WO2021068555A1 - Areca nut palm root rot fungicide prepared using lysinibacillus boronitolerans as base cells - Google Patents

Abstract

Disclosed is a use of Lysinibacillus boronitolerans in inhibiting the growth of Cerrena unicolor. Lysinibacillus boronitolerans can not only reduce the occurrence of areca nut palm root rot to a great extent, but also poses no threat to the ecological environment, is expected to be used as a high-quality base microorganism for synthetic biology research, and thus brings a new direction to the prevention and treatment of areca nut palm root rot.

Description

A kind of betel nut root rot fungicide prepared by using boron-resistant lysine bacillus as chassis cells Technical field

The invention relates to a synthetic biology modification and application of biocontrol bacteria, in particular to a betel nut root rot fungicide.

Background technique

Betel nut contains a variety of nutrients and beneficial compounds required by the human body, such as fat, betel nut oil, alkaloids, catechins, choline and other ingredients. Betel nut has a unique function of preventing miasma. It is the fruit of medicine used by doctors in the past dynasties to treat diseases. It also has the alias of "Cleaning Miasma Pill". Because the disease of miasma is generally related to irregular diet and stagnation of qi, betel nut can reduce qi, eliminate food and expectorant, so its medicinal properties have been widely concerned. At the same time, reports show that betel nut has anthelmintic effects, such as paralyzing liver fluke nervous system, inhibiting schistosomiasis liver migration, and repelling tapeworms, pinworms, worms, roundworms, etc. There are even reports showing that betel nut has varying degrees of resistance to pathogenic microorganisms, hypertension, and cancer.

Betel nut root rot bacteria can cause betel nut brown root disease, black streaked root disease, etc. After the root and stem base of betel nut are damaged, it will affect its ability to absorb and transport water and inorganic salts to varying degrees, and disrupt the normal physiological activities of betel nut. Sometimes it even causes the diseased tree to die in 1-2 years.

The prevention and control measures of common betel nut root rot bacteria include:

Agricultural measures: completely remove or poison the diseased tree stumps and roots in the forest land, root out dead or incurable diseased plants, and add fertilizers to enhance the resistance of betel nuts to diseases, thereby eliminating pathogen infection from the source; Check the condition regularly, find diseased plants, and deal with them in time. This control method requires a lot of energy, and it is difficult to completely remove all pathogenic bacteria in the soil. Once the pathogenic bacteria flood again, it will bring immeasurable losses.

Chemical control: Use chemicals to irrigate the soil around the diseased trees to kill the germs. Although the use of chemical agents has brought many conveniences and effects to the prevention and control of plant diseases, the production cost of chemical agents is inherently high, and after the use of chemical agents for sterilization, environmental pollution, excessive pesticide residues in agricultural products and pathogen resistance will follow. Negative effects such as the formation of AIDS have caused widespread concern in the society.

Therefore, in recent years, countries all over the world are striving to develop new methods that can replace traditional chemical agents to control plant diseases. Among them, the use of microorganisms and their metabolites for biological control is recognized as an environmentally friendly option. Furthermore, synthetic biology is used to transform a biological system that has the function of resisting betel nut root rot fungus, or a biocontrol bacteria that has a growth inhibitory function against betel nut root rot fungus, and transform it into a high-version model microbial chassis cell, making it the smallest cell The production of biological pesticides in factories will become the fastest and most effective way to control plant diseases and bring new directions for the prevention and control of important crop pathogens.

Summary of the invention

In view of the technical problems in the prior art, the present invention proposes the application of boron-resistant lysine bacillus to inhibit the growth of Cerrena unicolor, wherein the boron-resistant lysine bacillus (Lysinibacillus boronitolerans) ) Was deposited at the China Center for Type Culture Collection on October 8, 2019, referred to as CCTCC, and the deposit number is CCTCC NO.M 2019773.

A bactericide, comprising: a fermentation broth of Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans), the Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans) was deposited in the China Type Culture Collection on October 8, 2019 , Referred to as CCTCC, the deposit number is CCTCC NO.M 2019773; and accessories.

In the above-mentioned bactericide, the auxiliary material is one or more of water, liquid culture medium, solid culture medium, and glycerin.

The bactericide as described above, wherein the fermentation broth of Boron-resistant Lysine Bacillus is the fermentation broth of Lysinibacillus boronitolerans (Lysinibacillus boronitolerans) cultured for 24-96 hours, and the OD ₆₀₀ is 4.8-2.1.

Application of Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans) in the preparation of a fungicide for inhibiting the growth of Cerrena unicolor, wherein the Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans) was issued in October 2019 It was deposited at the China Center for Type Culture Collection on the 8th, referred to as CCTCC, and the deposit number is CCTCC NO.M 2019773.

Application of Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans) as chassis cells in the preparation of a fungicide for inhibiting rice blast, wherein the Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans) was deposited in China on October 8, 2019 The Type Culture Collection, referred to as CCTCC, has the deposit number CCTCC NO.M 2019773.

