CN113528370B - Bacillus belgii strain and application thereof - Google Patents
Bacillus belgii strain and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
- A23B7/155—Microorganisms; Enzymes; Antibiotics
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- 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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to a Bacillus velezensis (KL-2) strain and application thereof. The strain is Bacillus velezensis (Bacillus velezensis) KL-2, is preserved in a China Center for Type Culture Collection (CCTCC) in 2019, 10 months and 12 days, and has a preservation number of CCTCC NO: M2019817. The Bacillus subtilis KL-2 is derived from ripe red fruits of cherry tomatoes and is safer than other sources; the antibacterial agent not only can inhibit various pathogenic bacteria of cherry tomatoes, but also has good inhibition on common pathogenic bacteria of other fruits and vegetables, so that the antibacterial agent has broad-spectrum antibacterial property and ideal disease prevention effect, provides strain resources for biological prevention and control of fruits and vegetables, and has good application prospect.
Description
Technical Field
The invention relates to a Bacillus velezensis (KL-2) strain and application thereof.
Background
Spoilage by phytopathogenic fungi is one of the major causes of postharvest loss of fruits and vegetables. The loss caused by pathogenic fungi of the picked fruits and vegetables in developed countries is about 20-25%; in developing countries, post-harvest losses are often more severe, even up to over 50%, due to insufficient storage and transportation facilities.
The prevention and control research is mainly carried out on the aspects of postharvest preservation of fruits and vegetables at home and abroad from the following aspects: (1) Physical prevention, namely inhibiting the infection of pathogenic bacteria by physical methods such as heat treatment, refrigeration, air conditioning, ultraviolet irradiation treatment and the like; (2) Chemical control, which is a way of inhibiting the growth of pathogenic bacteria by using chemical bactericides such as pesticides and the like, is an obvious effect, but long-term and unreasonable use of chemical bactericides can easily cause drug resistance of fungi and is more easily harmful to human bodies and the environment; (3) Biological control, and controlling the proliferation of pathogenic microorganisms by using biocontrol microorganisms or metabolites thereof.
In the comprehensive management of fruits and vegetables before and after picking, the use of biocontrol microorganisms as biological control agents is very important, and the biocontrol microorganisms are widely applied to disease control of crops and are also a research focus of the current microbial ecological control. The biocontrol microorganism inhibits pathogenic bacteria by competing with them for nutrients and spaces, producing antibacterial substances, inducing plants to develop disease resistance and parasitic effects, which is effective in short, medium and long term, and does not cause harm to the environment and human and livestock.
Bacillus subtilis (Bacillus velezensis) is a new species of Bacillus, can produce various secondary metabolites, and has potential bacteriostatic activity and plant growth promoting effect. However, the application of the Bacillus belgii strain in the preservation and freshness of picked fruits and vegetables is still less at present, and no mature application scheme is available. Therefore, based on the biocontrol property of the bacillus beleisi, the biocontrol strain which is safe in source, harmless to the ecological environment and simple in culture conditions is provided, and the biocontrol strain has important biocontrol significance on the postharvest fresh-keeping of fruits and vegetables.
Disclosure of Invention
One of the objects of the present invention is to provide a Bacillus velezensis strain. The biocontrol strain can utilize the antagonistic action to play a role in the fresh-keeping of the picked fruits and vegetables, is a green, environment-friendly, non-toxic and harmless microbial control method, and has wide market prospect.
The second object of the present invention is to provide a method for culturing the strain.
The third purpose of the invention is to provide the application of the strain in inhibiting the proliferation of pathogenic microorganisms.
In order to achieve the purpose, the invention adopts the following technical scheme:
the Bacillus belgii strain is characterized in that the strain is Bacillus belgii (Bacillus velezensis) KL-2, is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 months and 12 days, and has a preservation number of CCTCC NO: M2019817.
The Bacillus belgii strain is characterized in that the 16S rDNA of the Bacillus belgii (Bacillus velezensis) KL-2 is a base sequence shown in SEQ ID NO. 1.
A method for cultivating the bacillus beleisi strain is characterized by comprising the following specific steps of: inoculating a bacillus beleisis strain into an LB culture solution, wherein the pH of the culture solution is = 5-9, and the culture temperature is as follows: at 20-50 deg.c and in stable growth period within 12-24 hr.
An application of the Bacillus belgii strain in inhibiting the proliferation of pathogenic microorganisms.
