CN111100806B - Areca-nut root rot bactericide prepared by taking boron-resistant lysine bacillus as underpan cells - Google Patents
Areca-nut root rot bactericide prepared by taking boron-resistant lysine bacillus as underpan cells Download PDFInfo
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Abstract
The invention relates to application of lysine bacillus borandii in inhibiting growth of betel nut rhizoctonia rot (Cerrena unicolor). The boron-resistant lysine bacillus (lysine bacillus boronitolerans) not only can reduce the occurrence of the betel nut root rot to a great extent, but also has no threat to the ecological environment, is expected to be used as a high-quality chassis microorganism for synthetic biology research, and brings a new direction for the prevention and treatment of the betel nut root rot.
Description
Technical Field
The invention relates to synthetic biology transformation and application of biocontrol bacteria, in particular to a bactericide for treating root rot of areca nuts.
Background
Arecae semen contains various nutrients and beneficial compounds required by human body, such as fat, arecae semen oil, alkaloid, catechin, choline, etc. Betel nut has a unique function of malaria, and is a herb of Miao miao, also called "miasma", which is a name for the treatment of diseases in physicians of all generations. Because the miasma is usually related to irregular diet and stagnation of qi, while Bing Lang can descend qi, promote digestion and dispel phlegm, its medicinal properties are widely noticed. Meanwhile, reports show that the betel nut has the function of expelling parasites, such as paralysis on the liver fluke nervous system, inhibition on schistosome liver metastasis, expelling tapeworm, pinworm, worm, ascaris and the like. Even reports show that the betel nut has different degrees of resistance to pathogenic microorganisms, hypertension, cancer and the like.
The betel nut rhizoctonia rot can cause betel nut brown root disease, black streak root disease and the like, so that the root and stem base of the betel nut are damaged, the capacity of absorbing and transporting water and inorganic salt is influenced to different degrees, the normal physiological activity of the betel nut is damaged, and the diseased tree even dies for 1 to 2 years in severe cases.
Common prevention and treatment measures for betel nut root rot include:
agricultural measures are as follows: thoroughly removing or poisoning diseased stumps and roots in the forest land, digging out dead plants or non-rescue diseased plants with roots, applying fertilizer, enhancing the resistance of the betel nuts to diseases, and further avoiding germ infection from the source; the disease condition is checked regularly, the diseased plant is found, and the treatment is carried out in time. The prevention and treatment method consumes a great deal of energy, and is difficult to completely remove all pathogenic bacteria in the soil, and once the pathogenic bacteria are overflowed again, immeasurable loss is brought.
Chemical control: chemical agents are used for irrigating the soil around the diseased trees to kill germs. Although the use of chemical agents brings great convenience and effect to the control of plant diseases, the production cost of chemical agents is high, and the negative effects of environmental pollution, overproof pesticide residues of agricultural products, the formation of drug resistance of pathogenic bacteria and the like caused by the sterilization of chemical agents are widely concerned by the society.
Therefore, in recent years, efforts have been made in various countries throughout the world to develop new methods for controlling plant diseases that can replace traditional chemical agents. Among them, the use of microorganisms and their metabolites for biological control is recognized as an environmentally friendly option. Furthermore, a biological system with the function of resisting betel nut root rot pathogen is reformed by utilizing synthetic biology, or biocontrol bacteria with the growth inhibition function on the betel nut root rot pathogen is reformed into high-version mode microbial chassis cells, so that the high-version mode microbial chassis cells are used as minimum cell factories to produce biopesticides, the path for controlling plant diseases is the most rapid and effective, and a new direction is brought to the prevention and control of pathogenic bacteria of important crops.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides an application of boron-resistant lysine bacillus in inhibiting the growth of betel nut rhizoctonia rot (Cerrena unicolor), wherein the boron-resistant lysine bacillus (Lysinibacillus boroniculatus) is preserved in the China Center for Type Culture Collection (CCTCC) in 2019, 10 and 8 months, and the preservation number is CCTCC NO. M2019773.
A biocide, comprising: a fermentation broth of boron-resistant lysine bacillus (lysine bacillus boronictolans), wherein the boron-resistant lysine bacillus (lysine bacillus boronictolans) is preserved in the China Center for Type Culture Collection (CCTCC) in 2019, 10 and 8 months, and the preservation number is CCTCC NO. M2019773; and an auxiliary material.
The bactericide as described above, the adjuvant is one or more of water, liquid culture medium, solid culture medium, and glycerol.
The bactericide as described above, wherein the fermentation liquid of the lysine bacillus borotolerant is Bacillus borolysine (Lysinibacillus boronitolorans) cultured for 24-96 hours, OD 600 4.8-2.1 hours.
Application of boron-resistant lysine bacillus (lysine bacillus boronicolorans) in preparation of a bactericide for inhibiting growth of betel nut rhizoctonia rot (Cerrena unicolor), wherein the boron-resistant lysine bacillus (lysine bacillus boronicolorans) is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 and 8 days, and the preservation number is CCTCC NO. M2019773.
Application of boron-resistant lysine bacillus (lysine bacillus boronictolans) as a basal disc cell in preparation of a bactericide for inhibiting rice blast, wherein the boron-resistant lysine bacillus (lysine bacillus boronictolans) is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 and 8 months, CCTCC for short, and the preservation number is CCTCC NO. M2019773.
