High-performance antagonistic bacterium RQH1 and application thereof in prevention and control of soft rot of konjak

Abstract

The invention discloses a high-performance antagonistic bacterium RQH1 and application of the high-performance antagonistic bacterium RQH1 in prevention and control of amorphophallus konjac soft rot. The high-performance antagonistic bacterium RQH1 is preserved in China Center for Type Culture Collection, the preservation number is CCTCC NO. M 2023719, the preservation address is Wuhan University, Wuhan, China, and the preservation time is May 10, 2023. According to the invention, a high-performance antagonistic bacterium RQH1 is obtained by directional screening in a healthy konjak plant body, and the strain not only shows broad-spectrum and high-efficiency bacteriostatic activity on various konjak soft rot pathogenic bacteria, but also obviously inhibits the pathogenic bacteria from growing and infecting; meanwhile, various growth promoting functions such as phosphorus dissolving, potassium dissolving and nitrogen fixing can be achieved, the soil nutrient condition can be improved, and konjak plants can be promoted to grow robustly. In addition, the composition and antibacterial mechanism of volatile metabolites of the RQH1 strain are systematically analyzed. Therefore, a high-value strain resource which is multifunctional, easy to culture and high in adaptability is provided for green prevention and control of the soft rot of the konjak, and various defects in the prior art are effectively overcome.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a high-performance antagonistic bacterium RQH1 and its application in the control of soft rot disease in konjac. Background Technology

[0002] Konjac( Amorphophallus konjac As a high-value-added economic crop, konjac is facing severe challenges in its industrialization and large-scale development. With the promotion of continuous cropping obstacles and intensive cultivation models, konjac soft rot, caused by the combined infection of soil-borne pathogens, has become a bottleneck restricting industrial upgrading. This disease is characterized by a short incubation period, complex transmission routes, and a short window for control, with a field incidence rate of over 60%, earning it the reputation of being a "destructive disease" for konjac. Currently, the control of this disease still relies mainly on chemical pesticides, but long-term overuse not only leads to increased drug resistance in pathogens but also causes environmental pollution and pesticide residues, resulting in high control costs and a continuous negative impact on the ecosystem.

[0003] In terms of biological control, existing antagonistic bacterial strains remain relatively limited. Most strains suffer from narrow antibacterial spectra and poor resistance, hindering large-scale application in actual production. Furthermore, existing strains often exhibit limited functionality, possessing only antibacterial activity and lacking growth-promoting functions such as phosphorus solubilization, potassium solubilization, and nitrogen fixation, thus failing to achieve a synergistic "disease prevention-growth promotion" effect. Simultaneously, systematic research on the volatile metabolites of antagonistic bacteria is relatively weak, and their composition, antibacterial mechanisms, and impact on pathogen motility remain unclear. Therefore, screening superior strains that exhibit significant antagonistic activity against the pathogen causing konjac soft rot, while simultaneously inducing systemic resistance in konjac and possessing growth-promoting functions, is of great significance for achieving green and sustainable control of konjac soft rot and is also an urgent need for current industrial development. Summary of the Invention

[0004] The purpose of this invention is to provide a high-performance antagonistic bacterium RQH1 and its application in the control of konjac soft rot. A high-performance antagonistic bacterium RQH1 was obtained by screening from healthy konjac plants. This strain not only exhibits broad-spectrum and highly efficient antibacterial activity against a variety of konjac soft rot pathogens, but also has multiple growth-promoting functions such as phosphorus solubilization, potassium solubilization, and nitrogen fixation.

[0005] The above-mentioned technical objective of the present invention is achieved through the following technical solution: A high-performance antagonistic bacterium ( Bacillus amyloliquefaciens RQH1, the strain is deposited at the China Center for Type Culture Collection, accession number CCTCC NO. M 2023719, deposit address: Wuhan University, Wuhan, China, deposit date: May 10, 2023.

[0006] More preferably, the strain has broad-spectrum antibacterial activity against the pathogen of konjac soft rot, and has significant antibacterial effects against at least the pathogenic bacteria Coccidioides HBY3-5 and the pathogenic fungus Fusarium solani.

[0007] More preferably, the strain possesses at least one of the following plant growth-promoting functions: phosphorus solubilization, potassium solubilization, or nitrogen fixation.

[0008] More preferably, the strain is capable of producing at least one volatile antibacterial metabolite selected from the following: 4-methyl-heptane, (1.alpha.,2.alpha.,3.beta.)-1,2,3-trimethyl-cyclohexane, (3-methyl-2-butenyl)-benzene, and nonanoic acid.

[0009] This invention also provides a high-performance antagonistic bacterium RQH1 and its application in the prevention and control of soft rot disease in konjac.

