CN113528370A - Bacillus belgii strain and application thereof - Google Patents
Bacillus belgii strain and application thereof Download PDFInfo
- Publication number
- CN113528370A CN113528370A CN202110434803.8A CN202110434803A CN113528370A CN 113528370 A CN113528370 A CN 113528370A CN 202110434803 A CN202110434803 A CN 202110434803A CN 113528370 A CN113528370 A CN 113528370A
- Authority
- CN
- China
- Prior art keywords
- strain
- bacillus
- bacillus belgii
- penicillium
- pathogenic bacteria
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- 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
-
- 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
-
- 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 (Bacillus velezensis) KL-2 strain and application thereof. The strain is 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 subtilis KL-2 is derived from ripe red fruits of cherry tomatoes and is safer than other sources; the antibacterial agent can inhibit various pathogenic bacteria of cherry tomatoes and 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 control of fruits and vegetables, and has good application prospect.
Description
Technical Field
The invention relates to a Bacillus velezensis (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 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 characteristic of Bacillus belgii, the biocontrol strain with safe source, no harm to the ecological environment and simple culture conditions is provided, and the biocontrol strain has important biocontrol significance for the postharvest preservation of fruits and vegetables.
Disclosure of Invention
One of the objects of the present invention is to provide a Bacillus velezensis strain. The biocontrol bacterium can utilize the antagonistic action to play a role in the postharvest fresh-keeping of fruits and vegetables, is a green, environment-friendly, nontoxic 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 subtilis strain is characterized in that the strain is Bacillus subtilis (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 belgii strain is characterized by comprising the following specific steps: inoculating a Bacillus belgii strain into an LB culture solution, wherein the pH value of the culture solution is 5-9, and the culture temperature is as follows: and the temperature is 20-50 ℃, and the growth period is stable within 12-24 hours.
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 salina, Ascomycetes, Colletotrichum gloeosporioides, Monilinia persicinum, 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. M2019817, and the preservation address is eight-path Loojia mountain in Wuchang district of Wuhan city, Hubei province.
The Bacillus beleisi KL-2 is a gram-staining negative bacterium, the somatic cells are short rod-shaped, the colony forms are regular and round, the edges are neat, the bacterial colonies are light yellow and dull, the bacterial colonies are opaque, and the bacterial colonies are separated from ripe red fruits of cherries and tomatoes picked from a certain original vegetable seed field in the Shanghai.
The Bacillus beleisi KL-2 of the invention has positive reaction in a V-P test, can not utilize citrate and propionate, can utilize D-xylose, L-arabinose and D-mannitol, can liquefy gelatin, can not grow at 7% NaCI, can grow at pH5.7, has positive nitrate reduction reaction, can hydrolyze starch, and can 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 belgii KL-2 has an inhibiting effect on other fruit and vegetable postharvest diseases caused by Monilinia fructicola, botrytis cinerea, Penicillium persicum, Penicillium citrinum, colletotrichum gloeosporioides and Alternaria armeniaca, and the inhibiting rate is higher than 58%, wherein the inhibiting rate on the Monilinia fructicola is maximum 80%.
The Bacillus velezensis KL-2 is derived from ripe red fruits of cherry tomatoes and is safer than other sources; the antibacterial agent can inhibit various pathogenic bacteria of cherry tomatoes and 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 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 shows a 16S rDNA sequence Neighbor-Joining phylogenetic tree of Bacillus belgii KL-2.
FIG. 5 shows the Neighbor-Joining phylogenetic tree of the gyrA sequence of Bacillus beilesiensis 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 melanogenum), Penicillium sp (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 life sciences of Shanghai university 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 Laurencia littoralis (Colletotrichum gloeosporioides).
(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 Biocide Strain
1 method of experiment
1.1 isolation and purification of the Strain
Weighing 10g of a mature red fruit sample of cherry tomatoes picked from a certain vegetable stock ground in Shanghai, adding the mature red fruit sample into a 90mLLB liquid culture medium, and performing shake culture at 37 ℃ and 150r/min for 48 h. Then 1mL of the bacterial solution was aspirated, and the concentration was diluted to 10 with sterilized distilled water-1To 10-7Respectively take 10-5、10-6、10-7After 0.1mL of the gradient dilution was applied to LB medium and cultured at 37 ℃ for 24 hours, single colonies on the plate were streaked to other fresh plates to purify the strain. Repeat 3 times.
1.2 preliminary screening
Placing 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.
The inhibition ratio (%) - (control colony diameter-treatment colony diameter)/control colony diameter X100%
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 an LB plate, cultured at 37 ℃ for 12 hours, and the color, morphology, transparency and the like of colonies were observed, and the cell morphology was observed by using an optical microscope and gram staining.
