CN112940979B

CN112940979B - Bacterial strain, extract, nano pesticide and application and preparation method thereof

Info

Publication number: CN112940979B
Application number: CN202110291229.5A
Authority: CN
Inventors: 郝凌云; 徐颖; 黄刚; 尚琛晶
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2021-03-18
Filing date: 2021-03-18
Publication date: 2022-07-19
Anticipated expiration: 2041-03-18
Also published as: CN112940979A

Abstract

The invention discloses a bacterial strain, an extract, a nano pesticide and an application and a preparation method thereof, wherein the bacterial strain is a strain R64 derived from mangrove, the classification name of the strain R64 is pseudomonas aeruginosa (Pseudomonas aeruginosa), and the preservation number of the strain R64 is CGMCC No. 21803. The extract of the strain R64 extracted from the bacterial strain R64 and the nano pesticide synthesized by the extract of the strain R64 have higher antibacterial activity and high efficiency and lower toxicity to environmental microorganisms. Meanwhile, the biological preparation method has the advantages of low cost, simple operation and relative environmental protection. The invention provides a new natural product pesticide, and simultaneously reduces the environmental toxicity of the natural product pesticide and increases the antibacterial activity and the high efficiency of the natural product pesticide by a method for preparing the nano pesticide.

Description

Bacterial strain, extract, nano pesticide and application and preparation method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a bacterial strain, an extract, a nano pesticide, and an application and a preparation method thereof.

Background

Plant diseases caused by pathogenic microorganisms, particularly fungal diseases on a large number of food crops seriously threaten agricultural production and are always limiting factors for restricting the safety and yield of agricultural products. However, most crops lack good resistant varieties against phytopathogens at present, and the control of the phytopathogens still depends on traditional chemical control means, including the use of agricultural antibiotics, inorganic copper, organic copper, thiazole preparations and the like. However, the use of conventional chemical pesticides not only easily causes problems such as environmental pollution, pesticide residue, high toxicity to environmental microorganisms, and the like, but also promotes the generation of drug-resistant strains.

Accordingly, the prior art is in need of improvement and development.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide a bacterial strain, an extract, a nano pesticide and an application and a preparation method thereof, and aims to solve the problems that the existing chemical pesticide easily causes environmental pollution and has high toxicity to environmental microorganisms.

The technical scheme of the invention is as follows:

a bacterial strain, wherein the bacterial strain is a mangrove-derived strain R64 which is classified and named as Pseudomonas aeruginosa (Pseudomonas aeruginosa), the bacterial strain is deposited in China general microbiological culture Collection center on No. 01 of No. 02 of 2021, and the deposit number of the bacterial strain R64 is CGMCC No. 21803.

The invention relates to an application of the bacterial strain in preparing pesticides for preventing and treating plant diseases.

An extract of a strain R64, wherein the extract of the strain R64 is extracted from the bacterial strain disclosed by the invention.

The invention relates to application of an extract of a strain R64 in preparing pesticides for preventing and treating plant diseases.

A preparation method of a nano pesticide comprises the following steps:

activating the strain R64 to obtain an activated R64 single colony;

sequentially fermenting and extracting the activated R64 single colonies to obtain an extract of a strain R64;

mixing the extract of the strain R64 with AgNO₃And mixing the solutions, and reacting to obtain the nano pesticide.

Optionally, the step of activating the strain R64 of the present invention specifically includes:

mixing the strain R64 with glycerol, and freezing and storing in a refrigerator;

putting the frozen bacterium solution into a marine bacterium agar culture medium, selecting the frozen bacterium solution, scribing by using a plate scribing method, and culturing in an incubator.

