CN114015616A - Bacillus amyloliquefaciens XJ-BV2007 as well as culture method and application thereof - Google Patents
Bacillus amyloliquefaciens XJ-BV2007 as well as culture method and application thereof Download PDFInfo
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- bacillus amyloliquefaciens
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- alternaria alternata
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Images
Classifications
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- 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 and a culture method and application thereof, wherein the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) XJ-BV2007 is preserved in 26 months 10 and 2021 in China general microbiological culture Collection center with the preservation number of CGMCC NO.23669, and the Bacillus amyloliquefaciens shows a good inhibition effect on the growth of alternaria alternate in a plate confronting test, also has an inhibition effect on the synthesis of alternaria alternate, and can be applied to biological control of alternaria alternate pollution of fruits and vegetables.
Description
Technical Field
The invention relates to the technical field of prevention and control of fruit and vegetable mycotoxin, and particularly relates to a bacillus amyloliquefaciens XJ-BV2007 as well as a culture method and application thereof.
Background
Tomatoes are generally classified into fresh tomatoes and processed tomatoes, the China is a large producing country of the processed tomatoes, and according to the food and agriculture organization data of the United nations, about 6276 ten thousand tons of tomatoes are provided in China in 2019, which account for about 35% of the global yield, and the demand of the market for tomatoes is gradually increased along with the improvement of the processing and manufacturing level. However, tomatoes are susceptible to contamination by pathogenic microorganisms during the processes of tomato processing, growth, harvesting, storage, transportation, processing and the like, and particularly during storage, the postharvest tomatoes are more rotten by Alternaria alternata (Alternaria alternata), which not only causes great yield loss, but also accumulates Alternaria toxin which is a potential threat to human health in the tomatoes and products thereof. Among them, the toxicity and the detected content of tenuazonic acid (TeA) are at the head of each alternariotoxin, and listed in the registration book of toxic chemicals by the U.S. food and drug administration, Alternariol (AOH) and Alternariol Monomethyl Ether (AME) have acute toxicity and can show synergistic effects (Pinto et al, 2017); the European food safety agency has recommended that AOH and AME be ingested at a daily dose of no more than 2.5ng/kg body weight, and tentoxin (TENTON, TEN) and TeA at no more than 1500ng/kg body weight in food.
At present, the spraying of chemical bactericides is a main measure for preventing and treating tomato fungal diseases and controlling mycotoxin pollution, but is limited by adverse factors such as high carcinogenicity, long degradation period, serious environmental pollution and the like, and the chemical synthetic bactericides are increasingly used in a standard way and even are limited in use. Relatively speaking, the biological bactericide which is safe, environment-friendly and efficient is more scientific, reasonable and urgent.
Disclosure of Invention
In view of the above, the main object of the present invention is to provide a bacillus amyloliquefaciens XJ-BV2007 capable of inhibiting the growth of alternaria alternate (a. alternate) and efficiently inhibiting the production of alternaria toxin, a culture method and applications thereof, which have the effects of inhibiting the growth of alternaria alternate in picked tomatoes and inhibiting the production of alternaria toxin in tomatoes.
The purpose of the invention and the technical problem to be solved are realized by adopting the following technical scheme. According to the Bacillus amyloliquefaciens strain XJ-BV2007 provided by the invention, the strain is preserved in China general microbiological culture Collection center at 26 th 10 th 2021, and the preservation number is CGMCC NO. 23669.
The Bacillus amyloliquefaciens XJ-BV2007 is firstly separated from soil of a tomato processing land in the basin of Yanghuang, and a biocontrol strain XJ-BV2007 with good inhibition effect on alternaria and alternaria toxin in tomatoes is identified as the Bacillus amyloliquefaciens by 16 srRNA.
The purpose of the invention and the technical problem to be solved can be realized by adopting the following technical scheme. The culture method of the bacillus amyloliquefaciens XJ-BV2007 provided by the invention comprises the following steps:
inoculating strain XJ-BV2007 hypha to NA solid culture medium by using an inoculating needle for activation, culturing for 12-24h in an incubator at 25-40 ℃, picking out a single colony of bacillus amyloliquefaciens by using the inoculating needle, transferring to NB liquid culture medium, culturing for 12-24h in a shaking incubator at 25-40 ℃ at the speed of 125-175r/min, sucking culture solution with the total volume of 1-10% of the liquid culture medium, transferring to fresh NB liquid culture medium, culturing for 24-36h at 25-40 ℃ at 125-175r/min to obtain XJ-BV2007 fermentation broth, wherein the viable count reaches (1-9) x 105CFU/mL。
The purpose of the invention and the technical problem to be solved can be realized by adopting the following technical scheme. The application of the bacillus amyloliquefaciens XJ-BV2007 provided by the invention in preventing and treating alternaria alternate contamination of fruits and vegetables.
Preferably, the application of the bacillus amyloliquefaciens XJ-BV2007 in preventing and controlling alternaria alternate contamination of fruits and vegetables is disclosed, wherein the application comprises the following steps:
the concentration is (1-9) × 105CFU/mL fermentation liquid of Bacillus amyloliquefaciens XJ-BV2007 is directly sprayed on the surface of a biological sample.
The purpose of the invention and the technical problem to be solved can be realized by adopting the following technical scheme. The invention provides a biocontrol preparation containing the bacillus amyloliquefaciens XJ-BV 2007.
Preferably, the biocontrol formulation containing the bacillus amyloliquefaciens XJ-BV2007 is in the form of liquid, powder spray, dry wettable powder or dry wettable granules.
Preferably, the biocontrol preparation containing the bacillus amyloliquefaciens XJ-BV2007 contains the XJ-BV2007 fermentation liquor.
Preferably, the biocontrol preparation containing the bacillus amyloliquefaciens XJ-BV2007 is liquid.
Preferably, the biocontrol preparation containing the bacillus amyloliquefaciens XJ-BV2007 contains the XJ-BV2007 fermentation liquor.
Preferably, the biocontrol preparation containing the bacillus amyloliquefaciens XJ-BV2007 is a biocontrol preparation, wherein the final concentration of the bacillus amyloliquefaciens XJ-BV2007 in the biocontrol preparation is (1-9) multiplied by 105CFU/mL。
The purpose of the invention and the technical problem to be solved can be realized by adopting the following technical scheme. The invention provides application of a biocontrol agent in preventing and treating alternaria alternate contamination of fruits and vegetables.
Preferably, the biocontrol agent is used for preventing and controlling alternaria alternata toxin pollution of fruits and vegetables, wherein the prevention and control of alternaria alternata toxin pollution of fruits and vegetables comprises at least one of alternaria alternata growth inhibition or alternaria alternata toxin synthesis inhibition.
Preferably, the application of the biocontrol agent in preventing and controlling the alternaria alternate contamination of fruits and vegetables is implemented, wherein the application comprises the following steps:
the biocontrol agent is uniformly sprayed on the surface of a biological sample to inhibit alternaria alternata and alternaria alternate toxin.
By the technical scheme, the bacillus amyloliquefaciens XJ-BV2007 and the culture method and the application thereof provided by the invention at least have the following advantages:
according to the invention, a biocontrol bacterium XJ-BV2007 with a good inhibition effect on alternaria and alternaria toxin in tomatoes is separated from soil for the first time, the biocontrol bacterium XJ-BV2007 is identified as bacillus amyloliquefaciens through 16srRNA, the thallus of the bacillus amyloliquefaciens XJ-BV2007 shows a good inhibition effect on the growth of alternaria in a plate confronting test, the inhibition rate is 68.40%, and meanwhile, the synthesis inhibition rate on the alternaria toxin TeA on a confronting plate is 40.25%, so that the biocontrol bacterium XJ-BV can be applied to the control of alternaria and alternaria toxin TeA.
When the bacillus amyloliquefaciens XJ-BV2007 and alternaria alternate are cultured in a liquid culture medium, the growth of pathogenic bacteria can be obviously inhibited, the inhibition rate is higher than 95% visually, and the synthesis inhibition rate of the alternaria alternate TeA is as high as 99.71%.
The bacillus amyloliquefaciens XJ-BV2007 can effectively inhibit the growth of alternaria alternata on tomatoes under the condition of normal-temperature storage, the inhibition rate reaches 88.04%, the prevention effect of the bacillus amyloliquefaciens XJ-BV2007 on alternaria alternata TeA pollution in tomatoes can reach 93.28%, the prevention and control effect is good, and therefore the bacillus amyloliquefaciens XJ-BV2007 can be used for preventing and controlling the alternaria solani pollution.