[0010] In summary, the present invention has the following beneficial effects: This invention obtains a high-performance antagonistic bacterium, RQH1, from healthy konjac plants through screening. This strain not only exhibits broad-spectrum and highly efficient antibacterial activity against various pathogens causing konjac soft rot, but also possesses multiple growth-promoting functions such as phosphorus solubilization, potassium solubilization, and nitrogen fixation. Furthermore, this invention systematically elucidates the composition of its volatile metabolites and its antibacterial mechanism. Therefore, it provides a high-value strain resource that is multifunctional, easily cultured, and highly adaptable for the green control of konjac soft rot, effectively overcoming many shortcomings of existing technologies. Attached Figure Description

[0011] Figure 1 This is a diagram showing the screening results of the RQH1 strain versus HBY3-5 strain in this invention; Figure 2 These are colony diagrams of the RQH1 strain in this invention (A: RQH1 colonies on the front of the entire plate; B: RQH1 colonies on the back of the entire plate). Figure 3 This is a phylogenetic tree of the RQH1 strain and closely related species in this invention; Figure 4 This is a diagram illustrating the antagonistic effects of strain RQH1 against different pathogens in this invention (A: Fusarium solani; B: Alternaria spp.; C: Fusarium oxysporum; D: Pseudomonas aeruginosa; E: Fusarium solani; F: Colletotrichum spp.; G: Stagonosporopsis vannaccii H: Alternaria alternata; I: Acanthopanax nigricans. Figure 5 This is a qualitative experimental diagram of the inorganic phosphorus solubility of strain RQH1 in this invention; Figure 6 This is a graph showing the potassium solubilization capacity of the RQH1 strain in this invention; Figure 7This is a graph showing the nitrogen fixation capacity of the RQH1 strain in this invention; Figure 8 This is a growth curve of the RQH1 strain in this invention; Figure 9 This is a graph showing the relationship between the absorbance of strain RQH1 and the concentration of the bacterial solution in this invention. Figure 10 This is a graph showing the effect of different initial pH culture media on the growth of RQH1 strain in this invention; Figure 11 This is a graph showing the effect of different carbon source culture media on the growth of RQH1 strain in this invention; Figure 12 This is a graph showing the effect of different nitrogen source culture media on the growth of RQH1 strain in this invention; Figure 13 This is a concentration curve of strain RQH1 in this invention; Figure 14 These are diagrams illustrating the antibacterial effects of crude extracts of RQH1 sterile fermentation broth extracted with different solvents in this invention (A: antibacterial effect of ethyl acetate extract; B: antibacterial effect of methanol extract; C: antibacterial effect of acetone extract). Figure 15 This is a diagram verifying the antibacterial effect of the volatile metabolites of strain RQH1 on HBY3-5 using the bipartite plate confrontation culture method in this invention. Figure 16 This is a diagram verifying the antibacterial effect of the volatile metabolites of strain RQH1 on HBY3-5 using the filter paper disc method in this invention; Figure 17 This is a diagram showing the effect of volatile metabolites of strain RQH1 on the migration of HBY3-5 in this invention; Figure 18 This is a diagram showing the effect of volatile metabolites of strain RQH1 on the clustering and movement of HBY3-5 in this invention; Figure 19 This invention relates to the effect of volatile metabolites of strain RQH1 on the twitching movement of HBY3-5. Figure 20 This is the TIC overlap diagram of mass spectrometry detection of volatile metabolites QC samples of strain RQH1 in this invention; Figure 21 This is a circular diagram of the RQH1 metabolites from the RQH1 strain in this invention; Figure 22 This is a differential metabolic circular diagram of the RQH1 strain HH group versus the HBY group in this invention; Figure 23 This is a differential metabolic volcano diagram of the RQH1 strain HH group versus the HBY group in this invention. Detailed Implementation

[0012] The present invention will be further described in detail below with reference to the accompanying drawings.

[0013] Example 1: A high-performance antagonistic bacterium ( Bacillus amyloliquefaciens RQH1, the strain is deposited at the China Center for Type Culture Collection, accession number CCTCC NO. M 2023719, deposit address: Wuhan University, Wuhan, China, deposit date: May 10, 2023.

[0014] The strain exhibits broad-spectrum antibacterial activity against the pathogen causing konjac soft rot, showing significant inhibitory effects against at least the pathogenic bacteria *Sacchariella coccidioides* HBY3-5 and the pathogenic fungus *Fusarium solani*. The strain also possesses at least one plant growth-promoting function selected from phosphorus solubilization, potassium solubilization, or nitrogen fixation. The strain can produce at least one volatile antibacterial metabolite selected from the following: 4-methyl-heptane, (1.alpha.,2.alpha.,3.beta.)-1,2,3-trimethyl-cyclohexane, (3-methyl-2-butenyl)-benzene, and nonanoic acid.

[0015] Example 2: Isolation, purification, and screening of antagonistic bacteria RQH1 I. Materials and Methods 1. Culture medium formulation LB liquid medium: 10g tryptone, 10g NaCl, 5g yeast extract, 1000mL water, pH=7. If making a solid medium, add 18g agar.

