CN116731892A - Antibacterial bacillus bailii Y103-16 and application thereof - Google Patents
Antibacterial bacillus bailii Y103-16 and application thereof Download PDFInfo
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- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses bacillus bailiiBacillus velezensis) Y103-16, the strain has been preserved in China center for type culture collection (CCTCC M20211545) in Wuhan City of Hubei province at 2021, 12 months and 13 days, and is classified and named as Bacillus belicusBacillus velezensis) Y103-16. The strain with stronger antibacterial activity is obtained by screening in apple resistant stock endophytes, the strain Y103-16 is subjected to further screening, domestication and breeding, morphological characteristics, 16S rDNA sequence determination and phylogenetic analysis, and antagonism of the strain is applied to apple ring spot, apple mould heart disease, cherry brown rot, cherry stem rot, grape anthracnose and grape gray moldThe bacteria have remarkable antibacterial effect, and the antibacterial activity of the fermentation liquor has good thermal stability, so that the bacteria have wide application fields.
Description
Technical Field
The invention relates to the technical field of biological control, in particular to bacillus bailii and the technical field of application of the bacillus bailii in fruit tree pathogenic fungi control.
Background
Chemical control is an important means for controlling plant diseases, however, a large amount of chemical pesticides are used for polluting the environment, and the long-term use of the chemical pesticides also seriously threatens the health of human beings. In order to reduce environmental pollution, promote plant growth and improve plant resistance to diseases, more and more microbial biocontrol microbial agents are gradually developed. The biocontrol bacteria can secrete various metabolites with antagonistic capability, and can inhibit or directly kill pathogenic bacteria. The resistant stock can improve the disease resistance and stress resistance of the fruit trees, and even can improve the continuous cropping resistance of the fruit trees. Therefore, the method for searching and screening the antibacterial endophyte from the fruit tree resistant stock is a novel method for screening and inhibiting the fungal diseases of the fruit tree.
Disclosure of Invention
Aiming at the problem that the prior art does not have researches on bacillus bailii (Bacillus velezensis) as antagonism bacteria of various fruit tree pathogenic fungi at home and abroad, the invention aims to provide an endophyte bacillus bailii (Bacillus velezensis) Y103-16 strain separated from apple resistant stocks and application thereof as antagonism bacteria of various fruit tree pathogenic fungi. Experiments prove that the bacteria with antagonism on the bacillus beijerinus (Bacillus velezensis) Y103-16 strain provided by the invention have outstanding antibacterial effects on alternaria mali, botrytis cinerea, anthracis viticola, brown rot of cherry and stem rot of cherry.
The technical scheme of the invention is as follows:
the strain with stronger antibacterial activity is obtained by screening antagonistic bacteria on apple resistant stock endophytes, and a strain with the number of Y103-16 is obtained through further screening, domestication and breeding. The classification status of the obtained strain is preliminarily determined by morphological characteristics and 16S rDNA sequence determination and phylogenetic analysis; meanwhile, the characteristics of bacteriostasis rate, stability and the like of the bacterial Y103-16 fermentation liquor are deeply studied, a new choice is provided for biologically preventing and treating fruit tree pathogenic fungi, and antagonism of the bacterial Y103-16 fermentation liquor has outstanding technical effects on solving apple ring rot, apple mould heart disease, grape gray mold, grape anthracnose, cherry brown rot and cherry stem rot.
The bacillus bailii (Bacillus velezensis) serving as antagonism of various fruit tree pathogenic fungi is obtained by separating, screening and culturing in apple resistance stocks, a batch of microbial strains serving as antagonism of fruit tree pathogenic fungi is obtained, a strain with the number of Y103-16 is screened out from the microbial strains, and the bacillus bailii has stable and obvious antagonism effects on alternaria mali, botrytis cinerea, anthracnose, cherry brown rot and cherry stem rot, and belongs to bacillus bailii (Bacillus velezensis) through microbiological classification and identification.
