A method for preventing chestnut blight by mixing a fermentation filtrate of biocontrol bacteria with ethyl aluminum bisphosphonate as an immune activator

Abstract

The present application relates to the technical field of plant protection, and particularly relates to a chestnut blight prevention and treatment method based on mixing of an ethoprop aluminum-containing immune activator and a biocontrol bacterial fermentation filtrate. The technical scheme comprises the following steps: preparing PDA culture mediums containing different concentrations of ethoprop aluminum and different dilution multiples of a B. badius B268 fermentation filtrate, transferring a bacterial cake for culture, performing indoor toxicity determination, selecting chestnut saplings, setting three treatment groups and two control groups, collecting leaf blades before spraying of the pesticide for storage, and using the leaf blades for subsequent enzyme activity analysis and MDA content determination, determining a spraying concentration according to the toxicity test result, spraying the treatment groups of the chestnut saplings, mixing the ethoprop aluminum and the B268 fermentation filtrate in the treatment group, and then performing inoculation experiments, collecting leaf blade samples periodically after inoculation for subsequent enzyme activity analysis and MDA content determination, and performing transcriptome sequencing. The present application can improve the bacteriostatic capacity of the prevention and treatment pesticide, activate the plant's own immune function, and reduce the amount of chemical pesticide application.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant protection, and particularly relates to a chestnut blight prevention method based on mixing a fermentation filtrate of biocontrol bacteria with aluminum ethylphosphonate as an immune activator. BACKGROUND

[0002] Chestnut blight, also known as chestnut dry rot or ketone dry rot, is a fungal disease mainly caused by Cryphonectria parasitica, which seriously damages the main stem and branches of chestnut, and sometimes affects the leaves. In the initial stage, many round or irregular lesions appear on the branches, with colors ranging from red-brown to purple-brown. The lesions are slightly raised, sunken when dry, and have dry shrinkage and cracks, with dense orange or brown small particles. The disease often occurs on the main stem or main branch about 1 meter above the ground. The initial water-logged spots on the diseased part gradually expand and surround the main stem, with wet and rotten tissues and possible overflow of brown juice. When the disease worsens, the lesion color deepens, which may cause the bark to shrink and crack longitudinally, and eventually die. The leaves on the infected main stem or branch become small and yellow, and in severe cases, the buds cannot germinate and the leaves turn brown and die.

[0003] Aluminum ethylphosphonate: As an organic phosphorus systemic fungicide with bidirectional conduction, its fungicidal effect not only lies in inhibiting spore germination or hindering the formation of mycelium and spores after entering the plant body, but also lies in stimulating the pathogen to release elicitors to induce the synthesis of phytoalexins. When the pathogen invades, the plant can rapidly synthesize and accumulate flavonoid phytoalexins, thereby greatly enhancing the plant's resistance to pathogenic bacteria.

[0004] Bacillus velezensis B268: Strain B268 was isolated from rhizosphere soil samples of Casuarina equisetifolia coastal protection forest in Yueqing City, Zhejiang Province (Na+ content is 7.119 mg per gram of dry soil), and the preservation number is CGMCC No. 13225. B268 was identified by multiphase classification method and determined as a new strain type of the same Bacillus velezensis group. Studies have found that B268 grows best in 3% (W / V) NaCl, and its growth range is between 0 and 15% (W / V) NaCl. The antibacterial activity of B268 was determined by viable cell counting and plate confrontation method. The strain has antagonistic activity against 9 kinds of tested pathogenic bacteria, and has research and development potential in plant salt damage and disease biological control.

[0005] At present, the main methods for preventing and treating chestnut blight include chemical control and biological control. Chemical control mainly uses fungicides for spraying, but long-term use may lead to drug resistance of pathogenic bacteria, pollution of the environment, and affect the quality of chestnut. Biological control mainly uses antagonistic microorganisms or their metabolites to prevent and control diseases, which has the advantages of environmental protection and safety, but the prevention and control effect is often not as good as chemical control.

[0006] Therefore, the application provides a chestnut blight prevention and treatment method based on a fermentation filtrate of biocontrol bacteria mixed with ethoprophos as an immune activator. SUMMARY

[0007] The application aims to solve the problems in the background art by providing a chestnut blight prevention and treatment method based on a fermentation filtrate of biocontrol bacteria mixed with ethoprophos as an immune activator.

