Antarctic deep sea salt-resistant rooting bacterium and application thereof in fertilizer

Abstract

The invention relates to an Antarctic deep sea salt-resistant rooting bacterium and an application thereof in a fertilizer, and the Antarctic deep sea salt-resistant rooting bacterium is characterized by comprising globulin extraction and microorganism extraction, the microorganism extracted from the microorganism is bacillus N311 which is preserved in the China Center for Type Culture Collection on October 15, 2015, and the preservation number of the China Center for Type Culture Collection is CCTCC M 2015607; the bacillus N311 is bacillus velezensis, and the bacillus N311 is bacillus velezensis. The glycinin extracting solution prepared by the invention not only has better antibacterial property, but also can promote the growth of crop roots.

Description

TECHNICAL FIELD

[0001] The application relates to an Antarctic deep-sea salt-tolerant rhizobium and application thereof to fertilizers. BACKGROUND

[0002] The Antarctic-derived Bacillus N311 has been preserved in the China Center for Type Culture Collection on October 15, 2015, and the preservation center preservation number is CCTCC M 2015607, the patent number of the strain applied for is 2015109992628, and the patent name is Antarctic-derived Bacillus N311 and application thereof to prevention and treatment of plant pathogenic fungi. The supernatant prepared from the Antarctic-derived Bacillus N311 has good bacteriostasis.

[0003] The company mainly engages in research and development, production and import and export business of microbial preparations, water-soluble fertilizers and the like. The company believes that the Antarctic has a special geographical environment, and the growth conditions of the strain are much more harsh than those in inland, so the strain has a better prospect in application to agriculture. After evaluation by the company, the Antarctic-derived Bacillus N311 has a good application prospect, and the company has the ability to transform the patent of the scientific research institute. After several negotiations, the company purchased the patent, and transferred the patent to the company on December 27, 2023. In order to further determine the functionality of the strain, the company applied for identification of the strain, and the identification result was issued on March 4, 2025. The strain is Bacillus velezensis.

[0004] The company has carried out direct application research and indirect application research on the Bacillus N311 to tap the potential of the strain in application to agriculture.

[0005] The present application is a research on the direct application of the Bacillus N311. Before the research of the company, no one has developed and researched the functions of the strain other than the known bacteriostasis. SUMMARY

[0006] The application provides an Antarctic deep-sea salt-tolerant rhizobium and application thereof to fertilizers, and solves the technical problem of developing and researching the functions of the Bacillus N311 other than the known bacteriostasis, so that the strain can better serve agriculture.

[0007] In order to solve the above technical problem, the following technical scheme is adopted in the application. The Antarctic deep-sea salt-tolerant rhizobium is the Bacillus N311, which has been preserved in the China Center for Type Culture Collection on October 15, 2015, and the preservation center preservation number is CCTCC M 2015607. The strain was identified as Bacillus velezensis by the College of Life Science and Technology, Huazhong Agricultural University and the Microbial Product Quality Inspection and Test Center of the Ministry of Agriculture and Rural Affairs (Wuhan) on March 4, 2025.

[0008] The physiological and biochemical characteristics of the Bacillus N311 are identified as, Anaerobic growth - V-P assay Nitrate reduction + Starch hydrolysis Gelatin liquefaction + Citrate utilization D-xylose acid production + L-arabinose acid production D-mannitol acid production + pH5.7 growth Propionate utilization - 7% sodium chloride growth +.

[0009] The Bacillus N311 is prepared according to the following steps, The preserved Bacillus N311 is inoculated into a seed culture medium, and cultured at 28-30°C for 24-48h to activate, to obtain activated Bacillus N311; The activated Bacillus N311 is inoculated into an LB culture medium, and cultured at 28-30°C with 180-220r / min shaking for 48-60h, to obtain Bacillus N311 seed liquid; The Bacillus N311 seed liquid is spray dried, to obtain Bacillus N311 seed powder; The Bacillus N311 includes one or both of Bacillus N311 seed liquid and Bacillus N311 seed powder; The seed culture medium is: sodium chloride 10g / L, tryptone 10g / L, yeast extract 5g / L, agar 20g / L, and the rest is water, sterilized at 121°C for 20min; The LB culture medium is: sodium chloride 10g / L, tryptone 10g / L, yeast extract 5g / L, and the rest is water, sterilized at 121°C for 20min; The spray drying temperature is not more than 60°C.

