Application of bacillus cereus as chlorantraniliprole synergist

Abstract

The invention discloses an application of bacillus cereus as a chlorantraniliprole synergist. According to the invention, the bacillus cereus is added into the nutrient solution applied with the chlorantraniliprole, so that the absorption of the plant root system on the chlorantraniliprole can be obviously promoted, and the transport and enrichment capabilities of the plant root system to the overground part of the plant are enhanced, so that the plant obtains more durable and efficient insect resistance, the utilization rate of the chlorantraniliprole is increased, and the yield of the plant is increased. The field application amount is reduced. Through plant growth index analysis, chlorantraniliprole under experimental concentration does not have an inhibition effect on corn plant height, root length and fresh weight. Meanwhile, on the seventh day after inoculation, a treatment group added with the bacillus cereus can promote the increase of the plant height and the fresh weight of the corn. Therefore, the bacillus cereus can be applied to agricultural production as a biological preparation with the synergistic and plant growth promoting functions of chlorantraniliprole. According to the technology, pesticide decrement application is achieved through a biological synergistic mechanism, and a new solution is provided for pesticide decrement and synergism and crop growth regulation and control.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and specifically, to the application of Bacillus cereus as a synergist for chlorantraniliprole. Background Art

[0002] With the increasing environmental pollution, the technology of reducing pesticide usage and increasing efficiency has become an important research direction for the sustainable development of agriculture. Approximately 100 million to 2.5 billion tons of pesticides are applied to farmland and towns globally each year, becoming an important source of the spread of environmental chemical pollutants. It is estimated that less than 1% of pesticides act on target organisms, and most of the rest enter the ecosystem, having adverse effects on human health and the ecological environment. Due to their lipophilic characteristics, pesticide residues are easily accumulated in the soil, seriously affecting the growth, development, and reproduction of non-target organisms in the soil environment. Data from the China Pesticide Information Network shows that the pesticide utilization rate of the three major staple crops (wheat, corn, and rice) in China in 2022 was only 41.8%. Therefore, improving the effective utilization rate of pesticides has become a key technical requirement for the green development of agriculture.

[0003] Chlorantraniliprole belongs to the class of o - formylaminobenzamide insecticides. It is enriched in tissues through the systemic action of plants and acts on the insect nervous system after being ingested by pests to achieve the insecticidal function. Its systemic property enables the drug to effectively penetrate into plant tissues after spraying and maintain the drug effect for a long time. This agent has low toxicity (the toxicity levels for birds, fish, bees, and mammals are low), a long-lasting effect of more than 15 days, resistance to rain washing, and the residues in agricultural products meet safety standards. It is now widely used for the prevention and control of agricultural pests. The control targets cover various pests such as Spodoptera litura, Ostrinia furnacalis, and Cnaphalocrocis medinalis, and it also has a significant control effect on the invasive species Spodoptera frugiperda. Under field conditions, chlorantraniliprole degrades rapidly on crops and has a short half-life, belonging to easily degradable pesticides. Correct application according to the recommended dosage can ensure that the residues in agricultural products meet the standards. However, it should be noted that its retention period in the soil is relatively long, and long-term application may lead to soil accumulation. Soil residues may pose ecological risks through two pathways: (1) affecting the physical and chemical properties of the soil by changing the microbial community structure; (2) posing a potential threat to human health and the aquatic ecosystem through food chain transfer or environmental migration (leaching, runoff). Therefore, it is very important to effectively utilize this part of chlorantraniliprole that remains in the soil for a long time.

[0004] The Bacillus cereus BCS1 strain was independently isolated by the present inventors, which can significantly promote the degradation of pyrethroid pesticides in the soil-plant system and exhibits good environmental adaptability (Huang Y, Yang L, Pan K, et al. Heavy metal-tolerant bacteria Bacillus cereus BCS1 degrades pyrethroid in a soil-plant system[J]. Journal of Hazardous Materials, 2024, 461. DOI: 10.1016 / j.jhazmat.2023.132594.). However, there has been no relevant technical report on using Bacillus cereus as a synergist for chlorantraniliprole to achieve the reduction of pesticide application by promoting the absorption and transport of chlorantraniliprole in plants. Summary of the Invention

[0005] The object of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and provide an application of Bacillus cereus as a synergist for chlorantraniliprole.

