CN114921368B - Bacillus mojavensis YL-RY0310 and culture method and application thereof - Google Patents
Bacillus mojavensis YL-RY0310 and culture method and application thereof Download PDFInfo
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- 241001249117 Bacillus mojavensis Species 0.000 title claims abstract description 80
- 238000012136 culture method Methods 0.000 title abstract description 10
- ZRWPUFFVAOMMNM-UHFFFAOYSA-N Patulin Chemical compound OC1OCC=C2OC(=O)C=C12 ZRWPUFFVAOMMNM-UHFFFAOYSA-N 0.000 claims abstract description 119
- 241001123663 Penicillium expansum Species 0.000 claims abstract description 43
- 230000012010 growth Effects 0.000 claims abstract description 27
- 235000012055 fruits and vegetables Nutrition 0.000 claims abstract description 18
- 238000009629 microbiological culture Methods 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 20
- 239000001963 growth medium Substances 0.000 claims description 18
- 230000000443 biocontrol Effects 0.000 claims description 15
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- 108020004414 DNA Proteins 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 240000008866 Ziziphus nummularia Species 0.000 description 2
- 235000015197 apple juice Nutrition 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
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- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 1
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- 244000241257 Cucumis melo Species 0.000 description 1
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 231100000678 Mycotoxin Toxicity 0.000 description 1
- 241001236817 Paecilomyces <Clavicipitaceae> Species 0.000 description 1
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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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
-
- 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
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- Chemical & Material Sciences (AREA)
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- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Nutrition Science (AREA)
- Genetics & Genomics (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
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- Environmental Sciences (AREA)
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Abstract
The invention provides a new separated Bacillus mojavensis (Bacillus mojavensis) YL-RY0310, a culture method and application thereof, wherein the Bacillus mojavensis (Bacillus mojavensis) YL-RY0310 is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.24646 at 4-6-2022, and the Bacillus mojavensis shows better inhibition effect on the growth of penicillium expansum in a plate counter test, and simultaneously has inhibition effect on the synthesis of patulin, so that the Bacillus mojavensis can be applied to biological control of the patulin pollution of fruits and vegetables.
Description
Technical Field
The invention relates to the technical field of prevention and control of mycotoxins of fruits and vegetables, in particular to bacillus mojavensis YL-RY0310 and a culture method and application thereof.
Background
China is the largest apple producing country worldwide, the yield accounts for more than 50% of the global apple yield, and is the fruit variety with the largest yield except melons, and has important roles in the fruit industry in China. However, the penicillium of apples caused by penicillium expansum is serious during picking, storing and transporting apples, and a large amount of Patulin (Patulin, PAT, C) which is a potential threat to human health is accumulated in the fruits while causing huge economic loss 7 H 6 O 4 )。
PAT was reported by the World Health Organization (WHO) as early as 2005 to be a genotoxic compound, and both WHO and the United states food and drug administration (USAFDA) limited the detection of PAT in foods to 50 μg/kg. Since pregnant women and infants belong to a high risk group exposed to PAT, the European Commission in Japan has increased this standard to 10. Mu.g/kg, and has limited the maximum allowable daily intake of PAT to 0.4. Mu.g/kg. Although the apple planting area and the apple yield in China are the first place in the world throughout the year, the export quantity is strictly limited due to the toxin detection rate. Studies have shown that 8% of the 25 apple juice samples detected the presence of patulin; the pollution rate of the patulin in apple juice samples in different processing seasons of Shaanxi reaches 96.7-100%, and the level of the patulin is about 6.30-8.90 mug/kg; in recent two years, researchers have investigated 137 fruit products sold and consumed in the market of China, and the pollution rate of the spanned penicillin is 30.7%, and the concentration range of the spanned penicillin is from 10 mu g/kg to 276.9 mu g/kg. Therefore, prevention and control of the pollution of the patulin to fruits and vegetables are the urgent problem to be solved.
Chemical bactericides have been an important method commonly used in preventing and controlling pathogen infection of fruits, but with intensive research, most chemical bactericides are found to have potential carcinogenic risks, and long-term use of the chemical bactericides can further exacerbate environmental pollution and cause pathogen resistance. In comparison, the adoption of the safe, environment-friendly and efficient biological bactericide is more scientific, reasonable and urgent.