Specifically, bacillus belicus serving as fruit tree pathogenic fungi antagonistic bacteria is provided by the invention, and the Latin name of a biological material is: bacillus velezensis, designated Y103-16. The strain has been preserved in China Center for Type Culture Collection (CCTCC) prior to the application date. Address: eight-path 299 university of Wuhan, hubei province, china center for type culture Collection, post code: 430072. the preservation date is 2021, 12 months and 06 days, and the preservation number is CCTCC NO. M20211545. Identified microbiologically as bacillus beliensis (Bacillus velezensis). The optimal growth conditions of the strain are as follows: the temperature is 30 ℃, the culture medium (LB) is 1 percent (W/V) of peptone, 0.5 percent (W/V) of yeast extract, 1 percent (W/V) of sodium chloride, 2 percent (W/V) of agar, the pH is 7.0-7.2, and the sterilization is carried out for 20 minutes at 121 ℃; culturing on LB plate at 30deg.C for 14-16 hr to obtain colony with medium size, milky white color, opaqueness, irregular edge, flatness, and dry surface; microscopic observation of the bacterial cells in a short rod shape, and the bacterial cells grow in spores; the strain Y103-16 was morphologically determined according to the ninth edition of Bergey's Manual of Systematic Bacterio-log, and the Manual of identification of bacterial systems, physiological and biochemical tests, to determine that the strain Y103-16 was Bacillus bailii (Bacillus velezensis). By BLAST homology alignment, the 16S rDNA sequence of strain Y103-16 was most similar to the 16S rDNA sequence of Bacillus velezensis, and thus strain Y103-16 was identified as Bacillus belicus (Bacillus velezensis).
Further, the invention relates to application of bacillus beijerinus (Bacillus velezensis) Y103-16 as antagonistic bacteria of alternaria mali, botrytis cinerea, botrytis anthracis, brown rot of cherry and stem rot of cherry.
Further, the activity of the fermentation broth of the Bacillus bailii (Bacillus velezensis) Y103-16 strain can still maintain the antibacterial rate of more than 80% after being treated for 15 minutes at 40 ℃, 60 ℃ and 100 ℃.
Furthermore, the fermentation liquor of the bacillus beijerinus (Bacillus velezensis) Y103-16 strain can still keep 82.93 percent of antibacterial rate after being treated for 15 minutes at the temperature of 100 ℃, and the thermal stability of active substances is high.
By implementing the specific invention content, the following beneficial effects can be achieved: the bacillus beleisii (Bacillus velezensis) Y103-16 is bacillus, has higher application value in the aspects of apple ring rot, apple mould heart disease, grape gray mold, grape anthracnose, cherry brown rot and cherry stem rot prevention and treatment, and particularly has positive effects on improving crop yield, reducing pesticide consumption and constructing a good ecological environment aiming at antagonism bacteria which are separated from local habitats and adapt to local climate and growth environment.
Drawings
FIG. 1 shows a colony morphology of Bacillus belicus (Bacillus velezensis) Y103-16.
FIG. 2 is a 16SrDNA phylogenetic tree of Bacillus beleiensis (Bacillus velezensis) Y103-16.
FIG. 3 shows the inhibition of Bacillus belicus (Bacillus velezensis) Y103-16 against the plates of Rhizoctonia cerealis, botrytis cinerea, monilinia cerealis and Pursh cherry.
FIG. 4 shows the inhibition of Bacillus belicus (Bacillus velezensis) Y103-16 sterile fermentation broth against Rhizoctonia cerealis and Bacillus viticola.
FIG. 5 is a graph showing the temperature sensitivity measurement of the antibacterial activity of Bacillus subtilis (Bacillus velezensis) Y103-16 sterile fermentation broth against Rhizoctonia cerealis after treatment at different temperatures.
Detailed Description
The present invention is illustrated by the following examples, but the present invention is not limited to the following examples.
