Microbial inoculant of pgpr for preventing soil compaction and preparation method thereof

Abstract

The present application relates to the technical field of soil improvement, and specifically relates to a PGPR microbial agent for preventing soil compaction and a preparation method thereof, which comprises the following raw materials in parts by weight: microencapsulated PGPR microorganism 50-80 parts, microencapsulated microbial activator 8-15 parts, negative charge nanofiber 10-20 parts, and coating agent 40-60 parts; the coating agent is a mixture of polyethylene glycol and polypropylene glycol in a weight ratio of (60-80):(20-40); and the coating agent is dissolved in a glycol ether solvent to form a film. In the present application, the PGPR microorganism and the microbial activator screened are subjected to microencapsulation treatment, and then embedded with the negative charge nanofiber by using a coating agent to obtain a microbial agent, which is put into soil, can effectively improve soil structure, enhance the coagulation of soil, significantly improve soil compaction, and can also prevent soil compaction for a long time, thereby providing a suitable soil environment for plant growth.

Description

Technical Field

[0001] This invention relates to the field of soil improvement technology, specifically to a microbial agent for preventing soil compaction using PGPR and its preparation method. Background Technology

[0002] PGPR (Polymeric Progenitor Rhizogenes) are beneficial microorganisms that live freely within plants, epiphytically on roots or in the rhizosphere soil. They exert biocontrol effects against pathogens, promote the absorption of minerals and other inorganic substances by plants, and produce compounds beneficial to plant growth. PGPR microorganisms have the ability to decompose organic matter, transforming organic residues in the soil, such as plant roots and fallen leaves, into humus and other organic matter. Humus increases soil organic matter content, improves soil physical properties, and provides nutrients for soil microorganisms, promoting their growth and reproduction, further enhancing soil fertility and structural stability. Furthermore, PGPR microorganisms participate in the transformation and cycling of nutrients such as nitrogen, phosphorus, and potassium in the soil, improving the availability of these nutrients and enabling plants to better absorb them, promoting plant growth. The growth of plant roots also has a positive impact on soil structure, helping to prevent soil compaction.

[0003] For example, Chinese patent CN114921370B discloses a microbial soil conditioner and remediation agent containing a compound microbial agent, which includes Bacillus vesiculosus ZLP-101, Bacillus subtilis BSD-2, silicate bacteria HM8841, and phosphate-solubilizing bacteria HM0332. This microbial soil conditioner and remediation agent can improve soil physical properties and microecological environment, enhance soil permeability, effectively improve soil compaction and acidification problems, and significantly kill underground pests, reducing their incidence. However, when this microbial soil conditioner and remediation agent is applied to the soil, although it can improve the physical properties of the soil, its effect is limited because it is easily lost in the soil and has a low retention rate. It cannot exert its effects for a long time, and it cannot stabilize the soil structure to prevent the recurrence of soil compaction. Summary of the Invention

[0004] To address the problems existing in the prior art, the purpose of this invention is to provide a microbial agent for preventing soil compaction using PGPR and its preparation method.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A microbial agent for preventing soil compaction using PGPR comprises the following raw materials in parts by weight: 50-80 parts of microencapsulated PGPR microorganisms, 8-15 parts of microencapsulated microorganism activator, 10-20 parts of negatively charged nanofibers, and 40-60 parts of coating agent.

[0007] The coating agent is a mixture of polyethylene glycol and polypropylene glycol in a weight ratio of (60-80):(20-40);

[0008] The coating agent is dissolved in a diol ether solvent to form a thin film.

[0009] As a further preferred embodiment of the present invention, the microencapsulated PGPR microorganisms are prepared by the following method:

[0010] S1 isolates microorganisms from the rhizosphere soil of soybean growing areas to obtain pure cultures of the strains;

[0011] S2 was subjected to mutagenesis screening under ultrasonic treatment combined with plasma treatment to obtain PGPR microorganisms with strong ability to degrade organophosphorus compounds.

[0012] S3 uses whey protein and trehalose as wall materials and positively charged hollow nanorods as functional materials to microencapsulate PGPR microorganisms, thus obtaining microencapsulated PGPR microorganisms.

[0013] Furthermore, the specific operation method in S1 is as follows:

[0014] 1) Add 10-15g of rhizosphere soil from the soybean growing area to 90-150mL of sterile PBS, shake at 30℃ and 180-260rpm for 30-50min, then centrifuge at 5000-8000rpm for 15-20min, remove the supernatant, then add 10-20mL of sterile PBS to the precipitate, shake to obtain a suspension;

[0015] 2) Add 2-5 mL of the suspension to 100-200 mL of LB liquid medium containing 1-3 mmol / L dipotassium hydrogen phosphate for selection culture. Culture at 30℃ and 180-260 rpm with shaking for two days. Then, subculture 5-6 times at an inoculum rate of 5-6%. Prepare 10n samples using a serial dilution method. –2 -10 –6 A series of dilutions were prepared, and 100-160 μL of each was spread onto LB solid medium. The medium was incubated at 30°C until colonies were well grown. Single colonies were picked and pure cultures of the strain were obtained by streak plating.

