A strain of Bacillus Velezii capable of degrading plastic mulch, a bacterial agent and its application

Abstract

The present invention belongs to the technical field of microbial degradation, and specifically relates to a strain of Bacillus velezensis for degrading plastic mulch, a bacterial agent and an application thereof. Bacillus velezensis NBL‑BQ185 for degrading plastic mulch has a deposit number of CCTCC M 2024855. Bacillus velezensis NBL‑BQ185 has the effect of efficiently degrading mulch, and has a good degradation effect on common agricultural mulch. The degradation rate of ordinary mulch after 60 days in the field is 15.74%, and the degradation rate of degradable mulch after 60 days is 16.23%. Bacillus velezensis NBL‑BQ185 can promote the growth of different crops, such as tomatoes and cucumbers, and is of great significance to the bioremediation of mulch degradation.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial degradation, and specifically relates to a strain of Bacillus Velezii for degrading plastic mulch, a bacterial agent and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Ground film has the functions of retaining moisture and increasing temperature in agriculture, which can promote agricultural production and efficiency. While polyethylene ground film brings economic benefits to agricultural production, it also causes varying degrees of pollution to the agricultural ecological environment. Studies have found that by 14 The researchers conducted follow-up monitoring on polyethylene mulch labeled with C and found that its degradation rate was only 0.2% to 0.5% after 10 years of landfilling in soil. The molecular weight of polyethylene mulch buried in soil for about 30 years was then calculated and the surface microscopic structure characteristics were analyzed. It was estimated that under field landfill conditions, it would take about 300 years for a polyethylene mulch with a thickness of 60 μm to be completely degraded.

[0004] Israeli scientists Hadad and others discovered a thermophilic Brevibacillus borstelensis strain that can reduce the mass and molecular weight of polyethylene plastics by 11% and 30% respectively within 30 days at 50°C. Bacillus cereus isolated from mangroves in Peninsular Malaysia caused the weight of PE, PET and PS to drop by 1.6%, 6.6% and 7.4% respectively within 40 days. Bacillus subtilis H1584 isolated from pelagic waters had a degradation rate of 1.75% for LDPE within 30 days. Bacillus cereus isolated from landfills had a degradation rate of 1.78% for HDPE within 30 days. Bacillus cereus isolated from cow dung had a degradation rate of 5.9% for HDPE within 83 days. Two strains of Bacillus amyloliquefaciens (BSM-1) and Bacillus amyloliquefaciens (BSM-2) isolated from soil had degradation rates of 11% and 16% for LDPE after 60 days of culture. Japanese scientists Kohei Oda et al. screened a new type of bacteria, Ideonella sakaiensis 201-F6, from sludge in a PET recycling station. It can produce 0.13 mg cm per day in an environment of 30°C. -2Degradation rate: Highly efficient in degrading PET. Yang Jun et al. isolated strains that can degrade polyethylene efficiently from waxworms and mealworms (Enterobacter asburiaeYT1 and Bacillus sp.YP1 had polyethylene degradation rates of 6.1±0.3% and 10.7±0.2% respectively within 60 days, and Exiguobacterium sp.YT2 had a degradation rate of 7.4±0.4% for polystyrene within 60 days). Streptomyces albogriseolus, a white light gray Streptomyces strain screened from soil by Feng Jing, can degrade polyethylene efficiently. Obvious small holes and damage were observed in polyethylene films under an electron microscope. Further research revealed that the bacteria that have the function of degrading polyethylene is a laccase. Chinese patent CN 111593000 A discloses the isolated and screened mulch-degrading bacteria Klebsiella pneumoniae N3-2, which achieves a high weight loss rate for polyethylene plastic mulch and PBAT plastic mulch. After 8 weeks of treatment with the strain, the weight loss rates of PE mulch and PBAT mulch reached 3.360±0.082% and 6.538±0.086%, respectively. Chinese patent CN 117327627 A discloses that after 15 days of treatment with a mulch-degrading bacteria, the polyethylene surface showed roughness, cracks and gullies, and its weight loss rate was as high as 6%. Chinese patent CN 116024104A discloses the use of a fungus in the degradation of polyethylene mulch. The fungus is Purpereocillim sp. FL-M-2, which treats the mulch for 30d and 60d, and cracks and cavities of varying degrees appear. As the degradation time increases, the cracks and cavities gradually become larger.

