Nitrogen-fixing microbial agent for improving soil nitrogen reserve and preparation method of nitrogen-fixing microbial agent

Abstract

The invention discloses an azotobacter agent for improving soil nitrogen reserves and a preparation method thereof, and belongs to the field of soil improvement. The nitrogen-fixing bacterial agent comprises nitrogen-fixing bacteria and an inducer, the nitrogen-fixing bacteria are non-pathogenic azospirillum and bacillus subtilis, and the inducer is trehalose. The preservation number of the nitrogen-fixing spirillum is CGMCC (China General Microbiological Culture Collection Center) No.6778. The nitrogen-fixing microbial agent prepared by compounding azospirillum, bacillus subtilis and trehalose can significantly improve the nitrogen reserve in soil, improve the thickness and sugar degree of grape skin, and improve the quality and yield of grapes.

Description

Technical Field

[0001] The present invention relates to the field of soil improvement, and in particular to a nitrogen-fixing bacterial agent for improving soil nitrogen reserves and a preparation method thereof. Background Art

[0002] Nitrogen-fixing bacteria refers to microbial fertilizers developed from bacteria that can fix nitrogen. By applying microbial fertilizers, microorganisms increase the nitrogen content in the soil that can be absorbed and utilized by plants through nitrogen fixation, promote plant growth, and increase plant yields. Compared with traditional chemical nitrogen fertilizers, microbial fertilizers are less polluting and more efficient, and meet the requirements of green, environmentally friendly and sustainable agricultural development.

[0003] Nitrogen-fixing bacteria mainly include autotrophic nitrogen-fixing bacteria, symbiotic nitrogen-fixing bacteria and associative nitrogen-fixing bacteria. Symbiotic nitrogen-fixing bacteria and associative nitrogen-fixing bacteria have high requirements for growth conditions and symbiotic plants, so it is difficult to be widely promoted and applied. Autotrophic nitrogen-fixing bacteria are the main strains used to produce microbial fertilizers. Composite strains have better nitrogen-fixing effects than single strains. However, because different strains have different effects such as promotion, antagonism or no correlation, the selection of composite strains is the key and difficulty in making microbial fertilizers.

[0004] Grapes are known as the top of the world's four major fruits. They are rich in nutrition, widely used, beautiful in color, fragrant, and delicious. They are the best fruits. They can be eaten fresh or made into wine, and their fruits, roots, and leaves can all be used as medicine. The whole body is a treasure. Therefore, they are planted in many parts of the world. Grapes themselves cannot fix atmospheric nitrogen, and they need to rely on exogenous fertilization to supply the required nitrogen to improve the quality and yield of grapes. However, chemical fertilization is still the main method at present, and long-term or excessive use of chemical nitrogen-containing fertilizers may cause nitrogen to be washed away with water, which is not conducive to plant absorption and utilization, resulting in nutrient deficiency, or nitrogen fertilizers are washed out from the roots of plants and infiltrated into deeper groundwater, causing environmental pollution. At present, there are many nitrogen-fixing microbial fertilizers on the market. When used alone in the process of grape planting, they cannot fully exert the nitrogen-fixing effect, it is difficult to ensure the nitrogen content and fertility in the soil, and it also affects the growth of grapes. Therefore, providing a nitrogen-fixing microbial fertilizer suitable for grape planting is of great significance for soil fertility improvement and grape production. Summary of the invention

[0005] The purpose of the present invention is to provide a nitrogen-fixing bacterial agent for increasing soil nitrogen reserves and a preparation method thereof, so as to solve the problems existing in the above-mentioned prior art. The nitrogen-fixing bacterial agent prepared by compounding Azospirillum, Bacillus subtilis and trehalose can significantly increase nitrogen reserves in the soil and improve grape quality and yield.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a nitrogen-fixing bacteria agent, comprising nitrogen-fixing bacteria and an inducer, wherein the nitrogen-fixing bacteria are non-pathogenic Azospirillum and Bacillus subtilis, and the inducer is trehalose.

