Complex microbial inoculant as well as preparation method and application thereof

Abstract

The invention discloses a complex microbial inoculant as well as a preparation method and application thereof. The complex microbial inoculant comprises trichoderma fungi, mucor fungi, irpex fungi and bacillus bacteria, the effective viable count of the prepared complex microbial inoculant is 2.3 * 10 < 8 > / mL, the cellulase activity is 38U / mL, and the complex microbial inoculant reaches the standard of agricultural microbial inoculants GB 20287-2006. The composite liquid microbial inoculum prepared by the invention has the characteristic of rapid decomposition, is suitable for promoting the decomposition of straws directly returned to the field, and can be used for degrading rape straws in the field and increasing humus; and when the complex microbial inoculant is diluted by 30 times for use, the complex microbial inoculant has a relatively good growth promoting effect on rape and corn seeds. The liquid complex microbial inoculant can accelerate crop straw decomposition and promote crop growth at the same time, and provides complex microbial resources and technical support for 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 biological resource. With the development of agricultural production, my country's grain output has increased significantly, and the amount of crop straw has also increased accordingly. In addition, the popularization and promotion of diversified combustion energy sources have resulted in a large amount of surplus straw in rural areas. In addition, soil degradation, soil organic carbon and nutrient loss, and soil fertility decline are the main problems facing my country's agricultural development. Healthy soil is important for crop growth, thereby protecting human health. More than half of the products of crop photosynthesis are present in straw, which is rich in nitrogen, phosphorus, potassium and organic matter. Returning straw to the field can improve the ecological environment of farmland to varying degrees and regulate the imbalance of soil nutrients. Adhering to the annual return of straw to the field not only has a significant increase in the fertilization stage, but also has an effect on increasing crop production. However, crop straw has a high crude fiber content (30%-40%) and contains lignin, etc. Direct return to the field usually leads to slow decomposition of straw, inability to quickly absorb and utilize it, poor improvement of soil organic matter, and reduced crop yields; in addition, crop straw also faces problems such as large volume, high recycling cost, and inconvenience for unified recycling. To solve this problem, the application of biodegradation technology in the process of returning straw to the field has attracted more and more attention. Biodegradation technology is an economical and environmentally friendly process. It uses functional bacteria to mediate the rapid humification of straw waste in response to the key issues of straw resource utilization and soil improvement. Compared with other crop straw resource utilization technologies, it has the advantages of low cost, simple process, low technical difficulty and harmlessness to the environment.

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

[0004] One of the purposes of the present invention is to provide a composite bacterial agent; the second purpose of the present invention is to provide a method for preparing the composite bacterial agent; the third purpose of the present invention is to provide an application of the composite bacterial agent in degrading straw; the 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 uses Trichoderma longifolia NT241, Mucor circinelloides GJ242, Psoralea corylifolia GJ243, and Bacillus subtilis GJ231 as active ingredients, and degrades the straw, thereby effectively achieving rapid straw decomposition and returning to the field on the spot, and promoting the growth of crops such as rape and corn. As a composite microbial agent 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, Irpexlacteus GJ243, and Bacillus subtilis GJ231, wherein:

[0008] The Mucor circinelloides GJ242 is deposited in the General Microbiology Center of the China Microbiological 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 Microbiological Culture Collection Administration, with a preservation date of April 7, 2024 and a preservation number of CGMCC No.41139; a patent has been applied for the strain, with the application number 202411675277.4.

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

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

[0012] Preferably, the dosages of Trichoderma longibrachiatum NT241, Mucor circinelloides GJ242, Pseudomonas albus 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, which comprises 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 fermented bacterial liquid after the fermentation is completed, which is the seed liquid of Bacillus subtilis GJ231;

[0016] Fermenting the liquid of Trichoderma longifolia NT241 until the logarithmic phase of the strain, and obtaining the fermented bacterial liquid after the fermentation is completed, which is the seed liquid of Trichoderma longifolia NT241;

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

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

[0019] Step 2: Preparation of bacterial solution:

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

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

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

[0023] The seed liquid of the white capsule rake tooth fungus GJ243 prepared in step 1 is inoculated into the fermentation tank liquid for fermentation at an inoculation rate of 3-5%. After the fermentation is completed, the fermented bacterial liquid is obtained, which is the bacterial agent of the white capsule 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] The Bacillus subtilis GJ231 is subjected to liquid fermentation until the strain reaches the logarithmic phase, wherein the medium for the liquid fermentation is LB liquid medium, and the liquid fermentation conditions are 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, and fermentation liquid is obtained after the fermentation is completed, which is the seed liquid of Bacillus subtilis GJ231;

