Clostridium butyricum for promoting growth of wheat in seedling stage and application of clostridium butyricum

Abstract

The invention discloses clostridium butyricum for promoting wheat seedling stage growth and application thereof, and relates to the technical field of microbial agents. The clostridium butyricum is clostridium butyricum JBH-BD1 and is preserved in the China Center for Type Culture Collection, and the preservation number of the clostridium butyricum is CCTCC NO: M 2022151; the fermentation liquor prepared from the clostridium butyricum by using the fermentation method disclosed by the invention is high in viable bacteria quality, strong in stability, low in cost and simple in preparation method; the microbial fertilizer prepared from the clostridium butyricum JBH-BD1 can increase the germination number of wheat, effectively promote the growth of wheat in the seedling stage, promote the fresh weight and dry weight of wheat roots in the seedling stage, the soil water content and the like, has the effect obviously superior to that of other bacterial fertilizers, and has outstanding application value in the aspect of wheat growth.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial agents, and in particular relates to Clostridium butyricum for promoting the growth of wheat seedlings and an application thereof. Background Art

[0002] Wheat is one of my country's traditional main grain crops and the staple food of my country's populous northern region. Its consumption accounts for more than half of the country's total grain consumption. In several national grain consumption surveys in recent years, total wheat consumption has ranked first. The role of wheat in stabilizing grain prices and farmers' income is becoming increasingly apparent.

[0003] The seedling stage is an important stage in the growth and development of crops, and wheat is no exception. Seedling wheat plays a vital role in the entire growth process. During the germination of seeds, seedling wheat can secrete various enzymes to help decompose complex organic matter such as starch and protein in the seeds, and release nutrients for wheat seedlings to absorb. The growth rate of wheat in the seedling stage is relatively fast, mainly in the root system. A well-developed root system is essential for the overall growth of the plant. By increasing the absorption area of ​​the root system, seedling wheat can absorb more water and nutrients to support the healthy growth of wheat in the later stages. Seedling wheat can secrete plant growth hormones and a series of antioxidants to enhance the self-repair ability of wheat, so that it can maintain a normal growth state under adverse conditions such as low temperature, drought, flooding, diseases and insect pests. Therefore, in wheat planting, enhancing the wheat seedling stage and ensuring that the wheat seedling stage fully plays its role and function can provide a solid foundation for high yield and high quality of wheat in the later stage. At present, the management of wheat seedlings only involves the application of chemical fertilizers and large amounts of watering, which not only causes environmental pollution but also wastes water resources.

[0004] The beneficial bacteria in the microbial fertilizer accelerate the decomposition of soil organic matter and promote the formation of granular structure, thereby breaking the soil compaction, regulating and improving the soil's water retention, fertilizer retention, and air permeability, making it more suitable for crop seedling growth. At the same time, it degrades pesticide and heavy metal residues in the soil and repairs the polluted soil environment. Clostridium butyricum has the characteristics of long logarithmic period, early acid production, large acid volume, high transsporulation rate, high enzyme activity, strong antibacterial, acid and alkali resistance, bile salt resistance, high temperature resistance, and high stability. As the "king of probiotics", it has the advantages of other probiotics. At the same time, the acetic acid, butyric acid, amylase, vitamin K and B vitamins produced during fermentation, as well as unused culture medium components, are all nutrients that promote plant seedling growth.

[0005] However, it is still unknown whether the bacterial fertilizer prepared using the fermentation broth of Clostridium butyricum can play a role in promoting the growth of wheat seedlings. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a Clostridium butyricum for promoting the growth of wheat seedlings and its application. The Clostridium butyricum is Clostridium butyricum JBH-BD1, and the fermentation liquid formed by fermentation of the Clostridium butyricum has high live bacteria quality, strong stability, low cost, simple preparation method, and can effectively promote the growth of wheat seedlings.