The invention relates to main raw materials, auxiliary materials, reagents and instrument equipment: the pathogenic bacteria to be tested, namely, alternaria mali (Botryosphaeria dothidea), apple heart germ (Trichothecium roseum), cherry brown rot germ (Monilinia fructicola), cherry stem rot germ (Phytophthora nicotiana), grape anthracnose germ (Colletotrichum gloeosporioides) and grape gray mold germ (Botrytis Cinerea) are separated, identified and stored by the smoke counter academy of sciences; separating and culturing endophytes from apple resistant stock branches which are independently cultivated by a tobacco stand academy of sciences; the culture medium adopts potato dextrose culture medium (PDA), LB culture medium; SPX-280B-Z biochemical incubator, YXQ-70A high-pressure steam sterilizing pot, SW-CJ-2F purifying workbench, SIGMA3K15 centrifuge, HZQ-F160 full-temperature oscillating table. Veriti96 PCR instrument, electrophoresis instrument Bio-Rad Mode 200/2.0, CX5 gel imager, PCR premix (TaKaRa Biotechnology) and the rest of the reagents are all analytically pure.
All of the raw materials, reagents and equipment selected for use in the present invention are well known in the art, and other reagents and equipment known in the art may be suitable for use in the practice of the following embodiments of the invention.
Embodiment one: isolation, screening and identification of Bacillus bailii (Bacillus velezensis) Y103-16
(1) Isolation and screening of antagonistic bacteria
Washing current annual branches of resistant stocks in an apple orchard of an agricultural scion under water for 15min, cutting into small sections with the length of 2-3 cm, sterilizing with 75% alcohol for 30s, soaking with 0.1% raw mercury solution for 8min, washing with sterile water for 3-5 times, removing the outermost layer of the washed branches, reserving xylem, cutting into small blocks, placing into a 2mL centrifuge tube containing small steel balls, adding 500 mu L of pure water into a vibration crusher to crush into slurry, pouring onto an LB flat plate, uniformly spreading with a spreader, placing into a 30 ℃ incubator for culturing until the separated matters grow out, scribing, separating and purifying bacterial colonies growing on the flat plate, and finally scribing and preserving the inclined plane for later use.
(2) Screening for antagonistic bacteria by plate counter method
Activating pathogenic bacteria of fruit trees on an LB culture medium plate by adopting a plate counter method, placing a puncher punching bacterial cake in the center of another fresh LB culture medium plate after hyphae grow out, inoculating 4 separated antagonistic bacterial strains to be selected around pathogenic bacterial sheets, culturing at a constant temperature of 30 ℃ in each dish, and judging the antagonistic effect according to the existence of a bacteriostasis ring and the size of a bacteriostasis zone. And simultaneously, carrying out a secondary counter experiment on the strain with the antagonism, and screening out the strain with stronger antagonism for subsequent experiments.
Separating a plurality of strains of bacteria from apple resistant stocks, judging whether the apple resistant stocks have antagonism according to the existence of a bacteriostasis zone by adopting a flat plate counter method, and primarily screening out strains with a certain antagonism to pathogenic bacteria of fruit trees. And (3) carrying out a secondary counter experiment on the strain with the antagonism, and screening out the strain with stronger antagonism for subsequent experiments.
The bacterial isolate with good antagonism is obtained by re-screening, is named as Y103-16, can obviously inhibit the growth of pathogenic bacteria hypha of fruit trees after being cultured for 72 hours, and has stable antibacterial effect through multiple experiments.
Referring to figure 1, the obtained Y103-16 bacteria are cultured for 14-16 hours at 30 ℃ on an LB culture medium plate, and the bacterial colony is of equal size, is milky white, opaque, irregular in edge, flat and dry in surface; microscopic observation of the bacterial cells in a short rod shape, and the bacterial cells grow in spores; the morphology of the Y103-16 strain was examined and the shape, size and physiological and biochemical reactions of the strain were examined according to the ninth edition of Bergey's Manual of Systematic Bacterio-log, and the biological properties of the Y103-16 strain of the present invention are shown in Table 1.