Disclosure of Invention
Accordingly, the main object of the present invention is to provide bacillus mojavensis YL-RY0310 which can inhibit the growth of penicillium expansum (P.expansum) and inhibit the production of patulin with high efficiency, and a culture method and application thereof, which have the effects of inhibiting the growth of penicillium expansum in apples after harvest and inhibiting the production of patulin in vivo.
The aim and the technical problems of the invention are realized by adopting the following technical proposal. The bacillus mojavensis (Bacillus mojavensis) YL-RY0310 provided by the invention is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 24646 in 2022, 4 and 6.
The bacillus mojavensis (Bacillus mojavensis) YL-RY0310 is a biocontrol strain YL-RY0310 which is separated from the soil of the jujube garden of Xinjiang and Tian Kunyu groups and has good inhibition effect on penicillium expansum and patulin in apples for the first time, and is identified as bacillus mojavensis by 16S rRNA.
The aim and the technical problems of the invention can be achieved by adopting the following technical proposal. The culture method of the bacillus mojavensis YL-RY0310 provided by the invention comprises the following steps:
inoculating strain YL-RY0310 onto NA solid culture medium with inoculating needle for activation, culturing in 25-40deg.C incubator for 24-48 hr, picking single colony of Bacillus mojavensis with inoculating needle, transferring to NB liquid culture medium, culturing in 25-40deg.C shaking incubator at 125-150r/min for 12-24 hr, sucking culture solution 1-5% of total volume of liquid culture medium, transferring to fresh NB liquid culture medium, and culturing at 25-40deg.C at 125-150r/minCulturing for 24-72 hr to obtain YL-RY0310 fermentation liquor, and the viable count reaches (1-10) x 10 7 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 mojavensis YL-RY0310 in preventing and treating fruit and vegetable patulin pollution.
Preferably, the application of the bacillus mojavensis YL-RY0310 in preventing and treating the patulin pollution of fruits and vegetables, wherein the application comprises the following steps:
the concentration is (1-10) multiplied by 10 7 The fermentation broth of CFU/mL Bacillus mojavensis YL-RY0310 was sprayed directly onto the surface of the biological sample.
The aim and the technical problems of the invention can be achieved by adopting the following technical proposal. The biocontrol agent provided by the invention contains the bacillus mojavensis YL-RY 0310.
Preferably, the biocontrol agent containing the bacillus mojavensis YL-RY0310 is in the form of liquid, powder spraying, dry wettable powder or dry wettable particles.
Preferably, the biocontrol agent containing the bacillus mojavensis YL-RY0310 contains the YL-RY0310 fermentation liquid.
Preferably, the biocontrol agent containing the bacillus mojavensis YL-RY0310 is liquid.
Preferably, the biocontrol agent containing the bacillus mojavensis YL-RY0310 contains the YL-RY0310 fermentation liquid.
Preferably, the aforementioned biocontrol agent containing the above Bacillus mojavensis YL-RY0310, wherein the final concentration of the Bacillus mojavensis YL-RY0310 in the biocontrol agent is (1-10). Times.10 7 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 a biocontrol agent in preventing and treating fruit and vegetable patulin pollution.
Preferably, the use of the aforementioned biocontrol formulation for controlling fruit and vegetable patulin contamination, wherein said controlling fruit and vegetable patulin contamination comprises at least one of inhibiting growth of penicillium expansum or inhibiting patulin synthesis.
Preferably, the application of the biocontrol agent in preventing and treating the fruit and vegetable patulin pollution comprises the following steps:
the biocontrol agent is uniformly sprayed on the surface of a biological sample to inhibit penicillium expansum and patulin.
By means of the technical scheme, the bacillus mojavensis YL-RY0310 and the culture method and application thereof provided by the invention have at least the following advantages:
the biocontrol strain YL-RY0310 with good inhibition effect on the penicillium expansum and the patulin in apples is separated from soil for the first time, and is identified as bacillus mojavensis by 16S rRNA, and the thallus of the bacillus mojavensis YL-RY0310 has good inhibition effect on the growth of the penicillium expansum in a plate counter test, the inhibition rate is 62.57%, and meanwhile, the synthesis inhibition rate on the patulin PAT on the counter plate is 99.81%, so that the biocontrol strain can be applied to the control of the penicillium expansum and the patulin PAT.