[0010] The prepared Antarctic deep-sea salt-tolerant root-forming bacteria are applied to a fertilizer, and the fertilizer includes soybean meal and decomposed manure; the preparation method is, The Bacillus N311 seed liquid is sprayed on the mixture of soybean meal and decomposed manure, to obtain the fertilizer containing the Antarctic deep-sea salt-tolerant root-forming bacteria; or The Bacillus N311 seed powder, soybean meal and decomposed manure are uniformly mixed, to obtain the fertilizer containing the Antarctic deep-sea salt-tolerant root-forming bacteria; The mass ratio of Bacillus N311, soybean meal and decomposed manure is 0.1-60:5-80:5-80; The organic matter content in the fertilizer is ≥40, and the water content is ≤30%.

[0011] The fertilizer also includes one or more of urea, ammonium sulfate, bentonite, potassium fulvate, mushroom residue, monoammonium phosphate, potassium chloride and potassium sulfate.

[0012] The application has the following beneficial technical effects: 1. The application develops and researches the functions of Bacillus N311 other than the known bacteriostasis.

[0013] 2. The application directly applies Bacillus N311 to agriculture, which also has the effect of promoting root growth, and is identified to grow in 7% sodium chloride, that is, Bacillus N311 has high salt resistance.

[0014] 3. Bacillus N311 of the application is also verified by tests to be able to inhibit the growth of nitrous acid bacteria, which in agriculture is to inhibit the conversion of ammonium nitrogen to nitrous acid nitrogen, and further inhibit the conversion of nitrous acid nitrogen to nitric acid nitrogen, thereby reducing the loss of nitrogen and increasing the utilization rate of nitrogen. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Figure 1 is a graph showing the bacteriostatic effect of Bacillus N311 seed liquid on nitrous acid bacteria. DETAILED DESCRIPTION

[0016] In order to better understand the above technical solutions, the technical solutions of the application will be described in detail below in combination with preferred examples. Example 1

[0017] A salt-tolerant root-growing bacterium in Antarctic deep sea is Bacillus N311, which has been preserved in China Typical Culture Collection Center on October 15, 2015, and the preservation center preservation number is CCTCC M 2015607, and its characteristics are that the strain was re-identified as Bacillus velezensis by the College of Life Science and Technology of Huazhong Agricultural University and the Microbial Product Quality Inspection and Test Center of the Ministry of Agriculture and Rural Affairs (Wuhan) on March 4, 2025.

[0018] The physiological and biochemical characteristics of the Bacillus N311 are identified as, Anaerobic growth - V-P assay + Nitrate reduction + starch hydrolysis + Gelatin liquefaction + citrate utilization + D-xylose acid production + L-arabinose acid production + D-mannitol acid production + pH5.7 growth + Propionate utilization - 7% sodium chloride growth +.

[0019] The seed liquid of the Bacillus N311 is prepared according to the following steps, The bacillus N311 is inoculated into a seed culture medium, and cultured at 28℃ for 24h to activate, and the activated bacillus N311 is obtained; The activated bacillus N311 is inoculated into an LB culture medium, and cultured at 28℃ with 180r / min oscillation for 48h, and the bacillus N311 seed liquid is obtained; The seed culture medium is: sodium chloride 10g / L, tryptone 10g / L, yeast extract 5g / L, agar 20g / L, and the rest is water, and high-pressure sterilization is carried out at 121℃ for 20min; The LB culture medium is: sodium chloride 10g / L, tryptone 10g / L, yeast extract 5g / L, and the rest is water, and high-pressure sterilization is carried out at 121℃ for 20min.

[0020] Application of the Antarctic deep-sea salt-tolerant root fungus in fertilizer, The bacillus N311 seed liquid is sprayed on the mixture of soybean meal, decomposed pig manure and urea, and the fertilizer containing the Antarctic deep-sea salt-tolerant root fungus is obtained; The fertilizer is composed of soybean meal, decomposed pig manure, urea and bacillus N311 seed liquid according to the mass ratio of 50:45:4.8:0.2. Example 2

[0021] An Antarctic deep-sea salt-tolerant root fungus is bacillus N311, which has been preserved in China Typical Culture Collection Center on October 15, 2015, and the preservation center preservation number is CCTCC M 2015607, and the characteristics are that the strain is identified as bacillus velezensis by the College of Life Science and Technology of Huazhong Agricultural University and the Microbial Product Quality Inspection and Test Center (Wuhan) of the Ministry of Agriculture and Rural Affairs on March 4, 2025.

[0022] The physiological and biochemical characteristics of the bacillus N311 are identified as, Anaerobic growth - V-P assay + Nitrate reduction + starch hydrolysis + Gelatin liquefaction + citrate utilization + D-xylose acid production + L-arabinose acid production + D-mannitol acid production + pH5.7 growth + Propionate utilization - 7% sodium chloride growth +.

[0023] The bacillus N311 seed liquid is prepared according to the following steps, The bacillus N311 is inoculated into a seed culture medium, and cultured at 28℃ for 24h to activate, and the activated bacillus N311 is obtained; The activated Bacillus N311 is inoculated into the LB culture medium, and cultured at 30℃ and 180r / min for 48h, to obtain the seed solution of Bacillus N311. The seed culture medium comprises 10g / L of sodium chloride, 10g / L of tryptone, 5g / L of yeast extract, 20g / L of agar, and the balance is water, and is sterilized at 121℃ for 20min. The LB culture medium comprises 10g / L of sodium chloride, 10g / L of tryptone, 5g / L of yeast extract, and the balance is water, and is sterilized at 121℃ for 20min.

[0024] Application of the Antarctic deep-sea salt-tolerant Rhizobium in fertilizers, The seed solution of Bacillus N311 is sprayed on the mixture of soybean meal, decomposed pig manure and monoammonium phosphate, to obtain the fertilizer containing the Antarctic deep-sea salt-tolerant Rhizobium. The fertilizer is composed of soybean meal, decomposed pig manure, monoammonium phosphate and the seed solution of Bacillus N311, and the mass ratio is 30:60:9:1. Example 3

[0025] The Antarctic deep-sea salt-tolerant Rhizobium is Bacillus N311, which has been preserved in the China Center for Type Culture Collection on October 15, 2015, and the preservation number is CCTCC M 2015607.

[0026] The physiological and biochemical characteristics of Bacillus N311 are identified as follows, Anaerobic growth - V-P assay + Nitrate reduction + Starch hydrolysis + Gelatin liquefaction + Citrate utilization + D-xylose acid production + L-arabinose acid production + D-mannitol acid production + Growth at pH 5.7 + Propionate utilization - 7% sodium chloride growth +.

[0027] The seed solution of Bacillus N311 is prepared according to the following steps, The Bacillus N311 is inoculated into the seed culture medium, and cultured at 30℃ for 24h to obtain the activated Bacillus N311; The activated Bacillus N311 is inoculated into the LB culture medium, and cultured at 30℃ and 200r / min for 48h, to obtain the seed solution of Bacillus N311; The seed culture medium is: sodium chloride 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, agar 20 g / L, the balance is water, high pressure sterilization at 121℃ for 20 min; The LB culture medium is: sodium chloride 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, the balance is water, high pressure sterilization at 121℃ for 20 min.

[0028] Application of Antarctic deep-sea salt-tolerant root fungus in fertilizer, The mixture of soybean meal, mature pig manure and monoammonium phosphate is sprayed with Bacillus N311 seed liquid to obtain a fertilizer containing Antarctic deep-sea salt-tolerant root fungus; The fertilizer is composed of soybean meal, mature pig manure, monoammonium phosphate and Bacillus N311 seed liquid in a mass ratio of 30:60:9:1. Example 4

[0029] An Antarctic deep-sea salt-tolerant root fungus is Bacillus N311, which has been preserved in China Center for Type Culture Collection on October 15, 2015, and the preservation center preservation number is CCTCC M 2015607, and the strain is identified as Bacillus velezensis by the College of Life Science and Technology, Huazhong Agricultural University and the Microbial Product Quality Inspection and Test Center (Wuhan) of the Ministry of Agriculture and Rural Affairs on March 4, 2025.

[0030] The physiological and biochemical characteristics of the Bacillus N311 are identified as, Anaerobic growth - V-P assay + Nitrate reduction + starch hydrolysis + Gelatin liquefaction + citrate utilization + D-xylose acid production + L-arabinose acid production + D-mannitol acid production + pH5.7 growth + Propionate utilization - 7% sodium chloride growth +.

