Growth-promoting drought-resisting silicon-decomposing complex microbial inoculant as well as preparation method and application thereof

Abstract

The invention discloses a growth-promoting, drought-resisting and silicon-decomposing complex microbial inoculant as well as a preparation method and application thereof, and belongs to the field of agricultural microbial preparations, the complex microbial inoculant is prepared from kluyvera medians L5N1, pseudomonas lini S2-1 and bacillus mucilaginosus JH3, the mass ratio of the L5N1 to the S2-1 to the JH3 is (1-3): (1-3): (1-5), and the mass ratio of the L5N1 to the S2-1 to the JH3 is (1-3): (1-5). The complex microbial inoculant can be loaded by adopting an organic fertilizer and / or attapulgite, and is used for promoting growth, resisting drought and decomposing silicon of the tobaccos; according to the invention, the silicon dissolving function, ACC deaminase production capacity, IAA production capacity, siderophore production capacity and phosphate solubilizing capacity of microorganisms are synergistically acted on plants, and the three microorganisms are used for jointly assisting the plants in resisting drought stress and promoting the growth of the plants.

Description

Technical Field

[0001] The present invention relates to the field of agricultural microbial preparations, and in particular to a growth-promoting, drought-resistant, and silicon-dissolving composite bacterial agent, and a preparation method and application thereof. Background Art

[0002] As an important cash crop, tobacco is an important industrial pillar of my country's tax revenue. Tobacco cultivation in southwest my country has a long history and is currently one of the largest tobacco leaf producing areas. However, in the past decade, frequent droughts in the southwestern tobacco area have led to ecological damage and serious economic losses. After tobacco suffers from drought stress, the absorption of soil nutrients by tobacco plants is limited, resulting in obvious potassium deficiency, malnutrition of tobacco plants, and development stunted. Drought can cause a large accumulation of reactive oxygen species (ROS). Generally speaking, plants have inherent natural physiological and molecular defense mechanisms, the most important of which is the antioxidant enzyme system, which plays a key role in removing ROS. However, under continuous drought stress, the plant's own drought resistance system is suppressed, and various organs cannot work normally. The middle and upper tobacco leaves often have thick epidermis, compact structure, and low water content. This type of tobacco leaves is difficult to turn yellow and difficult to dehydrate, the yield of green tobacco increases, and the output value of high-quality tobacco leaves decreases. Therefore, enhancing the drought resistance of tobacco plants, improving the absorption and utilization of nutrients in the soil by tobacco under drought stress, increasing the yield of dryland tobacco plants, and ensuring the quality of tobacco leaves are key issues that need to be urgently solved in the current tobacco planting process.

[0003] In order to improve the drought resistance of tobacco, many explorations have been carried out in this field. At present, the main technical means to improve the drought resistance of tobacco include the selection and breeding of excellent drought-resistant tobacco varieties and the addition of chemical water retaining agents during the planting process. However, the selected drought-resistant tobacco varieties are often difficult to achieve the preservation of both variety drought resistance and quality improvement. At the same time, plant breeding has problems such as long breeding cycle, unstable performance maintenance, and high investment; and the application of water retaining agents such as acrylates and cellulose substances often easily destroys the soil structure and affects the efficiency of plant nutrient utilization. Plants and microorganisms maintain a close connection during drought. Plants transmit signals to microorganisms through small molecule compounds secreted by the rhizosphere, and are selectively metabolized by rhizosphere microorganisms, thereby shaping the structure of their own root microbial community. The use of plant-microorganism symbiotic system to solve drought stress problems is effective. Microorganisms promote the development and elongation of the root system by producing plant growth hormones. Microorganisms can also dissolve insoluble phosphorus, potassium, and silicon in the soil. While improving the drought resistance of plants, they can increase the potassium ion content of tobacco leaves and reduce the starch content, improve tobacco quality, and increase tobacco yield.

