CN113215065A

CN113215065A - Bacillus aryabhattai resistant to heavy metal copper and application thereof

Info

Publication number: CN113215065A
Application number: CN202110697788.6A
Authority: CN
Inventors: 王桔红; 陈文�
Original assignee: Hanshan Normal University
Current assignee: Hanshan Normal University
Priority date: 2021-06-23
Filing date: 2021-06-23
Publication date: 2021-08-06
Anticipated expiration: 2041-06-23
Also published as: CN113215065B

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a heavy metal copper-resistant bacillus aryabhattai and application thereof. The method screens out the Bacillus aryabhattai JDD (Bacillus aryabhattai) which resists heavy metal copper from fresh rhizobium of pseudo-anethod, wherein the highest tolerant concentration of the Bacillus aryabhattai JDD to the heavy metal copper is more than 375 mg/L; the Bacillus aryabhattai JDD can effectively remove copper in a heavy metal polluted environment and can realize the restoration of heavy metal polluted soil; the JDD of the Bacillus aryabhattai has a remarkable growth promoting effect on seed germination under heavy metal copper pollution, and the germination rate of plant seeds dyed with the JDD of the Bacillus aryabhattai is kept between 76.7 and 91 percent under the stress of copper ions with various concentrations and is remarkably higher than that of an uncontaminated group; the Bacillus aryabhattai JDD can promote the increase of the plant height, the overground fresh weight and the underground fresh weight of seedlings, has obvious growth promotion effect on the growth of the seedlings polluted by heavy metal copper, and has wide application prospect.

Description

Bacillus aryabhattai resistant to heavy metal copper and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a heavy metal-resistant bacillus aryabhattai and application thereof.

Background

In recent years, the heavy metal pollution of soil is becoming more serious due to the development of industry and human activities. Heavy metals cannot be automatically decomposed in soil, have long residual time and great harm, and are easily absorbed by crops to influence the health of human bodies. Copper contamination is a common heavy metal contamination. The mining of copper ores, the accumulation of tailings, the discharge of industrial wastewater, the use of copper-containing pesticides and other factors can cause excessive accumulation of copper elements in soil. Copper element is absorbed by plants and stays at roots, so that the absorption of other components, particularly iron element, by roots is hindered, and the plants are poor in growth. And excessive copper enters the human body to cause the poisoning phenomena of nausea, vomiting, epigastric pain, acute hemolysis, renal tubular deformation and the like.

The traditional Chinese medicines for the current remediation technology of heavy metal soil pollution comprise physical remediation, chemical remediation, phytoremediation, microbial remediation, combined remediation and the like. The traditional physical repair mode has large project amount and high cost and is difficult to be applied to large-area soil repair treatment; the operation process of chemical remediation is relatively complex, and simultaneously, the soil structure can be damaged, so that the soil fertility is reduced, and secondary pollution is possible to exist; the phytoremediation is a green and environment-friendly soil heavy metal pollution remediation technology due to low cost and no secondary pollution, but has the defects of small biomass, long growth cycle and the like, and the ideal effect is difficult to achieve by single phytoremediation. Therefore, the microbial remediation technology is a common method for improving the heavy metal soil pollution.

Bacillus aryabhattai belongs to gram-positive bacteria of bacillus, is widely distributed in nature, has strong stress resistance, and is found in animal intestinal tracts, plant rhizosphere, deep sea and plateau. The Bacillus aryabhattai can degrade or synthesize macromolecular compounds, including decolorization, sewage degradation, synthetic perfume, synthetic bioflocculant and leukemia-resistant specific drugs; can also be symbiotic with plants and promote plant growth, including hormone synthesis, stress resistance and crop yield increase. At present, the strain resources of Bacillus aryabhattai are still deficient, and some varieties are not suitable for being applied to crops.

The method screens out the Bacillus aryabhattai JDD which resists the heavy metal copper from the fresh root nodules of the pseudo-ginseng plant body, wherein the highest tolerant concentration of the Bacillus aryabhattai JDD to the heavy metal copper is more than 375 mg/L; the Bacillus aryabhattai JDD can effectively remove copper ions in the heavy metal polluted environment and can realize the restoration of heavy metal polluted soil; the Bacillus aryabhattai JDD can improve the germination rate of the pseudo-ginseng seeds under normal and copper stress, promote the increase of the stem length, the plant height and the overground fresh weight of seedlings, has a remarkable growth promoting effect on the growth of the seedlings polluted by heavy metal copper, and has a wide application prospect.

Disclosure of Invention

In view of the above problems, the present invention aims to provide a heavy metal copper-resistant bacillus aryabhattai and an application thereof, specifically comprising the following contents:

in a first aspect, the present invention provides a Bacillus aryabhattai JDD, which is deposited at the guangdong province collection center of microbial cultures at 2021, 5/8, with the deposit numbers: GDMCC No: 61645.

in a second aspect, the present invention provides an application of the bacillus aryabhattai JDD of the first aspect, or a bacterial suspension of the bacillus aryabhattai JDD of the first aspect, or a microbial inoculum containing the bacillus aryabhattai JDD of the first aspect in remediation of heavy metal contaminated soil.

