CN113925064B

CN113925064B - Application of B-lysine-resistant bacillus in inhibition of growth of pitaya soft rot germs

Info

Publication number: CN113925064B
Application number: CN202111280400.9A
Authority: CN
Inventors: 李宏; 曹欣; 刘柱; 陈银华; 马香; 王丹; 唐燕琼; 高玉晓; 胡新文
Original assignee: Hainan University
Current assignee: Hainan University
Priority date: 2021-11-01
Filing date: 2021-11-01
Publication date: 2023-02-03
Anticipated expiration: 2041-11-01
Also published as: CN113925064A

Abstract

The invention discloses an application of boron-resistant lysine bacillus in inhibiting the growth of pitaya soft rot germs, wherein the boron-resistant lysine bacillus is preserved in a China center for type culture collection in 2019, 10 and 8 months, and the preservation number is CCTCC NO: m2019773. The boron-resistant lysine-resistant bacillus can reduce the occurrence and harm of soft rot of pitaya to a great extent, has no influence on 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 soft rot of the pitaya.

Description

Application of B-lysine-resistant bacillus in inhibition of growth of pitaya soft rot germs

Technical Field

The invention relates to synthetic biology transformation and application of biocontrol bacteria, in particular to application of boron-resistant lysine bacillus in inhibiting growth of pitaya soft rot bacteria.

Background

The pitaya is a low-energy fruit, is rich in water-soluble dietary fiber, has the effects of losing weight, reducing cholesterol, preventing constipation, colorectal cancer and the like, is rich in fiber, and can prevent constipation. Pitaya contains less vegetable albumin in common vegetables and fruits, and the albumin can be combined with heavy metal ions in human bodies to play a role in detoxification. It is rich in antioxidant vitamin C, and has effects in whitening skin and preventing black speck. In addition, the content of iron in the pitaya is very rich. The fruit of dragon fruit has high content of anthocyanin, especially red meat. Anthocyanins are effective antioxidants and are effective in preventing vascular sclerosis and thus preventing heart attacks and cerebral stroke due to blood clot formation; it also has effects in scavenging free radicals, and resisting aging; can also improve the prevention of brain cell degeneration and inhibit the occurrence of dementia.

The soft rot disease spots of the dragon fruits are infiltrated and semitransparent at the initial stage, and the soft rot disease tissues at the later stage appear. In the case of dampness, yellow bacteria are discharged from the affected part, which gives off a fishy smell and spreads to the whole stem node, leaving only the xylem in the center of the stem. The disease is mostly caused by the infection of wounds on tender nodes at the middle upper part of a plant and is related to insect bite and other wounds; but the damage to plants is serious, and disease-causing rottenness of nodes and even downward and upward spread to other stem nodes are often caused. If the seedling management is not good, the field soil humidity is too high, and the disease attack is common. The soft rot of dragon fruit usually starts to be developed from 10 months, the disease is in the prime stage from 1 to 3 months, and the disease is relieved when the temperature rises in 4 months in the next year. The disease has the characteristics of acute onset, rapid spread and great harm, and if effective prevention and treatment measures are not taken in time, the yield is reduced, and the dragon fruit production is seriously influenced.

The common prevention and treatment measures for the pitaya soft rot bacteria comprise the following steps:

(1) Field measures

The field is cleaned in time, and particularly, the diseased fruits are removed and taken out of the field to be burnt or buried deeply. Strong seedlings are cultivated, series of nutrition of Jiamei and Yinlai organic and inorganic combo is added, and the disease resistance of plants is enhanced. Timely planting and reasonably close planting. Drainage is carried out in time in rainy season, water is not accumulated particularly at the lower water head, ventilation is enhanced for protected field cultivation, overhigh humidity in the shed is prevented, and pesticides are sprayed in time to prevent and control fruit-eating pests such as cotton bollworms and the like.

(2) Prevention and treatment with medicament

Before and after rain, 4000 times of 72 percent agricultural streptomycin sulfate soluble powder or 4000 times of new phytomycin solution, 500 times of 50 percent wettable powder of copper succinate and copper ferulate, 500 times of 77 percent wettable particle powder, 800 to 1000 times of 47 percent Carrinone wettable powder and 400 times of 30 percent basic copper sulfate suspension are sprayed in time, and the pesticide application is stopped 3 days before harvesting.