[0004] Plant rhizosphere microorganisms play an important role in plant drought resistance. Plant growth-promoting rhizobacteria (PGPR) can establish beneficial connections with plants, activate unavailable nitrogen, phosphorus, potassium and other macronutrients in the soil, assist plants in efficiently utilizing nutrient resources, promote plant growth and increase crop yields. On the other hand, PGPR can induce plants to produce systemic resistance to biotic and abiotic stresses and improve their ability to resist adversity. The identified PGPR strains include Bacillus, Pseudomonas, Klyvera, Azotobacter, Enterobacter and Arthrobacter. However, the existing reports mainly focus on microorganisms promoting plant growth and resisting biotic stresses such as pests and diseases. Most PGPR strains have weak survival ability in drought environments, and the shelf life of liquid microbial agents is short. There are few reports on microbial strains that can simultaneously improve plant growth promotion and drought resistance.

[0005] Therefore, there is an urgent need in the art for a microbial preparation that can effectively enhance the drought resistance of plants. Summary of the invention

[0006] One of the purposes of the present invention is to provide a growth-promoting, drought-resistant and silicon-dissolving composite bacterial agent to solve the above-mentioned problems.

[0007] In order to achieve the above-mentioned purpose, the technical solution adopted by the present invention is as follows: a growth-promoting, drought-resistant, silicon-dissolving composite bacterial agent, the composite bacterial agent is composed of Klyvera intermedia L5N1, Pseudomonas lini S2-1 and Bacillus mucilaginosus subsp. siliceus JH3, wherein the mass ratio of L5N1, S2-1 and JH3 is 1-3:1-3:1-5.

[0008] As a preferred technical solution, the effective viable bacteria count of the composite bacterial agent is 10 8 cfu / mL~10 9 cfu / mL.

[0009] The growth-promoting, drought-resistant and silicon-dissolving composite bacterial agent of the present invention can be applied by root irrigation, leaf spraying or combined application of root irrigation and leaf spraying, and can also be applied in combination with improved mineral organic fertilizer.

[0010] As a preferred technical solution, the Kluyvera intermedia L5N1 was deposited in the China Center for Type Culture Collection on April 29, 2022, with a deposit number of CCTCC NO: M 2022531 and a NCBI number of ON533881; Pseudomonas lini S2-1 was deposited in the China Center for Type Culture Collection on April 29, 2022, with a deposit number of CCTCC NO: M 2022530 and a NCBI number of ON533882; Bacillus mucilaginosus subsp. siliceus JH3 was deposited in the China General Microbiological Culture Collection Center on March 9, 2023, with a deposit number of CGMCC No.26781 and a NCBI number of SRR27875362.

[0011] The second object of the present invention is to provide a method for preparing the composite bacterial agent mentioned above, the technical scheme adopted is to expand and culture the strains L5N1, S2-1, and JH3 in the corresponding expansion medium, and then compound them according to the mass ratio of 1-3:1-3:1-5, and the effective viable count is 10 8 cfu / mL~10 9 cfu / mL, that’s it.

[0012] The third object of the present invention is to provide the use of the above-mentioned composite bacterial agent in the preparation of a biological agent for promoting tobacco growth, resisting drought and dissolving silicon.

[0013] As a preferred technical solution, the composite bacterial agent is loaded with organic fertilizer, and the organic fertilizer and the composite bacterial agent are compounded in a mass ratio of 80% to 90%: 10% to 20% to prepare a biological organic fertilizer.

[0014] Organic fertilizer as a carrier can provide microorganisms with the necessary nutrients and living space, helping them survive better in adverse conditions.

[0015] The specific preparation method can be: after mixing the organic fertilizer with the composite bacterial agent, place it in a ventilated, dry and clean fermentation room for secondary fermentation, ferment at room temperature for 5-7 days, turn it over every 18 hours, and keep the moisture content less than 30wt% and the number of viable bacteria>2×10 7 Pieces / gram.

[0016] As a preferred technical solution, attapulgite is used to load the composite bacterial agent, and the attapulgite and the composite bacterial agent are compounded in a mass ratio of 80% to 90%: 10% to 20% to prepare a mineral biological fertilizer.

[0017] Attapulgite can provide more nutrients for microorganisms. Its loose and porous structure will carry more microorganisms and can improve the soil and prevent soil compaction.

[0018] The specific preparation method can be: after compounding the attapulgite with the composite bacterial agent, place it in a ventilated, dry and clean fermentation room for secondary fermentation, ferment at room temperature for 5-7 days, turn it over every 18 hours, and keep the moisture content less than 30wt% and the number of viable bacteria>2×10 7 Pieces / gram.

[0019] As a preferred technical solution, attapulgite and organic fertilizer are used to jointly load the composite bacterial agent. The attapulgite and organic fertilizer are first mixed in a mass ratio of 1-2:4-39 to obtain a mixed carrier, and then the mixed carrier and the composite bacterial agent are compounded in a mass ratio of 80%-90%:10%-20% to prepare an improved mineral biological fertilizer.

[0020] Combining the advantages of organic fertilizer and attapulgite, attapulgite and organic fertilizer can provide more nutrients for microorganisms, improve soil and avoid soil compaction.

[0021] The specific preparation method can be: after compounding attapulgite and organic fertilizer with microbial agent, place them in a ventilated and dry place for secondary fermentation for 5-7 days, the temperature should be maintained at 30-35℃, turn them over every 18 hours, and after fermentation, keep the moisture content less than 30%, and the number of viable bacteria>2×10 7 Pieces / gram.

[0022] The invention utilizes the growth-promoting and drought-resistant characteristics of three kinds of microorganisms to improve the drought resistance of tobacco and promote tobacco growth; provides more nutrients to plants through the microorganisms' efficient production of IAA, ACC deaminase, phosphorus dissolving and silicon dissolving capabilities, promotes tobacco growth, and assists tobacco plants in resisting drought stress. The invention utilizes the characteristics of microorganisms in producing plant hormones IAA, ACC deaminase and iron carriers to alleviate tobacco drought stress and promote plant growth in arid environments; microorganisms dissolve effective phosphorus in minerals to promote plant absorption of phosphorus and promote plant growth; and effective silicon dissolved by microorganisms provides a soluble silicon source for plants in arid environments.

[0023] Specifically, the mucilaginous Bacillus of the present invention has the ability to dissolve silicon. The mucilaginous Bacillus can produce organic acids such as malic acid and citric acid, which can dissolve insoluble silicon into soluble silicon for plant absorption. The application of exogenous silicon can improve the photosynthesis, antioxidant and osmotic regulation of tobacco under drought stress, alleviate the damage of drought to tobacco seedlings, and promote its growth. Silicon can stimulate the formation of a double-layer silicon dioxide cuticle in the epidermis of plant leaves, thereby reducing the water loss of leaves. At the same time, silicon can be deposited on the cell wall, maintaining the characteristics of the cell wall and guard cells under drought stress, ensuring the normal operation of plant photosynthesis, and helping plants resist drought.

[0024] Compared with the prior art, the advantages of the present invention are:

[0025] (1) The composite microbial agent of the present invention has strong growth-promoting and drought-resistant capabilities: Klyvera intermedia L5N1, Pseudomonas lini S2-1 and Paenibacillus lini JH3 are constructed into a composite preparation, which combines the capabilities of efficient phosphorus solubilization, silicon solubilization, IAA production, ACC deaminase and iron carrier. Silicon-solubilizing bacteria JH3 is used with microorganisms with high IAA, ACC, iron carrier and strong phosphorus solubilization to construct a silicon-solubilizing and drought-resistant microbial biological preparation: Klyvera intermedia L5N1 and Pseudomonas lini S2-1 are compounded to construct a drought-resistant, growth-promoting and silicon-solubilizing microbial biological preparation. Among them, L5N1 has the highest indoleacetic acid production and the strongest phosphorus solubilizing ability. The indoleacetic acid production of L5N1 is about 45.23 mg / L, and the phosphorus solubilizing ability is 0.62 g / L; S2-1 has the highest ability to produce siderophores and ACC deaminase, with the ability to produce siderophores of 0.51±0.01 g / L and the ability to produce ACC deaminase of 0.22±0.0110 g / L. IAA endogenous auxin produced by microorganisms can regulate plant growth and development and improve plant stress resistance, and also play a role as a signal in regulating plant-microorganism interactions; ACC deaminase can reduce the level of crop stress ethylene under drought conditions and improve the drought resistance of crops; siderophores can provide Fe elements for bacteria themselves, and can also provide nutrients for plants to promote plant growth; the phosphorus solubilizing ability of microorganisms helps plants get rid of nutritional restrictions under drought stress and enhance the tolerance of tobacco plants under drought conditions. Soluble silicon dissolved by microorganisms can help plants resist drought adversity, increase the content of photosynthetic pigments in leaves, enhance the activity of antioxidant enzymes, and reduce the content of malondialdehyde;

