Complex microbial inoculant as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a complex microbial inoculant as well as a preparation method and application thereof. The complex microbial inoculant is prepared from the following raw materials in percentage by weight: 55%-65% of complex microbial freeze-dried powder, 25%-35% of a solid-phase carrier, 5%-8% of methylcellulose, 0.1%-0.2% of cyclodextrin, 4%-6% of lignin cyclic acid sodium and 1%-2% of monopotassium phosphate, totaling 100%, the compound bacterium freeze-dried powder is prepared from compound bacteria and a compound bacterium freeze-drying protective agent in a mass ratio of 1: (4-5); the compound bacteria are composed of four kinds of bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is selected from the group consisting of bacillus amyloliquefaciens (Bacillus amyloliquefaciens). After the complex microbial inoculant is applied to crops, indexes such as the plant height, the root length, the whole plant fresh quality and the whole plant dry quality of the crops are all improved, so that the complex microbial inoculant can be used for improving the yield and the quality of the crops.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a composite bacterial agent and a preparation method and application thereof. Background Art

[0002] The long-term use of chemical fertilizers has seriously neglected the sustainable use of soil fertility. The soil fertility cannot be supplemented, resulting in soil compaction and acidification, which seriously affects the full development of crop roots. In my country, fruit trees and various crops, including facility agriculture, are also mainly fertilized with chemical fertilizers. In order to deal with the above problems, microbial fertilizers are currently commonly used. Microbial fertilizers have the following advantages: (1) Improving soil fertility: The beneficial bacteria in microbial agents can decompose organic matter in the soil and release nutrients. At the same time, they can also fix nitrogen, dissolve phosphorus and potassium, improve the effectiveness of nutrients such as nitrogen, phosphorus and potassium in the soil, and increase soil fertility. (2) Improving soil structure: The extracellular polysaccharides and other substances secreted by beneficial bacteria are the adhesives of soil aggregate structure, which can promote the formation of soil aggregate structure, make the soil loose and breathable, and improve the soil's water and fertilizer retention capacity and air permeability. (3) Stimulating plant growth and development: The beneficial bacteria in microbial agents can secrete active substances such as gibberellins, cytokinins, and auxins to stimulate, regulate and promote the growth and development of crops, which is beneficial to increasing crop yields. (4) Enhance the disease resistance and stress resistance of plants: Beneficial bacteria can produce antibiotics, antibacterial proteins and other substances, inhibit the growth and reproduction of pathogens in the soil, and reduce the probability of disease occurrence. At the same time, it can also enhance the stress resistance of plants, such as improving the drought resistance, cold resistance, lodging resistance, disease resistance and salt-alkali resistance of plants. (5) Reduce the amount of chemical fertilizers: Microbial agents can improve the utilization rate of soil nutrients, reduce the amount of chemical fertilizers used, reduce agricultural production costs, and also reduce the pollution of chemical fertilizers to the environment.

[0003] Although existing technical studies have shown that the disease inhibition and growth promotion effect of a single bacterium is not as good as that of a mixed bacterial community, considering the antagonistic and competitive relationship between microorganisms, not all combinations of microorganisms have a higher effect than a single bacterium. Therefore, there is an urgent need to provide a composite bacterial agent that has a synergistic growth-promoting effect on crops. Summary of the invention

[0004] The purpose of the present invention is to provide a composite bacterial agent and a preparation method and application thereof, which are used to improve the yield and quality of crops.

