Bacillus thuringiensis strain, fermentation culture method and application of bacillus thuringiensis strain

Abstract

The invention provides a bacillus thuringiensis strain, a fermentation culture method and application of the bacillus thuringiensis strain. The strain is preserved in the China Center for Type Culture Collection on September 28, 2011, the preservation address is No. 299 on eight road, Wuchang District, Wuhan City, Hubei Province, the preservation number is CCTCC No: M 2011334, and the preservation date is September 28, 2011. The strain cultured by the fermentation method has efficient, specific and stable insecticidal activity on culex, aedes and other mosquitoes, has the advantages of low preparation cost, environmental friendliness and the like, can be developed into a biological insecticide for preventing and killing mosquitoes and preventing and controlling malaria, dengue fever, chikungunya and other mosquito-borne infectious diseases, and meets the requirements of green and safe prevention and control in China.

Description

Technical Field

[0001] This disclosure belongs to the field of microbial technology, specifically relating to a fermentation culture method for Bacillus thuringiensis strains and its application. Background Technology

[0002] Mosquitoes are one of the most widespread and important insect groups globally, carrying and transmitting various pathogens such as bacteria, parasites, and viruses, making them the primary vectors for vector-borne infectious diseases worldwide. Mosquitoes can cause diseases such as malaria, dengue fever, and chikungunya, which have high morbidity, disability, and even mortality rates, seriously threatening public health. Mosquitoes are highly adaptable, able to survive and reproduce in diverse environments. Their eggs can even remain dormant for over two years under drought conditions, hatching rapidly upon contact with stagnant water. Numerous factors make mosquito control difficult, and while chemical insecticides remain the mainstay of emergency control, their drawbacks are becoming increasingly acute. On the one hand, they are significantly toxic to the environment; burning mosquito coils releases PM2.5 and formaldehyde, and long-term use can induce respiratory diseases. Pyrethroid insecticides pollute soil and water, causing fatal damage to non-target organisms such as bees and fish. On the other hand, the crisis of insecticide resistance is intensifying; several countries have already discovered mosquito species resistant to pyrethroids, and malaria transmission rates in some areas have rebounded by over 30%. Therefore, green and safe biological control methods are becoming increasingly popular.

[0003] Bacillus thuringiensis subsp. Israel ( Bacillus thuringiensis Bacillus thuringiensis (Bti) is a genus of Bacillus, an important subspecies of Bacillus thuringiensis. The bacteria have blunt, rounded ends, flagella, and are highly motile. The sporangia do not swell, and the spores are short-elliptical or oval. Parasporal crystals are nearly round, and the bacteria are Gram-positive. Bti exhibits strict host specificity, acting only against the larvae of Diptera such as mosquitoes and blackflies. It is highly effective and low in toxicity, thus commonly used for mosquito control. The main mechanism of action of Bti relies on the protoxins produced during the quiescent phase of endospore formation. These protoxins exist in crystalline form and include Cry and Cyt. Both dissolve in the alkaline conditions of the larval gut. Cry toxins bind to protein receptors on the outer membrane of intestinal cells, while Cyt toxins bind to phospholipids, disrupting the intestinal function of pests and leading to larval starvation and death. However, there are currently few highly toxic Bti strains, necessitating the discovery of new Bti strains with high mosquito-killing activity to enrich the variety of mosquito-killing strains. Furthermore, bio-fermentation methods often suffer from drawbacks such as low fermentation levels, unstable product quality, and difficulty in practical application. Summary of the Invention

[0004] In view of the above problems of shortage of high-efficiency mosquito-killing Bti strains and defects in fermentation application, the present disclosure provides a Bacillus thuringiensis strain, a fermentation culture method and application thereof. The Bacillus thuringiensis strain cultured by the fermentation method has high-efficiency, specific and stable insecticidal activity on mosquitoes such as Culex and Aedes, and has the advantages of low preparation cost and environmental friendliness, and can be developed as a biological insecticide for mosquito prevention and killing, prevention and control of mosquito-borne infectious diseases such as malaria, dengue fever and Kyasanur fever, and meets the demand of green and safe prevention and control in China.

