Bactericidal composition containing Aminopyrifen and Ipflufenoquin and application thereof

Abstract

The invention belongs to the field of agricultural bactericides, and particularly relates to a bactericidal composition containing Aminopyrifen and Ipflufenoquin and application of the bactericidal composition, and the mass ratio of Aminopyrifen to Ipflufenoquin in active ingredients of the bactericidal composition is (1: 4)-(2: 3) or 3: 2. Indoor biological assay, observation of conidium germination, observation of toxosome formation and field efficacy test results show that the composition has a synergistic effect and has excellent bacteriostasis and disease prevention effects on gibberellic disease bacteria and wheat scab caused by the gibberellic disease bacteria. The composition consists of effective components with different action mechanisms, is beneficial to overcoming and delaying the generation of drug resistance of pathogenic bacteria, and is safe to the growth of test crops.

Description

Technical Field

[0001] The invention belongs to the field of agricultural fungicides, and specifically relates to a fungicide composition containing Aminopyrifen and Ipflufenoquin and application thereof. Background Art

[0002] Wheat is an important commercial grain and main storage grain in my country, and its quality and safety are related to the national economy and people's livelihood. Since 2010, affected by factors such as climate warming, lack of disease-resistant varieties and large-scale return of straw to the fields, wheat fusarium rust caused by Fusarium graminearum species complex (FGSC) has frequently spread in the wheat-growing areas in the middle and lower reaches of the Yangtze River and the Huanghuai wheat-growing areas, and has become one of the most important diseases of wheat. The Ministry of Agriculture and Rural Affairs has included wheat fusarium rust in the "List of Class I Crop Diseases and Insect Pests" in September 2020. In addition to causing a reduction in grain production, wheat fusarium rust is more serious because the fusarium produces a variety of fusarium toxins in wheat grains, such as deoxynivalenol (DON) and zearalenone (ZEA), which seriously threaten food safety and human and animal health. However, there is currently no wheat variety that is both immune or highly resistant to fusarium head blight and suitable for large-scale cultivation. Spraying chemical fungicides during the heading and flowering period of wheat is still the main measure for the prevention and control of fusarium head blight, but long-term single spraying of chemical fungicides has led to the development of drug resistance in fusarium head blight, affecting the efficacy of the drug, and posing a huge challenge to the prevention and control of wheat fusarium head blight. Screening for highly efficient combined fungicides with dual effects of disease prevention and toxicity reduction, and delaying the development of fungicide resistance in pathogens, has practical value for the scientific prevention and control of wheat fusarium head blight.

[0003] Aminopyrifen is a broad-spectrum pyridine fungicide with the chemical name: (4-phenoxyphenyl)methyl-2-amino-6-methyl-3-pyridinecarboxylate. Aminopyrifen has a novel fungicide mechanism, which inhibits the activity of acyltransferase (GWT-1) and prevents the precursor biosynthesis of glycosylphosphatidylinositol to achieve fungicidal effects. Aminopyrifen has no cross-resistance with commonly used commercial fungicides and has good preventive effects on a variety of plant diseases such as gray mold, sclerotinia, powdery mildew, apple fusarium head blight, rice blast and anthracnose. The chemical structure of Aminopyrifen is as follows:

[0004]

[0005] Ipflufenoquin is a quinoline fungicide with a novel mechanism of action. The International Fungicide Resistance Action Committee (FRAC) has not yet classified its mechanism of action. Ipflufenoquin has a broad fungicide spectrum and is highly effective in controlling black spot, leaf spot, gray mold, anthracnose, sclerotinia, and rice leaf blight. It can also be used to control pathogens that have developed resistance to existing agents, including plant pathogenic fungi such as Black Spot, Botrytis, and Sclerotinia. The chemical name of Ipflufenoquin: 2-[(7,8-difluoro-2-methyl-3-quinolyl)oxy]-6-fluoro-a,a-dimethylbenzyl alcohol. The chemical structure of Ipflufenoquin is as follows:

[0006]

[0007] The International Fungicide Resistance Action Committee (FRAC) recommends that the combination of agents with different mechanisms of action can avoid or delay the development of resistance. The development of a fungicide combination of Aminopyrifen and Ipflufenoquin can not only effectively solve the problem of crop disease prevention and control, but also avoid or delay the development of resistance by pathogens. So far, there have been no reports on the combination of Aminopyrifen and Ipflufenoquin for disease prevention and control. Summary of the invention

[0008] The present invention aims to provide a highly efficient, low-toxic, environmentally friendly fungicide composition, which has excellent control effects on wheat yield reduction and toxin pollution caused by fusarium head blight, and can also control the development of drug resistance in pathogens and extend the service life of the agent. The specific technical scheme is as follows:

[0009] A bactericidal composition containing Aminopyrifen and Ipflufenoquin, wherein the active ingredients are composed of Aminopyrifen and Ipflufenoquin, and the mass ratio of Aminopyrifen and Ipflufenoquin is (1:4)-(2:3) or 3:2. The preferred mass ratio of Aminopyrifen and Ipflufenoquin is 1:4, 3:7, 2:3 or 3:2. The more preferred mass ratio of Aminopyrifen and Ipflufenoquin is 1:4.

