Bacillus amyloliquefaciens and application of bacillus amyloliquefaciens in blocking root infection of pear fire blight bacteria

Abstract

The invention discloses bacillus amyloliquefaciens and application of the bacillus amyloliquefaciens in blocking of root infection of pear fire blight bacteria, the bacillus amyloliquefaciens is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.28054, and the preservation date is July 28, 2023. The bacillus amyloliquefaciens has the advantages that the bacillus amyloliquefaciens can be applied to prevention of root infection of pear fire blight bacteria; the bacillus amyloliquefaciens provided by the invention solves the problems of trunk morbidity and tree withering caused by pear fire blight self-root infection.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms and agriculture, in particular to a strain of Mutant Bacillus amyloliquefaciens and application thereof in blocking the infection of Erwinia amylovora pathogen from roots. Background Art

[0002] In 2016, apricot fire blight first occurred in Xinjiang and caused devastating damage to the Xinjiang fragrant pear industry. The disease has various transmission pathways, including wounds, rain, aerosols, soil, and pollen. Among them, the infection of apricot fire blight from the root (stock) is an important pathway that causes the main trunk of the fragrant pear to become diseased, making prevention and control difficult and causing the tree to die. This is also the first new pathway for the transmission of apricot fire blight discovered internationally.

[0003] The existing prevention and control of fire blight of apricot mainly relies on chemical pesticides. Although they have certain preventive effects, they are easy to cause pesticide residues, endanger human health, and destroy the ecological balance. In addition, the long-term use of pesticides to prevent and control fire blight can easily make pathogens resistant, increasing the difficulty of fire blight prevention and control. Biological control uses secondary metabolites produced by microorganisms to prevent and control diseases, which can effectively solve the problem of pesticide residues, will not make pathogens resistant, is harmless to the human body, and will not cause environmental pollution. Studies have shown that Pantoea agglomerans E352 can effectively inhibit the growth and reproduction of fire blight of apricot by producing polyketide diphenhydramine and dipeptide pasteurium, but it is not easy to colonize in the field, especially in saline-alkali environments.

[0004] For example, Chinese authorized patent CN112358366B discloses a composite bacterial fertilizer for preventing and treating apricot fire blight, and its preparation method and application, by mixing Bacillus subtilis, Bacillus amyloliquefaciens, Pseudomonas fluorescens, and Candida guilliermondii in a specific mass ratio, and compounding a certain amount of cow dung, sheep dung, mushroom residue, fulvic acid, and grape dregs to prepare the composite bacterial fertilizer. The technical scheme of the composite bacterial fertilizer that can be used to prevent and treat apricot fire blight creatively proposed by the present invention, while forming a stable microecology and ensuring a high number of live bacteria through the compounding of bacterial species in a specific ratio, can not only promote plant growth but also have a significant effect on preventing and treating apricot fire blight, and can be applied in saline-alkali land, and can reduce the risk of pear trees being infected with apricot fire blight.

[0005] For another example, Chinese authorized patent CN115029269B discloses an antagonistic bacterium of amylovora that produces lipopeptide antibiotics, and its fermentation method and application, which belong to the field of agricultural microbial technology. The strain has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, and is classified and named Bacillus Velez, with a deposit number of CGMCC No. 24843. The lipopeptide antibiotics produced by the strain have significant antagonistic effects and excellent disease prevention effects on amylovora, have a certain tolerance to high temperature, acid and alkali, protease, and ultraviolet rays, and have a fast growth and reproduction speed, strong competitiveness, can be produced cheaply, and have good development and application prospects. The present invention also discloses a fermentation method for improving the production of lipopeptide antibiotics by the strain, which is low-cost and easy to operate, and can improve the effect in the biological control of amylovora, and provides a theoretical basis and technical reference for the industrial fermentation production of the strain and the research and development of amylovora biological pesticides.

[0006] However, the prevention and control methods disclosed in the prior art require the use of special strains, which are costly, and how to prevent and control the disease in the trunk is still a huge challenge. Based on this, the Xinjiang Academy of Forestry Sciences' Autonomous Region Fragrant Pear Industry Technology System post expert team has spent 5 years of experimental research and developed a new method for effectively preventing and controlling the main trunk lesions of pear fire blight, solving the problem of pear fire blight infecting the trunk of fragrant pear trees and causing tree death. Summary of the invention

[0007] In view of this, the present invention provides a strain of Bacillus amyloliquefaciens and its use in blocking the infection of Erwinia amylovora from the root. To achieve the above purpose, the present invention mainly provides the following technical solutions:

[0008] The present invention relates to a strain of Bacillus amyloliquefaciens, which is deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC No. 28054 and a deposit date of July 28, 2023.