[0005] According to a first aspect of the present disclosure, a Bacillus thuringiensis strain is provided, characterized in that the strain is preserved in the China Center for Type Culture Collection, located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with a preservation number of CCTCC No: M 2011334 and a preservation date of September 28, 2011.

[0006] According to a second aspect of the present disclosure, a fermentation medium for Bacillus thuringiensis is provided, comprising: Corn meal 20-30 g / L Soybean meal powder 10-20 g / L Corn steep liquor 10-30 g / L Glucose 2-10 g / L Amino acid 0.2-0.5 g / L Trace elements 0.01-0.08 g / L.

[0007] In some embodiments, the concentration of corn meal is 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, or any value between any two numerical values.

[0008] In some embodiments, the concentration of soybean meal powder is 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, or any value between any two numerical values.

[0009] In some embodiments, the concentration of corn steep liquor is 10 g / L, 12 g / L, 14 g / L, 16 g / L, 18 g / L, 20 g / L, 22 g / L, 24 g / L, 26 g / L, 28 g / L, 30 g / L, or any value between any two numerical values.

[0010] In some embodiments, the concentration of sucrose is 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, or any value between any two of these values.

[0011] In some embodiments, the micronutrient is selected from one or more of ferrous sulfate, manganese chloride, zinc chloride, cobalt chloride, and copper chloride. In a particular embodiment, the micronutrient is manganese chloride.

[0012] In some embodiments, the concentration of the micronutrient is 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, or any value between any two of these values.

[0013] In some embodiments, the amino acid is selected from one or more of L-lysine, L-leucine, L-phenylalanine, and L-valine. In a particular embodiment, the amino acid is L-leucine.

[0014] In some embodiments, the concentration of the amino acid is 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, or any value between any two of these values.

[0015] In some embodiments, the fermentation medium further comprises: potassium phosphate monobasic at a concentration of 0.05-0.5 g / L, magnesium sulfate at a concentration of 0.01-0.05 g / L, sodium chloride at a concentration of 0.1-0.5 g / L, and calcium chloride at a concentration of 0.01-0.05 / L.

[0016] In some embodiments, the concentration of potassium phosphate monobasic is 0.05 g / L, 0.1 g / L, 0.15 g / L, 0.2 g / L, 0.25 g / L, 0.3 g / L, 0.35 g / L, 0.4 g / L, 0.45 g / L, 0.5 g / L, or any value between any two of these values.

[0017] In some embodiments, the concentration of magnesium sulfate is 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, or any value between any two of these values.

[0018] In some embodiments, the concentration of sodium chloride is 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, or any value between any two of these values.

[0019] In some embodiments, the concentration of calcium chloride is 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, or any value between any two of the values.

[0020] In some embodiments, the solvent of the fermentation medium is water, in some embodiments, deionized water.

[0021] In some embodiments, the fermentation medium comprises: Corn meal 25 g / L Soybean meal powder 15 g / L Corn steep liquor 20 g / L Glucose 5 g / L Potassium dihydrogen phosphate 0.1 g / L Magnesium sulfate 0.5 g / L Calcium chloride 0.5 g / L Manganese chloride 0.04 g / L L-leucine 0.3 g / L.

[0022] According to a third aspect of the present disclosure, there is provided a fermentation culture method of Bacillus thuringiensis, comprising the following steps: 1) inoculating a seed liquid of Bacillus thuringiensis into a seed culture medium for culture to obtain a seed liquid; 2) inoculating the seed liquid obtained in step 1) into a fermentation culture medium for culture to obtain a fermentation culture liquid, In some embodiments, the Bacillus thuringiensis strain is deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC No: M 2011334.

[0023] In some embodiments, in step 2), the initial pH of the fermentation culture medium is 6.5-7.5; and / or the inoculation amount of the seed liquid is 2%-5% (w / w); and / or the fermentation culture time is 28-40 h; and / or the dissolved oxygen of the fermentation culture liquid is ≥ 30%.

