CN108148794B - Bacillus subtilis DYr3.3 with broad-spectrum antibacterial activity, and preparation method and application thereof - Google Patents
Bacillus subtilis DYr3.3 with broad-spectrum antibacterial activity, and preparation method and application thereof Download PDFInfo
- Publication number
- CN108148794B CN108148794B CN201810202589.1A CN201810202589A CN108148794B CN 108148794 B CN108148794 B CN 108148794B CN 201810202589 A CN201810202589 A CN 201810202589A CN 108148794 B CN108148794 B CN 108148794B
- Authority
- CN
- China
- Prior art keywords
- bacillus subtilis
- strain
- pear
- volume ratio
- rot
- 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.)
- Active
Links
Images
Classifications
-
- 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
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
-
- 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
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a bacillus subtilis DYr3.3 with broad-spectrum antibacterial activity, a preparation method and application thereof, and antagonistic bacteria bacillus subtilis DYr3.3 and CCTCC M2018024, and the steps are as follows: (1) collecting the roots of the purple branch roses, cutting the roots into tissues with different sizes, disinfecting the tissues by 70% alcohol, washing the tissues by sterile water, placing the tissues on a PDA culture medium for dark culture, transferring grown bacterial colonies onto a new PDA culture medium after culture, and streaking, separating and purifying single bacterial colonies; (2) after separation and purification, an antagonistic bacterium bacillus subtilis strain DYr3.3 is obtained, and the nucleotide sequence of the antagonistic bacterium bacillus subtilis strain DYr3.3 is shown as SEQ ID NO: 1, the application of the strain DYr3.3 in preparing a medicament for treating or preventing pear fruit rot. The DYr3.3 bacterial liquid obtained by liquid fermentation of the strain DYr3.3 can prevent pear fruit rot caused by pear ring rot and anthracnose, has a good disease prevention effect on the pear fruit rot, remarkably inhibits the pear fruit rot, has the prevention efficiency of 100 percent, and is widely applied to prevention and control of the pear fruit rot.
Description
Technical Field
The invention belongs to the technical field of biological control of plant diseases, and particularly relates to a bacillus subtilis DYr3.3 with broad-spectrum antibacterial activity, a preparation method of the bacillus subtilis DYr3.3 with broad-spectrum antibacterial activity, and application of the bacillus subtilis DYr3.3 with broad-spectrum antibacterial activity.
Background
Since the production places of the fruit and vegetable products are far away from cities and the maturation periods are relatively concentrated, a certain storage and transportation period is required, and a great amount of postharvest loss, particularly rotting of the fruits and vegetables, is caused in the period. It is reported that 10% -30% of fresh fruit is lost in the postharvest rot in developed countries, and that in developing countries lacking storage and transportation refrigeration equipment, the rate of spoilage is as high as 40% -50%. At present, the prevention and the treatment of the diseases are mainly carried out by chemical agents, which not only causes serious harm to people, livestock and ecological environment, but also increases the residual quantity of toxic chemical substances in agricultural products, and simultaneously is easy to induce the generation of drug resistance of plants to pathogenic bacteria. In recent years, researchers have replaced some traditional chemical pesticides with bacillus to provide new research ideas and methods for controlling postharvest diseases of fruits and vegetables [ Tianshihefangqing, biological technology for controlling postharvest diseases of fruits and vegetables, and botany report, 2000, 17 (3): 211- & ltCHEM & gt 217 ].
The pear fruits are often rotten during storage after being harvested, and the rotten during the storage after being harvested is often caused by infection of ring rot and anthracnose pathogen. Currently, studies have been made on the use of bacillus to control apple rot during storage, including the use of bacillus licheniformis to control apple rot [ gigaforest, etc. ] caused by apple ring rot and anthracnose, the inhibition of bacillus licheniformis to apple ring rot and anthracnose and its control of apple ring rot during storage, academy of fruit trees, 2008, 25 (2): 209-214 ], the disease prevention rate of the treated apple fruits reaches 33.0%; the bacillus amyloliquefaciens NCPSJ7 is adopted to prevent and treat the ring rot of picked apples [ Huangling and Ling ], the bacillus amyloliquefaciens NCPSJ7 is adopted to biologically prevent and treat the ring rot of picked apples, and Chinese food and nutrition 2015 and 21 (2): 20-24 ]; the bacillus subtilis B-903 is adopted to control the ring spot of the picked apples, the treatment effect of inoculating 10 times of B-903 bacterial liquid and 20 times of the B-903 bacterial liquid before germ invasion is better, and the inhibition effect of 7d reaches 64.4-68.2%; the inhibition effect of B-903 treatment after the invasion and the onset of pathogenic bacteria is 13.5-25.8% [ Zhao Baige pigeon, etc., the inhibition effect of Bacillus subtilis B-903 on apple ring rot bacteria and the control effect on diseases, the report of plant pathology, 1996, 27(3)213- ] 214 ]. In the research, various bacilli are mainly used for controlling ring spot and anthracnose of picked apples, but the effect of each strain is not ideal enough, and the working concentration is low (the stock solution or the solution is diluted by 20 times slightly), so that the practical application requirement in production cannot be met. Screening and utilizing bacillus subtilis to prevent and treat the pear ring rot after picking, separating 21 bacillus subtilis strains from a pear sample, inhibiting the rot of pear fruits inoculated with the pear ring rot, inhibiting the lesion spreading efficiency to be more than 40% [ butyrobeys ], screening and utilizing the bacillus subtilis to prevent and treat the pear ring rot, Jiangsu agriculture bulletin, 2009, 25 (5): 1002-1006), the requirements of practical application in production are far from being met.