[0008] To achieve the above-mentioned purpose, the application provides the following technical solution: a chestnut blight prevention and treatment method based on a fermentation filtrate of biocontrol bacteria mixed with ethoprophos as an immune activator, comprising the following steps:

[0009] Step 1: using PDA medium to culture chestnut blight and using a puncher to punch a fungus cake;

[0010] Step 2: using different concentrations of ethoprophos solution and different dilution multiples of fermentation filtrate of Bacillus velezensis B268 to configure PDA medium and culture the fungus cake in the medium for indoor virulence determination;

[0011] Step 3: selecting chestnut saplings, setting two control groups and three treatment groups: the positive control group is sprayed with sterile water and inoculated with a pathogenic fungus; the negative control group is sprayed with sterile water and inoculated with sterile PDA; the ethoprophos treatment group; the B268 fermentation filtrate treatment group and the ethoprophos+B268 fermentation filtrate treatment group, in the case of not spraying the agent, collecting the sapling leaves and storing them for subsequent defense enzyme activity analysis and MDA content determination;

[0012] Step 4: according to the results of step 2, using the concentration with better indoor bacteriostatic effect to uniformly spray the leaves and trunks of the three treatment groups, wherein the ethoprophos+B268 fermentation filtrate treatment group needs to be mixed, the mixing step is to configure the ethoprophos solution, calculate the volume of the fermentation filtrate added according to the volume of the ethoprophos solution, and perform ex vivo inoculation and in vivo inoculation experiments respectively after three days;

[0013] Step 5: on the 2nd, 4th, 8th and 16th day after inoculation, regularly collect leaf samples of each group and properly store them for subsequent defense enzyme activity analysis and MDA content determination, and on the 6th day after inoculation, select part of the samples for transcriptome sequencing analysis;

[0014] The preparation method of ethoprophos in the ethoprophos treatment group and the ethoprophos+B268 fermentation filtrate treatment group is as follows: preparing raw materials: 95% purity ethoprophos original drug and additives, the additives including 50% purity alkyl polyglycoside, dodecyl betaine, gum arabic as a binder, ammonium sulfate as a filler, then mixing, crushing, mixing with water, granulating and drying;

[0015] The above-mentioned Bacillus velezensis B268 is isolated from the rhizosphere soil of Casuarina equisetifolia forest, and its preservation number is CGMCC No.13225. The method for preparing the fermentation liquor of Bacillus velezensis B268 is as follows: first, a single colony of B268 strain is inoculated into a test tube containing 4 ml of YTGB medium, then the seed liquor is prepared by culturing at 28°C and 140 rpm for 24 hours, then 1% of the seed liquor is used for expansion culture for 48 hours to obtain the fermentation liquor, finally, the fermentation filtrate of B268 is obtained by centrifugal separation and filtering the supernatant with a 0.22 micron filter membrane.

[0016] The preservation unit of Bacillus velezensis B268 is the General Microbiological Center of China Microbial Culture Collection Management Committee;

[0017] The preservation address is No.3, Beichen West Road, Chaoyang District, Beijing;

[0018] The preservation registration number is CGMCC No.13225;

[0019] The taxonomic name is Bacillus siamensis;

[0020] The biological material (strain) was received by the preservation center on November 1, 2016 and registered.

[0021] Preferably, the ingredient ratio of the 4 ml YTGB medium is as follows: yeast extract 0.05%, tryptone 0.5%, glucose 1%, beef extract 0.3%, and the pH value is 7.0.

[0022] Preferably, the raw material preparation ratio of the aluminum ethylphosphonate is as follows: aluminum ethylphosphonate original drug 85-87%, alkyl glycoside 2-5%, dodecyl betaine 2-5%, gum arabic 1-5%, and ammonium sulfate 5-10%. Then the above ingredients are uniformly mixed, the particle size of the crushed material is controlled to be less than 23 microns, then water is added to the total amount of the material, the amount of water added is 7%, then the material is extruded and granulated by a screw extruder, finally, the material is dried by a vibration fluidized bed dryer, the gas phase temperature of the dryer is controlled to be less than 120°C, and the moisture content of the dried granules is less than 0.6%.