The bacillus amyloliquefaciens XJ-BV2007 is applied to prevention and control of alternaria solani pollution, and can overcome a series of problems caused by the use of chemical pesticides, so that the bacillus amyloliquefaciens is beneficial to pollution-free production of agricultural products and improves the product quality.
The foregoing is a summary of the present invention, and in order to provide a clear understanding of the technical means of the present invention and to be implemented in accordance with the present specification, the following is a detailed description of the preferred embodiments of the present invention.
Drawings
FIG. 1 is a graph showing the bacteriostatic properties of Bacillus amyloliquefaciens XJ-BV2007, Bacillus atrophaeus XJ-BV2001, Bacillus licheniformis XJ-BV2004, Bacillus altitudinis XJ-BV2006 and a control CK (only alternaria strain inoculated with no antagonistic bacteria) in example 1 of the present invention;
FIG. 2 is a colony morphology of Bacillus amyloliquefaciens XJ-BV2007 in example 1 of the present invention;
FIG. 3 shows the rDNA gene developmental clade of Bacillus amyloliquefaciens XJ-BV2007 in example 1 of the present invention;
FIG. 4 is a graph showing the antagonistic action of Bacillus amyloliquefaciens XJ-BV2007 on Alternaria alternata in a liquid medium in example 2 of the present invention (upper graph is a control group: only Alternaria alternata is liquid-cultured; lower graph is an experimental group: both Bacillus amyloliquefaciens XJ-BV2007 and Alternaria alternata are liquid-cultured)
FIG. 5 is a graph showing the antagonistic action of Bacillus amyloliquefaciens XJ-BV2007 on processed alternaria solani in example 3 of the present invention (upper graph is a control group: only alternaria alternata was inoculated; lower graph is an experimental group: Bacillus amyloliquefaciens XJ-BV2007 and alternaria alternata were inoculated at the same time).
Detailed Description
To further illustrate the technical means and effects of the present invention for achieving the predetermined objects, the following detailed description will be given to specific embodiments, structures, characteristics and effects of a bacillus amyloliquefaciens XJ-BV2007 and a culture method and an application thereof according to the present invention with reference to preferred embodiments.
The invention provides a strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) XJ-BV2007 which is preserved in China general microbiological culture Collection center (CGMCC for short, with the address of CGMCC, the institute of microbiology, China academy of sciences, 3, institute of Ministry of sciences, North road, No.1, North Cheng, south China, Beijing City, 10.26.1.8978, the preservation number is CGMCC NO.23669, and the strain is named as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) in classification.
The Bacillus amyloliquefaciens XJ-BV2007 is separated from soil of a tomato processing land in the basin of Yanqi in Xinjiang for the first time, is a biocontrol strain XJ-BV2007 with good inhibition effect on alternaria alternate (A.alternate) and alternaria toxin in tomatoes, is identified as the Bacillus amyloliquefaciens by 16srRNA, shows good inhibition effect on the growth of alternaria alternate in a solid plate confronting test, has the inhibition rate of 68.40 percent, and has the inhibition rate of 40.25 percent on alternaria tenuifolia keto acid (TeA) which is one of the alternaria alternate. In a liquid culture test, the compound also shows better inhibition effect on the growth of alternaria alternata, the inhibition rate is more than 95 percent visually, and the inhibition rate on alternaria alternata TeA is 99.71 percent.
The invention also provides a culture method of the bacillus amyloliquefaciens XJ-BV2007, which comprises the following steps:
inoculating strain XJ-BV2007 hypha to NA solid culture medium by using an inoculating needle for activation, culturing for 12-24h in an incubator at 25-40 ℃, picking out a single colony of bacillus amyloliquefaciens by using the inoculating needle, transferring to NB liquid culture medium, culturing for 12-24h in a shaking incubator at 25-40 ℃ at the speed of 125-175r/min, sucking culture solution with the total volume of 1-10% of the liquid culture medium, transferring to fresh NB liquid culture medium, culturing for 24-36h at 25-40 ℃ at 125-175r/min to obtain XJ-BV2007 fermentation broth, wherein the viable count reaches (1-9) x 105CFU/mL。
The invention also provides application of the bacillus amyloliquefaciens XJ-BV2007 in prevention and treatment of alternaria alternate contamination of fruits and vegetables.
