Paenibacillus hydrogeniformis and application of the paenibacillus hydrogeniformis in promoting crop growth

Abstract

The present application relates to the technical field of microorganism, and particularly relates to a paste-like Paenibacillus sp. HX-02 and application of the paste-like Paenibacillus sp. HX-02 in promoting crop growth. The preservation number of the paste-like Paenibacillus sp. HX-02 is CGMCC No. 30949. The application includes: improving one or more of plant height, branch number, weight or quantity of crops. The present application screens and separates a paste-like Paenibacillus sp. HX-02, which can decompose soil mineral lattice structure to release K, Fe, Mg, Si, Al and Mo, secrete cytokinin, gibberellin, kinetin, indole acetic acid and acetylsalicylic acid, promote crop growth, improve crop yield, and has important application value in the field of agricultural development.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a gelatinous Bacillus spp. HX-02 and its application in promoting crop growth. Background Technology

[0002] Plant hormones are crucial signaling molecules regulating plant growth and development, playing a vital role in agricultural production. Traditional synthetic hormone production can pollute the environment and adversely affect non-target organisms. Therefore, developing environmentally friendly and sustainable methods for plant hormone production has become a research hotspot. Currently reported plant hormone-producing microbial strains are mostly obtained by screening microorganisms in the natural environment or modifying known strains. However, the hormone-producing capacity of these strains is often unstable and easily lost. Therefore, researchers are constantly searching for new and more efficient plant hormone-producing microbial strains to achieve large-scale, low-cost, and environmentally friendly plant hormone production.

[0003] By decomposing minerals in the soil through microorganisms, dissolved trace elements that plants can absorb can be released, which can significantly improve the efficiency of plant absorption and utilization of these key nutrients, improve the nutritional status of plants, and thus promote plant growth and development, and increase crop yield and quality.

[0004] gelatinous spore-forming bacteria ( Paenibacillus mucilaginosus Belonging to the phylum Firmicutes, order Bacillus, family Bacillusaceae, and genus Bacillus, these are spore-forming Gram-negative bacteria. These bacteria possess the ability to form a polysaccharide matrix (also known as a gel) outside their cell walls, giving the bacterial surface a sticky and elastic quality. Gelatinous Bacillus species are widely found in various natural environments, including soil, water, and plant rhizospheres. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a gelatinous Bacillus thuringiensis HX-02 and its application in promoting crop growth.

[0006] This invention screens and isolates a gelatinous Bacillus from soil that promotes plant growth and increases plant yield, and then preserves it biologically. The preservation information is as follows: Preservation number: CGMCC No. 30949; Classification: Bacillus jellyii Paenibacillus mucilaginosus Depository Institution: China General Microbiological Culture Collection Center; Depository Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China; Depository Date: June 14, 2024.

[0007] In a first aspect, the present invention provides a microbial agent comprising the aforementioned gelatinous Bacillus HX-02 or its fermentation product.

[0008] The fermentation product described in this invention can be either the supernatant obtained from the fermentation of the microorganism (excluding the microorganism itself) or all products including the microorganism itself.

[0009] Further, the bacterial agent is a solid bacterial agent, a liquid bacterial agent, or a microbial bacterial agent, wherein the total viable count of the gelatinous Bacillus spp. HX-02 in the bacterial agent is 1.0 × 10⁻⁶. 7-10 cfu / g.

[0010] Secondly, the present invention provides the application of the aforementioned gelatinous Bacillus HX-02, or the aforementioned bacterial agent, in promoting plant growth.

[0011] Furthermore, the promotion of crop growth includes increasing one or more of the following: plant height, number of branches, weight, or quantity of the crop.

[0012] The present invention further provides the application of the aforementioned gelatinous Bacillus HX-02, or the aforementioned inoculant, in improving crop yield.

[0013] Furthermore, the crop is one or more of potatoes, soybeans, cotton, peanuts, watermelons, or apples.

[0014] The present invention further provides the application of the aforementioned gelatinous Bacillus HX-02, or the aforementioned bacterial agent, in the decomposition of soil mineral lattice structure to release K, Fe, Mg, Si, Al and Mo.

[0015] The present invention further provides the use of the aforementioned gelatinous Bacillus HX-02, or the aforementioned bacterial agent, in the production of cytokinins, gibberellins, indoleacetic acid, or acetylsalicylic acid.