Therefore, in recent years, new methods for controlling plant diseases, which can replace conventional chemical agents, have been developed in various countries throughout the world. Wherein the biological control by using microorganisms and metabolites thereof is recognized as an environment-friendly option. Furthermore, a biological system with the function of the pitaya soft rot fungi is reformed by utilizing synthetic biology, or biocontrol fungi with the growth inhibition function on the pitaya soft rot fungi 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 method becomes the most rapid and effective way for controlling plant diseases, 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 pitaya soft rot bacteria (Fusarium oxysporum), wherein the boron-resistant lysine bacillus (Lysinibacillus boroniculatus P42) is preserved in the China center for type culture collection, CCTCC for short, in 2019, 10, 8 days, and the preservation number is CCTCC NO: m2019773.

The boron-resistant lysine bacillus can be used for preparing a bactericide for inhibiting the growth of the pitaya soft rot bacteria.

The B-lysine-resistant bacillus can be used as a chassis cell for preparing a bactericide for inhibiting the growth of pitaya soft rot bacteria.

The boron-resistant lysine bacillus serving as a chassis cell-based modification module can be used for preparing a bactericide for inhibiting the growth of pitaya soft rot.

In the invention, the bactericide for inhibiting the growth of the pitaya soft rot bacteria comprises the boron-resistant lysine bacillus fermentation liquor and auxiliary materials, wherein the volume percentage content of the boron-resistant lysine bacillus fermentation liquor is 10-90%. Mixing the B-resistant lysine bacillus fermentation filtrate with auxiliary materials to prepare a drug-containing flat plate, and taking the flat plate without adding fermentation liquor as a reference; in general, the fermentation liquor of the boron-resistant lysine bacillus and auxiliary materials are mixed in a ratio of 1:9, namely 10 percent of fermentation liquor, and the proportion of the auxiliary materials can be adjusted according to the storage conditions of the bactericide, for example, the volume percentage of the auxiliary materials can be 10 percent, 25 percent, 50 percent, 75 percent and the like. Wherein: the auxiliary material is one or more of water, a liquid culture medium, a solid culture medium and glycerol, the boron-resistant lysine bacillus fermentation liquor is the OD obtained by culturing the boron-resistant lysine bacillus for 24-96 hours ₆₀₀ 4.8-2.5, specifically 200ml of bacterial liquid of B-resistant lysine bacillus (initial OD of 0.02 OD) ₆₀₀ The liquid filling amount of the LB culture medium per 500ml is cultured for 96 hours at 37 ℃ at the rotating speed of 150r/min, and the fermentation product is obtained when the OD value of the bacterial liquid reaches 2.5.

A method for preventing or treating soft rot of pitaya, comprising the steps of:

step one, obtaining a bactericide for inhibiting the growth of the soft rot germs of the dragon fruits;

and step two, mixing the bactericide for inhibiting the growth of the pitaya soft-rot germs obtained in the step one with a medium (a 0.8cm puncher is used for punching a fungus cake of the pitaya soft-rot germs), and further inhibiting the growth of pathogenic bacteria on the surface of the medium. Wherein the medium is a carrier carrying the pitaya soft rot pathogen, and the carrier is one or more of seeds, plants, soil for plant growth and a culture medium for culturing the plants.

Compared with the prior art, the invention has the following advantages:

the boron-resistant lysine bacillus can reduce the occurrence and harm of the soft rot of the dragon fruits to a great extent, has no influence on 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 soft rot of the dragon fruits.

Preservation information:

the preservation unit is fully called as follows: china center for type culture Collection;

the preservation unit is abbreviated as: CCTCC (China center for type communication);

and (4) storage address: wuhan, wuhan university;

the preservation date is as follows: 2019, 10 and 8 months;

the preservation number is: CCTCC NO: m2019773.

