Application of phenylglycine nitrile in the preparation of plant pathogen inhibitors

Abstract

The present invention relates to the application of phenylacetonitrile in the preparation of plant pathogen inhibitors. The phenylacetonitrile described in the present invention can effectively inhibit the growth of various plant pathogen genera such as Rhizoctonia, Botrytis cinerea, Sclerotinia, Fusarium, Pseudomonas, etc., and can be used for the development of biological pesticides, with broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease prevention and control, and particularly relates to application of anilinoacetonitrile in the preparation of plant pathogen inhibitors. Background Art

[0002] In recent years, bacterial and fungal plant diseases have become increasingly frequent, affecting nearly every plant, whether vegetables, fruit trees, or other crops. Multiple bacterial and fungal diseases can occur simultaneously on a single plant. These diseases are extremely devastating and difficult to control. Once an infection occurs, a growing area can become a lifelong epidemic zone. Their explosive, epidemic, and devastating nature often results in significant yield reductions. The long-term, high-volume application of chemical pesticides, including antibiotics, has led to increased resistance, making pest and disease control more difficult, increasing costs, leading to excessive pesticide residues, and reducing product quality. Therefore, there is an urgent need to develop low-toxic, non-resistant plant antimicrobial agents that are less likely to induce resistance in pathogens.

[0003] Phenylaminoacetonitril, with the molecular formula C8H8N2, is a khaki or yellowish-brown solid with no distinctive odor and is readily soluble in organic solvents. As an intermediate in the production of indigo, it is primarily used for denim dyeing. Currently, there are no reports on the use of phenylaminoacetonitrile in inhibiting plant pathogens. Summary of the Invention

[0004] Through more than 10,000 target screening tests and research and development, the applicant found that aniline acetonitrile has a significant inhibitory effect in inhibiting bacterial diseases such as rice bacterial leaf streak and tobacco wilt, as well as fungal diseases such as cucumber damping-off, and can be developed as a new potential fungicide resource.

[0005] In view of this, the object of the present invention is to provide a new use of anilinoacetonitrile.

[0006] The new application of the anilinoacetonitrile provided by the present invention is application of the anilinoacetonitrile in the preparation of plant pathogen inhibitors.

[0007] The plant pathogens of the present invention include plant fungi or bacteria.

[0008] The plant pathogenic bacteria of the present invention are of at least one genus selected from the group consisting of Rhizoctonia, Botrytis cinerea, Sclerotinia, Fusarium, and Pseudomonas.

[0009] Furthermore, the plant pathogenic bacteria are of at least one species selected from the group consisting of Rhizoctonia, Botrytis cinerea, Sclerotinia, and Pseudomonas.

[0010] The hosts of the plant pathogen include but are not limited to strawberry, grape, broad bean, onion, ginseng, clover, cereals, oats, eggplant, corn, etc.; the corn refers to corn.

[0011] Among the plant pathogens in the present invention, Botrytis belongs to at least one selected from Botrytis cinerea, Botrytis fragariae, Botrytis viticola, Botrytis fabae, Botrytis allii;

[0012] Among the Sclerotinia, it is at least one selected from Sclerotinia ginseng, Sclerotinia trifoliorum, Sclerotinia sclerotiorum, Sclerotinia minor, Sclerotinia asari;

[0013] Among the Rhizoctonia, it is at least one selected from Rhizoctonia oryzae, Rhizoctonia zeae, Rhizoctonia cerealis, Rhizoctonia solani;

[0014] Among the Fusarium, it is at least one selected from Fusarium avenaceum, Fusarium concolor, Fusarium culmorum, Fusarium graminearum, Fusarium moniliforme, Fusarium nivale, Fusarium oxysporum, Fusarium solani;

[0015] The Pseudomonas includes Pseudomonas solanacearum.

[0016] The dosage form of the plant pathogen inhibitor in the present invention is selected from at least one of powder, wettable powder, granule, water dispersible granule, suspension, emulsifiable concentrate, microemulsion, aqueous solution.

[0017] Furthermore, the dosage form of the plant pathogen inhibitor is aqueous solution.

[0018] The effective concentration of the phenylacetonitrile in the present invention during application is 10 - 500 ppm, further 50 - 150 ppm; more preferably 100 ppm.

[0019] The phenylacetonitrile in the present invention can also be used in combination with other agricultural fungicides, including but not limited to jinggangmycin, hymexazol, guanshengsu, etc.