Boron-resistant lysine bacillus (Lysinibacillus boronitolerans) is used as a chassis cell-based transformation module, wherein the boron-resistant lysine bacillus (Lysinibacillus boronitolerans) was deposited in the China Type Culture Collection on October 8, 2019 , Referred to as CCTCC, and the deposit number is CCTCC NO.M 2019773.

The application of the above-mentioned transformation module in the preparation of a fungicide for inhibiting the growth of Cerrena unicolor.

A method for preventing or treating betel nut root rot, comprising: obtaining the fungicide according to claim 2; and mixing the fungicide with a medium.

The method as described above, wherein the medium is a carrier carrying Cerrena unicolor.

The method as described above, wherein the carrier is one or more of a seed, a plant, the soil where the plant grows, and a medium for cultivating the plant.

Boron-resistant lysine bacillus (Lysinibacillus boronitolerans) can effectively prevent the betel nut from being infected with pathogenic bacteria, resulting in production reduction or even death, so that the yield of betel nut can be maintained. At the same time, the incorporation of boron-resistant lysine bacillus (Lysinibacillus boronitolerans) into the soil will not cause adverse effects on the soil environment, nor will it cause ecological pollution, and is an environmentally friendly disease prevention and control measure.

Description of the drawings

Hereinafter, the preferred embodiments of the present invention will be described in further detail with reference to the accompanying drawings, in which:

Fig. 1 is the identification of potential biocontrol bacteria isolated from soil according to an embodiment of the present application. Among them, Figure 1A is a gel electrophoresis separation map of bacterial genomic DNA separated and purified from soil; Figure 1B is a gel electrophoresis separation map of PCR products obtained by specific amplification of genomic DNA with 16S primers; Figure 1C is a through The phylogenetic tree of potential biocontrol bacteria obtained after sequence alignment, through sequence comparison analysis, identified the obtained potential biocontrol bacteria as Lysinibacillus boronitolerans (Lysinibacillus boronitolerans);

Fig. 2 shows the growth inhibitory effect of the fermentation broth of boron-lysine-resistant Bacillus at different stages on the betel rot fungus according to an embodiment of the present application. Among them, Figures 2A, 2B, 2C, 2D, and 2E are the experimental groups, in which the sterile fermentation filtrate obtained after fermentation of Bacillus boron-lysine for 24h, 48h, 72h, 96h, and 120h was added to the culture medium; 2F is the control group, without adding the sterile fermentation filtrate of Boron-Lysine-resistant Bacillus to its medium;

Fig. 3 shows the antibacterial activity detection of different concentrations of ethyl acetate extracts of the 96h fermentation broth of Lysinibacillus boronitolerans (Lysinibacillus boronitolerans) with different concentrations against Cerrena unicolor according to an embodiment of the present application. Wherein, the concentration of the crude extract is 1mg/ml, 5mg/ml, 10mg/ml and 15mg/ml; and

Figure 4 shows the antibacterial activity of extracts obtained by precipitation with ammonium sulfate with different saturations of 96h fermentation broth of Lysinibacillus boronitolerans (Lysinibacillus boronitolerans) according to an embodiment of the present application against Cerrena unicolor Detection.

Detailed ways

In order to make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments They are a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

In the following detailed description, reference may be made to the various drawings of the specification that are part of this application to illustrate specific embodiments of the application. Each specific embodiment of the present application is described in sufficient detail below, so that a person of ordinary skill with relevant knowledge and technology in the field can implement the technical solution of the present application. It should be understood that other embodiments may also be used or structural and logical changes to the embodiments of the present application may be made.

Those skilled in the art should understand that when it is stated that bacteria are fermented and cultured to a certain concentration, the said concentration is a concentration within a certain numerical range. For example, when the concentration is expressed by the OD value, if an OD ₆₀₀ of 4.8 appears in the application, then The actual OD value is about 4.8, such as the OD ₆₀₀ value is 4.8 ± 0.6. The time required to ferment and cultivate the bacteria to a certain level is a certain time that cannot be accurate to minutes and seconds, and the required time is related to the environmental temperature of bacterial fermentation, culture, and the type of culture medium, so it appears in this application The time related to bacterial fermentation, cultivation, growth, etc., are approximate times, not definite times.

Some nouns appearing in this application have the following interpretations:

The fermentation broth mentioned in this application refers to the liquid with fermentation products produced by bacteria after fermentation. For example, the fermentation broth of Lysinibacillus boronitolerans is the fermentation broth of Lysinibacillus boronitolerans with 200ml broth (initial OD). About 0.02OD ₆₀₀ /ml)/500ml LB medium filling volume, the fermentation product obtained by culturing at 37°C at 150r/min for about 24h, 48h, 72h, 96h or 120h. The fermentation time of boron-resistant lysine bacillus is about 24h, 48h, 72h, 96h, 120h, and the OD ₆₀₀ value of the bacterial solution is about 4.8, 4.5, 3.0, 2.5 and 2.1 respectively (OD value decreases with the extension of fermentation time) . After the fermentation, the precipitate is removed by centrifugation, the fermentation supernatant is collected, and then filtered with a 0.22 μm filter membrane to obtain a sterile fermentation filtrate.