[0016] Acetylsalicylic acid is an organic compound that plays a physiological role in plant growth and development. However, it is also the main component of aspirin and can be used in drug preparation. The gelatinous Bacillus HX-02 provided by this invention has the ability to directly secrete acetylsalicylic acid and can be used for the industrial production of acetylsalicylic acid or its drugs.

[0017] Thirdly, the present invention provides a method for promoting crop growth, comprising: Apply the described gelatinous Bacillus HX-02 or the described inoculant to the crop.

[0018] Furthermore, when the crop is potato, the application method is to spray it on the surface of the potato seed tubers, and the application rate is 200~400g / mu; When the crop is soybean, the application method is seed dressing, and the application rate is 100~200g / mu.

[0019] When the crop is cotton, the method of application is seed dressing, and the amount of seed dressing is 50~100g / kg.

[0020] When the crop is peanut, the application rate is 1-5 kg / mu.

[0021] When the crop is watermelon, the application rate is 1-5 kg / mu.

[0022] When the crop is apple, the application rate is 200-500g per tree.

[0023] The present invention has the following beneficial effects: This invention screens and isolates a gelatinous Bacillus that promotes plant growth and increases plant yield. It can secrete a variety of stimulants and decompose the soil mineral lattice structure, releasing elements such as K, Fe, Mg, Si, Al, and Mo to increase crop height, number of branches, weight or quantity, and thus increase crop yield per acre.

[0024] The gelatinous Bacillus HX-02 provided by this invention is isolated from soil and is harmless to humans, animals and the environment, meeting the needs of green plant protection and sustainable agricultural development. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0026] Figure 1 This is a chromatogram of gibberellin secreted by Bacillus jelly-like spores HX-02, as provided in Example 2 of this invention.

[0027] Figure 2 This is a chromatogram of kinetin secreted by Bacillus jelly-like spore-forming bacteria HX-02, provided in Example 2 of this invention.

[0028] Figure 3 This is a chromatogram of cytokinins secreted by Bacillus jelly-like spore-forming bacteria HX-02, as provided in Example 2 of this invention.

[0029] Figure 4 This is a chromatogram of indoleacetic acid secreted by Bacillus jelly-like spore-forming bacteria HX-02, as provided in Example 2 of the present invention.

[0030] Figure 5This is a chromatogram of acetylsalicylic acid secreted by Bacillus jelly-like spores HX-02, as provided in Example 2 of this invention. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0032] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0033] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0034] Example 1: Effect of Bacillus jellyii HX-02 on the decomposition of potassium aluminum acid 1. Experimental Methods 100 ml of liquid culture medium was placed in a 250 mL Erlenmeyer flask and sterilized at 121 °C for 30 min. When the temperature dropped below 45 °C, 5% of Bacillus thuringiensis HX-02 was inoculated. An equal volume of inactivated bacterial solution was added to the control. The flasks were then incubated at 30 °C on a rotating shaker at 190 r / min for 38 h before further treatment.

[0035] The Bacillus spp. culture medium was centrifuged at 6000 rpm for 20 min to remove bacterial cells and residue. The water-soluble potassium in the supernatant was determined. The residue was boiled in 1 mol / L HCl for 10 min, and the exchangeable potassium in the filtrate and the fixed potassium in the residue were determined. K was determined using a flame photometer. + Determination of Mg by GGX-2 type atomic absorption spectrometer 2+ and Zn 2+ Hitachi UV-22 OA colorimeter for measuring Al 3+ Fe 3+ and Si 4+ Mo was determined using an oscilloscope polarograph JP-1. 2+ .

[0036] 2. Experimental Results Using potassium aluminate as a substrate, *Bacillus spp.* HX-02 was inoculated and cultured for 38 h. After centrifugation to remove bacterial cells and residue, the water-soluble potassium in the supernatant, and the replaced and fixed potassium in the residue were measured. Four repeated measurements showed that the treated water-soluble potassium (1.80 mg) increased by 0.47 mg compared to the uninoculated control (1.33 mg), representing an increase of 35.34%, which was statistically significant.

[0037] The residual total potassium (including replaced potassium and fixed potassium) was measured at an average of 91.0 mg after four replicates, which was 0.70 mg less than the uninoculated control. Although some water-soluble potassium was converted to cellular potassium and adsorbed on the surface of solid particles, thus remaining in the residue, the fixed potassium in the treated sample was lower than the control. This increase in water-soluble potassium and decrease in fixed potassium demonstrates that strain HX-02 has excellent potassium-releasing ability.

