PGPR microbial agent for preventing soil hardening and preparation method of PGPR microbial agent

Abstract

The invention relates to the technical field of soil improvement, in particular to a PGPR soil hardening prevention microbial agent and a preparation method, the PGPR soil hardening prevention microbial agent comprises the following raw materials by weight: 50-80 parts of microencapsulated PGRG microorganisms, 8-15 parts of a microencapsulated microbial activator, 10-20 parts of negative charge nanofibers, and 40-60 parts of a coating agent; the coating agent is a mixture composed of polyethylene glycol and polypropylene glycol according to the weight ratio of (60-80): (20-40); the coating agent is dissolved in a glycol ether solvent to form a film. According to the invention, the screened PGRG microorganisms and the microbial activator are subjected to microencapsulation treatment, the microencapsulated PGRG microorganisms and the microbial activator are embedded together with the negative charge nanofibers through the coating agent to obtain the microbial agent, and the microbial agent is put into soil, so that the soil structure can be effectively improved, the coagulation effect of the soil can be enhanced, and the soil hardening can be obviously improved; soil hardening can be prevented for a long time, and a suitable soil environment is provided for plant growth.

Description

Technical Field

[0001] The present invention relates to the technical field of soil improvement, and specifically to a microbial agent for preventing soil compaction by PGPR and a preparation method thereof. Background Technique

[0002] PGPR are a class of beneficial bacteria that live freely in plants, attach to the roots or rhizosphere soil, have biocontrol effects on pathogenic bacteria, can promote plants to absorb inorganic substances such as minerals, and produce compounds beneficial to plant growth. PGRG microorganisms have the ability to decompose organic substances. They can decompose and transform organic residues in the soil, such as plant roots and fallen leaves, into organic substances such as humus. Humus can increase the content of soil organic matter, improve the physical properties of the soil, and at the same time provide nutrients for soil microorganisms, promote the growth and reproduction of microorganisms, and further enhance the fertility and structural stability of the soil. In addition, PGRG microorganisms also participate in the transformation and cycling processes of nutrients such as nitrogen, phosphorus, and potassium in the soil, improve the availability of these nutrients, enable plants to better absorb nutrients, promote plant growth, and the growth of plant roots will have a positive impact on the soil structure, helping to prevent soil compaction.

[0003] For example, Chinese Patent CN114921370B discloses a microbial soil improvement and repair agent containing a composite microbial agent, which includes Bacillus velezensis ZLP-101, Bacillus subtilis BSD-2, silicate bacteria HM8841, and phosphate-solubilizing bacteria HM0332; this microbial soil improvement and repair agent can improve the physical properties and microecological environment of the soil, enhance soil air permeability, effectively improve soil compaction and acidification problems, and can also significantly kill underground pests and reduce the incidence of underground pests; however, when this microbial soil improvement and repair agent is put into the soil for use, although it can improve the physical properties of the soil, due to its easy loss in the soil and low retention rate in the soil, its effect is limited, it cannot play a role for a long time, and it cannot stabilize the soil structure to prevent the recurrence of soil compaction. Summary of the Invention

[0004] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide a microbial agent for preventing soil compaction by PGPR and a preparation method thereof.

[0005] To achieve the above purpose, the present invention provides the following technical solutions: A microbial agent for preventing soil compaction by PGPR, comprising the following raw materials in parts by weight: 50-80 parts of microencapsulated PGRG microorganisms, 8-15 parts of microencapsulated microbial activator, 10-20 parts of negatively charged nanofibers, and 40-60 parts of coating agent; The coating agent is a mixture composed of polyethylene glycol and polypropylene glycol in a weight ratio of (60-80):(20-40); The coating agent is dissolved in a diethylene glycol ether solvent to form a thin film.

[0006] As a further preferred embodiment of the present invention, the microencapsulated PGRG microorganism is prepared as follows: S1 Isolate the microorganisms in the rhizosphere soil of the soybean planting area to obtain a pure culture of strains; S2 Under the action of ultrasound combined with plasma treatment, perform mutagenesis screening to obtain PGRG microorganisms with strong organophosphorus degradation ability; S3 Use whey protein and trehalose as wall materials and positively charged hollow nanorods as functional materials to microencapsulate the PGRG microorganisms, and the microencapsulated PGRG microorganisms can be obtained.

[0007] Furthermore, the specific operation method in S1 is as follows: 1) Add 10 - 15 g of rhizosphere soil from the soybean planting area to 90 - 150 mL of sterile PBS, shake at 30 °C and 180 - 260 rpm for 30 - 50 min, then centrifuge at 5000 - 8000 rpm for 15 - 20 min, remove the supernatant, and then add 10 - 20 mL of sterile PBS to the precipitate and shake to obtain a suspension; 2) Take 2 - 5 mL of the suspension and add it to 100 - 200 mL of LB liquid medium containing 1 - 3 mmol / L of dipotassium hydrogen phosphate for screening culture, shake and culture at 30 °C and 180 - 260 rpm for two days, then subculture 5 - 6 times according to an inoculation amount of 5 - 6%, and use the gradient dilution method to prepare 10 –2 -10 –6 series dilution solutions, respectively take 100 - 160 μL and coat them on the LB solid medium, culture at 30 °C until the colonies grow well, pick single colonies, and use the streaking method to obtain a pure culture of strains.

