C1) compounding with various mosquito trapping agents to prepare a microorganism trapping and killing mosquito killing agent;

(C2) can be used alone as mosquito killing agent.

6. The application of the multi-bacterium composite synergistic liquid mosquito-killing bactericide in combination with a mosquito-luring agent as claimed in claim 5, wherein: the microbial trapping and killing mosquito killer comprises, by weight, 6-12% of brown sugar, white sugar or honey, 3-6% of yeast powder, 1-3% of multi-bacterium composite synergistic liquid mosquito killing microbial inoculum and the balance of clear water.

7. The application of the multi-bacterium composite synergistic liquid mosquito-killing bactericide in combination with a mosquito-luring agent as claimed in claim 5, wherein: the microbe trapping and killing mosquito killer comprises, by weight, 6-12% of brown sugar, white sugar or honey, 3-6% of yeast powder, 1-3% of multi-bacterium composite synergistic liquid mosquito killing microbial inoculum, 30-50% of beer, and the balance of clear water.

8. The application of the multi-bacterium composite synergistic liquid mosquito-killing bactericide in combination with a mosquito-luring agent as claimed in claim 5, wherein: the microbe trapping and killing mosquito killer comprises, by weight, 6-12% of brown sugar, white sugar or honey, 3-6% of yeast powder, 1-3% of multi-bacterium composite synergistic liquid mosquito killing microbial inoculum, 10-30% of leftovers soup and the balance of clear water.

9. The application of the multi-bacterium composite synergistic liquid mosquito-killing bactericide in combination with a mosquito-luring agent as claimed in claim 5, wherein: the microbial trapping and killing mosquito killer comprises 2-20% of brown sugar or white sugar, 10-20% of sesame paste, 1-3% of sodium glutamate, 1-3% of multi-bacterium composite synergistic liquid mosquito killing microbial inoculum and the balance of clear water.

10. The application of the multi-bacterium composite synergistic liquid mosquito-killing bacterium agent as claimed in any one of claims 5 to 9 in combination with a mosquito attractant, characterized in that: the method comprises the following application processes:

(D1) one of the above-mentioned microbial trapping and killing mosquito-killing agents is selectively added into the mosquito-storage box of mosquito-killing lamp;

(D2) filling one of the above-mentioned microbial trapping and killing mosquitocides into a container such as a basin or a plate, dipping black cotton cloth and flatly hanging the container above, wherein the lower edge of the cotton cloth is dipped in the preparation liquid to keep the black cotton cloth moist, and mosquitoes can infect mosquito-killing bacteria and be killed as long as the mosquitoes touch the preparation liquid;

(D3) the bactericide is diluted by 100 to 1000 times with water alone or sprayed by an airplane, so that large-scale mosquito killing can be implemented;

(D4) the bactericide is diluted by 100 to 1000 times with water and sprayed to a water system, so that large-area mosquito larvae can be killed.

Technical Field

The invention belongs to the field of microbial control of mosquito-borne pests, and particularly relates to a multi-bacterium composite synergistic liquid mosquito-killing microbial agent as well as a preparation method and application thereof.

Background

Mosquito-mediated diseases can be transmitted to people by over 80 kinds, and most of the diseases are serious diseases with high lethal disability rate, such as malaria, cholera, dengue fever, filariasis, yellow fever, black speck disease, Japanese encephalitis and the like. Human combat with mosquitoes has not been a definitive victory but has become increasingly more intense over a hundred years. About half of the population today around the world faces the threat of mosquito-mediated virulent diseases, with up to 83 million people being deprived of mosquitoes each year. The world health organization in 2019 promulgated that malaria transmitted only by mosquito vectors is more than 2 hundred million people per year, and on average one child dies of malaria every one to two minutes. There are 32 billion people worldwide at risk for abuse. Mosquito vectors are so abusive that they pose a serious challenge to the advancement of modern technology and human civilization. The method can thoroughly kill mosquito vectors in large area and large scale, eliminate the infection sources of the serious diseases, does not damage the ecological environment, does not influence the survival and health of human beings, and is a great subject which needs to be solved urgently for people.

