Bacillus thuringiensis 07 for repairing cadmium-polluted soil and application of bacillus thuringiensis 07

Abstract

The invention relates to bacillus thuringiensis 07 for repairing cadmium-polluted soil and application of the bacillus thuringiensis 07, and belongs to the technical field of environmental pollution treatment. The invention provides bacillus thuringiensis 07 for repairing cadmium-polluted soil, the Latin name of the bacillus thuringiensis 07 is bacillus thuringiensis 07, the bacillus thuringiensis 07 is preserved in the Guangdong Microbial Culture Collection Center on December 2, 2024, the preservation address is the 5th floor, No. 59 building, No. 100 Courtyard, Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC (China General Microbiological Culture Collection Center): 65572. The bacillus thuringiensis 07 has the effect of repairing the cadmium-polluted soil, can promote crop growth, and has the advantages of being low in cost, high in repairing efficiency and simple and convenient in treatment step when being applied to repairing the cadmium-polluted soil together with the smooth vetch.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental pollution control, and in particular to Bacillus thuringiensis 07 for repairing cadmium-contaminated soil and application thereof. Background Art

[0002] The background content of (cadmium) Cd in the global soil is 0.01-2.00 mg / kg, with a median of 0.35 mg / kg. Heavy metal pollution in soil not only affects the soil environment itself, causing changes in soil physical and chemical properties, microbial communities, and enzyme activity, but also has a significant impact on plant growth, inhibiting plant growth and reducing aboveground biomass, causing damage to plant cell membranes, biomolecules, and organelles, and inhibiting plant photosynthesis. Therefore, the remediation of Cd-contaminated soil is urgent.

[0003] At present, the methods for soil Cd pollution remediation can be roughly divided into agricultural ecological restoration, physical restoration, chemical restoration and biological restoration. Among them, agricultural ecological restoration measures refer to the selection of farming management systems according to local conditions to reduce the harm of heavy metals. Agricultural ecological restoration measures can not only maintain soil fertility, but also promote the natural ecological cycle and coordinated operation of the system, but there are unfavorable factors such as long restoration time and slow effect; the main methods of physical restoration are soil import method, soil replacement method and soil turning method. The cadmium pollution in the soil is reduced by adding clean soil, removing old soil and deeply burying polluted soil. This method is simple and fast, but it does not really remove cadmium pollution from the soil and has potential hazards; chemical remediation refers to the addition of chemical modifiers to the contaminated soil to reduce the heavy metal pollution. It is a kind of fixed conversion, dissolution extraction and extraction separation, so as to reduce the content of heavy metals in contaminated soil and change the soil environmental conditions. Chemical remediation is carried out on the basis of contaminated soil and is simple and easy, but it only changes the form of cadmium in the soil and does not really remove cadmium pollution. There is a possibility of reactivation hazards and it is not a permanent remediation measure. Biological remediation refers to the use of certain habits of organisms to adapt to, inhibit and improve heavy metal pollution. Generally, there are animal remediation, plant remediation and microbial remediation. Among them, microbial cadmium contaminated soil remediation, as a green and environmentally friendly remediation technology, has attracted great attention from relevant research institutions at home and abroad and has broad application prospects, but the remediation is slow and the remediation effect is unstable, making it difficult to promote.

[0004] Based on this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a Bacillus thuringiensis 07 for repairing cadmium-contaminated soil and its application, so as to solve the problem that the bioremediation method in the prior art has a slow speed and unstable effect in repairing Cd-contaminated soil.

[0006] To achieve the above-mentioned invention objectives, the present invention provides the following technical solutions:

[0007] The present invention provides a Bacillus thuringiensis 07 for repairing cadmium-polluted soil. The Latin name of the Bacillus thuringiensis 07 is Bacillus thuringiensis, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on December 02, 2024. The deposit address is the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 65572.

[0008] The present invention provides a method for preparing a bacterial liquid of the Bacillus thuringiensis 07. The Bacillus thuringiensis 07 is inoculated into an LB liquid medium and cultured at a rotation speed of 180-220 rpm until the logarithmic growth phase.

[0009] Preferably, the temperature of the culture is 28-32 °C.

[0010] The present invention provides a method for preparing a bacterial suspension of the Bacillus thuringiensis 07, which includes the following steps:

[0011] (1) The Bacillus thuringiensis 07 is inoculated into an LB solid medium and cultured at 28-32 °C for 22-26 h to obtain the cultured bacterial strain;

[0012] (2) The cultured bacterial strain is inoculated into an LB liquid medium and cultured for 2-4 days to obtain a fermented bacterial liquid;

[0013] (3) The fermented bacterial liquid is centrifuged to obtain a precipitate, which is resuspended with water to obtain a bacterial suspension.

[0014] Preferably, the temperature of the culture in step (2) is 28-32 °C.

[0015] Preferably, the rotation speed of the culture in step (2) is 180-220 rpm.

[0016] Preferably, the concentration of the bacterial suspension in step (3) is 0.5-1.5×10 8 CFU / mL.

[0017] The present invention provides the application of the Bacillus thuringiensis 07 or the bacterial liquid prepared by the preparation method or the bacterial suspension prepared by the preparation method in the preparation of a product for repairing cadmium-polluted soil.

[0018] The present invention provides the application of the Bacillus thuringiensis 07 or the bacterial liquid prepared by the preparation method or the bacterial suspension prepared by the preparation method in the preparation of a product for promoting plant growth.

[0019] The present invention provides a method for repairing cadmium-polluted soil, which uses spraying a microbial suspension in combination with planting Vicia villosa Roth to repair cadmium-polluted soil;

[0020] The microbial suspension is the suspension prepared by the described preparation method.

[0021] The present invention has the following technical effects and advantages:

[0022] The present invention has screened out a Bacillus thuringiensis 07 for repairing cadmium-polluted soil. The Bacillus thuringiensis 07 not only has the function of repairing cadmium-polluted soil, but also can promote the growth of crops. Its combined application with Vicia villosa Roth in the repair of cadmium-polluted soil has the advantages of low cost, high repair efficiency, and simple treatment steps. Description of the Drawings

[0023] Figure 1 Colony morphology of strain 07 isolated in Example 1;

[0024] Figure 2 Phylogenetic tree of strain 07 isolated in Example 1;

[0025] Figure 3 Growth-promoting characteristics of Bacillus thuringiensis 07;

[0026] Figure 4 Removal rate of Cd by Bacillus thuringiensis 07 under different treatment conditions 2+ ;

[0027] Figure 5 Adsorption amount of Cd by Bacillus thuringiensis 07 under different treatment conditions 2+ ;

[0028] Figure 6 Effect of different initial cadmium concentrations on the growth of Bacillus thuringiensis 07;

[0029] Figure 7 Effect of different initial cadmium concentrations on the removal rate of Cd by Bacillus thuringiensis 07 2+ ;

[0030] Figure 8 Above-ground growth and underground root growth of Vicia villosa Roth in different treatment groups;

[0031] Figure 9 Plant height, root length, number of root nodules, above-ground part, fresh weight of roots, and heavy metal content in both above-ground and underground parts of Vicia villosa Roth in different treatment groups;

[0032] Figure 10Effect of Vicia villosa Roth on available cadmium (Cd) content in soil for different treatment groups;

[0033] Figure 11 Growth of aboveground and underground roots of Brassica chinensis in soil for different treatment groups;

[0034] Figure 12 Effect of soil on growth indexes of Brassica chinensis for different treatment groups;

[0035] Figure 13 Effect of soil on root morphology of Brassica chinensis for different treatment groups;

[0036] Figure 14 Effect of Cd content in soil and Brassica chinensis for different treatment groups.