[0024] Those skilled in the art should understand that the dissolved oxygen is the ratio of oxygen to the culture medium, and the unit is mg / L.

[0025] In some embodiments, the fermentation culture medium is adjusted to an initial pH of 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or any value between any two of the values.

[0026] In some embodiments, the inoculation amount is 2% (w / w), 2.5% (w / w), 3% (w / w), 3.5% (w / w), 4% (w / w), 4.5% (w / w), 5% (w / w), or any value between any two numerical values.

[0027] In some embodiments, the temperature of the fermentation culture is 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or any value between any two numerical values.

[0028] In some embodiments, the pH of the fermentation culture is 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or any value between any two numerical values.

[0029] In some embodiments, the duration of the fermentation culture is 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 h, 40 h, or any value between any two numerical values.

[0030] In some embodiments, the rotation speed is 200 r / min, 210 r / min, 220 r / min, 230 r / min, 240 r / min, 250 r / min, 260 r / min, 270 r / min, 280 r / min, 290 r / min, 300 r / min, or any value between any two numerical values.

[0031] In some embodiments, the dissolved oxygen is 30%, 35%, 40%, 45%, 50%, 55%, 60%, etc.

[0032] In some embodiments, the fermentation culture method comprises the following steps: (1) Preparation of seed liquid: the seed liquid of Bacillus thuringiensis is inoculated on Luria-Bertani (LB) liquid medium to obtain a seed liquid, and the concentration of the bacterial cells is 10 8 cfu / mL; (2) Fermentation culture: the seed liquid is inoculated in the sterilized fermentation medium, and the inoculation amount is 3% (w / w); the rotation speed is 300 r / min, the dissolved oxygen is ≥30%, the culture temperature is 30°C, and the fermentation time is 28 h.

[0033] In some embodiments, the fermentation culture method comprises the following steps: (1) Sterilization of the fermentation tank: the liquid volume of the fermentation medium is 50%-60% of the volume of the fermentation tank, the initial pH is adjusted to 7.0, and the high-pressure steam sterilization is performed at 121°C for 30 min. (2) Preparation of seed liquid: the Bacillus thuringiensis strain is inoculated on the LB liquid medium for culture, i.e. the seed liquid, and the bacterial concentration is 108cfu / mL; (3) Fermentation culture: the seed liquid is inoculated into the sterilized fermentation medium of step (1) at an inoculation amount of 3% (w / w); the rotation speed is 300 r / min, the dissolved oxygen is ≥30%, the culture temperature is 30°C, and the fermentation time is 28 h.

[0034] In some embodiments, the Bacillus thuringiensis strain in step (1) is Bacillus thuringiensis subspecies israelensis.

[0035] In some embodiments, the Bacillus thuringiensis subspecies israelensis is Bacillus thuringiensis subspecies israelensis with the accession number CCTCC No: M 2011334.

[0036] According to a fourth aspect of the present disclosure, a composition is provided, which comprises the fermentation culture liquid of the Bacillus thuringiensis strain of the first aspect.

[0037] According to a fifth aspect of the present disclosure, a preparation method of the composition of the fourth aspect is provided, which comprises the following steps: The fermentation culture liquid of the Bacillus thuringiensis strain of the first aspect is spray-dried to obtain the composition.

[0038] According to a sixth aspect of the present disclosure, the strain of the first aspect, the fermentation liquid obtained by the fermentation culture method of the third aspect, and the composition of the fourth aspect are applied in pest control.

[0039] In some embodiments, the composition is used for controlling dipteran insect pests.

[0040] In some embodiments, the dipteran insects include any one or more of the following: Aedes, Culex, and Anopheles. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 A phylogenetic tree of the strain provided by the present disclosure is shown under the background level of whole bacterial genome; Figure 2 A detailed protein-based phylogenetic tree of the strain provided by the present disclosure is shown; Figure 3 A CDS-based intraspecific phylogenetic tree of the strain provided by the present disclosure is shown. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical scheme and advantages of the present disclosure more clear, the present disclosure will be further described in detail below in combination with specific embodiments. The following specific embodiments are further descriptions of the technical scheme of the present disclosure, but the protection scope of the present disclosure is not limited to these embodiments. Any changes or equivalent replacements without departing from the concept of the present disclosure are included in the protection scope of the present disclosure.

