阅读说明：本技术 用于防治黄单胞菌的杀菌剂及其应用 (Bactericide for preventing and treating xanthomonas and application thereof ) 是由 王莉 武瑶 汪艳丽 孟繁凡 钱韦 黄贵修 时涛 于 2021-01-19 设计创作，主要内容包括：本发明涉及农业保护技术领域,公开了用于防治黄单胞菌的杀菌剂及其应用。所述杀菌剂含有麝香草酚和/或原儿茶酸。所述用于防治黄单胞菌的杀菌剂属于从植物中提取的植物源杀菌剂,具有高效、低毒、环境友好、持久性好以及可降解的特点。(The invention relates to the technical field of agricultural protection, and discloses a bactericide for preventing and treating xanthomonas and application thereof. The bactericide contains thymol and/or protocatechuic acid. The bactericide for preventing and treating xanthomonas belongs to a botanical bactericide extracted from plants, and has the characteristics of high efficiency, low toxicity, environmental friendliness, good durability and degradability.)

1. A bactericide for controlling Xanthomonas campestris, which comprises thymol and/or protocatechuic acid.

2. The bactericide for controlling xanthomonas according to claim 1, which further contains a production-acceptable adjuvant and a carrier.

3. The fungicide according to claim 2, wherein said productively allowable adjuvants and carriers include dispersants, wetting agents, emulsifiers, stickers, thickeners, antifreezes, antifoaming agents, diluents, solvents and other inert carriers.

4. The bactericide for controlling xanthomonas according to claim 1, which is in any solid or liquid form that is acceptable in terms of production.

5. The bactericide for controlling xanthomonas according to claim 1 or 4, wherein the bactericide is in the form of powder, soluble powder, wettable powder, granules, aqueous solution, emulsifiable concentrate, microemulsion, aqueous emulsion, water dispersible granules, suspension, smoke agent, sustained-release agent or oil agent.

6. The fungicide for controlling xanthomonas according to claim 1, wherein the xanthomonas comprises xanthomonas campestris and/or xanthomonas carpi.

7. The bactericide for preventing and treating Xanthomonas according to claim 1 or 6, wherein the concentration of thymol in the bactericide is not less than 0.48 mg/mL.

8. The bactericide for preventing and treating Xanthomonas according to claim 1 or 6, wherein the concentration of protocatechuic acid in the bactericide is not less than 0.96 mg/mL.

9. Use of the fungicide for controlling xanthomonas according to any one of claims 1 to 8 for controlling plant diseases caused by xanthomonas.

10. The use according to claim 9, wherein the application method of the bactericide comprises plant spraying, root irrigation and seed treatment.

Technical Field

The invention relates to the technical field of agricultural protection, in particular to a bactericide for preventing and treating xanthomonas and application thereof.

Background

Crop diseases caused by pathogenic microorganisms are important accidents which cause great loss of agricultural production in China and harm national food production safety. The traditional disease control technology has the defects of low efficiency, abuse and overuse of antibiotics, environmental pollution of chemical pesticides, potential safety hazards to people and livestock and the like, and can not meet the requirements of plant disease control on the whole. Therefore, it is necessary to realize green prevention and control of pathogenic bacteria aiming at important crop diseases, develop new technologies of plant-derived bactericides with small environmental pollution, obvious bactericidal effect and degradability.

Bacteria of the genus Xanthomonas (Xanthomonas) belong to the family Xanthomonas, order Xanthomonas, class Proteobacteria, a large genus of gram-negative bacteria. Xanthomonas bacteria include 27 plant-related pathogenic bacteria, which cause serious diseases of over 400 plants including important crops such as rice, citrus, Chinese cabbage, pepper and the like. The model species xanthomonas campestris (x.campestis pv. campestis, Xcc) in this genus infects all cruciferous plants, causing black rot, and pathogenic bacteria spread along the vascular bundle of the plant from the leaf margin to form V-type diseases. Bacterial blight of rice caused by X.oryzae pv. oryzae (Xoc) and X.oryzae pv. oryzae (Xoo) are two common bacterial diseases of rice, which occur in all rice areas in the world and are one of the main diseases of rice. The citrus canker caused by the citrus canker germ (X.citri pv.citri) has high morbidity, fast transmission and wide host range, and is considered as an important epidemic disease detection disease in the citrus production in the world. Bacterial wilting disease caused by xanthomonas carpi (x. axolopodis pv. manihot, Xam) seriously affects the yield of cassava as a tropical important food crop, and is a main disease in cassava production areas such as guangdong, Guangxi, Hainan and Yunnan China.