TABLE 1 physiological and biochemical characteristics of Strain Y103-16
| Project | Results | Project | Results |
| V-P | - | Contact enzyme | + |
| Methyl red | - | Oxidase enzyme | + |
| Starch-dissolving hydrolysis | + | Maltose | + |
| Nitrate reduction | - | Mannitol (mannitol) | + |
| Hydrogen sulfide test | + | Lactose and lactose | + |
| Gram staining | + | Sucrose | - |
(2) Antagonistic 16S rDNA sequence for PCR amplification and sequencing thereof
Antagonistic 16S rDNA sequences were amplified using colony PCR. The PCR amplification reaction system was 50. Mu.L, containing 24. Mu.L of premix Taq, 1. Mu.L of upstream primer, 1. Mu.L of downstream primer, and 24. Mu.L of sterile water with a small amount of bacteria as a template.
Amplification conditions: 94℃for 4min,94℃for 30s,55℃for 30s,72℃for 60s,32 cycles; and at 72℃for 10min. The amplified product (about 1500 bp) was identified by 1% agarose gel electrophoresis and the PCR product was directly subjected to two-way sequencing.
Upstream primer (27F): 5'-TCCTCCGCTTATTGATATGC-3';
downstream primer 2 (1492R): 5'-CAAACTTGGTCATTAGAGGA-3'.
16S rRNA sequence alignment and phylogenetic analysis
Sequencing the amplified product by agarose gel electrophoresis to detect target bands, analyzing the sequencing result by using BioEdit and MEGA7.0, performing BLAST comparison on NCBI website to determine strain classification, and finally constructing a phylogenetic tree for the obtained monoclonal strain, wherein the similarity of the strain Y106-13 to Bacillus bailii (Bacillus velezensis) is up to 99%, and the result is shown in figure 2.
Embodiment two: determination of bacteriostasis spectra
The bacteriostasis spectrum of the strain Y103-16 is detected by adopting a counter culture method. The antibacterial effects of the strain on the alternaria mali (Botryosphaeria dothidea), the apple heart germ (Trichothecium roseum), the cherry brown rot germ (Monilinia fructicola), the cherry stem rot germ (Phytophthora nicotiana), the grape anthracnose germ (Colletotrichum gloeosporioides) and the grape gray mold germ (Botrytis Cinerea) are respectively measured. Firstly, pathogenic bacteria are activated on a PDA flat plate, a pathogenic bacteria cake is manufactured by a 6mm puncher, inoculated in the center of the PDA flat plate, and 2 mu L of bacterial strain Y103-16 bacterial suspension (10) 9 CFU/mL) was cultured at a distance of 4cm from the cake at 30 ℃ for 4 to 7 days, the pathogenic bacteria diameters of the control group and the treatment group were measured, and the inhibition ratio was calculated as inhibition ratio= (control colony diameter-treatment group colony antagonistic bacteria direction diameter)/(control colony diameter-inoculated bacteria mass diameter) ×100%.
The results show that the strain Y103-16 has broad-spectrum antibacterial property, has better inhibition effects on 6 fruit tree fungus pathogenic bacteria, namely apple ring rot, apple heart disease, cherry brown rot, cherry stem rot, grape anthracnose and grape gray mold, and the inhibition rates are 68.18%, 93.33%, 50.00% and 47.06%, 60.71% and 70.37% respectively, and the bacterial colony diameter and the control bacterial colony diameter after the inhibition of the strain Y103-16 are shown in the table 2, and the antibacterial effect is shown in figure 3.
TABLE 2 bacterial colony diameter, control colony diameter and antibacterial Rate
Embodiment III: determination of bacteriostatic activity of fermentation liquor
2 pathogenic bacteria of the apple ring spot bacteria and the grape anthracnose bacteria are selected to detect the antibacterial activity of the fermentation liquor of the strain Y103-16. Culturing potato liquid culture medium with strain Y103-16 in shaking incubator at 30deg.C for 24 hr, centrifuging 12000r/min for 20min, collecting supernatant, and filtering with bacterial filter. The filtrate after filtration sterilization was plated to PDA medium at a ratio of 1:10. The pathogenic bacteria of the apple ring spot and the grape anthracnose after activation are beaten into bacterial cakes by a 6mm puncher, and then inoculated in the center of a PDA flat plate and cultured for 4-7d in a 30 ℃ incubator. The pathogen diameters of a control group and a treatment group are respectively measured, the inhibition rate is calculated, and the calculation formula is as follows: inhibition ratio = (control colony diameter-treatment group colony diameter)/(control colony diameter-inoculation pellet diameter) ×100%.