Because of the increase of the resistance of the mosquito vectors to the chemical insecticide, the pollution and damage of the chemical insecticide to the ecological environment in the manufacturing and using processes, and the damage of the chemical insecticide to non-standard organisms, the large-scale mosquito killing of the chemical insecticide is greatly limited.

Mosquito repellent incense and mosquito repellent contain pyrethroid toxin, which can cause inhalation harm to people and hurt the health of people, and is inconvenient for killing mosquitoes in a large amount and durably.

The long-wave ultraviolet rays in the mosquito killer lamp can penetrate through the dermis of human skin, can accelerate the aging of the skin after being used for a long time, and can cause eye injury of people; mosquito fragments and germs electrically shocked or air-dried by the mosquito killer lamp are diffused into the air, which is not good for human body.

In the current microbial liquid mosquito killing preparation, a single bacterium preparation host is limited, and the species of a mosquito killing medium is not completely impregnated; the preparation with mixed multiple bacteria is mixed with multiple bacteria to generate bacteria antagonism, the effective period is short, and the effect is poor; the influence of the variant strains on human health and ecological environment of individual strains subjected to genetic modification or grafting is difficult to popularize in a large area before scientific verification is carried out. Moreover, the liquid preparations have the common problems of short effective period, unstable performance and poor effect caused by mutual competition of oxygen nutrients by aerobic bacteria, mutual antagonism and increase of saturated malformation.

The conventional microbial aerobic bacteria (including bacteria, fungi and actinomycetes) agent is propagated for a generation within minutes to hours under a liquid state condition, the strain is continuously propagated to form saturated growth, the strain is antagonistic in a competition process of competing for nutrient oxygen and rapidly goes malformed and dies, the microbial inoculum becomes black and smelly, most of flora becomes malformed and weak after the microbial inoculum is diluted, the effective function of the microbial inoculum cannot be exerted, the effective period is short under the normal temperature condition, the general bacteria are only months, and the fungi are only days. If multiple aerobic bacteria are mixed, in the process of competing for oxygen and nutrients, the mutual antagonism of the multiple aerobic bacteria is more violent, and the decay and death of the microbial inoculum are quicker.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a multi-bacterium composite synergistic liquid mosquito-killing bacterium agent and a preparation method and application thereof. The invention selects five aerobic bacteria combinations which are respectively special for killing mosquitoes, and aims to solve the problem of poor single effect of strains; the invention also selects four anaerobic bacteria which can inhibit the abnormal growth of aerobic bacteria, and compounds the four anaerobic bacteria with five aerobic bacteria, and aims to solve the problem that the aerobic bacteria in the liquid microbial inoculum mutually antagonize and have short effective period and are easy to decay and lose efficacy; the invention also designs a method for combining and using with a plurality of mosquito luring agents and independently applying the mosquito luring agents, and aims to provide the possibility of practical application for multi-channel large-scale killing of mosquito vectors.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a multi-bacterium composite synergistic liquid mosquito killing microbial inoculum comprises a bacterial solution A consisting of five aerobic bacteria for killing mosquitoes and a bacterial solution B consisting of four antagonistic anaerobic bacteria;

the A bacterial liquid consists of the following five aerobic bacteria:

bacillus thuringiensis subspecies israelensis,

the mosquito-killing streptomyces is used for killing the mosquito,

the strain of the beauveria bassiana (balsamo) Vuillemin (Beauveria bassiana (balsamo) Vuillemin (Vuillemin) Vuillemin (,

the green muscardine fungus of the chafer,

isaria fumosorosea;

the B bacterial liquid consists of the following four anaerobic bacteria:

the bacterium Lactobacillus bulgaricus is a bacterium of the species Lactobacillus bulgaricus,

the bacteria of the lactobacillus acidophilus are selected,

the lactobacillus plantarum strain is a strain of lactobacillus plantarum,

lactobacillus delbrueckii.

