Screening method of magnaporthe oryzae toxin degrading bacteria

Abstract

The invention discloses a screening method of magnaporthe oryzae toxin degrading bacteria, and relates to the technical field of microbiological screening, the method comprises the following specific steps: sample collection and strain preliminary separation: collecting a plurality of samples in different rice field environments, and obtaining pure culture strains through a dilution coating method or a tissue isolation method through multiple times of streak separation; primary screening and HPLC secondary screening based on a culture medium: preparing a culture medium primary screening strain by taking the magnaporthe oryzae toxin as a unique carbon and nitrogen source, through the culture medium primary screening by taking the magnaporthe oryzae toxin as the unique carbon and nitrogen source, potential degradation strains can be rapidly enriched from numerous microorganisms, a large amount of invalid screening is avoided, and the degradation capability of the strains can be accurately determined through HPLC secondary screening; according to the method, efficient degradation strains are quickly locked, target strains are further screened by utilizing the infection relation between bacteriophages and germs through phage-mediated screening, the overall screening efficiency is remarkably improved, the screening period is greatly shortened, and the strains with the capacity of efficiently degrading the pyricularia oryzae toxins are obtained more quickly.

Description

Technical Field

[0001] This invention relates to the field of microbial screening technology, specifically a screening method for bacteria that degrade rice blast fungus toxin. Background Technology

[0002] Rice blast, a major disease that seriously threatens global rice production, is caused by toxins secreted by the rice blast fungus. These toxins can damage the cell structure and physiological functions of rice, leading to disease and reduced yield. Currently, chemical pesticides dominate the control of rice blast. Although they can control the disease to some extent, the environmental pollution, pesticide residues exceeding standards, and the increasing resistance of pathogens caused by long-term use are becoming increasingly prominent. Against this backdrop, biological control, with its green, environmentally friendly, and sustainable advantages, has become an important direction for rice blast control research. Screening out highly efficient toxin-degrading bacteria of the rice blast fungus is the core link in achieving the goal of biological control.

[0003] Existing methods for screening bacteria that degrade rice blast fungus toxin mostly employ traditional methods using culture media with the target toxin as the carbon and nitrogen source. While this traditional method has played a role in screening degrading bacteria, it also has several significant shortcomings. First, the screening efficiency is low. Due to limitations in culture medium composition and screening conditions, the number of strains that can adapt and grow on specific media is limited, and many potentially degrading strains may be missed. This makes the screening process time-consuming and labor-intensive, and it is difficult to quickly obtain a large number of target strains. Second, the screening conditions are singular. Relying solely on the carbon and nitrogen source conditions in the culture medium cannot comprehensively reflect the degradation and adaptability of strains in the actual environment. The strains screened may not perform well in practical applications. In addition, traditional methods are difficult to discover strains with special mechanisms of action. Some strains may degrade toxins through unique metabolic pathways or enzyme systems, but these strains may not be effectively identified and isolated under traditional screening conditions. This limits the development and utilization of degrading bacteria resources and is not conducive to in-depth research and application of biological control of rice blast. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a screening method for bacteria that degrade rice blast fungus toxin. This method utilizes a culture medium with rice blast fungus toxin as the sole carbon and nitrogen source for initial screening, followed by precise determination of the strain's degradation ability using HPLC for secondary screening. Furthermore, phage-mediated screening technology is introduced, opening up a new screening pathway from a virological perspective. Changes in reporter gene expression directly reflect the strain's impact on the phage-pathogen system. Finally, 16S rDNA sequence analysis accurately determines the strain's taxonomic position, significantly improving screening efficiency and accuracy. This provides new ideas and high-quality strain resources for the biological control of rice blast.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for screening bacteria that degrade rice blast fungus toxins, the method comprising the following specific steps:

[0006] Sample collection and preliminary isolation of strains: Multiple samples were collected from different paddy field environments. Pure culture strains were obtained by multiple streak separations using dilution plating or tissue isolation methods.

[0007] Primary screening based on culture medium and secondary screening by HPLC: The culture medium was prepared using rice blast fungus toxin as the sole carbon and nitrogen source for primary screening of strains. The degradation rate of the toxin by the primary screening strains was determined by high performance liquid chromatography for secondary screening, and strains with high degradation rates were selected.

[0008] Isolation, identification and modification of bacteriophages: Bacteriophages that can infect rice blast fungus are isolated from the samples, identified by morphological observation and nucleic acid sequencing, and then modified by integrating reporter genes into the bacteriophage genome using genetic engineering technology.