[0028] The long-branch Trichoderma NT241 is subjected to liquid fermentation until the strain reaches the logarithmic phase, wherein the medium for the liquid fermentation is PDB medium, and the liquid fermentation conditions are as follows: the fermentation temperature is 25-30° C., the rotation speed is 180-200 r / min, the pH value of the fermentation liquid is 6-7, the volume of the fermentation tank is 50 L, the pressure of the fermentation tank is 0.05-0.07 MPa, and the fermentation time is 2-3 days. After the fermentation is completed, the fermentation liquid is obtained, which is the seed liquid of the long-branch Trichoderma NT241;

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

[0030] 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: fermentation temperature is 25-30°C, rotation speed is 180-200r / min, fermentation liquid pH is 6-7, fermentation tank volume is 50L, fermentation tank pressure is 0.05-0.07Mpa, fermentation time is 3-3.5d, and 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 is inoculated into the fermentation tank for liquid fermentation at an inoculation rate of 3-5%. The liquid fermentation medium is LB liquid medium. The liquid fermentation conditions are as follows: fermentation temperature is 30-35°C, rotation speed is 150-180r / min, fermentation liquid pH is 7-8, fermentation tank volume is 500L, fermentation tank pressure is 0.05-0.07Mpa, fermentation time is 72h, defoamer is a polyether defoamer, and after the fermentation is completed, the fermented bacterial liquid is obtained, which is the bacterial agent of Bacillus subtilis GJ231; the number of effective live bacteria in the bacterial liquid is ≥2×10 8 Pieces / ml;

[0033] The long-branch Trichoderma NT241 seed liquid prepared in step 1 is inoculated into the fermentation tank for liquid fermentation at an inoculation rate of 3-5%. The liquid fermentation medium is PDB medium, and the liquid fermentation conditions are as follows: fermentation temperature is 25-30°C, rotation speed is 200-220r / min, fermentation liquid pH is 6-7, fermentation tank volume is 500L, fermentation tank pressure is 0.05-0.07Mpa, fermentation time is 5-6d, and defoamer is a polyether defoamer. After the fermentation is completed, the fermented bacterial liquid is obtained, which is the bacterial agent of long-branch Trichoderma NT241; the number of effective live bacteria in the bacterial liquid is ≥1×10 7 Pieces / ml;

[0034] The seed liquid of Mucor circinelloides GJ242 prepared in step 1 is inoculated into the fermentation tank for liquid fermentation at an inoculation rate of 3-5%, the liquid culture medium is PDB culture medium, and the liquid fermentation conditions are as follows: fermentation temperature is 25-30°C, rotation speed is 200-220r / min, fermentation liquid pH is 6-7, fermentation tank volume is 500L, fermentation tank pressure is 0.05-0.07Mpa, fermentation time is 5-6d, defoamer is a polyether defoamer, after fermentation, the fermented bacterial liquid is obtained, which is the bacterial agent of Mucor circinelloides GJ242; the number of effective live bacteria in the bacterial liquid is ≥1×10 7 Pieces / ml;

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

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

[0037] In a third aspect, the present invention provides application 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] 1. The composite microbial agent provided by the present invention is a new microbial agent prepared by using four high-quality microorganisms selected by the self-selected microorganisms, namely, Trichoderma longifolia NT241, Mucor circinelloides GJ242, Psoralea corylifolia GJ243, and Bacillus subtilis GJ231, as active ingredients. The composite microbial agent has a strong cellulase activity of 38 U / mL, and meets the agricultural microbial agent GB20287--2006 standard.

[0043] 2. The composite microbial agent of the present invention has the function of degrading straw, and the effect is remarkable. Through the test of using the composite microbial agent on rape straw, it was found that: (1) the composite microbial agent of the present invention has a degradation rate of 55.29% for hemicellulose, a degradation rate of 31.08% for cellulose, and a degradation rate of 21.09% for lignin; (2) the composite microbial agent of the present invention has a significantly better degree of humus conversion of rape straw than the blank group, indicating that the composite microbial agent of the present invention can promote the humus degree of straw.

