A composite bacterial agent and its preparation method and application

Abstract

The present invention discloses a composite bacterial agent and its preparation method and application. The composite bacterial agent comprises Trichoderma, Mucor, Rhacophyton, and Bacillus. The effective viable bacterial count of the prepared composite bacterial agent is 2.3×10 8 The composite liquid inoculant prepared by the present invention has rapid decomposition characteristics and is suitable for direct straw return to the field to promote decomposition. It can be used to degrade rapeseed straw and increase humus in the field. When the composite inoculant is diluted 30 times, it has a good growth-promoting effect on rapeseed and corn seeds. The liquid composite inoculant of the present invention can accelerate the decomposition of crop straw while promoting crop growth, providing composite bacterial resources and technical support for the rapid decomposition of crop straw.

Description

Technical Field

[0001] The present invention relates to the field of environmental microbial technology, and in particular to a composite bacterial agent and a preparation method and application thereof. Background Art

[0002] Crop straw is a multi-purpose, renewable bioresource. With the development of agricultural production, my country's grain output has increased significantly, and the amount of crop straw has also increased accordingly. Coupled with the widespread adoption of diversified combustion energy sources, a large amount of surplus straw has emerged in rural areas. Furthermore, soil degradation, loss of soil organic carbon and nutrients, and declining soil fertility are major challenges facing my country's agricultural development. Healthy soil is crucial for crop growth and, in turn, for human health. More than half of the products of crop photosynthesis are found in straw, which is rich in nitrogen, phosphorus, potassium, and organic matter. Returning straw to fields can improve farmland ecology and regulate soil nutrient imbalances to varying degrees. Regularly returning straw to fields not only significantly increases fertility during the cultivation phase but also boosts crop yields. However, crop straw has a high crude fiber content (30%-40%) and contains lignin. Directly returning straw to fields often results in slow decomposition, preventing rapid absorption and utilization, resulting in poor soil organic matter improvement and reduced crop yields. Furthermore, crop straw is bulky, expensive to recycle, and difficult to recycle and process uniformly. To address this issue, the application of biodegradation technology in the straw return process has attracted increasing attention. Biodegradation technology is an economical and environmentally friendly process. It addresses the key issues of straw resource utilization and soil improvement by using functional bacteria to mediate the rapid humification of straw waste. Compared with other crop straw resource utilization technologies, it has the advantages of lower cost, simple process, low technical difficulty, and environmentally friendly.

[0003] Therefore, in response to the needs of the above-mentioned prior art, in order to achieve rapid ripening and composting of straw and return it to the field on site and improve the ecological environment of farmland, the present invention aims to provide a liquid composite bacterial agent for degrading crop straw and its preparation method and application. Summary of the Invention

[0004] One of the purposes of the present invention is to provide a composite bacterial agent; a second purpose of the present invention is to provide a preparation method of the composite bacterial agent; a third purpose of the present invention is to provide an application of the composite bacterial agent in degrading straw; a fourth purpose of the present invention is to provide an application of the composite bacterial agent in promoting crop growth.

[0005] The composite microbial agent of the present invention utilizes Trichoderma longibrachiatum NT241, Mucor circinelloides GJ242, Trichoderma leucospermi GJ243, and Bacillus subtilis GJ231 as active ingredients. By degrading straw, it not only effectively achieves rapid straw composting and on-site field return, but also promotes the growth of crops such as rapeseed and corn. As a composite microbial fertilizer, it can be widely used in crop production and has a positive effect.

[0006] In order to achieve the purpose of the present invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a composite bacterial agent, wherein the active ingredients are prepared from four active ingredients: Trichoderma longibrachiatum NT241, Mucor circinelloides GJ242, Irpex lacteus GJ243, and Bacillus subtilis GJ231, wherein:

[0008] The Mucor circinelloides GJ242 was deposited in the General Microbiology Center of the China Culture Collection Administration, with a deposit date of April 7, 2024 and a deposit number of CGMCC No. 41140. This strain is a new strain.

[0009] The Trichoderma longibrachiatum NT241 was deposited in the General Microbiology Center of the China Culture Collection Administration, with a deposit date of April 7, 2024 and a deposit number of CGMCC No. 41139. The strain has been patented with the application number 202411675277.4.

[0010] The Irpex lacteus GJ243 was deposited in the General Microbiology Center of the China Culture Collection Administration, with a deposit date of April 7, 2024 and a deposit number of CGMCC No. 41281. The strain has been patented with the application number 202411302628.7.

[0011] The Bacillus subtilis GJ231 was deposited in the General Microbiology Center of the China Culture Collection Administration, with a deposit date of August 4, 2023 and a deposit number of CGMCC No. 28101. The strain has been patented, with the patent number ZL202311296123.X.

[0012] Preferably, the dosages of Trichoderma longibrachiatum NT241, Mucor circinelloides GJ242, Trichoderma leucospermum GJ243, and Bacillus subtilis GJ231 of the present invention are distributed in equal proportions.

