A fish sauce Bacillus and its microbial agent and application

Abstract

The present invention relates to the field of microorganisms, and discloses a fish sauce Bacillus and its microbial agent and application. The strain name of the fish sauce Bacillus is A01, and the preservation number is CGMCC No.32300. The fish sauce Bacillus A01 provided by the present invention can improve the salt-alkali tolerance of plants in salinized soil, promote the growth of plant nodules, and promote the growth of plants under salt stress conditions. This is the first time that a strain capable of promoting plant nodule growth and plant growth under salt stress has been found in the genus of fish sauce Bacillus.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a fish sauce bacillus and a microbial agent and application thereof. Background Art

[0002] With the continuous impact of global climate change and human activities, the problem of soil salinization is becoming increasingly serious and has become an important factor restricting agricultural production and ecological environment improvement. Improving and utilizing saline-alkali land is an important issue in the fields of agriculture and ecology.

[0003] As the "second genome" of plants, rhizosphere microorganisms have great potential in improving the ability of crops to resist saline-alkali stress and promoting the "suitability of crops for the right land". In recent years, the application of plant growth-promoting rhizobacteria (PGPR) in saline-alkali land improvement and crop production has gradually attracted attention, providing new ideas and methods for the sustainable utilization of saline-alkali land. PGPR improves the salt-alkali tolerance of plants through various mechanisms, including improving nutrient efficiency, regulating hormone homeostasis, enhancing plant induced resistance, and regulating the expression of transcription factors. These mechanisms help plants better adapt to saline-alkali environments, thereby improving crop yield and quality.

[0004] Fish sauce Bacillus spp. Jeotgalibacillus ) was proposed by Yoon et al. in 2001. Currently, there are few studies on this genus, and no strains have been reported that can promote nodule growth, improve plant salt-alkali tolerance, and thus promote plant growth under salt stress conditions. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a fish sauce Bacillus and its microbial agent and application. The fish sauce Bacillus A01 provided by the present invention can improve the salt-alkali tolerance of plants and promote the growth of plant nodules and plant growth under salt stress.

[0006] The specific technical scheme of the present invention is:

[0007] In a first aspect, the present invention provides a fish sauce Bacillus ( Jeotgalibacillus sp. ), the strain name of the fish sauce Bacillus is A01, and the preservation number is CGMCC No.32300.

[0008] The deposit information of the above strain (Bacillus subtilis A01) is as follows:

[0009] Name of depository: China General Microbiology Center of Microbiological Culture Collection Administration (abbreviated as “CGMCC”);

[0010] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0011] Deposit date: October 22, 2024;

[0012] Deposit number: CGMCC No.32300.

[0013] The strain provided by the present invention is a fish sauce Bacillus ( Jeotgalibacillus ) is a new species. Since the new species has not yet been named, the deposit certificate uses the term “ sp. ” refers to.

[0014] The strain (Bacillus spp. A01) provided by the present invention can play the following roles in salinized soil: improve the salt-alkali tolerance of plants, promote the growth of plant nodules, and promote the growth of plants under salt stress conditions. This provides a new strain resource for developing new biofertilizers and alleviating plant growth problems in saline-alkali soils.

[0015] In a second aspect, the present invention provides a microbial agent comprising the fish sauce Bacillus and auxiliary materials.

[0016] Preferably, the auxiliary material is a liquid culture medium.

[0017] Furthermore, the liquid culture medium contains 4-6 g / L of peptone, 0.5-1.5 g / L of yeast powder, 0.05-0.2 g / L of ferric citrate, 18-20 g / L of sodium chloride, 5-6.5 g / L of magnesium chloride, 3-4 g / L of sodium sulfate, 1.5-2 g / L of calcium chloride, 0.5-0.6 g / L of potassium chloride, 0.1-0.2 g / L of sodium carbonate, 0.05-0.1 g / L of potassium bromide, 0.03-0.05 g / L of strontium chloride, 0.01-0.03 g / L of boric acid, 0-0.0050.004 g / L of sodium silicate, 0-0.003 g / L of sodium fluoride, 0-0.002 g / L of sodium nitrate, and 0.005-0.01 g / L of disodium hydrogen phosphate.