Disclosure of Invention
The invention aims to provide a Bacillus subtilis DYr3.3 with broad-spectrum antibacterial activity, which has broad-spectrum antifungal activity and has remarkable antibacterial activity on main pathogenic fungi of pear trees, including bougainveria berangeriana, anthracnose (including Colletotrichiumc. In addition, the compound has good antagonistic activity on other common horticultural crop pathogenic fungi, such as colletotrichum japonicum (Colletotrichumcamelliae), phomopsis (Phomopsis), Pestalotiopsis theobroma and Penicillium citrinum (Penicillium citrinum).
Another purpose of the invention is to provide a preparation method of the bacillus subtilis with broad-spectrum bacteriostatic activity, the method is easy and convenient to operate, after liquid fermentation, a liquid preparation which is convenient to dissolve in water for use and is storable is obtained, and the spore concentration is measured to be 4.41 multiplied by 107cfu/mL
The invention also aims to provide application of the Bacillus subtilis DYr3.3 with broad-spectrum antibacterial activity in preparing a medicament for treating or preventing pear fruit rot, wherein a fermentation product of the strain has a good disease prevention effect on the pear fruit rot, the pear fruit rot is remarkably inhibited, and the prevention effect can reach 100%.
In order to achieve the purpose, the invention adopts the following technical measures:
the research discovers a bacillus subtilis strain DYr3.3 which can strongly inhibit the decay of pear fruits caused by ring rot fungi and anthrax, the effect can reach 100 percent, and the bacillus subtilis strain has obvious practical application potential.
A preparation method of broad-spectrum antibacterial activity bacillus subtilis comprises the following steps:
(1) collecting the root of the purple branch rose in Dazhu rose land of Huangshi city, Hubei province, cutting the root into a tissue with the size of about 0.5cm, sterilizing with 70% (volume ratio) alcohol for 1min, washing with sterile water for 2-3 times, placing on a PDA culture medium for dark culture at 24-26 ℃, transferring the grown bacterial colony to a new PDA culture medium for 34-38 h, and streaking, separating and purifying the single bacterial colony;
the preparation method of the PDA culture medium comprises the following steps: dissolving 200g of potatoes, 20g of cane sugar and 15g of agar powder in 1000mL of distilled water, and adjusting the pH value to about 6.8-7.2;
(2) after separation and purification, an antagonistic bacterium Bacillus subtilis strain DYr3.3 from the root of the purple branch rose is obtained, and the nucleotide sequence of the antagonistic bacterium Bacillus subtilis strain DYr3.3 is shown as SEQ ID NO: 1, the strain DYr3.3 is preserved in a China Center for Type Culture Collection (CCTCC) in 2018, 1 month and 12 days, the preservation number is CCTCC M2018024, and the DYr3.3 has strong antagonistic capability on ring rot pear bacteria (shown in figure 1) and various pathogenic bacteria (shown in figure 3) in a plate confrontation experiment and has good control effect on pear fruit rot;
the application of broad-spectrum bacteriostatic activity of bacillus subtilis DYr3.3 in preparing a medicament for treating or preventing pear fruit rot comprises the following steps:
A. a single colony of the DYr3.3 strain is selected in an aseptic environment, inoculated in a 250mL triangular flask filled with 90mL of an optimal culture solution A (3.0 percent (mass-volume ratio) of glucose, 0.5 percent (mass-volume ratio) of yeast extract powder, 0.3 percent (mass-volume ratio) of sodium chloride and 0.1 percent (mass-volume ratio) of magnesium sulfate, and the pH value is adjusted to be 6.0-6.5), and subjected to shaking culture at 28 ℃ and 150r/min for 24h to obtain a seed culture solution.
B. Preparing 10 500mL triangular bottles, filling 200mL culture solution A in each bottle, adding the seed culture solution obtained in the step A into each bottle according to the proportion of 1:100, placing the bottles in a large-amplitude constant-temperature shaking table, and carrying out shaking culture at the temperature of 28 ℃ for 48 hours at 150r/min to obtain DYr3.3 fermentation liquor with the concentration of 4.89 multiplied by 106cfu/mL。
C. And D, taking the fermentation liquor of the DYr3.3 strain obtained in the step B, diluting the fermentation liquor by 100 times with clear water, soaking the pear fruits for 30s, taking out the pear fruits, airing the pear fruits, and storing the pear fruits at a low temperature (4 ℃) or room temperature (20-25 ℃) which is the same as the below).
