CN119662474A - Bacillus amyloliquefaciens and application thereof in blocking root infection of erwinia amylovora - Google Patents

Abstract

The present invention discloses a strain of Bacillus amyloliquefaciens and its application in blocking the infection of fire blight from the root. The Bacillus amyloliquefaciens is deposited in the General Microbiological Center of China Microbiological Culture Collection Committee with a deposit number of CGMCC No. 28054 and a deposit date of July 28, 2023. The Bacillus amyloliquefaciens of the present invention solves the problem of trunk disease and tree death caused by the infection of fire blight from the root.

Description

Bacillus amyloliquefaciens and application thereof in blocking root infection of erwinia amylovora

Technical Field

The invention relates to the technical field of microorganisms and agriculture, in particular to bacillus amyloliquefaciens and application thereof in blocking root infection of erwinia amylovora.

Background

In 2016, pear fire epidemic disease occurs in Xinjiang for the first time and constitutes a destructive hazard to the Xinjiang bergamot pear industry, and the disease transmission route is various, and can be transmitted through the routes of wound, rainwater, aerosol, soil, pollen and the like. The infection of the erwinia amylovora from the root (stock) is an important way for causing the disease of the trunk of the bergamot pear to cause difficult control and dead tree, and is also a new way for discovering the spread of the erwinia amylovora for the first time internationally.

The existing control of the pear fire epidemic disease takes chemical pesticides as the main material, although the control has certain control effect, pesticide residues are easy to cause, the human health is damaged, the ecological balance is destroyed, and the pesticide is used for controlling the pear fire epidemic disease for a long time, so that pathogenic bacteria are easy to generate drug resistance, and the control difficulty of the pear fire epidemic disease is increased. The biological control utilizes the secondary metabolite produced by microorganisms to control diseases, can effectively solve the problem of pesticide residues, does not cause pathogenic bacteria to produce drug resistance and is harmless to human bodies, and environmental pollution is avoided. Studies show that Pantoea agglomerans E352,352 can effectively inhibit the growth and reproduction of Pyricularia pyrifolia by producing the polyketide-cefoxitin and the dipeptide-based Pasteur, but is not easy to colonize in fields, and is more difficult to survive in saline-alkali environments.

For example, chinese patent CN112358366B discloses a compound bacterial fertilizer for controlling pear fire blight, a preparation method and application thereof, and the compound bacterial fertilizer is prepared by mixing bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), pseudomonas fluorescens (Pseudomonas fluorescens) and candida mongolica (Candida guilliermondii) according to a specific mass ratio, and compounding and combining cow dung, sheep dung, mushroom residue, fulvic acid and wine residue with a certain content. The technical scheme of the compound bacterial fertilizer for preventing and treating the pear fire blight provided by the invention is that the strain with a specific proportion is compounded to form stable microecology and ensure a higher viable count, so that the compound bacterial fertilizer not only can promote plant growth, but also has an obvious pear fire epidemic prevention effect, is applicable to saline-alkali soil, and can reduce the risk of pear infection of pear fire epidemic.

For another example, chinese patent CN115029269B discloses a piroxicam antagonistic bacterium producing lipopeptide antibiotics, and a fermentation method and application thereof, and belongs to the technical field of agricultural microorganisms. The strain is preserved in China general microbiological culture Collection center, and is classified and named as bacillus bailii, and the preservation number is CGMCC No.24843. The lipopeptide antibiotics produced by the strain have remarkable antagonism and excellent disease prevention effect on the erwinia amylovora, have certain tolerance to high temperature, acid and alkali, protease and ultraviolet rays, have high growth and propagation speed and strong competitiveness, can be produced at low cost, and have good development and application prospects. The invention also discloses a fermentation method for improving the lipopeptide antibiotics produced by the strain, which has low cost and simple and convenient operation, can improve the effect in biological control of the pear fire blight, and provides theoretical basis and technical reference for industrial fermentation production of the strain and research and development of the pear fire blight biological pesticide.

