CN113430190B - Preparation method and application of composite microsphere containing paenibacillus polymyxa sodium alginate - Google Patents

Preparation method and application of composite microsphere containing paenibacillus polymyxa sodium alginate Download PDF

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CN113430190B
CN113430190B CN202110514297.3A CN202110514297A CN113430190B CN 113430190 B CN113430190 B CN 113430190B CN 202110514297 A CN202110514297 A CN 202110514297A CN 113430190 B CN113430190 B CN 113430190B
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paenibacillus polymyxa
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CN113430190A (en
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温立颖
薛新田
杨浩然
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Jiushengfeng Weifang Biotechnology Development Co ltd
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Abstract

A preparation method and application of a composite microsphere containing Paenibacillus polymyxa sodium alginate comprise the following steps: preparing a paenibacillus polymyxa bacterial suspension: carrying out logarithmic culture on paenibacillus polymyxa, and then centrifuging and collecting to obtain a bacterial suspension; preparing primary microcapsules: adding stabilizer and starch into the bacterial suspension, adding into sodium alginate to obtain intermediate solution, and placing the intermediate solution in low concentration CaCl 2 Preparing a primary microcapsule suspension; hardening the capsule: placing the primary microcapsule in high-concentration CaCl 2 Hardening is performed. The sodium alginate microspheres synthesized by sodium alginate and calcium chloride can adsorb spores of paenibacillus polymyxa, so that the spores are colonized in the propagation nest, the stress resistance of the spores is improved, the re-colonization of the spores is continued, and the sodium alginate microspheres can be used as the propagation nest and the propagation nest of the biocontrol strain. The addition of 4% gelatin in the process of preparing the microcapsule can change the disintegration time and the release speed of the capsule so as to adapt to different use occasions.

Description

Preparation method and application of composite microsphere containing paenibacillus polymyxa sodium alginate
Technical Field
The application relates to a preparation method and application of a composite microsphere containing paenibacillus polymyxa sodium alginate.
Background
Biological control is an effective and sustainable method for controlling plant diseases, and biocontrol bacteria such as trichoderma, pseudomonas, bacillus, streptomyces, certain endophytic bacteria and the like can obviously control various plant diseases, particularly plant soil-borne diseases. Among them, the gram-positive paenibacillus polymyxa and the like are particularly concerned in agricultural production due to high temperature resistance, drying resistance, radiation resistance and toxic chemical substances. The paenibacillus polymyxa mainly participates in nitrogen fixation, phosphorus dissolution, formation of plant hormones, antibacterial substances and the like, has broad-spectrum antagonistic activity, can effectively prevent various plant fungi, bacteria, nematodes and other diseases, and simultaneously promotes plant growth and improves crop yield. In recent years, there has been increasing interest in the role of Paenibacillus polymyxa as a source of rhizobacteria and potential bioactive metabolites that promote plant growth. However, the uncoated Paenibacillus polymyxa has the problems of difficult colonization, low survival rate, reduced stress resistance and the like in production practice.
Disclosure of Invention
In order to solve the problems, the application provides a preparation method of a composite microsphere containing paenibacillus polymyxa sodium alginate on one hand, and the preparation method comprises the following steps: preparing a paenibacillus polymyxa bacterial suspension: carrying out logarithmic culture on paenibacillus polymyxa, and then centrifuging and collecting to obtain a bacterial suspension; preparing primary microcapsules: adding stabilizer and starch into the bacterial suspension, adding into sodium alginate to obtain intermediate solution, and placing the intermediate solution in low concentration CaCl 2 Preparing a primary microcapsule suspension; hardening the capsule: placing the primary microcapsule in high-concentration CaCl 2 Hardening is performed.
Preferably, the strain number of the Paenibacillus polymyxa (Paenibacillus polymyxa) is BSYL-6, and the strain is preserved in China general microbiological culture Collection center (CGMCC) at 26.04.2020, and the preservation unit address is as follows: no. 3 of Xilu No.1 of Beijing, chaoyang, and the preservation number of the strain is CGMCC No.19736.
Preferably, the stabilizer is paraffin with low phase transition temperature, and the phase transition temperature of the paraffin is 25-30 ℃.