[0023] Preferably, the indoor virulence determination includes: preparing PDA medium with the concentration of aluminum ethylphosphonate of 0.1 mg / L, 1 mg / L, 10 mg / L, 100 mg / L and 200 mg / L and inoculating the Chinese chestnut blight fungus, then culturing under the condition of 28°C and light, when the mycelium of the control group covers 3 / 4 of the medium, the inhibitory effect is evaluated by the mycelial growth rate method, and the EC 50The fermentation filtrate of Bacillus velezensis B268 is diluted to 5 times, 10 times, 20 times, 40 times and 80 times, PDA culture medium is configured in this way and inoculated with chestnut blight, and the culture is carried out under the condition of illumination at 28 DEG C, when the mycelium of the control group grows to 3 / 4 of the culture medium, the inhibitory effect is determined by using the mycelium growth rate method, and the EC 50 value is calculated.

[0024] Preferably, the defense enzyme activity analysis comprises quantifying the functional activity of catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD).

[0025] Preferably, the in vitro inoculation is as follows: after three days of treatment, the leaf veins of the leaves of each treatment are wrapped with sterile cotton for moisturizing, a 5mm measuring area is marked on the middle part of the leaf veins with a vernier caliper, the area is scratched with a sterilized blade, then the pathogenic bacteria cultured for three days are used to make a fungus cake with a 5mm puncher, and the mycelium surface of the fungus cake is tightly attached to the wound of the leaf, and then the leaf is placed in a sterile fresh-keeping box with a filter paper soaked with sterile water at the bottom for moisturizing culture.

[0026] Preferably, the in vivo inoculation is as follows: a hole is created on the tree trunk by using a 7mm puncher, the phloem is removed, the pathogenic bacteria cultured for three days are used to make a fungus cake by using a 7mm puncher, and the mycelium surface of the fungus cake is attached to the xylem, then the hole is wrapped with a sealing film for moisturizing treatment, and the negative control group is inoculated with PDA culture medium without pathogenic bacteria.

[0027] Compared with the prior art, the application has the following beneficial effects:

[0028] The bactericidal mixture obtained by binary compounding of Bacillus velezensis B268 and phosphocarb has a synergistic effect on the prevention and treatment of chestnut blight;

[0029] The prevention and treatment effect is remarkable: the resistance of chestnut trees to chestnut blight can be effectively improved, the occurrence and expansion of disease spots are reduced, and the prevention and treatment effect is better than that of traditional biological control methods;

[0030] The bactericidal composition of the application utilizes the disease resistance function of biological fungicides and the immune activation function of phosphocarb, improves the disease resistance effect from the inside and outside of the plant body, and can achieve the ecological mode of reducing the amount of fungicide and improving the effect by compounding.

[0031] Mixing of phosphocarb and Bacillus velezensis B268 can improve the bacteriostatic capacity of the fungicide, activate the immune function of the plant itself, reduce the amount of chemical pesticide, and play the disease resistance induction function of phosphocarb and the bacteriostatic effect of the fermentation filtrate of Bacillus velezensis B268, so that the overall prevention and treatment effect is improved. BRIEF DESCRIPTION OF DRAWINGS

[0032] Fig. 1 A flowchart of the present application;

[0033] Fig. 2 A schematic diagram of the inhibition effect of B. velezensis B268 fermentation filtrate on PDA on the chestnut blight fungus;

[0034] Fig. 3 A schematic diagram of the inhibition effect of B. velezensis B268 fermentation filtrate on PDA on the chestnut blight fungus. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0036] Example 1

[0037] As shown in the drawings, Figs. 1-3 The present application proposes a chestnut blight prevention and treatment method based on ethyl aluminum phosphate as an immune activator mixed with biocontrol agent fermentation filtrate, which comprises the following steps:

[0038] B. velezensis B268 is isolated from rhizosphere soil samples of Casuarina equisetifolia coastal protection forest, and has a preservation number of CGMCC No.13225. The preparation method of the B. velezensis B268 fermentation broth is as follows: a single colony is picked on LB culture medium inoculated with B268 strain to a test tube containing 4 ml YTGB culture medium ((W / V): yeast extract (BD) 0.05%, trypticase peptone (BD) 0.5%, glucose 1%, beef powder 0.3%, pH 7.0), and then the test tube is cultured at 28°C and 140 r / min for 24 h to prepare a seed liquid. 1% of the seed liquid is taken for expansion culture for 48 h to obtain the fermentation broth. After centrifugation, the supernatant is filtered through a 0.22 μm filter membrane to obtain the B268 fermentation filtrate;

