Bacillus subtilis subsp. Natto YJ01 and application thereof

Abstract

The invention discloses bacillus subtilis subsp. Natto YJ01 and application of the bacillus subtilis subsp. Natto YJ01. The preservation number of the bacillus subtilis subsp. Natto YJ01 strain is GDMCC No: 64944, and the bacillus subtilis subsp. Natto YJ01 strain can be used for dissolving inorganic phosphorus and synthesizing various active substances such as indoleacetic acid, siderophore, protease, gamma-polyglutamic acid, exopolysaccharide and the like. The YJ01 can promote the germination of lavender seeds and the growth in the germination period, and promote the growth and development of corn and oil sunflower plants. Meanwhile, the YJ01 can tolerate a high saline-alkali environment and can improve the physical structure of saline-alkali soil, and key growth indexes such as biomass, leaf area and root development of the corn and the oil sunflower can be effectively improved by applying the YJ01 to the saline-alkali soil. Therefore, the application of the strain YJ01 not only provides a new method for crop growth promotion and saline-alkali soil improvement, but also has important significance for agricultural sustainable development.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and specifically relates to a Bacillus subtilis subsp. natto strain YJ01 and its application. Background Art

[0002] Saline-alkali land is a widespread land degradation problem globally. Its high salt content and alkaline environment severely limit the growth and development of crops, leading to a decline in agricultural productivity and having a profound impact on the ecological environment. The area of saline-alkali land is vast, and the problem of secondary salinization is particularly prominent, making it crucial to improve land use efficiency and amend saline-alkali soil for sustainable agricultural development. A series of saline-alkali soil treatment technologies have been implemented, including mainly water conservancy project improvement supplemented by agronomic improvement, chemical improvement, and biological improvement. Among these methods, biological improvement has received attention due to its good ecological benefits and is achieved by biological salt absorption, salt transfer, or improving the salt tolerance of crops.

[0003] In recent years, the application of functional microorganisms in saline-alkali soil improvement has received great attention and become a research hotspot. Specific microbial strains, such as plant growth-promoting rhizobacteria (PGPR) and arbuscular mycorrhizal fungi (AMF), have been proven to be able to improve soil structure, reduce soil salinity, and promote plant growth by functions such as phosphorus solubilization, nitrogen fixation, secretion of organic acids, and IAA. The application of microbial inoculants can effectively improve soil nutrients and fertility and improve the soil microbial community structure, thereby amending saline-alkali soil. However, the survival and colonization of inoculants are greatly affected by the saline-alkali environment, and the application effect is unstable.

[0004] Bacillus subtilis subsp. natto, namely natto bacillus (natto bacteria), has a long application history in the food industry for the traditional fermented food "natto" formed by fermenting soybeans and is a generally recognized as safe functional strain. Multiple active substances synthesized by natto bacteria, such as nattokinase, γ-polyglutamic acid (γ-PGA), and vitamin K, have received much attention. γ-PGA is a multifunctional biodegradable polymer material and has a wide range of applications in fields such as medicine, cosmetics, food industry, environmental protection, agriculture, health products, and sanitary products. γ-PGA not only has significant effects as a drug carrier, moisturizer, antioxidant, and flocculant, etc., but is also considered an environmentally friendly polymer material due to its non-toxicity and biodegradability to humans and the environment. Application research in agriculture shows that γ-PGA has the effects of saving fertilizers and increasing efficiency, improving crop yield and quality, and enhancing the disease and drought resistance of crops.

[0005] Bacillus natto has been widely used in biological fertilizers and soil conditioners due to its strong adaptability and various functions such as promoting plant growth and biological control. However, existing research has mainly focused on the application of natto bacteria or γ-polyglutamic acid under general soil conditions, with less research on special environments such as saline-alkali soils. The present invention aims to explore the plant growth-promoting effects of Bacillus natto in ordinary and saline-alkali environments, as well as its potential in improving saline-alkali soils. The Bacillus natto of the present invention can not only survive under high saline-alkali conditions, but also effectively improve the structure of saline-alkali soils, promote the formation of soil aggregates and enhance the water-stable structure, reduce salt content, promote seed germination and seedling growth, and improve the tolerance of crops to saline-alkali environments. Summary of the Invention

[0006] The object of the present invention is to provide a strain of Bacillus subtilis subsp. natto YJ01 that can synthesize various active substances such as indoleacetic acid, siderophore, γ-polyglutamic acid, and exopolysaccharide, and can dissolve inorganic phosphorus, tolerate high saline-alkali, and improve the structure of saline-alkali soil.