[0008] Furthermore, the specific operation method in S2 is as follows: 1) Pick the isolated and purified single colonies and inoculate them into the LB liquid medium for culture. Take 10 - 15 μL of the bacterial suspension cultured for 24 h, evenly coat it on the metal plate, and then place it in the sample treatment chamber of the ARTP mutagenesis system. Adjust the distance between the metal plate and the gas flow port to 2 - 5 mm. The working parameters of ARTP are that the radio frequency power input is 100 - 150 W, the flow rate of pure helium as the working gas is 10 - 15 L / min, and the treatment time is 20 - 90 s. During the treatment process, apply intermittent ultrasonic waves with a frequency of 0.1 - 1000 kHz and a power of 0.01 - 1000 W for 20 - 30 s, and then measure the lethality of the mutagenized strains to determine the optimal mutagenesis time; 2) Based on the mutagenesis time determined in step (4), the mutagenesis sample was eluted with sterile saline and gradiently diluted to 10 -6 -10 -8 100 μL of bacterial suspension was inoculated into LB solid medium with increasing concentration of dipotassium hydrogen phosphate to screen strains. The increasing gradient of dipotassium hydrogen phosphate concentration in LB solid medium was 10, 20, 30, 40, 50, 70, 100 and 120 mmol / L. After culture elimination, high-purity strains were screened; 3) Inoculate the highly pure strain into LB liquid medium for activation and shake on a shaker until the bacterial solution OD 600 The value is 0.8, aspirate 10-20µL of bacterial liquid and drop it on the organophosphorus solid culture medium, mark the strain, seal the organophosphorus solid culture medium and invert it in a 28℃ incubator for culture for 4-7 days. During this period, observe and record every day whether there is a transparent circle on the plate, select the colony with a transparent circle, measure the diameter D of the transparent circle and the diameter d of the colony, and judge the ability of the strain to degrade organophosphorus by the D / d ratio, so as to screen out PGRG microorganisms with strong organophosphorus degradation ability.

[0009] Going further, the specific operation methods in S3 are as follows: 1) Dissolve whey protein in sterilized distilled water, stir thoroughly, and pasteurize in a water bath at 80-85°C for 5-10 minutes to obtain a whey protein solution with a concentration of 80-100 g / L. Dissolve trehalose in sterile distilled water, and sterilize in a water bath at 80-85°C for 5-10 minutes to obtain a trehalose solution with a concentration of 20-40 g / L. 2) adding positively charged hollow nanorods to the whey protein solution and dispersing them evenly to obtain a dispersion having a solid content of 1.0-1.8%, fully mixing 75-100 mL of the dispersion, 75-100 mL of the trehalose solution and 50-80 mg of the PGRG microorganism, and then spray drying the mixture under the conditions of an air flow rate of 350-400 L / h, a feed flow rate of 0.3-0.5 L / h, an inlet air temperature of 120-123°C, an outlet air temperature of 65-70°C, and a carrier gas pressure of 0.1-0.2 MPa to obtain microencapsulated PGRG microorganisms.

[0010] As a further preferred embodiment of the present invention, the microencapsulated microorganism activator is prepared as follows: S1 adds trace elements and growth factors to deionized water, and then adds a small molecule organic carbon source to obtain a primary composite microbial activator, and then adds a pH buffer solution to the primary composite microbial activator to adjust the pH to 6.5 to obtain a composite microbial activator; S2 uses whey protein and trehalose as wall materials and positively charged hollow nanorods as functional materials to microencapsulate the composite microbial activator to obtain a microencapsulated composite microbial activator.

[0011] Furthermore, in S1, the ratio of the trace elements, growth factors, small molecule organic carbon source, pH buffer, and deionized water is (55-60) mL: (1-3) mL: (10-15) g: (1.0-1.5) mL: (900-1200) mL; The formula of the trace elements is 0.3-0.5 g / L zinc sulfate, 1.2-1.6 g / L copper sulfate, 0.2-0.3 g / L ammonium molybdate, 0.05-0.08 g / L sodium borate, 0.05-0.08 g / L magnesium sulfate, and 0.5-0.8 g / L iron sulfate; The growth factor formula is niacin 4-6g / L, pantothenic acid 5-7g / L, cobalamin 4-6g / L; The small molecule organic carbon source is selected from at least one of glucose, sodium citrate, sodium acetate and sucrose; The pH buffer solution is composed of 0.2 mol / L boric acid solution, 0.2 mol / L potassium chloride solution, and 0.1 mol / L sodium hydroxide solution in a volume ratio of (50-60): (50-60): 1.

[0012] Furthermore, the specific operation method in S2 is as follows: 1) Dissolve whey protein in sterilized distilled water, stir thoroughly, and pasteurize in a water bath at 80-85°C for 5-10 minutes to obtain a whey protein solution with a concentration of 80-100 g / L. Dissolve trehalose in sterile distilled water, and sterilize in a water bath at 80-85°C for 5-10 minutes to obtain a trehalose solution with a concentration of 20-40 g / L. 2) adding positively charged hollow nanorods to the whey protein solution and dispersing them uniformly to obtain a dispersion having a solid content of 1.0-1.8%, fully mixing 75-100 mL of the dispersion, 75-100 mL of the trehalose solution and 120-180 mg of the composite microbial activator, and then spray drying the mixture under the conditions of an air flow rate of 350-400 L / h, a feed flow rate of 0.3-0.5 L / h, an inlet air temperature of 120-123° C., an outlet air temperature of 65-70° C. and a carrier gas pressure of 0.1-0.2 MPa to obtain a microencapsulated microbial activator.

[0013] As a further preferred embodiment of the present invention, the positively charged hollow nanorods are prepared by the following method: S1 uses trimesic acid and bismuth nitrate as raw materials and methanol as solvent to obtain a precursor through hydrothermal reaction, then mixes the precursor with potassium bromide and adds it into deionized water, and then treats it in an oil bath to obtain hollow nanorods; S2 Amino-functionalized modification: The hollow nanorods are added to an aqueous methanol solution, the pH is adjusted to 4.5 - 5.0, ultrasonicated for 1 - 3 h, and while stirring, KH550 and glacial acetic acid are added, then ultrasonicated for another 1 - 3 h, followed by centrifugal separation, washing, and drying to obtain the product.

[0014] Furthermore, the ratio of trimesic acid, methanol, and bismuth nitrate is (0.7 - 1.2) g : (50 - 80) mL : (0.09 - 0.13) g; For the hydrothermal reaction, the temperature is 120 - 130 °C and the reaction time is 24 - 30 h; The ratio of the precursor, potassium bromide, and deionized water is (0.5 - 0.8) g : (1.0 - 1.5) g : (500 - 800) mL; For the oil bath treatment, the temperature is 90 - 93 °C and the treatment time is 1 - 2 h; The ratio of the hollow nanorods, methanol / aqueous solution, KH550, and glacial acetic acid is (1 - 3) g : (80 - 130) mL : (15 - 20) mL : (3 - 6) mL; The volume ratio of the methanol / aqueous solution is (80 - 85) : (15 - 20).

[0015] As a further preferred embodiment of the present invention, the negatively charged nanofibers are prepared as follows: 1) Commercially available chitin is soaked in dilute hydrochloric acid at room temperature for 12 - 15 h, washed repeatedly with distilled water, then soaked in sodium hydroxide solution for 12 - 15 h, and after repeated washing with distilled water, decolorized with 0.3 - 0.5 wt% sodium chlorite and dried to obtain chitin powder with an acetylation degree of 95%; 2) The chitin powder is dispersed in an aqueous solution of ammonium persulfate with a concentration of 1 mol / L, magnetically stirred at 60 - 65 °C for 20 - 24 h, and then negatively charged nanofibers are obtained by centrifugation; The concentration of the sodium hydroxide solution is 0.10 - 0.15 mol / L; The mass ratio of the chitin powder to the ammonium persulfate aqueous solution is (0.5 - 0.8) : (100 - 160).

[0016] A preparation method of a microbial agent for preventing soil compaction by PGPR, characterized in that it specifically includes the following steps: By weight, the coating agent is dissolved in a diethylene glycol ether solvent to obtain a coating agent solution with a concentration of 25 - 35 wt%, then the microencapsulated PGRG microorganisms, microencapsulated microbial activator, and negatively charged nanofibers are fully mixed, and then put into the coating agent solution, and spray-dried after sufficient stirring.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: In the present invention, the rhizosphere soil in the soybean planting area is used as the source of PGRG microorganisms. Through separation and screening culture in a medium containing dipotassium hydrogen phosphate, strains are obtained. Then, under the combined action of ultrasonic waves and plasma treatment, the strains are mutagenized. The ultrasonic wave action can promote the material exchange between microbial cells and the surrounding environment, accelerate the transmission of nutrients to the cell surface, and promote the metabolic activities of microorganisms. At the same time, plasma treatment can change the gene sequence of microorganisms, and high-yield strains with excellent traits and stable inheritance are screened out. Then, they are inoculated into a medium with an increasing concentration of dipotassium hydrogen phosphate to screen for highly phosphorus-tolerant strains, and through secondary screening on an organic phosphorus medium, PGRG microorganisms with strong organic phosphorus degradation ability are obtained. The PGRG microorganisms obtained through screening can effectively decompose organic substances in the soil, release nutrients, and at the same time produce some viscous substances to promote the aggregation of soil particles, improve the soil structure, especially can efficiently decompose the residual organic phosphorus in the soil and convert it into inorganic phosphorus, thereby promoting the absorption and utilization by plant roots. Thus, while effectively improving soil compaction, it also promotes the growth of plants. And in order to improve the utilization rate of PGRG microorganisms and enable them to play a long-term and efficient role in the soil, the present invention conducts microencapsulation treatment on PGRG microorganisms. Using whey protein and trehalose as wall materials, the PGRG microorganisms are encapsulated, and positively charged hollow nanorods are also added to the wall materials. The positively charged hollow nanorods have a hollow structure. In the wall materials, due to carrying positive charges, they repel each other under electrostatic action and can be evenly dispersed in the wall materials. And their rod-shaped structure can form a support framework in the wall materials, improving the resistance of the wall materials, thereby enhancing the resistance of the microcapsules to external forces and enabling them to maintain the integrity of the structure during subsequent processing. At the same time, their hollow structure provides a channel for the release of PGRG microorganisms, enabling a slow-release effect, thereby effectively improving their utilization rate. Further, in order to enhance the biological activity of PGRG microorganisms and promote growth after they are released into the soil, in the present invention, a composite microbial activator composed of trace elements, growth factors, and small molecule organic carbon sources is also microencapsulated, enabling it to be slowly released into the soil, thereby promoting the growth and metabolism of PGRG microorganisms, accelerating the reproduction and growth of PGRG microorganisms, and thus quickly improving the soil compaction phenomenon.

[0018] In order to use the microencapsulated PGRG microorganism and the microencapsulated microorganism activator in combination, in the present invention, chitosan is treated with acid and alkali to remove astringency, and then the chitosan is oxidized with ammonium persulfate to oxidize the hydroxymethyl groups on the chitosan molecular chain into carboxyl groups, thereby obtaining negatively charged nanofibers; then the coating agent is dissolved in the glycol ether to obtain a coating agent solution, and the microencapsulated PGRG microorganism, the microencapsulated microorganism activator and the negatively charged nanofibers are put into the coating agent solution and fully mixed. Since the microencapsulated PGRG microorganism and the microencapsulated microorganism activator both carry positive charges, they are evenly dispersed in the coating agent solution, and the negatively charged nanofibers are adsorbed on the surfaces of the microencapsulated PGRG microorganism and the microencapsulated microorganism activator under the action of electrostatics, and through sufficient mechanical stirring, the negatively charged nanofibers are continuously entangled and cross-linked to form a wrapped network structure, thereby encapsulating the microencapsulated PGRG microorganism and the microencapsulated microorganism activator. The microbial activator is wrapped, and the coating agent is coated on the surface, and spray-dried to obtain the microbial agent; by putting the microbial agent into the soil, the coating is continuously decomposed in the soil, thereby releasing the internal microencapsulated PGRG microorganisms and microencapsulated microbial activators into the soil. Due to the network structure formed by the negatively charged nanofibers, the surface is rougher and it is easy to adhere to the soil particles, so it is trapped in the soil and not easy to lose, thereby increasing its action time in the soil, promoting the agglomeration of soil particles for a long time, improving the soil structure, and preventing the soil from being compacted. Moreover, the microencapsulated PGRG microorganisms and microencapsulated microbial activators are both positively charged. Under the action of static electricity, they can be adsorbed together with the negatively charged soil colloids, thereby reducing the loss of soil colloids, helping to enhance the cohesion of the soil, thereby increasing the air permeability, water permeability and water retention of the soil, improving the physical properties of the soil, and achieving the improvement of the soil structure.