[0031] The Bacillus N311 seed liquid is prepared according to the following steps, The Bacillus N311 is inoculated into the seed culture medium, cultured at 30℃ for 24 h, activated, and the activated Bacillus N311 is obtained; The activated Bacillus N311 is inoculated into the LB culture medium, and cultured at 30℃ with 200 r / min shaking for 48 h, which is the Bacillus N311 seed liquid; The Bacillus N311 seed liquid is spray dried to obtain Bacillus N311 seed powder; The spray temperature is 60℃; The seed culture medium is: sodium chloride 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, agar 20 g / L, the balance is water, high pressure sterilization at 121 DEG C for 20 min; The LB culture medium is: sodium chloride 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, the balance is water, high pressure sterilization at 121 DEG C for 20 min.

[0032] Application of Antarctic deep-sea salt-tolerant Rhizobium in fertilizer, The mixture of Bacillus N311 seed powder, soybean meal, composted pig manure mushroom residue, ammonium sulfate and monoammonium phosphate is added to obtain the fertilizer containing Antarctic deep-sea salt-tolerant Rhizobium. The fertilizer is composed of soybean meal, composted pig manure, mushroom residue, ammonium sulfate, monoammonium phosphate and Bacillus N311 seed powder in a mass ratio of 25:39:9:15:8:4.

[0033] The beneficial effects of the present application are further illustrated below in combination with test data: Experiment 1 1.1 Test site: laboratory of Shandong Tudakuer Fertilizer Co., Ltd.

[0034] 1.2 Experimental detection: average number of roots (strips) and average length of roots (cm), average number of roots, calculation method: total number of roots / total number of plants; average length of roots, calculation method: total length of roots / root number.

[0035] 1.3 Test material: blank (water), comparison 1 (except that Bacillus vallismortis purchased from China Typical Culture Collection Center, preservation number is CCTCC AB 2016194, the other is consistent with example 1, the strain is derived from the soil of Xiaoshanjiao in Lichuan, Hubei, with broad-spectrum antibacterial activity), comparison 2 (except that Bacillus vallismortis purchased from China Typical Culture Collection Center, preservation number is CCTCC AB 205354, the other is consistent with example 1, the strain is derived from Xinjiang sand soil) and Bacillus seed liquid prepared in example 1.

[0036] 1.4 Test method: the plant test is Clivia nobilis; Clivia nobilis branches are purchased from the market, and the branches with basically consistent thickness are selected, cut to 45 cm each, 5 internodes are left at the lower part of the branch, 6 leaves are left at the upper part, the lower part of the branch is obliquely cut, and 0.5% potassium permanganate solution is used for disinfection for 5s, and then washed with clean water. The pruned Clivia nobilis is placed in a glass vase with a solution height of 22 cm, the vase is 29.5 cm high, the upper opening is 12.6 cm in diameter, and the lower opening is 10.4 cm in diameter, 4 Clivia nobilis are placed in each vase, and 10 parallel tests are made. The solution of blank is water, and the other is added with 5 ml of each treatment aqueous solution. The test period is 15 days.

[0037] The experiment is different in each treatment, and other treatments are consistent.

[0038] 2 Results and analysis The number of roots (strips), referred to as the number, and the length of the roots (cm), referred to as the length, are shown in Table 1 Table 1 As can be seen from Table 1, the prepared Example 1 of the present application has outstanding performance in both the number of roots and the length of roots, that is, Bacillus N311 has good root-promoting and root- forming properties.

[0039] Experiment two 1.1 Test site: Dali village, Jining Yutai county; corn variety: Longping 285.

[0040] 1.2 Experimental detection: yield (kg / acre), nitrogen content in corn stalks (%) and nitrogen content in corn kernels (%).

[0041] 1.3 Test materials: comparison 1 (except that Bacillus N311 is not added, other conditions are the same as Example 1), comparison 2 (except that soybean meal is replaced by mature pig manure, other conditions are the same as Example 1), comparison 2 (except that mature pig manure is replaced by soybean meal, other conditions are the same as Example 1) and Example 1 prepared fertilizer containing Antarctic deep-sea salt-tolerant root-forming bacteria. Among them, 4.8% urea is added to meet the requirement of total nutrient content >=4% in NY525-2021 organic fertilizer standard.

