Compound biocontrol bacterium immobilized microspheres as well as preparation method and application thereof

Abstract

The invention relates to a compound biocontrol bacterium immobilized microsphere as well as a preparation method and application thereof, and belongs to the technical field of biocontrol. In order to solve the problem of lack of compound biocontrol bacteria and an effective immobilization method in the prior art, the invention provides a compound biocontrol bacteria immobilized microsphere, which is formed by embedding compound biocontrol bacteria in sodium alginate-calcium chloride, and the compound biocontrol bacteria comprise bacillus velezensis and spiropolyspora pink. The bacillus velezensis and the spiropolyspora pink have an excellent inhibition effect on botrytis cinerea after being compounded, and the compound bacterial liquid is immobilized in the calcium alginate microspheres by virtue of an immobilization technology, so that the activity of microorganisms can be more effectively maintained, and the stability and the effect of biological control are remarkably improved. According to the invention, the compounding proportion and the immobilization preparation process of the biocontrol bacteria are optimized, so that the antibacterial effect of the compounded biocontrol bacteria immobilized microspheres reaches 76.19%, which is superior to that of a compounded microbial inoculum and a traditional chemical pesticide.

Description

Technical Field

[0001] The invention belongs to the technical field of biocontrol, and in particular relates to a composite biocontrol bacteria immobilized microsphere and a preparation method and application thereof. Background Art

[0002] Biocontrol agents play many important roles in agricultural production. They can not only inhibit pathogens, increase plant immunity, promote plant growth and improve fruit quality, but also protect the environment and reduce the use of chemical pesticides. However, most biocontrol agents often have weak colonization ability after being applied in the field and cannot form sufficient biological communities, which reduces the biocontrol effect. Colonized biocontrol bacteria are also easily affected by environmental factors such as temperature, humidity, light, and soil pH, resulting in limited survival time and stability, making it difficult to continuously exert biocontrol effects. In particular, the disease resistance of a single biocontrol agent is more susceptible to the adaptive changes and drug resistance of pathogens. Compound biocontrol agents expand the control spectrum through the synergistic effect between different types of biocontrol bacteria, thereby enhancing the biocontrol effect.

[0003] Immobilization can significantly improve the stability of biocontrol bacteria, making them more adaptable to the complex and changing environmental conditions in the field. However, for compound biocontrol agents, different strains may have different adaptability to immobilization carriers, and the selection of suitable immobilization carriers is crucial to ensure the stability and activity of the strains. The existing technology lacks systematic research in the screening, compounding ratio, and immobilization methods of compound biocontrol bacteria, resulting in the inability of biocontrol agents to fully exert their control effects. Summary of the invention

[0004] In order to solve the problem that the prior art lacks compound biocontrol bacteria and effective immobilization methods, the present invention provides compound biocontrol bacteria immobilized microspheres and a preparation method and application thereof.

[0005] The technical solution of the present invention:

[0006] The invention discloses a composite biocontrol bacteria immobilized microsphere, which is composed of composite biocontrol bacteria embedded in sodium alginate-calcium chloride. The composite biocontrol bacteria comprises Bacillus velezensis and Clonostachys rosea. The preservation number of Bacillus velezensis is CGMCC No.25497; the strain preservation number of Clonostachys rosea is CGMCC No.1977.

[0007] Furthermore, the ratio of the number of live bacteria of the Bacillus velez to that of the pink spiral polysporus is 1-2:1-2.

[0008] A method for preparing composite biocontrol bacteria immobilized microspheres comprises the following steps:

[0009] Step 1: inoculating Bacillus Velez in a liquid culture medium to obtain a Bacillus Velez bacterial solution, inoculating Pink Spiral Polysporus in a solid culture medium to obtain Pink Spiral Polysporus hyphae; mixing the obtained Bacillus Velez bacterial solution with the Pink Spiral Polysporus hyphae to obtain a composite bacterial solution;

[0010] Step 2: preparing a sodium alginate solution, mixing the composite bacterial solution obtained in step 1 with the sodium alginate solution to obtain a mixed system;

[0011] Step 3: Use a sterile syringe equipped with a needle to drop the mixed system obtained in step 2 into the calcium chloride solution drop by drop, and obtain the composite biocontrol bacteria immobilized microspheres after hardening treatment and washing.