At present, the xanthomonas disease control mainly adopts the measures of chemical sterilization and biological control. The most commonly used chemical biocides are copper and copper formulations, however, the use of copper formulations over large areas has caused serious resistance and environmental pollution problems over the last decades, while other biocides such as, for example, serthiazole, streptomycin, oxytetracycline, vernacin, and benzoyl-S-methyl, etc., have also developed more or less single and multiple drug resistance. In addition, biocontrol bacteria such as specific bacteria virus-bacteriophage, bacteria natural antagonist bacillus amyloliquefaciens MBI 600, bacillus pumilus QST 2808 and the like also obtain better xanthomonas sterilization effect. Nevertheless, the rapid occurrence of random mutations in bacteria limits the long-term use of bacteriophages and biocontrol bacteria. Therefore, the bactericide with high efficiency, low toxicity, environmental protection and degradable activity in the plant extract becomes a substitute method for green prevention and control of plant diseases.

Thymol (Thymol), also known as Thymol, and known by the chemical name 5-methyl-2-isopropyl phenol, is a monoterpene phenolic compound extracted from thyme oil, oregano oil, Ocimum gratissimum, and the like. Thymol not only has antifungal, antibacterial, antifungal, parasite killing effects, but also is a cadaver preservative. The relevant technical patents of monomer, binary or multicomponent mixing with thymol as the main component include: composition for preventing and treating bee mites and worms (KR20200122613A, binary mixture of quercetin and thymol for preventing and treating bee mites; GR1009197B, mixture of pyrethrum, geraniol and/or thymol as pesticide). Antibacterial and antifungal composition (CN110559423A, vancomycin and thymol are used simultaneously to remove biofilm-adhered bacteria; ZA201806217B, ternary mixture of thymol, terpineol and cationic phospholipid complex is a broad-spectrum bactericide; PL2687095(T3), mixture of carvacrol or thymol and copper preparation can prevent fungal infection; CN108719457A, thymol nano-emulsion can be used as a broad-spectrum bactericide; CN109463384A, mixture of thymol and carotenoid is suitable for preventing and treating gray mold of corn and vegetables; CN109819992A, binary combination of pyrrole ester bacteria ester and thymol is used for controlling powdery mildew, downy mildew, scab, leaf spot and plague of ornamental plants and crops such as fruit trees and vegetables; CN106511325A, thymol monomer is used for salmonella infection; CN111183991A, thymol, carvacrol and menthol ternary combination has strong killing effect on gray mold and powdery mildew of various crops; CN107306972A, the thymol, the benzothiazolinone and other pesticides are subjected to monomer mixing or binary mixing to control plant diseases including rice bacterial leaf blight; CN105123803A, thymol and kasugamycin are mixed in a binary way and are used for preventing and treating downy mildew, plague and other diseases of various crops such as grapes, cucumbers, tomatoes, potatoes and the like caused by fungi; CN104478607A, thymol, isopentenyl adenine and amino acid ternary mixture, which can prevent and treat powdery mildew of watermelon, gray mold of tomato, orange acute anthracnose, phytophthora capsici leonian, verticillium wilt of eggplant and the like; CN102106347A, a cinnamyl aldehyde, eugenol, thymol and tannin composite bactericide against escherichia coli, pseudomonas aeruginosa, salmonella, staphylococcus aureus and listeria; ZA200207674B, RU2002128741A, US2003175283A1, thymol, menthol and essential oil of menthol or its monoterpene component at proper ratio, and can be used as fourth generation herbal antibiotic preparation; CN110742876A, thymol monomer, for the treatment of psoriasis). Feed additive (CN110731422A, fermentation substance of thymol and live bacteria of enterococcus faecalis). Although patent application CN107306972A discloses that thymol in combination with benzothiazolinone or other pesticides can control bacterial blight of rice, the combination still contains antibiotics capable of generating drug resistance. The only technique as a monomer is the treatment of psoriasis.

