CN118126903B - A multifunctional acid-resistant and alkali-producing Bacillus velez, its microbial agent and its application - Google Patents

Abstract

The invention discloses an acid and alkali resistant multifunctional bacillus bailii, a microbial agent and application thereof, wherein the multifunctional bacillus bailii is named bacillus bailii (Bacillus velezensis) Y7 and is preserved in the China general microbiological culture Collection center (China general microbiological culture Collection center) for 7 months and 3 days in 2023, and the preservation number is: CGMCC No 27762. The active ingredient of the microbial agent is the bacillus bailii. The bacteria and the microbial agent can be applied to phosphate dissolution, potassium dissolution and nitrogen fixation, and can also be applied to promotion of plant growth under acid stress. In addition, the strain can be used as a soil conditioner, can slow down the acidification of soil, can increase the biomass of plants, can promote the growth of tobacco, can reduce the use of chemical fertilizers, and has wide application prospects based on the multifunctional biological characteristics of the strain Y7 and microbial agents thereof.

Description

A multifunctional acid-resistant and alkali-producing Bacillus Velezii, its microbial agent and its application

Technical Field

The invention relates to the technical field of microorganisms, in particular to a multifunctional acid-resistant and alkali-producing Bacillus velez, a microbial agent thereof and application thereof.

Background Art

Soil acidification is one of the main factors affecting agricultural development. It will lead to a decline in soil fertility and deterioration of soil structure, threatening the normal physiological development of crops and hindering agricultural development.

At present, there are three main problems in acidic soil cultivation. On the one hand, soil acidification leads to a decrease in fertilizer utilization by crops, resulting in a waste of resources. On the other hand, soil acidification leads to a deterioration in soil physical and chemical properties, promoting the precipitation of toxic ions, thus affecting the normal growth of crops. On the third hand, soil acidification also leads to changes in the structure of soil microbial communities, increasing the number of pathogenic bacteria in the soil, leading to frequent crop diseases and reduced yield and quality. Therefore, finding a green, safe and sustainable way to cultivate acidic soil crops and reduce or avoid the impact of soil acidification on plant growth has become the focus of attention.

Among them, microbial agents are an emerging biological improvement method that has obvious advantages of safety, high efficiency and environmental protection compared with traditional methods. Microbial agents directly improve the poor growth environment of the rhizosphere of plants in acidic soil by exerting the multiple functions of acid-resistant soil microorganisms, effectively alleviate the adverse effects of soil acidification on crop growth, and greatly improve the fertility of acidic soil. Among them, microbial agent improvement, as an emerging biological improvement measure, has great potential due to its advantages of environmental protection and high ecological benefits.

However, there are few strains of acid-resistant microbial agents that have been discovered so far, and they have problems such as single function and unstable effect. The unstable effect is mainly reflected in two aspects. On the one hand, the function of the strain is unstable. With the continuous subculture of the strain, the growth-promoting function of the strain will continue to degenerate, which will in turn affect the effect of the agent; on the other hand, the activity is unstable. Under harsh growth environment, the strain will be inactivated in large numbers. As the main body of the function, the large-scale inactivation of the strain will affect the function of the agent.

Therefore, it is of great significance and application value to screen out efficient and stable multifunctional bacteria and develop an acid-resistant and alkali-producing growth-promoting microbial agent.

Therefore, the prior art needs to be further improved.

Summary of the invention

In view of the above problems, the present invention provides a multifunctional acid-resistant and alkali-producing Bacillus Velez, a microbial agent and an application thereof. The multifunctional Bacillus Velez has multiple properties such as solubilizing phosphorus and potassium, fixing nitrogen, reducing acid, promoting plant growth under acid stress, increasing the pH value of the environment and improving the soil. The multifunctional Bacillus Velez has multiple properties, such as can be used to prepare a microbial agent, be promoted and applied in crop production practice, and improve soil utilization.

To solve the above problems, this application provides the following technical solutions:

In a first aspect, the present application provides a multifunctional acid-resistant and alkali-producing Bacillus velezensis, which is named Bacillus velezensis Y7 and has a deposit number of CGMCC No 27762.

