CN114015616B - Bacillus amyloliquefaciens XJ-BV2007 and culture method and application thereof - Google Patents
Bacillus amyloliquefaciens XJ-BV2007 and culture method and application thereof Download PDFInfo
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- 239000002689 soil Substances 0.000 description 5
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- 241000194108 Bacillus licheniformis Species 0.000 description 4
- CEBXXEKPIIDJHL-UHFFFAOYSA-N alternariol Chemical compound O1C(=O)C2=C(O)C=C(O)C=C2C2=C1C=C(O)C=C2C CEBXXEKPIIDJHL-UHFFFAOYSA-N 0.000 description 4
- LCSDQFNUYFTXMT-UHFFFAOYSA-N djalonensone Chemical compound C1=C(O)C=C2OC(=O)C3=C(O)C=C(OC)C=C3C2=C1C LCSDQFNUYFTXMT-UHFFFAOYSA-N 0.000 description 4
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- SIIRBDOFKDACOK-UHFFFAOYSA-N tentoxin Natural products CN1C(=O)C(CC(C)C)NC(=O)C(C)N(C)C(=O)CNC(=O)C1=CC1=CC=CC=C1 SIIRBDOFKDACOK-UHFFFAOYSA-N 0.000 description 2
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- CKDYDMSDCNQHEB-UHFFFAOYSA-N Dihydrokaempferide Natural products C1=CC(OC)=CC=C1C1C(O)C(=O)C2=C(O)C=C(O)C=C2O1 CKDYDMSDCNQHEB-UHFFFAOYSA-N 0.000 description 1
- LCGLNKUTAGEVQW-UHFFFAOYSA-N Dimethyl ether Chemical compound COC LCGLNKUTAGEVQW-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention provides a newly separated bacillus amyloliquefaciens (Bacillus amyloliquefaciens) XJ-BV2007, a culture method and application thereof, wherein the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) XJ-BV2007 is preserved in China general microbiological culture Collection center (CGMCC) No.23669 in 10-month 26 of 2021, and the bacillus amyloliquefaciens has a good inhibition effect on the growth of alternaria alternata in a flat plate counter experiment, and also has an inhibition effect on the synthesis of alternaria alternata toxin, so that the bacillus amyloliquefaciens can be applied to biological prevention and treatment of the pollution of the alternaria alternata.
Description
Technical Field
The invention relates to the technical field of prevention and control of mycotoxins of fruits and vegetables, in particular to bacillus amyloliquefaciens XJ-BV2007 and a culture method and application thereof.
Background
Tomatoes are generally classified into fresh tomatoes and processed tomatoes, china is a large production country of processed tomatoes, about 6276 ten thousand tons of tomatoes are provided in China in 2019 according to data of grain and agriculture organizations of united countries, the tomatoes account for about 35% of global yield, and the demands of the market for tomatoes are gradually increased along with the improvement of processing and manufacturing levels. However, tomatoes that are susceptible to contamination by pathogenic microorganisms during processing, growth, harvesting, storage, processing, etc., particularly during storage, decay is relatively heavy after harvesting due to alternaria alternata (Alternaria alternata), resulting in not only significant yield losses, but also accumulation of alternaria alternata toxins in tomatoes and their products that are potentially threatening to human health. Among them, tenuazonic acid (TeA) has toxicity and detected content, all of which are in the first place of each of the chain sporotoxins, and are listed in the toxic chemical substance registry by the U.S. food and drug administration, with acute toxicity and can show synergistic effect (Pinto et al 2017) of chain sporophenol (alternariol, AOH) and chain sporophenol monomethyl ether (alternariol monomethyl ether, AME); the European food Security agency has suggested that the daily intake of AOH and AME in foods does not exceed 2.5ng/kg body weight, and that Tentoxins (TENs) and TeAs do not exceed 1500ng/kg body weight.
At present, chemical bactericides are main measures for preventing and controlling tomato fungal diseases and controlling mycotoxin pollution, but are limited by adverse factors such as high carcinogenicity, long degradation period, heavy environmental pollution and the like, and chemical synthetic bactericides are increasingly used regularly and even limited. In comparison, the adoption of the safe, environment-friendly and efficient biological bactericide is more scientific, reasonable and urgent.
