Compound microbial inoculant for improving soil micro-ecology and promoting plant growth and application thereof

Abstract

The present application relates to the field of agricultural microorganism technology, and discloses a composite microbial agent for improving soil micro-ecology and promoting plant growth and application thereof.The composite microbial agent comprises 30-40 parts of Bacillus subtilis, 20-40 parts of Pseudomonas, 10-20 parts of nitrogen-fixing bacteria, 5-10 parts of photosynthetic bacteria, 5-10 parts of Trichoderma and 5-10 parts of Penicillium; wherein the Bacillus subtilis is Bacillus subtilis L11, which is preserved in the China General Microbiological Culture Collection Center on July 11, 2024, with the preservation number of CGMCC No.31274 and the classification name of Bacillus subtilis The present application adopts the composite microbial agent for improving soil micro-ecology and promoting plant growth, constructs a multi-bacterial synergistic community, utilizes the complementary metabolic advantages of different strains in the rhizosphere, improves nutrient transformation and stress resistance characteristics, and thus improves soil micro-ecology and promotes plant root development.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural microorganism technology, and in particular to a compound microbial agent for improving soil microecology and promoting plant growth and application thereof. BACKGROUND

[0002] With the popularization of organic agriculture and green food certification, microbial agents as "environmentally friendly" inputs have become an essential choice for producing high-quality agricultural products. Plant growth-promoting rhizobacteria (PGPR) refers to beneficial bacteria that live around plant roots and can promote plant growth and antagonize pathogenic bacteria. With the development of agricultural microbial agents, the research and application of PGPR with different functions have become a focus in modern agricultural production. Bacillus has strong adaptability to different environments and occupies a large share in agricultural microbial products. PGPR related to Bacillus genus makes important contributions to the survival ability, development, and yield of plants under biological and non-biological challenges.

[0003] Soil microbial communities are generally considered to be good indicators of soil quality, and play an important role in the services and functions of agricultural ecosystems by recycling soil nutrients, maintaining and improving soil microbiome, and antagonizing plant pests and pathogens. Although microorganisms have been used for soil improvement, biological control, and production of biological fertilizers for a long time, the study of the effects of microbial inoculants on microbial communities is relatively limited. Moreover, some microbial agents have relatively single function in improving soil and poor effect. The present application aims to overcome the deficiencies in the prior art and develop a compound microbial agent for improving soil microecology and promoting plant growth, enriching the understanding of the function and mechanism of microbial agents in agricultural applications, and providing a reference for the development of related microbial agents. SUMMARY

[0004] The purpose of the present application is to provide a compound microbial agent for improving soil microecology and promoting plant growth and application thereof. A multi-bacterial synergistic community is constructed, the complementary metabolic advantages of different strains in the rhizosphere are utilized, the nutrient transformation and stress resistance characteristics are improved, and the soil microbial community structure is adjusted, so as to improve the soil microecology and promote the development of plant roots.

[0005] To achieve the above-mentioned purpose, the present application provides a compound microbial agent for improving soil microecology and promoting plant growth, which comprises the following components by weight: Bacillus subtilis 30-40 parts, Pseudomonas 20-40 parts, nitrogen-fixing bacteria 10-20 parts, photosynthetic bacteria 5-10 parts, Trichoderma 5-10 parts, and Penicillium 5-10 parts.

[0006] The Bacillus subtilis is Bacillus subtilis L11, which is preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC No.31274 and a preservation date of July 11, 2024, and is classified and named as Bacillus subtilis L11.Bacillus subtilis

[0007] Pseudomonas is Pseudomonas marina Pseudomonas marianaensis CICC 24308;

[0008] Azotobacter is Azotobacter halophyticus Azotobacter salinestris CICC 10309;

[0009] Photosynthetic bacteria is oligotrophic shallow powder incomplete photosynthetic bacteria Roseateles oligotrophus CICC 24092;

[0010] Trichoderma is Trichoderma harzianum Trichoderma harzianum CICC 13056;

[0011] Penicillium is Penicillium crustosum Penicillium stecki i CICC 40693.

[0012] Further, the following components by weight are included: 32 parts of Bacillus subtilis, 24 parts of Pseudomonas, 18 parts of Azotobacter, 8 parts of photosynthetic bacteria, 7 parts of Trichoderma, and 5 parts of Penicillium.

[0013] Further, the complex microbial agent further includes a dispersing agent, an embedding agent, and a protective agent; the dispersing agent is nano-activated carbon powder; the embedding agent includes sodium alginate, starch, and meat and bone meal, and the mass ratio of sodium alginate: starch: meat and bone meal is 1-2: 1: 1, preferably 1.5: 1: 1; and the protective agent is activated carbon powder.

[0014] Further, the number of viable bacteria in the complex microbial agent is 1x10 8 ~1x10 10 CFU / g.