[0005] Although bacteria that can degrade mulch films have been obtained in the prior art, the degradation rate of the bacteria is still at a relatively low level, and it is impossible to determine whether the degradation bacteria have an adverse effect on plant growth. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a strain of Bacillus velezensis for degrading plastic mulch, a bacterial agent and its application. Bacillus velezensis NBL-BQ185 for degrading plastic mulch can effectively degrade mulch, promote plant growth, and has a wide range of environmental adaptability, and is expected to be widely used in the degradation of agricultural plastic waste.

[0007] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0008] In the first aspect, a strain of Bacillus velezensis NBL-BQ185 for degrading plastic mulch, with a deposit number of CCTCC M 2024855. Bacillus velezensis NBL-BQ185 was deposited in the China Center for Type Culture Collection, abbreviated as CCTCC, on April 30, 2024, with an address of: Wuhan University, Wuhan, China.

[0009] The film-degrading bacteria were screened using grain weevils as samples, and a strain Bacillus velezensis NBL-BQ185 was screened out that can degrade film, and can use film as the only carbon source to achieve rapid degradation of agricultural film. The 16S rDNA gene sequence analysis method was used for identification, and the 16S rDNA sequence of the strain was 99.93% homologous to the Bacillus velezensis sequence in the NCBI database, and was identified as Bacillus velezensis, named Bacillus velezensis NBL-BQ185, referred to as NBL-BQ185 or BQ185.

[0010] Preferably, its DNA sequence is shown as SEQ ID NO.1.

[0011] Preferably, the bacterial body is in the shape of a short rod, and the long rod is broken into a short rod; the colony is round, white and opaque, moist in texture, concave in the middle, and raised at the edge.

[0012] In a second aspect, a bacterial agent comprises the Bacillus velezensis NBL-BQ185 and / or its bacterial suspension and / or its culture as described in the first aspect.

[0013] Preferably, the culture comprises Bacillus velezensis NBL-BQ185 and its metabolites as described in the first aspect.

[0014] In a third aspect, use of Bacillus velezensis NBL-BQ185 as described in the first aspect and / or the bacterial agent as described in the second aspect in degrading plastic mulch.

[0015] Preferably, the degree of drought during the degradation of the plastic mulch film is no more than 40%.

[0016] Preferably, the salinity during the degradation of the plastic mulch film is no higher than 10%.

[0017] Preferably, the pH value during the degradation of the plastic mulch film is 5 to 9.

[0018] In a fourth aspect, use of Bacillus velezensis NBL-BQ185 as described in the first aspect and / or the bacterial agent as described in the second aspect in promoting plant growth.

[0019] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0020] Bacillus velezensis NBL-BQ185 has the effect of efficiently degrading mulch films, and has a good degradation effect on common agricultural mulch films. The degradation rate of ordinary mulch films in the field after 60 days is 15.74%, and the degradation rate of degradable mulch films after 60 days is 16.23%.