[0005] The technical solution adopted by the present invention is: the present invention provides a composite bacterial agent, which is composed of the following raw materials in weight percentage: 55% to 65% of composite bacterial freeze-dried powder, 25% to 35% of solid phase carrier, 5% to 8% of methyl cellulose, 0.1% to 0.2% of cyclodextrin, 4% to 6% of sodium lignin cyclodextrin, and 1% to 2% of potassium dihydrogen phosphate, which totals 100%; the composite bacterial freeze-dried powder is made of composite bacteria and composite bacterial freeze-dried protective agent in a mass ratio of 1:4 to 5; the composite bacteria is composed of 4 kinds of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), the amyloliquefaciens are DT2-1, KL9, KS7 and MR4, the mass ratio of the fermentation seed liquid of DT2-1, KL9, KS7 and MR4 in the composite bacteria is 1:1-2:1-2:1-2; the composite bacteria freeze-drying protective agent is made of sucrose, protein substances and tryptic soytone liquid culture medium, the mass ratio of the protein substances, sucrose and tryptic soytone liquid culture medium is 1:2-3:17-18; the protein substances are bovine serum albumin or wheat peptone.

[0006] Preferably, the OD of the fermentation seed liquid of DT2-1, KL9, KS7 and MR4 is 600 is 1.0.

[0007] Preferably, the solid phase carrier is any one of kaolinite, diatomaceous earth and attapulgite.

[0008] Preferably, the viable bacterial count of the composite bacterial agent is greater than 2.9×10 10 CFU / g.

[0009] Preferably, the composite bacterial agent is composed of the following raw materials in weight percentage: 60% composite bacterial freeze-dried powder, 29.9% solid phase carrier, 5% methyl cellulose, 0.1% cyclodextrin, 4% sodium lignin cyclodextrin, and 1% potassium dihydrogen phosphate.

[0010] The present invention also provides a method for preparing the composite bacterial agent, which is characterized by comprising the following steps:

[0011] The fermentation seed liquids of DT2-1, KL9, KS7 and MR4 are prepared respectively; the fermentation seed liquids of DT2-1, KL9, KS7 and MR4 are mixed in a mass ratio of 1:1-2:1-2:1-2, centrifuged, washed and precipitated to obtain composite bacteria; the composite bacteria and the composite bacteria freeze-drying protective agent are evenly mixed in a mass ratio of 1:4-5, freeze-dried to obtain composite bacteria freeze-dried powder; the composite bacteria freeze-dried powder, solid phase carrier, methyl cellulose, cyclodextrin, sodium lignin cyclophosphate and potassium dihydrogen phosphate, which are raw materials of the mass percentage, are mixed in weight percentage to prepare a composite bacterial agent.

[0012] Preferably, the freeze-drying temperature is no higher than -50°C.

[0013] Preferably, the composite bacterial agent is used to improve the yield and quality of crops.

[0014] Preferably, the crop is any one of cotton, wheat and corn.

[0015] Preferably, the mass is any one or more of plant height, root length, stem base diameter, fresh weight and dry weight.

[0016] Preferably, the method for using the composite bacterial agent is as follows: the composite bacterial agent is diluted with water to obtain a dilution of 0.5 g / L to 2 g / L, and the crops are irrigated, and the amount of the dilution for irrigation per crop is 50 mL to 60 mL.

[0017] Preferably, the composite bacterial agent is used in the cotton planting process in Xinjiang.

[0018] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a composite bacterial agent, which is composed of the following raw materials in percentage by weight:

[0019] The composite bacteria freeze-dried powder is 55% to 65%, the solid phase carrier is 25% to 35%, the methyl cellulose is 5% to 8%, the cyclodextrin is 0.1% to 0.2%, the sodium lignin cyclophosphate is 4% to 6%, and the potassium dihydrogen phosphate is 1% to 2%, which is 100% in total; the composite bacteria freeze-dried powder is made of the composite bacteria and the composite bacteria freeze-dried protective agent in a mass ratio of 1:4 to 5; the composite bacteria is composed of 4 kinds of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), the amyloliquefaciens are DT2-1, KL9, KS7 and MR4, the mass ratio of the fermented seed liquid of DT2-1, KL9, KS7 and MR4 in the composite bacteria is 1:1-2:1-2:1-2; the composite bacteria freeze-drying protective agent is made of sucrose, protein substances and tryptic soytone liquid culture medium, the mass ratio of protein substances, sucrose and tryptic soytone liquid culture medium is 1:2-3:17-18; the protein substances are bovine serum albumin or wheat peptone. After the composite bacterial agent of the present invention is applied to crops, the plant height, root length, whole plant fresh weight, whole plant dry weight and other indicators of the crops are all improved.

