Screening method of novel leuconostoc and bacteriostatic mechanism of leuconostoc on leaf mold of zanthoxylum bungeanum

Abstract

The invention discloses a novel leuconostoc screening method and an antibacterial mechanism of leuconostoc on leaf mold of zanthoxylum bungeanum, and relates to the technical field of pathogenic bacteria experiment and analysis. The novel leuconostoc screening method comprises the following steps that S1, materials are prepared, specifically, rotten Chinese prickly ash tree plants are selected, and tested strains in the Chinese prickly ash tree plants comprise plant pathogens of alternaria alternata, fusarium solani, fusarium layered, fusarium oxysporum, fusarium syphilis and colletotrichum pini, and are stored in a refrigerator of a laboratory at the temperature of 4 DEG C for standby application; in the invention, the leuconostoc also has an efficient inhibition effect on field crop pathogenic fungus alternaria alternata, and the research expands the biocontrol spectrum of lactic acid bacteria. Meanwhile, the invention discloses a molecular mechanism that the Leuconostoc suionicum HJ-L synergistically regulates and controls the synthesis of antibacterial substances through lipid metabolism, an ABC transport system and an autophagy pathway, and a theoretical basis and a practical basis are provided for the application of the strain in green prevention and control of the leaf mold of Chinese prickly ash.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of original bacteria experiments and analysis, and particularly relates to a screening method of a new Leuconostoc and an antibacterial mechanism of the Leuconostoc on pepper leaf mildew. BACKGROUND

[0002] Alternaria fungi are a kind of plant pathogenic fungi with wide distribution, which can infect many important crops such as wheat, soybean and potato. The toxins produced by the fungi not only reduce the yield and quality of crops, but also threaten human health, and are one of the important factors restricting global agricultural production and agricultural product trade. In recent years, the cultivation area has been expanding, but the leaf mildew caused by Alternaria fungi has become increasingly serious. At present, the disease is mainly controlled by chemical fungicides, and the development of green and efficient biological control technology is of great significance to ensure the sustainable development of the pepper industry and improve the safety of agricultural products.

[0003] Research shows that many lactic acid bacteria have significant inhibitory effect on plant pathogenic fungi. For example, Lactobacillus plantarum can inhibit Aspergillus niger and Aspergillus flavus; L. casei, L. coryniformis and Pediococcus pentosaceus show antagonistic activity against Penicillium citrinum, Aspergillus fumigatus and Candida respectively. In the postharvest disease control of fruits and vegetables, L. plantarum CUK501 can inhibit cucumber pathogenic bacteria, P. pentosaceus can effectively control apple penicillium disease, and the control efficiency of Lactobacillus CICC 23120 on gray mold disease reaches 79.3%. However, the existing research on the biological control effect of lactic acid bacteria mainly focuses on postharvest diseases of fruits and vegetables, and the prevention and control of field crops (such as pepper) is still blank. In addition, the antibacterial effect of lactic acid bacteria has strain specificity, and screening of high-efficiency antagonistic strains and analysis of the action mechanism are the key to the development of biological control agents. SUMMARY

[0004] The purpose of the present application is to provide a screening method of a new Leuconostoc and an antibacterial mechanism of the Leuconostoc on pepper leaf mildew, so as to solve the problems in the background art.

[0005] To achieve the above-mentioned purpose of the application, the following technical solutions are adopted: The present application provides a screening method of a new Leuconostoc, comprising the following steps: S1, material preparation: selecting rotten pepper tree plants, and the test strains in the pepper tree plants include Alternaria fungi, Fusarium solani, Fusarium proliferatum, Fusarium oxysporum, Fusarium reticulatum and plant pathogenic fungi of Phaeolus schweinitzii, and are stored in a 4℃ laboratory refrigerator for standby; S2, selection of medium, reagent and equipment: select the corresponding medium for the test strain, and dilute the medium to 1 000 mL with deionized water, adjust the corresponding pH, add 15.0 g / L agar when preparing solid medium, 121℃ high pressure sterilization for 20 min, the reagent selection is: bacterial genome extraction kit, PCR purification kit and agarose, the equipment selection is: stereoscopic microscopic imaging system, stainless steel positive pressure filtration device, ultraviolet spectrophotometer, constant temperature oscillation incubator and vertical high pressure steam sterilization device; S3, culture of antagonistic bacteria HJ-L with antibacterial activity to alternaria alternata: take a small piece of rotten Zanthoxylum bungeanum trunk, place it in a sterilized triangular flask, add 10 mL of sterile water, shake at 180 r for 30 min, dilute to 10-6, 10-7 and 10-8, take 100 μL and spread on LB solid medium, incubate at 32℃ for 24 h, then pick single colonies for liquid culture; S4, isolation of antagonistic bacteria HJ-L with antibacterial activity to alternaria alternata: the colonies in S3, place the alternaria alternata HJ-2 of Zanthoxylum bungeanum leaf blight pathogen in PDA solid medium and culture for 7 days, punch into a fungus cake with a puncher with a diameter of 6 mm, inoculate 10 μL of strain HJ-L culture liquid in a triangle shape at a distance of 2.0 cm from the fungus cake, and use a fungus plate without culture liquid as a control, incubate at 30℃ for 7 days, repeat each treatment 3 times, and measure the inhibition rate; S5, screening of antagonistic bacteria HJ-L with antibacterial activity to alternaria alternata: S4, inhibition rate (%) = [colony diameter (mm) of control group - colony diameter (mm) of test group] / [colony diameter (mm) of control group - 6 (mm)] x 100

[16] , select strain HJ-L by measuring the inhibition rate.

