Bacillus thuringiensis with camellia oleifera meal as main fermentation raw material, solid fermentation culture method and application

Abstract

The invention belongs to the technical field of microbial pesticides, and particularly relates to bacillus thuringiensis with camellia oleifera meal as a main fermentation raw material, a solid fermentation culture method and application. The bacillus thuringiensis which can be subjected to solid fermentation by taking oil tea meal as a main raw material is obtained from a microbial library through repeated screening, and the preservation number of the strain is CCTCC (China Center for Type Culture Collection) NO: M20241924. The number of spores fermented by the strain can stably exceed 8.0 * 10 < 9 > cfu / g. Therefore, according to the technical scheme, a new direction is provided for recycling of the oil-tea meal, the oil-tea meal is used as a main fermentation raw material to produce the biopesticide, the treatment method can reduce emission of agricultural wastes and use of chemical pesticides, and the method is very environment-friendly.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial pesticides, and in particular relates to a Bacillus thuringiensis using oil-tea camellia meal as a main fermentation raw material, a solid fermentation culture method and an application thereof. Background Art

[0002] Camellia oleifera meal is the powder of the pomace left after the camellia seeds are squeezed and crushed, and is a kind of agricultural waste. Camellia oleifera meal contains a variety of nutrients such as protein, sugar, tea saponin and minerals. Abandoning it not only wastes resources, but also easily pollutes the environment. Extracting tea saponin is a common method of recycling camellia oleifera meal, and its product has high added value, but the residue after extraction is difficult to reuse, and it also faces the problem of residue disposal. Camellia oleifera meal is a good choice as feed. Since camellia oleifera meal contains a high content of tea saponin, animals cannot eat it directly. It needs to be removed by fermentation as a supplement to feed, and a large amount of camellia oleifera meal residue cannot be fully utilized. After the camellia oleifera meal is fermented and used as a fertilizer, it can make full use of the camellia oleifera meal resources without causing environmental pollution, but its product added value is not high, which is not conducive to large-scale promotion and application.

[0003] In the private sector, some farmers use fermented tea-leaf meal to prevent field pests. Tea-leaf meal, as the main ingredient, is also widely used as a pond cleaning agent in the aquaculture industry, suggesting that tea-leaf meal can be used as a fermentation raw material for microbial pesticides. Solid fermentation has the advantages of simple operation, low energy consumption, easy control of the fermentation process, and relatively low requirements for sterility, and is particularly suitable for treating agricultural waste. At present, the solid-state fermentation of tea-leaf meal mostly uses fungi, such as Acropora terrestris (Bi Luanluan, Master's thesis of Central South University of Forestry and Technology, 2023), Aspergillus niger (Journal of Chinese Institute of Food Science and Technology, 2012, 12(06), 71-79) and yeast (China Feed, 2023, (03), 5-10), etc., or uses a variety of bacteria for mixed fermentation (CN115197010A, CN115197014A), and its uses are mostly feed or fertilizer. There is no relevant literature report on the solid fermentation of microbial pesticides using tea-leaf meal as the main fermentation matrix and a single bacterial strain.

[0004] The invention provides a Bacillus thuringiensis with oil-tea camellia meal as a main fermentation raw material, a solid fermentation medium and an application thereof. The solid fermentation medium uses oil-tea camellia meal as a main fermentation matrix, the fermentation method is simple and easy to operate, the product after fermentation has good insecticidal activity, can be directly used as a microbial insecticide, and provides a direction with promotion potential for agricultural waste oil-tea camellia meal. Summary of the invention

[0005] The invention provides a bacillus thuringiensis Bt-YCP-128 with oil-tea cake as main fermentation raw material. The preservation number of the strain is CCTCC NO: M20241924. The strain grows well on a solid culture medium with oil-tea cake as main matrix.

[0006] The invention also provides a solid fermentation culture method for Bacillus thuringiensis Bt-YCP-128 using oil-tea cake as a main matrix.