[0015] Still further, the application also provides a preparation method of the above-mentioned improved soil micro-ecological complex microbial agent for promoting plant growth, including the following steps:

[0016] Step 1, preparation of a bacterial powder mixture: weigh the bacterial powder of Bacillus subtilis, Pseudomonas, Azotobacter, and Trichoderma according to the proportion, mix uniformly, and obtain a bacterial powder mixture;

[0017] Step 2, activated carbon fixation: dissolve the bacterial powder mixture in deionized water, add nano-activated carbon powder, centrifuge after being placed in a shaking bed for 24 hours, and clean with sterile water, to obtain an activated carbon complex bacteria;

[0018] Step 3, preparation of a microbial agent microsphere wall material: dissolve sodium alginate, starch, and meat and bone meal in deionized water, fully stir uniformly, sterilize at 121°C for 20 minutes, and obtain a microbial agent microsphere wall material solution;

[0019] ​Step 4, add the activated carbon composite bacteria to the bacteria agent microsphere wall material solution, and culture at 34 DEG C for 1h under the condition of 180 r / min shaking table oscillation to obtain a mixed solution;

[0020] Step 5, the mixed solution in step 4 is taken by using a syringe, and the mixed solution in the syringe is slowly dropped into a 2% calcium chloride solution to crosslink for 2h to form embedding beads;

[0021] Step 6, the embedding beads are filtered out, washed for 2-3 times by using sterile water, and the surface water is wiped off by using sterile filter paper to obtain wet microspheres;

[0022] Step 7, the activated carbon powder loaded with photosynthetic bacteria and penicillium is uniformly coated on the surface of the wet microspheres, and low-temperature drying is carried out to obtain a composite bacteria agent.

[0023] Further, in step 2, the mass ratio of the bacteria powder mixture and the nano activated carbon powder is 1:1-2.

[0024] Further, in step 4, the mass-volume ratio of the activated carbon composite bacteria and the bacteria agent microsphere wall material solution is 10g:50-80mL.

[0025] Further, in step 7, the preparation method of the activated carbon powder loaded with photosynthetic bacteria and penicillium is as follows:

[0026] The photosynthetic bacteria and the penicillium are respectively activated and cultured in a liquid culture medium for 24h, then the activated bacteria liquid is inoculated in a sterilized liquid culture medium in an equal proportion, activated carbon powder is added, and after being cultured in a shaking table for 24h, centrifugal separation is carried out, the bacteria are washed by using sterile water, and low-temperature drying is carried out to obtain the activated carbon powder loaded with photosynthetic bacteria and penicillium.

[0027] Further, in step 7, after being cultured in a shaking table for 24h, the viable bacterial count of the photosynthetic bacteria and the penicillium is respectively 1x10 8 ~1x10 9 CFU / mL.

[0028] Further, the application also provides the application of the improved soil micro-ecological composite bacteria agent for promoting plant growth in improving soil micro-ecology and promoting plant growth, and the application is applied to promoting plant root growth.

[0029] The improved soil micro-ecological composite bacteria agent for promoting plant growth and the application thereof have the following advantages and positive effects:

[0030] 1. The application loads photosynthetic bacteria and penicillium in the activated carbon protection layer of the outermost layer of the composite microbial agent, solves the problem that the conventional microbial agent may be difficult to start the cycle in the extremely low soil organic matter and poor nutrition environment, and the oligotrophic photosynthetic bacteria can survive and start the initial ecological niche by using light energy and little nutrition, creates conditions for the colonization of subsequent microbial populations, the creased penicillium starts to decompose the residual and difficult-to-utilize organic matter in the soil first, starts the material cycle, and provides a material basis for the colonization of other microbial agents; with the slow release of internal microorganisms, photosynthetic bacteria and nitrogen-fixing bacteria perform photosynthesis and nitrogen fixation, not only provide C and N for the growth of plants, but also provide C source and N source for the growth of other strains, prolong the survival time of the bacteria, form a multi-bacterial synergistic community, and circulate. Therefore, the application constructs a multi-bacterial synergistic community, utilizes the complementary metabolic advantages of different strains in the rhizosphere, improves the nutrient transformation and stress resistance characteristics, thereby improving the soil microecology and promoting the development of plant roots.

[0031] 2. In the preparation process of the composite microbial agent, the microorganisms are fixed by activated carbon before embedding, so that the microorganisms are uniformly distributed in the activated carbon during embedding, and the problem of survival rate reduction caused by excessive concentration of microorganisms during embedding is avoided; and the activated carbon is used for protection after embedding, thereby reducing the problem that the strains are easily inactivated in the harsh environment. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 The L11 selected by the application Bacillus subtilis Plate colony morphology of L11;

[0033] Figure 2 The L11 selected by the application Bacillus subtilis Gram staining microscope morphology observation diagram of L11. DETAILED DESCRIPTION

[0034] The technical solutions of the application will be further described in detail through specific embodiments.

[0035] Unless otherwise defined, the technical terms or scientific terms used in the application should be understood as the usual meanings understood by those skilled in the art in the field of the application.

[0036] Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor belong to the protection scope of the application. The experimental methods not specified in the following embodiments are usually determined according to national standards. The experimental instruments, equipment and reagents not specified in the following embodiments are all commercially available raw materials.

[0037] Unless otherwise defined or specified, all professional and scientific terms used in the present application have the same meaning as familiar to those skilled in the art. In addition, any method and material similar or equivalent to those described can be used in the present application. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0038] Biological material preservation information: Bacillus subtilis L11, preserved in the China General Microbiological Culture Collection Center, with the preservation number CGMCC No. 31274, and the classification and naming date of July 11, 2024, Bacillus subtilis L11. Bacillus subtilis ).

[0039] The Bacillus subtilis L11 used in the present application is specifically screened as follows: Bacillus subtilis

[0040] 1. Strain screening

[0041] The soil sample was collected from the surface of the pepper straw after 2 months of returning to the field in the greenhouse of the Linyi University campus scientific research base in Linyi City, Shandong Province, and was fully mixed after removing impurities. 10 g of the collected soil sample was weighed, and sterile normal saline was added at a water-soil ratio of 10:1. The soil sample was fully mixed with sterile normal saline by shaking at 37°C and 150 rpm for 30 min, and then was allowed to stand for 15 min. The supernatant was taken, gradient diluted, and 10 -2 ~10 -7 dilution gradient soil supernatant suspension. 200 uL of the soil supernatant suspension with dilution gradients of 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 were taken and spread on a cooled cellulose Congo red culture medium plate, and each dilution gradient was repeated 3 times.

