Complex microbial inoculant for promoting heavy metal cadmium resistance of crops as well as 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 crops to resist heavy metal cadmium 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 24%-41%, bacillus subtilis with the cell count content of 30%-46%, pseudomonas fluorescens with the cell count content of 16%-32% and azotobacter chroococcum with the cell count content of 14%-25%. When the microbial agent is applied to root systems of plants growing in the heavy metal cadmium land, the cadmium resistance of the plants can be improved, and the plant height, the ground diameter, the fresh weight, the dry weight, the root length and the root number of the plants are remarkably increased; in addition, the complex microbial inoculant can effectively improve the growth of crops in the cadmium field and solve the problem of cadmium field repair in the field by improving the activity of antioxidant enzyme, increasing the chlorophyll content, helping plants maintain high photosynthetic activity and relieving the damage of cadmium stress to a plant photosynthesis system.

Description

Technical Field

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

[0002] Cadmium soil is a non-biological adversity problem that seriously limits crop production and affects the sustainable development of global agriculture. Heavy metal cadmium in cadmium soil can cause osmotic stress, ion toxicity and nutrient absorption disorders in crops, thereby inhibiting root development and affecting the normal growth and yield of crops. In addition, the problem of soil cadmification is further aggravated by unreasonable agricultural management measures such as climate change, improper irrigation and excessive fertilization. Traditional cadmium land improvement methods, such as drainage and cadmium washing and the application of chemical amendments, can improve the soil environment in the short term, but these methods are often costly and may cause secondary pollution to the ecological environment.

[0003] In recent years, the excavation and application of cadmium-resistant microbial agents have provided new solutions for the improvement of cadmium land. Studies have shown that specific beneficial microorganisms can significantly alleviate the adverse effects of cadmium 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 patents for the invention of microbial agents to promote crop cadmium resistance. For example, the invention of patent number CN117448006A discloses a composite passivation material and preparation method for the treatment of acidic cadmium-contaminated soil. The composition of the composite passivation material includes: straw biochar, silicon calcium potassium magnesium fertilizer, clay mineral, corn stalk, composite functional microbial agent (Bacillus subtilis, Bacillus licheniformis, Trichoderma harzianum, Paecilomyces lilacinus). The invention is mainly used for the treatment of acidic cadmium-contaminated land, the treatment scope is limited, and the invention requires a large number of raw materials, the cost is high, and the long-term application of fertilizers is not conducive to environmental protection.

[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 heavy metal cadmium, 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 heavy metal cadmium, wherein the effective live bacteria in the composite bacterial agent include Bacillus licheniformis with a cell content of 24-41%, Bacillus subtilis with a cell content of 30-46%, Pseudomonas fluorescens with a cell content of 16-32%, and Azotobacter nitrogen-fixing from brown spherical with a cell content of 14-25%.

[0008] Preferably, in the present invention, the effective live bacteria in the composite bacterial agent include Bacillus licheniformis with a cell content of 25-38%, Bacillus subtilis with a cell content of 32-40%, Pseudomonas fluorescens with a cell content of 18-28% and Azotobacter nitrofuga with a cell content of 15-22%.

[0009] Preferably, in the present invention, the effective live bacteria in the composite bacterial agent include Bacillus licheniformis with a cell content of 26±1%, Bacillus subtilis with a cell content of 34±1%, Pseudomonas fluorescens with a cell content of 22±1% and Azotobacter nitrofuga with a cell content of 18±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 0.50-7%.

[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 0.20-4%.

[0020] Preferably, in the present invention, the mass percentage of magnesium sulfate in the bacterial agent protective agent is 0.05-1.50%.

[0021] Preferably, in the present invention, the mass percentage of manganese sulfate in the bacterial agent protective agent is 0.05-0.60%.

[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 0.50-6.00%.

[0029] Preferably, in the present invention, the mass percentage of clove chips in the antioxidant is 0.50-6.00%.

[0030] Preferably, in the present invention, the mass percentage of Aconitum carmichaelii powder in the antioxidant is 0.10-2.00%.

[0031] Preferably, in the present invention, the mass percentage of gallic acid powder in the antioxidant is 0.01-1.00%.

