Complex microbial inoculant for promoting heavy salt and alkali resistance of crops, and preparation method and application of complex microbial inoculant

Abstract

The invention relates to the technical field of agricultural microbial preparations, in particular to a complex microbial inoculant for promoting severe salt and alkali resistance of crops as well as a preparation method and application of the complex microbial inoculant. Effective viable bacteria in the complex microbial inoculant comprise bacillus licheniformis with the cell count content of 26%-41%, bacillus subtilis with the cell count content of 31%-46% and pseudomonas fluorescens with the cell count content of 21%-36%. When the microbial agent is applied to plant root systems of soda saline-alkali soil containing sodium carbonate and sodium bicarbonate, the saline-alkali resistance of plants can be improved, and the plant height, ground diameter, fresh weight, dry weight, root length and root number of the plants are remarkably increased; besides, the complex microbial inoculant can effectively improve the growth of saline-alkali soil crops and solve the difficult problem of soda saline-alkali soil remediation in the field by improving the activity of antioxidant enzymes, increasing the content of chlorophyll, helping plants maintain high photosynthetic activity and relieving the damage of saline-alkali stress to a plant photosynthesis system.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural microbial preparations, and in particular to a composite microbial agent for promoting crop resistance to severe salinity and alkali, and a preparation method and application thereof. Background Art

[0002] Saline-alkali soil is an abiotic stress problem that severely limits crop production and affects the sustainable development of global agriculture. High salt and alkaline components in saline-alkali soil can cause osmotic stress, ion toxicity, and nutrient absorption disorders in crops, which in turn inhibit root development and affect the normal growth and yield of crops. In addition, the problem of soil salinization is further aggravated by unreasonable agricultural management measures such as climate change, improper irrigation, and excessive fertilization. Although traditional saline-alkali land improvement methods, such as drainage and salt washing and the application of chemical improvers, can improve the soil environment in the short term, these methods are often costly and may cause secondary pollution to the ecological environment.

[0003] In recent years, the exploration and application of salt-alkali resistant bacterial agents have provided new solutions for the improvement of saline-alkali land. Studies have shown that specific beneficial microorganisms can significantly alleviate the adverse effects of salt-alkali stress on crops by improving soil structure, increasing nutrient utilization, promoting plant growth and other mechanisms. This type of agricultural microbial preparation is considered to be a sustainable, environmentally friendly and long-term solution. At present, there are invention patents for promoting crop salt-alkali resistance microbial agents. For example, the invention patent with application number CN109486709A provides a strain of Fan's salt mononas JPT10-1 that improves the salt tolerance of corn and its bacterial agent and application. The strain can significantly improve the height, root length, wet weight and dry weight of corn seedlings. However, a single strain is not adaptable enough when facing a complex and changeable saline-alkali environment, and there is still a lot of room for improvement in practical application. At present, the microbial preparation products for saline-alkali adversity on the market are still relatively limited, and it is urgent to develop more efficient bacterial agents and their preparation methods to cope with the increasingly serious salinization problem and promote the sustainable development of agriculture.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a composite bacterial agent for promoting crop resistance to severe salinity and alkali, and a preparation method and application thereof.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a composite bacterial agent for promoting crop resistance to severe salinity and alkali, wherein the effective live bacteria in the composite bacterial agent include Bacillus licheniformis with a cell content of 26-41%, Bacillus subtilis with a cell content of 31-46%, and Pseudomonas fluorescens with a cell content of 21-36%.

[0008] Preferably, in the present invention, the effective live bacteria in the composite bacterial agent include Bacillus licheniformis with a cell content of 29-37%, Bacillus subtilis with a cell content of 33-42%, and Pseudomonas fluorescens with a cell content of 25-33%.

[0009] Preferably, in the present invention, the effective live bacteria in the composite bacterial agent include Bacillus licheniformis with a cell content of 34±1%, Bacillus subtilis with a cell content of 37±1% and Pseudomonas fluorescens with a cell content of 29±1%.

[0010] Preferably, in the present invention, the composite bacterial agent further comprises a bacterial agent protective agent and an antioxidant; the mass ratio of the bacterial agent protective agent to the antioxidant is 1:2-8.

[0011] Preferably, in the present invention, the mass of the effective live bacteria is 6-7 times the sum of the mass of the bacterial agent protective agent and the antioxidant.

[0012] Preferably, in the present invention, the bacterial agent protective agent is selected from one or more of glycerol, polyethylene glycol, peptone, Tween, trehalose, anhydrous sodium acetate, dipotassium hydrogen phosphate, magnesium sulfate, and manganese sulfate.

[0013] Preferably, in the present invention, the mass percentage of glycerol in the bacterial agent protective agent is 12-36%.

[0014] Preferably, in the present invention, the mass percentage of polyethylene glycol in the bacterial agent protective agent is 4-22%.

[0015] Preferably, in the present invention, the mass percentage of peptone in the bacterial agent protective agent is 2-12%.

[0016] Preferably, in the present invention, the mass percentage of Tween in the bacterial agent protective agent is 0.5-7%.

[0017] Preferably, in the present invention, the mass percentage of trehalose in the bacterial agent protective agent is 8-28%.

[0018] Preferably, in the present invention, the mass percentage of anhydrous sodium acetate in the bacterial agent protective agent is 2-18%.

[0019] Preferably, in the present invention, the mass percentage of dipotassium hydrogen phosphate in the bacterial agent protective agent is 0.2-4%.

[0020] Preferably, in the present invention, the mass percentage of magnesium sulfate in the bacterial agent protective agent is 0.05-1.5%.

[0021] Preferably, in the present invention, the mass percentage of manganese sulfate in the bacterial agent protective agent is 0.005-0.6%.

[0022] Preferably, in the present invention, the antioxidant is selected from one or more of shiitake mushroom scraps, white button mushroom scraps, celandine scraps, waste tea powder, salvia miltiorrhiza powder, licorice scraps, clove scraps, aconite powder, gallic acid powder, and superoxide dismutase SOD.

