Microbacterium ZWM4 and use thereof in degradation of salicylic acid

Abstract

A microbacterium ZWM4 and use thereof in the degradation of salicylic acid, pertaining to the technical field of microorganisms. The microbacterium ZWM4 is deposited in the China General Microbiological Culture Collection Center, with a deposit number of CGMCC NO: 30109. The microbacterium ZWM4 can effectively degrade salicylic acid, with a degradation rate of up to 37.47%. Using the microbacterium ZWM4 obtained through screening to degrade salicylic acid offers lower costs and higher efficiency, thereby further improving the crop yield, and producing good economic benefits.

Description

Microbacterium ZWM4 and application thereof in degrading salicylic acid TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial application, and particularly relates to a microbacterium ZWM4 and application thereof in degrading salicylic acid. BACKGROUND

[0002] Salicylic acid is a plant root excreted allelopathic autotoxic substance, which inhibits the growth of crops. By screening salicylic acid degrading bacteria, degrading salicylic acid, reducing the accumulation of autotoxic substances, and reducing the continuous cropping obstacles of crops, the yield of crops can be improved.

[0003] Microbial degradation technology, as a green and environmentally friendly pollution control method, has been widely applied. In terms of degrading salicylic acid, a variety of microorganisms have been found to have degrading ability. Among them, microbacterium, as a common bacterium, has the advantages of fast growth speed, strong adaptability and high degradation efficiency, so it has great application potential in the field of salicylic acid degradation.

[0004] Microbacterium is a kind of gram-positive, aerobic or facultative anaerobic bacillus. The bacteria of this genus are widely distributed in natural environment, such as soil, water and dairy products. Microbacterium has strong environmental adaptability, can grow and reproduce under various conditions, and shows good degradation ability to various organic pollutants. Studies have shown that some microbacterium species can use salicylic acid as the sole carbon source for growth and degrade it into harmless substances through a series of biochemical reactions.

[0005] However, the current research on microbacterium degrading salicylic acid is still in its infancy, and there are many deficiencies. First, the screening and identification of microbacterium species with high degradation capacity are not sufficient; second, the mechanism and pathway of microbacterium degrading salicylic acid are not clear; finally, the stability and efficiency of microbacterium in practical application need further research and optimization.

[0006] Therefore, the present application aims to screen microbacterium species with high salicylic acid degradation capacity by in-depth study of the physiological characteristics and degradation mechanism of microbacterium, and explore its application in degrading salicylic acid. Through the implementation of the present application, a new and efficient microbial degradation method for the degradation of plant root excreted allelopathic autotoxic substance salicylic acid is expected to be provided, which can further reduce the continuous cropping obstacles of crops and improve the yield of crops.

[0007] SUMMARY

[0008] The present application aims to provide a microbacterium ZWM4 and its application in degrading salicylic acid, which provides a new and efficient microbial degradation method for the treatment of salicylic acid pollution, and makes outstanding contributions to environmental protection and sustainable development.

[0009] The object of the present application is achieved by the following technical solutions.

[0010] The present application provides a microbacterium ZWM4, which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO. 30109.

[0011] Further, the 16S rDNA sequence of the microbacterium ZWM4 is shown in SEQ ID NO. 1.

[0012] The present application also provides a microbial agent containing the microbacterium ZWM4.

[0013] The present application also provides the use of the microbacterium ZWM4 or the microbial agent in the degradation of salicylic acid.

[0014] Further, the degradation conditions of salicylic acid are that the pH is 7, the temperature is 30℃, the inoculation amount of the microbacterium ZWM4 is 1%, and the degradation time is 7d.

[0015] The present application also provides the use of the microbacterium ZWM4 or the microbial agent in the preparation of a microbacterium bio-organic fertilizer.

[0016] Further, the microbacterium bio-organic fertilizer can alleviate the continuous cropping obstacles of crops by degrading salicylic acid, promote the growth and development of crops, and improve the yield.

[0017] The present application has the following beneficial effects.

[0018] The microbacterium ZWM4 in the present application can effectively degrade salicylic acid, and the degradation rate can reach 37.47%, which helps to reduce the accumulation of self-toxic substances salicylic acid in crops, alleviate the continuous cropping obstacles of crops, and improve the yield of crops. Using the microbacterium ZWM4 screened in the present application to degrade salicylic acid has lower cost and higher efficiency, further improves the yield of crops, and produces good economic benefits. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0020] Fig. 1 is a surface morphology diagram of the microbacterium ZWM4; DETAILED DESCRIPTION

[0021] The present application will now be described in detail with reference to a number of exemplary embodiments thereof, which description should not be considered as limiting the application, but rather as a description of certain aspects, features and embodiments thereof.

[0022] The technical solutions of the present application will be described in detail below in conjunction with specific examples. Obviously, the described examples are only a part of the embodiments of the present application, and not all the embodiments. Based on the examples in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0023] The present application will be further described below in conjunction with the drawings and examples.