[0044] 3. The composite microbial agent of the present invention has a significant effect in promoting crop growth. Through the germination test of corn and rapeseed seeds, it was found that the germination rates of rapeseed seeds and corn seeds treated with the composite microbial agent of the present invention were better than those of the blank group, and when the dilution multiple of the composite microbial agent was 30 times, the germination rates of rapeseed seeds and corn seeds were the best.

[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 a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0047] Figure 2 :Schematic diagram of 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 strain inoculants

[0053] Figure 8 :The effect of rape straw degradation in the field

[0054] Fig. 9 :Degradation effect of rape straw lignocellulose

[0055] Fig.10 :Changes in humus content

[0056] Fig.11 :Germination rate of rapeseed seeds

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

[0058] The present invention is described in detail below in conjunction with 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. For those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

[0059] The test materials used in the following examples of the present invention, unless otherwise specified, 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 peel, cut into small cubes with a side length of 2cm, add appropriate amount of distilled water, boil for 30min, and filter with 8 layers of gauze. Add 20g of agar powder and 20g of glucose to the filtrate, stir evenly, distilled water to 1000mL, pH natural, and sterilize at 121℃ for 25min.

[0061] PDB liquid culture medium: Wash and peel potatoes without germination, weigh 200g, cut into small cubes with a side length of 2cm, add appropriate amount of distilled water, boil for 30min, and filter with 8 layers of gauze. Add 20g of glucose to the filtrate, stir evenly, distilled water to 1000mL, pH natural, and sterilize at 121℃ for 25min.

[0062] LB liquid culture 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 longifolia NT241, Mucor circinelloides GJ242, Trichoderma leucophylla GJ243, and Bacillus subtilis GJ231 were all isolated independently by our laboratory in the early stage.

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

[0066] Obviously corrupted rape straw samples were collected from rape straw that had been naturally corrupted 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 cut a sample tissue of 0.5 cm × 0.5 cm in size and inoculate it into PDA culture medium, and culture it in a constant temperature incubator at 25°C for 3-4 days. When a small amount of hyphae grows out of the sample tissue, the hyphae at the edge of the tissue are selected according to different hyphae morphological characteristics for multiple purification to obtain pure strains.

[0068] The obtained pure strain was inoculated into PDA-Congo red agar medium and cultured in a 30°C constant temperature incubator for 3 days. The decolorization of the pure colony on the Congo red medium was observed and recorded, and the decolorization circle diameter was used to preliminarily determine 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 inverted and placed in a 30°C constant temperature incubator to observe the morphology of the colonies grown. Figure 2 As shown, strain GJ242 reproduced rapidly on the surface of solid PDA medium, the colony texture was loose, and the mycelium was white, dense, and fluffy.

[0070] The genomic DNA of strain GJ242 was extracted, and the 18S rRNA gene sequence was amplified and sequenced using the universal primers NS1 (5'-GTAGTCATATG CTTGTCTC-3') and NS6 (5'-GCATCACAGACCTGTTATTGCCTC-3'). The measured sequence was aligned and homologous to the sequence using the Basic Local Alignment Search Tool (BLAST) at NCBI to determine the accuracy of the sequencing results. After confirming that the sequence was correct, the required reference strain sequence was downloaded, and the phylogenetic tree was constructed using the maximum likelihood method using MEGA 7.0 software, and the phylogenetic analysis was completed by performing 1000 Bootstrap tests.

[0071] The sequencing results are as follows:

[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 verified that GJ242 is Mucor circinelloides at the molecular biological level.

[0075] Example 2 Determination of strain compatibility

[0076] The plate confrontation method was used to test the antagonism between bacteria and fungi. In a sterile operating table, the fungi obtained by screening were punched and inoculated into the center of the PDA culture medium. A ring of bacteria was picked up with an inoculation loop and inoculated at a distance of 2-3 cm from the fungi. The culture medium was placed in a 30°C constant temperature incubator and inverted for 3-5 days. The growth of the strains was observed. If there was no obvious boundary at the intersection of the strains and the growth of the two strains was not affected, it indicated that there was no antagonism between the strains.

[0077] The antagonistic activity of the strains used in the experiment was 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 longifolia NT241, 25% Mucor circinelloides GJ242, 25% Leucophyllum sphaeroides GJ243, 25% Bacillus subtilis GJ231.