The safety levels of the five aerobic bacteria are four levels, so that the five aerobic bacteria are safe and harmless to human, livestock and ecological environment, and have different infection and killing effects on mosquito vectors and larvae bacteria:

1. the strain name is as follows: bacillus thuringiensis subspecies israelensis, the name of latin: bacillus thuringiensis israelensis, Bti, bacteria and aerobic bacteria; the strain is preserved in China general microbiological culture Collection center with the following serial numbers: CGMCC1.1754, function: has strong poisoning effect on 38 kinds of mosquitoes and culex larvae;

2. the strain name is as follows: streptomyces mosquitocidus, the academic name of latin: streptomyces cubicilicius, bacteria, actinomycetes and aerobic bacteria; the strain is preserved in the China agricultural microbial Protection center, and the number is as follows: ACCC41132, function: has obvious poisoning effect on anopheles, culex and aedes larvae;

3. the strain name is as follows: beauveria bassiana, Latin name: beauveria bassiana, fungi, aerobic bacteria; the strain is preserved in China general microbiological culture Collection center with the following serial numbers: ACCC30110, function: the mosquito killer has good killing effect on culex, aedes and larvae;

4. the strain name is as follows: metarhizium anisopliae, Latin school name: metarhizium anisopliae, fungi, aerobic bacteria; the strain is preserved in China agricultural microorganism strain preservation center with the following serial numbers: ACCC30798, function: the mosquito killer has good killing effect on aedes and larvae;

5. the strain name is as follows: isaria fumosorosea, Latin's chemical name: fumosolosea wire, fungi, aerobic bacteria; the strain is preserved in China general microbiological culture Collection center with the following serial numbers: ACCC37775, function: can infect homopteran insects such as mosquitoes.

The safety levels of the four anaerobic bacteria are four levels, so that the four anaerobic bacteria are safe and harmless to human, livestock and ecological environment, and the saturation growth of aerobic bacteria can be inhibited;

1. the strain name is as follows: lactobacillus bulgaricus, the university name of latin: lactobacillus bulgaricus, bacteria, anaerobes; the strain is preserved in China agricultural microorganism strain preservation center with the following serial numbers: ACCC 19940;

2. the strain name is as follows: lactobacillus acidophilus, latin scientific name: lactobacillus acidophilus, bacteria, anaerobes; the strain preservation and China general microbiological culture Collection center, the number is: ATCC 4357;

3. the strain name is as follows: lactobacillus plantarum, latin scientific name: lactobacillus plantarum, bacteria, anaerobes; the strain preservation and China agricultural microbial strain preservation center has the following numbering: ACCC 1101;

4. the strain name is as follows: lactobacillus delbrueckii, latin scientific name: lactobacillus delbrueckii subsp, bacterial, anaerobic; the strain preservation and China general microbiological culture Collection center, the number is: ACCC 05468.

A preparation method of a multi-bacterium composite synergistic liquid mosquito-killing bacterium agent comprises the following steps:

the first step is as follows: respectively fermenting and culturing five aerobic bacteria in the bacteria liquid A into five liquid aerobic bacteria liquid,

(1) respectively preparing five liquid culture media of bacillus thuringiensis, streptomyces mosquitocidus, beauveria bassiana, metarhizium anisopliae and isaria fumosorosea, and inoculating corresponding strains into the five culture media; then carrying out primary amplification culture on the strains under the conditions of 25-37 ℃ and aerobic fermentation at the temperature of 120-;

(2) then respectively taking five aerobic bacteria liquid of primary fermentation, inoculating corresponding bacteria liquid into five liquid aerobic bacteria culture media, and carrying out secondary expansion culture on the bacteria liquid under the same conditions to obtain five secondary expansion aerobic fermentation bacteria liquid, wherein the inoculation amount of each primary bacteria liquid is 2-10% of the mass of the culture medium;

(3) respectively taking five aerobic bacteria liquid of secondary fermentation, inoculating corresponding bacteria liquid into five liquid aerobic bacteria culture media, and carrying out three-stage expanded culture on the bacteria liquid under the same conditions to obtain five bacteria liquid of three-stage expanded aerobic fermentation, wherein the inoculation amount of each secondary bacteria liquid is 2-10% of the mass of the culture medium;

(4) mixing the five bacteria solutions subjected to the three-stage culture according to the proportion of 10-30% respectively, standing and homogenizing for 24 hours to obtain the bacteria solution A, wherein the actual proportion of the five mosquito-killing aerobic bacteria solutions can be properly adjusted according to the species of mosquitoes and the specialty of mosquito-killing bacteria in the specific implementation;

the second step is that: fermenting and culturing the four anaerobic bacteria in the B bacteria liquid into anaerobic bacteria liquid mixed with four liquids,