Protocatechuic acid (PCA), also known as 3, 4-dihydroxybenzoic acid, is a natural phenolic acid compound with antioxidant effect in many fruits and vegetables, and has various physiological activities of resisting aging, inflammation, bacteria and virus, protecting heart, nerves, kidney, etc. In vitro experiments show that protocatechuic acid has inhibitory effect on Candida albicans, Staphylococcus aureus, Diplococcus pneumoniae, Pseudomonas aeruginosa, Escherichia coli, helicobacter pylori, etc. Protocatechuic acid can effectively reduce the urease activity of drug-resistant helicobacter pylori, and the Minimum Inhibitory Concentration (MIC) value of 90% of tested helicobacter pylori is 256 mug/mL. The minimum inhibitory concentration of protocatechuic acid for inhibiting salmonella typhimurium is 2.0mg/mL by microdilution method, and scanning electron microscope results show that the polarity end of bacteria treated by PCA is completely cracked, and the bacterial content is released to cause cell death, which may be because PCA destroys the net electrical balance of the bacterial outer membrane to cause the permeability change of the outer membrane, and may also cause the damage of membrane function and metabolic process due to the reduction of relative acidity around the bacterial membrane and the increase of oxidation degree. The protocatechuic acid separated and purified from Paenibacillus elgii HOA73 has bactericidal effect on botrytis cinerea which is a strawberry fruit fungus disease, and the bacteriostatic concentration of the protocatechuic acid is 64 mug/mL. The patent applications CN104523670A and CN102151256A introduce the application of protocatechuic acid in preventing and treating virus infectious diseases of livestock and poultry.

In summary, in the prior art, no other inventions mention or describe the use of thymol and protocatechuic acid monomers for the control of the phytopathogen xanthomonas.

Disclosure of Invention

The invention aims to solve the problems of poor sterilization effect, high toxicity, environmental pollution, nondegradable property and easy generation of drug resistance in the prior art, and provides a bactericide for preventing and treating xanthomonas and application thereof.

In order to achieve the above object, the present invention provides, in one aspect, a bactericide for controlling xanthomonas bacterium, characterized in that the bactericide contains thymol and/or protocatechuic acid.

Preferably, the bactericide also contains auxiliary agents and carriers allowed in production.

Preferably, the manufacturing approved adjuvants and carriers include dispersants, wetting agents, emulsifiers, stickers, thickeners, antifreeze, defoamers, diluents, solvents, and other inert carriers.

Preferably, the dosage form of the bactericide is any solid or liquid dosage form that is allowed in production.

Preferably, the dosage form of the bactericide comprises powder, soluble powder, wettable powder, granules, aqueous solution, missible oil, microemulsion, aqueous emulsion, water dispersible granules, suspending agent, smoke agent, sustained release agent or oil agent.

Preferably, the xanthomonas comprises the crucifer pathogen xanthomonas campestris and/or the euphorbiaceae plant cassava pathogen xanthomonas carpi.

Preferably, the concentration of thymol in the bactericide is more than or equal to 0.48 mg/mL.

Preferably, the concentration of protocatechuic acid in the bactericide is more than or equal to 0.96 mg/mL.

The second aspect of the invention provides the use of the above fungicide for controlling xanthomonas in the control of plant diseases caused by xanthomonas.

Preferably, the application method of the bactericide comprises plant spraying, root irrigation and seed treatment.

The bactericide for preventing and treating xanthomonas provided by the invention belongs to a botanical bactericide extracted from plants, and has the characteristics of high efficiency, low toxicity, environmental friendliness, good durability and degradability.