Preferably, the heavy metal is copper.

In a third aspect, the present invention provides an application of the JDD of the first aspect, or a bacterial suspension of the JDD of the first aspect, or a microbial preparation containing the JDD of the first aspect in promoting plant growth.

Preferably, the plant is a pseudo-ginseng.

In a fourth aspect, the present invention provides an application of the bacillus aryabhattai JDD of the first aspect, or a bacterial suspension of the bacillus aryabhattai JDD of the first aspect, or a microbial inoculum containing the bacillus aryabhattai JDD of the first aspect, in improving tolerance of plants to heavy metal stress environments.

Preferably, the plant is a pseudo-ginseng.

Preferably, the heavy metal is copper.

In a fifth aspect, the present invention provides an application of the bacillus aryabhattai JDD of the first aspect, or a bacterial suspension of the bacillus aryabhattai JDD of the first aspect, or a microbial inoculum containing the bacillus aryabhattai JDD of the first aspect, in promoting plant growth under a heavy metal stress environment.

Preferably, the plant is a pseudo-ginseng.

Preferably, the heavy metal is copper.

In a sixth aspect, the present invention provides a plant growth promoting agent, which contains the bacillus aryabhattai JDD of the first aspect, or a bacterial suspension of the bacillus aryabhattai JDD of the first aspect, or a microbial agent containing the bacillus aryabhattai JDD of the first aspect.

Preferably, the plant growth promoter is used as a plant growth promoter under heavy metal stress.

Preferably, the heavy metal is copper.

Preferably, the plant is a pseudo-ginseng.

In a seventh aspect, the present invention provides a soil remediation agent, wherein the soil remediation agent contains the bacillus aryabhattai JDD of the first aspect, or a bacterial suspension of the bacillus aryabhattai JDD of the first aspect, or a microbial inoculum containing the bacillus aryabhattai JDD of the first aspect.

Preferably, the soil remediation agent is used to remediate heavy metal contaminated soil.

Preferably, the heavy metal is copper.

The invention has the beneficial effects that:

(1) the method comprises the steps of screening a heavy metal copper-resistant Bacillus aryabhattai JDD from fresh rhizobium of pseudo-broom, wherein the highest tolerance concentration of the Bacillus aryabhattai JDD to the heavy metal copper is more than 375 mg/L; the Bacillus aryabhattai JDD can effectively remove heavy metal copper in a heavy metal polluted environment, the removal rate reaches 32.67% when the concentration of the heavy metal copper is 150mg/L, and the remediation of the heavy metal polluted soil can be realized;

(2) the Bacillus aryabhattai JDD can improve the germination rate of the pseudo-ginseng seeds under normal and copper stress, promote the increase of the stem length, the plant height and the overground fresh weight of seedlings, has a remarkable growth promotion effect on the growth of the seedlings polluted by heavy metal copper, and has a wide application prospect.

Drawings

FIG. 1 growth curves of Bacillus aryabhattai JDD;

FIG. 2A Bacillus licheniformis JDD micrograph;

FIG. 3 detection of PCR products by gel electrophoresis

FIG. 4 Congo Red test results for Bacillus aryabhattai JDD;

FIG. 5 results of methyl Red experiments with Bacillus aryabhattai JDD;

FIG. 6 Amylase hydrolysis results for Bacillus aryabhattai JDD;

FIG. 7 meat extract peptone growth results of Bacillus aryabhattai JDD;

FIG. 8 BTB culture results of Bacillus aryabhattai JDD;

FIG. 9 shows the results of the detection of the tolerance of Bacillus aryabhattai JDD to heavy metal copper;

FIG. 10 shows the result of removing rate of heavy metal copper by Bacillus aryabhattai JDD;

FIG. 11 shows the effect of Bacillus aryabhattai JDD on the germination rate of Phaseolus vulgaris seeds;

FIG. 12 fold increase in the germination rate of P.tonkinensis seeds after contamination;

FIG. 13 results of the high effect of Bacillus aryabhattai JDD on P.pseudolaris;

FIG. 14 results of the effect of Bacillus aryabhattai JDD on the above-ground fresh weight of P.pseudo-ginseng;

FIG. 15 results of the effect of Bacillus aryabhattai JDD on the underground fresh weight of P.pseudo-ginseng;

FIG. 16 underground fresh weight gain of Pseudobulbus Cremastrae Seu pleiones after contamination with bacteria.

Detailed Description

The scope of the present invention is described in detail below with reference to specific examples, but it should be noted that the scope of the present invention is not limited by the following examples.