[0010] The bactericidal composition of the present invention further comprises agricultural auxiliary components and is configured into a pesticide preparation. The total mass percentage of the active components is 5%-75%. The preferred total mass percentage of the active components is 10%-60%.

[0011] The bactericidal composition preparation of the present invention is a suspension, microemulsion, aqueous emulsion, wettable powder, water-dispersible granule or dispersible oil suspension, preferably a suspension or water-dispersible granule.

[0012] The fungicide composition of the present invention is used for preventing and controlling crop diseases, and preferably the crop disease is wheat fusarium head blight.

[0013] The fungicide composition of the present invention can inhibit the formation of toxin bodies of wheat fusarium.

[0014] Compared with the prior art, the present invention achieves the following excellent technical effects:

[0015] The bactericidal composition of the present invention has excellent prevention and control effects on wheat yield reduction and toxin pollution caused by fusarium scurf, and can also control the generation of drug resistance of pathogenic bacteria and prolong the service life of the agent.

[0016] When the mass ratio of Aminopyrifen and Ipflufenoquin is 1:4, 3:7, 2:3 or 3:2, it exhibits a significant synergistic effect on wheat fusarium head blight, which can effectively reduce the dosage of active ingredients and is safe for crops.

[0017] The study found that the fungicide composition can inhibit the formation of toxin bodies of wheat fusarium. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :Effects of Aminopyrifen and Ipflufenoquin combination on conidia germination of Gibberella fusca

[0019] Figure 2 :Effects of Aminopyrifen and Ipflufenoquin on the Toxisome Formation of Gibberella fusca DETAILED DESCRIPTION

[0020] The following examples are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0021] Example 1. Indoor toxicity test of the combination of Aminopyrifen and Ipflufenoquin against wheat fusarium

[0022] Test object: wheat fusarium (Fusarium graminearum), which was isolated and purified from wheat ears with natural fusarium graminearum in the field. After the strain was identified by morphology and pathogenicity, it was prepared into 15% glycerol bacteria and frozen in an ultra-low temperature refrigerator for use.

[0023] Test method: refer to the mycelium growth rate method in the "People's Republic of China Agricultural Industry Standard NY / T 1156.6-2006 Pesticide Indoor Bioassay Test Standard for Fungicides Part 6: Determination of the Combined Effect of Mixtures". Each single agent and each group of mixed agents are set at 5 mass concentrations according to the content of the active ingredient, and the inhibition rate of the growth of the pathogen is between 10% and 95%. The wheat fusarium in the frozen tube of the strain is activated on the PDA culture medium. When the colony size is about 4 / 5 of the culture dish, a sterilized stainless steel puncher is used to punch a 5mm diameter bacterial cake from the edge of the colony. The sterile inoculation needle transfers the bacterial cake to the center of the pre-prepared series of concentrations of the drug-containing PDA plate, and the plate is placed in an incubator with a temperature of 25℃ and a light cycle of 12L:12D for 3 days. The diameter of the colony is measured by the cross method to calculate the corrected inhibition percentage; then the EC of each agent is calculated based on the linear regression equation between the probability value of the inhibition rate and the logarithm of the series concentration. 50 The value was repeated 4 times for each treatment, and the treatment without drug was set as blank control. The synergistic coefficient (SR) was calculated according to the Wadley method, and a comprehensive evaluation of the mixing effect was made. The SR value was calculated according to the following formula:

[0024] EC 50 (Theoretical value) = (P A +P B ) / (P A / EC 50 A+P B / EC 50 B)

[0025] SR=EC 50 (Theoretical value) / EC 50 (Actual value)

[0026] Among them, P A is the percentage of drug A in the mixture, P B It is the percentage of drug B in the mixture. SR≤0.5 indicates antagonistic effect, 0.5<SR<1.5 indicates additive effect, and SR≥1.5 indicates synergistic effect.

