Application of Bacillus Velezii as a thiamethoxam synergist

Abstract

The present invention discloses the use of Bacillus velezensis as a thiamethoxam synergist. The present invention finds that treating corn treated with thiamethoxam with a Bacillus velezensis bacterial solution promotes the absorption and utilization of thiamethoxam by the corn, enhances the transport and accumulation of thiamethoxam to the aboveground parts of the corn, significantly increases the thiamethoxam content in the roots and leaves of the corn, improves the effective utilization rate of the pesticide, and promotes corn growth. The present invention provides a new approach to improving pesticide utilization and exemplifies the development of a microbial agent for efficient pesticide utilization. The microbial agent has broad application prospects as a thiamethoxam synergist for promoting crop growth and improving pesticide utilization.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to application of Bacillus velezensis as a thiamethoxam synergist. Background Art

[0002] The use of pesticides protects plants from various pests and diseases, ensuring normal agricultural production. my country is a major user of pesticides. Long-term, excessive, frequent, and unscientific use has increased production costs while leading to widespread pesticide residues in soil and water. This not only undermines soil ecological stability and poses a series of environmental risks, but also accumulates in the edible parts of crops through plant uptake and transport, posing a significant risk to human health. Faced with increasingly stringent pesticide pollution and widespread public concern about the ecological environment, there is an urgent need to develop new, efficient, and safe technologies and methods to improve the effective utilization of pesticides, reduce pesticide pollution, and safeguard the quality of agricultural products and the ecological environment.

[0003] Reducing pesticide use and increasing its efficiency is a key initiative in promoting quality-oriented and green agriculture. Improving pesticide utilization efficiency is crucial for this. Currently, research on improving pesticide efficiency primarily focuses on improving pesticide formulations, enhancing the performance of plant protection machinery, and promoting advanced application techniques. These efforts aim to enhance pesticide deposition on plant leaves, thereby improving pesticide efficiency. However, the impact of microorganisms in the rhizosphere on crop pesticide uptake has been overlooked. The rhizosphere is rich in microbial resources, and some rhizosphere bacteria can influence crop absorption and utilization of exogenous substances, even pesticides. Therefore, leveraging the regulatory effects of rhizosphere bacteria on crop absorption and utilization of exogenous substances and their growth-promoting effects, research and development of dual-action microbial agents that promote corn growth and pesticide uptake, along with field application techniques, could significantly improve pesticide efficiency and hold great promise for future applications, worthy of further research and development.

[0004] Patent WO2023138678A1 discloses a composition comprising Bacillus velez M173 and thiamethoxam, among others. This pesticide composition has a variety of uses, such as improving plant resistance to pathogens or adverse conditions, or promoting plant growth; and preventing and / or treating plant diseases caused by pathogens, or preventing and / or alleviating plant damage or necrosis caused by non-pathogenic conditions. The combined use of Bacillus velez and thiamethoxam has been commonly reported in promoting healthy plant growth, but there have been no reports on the use of Bacillus velez as a thiamethoxam synergist. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and provide the use of Bacillus velezensis 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-action bacterial agent that promotes 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 the absorption and utilization of thiamethoxam by plants.

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

[0009] The present invention finds that treating corn with a Bacillus velezensis liquid and then transplanting it into soil applied with thiamethoxam promotes the absorption and utilization of thiamethoxam by the corn, enhances the transport and accumulation of thiamethoxam to the aboveground part of the corn, significantly increases the content of thiamethoxam in the roots and leaves of the corn, enhances the insecticidal effect of thiamethoxam, improves the effective utilization rate of the pesticide, and can significantly promote the growth of the corn.

[0010] Therefore, the present invention provides the use of Bacillus velezensis 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 found that the treatment of adding a bacterial liquid of Bacillus Velez and applying thiamethoxam significantly increased the plant height, aboveground fresh weight and leaf area of ​​corn, indicating that the combined use of the strain and thiamethoxam has a significant promoting effect on corn growth; at the same time, it 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.

[0015] Therefore, the present invention also provides the use of Bacillus Velezii in the preparation of a dual-action bacterial agent that promotes plant growth and enables plants to absorb and utilize thiamethoxam.

