Paenibacillus mucilaginosus as well as related products and application thereof

Abstract

The invention discloses paenibacillus mucilaginosus as well as a related product and application thereof, and relates to the field of microorganisms. The paenibacillus mucilaginosus is a strain JH3, has high silicon dissolving capacity which can reach 44.71-47.87 mu g / mL, has multiple application modes, can be applied to agricultural production, can improve the drought resistance of plants, can promote plant growth, can reduce agricultural cost, and is green and environment-friendly.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a Paenibacillus subtilis and related products and applications thereof. Background Art

[0002] As a high-value-added economic crop, tobacco is usually grown in mountainous areas with limited water supply and is susceptible to drought. Drought has a serious impact on the entire life cycle of tobacco. Drought will inhibit seed germination and emergence during tobacco seedling cultivation, affecting later transplanting and planting; during the tobacco growth stage, drought affects tobacco tillering and root development, resulting in short tobacco plants, fewer leaves per plant, and smaller leaf area, reducing the photosynthetic capacity and stress resistance of tobacco leaves; during the tobacco leaf maturity stage, drought will cause tobacco leaves to mature prematurely, reduce the accumulation of effective chemical components in tobacco leaves, and affect the aroma and taste of tobacco leaves. Although some studies have focused on tobacco drought resistance, they currently focus on tobacco's own response, regulation and adaptation strategies to drought stress, and improve tobacco drought resistance through crop breeding, agronomic measures and chemical additives.

[0003] Microorganisms participate in tobacco's life activities such as water absorption, nutrient metabolism, hormone regulation, and stress defense through complex interactions, and are one of the important factors affecting tobacco's drought resistance. However, few studies have used microorganisms to improve tobacco's drought resistance. The bioavailable silicon in the soil can increase the plant's stress resistance, including resistance to environmental stresses such as drought, high temperature, low temperature, salinity, and heavy metals. However, silicon in the soil mostly exists in the form of insoluble silicates and cannot be directly absorbed and utilized by plants. Symbiosis between plants and microorganisms can promote the dissolution of mineral elements in plant soils. Bacillus mucilaginosus subsp. siliceus is a reported functional microorganism with clear silicon-dissolving activity. However, the main function of this type of strain is currently to dissolve potassium and phosphorus, and it has a certain nitrogen fixation ability. The silicon-dissolving activity of existing strains is low. The silicon-dissolving ability of P.mucilaginosus KN-18 strain (preservation number CCTCC NO.M2014321) in the fermentation broth is 26.8μg / mL, which is the strain with the strongest silicon-dissolving ability in existing research in the agricultural field.

[0004] Currently, the market lacks strains that can effectively improve plants' drought resistance.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a gelatinous bacillus and related products and applications thereof.

[0007] The present invention is achieved in that:

[0008] In a first aspect, an embodiment of the present invention provides a Paenibacillus mucilaginosus JH3, whose taxonomic name is Bacillus mucilaginosus subsp. siliceus and whose deposit number is CGMCC No.26781.

[0009] In a second aspect, an embodiment of the present invention provides a culture obtained by culturing the Paenibacillus subtilis JH3 described in the above embodiment.

[0010] In a third aspect, an embodiment of the present invention provides a biological bacterial agent, which contains the gelatinous Bacillus JH3 described in the above embodiment and / or the culture described in the above embodiment.

[0011] In a fourth aspect, embodiments of the present invention provide use of the Paenibacillus subtilis JH3 described in the preceding embodiments, or the culture described in the preceding embodiments, or the biological agent described in the preceding embodiments in the preparation of a fertilizer or a soil conditioner.

[0012] In a fifth aspect, an embodiment of the present invention provides a fertilizer, comprising: a carrier and the Bacillus subtilis JH3 described in the preceding embodiment or the culture described in the preceding embodiment or the biological agent described in the preceding embodiment; or obtained by fermenting a mixture of the carrier and the Bacillus subtilis JH3 described in the preceding embodiment or the culture described in the preceding embodiment or the biological agent described in the preceding embodiment.

[0013] In a sixth aspect, an embodiment of the present invention provides a soil conditioner, which contains: the Bacillus subtilis JH3 described in the preceding embodiment or the culture described in the preceding embodiment or the biological agent described in the preceding embodiment or the fertilizer described in the preceding embodiment.

[0014] In a seventh aspect, an embodiment of the present invention provides a use of the Bacillus subtilis JH3 described in the preceding embodiments, or the culture described in the preceding embodiments, or the biological agent described in the preceding embodiments, or the fertilizer described in the preceding embodiments, or the soil conditioner described in the preceding embodiments in any of the following: (1) plant planting; (2) promoting plant growth or preparing a product that promotes plant growth; (3) improving plant drought resistance or preparing a product that improves plant drought resistance; (4) soil conditioning.

[0015] In an eighth aspect, an embodiment of the present invention provides a screening culture medium, comprising the following components and final concentrations of the components: 1-10 g / L carbohydrates, 1-5 g / L Na2HPO4, 0.1-1 g / L MgSO4·7H2O, 0.001-0.01 g / L FeCl3, 0.01-0.5 g / L CaCO3, 0.1-5 g / L silicon source and 15-20 g / L agar; wherein the silicon source comprises a silicate soil conditioner.

[0016] In a ninth aspect, an embodiment of the present invention provides the use of the screening medium described in the above embodiment in screening silicon-dissolving bacteria. The present invention has the following beneficial effects:

[0017] (1) A new colloid bacillus JH3 was provided, which had a high silica-dissolving capacity of 44.71-47.87 μg / mL;

[0018] (2) Paenibacillus mucilaginosus JH3 can be used in agriculture to improve the drought resistance of plants;

[0019] (3) Paenibacillus mucilaginosus JH3 can reduce the use of silicon fertilizer, lower agricultural costs, and protect the environment;

[0020] (4) Paenibacillus subtilis JH3 can promote plant growth and improve soil quality.