[0013] In a second aspect, the present invention provides a method for preparing the composite bacterial agent, comprising the following steps:

[0014] Step 1, preparation of seed solution:

[0015] Fermenting the Bacillus subtilis GJ231 liquid until the strain reaches the logarithmic phase, and obtaining a fermentation liquid after the fermentation is completed, which is the seed liquid of Bacillus subtilis GJ231;

[0016] The liquid of Trichoderma longibrachiatum NT241 was fermented until the strain reached the logarithmic phase, and the fermentation liquid was obtained after the fermentation was completed, which was the seed liquid of Trichoderma longibrachiatum NT241;

[0017] Fermenting the liquid of Mucor circinelloides GJ242 until the strain reaches the logarithmic phase, and obtaining the fermented bacterial liquid after the fermentation is the seed liquid of Mucor circinelloides GJ242;

[0018] Fermenting the liquid of the white sac rake tooth fungus GJ243 until the strain reaches the logarithmic phase, and obtaining the fermented bacterial liquid after the fermentation is completed is the seed liquid of the white sac rake tooth fungus GJ243;

[0019] Step 2: Preparation of bacterial solution:

[0020] The Bacillus subtilis GJ231 seed liquid prepared in step 1 is inoculated into the fermentation tank liquid at an inoculum rate of 3-5% for fermentation. After the fermentation is completed, the fermentation liquid obtained is the Bacillus subtilis GJ231 inoculum;

[0021] The long-branch Trichoderma NT241 seed liquid prepared in step 1 is inoculated into the fermentation tank liquid at an inoculum rate of 3-5% for fermentation. After the fermentation is completed, the fermentation liquid obtained is the long-branch Trichoderma NT241 inoculum;

[0022] The seed liquid of Mucor circinelloides GJ242 prepared in step 1 is inoculated into the fermentation tank liquid at an inoculum rate of 3-5% for fermentation. After the fermentation is completed, the fermentation liquid obtained is the inoculum of Mucor circinelloides GJ242;

[0023] The seed liquid of the white sac rake tooth fungus GJ243 prepared in step 1 is inoculated into the fermentation tank liquid at an inoculum rate of 3-5% for fermentation. After the fermentation is completed, the fermentation liquid is obtained, which is the bacterial agent of the white sac rake tooth fungus GJ243;

[0024] Step 3: Preparation of composite bacterial agent:

[0025] The four bacterial agents prepared above, namely, Trichoderma longibrachiatum NT241, Mucor circinelloides GJ242, Trichoderma leucospermum GJ243, and Bacillus subtilis GJ231, were mixed in equal volumes to obtain a composite bacterial agent.

[0026] Preferably, the preparation of the seed solution in the preparation method of the present invention is specifically as follows:

[0027] Bacillus subtilis GJ231 is fermented in liquid form until the strain reaches the logarithmic phase. The liquid fermentation medium is LB liquid medium. The liquid fermentation conditions are as follows: fermentation temperature 30-35°C, rotation speed 150-180r / min, fermentation liquid pH 7-8, fermentation tank volume 50L, fermentation tank pressure 0.05-0.07Mpa, fermentation time 20-24h, and fermentation liquid is obtained after the fermentation is completed, which is the Bacillus subtilis GJ231 seed liquid;

[0028] The long-branch Trichoderma NT241 was fermented in liquid form until the strain reached the logarithmic phase. The liquid fermentation medium was PDB medium. The liquid fermentation conditions were as follows: fermentation temperature of 25-30°C, rotation speed of 180-200 r / min, fermentation liquid pH of 6-7, fermentation tank volume of 50 L, fermentation tank pressure of 0.05-0.07 MPa, and fermentation time of 2-3 days. After the fermentation, the fermentation liquid was obtained, which was the seed liquid of the long-branch Trichoderma NT241.

[0029] The liquid fermentation of Mucor circinelloides GJ242 was carried out until the strain reached the logarithmic phase. The liquid fermentation medium was PDB medium. The liquid fermentation conditions were as follows: fermentation temperature of 25-30°C, rotation speed of 180-200 r / min, pH of fermentation liquid of 6-7, fermentation tank volume of 50 L, fermentation tank pressure of 0.05-0.07 MPa, and fermentation time of 2-3 days. The fermentation liquid obtained after the fermentation was the seed liquid of Mucor circinelloides GJ242.

[0030] The white capsule rake tooth fungus GJ243 is subjected to liquid fermentation until the strain reaches the logarithmic phase. The liquid fermentation medium is PDB medium. The liquid fermentation conditions are as follows: fermentation temperature of 25-30°C, rotation speed of 180-200 r / min, fermentation liquid pH of 6-7, fermentation tank volume of 50L, fermentation tank pressure of 0.05-0.07Mpa, fermentation time of 3-3.5d. The fermentation liquid obtained after the fermentation is the seed liquid of the white capsule rake tooth fungus GJ243.

[0031] Preferably, the preparation of the bacterial solution in the preparation method of the present invention is specifically as follows:

[0032] The Bacillus subtilis GJ231 seed liquid prepared in step 1 was inoculated into a fermentation tank for liquid fermentation at an inoculum rate of 3-5%. The liquid fermentation medium was LB liquid medium. The liquid fermentation conditions were as follows: fermentation temperature of 30-35°C, rotation speed of 150-180 r / min, fermentation liquid pH of 7-8, fermentation tank volume of 500 L, fermentation tank pressure of 0.05-0.07 MPa, fermentation time of 72 h, and defoaming agent of polyether type. After the fermentation was completed, the fermentation liquid obtained was the bacterial agent of Bacillus subtilis GJ231. The number of effective viable bacteria in the bacterial liquid was ≥2×10 8 pcs / ml;

[0033] The long-branch Trichoderma NT241 seed liquid prepared in step 1 was inoculated into a fermentation tank at an inoculum size of 3-5% for liquid fermentation. The liquid fermentation medium was PDB medium. The liquid fermentation conditions were as follows: fermentation temperature of 25-30°C, rotation speed of 200-220 r / min, fermentation liquid pH of 6-7, fermentation tank volume of 500 L, fermentation tank pressure of 0.05-0.07 MPa, fermentation time of 5-6 d, and defoaming agent of polyether type. After the fermentation was completed, the fermentation liquid obtained was the inoculum of long-branch Trichoderma NT241; the effective viable bacteria count in the liquid was ≥1×10 7 pcs / ml;