[0037] Deposit Instructions

[0038] The Latin name of Bacillus thuringiensis 07 is Bacillus thuringiensis. It was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on December 02, 2024. The deposit address is the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 65572. Detailed Implementation Modes

[0039] The present invention provides a Bacillus thuringiensis 07 for repairing cadmium-polluted soil. The Latin name of the Bacillus thuringiensis 07 is Bacillus thuringiensis. It was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on December 02, 2024. The deposit address is the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 65572.

[0040] The present invention provides a method for preparing a bacterial liquid of the Bacillus thuringiensis 07. The Bacillus thuringiensis 07 is inoculated into an LB liquid medium and cultured to the logarithmic growth phase at a rotation speed of 180 - 220 rpm;

[0041] The preferred rotation speed of the culture is 200 rpm.

[0042] In the present invention, the temperature of the culture is 28 - 32 °C, preferably 30 °C.

[0043] The present invention provides a method for preparing a bacterial suspension of the Bacillus thuringiensis 07, including the following steps:

[0044] (1) The Bacillus thuringiensis 07 is inoculated into an LB solid medium and cultured at 28 - 32 °C for 22 - 26 h to obtain the cultured bacterial strain;

[0045] The culture temperature is preferably 30 °C, and the culture time is preferably 24 h;

[0046] (2) Inoculate the cultured strain into LB liquid medium and culture for 2 - 4 days to obtain a fermented bacterial liquid;

[0047] The culture time is preferably 3 days;

[0048] (3) Centrifuge the fermented bacterial liquid to obtain a precipitate, and resuspend it with water to obtain a bacterial suspension.

[0049] In the present invention, the culture temperature in step (2) is 28 - 32 °C, preferably 30 °C.

[0050] In the present invention, the culture rotation speed in step (2) is 180 - 220 rpm, preferably 200 rpm.

[0051] In the present invention, the concentration of the bacterial suspension in step (3) is 0.5 - 1.5×10 8 CFU / mL, preferably 1×10 8 CFU / mL.

[0052] The present invention provides the application of the Bacillus thuringiensis 07 as described above, or the bacterial liquid prepared by the preparation method as described above, or the bacterial suspension prepared by the preparation method as described above in the preparation of a product for repairing cadmium - contaminated soil.

[0053] The present invention provides the application of the Bacillus thuringiensis 07 as described above, or the bacterial liquid prepared by the preparation method as described above, or the bacterial suspension prepared by the preparation method as described above in the preparation of a product for promoting plant growth.

[0054] The present invention provides a method for repairing cadmium - contaminated soil, which uses spraying a microbial bacterial suspension in combination with planting Vicia villosa to repair the cadmium - contaminated soil;

[0055] The microbial bacterial suspension is the bacterial suspension prepared by the preparation method as described above.

[0056] The following combines examples to elaborate on the technical solutions provided by the present invention in detail, but they cannot be understood as limiting the protection scope of the present invention.

[0057] Example 1: Screening of cadmium (Cd) - tolerant endophytic bacteria in the roots of Vicia villosa

[0058] 1. Collection of Vicia villosa samples

[0059] The collection site of Vicia villosa is a moderately Cd - contaminated farmland in the south of China, and the growing Vicia villosa has certain tolerance and enrichment ability to Cd.

[0060] The collection method was to randomly dig up healthy Vicia villosa Roth plants with roots and soil. After the samples, together with the rhizosphere soil, were put into pre-prepared clean fresh-keeping bags, they were taken back to the laboratory and stored at 4°C for the isolation of root endophytic bacteria.

[0061] 2. Isolation of Cd-tolerant endophytic bacteria

[0062] The collected roots of Vicia villosa Roth were rinsed clean with sterilized water and air-dried indoors. Under sterile conditions, 10 g of the washed plant roots were weighed, surface-sterilized with 75% ethanol, cut into 2 - 3 cm segments with a sterile scalpel, soaked in 75% ethanol for 1 min, and then rinsed twice with sterile water. Then, they were soaked in 0.1% mercuric chloride for 30 s, rinsed three times with sterile water, put into a sterilized mortar containing 9 mL of sterile water, and a little sterilized quartz sand was added and ground evenly. After standing for 15 min, 1 mL was taken and diluted to 10 -4 、10 -5 、10 -6 concentration gradients. From each concentration gradient suspension, 0.1 mL of the solution was taken and spread on an LB solid medium (CdCl2 100 mg, agar 15 g / L, yeast powder 5.0 g / L, NaCl 10.0 g / L, tryptone 10.0 g / L, made up to 1000 mL with distilled water, adjusted to pH 7.0, sterilized at 121°C for 20 min) with a sterile spreading rod, where the Cd 2+ concentration was 100 mg / L. The plates were incubated upside down at 30°C, with the sterile water used to wash the samples last as a control. After continuously observing for 5 - 7 days, when colonies grew on the surface of the solid medium, single colonies with different morphologies and sizes were separately picked and inoculated onto new LB solid media for streak culture to isolate single-form strains. When colonies grew on the new solid media, they were purified two more times, and thus multiple single strains were isolated. After screening, a strain that could tolerate a Cd 2+ concentration of 100 mg / L was obtained, and this strain was named 07.

[0063] Example 2: Morphological, physiological and biochemical, and molecular biological identification of strain 07

[0064] 1. Morphological identification

[0065] The strain 07 isolated in Example 1 was inoculated into LB solid medium for ordinary culture and streak culture respectively. The colony morphology in ordinary culture was as shown in Figure 1 (A), and the streak culture was as shown in Figure 1 (B). Then, the characteristics of the strain were observed under an electron microscope, and the results were as shown in Figure 1 (C).

[0066] According to Figure 1 (A) and Figure 1(B) It can be seen that the colony characteristics of strain 07 are round light gray colonies, with a viscous surface, a raised and wrinkled middle, regular wavy edges, and a pungent odor. According to Figure 1 (C) It can be seen that under the electron microscope, the bacteria are oval in structure, and the surface of the bacteria is wrinkled and uneven.

[0067] 2. Identification of physiological and biochemical characteristics

[0068] The Biolog microplate method was used and carried out in accordance with the operation manual of Biolog GenⅢ microplate (Beijing Zhijie Fangyuan Technology Co., Ltd.). The specific operation was to inoculate the pure strain of strain 07 isolated in Example 1 into BUG agar medium and culture it at 33°C; then use a cotton swab to pick up the colonies from the plate, and then insert the end of the cotton swab into the bottom of the inoculation tube containing the inoculation liquid, and shake it up and down to release the bacteria into the inoculation liquid. After stirring evenly, a bacterial suspension was obtained, and it was measured with a turbidimeter and adjusted to the required concentration; then the bacterial suspension was added to the identification plate and the lid was covered. After 24h and 48h respectively, it was put into the Biolog reader for measurement. The Biolog identification results are shown in Table 1. In the table, "+" indicates positive and "-" indicates negative.