The toxic medium method mentioned in this application is also called the growth rate method, which is one of the conventional methods for determining the virulence of fungicides, and is suitable for fungi that do not grow spores but have faster hyphae growth. The virulence of the agent can be measured by the growth rate of the colony. Toxic medium method is to mix the test agent with the culture medium, and measure the toxicity of the medicine by the growth rate of the colony on the culture medium. Generally used for fungi that do not produce spores or have a small amount of spores and dense hyphae. The colony growth rate is generally expressed by the time (days or hours) required for the colony to reach a given size, or the size of the colony diameter per unit time.

The perforating method mentioned in this application refers to perforating a test plate with a sterilized perforator or steel pipe, and then injecting a certain amount of the sample to be tested into the hole, and measuring the inhibition zone after incubating for a period of time. A way of size.

The extraction method mentioned herein refers to the use of the difference in solubility or partition coefficient of a compound in two immiscible (or slightly soluble) solvents to transfer the compound from one solvent to another. After repeated extractions, most of the compounds are extracted. Ethyl acetate is a medium polar solvent, which can extract a variety of substances from the fermentation filtrate of bacteria, such as molecules with less polarity (such as glucose or glycosides), molecules with very small polarity (such as certain Paraffin), molecules containing salt structures (such as amino acids), etc.

The ammonium sulfate precipitation mentioned in this application refers to the technique of using different concentrations of ammonium sulfate solutions to precipitate and separate proteins. Commonly used to separate immunoglobulins. The ammonium sulfate precipitation method can be used to concentrate and partially purify proteins from a large number of crude preparations. The high concentration of salt ions in the protein solution can compete with the protein for water molecules, thereby destroying the hydration film on the surface of the protein, reducing its solubility, and making it precipitate out of the solution. The solubility of various proteins is different, so different concentrations of salt solutions can be used to precipitate different proteins. This method is called salting out. Salt concentration is usually expressed in terms of saturation. Ammonium sulfate is the most widely used because of its high solubility, low temperature coefficient and difficulty in denaturing protein.

The identification of bacteria with 16S primers in this application refers to the use of methods such as PCR and sequence comparison to determine the type of bacteria to be tested. 16S rDNA is a DNA sequence corresponding to 16S rRNA on bacterial chromosomes, which is present in all bacterial chromosomal genes. Its internal structure is composed of conserved regions and variable regions. The variable regions within the molecule show the specificity of bacteria at the level of different classification levels.

There are three types of ribosomal RNA in bacteria, namely 5S, 16S and 23S rRNA. Among them, 5S rRNA is easy to analyze, but the number of nucleotides is too small, consisting of only a few dozen nucleotides, resulting in insufficient genetic information and cannot be used for classification research; 23S rRNA is too large for its molecular weight and contains nucleosides. The amount of acid is almost twice that of 16S rRNA, and it is difficult to analyze and not select it for classification research. 16S rRNA is usually used for bacterial classification research.

First, 16S rRNA is ubiquitous in prokaryotes (the homologous molecule in eukaryotes is 18S rRNA). rRNA participates in the process of biological protein synthesis, its function is essential to any organism, and it remains unchanged during the long course of biological evolution, which can be regarded as the time clock of biological evolution. Secondly, 16S rRNA molecules contain both highly conserved sequence regions and moderately conserved and highly variable sequence regions. Therefore, it is suitable for the study of various biological relationships with different evolutionary distances. Third, the relative molecular weight of 16S rRNA is moderate, about 2kb nucleotides, which is convenient for sequence analysis. Therefore, it can be used as a good tool to measure the evolution and genetic relationship of various organisms.

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 difficult to store. Therefore, 16S rDNA is usually used to identify the type of bacteria.

The PCR, or polymerase chain reaction mentioned in this application, means that under the catalysis of DNA polymerase, the parent strand DNA is used as a template, and a specific primer is used as the starting point for extension. Through the steps of denaturation, annealing, extension, etc., it replicates with the parent strand in vitro. The process of DNA complementary to the template DNA. PCR is an in vitro synthetic amplification technology that can quickly and specifically amplify target DNA fragments in vitro. In the embodiments of the present application, the parent strand may be the genomic DNA of a monoclonal test bacterium.

The OD value mentioned in this application is the abbreviation of optical density (optical density), which represents the optical density absorbed by the test object. Measure the absorbance value of the bacterial culture solution at 600nm (expressed by OD ₆₀₀ ), you can measure the concentration of the bacterial culture solution to estimate the growth of bacteria, so the optical density value at 600nm can be used to express the cell density of the bacterial body, among which, The absorbance is proportional to the concentration of bacteria in the culture solution.