[0034] The seed liquid of Mucor circinelloides GJ242 prepared in step 1 was inoculated into a fermentation tank for liquid fermentation at an inoculum size of 3-5%. The liquid culture medium was PDB culture medium. The liquid fermentation conditions were as follows: fermentation temperature of 25-30°C, rotation speed of 200-220 r / min, fermentation liquid pH of 6-7, fermentation tank volume of 500 L, fermentation tank pressure of 0.05-0.07 MPa, fermentation time of 5-6 d, and defoaming agent of polyether type. After the fermentation was completed, the fermentation liquid obtained was the inoculum of Mucor circinelloides GJ242. The number of effective viable bacteria in the liquid was ≥1×10 7 pcs / ml;

[0035] The seed liquid of the white sac rake tooth fungus GJ243 prepared in step 1 is inoculated into a fermentation tank for liquid fermentation at an inoculum rate of 3-5%. The liquid culture medium is PDB culture medium. The liquid fermentation conditions are as follows: fermentation temperature of 25-30°C, rotation speed of 200-220r / min, fermentation liquid pH of 6-7, fermentation tank volume of 500L, fermentation tank pressure of 0.05-0.07Mpa, fermentation time of 6-7d, defoaming agent of polyether defoaming agent. After the fermentation is completed, the fermentation liquid obtained is the bacterial agent of the white sac rake tooth fungus GJ243; the effective viable bacteria count in the bacterial liquid is ≥1×10 7 pcs / ml.

[0036] The effective viable bacteria count of the composite bacterial agent prepared by the present invention is: 2.3×10 8 pcs / ml.

[0037] In a third aspect, the present invention provides the use of the above-mentioned composite bacterial agent in degrading straw.

[0038] Preferably, the composite bacterial agent of the present invention is used in degrading rape straw.

[0039] In a fourth aspect, the present invention provides the use of the above-mentioned composite bacterial agent in promoting crop growth.

[0040] Preferably, the composite bacterial agent of the present invention is used to promote the growth of rapeseed and corn crops.

[0041] Beneficial effects of the present invention:

[0042] The composite inoculant provided by this invention is a novel microbial inoculant prepared using four high-quality microorganisms selected independently: Trichoderma longibrachiatum NT241, Mucor circinelloides GJ242, Trichoderma leucospermi GJ243, and Bacillus subtilis GJ231. This composite inoculant exhibits a strong cellulase activity of 38 U / mL, meeting the GB20287-2006 standard for agricultural microbial inoculants.

[0043] 2. The composite inoculant of the present invention has a significant effect on straw degradation. Experiments on rapeseed straw revealed that: (1) the composite inoculant of the present invention had a hemicellulose degradation rate of 55.29%, a cellulose degradation rate of 31.08%, and a lignin degradation rate of 21.09%; (2) the composite inoculant of the present invention significantly improved the degree of humus conversion of rapeseed straw compared to the blank control, indicating that the composite inoculant of the present invention can promote the humus conversion of straw.

[0044] 3. The composite microbial agent of the present invention has a significant effect in promoting crop growth. Germination tests on corn and rapeseed seeds showed that the germination rates of rapeseed and corn seeds treated with the composite microbial agent of the present invention were superior to those of the blank control group. The germination rates of rapeseed and corn seeds were the highest when the composite microbial agent was diluted 30 times.

[0045] 4. This composite bacterial agent not only has a simple formula, low cost, and short preparation cycle, but is also suitable for industrial mass production, easy to promote and apply, can reduce the use of chemical fertilizers, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 : Schematic diagram of the Congo red decolorization results of Mucor circinelloides GJ242

[0047] Figure 2 :Schematic diagram of the morphological characteristics of Mucor circinelloides GJ242

[0048] Figure 3 :Analysis of the phylogenetic tree of Mucor circinelloides GJ242

[0049] Figure 4 :Schematic diagram of strain compatibility results

[0050] Figure 5 : Prepared composite bacterial agent

[0051] Figure 6 : Glucose standard curve

[0052] Figure 7 :Verification of cellulose degradation rate of single strain inoculants and composite bacteria inoculants

[0053] Figure 8 :Rape straw field degradation effect diagram

[0054] Figure 9 :Degradation effect of rape straw lignocellulose

[0055] Figure 10 : Changes in humus content

[0056] Figure 11 :Germination rate of rapeseed seeds

[0057] Figure 12 :Germination rate of corn seeds DETAILED DESCRIPTION

[0058] The present invention is described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. Those skilled in the art will be able to make several modifications and improvements without departing from the scope of the present invention, all of which fall within the scope of protection of the present invention.

[0059] Unless otherwise specified, the test materials used in the following examples of the present invention were purchased from conventional biochemical reagent stores. Unless otherwise specified, the methods in the examples are conventional methods in this field. The culture media and other materials used in the following examples are as follows:

[0060] PDA solid medium: Weigh 200g of potato peels and cut into 2cm cubes. Add an appropriate amount of distilled water and boil for 30 minutes. Filter through 8 layers of gauze. Add 20g of agar powder and 20g of glucose to the filtrate, stir thoroughly, and dilute to 1000mL with distilled water. Adjust the pH to natural and sterilize at 121°C for 25 minutes.

[0061] PDB liquid medium: Wash and peel unsprouted potatoes, weigh 200g, cut into 2cm cubes, add an appropriate amount of distilled water, boil for 30 minutes, and filter through 8 layers of gauze. Add 20g of glucose to the filtrate, stir thoroughly, and dilute to 1000mL with distilled water. Adjust the pH to natural, and sterilize at 121°C for 25 minutes.

[0062] LB liquid medium: 10 g tryptone, 5 g yeast powder, 10 g sodium chloride, 1000 mL distilled water, natural pH, high temperature sterilization at 121°C for 25 min.

[0063] Crop straw: Crop straw obtained from Guiyang City, Guizhou Province after grain harvest in April, with a moisture content of about 10-20%.