[0069] According to Table 1, in terms of physical and chemical properties, strain 07 shows Gram-positive staining, as Figure 1 (D) shows. In addition, strain 07 can also hydrolyze milk, starch, Tween 20, Tween 80, and cellulose, and is also positive in nitrate reduction, arginine dihydrolase, esculin hydrolysis, and gelatin hydrolysis, that is, strain 07 can grow normally under these conditions; in terms of carbon source utilization, strain 07 can decompose and utilize carbon sources such as D-glucose, N-acetylglucosamine, potassium gluconate, D-maltose, malic acid, and trisodium citrate, while it is negative for L-arabinose, D-mannose, D-dextromannose, caproic acid, adipic acid, phenylacetic acid, etc., that is, it cannot be fully utilized; in addition, the ZYM reagent conditions of strain 07 were tested. Strain 07 is positive for alkaline phosphatase, esterase (C4), lipase (C8), leucine arylamidase, and α-glucosidase, and negative for lipase (C14), valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, β-glucosidase, N-acetyl-glucosaminidase, α-mannosidase, and β-fucosidase. Generally speaking, strain 07 can adapt to a variety of environments and can decompose and utilize a variety of organic substances, which conforms to the physiological and biochemical characteristics of Bacillus.

[0070] Table 1 Biolog identification results of strain 07

[0071]

[0072]

[0073] 3. Molecular biological identification

[0074] The strain 07 isolated in Example 1 was identified by 16S rRNA amplification and sequence analysis method.

[0075] The specific method is as follows: The genomic DNA of the purified strain was extracted using the TSINGKE Plant DNA Extraction Kit (universal type), and the 16S rRNA of the strain was amplified using the following universal primers (synthesized by Beijing Tsingke Biotechnology Co., Ltd.): 27F (upstream primer, sequence: 5'-AGTTTGATCMTGGCTCAG-3', SEQ ID No.1) and 1492R (downstream primer, sequence 5'-GGTTACCTTGTTACGACTT-3', SEQ ID No.2), and then the amplified product was sent to Beijing Tsingke Biotechnology Co., Ltd. for sequencing. The amplification system of 16S rRNA is shown in Table 2, and the amplification conditions of 16S rRNA are shown in Table 3.

[0076] Table 2 Amplification system of 16S rRNA

[0077] Component Volume / μL 1×TSE101 Gold Mix 45 27F(10P) 2 1492R(10P) 2 DNA Template 1 Total Volume 50

[0078] Table 3 Amplification conditions of 16S rRNA

[0079]

[0080]

[0081] The sequencing results were uploaded to the NCBI database, and BLAST alignment was performed on the website, and the MEGA 11 software was used to construct a phylogenetic tree using the Neighbor-Joining method, as Figure 2 shown.

[0082] According to Figure 2 it can be seen that the strain 07 has the closest genetic relationship with Bacillus thuringiensis P2-27, clusters into one branch, and the sequence homology is 99%.

[0083] In summary, based on the culture characteristics, morphological characteristics, physiological and biochemical characteristics of strain 07 and the results of 16S rRNA sequence analysis, this strain was identified as Bacillus thuringiensis 07, with its Latin name being Bacillus thuringiensis. It was deposited at the Guangdong Microbial Culture Collection Center on December 02, 2024. The deposit address is the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 65572.

[0084] Example 3: Determination of the growth-promoting characteristics of Bacillus thuringiensis 07

[0085] 1. Determination of phosphorus-solubilizing ability

[0086] Prepare Pikovskava inorganic phosphorus-solubilizing solid medium:

[0087] Dissolve 0.5 g of (NH4)2SO4, 0.3 g of MgSO4·7H2O, 0.3 g of FeSO4·7H2O, 0.03 g of MnSO4·7H2O, 5 g of Ca3(PO4)2, 10 g of glucose, and 15 g of agar in deionized water, make up the volume to 1 L, and adjust the pH value to 7 to obtain Pikovskava inorganic phosphorus-solubilizing solid medium.

[0088] Prepare organic phosphorus-solubilizing solid medium:

[0089] Dissolve 10 g of glucose, 0.5 g of (NH4)2SO4, 0.3 g of NaCl, 0.3 g of KCl, 0.03 g of FeSO4·7H20, 0.03 g of MnSO4·4H2O, 0.2 g of egg yolk lecithin, 5 g of CaCO3, 0.4 g of yeast extract, and 15 g of agar in deionized water, make up the volume to 1 L, and adjust the pH value to 7; sterilize by high-pressure steam to obtain organic phosphorus-solubilizing solid medium.

[0090] Inoculate the preserved Bacillus thuringiensis 07 into the inorganic phosphorus-solubilizing solid medium and the organic phosphorus solid medium respectively. After culturing in an incubator at a constant temperature of 30°C for 7 days, observe whether there is a clear zone around the colony. If there is a clear zone, it is considered that Bacillus thuringiensis 07 has the ability to dissolve inorganic phosphorus and organic phosphorus, and the diameter of the clear zone is the size of the ability of the strain to dissolve inorganic phosphorus and organic phosphorus. If there is no clear zone, it is considered that Bacillus thuringiensis 07 does not have the ability to dissolve inorganic phosphorus and organic phosphorus.

[0091] 2. Determination of siderophore-producing ability

[0092] Siderophore refers to a substance secreted by bacteria into the surrounding environment under iron-deficient conditions for Fe 3+Substances with extremely strong specific chelating effects. The siderophore of Bacillus thuringiensis 07 was determined by the CAS plate method. The principle is that chrome azurol S (CAS), ferric ions, and cetyltrimethylammonium bromide (HTDMA) can form a sky-blue complex. The culture plate is stained with the CAS dye solution. When the strain releases ferric ions into the culture plate, the ternary complex is destroyed and turns yellow, that is, a yellow halo appears around the colony.

[0093] Prepare the CAS detection medium:

[0094] Dissolve 100 g of glucose, 20 g of peptone, 0.5 g of magnesium sulfate heptahydrate, 0.5 g of calcium chloride, 0.06 g of chrome azurol (CAS), 0.0027 g of ferric trichloride hexahydrate, and 0.073 g of cetyltrimethylammonium bromide in 200 mL of 10× buffer solution, and make up the volume to 1 L with deionized water. Adjust the pH value to 6.8 to obtain the CAS detection liquid medium. During the preparation process, add 15 g of agar to obtain the CAS detection solid medium.

[0095] Prepare the CDA liquid medium:

[0096] Dissolve 10 g of casein peptone, 1 g of glucose, 5 g of yeast extract, and 5 g of sodium chloride in deionized water, and make up the volume to 1 L. Adjust the pH value to 7 to obtain the CDA liquid medium.

[0097] Inoculate Bacillus thuringiensis 07 on the CAS detection solid medium, and incubate it in an incubator at a constant temperature of 30 °C for 7 days. Record the presence or absence of an orange halo on the medium to preliminarily determine whether the strain has the ability to produce siderophores. If the strain has the ability to produce siderophores, then quantitatively analyze the produced siderophores.