[0027] Test results: From the data in Table 1, it can be seen that Aminopyrifen and Ipflufenoquin both have excellent antibacterial effects on wheat fusarium; the combinations of Aminopyrifen and Ipflufenoquin in ratios of 1:4, 3:7, 2:3 and 3:2 show synergistic effects, among which the maximum synergistic ratio is 1:4.

[0028] Table 1 Results of indoor toxicity test of Aminopyrifen and Ipflufenoquin against wheat fusarium

[0029] Pharmacy Regression equation Correlation coefficient <![CDATA[EC 50 (μg / mL)]]> SR Aminopyrifen(APF) y=0.8867x+5.9882 0.9862 0.0768 / Ipflufenoquin(IFQ) y=1.4203x+5.3911 0.9955 0.5305 / APF+IFQ(1:9) y=0.5716x+5.1400 0.9464 0.5689 0.5862 APF+IFQ(1:4) y=0.5567x+5.5279 0.9852 0.1127 2.1578 APF+IFQ(3:7) y=0.5319x+5.5105 0.9409 0.1097 1.7444 APF+IFQ(2:3) y=0.5899x+5.6382 0.9933 0.0828 1.9051 APF+IFQ(1:1) y=1.6611x+6.6338 0.9350 0.1038 1.2926 APF+IFQ(3:2) y=0.7996X+5.9981 0.9932 0.0565 2.0661 APF+IFQ(7:3) y=0.5422x+5.3688 0.9725 0.2089 0.4945 APF+IFQ(4:1) y=0.6146x+5.6032 0.9939 0.1044 0.8874 APF+IFQ(9:1) y=1.0099x+6.1272 0.9771 0.0765 1.0978

[0030] Example 2. Effect of Aminopyrifen and Ipflufenoquin on the germination of conidia of wheat fusarium

[0031] Experimental method: Aminopyrifen and Ipflufenoquin single agent and their mixture with a ratio of 1:4 were set at 5 mass concentrations according to the content of active ingredients, and the inhibition rate of conidia germination of wheat fusarium was between 10% and 95%. The activated wheat fusarium was cultured on PDA medium for 3 days, and a 5mm diameter bacterial cake was punched from the edge of the colony with a sterilized stainless steel puncher. Five bacterial cakes were picked up with an inoculation needle and inoculated into a 50mL conical flask containing 30mL of induced spore production culture medium, and incubated in a shaker at 25℃, 12L:12D, and 180r / min. After shaking for 4 days, the conidia filtrate was collected by filtering with two layers of sterile gauze into a 50mL sterile centrifuge tube, and the conidia yield was counted by a hemocytometer, and the conidia were adjusted to a final concentration of 1×10 4 / mL. Aminopyrifen and Ipflufenoquin were added as single agents and their mixtures in a ratio of 1:4, respectively, and each treatment was repeated 3 times. The treatment without the drug was set as a blank control. The cells were placed at 25°C, 180 r / min, and in the dark for 3 hours, and the germination rate was counted. The germinated conidia were stained with fluorescent whitening agent (calcofluor white, CFW), and the conidia morphology in differential interference contrast (DIC) and excitation wavelength 405nm channel were observed under a laser confocal microscope, and 8-bit mode images were collected. Under the microscope, the length of the germ tube of the spores reaching the short diameter of the spore or more was considered to be germinated. 300 spores were randomly counted for each treatment, and the percentage of inhibition of conidia germination was calculated. Then, according to the linear regression equation between the probability value of the inhibition rate and the logarithm of the series concentration, the EC value of the drug on conidia germination was calculated. 50 value.

[0032] Test results: From Table 2 and Figure 1 From the data, it can be seen that both Aminopyrifen and Ipflufenoquin as single agents and their mixtures in a ratio of 1:4 have a strong inhibitory effect on the conidia germination of wheat fusarium, but the mixture of Aminopyrifen and Ipflufenoquin in a ratio of 1:4 has a more prominent inhibitory effect on the conidia germination of wheat fusarium.

[0033] Table 2 Results of the germination of conidia of wheat fusarium by the combination of Aminopyrifen and Ipflufenoquin

[0034] Pharmacy Regression equation Correlation coefficient <![CDATA[EC 50 (μg / mL)]]> Aminopyrifen(APF) y=0.7901x+5.7603 0.9863 0.1091 Ipflufenoquin(IFQ) y=1.4021x+5.3253 0.9933 0.5861 APF+IFQ(1:4) y=1.2149x+6.3818 0.9922 0.0729

[0035] Note: APF stands for Aminopyrifen and IFQ stands for Ipflufenoquin.