[0026] (2) The present invention selects materials suitable for being carriers of composite bacterial agents: In the present invention, organic fertilizer can be used as a microbial carrier, and functional microorganisms can be attached to organic fertilizers from various sources to prepare a new type of biological organic fertilizer, thereby enhancing the activity of growth-promoting and drought-resistant microorganisms, promoting tobacco growth, improving tobacco quality, enhancing drought resistance, and inhibiting the growth of harmful microorganisms in the soil; using attapulgite as a microbial carrier, functional microorganisms can be attached to attapulgite to prepare a new type of mineral biological bacterial fertilizer, maintaining the activity of microorganisms and providing nutrients such as potassium, phosphorus, and silicon, improving the soil, alleviating soil compaction, and promoting tobacco growth; using organic fertilizer and attapulgite as microbial carriers, a new type of mineral biological organic fertilizer can be prepared, thereby enhancing the activity of growth-promoting and drought-resistant microorganisms, providing nutrients, improving the soil, alleviating soil compaction, enhancing drought resistance, promoting tobacco growth, and inhibiting the growth of harmful microorganisms;

[0027] (3) The biological agent of the present invention can be applied by a variety of methods. For example, the composite bacterial agent can be applied by root irrigation, leaf spraying, or root irrigation and leaf spraying at the same time. The bacterial agent of the present invention can be used in multiple forms and multiple functions to help plants resist drought stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a graph showing the changes in high-quality tobacco leaves and total yields in field trials using different liquid bacterial agents in Example 1 of the present invention;

[0029] Figure 2 This is a photo of different liquid bacterial agents used in potted plant experiments in Example 2 of the present invention with water cutoff for 15 consecutive days;

[0030] Figure 3 This is a diagram showing changes in the enzyme system and reducing sugar content of the potted tobacco plants after the composite bacterial community JCL was applied to the potted tobacco plants in Example 2 of the present invention;

[0031] Figure 4 This is a diagram showing the effect of applying functional microorganism-loaded organic fertilizer to potted tobacco plants to promote tobacco plant growth in Example 3 of the present invention;

[0032] Figure 5 This is a diagram showing changes in the enzyme system and reducing sugar content of the potted tobacco plants after the functional microorganisms loaded with attapulgite were applied to the potted tobacco plants in Example 4 of the present invention;

[0033] Figure 6 This is a diagram showing the yield of tobacco leaves at different parts after the composite bacterial agent is applied in the field in Example 5 of the present invention;

[0034] Figure 7 This is a diagram showing the growth of tobacco plants after applying the improved mineral bio-organic fertilizer in Example 6 of the present invention;

[0035] Figure 8 This is the effect of different application methods on plant height in Example 7 of the present invention. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with the accompanying drawings.

[0037] In the following embodiments, the three strains were donated by Southwest Jiaotong University, and the three strains have been deposited separately: Klyvera intermedia L5N1 was deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2022531; Pseudomonas lini S2-1 was deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2022530; Bacillus mucilaginosus subsp. siliceus JH3 was deposited in China Center for General Microbiological Culture Collection with a deposit number of CGMCC No.26781.

[0038] Embodiment 1:

[0039] (1) Preparation of composite bacterial agent:

[0040] L5N1 and S2-1 were inoculated into LB medium at 30-35°C and 150 r / min respectively. After 24 h of culture, the number of viable bacteria reached 10 7 ~10 8 cfu / mL; JH3 was inoculated in nitrogen-free liquid culture medium, cultured at 30-35°C and 180 r / min for 36 h, and the number of viable bacteria was about 10 7 cfu / mL~10 8 cfu / mL, to obtain seed solution;

[0041] JH3, L5N1, and S2-1 were compounded in a mass ratio of 1:1:1 to obtain the silicon-dissolving growth-promoting and drought-resistant biological agent JCL;

[0042] L5N1 and S2-1 were prepared into the bacterial agent LS in a ratio of 1:1;

[0043] (2) Field trials:

[0044] In order to explore the application effects of the composite bacterial agent JCL, the single bacterial agent JH3, the two strains LS (all three were experimental groups) and the control group (CK group, i.e., no bacterial agent was applied) prepared in this example, the yield and total yield of high-quality tobacco leaves under different treatments were compared and analyzed.

