WO2021068556A1 - Fongicide contre le pathogène de la pyriculariose du riz préparé à l'aide de lysinibacillus boronitolerans en tant que cellule châssis - Google Patents

Fongicide contre le pathogène de la pyriculariose du riz préparé à l'aide de lysinibacillus boronitolerans en tant que cellule châssis Download PDF

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WO2021068556A1
WO2021068556A1 PCT/CN2020/097735 CN2020097735W WO2021068556A1 WO 2021068556 A1 WO2021068556 A1 WO 2021068556A1 CN 2020097735 W CN2020097735 W CN 2020097735W WO 2021068556 A1 WO2021068556 A1 WO 2021068556A1
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boron
resistant
medium
lysine
bacillus
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PCT/CN2020/097735
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Chinese (zh)
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刘柱
晏承梁
马香
唐燕琼
李宏
高玉晓
林敏�
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海南大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the invention relates to a synthetic biology modification and application of biocontrol bacteria, in particular to a rice blast fungicide.
  • Rice blast is one of the important diseases of rice, which can cause a substantial reduction in rice production, and in severe cases, it can lead to a reduction of 40% to 50%, or even no grain harvest.
  • Rice blast is caused by the parasitic fungus (such as rice blast fungus), and the spread of the disease is spread by conidia in the air.
  • the optimum temperature for the development of the pathogen is 25°C to 28°C. High humidity is good for conidia formation and germination, while high humidity for more than one day and night is good for the spread of the disease.
  • Magnaporthe grisea belongs to the subphylum Deuteromycotina. In addition to infecting rice and causing it to infect rice blast, it can also cause skin diseases in humans and some animals.
  • Drug control use antibacterial chemicals to soak seeds in advance, spray pesticides, etc.
  • 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 aroused widespread concern in the society.
  • the present invention proposes a boron-resistant lysine bacillus (Lysinibacillus boronitolerans), which 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.M 2019773.
  • CCTCC boron-resistant lysine bacillus
  • a bactericide includes the above-mentioned boron-resistant Lysine Bacillus fermentation broth (Lysinibacillus boronitolerans) and auxiliary materials.
  • the auxiliary material is one or more of water, liquid culture medium, solid culture medium, and glycerin.
  • the above bactericide wherein the fermentation broth of Boron-resistant Lysine Bacillus is the above-mentioned Lysinibacillus boronitolerans (Lysinibacillus boronitolerans) cultured for 24-96 hours, and the OD 600 is 4.8-2.5 hours Of fermentation broth.
  • Lysinibacillus boronitolerans Lysinibacillus boronitolerans
  • a boron-lysine-resistant bacillus as described above is used as a chassis cell-based transformation module.
  • a method for preventing or treating rice blast includes: obtaining the above-mentioned fungicide; and mixing the fungicide with a medium.
  • the medium is a vector carrying Magnaporthe oryzea.
  • the carrier is one or more of a seed, a plant, the soil where the plant grows, and a medium for cultivating the plant.
  • the bactericide as described above wherein the fermentation broth of Lysinibacillus boronitolerans is the fermentation broth of Lysinibacillus boronitolerans with 200ml broth (initial OD is 0.02OD 600 /ml)/500ml LB medium
  • the fermentation product is obtained when the OD value of the bacteria liquid reaches 4.8, 4.0, 2.5, and the OD value of the bacteria liquid reaches 4.8, 4.0, 2.5, and the culture is carried out at 150r/min at 37°C for 24h, 60h or 96h.
  • Rice blast is an important disease of rice, causing serious economic losses to agricultural production.
  • the extensive use of chemical pesticides also poses a huge threat to the environment and food.
  • Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans) can not only reduce the occurrence and damage of rice blast to a large extent, but also has no impact on the ecological environment. It is expected to be a high-quality chassis microorganism for synthetic biology research and bring to the control of rice blast. New direction.
  • Fig. 1 is the identification of potential biocontrol bacteria isolated from soil according to an embodiment of the present application.
  • 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 rice blast fungus according to an embodiment of the present application.
  • Figures 2A, 2B, and 2C are the experimental groups, in which the sterile fermentation filtrate obtained after fermentation with boron-resistant lysine Bacillus for 24h, 60h, and 96h was added to the culture medium;
  • Figure 2D is the control group. Add sterile fermentation filtrate of Boron-Lysine-resistant Bacillus to the medium;
  • Fig. 3 shows the antibacterial activity detection of different concentrations of ethyl acetate extracts of 96h fermentation broth of Lysinibacillus boronitolerans (Lysinibacillus boronitolerans) against Magnaporthe oryzea according to an embodiment of the present application.
  • the concentration of the crude extract is 1mg/ml, 5mg/ml, 10mg/ml and 15mg/ml;
  • Figure 4 shows the detection of antibacterial activity of extracts obtained by precipitation of ammonium sulfate with different saturations in 96h fermentation broth of Lysinibacillus boronitolerans according to an embodiment of the present application against Magnaporthe oryzea .