Drawings

FIG. 1 is a schematic of the MIC of thymol and protocatechuic acid pair Xam in example 1;

FIG. 2 is a graph of MIC measurements of thymol and protocatechuic acid versus Xcc for example 1;

FIG. 3 is a growth inhibition curve of thymol and protocatechuic acid against Xam and Xcc in example 2;

FIG. 4 is the results of the inhibition Xam and the Xcc biofilm activity assay of thymol and protocatechuic acid in example 3;

FIG. 5 shows the results of measuring the motility of Xam inhibited by thymol and protocatechuic acid in example 4;

FIG. 6 is a graph showing the bactericidal effect of protocatechuic acid and thymol on Xam and Xcc in example 5;

FIG. 7 is a graph of the effect of thymol and protocatechuic acid in inhibiting the growth of Xam and Xcc in the host plants cabbage and cassava of example 6.

Detailed Description

The following detailed description of embodiments of the invention refers to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

The invention discloses new application of thymol and protocatechuic acid, wherein the thymol and protocatechuic acid can be used as plant source effective sterilization components for preventing and treating xanthomonas, and are applied to prevention and treatment of plant diseases caused by the xanthomonas.

The invention provides a bactericide for preventing and treating xanthomonas, which is characterized by comprising thymol and/or protocatechuic acid.

In the invention, the bactericide also contains auxiliary agents and carriers which are allowed in production. The added adjuvant or carrier is not an essential active ingredient in the present application and is safe to crops after application.

In a preferred embodiment, the manufacturing approved adjuvants and carriers include dispersants, wetting agents, emulsifiers, stickers, thickeners, antifreeze agents, defoamers, diluents, solvents, and other inert carriers.

In the present invention, the formulation of the bactericide is any solid or liquid formulation that is allowed in terms of production.

In a preferred embodiment, the dosage form of the bactericide comprises powder, soluble powder, wettable powder, granules, aqueous solution, missible oil, microemulsion, aqueous emulsion, water dispersible granules, suspending agent, smoke agent, sustained-release agent or oil agent.

In a preferred embodiment, the xanthomonas comprises the crucifer pathogen xanthomonas campestris and/or the euphorbiaceae plant cassava pathogen xanthomonas carpi.

In a preferred embodiment, the concentration of thymol in the germicide is greater than or equal to 0.48 mg/mL.

In a preferred embodiment, the concentration of protocatechuic acid in the bactericide is 0.96mg/mL or more.

The second aspect of the invention provides the use of the above fungicide for controlling xanthomonas in the control of plant diseases caused by xanthomonas.

In a preferred embodiment, the method of application of the fungicide includes plant spraying, root watering and seed treatment.

In the invention, the two bactericidal monomers thymol and protocatechuic acid have good effect and small toxic and side effect, and can prevent and treat plant diseases caused by xanthomonas more durably. The bactericide can destroy the thallus structure to cause leakage of cytoplasm contents, thereby achieving the purpose of sterilization, can be used for preventing and treating bacterial wilt, and is a green substitute medicine for chemical pesticides and antibiotics. When the concentration of thymol is 0.48mg/mL and the concentration of protocatechuic acid is 0.96mg/mL, the growth of bacteria can be completely inhibited, and bacterial diseases can be prevented and treated. Especially has excellent antibacterial effect on crucifer pathogenic bacteria xanthomonas campestris (Xcc) and euphorbiaceae plant cassava pathogenic bacteria xanthomonas carpi (Xam).

The present invention will be described in detail by way of examples, but the scope of the present invention is not limited thereto.

The following examples, which take the cruciferous plant pathogen Xanthomonas campestris (Xcc) and the euphorbiaceae plant cassava pathogen Xanthomonas carpi (Xam) as the research objects, demonstrate the control effect of thymol and protocatechuic acid on plant diseases caused by Xanthomonas.

Example 1

The method for measuring the Minimum Inhibitory Concentration (MIC) by thymol comprises the following steps:

thymol was dissolved in 50% ethanol to prepare 77.10mg/mL of mother liquor, which was diluted to appropriate concentration with bacterial NYG medium (peptone 5g/L, yeast extract 3g/L, glycerol 20g/L, pH 7.0) at the time of use. And detecting the minimum inhibitory concentration by adopting a liquid double dilution method. mu.L of thymol solution with concentration of 15.42mg/mL was added to 96-well plates, 100. mu.L was diluted in duplicate with equal volumes of NYG medium to give compounds with final concentrations of 7.71, 3.85, 1.93, 0.96, 0.48, 0.24, 0.12, 0.06 mg/mL. Bacteria grown overnight at 28 ℃ were diluted 1000-fold with NYG medium to OD600 of 0.4 and 100 μ L was added to the plates. The cells were incubated at 28 ℃ for 24 hours. The OD600 value was measured by the microplate reader, and the MIC value was the lowest concentration at which no bacterial growth was visible.

