CN113826618A - Agricultural microbial hydrogel agent with disease resistance and yield increase - Google Patents
Agricultural microbial hydrogel agent with disease resistance and yield increase Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
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Abstract
The invention discloses an agricultural microbial hydrogel agent with disease resistance and yield increase, which is characterized by comprising gamma-polyglutamic acid-chitosan-gelatin composite hydrogel and microbial powder, wherein the microbial powder comprises one or two of fermentation powder of bacillus subtilis MES810 and fermentation powder of bacillus licheniformis MES 816. The microbial hydrogel agent can be used for crop seedling raising, agricultural product yield increase, bacteriostasis and soil and water conservation.
Description
Technical Field
The invention belongs to the field of manufacture and application of biological fertilizers, and relates to an agricultural microbial hydrogel microbial inoculum for disease resistance and yield increase.
Technical Field
The microbial agent used in the market at present is divided into solid and liquid, the application method is to dilute and spray the microbial agent on the surface of a plant or in soil, the microbial agent has the advantages of obvious effect and small influence on the environment, and after the microbial agent is used, the microbial environment in the soil can be adjusted, the fertility is enhanced, and the resistance to pathogenic bacteria is improved. However, the microbial inoculum has high requirements on the environment when in use, and the effect and the subsequent growth condition can be influenced by rainstorm in burning days. Meanwhile, the microbial agent contains live thalli, and the growth of the microbial agent can not be regulated by proper amount of water.
The hydrogel is a novel functional polymer material with good biocompatibility. Hydrogels are capable of swelling in water to draw up water and hold a large amount of water and are insoluble. The hydrogel can absorb a large amount of water because the structure of the hydrogel is a polymer network system formed by bonding polymer chains with physical force (hydrogen bond, coulomb force, coordination bond, etc.) or chemical bond (covalent bond). The hydrogel has the advantages of unique swelling performance, slow release performance, biocompatibility and the like, so that the hydrogel is more and more concerned in various fields in recent years.
Microbial fermentation factories can generate a large amount of industrial wastewater every day, wherein the industrial wastewater contains microbial polysaccharides, carbon-nitrogen sources and various inorganic salts, substances rich in the wastewater can promote the growth of microorganisms, but the wastewater cannot be directly discharged due to environmental protection requirements, and the industrial wastewater can be discharged after being subjected to multi-stage wastewater treatment, so that residual nutrient substances in the wastewater are wasted, and the difficulty is increased for the production operation of the factories.
Therefore, it is necessary to invent a multifunctional microbial hydrogel by combining the advantages of the hydrogel such as swelling property, slow release property and biocompatibility and the physiological function of the microbial agent. The invention not only can solve the problem of microbial fermentation wastewater treatment, but also has excellent use value.
Disclosure of Invention
Based on the problems, the invention aims to combine the advantages of the hydrogel such as swelling performance, slow release performance, biocompatibility and the like and the physiological function of a microbial agent to obtain a multifunctional agricultural microbial gel. The method can also utilize the microbial fermentation wastewater to prepare the microbial gel, and the prepared microbial fermentation wastewater microbial gel has good application prospect and can improve the disease-resistant and yield-increasing effects of the agricultural microbial agent.
In order to achieve the purpose, the technical scheme of the invention is to provide an agricultural microbial hydrogel agent with disease resistance and yield increase, which comprises gamma-polyglutamic acid-chitosan-gelatin composite hydrogel and microbial powder.
The gamma-polyglutamic acid-chitosan-gelatin composite hydrogel and the microbial powder are uniformly mixed according to the mass ratio of 10:1 to prepare the chitosan-gelatin composite hydrogel.
The bacterial powder is preferably one or two of the fermented bacterial powder of bacillus subtilis MES810 and the fermented bacterial powder of bacillus licheniformis MES 816.
If two kinds of mixed bacteria powder are selected, the weight ratio of the bacillus subtilis fermented bacteria powder to the bacillus licheniformis fermented bacteria powder is 1: 1.