[0006] As a preferred scheme of the application, in S2, the medium comprises: PDA medium, MRS medium, CM medium, TSB medium, NB medium, SI medium, LB medium, NAM medium and ATP medium.

[0007] As a preferred scheme of the application, the PDA medium comprises: glucose: 20.0 g, potato extract: 1L, agar: 15.0 g; MRS medium includes: peptone 10.0 g, beef extract: 10.0 g, yeast powder: 5.0 g, glucose: 20.0 g, K2HPO4: 2.0 g, diammonium citrate: 2.0 g, sodium acetate: 5.0 g, MgSO4·7H2O: 0.58 g, MnSO4·H2O: 0.14 g, Tween 80: 1.0 mL, pH: 6.2; CM medium includes: sucrose: 10.0 g, yeast extract: 10.0 g, peptone: 10.0 g, K2HPO4: 10.0 g, NaCl: 2.0 g, MgSO4·7H2O: 0.2 g, Tween 80: 0.5 mL; TSB medium includes: tryptone: 17.0 g, soybean peptone: 3.0 g, NaCl: 5.0 g, KH2PO4: 2.5 g, glucose: 2.5 g.

[0008] As a preferred scheme of the present application, the NB medium includes: yeast powder: 1.0 g, sucrose: 10.0 g, peptone: 5.0 g, beef extract: 3.0 g; SI medium includes: tryptone: 8.0 g, yeast powder: 3.0 g, NaCl: 5.0 g, glucose: 5.0 g, KH2PO4: 2.0 g, Tween: 20 5.0 mL; LB medium includes: tryptone: 10.0 g, yeast powder: 3.0 g, NaCl: 10.0 g; NAM medium includes: beef extract: 3.0 g, peptone: 10.0 g, NaCl: 5.0 g; ATP medium includes: tryptone: 10.0 g, yeast powder: 10.0 g, sodium citrate: 5.0 g, glucose: 10.0 g, K2HPO4: 5.0 g, NaCl: 5.0 g, MgSO4·7H2O: 0.8 g, MnCl2·4H2O: 0.14 g, FeSO4·7H2O: 0.04 g, Tween 80: 1.0 mL.

[0009] The present application also provides a bacteriostatic mechanism of pepper leaf blight, comprising the following steps: S1, place the co-cultured 7d bacteriostatic plate under 150 times body microscope to observe and analyze the influence of HJ-L strain on the mycelium growth of alternaria alternata. Observe the morphological changes of mycelium tip, including branch density, growth direction and swelling phenomenon; S2, a punch is used to prepare a 6 mm diameter fungus cake for testing, which is inoculated in the central part of the PDA culture medium plate, and then the HJ-L culture solution is point-inoculated on the surface of the PDA culture medium in a triangular layout, and placed in a 30℃ constant temperature incubator for culture for 7 days, and then the inhibition effect on different pathogenic fungi is determined, and three biological replicates are set for each treatment; S3, the activated HJ-L strain culture solution is taken with a sterile inoculation loop, and a three-zone streaking method is used on the surface of the solid CM culture medium for strain purification, and then placed in a 37℃ constant temperature incubator for culture for 24 hours, and when single colonies are formed, typical colonies are selected for Gram staining, and after staining, the morphology of the bacterial body is observed using an optical microscope; S4, the target strain is identified based on 16S rRNA gene sequence analysis: the strain with significant antagonistic effect in the inhibition preliminary screening experiment is selected, bacterial DNA is extracted, and 16S rRNA gene amplification is carried out using universal primers 27F and 1492R, and a 25μL standard reaction system is constructed: 2×Es Taq MasterMix: 12.5μL, forward / reverse primers: 1μL each, DNA template: 1μL, and sterile water is added to a final volume of 25μL. S5, based on the experimental screening results, the CM culture medium is determined as the best basic culture medium for the strain HJ-L: five parameters including initial pH, temperature, rotation speed, inoculation amount and salt concentration are investigated by single factor experiment design: the pH gradient is controlled by a phosphate-citric acid buffer system; the temperature gradient is cultured in an intelligent constant temperature shaking incubator; the rotation speed is carried out under the condition of 15mL liquid volume in a 50mL centrifugal tube; and the inoculation amount gradient is controlled by using logarithmic growth phase bacterial suspension.

[0010] As a preferred scheme of the application, in S4, the strain is subjected to an amplification procedure: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 55℃ annealing for 45s, 72℃ extension for 90s, a total of 35 cycles; and 72℃ final extension for 7min. The amplification product is verified by 1.0% agarose gel electrophoresis, the purified positive product completes sequence determination, and the effective sequence is submitted to the GenBank database for comparison and construction of a phylogenetic tree.

[0011] As a preferred scheme of the application, in S5, the experiment sets 9 different salt concentrations of 0.5%-14% to affect the growth of the strain, three replicates are set for each group of treatment, the bacterial liquid sample is taken after 22h of culture, the bacterial fragments are removed, the OD600 value is determined by using a UV-visible spectrophotometer, and the optimal growth parameter is determined.