[0042] Congo red cellulose medium: sodium nitrate 1.0 g, sodium hydrogen phosphate 1.2 g, potassium dihydrogen phosphate 0.9 g, magnesium sulfate 0.5 g, potassium chloride 0.5 g, yeast extract powder 0.5 g, acid hydrolyzed casein 1.0 g, Congo red 0.2 g, cellulose powder 5.0 g, and agar 15.0 g, and distilled water was added to 1 L.

[0043] ​The plate was inverted in a 37℃ constant temperature incubator for 48 h, and strains with rapid growth and obvious transparent circle on the cellulose Congo red medium were selected, and the plate streaking method was used for further purification. After obtaining single colonies, multiple subcultures were carried out, and strains with good growth and stable morphology were selected for subsequent experimental research. Finally, a white strain with rough colony morphology and no transparency on the culture dish was isolated (as shown in Figure 1 The strain was stained using gram staining method, and the strain was gram-positive bacteria, and the bacterial body was short rod-shaped under microscope (as shown in Figure 2 ).

[0044] The bacillus subtilis is bacillus subtilis L11, which is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.31274, the preservation date is July 11, 2024, and the classification and naming is bacillus subtilis L11 (as shown in Bacillus subtilis );

[0045] The liquid medium thereof is LB liquid medium;

[0046] The pseudomonas is pseudomonas marianae Pseudomonas marianaensis CICC 24308;

[0047] The liquid medium thereof is (1L): yeast extract powder 1.0 g, peptone 5.0 g, ferric citrate 0.1 g, NaCl 19.45 g, MgCl 25.9 g, KCl 0.55 g, Na2SO4 3.24 g, CaCl2 1.8 g, Na2CO3 0.16 g, KBr 0.08 g, SrCl2 4.0 mg, H3BO3 2.0 mg, NaSiO3 4.0 mg, NaF 2.4 mg, NH4NO3 1.6 mg, Na2HPO4 8.0 mg, distilled water 1000.0 mL, pH 7.6;

[0048] The nitrogen-fixing bacteria is azotobacter salinus Azotobacter salinestris CICC 10309;

[0049] The liquid medium thereof is (1L): yeast extract powder 0.5 g, mannitol 20.0 g, KH2PO4 0.2 g, K2HPO4 0.8 g, MgSO4·7H2O 0.2 g, CaSO4·2H2O 0.1 g, FeCl3 1.0 mg, Na2MoO4·2H2O 1.0 mg, distilled water 1000.0 mL, pH 7.2;

[0050] The photosynthetic bacteria is oligotrophic and shallow powder incomplete photosynthetic bacteria Roseateles oligotrophus CICC 24092;

[0051] Its liquid medium is (1L): yeast extract powder 0.5 g, peptone 0.5 g, casein hydrolysate 0.5 g, glucose 0.5 g, soluble starch 0.5 g, sodium pyruvate 0.3 g, KH2PO4 0.3 g, MgSO4·7H2O 0.05 g, distilled water 1000.0 mL, pH 7.2;

[0052] Trichoderma is Trichoderma harzianum Trichoderma harzianum CICC 13056;

[0053] Its liquid medium is (1L): potato extract 1000.0 mL, glucose 20.0 g, pH natural;

[0054] Penicillium is Penicillium frequentans Penicillium stecki i CICC 40693

[0055] Its liquid medium is (1L): malt extract powder 30.0 g, distilled water 1000.0 mL, pH 5.5.

[0056] Example 1

[0057] The complex microbial agent for improving soil microecology to promote plant growth comprises the following components by weight: Bacillus subtilis 30 parts, Pseudomonas 20 parts, nitrogen-fixing bacteria 10 parts, photosynthetic bacteria 5 parts, Trichoderma 5 parts, and Penicillium 5 parts.

[0058] The complex microbial agent further comprises a dispersing agent, an embedding agent, and a protective agent. The dispersing agent is nano-activated carbon powder. The embedding agent comprises sodium alginate, starch, and meat and bone meal, and the mass ratio of sodium alginate: starch: meat and bone meal is 1.5:1:1. The protective agent is activated carbon powder.

[0059] The number of viable bacteria in the complex microbial agent is 1×10 9 CFU / g.

[0060] The preparation method of the complex microbial agent comprises the following steps:

[0061] Step 1, preparation of a bacterial powder mixture: weigh the bacterial powder of Bacillus subtilis, Pseudomonas, nitrogen-fixing bacteria, and Trichoderma according to the proportion, mix uniformly, and obtain a bacterial powder mixture;

[0062] Step 2, activated carbon fixation: dissolve 5 g of the bacterial powder mixture in deionized water, add 5 g of nano-activated carbon powder, place in a shaking bed for 24 h, centrifuge, and wash with sterile water to obtain an activated carbon complex microbial agent.