The PDA medium described in this application refers to potato glucose medium, where P, D, and A are the abbreviations of Potato Dextrose Agar (Medium). PDA medium is a commonly used fungus medium such as yeast, mold, and mushroom, and it is a semi-synthetic medium.

The biocontrol bacteria or biocontrol bacteria mentioned in this application refer to one or more kinds of bacteria with biological control functions. It refers to the use of beneficial microorganisms to kill or reduce the number of pathogenic organisms to control the occurrence and development of plant diseases. Also known as "using bacteria to cure bacteria". Biological control is an important part of the integrated pest management system. It has the advantages of not polluting the environment, non-toxic to humans and animals, and no side effects to plants. It is especially suitable for the control of soil-borne diseases.

In the ecological environment, there are many mechanisms for one kind of microorganism to control the growth of other microorganisms. Different biocontrol bacteria and the same kind of biocontrol bacteria may have different biocontrol mechanisms when interacting with different plants. Taking Trichoderma as an example, the biocontrol mechanism of biocontrol bacteria can be roughly divided into competition. For example, Trichoderma is highly adaptable to the environment, grows much faster than pathogens, can compete with pathogens for nutrition or space, and effectively utilize plant surfaces. Or the concentration of nutrients near the entry point, quickly occupy the space to absorb nutrients, occupy the invasion site of pathogens without leaving a gap for the invasion of pathogens; antagonism, such as the non-volatile metabolites produced by Trichoderma can strongly inhibit cotton yellow The growth of wilt fungus causes the hyphae of the pathogenic fungus to appear cell cytoplasmic concentration and hyphae rupture; induce resistance, such as Trichoderma viride penetrates and colonizes the cotton root epidermis and outer skin tissues, and its peroxidase activity increases , The accumulation of terpenoids can control pathogen infection more effectively than plants that are not infected by Trichoderma viride, and induce disease resistance of cotton; parasitic effect; antibiotic effect, etc. Many biocontrol microorganisms play a biocontrol role through a single mechanism, and some microorganisms can work together by focusing on different mechanisms.

In this application, more than 100 strains of bacteria that may have biocontrol activity are isolated and purified from the soil, and the main fungal pathogens existing in the laboratory are used as targets to detect the biocontrol properties of the isolated bacteria.

Example 1. Isolation and purification of soil strains

According to an embodiment of the present application, bacteria can be separated and purified from any soil where biocontrol bacteria may exist, and their biocontrol properties can be tested. According to an embodiment of the present application, the tomato-rice rotation soil is taken, and the bacteria in it are separated and purified.

1. Prepare soil diluent. Weigh an appropriate amount of soil. The soil mentioned here is the soil that may contain biocontrol bacteria retrieved from the wild in advance. According to an embodiment of the present application, the soil can also be taken directly in the field. According to an embodiment of the present application, 10 g of soil can be weighed. Put the weighed soil into sterile water, break it, and then let it stand for more than 30 minutes to analyze the microorganisms in the soil. According to an embodiment of the present application, 10 g of soil is weighed and mixed with 100 mL of sterile water. The obtained supernatant is the soil stock solution.

Soil stock solution was then diluted 10-fold, ^{102-fold, 103-fold, 104-fold, 105-fold, 106-fold} and so on. According to an embodiment of the present application, dilute in the above-mentioned manner to obtain 1 g, 10 ^-1 g, 10 ^-2 g, 10 ^-3 g, 10 ^-4 g, 10 ^-5 g, 10 ^-6 g and other soils Microbial content. This step is convenient to obtain a single clone of the microorganisms in the soil. For example, according to an embodiment of the present application, suck 10 mL of the soil stock solution in a No. 0 test tube, and then suck 1 mL of the soil stock solution from this test tube, mix it in a No. 1 test tube with 9 mL of sterile water, and mix well, so that Dilute the soil stock solution 10 times. Operating sequentially, to obtain a stock solution was diluted ^102-fold Soil, ¹⁰³ times, ¹⁰⁴ times, ¹⁰⁵ times, ¹⁰⁶ times solution. Label the test tubes as 0, 1, 2, 3, 4, 5, and 6 according to the dilution multiple of the soil stock solution.

2. Paint the board. That is, the microorganisms in each test tube in step 1 are coated on the solid medium. According to an embodiment of the present application, the solution is respectively sucked from the 7 test tubes made in step 1, transferred to the surface of the bacterial solid culture medium, and the solution is evenly coated with a tool such as a spatula, a glass rod, and the like. After drying until the surface of the culture medium is relatively dry and no liquid state, the culture medium is packaged. According to an embodiment of the present application, a sealing film can be used for packaging. According to an embodiment of the present application, according to experimental and statistical requirements, the solution of each dilution can be coated on the surface of multiple solid culture media. For example, the solution of each dilution is spread on the surface of 3 solid culture media.

