CN112391311A

CN112391311A - Bacillus subtilis and influence thereof on soil nutrient content and enzyme activity

Info

Publication number: CN112391311A
Application number: CN202011276029.4A
Authority: CN
Inventors: 谭竹毅; 张佳
Original assignee: Qingdao Probex Biotechnology Co ltd
Current assignee: Qingdao Probex Biotechnology Co ltd
Priority date: 2020-11-16
Filing date: 2020-11-16
Publication date: 2021-02-23

Abstract

The invention is suitable for the technical field of microbial breeding, and provides a bacillus subtilis which is a bacillus subtilis PLBS015 and is preserved in China general microbiological culture Collection center (CGMCC) for 12-17 days in 2018, wherein the preservation number is CGMCC NO. 16953; the invention also provides application of the bacillus subtilis to soil nutrient content and enzyme activity. Therefore, the bacillus subtilis PLBS015 disclosed by the invention has better high-temperature tolerance, acid tolerance, antibacterial activity and safety, and a microbial agent containing the bacillus subtilis PLBS015 is applied to soil, so that soil nutrient imbalance and soil-borne disease aggravation can be avoided, and stable, healthy and sustainable development of crop growth and agricultural production is promoted.

Description

Bacillus subtilis and influence thereof on soil nutrient content and enzyme activity

Technical Field

The invention relates to the technical field of microbial breeding, in particular to bacillus subtilis and influence thereof on soil nutrient content and enzyme activity.

Background

The soil is a material foundation for the growth and development of crops and the survival of the crops, and the sustainable utilization of the soil is a precondition for the stable development of agricultural production. In a soil ecosystem, soil enzymes participate in biochemical processes such as decomposition of dead branches and fallen leaves of soil, decomposition and synthesis of various organic substances, nutrient release, circulation and the like, and the soil enzymes have high and low activity and directly or indirectly influence the conversion and circulation rate of various substances in the soil ecosystem. The factors influencing soil enzymes mainly comprise soil microorganisms, soil physical and chemical properties and nutrients, planted crops, fertilization measures and the like, the soil microorganisms and crop root systems are main sources of the soil enzymes, and the activity of the soil microorganisms and the crop root systems is directly influenced by the soil nutrients.

Due to long-term application of chemical fertilizers, soil is hardened, the content of organic matters is reduced, the microbial activity of the soil is reduced, substances are difficult to convert and degrade, soil nutrients are disordered, soil-borne diseases are aggravated, and the like, so that stable, healthy and sustainable development of agricultural production is restricted.

The microbial fertilizer is a novel fertilizer widely applied to production in recent years. The research shows that the microbial fertilizer contains microbial flora, active enzyme, organic matter, multiple trace elements and the like, and has a remarkable effect of promoting the activity of soil enzyme. The microbial fertilizer improves the growth environment and the nutritional condition of crops through microbial activities and related metabolites, stimulates the growth and development of the crops, promotes the conversion of soil nutrients and improves the soil nutrient condition, thereby improving the yield of agricultural products and improving the quality.

The bacillus subtilis is used as a microorganism in a microbial fertilizer and has important influence on the soil nutrient content and the enzyme activity.

Disclosure of Invention

In view of the above-mentioned drawbacks, the present invention aims to provide a bacillus subtilis with better high temperature tolerance, acid tolerance, antibacterial activity and safety. The microbial agent containing the bacillus subtilis PLBS015 is applied to soil, so that soil nutrient imbalance and soil-borne disease aggravation can be avoided, and the growth and development of crops and stable, healthy and sustainable development of agricultural production can be promoted.

In order to achieve the aim, the invention provides a bacillus subtilis which is a bacillus subtilis PLBS015 and is preserved in China general microbiological culture Collection center (CGMCC) for 12-17 th in 2018 with the preservation number of CGMCC NO. 16953.

According to the bacillus subtilis, the bacillus subtilis is taken from rice bran.