[0007] Another object of the present invention is to provide the role of Bacillus subtilis subsp. natto YJ01 in improving saline-alkali soils and protecting crops.

[0008] To achieve the object of the present invention, in the first aspect, the present invention provides a strain of Bacillus subtilis subsp. natto YJ01 isolated and purified from the rhizosphere soil of cotton collected from a saline-alkali soil in Yili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region. It is classified and named Bacillus subtilis subsp. natto, and has now been deposited with the Guangdong Microbial Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Guangdong Province, Institute of Microbiology, Guangdong Academy of Sciences, postal code 510070, deposit number GDMCC No: 64944, deposit date August 2, 2024.

[0009] In the second aspect, the present invention provides a bacterial agent containing the Bacillus subtilis subsp. natto YJ01.

[0010] In the third aspect, the present invention provides the application of the Bacillus subtilis subsp. natto YJ01 or its bacterial agent in fermenting natto.

[0011] In the fourth aspect, the present invention provides the application of the Bacillus subtilis subsp. natto YJ01 or its bacterial agent in improving the structure of saline-alkali soil or preparing a soil conditioner.

[0012] In the fifth aspect, the present invention provides the application of the Bacillus subtilis subsp. natto YJ01 or its bacterial agent in promoting plant growth or preparing a plant growth promoter.

[0013] Furthermore, the plant growth promotion is plant growth promotion in ordinary soil and / or saline-alkali soil.

[0014] The plants include, but are not limited to, corn, lavender, and sunflower.

[0015] In a sixth aspect, the present invention provides any one of the following applications of the Bacillus subtilis natto subsp. YJ01:

[0016] 1) For nattokinase production by natto fermentation;

[0017] 2) For γ-polyglutamic acid production;

[0018] 3) For exopolysaccharide production;

[0019] 4) For siderophore production;

[0020] 5) For IAA production;

[0021] 6) For dissolving inorganic phosphorus.

[0022] The Bacillus natto of the present invention can tolerate high salinity and alkalinity and improve the soil structure of saline-alkali land, thus opening up a new way for the improvement and sustainable utilization of saline-alkali soil, contributing to ecological restoration and sustainable development of agriculture, and having important scientific significance and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the phylogenetic tree of the 16S rRNA gene of the strain YJ01 of the present invention.

[0024] Figure 2 It is the colony, cell morphology characteristics and natto fermentation ability of the strain YJ01 of the present invention. (a) Colony morphology characteristics of the strain YJ01, (b) Cell morphology observed by TEM, (c) Natto produced by YJ01 fermenting soybeans.

[0025] Figure 3 It is the growth-promoting related traits of the strain YJ01 in the preferred embodiment of the present invention. (a) Phosphorus solubilization detection, (b) Siderophore detection, (c) IAA production, (d) γ-PGA and EPS production.

[0026] Figure 4 It is the growth-promoting effect of the strain YJ01 on maize test-tube seedlings in the preferred embodiment of the present invention. (a) Photos of maize seedlings in different treatment groups, (b) Stem length of maize seedlings, (c) Fresh and dry weights of above-ground parts, (d) Fresh and dry weights of underground parts. QCK is the control of soaking seeds with clear water, MCK is the control of soaking seeds with fermented culture solution, YJ01(10 6 ) is the treatment of soaking seeds with the YJ01 fermentation broth with a viable cell count of 1×10 6 cfu / mL, YJ01(10 7 ) is the treatment of soaking seeds with the YJ01 fermentation broth with a viable cell count of 1×10 7Seed soaking treatment with the fermentation broth of YJ01 at cfu / mL. The data in the figure are the mean values, and the error bars are the standard deviations (SD). One-way ANOVA was performed using Duncan's multiple comparison method, and different letters indicate significant differences at the P < 0.05 level.

[0027] Figure 5 This shows the promoting effect of strain YJ01 on the germination and seedling growth of lavender seeds in the preferred embodiment of the present invention. (a) Photos of lavender seed germination in different treatment groups, (b) germination rate, (c) lengths of the stems and roots of lavender seedlings, (d) fresh and dry weights of seedlings. QCK is the control group soaked with normal saline, "YJ01 low" is the treatment group soaked with the bacterial solution of YJ01 at a concentration of 1×10 7 cfu / mL, and "YJ01 high" is the treatment group soaked with the bacterial suspension of YJ01 at a concentration of 1×10 8 cfu / mL. The data in the figure are the mean values, and the error bars are the standard deviations (SD). One-way ANOVA was performed using Duncan's multiple comparison method, and different letters indicate significant differences at the P < 0.05 level.