[0021] (5) Paenibacillus subtilis JH3 can be applied in a variety of ways. It can be directly diluted and applied as root irrigation, or it can be applied to plant leaves by spraying.

[0022] (6) Improving the screening culture medium for silicon-dissolving bacteria shortens the screening cycle of silicon-dissolving bacteria compared to the existing technology, providing a new way to obtain silicon-dissolving bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 The colony morphology of strain JH3 after 48 hours of growth on the screening medium (left), and the observation picture of strain JH3 under a 1200x microscope (right);

[0025] Figure 2 To investigate the silicon-dissolving ability of silicon-dissolving strains on different culture media;

[0026] Figure 3 The bacterial load of different materials;

[0027] Figure 4 Circle map of the genome of strain JH3;

[0028] Figure 5 is the evolutionary tree of strain JH3;

[0029] Figure 6 KEGG pathway analysis for strain JH3;

[0030] Figure 7 For the analysis of the acid production pathway of strain JH3;

[0031] Figure 8 For the secondary metabolite gene cluster analysis of strain JH3;

[0032] Fig. 9 The strain JH3 was used in potted simulated drought experiments;

[0033] Fig.10 Effects of applying JH3 bacterial agent on tobacco plant growth (Note: JH3 is a liquid bacterial fertilizer); (a) Tobacco leaf fresh weight; (b) Tobacco leaf dry weight; (c) Tobacco leaf area; (d) Tobacco plant stem diameter;

[0034] Fig.11 Effects of applying JH3 bacterial agent on the content of available silicon in soil and silicon in plants (Note: JH3 is a liquid bacterial fertilizer); (a) the content of available silicon in soil; (b) the content of silicon in plants;

[0035] Fig.12 Effects of applying JH3 bacterial agent on enzyme activity and osmotic regulating substances of tobacco plants under drought conditions (Note: JH3 is a liquid bacterial fertilizer); (a) Changes in POD content of tobacco leaves during drought stress; (b) Changes in CAT content of tobacco leaves during drought stress; (c) Changes in reducing sugar content of tobacco leaves during drought stress; (d) Changes in MDA content of tobacco leaves during drought stress;

[0036] Fig.13 Effects of applying JH3 bacterial agent on chlorophyll content of tobacco plants (Note: JH3 is liquid bacterial fertilizer); (a) Changes in chlorophyll a content of tobacco leaves during drought stress; (b) Changes in chlorophyll b content of tobacco leaves during drought stress; (c) Changes in chlorophyll a / b content of tobacco leaves during drought stress;

[0037] Fig.14 The effect of applying JH3 bacterial agent on the water content of tobacco plants (Note: JH3 is a liquid bacterial fertilizer); (a) Changes in water content of tobacco leaves during drought stress; (b) Comparison of leaf conditions; (c) Comparison of whole plant conditions;

[0038] Fig.15 Effects of applying JH3 bacterial agent in the field on tobacco plant growth (Note: JH3 is a liquid bacterial fertilizer); (a) Fresh weight of tobacco leaves in each part; (b) Dry weight of tobacco leaves in each part; (c) Biomass of tobacco leaves in each part; (d) Plant height of tobacco leaves in each part;

[0039] Fig.16The effects of applying JH3 bacterial agent and organic fertilizer in the field on tobacco plant growth; (a) Fresh weight of tobacco leaves in each part; (b) Dry weight of tobacco leaves in each part; (c) Biomass of tobacco leaves in each part; (d) Plant height of tobacco leaves in each part; Note: FJ is a solid bacterial fertilizer obtained by combining silicon-dissolving bacteria JH3 with organic fertilizer;

[0040] Fig.17 The effects of applying JH3 bacterial agent and attapulgite soil in the field on the growth of tobacco plants; (a) Fresh weight of tobacco leaves in each part; (b) Dry weight of tobacco leaves in each part; (c) Biomass of tobacco leaves in each part; (d) Plant height of tobacco plants in each part; Note: ATJ is a solid bacterial fertilizer obtained by combining the silicon-dissolving bacteria JH3 with attapulgite;

[0041] Fig.18 Effects of field application of JH3 inoculant combined with attapulgite and organic fertilizer on tobacco plant growth; (a) Fresh weight of tobacco leaves of each part; (b) Dry weight of tobacco leaves of each part; (c) Biomass of tobacco leaves of each part; (d) Plant height of tobacco leaves of each part; Note: FAJ is a solid bacterial fertilizer obtained by combining the silicon-dissolving bacteria JH3 with organic fertilizer and attapulgite. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0043] In response to the problems of the prior art, the present application provides a new colloidal Bacillus JH3, which has excellent silicon-dissolving ability, ranging from 44.71 to 47.87 μg / mL, and can be used in agriculture to improve the drought resistance of plants. Specifically, the soluble silicon produced by JH3 can alleviate the decline in biomass indicators of plants caused by drought environments, promote the accumulation of osmotic regulating substances and photosynthetic pigments, and increase the activity of antioxidant enzymes; the soluble silicon produced by it can also stimulate the formation of a double-layer silica cuticle in the epidermis of plant leaves, thereby reducing leaf water loss; in addition, soluble 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, thereby enhancing the plant's resistance to drought stress.

[0044] Secondly, the application of colloidal Bacillus JH3 can reduce the use of silicon fertilizer, reduce agricultural costs, and protect the environment. At present, the mainstream silicon fertilizer products include calcium silicate and citrate-soluble silicon fertilizer made from industrial waste. The former is cheap but not easily absorbed by plants and has the risk of introducing other heavy metal ions, and the latter is expensive and difficult to produce and promote. The silicon-dissolving bacteria JH3 (Bacillus mucilaginosus subsp. siliceus) with strong silicon-dissolving ability provided by the embodiment of the present invention can not only effectively dissolve the insoluble silicon in the soil, but also promote plant absorption, transport soluble silicon to the stem and leaves through the roots, so that silicified cells are formed in the plant body, and it is enhanced to resist the damage of substances such as malondialdehyde, and the mechanical strength of the stem can be enhanced by the transportation of silicon, and the disease resistance of the plant is improved. Silicon can improve the photosynthetic pigments of plants in drought adversity to promote their growth and development in drought adversity, and improve the drought resistance of plants.

