Chryseobacterium sp. and application thereof

Abstract

The application relates to a Chryseobacterium sp. with a preservation number of CGMCC No. 25365 and a strain number of CP3; the Chryseobacterium sp. has the characteristics of promoting growth and degrading chlorpyrifos, can be prepared into a bacterial agent, can enhance the degradation of residual chlorpyrifos in rice, can promote the growth of rice, and is applied to the repair of chlorpyrifos residual pollution in a rice planting system; the Chryseobacterium sp. belongs to plant endophytic bacteria, can long-term colonize in rice, can reduce pesticide residues in crops on the premise of not affecting the quality of crops, can guarantee the quality and safety of agricultural products, and can realize green and safe production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbiology and food safety, in particular to a Chryseobacterium sp. and its application in degrading chlorpyrifos residue in rice and promoting rice growth. BACKGROUND

[0002] Chlorpyrifos is a broad-spectrum organophosphorus insecticide with low price, wide insecticidal spectrum and long persistence, which is widely used in crops such as rice to control rice planthoppers and other pests. It is reported that less than 1% of the applied chlorpyrifos can act on the target organisms. Long-term and unreasonable use has led to large-scale pollution of soil, groundwater, sediments and air. At present, chlorpyrifos is one of the pesticide varieties with the highest detection rate of residues in the environment, and the residue problem in fruits, vegetables and food crops has also been frequently reported. For non-target organisms such as mammals, the accumulation of chlorpyrifos residues in the environment or food can damage the function of the kidneys and liver, affect the development of the nervous system of infants, cause congenital defects and induce cancer. Therefore, it is of great significance to eliminate chlorpyrifos residues in the environment and plants to reduce ecological risk and ensure the quality and safety of agricultural products.

[0003] The degradation of chlorpyrifos in the environment is usually divided into biodegradation and abiotic degradation. Abiotic degradation mainly refers to physical and chemical methods, but these technologies often have the accumulation of toxic by-products and stubborn residues, and even can be transformed into secondary pollutants with higher toxicity, which need further treatment. In recent years, with the rise of microbial technology, some microorganisms with pesticide degradation function have been isolated and purified, and the use of microorganisms to digest residual chlorpyrifos is a new trend. Compared with physical and chemical methods, microbial combined with phytoremediation technology has the advantages of green, environmental protection, economy, high efficiency, etc., and has broad and good prospects in the remediation of chlorpyrifos residues in the environment and the realization of green production process of crops. There have been reports that the genera with chlorpyrifos degradation ability include Bacillus sp., Cronobacter sp., Cupriavidus sp., Sphingomonas sp., Sphingobacterium sp., Pseudomonas sp., etc., but there is no research on Chryseobacterium sp. in degrading chlorpyrifos.

[0004] Chryseobacterium sp. widely exists in nature. Existing researches show that Chryseobacterium sp. plays a role in the degradation of plasticizers, picolinic acid, aniline and herbicide nicosulfuron, but there is no report on Chryseobacterium sp. in the remediation of pesticide pollution. SUMMARY

[0005] To solve the above problems, the present application provides a Chryseobacterium sp. and its application, to enhance the degradation of chlorpyrifos residues in rice, promote the growth of rice, reduce the residues of chlorpyrifos in the environment and crops, and has the application prospect of green and safe production.

[0006] To achieve the above-mentioned purpose, the present application firstly provides a Chryseobacterium sp. with a preservation number of CGMCC NO. 25365, which is named CP3 by the applicant. The strain is isolated from the body of a plant of the family Poaceae, rice, and the strain is rod-shaped, does not have endospores, belongs to the genus Chryseobacterium, and is harmless to animals and plants. The applicant preserved the strain in the China General Microbiological Culture Collection Center (CGMCC) on July 20, 2022, address: No. 1, Beichen West Road, Beijing Chaoyang District, China Institute of Microbiology, Beijing, China, postcode: 100101.

[0007] Secondly, the present application provides the application of the Chryseobacterium sp. with a preservation number of CGMCC NO. 25365 in degrading chlorpyrifos in the environment or the body of a plant, especially in crops (such as rice).