Preferably, the particle size of the stabilizer is 1 to 10 μm.
Preferably, 2-4wt% of starch and 1-3wt% of stabilizing agent are added into the bacterial suspension, and the mixture is shaken uniformly to fully adsorb bacteria to obtain an intermediate mixed solution.
Preferably, 2-4wt% of gelatin is also included in the bacterial suspension.
Preferably, the intermediate mixed solution is added into 1.5 to 2.5 percent sodium alginate solution which is 8 to 10 times of the intermediate mixed solution, the mixture is evenly stirred to obtain intermediate solution, and the intermediate solution is continuously extruded into low-concentration CaCl with the molar concentration of 0.08 to 0.12mol/L by a syringe pump in a dropwise manner 2 A primary microcapsule suspension is obtained in the solution.
Preferably, the preparation of the paenibacillus polymyxa suspension is prepared as follows:
respectively activating the stored Paenibacillus polymyxa strains for 2-3 times with NB culture medium, inoculating in NB culture medium, culturing at 37 deg.C, centrifuging logarithmic phase culture solution, collecting thallus, making into Paenibacillus polymyxa suspension with viable count not less than 1.0 × 10 8 CFU/mL。
Preferably, the high concentration of CaCl 2 CaCl in solution 2 The mass concentration of (B) is 4-6wt%.
On the other hand, the application also discloses application of the composite microspheres prepared by the application as a colonization and propagation nest of a biocontrol strain.
On the other hand, the application also discloses application of the composite microspheres prepared by the application in preventing and treating crop soil-borne diseases.
This application can bring following beneficial effect: the sodium alginate microspheres synthesized by sodium alginate and calcium chloride can adsorb spores of paenibacillus polymyxa, so that the spores are colonized in the propagation nest, the stress resistance of the spores is improved, re-colonization of the spores is continued, and the sodium alginate microspheres can be used as the propagation nest and the propagation nest of a biocontrol strain. The addition of 4% gelatin in the process of preparing the microcapsule can change the disintegration time and the release speed of the capsule so as to adapt to different use occasions.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:
FIG. 1 is an image of the bacteriostatic activity of different products against Ralstonia solanacearum.
FIG. 2 is a graph showing the effect of different products on the colonization ability of Paenibacillus polymyxa in rhizosphere soil.
Detailed Description
In order to clearly explain the technical features of the present invention, the present application will be explained in detail by the following embodiments in combination with the accompanying drawings.
1. And (3) synthetic test:
activating the stored BSYL-6 strain with NB culture medium for 2-3 times, inoculating in NB culture medium, culturing at 37 deg.C, centrifuging logarithmic phase culture solution, collecting thallus, making thallus suspension with sterile water, and counting viable bacteria number of 10 8 CFU/mL。
The first scheme is as follows: adding starch into the bacterial suspension according to the amount of 4%, and shaking uniformly to make the starch fully adsorb the bacteria. Adding 0.5mL of the mixed solution into 4.5mL of 2% sodium alginate solution, uniformly stirring, continuously dripping by using an injection pump to uniformly extrude the mixed solution into 0.1mol/L CaCl 2 Observing whether spherical capsules are formed in the solution; after the microcapsules are formed, the microcapsules are put into 5wt% of CaCl 2 Hardening in the solution, rinsing with sterilized distilled water and sucking to dry.
Scheme two is as follows: adding starch 2wt% and stabilizer 1wt% into the bacterial suspension, and shaking uniformly to make it fully adsorb thallus. Adding 0.5mL of the mixed solution into 4.5mL of 2% sodium alginate solution, uniformly stirring, continuously and dropwise extruding the mixed solution into 0.08mol/LCaCl2 solution by using an injection pump at a constant speed to observe whether spherical capsules are formed or not; after the microcapsules are formed, the microcapsules are put into a CaCl2 solution with the concentration of 6wt% for hardening, and the microcapsules are rinsed and sucked dry by sterile distilled water.
The stabilizer is paraffin with low phase transition temperature, and the phase transition temperature of the paraffin is 25-30 ℃; the particle size of the stabilizer is 1-10 mu m.