It was identified that Bacillus velezensis Y7 belongs to the Bacillaceae family, Bacillus genus, and Bacillus velezensis species. On July 3, 2023, the strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number: CGMCC No 27762, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

The strain Y7 is a growth-promoting bacterium isolated by the inventor from soil samples collected from acidic soil in Weifang City. It enriches the resources of acid-resistant growth-promoting bacteria and lays a foundation for the research and development of growth-promoting bacterial agents. The morphological characteristics of the Velez subtilis Y7 are: Gram-positive bacteria, the strain is rod-shaped; the colony is milky white, colorless and transparent in the early growth stage, white in the middle and late growth stages, polysaccharide colloid is produced, wrinkles appear on the surface of the colony in the late growth stage, and spores are produced.

Experiments have shown that the strain Y7 not only has excellent growth-promoting effects such as solubilizing phosphorus, potassium and nitrogen, but also has the effect of lowering environmental pH and can be used to improve soil. Further studies have found that under acid stress, Bacillus Velez Y7 has good growth-promoting properties on tobacco, increasing the plant height, fresh weight, leaf area and stem diameter of tobacco by 64.43%, 25.91%, 45.73% and 34.94% respectively compared with the control group, which can significantly promote crop growth.

In a second aspect, the present application also provides a microbial agent, the active ingredient of which is the aforementioned Bacillus Velezii.

Based on the above-mentioned excellent comprehensive biological characteristics of Bacillus Velezii Y7, such as phosphate solubilization, potassium solubilization, nitrogen fixation, alkali production and growth promotion, it can be used as an active ingredient to prepare microbial agents, reduce the use of chemical fertilizers, and increase plant biomass. It can be directly processed into microbial agents for field application, and the microbial agents can use the fermentation liquid of the microorganism or microbial freeze-dried powder, etc.

In addition, the bacteria can be used to prepare microbial fertilizers. The above-mentioned Bacillus Velez subtilis Y7 can be directly fermented in a solid fermentation medium (or liquid culture medium) to prepare microbial fertilizers of different dosage forms directly or further processed (with carriers, other additives, etc.), or further added with other fertilizers to make compound fertilizers.

Optionally, in the microbial agent, the active ingredient is the aforementioned Bacillus Velezii cells, fermentation liquid or fermentation supernatant.

Optionally, the microbial agent is in the form of a wettable powder, a water dispersant, a water suspension or a dispersible oil suspension.

Preferably, in the microbial agent, the effective viable count of Bacillus Velezii Y7 is not less than 50 million/g.

In a third aspect, the present application provides the use of the aforementioned Bacillus Velez or the aforementioned microbial agent in phosphate solubilization, potassium solubilization, and nitrogen fixation.

Experiments have shown that Bacillus Velez Y7 can increase environmental pH, dissolve organic phosphorus, insoluble potassium and fix nitrogen. It obviously has certain growth-promoting properties and a strong potassium-solubilizing ability.

In a fourth aspect, the present application provides the use of the aforementioned Bacillus Velez or the aforementioned microbial agent in increasing the pH value of the environment and improving the soil.

The Bacillus Velezii Y7 not only has excellent acid resistance, but also has a wide adaptability to pH, can grow in a pH range of 3-9, and can still maintain a high bacterial concentration at a pH of 4; more importantly, the strain also has the ability to reduce acidity, increasing the pH value of the liquid LB culture medium from 5 to about 8.06 within 24 hours; it can also increase the pH value of the soil and improve the acidification of the soil.

Therefore, Bacillus Velez or the aforementioned microbial agents can be used to increase the pH value of the environment and applied to improve the soil.

In a fifth aspect, the present application provides the use of the aforementioned Bacillus Velez or the aforementioned microbial agent in promoting the growth of plants under acid stress.

Experiments have shown that the strain Y7 can increase the leaf area, number of leaves and plant height of tobacco under acid stress, indicating that the strain Y7 can alleviate the stress caused by acid stress on plants, promote the growth of plants (such as tobacco) under acid stress environment, and increase their yield; it also provides a basis for the role of the acid-resistant alkali-producing growth-promoting bacteria of the present invention in promoting growth and reducing acid.

Optionally, in the application, the plants include tobacco and pepper.