[0063] Step 3, preparation of the wall material of the microbial agent microspheres: sodium alginate, starch and meat and bone meal were dissolved in deionized water, stirred uniformly, sterilized at 121℃ for 20 min, to obtain a wall material solution of the microbial agent microspheres, wherein the concentration of sodium alginate was 1.5%, the concentration of starch was 1%, and the concentration of meat and bone meal was 1%;

[0064] Step 4, the activated carbon composite bacteria were added to 80 mL of the wall material solution of the microbial agent microspheres, and were cultured at 34℃ with shaking at 180 r / min for 1 h to obtain a mixed solution;

[0065] Step 5, the mixed solution in step 4 was taken by a syringe, and the mixed solution in the syringe was slowly dropped into a 2% calcium chloride solution for crosslinking for 2 h to form embedding beads;

[0066] Step 6, the embedding beads were filtered out, washed with sterile water for 2-3 times, and the surface water was wiped off with sterile filter paper to obtain wet microspheres;

[0067] Step 7, the activated carbon powder loaded with photosynthetic bacteria and penicillium was uniformly coated on the surface of the wet microspheres, and low-temperature drying was performed to obtain a composite microbial agent;

[0068] The preparation method of the activated carbon powder loaded with photosynthetic bacteria and penicillium is as follows:

[0069] The photosynthetic bacteria and penicillium were respectively activated and cultured in a liquid medium for 24 h, then the activated bacteria liquid was inoculated in the sterilized liquid medium in an equal proportion, activated carbon powder was added, and centrifugal separation was performed after being cultured in a shaking bed for 24 h, and the activated carbon powder loaded with photosynthetic bacteria and penicillium was obtained by washing with sterile water and low-temperature drying; after being cultured in a shaking bed for 24 h, the viable bacterial count of the photosynthetic bacteria and penicillium was 1×10 8 CFU / mL, respectively.

[0070] Example 2

[0071] The modified soil micro-ecological composite microbial agent for promoting plant growth comprises the following components in parts by weight: 32 parts of bacillus subtilis, 24 parts of pseudomonas, 18 parts of nitrogen-fixing bacteria, 8 parts of photosynthetic bacteria, 7 parts of trichoderma, and 5 parts of penicillium;

[0072] The composite microbial agent further comprises a dispersing agent, an embedding agent and a protective agent, the dispersing agent is nano-activated carbon powder, the embedding agent comprises sodium alginate, starch and meat and bone meal, and the mass ratio of sodium alginate: starch: meat and bone meal is 1.5:1:1; and the protective agent is activated carbon powder.

[0073] The viable bacterial count in the composite microbial agent is 1×10 9 CFU / g.

[0074] The preparation method of the composite microbial agent comprises the following steps:

[0075] Step 1, preparation of bacterial powder mixture: weigh Bacillus subtilis, Pseudomonas, Azotobacter, Trichoderma sp. bacterial powder according to the proportion, mix uniformly to obtain bacterial powder mixture;

[0076] Step 2, activated carbon fixation: dissolve 5g of bacterial powder mixture in deionized water, add 10g of nano activated carbon powder, centrifuge after 24h of shaking bed culture, and wash with sterile water to obtain activated carbon composite bacteria;

[0077] Step 3, preparation of bacterial agent microsphere wall material: dissolve sodium alginate, starch and meat and bone meal in deionized water, stir uniformly, sterilize at 121℃ for 20min to obtain bacterial agent microsphere wall material solution, wherein the concentration of sodium alginate is 1.5%, the concentration of starch is 1%, and the concentration of meat and bone meal is 1%;

[0078] Step 4, add activated carbon composite bacteria to 120mL of bacterial agent microsphere wall material solution, cultivate at 34℃, 180r / min shaking bed for 1h to obtain mixed solution;

[0079] Step 5, use a syringe to suck the mixed solution in step 4, slowly drop the mixed solution in the syringe into 2% calcium chloride solution, crosslink for 2h to form embedding beads;

[0080] Step 6, filter out the embedding beads, wash with sterile water for 2-3 times, wipe the surface water with sterile filter paper to obtain wet microspheres;

[0081] Step 7, uniformly coat the activated carbon powder loaded with photosynthetic bacteria and penicillium on the surface of the wet microspheres, and dry at low temperature to obtain composite bacterial agent;

[0082] The preparation method of the activated carbon powder loaded with photosynthetic bacteria and penicillium is as follows:

[0083] Cultivate photosynthetic bacteria and penicillium in liquid medium for 24h respectively, then inoculate the activated bacteria liquid in the sterilized liquid medium in a proper proportion, add activated carbon powder, centrifuge after 24h of shaking bed culture, wash with sterile water, and dry at low temperature to obtain activated carbon powder loaded with photosynthetic bacteria and penicillium; after 24h of shaking bed culture, the viable count of photosynthetic bacteria and penicillium is 1x10 8 CFU / mL.

[0084] Example 3

[0085] The modified soil micro-ecological composite bacterial agent for promoting plant growth comprises the following components by weight: Bacillus subtilis 40 parts, Pseudomonas 40 parts, Azotobacter 20 parts, photosynthetic bacteria 10 parts, Trichoderma sp. 10 parts, and penicillium 10 parts;

[0086] The complex bacterial agent also comprises a dispersing agent, an embedding agent and a protective agent, the dispersing agent is nano activated carbon powder, the embedding agent comprises sodium alginate, starch and meat and bone meal, the mass ratio of sodium alginate, starch and meat and bone meal is 1.5:1:1, and the protective agent is activated carbon powder.

[0087] The viable bacterial count in the complex bacterial agent is 1×10 10 CFU / g.