[0098] The quantitative analysis method is as follows:

[0099] Inoculate Bacillus thuringiensis 07 on the CDA liquid medium, and incubate it in an incubator at a constant temperature of 30 °C for 7 days. Centrifuge to obtain the supernatant. Mix the supernatant with the CAS iron detection liquid medium in equal volume, and perform color development in the dark box at 30 °C for 72 hours. After completion, measure the absorbance value at a wavelength of 630 nm. Compare the absorbance value with the absorbance value of the control (λ / λ0) to determine the quantitative situation of the siderophores produced by Bacillus thuringiensis 07.

[0100] 3. Determination of potassium solubilizing ability

[0101] Prepare the Aleksandrov silicate medium:

[0102] Dissolve 5 g of yeast extract powder, 0.1 g of calcium carbonate, 0.04 g of calcium chloride, 1.67 g of K2HPO4·3H2O, 0.87 g of KH2PO4, 0.004 g of FeCl3·6H2O, 0.1 g of MgSO4·7H2O, and 15.0 g of agar in deionized water, make up the volume to 1 L, adjust the pH value to 7, and sterilize by high-pressure steam to obtain Aleksandrov silicate medium.

[0103] Inoculate Bacillus thuringiensis 07 on Aleksandrov silicate medium, and culture it in an incubator at a constant temperature of 30 °C for 7 days. If Bacillus thuringiensis 07 can grow on the medium, it indicates that it has the ability to dissolve potassium.

[0104] 4. Determination of nitrogen fixation ability

[0105] Prepare Ashby's nitrogen-free medium:

[0106] Dissolve 10 g of mannitol, 0.2 g of KH2PO4, 0.2 g of MgSO4, 0.2 g of NaCl, 0.1 g of CaSO4, 5 g of CaCO3, and 15 g of agar in deionized water, make up the volume to 1 L, adjust the pH value to 7, and sterilize by high-pressure steam to obtain Ashby's nitrogen-free medium.

[0107] Inoculate Bacillus thuringiensis 07 on Ashby's nitrogen-free medium, and culture it in an incubator at a constant temperature of 30 °C for 7 days. If Bacillus thuringiensis 07 can grow on the medium, it indicates that it has the ability to fix nitrogen.

[0108] 5. Determination of the ability to produce indole-3-acetic acid (IAA)

[0109] Add L-tryptophan to LB liquid medium to obtain LB liquid medium containing L-tryptophan, so that the final concentration of L-tryptophan in the medium is 0.5 mg / mL.

[0110] Inoculate Bacillus thuringiensis 07 into LB liquid medium containing L-tryptophan, and culture it at 28 °C with a shaking speed of 180 rpm for 24 h, using LB liquid medium without tryptophan as a negative control. After the culture is completed, centrifuge at 8000 rpm for 10 min, take 1 mL of the supernatant into a test tube, add 2 mL of Salkowski's reagent (1 mL of 0.5 M FeCl3 solution and 50 mL of 35% HClO4 solution), and react in the dark for 20 min. Compared with the control, if a pink color appears in the test tube, it is determined that IAA is produced. If IAA is produced, further quantitative analysis of IAA is carried out.

[0111] The quantitative analysis method is as follows:

[0112] Weigh 0.1 g of IAA, dissolve it with a small amount of 95% ethanol, and make up the volume to 100 mL with ddH2O to prepare a stock solution with a concentration of 1000 mg / L, and store it at 4°C. Dilute the IAA stock solution to prepare IAA standard solutions with concentrations of 0, 5, 10, 20, 40, 80, and 100 μg / mL. Similarly, take 1 mL of the standard solution and react it with 2 mL of Salkowski's reagent in the dark for 20 min, and measure the OD 530 absorbance value of, and draw an IAA standard curve.

[0113] For the colored liquid, measure the OD 530 absorbance value of, and calculate the concentration of IAA produced by the strain through the IAA standard curve.

[0114] Through the determination of the growth-promoting characteristics of Bacillus thuringiensis 07, the results are as Figure 3 shown, and then quantitative analysis is carried out. The results are shown in Table 4. In the table, "+" indicates positive and "-" indicates negative.

[0115] Table 4 Growth-promoting characteristics of Bacillus thuringiensis 07

[0116]

[0117] According to Table 4 and Figure 3 it can be seen that Bacillus thuringiensis 07 has the ability to dissolve organic phosphorus and potassium, and can also produce siderophores and IAA.

[0118] Example 4: Screening of the culture conditions for the adsorption of Cd by Bacillus thuringiensis 07 2+

[0119] Dissolve CdCl2 in sterile water to prepare a Cd 2+ mother liquor with a concentration of 1000 mg / L for standby.

[0120] Inoculate Bacillus thuringiensis 07 into LB broth medium, and the culture conditions are 30°C and a rotation speed of 200 rpm. Cultivate until the logarithmic growth phase to obtain a Bacillus thuringiensis 07 bacterial solution.

[0121] For the OD of the Bacillus thuringiensis 07 bacterial solution 600Adjust the value to 0.8, then aliquot it into 50 mL sterile centrifuge tubes, centrifuge at 6000 rpm and 4 °C for 15 min, collect the bacterial cell precipitate by centrifugation, wash the centrifuged bacterial cell precipitate with sterile normal saline to remove the culture medium residues, and repeat the washing 2 - 3 times. Place the washed bacterial cells in a 50 mL centrifuge tube, put on a sterile breathable sealing film at the tube mouth, and place it in a -20 °C refrigerator for pre-freezing to completely freeze the sample. Put the pre-frozen sample into the freeze-drying chamber of a freeze dryer, and freeze-dry at -80 °C for 12 h. After freeze-drying, aliquot the freeze-dried bacterial cells into sterile freeze-drying tubes and store them in a -20 °C refrigerator for later use.

[0122] Add Cd 2+ stock solution to LB liquid medium so that the final concentration of Cd 2+ is 100 mg / L to obtain LB liquid medium containing Cd 2+ Take 50 mL of LB liquid medium containing Cd 2+ and adjust the pH using 1 M HCl or NaOH to obtain LB liquid media containing Cd 2+ with pH values of 4.0, 5.0, 6.0, 7.0, and 8.0 respectively.

[0123] Add 0.05 g of freeze-dried bacterial cells to LB liquid media containing Cd 2+ with different pH values, and react by shaking at 180 rpm at 30 °C for 48 h. Measure the removal rate and adsorption capacity of Bacillus thuringiensis 07 for Cd 2+ under different pH conditions; set the pH value of the LB liquid medium containing Cd 2+ to 7, add 0.05 g of freeze-dried bacterial cells to the LB liquid medium containing Cd 2+ , and react by shaking at 180 rpm at 22, 25, 28, 30, and 33 °C for 48 h respectively. Measure the removal rate and adsorption capacity of Bacillus thuringiensis 07 for Cd 2+ at different temperatures; set the pH value of the LB liquid medium containing Cd 2+ to 7, add 0.05 g of freeze-dried bacterial cells to the LB liquid medium containing Cd 2+ , and react by shaking at 30 °C at 130, 150, 180, 200, and 220 rpm for 48 h respectively. Measure the removal rate and adsorption capacity of Bacillus thuringiensis 07 for Cd 2+ at different rotation speeds; set the pH value of the LB liquid medium containing Cd 2+ to 7, and add 0.02, 0.05, 0.08, 0.10, and 0.15 g of freeze-dried bacterial cells to the LB liquid medium containing Cd 2+In the LB liquid medium, the reaction was oscillated at 180 rpm at 30 °C for 48 h, and the removal rate and adsorption capacity of Bacillus thuringiensis 07 for Cd were measured under different inoculum amounts. 2+ The removal rate and adsorption capacity.