[0012] Furthermore, the liquid culture medium in step 1 is LB culture medium, the culture conditions of Bacillus velez is culturing at 28°C and 200rpm for 24 hours, the solid culture medium is PDA culture medium, and the culture conditions of Polysporus rosiglita are culturing at 28°C for 7 days.

[0013] Furthermore, the number of viable bacteria of Bacillus velez in the composite bacterial solution in step 1 is 1×10 6 CFU / mL, the viable count of the pink spiral polysporon is 1×10 6 CFU / mL.

[0014] Furthermore, in step 2, the mass concentration of sodium alginate in the mixed system is 3-5%, and the mass concentration of the composite bacterial solution in the mixed system is 20-40%.

[0015] Furthermore, in step 3, the mass concentration of the calcium chloride solution is 1-3%, the temperature of the hardening treatment is 4° C. for 2-6 hours, and the washing is firstly washed with physiological saline and then rinsed with sterile water.

[0016] Furthermore, the average particle size of the composite biocontrol bacteria immobilized microspheres obtained in step three is 3.5 mm.

[0017] The invention discloses an application of composite biocontrol bacteria immobilized microspheres in the fields of agricultural disease prevention and control, plant growth promotion, soil improvement, environmental pollution control or food preservation and antiseptic treatment.

[0018] Furthermore, the agricultural disease control is the control of tomato gray mold.

[0019] Beneficial effects of the present invention:

[0020] The compounding of Bacillus Velezii and Pink Spiral Polysporon in the present invention has an excellent inhibitory effect on tomato gray mold. The present invention fixes the compounded bacterial solution in calcium alginate microspheres through immobilization technology, which significantly improves the stability and effect of biological control. The present invention optimizes the compounding ratio of biocontrol bacteria and the preparation process of compounded biocontrol bacteria immobilized microspheres through systematic research, so that the antibacterial effect of compounded biocontrol bacteria immobilized microspheres reaches 76.19%, which is better than the compounded bacterial agent itself and traditional chemical pesticides.

[0021] The composite biocontrol bacteria immobilized microspheres provided by the present invention provide a favorable living environment for the composite biocontrol bacteria, can more effectively maintain the activity of microorganisms, and prolong the storage validity period, thereby ensuring the stability and durability of the composite bacterial agent in practical applications.

[0022] The composite biocontrol bacteria immobilized microspheres provided by the present invention not only have significant antibacterial effects, but also can promote the growth of tomato plants. Experiments have shown that the composite bacterial agent and its immobilized microspheres can significantly improve the germination rate and radicle length of tomato seeds, while increasing morphological indicators such as plant height, stem thickness, fresh weight and dry weight of tomato seedlings. In addition, it can also significantly improve the photosynthesis efficiency of tomato plants, increase the SOD and POD enzyme activities of plants, enhance defense mechanisms, and further enhance their disease resistance, yield and quality.

[0023] Compared with traditional chemical pesticides, the biological agent and its immobilization technology used in the present invention are more environmentally friendly and reduce pesticide residues and environmental pollution problems. This is in line with the current trend of sustainable agricultural development and is conducive to protecting the ecological environment and food safety.

[0024] The preparation process of the composite biocontrol bacteria immobilized microspheres of the present invention is relatively simple, and the required raw materials are easily available and low in cost. By optimizing the preparation conditions, the microspheres can be ensured to have a uniform particle size, high stability, and be easy to store and transport. This is conducive to the promotion and application of the technology and provides a new solution for agricultural disease prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a real photo of the composite biocontrol bacteria immobilized microspheres prepared in Example 1;

[0026] Figure 2 The comparison diagram of the antibacterial bands of the composite bacterial solution prepared in step 1 of Example 1, Example 2 and Example 3, A is Example 2, B is Example 1, and C is Example 3;

[0027] Figure 3 The optimal prediction result of the BOX-BEHNKEN model for the number of viable bacteria in Example 4;