Disclosure of Invention
Therefore, the main purpose of the invention is to provide bacillus amyloliquefaciens XJ-BV2007 which can inhibit the growth of Alternaria alternata (A. Alternate) and inhibit the generation of Alternaria alternata with high efficiency, and a culture method and application thereof, and has the effects of inhibiting the growth of Alternaria alternata in tomatoes after harvest and inhibiting the generation of Alternaria alternata in tomatoes.
The aim and the technical problems of the invention are realized by adopting the following technical proposal. The bacillus amyloliquefaciens (Bacillus amyloliquefaciens) XJ-BV2007 provided by the invention is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 23669 in the 10 th month and 26 th year of 2021.
The bacillus amyloliquefaciens (Bacillus amyloliquefaciens) XJ-BV2007 is firstly separated from soil of tomato processing field of the astragalus basin in Xinjiang, and the biocontrol strain XJ-BV2007 with good inhibition effect on Alternaria alternata and Alternaria alternata is identified as bacillus amyloliquefaciens by 16 srRNA.
The aim and the technical problems of the invention can be achieved by adopting the following technical proposal. The culture method of the bacillus amyloliquefaciens XJ-BV2007 provided by the invention comprises the following steps:
inoculating mycelia of strain XJ-BV2007 to NA solid culture medium with inoculating needle for activation, culturing for 12-24 hr in incubator at 25-40deg.C, picking single colony of Bacillus amyloliquefaciens with inoculating needle, transferring to NB liquid culture medium, culturing for 12-24 hr at 125-175r/min in shaking incubator at 25-40deg.C, transferring culture solution with 1-10% total volume of liquid culture medium to fresh NB liquid culture medium, culturing for 24-36 hr at 125-175r/min at 25-40deg.C to obtain XJ-BV2007 fermentation broth with viable count of (1-9) ×10 5 CFU/mL。
The aim and the technical problems of the invention can be achieved by adopting the following technical proposal. The invention provides an application of bacillus amyloliquefaciens XJ-BV2007 in preventing and treating fruit and vegetable alternaria alternata toxin pollution.
Preferably, the application of the bacillus amyloliquefaciens XJ-BV2007 in preventing and treating the fruit and vegetable alternaria alternata toxin pollution comprises the following steps:
the concentration is (1-9). Times.10 5 The fermentation broth of CFU/mL bacillus amyloliquefaciens XJ-BV2007 is directly sprayed on the surface of a biological sample.
The aim and the technical problems of the invention can be achieved by adopting the following technical proposal. The biocontrol preparation containing the bacillus amyloliquefaciens XJ-BV2007 is provided by the invention.
Preferably, the biocontrol preparation containing the bacillus amyloliquefaciens XJ-BV2007 is in the form of liquid, powder spraying, dry wettable powder or dry wettable particles.
Preferably, the biocontrol agent containing the bacillus amyloliquefaciens XJ-BV2007 comprises the XJ-BV2007 fermentation broth.
Preferably, the biocontrol agent containing the bacillus amyloliquefaciens XJ-BV2007 is liquid.
Preferably, the biocontrol agent containing the bacillus amyloliquefaciens XJ-BV2007 comprises the XJ-BV2007 fermentation broth.
Preferably, the aforementioned biocontrol agent containing the above bacillus amyloliquefaciens XJ-BV2007, wherein the final concentration of the bacillus amyloliquefaciens XJ-BV2007 in the biocontrol agent is (1-9) x 10 5 CFU/mL。
The aim and the technical problems of the invention can be achieved by adopting the following technical proposal. The application of the biocontrol agent in preventing and treating the pollution of the fruit and vegetable alternaria alternata is provided by the invention.
Preferably, the application of the biocontrol agent in preventing and controlling the Alternatoxin pollution of fruits and vegetables is disclosed, wherein the prevention and control of Alternatoxin pollution of fruits and vegetables comprises at least one of inhibiting Alternatoxin growth or inhibiting Alternatoxin synthesis.
Preferably, the application of the biocontrol agent in preventing and treating the pollution of the fruit and vegetable alternaria alternata toxin comprises the following steps:
the biocontrol agent is uniformly sprayed on the surface of a biological sample to inhibit the alternaria alternata and the alternaria alternata toxin.