[0088] The preparation method of the complex bacterial agent comprises the following steps:

[0089] Step 1, preparation of bacterial powder mixture: weigh the bacterial powder of Bacillus subtilis, Pseudomonas, nitrogen-fixing bacteria and Trichoderma in proportion, mix uniformly to obtain the bacterial powder mixture;

[0090] Step 2, activated carbon fixation: dissolve 5g of the bacterial powder mixture in deionized water, add 8g of nano activated carbon powder, centrifuge after being placed in a shaking bed for 24h, and wash with sterile water to obtain the activated carbon complex bacteria;

[0091] Step 3, preparation of bacterial agent microsphere wall material: dissolve sodium alginate, starch and meat and bone meal in deionized water, fully stir uniformly, sterilize at 121℃ for 20min to obtain the bacterial agent microsphere wall material solution, wherein the concentration of sodium alginate is 1.5%, the concentration of starch is 1%, and the concentration of meat and bone meal is 1%;

[0092] Step 4, add the activated carbon complex bacteria to 100mL of the bacterial agent microsphere wall material solution, and cultivate at 34℃ and 180r / min in a shaking bed for 1h to obtain a mixed solution;

[0093] Step 5, use a syringe to suck the mixed solution in step 4, slowly drop the mixed solution in the syringe into a 2% calcium chloride solution, crosslink for 2h to form embedding beads;

[0094] Step 6, filter out the embedding beads, wash with sterile water for 2-3 times, wipe the surface water with sterile filter paper to obtain wet microspheres;

[0095] Step 7, uniformly wrap the activated carbon powder loaded with photosynthetic bacteria and penicillium on the surface of the wet microspheres, and dry at low temperature to obtain the complex bacterial agent;

[0096] The preparation method of the activated carbon powder loaded with photosynthetic bacteria and penicillium is as follows:

[0097] Respectively activate and cultivate the photosynthetic bacteria and the penicillium in a liquid culture medium for 24h, then inoculate the activated bacterial liquid in the sterilized liquid culture medium in an equal proportion, add activated carbon powder, centrifuge after being placed in a shaking bed for 24h, wash with sterile water, and dry at low temperature to obtain the activated carbon powder loaded with photosynthetic bacteria and penicillium; after being cultured in the shaking bed for 24h, the viable bacterial count of the photosynthetic bacteria and the penicillium is 1×10 8 CFU / mL.

[0098] Comparative Example 1

[0099] The complex microbial agent for improving soil micro-ecology and promoting plant growth comprises the following components by weight: 32 parts of Bacillus subtilis, 24 parts of Pseudomonas, 18 parts of nitrogen-fixing bacteria, 8 parts of photosynthetic bacteria, 7 parts of Trichoderma, and 5 parts of Penicillium;

[0100] The complex microbial agent further comprises an embedding agent and a protective agent; the embedding agent comprises sodium alginate, starch, and meat and bone meal, and the mass ratio of sodium alginate: starch: meat and bone meal is 1.5:1:1; the protective agent is activated carbon powder.

[0101] The viable bacterial count in the complex microbial agent is 1×10 9 CFU / g.

[0102] The preparation method of the complex microbial agent comprises the following steps:

[0103] Step 1: Preparation of a microbial powder mixture: weigh the microbial powders of Bacillus subtilis, Pseudomonas, nitrogen-fixing bacteria, and Trichoderma according to the proportions, and mix them uniformly to obtain a microbial powder mixture;

[0104] Step 2: Omit;

[0105] Step 3: Preparation of a microbial agent microsphere wall material: dissolve sodium alginate, starch, and meat and bone meal in deionized water, and stir them thoroughly and uniformly, sterilize them at 121°C for 20 min, and obtain a microbial agent microsphere wall material solution;

[0106] Step 4: add 5 g of the microbial powder mixture to 50 mL of the microbial agent microsphere wall material solution, and cultivate them at 34°C and 180 r / min on a shaking table for 1 h to obtain a mixed solution;

[0107] Step 5: use a syringe to suck the mixed solution in Step 4, and slowly drop the mixed solution in the syringe into a 2% calcium chloride solution, crosslink for 2 h, and form embedded small balls;

[0108] Step 6: filter out the embedded small balls, wash them with sterile water for 2-3 times, wipe off the surface water with sterile filter paper, and obtain wet microspheres;

[0109] Step 7: uniformly coat activated carbon powder loaded with photosynthetic bacteria and Penicillium on the surface of the wet microspheres, and dry them at low temperature to obtain a complex microbial agent;

[0110] The preparation method of the activated carbon powder loaded with photosynthetic bacteria and Penicillium is as follows:

[0111] The photosynthetic bacteria and the penicillium are respectively activated and cultured in a liquid medium for 24 hours, then the activated bacteria liquid is inoculated in the sterilized liquid medium in an equal proportion, activated carbon powder is added, and after being cultured in a shaking bed for 24 hours, centrifugal separation is performed, the activated carbon powder is washed with sterile water, and low-temperature drying is performed to obtain the activated carbon powder loaded with the photosynthetic bacteria and the penicillium; after being cultured in the shaking bed for 24 hours, the viable count of the photosynthetic bacteria and the penicillium is 1×10 8 CFU / mL.

[0112] Comparative example 2

[0113] The modified soil micro-ecological composite microbial agent for promoting plant growth comprises the following components in parts by weight: 32 parts of bacillus subtilis, 24 parts of pseudomonas, 18 parts of nitrogen-fixing bacteria, 8 parts of photosynthetic bacteria, 7 parts of trichoderma, and 5 parts of penicillium;

[0114] The composite microbial agent further comprises a dispersing agent and an embedding agent, the dispersing agent is nano-activated carbon powder, and the embedding agent comprises sodium alginate, starch, and meat and bone meal, and the mass ratio of sodium alginate, starch, and meat and bone meal is 1.5:1:1; the protective agent is activated carbon powder.

[0115] The viable count of the composite microbial agent is 1×10 9 CFU / g.