The application of the Bacillus belgii strain in inhibiting the proliferation of pathogenic microorganisms is characterized in that the pathogenic microorganisms are: alternaria tenuissima, aspergillus niger, penicillium notatum, trichoderma pseudokoningii, cladosporium salinum, ascomycetes, colletotrichum gloeosporioides, brown rot peach, botrytis cinerea, penicillium persicum, penicillium citrinum, colletotrichum litchi or alternaria apricot.
The Bacillus belgii strain is identified by 16S rDNA strain, is Bacillus belgii (Bacillus velezensis), has a preservation name of Bacillus velezensis KL-2, is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 months and 12 days, and has a preservation number of: CCTCC NO of M2019817, and the preservation address is eight-path Loojia mountain in Wuchang district, wuhan City, hubei province.
The Bacillus beleisi KL-2 is gram-staining negative bacteria, the somatic cells are short rod-shaped, the colony forms are regular and round, the edges are regular, the colony is faint yellow and dull, the colony is opaque, and the mature red fruits of the cherry tomatoes are separated from the original vegetable seed fields in Shanghai.
The Bacillus beleisi KL-2 of the present invention showed a positive reaction in the V-P test, could not use citrate, propionate, could use D-xylose, L-arabinose, D-mannitol, could liquefy gelatin, could not grow at 7% NaCI, could grow at pH5.7, was positive in the nitrate reduction reaction, could hydrolyze starch, could not grow under anaerobic conditions.
The Bacillus beleisi KL-2 has obvious inhibition effect on alternaria tenuissima, aspergillus niger, penicillium, trichoderma pseudokoningii, cladosporium halophilum, ascomycetes and colletotrichum gloeosporioides which cause cherry tomato postharvest diseases; but has no inhibitory effect on P.apicomplexa.
The Bacillus beilaisi KL-2 has an inhibiting effect on other fruit and vegetable postharvest diseases causing Monilinia fructicola, tomato botrytis cinerea, penicillium persicum, citrus penicillium digitatum, colletotrichum gloeosporioides and Alternaria prunifolia, and the inhibiting rate is higher than 58%, wherein the inhibiting rate on the Monilinia fructicola is 80% at most.
The Bacillus velezensis KL-2 is derived from ripe red fruits of cherry tomatoes and is safer than other sources; the antibacterial agent not only can inhibit various pathogenic bacteria of cherry tomatoes, but also has good inhibition on common pathogenic bacteria of other fruits and vegetables, so that the antibacterial agent has broad-spectrum antibacterial property and ideal disease prevention effect, provides strain resources for biological prevention and control of fruits and vegetables, and has good application prospect.
Drawings
FIG. 1 shows the inhibition rate of the strain KL-2 of the present invention against pathogenic bacteria.
FIG. 2 shows the colony morphology of strain KL-2 after 12h of plate culture in LB solid medium.
FIG. 3 shows the cell morphology of KL-2 under an optical microscope.
FIG. 4 is a phylogenetic tree of 16S rDNA sequence Neighbor-Joining of Bacillus belgii KL-2.
FIG. 5 shows the phylogenetic tree of the gyrA sequence Neighbor-Joining of Bacillus beiLeisi KL-2.
FIG. 6 is a growth curve of strain KL-2.
FIG. 7 is the effect of pH on the growth of strain KL-2.
FIG. 8 is the effect of temperature on the growth of strain KL-2.
FIG. 9 shows the bacteriostatic effect of strain KL-2.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The room temperature in the present invention means a temperature between the operations of carrying out the test, and is generally 25 ℃.
Pathogenic bacteria used in the present invention: alternaria tenuissima (Alternaria tenuissima), aspergillus niger (Aureobasidium melanogenesis), penicillium (Penicillium oxyalicum), sporotrichum acutum (Pleuropterum acutum), trichoderma koningii (Trichoderma koningiensis), cladosporium halophila (Cladosporium halolerans), ascomycetes (Ascomycota), colletotrichum gloeosporioides (Colletotrichum gloeosporioides) were screened from food quality and safety control laboratories of the national institute of Life sciences of Shanghai and stored. Monilinia fructicola (Monilinia fructicola), botrytis cinerea (Botrytis cinerea) from food quality and safety control laboratories of the institute of Life sciences of Shanghai university; penicillium persicum (Penicillium), penicillium citrinum (Penicillium digitatum) and Alternaria apricot (Alternaria) were purchased from China forestry microorganism strain preservation management center; college of agricultural sciences environmental and plant protection institute of Lasioderma litchi (Colletotrichum gloeosporioides) of China.
