Arthrobacter oxydans and application thereof

Abstract

The invention relates to application of pseudoarthrobacter oxydans D-14 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.31959 to degradation of carbendazim and promotion of growth of green vegetables, in particular to application of the pseudoarthrobacter oxydans D-14 to degradation of carbendazim. The strain can be prepared into a microbial agent, and after the strain is colonized in green vegetables, degradation of carbendazim in the green vegetables and soil can be effectively promoted, and increase of biomass and improvement of nutritional indexes of the green vegetables are promoted; the pseudoarthrobacter oxydans belongs to plant rhizosphere bacteria, can colonize roots of green vegetables, and can promote the growth of the vegetables on the basis of promoting the degradation of carbendazim. The use of the fungicide is beneficial to reducing carbendazim residues in the environment and crops, and is beneficial to guaranteeing the safety of the environment and agricultural products.

Description

Technical Field

[0001] The invention relates to the fields of microbiology and food safety, in particular to a strain of Pseudarthrobacter oxydans D-14 and application thereof in degrading carbendazim and promoting the growth of crops. Technical Background

[0002] Carbendazim is a widely used, highly effective, low-toxic, broad-spectrum systemic fungicide. It has good control effects on fungal diseases such as ergot, black spot, anthracnose, etc. of crops, so it is widely used in agricultural production. Since carbendazim is often used in the growth and development of plants, its residues are easily detected in common foods in daily life such as vegetables, fruits, grains, oilseeds and Chinese medicinal materials. Carbendazim has stable chemical properties, a long residual period in the natural environment, and is difficult to degrade. Continuous use for many years has caused soil, water and air pollution; and carbendazim can be absorbed by plants and enter the plant body, thereby affecting the growth and quality of crops, causing food safety hazards, and also leading to the deterioration of the agricultural environment. Timely removal of pesticide residues in the soil environment and crops is an urgent problem to be solved in agricultural production.

[0003] The degradation of carbendazim in the environment includes physical, chemical and microbial degradation. Microbial degradation is considered to be a promising degradation or detoxification method due to its high efficiency, economy and eco-friendliness. At present, there are few applications of Pseudomonas oxidans, such as alleviating crop continuous cropping obstacles and tolerating heavy metals. There is no literature report on the degradation of the pesticide carbendazim by Pseudomonas oxidans. Summary of the invention

[0004] In view of the above problems, the present invention provides a strain of Pseudomonas oxidans (D-14), which can degrade carbendazim in a green vegetable planting system, reduce the enrichment of carbendazim in the aboveground and underground parts of green vegetables, and promote the growth of green vegetables and improve nutritional indicators.

[0005] Specifically, the present application is implemented through the following technical solutions:

[0006] First, the present application provides a strain of Pseudarthrobacter oxydans with a preservation number of CGMCC No.31959. The strain is a Gram-negative bacterium, short rod-shaped, polymerized, and spore-free. The applicant named it D-14. The strain was isolated from the rhizosphere of green vegetables grown in pesticide-contaminated soil and is harmless to animals and plants. On September 12, 2024, the applicant deposited the strain in the General Microbiology Center of the China Microbiological Culture Collection Administration (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Postal Code: 100101; The preservation number is CGMCC No.31959.

[0007] Secondly, the present application also provides the use of Pseudoarthrobacter oxidans with a deposit number of CGMCC No. 31959 in the degradation of carbendazim, including the use of carbendazim in the degradation environment (such as soil) and in crops (such as green vegetables).

[0008] Furthermore, the application of the above-mentioned Pseudomonas oxidans in degrading carbendazim in green vegetables and planting environments, the specific steps are as follows: root irrigation of crops is carried out with Pseudomonas oxidans agent (D-14 agent) to degrade the residual carbendazim in crops and soil; the above-mentioned root irrigation refers to: root irrigation treatment once before transplanting crops and in the second week after transplanting, for a total of 2 treatments, each time 100 ml / crop.

[0009] Third, the present application also provides the use of Pseudomonas oxidans with a deposit number of CGMCC No. 31959 in promoting the growth of green vegetables and improving the biomass and quality (soluble sugar, soluble protein) of green vegetables. That is, the green vegetables are irrigated with the Pseudomonas oxidans agent (D-14 agent) to promote the growth of green vegetables and improve the biomass and quality of green vegetables; the above-mentioned root irrigation refers to: irrigating the roots once before transplanting the green vegetables and once in the second week after transplanting, for a total of 2 treatments, each time 100 ml / tree.