[0116] The preparation method of the composite microbial agent comprises the following steps:

[0117] Step 1, preparation of a bacterial powder mixture: the bacterial powder of bacillus subtilis, pseudomonas, nitrogen-fixing bacteria, photosynthetic bacteria, trichoderma, and penicillium is weighed according to the proportion, mixed uniformly, and a bacterial powder mixture is obtained;

[0118] Step 2, activated carbon fixation: 5 g of the bacterial powder mixture is dissolved in deionized water, 10 g of nano-activated carbon powder is added, centrifugal separation is performed after being cultured in a shaking bed for 24 hours, and the activated carbon composite bacteria are obtained by washing with sterile water;

[0119] Step 3, preparation of a microbial agent microsphere wall material: sodium alginate, starch, and meat and bone meal are dissolved in deionized water, fully stirred and uniformly mixed, sterilized at 121 ℃ for 20 minutes, and a microbial agent microsphere wall material solution is obtained, wherein the concentration of sodium alginate is 1.5%, the concentration of starch is 1%, and the concentration of meat and bone meal is 1%;

[0120] Step 4, the activated carbon composite bacteria are added to 120 mL of the microbial agent microsphere wall material solution, and the mixture is cultured in a shaking bed at 34 ℃ and 180 r / min for 1 hour to obtain a mixed liquid;

[0121] Step 5, the mixed liquid in the syringe is slowly dropped into a 2% calcium chloride solution, cross-linked for 2 hours, and an embedding small ball is formed;

[0122] Step 6, filter out the embedding beads, rinse with sterile water for 2-3 times, wipe off the surface water with sterile filter paper, and obtain wet microspheres;

[0123] Step 7, uniformly coat the activated carbon powder on the surface of the wet microspheres, and dry at low temperature to obtain the composite microbial agent;

[0124] Comparative Example 3

[0125] The modified soil micro-ecological composite microbial agent for promoting plant growth comprises the following components in parts by weight: Bacillus subtilis 32 parts, Pseudomonas 24 parts, nitrogen-fixing bacteria 18 parts, photosynthetic bacteria 8 parts, Trichoderma 7 parts, and Penicillium 5 parts.

[0126] The composite microbial agent further comprises a dispersing agent and an embedding agent. The dispersing agent is nano-activated carbon powder. The embedding agent comprises sodium alginate, starch, and meat and bone meal, and the mass ratio of sodium alginate: starch: meat and bone meal is 1.5: 1: 1.

[0127] The number of viable bacteria in the composite microbial agent is 1×10 9 CFU / g.

[0128] The preparation method of the composite microbial agent comprises the following steps:

[0129] Step 1, preparation of bacterial powder mixture: weigh the bacterial powder of Bacillus subtilis, Pseudomonas, nitrogen-fixing bacteria, photosynthetic bacteria, Trichoderma, and Penicillium according to the proportion, mix uniformly, and obtain the bacterial powder mixture;

[0130] Step 2, activated carbon fixation: dissolve 5 g of the bacterial powder mixture in deionized water, add 10 g of nano-activated carbon powder, centrifuge after being cultured in a shaking bed for 24 h, and wash with sterile water to obtain the activated carbon composite bacteria;

[0131] Step 3, preparation of microbial agent microsphere wall material: dissolve sodium alginate, starch, and meat and bone meal in deionized water, fully stir uniformly, sterilize at 121°C for 20 min, and obtain the microbial agent microsphere wall material solution, wherein the concentration of sodium alginate is 1.5%, the concentration of starch is 1%, and the concentration of meat and bone meal is 1%;

[0132] Step 4, add the activated carbon composite bacteria to 120 mL of the microbial agent microsphere wall material solution, and culture in a shaking bed at 34°C and 180 r / min for 1 h to obtain a mixed solution;

[0133] Step 5, use a syringe to suck the mixed solution in step 4, slowly drop the mixed solution in the syringe into a 2% calcium chloride solution, crosslink for 2 h, and form embedding beads;

[0134] Step 6, filter out the embedding beads, rinse with sterile water for 2-3 times, wipe off the surface water with sterile filter paper, and obtain wet microspheres;

[0135] Step 7, low-temperature drying to obtain the composite microbial agent;

[0136] Comparative Example 4

[0137] The complex microbial agent for improving soil micro-ecology and promoting plant growth comprises the following components by weight: 56 parts of Bacillus subtilis, 18 parts of nitrogen-fixing bacteria, 8 parts of photosynthetic bacteria, 7 parts of Trichoderma, and 5 parts of Penicillium.

[0138] The complex microbial agent further comprises a dispersing agent, an embedding agent, and a protective agent. The dispersing agent is nano-activated carbon powder. The embedding agent comprises sodium alginate, starch, and meat and bone meal, with a mass ratio of sodium alginate:starch:meat and bone meal = 1.5:1:1. The protective agent is activated carbon powder. The number of viable bacteria in the complex microbial agent is 1×10 9 CFU / g.

[0139] Comparative Example 5

[0140] The complex microbial agent for improving soil micro-ecology and promoting plant growth comprises the following components by weight: 40 parts of Bacillus subtilis, 24 parts of Pseudomonas, 18 parts of nitrogen-fixing bacteria, 7 parts of Trichoderma, and 5 parts of Penicillium.

[0141] The complex microbial agent further comprises a dispersing agent, an embedding agent, and a protective agent. The dispersing agent is nano-activated carbon powder. The embedding agent comprises sodium alginate, starch, and meat and bone meal, with a mass ratio of sodium alginate:starch:meat and bone meal = 1.5:1:1. The protective agent is activated carbon powder. The number of viable bacteria in the complex microbial agent is 1×10 9 CFU / g.