The strain DYr3.3 is subjected to liquid fermentation to obtain DYr3.3 bacterial liquid, which can prevent pear fruit rot caused by pear ring rot and anthracnose, and can be applied to prevention and control of pear fruit rot.
Compared with the prior art, the invention has the following advantages and effects:
(1) the pear fruits are often rotten during storage after being harvested, and the rotten during the storage after being harvested is often caused by infection of ring rot and anthracnose pathogen. At present, no bacillus biocontrol agent for effectively preventing pear fruit from rotting exists, and bacillus subtilis DYr3.3 has a remarkable effect of preventing and treating pear fruit from rotting and has the advantages and effects of being applied to fruit and vegetable fresh-keeping.
(2) The commercially developed bacillus subtilis strain is mainly used for field crop field disease control, but not fruit postpartum disease control, for example, the bacillus subtilis B-916 strain found by the plant protection research institute of Jiangsu agricultural academy of sciences is subjected to pesticide registration and is applied to field control of rice sheath blight; biocontrol bacterium B3 (trade name Meifengning) of Nanjing university of agriculture is applied to the field control of wheat sharp eyespot. Thus, strain DYr3.3 has a different direction of application than other strains of Bacillus subtilis which have already been commercialized.
(3) In the early stage, bacillus subtilis applied to preventing and treating the ring rot pathogen is screened. For example, there are researchers who isolated 21 bacillus subtilis strains from pear samples, which can inhibit lesion extension with an efficiency of about 40% [ see: dingxiang soldiers, screening and usingBacillus subtilis for preventing and treating pear ring rot, Jiangsu agricultural science, 2009, 25 (5): 1002-1006); the control effect of the biocontrol bacterium 11 on the pear ring rot is 23.83 percent (see the details: liumailang, etc., sodium bicarbonate (NaHCO)3) The fruit tree bulletin 2010,27(5): 757-; indoor inhibition efficiency of 2 stock solutions of bacillus subtilis Sf-19 and Sf-28 on pyricularia pyricularis bacteria is 71.97% and 64.54% [ see: zhali et al, 2-strain Bacillus subtilis indoor inhibitory action research on Ribes burejense, fruit tree bulletin, 2010,27(5): 823, 827. Similar research also adopts bacillus subtilis to control apple ring rot after picking, but the result shows that the prevention and control effect of the strain is poor, the inhibition effect of 7d under the treatment of 10 times liquid and 20 times liquid reaches 64.4-68.2 [ Zhao Baige pigeon, etc. ], the inhibition effect of bacillus subtilis B-903 on apple ring rot and the control effect on the apple ring rot are reported in the plant pathology, 1996, 27(3)213- "214 ]. In conclusion, the effect of the earlier-separated bacillus subtilis in the prevention and treatment experiment for preventing and treating the pear ring rot after picking is not obvious, so that the DYr3.3 strain has a better effect of preventing and treating the pear rot than the earlier-obtained bacillus subtilis strain.
(4) Other bacilli have also been studied to control apple rot during storage, including the use of bacillus licheniformis and bacillus amyloliquefaciens to control apple rot caused by ring rot and colletotrichum gloeosporioides, but the resulting strains have insignificant disease control effects [ see: the inhibition of bacillus licheniformis to apple ring spot and anthracnose and the prevention and treatment effect thereof to apple ring spot in storage period, such as meglin, 2008, 25 (2): 209 to 214; fructus syringae, etc., adopts bacillus amyloliquefaciens NCPSJ7 to perform biological control function on the ring rot of picked apples, and Chinese food and nutrition 2015, 21 (2): 20-24. Therefore, the bacillus subtilis strain DYr3.3 has better pear fruit rot control effect than other bacillus species strains obtained in the early stage.
Drawings
FIG. 1 shows the morphology of a Bacillus subtilis strain DYr3.3 and the effect of inhibiting Verticillium pyricularis.
Wherein, the middle of the PDA culture medium is a strain of Rhizopus pyricularis (Botryosphaeria berengiana), and the growth of the strain is inhibited by surrounding strains; several colonies of strain DYr3.3 were surrounded by PDA medium.
FIG. 2 is a graph showing the linear relationship between absorbance and concentration of a Bacillus subtilis strain DYr3.3 at a wavelength of 600 nm.
In the figure, a single colony of a strain DYr3.3 is picked and placed in a culture medium A (3.0% of glucose, 0.5% of yeast extract powder, 0.3% of sodium chloride and 0.1% of magnesium sulfate, the pH value is adjusted to 6.0-6.5), after culturing for 48 hours at 28 ℃, diluted according to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50 and 100 times, and the light absorption value under the wavelength of 600nm is measured. Diluting 50 μ L to 10-5、10-6And (3) doubling, inoculating the bacterial strain on the most suitable solid culture medium for plate coating, sealing, placing the bacterial strain in a constant-temperature incubator at 28 ℃ for culture, and periodically observing until clear and countable bacterial colonies appear, and then determining. Measuring OD600 light absorption value and corresponding cell biomass, showing that the light absorption value (x) and the cell amount (y) are in positive correlation, and the linear equation is that y is 45,607,035x-831,951 (R)2＝0.9884)。
FIG. 3 is a diagram showing the bacteriostatic effect of Bacillus subtilis DYr3.3 on 12 fungal strains on PDA medium.