[0064] The functional bacteria used in the microbial agent: Trichoderma longibrachiatum NT241, Mucor circinelloides GJ242, Trichoderma leucospermum GJ243, and Bacillus subtilis GJ231 were all obtained by our laboratory's independent isolation in the early stage.

[0065] Example 1 Isolation, screening and identification of Mucor circinelloides GJ242 strain

[0066] Straw samples with obvious decay were collected from rape straw that had been naturally decayed for more than two months, and the strains were isolated using conventional tissue separation methods.

[0067] The specific method is as follows: Use a sterile scalpel to excise a 0.5 cm x 0.5 cm sample tissue, inoculate it into PDA culture medium, and incubate it in a dark incubator at 25°C for 3-4 days. Once a small amount of hyphae has grown from the sample tissue, isolate hyphae from the edge of the tissue according to their morphological characteristics and perform multiple purifications to obtain a pure strain.

[0068] The obtained pure strain was inoculated into PDA-Congo red agar medium and cultured in a 30℃ constant temperature incubator for 3 days. The decolorization of the pure colony on the Congo red medium was observed and recorded. The decolorization zone diameter was used to preliminarily judge the lignocellulose degradation ability of the strain. Figure 1 As shown, the diameter of the Congo red hydrolysis zone of Mucor circinelloides GJ242 was about 6.89±0.21 cm, and the colony diameter was about 6.62±0.19 cm, indicating that it had a certain ability to degrade lignocellulose.

[0069] The strain GJ242 was further inoculated onto the PDA agar medium, and then the PDA agar medium was placed upside down in a 30°C constant temperature incubator to observe the morphology of the grown colonies. Figure 2 As shown, strain GJ242 reproduced rapidly on the surface of solid PDA medium, with loose colony texture and white, dense and fuzzy mycelium.

[0070] Genomic DNA from strain GJ242 was extracted, and the 18S rRNA gene sequence was amplified and sequenced using the universal fungal primers NS1 (5'-GTAGTCATATG CTTGTCTC-3') and NS6 (5'-GCATCACAGACCTGTTATTGCCTC-3'). The resulting sequence was aligned and analyzed for homology using the Basic Local Alignment Search Tool (BLAST) at NCBI to confirm the accuracy of the sequencing results. After confirming the sequence accuracy, the required reference strain sequences were downloaded, and a phylogenetic tree was constructed using the maximum likelihood method using MEGA 7.0 software. The phylogenetic analysis was completed with 1000 bootstrap tests.

[0071] The sequencing results are shown below:

[0072] >

[0073]

[0074] Phylogenetic tree analysis Figure 3 As shown, GJ242 forms a distinct branch with Mucor circinelloides. 18S rRNA sequence alignment and results further confirmed GJ242 as Mucor circinelloides at the molecular biological level.

[0075] Example 2 Determination of strain compatibility

[0076] Antagonism between bacteria and fungi is determined using the plate standoff method. Under a sterile operating table, the screened fungi are punched into the center of a PDA culture medium. A ring of bacteria is inoculated with an inoculating loop 2-3 cm from the fungus. The culture medium is then incubated upside down in a 30°C incubator for 3-5 days. The growth of the strains is observed. If there is no distinct boundary where the strains intersect, and the growth of both strains is unaffected, this indicates no antagonism between the strains.

[0077] The antagonistic activity of the strains used in the experiment was as follows Figure 4 As shown, there is no obvious antagonistic zone between the strains, and there is no inhibitory effect between strains NT241, GJ242, GJ243, and GJ231. The strains are compatible and can be used as materials for composite bacterial agents.

[0078] Example 3 Composite bacterial agent

[0079] Formula: 25% Trichoderma longibrachiatum NT241, 25% Mucor circinelloides GJ242, 25% Trichoderma leucospermum GJ243, and 25% Bacillus subtilis GJ231.

[0080] The Trichoderma longibrachiatum NT241 was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms on April 7, 2024, with a deposit number of CGMCC No. 41139.

[0081] The Mucor circinelloides GJ242 was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms on April 7, 2024, with a deposit number of CGMCC No. 41140.

[0082] The Irpex lacteus GJ243 was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms on April 7, 2024, with a deposit number of CGMCC No. 41281.

[0083] The Bacillus subtilis GJ231 is deposited in the General Microbiology Center of the China Culture Collection Administration, with a deposit date of August 4, 2023 and a deposit number of CGMCC No. 28101.

[0084] Example 4 Preparation of composite bacterial agent

[0085] (1) Preparation of seed solution

[0086] Bacillus subtilis GJ231, deposited with CGMCC No. 28101, was subjected to liquid fermentation until the strain reached its logarithmic phase. The liquid culture medium was LB liquid culture medium. The liquid fermentation conditions were: fermentation temperature of 30-35°C, rotation speed of 150-180 r / min, fermentation liquid pH of 7-8, fermentation tank volume of 50 L, fermentation tank pressure of 0.05-0.07 MPa, and fermentation time of 20-24 hours. After fermentation, the fermented bacterial liquid was obtained, which was the Bacillus subtilis GJ231 seed liquid.

[0087] Trichoderma longibrachiatum NT241 (CGMCC No. 41139) was subjected to liquid fermentation to the logarithmic phase in PDB medium. The fermentation conditions were: fermentation temperature of 25-30°C, fermentation speed of 180-200 r / min, fermentation broth pH of 6-7, fermentation tank volume of 50 L, fermentation tank pressure of 0.05-0.07 MPa, and fermentation time of 2-3 days. After fermentation, the resulting fermentation broth was used as the seed solution of Trichoderma longibrachiatum NT241.