According to the bacillus subtilis, the content of the bacillus subtilis in a microbial agent is more than or equal to 2 multiplied by 10⁹cfu/g。

According to the bacillus subtilis, the addition amount of the microbial agent in soil is 15-50 kg/hm²。

According to the bacillus subtilis, the addition amount of the microbial agent in soil is 25-32 kg/hm²。

According to the bacillus subtilis, the invention also provides a method for screening the bacillus subtilis, which comprises the following steps:

step one sample Collection

Crushing rice bran, uniformly mixing sterile PBS buffer solution and rice bran powder, heating in a water bath, and performing gradient dilution on supernate to obtain bacterial source diluents with different concentrations;

isolation of the strains in step two

Respectively absorbing the bacterial source diluent to inoculate on the surface of the TSA flat plate, and performing static culture to obtain single bacterial colonies; step three purification of isolate

And respectively picking the single colonies, streaking and inoculating a TSA plate, culturing for 24h, and repeating streaking, inoculating and culturing for 3 times to obtain pure cultured isolate bacteria.

The invention aims to provide bacillus subtilis PLBS015 which has good high-temperature tolerance, acid tolerance, antibacterial activity and safety. By applying the microbial agent containing the bacillus subtilis PLBS015 to soil, the content of organic matters in the soil can be improved to a certain extent, the contents of alkaline hydrolysis nitrogen, available phosphorus and quick-acting potassium in the soil are obviously improved, the activities of soil urease, sucrase and catalase are improved, soil nutrient imbalance and soil-borne disease aggravation are avoided, and the stable, healthy and sustainable development of crop growth and agricultural production is promoted.

Drawings

FIG. 1 is a colony morphology of Bacillus subtilis PLBS015 of the present invention;

FIG. 2 is a graph showing the growth of Bacillus subtilis PLBS015 according to the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The invention provides a bacillus subtilis with the following scientific name: bacillus subtilis; the bacillus subtilis is bacillus subtilis PLBS015 which is preserved in China general microbiological culture Collection center (CGMCC, address: No. 3 of Xilu No.1 of Beijing Kogyo North Chen-Yang) in 2018, 12 months and 17 days, and the preservation number is CGMCC NO. 16953.

The invention discloses a method for screening bacillus subtilis PLBS015, which is prepared from rice bran, and comprises the following steps:

step one sample Collection

Pulverizing rice bran, mixing sterile PBS buffer solution and rice bran powder at a ratio of 1g rice bran to 5mL PBS buffer solution, heating in 85 deg.C water bath for 20min, and performing 10-fold serial gradient dilution on 1mL supernatant to obtain 10^-1～10^-4A series of dilutions of bacterial source at different concentrations.

Isolation of the strains in step two

And (3) inoculating the bacterial source diluent with each dilution on the surface of the TSA plate, inoculating 3 dilutions on each plate, and performing static culture at 37 ℃ for 24 hours to obtain a single colony.

Step three purification of isolate

A single colony in the plate is picked, streaked and inoculated to a TSA plate, cultured at 37 ℃ for 24h, and streaked and inoculated repeatedly for 3 times to obtain a pure cultured isolate.

The invention also provides an identification method of the bacillus subtilis PLBS015, which comprises morphological identification and gene sequence identification.

Morphological identification

And (3) taking separated bacterium liquid, diluting the separated bacterium liquid with a proper amount of normal saline, smearing the smear, performing gram staining and spore staining, and observing the staining condition and morphological characteristics of the bacterium under a microscope. (see FIG. 1)

As can be seen from FIG. 1, the isolated bacteria were milky white, opaque, irregular-edged, wrinkled on the surface and easily picked up on the TSA plate.

Performing microscopic examination after gram staining, wherein the thallus is in a long rod shape, gram-positive bacteria are in a green spore staining state, and the spores are in a larger proportion and are basically positioned on one side; meets the characteristics of bacillus.

Identification of Gene sequences

The isolated bacterial strain is sent to the company of biological engineering (Shanghai) and Limited for sequencing, and is compared with the 16S database of NCBI according to the obtained 16S r RNA sequence information, and the parameter identification is set to be more than 95. And then selecting the first 30 RNA sequences with the highest identification (if the sequence is insufficient, the RNA sequences are completely taken), performing sequence multiple alignment by using muscle software, and constructing a phylogenetic tree by using FastTree software. The results show that the isolated strain is closest to the published Bacillus subtilis velezensis UCMB 5033. Therefore, the isolate strain was classified as Bacillus subtilis, designated Bacillus subtilis PLBS 015.

In order to verify the strain properties of the bacillus subtilis PLBS015, strain property tests are carried out, wherein the strain property tests comprise a growth curve of a strain, a high-temperature tolerance test, an acid tolerance test, an in-vitro bacteriostasis test and an animal safety test.