[0008] Preferably, the deposit number of the Azospirillum is CGMCC No.6778.

[0009] The present invention also provides a method for preparing the nitrogen-fixing bacterial agent, comprising the following steps:

[0010] The bacterial powders of Azospirillum and Bacillus subtilis are obtained respectively, and then the bacterial powders of Azospirillum, Bacillus subtilis and trehalose are mixed to prepare a nitrogen-fixing bacterial agent.

[0011] Preferably, the mass ratio of the Azospirillum powder, the Bacillus subtilis powder and the trehalose is (3-5):(1-3):(0.5-1.5).

[0012] Preferably, the mass ratio of the Azospirillum powder, the Bacillus subtilis powder and the trehalose is 3:1.25:1.

[0013] The present invention also provides application of the nitrogen-fixing bacteria agent in improving soil nitrogen reserves.

[0014] The invention also provides application of the nitrogen-fixing bacteria agent in improving grape quality and yield.

[0015] Preferably, the improving the quality of grapes comprises improving the quality of grapes by reducing the thickness of grape skin.

[0016] The present invention also provides a method for increasing soil nitrogen reserves or increasing grape quality and yield, comprising the step of applying the nitrogen-fixing bacteria agent to the soil.

[0017] Preferably, the application method is: during the grape seedling stage, the nitrogen-fixing bacteria agent is applied to the roots at a rate of 1-2 kg / mu, and during the fruit-setting stage, the nitrogen-fixing bacteria agent is applied to the roots at a rate of 3-5 kg / mu.

[0018] The present invention discloses the following technical effects:

[0019] The nitrogen-fixing bacteria agent composed of Azospirillum, Bacillus subtilis and trehalose of the present invention is not antagonistic to each other, and the use of trehalose can recruit the two bacteria to combine with beneficial bacteria in the soil to quickly form a dominant bacterial community, and the three cooperate with each other to have a significant synergistic effect. It is found through experiments that the nitrogen-fixing bacteria agent formed by the two bacteria and trehalose can significantly improve the nitrogen reserves in the soil, provide advantageous conditions for grapes to absorb and utilize nitrogen elements, and can significantly promote the growth of grapes and improve the yield and quality through the secretion and conversion of nitrogen elements and small molecular nutrients that are beneficial to the absorption and utilization of grapes by the dominant bacterial community. DETAILED DESCRIPTION

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0022] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0023] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0024] The words “include,” “including,” “have,” “contain,” etc. used in the present invention are open-ended terms, meaning including but not limited to.

[0025] The following examples involve Azospirillum humicireducens SgZ-5 T, whose deposit number is CGMCC No.6778, purchased from the General Microbiology Center of the China Microbiological Culture Collection Administration Committee; the bacteria have also been disclosed in the authorized patent "CN102965307B A strain of nitrogen-fixing spirulina and its microbial preparation". Bacillus subtilis fermentation powder is commercially available. The Bacillus subtilis used in the embodiment of the present invention is purchased from the official website of China Microbiological Strain (link https: / / www.biobw.org / China-strain / bio-67659.html), with a platform number of Bio-67659. Bacillus subtilis can also be obtained through other channels. The above strains can all be purchased from the above institutions or units.

[0026] Trehalose is commercially available, and the trehalose used in the examples of the present invention was purchased from Hebei Lihua Biotechnology Co., Ltd.

[0027] Azospirillum powder can be obtained as follows:

[0028] (1) Preparing a seed solution of Azospirillum: After activating Azospirillum, inoculate the solution with NB medium and culture at 30° C. and 150 rpm for 24 h to obtain a seed solution;

[0029] (2) preparing fermentation broth of Azospirillum: inoculating NB medium with the seed liquid at a 5% inoculum amount, culturing at 30° C. and 150 rpm for 4 days to obtain fermentation broth;

[0030] (3) The fermentation liquid is concentrated and freeze-dried to obtain Azospirillum powder.

[0031] The Bacillus subtilis powder directly adopts the Bacillus subtilis fermentation powder purchased above.