The boron-resistant lysine bacillus (lysine bacillus boronictolans) is used as a modification module based on chassis cells, wherein the boron-resistant lysine bacillus (lysine bacillus boronictolans) is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 and 8 months, CCTCC NO. M2019773 is abbreviated as CCTCC.
The modified module is applied to preparation of a bactericide for inhibiting growth of betel nut root rot (Cerrena unicolor).
A method for preventing or treating betel nut root rot, comprising: obtaining a germicide according to claim 2; and mixing the biocide with the medium.
The method as described above, wherein the medium is a carrier carrying betel nut root rot (Cerrena unicolor).
The method as described above, wherein the carrier is one or more of a seed, a plant, a soil where the plant grows, and a culture medium where the plant is cultured.
The boron-resistant lysine bacillus (lysine bacillus boronitolorans) can effectively prevent and treat betel nut from infecting germ to cause yield reduction and even death, so that the yield of the betel nuts can be maintained. Meanwhile, the boron-resistant lysine bacillus (lysine bacillus boronitolorans) is mixed into the soil, does not cause adverse effect on the soil environment, does not cause ecological pollution, and is an environment-friendly germ control measure.
Drawings
Preferred embodiments of the present invention will now be described in further detail with reference to the accompanying drawings, in which:
FIG. 1 is an identification of potential biocontrol bacteria isolated from soil according to one embodiment of the present application. Wherein, FIG. 1A is a gel electrophoresis separation map of bacterial genomic DNA isolated and purified from soil; FIG. 1B is a diagram showing a gel electrophoresis separation of a PCR product obtained by specific amplification of genomic DNA using a 16S primer; FIG. 1C is a phylogenetic tree of potential biocontrol bacteria obtained by sequence alignment, and the obtained potential biocontrol bacteria is identified to be boron-resistant lysine bacillus (Lysinibacillus boronotrolans) by sequence alignment analysis;
FIG. 2 is a graph showing the growth inhibitory effect of B.borotolerant L.lysimachiae fermentation broth on P.areca at different stages according to one embodiment of the present application. Wherein, FIGS. 2A, 2B, 2C, 2D, 2E are experimental groups, sterile fermentation filtrates obtained after B-lysine resistant bacillus fermentation for 24h, 48h, 72h, 96h, 120h are respectively added into the culture medium; FIG. 2F is a control group to which a sterile fermentation filtrate of B.borotolerant lysinibacillus was not added to the medium;
fig. 3 is a graph of the bacteriostatic activity of ethyl acetate extracts of 96h fermentation broth of b.borotolerant Lysinibacillus boroniterioderans (sorrena unicolor) against betel nut rhizoctonia species (Cerrena unicolor) according to one embodiment of the present application. Wherein the concentration of the crude extract is 1mg/ml, 5mg/ml, 10mg/ml and 15mg/ml respectively; and
fig. 4 is a graph showing the bacteriostatic activity of extracts obtained by precipitation of 96h fermentation broth of lysine bacillus boron tolerant (Lysinibacillus boroniclaris) with ammonium sulfate of different saturation levels on betel nut rhizoctonia rot (Cerrena unicolor) according to an embodiment of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
In the following detailed description, reference is made to the accompanying drawings that form a part hereof and in which is shown by way of illustration specific embodiments of the application. Various specific embodiments of the present application are described in sufficient detail to enable those skilled in the art, having the benefit of this disclosure, to practice the subject application. It is to be understood that other embodiments may be utilized and structural and logical changes may be made to the embodiments of the present application.
Those skilled in the art should understand thatWhen it is understood that when it is stated that the bacteria are fermented, cultured to a concentration, the concentration is in a range of values, for example when the concentration is expressed as OD, where OD is present 600 At 4.8, the actual OD is about 4.8, e.g., OD 600 The value was 4.8. + -. 0.6. The time required for fermenting and culturing the bacteria to a certain degree is determined time which cannot be accurately measured in minutes and seconds, and the required time is related to the bacterial fermentation, the environmental temperature of the culture, the type of the culture medium and the like, so that the time related to the bacterial fermentation, the culture, the growth and the like presented in the application is approximate time rather than determined time.
Some of the terms appearing in the present application have the following definitions:
the fermentation liquid as referred to herein means a liquid having a fermentation product produced by fermentation of a bacterium, for example, a B-resistant lysine bacillus (Lysinibacillus boronitolorans) fermentation liquid is 200ml of a bacterial liquid of B-resistant lysine bacillus (initial OD is about 0.02 OD) 600 The fermentation product obtained by culturing the raw materials at 37 ℃ for about 24h, 48h, 72h, 96h or 120h at a rotation speed of 150r/min in a liquid filling amount per 500ml of LB medium. The fermentation time of the B-lysine resistant bacillus is about 24h, 48h, 72h, 96h and 120h, and the OD of the bacterial liquid 600 The values were about 4.8, 4.5, 3.0, 2.5 and 2.1, respectively (OD decreases with increasing fermentation time). After the fermentation is finished, centrifuging to remove the precipitate, collecting the fermentation supernatant, and then filtering with a filter membrane of 0.22 mu m to obtain sterile fermentation filtrate.