[0043] The experimental methods used in the following embodiments are conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by commercial channels by those skilled in the art.

[0044] Example 1: Obtaining and identifying the strain (1) Isolation and purification of the strain The steps of isolation and purification of the strain are as follows: The soil is selected from the surrounding area of Nanhu Lake in Wuhan, Hubei. 10 g of soil sample is taken into a 90 mL sterile water triangle bottle, mixed uniformly at room temperature for 30 min, and then heat treated at 80°C water bath for 10 min to kill non-sporulated bacteria. Then, gradient dilution is performed according to 10, 10 2 , 10 3 gradient dilution, and then coated on LB solid medium plates. After 48 h of inverted culture at 30°C, single colonies are picked and gradient streaked on LB solid medium plates. After three times of streaking and purification, a strain of Bacillus is isolated. The strain is preserved in a -80°C refrigerator with 50% glycerol.

[0045] (2) Preservation of the strain Bacillus thuringiensis subspecies israelensis (Bti) is preserved in China Center for Type Culture Collection (CCTCC) at 299 Bajiyilu, Wuchang District, Wuhan City, Hubei Province, China. The preservation number is CCTCC No: M 2011334, and the preservation date is September 28, 2011. The survival state is alive.

[0046] (3) Physiological and biochemical characteristics identification of the strain The strain is cultured in a 30°C incubator for 48 h. The colony characteristics are observed and recorded, and an appropriate amount of colonies are picked for Gram staining. The physiological and biochemical characteristics of the strain are identified by referring to the relevant experimental methods of Bergey's Manual of Determinative Bacteriology (8th edition) and Microbiology Experiment Manual. The physiological and biochemical identification results are shown in Table 1.

[0047] Table 1: Physiological and biochemical characteristics identification results of the strain

[0048] (4) Phylogenetic tree analysis of the strain The whole genome level multi-gene phylogenetic tree analysis was performed on the strain. The protein sequences of the sequencing sample and the whole genomes of 2586 prokaryotes whose whole genome sequences have been published were aligned, the phylogenetic distance was calculated using CVTree, and the phylogenetic tree was constructed using the Neighbor Join method.

[0049] From Figure 1 The phylogenetic tree shown in the figure shows that the strain isolated by the present disclosure is located in Bacillus thuringiensis the branch, and the strains in the branch are Bacillus thuringiensis serovar israelensis As can be seen from the above, the whole genome level phylogenetic tree analysis result shows that the strain is Bacillus thuringiensis serovar israelensis .

[0050] All Bacillus thuringiensis were aligned, and Escherichia coli E. coli K-12 MG1655 was used as an outgroup for further phylogenetic analysis. It was found that the strain sample was relatively close to Bacillus thuringiensis serovar israelensis ATCC 35646 (as shown in Figure 2 and Figure 3 ). Compared with the whole genome reference sequence provided by NCBI, the sample genome sequence is closest to the whole genome sequence published by NCBI Bacillus thuringiensis serovar israelensis HD-789 strain, which is judged to belong to Bacillus thuringiensis serovar israelensis . In summary, Figures 1-3 the whole genome level phylogenetic tree analysis result shows that the strain is Bacillus thuringiensis serovar israelensis .

[0051] Example 2: Determination of the mosquito killing activity of the strain The isolated strain was spread on LB solid medium plates, and incubated at 30°C for 48 h. Then a single colony was picked from the LB plate and inoculated into 5 mL of LB liquid medium. After activation culture at 30°C, 200 rpm for 12 h, the strain was transferred into 200 mL of LB liquid medium at an inoculation amount of 1%. After 48 h, the culture solution was removed, centrifuged at 5000 rpm for 10 min to collect the bacterial slurry, and the bacterial slurry was configured into bacterial suspension with six concentration gradients of 0.5 μg / L, 1 μg / L, 2 μg / L, 3 μg / L, 5 μg / L and 10 μg / L using dechlorinated tap water according to different masses.

