Bacillus licheniformis ZWM1 and use thereof in degrading matrine

Abstract

Provided are Bacillus licheniformis ZWM1 and use thereof in degrading matrine, belonging to the technical field of microorganisms. The Bacillus licheniformis ZWM1 is deposited in the China General Microbiological Culture Collection Center, and the deposit number is CGMCC NO. 30106. The Bacillus licheniformis ZWM1 can effectively degrade matrine. Under the content of matrine up to 0.5 g / L, the 7 d degradation rate can reach 18.11%, which helps to reduce the accumulation of autotoxic substances of crops, alleviate continuous cropping obstacles of crops, promote crop growth and development, and improve the yield. As a new biodegrading bacterium, the Bacillus licheniformis ZWM1 overcomes the limitations of traditional degradation methods, and provides a new and more effective method to degrade matrine. This is not only a technical innovation, but also provides a new idea for solving the problem of matrine degradation.

Description

Bacillus licheniformis ZWM1 and application thereof in degrading matrine TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial application, and particularly relates to a Bacillus licheniformis ZWM1 and application thereof in degrading matrine. BACKGROUND

[0002] Matrine is mainly a self-toxic substance secreted by plant roots, which inhibits the growth of crops. By screening matrine-degrading bacteria, the accumulation of self-toxic substances can be reduced, the continuous cropping obstacles of crops can be alleviated, and the yield of crops can be improved.

[0003] As a common microorganism, Bacillus licheniformis has strong metabolic capacity and environmental adaptability, and has been widely used in the degradation process of various organic pollutants. The bacteria have the characteristics of producing endogenous spores, so they have strong heat resistance, cell morphology and arrangement in the form of rods, and other characteristics, which enable them to exist in the form of spores in extremely harsh environments, and exist in the form of growth in good environments. In addition, Bacillus licheniformis is a facultative anaerobe, which can grow well in aerobic and anaerobic environments, so it can be better used for the degradation of organic matter.

[0004] However, although Bacillus licheniformis has certain potential in the degradation of organic pollutants, the specific application and research of Bacillus licheniformis in the degradation of matrine are still relatively limited. Different types of Bacillus licheniformis may have different degradation abilities and mechanisms for matrine, so it is of great significance to screen strains with high degradation ability and further study the degradation mechanism for reducing the accumulation of self-toxic substance matrine in plants and improving yield.

[0005] SUMMARY

[0006] The purpose of the present application is to provide a Bacillus licheniformis ZWM1 and application thereof in degrading matrine. The Bacillus licheniformis ZWM1 can have a significant effect on degrading matrine when the concentration of matrine is as high as 0.5 g / L, the inoculation amount of the Bacillus licheniformis ZWM1 is 1%, and the degradation time is 7 days.

[0007] The purpose of the present application is achieved by the following technical solutions:

[0008] The present application provides a Bacillus licheniformis ZWM1, which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO.30106.

[0009] Further, the 16S rDNA sequence of the Bacillus licheniformis ZWM1 is shown in SEQ ID NO.1.

[0010] The application further provides a microbial agent, which comprises the bacillus licheniformis ZWM1.

[0011] The application further provides an application of the bacillus licheniformis ZWM1 or the microbial agent in degrading matrine.

[0012] Further, the degradation condition of the matrine is that the pH is 8, the temperature is 30 DEG C, the inoculation amount of the bacillus licheniformis ZWM1 is 1%, the concentration of the matrine is 0.5 g / L, and the degradation rate reaches 18.11% in 7 days.

[0013] The application further provides an application of the bacillus licheniformis ZWM1 or the microbial agent in preparing the bacillus licheniformis bio-organic fertilizer.

[0014] Further, the bacillus licheniformis bio-organic fertilizer can alleviate the continuous cropping obstacles of crops, promote the growth and development of crops, and improve the yield by degrading matrine.