3. Microbial culture. Place the bacteria-coated solid culture medium in a suitable environment, and take it out after the visible monoclonal bacteria grow. According to an embodiment of the present application, bacteria can be cultured in a 37-degree constant temperature environment, such as a 37-degree incubator, water bath, etc., for more than 12 hours. The bacteria can also be cultured in a 28 degree environment, such as a 28 degree incubator, water bath, etc., for more than 48 hours.

4. Count and description of colonies. Count the number of colonies on the surface of each solid medium, observe the morphological characteristics of the colonies, and record the results. For example, in test tube 0, 10 mL of soil stock solution is directly sucked and coated on a solid medium, and the number of colonies obtained by culturing is the number of bacteria in 1 g of soil; the liquid in test tube 1 is the dilution of the solution in test tube 0 It is obtained by 10 times, so it is coated on a solid medium, and the number of colonies obtained by culturing is the number of bacteria in 0.1g of soil, and other test tubes can be deduced by analogy.

5. The plate is scribed and separated. The colonies obtained in the above steps were streaked and re-cultured at a suitable temperature. This operation can not only isolate the monoclonal strains, but also multiply and store the monoclonal strains, and facilitate subsequent sequencing and classification experiments.

6. Bacteria preservation and identification.

Example 2. Identification of strains

According to an embodiment of the present application, 16S universal primers can be used for preliminary identification of strains. According to an embodiment of the present application, the strain can be directly used as a substrate for bacterial species identification, or genomic DNA can be extracted first, and genomic DNA can be used as a substrate for bacterial species identification. According to an embodiment of the present application, agarose gel electrophoresis is used to detect the content and quality of the extracted bacterial genomic DNA. Fig. 1A is an agarose gel electrophoresis diagram of bacterial genomic DNA extracted according to an embodiment of the present application. As shown in Fig. 1A, the bands of the genomic DNA are obvious, and the concentration meets the requirements of subsequent PCR experiments.

1. Design and select primers for PCR amplification of 16S rDNA. According to an embodiment of the present application, PCR primers for identifying strains can be designed by themselves. According to an embodiment of the present application, commonly used general primers for bacterial identification can also be selected for PCR amplification. According to an 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'.

According to the different selected DNA polymerases, select the appropriate PCR reaction system and reaction conditions. This application does not limit the use of any kind of DNA polymerase, nor does it limit the PCR reaction system and reaction conditions after using the same DNA polymerase. For example, when using Taq DNA polymerase for PCR amplification, you can refer to the reaction system listed in Table 1:

Table 1. PCR system table (12μL)

The reaction conditions of PCR are as follows: first carry out pre-denaturation at 94℃, 5min, and then enter the cycle program, each cycle program is 94℃ denaturation 30s, 55℃ annealing for 30s, 72℃ extension for 1min30s, for 30 cycles, and finally 72 Incubate at ℃ for 7min.

According to an embodiment of the present application, agarose gel electrophoresis is used to detect the content and quality of DNA fragments obtained after PCR using the extracted genomic DNA as a template through 16S universal primers. FIG. 1B is an agarose gel electrophoresis diagram of a DNA fragment obtained by performing PCR with 16S universal primers based on the extracted bacterial genomic DNA as a template according to an embodiment of the present application. As shown in Figure 1B, the 7 groups of experiments all obtained PCR products with a single band and a concentration that met the requirements for subsequent sequencing and identification.

2. Identification of strains. According to an embodiment of the present application, the PCR product obtained in the above experiment can be sequenced. The obtained sequencing results are compared with the sequences of the existing strains, and finally the types of the identified strains are obtained. For example, input the results obtained by sequencing to the NCBI website (https://www.ncbi.nlm.nih.gov) for blasting, and the identified strains can be obtained. Figure 1C is a phylogenetic tree obtained by PCR amplification of the isolated strains using 16s primers according to an embodiment of the present application, and the phylogenetic tree obtained after sequence alignment, and the biocontrol bacteria identified as boron-resistant lysine Acid Bacillus (Lysinibacillus boronitolerans). This strain was deposited in the China Center for Type Culture Collection on October 8, 2019, referred to as CCTCC, and the deposit number is CCTCC NO.M2019773. According to the phylogenetic tree shown in Figure 1C, the evolutionary relationship between the boron-resistant Lysine-resistant Bacillus (Lysinibacillus boronitolerans) and other bacterial species can be clearly seen.