Optionally, the steps of sequentially fermenting and extracting the activated R64 single colonies specifically include:

inoculating the activated R64 single colony in a culture medium, shaking and fermenting to obtain a strain R64 fermentation liquid;

adding ethyl acetate into the fermentation liquor of the strain R64 for extraction, oscillating and uniformly mixing by using a separating funnel, standing, discharging a lower-layer water phase, pouring an upper-layer organic phase into a distillation flask, and performing vacuum rotary evaporation until the ethyl acetate is completely evaporated;

after elution with methanol, the extract was concentrated by a vacuum centrifugal concentrator to obtain an extract of the strain R64.

Optionally, the extract of the strain R64 is mixed with AgNO₃Mixing the solutions, and reacting to obtain the nano pesticide, which specifically comprises the following steps:

adding the extract of the strain R64 into deionized water, adjusting the pH to 7 with NaOH, and adding AgNO₃Heating the solution under the condition of keeping out of the sun, and then stirring for reaction;

filtering with a filter membrane, centrifuging, collecting supernatant, adding water of the same volume, centrifuging, and repeating for three times to obtain precipitate;

transferring, freeze-drying and weighing the precipitate, and metering the volume by using sterile deionized water to obtain the nano pesticide.

The nano pesticide is prepared by the method.

The invention relates to an application of a nano pesticide in preparing a pesticide for preventing and treating plant diseases.

Has the advantages that: the invention provides a bacterial strain R64, extracts of the bacterial strain R64 are extracted from the bacterial strain R64, and the nano pesticide is synthesized by the extracts of the bacterial strain R64, and has high antibacterial activity and high efficiency, low toxicity to environmental organisms, low organic solvent content and environmental friendliness. Meanwhile, the biological preparation method has the advantages of low cost, simple operation and relative environmental protection.

Drawings

FIG. 1 is a graph showing the results of the inhibition ratio of an extract liquid of the strain R64 against the plant rice blast pathogenic fungus P131(Magnaporthe oryzae) as a function of concentration;

FIG. 2 is a UV spectrum of a nano pesticide R64-Ag;

FIG. 3 is a scanning electron microscope image of the nano pesticide R64-Ag;

FIG. 4 is a transmission electron microscope image of the nano pesticide R64-Ag;

FIG. 5 is a graph showing the results of the activity of the extract of the strain R64, the nano pesticide R64-Ag, and the nano silver standard AgNPs (particle size 20nm) against rice blast pathogenic fungus P131(Magnaporthe oryzae) in the experimental group at different concentrations;

FIG. 6 is a graph showing the results of tests on the antibacterial activity of the extract of the strain R64, nanosilver, and nano pesticide R64-Ag against various pathogenic fungi, i.e., Alternaria solani (HLJ), Colletotrichum Fructicola (CF), Cochliobolus zeae (Cochliobacter xylinosus), and Botrytis cinerea (Botrytis cinerea);

FIG. 7a is a graph showing the results of the antibacterial test of the pesticide R64-Ag nanoparticles against the pathogenic bacterium Pebacterium carotovorum (Pec);

FIG. 7b is a chart showing the antibacterial activity test results of the nano pesticide R64-Ag on pathogenic bacteria Methicillin-resistantStaphylococcus aureus (MRSA);

FIG. 7c is a graph showing the result of the antibacterial activity test of the nanopesticide R64-Ag against pathogenic bacteria Pseudomonas aeruginosa (PAO 1);

FIG. 7d is a graph showing the results of the antibacterial activity test of the nano pesticide R64-Ag against pathogenic bacteria Pseudomonas syringae pv. tomato (Pst);

FIG. 8 is a graph showing the toxicity test results of the extract of the strain R64, the nano pesticide R64-Ag, the nano silver standard substance, etc. to artemia.

Detailed Description

The invention provides a bacterial strain, an extract of the bacterial strain R64, a nano pesticide and an application and a preparation method thereof, and the invention is further detailed below in order to make the purpose, the technical scheme and the effect of the invention clearer and clearer. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The inventor researches and discovers that mangrove wetland forms a unique habitat due to the unique geographical position of the mangrove wetland, and has the characteristics which are obviously different from the land ecosystem, such as high humidity, high salt, relative oxygen deficiency and the like. Such extreme environmental conditions can stimulate the evolution of unique metabolic adaptations of environmental microorganisms therein, thereby enabling the production of a variety of unique active compounds. Most of the researches on natural active compounds derived from mangrove are focused on the medical field and have less related researches in the agricultural field, and meanwhile, the researches show that the natural products of mangrove soil bacteria have the potential of inhibiting crop diseases as environment-friendly bactericides, and more than one quarter of strains can generate active mixtures for inhibiting different plant pathogenic fungi. More significantly, the activity of the extracts of the strains is higher than that of the existing pesticides under the same concentration, the toxicity to environmental microorganisms such as algae is lower than that of commercial pesticides, and the extracts show obvious inhibition effects on different diseases in plant experiments, so the extracts have great potential in developing environment-friendly fungicides.

Further research finds that the extract of the mangrove-derived strain R64(Pseudomonas sp.) has high-efficiency anti-rice blast activity, and the toxicity to environmental organisms such as artemia and human macrophages is far lower than that of the existing chemical pesticides, so that the deep research on the extract of the strain R64 and the application potential of the extract as the biological pesticide can provide a new strategy for effectively preventing and treating plant diseases.

Based on the above, the embodiment of the invention provides a bacterial strain, wherein the bacterial strain is a strain R64 derived from mangrove forest, which is classified and named as Pseudomonas aeruginosa (Pseudomonas aeruginosa), and the preservation number of the strain R64 is CGMCC No. 21803.

The strain R64 of the embodiment is obtained by separating, purifying and identifying from mangrove forest soil samples.

The embodiment of the invention also provides application of the bacterial strain in preparing pesticides for preventing and treating plant diseases.

The embodiment of the invention also provides an extract of the strain R64, wherein the extract of the strain R64 is extracted from the bacterial strain.

The embodiment of the invention also provides application of the extract of the strain R64 in preparing pesticides for preventing and treating plant diseases.

Different from the traditional pesticide preparation, the nanometer material has the problems of high organic solvent content, poor dispersibility, easy loss and the like, and can change the physical and chemical properties of the pesticide into a stable preparation which is uniformly dispersed and easily dissolved in water, thereby improving the utilization rate of the pesticide, reducing the pesticide residue and reducing the environmental pollution. Among the numerous nanomaterials, nanosilver is the most common and most widely used. The silver has excellent antibacterial activity, and has the advantages of high efficiency, safety, wide antibacterial spectrum, difficult drug resistance and the like. However, silver ions are easily oxidized under light, heat, and the like, and easily form precipitates with chloride ions and the like, resulting in a decrease in activity. The nano silver has the advantages of both silver and nano materials, obtains higher stability and utilization rate under the condition of ensuring high-efficiency and safe antibacterial property, avoids the synthetic nano silver from being oxidized and inactivated due to the coating of the raw materials added in the synthetic process, has more stable and long-term antibacterial activity, and has various preparation methods of the nano silver, wherein compared with methods such as physical chemistry and the like, the biological preparation method has the advantages of low cost, simple operation, relative environmental protection and the like.

Based on the above, the embodiment of the invention also provides a preparation method of the nano pesticide, which comprises the following steps:

s10, activating the strain R64 to obtain an activated R64 single colony;

s20, sequentially fermenting and extracting the activated R64 single bacterial colonies to obtain an extract of a strain R64;

s30, mixing the extract of the strain R64 with AgNO₃And mixing the solutions, and reacting to obtain the nano pesticide.

The embodiment provides a strain R64 capable of producing natural antibacterial products, an extract of the strain R64 with antibacterial activity is obtained by means of activation, fermentation and extraction, and the extract of the strain R64 is used as a raw material to prepare a nano pesticide, so that the environmental toxicity of the extract of the strain R64 is effectively reduced, and the antibacterial activity and the high efficiency of the extract are improved.

In step S10, in one embodiment, the activating step of the strain R64 specifically comprises:

In step S20, in one embodiment, the step of sequentially fermenting and extracting the activated R64 single colony includes:

inoculating the activated R64 single colony in a culture medium for shake culture, and then fermenting to obtain a strain R64 fermentation liquid;

In one embodiment, the steps of sequentially fermenting and extracting the activated strain R64 specifically include:

inoculating the activated strain R64 in a culture medium, shaking at the speed of 200rpm for 24h at the temperature of 28 ℃ after single strain culture, and then shaking at the speed of 200rpm at the room temperature for 4 days for fermentation to obtain strain R64 fermentation liquor;

adding ethyl acetate with the volume of 3 times that of the fermentation liquor of the strain R64 to extract, oscillating and uniformly mixing a separating funnel, standing for 15min, discharging a lower-layer water phase, pouring an upper-layer organic phase into a distillation flask, and performing vacuum rotary evaporation until ethyl acetate is completely evaporated;

In one embodiment, the culture medium for fermenting the strain R64 is SGTYP consisting of 5.0g of soluble starch, 5.0g of glucose, 1g of microbial peptone, 1g of tryptone, 1g of yeast extract, 17g of artificial sea salt, and 1L of distilled water.

In step S30, in one embodiment, the extract of strain R64 is mixed with AgNO₃Mixing the solutions, and reacting to obtain the nano pesticide, which specifically comprises the following steps:

filtering with a filter membrane, centrifuging, taking the supernatant, adding water with the same volume, centrifuging, and repeating for three times to obtain a precipitate;

and transferring, freeze-drying and weighing the precipitate, and performing constant volume by using sterile deionized water to obtain the nano pesticide.

In one example, an extract of the strain R64 is mixed with AgNO₃Mixing the solutions, and carrying out a reaction (referred to as a reduction reaction) to obtain the nano pesticide, which specifically comprises the following steps:

adding the extract of the strain R64 into deionized water, adjusting pH to 7 with NaOH, and adding AgNO₃Heating the solution to the final concentration of 4mM under the condition of keeping out of the light to 60 ℃, and stirring and reacting for 60 hours on a magnetic stirrer at 700 rpm;

filtering with 0.22 μm filter membrane, centrifuging at 7500rpm for 30min, removing supernatant, adding equal volume of water into precipitate, centrifuging at the same speed for 30min, and repeating for three times;

then, the obtained precipitate was transferred to freeze-drying, weighed, and stored at 4 ℃ in the dark with sterile deionized water to a constant volume of 10 mg/ml.

In this example, the reduction reaction was more favorably carried out at pH 7, and the reaction was carried out under a light-shielding condition since silver nitrate was easily decomposed by light.

The embodiment of the invention also provides a nano pesticide, wherein the nano pesticide is prepared by the method.

In this embodiment, the particle size of the nano-pesticide is less than 300 nm. In one embodiment, the nano-pesticide has a particle size of 20nm to 300 nm.

The embodiment of the invention also provides application of the nano pesticide in preparation of pesticides for preventing and treating plant diseases.

The extract of the strain R64 extracted from the bacterial strain R64 and the nano pesticide R64-Ag synthesized by the extract of the strain R64 have high antibacterial activity and high efficiency, have low toxicity to environmental microorganisms, have extremely low organic solvent content and are environment-friendly. Meanwhile, the biological preparation method has the advantages of low cost, simple operation and relative environmental protection.

In the embodiment, the method for preparing the nano pesticide by using the extract of the strain R64 reduces the environmental toxicity of the extract of the strain R64 as the pesticide and simultaneously increases the antibacterial activity and the high efficiency of the extract.

The invention is further illustrated by the following specific examples.