In a sixth aspect, the present application also provides a method for preparing the aforementioned microbial agent, which comprises the following steps:

The aforementioned Bacillus Velezii is inoculated into a liquid culture medium, and cultured in a shaking flask at a temperature of 27-32° C. and a rotation speed of 130-190 r/min. When the strain grows to a logarithmic growth phase, the bacterial solution is diluted with sterile water or culture medium to obtain the microbial agent.

Preferably, the dilution range of the fermentation broth is such that the OD ₆₀₀ absorbance is between 1.5 and 1.0.

In a seventh aspect, the present application also provides a soil conditioner, which includes the aforementioned multifunctional Bacillus Velez.

Experiments have shown that Bacillus Velez Y7 has a significant ability to reduce acidity. It increased the pH value of liquid LB culture medium from 5 to about 8.06 within 24 hours. The treatment group with strain Y7 can increase the pH of tobacco acidic soil from 5.1 to about 5.3 within 28 days, while the pH of tobacco acidic soil in the control group decreased from 5.1 to about 4.7. Obviously, the above-mentioned Bacillus Velez Y7 can be used as an active ingredient to prepare soil conditioners for improving acidic soils.

The present invention has the following beneficial effects:

1. The present invention provides a newly isolated multifunctional Bacillus Velez subtilis Y7 and its application. The strain is a highly efficient and stable multifunctional bacterial strain screened from acidic soil where tobacco is grown all year round. The bacterium not only has excellent acid resistance, an optimum pH of 4-9, a wide range of adaptability, and stable performance; moreover, the bacterium also has multiple growth-promoting properties such as acid reduction, organic phosphorus decomposition, potassium decomposition, and nitrogen fixation. The Bacillus Velez subtilis Y7 bacterial agent can significantly improve the acid resistance of tobacco under acid stress, mitigate the impact of soil acidification, promote tobacco growth, and increase tobacco biomass; on the other hand, it reduces the use of chemical fertilizers.

Based on the above comprehensive biological characteristics of Bacillus Velezii Y7, this acid-resistant and alkali-producing multifunctional bacterial strain and its microbial agent have broad application prospects.

2. The preparation method of the microbial preparation of Bacillus Velezii provided in this application is simple, has high production efficiency, low requirements on culture conditions, low production cost, short cycle, is suitable for the needs of industrial mass production, and is easy to promote and use.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a colony morphology diagram of Bacillus velez Y7;

Figures 2A to 2D show the functional identification results of Bacillus velez Y7;

FIG3 is an analysis result of the acid resistance of Bacillus velez Y7;

FIG4 is an analysis of acid reduction of Bacillus velez Y7;

FIG5 is a stability test result of Bacillus velez Y7;

FIG6 is the electrophoresis result of 16SrDNA of Bacillus Velez Y7 and the phylogenetic tree constructed based on the sequence of 16SrDNA gene;

FIG7 shows the promoting effect of Bacillus Velez Y7 on tobacco growth under acid stress;

Figure 8 shows the effect of Bacillus Velez Y7 on pH of acidic soil for tobacco planting.

9A-9D show the effects of Bacillus Velez Y7 on acidity indexes of tobacco-planting acidic soil.

DETAILED DESCRIPTION

The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

Example 1 Isolation and screening of strain Y7

1. Experimental methods:

Soil samples were collected from acidic soils of perennial tobacco planting in Weifang, and different microorganisms were isolated from them. The strains were stored on LB solid culture medium plates by streaking for later use. 10% dilute sulfuric acid was added to the LB finished culture medium to prepare a liquid culture medium with a pH of 5. The strains activated for 24 hours were diluted into 1×10 ^-5 and 1×10 ^-6 bacterial liquids, and 1 mL of each was inoculated into LB liquid culture media with different pH values. The samples were cultured in a shaking flask at a temperature of 27-32°C and a rotation speed of 130-190 r/min for 24 hours. The acid resistance of the strains was identified by detecting OD ₆₀₀ , and the strains with good growth were repeatedly purified by streaking.

2. Experimental results and analysis:

After screening and purification, a single colony of acid-resistant strains was obtained, which was named strain Y7. The colony and bacterial morphology are shown in Figure 1.

