Paenibacillus mucilaginosus HX-02 and application thereof in promoting crop growth

Abstract

The invention relates to the technical field of microorganisms, in particular to paenibacillus mucilaginosus HX-02 and application of the paenibacillus mucilaginosus HX-02 in promoting crop growth. The preservation number of the paenibacillus mucilaginosus HX-02 is CGMCC (China General Microbiological Culture Collection Center) No. 30949. The application comprises increasing one or more of the plant height, the branch number, the weight or the number of crops. According to the invention, the paenibacillus mucilaginosus HX-02 is obtained through screening and separation, and can decompose lattice structures of soil minerals to release K, Fe, Mg, Si, Al and Mo; secreting cytokinin, gibberellin, kinetin, indoleacetic acid and acetylsalicylic acid; therefore, the invention has important application value in the field of agricultural development.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a jelly-like Paenibacillus HX-02 and an application thereof in promoting crop growth. Background Art

[0002] Plant hormones are important signaling molecules that regulate plant growth and development, and they play a vital role in agricultural production. Traditional synthetic hormones may pollute the environment and have adverse effects on non-target organisms. Therefore, the development of environmentally friendly and sustainable methods for producing plant hormones has become a research hotspot. Most of the microbial strains that produce plant hormones reported so far are obtained by screening microorganisms in the natural environment or modifying known strains. The hormone-producing ability of these strains is often unstable and easy to lose. Therefore, researchers have been looking for new and more efficient microbial strains that produce plant hormones in order to achieve large-scale, low-cost, and environmentally friendly production of plant hormones.

[0003] Microorganisms decompose minerals in the soil and release dissolved trace elements that can be absorbed by plants. This can also significantly improve the absorption and utilization efficiency of these key nutrients by plants, improve the nutritional status of plants, thereby promoting plant growth and development, and increasing crop yield and quality.

[0004] Paenibacillus jelly-like Paenibacillus mucilaginosus ) belongs to the Firmicutes, Bacilales, Bacillaceae, Bacillus genus, and is a type of spore-forming Gram-negative bacteria that has the ability to form a polysaccharide matrix (also called jelly) outside the cell wall, which gives the bacterial surface a sticky and elastic property. Jelly-like Bacillus is widely found in a variety of natural environments such as soil, water, and plant rhizosphere. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a jelly-like Paenibacillus HX-02 and an application thereof in promoting crop growth.

[0006] The present invention screened and separated a jelly-like bacillus having the function of promoting plant growth and increasing plant yield from soil, and made a biological deposit of the bacillus. The deposit information is as follows: Deposit number: CGMCC No.30949; Classification name: Paenibacillus jelly Paenibacillus mucilaginosus ; Depository: General Microbiology Center, China Culture Collection Administration; Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101; Deposit date: June 14, 2024.

[0007] In a first aspect, the present invention provides a bacterial agent, comprising the jelly-like Paenibacillus HX-02 or a fermentation product thereof.

[0008] The fermentation product of the present invention can be the supernatant obtained by fermentation of the microorganism (excluding the microorganism itself) or the whole product including the microorganism itself.

[0009] Furthermore, the bacterial agent is a solid bacterial agent, a liquid bacterial agent or a microbial bacterial agent, and the total viable count of the jelly-like Bacillus HX-02 in the bacterial agent is 1.0×10 7-10 cfu / g.

[0010] In a second aspect, the present invention provides the use of the jelly-like Bacillus HX-02 or the bacterial agent in promoting plant growth.

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

[0012] The present invention further provides the use of the jelly-like Paenibacillus HX-02 or the bacterial agent in increasing crop yield.

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

[0014] The present invention further provides the use of the jelly-like Bacillus HX-02 or the bacterial agent in decomposing the lattice structure of soil minerals to release K, Fe, Mg, Si, Al and Mo.

[0015] The present invention further provides the use of the jelly-like Paenibacillus HX-02 or the bacterial agent in producing cytokinins, gibberellins, indoleacetic acid or acetylsalicylic acid.

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

[0017] In a third aspect, the present invention provides a method for promoting crop growth, comprising: The jelly-like Paenibacillus HX-02 or the bacterial agent is applied to crops.

[0018] Furthermore, when the crop is potato, the application method is spraying on the surface of potato seed blocks, and the application amount is 200-400 g / mu; When the crop is soybean, the application method is seed dressing, and the application amount is 100-200g / mu.