Example 1 activation of bacteria and fungi

1. Separation, purification and identification of the strain R64:

obtaining a sample from surface soil (5cm deep) of red mangrove forest of cattail hong Kong, taking a part of the sample, soaking the part in sterile water, and taking a supernatant (leachate) after a period of time;

diluting the soil leachate by a multiple of 10, 100, 1000 and 10000, coating the diluted soil leachate on an MA culture medium by using an applicator, and culturing the diluted soil leachate in an incubator at 28 ℃ for 48 hours;

picking single colony, inoculating on new MA plate by plate marking method (if no single colony exists on the coated plate, continuously diluting with 10 times gradient until single colony appears), and culturing in 28 deg.C incubator for 48 h;

after PCR (two-way primer, 27F: AGAGTTTGATCMTGGCTCAG; 1492R: GGTTACCTTGCAGACTT) the sequence was determined by Oncorka.

Identification results after NCBI alignment, the genus R64 was found to be Pseudomonas, and the similarity to Pseudomonas aeruginosa was 100%.

2. Activation of strain R64:

the strain R64 and glycerol with the mass fraction of 20% are mixed in the same volume, and the mixture is frozen and stored in a refrigerator at the temperature of minus 80 ℃.

The frozen R64 bacterial liquid was taken out of the refrigerator and put into MA medium (BD Difco TM Marine Agar 2216,55.1g/L), a small amount of the frozen R64 bacterial liquid was picked up, streaked by the plate streaking method, and cultured in an incubator at 28 ℃ for 48 hours for use.

3. Activation of the bacteria pec (Pebactium carotovorum), PAO1(Pseudomonas aeruginosa), MRSA (Methicillin-resistant Staphylococcus aureus): taking out the three frozen bacterial liquids, respectively placing the three frozen bacterial liquids into LA culture media (10 g of tryptone, 5g of yeast extract, 10g of NaCl, 15g of agar and 1L of deionized water), respectively picking a small amount of ice cubes, streaking by using a streaking method, and culturing in an incubator at 37 ℃ for 48 hours for later use, wherein the three bacteria are pathogenic bacteria.

4. Activation of bacterial Pst (Pseudomonas syringaepv. tomato): the frozen glycerol-containing bacterial solution was removed and placed in King's B medium (peptone 20g, glycerol 10mL, K)₂HP0₄ 1.5g，MgS0₄·7H₂01.5 g, 15g of agar and 1000mL of water), a small amount of ice blocks are picked, streaked by a plate streaking method, and cultured in an incubator at 28 ℃ for 48 hours for later use, wherein the bacteria are plant pathogenic bacteria.

5. Activation of fungi:

a filter paper sheet with fungus hyphae is attached to the center of a PDA culture medium (Dickinson and company, Potato Dextrose Agar,23g/L PDB +15g/L Agar), and is subjected to illumination culture at 28 ℃ for 4 days for later use, wherein the fungi are Alternaria solani (HLJ), Colletotrichum Fructicola (CF), Cochliobacter zeae (Cochliobacter heperosporus), Botrytis cinerea (Botrytis cinerea), and the activated fungi are all plant pathogenic bacteria.

Example 2 preparation of extract of Strain R64

1. Fermentation of strain R64:

200mL of SGTYP medium was added to a 500mL Erlenmeyer flask, and after inoculating the activated R64 single strain, it was shaken at 200rpm for 24 hours at 28 ℃ before transferring 50mL into a 2L Erlenmeyer flask containing 800mL of SGTYP, and shaken at 200rpm at room temperature for 4 days. The SGTYP medium consisted of 5.0g of soluble starch, 5.0g of glucose, 1.0g of microbial peptone, 1.0g of tryptone, 1g of yeast extract, 17g of artificial sea salt, and 1L of distilled water (prepared in the laboratory, pH 7.6. + -. 0.2. the company producing starch and glucose is Avastin; the company producing microbial peptone, tryptone and yeast extract is OXOID; the company producing artificial sea salt is Yier.)