Example 3 Identification of biocontrol characteristics of purified bacteria

According to an embodiment of the present application, the toxic medium method can be used to identify the antibacterial activity of the potential biocontrol bacteria isolated in the present application. According to an embodiment of the present application, the specific operation of the toxic medium method is:

1. Configure the PDA solid medium and cool it to about 55°C; or heat and melt the solidified PDA medium and cool it to about 55°C;

2. Pick a single colony of Boron-resistant Lysine Bacillus and inoculate it in LB liquid medium, cultivate overnight at 37°C, 150r/min, transfer the bacterial solution the next day, and use the volume of 200ml bacterial solution/500ml LB medium , Until the bacterial concentration is 0.02 OD ₆₀₀ /ml, 150r/min rotation speed, placed in a constant temperature shaker 37 ℃ for about 24h, 48h, 72h, 96h, 120h, so that the OD ₆₀₀ value reaches 4.8, 4.5, 3.0, 2.5 and 2.1 respectively Around, collect the fermentation products at different time points, place them in a centrifuge, centrifuge at 8000r/min to remove the precipitates to obtain the fermentation supernatant, and then filter the fermentation supernatant with a 0.22μm filter membrane to obtain a sterile fermentation filtrate;

3. Mix the fermentation filtrate and the PDA medium in a volume ratio of 1:9, that is, the fermentation broth accounts for 10%, to prepare a drug-containing plate containing the antagonistic antibacterial fermentation broth;

4. After the culture medium mixed with the fermentation broth is cooled, inoculate 1 test pathogenic bacteria cake (diameter 8mm) in the center of the plate;

5. Place the inoculated medium in a 28°C constant temperature incubator for cultivation.

Take the PDA medium plate without fermentation broth as a control, repeat the above steps 1-4 at least three times, that is, do at least three antibacterial test experiments of biocontrol bacteria against specific bacteria.

In this application, the antibacterial properties of biocontrol bacteria are evaluated by the following formula:

Mycelial growth inhibition rate (%) = (control colony diameter-treated colony diameter) / (control colony diameter-0.8) × 100%

Figure 2 shows the growth inhibitory effect of Lysinibacillus boronitolerans fermentation filtrate on Cerrena unicolor according to an embodiment of the present application. Among them, Figures 2A, 2B, 2C, 2D, and 2E are the experimental groups, in which the sterile fermentation filtrate obtained after fermenting with Boron-Lysine Bacillus for about 24h, 48h, 72h, 96h and 120h was added to the culture medium; Figure 2F is a control group, without adding the sterile fermentation filtrate of Boron-Lysine-resistant Bacillus to its culture medium. The dot in the middle of the medium is the target pathogen. According to an embodiment of the present application, the target pathogen is Cerrena unicolor.

Combining Figure 2 and the above formula, it can be clearly seen that the growth of the pathogen of betel nut root rot is obviously inhibited by the fermentation filtrate of boron-resistant lysine bacillus. According to calculations, the antagonistic efficiency of the fermentation broth of Lysinibacillus boronitolerans for about 24h, 48h, 72h, 96h and 120h against Cerrena unicolor is about 53.33%, 60%, respectively. 76%, 82.67%, 77.33%. According to multiple experimental verifications in this application, when the fermentation time of Lysinibacillus boronitolerans is 96h and the OD ₆₀₀ is about 2.5, it can effectively inhibit the growth of Cerrena unicolor, and the antagonistic efficiency reaches About 82%.

Example 4 Identification of Biocontrol Characteristics of Crude Extracts of Isolated Bacterial Fermentation Broth

According to an embodiment of the present application, the ethyl acetate extraction method and the ammonium sulfate precipitation extraction method are used to crudely extract the bacterial fermentation broth with biocontrol effects, and the antibacterial activity of the crude extract is identified by the perforation method.

According to an embodiment of the present application, the specific operation steps of the punching method are as follows:

1) Prepare PDA solid medium and cool it to about 55°C; or heat and melt the solidified PDA medium and cool it to about 55°C;

2) After the medium is cooled, in the midline part of the medium, 3.5cm from the center point, use a 0.8cm puncher to carefully press and punch holes, and use the sterilized tweezers to grab the excess medium and place it in the center of the plate. Inoculate 1 test pathogenic bacteria cake (diameter 8mm);

3) Carefully inject the extract into the punched hole. In this experiment, the left hole is the control solvent, and the right is the extract.

According to an embodiment of the present application, the specific operation for preparing the crude extract is as follows:

(1) Preparation of crude extract by ethyl acetate extraction method

Incubate boron-resistant lysine bacillus at 37°C at a speed of 150r/min, then transfer the bacterial solution to 200ml bacterial solution/500ml LB medium to fill the volume to a final concentration of 0.02OD ₆₀₀ /ml, continue Rotate at 150r/min and incubate at 37℃ for 96h, so that the OD ₆₀₀ value reaches about 2.5. Collect the fermentation product, centrifuge at 8000r/min to remove the precipitate to obtain the fermentation supernatant, and then filter it with a 0.22μm filter to obtain a sterile Fermentation filtrate. Take 400 ml of the aseptic fermentation filtrate, add an equal volume of ethyl acetate solvent, shake and extract 4 times, after the extraction, use a separatory funnel to take out all the ethyl acetate in the upper layer to obtain the extract. Combine the four extracts and add them to a 500ml rotary evaporation flask for rotary evaporation concentration until the ethyl acetate is completely evaporated to dryness, and then add acetone to dissolve the remaining extract to make the concentration 1mg/ml, 5mg/ml, 10mg/ml, 15mg /ml, respectively test the antibacterial activity of the crude extract. The control solvent is acetone. Fig. 3 is an embodiment of the present application using ethyl acetate extraction to prepare a crude extract of the fermentation broth of boron-lysine-resistant Bacillus fermented for 96 hours, and test its inhibitory effect on the growth of betel rot fungus. As shown in the experimental results shown in Figure 3, the crude extracts of different concentrations all have different degrees of inhibition on betel nut root rot bacteria. Among them, when the concentration of the bold substance is 10mg/ml, the inhibitory effect on Cerrena unicolor is the most obvious.