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

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

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

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

[0023] The present invention has the following beneficial effects: The present invention screens and separates a jelly-like bacillus capable of promoting plant growth and increasing plant yield. The bacillus 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 the plant height, branch number, weight or quantity of crops, and increase the per-acre yield of crops.

[0024] The jelly-like Paenibacillus HX-02 provided by the present invention is separated from soil, is harmless to humans, animals and the environment, and meets the needs of green plant protection and sustainable agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 This is a chromatogram of gibberellins secreted by the jelly-like Bacillus HX-02 provided in Example 2 of the present invention.

[0027] Figure 2 This is a chromatogram of kinetin secreted by the jelly-like Bacillus HX-02 provided in Example 2 of the present invention.

[0028] Figure 3 This is a chromatogram of cytokinin secretion by the jelly-like Paenibacillus HX-02 provided in Example 2 of the present invention.

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

[0030] Figure 5This is a chromatogram of acetylsalicylic acid secreted by the jelly-like Bacillus HX-02 provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0033] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0034] Example 1 Effect of Paenibacillus jelly HX-02 on decomposing potassium aluminum acid 1. Experimental methods Place 100 ml of liquid culture medium in a 250 ml conical flask and sterilize at 121 °C for 30 min. When the temperature drops below 45 °C, inoculate 5% of the jelly-like Bacillus HX-02. Add an equal amount of inactivated bacterial solution to the control and culture on a rotary shaker at 30 °C and 190 r / min for 38 h before treatment.

[0035] The jelly-like culture solution of Paenibacillus was centrifuged at 6000r / min for 20min to remove the bacteria and residues. The water-soluble potassium in the supernatant was determined. The residue was boiled with 1mol / L HCl for 10min, and the exchangeable potassium in the filtrate and the fixed potassium in the residue were determined. K was determined by flame photometry. + ; Atomic absorption spectrophotometer GGX-2 for determination of Mg 2+ and Zn 2+ ; Hitachi UV-22 OA colorimeter to measure Al 3+ , Fe 3+ and Si 4+ ; Determination of Mo by oscillographic polarograph JP-1 2+ .

[0036] 2. Experimental results With potassium aluminate as substrate, inoculate jelly-like Bacillus HX-02, culture for 38 hours, centrifuge to remove bacteria and residues, and measure the water-soluble potassium in the supernatant, the replaceable potassium and fixed potassium in the residues. After 4 repeated measurements, the water-soluble potassium in the treatment was 1.80 mg, which was 0.47 mg higher than the uninoculated control (1.33 mg), with an increase rate of 35.34%, and the difference reached a significant level by biostatistics.

[0037] The total potassium in the residue (including exchangeable potassium and fixed potassium) was determined to be 91.0 mg on average after 4 treatments, which was 0.70 mg less than the uninoculated control. Although part of the water-soluble potassium was converted into cellular potassium and adsorbed on the surface of solid particles and could not be washed off, it was included in the residue, but the fixed potassium in the treatment was lower than that in the control. The increase in water-soluble potassium and the decrease in fixed potassium indicate that the HX-02 strain has a good potassium-solubilizing ability.

[0038] Table 1 Effect of HX-02 strain on decomposing potassium aluminum acid

[0039] It can be seen from Table 1 that the HX-02 strain can destroy the mineral lattice structure and release not only potassium, but also Fe, Mg, Si, Al, Mo and other elements. Except for Zn, the elements measured in the inoculated treatment increased to varying degrees compared with the non-inoculated treatment, among which Si increased the most, by 150%, followed by Mo, by 100%, and then Mg, by 59%. Fe and Al increased less, by 16.6% and 17.2% respectively.

[0040] Example 2 Study on the level of phytostimulants secreted by Paenibacillus jelly 1. Experimental methods 1.1 GA3 (gibberellin) detection method Accurately measure 100.0 mL of the fermentation broth of jelly-like Bacillus 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 detect GA3.

[0041] 1.2 Detection methods of KT (kinetin) and tZ (cytokinin) Accurately measure 100 mL of the jelly-like 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 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 detect tZ and KT.

[0042] 1.3 Detection method of IAA (indoleacetic acid) First, the jelly-like Bacillus fermentation liquid was diluted 20 times with pure water, and then 100 mL of the diluted jelly-like Bacillus fermentation liquid was measured and centrifuged at 7000 rpm for 15 min. 5 mL of the liquid was placed in a 15 mL centrifuge tube, and an appropriate amount of 1 mol / L citric acid aqueous solution was added to adjust the pH to 2-3. 5 mL of ethyl acetate was added for extraction twice, and the upper organic phase was transferred to a new EP tube, blown dry with nitrogen, and 0.2 mL of methanol was added to dissolve, mixed, filtered with a syringe filter, and then IAA was detected.