By means of the technical scheme, the bacillus amyloliquefaciens XJ-BV2007 and the culture method and application thereof have at least the following advantages:
the biocontrol strain XJ-BV2007 with good inhibition effect on the Alternaria alternata and Alternaria alternata in tomatoes is separated from soil for the first time, and is identified as the Bacillus amyloliquefaciens by 16srRNA, and the thallus of the Bacillus amyloliquefaciens XJ-BV2007 shows good inhibition effect on the Alternaria alternata growth in a plate opposite test, the inhibition rate is 68.40%, and meanwhile, the synthesis inhibition rate on Alternaria alternata on opposite plates is 40.25%, so that the biocontrol strain can be applied to the control of Alternaria alternata and Alternaria alternata.
When bacillus amyloliquefaciens XJ-BV2007 and alternaria alternata are cultured in a liquid culture medium, the growth of pathogenic bacteria can be obviously inhibited, the inhibition rate is higher than 95% by visual inspection, and the synthesis inhibition rate of alternaria alternata TeA is as high as 99.71%.
The bacillus amyloliquefaciens XJ-BV2007 disclosed by the invention can effectively inhibit the growth of Alternaria alternata on tomatoes under normal-temperature storage conditions, the inhibition rate reaches 88.04%, and the prevention effect of the bacillus amyloliquefaciens XJ-BV2007 on Alternaria alternata pollution in tomatoes can reach 93.28%, so that the prevention effect is good, and the pollution of the Alternaria alternata can be prevented and controlled by using the bacillus amyloliquefaciens XJ-BV 2007.
The bacillus amyloliquefaciens XJ-BV2007 disclosed by the invention is applied to prevention and control of Alternaria solani pollution, and can overcome a series of problems caused by using chemical pesticides, so that pollution-free production of agricultural products is facilitated, and the product quality is improved.
The foregoing description is only an overview of the present invention, and is intended to provide a more thorough understanding of the present invention, and is to be accorded the full scope of the present invention.
Drawings
FIG. 1 is a graph showing the antibacterial performance of Bacillus amyloliquefaciens XJ-BV2007, and Bacillus atrophaeus XJ-BV2001, bacillus licheniformis XJ-BV2004, and Bacillus highland XJ-BV2006 of control group CK (inoculated with Alternaria alternata strain alone without antagonistic bacteria);
FIG. 2 is a colony morphology diagram of Bacillus amyloliquefaciens XJ-BV2007 of example 1 of the present invention;
FIG. 3 shows the rDNA gene development phylogenetic tree of Bacillus amyloliquefaciens XJ-BV2007 of example 1 of the present invention;
FIG. 4 is a graph showing the antagonistic action of Bacillus amyloliquefaciens XJ-BV2007 of example 2 of the present invention on Alternaria alternata in liquid medium (upper graph is a control group: liquid-only Alternaria alternata; lower graph is an experimental group: liquid-simultaneous culture of Bacillus amyloliquefaciens XJ-BV2007 and Alternaria alternata)
FIG. 5 is a graph showing the antagonism of Bacillus amyloliquefaciens XJ-BV2007 of example 3 of the present invention against Alternaria solani (upper graph: seed Alternaria only; lower graph: seed Alternaria solani simultaneously XJ-BV2007 and Alternaria solani).
Detailed Description
In order to further describe the technical means and effects adopted for achieving the preset aim of the invention, the following is a detailed description of the specific implementation, structure, characteristics and effects of a bacillus amyloliquefaciens XJ-BV2007, a culture method and an application thereof according to the present invention in combination with the preferred embodiments.
The invention provides bacillus amyloliquefaciens (Bacillus amyloliquefaciens) XJ-BV2007, which is preserved in China general microbiological culture Collection center (CGMCC) for 10 months and 26 days in 2021, and has the preservation number of CGMCC NO.23669, namely, bacillus amyloliquefaciens (Bacillus amyloliquefaciens) classified by the national institute of China academy of sciences, including North Chen West Lu 1, god of Beijing, and national academy of sciences, including 3, and the address of the microorganism.
The bacillus amyloliquefaciens (Bacillus amyloliquefaciens) XJ-BV2007 is firstly separated from soil of a tomato field processed by a astragalus basin in Xinjiang, and the biocontrol strain XJ-BV2007 with good inhibition effect on the Alternaria alternata (A. Alternate) and Alternaria alternata is identified as the bacillus amyloliquefaciens by 16srRNA, and shows that the bacillus amyloliquefaciens has good inhibition effect on the Alternaria alternata growth in a solid plate counter experiment, the inhibition rate is 68.40%, and the inhibition rate on one of Alternaria alternata-tenascomycete (TeA) is 40.25%. In the liquid culture test, the compound also shows good inhibition effect on the growth of the alternaria alternata, the inhibition rate is visually detected to be more than 95%, and the inhibition rate on the alternaria alternata TeA is 99.71%.