Drawings

FIG. 1 is a diagram of a gel electrophoresis separation of bacterial genomic DNA isolated and purified from soil;

FIG. 2 is a diagram showing a gel electrophoresis separation of a PCR product obtained by specific amplification of genomic DNA using a 16S primer;

FIG. 3 is a phylogenetic tree of potential biocontrol bacteria obtained by sequence alignment, and the obtained potential biocontrol bacteria is identified to be B-resistant lysine bacillus by sequence alignment analysis;

FIG. 4 shows the growth inhibitory effect of B-resistant L-lysine bacillus fermentation broth on Pitaya soft rot pathogen, wherein A is an experimental group, and a sterile fermentation filtrate obtained after B-resistant L-lysine bacillus fermentation is mixed in a culture medium of the experimental group and fermented for 96 hours; b is a control group, in which the culture medium was not mixed with a sterile fermentation filtrate of B.borotolerant lysinibacillus.

Detailed Description

The technical solution of the present invention is further described below with reference to the accompanying drawings, but not limited thereto, and any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be covered by the protection scope of the present invention.

It will be understood by those skilled in the art that when bacteria are described as being fermented and cultured to a concentration, the concentration is within a range of values, for example when the concentration is expressed as OD, the OD if present in the invention 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 by minutes and seconds which cannot be accurately determinedThe time required is related to the bacterial fermentation, the environmental temperature of the culture, the kind of the medium, and the like, so that the time related to the bacterial fermentation, the culture, the growth, and the like, which appears in the present invention, is an approximate time, not a definite time.

Some of the terms appearing in the present invention have the following definitions:

the fermentation liquid refers to a liquid with a fermentation product generated after bacteria are fermented, for example, the boron-resistant lysine bacillus fermentation liquid is 200ml bacterial liquid (initial OD is about 0.02 OD) of boron-resistant lysine bacillus ₆₀₀ The resulting fermentation product was incubated at 37 ℃ for about 24h, 60h or 96h at 150r/min in a liquid content of LB medium/500 ml. When the fermentation time of the B-lysine resistant bacillus is about 24 hours, 60 hours and 96 hours, the OD values of the bacterial liquid are about 4.8, 4.0 and 2.5 respectively (the OD value is reduced along with the prolonging of the 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 of the present invention is also called growth rate method, is one of the conventional methods for measuring the toxicity of germicide, and is suitable for fungi with fast hypha growth and no spore growth. The toxicity of the agent can be measured by the growth speed of colonies. 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 by 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 identification of bacteria by the 16S primer refers to the determination of the type of bacteria to be detected by methods such as PCR, sequence comparison and the like. The 16S rDNA is a DNA sequence corresponding to the code 16S rRNA on the bacterial chromosome, exists in all bacterial chromosome genes, and the internal structure of the gene consists 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.

Three ribosomal RNAs are included in the bacteria, 5S, 16S and 23S rRNA respectively. 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 it, and therefore, 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 various biological genetic relationships with different evolutionary distances. Third, the relative molecular weight of 16S rRNA is moderate, about 2kb of nucleotides, which facilitates sequence analysis. Therefore, it can be used as a good tool for measuring the evolution and affinity relationship of various organisms.

The coding gene of 16S rRNA is 16S rDNA, and it is difficult to directly extract 16S rRNA from bacteria, and the extracted RNA is easy to degrade and difficult to store, so that the 16S rDNA is usually used for identifying the type of bacteria.

The OD value in the present invention is an abbreviation for optical density, and 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 value is proportional to the concentration of bacteria in the culture solution.

The biocontrol bacteria or biocontrol bacteria in the invention refer to one or more bacteria with biological control function, and refer to a measure for controlling the occurrence and development of plant diseases by killing or reducing the number of pathogenic organisms by using beneficial microorganisms, which is also called as bacteria control. 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.

The PDA culture Medium refers to a potato glucose culture Medium, wherein P, D, A is an abbreviation of Pota to 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.

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 growth speed far faster than that of pathogenic bacteria, can compete with the pathogenic bacteria in nutrition or space, effectively utilizes low-concentration nutrient substances on the surface of plants or near the invasion point to quickly occupy 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, such as that non-volatile metabolites which can be produced by trichoderma 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 function biocontrol by a single mechanism, and some may function by concentrating the association between different mechanisms.