The method for measuring the minimum inhibitory concentration of protocatechuic acid comprises the following steps:

protocatechuic acid was dissolved in water to prepare 77.10mg/mL of a mother liquor, which was diluted to an appropriate concentration with bacterial NYG medium (peptone 5g/L, yeast extract 3g/L, glycerol 20g/L, pH 7.0) at the time of use. And detecting the minimum inhibitory concentration by adopting a liquid double dilution method. 200 μ L of protocatechuic acid solution with concentration of 30.84mg/mL was added to 96-well plates, 100 μ L was diluted two-fold serially with equal volume of NYG medium to give final concentrations of 15.42, 7.71, 3.85, 1.93, 0.96, 0.48, 0.24, 0.12 mg/mL. Bacteria grown overnight at 28 ℃ were diluted 1000-fold with NYG medium to OD600 of 0.4 and 100 μ L was added to the plates. The cells were incubated at 28 ℃ for 24 hours. The OD600 value was measured by the microplate reader, and the MIC value was the lowest concentration at which no bacterial growth was visible.

The minimum inhibitory concentration is an activity index of the in-vitro bactericidal capacity of the medicine, and the smaller the MIC value is, the stronger the inhibitory action of the compound is. The minimum bacteriostatic concentration of thymol and protocatechuic acid is determined by adopting a gradient dilution method, and the thymol and protocatechuic acid are doubly diluted by an NYG culture medium on a 96-well plate, wherein the dilution multiple of the thymol is 5-1285 times, the dilution multiple of the protocatechuic acid is 2.5-642.5 times, and the concentration of a bacterial liquid is 10^-6～10^-7cultured at 28 ℃ for 24h between cfu/mL. The MIC of thymol and protocatechuic acid for Xam is shown schematically in FIG. 1, and the MIC determination curve for thymol and protocatechuic acid for Xcc is shown in FIG. 2. As can be seen, the minimum inhibitory concentration of thymol on Xam and Xcc is 0.48 mg/mL; the minimum inhibitory concentration of protocatechuic acid to Xam and Xcc was 0.96 mg/mL. The minimum inhibitory concentration of protocatechuic acid is higher than that of thymol.

Example 2

The growth curve reflects the inhibition of pathogenic bacteria growth over time, and the detection of the thymol inhibition bacterial growth curve comprises the following steps:

NYG medium, bacteria grown overnight at 28 ℃ were adjusted to OD600 0.4 with NYG medium, diluted 100-fold to OD600 0.004. Thymol is dissolved in 50% ethanol to prepare a mother liquor of 50.00 mg/mL. 1mL of diluted bacterial solution is put into a 1.5mL Eppendorf centrifuge tube, 20, 10, 5, 2 and 1 mu L of thymol mother liquor are respectively added to prepare bactericide mixed liquor with final concentration of 1.00, 0.50, 0.25, 0.10 and 0.05 mg/mL. Inoculating 200 μ L of the mixed solution to a growth curve determination plate, continuously culturing at 28 deg.C and 110rpm/min, and determining OD600 value with a microplate reader every 2h until the bacteria growth reaches a stationary phase.

The growth curve reflects the inhibition of pathogenic bacteria growth over time, and the detection of the protocatechuic acid inhibition of bacterial growth curve comprises the following steps:

NYG medium, bacteria grown overnight at 28 ℃ were adjusted to OD600 0.4 with NYG medium, diluted 100-fold to OD600 0.004. Protocatechuic acid is dissolved in water to prepare a mother liquor of 50.00 mg/mL. 1mL of diluted bacteria solution is put into a 1.5mL Eppendorf centrifuge tube, and 20, 10, 5, 2 and 1 mu L of protocatechuic acid mother liquor are respectively added to prepare bactericide mixed liquor with final concentrations of 1.00, 0.50, 0.25, 0.10 and 0.05 mg/mL. Inoculating 200 μ L of the mixed solution to a growth curve determination plate, continuously culturing at 28 deg.C and 110rpm/min, and determining OD600 value with a microplate reader every 2h until the bacteria growth reaches a stationary phase.