[0016] PDA solid medium: 200g potato, 20g glucose, 20g agar, 1000mL water, pH=7.

[0017] MH medium: 17.5g acid-hydrolyzed casein, 2g beef extract, 1.5g starch, 12g agar, 1000mL water.

[0018] 2. Test plants Healthy Amorphophallus konjac plants within 3 meters of Amorphophallus konjac plants infected with soft rot in Buliu Village, Fuyuan County, Qujing City, Yunnan Province.

[0019] 3. Test strains Pathogenic bacteria: HBY3-5 (Coccella sacchariformis) K. cowanii ).

[0020] Pathogenic fungi: 2-1-4 ( Stagonosporopsis vannaccii ), 13-2-5 (Grapevine stem spot fungus) Phomopsis viticda H2-1 (Fusarium solani) Fusarium solani ), 23-2 (Fusarium latifolium) Fusarium proliferatum ), 13-4-4 (Alternaria alternata) Alternaria alternata X2 (Fusarium oxysporum) Fusarium oxysporum ), 25-1-3 (Alternaria spp.) Alternaria tenuissima ), 9-2-6 (Black Cocci) Epicoccum nigrum ), 17-4-2 (Winter Wheat Spiny Discus) Colletotrichum liriopes ).

[0021] 4. Isolation and purification of antagonistic bacteria Endophytic bacteria were isolated using a tissue isolation method. Fresh, infected leaves, stems, and corms of *Amorphophallus konjac* were collected, cut with a sterile scalpel, and immersed in 75% alcohol for 30 seconds, then in sodium hypochlorite for 1 minute, and rinsed three times with sterile water for 10 seconds each time. After sterilization, the samples were air-dried on sterile filter paper and inoculated onto LB agar, with three aliquots per LB agar and three replicates for each site. After inoculation, the petri dishes were sealed with sealing film and incubated at 28°C for 3 days. Single colonies were streaked onto LB agar plates for purification three times.

[0022] 5. Primary and secondary screening of antagonistic bacteria Antagonistic bacteria capable of inhibiting the pathogen HBY3-5 were screened from endophytic bacteria using the double-layer plate method.

[0023] Initial screening: Six bacteria were spotted onto LB solid culture dishes and incubated at 28°C for 24 hours. The pathogenic bacteria HBY3-5 for konjac soft rot, cultured on LB, were then mixed in 0.9% physiological saline, followed by MH semi-solid medium. The mixture was poured onto the cultured test plates and incubated at 28°C for 24 hours. The presence of transparent inhibition zones was observed. Each plate was tested in triplicate.

[0024] Secondary screening: Bacteria exhibiting inhibition zones in the initial screening were selected and spotted onto LB solid culture dishes. Four spots were placed on each bacterium on a separate plate. After encapsulation, the plates were incubated at 28°C for 48 hours. Subsequent treatment was the same as the initial screening, and each plate was repeated three times. The inhibition rate was calculated after the appearance of inhibition zones.

[0025] 6. Identification of antagonistic bacteria 6.1 Observation of antagonistic bacteria morphology The selected antagonistic bacteria were cultured on LB solid culture dishes for 24 h using the streak method, and the morphology of the single colonies was observed for morphological identification.

[0026] 6.2 Physiological and biochemical tests of antagonistic bacteria Nine indicators were selected for physiological and biochemical tests of antagonistic bacteria, including Gram staining, starch hydrolysis test, 7% NaCl growth test, gelatin hydrolysis test, spore staining test, hydrogen sulfide production test, catalase test, phenylalanine deaminase test, and urease test.

[0027] 6.3 Molecular identification of antagonistic bacteria Antagonistic bacteria were inoculated onto LB agar plates and incubated at 28°C for 24 h. After single-cell droplets were observed, the samples were sent to Qingke Biotechnology for one-way sequencing. Sequence alignment was performed in NCBI to obtain highly similar sequences, and a phylogenetic tree was constructed using neighbor-joining in MEGA 7.0. PCR amplification conditions were: pre-denaturation at 98°C for 2 min, denaturation at 98°C for 10 s, annealing at 56°C for 10 s, extension at 72°C for 20 s, final extension at 72°C for 5 min, and hold at 4°C, for a total of 35 cycles. Primers used are listed in Table 1.

[0028] Table 1 PCR amplification primers 7. Broad-spectrum antibacterial experiment of antagonistic bacteria To evaluate the broad-spectrum antibacterial activity of antagonistic bacteria, the antibacterial spectrum of pathogenic fungi preserved in the laboratory was determined. Nine pathogenic fungi, including strains 2-1-4 and 13-2-5, were used as target bacteria. Using the plate confrontation method, pathogenic fungal discs were inoculated into the center of a PDA solid culture dish. Antagonistic bacteria were then picked up with a bamboo stick and connected to four points 2.5 cm from the center of the fungal disc using the streak method. A control group was used, with only pathogenic fungi inoculated. All treatment groups and the control group were repeated three times. The diameter of the pathogenic fungal discs was measured daily. The culture was terminated when the fungi in the control group completely covered the plate, and the inhibition rate was calculated.