[0032] Preferably, in the present invention, the mass percentage of superoxide dismutase SOD in the antioxidant is 0.01-1.00%.

[0033] Preferably, in the present invention, the bacterial agent protective agent includes 29±3% glycerol, 18±2% polyethylene glycol, 10±1% peptone, 5±0.50% Tween, 22±2% trehalose, 12±1% anhydrous sodium acetate, 2±0.20% dipotassium hydrogen phosphate, 1.50±0.20% magnesium sulfate and 0.50±0.10% manganese sulfate.

[0034] Preferably, in the present invention, the antioxidant comprises 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±0.30% licorice scraps, 3±0.30% clove scraps, 1±0.10% aconite powder, 0.50±0.10% gallic acid powder and 0.50±0.10% 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 heavy metal cadmium, 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 heavy metal cadmium.

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

[0041] Preferably, in the present invention, the performance of resistance to heavy metal cadmium includes at least one of the following:

[0042] (1) Improve the cadmium resistance of crops under cadmium stress;

[0043] (2) Improve crop energy conversion efficiency under cadmium stress;

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

[0045] (4) Improve crop resistance to cadmium stress;

[0046] (5) Improving at least one of the following parameters of crops under cadmium 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 heavy metal cadmium, and a preparation method and application thereof. 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%, Pseudomonas fluorescens with a cell content of 21-36%, and Azotobacter nitrofuga with a cell content of 14-25%. Applying the above-mentioned bacterial agent to the root system of plants in land containing heavy metal cadmium can improve the plant's ability to resist cadmium, and the plant height, ground diameter, fresh weight, dry weight, root length and root number of the plant 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 cadmium stress to the photosynthesis system of plants, effectively improve the growth of crops in cadmium land, and solve the problem of cadmium 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 cadmium 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 cadmium stress conditions.

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

[0053] Figure 4 These are the results of the effects of bacterial agents on the average chlorophyll and photosynthesis of corn seedlings under cadmium stress.

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

[0055] Figure 6 The results are of the phosphorus-solubilizing ability of the bacterial agent at different times.

[0056] Figure 7 The results of nitrogen fixation ability of bacterial agents 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 bacterial agent that can effectively promote crop growth under cadmium adverse conditions, and elaborates on its preparation method and application.

[0059] The invention firstly provides a composite bacterial agent for promoting the resistance of crops to heavy metal cadmium, wherein the effective live bacteria thereof include Bacillus licheniformis, Bacillus subtilis, Pseudomonas fluorescens and Nitrogen-fixing Bacterium chlorosphaeroides.

[0060] In the present invention, in the composite bacterial agent for promoting crop resistance to heavy metal cadmium, the cell number of Bacillus licheniformis accounts for 24-41%, preferably 25-38%, and more preferably 26%; the cell number of Bacillus subtilis accounts for 30-46%, preferably 32-40%, and more preferably 34%; the cell number of Pseudomonas fluorescens accounts for 16-32%, preferably 18-28%, and more preferably 22%; the cell number of Azotobacter nitrogen-fixing bacteria accounts for 14-25%, preferably 15-22%, and more preferably 18%.

[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 heavy metal cadmium environments to promote the stress resistance of crops.

[0062] The above-mentioned composite bacterial agent provided by the present invention uses a combination of four bacteria, namely Bacillus licheniformis, Bacillus subtilis, Pseudomonas fluorescens and Azotobacter chlororaphis, which has obvious synergy compared with the invention of application number CN1 17448006A, can effectively alleviate the damage of cadmium adversity to corn, and has a better effect on improving the growth of corn. Therefore, it has higher application value and promotion potential in promoting growth in cadmium fields.

[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; Azotobacter nitrofurantoides, with a deposit number of CGMCC No 1.5803. 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: 5-15g / L tryptone, 5-17g / L sodium chloride, and 1-9g / L yeast extract. More preferably, it includes 12g / L tryptone, 12g / L sodium chloride, and 6g / L yeast extract. In addition, the present invention preferably uses NaOH to adjust the pH of the culture medium, uses 1.00mol / L sodium carbonate solution (NaOH) to adjust the pH value to 7.00, and then sterilizes at 121°C for 20min, cools and uses, and after preparation, it is packaged and sterilized at 121°C for 20min 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 0.80 to 1.00.