[0012] Compared with the prior art, the screening method of a new type of Leuconostoc provided by the application and the inhibition mechanism of the Leuconostoc on pepper leaf mold have one or more of the following beneficial effects: The present study found that Leuconostocsuionicum has a high inhibitory effect on the field crop pathogenic fungus Alternaria alternata, which expands the biocontrol spectrum of lactic acid bacteria (LAB). At the same time, strain HJ-L showed inhibitory activity against the five pathogenic fungi tested, with an inhibition rate of 62% to 88%, of which the inhibition rate against Fusarium solani was the highest (88%), and the inhibition rate against Fusarium solani was relatively low (62%); The optimal growth conditions of strain HJ-L are pH 6.5, 30°C, and 180 r / min, which meet the basic requirements of industrial fermentation, and the crude extract has the potential to be directly used for the development of biological pesticide preparations. This study first reveals the molecular mechanism of Leuconostocsuionicum HJ-L in the synergistic regulation of the synthesis of antibacterial substances through lipid metabolism (alpha-linolenic acid, arachidonic acid pathway), ABC transport system and autophagy pathway, providing a theoretical basis and practical foundation for the application of the strain in the green control of pepper leaf mold. BRIEF DESCRIPTION OF DRAWINGS

[0013] The drawings accompanying the specification of this application serve to provide a further understanding of the application, and the illustrative embodiments of the application and their descriptions serve to explain the application, and do not constitute an improper limitation on the application.

[0014] Figure 1 is a schematic diagram of the antibacterial effect of strain HJ-L of the present application on five plant pathogenic fungi; Figure 2 is a schematic diagram of the antibacterial rate of strain HJ-L of the present application on five plant pathogenic fungi; Figure 3 is a schematic diagram of the phylogenetic tree of strain HJ-L of the present application; Figure 4 is a gradient diagram of the effect of different factors on the growth amount of strain HJ-L of the present application; Figure 5 is a schematic diagram of the effect of ethyl acetate extract of strain HJ-L of the present application on the mycelial growth of Alternaria alternata; Figure 6 is a metabolic pathway enrichment analysis of the present application; Figure 7 is a TIC overlay of the positive ion mode of the QC sample of the present application; Figure 8 is a TIC overlay of the negative ion mode of the QC sample of the present application. DETAILED DESCRIPTION

[0015] To enable persons skilled in the art to understand the features and effects of the present application, the following is a general description and definition of the terms and phrases mentioned in the specification and claims. Unless otherwise specified, all technical and scientific words used herein are understood to have the usual meaning to those skilled in the art of the present application, and in the event of conflict, the definitions in the specification shall prevail.

[0016] Theories and mechanisms described and disclosed herein, whether correct or not, should not be considered limiting of the scope of the present application, which is limited only by the claims. The present application can be implemented in a manner different than is described herein without departing from the scope of the application.

[0017] Herein, all features defined by numerical ranges or percentage ranges, such as numerical values, amounts, contents and concentrations, are for the sake of brevity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have encompassed and specifically disclosed all possible sub-ranges and individual numerical values within the range (including integers and fractions).

[0018] Herein, unless otherwise specified, "comprise", "include", "contain", "have" or similar words are intended to cover the meaning of "consist of" and "consist essentially of", for example, "A comprises a" covers the meaning of "A comprises a and other" and "A comprises only a".

[0019] Herein, for the sake of brevity, all possible combinations of the various technical features in the various embodiments or examples are not described. Therefore, the various technical features in the various embodiments or examples can be combined in any manner as long as there is no contradiction, and all possible combinations should be considered to be within the scope of the present specification.

[0020] The present application is further illustrated by the following specific examples. It is to be understood that these examples are only for the purpose of illustration and are not intended to limit the scope of the present application. Furthermore, it is to be understood that those skilled in the art can make various modifications or changes to the present application upon reading the content of the present specification, and such equivalent forms should be considered to fall within the scope of the appended claims.

[0021] In the following examples, the instruments and apparatuses of the art are used. In the following examples, the experimental methods not otherwise specified are usually performed according to the conventional conditions, or according to the conditions suggested by the manufacturers. In the following examples, various raw materials are used, and unless otherwise specified, the conventional commercially available products are used, and the specifications are the conventional specifications in the art. In the specification of the present application and in the following examples, unless otherwise specified, "%" means weight percent, "parts" means weight parts, and the ratio means weight ratio. Examples