However, the control methods disclosed in the prior art require the use of special strains, and are relatively costly, and how to control the disease, especially in the case of trunk attack, remains a great challenge. Based on the method, a novel method for efficiently preventing and treating the trunk disease spots of the bergamot pear fire blight is developed by a post expert team of an autonomous region bergamot pear industry technical system of Xinjiang forestry academy of sciences for 5 years, and the problem of dead tree caused by the infection of the trunk of the bergamot pear tree by the bergamot pear fire blight is solved.

Disclosure of Invention

In view of the above, the invention provides bacillus amyloliquefaciens and application thereof in blocking root infection of erwinia amylovora. In order to achieve the above purpose, the present invention mainly provides the following technical solutions:

The invention relates to bacillus amyloliquefaciens (Bacillus amyloliquefaciens), which is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.28054 and the preservation date of 2023, 7 and 28 days.

The invention also relates to application of the bacillus amyloliquefaciens in blocking root infection of erwinia amylovora.

In a preferred embodiment of the invention, the application comprises the steps of diluting bacillus amyloliquefaciens with clear water to obtain 300-400 times of diluent for standby, digging a ring pit with the depth of 30-40 cm and the width of 15-25 cm from the trunk of a tree body of 0.8-1.2 m, irrigating 40-60 liters of diluent for each tree to perform root irrigation to block the infection of erwinia amylovora from roots, and performing the first root irrigation for 4-5 months and the second root irrigation for 6-7 months each year for root irrigation time.

In a preferred embodiment of the invention, the application comprises the steps of root irrigation by a dropper system, wherein the dosage of the agent is 1200-1800g of bacillus amyloliquefaciens per mu, the root irrigation is performed for the first time in 4-5 months and the root irrigation is performed for the second time in 6-7 months.

Compared with the prior art, the invention has the beneficial effects that:

① The bacillus amyloliquefaciens solves the problems of trunk morbidity and dead tree body caused by root infection of pear fire epidemic disease;

② The method is simple, quick and efficient to operate, low in cost, and about 40 yuan for each mu of medicament, and the root infection incidence rate is 0%;

③ The invention can realize the integration of water, fertilizer and pesticide for the pear garden with the drip irrigation system, reduces the labor expense and reduces the prevention and treatment cost;

④ The invention is a pure biological agent, has no pollution and no residue, and realizes the protection of soil ecological environment and the green and organic realization of fruits and vegetables.

Detailed Description

In order to further describe the technical means and effects adopted by the present invention to achieve the preset purpose, the following detailed description is given below according to the specific embodiments, technical schemes, features and effects of the present invention. The particular features, structures, or characteristics of the various embodiments in the description below may be combined in any suitable manner.

Example 1

After a new transmission path of pear fire blight caused by root infection is found in 2019, a Xinjiang forestry academy of sciences expert team timely develops blocking technology research and development, indoor antibacterial tests are conducted on bacillus amyloliquefaciens separated and identified from pear garden pear trees, the result shows that the antibacterial effect of the bacillus amyloliquefaciens on pear fire epidemic pathogens reaches 63%, the antibacterial effect is obviously higher than that of similar biocontrol bacteria in the market, the antibacterial effect of the bacillus amyloliquefaciens is improved to the greatest extent, a bacillus amyloliquefaciens is obtained by cooperation with northwest university and adopting a high-temperature domestication technology, the transmission path of pear fire epidemic pathogens from root infection can be blocked by 300 times of underground root irrigation after 1000 times dilution, and the disease incidence of pear is greatly reduced. The bacillus amyloliquefaciens is preserved in China general microbiological culture Collection center (CGMCC) No.28054, and the preservation date is 2023, 7 and 28.

1.1 Detailed composition and proportions of the high temperature domesticated microbial preparation

(1) Seed culture medium (g/3L) comprises tryptone 3, complex protease 3, yeast extract 2, beef extract 1, naCl 10, and pH 7.5.