1.2 characterization of physiological and biochemical characteristics
Performing 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 determination on the strain KL-2. 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 the 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 5 min; denaturation at 95 ℃ for 45s, annealing at 57 ℃ for 45s, extension at 72 ℃ for 80s, and 35 cycles; extension at 72 ℃ for 10 min. 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 the classification and identification of bacteria at present, and the general similarity of more than 98 percent 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 beleisi (MK 780002.1); FIG. 5 shows that the genetic distance between KL-2 and Bacillus subtilis (NZ _ CP011937.1) 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. The 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. M2019817, and the preservation address is eight-path Loojia mountain in Wuchang district of Wuhan city, Hubei province.
Example 3 culture conditions of Bacillus belgii KL-2
1 method of experiment
1.1 preparation of seed culture
Inoculating KL-2 of a strain pre-activated on an LB solid mediumCulturing in sterilized LB liquid culture medium at 37 deg.C and 150r/min for 24 hr, and adjusting concentration to 1 × 10 with sterile distilled water8cfu/L is the seed culture solution of the strain KL-2.
1.2 determination of growth curves
1mL of the seed culture medium 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 48 h. 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 48 h. 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 shaking table 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 a logarithmic growth phase within 12 hours after being inoculated into the LB culture solution, is in a stable growth phase within 12-24 hours, and enters an aging phase after being inoculated for 24 hours.
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 large pH range, but the pH value has obvious influence on the growth of the strain KL-2, wherein the pH value is 5-9, the strain KL-2 is suitable for growth, and the optimum pH value 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 tend to increase firstly and then decrease 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.
The inhibition ratio (%) - (control colony diameter-treatment colony diameter)/control colony diameter X100%
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 the 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 (5)
1. 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 M2019817, CCTCC NO.
2. The Bacillus belgii strain according to claim 1, wherein the 16SrDNA of Bacillus belgii (Bacillus velezensis) KL-2 has the base sequence shown in SEQ ID No. 1.
3. A method for cultivating the Bacillus belgii strain of claim 1, comprising the steps of: inoculating a Bacillus belgii strain into an LB culture solution, wherein the pH value of the culture solution is 5-9, and the culture temperature is as follows: and the temperature is 20-50 ℃, and the growth period is stable within 12-24 hours.
4. Use of a strain of bacillus beijerinckii according to claim 1 or 2 for inhibiting the proliferation of pathogenic microorganisms.
5. Use of a strain of bacillus beijerinckii according to claim 4 for inhibiting the proliferation of pathogenic microorganisms, characterized in that the pathogenic microorganisms are: 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110434803.8A CN113528370B (en) | 2021-04-22 | 2021-04-22 | Bacillus belgii strain and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110434803.8A CN113528370B (en) | 2021-04-22 | 2021-04-22 | Bacillus belgii strain and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113528370A true CN113528370A (en) | 2021-10-22 |
| CN113528370B CN113528370B (en) | 2022-12-23 |
Family
ID=78095345
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110434803.8A Active CN113528370B (en) | 2021-04-22 | 2021-04-22 | Bacillus belgii strain and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113528370B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113943679A (en) * | 2021-11-08 | 2022-01-18 | 南京农业大学 | Bacillus beleister strain for sheep nasal cavity source and application thereof |
| 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 |
| CN115011504A (en) * | 2022-04-21 | 2022-09-06 | 湖南省蔬菜研究所 | Bacillus belgii XY40-1 and application thereof |
| CN115873770A (en) * | 2023-02-16 | 2023-03-31 | 潍坊科技学院 | Bacillus belgii and application thereof in prevention and treatment of tomato diseases |
| CN116121123A (en) * | 2022-12-09 | 2023-05-16 | 青岛农业大学 | Bacillus bailii and application thereof in grape gray mold and grape fresh-keeping |
| 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 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110184221A (en) * | 2019-06-05 | 2019-08-30 | 中国农业科学院蔬菜花卉研究所 | One plant of Bei Laisi bacillus and its application in prevention and treatment tomato phytophthora root rot |
| CN110699275A (en) * | 2019-09-03 | 2020-01-17 | 山西省农业科学院农产品贮藏保鲜研究所 | Bacterial strain for antagonizing botrytis cinerea pathogenic bacteria after fruit and vegetable picking and application thereof |
| CN111925966A (en) * | 2020-08-28 | 2020-11-13 | 山东省果树研究所 | Bacillus belgii and culture method and application thereof |
| CN111961613A (en) * | 2020-07-29 | 2020-11-20 | 华中农业大学 | Bacillus belgii and application thereof in preventing and treating peach brown rot |