[0016] Furthermore, the specific operation method in S2 is as follows:

[0017] 1) Pick a single colony after isolation and purification and inoculate it into LB liquid medium for culture. Take 10-15 μL of bacterial suspension cultured for 24 h and spread it evenly on a metal plate. Then place it in the sample processing chamber of the ARTP mutagenesis system. Adjust the distance between the metal plate and the gas flow port to 2-5 mm. The operating parameters of ARTP are: radio frequency power input of 100-150 W, flow rate of pure helium working gas of 10-15 L / min, and treatment time of 20-90 s. During the treatment, apply intermittent ultrasound with a frequency of 0.1-1000 kHz and a power of 0.01-1000 W for 20-30 s. Then determine the lethality of the mutagenic strain and determine the optimal mutagenesis time.

[0018] 2) Based on the mutagenesis time determined in step (4), the mutagenesis sample is eluted with sterile physiological saline and serially diluted to 10⁻⁶. -6 -10 -8 100 μL of bacterial suspension was inoculated into LB solid medium with increasing dipotassium hydrogen phosphate concentrations to screen strains. The dipotassium hydrogen phosphate concentrations in the LB solid medium were increasing at 10, 20, 30, 40, 50, 70, 100 and 120 mmol / L. After culturing and elimination, high-purity strains were obtained.

[0019] 3) Inoculate the high-purity bacterial strain into LB liquid medium for activation, and shake on a shaker until the bacterial solution reaches OD. 600 The value is 0.8. Take 10-20 µL of bacterial suspension and drop it onto organophosphate solid medium. Label the strain, seal the organophosphate solid medium and invert it in a 28℃ incubator for 4-7 days. During this period, observe and record whether a clear zone appears on the plate every day. Select colonies with clear zones and measure the diameter D of the clear zone and the diameter d of the colony. The D / d ratio is used to determine the ability of the strain to degrade organophosphates, thereby screening out PGPR microorganisms with strong organophosphate degradation capabilities.

[0020] Furthermore, the specific operation method in S3 is as follows:

[0021] 1) Dissolve whey protein in sterile distilled water, stir thoroughly, and pasteurize in a water bath at 80-85℃ for 5-10 minutes to obtain a whey protein solution with a concentration of 80-100 g / L. Then dissolve trehalose in sterile distilled water and sterilize in a water bath at 80-85℃ for 5-10 minutes to obtain a trehalose solution with a concentration of 20-40 g / L.

[0022] 2) Positively charged hollow nanorods were added to the whey protein solution and dispersed evenly to obtain a dispersion with a solid content of 1.0-1.8%. 75-100 mL of the dispersion, 75-100 mL of trehalose solution and 50-80 mg of PGPR microorganisms were thoroughly mixed. Then, spray drying was carried out under the conditions of air flow rate of 350-400 L / h, feed flow rate of 0.3-0.5 L / h, inlet air temperature of 120-123℃, outlet air temperature of 65-70℃ and carrier gas pressure of 0.1-0.2 MPa to obtain microencapsulated PGPR microorganisms.

[0023] As a further preferred embodiment of the present invention, the microencapsulated microbial activator is prepared by the following method:

[0024] S1 adds trace elements and growth factors to deionized water, then adds a small molecule organic carbon source to obtain a primary compound microbial activator. Then, a pH buffer solution is added to the primary compound microbial activator to adjust the pH to 6.5, thus obtaining a compound microbial activator.

[0025] S2 uses whey protein and trehalose as wall materials and positively charged hollow nanorods as functional materials to microencapsulate the composite microbial activator, thus obtaining the microencapsulated composite microbial activator.

[0026] 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;

[0027] The formula for the trace elements is as follows: zinc sulfate 0.3-0.5 g / L, copper sulfate 1.2-1.6 g / L, ammonium molybdate 0.2-0.3 g / L, sodium borate 0.05-0.08 g / L, magnesium sulfate 0.05-0.08 g / L, and ferric sulfate 0.5-0.8 g / L.

[0028] The growth factor formulation consists of 4-6 g / L nicotinic acid, 5-7 g / L pantothenic acid, and 4-6 g / L cobalamin.

[0029] The small molecule organic carbon source is selected from at least one of glucose, sodium citrate, sodium acetate, and sucrose;

[0030] The pH buffer solution is formulated as follows: 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.

[0031] Furthermore, the specific operation method in S2 is as follows:

[0032] 1) Dissolve whey protein in sterile distilled water, stir thoroughly, and pasteurize in a water bath at 80-85℃ for 5-10 minutes to obtain a whey protein solution with a concentration of 80-100 g / L. Then dissolve trehalose in sterile distilled water and sterilize in a water bath at 80-85℃ for 5-10 minutes to obtain a trehalose solution with a concentration of 20-40 g / L.

[0033] 2) Add positively charged hollow nanorods to the whey protein solution and disperse them evenly to obtain a dispersion with a solid content of 1.0-1.8%. Mix 75-100 mL of the dispersion, 75-100 mL of trehalose solution and 120-180 mg of composite microbial activator thoroughly. Then spray dry the mixture under the conditions of air flow rate of 350-400 L / h, feed flow rate of 0.3-0.5 L / h, inlet air temperature of 120-123℃, outlet air temperature of 65-70℃ and carrier gas pressure of 0.1-0.2 MPa to obtain microencapsulated microbial activator.