The center of the plate is streaked DYr3.3 bacteria liquid, and the two sides are inoculated fungus hypha blocks. CK (CK)-Control with no strain DYr3.3, but with only fungal hyphal blocks. The fungi and strains used are marked on top of the plate.
FIG. 4 is a bar graph of the antagonistic inhibition width of Bacillus subtilis strain DYr3.3 against 12 fungal strains on PDA medium. The ordinate is the bacteriostatic width, and the abscissa is the determination of fungi and strains. The significant difference analysis was performed using SPSS Statistics 21.0(WinWrap Basic;http://www.winwrap.com) Differential significance analysis was performed (Duncan's test, P ═ 0.05).
FIG. 5 is a graph showing the effect of DYr3.3 in inhibiting decay of pear fruit.
A, a left picture 'CK +' is a picture of fruit morbidity of inoculated pyricularia pyricularis (upper) and pyricularia pyricularis (lower) mycelium blocks; the right picture 'DYr3.3' is a picture that after the fermentation liquor of DYr3.3 is soaked, pear fruits are not rotten after the pear ring rot fungus hypha blocks (upper) and the pear anthrax fungus hypha blocks (lower); b, inoculating the pear ring rot fungus hypha block and the pear anthrax fungus hypha block separately and soaking DYr3.3 fermentation liquid, and inoculating the two kinds of bacteria to obtain a lesion length histogram.
Detailed Description
Example 1
A preparation method of broad-spectrum antibacterial activity bacillus subtilis comprises the following steps:
(1) collecting the root of the purple branch rose in Dazhu rose land of Huangshi city, Hubei province, cutting the root into a tissue with the size of about 0.5cm, sterilizing with 70% (volume ratio) alcohol for 1min, washing with sterile water for 2-3 times, placing on a PDA culture medium for dark culture at 25 ℃, transferring the grown bacterial colony to a new PDA culture medium for streaking, separating and purifying a single colony after culturing for 36 h;
the preparation method of the PDA culture medium comprises the following steps: dissolving 200g of potatoes, 20g of cane sugar and 15g of agar powder in 1000mL of distilled water, and adjusting the pH value to about 7.0;
(2) after separation and purification, an antagonistic bacterium Bacillus subtilis strain DYr3.3 from the root of the purple branch rose is obtained, and the nucleotide sequence of the antagonistic bacterium Bacillus subtilis strain DYr3.3 is shown as SEQ ID NO: 1, the strain DYr3.3 is preserved in a China Center for Type Culture Collection (CCTCC) in 2018, 1 month and 12 days, the preservation number is CCTCC M2018024, and the DYr3.3 has strong antagonistic capability on ring rot pear bacteria (shown in figure 1) and various pathogenic bacteria (shown in figure 3) in a plate confrontation experiment and has good control effect on pear fruit rot;
molecular characterization of antagonistic bacteria dyr 3.3:
extracting the genome DNA of antagonistic bacterium DYr3.3 [ see the details: identifying, disease prevention effect and growth promotion effect research of a potato ring rot biocontrol strain P1, a plant pathology report 2010, 40 (1): 66-73 ] using the extracted DNA as template, and BSF (5'-AGAGTTTGATCCTGGCTCAG-3') and BSR (5'-AAGGAGGTGATCCAGCCGCA-3') primers to amplify 16S rDNA, the expected size is 1500 bp.
And (3) PCR reaction system: 10 XPCR buffer (containing Mg)2+) 2.5. mu.L, 10mmol/L dNTPs 0.5. mu.L, 10. mu. mol homologous and complementary primers 0.5. mu.L each, Taq DNA polymerase 0.2. mu.L, DNA template 1. mu.L, deionized water 19.8. mu.L.
The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 4min, followed by denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 90s, 35 cycles, re-extension at 72 ℃ for 5min, and termination at 16 ℃.
The PCR products are analyzed by 1 percent agarose gel electrophoresis, and the target fragments are purified and sent to Shanghai biological engineering technology service company Limited for sequencing. The obtained sequence was subjected to similarity analysis with the sequences in the NCBI database using BLASTn, and the results showed that: the homology between the 16S rDNA sequence of the strain DYr3.3 and the 16S rDNA sequence of the Bacillus subtilis OPP 31 (sequence accession number: JQ308571.1) is 99% (coverage 98%, E value 0.0, identity 99%).
Biolog microorganism identification:
the strain is sent to the national emphasis laboratory of agricultural microbiology of university of agriculture in China to carry out Biolog microorganism identification, and 71 carbon source utilization rates and 23 chemosensitivity determinations are carried out. The method mainly comprises the following steps: the strain DYr3.3 is subjected to four-zone streak purification culture on a BUG plate, after overnight culture at 33 ℃ in the dark, a single colony is picked up and mixed in an inoculum IF-B, then the mixture is inoculated into a Biolog 96-hole identification plate, each hole is inoculated with 100 mu L, the identification plate is sealed and placed at 33 ℃ for dark culture for 16-24 hours, and then reading is carried out. The results show that the strain DYr3.3 has the highest similarity to Bacillus atrophaeus/subtilis, i.e., Bacillus atrophaeus and Bacillus subtilis (PROB 0.575; SIM 0.575; DIST 6.258; Organism Type GP-Rod-SB; specials Bacillus atrophaeus/subtilis). And D Yr3.3 is determined as Bacillus subtilis by combining the molecular identification result and the Biolog identification result.
Colony and morphological characteristics of antagonistic bacteria DYr3.3
On LB plates, the colonies of the Bacillus subtilis DYr3.3 strain were irregular, with irregular wavy projections on the edges, flat, milky white and opaque (see FIG. 1).
Culturing the antagonistic bacteria DYr3.3:
the culture conditions of the DYr3.3 strain are as follows: the strain activation adopts PDA culture medium, namely 200g of potato, 20g of cane sugar and 15g of agar powder are dissolved in 1000mL of distilled water, and the pH value is about 7.0. Taking a proper amount of bacterial liquid to perform streak culture on a PDA culture medium. After inoculation, the mixture is placed in a constant-temperature incubator at 28 ℃ for dark culture until bacterial colonies appear.
The culture medium A is used for fermentation culture of the strain DYr3.3 and determination of the number of spores, and the specific formula of the culture medium A is as follows: 3.0% of glucose (mass-volume ratio), 0.5% of yeast extract powder (mass-volume ratio), 0.3% of sodium chloride (mass-volume ratio) and 0.1% of magnesium sulfate (mass-volume ratio), and adjusting the pH value to 6.0-6.5; wherein 7-8 g of agar (per 500mL of culture medium) is added into the solid culture medium, and the culture medium is sterilized by high-pressure steam at 121 ℃ for 20 min.
And adding the culture medium A into the fermentation liquor obtained by culture to dilute the fermentation liquor to different times to obtain sample bacterial suspensions with the dilution times of 1, 2, 3, 4, 5, 10, 20, 30, 40, 50 and 100 times, and taking the culture medium A as a blank control. Measurement of sample OD at different dilution fold Using UITROSPEC nucleic acid protein Detector at wavelength of 600nm600The value is obtained. To improve the accuracy of the experimental data, 3 sets of replicates were prepared.
Preparing to dilute to 10-3、10-4、10-5、10-6Multiple sample suspensions for spore number determination. Diluting 50 μ L to 10-5、10-6Doubling the suspension of the DYr3.3 sample and diluting to 10-4、10-5And respectively inoculating the multiplied bacterial suspensions on the most suitable solid culture medium for plate coating, sealing, placing in a constant-temperature incubator at 28 ℃ for culture, and periodically observing until clear and countable bacterial colonies appear, and then determining. To improve the accuracy of the assay, 5 replicates of each dilution sample were made. The linear equation for measuring OD600 absorbance and biomass of the cells is y-45,607,035 x-831,951 (R)20.9884) (fig. 2).
Antagonism experiment of antagonistic bacteria
The antagonistic activity of the strain DYr3.3 on fungi is evaluated by adopting a plate confronting method: taking 10 mu L of shake-cultured bacterial liquid on a PDA culture medium, smearing the liquid in the middle of a culture dish, connecting a mycelium block with the diameter of 5mm at a position 4cm away from a bacterial line, setting non-inhibition bacterial treatment as a control, repeating 3 culture dishes for each treatment, carrying out dark culture at 25 ℃, measuring the size of an inhibition zone of each treatment when a control group just grows over the whole culture dish, and judging the antagonistic effect according to the existence of the inhibition zone and the size of the inhibition zone.
The antagonistic strains tested included Verticillium pyricularis (Botryosphaeria berengiana), Colletotrichum fructicola (Colletotrichumfructicola), Valsaxal var. pyri, Ralstonia pearicola (Phomopsis fukushi), Colletotrichum theacea (Colletotrichumcalaceae), Pestalotiopsis theobroma (Pestalotiopsis _ the), Phomopsis (Phomopsis), Penicillium italicum (Mucorracemus), and Rhizopus nigricans (Rhizopus nigricans), each of which was isolated in advance in this laboratory. The result shows that DYr3.3 has strong bacteriostatic action on other pathogenic fungi except for no obvious bacteriostatic action on rhizopus nigricans, the bacteriostatic range is 5.3-11.5mm, and the bacteriostatic effects on different pathogenic fungi have obvious difference.
Example 2
The application of Bacillus subtilis with broad-spectrum antibacterial activity in preparing or preventing pear fruit rot disease medicines comprises the following steps:
A. a single colony of the DYr3.3 strain is selected in an aseptic environment, inoculated in a 250mL triangular flask filled with 90mL of an optimal culture solution A (3.0 percent (mass-volume ratio) of glucose, 0.5 percent (mass-volume ratio) of yeast extract powder, 0.3 percent (mass-volume ratio) of sodium chloride and 0.1 percent (mass-volume ratio) of magnesium sulfate, and the pH value is adjusted to be 6.0-6.5), and subjected to shaking culture at 28 ℃ and 150r/min for 24h to obtain a seed culture solution.