The biological material MES810 is classified and named as Bacillus subtilis, and is preserved in China general microbiological culture collection center with the preservation address of No. 3 Xilu-Beijing north Chenyang district, the preservation date of No. 8 months and 10 days in 2017, and the preservation number of the biological material MES810 is: CGMCC No. 14514; the biological material MES816 is classified and named as Bacillus licheniformis, and is preserved in China general microbiological culture Collection center with the preservation address of No. 3 Xilu No.1 Beijing, Chaoyang, the preservation date of 5.8.2018 and the preservation number of: CGMCC No. 15744.
Carrying out shake flask culture, primary seed culture, secondary seed culture, fermentation, concentration and spray drying on the fermentation bacteria powder of bacillus subtilis MES810 and the fermentation bacteria powder of bacillus licheniformis MES816 to prepare corresponding bacteria powder, and then respectively subpackaging; the effective viable count of the powder of the bacillus subtilis MES810 is not less than 10 multiplied by 1011fu/g, and the effective viable count of the powder of the bacillus licheniformis MES816 zymocyte is not less than 10 multiplied by 1011 fu/g.
Wherein, the preparation methods of the bacillus subtilis zymocyte powder and the bacillus licheniformis zymocyte powder refer to, and the patent application No. CN201911410610.8 discloses a composite microbial inoculum for preventing and treating pond lichen, a preparation method and a preparation method of the bacillus subtilis zymocyte powder and the bacillus licheniformis zymocyte powder in the application.
In the gamma-polyglutamic acid-chitosan-gelatin composite hydrogel, the weight portions are as follows: the mass ratio of the chitosan to the gelatin is 1-8: 23-16.
The gamma-polyglutamic acid-chitosan-gelatin composite hydrogel is prepared by the following steps:
(1) dissolving chitosan in 20ml of deionized water containing 1 wt% (mass percent) of acetic acid at room temperature, and stirring at 60 ℃ until the chitosan is completely dissolved to obtain a chitosan solution;
(2) dissolving gelatin in 20ml of deionized water containing 1 wt% (mass percent) of acetic acid at room temperature, and stirring at 60 ℃ until the gelatin is completely dissolved to obtain a gelatin solution;
(3) pouring the chitosan solution in the step (1) and the gelatin solution in the step (2) into the same beaker, uniformly mixing, and putting the beaker into an ultrasonic oscillator for eliminating bubbles by ultrasonic waves;
(4) pouring the mixed solution after eliminating bubbles into a mould, and standing at room temperature to form chitosan-gelatin composite hydrogel;
(5) immersing the chitosan-gelatin composite hydrogel into 1% of gamma-polyglutamic acid solution, and obtaining the gamma-polyglutamic acid-chitosan-gelatin composite hydrogel at the pH of 7.0 and at room temperature for 24 hours;
wherein the weight ratio of chitosan: gelatin: glacial acetic acid: the deionized water is prepared from 1-8 mass percent: 23-16: 1:200.
The preparation method can adopt fermentation wastewater to replace deionized water to prepare the gamma-polyglutamic acid-chitosan-gelatin composite hydrogel.
(1) Dissolving chitosan in 20ml of microbial fermentation wastewater containing 1 wt% (mass percentage) acetic acid at room temperature, and stirring at 60 ℃ until the chitosan is completely dissolved to obtain a chitosan solution;
(2 dissolving gelatin in 20ml of microbial fermentation wastewater containing 1 wt% (mass percentage) of acetic acid at room temperature, and stirring at 60 ℃ until the gelatin is completely dissolved to obtain a gelatin solution;
(3) pouring the chitosan solution in the step (1) and the gelatin solution in the step (2) into the same beaker, uniformly mixing, and putting the beaker into an ultrasonic oscillator for eliminating bubbles by ultrasonic waves;
(4) pouring the mixed solution after eliminating bubbles into a mould, and standing at room temperature to form chitosan-gelatin composite hydrogel;
(5) the chitosan-gelatin composite hydrogel is immersed in 1% of gamma-polyglutamic acid solution and is subjected to 24 hours at the pH value of 7.0 and the room temperature to obtain the gamma-polyglutamic acid-chitosan-gelatin composite hydrogel.