[0034] As a further preferred embodiment of the present invention, the positively charged hollow nanorods are prepared by the following method:

[0035] S1 uses pyromellitic acid and bismuth nitrate as raw materials and methanol as solvent to obtain a precursor through hydrothermal reaction. Then, the precursor is mixed with potassium bromide and added to deionized water. After oil bath treatment, hollow nanorods are obtained.

[0036] S2 amino-functionalized modification: Hollow nanorods were added to a methanol aqueous solution, the pH was adjusted to 4.5-5.0, and the mixture was sonicated for 1-3 hours. KH550 and glacial acetic acid were added while stirring, and the mixture was sonicated for another 1-3 hours. The mixture was then centrifuged, washed, and dried to obtain the final product.

[0037] Furthermore, the ratio of pyromellitic acid, methanol, and bismuth nitrate is (0.7-1.2) g : (50-80) mL : (0.09-0.13) g;

[0038] The hydrothermal reaction is carried out at a temperature of 120-130℃ for 24-30 hours.

[0039] The ratio of the precursor, potassium bromide, and deionized water is (0.5-0.8) g : (1.0-1.5) g : (500-800) mL;

[0040] The oil bath treatment is performed at a temperature of 90-93℃ for 1-2 hours.

[0041] The ratio of hollow nanorods, methanol / water solution, KH550, and glacial acetic acid is (1-3) g: (80-130) mL: (15-20) mL: (3-6) mL;

[0042] The methanol / water solution has a volume ratio of (80-85):(15-20).

[0043] As a further preferred embodiment of the present invention, the negatively charged nanofibers are prepared by the following method:

[0044] 1) Soak commercially available chitin in dilute hydrochloric acid at room temperature for 12-15 hours, wash repeatedly with distilled water, soak in sodium hydroxide solution for 12-15 hours, wash repeatedly with distilled water, decolorize with 0.3-0.5wt% sodium chlorite, and dry to obtain chitin powder with an acetylation degree of 95%.

[0045] 2) Chitosan powder was dispersed in a 1 mol / L ammonium persulfate aqueous solution and magnetically stirred at 60-65℃ for 20-24 h. Then, negatively charged nanofibers were obtained by centrifugation.

[0046] The sodium hydroxide solution has a concentration of 0.10-0.15 mol / L;

[0047] The mass ratio of chitin powder to ammonium persulfate aqueous solution is (0.5-0.8):(100-160).

[0048] A method for preparing a microbial agent for preventing soil compaction using PGPR (Potentially Produced Residue Preservative), characterized by comprising the following steps:

[0049] The coating agent is dissolved in a diol ether solvent according to the weight percentage to obtain a coating agent solution with a concentration of 25-35 wt%. The microencapsulated PGPR microorganisms, microencapsulated microbial activators and negatively charged nanofibers are then thoroughly mixed and added to the coating agent solution. After thorough stirring, the mixture is spray-dried.

[0050] Compared with the prior art, the beneficial effects of the present invention are:

[0051] In this invention, rhizosphere soil from soybean growing areas serves as the source of PGPR microorganisms. Strains are obtained through isolation and screening in a dipotassium hydrogen phosphate (DHP) medium. These strains are then mutagenized using a combination of ultrasound and plasma treatment. Ultrasound promotes material exchange between microbial cells and the surrounding environment, accelerates nutrient transport to the cell surface, and promotes microbial metabolic activity. Simultaneously, plasma treatment alters the gene sequence of the microorganisms, allowing for the selection of high-yielding strains with superior traits and stable inheritance. These strains are then inoculated into a medium with progressively increasing DHP concentrations to screen for highly phosphorus-tolerant strains. A secondary screening on an organic phosphorus medium yields PGPR microorganisms with strong organic phosphorus degradation capabilities. The selected PGPR microorganisms effectively decompose organic matter in the soil, releasing nutrients and producing sticky substances that promote soil particle aggregation and improve soil structure. In particular, they efficiently decompose residual organic phosphorus in the soil, converting it into inorganic phosphorus, thereby promoting absorption and utilization by plant roots. This effectively improves soil compaction while also promoting plant growth. Furthermore, to enhance the utilization rate of PGPR microorganisms, they are further developed... This invention allows for long-term and efficient operation in soil. It involves microencapsulation of PGPR microorganisms using whey protein and trehalose as wall materials. Positively charged hollow nanorods are also incorporated into the wall material. These hollow nanorods, due to their positive charge, repel each other under electrostatic forces, allowing them to disperse uniformly within the wall material. Their rod-like structure forms a supporting framework, enhancing the wall material's resistance and thus strengthening the microcapsules' ability to withstand external forces. This ensures the microcapsules maintain their structural integrity during subsequent treatments. Its hollow structure provides a channel for the release of PGPR microorganisms, enabling a slow-release effect and effectively improving their utilization rate. Furthermore, in order to enhance the biological activity and promote the growth of PGPR microorganisms after they are released into the soil, the present invention also microencapsulates a composite microbial activator composed of trace elements, growth factors, and small molecule organic carbon sources, so that it can also be slowly released into the soil, thereby promoting the growth and metabolism of PGPR microorganisms, accelerating their reproduction and growth, and thus quickly improving soil compaction.

