Application of bacillus velezensis as thiamethoxam synergist

Abstract

The invention discloses an application of bacillus velezensis as a thiamethoxam synergist, and finds that after being treated by a bacillus velezensis liquid for corn to which thiamethoxam is applied, the bacillus velezensis liquid can promote the absorption and utilization of the corn to the thiamethoxam and enhance the transfer and enrichment of the thiamethoxam to the overground part of the corn, so that the effect of improving the corn yield is achieved. The content of thiamethoxam in corn roots and leaves is remarkably increased, the effective utilization rate of pesticides is increased, and meanwhile corn growth is promoted. The invention provides a new thought for improving the utilization rate of the pesticide, provides an example for developing a fungicide for efficiently utilizing the pesticide, and the fungicide can be used as a thiamethoxam synergist and has a wide application prospect in promoting the growth of crops and improving the utilization rate of the pesticide.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to application of Bacillus velezine as a thiamethoxam synergist. Background Art

[0002] The use of pesticides protects plants from various diseases, insects and weeds, and ensures normal agricultural production. my country is a major user of pesticides. Long-term excessive, frequent and unscientific use of pesticides has increased the cost of pesticide production and led to widespread pesticide residues in soil and water. This not only destroys the ecological stability of the soil and brings a series of environmental risks, but also accumulates in the edible parts of crops through plant absorption and transport, which can easily cause risks to human health. In the face of increasingly severe pesticide pollution and the public's widespread concern about the ecological environment, it is urgent to develop new, efficient and safe technologies and methods to improve the effective utilization rate of pesticides, reduce pesticide pollution, and ensure the quality of agricultural products and the safety of the ecological environment.

[0003] Reducing the amount of pesticides and increasing their efficiency is an important measure to promote quality and green agriculture. Improving the utilization rate of pesticides in crops is the key to reducing the amount of pesticides and increasing their efficiency. At present, the research on improving the effective utilization rate of pesticides is mainly to improve the types of pesticide formulations, improve the performance of plant protection machinery, promote advanced pesticide application processes, etc., and to increase the deposition of liquid pesticides on the surface of plant leaves to improve the effective utilization rate of pesticides. However, the effect of microorganisms in the rhizosphere soil on crop absorption of pesticides is ignored. In comparison, the microbial resources in the rhizosphere of crops are extremely rich, and some rhizosphere bacteria can affect the absorption and utilization of exogenous substances by crops, and even affect the absorption and utilization of pesticides by crops. Therefore, based on the regulatory effect of rhizosphere bacteria on the absorption and utilization of exogenous substances by crops and the promotion of crop growth, the research and development of dual-effect microbial agents that promote corn growth and pesticide absorption and their field application technology have a significant effect on improving the effective utilization rate of pesticides, and the application prospects are very broad, which is worthy of in-depth research and development.

[0004] Patent WO2023138678A1 discloses a composition comprising Bacillus Velezii M173 and thiamethoxam, etc. The pesticide composition has multiple uses, such as use in improving plant resistance to pathogens or adverse conditions, or use in promoting plant growth; and use in preventing and / or treating plant diseases caused by pathogens, or use in preventing and / or alleviating plant damage or necrosis caused by non-pathogenic conditions. Among them, the combined use of Bacillus Velezii and thiamethoxam has been commonly reported in promoting healthy plant growth, but there has been no report on the use of Bacillus Velezii as a thiamethoxam synergist. Summary of the invention

[0005] The purpose of the present invention is to overcome the above defects and shortcomings in the prior art and provide the use of Bacillus velez as a thiamethoxam synergist.

[0006] The second object of the present invention is to provide the use of Bacillus Velez in the preparation of a dual-effect bacterial agent for promoting plant growth and plant absorption and utilization of thiamethoxam.

[0007] The third object of the present invention is to use a microbial agent in promoting plant growth and promoting plant absorption and utilization of thiamethoxam.

[0008] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0009] The present invention finds that after corn is treated with a Bacillus Velezii liquid and then transplanted into soil applied with thiamethoxam, the absorption and utilization of thiamethoxam by corn can be promoted, the transport and enrichment of thiamethoxam to the aboveground part of corn can be enhanced, the content of thiamethoxam in the roots and leaves of corn can be significantly increased, the insecticidal effect of thiamethoxam can be enhanced, the effective utilization rate of pesticides can be improved, and the growth of corn can be significantly promoted.

[0010] Therefore, the present invention provides the use of Bacillus Velezii as a thiamethoxam synergist.

[0011] Furthermore, the thiamethoxam synergist has at least one of the following functions (1) to (2):

[0012] (1) Promote plant absorption of thiamethoxam;

[0013] (2) Enhance the transport and accumulation of thiamethoxam to the aboveground parts of plants.

[0014] The present invention finds that the treatment of adding Bacillus Velez subtilis liquid and applying thiamethoxam significantly increases the plant height, aboveground fresh weight and leaf area of ​​corn, indicating that the combination of the strain and thiamethoxam has a significant promoting effect on corn growth; at the same time, the content of thiamethoxam in the roots and leaves of corn can be significantly increased, indicating that Bacillus Velez subtilis can promote the absorption and utilization of thiamethoxam by corn.

[0015] Therefore, the present invention also provides the use of Bacillus Velezii in preparing a dual-effect bacterial agent for promoting plant growth and plant absorption and utilization of thiamethoxam.

[0016] Furthermore, the application is to use the Bacillus Velez subtilis liquid to irrigate or dip the roots of plants, and apply thiamethoxam at the same time.