Example 2 Identification of strain Y7

1. Morphological identification

As shown in Figure 1, Y7 is a Gram-positive bacterium with a rod-shaped strain. The colonies are milky white, colorless and transparent in the early growth stage, white in the middle and late growth stages, and polysaccharide colloid is produced. In the late growth stage, wrinkles appear on the surface of the colonies and spores are produced.

2. Physiological and biochemical identification

The physiological and biochemical identification of Y7 was carried out according to the Manual of Identification of Common Bacterial Systems.

The results of physiological and biochemical identification are shown in Table 1. As shown in Table 1, the V-P test of the bacteria is positive, and the bacteria can hydrolyze mannitol and starch, liquefy gelatin, produce hydrogen sulfide and nitrate reductase, etc.

Table 1 Physiological and biochemical identification of strain Y7

3. Molecular Biological Identification

Based on the above morphological, physiological and biochemical identifications, strain Y7 was further identified by molecular biology.

(1) Experimental methods:

The DNA of strain Y7 was extracted and used as a template. The 16S rDNA universal upstream primer 27F and downstream primer 1492R were used to amplify the 16S rDNA nucleotide fragment of the bacterium. The amplification system is shown in Table 2. The amplified product was sent to Sangon Co., Ltd. for sequencing.

The primer sequences are as follows:

Upstream primer 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ (SEQ ID No: 1);

Downstream primer 1492R: 5′-TACGGTTACCTTGTTACGACTT-3′ (SEQ ID No: 2).

Table 2 16S rDNA PCR system

(2) Experimental results and analysis

The sequencing results showed that the length of 16SrDNA of strain Y7 was 1436 bp, and the sequence was shown in SEQ ID No: 3 in the sequence table. The sequence obtained was submitted to NCBI for BLAST comparison, and the strain sequence with higher similarity was selected for download. The phylogenetic tree was constructed using the software MEGA 6.0, as shown in Figure 6. The sequence comparison results of 16SrDNA showed that the sequence similarity between strain Y7 and Bacillus velezensis was the highest.

Therefore, combined with the morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis results of the bacteria, it can be determined that the strain is Bacillus velezensis, and it is named Bacillus velezensis Y7.

Example 3 Qualitative identification of the function of strain Y7

The purpose of this example is to analyze the application potential of strain Y7 in increasing the content of soluble potassium, soluble organic phosphorus, soluble inorganic phosphorus and nitrogen fixation, and to determine the performance of strain Y7 in dissolving organic phosphorus, insoluble potassium and nitrogen fixation.

1. The culture medium and its formula used in this experiment:

Organic phosphorus culture medium: glucose 10.0g/L, ammonium sulfate 0.5g/L, yeast extract powder 0.5g/L, chloride 0.3g/L, potassium chloride 0.3g/L, magnesium sulfate 0.3g/L, ferrous sulfate 0.03g/L, manganese sulfate 0.03g/L, lecithin 0.2g/L, calcium carbonate 1.0g/L, agar 15g/L, pH 7.0.

Inorganic phosphorus culture medium: glucose 10.0 g/L, ammonium sulfate 0.5 g/L, yeast extract powder 0.5 g/L, sodium chloride 0.3 g/L, potassium chloride 0.3 g/L, magnesium sulfate 0.3 g/L, ferrous sulfate 0.03 g/L, manganese sulfate 0.03 g/L, calcium phosphate 5.0 g/L, agar 15.0 g/L, pH 7.0.

Ashurst medium: potassium dihydrogen phosphate 0.2 g/L, magnesium sulfate 0.2 g/L, sodium chloride 0.2 g/L, calcium carbonate 5.0 g/L, mannitol 10.0 g/L, calcium sulfate 0.1 g/L, agar 15 g/L, pH 7.0.

Potassium-dissolving culture medium: glucose 10g/L, disodium hydrogen phosphate 0.2g/L, magnesium sulfate 0.2g/L, sodium chloride 0.2g/L, calcium sulfate 0.2g/L, calcium carbonate 5g/L, potassium feldspar powder 25g/L, agar 20g/L, pH 7.2.

