Bacillus atrophaeus, application of bacillus atrophaeus, microbial agent for saline-alkali soil and application of microbial agent

Abstract

The invention relates to the technical field of microorganism application, and particularly discloses bacillus atrophaeus and application thereof, and a microbial agent for saline-alkali soil and application thereof. The preservation number of the bacillus atrophaeus is CGMCC (China General Microbiological Culture Collection Center) Meanwhile, the invention further provides a microbial agent prepared from the bacillus atrophaeus, bacillus subtilis, bacillus licheniformis and bacillus velezensis, the preservation number of the bacillus subtilis is CGMCC No.29774, and the preservation number of the bacillus licheniformis is CGMCC No.29774. The preservation number of the bacillus licheniformis is CGMCC (China General Microbiological Culture Collection Center) The preservation number of the bacillus velezensis is CGMCC (China General Microbiological Culture Collection Center) The microbial agent for saline-alkali soil provided by the invention can significantly improve the emergence rate of corn seeds, the plant height and the root fresh weight of corn plants under the condition of saline-alkali soil, and can effectively improve the yield of crops under severe conditions.

Description

Technical Field

[0001] The present application relates to the technical field of microbial application, and more specifically, to a Bacillus atrophaeus and its application, and a microbial agent for saline-alkali land and its application. Background Art

[0002] Saline-alkali land is a general term for various types of salinized and alkaline soils. The soil in saline-alkali land usually has the characteristics of high salt and high alkalinity. The salt contained in saline-alkali soil will affect the normal growth of crops. The improvement of saline-alkali land can not only improve land utilization, but also is an important guarantee for food security.

[0003] At present, saline-alkali land improvement is mainly divided into physical, chemical and biological methods. Biological method can not only improve the soil microecological environment, but also enhance the stress resistance of crops, which is one of the important measures to improve saline-alkali land.

[0004] Therefore, in order to better improve saline-alkali land, it is urgent to find new bacterial community structures to improve the arable land environment through biological methods, enhance crop resistance, and ensure food production. Summary of the invention

[0005] The present application provides a bacillus atrophaeus and its application, a microbial agent for saline-alkali land and its application. The bacillus atrophaeus and the microbial agent for saline-alkali land comprising bacillus atrophaeus, bacillus subtilis, bacillus licheniformis and bacillus velez provided in the present application can significantly improve the emergence rate of corn seeds, the plant height and the fresh root weight of corn plants in saline-alkali land, and can effectively improve the yield of crops under adverse conditions.

[0006] In a first aspect, the present application provides a Bacillus atrophaeus, using the following technical solution: The atrophic Bacillus provided by the present application ( Bacillus atrophaeus ) was deposited in the General Microbiology Center of China Microorganism Culture Collection on August 23, 2023, with the deposit number CGMCC No. 28234, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0007] The strain was isolated from saline-alkali soil samples in Tacheng, Xinjiang. The strain is oval, with a bulge in the middle of the colony, white, irregularly wrinkled and dull, and with uneven edges. The strain can grow normally under 15-18% NaCl conditions and pH 10.

[0008] This strain can increase the germination rate of corn seeds, the plant height and fresh root weight of corn plants in saline-alkali land, and can effectively increase the yield of crops under adverse conditions.

[0009] In a second aspect, the present application provides a culture comprising the above-mentioned Bacillus atrophaeus.

[0010] In a third aspect, the present application provides a bacterial suspension comprising the above-mentioned Bacillus atrophaeus.

[0011] In a fourth aspect, the present application provides a bacterial agent, which includes the above-mentioned Bacillus atrophaeus.

[0012] In a fifth aspect, the present application provides a bacterial powder comprising the above-mentioned Bacillus atrophaeus.

[0013] In a sixth aspect, the present application provides a microbial agent for saline-alkali land, which adopts the following technical solution: A microbial agent for saline-alkali land comprises the above-mentioned Bacillus atrophaeus as well as Bacillus subtilis, Bacillus licheniformis and Bacillus velezii.

[0014] Wherein, the Bacillus subtilis ( Bacillus subtilis ) was deposited in the General Microbiology Center of China Microorganism Culture Collection on January 25, 2024, with the deposit number CGMCC No. 29774, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0015] Wherein, the Bacillus licheniformis ( Bacillus licheniformis ) was deposited in the General Microbiology Center of China Microorganism Culture Collection on January 25, 2024, with the deposit number CGMCC No. 29773, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0016] Wherein, the Bacillus Velezii ( Bacillus velezensis ) was deposited in the General Microbiology Center of China Microorganism Culture Collection on January 25, 2024, with the deposit number CGMCC No. 29772, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0017] The above-mentioned Bacillus subtilis was isolated from a saline-alkali soil sample in Bayannaoer, Inner Mongolia. The strain is straight rod-shaped, with a bulge in the middle of the colony, white, irregularly wrinkled and dull, and neat edges. The strain can grow normally under 15-18% NaCl conditions and pH 10.