[0009] Phage-mediated screening: Recombinant phages carrying reporter genes were mixed with rice blast fungus, and inoculated into secondary screening strains for culture. By detecting differences in reporter gene expression, strains with potential toxin degradation affecting the phage-pathogen system were screened.

[0010] Strain identification: Genomic DNA was extracted from the screened potential strains using 16S rDNA sequence analysis, amplified by PCR, and sequenced. The strains were then compared with the database to determine their taxonomic position.

[0011] Furthermore, in the sample collection and preliminary strain isolation steps, samples of rice rhizosphere soil, diseased rice plant tissue, surrounding soil, and water were collected from paddy fields in different regions and ecological environments. The sample size for rhizosphere soil and surrounding soil was 80-100g, for diseased plant tissue 5-10g, and for water 500mL. After collection, the samples were immediately placed in sterile bags or containers and stored at low temperature. In the laboratory, for soil and water samples, the dilution-spreading method was used for strain isolation. The samples were added to Erlenmeyer flasks containing sterile water, shaken thoroughly for 30 minutes to disperse the microorganisms, and then serially diluted 10-fold. 0.1mL of the bacterial suspension at an appropriate dilution was spread onto beef extract peptone agar plates. The dilutions included 10... -3 10 -4 10 -5 Three replicate plates were set up for each dilution and incubated in a constant temperature incubator at 30℃ for 2-3 days. For rice diseased plant tissue samples, the tissue isolation method was used. The diseased plant tissue was rinsed with sterile water, then disinfected with 75% alcohol for 30 seconds, rinsed with sterile water 3 times, cut into small pieces, placed on beef extract peptone medium plates, and incubated at 30℃ for 3 days. After the incubation, colonies with different characteristics such as morphology, color, and size were picked and streaked multiple times until pure culture strains were obtained.

[0012] Furthermore, in the primary screening and HPLC secondary screening steps based on culture medium, a culture medium is prepared using rice blast toxin as the sole carbon and nitrogen source for the primary screening strains. A screening culture medium is prepared using rice blast toxin as the sole carbon and nitrogen source, comprising 1g of rice blast toxin extract, 1g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, 0.5g of sodium chloride, and 20g of agar. Distilled water is added to 1000mL, and the pH is adjusted to 7.0-7.2. The medium is then autoclaved at 121℃ for 20 minutes. The obtained pure culture strains are inoculated onto screening culture medium plates, one strain per plate. A blank control plate without inoculated strains is also provided. The plates are incubated at 30℃ for 3-5 days, and the growth of the strains is observed. Strains that can grow on the screening culture medium are preliminarily identified as strains with the potential to degrade rice blast toxin.

[0013] Furthermore, in the culture medium-based primary screening and HPLC secondary screening steps, the strains obtained from the primary screening are secondary screened. A liquid culture medium containing rice blast fungus toxin is prepared, and the primary screening strains are inoculated into this liquid culture medium, with three bottles of each strain inoculated, each bottle containing 100 mL. The culture is carried out at 30℃ and 150 r / min with shaking for 5-7 days. After the culture is completed, the culture medium is centrifuged at 8000 r / min for 10 min, and the supernatant is collected for analysis by high performance liquid chromatography (HPLC). The HPLC analysis conditions are: a 250 mm × 4.6 mm C18 column with a particle size of 5 μm, a mobile phase of acetonitrile to water ratio of 40:60, a flow rate of 1.0 mL / min, a detection wavelength of 254 nm, and a column temperature of 30℃. Based on the HPLC detection results, the degradation rate of rice blast fungus toxin by each strain is calculated, and strains with high degradation rates are selected for the next screening step.

[0014] Furthermore, in the primary screening and HPLC secondary screening steps based on culture medium, a liquid culture medium containing rice blast fungus toxin is prepared, the components of which include 2g of rice blast fungus toxin extract, 1g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, 0.5g of sodium chloride, distilled water added to 1000mL, and the pH adjusted to 7.0-7.2, and then autoclaved at 121℃ for 20min.