[0010] The above-mentioned Pseudomonas oxidans agent (D-14 agent) is composed of MSM culture medium and Pseudomonas oxidans with a preservation number of CGMCC No. 31959; the components of each liter of MSM culture medium are as follows: 0.4g MgSO4·7H2O, 0.2g FeSO4·7H2O, 0.2g K2HPO4, 0.2g (NH4) 2SO4, 0.08g CaSO4, 1L deionized water, pH 7.0; in the above-mentioned D-14 agent, the final concentration of Pseudomonas oxidans is preferably 10 7 cfu / mL.

[0011] This application is the first to discover Pseudoarthrobacter oxidans that can degrade carbendazim in green vegetable plants grown in carbendazim-contaminated soil, and to study, identify and preserve them. Experiments have shown that this strain can colonize in the rhizosphere and roots of green vegetables, reduce the amount of carbendazim residues in the soil and the aboveground and underground parts of green vegetables, and reduce the carbendazim residues in crops and the environment; at the same time, it can promote the growth of green vegetables and improve quality indicators. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is an electron microscope scanning image of Pseudomonas oxidans D-14.

[0013] Figure 2 Schematic diagram of the in vitro degradation function verification results of Pseudomonas oxidans D-14 on carbendazim.

[0014] Figure 3 Schematic diagram of the degradation results of carbendazim residues in soil and green vegetables by D-14 bacterial agent.

[0015] Figure 4 This is a schematic diagram of the growth promotion index data of Pseudomonas oxidans D-14. DETAILED DESCRIPTION

[0016] The culture medium involved in the embodiment:

[0017] LB medium / LB solid plate: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15-20 g / L, adjust pH to 7.0 with NaOH;

[0018] Inorganic salt liquid culture medium (MSM): MgSO4·7H2O (0.4 g), FeSO4·7H2O (0.2 g), K2HPO4 (0.2 g), (NH4)2SO4 (0.2 g), CaSO4 (0.08 g), deionized water 1 L, pH 7.0.

[0019] MSM solid culture medium: Add agar with a final concentration of 20 g / L to the inorganic salt liquid culture medium.

[0020] King B medium: 1000 mL of deionized water, 32 g of tryptone, 1.15 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate heptahydrate, 10 mL of glycerol, plus 1 g of L-tryptophan.

[0021] The nitrogen-fixing medium was purchased from Coolaber, and the iron carrier-producing medium (CAS) was purchased from Xianghai Biological Co., Ltd. The above culture media were prepared according to the instructions for use of the culture media.

[0022] All culture media must be sterilized at 121°C for 20 min before use.

[0023] Unless otherwise specified, all reagents involved in the following examples were purchased from commercial channels.

[0024] Example 1 Isolation and identification of Pseudomonas oxidans D-14 and verification of its degradation function

[0025] 1. Isolation and identification of strain D-14 (Pseudarthrobacter oxydans D-14)

[0026] In June 2023, the applicant collected green vegetables grown in pesticide-contaminated soil in the experimental field of Jiangsu Academy of Agricultural Sciences (E118°86′, N32°03′) and isolated and screened rhizospheric bacteria with carbendazim degradation function. Rhizospheric bacteria isolation and screening method: Sterile LB medium containing 50ppm carbendazim was added to the rhizospheric soil sample, and cultured at 30°C and 150r / min shaking for 1d; then the bacterial solution was taken for 10 4 , 10 5 , 10 6 Dilute by 100 μl, spread 100 μl of the dilution evenly on a LB solid plate containing 50 ppm carbendazim, and culture at 30°C in the dark for 1 to 3 days. Select a single colony with good growth on the LB plate and streak it on an MSM plate containing 20 mg / kg carbendazim, and culture the plate at 30°C for 1 day. Continue to purify the colonies on the MSM plate until a monoclonal strain is obtained. A rhizosphere bacterium with carbendazim degradation characteristics is obtained through screening. The electron microscope scanning image is shown in the figure below. Figure 1 As shown, the strain is short rod-shaped, arranged in multiple clusters, and has no spores. The applicant named the strain D-14.

[0027] Through physiological and biochemical characteristics and 16SrDNA conservative sequence comparison, the D-14 strain was identified as Pseudarthrobacter oxydans. The applicant deposited the strain in the General Microbiology Center (CGMCC) of China Microorganism Culture Collection Administration on September 10, 2024, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, the deposit number is CGMCC No. 22887, and the classification name is Pseudarthrobacter oxydans.