[0021] Bacillus velezensis NBL-BQ185 can promote the growth of different crops, such as tomatoes and cucumbers, and is of great significance for the bioremediation of mulch film degradation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0023] Figure 1 The photos of different mulch-film degrading bacteria on mulch film degradation in Example 1, A is CK group, B is NBL-BQ180 group, C is NBL-BQ185 group, D is NBL-BQ186 group, E is NBL-BQ187 group, and F is NBL-BQ188 group;

[0024] Figure 2 These are the apparent morphological images of the mulch films treated with Bacillus velezensis NBL-BQ185 and the control group in Example 1 under a stereo microscope, A is a surface morphological image of the mulch film treated with Bacillus velezensis NBL-BQ185 under a low-power microscope (1×), B is a surface morphological image of the mulch film treated with Bacillus velezensis NBL-BQ185 under a high-power microscope (4.5×), and C is a surface morphological image of the mulch film of the control group under a low-power microscope (1×);

[0025] Figure 3 The colony morphology of Bacillus velezensis NBL-BQ185 in Example 1 is shown;

[0026] Figure 4 The bacterial morphology of Bacillus velezensis NBL-BQ185 in Example 1;

[0027] Figure 5 is a growth curve diagram of Bacillus velezensis NBL-BQ185 in Example 1;

[0028] Figure 6 This is the evaluation of the degradation ability of different metabolites of Bacillus velezensis NBL-BQ185 on mulch film in Example 2;

[0029] Figure 7 The results of the determination of germination rates of cucumber seeds treated with different concentrations of Bacillus velezensis NBL-BQ185, 100-fold diluted NB medium and clean water in Example 3;

[0030] Figure 8 These are photos of cucumber seeds treated with different concentrations of Bacillus velezensis NBL-BQ185, 100-fold diluted NB medium, and clean water in Example 3;

[0031] Fig. 9 The photographs of Bacillus velezensis NBL-BQ185, 100-fold diluted NB medium and water-promoted tomato in Example 3;

[0032] Fig.10 (A) plant height and (B) plant stem diameter of Bacillus velezensis NBL-BQ185, 100-fold diluted NB medium and water-promoted tomato in Example 3;

[0033] Fig.11 The results of the simulated drought tolerance test of Bacillus velezensis NBL-BQ185 in Example 4 are as follows;

[0034] Fig.12 The results of the salinity tolerance test of Bacillus velezensis NBL-BQ185 in Example 4 are as follows;

[0035] Fig.13 The results of the ultraviolet radiation tolerance test of Bacillus velezensis NBL-BQ185 in Example 4 are as follows;

[0036] Fig.14 The results of the acid-base tolerance test of Bacillus velezensis NBL-BQ185 in Example 4 are shown. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments and comparative examples.

[0038] The mulch film, grain weevils and reagents used in the examples were purchased through conventional channels unless otherwise specified, and the culture medium formula used was as follows.

[0039] Test medium: Inorganic salt medium: K2HPO4 0.7g, KH2PO4 0.7g, MgSO4 . 7H2O 0.7g, NH4NO31g, NaCl 0.005g, FeSO4 . 7H2O 0.002g, ZnSO4 . 7H2O 0.002g, MgSO4 . H2O 0.001g, distilled water 1L, natural pH, add 18-20g agar to the solid culture medium, and sterilize at 121℃ for 25min.

[0040] Enrichment medium A (g / L): yeast extract 10.0, K2HPO4 0.7, KH2PO4 0.7, MgSO4·7H2O 0.7, NH4NO3 1.0, NaCl 0.005, FeSO4·7H2O 0.002, ZnSO4·7H2O 0.002, MnSO4·H2O 0.001, pH 7.2.

[0041] Enrichment medium B (g / L): beef extract 3.0, peptone 3.0, yeast extract 5.0, K2HPO4 0.7, KH2PO4 0.7, MgSO4·7H2O 0.7, NH4NO3 1.0, NaCl 0.005, FeSO4·7H2O 0.002, ZnSO4·7H2O 0.002, MnSO4·H2O 0.001, pH 7.2.

[0042] NB liquid culture medium: NB solid culture medium 18 g, distilled water 1 L, high pressure sterilization at 121℃ for 20 min.

[0043] NA solid plate medium and slant storage medium: 18g NA solid medium, add 20g agar, 1L distilled water, natural pH, autoclave at 121℃ for 20min, take out at 60-70℃.