[0019] In the present invention, the screened PGRG microorganisms and microbial activators are subjected to microencapsulation treatment, and then embedded together with negatively charged nanofibers through a coating agent to obtain a microbial agent, which is then put into the soil. This can effectively improve the soil structure, enhance the soil cohesion, significantly improve soil compaction, and prevent the occurrence of soil compaction for a long time, thereby providing a suitable soil environment for plant growth. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] In the embodiment of the present invention, the screening and cultivation of PGRG microorganisms is carried out as follows: 1) Add 10 g of rhizosphere soil from the soybean planting area to 90 mL of sterile PBS, shake at 30 °C and 260 rpm for 30 min, then centrifuge at 5000 rpm for 15 min, discard the supernatant, and then add 10 mL of sterile PBS to the precipitate and shake to obtain a suspension; 2) Take 2 mL of the suspension and add it to 100 mL of LB liquid medium containing 1 mmol / L of dipotassium hydrogen phosphate for screening culture. Shake at 30 °C and 180 rpm for two days, and then subculture 5 times according to an inoculation amount of 5%. Then, use the gradient dilution method to prepare 10 –2 -10 –6 series of dilution solutions. Take 100 μL each and spread them on LB solid medium, culture at 30 °C until the colonies grow well, pick a single colony, and use the streaking method to obtain a pure culture of the strain; 3) Pick the isolated and purified single colony and inoculate it into LB liquid medium for culture. Take 10 μL of the bacterial suspension cultured for 24 h, spread it evenly on a metal plate, and then place it in the sample treatment chamber of the ARTP mutagenesis system. Adjust the distance between the metal plate and the gas flow port to 2 mm. The working parameters of ARTP are as follows: the radio frequency power input is 100 W, the flow rate of pure helium as the working gas is 10 L / min, and the treatment time is 30 s. During the treatment process, apply intermittent ultrasonic waves with a frequency of 40 kHz and a power of 20 W for 20 s. Then, measure the lethality rate of the mutagenized strain to determine the optimal mutagenesis time; 4) Based on the mutagenesis time determined in step (4), elute the mutagenized sample with sterile normal saline and gradient dilute it into 10 -6 -10 -8 bacterial suspension. Take 100 μL of the bacterial suspension and inoculate it into LB solid medium with an increasing concentration of dipotassium hydrogen phosphate to screen the strain. The increasing gradient of the dipotassium hydrogen phosphate concentration in the LB solid medium is 10, 20, 30, 40, 50, 70, 100, and 120 mmol / L. After culturing and elimination, a high-purity strain is screened; 5) Inoculate the high-purity strain into LB liquid medium for activation, shake it on a shaker until the OD 600 value of the bacterial liquid is 0.8. Pipette 10 µL of the bacterial liquid and drop it on the organic phosphorus solid medium, mark the strain, seal the organic phosphorus solid medium and place it upside down in an incubator at 28 °C for 4 days. Observe and record every day whether a transparent circle appears on the plate during this period. Select the colonies with a transparent circle, measure the diameter D of the transparent circle and the diameter d of the colony, and judge the ability of the strain to decompose organic phosphorus through the D / d ratio, so as to screen out the PGRG microorganism with strong ability to degrade organic phosphorus.

[0022] In the embodiment of the present invention, the compound microbial activator is prepared as follows: Trace elements and growth factors are added to deionized water, and then a small molecule organic carbon source is added to obtain a primary composite microbial activator. Then, a pH buffer solution is added to the primary composite microbial activator to adjust the pH to 6.5 to obtain a composite microbial activator; The ratio of trace elements, growth factors, small molecule organic carbon source, pH buffer, and deionized water is 55 mL: 1 mL: 10 g: 1.0 mL: 900 mL; The formulation of the trace elements is zinc sulfate 0.3 g / L, copper sulfate 1.2 g / L, ammonium molybdate 0.2 g / L, sodium borate 0.05 g / L, magnesium sulfate 0.05 g / L, and ferric sulfate 0.5 g / L; The formulation of the growth factors is nicotinic acid 4 g / L, pantothenic acid 5 g / L, and cobalamin 4 g / L; The small molecule organic carbon source is selected as glucose; The formulation of the pH buffer solution is composed of 0.2 mol / L boric acid solution, 0.2 mol / L potassium chloride solution, and 0.1 mol / L sodium hydroxide solution in a volume ratio of 50:50:1.

[0023] In the embodiment of the present invention, the preparation method of the negatively charged nanofibers is as follows: 1) Commercially available chitin is soaked in dilute hydrochloric acid at room temperature for 12 h, washed repeatedly with distilled water, then soaked in 0.10 mol / L sodium hydroxide solution for 12 h, and then washed repeatedly with distilled water. The pigment is removed with 0.3 wt% sodium chlorite and dried to obtain chitin powder with an acetylation degree of 95%; 2) 0.5 g of chitin powder is dispersed in 100 g of an aqueous solution of ammonium persulfate with a concentration of 1 mol / L, and magnetically stirred at 60 °C for 20 h to oxidize the hydroxymethyl on the chitin molecular chain into a carboxyl group, and then negatively charged nanofibers are obtained by centrifugation.