[0006] The second object of the present invention is to provide an application of a bacterial suspension or bacterial agent of Bacillus cereus as a synergist for chlorantraniliprole.

[0007] The third object of the present invention is to provide an application of Bacillus cereus in the preparation of a dual-effect bacterial agent that can both promote plant growth and enhance the absorption and utilization of chlorantraniliprole.

[0008] The above objects of the present invention are achieved by the following technical solutions:

[0009] Chlorantraniliprole is a novel anthranilic diamide insecticide that acts on the insect ryanodine receptor. It has the characteristics of high insecticidal activity, broad insecticidal spectrum, long residual period, low resistance development in pests, safety to non-target organisms, and no cross-resistance with conventional pesticides. It is widely used in the control of Lepidoptera pests on crops such as rice and vegetables. Field test results show that chlorantraniliprole degrades rapidly and has a short half-life on crops, belonging to easily degradable pesticides. When applied correctly according to the recommended dose, it will not cause residues on crops. However, excessive application will lead to the accumulation and residue of chlorantraniliprole on the soil surface. Through rainwashing, surface runoff will enter the lower layer of the soil, and its retention period in the soil is relatively long. Therefore, how to utilize this part of pesticides is crucial. The present invention discovers that exogenous application of Bacillus cereus in the nutrient solution containing chlorantraniliprole can promote the absorption of chlorantraniliprole by plant roots, and promote the transport and enrichment of chlorantraniliprole from plant roots to the above-ground parts of plants, enabling plants to have longer and more efficient insect resistance, improving the utilization rate of chlorantraniliprole, and reducing the use of pesticides in the field. Through the analysis of plant growth indicators, chlorantraniliprole at the experimental concentration did not inhibit the plant height, root length, and fresh weight of maize. At the same time, it was found that on the 7th day after inoculation, the treatment group with the addition of Bacillus cereus could promote the increase of maize plant height and fresh weight.

[0010] Therefore, the present invention provides the application of Bacillus cereus as a synergist for chlorantraniliprole.

[0011] The present invention also provides the application of the bacterial suspension or bacterial agent of Bacillus cereus as a synergist for chlorantraniliprole.

[0012] Furthermore, the synergist has at least one of the following functions (1) - (2):

[0013] (1) Promote the absorption of chlorantraniliprole by plant roots;

[0014] (2) Promote the transport and enrichment of chlorantraniliprole from plant roots to the above-ground parts of plants.

[0015] The present invention provides the application of Bacillus cereus in the preparation of a dual-effect bacterial agent that can both promote plant growth and enhance the absorption and utilization of chlorantraniliprole.

[0016] Furthermore, the application is to apply Bacillus cereus to the roots of plants sprayed with chlorantraniliprole.

[0017] Furthermore, the bacterial suspension is prepared by picking fresh single colonies of Bacillus cereus on an LB plate, pre-culturing them in LB liquid medium for 12 h, centrifuging the obtained bacterial solution at 4000 rpm at 4 °C for 5 min, discarding the supernatant, and resuspending it with an appropriate amount of sterile water.

[0018] Furthermore, the application concentration of Bacillus cereus is OD600 The seed liquid with an OD of 0.5 - 0.7 is diluted at a volume ratio of 1:50 - 70.

[0019] Furthermore, the application concentration of the Bacillus cereus is OD 600 The seed liquid with an OD of 0.6 is diluted at a volume ratio of 1:60.

[0020] Further, the application concentration of the Bacillus cereus is expressed by OD 600 The bacterial suspension with an OD of 0.6 is inoculated into 600 mL of nutrient solution at a ratio of the volume of the suspension (mL) to the volume of the nutrient solution (mL) of 1:60.

[0021] Further, the application concentration of the chlorantraniliprole is 4 - 6 mg / L.

[0022] Furthermore, the application concentration of the chlorantraniliprole is 5 mg / L

[0023] Further, the Bacillus cereus is Bacillus cereus BCS1.

[0024] Further, the plant is corn.

[0025] Further, the above-ground part is the corn stem and / or leaves.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention provides the application of Bacillus cereus as a synergist for chlorantraniliprole. The present invention discovers that adding Bacillus cereus to the nutrient solution for applying chlorantraniliprole can significantly promote the absorption of chlorantraniliprole by plant roots, enhance the transport and enrichment ability of chlorantraniliprole to the above-ground part of the plant, enable the plant to obtain long-lasting and high-efficiency insect resistance, improve the utilization rate of chlorantraniliprole, and thus reduce the field application amount. Through the analysis of plant growth indexes, it shows that chlorantraniliprole at the experimental concentration does not have an inhibitory and toxic effect on the root length, plant height, and fresh weight of corn. At the same time, it is found that the treatment group with Bacillus cereus can promote the increase of corn plant height and fresh weight on the 7th day after inoculation. Therefore, Bacillus cereus can be used in the field as both a synergist for chlorantraniliprole and a plant growth promoter. This technology realizes the reduction of pesticide application through a biological synergistic mechanism, providing a new solution for pesticide reduction and efficiency enhancement and crop growth regulation. Description of the Drawings

[0028] Figure 1Phylogenetic analysis of Bacillus cereus strain BCS1. Note: A neighbor-joining phylogenetic tree was constructed based on the 16S rDNA sequence, showing the evolutionary relationship between BCS1 and related strains. The phylogenetic tree was constructed using the neighbor-joining method. The numbers in parentheses represent the GenBank accession numbers.

[0029] Figure 2 Effect of adding Bacillus cereus BCS1 on the concentration of chlorantraniliprole in maize roots. Note: The treatment group was inoculated with a BCS1 bacterial suspension with an OD 600 of 0.6 (volume ratio of bacterial solution: nutrient solution = 1:60, total volume 600 mL). The data were analyzed by ANOVA. ns indicates P>0.05, no significant difference; * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001.

[0030] Figure 3 Effect of adding Bacillus cereus BCS1 on the concentration of chlorantraniliprole in maize stems. Note: The treatment method and data analysis were the same as Figure 2 .

[0031] Figure 4 Effect of adding Bacillus cereus BCS1 on the concentration of chlorantraniliprole in maize leaves. Note: The treatment method and data analysis were the same as Figure 2 .

[0032] Figure 5 Effect of adding Bacillus cereus BCS1 on the concentration of chlorantraniliprole in nutrient solution. Note: The treatment method and data analysis were the same as Figure 2 .

[0033] Figure 6 Effect of adding Bacillus cereus BCS1 on the root length of maize cultivated in nutrient solution containing chlorantraniliprole. Note: The treatment method and data analysis were the same as Figure 2 .

[0034] Figure 7 Effect of adding Bacillus cereus BCS1 on the plant height of maize cultivated in nutrient solution containing chlorantraniliprole. Note: The treatment method and data analysis were the same as Figure 2 .

[0035] Figure 8 Effect of adding Bacillus cereus BCS1 on the fresh weight of maize cultivated in nutrient solution containing chlorantraniliprole. Note: The treatment method and data analysis were the same as Figure 2 . Specific implementation methods

[0036] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments. However, the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0037] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0038] Table 1 Nutrient solution formula

[0039]

[0040] Note: Adjust the pH to 6.5.

[0041] Example 1 Isolation and identification of Bacillus cereus

[0042] I. Experimental method

[0043] According to the literature (Huang Y, Yang L, Pan K, et al. Heavy metal-tolerant bacteria Bacillus cereus BCS1 degrades pyrethroid in a soil-plant system [J]. Journal of Hazardous Materials, 2024, 461. DOI: 10.1016 / j.jhazmat.2023.132594.), the method for isolating and identifying Bacillus cereus BCS1 of the present invention is as follows:

[0044] (1) 5 g of soil samples collected from farmland in Guangzhou, China (the pH of the soil sample was 6.7, the temperature was 24 °C. The water content of the soil sample was 23.6%, the water holding capacity was 39.0%, and the organic matter content was 4.6%. It belonged to the clay loam texture category) was added to 50 mL of MSM containing 20 mg·L -1 β-CP (a pyrethroid pesticide, beta-cypermethrin), and incubated at 200 rpm and 30 °C for 3 d (days). Subsequently, the culture was spread on an MSM plate containing 20 mg·L -1 β-CP as the sole carbon source for isolating single colonies. The strain with the highest β-CP degradation activity was further characterized and identified by morphology and 16S rDNA gene analysis.

[0045] (2) 16S rDNA molecular biological identification: Using the Ezup column bacterial genomic DNA extraction reagent from Sangon Biotech (product numbers SK8255 / SK8256), the genomic DNA of Bacillus cereus BCS1 was extracted according to the instruction manual. Using the 16S rDNA universal primers for bacteria (27F: 5’-AGTTTGATCMTGGCTCAG-3’; 1492R: 5’-GGTTACCTTGTTACGACTT-3’), PCR amplification was carried out, and sequencing was performed by Sangon Biotech Co., Ltd. (Shanghai). The 16S rDNA sequence of strain BCS1 was subjected to blast alignment analysis on the ribosome database to retrieve the 16S rDNA gene sequences of strains with higher sequence homology to it.

[0046] II. Experimental results

[0047] According to the isolation results, the strain with higher degradation activity was named BCS1.

[0048] (1) Morphological identification: After incubating strain BCS1 in LB medium for 7 days, strain BCS1 showed Gram-positive characteristics. After incubating at 30 °C for 72 h, the colonies formed on the LB agar plate were approximately circular, soft, opaque, and slightly bright white in appearance.

[0049] (2) The 16S rDNA gene of strain BCS1 (1499 bp; GenBank accession number: OR418499) had a high sequence similarity with Bacillus cereus ATCC14579 and Bacillus cereus YQ15, both with a similarity of 99.93%. In addition, according to the 16S rDNA dendrogram, the phylogenetic position of BCS1 was closest to Bacillus cereus ATCC14579 ( Figure 1 ). Based on these results, strain BCS1 was identified as Bacillus cereus.

[0050] Example 2 Effect of BCS1 on the absorption and translocation of pesticides by maize plants

[0051] I. Experimental methods

[0052] (1) Strain activation and preparation

[0053] Strain activation: Pick a fresh single colony of Bacillus cereus BCS1 from an LB solid plate and inoculate it into 100 mL of LB liquid medium. Incubate with shaking at 30 °C and 180 rpm for 5 - 6 h until the logarithmic growth phase. Bacterial cell collection: Transfer the bacterial liquid to a 50 mL centrifuge tube, centrifuge at 4000 rpm for 5 min, and discard the supernatant; add sterile water to resuspend the bacterial cells. After vortexing for 1 min, centrifuge at 6000 rpm for 5 min, and repeat the washing once. Finally, resuspend with 50 mL of sterile water. Take 200 μL of the bacterial liquid and add it to a 96-well plate, and measure the OD 600 value using an enzyme-linked immunosorbent assay (ELISA) reader (wavelength 600 nm), and calculate the inoculation volume according to the formula C1×V1 = C2×V2 (C1: OD value of the bacterial liquid, V1: inoculation volume, C2: OD value of the target concentration, V2: volume of the nutrient solution). 600 value, V1: inoculation volume, C2: OD 600 value, V2: volume of the nutrient solution).

[0054] (2) Inoculation treatment

[0055] Experimental group: Inoculate the bacterial suspension with an OD 600 value of 0.6 into 600 mL of nutrient solution containing 5 mg / L chlorantraniliprole at a volume ratio of 1:60.

[0056] Control group: Use an equal volume (600 mL) of sterile nutrient solution with the same concentration of chlorantraniliprole.

[0057] Analysis of chlorantraniliprole concentration: Collect corn root, stem, and leaf tissues on the 3rd and 7th days after inoculation, and measure the pesticide concentration.

[0058] (3) Pretreatment of corn plants

[0059] Sample homogenization: Accurately weigh 0.5 g of each of the corn root, stem, and leaf tissues using an analytical balance, place them in a 5 mL grinding tube, add 3 grinding beads with a diameter of 5 mm, pre-cool with liquid nitrogen, and grind at a frequency of 60 Hz for 6 min. Centrifuge at 9000 rpm for 30 s. Pesticide extraction: Add 1 mL of acetonitrile to the grinding tube, vortex for 1 min, then perform ultrasonic-assisted extraction for 30 min. Add 1 g of NaCl to saturate the aqueous solution, vortex for 1 min, centrifuge at 4000 rpm for 5 min, and transfer the supernatant to a 2 mL centrifuge tube. Sample purification: Add 50 mg of PSA (primary secondary amine, N-propylethylenediamine) to the root and stem samples, and add 100 mg of PSA to the leaf samples. After shaking for 1 min, centrifuge at 4000 rpm for 5 min, and transfer the supernatant through a 0.22 μm organic filter membrane to a brown injection vial for testing.

[0060] (3) Detection method for chlorantraniliprole

[0061] Instrument configuration: Agilent 1260 high performance liquid chromatography system; Chromatographic column: Agilent TC-C18(2), 5μm, 4.6*250mm; Flow rate: 0.8 mL / min; Column temperature: 40 °C; Injection volume: 10 μL; Mobile phase: acetonitrile / water (50:50, V / V); Detection wavelength: 200 nm.

[0062] II. Experimental results

[0063] It can be seen from Figure 2-5 that on the 3rd day after inoculation and pesticide application, there was no significant difference in the concentration of chlorantraniliprole in the root, leaf tissues and nutrient solution of the inoculated group compared with the control group ( Figure 2 , Figure 4 , Figure 5 ); the concentration in the stem of the inoculated group was significantly higher than that of the control group ( Figure 3 ). On the 7th day after pesticide application, the concentrations of chlorantraniliprole in the roots, stems and leaves of the inoculated group were all significantly higher than those of the control group ( Figure 2 , Figure 3 , Figure 4 ); there was no significant difference in the concentration of chlorantraniliprole in the nutrient solution of the inoculated group compared with the control group ( Figure 5 ) (presumably because the volume of the nutrient solution is large, even if there are differences in the accumulated concentration of pesticides in the corn roots at 7 days, it is not significant enough in the nutrient solution).

[0064] Conclusion: Applying BCS1 to the roots of corn can significantly improve the absorption of chlorantraniliprole by corn roots, promote the transport of chlorantraniliprole in the roots to the above-ground parts, improve the utilization rate of chlorantraniliprole, and reduce the usage amount of chlorantraniliprole.

[0065] Example 3 Growth-promoting effect of BCS1 on corn plants

[0066] I. Experimental method

[0067] As shown in the experimental method of Example 2, the plant height, root length and fresh weight of corn were measured on the 3rd day and the 7th day after adding the bacterial agent respectively to evaluate the effect of corn's absorption of chlorantraniliprole on the plant growth status.

[0068] II. Experimental results

[0069] The results are as Figure 6-8 shown. On the 3rd day after inoculation and pesticide application, there was no significant difference in the plant height and fresh weight of the inoculated group compared with the control group ( Figure 7 , Figure 8 ); the root length of the inoculated group was significantly lower than that of the control group ( Figure 6 ). On the 7th day after inoculation and pesticide application, the plant height and fresh weight of the inoculated group were both higher than those of the control group ( Figure 7 , Figure 8);The root length of the inoculated group of plants was not significantly different from that of the control group( Figure 6 ). The results showed that during the time when Bacillus cereus BCS1 promoted the absorption of chlorantraniliprole by plants, the accumulated chlorantraniliprole did not cause toxicity to plants, and BCS1 could promote the increase in the plant height and fresh weight of maize on the 7th day after inoculation and application of the drug.

[0070] In summary, Bacillus cereus BCS1 can be used as a synergist for chlorantraniliprole and a plant growth promoter in the field.

Claims

1. Use of Bacillus cereus as a synergist for chlorantraniliprole.

2. Use of a bacterial suspension or bacterial agent of Bacillus cereus as a synergist for chlorantraniliprole.

3. The application according to any one of claims 1 or 2, characterized in that The synergist has at least one of the following functions (1) to (2): (1) Promote the absorption of chlorantraniliprole by plant roots; (2) Promote the transport and enrichment of chlorantraniliprole from plant roots to the above-ground parts of plants.

4. Use of Bacillus cereus in the preparation of a dual-effect bacterial agent that both promotes plant growth and enhances the absorption and utilization of chlorantraniliprole.

5. According to any one of the applications described in claims 1 to 2 or 4, it is characterized in that, The application is to apply Bacillus cereus to the roots of plants sprayed with chlorantraniliprole.

6. The application according to claim 5, wherein The application concentration of the Bacillus cereus is the seed liquid with an OD 600 value of 0.5 - 0.7 diluted at a volume ratio of 1:50 - 70.

7. The application according to claim 5, characterized in that The application concentration of chlorantraniliprole is 4 - 6 mg / L.

8. The use according to any one of claims 1 to 2 or 4, characterized in that: The Bacillus cereus is Bacillus cereus BCS1.

9. The application according to any one of claims 3 or 4, characterized in that The plant is corn.

10. The application according to any one of claims 3 or 4, characterized in that The above-ground parts of the plant are corn stalks and / or leaves.

Images