(2) Fermentation medium (g/3L) comprises tryptone 10, complex protease hydrolysate 8, yeast extract 6, beef extract 5, naCl 10, and pH 7.5. 15mL of a 50% dextrose solution, 1MCa (NO 3) 5mL,0.1M MnCl25mL,1mM FeSO4 5mL, was added after sterilization. Culture broth (A) (g/2L) was fed with tryptone 30, proteolytic enzyme 15, yeast extract 20, naCl fed with liquid (B) 4MHCl 300mL fed with liquid (C) 10% NaOH 100mL.

1.2 Production Process of microbial preparation

1.2.1 Preparation of seed solution

Isolation of the disease from pear trees Bacillus amyloliquefaciens was stored at-20℃and the Bacillus amyloliquefaciens strain was inoculated on an activation medium solid plate and cultured at 37℃for 24hrs. The strain lawn was rinsed with sterilized physiological saline (0.1% Tween-80 added) to prepare a bacterial suspension. A suitable amount of the bacterial suspension was inoculated into 300mL of seed medium, and the culture was carried out at 45℃and 200rpm for 12hrs with a cell turbidity OD600 of 0.1.

1.2.2 Sterilization and inoculation of fermentation Medium

The 3L fermentation medium is sterilized in a fermenter at 121 ℃ for 20min, and the dissolved oxygen standard value is strictly corrected in the sterilization process. After sterilization, 15mL of a 50% glucose solution and 1M Ca (NO 3) 5mL,0.1MMnCl25mL,1mM FeSO4 5mL were added to maintain the temperature of the fermentation broth at 45 ℃. The feeding system equipped with the fermentation tank is used for feeding the liquid, and the pH value of the final fermentation liquor is adjusted to 7.5 by adjusting the feeding amount of the liquid feeding B and C. And adding the cultured seed liquid into a fermentation medium.

1.2.3 Control of fermentation Process

The air flow rate is 5L/min, the OD value is 25% and the rotating speed is a random variable by utilizing the self dissolved oxygen linkage module of the fermentation tank. Along with the fermentation, the dissolved oxygen value of the fermentation liquid is gradually reduced to a set value, and at the moment, mechanical stirring is started to maintain the dissolved oxygen concentration of the fermentation liquid, so that the growth and metabolism of cells are satisfied. When the turnover reaches the highest value and is maintained for a certain time, the turnover starts to descend, at the moment, a feeding module is started, the culture solution (A) is fed into the fermentation liquid at a certain speed, and on the premise of maintaining stirring turnover, the turnover of the fed culture solution is finely adjusted in real time along with the change of cell physiology and metabolism. And when the pH value is raised to above 8.5, pouring and collecting the fermentation liquor.

1.2.4 Detection of bacteriostatic Effect of microbial Agents

The fermentation broth was centrifuged to prepare a supernatant, and a sterilized filter paper disc (diameter 5 mm) was soaked in the supernatant and dried at 45 ℃. Meanwhile, several commercial bacteriostats are taken to prepare filter paper discs by the same method and serve as a control. Inoculating Xinjiang pear bacterial disease pathogenic bacteria on an LB solid plate for culturing for 16hrs, scraping a lawn to physiological saline by using a cotton swab, and preparing bacterial suspension. The bacterial suspension is coated on an LB solid flat plate by a cotton swab, the prepared filter paper sheet is stuck on the surface of a culture medium, the culture is carried out for 24hrs at 37 ℃, and the antibacterial effect of the fermentation broth is evaluated by the size of an antibacterial circle formed at the edge of the filter paper sheet. When the diameter of the inhibition zone reaches more than 3cm, the microbial preparation is regarded as qualified microbial preparation.