[0015] Furthermore, in the steps of phage isolation, identification, and modification, phages capable of infecting *Strombus rice* are isolated from collected paddy field water samples and soil leachate samples. The samples are filtered through a 0.45 μm filter membrane, and 50 mL of the filtrate is added to an Erlenmeyer flask containing 5 mL of logarithmic growth phase *Strombus rice* culture medium. The flask is incubated at 30°C with shaking for 6 hours. After incubation, the mixture is centrifuged at 8000 r / min for 10 min, and the supernatant is used as the phage stock solution. The phage stock solution is then serially diluted 10-fold, and 10 μL of each solution is used as the final product. -3 10 -4 10-5 0.1 mL of the dilution buffer was mixed with 0.2 mL of logarithmic growth stage rice blast fungus and added to 3 mL of semi-solid medium containing 0.7% agar. After mixing, the mixture was poured into plates containing solid medium and incubated at 30℃ for 12-16 hours after the semi-solid medium solidified. Plaque formation was observed, and a single plaque was picked and purified three times to obtain purified bacteriophages. The morphology of the bacteriophages was observed using transmission electron microscopy, and the bacteriophage nucleic acid was extracted and sequenced. The bacteriophage species were identified by comparison with known bacteriophage sequences. The identified bacteriophages were modified using genetic engineering techniques. Primers were designed to amplify the green fluorescent protein gene. Simultaneously, integration sites were selected in the bacteriophage genome, and homologous arm primers were designed. The reporter gene was ligated to the homologous arm by overlap extension PCR to construct a recombinant DNA fragment. The recombinant DNA fragment was introduced into the bacteriophage host bacteria by electroporation. Recombinant bacteriophages carrying the reporter gene were obtained through resistance screening and PCR identification. These bacteriophages were then cultured, purified, and their infection titers were determined.

[0016] Furthermore, in the phage-mediated screening step, the obtained recombinant phage is mixed with rice blast fungus at a ratio of MOI = 10 and added to liquid culture medium to form a phage-pathogen culture system. Candidate strains with high degradation rates obtained from secondary screening are inoculated into the culture system, with three replicates for each candidate strain. A phage-pathogen culture system without candidate strains is set up as a control. The culture system is placed at 30℃ and 150 r / min and shaken for 24-48 hours. After the culture is completed, the expression of the reporter gene in the culture system is detected using equipment such as fluorescence microscopy or flow cytometry. If there are microorganisms in the candidate strains that can degrade toxins, their degradation will affect the phage infection process or the physiological state of rice blast fungus, thereby causing changes in the expression of the reporter gene. The candidate strains corresponding to the culture systems with significant differences in reporter gene expression compared with the control are screened as potential strains with toxin degradation capabilities.

[0017] Furthermore, in the strain identification step, the screened potential strains are identified using 16S rDNA sequencing. Genomic DNA of the strains is extracted using a bacterial genomic DNA extraction kit, and PCR amplification is performed using universal 16S rDNA primers 27F and 1492R. The PCR reaction system is as follows: 12.5 μL of 2×TaqPCRMasterMix, 1 μL each of 10 μM forward and reverse primers, 1 μL of template DNA, and 9.5 μL of ddH2O. The PCR reaction conditions are: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 30 cycles; and a final extension at 72℃ for 10 min. The PCR amplification products are sequenced, and the sequencing results are compared with known sequences in the GenBank database. The taxonomic position of the strain is determined based on the comparison results.

[0018] Compared with existing technologies, this screening method for rice blast fungus toxin-degrading bacteria has the following advantages:

[0019] I. This invention utilizes a culture medium with rice blast fungus toxin as the sole carbon and nitrogen source for initial screening, which can rapidly enrich potential degrading strains from numerous microorganisms, avoiding a large number of ineffective screenings. Furthermore, HPLC secondary screening can accurately determine the degradation ability of strains, quickly identifying highly efficient degrading strains. Phage-mediated screening utilizes the infection relationship between bacteriophages and pathogens to further screen target strains, significantly improving overall screening efficiency, greatly shortening the screening cycle, and obtaining strains with highly efficient rice blast fungus toxin degradation capabilities more quickly, thus saving valuable time for subsequent research and applications.

[0020] Second, this invention uses 16S rDNA sequence analysis to identify screened strains, which can accurately determine the taxonomic position of the strains and avoid misjudgment due to strain similarity. In phage-mediated screening, changes in reporter gene expression directly reflect the influence of the strain on the phage-pathogen system. If the strain has toxin degradation ability, it will cause significant differences in reporter gene expression, making the screening results more reliable. This provides a solid foundation for further in-depth research on strain characteristics and development and application, and reduces the risks and costs caused by inaccurate screening.