[0124] Each experiment was repeated 3 times, and a blank control was set. After the shaking reaction was completed, the reaction solution was centrifuged at 10,000 rpm for 15 min, and the concentration of Cd in the supernatant was measured by ICP-MS. 2+ Concentration, calculate the removal rate and adsorption capacity of Cd. The removal rate of Bacillus thuringiensis 07 for Cd under different treatment conditions is shown as 2+ follows. In the figure, (A) represents the removal rate of Bacillus thuringiensis 07 for Cd under different pH conditions, (B) represents the removal rate of Bacillus thuringiensis 07 for Cd under different temperatures, (C) represents the removal rate of Bacillus thuringiensis 07 for Cd under different rotation speeds, and (D) represents the removal rate of Bacillus thuringiensis 07 for Cd under different inoculum amounts; the adsorption capacity of Bacillus thuringiensis 07 for Cd under different treatment conditions is shown as 2+ follows. In the figure, (A) represents the adsorption capacity of Bacillus thuringiensis 07 for Cd under different pH conditions, (B) represents the adsorption capacity of Bacillus thuringiensis 07 for Cd under different temperatures, (C) represents the adsorption capacity of Bacillus thuringiensis 07 for Cd under different rotation speeds, and (D) represents the adsorption capacity of Bacillus thuringiensis 07 for Cd under different inoculum amounts. Figure 4 shown. In the figure, (A) represents the removal rate of Bacillus thuringiensis 07 for Cd 2+ under different pH conditions, (B) represents the removal rate of Bacillus thuringiensis 07 for Cd 2+ under different temperatures, (C) represents the removal rate of Bacillus thuringiensis 07 for Cd 2+ under different rotation speeds, and (D) represents the removal rate of Bacillus thuringiensis 07 for Cd 2+ under different inoculum amounts; the adsorption capacity of Bacillus thuringiensis 07 for Cd 2+ under different treatment conditions is shown as Figure 5 shown. In the figure, (A) represents the adsorption capacity of Bacillus thuringiensis 07 for Cd 2+ under different pH conditions, (B) represents the adsorption capacity of Bacillus thuringiensis 07 for Cd 2+ under different temperatures, (C) represents the adsorption capacity of Bacillus thuringiensis 07 for Cd 2+ under different rotation speeds, and (D) represents the adsorption capacity of Bacillus thuringiensis 07 for Cd 2+ under different inoculum amounts.

[0125] The formula for calculating the removal rate is:

[0126]

[0127] The formula for calculating the adsorption capacity is:

[0128]

[0129] In the formula, C0 represents the concentration of Cd in the medium before the reaction, C 2+ represents the concentration of Cd in the medium after the reaction, m (g) represents the mass of the freeze-dried bacteria, and V (L) represents the volume of the medium after the reaction. e represents the concentration of Cd in the medium after the reaction, m (g) represents the mass of the freeze-dried bacteria, and V (L) represents the volume of the medium after the reaction. 2+ According to

[0130] According to Figures 4 - 5 it can be seen that the pH value has an impact on the removal of Cd by Bacillus thuringiensis 072+ has a significant impact on the effect. The experimental results show that Bacillus thuringiensis 07 has the highest removal rate of Cd at a pH value of 5 2 +, and too high or too low pH values will reduce its adsorption capacity. Temperature also has a significant impact on the ability of Bacillus thuringiensis 07 to remove Cd 2+ , and the experimental results show that Bacillus thuringiensis 07 has the highest removal rate of Cd at 33°C 2+ , and too high or too low temperatures will affect its biological activity. The rotation speed affects the contact efficiency between bacteria and Cd 2+ , and the experiment shows that when the rotation speed is 200 rpm, Bacillus thuringiensis 07 has the highest removal rate of Cd 2 +, and too high a rotation speed may affect the attachment and growth of bacteria. The amount of inoculum directly affects the removal rate of Cd 2 + by bacteria. The experiment shows that when the inoculum amount is 0.1 g, Bacillus thuringiensis 07 has the highest removal rate of Cd 2+ , and there is a significant difference compared with other groups (P < 0.05). In addition, when the inoculum amount is 0.02 g, Bacillus thuringiensis 07 has the highest adsorption amount of Cd 2+ , and there is a significant difference compared with other groups (P < 0.05). This difference is attributed to the different effects of the inoculum amount on the adsorption amount, resulting in the difference. Since the removal rate of Cd by bacteria is relatively low when the inoculum amount is 0.02 g 2+ , the adsorption capacity and adsorption efficiency of bacteria must be considered simultaneously in the actual adsorption scheme. Therefore, considering comprehensively, the inoculum amount of 0.1 g is selected as the optimal inoculum amount of Bacillus thuringiensis 07. In summary, the optimal culture conditions for Bacillus thuringiensis 07 to adsorb Cd 2+ are: pH value of 5, culture temperature of 33°C, rotation speed of 200 rpm, and inoculum amount of 0.1 g.

[0131] Example 5: Effects of different initial cadmium concentrations on the adsorption of Cd by the strain 2+ by the strain

[0132] Using 250 mL conical flasks as containers to study the effects of different initial concentrations of Cd 2+ on the growth state of Bacillus thuringiensis 07 and Cd adsorption.

[0133] Prepare a Cd 2+ stock solution with a mass concentration of 1000 mg / L using CdCl2, and add the Cd 2+ stock solution to the LB liquid medium to make the Cd 2+ concentration in the LB liquid medium be 25, 50, 75, 100, and 125 mg / L, with no addition of Cd 2+The LB liquid medium of the mother liquor was used as a blank control. After autoclaving at 121 °C for 30 min, Bacillus thuringiensis 07 adsorbed Cd as determined in Example 4 2+ The best culture conditions were set as follows: the adsorption system had a pH value of 5, a culture temperature of 33 °C, an input amount of freeze-dried bacteria of 0.1 g, and continuous culture in a constant-temperature shaking incubator at a rotation speed of 200 rpm. After the above biological adsorption process was completed, sampling was set at 6, 12, 24, 48, 72, 120, 144, and 192 h. Centrifugation was carried out at 10,000 rpm for 15 min, and the Cd in the supernatant was measured by ICP-MS 2+ concentration. The experiment was set with 3 repeated treatments, and a blank control was also set. Calculate the Cd 2+ removal rate; measure the OD of the culture solution with a visible-ultraviolet spectrophotometer 600 value, measure the pH value with a pH meter. The effects of different initial cadmium concentrations on the growth of Bacillus thuringiensis 07 are as Figure 6 shown. In the figure, (A) shows the results of the change in pH value over time of Bacillus thuringiensis 07 in LB liquid medium containing different concentrations of Cd 2+ and (B) shows the results of the change in growth amount over time of Bacillus thuringiensis 07 in LB liquid medium containing different concentrations of Cd 2+ .