[0016] Furthermore, the application is to use the Bacillus Velez subtilis liquid to perform root irrigation or root dipping treatment on the plants, and to apply thiamethoxam at the same time.

[0017] Preferably, the plants are treated with a Bacillus velezensis solution for root irrigation during planting. Applying microbial agents in corn fields, both through root irrigation and root dipping, significantly increased the thiamethoxam content in corn leaves, indicating that microbial agents have a strong promoting effect on corn's absorption of thiamethoxam under farmland conditions, and that applying microbial agents through root irrigation is more effective.

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

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

[0020] The present invention further uses MSM culture medium with four carbon sources, namely corn flour, soybean meal, wheat flour and soybean flour, to ferment Bacillus Velez. It is found that after 6 to 10 days, the number of effective viable bacteria of Bacillus Velez in the microbial fermentation medium using soybean flour as the carbon source is the highest; 5% soybean flour addition is the most suitable carbon source addition amount for the microbial fermentation medium; and 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 velezensis as the sole active ingredient and further 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 viable Bacillus velezensis 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 absorbed thiamethoxam and transported and accumulated it to various parts of the plant. Bacillus velezensis not only increased the thiamethoxam content in maize roots and leaves, but also improved the pesticide transport coefficient of maize. This suggests that the strain can promote thiamethoxam uptake in maize while enhancing its transport and accumulation to the aboveground parts, thereby increasing its insecticidal efficacy.

[0027] Specifically, 10 mL of bacterial agent (with a live bacterial count of approximately 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 conditions, transplanted corn was treated with root irrigation and root dipping with a 1-200-fold diluted fungal agent, with water treatment as the control. Ten days after transplanting, 50 mL of 40 mg / L thiamethoxam was applied to the roots of corn by root irrigation. 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 use of Bacillus Velez-Pinus as a thiamethoxam synergist. The present invention finds that treating corn that has been treated with thiamethoxam with a Bacillus Velez-Pinus bacterial solution promotes the absorption and utilization of thiamethoxam by the corn, enhances the transport and accumulation of thiamethoxam to the aboveground parts of the corn, significantly increases the thiamethoxam content in the corn roots and leaves, improves the effective utilization rate of the pesticide, and significantly promotes corn growth. This invention provides a new approach to improving pesticide utilization and exemplifies the development of highly efficient pesticide utilization agents. This agent has broad application prospects as a thiamethoxam synergist for promoting crop growth and improving pesticide utilization. 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 velezensis 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 represents maize plant height; B represents aboveground fresh weight of maize; C represents SPAD value of maize leaves; and D represents leaf area of ​​maize leaves. Note: CK represents no culture supplemented with growth-promoting bacteria to the rhizosphere; Baillius velezensis represents culture supplemented with Bacillus velezensis; Rhodococcus sp represents culture supplemented with Rhodococcus sp; and Bacilius methylotrophicus represents culture supplemented with Bacillus methylotrophicus. Different lowercase letters indicate significant differences in maize growth indicators at the same time (p < 0.05, one-way ANOVA).

[0032] Figure 3 The effect of growth-promoting bacteria on the content of thiamethoxam in maize roots and leaves. Figure 3A represents the thiamethoxam concentration in maize roots; B represents the thiamethoxam concentration in maize leaves. Note: CK represents the culture without growth-promoting bacteria added to the rhizosphere; Baillius velezensis represents the culture with Bacillus velezensis; Rhodococcus sp represents the culture with Rhodococcus sp; and Bacilius methylotrophicus represents the culture with Bacillus methylotrophicus. Different lowercase letters indicate significant differences in thiamethoxam concentrations between maize roots and leaves at the same time (p < 0.05, one-way ANOVA).

[0033] Figure 4 This represents the pesticide transport coefficient for corn grown in the rhizosphere growth-promoting bacteria experiment. Note: CK represents the culture without rhizosphere growth-promoting bacteria, Baillius velezensis represents the culture with Bacillus velezensis, Rhodococcus sp represents the culture with Rhodococcus sp, and Bacilius methylotrophicus represents the culture with Bacillus methylotrophicus. Different lowercase letters indicate significant differences in the pesticide transport coefficient for corn grown at the same time (p < 0.05, one-way analysis of variance).