[0021] Other advantages, objects and features of the present invention will be described in part in the following description and, in part, will be apparent to those skilled in the art based on an examination of the following or may be learned from the practice of the invention. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0023] Figure 1 Flowchart of the screening method for bacteria that degrade rice blast fungus toxin;

[0024] Figure 2 A flowchart illustrating the screening method for bacteria that degrade rice blast fungus toxin. Detailed Implementation

[0025] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0026] Example 1

[0027] In a humid paddy field area in southern my country, where the climate is warm and humid with an average annual rainfall of over 1500 mm, six sampling points were randomly selected based on a grid-like distribution principle. Each sampling point was approximately 50 meters apart to ensure representativeness of the samples. Using a sterile sampling shovel, approximately 80g of soil was collected from the rice root zone, reaching a depth of 10-15 cm. Simultaneously, based on typical symptoms of rice blast (spindle-shaped lesions with a grayish-white center, brown edges, and necrotic lines extending from them), ten rice plants exhibiting obvious blast symptoms were selected. Diseased leaves, stems, and other plant tissues were cut off using sterile scissors. Additionally, three sampling points were selected near ditches and field ridges around the paddy field, collecting 100g of soil from each point. A 500mL water sample was also collected from the paddy field using a sterile water sampler. All samples were promptly placed in sterile sealed containers, labeled with the sampling time, location, and sample type, and stored in a 4℃ portable refrigerator for up to six hours.

[0028] In the laboratory, soil and water samples were prepared using a dilution-spreading method. 5g of soil sample was added to a 45mL sterile Erlenmeyer flask containing glass beads. The flask was shaken at 200 rpm for 30 minutes to ensure thorough dispersion of microorganisms, resulting in a bacterial suspension. This suspension was then serially diluted 10-fold. 10... -3 10 -4 10 -50.1 mL of bacterial suspension at each dilution was evenly spread onto beef extract peptone agar plates using a sterile spreader. Three replicates were set up for each dilution. The plates were incubated upside down in a 30°C incubator for 2 days. Water samples were also serially diluted and spread in the same way. After incubation, colonies of different morphologies were observed, including round, oval, and irregular shapes, with colors such as white, milky white, and pale yellow. For diseased tissue samples, the samples were gently rinsed three times with sterile water to remove surface impurities, then immersed in 75% alcohol for 30 seconds for disinfection, and then rinsed three times with sterile water for detoxification. The disinfected tissue was cut into 5 mm × 5 mm pieces and evenly placed on beef extract peptone agar plates. The plates were incubated at 30°C for 3 days. Colonies of different morphologies were picked from the colonies that grew around the tissue pieces and isolated by streak plating three times. Finally, 65 pure culture strains with different morphological and physiological characteristics were obtained.

[0029] Next, initial screening was conducted. A screening medium was prepared strictly according to the formula, using rice blast fungus toxin as the sole carbon and nitrogen source. To ensure the quality of the medium, a high-precision electronic balance was used to weigh each component during preparation, and the pH was precisely adjusted to 7.1 using a pH meter. Sixty-five pure culture strains were inoculated onto plates, with each strain inoculated into three replicate plates. Five blank control plates without inoculated strains were also set up to eliminate environmental interference. The plates were incubated at 30°C for four days, and the growth of the strains was observed and recorded daily. Twelve strains were found to grow on the screening medium and were preliminarily identified as strains with the potential to degrade rice blast fungus toxin based on the screening principle. These 12 strains were then subjected to secondary screening. A liquid medium containing rice blast fungus toxin was prepared. To ensure experimental accuracy, the medium was autoclaved and cooled to room temperature before inoculation. Three 100mL bottles were inoculated for each strain, and the medium was incubated at 30°C and 150 rpm with shaking for six days. The OD of the culture medium was monitored daily during the incubation process. 600 After the culture was completed, the culture medium was centrifuged at 8000 r / min for 10 min, and the supernatant was analyzed by high performance liquid chromatography (HPLC). To ensure the reliability of the detection results, the instrument was fully calibrated and blank was tested before HPLC analysis. Finally, three strains with high degradation rates were screened out.