[0008] Further, the application of the strain CGMCC NO. 25365 in chlorpyrifos residues in the body of a plant refers to preparing the above-mentioned Chryseobacterium sp. into a bacterial liquid with a bacterial content of 10 8 CFU / mL (OD 600 =1), and performing root soaking treatment on the roots of the plant for 12h to enhance the degradation of chlorpyrifos residues in the body of the plant. Preferably, the root soaking treatment is performed on the roots of rice at the three-leaf-one-heart stage for 12h to enhance the degradation of chlorpyrifos residues in the body of the rice.

[0009] Thirdly, the present application also provides the application of the above-mentioned Chryseobacterium sp. with a preservation number of CGMCC NO. 25365 in promoting the growth of rice and increasing the biomass (i.e. dry weight) of rice. The application specifically refers to preparing the above-mentioned Chryseobacterium sp. into a bacterial agent with a bacterial content of 10 8 CFU / mL (OD 600 =1), and performing root soaking treatment on the rice to promote the growth of the rice and increase the biomass of the rice.

[0010] Fourthly, the present application also provides a bacterial agent containing the Chryseobacterium sp. with a preservation number of CGMCC NO. 25365; the bacterial agent also includes sterile physiological saline. The bacterial content of the Chryseobacterium sp. in the bacterial agent is preferably 10 8 CFU / mL.

[0011] Compared with the prior art, the Chryseobacterium sp. provided by the application is isolated from plants, and belongs to Chryseobacterium sp. The strain can colonize in rice, promote the degradation of chlorpyrifos in rice, reduce the residue of chlorpyrifos in crops, promote the growth of rice, and increase the biomass of crops. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 It is a scanning electron microscope graph of Chryseobacterium sp. CP3.

[0013] Figure 2 It is a schematic diagram of the degradation function verification result of Chryseobacterium sp. CP3 on chlorpyrifos.

[0014] Figure 3 It is a schematic diagram of the growth promotion function verification result of Chryseobacterium sp. CP3; wherein A is a color reaction photo of the experimental group (CP3) and the control group IAA; B is the IAA content produced by the CP3 strain; and C is a schematic diagram of the phosphorus solubilization ability of the CP3 strain.

[0015] Figure 4 It is a schematic diagram of the degradation result of the CP3 bacterial agent on the residual chlorpyrifos in rice. DETAILED DESCRIPTION

[0016] The medium involved in the example is as follows:

[0017] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0;

[0018] LB solid medium: add agar with a final concentration of 20 g / L to the LB liquid medium;

[0019] Inorganic phosphorus bacterial medium (PVK): glucose (10.0 g), (NH4)2SO4 (0.5 g), NaCl (0.3 g), MgSO4·7H2O (0.03 g), KCl (0.3 g), FeSO4·7H2O (0.03 g), Ca3(PO4)2 (5.0 g), agar (15.0 g), deionized water 1 L;

[0020] Inorganic salt (MSM) liquid medium: 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;

[0021] Rice medium: (NH4)2SO4(2.379 g), MgSO4·7H2O (6.655 g), KNO3(0.910 g), CaNO3·4H2O (4.251 g), K2HPO4(1.225 g), FeSO4+Na2EDTA (0.557 g+0.745 g), MnCl2·2H2O (0.0099 g), Na2MoO4·2H2O (0.0185 g), H3BO3(0.12358 g), ZnSO4·7H2O (0.0115 g), CuSO4·5H2O (0.00498 g), add water to 1 L, pH to 5.6.

[0022] Inorganic salt (MSM) solid medium: add agar with a final concentration of 20 g / L to the inorganic salt liquid medium; strain activation medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0.

[0023] The reagents involved in the following examples are purchased from commercial channels unless otherwise specified.

[0024] Example 1 Isolation and identification of Chryseobacterium sp. CP3

[0025] In July 2021, the applicant collected rice plants sprayed with chlorpyrifos from the experimental field of Jiangsu Academy of Agricultural Sciences, then performed surface sterilization on the rice roots and ground them under sterile conditions, took 100 μL of the solution and coated it on an inorganic salt solid medium with chlorpyrifos as the only carbon and nitrogen source, and screened a plant endophyte with chlorpyrifos degradation characteristics. The applicant named the strain CP3, which was rod-shaped and had no endospores. The electron microscope picture of the strain is shown in Figure 1 .