[0018] The above-mentioned Bacillus licheniformis was isolated from a saline-alkali soil sample in Bayannaoer, Inner Mongolia. The strain is straight rod-shaped, with a bulge in the middle of the colony, white, irregularly wrinkled and dull, and neat edges. The strain can grow normally under 15-18% NaCl conditions and pH 10.

[0019] The above-mentioned Bacillus Velezii was isolated from saline-alkali soil samples in Tacheng, Xinjiang. The strain is rod-shaped, the colony is flat in the middle, white, shiny on the surface, and has irregular edges. The strain can grow normally under 15-18% NaCl conditions and pH 10.

[0020] The microbial agent for saline-alkali land provided in the present application can significantly improve the emergence rate of corn seeds, the plant height and fresh root weight of corn plants in saline-alkali land, and can effectively increase the yield of crops under adverse conditions.

[0021] Optionally, the weight proportions of each bacterial species in the microbial agent for saline-alkali land are as follows: 1-2 parts of Bacillus atrophaeus; 1-2 parts of Bacillus subtilis; 1-2 parts of Bacillus licheniformis; and 1-2 parts of Bacillus velezii.

[0022] In a seventh aspect, the present application provides an application of the above-mentioned Bacillus atrophaeus, culture, bacterial suspension, bacterial agent, bacterial powder or microbial agent for saline-alkali land in saline-alkali land crop breeding.

[0023] Optionally, the saline-alkali land microbial agent can increase the emergence rate of crop seeds, crop plant height and root fresh weight.

[0024] Optionally, when the weight ratio of the Bacillus subtilis, the Bacillus licheniformis, the Bacillus atrophaeus and the Bacillus velez is 1:1:2:2, the germination rate of corn seeds can reach 69.8%.

[0025] Optionally, when the usage of the microbial agent for saline-alkali land is 6-9 kg / mu, the plant height of corn plants can be increased by more than 75%, and the fresh weight of roots can be increased by more than 31%.

[0026] In summary, this application has the following beneficial effects: 1. The present application provides a Bacillus atrophaeus strain, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on August 23, 2023, with a deposit number of CGMCC No. 28234. The strain can grow normally under 15-18% NaCI conditions and a pH of 10.

[0027] 2. The atrophic Bacillus can increase the germination rate of crop seeds, the height of crop plants and the fresh weight of roots in saline-alkali land, and can effectively increase the yield of crops under adverse conditions. In particular, it can increase the germination rate of corn seeds, the height of corn plants and the fresh weight of roots.

[0028] 3. The present application also provides a microbial agent for saline-alkali land, which includes the above-mentioned Bacillus atrophaeus, Bacillus subtilis, Bacillus licheniformis and Bacillus velez.

[0029] In addition to the aforementioned Bacillus atrophaeus, Bacillus subtilis ( Bacillus subtilis ) is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No. 29774; Bacillus licheniformis ( Bacillus licheniformis ) is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No. 29773; Bacillus velez ( Bacillus velezensis ) is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No. 29772. The above strains can grow normally under the conditions of 15-18% NaCl and pH 10.

[0030] 4. The saline-alkali land microbial agent can significantly increase the germination rate of crop seeds, the height of crop plants and the fresh weight of roots in saline-alkali land, and can effectively increase the yield of crops under adverse conditions. In particular, it can increase the germination rate of corn seeds, the height of corn plants and the fresh weight of roots. DETAILED DESCRIPTION

[0031] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are only used for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the terms belong.

[0032] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0033] In the present application, the terms "comprise" or "include" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0034] The present application provides a Bacillus atrophaeus ( Bacillus atrophaeus ), the Bacillus atrophaeus is deposited in the General Microbiological Center of China Microorganism Culture Collection Committee, and the deposit number is CGMCC No. 28234. In addition, the application also provides the isolation, purification and cultivation methods of the strain. And also provides the culture, bacterial suspension, bacterial powder and bacterial agent including the strain.

[0035] The present application also provides the use of the above-mentioned Bacillus atrophaeus and the culture, bacterial suspension, bacterial powder and bacterial agent comprising the strain in saline-alkali land crop breeding.