[0007] The Clostridium butyricum JBH-BD1 used in the present invention is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2022151, a deposit date of February 25, 2022, and a deposit address of Wuhan University, Wuhan, China, and has been published in patent application CN116103200A. The Clostridium butyricum in the present invention is screened from intestinal feces samples of healthy piglets, is natural, safe, acid-resistant, alkali-resistant, and high-temperature-resistant, and has strong protease, amylase, and organic acid production capabilities, and has an antibacterial effect on Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens, porcine Pasteurella multocida, and Actinobacillus pleuropneumoniae.

[0008] Patent CN116103200A discloses the application of Clostridium butyricum in iron enrichment. However, the application potential of Clostridium butyricum in wheat seedling stage has not been reported in the prior art. The present invention uses Clostridium butyricum to promote the growth of wheat seedlings for the first time, and confirms for the first time through experiments that compared with conventional Bacillus subtilis, lactic acid bacteria, compound fertilizers, etc., the Clostridium butyricum provided by the present invention has a stronger ability to promote the growth of wheat seedlings, so it can be used to promote the growth of wheat seedlings or to prepare microbial fertilizers, and has outstanding application value in wheat growth.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] One of the purposes of the present invention is to provide an application of Clostridium butyricum JBH-BD1 in promoting the growth of wheat seedlings. The Clostridium butyricum JBH-BD1 is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2022151.

[0011] The second object of the present invention is to provide an application of a microbial agent in promoting the growth of wheat seedlings, wherein the microbial agent comprises the Clostridium butyricum JBH-BD1 and / or its culture product.

[0012] The microbial agent is a microbial fertilizer, which can be in the form of a liquid preparation or a powder.

[0013] Furthermore, the preparation method of the microbial agent comprises the following steps: (1) taking the Clostridium butyricum and culturing it on a plate on a modified CM culture medium to activate the strain to obtain an activated strain; (2) inoculating the activated strain into the modified CM culture medium, culturing it at 32 to 38° C. for 12 to 18 hours, and obtaining a seed solution; (3) inoculating the seed solution obtained in step (2) into the modified CM culture medium at an inoculation amount of 1% to 3% by volume, and fermenting and culturing it at 30 to 38° C. for 35 to 40 hours. The obtained fermentation liquid or the powder prepared therefrom is the microbial agent.

[0014] Furthermore, the composition of the improved CM medium is as follows, per liter of culture medium: glucose 1.3-1.6 g / L, corn starch 0.4-0.6 g / L, peptone 2.5-3.5 g / L, yeast powder 2-3 g / L, K2HPO4 0.06-0.09 g / L, MgSO4·7H2O 0.04-0.06 g / L, MnSO4·H2O 0.004-0.006 g / L, and the balance is water.

[0015] Furthermore, the bacterial activation in step (1) is carried out by culturing at 33-37° C. for 12-18 hours.

[0016] Furthermore, the culture processes in steps (1), (2) and (3) are all carried out under anaerobic conditions.

[0017] The preparation method of the present invention has simple process and low preparation cost. In addition, the microbial agent prepared by the method has active ingredients including Clostridium butyricum and the culture product of the bacteria, and has an excellent effect on promoting the growth of wheat seedlings.

[0018] Furthermore, the application in promoting the growth of wheat seedlings is specifically embodied as an application in increasing the number of wheat germinations.

[0019] Furthermore, the application in promoting the growth of wheat seedlings is specifically embodied as the application in promoting the growth of wheat roots.

[0020] Furthermore, the application in promoting the growth of wheat seedlings is specifically embodied in the application in increasing the fresh weight and / or dry weight of wheat seedlings.

[0021] Furthermore, the application in promoting the growth of wheat seedlings is specifically embodied in increasing the soil moisture content of wheat seedlings.

[0022] The microbial agent of the present invention is used in the following method: take the liquid microbial agent and dilute it to a level where the number of viable Clostridium butyricum is (1-3)×10 7cfu / mL, and then spray the diluted liquid preparation on the roots of wheat seedlings; or first make the liquid microbial agent into a powder, and then sprinkle the powder into the field before wheat sowing. The number of viable bacteria in the powder preparation is (1-3)×10 10 cfu / mL, the application amount of the liquid preparation is 3-5L / mu; the application amount of the powder is 20-30kg / mu.