[0022] See Figures 1-8The application discloses a screening method of a novel Leuconostoc, and comprises the following steps. S1, material preparation: rotten Zanthoxylum bungeanum plants are selected, and plant pathogenic fungi including Alternaria alternata, Fusarium solani, Fusarium proliferatum, Fusarium oxysporum, Fusarium solani pisi and Guignardia tsugae are selected as test strains and are stored in a 4 DEG C laboratory refrigerator for standby; S2, selection of culture medium, reagent and equipment: corresponding culture media are selected according to the test strains, the culture media are made up to 1,000 mL with deionized water, the pH is adjusted, 15.0 g / L agar is additionally added when solid culture medium is prepared, 121 DEG C high-pressure sterilization is carried out for 20 min, the reagents are selected to be a bacterial genome extraction kit, a PCR purification kit and agarose, and the equipment is selected to be a stereoscopic micro-imaging system, a stainless steel positive pressure filtration device, an ultraviolet spectrophotometer, a constant-temperature oscillation incubator and a vertical high-pressure steam sterilization device; S3, culture of antagonistic bacteria HJ-L with antibacterial activity on Alternaria alternata: a small piece of rotten Zanthoxylum bungeanum bark is taken, placed in a sterilized triangular flask, 10 mL of sterile water is added, and after 180 r shaking bed culture for 30 min, dilution gradients of 10-6, 10-7 and 10-8 are obtained, 100 μL is taken and inoculated on LB solid culture medium, and after 32 DEG C constant-temperature culture for 24 h, single colonies are picked and subjected to liquid culture; S4, separation of antagonistic bacteria HJ-L with antibacterial activity on Alternaria alternata: the colonies in S3 are placed in PDA solid culture medium and cultured for 7 days, a puncher with a diameter of 6 mm is used to punch into a fungus cake, and the fungus cake is inoculated in the center of newly prepared PDA culture medium, 10 μL of the culture liquid of the strain HJ-L is inoculated in a triangular shape at a distance of 2.0 cm from the fungus cake, a fungus plate without the culture liquid is used as a control, 30 DEG C constant-temperature culture is carried out for 7 days, each treatment is repeated for three times, and the antibacterial rate is measured; S5, screening of antagonistic bacteria HJ-L with antibacterial activity on Alternaria alternata: according to S4, the antibacterial rate (%) = [diameter (mm) of the colony in the control group-diameter (mm) of the colony in the test group] / [diameter (mm) of the colony in the control group-6 (mm)]*100

[16] , and the strain HJ-L is screened through the determination of the antibacterial rate.

[0023] Further, in S2, the culture medium comprises PDA culture medium, MRS culture medium, CM culture medium, TSB culture medium, NB culture medium, SI culture medium, LB culture medium, NAM culture medium and ATP culture medium.

[0024] In the above scheme, the PDA culture medium comprises the following components: 20.0 g of glucose, 1 L of potato extract and 15.0 g of agar. MRS medium includes: peptone 10.0 g, beef extract: 10.0 g, yeast powder: 5.0 g, glucose: 20.0 g, K2HPO4: 2.0 g, diammonium citrate: 2.0 g, sodium acetate: 5.0 g, MgSO4·7H2O: 0.58 g, MnSO4·H2O: 0.14 g, Tween 80: 1.0 mL, pH: 6.2; CM medium includes: sucrose: 10.0 g, yeast extract: 10.0 g, peptone: 10.0 g, K2HPO4: 10.0 g, NaCl: 2.0 g, MgSO4·7H2O: 0.2 g, Tween 80: 0.5 mL; TSB medium includes: tryptone: 17.0 g, soybean peptone: 3.0 g, NaCl: 5.0 g, KH2PO4: 2.5 g, glucose: 2.5 g; NB medium includes: yeast powder: 1.0 g, sucrose: 10.0 g, peptone: 5.0 g, beef extract: 3.0 g; SI medium includes: tryptone: 8.0 g, yeast powder: 3.0 g, NaCl: 5.0 g, glucose: 5.0 g, KH2PO4: 2.0 g, Tween: 20 5.0 mL; LB medium includes: tryptone: 10.0 g, yeast powder: 3.0 g, NaCl: 10.0 g; NAM medium includes: beef extract: 3.0 g, peptone: 10.0 g, NaCl: 5.0 g; ATP medium includes: tryptone: 10.0 g, yeast powder: 10.0 g, sodium citrate: 5.0 g, glucose: 10.0 g, K2HPO4: 5.0 g, NaCl: 5.0 g, MgSO4·7H2O: 0.8 g, MnCl2·4H2O: 0.14 g, FeSO4·7H2O: 0.04 g, Tween 80: 1.0 mL. Example

[0025] See Figures 1-8 , the antibacterial mechanism of Zanthoxylum bungeanum leaf blight, including the following steps: S1, place the co-cultured 7d antibacterial plate under 150 times body microscope and observe to analyze the influence of HJ-L strain on Alternaria alternata mycelial growth. Observe the morphological changes of mycelial tip, including branch density, growth direction and swelling phenomenon; S2, using puncher to prepare 6 mm diameter of the test fungus cake, inoculate the central PDA medium plate, then point the HJ-L culture liquid in the form of a triangle on the surface of the PDA medium, and place it in a 30℃ constant temperature incubator for 7d, then measure the inhibition effect on different pathogens, and set 3 biological repeats for each treatment; S3, using a sterile inoculation ring to dip the activated HJ-L strain culture liquid, and using a three-zone streak method on the surface of solid CM medium to purify the strain, then place it in a 37℃ constant temperature incubator for 24h, when single colonies are formed, select typical colonies for Gram staining, and then use an optical microscope to observe the morphology of the bacteria after staining; S4, based on 16S rRNA gene sequence analysis method to systematically identify the target strain: select the strain with significant antagonistic effect in the inhibition preliminary screening experiment, extract bacterial DNA, and use universal primers 27F and 1492R for 16S rRNA gene amplification, construct a 25μL standard reaction system: 2×Es Taq MasterMix: 12.5μL, forward / reverse primer: 1μL each, DNA template: 1μL, and sterile water to a final volume of 25μL; S5, based on the experimental screening results, determine that the CM medium is the best basic medium for strain HJ-L: use single factor experiment design to investigate five parameters: initial pH, temperature, rotation speed, inoculum size and salt concentration: pH gradient is controlled by phosphate-citric acid buffer system; temperature gradient is cultured in an intelligent constant temperature shaking incubator; rotation speed is performed under the condition of 15mL liquid volume in a 50mL centrifuge tube; inoculum size gradient is controlled by logarithmic growth phase bacterial suspension.