[0052] In 12 cm open flat dish pour different dilution bacteria liquid 100 mL, each flat dish put in 30 pieces of 3 age Aedes larvae, after 26℃ constant temperature culture 24 h check live insect number. Each concentration gradient set 3 parallel and set blank control. Use Abbott formula to calculate correction mortality and find probability value, adopt least square method to calculate lethal concentration LC 50 .

[0053] Table 2 Strain activity determination on Aedes

[0054] Strain biological activity determination result on 3 age Aedes is shown in Table 2, from which can know that the LC 50 of strain on Aedes is 6.51±0.14 μg / L, has higher virulence.

[0055] Example 3: Medium screening 1) Microelement kind screening Conventional medium: corn flour 25 g / L, soybean meal powder 15 g / L, corn slurry 20 g / L, glucose 5 g / L, potassium dihydrogen phosphate 0.1 g / L, magnesium sulfate 0.5 g / L, calcium chloride 0.5 g / L, the rest is sterile water, pH 7.0.

[0056] 1st medium: compared with conventional medium, the difference is only that 0.02 g / L (final concentration) ferrous sulfate is added, the rest is same; 2nd medium: compared with conventional medium, the difference is only that 0.02 g / L (final concentration) manganese chloride is added, the rest is same; 3rd medium: compared with conventional medium, the difference is only that 0.02 g / L (final concentration) zinc chloride is added, the rest is same; 4th medium: compared with conventional medium, the difference is only that 0.02 g / L (final concentration) cobalt chloride is added, the rest is same; 5th medium: compared with conventional medium, the difference is only that 0.02 g / L (final concentration) copper chloride is added, the rest is same.

[0057] The above medium materials are dissolved in sterile water in proportion, adjust pH to 7.0 as appropriate, according to 1 / 5 of effective volume of flask, 121℃ sterilization 30 min. Use LB medium to culture strain 12 h, bacterial number 10 8CFU / mL culture solution as seed liquid (the seed liquid is used for inoculation in the following examples), inoculate the seed liquid into the sterilized and cooled culture medium at 3% inoculation amount, carry out shake flask fermentation under the condition of 30°C and 200r / min, take sample after 28h to determine the biological titer of the fermentation broth. The biological titer is detected by Q / KNHB005-2024 enterprise standard.

[0058] Table 3 Influence of different trace elements on the strain

[0059] The experimental results are shown in Table 3. From the results, it can be seen that Mn 2+ and Co 2+ improve the biological titer of the strain fermentation broth, among which Mn 2+ has a more significant promoting effect on the biological titer of the strain; Fe 2+ and Zn 2+ have no obvious influence on the biological titer of the strain fermentation broth, and Cu 2+ has an inhibitory effect on the biological titer of the strain fermentation broth. Therefore, the present disclosure selects to add manganese chloride in the culture medium to improve the biological titer level of the strain fermentation broth.

[0060] 2) Manganese chloride content exploration Different contents of manganese chloride are added to the conventional culture medium to obtain treatment groups 1-5, and the control is the conventional culture medium (corn meal 25g / L, soybean meal 15g / L, corn syrup 20g / L, glucose 5g / L, potassium dihydrogen phosphate 0.1g / L, magnesium sulfate 0.5g / L, calcium chloride 0.5g / L, the rest is sterile water, pH 7.0). The above shake flask fermentation method is adopted for fermentation, and the biological titer is determined.

[0061] Table 4 Influence of different contents of manganese chloride on the strain

[0062] The experimental results are shown in Table 4. With the increase of the content of manganese chloride, the biological titer of the strain fermentation broth also increases, and when the content of manganese chloride is 0.04g / L, the biological titer of the strain fermentation broth is the highest, which is 212 ITU / mg, and then the biological titer begins to decrease.