The method containing toxic medium is also called growth rate method, is one of the conventional methods for measuring the toxicity of the bactericide, and is suitable for fungi which do not grow spores and have fast hypha growth. The virulence of the medicament can be measured by the growth speed of the colony. The medium-containing method is to mix the reagent with the culture medium and measure the toxicity of the reagent according to the growth rate of bacterial colony on the culture medium. Generally, the method is mainly used for fungi which do not produce spores or have less spores and dense hyphae. The colony growth rate is generally expressed in terms of the time (days or hours) required for a colony to reach a given size, or the size of the diameter of the colony per unit time.
The perforation method is a method of perforating a test plate with a sterilized perforator or steel tube, injecting a certain amount of sample to be tested into the hole, and culturing for a period of time to determine the size of the zone of inhibition.
The extraction method as used herein refers to the transfer of a compound from one solvent to another solvent by utilizing the difference in solubility or partition coefficient of the compound in two mutually immiscible (or sparingly soluble) solvents. Extracting most compounds repeatedly. Ethyl acetate is a medium-polar solvent and can extract a variety of substances from the bacterial fermentation filtrate, such as molecules with less polarity (e.g., glucose or glycosides with sugars), molecules with very low polarity (e.g., certain paraffins), molecules containing salt structures (e.g., amino acids), and the like.
The ammonium sulfate precipitation as referred to herein refers to a technique for precipitating and separating proteins with ammonium sulfate solutions of different concentrations. Are commonly used for the isolation of immunoglobulins. Ammonium sulfate precipitation can be used to concentrate and partially purify proteins from bulk crude preparations. High concentration salt ions compete with protein for water molecules in protein solution, thus destroying hydrated film on protein surface, reducing its solubility and making it precipitate out of solution. The solubility of each protein is different and thus different proteins can be precipitated using different concentrations of salt solution. This method is called salting out. Salt concentration is usually expressed in terms of saturation. Ammonium sulfate is most widely used because of its high solubility, low temperature coefficient and low tendency to denature proteins.
The 16S primer for identifying bacteria refers to determining the type of bacteria to be detected by using methods such as PCR (polymerase chain reaction), sequence comparison and the like. The 16S rDNA is a DNA sequence corresponding to the coding 16S rRNA on a bacterial chromosome, exists in all bacterial chromosome genes, and has an internal structure consisting of two parts, namely a conserved region and a variable region. The variable region exists in the molecule, and shows the specificity of the bacteria on different classification grade levels.
The bacterium contains three ribosomal RNAs, 5S, 16S and 23S rRNA. Although 5S rRNA is easy to analyze, the number of nucleotides is too small, and the nucleotides consist of only dozens of nucleotides, so that genetic information is insufficient and the 5S rRNA cannot be used for classification research; the 23S rRNA has a molecular weight too large and contains almost twice as many nucleotides as the 16S rRNA, and thus it is difficult to analyze the DNA, and thus it is not selected for classification studies. 16S rRNA is commonly used for bacterial taxonomic studies.
First, 16S rRNA is ubiquitous in prokaryotes (the homologous molecule in eukaryotes is 18S rRNA). rRNA is involved in the synthesis of biological proteins, and its function is essential for any organism, and remains unchanged in the long course of biological evolution, and can be regarded as the time clock for biological evolution. Secondly, the 16S rRNA molecule contains a highly conserved sequence region and a moderately conserved and highly variable sequence region, so that the method is suitable for researching the biological genetic relationship of various types with different evolutionary distances. Thirdly, the relative molecular weight of 16S rRNA is moderate, about 2kb of nucleotides, which is convenient for sequence analysis. Therefore, it can be used as a good tool for measuring the evolution and affinity relationship of various organisms.
Since the coding gene of 16S rRNA is 16S rDNA, it is difficult to directly extract 16S rRNA from bacteria, and the extracted RNA is easily degraded and not easily preserved, etc., 16S rDNA is generally used to identify the type of bacteria.
The PCR, i.e., the polymerase chain reaction, as used herein, refers to a process of in vitro replication of daughter strand DNA complementary to the DNA of a parent strand template by denaturing, annealing, extending, etc., using the parent strand DNA as a template and a specific primer as an extension origin, under the catalysis of DNA polymerase. PCR is an in vitro synthesis amplification technology, and can rapidly and specifically amplify a target DNA fragment in vitro. In embodiments of the present application, the parent strand may be the genomic DNA of a monoclonal test bacterium.
The OD value described herein is an abbreviation for optical density, which indicates the optical density absorbed by the test object. Measuring the absorbance (by OD) of the culture at 600nm 600 Expressed), the concentration of the culture solution of bacteria can be measured to estimate the growth of bacteria, so the optical density value at 600nm can be used to represent the somatic cell density, wherein the absorbance is proportional to the concentration of bacteria in the culture solution.
The PDA culture Medium refers to a Potato glucose culture Medium, wherein P, D, A is the abbreviation of Potato Dextrose Agar (Medium). The PDA culture medium is a semi-synthetic culture medium, and is a commonly used culture medium for fungi such as yeast, mold, mushroom, etc.
The biocontrol bacteria or biocontrol bacteria as referred to herein are one or more bacteria having a biocontrol function. Refers to a kind of measures for controlling the occurrence and development of plant diseases by utilizing beneficial microorganisms to kill or reduce the number of pathogenic organisms. Also called as "treating bacteria with bacteria". Biological control is an important part of a comprehensive pest control system. It has the advantages of no environmental pollution, no toxicity to human and livestock, no side effect to plant, etc., and is especially suitable for controlling soil-borne diseases.
In an ecological environment, the action mechanism of one microorganism for controlling the growth of other microorganisms is very diverse, different biocontrol bacteria and the same biocontrol bacteria can have different biocontrol mechanisms when acting with different plants. Taking trichoderma as an example, the biocontrol mechanism of biocontrol bacteria can be roughly divided into competitive action, for example, trichoderma has strong adaptability to the environment, has a much higher growth speed than pathogenic bacteria, can compete with the pathogenic bacteria for nutrition or space, effectively utilizes the concentrated nutrient substances on the surface of plants or near the invasion point, quickly occupies the space to absorb nutrition, occupies the invasion site of the pathogenic bacteria and does not leave a gap for the invasion of the pathogenic bacteria; antagonism, for example, the non-volatile metabolite produced by trichoderma can strongly inhibit the growth of verticillium dahliae, so that pathogenic bacteria hypha has the phenomena of cell protoplasm concentration, hypha breakage and the like; inducing resistance, such as trichoderma viride penetrating and colonizing in the cotton root epidermis and cortex tissue, its peroxidase activity is raised, terpenoid accumulates, has controlled the infection of pathogenic bacteria more effectively than the plant not infected by trichoderma viride, has induced the disease resistance of the cotton; parasitic effects; antibiotic action, etc. Many biocontrol microorganisms are biocontrol by a single mechanism, and some microorganisms may function in combination by concentrating different mechanisms.
The method detects the biocontrol characteristics of separated bacteria by separating and purifying about 100 strains of bacteria possibly having biocontrol activity from soil and taking the existing main fungal pathogenic bacteria in a laboratory as targets.
Example 1 isolation and purification of soil Strain
According to one embodiment of the present application, bacteria can be isolated, purified from any soil where biocontrol bacteria may be present, and tested for their biocontrol properties. According to one embodiment of the application, tomato-rice crop rotation soil is taken, and bacteria in the tomato-rice crop rotation soil are separated and purified.
1. And (4) preparing a soil diluent. And weighing a proper amount of soil. The soil is taken back from the field in advance and may contain biocontrol bacteria. According to one embodiment of the application, the soil can also be directly weighed out in the field. According to one embodiment of the present application, 10g of soil may be weighed. And (3) putting the weighed soil into sterile water, smashing, and standing for more than 30 minutes to fully separate out microorganisms in the soil. According to one example of the application, 10g of soil was weighed and mixed in 100mL of sterile water. The obtained supernatant is the soil stock solution.
Then diluting the soil stock solution by 10 times and 10 times 2 10 times of 3 10 times of 4 10 times of 5 10 times of 6 Doubling, etc. According to one embodiment of the present application, dilution in the above manner provides 1g, 10g -1 g、10 -2 g、10 -3 g、 10 -4 g、10 -5 g、10 -6 g, and the content of microorganisms in the soil. This step facilitates the obtaining of a single clone of the microorganism in the soil. For example, according to one embodiment of the present application, 10mL of the soil stock solution is pipetted into test tube No. 0, 1mL of the soil stock solution is pipetted from the test tube, and the pipetted soil stock solution is mixed with 9mL of sterile water in test tube No. 1, and the mixture is thoroughly mixed to dilute the soil stock solution by 10 times. Sequentially operating to obtain the dilution 10 of the soil stock solution 2 10 times of 3 10 times of 4 10 times of 5 10 times of 6 Double solution. The test tubes are respectively marked as 0,1,2,3,4,5,6 according to the dilution multiple of the soil stock solution.
2. And (6) coating the plate. That is, the microorganisms in the test tubes in step 1 are applied to a solid medium. According to an embodiment of the present application, the solution is sucked from each of the 7 test tubes prepared in step 1, transferred to the surface of the bacterial solid medium, and uniformly coated by using a scraper, a glass rod or the like. Drying in the sun until the surface of the culture medium is dry and no liquid is present, and packaging the culture medium. According to one embodiment of the present application, a sealing film may be used for encapsulation. According to one embodiment of the present application, each diluted solution can be spread on multiple solid medium surfaces according to experimental and statistical requirements. For example, each dilution of the solution is spread on the surface of 3 solid media.
3. And (4) culturing microorganisms. And (3) placing the solid culture medium coated with the bacteria in a proper environment, and taking out after the visible monoclonal bacteria grow out. According to one embodiment of the present application, the bacteria can be cultured in a 37-degree constant temperature environment, such as a 37-degree incubator, a water bath, etc., for more than 12 hours. The bacteria can also be cultured in 28 deg.C environment, such as 28 deg.C incubator, water bath, etc., for more than 48 hr.
4. Counting and colony description. And calculating the number of colonies on the surface of each solid medium, observing the colony morphology characteristics, and recording the results. For example, 10mL of directly aspirated soil stock solution is contained in test tube No. 0, the soil stock solution is coated on a solid culture medium, and the number of colonies obtained by culture is 1g of the number of bacteria in the soil; the liquid in the test tube No. 1 is obtained by diluting the solution in the test tube No. 0 by 10 times, so the liquid is coated on a solid culture medium, the number of the bacterial colonies obtained by culture is 0.1g of the number of bacteria in soil, and the rest of the test tubes are analogized.