[0018] Preferably, the preparation method of the microbial agent comprises the following steps:

[0019] S1: streaking the fish sauce Bacillus on a solid culture medium until a single colony grows;

[0020] S2: Pick a single colony and inoculate it into a liquid culture medium for the first expansion culture to obtain a primary bacterial liquid;

[0021] S3: inoculating the bacterial liquid into a liquid culture medium, and performing a second expansion culture to obtain the microbial agent.

[0022] Further, in step S1, the solid culture medium contains 4-6 g / L of peptone, 0.5-1.5 g / L of yeast powder, 0.05-0.2 g / L of ferric citrate, 18-20 g / L of sodium chloride, 5-6.5 g / L of magnesium chloride, 3-4 g / L of sodium sulfate, 1.5-2 g / L of calcium chloride, 0.5-0.6 g / L of potassium chloride, 0.1-0.2 g / L of sodium carbonate, 0.05-0.1 g / L of potassium bromide, 0.03-0.05 g / L of strontium chloride, 0.01-0.03 g / L of boric acid, 0-0.005 g / L of sodium silicate, 0-0.003 g / L of sodium fluoride, 0-0.002 g / L of sodium nitrate, 0.005-0.01 g / L of disodium hydrogen phosphate, and 15-20 g / L of agar.

[0023] Furthermore, in step S1, the temperature of the streaking culture is 25-30°C.

[0024] Furthermore, in step S2, the temperature of the first expansion culture is 25-30°C and the time is 8-15 h.

[0025] Furthermore, in step S3, when the bacterial liquid is inoculated into the liquid culture medium, the volume ratio of the bacterial liquid to the liquid culture medium is 1:50-100; the temperature of the second expansion culture is 25-30° C., and the time is 8-15 h.

[0026] In a third aspect, the present invention provides the use of the fish sauce Bacillus or the microbial agent in improving the salt-alkali tolerance of plants.

[0027] Preferably, the improving the salt-alkali tolerance of plants includes promoting the growth of plant nodules and the growth of plants under salt stress.

[0028] Preferably, the application comprises the following steps: planting the plants in saline-alkali soil and inoculating the fish extract Bacillus by root irrigation.

[0029] Furthermore, the specific step of inoculating the fish sauce Bacillus by root irrigation includes: applying a liquid consisting of the fish sauce Bacillus and sterile water and having an absorbance value of 1.0-1.5 at a wavelength of 600 nm to the soil at the roots of the plants.

[0030] In a fourth aspect, the present invention provides the use of the fish sauce Bacillus or the microbial agent in promoting the growth of plant nodules.

[0031] Preferably, the promoting plant nodule growth is promoting plant nodule growth under salt stress.

[0032] Preferably, the application comprises the following steps: planting the plants in saline-alkali soil and inoculating the fish extract Bacillus by root irrigation.

[0033] Furthermore, the specific step of inoculating the fish sauce Bacillus by root irrigation includes: applying a liquid consisting of the fish sauce Bacillus and sterile water and having an absorbance value of 1.0-1.5 at a wavelength of 600 nm to the soil at the roots of the plants.

[0034] Compared with the prior art, the present invention has the following advantages:

[0035] The fish sauce Bacillus A01 provided by the present invention is a fish sauce Bacillus genus ( Jeotgalibacillus ); moreover, this strain can improve the tolerance of plants to salt stress and promote the growth of plant nodules and plant growth under salt stress. This is the first time that a strain has been found in the genus Bacillus that can promote the growth of plant nodules and plant growth under salt stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 These are photos of corn plants in different treatment groups in Example 3. Notes: Figure 1 The above picture is the overall photo of the potted plant experiment. Figure 1 The following figure shows a photograph of representative plants selected from the pot experiment.

[0037] Figure 2 is the plant height of corn plants in different treatment groups in Example 3. Note: "*" indicates p<0.05, that is, the comparison between the two groups is statistically significant; "***" indicates p<0.001, that is, the comparison between the two groups is extremely statistically significant.