In some embodiments of the invention, wherein the applying may comprise the steps of:
the concentration is (1-9) × 105CFU/mL fermentation liquid of Bacillus amyloliquefaciens XJ-BV2007 is directly sprayed on the surface of a biological sample, so as to prevent and control alternaria toxin pollution of the biological sample. The spraying manner in this embodiment may be spraying by an automatic sprayer (the spraying rate may be 0.5L/min, for example), or spraying by a plant protection unmanned aerial vehicle (the spraying rate may be 0.43L/min (a single nozzle, taking water as an example), and is not limited specifically herein.
In some embodiments of the invention, wherein the applying may comprise the steps of:
directly inoculating the surface of the biological sample with the concentration of (1-9) × 105CFU/mL of fermentation broth of Bacillus amyloliquefaciens XJ-BV2007, so as to prevent and control alternaria toxin pollution of biological samples. The amount of the 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 is in the form of a liquid, a dusting powder, a dry wettable powder or a dry wettable granule. In view of cost saving, the form of the biocontrol agent is preferably made in the form of a liquid.
In some embodiments of the invention, wherein the biocontrol formulation comprises the XJ-BV2007 fermentation broth described above.
In some embodiments of the invention, wherein the final concentration of bacillus amyloliquefaciens XJ-BV2007 in the biocontrol formulation is (1-9) × 105CFU/mL。
The invention also provides application of the biocontrol agent in preventing and treating alternaria alternate contamination of fruits and vegetables.
In some embodiments of the present invention, wherein the controlling alternaria alternata toxin contamination in fruits and vegetables may comprise at least one of inhibiting alternaria growth or inhibiting alternaria synthesis, such as inhibiting alternaria TeA synthesis.
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 the whole fruit sample, particularly on the parts susceptible to alternaria alternata, such as fruit stalks, fruit bottoms and the like, so as to prevent alternaria alternata toxin pollution of the biological sample. The spraying manner in this embodiment may be spraying by an automatic sprayer (the spraying rate may be 0.5L/min, for example), or spraying by a plant protection unmanned aerial vehicle (the spraying rate may be 0.43L/min (a single nozzle, taking water as an example), and is not limited specifically herein.
Unless otherwise specified, the following materials, reagents and the like are commercially available products well known to those skilled in the art; unless otherwise specified, all methods are well known in the art. Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.
The present invention will be further described with reference to the following specific examples, which should not be construed as limiting the scope of the invention, but rather as providing those skilled in the art with certain insubstantial modifications and adaptations of the invention based on the teachings of the invention set forth herein.
Example 1 isolation, screening and identification of Bacillus amyloliquefaciens XJ-BV2007
1. Separating and screening strains
Separating antagonistic bacteria from the soil of the tomato field: weighing 5g of soil, adding 45mL of sterile water, shaking the soil at 27 ℃, culturing the soil at 150r/min for 30min, and carrying out shake culture on the obtained liquid by 10-1-10-6By gradient dilution of (2), pipetting 100. mu.L, 10. mu.L, respectively-1-10-6The dilution of (6) was applied to NA medium and cultured in an incubator (150r/min) at 27 ℃ for 24 hours. Individual colonies on the plates were picked and streaked on NA medium.
And screening the antagonistic ability by adopting a plate confronting method. Placing a 7-day-old Alternaria alternata mycelium disk with the diameter of 5mm (the strain is purchased from China agricultural microbial strain preservation management center and is numbered ACCC 37489) in the center of a PDA culture medium, inoculating antagonistic bacteria with the diameter of 5mm on the same flat plate at a position 2cm away from the Alternaria alternata mycelium disk, observing whether the growth of the mycelium is inhibited or not by taking the flat plate which is not inoculated with the antagonistic bacteria and is only inoculated with the Alternaria alternata as a control, measuring the diameter of the Alternaria alternata on the 7 th day, and calculating the inhibition rate according to the radial growth diameter, wherein each strain has three copies. Selecting the bacterial strain with the bacteriostatic rate of more than 60 percent, and storing for later use.