(II) culture Medium used in the present invention
LB culture medium: 5g of beef extract, 5g of yeast extract, 5g of sodium chloride, 18g of agar, 10g of fish meal peptone and 1L of water.
LB liquid medium: 5g of beef extract, 5g of yeast extract, 5g of sodium chloride, 10g of fish meal peptone and 1L of water.
PDA culture medium: 200g peeled potato, 20g glucose, 20g agar, 1L water.
Example screening of a biocontrol Strain
1 Experimental method
1.1 isolation and purification of the Strain
Weighing 10g of mature red fruit sample of cherry tomato picked from a certain vegetable stock ground in Shanghai, adding into 90mLLB liquid culture medium, and shake culturing at 37 deg.C and 150r/min for 48h. Then 1mL of the bacterial solution was aspirated, and the concentration was diluted to 10 with sterile distilled water -1 To 10 -7 Respectively take 10 -5 、10 -6 、10 -7 Gradient diluent0.1mL of the suspension was spread on LB medium, and after culturing at 37 ℃ for 24 hours, a single colony on the plate was streaked onto another fresh plate to purify the strain. Repeat 3 times.
1.2 preliminary screening
Placing the pathogenic bacteria block of Alternaria tenuissima in the center of a PDA (personal digital assistant) plate, inoculating purified bacteria to be screened on two sides 2cm away from the pathogenic bacteria block, culturing for 4d in a constant-temperature incubator at 28 ℃, observing the growth condition of bacterial colonies, and screening out strains which generate inhibition zones and inhibit pathogenic bacteria.
1.3 double sifting
Placing eight pathogenic bacteria blocks in the center of a PDA (personal digital assistant) plate, selecting bacteria with an antibacterial effect by using an inoculating loop, inoculating the bacteria on 4 angular points 3cm away from the center of the plate, placing the plate into an incubator for constant-temperature culture at 28 ℃ for 4d, observing the growth state of the pathogenic bacteria by using a PDA plate culture medium only inoculated with the pathogenic bacteria as a contrast, measuring the diameters of the bacterial colonies of the pathogenic bacteria, and calculating the inhibition rate of the strains according to the following formula.
Inhibition (%) = (control colony diameter-treated colony diameter)/control colony diameter × 100%
2 results and analysis of the experiments
By taking the ripe red fruits of the cherry tomatoes as samples and taking the most common pathogenic fungus Alternaria tenuissima in the cherry tomatoes as an indicator bacterium, an antagonistic bacterium with obvious bacteriostatic effect is obtained by screening the ripe red fruits of the cherry tomatoes through a flat plate antagonistic test and is named as KL-2. The inhibitory effects of the strain KL-2 on the above eight pathogenic bacteria are shown in FIG. 1. Has obvious inhibiting effect on alternaria tenuissima, aspergillus niger, penicillium, trichoderma pseudokoningii, cladosporium halophila, ascomycetes and colletotrichum gloeosporioides, and a strain KL-2; however, the strain has no inhibition effect on the P.multiplex, which shows that the strain KL-2 can inhibit various pathogenic bacteria in tomato.
Example 2 identification of Strain KL-2
1 method of experiment
1.1 morphological characterization
The strain KL-2 was streaked on LB plate, cultured at 37 ℃ for 12 hours, and the color, morphology, transparency and other characteristics of the colonies were observed, and the cell morphology was observed by using an optical microscope and gram stain.
1.2 characterization of physiological and biochemical Properties
The strain KL-2 was subjected to V-P test, citrate utilization, propionate utilization, D-xylose utilization, L-arabinose utilization, D-mannitol utilization, gelatin liquefaction test, 7% NaCl growth, pH5.7 growth, nitrate reduction, starch hydrolysis, and anaerobic assay. This was repeated three times.
1.3 molecular biological identification
The strain KL-2 is sent to a biological engineering (Shanghai) corporation for sequencing, and a sequencing result is input into an NCBI database for homology analysis after sequencing. Alignment was performed using BLAST software, and phylogenetic trees were constructed using MEGA7.0 software.
The gyrA gene is subjected to PCR amplification by using corresponding universal primers (table 1), and the PCR reaction program of a gyrA-F + gyrA-R primer pair is that the gyrA-F + gyrA-R primer pair is pre-denatured at 95 ℃ for 5min; denaturation at 95 ℃ for 45s, annealing at 57 ℃ for 45s, extension at 72 ℃ for 80s,35 cycles; extension at 72 ℃ for 10min. The PCR amplification product was sent to Biotechnology engineering (Shanghai) Co., ltd for sequencing. The sequencing products were uploaded to NCBI, where a BLAST homology alignment was performed. The highest similarity sequence of the strain sequence and the sequence of the model strain are selected as reference sequences, and multi-sequence alignment is carried out by using Clustalw in MEGA 7.0. And (4) constructing the phylogenetic tree by using an N-J method. The confidence of each branch of the phylogenetic tree is analyzed 1000 times by repeated sampling.