When the bacillus mojavensis YL-RY0310 and the penicillium expansum 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 patulin PAT is up to 71.66%.
The bacillus mojavensis YL-RY0310 can effectively inhibit the growth of the penicillium expansum on apples under the normal-temperature storage condition, the inhibition rate reaches 64.71%, the control effect of the bacillus mojavensis YL-RY0310 on the patulin PAT pollution in apples can reach 58.01%, and the control effect is good, so that the bacillus mojavensis YL-RY0310 can be used for controlling the pollution of the patulin in apples.
The bacillus mojavensis YL-RY0310 is applied to the prevention and control of the penicillium expansium pollution, and can overcome a series of problems caused by the use of chemical pesticides, thereby being beneficial to the pollution-free production of agricultural products and improving the product quality.
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 properties of Bacillus mojavensis YL-RY0310, and YL-RY0304, YL-RY0309, YL-RA0201, YL-RA0204, YL-RA0208, YL-RA0210, and control group CK (which is not inoculated with antagonistic bacteria but only with penicillium expansum strain) of example 1 of the present invention;
FIG. 2 colony morphology of Bacillus mojavensis YL-RY0310 of example 1 of the present invention;
FIG. 3 a 16S rRNA gene developmental tree of Bacillus mojavensis YL-RY0310 of example 1 of the present invention;
FIG. 4 is a graph showing antagonism of Bacillus mojavensis YL-RY0310 in the liquid medium against Penicillium expansum (CK is a control group: liquid culture of Penicillium expansum alone; experimental group is a mixed solution of YL-RY0310 bacterial suspension and Penicillium expansum) according to example 2 of the present invention
FIG. 5 is a graph showing antagonism of Bacillus mojavensis YL-RY0310 against Penicillium expansium in example 3 of the present invention.
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 specific implementation, structure, characteristics and effects of a bacillus mojavensis YL-RY0310, a culture method and an application thereof according to the present invention in combination with the preferred embodiments.
The invention provides a bacillus mojavensis (Bacillus mojavensis) YL-RY0310 which has been preserved in China general microbiological culture Collection center (CGMCC, address: national institute of microbiology, national academy of sciences, 3 rd, national academy of sciences, 1 st area, G.Chen) in 2022, 4 months and 6 days, with a preservation number of CGMCC NO.24646, and classified and named as bacillus mojavensis (Bacillus mojavensis).
The bacillus mojavensis (Bacillus mojavensis) YL-RY0310 is separated from soil of a jujube garden with Xinjiang and Tian Kunyu groups for the first time, the biocontrol strain YL-RY0310 with good inhibition effect on penicillium expansum (P.expansum) and patulin in apples is identified as bacillus mojavensis by 16S rRNA, and the bacillus mojavensis has good inhibition effect on the growth of penicillium expansum in a solid plate counter test, the inhibition rate is 62.57%, and the inhibition rate on PAT is 99.81%. In the liquid culture test, the inhibition effect on the growth of the penicillium expansum is better, the inhibition rate is more than 95% visually, and the inhibition rate on the patulin is 71.66%.
The invention also provides a culture method of the bacillus mojavensis YL-RY0310, which comprises the following steps:
inoculating strain YL-RY0310 onto NA solid culture medium with inoculating needle for activation, culturing in 25-40deg.C incubator for 24-48 hr, picking single colony of Bacillus mojavensis with inoculating needle, transferring to NB liquid culture medium, culturing in 25-40deg.C shaking incubator at 125-150r/min for 12-24 hr, sucking culture solution 1-5% of total volume of liquid culture medium, transferring to fresh NB liquid culture medium, culturing at 25-40deg.C at 125-150r/min for 24-72 hr to obtain YL-RY0310 fermentation broth with viable count reaching (1-10) ×10 7 CFU/mL。
The invention also provides an application of the bacillus mojavensis YL-RY0310 in preventing and treating patulin pollution in fruits and vegetables.