2. Experimental methods:

Y7 was cultured in shake flasks for activation and propagation. Potassium-solubilizing, phosphorus-solubilizing and nitrogen-fixing solid culture media were prepared according to the above formulas respectively. Sterilized plates were prepared. After the culture media was sterilized, they were poured onto the plates. After solidification, 1 μL of the bacterial solution was aspirated and spotted onto various plates. The plates were sealed and placed inverted at 28°C for culture. When transparent hydrolysis circles or colonies appeared, the experimental results were observed.

Evaluation criteria for potassium and phosphorus solubility: diameter ratio = transparent circle diameter / plaque diameter

Nitrogen fixation evaluation criteria: Determination of colony diameter.

Table 3 Analysis of potassium-solubilizing and nitrogen-fixing properties of strain Y7

3. Experimental results and analysis

From the results in Table 3, it can be seen that the strain has certain growth-promoting properties of solubilizing potassium, organic phosphorus, inorganic phosphorus and nitrogen fixation, among which the ability of solubilizing potassium is relatively strong, and its solubility index can reach 7.38.

Example 4 Acid resistance test of strain Y7

1. Experimental methods:

The bacterial liquid of strain Y7 was inoculated into LB liquid culture media with different pH values. In this embodiment, the pH value of the liquid culture medium was 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. The culture was carried out in a shaking flask at a temperature of 28°C and a rotation speed of 130-190 r/min for 24 h. The growth of the strain under different pH conditions was analyzed by detecting OD ₆₀₀ to determine the acid resistance of the acid-resistant and alkali-producing growth-promoting bacteria strain. The results are shown in FIG3 .

2. Experimental results and analysis

The results in Figure 3 show that the acid-resistant and alkali-producing growth-promoting bacteria strain Y7 has a large adaptability to pH. It can grow in the range of pH 3-9 and has strong activity in the range of pH 4-8. This indicates that strain Y7 can tolerate a large pH range and has strong acid and alkali resistance.

Example 5 Determination of acid-reducing ability of strain Y7

1. Experimental methods

In order to study the acid-reducing ability of strain Y7, LB liquid culture medium with a pH of 5 was prepared, and strain Y7 was inoculated into the culture medium at an inoculum size of 1%. After culturing at 28°C for 24 h, 36 h and 48 h, respectively, the change in pH of the liquid culture medium was detected using a pH meter.

2. Experimental results and analysis

As shown in the results of Table 4, strain Y7 increased the pH of the LB liquid culture medium from 5 to 7.92 within 24 hours, and strain Y7 increased the pH of the LB liquid culture medium to 8.04 within 48 hours, which indicates that the strain has a strong alkali-producing ability and significantly increases the pH value of the culture medium.

Table 4 pH changes of liquid culture medium inoculated with strain Y7

Example 6 Stability test of strain Y7

1. Experimental methods

In order to study the stability of the acid-reducing ability of strain Y7, LB liquid culture medium with a pH of 5 was prepared, and strain Y7 was inoculated at an inoculation size of 1%. After shaking flask culture at 28°C for 24 h, subculture was performed, and the changes in the pH of the liquid culture medium were detected using a pH meter.

In order to study the stability of the growth-promoting ability of strain Y7, the subcultured strain was inoculated into potassium-solubilizing, phosphorus-solubilizing and nitrogen-fixing solid culture media, cultured upside down at 28°C, and the size of the transparent hydrolysis zone or colony was recorded.

2. Experimental results and analysis

As shown in the results of Figure 5, after 40 subcultures, strain Y7 can still increase the pH of the LB liquid culture medium from 5 to about 8.12, which shows that the strain not only has a strong alkali production ability, but also has extremely high stability, and can still effectively increase the pH of the culture medium after multiple subcultures.

As shown in the results of Table 5, after 40 subcultures, strain Y7 was inoculated into the corresponding culture medium, and the colony and hydrolysis zone size were observed. It was found that the potassium, organic phosphorus, inorganic phosphorus and nitrogen fixation abilities of strain Y7 did not deteriorate significantly, which indicates that it has good stability.