[0017] Preferably, the plant is planted with a Bacillus Velezii solution for root irrigation. The use of microbial agents in corn fields by root irrigation and root dipping significantly increased the content of thiamethoxam in corn leaves, indicating that microbial agents have a good promoting effect on corn absorption of thiamethoxam under farmland environmental conditions, and the effect of applying microbial agents by root irrigation is better.

[0018] Furthermore, the dosage of the Bacillus Velezii liquid is 8-12 mL, and the amount of live bacteria is 1×10 8 ~4×10 9 CFU / mL.

[0019] Furthermore, the dosage of thiamethoxam is 1.8 to 2 mg / kg.

[0020] The present invention further uses MSM culture medium with four carbon sources of corn flour, soybean meal, wheat flour and soybean flour to ferment Bacillus Velez, and finds that at 6 to 10 days, the number of effective live bacteria of Bacillus Velez in the microbial fermentation medium using soybean flour as the carbon source is the largest; 5% soybean flour addition is the most suitable carbon source addition amount for the microbial fermentation medium; 0.1% sodium alginate solution has the best protective effect on the strains in the bacterial agent.

[0021] Therefore, the present invention also provides an application of a microbial agent in promoting plant growth and promoting plant absorption and utilization of thiamethoxam. The microbial agent uses Bacillus Velezii as the only active ingredient and also comprises a basal salt culture medium, a carbon source material soybean powder and a protective agent sodium alginate.

[0022] Furthermore, the carbon source material is 5% soybean powder; and the protective agent is 0.1% sodium alginate.

[0023] Furthermore, the amount of live bacteria of Bacillus Velez in the microbial agent is 1×10 8 ~4×10 9 CFU / mL.

[0024] Furthermore, the Bacillus velezensis is Bacillus velezensis

[0025] Furthermore, the plant is corn.

[0026] Furthermore, the plant absorbs thiamethoxam and transports and accumulates it to various parts of the plant. Bacillus Velezii can not only increase the content of thiamethoxam in the roots and leaves of corn, but also increase the pesticide transport coefficient of corn, indicating that the strain can promote corn to absorb thiamethoxam while enhancing the transport and enrichment of thiamethoxam to the aboveground part, thereby enhancing the insecticidal effect of thiamethoxam.

[0027] Specifically, 10 mL of bacterial agent (with a live bacterial count of about 2×10 9 CFU / mL), thiamethoxam was applied at the recommended dose of 2 mg / kg, and the utilization rate of thiamethoxam in corn increased by 43.94-97.30% after 10 days. Under field environmental conditions, the transplanted corn was treated with root irrigation and root dipping with the bacterial agent diluted 1-200 times, and clean water treatment was used as the control. 40 mg / L thiamethoxam 50 mL was applied to the roots of corn by root irrigation 10 days after transplanting. Ten days after application, the utilization rates of thiamethoxam in corn treated with root irrigation and root dipping increased by 79.86-122.67% and 39.72%-94.15%, respectively.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention provides the application of Bacillus velez as a thiamethoxam synergist. The present invention finds that after corn to which thiamethoxam is applied is treated with Bacillus velez bacterial solution, the absorption and utilization of thiamethoxam by corn is promoted, the transport and enrichment of thiamethoxam to the aboveground part of corn is enhanced, the content of thiamethoxam in the roots and leaves of corn is significantly increased, the effective utilization rate of pesticides is increased, and the growth of corn can be significantly promoted. The present invention provides a new idea for improving the utilization rate of pesticides and provides an example for the development of a microbial agent for efficient utilization of pesticides. The microbial agent can be used as a thiamethoxam synergist in promoting crop growth and improving the utilization rate of pesticides, and has a wide application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The growth curves of three growth-promoting bacteria are shown in Figure 2. Figure 1 A is Bacillus velez 20025; B is Rhodococcus 20603; C is methylotrophic Bacillus 20839.

[0031] Figure 2 It is the corn growth index of the root zone growth-promoting bacteria experiment. Figure 2 A is the height of corn plant; B is the fresh weight of corn aboveground part; C is the SPAD value of corn leaf; D is the leaf area of ​​corn leaf. Note: CK represents the culture without adding root zone growth-promoting bacteria, Baillius velezensis represents the culture with the addition of Velez bacillus liquid, Rhodococcus sp represents the culture with the addition of Rhodococcus liquid, and Bacilius methylotrophicus represents the culture with the addition of methylotrophic bacillus liquid. Different lowercase letters indicate significant differences in corn growth indicators at the same time (p<0.05, one-way analysis of variance).

[0032] Figure 3 The effect of growth-promoting bacteria on the content of thiamethoxam in maize roots and leaves. Figure 3A is the thiamethoxam content in the corn root; B is the thiamethoxam content in the corn leaf. Note: CK represents the culture without adding growth-promoting bacteria to the root zone, Bailliusvelezensis represents the culture with the addition of Bacillus velezensis, Rhodococcus sp represents the culture with the addition of Rhodococcus sp, and Bacilius methylotrophicus represents the culture with the addition of methylotrophic Bacillus sp. Different lowercase letters indicate that the thiamethoxam concentrations in the corn root and leaf at the same time are significantly different (p<0.05, one-way ANOVA).

[0033] Figure 4 is the maize pesticide transport coefficient of the root zone growth-promoting bacteria experiment. Note: CK represents the culture without the addition of root zone growth-promoting bacteria, Baillius velezensis represents the culture with the addition of Bacillus velezensis, Rhodococcus sp represents the culture with the addition of Rhodococcus, and Bacilius methylotrophicus represents the culture with the addition of methylotrophic Bacillus. Different lowercase letters indicate that the maize pesticide transport coefficient is significantly different at the same time (p<0.05, one-way ANOVA).