[0023] Preferably, in the present invention, the mass percentage of shiitake mushroom scraps in the antioxidant is 5-32%.

[0024] Preferably, in the present invention, the mass percentage of white mushroom scraps in the antioxidant is 3-30%.

[0025] Preferably, in the present invention, the mass percentage of celandine chips in the antioxidant is 2-18%.

[0026] Preferably, in the present invention, the mass percentage of waste tea leaf powder in the antioxidant is 5-30%.

[0027] Preferably, in the present invention, the mass percentage of Danshen powder in the antioxidant is 1-12%.

[0028] Preferably, in the present invention, the mass percentage of liquorice scraps in the antioxidant is 0.5-6.0%.

[0029] Preferably, in the present invention, the mass percentage of clove chips in the antioxidant is 0.5-6.0%.

[0030] Preferably, in the present invention, the mass percentage of Aconitum carmichaelii powder in the antioxidant is 0.1-2.0%.

[0031] Preferably, in the present invention, the mass percentage of gallic acid powder in the antioxidant is 0.01-1.0%.

[0032] Preferably, in the present invention, the mass percentage of superoxide dismutase SOD in the antioxidant is 0.01-1.0%.

[0033] Preferably, in the present invention, the bacterial agent protective agent includes 29±3% glycerol, 18±2% polyethylene glycol, 10±1% peptone, 5±0.5% Tween, 22±2% trehalose, 12±1% anhydrous sodium acetate, 2±0.2% dipotassium hydrogen phosphate, 1.5±0.2% magnesium sulfate and 0.5±0.1% manganese sulfate.

[0034] Preferably, in the present invention, the antioxidant includes 27±3% shiitake mushroom scraps, 21±2% white mushroom scraps, 11±1% celandine scraps, 26±3% waste tea powder, 7±1% salvia miltiorrhiza powder, 3±v.3% licorice scraps, 3±0.3% clove scraps, 1±0.1% aconite powder, 0.5±0.1% gallic acid powder and 0.5±0.1% superoxide dismutase SOD.

[0035] Preferably, in the present invention, the mass ratio of the bacterial agent protective agent, the antioxidant and the effective live bacteria is 1:2-4:21-30.

[0036] Preferably, in the present invention, the mass ratio of the bacterial agent protective agent, the antioxidant and the effective live bacteria is 1:3:28.

[0037] In a second aspect, the present invention provides a method for preparing the composite bacterial agent for promoting crop resistance to severe salinity and alkali, comprising the following steps: spray-drying a mixed raw material containing a bacterial agent protective agent, an antioxidant and effective live bacteria at a low temperature, with an air inlet temperature of 80-90°C.

[0038] Preferably, in the present invention, silica gel desiccant is added after low-temperature spray drying; the mass ratio of silica gel desiccant to effective live bacteria is 1-3:100.

[0039] In a third aspect, the present invention provides the use of the composite bacterial agent or the composite bacterial agent prepared by the preparation method in nitrogen fixation, phosphorus solubilization and promoting crop resistance to severe salinity and alkali.

[0040] Preferably, in the present invention, the crop comprises corn.

[0041] Preferably, in the present invention, the performance of severe salt and alkali resistance includes at least one of the following:

[0042] (1) Improve the salt resistance of crops under saline-alkali stress;

[0043] (2) Improve crop energy conversion efficiency under saline-alkali stress;

[0044] (3) Improve the antioxidant enzyme activity of crops under saline-alkali stress;

[0045] (4) Improve crop resistance to saline-alkali stress;

[0046] (5) Improving at least one of the following parameters of crops under saline-alkali stress: plant height, ground diameter, number of roots, root length, wet weight, dry weight, chlorophyll content, net photosynthetic rate, POD enzyme activity, CAT enzyme activity and SOD enzyme activity.

[0047] Beneficial effects:

[0048] The present invention provides a composite bacterial agent for promoting crop resistance to severe salinity and its preparation method and application. The effective live bacteria in the composite bacterial agent of the present invention include Bacillus licheniformis with a cell content of 26-41%, Bacillus subtilis with a cell content of 31-46%, and Pseudomonas fluorescens with a cell content of 21-36%. Applying the above-mentioned bacterial agent to the root system of plants in soda saline-alkali land containing sodium carbonate and sodium bicarbonate can improve the salt-alkali resistance of plants, and the plant height, ground diameter, fresh weight, dry weight, root length and root number of the plants are significantly increased; in addition, the composite bacterial agent can help plants maintain a high photosynthetic activity by increasing the activity of antioxidant enzymes and increasing the chlorophyll content, alleviate the damage of salt-alkali stress to the photosynthesis system of plants, effectively improve the growth of crops in saline-alkali land, and solve the problem of soda saline-alkali land restoration in this field. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0050] Figure 1 These are the results of the effect of bacterial agents on the average plant height and ground diameter of corn seedlings under saline-alkali stress conditions.

[0051] Figure 2 These are the results of the effect of bacterial agents on the average root number and root length of corn seedlings under saline-alkali stress conditions.

[0052] Figure 3 These are the results of the effect of bacterial agents on the average wet weight and dry weight of corn seedlings under saline-alkali stress conditions.

[0053] Figure 4 This is the result of the effect of bacterial agent on the average chlorophyll and photosynthesis of corn seedlings under saline-alkali stress conditions.

[0054] Figure 5 These are the average POD, CAT and SOD results of the bacterial agent on corn seedlings under saline-alkali stress conditions.

[0055] Figure 6 The results of pH value and phosphate solubilization ability of the bacterial agent at different times.

[0056] Figure 7 The pH value and nitrogen fixation ability of the bacterial agent at different times.

[0057] Figure 8 It is an antagonistic relationship between bacterial agents. DETAILED DESCRIPTION

[0058] The present invention is dedicated to providing a microbial agent that can effectively promote crop growth under saline-alkali adverse conditions, and elaborates on its preparation method and application.

[0059] The present invention firstly provides a composite bacterial agent for promoting crop resistance to severe salinity and alkali, wherein the effective live bacteria thereof include Bacillus licheniformis, Bacillus subtilis and Pseudomonas fluorescens.