| 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 |
| CN112458012A (en) * | 2020-11-24 | 2021-03-09 | 新疆农业科学院微生物应用研究所（中国新疆-亚美尼亚生物工程研究开发中心） | Bacillus belgii microbial agent and application thereof |
-
2021
- 2021-04-22 CN CN202110434803.8A patent/CN113528370B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110184221A (en) * | 2019-06-05 | 2019-08-30 | 中国农业科学院蔬菜花卉研究所 | One plant of Bei Laisi bacillus and its application in prevention and treatment tomato phytophthora root rot |
| CN110699275A (en) * | 2019-09-03 | 2020-01-17 | 山西省农业科学院农产品贮藏保鲜研究所 | Bacterial strain for antagonizing botrytis cinerea pathogenic bacteria after fruit and vegetable picking and application thereof |
| CN111961613A (en) * | 2020-07-29 | 2020-11-20 | 华中农业大学 | Bacillus belgii and application thereof in preventing and treating peach brown rot |
| 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 |
| CN112458012A (en) * | 2020-11-24 | 2021-03-09 | 新疆农业科学院微生物应用研究所（中国新疆-亚美尼亚生物工程研究开发中心） | Bacillus belgii microbial agent and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 沙月霞 等: "贝莱斯芽孢杆菌E69预防稻瘟病等多种真菌病害的潜力", 《中国农业科学》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113943679A (en) * | 2021-11-08 | 2022-01-18 | 南京农业大学 | Bacillus beleister strain for sheep nasal cavity source and application thereof |
| 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 |
| CN115011504A (en) * | 2022-04-21 | 2022-09-06 | 湖南省蔬菜研究所 | Bacillus belgii XY40-1 and application thereof |
| CN115011504B (en) * | 2022-04-21 | 2023-08-11 | 湖南省蔬菜研究所 | Bacillus bailii XY40-1 and application thereof |
| CN116121123A (en) * | 2022-12-09 | 2023-05-16 | 青岛农业大学 | Bacillus bailii and application thereof in grape gray mold and grape fresh-keeping |
| 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 |
| CN115873770A (en) * | 2023-02-16 | 2023-03-31 | 潍坊科技学院 | Bacillus belgii and application thereof in prevention and treatment of tomato diseases |
| 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 |
| CN116987613B (en) * | 2023-04-03 | 2024-04-26 | 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 |
| CN117264847B (en) * | 2023-11-01 | 2024-04-26 | 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 YTQ, compound agent and application thereof in improving low-temperature freeze injury resistance of plants |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113528370B (en) | 2022-12-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113528370B (en) | Bacillus belgii strain and application thereof | |
| CN109022315B (en) | Broad-spectrum disease-resistant biocontrol bacillus and application thereof | |
| CN103173397B (en) | Broad-spectrum antibacterial bacillus amyloliquefaciens strain and application thereof | |
| CN102965320B (en) | Bacillus subtilis for preventing and controlling plant fungal disease and application of bacillus subtilis | |
| CN105670958B (en) | One plant of low temperature biocontrol bacterial strain and its application | |
| CN113151062B (en) | Bacillus belgii LJBV19 and application thereof | |
| CN106479938B (en) | A kind of Brevibacillus brevis bacterial strain and its application | |
| CN109439577A (en) | One plant of broad-spectrum antiseptic bacillus amyloliquefaciens and its application | |
| CN109456921B (en) | Paenibacillus polymyxa, application thereof, microbial agent, powder and granules | |
| CN113969247A (en) | Bacterium for inhibiting tobacco disease pathogenic bacteria and application thereof | |
| Nursyirwani et al. | Isolation of cellulolytic bacteria from mangrove sediment in Dumai marine station Riau and the antibacterial activity against pathogens | |
| CN111286477B (en) | Pseudomonas syringae and application thereof in prevention and treatment of apple ring rot | |
| CN112458011A (en) | Bacillus amyloliquefaciens ZJK1 and application thereof | |
| CN113699065B (en) | Bacillus vallismortis and application thereof | |
| Toppo et al. | Isolation and characterization of bacterial antagonist to plant pathogenic fungi (Fusarium spp.) from agro based area of Bilaspur | |
| CN108118004B (en) | Application of pichia stipitis in preventing and treating postharvest diseases of fruits | |
| CN102604869A (en) | Bio-control bacteria strain 1JN2 for preventing and treating bacterial fruit blotches of watermelons and application thereof | |
| KR20080045346A (en) | Bacillus subtilis m27 and biological control of sclerotinia rot by using the same | |
| KR101139794B1 (en) | Biological control of plant diseases by mixed Bacillus subtilis M27 and 5M43 | |
| CN114085788B (en) | Pseudomonas amazonensis strain and application thereof | |
| CN116731892A (en) | Antibacterial bacillus bailii Y103-16 and application thereof | |
| CN107058180B (en) | Endophytic bacterium bacillus subtilis strain and microbial agent of pasture in alpine grassland as well as preparation method and application of strain and microbial agent | |
| WO2022095127A1 (en) | Biocontrol strain yw-1, preparation of biocontrol agent using biocontrol strain yw-1, and application of biocontrol agent | |
| CN114467975A (en) | Application of staphylococcus equi in prevention and treatment of fruit and vegetable diseases | |
| CN107488608B (en) | Streptomyces albidoflavus Z9 and application thereof in preventing and treating sunflower sclerotiniose |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |