A strain of Bacillus subtilis and its application in the resistance of ginseng to aluminum stress

Abstract

The present invention relates to the field of ginseng planting, and in particular, to a strain of Bacillus subtilis and its application in ginseng resistance to aluminum stress. The Bacillus subtilis YX1 of the present invention is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, and the deposit number is CGMCC No.31921. The Bacillus subtilis YX1 is separated from the rhizosphere soil of mountain ginseng under the forest, exhibits strong aluminum resistance, can produce exopolysaccharides and effectively colonize in the ginseng root system, and has a positive effect on ginseng resistance to salt stress, especially aluminum stress.

Description

Technical Field

[0001] The invention relates to the field of ginseng planting, and in particular to a bacillus subtilis strain and application thereof in ginseng resistance to aluminum stress. Background Art

[0002] Ginseng (Panax ginseng CAMey) is a perennial herbaceous plant belonging to the Araliaceae family. Usually, it takes 4 to 6 years to harvest ginseng in farmland. Under the long-term single cultivation mode, the soil in the ginseng field becomes acidic. When the soil pH is less than 5, aluminum (Al) will be dissolved into soluble Al 3+ , when Al 3+ When the concentration increases, it will interfere with the metabolic activities of soil microbial communities and have a toxic effect on plants. 3+ The content gradually increases. When it exceeds a certain threshold, Al 3+ It will be toxic to ginseng and will become one of the main factors limiting ginseng growth in acidic soil.

[0003] Rhizosphere microorganisms are called the second genome of plants and also have a variety of aluminum-tolerant characteristics, which may play an important role in alleviating plant growth stress. Studies have shown that rhizosphere microorganisms that can tolerate high concentrations of aluminum (such as more than 100μM or up to 200mM) are called aluminum-tolerant microorganisms. Currently, in agricultural ecosystems, screening and applying aluminum-tolerant microorganisms to alleviate the problem of aluminum toxicity in acidic soils has become one of the main strategies for the green and sustainable development of the cultivation industry of medicinal plants such as ginseng.

[0004] Existing technologies for improving the impact of aluminum in acidic soil on plants include the application of chemical fertilizers such as lime. Although it can significantly increase soil pH, the amount of application should not be controlled. Once too much is applied, the soil will become alkaline, which is not good for plants that prefer slightly acidic soils, such as ginseng. At the same time, the application of lime will fix the nutrients in the soil, such as phosphorus, iron, and zinc, resulting in a decrease in the effectiveness of these beneficial elements and affecting the absorption of these elements by ginseng. The application of lime may also lead to the deterioration of soil structure and have adverse effects on beneficial microorganisms in the soil, which will not only increase the economic burden of agricultural growth, but may also affect water quality and have serious impacts on the environment.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide a Bacillus subtilis strain and its application in ginseng resistance to aluminum stress. The Bacillus subtilis YX1 is separated from the rhizosphere soil of wild ginseng under the forest, exhibits strong aluminum resistance, can produce exopolysaccharides and effectively colonize in the ginseng root system, and has a positive effect on ginseng's resistance to salt stress, especially aluminum stress.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] One aspect of the present invention relates to a strain of Bacillus subtilis YX1, which is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with a deposit number of CGMCC No.31921.

[0009] Bacillus subtilis is a Gram-positive bacterium widely found in soil and plant rhizosphere. Due to its many species, fast growth and easy reproduction, it has become one of the bacteria with high safety and strong application potential. During its growth, Bacillus subtilis can secrete substances such as subtilisin and nystatin, thereby inhibiting pathogens. This study found that Bacillus subtilis can produce extracellular polysaccharides, easily colonize in the root system of ginseng, and show strong aluminum resistance. The Bacillus subtilis YX1 of the present invention is separated from the rhizosphere soil of wild ginseng under the forest of Yunxiu Ginseng Industry Technology Co., Ltd. in Dongfeng County, Liaoyuan City, Jilin Province (125°14'23"E, 42°22'40"N, 435m). By applying Bacillus subtilis YX1 or a beneficial microbial agent containing Bacillus subtilis YX1, the aluminum resistance of ginseng can be improved, and the growth of ginseng in an acidic environment can be promoted, which is of great significance to the healthy and sustainable development of the cultivation industry of medicinal plants such as ginseng.