[0060] In the present invention, in the composite bacterial agent for promoting crop resistance to severe salt and alkali, the cell number of Bacillus licheniformis accounts for 26-41%, preferably 29-37%, and more preferably 34%; the cell number of Bacillus subtilis accounts for 31-46%, preferably 33-42%, and more preferably 37%; the cell number of Pseudomonas fluorescens accounts for 21-36%, preferably 25-33%, and more preferably 29%.

[0061] The composite bacterial agent provided by the present invention has good nitrogen fixation and phosphorus solubilization capabilities under mixed culture conditions, and can be applied to severe saline-alkali environments to promote the stress resistance of crops.

[0062] The composite bacterial agent provided by the present invention uses a combination of three bacteria, namely, Bacillus licheniformis, Bacillus subtilis and Pseudomonas fluorescens, and compared with the invention of application number CN109486709A, the root length is increased by 3%, the wet weight is increased by 3%, and the dry weight is increased by 5%. The above three bacteria have obvious synergistic effects, can effectively alleviate the damage of saline-alkali adversity to corn, and have a better effect on improving the growth of corn. Therefore, it has higher application value and promotion potential in promoting growth in saline-alkali land.

[0063] Unless otherwise specified, the present invention does not require the sources of the above-mentioned strains, and they can be obtained from conventional commercial channels. In a more specific and preferred embodiment of the present invention, strains from the following sources are used: Bacillus licheniformis, with a deposit number of CGMCC No.1.10314; Bacillus subtilis, with a deposit number of CGMCC No.1.15792; Pseudomonas fluorescens, with a deposit number of CGMCC No.1.15059. The above-mentioned microorganisms with deposit numbers can be purchased from the corresponding deposit units. The deposit institutions and address information involved are: China General Microbiological Culture Collection Center (CGMCC), the address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0064] The present invention does not impose any particular limitation on the method for preparing the above-mentioned bacterial agent, and the above-mentioned bacterial flora obtained by conventional methods in the art can play a corresponding role.

[0065] In an alternative scheme of the present invention: the independent bacterial agents of the above-mentioned bacteria are purchased separately, and then mixed according to the cell number ratio to obtain the above-mentioned bacterial population.

[0066] In another alternative embodiment of the present invention, the above-mentioned bacterial community is obtained by culturing with a bacterial agent.

[0067] In a more preferred and specific embodiment, the culture medium for bacterial agent culture includes: 5-15 g / L tryptone, 5-17 g / L sodium chloride, and 1-9 g / L yeast extract. More preferably, it includes 12 g / L tryptone, 12 g / L sodium chloride, and 6 g / L yeast extract. In addition, the present invention preferably uses NaOH to adjust the pH of the culture medium, uses 1.0 mol / L sodium carbonate solution (NaOH) to adjust the pH value to 7.0, and then sterilizes at 121°C for 20 minutes, cools for use, and after preparation, it is packaged and sterilized at 121°C for 20 minutes using a high-pressure steam autoclave. The present invention does not specifically limit the sources of the above-mentioned components, and conventional commercial products in this field are all acceptable.

[0068] In the present invention, the temperature of the bacterial agent culture is preferably 20 to 37° C.; the time of the bacterial agent culture is preferably 12 h to 48 h.

[0069] In the present invention, the bacterial agent can be cultured to obtain bacterial liquid. Alternatively, the bacterial agent can be obtained by centrifugation; the speed of the centrifugation is preferably 4000 rpm; and the time of the centrifugation is preferably 5 min.

[0070] Preferably, the viable bacteria concentration OD of the bacterial solution is 600nm The value of the bacterial solution is about 0.8 to 1.0.

[0071] Preferably, the method of applying the composite bacterial agent includes one or more of spraying, irrigation, soaking, root application, drip irrigation, soil mixing or other suitable application methods.

[0072] Preferably, the above method can also improve the stress resistance of the plant by spraying the bacterial agent on the above-ground parts of the plant (such as leaves). This spraying method helps to improve the overall health level and adversity adaptability of the plant.

[0073] Preferably, the bacterial agent can be widely used in various crops such as dicotyledons, monocotyledons, grasses, and solanaceae.

[0074] The present invention preferably applies the composite bacterial agent to heavy saline-alkali stress, which can improve the salt resistance of crops such as corn, including but not limited to improving root growth, enhancing plant stress resistance, promoting nutrient absorption and improving water use efficiency.

[0075] Preferably, the severe saline-alkali environment is composed of soda saline-alkali soil mainly composed of sodium carbonate and sodium bicarbonate. For example, a saline-alkali soil environment after the soil is soaked in saline-alkali solution with a saline concentration of 80mmoL / L (pH>9.5) and a sodium carbonate and sodium bicarbonate content of Na2CO3:NaHCO3=1:9 (Na2CO3:8mM / L and NaHCO3:72mM / L).

[0076] In a more preferred and specific embodiment, after the bacterial cells are obtained by culture, the composite microbial cells are mixed with a bacterial agent protective material to obtain a composite bacterial agent mixture.

[0077] In the present invention, the microbial agent protective material includes a microbial agent protective agent and an antioxidant.

[0078] Among them, the bacterial agent protective agent preferably includes 12-36% glycerol, more preferably 29% glycerol; preferably includes 4-22% polyethylene glycol, more preferably 18% polyethylene glycol; preferably includes 2-12% peptone, more preferably 10% peptone; preferably includes 0.5-7% Tween, more preferably 5% Tween; preferably includes 8-28% trehalose, more preferably 22% trehalose; preferably includes 2-18% anhydrous sodium acetate, more preferably 12% anhydrous sodium acetate; preferably includes 0.2-4% dipotassium hydrogen phosphate, more preferably 2% dipotassium hydrogen phosphate; preferably includes 0.05-1.5% magnesium sulfate, more preferably 1.5% magnesium sulfate; preferably includes 0.005-0.6% manganese sulfate, more preferably 0.5% manganese sulfate.