Thymol and protocatechuic acid were added to the culture of pathogenic bacteria at various concentrations and growth inhibition of Xam and Xcc was observed at various time points, and the growth inhibition curves of Xam and Xcc are shown in FIG. 3. As can be seen from FIG. 3, thymol showed inhibition of both bacteria at a concentration of 0.10mg/mL, completely inhibiting their growth after 20 h. The protocatechuic acid concentration is 1.00mg/mL, and the growth of the two bacteria can be completely inhibited within 30 hours.

Example 3

The detection process of the thymol for inhibiting the activity of the bacterial biofilm is as follows:

NYG medium, overnight grown bacteria at 28 ℃, centrifuged at 10,000rpm at room temperature to pellet, and cultured in MMX minimal medium (4.0g/L K)₂HPO₄，6.0g/L KH₂PO₄，2.0g/L(NH₄)₂SO₄1.0g/L trisodium citrate, 0.2g/L MgSO₄·7H₂O, 5.0g/L glucose, pH 7.0) was washed twice, resuspended to MMX OD600 0.4, and diluted 10-fold. Thymol is dissolved in 50% ethanol to prepare a mother liquor of 50.00 mg/mL. Taking 1mL of diluted bacteria solution to a 1.5mL Eppendorf centrifuge tube, adding 10, 5 and 2 mu L of thymol mother liquor respectively,to prepare a bactericide mixed solution with final concentrations of 0.50, 0.25 and 0.10 mg/mL. Putting 200 mu L of the suspension into a 96-well PVC (polyvinyl carbonate) cell culture plate, standing and culturing at 28 ℃ for 48 hours, measuring OD600, slightly pouring off the supernatant, washing the plate with distilled water for 2-3 times, and naturally drying in the air. Adding 200 μ L of 0.1% crystal violet solution, shaking gently at 100rpm for 30min, pouring out the crystal violet solution, washing with distilled water for 2 times, air drying, adding 200 μ L of 95% ethanol, shaking gently at 100rpm for 10min, and measuring the absorbance value of OD 590. The ability to biofilm formation was expressed as: OD590/OD 600.

The protocatechuic acid inhibition bacterial biofilm activity detection process is as follows:

NYG medium, overnight grown bacteria at 28 ℃, centrifuged at 10,000rpm at room temperature to pellet, and cultured in MMX minimal medium (4.0g/L K)₂HPO₄，6.0g/L KH₂PO₄，2.0g/L(NH₄)₂SO₄1.0g/L trisodium citrate, 0.2g/L MgSO₄·7H₂O, 5.0g/L glucose, pH 7.0) were washed twice, resuspended to MMX to adjust OD600 to 0.4, and diluted to OD600 to 0.04. Protocatechuic acid is dissolved in water to prepare mother liquor of 80.00 mg/mL. 1mL of diluted bacteria solution is taken to be put into a 1.5mL Eppendorf centrifuge tube, 10, 5 and 1.25 mu L of protocatechuic acid mother liquor are respectively added to prepare bactericide mixed liquor with final concentration of 0.80, 0.40 and 0.10 mg/mL. Putting 200 mu L of the suspension into a 96-well PVC (polyvinyl carbonate) cell culture plate, standing and culturing at 28 ℃ for 48 hours, measuring OD600, slightly pouring off the supernatant, washing the plate for 2-3 times by using distilled water, and naturally drying in the air. Adding 200 μ L of 0.1% crystal violet solution, shaking gently at 100rpm for 30min, pouring out the crystal violet solution, washing with distilled water for 2 times, air drying, adding 200 μ L of 95% ethanol, shaking gently at 100rpm for 10min, and measuring the absorbance value of OD 590. The ability to biofilm formation was expressed as: OD590/OD 600.