[0036] The present application also provides a microbial agent for saline-alkali land. The microbial agent for saline-alkali land includes the above-mentioned Bacillus atrophaeus and Bacillus subtilis, Bacillus licheniformis and Bacillus velez. Among them, Bacillus subtilis ( Bacillus subtilis ) is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No. 29774; Bacillus licheniformis ( Bacillus licheniformis ) is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No. 29773; Bacillus velez ( Bacillus from Velez ) is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number being CGMCC No. 29772.

[0037] The present application also provides the use of the above-mentioned microbial agent for saline-alkali land in saline-alkali land crop breeding.

[0038] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application. The embodiments described below are exemplary and are only used to explain the present application, and cannot be interpreted as limiting the present application.

[0039] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0040] In the following embodiments, the formula of the LB solid culture medium involved is as follows: based on a volume of 1 L, it includes 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 15 g of agar; the formula of the LB liquid culture medium involved is as follows: based on a volume of 1 L, it includes 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl.

[0041] The present application is further described in detail below in conjunction with the embodiments and test results. Example Example 1

[0042] This example provides the separation and purification process of new strains of Bacillus atrophaeus, Bacillus subtilis, Bacillus licheniformis and Bacillus velez.

[0043] The details are as follows: (1) Soil sample collection The saline-alkali soil in Bayannur, Inner Mongolia and the saline-alkali soil in Tacheng, Xinjiang were collected respectively. There were no less than 5 sampling points in each of the above regions, and the soil profile depth was no less than 30cm. The soil samples from each of the above regions were mixed to form 5kg of soil samples. A total of 10kg of soil samples were collected from the two regions, stored at 4℃ and brought back to the laboratory for subsequent strain screening experiments.

[0044] (2) Isolation of target bacteria Step 1: Divide the soil from each area into 5 portions, 1 kg each, for a total of 10 portions; add them to a 5L triangular flask containing 2.5L sterile water in advance under sterile conditions, stir at room temperature for 30 minutes, let stand for 1 hour, and collect all the supernatant. According to this scheme, a total of 10 portions of supernatant were collected.

[0045] Take 500 mL of each supernatant and add it to a 5L Erlenmeyer flask containing 2.5L of LB liquid culture medium (pH 10) under sterile conditions, and culture it at 37°C for 24 hours. Dilute the supernatant to 10 -6 , select a dilution concentration of 10 -4 , 10 -5 , 10 -6 The supernatant was streaked on LB solid medium and inverted for 48 h at 37°C, and a total of 18 strains were obtained.

[0046] Step 2: The 18 strains obtained from the above screening were inoculated into a pre-sterilized 1L Erlenmeyer flask containing 500mL of LB liquid culture medium (NaCI concentration of 15%) under sterile conditions, and cultured at 37°C for 24h. The supernatant was spread on the LB solid culture medium and inverted for 48h at 37°C to obtain a total of 8 strains.

[0047] Step 3: The 8 strains obtained from the above screening were inoculated under sterile conditions into a pre-sterilized 1L Erlenmeyer flask containing 500mL of LB liquid culture medium (pH 10, NaCI concentration of 15%), and cultured at 37°C for 24h. The supernatant was spread on the LB solid culture medium and cultured inverted at 37°C for 48h. A total of 6 strains were obtained, named Strain No. 1, Strain No. 2, Strain No. 3, Strain No. 4, Strain No. 5 and Strain No. 6 respectively.

[0048] Step 4: The 6 strains obtained by the above screening were inoculated into 1L Erlenmeyer flasks containing 500mL of different types of LB liquid culture medium in advance under sterile conditions, and cultured at 37°C for 24h to measure the growth of each strain in different types of LB liquid culture medium. The results are shown in Table 1.

[0049] There are 9 different types of LB liquid culture media, namely: 1) Ordinary LB liquid medium; 2) LB liquid medium with a pH of 9 and a NaCl concentration of 10%; 3) LB liquid medium with a pH of 10 and a NaCl concentration of 10%; 4) LB liquid medium with a pH of 9 and a NaCl concentration of 15%; 5) LB liquid medium with a pH of 10 and a NaCl concentration of 15%; 6) LB liquid medium with a pH of 10 and a NaCl concentration of 18%; 7) LB liquid medium with a pH of 11 and a NaCl concentration of 18%; 8) LB liquid medium with a pH of 10 and a NaCl concentration of 20%; 9) LB liquid medium with a pH of 11 and a NaCl concentration of 20%.

[0050] Table 1 Growth of the 6 strains screened in different types of LB liquid culture media

[0051] As shown in Table 1, strain No. 2, strain No. 3, strain No. 4 and strain No. 6 grew well under different saline-alkali conditions, so the above 4 strains with salt-alkali resistance were finally screened out.