[0026] Further, in S4, the strain is subjected to an amplification procedure: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 55℃ annealing for 45s, 72℃ extension for 90s, a total of 35 cycles; 72℃ final extension for 7min. The amplification product is verified by 1.0% agarose gel electrophoresis, the purified positive product completes sequence determination, and the effective sequence is submitted to GenBank database for comparison and construction of phylogenetic tree.

[0027] Further, in S5, the experiment sets 9 different salt concentrations of 0.5%-14% to affect the growth of the strain, each group of treatment sets three repeats, takes the bacterial liquid sample after 22h of culture, removes the bacterial debris, and uses a UV-visible spectrophotometer to measure the OD600 value to determine the optimal growth parameter. EMBODIMENT

[0028] 1) Effect of antagonistic bacteria HJ-L ethyl acetate extract on Alternaria alternata mycelial growth Ethyl acetate extract preparation: Take 500 mL of sterile fermentation broth, add an equal volume of extraction solvent, mix well, and let stand at room temperature for 48 hours. After the mixture is fully layered, use a separatory funnel to separate and collect the organic phase. Place the organic phase in a rotary evaporator and perform vacuum distillation at 40°C: when the organic phase is concentrated to dryness. Then redissolve the organic phase concentrate in 5-10 mL of methanol, which is used as the antibacterial active extract for subsequent determination of the inhibitory effect on Alternaria alternata mycelial growth.

[0029] The antifungal activity of HJ-L strain ethyl acetate phase crude extract was determined by mycelial growth rate method. The specific operation is as follows: the freeze-dried extract was dissolved in sterile 10% (v / v) methanol solution (filtered through 0.22 μm PVDF membrane to remove bacteria), a series of concentrations of 20.00, 10.00, 5.00, 2.50, 1.25, 0.65 and 0.32 mg / mL were prepared by double gradient dilution method (error range ± 2%), and blank matrix control containing equal volume of sterile methanol was set. Using a sterile puncher to obtain a 6.0 mm edge growth area of Alternaria alternata mycelial cake, and placing it in the geometric center of the drug-containing plate with sterile operation, 28°C constant temperature culture for 7 days, three replicates were set for each treatment. Record the mycelial expansion dynamics every day, and measure the maximum expansion diameter of the colony by cross method. Further calculate the inhibition rate of antagonistic bacteria HJ-L. Inhibition rate (%) = (control colony diameter - treatment colony diameter) / (control colony diameter - mycelial cake diameter) x 100. After 2 days of inoculation, the minimum drug concentration that completely inhibits the growth of Alternaria alternata mycelium is the minimum inhibitory concentration; after 5 days of inoculation, the minimum drug concentration that causes 99.9% inactivation of Alternaria alternata is the minimum bactericidal concentration.

[0030] 2) Observation of the effect of the extract on the mycelial morphology of Alternaria alternata The effect of the extract on the ultrastructure of Alternaria alternata was analyzed by microslide culture technique: a sterile cover glass was inserted at a 45° angle in a PDA plate, and a freshly prepared Alternaria alternata mycelial cake (Φ = 6.00 ± 0.05 mm) was inoculated in the center of the medium. After 28°C culture for 72 h, the mycelial front extended to 2 / 3 of the area of the cover glass, the cover glass was taken out aseptically and transferred to PDB liquid medium (50 mL conical flask with 20 mL liquid) containing MIC concentration of extract, and PDB medium without extract was set as blank control, and cultured at 150 r / min for 24 h. After washing with sterile water for 3 times, the mycelial morphology was observed under a light microscope.

[0031] 3) 1.2.8 Analysis of the antibacterial active ingredients in the ethyl acetate extract organic phase of the fermentation broth of antagonistic bacteria HJ-L Bioactive components in the ethyl acetate extract were analyzed using untargeted LC-MS metabolomics. The freeze-dried active extract was reconstituted in chromatographic-grade methanol (containing 0.1% formic acid), filtered through a 0.22 μm nylon membrane, and then mass spectrometrically acquired using a detection platform. Chromatographic separation was performed using an ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) with a gradient elution of 0.1% formic acid aqueous solution (A) and acetonitrile (B) (0–15 min, 5–95% B). Mass spectrometry was performed in ESI± mode, acquiring data in the m / z range of 50–1500. Raw mass spectrometry data were preprocessed using Progenesis QI 3.0 software, and metabolites were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Lipid Metabolites and Pathways Strategy (LIPID MAPS) databases.

[0032] Furthermore, the statistical analysis of the experimental data was performed using SPSS 17.0 and Excel 2007 software. Example

[0033] Results Analysis 1) Morphology of HJ-L strain Colony morphology observation showed that the target strain formed small, smooth, round, milky white, translucent or opaque colonies on solid culture medium. After Gram staining, the cells turned purple and were Gram positive.