[0045] The specific method is: after transplanting tobacco, the fungi JH3, LS, and JCL are applied by root irrigation. The tobacco leaves are picked from the middle and upper parts during maturity and flue-cured tobacco is made. By measuring the fresh weight and dry weight of tobacco leaves in different parts, it was found that the fresh weight and dry weight of each part of the experimental group with the application of the fungi were significantly higher than those of the control group, and the number of upper tobacco leaves in the experimental group was significantly higher than that of the control group, and the upper tobacco leaves were more likely to produce high-quality tobacco leaves. After applying the composite fungi JCL in the field, under drought conditions, the total yield of the application of the fungi JCL was increased by 31.17%, 15.98%, and 1.65% compared with CK, the application of the LS fungi, and the JH3 fungi, respectively (such as Figure 1 As shown). After applying JCL, the yield of high-quality tobacco leaves increased by 47.58%, 33.74% and 14.83% compared with CK, LS and JH3 respectively; this proves that the three bacteria of the present invention synergize with each other, rather than simply superimposing the three.

[0046] Example 2

[0047] (1) Preparation of composite bacterial agent:

[0048] L5N1 and S2-1 were inoculated into LB medium at 30-35°C and 150 r / min respectively. The culture time was 24 h until the number of viable bacteria reached 10 7 ~10 8 cfu / mL; JH3 was inoculated in a liquid culture medium with silicon-dissolving nitrogen-free bacteria and cultured at a temperature of 30-35°C and a rotation speed of 180 r / min. After 36 h of culture, the number of viable bacteria was about 10 7 cfu / mL~10 8 cfu / mL, to obtain seed solution,

[0049] L5N1, S2-1 and JH3 were compounded in a mass ratio of 1:1:3 to obtain the silicon-dissolving growth-promoting and drought-resistant biological agent JCL;

[0050] L5N1 and S2-1 were compounded in a mass ratio of 1:1 to obtain LS.

[0051] (2) Potted plant experiment

[0052] In order to explore the application effects of the composite bacterial agent JCL, the single bacterial agent JH3, the two strains of bacteria LS (all three are experimental groups) and the control group (CK group, i.e., no bacterial agent is applied except normal base fertilizer) prepared in this example, a potted plant experiment was conducted:

[0053] The compound microbial agent was added when the seedlings were transplanted; each pot (21 cm * 19 cm * 17 cm) was loaded with 3.5 kg of soil in the pot experiment; the functional microbial agent was applied by root irrigation;

[0054] Drought was simulated by continuous drought for 15 days. The growth of plants during the experiment was shown in Figure 2 The effect of applying the bacterial agent JCL and the bacterial agent JH3 was better. After 15 days of water shortage, the leaves were still green and firm. After 15 days of continuous water shortage, the POD content in the tobacco fresh leaves increased by 241.51% ( Figure 3 a); CAT content in fresh tobacco leaves increased by 233.33% compared with the control group ( Figure 3 b); MDA in fresh tobacco leaves was 39.58% lower than that in the control group ( Figure 3 d); the reducing sugar content increased by 0.64% compared with the control group ( Figure 3 c).

[0055] Example 3

[0056] Preparation and potted experiment of silicon-dissolving growth-promoting and drought-resistant biological organic fertilizer

[0057] (1) Preparation of silicon-dissolving growth-promoting and drought-resistant biological organic fertilizer:

[0058] First, prepare the seed solution: inoculate L5N1 and S2-1 into LB medium respectively, at a temperature of 30-35°C, a rotation speed of 150 r / min, and culture for 24 h until the number of viable bacteria reaches 10 7 ~10 8 cfu / mL; JH3 was inoculated in a liquid culture medium with silicon-dissolving nitrogen-free bacteria and cultured at a temperature of 30-35°C and a rotation speed of 180 r / min. After 36 h of culture, the number of viable bacteria was about 10 7 cfu / mL~10 8 cfu / mL, to obtain seed solution;