[0032] The viable bacterial counts of Azospirillum and Bacillus subtilis in the bacterial powders of the following examples are respectively above 3 billion / g and above 1 billion / g.

[0033] Example 1 Preparation of nitrogen-fixing bacteria agent

[0034] The nitrogen-fixing bacteria agent includes the following components: Azospirillum powder, Bacillus subtilis powder and trehalose, with a mass ratio of 3:1:0.5.

[0035] The preparation method of the nitrogen-fixing bacterial agent is as follows: firstly, the powder of Azospirillum and the powder of Bacillus subtilis are prepared respectively, and then the powders are evenly mixed with trehalose according to the dosage ratio to prepare the nitrogen-fixing bacterial agent.

[0036] Example 2 Preparation of nitrogen-fixing bacteria agent

[0037] The nitrogen-fixing bacteria agent includes the following components: Azospirillum powder, Bacillus subtilis powder and trehalose, with a mass ratio of 3.5:1:0.5.

[0038] The preparation method of the nitrogen-fixing bacteria agent is the same as that in Example 1.

[0039] Example 3 Preparation of nitrogen-fixing bacteria agent

[0040] The nitrogen-fixing bacteria agent includes the following components: Azospirillum powder, Bacillus subtilis powder and trehalose, with a mass ratio of 4:1.5:0.5.

[0041] The preparation method of the nitrogen-fixing bacteria agent is the same as that in Example 1.

[0042] Example 4 Preparation of nitrogen-fixing bacteria agent

[0043] The nitrogen-fixing bacteria agent includes the following components: Azospirillum powder, Bacillus subtilis powder and trehalose, with a mass ratio of 4:1.5:1.5.

[0044] The preparation method of the nitrogen-fixing bacteria agent is the same as that in Example 1.

[0045] Example 5 Preparation of nitrogen-fixing bacteria agent

[0046] The nitrogen-fixing bacteria agent includes the following components: Azospirillum powder, Bacillus subtilis powder and trehalose, with a mass ratio of 3:3:1.5.

[0047] The preparation method of the nitrogen-fixing bacteria agent is the same as that in Example 1.

[0048] Example 6 Preparation of nitrogen-fixing bacteria agent

[0049] The nitrogen-fixing bacteria agent includes the following components: Azospirillum powder, Bacillus subtilis powder and trehalose, with a mass ratio of 3:1.25:1.

[0050] The preparation method of the nitrogen-fixing bacteria agent is the same as that in Example 1.

[0051] Example 7 Preparation of nitrogen-fixing bacteria agent

[0052] The nitrogen-fixing bacteria agent includes the following components: Azospirillum powder, Bacillus subtilis powder and trehalose, with a mass ratio of 3:1:1.

[0053] The preparation method of the nitrogen-fixing bacteria agent is the same as that in Example 1.

[0054] Example 8 Preparation of nitrogen-fixing bacteria agent

[0055] The nitrogen-fixing bacteria agent includes the following components: Azospirillum powder, Bacillus subtilis powder and trehalose, with a mass ratio of 5:2:1.

[0056] The preparation method of the nitrogen-fixing bacteria agent is the same as that in Example 1.

[0057] Example 9 Preparation of nitrogen-fixing bacteria agent

[0058] The nitrogen-fixing bacteria agent includes the following components: Azospirillum powder, Bacillus subtilis powder and trehalose, with a mass ratio of 5:3:1.5.

[0059] The preparation method of the nitrogen-fixing bacteria agent is the same as that in Example 1.

[0060] Comparative Example 1

[0061] Compared with Example 6, the difference is that the nitrogen-fixing bacteria agent does not contain Azospirillum. The other method steps are the same.

[0062] Comparative Example 2

[0063] Compared with Example 6, the difference is that the nitrogen-fixing bacterial agent does not contain Bacillus subtilis. The other method steps are the same.

[0064] Comparative Example 3

[0065] Compared with Example 6, the difference is that the nitrogen-fixing bacterial agent does not contain trehalose. The other method steps are the same.