(1) respectively preparing liquid anaerobic bacteria culture media of lactobacillus bulgaricus, lactobacillus acidophilus, lactobacillus plantarum and lactobacillus delbrueckii, respectively inoculating respective corresponding strains to the four liquid anaerobic bacteria culture media, standing and fermenting hermetically for 10-30 days at 25-37 ℃, and performing primary amplification culture on the strains to obtain four primary anaerobic bacteria liquid;

(2) mixing the four anaerobic bacteria liquids of primary culture according to the proportion of 25 percent, sealing and standing for 24 hours to obtain anaerobic mixed bacteria liquid of primary fermentation;

(3) preparing a mixed fermentation liquid anaerobic bacteria culture medium, inoculating the anaerobic mixed bacteria liquid subjected to primary fermentation into the mixed fermentation liquid anaerobic bacteria culture medium, wherein the inoculation amount is 5-10% of the mass of the culture medium, standing the culture medium at the temperature of 30-37 ℃, performing closed anaerobic fermentation for 10-30 days, and performing secondary expansion culture on the strains to obtain a mixed fermentation secondary anaerobic bacteria liquid;

(4) repeating the step (3) to obtain a third-level anaerobic bacterial liquid of mixed fermentation, and obtaining a mixed bacterial liquid B after measuring the pH value to be less than or equal to 5;

the third step: proportioning: according to the proportion of 50-70% of the bacterial liquid A and 50-30% of the bacterial liquid B, slowly injecting the five bacterial liquids A into the mixed bacterial liquid B, uniformly stirring, standing for 24 hours, homogenizing and stabilizing to obtain the multi-bacterium composite synergistic liquid mosquito-killing microbial inoculum.

In the technical scheme, the fermentation medium of the five bacterial liquids forming the bacterial liquid A has the following formula:

(a1) the formula of the bacillus thuringiensis culture medium comprises the following components:

20g of sucrose, 5g of peptone, 5g of yeast extract, 0.3g of dipotassium phosphate, 1g of disodium phosphate, 1g of magnesium sulfate, 0.02g of ferrous sulfate, 0.02g of calcium chloride and the balance of sterile water to 1L; the culture conditions are as follows: the initial pH value is 7.2, the temperature is 25-37 ℃, the temperature is 120-;

(a2) the formula of the mosquito-killing streptomyces culture medium comprises the following components:

20g of soluble starch, 1g of potassium nitrate, 0.5g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate, 0.5g of sodium chloride, 0.01g of ferrous sulfate and the balance of sterile water to 1L; the culture conditions are as follows: the initial pH value is 7.0, the light is shielded, the temperature is 25-37 ℃, the temperature is 120-;

(a3) the formula of the beauveria bassiana culture medium comprises the following components:

50g of cane sugar, 1g of dipotassium hydrogen phosphate and 1 percent of corn leaching liquor, wherein 1L of the corn leaching liquor is complemented; the culture conditions are as follows: the initial pH value is 7.0, the light is shielded, the temperature is 25-37 ℃, the temperature is 120-;

(a4) the formula of the metarhizium anisopliae culture medium comprises the following components:

50g of cane sugar, 1g of dipotassium hydrogen phosphate and 1 percent of corn leaching liquor, wherein 1L of the corn leaching liquor is complemented; the culture conditions are as follows: the initial pH value is 7.0, the light is shielded, the temperature is 25-37 ℃, the temperature is 120-;

(a5) the formula of the Isaria fumosorosea culture medium comprises the following components:

1L of 20% potato extract and 20g of sucrose; the culture conditions are as follows: initial pH value is 7.0, shading is carried out at 25-37 ℃, temperature is 120-.

Further, the 1% corn extract is prepared by adding water 1L into 10g of fresh corn kernels, boiling for 30 minutes, filtering to remove residues, and supplementing sterile water to 1L; the 20% potato extractive solution is prepared from peeled potato 200g, cutting into small pieces, adding water 1L, boiling for 30 min, filtering to remove residue, and supplementing sterile water to 1L.