[0079] In the present invention, the antioxidant preferably includes 5-32% of shiitake mushroom scraps, more preferably 27% of shiitake mushroom scraps; preferably includes 3-30% of white button mushroom scraps, more preferably 21% of white button mushroom scraps; preferably includes 2-18% of celandine scraps, more preferably 11% of celandine scraps; preferably includes 5-30% of waste tea leaf powder, more preferably 26% of waste tea leaf powder; preferably includes 1-12% of salvia miltiorrhiza powder, more preferably 7% of salvia miltiorrhiza powder; preferably includes 0.5-6 .0% of licorice debris, more preferably 3% of licorice debris; preferably includes 0.5-6.0% of clove debris, more preferably 3% of clove debris; preferably includes 0.1-2.0% of Aconitum powder, more preferably 1% of Aconitum powder; preferably includes 0.01-1.0% of gallic acid powder, more preferably 0.5% of gallic acid powder; preferably includes 0.01-1.0% of superoxide dismutase SOD, more preferably 0.5% of superoxide dismutase SOD.

[0080] In the present invention, the mass ratio of the bacterial agent protective agent to the oxidant is preferably 1:(2-8); the mass of the bacteria in the composite bacterial agent is preferably 6-7 times the sum of the mass of the bacterial agent protective agent and the antioxidant.

[0081] In a more preferred and specific embodiment, a protective material is added to the bacterial body of the composite bacterial agent of the present invention: the mass ratio of the bacterial agent protective agent, the antioxidant, and the composite bacterial agent bacterial body is 1:3:28; a silica gel desiccant is further used to maintain the product dry, and the ratio of the silica gel desiccant to the composite bacterial agent bacterial body is about 1-3:100. The present invention preferably vacuum spray-dries the bacterial agent mixture to obtain the bacterial agent powder. In the present invention, the spray drying air inlet temperature is preferably 75-110°C, more preferably 90°C.

[0082] The present invention is the first to compound and obtain a composite bacterial agent that has such a significant effect on promoting crop resistance to salt and alkali. Under 80mmoL / L salt-alkali stress, the bacterial agent significantly improves the salt resistance of corn. Compared with the control group, all indicators have been improved: plant height, ground diameter, root number, root length, wet weight and dry weight increased by 30.5%, 62.8%, 31.8%, 77.6%, 61.0% and 68.4% respectively. The present invention uses these beneficial bacterial agents, and these achievements show the practical application value and potential impact of the bacterial agents. The promotion effect of the bacterial agents on plant roots deepens the understanding of the interaction between plant roots and soil microorganisms, especially the improvement of root structure of microorganisms in severe saline-alkali environments, and improves nutrient absorption efficiency. This means that the technology can be directly applied to the growth of plants in saline-alkali lands, especially in areas with severe salinization, which can help crops overcome adversity and increase yields, thereby effectively improving the agricultural growth efficiency of saline-alkali lands.

[0083] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0084] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0085] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0086] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0087] Example 1

[0088] This embodiment provides a composite bacterial agent for promoting crop resistance to severe salinity and alkali, wherein the effective live bacteria include Bacillus licheniformis with a cell content of 33-34%, Bacillus subtilis with a cell content of 36-37%, and Pseudomonas fluorescens with a cell content of 29%.

[0089] This example studies and confirms that the composite bacterial agent for promoting crop resistance to severe salinity and alkali has the following characteristics under salinity and alkali stress: improving crop salt resistance, improving crop photosynthetic efficiency, improving crop antioxidant enzyme activity, and enhancing crop stress resistance.

[0090] The specific research contents of this embodiment include:

[0091] 1) The composite bacterial agent improves the salt resistance of corn under saline-alkali stress.

[0092] (1) The composite bacterial agent was obtained by inoculating into LB liquid culture medium and then culturing, and the cell number ratio of the bacterial agent was: Bacillus licheniformis 34%, Bacillus subtilis 37%, and Pseudomonas fluorescens 29%.

[0093] (2) Saline-alkali soil uses soda saline-alkali soil, and its salt-alkali molar ratio is: Na2CO3:NaHCO3=1:9.

[0094] (3) Corn planting conditions were set as follows: temperature 20℃~25℃, 12h light / 12h dark, and light intensity of 12000Lx.

[0095] After the corn seeds germinate 2-3 cm, they are transplanted into pots filled with nutrient soil, with 5 seedlings planted in each pot, for a total of 60 seedlings, divided into 12 pots.

[0096] (4) The seedlings were divided into a salt-free group and a salt-free group, and each group was further divided into a bacterial group and a sterile group. The salt-free group was treated once every 7 days for three times, and each time the saline solution (total molar concentration of saline solution was 80mmoL / L, pH>9.5; the molar concentration of Na2CO3 was 8mM / L; the molar concentration of NaHCO3 was 72mM / L) was poured with 66.7ml / pot, a total of 200mL / pot, and 200g of soil per pot; the bacterial group was poured with 25mL of bacterial solution per pot; the salt-free group and the sterile group used water instead of saline solution and bacterial solution; each group was repeated three times, a total of 12 pots, and cultured for 30 days.

[0097] The results are shown in Tables 1-3 and Figure 1-3 .

[0098] Table 1 Results of salt resistance of corn plant height and ground diameter under saline-alkali stress by bacterial inoculants

[0099]

[0100] Table 2 Results of salt resistance of maize root number and root length under saline-alkali stress by bacterial inoculants

[0101]

[0102]

[0103] Table 3 Results of salt resistance of corn wet weight and dry weight under saline-alkali stress

[0104]

[0105] From Table 1 and Figure 1 It can be seen that under saline-alkali conditions, the composite microbial agent significantly increased the average plant height of corn (increased by 30.4%) and the average ground diameter (increased by 45.0%) compared with the control group. The composite microbial agent improves the rhizosphere microenvironment, promotes plant nutrient absorption and hormone balance, and improves the adaptability of corn to saline-alkali adversity. This helps to alleviate the adverse effects of saline-alkali stress on corn growth and development.