2. Extraction of extract of strain R64:

adding 3 times of ethyl acetate into fermented liquid of the strain R64, oscillating and uniformly mixing by using a separating funnel, standing for 15min, discharging a lower-layer aqueous phase, pouring the upper-layer organic phase into a distillation flask, and performing vacuum rotary evaporation until the ethyl acetate is completely evaporated; after methanol elution, the extract was concentrated in a vacuum centrifugal concentrator and weighed after 24 hours to obtain an extract of the strain R64. The extract of strain R64 was made up to 50mg/mL with dimethyl sulfoxide (DMSO) for further use.

FIG. 1 is a graph showing the result of the inhibition rate of the extract liquid of the strain R64 on the fungus P131 varying with the concentration, and when the concentration of the extract liquid of the strain R64 reaches 500. mu.g/mL, the inhibition rate of the extract liquid of the strain R64 on the fungus P131 can reach 70%.

Example 3 preparation of a Nanoparticulate pesticide R64-Ag

And (3) synthesis of nano pesticide R64-Ag:

90mL of deionized water was taken, 2mL of the crude extract of the active mixture of R64 was added thereto, 4mL of the crude extract of the active mixture of R64 was added thereto, the pH was adjusted to 7 with NaOH under a pH meter, and 4mL of 0.1M AgNO was added thereto₃When the volume of the solution is 100mL, the concentration of the extract of the strain R64 is 1mg/mL and 2mg/mL respectively. Heating to 60 ℃ under the condition of keeping out of the sun, stirring on a magnetic stirrer at 700rpm, and reacting for 60 hours;

after 60 hours, the mixture was filtered through a 0.22 μm filter, centrifuged at 7500rpm for 30min, the supernatant was removed, and the precipitate was washed with an equal volume of water and centrifuged at equal speed for 30min, and this operation was repeated three times. Then transferring and freeze-drying the precipitate, weighing, and fixing the volume to 10mg/mL by using sterile deionized water and storing at 4 ℃ in the dark for later use.

FIG. 2 is a UV spectrum of the nano pesticide R64-Ag: the reacted nano pesticide is diluted to 750 mu g/mL, 200mL is added into a 96-well plate, an ultraviolet absorption peak of 300-600nm is measured by a microplate reader, a higher characteristic absorption peak appears around 430nm, the generation of nano silver is shown, when the concentration of the extract liquid of the strain R64 is 1mg/mL (namely R64 in the figure is 1mg/mL), the absorption peak of the nano pesticide R64-Ag generated in the same reaction time is far lower than that when the concentration of the extract liquid of the strain R64 is 2mg/mL (namely R64 in the figure is 2 mg/mL).

FIG. 3 is an SEM image of the nano pesticide R64-Ag: after the nano pesticide after the reaction is diluted 1000 times, the nano pesticide is dripped on conductive gel, and the shape is observed by magnifying the shape by 100000 times by using a Scanning Electron Microscope (Thermo Scientific, High Resolution Scanning Electron Microscope APREO S), so that the shape of the nano pesticide R64-Ag is mostly sheet-shaped, spherical and rod-shaped, and the length or the width is between 20 and 300 nm.

FIG. 4 is a TEM image of the nano-pesticide R64-Ag, in which the nano-pesticide after reaction is diluted 1000 times, dropped on a copper mesh, and observed by a transmission electron microscope (tungsten filament transmission electron microscope HITACH HT7700) with magnification of 300000 times, and the nano-pesticide R64-Ag is mostly in the form of sheet, sphere and rod, and has a particle size of 20-300 nm.

Example 4 antifungal Activity assay

Taking 25mL of sterilized PDA culture medium cooled to 50-60 ℃, adding a certain volume of extract of the strain R64 with constant volume, nano pesticide R64-Ag and nano silver standard substance AgNPs (purchased from Shanghai ultra Wei nano Co., Ltd., 20nm), wherein each sample has three concentrations of 50 mug/mL, 100 mug/mL and 200 mug/mL respectively, punching the activated rice blast bacterium plate along the hypha edge by using a sterile hollow nuclear magnetic tube, placing the punched bacterium cake in the center of the drug-containing plate, and placing the drug-containing plate in an incubator at 28 ℃ for culture for 5 d. If the growth of the hypha of the fungus is inhibited, the fungus is proved to have antifungal activity, the diameter of the inhibition zone is measured for 3 times and recorded, and the inhibition rate is calculated, wherein the inhibition rate is [ (Control diameter-containing plate diameter)/Control diameter ]. 100%.