[0028] 2. Verification of the ability of strain D-14 (Pseudarthrobacter oxydans D-14) to degrade carbendazim

[0029] The D-14 strain obtained in step 1 was first cultured in LB medium for 12 hours. The bacterial solution was centrifuged (6000 rpm, 5 min, 20°C) and resuspended and washed three times with sterile inorganic salt solution (pH 7.0). The OD 600=1.0); then, the D-14 bacterial suspension was added to an inorganic salt liquid medium with carbendazim as the sole carbon source (10 μg / mL) at a volume ratio of 2% and cultured for 3, 7, and 14 days; the residual amount of carbendazim in the inorganic salt liquid medium at different culture times was detected by liquid chromatography-mass spectrometry (LC-MS), and compared with the blank group (CK), the degradation rate of the D-14 strain was calculated:

[0030] C=(AB) / A*100%

[0031] A = carbendazim concentration in CK, mg / L;

[0032] B = carbendazim concentration in experimental group, mg / L;

[0033] C = carbendazim degradation rate

[0034] The in vitro degradation capacity of carbendazim of D-14 was determined by calculation using this formula. The LC-MS parameters are as follows:

[0035] The analysis was performed by Shimadzu high performance liquid chromatography coupled with AB Sciex QTRAP 5500+ mass spectrometry system. The chromatographic column was Kinetex C18 column (3.0×100mm, 2.6μm). 0.1% formic acid aqueous solution (A) and acetonitrile (B) were the mobile phases. The chromatographic gradient program started from 90% A, maintained at this ratio for 1 min, then decreased to 10% A within 3 min and maintained for 1 min, then returned to 90% A at 5.01 min and maintained at this ratio for 0.9 min. The flow rate was 0.3 mL / min, the column temperature was 30°C, and the injection volume was 2μL. The scanning mode was positive ion mode. The electrospray voltage was +5.50 kV. The auxiliary gas (N2) and nebulizer gas (N2) were 55 psi, the ion source temperature was 550°C, and the determination method was multiple reaction monitoring (MRM).

[0036] Test results such as Figure 2 As shown, within 14 days, D-14 bacterial solution (OD 600 =1.0) The degradation rate of carbendazim at a concentration of 10 mg / L was 28.6%, which verified that D-14 had the degradation property for carbendazim.

[0037] Example 2D-14 Preparation of bacterial agent

[0038] The specific preparation steps of D-14 bacterial agent in this embodiment are as follows:

[0039] 1) The D-14 strain isolated in Example 1 was inoculated into an LB medium containing 10 mg / L (final concentration) of carbendazim for enrichment, and streaked on an MSM solid plate containing 10 mg / L (final concentration) of carbendazim and an LB solid plate containing 10 mg / L (final concentration) of carbendazim, and then a single colony was picked from the plate and placed in an LB liquid medium, and cultured on a shaker at 30° C. and 150-220 rpm for 12 h, and activated twice in succession to obtain an activated bacterial solution;

[0040] 2) Place the activated bacterial solution in a 50 ml sterilized centrifuge tube in a clean bench, centrifuge at 20°C and 6000 rpm for 5 min to precipitate the bacteria, sterilize with high pressure steam at 121°C for 20 min, rinse with MSM liquid medium 3 times, and then add MSM liquid medium again to adjust the OD value of the bacterial solution 600 =1(the bacterial solution concentration is about 10 10 cfu / mL), and the D-14 bacterial agent was obtained.

[0041] Example 3D-14 Experiment on promoting the degradation of carbendazim in green vegetables and soil environment and promoting the growth of green vegetables

[0042] The soil was collected from the fields of Jiangsu Academy of Agricultural Sciences, and after drying, it was hammered and crushed, and passed through a 20-mesh sieve to remove impurities for later use. Composition: organic matter 4%; silt 49%; clay 22%; sand 25%. Original soil nutrients: total organic carbon 14.86 mg / kg; available potassium 0.14 g / kg; available phosphorus 0.05 g / kg; total nitrogen 1.65 g / kg; pH 6.7.

[0043] Carbendazim treatment: Carbendazim original drug was diluted and mixed with soil. After the soil was aged for one week, a vegetable planting experiment was carried out (the carbendazim concentration in the soil was about 5 ppm during planting). 1.5 kg of soil was placed in each pot, and a bacterial agent treatment group and a control group were set up, with 3 replicates for each treatment.