[0009] Another aspect of the present invention relates to the use of the above-mentioned Proteobacterium amyloliquefaciens in blocking the infection of Erwinia amylovora from the roots.

[0010] In a preferred embodiment of the present invention, the application comprises the following steps: diluting Bacillus amyloliquefaciens with clean water to obtain a 300-400-fold dilution for standby use; digging a circular pit with a depth of 30-40 cm and a width of 15-25 cm at a distance of 0.8-1.2 meters from the main trunk of the tree, and watering each tree with 40-60 liters of the dilution to irrigate the roots to block the infection of the fire blight bacteria from the roots; the root irrigation time is the first root irrigation in April-May each year and the second root irrigation in June-July.

[0011] In a preferred embodiment of the present invention, the application comprises the following steps: root irrigation is carried out through a drip irrigation system, the dosage of the agent is 1200-1800g of Bacillus amyloliquefaciens per mu, the first root irrigation is carried out in April-May each year, and the second root irrigation is carried out in June-July.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] ① The Bacillus amyloliquefaciens of the present invention solves the problem of fire blight infecting the root causing the trunk to become diseased and the tree to die;

[0014] ② The method of the present invention is simple, fast, efficient and low-cost. The cost of the agent is about 40 yuan per mu, and the incidence of root infection is 0%;

[0015] ③ The present invention can realize the integration of water, fertilizer and medicine for pear orchards equipped with drip irrigation systems, thus reducing labor expenses and prevention and control costs;

[0016] ④ The present invention is a pure biological preparation, which is pollution-free and residue-free, and achieves the protection of the soil ecological environment and the realization of green and organic fruits and vegetables. DETAILED DESCRIPTION

[0017] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation methods, technical solutions, features and effects of the present invention application are described in detail below. The specific features, structures, or characteristics of the multiple embodiments in the following description can be combined in any suitable form.

[0018] Example 1

[0019] Since the discovery of a new transmission route of fire blight from the roots that caused the main trunk of the fragrant pear to become ill and the tree to die in 2019, the expert team of Xinjiang Academy of Forestry Sciences has promptly carried out research and development of blocking technology, and conducted indoor antibacterial tests on a type of Bacillus amyloliquefaciens isolated and identified from the fragrant pear trees in the diseased pear orchard. The results showed that the antibacterial effect of Bacillus amyloliquefaciens on fire blight was 63%, which was significantly higher than the antibacterial effect of similar biocontrol bacteria on the market. In order to maximize the antibacterial effect of the Bacillus amyloliquefaciens, a strain of Bacillus amyloliquefaciens was obtained by high-temperature acclimation technology in cooperation with Northwest University. The prevention effect of crown spraying after 1000 times dilution reached 84.3%, and 300 times dilution underground root irrigation could block the transmission route of fire blight from the roots, greatly reducing the incidence of the main trunk of the fragrant pear. The Bacillus amyloliquefaciens was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCC No.28054 and the deposit date July 28, 2023.

[0020] 1.1 Detailed composition and proportion of high temperature acclimated microbial preparations

[0021] (1) Seed culture medium (g / 3L): 3 g of tryptone, 3 g of chromatin hydrolysate, 2 g of yeast extract, 1 g of beef extract, 10 g of NaCl, pH 7.5.

[0022] (2) Fermentation medium (g / 3L): tryptone 10, chromatin hydrolysate 8, yeast extract 6, beef extract 5, NaCl 10, pH 7.5. After sterilization, add 50% glucose solution 15mL, 1MCa(NO3) 5mL, 0.1M MnCl25mL, 1mMFeSO4 5mL. Feed culture solution (A) (g / 2L): tryptone 30, chromatin hydrolysate 15, yeast extract 20, NaCl feed solution (B): 4MHCl 300mL feed solution (C): 10% NaOH 100mL.