[0059] Then, L5N1, S2-1 and JH3 were compounded in a ratio of 3:1:5 to obtain a composite bacterial agent; and organic fertilizer and the composite bacterial agent were compounded in a mass ratio of 9:1 and placed in a ventilated, dry and clean fermentation room for fermentation at room temperature for 5-7 days. The humidity should not be less than 30%, and the fermentation should be turned over every 18 hours. After fermentation, the moisture should be kept less than 30%, and the number of viable bacteria should be >2×10 7 cfu / g, and obtain silicon-dissolving, growth-promoting and drought-resistant biological organic fertilizer.

[0060] (2) Potted plant experiment:

[0061] Add when transplanting seedlings. In potted experiments, each pot (21 cm * 19 cm * 17 cm) was loaded with 3.5 kg of soil;

[0062] CK is a blank control group, OF is a conventional experimental group in which only organic fertilizer (the organic fertilizer in this embodiment is sheep manure organic fertilizer) is applied without compounding the composite bacterial agent (50 g of biological organic fertilizer is applied in the tobacco pit before transplanting the tobacco seedlings), and OF-JCL is a silicon-dissolving growth-promoting and drought-resistant biological organic fertilizer prepared in this embodiment (50 g is applied in the tobacco pit before transplanting the tobacco seedlings);

[0063] After 15 days of continuous water shortage, the results were as follows Figure 4 As shown. When the functional strain used in the present invention is applied, the fresh weight of tobacco leaves under drought stress is increased by 37.29% compared with the control group ( Figure 4 c); The dry weight of tobacco leaves increased by 110.74% compared with the control group ( Figure 4 a); The tobacco leaf area increased by 140.99% compared with the control group ( Figure 4 d); The stem diameter of tobacco plants increased by 50% compared with the control group ( Figure 4 b).

[0064] Example 4

[0065] Potted experiment of mineral biological fertilizer

[0066] (1) Preparation of mineral biological fertilizer:

[0067] First, prepare the seed solution: inoculate L5N1 and S2-1 into LB medium respectively, at a temperature of 30-35°C, a rotation speed of 150 r / min, and culture for 24 h until the number of viable bacteria reaches 10 7 ~10 8 cfu / mL; JH3 was inoculated in a liquid culture medium with silicon-dissolving nitrogen-free bacteria and cultured at a temperature of 30-35°C and a rotation speed of 180 r / min. After 36 h of culture, the number of viable bacteria was about 10 7 cfu / mL~10 8 cfu / mL, to obtain seed solution,

[0068] Then, L5N1, S2-1 and JH3 were compounded in a ratio of 2:2:3 to obtain a composite bacterial agent; then, attapulgite (attapulgite in this embodiment was purchased from Hengyuan New Materials) and the composite bacterial agent were configured in a mass ratio of 8.5:1.5, and placed in a ventilated and dry place for room temperature fermentation for 5-7 days, turning over every 18 hours, and keeping the moisture content less than 30% after fermentation, and the number of viable bacteria>10 7 cfu / g

[0069] (2) Pot experiment: Add soil when transplanting seedlings. In the pot experiment, each pot (21 cm * 19 cm * 17 cm) was loaded with 3.5 kg of soil. The AT-JCL group was added with 50 g of the mineral bio-organic fertilizer prepared in this example, the CK was the blank control group, and the AT group was added with only 50 g of attapulgite. The results are shown in Figure 5As shown, after the application of the functional strain used in the present invention, the POD content in the tobacco fresh leaves was increased by 279.25% ( Figure 5 a); The CAT content in fresh tobacco leaves increased by 281.48% compared with the control group ( Figure 5 c); MDA in fresh tobacco leaves was 50.13% lower than that in the control group ( Figure 5 b); the reducing sugar content increased by 5.18% compared with the control group ( Figure 5 d);

[0070] It is proved that the tobacco growth-promoting and drought-resistant functional microorganisms developed in this example can help tobacco resist drought stress and reduce the damage caused by drought to plants by increasing the activity of antioxidant enzymes.