[0088] Mucor circinelloides GJ242 (CGMCC accession number 41140) was fermented in liquid medium until the strain reached the logarithmic phase. The liquid culture medium was PDB medium. The fermentation conditions were: fermentation temperature of 25-30°C, fermentation speed of 180-200 rpm, fermentation broth pH of 6-7, fermentation tank volume of 50 L, fermentation tank pressure of 0.05-0.07 MPa, and fermentation time of 2-3 days. After fermentation, the fermented broth obtained was the seed liquid of Mucor circinelloides GJ242.

[0089] White capsule spore GJ243 (CGMCC No. 41281) was subjected to liquid fermentation to the logarithmic phase using PDB medium. The fermentation conditions were: fermentation temperature of 25-30°C, rotation speed of 180-200 rpm, fermentation broth pH of 6-7, fermentation tank volume of 50 L, fermentation tank pressure of 0.05-0.07 MPa, and fermentation time of 3-3.5 days. The fermented bacterial liquid obtained after fermentation was the seed liquid of White capsule spore GJ243.

[0090] (2) Preparation of microbial agents

[0091] The Bacillus subtilis GJ231 seed liquid prepared in (1) is inoculated into a fermentation tank for liquid fermentation at an inoculation rate of 3-5%. The liquid culture medium is LB liquid culture medium. The liquid fermentation conditions are as follows: fermentation temperature of 30-35°C, rotation speed of 150-180r / min, fermentation liquid pH of 7-8, fermentation tank volume of 500L, fermentation tank pressure of 0.05-0.07Mpa, fermentation time of 48-72h, and defoaming agent of polyether type. After the fermentation is completed, the fermentation liquid is obtained, which is the bacterial agent of Bacillus subtilis GJ231. The number of effective viable bacteria in the bacterial liquid is ≥2×10 8 pcs / ml.

[0092] The seed liquid of Trichoderma longifolia NT241 with the accession number CGMCC No.41139 prepared in (1) was inoculated into a fermentation tank at an inoculum rate of 3-5% for liquid fermentation. The liquid culture medium was PDB culture medium. The liquid fermentation conditions were as follows: fermentation temperature of 25-30°C, rotation speed of 200-220 r / min, fermentation liquid pH of 6-7, fermentation tank volume of 500 L, fermentation tank pressure of 0.05-0.07 MPa, fermentation time of 5-6 days, and a polyether defoamer. After the fermentation was completed, the fermentation liquid was obtained, which was the inoculum of Trichoderma longifolia NT241. The number of effective viable bacteria in the liquid was ≥1×10 7 pcs / ml.

[0093] The seed liquid of Mucor circinelloides GJ242 with the accession number CGMCC No. 41140 prepared in (1) is inoculated into a fermentation tank for liquid fermentation at an inoculum rate of 3-5%. The liquid culture medium is PDB culture medium. The liquid fermentation conditions are as follows: fermentation temperature of 25-30°C, rotation speed of 200-220 r / min, fermentation liquid pH of 6-7, fermentation tank volume of 500 L, fermentation tank pressure of 0.05-0.07 MPa, fermentation time of 5-6 days, and a polyether defoamer. After the fermentation is completed, the fermentation liquid is obtained, which is the inoculum of Mucor circinelloides GJ242, and the effective viable bacteria count in the liquid is ≥1×10 7 pcs / ml.

[0094] The seed liquid of the white sac rake tooth fungus GJ243 with the accession number CGMCC No.41281 prepared in (1) is inoculated into a fermentation tank at an inoculum amount of 3-5% for liquid fermentation. The liquid culture medium is PDB culture medium. The liquid fermentation conditions are as follows: fermentation temperature of 25-30°C, rotation speed of 200-220r / min, fermentation liquid pH of 6-7, fermentation tank volume of 500L, fermentation tank pressure of 0.05-0.07Mpa, fermentation time of 6-7d, and defoaming agent of polyether type. After the fermentation is completed, the fermentation liquid is obtained as the inoculum of white sac rake tooth fungus GJ243, and the effective viable bacteria count in the liquid is ≥1×10 7 pcs / ml.

[0095] (3) Mix equal volumes of Trichoderma longibrachiatum NT241, Mucor circinelloides GJ242, Trichoderma leucospermum GJ243, and Bacillus subtilis GJ231 to obtain a composite bacterial agent, such as Figure 5 shown.

[0096] Example 5 Detection of effective viable bacteria count of composite bacterial agent

[0097] The effective viable bacterial count was determined by plate count method. 10.0 mL of the composite bacterial agent prepared in Example 4 was added to 90 mL of sterile water, allowed to stand for 20 min, and fully shaken on a rotary shaker at 200 r / min for 30 min to form a mother liquid bacterial suspension (1×10 1 ). Use a sterile pipette to draw 0.5mL of the above-mentioned mother liquid bacterial suspension, add it to 4.5mL of sterile water, and perform a series of dilutions from 1 to 10. Use a sterile pipette to draw 0.1mL of the bacterial suspension diluted 4, 5, and 6 times respectively, and add it to the pre-prepared PDA culture medium plate. Use a sterile stick to evenly spread the bacterial suspension of different dilutions on the agar surface. Repeat each dilution 3 times, and use sterile water (or saline) as a blank control. Culture at 30°C. Use the dilution plate with 20 to 300 colonies as the counting standard (10 to 150 colonies for filamentous fungi), and count the number of effective live bacteria and the number of miscellaneous bacteria respectively. Calculate according to the following formula:

[0098]

[0099] Where X is the average number of colonies, in units; k is the dilution factor; V1 is the volume of the base solution, in milliliters (mL); V2 is the amount of bacterial suspension added, in milliliters (mL).

[0100] Results: The effective viable bacteria count of the bacterial agent was 2.3×10 8 / mL, reaching the standard of agricultural microbial agents GB20287-2006.

[0101] Implementation Case 6: Cellulase Activity Detection of Composite Bacterial Agent

[0102] (1) Drawing of standard curve

[0103] Take 5 large test tubes and use a pipette to accurately draw the standard glucose solution and sodium phosphate buffer solution according to Table 1 and mix them evenly to obtain standard glucose solutions of various concentrations.