[0052] (3) Preliminary identification: The four strains isolated and purified above were examined and observed under a microscope. Based on the results of the microscopic examination, strain No. 2 was preliminarily identified as Bacillus subtilis, strain No. 3 as Bacillus licheniformis, strain No. 4 as Bacillus atrophaeus, and strain No. 6 as Bacillus velez. Example 2

[0053] This example provides the properties of strain No. 4 isolated and purified in Example 1 and 16S The results of rDNA sequencing are as follows: 1. Properties of strains (1) Morphological characteristics The strain is oval in shape, with a bulge in the middle of the colony, white in color, with irregular wrinkles and dull luster on the surface, and irregular edges.

[0054] (2) Cultivation characteristics The optimal growth conditions for this strain are: pH=7-10, temperature 35-37℃, and rotation speed 150-180r / min.

[0055] (3) Functional characteristics The strain can grow normally under 15-18% NaCl conditions and pH 10.

[0056] (4) The strain was identified using 16S rDNA sequencing technology, and the 16S rDNA sequence of the strain was obtained and compared. The phylogenetic tree was constructed using the neighbor-joining method using MEGA5.0 software to determine the position of the strain in the phylogenetic tree. The identification and comparison results showed that the strain was Bacillus atrophaeus ( Bacillus atrophaeus ).

[0057] Bacillus atrophaeus ( Bacillus atrophaeus ) was deposited in the General Microbiology Center of China Microorganism Culture Collection on August 23, 2023, with the deposit number CGMCC No. 28234, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. Example 3

[0058] This embodiment provides a fermentation broth of Bacillus atrophaeus.

[0059] The preparation method of the above fermentation liquid is as follows: The Bacillus atrophaeus separated and purified in Example 1 was activated, transferred to LB liquid culture medium with a pH of 7, and cultured at 35° C. and 180 r / min for 24 hours to obtain a fermentation broth of Bacillus atrophaeus. Example 4

[0060] This embodiment provides a fermentation supernatant of Bacillus atrophaeus.

[0061] The preparation method of the above fermentation supernatant is as follows: The Bacillus atrophaeus separated and purified in Example 1 was activated and transferred to an LB liquid culture medium with a pH of 7, and cultured at 35° C. and 180 r / min for 24 h to obtain a fermentation broth of Bacillus atrophaeus; the fermentation broth was centrifuged and the supernatant was retained, which was the fermentation supernatant of Bacillus atrophaeus. Example 5

[0062] This embodiment provides a bacterial suspension of Bacillus atrophaeus.

[0063] The preparation method of the above bacterial suspension is as follows: The Bacillus atrophaeus separated and purified in Example 1 was activated and transferred to an LB liquid culture medium with a pH of 7, and cultured at 35° C. and 180 r / min for 24 h to obtain a fermentation broth of Bacillus atrophaeus; the fermentation broth was centrifuged to retain the precipitate, and the precipitate was resuspended with sterile water to obtain a bacterial suspension of Bacillus atrophaeus. Example 6

[0064] This embodiment provides a bacterial agent of Bacillus atrophaeus.

[0065] The preparation process of the above-mentioned microbial agent is as follows: The activated strain plate was used to inoculate the Bacillus atrophaeus isolated and purified in Example 1 into a 500 mL conical flask with a liquid fermentation seed liquid culture medium, and cultured in a constant temperature shaker at 35° C. and 180 rpm for 24 h to obtain a cultured seed liquid for standby use.

[0066] The cultured seed liquid is inoculated into a 100L fermentation tank, the fermentation liquid volume of the fermentation tank is 70% of the total volume of the fermentation tank, the fermentation inoculation amount is 1%, the rotation speed is 180rpm, the temperature is 35℃, the ventilation volume is 1vvm, and the culture is carried out for 24 hours. When the spore rate reaches more than 90%, the tank is removed. This is the bacterial agent of Bacillus atrophaeus. Example 7

[0067] This embodiment provides a bacterial powder of Bacillus atrophaeus.

[0068] The specific preparation process of the above bacterial powder is as follows: the bacterial agent prepared in Example 6 is spray-dried to obtain bacterial powder, that is, the bacterial powder of Bacillus atrophaeus is obtained. Example 8

[0069] This example provides the properties of strain No. 2 isolated and purified in Example 1 and 16S The results of rDNA sequencing are as follows: 1. Properties of strains (1) Morphological characteristics The strain is straight rod-shaped, with a bulge in the middle of the colony, white in color, with irregular wrinkles and dull surface, and neat edges.