[0052] To combine microencapsulated PGPR microorganisms and microencapsulated microbial activators, this invention involves deastringing chitin after acid-base treatment, followed by oxidation with ammonium persulfate to oxidize the hydroxymethyl groups on the chitin molecular chain into carboxyl groups, thereby obtaining negatively charged nanofibers. A coating agent is then dissolved in a diol ether to obtain a coating agent solution. The microencapsulated PGPR microorganisms, microencapsulated microbial activators, and negatively charged nanofibers are then added to the coating agent solution and thoroughly mixed. Since both the microencapsulated PGPR microorganisms and microencapsulated microbial activators carry a positive charge, they are uniformly dispersed in the coating agent solution. Under electrostatic attraction, the negatively charged nanofibers adsorb onto the surface of the microencapsulated PGPR microorganisms and microencapsulated microbial activators. Through thorough mechanical stirring, the negatively charged nanofibers continuously entangle and cross-link, forming a wrapped network structure, thereby encapsulating the microencapsulated PGPR microorganisms and microencapsulated microbial activators. Microbial activators are encapsulated, and a coating agent is applied to the surface. After spray drying, a microbial agent is obtained. By introducing the microbial agent into the soil, the coating decomposes continuously, releasing the microencapsulated PGPR microorganisms and microencapsulated microbial activators into the soil. Due to the network structure formed by negatively charged nanofibers, the surface is rougher and easily adheres to soil particles, thus retaining them in the soil and preventing loss. This increases their duration of action in the soil, promoting long-term soil particle aggregation, improving soil structure, and preventing soil compaction. Moreover, both the microencapsulated PGPR microorganisms and microencapsulated microbial activators carry a positive charge. Under electrostatic effects, they can adsorb with negatively charged soil colloids, reducing the loss of soil colloids and enhancing soil cohesion. This increases soil aeration, permeability, and water retention, improves soil physical properties, and ultimately improves soil structure.

[0053] In this invention, microencapsulated PGPR microorganisms and microbial activators obtained through screening are encapsulated together with negatively charged nanofibers and then coated with a coating agent to obtain microbial agents. When these agents are introduced into the soil, they can effectively improve soil structure, enhance soil cohesion, significantly improve soil compaction, and prevent soil compaction in the long term, thus providing a suitable soil environment for plant growth. Detailed Implementation

[0054] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0055] In this embodiment of the invention, the method for screening and culturing PGPR microorganisms is as follows:

[0056] 1) Add 10g of rhizosphere soil from the soybean planting area to 90mL of sterile PBS, shake at 30℃ and 260rpm for 30min, then centrifuge at 5000rpm for 15min, remove the supernatant, then add 10mL of sterile PBS to the precipitate, shake to obtain a suspension;

[0057] 2) Add 2 mL of the suspension to 100 mL of LB liquid medium containing 1 mmol / L dipotassium hydrogen phosphate for selection culture. Culture at 30℃ and 180 rpm for two days with shaking. Then, subculture 5 times at a 5% inoculum rate. Prepare 10n samples using a serial dilution method. –2 -10 –6 Each series of dilutions was spread at 100 μL onto LB solid medium and incubated at 30°C until colonies were well grown. Single colonies were picked and pure cultures of the strain were obtained by streak plating.

[0058] 3) Pick a single colony after isolation and purification and inoculate it into LB liquid medium for culture. Take 10 μL of bacterial suspension that has been cultured for 24 h and spread it evenly on a metal plate. Then place it in the sample processing chamber of the ARTP mutagenesis system. Adjust the distance between the metal plate and the gas flow port to 2 mm. The operating parameters of ARTP are: radio frequency power input of 100 W, working gas pure helium flow rate of 10 L / min, and processing time of 30 s. During the processing, apply intermittent ultrasound with a frequency of 40 kHz and a power of 20 W for 20 s. Then determine the lethality of the mutagenized strain and determine the optimal mutagenesis time.

[0059] 4) Based on the mutagenesis time determined in step (4), the mutagenesis sample is eluted with sterile physiological saline and serially diluted to 10⁻⁶. -6 -10 -8 100 μL of bacterial suspension was inoculated into LB solid medium with increasing dipotassium hydrogen phosphate concentrations to screen strains. The dipotassium hydrogen phosphate concentrations in the LB solid medium were increasing at 10, 20, 30, 40, 50, 70, 100 and 120 mmol / L. After culturing and elimination, high-purity strains were obtained.

[0060] 5) Inoculate the high-purity bacterial strain into LB liquid medium for activation, and shake on a shaker until the bacterial solution reaches OD. 600 The value was 0.8. 10 µL of bacterial suspension was dropped onto organophosphate solid medium, the strain was labeled, the organophosphate solid medium was sealed and inverted in a 28℃ incubator for 4 days. During this period, the appearance of a clear zone on the plate was observed and recorded every day. Colonies with clear zones were selected, and the diameter of the clear zone D and the diameter of the colony d were measured. The D / d ratio was used to determine the ability of the strain to degrade organophosphates, thereby screening out PGPR microorganisms with strong organophosphate degradation capabilities.

[0061] In this embodiment of the invention, the compound microbial activator is prepared as follows:

[0062] Trace elements and growth factors are added to deionized water, and then a small molecule organic carbon source is added to obtain a primary compound microbial activator. Then, a pH buffer solution is added to the primary compound microbial activator to adjust the pH to 6.5, thus obtaining a compound microbial activator.