Growth curve of the Strain

After the separated bacterial strains are activated, the separated bacterial strains are respectively inoculated into sterilized beef extract peptone culture media according to the inoculation amount of 5 percent, and the beef extract peptone culture media are placed into a shaker for culture at the temperature of 30 ℃. Sampling every 2h at the initial stage of culture, measuring the absorbance value of the bacterial liquid by using a spectrophotometer, and drawing a strain growth curve. (see FIG. 2)

As can be seen from FIG. 2, Bacillus subtilis PLBS015 grew rapidly in beef extract peptone medium; the growth is rapid within 0-14h, which can be regarded as logarithmic growth phase, the concentration is maintained at a higher level and reaches a stable state within 14-20h, and the growth speed is gradually reduced after 20 h.

High temperature resistance test

Culturing the isolated strain with liquid LB culture medium for 48h, centrifuging to obtain supernatant, diluting with aseptic technique, placing in water bath at 80, 90 and 100 deg.C for 30min, cooling with flowing water, culturing at room temperature without water bath as control, and calculating the survival rate of the strain after water bath by plate counting method.

As can be seen from Table 1, the survival rate of the isolated strain at 80 ℃ is higher than 85%, which indicates that the high temperature tolerance of the Bacillus subtilis PLBS015 is strong.

TABLE 1 survival Rate of Bacillus subtilis at different bath temperatures%

Acid tolerance test

Inoculating the isolated bacterial strains into liquid LB culture medium with pH of 3.0, 4.0, 5.0 respectively to make the initial concentration of isolated bacterial strains about 1 × 10⁶cfu/m L, cultured in a shaker at 37 ℃ and plated on a solid medium for plate counting, and the survival rate was calculated.

As can be seen from Table 2, the survival rate of the isolated strain under the condition of pH 3.0 is higher than 95%, which indicates that the acid tolerance of the Bacillus subtilis PLBS015 is stronger.

TABLE 2 survival Rate of Bacillus subtilis under different pH conditions%

In vitro bacteriostasis test

The antimicrobial activity of the isolate strain was determined by oxford cup method. By Escherichia coli, Staphylococcus aureus, Aeromonas hydrophila and Aeromonas sobriaPerforming bacteria inhibition analysis, adjusting the concentration of indicator bacteria and isolate bacteria strain to 10⁶cfu/m L, uniformly coating indicator bacteria on a solid LB plate, placing sterile Oxford cups on the indicator bacteria, and enabling the indicator bacteria to cling to the culture medium, wherein 0.2mL of bacteria separating liquid is added into each Oxford cup. And (3) placing the flat plate in a constant temperature box for culturing for 24h, measuring the diameter of the inhibition zone, and calculating the ratio of the inhibition zone to the diameter of the inhibition zone.

The diameter of the inhibition zone can reflect the strength of the in-vitro inhibition capacity of the isolate strain to pathogenic bacteria.

As can be seen from Table 3, the isolate strains have inhibitory effects on Escherichia coli, Staphylococcus aureus, Aeromonas hydrophila and Aeromonas sobria, but have strong inhibitory effects on Escherichia coli, Staphylococcus aureus and Aeromonas sobria; therefore, the bacillus subtilis PLBS015 has better antibacterial activity.

TABLE 3 antagonistic action of Bacillus subtilis on indicator bacteria

Animal safety test

Mice were divided into 2 groups, group 1 being the test group and group 2 being the control group. The test groups were each fed with a separate strain of isolate (concentration approximately 1.0X 10)⁸Diluting the solution with CFU/mL according to a ratio of 1:10, and injecting drinking water for mixing uniformly); the control group was fed with physiological saline. The state of the mice was observed, and the onset and death of the mice were recorded, and the test period was 4 weeks.

The test result shows that compared with the control group, the mice in the test group all feed normally and have good spirit and no death during the test period, which indicates that the bacillus subtilis PLBS015 has good safety.

The bacillus subtilis PLBS015 has better high-temperature tolerance, acid tolerance, bacterial inhibition and safety, so the bacillus subtilis PLBS015 is applied to soil, and in order to verify the influence of the bacillus subtilis PLBS015 on the soil nutrient content and the enzyme activity, the influence is verified.

