Method for preventing and treating cryphonectria parasitica based on blending of aliette as immune activator and biocontrol bacterium fermentation filtrate

Abstract

The invention relates to the technical field of plant protection, in particular to a cryphonectria parasitica prevention and control method based on a mixture of fosetyl-aluminum as an immune activator and a biocontrol bacterium fermentation filtrate. According to the technical scheme, the method comprises the steps that a PDA culture medium containing validamycin and bacillus velezensis B268 is prepared, fosetyl-aluminum with different concentrations and bacillus velezensis B268 fermentation filtrate with different dilution ratios are added into the culture medium, pathogenic bacterium cakes are inoculated for culture, indoor toxicity determination is conducted, three-year-old Chinese chestnut tree seedlings are selected, and the three-year-old Chinese chestnut tree seedlings are selected. The method comprises the following steps: setting three treatment groups and two control groups, collecting leaves before spraying a medicament, storing the leaves for subsequent enzyme activity and MDA content determination, determining the spraying concentration according to a toxicity test result, spraying Chinese chestnut seedlings in the treatment groups, mixing fosetyl-aluminum + B268 fermentation filtrate in the treatment groups, then carrying out an inoculation experiment, and regularly collecting leaf samples. The kit is used for detecting the activity of a defensive enzyme and the content of Malrondialdehyde (MDA, malondialdehyde) and carrying out transcriptome sequencing. The invention can improve the bacteriostatic ability of the prevention and treatment agent, activate the autoimmune function of plants, and reduce the application amount of chemical pesticides.

Description

Technical Field

[0001] The invention relates to the technical field of plant protection, and in particular to a method for preventing and controlling chestnut blight based on ethyl phosphite aluminum as an immune activator mixed with biocontrol bacteria fermentation filtrate. Background Art

[0002] Chestnut blight, also known as chestnut blight or ketone blight, is a fungal disease caused mainly by Cryphonectria parasitica. This disease causes serious damage to the trunk and branches of chestnuts, and sometimes affects the leaves. In the initial stage, many round or irregular lesions will appear on the branches, ranging from reddish brown to purple-brown in color. The lesions are slightly raised, sinking when dry, shrinking and cracking, and densely covered with orange-yellow or brown small particles. The disease often occurs in the trunk or main branch about 1 meter above the ground. Water-soaked spots appear in the diseased part at first, gradually expanding to surround the trunk, the tissue is wet and rotten, and brown juice may overflow. When the disease worsens, the color of the lesions deepens, which may cause the bark to shrink and crack longitudinally, and eventually die. The leaves on the infected trunk or branches will become smaller and yellower. In severe cases, the buds cannot germinate, and the leaves turn brown and die.

[0003] Aluminum fosetyl: Aluminum fosetyl is a bidirectional organophosphorus systemic fungicide. Its fungicidal effect is not only reflected in the fact that it can inhibit spore germination or hinder the formation of mycelium and spores after entering the plant body, but its metabolite phosphate can stimulate pathogens to release elicitors to induce the synthesis of phytoalexins, and when pathogens invade, it can enable plants to quickly synthesize and accumulate flavonoid phytoalexins, thereby greatly enhancing the plant's resistance to pathogens.

[0004] Bacillus Velezii B268: Strain B268 was isolated from the rhizosphere soil sample of Casuarina equisetifolia coastal defense forest in Yueqing City, Zhejiang Province (Na+ content was 7.119 mg per gram of dry soil), with the deposit number CGMCC No.13225. B268 was identified by multiphase classification and determined to be a new phylotype of the same Bacillus Velezii group. The study found that B268 grew best under 3% (W / V) NaCl conditions, and its growth range was between 0 and 15% (W / V) NaCl. The antibacterial activity of B268 was determined by viable bacterial counts and plate confrontation methods. The strain had antagonistic activity against 9 test pathogens and has research and development potential in the biological control of plant salt damage and diseases.

[0005] At present, the main methods for preventing and controlling chestnut blight include chemical control and biological control. Chemical control mainly uses fungicides for spraying, but long-term use will cause pathogens to develop drug resistance, pollute the environment, and affect the quality of chestnuts. Biological control mainly uses antagonistic microorganisms or their metabolites to prevent and control diseases. It has the advantages of environmental protection and safety, but the prevention effect is often not as good as chemical control.