[0028] Figure 4The following is a comparison chart of the influence of various factors on the immobilization rate of the composite biocontrol bacteria immobilized microspheres in Example 4, wherein the ordinates are all immobilization rates, the abscissas of A are calcium chloride concentration and sodium alginate concentration, the abscissas of B are the amount of bacteria and the sodium alginate concentration, the abscissas of C are the hardening time and the sodium alginate concentration, the abscissas of D are the hardening time and the calcium chloride concentration, the abscissas of E are the amount of bacteria and the calcium chloride concentration, and the abscissas of F are the amount of bacteria and the hardening time;

[0029] Figure 5 These are morphological photos of tomato seedlings in each group in Example 5;

[0030] Figure 6 is the root system diagram of each group of tomato seedlings in Example 5;

[0031] Figure 7 This is a comparison chart of the photosynthetic efficiency of each group of tomato seedlings in Example 5, A is the Fv / Fm capacity, B is the photosynthetic system II Y (NPQ) value, C is the photochemical quenching, and D is the ETR (II) capacity;

[0032] Figure 8 This is a comparison chart of SOD activity and POD activity of each group of tomato seedlings in Example 5;

[0033] Fig. 9 This is a comparison chart of the control effects of various groups of tomato seedlings on tomato gray mold in Example 6, A is the pyrimethanil group, B is the blank control group, C is the blank microsphere group, D is the composite bacterial solution group, E is the composite biocontrol bacteria immobilized microsphere group, and F is the control biocontrol bacteria group. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described below in conjunction with the embodiments, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the protection scope of the present invention. The process equipment or devices not specifically noted in the following embodiments are all conventional equipment or devices in the art. If not specifically specified, the raw materials used in the embodiments of the present invention can be obtained commercially; if not specifically specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0035] Example 1

[0036] This embodiment provides a composite biocontrol bacteria immobilized microsphere and a preparation method thereof.

[0037] The composite biocontrol bacteria immobilized microspheres of this embodiment are composed of composite biocontrol bacteria embedded in sodium alginate-calcium carbonate, and the composite biocontrol bacteria include Bacillus velezensis and Polysporus pinkus at a live bacteria ratio of 1:1.

[0038] The Bacillus Velez in this embodiment was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on August 5, 2022, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 25497.

[0039] In this embodiment, the pink spiral polysporus was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, and the culture collection number is: CGMCC No.1977.

[0040] The preparation method of the composite biocontrol bacteria immobilized microspheres of this embodiment comprises the following steps:

[0041] Step 1: Inoculate Bacillus Velez in LB liquid culture medium, and culture at 28°C and 200 rpm for 24 h to obtain Bacillus Velez bacterial solution; inoculate Pink Spiral Polysporon in PDA solid culture medium, and culture at 28°C for 7 days to obtain Pink Spiral Polysporon hyphae; mix the obtained Bacillus Velez bacterial solution with the pink Spiral Polysporon hyphae to obtain a composite bacterial solution, wherein the number of viable Bacillus Velez in the composite bacterial solution is 1×10 6 CFU / mL, the viable count of the pink spiral polysporon CR is 1×10 6 CFU / mL.

[0042] Step 2: preparing a sodium alginate solution, mixing the composite bacterial solution obtained in step 1 with the sodium alginate solution to obtain a mixed system, wherein the mass concentration of sodium alginate in the mixed system is 3.9%, and the mass concentration of the composite bacterial solution in the mixed system is 28%;

[0043] Step 3: Use a sterile syringe equipped with a needle to dropwise add the mixed system obtained in step 2 into a 1.8% calcium chloride solution, harden at 4°C for 4.8 hours, collect the hardened microspheres, wash them in sterile saline, and then rinse with sterile water to obtain the composite biocontrol bacteria immobilized microspheres, which are stored at 4°C for use.

[0044] Example 2

[0045] This embodiment provides a composite biocontrol bacteria immobilized microsphere and a preparation method thereof.

[0046] The composite biocontrol bacteria immobilized microspheres of this embodiment are composed of composite biocontrol bacteria embedded in sodium alginate-calcium carbonate, and the composite biocontrol bacteria include Bacillus velezensis and Polysporus pinkus at a live bacteria count ratio of 2:1.

[0047] The Bacillus Velez in this embodiment was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on August 5, 2022, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 25497.