[0070] (2) Cultivation characteristics The optimal growth conditions for this strain are: pH=7-10, temperature 35-37℃, and rotation speed 180-200r / min.

[0071] (3) Functional characteristics The strain can grow normally under 15-18% NaCl conditions and pH 10.

[0072] (4) The strain was identified using 16S rDNA sequencing technology, and the 16S rDNA sequence of the strain was obtained and compared. The phylogenetic tree was constructed using the neighbor-joining method using MEGA5.0 software to determine the position of the strain in the phylogenetic tree. The identification and comparison results showed that the strain was Bacillus subtilis ( Bacillus subtilis ).

[0073] Bacillus subtilis ( Bacillus subtilis ) was deposited in the General Microbiology Center of China Microorganism Culture Collection on January 25, 2024, with the deposit number CGMCC No. 29774, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. Example 9

[0074] This embodiment provides a fermentation broth of Bacillus subtilis.

[0075] The preparation method of the above fermentation liquid is as follows: The Bacillus subtilis isolated and purified in Example 1 was activated, transferred to LB liquid culture medium with a pH of 7, and cultured at 35° C. and 200 r / min for 24 hours to obtain a fermentation liquid of Bacillus subtilis. Example 10

[0076] This embodiment provides a fermentation supernatant of Bacillus subtilis.

[0077] The preparation method of the above fermentation supernatant is as follows: The Bacillus subtilis separated and purified in Example 1 was activated and transferred to an LB liquid culture medium with a pH of 7, and cultured at 35° C. and 200 r / min for 24 h to obtain a fermentation broth of Bacillus subtilis; the fermentation broth was centrifuged to retain the supernatant, which was the fermentation supernatant of Bacillus subtilis. Embodiment 11

[0078] This embodiment provides a bacterial suspension of Bacillus subtilis.

[0079] The preparation method of the above bacterial suspension is as follows: The Bacillus subtilis isolated and purified in Example 1 was activated and transferred to an LB liquid culture medium with a pH of 7, and cultured at 35° C. and 200 r / min for 24 h to obtain a fermentation broth of Bacillus subtilis; the fermentation broth was centrifuged to retain the precipitate, and the precipitate was resuspended with sterile water to obtain a bacterial suspension of Bacillus subtilis. Example 12

[0080] This embodiment provides a bacterial agent of Bacillus subtilis.

[0081] The preparation process of the above-mentioned microbial agent is as follows: Using the activated strain plate, the Bacillus subtilis isolated and purified in Example 1 was inoculated into a 500 mL conical flask with a liquid fermentation seed liquid culture medium, and cultured in a constant temperature shaker at 35° C. and 200 rpm for 24 hours to obtain a cultured seed liquid for standby use.

[0082] The cultured seed liquid is inoculated into a 100L fermentation tank, the fermentation liquid volume of the fermentation tank is 70% of the total volume of the fermentation tank, the fermentation inoculation amount is 1%, the rotation speed is 200rpm, the temperature is 35℃, the ventilation volume is 1vvm, and the culture is carried out for 24 hours. When the spore rate reaches more than 90%, the tank is removed. This is the inoculum of Bacillus subtilis. Embodiment 13

[0083] This embodiment provides a bacterial powder of Bacillus subtilis.

[0084] The specific preparation process of the above-mentioned bacterial powder is as follows: the bacterial agent prepared in Example 12 is spray-dried to obtain bacterial powder, that is, the bacterial powder of Bacillus subtilis is obtained. Embodiment 14

[0085] This example provides the properties of strain No. 3 isolated and purified in Example 1 and 16S The results of rDNA sequencing are as follows: 1. Properties of strains (1) Morphological characteristics The strain is straight rod-shaped, with a bulge in the middle of the colony, white in color, with irregular wrinkles and dull surface, and neat edges.

[0086] (2) Cultivation characteristics The optimal growth conditions for this strain are: pH=7-10, temperature 35-37℃, and rotation speed 180-200r / min.

[0087] (3) Functional characteristics The strain can grow normally under 15-18% NaCl conditions and pH 10.

[0088] (4) The strain was identified using 16S rDNA sequencing technology, and the 16S rDNA sequence of the strain was obtained and compared. The phylogenetic tree was constructed using the neighbor-joining method using MEGA5.0 software to determine the position of the strain in the phylogenetic tree. The identification and comparison results showed that the strain was Bacillus licheniformis ( Bacillus lichen-like ).

[0089] Bacillus licheniformis ( Bacillus licheniformis) was deposited in the General Microbiology Center of China Microorganism Culture Collection on January 25, 2024, with the deposit number CGMCC No. 29773, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. Embodiment 15

[0090] This embodiment provides a fermentation broth of Bacillus licheniformis.