[0134] According to Figure 6 it can be seen that from the change in pH value, as the initial cadmium concentration increases, the pH value of the medium all rises in the initial stage of culture, but tends to be stable at 192 hours. Specifically, at Cd concentrations of 25 mg / L and 50 mg / L 2+ , the pH values at 192 hours are 8.93 and 8.98 respectively, indicating that at lower concentrations, the pH value changes less and tends to be stable; at higher concentrations (75 mg / L, 100 mg / L, and 125 mg / L), the pH values at 192 hours are 8.84, 8.51, and 8.42 respectively, indicating that as the cadmium concentration increases, the pH value rises more significantly, but the rising trend is relatively slow at the highest concentration (125 mg / L). From the change in growth amount, the growth amount (OD 600 ) of the strain increases with time at all concentrations, but the growth rate slows down at higher concentrations. At Cd concentrations of 25 mg / L and 50 mg / L 2+ , the OD 600 values at 192 hours are 1.831 and 1.496 respectively, indicating that at lower concentrations, the growth amount of the strain is higher and the growth rate is faster; at higher concentrations (75 mg / L, 100 mg / L, and 125 mg / L), the OD 600The values ​​were 1.778, 0.978 and 1.893, respectively, indicating that with the increase of cadmium concentration, the growth amount of the strain decreased and the growth rate slowed down significantly, but at 75 mg / L and 125 mg / L concentrations, the OD 600 The increase in the value was large, indicating that the strain could still grow well under these concentrations. Comprehensive analysis showed that Bacillus thuringiensis 07 showed good adaptability and growth ability in the cadmium environment with lower concentrations (25mg / L and 50mg / L), with small changes in pH value and high growth. At higher concentrations (75mg / L, 100mg / L and 125mg / L), although the strain could still grow, the pH value increased more significantly, the growth amount decreased and the growth rate slowed down, indicating that high concentrations of cadmium had a certain inhibitory effect on the growth of the strain. These results show that Bacillus thuringiensis 07 has good remediation potential in a low concentration Cd pollution environment and can be effectively used in the remediation of cadmium-contaminated soil. At the same time, even at higher concentrations, the strain can still maintain a certain growth ability, showing its adaptability and application prospects under different pollution levels.

[0135] Effect of different initial cadmium concentrations on the resistance of Bacillus thuringiensis 07 to Cd 2+ The removal rate of Figure 7 As shown in the figure, (A) shows the effect of Bacillus thuringiensis 07 on Cd when the initial cadmium concentration is 25 mg / L. 2+ (B) represents the removal rate of Cd by Bacillus thuringiensis 07 when the initial cadmium concentration is 50 mg / L 2+ The removal rate of Cd is shown in Table 1. (C) indicates the removal rate of Cd by Bacillus thuringiensis 07 when the initial cadmium concentration is 75 mg / L. 2+ The removal rate of Cd by Bacillus thuringiensis 07 is 100 mg / L. 2+ The removal rate of Cd was (E) when the initial cadmium concentration was 125 mg / L. 2+ removal rate.

[0136] according to Figure 7 It can be seen that at 25 mg / L Cd 2+ At the concentration, Bacillus thuringiensis 07 showed a more significant Cd 2+ Removal effect. As time went by, the removal rate showed a steady upward trend. At 192h, the removal rate reached a maximum of 92.27%, indicating that Bacillus thuringiensis 07 was effective in removing Cd at low concentrations. 2+ It has good adsorption performance and continuous removal ability in the environment, and can effectively reduce low concentration Cd in the soil 2+ The content of Cd provides strong support for the restoration of slightly Cd-contaminated soil. 2+When the concentration was increased to 50 mg / L, Bacillus thuringiensis 07 still maintained a high removal rate, and reached the highest of 76.49% at 120 h, with the removal rate being particularly prominent. This indicates that Bacillus thuringiensis 07 has strong adaptability and adsorption efficiency to medium-concentration Cd 2+ and can alleviate the problem of medium-concentration Cd pollution in soil to a certain extent, reducing the accumulation of Cd in soil and its toxicity to plants. In the face of a relatively high concentration of 75 mg / L Cd 2+ , the removal rate of Bacillus thuringiensis 07 increased rapidly in the initial stage of the experiment and remained at a relatively high level in the subsequent time, and reached the highest of 68.72% at 120 hours. This result highlights the high-efficiency adsorption ability of Bacillus thuringiensis 07 in an environment of relatively high concentration Cd 2+ and can quickly capture and fix Cd 2+ , reducing its activity and mobility in soil, providing an effective solution for treating moderately Cd-polluted soil and helping to improve soil environmental quality. Under the condition of a high concentration of 100 mg / L Cd 2 +, the removal rate of Bacillus thuringiensis 07 also performed well, and with the extension of time, the removal rate increased steadily, reaching the highest of 47.02% at 120 h. This shows that Bacillus thuringiensis 07 has the ability to continuously work in an environment of high concentration Cd 2+ and can gradually reduce the pollution degree of high-concentration Cd in soil, reducing the adverse effects of Cd on the soil ecosystem and plant growth, which is of great significance for treating severely Cd-polluted soil. Even in an environment of an ultra-high concentration of 125 mg / L Cd 2+ , Bacillus thuringiensis 07 still showed a certain removal rate, and the removal rate increased in the later stage of the experiment, and reached the highest of 40.26% at 144 h. This fully proves that Bacillus thuringiensis 07 has strong stress resistance and adaptability, can survive and play an adsorption role under extreme Cd 2+ concentration conditions, providing a potential remediation method for dealing with severely Cd-polluted soil and helping to reduce the total amount and toxicity of Cd in soil.

[0137] Example 6: Pot experiment on the remediation of Cd-polluted soil by Bacillus thuringiensis 07 combined with Vicia villosa

[0138] 8 kg of Cd-polluted soil was fully mixed and filled into polyethylene plastic flowerpots. Four treatment groups were set up (Treatment 1 group: no treatment, as a blank control; Treatment 2 group: only Vicia villosa was planted; Treatment 3 group: only the bacterial suspension of Bacillus thuringiensis 07 was irrigated; Treatment 4 group: Vicia villosa was planted and the bacterial suspension of Bacillus thuringiensis 07 was irrigated at the same time), and each treatment was repeated three times.

[0139] Germination and disinfection of Vicia villosa Roth seeds: Vicia villosa Roth seeds were disinfected with 1% sodium hypochlorite solution for 30 min, then rinsed 5 times with sterile water. Next, the seeds were soaked in sterile water at room temperature for 12 h to break seed dormancy. Then, Vicia villosa Roth seeds were evenly sown in flower pots at a seeding rate of 12 plants per pot in Treatment 2 and Treatment 4, and the planting depth was 1 cm. Subsequently, distilled water was used for irrigation every 3 days to ensure normal plant growth. No additional nutrients were added during the growth period of Vicia villosa Roth, and the light, temperature and other conditions were kept consistent for all treatments.