[0023] 1.2 Production process of microbial preparations

[0024] 1.2.1 Preparation of seed solution

[0025] Bacillus amyloliquefaciens isolated from the pear trees in the diseased pear orchard was stored at -20°C, and the Bacillus amyloliquefaciens strain was inoculated on the activated medium solid plate and cultured at 37°C for 24 hours. The bacterial moss of the strain was washed with sterilized saline (with 0.1% Tween-80) to prepare a bacterial suspension. An appropriate amount of the bacterial suspension was inoculated into 300 mL of seed culture medium to make the cell turbidity OD600 0.1, and cultured at 45°C and 200rpm for 12 hours.

[0026] 1.2.2 Sterilization and inoculation of fermentation medium

[0027] Sterilize the above 3L fermentation medium in a fermenter at 121℃ for 20min, and strictly calibrate the dissolved oxygen standard value during the sterilization process. After sterilization, add 15mL of 50% glucose solution, 5mL of 1M Ca(NO3), 5mL of 0.1MMnCl25mL, 5mL of 1mM FeSO4 and other ingredients to maintain the fermentation liquid temperature at 45℃. Use the feed system equipped with the fermenter to connect the flow feed liquid, and adjust the feed amount of flow feed liquid B and C to adjust the final fermentation liquid pH to 7.5. Add the above cultured seed liquid to the fermentation medium.

[0028] 1.2.3 Fermentation process control

[0029] The dissolved oxygen linkage module of the fermenter is used to set the air flow rate to 5L / min, the OD value to 25%, and the rotation speed to a random variable. As the fermentation proceeds, the dissolved oxygen value of the fermentation liquid gradually decreases to the set value. At this time, mechanical stirring is started to maintain the dissolved oxygen concentration of the fermentation liquid to meet the growth and metabolism of the cells. When the concentration reaches the highest value and is maintained for a certain period of time, the concentration begins to decrease. At this time, the feeding module is started to flow the culture liquid (A) into the fermentation liquid at a certain speed. At the same time, the concentration of the culture liquid is fine-tuned in real time with the changes in cell physiology and metabolism, while maintaining the stirring concentration. When the pH rises above 8.5, the fermentation liquid is collected.

[0030] 1.2.4 Detection of antibacterial effect of microbial preparations

[0031] The fermentation liquid was centrifuged to prepare the supernatant, and a sterilized filter paper disc (5 mm in diameter) was soaked in the supernatant and dried at 45°C. At the same time, several commercial antibacterial agents were used to prepare filter paper discs in the same way as controls. The pathogens of bacterial diseases of Xinjiang pear trees were inoculated on LB solid plates and cultured for 16 hours. The bacterial moss was scraped into physiological saline with a cotton swab to prepare a bacterial suspension. The bacterial suspension was applied to the LB solid plate with a cotton swab, and the prepared filter paper was attached to the surface of the culture medium and cultured at 37°C for 24 hours. The antibacterial effect of the fermentation liquid was evaluated by the size of the antibacterial circle formed on the edge of the filter paper. When the diameter of the antibacterial circle reaches 3 cm or more, it is considered a qualified microbial preparation.

[0032] 2 Identification of Bacillus amyloliquefaciens

[0033] 2.1 Isolation and cultivation process of Bacillus amyloliquefaciens

[0034] (1) Pick a single colony with the same morphological characteristics from the plate, place it in a 250 mL conical flask with 50 mL of sterile LB liquid medium, pipette it evenly, and culture it in a shaking incubator at 30°C for 12 hours. After 12 hours, dilute the bacterial solution and measure the OD 600 , make the OD between 0.2-0.7, according to 1OD≈10 8 Cells / mL, estimate the cell concentration of the dilution solution, and then multiply by the dilution factor to calculate the concentration of the original bacterial solution. Bacteria preservation: Mix 300μL of 50% glycerol and 700μL of bacterial solution (clean bench) and store at low temperature.

[0035] 2.2 Identification process

[0036] 2.2.1 DNA extraction

[0037] (1) Take bacterial solution (the total amount should not exceed 10 8 The cells were centrifuged at 12000 rpm for 1 min in a centrifuge tube and the supernatant was aspirated.

[0038] (2) Add 180 μL Enzymatic Lysis Buffer to resuspend the cells.

[0039] (3) Incubate at 37°C for 30 min.

[0040] (4) Add 20 μL Proteinase K and vortex to mix. Add 200 μL Buffer GL and vortex to mix.