[0071] Example 5

[0072] Field test of compound microbial agent

[0073] (1) Preparation of composite bacterial agent:

[0074] First, prepare the seed solution: inoculate L5N1 and S2-1 into LB medium respectively, at a temperature of 30-35°C, a rotation speed of 150 r / min, and culture for 24 h until the number of viable bacteria reaches 10 7 ~10 8 cfu / mL; JH3 was inoculated in a liquid culture medium with silicon-dissolving nitrogen-free bacteria and cultured at a temperature of 30-35°C and a rotation speed of 180 r / min. After 36 h of culture, the number of viable bacteria was about 10 7 cfu / mL~10 8 cfu / mL, to obtain seed solution;

[0075] Then L5N1, S2-1 and JH3 were compounded in a mass ratio of 3:3:5 to obtain the composite bacterial agent JCL;

[0076] (2) Field trials:

[0077] The composite bacterial agent was diluted 50 times by volume with tap water, and applied to the roots at a ratio of 50 mL / plant / seedling, and the same tap water was added as a control (CK) for field testing;

[0078] After tobacco was transplanted, the fungus JCL was applied by root irrigation. The tobacco leaves were picked at the middle and upper parts during the maturity period and made into flue-cured tobacco. Figure 6 As shown in the figure, the yield of lower tobacco leaves increased by 76.24% compared with the control group; the yield of middle tobacco leaves increased by 118.44% compared with the control group; and the yield of upper tobacco leaves increased by 104.02%. After adding functional microorganisms, the yield of middle and upper tobacco leaves was significantly increased, which helped to improve the quality of tobacco leaves and increase tobacco yield.

[0079] Example 6

[0080] Field application test of improved mineral bio-organic fertilizer

[0081] (1) Preparation of improved mineral bio-organic fertilizer:

[0082] First, prepare the seed solution: inoculate L5N1 and S2-1 into LB medium respectively, at a temperature of 30-35°C, a rotation speed of 150 r / min, and culture for 24 h until the number of viable bacteria reaches 10 7 ~10 8 cfu / mL; JH3 was inoculated in a liquid culture medium with silicon-dissolving nitrogen-free bacteria and cultured at a temperature of 30-35°C and a rotation speed of 180 r / min. After 36 h of culture, the number of viable bacteria was about 10 7 cfu / mL~10 8 cfu / mL, to obtain seed solution;

[0083] Then, L5N1, S2-1 and JH3 were compounded in a ratio of 2:2:3 to obtain the composite bacterial agent JCL; then, attapulgite and organic fertilizer were mixed in a mass ratio of 1:39, and then the organic fertilizer-attapulgite mixture was loaded with the composite bacterial agent (compounded in a mass ratio of organic fertilizer-attapulgite: composite bacterial agent = 8:2), and placed in a ventilated and dry place for fermentation at room temperature for 5-7 days, turned over every 18 hours, and after fermentation, the moisture was kept less than 30%, and the number of effective live bacteria was>10 7 cfu / g, and the improved mineral bio-organic fertilizer OF-AT-JCL was obtained;

[0084] (2) Field application test:

[0085] After the tobacco was transplanted, the composite bacterial agent JCL prepared in step (1) of this embodiment was applied by root irrigation. The tobacco leaves were picked at the middle and upper parts at maturity and flue-cured tobacco was produced. Before the tobacco seedlings were transplanted, 50 g of the improved mineral bio-organic fertilizer prepared in step (1) of this embodiment was applied to the tobacco nest. A blank control group CK without adding the composite bacterial agent and the improved mineral bio-organic fertilizer was used. The preliminary results of agronomic traits during the barley period are as follows: Figure 7 As shown, the results showed that the stem diameter of the group treated with OF-AT-JCL increased by 76.18% compared with CK; the stem height increased by 49.72% compared with CK, and the maximum leaf area increased by 130.01% compared with CK.