TABLE 1 PCR primer sequences
2 results and analysis of the experiments
2.1 morphological characterisation
The morphological characteristics of the strain KL-2 are as follows: gram-negative bacteria, which have short rod-shaped somatic cells, regular round colony morphology, regular edges, pale yellow color, no luster, and opacity, as shown in fig. 2 and 3.
2.2 physiological and Biochemical identification
The results of physiological and biochemical characteristics of the strain KL-2 are shown in Table 2.
TABLE 2 physiological and biochemical characteristics of Bacillus belgii KL-2
Note: "+" indicates positive, and "-" indicates negative.
2.3 molecular biological identification
Firstly, the strain KL-2 has more than 99 percent of homology with a part of the known strain Bacillus velezensis through 16S rDNA fragment sequence comparison. 16S rDNA is a recognized ruler for classifying and identifying bacteria at present, and the general similarity is more than 98 percent, so that the bacteria can be regarded as a genus. However, the 16S rDNA sequence cannot distinguish and identify closely related species in the Bacillus, and the Bacillus can be effectively distinguished at the seed or subspecies level through the single gene phylogenetic analysis of housekeeping genes (gyrA) with larger sequence variation degrees. FIG. 4 shows a phylogenetic tree based on the 16S rDNA gene sequence, and shows that the strain KL-2 is closest to the genetic distance of Bacillus beielii (MK 780002.1); FIG. 5 shows that the genetic distance between the strain KL-2 and Bacillus subtilis (NZ _ CP 011937.1) is closest, and the strain is identified as Bacillus subtilis (Bacillus velezensis) by integrating the measurement results of thallus morphology, colony characteristics and physiological and biochemical indicators, as shown in the phylogenetic tree based on the gyrA gene sequence. 1483bp sequence is as follows:
TCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGT
the strain Bacillus velezensis KL-2 is preserved in a China Center for Type Culture Collection (CCTCC) in 2019, 10 months and 12 days, and the preservation number is as follows: CCTCC NO of M2019817, and the preservation address is eight-way Lojia mountain in Wuchang district, wuhan City, hubei province.
Example 3 culture conditions of Bacillus belgii KL-2
1 Experimental method
1.1 preparation of seed culture
Inoculating strain KL-2 pre-activated on LB solid medium into sterilized LB liquid medium, culturing at 37 deg.C and 150r/min for 24 hr, and adjusting concentration to 1 × 10 with sterile distilled water 8 cfu/L is the seed culture solution of the strain KL-2.
1.2 determination of growth curves
1mL of the seed culture solution was inoculated into 100mL of LB liquid medium and cultured on a shaker at 37 ℃ and 150 r/min. Taking 5mL of the thallus culture solution, measuring the absorbance at the wavelength of 600nm by using a spectrophotometer every 3h, observing the curve condition, and measuring for at least 48h. This was repeated three times.
1.3 determination of optimum growth pH
Adjusting the pH value of LB liquid culture medium to 100mL with NaOH to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 respectively, sterilizing, inoculating 1mL seed culture solution into 100mL LB liquid culture medium adjusted to different pH values, culturing in a shaking table at 37 ℃ and 150r/min, and measuring the absorbance of the culture medium at 600nm wavelength by using an ultraviolet-visible spectrophotometer after 48h. This was repeated three times.
1.4 determination of optimum growth temperature
Inoculating 1mL seed culture solution into 100mL LB liquid culture medium with pH of 7.0, culturing in shaker at 20 deg.C, 28 deg.C, 300 deg.C, 37 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 150r/min, and measuring absorbance at 600nm wavelength with spectrophotometer after 48 hr. This was repeated three times.
2 results and analysis of the experiments
2.1 growth Curve of Strain KL-2
The growth curve of the strain KL-2 is shown in figure 6, the strain KL-2 is in the logarithmic growth phase within 12h after being inoculated into the LB culture solution, is in the stationary growth phase within 12-24 h, and enters the aging phase 24h after being inoculated.
