A composite microbial agent with thiamethoxam promoting and degrading functions and application

Abstract

The application discloses a kind of composite microbial inoculant with the function of promoting growth and degrading thiamethoxam and application, the composite functional inoculant is composed of 2 thiamethoxam-degrading bacteria Enterobacter cloacae and Enterobacter cloacae and 1 growth-promoting enterobacter, and is all plant endophytic bacteria, the composite inoculant can be applied to soil source thiamethoxam pollution leaf vegetable system, can effectively reduce thiamethoxam residue in leaf vegetable while promoting plant growth, realizes green and safe production.

Description

Technical Field

[0001] This application relates to the fields of microbiology and food safety, and in particular to a compound microbial agent that combines growth promotion and thiamethoxam degradation functions and its application. Technical Background

[0002] Vegetables, as a leading sector in agriculture, play a vital role in crop cultivation. Pesticides are indispensable in vegetable production to control pests, diseases, and weeds, and to ensure yields and increase income. However, due to the irrational use of pesticides, pesticide residues in vegetables are frequently observed, with neonicotinoid pesticides being among the most frequently detected. Thiamethoxam, a second-generation neonicotinoid insecticide with low toxicity, is used for foliar spraying and soil drenching. After application, it is rapidly translocated to all parts of the plant, controlling pests such as aphids through stomach poisoning and contact action. Simultaneously, thiamethoxam also acts on non-target organisms, potentially reducing pollinators such as bees and increasing the incidence of liver tumors in mice. Furthermore, thiamethoxam has high water solubility and systemic activity, leading to accumulation and depletion of applied thiamethoxam within vegetables, affecting plant growth, agricultural product quality and safety, and even posing risks to human health. Therefore, under thiamethoxam stress, improving plant resistance and enhancing the degradation of residual thiamethoxam in the environment and within plants is particularly important.

[0003] Microbial remediation, characterized by high efficiency, low cost, and environmental friendliness, has become one of the main approaches to remediating pesticide residues in the environment. Currently, microbial degradation of thiamethoxam mainly focuses on single-strain organisms with degrading functions. Chinese patents "A strain of *Enterobacter mulberryii* and its application in degrading thiamethoxam" (CN110172430A) and "A strain of *Enterobacter cloacae* and its application in degrading thiamethoxam" (CN110106116A) disclose the application of *Enterobacter mulberryii* and *Enterobacter cloacae* in degrading thiamethoxam. However, single-strain organisms have limited efficiency, thus necessitating the combination of functional single strains to obtain multifunctional compound microbial agents to alleviate the drawbacks of single agents. However, to date, there are no reports on the application of multifunctional compound microbial agents in degrading thiamethoxam. Summary of the Invention

[0004] To address the above problems, this invention provides a compound microbial agent that combines growth promotion and thiamethoxam degradation functions. Targeting leafy vegetable systems contaminated with soil-source thiamethoxam, this compound microbial agent is applied to the growth of leafy vegetables and the degradation of thiamethoxam accumulated within the vegetables. This reduces the absorption and accumulation of thiamethoxam in the soil by leafy vegetables, lowers the residue of thiamethoxam in the vegetables, and does not introduce other pollution sources. It is highly efficient and environmentally friendly, ensuring safe vegetable production.

[0005] To achieve the above objectives, this application provides the following solution:

[0006] First, this application provides a composite microbial agent that combines growth promotion and thiamethoxam degradation functions, the composite microbial agent including *Enterobacter mulberryii* (… Enterobacter mori) Enterobacter cloacae ( Enterobacter cloacae) and Enterobacteriaceae ( Enterobacter sp. The complex microbial community consists of CS45 strains, and the three strains have the same bacterial content, with no antagonistic effect among them.

[0007] Furthermore, the preferred viable count of the above-mentioned compound microbial agent is 10. 8 CFU / mL; preferably, the bacterial agent also includes MSM liquid culture medium; the liquid MSM culture medium is prepared as follows: 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 to 1 L, pH 7.0.

[0008] Furthermore, the preferred *Enterobacterium mulberryense* strain is *Enterobacterium mulberryense* TMX-13 with accession number CGMCC No. 16236. This strain was deposited on August 8, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, and classified as *Enterobacterium mulberryense* (…). Enterobacter mori ).

[0009] The preferred strain of *Enterobacter cloacae*, TMX-6, with accession number CGMCC No. 16235, was deposited on August 8, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, and classified as *Enterobacter cloacae* (CGMCC). Enterobacter cloacae) .

[0010] Enterobacteriaceae ( Enterobacter Enterobacter CS45, with accession number CGMCC No. 29748, is a growth-promoting bacterium isolated from rice roots. It was deposited on January 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, and classified as Enterobacter (sp.). Enterobacter sp.).

[0011] Secondly, this invention provides the application of the aforementioned composite microbial agent, which combines growth-promoting and thiamethoxam-degrading functions, in promoting growth and degrading thiamethoxam in leafy vegetables. Specifically, it utilizes a microbial content of 10... 8The roots of leafy vegetables were soaked in a compound microbial agent at a concentration of CFU / mL for 12 hours before cultivation to enhance the degradation of thiamethoxam residues in the edible parts of the leafy vegetables and promote their growth.