[0030] Then, phage-related operations were performed to isolate phages from the collected paddy field water samples. The water samples were first filtered through a 0.45 μm filter membrane to remove larger impurities. 50 mL of the filtrate was then added to a solution containing 5 mL of phages in the logarithmic growth phase (OD2). 600 In a triangular flask containing 0.5% (r=0.5%) rice blast fungus culture medium, the mixture was incubated at 30℃ and 180 rpm for 6 hours with shaking. After incubation, the mixture was centrifuged at 8000 rpm for 10 minutes, and the supernatant was used as the phage stock solution. The phage stock solution was then serially diluted 10-fold, and 10 μL of each solution was used as the final product. -3, 10 -4 , 10 -5 0.1 mL of the diluent was mixed with 0.2 mL of logarithmic growth stage rice blast fungus, and then added to 3 mL of semi-solid culture medium (containing 0.7% agar). The mixture was quickly mixed and poured into a plate containing solid culture medium. After the semi-solid medium solidified, the plate was inverted and incubated at 30°C for 12-16 hours. Regularly shaped, clearly defined, round, transparent phage plaques were observed. A single plaque was picked, and the purification steps were repeated three times to obtain purified bacteriophages. The morphology was observed using transmission electron microscopy, and nucleic acid was extracted and sequenced, identifying it as a novel bacteriophage. To ensure the stability and functionality of the modified bacteriophage, a conserved region of the bacteriophage genome that does not affect its basic function was selected as the reporter gene integration site during the genetic engineering process. The green fluorescent protein gene was ligated to its homologous arm using overlap extension PCR to construct a recombinant DNA fragment. This fragment was then introduced into the bacteriophage host bacteria using electroporation. Recombinant bacteriophages carrying the green fluorescent protein gene were obtained through antibiotic selection and PCR identification. The recombinant bacteriophages were cultured and purified, and their infection titer was determined to be 1.2 × 10⁻⁶. 9 The PFU / mL titer was similar to that of the original phage, indicating that the modification did not significantly affect its infectivity. The recombinant phage was mixed with rice blast fungus at MOI=10 to form a culture system, and three candidate strains obtained from the rescreening were inoculated into it. At the same time, five phage-pathogen culture systems without candidate strains were set up as controls. Each system had three biological replicates. After shaking culture at 30℃ and 150r / min for 24 hours, fluorescence microscopy with a high-sensitivity fluorescence detection system was used to detect the green fluorescence intensity of one strain's culture system, which was found to be significantly lower than that of the control, and the number of fluorescent cells was reduced by about 40%. Based on the preliminary judgment principle, this strain was identified as a potential degrading bacterium.

[0031] Finally, the strain was identified using 16S rDNA sequencing. Genomic DNA was extracted from the strain using a dedicated bacterial genomic DNA extraction kit. To ensure DNA extraction quality, the kit instructions were strictly followed, and DNA concentration and purity were measured. PCR amplification was performed using universal 16S rDNA primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'). The PCR reaction system was 2×Taq. PCR Master Mix 12.5 μL, forward and reverse primers (10 μM) 1 μL each, template DNA 1 μL, ddH2O 9.5 μL. PCR reaction conditions were: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 30 cycles, and a final extension at 72℃ for 10 min. The PCR amplification products were sequenced, and the sequencing results were compared with known sequences in the GenBank database. BLAST analysis confirmed that the strain belonged to the genus Bacillus.

[0032] Example 2

[0033] In a drought-stricken paddy field area in northern China, where the average annual rainfall is less than 400 mm and irrigation relies on groundwater, the temperature difference between day and night is large, and the rice growing season is hot and dry. During the sampling phase, considering the differences in soil microbial distribution in arid paddy fields, five sampling points were selected in the paddy field using a diagonal method, with each sampling point about 80 meters apart. A sterile sampler was used to collect soil samples from the rhizosphere of the rice plants at a depth of 15-20 cm. 90g of soil was collected from each sampling point. Eight rice plants showing typical symptoms of rice blast (such as small brown spots on leaves that gradually expand into spindle-shaped lesions) were carefully searched in the paddy field. Diseased leaves and stem tissues were collected from these plants using sterile tools. Three sampling areas were set up near ponds and grassy areas around the paddy field, with 100g of soil collected from each area. 500mL of paddy field water samples were also collected using sterile containers. All samples were immediately placed in sterile sealed bags or containers, labeled with sampling information, and then placed in a low-temperature transport box equipped with an ice box. The samples were transported to the laboratory within 4 hours.