[0080] The Trichoderma longibrachiatum NT241 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit date of April 7, 2024 and a deposit number of CGMCC No.41139;

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

[0082] The Irpex lacteus GJ243 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit date of April 7, 2024 and a deposit number of CGMCC No.41281;

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

[0084] Example 4 Preparation of composite bacterial agent

[0085] (1) Preparation of seed solution

[0086] The Bacillus subtilis GJ231 with a deposit number of CGMCC No. 28101 is subjected to liquid fermentation until the strain is in the logarithmic phase. The liquid culture medium is an LB liquid culture medium. The liquid fermentation conditions are as follows: the fermentation temperature is 30-35° C., the rotation speed is 150-180 r / min, the pH value of the fermentation liquid is 7-8, the volume of the fermentation tank is 50 L, the pressure of the fermentation tank is 0.05-0.07 Mpa, and the fermentation time is 20-24 h. After the fermentation is completed, the fermented bacterial liquid is obtained, which is the seed liquid of Bacillus subtilis GJ231.

[0087] The long-branch Trichoderma NT241 with a preservation number of CGMCC No.41139 was subjected to liquid fermentation until the strain was in the logarithmic phase. The liquid culture medium was PDB culture medium. The liquid fermentation conditions were as follows: fermentation temperature of 25-30°C, rotation speed of 180-200r / min, pH of fermentation liquid of 6-7, fermentation tank volume of 50L, fermentation tank pressure of 0.05-0.07Mpa, and fermentation time of 2-3d. After the fermentation was completed, the fermentation liquid obtained was the seed liquid of the long-branch Trichoderma NT241.

[0088] The liquid fermentation of Mucor circinelloides GJ242 with the accession number of CGMCC No.41140 was carried out to the logarithmic phase of the strain, the liquid culture medium was PDB culture medium, the liquid fermentation conditions were: fermentation temperature of 25-30°C, rotation speed of 180-200r / min, pH of fermentation liquid of 6-7, fermentation tank volume of 50L, fermentation tank pressure of 0.05-0.07Mpa, and fermentation time of 2-3d. After the fermentation, the obtained fermentation liquid was the seed liquid of Mucor circinelloides GJ242.

[0089] The white capsule rake tooth fungus GJ243 with a preservation number of CGMCC No.41281 is subjected to liquid fermentation to the logarithmic phase of the strain, the liquid culture medium is PDB culture medium, and the liquid fermentation conditions are: fermentation temperature of 25-30°C, rotation speed of 180-200r / min, fermentation liquid pH of 6-7, fermentation tank volume of 50L, fermentation tank pressure of 0.05-0.07Mpa, and fermentation time of 3-3.5d. After the fermentation is completed, the fermented bacterial liquid obtained is the seed liquid of the white capsule rake tooth fungus GJ243.

[0090] (2) Preparation of bacterial agents

[0091] The Bacillus subtilis GJ231 seed liquid prepared in (1) is inoculated into the 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: 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 a polyether defoamer. After the fermentation is completed, the fermented bacterial liquid is obtained, which is the bacterial agent of Bacillus subtilis GJ231. The number of effective live bacteria in the bacterial liquid is ≥2×10 8 Pieces / ml.

[0092] The seed liquid of Trichoderma longifolia NT241 with the accession number of CGMCC No.41139 prepared in (1) is inoculated into the fermentation tank for liquid fermentation at an inoculation rate of 3-5%, the liquid culture medium is PDB culture medium, and the liquid fermentation conditions are: fermentation temperature is 25-30°C, rotation speed is 200-220r / min, fermentation liquid pH is 6-7, fermentation tank volume is 500L, fermentation tank pressure is 0.05-0.07Mpa, fermentation time is 5-6d, and polyether defoamer is used as defoamer. After the fermentation is completed, the fermented bacterial liquid is obtained, which is the bacterial agent of Trichoderma longifolia NT241, and the number of effective live bacteria in the bacterial liquid is ≥1×10 7 Pieces / ml.

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

[0094] The seed liquid of the white capsule rake tooth fungus GJ243 with the preservation number of CGMCC No.41281 prepared in (1) is inoculated into the fermentation tank for liquid fermentation at an inoculation rate of 3-5%, the liquid culture medium is PDB culture medium, and the liquid fermentation conditions are: fermentation temperature is 25-30°C, rotation speed is 200-220r / min, fermentation liquid pH is 6-7, fermentation tank volume is 500L, fermentation tank pressure is 0.05-0.07Mpa, fermentation time is 6-7d, and polyether defoamer is used as defoamer. After the fermentation is completed, the fermented bacterial liquid is obtained, which is the bacterial agent of the white capsule rake tooth fungus GJ243, and the number of effective live bacteria in the bacterial liquid is ≥1×10 7 Pieces / ml.