[0028] Figure 6 This shows the effects of strain YJ01 on the growth, stress resistance and soil quality of maize in saline-alkali soil in the preferred embodiment of the present invention. (a) Photos of maize plants in different treatment groups, (b) plant height and stem diameter of maize plants, (c) fresh and dry weights of the above-ground parts, (d) fresh and dry weights of the underground parts, (e) contents of proline (Pro) (unit: μg / g), malondialdehyde (MDA) (unit: nmol / g) and superoxide dismutase (SOD) enzyme activity (unit: U / g) in maize leaves, (f) EC, pH and water-soluble total salt content of the soil before and after maize planting, (g) changes in soluble salt content of the soil before and after maize planting. QCK is the control group with clear water, MCK is the control group with culture solution, and YJ01 is the treatment group inoculated with the fermentation broth. The data in the figure are the mean values ± standard deviations (SD). One-way ANOVA was performed using Duncan's multiple comparison method, and different letters indicate significant differences at the P < 0.05 level.

[0029] Figure 7Effects of strain YJ01 on the growth, stress resistance and soil quality of sunflowers in saline-alkali soil in the preferred embodiment of the present invention. (a) Photos of sunflower plants in different treatment groups, (b) plant height and stem diameter of sunflowers, (c) fresh weights of aboveground and underground parts, (d) dry weights of aboveground and underground parts, (e) contents of proline (Pro) (unit: μg / g), malondialdehyde (MDA) (unit: nmol / g) and superoxide dismutase (SOD) enzyme activity (unit: U / g) in sunflower leaves, (f) enzyme activities of soil urease (unit: μg / g), sucrase (unit: mg / g) and catalase (unit: U / kg). QCK is the clear water control, MCK is the culture solution control, and YJ01 is the treatment with inoculation of YJ01 bacterial solution. The data in the figure are mean ± standard deviation (SD), and the variance analysis uses the Duncan multiple comparison method. Different letters indicate significant differences at the P<0.05 level. Detailed implementation manners

[0030] The following examples are used to illustrate the present invention, but do not limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0031] Example 1 Isolation, identification and functional substance synthesis of Bacillus natto strain YJ01

[0032] Collect rhizosphere soil of cotton from saline-alkali soil in Yili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region. Take 5 g of soil and add 95 mL of sterile normal saline, shake and extract at 25 °C and 150 rpm for 30 min. After 10-fold serial dilution, take 100 μL and spread it on an LB plate supplemented with 10% sodium chloride (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 15 g / L, pH 7.0), and culture at 28 °C for 2 days to screen salt-tolerant strains. A Bacillus subtilis strain YJ01 capable of synthesizing γ-polyglutamic acid and nattokinase was isolated therefrom.

[0033] (1) 16S rRNA gene phylogenetic analysis

[0034] The genomic DNA of strain YJ01 was extracted using a bacterial DNA extraction kit (DL111-01, BMamp), and its 16S rRNA gene (SEQ ID NO:1) was amplified with primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’). The EzBioCloud analysis platform (www.ezbiocloud.net) was used for 16S rRNA gene sequence similarity alignment analysis, and it was found that YJ01 had the highest similarity with Bacillus strains. Among them, the sequence similarity with Bacillus velezensis CR-502 T 、Bacillus tequilensisKCTC 13622 T 、BacilluscabrialesiiTE3 T 、Bacillus inaquosorum KCTC 13429 T 、BacillussubtilisNCIB 3610 T and other Bacillus strains exceeded 98.7%. The phylogenetic tree of the 16S rRNA gene of strain YJ01 drawn using the neighbour-joining method with MEGA software is shown in Figure 1 , and the results showed that strain YJ01 clustered with Bacillus subtilis and Bacillus natto in one branch.

[0035] (2) Physiological and biochemical characteristics and functional substance synthesis

[0036] The Gram staining result of strain YJ01 was positive, it produced spores, the colonies on TSA medium were opaque, the surface was rough and wrinkled, and no pigment was produced ( Figure 2 a). The cells were rod-shaped, 2–3 μm in length and 0.7–1.0 μm in width ( Figure 2 b). It was negative for oxidase and positive for catalase. It could tolerate up to 13% NaCl. In addition, YJ01 had the ability to ferment natto, and the fermented natto was light yellow, had a good stretching state, and had no peculiar smell ( Figure 2 c).