[0045] In addition, the colloidal bacillus JH3 has a variety of application methods, can be directly diluted and root irrigated, or applied to plant foliage spraying, promote plant absorption and utilization of effective silicon; it can also be compounded with a variety of materials (attapulgite, organic fertilizer or its combination) to improve the effect of application, for example, compounding it with organic fertilizer to obtain biological organic fertilizer, which can be used to improve soil, balance soil acid and alkali, promote the growth of beneficial microorganisms in the soil, increase soil enzyme activity, and promote plant growth. Compared with inorganic fertilizers on the market, the organic fertilizer after the JH3 bacterial solution and organic fertilizer compound of this application has long-term effectiveness, and the efficient silicon dissolving effect of JH3 can sustainably release soluble silicon, promote plant absorption, recruit beneficial microorganisms, and provide a better living environment for plant growth; for another example, the JH3 bacterial solution is compounded with attapulgite to obtain a mineral biological bacterial agent. Applying attapulgite to the soil can improve soil structure, increase soil aeration and water holding capacity, and be beneficial to the growth and reproduction of microorganisms. Mixing JH3 with attapulgite can efficiently release the effective silicon in the attapulgite, providing plants with absorbable exogenous silicon. Its rich nitrogen, phosphorus and potassium can provide nutrients for plant growth, increase soil water retention, increase the void structure, and facilitate the respiration of plant roots.

[0046] Specific technical solutions

[0047] On the one hand, an embodiment of the present invention provides a Paenibacillus mucilaginosus JH3, whose taxonomic name is Bacillus mucilaginosus subsp. siliceus, and whose deposit number is CGMCC No.26781.

[0048] Bacillus mucilaginosus subsp. siliceus JH3 was deposited in the China General Microbiological Culture Collection on March 9, 2023. The deposit address is No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101, and the deposit number is: CGMCC No.26781.

[0049] On the other hand, an embodiment of the present invention further provides a culture obtained by culturing the Paenibacillus subtilis JH3 described in the above embodiment.

[0050] The present application has no special restrictions on the culture conditions of JH3, and the culture medium and culture conditions currently used for Bacillus mucilaginosus can be used.

[0051] In some embodiments, the culture conditions include: a temperature of 30-35°C, specifically any one of 30, 31, 32, 33, 34, 35°C or a range between any two thereof.

[0052] On the other hand, an embodiment of the present invention further provides a biological bacterial agent, which contains the Bacillus subtilis JH3 described in the above embodiment and / or the culture described in the above embodiment.

[0053] On the other hand, an embodiment of the present invention further provides use of the Bacillus subtilis JH3 described in any of the foregoing embodiments, or the culture described in any of the foregoing embodiments, or the biological agent described in any of the foregoing embodiments in the preparation of a fertilizer or a soil conditioner.

[0054] On the other hand, an embodiment of the present invention further provides a fertilizer, comprising: a carrier and the Paenibacillus subtilis JH3 described in any of the preceding embodiments, or the culture described in any of the preceding embodiments, or the biological agent described in any of the preceding embodiments; or obtained by fermenting a mixture of the carrier and the Paenibacillus subtilis JH3 described in any of the preceding embodiments, or the culture described in any of the preceding embodiments, or the biological agent described in any of the preceding embodiments.

[0055] In some embodiments, the mass ratio of the carrier to the Paenibacillus mucilaginosus JH3 or the culture or the biological agent is 8 to 9: 1 to 2. The mass ratio can be any one of 8: 1, 8: 1.2, 8: 1.4, 8: 1.6, 8: 1.8, 8: 2, 8.5: 1, 8.5: 1.2, 8.5: 1.4, 8.5: 1.6, 8.5: 1.8, 8.5: 2, 9: 1, 9: 1.2, 9: 1.4, 9: 1.6, 9: 1.8, 9: 2, or any two thereof.

[0056] In some embodiments, the carrier includes any one or more combinations of organic fertilizer and silicate soil conditioner.

[0057] In some embodiments, the silicate soil conditioner includes: any one or more combinations of attapulgite, montmorillonite, and zeolite.

[0058] In some embodiments, when the carrier includes organic fertilizer and attapulgite, the mass ratio of organic fertilizer to attapulgite is 1 to 2:4 to 39. The mass ratio can be any one of 1:4, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:39, 2:4, 2:10, 2:15, 2:20, 2:25, 2:30, 2:35, 2:39 or any two thereof.

[0059] In some embodiments, the fermentation conditions include: 30-35° C., 5-7 days. The fermentation temperature can be any one of 30, 31, 32, 33, 34, 35° C. or a range between any two thereof. The fermentation time can be any one of 5, 5.5, 6, 6.5, 7 days or a range between any two thereof.

[0060] In some embodiments, the fermentation further comprises: controlling the moisture content of the fermentation product to ≤ 30%. The method of controlling the moisture content can be through low-temperature drying technology, turning the fermentation product every 8 hours, and measuring the moisture content of the fermentation product at intervals.

[0061] In some embodiments, the fertilizer is an agricultural fertilizer.

[0062] The present invention has no special limitation on the organic fertilizer, and the organic fertilizer can be prepared by using existing commercially available organic fertilizer or based on existing conventional technical knowledge.

[0063] On the other hand, an embodiment of the present invention further provides a soil conditioner, which contains: the Bacillus subtilis JH3 described in any of the preceding embodiments, or the culture described in any of the preceding embodiments, or the biological agent described in any of the preceding embodiments, or the fertilizer described in any of the preceding embodiments.