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

[0035] Figure 6 Effects of different soybean powder addition amounts on the viable count of Bacillus velezensis. Note: 1%, 3%, 5%, 7%, and 9% represent cultures grown in basal salt medium with different mass fractions of soybean powder.

[0036] Figure 7 Effects of different protective agents on the preservation of Bacillus velezensis. Note: H1, H3, and H5 represent treatments with 0.1%, 0.3%, and 0.5% sodium alginate solutions, M1, M3, and M5 represent treatments with 1%, 3%, and 5% skim milk powder solutions, and S1, S3, and S5 represent treatments with 0.1%, 0.3%, and 0.5% sodium carboxymethyl cellulose solutions.

[0037] Figure 8 These are maize growth indicators for the microbial inoculum pot experiment. Note: CK represents the blank control group, and "inoculum" represents inoculation with microbial inoculum. "*" indicates significant differences in maize plant height, aboveground fresh weight, SPAD value, and leaf area between the treatment and control groups at the same time (p < 0.05, two-way ANOVA).

[0038] Figure 9Thiamethoxam concentrations in corn leaves from a potted microbial inoculum experiment. Note: CK represents a blank control group, and "Inoculum" represents inoculation with microbial inoculum. "*" indicates a significant difference in thiamethoxam concentrations in corn leaves between the treated and control groups at the same time (p < 0.05, two-way ANOVA).

[0039] Figure 10 These are maize growth indicators from the microbial inoculant field trial. Note: CK represents culture without microbial inoculant addition, "Y" represents root irrigation with microbial inoculant, and "P" represents root dipping with microbial inoculant. Different lowercase letters indicate significant differences in maize plant height, aboveground fresh weight, leaf SPAD value, and leaf area at the same time (p < 0.05, one-way ANOVA).

[0040] Figure 11 A comparison chart of corn growth effects from a field trial using microbial agents. Note: CK represents culture without microbial agents, "Y" represents root irrigation with microbial agents, and "P" represents root dipping with microbial agents.

[0041] Figure 12 Thiamethoxam concentrations in corn leaves from a field experiment with microbial inoculants. Note: CK represents incubation without microbial inoculants, "Y" represents root irrigation with microbial inoculants, and "P" represents immersion with microbial inoculants. Different lowercase letters indicate significant differences in thiamethoxam concentrations in corn leaves at the same time (p < 0.05, one-way ANOVA). DETAILED DESCRIPTION

[0042] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. 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 were commercially available.

[0044] 1. Materials

[0045] 1.1 Culture medium

[0046] Basal salt medium: (NH4)2SO4 2g, MgSO4·7H2O 0.2g, CaCl2·2H2O 0.01g, FeSO4·7H2O0.001g, Na2HPO4·12H2O 1.5g, KH2PO4 1.5g, H2O 1L.

[0047] Luria-Bertani medium: 10 g trypsin, 5 g yeast extract, 10 g NaCl, 1 L H2O. To prepare solid culture medium, add 2% agar.

[0048] Potato Dextrose Broth (PDB): 5 g potato extract, 20 g glucose, 1 L H2O. To prepare solid media, add 2% agar. Adjust the final pH of all media to 7 and autoclave at 121°C for 20 min.

[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, scavenger N-propylethylenediamine (PSA), and graphitized carbon black (GCB) were purchased from Shanghai Anpu Technology Co., Ltd.; sodium chloride was purchased from Tianjin Damao Chemical Reagent Factory; sodium alginate, carboxymethyl cellulose, skim milk powder, and urea were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; tryptone and yeast extract were purchased from OXOID Company, and agarose was purchased from Beijing Qingke Biotechnology Co., Ltd.

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

[0054] 0.9% saline: Accurately weigh 9 g of NaCl, add 1000 mL of distilled water, stir with a glass rod to fully dissolve, pour into a volumetric flask, seal and sterilize 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 of which was Huameitian 368, purchased from Guangdong Huanong University Seed Co., Ltd.