[0037] In addition, strain YJ01 could synthesize a variety of functional substances related to nutrient utilization, plant growth promotion and stress resistance. It could dissolve insoluble inorganic phosphorus (tricalcium phosphate) ( Figure 3 a), produce siderophores ( Figure 3 b), and synthesize 1.52 mg / L and 1.86 mg / L of indole-3-acetic acid (IAA) in DF and DF+ media respectively ( Figure 3c). In the γ-PGA fermentation medium (20 g / L glucose, 10 g / L sodium L-glutamate, NH 4 Cl 1 g / L, 1 g / L yeast extract, K 2 HPO 4 3 g / L, MgSO 4 ·7H 2 O 0.2 g / L, MnSO 4 0.05 g / L, CaCl 2 0.1 g / L, pH 7.0, sterilized at 121 °C for 20 min), cultured at 37 °C for 2 days, strain YJ01 synthesized 8.5 g / L of γ-PGA ( Figure 3 d). In the polysaccharide fermentation medium (20.0 g / L glucose, 20.0 g / L peptone, MgSO 4 0.05 g / L, Na 2 HPO 4 ·12H 2 O 2.0 g / L, NaH 2 PO 4 ·2H 2 O 1.0 g / L, pH 7.0, sterilized by high-pressure steam at 121 °C for 30 minutes), cultured at 28 °C for 2 days, YJ01 synthesized 1.4 g / L of extracellular polysaccharide ( Figure 3 d).

[0038] According to the sequencing results (the 16S rRNA gene sequence of strain YJ01 is shown in SEQ ID NO: 1), as well as the above-mentioned microbiological characteristics and physiological and biochemical properties, strain YJ01 was identified as Bacillus subtilis subsp. natto, and the preservation number is GDMCC No: 64944.

[0039] Example 2 Growth promotion effect of strain YJ01 on test-tube maize

[0040] Pick a single colony of YJ01 and inoculate it into an LB test tube, shake culture at 28 °C and 150 rpm for 12 h for activation, adjust OD 600 to 0.8 as the seed solution, and transfer it to a shake flask of polysaccharide fermentation medium according to an inoculation amount of 1% (20.0 g / L glucose, 20.0 g / L peptone, MgSO 4 0.05 g / L, Na 2 HPO 4 ·12H 2 O 2.0 g / L, NaH 2 PO 4 ·2H 2O 1.0 g / L, pH 7.0, autoclaved at 121 °C for 30 minutes), cultured at 28 °C and 150 rpm for 2 days, and the bacterial solution was diluted to 1×10 6 cfu / mL (low concentration) and 1×10 7 cfu / mL (high concentration) at two concentrations. Select plump Zhengdan 958 corn seeds, place them in 75% alcohol for disinfection for 5 minutes, and then use 5% H 2 O 2 for disinfection for 1.5 hours, rinse 3 times with sterile water, wrap with double-layer gauze and place in a petri dish, moisten with water, and place at 28 °C for germination until the seeds show white tips. Select corn seeds with neat white tips, divide them into 4 groups, and place them in equal amounts of clear water (QCK), fermentation culture medium (MCK), YJ01 low-concentration bacterial solution (10 6 ), and YJ01 high-concentration bacterial solution (10 7 ) for seed soaking treatment for 3 hours.

[0041] Roll the double-layer filter paper into a cylinder, place it in a large test tube containing 1 / 3 volume of Hoagland nutrient solution, and quantitatively add the bacterial solution around the tube wall with a syringe until the filter paper is completely soaked with the bacterial solution. Place the soaked corn seeds between the double-layer filter papers, 2 seeds per tube, with 5 tubes repeated for each group, place them in an artificial climate chamber at 26 °C, first cultivate in the dark for 2 days, and then cultivate under light for 3 days for harvesting. Carefully remove the seedlings from above the filter paper to avoid damaging the root tissue, and measure the corn stem length, above-ground fresh weight, above-ground dry weight, underground fresh weight, and underground dry weight respectively.