The third scheme is as follows: adding starch in an amount of 3% and a stabilizer in an amount of 2wt% into the bacterial suspension, and shaking uniformly to enable the bacterial suspension to fully adsorb bacteria. Adding 0.5mL of the mixed solution into 4.5mL of 2% sodium alginate solution, uniformly stirring, continuously and dropwise extruding the mixed solution into 0.1mol/LCaCl2 solution by using an injection pump at a constant speed to observe whether spherical capsules are formed or not; after the microcapsules are formed, the microcapsules are put into 5wt% CaCl2 solution for hardening, and the microcapsules are rinsed and sucked dry by sterile distilled water.
The stabilizer is paraffin with low phase transition temperature, and the phase transition temperature of the paraffin is 25-30 ℃; the particle size of the stabilizer is 1-10 mu m.
And the scheme is as follows: adding starch in an amount of 4wt% and a stabilizer in an amount of 3wt% into the bacterial suspension, and shaking uniformly to enable the bacterial suspension to fully adsorb bacteria. Adding 0.5mL of the mixed solution into 4.5mL of 2% sodium alginate solution, uniformly stirring, continuously extruding the mixed solution one drop by using an injection pump into 0.12mol/LCaCl2 solution at a constant speed, and observing whether a spherical capsule is formed or not; after the microcapsules are formed, the microcapsules are put into a CaCl2 solution with the concentration of 4wt% for hardening, and the microcapsules are rinsed and sucked dry by sterile distilled water.
The stabilizer is paraffin with low phase transition temperature, and the phase transition temperature of the paraffin is 25-30 ℃; the particle size of the stabilizer is 1-10 mu m.
And a fifth scheme: adding starch into the bacterial suspension according to the amount of 4%, and shaking uniformly to make the starch fully adsorb the bacteria. Adding 0.5mL of the mixed solution into 4.5mL of 2% sodium alginate solution, stirring uniformly, adding 2.5mL of 4% gelatin solution, stirring uniformly, and extruding the mixed solution into 0.1mol/LCaCl at uniform speed by using an injection pump for one drop 2 Observing whether spherical capsules are formed in the solution; after the microcapsules are formed, the microcapsules are put into 5wt% of CaCl 2 Hardening in the solution, rinsing with sterilized distilled water and sucking to dry.
Scheme six: adding starch 2%, stabilizer 1wt% and gelatin 4wt% into the bacterial suspension, and shaking uniformly to make it fully adsorb thallus. Adding 0.5mL of the mixed solution into 4.5mL of 2% sodium alginate solution, uniformly stirring, continuously dripping by using an injection pump to uniformly extrude the mixed solution into 0.08mol/LCaCl 2 Observing whether spherical capsules are formed in the solution; after the microcapsules are formed, the microcapsules are put into 6wt% of CaCl 2 In solutionHardening, rinsing with sterilized distilled water and sucking to dry.
The stabilizer is paraffin with low phase transition temperature, and the phase transition temperature of the paraffin is 25-30 ℃; the particle size of the stabilizer is 1-10 mu m.
The seventh scheme comprises the following steps: adding starch in an amount of 3%, a stabilizer in an amount of 2% and gelatin in an amount of 2% by weight into the bacterial suspension, and shaking uniformly to enable the bacterial suspension to fully adsorb bacteria. Adding 0.5mL of the mixed solution into 4.5mL of 2% sodium alginate solution, uniformly stirring, continuously dripping by using an injection pump to uniformly extrude the mixed solution into 0.1mol/LCaCl 2 Observing whether spherical capsules are formed in the solution; after the microcapsules are formed, the microcapsules are put into 5wt% of CaCl 2 Hardening in the solution, rinsing with sterilized distilled water and sucking to dry.
The stabilizer is paraffin with low phase transition temperature, and the phase transition temperature of the paraffin is 25-30 ℃; the particle size of the stabilizer is 1-10 mu m.