2. Strain re-screening
1) Preparation of strain XJ-BV2007 fermentation liquor
The antibacterial rate is measured by an inoculating needle>Inoculating 60% of strain XJ-BV2007 to an NA solid culture medium for activation, culturing for 24h in an incubator at 37 ℃, picking a single colony of bacillus amyloliquefaciens by using an inoculating needle, transferring to an NB culture medium, culturing for 24h at a speed of 150r/min in a shaking incubator at 37 ℃, sucking 5% of the total volume of a liquid culture medium, transferring to a fresh NB culture medium, culturing for 36h at a speed of 150r/min in the shaking incubator at 37 ℃ to obtain a fermentation liquid of antagonistic strain XJ-BV2007, wherein the viable count (concentration) is 5 × 105CFU/mL。
2) Determination of bacteriostatic Properties
And screening the antagonistic capacity of the fermentation liquor of the antagonistic bacteria by adopting a flat plate confronting method. Placing a 7-day-old 5 mm-diameter Alternaria alternata mycelium disk (the strain is purchased from China agricultural microorganism culture Collection center and is numbered ACCC 37489) in the center of PDA culture medium, digging off 5 mm-diameter PDA culture medium in 4 directions at a distance of 2cm from the mycelium disk on the same plate, and adding 200 μ L of antagonistic bacteria fermentation broth (the viable count is 5 × 10)5CFU/mL) as experimental group XJ-BV 2007; similarly, digging out PDA culture medium with diameter of 5mm from 4 directions 2cm away from the mycelium disk on the other 3 same plates, respectively, adding 200 μ L Bacillus atrophaeus fermentation broth (viable count of 5 × 10)5CFU/mL), 200 μ L Bacillus licheniformis fermentation liquid (viable count 5 × 10)5CFU/mL), 200. mu.L of Geobacillus altivelis fermentation broth (viable count 5X 10)5CFU/mL) as experimental group XJ-BV2001, experimental group XJ-BV2004 and experimental group XJ-BV2006, and using a plate which is not inoculated with antagonistic bacteria and only inoculated with alternaria strain as a control group CK to observe bacterial plaqueWhether growth was inhibited or not was determined by taking the diameter of alternaria alternata on day 7 and calculating the inhibition rate from its radial growth diameter, in triplicate for each strain. The test results are shown in FIG. 1 and Table 1. Wherein, the experimental group XJ-BV2001 shows that Alternaria alternata strain and Bacillus atrophaeus are inoculated at the same time; experimental group XJ-BV2004 shows the inoculation of both Alternaria strain and Bacillus licheniformis; experimental group XJ-BV2006 shows the inoculation of Alternaria alternata strain and Bacillus altitudinis at the same time; experimental group XJ-BV2007 shows the inoculation of Alternaria strain and Bacillus amyloliquefaciens simultaneously.
The inhibition ratio (%) - (C1-C2)/C1X 100%
C1 diameter of radial growth of Alternaria alternata of control group
C2 diameter of radial growth of Alternaria alternata of Experimental group
TABLE 1 Flat-plate confrontation rescreening
As can be seen from the experimental results shown in FIG. 1 and Table 1, the inhibition rate of the XJ-BV2001 experimental group on the growth of Alternaria alternata strain is about 64.51%, the inhibition rate of the XJ-BV2004 on the growth of Alternaria alternata strain is about 60.19%, the inhibition rate of the XJ-BV2006 on the growth of Alternaria alternata strain is about 60.76%, and the inhibition rate of the XJ-BV2007 on the growth of Alternaria alternata strain is about 68.40%, which shows that Bacillus atrophaeus, Bacillus licheniformis paroeniformis, Bacillus altidinis and Bacillus amyloliquefaciens all have the capability of inhibiting the growth of Alternaria alternata and have the best inhibition capability of Bacillus amyloliquefaciens.
3) Toxicity inhibiting assay
Placing the culture medium on the 8 th balance plate of the experimental group XJ-BV2007 in the 2) in a mortar, adding about 75mL of liquid nitrogen to quickly grind and crush the culture medium to 200 meshes, extracting the culture medium by using a dispersive solid-phase extraction (QuEChERS) and an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method, and detecting the content of alternaria toxin in the culture medium, wherein the inhibition rate of the experimental group XJ-BV2007 on alternaria toxin TeA is 40.25% compared with the control group CK.