The high activity of the biological membrane can reflect the sterilization effect of the bactericide. The biofilm formation ability was measured by crystal violet staining absorptiometry. The results of the Xam and Xcc biofilm activity assays inhibited by thymol and protocatechuic acid are shown in fig. 4, where thymol significantly inhibited Xam biofilm formation at a concentration of 0.10mg/mL, and for Xcc, there was a significant difference in biofilm formation at a concentration of 0.25 mg/mL. Biofilm yields of Xam and Xcc gradually decreased with increasing protocatechuic acid concentration, with biofilm formation decreasing most significantly at 0.80 mg/mL.

Example 4

The process for detecting the bacterial motility of thymol inhibition is as follows:

thymol is dissolved in 50% ethanol to prepare a mother liquor of 50.00 mg/mL. Preparing an NYG semisolid culture medium (5 g/L of peptone, 3g/L of yeast extract, 20g/L of glycerol, 0.3% agar and pH 7.0), autoclaving, cooling to 50-60 ℃, adding 125 mu L of thymol mother liquor into 25mL of the culture medium to obtain a final concentration of 0.25mg/mL, uniformly mixing, pouring into a sterile culture dish, and solidifying the culture medium for use. NYG medium, overnight grown bacteria at 28 ℃, centrifuged at 10,000rpm at room temperature to collect the biomass, 10mM MgCl₂Washing twice, and adjusting OD600 to 0.4. mu.L of the suspension was spotted on NYG semisolid medium containing thymol (0.25mg/mL) and cultured at 28 ℃ for 72h, and colony morphology and size were observed.

The protocatechuic acid inhibits bacterial motility and is detected as follows:

protocatechuic acid is dissolved in water to prepare mother liquor of 80.00 mg/mL. Preparing an NYG semisolid culture medium (5 g/L of peptone, 3g/L of yeast extract, 20g/L of glycerol, 0.3% agar and pH 7.0), autoclaving, cooling to 50-60 ℃, adding 125 mu L of protocatechuic acid mother liquor into 25mL of the culture medium to obtain a final concentration of 0.40mg/mL, uniformly mixing, pouring into a sterile culture dish, and solidifying the culture medium for use. NYG medium, overnight grown bacteria at 28 ℃, centrifuged at 10,000rpm at room temperature to collect the biomass, 10mM MgCl₂The cells were washed twice and OD600 was adjusted to 0.4. And (3) inoculating 1.5 mu L of bacterial liquid to a semisolid culture medium containing a bactericide, culturing for 72h at 28 ℃, and observing the colony morphology and the colony size.

Xam is cultured on a NYG solid plate with 0.3 percent of sub-lethal bactericide concentration for 3 to 4 days, and the growth capacity of bacteria and the change of thallus morphology are observed. The results of measuring the migration of pathogenic bacterium Xam by thymol and protocatechuic acid are shown in FIG. 5 (using concentration: 0.25mg/mL thymol, 0.40mg/mL protocatechuic acid). As can be seen, Xam without the bactericide has the advantages of high growth speed, large, full and milky colony and smooth surface. After 0.25mg/mL thymol is added, the growth of bacteria is seriously hindered, the colony morphology is small, and the color is slightly dark after dry stir-frying. The bacterial growth of the protocatechuic acid added in 0.40mg/mL is not greatly influenced, the colony morphology is still milky white, and the surface is smooth. Protocatechuic acid is less able to inhibit bacterial motility than thymol.

Example 5

The procedure of transmission electron microscopy for the observation of bacterial damage by thymol is as follows:

thymol is dissolved in 50% ethanol to prepare a mother liquor of 50.00 mg/mL. And (3) carrying out autoclaving on an NYG solid culture medium, cooling to 50-60 ℃, adding 125 mu L of thymol mother liquor into 25mL of the culture medium to obtain a final concentration of 0.25mg/mL, uniformly mixing, pouring into a sterile culture dish, and carrying out streak inoculation after the culture medium is solidified. Standing at 28 deg.C for 2 days, dipping a small amount of bacteria, suspending in water, dripping the suspension on copper grid with membrane, standing for 2min, and removing the excess solution with filter paper. Dyeing is carried out for 2-3 minutes by using 1% phosphotungstic acid (PTA) negative staining solution, and redundant PTA is carefully removed by using filter paper. After the grid is dried, the structural change of the bacteria is checked by a transmission electron microscope (JOEL JEM-1400, Japan) to verify the sterilization effect.