[0010] Common beneficial microorganisms in the rhizosphere soil of medicinal plants such as ginseng include Bacillus, Pseudomonas, Streptomyces, etc. The basis and premise for the role of microorganisms is that they can adhere, colonize and form biofilms in the environment. At present, there are few studies on the mechanism of the colonization process of beneficial microorganisms. The present invention is a strain of Bacillus subtilis YX1 isolated, cultured and identified from the rhizosphere soil of mountain ginseng under the forest. It can form a biofilm and can effectively colonize in the root system of ginseng. By producing extracellular polysaccharides and aluminum, it promotes the growth of ginseng, effectively solves the problem of ginseng being stressed by aluminum, and effectively alleviates the toxic effects of aluminum on ginseng. The strain has a positive effect on ginseng in resisting aluminum stress.

[0011] Another aspect of the present invention also relates to a microbial agent, including the Bacillus subtilis YX1.

[0012] The use of the microbial agent to treat ginseng roots by dipping or irrigating the roots can effectively improve the aluminum resistance of ginseng and promote the growth of ginseng in a salt stress environment, especially aluminum stress.

[0013] The present invention does not specifically limit the dosage form of the microbial agent, and any conventional dosage form in the art can be used as the dosage form of the microbial agent, and different dosage forms do not affect the efficacy of the microbial agent. In some specific embodiments, the microbial agent includes but is not limited to: a solid agent or a liquid agent.

[0014] Furthermore, the microbial agent further comprises: auxiliary materials, which include but are not limited to conventional carriers, solvents or coating agents in the art.

[0015] Another aspect of the present invention also relates to a method for ginseng to resist salt stress, which comprises applying the Bacillus subtilis YX1 or the microbial agent to ginseng.

[0016] The method for making ginseng resistant to salt stress comprises applying Bacillus subtilis YX1 or a microbial agent to ginseng, utilizing the characteristics of Bacillus subtilis YX1, such as strong aluminum resistance, colonization in the root system of ginseng, and secretion and production of a large amount of extracellular polysaccharides, to promote the ability of ginseng to resist salt stress, especially aluminum stress.

[0017] Furthermore, the application method includes but is not limited to: root dipping and / or root irrigation.

[0018] Root dipping refers to the process of immersing the roots of crops in fertilizer or pesticide diluted to a certain concentration.

[0019] Root irrigation is a form of irrigation. Specifically, it involves pouring medicine or fertilizer on the roots of plants.

[0020] Furthermore, the salt stress includes but is not limited to aluminum stress.

[0021] Furthermore, the aluminum stress includes but is not limited to: 3+ Caused soil acidification.

[0022] Another aspect of the present invention also relates to the use of the Bacillus subtilis YX1 or the microbial agent in the resistance of ginseng to salt stress.

[0023] Furthermore, the salt stress includes: aluminum stress.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The Bacillus subtilis YX1 provided by the present invention was isolated from the rhizosphere soil of wild ginseng under the forest of Yunxiu Ginseng Technology Co., Ltd. in Dongfeng County, Liaoyuan City, Jilin Province (125°14'23"E, 42°22'40"N, 435m), and exhibited strong aluminum resistance. It can produce exopolysaccharides and effectively colonize in the ginseng root system, and has a positive effect on ginseng's resistance to salt stress, especially aluminum stress.

[0026] (2) The microbial agent provided by the present invention can effectively improve the aluminum resistance of ginseng by dipping or irrigating the roots of ginseng, and promote the growth of ginseng in a salt stress environment, especially aluminum stress.

[0027] (3) The method for ginseng resistance to salt stress provided by the present invention comprises applying Bacillus subtilis YX1 or a microbial agent to ginseng, utilizing the characteristics of Bacillus subtilis YX1, such as strong aluminum resistance, colonization in the root system of ginseng, and secretion of a large amount of extracellular polysaccharides, to enhance the ability of ginseng to resist salt stress, especially aluminum stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 is the morphological diagram of Bacillus subtilis YX1;

[0030] Figure 2 is the phylogenetic tree of Bacillus subtilis YX1;

[0031] Figure 3 The morphological diagram of Bacillus subtilis YX1 under different concentrations of aluminum treatment;

[0032] Figure 4 This is a graph showing the aluminum resistance analysis results of Bacillus subtilis YX1;

[0033] Figure 5 This is a laser confocal microscope image of biofilm formation of Bacillus subtilis YX1 under aluminum treatment;

[0034] Figure 6 This is the result of the extracellular polysaccharide content produced by Bacillus subtilis YX1 under different concentrations of aluminum treatment;