[0064] On the other hand, the embodiments of the present invention further provide the use of the Bacillus mucilaginosus JH3 described in any of the foregoing embodiments, or the culture described in any of the foregoing embodiments, or the biological agent described in any of the foregoing embodiments, or the fertilizer described in any of the foregoing embodiments, or the soil conditioner described in any of the foregoing embodiments in any of the following:

[0065] (1) Plant cultivation; (2) Promoting plant growth or preparing products that promote plant growth; (3) Improving plant drought resistance or preparing products that improve plant drought resistance; (4) Soil conditioning.

[0066] In some embodiments, the plant includes any one or more of tobacco, tomato, and soybean.

[0067] In some embodiments, the fertilizer comprises agricultural fertilizer.

[0068] In some embodiments, the soil conditioning includes: improving the soil, adjusting the soil pH, increasing soil nutrients, and avoiding or reducing soil compaction.

[0069] On the other hand, an embodiment of the present invention also provides a screening culture medium, which includes the following components and the final concentrations of the components: 1-10 g / L sugar, 1-5 g / L Na2HPO4, 0.1-1 g / LMgSO4·7H2O, 0.001-0.01 g / LFeCl3, 0.01-0.5 g / L CaCO3, 0.1-5 g / L silicon source and 15-20 g / L agar; wherein the silicon source includes a silicate soil conditioner.

[0070] In some embodiments, the silicate soil conditioner includes any one or more of attapulgite, montmorillonite and zeolite.

[0071] The "attapulgite" in this article, also known as palygorskite, has a large specific surface area and adsorption capacity, good rheological and catalytic properties. At the same time, it has ideal colloidal properties and heat resistance. It is a rare mineral.

[0072] In some embodiments, the final concentration of the sugar in the screening medium can be any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 g / L or a range between any two of them. The final concentration of Na2HPO4 can be any one of 1, 2, 3, 4, 5 g / L or a range between any two of them. The final concentration of MgSO4·7H2O can be any one of 0.1, 0.2, 0.4, 0.6, 0.8, 1 g / L or a range between any two of them. The final concentration of FeCl3 can be any one of 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01 g / L or a range between any two of them. The final concentration of CaCO3 can be any one of 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5 g / L or any two thereof. The final concentration of the silicon source can be any one of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 g / L or any two thereof. The final concentration of agar can be any one of 15, 18, 20 g / L or any two thereof.

[0073] Compared with the existing screening culture medium for silicate-dissolving bacteria, the screening culture medium provided by the embodiment of the present invention is more conducive to the growth of silicate bacteria. It can shorten the culture time of the strain and improve the screening efficiency. It can shorten the screening cycle of microorganisms from 4 to 6 days to 2 to 4 days. The silicon-dissolving ability of the strains obtained by screening is also improved, which provides a new way for the effective screening of silicate-dissolving bacteria.

[0074] In some embodiments, the mesh size of the silicate soil conditioner (attapulgite, montmorillonite or zeolite) is 100-200 meshes, specifically any one of 100, 120, 140, 160, 180, 200 meshes or a range between any two of them.

[0075] In some embodiments, the sugars include any one or more of sucrose, glucose and mannitol.

[0076] In some embodiments, the pH of the screening medium is 7.0-7.5, specifically any one of 7.0, 7.1, 7.2, 7.3, 7.4, 7.5 or a range between any two of them.

[0077] In addition, an embodiment of the present invention also provides the use of the screening culture medium as described in any of the above embodiments in screening silicon-dissolving bacteria.

[0078] In some embodiments, the step of screening silicon-dissolving bacteria includes: inoculating candidate strains into a screening medium for culturing, and screening strains with strong silicon-dissolving ability.

[0079] In some embodiments, the culture conditions are the same as the culture conditions of strain JH3 described in any of the preceding embodiments and are not described in detail.

[0080] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0081] Example 1 Screening of silicon-dissolving bacteria

[0082] Verify the effect of different silicon sources on screening medium:

[0083] This embodiment provides an improved silica-dissolving bacteria screening medium, the ingredients of which are: sucrose 5g, Na2HPO4 2g, MgSO4·7H2O 0.5g, FeCl3 0.005g, CaCO3 0.1g, attapulgite 0.5g (200 mesh), agar 20g, H2O 1000mL, pH: 7.0.

[0084] Screening of silicon-dissolving bacteria based on improved silicon-dissolving bacteria screening medium:

[0085] Take 1g of tobacco rhizosphere soil from arid areas, add it into 99mL of sterile water in a clean bench, shake it and add it into 9mL sterile water test tubes in sequence for gradient dilution, dilute it into 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , shake it well and apply it to the improved silicon-dissolving bacteria screening medium, and place it in a constant temperature culture at 32°C for 2 days. The microorganisms can produce obvious colonies in the screening medium after growing for 2 days.

[0086] Based on the above screening culture medium, silicon-dissolving bacteria with better performance are screened and domesticated multiple times to obtain silicon-dissolving bacteria with stronger silicon-dissolving ability.

[0087] On the silica-dissolving bacteria screening medium, strain JH3 formed smooth, round, raised, sticky, elastic, translucent and colorless colonies ( Figure 1 ). After 48 hours of culture, the average diameter of the colonies of strain JH3 was 6.83 mm. The transparent hemispherical and viscous bacteria were selected for purification. The colonies after multiple purifications were expanded and the silicon-dissolving ability was quantitatively determined using the silicon-molybdenum blue colorimetric method. The strain with the strongest silicon-dissolving ability was screened out by colorimetry.