Table 5 Analysis of growth-promoting function of strain Y7 after multiple subcultures

Example 7 Effect of strain Y7 on tobacco growth under acid stress

1. Experimental methods:

Tobacco seeds were sown in a plug tray filled with peat soil, 2 seeds per hole were sown, covered with 0.5 cm of peat soil, the plug tray was placed in a tray, 1 L of water was added, and the seeds were germinated. Watering was done twice a week, 1 L of water each time. When the tobacco seedlings grew to the four-leaf and one-heart stage, they were transplanted into flower pots (10 cm high, 10 cm in diameter at the top), and then transplanted into large flower pots (20 cm high, 20 cm in diameter at the top) one week later for potted experiments. The tobacco pot experiment was conducted in the greenhouse of Qingdao Agricultural University, with a temperature of 28°C, a photoperiod of 14 h/10 h, a light intensity of 1000 Lux, an initial pH of 5.1 for acidic soil, and an initial pH of 6.1 for normal soil.

The following three treatment groups were set up for tobacco:

①Blank control group (normal soil with pH 6.1);

② Acid stress treatment group (acidic soil with pH 5.1);

③ Treat acidic soil and Y7 bacterial solution together (28℃, after 24 hours of culture, the bacterial solution is diluted 10 times and used for root irrigation, and the amount used is based on a water-soil mass ratio of 1:10).

Seven days after transplanting, the tobacco seedlings of each treatment were irrigated with diluted bacterial solution at a fixed water-soil ratio of 1:10. During this period, pymetrozine and nitenpyram were sprayed to prevent pests and diseases. After the tobacco had grown for 5 weeks, its growth index was measured.

The determination method of various growth indicators is as follows: after 35 days of growth, the natural height of the tobacco is measured with a ruler, and the length and width of the maximum leaf area are determined, and the leaf area is calculated by weighing; the chlorophyll content of the third leaf from bottom to top is determined with a chlorophyll meter, and the roots are rinsed and cleaned, and then the length and surface area of the roots and other indicators are determined with a root scanner.

2. Experimental results and analysis

As shown in Figure 7 and Table 6, the acid-tolerant alkaline-producing strain Y7 can effectively promote the growth of tobacco under acid stress conditions; compared with the acid stress treatment group, the plant height, fresh weight, leaf area and stem diameter of tobacco in the Y7 bacterial solution treatment group under acid stress increased significantly, increasing by 64.43%, 25.91%, 45.73% and 34.94% respectively. The above experimental results show that strain Y7 can effectively alleviate the effect of acid stress on plant growth and has a promoting effect on plant growth.

Table 6 Effect of Y7 on tobacco growth

Plant height (cm) Fresh weight (g) Leaf area (cm ² ) Stem diameter (cm) Number of blades Normal soil 25.57 85.13 184.56 1.20 7 Acidic soil 15.35 61.73 105.67 0.83 6 Acidic soil + Y7 24.78 77.73 153.99 1.12 6

Example 8 Effect of strain Y7 on tobacco-growing acidic soil

1. Experimental methods:

The tobacco seedlings at the four-leaf and one-heart stage were transplanted into pots (10 cm high, 10 cm in diameter at the top), and then transplanted into large pots (20 cm high, 20 cm in diameter at the top) one week later for a pot experiment. Soil samples were taken every seven days to test the pH changes in the soil. After five weeks of growth, soil samples were taken to test the acidity index changes. The following three treatment groups were set up:

①Blank control group (normal soil with pH 6.1);

② Acid stress treatment group (acidic soil with pH 5.1);

③ Treat acidic soil and Y7 bacterial solution together (28℃, the bacterial solution was diluted 10 times after 24 hours of cultivation).

Seven days after transplanting, the tobacco seedlings in each treatment were irrigated at a fixed rate of 1:10 in terms of water-soil ratio. During this period, pymetrozine and nitenpyram were sprayed to prevent pests and diseases. The initial pH of acidic soil was 5.1, and the initial pH of normal soil was 6.1.

2. Experimental results and analysis

(1) As shown in Figure 8, the treatment with strain Y7 increased the pH of acidic soil from 5.1 to about 5.3 within 28 days, while the pH of the soil without strain Y7 decreased. The pH of normal soil decreased from 6.1 to about 5.3, and the pH of acidic soil decreased from 5.1 to about 4.7. The possible reason is that the roots of tobacco produce acidic substances during growth, leading to soil acidification, and the addition of strain Y7 can alleviate this phenomenon.