[0038] Figure 3 It is the aboveground fresh weight of corn plants in different treatment groups in Example 3. Note: "*" indicates p<0.05, that is, the results of the two groups are statistically significant; "***" indicates p<0.001, that is, the results of the two groups are extremely statistically significant.

[0039] Figure 4 It is the root fresh weight of corn plants in different treatment groups in Example 3. Note: "*" indicates p<0.05, that is, the results of the two groups are statistically significant; "***" indicates p<0.001, that is, the results of the two groups are extremely statistically significant.

[0040] Figure 5 These are photos of soybean plants in different treatment groups in Example 4. Notes: Figure 5 The above picture is the overall photo of the potted plant experiment. Figure 5 The following figure shows a photograph of representative plants selected from the pot experiment.

[0041] Figure 6 It is the plant height of soybean plants in different treatment groups in Example 4. Note: "*" indicates p<0.05, that is, the comparison results between the two groups are statistically significant.

[0042] Figure 7 It is the aboveground fresh weight of soybean plants in different treatment groups in Example 4. Note: "*" indicates p<0.05, that is, the results of the two groups are statistically significant, and "**" indicates p<0.01, that is, the results of the two groups are statistically significant.

[0043] Figure 8 It is the root fresh weight of soybean plants in different treatment groups in Example 4. Note: "ns" means that the results of the two groups are not statistically significant. DETAILED DESCRIPTION

[0044] The present invention will be further described below in conjunction with the embodiments.

[0045] First, the present invention relates to a fish sauce Bacillus ( Jeotgalibacillus sp. ), the strain name of the fish sauce Bacillus is A01, and the preservation number is CGMCC No.32300. The strain is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (referred to as "CGMCC"), the address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the preservation date is October 22, 2024.

[0046] The strains were able to improve the salt-alkaline tolerance of plants in saline-alkali soils, promote the growth of plant root nodules, and promote plant growth under salt stress conditions - this is the first time that the strains have been found in the genus Bacillus ( Jeotgalibacillus ) were found to have strains that could promote plant nodule growth and plant growth under salt stress, providing new bacterial resources for developing new bio-fertilizers and alleviating plant growth problems in saline-alkali soils.

[0047] Second, the present invention relates to a microbial agent containing the fish sauce Bacillus and auxiliary materials.

[0048] In some specific embodiments, the auxiliary material is a liquid culture medium; the liquid culture medium contains 4-6 g / L of peptone, 0.5-1.5 g / L of yeast powder, 0.05-0.2 g / L of ferric citrate, 18-20 g / L of sodium chloride, 5-6.5 g / L of magnesium chloride, 3-4 g / L of sodium sulfate, 1.5-2 g / L of calcium chloride, 0.5-0.6 g / L of potassium chloride, 0.1-0.2 g / L of sodium carbonate, 0.05-0.1 g / L of potassium bromide, 0.03-0.05 g / L of strontium chloride, 0.01-0.03 g / L of boric acid, 0-0.005 g / L of sodium silicate, 0-0.003 g / L of sodium fluoride, 0-0.002 g / L of sodium nitrate, and 0.005-0.01 g / L of disodium hydrogen phosphate.

[0049] In some specific embodiments, the method for preparing the microbial agent comprises the following steps:

[0050] S1: streak culture the fish sauce Bacillus on a solid culture medium at 25-30° C. until a single colony grows; the solid culture medium contains 4-6 g / L peptone, 0.5-1.5 g / L yeast powder, 0.05-0.2 g / L ferric citrate, 18-20 g / L sodium chloride, 5-6.5 g / L magnesium chloride, 3-4 g / L sodium sulfate, 1.5-2 g / L calcium chloride, 0.5-0.6 g / L potassium chloride, 0.1-0.2 g / L sodium carbonate, 0.05-0.1 g / L potassium bromide, 0.03-0.05 g / L strontium chloride, 0.01-0.03 g / L boric acid, 0-0.005 g / L sodium silicate, 0-0.003 g / L sodium fluoride, 0-0.002 g / L sodium nitrate, and 0.005-0.01 g / L disodium hydrogen phosphate. g / L, agar 15~20 g / L;