2 Identification of Bacillus amyloliquefaciens

2.1 Bacillus amyloliquefaciens isolation and culture Processes

(1) Single colonies with consistent morphological characteristics were picked from the plates, placed in 250mL Erlenmeyer flasks with 50mL of sterile LB liquid medium, blown evenly, and shake cultivated at 30℃for 12 hours. And (3) diluting the bacterial liquid for 12 hours, measuring the OD ₆₀₀, enabling the OD to be between 0.2 and 0.7, estimating the cell concentration of the diluted liquid according to the OD of 1 to be approximately equal to 10 ⁸ cells/mL, and multiplying the diluted liquid by the dilution multiple to calculate the concentration of the original bacterial liquid. The bacteria are preserved, 50% glycerol 300 mu L and 700 mu L bacteria liquid are mixed (super clean bench) and preserved at low temperature.

2.2 Authentication procedure

2.2.1 DNA extraction

(1) The bacterial liquid (the total amount of cells is not more than 10 ⁸) is taken and centrifuged at 12000rpm for 1min in a centrifuge tube, and the supernatant is sucked.

(2) The cells were resuspended by addition of 180. Mu. L Enzymatic Lysis Buffer.

(3) Incubate at 37 ℃ for 30min.

(4) Add 20. Mu LProteinase K vortex and mix well. 200 mu LBuffer GL of the mixture was added and mixed by vortex shaking.

(5) Incubate at 56 ℃ for 30min. Can be added with 4 mu L of RNaseA solution with the concentration of 100mg/mL

(6) 200 Mu L of absolute ethyl alcohol is added, and vortex vibration is carried out for uniform mixing.

(7) And (3) adding the solution obtained in the step (6) into an adsorption column filled in a collection pipe, and transferring the solution into the collection pipe for multiple times. Centrifuge at 12000rpm for 1min, and discard the waste liquid.

(8) 500. Mu.L Buffer GW1 (check whether absolute ethanol is added) was added to the adsorption column, 12000rpm,1min, and the waste liquid was discarded.

(9) 500 Mu LBuffer GW (check absolute ethanol), 12000rpm,1min were added and the waste solution was discarded. This step was repeated once.

(10) 12000Rpm,2min, discarding the waste liquid, and air drying the adsorption column

(11) Loading the adsorption column into a new centrifuge tube, suspending 50 mu LBuffer GE in the middle of the adsorption column, standing for 2min at 12000rpm for 1-2min. The solution in the tube was again suspended and added dropwise to the center of the column at 12000rpm for 2min.

(12) The DNA concentration was checked, and agarose gel electrophoresis was performed to verify the DNA integrity and preservation was performed at-20 ℃.

2.2.2PCR amplification of 16S rDNA

PCR system

PCR reaction procedure

Denaturation, annealing and extension for 25-35 cycles

2.2.316S rDNA recovery

SWP Buffer was prepared in advance and the water bath was set to 55℃in advance.

(1) Preparing macroporous agarose gel, and adding TAE into the macroporous agarose gel to discharge air in the pores.

(2) Spotting, 50. Mu.L of the system was added with 7.5. Mu.L of fluorescent dye and 7.5. Mu.L (5X) Loading Buffer, mixed well and spotted in one well at about 35. Mu.L.

(3) Selecting markers according to the sizes of the fragments, adding the markers, and running electrophoresis.

(4) Finding out the target strip in the glue under ultraviolet light, cutting off the target strip completely, and transferring the target strip to a clean centrifuge tube.

(5) The cut gum pieces were weighed and XP2 was added 1:1.

(6) And using a constant-temperature water bath to carry out sol at 55 ℃ for 10min, reversing the centrifuge tube for several times every 1-2 min during the process, and accelerating dissolution until the gel blocks disappear.

(7) The solution is transferred to a DNA adsorption column of a matched collecting pipe, and 700 mu L is added at most once, so that the transfer can be performed for multiple times. Centrifuge on a high speed centrifuge at 12000rpm for 1min. Pouring the liquid in the collecting pipe back into the adsorption column, centrifuging at 12000rpm for 1min, and pouring the liquid in the collecting pipe.