[0012] Compared with existing technologies, the compound microbial agent provided by this invention contains *Enterobacter mulberryii* (TMX-13), *Enterobacter cloacae* (TMX-6), and the growth-promoting *Enterobacter* (CS45), all of which are isolated from plants and belong to plant endophytes; they colonize more stably in leafy vegetables. Furthermore, the compound microbial agent contains growth-promoting bacteria and thiamethoxam-degrading bacteria, which promote the growth of leafy vegetables while improving their stress resistance (i.e., enhancing their resistance to abiotic stresses such as thiamethoxam, including by promoting pesticide degradation in plants). Through the synergistic effect of the strains and the plant, it reduces thiamethoxam residues in the edible parts of leafy vegetables, ensuring safe production of leafy vegetables without introducing other sources of pollution. This invention is characterized by high efficiency, economy, and environmental friendliness. Attached Figure Description

[0013] Figure 1 This is a schematic diagram showing the compatibility test results of each strain.

[0014] Figure 2 This is a schematic diagram showing the effect of the compound microbial agent on reducing thiamethoxam contamination in leafy vegetables.

[0015] Figure 3 This is a schematic diagram showing the effect of the compound microbial agent on reducing the malondialdehyde content in leafy vegetables.

[0016] Figure 4 This is a schematic diagram showing the effect of the compound microbial agent on promoting the growth of leafy vegetables. Detailed Implementation

[0017] The culture medium involved in the examples:

[0018] LB liquid medium: containing 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride, with deionized water added to bring the volume to 1L, pH 7.0;

[0019] LB solid medium: LB liquid medium with agar added to a final concentration of 20 g / L;

[0020] Inorganic salt (MSM) liquid culture 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 to 1 L, pH 7.0.

[0021] The above three cultures are prepared by sterilization at 121℃ for 20 min before use.

[0022] Unless otherwise specified, all reagents used in the following examples were purchased commercially.

[0023] Example 1: Preparation of Compound Microbial Agent

[0024] Bacillus mulberryis ( Enterobacter mori TMX-13), Enterobacter cloacae ( Enterobacter cloacae TMX-6) and Enterobacteriaceae ( Enterobacter sp. The three strains (CS45) were inoculated into LB solid medium for activation. Single colonies were picked from the activated plates and transferred to sterile LB liquid medium. The culture was amplified and cultured at 30 °C and 200 rpm for 12 h to obtain activated cultures. These activated cultures were then inoculated into a fermenter and fermented at 30 °C and 200 rpm for 24 h to obtain the fermentation broth. The fermentation broth was then... o Centrifuge at 5000 rpm for 10 min, wash the precipitated bacterial cells three times with sterile MSM liquid medium, and then adjust to OD using sterile MSM liquid medium. 600 The value is 1.0, meaning that the number of viable bacteria obtained is 10. 8 TMX-13, TMX-6, and CS45 single-strain agents at CFU / mL.

[0025] Three single-strain agents were mixed in equal proportions to obtain a bacterial count of 10. 8 CFU / mL compound microbial agents were prepared by adding single-strain agents and compound-strain agents to sterile glycerol at a 1:1 (volume ratio) ratio for preservation. The compatibility between strains was tested using LB agar. Each strain was streaked across LB plates and incubated at 30 °C for 2 days. The growth of each strain at the streak intersection was observed to determine whether there was antagonistic interaction between the strains. If the strain grew at the intersection, there was no antagonistic interaction between the two strains; otherwise, there was antagonistic interaction.

[0026] The results are as follows Figure 1 As shown, Figure 1 In the middle, A and B represent TMX-6 and TMX-13, CS45 respectively; the results of the streak detection of TMX-13 and TMX-6, CS45 show that the growth of each strain was not inhibited at the intersection, that is, there is no antagonism among the three strains.

[0027] Example 2: Test on the ability of compound microbial inoculants to promote the degradation of thiamethoxam in leafy vegetables

[0028] Soil: Yellow-brown soil from farmland in Nanjing, Jiangsu Province was collected. After the soil sample was naturally air-dried, large stones and plant roots were removed, and the sample was passed through a 2 mm sieve for later use. Its basic physicochemical properties are: pH 7.24, organic matter content 24.61 g / kg, and organic carbon content 7.44 g / kg.