[0044] The Shanghai green seedlings were transplanted at the three true leaf stage. The specific experimental steps of the microbial agent treatment group were as follows: the strain was inoculated into LB medium, cultured in a shaking incubator at 30°C and 180 rpm for 24 h, then centrifuged and washed three times with MSM medium. The bacterial cells were collected and diluted with MSM medium, and the bacterial solution density was adjusted to OD 600 Reach about 0.2 (the density of bacteria counted on the plate is about 1×10 7 cfu / mL), and the bacterial solution is the inoculant. The inoculant was used for irrigation one day before and two weeks after the green vegetables were transplanted, with each irrigation volume of 100 mL. The control group was treated with the same volume of non-bacterial MSM medium. The other management methods of the two groups were the same.

[0045] The plants were harvested 30 days after transplanting and soil and vegetable samples were collected. The parallel treatment was n=3. Figure 3shown.

[0046] Enrichment and degradation of carbendazim: At harvest, the carbendazim concentration in the soil of the inoculant treatment group was 4.15 mg / kg, and the carbendazim concentration in the control soil was 5.95 mg / kg. The removal rate of carbendazim in the soil by the inoculant treatment group was 30.25%. The carbendazim content in the green vegetable plants was tested. The carbendazim concentrations in the aboveground and underground parts of the green vegetables in the inoculant treatment group were 2.34 and 3.41 mg / kg, respectively, and those in the control group were 4.17 and 5.36 mg / kg, respectively. The carbendazim concentrations in the aboveground and underground parts of the green vegetables in the inoculant treatment group were reduced by 43.88% and 36.38%, respectively. It can be seen that the strain has a good degradation effect on the carbendazim residual in the soil and the carbendazim in the crop plants.

[0047] D-14 improves the biomass and quality of green vegetables as shown in Table 1:

[0048] Table 1. Results of biomass and quality test of green vegetables

[0049]

[0050] The test results of D-14 improving the quality indexes of green vegetables are shown in Table 1. The fresh weight of green vegetables treated with D-14 under carbendazim stress was significantly increased compared with the CK group at the 30th day of harvest, and the fresh weight was increased by 30.97% compared with the control group. The soluble sugar and soluble protein of green vegetables treated with D-14 under carbendazim stress were increased by 10.94% and 14.29% respectively at the 30th day of harvest compared with the CK group.

[0051] Example 4D-14 Growth-promoting properties and experiment on the growth-promoting and quality-improving effects of green vegetables under carbendazim stress

[0052] 1. IAA: The D-14 bacterial agent prepared in Example 2 was inoculated into King B medium containing L-tryptophan (1 g) (precursor of IAA synthesis), and the King B medium without bacteria was used as the control group (CK). The culture was shaken at 32°C and 180 r / min for 12 h. Centrifuged at 8000 rpm for 5 min, the supernatant was mixed with Salkowski reagent (50 mL of 35% perchloric acid plus 1 mL of 0.5 mol / L ferric chloride) at a ratio of 1:2, and placed in a dark room for reaction for 30 min. The D-14 group produced dark red substances, indicating that the bacteria can produce IAA (see Figure 4 (shown in a).

[0053] 2. Nitrogen fixation ability: When D-14 bacterial solution is inoculated into nitrogen fixation medium, a transparent circle can be observed around D-14, indicating that D-14 has a certain nitrogen fixation ability (see Figure 4 (as shown in b).

[0054] 3. CAS production ability: D-14 bacterial solution was inoculated into CAS medium for culture. The strain grew normally and formed a yellow transparent circle around it, indicating that D-14 has the ability to produce iron carriers (see Figure 4 (as shown in c).

[0055] The above experimental results show that the D-14 bacterial agent has a good growth-promoting effect and can improve the biomass and quality of green vegetables.

Claims

1. A strain of Pseudomonas oxydans ( Pseudarthrobacter oxydans ), its deposit number is CGMCC No. 3195.

2. Use of Pseudomonas oxidans in the degradation of carbendazim as claimed in claim 1.

3. Use of Pseudomonas oxidans in promoting the growth of green vegetables as claimed in claim 1.

4. The use according to claim 2, characterized in that: The application refers to the use of Pseudomonas oxidans in degrading carbendazim in the environment and crop plants.

5. The use according to claim 3, characterized in that The application refers to the use of a bacterial agent containing Pseudomonas oxidans to irrigate the roots of green vegetables to increase the biomass, soluble sugar content and soluble protein content of the green vegetables.

6. The use according to claim 5, characterized in that: The bacterial agent containing Pseudomonas oxidans includes MSM culture medium and Pseudomonas oxidans.

7. A bacterial agent containing Pseudomonas oxidans with a preservation number of CGMCC No. 3195.

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