[0048] In this embodiment, the pink spiral polysporus was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, and the culture collection number is: CGMCC No.1977.

[0049] The preparation method of the composite biocontrol bacteria immobilized microspheres of this embodiment comprises the following steps:

[0050] Step 1: Inoculate Bacillus Velez in LB liquid culture medium, and culture at 28°C and 200 rpm for 24 h to obtain Bacillus Velez bacterial solution; inoculate Pink Spiral Polysporon in PDA solid culture medium, and culture at 28°C for 7 days to obtain Pink Spiral Polysporon hyphae; mix the obtained Bacillus Velez bacterial solution with the pink Spiral Polysporon hyphae to obtain a composite bacterial solution, wherein the number of viable Bacillus Velez in the composite bacterial solution is 2×10 6 CFU / mL, the viable count of the pink spiral polysporon CR is 1×10 6 CFU / mL.

[0051] Step 2: preparing a sodium alginate solution, mixing the composite bacterial solution obtained in step 1 with the sodium alginate solution to obtain a mixed system, wherein the mass concentration of sodium alginate in the mixed system is 3.9%, and the mass concentration of the composite bacterial solution in the mixed system is 28%;

[0052] Step 3: Use a sterile syringe equipped with a needle to dropwise add the mixed system obtained in step 2 into a 1.8% calcium chloride solution, harden at 4°C for 4.8 hours, collect the hardened microspheres, wash them in sterile saline, and then rinse with sterile water to obtain the composite biocontrol bacteria immobilized microspheres, which are stored at 4°C for use.

[0053] Example 3

[0054] This embodiment provides a composite biocontrol bacteria immobilized microsphere and a preparation method thereof.

[0055] The composite biocontrol bacteria immobilized microspheres of this embodiment are composed of composite biocontrol bacteria embedded in sodium alginate-calcium carbonate, and the composite biocontrol bacteria include Bacillus velezensis and Polysporus pinkus at a live bacteria ratio of 1:2.

[0056] The Bacillus Velez in this embodiment was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on August 5, 2022, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 25497.

[0057] In this embodiment, the pink spiral polysporus was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, and the culture collection number is: CGMCC No.1977.

[0058] The preparation method of the composite biocontrol bacteria immobilized microspheres of this embodiment comprises the following steps:

[0059] Step 1: Inoculate Bacillus Velez in LB liquid culture medium, and culture at 28°C and 200 rpm for 24 h to obtain Bacillus Velez bacterial solution; inoculate Pink Spiral Polysporon in PDA solid culture medium, and culture at 28°C for 7 days to obtain Pink Spiral Polysporon hyphae; mix the obtained Bacillus Velez bacterial solution with the pink Spiral Polysporon hyphae to obtain a composite bacterial solution, wherein the number of viable Bacillus Velez in the composite bacterial solution is 1×10 6 CFU / mL, the viable count of the pink spiral polysporon CR is 2×10 6 CFU / mL.

[0060] Step 2: preparing a sodium alginate solution, mixing the composite bacterial solution obtained in step 1 with the sodium alginate solution to obtain a mixed system, wherein the mass concentration of sodium alginate in the mixed system is 3.9%, and the mass concentration of the composite bacterial solution in the mixed system is 28%;

[0061] Step 3: Use a sterile syringe equipped with a needle to dropwise add the mixed system obtained in step 2 into a 1.8% calcium chloride solution, harden at 4°C for 4.8 hours, collect the hardened microspheres, wash them in sterile saline, and then rinse with sterile water to obtain the composite biocontrol bacteria immobilized microspheres, which are stored at 4°C for use.

[0062] The composite bacterial solution prepared in step 1 of Example 1, Example 2 and Example 3 was subjected to a plate antibacterial test on tomato gray mold pathogens. The results are as follows: Figure 2 And as shown in Table 1.

[0063] Table 1

[0064]

[0065] It can be seen from the data in Table 1 that when the ratio of the number of live bacteria of Bacillus velez to that of Polysporum rosenbergii is 1:1, the width of the inhibition zone is the largest, indicating that the compound antibacterial effect at this ratio is the best.