[0044] Example 1

[0045] The grain weevils were placed in a feeding box, starved for 2 days, and then fed with shredded polyethylene film for 10 days. The fed and domesticated insect bodies were placed in an ether solution and soaked for 10 seconds, taken out and soaked in a 0.1% mercuric chloride solution for 2-3 minutes, then soaked in 100% anhydrous ethanol for 15 seconds, and finally moved to sterile water for washing 2-3 times for surface sterilization. In the clean bench, the sterile grain weevils were placed in a sterile mortar and 1-3 mL of sterile water was added to fully grind, the grinding liquid was transferred to a sterilized 2 mL centrifuge tube, centrifuged at 8000 r / min for 1 minute, and the supernatant was aspirated as the grain weevils grinding liquid stock solution. 0.5 mL of the grain weevils grinding liquid stock solution was added to 250 mL triangular flasks containing 50 mL of enrichment medium A and enrichment medium B, respectively, and placed in a constant temperature oscillator at 30 ° C and 160 r / min for enrichment culture for 3 days. Three parallels were made for each gradient. Add 5 mL of the enriched culture solution of grain weevils to the inorganic salt medium containing quantitative PE powder (10 g / L), and acclimate and co-cultivate at 30°C and 160 r / min for 10 days. After that, take 5% of the previous culture solution every 10 days and inoculate it into a fresh liquid medium with PE as the only carbon source for further acclimatization, and co-cultivate twice. Add sterilized PE powder (10 g / L) to the basic inorganic salt medium plate, add 0.1 mL of grain weevils acclimatization culture solution to the surface of the plate for coating, set up 5 parallel plates for each sample, and culture in a constant temperature incubator at 30°C for 5 to 7 days. Pick the strains that can grow in the basic medium with PE powder as the only carbon source; repeatedly streak and separate on the medium, purify to obtain single colonies, and store at 4°C for use.

[0046] Seven mulch film degrading bacteria with degradation ability were screened and named NBL-BQ180, NBL-BQ185, NBL-BQ186, NBL-BQ187, NBL-BQ188, GX-1 and GX-3. The above 7 strains were inoculated into an inorganic salt medium containing 7% PE powder and cultured in a constant temperature incubator at 30°C for 5-7 days. The strains that can grow in the basic medium with PE powder as the only carbon source were selected; the single colonies were obtained by repeated streaking and purification on the medium and stored at 4°C for later use.

[0047] Five strains of mulch film degrading bacteria with degradation ability were obtained through repeated screening, namely NBL-BQ180, NBL-BQ185, NBL-BQ186, NBL-BQ187 and NBL-BQ188, abbreviated as BQ180, BQ185, BQ186, BQ187 and BQ188.

[0048] Three pieces of sterile mulch were added to NB medium (50mL / 250mL) as control CK. The mulch-degrading bacteria obtained by repeated screening were added to fresh NB medium and cultured in a constant temperature shaker at 30℃ and 180r / min for 24h. The experimental group inoculated the shaking culture fluid of mulch-degrading bacteria (BQ180, BQ185, BQ186, BQ187 and BQ188) in fresh NB medium (50mL / 250mL) at a 5% inoculation rate and cultured in a constant temperature shaker at 30℃ and 180r / min for 14d. The photos of each group are shown in Figure 2. Figure 1 As shown. After 14 days, the mulch was taken out, soaked in 2% SDS solution for 4 hours, and then washed repeatedly with sterile water to remove bacteria attached to the surface. After drying at 60°C, the weight was weighed using a 1 / 10,000 balance to determine the mulch weight loss rate. The mulch weight loss rate = ((weight before mulch degradation - weight after mulch degradation) / weight before mulch degradation).