[0036] Example 3. Effect of the combination of Aminopyrifen and Ipflufenoquin on the toxin formation of wheat fusarium

[0037] Experimental method: It has been reported (see literature: Boenisch MJ, Broz KL, Purvine SO, Chrisler WB, Nicora CD, Connolly LR, Freitag M, Baker SE, Kistler HC. 2017. Structural reorganization of the fungal endoplasmic reticulum upon induction of mycotoxin biosynthesis. Scientific Reports, 7: 44296, Tang GF, Chen Y, Xu JR, Kistler HC, Ma ZH. 2018. The fungal myosin I is essential for Fusarium toxisomeformation. PLoS Pathogens, 14(1): e1006827) that the biosynthesis of DON toxin of Fusarium fusarium is carried out in a special cell structure called "toxin body". Therefore, we activated the strain PH-1∷FgTri1-GFP and grew it on PDA medium for 3 days. We used a stainless steel cork puncher with a diameter of 5 mm to punch 4 bacterial cakes at the edge of the colony, inoculated them into 200 mL YEPD culture medium, and cultured them at 25°C and 180 r / min for 24 h. The inoculation needle picked up a mycelium ball and placed it in a 2 mL centrifuge tube and added a 5 mm diameter steel ball. The tissue grinder was oscillated at 65 Hz for 1 min, and 100 μL of bacterial solution was inoculated into 30 mL TBI culture medium. Aminopyrifen, Ipflufenoquin single agent and their 1:4 mixture were added respectively. The final concentration of each agent was 0.07 μg / mL. The treatment without agent was set as a blank control, wrapped in tin foil to avoid light, and cultured in a shaker at 25°C and 180 r / min for 48 h. The hyphae were aspirated and placed under a laser confocal microscope to observe the differential interference contrast (DIC) and green fluorescence signals in the 488 nm excitation wavelength channel, and the images in 8-bit mode were collected.

[0038] Test results: Figure 2It can be seen that when only the solvent DMSO was added to the TBI culture medium, a large number of toxin bodies were produced in the hyphae of wheat fusarium. The more toxin bodies there were, the greater the amount of DON toxin produced by the strain; after the fusarium was treated with 0.07μg / mLAminopyrifen, the green fluorescence intensity of FgTri1-GFP and the number of toxin bodies in the hyphae were significantly reduced; after being treated with a mixture of Aminopyrifen and Ipflufenoquin in a ratio of 1:4, the expression of FgTri1-GFP in the fusarium was extremely weak and toxin bodies could not be formed normally; and after the fusarium was treated with 0.07μg / mLIpflufenoquin, the green fluorescence intensity of FgTri1-GFP and the number of toxin bodies in the hyphae were significantly increased.

[0039] Example 4. Field efficacy of the combination of Aminopyrifen and Ipflufenoquin against wheat scab

[0040] Test method: The field efficacy test of the combination of Aminopyrifen and Ipflufenoquin against wheat scab was conducted in the High-tech Agricultural Park in Hefei City, Anhui Province and the Modern Agricultural Science and Technology Demonstration Park in Fengtai County, Anhui Province. The wheat varieties were Luyuan 502 and Yannong 5286 (both susceptible varieties). According to the provisions of the "Agricultural Industry Standard of the People's Republic of China NY / T1464.15-2007 Pesticide Field Efficacy Test Guidelines Part 15: Fungicide Control of Wheat Scab", the test was designed with 5 treatments, including 4 treatments and 1 blank control. Each treatment was repeated in 3 plots, and each plot was 30m 2 (3m×10m), the treatment groups were randomly arranged, and adjacent treatments were set up with 1m protection rows. The pesticide was applied once at the beginning of wheat heading and flowering, and then applied again after an interval of 6 days. The results were investigated at the milky stage. Each experimental plot was sampled at 5 points on the diagonal line, and 100 ears were investigated at each point. The percentage of dead ear area to the entire wheat ear area was used for classification, and the number of diseased ears and the total number of ears at each level were recorded. The disease classification standards are as follows:

[0041] Level 0: The whole ear is disease-free;

[0042] Level 1: The dead ear area accounts for less than 1 / 4 of the total ear area;

[0043] Level 3: The dead ear area accounts for 1 / 4 to 1 / 2 of the whole ear area;

[0044] Level 5: The dead ear area accounts for 1 / 2 to 3 / 4 of the whole ear area;

[0045] Level 7: The area of ​​dead ears accounts for more than 3 / 4 of the total ear area.

[0046] According to the survey results, the disease index and drug efficacy were calculated according to the following formula:

[0047]

[0048]

[0049] Test drug: All percentages in the preparation ratios are by mass.

[0050] (1) 20% Aminopyrifen suspension

[0051] Weigh 20% Aminopyrifen, 2.5% sodium polynaphthaldehyde sulfonate, 2% TERSPERSE 2425, 2.5% Nongru 2201#, 0.1% xanthan gum, 4% polyethylene glycol 4000, 0.3% sodium benzoate, and 0.1% phosphate defoamer, and add deionized water to 100% by mass.

[0052] According to the formula ratio, with water as the medium, add the original drug, dispersant, antifreeze and defoamer into the batching kettle and mix evenly. Then, ball mill it for 30 minutes, pump it into the dispersion tank for high-speed shearing for 3 minutes, and grind it fully with a sand mill to make a suspension.

[0053] (2) 20% AFP·IFQ suspension

[0054] Weigh 20% Ipflufenoquin, 2.5% sodium polynaphthaldehyde sulfonate, 2% TERSPERSE 2425, 2.5% Nongru 2201#, 0.1% xanthan gum, 4% polyethylene glycol 4000, 0.3% sodium benzoate, and 0.1% phosphate defoamer, and add deionized water to 100% by mass.

[0055] According to the formula ratio, with water as the medium, add the original drug, dispersant, antifreeze and defoamer into the batching kettle and mix evenly. Then, ball mill it for 30 minutes, pump it into the dispersion tank for high-speed shearing for 3 minutes, and grind it fully with a sand mill to make a suspension.

[0056] (3) 20% AFP·IFQ suspension

[0057] Weigh 4% Aminopyrifen, 16% Ipflufenoquin, 2.5% sodium polynaphthaldehyde sulfonate, 2% TERSPERSE 2425, 2.5% Nongru 2201#, 0.1% xanthan gum, 4% polyethylene glycol 4000, 0.3% sodium benzoate, and 0.1% phosphate defoamer, and add deionized water to 100% by mass.

[0058] According to the formula ratio, with water as the medium, add the original drug, dispersant, antifreeze and defoamer into the batching kettle and mix evenly. Then, ball mill it for 30 minutes, pump it into the dispersion tank for high-speed shearing for 3 minutes, and grind it fully with a sand mill to make a suspension.

[0059] The test results are shown in Table 3.

[0060] Table 3 Field efficacy results of Aminopyrifen and Ipflufenoquin combined with wheat fusarium head blight

[0061]

[0062] Note: APF stands for Aminopyrifen and IFQ stands for Ipflufenoquin.

[0063] *Different lowercase letters after the data in the same column indicate significant differences at the P<0.05 level.

[0064] From the results of the field efficacy test on the prevention and control of wheat fusarium fusarium (Table 3), it can be seen that the combination of Aminopyrifen and Ipflufenoquin can significantly improve the control effect of wheat fusarium fusarium. Under the same dosage, the compound agent is significantly better than the single agent in terms of prevention effect, and is safe for wheat growth within the test range.

[0065] In summary, the results of indoor bioassay, observation of conidia germination, observation of toxin formation and field efficacy test show that the composition of the present invention has a synergistic effect and has excellent antibacterial and disease prevention effects on Fusarium fusarium and wheat Fusarium fusarium caused by Fusarium fusarium. The composition is composed of effective ingredients with different action mechanisms, which is conducive to overcoming and delaying the development of drug resistance of pathogens and is safe for the growth of test crops.

Claims

1. A bactericidal composition containing Aminopyrifen and Ipflufenoquin, characterized in that: The active ingredients are composed of Aminopyrifen and Ipflufenoquin, and the mass ratio of Aminopyrifen and Ipflufenoquin is (1:4)-(2:3) or 3:

2.

2. The bactericidal composition according to claim 1, characterized in that The mass ratio of Aminopyrifen to Ipflufenoquin is 1:4, 3:7, 2:3 or 3:

2.

3. The bactericidal composition according to claim 1 or 2, characterized in that: Contains agricultural auxiliary ingredients and is formulated into pesticide preparations.

4. The bactericidal composition according to claim 3, characterized in that: The total mass percentage of the active ingredients is 5%-75%.

5. The bactericidal composition according to claim 3, characterized in that: The total mass percentage of the active ingredients is 10%-60%.

6. The bactericidal composition according to claim 4, characterized in that: The pesticide formulation is a suspension, microemulsion, emulsion in water, wettable powder, water dispersible granule or dispersible oil suspension.

7. Use of the fungicidal composition according to any one of claims 1 to 7 for preventing and controlling crop diseases.

8. The use according to claim 7, characterized in that The crop disease is wheat scab.

9. Use of the fungicidal composition according to any one of claims 1 to 7 for inhibiting the formation of toxin bodies of wheat fusarium.

Images