[0023] The microbial agent provided by the present invention is a biological agent that can efficiently promote the growth of wheat seedlings. The agent and its matching application method have extremely high application value in the wheat seedling stage.

[0024] Compared with the prior art, the present invention has the following technical effects:

[0025] The fermentation liquid prepared by the fermentation method of the present invention using Clostridium butyricum JBH-BD1 has high live bacteria quality, strong stability, low cost and simple preparation method; the microbial fertilizer prepared by the present invention can increase the number of wheat germinations, effectively promote the growth of wheat in the seedling stage, and promote the root and seedling fresh weight, dry weight and soil water content of wheat, etc., and the effect is obviously better than other microbial fertilizers, and has outstanding application value in wheat growth.

[0026] Description of the deposit certificate:

[0027] Depository: China Center for Type Culture Collection;

[0028] Deposit number: CCTCC NO: M 2022151;

[0029] Deposit date: February 25, 2022;

[0030] Deposit address: Wuhan University, Wuhan, China;

[0031] Taxonomic nomenclature: Clostridium butyricum JBH-BD1. DETAILED DESCRIPTION

[0032] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the method, steps or conditions of the present invention all belong to the scope of the present invention. The reagents, products, instruments, etc. used in the following examples can all be obtained commercially, and the methods used in the examples are consistent with the conventional methods unless otherwise specified.

[0033] The present invention uses the Clostridium butyricum JBH-BD1 to provide a method for preparing a microbial agent, comprising the following steps:

[0034] (1) taking the Clostridium butyricum and culturing it on a plate on a modified CM medium to activate the strain, thereby obtaining an activated strain;

[0035] (2) inoculating the activated strain into a modified CM medium and culturing at 32 to 38° C. for 12 to 18 hours to obtain a seed solution;

[0036] (3) Inoculating the seed solution obtained in step (2) into the improved CM culture medium at an inoculation rate of 1% to 3% by volume, and fermenting and culturing at 30 to 38° C. for 35 to 40 hours. The obtained fermentation liquid or the powder prepared therefrom is the microbial agent.

[0037] The composition of the improved CM culture medium is as follows, per liter of culture medium: 1.3-1.6 g / L of glucose, 0.4-0.6 g / L of corn starch, 2.5-3.5 g / L of peptone, 2-3 g / L of yeast powder, 0.06-0.09 g / L of K2HPO4, 0.04-0.06 g / L of MgSO4·7H2O, 0.004-0.006 g / L of MnSO4·H2O, and the balance is water.

[0038] The bacterial activation in step (1) is specifically carried out by culturing the activated bacteria at 33-37° C. for 12-18 hours.

[0039] The cultivation processes in steps (1), (2) and (3) are all carried out under anaerobic conditions.

[0040] The technical solution of the present invention is further described in detail below in conjunction with embodiments.

[0041] Example 1

[0042] This embodiment provides a method for preparing the Clostridium butyricum biological agent for promoting the growth of wheat seedlings, comprising the following steps:

[0043] (1) taking the cryopreserved Clostridium butyricum JBH-BD1, with a volume inoculation amount of 1%, and anaerobically culturing it in a modified CM medium (pH 7.20±0.20, plate medium) at 35°C for 16 h to obtain an activated strain;

[0044] (2) Using a sterile pipette, take 100 mL of the activated bacteria obtained in step (1), inoculate it into a modified CM medium (pH 7.20±0.20, liquid medium), and culture it at 35° C. under anaerobic conditions for 16 h to obtain a seed solution;

[0045] (3) inoculating the seed solution obtained in step (2) into a modified CM medium (pH value is 7.20±0.20, liquid medium) at an inoculum volume ratio of 1%, and fermenting and culturing at 35° C. under anaerobic conditions for 36 hours to obtain a fermentation liquid; detecting the number of bacteria and spores in the fermentation liquid, and stopping the fermentation and culturing when the mature spores in the fermentation liquid account for 90% of the total number of spores and bacteria, thereby obtaining a biological agent.