[0061] The soil for the potted experiments was collected from the Zengcheng Experimental Base of South China Agricultural University. During the collection process, 5-20 cm of soil was randomly collected, 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 test 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, single colonies were picked and separated and purified and placed in PDB medium for 24 hours. The obtained bacterial solution was centrifuged at 4000r / min, the supernatant was discarded, and it was rinsed with 0.9% saline and resuspended to make the OD600 value 1 as an inoculum. The inoculum was transferred to freshly sterilized LB liquid culture medium and three replicates were set. It was cultured in a constant temperature shaker at 30°C and 180r / min. The OD600 of the culture solution was measured every 4 hours until 36 hours. The growth curve of the strain was drawn 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 1 As shown in the figure, the lag phase for Bacillus Velezii lasts from 0 to 4 hours, during which sufficient enzymes and energy are stored for cell expansion, metabolic activity, and cell proliferation. The logarithmic growth phase lasts from 4 to 20 hours, during which the bacteria consume nutrients in the culture medium and undergo rapid metabolic activity, rapidly expanding their numbers. The stationary phase lasts from 20 to 52 hours, during which bacterial growth slows due to nutrient consumption and the accumulation of toxic substances produced by metabolism. The lag phase for Rhodococcus lasts from 0 to 4 hours, the logarithmic growth phase from 4 to 24 hours, and the stationary phase from 24 to 52 hours. The lag phase for Methylotrophic Bacillus lasts from 0 to 4 hours, the logarithmic growth phase from 4 to 20 hours, and the stationary phase from 20 to 52 hours. The OD600 values ​​for all three strains are approximately 1 when they reach the stationary phase.

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

[0073] 1. Experimental Methods

[0074] 1. Sow corn seeds in seedling trays and raise the corn seedlings to the three-leaf stage (approximately 10 cm plant height). Weigh 900 g of sieved soil, add 1 mL of an 1800 mg / L thiamethoxam preparation and 50 mL of distilled water to a conical flask, mix thoroughly, and add to the soil to achieve an initial thiamethoxam concentration of 2 mg / kg. Place the mixed soil in pots (13.5 cm diameter, 13 cm depth). Select four evenly growing corn seedlings and transplant them into the pots. Plant six pots per treatment. All pots were placed outdoors for cultivation. Soil moisture was regularly checked and maintained with manual watering. The experiment included a control (CK) and three nitrogen-fixing bacteria treatments. After transplanting the corn, the three nitrogen-fixing bacteria were cultured in PDB medium for 24 hours. The OD600 value of the bacterial suspension was adjusted to 1. The bacterial suspension was then applied to the corn roots by root irrigation, with 10 mL of the bacterial suspension applied to each corn seedling. Three replicates were set up for each treatment.

[0075] Samples were collected from corn plants and root zone soil 7, 10, 12, and 14 days after transplanting. Corn roots were washed four times with distilled water to ensure complete removal of soil and pesticides adhering to the root surface. The plant surface was wiped dry with filter paper. Corn growth indicators and thiamethoxam levels in the corn plants and soil were then 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. The corn plants were then 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 between the growth point at the base of the stem and the tip of the uppermost expanded leaf. The root elongation refers to the distance between the root tip of the longest root and the growth point at the base of the stem.

[0079] The fresh weight of rice shoots and roots was measured using an analytical balance.

[0080] The SPAD value of corn leaves was measured using a SPAD instrument, and the measurement was performed three times to obtain the average value.

[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-performance liquid chromatography-mass spectrometry (UPLC-MS) detection, Waters ACQUITY column An HSS C18 column (2.1 mm × 100 mm, 1.8 μm) was used with a column temperature of 40°C, a flow rate of 0.2 mL / min, and an injection volume of 1 μL. The mobile phases consisted of methanol (A) and 0.1% formic acid in water (B). A gradient elution scheme was used, as shown in Table 3. Mass spectrometry analysis was performed using an XEVO TQD triple quadrupole in positive ion mode using an electrospray ionization (ESI) source with multiple reaction monitoring (MRM). The capillary voltage was 1.5 kV, the cone voltage was 39 V, the desolvation temperature was 350°C, and the desolvation gas flow rate was 650 L / Hr. Thiamethoxam was monitored by mass spectrometry with a precursor ion of 292 m / z and product ions of 181.09 and 211.01 m / z, respectively, at collision energies of 23 V and 12 V, respectively. The 292 / 181.09 m / z pair was the quantification ion.

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

[0086]

[0087] (2) Standard curve and linear range

[0088] Thiamethoxam concentration was determined using the external standard method, with concentration and liquid phase peak area correlated using a linear equation. Thiamethoxam stock solution was diluted with acetonitrile to prepare five concentrations: 0.5, 0.1, 0.05, 0.01, and 0.005 mg / L. A standard curve was plotted using peak area as the ordinate and standard solution concentration as the abscissa, and a 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 place it in an ultrasonic cleaner and sonicate for 30 min. Add 3 g of sodium chloride and 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 0.5 g of sample into a 5 mL grinding tube, add grinding beads, and add 3 mL of mass spectrometry acetonitrile. Vortex for 1 minute, then ultrasonically extract for 30 minutes. Add 1.0 g of sodium chloride, vortex for 1 minute, and centrifuge at 9,000 rpm for 3 minutes. Transfer 1 mL of supernatant to a 2 mL tube containing 50 mg of PSA and 10 mg of GCB. Vortex for 1 minute, centrifuge at 10,000 rpm for 5 minutes, aspirate the supernatant using a disposable syringe, filter through a 0.22 μm microporous organic filter membrane, and collect in a brown injection bottle for analysis.

[0092] Corn root: Accurately weigh 0.5 g of sample into a 5 mL grinding tube, add grinding beads, and add 3 mL of mass spectrometry acetonitrile. Vortex for 1 minute, then ultrasonically extract for 30 minutes. Add 1.0 g of sodium chloride, vortex for 1 minute, and centrifuge at 9,000 rpm for 3 minutes. Transfer 1 mL of the supernatant to a 2 mL tube containing 50 mg of PSA, vortex for 1 minute, and centrifuge at 10,000 rpm for 5 minutes. Aspirate the supernatant using a disposable syringe, filter through a 0.22 μm microporous organic filter membrane, and collect in a brown injection bottle for analysis.

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

[0094] Uncontaminated soil, corn leaf, and root samples were weighed into 50-mL centrifuge tubes according to the mass requirements of each component in 2.3. 10 μL of thiamethoxam standard solution of varying concentrations was added to the centrifuge tubes to achieve final thiamethoxam concentrations of 5, 0.5, and 0.05 mg / L in the soil, and 5, 0.5, and 0.05 mg / kg in the corn leaf and root samples, respectively. Extraction and determination were performed as described above, and the spiked recovery was calculated.

[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 2 Compared with the blank control group, corn plant height increased by 7.61-16.63% within 7-11 days of treatment with the Bacillus Velez bacteria solution, and corn plant height increased by 7.33% after 7 days of treatment with the methylotrophic Bacillus Velez bacteria solution. The aboveground fresh weight of corn increased by 28.38% and 16.35% after 9 and 13 days of treatment with the Bacillus Velez bacteria solution, and the leaf area of ​​corn increased by 20.58% and 11.82% after 9 and 11 days of treatment with the Bacillus Velez bacteria solution. Treatment with the Bacillus Velez bacteria solution significantly increased corn plant height, aboveground fresh weight, and leaf area, indicating that the strain has a significant growth-promoting effect on corn.

[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 3Compared with the blank control, treatment with Bacillus Velez-Pinus increased thiamethoxam content in maize roots by 114.25% 13 days after treatment, while treatment with Rhodococcus increased thiamethoxam content in maize roots by 20.23% and 135.37% 9 and 13 days after treatment. Treatment with Bacillus Velez-Pinus increased thiamethoxam content in maize leaves by 26.34-35.39% between 9 and 13 days after treatment. Treatment with Bacillus Velez-Pinus significantly increased thiamethoxam content in maize roots and leaves, indicating that Bacillus Velez-Pinus can enhance maize's absorption and utilization of thiamethoxam. In summary, Bacillus Velez-Pinus can promote maize growth and enhance its absorption and utilization.

[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 to thiamethoxam. Figure 4 Compared with the blank control group, the Bacillus Velez-like bacteria treated with the liquid solution increased the pesticide transport coefficient by 40.41% 9 days after application, and the methylotrophic Bacillus bacteria treated with the liquid solution increased the pesticide transport coefficient by 62.17% 11 days after application. This indicates that these two growth-promoting bacteria can enhance the pesticide transport coefficient of maize and promote the translocation of thiamethoxam from maize roots to aboveground parts. Bacillus Velez-like bacteria can both increase the concentration of thiamethoxam in maize roots and leaves and improve the pesticide transport coefficient, indicating that this strain can promote thiamethoxam uptake in maize while enhancing its translocation and accumulation to aboveground parts, thereby enhancing the insecticidal efficacy of thiamethoxam.

[0099] 4. As can be seen from Table 4, the recoveries of thiamethoxam in soil, corn roots, and corn leaves were 81.84-97.88%, 85.02-96.51%, and 93.08-107.46%, respectively. The relative standard deviations (RSDs) were all less than 10%, which met the requirements of the "NY / T 788-2018 Test Guidelines for Pesticide Residues in Crops". This shows that the extraction method is stable and reliable and can 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 for microbial fermentation medium

[0103] 1. Experimental Methods

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

[0105] A single-factor experiment was conducted using corn flour, soybean flour, bagasse, soybean meal, and wheat flour as carbon sources, with potato broth (PDB) as a control. Five carbon sources were added to MSM medium at a 5% mass fraction. 1 mL of a nitrogen-fixing bacterial suspension (OD600 value of 1) was then inoculated into the MSM and PDB medium supplemented with each carbon source. The cultures were shaken at 30°C and 150 rpm for 4, 6, 8, and 10 days. The viable count of nitrogen-fixing bacteria in the culture medium was determined using the dilution spread plate method. Three replicates were used for each treatment.

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

[0107] The optimal carbon source was selected and its addition amount was varied to set up a single-factor experiment. MSM medium was added with carbon source at concentrations of 1%, 3%, 5%, 7%, and 9% by mass. Then, 1 mL of resuspended bacterial suspension (OD600 value of 1) was inoculated and cultured in a shaker at 30°C and 150 rpm for 1, 2, 3, and 4 days. The effective viable count of nitrogen-fixing bacteria in the culture medium was determined using the dilution spread plate method. Three replicates were set for each treatment.

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

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

[0110] Prepare solutions of 0.1%, 0.3%, and 0.5% sodium carboxymethylcellulose (CMC) as a protective agent; 0.1%, 0.3%, and 0.5% sodium alginate as a protective agent; and 1%, 3%, and 5% skim milk powder as a protective agent. Sterilize the solutions in an autoclave at 121°C for 20 minutes. Inject 15 mL of each sterilized protective agent solution into the bacterial cells, shake for 15-20 minutes to disperse the cells and thoroughly mix with the protective agent, then store at room temperature. Samples were collected on days 1, 3, 5, and 7, and OD600 values ​​were measured. Three replicates were used 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 five carbon sources such as corn flour and soybean meal were added at a mass fraction of 5% on the basis of basal salt medium (MSM). The most suitable carbon source was determined by comparing the effective viable bacteria counts in various culture media. Figure 5As shown in the results, compared with PDB medium, the fermentation of Bacillus velez 20025 using MSM medium with four carbon sources, 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 using soybean meal as the carbon source was the highest, at 2.93×10 9 CFU / mL; at 6d, 8d, and 10d, the number of effective viable bacteria of Bacillus velez in the microbial fermentation medium using 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 amount of carbon source most suitable 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. Figure 6 It was found that on the 3rd day 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 3.18×10 9 CFU / mL, and remained stable in the following period; on the 4th day of fermentation, the effective viable bacterial count of the culture medium with a 7% addition amount reached a peak of 3.02×10 9 CFU / mL, and remained stable in the following period; at 5 days, the effective viable bacterial count of the culture medium with an addition of 9% reached a peak of 3.74×10 9 CFU / mL and remained stable thereafter. Between days 1 and 4 of fermentation, the effective viable bacterial count in the medium with a 5% addition was significantly higher than in the medium with other additions. Between days 5 and 7 of fermentation, there was no significant difference between the 5% and 7% additions, while the effective viable bacterial count in the medium with a 9% addition was significantly higher than in the medium with other additions. Taking all factors into consideration, a 5% mass fraction of soybean flour is the optimal carbon source addition for microbial fermentation. A 5% mass fraction of soybean flour was determined to be used in combination with a basal salt medium as the initial fermentation medium for Bacillus velezensis.

[0114] 3. After 4 days of fermentation of Bacillus velezensis using 5% by mass soybean powder and basal salt culture medium as the initial fermentation medium, a protective agent was added to the fermentation liquid to extend the shelf life of the microbial agent, and a microbial agent was prepared. 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] Corn seeds were sown in seedling trays and raised to the three-leaf stage (plant height approximately 10 cm). 900 g of sieved soil was weighed, and 1 mL of an 1800 mg / L thiamethoxam preparation and 50 mL of distilled water were added to a conical flask. The mixture was then added to the soil and mixed thoroughly to achieve an initial thiamethoxam concentration of 2 mg / kg. The mixed soil was placed in a flowerpot (13.5 cm diameter, 13 cm depth). Four evenly growing corn seedlings were selected and transplanted into the flowerpots. Six pots were planted per treatment. All pots were placed outdoors for cultivation. Soil moisture was regularly checked and maintained by manual watering. The experiment included a control (CK) and three nitrogen-fixing bacteria treatments: After transplanting the corn, the microbial inoculum was diluted 20-fold and applied to the corn roots via root irrigation. 10 mL of the inoculum was applied to each corn seedling, with three replicates per treatment.

[0118] Samples were collected from corn plants and root zone soil 7, 10, 12, and 14 days after transplanting. Corn roots were washed four times with distilled water to completely remove any soil and pesticides adhering to the root surface. The plant surface was then wiped dry with filter paper. Corn growth indicators were then measured, and the pesticide thiamethoxam was extracted 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) the addition of microbial agents were set up. 10 mL of a 20-fold diluted microbial agent was added to the roots of each corn plant by 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 8As shown in the results, compared with the blank control group, the microbial agent treatment increased maize plant height by 15.20%, 8.93%, and 8.09% at 7, 10, and 14 days after application; the aboveground fresh weight of maize increased by 17.81% to 27.29% at 10 to 14 days after application; the SPAD value of maize leaves increased by 5.62% to 10.88% at 10 to 14 days after application; and the leaf area of ​​maize leaves increased by 26.48% to 34.09% at 10 to 14 days after application. The addition of microbial agents significantly increased maize plant height, aboveground fresh weight, SPAD value, and leaf area, indicating that microbial agents have a significant promoting effect on maize growth.

[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. 10 mL of a 20-fold diluted microbial agent was added to the roots of each corn plant by irrigation. The content of thiamethoxam in the corn leaves was compared to clarify the effect of microbial agents on the absorption of pesticides by corn. Figure 9 As shown in the data, after thiamethoxam application, thiamethoxam concentrations in corn leaves initially increased and then decreased. With increasing application time, the accumulated concentration of thiamethoxam in corn leaves gradually increased, reaching a peak around day 10, and then gradually decreased. Compared with the blank control group, microbial inoculant treatment increased thiamethoxam content in corn leaves by 43.94% to 97.30% within 10 to 12 days after application. The addition of microbial inoculants significantly increased thiamethoxam content in corn leaves, indicating that Bacillus velezensis can enhance the absorption and utilization of thiamethoxam in corn. In summary, microbial inoculants can promote corn growth while also enhancing its absorption and utilization.

[0122] Example 5 Field Verification of Microbial Agent Function

[0123] 1. Experimental Methods

[0124] Corn seeds were sown in seedling trays and grown to the three-leaf stage (approximately 10 cm tall). The corn, which showed consistent growth, was then transplanted to farmland in Zengcheng. The field experiment included three treatments: a blank control, a root irrigation treatment (Y), and a root dipping treatment (P). Each treatment consisted of two plots, each 15 m long and 0.8 m wide, with a 65 cm row spacing and a 30 cm plant spacing. A 2-m-long protective row was placed between the plots.

[0125] Root irrigation treatment with microbial agent: After transplanting corn, apply 50 mL of 20-fold diluted microbial agent to the roots of each corn plant.

[0126] Root dipping 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.

[0127] After treatment, each corn plant was watered with 150 mL of water. Ten days after treatment, 1.2 g of 25% water-dispersible granules of thiamethoxam was diluted with 18 L of water to prepare a 40 mg / L thiamethoxam solution (the recommended dosage for corn fields). This solution was then irrigated to the roots of each corn plant with 50 mL of the 40 mg / L thiamethoxam solution.

[0128] Samples were collected from corn plants and root zone soil 10, 12, 14, and 16 days after application. The corn roots were washed four times with distilled water to ensure complete removal of soil and pesticide adhering to the root surface. The plant surface was wiped dry with filter paper. Corn growth indicators were then measured, and the thiamethoxam content in the corn plants was assayed.

[0129] 2. Experimental Results

[0130] 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. Figure 10 As shown in the results, compared with the control group, the height of corn plants in the microbial agent root irrigating treatment increased by 12.14-20.22% within 10-16 days after application, and the height of corn plants in the microbial agent root dipping treatment increased by 11.21-20.03% within 10-16 days after application; the fresh weight of corn aboveground part in the microbial agent root irrigating treatment increased by 42.57-51.20% within 10-16 days after application, and the fresh weight of corn plants in the microbial agent root dipping treatment increased by 35.90-69.8 1%; root irrigation with the microbial agent increased the SPAD value of corn leaves by 10.38-17.16% within 10-16 days after application, and the root dipping treatment increased the SPAD value of corn leaves by 12.14-17.22% within 10-16 days after application; the root irrigation with the microbial agent increased the leaf area of ​​corn leaves by 42.27-54.92% within 10-16 days after application, and the root dipping treatment increased the leaf area of ​​corn leaves by 41.59-51.86% within 10-16 days after application. The application of microbial agents in corn fields through root irrigation and root dipping significantly increased the plant height, aboveground fresh weight, leaf SAPD value, and leaf area of ​​corn, indicating that microbial agents have the function of promoting corn growth.

[0131] like Figure 11 As shown in the figure, after application, the corn plants treated with root irrigation of the bacterial solution grew the best, followed by the corn plants treated with root dipping of the bacterial solution, and the corn plants in the water control group grew the worst. This shows that both microbial agent treatments promoted corn growth under field conditions.

[0132] In order to explore the effect of microbial agents on the absorption of pesticides by corn under farmland environmental conditions, the pesticide was 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. Figure 12 As shown in the data, after thiamethoxam application, thiamethoxam concentrations in corn leaves showed a gradual downward trend. This is presumably because corn absorbs and utilizes thiamethoxam more efficiently under farmland conditions. The thiamethoxam concentration in corn leaves peaked before 10 days, leading to a gradual decline in thiamethoxam concentrations over time within 10 to 16 days. Compared with the blank control, thiamethoxam concentrations in corn leaves increased by 79.86% to 122.67% within 10 to 16 days after microbial inoculant root dredging, and by 39.72% to 94.15% within 10 to 16 days after microbial inoculant root dipping. Both root dredging and root dipping significantly increased thiamethoxam concentrations in corn leaves, indicating that microbial inoculants effectively promote thiamethoxam absorption in corn under farmland conditions, with root dredging being more effective.

Claims

1. Use of Bacillus velezensis as a thiamethoxam synergist, characterized in that: The Velez Bacillus is Velez Bacillus Bacillus velezensis CICC ® 20025.

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

3. The use of Bacillus velez in the preparation of a dual-action bacterial agent for promoting plant growth and plant absorption and utilization of thiamethoxam, characterized in that: The Velez Bacillus is Velez Bacillus Bacillus velezensis CICC ® 20025.

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 to apply thiamethoxam at the same time.

5. The use 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 application 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 further comprises a basic salt culture medium, soybean powder as a carbon source, and sodium alginate as a protective agent; the Bacillus velezii is Bacillus velezii Bacillus velezensis CICC ® 20025.

8. The application 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 3 or 7, characterized in that: The plant is corn.

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