[0091] The preparation method of the above fermentation liquid is as follows: The Bacillus licheniformis isolated and purified in Example 1 was activated, transferred to an LB liquid culture medium with a pH of 7, and cultured at 35° C. and 200 r / min for 24 hours to obtain a fermentation liquid of Bacillus licheniformis. Example 16

[0092] This embodiment provides a fermentation supernatant of Bacillus licheniformis.

[0093] The preparation method of the above fermentation supernatant is as follows: The Bacillus licheniformis isolated and purified in Example 1 was activated and transferred to an LB liquid culture medium with a pH of 7, and cultured at 35° C. and 200 r / min for 24 h to obtain a fermentation broth of Bacillus licheniformis; the fermentation broth was centrifuged and the supernatant was retained, which was the fermentation supernatant of Bacillus licheniformis. Embodiment 17

[0094] This embodiment provides a bacterial suspension of Bacillus licheniformis.

[0095] The preparation method of the above bacterial suspension is as follows: The Bacillus licheniformis isolated and purified in Example 1 was activated and transferred to an LB liquid culture medium with a pH of 7, and cultured at 35° C. and 200 r / min for 24 h to obtain a fermentation broth of Bacillus licheniformis; the fermentation broth was centrifuged to retain the precipitate, and the precipitate was resuspended with sterile water to obtain a bacterial suspension of Bacillus licheniformis. Embodiment 18

[0096] This embodiment provides a bacterial agent of Bacillus licheniformis.

[0097] The preparation process of the above-mentioned microbial agent is as follows: The activated strain plate was used to inoculate the Bacillus licheniformis isolated and purified in Example 1 into a 500 mL conical flask with a liquid fermentation seed liquid culture medium, and cultured in a constant temperature shaker at 35° C. and 200 rpm for 24 h to obtain a cultured seed liquid for standby use.

[0098] The cultured seed liquid is inoculated into a 100L fermentation tank, the fermentation liquid volume of the fermentation tank is 70% of the total volume of the fermentation tank, the fermentation inoculation amount is 1%, the rotation speed is 200rpm, the temperature is 35℃, the ventilation volume is 1vvm, and the culture is carried out for 24 hours. When the spore rate reaches more than 90%, the tank is removed. This is the inoculum of Bacillus licheniformis. Embodiment 19

[0099] This embodiment provides a bacterial powder of Bacillus licheniformis.

[0100] The specific preparation process of the above-mentioned bacterial powder is as follows: the bacterial agent prepared in Example 18 is spray-dried to obtain bacterial powder, that is, the bacterial powder of Bacillus licheniformis is obtained. Embodiment 20

[0101] This example provides the properties of strain No. 6 isolated and purified in Example 1 and 16S The results of rDNA sequencing are as follows: 1. Properties of strains (1) Morphological characteristics The strain is rod-shaped, with a flat center, white color, shiny surface and irregular edges.

[0102] (2) Cultivation characteristics The optimal growth conditions for this strain are: pH=7-10, temperature 35-37℃, and rotation speed 180-200r / min.

[0103] (3) Functional characteristics The strain can grow normally under 15-18% NaCl conditions and pH 10.

[0104] (4) The strain was identified using 16S rDNA sequencing technology, and the 16S rDNA sequence of the strain was obtained and compared. The phylogenetic tree was constructed using the neighbor-joining method using MEGA5.0 software to determine the position of the strain in the phylogenetic tree. The identification and comparison results showed that the strain was Bacillus velezinsis ( Bacillus velezensis ).

[0105] Bacillus velez Bacillus velezensis ) was deposited in the General Microbiology Center of China Microorganism Culture Collection on January 25, 2024, with the deposit number CGMCC No. 29772, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. Embodiment 21

[0106] This embodiment provides a fermentation broth of Bacillus Velezii.

[0107] The preparation method of the above fermentation liquid is as follows: The Bacillus Velez subtilis isolated and purified in Example 1 was activated and transferred to an LB liquid culture medium with a pH of 7, and cultured at 35° C. and 180 r / min for 24 hours to obtain a fermentation broth of Bacillus Velez subtilis. Embodiment 22

[0108] This embodiment provides a fermentation supernatant of Bacillus Velezii.

[0109] The preparation method of the above fermentation supernatant is as follows: The Bacillus Velez subtilis separated and purified in Example 1 was activated and transferred to an LB liquid culture medium with a pH of 7, and cultured at 35° C. and 180 r / min for 24 h to obtain a fermentation broth of Bacillus Velez subtilis; the fermentation broth was centrifuged and the supernatant was retained, which was the fermentation supernatant of Bacillus Velez subtilis. Embodiment 23

[0110] This embodiment provides a bacterial suspension of Bacillus Velezii.