[0051] S2: Pick a single colony and inoculate it into liquid culture medium, and carry out the first expansion culture at 25-30℃ for 8-15 hours to obtain a primary bacterial liquid;

[0052] S3: inoculating the bacterial liquid into the liquid culture medium, with the volume ratio of the bacterial liquid to the liquid culture medium being 1:50-100, and performing a second expansion culture at 25-30° C. for 8-15 h to obtain the microbial agent;

[0053] In steps S2 and S3, the liquid culture medium contains 4-6 g / L of peptone, 0.5-1.5 g / L of yeast powder, 0.05-0.2 g / L of ferric citrate, 18-20 g / L of sodium chloride, 5-6.5 g / L of magnesium chloride, 3-4 g / L of sodium sulfate, 1.5-2 g / L of calcium chloride, 0.5-0.6 g / L of potassium chloride, 0.1-0.2 g / L of sodium carbonate, 0.05-0.1 g / L of potassium bromide, 0.03-0.05 g / L of strontium chloride, 0.01-0.03 g / L of boric acid, 0-0.005 g / L of sodium silicate, 0-0.003 g / L of sodium fluoride, 0-0.002 g / L of sodium nitrate, and 0.005-0.01 g / L of disodium hydrogen phosphate.

[0054] Thirdly, the present invention relates to the application of the fish sauce Bacillus or the microbial agent in improving the salt-alkali tolerance of plants.

[0055] In the above application, specifically:

[0056] In some specific embodiments, improving the salt-alkali tolerance of plants includes promoting the growth of plant nodules and the growth of plants under salt stress.

[0057] In some specific embodiments, the application includes the following steps: planting the plant in saline-alkali soil and inoculating the fish extract bacillus by root irrigation. Optionally or preferably, the specific step of inoculating the fish extract bacillus by root irrigation includes: applying a liquid composed of the fish extract bacillus and sterile water and having an absorbance value of 1.0 to 1.5 at a wavelength of 600 nm to the soil at the root of the plant.

[0058] In some embodiments, the plant is corn or soybean.

[0059] Fourthly, the present invention relates to the use of the fish sauce Bacillus or the microbial agent in promoting the growth of plant root nodules.

[0060] In the above application, specifically:

[0061] In some specific embodiments, the promoting plant nodule growth is promoting plant nodule growth under salt stress.

[0062] In some specific embodiments, the application includes the following steps: planting the plant in saline-alkali soil and inoculating the fish extract bacillus by root irrigation. Optionally or preferably, the specific step of inoculating the fish extract bacillus by root irrigation includes: applying a liquid composed of the fish extract bacillus and sterile water and having an absorbance value of 1.0 to 1.5 at a wavelength of 600 nm to the soil at the root of the plant.

[0063] In some embodiments, the plant is corn or soybean.

[0064] The present invention is described below by specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims and any equivalents thereof are the protection scope of the present invention.

[0065] Example 1: Obtaining fish sauce Bacillus A01

[0066] Sample pretreatment: Seawater is centrifuged to remove particles such as silt.

[0067] Dilution: The pretreated seawater samples were serially diluted with sterile distilled water, 10 -1 , 10 -2 , 10 -3 , 10 -4 Not waiting.

[0068] Spreading: Take 150 μL of the dilution and spread it evenly on the surface of the culture medium.

[0069] Culture: Place the plate upside down in a 28°C incubator for culture.

[0070] Purification: Observe the characteristics of the colonies, such as size, shape, color, glossiness, etc., select representative colonies and purify them on new plates by continuous streaking method.

[0071] Storage: Pick a single colony after purification, culture it overnight at 28°C and 200 rpm, and store it in a lyophilized tube for subsequent research.

[0072] The obtained strain was numbered A01.