[0095] (3) Mix equal volumes of Trichoderma longifolia NT241, Mucor circinelloides GJ242, Psoralea corylifolia 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 bacteria 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 take 0.5mL of the above 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 take 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 apply 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 plate with a dilution of 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; V is the average number of colonies, in units; 1 —basal fluid volume, in milliliters (mI); V 2 —The amount of bacterial suspension added, in milliliters (mL).

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

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

[0102] (1) Drawing of standard curve

[0103] Take 5 large test tubes and use a pipette to accurately pipette 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 Glucose amount in 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 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 micrograms of glucose as the horizontal axis and OD value as the vertical axis.

[0107] (2) Preparation of original enzyme solution

[0108] 10.0 mL of the composite bacterial agent prepared in Example 4 was measured and added to a triangular flask containing glass beads, and then a certain volume of distilled water was added for dilution, allowed to stand for 20 min, shaken at 200 r / min for 30 min, and then filtered through four layers of gauze, and the filtrate was centrifuged at 3 000 r / min for 10 min. The supernatant after centrifugation was diluted to an appropriate concentration according to the enzyme activity, which was the original enzyme solution for testing.

[0109] (3) Measurement steps

[0110] Take 3 large test tubes, 1 as a blank control, and the other 2 as parallel sample tubes. Add 1.0mL of original enzyme solution to the sample tube, then add 4.0mL of CMC buffer preheated to 60℃ to each of the 3 test tubes, react in a 60℃ water bath for 20min, take out, immediately add 3.0mL of DNS colorimetric solution to each tube, shake well, and then add 1.0mL of original enzyme solution to the control tube. Put the 3 test tubes in a boiling water bath, take out immediately after color development for 5min, cool with running water, and measure the OD value at 490nm with a spectrophotometer.

[0111] (4) Calculation of cellulase activity

[0112] The measured OD value was converted into micrograms of glucose according to the standard curve.

[0113]

[0114] Calculation of enzyme activity: 1 mL of original enzyme solution produces 1 ug of glucose in 1 min, 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 >0.95, which proves that the linear relationship between its absorbance and glucose concentration is reliable and can be used as a standard curve. Finally, the cellulase activity of the composite microbial agent was measured to reach 38U / mL, reaching the standard of agricultural microbial agents GB 20287-2006.

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

[0118] The harvested rape straw was naturally air-dried and mechanically treated to 1-2 cm, and 30 g was weighed and placed in a nylon mesh bag and sterilized at 121°C for 20 min for use. After sterilization and cooling, sterile water was sprayed to keep its moisture content at 60%-70%. Five groups were designed in the experiment, and the single-strain inoculants of NT241, GJ242, GJ243, and GJ231 prepared in Implementation Case 4 and the composite inoculants were added to the rape straw. The inoculant addition amount was 5%, and the mixture was evenly mixed and cultured at 30°C for 7 days. After the culture was completed, the degradation ability of each strain was evaluated by measuring the changes in the cellulose content of the rape straw before and after degradation.

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

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

[0121] The experiment set up two groups, one experimental group and one control group, with three parallel groups in each group. Each group took about 200 kg of rapeseed straw from which rapeseed had been collected and placed on the same piece of land. The straw inoculated with the composite microbial agent was the experimental group, and water was used as the blank control group. The stacking of straw requires that the roots of the straw be placed in the middle, the top part faces outward, and the straw is stacked in a circular shape. After each stacking height of 30 to 35 cm, the microbial agent is evenly sprayed and the roots are broken and compacted with a hoe. The stacking height is about 1.3 to 1.5 meters before filming, and the width is about 1.5 to 2.0 meters. During the stacking process, the moisture content of the straw is guaranteed to be between 60 and 70%. After the stacking is completed, a black 0.01 mm recyclable polyethylene blown agricultural mulch is used to cover the straw. When covering the film, all the straw is covered without leaving any gaps, which can ensure the temperature stability during the composting process and increase the survival rate of the functional strains in the composite microbial agent. The height of the straw stacking after filming is about 1.2 to 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 sag. When the film was opened, the temperature inside the experimental group was high, and hot air was emitted. The straw was black in color, soft in texture, without obvious odor, and had obvious decomposition ( Figure 8a and b). The blank group did not have obvious sagging as a whole. The film was opened, the temperature was low, there was no heat emission, there was obvious mold growth, the moldy smell was serious, 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 Fig. 9 As shown in the figure, the degradation rates of hemicellulose were 55.29% and 16.39%, the degradation rates of cellulose were 31.08% and 20.09%, and the degradation rates of lignin were 21.09% and 10.09%, respectively. The degradation rates of the experimental groups were 38.89%, 11.00%, and 11.01% higher than those of the blank group. The addition of composite microbial agents had the best effect on the degradation of hemicellulose in rape 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 Fig.10 As shown: 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±12.80g / 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 straw composite bacterial agent could promote the humification degree of straw.