[0042] The results are shown in Figure 4 , compared with the clear water control (QCK), seed soaking with the fermentation culture medium (MCK) significantly inhibited the growth of corn seedlings, with a decrease in stem length and biomass, which may be due to the salts in the culture medium, while seed soaking with the YJ01 bacterial solution significantly promoted the growth of corn seedlings ( Figure 4 a). Among them, when the bacterial solution concentration was 1×10 6 cfu / mL, the growth of corn seedlings was the best. Compared with MCK, the stem length increased by 44% ( Figure 4 b), the above-ground fresh weight and dry weight increased by 98% and 86% respectively ( Figure 4 c), and the underground fresh weight and dry weight increased by 62% and 106% respectively ( Figure 4 d). When the bacterial solution concentration was 1×10 7 cfu / mL, the overall growth promotion effect on corn seedlings was slightly lower than that at the concentration of 1×10 6 cfu / mL, which may be due to the higher IAA concentration in the high-concentration bacterial solution resulting in growth inhibition.

[0043] Promoting Effect of Strain YJ01 on Lavender Seed Germination in Example 3

[0044] Pick a single colony of YJ01 and inoculate it into an LB test tube. Shake and culture it at 28 °C and 150 rpm for 12 h for activation. Adjust OD 600 to 0.8 as the seed liquid. Transfer it to an LB culture medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH 7.0) shake flask according to an inoculation amount of 1%, and culture it at 28 °C and 150 rpm for 24 h. Centrifuge to collect the thalli, discard the culture supernatant, wash the thalli 3 times with sterile water and then resuspend them, and dilute the bacterial solution to 1×10 8 cfu / mL (high concentration) and 1×10 7 cfu / mL (low concentration) at two concentrations. Select plump "French Blue" lavender seeds, rinse the surface with sterile water, soak them in 75% alcohol for 2 min, rinse them three times with sterile water, then soak them in 2% sodium hypochlorite for 10 min, and rinse them three times with sterile water for seed surface disinfection treatment. Divide the seeds into 4 groups and soak them in sterile water (QCK), low-concentration bacterial solution (YJ01-low), high-concentration bacterial solution (YJ01-high), and 300 mg / L gibberellin solution (GA) for 3 hours. Sow the soaked seeds in a petri dish containing 3 layers of filter paper, sow 30 seeds in each dish, repeat each treatment in 3 dishes, add 7 mL of distilled water, and culture them in an artificial climate chamber at 25 °C for 20 days. Take the hypocotyl reaching 1 / 2 of the seed size as the lavender seed germination standard, observe and record the seed germination situation every day until germination is completed. And add sterile water in a timely manner to keep the filter paper moist. After culturing for 20 days, count the number of germinated seeds, and carefully take out the seedlings from above the filter paper to avoid damaging the root tissue, and measure the stem length, root length, and biomass respectively. Calculate the germination rate of the seeds in each treatment according to the following formula:

[0045] Germination rate (%) = (Number of germinated seeds / Total number of seeds) × 100%

[0046] The results are shown in Figure 5 . Compared with the physiological saline control, the seed soaking treatments with strain YJ01 and gibberellin significantly improved the germination rate and growth status of lavender seeds. Among them, the high-concentration bacterial solution treatment had the best promoting effect on the germination rate and dry weight of lavender seeds, which was equivalent to the effect of gibberellin seed soaking. Although the low-concentration bacterial solution treatment group had a relatively poor promoting effect on the germination rate, it had a better promoting effect on the stem length and root length (no significant difference from the gibberellin seed soaking treatment).

[0047] Effect of Strain YJ01 on Aggregate Formation and Water-Stable Structure of Saline Soil in Example 4

[0048] Collect saline soil samples (with a water-soluble total salt content of 5.8 g / kg) to conduct soil structure stability experiments. Inoculate strain YJ01 into LB culture medium and shake-culture at 28 °C and 150 rpm for 12 h, adjust OD 600 to 0.8 as the seed liquid. Inoculate the seed liquid into the polysaccharide fermentation medium (20.0 g / L glucose, 20.0 g / L peptone, 0.05 g / L MgSO 4 , Na 2 HPO 4 ·12H 2 O 2.0 g / L, NaH 2 PO 4 ·2H 2 O 1.0 g / L, pH 7.0, sterilize at 121 °C under high-pressure steam for 30 minutes), shake-culture at 28 °C and 150 rpm for 2 days, and then dilute the bacterial liquid concentration to 1×10 8 cfu / mL with the fermentation medium.