[0073] Example 2: Identification of fish sauce Bacillus A01

[0074] The 16S rDNA gene sequence of strain A01 is 1449 bp in length, and the sequence is shown in SEQ ID NO: 1, that is, specifically as follows:

[0075]

[0076] The 16S rDNA gene was compared in the NCBI database, and the strain with the highest similarity belonged to Jeotgalibacillus terrae , with a similarity of 98.29%. The detailed comparison results are shown in Table 1. According to the double cross-validation statistics of Mincheol Kim et al., the standard for judging a new species is a 16S rRNA gene sequence similarity of less than 98.65% (KimM, Oh HS, Park SC, et al. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for speciesdemarcation of prokaryotes[J]. International Journal of Systematic andEvolutionary Microbiology, 2014, 64(Pt 2):346-351. DOI:10.1099 / ijs.0.059774-0.). Therefore, the sequence comparison results show that strain A01 belongs to the genus Bacillus ( Jeotgalibacillus ) a new species.

[0077] Table 1 16S rDNA gene comparison results

[0078]

[0079] The strain A01 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (abbreviated as "CGMCC"), the address of the depository is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is October 22, 2024, and the deposit number is CGMCC No.32300.

[0080] Example 3: Effect of fish sauce Bacillus A01 on improving salt-alkali tolerance of corn

[0081] 3.1 Activation and expansion of bacterial strains

[0082] 2216 liquid culture medium was prepared according to the following formula: 5.0 g / L peptone, 1.0 g / L yeast powder, 0.1 g / L ferric citrate, 19.45 g / L sodium chloride, 5.98 g / L magnesium chloride, 3.24 g / L sodium sulfate, 1.8 g / L calcium chloride, 0.55 g / L potassium chloride, 0.16 g / L sodium carbonate, 0.08 g / L potassium bromide, 0.034 g / L strontium chloride, 0.022 g / L boric acid, 0.004 g / L sodium silicate, 0.0024 g / L sodium fluoride, 0.0016 g / L sodium nitrate, and 0.008 g / L disodium hydrogen phosphate.

[0083] 2216 solid culture medium was prepared according to the following formula: 5.0 g / L peptone, 1.0 g / L yeast powder, 0.1 g / L ferric citrate, 19.45 g / L sodium chloride, 5.98 g / L magnesium chloride, 3.24 g / L sodium sulfate, 1.8 g / L calcium chloride, 0.55 g / L potassium chloride, 0.16 g / L sodium carbonate, 0.08 g / L potassium bromide, 0.034 g / L strontium chloride, 0.022 g / L boric acid, 0.004 g / L sodium silicate, 0.0024 g / L sodium fluoride, 0.0016 g / L sodium nitrate, 0.008 g / L sodium hydrogen phosphate, and 15 g / L agar.

[0084] The fish sauce Bacillus A01 glycerol strain stored in a -80℃ refrigerator was streaked on 2216 solid culture medium, placed in an incubator, and cultured overnight at 28℃. Subsequently, a single colony was picked and inoculated into 5 mL of 2216 liquid culture medium, and cultured overnight with shaking at 28℃ and 200 r / min. The bacterial solution obtained after the culture was transferred to a conical flask containing 2216 liquid culture medium at a ratio of 1 / 50 (i.e., the volume ratio of the bacterial solution to the liquid culture medium was 1:50), and continued to be cultured overnight with shaking at 28℃ and 200 r / min. Next, centrifuge at 4000 rpm for 15 min to collect the cells, wash the cells once with sterile water, and finally resuspend the cells with sterile water to an absorbance value (OD) at a wavelength of 600 nm. 600 ) = 1, and obtain the fish sauce Bacillus A01 liquid bacterial agent for use.

[0085] 3.2 Test soil

[0086] The test soil 1 is a non-salinized matrix from Jiangsu Xingnong Matrix Technology Co., Ltd. The soil conditions are as follows (the results of multi-point sampling tests): dry weight 0.2~0.5 g / cm 3, total porosity 60~80%, large and small void ratio 1:4~8, pH value 5.8~7.0, EC 2~3 ms / cm, organic matter content ≥35%, total nitrogen, phosphorus and potassium content 3~5%, total effective nitrogen, phosphorus and potassium content 6.0~10.0%.