[0024] Example 1 A microbial agent for preventing soil compaction by PGPR includes the following raw materials in parts by weight: 50 parts of microencapsulated PGRG microorganisms, 8 parts of microencapsulated microbial activator, 10 parts of negatively charged nanofibers, and 40 parts of coating agent; The coating agent is a mixture composed of polyethylene glycol and polypropylene glycol in a weight ratio of 60:40; The coating agent is dissolved in a diethylene glycol ether solvent to form a film; The preparation method of the microbial agent specifically includes the following steps: According to the parts by weight, the coating agent is dissolved in a diethylene glycol ether solvent to obtain a coating agent solution with a concentration of 25 wt%. Then, the microencapsulated PGRG microorganisms, the microencapsulated microbial activator, and the negatively charged nanofibers are fully mixed and then put into the coating agent solution. After sufficient stirring, spray drying is carried out.

[0025] Among them, the microencapsulated PGRG microorganism is prepared as follows: 1) Dissolve whey protein in sterilized distilled water, stir thoroughly, and pasteurize in a water bath at 80°C for 5 minutes to obtain a whey protein solution with a concentration of 80 g / L. Then, dissolve trehalose in sterile distilled water, and sterilize in a water bath at 80°C for 5 minutes to obtain a trehalose solution with a concentration of 20 g / L. 2) Positively charged hollow nanorods were added to the whey protein solution and dispersed evenly to obtain a dispersion with a solid content of 1.0%. 75 mL of the dispersion, 75 mL of the trehalose solution and 50 mg of the PGRG microorganism were fully mixed, and then spray-dried under the conditions of an air flow rate of 350 L / h, a feed flow rate of 0.3 L / h, an inlet air temperature of 120°C, an outlet air temperature of 65°C, and a carrier gas pressure of 0.1 MPa to obtain microencapsulated PGRG microorganisms.

[0026] Among them, the microencapsulated microbial activator is prepared by the following method: 1) Dissolve whey protein in sterilized distilled water, stir thoroughly, and pasteurize in a water bath at 80°C for 5 minutes to obtain a whey protein solution with a concentration of 80 g / L. Then, dissolve trehalose in sterile distilled water, and sterilize in a water bath at 80°C for 5 minutes to obtain a trehalose solution with a concentration of 20 g / L. 2) Positively charged hollow nanorods were added to the whey protein solution and dispersed evenly to obtain a dispersion with a solid content of 1.0%. 75 mL of the dispersion, 75 mL of the trehalose solution and 120 mg of the composite microbial activator were fully mixed, and then spray-dried under the conditions of an air flow rate of 350 L / h, a feed flow rate of 0.3 L / h, an inlet air temperature of 120°C, an outlet air temperature of 65°C, and a carrier gas pressure of 0.1 MPa to obtain a microencapsulated microbial activator.

[0027] The above-mentioned positively charged hollow nanorods are prepared as follows: 1) Dissolve 0.7 g of trimesic acid in 50 mL of methanol, then add 0.09 g of bismuth nitrate, stir well and transfer to a reactor, react at 120 ° C for 24 h, centrifuge after the reaction is completed, wash repeatedly with methanol, and dry to obtain a precursor; 2) Add 0.5g of the precursor and 1.0g of potassium bromide into 500mL of deionized water, stir at 500r / min for 30min, and then place in a 90℃ oil bath for 1h. After the treatment, wash the product repeatedly with deionized water and ethanol, and dry it to obtain hollow nanorods; 3) Add 1 g of hollow nanorods to 80 mL of a methanol / water solution with a volume ratio of 80:20, adjust the pH value to 4.5, and ultrasonically disperse at 200 W for 2 h. Then, add 15 mL of KH550 and 3 mL of glacial acetic acid under stirring at 150 r / min, and ultrasonically treat for another 2 h. After centrifuging the obtained product, repeatedly wash it with pure water and acetone, and dry it to obtain positively charged hollow nanorods.

[0028] Example 2 A PGPR microbial agent for preventing soil compaction, comprising the following raw materials in parts by weight: 70 parts of microencapsulated PGRG microorganisms, 12 parts of microencapsulated microbial activators, 15 parts of negatively charged nanofibers, and 50 parts of coating agents; The coating agent is a mixture of polyethylene glycol and polypropylene glycol in a weight ratio of 70:30; The coating agent is dissolved in the glycol ether solvent to form a film; The preparation method of the microbial agent specifically comprises the following steps: According to the weight percentage, the coating agent is dissolved in the glycol ether solvent to obtain a coating agent solution with a concentration of 30wt%, and then the microencapsulated PGRG microorganisms, microencapsulated microorganism activator and negatively charged nanofibers are fully mixed and then put into the coating agent solution, and then spray-dried after being fully stirred.

[0029] Among them, the microencapsulated PGRG microorganism is prepared as follows: 1) Dissolve whey protein in sterilized distilled water, stir thoroughly, and pasteurize in a water bath at 82°C for 7 min to obtain a whey protein solution with a concentration of 90 g / L. Then, dissolve trehalose in sterile distilled water, and sterilize in a water bath at 82°C for 7 min to obtain a trehalose solution with a concentration of 30 g / L. 2) Positively charged hollow nanorods were added to the whey protein solution and dispersed evenly to obtain a dispersion with a solid content of 1.5%. 80 mL of the dispersion, 80 mL of the trehalose solution and 65 mg of the PGRG microorganism were fully mixed, and then spray-dried under the conditions of an air flow rate of 380 L / h, a feed flow rate of 0.4 L / h, an inlet air temperature of 121°C, an outlet air temperature of 67°C, and a carrier gas pressure of 0.1 MPa to obtain microencapsulated PGRG microorganisms.