The invention also provides a culture method of the bacillus amyloliquefaciens XJ-BV2007, which comprises the following steps:
inoculating mycelia of strain XJ-BV2007 to NA solid culture medium with inoculating needle for activation, culturing for 12-24 hr in incubator at 25-40deg.C, picking single colony of Bacillus amyloliquefaciens with inoculating needle, transferring to NB liquid culture medium, culturing for 12-24 hr at 125-175r/min in shaking incubator at 25-40deg.C, transferring culture solution with 1-10% total volume of liquid culture medium to fresh NB liquid culture medium, culturing for 24-36 hr at 125-175r/min at 25-40deg.C to obtain XJ-BV2007 fermentation broth with viable count of (1-9) ×10 5 CFU/mL。
The invention also provides application of the bacillus amyloliquefaciens XJ-BV2007 in preventing and treating the pollution of the fruit and vegetable alternaria alternata toxin.
In some embodiments of the invention, wherein the application may comprise the steps of:
the concentration is (1-9). Times.10 5 The fermentation liquid of the bacillus amyloliquefaciens XJ-BV2007 of CFU/mL is directly sprayed on the surface of a biological sample, so that the contamination of the biological sample with the alternaria alternata is prevented. The spraying manner in this embodiment may be an automatic sprayer (the spraying rate may be, for example, 0.5L/min), or may be a plant protection unmanned aerial vehicle (the spraying rate may be 0.43L/min (a single nozzle, for example, water), which is not limited herein.
In some embodiments of the invention, wherein the application may comprise the steps of:
inoculating the biological sample directly onto the surface with a concentration of (1-9) ×10 5 The fermentation liquid of CFU/mL bacillus amyloliquefaciens XJ-BV2007 is used for preventing and treating the alternaria alternata toxin pollution of biological samples. The amount of inoculation may be, for example, 10. Mu.L.
The invention also provides a biocontrol preparation containing the bacillus amyloliquefaciens XJ-BV 2007.
In some embodiments of the invention, wherein the biocontrol formulation may be in the form of a liquid, a powder spray, a dry wettable powder or a dry wettable granule. The form of the biocontrol agent is preferably made in a liquid form in view of cost saving.
In some embodiments of the present invention, wherein the biocontrol formulation comprises the XJ-BV2007 fermentation broth described above.
In some embodiments of the present invention, wherein the final concentration of Bacillus amyloliquefaciens XJ-BV2007 in the biocontrol formulation is (1-9). Times.10 5 CFU/mL。
The invention also provides application of the biocontrol agent in preventing and treating the pollution of the fruit and vegetable alternaria alternata toxin.
In some embodiments of the invention, wherein said controlling the contamination with alternaria alternata may comprise at least one of inhibiting the growth of alternaria alternata or inhibiting the synthesis of alternaria alternata, such as inhibiting the synthesis of alternaria alternata TeA.
In some embodiments of the invention, wherein the applying comprises the steps of:
the biocontrol agent is uniformly sprayed on the surface of a biological sample, such as the surface of a whole fruit sample, especially the positions of fruit stalks, fruit bottoms and the like which are easy to infect the Alternaria alternata, so that the Alternaria alternata toxin pollution of the biological sample is prevented and treated. The spraying manner in this embodiment may be an automatic sprayer (the spraying rate may be, for example, 0.5L/min), or may be a plant protection unmanned aerial vehicle (the spraying rate may be 0.43L/min (a single nozzle, for example, water), which is not limited herein.
Unless otherwise indicated, materials, reagents, and the like referred to below are commercially available products well known to those skilled in the art; unless otherwise indicated, the methods are all methods well known in the art. Unless otherwise defined, technical or scientific terms used should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.
The invention will be further described with reference to specific examples, which are not to be construed as limiting the scope of the invention, but rather as falling within the scope of the invention, since numerous insubstantial modifications and adaptations of the invention will now occur to those skilled in the art in light of the foregoing disclosure.