The invention 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: separation and purification of soil strain

Bacteria can be isolated, purified from any soil in which they may be present, and tested for their biocontrol properties. In this example, tomato-rice crop rotation soil was taken, and bacteria in the soil were separated and purified.

Step 1, 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, and the soil can also be directly weighed in the field. Weighing 10g of soil, mixing the soil in 100mL of sterile water, smashing, standing for more than 30 minutes, and fully separating out microorganisms in the soil to obtain supernatant, namely the soil stock solution.

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. to obtain 1g and 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, 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 so that the soil stock solution is diluted 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 and 2,3,4,5,6 according to the dilution multiple of the soil stock solution.

And 2, coating the plate. And (3) respectively sucking the solutions from the 7 test tubes prepared in the step (1), transferring the solutions to the surface of the bacterial solid culture medium, and uniformly coating the solutions by using a scraper, a glass rod and other tools. And (5) drying until the surface of the culture medium is dry and is in a liquid-free state, and then packaging the culture medium by using a sealing film. Depending on experimental and statistical requirements, each dilution of the solution can be applied to multiple solid medium surfaces. For example, each dilution of the solution is applied to 3 solid medium surfaces.

And 3, culturing microorganisms. The bacteria are cultured in a 37-degree constant temperature environment, such as a 37-degree constant temperature box, a water bath and the like, for more than 12 hours. The bacteria can also be cultured in 28-degree environment, such as 28-degree incubator, water bath, etc., for more than 48 hours.

Step 4, enumeration 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 No. 1 test tube is obtained by diluting the solution in the No. 0 test tube 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 the soil, and the rest can be analogized in other test tubes.

And 5, scribing and separating the flat plate. 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.

And 6, preserving and identifying strains.

Example 2: identification of the Strain species

The 16S universal primer is used for carrying out preliminary identification on the strain type, the strain can be directly used as a substrate for carrying out strain identification, or genomic DNA is extracted firstly and used as a substrate for carrying out strain identification. In this example, the content and quality of the extracted bacterial genomic DNA were examined by agarose gel electrophoresis. FIG. 1 is an agarose gel electrophoresis chart of the extracted bacterial genomic DNA of the present example, as shown in FIG. 1, the genomic DNA has a significant band and the concentration meets the requirements of the subsequent PCR experiment.

1. The selection primers were designed for PCR amplification of 16S rDNA. The PCR primers for identifying the strain species can be designed by self, and the common bacteria identification universal primers can be selected for PCR amplification. In this example, universal primers 27F and 1492R were 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 invention is not limited to the use of any kind of DNA polymerase, nor to 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 under the condition of 94 ℃ and 5min, then entering a cycle program, wherein each cycle program comprises 30s for 94 ℃ denaturation, 30s for 55 ℃ annealing, 30s for 72 ℃ extension for 1min, performing 30 cycles, and finally keeping the temperature at 72 ℃ for 7min.

In this example, agarose gel electrophoresis was used to detect the content and quality of DNA fragments obtained by PCR using the extracted genomic DNA as a template with 16S universal primers. FIG. 2 is an agarose gel electrophoresis image of a DNA fragment obtained by PCR using 16S universal primers based on the extracted bacterial genomic DNA as a template according to this example. As shown in FIG. 2, 7 sets of experiments all obtained PCR products with single bands and concentrations meeting the requirements of subsequent sequencing identification.

2. And (5) identifying the strain type. The PCR products obtained from the above experiments were subjected to sequencing treatment. 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. 3 shows that 16S rDNA is obtained by PCR amplification of the separated strain with 16S primer, and the obtained biocontrol bacterium is B-resistant lysine bacillus (Lysinibacillus boronicteromolecules P42) through the phylogenetic tree obtained after sequence comparison. 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 of the present invention with other bacterial species can be clearly seen from the phylogenetic tree shown in FIG. 3.