[0140] When the Vicia villosa Roth in the flower pots grew to a height of 3 cm, a bacterial suspension of Bacillus thuringiensis 07 was prepared. The Bacillus thuringiensis 07 preserved in glycerol at ultra-low temperature (-80 °C) was activated: Bacillus thuringiensis 07 was inoculated onto LB solid medium and cultured at 30 °C for 24 h by the streaking method. Then, single colonies were picked with a sterile inoculation loop and inoculated into LB liquid medium, and cultured with shaking at 200 rpm at 30 °C for 3 days to obtain the strain fermentation broth. Then, the strain fermentation broth was centrifuged to remove the LB liquid medium, and the cell precipitate was retained. The strain was then rinsed clean with sterile water, and the cell precipitate was formulated into a Bacillus thuringiensis bacterial suspension of 1×10 8 CFU / mL for standby.

[0141] In the flower pots of Treatment 3 and Treatment 4, 1 L of Bacillus thuringiensis bacterial suspension with a concentration of 1×10 8 CFU / mL was poured into the soil of each treatment. 1 L of sterile water was poured in Treatment 1 and Treatment 2. It was inoculated once a month, and the test cycle was 3 months, with a total of 3 inoculations. The entire pot experiment was carried out at the experimental base of the Yunnan Academy of Agricultural Sciences. The placement positions were randomly changed at regular intervals. During this period, the plant growth conditions such as light and water content were kept consistent for each treatment. Pest and disease control should be noted during the plant growth period, and other field management measures were carried out according to the conventional methods.

[0142] 1. Growth of Vicia villosa Roth in different treatment groups

[0143] When Vicia villosa Roth was harvested at the full-bloom stage, the above-ground growth and underground root growth of Vicia villosa Roth in different treatment groups were observed. The results are as Figure 8 shown. In the figure, (A) represents the above-ground growth of Vicia villosa Roth in Treatment 2 group, (B) represents the above-ground growth of Vicia villosa Roth in Treatment 3 group, (C) represents the root growth of Vicia villosa Roth in Treatment 2 group, and (D) represents the root growth of Vicia villosa Roth in Treatment 3 group.

[0144] According to Figure 8It can be seen that the growth of the vetch in Treatment Group 3 irrigated with the Bacillus thuringiensis 07 bacterial suspension was more vigorous, and the plant height and overall growth condition were better than those in Treatment Group 2. The root system of the vetch in Treatment Group 3 irrigated with the Bacillus thuringiensis 07 bacterial suspension was more developed, and both the root length and the number of root nodules were more than those in Treatment Group 2.

[0145] 2. Effects of different treatment groups on the growth indexes of vetch

[0146] At the full-bloom stage of vetch, the plant height, root length, number of root nodules, aboveground part, fresh weight of roots, and heavy metal contents in both the aboveground and underground parts of vetch in different treatment groups were recorded. The results are as Figure 9 shown. In the figure, (A) represents the plant height of vetch in different treatment groups, (B) represents the root length of vetch in different treatment groups, (C) represents the fresh weight of the aboveground part of vetch in different treatment groups, (D) represents the fresh weight of the underground part of vetch in different treatment groups, (E) represents the number of root nodules of vetch in different treatment groups, and (F) represents the Cd contents in the aboveground and underground parts of vetch in different treatment groups.

[0147] According to Figure 9 it can be seen that the vetch in Treatment Group 3 irrigated with the Bacillus thuringiensis 07 bacterial suspension was significantly higher than that in Treatment Group 2 in terms of plant height, root length, fresh weight of the aboveground part, fresh weight of the underground part, number of root nodules, and Cd contents in the aboveground and underground parts. This shows that Bacillus thuringiensis 07 promoted the growth of the aboveground part of vetch, helped the extension and development of the vetch root system, also promoted the biomass accumulation of the aboveground part of vetch and the growth of the underground part, increased the absorption of Cd by vetch, reduced the accumulation of Cd in the soil, thereby reducing the risk of Cd entering the human body through the food chain and ensuring the quality of agricultural products and the safety of the ecological environment.

[0148] 3. Effects of different treatment groups of vetch on the content of available cadmium (Cd) in soil

[0149] The remaining vetch plants collected in "2. Growth indexes of vetch in different treatment groups" were shredded and turned over and pressed into the soil in situ for natural decomposition. During this period, the soil water management was the same as that during the plant growth period. After 15 days of natural decomposition, the soil was sampled destructively and further analyzed. The results are as Figure 10 shown.

[0150] According to Figure 10It can be seen that before and after the incorporation of the first treatment group (CK), there was no significant change in the available Cd content in the soil, indicating that the bioavailability of Cd in the soil was relatively stable under natural conditions. After the incorporation of the second treatment group (Vicia villosa), the available Cd content in the soil decreased. At the same time, after the incorporation of the fourth treatment group (Vicia villosa + 07), the available Cd content in the soil decreased significantly, and the effect was better than that of using only the second treatment group or the third treatment group alone. This shows that the combined use of the Cd-tolerant endophytic bacterium Bacillus thuringiensis 07 and Vicia villosa can not only significantly promote the growth of Vicia villosa and increase its biomass, but also effectively reduce the bioavailability of Cd in the soil. This technology has significant advantages in the remediation of Cd-polluted soil, not only reducing the activity of Cd in the soil, but also reducing the risk of its entry into the human body through the food chain, ensuring the quality of agricultural products and the safety of the ecological environment. In addition, this method is low-cost and easy to operate, with important agricultural application value and environmental protection significance, providing new ideas and solutions for the remediation of Cd-polluted soil.

[0151] Example 7: Study on Cd stress of pakchoi after remediation by combining Bacillus thuringiensis 07 and Vicia villosa

[0152] Referring to the seed treatment process in Example 6, the pretreated pakchoi seeds were transferred to a seedling tray filled with soilless substrate and cultivated at room temperature, and watered irregularly to ensure seed germination. Two weeks after seeding, pakchoi seedlings with strong growth and uniform growth were selected and transplanted into the soil repaired in Example 6, with a planting density of 3 plants per pot. Before transplantation, 15 g of basal fertilizer (N:P:K = 1:1:1) was applied to each flower pot, and other field management measures were carried out according to industry standards.

[0153] 1. Effects of soils in different treatment groups on the growth of pakchoi

[0154] The above-ground growth and underground root growth of pakchoi in soils of different treatment groups were observed, and the results are as Figure 11 shown. In the figure, (A) represents the above-ground growth of pakchoi in the soil of treatment group 1, (B) represents the above-ground growth of pakchoi in the soil of treatment group 3, (C) represents the low above-ground growth of pakchoi in the soil of treatment group 2, (D) represents the above-ground growth of pakchoi in the soil of treatment group 4, (E) represents the underground root growth of pakchoi in the soil of treatment group 1, (F) represents the underground root growth of pakchoi in the soil of treatment group 3, (G) represents the underground root growth of pakchoi in the soil of treatment group 2, and (H) represents the underground root growth of pakchoi in the soil of treatment group 4.