[0020] The present invention adds a freeze-drying protective agent and a solid phase carrier in the process of preparing the composite microbial agent, which not only facilitates the storage of the composite microbial agent, but also realizes the slow release of fertilizer effect. In view of the environmentally friendly characteristics of microbial fertilizers, the composite microbial agent of the present invention can also be used as a fermentation biofertilizer and pesticide for development, which can not only promote the growth of all crops, inhibit soil-borne diseases, but also has a slow release effect of fertilizer effect; it can also centrally repair soil and water pollution in a long-term and eco-friendly manner. Therefore, the composite microbial agent of the present invention has a good application prospect.

[0021] In addition, since KL9, KS7 and MR4 cotton wilt pathogens have strong antibacterial functions and DT2-1 is cold-resistant, the composite bacterial agent of the present invention can also improve the stress resistance of crops, inhibit diseases, especially reduce the occurrence of cotton wilt during cotton growth, so as to alleviate the problem of soil degradation caused by excessive use of chemical fertilizers and the problems of crop resistance to late spring cold, continuous high temperature in summer, poor disease resistance, yield and quality, so as to achieve sustainable agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 These are the composite bacteria collected in Example 1, and both A and B are composite bacteria.

[0023] Figure 2 These are two lyophilized protective agents prepared in Example 1 and Example 4, A is Reagent 18 prepared in Example 1, and B is Reagent PS prepared in Example 4.

[0024] Figure 3 The lyophilized composite bacteria powders prepared in Example 1 and Example 4 are: A and B are the lyophilized composite bacteria powders prepared in Example 1; C and D are the lyophilized composite bacteria powders prepared in Example 4.

[0025] Figure 4 The results of coating the composite bacteria freeze-dried powder prepared in Example 1 and Example 4 using the dilution coating method before and after freeze-drying are shown, from left to right, the composite bacteria freeze-dried powder of Example 1 before freeze-drying, the composite bacteria freeze-dried powder of Example 1 after freeze-drying, the composite bacteria freeze-dried powder of Example 4 before freeze-drying, and the composite bacteria freeze-dried powder of Example 4 after freeze-drying.

[0026] Figure 5 The composite bacterial agents prepared in the present invention, A to F are respectively: the composite bacterial agents prepared in Examples 1 to 6.

[0027] Figure 6 The cotton seedlings were treated with the freeze-dried powder of the composite bacteria prepared in Example 1 and Example 4. From left to right: the first and second strains were treated with the freeze-dried powder of the composite bacteria in Example 1, the third and fourth strains were treated with the freeze-dried powder of the composite bacteria in Example 4, and the fifth and sixth strains were treated with the control group in parallel.

[0028] Figure 7 The effect of freeze-dried powder of composite bacteria prepared in Example 1 and Example 4 on the height of cotton seedlings.

[0029] Figure 8 The effect of freeze-dried powder of composite bacteria prepared in Example 1 and Example 4 on the root length of cotton seedlings.

[0030] Fig. 9 The effect of freeze-dried powder of composite bacteria prepared in Example 1 and Example 4 on the stem base diameter of cotton seedlings.

[0031] Fig.10 The effect of freeze-dried powder of composite bacteria prepared in Example 1 and Example 4 on the fresh weight of cotton seedlings.

[0032] Fig.11 The effect of freeze-dried powder of composite bacteria prepared in Example 1 and Example 4 on the dry weight of cotton seedlings.