[0086] Example 7

[0087] Experiment on the influence of different application methods on the application effect of compound bacterial agents

[0088] (1) Preparation of composite bacterial agent:

[0089] First, prepare the seed solution: inoculate L5N1 and S2-1 into LB medium respectively, at a temperature of 30-35°C, a rotation speed of 150 r / min, and culture for 24 h until the number of viable bacteria reaches 10 7 ~10 8 cfu / mL; JH3 was inoculated in a liquid culture medium with silicon-dissolving nitrogen-free bacteria and cultured at a temperature of 30-35°C and a rotation speed of 180 r / min. After 36 h of culture, the number of viable bacteria was about 10 7 cfu / mL~10 8 cfu / mL, to obtain seed solution;

[0090] Then L5N1, S2-1 and JH3 were compounded in a mass ratio of 1:1:3 to obtain the drought-resistant and growth-promoting composite bacterial agent JCL;

[0091] (2) Field trials:

[0092] The compound bacterial agent was diluted 50 times in volume and applied to the roots at a ratio of 50 mL / plant. The same tap water was added as a control. Field tests were conducted using foliar spray-JCL, root irrigation-JCL, foliar spray-JCL+root irrigation-JCL, and foliar spray-JCL+OF-JCL. The plant heights of plants with different application methods during growth were measured ( Figure 8 ), the results showed that the plant height of the plant using only leaf spray of compound microbial agent was 27.55% higher than that of the CK blank control; the plant height of the plant using only root irrigation of compound microbial agent was 15.59% higher than that of the CK blank control; the plant height of the plant using leaf spray and root irrigation of compound microbial agent was 36.36% higher than that of the CK blank control; and the plant height after leaf spray of compound microbial agent and biological organic fertilizer was 93.20% higher than that of the CK blank control.

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A growth-promoting, drought-resistant and silicon-dissolving composite bacterial agent, characterized in that: The composite bacterial agent consists of Klyvera intermedia L5N1, Pseudomonas lini S2-1 and Bacillus mucilaginosus subsp. siliceus JH3, wherein the mass ratio of L5N1, S2-1 and JH3 is 1-3:1-3:1-5.

2. The growth-promoting, drought-resistant and silicon-dissolving composite bacterial agent according to claim 1, characterized in that: The effective viable bacteria count of the composite bacterial agent is 10 8 cfu / mL~10 9 cfu / mL.

3. The growth-promoting, drought-resistant and silicon-dissolving composite bacterial agent according to claim 1, characterized in that: The Kluyvera intermedia L5N1 was deposited in the China Center for Type Culture Collection on April 29, 2022, with a deposit number of CCTCC NO: M 2022531 and a NCBI number of ON533881; Pseudomonas lini S2-1 was deposited in the China Center for Type Culture Collection on April 29, 2022, with a deposit number of CCTCC NO: M2022530 and a NCBI number of ON533882; Bacillus mucilaginosussubsp.siliceus JH3 was deposited in the China General Microbiological Culture Collection Center on March 9, 2023, with a deposit number of CGMCC No.26781 and a NCBI number of SRR27875362.

4. The method for preparing the composite bacterial agent according to claim 1, 2 or 3, characterized in that: The strains L5N1, S2-1, and JH3 were expanded and cultured in the corresponding expansion medium, and then compounded in a mass ratio of 1-3:1-3:1-5, and the effective viable count was 10 8 cfu / mL~10 9 cfu / mL, that’s it.

5. Use of the composite bacterial agent according to claim 1, 2 or 3 in the preparation of a biological agent for promoting tobacco growth, resisting drought and dissolving silicon.

6. The use according to claim 5, characterized in that: The composite bacterial agent is loaded with organic fertilizer, and the organic fertilizer and the composite bacterial agent are compounded in a mass ratio of 80% to 90%: 10% to 20% to prepare a biological organic fertilizer.

7. The use according to claim 5, characterized in that: The composite bacterial agent is loaded on attapulgite, and the attapulgite and the composite bacterial agent are compounded in a mass ratio of 80% to 90%: 10% to 20% to prepare a mineral biological bacterial fertilizer.

8. The use according to claim 5, characterized in that: The composite bacterial agent is loaded with attapulgite and organic fertilizer. The attapulgite and organic fertilizer are first mixed in a mass ratio of 1-2:4-39 to obtain a mixed carrier. The mixed carrier is then compounded with the composite bacterial agent in a mass ratio of 80%-90%:10%-20% to prepare an improved mineral biological bacterial fertilizer.

9. The use according to claim 5, characterized in that: The composite microbial agent is used by foliar spraying, root irrigation, or a combination of foliar spraying and root irrigation.

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