[0063] 3) Amino acid type screening Fermentation is carried out in a 15L tank, and the fermentation culture medium is as follows: corn meal 25g / L, soybean meal 15g / L, corn syrup 20g / L, glucose 5g / L, potassium dihydrogen phosphate 0.1g / L, magnesium sulfate 0.5g / L, calcium chloride 0.5g / L, manganese chloride 0.04g / L, the rest is sterile water, pH 7.0.

[0064] The fermentation process parameters are as follows: the medium is prepared according to the loading coefficient of 60% of the 15L fermenter, sterilized at 121°C for 30min, the inoculation amount is 3%, the culture temperature is 30°C, the stirring speed is 300r / min, and the dissolved oxygen content is ≥30%.

[0065] Different amino acids are added to the content of 0.2g / L at 10h of fermentation, and the fermentation is completed at 28h of fermentation.

[0066] The amino acids are L-lysine, L-leucine, L-phenylalanine and L-valine, respectively, to obtain treatment groups 6-9, and the control treatment group does not add amino acids, and the biological titer is determined at the end of fermentation.

[0067] Table 5 Effect of different amino acids on the strain

[0068] The experimental results are shown in Table 5. From the results, it can be seen that L-lysine, L-leucine and L-valine have a promoting effect on the biological titer of the strain, among which L-leucine has the most significant effect on the titer of the strain, and L-phenylalanine has a slight inhibitory effect on the strain.

[0069] 4) L-leucine content exploration The fermentation is carried out by the 15L tank feeding method in the amino acid screening, different contents of L-leucine are added, respectively, to obtain treatment groups 10-14, and the control treatment group has no feeding addition. The biological titer is determined after fermentation.

[0070] Table 6 Effect of different contents of L-leucine on the strain

[0071] The experimental results are shown in Table 6. From the results, it can be seen that with the increase of L-leucine content, the titer of the strain shows a trend of first increasing and then decreasing. When the content of L-leucine is 0.3g / L, the titer of the strain is the largest, which is 287 ITU / mg.

[0072] Example 4: Evaluation of fermentation method stability The culture medium is corn flour 25 g / L, soybean meal powder 15 g / L, corn syrup 20 g / L, glucose 5 g / L, potassium dihydrogen phosphate 0.1 g / L, magnesium sulfate 0.5 g / L, calcium chloride 0.5 g / L, manganese chloride 0.04 g / L, and the rest is sterile water, pH 7.0. The fermentation process parameters are as follows: the culture medium is prepared according to the 60% loading coefficient of the 15 L fermenter, sterilized at 121°C for 30 min, the inoculation amount is 3%, the culture temperature is 30°C, the stirring speed is 300 r / min, the dissolved oxygen is ≥30%, L-leucine is added to 0.3 g / L at 10 h of fermentation, and the fermentation is completed at 28 h. The strain is fermented in three batches of 15 L fermenter, and the biological titer of different batches is shown in Table 7. The titer is between 280-290 ITU / mg under the fermentation method, and the batch stability is good.

[0073] Table 7 Spore yield and mosquito killing effect of different batches of strains

[0074] Example 5: Preparation containing strain and titer The fermentation method of Example 4 is adopted, and the fermentation culture solution of the strain is obtained. The fermentation filtrate is obtained by filtering the fermentation culture solution with an 80 mesh screen. The fermentation filtrate is centrifuged (5000 r / min, 10 min) to collect the bacterial slurry, which is spray dried (the tower top inlet temperature is 140-150°C, and the outlet temperature is maintained at 80-85°C) to obtain the preparation containing the strain. The titer of the preparation containing the strain is 12883 ITU / mg.

[0075] Example 6: Mosquito killing effect experiment of strain preparation Referring to the activity verification method in Example 2, the test objects are set as Aedes, Culex and Anopheles, respectively, to detect the application effect of the strain preparation in Example 5. The results are shown in Table 8. The preparation has good mosquito killing effect on different types of mosquitoes, and can be used as a biological mosquito killer for preventing and controlling mosquito-borne infectious diseases such as malaria, dengue fever and Kyasanur fever, which meets the demand of green and safe prevention and control in China.