[0066] Application Example 1: Nitrogen-fixing bacteria improve soil nitrogen reserves

[0067] 13 plots were randomly selected in Xiongyue Town, Yingkou City, Liaoning Province, each with an area of ​​1 mu, and were numbered A1-A13. The ammonium nitrogen content in the soil was measured before spring plowing as a control; the nitrogen-fixing bacteria prepared in Examples 1-9 were applied to plots A1-A9 at 5 kg per mu during spring plowing, and the nitrogen-fixing bacteria prepared in Comparative Examples 1-3 were applied to plots A10-A12 at 5 kg per mu during spring plowing, and the changes in the content of ammonium nitrogen in the soil were measured after 2 months. A13 did not apply any nitrogen-fixing bacteria before and after spring plowing as a blank control.

[0068] The same amount of soil was taken according to the five-point sampling method, and then the soil samples sampled at the five points were mixed evenly and tested using a commercially available soil ammonium nitrogen kit.

[0069] Increase rate of ammonium nitrogen content (%) = (ammonium nitrogen content in soil two months after spring ploughing - ammonium nitrogen content in soil before spring ploughing) / ammonium nitrogen content in soil before spring ploughing

[0070] The statistical results of the increase rate of ammonium nitrogen content are shown in Table 1.

[0071] Table 1

[0072] Experimental site Increase rate of ammonium nitrogen content (%) A1 14.65 A2 12.04 A3 12.32 A4 13.44 A5 20.23 A6 30.12 A7 18.25 A8 16.75 A9 13.10 A10 6.52 A11 8.78 A12 10.08 A13 0.04

[0073] A13 in the table is used as a blank control. The ammonium nitrogen content in the soil before and after spring plowing has almost no change, indicating that the microbial community contained in the experimental soil itself has no effect on the increase of ammonium nitrogen content. The nitrogen-fixing bacteria prepared by applying nitrogen-fixing spirillum, Bacillus subtilis and trehalose to the soil can significantly improve soil nutrients and increase nitrogen reserves in the soil. Among them, nitrogen-fixing spirillum is a highly safe strain that can be applied to biological agriculture. It has a motile type, oxidase activity, catalase activity, etc., which can promote the formation of dominant beneficial bacteria in the soil and improve soil structure. Bacillus subtilis is also a highly safe strain that is non-toxic and harmless to humans and animals. The bacteria has strong adaptability and stable performance. It can secrete a variety of antibacterial peptides, biological enzymes and other ingredients. It can improve soil fertility and fertilizer utilization, and can also inhibit the absorption of nitrate nitrogen, heavy metals and pesticides by plants, etc., which can significantly improve soil structure. Trehalose itself acts as a carbon source to recruit microorganisms, prompting Azospirillum and Bacillus subtilis to combine with beneficial bacteria in the soil to form a dominant bacterial community, improve the microenvironment for microbial growth, and provide a basis for the production of soil nitrogen transformation-related microorganisms and key enzyme activities, thereby achieving the purpose of regulating the ammonium nitrogen content in the soil.

[0074] Application Example 2: Nitrogen-fixing bacteria improve grape quality and yield

[0075] Dengta grapes are a specialty of Dengta City, Liaoyang City, Liaoning Province, and a national agricultural product geographical indication, providing people with a special product. However, Dengta grapes have thick skin, are acidic, and have low sugar content, which also limits their popularity in the market. The thick skin and low sweetness of grapes are likely caused by poor water and soil or insufficient sunlight. The present invention uses the nitrogen-fixing bacteria agent of Example 6 of the present invention during grape planting, and it is found that the problems of thick grape skin and poor sweetness can be significantly improved.