[0049] (1) Soil aggregate analysis

[0050] Spray clear water (QCK), fermentation culture solution (MCK), and YJ01 bacterial liquid (YJ01) evenly onto 150 g of saline soil according to a mass ratio of 5% respectively, put them into 200 mL wide-mouth soil culture bottles, and incubate in the dark at 28 °C and 70% relative humidity for 30 days to analyze the effect of inoculating Bacillus natto YJ01 on the structure of saline soil.

[0051] Soil aggregates were measured by the wet sieving method. Weigh 30 g of air-dried undisturbed saline soil and spread it evenly on a 2 mm sieve. Place 250 μm, 53 μm, and 20 μm sieves in sequence below. Put the nested sieves into the iron bucket supporting the aggregate analyzer, add deionized water to just submerge the soil samples in the sieves and soak for 2 minutes, and then vibrate vertically at a frequency of 40 times per minute for 2 minutes. After sieving, take out the nested sieves from the water, and at the same time, wash the soil particles in each sieve into aluminum boxes that have been dried and weighed in advance. Put the aluminum boxes into the oven and dry them to a constant weight at 105 °C, weigh them, and calculate the content of aggregates with different particle sizes. According to the particle size, the aggregates are divided into the following categories: larger macroaggregates (2–8 mm), smaller macroaggregates (0.25–2 mm), microaggregates (0.053–0.25 mm), and silt plus clay fractions (<0.053 mm). The average equivalent diameter (mean weight diameter, MWD) of the soil aggregates separated by the wet sieving method is used as an evaluation index for soil structure stability. The calculation formula of MWD is as follows:

[0052]

[0053] Among them, MWD is the average equivalent diameter, which is the average diameter (mm) of the aggregates in the i-th particle size range, and wi is the proportion of the aggregates in the i-th particle size range in the total aggregate distribution (after sand grain correction).

[0054] (2) Analysis of soil water-stable structure

[0055] According to the same method, 5% by mass of clear water (QCK), fermentation culture solution (MCK), and YJ01 bacterial solution were respectively inoculated into 250 g of saline soil, filled into PVC pipes. The lower part of the PVC pipe was first wrapped and sealed with plastic wrap, and cultured in the dark at 28 °C and 70% relative humidity for 30 days. Then, the plastic wrap at the lower part of the soil column was slowly removed without damaging the soil structure. Then, a qualitative filter paper of appropriate size was placed, fixed with tape, and 5 holes were evenly punched with toothpicks. The soil column was vertically fixed on the shelf to ensure that the leaching solution fell vertically. Then, the soil samples were leached, and the infiltration rate of the leaching solution (the time when the leaching solution infiltration was completed was recorded regularly, including the time when the first drop of leaching solution fell from the soil column into the triangular flask and the time when all the leaching solution was added to the soil column) and the EC value of the leaching solution were respectively detected.

[0056] The MWD of soil aggregates reflects the stability of soil structure, and the increase in soil salt content usually hinders the aggregation of clay particles into aggregates, resulting in poor soil drainage. The results show that after inoculation with YJ01 bacterial solution, the MWD of saline soil aggregates increased by 106% and 67% compared with the QCK and MCK controls respectively (Table 1), indicating that the soil aggregate structure was significantly improved after treatment with YJ01 bacterial solution. In addition, the results of the soil leaching experiment show that after inoculation with YJ01, the drainage performance of saline soil was significantly improved. Compared with the QCK and MCK control groups, the falling time of the first drop of leaching solution and the total falling time of 500 mL of leaching solution in the YJ01 bacterial solution treatment group were greatly shortened (Table 1).

[0057] Table 1 Effects of Bacillus natto YJ01 on the formation and water-stable structure of saline soil aggregates

[0058]

[0059] Note: QCK is the control with clear water application, MCK is the culture solution control, and YJ01 is the treatment with YJ01 bacterial solution at an inoculation concentration of 1×10 8 cfu / mL. The data in the table are the mean ± standard deviation (SD). One-way ANOVA was performed using the Duncan multiple comparison method, and different letters indicate significant differences at the P < 0.05 level.