[0076] Table 8

[0077] In the description of the specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present disclosure. In the specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples without contradiction.

[0078] Although the embodiments of the present disclosure have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present disclosure, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present disclosure.

Claims

1. A Bacillus thuringiensis strain, characterized in that, The strain is preserved in China Center for Type Culture Collection, Wuhan, Hubei, China, and the preservation number is CCTCC No: M 2011334, and the preservation date is September 28, 2011.

2. A fermentation medium of Bacillus thuringiensis, characterized in that, The fermentation medium comprises: Corn powder 20-30 g / L Soybean meal powder 10-20 g / L Corn steep liquor 10-30 g / L Glucose 2-10 g / L Trace elements 0.01-0.08 g / L Amino acids 0.2-0.5 g / L Preferably, the trace elements are selected from one or more of ferrous sulfate, manganese chloride, zinc chloride, cobalt chloride and copper chloride; Preferably, the amino acids are selected from one or more of L-lysine, L-leucine, L-phenylalanine and L-valine.

3. The fermentation medium of claim 2, wherein, The fermentation medium further comprises potassium dihydrogen phosphate at a concentration of 0.05-0.5 g / L, magnesium sulfate at a concentration of 0.1-0.8 g / L and calcium chloride at a concentration of 0.1-0.8 / L; Preferably, the solvent of the fermentation medium is water, more preferably deionized water; Preferably, the fermentation medium comprises: Corn powder 25 g / L Soybean meal powder 15 g / L Corn steep liquor 20 g / L Glucose 5 g / L Potassium dihydrogen phosphate 0.1 g / L Magnesium sulfate 0.5 g / L Calcium chloride 0.5 g / L Manganese chloride 0.04 g / L L-leucine 0.3 g / L.

4. A fermentation culture method of Bacillus thuringiensis, characterized by, The culture method comprises: 1) inoculating the seed liquid of Bacillus thuringiensis into a seed culture medium for culture to obtain a seed liquid; 2) inoculating the seed liquid obtained in step 1) into a fermentation culture medium for culture to obtain a fermentation culture liquid, wherein the Bacillus thuringiensis strain is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC No: M 2011334.

5. The fermentation culture method according to claim 4, characterized by, In step 2), the initial pH of the fermentation culture medium is 6.5-7.5; and / or the inoculation amount of the seed liquid is 2%-5% (w / w); and / or the fermentation culture time is 28-40 h; and / or the dissolved oxygen of the fermentation culture liquid is ≥30%; Preferably, the fermentation culture method comprises the following steps: (1) Preparation of seed liquid: the seed liquid of Bacillus thuringiensis was inoculated on Luria-Bertani liquid medium to obtain seed liquid, and the concentration of the bacterial body was 10 8 cfu / mL; (2) fermentation culture: inoculating the seed liquid into the sterilized fermentation culture medium at an inoculation amount of 3% (w / w); the rotation speed is 200-300 r / min, the dissolved oxygen is ≥30%, the culture temperature is 30°C, and the fermentation time is 28 h.

6. The fermentation culture method according to claim 5, characterized by, The Bacillus thuringiensis strain in step (1) is Bacillus thuringiensis subspecies israelensis; Preferably, the Bacillus thuringiensis subspecies israelensis is the Bacillus thuringiensis subspecies israelensis with the preservation number of CCTCC No: M 2011334.

7. A composition characterized in that, The composition comprises the fermentation culture liquid of the Bacillus thuringiensis strain of claim 1.

8. A process for the preparation of a composition according to claim 7, characterized in that, The composition is obtained by spray drying the fermentation culture liquid of the Bacillus thuringiensis strain of claim 1.

9. Use of the strain of claim 1, the fermentation broth obtained by the fermentation culture method of any one of claims 4-6, or the composition of claim 7 in pest control.

10. Use according to claim 9, characterized in that, The composition is used for controlling dipteran pests; Preferably, the dipteran pests include any one or more of Aedes, Culex, and Anopheles.

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