[0066] Example 4

[0067] The immobilization rate of immobilized microspheres refers to the efficiency of fixing the compound biocontrol bacteria on the microsphere carrier by the immobilization technology. In order to further explore the important role of the proportion of carrier materials in the preparation process of compound biocontrol bacteria immobilized microspheres, this embodiment adopts the BOX-BEHNKEN model for analysis, and a secondary regression analysis is performed accordingly. Through the BOX-BEHNKEN model analysis, this embodiment obtains the regression equations of the four key factors of sodium alginate concentration (A), calcium chloride concentration (B), bacterial dosage (C) and hardening time (D), which are specifically:

[0068] Y=90.22-4.82A-2.50B+3.07C-0.28D-1.09AB+3.46AC+0.063AD+0.11BC+0.080BD-1.72CD-15.46A2-5.20B2-3.16C2-3.70D2

[0069] The F value of the model is as high as 33.67, and the overall P value is less than 0.0001, reaching a very significant level. At the same time, the values ​​of R2adj are close to 1, 0.9712 and 0.9423, respectively, and the difference between R2pred is less than 0.2. These statistical indicators together show that the model can more accurately reflect the relationship between the four factors alone or their interactions and the fixation rate.

[0070] In addition, the coefficient of variation (CV%) of the model is 2.69%, which is far below the threshold of 10%, further proving the reliability and predictive ability of the model. Using Design-expert 10 software for analysis and prediction, this embodiment obtained the optimal conditions for the immobilization rate of each factor: the amount of bacteria was 28.33%, the hardening time was 4.881 hours, the sodium alginate concentration was 3.903%, and the calcium chloride concentration was 1.860%. Under these optimized conditions, the predicted value of the immobilization rate of the composite biocontrol bacteria immobilized microspheres was as high as 90.224%.

[0071] However, considering the experimental error and the feasibility of actual operation, the present embodiment makes appropriate corrections to these immobilization conditions: the amount of bacteria is adjusted to 28%, the hardening time is set to 4.8 hours, the sodium alginate concentration is set to 3.9%, and the calcium chloride concentration is set to 1.8%. Based on these corrected conditions, the present embodiment conducts a verification test. The results of the three tests show that the immobilization rates are 87.34%, 90.28% and 91.04% respectively, and the average immobilization rate is 89.55%, which is highly consistent with the predicted model value of 90.224%.

[0072] In order to more intuitively display the relationship between the variables, this embodiment also generates a three-dimensional surface diagram of all variables, such as Figure 4As shown. These charts reveal that the relationship between calcium chloride concentration, hardening time and bacterial dosage is approximately circular, indicating that the interaction between them is not significant. The effect of bacterial dosage on the immobilization rate of composite biocontrol bacteria immobilized microspheres is slightly greater than that of hardening time, and the contour map shows that there is a more obvious interaction between hardening time and bacterial dosage. Based on the above analysis, this embodiment can conclude that the order of the influence of the four conditions on the microsphere immobilization rate is: sodium alginate concentration>calcium chloride concentration>bacterial dosage>hardening time.

[0073] Example 5

[0074] This example investigates the growth-promoting effect of composite biocontrol bacteria immobilized microspheres on tomatoes.

[0075] The experiment in this example has a total of 5 treatments, namely the composite biocontrol bacteria immobilized microsphere group prepared in Example 1, the composite bacterial solution group obtained in step 1 of Example 1, the blank control CK group, the blank microsphere group and the control biocontrol bacteria group, wherein the control biocontrol bacteria is Bacillus siamensis, which is preserved by the horticultural crop biotechnology research group of Northeast Agricultural University.

[0076] The specific experimental method of this embodiment is as follows:

[0077] After sowing the tomato seeds, they were cultured for 14 days, and then the corresponding reagents were added 1 cm from the roots of the seedlings in each group:

[0078] (1) Add about 45 to 55 microspheres of immobilized compound biocontrol bacteria to the seedlings 1 cm from the root of the immobilized compound biocontrol bacteria microspheres group.

[0079] (2) Add 1 mL of the composite bacterial solution at 1 cm from the root of the seedlings in the composite bacterial solution group. The number of viable Bacillus Velezii in the composite bacterial solution was 1×10 6 CFU / mL, the viable count of the pink spiral polysporon CR is 1×10 6 CFU / mL.