[0030] Among them, the microencapsulated microbial activator is prepared by the following method: 1) Dissolve whey protein in sterilized distilled water, stir thoroughly, and pasteurize in a water bath at 82°C for 7 min to obtain a whey protein solution with a concentration of 90 g / L. Then, dissolve trehalose in sterile distilled water, and sterilize in a water bath at 82°C for 7 min to obtain a trehalose solution with a concentration of 30 g / L. 2) Positively charged hollow nanorods were added to the whey protein solution and dispersed evenly to obtain a dispersion with a solid content of 1.5%. 80 mL of the dispersion, 80 mL of the trehalose solution and 150 mg of the composite microbial activator were fully mixed, and then spray-dried under the conditions of an air flow rate of 370 L / h, a feed flow rate of 0.4 L / h, an inlet air temperature of 121°C, an outlet air temperature of 67°C and a carrier gas pressure of 0.1 MPa to obtain a microencapsulated microbial activator.

[0031] The above-mentioned positively charged hollow nanorods are prepared as follows: 1) Dissolve 0.9 g of trimesic acid in 65 mL of methanol, then add 0.12 g of bismuth nitrate, stir well and transfer to a reactor, react at 125 °C for 28 h, centrifuge after the reaction is complete, wash repeatedly with methanol, and dry to obtain a precursor; 2) 0.7 g of the precursor and 1.3 g of potassium bromide were added to 700 mL of deionized water, stirred at 700 r / min for 40 min, and then placed in a 92°C oil bath for 1.5 h. After the treatment, the product was repeatedly washed with deionized water and ethanol, and dried to obtain hollow nanorods; 3) Add 2 g of hollow nanorods to 100 mL of a methanol / water solution with a volume ratio of 85:15, adjust the pH value to 5.0, and ultrasonically disperse at 250 W for 2.5 h. Then, add 18 mL of KH550 and 5 mL of glacial acetic acid under stirring at 180 r / min, and ultrasonically treat for another 2.5 h. After centrifuging the obtained product, repeatedly wash it with pure water and acetone, and dry it to obtain positively charged hollow nanorods.

[0032] Example 3 A PGPR microbial agent for preventing soil compaction, comprising the following raw materials in parts by weight: 80 parts of microencapsulated PGRG microorganisms, 15 parts of microencapsulated microbial activators, 20 parts of negatively charged nanofibers, and 60 parts of coating agents; The coating agent is a mixture of polyethylene glycol and polypropylene glycol in a weight ratio of 80:20; The coating agent is dissolved in the glycol ether solvent to form a film; The preparation method of the microbial agent specifically comprises the following steps: According to the weight percentage, the coating agent is dissolved in the glycol ether solvent to obtain a coating agent solution with a concentration of 35wt%, and then the microencapsulated PGRG microorganisms, microencapsulated microorganism activator and negatively charged nanofibers are fully mixed and then put into the coating agent solution, and spray-dried after being fully stirred.

[0033] Among them, the microencapsulated PGRG microorganism is prepared as follows: 1) Dissolve whey protein in sterilized distilled water, stir thoroughly, and pasteurize in a water bath at 85°C for 10 min to obtain a whey protein solution with a concentration of 100 g / L. Then, dissolve trehalose in sterile distilled water, and sterilize in a water bath at 85°C for 10 min to obtain a trehalose solution with a concentration of 40 g / L. 2) Positively charged hollow nanorods were added to the whey protein solution and dispersed evenly to obtain a dispersion with a solid content of 1.8%. 100 mL of the dispersion, 100 mL of the trehalose solution and 80 mg of the PGRG microorganism were fully mixed, and then spray-dried under the conditions of an air flow rate of 400 L / h, a feed flow rate of 0.5 L / h, an inlet air temperature of 123°C, an outlet air temperature of 70°C, and a carrier gas pressure of 0.2 MPa to obtain microencapsulated PGRG microorganisms.

[0034] Among them, the microencapsulated microbial activator is prepared by the following method: 1) Dissolve whey protein in sterilized distilled water, stir thoroughly, and pasteurize in a water bath at 85°C for 10 min to obtain a whey protein solution with a concentration of 100 g / L. Then, dissolve trehalose in sterile distilled water, and sterilize in a water bath at 85°C for 10 min to obtain a trehalose solution with a concentration of 40 g / L. 2) Positively charged hollow nanorods were added to the whey protein solution and dispersed evenly to obtain a dispersion with a solid content of 1.8%. 100 mL of the dispersion, 100 mL of the trehalose solution and 180 mg of the composite microbial activator were fully mixed, and then spray-dried under the conditions of an air flow rate of 400 L / h, a feed flow rate of 0.5 L / h, an inlet air temperature of 123°C, an outlet air temperature of 70°C, and a carrier gas pressure of 0.2 MPa to obtain a microencapsulated microbial activator.

[0035] The above-mentioned positively charged hollow nanorods are prepared as follows: 1) Dissolve 1.2 g of trimesic acid in 80 mL of methanol, then add 0.13 g of bismuth nitrate, stir well and transfer to a reactor, react at 130 ° C for 30 h, centrifuge after the reaction is completed, wash repeatedly with methanol, and dry to obtain a precursor; 2) 0.8 g of the precursor and 1.5 g of potassium bromide were added to 800 mL of deionized water, stirred at 800 r / min for 50 min, and then placed in a 93°C oil bath for 2 h. After the treatment, the product was repeatedly washed with deionized water and ethanol, and dried to obtain hollow nanorods; 3) Add 3 g of hollow nanorods into 130 mL of methanol / water solution with a volume ratio of 85:15, adjust the pH value to 5.0, ultrasonically disperse for 3 h at 300 W, then add 20 mL of KH550 and 6 mL of glacial acetic acid under stirring at 200 r / min, and ultrasonically treat for another 3 h. After centrifuging the obtained product, wash it repeatedly with pure water and acetone, and dry it to obtain positively charged hollow nanorods.