[0006] To this end, we proposed a method for preventing and controlling chestnut blight based on ethylene phosphite aluminum as an immune activator mixed with biocontrol bacteria fermentation filtrate. Summary of the invention

[0007] The purpose of the present invention is to address the problems existing in the background technology and to propose a method for preventing and controlling chestnut blight based on ethyl phosphite aluminum as an immune activator mixed with biocontrol bacteria fermentation filtrate.

[0008] To achieve the above-mentioned purpose, the present invention provides the following technical scheme: a method for preventing and controlling chestnut blight based on fosetyl aluminum as an immune activator mixed with biocontrol bacteria fermentation filtrate, comprising the following steps, the step 1: using PDA culture medium to culture chestnut blight pathogens and using a puncher to make bacterial cakes, the step 2: using fosetyl aluminum solutions of different concentrations and different dilutions of Bacillus Velez B268 fermentation filtrate to prepare PDA culture medium and transferring the bacterial cakes to the culture medium for culturing, and performing indoor toxicity determination, the step 3: selecting chestnut seedlings, setting two control groups and three treatment groups: a positive control group (sprayed with sterile water and inoculated with pathogens), a negative control group (sprayed with sterile water and inoculated with sterile PDA), a fosetyl aluminum treatment group, a B268 fermentation filtrate treatment group and a fosetyl aluminum + B268 fermentation In the filtrate treatment group, without spraying the agent, the leaves of the seedlings were collected and preserved for subsequent determination of defense enzyme activity and MDA content. The step 4: according to the result of step 2, the chestnut seedlings and trunks of the three treatment groups were evenly sprayed with a concentration with good indoor antibacterial effect, wherein the ethylene phosphate aluminum + B268 fermentation filtrate treatment group needed to be mixed, and the mixing step was to prepare the ethylene phosphate aluminum solution, and the volume of the fermentation filtrate added was calculated according to the volume of the ethylene phosphate aluminum solution. After three days, in vitro inoculation and in vivo inoculation experiments were carried out respectively. The step 5: on the 0th, 2nd, 4th, 8th and 16th days, leaf samples of each group were regularly collected and properly preserved for subsequent detection of defense enzyme activity and MDA content. On the 8th day, some samples were selected for transcriptome sequencing analysis. Depository: General Microbiology Center of China Culture Collection Administration; Deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; Deposit registration number: CGMCC No.13225; Taxonomic name: Bacillussiamensis; The biological material (strain) was received and registered by this deposit center on November 1, 2016.

[0009] Preferably, the Bacillus Velez subsp. B268 is isolated from the rhizosphere soil of the Casuarina coastal defense forest, and its preservation number is CGMCC No. 13225. The method for preparing the fermentation broth of Bacillus Velez subsp. B268 is as follows: first, a single colony of the B268 strain is inoculated into a test tube containing 4 ml of YTGB medium (the component ratio is: 0.05% yeast extract, 0.5% tryptone, 1% glucose, 0.3% beef extract powder, pH value is 7.0), and then cultured at 28°C and 140 rpm for 24 hours to prepare a seed solution, then, 1% of the seed solution is taken for expanded culture for 48 hours to obtain a fermentation broth, and finally, the supernatant is filtered by centrifugation and using a 0.22-micron filter membrane to obtain the fermentation filtrate of B268.

[0010] Preferably, the preparation method of the ethylene phosphate aluminum injection agent is as follows: raw material preparation: 95% purity ethylene phosphate aluminum technical and auxiliary agents, the auxiliary agents include 50% purity alkyl glycoside, dodecyl betaine, gum arabic as a binder, ammonium sulfate as a filler, and then mixing, crushing, adding water to mix, granulating and drying.

[0011] Preferably, the formula ratio of the preparation is: 85-87% of ethylene phosphite aluminum technical, 2-5% of alkyl glycoside, 2-5% of dodecyl betaine, 1-5% of gum arabic, and 5-10% of ammonium sulfate. The above ingredients are then mixed evenly, crushed to control the fineness of the crushed material to be less than 23 microns, then water is added to mix the material in an amount of 7% of the total amount of the material, then extruded and granulated by a screw extruder granulator, and finally dried by a vibrating fluidized bed dryer, the gas phase temperature of the dryer is controlled to be less than 120° C., and the moisture content of the granules after drying is less than 0.6%.