2.2 Effect of pH on growth of Strain KL-2
The influence of pH on the strain KL-2 is shown in figure 7, the strain KL-2 can grow in a larger pH range, but the pH value has obvious influence on the growth of the strain KL-2, wherein the pH = 5-9 is suitable for the growth of the strain KL-2, and the optimum pH is about 7.0.
2.3 Effect of temperature on growth of Strain KL-2
The influence of temperature on the strain KL-2 is shown in figure 8, the temperature has obvious influence on the growth and the reproduction of the strain KL-2, the growth and the reproduction rate of the strain KL-2 are in a trend of increasing firstly and then decreasing in the range of 20-50 ℃, and the optimal temperature is about 30 ℃.
Example 4 application of Bacillus belgii KL-2 in the field of biocontrol
1 method of experiment
Six pathogenic bacteria blocks are placed in the center of a PDA flat plate, a strain KL-2 is picked by an inoculating loop and is point-connected on 4 angular points 3cm away from the center of the flat plate, the flat plate is placed in an incubator for constant-temperature culture for 4 days at 28 ℃, three groups of treatment are performed in parallel, and the experiment is repeated twice. And (3) taking a PDA (potato dextrose agar) plate culture medium inoculated with only the pathogenic bacteria as a reference, observing the growth state of the pathogenic bacteria, measuring the colony diameter of the pathogenic bacteria, and calculating the inhibition rate of the strain.
Inhibition (%) = (control colony diameter-treated colony diameter)/control colony diameter × 100%
2 results and analysis of the experiments
In order to research the biocontrol efficacy of Bacillus beilesiensis KL-2, six indicator bacteria, namely Monilinia persica, botrytis cinerea, penicillium persicum, penicillium citrinum, colletotrichum gloeosporioides and Alternaria pruni, are selected, and the influence of the strain KL-2 on pathogenic bacteria of picked fruits and vegetables is researched. As shown in figure 9, the strain KL-2 has obvious inhibition effect on six pathogenic bacteria, the most obvious inhibition effect is on Monilinia fructicola, and the inhibition rate can reach 80%; the penicillium persicum is immediately followed, and the inhibition rate of the penicillium persicum can reach 77%; the subsequent steps are tomato gray mold, litchi colletotrichum gloeosporioides and apricot crosslinked sporotrichum, the inhibition rates of the three pathogenic bacteria are respectively 66 percent, 69 percent and 62 percent, the inhibition rates are all over 60 percent, and finally, the inhibition effect of the citrus penicillium digitatum is slightly weaker than that of the former pathogenic bacteria, but the inhibition rate also reaches 54 percent. The bacterial strain KL-2 can inhibit various pathogenic fungi causing fruit and vegetable diseases and has broad-spectrum bacteriostasis.
Sequence listing
<110> university at Shanghai
<120> Bacillus belgii strains and uses thereof
<141> 2021-04-22
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3
<212> DNA
<213> Bacillus belgii (Bacillus velezensis)
<400> 1
TCAGGACGAA CGCTGGCGGC GTGCCTAATA CATGCAAGTC GAGCGGACAG ATGGGAGCTT
GCTCCCTGAT GTTAGCGGCG GACGGGTGAG TAACACGTGG GTAACCTGCC TGTAAGACTG
GGATAACTCC GGGAAACCGG GGCTAATACC GGATGGTTGT TTGAACCGCA TGGTTCAGAC
ATAAAAGGTG GCTTCGGCTA CCACTTACAG ATGGACCCGC GGCGCATTAG CTAGTTGGTG
AGGTAACGGC TCACCAAGGC GACGATGCGT AGCCGACCTG AGAGGGTGAT CGGCCACACT
GGGACTGAGA CACGGCCCAG ACTCCTACGG GAGGCAGCAG TAGGGAATCT TCCGCAATGG
ACGAAAGTCT GACGGAGCAA CGCCGCGTGA GTGATGAAGG TTTTCGGATC GTAAAGCTCT