B. Preparing 10 triangular flasks of 500mL each containing 200mL of fermentation broth, adding seed culture broth into each flask at a ratio of 1:100 (volume ratio), placing in a large-amplitude constant-temperature freezing shaker, and performing shaking culture at 28 deg.C for 48 hr at 150r/min to obtain DYr3.3 fermentation broth with concentration of 4.89 × 106cfu/mL。
Taking Bacillus subtilis DYr3.3 strain fermentation liquor, diluting with clear water by 100 times, soaking pear fruits for 30s, taking out, air drying, and storing at low temperature (4-8 ℃) or normal temperature (20-25 ℃).
Example 3:
experiment for preventing pear fruit rot by antagonistic bacteria
Diluting Bacillus subtilis strain DYr3.3 by 100 times to obtain a solution with a concentration of 4.89 × 106The fully mature pear fruits (crown pears in Hebei, Pyrus bretscherreihd. var. Huanggguanli) purchased from the supermarket are respectively soaked in the prepared bacterial suspension for about 30s, so that the surfaces of the pear fruits are fully and uniformly stained with bacteria, and then the pear fruits are taken out and dried. 6 replicates of each treatment group were inoculated with equal intervals of activated pyricularia piricola cakes (5 mm in diameter) at the waist of the fruits and the inoculated sites were covered with moistened cotton pieces soaked with sterile water. Taking another 2 pears which are not soaked by the bacterial suspension as CK+And (4) a control group. Placing the treated pears in plastic trays respectively paved with a layer of moist gauze and a preservative film, keeping the relative humidity in the trays at any time, sealing the plastic trays with the preservative film, and culturing at 30 ℃. And recording the result after the positive control is seriously attacked.
The results show that the DYr3.3 fermentation liquor is adopted to spray fruits and then inoculated with the mycelial block of pyrenoid (Botryosphaeria berengiana) to carry out a disease prevention experiment; meanwhile, the hypha blocks are inoculated as a control after sterile water is sprayed. The results show that after 2 days of inoculation, the control group sprayed with clear water and inoculated with the mycelium block starts to start pathogenic bacteria, the disease spots gradually expand to 5 days, the diseased fruits are basically all rotten, while the fruits treated by the DYr3.3 fermentation liquor have no disease, and the results are similar after three times of repetition.
Meanwhile, spraying the fermentation liquor of DYr3.3, inoculating mycelia of colletotrichum gloeosporioides (Botryosphaeria bergeriana) to the fruits, performing a disease prevention experiment, spraying sterile water to inoculate mycelia as a control, repeating the steps for 6 times, and counting the results after the fruits of the control group are completely rotten. The results show that after 7 days of inoculation, the hypha block inoculated after spraying clear water causes serious morbidity of the control group, the diseased fruits are basically all rotted, and the fruits treated by the strain DYr3.3 do not cause morbidity (see figure 5).
Sequence listing
<110> university of agriculture in Huazhong
<120> broad-spectrum bacteriostatic activity of bacillus subtilis DYr3.3, preparation method and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1488
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gccaaggtct gggagtctat aatgcagtcg agcggacaga tgggagcttg ctccctgata 60
ttagcggcgg acgggtgagt aacacgtggg taacctgcct gtaagactgg gataactccg 120
ggaaaccggg gctaataccg gatggttgtc tgaaccgcat ggttcagaca taaaaggtgg 180
cttcggctac cacttacaga tggacccgcg gcgcattagc tagttggtga ggtaacggct 240
caccaaggcg acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac 300
acggcccaga ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg 360
acggagcaac gccgcgtgag tgatgaaggt tttcggatcg taaagctctg ttgttaggga 420
agaacaagtg ccgttcaaat agggcggcac cttgacggta cctaaccaga aagccacggc 480
taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gaattattgg 540
gcgtaaaggg ctcgcaggcg gtttcttaag tctgatgtga aagcccccgg ctcaaccggg 600
gagggtcatt ggaaactggg gaacttgagt gcagaagagg agagtggaat tccacgtgta 660
gcggtgaaat gcgtagagat gtggaggaac accagtggcg aaggcgactc tctggtctgt 720
aactgacgct gaggagcgaa agcgtgggga gcgaacagga ttagataccc tggtagtcca 780
cgccgtaaac gatgagtgct aagtgttagg gggtttccgc cccttagtgc tgcagctaac 840
gcattaagca ctccgcctgg ggagtacggt cgcaagactg aaactcaaag gaattgacgg 900
gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca 960
ggtcttgaca tcctctgaca atcctagaga taggacgtcc ccttcggggg cagagtgaca 1020
ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1080
cgcaaccctt gatcttagtt gccagcattc agttgggcac tctaaggtga ctgccggtga 1140
caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca 1200
cacgtgctac aatggacaga acaaagggca gcgaaaccgc gaggttaagc caatcccaca 1260
aatctgttct cagttcggat cgcagtctgc aactcgactg cgtgaagctg gaatcgctag 1320
taatcgcgga tcagcatgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1380
acaccacgag agtttgtaac acccgaagtc ggtgaggtaa cctttatggt agccagccgc 1440
cgaaggtggg acagatgatt ggggtgaagt cgaagcaacg ttgcatag 1488
Claims (2)
1. Broad-spectrum bacteriostatic activity of bacillus subtilis (Bacillus subtilis)Bacillus subtilis) DYr3.3, characterized in that: antagonistic bacterium Bacillus subtilis (Bacillus subtilis) The strain DYr3.3 has a preservation number of CCTCC NO: M2018024.