[0012] Preferably, the indoor toxicity determination includes: diluting the ethylenediaminetetraacetic acid raw material with sterile water to the required concentration, adding the corresponding volume of liquid medicine according to the total volume of the PDA culture medium, and preparing PDA culture medium containing different concentrations of jinggangmycin, specifically 0.1 mg / L, 1 mg / L, 10 mg / L, 100 mg / L and 200 mg / L. Add an equal amount of sterile water to the control culture medium, inoculate chestnut blight pathogen after the preparation, and then culture under light conditions at 28°C. When the mycelium of the control group covers 3 / 4 of the culture medium, evaluate its inhibitory effect by the mycelium growth rate method, and calculate the EC 50 The fermentation filtrate of Bacillus Velezii B268 was diluted to 5 times, 10 times, 20 times, 40 times, and 80 times with PDA medium. No solution was added to the control group. After the preparation was completed, chestnut blight pathogen was inoculated and cultured under the same light conditions of 28°C. When the mycelium of the control group grew to 3 / 4 of the culture medium, the mycelium growth rate method was used to determine its inhibitory effect, and the EC 50 value.

[0013] Preferably, the defense enzyme activity analysis performed comprises quantifying the functional activities of catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD).

[0014] Preferably, the in vitro inoculation: after three days of treatment, take the leaves of each treatment for indoor in vitro inoculation experiments, wrap the end of the leaf vein with sterile cotton to keep it moist, and use a vernier caliper to mark a 5 mm measuring area at the position of the leaf vein in the middle of the leaf, scratch the area with a sterilized blade, then take the pathogen cultured for three days, use a 5 mm puncher to make a bacterial cake, and put its mycelium surface close to the leaf wound, and then place it in a sterile fresh-keeping box with a sterile water-soaked filter paper on the bottom for moisturizing and culturing.

[0015] Preferably, the live inoculation is as follows: holes are created on the trunk using a 7 mm borer, the phloem layer is removed, pathogens cultured for three days are taken, a fungus cake is made using a 7 mm borer, and its mycelium surface is attached to the xylem, and then a sealing film is used to surround the holes for moisturizing treatment, and the negative control group is inoculated with a PDA culture medium that does not contain pathogens.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] By compounding Bacillus Velezii B268 and ethyl phosphite aluminum in a binary manner, a fungicide mixture is obtained within a certain mass ratio range, which has a synergistic effect in preventing and controlling chestnut blight.

[0018] Significant prevention effect: It can effectively improve the resistance of chestnut trees to chestnut blight, reduce the occurrence and spread of lesions, and its prevention effect is better than traditional biological control methods;

[0019] The bactericidal composition of the present invention utilizes the disease resistance function of the biological agent and the immune activation function of ethyl phosphite aluminum at the same time, thereby improving the disease resistance effect from both the inside and outside of the plant body, and can achieve an ecological mode of reducing the amount of application and increasing the effect through compounding;

[0020] Mixing ethylene fosetyl aluminum with Bacillus Velez B268 can improve the antibacterial ability of the control agent, while activating the plant's own immune function, reducing the application of chemical pesticides, and exerting the disease-resistant inducing function of ethylene fosetyl aluminum and the antibacterial effect of the fermentation filtrate of Bacillus Velez B268. The combined use can improve the overall prevention effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic diagram of the process of the present invention;

[0022] Figure 2 It is a schematic diagram of the inhibitory effect of the fermentation filtrate of Bacillus Velezii B268 of the present invention on chestnut blight pathogen on PDA;

[0023] Figure 3 The schematic diagram is a schematic diagram of the inhibitory effect of the fermentation filtrate of Bacillus Velezii B268 on chestnut blight pathogen on PDA. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Embodiment 1

[0026] like Figure 1-Figure 3 As shown, the present invention proposes a method for preventing and controlling chestnut blight based on fosetyl aluminum as an immune activator mixed with biocontrol bacteria fermentation filtrate, comprising the following steps:

[0027] Bacillus Velez B268 is a rhizosphere soil sample isolated from a Casuarina equisetifolia coastal defense forest, and its accession number is CGMCC No.13225. The preparation method of the fermentation liquid of Bacillus Velez B268 is as follows: a single colony is picked from the LB culture medium inoculated with the B268 strain and placed in a test tube containing 4 ml of YTGB culture medium ((W / V): yeast powder (yeast extract, BD) 0.05%, trypticase peptone (trypticase peptone, BD) 0.5%, glucose 1%, beef powder 0.3%, pH 7.0), and the culture is carried out at 28°C and 140r / min for 24h to prepare a seed liquid, 1% of the seed liquid is taken for expansion culture for 48h to obtain the fermentation liquid, and the supernatant is filtered through a 0.22μm filter membrane after centrifugation to obtain the B268 fermentation filtrate.

[0028] The preparation method of ethylene phosphate aluminum dry injection agent comprises the following steps: raw material preparation: ethylene phosphate aluminum original medicine with a purity of 95% and auxiliary agents, wherein the auxiliary agents include alkyl glycoside with a purity of 50%, dodecyl betaine, gum arabic as a binder, and ammonium sulfate as a filler, and then the raw materials are mixed, crushed, mixed with water, granulated and dried, and the prepared formula ratio is: ethylene phosphate aluminum original medicine: 85-87%, alkyl glycoside: 2-5%, dodecyl betaine: 2-5%, gum arabic: 1-5%, and ammonium sulfate: 5-10%, and then the above components are evenly mixed, crushed to control the fineness of the crushed material to be less than 23 microns, then water is added to mix and the amount of water added accounts for 7% of the total amount of the material, and then extrusion granulation is performed through a screw extrusion granulator, and finally drying is performed by a vibrating fluidized bed dryer, and the gas phase temperature of the dryer is controlled to be less than 120° C. After drying, the moisture content of the granules is less than 0.6%.

[0029] Step 1: Cultivate chestnut blight pathogen on PDA medium for 3 days. Under sterile conditions, use a 5mm sterile puncher to punch out bacterial cakes from the edge of the colony where mycelium grows vigorously.

[0030] Step 2: Prepare PDA medium with different concentrations of ethylene phosphite aluminum solution and dilute the fermentation filtrate of Bacillus velezensis B268 at different times and transfer it to chestnut blight pathogen culture.

[0031] Then carry out indoor toxicity test, the steps are as follows:

[0032] Use PDA medium containing 0.1 mg / L, 1 mg / L, 10 mg / L, 100 mg / L, and 200 mg / L of jinggangmycin and inoculate chestnut blight pathogen. Use medium to dilute the fermentation filtrate of Bacillus Velezii B268 to 5 times, 10 times, 20 times, 40 times, and 80 times to prepare PDA medium. Add an equal amount of sterile water to the control medium. After the preparation is completed, inoculate chestnut blight pathogen and culture under light at 28°C. When the mycelium of the control group fills 3 / 4 of the medium, the mycelium growth rate method is used to determine its inhibitory effect, and the EC is calculated. 50 value.

[0033] The specific implementation is as follows:

[0034] Aluminium phosphite group:

[0035] Weigh an appropriate amount of ethylene fosetyl aluminum original drug and dilute it in a gradient manner with sterile water to prepare ethylene fosetyl aluminum solutions of 0.1 mg / L, 1 mg / L, 10 mg / L, 100 mg / L, and 200 mg / L respectively.

[0036] Weigh an appropriate amount of potato dextrose agar (PDA) powder, add distilled water according to the ratio in the instructions, heat to dissolve, and dispense the dissolved PDA culture medium into sterile culture dishes, about 15-20 ml per dish. When the PDA culture medium cools to 50-60℃, add different concentrations of ethylene phosphite aluminum solution and mix thoroughly.

[0037] The chestnut blight pathogen was inoculated on PDA medium containing different concentrations of ethylene phosphite aluminum, with 5 replicates for each concentration, and an equal amount of sterile water was added to the control group.

[0038] Bacillus Velezii B268 fermentation filtrate group:

[0039] Prepare according to the formula of PDA culture medium and make it into the same volume, sterilize and cool to 50-60℃.