[0034] Figure 5 The effect of different carbon sources on the viable count of Bacillus velez. Note: PDB stands for potato liquid medium, and MSM stands for basal salt medium.

[0035] Figure 6 The effect of different soybean powder addition amounts on the viable count of Bacillus Velez. Note: 1%, 3%, 5%, 7% and 9% represent the culture of basal salt medium with different mass fractions of soybean powder.

[0036] Figure 7 Note: H1, H3 and H5 represent the treatment with 0.1%, 0.3% and 0.5% sodium alginate solution, M1, M3 and M5 represent the treatment with 1%, 3% and 5% skim milk powder solution, S1, S3 and S5 represent the treatment with 0.1%, 0.3% and 0.5% sodium carboxymethyl cellulose solution.

[0037] Figure 8 It is the corn growth index of the microbial agent pot experiment. Note: CK represents the blank control group, and "agent" represents the cultivation with the addition of microbial agents. "*" indicates that the corn plant height, aboveground fresh weight, SPAD value and leaf area of ​​the treatment group and the control group at the same time are significantly different (p<0.05, two-way analysis of variance).

[0038] Fig. 9The thiamethoxam content of corn leaves in the microbial agent pot experiment. Note: CK represents the blank control group, and "microbial agent" represents the culture with the addition of microbial agents. "*" indicates that the thiamethoxam content of corn leaves in the treatment group and the control group at the same time is significantly different (p<0.05, two-way analysis of variance).

[0039] Fig.10 It is the corn growth index of the microbial agent field test. Note: CK represents the culture without adding microbial agents, "Y" represents the microbial agent root irrigation treatment, and "P" represents the microbial agent root dipping treatment. Different lowercase letters indicate that the corn plant height, aboveground fresh weight, leaf SPAD value and leaf area at the same time are significantly different (p<0.05, one-way analysis of variance).

[0040] Fig.11 This is a comparison chart of corn growth effects in the field test of microbial agents. Note: CK represents cultivation without adding microbial agents, "Y" represents root irrigation treatment with microbial agents, and "P" represents root dipping treatment with microbial agents.

[0041] Fig.12 The thiamethoxam content of corn leaves in the field experiment of microbial agents. Note: CK represents the culture without adding microbial agents, "Y" represents the root irrigation treatment of microbial agents, and "P" represents the immersion treatment of microbial agents. Different lowercase letters indicate that the thiamethoxam content of corn leaves at the same time is significantly different (p<0.05, one-way analysis of variance). DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0043] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0044] 1. Materials

[0045] 1.1 Culture medium

[0046] Basal salt medium: (NH 4 ) 2 SO 4 2g, MgSO 4 7H 2 O 0.2g, CaCl 2 ·2H 2 O 0.01g, FeSO 4 7H 2 O0.001g, Na 2 HPO 4 12H 2O 1.5g, KH 2 PO 4 1.5g, H 2 O 1L.

[0047] Luria-Bertani medium: trypsin 10 g, yeast extract 5 g, NaCl 10 g, H 2 O 1 L. When preparing solid culture medium, add 2% agar to the above medium.

[0048] Potato dextrose medium (PDB): 5 g potato extract, 20 g glucose, H 2 O 1L. When preparing solid culture medium, add 2% agar to the above medium. The final pH of all media is adjusted to 7 and then sterilized by autoclave at 121℃ for 20min.

[0049] 1.2 Drug and reagent configuration

[0050] Thiamethoxam technical drug: purity 95%, purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0051] Thiamethoxam water dispersible granules: active ingredient content 25%, registration number: PD20171923, purchased from Shandong Bainongsida Biotechnology Co., Ltd.

[0052] Mass spectrometry grade methanol, mass spectrometry grade acetonitrile, mass spectrometry grade formic acid, purifier N-propylethylenediamine (PSA), and graphitized carbon black (GCB) were all purchased from Shanghai Anpu Technology Co., Ltd.; sodium chloride was purchased from Tianjin Damao Chemical Reagent Factory; sodium alginate, carboxymethyl cellulose, skimmed milk powder, and urea were all purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; tryptone and yeast extract were purchased from OXOID, and agarose was purchased from Beijing Qingke Biotechnology Co., Ltd.

[0053] Preparation of thiamethoxam mother solution: Weigh 0.1 g of thiamethoxam original drug, add organic solvent acetonitrile to fully dissolve it, and finally make up to volume in a 100 mL brown bottle. The concentration of the mother solution is 1000 mg / L.

[0054] 0.9% saline: Accurately weigh 9 g NaCl, add 1000 mL distilled water, stir with a glass rod to make it fully dissolved, pour into a volumetric flask, seal it and sterilize it at high temperature.

[0055] 0.1% formic acid aqueous solution: Add 1 mL of formic acid and dissolve in 1000 mL of triple distilled water.

[0056] 1.3 Test instruments and equipment

[0057] Table 1 Main test instruments and their manufacturers

[0058]

[0059] 1.4 Test crops and soil

[0060] The test crop was corn, the variety was Huameitian 368, purchased from Guangdong Hua Nong University Seed Co., Ltd.

[0061] The soil for the potted plant experiments was collected from the Zengcheng Experimental Base of South China Agricultural University. During the collection process, 5-20 cm of soil was randomly selected and then dried, crushed and sieved (8 mesh) to remove plant impurities, animal remains and other impurities.

[0062] The field experiment was carried out at the Zengcheng Experimental Base of South China Agricultural University.