[0111] The preparation method of the above bacterial suspension is as follows: The Bacillus Velez subtilis obtained by separation and purification in Example 1 was activated and transferred to an LB liquid culture medium with a pH of 7, and cultured at 35° C. and 180 r / min for 24 h to obtain a fermentation broth of Bacillus Velez subtilis; the fermentation broth was centrifuged to retain the precipitate, and the precipitate was resuspended with sterile water to obtain a bacterial suspension of Bacillus Velez subtilis. Embodiment 24

[0112] This embodiment provides a bacterial agent of Bacillus Velezii.

[0113] The preparation process of the above-mentioned microbial agent is as follows: The activated strain plate was used to inoculate the Bacillus Velezii isolated and purified in Example 1 into a 500 mL conical flask with a liquid fermentation seed liquid culture medium, and cultured in a constant temperature shaker at 35° C. and 180 rpm for 24 h to obtain a cultured seed liquid for standby use.

[0114] The cultured seed liquid is inoculated into a 100L fermentation tank, the fermentation liquid volume of the fermentation tank is 70% of the total volume of the fermentation tank, the fermentation inoculation amount is 1%, the rotation speed is 180rpm, the temperature is 35℃, the ventilation volume is 1vvm, and the culture is carried out for 24 hours. When the spore rate reaches more than 90%, the tank is removed. This is the inoculum of Bacillus Velezii. Embodiment 25

[0115] This embodiment provides a bacterial powder of Bacillus Velezii.

[0116] The specific preparation process of the above-mentioned bacterial powder is as follows: the bacterial agent prepared in Example 18 is spray-dried to obtain bacterial powder, that is, the bacterial powder of Bacillus Velezii is obtained. Embodiment 26

[0117] This embodiment provides a microbial agent for saline-alkali land.

[0118] The microbial agent for saline-alkali land includes the powder of Bacillus subtilis prepared in Example 13, the powder of Bacillus licheniformis prepared in Example 19, the powder of Bacillus atrophaeus prepared in Example 7, and the powder of Bacillus velezii prepared in Example 25. The bacterial content of each type of powder is 2 billion / g.

[0119] In this embodiment, the above-mentioned types of bacterial powder are compounded in a weight ratio of 1:1:1:1. Examples 27-32

[0120] Examples 27-32 respectively provide a microbial agent for saline-alkali land.

[0121] The difference between the above-mentioned microbial agent for saline-alkali land and Example 26 is that the weight ratio of each type of bacterial powder is as shown in Table 2. The rest is consistent with Example 26.

[0122] Table 2 Weight ratio of various types of bacterial powder in microbial agents for different saline-alkali lands Performance test 1

[0123] The single strain bacterial powder and the microbial agent prepared by compounding bacterial powder were respectively used for crop planting test in saline-alkali land. The crop in this application is corn, and the method of use is seed dressing. The details are as follows: (1) Experimental field: Ordos corn planting base in Inner Mongolia.

[0124] (2) Seed mixing process: Mix 5g of bacterial powder or compound bacterial agent with 1500g of seeds at a weight ratio of 1:300.

[0125] (3) The mixed corn seeds were planted in the test field (1500g seeds / mu) and a small field test was conducted to verify the effects of single strain powder and microbial agents prepared by compound powder on corn seed germination. At the same time, corn seeds that had not been mixed were used as the control group. The results were statistically analyzed after 15 days.

[0126] The results are shown in Table 3.

[0127] Table 3 Effects of different single strains of bacterial powder and compound microbial agents on corn seed germination in saline-alkali land

[0128] As shown in Table 3, the Bacillus atrophaeus, Bacillus subtilis, Bacillus licheniformis and Bacillus velez screened and obtained in this application can all improve the emergence rate of corn seeds in saline-alkali soil conditions, but the effects of Bacillus subtilis, Bacillus licheniformis and Bacillus velez are significantly lower than that of Bacillus atrophaeus. The Bacillus atrophaeus (deposit number CGMCC No. 28234) in this application is the first Bacillus atrophaeus discovered to be able to improve the emergence rate of corn seeds in saline-alkali soil conditions.

[0129] At the same time, the microbial inoculant prepared using the powder of Bacillus atrophaeus, Bacillus subtilis, Bacillus licheniformis and Bacillus velezii in the present application can significantly improve the germination rate of corn seeds in saline-alkali land, and the improvement effect is greater than the improvement effect of the powder of Bacillus atrophaeus, Bacillus subtilis, Bacillus licheniformis and Bacillus velezii alone.