[0040] The fermentation filtrate of Bacillus Velezii B268 was added to the PDA medium according to the dilution multiples, so as to obtain dilutions of 5 times, 10 times, 20 times, 40 times and 80 times respectively for use.

[0041] Pour the PDA medium added with B268 fermentation filtrate into culture dishes, about 15-20 ml per dish, set 5 replicates for each concentration, and do not add any solution to the control group.

[0042] Transfer the prepared culture medium to the pathogen cake. After the transfer is completed:

[0043] The culture dish was placed in an incubator at 28°C under light conditions.

[0044] The mycelial growth was observed every day, and when the mycelium of the control group filled 3 / 4 of the culture medium, the colony diameters of each treatment group were measured.

[0045] Use a ruler or vernier caliper to measure the diameter of the colony growth in each treatment group.

[0046] Calculate the mycelial growth rate of each treatment group (mycelial growth diameter / culture days):

[0047] The mycelium growth inhibition rate of each treatment group was calculated according to the following formula: inhibition rate (%) = (colony diameter of control group - colony diameter of treatment group) / (colony diameter of control group - diameter of bacterial cake) × 100%.

[0048] EC 50 The value refers to the concentration of fermentation filtrate required to inhibit mycelial growth by 50%. Then, the statistical software SPSS was used to process the data and calculate the EC 50 The above steps can be used to calculate the concentration of ethylene phosphite aluminum solution and the concentration of Bacillus velezensii B268 fermentation filtrate with better indoor antibacterial effect.

[0049] Step 3: Select chestnut saplings of uniform age and growth, set up two control groups: a positive control group and a negative control group, and set up three treatment groups: ethylene aluminum treatment group, B268 fermentation filtrate treatment group, and ethylene aluminum + B268 fermentation filtrate treatment group. Collect leaves when no pesticide is sprayed and preserve them for subsequent determination of defense enzyme activity and MDA content. Collect leaves when no pesticide is sprayed and preserve them for subsequent determination of defense enzyme activity and MDA content.

[0050] Step 4: According to the result of step 2, the concentration with better indoor antibacterial effect is used as the spraying concentration. For the three treatment groups: ethylene phosphate aluminum treatment group, B268 fermentation filtrate treatment group, ethylene phosphate aluminum + B268 fermentation filtrate treatment group, the corresponding concentration solutions are used to evenly spray the leaves and trunks. Among them, the ethylene phosphate aluminum + B268 fermentation filtrate treatment group needs to be mixed. The mixing step is to dilute the B268 fermentation filtrate with water to the corresponding concentration, and then dilute the ethylene phosphate aluminum original drug with the diluted B268 fermentation filtrate to obtain the mixed agent of the corresponding concentration. Three days later, in vitro inoculation and in vivo inoculation are carried out respectively.

[0051] Specific steps for in vitro inoculation:

[0052] Three days later, leaves of each group (positive control group, fosetyl aluminum treatment group, B268 fermentation filtrate treatment group, fosetyl aluminum + B268 fermentation filtrate treatment group) were collected.

[0053] Wrap the end of the main vein of the collected leaves with sterile cotton to keep the leaves moist, use a vernier caliper to measure a 5 mm area at the main vein in the middle of the leaf, and mark it with a marker.

[0054] The marked 5 mm area on the leaf was gently scratched with a sterilized razor blade to expose the wound.

[0055] Use the mushroom cake made in step 1, place the mycelium side of the cake downwards, and stick it to the wound area of ​​the leaf to ensure that the mushroom cake is in close contact with the wound.

[0056] The inoculated leaves were placed in a sterile fresh-keeping box with filter paper soaked in sterile water at the bottom for moisturizing culture.

[0057] Regularly observe the growth of pathogens on the leaves and record data such as the spread rate of the pathogens and the severity of the disease.

[0058] The leaf disease is calculated as follows:

[0059] After inoculation, observe the growth of pathogens on the leaves at regular intervals (24 hours, 48 ​​hours, 72 hours), use a magnifying glass or microscope to assist in observation, and record the expansion of hyphae;

[0060] Use a camera or mobile phone to take photos to record the disease development of the leaves for subsequent analysis, and record the diameter or area of ​​the pathogen at each inoculation point.