[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 cadmium stress, which can improve the cadmium 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 heavy metal cadmium environment is a cadmium soil environment with a cadmium concentration of 30 mmoL / 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 0.50-7% 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 0.20-4% dipotassium hydrogen phosphate, more preferably 2% dipotassium hydrogen phosphate; preferably includes 0.05-1.50% magnesium sulfate, more preferably 1.50% magnesium sulfate; preferably includes 0.05-0.60% manganese sulfate, more preferably 0.50% 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 0.50-6.00% licorice crumbs, more preferably 3% licorice crumbs; preferably 0.50-6.00% clove crumbs, more preferably 3% clove crumbs; preferably 0.10-2.00% aconite powder, more preferably 1% aconite powder; preferably 0.01-1.00% gallic acid powder, more preferably 0.50% gallic acid powder; preferably 0.01-1.00% superoxide dismutase SOD, more preferably 0.50% 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 composite bacterial agent bacteria 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 cadmium. Under 30mmoL / L cadmium stress, the bacterial agent significantly improves the cadmium 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 are increased by 32.60%, 26.90%, 37%, 64.90%, 39.30% and 30.80% respectively. The present invention uses these beneficial bacterial agents, and these results show the practical application value and potential impact of the bacterial agents. The bacterial agent promotes the plant root system, deepens the understanding of the interaction between the plant root system and soil microorganisms, especially the improvement of the root structure of microorganisms in the heavy metal cadmium environment, and improves the nutrient absorption efficiency. This means that the technology can be directly applied to the growth of plants in cadmium land, especially in areas with severe cadmium, which can help crops overcome adversity and increase yields, thereby effectively improving the agricultural growth efficiency of cadmium land.

[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] The present embodiment provides a composite bacterial agent for promoting crop resistance to heavy metal cadmium. The effective live bacteria in the composite bacterial agent of the present invention include Bacillus licheniformis with a cell content of 24-41%, Bacillus subtilis with a cell content of 30-46%, Pseudomonas fluorescens with a cell content of 16-32%, and Azotobacter nitrogen-fixing from brown spherical with a cell content of 14-25%.

[0089] This example studies and confirms that the composite bacterial agent for promoting crop resistance to heavy metal cadmium has the following characteristics under cadmium stress: improving crop resistance to cadmium, 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 cadmium resistance of corn under cadmium 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 26%, Bacillus subtilis 34%, Pseudomonas fluorescens 22%, and Nitrogen-fixing Bacterium chlororaphis 18%.

[0093] (2) The total molar concentration of cadmium in cadmium soil is 30mmoL / L.

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

[0095] After germination, the corn seeds were 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 cadmium-free group and a cadmium-containing group, and each group was further divided into a bacterial group and a sterile group. The cadmium-containing group was treated once every 7 days for three times, and each time 66.7 ml of cadmium solution (total molar concentration of cadmium was 30 mmoL / L) was poured per pot, for a total of 200 ml / pot, with 200 g of soil per pot; the bacterial group was poured with 25 ml of bacterial solution per pot; the cadmium-free group and the sterile group used water instead of cadmium solution and bacterial solution; each group was repeated three times, for 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 cadmium resistance of maize plant height and ground diameter under cadmium stress by bacterial inoculants

[0099]

[0100] Table 2 Results of cadmium resistance of maize root number and root length under cadmium stress by bacterial inoculants

[0101]

[0102] Table 3 Results of wet and dry weight cadmium resistance of corn under cadmium stress by bacterial inoculants

[0103]

[0104] From Table 1 and Figure 1 It can be seen that under cadmium conditions, the composite microbial agent significantly increased the average plant height of corn (increased by 32.60%) and the average ground diameter (increased by 26.90%) 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 cadmium adversity. This helps to alleviate the adverse effects of cadmium stress on corn growth and development.

[0105] From Table 2 and Figure 2 It can be seen that under the condition of cadmium treatment, the composite microbial agent significantly increased the average root number of corn (increased by 37.00%) and the average root length (increased by 64.90%) 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 was 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 continuous growth of corn under cadmium stress.