[0087] Test soil 2 is a salinized matrix. Sodium chloride was added to test soil 1. The soil quality is as follows (the results were measured after multi-point sampling): dry weight 0.2~0.5 g / cm 3 , total porosity 60~80%, large and small void ratio 1:4~8, pH value 5.8~7.0, EC 2~3 ms / cm, organic matter content ≥35%, total nitrogen, phosphorus and potassium content 3~5%, total effective nitrogen, phosphorus and potassium content 6.0~10.0%, NaCl content 0.3~0.5%.

[0088] 3.3 Application of strains in improving salt-alkali tolerance of corn

[0089] Group #1 (denoted as "NaCl+A01"): Corn seeds were surface sterilized by soaking in 1% NaClO solution for 20 min, followed by rinsing with sterile water 5 times. The sterilized corn seeds were sown in pre-moistened vermiculite pots and placed in a light incubator at 25°C with a day / night ratio of 16h / 8h to promote seed germination. After 5 days of seed germination, seedlings with consistent growth conditions were selected and transplanted into pots filled with experimental soil 2. Two days after transplanting, the liquid inoculant of Bacillus spp. A01 was applied to the pots by root inoculation at a rate of 100 mL / plant.

[0090] Group #2 (referred to as "blank control"): The only difference from the experimental group is that the test soil 2 was replaced by the test soil 1, and the fish sauce Bacillus A01 liquid inoculant was replaced by water.

[0091] Group #3 (denoted as “A01”): The only difference from the experimental group is that test soil 2 was replaced by test soil 1.

[0092] Group #4 (denoted as "NaCl"): The only difference from the experimental group was that the fish sauce Bacillus A01 liquid inoculum was replaced with water.

[0093] 3.4 Test results

[0094] After being transplanted into pots and cultivated for 2 weeks, the growth of corn plants in each group was as follows: Figure 1 The growth indicators of corn plants (including plant height, aboveground fresh weight and root fresh weight) were tested, and the results are as follows Figure 2~Figure 4 As shown. Figure 1~Figure 4It can be seen that: compared with the "blank control" group, the plant height, aboveground fresh weight and root fresh weight of corn in the "A01" group increased, indicating that under normal conditions (non-salinized soil), Bacillus subtilis A01 can promote the growth of corn plants to a certain extent; compared with the "blank control" group, the plant height, aboveground fresh weight and root fresh weight of corn in the "NaCl" group were significantly reduced, indicating that under salt stress, the growth of corn plants was inhibited; compared with the "NaCl" group, the plant height, aboveground fresh weight and root fresh weight of corn in the "NaCl+A01" group were significantly increased, and the plant height and root fresh weight were close to those of the "blank control" group, indicating that Bacillus subtilis A01 has good salt-alkali tolerance, can play a role in salinized soil, improve the salt-alkali tolerance of corn, and promote the growth of corn plants under salt stress.

[0095] Example 4: Effect of Bacillus subtilis A01 on improving the salt-alkali tolerance of soybeans

[0096] 4.1 Activation and expansion of bacterial strains

[0097] 2216 liquid culture medium was prepared according to the following formula: 5.0 g / L peptone, 1.0 g / L yeast powder, 0.1 g / L ferric citrate, 19.45 g / L sodium chloride, 5.98 g / L magnesium chloride, 3.24 g / L sodium sulfate, 1.8 g / L calcium chloride, 0.55 g / L potassium chloride, 0.16 g / L sodium carbonate, 0.08 g / L potassium bromide, 0.034 g / L strontium chloride, 0.022 g / L boric acid, 0.004 g / L sodium silicate, 0.0024 g / L sodium fluoride, 0.0016 g / L sodium nitrate, and 0.008 g / L disodium hydrogen phosphate.

[0098] 2216 solid culture medium was prepared according to the following formula: 5.0 g / L peptone, 1.0 g / L yeast powder, 0.1 g / L ferric citrate, 19.45 g / L sodium chloride, 5.98 g / L magnesium chloride, 3.24 g / L sodium sulfate, 1.8 g / L calcium chloride, 0.55 g / L potassium chloride, 0.16 g / L sodium carbonate, 0.08 g / L potassium bromide, 0.034 g / L strontium chloride, 0.022 g / L boric acid, 0.004 g / L sodium silicate, 0.0024 g / L sodium fluoride, 0.0016 g / L sodium nitrate, 0.008 g / L sodium hydrogen phosphate, and 15 g / L agar.