[0043] 1.4 Detection method of acetylsalicylic acid Take 100 mL of jelly-like Bacillus fermentation liquid, centrifuge at 7000 rpm for 15 minutes, take 5 mL of liquid and place it in a 15 mL centrifuge tube, add an appropriate amount of trichloroacetic acid aqueous solution to adjust to weak acidity, mix and shake for 1 minute. Add 5 mL of a mixture of ethyl acetate and cyclohexane (1:1 v / v) 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, filter with a syringe filter and then test.

[0044] 1.5 Liquid chromatography conditions Chromatographic column: Compass C18 (2) reverse phase column (250 mm*4.6 mm, 5 μm).

[0045] Column temperature: 35°C.

[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 210nm; 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; Determination of IAA: 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 GA3 (gibberellin) standard sample standard curve and sample determination The standard curve of GA3 standard sample was obtained by determination. Figure 1 ) shows that the retention time is 18.834min. Table 2 shows that the content of GA3 in the jelly-like Paenibacillus fermentation broth is 0.7168μg / mL.

[0049] 2.2 KT (kinetin) and tZ (cytokinin) standard sample standard curve and sample determination The KT and standard sample standard curves were obtained by determination. Figure 2 ) showed that the retention time of the stimulant was 8.759 min. Since the peak of the hormone did not appear in the liquid chromatography of the fermentation broth of Bacillus gelatinosa, the strain did not secrete KT.

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

[0051] 2.3 IAA (indoleacetic acid) standard sample standard curve and sample determination The standard curve of IAA standard sample was obtained by determination. From the IAA determination chromatogram ( Figure 4 ) shows that the retention time of the stimulant is 15.887min. Table 2 shows that the content of IAA in the jelly-like Paenibacillus fermentation broth is 397.12μg / mL. 2.4 Detection method of acetylsalicylic acid The standard curve of acetylsalicylic acid standard sample was obtained by determination. Figure 5 ) shows that the retention time is 8.753 min. Table 2 shows that the content of acetylsalicylic acid in the jelly-like Paenibacillus fermentation broth is 9.94 μg / mL.

[0052] Table 2 Determination of hormone content produced by jelly-like Paenibacillus

[0053] Example 3 Field Fertilizer Efficacy Test of Microbial Agents on Potatoes and Soybeans 1. Experimental methods Jelly-like Bacillus HX-02 microbial agent: powder, 500g / bag; bacterial content ≥50.0×10 8 cfu / g, water content ≤8%; Crops: Potato variety Jinshu No. 8, soybean variety Jindou 20.

[0054] Potato: Dilute the microbial agent with water at a ratio of 1:1. Spread the potato pieces cut into pieces with germs evenly when applying. Shake the diluted bacterial solution evenly and use a sprayer to evenly sprinkle it on the surface of the germ pieces. Turn it over lightly and spray it again. After the spraying of the microbial agent is completed, dry it in the shade and then sow it. The test area is 1.0 mu for high-efficiency microbial agent and 0.5 mu for blank control. The dosage per mu is 2kg / mu.

[0055] Cultivation and management: The experiment was conducted according to local practices. The experimental site was flat and fertile. 500 kg of farmyard manure was applied per mu, the planting density was 2470 plants / mu, sowing was done in late April, harvesting was done in early October, and yield was calculated by region at harvest time.

[0056] Soybean: Dilute the microbial agent with water in a 1:1 ratio. Use the seed mixing method to mix 500g of the agent directly on 1.0 mu of soybean seeds. Dry in the shade and then sow.

[0057] Cultivation and management: The experiment was conducted according to local practices. The experimental site was flat and fertile. 500 kg of farmyard manure was applied per mu, the land was prepared before sowing, and 15,800 seedlings were left per mu. Sowing was carried out on April 21 and harvesting was carried out on October 14. Each district calculated the yield based on a single harvest.

[0058] 2. Experimental results 2.1 Effects of different treatments on potato biological traits Table 3 Effects of HX-02 microbial agent on biological characteristics of potato

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

[0060] It can be seen from the above table that after applying HX-02 microbial agent, the potato yield per mu increased by 205 kg, and the increase rate reached 10.5%.