[0104] Table 1 Preparation of standard glucose solutions of different concentrations

[0105] Test tube number Standard glucose solution / mL Sodium phosphate buffer / mL Amount of glucose in the test tube / ug 1 0 5.0 0 2 0.4 4.6 400 3 0.8 4.2 800 4 1.6 3.4 1600 5 3.2 1.8 3200

[0106] Add 3.0 mL of DNS colorimetric solution to each tube, shake well, heat accurately in a boiling water bath for 5.0 min, cool with running water, shake well, and measure the OD value of each tube at 490 nm. Draw a standard curve with the micrograms of glucose as the horizontal axis and the OD value as the vertical axis.

[0107] (2) Preparation of original enzyme solution

[0108] Measure 10.0 mL of the composite bacterial agent prepared in Example 4 and add it to a conical flask containing glass beads. Dilute with a predetermined volume of distilled water, let it rest for 20 minutes, shake it at 200 rpm for 30 minutes, and then filter it through four layers of gauze. Centrifuge the filtrate at 3000 rpm for 10 minutes. Dilute the supernatant after centrifugation to an appropriate concentration based on enzyme activity. This serves as the original enzyme solution for testing.

[0109] (3) Measurement steps

[0110] Prepare three large test tubes, one as a blank control and the other two as parallel sample tubes. Add 1.0 mL of the original enzyme solution to the sample tube. Then, add 4.0 mL of CMC buffer, preheated to 60°C, to each of the three test tubes. Incubate in a 60°C water bath for 20 minutes. Remove from the bath and immediately add 3.0 mL of DNS colorimetric solution to each tube. Shake well, then add 1.0 mL of the original enzyme solution to the control tube. Place the three test tubes in a boiling water bath. After 5 minutes of color development, remove from the bath, cool under running water, and measure the OD value at 490 nm using a spectrophotometer.

[0111] (4) Calculation of cellulase activity

[0112] According to the standard curve, the measured OD value is converted into micrograms of glucose.

[0113]

[0114] Calculation of enzyme activity: 1 mL of original enzyme solution produces 1 μg of glucose in 1 minute, which is defined as 1 enzyme activity unit (U).

[0115] Where: U is the enzyme activity of the sample, in micrograms per milliliter (ug / mL); k is the sample dilution factor; m1 is the amount of glucose in the sample, in micrograms (ug); m0 is the amount of glucose in the control, in micrograms (ug); 20 is the reaction time between the enzyme and the substrate, in minutes (min).

[0116] The obtained glucose standard curve is as follows: Figure 6 As shown, R 2 The absorbance was >0.95, demonstrating a reliable linear relationship between the absorbance and glucose concentration, which could be used as a standard curve. The final measured cellulase activity of the composite inoculant reached 38 U / mL, meeting the GB 20287-2006 standard for agricultural microbial inoculants.

[0117] Example 7: Verification of the degradation ability of bacterial agents

[0118] After harvest, rapeseed straw was naturally air-dried and mechanically processed to 1-2 cm. 30 g of the straw was weighed and placed in a nylon mesh bag and sterilized at 121°C for 20 minutes. After sterilization and cooling, sterile water was sprayed to maintain a moisture content of 60% to 70%. Five groups were designed, each adding 5% of the NT241, GJ242, GJ243, and GJ231 single-strain inoculants prepared in Example 4, as well as the composite inoculant, to the rapeseed straw. After mixing evenly, the straw was incubated at 30°C for 7 days. After incubation, the degradation capacity of each strain was evaluated by measuring the changes in cellulose content in the rapeseed straw before and after degradation.

[0119] The results are as follows Figure 7 As shown in the results, each strain has a certain ability to degrade cellulose. The cellulose degradation rate of the composite agent is about 22.8% after 7 days. The strain GJ243 has the highest degradation rate of the single strain, which is about 16%. The strain GJ231 has the lowest degradation rate, which is about 10%. The cellulose degradation effect of each single strain is lower than that of the composite agent.

[0120] Example 8 Application of composite bacterial agent in field degradation of rape straw

[0121] The experiment involved two groups: one experimental group and one control group, with three parallel groups in each. Approximately 200 kg of rapeseed straw, harvested from the rapeseed, was placed on the same plot of land in each group. The straw inoculated with the composite microbial inoculant served as the experimental group, while the control group consisted of water. The straw was stacked in a circular pattern, with the roots centered and the tips facing outward. After each 30-35 cm pile, the inoculant was evenly sprayed and the roots were broken and compacted with a hoe. The height of the stack before mulching was approximately 1.3-1.5 meters, and the width was approximately 1.5-2.0 meters. During stacking, the moisture content of the straw was maintained between 60% and 70%. After stacking, the straw was covered with a 0.01 mm black, recyclable polyethylene blown plastic film. Covering the straw completely with film, leaving no gaps, ensured a stable temperature during composting and increased the survival rate of the functional strains in the composite microbial inoculant. The height of the mulched straw was approximately 1.2-1.4 meters. After the composting is completed (about 17 to 20 days), the degradation effect is judged by the degree of collapse of the straw appearance, odor, temperature, color, cellulose content and humus content.

[0122] The results showed that compared with the blank group, the experimental group had a more obvious overall decline. When the film was opened, the temperature inside the experimental group was high, and hot air was emitted. The straw turned black, had a soft texture, had no obvious odor, and showed obvious decomposition. Figure 8a and b). The blank group had no obvious sagging. The film was opened, the temperature was low, there was no heat emission, there was obvious mold growth, the moldy smell was strong, the overall texture of the straw was hard, and the decomposition phenomenon was not obvious ( Figure 8 c and d)).