[0029] II. Results and Analysis 1. Results of endophytic bacteria isolation and purification A total of 22 endophytic bacteria strains were isolated and purified, which were used for screening endophytic antagonistic bacteria in konjac.

[0030] 2. Screening results of antagonistic bacteria Six antagonistic bacteria capable of inhibiting the pathogenic bacterium HBY3-5 of konjac soft rot were screened from 22 endophytic bacteria using a double-layer plate confrontation experiment. (See Table 2 and...) Figure 1 It can be seen that RQH1 has a significantly higher inhibitory effect on HBY3-5 than the other 5 antagonistic bacteria, with an inhibition zone diameter of 23 mm and an inhibition rate of 27.39%. The other 5 antagonistic bacteria also have a certain inhibitory effect on HBY3-5.

[0031] Table 2. Antibacterial effect of strain RQH1 against pathogens 3. Identification results of the tested antagonistic bacteria 3.1 Colony morphology of the tested antagonistic bacteria RQH1 colony morphology characteristics, such as Figure 2 As shown, white colonies appear on LB medium, with raised areas, rough edges, and a dry, opaque surface.

[0032] 3.2 Physiological and biochemical characteristics of the tested antagonistic bacteria Nine physiological indicators, including Gram staining and starch hydrolysis, were measured. The results are shown in Table 3.

[0033] Table 3 Physiological and biochemical characteristics of strain RQH1 3.3 Molecular biological identification of the tested antagonistic bacteria Sequencing results of the conserved 16S rDNA gene of the RQH1 antagonistic bacteria were imported into NCBI and subjected to BLAST alignment. The results showed that the RQH1 strain sequence matched that in GenBank. B. amyloliquefaciens The similarity is as high as 99.42%, such as Figure 3 As shown, a phylogenetic tree of strain RQH1 was constructed using the neighbor-joining method in MEGA 7.0. Based on the morphological characteristics and physiological and biochemical properties of strain RQH1, combined with sequence alignment results, RQH1 was identified as Bacillus amyloliquefaciens (Bacillus). B. amyloliquefaciens ).

[0034] 4. Results of the determination of the antibacterial spectrum of the tested antagonistic bacteria From Table 4 and Figure 4 It can be seen that strain RQH1 has antagonistic effects on all nine pathogenic fungi, and can produce significant inhibitory effects, showing good broad-spectrum antibacterial activity.

[0035] Table 4. Antagonistic effects of strain RQH1 against 9 pathogenic fungi III. Summary (1) A total of 22 endophytic bacteria were isolated from healthy Amorphophallus konjac plants. One antagonistic bacterium HBY3-5, the pathogen of Amorphophallus soft rot, was screened using the double-layer plate method. After morphological identification, physiological and biochemical identification and molecular biological identification of the 16S rDNA gene, RQH1 was identified as Bacillus amyloliquefaciens (Bacillus). B. amyloliquefaciens ).

[0036] (2) Using nine pathogenic fungi as target bacteria, the broad-spectrum antibacterial activity of six antagonistic bacteria was determined. They all showed good broad-spectrum antibacterial activity against the nine pathogenic fungi.

[0037] Example 3: Study on the antibacterial and growth-promoting properties of antagonistic bacterium RQH1 I. Materials and Methods 1. Culture medium formulation LB liquid medium: 10g tryptone, 10g NaCl, 5g yeast extract, 1000mL water, pH=7. If making a solid medium, add 18g agar.

[0038] Inorganic salt culture medium: (NH4)SO4 2g, K2HPO4·3H2O 2g, NaH2PO4·H2O 0.6g, NaCl 1g, CaCl2 0.02g, water 1000mL.

[0039] Beef extract peptone (BPM) medium: 3g beef extract, 10g peptone, 5g NaCl, 20g agar, 1000mL water, pH=7.

[0040] Pikovskava inorganic phosphate-solubilizing medium: (NH4)2SO4 0.5g, MgSO4·7H2O 0.3g, FeSO4·7H2O 0.3g, MnSO4·7H2O 0.03g, Ca3(PO4)2 5g, glucose 10g, agar 15g, water 1000mL, pH=7.

[0041] Organophosphorus egg yolk medium: glucose 10g, (NH4)2SO4 0.5g, NaCl 0.3g, KCl 0.3g, FeSO4·7H2O 0.03g, MnSO4·4H2O 0.03g, egg yolk lecithin 0.2g, CaCO3 5g, yeast extract 0.4g, agar 15g, water 1000mL, pH=7.