In some embodiments of the invention, wherein the application may comprise the steps of:
the concentration is (1-10) multiplied by 10 7 The fermentation liquor of CFU/mL bacillus mojavensis YL-RY0310 is directly sprayed on the surface of the biological sample, so as to prevent and treat the patulin pollution of the biological sample. 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 (1-10) x 10 7 CFU/mL fermentation broth of Bacillus mojavensis YL-RY0310, to control patulin contamination 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 YL-RY 0310.
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 above-described YL-RY0310 fermentation broth.
In some embodiments of the present invention, wherein the final concentration of Bacillus mojavensis YL-RY0310 in the biocontrol agent is (1-10). Times.10 7 CFU/mL。
The invention also provides application of the biocontrol agent in preventing and treating the fruit and vegetable patulin pollution.
In some embodiments of the invention, wherein said controlling patulin contamination of fruits and vegetables may comprise at least one of inhibiting patulin growth or inhibiting patulin synthesis, such as inhibiting patulin synthesis.
In some embodiments of the invention, wherein the applying comprises the steps of:
the biocontrol preparation 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 and spread penicillium, so as to prevent and treat the patulin pollution of the biological sample. 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 identification of Bacillus mojavensis YL-RY0310
1. Bacterial separation and screening
Isolation of antagonistic bacteria from date garden soil and Tian Kunyu 244 clusters: evenly separating by a quartering method, weighing 5g of soil, adding 45mL of sterile water, shaking and culturing at 27 ℃ for 30min at 150r/min, and subjecting the obtained liquid to 10 -1 -10 -6 Is diluted in a gradient of 100. Mu.L, 10 -1 -10 -6 The dilutions (6 gradients) were plated onto NA medium and incubated for 24h in an incubator at 27 ℃. Individual colonies on the plates were picked and streaked on NA medium.
1) Determination of hypha growth inhibition rate
The antagonistic ability was screened by the plate counter method. And (3) activating the separated and purified strain on the NA culture medium for 24 hours, gently scraping single colony on the surface of the flat plate by using an inoculating loop, inoculating the single colony into the NB culture medium, and carrying out liquid shake culture at 28 ℃ and 130r/min for 24 hours to obtain a strain suspension. Preparing spore suspension from activated penicillium expansum (strain purchased from China center for type culture collection of agricultural microorganisms, number ACCC 36188), sucking 10 μl of raw liquid drop at the center of NA culture medium, and inoculating 10 μl of activated antagonistic bacterial suspension on the same plate at a distance of 2.5cm from the center for 24 hr; the control was a plate without antagonistic bacteria inoculated with only penicillium expansum, and it was observed whether the hypha growth was inhibited, the diameter of penicillium expansum on day 7 was measured, and the inhibition rate was calculated from the radial growth diameter thereof, in triplicate for each strain. Selecting the strain with the highest bacteriostasis rate, and preserving for later use. The inhibition rate was calculated according to the following formula, and the test results are shown in Table 1 and FIG. 1
Inhibition (%) = (C1-C2)/c1×100%
C1 radial growth diameter of Penicillium expansum of control group
C2 radial growth diameter of Penicillium expansum of the experimental group
Table 1: inhibition effect of penicillium expansum on flat plate
As can be seen from the experimental results in FIG. 1 and Table 1, the growth inhibition rate of YL-RY0304 experimental group against the strain of Penicillium expansum was about 37.45%, the growth inhibition rate of YL-RY0309 experimental group against the strain of Penicillium expansum was about 60.60%, the growth inhibition rate of YL-RY0310 against the strain of Penicillium expansum was about 62.57%, the growth inhibition rate of YL-RA0201 against the strain of Penicillium expansum was about 42.75%, the growth inhibition rate of YL-RA0204 experimental group against the strain of Penicillium expansum was about 57.82%, the growth inhibition rate of YL-RA0208 experimental group against the strain of Penicillium expansum was about 53.94%, and the growth inhibition rate of YL-RA0210 against the strain of Penicillium expansum was about 57.37%. Indicating that YL-RY0309, YL-RA0204, YL-RA0208, YL-RA0210 and Bacillus mojavensis Bacillus mojavensis YL-RY0310 all have the ability to inhibit the growth of penicillium, and the inhibition ability of Bacillus mojavensis Bacillus mojavensis YL-RY0310 is optimal.