[0024] Example 1 Identification of Microbacterium ZWM4

[0025] (1) Morphology and physiological and biochemical identification of Microbacterium ZWM4

[0026] The Microbacterium ZWM4 of the present application is isolated by screening in natural water bodies. Its morphological characteristics are as follows: after growing on LB solid 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 colonies are yellow, smooth and transparent, as shown in Figure 1. Under a microscope, the bacterial bodies are slender and irregular rod-shaped, with a size of 0.4-0.8 μm (width) x 1.0-4.0 μm (length).

[0027] The physiological and biochemical characteristics of the Microbacterium ZWM4 of the present application are shown in Table 1

[0028] Table 1 Physiological and biochemical characteristics of Microbacterium ZWM4

[0029] As can be seen, the Microbacterium ZWM4 is a gram-positive bacterium, and the results of V-P test, indole reaction, citrate utilization test, gelatin liquefaction test and lipase detection experiment are all negative, and the result of sugar fermentation experiment is positive, i.e. the Microbacterium ZWM4 has sugar fermentation ability.

[0030] (2) Molecular biological identification of Microbacterium ZWM4

[0031] Bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit (Ezup column bacterial genomic DNA extraction kit, Shenguo Bioengineering) according to the kit operation manual. The bacterial DNA was amplified by primers, and the gel was run for verification. The primer sequences were 27F: AGAGTTTGATCMTGGCTCAG and 1492R: GGTTACCTTGTTACGACTT. The sequence of the amplified 16S rRNA gene of the Microbacterium ZWM4 obtained by sequencing is shown in 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 Microbacterium sp., with a similarity of 98.92%, and was named Microbacterium sp. ZWM4. It was preserved in the China General Microbiological Culture Collection Center (CGMCC) on March 25, 2024, and the preservation number was CGMCC NO. 30109.

[0032] Example 2 Determination of salicylic acid degradation rate by high performance liquid chromatography

[0033] 1. Materials and equipment

[0034] The experimental materials used were salicylic acid standard with a purity of more than 99%, analytical pure methanol, formic acid and ethanol solvents.

[0035] 2. Test method

[0036] 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 of salicylic acid ((NH4)2SO42 g, Na2HPO41.3 g, KH2PO42 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 taken and 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 salicylic acid but no degrading bacteria; all test treatments were repeated three times.

[0037] An ultra-high performance liquid chromatograph was used, with an Agilent C18 (4.6 x 250 mm, 0.5 μm) chromatographic column, a chromatographic column temperature of 30°C, a flow rate of 1 mL / min, a sample injection amount of 10 μL, a mobile phase of methanol and 0.1% formic acid, and a detection wavelength of 260 mm.

[0038] Calculation of degradation rate (AU value): degradation rate = (AU value of salicylic acid in the control group - AU value of residual salicylic acid in the medium after 7 days of culture) / AU value of salicylic acid in the control group * 100%.

[0039] 3. Experimental results

[0040] According to the calculation formula of the degradation rate, the degradation rate of salicylic acid after 7 days can reach 37.47%.

[0041] Example 3 Preparation of Bacillus licheniformis liquid inoculum

[0042] The micrococcus was inoculated into LB liquid medium, and cultured at 28°C, 150 rpm for 48h (OD600 value greater than 0.5, i.e. the concentration of microorganisms can reach 10 9 ) to obtain the micrococcus liquid inoculum, which can be used for the preparation of microbial organic fertilizer and also can be used as liquid inoculum.

[0043] Example 4 Preparation of micrococcus bio-organic fertilizer

[0044] The bacteria liquid cultured in LB liquid medium was inoculated into the compost at 10% (v / m), and fermented at room temperature for 7 days to obtain the micrococcus bio-organic fertilizer, which can relieve the continuous cropping obstacles, promote the growth and development of crops, and improve the yield.

[0045] Of course, the above description is not limited to the above examples, and the technical features not described in the present application can be realized by or using the prior art, which will not be described here: the above examples and drawings are only used to illustrate the technical solutions of the present application and are not limited to the present application, and the preferred embodiments of the present application are described in detail, and those skilled in the art should understand that the changes, modifications, additions or substitutions made by those skilled in the art within the essential scope of the present application do not deviate from the spirit of the present application, and also belong to the protection scope of the claims of the present application.

[0046]

Claims

1. A microbacterium ZWM4, wherein the microbacterium ZWM4 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO:30109.

2. The microbacterium ZWM4 as described in claim 1, characterized in that, The 16S rDNA sequence of the microbacterium ZWM4 is shown in SEQ ID NO.

1.

3. A microbial inoculant, characterized in that, The microbial agent includes the microbacterium ZWM4 as described in claim 1.

4. The application of the microbacterium ZWM4 as described in any one of claims 1-2 or the microbial agent as described in claim 3 in the degradation of salicylic acid.

5. The application as described in claim 4, characterized in that, The conditions for degrading salicylic acid were: pH 7, temperature 30℃, inoculum size of Microbacterium ZWM4 1%, and salicylic acid concentration of 0.5 g / L. The degradation rate reached 37.47% after 7 days.

6. The application of Microbacterium ZWM4 as described in any one of claims 1-2 or the microbial agent as described in claim 3 in the preparation of Microbacterium bio-organic fertilizer.

7. The application as described in claim 6, characterized in that, The microbial bio-organic fertilizer can alleviate crop rotation obstacles by degrading salicylic acid, promote crop growth and development, and increase yield.

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