5. The plates were scribed for separation. The colonies obtained in the above step are streaked and re-cultured at a suitable temperature. By the operation, the monoclonal strains can be separated, can be propagated and stored, and is favorable for subsequent sequencing and classification tests.
6. And (5) preserving and identifying strains.
Example 2 identification of the Strain species
According to one embodiment of the present application, the 16S universal primers can be used for the preliminary identification of strain species. According to one embodiment of the present application, the strain may be directly used as a substrate for species identification, or the genomic DNA may be extracted first and the genomic DNA may be used as a substrate for species identification. According to one embodiment of the present application, agarose gel electrophoresis is used to detect the content and quality of extracted bacterial genomic DNA. FIG. 1A is an agarose gel electrophoresis of bacterial genomic DNA extracted according to an embodiment of the present application, as shown in FIG. 1A, the genomic DNA has a distinct band and a concentration that meets the requirements of a subsequent PCR experiment.
1. The selection primers were designed for PCR amplification of 16S rDNA. According to one embodiment of the present application, PCR primers for identifying the strain species can be designed by itself. According to one embodiment of the present application, commonly used bacterial identification universal primers can also be selected for PCR amplification. According to one embodiment of the present application, universal primers 27F and 1492R are selected for PCR amplification, wherein the DNA sequences of 27F and 1492R are as follows:
27F：5’-AGAGTTTGATCCTGGCTCAG-3’；
1492R：5’-GGTTACCTTGTTACGACTT-3’。
selecting a proper PCR reaction system and reaction conditions according to the selected different DNA polymerases. The present application does not limit the use of any kind of DNA polymerase, nor the PCR reaction system and reaction conditions after the use of the same kind of DNA polymerase. For example, when Taq DNA polymerase is selected for PCR amplification, reference is made to the reaction system as listed in Table 1:
TABLE 1 PCR systems Table (12. Mu.L)
The reaction conditions of PCR were: firstly, performing pre-denaturation at 94 ℃ for 5min, then entering a circulation program, wherein each circulation program comprises 94 ℃ denaturation 30s,55 ℃ annealing 30s,72 ℃ extension 1min 30s, performing 30 cycles, and finally, keeping the temperature at 72 ℃ for 7min.
According to one embodiment of the present application, agarose gel electrophoresis was used to detect the content and quality of DNA fragments obtained after PCR with 16S universal primers using extracted genomic DNA as a template. FIG. 1B is an agarose gel electrophoresis of a DNA fragment obtained after PCR with 16S universal primers based on extracted bacterial genomic DNA as a template according to one embodiment of the present application. As shown in FIG. 1B, 7 experiments all obtained PCR products with single bands and concentrations meeting the requirements of subsequent sequencing identification.
2. And (5) identifying the strain type. According to one embodiment of the present application, the PCR products obtained from the above experiments can be sequenced. And comparing the obtained sequencing result with the existing strain sequence to finally obtain the variety of the identified strain. For example, the sequencing results are input to NCBI website (https:// www.ncbi.nlm.nih.gov) for blast to obtain the identified strain species. FIG. 1C shows a schematic diagram of a phylogenetic tree obtained by PCR amplification of the isolated strains with 16S primers to obtain 16S rDNA and sequence alignment, and the obtained biocontrol bacteria is identified to be B-resistant lysine bacillus (Lysinibacillus boroniculatus) according to an embodiment of the present application. The strain is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 months and 8 days, and the preservation number is CCTCC NO. M2019773. The evolutionary relationship of B.borotolerant Lysinibacillus (Lysinibacillus boronicterorans) of the present application to other bacterial species can be clearly seen from the phylogenetic tree shown in FIG. 1C.
Example 3 biocontrol characterization of purified bacteria
According to one embodiment of the present application, the bacteriostatic activity of the potential biocontrol bacteria isolated by the present application can be identified by a toxic medium-containing method. According to one embodiment of the present application, the toxic medium-containing method specifically operates as follows:
1. preparing a PDA solid culture medium, and cooling the PDA solid culture medium to about 55 ℃; or heating and melting the solidified PDA culture medium and cooling to about 55 ℃;
2. selecting a single colony of the B-resistant lysine bacillus, inoculating the single colony in an LB liquid culture medium, culturing at 37 ℃ and 150r/min overnight, transferring bacterial liquid in the next day, and filling the liquid with 200ml of bacterial liquid/500 ml of LB culture medium until the bacterial concentration is 0.02OD 600 Putting the mixture into a constant temperature shaking table at 37 ℃ for culturing for 24h, 48h, 72h, 96h and 120h at the rotation speed of 150r/min so as to obtain OD 600 The values respectively reach about 4.8, 4.5, 3.0, 2.5 and 2.1, and hair is respectively collected at different time pointsPlacing the fermentation product in a centrifuge, centrifuging at 8000r/min to remove precipitate to obtain fermentation supernatant, and filtering the fermentation supernatant with 0.22 μm filter membrane to obtain sterile fermentation filtrate;
3. the fermentation filtrate was mixed with PDA medium at a ratio of 1:9, namely 10 percent of fermentation liquor, and preparing a drug-containing flat plate containing the antagonistic bacteria fermentation liquor;
4. after the culture medium mixed with the fermentation liquid is cooled, 1 pathogenic bacteria cake (the diameter is 8 mm) to be tested is inoculated in the center of the flat plate;
5. and placing the inoculated culture medium in a constant-temperature incubator at 28 ℃ for culture.