[0033] Fig.12 The cotton seedlings were treated with the composite microbial agents prepared in Examples 4 to 6, from left to right: the first and second strains were cotton seedlings treated in parallel with the composite microbial agents of Example 4, the third and fourth strains were cotton seedlings treated in parallel with the composite microbial agents of Example 5, the fifth and sixth strains were cotton seedlings treated in parallel with the composite microbial agents of Example 6, and the seventh and eighth strains were cotton seedlings treated in parallel with the control group.

[0034] Fig.13 The effect of the composite bacterial agent prepared in Examples 4 to 6 on the height of cotton seedlings.

[0035] Fig.14 The effect of the composite bacterial agent prepared in Examples 4 to 6 on the root length of cotton seedlings.

[0036] Fig.15 The effect of the composite bacterial agent treatment prepared in Examples 4 to 6 on the stem base diameter of cotton seedlings.

[0037] Fig.16 The effect of the composite bacterial agent prepared in Examples 4 to 6 on the fresh weight of cotton seedlings.

[0038] Fig.17 The effect of the composite bacterial agent prepared in Examples 4 to 6 on the dry weight of cotton seedlings. DETAILED DESCRIPTION

[0039] The present invention is further described below by specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0040] The inventive concept of the present invention is as follows:

[0041] According to the salinity of the land in southern Xinjiang, the present invention screens a group of salt-alkali, low-temperature and drought-resistant strains in the soil with high salinity to form a strain resource library. The strains in these strain libraries have different characteristics such as salt-alkali resistance, low-temperature resistance, drought resistance, phosphorus and potassium solubility, iron carrier production, growth promotion, and disease resistance, including wild medicinal plant endophytes DT2-1, KL9, KS7 and rhizosphere bacteria MR4.

[0042] In the reference "Dai Xianxing. Screening, identification and preliminary study of the antibacterial mechanism of antagonistic bacteria against cotton wilt and verticillium wilt [D]. Tarim University, 2024.", researchers mixed the bacterial suspension of KL9, KS7 and MR4 with attapulgite to prepare a solid bacterial agent for cotton growth promotion research. The solid bacterial agent mentioned in the article has good stability in short-term storage, but if it is stored for a long time, its stability and viable bacteria count will drop significantly. And considering the antagonistic and competitive relationship between microorganisms, not any combination of several microorganisms has a higher effect than a single bacterium. For example, the reference Xia, L., Miao, Y., Cao, A. et al. Biosynthetic gene cluster profiling predicts the positive association between antagonism and phylogeny in Bacillus. Nat Commun 13, 1023 (2022). mentioned that Bacillus SQR9 has an inhibitory effect on other Bacillus of the same genus. In addition, the prior art found that the solid bacterial agent prepared using KL9, KS7 and MR4 can inhibit the growth of cotton blight and Verticillium wilt pathogens, and can also promote the growth of cotton. Considering that the test site of the present invention is located in the high-latitude Xinjiang region, during the cotton planting period, the lowest temperature can reach 4°C, but KL9, KS7 and MR4 do not have the characteristics of low temperature resistance, so in a low temperature environment, KL9, KS7 and MR4 are difficult to play a growth-promoting role.

[0043] The above reasons bring many inconveniences to ordinary agricultural production. How to further improve the growth of cotton under low temperature conditions is a technical problem to be solved.