[0039] The preparation method of ethoprophos in the ethoprophos treatment group and the ethoprophos+B268 fermentation filtrate treatment group is as follows: 95% pure ethoprophos raw material and adjuvants, including 50% pure alkyl glycoside, dodecyl betaine, gum arabic as a binder, and ammonium sulfate as a filler, are mixed, crushed, mixed with water, granulated, and dried. The proportion of raw material preparation is as follows: ethoprophos raw material: 85-87%, alkyl glycoside: 2-5%, dodecyl betaine: 2-5%, gum arabic: 1-5%, and ammonium sulfate: 5-10%. Then, the above ingredients are uniformly mixed, crushed to control the fineness of the crushed material to be less than 23 microns, then mixed with water in an amount of 7% of the total amount of the material, then extruded and granulated by a screw extrusion granulator, and finally dried by a vibrating fluidized bed dryer with a gas phase temperature less than 120°C. The moisture content of the dried granules is less than 0.6%.

[0040] Step 1: Cultivate the chestnut blight fungus on PDA medium for 3 days to generate a fungus cake. Under sterile conditions, use a 5mm sterile punch to punch the fungus cake from the edge of the colony where the mycelium is growing vigorously.

[0041] Step 2: Use different concentrations of ethoprophos solution and different dilutions of Bacillus velezensis B268 fermentation filtrate to configure PDA medium and transfer the chestnut blight fungus culture.

[0042] Then perform indoor toxicity determination, with the following steps:

[0043] Use concentrations of 0.1mg / L, 1mg / L, 10mg / L, 100mg / L, and 200mg / L of ethoprophos to configure PDA medium and inoculate the chestnut blight fungus. Dilute the Bacillus velezensis B268 fermentation filtrate to 5 times, 10 times, 20 times, 40 times, and 80 times to configure PDA medium and inoculate the chestnut blight fungus. Incubate at 28°C under light. When the mycelium of the control group grows to 3 / 4 of the medium, use the mycelial growth rate method to determine the inhibition effect and calculate the EC 50 value.

[0044] The specific implementation method is as follows:

[0045] Ethoprophos group:

[0046] Dissolve an appropriate amount of ethoprophos solid particles in sterile water to prepare 0.1mg / L, 1mg / L, 10mg / L, 100mg / L, and 200mg / L ethoprophos solutions, respectively.

[0047] Take the appropriate amount of potato dextrose agar (PDA) powder, according to the instructions, add distilled water, heat and dissolve, and then pour the dissolved PDA medium into sterile culture dishes, about 15-20 ml per dish. When the PDA medium cools to 50-60℃, add different concentrations of ethylphosphonate solution and mix well.

[0048] Inoculate the chestnut blight fungus on PDA medium containing different concentrations of ethylphosphonate, with 5 replicates for each concentration. The control group does not add any solution and only inoculates the chestnut blight fungus.

[0049] Bacillus velezensis B268 fermentation filtrate group:

[0050] Prepare PDA medium according to the formula and adjust the volume to the same volume. After sterilization, wait until it cools to 50-60℃.

[0051] Add the Bacillus velezensis B268 fermentation filtrate to the PDA medium according to the dilution factor to obtain a dilution of 5 times, 10 times, 20 times, 40 times, and 80 times, respectively, for standby.

[0052] Pour the PDA medium with B268 fermentation filtrate into culture dishes, about 15-20 ml per dish. Set 5 replicates for each concentration. The control group does not add any solution and only inoculates the chestnut blight fungus.

[0053] After the above ethylphosphonate group and Bacillus velezensis B268 fermentation filtrate group are transferred, the following steps are taken:

[0054] Place the inoculated culture dishes in a 28℃, light incubator for incubation.

[0055] Observe the mycelial growth every day. When the control group's mycelium covers 3 / 4 of the medium, start measuring the colony diameter of each treatment group.

[0056] Use a ruler or vernier caliper to measure the colony growth diameter of each treatment group.

[0057] Calculate the mycelial growth rate of each treatment group (mycelial growth diameter / culture days):

[0058] Calculate the mycelial growth inhibition rate of each treatment group according to the following formula: inhibition rate (%) = (control group colony diameter - treatment group colony diameter) / (control group colony diameter - colony cake diameter) x 100%.