[0042] Silicate medium: 10g sucrose, 0.5g yeast extract, 1g (NH4)2SO4, 2g Na2HPO4, 0.03g MgSO4·7H2O, 1g CaCO3, 1g potassium feldspar powder, 15g agar, 1000mL water.

[0043] Ashby's nitrogen-free medium: mannitol 10g, KH2PO4 0.2g, MgSO4 0.2g, NaCl 0.2g, CaSO4 0.1g, CaCO3 5g, water 1000mL, pH=7; when used as a solid medium, add 1.5% agar.

[0044] 2. Test strain: RQH1.

[0045] 3. Determination of the phosphorus-solubilizing, potassium-solubilizing, and nitrogen-fixing abilities of antagonistic bacteria 3.1 Determination of phosphate-solubilizing ability of antagonistic bacteria Prepare Pikovskava inorganic and organic phosphorus-solubilizing solid media. Use a pipette tip to punch wells in the solid media, with four wells per plate as four replicates. Place bacterial suspensions cultured in LB medium for 1 day into the wells and incubate at 28°C for 7 days. The appearance of a clear zone around the well indicates that the strain has the ability to solubilize inorganic or organic phosphorus. Each treatment is repeated three times.

[0046] 3.2 Determination of potassium-solubilizing ability of antagonistic bacteria Single colonies of antagonistic bacteria were selected and inoculated at four points 2.5 cm from the center of a silicate petri dish as four replicates. The cultures were incubated at 28°C for 5 days. The appearance of a clear zone around each well indicated that the strain possessed potassium-solubilizing ability. The width of the hydrolysis zone was calculated by subtracting the bacterial cell diameter from the diameter of the clear zone using the cross-multiplication method. Strains with good potassium-solubilizing effects were screened by comparing the width of the hydrolysis zone and the solubility index. Each treatment was repeated three times.

[0047] 3.3 Determination of nitrogen fixation capacity of antagonistic bacteria Single colonies of antagonistic bacteria were picked and inoculated onto solid Ashby nitrogen-free medium. Four inoculation points, 2.5 cm from the center of the petri dish, were used as four replicates. The cultures were incubated at 28°C for 7 days. Strains that could grow normally on nitrogen-free medium were considered to have nitrogen-fixing ability. The growth of the strains was recorded daily, and each treatment was repeated three times.

[0048] 4. Determination of growth conditions for antagonistic bacteria 4.1 Determination of growth curves of antagonistic bacteria Single colonies of the purified antagonistic bacteria were picked and placed in LB liquid medium, then incubated in a shaker at 180 r / min and 25℃. Every 3 h, the culture solution was collected and measured using a spectrophotometer to determine the OD. 600 Take OD 600 The growth curve of the antagonistic bacteria was plotted with the value on the ordinate and the time point on the abscissa. The measurement was conducted for a total of 48 hours. If the trend was still unstable after 48 hours, the measurement was continued.

[0049] 4.2 Preparation of Seed Liquid The purified antagonistic bacteria were inoculated into a conical flask containing 50 mL of LB culture medium and cultured at 28 °C and 180 r / min in a shaker until the optimal growth time was reached for use.

[0050] 4.3 Effects of different initial pH media on the growth of antagonistic bacteria Seed culture was inoculated into 100 mL of LB culture medium with initial pH values ​​of 5, 6, 7, 8, and 9 for culture and measurement.

[0051] 4.4 Determination of the optimal carbon source for antagonistic bacterial culture Seed culture was inoculated into inorganic salt culture media containing different carbon sources: sucrose, lactose, maltose, and glucose, and cultured for analysis.

[0052] 4.5 Determination of the optimal nitrogen source for antagonistic bacterial culture Seed culture was inoculated into inorganic salt culture media containing different nitrogen sources: yeast powder, beef extract, tryptone, ammonium sulfate, ammonium chloride, and potassium nitrate, and cultured for analysis.

[0053] II. Results and Analysis 1. Study on the phosphorus-solubilizing, potassium-solubilizing, and nitrogen-fixing abilities of the tested antagonistic bacteria Depend on Figure 5-7 It is known that the antagonistic bacteria RQH1 has the ability to dissolve inorganic phosphorus, solubilize potassium, and fix nitrogen.

[0054] 2. Study on growth conditions of the tested antagonistic bacteria 2.1 Growth curves of the tested antagonistic bacteria OD at different time points of antagonistic bacteria RQH1 600 The value was obtained by spectrophotometer measurement and expressed as OD. 600 The values ​​are plotted on the ordinate and time points on the abscissa to create growth curves for the antagonistic bacteria. The resulting growth curves are shown in [Figure number missing]. Figure 8 As shown in the figure: RQH1 has a delayed phase of 0-6 hours, during which the strain adapts to the environment; a logarithmic growth phase of 6-27 hours, during which the strain rapidly multiplies and exhibits vigorous metabolism; and a stationary phase of 27-51 hours, during which OD... 600 The basics remain unchanged.