[0063] The ratio of trace elements, growth factors, small molecule organic carbon sources, pH buffers, and deionized water is 55mL:1mL:10g:1.0mL:900mL;

[0064] The trace element formula 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.

[0065] The growth factor formula is 4g / L nicotinic acid, 5g / L pantothenic acid, and 4g / L cobalamin;

[0066] Glucose was chosen as the small molecule organic carbon source.

[0067] The pH buffer solution is formulated with 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.

[0068] In this embodiment of the invention, the method for preparing negatively charged nanofibers is as follows:

[0069] 1) Commercially available chitosan was soaked in dilute hydrochloric acid at room temperature for 12 hours, washed repeatedly with distilled water, soaked in 0.10 mol / L sodium hydroxide solution for 12 hours, washed repeatedly with distilled water, depigmented with 0.3 wt% sodium chlorite, and dried to obtain chitosan powder with an acetylation degree of 95%.

[0070] 2) Disperse 0.5g of chitin powder in 100g of 1mol / L ammonium persulfate aqueous solution and stir magnetically at 60℃ for 20h to oxidize the hydroxymethyl groups on the chitin molecular chain to carboxyl groups. Then, obtain negatively charged nanofibers by centrifugation.

[0071] Example 1

[0072] A microbial agent for preventing soil compaction using PGPR comprises the following raw materials in parts by weight: 50 parts microencapsulated PGPR microorganisms, 8 parts microencapsulated microorganism activator, 10 parts negatively charged nanofibers, and 40 parts coating agent.

[0073] The coating agent is a mixture of polyethylene glycol and polypropylene glycol in a weight ratio of 60:40;

[0074] The coating agent is dissolved in a diol ether solvent to form a thin film;

[0075] The preparation method of this microbial agent specifically includes the following steps:

[0076] The coating agent is dissolved in a diol ether solvent according to the weight percentage to obtain a coating agent solution with a concentration of 25 wt%. The microencapsulated PGPR microorganisms, microencapsulated microbial activators and negatively charged nanofibers are then thoroughly mixed and added to the coating agent solution. After thorough stirring, the mixture is spray-dried.

[0077] The preparation method of the microencapsulated PGPR microorganism is as follows:

[0078] 1) Dissolve whey protein in sterile distilled water, stir thoroughly, and pasteurize in an 80°C water bath 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 an 80°C water bath for 5 minutes to obtain a trehalose solution with a concentration of 20 g / L.

[0079] 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 trehalose solution and 50 mg of PGPR microorganisms were thoroughly mixed and then spray-dried under the conditions of air flow rate of 350 L / h, feed flow rate of 0.3 L / h, inlet air temperature of 120℃, outlet air temperature of 65℃ and carrier gas pressure of 0.1 MPa to obtain microencapsulated PGPR microorganisms.

[0080] The microencapsulated microbial activator is prepared as follows:

[0081] 1) Dissolve whey protein in sterile distilled water, stir thoroughly, and pasteurize in an 80°C water bath 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 an 80°C water bath for 5 minutes to obtain a trehalose solution with a concentration of 20 g / L.

[0082] 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 trehalose solution and 120 mg of composite microbial activator were thoroughly mixed and then spray-dried under the conditions of air flow rate of 350 L / h, feed flow rate of 0.3 L / h, inlet air temperature of 120℃, outlet air temperature of 65℃ and carrier gas pressure of 0.1 MPa to obtain microencapsulated microbial activator.

[0083] The above-mentioned positively charged hollow nanorods are prepared by the following method:

[0084] 1) Dissolve 0.7g of trimesic acid in 50mL of methanol, then add 0.09g of bismuth nitrate, stir thoroughly and transfer to a reaction vessel, react at 120℃ for 24h, centrifuge after the reaction is completed, wash repeatedly with methanol, and dry to obtain the precursor;

[0085] 2) Add 0.5g of precursor and 1.0g of potassium bromide to 500mL of deionized water, stir at 500r / min for 30min, and then treat in an oil bath at 90℃ for 1h. After treatment, wash the product repeatedly with deionized water and ethanol, and dry to obtain hollow nanorods.

[0086] 3) Add 1g of hollow nanorods to 80mL of methanol / water solution with a volume ratio of 80:20, adjust the pH to 4.5, and sonicate at 200W for 2h. Then, add 15mL of KH550 and 3mL of glacial acetic acid while stirring at 150r / min, and sonicate for another 2h. After centrifuging the obtained product, wash it repeatedly with pure water and acetone, and dry it to obtain positively charged hollow nanorods.

[0087] Example 2

[0088] A microbial agent for preventing soil compaction using PGPR comprises the following raw materials in parts by weight: 70 parts microencapsulated PGPR microorganisms, 12 parts microencapsulated microbial activator, 15 parts negatively charged nanofibers, and 50 parts coating agent.

[0089] The coating agent is a mixture of polyethylene glycol and polypropylene glycol in a weight ratio of 70:30;

[0090] The coating agent is dissolved in a diol ether solvent to form a thin film;

[0091] The preparation method of this microbial agent specifically includes the following steps:

[0092] The coating agent is dissolved in a diol ether solvent according to the weight percentage to obtain a 30wt% coating agent solution. The microencapsulated PGPR microorganisms, microencapsulated microbial activator and negatively charged nanofibers are then thoroughly mixed and added to the coating agent solution. After thorough stirring, the mixture is spray-dried.