[0106] From Table 2 and Figure 2 It can be seen that under saline-alkali treatment conditions, the composite microbial agent significantly increased the average root number (increased by 31.9%) and average root length (increased by 77.6%) of corn compared with the control group. The composite microbial agent assisted the increase in root number and root length by improving the rhizosphere microenvironment, indicating that the underground part of the plant has been significantly improved. The development of the root system not only helps to improve the ability to absorb water and nutrients, but also enhances the stability of the plant. The microbial agent provides a guarantee for the continued growth of corn under alkaline stress.

[0107] From Table 3 and Figure 3 It can be seen that under saline-alkali treatment conditions, the experimental group significantly increased the average wet weight of corn (increased by 50.7%) and the average dry weight (increased by 40.3%) compared with the control group. The increase in wet weight and dry weight indicates a significant increase in the overall biomass of corn, which is directly related to the increase in yield. The increase in dry weight especially reflects the effective accumulation of photosynthesis products (such as carbohydrates), indicating that the plant can still achieve a high energy conversion efficiency under saline-alkali stress.

[0108] 2) The composite bacterial agent improves the photosynthetic efficiency of corn and the activity of antioxidant enzymes under saline-alkali stress, thereby enhancing its stress resistance.

[0109] (1) Under the same culture conditions as above, the cell numbers of the composite bacterial agent were as follows: Bacillus licheniformis 33%, Bacillus subtilis 36%, and Pseudomonas fluorescens 29%.

[0110] (2) Saline-alkali soil uses soda saline-alkali soil, and its salt-alkali molar ratio is: Na2CO3:NaHCO3=1:9.

[0111] (3) Corn planting conditions were set as follows: temperature 20℃~25℃, 12h light / 12h dark, and light intensity of 12000Lx.

[0112] After the corn seeds germinate 2-3 cm, they are transplanted into pots filled with nutrient soil, with 5 seedlings planted in each pot, for a total of 60 seedlings, divided into 12 pots.

[0113] (4) The seedlings were divided into a salt-free group and a salt-free group, and each group was further divided into a bacterial group and a sterile group. The salt-free group was treated once every 7 days for three times, and each time the saline solution (total molar concentration of saline solution was 80mmoL / L, pH>9.5; the molar concentration of Na2CO3 was 8mM / L; the molar concentration of NaHCO3 was 72mM / L) was poured with 66.7ml / pot, a total of 200mL / pot, and 200g of soil per pot; the bacterial group was poured with 25mL of bacterial solution per pot; the salt-free group and the sterile group used water instead of saline solution and bacterial solution; each group was repeated three times, a total of 12 pots, and cultured for 30 days.

[0114] The results are shown in Tables 4-5 and Figure 4-5 .

[0115] Table 4 Results of chlorophyll content and photosynthesis salt resistance of bacterial agents under saline-alkali stress

[0116]

[0117] Table 5 Results of salt resistance of corn POD, CAT and SOD enzyme activities under saline-alkali stress by bacterial agents

[0118]

[0119] From Table 4 and Figure 4 It can be seen that under saline-alkali treatment conditions, the composite bacterial agent significantly increased the chlorophyll content and net photosynthetic rate of corn compared with the control group, increasing by 34.1% and 38.4% respectively. The composite bacterial agent helps plants maintain a high level of photosynthetic activity by increasing the chlorophyll content, alleviating the damage of saline-alkali stress to the photosynthesis system. This effect further enhances the stress resistance of corn, allowing the plants to maintain a healthy growth state under adverse conditions. In saline-alkali adversity, the bacterial agent promotes the activity of rhizosphere microorganisms and improves the availability of nutrients. The enhancement of photosynthesis not only promotes the growth of the aboveground part, but also improves the nutrient absorption efficiency through the synergistic effect of roots, stems and leaves, providing more stable growth conditions for the entire plant.

[0120] From Table 5 and Figure 5 It can be seen that under saline-alkali treatment conditions, the composite microbial agent significantly increased the POD, CAT and SOD enzyme activities of corn compared with the control group, increasing by 38.4%, 39.8% and 38.9% respectively. By increasing the activity of antioxidant enzymes, the composite microbial agent enables corn to adapt to saline-alkali adversity more effectively. This enhanced stress resistance is manifested in many aspects such as plant growth, photosynthesis and nutrient absorption, enabling corn to maintain a high level of growth and development under adverse conditions. After the activity of POD, CAT and SOD enzymes is increased, the redox balance in plant cells is maintained, and metabolic activities are more efficient and stable. This provides more favorable growth conditions for corn under saline-alkali stress and promotes the overall development of the plant.

[0121] Example 2

[0122] This example uses the composite bacterial agent for promoting crop resistance to severe salinity and alkali provided in Example 1 as experimental material, and studies and confirms that it has the ability to fix nitrogen and solubilize phosphorus.

[0123] The specific research contents of this embodiment include:

[0124] 1) The strains in the composite microbial agent that promotes crop resistance to severe salinity and alkali have the ability to solubilize phosphate after mixed cultivation.

[0125] (1) Bacillus licheniformis, Bacillus subtilis, Pseudomonas fluorescens and the composite bacterial agent provided in Example 1 were inoculated into LB liquid culture medium and cultured at 37°C for 12 h to obtain OD 600 The value of the bacterial solution is about 0.8-1.0.

[0126] (2) Determination of phosphate solubilization ability: The bacterial solution obtained in step (1) was inoculated into NBRIP liquid culture medium, and cultured on a shaker at 28°C and 130 r / min for 7 days. 1.5 ml of fermentation liquid was collected at 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 108 h, and 120 h, centrifuged at 10,000 r / min for 5 min, filtered, and placed in a -20°C refrigerator for standby use, and repeated three times.

[0127] (3) Statistic the pH value and soluble phosphorus content of each group at different times and take the average value.

[0128] The results are shown in Tables 6-7 and Figure 6 .