2. The use of the broad-spectrum bacteriostatic bacillus subtilis DYr3.3 of claim 1 for preventing pear fruit rot, which comprises the following steps:
A. selecting a single colony of the DYr3.3 strain in an aseptic environment, inoculating the single colony in a 250mL triangular flask filled with 90mL of the optimal culture solution A, and performing shaking culture at 28 ℃ and 150r/min for 24h to obtain a seed culture solution;
B. preparing 10 500mL triangular bottles, filling 200mL culture solution A in each bottle, adding the seed culture solution obtained in the step A into each bottle according to the volume ratio of 1:100, placing the bottles in a large-amplitude constant-temperature shaking table, and performing shaking culture at the temperature of 28 ℃ for 48 hours at 150r/min to obtain a DYr3.3 fermentation solution with the concentration of 4.89 multiplied by 106cfu/mL；
C. B, taking the fermentation liquor of the DYr3.3 strain obtained in the step B, diluting the fermentation liquor by 100 times with clear water, soaking the pear fruits for 30s, taking out the pear fruits, airing the pear fruits, and storing the pear fruits at low temperature or room temperature;
the preparation method of the culture solution A comprises the following steps: 3.0% of glucose by mass and volume ratio, 0.5% of yeast extract powder by mass and volume ratio, 0.3% of sodium chloride by mass and volume ratio, 0.1% of magnesium sulfate by mass and volume ratio, and adjusting the pH value to 6.0-6.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810202589.1A CN108148794B (en) | 2018-03-13 | 2018-03-13 | Bacillus subtilis DYr3.3 with broad-spectrum antibacterial activity, and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810202589.1A CN108148794B (en) | 2018-03-13 | 2018-03-13 | Bacillus subtilis DYr3.3 with broad-spectrum antibacterial activity, and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108148794A CN108148794A (en) | 2018-06-12 |
| CN108148794B true CN108148794B (en) | 2020-12-22 |
Family
ID=62456663
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810202589.1A Active CN108148794B (en) | 2018-03-13 | 2018-03-13 | Bacillus subtilis DYr3.3 with broad-spectrum antibacterial activity, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108148794B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109810922B (en) * | 2019-02-26 | 2020-10-30 | 武威市林业科学研究院 | Pear dry blight antagonistic bacterium, biological agent and application thereof |
| CN109929782B (en) * | 2019-04-12 | 2022-10-11 | 江西省科学院微生物研究所 | Bacillus subtilis for inhibiting phomopsis and application thereof |
| CN110037100A (en) * | 2019-05-21 | 2019-07-23 | 河北农业大学 | It is a kind of prevent and treat fruit postharvest decay composition and its application |
| CN110583664B (en) * | 2019-09-09 | 2021-09-03 | 南通大学 | Application of 2,6-dicarboxy pyridine |
| CN112625966A (en) * | 2020-12-30 | 2021-04-09 | 天津开发区坤禾生物技术有限公司 | Microbial fertilizer for effectively preventing and treating pear tree rot and prevention and treatment method |
| CN112940994B (en) * | 2021-04-25 | 2022-08-23 | 金禾佳农（北京）生物技术有限公司 | Bacillus subtilis, culture method and application |
| CN113528377B (en) * | 2021-06-21 | 2023-02-03 | 山东碧蓝生物科技有限公司 | Bacillus subtilis with growth promoting and corrosion preventing effects and application thereof |
| CN114410528B (en) * | 2022-01-25 | 2023-07-21 | 山西农业大学食品科学与工程学院(山西省农业科学院农产品贮藏保鲜研究所) | Biocontrol bacterial liquid for storage period of Yulu fragrant pears and preparation method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695360A (en) * | 2014-01-13 | 2014-04-02 | 淮海工学院 | Separating method and applications of marine bacillus amyloliquefaciens 1A |
| CN104164394A (en) * | 2014-08-04 | 2014-11-26 | 北京林业大学 | Antagonistic phytopathogen strain and application thereof |
| CN104946567A (en) * | 2015-07-02 | 2015-09-30 | 北京市农林科学院 | Bacillus atrophaeus and application thereof |
| WO2016088747A1 (en) * | 2014-12-01 | 2016-06-09 | 日産化学工業株式会社 | Hydrazone derivative and pest control agent |
| CN106906172A (en) * | 2017-05-04 | 2017-06-30 | 陕西枫丹百丽生物科技有限公司 | One plant of Streptomycesalbidoflhaving and its application in terms of apple tree canker preventing and treating |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101870959B (en) * | 2010-06-11 | 2012-06-06 | 河北省科学院生物研究所 | Application of bsacillus subtilis, microbial inoculum thereof and preparations thereof in fruit fresh keeping field |
| KR20120010791A (en) * | 2010-07-27 | 2012-02-06 | 안동대학교 산학협력단 | Antifungal activity of Bacillus sp. AM44 against to multiple apple mold diseases and the improvement in the quality of apples |