[0026] The applicant preliminarily identified the CP3 strain as Chryseobacterium sp. through physiological and biochemical characteristics and 16S rDNA conservative sequence comparison, and preserved the strain in the China General Microbiological Culture Collection Center (CGMCC) on July 20, 2022, address: No. 1, Beichen West Road, Chaoyang District, Beijing, China, postcode: 100101, preservation number: CGMCC No. 25365, and classified and named it as Chryseobacterium sp.

[0027] Example 2 Functional verification of chlorpyrifos degradation by strain CP3 (Chryseobacterium sp. CP3)

[0028] The identified CP3 was cultured overnight using LB liquid medium, the bacterial solution was centrifuged at 5000 rpm for 5 min, the supernatant was removed, the bacterial solution was washed 4 times with PBS buffer (pH 7.0), and then resuspended with MSM liquid medium. The bacterial suspension was added to the inorganic salt liquid medium containing chlorpyrifos (1 mg / L) as the only carbon and nitrogen source at a bacterial inoculation amount of 1%, and then shaken and cultured for 2, 12, 24, 36 and 48 h. Finally, the content of chlorpyrifos in the inorganic salt liquid medium was detected by gas chromatography (GC) (reference: Feng F, Ge J, Li Y, He S, Zhong J, Liu X, Yu X. Enhanced degradation of chlorpyrifos in rice (Oryza sativa L.) by five strains of endophytic bacteria and their plant growth promotional ability. Chemosphere, 2017, 184: 505-513.), and the inorganic salt medium without bacteria was used as a control group (1% MSM liquid medium was added). The degradation rate of the strain was calculated according to the following formula:

[0029] Chlorpyrifos degradation rate = (C S -C0) / C0*100%, wherein C S is the content of chlorpyrifos in the experimental group, C0 is the content of chlorpyrifos in the control group, and the degradation function of Chryseobacterium sp. CP3 on chlorpyrifos is determined.

[0030] The results are shown in Table 1. Figure 2 In the MSM liquid medium with 1 mg / L chlorpyrifos as the only carbon and nitrogen source, the residual chlorpyrifos in the experimental group was 0.21 mg / L after shaking and culturing for 48 h, and the degradation rate was 71.6%. The degradation ability of Chryseobacterium sp. CP3 on chlorpyrifos was determined.

[0031] Example 3: Verification of the growth-promoting function of Chryseobacterium sp. CP3

[0032] 1. Verification of the IAA production ability of CP3

[0033] After the CP3 bacteria were cultured in LB liquid medium containing L-tryptophan (IAA precursor) for 1 day, centrifuged at 8000 rpm for 5 min, the supernatant was added to the centrifuge tube containing Salkowski color developing agent at a ratio of 1:1, and the LB solution medium containing L-tryptophan (IAA precursor) without bacteria was used as a control group. After 10 min of light-free reaction, the color reaction was observed and the IAA content was measured (Reference: Liu X P, Zang S Y, Zhi G, Qu F T. Screening of halophytic bacteria and study on their plant growth-promoting ability. Soil Bulletin, 2022, 53(03):567-576.).

[0034] The results are shown in A of Figure 3 Compared with the control group, red liquid was generated in the experimental group (CP3). Further determination of IAA content is shown in B of Figure 3 , indicating that CP3 can produce IAA up to 25.9 mg / L, verifying that CP3 has strong IAA production function.

[0035] 2. Verification of the phosphorus solubilization function of CP3

[0036] The overnight cultured CP3 was inoculated on the PVK plate, and the hydrolysis circle was observed after 4 days of culture at 28°C. The results are shown in C of Figure 3 , indicating that CP3 has obvious hydrolysis circle around it, verifying that CP3 has the function of phosphorus solubilization, which can convert insoluble phosphorus into available phosphorus and improve the utilization of available phosphorus.