[0106] From Table 3 and Figure 3 It can be seen that under the cadmium treatment condition, the experimental group significantly increased the average wet weight of corn (increased by 39.30%) and the average dry weight (increased by 30.80%) 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 photosynthetic products (such as carbohydrates), indicating that the plant can still achieve a high energy conversion efficiency under cadmium stress.

[0107] 2) The composite bacterial agent improves the photosynthetic efficiency of corn and the activity of antioxidant enzymes under cadmium stress, thereby enhancing its resistance to stress.

[0108] (1) Under the same culture conditions as above, the cell numbers of the composite bacterial agent were as follows: 26% for Bacillus licheniformis, 34% for Bacillus subtilis, 22% for Pseudomonas fluorescens, and 18% for Azotobacter nitrofuga.

[0109] (2) Cadmium soil uses soda cadmium soil, and its cadmium molar ratio is: 30mmoL / L.

[0110] (3) Corn planting conditions were set as follows: temperature 25°C-35°C, 12 h light / 12 h dark, and light intensity of 12000 Lx.

[0111] After germination, the corn seeds were transplanted into pots filled with nutrient soil, with 5 seedlings planted in each pot, for a total of 60 seedlings, divided into 12 pots.

[0112] (4) The seedlings were divided into a cadmium-free group and a cadmium-containing group, and each group was divided into a bacterial group and a sterile group. The cadmium-containing group was treated once every 7 days for three times, and each time 66.7 ml of cadmium solution (total molar concentration of cadmium was 30 mmoL / L) was poured per pot, a total of 200 mL / pot, and 200 g of soil per pot; the bacterial group was poured with 25 mL of bacterial solution per pot; the cadmium-free group and the sterile group used water instead of cadmium solution and bacterial solution; each group was repeated three times, a total of 12 pots, and cultured for 30 days. The results are shown in Tables 4-5 and Figure 4-5 .

[0113] Table 4 Results of chlorophyll content and photosynthetic resistance to cadmium in the bacterial inoculants under cadmium stress

[0114]

[0115] Table 5 Results of cadmium resistance of corn POD, CAT and SOD enzyme activities under cadmium stress by bacterial agents

[0116]

[0117] From Table 4 and Figure 4 It can be seen that under the condition of cadmium treatment, the composite bacterial agent significantly increased the chlorophyll content and net photosynthetic rate of corn compared with the control group, increasing by 34.80% and 40.50% respectively. The composite bacterial agent helps plants maintain a high photosynthetic activity by increasing the chlorophyll content and alleviates the damage of cadmium stress to the photosynthetic system. This effect further enhances the stress resistance of corn, allowing the plants to maintain a healthy growth state under adverse conditions. In cadmium 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.

[0118] From Table 5 and Figure 5It can be seen that under the condition of cadmium treatment, the composite microbial agent significantly increased the POD, CAT and SOD enzyme activities of corn compared with the control group, increasing by 45.50%, 48.10% and 43.01% respectively. The composite microbial agent can adapt corn to cadmium adversity more effectively by increasing the activity of antioxidant enzymes. 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 cadmium stress and promotes the overall development of the plant.

[0119] Example 2

[0120] This example uses the composite bacterial agent for promoting crop resistance to heavy metal cadmium provided in Example 1 as experimental material, and studies and confirms that it has the ability to fix nitrogen and solubilize phosphorus.

[0121] The specific research contents of this embodiment include:

[0122] 1) The strains in the composite bacterial agent that promotes crop resistance to heavy metal cadmium have the ability to solubilize phosphate after mixed cultivation.

[0123] (1) Bacillus licheniformis, Bacillus subtilis, Pseudomonas fluorescens, Azotobacter sphaeroides and the composite bacterial agent provided in Example 1 were inoculated into LB liquid medium and cultured at 37°C for 12 h to obtain OD 600 The value of the bacterial solution is about 0.80-1.00.

[0124] (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. 1.50 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.

[0125] (3) The pH value and soluble phosphorus content of each group at different times were counted and the average value was taken.

[0126] The results are shown in Table 6 and Figure 6 .