[0020] The present invention also provides an agricultural fungicide composition, whose components include but not limited to the active ingredient phenylacetonitrile, auxiliary materials, solvents;

[0021] Furthermore, it includes 0.1 - 20 wt% phenylacetonitrile;

[0022] Furthermore, the auxiliary materials include but not limited to emulsifiers; more specifically, it includes 0.5 - 30 wt% emulsifiers;

[0023] Furthermore, the solvent is an inorganic solvent, an organic solvent; the function of the solvent selected in the present invention is to dissolve phenylacetonitrile; more specifically, it is an organic solvent; the mass percentage of the organic solvent is 0.5 - 30 wt%.

[0024] Furthermore, the organic solvent is an alcohol solvent, acetone; the emulsifier is Tween - 80.

[0025] Further, the alcohol solvent is methanol or ethanol.

[0026] Further, in the present invention, the agricultural fungicide composition further comprises at least one component selected from jinggangmycin, hymexazol, and guansheng element; more preferably, it is jinggangmycin and / or hymexazol.

[0027] Beneficial effects of the present invention: Compared with common chemical pesticides, the phenylacetonitrile in the present invention, as a novel potential fungicide, is not likely to cause phytotoxicity to crops, has a low dosage, and is not likely to develop resistance. The effective concentration of general chemical pesticides is about 1000 ppm, while the antibacterial effective concentration of phenylacetonitrile in the present invention is only about 100 ppm. It can be used for the development of biological pesticides and has broad application prospects. Description of the Drawings

[0028] Figure 1 It is the antibacterial effect diagram of the present invention and other antibacterial agents. A, B, C, and D are Rhizoctonia solani, Botrytis cinerea, Sclerotinia sclerotiorum, and Fusarium graminearum in sequence. In each figure, from left to right and from top to bottom, they represent CK, solvent control, 1% aqueous solution of phenylacetonitrile, and 98% hymexazol in sequence. Detailed Embodiments

[0029] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0031] It should be clear that the experimental methods used in the following embodiments are all conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can all be obtained from commercial channels unless otherwise specified.

[0032] As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover non-exclusive inclusion. For example, a composition, step, method, or article containing the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, or article.

[0033] The 98% hymexazol soluble powder applied in the present invention (Tianjin Lvheng Chemical Co., Ltd., pesticide registration number: PD20190146, production date: 20211008); Zhongshengmycin (effective content 3%, Fujian Kaili Biological Products Co., Ltd., pesticide registration number: PD20190224; production batch number: 20210315); Jinggangmycin (effective content 13%, Tonglu Huifeng Biotechnology Co., Ltd., Zhejiang Province, pesticide registration number: PD20130887; production batch number: 20220708).

[0034] Example 1

[0035] An agricultural fungicide composition comprising the following components: 1 wt% anilinoacetonitrile, 5 wt% organic solvent ethanol, 5 wt% emulsifier Tween-80, and the balance being water.

[0036] The emulsifier used in the present invention will not affect the application effect of the preparation.

[0037] Preparation method of the preparation: The active ingredient anilinoacetonitrile is mixed with the organic solvent and water according to the above dosage percentages and completely dissolved, and then the emulsifier is added, and stirred evenly to obtain an aqueous solution.

[0038] Example 2

[0039] Detect the antifungal ability of the prepared 1% anilinoacetonitrile aqueous solution

[0040] Method: The growth rate method is used for the activity test, and the specific steps are as follows:

[0041] (1) Under a sterile environment, the fungal strains stored at -80 °C are activated respectively. Among them, Botrytis cinerea Pers.ex Fr. (cucumber leaves), Rhizoctonia solani, Colletotrichum gloeosporioides and other fungi are inoculated onto PDA medium and cultured at 25 °C for 3 d to 5 d, and detected according to the growth characteristics of the strains.

[0042] (2) Prepare the medicament under a sterile environment, including the aqueous solution of Example 1 and the existing fungicide hymexazol. The whole experimental process is carried out under sterile operation, and all consumables need to be sterilized by high temperature and high pressure and are not reused; Weigh the drugs according to the set concentration, add them to the sterilized centrifuge tubes, add sterile water to make up to 10 mL, and shake well for standby.

[0043] (3) Heat and melt the prepared rye medium and PDA medium, cool to 45 °C to 50 °C, take 1 mL of each prepared sample in a petri dish respectively, add 9 mL of the medium, shake gently, make marks, and place horizontally to cool.