(2) As shown in Figure 9, the treatment with strain Y7 reduced the hydrolyzable total acid, exchangeable hydrogen, exchangeable acid and exchangeable aluminum by 43.47%, 97.52%, 35.67% and 20.57% respectively compared with the treatment without strain Y7 in acidic soil. The above experimental results show that strain Y7 can effectively alleviate soil acidification and effectively reduce soil acidity indicators.

Example 9 Effect of strain Y7 on pepper growth

1. Experimental methods

Pepper seeds were sown in double seed sowing methods in plug trays for seedling cultivation. After the seeds germinated, they were watered twice a week with 1L of water each time. After the pepper seedlings were 4 weeks old, seedlings with consistent growth conditions were selected and transplanted into pots (10 cm high, 10 cm in diameter at the top). After one week, they were transplanted into large pots (20 cm high, 20 cm in diameter at the top) again for potted experiments.

The pepper pot experiment was carried out in the greenhouse of Qingdao Agricultural University with a temperature of 28°C, a photoperiod of 14 h/10 h, a light intensity of 1000 Lux, an initial pH of 5.1 for acidic soil, and an initial pH of 6.1 for normal soil.

The following three treatment groups were set up for tobacco:

①Blank control group (normal soil with pH 6.1);

② Acid stress treatment group (acidic soil with pH 5.1);

③ Treat acidic soil and Y7 bacterial solution together (28℃, the bacterial solution was diluted 10 times after 24 hours of cultivation).

Seven days after transplanting the pepper seedlings of each treatment, the roots were irrigated with 150 ml/plant. Growth indicators of the peppers were measured after they had grown for 5 weeks.

2. Experimental results and analysis

As shown in Table 7, the use of bacterial solution treatment can effectively promote the growth of peppers and the ripening of fruits. Compared with acidic soil, the treatment with the addition of Y7 bacterial solution increased the plant height, maximum leaf area, and length and fresh weight of pepper fruits by 12.2%, 19.6%, 38.1% and 53.1%, respectively. The bacterial solution treatment effectively alleviated the stress of acidic soil on the pepper seedlings themselves, and also significantly promoted the growth and development of pepper fruits.

Table 7 Effect of Y7 on pepper growth

Plant height (cm) Stem diameter (cm) Leaf area (cm ² ) Fresh weight of pepper (g) Pepper length (cm) Normal soil 31.2 0.6 59.4 3.2 11.2 Acidic soil 25.4 0.5 44.3 2.1 6.4 Acidic soil + Y7 28.7 0.5 53.6 2.9 9.8

Claims (8)

1. The multifunctional bacillus beliae with acid and alkali resistance is characterized in that the bacillus beliae is named bacillus beliae (Bacillus velezensis) Y7, and the preservation number of the bacillus beliae is CGMCC No 27762.

2. A microbial agent characterized in that the active ingredient is the bacillus beijerinckii thallus or fermentation broth of claim 1.

3. The microbial agent of claim 2, wherein the microbial agent is in the form of a wettable powder, a water-dispersible agent, an aqueous suspension agent, or a dispersible oil suspension agent.

4. Use of bacillus belicus according to claim 1 or a microbial agent according to claim 2 for phosphate, potassium and nitrogen dissolution.

5. Use of bacillus belicus according to claim 1 or a microbial agent according to claim 2 for increasing the PH of acidic soil and slowing down the acidification of the soil.

6. Use of bacillus beljavensis according to claim 1 or of a microbial agent according to claim 2 for promoting the growth of plants under acid stress, said plants being tobacco or capsicum.

7. A method for preparing the microbial agent according to claim 2, comprising the steps of:

Inoculating the bacillus belicus according to claim 1 into a liquid culture medium, culturing in a shake flask at the temperature of 27-32 ℃ and the rotation speed of 130-190 r/min, and diluting a bacterial liquid when the strain grows to a logarithmic phase to obtain the microbial inoculum.

8. A soil conditioner comprising the multifunctional bacillus belicus according to claim 1.