[0038] Table 1. Effect of strain HX-02 on the decomposition of potassium aluminum acid

[0039] As shown in Table 1, strain HX-02 can disrupt the mineral crystal structure, releasing not only potassium but also elements such as Fe, Mg, Si, Al, and Mo. Except for Zn, the inoculated treatment showed varying degrees of increase in the measured elements compared to the uninoculated treatment. Si saw the largest increase at 150%, followed by Mo at 100%, and then Mg at 59%. Fe and Al showed smaller increases, at 16.6% and 17.2%, respectively.

[0040] Example 2: Study on the level of phytostimulants secreted by Bacillus jelly-like bacteria 1. Experimental Methods 1.1 Detection method for GA3 (gibberellin) Accurately measure 100.0 mL of the fermentation broth of Bacillus thuringiensis HX-02, centrifuge at 7000 rpm for 15 min, take 5.0 mL of the supernatant and place it in a 15 mL centrifuge tube, add an appropriate amount of 1.0 mol / L citric acid aqueous solution to adjust the pH to 2-3, add 5.0 mL of ethyl acetate to extract twice, transfer the upper organic phase to a new EP tube, blow dry with nitrogen, add 0.2 mL of methanol to dissolve, mix well, filter with a syringe filter and then perform GA3 detection.

[0041] 1.2 Detection methods for KT (kinetin) and tZ (cytokinin) Accurately measure 100 mL of the gelatinous Bacillus fermentation broth, centrifuge at 7000 rpm for 15 min, take 5 mL of the supernatant and place it in a 15 mL centrifuge tube, add 5 mL of ethyl acetate for extraction twice, transfer the upper organic phase to a new EP tube, blow dry with nitrogen, add 0.2 mL of methanol to dissolve, mix well, filter with a syringe filter, and then perform tZ and KT detection.

[0042] 1.3 Detection method for IAA (indoleacetic acid) First, dilute the Bacillus jelly-like fermentation broth 20 times with purified water. Then, measure 100 mL of the diluted Bacillus jelly-like fermentation broth, centrifuge at 7000 rpm for 15 min, take 5 mL of the liquid and place it in a 15 mL centrifuge tube. Add an appropriate amount of 1 mol / L citric acid aqueous solution to adjust the pH to 2-3, add 5 mL of ethyl acetate for extraction twice, transfer the upper organic phase to a new EP tube, blow dry with nitrogen, add 0.2 mL of methanol to dissolve, mix well, filter with a syringe filter, and then perform IAA detection.

[0043] 1.4 Detection method for acetylsalicylic acid Take 100 mL of the gelatinous Bacillus fermentation broth, centrifuge at 7000 rpm for 15 min, transfer 5 mL of the liquid to a 15 mL centrifuge tube, add an appropriate amount of trichloroacetic acid aqueous solution to adjust to weak acidity, mix and shake for 1 min. Add 5 mL of a mixture of ethyl acetate and cyclohexane (1:1 v / v) for extraction twice, transfer the upper organic phase to a new EP tube, dry under nitrogen, add 0.2 mL of methanol to dissolve, mix well, filter with a syringe filter and then analyze.

[0044] 1.5 Liquid Chromatography Conditions Chromatographic column: Compass C18 (2) reversed-phase column (250mm*4.6mm, 5μm).

[0045] Column temperature: 35℃.

[0046] Flow rate: 1 mL / min, injection volume: 10 μL.

[0047] Mobile phase: GA3 determination: 0.1% phosphoric acid aqueous solution: acetonitrile = 85:15 (V / V), wavelength 210 nm; Determination of tZ: 1% acetic acid aqueous solution: methanol = 70:30 (V / V), wavelength 270nm; Determination of KT: 1% acetic acid aqueous solution: methanol = 70:30 (V / V), wavelength 254nm; IAA determination: 1% acetic acid aqueous solution: methanol = 65:35 (V / V), wavelength 275nm; Determination of acetylsalicylic acid: 1% acetic acid aqueous solution: methanol = 50:50 (V / V), wavelength 294nm.

[0048] 2. Experimental Results 2.1 Standard Curve and Sample Determination of GA3 (Gibberellin) Standard Samples A standard curve for GA3 standard samples was obtained. From the GA3 chromatogram ( Figure 1 The results show that the retention time was 18.834 min. Table 2 shows that the GA3 content in the Bacillus jelly-like fermentation broth was 0.7168 μg / mL.