[0034] 2) Effects of antagonistic bacteria HJ-L on the hyphal morphology of pathogenic fungi Using pathogen cultures cultured for 7 days as experimental controls, strain HJ-L showed a significant antagonistic effect against Alternaria alternata, with a hyphal growth inhibition rate of 83.2±0.5%. Stereoscopic observation revealed that the hyphae in the control group exhibited a typical radial pattern at the tips, with uniform hyphae thickness and a dense, villous structure in the swollen apical area; while the hyphae in the HJ-L treatment group showed significant ultrastructural distortions, manifested as apical swelling, cell wall collapse, and stunted elongation growth.

[0035] The above results indicate that strain HJ-L has a significant inhibitory effect on the growth of Alternaria mycelia.

[0036] 3) Broad-spectrum antagonistic activity of HJ-L strain For details, please refer to the following: Figure 1 and Figure 2Strain HJ-L could inhibit the mycelial growth of five plant pathogenic fungi, but the inhibition rate was different. The inhibition rate of Fusarium proliferatum was 65%, the inhibition effect of Fusarium oxysporum was 72%, the inhibition effect of Fusarium solani was 88%, the inhibition rate of Fusarium solani was 62%, and the inhibition rate of Dothidella pini was 83%. The inhibition effect of the strain on different pathogenic fungi was significantly different, among which the inhibition ability on Fusarium solani was the strongest, and the inhibition effect on Fusarium solani was relatively weak.

[0037] 4) Construction of the phylogenetic tree of antagonistic bacteria HJ-L Specific reference Figure 3 Phylogenetic analysis showed that the strain had 100% sequence similarity with Leuconostoc suionicum strain M6S3B6, and formed an independent branch with Leuconostoc suionicum group in the phylogenetic tree. Finally, HJ-L was identified as Leuconostoc.

[0038] 5) Biological growth characteristics of antagonistic bacteria HJ-L Specific reference Figure 4 Through medium screening experiment, it was determined that CM was the best culture medium for strain HJ-L. NaCl concentration had a significant effect on its growth, and when the NaCl content exceeded 1%, the growth of the bacteria was inhibited, and the optimal salt concentration condition (1%) OD600 value reached 1.34. Temperature gradient experiment showed that within the range of 28-32°C, the biomass showed a trend of first increasing and then decreasing, and the peak value of OD600 was 1.31 at 30°C. The strain could normally proliferate within the pH range of 5.5-7.5, and the OD600 value was 0.78 at pH 6.5, while the acidic or alkaline environment significantly reduced its growth efficiency. Inoculum size test showed that 1%-7% inoculum size interval could support the growth of the strain, and the OD600 reached the highest value of 0.86 at 3% inoculum size. In the shaking speed experiment, within the range of 140-200 r / min, the biomass accumulation was positively correlated with the rotation speed, and the OD600 value increased to 1.96 at 180 r / min.

[0039] 6) Determination of MIC and MFC of ethyl acetate extract of antagonistic bacteria HJ-L on Alternaria alternata Specific reference Figure 4 After 2 days of culture, the mycelial growth of Alternaria alternata in the treatment groups of 20.00, 10.00 and 5.00 mg / mL concentration of ethyl acetate extract of strain HJ-L was completely inhibited, and the minimum inhibitory concentration of the strain on Alternaria alternata was determined to be 5.00 mg / mL; after 5 days of culture, the treatment groups of 20.00 and 10.00 mg / mL still showed complete growth inhibition, so the minimum bactericidal concentration of the strain on Alternaria alternata was determined to be 10.00 mg / mL.

[0040] 7) Effect of antagonistic bacteria HJ-L ethyl acetate extract on Alternaria alternata mycelial morphology Microscopic observation showed that the mycelial morphology of A. alternata was significantly changed after treatment with the ethyl acetate extract of strain HJ-L. The mycelia of the control group presented a typical normal structure: thick and dense mycelia, uniform cytoplasm, and smooth surface. The mycelial network of the treatment group was sparse, and abnormal swelling vacuolization phenomenon appeared locally.

[0041] The above results showed that the extract interfered with the normal growth and development of A. alternata by damaging the integrity of the mycelial cell membrane, ultimately inducing ultrastructural pathological damage.

[0042] 8) Analysis of the antimicrobial active ingredients in the organic phase of the ethyl acetate extract of antagonistic bacteria HJ-L For details, see Figure 5 LC-MS detection results showed that a total of 6003 metabolites were annotated in the ethyl acetate extract sample of strain HJ-L fermentation broth. Among them, there were 2966 metabolites in the positive ion mode and 3037 metabolites in the negative ion mode.

[0043] According to literature review, there were 20 substances related to inhibiting plant pathogens in the metabolites annotated in the positive ion mode. There were 7 substances related to inhibiting plant pathogens in the metabolites annotated in the negative ion mode.

[0044] The above results further confirmed that the antibacterial effect of HJ-L strain was related to the antibacterial components of its metabolites.

[0045] Metabolic pathway enrichment visualization analysis showed that the enrichment factor was positively correlated with the significance level of the metabolic pathway (P < 0.05). The diameter of the bubbles in the scatter plot directly reflected the order of magnitude difference in the number of enriched metabolites in a specific pathway, and the distribution characteristics showed that the core metabolic network of strain HJ-L presented significant enrichment characteristics. Strain HJ-L showed high enrichment (Rich Factor > 1.0) in 2-ketocarboxylic acid metabolism, ABC transporter system, alpha-linolenic acid metabolism, and arachidonic acid metabolism, indicating that these pathways played a dominant role in the metabolic activity of the strain. The significant enrichment of Autophagy-other and Autophagy-yeast (P-value < 0.25) suggested that strain HJ-L might regulate energy metabolism or stress response through the autophagy mechanism.