[0034] In the laboratory, bacterial strains were isolated. For soil samples, 5g was added to an Erlenmeyer flask containing 45mL of sterile water and glass beads. The flask was shaken at 180r / min for 35min to promote the full release of microorganisms. Subsequently, a 10-fold serial dilution was performed, and 10... -3 10 -4 10 -50.1 mL of bacterial suspension at each dilution was evenly spread on beef extract peptone agar plates, with three replicates for each dilution. The plates were incubated at 30°C for two days. Water samples were treated similarly. After incubation, diverse colony morphologies were observed, including smooth, round colonies and wrinkled colonies. Diseased tissue was first rinsed with sterile water to remove surface dust, then disinfected by immersion in 75% alcohol for 35 seconds. After rinsing with sterile water four times, the tissue was cut into 4 mm × 4 mm pieces and laid flat on agar plates. The plates were incubated at 30°C for three days. Colonies of different morphologies were selected from those growing around the tissue pieces. Through three consecutive streak plate separation operations, 58 pure culture strains were finally obtained.

[0035] During the initial screening, a screening medium with rice blast fungus toxin as the sole carbon and nitrogen source was strictly prepared. Each component was accurately weighed using an electronic analytical balance, and the pH of the medium was adjusted to 7.2 using a precision pH meter. Fifty-eight pure culture strains were inoculated onto screening medium plates, with three replicates per strain. Six blank control plates were also set up. The plates were incubated at 30℃ for 5 days, with daily observations and records. Ten strains were found to grow and were preliminarily identified as strains with degradation potential. For the secondary screening, a liquid medium containing rice blast fungus toxin was prepared. After autoclaving and cooling, the 10 initially screened strains were inoculated into three 100mL liquid medium bottles and cultured at 30℃ and 150 rpm for 6 days with shaking. During this period, the OD of the culture medium was measured daily at the same time using a spectrophotometer. 600 The growth curve was plotted, and after the culture was completed, the supernatant was collected by centrifugation at 8000 r / min for 10 min and analyzed by high performance liquid chromatography. Before the analysis, the HPLC instrument was calibrated with standard and baseline stability was tested. Finally, three strains with high degradation rates were screened out.

[0036] Regarding phage-related operations, phages were isolated from collected paddy field water samples. The water samples were first filtered through a 0.45 μm filter membrane, and 50 mL of the filtrate was mixed with 5 mL of logarithmic growth phase (OD) solution. 600 Mix the rice blast fungus culture medium (0.5%) and incubate at 30℃ and 180 rpm for 6 hours with shaking. Centrifuge the mixture at 8000 rpm for 10 minutes and collect the supernatant as the phage stock solution. Perform 10-fold serial dilutions and take 10... -3 10 -4 10 -50.1 mL of the dilution buffer was mixed with 0.2 mL of logarithmic growth stage rice blast fungus, and 3 mL of semi-solid culture medium (containing 0.7% agar) was added. The mixture was quickly mixed and poured into solid culture plates. After solidification, the plates were inverted and incubated at 30°C for 12-16 hours. Circular phage plaques with neat edges were observed. Individual plaques were picked and purified three times. Morphology was observed using transmission electron microscopy, and nucleic acid was extracted and sequenced. The phage was identified as a novel bacteriophage. When modifying the phage, regions of its genome that did not affect key functions were selected. The reporter gene was ligated to the homologous arm using overlap extension PCR to construct a recombinant DNA fragment. This fragment was introduced into host bacteria using electroporation. After resistance selection and PCR identification, recombinant phages carrying the reporter gene were obtained, and their infection titer was determined to be 1.1 × 10⁻⁶. + PFU / mL, the recombinant phage was mixed with rice blast fungus at MOI=10 to construct a culture system, and the three candidate strains selected for rescreening were inoculated. Six control culture systems were set up, with three replicates in each system. After culturing at 30℃ and 150r / min for 24 hours, the expression of reporter gene was detected by flow cytometry. It was found that the expression of reporter gene in the culture system of one strain was significantly lower than that in the control, and it was screened as a potential degrading bacterium.

[0037] Finally, potential degrading bacteria were identified. Genomic DNA of the strain was extracted using a high-quality bacterial genomic DNA extraction kit. After extraction, the DNA concentration and purity were detected by a nucleic acid concentration analyzer. PCR amplification was performed using the universal 16S rDNA primers 27F and 1492R. The amplified products were sequenced, and the sequencing results were compared with the GenBank database. Analysis confirmed that the strain belonged to the genus Pseudomonas sp.