[0080] (3) Add 1 mL of sterile water 1 cm from the root of the seedlings in the blank control CK group.

[0081] (4) Add about 45 to 55 blank microspheres without the compound biocontrol bacteria at 1 cm from the root of the seedlings in the blank microsphere group.

[0082] (5) Add 1 mL of conventional culture solution of Bacillus siamensis to the seedlings of the control biocontrol bacteria group 1 cm from the root. The number of viable bacteria of Bacillus siamensis in the culture solution is 1×10 6 CFU / mL.

[0083] After 30 days of cultivation, the morphological indexes of seedlings and related indexes of root system were evaluated in detail. Each group was repeated three times, and the stem diameter, plant height, dry weight and fresh weight of plants were measured. The results are as follows Figure 5 , Figure 6 And as shown in Table 2.

[0084] Table 2

[0085]

[0086]

[0087] As shown in Table 2, the compound bacterial solution group was superior to the control biocontrol bacteria group, blank microsphere group and blank control group in terms of plant height, stem thickness, fresh weight and dry weight. This result shows that the compound bacterial solution has a significant promoting effect on the growth of tomato seedlings. At the same time, the data of the blank microsphere group was also better than that of the blank control group, which suggests that the calcium ions in the microspheres have a certain weak promoting effect on the growth of tomato seedlings. It is worth noting that the performance of the compound biocontrol bacteria immobilized microsphere group in terms of plant height, stem thickness, fresh weight and dry weight exceeded that of other treatment groups, which fully demonstrates that the immobilized microspheres have a more significant growth-promoting effect.

[0088] In order to more fully understand the impact of each treatment group on the root system of tomato seedlings, the present embodiment also measures the relevant indexes of the root system and takes scanned images of the root system. As can be seen from the data, the tomato seedlings after the treatment of the compound bacterial liquid group are superior to the control biocontrol group in various root system indicators. At the same time, the performance of the blank microsphere group is also better than that of the blank control group, which further confirms the positive impact of the microsphere structure on the growth of the root system of tomato seedlings. Among all treatment groups, the effect of the compound biocontrol bacteria immobilized microsphere group is the most significant, and its root length, surface area, number of nodes and number of root tips are all about 2 times that of the blank control group. This result not only proves that the compound bacterial liquid and the compound biocontrol bacteria immobilized microsphere have good growth-promoting ability on the root system, but also shows that the growth environment of the compound biocontrol bacteria immobilized microsphere in the soil is better than that of the compound bacterial liquid group, thereby providing more favorable growth conditions for it.

[0089] This example investigates the photosynthesis efficiency of each group of tomato seedlings. The results are as follows Figure 7As shown in the figure, the maximum photosynthetic efficiency value Fv / Fm of each treatment group showed certain differences, which were 0.55, 0.77, 0.72, 0.57 and 0.63 respectively. Specifically, the photosynthetic efficiency of the immobilized microspheres of the compound biocontrol bacteria group and the compound bacterial solution group did not show significant differences, but both were significantly higher than the blank microsphere group, the blank control group and the control biocontrol bacteria group. At the same time, it is worth noting that there was no significant difference in the photosynthetic efficiency between the blank microsphere group and the blank control group. This phenomenon shows that the immobilized compound biocontrol bacteria microspheres can provide a more superior living environment for the compound biocontrol bacteria in the soil than the compound bacterial solution, so that the compound biocontrol bacteria can be enriched more in a certain space, thereby maintaining a higher number, thereby enhancing the maximum photosynthetic efficiency of the plant.

[0090] The higher the value of Y(NPQ), the stronger the light protection ability of the plant. The Y(NPQ) values ​​of each treatment group were 0.382, 0.544, 0.513, 0.418 and 0.473, respectively. Among them, the light protection ability of the compound biocontrol bacteria immobilized microsphere group and the compound bacterial solution group exceeded that of the blank control group, the blank microsphere group and the control biocontrol bacteria group. This shows that the compound bacterial solution can improve the light saturation ability of the plant, and the immobilized compound biocontrol bacteria microspheres are more capable in this regard.