[0049] As shown in Table 1, the film degradation rate of BQ185 was 17.49%, which was about 36 times higher than that of the control group CK. At the same time, the degradation rates of the five film-degrading bacteria BQ180, BQ185, BQ186, BQ187, and BQ188 were 2.87%, 17.49%, 4.31%, 1.68%, and 4.61%, respectively.

[0050] Table 1 The degradation effect of mulch film by mulch film degrading bacteria

[0051]

[0052] The mulch film treated with mulch film degrading bacteria NBL-BQ185 for 14 days was soaked in 2% SDS solution for 4 hours, then washed repeatedly with sterile water to remove bacteria attached to the surface, dried at 60℃, and observed under a stereo microscope for surface morphology and compared with the control group. Figure 2 As shown in the images obtained by stereomicroscope observation, the surface of the mulch in the control group that was not inoculated with bacteria was relatively smooth, while the surface of the mulch treated with mulch-degrading bacteria NBL-BQ185 became rough and soft, with obvious holes and cracks, indicating that the mulch-degrading bacteria NBL-BQ185 can effectively degrade the mulch.

[0053] A single colony of NBL-BQ185 was streaked onto a NA solid plate medium, and the plate was inverted in a constant temperature incubator and cultured at 30°C for 24 h. Figure 3 The colonies of NBL-BQ185 are round, opaque white, moist, concave in the middle, and raised at the edges. Figure 4 As shown, the strain NBL-BQ185 was observed under a microscope to be in the shape of short rods, and the long rods were broken into short rods.

[0054] The 16S rDNA gene sequence analysis method was used to identify the mulch film degradation strain NBL-BQ185. The 16S rDNA gene sequence had a homology of 99.93% with the Bacillus velezensis sequence in the NCBI database. The strain NBL-BQ185 was identified as Bacillus velezensis and named Bacillus velezensis NBL-BQ185.

[0055] The complete sequence of Bacillus velezensis NBL-BQ185 gene is shown below:

[0056]

[0057] Bacillus velezensis NBL-BQ185 was added to fresh NB medium and cultured in a constant temperature shaker at 30°C and 180 r / min for 24 h. It was then inoculated into fresh NB medium (100 mL / 500 mL) at a 5% inoculum and cultured in a constant temperature shaker at 30°C and 180 r / min for 36 h. Samples were taken every 2 h, and 3 mL was sampled each time for OD 600 , viable count determination, and finally obtain the OD at a series of time points 600 The curve diagram of the value and the number of viable bacteria. Figure 5 As shown, the OD of Bacillus velezensis NBL-BQ185 4 hours after inoculation with NB medium 600 The value rises rapidly, 4 to 12 hours is the logarithmic growth period, 12 to 36 hours is the stable period, among which the strain has vigorous metabolism and fast reproduction speed between 4 and 12 hours.

[0058] Example 2

[0059] Three pieces of sterile mulch were added to the inorganic salt culture medium (100mL / 500mL), and sterile water and NB culture medium were added at a 5% inoculation rate to serve as the control groups CK1 and CK2. The highly efficient mulch film degrading bacteria Bacillus velezensis NBL-BQ185 were added to the fresh NB culture medium and cultured in a constant temperature shaker at 30°C and 180r / min for 24h. In the experimental group, the shaken culture solution of the mulch film degrading bacteria NBL-BQ185 was divided into BQ185 cells, BQ185 supernatant, sterilized BQ185 supernatant and sterilized BQ185 cells, and inoculated into the inorganic salt culture medium (100mL / 500mL) with mulch film as the only carbon source at a 5% inoculation rate, and cultured in a constant temperature shaker at 30°C and 180r / min for 14d. The photos of each group are shown in the figure. Figure 6 As shown. After 14 days, take out the mulch film, soak it in 2% SDS solution for 4 hours, and then wash it repeatedly with sterile water to remove bacteria attached to the surface. After drying at 60℃, weigh it with a 1 / 10,000 balance to judge the degradation effect of the mulch film by the mulch film weight loss rate. The mulch film weight loss rate = ((weight before mulch film degradation - weight after mulch film degradation) / weight before mulch film degradation).