[0127] Table 6 Results of soluble phosphorus content of bacterial agents at different times

[0128]

[0129] Furthermore, from Table 6 and Figure 6It can be seen that the soluble phosphorus content of the inoculant changes at different times: the soluble phosphorus content of Bacillus licheniformis reaches 198.15 mg / L from 12h to 96h, decreases slightly after 96h, but reaches a peak of 217.29 mg / L at 120h; the soluble phosphorus content of Bacillus subtilis increases slowly in the initial stage (12h to 48h), increases rapidly after 48h, reaches a peak of 262.09 mg / L at 108h, and begins to decrease by 241.49 mg / L after 120h. g / L; the soluble phosphorus content of Pseudomonas fluorescens increased steadily over time, reaching a peak of 283.58 mg / L at 120 h, showing a sustained and stable phosphorus solubilizing ability as a whole; the soluble phosphorus content of brown ball nitrogen-fixing bacteria reached 182.16 mg / L during 12 h to 36 h; the soluble phosphorus content of the mixed agent of the four bacteria quickly reached a peak of 247.28 mg / L from 12 h to 72 h, and then dropped rapidly to 156.71 mg / L at 120 h. The experimental results show that the composite agent can continuously and effectively release phosphorus, and through the synergistic effect of different strains, it shows excellent phosphorus solubilization ability, provides a stable nutrient supply for crops, and has important agricultural application value, especially in improving the effectiveness of soil phosphorus and improving cadmium soil, and has significant potential.

[0130] 2) The strains in the composite bacterial agent that promotes crop resistance to severe cadmium have the ability to fix nitrogen under mixed culture conditions.

[0131] (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.

[0132] (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.

[0133] (3) Count the nitrogen fixation capacity of each group at different times and take the average value.

[0134] Depend on Figure 7It can be seen that the changes in the ammonium nitrogen content of the inoculants at different times: the ammonium nitrogen content of Bacillus licheniformis gradually increased from 40.90 mg / L at 12h, reached a peak of 120.26 mg / L at 96h, and decreased after 96h, but slightly rebounded to 118.53 mg / L at 120h; the ammonium nitrogen content of Bacillus subtilis increased slowly in the early stage (12h to 48h), remained stable at about 48h, began to increase significantly at 86h, and reached 81.90 mg / L at 120h, showing a continuous nitrogen-increasing effect; the ammonium nitrogen content of Pseudomonas fluorescens increased rapidly in the early stage (12h to 36h), increasing from 5.00 mg / L to 15.63 mg / L, There was a significant increase from 72h, and reached a peak of 36.90mg / L at 120h; the nitrogen fixation capacity of brown ball nitrogen-fixing bacteria was 31.66mg / L at 12h, and the nitrogen fixation capacity gradually increased with the extension of culture time, reaching 116.93mg / L at 72h, which was the highest value. However, at 120h, the nitrogen fixation capacity dropped to 36.16mg / L. Overall, the accumulation capacity of ammonium nitrogen was lower than that of other bacterial agents, but it had certain potential in the later period. The ammonium nitrogen content of the mixed bacterial agent of the three bacteria increased slowly from 12h to 24h, from 14.53mg / L to 31.03mg / L, and dropped rapidly after 36h, leaving only 13.46mg / L at 120h. The experiment showed that the mixed bacterial agent had a certain nitrogen fixation effect.

[0135] Example 3

[0136] This example uses the composite bacterial agent for promoting crop resistance to heavy metal cadmium provided in Example 1 as the experimental material, and studies and confirms that there is no antagonistic relationship between the various bacterial species.

[0137] The specific research contents of this embodiment include:

[0138] 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.

[0139] Results Figure 8 : There was no antagonism between the strains shown.

[0140] The present invention prepares a composite bacterial agent with four kinds of bacteria: among them, Bacillus licheniformis and brown ball nitrogen-fixing bacteria have 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.

[0141] Example 4

[0142] In this embodiment, based on the composite bacterial agent for promoting crop resistance to heavy metal cadmium 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.

[0143] 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.

[0144] This embodiment provides a method for preparing the above product, the steps are:

[0145] (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 7.00, 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.

[0146] (2) Bacterial agent culture: Bacillus licheniformis, Bacillus subtilis, Pseudomonas fluorescens, and Azotobacter chlororaphis 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 26%, Bacillus subtilis 34%, Pseudomonas fluorescens 22%, and Azotobacter chlororaphis 18%. The bacterial agent cells were obtained by centrifugation at 4000 rpm for 5 min.