The YMA medium described in the following examples of the present invention consists of: mannitol 6g, yeast extract 1.6g, K₂HPO₃0.2g、 MgSO₄·7H₂O 0.08g、NaCl 0.04g、CaCl₂0.02g, 8.8g of agar, 1.6mL of Rh trace element and 400mL of distilled water; the pH value is 6.8-7.0. (Rh trace element solution: H)₃BO₃ 5g、Na₂MnO₄ 5g；H₂O 1000mL)。

Example 1 isolation and preservation of Bacillus aryabhattai JDD

1. Isolation of Strain JDD

Collecting Phaseolus vulgaris (Desmodium heterophyllum) in Hongshan forest of Guangdong Chaozhou city of 9 Yue 2019, removing stems and leaves, cleaning roots with clear water, selecting large, full and fresh root nodules at the roots, washing off impurities, washing, soaking in 95% ethanol for 3-5 min, and adding 5% sodium hypochlorite (0.1% HgCl)₂) Surface sterilization for 3min, followed by 10 washes with sterile water under sterile conditions. On an ultra-clean workbench sterilized by an ultraviolet lamp, shaking up sterile water flowing down from the root nodules washed for the last time, pouring a small amount of sterile water on the YMA culture medium for uniform coating, carrying out inverted culture at 28 ℃ for 3-5 days, and observing the growth condition of the strains on the culture medium. If no colony appears, the root nodule is proved to be cleaned and disinfected, otherwise, the reason for the colony appearing is analyzed, and the cleaning and disinfection of the root nodule are carried out again.

The single nodule was cut in half under ultraclean bench aseptic conditions, the half nodule was clamped with sterile forceps, the cut was streaked facing the YMA (prepared and sterilized in advance) surface, and cultured upside down in a 28 ℃ incubator. After 48h of culture, single colonies were picked for purification culture to obtain a pure culture, which was designated JDD.

The optimal growth temperature of the JDD bacteria is 28 ℃, and the optimal pH is 6-7; the bacterial colony can grow up after 24-48h at 28 ℃, and the growth is fast; the colony is smooth, round, semi-transparent and large-viscosity milky rhinorrhea-shaped heaves.

2. Preparation of JDD growth curve

Preparing three 100ml YMA culture media, inoculating a ring of JDD bacteria under aseptic condition after autoclaving, culturing by using a shaking table (the rotating speed is 120r/min and the temperature is 28 ℃), measuring the OD value (the wavelength is 600nm) of the bacteria liquid every 2 hours, calculating three repeated average values by using a blank liquid culture medium as a reference, and obtaining the growth log phase of the strain JDD according to the variation curve of the absorbance value.

The curve of change in OD value of JDD bacterial liquid cultured for 4-14h is shown in FIG. 1, in which the curve from 4-12h rises slowly, the time is lag phase, the curve from 12-14h grows fastest, and is exponential growth phase of JDD bacteria, at this time, the JDD bacterial strain has fastest propagation speed, active growth and strong metabolic capacity, and can be used to prepare bacterial suspension with best inoculation effect.

3. Identification of Strain JDD

3.1 microscopic Observation of Strain JDD

Taking a little bacteria by inoculating loop near the flame of alcohol lamp, placing in the center of a drop of sterile water, making into thin and uniform bacterial smear, naturally drying, fixing on the flame of alcohol lamp for 2-3 times, dyeing with safranin dye solution for 1-2min, washing with fine water flow, removing residual water, and performing microscopic examination. As shown in FIG. 2, JDD bacteria were observed to be Brevibacterium having a strong refractive index under a 100-fold objective lens (oil scope).

3.2 sequencing and identification of JDD Gene of Strain

(1) Extraction of genomic DNA from strain JDD

Placing the JDD strain on the culture medium beside the flame of an alcohol burner in a mortar, and grinding by liquid nitrogen; adding the ground bacteria into a 1.5mL centrifuge tube, marking the name of the strain, adding 0.6mL TE (pH 8.0), and sucking and beating uniformly by using a gun head to fully suspend the bacteria; add 250. mu.L 10% SDS, mix by gentle inversion; adding 3 μ L proteinase K (20ng/μ L), mixing gently, and water bath at 37 deg.C for 1 h; adding 150 mu L of 5mol/L NaCl, and gently mixing; adding 150 μ L of 2% CTAB, mixing gently, and water-bathing at 65 deg.C for 20 min; 12000rpm, centrifuging for 20min at normal temperature; carefully sucking the supernatant into a new 1.5mL centrifuge tube, adding isopropanol with the same volume, fully and uniformly mixing, standing at room temperature for 30min, 12000rpm, and centrifuging at 4 ℃ for 10 min; sucking off the supernatant, draining the liquid on absorbent paper, adding 750 μ L70% ethanol, flicking the tube wall, suspending the precipitate, repeatedly reversing for several times, centrifuging at 12000rpm at 4 deg.C for 2 min; add 30. mu.L of purified water to each tube to dissolve the precipitate (RNase in water, final concentration 10 ng/. mu.L), flick the tube wall by hand and dissolve overnight at 4 ℃.

(2) And (3) DNA electrophoresis detection:

and (3) PCR amplification:

16s primer sequence: primer1(27 f): AGAGTTTGATCMTGGCTCAG, respectively; primer2 (1492R): TACGGYTACCTTGTTACGACTT, respectively;

reaction system: h₂O, 17.8 μ L; buffer, 3 μ L; dNTP, 2. mu.L; primer1, 3 μ L; primer2, 3 μ L; DNA template, 1. mu.L; enzyme, 0.2 μ L; the total volume is 30 mu L;

reaction conditions are as follows: 95 ℃ for 5 min; 95 ℃ for 30 sec; 55 ℃ for 30 sec; 72 ℃ for 1 min; 35 cycles; 72 ℃ for 10 min; at 12 ℃, forever;

(3) results

The results of detecting PCR products by gel electrophoresis are shown in FIG. 3, wherein 3 corresponds to the strain JDD. Electrophoresis conditions: 3 μ L of sample + 1% agarose gel, Marker band composition: 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp and 5000 bp. The 750bp band concentration of 60 ng/3. mu.L is shown as a highlight band, and the remaining band concentrations are all 30 ng/3. mu.L. The sequence of the 16S rDNA gene of the obtained strain JDD is shown in SEQ ID NO. 1.

Through BLAST homology comparison and evolutionary analysis, the result shows that the strain JDD has the closest affinity relationship with Bacillus aryabhattai, and the strain JDD is determined to be Bacillus aryabhattai (Bacillus aryabhattai) which is totally called as Bacillus aryabhattai JDD. The Bacillus aryabhattai JDD is preserved in Guangdong province microorganism strain preservation center at 2021, 5, 8 days, and the preservation numbers are as follows: GDMCC No: 61645, respectively; the preservation address is as follows: building No. 59, building No. 5 of the first-furious Zhonglu 100 yard in Guangzhou city; and (4) contacting the telephone: 020-87137633.

4. Determination of JDD physiological and biochemical characteristics of Bacillus aryabhattai

The JDD strain of Bacillus aryabhattai was streaked on a solid YMA medium and then cultured in an incubator at 28 ℃ for 3 days until the lawn was full. And (3) selecting a loopful of bacteria from the full-fleshed lawn by using an inoculating loop, inoculating the loopful of bacteria into 80mL of YMA liquid culture medium, and culturing for 3d on a shaking table with the rotating speed of 150r/min and the temperature of 28 ℃ to obtain the JDD bacteria suspension of the Bacillus aryabhattai. Preparing a methyl red culture medium, a Congo red culture medium, a hydrolyzed starch culture medium, a meat extract peptone growth culture medium and a BTB culture medium.

4.1 Congo Red experiment

Congo Red assay was used to determine whether the cell surface components contained cellulose. Congo red can dye cellulose into red compound, so bacterial colony turns red, which is a positive reaction and shows that cellulose exists on the surface of cells; when cellulose is decomposed by cellulase, congo red-cellulose compound can not be formed, and a transparent ring taking cellulose decomposing bacteria as a center appears in the culture medium, and the cellulose decomposing bacteria are screened by judging whether the transparent ring is generated or not. Bacillus aryabhattai JDD was inoculated onto Congo red medium and cultured at 28 ℃ for 3d, three replicates per treatment. The results are shown in fig. 4, the congo red medium inoculated with bacillus aryabhattai JDD did not produce a transparent ring, and was a negative reaction, indicating that bacillus aryabhattai JDD could not produce cellulase to decompose cellulose, and was not a cellulolytic bacterium.

4.2 methyl Red experiment (MR experiment)

Some bacteria have the ability to perform mixed acid fermentation of glucose in MR-VP medium. The products of mixed acid fermentation are mainly a complex mixture of lactic acid, acetate, succinic acid and formic acid and ethanol and equal amounts of hydrogen and carbon dioxide, making the medium acidic. Methyl red is a pH indicator, remains red when the pH is equal to or less than 4.4, and is positive; otherwise, the result is negative. Bacillus aryabhattai JDD was inoculated on methyl red medium and cultured at 28 ℃ for 3d, three replicates for each treatment. As a result, as shown in FIG. 5, the JDD of Bacillus aryabhattai did not turn red after 3 days of culture in methyl red medium, and was a negative reaction, i.e., no acid was produced in the medium, indicating that the JDD of Bacillus aryabhattai did not have the ability to ferment glucose mixed acid.