[0060] As shown in Table 2, the degradation rate of mulch film by the supernatant of Bacillus velezensis NBL-BQ185 fermentation liquid was the highest at 13.12%, followed by the supernatant after sterilization at 6.41%, the bacterial cells and the sterilized bacterial cells at 5.47% and 4.64%, respectively, and the degradation rates of CK1 and CK2 at 0.91% and 1.14%, respectively. In summary, the substances that can degrade mulch film mainly exist in the supernatant of Bacillus velezensis NBL-BQ185 fermentation liquid and are not resistant to high temperatures.

[0061] Table 2 Degradation efficiency of different metabolites of Bacillus velezensis NBL-BQ185

[0062]

[0063]

[0064] Bacillus velezensis NBL-BQ185 was inoculated into fresh NB medium and cultured in a constant temperature shaker at 30°C and 180 r / min for 24 h. The fermentation broth of mulch film degrading bacteria was divided into supernatant, fermentation broth and bacterial cells, and the enzyme activity was determined using a purchased kit (Solarbio, catalog number: BC0090). As shown in Table 3, the peroxidase activity of the supernatant, fermentation broth and bacterial cells of Bacillus velezensis NBL-BQ185 was 0.32 U / 10 4 cell, 0.28U / 10 4 cell, 0.28U / 10 4 cell; the conversion units are 15.69U / mg, 13.55U / mg, and 13.55U / mg respectively.

[0065] Table 3 Peroxidase activity of supernatant, fermentation broth and cells of Bacillus velezensis NBL-BQ185

[0066]

[0067] Bacillus velezensis NBL-BQ185 was inoculated into fresh NB medium and cultured in a constant temperature shaker at 30°C and 180 r / min for 24 h. The fermentation broth of the mulch film degrading bacteria was divided into supernatant, fermentation broth and bacterial cells, and the enzyme activity was determined using a purchased kit (Solarbio, catalog number: BC3595). As shown in Table 4, the catalase activity of the supernatant, fermentation broth and bacterial cells of Bacillus velezensis NBL-BQ185 was 0.053 U / 10 4 cell、0.081U / 104 cell、0.099U / 10 4 cell; the conversion units are 3.6U / mg, 5.5U / mg, and 6.7U / mg respectively.

[0068] Table 4 Catalase activity of supernatant, fermentation broth and cells of Bacillus velezensis NBL-BQ185

[0069]

[0070] Example 3

[0071] Bacillus velezensis NBL-BQ185 was added to fresh NB medium and cultured in a constant temperature shaker at 30°C and 180 r / min for 24 h. The experimental group used NBL-BQ185 fermentation broth with a concentration gradient of 10 6 , 10 7 , 10 8 CFU / mL, soak cucumber seeds for 24 hours, place the seeds in groups on a plate containing sterile filter paper, and measure the seed germination rate after 4 days. NB medium diluted 100 times and water were used as control groups. Figure 7 As shown, the germination rate of the water group was 42.5%, the germination rate of the NB group diluted 100 times was 17.5%, and the germination rate of the NB group diluted 100 times was 17.5%. 6 The best germination rate of Bacillus velezensis NBL-BQ185 group was 55%, 10 7 The germination rate of Bacillus velezensis NBL-BQ185 group was 42%, 10 8 The germination rate of the Bacillus velezensis NBL-BQ185 group was 20%. Figure 8 In summary, the film-degrading bacteria Bacillus velezensis NBL-BQ185 at a certain concentration (10 6 CFU / mL) can promote cucumber seed germination.