[0046] Therefore, the metabolic characteristics of the strain were centered on transmembrane transport, lipid metabolism, and stress response, and it also had the ability to degrade exogenous substances. Its multi-pathway cooperative mode might provide a metabolic basis for environmental adaptability.

[0047] The main metabolic pathways were obtained, mainly focusing on the core metabolic pathway with the highest proportion, and the secondary metabolic ability was outstanding, showing that the strain had the potential to produce active substances. The metabolism of microorganisms in various environments, glycerophospholipid metabolism, and arachidonic acid metabolism provided important information for the study of the antibacterial mechanism of strain HJ-L in the later stage.

[0048] 9)Conclusion and discussion In this study, we successfully screened a lactic acid bacterial strain Leuconostoc suionicum HJ-L with high inhibitory effect on the pathogen of pepper leaf blight, with an inhibition rate of 83.2% ± 0.5%. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MFC) of the ethyl acetate extract of the strain on the pathogen were 5.00 mg / mL and 10.00 mg / mL, respectively. Previous studies have focused on the genus Lactobacillus.

[0049] Through metabolomics analysis, 27 metabolites with antibacterial activity were identified in the ethyl acetate extract sample of strain HJ-L fermentation broth, including polyketides and lipopeptides that can destroy fungal cell membranes. The difference in inhibition rate is related to the targeting of HJ-L metabolites: the cell membrane of Fusarium solani is rich in sterols, which is easily destroyed by lipopeptides; while the cell wall structure of F. solani is more dense, which resists the penetration of metabolites. Microscopic observation showed that HJ-L treatment caused the swelling of the pathogen hyphal tip and cytoplasm leakage, and its mechanism may include the interference of lipopeptides with chitin synthase to cause cell wall deformity, and the inhibition of pathogen energy metabolism in an acidic microenvironment. In addition, the linoleic acid produced by HJ-L may play a role in inhibiting bacteria by destroying the sterol structure of the fungal cell membrane, which is consistent with the lipid-mediated antibacterial mechanism proposed by Quattrini et al. The enrichment characteristics of the α-linolenic acid metabolic pathway of HJ-L are consistent with the report of Lactobacillus plantarum FS-1, but its unique arachidonic acid metabolism may further enhance the secretion efficiency of antibacterial substances by enhancing membrane fluidity.

[0050] Therefore, any person skilled in the art within the technical scope disclosed by the present application, according to the technical solutions and inventive concepts of the present application, can make equivalent replacements or changes, which should be covered within the protection scope of the present application.

Claims

1. A novel screening method for Leuconostoc mesenteroides, characterized in that, Includes the following steps: S1. Material preparation: Select rotten pepper trees. The test strains in the pepper trees include Alternaria, Fusarium solani, Fusarium moniliforme, Fusarium oxysporum, Fusarium syphilis, and Plasmodium pineis, which are plant pathogens. Store them in a laboratory refrigerator at 4°C for later use. S2. Selection of Culture Media, Reagents and Equipment: Select the appropriate culture media for the test strain, and bring the culture media to a final volume of 1000 mL with deionized water, adjust the pH accordingly, and add an additional 15.0 g / L agar when preparing solid culture media. Autoclave at 121 ℃ for 20 min. The reagents selected are: bacterial genome extraction kit, PCR purification kit and agarose. The equipment selected are: stereomicroscopy imaging system, stainless steel positive pressure filter, ultraviolet spectrophotometer, constant temperature shaking incubator and vertical autoclave. S3. Culture of HJ-L, an antagonistic bacterium with antibacterial activity against Alternaria: Take a small piece of rotten, peeled Sichuan pepper trunk, place it in a sterile Erlenmeyer flask, add 10 mL of sterile water, and incubate at 180 r for 30 min on a shaker. Then, dilute the culture to 10⁻⁶, 10⁻⁷ and 10⁻⁸, and take 100 μL of each culture and spread it on LB solid medium. After incubation at 32℃ for 24 h, pick single colonies for liquid culture. S4. Isolation of antagonistic bacteria HJ-L with antibacterial activity against Alternaria: The colonies in S3 were obtained by culturing Alternaria hJ-2, the pathogen of leaf mold of Zanthoxylum bungeanum, in PDA solid medium for 7 days. The colonies were then broken into mycelial cakes using a 6 mm diameter punch and inoculated into the center of freshly prepared PDA medium. 10 μL of HJ-L culture solution was inoculated in a triangular pattern at a distance of 2.0 cm from the mycelial cake. A fungal plate without culture solution was used as a control. The culture was kept at 30℃ for 7 days. Each treatment was repeated 3 times, and the inhibition rate was determined. S5. Screening of antagonistic bacteria HJ-L with antibacterial activity against Alternaria: In S4, the inhibition rate (%) = [colon diameter of control group (mm) - colony diameter of test group (mm)] / [colon diameter of control group (mm) - 6 (mm)] × 100 [16], and strain HJ-L was screened by measuring the inhibition rate.