[0147] (3) Add strain protective agent and antioxidant to the composite bacterial agent. The formula of strain protective agent is: 29% glycerol, 18% polyethylene glycol, 10% peptone, 5% Tween, 22% trehalose, 12% anhydrous sodium acetate, 2% dipotassium hydrogen phosphate, 1.50% magnesium sulfate and 0.50% manganese sulfate; the formula of 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, 0.50% gallic acid powder and 0.50% superoxide dismutase SOD. The mass ratio of bacterial agent protective agent, antioxidant and bacterial agent bacterial body is 1:3:28.

[0148] (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 cadmium-resistant bacterial agent lignocellulose decomposing bacterial powder.

[0149] (5) Use of desiccant: The mass ratio of silica gel desiccant to bacterial cells is 1-3:100.

[0150] (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.

[0151] This example measures the shelf life of the above products, and the shelf life under different storage conditions is as follows:

[0152] Refrigerated storage (4°C):

[0153] 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.

[0154] Storage at room temperature (about 25°C, light-free, dry environment):

[0155] 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.

[0156] High temperature storage (>35°C, high humidity environment):

[0157] 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.

[0158] Innovation Advantage Analysis

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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 heavy metal cadmium, characterized in that: The effective live bacteria in the composite bacterial agent include Bacillus licheniformis with a cell content of 24-41%, Bacillus subtilis with a cell content of 30-46%, Pseudomonas fluorescens with a cell content of 16-32% and Azotobacter nitrogen-fixing from brown spherical with a cell content of 14-25%.

2. The composite bacterial agent for promoting crop resistance to heavy metal cadmium according to claim 1, characterized in that: 25-38% of Bacillus licheniformis, 32-40% of Bacillus subtilis, 18-28% of Pseudomonas fluorescens, and 15-22% of Azotobacter nitrofugae; Preferably, in the present invention, the effective live bacteria in the composite bacterial agent include Bacillus licheniformis with a cell content of 26±1%, Bacillus subtilis with a cell content of 34±1%, Pseudomonas fluorescens with a cell content of 22±1% and Azotobacter nitrofuga with a cell content of 18±1%.

3. The composite bacterial agent for promoting crop resistance to heavy metal cadmium 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 heavy metal cadmium 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 heavy metal cadmium 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 0.50-7%; 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 0.20-4%; And / or, the mass percentage of magnesium sulfate in the bacterial agent protective agent is 0.05-1.50%; And / or, the mass percentage of manganese sulfate in the bacterial agent protective agent is 0.05-0.60%.

6. The composite bacterial agent for promoting crop resistance to heavy metal cadmium 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 0.50-6.00%; and / or, the mass percentage of clove crumbs in the antioxidant is 0.50-6.00%; and / or, the mass percentage of Wufuzi powder in the antioxidant is 0.10-2.00%; and / or, the mass percentage of gallic acid powder in the antioxidant is 0.01-1.00%; And / or, the mass percentage of superoxide dismutase SOD in the antioxidant is 0.01-1.00%.

7. The composite bacterial agent for promoting crop resistance to heavy metal cadmium 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±0.50% Tween, 22±2% trehalose, 12±1% anhydrous sodium acetate, 2±0.20% dipotassium hydrogen phosphate, 1.50±0.20% magnesium sulfate and 0.50±0.10% 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±0.30% liquorice scraps, 3±0.30% clove scraps, 1±0.10% aconite powder, 0.50±0.10% gallic acid powder and 0.50±0.10% superoxide dismutase SOD.

8. The composite bacterial agent for promoting crop resistance to heavy metal cadmium 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 heavy metal cadmium 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 heavy metal cadmium; Preferably: the crop includes corn; Preferably, the performance of resistance to heavy metal cadmium includes at least one of the following: (1) Improve the cadmium resistance of crops under cadmium stress; (2) Improve crop energy conversion efficiency under cadmium stress; (3) Improve the antioxidant enzyme activity of crops under cadmium stress; (4) Improve crop resistance to cadmium stress; (5) Improving at least one of the following parameters of crops under cadmium 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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