[0038] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for screening bacteria that degrade rice blast fungus toxin, characterized in that, The method includes the following specific steps: Sample collection and preliminary isolation of strains: Multiple samples were collected from different paddy field environments. Pure culture strains were obtained by multiple streak separations using dilution plating or tissue isolation methods. Primary screening based on culture medium and secondary screening by HPLC: The culture medium was prepared using rice blast fungus toxin as the sole carbon and nitrogen source for primary screening of strains. The degradation rate of the toxin by the primary screening strains was determined by high performance liquid chromatography for secondary screening, and strains with high degradation rates were selected. Isolation, identification and modification of bacteriophages: Bacteriophages that can infect rice blast fungus are isolated from the samples, identified by morphological observation and nucleic acid sequencing, and then modified by integrating reporter genes into the bacteriophage genome using genetic engineering technology. Phage-mediated screening: Recombinant phages carrying reporter genes were mixed with rice blast fungus, and inoculated into secondary screening strains for culture. By detecting differences in reporter gene expression, strains with potential toxin degradation affecting the phage-pathogen system were screened. Strain identification: Genomic DNA was extracted from the screened potential strains using 16S rDNA sequence analysis, and then amplified by PCR and sequenced. The strains were then compared with the database to determine their taxonomic position.

2. The screening method for rice blast fungus toxin-degrading bacteria according to claim 1, characterized in that, In the sample collection and preliminary strain isolation steps, samples were collected from rice rhizosphere soil, diseased rice plant tissue, surrounding soil, and water from paddy fields in different regions and ecological environments. The sample size for rhizosphere soil and surrounding soil was 80-100g, for diseased plant tissue 5-10g, and for water 500mL. Immediately after collection, the samples were placed in sterile bags or containers and stored at low temperature. In the laboratory, for soil and water samples, the dilution-spreading method was used for strain isolation. The samples were added to Erlenmeyer flasks containing sterile water, shaken thoroughly for 30 minutes to disperse the microorganisms, and then serially diluted 10-fold. 0.1mL of the appropriate dilution suspension was spread onto beef extract peptone agar plates. The dilutions included 10... -3 10 -4 10 -5 Three replicate plates were set up for each dilution and incubated in a constant temperature incubator at 30℃ for 2-3 days. For rice diseased plant tissue samples, the tissue isolation method was used. The diseased plant tissue was rinsed with sterile water, then disinfected with 75% alcohol for 30 seconds, rinsed with sterile water 3 times, cut into small pieces, placed on beef extract peptone medium plates, and incubated at 30℃ for 3 days. After the incubation, colonies with different characteristics such as morphology, color, and size were picked and streaked multiple times until pure culture strains were obtained.

3. The screening method for rice blast fungus toxin-degrading bacteria according to claim 1, characterized in that, In the primary screening and HPLC secondary screening steps based on culture medium, a culture medium for primary screening strains is prepared using rice blast toxin as the sole carbon and nitrogen source. A screening culture medium is prepared using rice blast toxin as the sole carbon and nitrogen source, comprising 1g of rice blast toxin extract, 1g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, 0.5g of sodium chloride, and 20g of agar. Distilled water is added to 1000mL, and the pH is adjusted to 7.0-7.

2. The medium is then autoclaved at 121℃ for 20 minutes. The obtained pure culture strains are inoculated onto screening culture medium plates, one strain per plate. A blank control plate without inoculated strains is also provided. The plates are incubated at 30℃ for 3-5 days, and the growth of the strains is observed. Strains that can grow on the screening culture medium are preliminarily identified as strains with the potential to degrade rice blast toxin.

4. The screening method for rice blast fungus toxin-degrading bacteria according to claim 1, characterized in that, In the culture medium-based primary screening and HPLC secondary screening steps, the strains obtained from the primary screening are secondary screened. A liquid culture medium containing rice blast fungus toxin is prepared, and the primary screening strains are inoculated into this liquid culture medium, with 3 bottles of each strain and a volume of 100 mL per bottle. The culture is carried out at 30℃ and 150 r / min with shaking for 5-7 days. After the culture is completed, the culture medium is centrifuged at 8000 r / min for 10 min, and the supernatant is collected for analysis by high performance liquid chromatography (HPLC). The HPLC analysis conditions are: a 250 mm × 4.6 mm C18 column with a particle size of 5 μm, a mobile phase of acetonitrile to water ratio of 40:60, a flow rate of 1.0 mL / min, a detection wavelength of 254 nm, and a column temperature of 30℃. Based on the HPLC detection results, the degradation rate of rice blast fungus toxin by each strain is calculated, and strains with high degradation rates are selected for the next screening step.