[0044] In order to improve the utilization rate of KL9, KS7 and MR4 solid bacterial agents, and to achieve further growth of cotton by KL9, KS7 and MR4 under low temperature conditions, the present invention proposes a novel composite bacterial agent, namely: on the basis of KL9, KS7 and MR4 solid bacterial agents, the following are added: (1) a freeze-dried protective agent, which not only protects the composite bacteria from cell damage caused by low temperature during the freezing process of the composite bacteria, thereby increasing the number of live bacteria in the composite bacterial agent, but also the freeze-dried powder of the composite bacteria formed by freezing the bacterial bodies used in the present invention is more stable than the unfreeze-dried bacterial bodies; (2) a solid phase carrier such as kaolinite, attapulgite or diatomaceous earth and a chemical growth-promoting substance such as methyl cellulose, cyclodextrin, sodium lignin cyclophosphate and potassium dihydrogen phosphate, and the amyloliquefaciens is mixed with the solid phase carrier and the chemical growth-promoting substance, wherein the solid phase carrier is used to load the strain. By taking advantage of the differences in the physiological responses of crops, pathogens and Bacillus amyloliquefaciens to different nutrients, adding appropriate amounts of nutrients as synergistic factors can improve plant resistance, strengthen the biological control effect, promote plant growth, and have the dual functions of disease prevention and yield increase; (3) DT2-1, DT2-1 is salt-tolerant and cold-tolerant, and can restore the growth-promoting effects of KL9, KS7 and MR4 on cotton under low temperature conditions. In general, the synergistic effect of these four bacteria, combined with solid phase carriers and other components, on cotton growth is obvious under low temperature conditions.

[0045] In order to enable those skilled in the art to better understand the technical solution of the present invention and implement it, the present invention is further described below in conjunction with specific embodiments. In the description of the present invention, unless otherwise specified, the reagents used are all commercially available, and the methods used are all conventional techniques in the art.

[0046] The invention provides a method for preparing a composite bacterial agent, comprising three processes of preparing composite bacteria, preparing composite bacterial freeze-dried powder and preparing the composite bacterial agent.

[0047] The four strains DT2-1, KL9, KS7 and MR4 used in the present invention belong to the genus Bacillus amyloliquefaciens. These four strains have been disclosed in relevant literature. DT2-1 is derived from the reference "Zulihumar Metikadir. Screening, identification and antibacterial and growth-promoting properties of antagonistic bacteria against cotton wilt [D]. Tarim University, 2024."; KL9, KS7 and MR4 are derived from the reference "Dai Xianxing. Screening, identification and preliminary study of the antibacterial mechanism of antagonistic bacteria against cotton wilt and verticillium wilt [D]. Tarim University, 2024.".

[0048] Example 1

[0049] A method for preparing a composite bacterial agent, the method is as follows:

[0050] S1. Preparation of composite bacteria, the process is as follows:

[0051] (1) Activate DT2-1, KL9, KS7 and MR4 respectively, as follows;

[0052] Take out the stored DT2-1, KL9, KS7 and MR4 bacterial suspension from the -80℃ refrigerator, let it melt naturally on the ice box, use an inoculation loop to dip the bacterial suspension on the solid tryptic soy agar medium TSA medium on a sterile operating table, seal it with sealing film, place it in a 30℃ constant temperature incubator and culture it upside down. Single colonies will grow overnight.

[0053] (2) DT2-1, KL9, KS7 and MR4 were cultured separately to prepare fermentation seed liquid, the steps are as follows:

[0054] Single colonies were picked and inoculated into 150 mL TSB liquid medium in a triangular flask, and cultured at 30°C, 180 rpm / min in a shaking incubator for 24 hours. The OD 600 The fermentation seed solution is 1.0. DT2-1, KL9, KS7 and MR4 were all prepared according to the method described in this paragraph to obtain DT2-1 fermentation seed solution, KL9 fermentation seed solution, KS7 fermentation seed solution and MR4 fermentation seed solution, respectively.

[0055] (3) Collecting composite bacteria, the steps are as follows:

[0056] The fermentation seed liquids of DT2-1, KL9, KS7 and MR4 were mixed in a mass ratio of 1:1:1:1, centrifuged at 8000 rpm for 5 min at 4°C, the supernatant was discarded, and the precipitate was washed with sterile water, centrifuged and the supernatant was discarded, and the washing was repeated 3 times and the precipitate was collected to obtain the composite bacteria. The composite bacteria were as follows Figure 1 shown.