[0099] The fish sauce Bacillus A01 glycerol strain stored in a -80℃ refrigerator was streaked on 2216 solid culture medium, placed in an incubator, and cultured overnight at 28℃. Subsequently, a single colony was picked and inoculated into 5 mL of 2216 liquid culture medium, and cultured overnight with shaking at 28℃ and 200 r / min. The bacterial solution obtained after the culture was transferred to a conical flask containing 2216 liquid culture medium at a ratio of 1 / 50 (i.e., the volume ratio of the bacterial solution to the liquid culture medium was 1:50), and continued to be cultured overnight with shaking at 28℃ and 200 r / min. Next, centrifuge at 4000 rpm for 15 min to collect the cells, wash the cells once with sterile water, and finally resuspend the cells with sterile water to an absorbance value (OD) at a wavelength of 600 nm. 600 ) = 1, and obtain the fish sauce Bacillus A01 liquid bacterial agent for use.

[0100] 4.2 Test soil

[0101] The test soil 1 is a normal non-salinized matrix from Jiangsu Xingnong Matrix Technology Co., Ltd. The soil quality is as follows (the results of multi-point sampling tests): dry weight 0.2~0.5 g / cm 3 , total porosity 60~80%, large and small void ratio 1:4~8, pH value 5.8~7.0, EC 2~3 ms / cm, organic matter content ≥35%, total nitrogen, phosphorus and potassium content 3~5%, total effective nitrogen, phosphorus and potassium content 6.0~10.0%.

[0102] Test soil 2 is a salinized matrix. Sodium chloride was added to test soil 1. The soil quality is as follows (the results were measured after multi-point sampling): dry weight 0.2~0.5 g / cm 3 , total porosity 60~80%, large and small void ratio 1:4~8, pH value 5.8~7.0, EC 2~3 ms / cm, organic matter content ≥35%, total nitrogen, phosphorus and potassium content 3~5%, total effective nitrogen, phosphorus and potassium content 6.0~10.0%, NaCl content 0.3~0.5%.

[0103] 4.3 Application of strains in improving soybean salt-alkali tolerance

[0104] Group #1 (denoted as "NaCl+A01"): Soybean seeds were surface sterilized by soaking in 1% NaClO solution for 20 min, followed by rinsing with sterile water 5 times. The sterilized soybean seeds were sown in pre-moistened vermiculite pots and placed in a light incubator at 25°C with a day / night ratio of 16h / 8h to promote seed germination. Seven days after seed germination, seedlings with consistent growth conditions were selected and transplanted into pots filled with experimental soil 2. Two days after transplanting, the liquid inoculant of Bacillus spp. A01 was applied to the pots by root inoculation at a rate of 100 mL / plant.

[0105] Group #2 (referred to as "blank control"): The only difference from the experimental group is that the test soil 2 was replaced by the test soil 1, and the fish sauce Bacillus A01 liquid inoculant was replaced by water.

[0106] Group #3 (denoted as “A01”): The only difference from the experimental group is that test soil 2 was replaced by test soil 1.

[0107] Group #4 (denoted as "NaCl"): The only difference from the experimental group was that the fish sauce Bacillus A01 liquid inoculum was replaced with water.

[0108] 4.4 Test results

[0109] After being transplanted into pots and cultivated for 2 weeks, the growth of soybean plants in each group was as follows: Figure 5 The growth indicators of soybean plants (including plant height, aboveground fresh weight and root fresh weight) were tested, and the results are as follows Figure 6~Figure 8 As shown. Figure 5~Figure 8 It can be seen that: compared with the "blank control" group, the soybean plant height and aboveground fresh weight of the "A01" group increased, indicating that under normal conditions (non-salinized soil), Bacillus subtilis A01 can promote the growth of soybean plants to a certain extent; compared with the "blank control" group, the soybean plant height, aboveground fresh weight and root fresh weight of the "NaCl" group were significantly reduced, indicating that under salt stress, the growth of soybean plants was inhibited; compared with the "NaCl" group, although there was no significant difference in the root fresh weight of soybeans in the "NaCl+A01" group, the plant height and aboveground fresh weight were significantly increased, indicating that Bacillus subtilis A01 has good salt-alkali tolerance and can play a role in salinized soil, improve the salt-alkali tolerance of soybeans, and promote the growth of soybean plants under salt stress.