EXAMPLE 1 isolation, screening and characterization of Bacillus amyloliquefaciens XJ-BV2007
1. Bacterial separation and screening
Isolation of antagonistic bacteria from tomato soil: weighing 5g of soil, adding 45mL of sterile water, shaking flask culture at 27deg.C and 150r/min for 30min, and subjecting the obtained liquid to 10 -1 -10 -6 Is diluted in a gradient of 100. Mu.L, 10 -1 -10 -6 Is plated onto NA medium and incubated in an incubator (150 r/min) at 27℃for 24h. Individual colonies on the plates were picked and streaked on NA medium.
The antagonistic ability was screened by the plate counter method. A7-day-old Alternaria alternata mycelium tray (strain is purchased from China center for agricultural microbiological culture collection center, and is numbered ACCC 37489) with the diameter of 5mm is placed in the center of a PDA culture medium, antagonistic bacteria with the diameter of 5mm are inoculated on the same plate at a position which is 2cm away from the Alternaria alternata mycelium tray, the antagonistic bacteria are not inoculated, a plate which is only inoculated with Alternaria alternata is used as a control, whether the growth of mycelium is inhibited or not is observed, the diameter of Alternaria alternata on day 7 is measured, the inhibition rate is calculated through the radial growth diameter of the Alternaria alternata, and each strain is in triplicate. Selecting a strain with a bacteriostasis rate of more than 60%, and preserving for later use.
2. Bacterial re-screening
1) Preparation of bacterial strain XJ-BV2007 fermentation broth
Antibacterial rate by inoculating needle>60% of strain XJ-BV2007 was inoculated into NA solid mediumThe strain is activated, cultured for 24 hours in an incubator at 37 ℃, single colony of bacillus amyloliquefaciens is picked by an inoculating needle, transferred into NB culture medium, cultured for 24 hours at 150r/min in a shaking incubator at 37 ℃,5 percent of the total volume of liquid culture medium is sucked into fresh NB culture medium, cultured for 36 hours at 150r/min in a shaking incubator at 37 ℃ to obtain fermentation liquor of antagonistic strain XJ-BV2007, and the number (concentration) of viable bacteria is 5 multiplied by 10 5 CFU/mL。
2) Antibacterial property measurement
The antagonistic fermentation broth is screened by adopting a flat plate counter method. A7-day-old mycelium plate of Alternaria alternata (strain is purchased from China center for culture collection of agricultural microorganisms, and is numbered ACCC 37489) with a diameter of 5mm is placed in the center of PDA culture medium, PDA culture medium with a diameter of 5mm is dug out in 4 directions on the same plate 2cm away from the mycelium plate, and 200 μl of antagonistic bacteria fermentation broth (viable count is 5×10) is added 5 CFU/mL) as experimental group XJ-BV 2007; similarly, PDA culture medium with diameter of 5mm was dug out in 4 directions on the other 3 same plates 2cm away from mycelium trays, and 200. Mu.L of Bacillus atrophaeus fermentation broth (viable count 5×10) was added respectively 5 CFU/mL), 200. Mu.L of Bacillus licheniformis fermentation broth (viable count 5X 10) 5 CFU/mL), 200. Mu.L of Bacillus subtilis broth (viable count 5X 10) 5 CFU/mL) was used as the experimental group XJ-BV2001, the experimental group XJ-BV2004, and the experimental group XJ-BV2006, and the plate without inoculating antagonistic bacteria, which was inoculated with Alternaria alternata only, was used as the control group CK, and whether the plaque growth was inhibited was observed, the diameter of Alternaria alternata on day 7 was measured, and the inhibition ratio was calculated from the radial growth diameter thereof, each strain was in triplicate. The test results are shown in FIG. 1 and Table 1. Wherein, the experimental group XJ-BV2001 represents simultaneous inoculation of Alternaria alternata strain and Bacillus atrophaeus Bacillus atrophaeus; experimental group XJ-BV2004 represents simultaneous inoculation of the alternaria alternata strain and bacillus licheniformis Bacillus paralicheniformis; experimental group XJ-BV2006 represents simultaneous inoculation of the alternaria alternata strain and geobacillus highland Bacillus altitudinis; the experimental group XJ-BV2007 indicated simultaneous inoculation of Alternaria alternata strain and Bacillus amyloliquefaciens Bacillus amyloliquefaciens.