(8) 300. Mu.L XP2 was added to the column and allowed to stand for 1min. The liquid in the collection tube was then discarded by centrifugation at 12000rpm for 1min.

(9) 700. Mu.L SWP Buffer (diluted with absolute ethanol) was added to the column, centrifuged at 12000rpm for 1min, the filtrate was discarded, and repeated.

(10) The collection tube was emptied and the empty tube was centrifuged at 12000rpm for 2min.

(11) The adsorption column is sleeved into a new centrifuge tube, and the centrifuge tube is uncapped and dried for 5min by a 55 ℃ oven. Note that the cover is marked.

(12) 30 Mu L of EB or ultrapure water is dripped into the center of the adsorption column membrane, and the mixture is kept stand for 2min. Then centrifuged at 12000rpm for 2min. The liquid in the centrifuge tube was then re-dripped onto the membrane of the adsorption column and centrifuged at 12000rpm for 2min.

(13) DsDNA concentration was measured using an ultra-micro spectrophotometer, marked on the centrifuge tube wall and samples were stored at-20 ℃.

2.2.416S rDNA purification

Prevent the sample from being polluted by exogenous DNA and can be used for DNA purification. Is prepared in advance DNAWB

(1) Adding 4-5 times of CP Buffer with the volume of PCR reaction into the liquid, uniformly mixing, and adding into an adsorption column sleeved with a collection pipe.

(2) Standing for 1min, and centrifuging at 12000rpm for 1min. The liquid in the collection tube was then poured back into the column and centrifuged at 12000rpm for 1min.

(3) The filtrate was discarded, and 700. Mu. LDWB was added to the column and centrifuged at 12000rpm for 1min. Repeating once.

(4) The filtrate was discarded and the empty column was centrifuged at 12000rpm for 2min.

(5) The adsorption column is sleeved into a new centrifuge tube, and the centrifuge tube is uncapped and dried for 5min at 55 ℃.

(6) 35. Mu.L of ultrapure water (heated in a water bath) was added dropwise to the center of the adsorption column film, and the mixture was allowed to stand for 2 minutes. Centrifuge at 12000rpm for 2min. The liquid in the centrifuge tube was then re-dripped onto the membrane of the adsorption column and centrifuged at 12000rpm for 2min.

(7) DsDNA concentration was measured and stored at-20 ℃.

2.2.5, Sample-feeding sequencing and alignment

The 16srDNA sequence of the bacillus amyloliquefaciens Bacillus amyloliquefaciens B310,310 obtained by high-temperature domestication of the invention is obtained by sequencing the 16 srDNA:

ATGGAGAATATATTGGATCTTTGGAATCAAGCCTTAGCTCAAATTGAGAAAAAGCTAAGCAAACCGAGCTTCGAAACTTGGATGAAGTCGACGAAAGCCCATTCGCTGCAAGGAGATACCTTAACCATCACCGCTCCCAATGAATTTGCCAGAGACTGGCTGGAGTCCAGGTACTTACACCTCATTGCAGATACCATATATGAATTAACCGGGGAAGAATTGAGCGTCAAATTTGTCATTCCTCAAAATCAGGATGAAGAAAACTTTATGCCGAAACCTCAAGTGAAAAAAGCGGCAAAAGAGGAACCGTCTGATTTTCCCCAAAGCATGCTCAACCCGAAATATACATTCGACACATTTGTCATCGGATCGGGAAACCGATTCGCCCATGCAGCATCACTGGCTGTGGCGGAAGCACCCGCTAAAGCGTACAACCCTTTATTTATTTACGGGGGGGTCGGCTTAGGAAAAACCCACTTAATGCACGCAATCGGCCATTATGTCATTGATCATAATCCTTCAGCCAAAGTGGTGTACCTGTCTTCTGAAAAATTCACAAATGAATTCATCAACTCAATCCGCGACAATAAAGCCGTTGATTTTCGCAACCGCTACCGGAATGTTGATGTACTTTTGATAGATGATATTCAATTTTTAGCCGGAAAAGAACAAACACAGGAAGAGTTTTTCCATACATTCAACACGCTTCACGAAGAAAGCAAACAAATCGTGATCTCAAGCGACAGACCGCCAAAAGAGATTCCGACCCTTGAAGACCGGCTGCGTTCGCGGTTCGAATGGGGACTGATTACGGACATCACCCCGCCGGATCTGGAAACGCGGATTGCCATCCTGAGAAAGAAAGCGAAGGCGGAAGGCCTTGATATTCCGAATGAAGTCATGCTTTACATCGCGAATCAGATTGACAGCAACATCCGCGAACTGGAAGGGGCACTAATCCGGGTTGTTGCCTATTCATCTTTAATCAATAAAGACATTAATGCGGATCTGGCTGCTGAAGCATTGAAAGATATTATTCCGTCTTCAAAGCCGAAGGTCATTACGATAAAAGAGATTCAGCGGATTGTCGGACAGCAGTTCAACATTAAGCTTGAAGATTTCAAAGCGAAAAAGCGTACAAAATCCGTCGCGTTTCCAAGACAGATTGCGATGTATCTTTCCAGAGAAATGACGGATTCTTCTTTGCCCAAAATCGGGGAAGAATTTGGCGGGAGAGACCATACAACCGTCATTCACGCTCATGAAAAGATTTCAAAACTGCTGATTGATGACGAGCAGCTTCAGCAGCAGGTGAAAGAAATAAAAGAACAGCTTAAATAA, Identifying the bacillus amyloliquefaciens.

The separated bacillus amyloliquefaciens Bacillus amyloliquefaciensB310,310 is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.28054 and the preservation date of 2023, 7 and 28 days

3 Field experiments

Implementation method of pear garden without drip irrigation system

The medicine liquid is prepared by preparing the bacillus amyloliquefaciens Bacillus amyloliquefaciens B31,500 g/bag, and diluting with clear water to obtain 300-400 times of diluent for later use. Digging ring pits with a depth of 30-40 cm and a width of 20 cm at a distance of about 1m from the trunk of the tree body, and irrigating about 50 liters of diluent for each tree according to the age or crown size of the tree to irrigate roots and block the infection of the Pyricularia pyrifolia disease germs from the roots.

The root irrigation time is that the first root irrigation is carried out in 4-5 months and the second root irrigation is carried out in 6-7 months each year, and the root irrigation twice can prevent the main trunk of the tree from being infected by the pear fire epidemic disease.

Implementation method of pear garden with drip-free irrigation system

According to the tree age or the tree crown size, root irrigation is carried out through a dropper system, the dosage of the medicament is 1500 g/mu, namely 3 bags/mu, the root irrigation is carried out for the first time in 4-5 months and the root irrigation is carried out for the second time in 6-7 months each year, and the root irrigation for the second time can block the trunk of the tree caused by root infection of the pear fire epidemic disease.

100 Mu of pear demonstration garden is established, and the pear demonstration garden is divided into a treatment area and a control area, wherein the treatment area adopts 400 times liquid bacillus amyloliquefaciens Bacillus amyloliquefaciens B to 310 (100 hundred million/g) through underground root irrigation and burette-free pear garden, and is controlled through pit opening or ditching under the tree, and the dosage of each tree liquid medicine is 35-50L/plant. The pear garden with drip irrigation tape is used for root irrigation prevention and control according to the dosage of 1500 g/mu, and a water, fertilizer and pesticide integrated efficient prevention and control technical system can be formed. The control area was treated with the same amount of bacillus amyloliquefaciens produced by wuhan in hubei to the company limited. After half a year, the results of the trunk analysis are counted, and show that the morbidity of the treatment area is obviously different from that of the control area, the morbidity of the trunk of the treatment area is 0%, and the morbidity of the trunk of the control area is 6%.

The foregoing disclosure is merely illustrative of specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art will readily appreciate variations or substitutions within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (4)

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, 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.