Example 3: biocontrol property identification of purified bacteria

The bacteriostatic activity of the potential biocontrol bacteria separated by the method is identified by adopting a toxic medium-containing method. In this embodiment, the toxic medium-containing method specifically includes the following operations:

1. preparing a PDA solid culture medium, and cooling 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 ℃ overnight at 150r/min, transferring bacterial liquid the next day, and filling liquid in 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 about 24-96 h at the rotating speed of 150r/min so as to obtain OD ₆₀₀ Collecting fermentation products at different time points respectively when the value reaches about 4.8-2.5, placing the fermentation products in a centrifuge, centrifuging at 8000r/min to remove precipitates to obtain fermentation supernatant, and filtering the fermentation supernatant by using a filter membrane of 0.22 mu m 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. 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 1-5 for at least three times, and then carrying out at least three tests of the bacteriostasis of the biocontrol bacteria to specific bacteria.

In the invention, 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. 4 shows the growth inhibition effect of B-lysine-resistant Bacillus subtilis fermentation broth on Pitaya soft rot. Wherein A is an experimental group, and a sterile fermentation filtrate obtained after boron-resistant lysine bacillus is mixed in a culture medium and fermented for 96 hours; b is a control group, sterile fermentation filtrate of boron-resistant lysine bacillus is not mixed in a culture medium, a round point in the middle of the culture medium is a target pathogenic bacterium which is dripped in, and the target pathogenic bacterium is pitaya soft rot.

It is apparent from the above formula in conjunction with fig. 4 that the growth of pitaya soft rot is significantly inhibited by lysinibacillus borealis. According to measurement and calculation, the antagonism efficiency of the fermentation liquid of the lysine bacillus boracium resistant fermentation for about 96 hours to the pitaya soft rot pathogen is about 79.1%. According to multiple experiments of the invention, the fermentation time of the B-resistant lysine bacillus is 96h ₆₀₀ When the concentration is about 2.5, the compound can effectively inhibit the growth of the pitaya soft rot germs, and the antagonistic efficiency reaches about 79 percent.

In this embodiment, biocontrol bacteria (e.g., the B-tolerant L-lysine bacillus of the present invention) may be mixed with a medium to inhibit the growth of pathogenic bacteria, thereby preventing the infection of plants with pathogenic bacteria or treating plants infected with pathogenic bacteria. In this embodiment, the medium is any carrier capable of carrying pathogenic bacteria, for example, the medium may include one or more of a seedling of the dragon fruit, soil in which the dragon fruit grows, an aquatic environment, and even a culture medium for culturing plants. The pathogenic bacteria can be Pitaya soft rot bacteria. Of course, the kind of pathogenic bacteria should not be limited, and bacteria, fungi, etc. that the B.borotolerant Bacillus of the present invention can inhibit its growth to some extent should be regarded as the pathogenic bacteria of the present invention.

According to the embodiment, various components in the B-lysine-resistant bacillus fermentation filtrate have different degrees of inhibition effects on the pitaya soft rot bacteria. Therefore, the boron-resistant lysine bacillus can be used as a chassis microorganism chassis cell to be used for mass production of fermentation filtrate with bacteriostatic activity, thereby meeting the requirements of agriculture and production life on bactericides.

According to the embodiment, the boron-resistant lysine bacillus can be further improved on the basis of being a modified module which can produce one or more proteins or other substances with specific antibacterial activity in a large scale, so that the function of the bactericide for inhibiting the growth of the pitaya soft-rot germs prepared by the boron-resistant lysine bacillus is enhanced, and the efficiency of the bactericide for inhibiting the growth of the pitaya soft-rot germs prepared by the boron-resistant lysine bacillus is improved.

According to the embodiment, the boron-resistant lysine bacillus can be prepared into a bactericide for inhibiting the growth of pathogenic bacteria such as pitaya soft rot. The bactericide comprises boron-resistant lysine-resistant bacillus and auxiliary materials, wherein: the fermentation time of the B-lysine-resistant bacillus fermentation liquor in the bactericide is 96h ₆₀₀ When the concentration is about 2.5, the growth of the pitaya soft rot bacteria can be effectively inhibited; the adjuvants are one or more of water, liquid culture medium, solid culture medium, and glycerol, and the glycerol ratio can be adjusted according to storage conditions of bactericide, such as volume ratio of glycerol of 10%, 25%, 50%, 75%, etc.