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

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

[0063] It can be seen from the above table that after applying HX-02 microbial agent, soybean yield per mu increased by 14.0 kg, and the increase rate reached 7.8%.

[0064] Example 4 Field Fertilizer Efficacy Test of HX-02 Microbial Agent on Cotton 1. Experimental methods The cotton variety is smooth-leaf cotton. The cotton seeds are germinated and then mixed with seeds. A simple comparative method is used for the test. 500 g of microbial agent (Bacillus jelly HX-02 microbial agent, the same as in Example 3) is mixed with 8 kg of seeds for treatment, and no mixing is used as the control.

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

[0066] Mixing cotton seeds with HX-02 microbial agent before sowing can supplement the lack of potassium fertilizer in the soil and during the growth and development of cotton, showing that the plants are stable and the leaves are dark green. Applying microbial agent can increase the number and weight of bolls. The average boll per plant is 4.4, an increase of 18.9% over the control; the average single boll weight is 4.31 grams, an increase of 6.4% over the control. Improve the yield of cotton per unit area. The yield per mu of microbial agent application is 500.5 kg of lint, an increase of 18.9% over the control. Promote the stable growth of cotton and early maturity. The average pre-frost flower of cotton treated with microbial agent is 70%, 0.6% higher than the control.

[0067] Example 5 Field Fertilizer Efficacy Test of HX-02 Microbial Agent on Peanuts, Watermelons and Apples 1. Experimental methods For peanuts, 2 kg of microbial agent was applied per mu. For watermelons, 2 kg of microbial agent was applied in holes during sowing or planting. For apples (Red Fuji), 250 g of microbial agent was applied in the tree furrow around each tree. The microbial agent used was the jelly-like Bacillus HX-02 microbial agent, which was the same as in Example 3.

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

[0069] Compared with the control, the application of HX-02 microbial agent on peanuts increased the height of peanut plants by 20%, the biomass of single plants and the main root length by 41.5% and 36.6% respectively, the fruit full rate by 21.9%, and the yield per mu by 21.5%.

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

[0071] The sugar content of watermelons treated with HX-02 microbial agent increased by an average of 1.46, with an output of 4761 kg per mu, while the yield of watermelon per mu in the control field was 3864 kg. The application of biological potassium increased the watermelon yield by 897 kg per mu compared with the control field, with an increase rate of 23.2%. The increase in the weight of each watermelon plant was the main factor for the increase in yield.

[0072] Compared with the control trees, the inoculated trees at maturity had a significant effect on the coloring of apples, with the coloring of each apple on the inoculated trees reaching more than 70%, while the control trees were 45-50%. The apples treated with microbial agents were large and beautiful, with an output of 2,250 kg per mu, an increase of 25%.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A jelly-like bacillus ( Paenibacillus mucilaginosus ) HX-02, characterized in that, The deposit number of the jelly-like Bacillus HX-02 is: CGMCC No.30949.

2. A microbial agent, characterized in that: The invention comprises the jelly-like Paenibacillus sp. HX-02 or a fermentation product thereof as described in claim 1.

3. The bacterial agent according to claim 2, characterized in that The bacterial agent is a solid bacterial agent, a liquid bacterial agent or a microbial bacterial agent. In the bacterial agent, the total viable count of the jelly-like Bacillus HX-02 is 1.0×10 7-10 cfu / g.

4. Use of the jelly-like Paenibacillus HX-02 according to claim 1, or the bacterial agent according to claim 2 or 3 in promoting plant growth.

5. The use according to claim 4, characterized in that: The promoting crop growth includes: increasing one or more of the plant height, branch number, weight or quantity of the crop.

6. Use of the jelly-like Paenibacillus HX-02 according to claim 1, or the bacterial agent according to claim 2 or 3 in increasing crop yield.

7. The use according to any one of claims 4 to 6, characterized in that: The crops are one or more of potatoes, soybeans, cotton, peanuts, watermelons, or apples.

8. Use of the jelly-like Bacillus HX-02 according to claim 1, or the bacterial agent according to claim 2 or 3 in decomposing the lattice structure of soil minerals to release K, Fe, Mg, Si, Al and Mo.

9. Use of the jelly-like Paenibacillus HX-02 according to claim 1, or the bacterial agent according to claim 2 or 3 in the production of cytokinins, gibberellins, indole-containing acid or acetylsalicylic acid.

10. A method for promoting crop growth, characterized in that: include: The jelly-like Paenibacillus HX-02 according to claim 1 or the bacterial agent according to claim 2 is applied to crops.

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