First, test materials

The contents of the nutrient substances in the test soil are respectively as follows: 27.6g/kg of organic matter, 1.25g/kg of total nitrogen, 0.71g/kg of total phosphorus, 13.2g/kg of total potassium, 110.4mg/kg of alkaline hydrolysis nitrogen, 64.2mg/kg of available phosphorus, 182.6mg/kg of quick effect and 5.75 of pH; the strains in the test soil are respectively as follows: bacterium 0.11X 10⁷cfu/g, actinomycete 0.96X 10⁶cfu/g, fungus 1.60X 10⁴cfu/g。

Planting corns in test soil, wherein the row spacing is 55-75 cm, the plant spacing is 35-50 cm, and the nitrogen application amount is 105kg/hm². The content of the bacillus subtilis PLBS015 in the microbial agent is more than or equal to 2 multiplied by 10⁹cfu/g。

Second, design and process of experiment

The test soil is divided into 4 experimental groups, wherein one control group and three experimental groups are adopted, the addition amount of the microbial agent in the soil of the control group is 0, and the addition amount of the microbial agent in the soil of the experimental groups is 15-50 kg/hm²。

The microbial agent is applied by a hole application method along with the base fertilizer 3d before transplanting.

Third, items and methods of measurement

Soil nutrient determination

Collecting the middle ridge bodies of two corn plants by a sampler in a 5-point sampling method at 30, 45 and 60 days after the corn seedlings emerge respectively, taking a plough layer soil sample with the depth of 0-20 cm as about 1kg, uniformly mixing the soil samples by taking a cell as a unit, packaging the mixture by using sterile self-sealing bags, naturally drying the mixture in the absence of direct sunlight, grinding and removing impurities and residual roots of the corn, and determining the soil nutrients after sieving.

The organic matter adopts a hydration thermogravimetric potassium chromate oxidation colorimetric method; alkaline hydrolysis nitrogen is subjected to alkali de-spreading; the effective phosphorus adopts a sodium bicarbonate method; the quick-acting potassium is obtained by flame photometry.

Soil enzyme activity assay

And (3) collecting 0-20 cm soil layers of each experimental group by a 5-point method, removing impurities and stones, mixing and preparing samples, and screening the samples by a 1mm sieve for later use after the samples are naturally air-dried.

The catalase activity is determined by potassium permanganate titration method, and the result shows that 0.02mol/L KMnO is consumed by 1g of soil₄Expressed in volume (mL); the soil urease activity is measured by adopting a phenol-sodium hypochlorite colorimetric method, and the result is that NH is contained in 1g of soil after 24 hours₄ ⁺-mass of N (mg); the sucrase is measured by a 3, 5-dinitrosalicylic acid colorimetric method, and the enzyme activity is expressed by the mass (mg) of glucose in 1g of soil after 24 hours.

In order to further verify the influence of the bacillus subtilis PLBS015 on the soil nutrient content and the enzyme activity, the bacillus subtilis PLBS015 is applied to the soil by the method, the following examples are set, and the soil nutrient content and the enzyme activity in each example are measured.

The addition amount of the microbial agent in each example is shown in Table 4; the results of the soil indexes in the examples are shown in tables 5 and 6.

Comparative example

The addition amount of the microbial agent in the soil is 0.

After the soil nutrient content and the enzyme activity are measured, the content of organic matters in the soil is 26.63g/kg, the content of alkaline hydrolysis nitrogen is 138.25mg/kg, the content of available phosphorus is 76.06mg/kg, and the content of quick-acting potassium is 220.17 mg/kg; the soil was found to have urease activity of 3.06 mg/g.d, catalase activity of 2.26 mg/g.h, and invertase activity of 42.64 mg/g.d.

Example 1

The addition amount of the microbial inoculum in the soil is 25kg/hm²。

After the soil nutrient content and the enzyme activity are measured, the content of organic matters in the soil is 29.55g/kg, the content of alkaline hydrolysis nitrogen is 180.78mg/kg, the content of available phosphorus is 90.64mg/kg, and the content of quick-acting potassium is 310.65 mg/kg; the soil was found to have urease activity of 5.12 mg/g.d, catalase activity of 3.59 mg/g.h, and invertase activity of 82.40 mg/g.d.