The chitosan: gelatin: glacial acetic acid: the proportion of the fermentation wastewater is 1-8 by mass: 23-16: 1:200.
The microbial fermentation wastewater is mainly wastewater produced by fermenting bacillus subtilis MES810 and bacillus licheniformis MES816 in a microbial fermentation factory, and is rich in microbial polysaccharides, carbon and nitrogen sources and various inorganic salts.
Compared with the prior art, the invention has the following advantages:
the multifunctional microbial hydrogel microbial inoculum obtained by the invention can absorb water which is many times of the self weight in a short time, can be slowly released, and has excellent water-retaining and drought-resisting performance. After acting on the surface of the plant, the plant cleaning agent has good disease treatment capability and retention capability, and can not be washed away due to a small amount of rainfall to lose the using effect. The multifunctional microbial fermentation wastewater gel microbial inoculum obtained by the invention can also utilize fermentation wastewater, save manpower and material resources for wastewater treatment and increase the efficacy of a hydraulic microbial inoculum.
The water-retaining and bacteriostatic microbial hydrogel obtained by the invention has good water-retaining property and mechanical strength, and has practical application effects in the aspects of slope ecology protection and soil erosion prevention.
The invention has practical application value in the fields of agricultural production, desertification control, side slope ecological protection, water and soil loss prevention and control and the like, and is suitable for popularization and application. The application prospect is considerable.
Drawings
FIG. 1 is a graph comparing swelling properties of gamma-polyglutamic acid-chitosan-gelatin composite hydrogel according to the present invention;
FIG. 2 is a graph showing the comparison of the compression properties of the gamma-polyglutamic acid-chitosan-gelatin composite hydrogel of the present invention.
Detailed Description
The invention will now be further described by way of specific examples, which are not intended to limit the scope of the invention. It will be understood by those skilled in the art that equivalent substitutions for the technical features of the present invention, or corresponding modifications, can be made within the scope of the present invention.
The instruments, reagents, materials and the like used in the following examples are, unless otherwise specified, conventional instruments, reagents, materials and the like known in the art, and are commercially available, and the experimental methods, detection methods and the like used in the following examples are, unless otherwise specified, conventional experimental methods, detection methods and the like known in the art.
EXAMPLE 1 preparation of hydrogel
According to the preparation method of the invention, the gamma-polyglutamic acid-chitosan-gelatin composite hydrogel is prepared according to the formula shown in the table I.
TABLE 1 preparation of Gamma-polyglutamic acid-chitosan-gelatin composite hydrogel
| Numbering | Chitosan, gelatin: glacial acetic acid: |
| 1 | 1:23:1:200 |
| 2 | 2:22:1:200 |
| 3 | 4:20:1:200 |
| 4 | 8:16:1:200 |
The results show that the hydrogel can be formed in four proportions. As can be seen from the figure, the gamma-polyglutamic acid-chitosan-gelatin composite hydrogel becomes more yellow as the ratio of chitosan to gelatin increases. When preparing the hydrogel, it was observed that the volume of the prepared gamma-polyglutamic acid-chitosan-gelatin composite hydrogel was also larger and larger as the ratio of chitosan to gelatin was increased. This is probably because the more chitosan reacts with the gamma-polyglutamic acid solution, the more severe the reaction with the gamma-polyglutamic acid solution, so the synthesized gamma-polyglutamic acid-chitosan-gelatin composite hydrogel becomes larger and larger in volume as the chitosan increases. The hydrogel prepared in formulation 4 had the largest volume.
EXAMPLE 2 hydrogel Performance testing
2.1 hydrogel swelling Performance test
The method for measuring the swelling rate of the hydrogel in the test adopts a tea bag method.