[0129] Table 6 pH value results of bacterial agents at different times

[0130]

[0131] Table 7 Results of soluble phosphorus content of bacterial agents at different times

[0132]

[0133] From Table 6 and Figure 6 It can be seen that the pH value of the inoculum changes at different times in the NBRIP liquid culture medium: the pH of Bacillus licheniformis drops rapidly from 7.0 to 5.20 from 0h to 60h, and then the pH fluctuates slightly, finally reaching the lowest value of 4.90 at 120h; the initial pH value of Bacillus subtilis is also 7.0, which is in a neutral environment. Within 12 hours, the pH rises (7.45). This indicates that Bacillus subtilis may not have significant metabolic activity during this period, or its metabolites do not immediately cause acidification. However, from 24h to 60h, the pH drops significantly from 7.30 to 4.75, indicating that Bacillus subtilis begins to produce acidic substances after a longer period of culture. At 120 hours, the pH continued to remain at a low value (4.70), indicating that the metabolic activity of this strain will lead to acidification of the environment in the later stage, but overall, it has good tolerance in acidic environments; the pH value of Pseudomonas fluorescens slowly decreased in the first 60 hours, from 7.0 to 6.00, and the rate of decline accelerated from 60 hours, eventually reaching the lowest value of 4.60 at 120 hours; the pH of the mixed inoculant of the three bacteria dropped rapidly to 4.80 within the first 48 hours, and then gradually recovered to 6.05 (108 hours and 120 hours). The composite inoculant caused a greater drop in pH in the initial stage, which may be related to the production of organic acids by its metabolism.

[0134] Furthermore, from Table 7 and Figure 6It can be seen that the soluble phosphorus content of the inoculants changes at different times: the soluble phosphorus content of Bacillus licheniformis increased steadily from 12h to 96h, reaching 198.147mg / L, and decreased slightly after 96h, but reached a peak of 217.28mg / L at 120h; the soluble phosphorus content of Bacillus subtilis increased slowly in the initial stage (12h to 48h), rose rapidly after 48h, reached a peak of 262.09mg / L at 108h, and began to decrease to 241.48mg / L after 120h; the soluble phosphorus content of Pseudomonas fluorescens increased steadily with time, reaching a peak of 283.58mg / L at 120h, showing a continuous and stable phosphorus solubilizing ability as a whole; the soluble phosphorus content of the mixed inoculant of the three bacteria quickly reached a peak of 247.28mg / L from 12h to 72h, and then decreased rapidly to 156.713mg / L at 120h. The experimental results show that the composite bacterial agent can release phosphorus continuously and effectively. Through the synergistic effect of different bacterial species, it demonstrates excellent phosphorus-solubilizing ability and provides a stable nutrient supply for crops. It has important agricultural application value, especially in improving the effectiveness of soil phosphorus and improving saline-alkali soils. It has significant potential.

[0135] 2) The strains in the composite bacterial agent that promotes crop resistance to severe salinity and alkali have the ability to fix nitrogen under mixed culture conditions.

[0136] (1) The composite bacterial agent provided in Example 1 was inoculated into LB liquid culture medium and cultured at 37°C for 12 h to obtain a microbial agent with a viable bacterial concentration of OD 600 The value of the bacterial solution is about 0.8-1.0.

[0137] (2) Determination of nitrogen fixation ability: The bacterial solution obtained in step (1) was added to Ashby liquid medium and cultured at 37°C and 160 r / min for 7 days. 1.5 ml of the fermentation liquid was collected at 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 108 h, and 120 h, centrifuged at 10,000 r / min for 5 min, filtered, and placed in a -20°C refrigerator for use, and repeated three times.

[0138] (3) Count the pH value and nitrogen fixation capacity of each group at different times and take the average value.

[0139] Depend on Figure 7It can be seen that the pH value of the inoculant changes at different times in the Ashby liquid medium: the pH of the mixed inoculant of the three bacteria steadily decreases to 6.7 in the first 36 hours; it drops to a minimum of 6.6 at 60 hours; it gradually recovers from 72 hours and reaches a peak of 7.4-7.5 at 108 hours to 120 hours. The mixed inoculant of the three bacteria shows a strong nitrogen fixation ability. By secreting acidic or alkaline metabolites to adjust the pH, microorganisms can play a nitrogen fixation role in the optimal environment. The synergistic effect of the mixed inoculant may enhance its nitrogen fixation potential at certain stages, help increase the nitrogen supply in the soil, and thus promote crop growth.

[0140] Depend on Figure 7 It can be seen that the ammonium nitrogen content of the inoculants changes at different times: the ammonium nitrogen content of the mixed inoculant of the three bacteria increases slowly from 12h to 24h, from 14.53mg / L to 31.03mg / L, and then decreases rapidly after 36h, leaving only 13.46mg / L at 120h. The experiment shows that the mixed inoculant has a certain nitrogen fixation effect.

[0141] Example 3

[0142] This example uses the composite bacterial agent for promoting crop resistance to severe salinity and alkali provided in Example 1 as the experimental material, and studies and confirms that there is no antagonistic relationship between the various bacterial species.

[0143] The specific research contents of this embodiment include:

[0144] After the bacterial agent is in LB culture medium, the screened strains are cross-streaked on beef extract peptone culture medium plates using the plate confrontation method, and cultured upside down in an incubator at 30°C for 3 to 5 days. The growth of the strains at the intersections is observed during the culture period.

[0145] result Figure 8 It can be seen that there is no antagonism between the strains shown.

[0146] The present invention prepares a composite bacterial agent with three kinds of bacteria: among them, Bacillus licheniformis has the ability to convert nitrogen in the atmosphere into a form that can be absorbed by plants, thereby increasing the nitrogen content in the soil and enhancing the growth of crops; organic matter decomposition and nutrient release; Bacillus subtilis can decompose complex organic matter in the soil and release nutrients that can be absorbed by plants, especially in an environment where soil organic matter is scarce, this role is particularly important to improve soil fertility; Pseudomonas fluorescens can secrete plant growth regulators such as indoleacetic acid (IAA), significantly promote plant root development, enhance root absorption capacity, thereby improving plant absorption efficiency of water and nutrients. The organic acids, enzymes and other metabolites produced by the metabolic activities of the above-mentioned strains can improve the physical and chemical properties of the soil, improve the aggregate structure of the soil, enhance air permeability and water retention, and create a rhizosphere environment that is conducive to plant growth. At the same time, the strains work synergistically in various ways such as nitrogen fixation, decomposition of organic matter, promotion of root development and disease inhibition, and can comprehensively improve the growth vitality and stress resistance of plants, thereby improving the yield and quality of crops. As an ideal composition of the composite microbial agent, the composite microbial agent provided by the present invention has broad application prospects in agricultural production.