[0057] S2, mixing the composite bacteria and the composite bacteria freeze-drying protective agent evenly, freeze-drying, and obtaining the composite bacteria freeze-dried powder, the process is as follows:

[0058] The present invention provides a freeze-drying protective agent, Reagent 18 freeze-drying protective agent, which is used to help the composite bacteria resist the risks of low temperature, oxidation and dehydration, and ensure the number of live bacteria of the bacteria. The formula is as follows:

[0059] The mass ratio of Reagent 18 lyophilization protective agent: bovine serum albumin: sucrose and tryptic soy bean liquid medium TSB is 1:2:17. Figure 2 shown.

[0060] Take the composite bacteria prepared in S1, add it to Reagent 18 lyophilized protective agent, use a vortex mixer to mix at room temperature for 5 minutes, and the mass ratio of the composite bacteria to the lyophilized protective agent is 1:4. Pipette 1 mL of the mixture of the composite bacteria and lyophilized protective agent, then graded dilution and plate coating, and culture at 30°C for 24 hours to count the viable bacteria, which is recorded as the number of viable bacteria before lyophilization. Pre-freeze the mixture made of the composite bacteria and lyophilized protective agent at -80°C for 12 hours. Transfer the pre-frozen mixture to a LABCONCO freeze dryer and freeze-dry at -50°C until there is no liquid crystal, to obtain the composite bacteria lyophilized powder, such as Figure 3 shown.

[0061] In order to facilitate the storage of the composite bacteria freeze-dried powder, the composite bacteria freeze-dried powder is subjected to S3 treatment.

[0062] S3, the composite bacteria freeze-dried powder, solid phase carrier, methyl cellulose, cyclodextrin, sodium lignin cyclophosphate and potassium dihydrogen phosphate are mixed to prepare a composite bacterial agent, and the process is as follows:

[0063] The composite bacteria freeze-dried powder prepared in S2 and the solid phase carrier kaolinite, methyl cellulose, cyclodextrin, sodium lignin cyclophosphate, and potassium dihydrogen phosphate were uniformly mixed to obtain a composite bacterial agent named Reagent 18WP-KAO. The weight percentage of each component of the composite bacterial agent is: 60% of composite bacteria freeze-dried powder, 29.9% of solid phase carrier, 5% of methyl cellulose, 0.1% of cyclodextrin, 4% of sodium lignin cyclophosphate, and 1% of potassium dihydrogen phosphate.

[0064] Example 2

[0065] A method for preparing a composite bacterial agent, the method is as follows:

[0066] The solid phase carrier of the composite bacterial agent was diatomaceous earth, and the other conditions were exactly the same as those in Example 1 to prepare a composite bacterial agent named Reagent 18WP-PE.

[0067] Example 3

[0068] A method for preparing a composite bacterial agent, the method is as follows:

[0069] The solid phase carrier of the composite bacterial agent was attapulgite, and the other conditions were exactly the same as those in Example 1 to prepare a composite bacterial agent named Reagent 18WP-ATP.

[0070] Example 4

[0071] A method for preparing a composite bacterial agent, the method is as follows:

[0072] The present invention also provides a freeze-drying protective agent, namely ReagentPS freeze-drying protective agent, which is used to help the composite bacteria resist the risks of low temperature, oxidation and dehydration and ensure the number of live bacteria of the bacteria. The formula is as follows:

[0073] Reagent PS freeze-drying protective agent, wheat peptone: sucrose and TSB mass ratio is 1:2:17, Reagent PS freeze-drying protective agent is as follows Figure 2 shown.

[0074] The composite bacteria freeze-dried powder was prepared from the composite bacteria and ReagentPS freeze-drying protective agent. The other experimental conditions were exactly the same as those in Example 1. A composite bacterial agent was prepared and named ReagentPS WP-KAO.