[0059] EC 50 The value refers to the concentration of fermentation filtrate required to inhibit mycelial growth by 50%. Then use statistical software SPSS to process the data and calculate the EC 50 value. Through the above steps, the concentration of ethylphosphonate solution and the concentration of Bacillus velezensis B268 fermentation filtrate that have better indoor antibacterial effect can be calculated.

[0060] Step 3: Selecting chestnut seedlings with consistent age and growth vigor, and setting two control groups:

[0061] Positive control group: spraying sterile water and inoculating pathogenic fungi;

[0062] Negative control group: spraying sterile water and inoculating sterile PDA,

[0063] Three treatment groups: aluminum phosphide treatment group, B268 fermentation filtrate treatment group, and aluminum phosphide + B268 fermentation filtrate treatment group. Collect leaf blades when no pesticide is sprayed and save for subsequent defense enzyme activity analysis and MDA content determination.

[0064] Step 4: According to the results of step 2, use the concentration with better indoor antibacterial effect as the spraying concentration. Spray the corresponding concentration solution uniformly on the leaf surface and trunk for the three treatment groups: aluminum phosphide treatment group, B268 fermentation filtrate treatment group, and aluminum phosphide + B268 fermentation filtrate treatment group. The aluminum phosphide + B268 fermentation filtrate treatment group needs to be mixed. The mixing steps are to prepare the aluminum phosphide solution, calculate the volume of the fermentation filtrate added according to the volume of the aluminum phosphide solution, and perform ex vivo inoculation and in vivo inoculation after three days.

[0065] The mixing step is to calculate the volume of the fermentation filtrate added according to the volume of the aluminum phosphide solution, and perform ex vivo inoculation and in vivo inoculation after three days. According to the determined mixing ratio, slowly add Bacillus velezensis B268 fermentation filtrate to the aluminum phosphide solution, gently stir the mixed solution with a sterile stirring rod to ensure that the two liquids are fully mixed and uniform.

[0066] The specific steps of ex vivo inoculation are as follows:

[0067] After three days, collect the leaves of each group (positive control group, aluminum phosphide treatment group, B268 fermentation filtrate treatment group, and aluminum phosphide + B268 fermentation filtrate treatment group).

[0068] Wrap the collected leaf blades with sterile cotton at the main vein end, keep the leaves moist, and use a vernier caliper to measure a 5mm area at the main vein of the middle part of the leaf, and mark it with a marker.

[0069] Gently scratch the marked 5mm area on the leaf with a sterilized blade to expose the wound.

[0070] Use the fungus cake prepared in step 1, with the fungus cake mycelium facing down, attached to the wound area of the leaf, and ensure that the fungus cake is in close contact with the wound.

[0071] Place the inoculated leaves in a sterile fresh-keeping box with a bottom covered with a filter paper soaked with sterile water for moist culture.

[0072] Periodically observe the growth of the pathogen on the leaves, record the expansion speed of the pathogen, the degree of disease, and other data.

[0073] The leaf disease calculation method is as follows:

[0074] Observe the growth of the pathogen on the leaves at certain intervals (24 hours, 48 hours, 72 hours) after inoculation, use a magnifying glass or microscope to assist observation, and record the expansion of the mycelium;

[0075] Use a camera or mobile phone to take pictures to record the development of the disease on the leaves for subsequent analysis, and record the diameter or area of the pathogen at each inoculation point.

[0076] According to the degree of expansion of the pathogen on the leaves, the disease can be divided into several grades:

[0077] 0: No disease spot

[0078] 1: Disease spot diameter less than 1mm

[0079] 3: Disease spot diameter between 1-2mm

[0080] 5: Disease spot diameter between 2-5mm

[0081] 7: Disease spot diameter greater than 5mm

[0082] Disease index is a quantitative indicator reflecting the severity of the disease, and the calculation formula is as follows:

[0083]

[0084] Collect data from each group of leaves.

[0085] According to the calculation results, the disease index of each group is obtained.

[0086] According to the disease index of the in vitro leaves, the lower the disease index, the milder the disease of the plant; the higher the disease index, the more serious the disease of the plant, thereby determining the influence of different control groups on the growth and incidence of chestnut blight, indicating that ethoprophos and Bacillus velezensis B268 fermentation filtrate have a synergistic effect.

[0087] The specific steps of in vivo inoculation are as follows:

[0088] After three days, prepare the chestnut seedlings of each group (positive control group, ethoprophos treatment group, B268 fermentation filtrate treatment group, ethoprophos + B268 fermentation filtrate treatment group).