[0007] The last object of the present invention is to provide an application of the solid fermentation product of Bacillus thuringiensis Bt-YCP-128 in agricultural pest control. The culture after solid fermentation can be directly used for the control of agricultural pests, including pests of the order Lepidoptera.

[0008] In order to achieve the above object, the present invention adopts the following technical measures:

[0009] The applicant has screened out a strain of Bacillus thuringiensis suitable for solid fermentation with tea seed meal as the main raw material from the microbial resource library built by itself. The Bacillus thuringiensis has been sent to the China Center for Type Collection for preservation on September 5, 2024, and the classification name is: Bacillus thuringiensis Bt-YCP-128, the preservation number is: CCTCC NO: M20241924, and the address is: Wuhan University, Wuhan, China.

[0010] The strain Bt-YCP-128 was well cultured and grown in LB liquid medium. The vegetative cells were rod-shaped, with blunt ends and peritrichous flagella. The sporangium did not swell, the spores were lateral, and paraspore crystals were formed at the other end. After 24 hours of culture, the spores and paraspore crystals fell off in large quantities, and the crystal morphology was mainly rhombuses of different sizes. The acetylmethyl carbinol test, lecithinase test, and starch hydrolysis test were positive, and the bacterial film could be formed, but sucrose and mannose could not be utilized.

[0011] A solid fermentation culture method for Bacillus thuringiensis Bt-YCP-128 using tea seed meal as a main substrate, comprising inoculating Bacillus thuringiensis Bt-YCP-128 into a solid fermentation medium for solid fermentation;

[0012] The formula of the solid fermentation medium includes: support material, corn flour or soybean flour, tea seed meal and water;

[0013] The mass ratio of the supporting material, corn flour or soybean flour, and tea seed meal is: 2-5:1-2:2-6;

[0014] The supporting material comprises: a mixture of one or more of bran, peat, coconut shell, rice husk, vermiculite, perlite and / or sawdust;

[0015] The water accounts for 40-70% of the total mass of the supporting material, corn flour or soybean flour and oil tea meal.

[0016] In the above method, preferably, the water contains 0.5-1.5% potassium salt;

[0017] In the above method, preferably, the solid-state fermentation method is flat-laying static fermentation, the culture medium is spread flat in the container, the height of the spread is 2-5 cm, the inoculation amount of Bacillus thuringiensis Bt-YCP-128 is 5-10% of the strain, and the fermentation is carried out at 25-30°C for 3-7 days

[0018] The protection scope of the present invention also includes the use of the solid fermentation product prepared by the above-mentioned culture method in preventing and controlling lepidopteran pests;

[0019] Application of the solid fermentation product prepared by the above-mentioned culture method in the preparation of biological agents for controlling lepidopteran pests;

[0020] In the above applications, the lepidoptera pests include: cabbage looper, rice sheathworm, looper, pine caterpillar, tobacco budworm, corn borer, rice leaf folder, etc.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] 1. The present invention obtained a strain of Bacillus thuringiensis from a microbial library through repeated screening, which can be used for solid fermentation with tea seed meal as the main raw material, and the number of fermented spores can be more than 8.0╳10 9 Therefore, the technical solution of the present invention provides a new direction for the recycling of camellia oleifera meal. The camellia oleifera meal is used as the main fermentation raw material to produce biological pesticides. This treatment method can reduce the discharge of agricultural waste and the use of chemical pesticides, and is very environmentally friendly.

[0023] 2. The present invention has a simple operation method, uses a single strain of bacteria, does not require large-scale equipment, and has cheap and readily available raw materials. The solid-state fermentation culture obtained by the present invention can be directly used as a biological control agent for lepidopteran pests without further treatment, and is suitable for large-scale promotion and application. DETAILED DESCRIPTION

[0024] The present invention is further described below by way of examples, so that the technical personnel in the field can more fully understand the present invention, but the present invention is not limited to the following examples. The technical solutions described in the present invention, unless otherwise specified, are conventional solutions in the art; the reagents or materials, unless otherwise specified, are all from commercial channels.