3. Morphological characteristics
Bacterial colony of the strain XJ-BV2007 on an 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 morphology of the bacterial colony is shown in figure 1; the microscopic morphology is shown in FIG. 2, gram-positive, rod-like, spore-forming.
4. Physiological and biochemical characteristics
The strain XJ-BV2007 is suitable for growing on Nutrient Agar (NA) and Nutrient Broth (NB) culture media, the suitable temperature range for growth is 4-45 ℃, the optimum temperature is 37 ℃, the suitable pH value for growth is 3-9, and the optimum pH value is 7.3.
5. Characteristics of molecular biology
Extracting DNA of antagonistic bacteria XJ-BV2007 by using a bacterial DNA extraction kit according to the instruction, and performing PCR amplification by using the DNA as a template and 16S rDNA universal primers 27F (5 '-AGAGTTGATVATGGCTCAG-3') and 1492R (5'-CTACGGTTACCTTGTTACGAC-3'), wherein the amplification conditions are as follows: at 94 ℃ for 260 seconds, at 55 ℃ for 20 seconds and at 72 ℃ for 700 seconds, and finally cooled to 4 ℃ for 35 cycles. The obtained fragment is submitted to Shanghai workers for sequence determination to obtain a gene with the length of 1362bp, adjacent species are analyzed by BLAST on NCBI, and a phylogenetic tree is constructed by using MEGA7.0, as shown in figure 3, the homology of antagonistic bacteria XJ-BV2007 and SGD isolate 4(Bacillus amyloliquefaciens partial 16S rRNA gene strain SGD isolate 4) of a 16S rRNA gene of a Bacillus amyloliquefaciens part reaches 96%, and the strain XJ-BV2007 can be finally identified as the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) by combining morphological characteristics, physiological and biochemical characteristics of the strain and the result of the BLAST system analysis. The 16S rDNA sequence of the strain XJ-BV2007 is shown in SEQ ID NO. 1.
Example 2 test of the Effect of Bacillus amyloliquefaciens XJ-BV2007 on Alternaria alternata and its toxins on liquid culture Medium
The viable cell count (concentration) of 6mL in example 1 was set to 9X 105CFU/mL Bacillus amyloliquefaciens XJ-BV2007 fermentation broth and 6mL of 9X 105CFU/mL Alternaria alternata spore suspension is mixed and added into 6mL Potato Dextrose Broth (PDB) liquid culture medium (the liquid composition of a control group and an experimental group is shown in Table 2), the mixture is cultured for 5 days at the temperature of 28 ℃ at 120r/min by a shaking table, the growth condition of pathogenic bacteria is observed, QuEChERS-UPLC-MS/MS method is used for extracting and detecting Alternaria alternata toxin TeA in the mixed culture solution, each group is carried out in 3 parallels, the inhibition rate is calculated according to the following formula, and the test result is shown in Table 4 and Table 3.
Toxin inhibition (%) is ═ 100% of control peak area-experimental peak area)/control peak area
TABLE 2 test grouping of Bacillus amyloliquefaciens XJ-BV2007 on liquid culture Medium
TABLE 3 inhibition of TeA by antagonism 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 obvious inhibition effect on the growth of liquid culture Alternaria alternata, and the inhibition effect can only be visually observed by phenomena to exceed 95% because the inhibition rate cannot be directly calculated by liquid.
As shown in Table 3, the QuEChERS-UPLC-MS/MS method is used for detecting the content of the alternaria toxin TeA in the shake culture liquid on the 5 th day, and compared with the CK of the control group, the inhibition rate of the XJ-BV2007 bacterial suspension in the experimental group on the alternaria toxin TeA reaches 99.71%.