[0046] Among them, the composition and dosage ratio (g / L) of the improved CM medium are as follows: glucose 1.5 g / L, corn starch 0.5 g / L, peptone 3 g / L, yeast powder 2.5 g / L, K2HPO4 0.08 g / L, MgSO4·7H2O 0.05 g / L, MnSO4·H2O 0.005 g / L, and the balance is water.

[0047] Comparative Example 1

[0048] This comparative example provides a method for preparing a liquid compound fertilizer, comprising the following steps:

[0049] (1) Weigh about 430 g of urea, about 420 g of diammonium phosphate, and about 340 g of potassium sulfate.

[0050] (2) Add an appropriate amount of water to a clean container, add urea, diammonium phosphate, and potassium sulfate in sequence, and stir until completely dissolved.

[0051] (3) Use Ph test paper to test the Ph value of the solution. The Ph value is around 6.5 and can be adjusted with citric acid or baking soda.

[0052] (4) Add pure water to make up to 1000 mL.

[0053] Comparative Example 2

[0054] This comparative example provides a Bacillus subtilis biological agent, the content of Bacillus subtilis is 1.0×10 9 CFU / mL. Its preservation certificate is as follows:

[0055] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0056] Deposit number: CGMCC No.19440;

[0057] Deposit date: March 2, 2020;

[0058] Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0059] Taxonomic name: Bacillus subtilis.

[0060] Comparative Example 3

[0061] This comparative example provides a Lactobacillus acidophilus biological agent, the content of Lactobacillus acidophilus is 1.0×10 9 CFU / mL, it was purchased from the Agricultural Microbiology Center of China Microorganism Collection Administration on January 23, 2015, its strain collection number is ACCC05489, its genus name is Lactobacillus, and its species name is acidophilus.

[0062] Example 2

[0063] In the culture room, four cultivation boxes of the same size were selected and numbered ABCD. Equal amounts of soil were weighed and placed in the cultivation boxes ABCD. 200 grains of wheat (Zhoumai 36) were planted. On the same day, 300 ml of the homemade liquid compound fertilizer in Comparative Example 1 was sprayed in cultivation box A, and 300 ml of the diluted bacterial count of 1×10 in Example 1 was sprayed in cultivation box B. 7 cfu / mL of Clostridium butyricum fermentation liquid, 300mL of the diluted bacteria in comparative example 2 was sprayed in cultivation box C to obtain 1×10 7 cfu / mL of Bacillus subtilis liquid, 300mL of the diluted bacteria count in Comparative Example 3 was 1×10 7 cfu / mL of lactic acid bacteria liquid, and the number of wheat sprouts was observed after 10 days. The experimental temperature and humidity were consistent with the indoor temperature and humidity, and no condition control was taken. The results are shown in Table 1.

[0064] Table 1 Determination of wheat germination number

[0065] Experimental Group A B C D Number of germination <![CDATA[179.33±1.25 d ]]> <![CDATA[196.33±0.47 a ]]> <![CDATA[191±0.81 b ]]> <![CDATA[184.33±1.25 c ]]>

[0066] The experimental results in Table 1 show that the bacterial fertilizer prepared by using Clostridium butyricum in the present invention can increase the number of wheat germinations, and the effect is better than that of Bacillus subtilis, lactic acid bacteria and compound fertilizer.

[0067] Example 3

[0068] In the culture room, four cultivation boxes of the same size were selected and numbered ABCD. Equal amounts of soil were weighed and placed in the cultivation boxes ABCD. 200 grains of wheat (Zhoumai 36) were planted. On the same day, 300 ml of the homemade liquid compound fertilizer in Comparative Example 1 was sprayed in cultivation box A, and 300 ml of the diluted bacterial count of 1×10 in Example 1 was sprayed in cultivation box B. 7 cfu / mL of Clostridium butyricum fermentation liquid, 300mL of the diluted bacteria in comparative example 2 was sprayed in cultivation box C to obtain 1×10 7 cfu / mL of Bacillus subtilis liquid, 300mL of the diluted bacteria count in Comparative Example 3 was 1×10 7cfu / mL of lactic acid bacteria liquid, and 20 days later when the wheat had two leaves and one heart, the roots of the wheat seedlings were measured, the length of the wheat leaves, the fresh weight and dry weight of the wheat, and the soil moisture content were measured. The results are shown in Table 2.