[0088] Table 1 Silicon dissolving capacity of different silicon dissolving bacteria

[0089] Strain information Dissolved silicon content range μg / mL Dissolved silicon content μg / mL L5N1 2.08～3.22 2.56 S2-1 4.07～10.19 6.82 JH1 33.27～40.29 36.96 JH2 32.60～37.32 35.65 JH3 44.71～47.78 46.25 JH4 24.08～25.02 24.59 JH5 36.23～41.33 38.48 MJH 35.96～39.73 36.81

[0090] The silica-dissolving bacteria JH3 has the strongest silica-dissolving ability, which is 44.71-47.87μg / mL, which is 66.83-78.61% higher than the highest silica-dissolving ability of 26.8μg / mL reported for agricultural production. JH3 was expanded and cultured in Axubei medium, the medium composition of which is: KH2PO4 0.2g, MgSO4·7H2O 0.2g, NaCl 0.2g, CaCO3 0.1g, attapulgite (200 mesh) 0.5g, agar 20g, H2O 1000mL, pH: 7.0. The bacteria are oligotrophic and are easier to survive in arid areas and sandy soils than other microorganisms.

[0091] The physiological and biochemical characteristics of the microorganism JH3 with silicon-dissolving ability were tested. The results showed that the silicon-dissolving bacteria JH3 was negative for oxidase and phenylalanine dehydrogenase activities; negative for methyl red and VP tests; able to hydrolyze starch, liquefy gelatin and positive for indole test, but poor in citrate utilization. JH3 had good utilization of glucose, mannitol, sucrose, fructose, lactose and maltose, but could not utilize raffinose, xylose and arabinose (Table 2).

[0092] Table 2 Physiological and biochemical characteristics of JH3

[0093] Physiological and biochemical indicators Test results Physiological and biochemical indicators Test results Oxidase － Gelatin liquefaction test + Methyl red test － Citrate utilization capacity － VP test － Indole test + Starch hydrolysis test + Phenylalanine dehydrogenase － Glucose utilization capacity + Maltose utilization capacity + Mannitol utilization capacity + Raffinose utilization capacity － Sucrose utilization capacity + Xylose utilization capacity － Fructose utilization ability + Arabinose utilization ability － Lactose utilization ability +

[0094] The strain Bacillus mucilaginosus subsp. siliceus JH3 was deposited in the China General Microbiological Culture Collection Center on March 9, 2023, with the deposit number: CGMCC No.26781.

[0095] Based on the above-mentioned improved silicon-dissolving bacteria screening medium, three groups of control culture media were set up. The control culture media were roughly the same as the screening culture media, except that the silicon sources in the culture media were different. The silicon source of the screening culture media was attapulgite, and the silicon source of the control screening culture media was glass powder, montmorillonite, and zeolite.

[0096] The silicon source in the silicon-dissolving bacteria screening medium was used to culture and domesticate the strain using four materials: attapulgite, glass powder, montmorillonite, and zeolite. The growth of silicon-dissolving bacteria JH3 was observed, and the silicon-dissolving ability of silicon-dissolving bacteria JH3 after being domesticated with different "silicon-dissolving bacteria screening medium" was quantitatively analyzed by silico-molybdenum blue colorimetry. By comparison, it can be seen that attapulgite as a silicon source can significantly improve the silicon-dissolving ability of microorganisms, such as Figure 2 shown.

[0097] At the same time, the loading capacity of glass powder, montmorillonite, zeolite and attapulgite on microorganisms was measured. Figure 3 As shown, it was found that with the increase of time, the ability of the material to load microorganisms showed a trend of first increasing and then decreasing. The bacterial load of attapulgite was relatively high at each stage. It can be seen that attapulgite as a silicon source can help improve the silicon-dissolving ability of microorganisms, and the ability of attapulgite to load microorganisms is stronger, so it can be used as a strain immobilization material.

[0098] Example 2 Performance study and function prediction of strain JH3

[0099] After strain JH3 was cultured in the culture medium for 24 hours, the cells in the logarithmic growth phase were collected by refrigerated centrifugation (8000r / min×15min) and quickly frozen. The culture medium composition was: 2.5g glucose, 4g starch, 1g (NH4)2SO4, 2g K2HPO4, 0.5g MgSO4·7H2O, 0.2g NaCl, 0.005g FeCl3, and the volume was adjusted to 1L with deionized water, and the pH was 7.0.

[0100] The second-generation and third-generation whole genome sequencing of Paenibacillus colloids JH3 was performed. The second-generation sequencing platform was Illumina NovaSeq, and the third-generation sequencing platform was Oxford Nanopore ONT. The results showed that the genomic DNA of Paenibacillus colloids JH3 was circular, without plasmids, with a genome size of 8.59 Mb and a GC content of 58.59%. A total of 40 ribosomal RNA (rRNA) genes and 156 transfer RNAs (tRNAs) were detected. The whole genome map is shown in Figure 4 The evolutionary tree of silicon-dissolving bacteria is shown in Figure 5 .

[0101] like Figure 6 As shown in the figure, through KEGG analysis, strain JH3 has more genes related to metabolism, providing energy for life and promoting its growth. Analysis of the metabolic pathways of the strain revealed that the strain has more genes related to synthases in the production of ascorbic acid, malic acid, and nicotine degradation. When applied to crops, it helps plants eliminate excessive reactive oxygen caused by drought stress, promotes microorganisms to dissolve minerals, and releases effective silicon, fast-acting phosphorus and other components, which helps provide plants with nutrients for their growth ( Figure 7 ).

[0102] like Figure 8As shown in the figure, using antiSMASH software, 12 secondary metabolite synthesis gene clusters were predicted, including 1 polyketide (PKS) type, 6 non-ribosomal peptide (NRPS) types, 2 terpene, and 3 NRPS and PKS hybrid types. Among them, the gene clusters of polyketide (PKS) and non-ribosomal peptide (NRPS) (including hybrid types) are abundant, accounting for more than 80% of the total gene clusters. The similarity of 1 gene cluster with the known metabolic (tridecaptin M) gene cluster is 100%, and the similarity of 2 gene clusters is between 8% and 60%. Whole genome analysis shows that strain JH3 has the ability to synthesize secondary metabolites, which also indicates that the strain has good research potential in agricultural fertilizer mining.