As shown in FIG. 5, R64-50, R64-100, and R64-200 respectively represent the inhibition rates of the crude product of strain R64 at working concentrations of 50, 100, and 200. mu.g/mL; ag-50, Ag-100 and Ag-200 respectively represent the inhibition rates of the nano-silver standard substance at working concentrations of 50, 100 and 200 mu g/mL; R64-Ag-50 and R64-Ag-100 respectively represent the inhibition rates of the nano pesticide synthesized by taking the R64 extract and silver nitrate as raw materials and reacting for 48 hours at working concentrations of 50 and 100 mu g/mL. Therefore, under the same concentration, the inhibition rate of the nano pesticide R64-Ag on the rice blast fungi is higher than that of the extract of the strain R64 and the nano silver standard substance.

As shown in fig. 6, the concentrations of the extract of the strain R64, R64-Ag and nano-silver AgNPs standard were 50 μ g/mL, in the figure, R64 represents the extract of the strain R64, and 2-7 represents R64-Ag (i.e., the synthesis conditions were that the concentration of the added R64 extract was 2mg/mL, and the pH was 7). Compared with the R64 extract and the nano silver standard solution, the nano pesticide R64-Ag has the highest inhibition rate on different pathogenic fungi under the same concentration.

Example 5 bacterial growth Curve detection

1. Shaking culture of bacteria: 3-5mL of LB medium was taken into a 15mL centrifuge tube, and single colonies of PAO1, MRSA, Pec, and Pst were added thereto, followed by shaking overnight at 37 ℃ (12 h). 3-5mL King' B medium was taken into a 15mL centrifuge tube, and single Pst colonies were added thereto and shaken overnight at 28 deg.C (12 h). After 12 hours, each was diluted 10-fold, and 237. mu.L of each was transferred to a 96-well plate at OD_595nmAnd measuring the absorption value, calculating the concentration of the bacterial liquid according to the proportion of 0.1OD (origin-to-diameter) 1 (origin-to-diameter) 10 (origin-to-diameter) 8, and adjusting the concentration of the bacterial liquid to 1 (origin-to-diameter) 10 (origin-to-diameter) 5.

2. Measurement of growth curves: taking 1mL of 10^5 bacteria liquid to a 1.5mL centrifuge tube, and respectively adding a certain volume of nano pesticide R64-Ag with constant volume into the centrifuge tube to ensure that the final concentrations are 0, 25 and 100 mu g/mL respectively. The treated bacterial liquid is added into 96-well plates respectively, 4 wells are added in each group, and 200 mu L of each well is added. The absorbance at 595nm was measured at 0, 3, 6, 12, 24h, respectively. And drawing a growth curve according to the measured data, wherein the test result is shown in figures 7a-7d, and when the concentration of the nano pesticide R64-Ag is 25 mu g/mL or more, the nano pesticide can completely inhibit the growth of bacteria.

Example 6 artemia toxicity measurement

1. And (3) artemia breeding: adding 30g of artificial sea salt into 1L of deionized water, adjusting the pH value to 8.5 with NaOH, weighing 2g of artemia cysts, adding the artemia cysts into the prepared artificial sea water, and culturing for 24 hours at the temperature of 28 ℃ under illumination.