[0091] The photochemical quenching values ​​of each treatment group were 0.67, 0.83, 0.87, 0.84 and 0.75, respectively. Among them, the photochemical quenching values ​​of the compound biocontrol bacteria immobilized microsphere group and the compound bacterial solution group were higher than those of the blank microsphere group, the blank control group and the control biocontrol bacteria group. It is particularly noteworthy that the photochemical quenching value of the compound biocontrol bacteria immobilized microsphere group was the highest among all treatment groups. This further illustrates that the compound bacterial solution has the ability to enhance the efficiency of plant photoelectron transfer, and the immobilized compound biocontrol bacteria microspheres are more powerful in this regard.

[0092] The higher the ETR(II) value, the better the efficiency of tomato seedlings in transferring light energy. The results showed that the relative electron transfer rates of the compound biocontrol bacteria immobilized microsphere group and the compound bacterial solution group were the best, at 62.78% and 58.34%, respectively. Both performed significantly better than the blank control group, blank microsphere group and control biocontrol bacteria group, and the immobilized compound biocontrol bacteria microspheres were more capable in this regard.

[0093] This example investigates the SOD and POD enzyme activities of tomato seedlings in each group. Figure 8As shown in the figure, the SOD enzyme activity and POD enzyme activity of the compound biocontrol bacteria immobilized microsphere group were higher than those of the compound bacterial solution group, the control biocontrol bacteria group, the blank control group and the blank microsphere group. This shows that the compound bacterial solution can increase the enzyme activity of plant SOD and POD and enhance the defense mechanism of the plant, and the immobilized compound biocontrol bacteria microspheres have better enhancement effect.

[0094] Example 6

[0095] This example investigates the greenhouse control effect of immobilized microspheres of composite biocontrol bacteria on tomato gray mold.

[0096] The experiment in this embodiment has a total of 6 treatments, namely the composite biocontrol bacteria immobilized microsphere group prepared in Example 1, the composite bacterial solution group obtained in step 1 of Example 1, the pyrimethanil control group, the blank control CK group, the blank microsphere group and the control biocontrol bacteria group, wherein the control biocontrol bacteria is Bacillus siamensis, which is preserved by the horticultural crop biotechnology research group of Northeast Agricultural University.

[0097] The specific experimental method of this embodiment is as follows:

[0098] After sowing the tomato seeds, they were cultured for 14 days, and then the corresponding reagents were added 1 cm from the roots of the seedlings in each group:

[0099] (1) Add about 45 to 55 microspheres of immobilized compound biocontrol bacteria to the seedlings 1 cm from the root of the immobilized compound biocontrol bacteria microspheres group.

[0100] (2) Add 1 mL of the composite bacterial solution at 1 cm from the root of the seedlings in the composite bacterial solution group. The number of viable Bacillus Velezii in the composite bacterial solution was 1×10 6 CFU / mL, the viable count of the pink spiral polysporon CR is 1×10 6 CFU / mL.

[0101] (3) Specific treatment method for the pyrimethanil control group: spray 1 mL of a solution prepared from 40% pyrimethanil powder at a concentration of 0.5 g / L using a leaf atomizer.

[0102] (4) Add 1 mL of sterile water 1 cm from the root of the seedlings in the blank control CK group.

[0103] (5) Add about 45 to 55 blank microspheres without the compound biocontrol bacteria at 1 cm from the root of the seedlings in the blank microsphere group.

[0104] (6) Add 1 mL of conventional culture solution of Bacillus siamensis to the seedlings of the control biocontrol bacteria group 1 cm from the root. The number of viable bacteria of Bacillus siamensis in the culture solution is 1×10 6 CFU / mL.

[0105] The spraying amount of the tomato gray mold pathogen spore suspension in the above treatment groups was the same, and the concentration of the tomato gray mold pathogen spore suspension sprayed was 1×10 5 cfu / mL.

[0106] The tomatoes treated in the above groups were placed in a 25℃ moisturizing culture and the disease conditions of the tomato plants were observed on the 20th day. Fig. 9 According to the classification standard of tomato gray mold, the leaf damage level was investigated, and the disease index and control effect were counted and calculated. The calculation formula is as follows:

[0107]

[0108]

[0109] The grading standards for tomato gray mold are shown in Table 3.