[0072] NB medium and water diluted 100 times were used as control groups, namely CK1 and CK2. Bacillus velezensis NBL-BQ185 was added to fresh NB medium and cultured in a constant temperature shaker at 30°C and 180 r / min for 24 h. The experimental group used NBL-BQ185 fermentation broth for 10 6CFU / mL was used for inoculation. Two tomato plants were set in each pot, and three parallels were set in each group. The inoculation was repeated twice and cultured for 15 days. The plant height and stem thickness of the tomato seeds after 15 days of culture were counted. The plant height was measured from the surface of the plug tray matrix to the growth point; the stem thickness was measured from two centimeters from the base.

[0073] like Fig. 9 As shown in Figure 2, the plant height and stem diameter of tomatoes treated with Bacillus velezensis NBL-BQ185 were significantly higher than those of the control groups CK1 and CK2, where the plant height and stem diameter were 15.4 cm and 3.69 mm, respectively (Figure 2). Fig.10 It has obvious plant growth-promoting function.

[0074] Example 4

[0075] Bacillus velezensis NBL-BQ185 was added to fresh NB medium and cultured in a constant temperature shaker at 30°C and 180r / min for 24h. The weighed PEG6000 was placed in a clean bench and irradiated with UV for 4h, and then added to the sterilized NB medium to make the PEG6000 gradient 0%, 10%, 20%, 25%, 30%, 35% and 40%. Two parallels were set for each gradient, inoculated at 5% inoculation amount, and cultured at 30°C for 24h, and the OD was measured. 600 , and compared the growth of NBL-BQ185 under different drought conditions. Fig.11 As shown in Figure 2, as the PEG6000 concentration (drought level) increased, the OD of NBL-BQ185 600 The value decreased, and NBL-BQ185 could at least tolerate 40% PEG6000 (moderate drought condition). 600 It is 0.858.

[0076] Bacillus velezensis NBL-BQ185 was added to fresh NB medium and cultured in a constant temperature shaker at 30°C and 180r / min for 24h. The salinity gradient was set to 0%, 2%, 4%, 6%, 8%, 10%, 12% and 14%, and two parallels were set for each gradient. The inoculation was inoculated at 5% and the OD was measured after culturing at 30°C for 24h. 600 , and compare the growth of NBL-BQ185 under different salinity conditions. Fig.12 As shown, NBL-BQ185 can tolerate at least 10% salinity, OD 600 It is 1.639.

[0077] Bacillus velezensis NBL-BQ185 was added to fresh NB medium and cultured in a constant temperature shaker at 30°C and 180 r / min for 24 h. The fermentation liquid was adjusted to OD with physiological saline. 600 0.8~1.0, diluted 10 times to 10 -1 ~10 -7 100 μL was taken for coating, and each coating was repeated twice. The plate was opened on the clean bench and irradiated vertically for 0 min, 1 min, 3 min, 5 min, 10 min, 15 min and 20 min in sequence. -5 , 10 -6 and 10 -7 The plate was used as a control and placed in a constant temperature incubator at 30°C. After 24 hours, the results were observed and recorded. The countable colonies were counted and the number of viable bacteria and the lethality were calculated. Fig.13 As shown, the mortality rates of Bacillus velezensis NBL-BQ185 after irradiation for 1min, 3min, 5min, 10min, 15min and 20min were 73.82%, 94.12%, 97.35%, 97.94%, 98.53% and 98.53%, respectively. The sterilization rate was as high as 95% when ultraviolet irradiation was 3min or more.

[0078] Bacillus velezensis NBL-BQ185 was added to fresh NB medium and cultured in a constant temperature shaker at 30°C and 180 r / min for 24 h. The pH concentration gradient was set to no adjustment, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0, and two parallels were set for each gradient. The inoculation was inoculated at 5% and the OD was measured after culturing at 30°C for 24 h. 600 .like Fig.14 As shown in the figure, NBL-BQ185 can tolerate at least pH 9.0, at which OD 600 is 1.70.