2. And (3) toxicity detection: taking a 24-hole plate, adding 10 artemia in each hole, adding 1mL of artificial seawater, adding 1-10 muL of extract of the strain R64 with constant volume, the nano pesticide R64-Ag, the commercially available chemical pesticide amikawis (azoxystrobin), prochloraz and the nano silver standard AgNPs (purchased from Shanghai ultra Wei Nano Co., Ltd., 20nm) according to the concentration, and shaking uniformly to make the final concentration of the obtained product to be 50, 30 and 10 mug/mL. After 24 hours of illumination culture at 28 ℃, the number of the living artemia is counted, the survival rate is counted (the number of the living artemia/the total artemia number is 100%), the test result is shown in figure 8, in the figure, R64-50 indicates that the concentration of the extract of the strain R64 is 50 mug/mL, R64-30 and R64-10 respectively indicate that the concentration of the extract of the strain R64 is 30 and 10 mug/mL, and the rest groups are analogized in turn, so that the toxicity of R64-Ag of the nano pesticide to the artemia is far lower than that of the existing chemical pesticide and nano silver standard.

In conclusion, the invention provides the bacterial strain, the extract of the bacterial strain R64, the nano pesticide and the application and preparation method thereof, and the extract of the bacterial strain R64 extracted from the bacterial strain and the nano pesticide synthesized by the extract of the bacterial strain R64 have higher antibacterial activity and high efficiency and lower toxicity to environmental microorganisms. Meanwhile, the biological preparation method has the advantages of low cost, simple operation and relative environmental protection. The invention provides a new natural product pesticide, and simultaneously reduces the environmental toxicity of the natural product pesticide and increases the antibacterial activity and the high efficiency of the natural product pesticide by a method for preparing the nano pesticide.

It will be understood that the invention is not limited to the examples described above, but that modifications and variations will occur to those skilled in the art in light of the above teachings, and that all such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.

Claims

1. A preparation method of a nano pesticide is characterized by comprising the following steps:

providing a strain R64, wherein the classification name of the strain R64 is Pseudomonas aeruginosa (Pseudomonas aeruginosa), and the preservation number of the strain R64 is CGMCC number 21803;

activating the strain R64 to obtain an activated R64 single colony;

sequentially fermenting and extracting the activated R64 single colony to obtain an extract of a strain R64;

mixing the extract of the strain R64 with AgNO3 solution for reaction to obtain the nano pesticide;

and sequentially fermenting and extracting the activated R64 single colonies, and specifically comprising the following steps:

inoculating the activated R64 single colony in a culture medium for shake flask culture, and then fermenting to obtain a strain R64 fermentation liquid;

eluting with methanol, and concentrating with vacuum centrifugal concentrator to obtain extract of the strain R64;

the culture medium is SGTYP, and the SGTYP consists of 5.0g of soluble starch, 5.0g of glucose, 1g of microbial peptone, 1g of tryptone, 1g of yeast extract, 17g of artificial sea salt and 1L of distilled water;

mixing the extract of the strain R64 with AgNO3 solution, and reacting to obtain the nano pesticide, which specifically comprises the following steps:

adding the extract of the strain R64 into deionized water, adjusting the pH to 7 by NaOH, adding AgNO3 solution until the final concentration is 4mM, heating to 60 ℃ under the condition of keeping out of the sun, and stirring and reacting for 60 hours on a magnetic stirrer at 700 rpm;

filtering with 0.22 μm filter membrane, centrifuging at 7500rpm for 30min, removing supernatant, adding water with equal volume to the precipitate, centrifuging at 7500rpm for 30min, repeating for three times to obtain precipitate;

and transferring, freeze-drying and weighing the precipitate, and fixing the volume to 10mg/ml by using sterile deionized water to obtain the nano pesticide.

2. The method for preparing nano pesticide according to claim 1, wherein the step of activating the strain R64 specifically comprises the following steps: putting the frozen bacterium solution into a marine bacterium agar culture medium, selecting the frozen bacterium solution, scribing by using a plate scribing method, and culturing in an incubator.

3. A nano pesticide, characterized in that the nano pesticide is prepared by the method of any one of claims 1-2.

4. An application of the nano-class agricultural chemical of claim 3 in preparing the agricultural chemical for preventing and treating plant diseases.