[0089] Prepare a 7mm puncher, sealing film, sterile PDA medium, and pathogen, use a 7mm puncher to punch the surface of the trunk, the depth should reach the removal of phloem, exposing the xylem, ensuring that the hole is clean and no debris is left.

[0090] Using the fungus cake made in step 1, the mycelium surface is pasted into the hole of the trunk xylem, and the fungus cake is pressed gently to ensure that the mycelium is in close contact with the xylem.

[0091] Use sealing film to wrap and seal the inoculation site to maintain humidity and promote the growth of pathogenic bacteria.

[0092] On another group of trees, use the same method to punch holes and inoculate sterile PDA medium as a negative control, with the same steps as inoculating pathogenic bacteria.

[0093] After inoculation, observe and record the reaction of the inoculation site regularly, such as the formation and expansion of lesions, and record the differences between different treatment groups. The specific observation method is as follows:

[0094] Cover the lesion with transparent grid paper and calculate the number of grid squares occupied by the lesion to estimate the lesion area.

[0095] Use image processing software Photoshop to measure the lesion area.

[0096] According to the proportion of lesion area to the total area of inoculation site, set the disease grade;

[0097] No lesion: 0 grade

[0098] Lesion area ≤5%: 1 grade

[0099] 5% < lesion area ≤10%: 3 grade

[0100] 10% < lesion area ≤20%: 5 grade

[0101] Lesion area >20%: 7 grade,

[0102] By collecting the lesion data of the trunk of each control group, calculate the disease index by the disease index calculation formula.

[0103] According to the calculation results, the disease index of the three control groups is obtained.

[0104] According to the disease index of the trunk lesion, the lower the disease index, the lighter the disease of the plant; the higher the disease index, the more serious the disease of the plant, so as to determine the influence of different control groups on the growth and incidence of chestnut blight, indicating that ethoprophos and Bacillus velezensis B268 fermentation filtrate have a synergistic effect.

[0105] Step 5: After 2 days, 4 days, 6 days, 8 days, and 16 days of inoculation, prepare sterile scissors, tweezers, and labeled sterile sampling bags, and randomly select leaves with consistent growth from each group of plants using sterile scissors.

[0106] Put the cut leaves into sterile sampling bags, and label them (including group, inoculation time, sampling time, etc.).

[0107] Put the sampling bags into liquid nitrogen for quick freezing, and store them in a -80℃ refrigerator.

[0108] At this time, take out the leaf samples stored for 2 days, 4 days, 8 days, and 16 days in step 3 and step 5 from the -80℃ refrigerator:

[0109] Catalase (CAT) activity determination:

[0110] First, mix the plant tissue with phosphate buffer (pH 7.0-7.4) and grind into homogenate in an ice bath.

[0111] Then centrifuge the homogenate at 12000g at 4℃, and take the supernatant for enzyme activity testing.

[0112] Prepare the reaction mixture in a test tube, containing 50mM phosphate buffer, 10mM H2O2 solution, and enzyme extract.

[0113] Add H2O2 to start the reaction, record the consumption of H2O2, and end the reaction with ammonium sulfate solution.

[0114] Finally, measure the reduction of H2O2 by spectrophotometry to calculate the activity of CAT.

[0115] Superoxide dismutase (SOD) activity determination:

[0116] Take the plant tissue, add a certain proportion of phosphate buffer (pH 7.0-7.4), and grind into homogenate in an ice bath;

[0117] Use the nitrogen blue tetrazolium (NBT) photochemical reduction method, and the reaction mixture includes:

[0118] 50mM phosphate buffer (pH 7.8), 13mM methionine, 75μM NBT, 0.1mM EDTA, 2μM riboflavin, enzyme extract;

[0119] Place the reaction mixture under a daylight lamp for photochemical reaction;

[0120] Immediately after the end of the light, terminate the reaction in the dark;

[0121] Determine the absorbance at 560nm, and calculate the SOD activity.

[0122] Peroxidase (POD) activity determination:

[0123] First, mix the plant tissue with phosphate buffer (pH 7.0-7.4) and grind into homogenate in an ice bath.

[0124] Then, a reaction mixture containing 25 mM phosphate buffer, 20 mM guaiacol, 5% H2O2 and enzyme extract was prepared.

[0125] The reaction was initiated by the addition of H2O2 and the change in absorbance was recorded at 470 nm using a spectrophotometer to calculate the activity of POD.