[0025] Embodiment 1:

[0026] Bacillus thuringiensis screening:

[0027] (1) Preparation of Bacillus thuringiensis inoculum: Bacillus thuringiensis (Bt) was obtained from the strain collection of this center, with a total of 215 strains. The frozen Bacillus thuringiensis was transferred to LB medium plates (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 20 g / L), and then placed in a 37°C incubator for 24 h. The colonies were picked and placed in LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, inoculum size 5%), and cultured at 37°C and 180 r / min for 12 h to prepare a bacterial suspension (10 8 cfu / mL).

[0028] (2) Bacillus solid fermentation: Bran, corn flour and tea seed meal (provided by Anhui Qianshan County Meilin Ecological Agriculture Development Co., Ltd.) powders were sieved through a 20-mesh sieve, mixed at a mass ratio of 1:1:1, added with 0.5 times the total mass of water, and divided into 150 mL conical flasks, spread to a height of 2 cm, and sterilized at 121°C for 30 min. After the substrate was cooled, 5% of the prepared bacterial suspension was added, mixed, and placed at 28°C for fermentation.

[0029] (3) Observation of Bacillus growth: Samples grown in solid fermentation for 5 days were taken for microscopic observation and spore counting to determine the Bacillus thuringiensis strains that are adapted to tea-leaf meal as the matrix. The spore count was calculated as follows: 10.00 g of solid culture was accurately weighed and placed in a 500 mL conical flask containing 90 mL of sterile water (0.1% Tween-80) and a small amount of glass beads. The flask was placed on a shaker and shaken at 180 r / min for 60 min to obtain a spore suspension. The spore count was determined by the dilution plate counting method. The suspension dilution was spread on the LB plate and placed in an 80°C water bath for 20 minutes to kill the vegetative bodies therein. Among the selected strains, about 5% of the strains could grow well on the solid culture medium with tea-leaf meal as the matrix and form a large number of spores (the number of spores exceeded 10 9 cfu / g, see Table 1), among which Bt-YCP-44 and Bt-YCP-128 had the largest number of spores. Bt-YCP-44 and Bt-YCP-128 were fermented repeatedly (5 times), and the number of spores of Bt-YCP-44 was less than 3 in two fermentations. ╳ 10 9 cfu / g, while the strain Bt-YCP-128 formed spores stably, and the number of spores exceeded 3╳10 in all five times. 9 cfu / g, which is more suitable for industrial production. Further fermentation process optimization research will be carried out on Bt-YCP-128.

[0030] Table 1 Production of some Bacillus spores

[0031]

[0032] Embodiment 2:

[0033] Screening of solid fermentation matrix:

[0034] (1) Preparation of Bacillus inoculum solution: Bacillus thuringiensis (Bt-YCP128) was screened and obtained in Example 1. The preparation method of Bacillus inoculum solution was the same as that in Example 1.

[0035] (2) Preparation of solid fermentation matrix: A total of 4 combinations were selected, and all components were mixed in the following proportions by weight:

[0036] Matrix No. 1: bran, corn flour and tea seed meal (1:1:1);

[0037] Matrix No. 2: bran, soybean powder and camellia oleifera meal (1:1:1);

[0038] Matrix No. 3: bran, soybean meal and camellia meal (1:1:1);

[0039] Matrix No. 4: bran, rice bran and tea oil meal (1:1:1).

[0040] Camellia oleifera meal was provided by Anhui Qianshan County Meilin Ecological Agriculture Development Co., Ltd. All materials were crushed and passed through a 20-mesh sieve for later use.

[0041] (3) Fermentation of strains: Add 0.5 times the weight of water to the prepared substrate and mix well, then spread it on a stainless steel shallow plate (20×30 cm) at a height of 1.5 cm. Sterilize all materials at 121°C for 30 min. After the substrate cools, add 5% (ml / g) of the prepared strain suspension and ferment it at room temperature of 28°C for 5 days, turning the substrate 2-3 times during the fermentation.