[0076] The specific implementation method is as follows: 30 grapes (3 years old) from a grape grower's planting base in Dengta City were randomly selected and randomly divided into an experimental group and a control group, with 15 grapes in each group and 400 grapes per mu. The experimental group applied 2.5g of nitrogen-fixing bacteria to the roots of each grape during the seedling stage, once; and 10g of nitrogen-fixing bacteria to the roots of each grape during the fruit-setting stage. The control group did not use any nitrogen-fixing bacteria during the seedling and fruit-setting stages. Management was carried out according to conventional grape planting. The grapes were harvested at maturity, and the grape yield, tannin content in the grape skin, and glucose content were measured.

[0077] Table 2 Grape yield

[0078] Grouping Experimental Group Control group Yield 5.12kg / plant 4.31kg / plant

[0079] The tannin content in grape skin was determined by Folin phenol reagent method. The results are shown in Table 3 below.

[0080] Table 3 Grape skin tannin content

[0081] Grouping Experimental Group Control group Tannin content 28mg / g 42mg / g

[0082] The amount of tannin is closely related to the thickness of the grape skin, so the determination of the tannin content in the grape skin can reflect the thickness of the grape skin to a certain extent. The results in Table 3 show that the tannin content in the grape skin of the experimental group is significantly lower than that of the control group, indicating that the grape skin of the experimental group is significantly thinner than that of the control group.

[0083] Three bunches of grapes were randomly selected from each group, and five grapes were collected from the upper, middle and lower parts, peeled, crushed into puree, and the sugar content was measured using a saccharimeter. The results are shown in Table 4 below.

[0084] Table 4 Glucose content

[0085] Grouping Experimental Group Control group Brix 23％ 19％

[0086] The results in Table 4 show that compared with the control group, the glucose level in the experimental group was significantly increased.

[0087] The above results indicate that the microbial agent prepared by the present invention has no antagonistic effect between the Azospirillum and Bacillus subtilis contained therein, and can effectively improve soil nitrogen reserves through the recruitment effect of trehalose, thereby laying a foundation for the formation and colonization of beneficial microbial flora, secreting and converting nitrogen elements and other small molecule nutrients that are beneficial to the absorption and utilization of grapes, thereby creating favorable conditions for improving the skin thickness, sweetness and yield of grapes.

[0088] 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. A nitrogen-fixing bacterial agent, characterized in that: The invention comprises nitrogen-fixing bacteria and an inducer, wherein the nitrogen-fixing bacteria are non-pathogenic Azospirillum and Bacillus subtilis, and the inducer is trehalose.

2. The nitrogen-fixing bacterial agent according to claim 1, characterized in that The deposit number of the Azospirillum is CGMCC No.6778.

3. A method for preparing the nitrogen-fixing bacterial agent according to claim 1 or 2, characterized in that: The following steps are involved: The bacterial powders of Azospirillum and Bacillus subtilis are obtained respectively, and then the bacterial powders of Azospirillum, Bacillus subtilis and trehalose are mixed to prepare a nitrogen-fixing bacterial agent.

4. The preparation method according to claim 3, characterized in that: The mass ratio of the Azospirillum powder, the Bacillus subtilis powder and the trehalose is (3-5):(1-3):(0.5-1.5).

5. The preparation method according to claim 4, characterized in that: The mass ratio of the Azospirillum powder, the Bacillus subtilis powder and the trehalose is 3:1.25:

1.

6. Use of the nitrogen-fixing bacterial agent as claimed in claim 1 or 2 in increasing soil nitrogen reserves.

7. Use of the nitrogen-fixing bacteria agent as claimed in claim 1 or 2 in improving grape quality and yield.

8. The use according to claim 7, characterized in that The improving of grape quality comprises improving grape quality by reducing the thickness of grape skin.

9. A method for increasing soil nitrogen reserves or improving grape quality and yield, characterized in that: The method comprises the step of applying the nitrogen-fixing bacteria agent according to claim 1 or 2 to the soil.

10. The method according to claim 9, characterized in that The application method is as follows: during the grape seedling stage, the nitrogen-fixing bacteria agent is applied to the roots at a rate of 1-2 kg / mu; during the fruit setting stage, the nitrogen-fixing bacteria agent is applied to the roots at a rate of 3-5 kg / mu.