And (3) taking a PDA culture medium plate without fermentation liquor as a control, repeating the steps 1-4 at least three times, and then carrying out at least three tests of the bacteriostasis of the biocontrol bacteria to specific bacteria.
In the application, the bacteriostatic property of the biocontrol bacteria is evaluated by the following formula:
hypha growth inhibition (%) = (control colony diameter-treated colony diameter)/(control colony diameter-0.8) × 100%
FIG. 2 shows the growth inhibitory effect of B.borotolerant Lysinibacillus boronitolorans (Lysinibacillus boronitolorans) fermentation filtrate on P.areca (Cerrena unicolor) according to one embodiment of the present application. Wherein, FIGS. 2A, 2B, 2C, 2D, 2E are experimental groups, sterile fermentation filtrates obtained after fermentation for about 24h, 48h, 72h, 96h and 120h by adding B.boratabensis into the culture medium respectively; FIG. 2F is a control group to which a sterile fermentation filtrate of B.borotolerant lysinibacillus was not added to the medium. The round point in the middle of the culture medium is the accessed target pathogenic bacteria. According to one embodiment of the present application, the target pathogen is betel nut root rot (Cerrena unicolor).
It can be clearly seen by combining fig. 2 and the above formula that the growth of betel nut root rot pathogenic bacteria is obviously inhibited by the B-lysine resistant bacillus fermentation filtrate. According to measurement and calculation, the antagonism efficiency of fermentation liquor of boron-resistant lysine bacillus (Lysinibacillus boronitolorans) after fermentation for about 24 hours, 48 hours, 72 hours, 96 hours and 120 hours on the betel nut root rot pathogen (Cerrena unicolor) is respectively about 53.33%, 60%, 76%, 82.67%77.33%. According to multiple experiments in the application, the fermentation time of the boron-resistant lysine bacillus (Lysinibacillus boronitilerans) is 96h 600 About 2.5, it can effectively inhibit the growth of betel nut root rot (Cerrena unicolor), and the antagonistic efficiency reaches about 82%.
EXAMPLE 4 identification of biocontrol Properties in isolation of crude extracts of bacterial fermentation broths
According to one embodiment of the application, the bacteria fermentation liquor with the biocontrol effect is subjected to coarse extraction by adopting an ethyl acetate extraction method and an ammonium sulfate precipitation extraction method, and the bacteriostatic activity of the coarse extract is identified by adopting a punching method.
According to one embodiment of the application, the specific operation steps of the punching method are as follows:
1) Preparing a PDA solid culture medium, and cooling the PDA solid culture medium to about 55 ℃; or heating and melting the solidified PDA culture medium and cooling to about 55 ℃;
2) After the culture medium is cooled, punching holes 3.5cm away from the central point of the central line part of the culture medium respectively by using a 0.8cm puncher, clamping the redundant culture medium by using sterilized tweezers, and inoculating 1 pathogenic bacteria cake (the diameter is 8 mm) to be tested in the center of a flat plate;
3) Carefully inject the extract into the punched wells. The control solvent was in the left well and the extract was in the right well.
According to one embodiment of the present application, the specific operation for preparing the crude extract is as follows:
(1) Ethyl acetate extraction method for preparing crude extract
Culturing B-lysine-resistant bacillus at 37 deg.C and 150r/min, transferring the bacterial liquid, and loading 200ml bacterial liquid/500 ml LB culture medium to final concentration of 0.02OD 600 The culture was continued at a rotation speed of 150r/min for 96h at 37 ℃ to obtain OD 600 Collecting fermentation product, centrifuging at 8000r/min to remove precipitate to obtain fermentation supernatant, and filtering with 0.22 μm filter membrane to obtain sterile fermentation filtrate. Adding equal volume of ethyl acetate solvent into 400ml of sterile fermentation filtrate, extracting for 4 times by oscillation, and separating the filtrate with a separating funnelThe ethyl acetate in the layer was taken out completely to obtain an extract. Combining the 4 times of extraction liquid, adding the extraction liquid into a 500ml rotary evaporation bottle for rotary evaporation concentration until ethyl acetate is completely evaporated to dryness, then adding acetone to dissolve residual extract to make the concentration of the residual extract to be 1mg/ml, 5mg/ml, 10mg/ml and 15mg/ml, and respectively testing the bacteriostatic activity of the crude extract. The control solvent was acetone. FIG. 3 shows that according to one embodiment of the application, a crude fermentation liquid extract of B-lysine-resistant bacillus fermented for 96 hours is prepared by an ethyl acetate extraction method, and the inhibition effect of the crude fermentation liquid extract on the growth of betel nut rhizoctonia solani is detected. As shown in FIG. 3, the crude extracts with different concentrations all have different degrees of inhibition effects on betel nut root rot. Wherein, when the concentration of the crude substances is 10mg/ml, the inhibition effect on the betel nut root rot pathogen (Cerrena unicolor) is most obvious.