[0041] (5) Incubate at 56°C for 30 min. Add 4 μL of 100 mg / mL RNase A solution.

[0042] (6) Add 200 μL of anhydrous ethanol and vortex to mix.

[0043] (7) Add the solution obtained in (6) to the adsorption column in the collection tube. The solution can be added in multiple times. Centrifuge at 12000 rpm for 1 min and discard the waste liquid.

[0044] (8) Add 500 μL of Buffer GW1 to the adsorption column (check whether anhydrous ethanol has been added), incubate at 12,000 rpm for 1 min, and discard the waste liquid.

[0045] (9) Add 500 μL Buffer GW2 (check anhydrous ethanol), 12000 rpm, 1 min, discard the waste liquid. Repeat this step once.

[0046] (10) 12000rpm, 2min, discard waste liquid, and dry the adsorption column

[0047] (11) Place the adsorption column in a new centrifuge tube, add 50 μL of Buffer GE to the center of the adsorption column, let it stand for 2 min, and incubate at 12,000 rpm for 1-2 min. Resuspend the solution in the tube and drip it to the center of the column, and incubate at 12,000 rpm for 2 min.

[0048] (12) Detect DNA concentration and verify DNA integrity by agarose gel electrophoresis and store at -20°C.

[0049] 2.2.2 PCR amplification of 16S rDNA

[0050] PCR system

[0051]

[0052] PCR reaction procedure

[0053]

[0054] Denaturation, annealing, extension 25-35 cycles

[0055] 2.2.3 16S rDNA recovery

[0056] Prepare SWP Buffer in advance and set the water bath to 55°C.

[0057] (1) Prepare macroporous agarose gel and add TAE into the pores of the macroporous gel to expel the air in the pores.

[0058] (2) Spotting: Add 7.5 μL of fluorescent dye and 7.5 μL (5x) Loading Buffer to 50 μL of the system, mix well, and spot about 35 μL in one well.

[0059] (3) Select a marker based on the fragment size and run electrophoresis after adding the marker.

[0060] (4) Find the target band in the gel under ultraviolet light, cut it off completely, and transfer it to a clean centrifuge tube.

[0061] (5) Weigh the cut rubber block and add XP2 in a 1:1 ratio.

[0062] (6) Use a constant temperature water bath at 55°C to dissolve the gel for 10 min. During this time, invert the centrifuge tube several times every 1 to 2 min to accelerate the dissolution until the gel lumps disappear.

[0063] (7) Transfer the solution to the DNA adsorption column in the matching collection tube. Add up to 700 μL at a time. This can be repeated multiple times. Centrifuge at 12,000 rpm for 1 min in a high-speed centrifuge. Pour the liquid in the collection tube back into the adsorption column and centrifuge at 12,000 rpm for 1 min. Pour out the liquid in the collection tube.

[0064] (8) Add 300 μL of XP2 to the adsorption column and let it stand for 1 min. Then centrifuge at 12,000 rpm for 1 min and discard the liquid in the collection tube.

[0065] (9) Add 700 μL SWP Buffer (diluted with anhydrous ethanol) to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the filtrate, and repeat once.

[0066] (10) Empty the collection tube and centrifuge the empty tube at 12,000 rpm for 2 min.

[0067] (11) Place the adsorption column into a new centrifuge tube and dry it in a 55°C oven with the lid open for 5 min. Make a mark on the lid.

[0068] (12) Add 30 μL of EB or ultrapure water to the center of the adsorption column membrane and let it stand for 2 minutes. Then centrifuge at 12,000 rpm for 2 minutes. Then drop the liquid in the centrifuge tube back onto the membrane of the adsorption column and centrifuge at 12,000 rpm for another 2 minutes.

[0069] (13) Measure the dsDNA concentration using an ultra-micro spectrophotometer, mark the wall of the centrifuge tube, and store the sample at -20°C.

[0070] 2.2.4 16S rDNA purification

[0071] To prevent the sample from being contaminated by exogenous DNA, DNA purification can be performed again. Prepare DNA WB in advance

[0072] (1) Add 4 to 5 times the PCR reaction volume of CP Buffer to the liquid, mix well, and add to the adsorption column in the collection tube.

[0073] (2) Let stand for 1 minute, centrifuge at 12,000 rpm for 1 minute. Pour the liquid in the collection tube back into the adsorption column and centrifuge at 12,000 rpm for 1 minute.