[0103] Example 3 Effect of potted application of strain JH3 on growth promotion and drought resistance of tobacco seedlings

[0104] The strain JH3 was added when the seedlings were transplanted. In the potted experiment, each pot (21cm×19cm×17cm) was loaded with 3.5kg of soil. An example group and a control group were set up. The example group included the application of strain JH3 alone, while the control group did not apply other fertilizers except normal base fertilizer. The JH3 bacterial solution was applied by root irrigation. The specific steps were as follows: the silicon-dissolving bacteria JH3 was inoculated into the nitrogen-free bacterial culture medium (Axubei culture medium), and fermented at a temperature of 35°C and a speed of 180r / min for 48h. When the number of live bacteria in the bacterial solution reached 1×10 8 ~5×10 8 The microbial agent was obtained by 50 times dilution when applied, and the diluted bacterial solution was applied to the roots at a ratio of 14.28 mL / kg·soil. The same amount of tap water was added as a control, and water was cut off for 15 days.

[0105] The drought experiment was carried out two weeks after the tobacco seedlings were grown, and the drought in the arid area was simulated by continuous water cut-off. Before the water cut-off began, the moisture content of the potted plants was adjusted to 90%, and the drought experiment lasted for 15 days. The potted control group and the example group had the same experimental conditions except whether the strain JH3 was added during the experiment.

[0106] By measuring the fresh weight, dry weight, stem diameter, leaf area and other aspects of plant growth of the above-ground and underground parts of tobacco plants, it was found that the growth indicators of the example group were better than those of the control group ( Fig. 9 and Fig.10 As shown in Figure 2, under drought stress, the fresh weight of tobacco leaves increased by 54.02% compared with the control group ( Fig.10 a); tobacco leaf dry weight increased by 100.95% compared with the control group ( Fig.10 b); tobacco leaf area increased by 132.10% compared with the control group ( Fig.10 c); tobacco stem diameter increased by 87.07% compared with the control group ( Fig.10(d) This indicates that applying JH3 can effectively promote better growth of plants under drought conditions, significantly increase plant biomass, promote plant leaf growth, promote plant photosynthesis, increase plant stem thickness, and facilitate the transportation of nutrients.

[0107] Fig.11 As shown in a, the available silicon content in the soil after applying strain JH3 first increased and then decreased. After 5 days of drought, the available silicon content of the soil applied with strain JH3 increased by 9.51% compared with that of the CK soil; after 10 days of drought, the available silicon content of the soil applied with strain JH3 increased by 11.8% compared with that of the CK soil; and after 15 days of drought, the potted soil applied with strain JH3 decreased by 21.42% compared with that of the CK potted soil. The available silicon in the soil decreased significantly, and the available silicon content in the plants should be analyzed together. Fig.11 As shown in b, after 15 days of drought, the silicon content in the roots of plants treated with strain JH3 was 427.88% higher than that in the roots of plants treated with CK, and the silicon content in the leaves of plants treated with strain JH3 was 300.13% higher than that in the leaves of plants treated with CK. Combined with the fact that the silicon content in the plants after the application of JH3 was much higher than that in the CK group, strain JH3 not only has the ability to efficiently dissolve silicon, but also can promote the absorption and utilization of effective silicon by plants, prevent and control plant water loss under drought stress, and keep them in a relatively good state.

[0108] The relative water content of leaves, POD, CAT, MDA, and chlorophyll content of leaves were measured, and by comparison, it was found that the photosynthetic rate in the example group was significantly higher than that in the control group. The malondialdehyde content in tobacco leaves of the inoculated example group and the control group was lower than that in the control group. The malondialdehyde content is a reflection of the degree of peroxidation of plant cell membranes. A high malondialdehyde content indicates that the degree of peroxidation of plant cell membranes is high and the cell membranes are seriously injured. This shows that during the drought stress process, the inoculated example group can protect the cell membrane of tobacco plants and reduce the damage caused by drought adversity. Fig.12 As shown, after the application of strain JH3 for 15 days of continuous water deprivation, the POD content in tobacco fresh leaves increased by 120.75% ( Fig.12 a); the CAT content in fresh tobacco leaves increased by 283.33% compared with the control group ( Fig.12 b); MDA in tobacco leaves was 65.82% lower than that in the control group ( Fig.12 d); the reducing sugar content increased by 11.43% compared with the control group ( Fig.12 (c). Fig.13 As shown, the chlorophyll a in tobacco leaves increased by 250% compared with the control group; the chlorophyll b increased by 88.26% compared with the control group; and the chlorophyll a / b ratio increased by 87.93%. Leaf water content ( Fig.14), the water content increased significantly by 522.71%. The activities of catalase (CAT) and peroxidase (POD) in the tobacco leaves of the inoculated group were higher than those of the control group, indicating that the inoculation of functional microorganisms can improve the drought resistance of tobacco by enhancing the activity of the antioxidant enzyme POD in tobacco. POD is an important protective enzyme in plants, which participates in the removal of active oxygen produced in plants. The higher its activity, the more conducive it is for plants to resist drought.

[0109] The strain JH3 of the invention has the ability to improve the activity of plant antioxidant enzymes, reduce the content of malondialdehyde, reduce the damage to plant cell membranes, increase the chlorophyll content of tobacco leaves, and promote plant growth.

[0110] Example 4 Field application effect of strain JH3

[0111] JH3 was inoculated into the Axubei culture medium, and the culture temperature was 35°C and the rotation speed was 180 r / min. After 36 h of culture, the number of viable bacteria was about 10 8 cfu / mL~10 9 cfu / mL, and obtain the microbial preparation.

[0112] The example group and the control group were set up. The example group included the application of JH3 (microbial preparation), and the control group did not apply other fertilizers except normal base fertilizer. The functional JH3 bacterial solution (microbial preparation) was applied by root irrigation. The specific operation was: dilute the microbial preparation 50 times, apply it by root irrigation at a ratio of 50mL / plant tobacco seedling, add the same amount of tap water as a control, and conduct field tests.