(2) Preparation of crude extract by ammonium sulfate precipitation
First, an optimum ammonium sulfate concentration is determined. Obtaining a large amount of fermentation liquor of the boron-resistant lysine bacillus under the optimal fermentation condition of 96h, collecting the fermentation liquor, and centrifuging at 4 ℃ and 8000rpm for 10min to remove thalli. Taking 200ml of each supernatant, respectively and slowly adding ammonium sulfate until the saturation degree is 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, carrying out overnight precipitation at 4 ℃, centrifuging at 8000rpm for 10min, collecting the precipitate, dissolving the precipitate with PBS (pH7.0), fixing the volume to the same volume, measuring the antibacterial activity of the precipitate, repeating the treatment for 3 times, wherein the saturation degree of ammonium sulfate of the component with the strongest activity is the optimum saturation degree of ammonium sulfate required for preparing the antibacterial crude extract.
Then, the crude extract was prepared by ammonium sulfate precipitation. Taking 400ml of fermentation supernatant of the boron-resistant lysine bacillus under the optimal fermentation condition of 96h, adding ammonium sulfate into the supernatant while stirring to ensure that the saturation degree reaches 50%, 60%, 70% or 80%, and precipitating overnight at 4 ℃. Centrifuging at 8000rpm for 10min, collecting precipitate, dissolving the precipitate with PBS (pH7.0), diluting to 10ml, and determining its antibacterial activity, wherein the control solvent is PBS (pH7.0). FIG. 4 shows that the crude extract of fermentation broth obtained by fermenting B.borotolerant L.lysimachiae for 96h by ammonium sulfate precipitation was tested for its inhibitory effect on the growth of P.areca according to one embodiment of the present application.
According to one embodiment of the present application, the growth of pathogens may be inhibited by mixing biocontrol bacteria, such as lysine bacillus boron tolerant bacteria (Lysinibacillus boronitolorans) of the present application, with a medium, thereby preventing the plant from infecting the pathogens or treating the plant already infected with the pathogens. According to an embodiment of the present application, the medium is any carrier that can carry pathogenic bacteria, for example, the medium may include one or more of areca seed, fruit, plant itself, soil in which areca is growing, water environment, even culture medium in which plant is cultured, and the like. According to one embodiment of the present application, the pathogenic bacteria may be betel nut root rot (Cerrena unicolor). Of course, the kind of pathogenic bacteria should not be limited, and bacteria, fungi, etc. that the boron-tolerant lysine bacillus (Lysinibacillus boronicterorans) of the present application can inhibit its growth to some extent should be considered as the pathogenic bacteria of the present application.
According to the experimental results shown in this example and fig. 3 and 4, it is known that various components in the fermentation filtrate of lysine bacillus boron tolerant bacteria (Lysinibacillus boronicterorans) have different degrees of inhibitory effects on betel nut root rot (Cerrena unicolor). Therefore, the boron-resistant lysine bacillus (lysine bacillus boronolans) can be used as the chassis microbial chassis cells for mass production of fermentation filtrate with bacteriostatic activity, thereby meeting the requirements of agriculture and production life on bactericides. According to an embodiment of the application, the lysine bacillus bororesistant bacteria (Lysinibacillus boronitolerans) can be further improved to be a modified module which can produce one or more proteins or other substances with specific bacteriostatic activity in a large scale, so that the function of the bactericide for inhibiting the growth of betel nut root rot (Cerrena unicolor) prepared by the module is enhanced, and the efficiency of the bactericide for inhibiting the growth of rice blast fungus (Magnaporthe oryzae) is improved.
According to an embodiment of the application, the boron-resistant lysine bacillus (Lysinibacillus boronitians) can be also prepared into a bactericide for inhibiting the growth of pathogenic bacteria such as betel nut root rot (Cerrena unicolor). According to one embodiment of the present application, the bactericide comprises B-tolerant lysine bacillus (Lysinibacillus boronictolans) and adjuvants. According to one embodiment of the present application, the bactericide comprises a fermentation broth prepared by a cell modification module based on B.borotolerant Lysinibacillus boronitolorans and an adjuvant. According to one embodiment of the application, the fermentation time of the B-tolerant lysine bacillus (Lysinibacillus boronitolorans) fermentation liquor in the bactericide is 96 hours, and the crude extract is obtained by an ethyl acetate extraction method and an ammonium sulfate precipitation method. According to one embodiment of the application, the engineered module is one that allows for the mass production of one or more proteins or other substances with specific bacteriostatic activity. According to one embodiment of the application, the auxiliary material is one or more of water, liquid medium, solid medium and glycerol. According to an embodiment of the present application, the ratio of glycerin may be adjusted according to the storage conditions of the bactericide, for example, the ratio of glycerin may be 10%, 25%, 50%, 75%, etc. by volume.
According to an embodiment of the application, the boron-resistant lysine bacillus can be mixed with other bacteria and fungi with biological control functions to prepare a preparation with good inhibition effect on various pathogenic bacteria.