[0123] The degradation effects of rapeseed straw lignocellulose in the experimental group and the blank group are as follows Figure 9 The results showed that the degradation rates of hemicellulose, cellulose, and lignin in the experimental groups were 55.29% and 16.39%, 31.08% and 20.09%, and 21.09% and 10.09%, respectively, increasing by 38.89%, 11.00%, and 11.01%, respectively, compared with the blank group. The addition of the composite inoculant had the best effect on the degradation of hemicellulose in rapeseed straw, followed by cellulose and lignin.

[0124] The results of rape straw humus transformation in the experimental group and the blank group are as follows Figure 10 As shown in the figure, the humus index of the experimental group and the blank group were 1.23±0.06 and 0.98±0.01, respectively, and the total humus content was 364.59±12.80 g / kg and 344.16±17.99, respectively. The humification degree of the experimental group was higher than that of the blank group, and the addition of the straw composite inoculant can promote the humification degree of straw.

[0125] Example 9: Germination of corn and rapeseed seeds with composite microbial agent

[0126] Corn and rapeseed seeds with plump, shiny grains, no insect damage or mechanical damage, and similar size were selected. Seed pretreatment included disinfection and germination. Seed disinfection was performed by soaking in a 0.1% NaClO solution at room temperature for 15 minutes and rinsing with distilled water five times. The disinfected seeds were soaked continuously at 25°C for 12 hours, with the water changed every 5 hours for soaking and germination. A double layer of filter paper was placed at the bottom of the disinfected petri dish, and the surface moisture of the disinfected and soaked seeds was absorbed. The seeds were neatly arranged in the petri dish, and the same number of seeds were placed in each petri dish. The experiment set up a gradient treatment of the composite bacterial agent with 5 concentrations (dilution factors of 5, 10, 15, 30, and 60, respectively). Sterile distilled water was used as a blank control. Other environmental factors were the same. Three parallels were set for each treatment, and each group of experiments was repeated three times. The number of germinations was recorded every day during the same time period for 7 consecutive days, and the germination rate of the seeds was calculated based on the number of germinations.

[0127] Results: The germination rates of rapeseed and corn seeds were as follows: Figure 11 and Figure 12As shown, seeds of both crops began to germinate after two days and completed germination in about a week, showing an increasing trend in germination. When the composite inoculant was diluted 30 times, rapeseed and corn seeds had the best germination rates, maintaining a leading trend from the start of incubation. By the seventh day of incubation, the germination rates of rapeseed and corn seeds reached a maximum of 90.83% and 77.27%, respectively, representing increases of 2.5% and 1.5% compared to the control group. Adding the straw composite inoculant at a dilution of 30 promoted germination of both rapeseed and corn seeds.

[0128] Although the present invention has been described in detail above using general explanations, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A composite bacterial agent, characterized in that: It is prepared from four active ingredients: Trichoderma longibrachiatum NT241, Mucor circinelloides GJ242, Irpex lacteus GJ243, and Bacillus subtilis GJ231, among which: The Mucor circinelloides GJ242 was deposited in the General Microbiology Center of the China Culture Collection Administration, with a deposit date of April 7, 2024 and a deposit number of CGMCC No. 41140.

2. A composite bacterial agent according to claim 1, characterized in that The dosages of the Trichoderma longibrachiata NT241, Mucor circinelloides GJ242, Trichoderma leucospermum GJ243 and Bacillus subtilis GJ231 are distributed in equal proportions.

3. A method for preparing the composite bacterial agent according to claim 1, characterized in that: The steps include: Step 1, preparation of seed solution: Fermenting the Bacillus subtilis GJ231 liquid until the strain reaches the logarithmic phase, and obtaining a fermentation liquid after the fermentation is completed, which is the seed liquid of Bacillus subtilis GJ231; The liquid of Trichoderma longibrachiatum NT241 was fermented until the strain reached the logarithmic phase, and the fermentation liquid was obtained after the fermentation was completed, which was the seed liquid of Trichoderma longibrachiatum NT241; Fermenting the liquid of Mucor circinelloides GJ242 until the strain reaches the logarithmic phase, and obtaining the fermented bacterial liquid after the fermentation is the seed liquid of Mucor circinelloides GJ242; Fermenting the liquid of the white sac rake tooth fungus GJ243 until the strain reaches the logarithmic phase, and obtaining the fermented bacterial liquid after the fermentation is completed is the seed liquid of the white sac rake tooth fungus GJ243; Step 2: Preparation of bacterial solution: The Bacillus subtilis GJ231 seed liquid prepared in step 1 is inoculated into the fermentation tank liquid at an inoculum rate of 3-5% for fermentation. After the fermentation is completed, the fermentation liquid obtained is the Bacillus subtilis GJ231 inoculum; The long branch Trichoderma NT241 seed liquid prepared in step 1 is inoculated into the fermentation tank liquid at an inoculum rate of 3-5% for fermentation. After the fermentation is completed, the fermentation liquid obtained is the long branch Trichoderma NT241 inoculum; The seed liquid of Mucor circinelloides GJ242 prepared in step 1 is inoculated into the fermentation tank liquid for fermentation at an inoculum rate of 3-5%. After the fermentation is completed, the fermentation liquid obtained is the inoculum of Mucor circinelloides GJ242; The seed solution of the white sac rake tooth fungus GJ243 prepared in step 1 is inoculated into the fermentation tank liquid at an inoculum rate of 3-5% for fermentation. After the fermentation is completed, the fermentation liquid obtained is the bacterial agent of the white sac rake tooth fungus GJ243; Step 3: Preparation of composite bacterial agent: The four bacterial agents prepared above, namely, Trichoderma longibrachiatum NT241, Mucor circinelloides GJ242, Trichoderma leucospermum GJ243, and Bacillus subtilis GJ231, were mixed in equal volumes to obtain a composite bacterial agent.