Example 3 application of Bacillus amyloliquefaciens XJ-BV2007 in prevention and treatment of alternaria toxin contamination of fruits and vegetables
Pricking mature fresh processed tomato (tested processed tomato variety is Henschel 1015, harvested in two six towns of Changji City of Changji Hui) with needle at 5mm depth, inoculating 10 μ L of live bacteria number of 5 × 105CFU/mL XJ-BV2007 bacterial suspension, 10 mu L of sterile water is inoculated as a control group, and after 30min, 10 mu L of 1 × 10 bacterial suspension is inoculated on the wound of each of the experimental group CK and the control group XJ-BV2007 (each of 5 tomatoes)5CFU/mL Alternaria spore suspension. All tomatoes were placed at room temperature of 37 ℃, the diameter of the lesions was measured on day 8 of culture, and alternarin TeA in the test tomatoes was extracted using the QuEChERS-UPLC-MS/MS method, 5 tomatoes per group were inoculated. The inhibition ratios were calculated according to the following formulas (1) and (2), and the test results are shown in table 4, table 5 and fig. 5.
Fungal growth inhibition (%) - (C1-C2)/C1X 100% (1)
Toxin inhibition (%) (control peak area-experimental peak area)/control peak area x 100% (2)
C1 radial growth diameter of control group Alternaria alternata
C2 diameter of radial growth of Alternaria alternata of Experimental group
TABLE 4 inhibition of growth of Alternaria solani 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 alternaria alternata on processed tomatoes is about 88.04%, indicating that the bacillus amyloliquefaciens has a good effect on inhibiting the growth of alternaria alternata on tomatoes.
As shown in Table 5, the content of alternaria toxin TeA in processed tomatoes on day 8 is detected by using a QuEChERS-UPLC-MS/MS method, and compared with a control group CK, the inhibition rate of an experimental group XJ-BV2007 on the alternaria toxin TeA reaches 93%.
TABLE 5 inhibition of TeA on tomato 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 tomato does not substantially grow any longer.
In the description of the present invention, numerous specific details are set forth. It is understood, however, 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.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention are still within the scope of the technical solution of the present invention.
SEQUENCE LISTING
<110> Sinkiang academy of agricultural sciences
<120> Bacillus amyloliquefaciens XJ-BV2007 as well as 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. A strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) XJ-BV2007 was deposited in China general microbiological culture Collection center (CGMCC) at 26 months 10 in 2021 with the deposit number of CGMCC NO. 23669.
2. The method for culturing bacillus amyloliquefaciens XJ-BV2007 according to claim 1, which is characterized by comprising the following steps:
inoculating strain XJ-BV2007 to NA solid culture medium by using an inoculating needle for activation, culturing for 12-24h in an incubator at 25-40 ℃, picking out a single colony of bacillus amyloliquefaciens by using the inoculating needle, transferring to NB liquid culture medium, culturing for 12-24h in a shaking incubator at 25-40 ℃ at the speed of 125-175r/min, sucking culture solution with the total volume of 1-10% of the liquid culture medium, transferring to fresh NB liquid culture medium, culturing for 24-36h at 25-40 ℃ at 125-175r/min to obtain XJ-2007 fermentation broth, wherein the viable count reaches (1-9) x 10 BV5CFU/mL。
3. The use of bacillus amyloliquefaciens XJ-BV2007 in the prevention and treatment of alternaria alternate contamination of fruits and vegetables according to claim 1.
4. The application of claim 3, wherein the application comprises the steps of:
the concentration is (1-9) × 105CFU/mL fermentation liquid of Bacillus amyloliquefaciens XJ-BV2007 is directly sprayed on the surface of a biological sample.
5. A biocontrol formulation containing bacillus amyloliquefaciens XJ-BV2007 as claimed in claim 1.
6. The biocontrol formulation of claim 5, wherein said biocontrol formulation is in the form of a liquid, dusting powder, dry wettable powder or dry wettable granules.
7. The biocontrol formulation of claim 5, wherein said biocontrol formulation comprises the XJ-BV2007 fermentation broth described above.
8. Use of a biocontrol agent according to any one of claims 5-7 for controlling alternaria alternata toxin contamination of fruits and vegetables.
9. The use of claim 8, wherein the control of alternaria alternata toxin contamination in fruits and vegetables comprises at least one of inhibition of alternaria alternata growth and inhibition of alternaria alternata toxin synthesis.
10. The application of claim 9, wherein the application comprises the steps of:
the biocontrol agent is sprayed on the surface of a biological sample to inhibit alternaria alternata and alternaria alternata toxin.
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