[0029] Thiamethoxam degradation and malondialdehyde (MDA) content: After Shanghai bok choy reached the three-leaf stage, Shanghai bok choy with uniform growth were selected and their roots were completely immersed in the compound microbial agent obtained in Case 1 and three single-strain agents at room temperature. The control group was immersed in MSM liquid medium. After 6 hours of inoculation treatment, the plants were transplanted into the above-mentioned farmland soil. After 14 days of cultivation, thiamethoxam (purchased from Jiangsu Huifeng Bio-Agriculture Co., Ltd.) was sprayed on the leaves of Shanghai bok choy at the dosage recommended in the product instructions. After 4 days of cultivation, samples were collected, and the thiamethoxam residue content in the edible parts of the leafy vegetables was detected by high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) (the detection method is referred to in the reference: Liu J, Cheng J, Zhou C, Ma L, Chen X, Li Y, SunX, Yan X, Geng R, Wan, Q, Yu X. Uptake kinetics and subcellular distribution of three classes of typical pesticides in rice plants. Science of The Total Environment, 2023, 858, 159826.) and the MDA content (Ma LY, Zhang SH, Zhang JJ, Zhang AP, Li N, Wang XQ, Yu QQ, Yang H. Jasmonic acids facilitate thedegradation and detoxification of herbicide isoproturon residues in wheatcrops ( Triticum aestivum ). Chemical Research in Toxicology, 2018, 31: 752-761.).

[0030] Thiamethoxam content in each group as follows Figure 2The results showed that the thiamethoxam content in Shanghai bok choy treated with compound microbial agents and single microbial agents was 0.36 mg / kg and 0.83-1.42 mg / kg, respectively, significantly lower than the control group (uninoculated Shanghai bok choy, 2.05 mg / kg). The thiamethoxam content in the compound microbial agent group was only 25.4-43.4% of that in the single microbial agent group, indicating that the application of compound microbial agents promoted the degradation of thiamethoxam, significantly reducing thiamethoxam residues in the edible parts of Shanghai bok choy, and the effect was superior to that of single microbial agents. Malondialdehyde, as a product of cell membrane lipid peroxidation, is often used to evaluate the degree of oxidative stress in plants (Ma LY, Zhang SH, Zhang JJ, Zhang AP, Li N, Wang XQ, Yu QQ, Yang H. Jasmonic acids facilitate the degradation and detoxification of herbicide isoproturon residues in wheat crops). Triticum aestivum ). Chemical Research in Toxicology, 2018, 31: 752-761.), Figure 3 The malondialdehyde (MDA) content in the compound microbial agent, single microbial agent, and control group were 3.15 μmol / L, 3.88-4.13 μmol / L, and 4.67 μmol / L, respectively. This indicates that the MDA content in Shanghai pine was significantly lower than that in the control group after applying the compound microbial agent and single microbial agent. The MDA content in Shanghai pine treated with the compound microbial agent was the lowest, suggesting that the application of the compound microbial agent reduced the oxidative stress of thiamethoxam on Shanghai pine.

[0031] Example 3: Experiment on the effect of compound microbial inoculant on promoting the growth of leafy vegetables

[0032] Once the Shanghai bok choy has grown to the stage of three leaves and one heart, select Shanghai bok choy plants with uniform growth and completely immerse their roots in the compound microbial inoculant obtained in Case 1, the three groups of single-strain inoculants, and the control group in MSM liquid culture medium. After 6 hours of immersion and inoculation treatment, transplant them into the above-mentioned farmland soil. After 14 days of cultivation, collect Shanghai bok choy samples and measure physiological indicators.

[0033] The results are as follows Figure 4As shown, the fresh weight of Shanghai bok choy after applying compound microbial agents and single microbial agents was 18.47 g and 10.33-12.33 g, respectively, significantly higher than the control group (5.79 g). Simultaneously, the dry weight of Shanghai bok choy after applying compound microbial agents and single microbial agents was 2.01 g and 0.92-1.33 g, respectively, significantly higher than the control group (0.48 g). The results indicate that the application of both single and compound microbial agents significantly increased the fresh and dry weight of Shanghai bok choy, and the biomass of Shanghai bok choy was highest after applying the compound microbial agent. This suggests that the compound microbial agent can promote the growth and increase the biomass of Shanghai bok choy, and its growth-promoting effect is stronger than that of single microorganisms.

Claims

1. A composite microbial agent with both growth-promoting and thiamethoxam-degrading functions, characterized in that, The bacterial agent comprises *Enterobacter mulberryii*, *Enterobacter cloacae*, and *Enterobacter* with the same bacterial content; the *Enterobacter mulberryii* is *Enterobacter mulberryii* with accession number CGMCC No. 16236. (Enterobacter mori) TMX-13; Enterobacter cloacae is Enterobacter cloacae with accession number CGMCC No. 16235. (Enterobacter cloacae) TMX-6; Enterobacter is Enterobacter with accession number CGMCC No. 29748. (Enterobacter sp.) CS45.

2. The composite microbial agent with both growth-promoting and thiamethoxam-degrading functions according to claim 1, characterized in that, The compound microbial agent also includes MSM liquid culture medium.

3. The application of the compound microbial agent with both growth-promoting and thiamethoxam-degrading functions as described in claim 1 or 2 in promoting the growth of leafy vegetables and degrading thiamethoxam in leafy vegetables; wherein the leafy vegetable is Shanghai bok choy.

4. The application according to claim 3, characterized in that, The content of beneficial bacteria is 10. 8 A compound microbial agent with a concentration of CFU / mL was used to soak the roots of leafy vegetables for 12 hours before cultivation to enhance the degradation of thiamethoxam in leafy vegetables and promote their growth.

Images