According to the embodiment, the B-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 this embodiment, the method for preventing or treating a pathogen from infecting a plant comprises the steps of: the fermentation liquor containing the lysine bacillus resisting boron is mixed with a medium to inhibit the growth of pathogenic bacteria on the surface of the medium, or the lysine bacillus resisting boron can be directly cultured and mixed with the medium after being prepared to the required concentration. The medium is any carrier capable of carrying pathogenic bacteria, for example, the medium may include one or more of dragon fruit seedlings, soil for growing the dragon fruit, water environment, even culture medium for culturing plants, and the like; the pathogenic bacteria can be Pitaya soft rot bacteria. Of course, the kind of pathogenic bacteria should not be limited, and the boron-tolerant lysine bacillus of the present invention and other bacteria, fungi, etc. mixed with it that can inhibit the growth thereof to some extent should be regarded as the pathogenic bacteria of the present invention.

The soft rot of the dragon fruit is an important disease of the dragon fruit, and causes serious economic loss to agricultural production. The use of chemical pesticides in large quantities also poses a great threat to the environment and food. The boron-resistant lysine bacillus can reduce the occurrence of soft rot of pitaya to a great extent, and has no threat to the ecological environment. 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, and a new direction is brought to the prevention and control of the soft rot of the dragon fruit.

Claims

1. Boron-resistant lysine bacillusLysinibacillus boronitoleransInhibiting Pitaya soft rot pathogenFusarium oxysporumThe application in growth aspect, the boron-resistant lysine bacillus is preserved in the China center for type culture Collection in 2019, 10, 8 and the preservation number is CCTCC NO: m2019773.

2. The use of lysine bacillus borotolerant to boron as claimed in claim 1 for the preparation of a fungicide for inhibiting the growth of soft rot of pitaya.

3. The application of the boron-resistant lysine bacillus in preparing the bactericide for inhibiting the growth of the pitaya soft rot bacteria according to claim 2, wherein the bactericide comprises a boron-resistant lysine bacillus fermentation liquid and an auxiliary material, and the volume percentage of the boron-resistant lysine bacillus fermentation liquid is 10-90%.

4. The application of the boron-resistant lysine bacillus in preparing the bactericide for inhibiting the growth of the pitaya soft rot bacteria according to claim 3, wherein the auxiliary materials are one or more of water, a liquid culture medium, a solid culture medium and glycerol.

5. The application of the boron-resistant lysine bacillus in the preparation of the bactericide for inhibiting the growth of the soft rot of pitaya according to claim 3, wherein the fermentation liquor of the boron-resistant lysine bacillus is OD (origin-destination) of the boron-resistant lysine bacillus cultured for 24 to 96 hours ₆₀₀ 4.8 to 2.5 hours.

6. The application of the B-resistant lysine bacillus in preparing the bactericide for inhibiting the growth of Pitaya sorghi according to claim 5, wherein the B-resistant lysine bacillus fermentation liquid is a fermentation product obtained by culturing the B-resistant lysine bacillus at 37 ℃ and 150r/min for 96h at the liquid loading of 200ml of liquid/500 ml of LB culture medium and when the OD value of the liquid reaches 2.5.

7. A method for preventing or treating soft rot of pitaya, characterized in that the method comprises the steps of:

step one, obtaining and inhibiting soft rot germs of pitayaFusarium oxysporumA growing fungicide comprising B-lysine resistant BacillusLysinibacillus boronitoleransFermentation liquor and auxiliary materials, wherein: the boron-resistant lysine bacillus is preserved in the China Center for Type Culture Collection (CCTCC) in 2019, 10 months and 8 days, and the preservation number is CCTCC NO: m2019773, wherein the volume percentage content of the boron-resistant lysine bacillus fermentation liquor is 10 to 90 percent;

and step two, mixing the bactericide for inhibiting the growth of the pitaya soft rot bacteria obtained in the step one with a medium, and further inhibiting the growth of pathogenic bacteria on the surface of the medium.

8. The method for preventing or treating soft rot of pitaya according to claim 7, wherein the medium is a carrier carrying the soft rot of pitaya, and the carrier is one or more of a seed, a plant, soil where the plant grows, and a culture medium in which the plant is cultured.