[0061] Depending on the extent of the pathogen's spread on the leaves, the disease can be divided into several levels:

[0062] Level 0: No lesions

[0063] Level 1: The diameter of the lesion is less than 1 mm

[0064] Level 3: The diameter of the lesion is between 1-2 mm

[0065] Level 5: The diameter of the lesion is between 2-5 mm

[0066] Level 7: The diameter of the lesion is greater than 5 mm

[0067] The disease index is a quantitative indicator that reflects the severity of the disease. The calculation formula is as follows:

[0068]

[0069] By collecting data from each group of leaves.

[0070] According to the calculation results, the disease index of each group was obtained.

[0071] According to the disease index of detached leaves, 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, thereby determining the effects of different control groups on the growth and incidence of chestnut blight, indicating that ethylene phosphite aluminum and the fermentation filtrate of Bacillus Velezii B268 have a synergistic effect.

[0072] The specific steps of live inoculation are as follows:

[0073] Three days later, chestnut seedlings of each group (positive control group, ethylene fosetyl aluminum treatment group, B268 fermentation filtrate treatment group, ethylene fosetyl aluminum + B268 fermentation filtrate treatment group) were prepared.

[0074] Prepare a 7mm cork borer, sealing film, sterile PDA culture medium, and pathogens. Use the 7mm cork borer to make holes on the surface of the trunk. The depth should be enough to remove the phloem and expose the xylem. Make sure the holes are clean and no debris remains.

[0075] Use the mushroom cake made in step 1, stick the mycelium surface into the holes in the wood of the tree trunk, and gently press the mushroom cake to ensure that the mycelium is in close contact with the wood.

[0076] Use sealing film to wrap and seal the inoculation site to maintain humidity and promote the growth of pathogens.

[0077] In another group of trees, holes were punched using the same method and inoculated with sterile PDA medium as a negative control, and the steps were the same as those for inoculating pathogens.

[0078] After inoculation, regularly observe and record the reaction of the inoculation site, such as the formation and expansion of lesions, and record the differences between different treatment groups. The specific observation methods are as follows:

[0079] Use transparent grid paper to cover the lesions, calculate the number of grids occupied by the lesions, and estimate the area of ​​the lesions.

[0080] The lesion area was measured using the image processing software Photoshop.

[0081] The disease grade is set according to the ratio of the lesion area to the total area of ​​the inoculated site;

[0082] No disease spots: Level 0

[0083] Lesion area ≤ 5%: Level 1

[0084] 5%<lesion area≤10%: Level 3

[0085] 10%<lesion area≤20%: Level 5

[0086] Lesion area>20%:7 level,

[0087] The disease index was calculated by collecting the data of tree trunk lesions in each control group using the disease index calculation formula.

[0088] According to the calculation results, the disease indexes of the three control groups were obtained.

[0089] According to the disease index of trunk lesions, 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 effects of different control groups on the growth and incidence of chestnut blight, indicating that ethylene phosphite aluminum and Bacillus Velez B268 fermentation filtrate have a synergistic effect.

[0090] Step 5: On the 0th, 2nd, 4th, 8th, and 16th day after the start of the experiment, prepare sterile scissors, tweezers, and labeled sterile sampling bags, randomly select leaves with consistent growth from each group of plants, and cut them with sterile scissors.

[0091] Place the cut leaves into a sterile sampling bag and mark them (including group, inoculation time, sampling time, etc.).

[0092] The sampling bags were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator.

[0093] At this time, take out the leaf samples stored in steps 3 and 5 for 0 days, 2 days, 4 days, 8 days, and 16 days from the -80°C refrigerator:

[0094] Catalase (CAT) activity assay:

[0095] First, the plant tissue was mixed with phosphate buffer (pH 7.0-7.4) and ground into a homogenate in an ice bath.

[0096] The homogenate was then centrifuged at 12000 g at 4 °C, and the supernatant was taken for enzyme activity test.

[0097] A reaction mixture was prepared in a test tube, comprising 50 mM phosphate buffer, 10 mM H2O2 solution and enzyme extraction solution.

[0098] The reaction was started by adding H2O2, the consumption of H2O2 was recorded, and the reaction was terminated with ammonium sulfate solution.