[0049] 2.2 Standard curves and sample determination of KT (kinetin) and tZ (cytokinin) standard samples The standard curves for KT and standard samples were obtained. The chromatogram of KT was determined (…). Figure 2 The results show that the retention time of the hormone is 8.759 min. Since the peak of this hormone did not appear in the liquid chromatography of the Bacillus jelly-like fermentation broth, this strain does not secrete KT.

[0050] The standard curve of the tZ standard sample was obtained by determination. The chromatogram of the tZ standard sample was determined. Figure 3 The results show that the retention time of this stimulant is 4.130 min. Table 2 shows that the tZ content in the Bacillus jelly-like fermentation broth is 0.0471 μg / mL.

[0051] 2.3 Standard Curve and Sample Determination of IAA (Indoleacetic Acid) Standard Samples The standard curve of the IAA standard sample was obtained. The chromatogram of the IAA was determined. Figure 4 The results show that the retention time of this stimulant is 15.887 min. Table 2 shows that the IAA content in the Bacillus jelly-like fermentation broth is 397.12 μg / mL. 2.4 Detection method for acetylsalicylic acid A standard curve for acetylsalicylic acid standard samples was obtained. The chromatogram of acetylsalicylic acid determination is shown below. Figure 5 The results show that the retention time was 8.753 min. Table 2 shows that the acetylsalicylic acid content in the Bacillus jelly-like fermentation broth was 9.94 μg / mL.

[0052] Table 2. Determination of hormone content in Bacillus jelly-like bacteria.

[0053] Example 3: Field Fertilizer Effect Test of Microbial Inoculants on Potatoes and Soybeans 1. Experimental Methods Bacillus jellyii HX-02 microbial inoculant: powder, 500g / bag; bacterial count ≥50.0×10⁻⁶ 8 cfu / g, moisture content ≤8%; Crops: Potato variety Jinshu 8 and soybean variety Jindou 20.

[0054] Potatoes: Dilute the microbial inoculant with water at a 1:1 ratio. When applying, spread the potato pieces with sprouts evenly, shake well, and then spray the diluted inoculant solution evenly on the surface of the sprouted pieces. Gently turn over and spray again. After the inoculant has been sprayed, let the potatoes air dry before planting. The experimental area was 1.0 mu of high-efficiency microbial inoculant and 0.5 mu of blank control, with a dosage of 2 kg / mu.

[0055] Cultivation and Management: The experiment was conducted according to local practices. The experimental plot was flat and had uniform fertility. 500 kg of farmyard manure was applied per mu (667 square meters), with a planting density of 2470 plants per mu. Sowing took place in late April, and harvesting in early October. Yield was calculated by zone at harvest time.

[0056] Soybeans: Dilute the microbial inoculant with water at a 1:1 ratio, and use the seed dressing method to mix 500g of inoculant directly with soybean seeds on 1.0 acre of land, dry in the shade, and then sow.

[0057] Cultivation and Management: The experiment was conducted according to local practices. The experimental plot was level and had uniform fertility. 500 kg of farmyard manure was applied per mu (667 square meters). The land was prepared before sowing, and 15,800 seedlings were retained per mu. Sowing was on April 21, and harvesting was on October 14. Yield was calculated based on a single harvest in each region.

[0058] 2. Experimental Results 2.1 Effects of different treatments on the biological traits of potatoes Table 3 Effects of HX-02 microbial inoculant on biological traits of potato

[0059] 2.2 Effects of different treatments on potato yield Table 4. Effects of HX-02 microbial inoculant on potato yield

[0060] As shown in the table above, the application of HX-02 microbial inoculant increased potato yield by 205 kg per mu, representing a yield increase of 10.5%.

[0061] 2.3 Effects of different treatments on the biological traits of soybean Table 5 Effects of HX-02 microbial inoculant on biological traits of soybeans

[0062] 2.4 Effects of different treatments on soybean yield Table 6. Effects of HX-02 microbial inoculant on soybean yield

[0063] As shown in the table above, the application of HX-02 microbial inoculant increased soybean yield by 14.0 kg per mu, representing a yield increase of 7.8%.

[0064] Example 4: Field Fertilizer Efficacy Test of HX-02 Microbial Inoculant in Cotton 1. Experimental Methods The cotton variety was cotton slicker. After the cotton seeds were germinated, they were treated with a simple comparative method. 8 kg of seeds were treated with 500g of microbial agent (Bacillus spp. HX-02 microbial agent, the same as in Example 3) as a treatment, and the control was not treated with seeds.

[0065] 2. Experimental Results Table 7 Effects of HX-02 microbial inoculant on cotton traits and yield.