[0069] Table 2 Statistics of wheat seedling growth

[0070] Experimental Group A B C D Root length (cm) <![CDATA[8.06±0.09 d ]]> <![CDATA[14.23±0.09 a ]]> <![CDATA[13.43±0.16 b ]]> <![CDATA[10.43±0.09 c ]]> Fresh weight of wheat (g) <![CDATA[3.7±0.012 d ]]> <![CDATA[6.31±0.016 a ]]> <![CDATA[6.05±0 b ]]> <![CDATA[4.84±0.008 c ]]> Wheat dry weight (g) <![CDATA[0.57±0.005 d ]]> <![CDATA[0.91±0.008 a ]]> <![CDATA[0.80±0.008 b ]]> <![CDATA[0.74±0.008 c ]]> Soil moisture content <![CDATA[12.67±0.005 d ]]> <![CDATA[14.02±0.014 a ]]> <![CDATA[13.20±0 b ]]> <![CDATA[13.00±0.002 c ]]>

[0071] The experimental results in Table 2 show that the bacterial fertilizer prepared by using Clostridium butyricum in the present invention can effectively promote the growth of wheat seedlings, and promote the fresh weight, dry weight and soil moisture content of wheat roots and seedlings, and the effect is better than other bacteria, fertilizers, etc.

[0072] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of Clostridium butyricum JBH-BD1 in promoting the growth of wheat seedlings, characterized in that: The Clostridium butyricum JBH-BD1 is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M2022151.

2. An application of a microbial agent in promoting the growth of wheat seedlings, characterized in that: The microbial agent comprises Clostridium butyricum JBH-BD1 and / or its culture product in the application of claim 1.

3. The use according to claim 2, characterized in that: The preparation method of the microbial agent comprises the following steps: (1) taking the Clostridium butyricum and culturing it on a plate on a modified CM medium to activate the strain, thereby obtaining an activated strain; (2) inoculating the activated strain into a modified CM medium and culturing at 32 to 38° C. for 12 to 18 hours to obtain a seed solution; (3) Inoculating the seed solution obtained in step (2) into the improved CM culture medium at an inoculation rate of 1% to 3% by volume, and fermenting and culturing at 30 to 38° C. for 35 to 40 hours. The obtained fermentation liquid or the powder prepared therefrom is the microbial agent.

4. The use according to claim 3, characterized in that: The composition of the improved CM medium is as follows: glucose 1.3-1.6 g / L, corn starch 0.4-0.6 g / L, peptone 2.5-3.5 g / L, yeast powder 2-3 g / L, K2HPO4 0.06-0.09 g / L, MgSO4·7H2O 0.04-0.06 g / L, MnSO4·H2O 0.004-0.006 g / L, and the balance is water.

5. The use according to claim 3, characterized in that: The bacterial activation in step (1) is carried out by culturing the activated bacteria at 33-37° C. for 12-18 hours.

6. The use according to claim 3, characterized in that: The cultivation processes in steps (1), (2) and (3) are all carried out under anaerobic conditions.

7. The use according to any one of claims 1 to 6, characterized in that: The application is application in increasing the number of wheat germinations.

8. The use according to any one of claims 1 to 6, characterized in that: The application is application in promoting root growth of wheat.

9. The use according to any one of claims 1 to 6, characterized in that: The application is application in increasing the fresh weight and / or dry weight of wheat at the seedling stage.

10. The use according to any one of claims 1 to 6, characterized in that: The application is to improve the soil moisture content during the wheat seedling stage.