[0055] Figure 9 The curve shows the relationship between the absorbance of the antagonistic bacterium RQH1 and the concentration of the bacterial solution.

[0056] 2.2 Effect of different initial pH culture media on the growth of the tested antagonistic bacteria Depend on Figure 10 It can be seen that RQH1 has a significantly higher growth capacity at pH 7 than at other pH values, and the OD at this initial pH value in the culture medium is significantly higher. 600 Reaching the highest level.

[0057] 2.3 Study on the optimal carbon source for culturing the tested antagonistic bacteria Depend on Figure 11 It can be seen that RQH1 exhibits the strongest growth ability when glucose is the carbon source, and the OD value in the culture medium with this carbon source is [missing information]. 600 Reaching the highest level.

[0058] 2.4 Study on the optimal nitrogen source for culturing the tested antagonistic bacteria Depend on Figure 12 It can be seen that RQH1 exhibits the strongest growth ability when peptone is the nitrogen source, and the OD value in the medium with this nitrogen source is [missing information]. 600 RGH1 reaches its maximum growth rate when yeast extract is the nitrogen source, and its OD value is highest in this nitrogen source medium. 600 Reaching the highest level.

[0059] III. Summary Growth curve analysis revealed that the RQH1 antagonistic bacteria exhibited three growth phases: a lag phase, a logarithmic growth phase, and a stationary phase. Growth condition analysis showed that pH, carbon source, and nitrogen source all had varying degrees of influence on the growth of the RQH1 antagonistic bacteria. Phosphorus solubilization, potassium solubilization, and nitrogen fixation capacity measurements revealed that the RQH1 antagonistic bacteria possessed a certain potassium solubilization capacity, and all also exhibited some phosphorus solubilization and nitrogen fixation capabilities, suggesting that the RQH1 antagonistic bacteria all possess a certain growth-promoting potential.

[0060] Example 4: Study on the antibacterial effect and mechanism of antagonistic bacterial metabolites I. Materials and Methods 1. Culture medium formulation LB liquid medium: 10g tryptone, 10g NaCl, 5g yeast extract, 1000mL water, pH=7. If making a solid medium, add 18g agar.

[0061] NB solid medium: 10g peptone, 10g NaCl, 5g beef extract, 18g agar, pH=7, 1000mL water.

[0062] 2. Test strains Antagonistic bacteria: RQH1. Pathogenic bacteria: HBY3-5.

[0063] 3. Determination of antagonistic bacteria concentration curve Single colonies of RQH1 antagonistic bacteria were picked and placed in an Erlenmeyer flask containing 100 mL of LB culture medium for incubation. The culture medium was placed in a refrigerated high-speed centrifuge and centrifuged at 12000 r / min for 10 min. The supernatant was removed, and the bacterial precipitate was washed three times with sterile water. The weight of the precipitate was measured, and then the precipitate was dissolved in 5 mL of sterile water in a laminar flow hood. The absorbance of the dissolved solution was measured with sterile water as a control.

[0064] Dilute the solution to 10. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 Eight concentrations were added to a hemocytometer, and the cells were counted and observed under an optical microscope to calculate the cell concentration, which was used to prepare a curve showing the relationship between dilution and cell concentration.

[0065] 4. Study on the antibacterial effect of crude extracts of antagonistic bacteria sterile fermentation broth obtained with different extraction solvents Ethyl acetate, acetone, and methanol were selected as three organic solvents for extraction from the sterile fermentation broth of antagonistic bacteria. The antibacterial activity of the crude extracts from the sterile fermentation broth extracted with different organic solvents was studied using the Oxford cup method. The antibacterial effect of the crude extracts obtained from the sterile fermentation broth with different extraction solvents was compared by measuring the diameter of the inhibition zone.

[0066] 5. Effects of antagonistic bacteria aseptic fermentation broth on pathogen growth (1) Pretreatment: Pick single colonies of antagonistic bacteria and single colonies of pathogenic bacteria and place them into 50 mL sterile centrifuge tubes containing 45 mL LB culture medium and incubate in a shaker at 180 r / min for 24 h.

[0067] (2) Sample preparation: The specific treatment method is shown in Table 5. Each treatment also needs to be allocated 4 time points of 12 h, 24 h, 48 h and 72 h, and the aseptic fermentation broth is divided into two treatment groups, treated and untreated, based on whether it has undergone high temperature treatment.

[0068] The cultured antagonistic bacterial suspension was centrifuged three times at 12000 r / min and 4℃ in a refrigerated high-speed centrifuge. The broth was then filtered through a 0.22 μm sterile filter to obtain a sterile fermentation broth of the antagonistic bacteria. The sterile fermentation broth was then diluted to 1 / 2, 1 / 4, and 1 / 8 with LB broth and aliquoted into 50 mL centrifuge tubes to obtain different concentrations of sterile fermentation broth. The pathogenic bacterial suspension was added to the unsterilized main tube of the sterile fermentation broth, aliquoted into corresponding 10 mL centrifuge tubes, and incubated at 28℃. After sterilization, the pathogenic bacterial suspension was added to the sterile fermentation broth, aliquoted into corresponding 10 mL centrifuge tubes, and incubated at 28℃. LB broth was used as a negative control.