[0066] Mixing cotton seeds with HX-02 microbial inoculant before sowing can supplement the lack of potassium fertilizer in the soil and during cotton growth and development, resulting in robust plants and dark green leaves. Applying the microbial inoculant increases the number and weight of bolls, with an average of 4.4 bolls per plant, an increase of 18.9% compared to the control; the average weight of a single boll is 4.31 grams, an increase of 6.4% compared to the control. It also increases cotton yield, with a yield of 500.5 kg of lint per mu (approximately 0.067 hectares) after applying the microbial inoculant, an increase of 18.9% compared to the control. Furthermore, it promotes stable growth and earlier maturity of cotton. The average pre-frost boll opening rate for cotton treated with the microbial inoculant is 70%, 0.6% higher than the control.

[0067] Example 5: Field fertilizer effect test of HX-02 microbial inoculant on peanuts, watermelons, and apples. 1. Experimental Methods Apply 2 kg of microbial inoculant per mu (667 square meters) to peanuts. Apply 2 kg / mu of microbial inoculant per hole when sowing or transplanting watermelons. Apply 250 g of microbial inoculant per tree in the circumferential trench. The microbial inoculant used is Bacillus spp. HX-02, the same as in Example 3.

[0068] 2. Test Results Table 8 Effects of HX-02 microbial inoculant on peanut traits and yield

[0069] Compared to the control group, the application of HX-02 microbial inoculant to the peanut plant base resulted in a 20% increase in plant height, a 41.5% increase in biomass per plant, a 36.6% increase in taproot length, a 21.9% increase in pod filling rate, and a 21.5% increase in yield per acre.

[0070] Table 9 Effects of HX-02 microbial inoculant on watermelon traits and yield

[0071] Watermelons treated with HX-02 microbial inoculant showed an average sugar content increase of 1.46 and a yield of 4761 kg / mu, compared to 3864 kg / mu in the control field. Applying bio-potassium resulted in an additional 897 kg / mu of watermelon compared to the control, representing a yield increase of 23.2%. The increase in the weight of individual watermelon plants was the main factor contributing to the yield increase.

[0072] Compared to control trees, inoculated trees showed a significant improvement in apple coloring at maturity, with each apple on inoculated trees achieving over 70% coloring, while the control trees only achieved 45-50%. Apples treated with microbial agents were larger, more beautifully colored, and yielded 2250 kg per mu (approximately 0.16 acres), representing a 25% increase in yield.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of gelatinous Bacillus HX-02, or its inoculants, in promoting crop growth; The preservation number of the gelatinous Bacillus HX-02 is: CGMCC No. 30949; The bacterial agent includes the gelatinous Bacillus HX-02; The crop is one or more of the following: potato, soybean, cotton, peanut, watermelon, or apple.

2. The application according to claim 1, characterized in that, The promotion of crop growth includes increasing one or more of the following: plant height, number of branches, or weight.

3. Application of gelatinous Bacillus HX-02, or its inoculants, in increasing crop yield; The preservation number of the gelatinous Bacillus HX-02 is: CGMCC No. 30949; The bacterial agent includes the gelatinous Bacillus HX-02; The crop is one or more of the following: potato, soybean, cotton, peanut, watermelon, or apple.

4. Application of gelatinous Bacillus HX-02, or its agents, in the decomposition of soil mineral lattice structures to release K, Fe, Mg, Si, Al, and Mo; The preservation number of the gelatinous Bacillus HX-02 is: CGMCC No. 30949; The bacterial agent includes the gelatinous Bacillus spp. HX-02.

5. The application of gelatinous Bacillus HX-02, or its agents, in the production of cytokinins, gibberellins, indoleacetic acid, or acetylsalicylic acid; The preservation number of the gelatinous Bacillus HX-02 is: CGMCC No. 30949; The bacterial agent includes the gelatinous Bacillus spp. HX-02.

6. The application according to any one of claims 1-5, characterized in that, The bacterial agent is a solid or liquid bacterial agent, and the total viable count of the gelatinous Bacillus spp. HX-02 in the bacterial agent is 1.0 × 10⁻⁶. 7-10 cfu / g.

7. A method for promoting crop growth, characterized in that, include: Apply gelatinous Bacillus HX-02 or other bacterial agents to crops; The preservation number of the gelatinous Bacillus HX-02 is: CGMCC No. 30949; The bacterial agent includes the gelatinous Bacillus HX-02; The crop is one or more of the following: potato, soybean, cotton, peanut, watermelon, or apple.

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