[0110] Table 3

[0111]

[0112] The greenhouse control effects of each group of tomatoes on tomato gray mold are shown in Table 4.

[0113] Table 4

[0114]

[0115] The test results showed that the disease severity index of tomato seedlings in the blank control group was as high as 77.78% after being infected with tomato gray mold, which was quite serious. In contrast, both the compound bacterial solution and the compound biocontrol bacteria immobilized microspheres showed stronger protective effects than the blank control group. It is worth mentioning that the protective effect of the compound biocontrol bacteria immobilized microspheres was more significant, reaching 76.19%.

Claims

1. A composite biocontrol bacteria immobilized microsphere, characterized in that: The invention is composed of sodium alginate-calcium chloride embedded composite biocontrol bacteria, wherein the composite biocontrol bacteria comprises Bacillus velezensis and Clonostachys rosea; the preservation number of the Bacillus velezensis is CGMCC No.25497; the strain preservation number of the Clonostachys rosea is CGMCC No.1977.

2. The composite biocontrol bacteria immobilized microspheres according to claim 1, characterized in that: The ratio of the number of live bacteria of the Bacillus velez to that of the pink spiral polysporus is 1-2:1-2.

3. A method for preparing the composite biocontrol bacteria immobilized microspheres as claimed in claim 1 or 2, characterized in that: The steps include: Step 1: inoculating Bacillus Velez in a liquid culture medium to obtain a Bacillus Velez bacterial solution, inoculating Pink Spiral Polysporus in a solid culture medium to obtain Pink Spiral Polysporus hyphae; mixing the obtained Bacillus Velez bacterial solution with the Pink Spiral Polysporus hyphae to obtain a composite bacterial solution; Step 2: preparing a sodium alginate solution, mixing the composite bacterial solution obtained in step 1 with the sodium alginate solution to obtain a mixed system; Step 3: Use a sterile syringe equipped with a needle to drop the mixed system obtained in step 2 into the calcium chloride solution drop by drop, and obtain the composite biocontrol bacteria immobilized microspheres after hardening treatment and washing.

4. The method for preparing the composite biocontrol bacteria immobilized microspheres according to claim 3, characterized in that: In step 1, the liquid culture medium is LB culture medium, the culture conditions of Bacillus Velezii are cultured at 28°C and 200rpm for 24 hours, the solid culture medium is PDA culture medium, and the culture conditions of Polysporus rosiglita are cultured at 28°C for 7 days.

5. The method for preparing the composite biocontrol bacteria immobilized microspheres according to claim 3 or 4, characterized in that: The number of viable bacteria of Bacillus Velez in the composite bacterial solution of step 1 is 1×10 6 CFU / mL, the viable count of the pink spiral polysporon is 1×10 6 CFU / mL.

6. The method for preparing the composite biocontrol bacteria immobilized microspheres according to claim 5, characterized in that: In step 2, the mass concentration of sodium alginate in the mixed system is 3-5%, and the mass concentration of the composite bacterial solution in the mixed system is 20-40%.

7. The method for preparing the composite biocontrol bacteria immobilized microspheres according to claim 8, characterized in that: In step 3, the mass concentration of the calcium chloride solution is 1-3%, the temperature of the hardening treatment is 4° C. for 2-6 hours, and the washing is firstly washed with physiological saline and then rinsed with sterile water.

8. The method for preparing the composite biocontrol bacteria immobilized microspheres according to claim 7, characterized in that: The average particle size of the composite biocontrol bacteria immobilized microspheres obtained in step 3 is 3.5 mm.

9. Use of the composite biocontrol bacteria immobilized microspheres as claimed in claim 1 or 2 in the fields of agricultural disease control, plant growth promotion, soil improvement, environmental pollution control or food preservation.

10. The use of the composite biocontrol bacteria immobilized microspheres according to claim 9 in the fields of agricultural disease control, plant growth promotion, soil improvement, environmental pollution control or food preservation and antiseptic treatment, characterized in that: The agricultural disease control is the control of tomato gray mold.

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