[0093] The preparation method of the microencapsulated PGPR microorganism is as follows:

[0094] 1) Dissolve whey protein in sterile distilled water, stir thoroughly, and pasteurize in a water bath at 82°C for 7 minutes 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 minutes to obtain a trehalose solution with a concentration of 30 g / L.

[0095] 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 trehalose solution and 65 mg of PGPR microorganisms were thoroughly mixed and then spray-dried under the conditions of air flow rate of 380 L / h, feed flow rate of 0.4 L / h, inlet air temperature of 121℃, outlet air temperature of 67℃ and carrier gas pressure of 0.1 MPa to obtain microencapsulated PGPR microorganisms.

[0096] The microencapsulated microbial activator is prepared as follows:

[0097] 1) Dissolve whey protein in sterile distilled water, stir thoroughly, and pasteurize in a water bath at 82°C for 7 minutes 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 minutes to obtain a trehalose solution with a concentration of 30 g / L.

[0098] 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 trehalose solution and 150 mg of composite microbial activator were thoroughly mixed and then spray-dried under the conditions of air flow rate of 370 L / h, feed flow rate of 0.4 L / h, inlet air temperature of 121℃, outlet air temperature of 67℃ and carrier gas pressure of 0.1 MPa to obtain microencapsulated microbial activator.

[0099] The above-mentioned positively charged hollow nanorods are prepared by the following method:

[0100] 1) Dissolve 0.9 g of trimesic acid in 65 mL of methanol, then add 0.12 g of bismuth nitrate, stir thoroughly and transfer to a reaction vessel, react at 125 °C for 28 h, centrifuge after reaction, wash repeatedly with methanol, and dry to obtain the precursor;

[0101] 2) Add 0.7g of precursor and 1.3g of potassium bromide to 700mL of deionized water, stir at 700r / min for 40min, and then treat in an oil bath at 92℃ for 1.5h. After treatment, wash the product repeatedly with deionized water and ethanol, and dry to obtain hollow nanorods.

[0102] 3) Add 2g of hollow nanorods to 100mL of methanol / water solution with a volume ratio of 85:15, adjust the pH to 5.0, and sonicate at 250W for 2.5h. Then, add 18mL of KH550 and 5mL of glacial acetic acid while stirring at 180r / min, and sonicate for another 2.5h. After centrifuging the obtained product, wash it repeatedly with pure water and acetone, and dry it to obtain positively charged hollow nanorods.

[0103] Example 3

[0104] A microbial agent for preventing soil compaction using PGPR comprises the following raw materials in parts by weight: 80 parts microencapsulated PGPR microorganisms, 15 parts microencapsulated microbial activator, 20 parts negatively charged nanofibers, and 60 parts coating agent.

[0105] The coating agent is a mixture of polyethylene glycol and polypropylene glycol in a weight ratio of 80:20;

[0106] The coating agent is dissolved in a diol ether solvent to form a thin film;

[0107] The preparation method of this microbial agent specifically includes the following steps:

[0108] The coating agent is dissolved in a diol ether solvent according to the weight percentage to obtain a coating agent solution with a concentration of 35 wt%. The microencapsulated PGPR microorganisms, microencapsulated microbial activators and negatively charged nanofibers are then thoroughly mixed and added to the coating agent solution. After thorough stirring, the mixture is spray-dried.

[0109] The preparation method of the microencapsulated PGPR microorganism is as follows:

[0110] 1) Dissolve whey protein in sterile distilled water, stir thoroughly, and pasteurize in an 85°C water bath for 10 minutes to obtain a whey protein solution with a concentration of 100 g / L. Then dissolve trehalose in sterile distilled water and sterilize in an 85°C water bath for 10 minutes to obtain a trehalose solution with a concentration of 40 g / L.

[0111] 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 trehalose solution and 80 mg of PGPR microorganisms were thoroughly mixed and then spray-dried under the conditions of air flow rate of 400 L / h, feed flow rate of 0.5 L / h, inlet air temperature of 123℃, outlet air temperature of 70℃ and carrier gas pressure of 0.2 MPa to obtain microencapsulated PGPR microorganisms.

[0112] The microencapsulated microbial activator is prepared as follows:

[0113] 1) Dissolve whey protein in sterile distilled water, stir thoroughly, and pasteurize in an 85°C water bath for 10 minutes to obtain a whey protein solution with a concentration of 100 g / L. Then dissolve trehalose in sterile distilled water and sterilize in an 85°C water bath for 10 minutes to obtain a trehalose solution with a concentration of 40 g / L.

[0114] 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 trehalose solution and 180 mg of composite microbial activator were thoroughly mixed and then spray-dried under the conditions of air flow rate of 400 L / h, feed flow rate of 0.5 L / h, inlet air temperature of 123℃, outlet air temperature of 70℃ and carrier gas pressure of 0.2 MPa to obtain microencapsulated microbial activator.