[0044] (4) Use a punch to make holes in the activated medium in concentric circles; then inoculate it onto the medium containing the agent and incubate it in an incubator at 25°C. When the blank mycelium grows to cover 2 / 3 of the petri dish or when the two fungal colonies grow close to each other, use the cross method to measure the diameter of the colony, calculate the average diameter, and calculate the control effect of the agent.

[0045] Among them, the formula for calculating the control effect is: I =

(D0 - Dt) / (D0 - 4)

[0046] I: Mycelial growth inhibition rate;

[0047] D0: Diameter of the blank colony in mm;

[0048] Dt: Diameter of the colony treated with the agent in mm.

[0049] Measure the antibacterial effects of the examples and the existing chemical fungicide hymexazol on fungi, where CK represents the treatment with clear water.

[0050] Among them, hymexazol is a common chemical control for fungal diseases in current agriculture, which is widely used and has a broad spectrum of targets. Diluting it 3000 times is the recommended application concentration in the product manual.

[0051] Table 1 Antibacterial effects of 1% anilinoacetonitrile aqueous solution of the present invention (%)

[0052]

[0053] Note: For the genus Rhizoctonia in Table 1, Rhizoctonia cerealis was selected; for the genus Botrytis, Botrytis cinerea was selected; for the genus Sclerotinia, Sclerotinia sclerotiorum was selected; for the genus Fusarium, Fusarium solani was selected.

[0054] Example 3

[0055] Based on the above test results, in this embodiment, through a large number of plate tests, the inhibition rules of 1% anilinoacetonitrile aqueous solution against different pathogenic bacteria are screened. It has a significant inhibitory effect on fungi of the genus Rhizoctonia, Botrytis, and Sclerotinia, but has no significant inhibitory effect on fungi of the genus Fusarium. Therefore, in this embodiment, each genus is refined into different species, and antibacterial tests are carried out separately to verify its antibacterial rules. The test method refers to Example 2. Botrytis cinerea, Botrytis fragariae, Botrytis sinoviticola, Botryotinia fabae, and Botrytis aclada in the genus Botrytis; Sclerotinia schinseng, Sclerotinia trifoliorum, Sclerotinia sclerotiorum, Sclerotinia minor, and Sclerotinia asari in the genus Sclerotinia; Rhizoctonia oryzae, Rhizoctonia zeae, Rhizoctonia cerealis, and Rhizoctonia solani in the genus Rhizoctonia; Fusarium avenaceum, Fusarium concolor, Fusarium culmorum, Fusarium graminearum, Fusarium moniliforme, Fusarium nivale, Fusarium oxysporum, and Fusarium solani in the genus Fusarium are respectively selected.

[0056] Table 2 Antibacterial effect of 1% anilinoacetonitrile aqueous solution of the present invention against the genus Rhizoctonia (%)

[0057]

[0058] Table 3 Antibacterial effect of 1% anilinoacetonitrile aqueous solution of the present invention against the genus Botrytis (%)

[0059]

[0060] Table 4 Antibacterial effect of 1% anilinoacetonitrile aqueous solution of the present invention against the genus Sclerotinia (%)

[0061]

[0062] Note: The negative values of the control effect in the table indicate that the reagent promotes the growth of pathogenic bacteria and has no inhibitory effect.

[0063] Table 5 Antibacterial effect of 1% anilinoacetonitrile aqueous solution of the present invention against Fusarium spp. (%)

[0064]

[0065]

[0066] The above test results show that the 1% anilinoacetonitrile aqueous solution of the present invention has a relatively significant inhibitory effect on Rhizoctonia oryzae, Rhizoctonia zeae, Rhizoctonia solani, Rhizoctonia cerealis in Rhizoctonia, Botrytis cinerea, Botrytis fragariae, Botrytis vinifera, Botrytis fabae, Botrytis allii in Botrytis, Sclerotinia ginseng, Sclerotinia trifoliorum, Sclerotinia sclerotiorum, Sclerotinia minor, Sclerotinia asari in Sclerotinia, and can be used to prepare antibacterial agents, but has an insignificant inhibitory effect on fungi such as Fusarium avenaceum, Fusarium concolor, Fusarium culmorum, Fusarium moniliforme, Fusarium oxysporum, Fusarium nivale, Fusarium oxysporum f. sp. lycopersici, Fusarium solani in Fusarium.