[0069] Table 5. Antibacterial treatment groups of aseptic fermentation broth (3) 96-well plate spotting: At the corresponding time point, take out the corresponding 10 mL centrifuge tube, and then use a pipette to add the corresponding liquid to the corresponding well. Add 6 wells to each treatment tube, and prepare 3 treatment tubes for each treatment group. Add 100 μL of sample to each well. Only measure LB culture medium at 12 h, and add 12 wells.

[0070] (4) Data determination: Place the 96-well plate with the sample added into the microplate reader that has been preheated for more than half an hour, adjust the OD value to 600, measure the absorbance, and calculate the antibacterial rate.

[0071] 6. Antibacterial activity of volatile antibacterial metabolites of antagonistic bacteria Using the RQH1 antagonistic bacteria obtained from previous screening as material, the antibacterial activity of volatile organic compounds produced by the antagonistic bacteria against the pathogenic bacterium HBY3-5 was evaluated on LB medium using the two-part plate confrontation culture method and the filter paper disc method.

[0072] 7. Effects of volatile antimicrobial metabolites of antagonistic bacteria on the motility of pathogens Inoculate 20 µL of antagonistic bacterial suspension (concentration approximately 1×10⁻⁶) onto the LB side of the septate culture dish. 9 CFU / mL), 2 μL of freshly prepared pathogenic bacterial suspension (adjusted to approximately 1×10⁻⁶ CFU / mL) was inoculated on the NB side of the culture dish. 6 The agar content in the NB-side medium should be set to 0.3%, 0.7%, and 1.6%, respectively, for the determination of swimming, swarming, and twitching.

[0073] 8. Identification of volatile metabolites of antagonistic bacteria 8.1 Sample Preparation Single colonies of both antagonistic and pathogenic bacteria were picked and placed in centrifuge tubes containing 45 ml of LB medium. The culture was carried out at 180 r / min and 28℃ for 24 h. Then, three treatment groups were prepared: single culture of antagonistic bacteria, single culture of pathogenic bacteria, and mixed solution. After incubation at 180 r / min and 28℃ for 48 h, the samples were frozen on dry ice and then sent for delivery.

[0074] 8.2 Determination of Metabolites Sample extraction: (1) Take the sample out of the -80℃ freezer and grind it with liquid nitrogen. Mix it evenly by vortexing. Weigh 0.2 mL of each sample into a headspace vial; (2) Add 0.2 g NaCl powder and 20 μL (10 μg / mL) internal standard solution to each sample; (3) Extract the sample using fully automated headspace solid phase microextraction (HS-SPME) for GC-MS analysis.

[0075] Chromatographic and mass spectrometry acquisition conditions: (1) HS-SPME extraction conditions: Under constant temperature of 60℃, shake for 5 min, insert a 120 μm DVB / CWR / PDMS extraction head into the sample headspace vial, perform headspace extraction for 15 min, desorb at 250℃ for 5 min, and then perform GC-MS separation and identification. Before sampling, the extraction head needs to be aged at 250℃ for 5 min in the Fiber Conditioning Station. (2) Chromatographic conditions: DB-5MS capillary column (30 mm × 0.25 mm × 0.25 μm), carrier gas is high-purity helium, constant flow rate is 1.2 mL / min, injection port temperature is 250℃, and solvent delay is 3.5 min. Program temperature rise: 40℃ for 3.5 min, rise to 100℃ at 10℃ / min, then rise to 180℃ at 7℃ / min, and finally rise to 280℃ at 25℃ / min, and hold for 5 min; (3) Mass spectrometry conditions: electron bombardment ion source, ion source temperature 230℃, quadrupole temperature 150℃, mass spectrometry interface temperature 280℃, electron energy 70 eV, scanning mode is selected ion detection mode, qualitative and quantitative ion precise scanning.

[0076] 8.3 Screening for Differential Metabolites Based on the Maiwei Metabolism Self-built Database, qualitative and quantitative analyses of metabolites in the samples were performed. Mass spectrometry files of the samples were opened using MassHunter quantitative software for integration and calibration. T-tests and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to identify differentially metabolites. Substances meeting the criteria of VIP>1, FC≥1.5, and FC≤0.67, with P<0.05, were identified as differentially metabolites in both analyses.

[0077] II. Results and Analysis 1. Analysis of the concentration curve of the tested antagonistic bacteria The precipitate after centrifugation and washing of 3 mL of RQH1 bacterial culture was 0.1 g, and this weight was used as the standard for preparing lysates in subsequent treatments. The concentration curve of the antagonistic bacterium RQH1 is shown below. Figure 13 .