(2) Preparation of crude extract by ammonium sulfate precipitation method

First, determine the optimal ammonium sulfate concentration. Obtain a large amount of fermentation broth of boron-resistant lysine bacillus under the optimal fermentation conditions for 96 hours, collect the fermentation broth, and centrifuge at 4°C and 8000 rpm for 10 minutes to remove the bacteria. Take 200ml each of the supernatants, slowly add ammonium sulfate to the saturation of 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, precipitate overnight at 4°C, and collect the precipitate by centrifugation at 8000 rpm for 10 minutes , The precipitate is dissolved in PBS (pH 7.0) and dilute to the same volume to determine its antibacterial activity. Each treatment is repeated 3 times. The ammonium sulfate saturation of the most active component is required for the preparation of the crude antibacterial extract. The optimum saturation of ammonium sulfate.

Next, the crude extract was prepared by the ammonium sulfate precipitation method. Take 400ml of the fermentation supernatant of Boron-Lysine-resistant Bacillus under the optimal fermentation conditions for 96 hours, and add ammonium sulfate to the supernatant while stirring to make the saturation reach 50%, 60%, 70% or 80%. Precipitation overnight at 4°C. The precipitate was collected by centrifugation at 8000 rpm for 10 min. The precipitate was dissolved in PBS (pH 7.0) and the volume was adjusted to 10 ml to determine its antibacterial activity. The control solvent was PBS (pH 7.0). Fig. 4 shows a 96-hour fermentation broth crude extract of boron-lysine-resistant Bacillus fermented by an ammonium sulfate precipitation method prepared according to an embodiment of the present application, and its inhibitory effect on the growth of betel rot fungus is tested.

According to an embodiment of the present application, the biocontrol bacteria (for example, Lysinibacillus boronitolerans of the present application) can be mixed with the medium to inhibit the growth of pathogenic bacteria, thereby preventing plant infection with pathogenic bacteria or treating Plants that have been infected with pathogenic bacteria. According to an embodiment of the present application, the medium is any carrier that can carry pathogenic bacteria. For example, the medium can include betel nut seeds, fruits, and the plant itself, as well as the soil and water environment where betel nut grows, and even the medium for cultivating plants. One or more of. According to an embodiment of the present application, the pathogenic bacteria may be Cerrena unicolor. Of course, the types of pathogenic bacteria should not be limited. The bacteria and fungi that can inhibit the growth of Lysinibacillus boronitolerans in this application to a certain extent should be regarded as pathogenic bacteria mentioned in this application.

According to the present example and the experimental results shown in Figs. 3 and 4, it can be seen that various components in the fermentation filtrate of Lysinibacillus boronitolerans (Lysinibacillus boronitolerans) have different degrees of inhibition on Cerrena unicolor. Therefore, Lysinibacillus boronitolerans can be used as chassis microbial chassis cells for mass production of fermentation filtrate with antibacterial activity, so as to meet the demand for fungicides in agriculture, production and life. According to an embodiment of the present application, it can be further improved on the basis of Lysinibacillus boronitolerans, which can be used for mass production of one or more proteins or other substances with specific antibacterial activity The modification module further enhances the function of the fungicide that inhibits the growth of Cerrena unicolor, and improves the efficiency of the fungicide that inhibits the growth of Magnaporthe oryzea.

According to an embodiment of the present application, Lysinibacillus boronitolerans can also be made into a fungicide to inhibit the growth of pathogenic bacteria such as Cerrena unicolor. According to an embodiment of the present application, the bactericide includes Lysinibacillus boronitolerans and auxiliary materials. According to an embodiment of the present application, the bactericide includes a fermentation broth prepared by a cell modification module based on Lysinibacillus boronitolerans (Lysinibacillus boronitolerans) and auxiliary materials. According to an embodiment of the present application, the fermentation time of the fermentation broth of Lysinibacillus boronitolerans (Lysinibacillus boronitolerans) in the fungicide is 96 hours, and the crude extract is an extract obtained by an ethyl acetate extraction method and an ammonium sulfate precipitation method. According to an embodiment of the present application, the modification module is a modification module that can mass-produce one or more proteins or other substances with specific antibacterial activity. According to an embodiment of the present application, the auxiliary material is one or more of water, liquid culture medium, solid culture medium, and glycerol. According to an embodiment of the present application, the proportion of glycerin can be adjusted according to the storage conditions of the bactericide. For example, the volume ratio of glycerin can be 10%, 25%, 50%, 75%, etc.