The process of the damage of protocatechuic acid to bacteria observed by a transmission electron microscope is as follows:

protocatechuic acid is dissolved in water to prepare mother liquor of 80.00 mg/mL. And (3) carrying out autoclaving on an NYG solid culture medium, cooling to 50-60 ℃, adding 125 mu L of protocatechuic acid mother liquor into 25mL of the culture medium to obtain a final concentration of 0.40mg/mL, uniformly mixing, pouring into a sterile culture dish, and carrying out streak inoculation after the culture medium is solidified. Standing at 28 deg.C for 2 days, dipping a small amount of bacteria, suspending in water, dripping the suspension on copper grid with membrane, standing for 2min, and removing the excess solution with filter paper. Dyeing is carried out for 2-3 minutes by using 1% phosphotungstic acid (PTA) negative staining solution, and redundant PTA is carefully removed by using filter paper. After the grids were dried, the structural change of the bacteria was examined by a transmission electron microscope (JOEL JEM-1400, Japan) to observe the bactericidal effect of the bactericide.

The bactericidal effect of protocatechuic acid and thymol on pathogenic bacteria Xam and Xcc is observed as shown in fig. 6. As can be seen from the figure, Xam without adding bactericide is in the shape of a short rod, the two ends are thinner, the thallus shape is complete, the surface is smooth, the thallus is full, no fold is formed, and the thallus is not damaged totally; after being treated by 0.25mg/mL thymol, the thallus structure is damaged in a large range, the cell surface is cracked, and the phenomenon of obvious bacterial cytoplasm leakage is caused; although 0.40mg/mL protocatechuic acid did not affect Xam motility, colony morphology was still significantly deformed and damaged, and cell contents were also leaked in large quantities. FIG. 6 shows that the Xcc without added biocide is long rod shaped, smooth in surface, full, without wrinkles; after being treated by 0.25mg/mL thymol, the thallus structure of individual bacteria is damaged, the cell surface is cracked and unsmooth, and the bacteria die due to the leakage of cell contents; after 0.40mg/mL of protocatechuic acid treatment, the surface of the Xcc cells had dispersed bubble-like projections, which might be the leakage points of the contents.

Example 6

Experimental groups: treating with 0.48mg/mL thymol and 0.96mg/mL protocatechuic acid for Xam hr respectively, cutting leaf, staining cassava leaf, culturing at 25 deg.C for 10 days under 16 hr illumination, and observing plant disease degree. Xcc was treated with 0.48mg/mL thymol and 0.96mg/mL protocatechuic acid for two hours, respectively, and then leaf-cut and cabbage leaf-stained, cultured at 25 ℃ under 16-hour light conditions for 10 days, and then the degree of plant disease was observed.

Control group: xam and Xcc were not treated and were directly impregnated with cassava leaves and cabbage leaves.

The effect of thymol and protocatechuic acid on inhibiting the growth of pathogenic bacteria Xam and Xcc in the host plants cabbage and cassava is shown in fig. 7, from which it can be observed that the pathogenic bacteria treated with thymol and protocatechuic acid have a seriously weakened pathogenic ability and do not cause wilting disease of plants, compared with the pathogenic bacteria not treated.

As can be seen from the above examples, the bactericide of the present invention has the object of sterilizing by destroying the bacterial structure, causing leakage of cytosolic contents, and inhibiting the formation of bacterial biofilm, and thymol also affects the mobility of bacteria Xam. The bactericide has broad-spectrum bactericidal capability on xanthomonas bacteria and has stronger capability of killing xanthomonas carpi and xanthomonas campestris. The bactericide of the invention has low thymol and protocatechuic acid toxicity, is safe to human and livestock, has little environmental pollution and relatively stable physicochemical property, can be stored for a long time, and is an effective botanical bactericide.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.