[0036] Comparative Example 1: This comparative example is basically the same as Example 1, except that it does not contain the microencapsulated microbial activator.

[0037] Comparative Example 2: This comparative example is basically the same as Example 1, except that the PGRG microorganisms are not microencapsulated.

[0038] Comparative Example 3: This comparative example is basically the same as Example 1, except that the microbial activator is not microencapsulated.

[0039] Comparative Example 4: This comparative example is basically the same as Example 1, except that it does not contain negatively charged nanofibers.

[0040] Comparative Example 5: This comparative example is basically the same as Example 1, except that it does not contain positively charged hollow nanorods for both the microencapsulated PGRG microorganisms and the microencapsulated microbial activator.

[0041] Test experiment: 1. Soil preparation Select a field with soil compaction and build a greenhouse. The soil bulk density of the selected field is 1.5 - 1.6, the total porosity is 25 - 30%, and the aeration porosity is 7 - 9%.

[0042] 2. Seedling raising The seeds are soaked in warm water at a water temperature of 55 - 65 °C for 30 minutes, stirring constantly during soaking.

[0043] Agent treatment: Soak the seeds in 1% trisodium phosphate for 30 minutes and in 100 - fold diluted formalin for 20 - 30 minutes, and set aside.

[0044] Put the treated seeds into a plug tray filled with substrate for seedling raising, with one seed in each plug hole.

[0045] The substrate is made by mixing attapulgite powder, shell powder, vermiculite powder, peanut shell powder, pine bark powder, plant ash, blue crushed stone, and isatis root powder in a mass ratio of 2:3:2:1:1.5:4:1:0.02, disinfecting it, and having a moisture content of 50 - 60%.

[0046] When the seedlings in the plug tray grow to 30 - 50 cm in height, prepare for transplanting and planting.

[0047] 3. Planting When the seedling height reaches 30 - 50 cm, transplant it into the greenhouse, usually in January. When transplanting, dig seedling holes in the planting area, one seedling per hole, with a spacing of 20 - 30 cm between each plant and a row spacing of 50 - 60 cm.

[0048] After transplanting, water gently in time. Do not flood the field with large amounts of water to prevent the newly transplanted chili peppers from being washed down.

[0049] 4. Field management: The roots of chili peppers are not well-developed and have shallow roots. Therefore, when watering, the time should not be too long, and flooding is strictly prohibited. Furrow irrigation or drip irrigation must be adopted.

[0050] Irrigation amount: Irrigate 5 - 6 times during the whole growth period, with each irrigation amount of 50 - 60 m 3 / mu, and keep the soil moist. It should follow the principle of frequent irrigation with small amounts of water. Flooding the field with large amounts of water and waterlogging in the field are strictly prohibited. Do not water at noon when it is hot or on rainy days. This is also the main measure to prevent chili pepper blight.

[0051] 5. Fertilization Mix the microbial inoculants obtained in Examples 1 - 3 and Comparative Examples 1 - 5 with potassium sulfate compound fertilizer respectively and then apply them. The amount of microbial inoculant is 2 kg / mu, and the amount of potassium sulfate compound fertilizer is 40 kg / mu.

[0052] 6. Disease control After transplanting pepper seedlings into the greenhouse, they are prone to chili pepper blight. When blight occurs, 600 - fold liquid of 50% copper methanate wettable powder or 600 - fold liquid of 30% metalaxyl - mancozeb can be used to drench the roots of diseased plants and surrounding plants. The amount of liquid medicine per plant is 250 grams, and drench 1 - 2 times with an interval of 5 - 7 days.

[0053] Through experiments, it is found that using the microbial inoculant of the present invention can significantly improve the soil, and the effect of growing chili peppers is good. The specific effect indicators are shown in Table 1.

[0054] Table 1 It can be seen from Table 1 that the microbial inoculant in the present invention can effectively improve the soil structure, enhance the cohesion of the soil, significantly improve soil compaction, and at the same time, can prevent the occurrence of soil compaction for a long time, provide a suitable soil environment for plant growth, and achieve the effect of increasing production and income.

[0055] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of this specification. These embodiments are selected and specifically described in this specification to better explain the principles and practical applications of the present invention, so that those skilled in the art can well understand and utilize the present invention. The present invention is only limited by the claims and their full scope and equivalents.

Claims

1. A microbial inoculant of PGPR for preventing soil compaction, characterized in that, It comprises the following raw materials in parts by weight: 50 - 80 parts of microencapsulated PGRG microorganisms, 8 - 15 parts of microencapsulated microbial activator, 10 - 20 parts of negatively charged nanofibers, and 40 - 60 parts of coating agent; The coating agent is a mixture composed of polyethylene glycol and polypropylene glycol in a weight ratio of (60 - 80):(20 - 40); The coating agent is dissolved in a diethylene glycol ether solvent to form a film.

2. The microbial inoculum for preventing soil compaction by PGPR according to claim 1, characterized in that, For the microencapsulated PGRG microorganisms, its preparation method is as follows: S1 Isolate the microorganisms in the rhizosphere soil of the soybean planting area to obtain a pure culture of strains; S2 Under the action of ultrasound combined with plasma treatment, perform mutagenesis screening to obtain PGRG microorganisms with strong ability to degrade organic phosphorus; S3 Use whey protein and trehalose as wall materials and positively charged hollow nanorods as functional materials to microencapsulate the PGRG microorganisms, and the microencapsulated PGRG microorganisms can be obtained.