In the technical scheme, the fermentation culture medium of four bacterial liquids forming the bacterial liquid B has the following formula:

(b1) the formula of the lactobacillus bulgaricus culture medium comprises the following components:

20g of sucrose, 35g of peptone, 1.5g of calcium carbonate, 30g of yeast extract, 2g of dipotassium hydrogen phosphate, 1.5g of magnesium sulfate, 1g of calcium carbonate, 0.02g of manganese sulfate, 1.5g of ammonium hydrogen phosphate and the balance of sterile water to 1L; the culture conditions are as follows: initial pH value of 6.0, 30-37 deg.C, shading, sealing, standing for anaerobic culture for 10-30 days;

(b2) the lactobacillus acidophilus culture medium formula comprises:

20g of sucrose, 20g of peptone, 1g of sodium chloride, 0.01g of manganese sulfate, 0.02g of magnesium sulfate, 0.5g of sodium acetate and the balance of sterile water to 1L; the culture conditions are as follows: initial pH value of 6.0, 30-37 deg.C, shading, sealing, standing for anaerobic culture for 10-30 days;

(b3) the lactobacillus plantarum culture medium formula comprises the following components:

20g of sucrose, 25g of yeast extract, 2g of dipotassium phosphate and sterile water for complementing 1L; the culture conditions are as follows: initial pH value of 6.0, 30-37 deg.C, shading, sealing, standing for anaerobic culture for 10-30 days;

(b4) the formula of the lactobacillus delbrueckii culture medium is as follows:

20g of sucrose, 35g of peptone, 1.5g of calcium carbonate, 30g of yeast extract, 2g of dipotassium hydrogen phosphate, 1.5g of magnesium sulfate, 1g of calcium carbonate, 0.02g of manganese sulfate, 1.5g of ammonium hydrogen phosphate and the balance of sterile water to 1L; the culture conditions are as follows: initial pH value of 6.0, 30-37 deg.C, shading, sealing, and standing for anaerobic culture for 10-30 days.

In the technical scheme, the formula of the mixed fermentation medium of four bacterial liquids of the B bacterial liquid is as follows:

20g of sucrose, 35g of peptone, 1.5g of calcium carbonate, 30g of yeast extract, 2g of dipotassium hydrogen phosphate, 1.5g of magnesium sulfate, 1g of calcium carbonate, 0.02g of manganese sulfate, 1.5g of ammonium hydrogen phosphate and the balance of sterile water to 1L; the culture conditions are as follows: initial pH value of 6.0, 30-37 deg.C, shading, sealing, and standing for anaerobic culture for 10-30 days.

The invention also provides an application of the multi-bacterium composite synergistic liquid mosquito killing bactericide in mosquito trapping, which specifically comprises the following steps:

(C1) compounding with various mosquito trapping agents to prepare a microorganism trapping and killing mosquito killing agent;

(C2) can be used alone as mosquito killing microbial inoculum.

Correspondingly, the invention provides a microbe trapping and killing mosquito killer containing the multi-bacterium composite synergistic liquid mosquito killing microbial inoculum, which comprises 6-12% of brown sugar, white sugar or honey, 3-6% of yeast powder, 1-3% of the multi-bacterium composite synergistic liquid mosquito killing microbial inoculum and the balance of clear water in percentage by weight.

Correspondingly, the invention provides a microbe trapping and killing mosquito killer containing the multi-bacterium composite synergistic liquid mosquito killing microbial inoculum, which comprises 6-12% of brown sugar, white sugar or honey, 3-6% of yeast powder, 1-3% of the multi-bacterium composite synergistic liquid mosquito killing microbial inoculum, 30-50% of beer and the balance of clear water in percentage by weight.

Correspondingly, the invention provides a microbe trapping and killing mosquito killer containing the multi-bacterium composite synergistic liquid mosquito killing microbial inoculum, which comprises 6-12% of brown sugar, white sugar or honey, 3-6% of yeast powder, 1-3% of the multi-bacterium composite synergistic liquid mosquito killing microbial inoculum, 10-30% of leftovers soup and the balance of clear water in percentage by weight.

Correspondingly, the invention provides a microbial bacterium trapping and killing mosquito killer containing the multi-bacterium composite synergistic liquid mosquito killer, which comprises 2-20% of brown sugar or white sugar, 10-20% of sesame paste, 1-3% of sodium glutamate, 1-3% of the multi-bacterium composite synergistic liquid mosquito killer and the balance of clear water in percentage by weight.