[0035] Figure 7 This is the result of IAA content produced by Bacillus subtilis YX1 under different concentrations of aluminum treatment;

[0036] Figure 8 This is the phenotype diagram of the effect of Bacillus subtilis YX1 on ginseng growth under aluminum treatment;

[0037] Fig. 9 This is a graph showing the results of the promoting effect of Bacillus subtilis YX1 on ginseng growth under aluminum treatment. DETAILED DESCRIPTION

[0038] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0039] Example 1 Isolation and cultivation of Bacillus subtilis YX1

[0040] The present invention isolated a strain of Bacillus subtilis YX1 from the rhizosphere soil of wild ginseng in the forest of Yunxiu Ginseng Industry Technology Co., Ltd., Dongfeng County, Liaoyuan City, Jilin Province (125°14'23"E, 42°22'40"N, 435m). The specific isolation and cultivation method is as follows:

[0041] (1) Weigh 10 g of rhizosphere soil, add 90 mL of sterile water into a 500 mL conical flask, shake thoroughly at 28 °C and 180 rpm for 30 min, and let stand for 10 min to obtain a dilution of 10. -1 Soil suspension. Use sterile water to dilute the suspension in a 10-fold concentration gradient. -3 , 10 -4 , 10 -5 The soil suspension is set aside.

[0042] (2) Pour the sterilized 100 mM Al2(SO4)3 solution and the prepared TSA medium into the culture dish, so that it covers the bottom of the culture dish and solidifies; take 100 μL of soil suspensions with different concentration gradients and add them to the TSA medium respectively, use a spreading rod to evenly spread the soil dilution on the entire culture medium, seal the culture dish with a sealing film, and place it in a constant temperature culture at 28°C for 7 days.

[0043] (3) After colonies grow on the TSA medium, select single colonies with different characteristics, colors, and sizes, and place them in the TSA medium with grid lines on the back. Continue to culture in a 28°C incubator for 7 days, then place the single colony in a 1.5 mL centrifuge tube containing 1 mL of TSB liquid medium and incubate at 180 r / min. -1 Incubate on a shaker at 28°C until ready for use.

[0044] Example 2 Purification and preservation of Bacillus subtilis YX1

[0045] The isolated strains were purified by plate streak method before identification. Take the YX1 turbid bacterial solution prepared in advance, use a sterilized inoculation loop to dip the bacterial solution, make a "Z"-shaped streak on the TSA plate, and freeze the remaining bacterial solution in a -80℃ refrigerator for later use; the inoculated TSA plate was inverted and cultured in a 28℃ constant temperature incubator for 7 days, and the growth of the colonies was observed during the period to see if there was any contamination by other bacteria.

[0046] Example 3 Preliminary identification of Bacillus subtilis YX1

[0047] Inoculate the strain on TSA culture medium, invert and culture in a 28℃ incubator for 2-3 days. After a single colony appears, observe the colony texture, shape and size, whether the edges are neat, whether the surface is smooth, whether there are ridges, transparency, and the color of the colony and culture medium. Figure 1 It can be seen that the YX1 strain is slightly yellow, nearly round, with irregular edges, and the colonies are rough and wrinkled.

[0048] Example 4 Molecular Biological Identification of Bacillus subtilis YX1

[0049] 4 μL of bacterial solution was used as a template for PCR amplification, and the universal primers 799F (5′-AACMGGATTAGATACCCKG-3′) and 1193R (5′-ACGTCATCCCCACCTTCC-3′) of bacterial 16S rDNA were used to amplify the strain sequence. The 50 μL PCR reaction system was 4 μL of bacterial solution template, 25 μL of 2×Taq MasterMix (GenStar), 1.7 μL of 799F / 1193R, and 17.6 μL of dd H2O. The PCR reaction conditions were 94°C pre-denaturation for 2 min, 94°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 1 min, a total of 30 cycles, and finally 72°C extension for 5 min. The obtained PCR products were subjected to 1% agarose electrophoresis, and the PCR original bacterial solution with detectable target bands was sent to Shanghai Biotech Co., Ltd. for sequencing. The measured sequences were input into NCBI, and the Blast and DNAMAN software were used for comparison analysis. Similar strain sequences with very high homology were found in GenBank. The online BLAST results showed that strain YX1 was most closely related to Bacillus subtilis subsp.inaquosorum, with a similarity of 96.18%, followed by Bacillus subtilis subsp.stercoris corrig and Bacillus subtilissubsp.spizizenii str.TU-B-10, both with a similarity of 96.08%. Phylogenetic tree analysis based on 16S rRNA gene sequences and using MEGA7.0 showed that strain YX1 and NR 116017Bacillus subtilis strain BCRC10255 were in the same large branch. In summary, YX1 was finally determined to be a member of the genus Bacillus subtilis and was named Bacillus subtilis YX1. Figure 2 .