4.3 hydrolysis of starch experiment

If the microorganism can produce amylase (extracellular enzyme), the starch in the culture medium can be hydrolyzed into maltose, glucose and the like, and then the maltose, the glucose and the like are absorbed and utilized by cells, and the starch does not turn blue when meeting iodine after being hydrolyzed, so that the existence of the starch decomposing capability of certain bacteria is judged. Inoculating Bacillus aryabhattai JDD on a starch culture medium, culturing for 3d at 28 ℃, repeating three times of treatment, dropwise adding a proper amount of iodine solution, and observing the result. As a result, as shown in FIG. 6, after Bacillus aryabhattai JDD was cultured in starch medium for 3 days, a proper amount of iodine solution was added dropwise, and the medium did not turn blue, indicating that the starch in the medium was decomposed by amylase, i.e., Bacillus aryabhattai JDD could produce amylase, which could hydrolyze starch.

4.4 meat extract peptone assay

Some bacteria produce tryptophanase in peptone, which decomposes to produce indole and pyruvate. The indole combines with p-dimethylaminobenzaldehyde to form a red rose indole. Adding a proper amount of ether into a peptone culture medium, then adding a p-dimethylaminobenzaldehyde reagent along the wall, judging whether indole is generated in the growth and metabolism of the microorganism according to whether a red reaction layer is generated, and further judging whether the microorganism has the capability of decomposing tryptophan. The red color formed was positive, while the red color formed was negative. Bacillus aryabhattai JDD was inoculated into meat extract peptone growth medium and incubated at 28 ℃ for 3 days, 3 replicates of each treatment. As a result, as shown in FIG. 7, Bacillus aryabhattai JDD did not show red color on the broth peptone medium, and the reaction was negative and could not decompose tryptophan to produce indole, indicating that Bacillus aryabhattai JDD could not produce tryptophanase to decompose tryptophan.

4.5BTB assay

BTB is blue-green and reacts with carbon dioxide to turn yellow. Inoculating Bacillus aryabhattai JDD on YMA culture medium containing 0.5% bromothymol blue, and culturing in 28 deg.C constant temperature incubator for 3d, wherein each treatment is repeated three times; and (6) observing the result. The results are shown in FIG. 8, after inoculating Bacillus aryabhattai JDD in BTB medium and culturing, the medium changed from green to yellow, which indicates that Bacillus aryabhattai JDD produces acid on BTB medium and belongs to fast-growing strains.

In conclusion, the Bacillus aryabhattai JDD of the invention can not produce cellulase to decompose cellulose, but does not produce cellulolytic bacteria; has no glucose mixed acid fermentation capacity; can produce amylase, can hydrolyze starch; the tryptophan can not be decomposed by the tryptophanase; produce acid on a BTB culture medium, and belong to fast-growing strains. Specifically, the results are shown in Table 1.

TABLE 1 detection of physiological and biochemical characteristics of rhizobia of Phaseolus vulgaris

Note: "-" indicates negative, and "+" indicates positive

Example 2 detection of copper tolerance by Bacillus aryabhattai JDD

(1) Activation of bacillus aryabhattai JDD: firstly, streaking a JDD strain of Bacillus aryabhattai on a solid YMA culture medium for inoculation, and then culturing for 3 days in an incubator at the temperature of 28 ℃ until the lawn is full;

(2) preparing a bacterial suspension: selecting a ring colony in 80mL YMA liquid culture medium (250mL conical flask) by using an inoculating loop in the cultured full lawn, and fully oscillating for 15h by using a shaking table (the rotating speed is 120r/min, the temperature is 28 ℃) to obtain a JDD bacterial suspension of the Bacillus aryabhattai;

(3) determination of the highest tolerated concentration of copper:

YMA solid medium containing copper (0, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400mg/L) was prepared in 3 replicates per concentration setting.

And (3) respectively coating 1mL of the bacterial suspension prepared in the step (2) on the copper-containing YMA solid culture medium, placing the medium in a thermostat at 28 ℃ for culturing for 12h, observing and recording the growth condition of a colony every 4h, counting the growth condition of the JDD of the Bacillus aryabhattai on the YMA culture medium with different copper concentrations, and detecting the copper tolerance of the JDD of the Bacillus aryabhattai.

(4) And (3) detection results: as shown in FIG. 9, the JDD colonies of B.aryabhattai reached 100% and the growth was good when the YMA medium contained no copper. When the copper concentration is 75-100mg/L, the colony amount is more than 75%. When the copper concentration is 150-350mg/L, the colony amount cultured for 12h is less than 20 percent, and the colony amount cultured for 22h is 18-40 percent; the colony amount after 36h culture is 20-50%. When the copper concentration is 375mg/L, the colony amount cultured for 36h is less than 5 percent, and the colony amount cultured for 40-53h reaches 10-12 percent. When the copper concentration is more than 400mg/L, a germ-free colony grows. The result shows that the Bacillus aryabhattai JDD has obvious tolerance to heavy metal copper, and the highest tolerance concentration is more than 375 mg/L.