[0063] 1.5 List of tested strains

[0064] Table 2 List of nitrogen-fixing strains

[0065]

[0066] Example 1 Determination of the growth curve of root zone growth-promoting bacteria

[0067] 1. Experimental Methods

[0068] (1) Determination of the growth curve of root-zone growth-promoting bacteria

[0069] For the three types of root zone growth-promoting bacteria (nitrogen-fixing bacteria) collected, pick out the isolated and purified single colonies in PDB medium and culture them for 24 hours. After the obtained bacterial solution is centrifuged at 4000r / min, the supernatant is discarded, and it is rinsed with 0.9% saline and resuspended so that the OD600 value is 1 as an inoculum. Transfer the inoculum to a freshly sterilized LB liquid culture medium and set up three replicates. Culture it in a constant temperature shaker at 30°C and 180r / min. Measure the OD600 of the culture solution every 4 hours until 36 hours. Draw the growth curve of the strain with the OD600 value as the vertical axis and the culture time as the horizontal axis.

[0070] 2. Experimental Results

[0071] By testing the OD600 value of microorganisms for 36 hours, the growth curves of three growth-promoting bacteria were obtained as follows: Figure 1As shown. The lag phase of Bacillus Velezii lasts from 0 to 4 hours, which reserves sufficient enzymes and energy for cell volume increase, metabolic activities, cell proliferation, etc. The logarithmic growth phase lasts from 4 to 20 hours, during which bacteria consume nutrients in the culture medium for rapid metabolic activities, rapidly proliferate and expand the number of bacteria. The stable phase lasts from 20 to 52 hours, and the bacterial reproduction rate decreases due to the consumption of nutrients in the culture medium and the accumulation of toxic substances produced by metabolism. The lag phase of Rhodococcus lasts from 0 to 4 hours, the logarithmic growth phase from 4 to 24 hours, and the stable phase from 24 to 52 hours. The lag phase of Methylotrophic Bacillus lasts from 0 to 4 hours, the logarithmic growth phase from 4 to 20 hours, and the stable phase from 20 to 52 hours. The OD600 values ​​of the three bacteria are all approximately 1 when they reach the stable phase.

[0072] Example 2 Effects of root zone growth-promoting bacteria on corn growth and corn absorption and utilization of thiamethoxam

[0073] 1. Experimental Methods

[0074] 1. Sow corn seeds in a seedling tray and raise corn seedlings to the three-leaf stage (plant height of about 10 cm). Weigh 900g of sieved soil, add 1mL of 1800mg / L thiamethoxam preparation and 50mL of distilled water in a conical flask, mix well and add to the soil to make the initial concentration of thiamethoxam in the soil reach 2mg / kg. Put the mixed soil into a flowerpot (diameter 13.5cm, depth 13cm), select 4 corn seedlings with uniform growth and transplant them into the flowerpot, plant 6 pots for each treatment, and place all flowerpots outdoors for cultivation. Check the soil moisture regularly and water it manually to maintain humidity. The experiment set up a control (CK) and three nitrogen-fixing bacteria treatments: after corn transplanting, the three nitrogen-fixing bacteria were cultured with PDB medium for 24h, and the OD600 value of the bacterial suspension was adjusted to 1. Then, the bacterial suspension was applied to the roots of corn by root irrigation. 10mL of bacterial suspension was applied to each corn seedling, and 3 replicates were set for each treatment.

[0075] Samples were collected at 7d, 10d, 12d, and 14d after corn transplanting. Corn plant samples and corn root zone soil were collected. The corn roots were washed 4 times with distilled water to ensure that the soil and drugs attached to the root surface were completely removed. The surface moisture of the plant was wiped dry with filter paper, and then the corn growth index and the thiamethoxam content in the corn plant and soil were measured.

[0076] 2. Determination of corn growth indicators

[0077] Whole corn plants were randomly collected, washed with distilled water, and the surface moisture was absorbed with filter paper, and the corn plants were divided into the aboveground part and the root part.

[0078] Use a ruler to measure the plant height and root elongation of corn. The plant height refers to the distance from the growth point at the base of the stem to the tip of the uppermost expanded leaf, and the root elongation refers to the distance from the tip of the longest root to the growth point at the base of the stem.

[0079] The fresh weight of the aboveground part and root of rice was measured using an analytical balance.

[0080] The SPAD value of corn leaves was measured using a SPAD instrument. The measurements were performed three times and the average value was taken.

[0081] The leaf area of ​​each corn leaf was measured using a leaf area meter, and then the total leaf area of ​​all corn leaves was calculated.

[0082] 3. Thiamethoxam detection

[0083] (1) Thiamethoxam detection method

[0084] Waters ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) detection, Waters ACQUITY column HSS C18 (2.1mm×100mm, 1.8μm), column temperature 40℃, flow rate 0.2mL / min, injection volume 1μL, mobile phase methanol (A) and 0.1% formic acid water (B), elution mode using gradient elution, the program is shown in Table 3. XEVO TQD triple quadrupole was used for mass spectrometry analysis, electrospray ion source (ESI) was used in positive ion mode, multiple reaction monitoring (MRM) was used, capillary voltage 1.5KV, cone voltage 39V, desolvation temperature 350℃, desolvation gas flow rate 650L / Hr. The parent ion of thiamethoxam mass spectrometry monitoring was 292m / z, and the daughter ions were 181.09 and 211.01m / z. The collision energies were 23 and 12V, respectively, of which 292 / 181.09m / z was the quantitative ion pair.