  • the concentration is a concentration within a certain numerical range.
  • concentration is expressed by the OD value
  • OD 600 of 4.8 appears in the application
  • 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.
  • the fermentation broth mentioned in this application refers to the fermentation product-containing liquid produced by bacteria after fermentation.
  • the fermentation broth of Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans) is a 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, 60h or 96h.
  • the fermentation time of boron-resistant lysine bacillus was about 24, 60h and 96h
  • the OD value of the bacterial solution was about 4.8, 4.0 and 2.5 (OD value decreased with the extension of fermentation time).
  • the precipitate is removed by centrifugation, the fermentation supernatant is collected, and then filtered with a 0.22 ⁇ m 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 the determination of 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 virulence of the medicine by the growth rate of the colony on the culture medium.
  • 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.
  • 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.
  • ribosomal RNA in bacteria There are three types of ribosomal RNA in bacteria, namely 5S, 16S and 23S rRNA.
  • 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.
  • 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 indispensable 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.
  • 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.
  • 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.
  • 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.
  • PCR is an in vitro synthetic amplification technology that can quickly and specifically amplify target DNA fragments in vitro.
  • the parent strand may be the genomic DNA of a monoclonal test bacterium.
  • optical density optical density
  • 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.
  • PDA medium 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.
  • biocontrol bacteria or biocontrol bacteria mentioned in this application refers 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.
  • Trichoderma 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.
  • 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.
  • bacteria can be separated and purified from any soil where biocontrol bacteria may exist, and their biocontrol properties can be tested.
  • the tomato-rice rotation soil is taken, and the bacteria in it are separated and purified.
  • the 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.
  • the soil can also be directly weighed in the field.
  • 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.
  • 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.
  • This step is convenient to obtain a single clone of the microorganisms in the soil.
  • test tube with 9 mL of sterile water, and mix well, so that Dilute the soil stock solution 10 times.
  • 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.
  • the microorganisms in each test tube in step 1 are coated on the solid medium.
  • the solution is respectively sucked from the 7 test tubes made in step 1, and 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.
  • a sealing film can be used for packaging.
  • 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.
  • bacteria can be cultured in a 37°C constant temperature environment, such as a 37°C constant temperature box, water bath, etc., for more than 12 hours.
  • the bacteria can also be cultured in an environment of 28°C, such as a 28°C thermostat, water bath, etc., for more than 48 hours.
  • 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.
  • the bacterial 16S universal primer can be used for preliminary identification of strain species.
  • the strain can be directly used as a template for bacterial species identification, or genomic DNA can be extracted first, and genomic DNA can be used as a template for bacterial species identification.
  • 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.
  • PCR primers for identifying strains can be designed by themselves.
  • commonly used general primers for bacterial identification can also be selected for PCR amplification.
  • universal primers 27F and 1492R are selected for PCR amplification, wherein the DNA sequences of 27F and 1492R are as follows:
  • reaction conditions of PCR are as follows: first carry out pre-denaturation at 94°C, 5min, and then enter the cycle program, each cycle program is 94°C denaturation 30s, 55°C annealing for 30s, 72°C extension for 1min30s, for 30 cycles, and finally 72 Incubate at °C for 7min.
  • 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.
  • 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.
  • input the results of sequencing to the NCBI website (https://www.ncbi.nlm.nih.gov) for blasting, and then the identified strains can be obtained.
  • Fig. 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.M 2019773.
  • CCTCC boron-resistant lysine-resistant Bacillus
  • the evolutionary relationship between the boron-resistant lysine-resistant Bacillus (Lysinibacillus boronitolerans) and other bacterial species can be clearly seen.
  • the toxic medium method can be used to identify the antibacterial activity of the potential biocontrol bacteria isolated in the present application.
  • the specific operation of the toxic medium method is:
  • test pathogenic bacteria cake (diameter 8mm) in the center of the plate
  • biocontrol 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 the fermentation filtrate of Lysinibacillus boronitolerans on Magnaporthe oryzea according to an embodiment of the present application.
  • Figures 2A, 2B, and 2C are the experimental groups, in which the sterile fermentation filtrate obtained after fermentation with boron-resistant lysine Bacillus for about 24h, 60h, and 96h was added to the culture medium;
  • Figure 2D is the control group, not in The aseptic fermentation filtrate of Boron-Lysine-resistant Bacillus is added to the culture medium.
  • the dots in the middle of the medium are the target pathogens dropped in.
  • the target pathogen is Magnaporthe oryzea.
  • 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.
  • the specific operation steps of the punching method are as follows:
  • the specific operation for preparing the crude extract is as follows:
  • FIG. 3 shows a 96-hour fermentation broth crude extract of boron-lysine-resistant Bacillus fermented by an ethyl acetate extraction method prepared according to an embodiment of the present application, and its inhibitory effect on the growth of Pyricularia oryzae is tested.
  • the crude extracts of different concentrations have different degrees of inhibition on the rice blast fungus.
  • 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.
  • 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 Pyricularia oryzae is tested.
  • Figure 4 in the precipitation experiments with different ammonium sulfate saturation degrees, the crude extract of the fermentation broth with 80% ammonium sulfate saturation has the most obvious inhibitory effect on the growth of Pyricularia oryzae.
  • the sterile fermentation filtrate or crude extract of biocontrol bacteria can be combined with the medium Mix to inhibit the growth of pathogenic bacteria, thereby preventing plants from infecting pathogenic bacteria or treating plants that have been infected with pathogenic bacteria.
  • the medium is any carrier that can carry pathogenic bacteria.
  • the medium may include rice seeds, the plant itself (that is, seedlings at various stages of rice growth), and may also include the soil and water environment where the rice grows. , Even one or more of the medium for cultivating plants.
  • the pathogenic bacteria may be Magnaporthe oryzea.
  • 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.
  • 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.
  • the modification module further enhances the function of the fungicide for inhibiting the growth of Magnaporthe oryzea, and improves the efficiency of preparing the fungicide for inhibiting the growth of Magnaporthe oryzea.
  • Lysinibacillus boronitolerans can also be made into a fungicide to inhibit the growth of pathogenic bacteria such as Magnaporthe oryzea.
  • the bactericide includes the fermentation filtrate of Lysinibacillus boronitolerans (Lysinibacillus boronitolerans) or its crude extract and auxiliary materials.
  • the bactericide includes a fermentation broth prepared by a cell modification module based on Lysinibacillus boronitolerans (Lysinibacillus boronitolerans) and auxiliary materials.
  • 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.
  • the modification module is a modification module that can mass-produce one or more proteins or other substances with specific antibacterial activity.
  • the auxiliary material is one or more of water, liquid culture medium, solid culture medium, and glycerol.
  • 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.
  • 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.
  • 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.
  • the medium is any carrier that can carry pathogenic bacteria.
  • the medium may include rice seeds, the plant itself (that is, seedlings at various stages of rice growth), and may also include the soil and water environment where the rice grows.
  • the pathogenic bacteria may be Magnaporthe oryzea.
  • 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.
  • Rice blast is an important disease of rice, causing serious economic losses to agricultural production.
  • the extensive use of chemical pesticides also poses a huge threat to the environment and food.
  • Boron-resistant lysine bacillus (Lysinibacillus boronitolerans) can not only reduce the occurrence of rice blast to a large extent, but also has no threat to the ecological environment.

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Abstract

L'invention concerne une application de Lysinibacillus boronitolerans pour inhiber la croissance de Magnaporthe oryzea. La Lysinibacillus boronitolerans. peut non seulement réduire considérablement l'apparition et les dommages causés par la pyriculariose du riz, mais est également respectueuse de l'environnement. Il est probable que la Lysinibacillus boronitolerans joue le rôle de micro-organisme de châssis de haute qualité dans des recherches de biologie synthétique, de manière à amener une nouvelle perspective à la prévention et à la lutte contre la pyriculariose du riz.
PCT/CN2020/097735 2019-10-11 2020-06-23 Fongicide contre le pathogène de la pyriculariose du riz préparé à l'aide de lysinibacillus boronitolerans en tant que cellule châssis WO2021068556A1 (fr)

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CN201910963020.1A CN111187726B (zh) 2019-10-11 2019-10-11 一种耐硼赖氨酸芽孢杆菌作为底盘细胞制备的稻瘟病杀菌剂
CN201910963020.1 2019-10-11

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