[0079] Example 5

[0080] NB medium diluted 100 times and water were used as control groups, namely CK1 and CK2. Bacillus velezensis NBL-BQ185 was added to fresh NB medium and cultured in a constant temperature shaker at 30°C and 180 r / min for 24 h. Each plot had an area of ​​1 m 2 The experimental group used Bacillus velezensis NBL-BQ185 fermentation broth for 10 6CFU / mL was used for inoculation, and the bacterial solution was used once a week for a total of 5 times for continuous culture. After 60 days of culture, the mulch was taken out, soaked in 2% SDS solution for 4 hours, and then washed repeatedly with sterile water to remove the bacteria attached to the surface. After drying at 60°C, its weight was weighed using a 1 / 10,000 balance. As shown in Table 5, after 60 days of Bacillus velezensis NBL-BQ185 treatment of the mulch, the degradation rate of the ordinary mulch reached 15.74%, which was 225 times that of the mulch in the CK1 group and 394 times that of the CK2 group.

[0081] Table 5 Degradation effect of Bacillus velezensis NBL-BQ185 on ordinary mulch film

[0082]

[0083] The NB medium diluted 100 times and clean water were used as the control group, namely CK1 and CK2. The mulch film degrading bacteria Bacillus velezensis NBL-BQ185 was added to the fresh NB medium and cultured in a constant temperature shaker at 30°C and 180r / min for 24h. The area of ​​each plot was 1m 2 The experimental group used the fermentation broth of the mulch film degrading bacteria Bacillus velezensis NBL-BQ185 for 10 6 CFU / mL was used for inoculation, and the bacterial solution was used once a week for a total of 5 times for continuous culture. After 60 days of culture, the mulch was taken out, soaked in 2% SDS solution for 4 hours, and then washed repeatedly with sterile water to remove the bacteria attached to the surface. After drying at 60°C, its weight was weighed using a 1 / 10,000 balance. As shown in Table 6, after 60 days of treatment of the mulch with Bacillus velezensis NBL-BQ185, the degradation rate of the degradable mulch reached 16.23%, which was 3.71 times the degradation rate of the NB mulch in the control group and 4.42 times that of the clean water treatment group. In summary, Bacillus velezensis NBL-BQ185 can accelerate the degradation of degradable mulch.

[0084] Table 6 Degradation effect of Bacillus velezensis NBL-BQ185 on degradable mulch film

[0085]

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A strain of Bacillus Velezii that degrades plastic mulch ( Bacillus velezensis ) NBL-BQ185, characterized in that, Its deposit number is CCTCC M 2024855.

2. The Bacillus Velezii as claimed in claim 1 Bacillus velezensis NBL-BQ185, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

1.

3. The Bacillus Velezii as claimed in claim 1 Bacillus velezensis NBL-BQ185, characterized in that The bacteria are in the shape of short rods, with long rods broken into short rods; the colonies are round, opaque white, moist in texture, concave in the middle, and raised at the edges.

4. A bacterial agent, characterized in that Comprising the Bacillus Velezii according to any one of claims 1 to 3 Bacillus velezensis NBL-BQ185 and / or its bacterial suspension and / or its culture; The culture comprises the Bacillus Velezii according to any one of claims 1 to 3 Bacillus velezensis NBL-BQ185 and its metabolites.

5. The Bacillus Velezii according to any one of claims 1 to 3 Bacillus velezensis Use of NBL-BQ185 and / or the bacterial agent as claimed in claim 4 in degrading plastic mulch.

6. The use according to claim 5, characterized in that The degree of drought during the degradation of plastic mulch is no more than 40%.

7. The use according to claim 5, characterized in that The salinity during the degradation of plastic mulch is not higher than 10%.

8. The use according to claim 5, characterized in that The pH value during the degradation of plastic mulch is 5 to 9.

9. The Bacillus Velezii according to any one of claims 1 to 3 Bacillus velezensis Use of NBL-BQ185 and / or the bacterial agent as claimed in claim 4 in promoting plant growth.

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