Example 2

The addition amount of the microbial inoculum in the soil is 28kg/hm²。

After the soil nutrient content and the enzyme activity are measured, the content of organic matters in the soil is 28.92g/kg, the content of alkaline hydrolysis nitrogen is 182.82mg/kg, the content of available phosphorus is 91.58mg/kg, and the content of quick-acting potassium is 289.72 mg/kg; the soil was found to have a urease activity of 4.83 mg/g.d, a catalase activity of 3.46 mg/g.h, and a sucrase activity of 80.16 mg/g.d.

Example 3

The addition amount of the microbial inoculum in the soil is 32kg/hm 2.

After the soil nutrient content and the enzyme activity are measured, the content of organic matters in the soil is 29.19g/kg, the content of alkaline hydrolysis nitrogen is 181.89mg/kg, the content of available phosphorus is 92.04mg/kg, and the content of quick-acting potassium is 297.48 mg/kg; the soil was found to have urease activity of 5.05 mg/g.d, catalase activity of 3.23 mg/g.h, and invertase activity of 79.64 mg/g.d.

Because the implementation processes of the embodiments are the same, the specific processes of other embodiments are not listed, and only the three embodiments with better effects are listed.

TABLE 4 addition amount of microbial inoculum in soil of each example kg/hm²

TABLE 5 nutrient content in soil of each example

TABLE 6 soil enzyme Activity of the examples

The embodiment shows that the application of the microbial agent containing the bacillus subtilis PLBS015 in the soil can improve the content of organic matters in the soil to a certain extent and obviously improve the content of alkaline hydrolysis nitrogen, available phosphorus and quick-acting potassium in the soil; the bacillus subtilis PLBS015 has a certain effect on improvement of soil organic matters and quick-acting nitrogen, phosphorus and potassium, and the bacillus subtilis PLBS015 can activate soil nutrients, prevent soil hardening, inhibit residual germs in soil, promote growth and development of plant seeds and seedlings and enhance root activity.

The above examples show that the application of the microbial agent containing the bacillus subtilis PLBS015 in soil can improve the activities of soil urease, sucrase and catalase, and show that the generated high-activity decomposition enzyme decomposes organic matters in the growth process of the bacillus subtilis PLBS015, and the generated metabolic products such as organic acids, enzymes and physiological active substances are easily absorbed by animals, plants and microorganisms. The bacillus subtilis PLBS015 can accelerate the conversion and circulation rate of various substances in a soil ecosystem, and promote the growth and development of crops.

In addition, the above examples show that the addition amount of the microbial agent containing the Bacillus subtilis PLBS015 in the soil is 25-32 kg/hm²In the process, the soil nutrient content and the soil enzyme activity are the best, so that the optimal addition amount of the microbial agent containing the bacillus subtilis PLBS015 in the soil is 25-32 kg/hm²。

In conclusion, the bacillus subtilis PLBS015 provided by the invention has better high-temperature tolerance, acid tolerance, antibacterial activity and safety. By applying the microbial agent containing the bacillus subtilis PLBS015 to soil, the content of organic matters in the soil can be improved to a certain extent, the contents of alkaline hydrolysis nitrogen, available phosphorus and quick-acting potassium in the soil are obviously improved, the activities of soil urease, sucrase and catalase are improved, soil nutrient imbalance and soil-borne disease aggravation are avoided, and the stable, healthy and sustainable development of crop growth and agricultural production is promoted.

The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it should be understood that various changes and modifications can be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. The bacillus subtilis is a bacillus subtilis PLBS015 which is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 12 months and 17 days, and the preservation number is CGMCC NO. 16953.

2. A bacillus subtilis according to claim 1 wherein the bacillus subtilis is taken from rice bran.

3. The Bacillus subtilis of claim 1, wherein the Bacillus subtilis is present in an amount of at least 2 x 10 in the microbial inoculant⁹cfu/g。

4. The bacillus subtilis according to claim 3, wherein the microbial agent is added in the soil in an amount of 15-50 kg/hm²。

5. The bacillus subtilis according to claim 4, wherein the microbial agent is added in the soil in an amount of 25-32 kg/hm²。

6. A method for screening the Bacillus subtilis of claim 1, comprising the steps of:

step one sample Collection

isolation of the strains in step two

Respectively absorbing the bacterial source diluent to inoculate on the surface of the TSA flat plate, and performing static culture to obtain single bacterial colonies;

step three purification of isolate