[0060] Example 5 Effects of strain YJ01 on the growth of maize in saline soil and soil physical and chemical properties

[0061] Pick a single colony of YJ01 and inoculate it into a shake flask containing fermentation culture medium (20.0 g of glucose, 20.0 g of tryptone, Na 2 HPO 4 12H 2 O 2.0 g, NaH 2 PO 4 2H 2 O 1.0 g, MgSO 4 0.05 g, 1000 mL of distilled water, pH 7.0, autoclaved at 121 °C for 30 min). Incubate at 28 °C and 180 rpm for 24 h, and dilute the bacterial liquid concentration to 1×10 6 cfu / mL to obtain a bacterial suspension. Select plump and uniform-sized Zhengdan 958 corn seeds, soak them in sterile water for 6 h, then soak them in 75% ethanol for 3 min and 30% H 2 O 2 for 10 min, and then wash them repeatedly with sterile water 3 times. Place them in a petri dish containing gauze and filter paper, moisten with water, and incubate in an incubator at 28 °C for germination. Take corn seeds with consistent radicle emergence, divide them into three groups, and soak them in distilled water (QCK), fermentation culture medium (MCK), and YJ01 bacterial liquid (YJ01) for 3 h respectively.

[0062] Conduct a pot experiment of corn in saline soil. The pH value of the collected saline soil is 8.77, the EC value is 0.94 mS / cm, the organic matter is 13.7 mg / kg, and the available nitrogen, available phosphorus, and available potassium are 192.5, 142.3, and 235.3 mg / kg respectively. The water-soluble total salt content is 2.67 g / kg (among them, the contents of water-soluble ions Na + 、Ca 2+ 、Mg 2+ 、Cl - are 472.4, 73.8, 13.6, and 184.3 mg / kg respectively). Air-dry the soil sample, grind it, and pass it through a 2-mm sieve. Fill 1.5 kg of soil into each pot, add 75 mL of the above YJ01 bacterial suspension, and add an equal amount of distilled water or fermentation culture medium to the control group. Sow 3 germinated corn seeds with consistent germination in each pot, with a sowing depth of about 3 cm. Water 250 mL every two days after sowing. Thin out the seedlings at the three-leaf stage, and keep two evenly sized corn seedlings in each pot. Collect samples on the 23rd day after sowing, and measure the plant height, stem diameter, aboveground dry weight, and underground dry weight of the corn seedlings respectively. Measure the contents of proline (Pro), malondialdehyde (MDA), and superoxide dismutase (SOD) enzyme activity in the leaves. And collect the rhizosphere soil of the corn, and measure the soil EC value, water-soluble total salt, Na + 、Cl - 、water-soluble Ca 2+ and water-soluble Mg 2+ contents respectively.

[0063] The results of the pot experiment showed that the root development of maize was significantly inhibited in saline soil, and the roots were underdeveloped ( Figure 6 a). Compared with the control group with clear water (QCK), the plant height and aboveground biomass of the control group with fermentation culture solution (MCK) decreased significantly, which might be due to the salt ions contained in the culture solution leading to the further aggravation of salt stress ( Figure 6 , b and c). Compared with MCK, after inoculation with the YJ01 bacterial solution, the growth of maize was restored, and the plant height and stem diameter increased by 10% ( Figure 6 a), and the fresh weight and dry weight of the aboveground part increased by 90% and 64% respectively ( Figure 6 c), while there was no significant difference in the underground biomass ( Figure 6 d). Compared with MCK, after applying the YJ01 bacterial solution, the Pro content in maize leaves returned to the level equivalent to that of QCK ( Figure 6 e), while the SOD enzyme activity in the leaves increased significantly, indicating that YJ01 improved the stress resistance level of the plants ( Figure 6 e).

[0064] By analyzing the physical and chemical properties of the soil before and after maize planting, it was found that compared with QCK, the introduction of the fermentation culture solution (MCK) led to the further increase of water-soluble salt ions in the soil from 2.67 g / L before planting to 4.65 g / L, while inoculation with the YJ01 bacterial solution effectively reduced the soil water-soluble salt content and soil conductivity ( Figure 6 f). Compared with MCK, the soil EC value, water-soluble total salt, and the contents of Cl - , Na + , Ca 2+ and Mg 2+ in the soil treatment group with YJ01 bacterial solution decreased by 34%, 44%, 52%, 39%, 56% and 74% respectively ( Figure 6 , f and g). These results indicated that the strain YJ01 had a significant improvement effect on saline soil and could effectively alleviate the impact of saline-alkali stress on crop growth.