2. The novel screening method for Leuconostoc mesenteroides according to claim 1, characterized in that: In S2, the culture media include: PDA medium, MRS medium, CM medium, TSB medium, NB medium, SI medium, LB medium, NAM medium and ATP medium.

3. The novel screening method for Leuconostoc mesenteroides according to claim 3, characterized in that: The PDA culture medium comprises: glucose: 20.0 g, potato extract: 1 L, agar: 15.0 g; MRS medium consists of: 10.0 g peptone, 10.0 g beef extract, 5.0 g yeast extract, 20.0 g glucose, 2.0 g K₂HPO₄, 2.0 g diammonium hydrogen citrate, 5.0 g sodium acetate, 0.58 g MgSO₄·7H₂O, 0.14 g MnSO₄·H₂O, 1.0 mL Tween 80, pH 6.2; CM medium consists of: sucrose: 10.0 g, yeast extract: 10.0 g, peptone: 10.0 g, K2HPO4: 10.0 g, NaCl: 2.0 g, MgSO4·7H2O: 0.2 g, Tween 80: 0.5 mL; TSB medium consists of: tryptone: 17.0 g, soybean peptone: 3.0 g, NaCl: 5.0 g, KH2PO4: 2.5 g, and glucose: 2.5 g.

4. The novel screening method for Leuconostoc mesenteroides according to claim 2, characterized in that: The NB culture medium comprises: yeast extract: 1.0 g, sucrose: 10.0 g, peptone: 5.0 g, and beef extract: 3.0 g; SI medium consists of: tryptone: 8.0 g, yeast extract: 3.0 g, NaCl: 5.0 g, glucose: 5.0 g, KH2PO4: 2.0 g, Tween: 20 5.0 mL; LB medium consists of: tryptone: 10.0 g, yeast extract: 3.0 g, NaCl: 10.0 g; NAM medium consists of: beef extract: 3.0 g, peptone: 10.0 g, NaCl: 5.0 g; The ATP medium consisted of: tryptone: 10.0 g, yeast extract: 10.0 g, sodium citrate: 5.0 g, glucose: 10.0 g, K2HPO4: 5.0 g, NaCl: 5.0 g, MgSO4·7H2O: 0.8 g, MnCl2·4H2O: 0.14 g, FeSO4·7H2O: 0.04 g, and Tween 80: 1.0 mL.

5. The antibacterial mechanism against leaf mold in Sichuan pepper, specifically the screening method for a novel Leuconostoc mesenteroides according to any one of claims 1-4, characterized in that... Includes the following steps: S1. The antibacterial plates co-cultured for 7 days were observed under a 150x stereomicroscope to analyze the effect of strain HJ-L on the mycelial growth of Alternaria alternata. Changes in the morphology of the hyphal tips were observed, including branching density, growth direction, and swelling. S2. Prepare fungal cakes with a diameter of 6 mm using a punch, inoculate them in the center of PDA medium plates, and then spot HJ-L culture solution on the surface of PDA medium in a triangular pattern. After culturing in a 30℃ constant temperature incubator for 7 days, determine their antibacterial effect against different pathogens. Each treatment is set up with 3 biological replicates. S3. Use a sterile inoculation loop to pick up the activated HJ-L strain culture medium and purify the strain by streak plating on the surface of solid CM medium. Then, incubate in a 37℃ constant temperature incubator for 24 hours. When a single colony is formed, select a typical colony for Gram staining. After staining, use an optical microscope to observe the morphology of the bacterial cells. S4. Systematic identification of target strains based on 16S rRNA gene sequence analysis: Select strains with significant antagonistic effects in the initial antibacterial screening experiment, extract bacterial DNA, and amplify 16S rRNA genes using universal primers 27F and 1492R. Construct a 25μL standard reaction system: 2×Es Taq MasterMix: 12.5μL, forward / reverse primers: 1μL each, DNA template: 1μL, and add sterile water to a final volume of 25μL. S5. Based on the experimental screening results, CM medium was determined to be the optimal basal medium for strain HJ-L. A single-factor experimental design was used to investigate five parameters: initial pH, temperature, rotation speed, inoculum size, and salt concentration. The pH gradient was regulated using a phosphate-citric acid buffer system. Temperature gradients were used in an intelligent constant temperature shaking incubator; rotation speed was set at 15 mL liquid volume in 50 mL centrifuge tubes; inoculum size gradients were controlled using logarithmic growth phase bacterial suspensions.

6. The antibacterial mechanism against leaf mold of Sichuan pepper according to claim 5, characterized in that: In S4, the strain underwent an amplification program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 45 s, 72℃ extension for 90 s, for a total of 35 cycles; and a final extension at 72℃ for 7 min. The amplified products were verified by 1.0% agarose gel electrophoresis. The purified positive products were sequenced, and the valid sequences were submitted to the GenBank database for alignment and phylogenetic tree construction.

7. The antibacterial mechanism against leaf mold of Sichuan pepper according to claim 5, characterized in that: In the experiment described in S5, nine different salt concentrations ranging from 0.5% to 14% were set up to show the effect of these concentrations on the growth of the bacterial strain. Each treatment group was replicated three times. After culturing for 22 hours, bacterial culture samples were taken, bacterial fragments were removed, and the OD600 value was measured using a UV-Vis spectrophotometer to determine the optimal growth parameter 2.

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