[0126] Malondialdehyde (MDA) content determination:

[0127] First, the tissue was mixed with phosphate buffer (pH 7.0-7.4) and ground into a homogenate in an ice bath.

[0128] Then, an equal amount of 20% TCA solution was added to the supernatant of the homogenate, mixed, and centrifuged at 12000g at 4°C for 10 minutes.

[0129] Next, the supernatant was mixed with an equal amount of 0.6% TBA solution, heated in a boiling water bath for 15 minutes, quickly cooled and centrifuged.

[0130] Finally, the content of MDA was determined by measuring the absorbance at 532 nm and 600 nm.

[0131] According to the calculation results, the effects of different control groups on the content of catalase, superoxide dismutase, peroxidase and malondialdehyde in plants were determined.

[0132] On the 6th day after inoculation, the leaf samples of step 5 for 6 days were taken for transcriptome sequencing,

[0133] Data analysis was performed on the sequencing data to better understand the effects of aluminum phosphide + B268 fermentation filtrate treatment on the differences in chestnut gene expression before and after treatment.

[0134] By comparing the above control groups, it can be concluded that the aluminum phosphide + B268 fermentation filtrate control group has good control effect.

[0135] According to the above method for evaluating the control effect, the synergistic effect of the aluminum phosphide solution and the B268 fermentation filtrate is obtained, thereby providing a chestnut blight control method based on the aluminum phosphide as an immune activator mixed with the fermentation filtrate of the biocontrol bacteria.

[0136] The above specific embodiments are only a few preferred embodiments of the present application, and based on the technical solutions of the present application and the related inspiration of the above embodiments, those skilled in the art can make various alternative improvements and combinations to the above specific embodiments.

[0137] It is apparent for a person skilled in the art that the present application is not limited to the details of the above described exemplary embodiments, but that it can be implemented in other concrete forms without departing from the spirit or essential characteristics of the application. Therefore, the embodiments should be considered in all respects as illustrative and not restrictive, the scope of the application being defined by the appended claims rather than the above description, and all changes coming within the meaning and equivalency range of the claims are intended to be embraced therein.

Claims

1. A method for preventing chestnut blight by using a fermentation filtrate of biocontrol bacteria mixed with ethoprophos as an immune activator, characterized in that, The method comprises the following steps: Step 1: culture the chestnut blight fungus by using a PDA culture medium and punch the fungus cake by using a puncher; Step 2: prepare PDA culture mediums by using different concentrations of ethoprophos solutions and different dilution multiples of the B. berlesianum B268 fermentation filtrate, and culture the fungus cake in the culture mediums to perform indoor toxicity determination; Step 3: select chestnut saplings, and set two control groups and three treatment groups: the positive control group is sprayed with sterile water and inoculated with the pathogenic fungus; the negative control group is sprayed with sterile water and inoculated with sterile PDA; the ethoprophos treatment group, the B. berlesianum B268 fermentation filtrate treatment group and the ethoprophos + B. berlesianum B268 fermentation filtrate treatment group are not sprayed with the agent, and the leaves of the saplings are collected and stored for subsequent determination of the defense enzyme activity and the MDA content; Step 4: according to the results of Step 2, the leaves and stems of the saplings in the three treatment groups are uniformly sprayed with the concentration with better indoor bacteriostatic effect, and the ethoprophos + B. berlesianum B268 fermentation filtrate treatment group needs to be mixed, the mixing step is to prepare the ethoprophos solution, add the fermentation filtrate according to the volume of the ethoprophos solution, and perform the in vitro inoculation and in vivo inoculation experiments after three days; Step 5: on the 2nd, 4th, 8th and 16th days after inoculation, the leaf samples in each group are regularly collected and properly stored for subsequent determination of the defense enzyme activity and the MDA content, and on the 6th day after inoculation, part of the samples are selected for transcriptome sequencing analysis; The preparation method of the ethoprophos in the ethoprophos treatment group and the ethoprophos + B. berlesianum B268 fermentation filtrate treatment group comprises the following steps: preparing raw materials, i.e., 95% pure ethoprophos and additives, the additives comprising 50% pure alkyl polyglycoside, dodecyl betaine, gum arabic as a binder and ammonium sulfate as a filler, then mixing, crushing, mixing with water, granulating and drying; The B. berlesianum B268 is separated from the rhizosphere soil of Casuarina equisetifolia coastal forest, and the preservation number is CGMCC No.13225, and the preparation method of the B. berlesianum B268 fermentation liquid is as follows: firstly, inoculate the single colony of the B268 strain into a test tube containing 4 ml of YTGB culture medium, then culture at 28℃ and 140 rpm for 24 hours to prepare a seed liquid, then take 1% of the seed liquid to perform expansion culture for 48 hours to obtain a fermentation liquid, and finally, centrifugalize and filter the supernatant by using a 0.22-micron filter membrane to obtain the fermentation filtrate of the B268.