[0042] (4) Spore count: The statistical method was the same as that in Example 1. The fermentation results showed that Bacillus thuringiensis grew well with corn flour and soybean flour as carbon and nitrogen sources, and the spore counts were the largest, which were 5.2 and 1.3, respectively. ╳ 10 9 cfu / g,4.8 ╳ 10 9 cfu / g, soybean meal and rice bran were used as carbon and nitrogen sources, the growth of Bacillus thuringiensis was not stable, and they were not suitable as solid culture medium for Bacillus thuringiensis. Corn flour was selected for subsequent experiments due to its cost advantage.

[0043] Embodiment 3:

[0044] Screening and optimization of solid fermentation conditions

[0045] (1) Screening of fermentation substrate ratio:

[0046] Five mixing ratios (2:1:7, 3:1:6, 4:1:5, 5:1:4, 1:1:1) were set with bran, corn flour and tea-leaf meal as the matrix. Fermentation was carried out according to the solid fermentation method in Example 2. After 5 days, the number of spores in different component ratios was determined.

[0047] The fermentation results showed that as the proportion of camellia oleifera meal in the matrix increased, the number of spores gradually decreased. When the proportion of camellia oleifera meal increased to 70%, the number of spores was less than 1. ╳ 10 9 cfu / g, which does not meet the standard for the use of later biological pesticides. Therefore, in the formulation, the proportion of tea seed meal should not exceed 60%. When bran, corn flour and tea seed meal were prepared as fermentation matrix at a ratio of 3:1:6, 4:1:5, 5:1:4 and 1:1:1 respectively, after five days of solid-state fermentation, the number of Bt-YCP-128 spores was 4.3╳10 9 cfu / g, 5.2╳10 9 cfu / g, 5.5╳10 9 cfu / g, 5.6╳10 9 cfu / g.

[0048] (2) Screening of fermentation water: Solid fermentation was carried out with a substrate ratio of bran, corn flour and tea meal (4:1:5), and water (0.7, 0.6, 0.5, 0.4, 0.3) times the weight of the substrate was added respectively. After mixing, the mixture was sterilized under high pressure. After 7 days of fermentation, the number of spores at different water addition ratios was measured. The fermentation results showed that too much water in the fermentation feed had high viscosity, which was not conducive to air circulation. Too little water in the fermentation feed could not meet the growth requirements of the strain. When the feed-water ratio was 0.5 and 0.6, the spore yield was the highest, with the number of spores being 5.5 and 5.3, respectively. ╳ 10 9 cfu / g, increasing or decreasing the addition of feed water will lead to a significant decrease in the number of spores.

[0049] (3) Investigation of pile height: After evenly mixing bran, corn flour and tea-leaf meal (4:1:5), add water 0.5 times the weight of the matrix, spread it flat on a stainless steel shallow plate (20×30cm), set different paving heights (2cm, 3cm, 4cm, 5cm, 6cm), sterilize it under high pressure, and use an inoculation amount of 5%. After 7 days of fermentation, measure the number of spores at different paving heights. The higher the pile height, the more spores are harvested per unit area. However, increasing the pile height is not conducive to the transfer of substances and affects fermentation. When the pile height exceeds 5cm, the number of spores decreases significantly. In the actual production process, the pile height should not exceed 5cm. The pile heights are 2cm, 3cm, 4cm, and 5cm, respectively. After seven days of fermentation, the number of spores obtained is 5.4 ╳ 10 9 cfu / g, 5.2╳10 9 cfu / g, 5.2╳10 9 cfu / g and 4.4╳10 9 cfu / g.

[0050] (4) Screening of inorganic salts: After bran, corn flour and tea seed meal (4:1:5) are evenly mixed, water 0.5 times the weight of the matrix is ​​added, and different inorganic salts (sodium chloride, calcium carbonate, magnesium sulfate, potassium dihydrogen phosphate) are added to the water, with the content of each being 1% (i.e., the mass percentage of inorganic salt to water). The mixture is spread flat on a stainless steel shallow plate (20×30 cm) with a spread height of 4 cm. The mixture is sterilized under high pressure with an inoculation amount of 5%. After 7 days of fermentation, the number of Bt-YCP-128 spores under different concentrations of inorganic salts is determined.