The application process of preparing the microorganism trapping and killing mosquito killer comprises the following two steps:

(D1) one of the above-mentioned microbial trapping and killing mosquito-killing agents is selectively added into the mosquito-storage box of mosquito-killing lamp;

(D2) one of the above-mentioned microbial trapping and killing mosquito-killing agents is filled in a container such as a basin or a plate, and is dipped with black cotton cloth and is flatly hung above the container, the lower edge of the cotton cloth is soaked in the preparation liquid, so that the wetting of the black cotton cloth is kept, and mosquitoes can be killed by infecting mosquito-killing bacteria only by touching the black cotton cloth.

The application process of the multi-bacterium composite synergistic liquid mosquito killing microbial inoculum used as the mosquito killing microbial inoculum independently comprises the following two steps:

(D3) the bactericide is diluted by 100 to 1000 times with water alone or sprayed by an airplane, so that large-scale mosquito killing can be implemented;

(D4) the bactericide is diluted by 100 to 1000 times with water and sprayed to a water system, so that large-area mosquito larvae can be killed.

Compared with the prior art, the invention has the following beneficial effects:

(1) the microbial inoculum provided by the invention is prepared by compounding five aerobic bacteria (A bacterial liquid) which kill mosquitoes and have respective specialities with four acid-producing anaerobic bacteria (B bacterial liquid), A, B two types of bacterial liquids are fermented respectively and then compounded in proportion, after compounding, under a weak acid environment, the growth condition of flora is inhibited, simultaneously the nutrient and metabolic energy are complemented, and a good symbiotic environment with dependence symbiosis and non-antagonism is formed among flora. Therefore, the preparation inhibits the saturation growth of flora, and the growth of the flora is slowed down under the condition of liquid normal temperature, most strains form a spore state, and the vitality of the strains is maintained and is in a dormant state temporarily. Under the condition of normal temperature, the storage period of validity of the multi-bacterium composite synergistic liquid mosquito-killing bacterium agent can reach more than 12 months, and the storage period of validity at 4 ℃ can reach more than 18 months. After dilution, the strain can quickly recover and act.

(2) The five aerobic bacteria in the A bacteria liquid adopt a method of respectively fermenting, compounding and symbiosis, so that the defects of different species, conditions and degrees of single bacteria for killing mosquitoes are overcome, and multiple bacteria are enabled to jointly play roles, have complementary functions and are combined to increase the efficiency.

(3) Because the chemical mosquito attractant is added with the insecticide which is alkaline, the smell of the chemical mosquito attractant is not favorable for attracting mosquitoes, and many chemical mosquito attractants need to be inhaled by a mosquito mouth to be poisoned, so the effect is often poor. The preparation of the invention is also added with the B microbial inoculum in a combined way, the pH value of the preparation is in a weak acid range of 4-6, and the smell of the weak acid can attract mosquitoes, thereby being beneficial to the exertion of the function of the mosquito-luring agent.

(4) The strains adopted by the multi-bacterium composite synergistic liquid mosquito-killing bactericide are screened, purified and stored in a national certified microbial conservation center or a scientific research institution from natural bacteria existing in natural environment, the safety categories of the strains are determined to be four types, the strains do not harm natural enemies of human, livestock, poultry and mosquitoes and ecological environment, and the mosquito vectors do not generate resistance, and can be widely used indoors and outdoors and sprayed in a large area. Chemical insecticides can be responsible for resistance to mosquito vectors and cause damage to non-target organisms. The variant microbial mosquito killing microbial inoculum developed by using genetically modified or artificially grafted strains can be popularized and used after scientific demonstration and inspection on the variant strains on the premise of ensuring the human health and ecological environment.

(5) The preparation is only added in a trace amount in the mosquito attractant, mosquitoes can be infected and attack by infection slightly, and can live with bacteria for 1-7 days before being poisoned and killed, and the infected mosquitoes become mosquito vectors of the microbial inoculum in the period and infect mosquito killing bacteria to more mosquitoes; chemical insecticides are most often sudden death-type agents and cannot be transmitted to mosquitoes.

Detailed Description

The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.