[0142] Comparative Example 6

[0143] The complex microbial agent for improving soil micro-ecology and promoting plant growth comprises the following components by weight: 37 parts of Bacillus subtilis, 24 parts of Pseudomonas, 18 parts of nitrogen-fixing bacteria, 8 parts of photosynthetic bacteria, and 7 parts of Trichoderma.

[0144] The complex microbial agent further comprises a dispersing agent, an embedding agent, and a protective agent. The dispersing agent is nano-activated carbon powder. The embedding agent comprises sodium alginate, starch, and meat and bone meal, with a mass ratio of sodium alginate:starch:meat and bone meal = 1.5:1:1. The protective agent is activated carbon powder. The number of viable bacteria in the complex microbial agent is 1×10 9 CFU / g.

[0145] Plant growth promotion test

[0146] Tomato seeds were placed on sterile wet filter paper to promote germination. Sterile water was added every 2 days to keep the filter paper moist. After 3 days, the seeds germinated, and the germinated tomato seeds were cultivated using small black square pots (upper diameter 7 x 7 cm, height 8 cm, lower bottom diameter 5 x 5 cm) with commercialized seedling substrate. When the first true leaf of the seedling was fully unfolded, the complex microbial agent was applied, 1-3 g was applied, and only once during cultivation. The application method was as follows: at the edge of the cultivation substrate, 2 small holes were dug to the middle of the pot with a tool. 1 g of embedding agent was placed in each hole, and after covering the soil, it was watered thoroughly. 10 biological replicates were set for each group, and the seedlings were randomly selected for data collection and measurement. The grouping and application of the complex microbial agent are shown in Table 1:

[0147] Table 1 Grouping and application of the complex microbial agent

[0148]

[0149] On the 40th day of tomato seedling growth, 6 seedlings were selected for sampling and measurement of different growth indicators (average value) in each treatment. The height of the seedling was measured using a tape measure (PH), which measured the height from 1 cm below the cotyledon to the growth point of the seedling. The stem diameter was measured using a vernier caliper (SD), which measured the stem diameter at about 1 cm from the ground as the stem diameter. The root structure indicators of tomato seedlings were measured. The root system of the seedling was scanned using a scanner (EPSON V800) to obtain the total root length of the root system cm (TRL), root surface area cm 2 (RSA), root volume cm 3 (RV), and root average diameter mm (RD). The results are shown in Table 2.

[0150] Table 2 Growth indicators

[0151]

[0152] As shown in Table 2, the complex microbial agents prepared in Examples 1-3 can significantly improve the growth of tomato seedlings, especially the promotion of plant height and stem diameter, and root growth. Compared with Examples 1-3, the complex microbial agents prepared in Comparative Examples 1-3 have reduced growth-promoting effects on plants. This shows that the addition of dispersants and protective agents helps to improve the growth-promoting effect of the complex microbial agent.

[0153] Compared with examples 1-3, 1-3, the growth of the composite microbial inoculant prepared in comparative examples 4-6 on the growth of tomato seedlings, especially the plant height, stem diameter and root system, is obviously reduced, which indicates that the lack of one kind of bacteria will reduce the growth of the composite microbial inoculant on the plant, and destroy the synergistic growth of the bacteria in the microbial inoculant. The application of the composite microbial inoculant in the application constructs a multi-bacterial synergistic community, utilizes the complementary metabolic advantages of different strains in the rhizosphere, improves the nutrient transformation and stress resistance characteristics, and adjusts the soil microbial community structure, so as to improve the soil microecology and promote the root development of the plant.

[0154] The application has the following advantages:

[0155] 1. In the application, the salt-dwelling nitrogen-fixing bacteria directly convert the free nitrogen in the air into ammonia available to plants, providing nitrogen sources for the growth of plants and other strains; the photosynthetic bacteria fix carbon dioxide by using light energy, synthesize organic matter, and consume harmful gases (such as hydrogen sulfide) secreted by the root system, providing carbon sources for the growth of plants and other strains; not only the survival rate of microorganisms in the composite microbial inoculant is improved, but also nutrients are provided for the plants.

[0156] 2. In the application, Bacillus subtilis and Pseudomonas can secrete organic acids and enzymes to strongly decompose elements such as phosphorus, potassium and silicon fixed in the soil, and convert them from ineffective states into available states for plants to absorb; at the same time, photosynthetic bacteria and Pseudomonas can also decompose and utilize residual organic molecules in the soil, and mineralize them into inorganic nutrients for plants to absorb.

[0157] 3. In the application, Trichoderma harzianum can directly wrap, penetrate and parasitize various soil-borne pathogenic fungi (such as Fusarium and Rhizoctonia), and at the same time, secrete powerful chitinase to "dissolve" the cell wall of the pathogenic bacteria; at the same time, Bacillus subtilis can directly inhibit or kill pathogenic bacteria and fungi by producing lipopeptide antibiotics, thereby solving the problem of plant diseases.

[0158] 4. In the application, the exopolysaccharides and gums secreted by Bacillus subtilis, photosynthetic bacteria and the like can bond small soil particles into stable aggregate structures, coordinate water and fertilizer, and fertilize the soil; at the same time, Pseudomonas, Bacillus subtilis and photosynthetic bacteria can produce plant growth hormones (such as IAA and cytokinin), directly stimulate root growth, form a larger and stronger root group, and enhance the absorption capacity; the salt-dwelling nitrogen-fixing bacteria help plants to improve the tolerance to salt and alkali stress. Photosynthetic bacteria, Pseudomonas and Bacillus subtilis can degrade or transform harmful substances (such as phenol and hydrogen sulfide) secreted by the root system and some residual pesticides, and purify the rhizosphere environment.