Inhibition (%) = (C1-C2)/c1×100%
C1 radial growth diameter of Alternaria alternata of control group
C2 radial growth diameter of Alternaria alternata of the experimental group
Table 1 Flat counter screen
As can be seen from the experimental results in FIG. 1 and Table 1, the growth inhibition rate of XJ-BV2001 experimental group on the Alternaria alternata strain is about 64.51%, the growth inhibition rate of XJ-BV2004 experimental group on the Alternaria alternata strain is about 60.19%, the growth inhibition rate of XJ-BV2006 experimental group on the Alternaria alternata strain is about 60.76%, and the growth inhibition rate of XJ-BV2007 on the Alternaria alternata strain is about 68.40%, which indicates that Bacillus atrophaeus Bacillus atrophaeus, bacillus licheniformis Bacillus paralicheniformis, bacillus highland Bacillus altitudinis and Bacillus amyloliquefaciens Bacillus amyloliquefaciens all have the ability of inhibiting Alternaria alternata growth, and the inhibition ability of Bacillus amyloliquefaciens Bacillus amyloliquefaciens is optimal.
3) Determination of toxicity inhibition energy
The culture medium on the 8 th day plate of the experimental group XJ-BV2007 in the 2) is placed in a mortar, about 75mL of liquid nitrogen is added for rapid grinding and crushing to 200 meshes, the culture medium is extracted by using a dispersive solid phase extraction (QuEChERS) method combined with an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method, and the content of the alternaria toxin in the culture medium is detected, and compared with a control group CK, the inhibition rate of the experimental group XJ-BV2007 on the alternaria toxin TeA reaches 40.25%.
3. Morphological features
Bacterial colony of the strain XJ-BV2007 on the NA culture medium is round, white, convex, smooth in surface, neat in edge and easy to pick up, the diameter of the bacterial colony is 1-2mm, and the bacterial colony is shown in figure 1; the microscopic morphology is shown in figure 2, and is gram positive, rod-shaped and sporulated.
4. Physiological and biochemical characteristics
The strain XJ-BV2007 is suitable for growth on Nutrient Agar (NA) and Nutrient Broth (NB) culture media, the suitable temperature range for growth is 4-45 ℃, the optimal temperature is 37 ℃, the suitable pH value for growth is 3-9, and the optimal pH value is 7.3.
5. Molecular biological characteristics
The bacterial DNA extraction kit was used, and the DNA of antagonistic bacteria XJ-BV2007 was extracted with reference to the instructions for use, and PCR amplification was performed using the universal primers 27F (5 '-AGAGTTGATVATGGCTCAG-3') and 1492R (5'-CTACGGTTACCTTGTTACGAC-3') of 16S rDNA as templates under the following amplification conditions: 94℃for 260 seconds, 55℃for 20 seconds, 72℃for 700 seconds, and finally cooled to 4℃and cycled 35 times. The resulting fragment was submitted to sequencing by Shanghai worker to obtain a 1362bp length gene, adjacent species were analyzed by BLAST on NCBI, and phylogenetic tree was constructed using MEGA7.0, as shown in FIG. 3, the homology of antagonistic bacterium XJ-BV2007 to SGD isolate 4 (Bacillus amyloliquefaciens partial 16S rRNA gene strain SGD isolate 4) of Bacillus amyloliquefaciens part 16S rRNA gene was 96%, and strain XJ-BV2007 was finally identified as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) by combining morphological characteristics, physiological and biochemical characteristics of the strain and the results of the above BLAST system analysis. The 16S rDNA sequence of the strain XJ-BV2007 is shown as SEQ ID NO. 1.
EXAMPLE 2 testing of the action of Bacillus amyloliquefaciens XJ-BV2007 on Alternaria alternata and its toxins on liquid Medium
The number (concentration) of viable bacteria of 6mL in example 1 was 9X 10 5 CFU/mL fermentation broth of Bacillus amyloliquefaciens XJ-BV2007 with 6mL 9×10 5 CFU/mL of the suspension of alternaria spores was mixed and added to 6mL of Potato Dextrose Broth (PDB) liquid medium (liquid compositions of control group and experimental group are shown in table 2), shake cultivation was performed at 28 ℃ for 5 days at 120r/min, growth of pathogenic bacteria was observed, and the alternaria toxins TeA in the mixed culture solution was extracted and detected by using the QuEChERS-UPLC-MS/MS method, 3 replicates were made for each group, inhibition ratios were calculated according to the following formula, and test results are shown in fig. 4 and table 3.