Example 3 measurement of the copper ion removal Rate by Bacillus aryabhattai JDD

(1) Preparing a bacterial suspension: selecting a ring colony in 80mL YMA liquid culture medium (250mL conical flask) by using an inoculating loop in the cultured full lawn, and fully oscillating for 15h by using a shaking table (the rotating speed is 120r/min, the temperature is 28 ℃) to obtain a JDD bacterial suspension of the Bacillus aryabhattai;

(2) preparing a sample solution: respectively preparing 100mL of YMA liquid culture medium containing metal copper (the copper concentration is 0, 25, 50, 75, 100, 125 and 150mg/L), respectively adding 3mL of bacterial suspension, then placing the mixture in a shaking incubator at 28 ℃ and 120 r.min < -1 > for culturing for 28h, then taking out, filtering the mixture by using a microfiltration membrane with the radius of 0.45 mu m to obtain a solution to be detected, reducing the solution to be detected by 16 times by using 1% nitric acid to prepare sample solutions within a measurable concentration range, and respectively taking 7mL of the sample solutions into a centrifuge tube (the centrifuge tube and the microfiltration membrane are firstly placed in 10% nitric acid for soaking for 12-24h, then washed by using clear water, and finally rinsed by using deionized water for later use).

(3) Drawing a standard curve: preparing a copper standard solution with a certain concentration gradient by using a 1% dilute nitric acid solution, and placing the copper standard solution in a flame atomic absorption spectrophotometer to measure the OD value of the copper standard solution. And (3) drawing a standard curve by taking the concentration of copper ions as an abscissa and the absorbance value of the standard solution as an ordinate, and obtaining a regression equation of the standard curve: y 0.1522x-0.003, degree of fit R²A standard curve is plotted, 0.99215.

(4) Determination of copper removal: placing the prepared sample solution (1) in a flame atomic absorption spectrophotometer to measure the OD value, and calculating the Cu in the JDD sample solution of the Bacillus aryabhattai cultured for 28 hours by using a standard curve formula and the measured OD value of the sample solution²⁺The concentration is calculated and the following calculation formula of the removal rate is utilized to calculate the JDD of the Bacillus aryabhattai to Cu²⁺The removal rate of (2):

(5) and (3) measuring results: the result is shown in fig. 10, in the range of the heavy metal copper concentration of 25-150mg/L, the bacillus aryabhattai JDD can remove the copper ions in the environment to a certain extent, i.e. the bacillus aryabhattai JDD has a certain removal effect on the copper ions, and when the copper concentration is 150mg/L, the removal rate reaches 32.67%.

Example 4 Effect of Bacillus aryabhattai JDD on seed Germination and seedling growth

1. Seed treatment

Soaking herba Viburni Diversifoliae seed in 75 deg.C warm water for 15min to break seed dormancy, and adding 0.1% HgCl₂Sterilizing the seed surface with the solution for 5min, sterilizing with 95% ethanol for 5min, washing with sterile water for 5 times in sterile operating platform, and drying with sterilized absorbent paper.

Selecting a ring colony in 80mL YMA liquid culture medium (250mL conical flask) by using an inoculating loop in the cultured full lawn, and fully oscillating for 15h by using a shaking table (the rotating speed is 120r/min, the temperature is 28 ℃) to obtain a JDD bacterial suspension of the Bacillus aryabhattai;

and (3) placing the inoculated group seeds in the bacterial suspension for soaking for 6h, keeping the inoculated group seeds in contact with the air in the soaking process, and naturally drying in the shade for 1 h.

2. Heavy metal stress and contamination culture

Preparing solution containing heavy metal copper with concentration of 0, 50, 100, 150, 200, 250, 300, 350, 400, 450 and 500 mg/L. The pseudo-kidney bean seeds not inoculated with Bacillus aryabhattai JDD were used as a control group, and the pseudo-kidney bean seeds inoculated with Bacillus aryabhattai JDD were used as an experimental group, and 30 seeds were treated for each, and 3 replicates were used. To make a simple culture environment with filter paper and culture dish. When the concentration of the heavy metal solution is prepared, the heavy metal solution is firstly concentrated by one time for preparation, and then the heavy metal solution and the diluted plant nutrient solution are prepared according to the proportion of 1:1, namely the concentration of the heavy metal in the culture solution is the designed concentration. Only adding plant nutrient solution into the control group, wherein the culture solution just does not contain filter paper; the experimental group was added with the same amount of plants as the control groupCulturing the culture solution for 24h, mixing the culture solution uniformly according to the proportion of heavy metal to plant nutrient solution (1: 1) after the JDD of the Bacillus aryabhattai is impregnated to seeds, adding the mixture into an experimental group and a control group in equal amount, covering a dish cover (the concentration of the prepared heavy metal is twice that of the designed heavy metal copper so as to avoid diluting the plant nutrient solution mother solution), adding the mixture into the dish cover, covering the dish cover, and placing the dish cover at the temperature of 28 ℃, the relative humidity of 50% and the illumination time of 10h d^-1The intelligent artificial climate box can keep good ventilation. In order to avoid the change of the concentration of the culture solution in the dish, the filter paper is replaced every two days, and the mixed culture solution of the heavy metal solution and the plant nutrient solution is added again.