[0099] Finally, the reduction of H2O2 was measured spectrophotometrically to calculate the activity of CAT.

[0100] Superoxide dismutase (SOD) activity assay:

[0101] Take plant tissue, add a certain proportion of phosphate buffer (pH7.0-7.4), and grind into a homogenate in an ice bath;

[0102] Using the nitroblue tetrazolium (NBT) photoreduction method, the reaction mixture includes:

[0103] 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 μM NBT, 0.1 mM EDTA, 2 μM riboflavin, enzyme extract;

[0104] The reaction mixture is placed under a fluorescent lamp for light reaction;

[0105] After the illumination ends, the reaction is terminated immediately by avoiding light;

[0106] The absorbance at 560 nm was measured and the SOD activity was calculated.

[0107] Peroxidase (POD) activity assay:

[0108] First, plant tissues were mixed with phosphate buffer (pH 7.0-7.4) and ground into a homogenate in an ice bath.

[0109] Next, a reaction mixture containing 25 mM phosphate buffer, 20 mM guaiacol, 5% H2O2 and an enzyme extract was prepared.

[0110] The reaction was initiated by adding H2O2, and the change in absorbance was recorded at 470 nm using a spectrophotometer to calculate the activity of POD.

[0111] Malondialdehyde (MDA) content determination:

[0112] The tissue was first mixed with phosphate buffer (pH 7.0-7.4) and ground into a homogenate in an ice bath.

[0113] Then, an equal amount of 20% TCA solution was added to the supernatant of the homogenate, mixed, and centrifuged at 12,000 g for 10 minutes at 4°C.

[0114] Next, the supernatant was mixed with an equal amount of 0.6% TBA solution, heated in a boiling water bath for 15 minutes, and then rapidly cooled and centrifuged.

[0115] Finally, the content of MDA was determined by measuring the absorbance at wavelengths of 532 nm and 600 nm.

[0116] According to the above calculation results, the effects of different control group treatments on catalase, superoxide dismutase, peroxidase and malondialdehyde in plants were determined.

[0117] On day 8, transcriptome sequencing was performed on leaf samples.

[0118] Data analysis was performed on the sequencing data to better understand the effect of fosetyl aluminum + B268 fermentation filtrate treatment on the difference in chestnut gene expression before and after treatment.

[0119] By comparing with the above control groups, it can be concluded that the control group of ethylene aluminum + B268 fermentation filtrate has a good prevention and treatment effect.

[0120] The control effect was evaluated according to the above method, and the synergistic effect of the mixture of ethylene phosphate aluminum solution and B268 fermentation filtrate was obtained, which was used for the chestnut blight control method based on ethylene phosphate aluminum as an immune activator mixed with biocontrol bacteria fermentation filtrate.

[0121] The above-mentioned specific embodiments are only several preferred embodiments of the present invention. Based on the technical solutions of the present invention and the relevant inspirations of the above-mentioned embodiments, those skilled in the art can make various alternative improvements and combinations to the above-mentioned specific embodiments.

[0122] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A method for preventing and controlling chestnut blight based on fosetyl aluminum as an immune activator mixed with biocontrol bacteria fermentation filtrate, comprising the following steps, characterized in that: The step 1: using PDA culture medium to culture chestnut blight pathogens and using a puncher to make fungus cakes, step 2: using different concentrations of ethylene phosphate aluminum solution and different dilutions of Velez Bacillus B268 fermentation filtrate to prepare PDA culture medium and transferring the fungus cakes to the culture medium for culturing, and performing indoor toxicity determination, the step 3: selecting chestnut seedlings, setting two control groups and three treatment groups: a positive control group (spraying sterile water and inoculating pathogens), a negative control group (spraying sterile water and inoculating sterile PDA), an ethylene phosphate aluminum treatment group, a B268 fermentation filtrate treatment group and an ethylene phosphate aluminum + B268 fermentation filtrate treatment group, collecting and preserving the seedling leaves without spraying the agent, and then Preparation for subsequent determination of defense enzyme activity and MDA content, step 4: according to the result of step 2, use a concentration with good indoor antibacterial effect to evenly spray the leaves and trunks of the chestnut seedlings in the three treatment groups, wherein the ethylene phosphate aluminum + B268 fermentation filtrate treatment group needs to be mixed, and the mixing step is to prepare the ethylene phosphate aluminum solution, and the volume of the fermentation filtrate added is calculated according to the volume of the ethylene phosphate aluminum solution. After three days, in vitro inoculation and in vivo inoculation experiments are carried out respectively, and step 5: on the 0th, 2nd, 4th, 8th, and 16th days, leaf samples of each group are regularly collected and properly preserved for subsequent detection of defense enzyme activity and MDA content, and on the 8th day, some samples are selected for transcriptome sequencing analysis.