[0125] Example 9 Composite bacterial agent for germination of corn and rapeseed seeds

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

[0127] Results: The germination rates of rapeseed and corn seeds were as follows: Fig.11 and Fig.12As shown in the figure, the seeds of the two crops began to germinate after 2 days and ended germination in about a week, and the germination showed an upward trend. Among them, when the dilution multiple of the composite microbial agent was 30 times, the germination rate of rapeseed seeds and corn seeds was the best. From the beginning of cultivation, they have been in a leading trend. On the 7th day of cultivation, the germination rate of rapeseed seeds can reach up to 90.83%, and the germination rate of corn seeds can reach up to 77.27%, which are 2.5% and 1.5% higher than the CK group, respectively. The use of straw composite microbial agent diluted by 30 times can promote the germination of rapeseed seeds and corn seeds.

[0128] Although the present invention has been described in detail above by means of general description, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

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 is deposited in the General Microbiology Center of the China Microbiological 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 long-branched Trichoderma NT241, the circinelloides Mucor GJ242, the white capsule spores of the ...

3. A method for preparing the composite bacterial agent as claimed in 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 fermented bacterial liquid after the fermentation is completed, which is the seed liquid of Bacillus subtilis GJ231; Fermenting the liquid of Trichoderma longifolia NT241 until the strain reaches the logarithmic phase, and obtaining the fermented bacterial liquid after the fermentation is completed, which is the seed liquid of Trichoderma longifolia NT241; Fermenting the liquid of Mucor circinelloides GJ242 until the logarithmic phase of the strain, and obtaining the fermented bacterial liquid after the fermentation is completed is the seed liquid of Mucor circinelloides GJ242; Fermenting the liquid of the white capsule rake tooth fungus GJ243 until the strain reaches the logarithmic phase, and the fermented bacterial liquid obtained after the fermentation is completed is the seed liquid of the white capsule rake tooth fungus GJ243; Step 2: Preparation of bacterial solution: Inoculate the Bacillus subtilis GJ231 seed liquid prepared in step 1 into the fermentation tank liquid for fermentation at an inoculation rate of 3-5%. After the fermentation is completed, the fermentation liquid is obtained, which is the bacterial agent of Bacillus subtilis GJ231; The long-branch Trichoderma NT241 seed solution prepared in step 1 is inoculated into the fermentation tank liquid for fermentation at an inoculation rate of 3-5%. After the fermentation is completed, the fermented bacterial solution is obtained, which is the long-branch Trichoderma NT241 bacterial agent; The seed liquid of Mucor circinelloides GJ242 prepared in step 1 is inoculated into the fermentation tank at an inoculation rate of 3-5% for liquid fermentation. After the fermentation is completed, the fermented bacterial liquid is obtained, which is the bacterial agent of Mucor circinelloides GJ242; The seed liquid of the white capsule rake tooth fungus GJ243 prepared in step 1 is inoculated into the fermentation tank liquid for fermentation at an inoculation rate of 3-5%. After the fermentation is completed, the fermented bacterial liquid is obtained, which is the bacterial agent of the white capsule 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, characterized in that: The preparation of the seed solution in step 1 is specifically as follows: The Bacillus subtilis GJ231 is subjected to liquid fermentation until the strain reaches the logarithmic phase, wherein the medium for the liquid fermentation is LB liquid medium, and the liquid fermentation conditions are 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, and fermentation liquid is obtained after the fermentation is completed, which is the seed liquid of Bacillus subtilis GJ231; The long-branch Trichoderma NT241 is subjected to liquid fermentation until the strain reaches the logarithmic phase, wherein the medium for the liquid fermentation is PDB medium, and the liquid fermentation conditions are as follows: the fermentation temperature is 25-30° C., the rotation speed is 180-200 r / min, the pH value of the fermentation liquid is 6-7, the volume of