According to one embodiment of the present application, a method for preventing or treating pathogenic bacteria infection of a plant comprises mixing a bactericide comprising a B.borotolerant lysine bacillus (Lysinibacillus boronitolorans) fermentation broth or a crude extract thereof as described above with a medium to thereby inhibit the growth of pathogenic bacteria on the surface of the medium. According to one embodiment of the present application, it is also possible to directly culture and adjust the boron-tolerant lysine bacillus (Lysinibacillus boroniticola) to a desired concentration and mix it with the medium. According to an embodiment of the present application, the medium is any carrier that can carry pathogenic bacteria, for example, the medium may include one or more of areca seed, fruit, plant itself, soil in which areca is growing, water environment, even culture medium in which plant is cultured, and the like. According to one embodiment of the present application, the pathogenic bacteria may be betel nut root rot (Cerrena unicolor). Of course, the species of pathogenic bacteria should not be limited, and the boron-tolerant lysine bacillus (Lysinibacillus boronictolans) of the present application, and other bacteria, fungi, etc. mixed therewith, which may inhibit the growth thereof to some extent, should be considered as pathogenic bacteria of the present application.
Therefore, if the boron-resistant lysine bacillus (Lysinibacillus borniticola) is mixed in the soil for planting the areca-nut trees, the growth of areca-nut root rot germs (Cerrena unicolor) in the soil can be completely inhibited, the areca-nut trees are effectively prevented from being infected by the germs, the yield is reduced, and even the areca-nut trees die, and the yield of the areca-nuts is maintained. Meanwhile, the boron-resistant lysine bacillus (lysine bacillus boronitolorans) is mixed into the soil, does not cause adverse effect on the soil environment, does not cause ecological pollution, and is an environment-friendly germ control measure. Furthermore, on the basis of the synthetic element and the synthetic device of the biological and antibacterial metabolites, the rational modification of the pathway, the synthetic module and the module or the system can be carried out, thereby generating stronger antibacterial activity; or by disclosing a gene circuit and a regulation and control network, a logic gene circuit and a functional gene circuit are reasonably designed or optimized to become a minimum cell factory so as to generate strong bacteriostatic activity, so that a new direction can be brought to the prevention and treatment of the betel nut root rot.
The above embodiments are provided only for illustrating the present invention and not for limiting the present invention, and those skilled in the art can make various changes and modifications without departing from the scope of the present invention, and therefore, all equivalent technical solutions should also fall within the scope of the present disclosure.
Claims (8)
1. B-tolerant lysine bacillus (B) ((B))Lysinibacillus boronitolerans) For inhibiting betel nut root rot bacteria (Cerrena unicolor) The application in growth, wherein, the B-lysine resistant bacillus (B.boratabensis) ((B.boratabensis))Lysinibacillus boronitolerans) Is preserved in China center for type culture Collection, CCTCC for short, in 2019, 10, month and 8, with the preservation number of CCTCC NO. M2019773.
2. B. tolerant L.lysimachiae (B.), (B.lysimachiae)Lysinibacillus boronitolerans) In the preparation of medicine for inhibiting areca rootRot fungus (Cerrena unicolor) Use in a growing fungicide, wherein said B-tolerant L-lysine bacillus (B.borreligious)Lysinibacillus boronitolerans) The strain is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 and 8 months, and the preservation number is CCTCC NO. M2019773.
3. B-tolerant lysine bacillus (B) ((B))Lysinibacillus boronitolerans) Application of the B-lysine-resistant bacillus (B.borreligious) as underpan cells in preparing bactericide for inhibiting betel nut root rotLysinibacillus boronitolerans) The strain is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 and 8 months, and the preservation number is CCTCC NO. M2019773.
4. A method for preventing or treating betel nut root rot, comprising:
the acquisition comprises: b-tolerant lysine bacillus (B) ((B))Lysinibacillus boronitolerans) The B-tolerant lysine bacillus strain of (a), (b)Lysinibacillus boronitolerans) The culture is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 and 8 months, wherein the preservation number is CCTCC NO. M2019773; and a bactericide of an auxiliary material; and
mixing the germicide with the medium.
5. The method of claim 4, wherein the medium is Argyrolobium arecanum (L.) Beauveria areca ® ™Cerrena unicolor) The vector of (1).
6. The method of claim 5, wherein the carrier is one or more of a seed, a plant, soil in which a plant is growing, a medium in which a plant is cultured.
7. The method of claim 4, wherein the adjuvant is one or more of water, liquid medium, solid medium, and glycerol.
8. The method of claim 4Wherein the fermentation liquid of the B-resistant lysine bacillus is B-resistant lysine bacillus (B-resistant lysine bacillus: (B)Lysinibacillus boronitolerans) OD after 24-120 hours of culture 600 4.8-2.1 times of fermentation broth.
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| CN113416670B (en) * | 2021-06-10 | 2022-08-12 | 江苏省农业科学院 | Boron-resistant lysine bacillus with bacteriostatic ability and application thereof |
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| CN113925064B (en) * | 2021-11-01 | 2023-02-03 | 海南大学 | Application of B-lysine-resistant bacillus in inhibition of growth of pitaya soft rot germs |
| CN114403158B (en) * | 2021-12-14 | 2022-07-05 | 海南大学 | B-lysine-resistant bacillus bacteriocin analogue and application thereof in inhibition of pseudomonas syringae tomato pathogenic varieties |
| CN114209811B (en) * | 2021-12-15 | 2024-03-29 | 海南大学 | Preparation method of boron-resistant lysine bacillus bacteriocin analogue and application of boron-resistant lysine bacillus bacteriocin analogue in inhibition of bacillus pumilus |
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