And a eighth scheme: adding starch 4%, stabilizer 3% and gelatin 2% into the bacterial suspension, and shaking uniformly to make it fully adsorb thallus. Adding 0.5mL of the mixed solution into 4.5mL of 2% sodium alginate solution, uniformly stirring, continuously dripping by using an injection pump to uniformly extrude the mixed solution into 0.12mol/LCaCl 2 Observing whether spherical capsules are formed in the solution; after the microcapsules are formed, the microcapsules are put into 4wt% of CaCl 2 Hardening in the solution, rinsing with sterilized distilled water and sucking to dry.
The stabilizer is paraffin with low phase transition temperature, and the phase transition temperature of the paraffin is 25-30 ℃; the particle size of the stabilizer is 1-10 mu m.
The eight schemes respectively take 0.1g of capsules, put the capsules into an EP tube to be smashed, suck 100 mu L of bacterial liquid, and use the bacterial liquid as 10 -1 、10 -2 、10 -3 、……、10 -10 、10 -11 And 10 -12 Diluting, taking 100 μ L from the last 3 dilutions, respectively, uniformly spreading on NA culture medium, repeating each dilution for 2 times, culturing at 37 deg.C for 24h, selecting 30-300 plates for colony count, and calculating the colony count, the results are shown in the following table:
serial number Number of colonies (x 10) 7 CFU/mL)
Scheme one 1.5
Scheme two 2.1
Scheme three 2.4
Scheme four 2.3
Scheme five 2.7
Scheme six 3.4
Scheme seven 3.1
Scheme eight 3.2
This shows that the sodium alginate composite microspheres can adsorb spores of paenibacillus polymyxa and enable the spores to colonize in the propagation nest. The number of colonies is obviously increased after the stabilizer is added, and the temperature of a microenvironment is presumed to be more stable in the actual synthesis and use processes, so that the development of the colonies is promoted.
2. Paenibacillus polymyxa assay:
2.1 paper dish method:
and testing the stress resistance of the paenibacillus polymyxa in the sodium alginate composite microspheres by using a paper disc method. Uniformly placing 3 paper discs (diameter of 6 mm) on a prepared NA culture medium, dripping 10 μ L sodium alginate composite micrococcus suspension (synthesized by scheme II) on the paper discs, and spraying with a spray can to obtain a concentration of 1.0 × 10 after the antagonistic bacterium solution is sufficiently absorbed by the culture medium 7 cfu/mL of bacterial suspension of ralstonia solanacearum, 10 mu L of sodium alginate composite micrococcus suspension (synthesized in the first scheme) is dripped on a paper disc to serve as a positive control, a blank control (no inoculation) is arranged, each treatment is repeated three times, and the mixture is inversely placed in an incubator for culture at 28 ℃. After 48 days, the observation results are shown in figure 1, the bacterial suspensions synthesized in the scheme I (the leftmost side) and the scheme II (the middle part) and the bacterial suspensions coated by the same have good bacteriostatic effects on ralstonia solanacearum, the diameter of the bacteriostatic zone of the bacterial suspensions is 3.1cm, and the bacteriostatic zone of the bacterial suspensions is relatively large and reaches 4.0cm. The rightmost side is blank. The method shows that the bacillus polymyxa is treated by the stabilizer after being coated by the sodium alginate composite microspheres, so that the stress resistance of spores is better improved, and the bacteriostatic action on pseudomonas solanacearum is enhanced.
2.2 potting test:
and testing the disease prevention effect of the paenibacillus polymyxa in the microcapsule by adopting a pot experiment. And (4) performing hole application on the tobacco seedlings with 6-8 leaves K326 by using the product prepared by the scheme II, wherein 0.067g of tobacco seedlings is planted in each plant, and the seedling culture substrate is kept dry before hole application. And performing hole application again after 5d, and inoculating the tobacco ralstonia solanacearum suspension into the rhizosphere soil of the tobacco seedling of each treatment group for 10 mL/plant after 2d of hole application for the second time. At the same time, the same treatment was carried out using the product prepared in scheme five, 0.067 g/strain/time, using BSYL-6 bacterial suspension (1.0X 10) 7 CFU/mL) as a positive control, 10 mL/plant/time, and a morbidity control CK1 (only bacterial wilt) and a blank control CK2 (no bacterial inoculation) which are the same as the others. Each treatment was performed 3 times at 5 strains. And 7d, inoculating ralstonia solanacearum, and investigating the disease rate (%), disease index and relative prevention and treatment effect (%) of each tobacco seedling.