[0159] In summary, the components of the composite microbial inoculant in the application synergize with each other and jointly play a role, thereby achieving the effect of promoting the growth of plants.

[0160] Therefore, the application adopts the improved soil micro-ecological composite microbial inoculant for promoting plant growth and its application to construct a multi-bacterial synergistic community, utilize the complementary metabolic advantages of different strains in the rhizosphere, improve the nutrient transformation and stress resistance characteristics, and adjust the soil microbial community structure, so as to improve the soil micro-ecology and promote the root development of plants.

[0161] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit them, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that: the technical solutions of the present application can still be modified or replaced by equivalents, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A complex microbial agent for improving soil micro-ecology to promote plant growth, characterized in that, The composition comprises the following components by weight: 30-40 parts of Bacillus subtilis, 20-40 parts of Pseudomonas, 10-20 parts of Azotobacter, 5-10 parts of photosynthetic bacteria, 5-10 parts of Trichoderma, and 5-10 parts of Penicillium; wherein the Bacillus subtilis is Bacillus subtilis L11, which is preserved in the China General Microbiological Culture Collection Center, has a preservation number of CGMCC No.31274, a preservation date of July 11, 2024, and a classification name of Bacillus subtilis (Bacillus subtilis) L11. Bacillus subtilis ​ The pseudomonad is *Pseudomonas mariana* ( Pseudomonas marianaensis CICC 24308; Azotobacter sp. is a halophilic azotobacter Azotobacter salinestris ) CICC 10309; The photosynthetic bacteria is oligotrophic shallow pink incomplete photosynthetic bacteria (Rhodopseudomonas palustris) Roseateles oligotrophus ) CICC 24092; Trichoderma is Trichoderma harzianum (Thompson) Harz (ATCC 20847) Trichoderma harzianum ) CICC 13056; Penicillium was Penicillium crustosum Penicillium steckii ) CICC 40693; The preparation method of the composite microbial agent comprises the following steps: Step 1, preparation of the bacterial powder mixture: the bacterial powder of Bacillus subtilis, Pseudomonas marianaensis, Azotobacter salinestris, Trichoderma harzianum and Penicillium steckii is weighed according to the proportion, mixed uniformly, and the bacterial powder mixture is obtained; Step 2, active carbon fixation: the bacterial powder mixture is dissolved in deionized water, nano active carbon powder is added, and then the mixture is placed in a shaking table for 24 hours, centrifuged, and washed with sterile water to obtain the active carbon composite bacteria; Step 3, preparation of the microbial agent microsphere wall material: sodium alginate, starch and bone meal are dissolved in deionized water, fully stirred and uniformly mixed, sterilized at 121 DEG C for 20 minutes to obtain the microbial agent microsphere wall material solution, and the mass ratio of sodium alginate, starch and bone meal is 1-2:1:1; Step 4, the active carbon composite bacteria is added to the microbial agent microsphere wall material solution, and the mixture is cultured in a shaking table at 34 DEG C and 180 r / min for 1 hour to obtain a mixed solution; Step 5, the mixed solution in the syringe is slowly dropped into a 2% calcium chloride solution, and cross-linked for 2 hours to form an embedding bead; Step 6, the embedding bead is filtered out, washed with sterile water for 2-3 times, and the surface water is wiped off with sterile filter paper to obtain a wet microsphere; Step 7, the active carbon powder loaded with photosynthetic bacteria and penicillium is uniformly coated on the surface of the wet microsphere, and low-temperature drying is performed to obtain the composite microbial agent.

2. The complex microbial agent for improving soil micro-ecology and promoting plant growth according to claim 1, characterized in that, The composite microbial agent comprises the following components by weight: 32 parts of Bacillus subtilis, 24 parts of Pseudomonas marianaensis, 18 parts of Azotobacter salinestris, 8 parts of photosynthetic bacteria, 7 parts of Trichoderma harzianum and 5 parts of Penicillium steckii.

3. The complex microbial agent for improving soil micro-ecological balance and promoting plant growth according to claim 1, characterized in that: The viable cell count in the complex microbial agent is 1 x 10 8 ~1 x 10 10 CFU / g.

4. The complex bacterial agent according to claim 1, characterized by: In step 2, the mass ratio of the bacterial powder mixture and the nano active carbon powder is 1:1-2.

5. The complex bacterial agent according to claim 1, characterized in that: In step 4, the mass-volume ratio of the active carbon composite bacteria and the microbial agent microsphere wall material solution is 10g:50-80mL.

6. The complex bacterial agent according to claim 1, characterized by, In step 7, the preparation method of the active carbon powder loaded with photosynthetic bacteria and penicillium is as follows: the photosynthetic bacteria and penicillium are respectively activated and cultured in a liquid culture medium for 24 hours, then the activated bacteria liquid is inoculated in the sterilized liquid culture medium in a proper proportion, the active carbon powder is added, and then the mixture is placed in a shaking table for 24 hours, centrifuged, washed with sterile water, and low-temperature dried to obtain the active carbon powder loaded with photosynthetic bacteria and penicillium.

7. The complex bacterial agent according to claim 6, characterized in that: After 24 h of shaking culture, the viable cell counts of photosynthetic bacteria and Penicillium were 1 x 10 8 9 CFU / mL, respectively.​ 8. The use of the improved soil micro-ecological promoting plant growth complex microbial agent according to any one of claims 1-7 in improving soil micro-ecology and promoting plant growth, characterized in that: It is applied to promote the growth of plant root system.

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