3. Results

3.1 Effect of Bacillus aryabhattai JDD on seed Germination Rate

As can be seen from FIG. 11, the germination rate of the uninfected pseudo-ginseng seeds tends to decrease first and then increase and then decrease as the concentration of the heavy metal copper increases, and the germination rate of the experimental group (inoculated with Bacillus aryabhattai JDD) is significantly higher than that of the control group (unawarrior Bacillus JDD).

When the concentration of heavy metal copper is 300, 350, 400, 450 and 500mg/L, the germination rate of the seed without the contaminated bacteria is respectively reduced to 58.8 percent, 36.7 percent, 21 percent, 55 percent and 39 percent from 73.3 percent when the seed is not contaminated by copper. The germination of the infected pseudo-ginseng seeds is kept high and stable, and when the infected pseudo-ginseng seeds are not polluted by copper, the germination rate of the infected seeds is 90 percent; when the concentration of the heavy metal copper is 50-150 mg/L, the germination rate of the pseudo-ginseng seeds is more than 90%; when the concentration of heavy metal copper is 200-350 mg/L, the germination rate of the pseudo-ginseng seeds is 81.1-91%; when the concentration of heavy metal copper is 400, 450 and 500mg/L, the germination rate of the pseudo-kidney bean seeds is 78.9%, 76.7% and 77.8%, which shows that the germination rate of the pseudo-kidney bean seeds inoculated with the Bacillus aryabhattai JDD is obviously higher than that of a control group (non-Bacillus aryabhattai JDD) under various copper concentrations, and particularly under the copper pollution of 300, 350, 400, 450 and 500mg/L, the germination rate of the inoculated seeds still keeps a stable state, and the germination rates are respectively 1.45 times, 2.21 times, 3.73 times and 1.37 times and 1.94 times of the non-infected seeds (as shown in figure 12).

The results show that the Bacillus aryabhattai JDD can improve the germination rate of the pseudo-ginseng seeds, and the germination rate of the infected seeds is kept between 76.7 and 91 percent along with the increase of the copper concentration, and is obviously higher than that of a control group. The stress effect of heavy metal copper on the pseudo-ginseng can be effectively relieved after the bacillus aryabhattai JDD is infected with the pseudo-ginseng, and the seed germination rate is improved.

3.2 Effect of Bacillus aryabhattai JDD on seedling growth

(1) The result of the effect of Bacillus aryabhattai JDD on the plant height of the pseudo-ginseng is shown in FIG. 13, and the growth invasion effect of the pseudo-ginseng seedlings is obvious along with the increase of the concentration of heavy metal copper in the culture solution; compared with a control group, the height of the pseudomonas pseudolari of the experimental group inoculated with the Bacillus aryabhattai JDD is obviously increased, and when the concentration of the heavy metal copper is 400, 450 and 500mg/L, the plant height is obviously greater than that of the control group without being infected with the bacillus pseudoradaiti JDD, so that the Bacillus aryabhattai JDD has a certain growth promoting effect on the plant height of the seedling polluted by the heavy metal copper.

(2) The result of the effect of Bacillus aryabhattai JDD on the overground fresh weight of the pseudo-ginseng is shown in FIG. 14, and the growth invasion effect on the seedling of the control group pseudo-ginseng is obvious along with the increase of the concentration of heavy metal copper in the culture solution; compared with a control group, the overground fresh weight of the pseudo-ginseng of the experimental group inoculated with the Bacillus aryabhattai JDD is obviously increased, and when the concentration of the heavy metal copper is 400, 450 and 500mg/L, the overground fresh weight of a plant body is obviously larger than that of the control group without being infected with bacteria, so that the Bacillus aryabhattai JDD has a certain growth promoting effect on the overground fresh weight of seedlings polluted by the heavy metal copper.

(3) The result of the effect of the Bacillus aryabhattai JDD on the underground fresh weight of the pseudo-geotrichum is shown in FIG. 15, and the invasion effect on the underground part of the seedling of the control pseudo-geotrichum is obvious along with the increase of the concentration of heavy metal copper in the culture solution; compared with the control group, the underground fresh weight of the pseudomonas putida inoculated with the bacillus aryabhattai JDD in the experimental group is obviously increased, and particularly, when the concentration of the heavy metal copper is 400, 450 and 500mg/L, the underground fresh weight of the infected plant body is respectively 5 times, 4 times and 4.3 times that of the uninfected plant body (as shown in figure 16). The fact shows that the Bacillus aryabhattai JDD has obvious growth promotion effect on the underground part of the seedling polluted by heavy metal copper.

The results of 3.1 and 3.2 show that the Bacillus aryabhattai JDD can promote the germination of the pseudo-georgette seeds under the stress of copper, improve the germination rate and have obvious growth promoting effect on the seed germination; the Bacillus aryabhattai JDD can promote the growth of the plant height, the fresh weight on the ground and the fresh weight under the ground of seedlings and plays a certain role in promoting the growth of the seedling of the pseudo-geotrichum japonicum.