[0147] Example 4

[0148] In this embodiment, based on the composite bacterial agent for promoting crop resistance to severe salinity and alkali provided in Example 1, a bacterial agent protective agent and an antioxidant are further added, thereby providing a composite bacterial agent product with a long shelf life and good stability, which is suitable for market promotion.

[0149] The product formula provided in this embodiment is: a bacterial agent protective agent, an antioxidant, and a composite bacterial agent cell, which are compounded in a mass ratio of 1:3:28; and supplemented with a silica gel desiccant, and the mass ratio of the silica gel desiccant to the bacterial agent cell is 1-3:100.

[0150] This embodiment provides a method for preparing the above product, the steps are:

[0151] (1) Preparation of straw decomposition medium: Prepare a straw decomposition medium containing 12 g / L tryptone, 12 g / L sodium chloride, and 6 g / L yeast extract, adjust the pH to 7.0, and dispense into 500 mL Erlenmeyer flasks, with 300 mL of the medium in each flask, and place at room temperature of 20 to 37°C for later use.

[0152] (2) Bacterial agent culture: Bacillus licheniformis, Bacillus subtilis, and Pseudomonas fluorescens were inoculated into straw decomposition medium and cultured at 20-37°C for 24 h. The cell number percentage of each bacterial species was determined: Bacillus licheniformis 34%, Bacillus subtilis 37%, and Pseudomonas fluorescens 29%. The bacterial agent cells were obtained by centrifugation at 4000 rpm for 5 min.

[0153] (3) Add strain protective agent and antioxidant to the composite bacterial agent. The formula of the strain protective agent is: 29% glycerol, 18% polyethylene glycol, 10% peptone, 5% Tween, 22% trehalose, 12% anhydrous sodium acetate, 2% dipotassium hydrogen phosphate, 1.5% magnesium sulfate and 0.5% manganese sulfate; the formula of the antioxidant is: 27% shiitake mushroom scraps, 21% white mushroom scraps, 11% celandine scraps, 26% waste tea powder, 7% salvia miltiorrhiza powder, 3% licorice scraps, 3% clove scraps, 1% aconite powder, 0.5% gallic acid powder and 0.5% superoxide dismutase SOD. The mass ratio of the bacterial agent protective agent, antioxidant and bacterial agent bacterial agent is 1:3:28.

[0154] (4) Preparation of bacterial powder: The bacterial bodies containing the protective material are spray-dried at low temperature to obtain bacterial powder. The air inlet temperature is 80-90°C to obtain salt-alkali resistant bacterial agent lignocellulose decomposing bacterial powder.

[0155] (5) Use of desiccant: The mass ratio of silica gel desiccant to bacterial cells is 1-3:100.

[0156] (6) Vacuum sealed packaging: The bacterial agent is placed in a sealed packaging bag protected by vacuum or nitrogen to reduce contact with oxygen and delay the oxidation process.

[0157] This example measures the shelf life of the above products, and the shelf life under different storage conditions is as follows:

[0158] Refrigerated storage (4°C):

[0159] The composite bacterial agent of this embodiment has a shelf life of up to 18-24 months, while other similar products generally have a shelf life of 12-18 months due to insufficient bacterial stability or low performance of protective materials.

[0160] Storage at room temperature (about 25°C, light-free, dry environment):

[0161] The storage period of the composite bacterial agent of this embodiment is 12-18 months, which is significantly better than the storage period of only 9-12 months of the products on the market. This is due to the effective inhibition of oxidation of bacterial metabolites by trehalose and anhydrous sodium acetate in the bacterial strain protective agent and shiitake mushroom scraps and waste tea powder in the antioxidant.

[0162] High temperature storage (>35°C, high humidity environment):

[0163] Under high temperature and high humidity conditions, the composite bacterial agent of this embodiment can still maintain a shelf life of 6-9 months, while other commercially available products usually have a shelf life of only 3-6 months. This is because this embodiment uses low-temperature spray drying technology combined with silica gel desiccant to effectively isolate moisture and delay bacterial oxidation and inactivation.

[0164] Innovation Advantage Analysis

[0165] Bacteria protectant: In the protectant formula of this embodiment, trehalose and glycerol work together to provide good anti-drying protection for the bacteria; polyethylene glycol and peptone further enhance the stability of the bacterial membrane. Compared with the commercially available products that generally use a single ingredient (such as sucrose, maltodextrin) protectant, the effect is significantly improved.

[0166] Antioxidants: The antioxidant components of this embodiment, such as waste tea powder and salvia miltiorrhiza powder, have excellent antioxidant properties. Compared with other products that usually only contain vitamin C or a single plant extract, the compounding effect is better and significantly extends the shelf life of bacterial activity.

[0167] Process optimization: The combination of low-temperature spray drying technology and vacuum-sealed packaging not only effectively controls the loss of bacteria, but also further reduces the impact of high temperature on bacterial activity.

[0168] Through the above comparison, it is clear that the composite bacterial agent of this embodiment shows significant stability and extended shelf life under different storage conditions, especially in adverse environments such as high temperature and high humidity. This not only improves the market promotion potential of the product, but also lays the foundation for subsequent application in harsh environments.

[0169] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composite bacterial agent for promoting crop resistance to severe salinity and alkali, characterized in that: The effective live bacteria in the composite bacterial agent include Bacillus licheniformis with a cell content of 26-41%, Bacillus subtilis with a cell content of 31-46% and Pseudomonas fluorescens with a cell content of 21-36%.