[0042] 1.4 Test method: Take two acres of corn land similar to the plot, divide it into six small areas, each small area is 180 square meters, the plant spacing of corn is 25*60 cm, the base application of each small area is 40 kg of fertilizer containing Antarctic deep-sea salt-tolerant root-forming bacteria, and 10 kg of 15-15-15 potassium chloride compound fertilizer is applied as a base.

[0043] 1.5 Detection method: uniform planting and uniform harvesting for each treatment, and weighing each small area separately. Kjeldahl nitrogen determination method is used for nitrogen content detection.

[0044] The harvest time is October 8, 2025.

[0045] The experiment is different in each treatment, and other treatments are consistent.

[0046] 2 Results and analysis Yield (kg / acre) is shown in Table 2 Table 2 As can be seen from Table 2, Bacillus N311, soybean meal and decomposed manure are indispensable. As can be seen from the comparison of the data of Comparative Example 1, Comparative Example 2, Comparative Example 3 and Example 1, Bacillus N311 has the effect of improving nitrogen utilization, which corresponds to the fact that Bacillus N311 can inhibit the growth of nitrite bacteria in the experiment.

Claims

1. A halotolerant root fungus in Antarctic deep sea, which is Bacillus N311, has been preserved in China Center for Type Culture Collection on October 15, 2015, and the preservation center preservation number is CCTCC M 2015607, and the characteristics are that, The strain is re-identified as Bacillus velezensis by the College of Life Sciences and Technology, Huazhong Agricultural University and the Microbial Product Quality Inspection and Test Center (Wuhan) of the Ministry of Agriculture and Rural Affairs on March 4, 2025.

2. The Antarctic deep sea halotolerant root fungus according to claim 1, characterized in that, The physiological and biochemical characteristics of the Bacillus N311 are identified as, Anaerobic growth - V-P assay + Nitrate reduction + Starch hydrolysis + Gelatin liquefaction + Citrate utilization + D-xylose acid production + L-arabinose acid production + D-mannitol acid production + pH5.7 growth + Propionate utilization - 7% sodium chloride growth +.

3. The Antarctic deep sea halotolerant root fungus according to claim 1, wherein The Bacillus N311 is prepared according to the following steps, The preserved Bacillus N311 is inoculated into a seed culture medium, cultured at 28-30 DEG C for 24-48 h to activate, and the activated Bacillus N311 is obtained; The activated Bacillus N311 is inoculated into an LB culture medium, cultured at 28-30 DEG C with 180-220 r / min shaking for 48-60 h, and the Bacillus N311 seed liquid is obtained; The Bacillus N311 seed liquid is spray dried, and the Bacillus N311 seed powder is obtained; The Bacillus N311 includes one or both of the Bacillus N311 seed liquid and the Bacillus N311 seed powder; The seed culture medium is: sodium chloride 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, agar 20 g / L, and the balance is water, sterilized at 121 DEG C for 20 min; The LB culture medium is: sodium chloride 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, and the balance is water, sterilized at 121 DEG C for 20 min; The spray drying temperature is not more than 60 DEG C.

4. Use of the Antarctic deep sea halotolerant root fungus prepared according to any one of claims 1 to 4 on fertilizers, characterized in that, The fertilizer includes soybean meal and decomposed manure; and the preparation method is, The Bacillus N311 seed liquid is sprayed on the mixture of soybean meal and decomposed manure to obtain the fertilizer containing the Antarctic deep-sea salt-tolerant rooting fungus; or The Bacillus N311 seed powder, soybean meal and decomposed manure are uniformly mixed to obtain the fertilizer containing the Antarctic deep-sea salt-tolerant rooting fungus; The mass ratio of the Bacillus N311, soybean meal and decomposed manure is 0.1-60:5-80:5-80; The organic matter content in the fertilizer is >=40, and the water content is <=30%.

5. Use of the Antarctic deep sea halotolerant endophytic bacterium prepared according to claim 4 in agriculture, characterized in that, The fertilizer further includes one or more of urea, ammonium sulfate, bentonite, potassium fulvate, mushroom residue, monoammonium phosphate, potassium chloride and potassium sulfate.

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