The swelling rate of the hydrogel was measured by the tea bag method as follows:
(1) the mass of the prepared hydrogel sample was weighed as W0.
(2) Using polyamide woven fabric with poor water absorption to sew a square tea bag with an opening at one end, soaking the tea bag in a swelling medium, wiping the surface water of the tea bag with filter paper, and weighing the tea bag to be U.
(3) The tea bag containing the sample was completely immersed in the swelling medium, taken out after twenty-four hours and weighed to a mass of W1.
We then expressed the swelling properties of the hydrogels by the values obtained after W1-W0-U, and the specific swelling properties are shown in FIG. 1.
The result shows that the swelling performance of the No. 2 mixture ratio is optimal.
2.2 hydrogel compression Performance test
The gamma-polyglutamic acid-chitosan-gelatin composite hydrogel is prepared into a cylindrical sample with the height of 1.6cm and the diameter of 1.5 cm. Compression testing was then performed at room temperature in air using a general purpose stretcher (Instron model 5567). The compression rate was 10mm/min, and the mechanical strength was examined. The results of the mechanical strength test are shown in fig. 2.
Keeping other factors unchanged in the synthesis process of the gamma-polyglutamic acid-chitosan-gelatin composite hydrogel, and changing the ratio of the added chitosan to gelatin. The ratio of chitosan to gelatin for sample No.1 was 1:23, the ratio of chitosan to gelatin for sample No. 2 was 1:11, the ratio of chitosan to gelatin for sample No. 3 was 1:5, and the ratio of chitosan to gelatin for sample No. 4 was 1: 2.
As can be seen from the figure, sample No. 2 has the highest compressive strength and can bear the maximum compressive stress of 0.04772 MPa. Sample No.1 can withstand a maximum compressive stress of 0.03985 MPa. Sample No. 3 can withstand a maximum compressive stress of 0.02504 MPa. Sample No. 4 can withstand a maximum compressive stress of 0.01685 MPa.
And (4) conclusion: the three test results are combined to show that the mixture ratio 2 not only has better forming capability, but also has better swelling performance and mechanical strength than other mixture ratios. Mixing the mixture with bacterial powder (one or two kinds of bacterial powder) 10:1 according to the formula of the ratio 2 to obtain the final multifunctional microbial hydrogel agent, and carrying out further performance tests. The microbial hydrogel microbial inoculum and the microbial fermentation wastewater gel microbial inoculum obtained by the technical scheme of the invention have excellent water retention and bacteriostasis effects, and have outstanding contribution to the aspects of disease resistance and yield increase of agricultural products.
2.3 Water-retaining and bacteriostatic test of microbial hydrogel
2.3.1 Water-retaining bacteriostatic- -seedling test of microbial hydrogel
Selecting two parts of completely same soil for sterilization treatment, wherein one part is mixed with 10% of multifunctional microbial fermentation wastewater gel microbial inoculum, the other part is mixed with 10% of hydrogel without bacterial powder, the last part is a blank control group, then cucumber seeds are planted in the blank control group, watering is carried out every day, observation is carried out after 7 days, cucumber seedlings of 3 groups of experiments grow and emerge, the blank control group seedlings are 9cm long, the cucumber seedlings mixed with the hydrogel without bacterial powder are 11cm long, and the cucumber seedlings obviously grow more quickly and robustly after the microbial fermentation wastewater gel microbial inoculum is added, and the seedling length is 21 cm. And stopping watering the melon seedlings after the measurement is finished, wherein the melon seedlings of the blank control group begin to wither after 1 day, the melon seedlings added with the microbial fermentation wastewater gel fungicide and the hydrogel without the bacterial powder still grow normally, the melon seedlings added with the hydrogel without the bacterial powder begin to wither after 4 days, the melon seedlings of the blank control group completely wither, the melon seedlings using the hydrogel without the bacterial powder basically completely wither after 7 days, and the melon seedlings added with the microbial fermentation wastewater gel fungicide only have the phenomenon of water shortage and atrophy. Therefore, the microbial fermentation wastewater gel bacterial agent not only has the obvious effect of rooting and seedling promotion, but also can keep the moisture contained in the plants for a longer time in a water-deficient state of the plants, such as long-distance transportation, maintain the activity of the root system and play a great role.