[0051] The experimental results show that sodium salt, calcium salt and magnesium salt have little effect on spore production. After adding sodium chloride, calcium carbonate and magnesium sulfate, the spore production was 5.1, 5.4 and 5.8 respectively. ╳ 10 9 cfu / g, adding potassium salt (potassium dihydrogen phosphate) can significantly increase the number of spores to 8.3 ╳ 10 9 cfu / g.

[0052] (5) Investigation of fermentation time: After bran, corn flour and tea seed meal (4:1:5) were mixed evenly, 0.5 times the weight of the matrix was added with water (containing 1% potassium dihydrogen phosphate), and the mixture was spread on a stainless steel shallow plate (20×30 cm) with a height of 4 cm. The mixture was sterilized by high pressure and fermented for 3, 4, 5, 6, and 7 days respectively. The number of spores at different fermentation days was measured. Bacillus grew rapidly and could reproduce in large numbers on the 3rd day and began to form spores (the number of spores was 1.9 ╳ 10 9 cfu / g, and then the spore count increased rapidly (the spore count on the fourth day was 4.2╳ 10 9 cfu / g), and on the 5th day, the spore count was about 8.5 ╳ 10 9 cfu / g, the spore count tended to be stable on the 6th and 7th days (the spore count was 8.6╳10 9 cfu / g, 8.3╳10 9 cfu / g). To save production costs, the solid fermentation time should generally not exceed 7 days, with 5 days being the best.

[0053] Embodiment 4:

[0054] Insecticidal activity assay:

[0055] (1) Fermentation of Bacillus thuringiensis: Bacillus thuringiensis was transferred to LB liquid medium and cultured at 37°C and 180 rpm for 12 h to prepare a bacterial suspension (10 8 cfu / mL). Bran, corn flour and tea seed meal (4:1:5) were used as fermentation matrix, crushed and sieved, 0.5 times the weight of water (containing 1% potassium dihydrogen phosphate) was added, mixed evenly, and spread on a stainless steel shallow plate (20×30cm) with a height of 4cm. All materials were sterilized at 121℃ for 30min. After the matrix was cooled, 5% of the prepared bacterial suspension was added, mixed evenly, and fermented at room temperature of 28℃ for 5 days. Solid fermentation powder of Bacillus thuringiensis (spore count 8.6╳10 9 cfu / g).

[0056] (2) Determination of activity against diamondback moth: Referring to the National Standard of the People's Republic of China GB / T 19567.1-2004, healthy 2nd-instar diamondback moth larvae with uniform growth were selected as test insects, and the toxicity of the solid fermentation product of Bacillus thuringiensis was determined.

[0057] Bacillus thuringiensis solid fermentation powder (spore count 8.6 ╳ 10 9cfu / g) was dissolved in 1% Tween aqueous solution and mixed with feed in proportion. A total of 7 concentration gradients were set up, and three replicates were set up for each gradient. The group fed with mixed feed containing the same volume of 1% Tween aqueous solution was used as the control. After 4 hours, the number of dead insects of different concentrations of the drug was checked and counted. The criterion for judging the dead test insects was that the insect body was lightly touched with a brush, and it was considered dead if it could not crawl normally. The corrected mortality rate of each treatment was calculated based on the statistical data. If the control mortality rate was <5%, no correction was required; if the control mortality rate was between 5% and 20%, correction was performed according to the formula: Corrected mortality rate = (treatment mortality rate – blank control mortality rate) / (1 – blank control mortality rate) * 100; if the control mortality rate was >20%, the experiment needed to be repeated. The results showed that Bacillus thuringiensis solid fermentation powder had high toxic activity against Plutella xylostella, and the LC 50 The value is 0.28 mg / g, therefore, the solid fermentation product of the present invention can be directly used as a biological pesticide mother drug.