[0115] The above-mentioned positively charged hollow nanorods are prepared by the following method:

[0116] 1) Dissolve 1.2g of trimesic acid in 80mL of methanol, then add 0.13g of bismuth nitrate, stir thoroughly and transfer to a reaction vessel, react at 130℃ for 30h, centrifuge after the reaction is completed, wash repeatedly with methanol, and dry to obtain the precursor;

[0117] 2) Add 0.8g of precursor and 1.5g of potassium bromide to 800mL of deionized water, stir at 800r / min for 50min, and then treat in an oil bath at 93℃ for 2h. After treatment, wash the product repeatedly with deionized water and ethanol, and dry to obtain hollow nanorods.

[0118] 3) Add 3g of hollow nanorods to 130mL of methanol / water solution with a volume ratio of 85:15, adjust the pH to 5.0, and sonicate at 300W for 3h. Then, add 20mL of KH550 and 6mL of glacial acetic acid while stirring at 200r / min, and sonicate for another 3h. After centrifuging the obtained product, wash it repeatedly with pure water and acetone, and dry it to obtain positively charged hollow nanorods.

[0119] Comparative Example 1: This comparative example is basically the same as Example 1, except that it does not contain microencapsulated microbial activators.

[0120] Comparative Example 2: This comparative example is basically the same as Example 1, except that the PGPR microorganisms are not microencapsulated.

[0121] Comparative Example 3: This comparative example is basically the same as Example 1, except that the microbial activator is not microencapsulated.

[0122] Comparative Example 4: This comparative example is basically the same as Example 1, except that it does not contain negatively charged nanofibers.

[0123] Comparative Example 5: This comparative example is basically the same as Example 1, except that the microencapsulated PGPR microorganisms and microencapsulated microbial activators do not contain positively charged hollow nanorods.

[0124] Test experiment:

[0125] 1. Soil preparation

[0126] Select fields with compacted soil and build greenhouses. The selected fields should have a soil bulk density of 1.5-1.6, a total porosity of 25-30%, and an aeration porosity of 7-9%.

[0127] 2. Seedling raising

[0128] Seeds are soaked in warm water at a temperature of 55-65℃ for 30 minutes, stirring constantly while soaking.

[0129] Chemical treatment: Soak seeds in 1% trisodium phosphate for 30 minutes, then soak seeds in 100 times diluted formalin solution for 20-30 minutes, and set aside.

[0130] Place the treated seeds into seedling trays filled with substrate, one seed in each cell.

[0131] The matrix is ​​made by mixing attapulgite powder, shell powder, vermiculite powder, peanut shell powder, pine bark powder, wood ash, blue gravel, and isatis root powder in a mass ratio of 2:3:2:1:1.5:4:1:0.02 and then sterilizing it, with a moisture content of 50-60%.

[0132] When the seedlings in the plug trays reach a height of 30-50cm, prepare them for transplanting and planting.

[0133] 3. Planting

[0134] When the seedlings reach a height of 30-50cm, they are transplanted into the greenhouse, usually in January. When transplanting, dig seedling holes in the planting area, one seedling per hole, with a spacing of 20-30cm between each seedling and a row spacing of 50-60cm.

[0135] Water lightly after transplanting, but avoid flooding to prevent the newly transplanted chili peppers from being washed away.

[0136] 4. Field management: Chili peppers have underdeveloped and shallow root systems, so watering should not be prolonged and flooding is strictly prohibited. Furrow irrigation or drip irrigation must be used.

[0137] Irrigation volume: Irrigate 5-6 times throughout the growing season, with each irrigation volume being 50-60m³. 3 Keep the soil moist. Adhere to the principle of frequent, light irrigation, strictly prohibiting flooding and waterlogging. Avoid watering during midday heat and on rainy days; these are also key measures for preventing and controlling blight in chili peppers.

[0138] 5. Fertilizing

[0139] The microbial agents obtained in Examples 1-3 and Comparative Examples 1-5 were mixed with potassium sulfate compound fertilizer and then applied, with 2 kg / mu of microbial agent and 40 kg / mu of potassium sulfate compound fertilizer.

[0140] 6. Disease prevention and control

[0141] After pepper seedlings are transplanted into greenhouses, they are susceptible to pepper blight. When blight occurs, drench the roots of the diseased plants and surrounding plants with a 600-fold dilution of 50% metalaxyl-copper wettable powder or a 600-fold dilution of 30% metalaxyl-mancozeb. Use 250 grams of solution per plant, drench once or twice, with an interval of 5 to 7 days.

[0142] Experiments have shown that the microbial agent of this invention can significantly improve the soil and has a good effect on planting chili peppers. Specific effect indicators are shown in Table 1.

[0143] Table 1

[0144]

[0145] As shown in Table 1, the microbial inoculant of this invention can effectively improve soil structure, enhance soil cohesion, significantly improve soil compaction, and prevent soil compaction in the long term, providing a suitable soil environment for plant growth and achieving the effect of increasing yield and income.