[0037] Example 4 Preparation of Chryseobacterium sp. CP3 bacterial agent

[0038] 1. The CP3 identified and separated in Example 1 was inoculated in LB culture medium, and repeated operations such as streaking and picking single colony culture were performed twice. The single colony was picked and cultured in LB liquid medium at 30°C for 24 h to obtain CP3 strain;

[0039] 2. The activated CP3 strain of step 1 was inoculated in a fermenter containing seed culture medium at an inoculation amount of 1%, and cultured at 25-38°C for 16-24 h with air to obtain liquid seed;

[0040] 3. The liquid seed was inoculated in a fermenter containing seed culture medium at an inoculation amount of 1% (volume ratio) and cultured at 30-35°C, 200 rpm in the dark to the logarithmic phase to obtain active bacterial culture;

[0041] 4. 50 mL of active bacterial culture of step 2 was centrifuged at 4°C, 5000 rpm for 20 min, the supernatant was removed, the bacterial bodies were washed with sterile physiological saline for 3 times, and the bacterial liquid was adjusted to OD 600 = 1 (bacterial content about 108 cfu / mL), i.e. to obtain CP3 inoculum, another inoculum was preserved in sterile glycerol at a ratio of 1:1 (volume ratio).

[0042] Example 5 CP3 inoculum enhances chlorpyrifos degradation and growth promotion in rice

[0043] The soil was obtained from the Jiangsu Academy of Agricultural Sciences, and after being exposed to sunlight and dried, it was passed through a 30-mesh sieve to remove large stones. The soil composition was as follows: pH = 5.97, organic matter content 66.8 g / kg of dry soil, clay content 2.33%, silt content 16.1%, and sand content 81.5%.

[0044] Rice seedlings (variety Nangjing 5055) with uniform growth and three-leaf-one-heart were selected, and the roots were soaked in the CP3 inoculum obtained in Example 4 for 6 hours. Non-inoculated rice was used as a control group (soaked in sterile saline). The rice seedlings were transplanted into the above soil and sprayed with chlorpyrifos to a leaf surface wetting state (purchased from Jiangsu Suzhou Jiaguang Chemical Co., Ltd., recommended dosage). After 3 days, rice samples were taken, the rice surface was cleaned, and the content of residual chlorpyrifos in the rice was determined by gas chromatography (GC) (reference: Feng F, Zhan H, Wan Q, Wang Y, Li Y, Ge J, Sun X, Zhu H, Yu X. Rice recruits sphingomonas strain HJY-rfp via root exudate regulation to increase chlorpyrifos tolerance and boost residual catabolism. Journal of Experimental Botany, 2021, 72(15): 5673-5686.). The results are shown in Figure 4 As shown in Table 1, after application of the CP3 inoculum, the chlorpyrifos residue in the rice decreased from 4.24 mg / kg to 3.16 mg / kg, demonstrating that the CP3 inoculum can significantly reduce the chlorpyrifos residue in the rice.

[0045] The application of Chlorpyrifos and the CP3 inoculum of Chryseobacterium was the same as above. After 14 days of application, rice samples were collected, and growth indicators were measured. The results are shown in Table 1.

[0046] Table 1 Results of CP3 inoculum increasing rice biomass

[0047]

[0048] As shown in Table 1, compared with the control group, the plant height, root length and dry weight of rice treated with CP3 bacterial agent were significantly increased, which proved that CP3 bacterial agent could promote the growth of rice and increase the biomass of rice.

Claims

1. A Chryseobacterium sp. with a preservation number of CGMCC NO. 25365. Chryseobacterium sp. with a preservation number of CGMCC NO. 25365.

2. The use of the Chryseobacterium in degrading chlorpyrifos in the environment or in the plant body according to claim 1.

3. The use of the Chryseobacterium in promoting the growth of rice according to claim 1.

4. The use according to claim 2, wherein the compound is ###0002### The degradation of chlorpyrifos in plants refers to the use of a bacterium containing 10 8 CFU / mL of the bacterium Xanthomonas to treat the roots of plants, which can degrade the residual chlorpyrifos in the plants.

5. The use according to claim 3, wherein the compound is ###0002### The application of the Chryseobacterium in promoting rice growth refers to using Chryseobacterium bacterial agent with a bacterial content of 10 8 CFU / mL to perform root soaking treatment on rice to promote rice growth.

6. A microbial agent containing Chryseobacterium with the preservation number of CGMCC NO. 25365.

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