[0130] In particular, when the weight ratio of Bacillus subtilis powder, Bacillus licheniformis powder, Bacillus atrophaeus powder and Bacillus velez powder in the microbial agent is 1:1:2:2, the germination rate of corn seeds can reach 69.8%. Embodiment 33

[0131] This embodiment provides a microbial agent for saline-alkali land.

[0132] The microbial agent for saline-alkali land includes the bacterial suspension of Bacillus subtilis prepared in Example 11, the bacterial suspension of Bacillus licheniformis prepared in Example 17, the bacterial suspension of Bacillus atrophaeus prepared in Example 5, and the bacterial suspension of Bacillus velezii prepared in Example 23. The bacterial content of each type of bacterial suspension is 2 billion cfu / ml.

[0133] In this embodiment, the above-mentioned various types of bacterial suspensions are compounded according to a volume ratio of 1:1:2:2. Performance test 2

[0134] The saline-alkali soil microbial agent prepared in Example 33 was used in the saline-alkali soil to grow crops, and the effect of the microbial agent on the growth of crops was investigated. In this application, the crop was corn, and the microbial agent was used by dripping. The details are as follows: (1) Test field: Same as performance test 1.

[0135] (2) Use by dropper: The saline-alkali soil microbial agent prepared in Example 33 was diluted with organic fertilizer (AMS, water-soluble organic fertilizer, organic matter ≥ 50 g / L) at a volume ratio of 1:3. The amount of the diluted liquid used is shown in Table 4. At the same time, the test results using the same amount of organic fertilizer were used as a control.

[0136] (3) According to the dosage of microbial agents, add the microbial agents to the corn plants after emergence with a dropper to explore the effect of the microbial agents prepared by bacterial suspension on the growth of corn plants. At the same time, corn plants that were not treated with microbial agents were used as the control group. The results were statistically analyzed after 15 days.

[0137] The results are shown in Table 4.

[0138] Table 4 Effects of microbial agents on the growth of corn plants in saline-alkali land

[0139] As shown in Table 4, the microbial agent for saline-alkali land prepared by using the bacterial suspension of Bacillus atrophaeus, the bacterial suspension of Bacillus subtilis, the bacterial suspension of Bacillus licheniformis and the bacterial suspension of Bacillus velezii in the present application can significantly increase the plant height and root fresh weight of corn plants in saline-alkali land. In particular, when the usage of the microbial agent for saline-alkali land is 6.25 kg / mu, the plant height of the corn plant can be increased by 75.66%, and the root fresh weight can be increased by 33.65%.

[0140] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0141] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application 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 application.

Claims

1. A Bacillus atrophaeus, characterized in that: The Bacillus atrophaeus ( Bacillus atrophaeus ) is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No. 28234.

2. A culture, characterized in that The culture comprises the Bacillus atrophaeus of claim 1.

3. A bacterial suspension, characterized in that The bacterial suspension comprises the Bacillus atrophaeus according to claim 1.

4. A bacterial agent, characterized in that The bacterial agent includes the Bacillus atrophaeus according to claim 1.

5. A bacterial powder, characterized in that: The bacterial powder includes the Bacillus atrophaeus according to claim 1.

6. A microbial agent for saline-alkali land, characterized in that: The microbial agent for saline-alkali land includes the Bacillus atrophaeus as claimed in claim 1, as well as Bacillus subtilis, Bacillus licheniformis and Bacillus velez; The Bacillus subtilis ( Bacillus subtilis ) is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No. 29774; The Bacillus licheniformis ( Bacillus licheniformis ) is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number being CGMCC No. 29773; The Bacillus Velez Bacillus velezensis ) is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No. 29772.

7. The microbial agent for saline-alkali land according to claim 6, characterized in that: The weight proportions of the various bacterial species in the saline-alkali land microbial agent are as follows: 1-2 parts of Bacillus atrophaeus; 1-2 parts of Bacillus subtilis; 1-2 parts of Bacillus licheniformis; and 1-2 parts of Bacillus velezii.

8. Use of the Bacillus atrophaeus according to claim 1, the culture according to claim 2, the bacterial suspension according to claim 3, the bacterial agent according to claim 4, the bacterial powder according to claim 5, or the microbial agent for saline-alkali land according to any one of claims 6 to 7 in saline-alkali land crop breeding.

9. The use according to claim 8, characterized in that: The microbial agent for saline-alkali land can improve the emergence rate of crop seeds, plant height and fresh root weight of crops.

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