[0146] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A microbial agent for preventing soil compaction using PGPR, characterized in that, The raw materials include the following parts by weight: 50-80 parts of microencapsulated PGPR microorganisms, 8-15 parts of microencapsulated microorganism activator, 10-20 parts of negatively charged nanofibers, and 40-60 parts of coating agent. The coating agent is a mixture of polyethylene glycol and polypropylene glycol in a weight ratio of (60-80):(20-40); The coating agent is dissolved in a diol ether solvent to form a thin film. The microencapsulated PGPR microorganisms are prepared as follows: S1 isolates microorganisms from the rhizosphere soil of soybean growing areas to obtain pure cultures of the strains; S2 was subjected to mutagenesis screening under ultrasonic treatment combined with plasma treatment to obtain PGPR microorganisms with strong ability to degrade organophosphorus compounds. S3 uses whey protein and trehalose as wall materials and positively charged hollow nanorods as functional materials to microencapsulate PGPR microorganisms, thus obtaining microencapsulated PGPR microorganisms. The microencapsulated microbial activator is prepared as follows: S1 adds trace elements and growth factors to deionized water, then adds a small molecule organic carbon source to obtain a primary compound microbial activator. Then, a pH buffer solution is added to the primary compound microbial activator to adjust the pH to 6.5, thus obtaining a compound 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, thus obtaining the microencapsulated composite microbial activator. The positively charged hollow nanorods are prepared by the following method: S1 uses pyromellitic acid and bismuth nitrate as raw materials and methanol as solvent to obtain a precursor through hydrothermal reaction. Then, the precursor is mixed with potassium bromide and added to deionized water. After oil bath treatment, hollow nanorods are obtained. S2 amino-functionalized modification: Hollow nanorods were added to a methanol aqueous solution, the pH was adjusted to 4.5-5.0, and the mixture was sonicated for 1-3 hours. KH550 and glacial acetic acid were added while stirring, and the mixture was sonicated for another 1-3 hours. The mixture was then centrifuged, washed, and dried to obtain the final product.

2. The microbial agent for preventing soil compaction using PGPR according to claim 1, characterized in that, The ultrasonic action is performed under intermittent ultrasonic waves with a frequency of 0.1-1000kHz and a power of 0.01-1000W for a duration of 20-30s. The plasma treatment involves placing a metal plate in the sample processing chamber of the ARTP mutagenesis system, adjusting the distance between the metal plate and the gas flow port to 2-5 mm, and setting the ARTP operating parameters as follows: RF power input of 100-150 W, pure helium flow rate of 10-15 L / min, and processing time of 20-90 s.

3. The microbial agent for preventing soil compaction using PGPR according to claim 1, characterized in that, The mutagenesis screening involves culturing the mutated strains in LB solid medium with progressively increasing dipotassium hydrogen phosphate concentrations to obtain high-purity strains, which are then screened on organic phosphorus solid medium. The concentration of dipotassium hydrogen phosphate increases in a gradient of 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.

4. The microbial agent for preventing soil compaction using PGPR according to claim 1, characterized in that, The ratio of trace elements, growth factors, small molecule organic carbon sources, pH buffers, and deionized water is (55-60) mL : (1-3) mL : (10-15) g : (1.0-1.5) mL : (900-1200) mL; The formula for the trace elements is as follows: zinc sulfate 0.3-0.5 g / L, copper sulfate 1.2-1.6 g / L, ammonium molybdate 0.2-0.3 g / L, sodium borate 0.05-0.08 g / L, magnesium sulfate 0.05-0.08 g / L, and ferric sulfate 0.5-0.8 g / L. The growth factor formulation consists of 4-6 g / L nicotinic acid, 5-7 g / L pantothenic acid, and 4-6 g / L cobalamin. 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 formulated as follows: 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.

5. A microbial agent for preventing soil compaction using PGPR according to claim 1, characterized in that, The ratio of pyromellitic acid, methanol, and bismuth nitrate is (0.7-1.2) g : (50-80) mL : (0.09-0.13) g; The hydrothermal reaction is carried out at a temperature of 120-130℃ for 24-30 hours. The ratio of the precursor, potassium bromide, and deionized water is (0.5-0.8) g : (1.0-1.5) g : (500-800) mL; The oil bath treatment is performed at a temperature of 90-93℃ for 1-2 hours. The ratio of hollow nanorods, methanol / water solution, KH550, and glacial acetic acid is (1-3) g: (80-130) mL: (15-20) mL: (3-6) mL; The methanol / water solution has a volume ratio of (80-85):(15-20).

6. A microbial agent for preventing soil compaction using PGPR according to claim 1, characterized in that, The negatively charged nanofibers are prepared by the following method: 1) Soak commercially available chitin in dilute hydrochloric acid at room temperature for 12-15 hours, wash repeatedly with distilled water, soak in sodium hydroxide solution for 12-15 hours, wash repeatedly with distilled water, decolorize with 0.3-0.5wt% sodium chlorite, and dry to obtain chitin powder with an acetylation degree of 95%. 2) Chitosan powder was dispersed in a 1 mol / L ammonium persulfate aqueous solution and magnetically stirred at 60-65℃ for 20-24 h. Then, negatively charged nanofibers were obtained by centrifugation. The sodium hydroxide solution has a concentration of 0.10-0.15 mol / L; The mass ratio of chitin powder to ammonium persulfate aqueous solution is (0.5-0.8):(100-160).

7. A method for preparing a microbial inoculant for preventing soil compaction according to claim 1, characterized in that, Specifically, the steps include the following: The coating agent is dissolved in a diol ether solvent according to the weight percentage to obtain a coating agent solution with a concentration of 25-35 wt%. The microencapsulated PGPR microorganisms, microencapsulated microbial activators and negatively charged nanofibers are then thoroughly mixed and added to the coating agent solution. After thorough stirring, the mixture is spray-dried.

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