2. The composite bacterial agent for promoting crop resistance to severe salinity and alkali according to claim 1, characterized in that: The effective live bacteria in the composite bacterial agent include Bacillus licheniformis with a cell content of 29-37%, Bacillus subtilis with a cell content of 33-42%, and Pseudomonas fluorescens with a cell content of 25-33%. Preferably, the effective live bacteria in the composite bacterial agent include Bacillus licheniformis with a cell content of 34±1%, Bacillus subtilis with a cell content of 37±1% and Pseudomonas fluorescens with a cell content of 29±1%.

3. The composite bacterial agent for promoting crop resistance to severe salinity and alkali according to claim 1 or 2, characterized in that: The composite bacterial agent also includes a bacterial agent protective agent and an antioxidant; the mass ratio of the bacterial agent protective agent to the antioxidant is 1:2-8.

4. The composite bacterial agent for promoting crop resistance to severe salinity and alkali according to claim 3, characterized in that: The mass of the effective live bacteria is 6-7 times the sum of the mass of the bacterial agent protective agent and the antioxidant.

5. The composite bacterial agent for promoting crop resistance to severe salinity and alkali according to claim 3 or 4, characterized in that: The bacterial agent protective agent is selected from one or more of glycerol, polyethylene glycol, peptone, Tween, trehalose, anhydrous sodium acetate, dipotassium hydrogen phosphate, magnesium sulfate, and manganese sulfate; Preferably: the mass percentage of glycerol in the bacterial agent protective agent is 12-36%; And / or, the mass percentage of polyethylene glycol in the bacterial agent protective agent is 4-22%; And / or, the mass percentage of peptone in the bacterial agent protective agent is 2-12%; And / or, the mass percentage of Tween in the bacterial agent protective agent is 0.5-7%; And / or, the mass percentage of trehalose in the bacterial agent protective agent is 8-28%; And / or, the mass percentage of anhydrous sodium acetate in the bacterial agent protective agent is 2-18%; And / or, the mass percentage of dipotassium hydrogen phosphate in the bacterial agent protective agent is 0.2-4%; And / or, the mass percentage of magnesium sulfate in the bacterial agent protective agent is 0.05-1.5%; And / or, the mass percentage of manganese sulfate in the bacterial agent protective agent is 0.005-0.6%.

6. The composite bacterial agent for promoting crop resistance to severe salinity and alkali according to any one of claims 3 to 5, characterized in that: The antioxidant is selected from one or more of shiitake mushroom scraps, white mushroom scraps, celandine scraps, waste tea powder, salvia miltiorrhiza powder, licorice scraps, clove scraps, aconite powder, gallic acid powder, and superoxide dismutase SOD; Preferably: the mass percentage of mushroom scraps in the antioxidant is 5-32%; and / or, the mass percentage of white mushroom scraps in the antioxidant is 3-30%; and / or, the mass percentage of celandine chips in the antioxidant is 2-18%; and / or, the mass percentage of waste tea leaves powder in the antioxidant is 5-30%; and / or, the mass percentage of salvia miltiorrhiza powder in the antioxidant is 1-12%; and / or, the mass percentage of liquorice crumbs in the antioxidant is 0.5-6.0%; and / or, the mass percentage of clove crumbs in the antioxidant is 0.5-6.0%; and / or, the mass percentage of Wufuzi powder in the antioxidant is 0.1-2.0%; and / or, the mass percentage of gallic acid powder in the antioxidant is 0.01-1.0%; And / or, the mass percentage of superoxide dismutase SOD in the antioxidant is 0.01-1.0%.

7. The composite bacterial agent for promoting crop resistance to severe salinity and alkali according to any one of claims 3 to 6, characterized in that: The bacterial agent protective agent includes 29±3% glycerol, 18±2% polyethylene glycol, 10±1% peptone, 5±0.5% Tween, 22±2% trehalose, 12±1% anhydrous sodium acetate, 2±0.2% dipotassium hydrogen phosphate, 1.5±0.2% magnesium sulfate and 0.5±0.1% manganese sulfate; And / or, the antioxidant includes 27±3% shiitake mushroom scraps, 21±2% white mushroom scraps, 11±1% celandine scraps, 26±3% waste tea leaf powder, 7±1% salvia miltiorrhiza powder, 3±0.3% liquorice scraps, 3±0.3% clove scraps, 1±0.1% aconite powder, 0.5±0.1% gallic acid powder and 0.5±0.1% superoxide dismutase SOD.

8. The composite bacterial agent for promoting crop resistance to severe salinity and alkali according to any one of claims 3 to 7, characterized in that: The mass ratio of the microbial protective agent, antioxidant and effective live bacteria is 1:2-4:21-30; Preferably, the mass ratio of the bacterial agent protective agent, the antioxidant and the effective live bacteria is 1:3:

28.

9. The method for preparing the composite bacterial agent for promoting crop resistance to severe salinity and alkali according to any one of claims 1 to 8, characterized in that: The mixed raw material containing the bacterial agent protective agent, antioxidant and effective live bacteria is spray-dried at low temperature, and the air inlet temperature is 80-90°C; Preferably, silica gel desiccant is added after low-temperature spray drying; the mass ratio of silica gel desiccant to effective live bacteria is 1-3:

100.

10. Use of the composite bacterial agent according to any one of claims 1 to 8 or the composite bacterial agent prepared by the preparation method according to claim 9 in nitrogen fixation, phosphorus solubilization and promoting crop resistance to severe salinity and alkali; Preferably: the crop includes corn; Preferably, the performance of severe salt and alkali resistance includes at least one of the following: (1) Improve the salt resistance of crops under saline-alkali stress; (2) Improve crop energy conversion efficiency under saline-alkali stress; (3) Improve the antioxidant enzyme activity of crops under saline-alkali stress; (4) Improve crop resistance to saline-alkali stress; (5) Improving at least one of the following parameters of crops under saline-alkali stress: plant height, ground diameter, number of roots, root length, wet weight, dry weight, chlorophyll content, net photosynthetic rate, POD enzyme activity, CAT enzyme activity and SOD enzyme activity.

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