The results show that the fermentation broth of the strain Y103-16 has good antibacterial activity, the inhibition rates of the strain Y103-16 on the colletotrichum apple ring spot and the colletotrichum grape anthracnose are 91.56% and 57.50%, and the bacterial colony diameter, the control bacterial colony diameter and the antibacterial rate after the fermentation broth of the strain Y103-16 is inhibited are shown in the table 3, and the antibacterial effect is shown in figure 4.
TABLE 3 diameter of bacterial colony after inhibition of Y103-16 fermentation broth, diameter of control colony and antibacterial rate
Embodiment four: temperature sensitivity of bacteriostatic activity of fermentation broth
Selecting the apple ring spot germ detection strain Y103-16 sterile fermentation broth for antibacterial activity. Strain Y103-16 was inoculated into 200mL of liquid LB medium, cultured with shaking at 30 ℃ for 24 hours at 200r/min, centrifuged at 12000r/min for 10min, and the supernatant was collected and sterilized by filtration with a bacterial filter. The supernatant after filtration and sterilization is respectively treated in a water bath kettle with the temperature of 20 ℃, 40 ℃, 60 ℃ and 100 ℃ for 15min. Mixing the treated supernatant with PDA culture medium according to the ratio of 1:10 to prepare a flat plate, inoculating apple ring rot pathogen cake at the center of a culture dish after the culture medium is solidified, culturing for 4-7 days in a 30 ℃ incubator, respectively measuring the diameters of pathogenic bacteria of a control group and a treatment group, and calculating the inhibition rate, wherein the calculation formula is as follows: inhibition ratio = (control colony diameter-treatment group colony diameter)/(control colony diameter-inoculation pellet diameter) ×100%.
The bacterial strain Y103-16 has strong high temperature resistance. The diameters of all treated colonies after being treated in a water bath kettle for 15min at 20 ℃, 40 ℃, 60 ℃ and 100 ℃ are respectively 1.3cm, 1.46cm, 1.47cm and 2cm, the diameters of control colonies are respectively 8.8cm, the antibacterial rate of fermentation liquor after being treated at 20 ℃, 40 ℃, 60 ℃ and 100 ℃ is 91.46%, 89.51%, 89.39% and 82.93%, and the antibacterial activity is still high-efficiency after being treated at 100 ℃ for 15min, which indicates that the heat stability of the antibacterial active substances of the fermentation liquor is good, and fig. 5 is an inhibition picture of apple ring rot bacteria after being treated at different temperatures.
Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.
Claims (5)
1. Antibacterial bacillus bailiiBacillus velezensis) Y103-16, characterized in that: the preservation number is CCTCC M20211545.
2. An antibacterial bacillus bailii according to claim 1Bacillus velezensis) The Y103-16 can be used as antagonistic bacteria of apple ring rot, apple moldy heart disease, cherry brown rot, cherry stem rot, grape anthracnose and grape gray mold pathogen.
3. An antibacterial bacillus bailii according to claim 2Bacillus velezensis) Y103-16, characterized in that: the strain fermentation liquor has stronger antibacterial effect on the colletotrichum gloeosporioides and the colletotrichum gloeosporioides.
4. An antibacterial bacillus bailii according to claim 3Bacillus velezensis) The use of Y103-16, characterized in that: the activity of the fermentation broth of the strain is treated at 40 ℃, 60 ℃ and 100 ℃ for 15 minutes, and the antibacterial rate is maintained to be more than 80%.
5. An antibacterial bacillus bailii according to claim 4Bacillus velezensis) Y103-16, characterized in that: the bacterial strain fermentation liquor is treated at 100 ℃ for 15 minutes to keep 82.93 percent of bacteriostasis rate, and the thermal stability of active substances is high.
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| 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 |
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