[0067] Example 4

[0068] Antibacterial test of 1% anilinoacetonitrile aqueous solution prepared in Example 1 against bacterial pathogens

[0069] Method: The OD method was used for the activity test.

[0070] Prepare Medium B: Yeast extract 1 g / L, Bacto peptone 10 g / L, Casein amino acids 1 g / L; 1000 mL of water was heated and dissolved (15 g / L of agar was added for solid medium), dispensed and sterilized at 121 °C for 30 min by moist heat.

[0071] (1) Under sterile conditions, the tobacco bacterial wilt pathogen, citrus canker pathogen, and rice bacterial leaf streak pathogen stored at low temperature were activated. The frozen strains were inoculated into Medium B on a superclean workbench, and cultured in a constant temperature shaker at 180 rpm / min and 30 °C until OD600 = 1.0. After streaking on the Medium B plate, it was inverted and cultured in a constant temperature incubator at 30 °C for 24 h to obtain single colonies. Inoculated into PDA medium and cultured at 25 °C for 3 d - 5 d, and detected according to the growth characteristics of the strains.

[0072] (2) Prepare the reagent under sterile conditions. The whole experiment was carried out under aseptic operation, and all consumables needed to be sterilized by high temperature and high pressure and not reused. Weigh the drug according to the set concentration, add it to the sterilized measuring cylinder, quantitatively add 30 mL of Medium B to the sterilized measuring cylinder, and add 300 μL of the test reagent to each bottle of medium, shake well and set aside.

[0073] (3) Take a sterilized conical flask, add 20 mL of liquid medium B, pick a well-grown colony (the center of the colony is pink and there is a wide white band at the edge with strong fluidity) from the cultured single-colony plate into the medium, and culture it in a constant-temperature shaker at 30 °C and 180 rpm / min until OD600 = 1.0 to prepare the inoculum mother liquor of the bacterial solution. Add the inoculum according to the ratio of medicated culture solution: inoculum = 200:1, and culture it in a constant-temperature shaker at 180 rpm / min and 30 °C.

[0074] (4) Incubate at a constant temperature for 24 h, measure the OD600 of the bacterial solution with a UV spectrophotometer, and calculate the growth inhibition rate of the medicament.

[0075] Formula for calculating the bacteriostatic rate: I =

(D0 - Dt) / (D0)

[0076] I: Growth inhibition rate

[0077] D0: OD600 of the bacterial solution in the blank group

[0078] Dt: OD600 of the bacterial solution treated with the medicament

[0079] Table 6 Control effect of 1% anilinoacetonitrile aqueous solution of the present invention against bacterial pathogens

[0080]

[0081] Note: Diluting Zhongshengmycin 3% by 500 times is the recommended application concentration in the product instruction manual.

[0082] In summary, except for Fusarium, the 1% anilinoacetonitrile aqueous solution of the present invention has a significant inhibitory effect on other fungi and bacteria. As a commonly used dye, anilinoacetonitrile has not been found to have bacteriostatic function and can be developed as a new potential fungicide in the future.

[0083] Example 5

[0084] Regarding the bacteriostatic experiment of the 1% anilinoacetonitrile aqueous solution prepared in Example 1

[0085] Method: An indoor in-vivo potted plant experiment was adopted to determine the control effect of the medicament on cucumber damping-off.

[0086] (1) Bacterial strain culture: Transfer the cucumber damping-off pathogen to a PDA plate for activation for 3 days, and then transfer it to a PDB liquid medium, 6 pieces per bottle, culture at 25 °C and 200 rpm for 3 days;

[0087] (2) Inoculation: The cultured mycelium was crushed into a mycelium suspension with a mycelium grinder for 5 min. The treated different mycelium suspensions were mixed with a certain amount of planting soil (earthworm manure + vermiculite), and cucumber seeds of the same pre-germinated size were planted. 15 mL of the prepared diluted liquid medicine was uniformly added to each treatment according to the test requirements. Among them, for the cucumber damping-off control agent, the 11% metalaxyl + pyrimethanil suspension seed coating agent was used to coat the cucumber seeds in advance according to the required seed coating agent concentration. Then, the soil was covered and cultured normally. On the 4th day after the first treatment, 15 mL of the liquid medicine was added to each treatment according to the test requirements, and the control agent, the 11% metalaxyl + pyrimethanil suspension seed coating agent, was added with 15 mL of the liquid medicine at a dilution of 1500 times. After all the treatments emerged completely, proper moisture was maintained for cultivation until the disease investigation results were obtained.