Toxin inhibition (%) = (control peak area-experimental peak area)/control peak area×100%
TABLE 2 test grouping of Bacillus amyloliquefaciens XJ-BV2007 on liquid Medium
TABLE 3 inhibition of TeA by antagonism test of Bacillus amyloliquefaciens XJ-BV2007 on liquid Medium
| Group of | Peak area of TeA | Inhibition rate |
| Control group CK | 3819502.00 | / |
| Experimental group XJ-BV2007 | 11239.44 | 99.71％ |
As shown in FIG. 4, bacillus amyloliquefaciens XJ-BV2007 has a remarkable inhibition effect on the growth of liquid-cultured Alternaria alternata, and since the inhibition rate cannot be directly calculated by liquid, the inhibition effect can only be visually detected by phenomenon to exceed 95%.
As shown in Table 3, the QuEChERS-UPLC-MS/MS method is used for detecting the content of the Alternaria alternata TeA in the shake culture liquid on the 5 th day, and compared with the CK of the control group, the XJ-BV2007 bacterial suspension in the experimental group has the inhibition rate of 99.71 percent on the Alternaria alternata TeA.
Example 3 application of Bacillus amyloliquefaciens XJ-BV2007 in prevention and treatment of Alternaria alternata contamination of fruits and vegetables
The surface (middle) of mature fresh processed tomato (the tested processed tomato variety is Henry 1015, and is collected in two six town of Changji City, changji-back, autonomous) is needled to 5mm depth, and then inoculated with 10 μl with viable count of 5×10 5 CFU/mL XJ-BV2007 bacterial suspension was inoculated with 10. Mu.L of sterile water as a control group, and after 30min, the experimental group CK and the control group XJ-BV2007 (5 tomatoes each) were inoculated with 10. Mu.L, 1×10 at the wound site 5 CFU/mL suspension of alternaria alternata spores. All tomatoes were placed at room temperature of 37 ℃, the diameter of the lesions was measured on day 8 of culture, and the alternaria toxin teas in tomatoes were detected by extraction using the QuEChERS-UPLC-MS/MS method, each group being inoculated with 5 tomatoes. The inhibition ratio was calculated according to the following formulas (1) and (2), and the test results are shown in tables 4, 5 and fig. 5.
Fungal growth inhibition (%) = (C1-C2)/c1×100% (1)
Toxin inhibition (%) = (control group peak area-experimental group peak area)/control group peak area x 100% (2)
C1 control Alternaria alternata radial growth diameter
C2 radial growth diameter of Alternaria alternata in experimental group
TABLE 4 inhibition of Alternaria solani growth by Bacillus amyloliquefaciens XJ-BV2007
From the experimental results in table 4, it can be seen that the inhibition rate of bacillus amyloliquefaciens XJ-BV2007 on processing tomatoes is about 88.04%, which shows that the bacillus amyloliquefaciens has good effect on inhibiting the growth of alternaria solani on tomatoes.
As shown in Table 5, the QuEChERS-UPLC-MS/MS method was used to detect the content of the Alternaria alternata in the 8 th-day processed tomatoes, and compared with the control group CK, the inhibition rate of the Alternaria alternata by the experimental group XJ-BV2007 reaches 93%.
TABLE 5 inhibition of TeA on tomatoes by Bacillus amyloliquefaciens XJ-BV2007
| Group of | Peak area of TeA | Inhibition rate |
| Control group CK | 422262.24 | / |
| Experimental group XJ-BV2007 | 28360.04 | 93.28％ |
As can be seen from FIG. 5, alternaria alternata in tomatoes does not grow substantially.
In the description of the present invention, numerous specific details are set forth. However, it is understood that embodiments of the invention may be practiced without these specific details. In some embodiments, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.
The above description is only of the preferred embodiments of the present invention, and is not intended to limit the present invention in any way, but any simple modification, equivalent variation and modification made to the above embodiments according to the technical substance of the present invention still fall within the scope of the technical solution of the present invention.