GTTGTTAGGG AAGAACAAGT GCCGTTCAAA TAGGGCGGCA CCTTGACGGT ACCTAACCAG
AAAGCCACGG CTAACTACGT GCCAGCAGCC GCGGTAATAC GTAGGTGGCA AGCGTTGTCC
GGAATTATTG GGCGTAAAGG GCTCGCAGGC GGTTTCTTAA GTCTGATGTG AAAGCCCCCG
GCTCAACCGG GGAGGGTCAT TGGAAACTGG GGAACTTGAG TGCAGAAGAG GAGAGTGGAA
TTCCACGTGT AGCGGTGAAA TGCGTAGAGA TGTGGAGGAA CACCAGTGGC GAAGGCGACT
CTCTGGTCTG TAACTGACGC TGAGGAGCGA AAGCGTGGGG AGCGAACAGG ATTAGATACC
CTGGTAGTCC ACGCCGTAAA CGATGAGTGC TAAGTGTTAG GGGGTTTCCG CCCCTTAGTG
CTGCAGCTAA CGCATTAAGC ACTCCGCCTG GGGAGTACGG TCGCAAGACT GAAACTCAAA
GGAATTGACG GGGGCCCGCA CAAGCGGTGG AGCATGTGGT TTAATTCGAA GCAACGCGAA
GAACCTTACC AGGTCTTGAC ATCCTCTGAC AATCCTAGAG ATAGGACGTC CCCTTCGGGG
GCAGAGTGAC AGGTGGTGCA TGGTTGTCGT CAGCTCGTGT CGTGAGATGT TGGGTTAAGT
CCCGCAACGA GCGCAACCCT TGATCTTAGT TGCCAGCATT CAGTTGGGCA CTCTAAGGTG
ACTGCCGGTG ACAAACCGGA GGAAGGTGGG GATGACGTCA AATCATCATG CCCCTTATGA
CCTGGGCTAC ACACGTGCTA CAATGGACAG AACAAAGGGC AGCGAAACCG CGAGGTTAAG
CCAATCCCAC AAATCTGTTC TCAGTTCGGA TCGCAGTCTG CAACTCGACT GCGTGAAGCT
GGAATCGCTA GTAATCGCGG ATCAGCATGC CGCGGTGAAT ACGTTCCCGG GCCTTGTACA
CACCGCCCGT CACACCACGA GAGTTTGTAA CACCCGAAGT CGGTGAGGTA ACCTTTTAGG
AGCCAGCCGC CGAAGGTGGG ACAGATGATT GGGGTGAAGT CGT
Claims (2)
1. A Bacillus belgii strain is characterized in that the strain is Bacillus belgii (II)Bacillus velezensis) KL-2 is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 months and 12 days, and the preservation number is CCTCC NO: M2019817.
2. Use of a strain of bacillus beijerinckii according to claim 1 for the preparation of a biocontrol agent for inhibiting the proliferation of pathogenic microorganisms: alternaria tenuissima, aspergillus niger, penicillium notatum, trichoderma pseudokoningii, cladosporium salina, ascomycetes, colletotrichum gloeosporioides, monilinia persicinum, botrytis cinerea, penicillium persicum, penicillium citrinum, colletotrichum litchi or Alternaria apricot.
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| CN114208851A (en) * | 2021-12-31 | 2022-03-22 | 江西农业大学 | Compound bactericide suitable for preventing and treating greening mildew after citrus picking and preparation method thereof |
| CN115011504B (en) * | 2022-04-21 | 2023-08-11 | 湖南省蔬菜研究所 | Bacillus bailii XY40-1 and application thereof |
| CN116121123B (en) * | 2022-12-09 | 2024-04-09 | 青岛农业大学 | Bacillus bailii and application thereof in grape gray mold and grape fresh-keeping |
| CN115873770B (en) * | 2023-02-16 | 2023-04-25 | 潍坊科技学院 | Bacillus bailii and application thereof in preventing and controlling tomato diseases |
| CN116987613A (en) * | 2023-04-03 | 2023-11-03 | 吉林农业大学 | Bacillus bailii YZ-375 and application thereof |
| CN117264847A (en) * | 2023-11-01 | 2023-12-22 | 山东省烟台市农业科学研究院(山东省农业科学院烟台市分院) | Bacillus bailii YTQ3, compound agent and application thereof in improving low-temperature freeze injury resistance of plants |
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| CN111925966A (en) * | 2020-08-28 | 2020-11-13 | 山东省果树研究所 | Bacillus belgii and culture method and application thereof |
| CN112029691A (en) * | 2020-09-28 | 2020-12-04 | 广西壮族自治区农业科学院植物保护研究所 | Application of Bacillus beleisi YM-11-C in prevention and treatment of mango anthracnose |
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| CN111925966A (en) * | 2020-08-28 | 2020-11-13 | 山东省果树研究所 | Bacillus belgii and culture method and application thereof |
| CN112029691A (en) * | 2020-09-28 | 2020-12-04 | 广西壮族自治区农业科学院植物保护研究所 | Application of Bacillus beleisi YM-11-C in prevention and treatment of mango anthracnose |
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