[0110] After being transplanted into pots and cultured for 2 weeks, the number of soybean nodules and fresh weight of nodules in each group were counted, and the results are shown in Table 2. According to Table 2, it can be seen that compared with the "blank control" group, the number of soybean nodules and fresh weight of nodules in the "A01" group increased, indicating that under normal conditions (non-salinized soil), Bacillus subtilis A01 can promote the growth of soybean nodules to a certain extent; compared with the "blank control" group, the number of soybean nodules and fresh weight of nodules in the "NaCl" group decreased significantly, indicating that under salt stress, the growth of soybean nodules was inhibited; compared with the "NaCl" group, the number of soybean nodules and fresh weight of nodules in the "NaCl+A01" group increased, indicating that Bacillus subtilis A01 has good salt-alkali tolerance and can play a role in salinized soil to promote the growth of soybean nodules.

[0111] Table 2 Nodule number and fresh weight of soybean in different treatment groups

[0112]

[0113] Unless otherwise defined, all technical terms and scientific terms used in the present invention have the same meanings as those commonly understood by ordinary technicians in the field to which the present disclosure belongs. Unless otherwise specified, the raw materials and equipment used in the present invention are conventional raw materials and equipment in the field and can be obtained from conventional commercial channels; unless otherwise specified, the methods used in the present invention are conventional methods in the field.

[0114] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A strain of Bacillus spores ( Jeotgalibacillus sp. ), characterized in that, The strain name of the fish sauce Bacillus is A01, and the preservation number is CGMCC No.32300.

2. A microbial agent, characterized in that: The invention comprises the fish sauce Bacillus according to claim 1 and auxiliary materials.

3. The microbial agent according to claim 2, characterized in that: The auxiliary material is liquid culture medium.

4. The microbial agent according to claim 3, characterized in that The liquid culture medium contains 4-6 g / L of peptone, 0.5-1.5 g / L of yeast powder, 0.05-0.2 g / L of ferric citrate, 18-20 g / L of sodium chloride, 5-6.5 g / L of magnesium chloride, 3-4 g / L of sodium sulfate, 1.5-2 g / L of calcium chloride, 0.5-0.6 g / L of potassium chloride, 0.1-0.2 g / L of sodium carbonate, 0.05-0.1 g / L of potassium bromide, 0.03-0.05 g / L of strontium chloride, 0.01-0.03 g / L of boric acid, 0-0.005 g / L of sodium silicate, 0-0.003 g / L of sodium fluoride, 0-0.002 g / L of sodium nitrate, and 0.005-0.01 g / L of disodium hydrogen phosphate.

5. Use of the fish sauce Bacillus according to claim 1 or the microbial agent according to any one of claims 2 to 4 in improving the salt-alkali tolerance of plants, characterized in that: The plant is corn or soybean.

6. The use according to claim 5, characterized in that: The method for improving the salt-alkali tolerance of plants includes promoting the growth of plant nodules and the growth of plants under salt stress, and the plant is soybean.

7. The use according to claim 5 or 6, characterized in that: The following steps are involved: The plants were planted in saline-alkali soil and inoculated with the fish extract Bacillus by root irrigation.

8. The use according to claim 7, characterized in that The specific steps of inoculating the fish extract bacillus by root irrigation include: applying a liquid consisting of the fish extract bacillus and sterile water and having an absorbance value of 1.0-1.5 at a wavelength of 600 nm to the soil at the roots of the plants.

9. Use of the fish sauce Bacillus according to claim 1 or the microbial agent according to any one of claims 2 to 4 in promoting the growth of plant nodules, characterized in that: The plant is soybean.

10. The use according to claim 9, characterized in that: The promoting plant nodule growth is promoting plant nodule growth under salt stress.

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