3. The microbial inoculant for preventing soil compaction by PGPR according to claim 2, characterized in that, In S2, for the ultrasound action, it is under intermittent ultrasonic waves with a frequency of 0.1 - 1000 kHz and a power of 0.01 - 1000 W, and the duration is 20 - 30 s; For the plasma treatment, place the metal plate in the sample treatment chamber of the ARTP mutagenesis system, adjust the distance between the metal plate and the gas flow port to 2 - 5 mm, and the working parameters of the ARTP are that the radio frequency power input is 100 - 150 W, the flow rate of pure helium as the working gas is 10 - 15 L / min, and the treatment time is 20 - 90 s.

4. A microbial agent for preventing soil compaction by PGPR according to claim 2, characterized in that, In S2, for the mutagenesis screening, culture the mutagenized strains in an LB solid medium with a gradually increasing concentration of dipotassium hydrogen phosphate to obtain high-purity strains, and then perform screening on an organic phosphorus solid medium; The concentration of dipotassium hydrogen phosphate increases in a gradient, and the gradient is 10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 70 mmol / L, 100 mmol / L, and 120 mmol / L.

5. A microbial inoculant for preventing soil compaction by PGPR according to claim 1, characterized in that, For the microencapsulated microbial activator, its preparation method is as follows: S1 Add trace elements and growth factors to deionized water, then add a small molecule organic carbon source to obtain a primary composite microbial activator, and then add a pH buffer solution to the primary composite microbial activator to adjust the pH to 6.5 to obtain a composite microbial activator; S2 Use whey protein and trehalose as wall materials and positively charged hollow nanorods as functional materials to microencapsulate the composite microbial activator, and the microencapsulated composite microbial activator can be obtained.

6. The microbial inoculum for preventing soil compaction by PGPR according to claim 5, characterized in that, In S1, the ratio of the trace elements, growth factors, small molecule organic carbon source, pH buffer, and deionized water is (55 - 60) mL:(1 - 3) mL:(10 - 15) g:(1.0 - 1.5) mL:(900 - 1200) mL; The formula of the trace elements is 0.3 - 0.5 g / L of zinc sulfate, 1.2 - 1.6 g / L of copper sulfate, 0.2 - 0.3 g / L of ammonium molybdate, 0.05 - 0.08 g / L of sodium borate, 0.05 - 0.08 g / L of magnesium sulfate, and 0.5 - 0.8 g / L of ferric sulfate; The formula of the growth factor is 4 - 6 g / L of nicotinic acid, 5 - 7 g / L of pantothenic acid, and 4 - 6 g / L of cobalamin; The small molecule organic carbon source is selected from at least one of glucose, sodium citrate, sodium acetate, and sucrose; The formula of the pH buffer solution is composed of 0.2 mol / L boric acid solution, 0.2 mol / L potassium chloride solution, and 0.1 mol / L sodium hydroxide solution in a volume ratio of (50 - 60):(50 - 60):

1.

7. A microbial agent for preventing soil compaction by PGPR according to any one of claims 2 or 5, characterized in that For the positively charged hollow nanorods, the preparation method is as follows: S1 Using trimesic acid and bismuth nitrate as raw materials, methanol as the solvent, a precursor is obtained through a hydrothermal reaction. Then, the precursor and potassium bromide are mixed and added to deionized water, and after oil bath treatment, hollow nanorods are obtained; S2 Amino-functionalized modification: The hollow nanorods are added to a methanol aqueous solution, the pH is adjusted to 4.5 - 5.0, ultrasonic treatment is carried out for 1 - 3 h, KH550 and glacial acetic acid are added while stirring, ultrasonic treatment is continued for 1 - 3 h, and then centrifugation, washing, and drying are carried out to obtain the product.

8. The microbial inoculum for preventing soil compaction by PGPR according to claim 7, characterized in that, The ratio of trimesic acid, methanol, and bismuth nitrate is (0.7 - 1.2) g:(50 - 80) mL:(0.09 - 0.13) g; For the hydrothermal reaction, the temperature is 120 - 130 °C and the reaction time is 24 - 30 h; The ratio of the precursor, potassium bromide, and deionized water is (0.5 - 0.8) g:(1.0 - 1.5) g:(500 - 800) mL; For the oil bath treatment, the temperature is 90 - 93 °C and the treatment time is 1 - 2 h; The ratio of the hollow nanorods, methanol / aqueous solution, KH550, and glacial acetic acid is (1 - 3) g:(80 - 130) mL:(15 - 20) mL:(3 - 6) mL; For the methanol / aqueous solution, the volume ratio is (80 - 85):(15 - 20).

9. A microbial agent for preventing soil compaction by PGPR according to claim 1, characterized in that, For the negatively charged nanofibers, the preparation method is as follows: 1) Soak commercially available chitin in dilute hydrochloric acid at room temperature for 12 - 15 h, wash it repeatedly with distilled water, then soak it in sodium hydroxide solution for 12 - 15 h, and after washing it repeatedly with distilled water, decolorize it with 0.3 - 0.5 wt% sodium chlorite and dry it to obtain chitin powder with an acetylation degree of 95%; 2) Disperse the chitin powder in an aqueous solution of ammonium persulfate with a concentration of 1 mol / L, magnetically stir it at 60 - 65 °C for 20 - 24 h, and then obtain negatively charged nanofibers through centrifugation; The concentration of the sodium hydroxide solution is 0.10 - 0.15 mol / L; The mass ratio of the chitin powder to the ammonium persulfate aqueous solution is (0.5 - 0.8):(100 - 160).

10. A preparation method of a microbial inoculum for preventing soil compaction by PGPR according to claim 1, characterized in that, Specifically, it includes the following steps: By weight, dissolve the coating agent in a glycol ether solvent to obtain a coating agent solution with a concentration of 25 - 35 wt%, then fully mix the microencapsulated PGRG microorganisms, microencapsulated microorganism activators, and negatively charged nanofibers, and then put them into the coating agent solution. After sufficient stirring, spray drying can be carried out.

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