[0075] Example 5

[0076] A method for preparing a composite bacterial agent, the method is as follows:

[0077] The composite bacteria freeze-dried powder is prepared from the composite bacteria and ReagentPS freeze-dried protective agent. The formula of ReagentPS freeze-dried protective agent is the same as that of Example 4, and the other experimental conditions are exactly the same as those of Example 2. The composite bacteria agent is prepared and named ReagentPS WP-PE.

[0078] Example 6

[0079] A method for preparing a composite bacterial agent, the method is as follows:

[0080] The composite bacteria freeze-dried powder is prepared from the composite bacteria and ReagentPS freeze-dried protective agent. The formula of the ReagentPS freeze-dried protective agent is the same as that of Example 4, and the other experimental conditions are exactly the same as those of Example 3. A composite bacterial agent is prepared and named ReagentPS WP-ATP.

[0081] Weigh 1 g of the composite bacterial freeze-dried powder obtained in Example 1 and Example 4, rehydrate it in sterile water, dilute the rehydrated bacteria in a gradient manner, spread them on a plate, and culture them at 30°C for 24 hours to count the live bacteria, which is recorded as the live bacteria count after freeze-drying. 7 The results are as follows Figure 4 shown.

[0082] The calculation formula of the survival rate of compound bacteria freeze-dried powder is:

[0083]

[0084] In the formula, A1: the number of viable bacteria after freeze-drying; A0: the number of viable bacteria before freeze-drying, and the unit of viable bacteria is CFU / g. The number of viable bacteria before and after freeze-drying and the freeze-drying survival rate are shown in Table 1, which shows that ReagentPS has a better freeze-drying protection effect.

[0085] Table 1 Freeze-dried survival rate

[0086]

[0087]

[0088] According to the difference of freeze-drying protective agent and solid phase carrier, the composite bacterial agent can be divided into 6 types, namely, Example 1-

[0089] The composite bacterial agent prepared in Example 6 is as follows Figure 5 As shown; plus two kinds of composite bacteria freeze-dried powder, namely the composite bacteria freeze-dried powder prepared in Example 1 and Example 4, a total of 8 kinds, the number of viable bacteria of the 8 kinds of bacterial agents were counted respectively, and the results are shown in Table 2.

[0090] Table 2 Number of viable bacteria of composite bacterial agent

[0091]

[0092] Example 7

[0093] Application of a composite bacterial agent

[0094] After the cotton seeds were raised in the seedling tray for 5 days, the cotton seedlings with uniform growth were selected and transplanted into a matrix prepared according to the mass ratio of peat soil to vermiculite of 3:1. The composite bacteria freeze-dried powder prepared in Example 1 and Example 4 was used for root irrigation treatment. The composite bacteria freeze-dried powder was prepared according to 1g freeze-dried powder and 1L water, 50mL per plant, and the seedling growth index was counted after 30 days of treatment. Compared with the control group with irrigation, the composite bacteria freeze-dried powder treatment showed a significant growth-promoting effect on the indicators such as plant height, root length, fresh weight of the whole plant, and dry weight of the whole plant of cotton seedlings. Figure 6 to Figure 11 As shown, the composite bacterial freeze-dried powder prepared with ReagentPS protective agent had a more obvious growth-promoting effect, so the composite bacterial agent prepared with ReagentPS protective agent was selected in subsequent experiments.

[0095] After the cotton seeds were raised in the seedling tray for 5 days, the cotton seedlings with uniform growth were selected and transplanted into a matrix prepared according to the mass ratio of peat soil: vermiculite = 3:1. The above results were selected. ReagentPS WP-KAO, ReagentPS WP-PE and ReagentPS WP-ATP composite bacterial agents prepared in Examples 4 to 6 were selected for root irrigation treatment. The composite bacterial agent powder was prepared by adding 1g of freeze-dried powder to 1L of water, and 50mL was irrigated for each plant. The seedling growth indicators were counted after 45 days of treatment. Compared with the control group, the composite bacterial agent treatment showed a significant growth-promoting effect on the indicators such as plant height, root length, fresh weight of the whole plant, and dry weight of the whole plant of cotton seedlings. Figure 12 to Figure 17 shown.