[0015] The application has the beneficial effects that:

[0016] The bacillus licheniformis ZWM1 in the application can effectively degrade matrine, and under the premise that the means for degrading matrine in the prior art are scarce, the strain and the microbial agent in the application break the limitation of the traditional degradation means, provide a new and more efficient method for degrading matrine. This is not only a technical innovation, but also provides a new idea for solving the problem of degrading matrine. The bacillus licheniformis ZWM1 in the application can effectively degrade matrine in 7 days under the condition that the content of matrine is 0.5 g / L and the inoculation amount of the bacillus licheniformis ZWM1 is 1%, and the degradation rate can reach 18.11%, which is helpful to reduce the accumulation of self-toxic substances of crops, alleviate the continuous cropping obstacles of crops, promote the growth and development of crops, and improve the yield. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description only show some embodiments of the application, and other drawings can be obtained by those skilled in the art without any creative effort.

[0018] Fig. 1 is a colony morphology diagram of the bacillus licheniformis ZWM1. DETAILED DESCRIPTION

[0019] The various exemplary embodiments of the application will be described in detail below, and the detailed description should not be considered as limiting the application, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the application.

[0020] The technical solutions of the present application will be described clearly and completely in combination with specific embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the protection scope of the present application.

[0021] The present application will be further described in combination with the drawings and embodiments.

[0022] Example 1 Identification of Bacillus licheniformis ZWM1

[0023] (1) Morphology and physiological and biochemical identification of Bacillus licheniformis ZWM1

[0024] The Bacillus licheniformis ZWM1 of the present application is isolated by screening in natural water body. Its morphological characteristics are as follows: after growing on LB solid culture medium (ingredients: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L, pH 7.0-7.2, agar 15-20 g / L) for 2 days, the colony is pink, the surface is convex and opaque, and the specific appearance is shown in Figure 1. Under a microscope, the bacterial body is rod-shaped, the length of a single cell is about 1-3 μm, and the diameter is about 0.5 μm.

[0025] The physiological and biochemical characteristics of the Bacillus licheniformis ZWM1 of the present application are shown in Table 1

[0026] Table 1 Physiological and biochemical characteristics of Bacillus licheniformis ZWM1

[0027] It can be seen that the Bacillus licheniformis ZWM1 is a gram-positive bacterium, and the results of gelatin liquefaction test and lipase detection experiment are both negative, and the results of V-P test, indole reaction, citrate utilization test and glycolysis experiment are all positive, that is, the Bacillus licheniformis ZWM1 can decompose glycerol into pyruvic acid and further decarboxylate to generate acetaldehyde, can decompose tryptophan in the culture medium to generate indole, can utilize citrate as a carbon source for growth, and also has the ability of glycolysis.

[0028] (2) Molecular biology identification of Bacillus licheniformis ZWM1

[0029] Bacterial genomic DNA extraction kit (Ezup column bacterial genomic DNA extraction kit, Shenguo Bioengineering) was used to extract bacterial DNA according to the kit operation instruction. The bacterial DNA was amplified by primers, and the gel was verified. The primer sequences were 27F: AGAGTTTGATCMTGGCTCAG and 1492R: GGTTACCTTGTTACGACTT. The sequence of the amplified 16S rRNA gene of Bacillus licheniformis ZWM1 obtained by sequencing is shown as SEQ ID NO. 1. By comparing the gene sequence through the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), it was identified that the closest species of the strain was Bacillus licheniformis, with a similarity of 100%, and was named Bacillus licheniformis ZWM1. It was preserved in the China General Microbiological Culture Collection Center (CGMCC) on March 25, 2024, with the preservation number CGMCC NO. 30106.

[0030] Example 2 Matrine degradation rate analysis

[0031] 1. Materials and equipment

[0032] The experimental materials used were matrine standard with a purity of more than 99%, analytical pure methanol, acetonitrile, ethanol and H2SO4 solvents, sterile water and ultrapure water.