[0074] (3) Discard the filtrate, add 700 μL DWB to the adsorption column, and centrifuge at 12,000 rpm for 1 min. Repeat once.

[0075] (4) Discard the filtrate and centrifuge the empty column at 12000 rpm for 2 min.

[0076] (5) Place the adsorption column into a new centrifuge tube and dry it at 55°C for 5 min with the lid open.

[0077] (6) Add 35 μL of ultrapure water to the center of the adsorption column membrane (heated in a water bath at the same time), let it stand for 2 minutes, centrifuge at 12,000 rpm for 2 minutes. Then drop the liquid in the centrifuge tube onto the membrane of the adsorption column again, and centrifuge at 12,000 rpm for 2 minutes.

[0078] (7) Measure dsDNA concentration and store at -20°C.

[0079] 2.2.5. Samples for sequencing and comparison

[0080] Through 16srDNA sequencing, the 16srDNA sequence of Bacillus amyloliquefaciens B310 obtained by high temperature acclimation of the present invention is obtained as follows:

[0081]

[0082] The isolated Bacillus amyloliquefaciens B310 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with the deposit number CGMCC No.28054 and the deposit date July 28, 2023.

[0083] 3 Field experiments

[0084] Implementation Method of Dripless Irrigation System in Pear Orchard

[0085] Solution preparation: Bacillus amyloliquefaciens B31, 500g / bag, dilute with water to obtain 300-400 times diluted solution for standby use. Dig a circular pit about 30-40cm deep and 20cm wide about 1m away from the main trunk of the tree, and irrigate each tree with about 50 liters of diluted solution according to the age of the tree or the size of the crown to prevent the apricot fire blight pathogen from infecting the roots.

[0086] The first root irrigation is carried out in April-May and the second root irrigation is carried out in June-July every year. Two root irrigations can block the pear fire blight from infecting the roots and causing disease in the main trunk of the tree.

[0087] Implementation method of pear orchard with dripless irrigation system

[0088] Depending on the age of the tree or the size of the crown, root irrigation is carried out through the drip irrigation system. The dosage of the agent is 1500g / mu, that is, 3 bags / mu. The first root irrigation is carried out in April-May each year, and the second root irrigation is carried out in June-July. Two root irrigations can block the pear fire blight from infecting the roots and causing disease in the tree trunk.

[0089] A 100-mu Xiangli demonstration garden was established, divided into a treatment area and a control area of ​​50 mu each. The treatment area used 400 times the liquid of Bacillus amyloliquefaciens B310 (10 billion / g) through underground root irrigation without drip irrigation. The root irrigation was carried out by digging holes or trenches under the trees. The dosage of each tree was 35-50L / plant. The pear orchard with drip irrigation belts was irrigated at a dosage of 1500g / mu for root prevention and control, which can form an integrated water, fertilizer and medicine efficient prevention and control technology system. The control area was treated with the same amount of Bacillus amyloliquefaciens produced by Hubei Wuhan Zhongxiang Co., Ltd. Six months later, the main trunk analysis showed that the incidence rate in the treatment area and the control area was significantly different, with the main trunk incidence rate in the treatment area being 0% and the main trunk incidence rate in the control area being 6%.

[0090] The above disclosure is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the above claims.

Claims

1. A strain of Bacillus amyloliquefaciens, which is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.28054 and a deposit date of July 28, 2023.

2. Use of the Proteobacterium amyloliquefaciens described in claim 1 in blocking the infection of Erwinia amylovora from the roots.

3. The use according to claim 2, comprising the following steps: diluting the Bacillus amyloliquefaciens with clean water to obtain a 300-400-fold dilution for standby use; digging a circular pit with a depth of 30-40 cm and a width of 15-25 cm at a distance of 0.8-1.2 m from the main trunk of the tree, and pouring 40-60 liters of the dilution for root irrigation for each tree to block the infection of the amylopectin disease bacteria from the roots; the root irrigation time is the first root irrigation in April-May each year and the second root irrigation in June-July each year.

4. The use according to claim 2, comprising the following steps: root irrigation through a drip irrigation system, with the dosage of the agent being 1200-1800g of Bacillus amyloliquefaciens per mu, with the first root irrigation being carried out in April-May each year and the second root irrigation being carried out in June-July each year.

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