SEQUENCE LISTING
<110> Xinjiang academy of agricultural science
<120> Bacillus amyloliquefaciens XJ-BV2007 and culture method and application thereof
<130> P2113804
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1362
<212> DNA
<213> Bacillus amyloliquefaciens (Bacillus amyloliquefaciens)
<400> 1
cttgctccct gatgttagcg gcggacgggt gagtaacacg tgggtaacct gcctgtaaga 60
ctgggataac tccgggaaac cggggctaat accggatggt tgtttgaacc gcatggttca 120
gacataaaag gtggcttcgg ctaccactta cagatggacc cgcggcgcat tagctagttg 180
gtgaggtaac ggctcaccaa ggcgacgatg cgtagccgac ctgagagggt gatcggccac 240
actgggactg agacacggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa 300
tggacgaaag tctgacggag caacgccgcg tgagtgatga aggttttcgg atcgtaaagc 360
tctgttgtta gggaagaaca agtgccgttc aaatagggcg gcaccttgac ggtacctaac 420
cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg 480
tccggaatta ttgggcgtaa agggctcgca ggcggtttct taagtctgat gtgaaagccc 540
ccggctcaac cggggagggt cattggaaac tggggaactt gagtgcagaa gaggagagtg 600
gaattccacg tgtagcggtg aaatgcgtag agatgtggag gaacaccagt ggcgaaggcg 660
actctctggt ctgtaactga cgctgaggag cgaaagcgtg gggagcgaac aggattagat 720
accctggtag tccacgccgt aaacgatgag tgctaagtgt tagggggttt ccgcccctta 780
gtgctgcagc taacgcatta agcactccgc ctggggagta cggtcgcaag actgaaactc 840
aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc 900
gaagaacctt accaggtctt gacatcctct gacaatccta gagataggac gtccccttcg 960
ggggcagagt gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta 1020
agtcccgcaa cgagcgcaac ccttgatctt agttgccagc attcagttgg gcactctaag 1080
gtgactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta 1140
tgacctgggc tacacacgtg ctacaatgga cagaacaaag ggcagcgaaa ccgcgaggtt 1200
aagccaatcc cacaaatctg ttctcagttc ggatcgcagt ctgcaactcg actgcgtgaa 1260
gctggaatcg ctagtaatcg cggatcagca tgccgcggtg aatacgttcc cgggccttgt 1320
acacaccgcc cgtcacacca cgagagtttg taacacccga ag 1362
Claims (10)
1. Bacillus amyloliquefaciens strainBacillus amyloliquefaciens) XJ-BV2007 is preserved in China general microbiological culture Collection center (CGMCC) at 10 and 26 months of 2021, and the preservation number is CGMCC NO.23669.
2. The method for culturing bacillus amyloliquefaciens XJ-BV2007 according to claim 1, comprising the steps of:
inoculating strain XJ-BV2007 onto NA solid culture medium with inoculating needle for activation, culturing for 12-24 hr in incubator at 25-40deg.C, picking single colony of Bacillus amyloliquefaciens with inoculating needle, transferring to NB liquid culture medium, culturing for 12-24 hr at 125-175r/min in shaking incubator at 25-40deg.C, transferring culture solution with 1-10% total volume of liquid culture medium into fresh NB liquid culture medium, culturing for 24-36 hr at 125-175r/min at 25-40deg.C to obtain XJ-BV2007 fermentation broth with viable count of (1-9) ×10 5 CFU/mL。
3. The use of bacillus amyloliquefaciens XJ-BV2007 in the control of alternaria alternata-produced contamination of fruit and vegetable alternaria alternata.
4. The application of claim 3, wherein the application comprises the steps of:
the concentration is (1-9). Times.10 5 The fermentation liquor of the bacillus amyloliquefaciens XJ-BV2007 of CFU/mL is directly sprayed on the surfaces of fruits and vegetables.
5. A biocontrol formulation comprising the bacillus amyloliquefaciens XJ-BV2007 of claim 1.
6. The biocontrol formulation of claim 5, wherein the biocontrol formulation is in the form of a liquid, powder spray, dry wettable powder or dry wettable particles.
7. The biocontrol formulation of claim 5, wherein said biocontrol formulation comprises the above XJ-BV2007 fermentation broth.
8. Use of a biocontrol formulation according to any one of claims 5-7 for controlling contamination with alternaria alternata produced fruit and vegetable alternaria alternata.
9. The use according to claim 8, wherein controlling contamination with alternaria alternata production fruit and vegetable alternaria alternata comprises at least one of inhibiting growth of alternaria alternata and inhibiting synthesis of alternaria alternata.
10. The application according to claim 9, wherein the application comprises the steps of:
spraying the biocontrol agent on the surfaces of fruits and vegetables to inhibit Alternaria alternata and Alternaria alternata toxin.
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