[0033] 2. Test method

[0034] A bacterial suspension with an OD600 value of 1.0 was prepared, and 0.12 ml of the suspension was transferred to an inorganic salt liquid medium containing 0.5 g / L matrine ((NH4)2SO4 2 g, Na2HPO4 1.3 g, KH2PO4 2 g, NaCl 5 g, ultrapure water 1000 ml, pH 7.2). After incubation at 28°C and 170 rpm for 7 days, 1 ml was centrifuged at 13000 x g for 5 min, resuspended with 1 ml of sterile water (ddH2O), and the supernatant was mixed with an equal volume of methanol. 0.1 ml of the mixed solution was added to 1.9 ml of 50% methanol solution, filtered and sterilized with a filter membrane with a pore size of 0.22 μm, and then subjected to HPLC analysis. The control group was an MSM solution containing matrine but not containing degrading bacteria; all test treatments were repeated three times.

[0035] Ultra-high performance liquid chromatography (UHPLC) was used with an Agilent C18 column (4.6 × 250 mm, 0.5 μm) at 30 °C. The flow rate was 1 mL / min, the injection volume was 10 μL, and the mobile phase was acetonitrile-anhydrous ethanol-3% H₂PO₄ (80:10:10) and water. The detection wavelength was 220 nm. The lowest detectable concentration of matrine in the water sample was 0.01 mg, meeting the residue detection requirements.

[0036] Degradation rate calculation (AU value): Degradation rate = (AU value of matrine in the control group - AU value of matrine remaining in the culture medium after 7 days of culture) / AU value of matrine in the control group * 100%.

[0037] 3. Experimental Results

[0038] Based on the degradation rate calculation formula above, it was found that the degradation rate of matrine can reach 18.11% after 7 days.

[0039] Example 3: Preparation of Bacillus licheniformis liquid inoculant

[0040] Bacillus licheniformis was inoculated into LB liquid medium and incubated at 28°C and 150 rpm for 48 h (OD). 600 A value greater than 0.5 indicates a microbial concentration that can reach 10. 9 This yields a liquid Bacillus licheniformis inoculant, which can be used in the preparation of microbial organic fertilizers or applied as a liquid inoculant.

[0041] Example 4: Preparation of Lichen Bacillus Bio-organic Fertilizer

[0042] The bacterial culture prepared in LB liquid medium is inoculated into well-rotted compost at 10% (v / m) and fermented at room temperature for 7 days to obtain Bacillus licheniformis bio-organic fertilizer, which can alleviate crop continuous cropping obstacles, promote crop growth and development, and increase yield.

[0043] Of course, the above description is not limited to the examples above. Technical features not described in this invention can be implemented by or using existing technology, and will not be repeated here. The above embodiments and drawings are only used to illustrate the technical solutions of this invention and are not intended to limit this invention. This invention has been described in detail with reference to preferred embodiments. Those skilled in the art should understand that any changes, modifications, additions or substitutions made by those skilled in the art within the scope of this invention do not depart from the spirit of this invention and should also fall within the scope of protection of the claims of this invention.

Claims

1. A Bacillus licheniformis ZWM1, which is deposited with the China General Microbiological Culture Collection Center under accession number CGMCC NO.30106.

2. The B. licheniformis ZWM1 of claim 1, wherein, The 16S rDNA sequence of the Bacillus licheniformis ZWM1 is shown as SEQ ID NO.

1.

3. A microbial inoculant, characterized in that, The microbial agent comprises the Bacillus licheniformis ZWM1 of claim 1. 4.Use of the Bacillus licheniformis ZWM1 of any one of claims 1-2 or the microbial agent of claim 3 in degrading matrine.

5. The use according to claim 4, wherein the compound is ###0002### The degradation conditions of matrine are as follows: pH 8, temperature 30℃, inoculation amount of Bacillus licheniformis ZWM1 1%, and matrine concentration 0.5 g / L, and the degradation rate is 18.11% in 7 days. 6.Use of the Bacillus licheniformis ZWM1 of any one of claims 1-2 or the microbial agent of claim 3 in preparing a Bacillus licheniformis bio-organic fertilizer.

7. Use according to claim 6, wherein The Bacillus licheniformis bio-organic fertilizer can alleviate the continuous cropping obstacles of crops, promote the growth and development of crops, and improve the yield by degrading matrine.

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