[0113] After tobacco is transplanted, there will be a period of mild drought. After entering the clustering stage and the vigorous growth stage, the water demand of the tobacco plants increases, but at the same time, the precipitation is insufficient, which will seriously affect the growth of the tobacco plants. In this experiment, the functional microorganism JH3 was added during the tobacco transplanting period and supplemented during the clustering period. At maturity, the tobacco leaves were picked from the middle and upper parts and flue-cured tobacco was 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 example group with functional microorganisms were significantly higher than those of the control group, and the number of upper tobacco leaves in the example group was significantly more than that of the control group, and the upper tobacco leaves were more likely to produce high-quality tobacco leaves.

[0114] The results are as follows Fig.15 As shown. The application of silicon-dissolving bacteria JH3 increased the fresh weight of the upper tobacco leaves by 22.17% compared with the control group ( Fig.15 a), the fresh weight of tobacco leaves in the middle part increased by 8.56% compared with the control group ( Fig.15 In a), the fresh weight of the lower tobacco leaves decreased by 8.56% compared with the control group ( Fig.15 a); the upper tobacco leaf dry weight increased by 23.29% compared with the control group ( Fig.15 b), the dry weight of tobacco leaves in the middle increased by 8.59% compared with the control group ( Fig.15 b), the dry weight of the lower tobacco leaves decreased by 20.05% compared with the control group ( Fig.15 b); the yield of upper tobacco leaves increased by 17.78% compared with the control group ( Fig.15 c), the yield of tobacco leaves in the middle part increased by 11.31% compared with the control group ( Fig.15 c), the yield of lower tobacco leaves decreased by 9.72% compared with the control group ( Fig.15 c); the tobacco plant height increased by 28.86% compared with the control group ( Fig.15 (d)

[0115] The application of strain JH3 in the field can promote tobacco growth, increase the biomass of tobacco plants, increase the yield of upper tobacco leaves, and effectively improve tobacco quality.

[0116] Example 5 Field application effect of JH3 compounded with organic fertilizer

[0117] The method for obtaining JH3 bacterial solution is described in Example 3; the organic fertilizer and JH3 bacterial solution are mixed evenly, wherein the mass of the organic fertilizer accounts for 85% of the mass of the biological organic fertilizer, and the mass of the JH3 bacterial solution accounts for 15% of the mass of the biological organic fertilizer (the number of bacteria in the bacterial solution should reach 10 8 cfu / mL-10 9 cfu / mL); the mixing method includes: taking out a small amount of organic fertilizer in batches, adding bacterial solution in a sterile environment, mixing, and then putting it back into the original organic fertilizer to mix, and then repeating the above steps until the bacterial solution and organic fertilizer are completely mixed, and then performing secondary fermentation, fermenting at room temperature for 7 days, turning over every 18 hours, and keeping the moisture content less than 30% after fermentation, and the number of viable bacteria>2×10 7 cfu / g, and obtain biological organic fertilizer.

[0118] Before transplanting tobacco seedlings, 50g of bio-organic fertilizer was applied to the tobacco nest, with conventional treatment as the control group. The results are as follows Fig.16 As shown in the figure. After JH3 bacterial solution was mixed with organic fertilizer and applied, the fresh weight of the upper tobacco leaves increased by 195.15% compared with the control group ( Fig.16 a), the fresh weight of tobacco leaves in the middle part increased by 142.41% compared with the control group ( Fig.16 In a), the fresh weight of the lower tobacco leaves decreased by 31.88% compared with the control group ( Fig.16 a); the dry weight of the upper tobacco leaves increased by 183.10% compared with the control group ( Fig.16 b), the dry weight of tobacco leaves in the middle increased by 144.37% compared with the control group ( Fig.16 b), the dry weight of the lower tobacco leaves decreased by 31.98% compared with the control group ( Fig.16 b); the yield of upper tobacco leaves increased by 142.69% compared with the control group ( Fig.16c), the yield of tobacco leaves in the middle part increased by 75.31% compared with the control group ( Fig.16 c), the dry weight of the lower tobacco leaves decreased by 29.74% compared with the control group ( Fig.16 c); the tobacco plant height increased by 38.21% compared with the control group ( Fig.16 (d)

[0119] It can be seen that the application of JH3 mixed with organic fertilizer in the field can significantly promote tobacco growth, increase the biomass of tobacco plants, increase the yield of upper tobacco leaves, and effectively improve tobacco quality.

[0120] Example 6 Field application effect of strain JH3 compounded with attapulgite

[0121] The method for obtaining JH3 bacterial solution is described in Example 3; the attapulgite and JH3 bacterial solution are mixed evenly, wherein the mass of the attapulgite accounts for 85% of the mass of the biological organic fertilizer, and the mass of the JH3 bacterial solution accounts for 15% of the mass of the biological organic fertilizer (the number of bacteria in the bacterial solution should reach 10 8 cfu / mL-10 9 cfu / mL), the mixing method includes: taking out a small amount of attapulgite in batches, adding bacterial solution under a sterile environment, mixing, and then putting it back into the original attapulgite to mix, and then repeating the above steps until the bacterial solution and attapulgite are completely mixed, and then performing secondary fermentation, fermenting at room temperature for 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, and obtain biological organic fertilizer.

[0122] The results are as follows Fig.17 As shown in the figure, the fresh weight of the upper tobacco leaves increased by 146.07% ( Fig.17 a), the fresh weight of tobacco leaves in the middle part increased by 54.83% compared with the control group ( Fig.17 In a), the fresh weight of the lower tobacco leaves decreased by 11.02% compared with the control group ( Fig.17 a); the upper tobacco leaf dry weight increased by 133.96% compared with the control group ( Fig.17 b), the dry weight of tobacco leaves in the middle increased by 53.37% compared with the control group ( Fig.17 b), the dry weight of the lower tobacco leaves decreased by 10.65% compared with the control group ( Fig.17 b); the yield of upper tobacco leaves increased by 83.79% compared with the control group ( Fig.17 c), the yield of tobacco leaves in the middle part increased by 50.15% compared with the control group ( Fig.17 c), the dry weight of the lower tobacco leaves decreased by 2.87% compared with the control group ( Fig.17 c); the tobacco plant height increased by 30.28% compared with the control group ( Fig.17 (d)

[0123] After the strain JH3 provided by the present invention is compounded with attapulgite soil, application in the field can significantly promote tobacco growth, increase the biomass of tobacco plants, increase the yield of upper tobacco leaves, and effectively improve tobacco quality.