[0065] Example 6 Effects of strain YJ01 on the growth of sunflower in saline soil and the physical and chemical properties of the soil

[0066] Pick a single colony of YJ01 and inoculate it into the fermentation culture solution (20.0 g of glucose, 20.0 g of tryptone, Na 2 HPO 4 12H 2 O 2.0 g, NaH 2 PO 4 2H 2 O 1.0 g, MgSO 40.05 g, 1000 mL of distilled water, pH 7.0, autoclaved at 121 °C for 30 min), shaken at 28 °C and 180 rpm for 24 h, and the bacterial concentration was diluted to 1×10 6 cfu / mL to obtain a bacterial suspension.

[0067] A pot experiment of sunflower in saline soil was set up. The pH value of the collected saline soil was 8.35, the EC value was 0.85 mS / cm, the organic matter was 18.1 mg / kg, and the available nitrogen, available phosphorus, and available potassium were 263.3, 212.4, and 287.5 mg / kg respectively. The water-soluble total salt content was 2.33 g / kg (among which, the contents of water-soluble ions Na + , Ca 2+ , Mg 2+ , Cl - were 158.9, 6.7, 8.7, and 93.6 mg / kg respectively). After air-drying the soil samples, they were ground and passed through a 2 mm sieve. 2.5 kg of soil was filled in each pot, and 125 mL of the above-mentioned bacterial suspension was added. The control group was added with an equal amount of clear water (QCK) or fermentation culture solution (MCK). Seeds of sunflower variety Jingshu S606 were selected, and 3 seeds were sown in each pot at a sowing depth of about 3 cm. After sowing, the soil was watered 1-2 times a day. Thinning was carried out three weeks later, and only one sunflower plant was retained in each pot. Six replicates were set for each treatment. At 56 days after sowing, plant and soil samples were collected to measure the plant height, stem diameter, and biomass of sunflower plants, and to measure the contents of proline (Pro), malondialdehyde (MDA), and superoxide dismutase (SOD) enzyme activity in leaves. And enzyme activity indexes such as soil urease, sucrase, and catalase were measured.

[0068] The results of the sunflower pot experiment are shown in Figure 7 . Compared with the pot experiment of corn in saline soil, the water-soluble salt content of the saline soil used in the sunflower pot experiment was lower, and the salt tolerance of sunflower was higher. Therefore, the growth and root development of sunflower plants were less affected in saline soil ( Figure 7 a). Compared with QCK, there was no significant difference in plant height and biomass of MCK ( Figure 7 , b, c, and d). Compared with QCK, the application of YJ01 bacterial liquid significantly promoted the growth of sunflower. The plant height increased by 22% ( Figure 7 , a and b), the above-ground dry weight and underground dry weight increased by 34% and 21% respectively ( Figure 7 d), the MDA content in leaves decreased slightly, the SOD enzyme activity increased slightly, and the Pro content did not change significantly ( Figure 7 e). By analyzing the physical and chemical properties of the soil before and after planting, it was found that the application of strain YJ01 significantly increased the enzyme activities of soil urease, sucrase, and catalase ( Figure 7f), especially invertase, showed an increase of more than 3 times compared with the MCK group, indicating that the inoculation of YJ01 improved soil fertility and nutrient transformation efficiency.

[0069] Although the present invention has been described in detail with general descriptions and specific embodiments above, some modifications or improvements can be made based on the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. Bacillus subtilis subsp. natto YJ01, deposit number GDMCC No: 64944.

2. A bacterial agent containing the Bacillus subtilis natto subspecies YJ01 according to claim 1.

3. Use of the Bacillus subtilis natto subspecies YJ01 or its bacterial agent according to claim 1 in fermenting natto.

4. Use of the Bacillus subtilis natto subspecies YJ01 or its bacterial agent according to claim 1 in improving the soil structure of saline-alkali land or preparing a soil conditioner.

5. Use of the Bacillus subtilis natto subspecies YJ01 or its bacterial agent according to claim 1 in promoting plant growth or preparing a plant growth promoter.

6. The use according to claim 5, characterized in that: The plant growth promotion is plant growth promotion in ordinary soil and saline-alkali soil.

7. The use according to claim 5 or 6, characterized in that: The plants include corn, lavender and sunflower.

8. Any of the following uses of the Bacillus subtilis natto subspecies YJ01 of claim 1: 1) Used for natto fermentation to produce nattokinase; 2) Used to produce γ-polyglutamic acid; 3) Used to produce extracellular polysaccharides; 4) Used to produce siderophore; 5) Used to produce IAA; 6) Used to dissolve inorganic phosphorus.

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