2.3.2 Water-holding bacteriostasis-bacteriostasis test of microbial hydrogel
Uniformly mixing the microbial fermentation wastewater gel microbial inoculum with water according to the concentration of 1 percent, and carrying out an antagonistic experiment with fusarium oxysporum and fusarium solani:
respectively inoculating fusarium oxysporum and fusarium solani on a PDA (personal digital assistant) flat plate, then culturing at 37 ℃, after pathogenic bacteria grow on the surface of the flat plate, immersing a filter paper sheet into an aqueous solution of a microbial fermentation wastewater gel microbial inoculum with the concentration of 1%, then placing the filter paper sheet on the surface of the pathogenic bacteria flat plate, culturing for 3 days at 37 ℃, and observing to find that a bacteriostatic ring appears on the surface of the flat plate. The microbial fermentation wastewater gel microbial inoculum has an inhibiting effect on two pathogenic bacteria.
Then, spraying the aqueous solution of the microbial hydrogel added with 1% of water retention and bacteriostasis on the surface of the banana leaf, spraying the aqueous solution mixed with the water retention and bacteriostasis hydrogel prepared from thousandth of bacteria powder and 1% of distilled water on the surface of the banana leaf, spraying the aqueous solution mixed with the thousandth of bacteria powder on the surface of the banana leaf as a contrast, then spraying the aqueous solution with purified water every day, and then measuring the number of bacteria remained on the leaf.
Table 2: comparison data of microbial hydrogel bacteriostasis test
The number of the residual bacteria of the plantain leaves sprayed by using the microbial fermentation wastewater gelling agent in the table is far higher than that of the control group. The gel microbial inoculum for the microbial fermentation wastewater has good retention property, can better stay on the surface of a plant, and achieves the effects of inhibiting bacteria and treating diseases. Meanwhile, the microbial fermentation wastewater gel microbial inoculum contains more nutrients and viscous substances, so that the bacteria powder contained in the microbial fermentation wastewater gel microbial inoculum can germinate and grow more quickly, and the antibacterial and protective effects are achieved.
2.3.2 test of water retention, bacteriostasis, and prevention of soil erosion by microorganism hydrogel
Selecting a piece of test field with slope, mixing 1/3 microbial fermentation wastewater gel microbial inoculum in the test field, adding 1/3 common hydrogel, finally 1/3 as a blank control group, measuring the slope of three pieces of land with the slope of 19% and the slope length of 5m, measuring the piece of land again after rainy season, wherein the result shows that the slope length of the slope of the land mixed with the microbial fermentation wastewater gel microbial inoculum is not changed, the slope of the common hydrogel group is changed into 17%, the slope length is changed into 5.7 m, the slope of the blank control group is changed into 15%, and the slope length is 6.3 m, which shows that the microbial fermentation wastewater gel microbial inoculum has actual effects in the aspects of slope prevention, water and soil loss prevention and the like. Further investigate the root growth condition of the plants in the slope test field.
Table 3: plant growth condition comparison data in 3 lands
Obviously, the root system of the plant is stronger and more developed after the microbial fermentation wastewater gel microbial inoculum is used, and the effect of protecting the land is better.
Claims (10)
1. An agricultural microbial hydrogel agent for disease resistance and yield increase is characterized by comprising gamma-polyglutamic acid-chitosan-gelatin composite hydrogel and microbial powder.
2. The agricultural microbial hydrogel agent for disease resistance and yield increase according to claim 1, which is prepared by uniformly mixing gamma-polyglutamic acid-chitosan-gelatin composite hydrogel and microbial powder according to a mass ratio of 10: 1.