4. The preparation method according to claim 3, wherein The preparation of the seed solution in step 1 is specifically as follows: Bacillus subtilis GJ231 was fermented in liquid form until the strain reached the logarithmic phase. The liquid fermentation medium was LB liquid medium. The liquid fermentation conditions were as follows: fermentation temperature 30-35° C., rotation speed 150-180 r / min, fermentation liquid pH 7-8, fermentation tank volume 50 L, fermentation tank pressure 0.05-0.07 MPa, fermentation time 20-24 h. After the fermentation was completed, a fermentation liquid was obtained, which was the Bacillus subtilis GJ231 seed liquid. The long-branch Trichoderma NT241 was fermented in liquid form until the strain reached the logarithmic phase. The liquid fermentation medium was PDB medium. The liquid fermentation conditions were as follows: fermentation temperature of 25-30°C, rotation speed of 180-200 r / min, fermentation liquid pH of 6-7, fermentation tank volume of 50 L, fermentation tank pressure of 0.05-0.07 MPa, fermentation time of 2-3 days, and after the fermentation, a fermentation liquid was obtained, which was the seed liquid of the long-branch Trichoderma NT241. The liquid fermentation of Mucor circinelloides GJ242 was carried out until the logarithmic phase of the strain, wherein the liquid fermentation medium was PDB medium, and the liquid fermentation conditions were as follows: fermentation temperature of 25-30°C, rotation speed of 180-200 r / min, pH of fermentation liquid of 6-7, fermentation tank volume of 50 L, fermentation tank pressure of 0.05-0.07 MPa, fermentation time of 2-3 days, and the fermentation liquid obtained after the fermentation was the seed liquid of Mucor circinelloides GJ242; The white capsule rake tooth fungus GJ243 is liquid fermented to the logarithmic phase of the strain. The liquid fermentation medium is PDB medium. The liquid fermentation conditions are as follows: fermentation temperature of 25-30°C, rotation speed of 180-200r / min, fermentation liquid pH of 6-7, fermentation tank volume of 50 L, fermentation tank pressure of 0.05-0.07 Mpa, fermentation time of 3-3.5 days. The fermentation liquid obtained after the fermentation is the seed liquid of the white capsule rake tooth fungus GJ243.

5. The preparation method according to claim 3, wherein The preparation of the bacterial solution in step 2 is specifically as follows: The Bacillus subtilis GJ231 seed liquid prepared in step 1 was inoculated into a fermentation tank for liquid fermentation at an inoculum rate of 3-5%. The liquid fermentation medium was LB liquid medium. The liquid fermentation conditions were as follows: fermentation temperature of 30-35°C, rotation speed of 150-180 r / min, fermentation liquid pH of 7-8, fermentation tank volume of 500 L, fermentation tank pressure of 0.05-0.07 Mpa, fermentation time of 72 h, defoaming agent of polyether type. After the fermentation was completed, the fermentation liquid obtained was the bacterial agent of Bacillus subtilis GJ231. The number of effective viable bacteria in the bacterial liquid was ≥2×10 8 Pieces / ml; The long-branch Trichoderma NT241 seed liquid prepared in step 1 was inoculated into a fermentation tank for liquid fermentation at an inoculum rate of 3-5%. The liquid fermentation medium was PDB medium. The liquid fermentation conditions were as follows: fermentation temperature of 25-30°C, rotation speed of 200-220 r / min, fermentation liquid pH of 6-7, fermentation tank volume of 500 L, fermentation tank pressure of 0.05-0.07 Mpa, fermentation time of 5-6 d, defoaming agent of polyether type. After the fermentation was completed, the fermentation liquid obtained was the inoculum of long-branch Trichoderma NT241; the effective viable bacteria count in the liquid was ≥1×10 7 Pieces / ml; The seed liquid of Mucor circinelloides GJ242 prepared in step 1 was inoculated into a fermentation tank for liquid fermentation at an inoculum rate of 3-5%. The liquid culture medium was PDB culture medium. The liquid fermentation conditions were as follows: fermentation temperature of 25-30°C, rotation speed of 200-220 r / min, fermentation liquid pH of 6-7, fermentation tank volume of 500 L, fermentation tank pressure of 0.05-0.07 Mpa, fermentation time of 5-6 d, and defoaming agent of polyether type. After the fermentation was completed, the fermentation liquid obtained was the inoculum of Mucor circinelloides GJ242; the effective viable bacteria count in the bacterial liquid was ≥1×10 7 Pieces / ml; The seed liquid of the white sac rake tooth fungus GJ243 prepared in step 1 was inoculated into the fermentation tank for liquid fermentation at an inoculum rate of 3-5%. The liquid culture medium was PDB culture medium. The liquid fermentation conditions were as follows: fermentation temperature of 25-30°C, rotation speed of 200-220 r / min, fermentation liquid pH of 6-7, fermentation tank volume of 500 L, fermentation tank pressure of 0.05-0.07 Mpa, fermentation time of 6-7 d, defoaming agent of polyether defoaming agent. After the fermentation was completed, the fermentation liquid obtained was the bacterial agent of the white sac rake tooth fungus GJ243. The number of effective viable bacteria in the bacterial liquid was ≥1×10 7 pcs / ml.

6. The preparation method according to claim 3, wherein The effective viable bacterial count of the composite bacterial agent in step 3 is: 2.3×10 8 pcs / ml.

7. Use of the composite bacterial agent according to any one of claims 1 to 2 or the composite bacterial agent prepared by the preparation method according to any one of claims 3 to 6 in degrading straw.

8. The use according to claim 7, characterized in that The straw is rape straw.

9. Use of the composite microbial agent according to any one of claims 1 to 2 or the composite microbial agent prepared by the preparation method according to any one of claims 3 to 6 in promoting the growth of rapeseed and corn.

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