2) Determination of toxicity inhibition Property of Flat mycelium growth
Placing the plate culture medium with the bacteriostasis rate of more than 50% and mycelium growth of 7 days in a mortar, adding about 75mL of liquid nitrogen, quickly grinding and crushing to 200 meshes, uniformly mixing the parallel groups, randomly sampling, and extracting and detecting the content of the patulin by referring to the method of patulin determination GB5009.185-2016 in food safety national standard food.
The conditions are as follows: using an XSelect HSS T3 column (5 μm, 4.6X1250 mm), the mobile phase was water and acetonitrile (77:23, v/v), flow rate 1.0mL/min, isocratic elution. The column temperature was 40℃and the run time of each sample was 10min with a chromatographic peak of 276nm. Meanwhile, a standard curve is manufactured according to the patulin standard substances with different concentrations, the toxin content in each experimental group is calculated according to the standard curve, and the inhibition rate is calculated. The toxin content and inhibition rate were calculated according to the following formulas (1) and (2), and the test results are shown in Table 2.
y＝56.828x-637.32 R 2 ＝0.9992 (1)
y: peak area x: concentration (ng/ml)
Toxin inhibition (%) = (control toxin content-experimental toxin content)/control toxin content x 100% (2)
Table 2: inhibition of patulin in plates
As can be seen from Table 2, the experimental groups YL-RY0309, YL-RY0310, YL-RA0204, YL-RA0208 and YL-RA0210 all have obvious inhibition effect on patulin synthesized by penicillium expansum, compared with the control group CK, wherein the inhibition effect of the Bacillus mojavensis Bacillus mojavensis YL-RY0310 is the best, and the inhibition effect is 99.81%.
3. Morphological features
Bacterial colony of the strain YL-RY0310 is round, white, large in bacterial colony, low in smoothness and dryness on the surface, convex in the middle, irregular in edges, opaque, flat and easy to pick up on NA culture medium. The colony diameter was 2-3mm, and the colony morphology was as shown in FIG. 2. Gram positive, rod-shaped, sporulation.
4. Physiological and biochemical characteristics
The strain YL-RY0310 is suitable for growth on Nutrient Agar (NA) and Nutrient Broth (NB) medium, the suitable temperature range for growth is 4-45 ℃, the optimal temperature is 37 ℃, the suitable pH for growth is 3-9, and the optimal pH is 7.3.
5. Molecular biological characteristics
Using a bacterial DNA extraction kit, referring to the DNA of the antagonistic bacterium YL-RY0310 which was extracted by the instructions, adjacent species were analyzed by BLAST on NCBI, and phylogenetic tree was constructed by using MEGA 11.0, as shown in FIG. 3, the antagonistic bacterium YL-RY0310 had a homology of 96% with the Bacillus mojavensis part 16S rRNA gene (Bacillus mojavensis partial 16S rRNA gene strain ifo 15718), and the strain YL-RY0310 could be finally identified as Bacillus mojavensis (Bacillus mojavensis) by combining the morphological characteristics, physiological and biochemical characteristics of the strain and the results of the BLAST system analysis described above. The 16S rRNA sequence of the strain YL-RY0310 is shown as SEQ ID NO. 1.
EXAMPLE 2 Effect test of Bacillus mojavensis YL-RY0310 on liquid Medium for spreading Penicillium and toxins thereof
6mL of the bacterial suspension of Bacillus mojavensis YL-RY0310 which had been activated for 48 hours was aspirated with 6mL of 1X 10 5 CFU/mL of the suspension of the extended penicillium spores was mixed and added into different liquid media containing 6mL of Potato Dextrose Broth (PDB) (the liquid compositions of a control group CK and an experimental group are shown in Table 3), shake cultivation is carried out for 5 days at 28 ℃ at 120r/min, the growth condition of pathogenic bacteria is observed, and the patulin PAT in the mixed culture solution is extracted and detected by an HPLC method, wherein 3 parallel patulin groups are used, the inhibition rate is calculated according to the following formula, and the test results are shown in FIG. 4 and Table 4.
Toxin inhibition (%) = (control peak area-experimental peak area)/control peak area×100%
Table 3: test group of Bacillus mojavensis YL-RY0310 on liquid medium
Table 4: inhibition of PAT by antagonism test of Bacillus mojavensis YL-RY0310 on liquid medium
As shown in FIG. 4, bacillus mojavensis YL-RY0310 has a remarkable inhibitory effect on the growth of liquid-cultured Penicillium extenhilum, and since the inhibition rate cannot be directly calculated by liquid, the inhibitory effect can only be visually detected by phenomenon to exceed 95%.