[0124] Example 7 Field application effect of JH3 compounded with attapulgite and organic fertilizer

[0125] The method for obtaining JH3 bacterial solution is described in Example 3; attapulgite and organic fertilizer are mixed at a mass ratio of 2:39, and then the mixture of organic fertilizer-attapulgite and JH3 bacterial solution are mixed to prepare biological organic fertilizer, and the JH3 bacterial solution accounts for 15% of the mass of the biological organic fertilizer (the number of bacteria in the bacterial solution should reach 10 8 cfu / mL-10 9 cfu / mL), the mixing method is to take out a small amount of the mixture of organic fertilizer and attapulgite in batches, add bacterial solution in a sterile environment, mix well, and then put it back into the mixture to mix well, repeat the above steps after mixing until the bacterial solution and the mixture are completely mixed, and then carry out secondary fermentation, ferment at room temperature for 7 days, turn over once every 18 hours, keep the moisture content less than 30% after fermentation, and the effective viable bacteria count is greater than 107 cfu / g to obtain biological organic fertilizer.

[0126] Before transplanting tobacco seedlings, 50g of bio-organic fertilizer was applied to the tobacco nest, and the conventional treatment was used as the control group. Fig.18 As shown, the fresh weight of upper tobacco leaves increased by 145.91% compared with the control group ( Fig.18 a), the fresh weight of tobacco leaves in the middle increased by 76.66% compared with the control group ( Fig.18 In a), the fresh weight of lower tobacco leaves decreased by 13.46% compared with the control group ( Fig.18 a); the dry weight of the upper tobacco leaves increased by 142.68% compared with the control group ( Fig.18 b), the dry weight of tobacco leaves in the middle increased by 77.89% compared with the control group ( Fig.18 b), the dry weight of the lower tobacco leaves decreased by 13.27% compared with the control group ( Fig.18 b); the yield of upper tobacco leaves increased by 83.66% compared with the control group ( Fig.18 c), the yield of tobacco leaves in the middle part increased by 42.59% compared with the control group ( Fig.18 c), the dry weight of the lower tobacco leaves decreased by 23.01% compared with the control group ( Fig.18 c); the height of tobacco plants increased by 39.43% compared with the control group ( Fig.18 (d)

[0127] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A gelatinous Bacillus JH3, characterized in that Its taxonomic name is Bacillus mucilaginosussubsp.siliceus, and its deposit number is CGMCC No.26781.

2. A culture, characterized in that The method is obtained by culturing the Paenibacillus subtilis JH3 described in claim 1.

3. A biological bacterial agent, characterized in that: It contains the Paenibacillus mucilaginosus JH3 according to claim 1 and / or the culture according to claim 2.

4. Use of the gelatinous Bacillus JH3 according to claim 1, the culture according to claim 2, or the biological agent according to claim 3 in the preparation of a fertilizer or a soil conditioner.

5. A fertilizer, characterized in that: It includes: A carrier and the Paenibacillus sp. JH3 according to claim 1 or the culture according to claim 2 or the biological agent according to claim 3; or obtained by fermenting a mixture of the carrier and the Paenibacillus sp. JH3 according to claim 1 or the culture according to claim 2 or the biological agent according to claim 3; Optionally, the mass ratio of the carrier to the Bacillus subtilis JH3 or the culture or the biological agent is 8-9:1-2; Optionally, the carrier includes: any one or more of organic fertilizer and silicate soil conditioner; Optionally, the silicate soil conditioner includes: any one or more combinations of attapulgite, montmorillonite, and zeolite; Optionally, when the carrier includes organic fertilizer and attapulgite, the mass ratio of the organic fertilizer to the attapulgite is 1-2:4-39; Optionally, the fermentation conditions include: 30-35° C., 5-7 days.

6. A soil conditioner, characterized in that: It contains: the Paenibacillus subtilis JH3 according to claim 1, or the culture according to claim 2, or the biological agent according to claim 3, or the fertilizer according to claim 5.

7. Use of the Paenibacillus subtilis JH3 according to claim 1, the culture according to claim 2, the biological agent according to claim 3, the fertilizer according to claim 5, or the soil conditioner according to claim 6 in any of the following: (1) Plant cultivation; (2) Promoting plant growth or preparing products that promote plant growth; (3) Improving plant drought resistance or preparing products that improve plant drought resistance; (4) Soil conditioning.

8. The use according to claim 7, characterized in that: The plants include any one or more of tobacco, tomato and soybean.

9. A screening medium, characterized in that The components and final concentrations of the components are as follows: 1-10 g / L sugar, 1-5 g / L Na2HPO4, 0.1-1 g / L MgSO4·7H2O, 0.001-0.01 g / L FeCl3, 0.01-0.5 g / L CaCO3, 0.1-5 g / L silicon source and 15-20 g / L agar; wherein the silicon source comprises a silicate soil conditioner; Optionally, the silicate soil conditioner includes any one or more of attapulgite, montmorillonite and zeolite; Optionally, the mesh size of the attapulgite, montmorillonite or zeolite is 100 to 200 meshes; Optionally, the sugar includes: any one or more of sucrose, glucose and trehalose; Optionally, the pH of the screening medium is 7.0-7.

5.

10. Use of the screening culture medium as claimed in claim 9 in screening silicon-dissolving bacteria.

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