3. The agricultural microbial hydraulic inoculant according to claim 2, wherein the microbial inoculant comprises one or both of a fermentation inoculant powder of bacillus subtilis MES810 and a fermentation inoculant powder of bacillus licheniformis MES 816.
4. The disease-resistant yield-increasing agricultural microbial hydraulic inoculant according to claim 3, wherein the bacillus subtilis MES810 is preserved in the China general microbiological culture Collection center with the preservation address of No. 3 Xilu No.1 Beijing north Chen, the south-facing-the-south, the preservation date of 2017, 8 months and 10 days, and the preservation numbers are as follows: CGMCC No. 14514; bacillus licheniformis MES816 is preserved in China general microbiological culture Collection center with the preservation address of No. 3 Xilu No.1 Beijing north Chen of the sunward area, the preservation date of 2018, 5 months and 8 days, and the preservation numbers are as follows: CGMCC No. 15744.
5. The agricultural microbial hydraulic microbial inoculant according to claim 3, wherein the number of effective viable bacteria in the Bacillus subtilis zymocyte powder is equal to or less than the number of effective viable bacteria in the Bacillus subtilis zymocyte powderNot less than 10 × 1011fu/g, effective viable count of not less than 10 × 10 in Bacillus licheniformis zymocyte powder11fu/g, the weight ratio of the fermentation bacteria powder of the bacillus subtilis MES810 to the fermentation bacteria powder of the bacillus licheniformis MES816 is 1: 1.
6. The agricultural microbial hydraulic inoculant for disease resistance and yield increase according to claim 5, wherein corresponding inoculant powders are prepared from the fermentation inoculant powder of bacillus subtilis MES810 and the fermentation inoculant powder of bacillus licheniformis MES816 through shake flask culture, primary seed culture, secondary seed culture, fermentation, concentration and spray drying.
7. The disease-resistant yield-increasing agricultural microbial hydrogel agent according to claim 1, wherein in the gamma-polyglutamic acid-chitosan-gelatin composite hydrogel, the mass ratio of chitosan to gelatin is 1-8: 23-16.
8. The disease-resistant yield-increasing agricultural microbial hydrogel agent according to claim 1, wherein the gamma-polyglutamic acid-chitosan-gelatin composite hydrogel is prepared by the following method:
(1) dissolving chitosan in 20ml of deionized water containing 1 wt% of acetic acid at room temperature, and stirring at 60 ℃ until the chitosan is completely dissolved to obtain a chitosan solution;
(2) dissolving gelatin in 20ml of deionized water containing 1 wt% of acetic acid at room temperature, and stirring at 60 ℃ until the gelatin is completely dissolved to obtain a gelatin solution;
(3) pouring the chitosan solution in the step (1) and the gelatin solution in the step (2) into the same beaker, uniformly mixing, and putting the beaker into an ultrasonic oscillator for eliminating bubbles by ultrasonic waves;
(4) pouring the mixed solution after eliminating bubbles into a mould, and standing at room temperature to form chitosan-gelatin composite hydrogel;
(5) immersing the chitosan-gelatin composite hydrogel into 1% gamma-polyglutamic acid solution, and obtaining the gamma-polyglutamic acid-chitosan-gelatin composite hydrogel at the pH of 7.0 and room temperature for 24 hours;
the chitosan: gelatin: glacial acetic acid: the proportion of the fermentation wastewater is 1-8 by mass: 23-16: 1:200.
9. The agricultural microbial hydrogel agent for resisting diseases and increasing yield according to claim 8, wherein deionized water is replaced by microbial fermentation wastewater, and the microbial fermentation wastewater is preferably wastewater generated in fermentation for preparing bacillus subtilis MES810 and bacillus licheniformis MES 816.
10. The agricultural microbial hydraulic fungicide for disease resistance and yield increase according to any one of claims 1 to 9 is applied to agricultural production, desertification treatment, slope ecological protection and water and soil loss prevention and treatment, and is beneficial to crop seedling raising, agricultural product yield increase, bacteriostasis and soil and water conservation.
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