Sequence listing

<110> Han mountain teaching college

<120> heavy metal copper-resistant bacillus aryabhattai and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1421

<212> DNA

<213> Pseudobulbus Virginianum (Desmodium heterophyllum)

<400> 1

aggtacgcta gctccttacg gttactccac cgacttcggg tgttacaaac tctcgtggtg 60

tgacgggcgg tgtgtacaag gcccgggaac gtattcaccg cggcatgctg atccgcgatt 120

actagcgatt ccagcttcat gtaggcgagt tgcagcctac aatccgaact gagaatggtt 180

ttatgggatt ggcttgacct cgcggtcttg cagccctttg taccatccat tgtagcacgt 240

gtgtagccca ggtcataagg ggcatgatga tttgacgtca tccccacctt cctccggttt 300

gtcaccggca gtcaccttag agtgcccaac taaatgctgg caactaagat caagggttgc 360

gctcgttgcg ggacttaacc caacatctca cgacacgagc tgacgacaac catgcaccac 420

ctgtcactct gtcccccgaa ggggaacgct ctatctctag agttgtcaga ggatgtcaag 480

acctggtaag gttcttcgcg ttgcttcgaa ttaaaccaca tgctccaccg cttgtgcggg 540

cccccgtcaa ttcctttgag tttcagtctt gcgaccgtac tccccaggcg gagtgcttaa 600

tgcgttagct gcagcactaa agggcggaaa ccctctaaca cttagcactc atcgtttacg 660

gcgtggacta ccagggtatc taatcctgtt tgctccccac gctttcgcgc ctcagcgtca 720

gttacagacc aaaaagccgc cttcgccact ggtgttcctc cacatctcta cgcatttcac 780

cgctacacgt ggaattccgc ttttctcttc tgcactcaag ttccccagtt tccaatgacc 840

ctccacggtt gagccgtggg ctttcacatc agacttaaga aaccgcctgc gcgcgcttta 900

cgcccaataa ttccggataa cgcttgccac ctacgtatta ccgcggctgc tggcacgtag 960

ttagccgtgg ctttctggtt aggtaccgtc aaggtacgag cagttactct cgtacttgtt 1020

cttccctaac aacagagttt tacgacccga aagccttcat cactcacgcg gcgttgctcc 1080

gtcagacttt cgtccattgc ggaagattcc ctactgctgc ctcccgtagg agtctgggcc 1140

gtgtctcagt cccagtgtgg ccgatcaccc tctcaggtcg gctatgcatc gttgccttgg 1200

tgagccgtta cctcaccaac tagctaatgc accgcgggcc catctgtaag tgatagccga 1260

aaccatcttt caatcatctc ccatgaagga gaagatccta tccggtatta gcttcggttt 1320

cccgaagtta tcccagtctt acaggcaggt tgcccacgtg ttactcaccc gtccgccgct 1380

aacgtcatag aagcaagctt ctaatcagtt cgctcgactg c 1421

Claims

1. A Bacillus aryabhattai (Bacillus aryabhattai) JDD, which was deposited at the Guangdong province culture Collection on 8/5.2021 with the deposit number: GDMCC No: 61645.

2. the application of the JDD of the bacillus aryabhattai or the suspension of the JDD of the bacillus aryabhattai or the microbial inoculum containing the JDD of the bacillus aryabhattai in remediation of the heavy metal polluted soil.

3. Use according to claim 2, wherein the heavy metal is copper.

4. The use of a JDD of bacillus aryabhattai, or a suspension of JDD of bacillus aryabhattai, or a microbial preparation comprising JDD of bacillus aryabhattai, as defined in claim 1, for promoting plant growth.

5. The use of the JDD of bacillus aryabhattai, or the suspension of JDD of bacillus aryabhattai, or the microbial preparation comprising JDD of bacillus aryabhattai for increasing the tolerance of plants to heavy metal stress environments according to claim 1.

6. The application of the JDD of Bacillus aryabhattai, the suspension of the JDD of Bacillus aryabhattai or the microbial inoculum containing the JDD of Bacillus aryabhattai in promoting the growth of plants in the environment with heavy metal stress.

7. Use according to any one of claims 4 to 6, wherein the plant is a Phaseolus vulgaris.

8. Use according to any one of claims 5 to 6, wherein the heavy metal is copper.

9. A plant growth promoting agent comprising the Bacillus aryabhattai JDD of claim 1, or a suspension of the Bacillus aryabhattai JDD, or a microbial agent comprising the Bacillus aryabhattai JDD.

10. A soil remediation agent comprising the Bacillus aryabhattai JDD of claim 1, or a suspension thereof, or a microbial inoculum containing the Bacillus aryabhattai JDD.