[0155] According to Figure 11It can be seen that the growth of pakchoi in the soil of treatment group 1 was average, with the plant size, leaf number and root development all at a medium level; the growth of pakchoi in the soil of treatment group 3 improved, with the plant height and leaf number increasing compared to the blank control group, and the root development being better; the growth of pakchoi in the soil of treatment group 2 was also better than that of the blank control group, with the plants being more robust, the leaves being more lush, and the roots being more developed; the growth of pakchoi in the soil of treatment group 4 was the best, with the plants being tall and the leaves being lush, and the root development being the most perfect. It shows that the soil repaired by the combined use of Bacillus thuringiensis 07 and Vicia villosa can significantly promote the growth of Chinese cabbage.

[0156] 2. Effects of soils of different treatment groups on the growth indexes of pakchoi

[0157] Pakchoi was harvested at the mature stage, and plant samples were collected. After washing the soil with tap water and then cleaning and draining with deionized water, the plant yield per plant, leaf length, leaf width, leaf number, aboveground dry weight and underground dry weight of each group were measured. The results are as Figure 12 shown. In the figure, (A) represents the effect of soils of different treatment groups on the yield of pakchoi, (B) represents the effect of soils of different treatment groups on the leaf length of pakchoi, (C) represents the effect of soils of different treatment groups on the leaf width of pakchoi, (D) represents the effect of soils of different treatment groups on the leaf number of pakchoi, (E) represents the effect of soils of different treatment groups on the aboveground dry weight of pakchoi, and (F) represents the effect of soils of different treatment groups on the underground dry weight of pakchoi.

[0158] According to Figure 12 it can be seen that in terms of yield, leaf length, leaf width, leaf number, aboveground dry weight and underground dry weight, the indexes of pakchoi grown in the soil of treatment group 4 were significantly higher than those of other treatment groups, indicating that the soil repaired by the combined use of Bacillus thuringiensis 07 and Vicia villosa can significantly promote the growth of Chinese cabbage.

[0159] 3. Effects of soils of different treatment groups on the root morphology of pakchoi

[0160] Pakchoi was harvested at the mature stage, and plant samples were collected. After washing the soil with tap water and then cleaning and draining with deionized water, the roots of pakchoi were scanned with an EPSON PERFECTION V 700 instrument, and the root length, root surface area, root volume, average root diameter, root tip number and branch number were measured with the root analysis software WinRHIZO - Pro2013 (Regent Instruments Inc.). The results are as Figure 13As shown in the figure, (A) shows the effect of soils in different treatment groups on the root length of pakchoi, (B) shows the effect of soils in different treatment groups on the root surface area of pakchoi, (C) shows the effect of soils in different treatment groups on the number of branches of pakchoi, (D) shows the effect of soils in different treatment groups on the number of root tips of pakchoi, (E) shows the effect of soils in different treatment groups on the average root diameter of pakchoi, and (F) shows the effect of soils in different treatment groups on the root volume of pakchoi.

[0161] According to Figure 13 it can be seen that in terms of root length, root surface area, root volume, average root diameter, number of root tips and number of branches, the indicators of pakchoi grown in the soil of treatment group 4 are significantly higher than those of other treatment groups, indicating that the soil repaired by the combined use of Bacillus thuringiensis 07 and Vicia villosa can significantly promote the root growth of pakchoi.

[0162] 4. Effects of Soils in Different Treatment Groups on Cd Content in Pakchoi

[0163] The Cd contents in the soils of different treatment groups, as well as in the above-ground and underground parts of pakchoi, were measured, and the results are as Figure 14 it can be seen that in the figure, (A) shows the Cd content in the soils of different treatment groups, and (B) shows the Cd contents in the above-ground and underground parts of pakchoi grown in different soils.

[0164] According to Figure 14 it can be seen that the Cd content in the soil of treatment group 4 is significantly lower than that of other treatment groups, and the Cd contents in the above-ground and underground parts of pakchoi grown in the soil of treatment group 4 are also significantly lower than those of other treatment groups. This indicates that the combined use of Bacillus thuringiensis 07 and Vicia villosa can not only effectively repair Cd-polluted soil, but also significantly reduce the absorption of Cd by subsequent crops, improve the safety of agricultural products, reduce the risk of Cd entering the human body through the food chain, and ensure the quality of agricultural products and the safety of the ecological environment. This technology has broad prospects in agricultural applications and provides new ideas and solutions for solving the problem of Cd-polluted soil.

[0165] It can be seen from the above examples that the present invention provides a Bacillus thuringiensis 07 for repairing cadmium-polluted soil. The Latin name of the Bacillus thuringiensis 07 is Bacillus thuringiensis, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on December 02, 2024. The deposit address is the 5th floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 65572. The Bacillus thuringiensis 07 of the present invention has the function of repairing cadmium-polluted soil and can also promote the growth of crops. The combined application of it and Vicia villosa in the repair of cadmium-polluted soil has the advantages of low cost, high repair efficiency and simple treatment steps.

[0166] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A Bacillus thuringiensis 07 for repairing cadmium-polluted soil, characterized in that, The Latin name of the Bacillus thuringiensis 07 is Bacillus thuringiensis, which was deposited in the Guangdong Microbial Culture Collection Center on December 02, 2024. The deposit address is the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 65572.

2. The preparation method of the bacterial liquid of Bacillus thuringiensis 07 according to claim 1, characterized in that, Inoculate Bacillus thuringiensis 07 into LB liquid medium and culture it at a rotation speed of 180 - 220 rpm until the logarithmic growth phase.

3. The preparation method according to claim 2, wherein The temperature of the culture is 28 - 32 °C.

4. The preparation method of the bacterial suspension of Bacillus thuringiensis 07 according to claim 1, characterized in that, It includes the following steps: (1) Inoculate Bacillus thuringiensis 07 into LB solid medium and culture it at 28 - 32 °C for 22 - 26 h to obtain the cultured strain. (2) Inoculate the cultured strain into LB liquid medium and culture it for 2 - 4 days to obtain the fermented broth. (3) Centrifuge the fermented broth to collect the precipitate and resuspend it with water to obtain the bacterial suspension.

5. The preparation method according to claim 4, characterized in that, In step (2), the temperature of the culture is 28 - 32 °C.

6. The preparation method according to claim 4, characterized in that, In step (2), the rotation speed of the culture is 180 - 220 rpm.

7. The preparation method according to claim 4, characterized in that, The concentration of the bacterial suspension described in step (3) is 0.5 to 1.5×10 8 CFU / mL.

8. Application of the bacterial liquid prepared by the Bacillus thuringiensis 07 described in claim 1 or the preparation method described in any one of claims 2 - 3, or the bacterial suspension prepared by the preparation method described in any one of claims 4 - 7 in the preparation of products for repairing cadmium - contaminated soil.

9. Application of the bacterial liquid prepared by the Bacillus thuringiensis 07 described in claim 1 or the preparation method described in any one of claims 2 - 3, or the bacterial suspension prepared by the preparation method described in any one of claims 4 - 7 in the preparation of products for promoting plant growth.

10. A method for remediating cadmium-polluted soil, characterized in that, Spray the microbial bacterial suspension and combine it with planting Vicia villosa to repair cadmium - contaminated soil. The microbial bacterial suspension is the bacterial suspension prepared by the preparation method described in any one of claims 4 - 7.

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