[0050] Bacillus subtilis YX1 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No.31921 and the deposit date of September 10, 2024.

[0051] The 16S rDNA sequencing result is shown in SEQ ID No.1:

[0052] SEQ ID No.1:

[0053]

[0054] Example 5 Determination of aluminum resistance of Bacillus subtilis YX1

[0055] The YX1 strain was placed in 30 mL LB liquid medium with aluminum concentrations of 25 mM, 50 mM, 75 mM, 100 mM, 150 mM, and 200 mM, respectively, and the culture medium was rotated at 180 r·min -1 Cultivate on a shaker at 28℃; use a pipette to draw the prepared YX1 bacterial solution and spot it on LB plates with aluminum concentrations of 25mM, 50mM, 75mM, 100mM, 150mM, and 200mM, and culture at 28℃ for 2 days. Take photos of the colony morphology and measure the OD of the shaken bacterial solution. 600 , the results are as follows Figure 3 and Figure 4 The results showed that as the aluminum concentration increased, the growth of the YX1 strain was first promoted and then inhibited; when the aluminum concentration was 200mM, the growth of the YX1 strain was inhibited; when the aluminum concentration was 100mM, the YX1 strain had a strong aluminum resistance.

[0056] Example 6 Determination of the ability of Bacillus subtilis YX1 to produce biofilm

[0057] The YX1 strain was transferred to TSB liquid medium and cultured in a shaking incubator at 28°C and 180 rpm for 3 days. The bacterial suspension was streaked onto tryptone soy agar (TSA) plates for activation. Single colonies were picked and shaken in tryptone soy broth (TSB). The bacterial suspension was diluted with saline to an OD of 600 The concentration of aluminum was 0.5, and the diluted bacterial solution was added to the bottom of the six-well plate and a cover glass was placed (soaked in concentrated sulfuric acid overnight and soaked in 75% ethanol for standby use), and then cultured in a constant temperature incubator at 37°C; double fluorescence staining was performed using concanavalin A (FITC-ConA) labeled with fluorescein isothiocyanate (FITC) and propidium iodide (PI). After 12 hours, the slide was removed, repeatedly rinsed with PBS, and excess water was absorbed with absorbent paper. It was fixed with 2.5% glutaraldehyde for 1.5 hours, sealed with 40% glycerol on a clean slide, and observed and collected images using a laser scanning confocal microscope. The results showed that when the aluminum concentration was 100mM, the ability of the YX1 strain to form a biofilm was much higher than that of the control, and was significantly enhanced ( Figure 5 ).

[0058] Example 7 Determination of the ability of Bacillus subtilis YX1 to produce extracellular polysaccharides

[0059] After the YX1 strain was cultured in a shaker for 48 hours, crude polysaccharides were extracted from the fermentation broth using the ethanol precipitation method; 10 mL of the fermentation broth was placed in a 50 mL centrifuge tube, centrifuged at low temperature to remove the bacteria, and then the supernatant was added with 2 times the volume of pre-cooled ethanol, and then allowed to stand at 4°C for 12 hours and centrifuged at 10,000 r / min for 10 minutes. The resulting precipitate was dissolved in distilled water to a fixed volume to obtain the crude polysaccharide solution; the OD was determined by the phenol-sulfuric acid method 490 Absorbance value; glucose standard curve was prepared with different concentrations of glucose standard solution (0.20mg / mL, 0.40mg / mL, 0.60mg / mL, 0.80mg / mL, 1.00mg / mL), and the polysaccharide content of the strain was calculated according to the standard curve. The results showed that as the aluminum concentration increased, the EPS content of the strain increased first and then decreased; when the aluminum concentration was 100mM, the EPS content was the largest ( Figure 6 ).