[0058] Table 1 Toxic activity of solid fermentation powder of Bacillus thuringiensis Bt-YCP128 against larvae of Spodoptera frugiperda

[0059]

[0060] Embodiment 5:

[0061] Insecticidal field experiment:

[0062] (1) Preparation of Bacillus thuringiensis oil suspension: After the white oil emulsifier (the amount added is 10% of the total mass of the suspension) and No. 9 white oil (the amount added is 59.5% of the total mass of the suspension) are mixed evenly, Bacillus thuringiensis solid fermentation powder (the amount added is 30% of the total mass of the suspension) and organic bentonite (the amount added is 0.5% of the total mass of the suspension) are added in sequence, and all the materials are evenly dispersed using a high-speed homogenizer. The homogenized material is put into a sand mill and ground for 30 minutes to obtain an oil suspension sample (2.5 ╳ 10 9 cfu / ml).

[0063] (2) Experimental design: The drug was applied from the peak hatching period of Plutella xylostella eggs to the early larval stage. The dosage of Bacillus thuringiensis oil suspension was set at 200 ml / mu, 400 ml / mu, and 600 ml / mu. At the same time, a clear water control group was set. The drug application method and water dosage were conventional spray method, 30 L / mu.

[0064] (3) Survey method: Randomly mark 10 cabbage plants in each plot, with 4 replicates for each treatment, and investigate the number of live larvae of different ages on the whole cabbage plant. Investigate the insect population base before applying the pesticide, and investigate the number of residual insects on the fixed plants and crop safety 4 and 7 days after applying the pesticide. Based on the survey results, calculate the control effect according to the following formula: Control effect = (total number of live insects before application - total number of live insects after application) / total number of live insects before application × 100%.

[0065] (4) Experimental results: The experimental results are shown in Table 2. The results show that all dosages can significantly reduce the number of pests. The 7-day control rate of 600 ml / mu can reach about 89%, which has a good control effect. In actual use, it is recommended to use a dosage of 600 ml / mu.

[0066] Table 2 Control effects of different treatments on Plutella xylostella

[0067]

Claims

1. An isolated Bacillus thuringiensis strain Bt-YCP-128, the deposit number of the strain is: CCTCC NO: M20241924.

2. A solid fermentation culture method for Bacillus thuringiensis Bt-YCP-128 using tea seed meal as a main substrate, comprising inoculating Bacillus thuringiensis Bt-YCP-128 into a solid fermentation medium for solid fermentation. The deposit number of the strain is: CCTCC NO: M20241924.

3. The formula of the solid fermentation medium comprises: Support material, corn flour or soybean flour, tea seed meal and water; The mass ratio of the supporting material, corn flour or soybean flour, and tea seed meal is: 2-5:1-2:2-6; The supporting material comprises: a mixture of one or more of bran, peat, coconut shell, rice husk, vermiculite, perlite and / or sawdust; The water accounts for 40-70% of the total mass of the supporting material, corn flour or soybean flour and tea seed meal.

4. The method according to claim 2, characterized in that: The water contains 0.5-1.5% potassium salt.

5. The method according to claim 2, characterized in that: The solid-state fermentation method is flat-lay static fermentation, that is, the culture medium is spread flat in a container, the height of the spread is 2-5 cm, the inoculation amount of Bacillus thuringiensis Bt-YCP-128 is 5-10% of the strain, and the fermentation is carried out at 25-30°C for 3-7 days.

6. Use of the solid-state fermentation product prepared by the cultivation method according to claim 2 in controlling lepidopteran pests.

7. Use of the solid-state fermentation product prepared by the cultivation method according to claim 2 in the preparation of biological agents for controlling lepidopteran pests.

8. The use according to claim 5 or 6, wherein the lepidoptera pests include: Cabbage looper, rice stem borer, looper, pine caterpillar, tobacco budworm, corn borer, and rice leaf folder.

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