[0088] (3) After the blank control was fully diseased, the disease grading investigation was carried out, and the disease index and control effect were calculated. Severity grading standards for cucumber fusarium wilt, root rot, and damping-off:

[0089] Grade 0: No symptoms;

[0090] Grade 1: Slight symptoms on the stems and leaves;

[0091] Grade 2: The plants were slightly wilted, necrosis spots appeared on the stems, and the leaves were yellowed;

[0092] Grade 3: The plants were moderately wilted, and the leaves drooped and yellowed;

[0093] Grade 4: The plants were severely wilted, lodged, and died.

[0094] Disease index = ∑(disease grade of diseased plants × number of plants at that disease grade) / (total number of investigated plants × highest disease grade) × 100% was used to calculate the disease index;

[0095] Relative control effect (%) = (disease index of the control group - disease index of the treatment group) / disease index of the control group × 100% was used to calculate the relative control effect.

[0096] The 1% cyanoacetamide aniline aqueous solution was compounded with the common fungicides 98% hymexazol and 13% jinggangmycin respectively, and the test results are shown in the following table.

[0097] Table 7 Control effects of different formulations on cucumber damping-off

[0098]

[0099] Both 98% hymexazol and 13% jinggangmycin are common control agents against damping-off. The results of this experiment show that when 98% hymexazol diluted 1500 times and 13% jinggangmycin diluted 1500 times are used alone, the control effects are relatively low, which are 41.8% and 40.8% respectively. After being compounded with the 1% phenylacetonitrile aqueous solution of the present invention, the control effect can be increased by about 35%. This shows that the preparation of the present invention has a significant synergistic effect when compounded with hymexazol and jinggangmycin in the field and has great application potential.

[0100] The foregoing examples are illustrative only and are used to explain some of the features of the method of the present invention. The appended claims are intended to claim as broad a scope as can be conceived, and the embodiments presented herein are merely illustrative of selected embodiments from all possible combinations of embodiments. Therefore, the intention of the applicant is that the appended claims should not be limited by the selection of examples that illustrate the features of the present invention. Some of the numerical ranges used in the claims also include sub-ranges within them, and variations within these ranges should also be construed as being covered by the appended claims where possible.

Claims

1. Use of phenylglycine nitrile in the preparation of plant pathogen inhibitors, characterized in that, The genus of the plant pathogen is selected from at least one of the genera Rhizoctonia, Botrytis, Sclerotinia, Fusarium, and Pseudomonas.

2. The application according to claim 1, characterized in that, The host of the plant pathogen is selected from at least one of strawberry, grape, broad bean, onion, ginseng, clover, cereal, eggplant, and maize.

3. The application according to claim 2, wherein The Botrytis in the plant pathogen is selected from at least one of Botrytis cinerea f. sp. fragariae, Botrytis viniferae, Botrytis fabae, and Botrytis allii. The Sclerotinia in the plant pathogen is selected from at least one of Sclerotinia ginseng, Sclerotinia trifoliorum, Sclerotinia sclerotiorum, and Sclerotinia asari. The Rhizoctonia in the plant pathogen is selected from at least one of Rhizoctonia oryzae, Rhizoctonia zeae, Rhizoctonia cerealis, and Rhizoctonia solani. The Fusarium in the plant pathogen is selected from at least one of Fusarium avenaceum, Fusarium concolor, Fusarium culmorum, Fusarium graminearum, Fusarium moniliforme, Fusarium nivale, Fusarium oxysporum, and Fusarium solani. The Pseudomonas in the plant pathogen includes Pseudomonas solanacearum.

4. The application according to claim 1, characterized in that, The dosage form of the plant pathogen inhibitor is selected from at least one of powder, wettable powder, granule, water dispersible granule, suspension, emulsifiable concentrate, microemulsion, and aqueous solution.

5. The application according to claim 4, characterized in that, The dosage form of the plant pathogen inhibitor is aqueous solution.

6. The application according to claim 1, characterized in that, The effective concentration of the phenylacetonitrile is 10 - 500 ppm.

7. The application according to claim 6, characterized in that, The effective concentration of the phenylacetonitrile is 50 - 150 ppm.

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