2. A method for preventing and controlling chestnut blight based on fosetyl aluminum as an immune activator mixed with biocontrol bacteria fermentation filtrate according to claim 1, characterized in that: The component ratio of the 4 ml YTGB culture medium is as follows: 0.05% of yeast extract, 0.5% of tryptone, 1% of glucose and 0.3% of beef extract powder, and the pH value is 7.

0.

3. The method for preventing and controlling chestnut blight based on fosetyl aluminum as an immune activator mixed with biocontrol bacteria fermentation filtrate according to claim 1, characterized in that: The raw material ratio of the phosethyl-Al in the phosethyl-Al treatment group and the phosethyl-Al+B268 fermentation filtrate treatment group: phosethyl-Al original drug: 85-87%, alkyl glycoside: 2-5%, dodecyl betaine: 2-5%, gum arabic: 1-5%, ammonium sulfate: 5-10%, then the above ingredients are mixed uniformly, the material fineness after crushing is controlled to be less than 23 microns, then water is added, the water addition amount accounts for 7% of the total amount of the material, then extrusion granulation is carried out through a screw extrusion granulator, finally, drying is carried out by using a vibration fluidized bed dryer, the gas phase temperature of the dryer is controlled to be less than 120℃, and the moisture content of the dried granules is less than 0.6%.

4. The method for preventing chestnut blight according to claim 1, wherein the immune activator is ethyl phosphonate aluminum. 1 The indoor virulence assay includes: preparing PDA culture medium with the concentration of 0.1 mg / L, 1 mg / L, 10 mg / L, 100 mg / L, 200 mg / L of phosphorus aluminum concentration and inoculating chestnut blight, and then culturing under the condition of 28℃ light, when the mycelium of the control group covers 3 / 4 of the culture medium, the inhibitory effect is evaluated by mycelial growth rate method, and the EC 50 value is calculated; the fermentation filtrate of bacillus velezensis B268 is diluted by 5 times, 10 times, 20 times, 40 times, 80 times, and PDA culture medium is configured in this way and inoculated with chestnut blight, and also cultured under the condition of 28℃ light, when the mycelium of the control group grows to 3 / 4 of the culture medium, the inhibitory effect is determined by mycelial growth rate method, and the EC 50 value is calculated.

5. The method for preventing and controlling chestnut blight based on fosetyl aluminum as an immune activator mixed with biocontrol bacteria fermentation filtrate according to claim 1, characterized in that: The defense enzyme activity analysis includes quantifying the functional activity of catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD).

6. The method for preventing chestnut blight according to claim 1, wherein the immune activator is ethyl phosphonate aluminum. 6 The in vitro inoculation: after three days of treatment, the leaves of each treatment are taken for indoor in vitro inoculation experiment, the end of the leaf veins of the leaves is wrapped with sterile cotton for moisturizing, and a 5mm measuring area is marked in the middle of the leaf veins of the leaves by using a vernier caliper, the area is scratched by using a sterilized blade, then the pathogen cultured for three days is taken, a fungus cake is made by using a 5mm puncher, and the mycelium surface is tightly attached to the leaf wound, then the fungus cake is placed in a sterile fresh-keeping box with a filter paper soaked in sterile water at the bottom for moisturizing culture.

7. The method for preventing and controlling chestnut blight based on fosetyl aluminum as an immune activator mixed with biocontrol bacteria fermentation filtrate according to claim 1, characterized in that: The in vivo inoculation: a hole is created on the trunk by using a 7mm puncher, the phloem is removed, the pathogen cultured for three days is taken, a fungus cake is made by using a 7mm puncher, and the mycelium surface is attached to the xylem, then the hole is surrounded by a sealing film for moisturizing treatment, and the negative control group is inoculated with PDA culture medium without pathogen.

Images