[0096] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0097] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, 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.

Claims

1. A composite bacterial agent, characterized in that: The composite bacterial agent is composed of the following raw materials in percentage by weight: Composite bacteria freeze-dried powder 55% to 65%, solid phase carrier 25% to 35%, methyl cellulose 5% to 8%, cyclodextrin 0.1% to 0.2%, sodium lignin cyclodextrin 4% to 6%, potassium dihydrogen phosphate 1% to 2%, totaling 100%; The composite bacteria freeze-dried powder is prepared from composite bacteria and composite bacteria freeze-dried protective agent in a mass ratio of 1:4-5; The composite bacteria is composed of four Bacillus amyloliquefaciens, wherein the Bacillus amyloliquefaciens are DT2-1, KL9, KS7 and MR4, and the mass ratio of the fermentation seed liquid of DT2-1, KL9, KS7 and MR4 in the composite bacteria is 1:1-2:1-2:1-2; The composite bacteria freeze-drying protective agent is made of sucrose, protein substances and tryptic soytone liquid culture medium, and the mass ratio of the protein substances, sucrose and tryptic soytone liquid culture medium is 1:2-3:17-18; The protein substance is bovine serum albumin or wheat peptone.

2. A composite bacterial agent according to claim 1, characterized in that: The solid phase carrier is any one of kaolinite, diatomaceous earth and attapulgite.

3. A composite bacterial agent according to claim 1, characterized in that: The viable bacterial count of the composite bacterial agent is greater than 2.9×10 10 CFU / g.

4. A composite bacterial agent according to claim 1, characterized in that: The composite bacterial agent is composed of the following raw materials in percentage by weight: The composite bacteria freeze-dried powder is 60%, the solid phase carrier is 29.9%, the methyl cellulose is 5%, the cyclodextrin is 0.1%, the sodium lignin cyclodextrin is 4%, and the potassium dihydrogen phosphate is 1%.

5. The method for preparing a composite bacterial agent according to claim 1, characterized in that: The following steps are involved: The fermentation seed liquids of DT2-1, KL9, KS7 and MR4 were prepared respectively; The fermented seed liquids of DT2-1, KL9, KS7 and MR4 were mixed in a mass ratio of 1:1-2:1-2:1-2, centrifuged, washed and the precipitate was collected to obtain a composite bacteria; The composite bacteria and the composite bacteria freeze-dried protective agent are mixed evenly at a mass ratio of 1:4-5, and freeze-dried to obtain the composite bacteria freeze-dried powder; The composite bacterial freeze-dried powder, solid phase carrier, methyl cellulose, cyclodextrin, sodium lignin cyclophosphate and potassium dihydrogen phosphate, which are raw materials of the same mass percentage, are mixed according to the weight percentage to prepare the composite bacterial agent.

6. The method for preparing a composite bacterial agent according to claim 5, characterized in that: The freeze-drying temperature is no higher than -50°C.

7. The use of a composite bacterial agent according to claim 1, characterized in that: The composite bacterial agent is used for improving the yield and quality of crops.

8. The use of a composite bacterial agent according to claim 7, characterized in that: The crop is any one of cotton, wheat and corn.

9. The use of a composite bacterial agent according to claim 7, characterized in that: The mass is any one or more of plant height, root length, stem base diameter, fresh weight and dry weight.

10. The use of a composite bacterial agent according to claim 7, characterized in that: The method of using the composite bacterial agent is as follows: The composite bacterial agent is diluted with water to obtain a dilution of 0.5 g / L to 2 g / L, and the crops are irrigated. The amount of the dilution for irrigation of each crop is 50 mL to 60 mL.

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