The control effect on tobacco black shank is shown in the following table:
Figure RE-GDA0003226421550000071
3. determination of delayed release characteristics:
and (3) utilizing soil of a pot experiment, respectively selecting a pot of tobacco seedlings at 1d, 5d, 10d, 15d, 30d and 60d after the second inoculation, and respectively carrying out quantitative detection on BSYL-6 in the tobacco rhizosphere soil by adopting RT-PCR. As can be seen from FIG. 2, BSYL-6 grows and breeds rapidly within 0-15d after inoculation, reaches the highest value at 15d, and then shows a descending trend; in a whole, the content of BSYL-6 in rhizosphere soil of the tobacco plant inoculated with the composite microsphere is higher than that of BSYL-6 inoculated bacterial suspension. The result shows that the recolonization of the spores is continued after the paenibacillus polymyxa is coated by the sodium alginate composite microspheres, and the colonization and propagation in the soil are facilitated. In addition, the disintegration time and the release speed of the two capsules are different, and the disintegration and release speed of the five capsules is slow, so that the five capsules have a slow control effect. Therefore, the addition of the gelatin in the process of preparing the microcapsule can change the disintegration time and the release speed of the capsule so as to adapt to different use occasions.
The embodiments in the present specification are described in a progressive manner, and the same and similar parts among the embodiments are referred to each other, and each embodiment focuses on the differences from the other embodiments. In particular, for the system embodiment, since it is substantially similar to the method embodiment, the description is simple, and for the relevant points, reference may be made to the partial description of the method embodiment.
The above are merely examples of the present application and are not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement or the like made within the spirit and principle of the present application shall be included in the scope of the claims of the present application.

Claims (5)

1. A preparation method of a composite microsphere containing Paenibacillus polymyxa sodium alginate is characterized in that: the method comprises the following steps:
preparing a paenibacillus polymyxa bacterial suspension: carrying out logarithmic culture on paenibacillus polymyxa, and then centrifuging and collecting to obtain a bacterial suspension;
preparing primary microcapsules: adding 2-4wt% of starch and 1-3wt% of stabilizing agent into the bacterial suspension, and shaking uniformly to fully adsorb bacteria to obtain an intermediate mixed solution; adding the intermediate mixed solution into 8-10 times of 1.5-2.5% sodium alginate solution, stirring to obtain intermediate solution, and continuously extruding the intermediate solution dropwise into low-concentration CaCl with molar concentration of 0.08-0.12mol/L by using injection pump 2 Obtaining primary microcapsule suspension in the solution; the stabilizer is paraffin with low phase transition temperature, and the phase transition temperature of the paraffin is 25-30 ℃; the particle size of the stabilizer is 1-10 mu m;
hardening the capsule: placing the primary microcapsule in high-concentration CaCl 2 Hardening is carried out; the high concentration of CaCl 2 CaCl in solution 2 The mass concentration of (A) is 4-6wt%;
the strain code of the paenibacillus polymyxa is BSYL-6, the strain is preserved in China general microbiological culture Collection center in 26.04.2020, and the strain preservation number is CGMCC No.19736.
2. The preparation method of the composite microsphere containing paenibacillus polymyxa sodium alginate according to claim 1, wherein the preparation method comprises the following steps: the bacterial suspension also comprises 2-4wt% of gelatin.
3. The preparation method of the composite microsphere containing paenibacillus polymyxa sodium alginate according to claim 1, which is characterized in that: the preparation of the paenibacillus polymyxa suspension is prepared as follows:
respectively activating the stored Paenibacillus polymyxa strains for 2-3 times with NB culture medium, inoculating in NB culture medium, culturing at 37 deg.C, centrifuging logarithmic phase culture solution, collecting thallus, making into Paenibacillus polymyxa suspension with viable count not less than 1.0 × 10 8 CFU/mL。
4. Use of the composite microspheres prepared by the method of any one of claims 1 to 3 as a colonization and propagation nest for biocontrol strains.
5. Use of composite microspheres prepared by the method of any one of claims 1-3 for controlling soil-borne diseases of crops.
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