5. The screening method for rice blast fungus toxin-degrading bacteria according to claim 4, characterized in that, In the primary screening and HPLC secondary screening steps based on culture medium, a liquid culture medium containing rice blast fungus toxin is prepared. The components include 2g of rice blast fungus toxin extract, 1g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, 0.5g of sodium chloride, distilled water added to 1000mL, and the pH adjusted to 7.0-7.

2. The medium is then autoclaved at 121℃ for 20min.

6. The screening method for rice blast fungus toxin-degrading bacteria according to claim 1, characterized in that, In the steps of phage isolation, identification, and modification, phages capable of infecting *Strombus rice* are isolated from collected paddy field water samples and soil leachate samples. The samples are filtered through a 0.45 μm filter membrane, and 50 mL of the filtrate is added to an Erlenmeyer flask containing 5 mL of logarithmic growth phase *Strombus rice* culture medium. The flask is incubated at 30°C with shaking for 6 hours. After incubation, the mixture is centrifuged at 8000 rpm for 10 minutes, and the supernatant is used as the phage stock solution. The phage stock solution is then serially diluted 10-fold, and 10 μL of each solution is used as the final product. -3 10 -4 10 -5 0.1 mL of the dilution buffer was mixed with 0.2 mL of logarithmic growth stage rice blast fungus and added to 3 mL of semi-solid medium containing 0.7% agar. After mixing, the mixture was poured into plates containing solid medium and incubated at 30℃ for 12-16 hours after the semi-solid medium solidified. Plaque formation was observed, and a single plaque was picked and purified three times to obtain purified bacteriophages. The morphology of the bacteriophages was observed using transmission electron microscopy, and the bacteriophage nucleic acid was extracted and sequenced. The bacteriophage species were identified by comparison with known bacteriophage sequences. The identified bacteriophages were modified using genetic engineering techniques. Primers were designed to amplify the green fluorescent protein gene. Simultaneously, integration sites were selected in the bacteriophage genome, and homologous arm primers were designed. The reporter gene was ligated to the homologous arm by overlap extension PCR to construct a recombinant DNA fragment. The recombinant DNA fragment was introduced into the bacteriophage host bacteria by electroporation. Recombinant bacteriophages carrying the reporter gene were obtained through resistance screening and PCR identification. These bacteriophages were then cultured, purified, and their infection titers were determined.

7. The screening method for rice blast fungus toxin-degrading bacteria according to claim 1, characterized in that, In the phage-mediated screening step, the obtained recombinant phage is mixed with rice blast fungus at a ratio of MOI = 10 and added to liquid culture medium to form a phage-pathogen culture system. Candidate strains with high degradation rates obtained from secondary screening are inoculated into the culture system, with three replicates for each candidate strain. A phage-pathogen culture system without candidate strains is set up as a control. The culture system is placed at 30℃ and 150 r / min and shaken for 24-48 hours. After the culture is completed, the expression of reporter genes in the culture system is detected using equipment such as fluorescence microscopy or flow cytometry. If there are microorganisms in the candidate strains that can degrade toxins, their degradation will affect the phage infection process or the physiological state of rice blast fungus, thereby causing changes in the expression of reporter genes. Candidate strains corresponding to culture systems with significant differences in reporter gene expression compared with the control are screened as potential strains with toxin degradation capabilities.

8. The screening method for rice blast fungus toxin-degrading bacteria according to claim 1, characterized in that, In the strain identification step, the screened potential strains are identified using 16S rDNA sequencing. Genomic DNA of the strains is extracted using a bacterial genomic DNA extraction kit. PCR amplification is performed using universal 16S rDNA primers 27F and 1492R. The PCR reaction system is: 12.5 μL of 2×TaqPCRMasterMix, 1 μL each of 10 μM forward and reverse primers, 1 μL of template DNA, and 9.5 μL of ddH2O. The PCR reaction conditions are: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 30 cycles; and a final extension at 72℃ for 10 min. The PCR amplification products are sequenced, and the sequencing results are compared with known sequences in the GenBank database. The taxonomic position of the strain is determined based on the comparison results.

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