[0060] Example 8 Determination of the ability of Bacillus subtilis YX1 to produce auxin

[0061] Determine the OD of bacterial suspension by spectrophotometry 600 The bacterial suspension was then centrifuged at 10,000 × g for 10 min, and the supernatant was added with an equal volume of Salkowski colorimetric solution and allowed to stand in the dark for 30 min. The OD was determined by spectrophotometry. 530 Calculate the bacterial concentration OD 600 When the value is 1, the amount of bacterial secretion of auxin (IAA) per unit volume of bacterial suspension. As the aluminum concentration increases, the IAA content of the YX1 strain increases first and then decreases, and when the aluminum concentration is 25mM, the IAA content of the YX1 strain is the highest, and there is no significant difference in the IAA content of the YX1 strain under different aluminum concentrations ( Figure 7 ).

[0062] Example 9 Experiment on promoting ginseng growth by Bacillus subtilis YX1

[0063] (1) Activation of Bacillus subtilis YX1: The strain YX1 stored at 4°C was activated, 10 μL of the frozen bacterial solution was inoculated into 1 mL of new TSB liquid medium, and the culture medium was incubated at 28°C at 180 rpm / min. -1 The culture was shaken for 7 days until the bacterial solution became turbid; 2 μL of the activated bacterial solution was taken out and the OD value at 600 nm was measured on a spectrophotometer, and the OD value of the bacterial solution was adjusted to 0.2 with sterile water for later use.

[0064] (2) Inoculate the ginseng root system with Bacillus subtilis YX1: Select one-year ginseng seedlings of uniform size, undamaged spores and surface disinfected, rinse with distilled water, and soak the ginseng seedlings in a bacterial solution with an OD value of 0.2 for 30 minutes in advance. After the soil is sterilized, put it into a flower pot with a diameter of 18 cm, and each pot contains 1.5 kg of soil. Transplant the ginseng that has been soaked in the bacterial solution in advance into the soil, and after transplanting, take 10 mL of the bacterial suspension for root irrigation. The four treatments are: the control group soil is treated with an equal amount of sterile water, the ginseng seedlings are inoculated with YX1 bacterial solution alone, the ginseng seedlings are treated with 100 mM aluminum, and the ginseng seedlings are inoculated with bacterial solution and 100 mM aluminum. Water is poured every 7 days and observed.

[0065] (3) Effect of Bacillus subtilis YX1 on ginseng growth under aluminum treatment: Ginseng seedlings treated with the above four treatments were photographed after 90 days of growth. Figure 8 At the same time, the ginseng seedlings were divided into the aboveground part and the underground part, and the ginseng plant height, aboveground fresh weight, root length, and underground fresh weight were measured. The results showed that compared with the control, the addition of YX1 strain alone could significantly promote the growth of ginseng seedlings, and 100mM aluminum treatment significantly inhibited ginseng growth. Inoculation of ginseng with YX1 strain under 100mM aluminum treatment could alleviate the inhibitory effect of aluminum on ginseng growth ( Fig. 9 ).

[0066] Therefore, the present invention believes that the reason why the YX1 strain promotes the growth of ginseng under aluminum treatment is that the strain secretes extracellular polysaccharides to form a biofilm, and the adsorption of extracellular polysaccharides reduces the concentration of aluminum ions, thereby alleviating the inhibitory effect of aluminum on ginseng growth.

[0067] Although the present invention has been illustrated and described with specific embodiments, it should be appreciated that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents without departing from the spirit and scope of the present invention. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. Therefore, this means that all such replacements and modifications within the scope of the present invention are included in the appended claims.

Claims

1. A strain of Bacillus subtilis YX1, deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No.31921.

2. A microbial agent, characterized in that: It includes the Bacillus subtilis YX1 described in claim 1.

3. The microbial agent according to claim 2, characterized in that: The microbial agent includes: a solid agent or a liquid agent.

4. The microbial agent according to claim 2 or 3, characterized in that: The microbial agent also includes auxiliary materials.

5. A method for ginseng to resist salt stress, characterized in that: Applying the Bacillus subtilis YX1 of claim 1 or the microbial agent of any one of claims 2 to 4 to ginseng; The salt stress is aluminum stress.

6. The method for ginseng resistance to salt stress according to claim 5, characterized in that: The application methods include: root dipping and / or root irrigation.

7. The method for ginseng resistance to salt stress according to claim 5, characterized in that: The aluminum stress is: 3+ Caused soil acidification.

8. Use of the Bacillus subtilis YX1 according to claim 1 or the microbial agent according to any one of claims 2 to 4 in the resistance of ginseng to salt stress; The salt stress is aluminum stress.

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