As shown in Table 4, the inhibition ratio of the YL-RY0310 bacterial suspension in the experimental group to the patulin PAT reaches 71.66% compared with the control group CK by detecting the patulin PAT content in the 5 th-day shake culture liquid by HPLC method.
Example 3 application of Bacillus mojavensis YL-RY0310 in preventing and treating Paecilomyces checanensis contamination of fruits and vegetables
After wiping and sterilizing the surface of a picked crispy apple (harvested from the co-invasive forest fruit of Bohal county, islamic, N.A.), sequentially using 75% alcohol and sterile water, a 5mm depth needle was used to puncture the equatorial surface of the apple with a sterile inoculating needle, followed by 10. Mu.L of an activated 72h YL-RY0310 bacterial suspension (designated as the experimental group). The control group CK was inoculated with 10. Mu.L of sterile water. After 30min, the control group and the experimental group respectively have a dose of 2×10 5 The CFU/mL spore suspension was re-inoculated with 10. Mu.L at the apple wound and each group was repeated four times. All apples were incubated for 8 days in a ventilated dark room temperature environment, then the diameter of the lesions was measured and the patulin PAT content of each group was detected by extraction using HPLC method, and the bacteriostatic and antitoxic effects were calculated. The inhibition ratio was calculated according to the following formulas (1) and (2), and the test results are shown in tables 5, 6 and fig. 5.
Fungal growth inhibition (%) = (C1-C2)/c1×100% (1)
Toxin inhibition (%) = (control toxin content-experimental toxin content)/control toxin content x 100% (2)
C1 control group expanded radial growth diameter of Penicillium
C2 test group radial growth diameter of Penicillium
Table 5: influence of Bacillus mojavensis YL-RY0310 on growth of Penicillium extending at different concentrations on apples
As can be seen from the experimental results in Table 5, the inhibition rate of the Bacillus mojavensis YL-RY0310 on the penicillium expansum on the apples is 64.34%, which shows that the YL-RY0310 has a good inhibition effect on the growth of the penicillium expansum in the apples.
Table 6: inhibition rate of Bacillus mojavensis YL-RY0310 on synthesizing PAT by penicillium expansum with different concentrations on apples
As shown in Table 6, the content of patulin PAT in the 8 th day apple was measured by HPLC method when the spore concentration of patulin was 2X 10 5 When CFU/mL is used, the inhibition rate of YL-RY0310 to PAT is 58.01%, and the inhibition effect is good.
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 mojavensis YL-RY0310, and culture method and application thereof
<130>
<160>
<170> PatentIn version 3.5
<210> 1
<211> 1424
<212> DNA
<213> Bacillus mojavensis (Bacillus mojavensis)
<400> 1
tgtgtcacct tcggcggctg gctccataaa ggttacctca ccgacttcgg gtgttacaaa 60
ctctcgtggt gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc gcggcatgct 120
gatccgcgat tactagcgat tccagcttca cgcagtcgag ttgcagactg cgatccgaac 180
tgagaacaga tttgtgggat tggcttaacc tcgcggtttc gctgcccttt gttctgtcca 240
ttgtagcacg tgtgtagccc aggtcataag gggcatgatg atttgacgtc atccccacct 300
tcctccggtt tgtcaccggc agtcacctta gagtgcccaa ctgaatgctg gcaactaaga 360
tcaagggttg cgctcgttgc gggacttaac ccaacatctc acgacacgag ctgacgacaa 420
ccatgcacca cctgtcactc tgcccccgaa ggggacgtcc tatctctagg attgtcagag 480
gatgtcaaga cctggtaagg ttcttcgcgt tgcttcgaat taaaccacat gctccaccgc 540
ttgtgcgggc ccccgtcaat tcctttgagt ttcagtcttg cgaccgtact ccccaggcgg 600
agtgcttaat gcgttagctg cagcactaag gggcggaaac cccctaacac ttagcactca 660
tcgtttacgg cgtggactac cagggtatct aatcctgttc gctccccacg ctttcgctcc 720