| CN103131658B (en) * | 2013-03-15 | 2015-02-11 | 中国农业大学 | Bacillus and application thereof in control of apple ring spot |
| CN103173397B (en) * | 2013-04-02 | 2015-02-04 | 山东省农业科学院农产品研究所 | Broad-spectrum antibacterial bacillus amyloliquefaciens strain and application thereof |
| CN105907662A (en) * | 2016-04-08 | 2016-08-31 | 中国农业大学 | Bacillus subtillis and application thereof |
| CN107267423B (en) * | 2017-07-28 | 2020-09-01 | 中国农业科学院果树研究所 | Bacillus amyloliquefaciens L-1 for antagonizing pear diseases and application thereof |
-
2018
- 2018-03-13 CN CN201810202589.1A patent/CN108148794B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695360A (en) * | 2014-01-13 | 2014-04-02 | 淮海工学院 | Separating method and applications of marine bacillus amyloliquefaciens 1A |
| CN104164394A (en) * | 2014-08-04 | 2014-11-26 | 北京林业大学 | Antagonistic phytopathogen strain and application thereof |
| WO2016088747A1 (en) * | 2014-12-01 | 2016-06-09 | 日産化学工業株式会社 | Hydrazone derivative and pest control agent |
| CN104946567A (en) * | 2015-07-02 | 2015-09-30 | 北京市农林科学院 | Bacillus atrophaeus and application thereof |
| CN106906172A (en) * | 2017-05-04 | 2017-06-30 | 陕西枫丹百丽生物科技有限公司 | One plant of Streptomycesalbidoflhaving and its application in terms of apple tree canker preventing and treating |
Non-Patent Citations (2)
| Title |
|---|
| Effects of rhizobacteria Paenibacillus polymyxa APEC136 and Bacillus subtilis APEC170 on biocontrol of postharvest pathogens of apple fruits;Young Soo KIM等;《Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology)》;20161213;第17卷(第12期);931-940 * |
| Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens;Arrebola, E等;《Journal of Applied Microbiology》;20101231;第108卷(第2期);386-395 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108148794A (en) | 2018-06-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108148794B (en) | Bacillus subtilis DYr3.3 with broad-spectrum antibacterial activity, and preparation method and application thereof | |
| CN111172080B (en) | Bacillus belgii and application thereof | |
| KR101569737B1 (en) | Novel endophytic bacteria Bacillus oryzicola isolated from rice rhizosphere and development of a natural biopesticide and plant strengthener using same | |
| CN111100820B (en) | Bacillus belgii HMB28023 and application thereof | |
| CN108148793B (en) | Paenibacillus polymyxa DYr4.4 with broad-spectrum antibacterial activity and preparation method and application thereof | |
| CN112175888B (en) | Bacillus belgii Hsg1949 and application thereof | |
| CN113717901B (en) | Bacillus bailii and application thereof in preventing and controlling various vegetable diseases | |
| CN109456921B (en) | Paenibacillus polymyxa, application thereof, microbial agent, powder and granules | |
| CN107099484B (en) | Lysobacter enzymogenes and application thereof | |
| CN112322560B (en) | Bacillus belgii and application thereof in prevention and control of pear diseases | |
| CN111040976B (en) | Bacillus amyloliquefaciens and application thereof | |
| CN113005056A (en) | Bacillus belgii HY19 and application thereof | |
| CN112481160B (en) | Paenibacillus polymyxa and application thereof in prevention and control of pear diseases | |
| CN113604376B (en) | Sugarcane endophytic bacillus subtilis and application thereof | |
| CN113151044B (en) | Pseudomonas for preventing and treating diseases of radix pseudostellariae and separation, screening and identification method and application | |
| CN110317747A (en) | A kind of bacillus amyloliquefaciens JT68 and its application in prevention and treatment tea anthracnose | |
| CN112358991B (en) | Paenibacillus CL01 and application thereof | |
| CN117165494A (en) | Kiwi fruit canker biocontrol strain Wq-1 and application thereof | |
| CN115960777B (en) | Bacillus pseudomycoides and application thereof in prevention and treatment of vegetable epidemic disease | |
| CN116536207A (en) | Bacillus atrophaeus WLKYSY-4, biological microbial inoculum and application thereof | |
| CN108102992B (en) | Microbacterium aurantiacus and application thereof in prevention and treatment of tomato root-knot nematodes | |
| CN115029261A (en) | Biocontrol compound microbial agent as well as preparation method and application thereof | |
| CN111363695B (en) | Apple tree rot biocontrol microbial inoculum and preparation method and application thereof | |
| CN112322561A (en) | Klebsiella and application thereof in prevention and control of fruit tree pear fire blight | |
| CN117535178B (en) | Biological control composite bacteria for bacterial soft rot and application thereof |
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 |