[0085] Table 3 Mobile phase ratio and flow rate for UPLC detection

[0086]

[0087] (2) Standard curve and linear range

[0088] The concentration of thiamethoxam was detected by the external standard method, and the concentration was related to the liquid phase peak area by a linear equation. The mother solution of thiamethoxam was diluted with acetonitrile to prepare five concentrations of 0.5, 0.1, 0.05, 0.01, and 0.005 mg / L. The standard curve was drawn with the peak area as the ordinate and the concentration of the standard solution as the abscissa, and the regression equation was fitted.

[0089] (3) Thiamethoxam extraction methods for different samples

[0090] Soil: Accurately weigh 5 g of soil into a 50 mL centrifuge tube, add 10 mL of mass spectrometry acetonitrile to the centrifuge tube and vortex for 1 min, then put it into an ultrasonic cleaner for 30 min, add 3 g of sodium chloride, vortex for 1 min, centrifuge at 5000 rpm for 3 min at room temperature, aspirate the supernatant with a disposable syringe, filter it through a 0.22 μm microporous organic filter membrane, and collect it in a brown injection bottle for testing.

[0091] Corn leaves: Accurately weigh 0.5g of sample into a 5mL grinding tube, add grinding beads, add 3mL of mass spectrometry acetonitrile, oscillate for 1min, and ultrasonically extract for 30min. Add 1.0g of sodium chloride, oscillate for 1min, and centrifuge at 9000rpm for 3min. Take 1mL of supernatant into a 2mL test tube containing 50mg PSA and 10mg GCB, oscillate for 1min, centrifuge at 10000rpm for 5min, and use a disposable syringe to absorb the centrifugal supernatant, filter it through a 0.22μm microporous organic filter membrane, and collect it in a brown injection bottle for testing.

[0092] Corn root: Accurately weigh 0.5g sample into a 5mL grinding tube, add grinding beads, add 3mL mass spectrometry acetonitrile, oscillate for 1min, and ultrasonically extract for 30min. Add 1.0g sodium chloride, oscillate for 1min, and centrifuge at 9000rpm for 3min. Take 1mL of supernatant into a 2mL test tube containing 50mg PSA, oscillate for 1min, centrifuge at 10000rpm for 5min, and use a disposable syringe to absorb the centrifugal supernatant, filter it through a 0.22μm microporous organic filter membrane, and collect it in a brown injection bottle for testing.

[0093] (4) Determination of recovery rate of thiamethoxam addition

[0094] Take soil, corn leaf and root samples that are not contaminated by pesticides, weigh them into 50mL centrifuge tubes according to the mass requirements of each component in 2.3, and add 10μL of thiamethoxam standard solution of different concentrations into the centrifuge tubes, so that the final concentration of thiamethoxam in the soil reaches 5, 0.5, and 0.05mg / L, respectively, and the final concentration of thiamethoxam in the corn leaf and root reaches 5, 0.5, and 0.05mg / kg, respectively. Extract and determine according to the above method, and calculate the added recovery rate.

[0095] 2. Experimental Results

[0096] 1. By adding bacterial solutions of different nitrogen-fixing strains to the roots of corn, the differences in corn growth indicators (plant height, aboveground fresh weight, leaf SPAD value and leaf area) treated with different nitrogen-fixing bacteria were compared to clarify the effects of root-zone growth-promoting bacteria on corn growth. Figure 2It can be seen that compared with the blank control group, the height of corn increased by 7.61-16.63% within 7-11 days after treatment with Bacillus Velez, and the height of corn increased by 7.33% after 7 days of treatment with methylotrophic Bacillus Velez; the aboveground fresh weight of corn increased by 28.38% and 16.35% after 9 and 13 days of treatment with Bacillus Velez; the leaf area of ​​corn increased by 20.58% and 11.82% after 9 and 11 days of treatment with Bacillus Velez. The treatment with Bacillus Velez significantly increased the plant height, aboveground fresh weight and leaf area of ​​corn, indicating that the strain has a significant promoting effect on corn growth.

[0097] 2. In order to explore the effect of nitrogen-fixing root-zone growth-promoting bacteria on the absorption of pesticides, different nitrogen-fixing strains of bacteria were added to the roots of corn, and the differences in the concentration of thiamethoxam in corn plants treated with different nitrogen-fixing bacteria were compared to clarify the effect of root-zone growth-promoting bacteria on the absorption of pesticides by corn. Figure 3 It can be seen that compared with the blank control group, the thiamethoxam content in the corn root treated with Bacillus Velez increased by 114.25% at 13 days after the drug application, and the thiamethoxam content in the corn root treated with Rhodococcus increased by 20.23% and 135.37% at 9 and 13 days after the drug application; the thiamethoxam content in the corn leaf treated with Bacillus Velez increased by 26.34-35.39% within 9-13 days after the drug application. Adding Bacillus Velez bacterial solution can significantly increase the content of thiamethoxam in the roots and leaves of corn, indicating that Bacillus Velez can promote the absorption and utilization of thiamethoxam by corn. In summary, Bacillus Velez can promote the absorption and utilization of thiamethoxam by corn while promoting the growth of corn.

[0098] 3. By analyzing the pesticide transport coefficient of thiamethoxam in corn plants, explore whether adding root zone growth-promoting bacteria affects the transport capacity of corn for thiamethoxam. Figure 4 It can be seen that compared with the blank control group, the pesticide transport coefficient of the treatment with Bacillus Velezii increased by 40.41% at 9 days after the application, and the pesticide transport coefficient of the treatment with methylotrophic Bacillus Velezii increased by 62.17% at 11 days after the application. This shows that these two growth-promoting bacteria can increase the pesticide transport coefficient of corn and promote the transport of thiamethoxam in the root system of corn to the aboveground part. Bacillus Velezii can not only increase the content of thiamethoxam in the roots and leaves of corn, but also increase the pesticide transport coefficient of corn, indicating that the strain can promote the absorption of thiamethoxam by corn while enhancing the transport and enrichment of thiamethoxam to the aboveground part, thereby enhancing the insecticidal effect of thiamethoxam.

[0099] 4. As can be seen from Table 4, the recovery rates of thiamethoxam in soil, corn roots and corn leaves were 81.84-97.88%, 85.02-96.51% and 93.08-107.46%, respectively, and the relative standard deviations (RSDs) were all less than 10%, which met the requirements of "NY / T 788-2018 Experimental Guidelines for Pesticide Residues in Crops", indicating that the extraction method was stable and reliable and could be used for the extraction of thiamethoxam in soil and corn plants.

[0100] Table 4 Thiamethoxam UPLC-MS detection recovery

[0101]

[0102] Example 3 Screening of Carbon Sources in Microbial Fermentation Medium

[0103] 1. Experimental Methods

[0104] 1. Selection of carbon source for microbial agents

[0105] A single factor experiment was set up with corn flour, soybean flour, bagasse, soybean meal, and wheat flour as carbon sources and potato liquid medium (PDB) as a control. Five carbon sources with a mass fraction of 5% were added to the MSM medium, and then 1 mL of nitrogen-fixing bacteria resuspended bacterial solution (OD600 value was 1) was inoculated into the MSM medium and PDB medium added with various carbon sources, and cultured in a shaker at 30°C and 150rpm for 4d, 6d, 8d, and 10d. The effective viable number of nitrogen-fixing bacteria in the culture medium was determined by the dilution plate method. Three replicates were set for each treatment.

[0106] 2. Selection of the amount of carbon source added to microbial agents

[0107] Select the most suitable carbon source and set up a single factor experiment by changing its addition amount. Add carbon source substances with mass fractions of 1%, 3%, 5%, 7%, and 9% to MSM medium, then inoculate 1mL of resuspended bacterial solution (OD600 value is 1), and culture in a shaker at 30°C and 150rpm for 1d, 2d, 3d, and 4d. Use the dilution plate method to determine the effective viable number of nitrogen-fixing bacteria in the culture medium. Set up 3 replicates for each treatment.

[0108] 3. Effects of different protective agents on strain preservation

[0109] Use 5% soybean powder with MSM culture medium as the fermentation broth of the microbial agent. After 4 days of cultivation, take 30mL of the fermentation broth into a sterilized 50mL centrifuge tube, centrifuge at 5000r / min for 3min, discard the supernatant, wash twice with sterile water, and collect the bacteria.

[0110] The protective agent sodium carboxymethyl cellulose (CMC) was prepared into solutions with concentrations of 0.1%, 0.3%, and 0.5%; the protective agent sodium alginate was prepared into solutions with concentrations of 0.1%, 0.3%, and 0.5%; the protective agent skimmed milk powder was prepared into solutions with concentrations of 1%, 3%, and 5%, and sterilized in a high-pressure steam sterilizer at 121°C for 20 minutes. 15mL of various sterilized protective agent solutions were injected into the bacteria, shaken for 15-20 minutes to break up the bacteria and mix them thoroughly with the protective agent, and stored at room temperature. Samples were taken on the 1st, 3rd, 5th, and 7th days, and the OD600 values ​​were measured. Three replicates were set for each treatment.

[0111] 2. Experimental Methods

[0112] 1. In order to screen the most suitable carbon source for the fermentation of Bacillus Velez 20025, potato liquid medium (PDB) was used as a control, and 5% of corn flour, soybean meal and other 5 carbon sources were added to the basal salt medium (MSM). The most suitable carbon source was determined by comparing the effective viable bacteria counts in various culture media. Figure 5 As shown in the figure, compared with PDB medium, the fermentation of Bacillus Velez 20025 using MSM medium with four carbon sources of corn flour, soybean meal, wheat flour and soybean flour can significantly increase the number of effective viable bacteria of Bacillus Velez in the fermentation broth. At 4 days of fermentation, the number of effective viable bacteria of Bacillus Velez in the microbial fermentation medium with soybean meal as the carbon source was the highest, which was 2.93×10 9 CFU / mL; at 6d, 8d and 10d of fermentation, the number of effective viable bacteria of Bacillus Velez in the microbial fermentation medium with soybean powder as the carbon source was the highest, which was 2.73×10 9 CFU / mL, 3.58×10 9 CFU / mL and 3.85×10 9 CFU / mL. After comprehensive consideration, soybean powder was selected as the carbon source of microbial fermentation medium, and used with basal salt medium as the initial fermentation medium for Bacillus Velezii.

[0113] 2. After determining that soybean powder is the best carbon source for the fermentation medium of Bacillus Velez, in order to explore the addition amount of the most suitable carbon source for the fermentation of Bacillus Velez, different amounts of soybean powder were added to the basal salt medium, and the effective viable bacteria count in the medium with different mass fractions of soybean powder was compared to determine the most suitable amount of carbon source addition. Figure 6 It was found that at 3 days of fermentation, the effective viable bacteria count of the culture medium with an addition amount of 1-5% reached a peak value of 8.60×10 8 CFU / mL, 2.12×10 9 CFU / mL and 3.18×109 CFU / mL, and remained stable in the following period. At 4 days of fermentation, the effective viable bacteria count of the culture medium with an addition amount of 7% reached a peak of 3.02×10 9 CFU / mL, and remained stable in the following period; at 5 days, the effective viable count of the culture medium with an addition of 9% reached a peak of 3.74×10 9 CFU / mL, and remained stable in the following period. During the 1st to 4th day of fermentation, the number of effective viable bacteria in the medium with a 5% addition was significantly higher than that in the medium with other additions; during the 5th to 7th day of fermentation, there was no significant difference between the medium with a 5% addition and the 7% addition, while the number of effective viable bacteria in the medium with a 9% addition was significantly higher than that in the medium with other additions. Comprehensively considered, the addition of 5% soybean powder by mass is the most suitable addition of carbon source material to the microbial fermentation medium. It was determined that the use of 5% soybean powder by mass with the basal salt medium was used as the initial fermentation medium for Bacillus Velezii.

[0114] 3. After fermenting Bacillus Velez with 5% soybean powder and basal salt medium as the initial fermentation medium for 4 days, a protective agent was added to the fermentation liquid to extend the shelf life of the microbial agent and prepare it into a microbial agent. In order to explore the effects of different types and concentrations of protective agents on the preservation of strain 20025 in the microbial agent, the most suitable concentration and type of protective agent were determined by comparing the changes in OD600 values ​​after adding different concentrations of three protective agents - sodium alginate, skimmed milk powder and sodium carboxymethyl cellulose. Figure 7 It can be seen that the OD600 value of 0.1% sodium alginate treatment is the highest, and is much higher than other concentrations and types of protective agents, indicating that 0.1% sodium alginate solution has the best protective effect on strain 20025 in the bacterial agent.

[0115] Example 4 Effect of microbial agents on corn absorption of thiamethoxam

[0116] 1. Experimental Methods

[0117] Sow corn seeds in a seedling tray and raise corn seedlings to the three-leaf stage (plant height of about 10 cm). Weigh 900g of sieved soil, add 1mL of 1800mg / L thiamethoxam preparation and 50mL of distilled water in a conical flask, mix well and add to the soil to make the initial concentration of thiamethoxam in the soil reach 2mg / kg. Put the mixed soil into a flowerpot (diameter 13.5cm, depth 13cm), select 4 corn seedlings with uniform growth and transplant them into the flowerpot, plant 6 pots for each treatment, and all flowerpots are placed outdoors for cultivation. Check the soil moisture regularly and water it manually to maintain humidity. The experiment sets up a control (CK) and three nitrogen-fixing bacteria treatments: after corn transplanting, the microbial agent is diluted 20 times and then applied to the roots of corn by root irrigation. 10mL of bacterial solution is applied to each corn seedling, and 3 replicates are set for each treatment.

[0118] Samples were collected 7, 10, 12, and 14 days after corn transplanting. Corn plant samples and soil from the corn root zone were collected. The corn roots were washed 4 times with distilled water to ensure that the soil and drugs attached to the root surface were completely removed. The surface moisture of the plant was wiped dry with filter paper, and then the corn growth index was measured to extract the pesticide thiamethoxam from the corn plants.

[0119] 2. Experimental Results

[0120] In order to explore the effect of microbial agents on corn growth, treatments with (or without) added agents were set up, and 10 mL of 20-fold diluted microbial agents were added to the roots of each corn plant by root irrigation. The differences in corn growth indicators (plant height, fresh weight, leaf SPAD value, and leaf area) were compared to clarify the growth-promoting effect of microbial agents on corn growth. Figure 8 As shown in the results, compared with the blank control group, the height of corn increased by 15.20%, 8.93% and 8.09% at 7d, 10d and 14d after the treatment with microbial agents; the fresh weight of the aboveground part of corn increased by 17.81-27.29% at 10-14d after the treatment; the SPAD value of corn leaves increased by 5.62-10.88% at 10-14d after the treatment; and the leaf area of ​​corn leaves increased by 26.48-34.09% at 10-14d after the treatment. The addition of microbial agents significantly increased the plant height, aboveground fresh weight, leaf SPAD value and leaf area of ​​corn, indicating that microbial agents have a significant promoting effect on the growth of corn.

[0121] In order to explore the effect of microbial agents on the absorption of pesticides by corn, we set up treatments with (or without) the addition of microbial agents, added 10 mL of 20-fold diluted microbial agents to the roots of each corn plant by root irrigation, and compared the content of thiamethoxam in corn leaves to clarify the effect of microbial agents on the absorption of pesticides by corn. Fig. 9As shown in the figure, after the application of thiamethoxam, the concentration of thiamethoxam in corn leaves showed a trend of first rising and then falling. With the increase of the application time, the concentration of thiamethoxam accumulated in corn leaves gradually increased, reaching a peak at about 10 days, and then gradually decreased. Compared with the blank control group, the thiamethoxam content of corn leaves treated with microbial agents increased by 43.94-97.30% within 10-12 days after the application. The addition of microbial agents can significantly increase the content of thiamethoxam in corn leaves, indicating that Bacillus Velezii can promote the absorption and utilization of thiamethoxam by corn. In summary, microbial agents can promote the absorption and utilization of thiamethoxam by corn while promoting the growth of corn. Example 5 Field verification of the function of microbial agents

[0122] 1. Experimental Methods

[0123] Corn seeds were sown in seedling trays, and after the corn was grown to the three-leaf stage (plant height of about 10 cm), the corn with consistent growth was transplanted to the farmland in Zengcheng. The field experiment set up three treatments, namely blank control, root irrigation treatment with bacterial agent (Y), and root dipping treatment with bacterial agent (P). Two plots were set up for each treatment. Each plot was 15m long and 0.8m wide, with a corn row spacing of 65cm and a plant spacing of 30cm. A protective row with a length of 2m was set between the plots.

[0124] Root irrigation treatment with bacterial agent: After corn is transplanted, apply 50 mL of 20-fold diluted bacterial agent to the roots of each corn plant.

[0125] Root treatment with fungal agent: Before transplanting corn, soak the corn in 20 times diluted fungal agent. After soaking for 30 minutes, transplant the corn seedlings into the farmland.

[0126] After the treatment, water each corn plant with 150 mL of water. 10 days after the bacterial agent treatment, add 1.2 g of 25% water-dispersible granules of thiamethoxam to 18 L of water to make a 40 mg / L thiamethoxam solution (recommended dosage for corn fields), and irrigate the roots of each corn plant with 50 mL of 40 mg / L thiamethoxam solution.

[0127] Samples were collected at 10, 12, 14, and 16 days after application. Corn plant samples and soil from the corn root zone were collected. The corn roots were washed 4 times with distilled water to ensure that the soil and drugs attached to the root surface were completely removed. The surface moisture of the plants was wiped dry with filter paper, and then the corn growth index was measured, and the thiamethoxam content in the corn plants was tested.

[0128] 2. Experimental Results

[0129] In order to explore the effect of microbial agents on corn growth under farmland environmental conditions, the pesticides were applied with or without the addition of microbial agents, and the differences in corn growth indicators were compared to clarify the growth-promoting effect of microbial agents on corn growth under field conditions. Fig.10 As shown in the figure, compared with the control group, the height of corn plants in the treatment of microbial root irrigation increased by 12.14-20.22% within 10-16 days after the application of microbial agents, and the height of corn plants in the treatment of microbial root dipping increased by 11.21-20.03% within 10-16 days after the application of microbial agents; the fresh weight of the aboveground part of corn in the treatment of microbial root irrigation increased by 42.57-51.20% within 10-16 days after the application of microbial agents, and the fresh weight of the aboveground part of corn in the treatment of microbial root dipping increased by 35.90-69.8 1%; the SPAD value of corn leaves increased by 10.38-17.16% in the treatment of root irrigation after application of microorganisms, and the SPAD value of corn leaves increased by 12.14-17.22% in the treatment of root dipping after application of microorganisms; the leaf area of ​​corn leaves increased by 42.27-54.92% in the treatment of root irrigation after application of microorganisms, and the leaf area of ​​corn leaves increased by 41.59-51.86% in the treatment of root dipping after application of microorganisms. The use of microorganisms in corn fields by root irrigation and root dipping significantly increased the plant height, aboveground fresh weight, leaf SAPD value and leaf area of ​​corn, indicating that microorganisms have the function of promoting corn growth.

[0130] like Fig.11 As shown in the figure, after the application of the pesticide, the corn plants treated with the bacterial solution root irrigation had the best growth, followed by the corn plants treated with the bacterial solution root dipping, and the corn plants in the water control group had the worst growth. This shows that the two microbial agent treatments promoted the growth of corn under field conditions.

[0131] In order to explore the effect of microbial agents on the absorption of pesticides by corn under farmland environmental conditions, the pesticides were applied with or without the addition of microbial agents, and the content of thiamethoxam in corn leaves was compared to clarify the effect of microbial agents on the absorption of pesticides by corn under field conditions. Fig.12As shown in the figure, after applying thiamethoxam, the thiamethoxam concentration in corn leaves showed a trend of gradual decline. It is speculated that under farmland environmental conditions, corn absorbs and utilizes thiamethoxam more efficiently, and the thiamethoxam concentration in corn leaves reaches a peak before 10 days. Therefore, within 10 to 16 days, the thiamethoxam concentration in the leaves gradually decreases with time. Compared with the blank control group, the thiamethoxam content in corn leaves increased by 79.86% to 122.67% within 10 to 16 days after the microbial agent was irrigated with roots, and the thiamethoxam content in corn leaves increased by 39.72% to 94.15% within 10 to 16 days after the microbial agent was dipped in roots. The use of microbial agents in corn fields by root irrigation and root dipping significantly increased the content of thiamethoxam in corn leaves, indicating that microbial agents have a good promoting effect on corn absorption of thiamethoxam under farmland environmental conditions, and the effect of applying microbial agents by root irrigation is better.

Claims

1. Application of Bacillus velez as a thiamethoxam synergist.

2. The use according to claim 1, characterized in that: The thiamethoxam synergist has at least one of the following functions (1) to (2): (1) Promote plant absorption of thiamethoxam; (2) Enhance the transport and accumulation of thiamethoxam to the aboveground parts of plants.

3. Application of Bacillus Velezii in the preparation of a dual-effect bacterial agent that promotes plant growth and plant absorption and utilization of thiamethoxam.

4. The use according to any one of claims 1 to 3, characterized in that: The application is to use the Bacillus Velez bacteria liquid to perform root irrigation or root dipping treatment on the plants, and at the same time apply thiamethoxam.

5. The application according to claim 4, characterized in that: The dosage of the Bacillus Velezii liquid is 8-12 mL, and the amount of live bacteria is 1×10 8 ~4×10 9 CFU / mL.

6. The use according to claim 4, characterized in that: The dosage of thiamethoxam is 1.8-2 mg / kg.

7. Use of a microbial agent in promoting plant growth and promoting plant absorption and utilization of thiamethoxam, characterized in that: The microbial agent uses Bacillus Velezii as the only active ingredient and also comprises a basic salt culture medium, soybean powder as a carbon source and sodium alginate as a protective agent.

8. The use according to claim 7, characterized in that: The carbon source material is 5% soybean powder; the protective agent is 0.1% sodium alginate.

9. The use according to any one of claims 1, 3 or 7, characterized in that: The Bacillus velezensis is Bacillus velezensis 20025.

10. The use according to any one of claims 3 or 7, characterized in that: The plant is corn.

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