the fermentation tank is 50 L, the pressure of the fermentation tank is 0.05-0.07 MPa, and the fermentation time is 2-3 days. After the fermentation is completed, the fermentation liquid is obtained, which is 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 medium for the liquid fermentation was PDB medium, and the liquid fermentation conditions were as follows: the fermentation temperature was 25-30°C, the rotation speed was 180-200r / min, the pH value of the fermentation liquid was 6-7, the volume of the fermentation tank was 50L, the pressure of the fermentation tank was 0.05-0.07Mpa, and the fermentation time was 2-3d. After the fermentation was completed, the fermentation liquid obtained 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: fermentation temperature is 25-30°C, rotation speed is 180-200r / min, fermentation liquid pH is 6-7, fermentation tank volume is 50L, fermentation tank pressure is 0.05-0.07Mpa, fermentation time is 3-3.5d, and 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, characterized in that: The preparation of the bacterial solution in step 2 is specifically as follows: The Bacillus subtilis GJ231 seed liquid prepared in step 1 is inoculated into the fermentation tank for liquid fermentation at an inoculation rate of 3-5%. The liquid fermentation medium is LB liquid medium. The liquid fermentation conditions are as follows: fermentation temperature is 30-35°C, rotation speed is 150-180r / min, fermentation liquid pH is 7-8, fermentation tank volume is 500L, fermentation tank pressure is 0.05-0.07Mpa, fermentation time is 72h, defoamer is a polyether defoamer, and after the fermentation is completed, the fermented bacterial liquid is obtained, which is the bacterial agent of Bacillus subtilis GJ231; the number of effective live bacteria in the bacterial liquid is ≥2×10 8 Pieces / ml; The long-branch Trichoderma NT241 seed liquid prepared in step 1 is inoculated into the fermentation tank for liquid fermentation at an inoculation rate of 3-5%. The liquid fermentation medium is PDB medium, and the liquid fermentation conditions are as follows: fermentation temperature is 25-30°C, rotation speed is 200-220r / min, fermentation liquid pH is 6-7, fermentation tank volume is 500L, fermentation tank pressure is 0.05-0.07Mpa, fermentation time is 5-6d, and defoaming agent is a polyether defoaming agent. After the fermentation is completed, the fermented bacterial liquid is obtained, which is the bacterial agent of long-branch Trichoderma NT241; the number of effective live bacteria in the bacterial liquid is ≥1×10 7 Pieces / ml; The seed liquid of Mucor circinelloides GJ242 prepared in step 1 is inoculated into the fermentation tank for liquid fermentation at an inoculation rate of 3-5%, the liquid culture medium is PDB culture medium, and the liquid fermentation conditions are as follows: fermentation temperature is 25-30°C, rotation speed is 200-220r / min, fermentation liquid pH is 6-7, fermentation tank volume is 500L, fermentation tank pressure is 0.05-0.07Mpa, fermentation time is 5-6d, defoamer is a polyether defoamer, after fermentation, the fermented bacterial liquid is obtained, which is the bacterial agent of Mucor circinelloides GJ242; the number of effective live bacteria in the bacterial liquid is ≥1×10 7 Pieces / ml; The seed liquid of the white capsule rake tooth fungus GJ243 prepared in step 1 is inoculated into the fermentation tank for liquid fermentation at an inoculation rate of 3-5%, the liquid culture medium is PDB culture medium, and the liquid fermentation conditions are: fermentation temperature is 25-30°C, rotation speed is 200-220r / min, fermentation liquid pH is 6-7, fermentation tank volume is 500L, fermentation tank pressure is 0.05-0.07Mpa, fermentation time is 6-7d, defoaming agent is a polyether defoaming agent, after the fermentation is completed, the fermented bacterial liquid is obtained, which is the bacterial agent of the white capsule rake tooth fungus GJ243; the number of effective live bacteria in the bacterial liquid is ≥1×10 7 Pieces / ml.

6. The preparation method according to claim 3, characterized in that: The effective viable bacteria count of the composite bacterial agent in step 3 is: 2.3×10 8 Pieces / 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 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 promoting crop growth.

10. The use according to claim 9, characterized in that The crops include but are not limited to: rapeseed and corn.

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