According to an embodiment of the present application, the boron-lysine-resistant bacillus of the present application can also be mixed with other bacteria and fungi with biological control functions to prepare a preparation that has a good inhibitory effect on a variety of pathogenic bacteria.

According to an embodiment of the present application, the method for preventing or treating pathogenic bacteria infecting plants includes applying the aforementioned fungicide containing the fermentation broth or crude extract of Lysinibacillus boronitolerans to Mix with the medium to inhibit the growth of pathogenic bacteria on the surface of the medium. According to an embodiment of the present application, it is also possible to directly cultivate and mix Lysinibacillus boronitolerans to a desired concentration, and then 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 can include betel nut seeds, fruits, and the plant itself, as well as the soil and water environment where betel nut grows, and even the medium for cultivating plants. One or more of. According to an embodiment of the present application, the pathogenic bacteria may be Cerrena unicolor. Of course, the types of pathogenic bacteria should not be limited. The Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans) of this application, as well as other biocontrol bacteria mixed with it, which can inhibit their growth to a certain extent, should be regarded as bacteria and fungi. The pathogenic bacteria mentioned in this application.

Therefore, if Lysinibacillus boronitolerans is mixed into the soil where betel nut trees are grown, the growth of Cerrena unicolor in the soil can be completely inhibited, and the infection of betel nut trees can effectively prevent the infection of betel nut trees, which may lead to production reduction or even production reduction. Death allows the production of betel nut to be maintained. At the same time, the incorporation of boron-resistant lysine bacillus (Lysinibacillus boronitolerans) into the soil will not cause adverse effects on the soil environment, nor will it cause ecological pollution, and is an environmentally friendly disease prevention and control measure. Further, for example, it is possible to assemble pathways, synthetic modules, and rationally modify modules or systems on the basis of synthetic elements and devices of biocontrol bacteria metabolites to produce more potent antibacterial activity; or by revealing gene circuits and Regulating the network, rationally designing or optimizing the logical gene circuit and functional gene circuit, making it the smallest cell factory to produce potent antibacterial activity, can bring a new direction for the prevention and treatment of betel nut root rot.

The above-mentioned embodiments are only used to illustrate the present invention, and are not intended to limit the present invention. Those of ordinary skill in the relevant technical fields can also make various changes and modifications without departing from the scope of the present invention. Therefore, all The equivalent technical solutions should also belong to the scope of the disclosure of the present invention.

Claims (11)

1. Application of boron-resistant lysine bacillus in inhibiting the growth of Cerrena unicolor, wherein the boron-resistant lysine bacillus (Lysinibacillus boronitolerans) was deposited in a Chinese type culture on October 8, 2019 The Depository Center, referred to as CCTCC, has the deposit number CCTCC NO.M 2019773.
2. A bactericide, comprising: a fermentation broth of Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans), the Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans) was deposited in the China Type Culture Collection on October 8, 2019 , Referred to as CCTCC, the deposit number is CCTCC NO.M 2019773; and accessories.
3. The bactericide according to claim 2, wherein the auxiliary material is one or more of water, liquid culture medium, solid culture medium, and glycerin.
4. The fungicide according to claim 2, wherein the fermentation broth of the boron-resistant bacillus lysine is the fermentation of Lysinibacillus boronitolerans (Lysinibacillus boronitolerans) cultured for 24-96 hours, and the OD ₆₀₀ is 4.8-2.1. liquid.
5. Application of Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans) in the preparation of a fungicide for inhibiting the growth of Cerrena unicolor, wherein the Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans) was issued in October 2019 It was deposited at the China Center for Type Culture Collection on the 8th, referred to as CCTCC, and the deposit number is CCTCC NO.M 2019773.
6. Application of Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans) as chassis cells in the preparation of a fungicide for inhibiting rice blast, wherein the Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans) was deposited in China on October 8, 2019 The Type Culture Collection, referred to as CCTCC, has the deposit number CCTCC NO.M 2019773.
7. Boron-resistant lysine bacillus (Lysinibacillus boronitolerans) is used as a chassis cell-based transformation module, wherein the boron-resistant lysine bacillus (Lysinibacillus boronitolerans) was deposited in the China Type Culture Collection on October 8, 2019 , Referred to as CCTCC, and the deposit number is CCTCC NO.M 2019773.
8. The application of the transformation module according to claim 8 in the preparation of a fungicide for inhibiting the growth of Cerrena unicolor.
9. A method for preventing or treating betel nut root rot, including:

   Obtain the fungicide according to claim 2; and

   The bactericide is mixed with the medium.
10. The method according to claim 9, wherein the medium is a vector carrying Cerrena unicolor.
11. The method according to claim 10, wherein the carrier is one or more of a seed, a plant, the soil in which the plant grows, and a medium for cultivating the plant.

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