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 Velez Bacillus B268 is isolated from the rhizosphere soil of the Casuarina equisetifolia coastal defense forest, and its deposit number is CGMCC No. 13225. The method for preparing the fermentation liquid of Velez Bacillus B268 is as follows: first, a single colony of the B268 strain is inoculated into a test tube containing 4 ml of YTGB medium (the component ratio is: 0.05% yeast extract, 0.5% tryptone, 1% glucose, 0.3% beef extract powder, and pH value is 7.0), and then cultured for 24 hours at 28° C. and 140 rpm to prepare a seed liquid, then, 1% of the seed liquid is taken for expansion culture for 48 hours to obtain a fermentation liquid, and finally, the supernatant is filtered by centrifugation and using a 0.22 micron filter membrane to obtain the fermentation filtrate of B268.

3. 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 preparation method of the ethylene aluminum injection dry agent comprises the following steps: preparing raw materials: ethylene aluminum technical with a purity of 95% and auxiliary agents, wherein the auxiliary agents include alkyl glycoside with a purity of 50%, dodecyl betaine, gum arabic as a binder, and ammonium sulfate as a filler, and then mixing, crushing, adding water to mix, granulating and drying.

4. 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 3, characterized in that: The preparation formula ratio is: 85-87% of ethylene aluminum phosphate technical, 2-5% of alkyl glycoside, 2-5% of dodecyl betaine, 1-5% of gum arabic, and 5-10% of ammonium sulfate. The above ingredients are then mixed evenly, crushed to control the fineness of the crushed material to be less than 23 microns, then water is added to mix the material 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, the gas phase temperature of the dryer is controlled to be less than 120° C., and the moisture content of the granules after drying is less than 0.6%.

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 indoor toxicity determination includes: using PDA culture medium containing 0.1 mg / L, 1 mg / L, 10 mg / L, 100 mg / L, and 200 mg / L of ethylene phosphatenic acid aluminum, adding an equal amount of sterile water to the control group, inoculating chestnut blight pathogens, and then culturing under light conditions at 28°C. When the mycelium of the control group covers 3 / 4 of the culture medium, the mycelium growth rate method is used to evaluate its inhibitory effect, and the EC is calculated. 50 The fermentation filtrate of Bacillus Velezii B268 was diluted to 5 times, 10 times, 20 times, 40 times, and 80 times with PDA medium. The control group was inoculated with chestnut blight pathogen without adding any solution. The control group was also cultured under the light condition of 28°C. When the mycelium of the control group grew to 3 / 4 of the culture medium, the mycelium growth rate method was used to determine its inhibitory effect, and the EC 50 value.

6. 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 defense enzyme activity analysis performed included quantification of the functional activities of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD).

7. 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 in vitro inoculation: After three days of treatment, take the leaves of each treatment for indoor in vitro inoculation experiments, wrap the end of the leaf vein with sterile cotton to keep it moist, and use a vernier caliper to mark a 5 mm measuring area at the position of the leaf vein in the middle of the leaf, scratch the area with a sterilized blade, then take the pathogen cultured for three days, use a 5 mm puncher to make a bacterial cake, and put its mycelium surface close to the leaf wound, and then place it in a sterile preservation box with a sterile water-soaked filter paper on the bottom for moisturizing and culturing.

8. 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 live inoculation: holes were created on the tree trunk using a 7 mm puncher, the phloem layer was removed, pathogens cultured for three days were taken, fungus cakes were made using a 7 mm puncher, and the mycelium surface was attached to the wood, and then the holes were surrounded by sealing film for moisture retention. The negative control group was inoculated with a PDA culture medium without pathogens.

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