tcagcgtcag ttacagacca gagagtcgcc ttcgccactg gtgttcctcc acatctctac 780
gcatttcacc gctacacgtg gaattccact ctcctcttct gcactcaagt tccccagttt 840
ccaatgaccc tccccggttg agccgggggc tttcacatca gacttaagga accgcctgcg 900
agccctttac gcccaataat tccggacaac gcttgccacc tacgtattac cgcggctgct 960
ggcacgtagt tagccgtggc tttctggtta ggtaccgtca aggtaccgcc ctattcgaac 1020
ggtacttgtt cttccctaac aacagagctt tacgatccga aaaccttcat cactcacgcg 1080
gcgttgctcc gtcagacttt cgtccattgc ggaagattcc ctactgctgc ctcccgtagg 1140
agtctgggcc gtgtctcagt cccagtgtgg ccgatcaccc tctcaggtcg gctacgcatc 1200
gttgccttgg tgagccatta cctcaccaac tagctaatgc gccgcgggtc catctgtaag 1260
tggtagccga agccaccttt tatgtttgaa ccatgcggtt caaacaagca tccggtatta 1320
gccccggttt cccggagtta tcccagtctt acaggcaggt tacccacgtg ttactcaccc 1380
gtccgccgct aacatcaggg agcaagctcc catctgtccg ctcgacttgc atgtatagca 1420
cgcc 1424
Claims (10)
1. Bacillus mohaiensis strainBacillus mojavensis) YL-RY0310 was deposited at the China general microbiological culture Collection center, with the accession number CGMCC No.24646, at 4 and 6 of 2022.
2. The method for culturing bacillus mojavensis YL-RY0310 according to claim 1, comprising the steps of:
inoculating strain YL-RY0310 onto NA solid culture medium with inoculating needle for activation, culturing 24-48h in incubator at 25-40deg.C, picking single colony of Bacillus mojavensis with inoculating needle, transferring to NB liquid culture medium, culturing 12-24h in shaking incubator at 25-40deg.C at 125-150r/min, transferring culture solution 1-5% of total volume of liquid culture medium to fresh NB liquid culture medium, culturing 24-72h at 125-150r/min at 25-40deg.C to obtain YL-RY0310 fermentation liquid, and viable count reaching (1-10) ×10 7 CFU/mL。
3. The use of bacillus mojavensis YL-RY0310 according to claim 1 for preventing and controlling penicillium expansum contamination of fruits and vegetables.
4. The application of claim 3, wherein the application comprises the steps of:
the concentration is (1-10) multiplied by 10 7 The fermentation liquor of CFU/mL bacillus mojavensis YL-RY0310 is directly sprayed on the surface of fruits and vegetables.
5. A biocontrol formulation comprising bacillus mojavensis YL-RY0310 as defined in 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 YL-RY0310 fermentation broth.
8. Use of a biocontrol formulation as claimed in any one of claims 5-7 for controlling patulin contamination of fruits and vegetables caused by penicillium expansum.
9. The use according to claim 8, wherein said controlling of patulin contamination of fruits and vegetables by penicillium expansum comprises inhibiting growth of penicillium expansum.
10. The application according to claim 9, wherein the application comprises the steps of:
the biocontrol agent is sprayed on the surfaces of fruits and vegetables to inhibit penicillium expansum and patulin.
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| CN105578884A (en) * | 2013-07-24 | 2016-05-11 | 拜耳作物科学股份公司 | Binary fungicidal composition |
| KR102073487B1 (en) * | 2018-08-31 | 2020-02-04 | 바래봉비료영농조합법인 | Method for manufacturing animal waste compost pellet inoculating bacillus vallismortis bs07m |
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| CN105578884A (en) * | 2013-07-24 | 2016-05-11 | 拜耳作物科学股份公司 | Binary fungicidal composition |
| WO2015036379A1 (en) * | 2013-09-13 | 2015-03-19 | Bayer Cropscience Ag | Fungicidal compositions containing thiazolylisoxazoline fungicide and biological fungicide |
| KR102073487B1 (en) * | 2018-08-31 | 2020-02-04 | 바래봉비료영농조합법인 | Method for manufacturing animal waste compost pellet inoculating bacillus vallismortis bs07m |
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