[0078] 2. Results of antibacterial activity of crude extracts of RQH1 aseptic fermentation broth obtained with different extraction solvents Based on the screening results of antagonistic bacteria RQH1, RQH1 with the best antibacterial effect against HBY3-5, the pathogen of konjac soft rot, was selected as the sample for experimentation. The results are as follows: Figure 14 As shown in the figure, the three crude extracts of RQH1 sterile fermentation broth after extraction with ethyl acetate, methanol, and acetone had no inhibitory effect on HBY3-5.

[0079] 3. Antibacterial effect of antagonistic bacteria RQH1 aseptic fermentation broth on pathogenic bacteria. Using HBY3-5 as the target bacterium, the antibacterial effects of the sterile fermentation broth of RQH1 and the sterile fermentation broth treated with high temperature to remove volatile metabolites were compared. Table 6 shows that the antibacterial effect of the untreated sterile fermentation broth of RQH1 against HBY3-5 was significantly higher than that of the RQH1 sterile fermentation broth at dilutions of 2, 4, and 8 at 12 h, 24 h, 48 h, and 72 h. The treated sterile fermentation broth of RQH1 showed no significant antibacterial effect at different times and concentrations.

[0080] Table 6. Antibacterial effect of RQH1 aseptic fermentation broth at different dilution ratios on HBY3-5 Based on the results of the antibacterial effect, the untreated sterile fermentation broth of RQH1 was selected to compare the antibacterial effect at different time points. As shown in Table 7, the antibacterial effect of the untreated sterile fermentation broth of RQH1 on HBY3-5 was significantly higher at 48 h than at 12 h, 24 h and 72 h. Therefore, 48 h was selected as the culture time for the mixed and single culture media in the subsequent metabolomics sample delivery experiment.

[0081] Table 7. Antibacterial effect of RQH1 aseptic fermentation broth on HBY3-5 at different time points 4. Antibacterial activity of volatile antibacterial metabolites of the tested antagonistic bacteria 4.1 Results of the bipartite plate confrontation culture method From Table 8 and Figure 15 It can be seen that the volatile metabolites of RQH1 antagonistic bacteria have antibacterial effects on pathogenic bacteria HBY3-5. Moreover, the growth of HBY3-5 after treatment with the volatile metabolites of RQH1 bacterial solution is significantly lower than that of HBY3-5 after treatment with other antagonistic bacteria, followed by RGH1.

[0082] Table 8. Verification of the antibacterial effect of RQH1 antagonizing bacterial volatile metabolites on HBY3-5. 4.2 Results of the filter paper method Depend on Figure 16 It can be seen that the volatile metabolites of RQH1 antagonistic bacteria have antibacterial effects on pathogenic bacteria HBY3-5, and the growth of HBY3-5 treated with the volatile metabolites of RQH1 bacterial solution is significantly lower than that of HBY3-5 treated with other antagonistic bacteria.

[0083] Combining the experimental results of the bipartite plate confrontation culture method and the filter paper disc method, the volatile metabolites of RQH1 showed a significantly higher antibacterial effect against HBY3-5 than the other five antagonistic bacteria.

[0084] 5. Effects of volatile antimicrobial metabolites of the tested antagonistic bacteria on the motility of pathogens. Depend on Figure 17 It can be seen that RQH1 antagonistic bacteria can affect the swimming of pathogenic bacteria HBY3-5, and the volatile metabolites of RQH1 have a significant effect on the swimming of HBY3-5.

[0085] Depend on Figure 18 It is known that RQH1 antagonistic bacteria can affect the clustering movement of HBY3-5, and the volatile metabolites of RQH1 have a significant effect on the clustering movement of HBY3-5.

[0086] Depend on Figure 19 It is known that RQH1 antagonistic bacteria can affect the twitching movement of HBY3-5, while the volatile metabolites of RGH1 have a significant effect on the twitching movement of HBY3-5.

[0087] 6. Identification results of volatile metabolites of RQH1 6.1 GC-MS Data Quality Control and Evaluation like Figure 20 The retention time and peak intensity of metabolite detection remained consistent, and the total current ion curves showed high overlap, indicating that the mass spectrometer had good signal stability and high data repeatability when detecting the same sample at different times.

[0088] 6.2 Identification of volatile metabolites of RQH1 GC-MS analysis showed that RQH1 produced a total of 634 volatile metabolites, such as... Figure 21 As shown, there are four categories of substances that account for more than 10%: heterocyclic compounds, hydrocarbons, esters, and ketones. There are 12 categories of substances that account for less than 10%: alcohols, aromatics, aldehydes, terpenes, amines, acids, phenols, nitrogen-containing compounds, halogenated hydrocarbons, other substances, sulfur-containing compounds, and ethers.

[0089] 6.3 Analysis of antibacterial volatile metabolites of RQH1 Differential analysis of volatile metabolites in the fermentation broths of the mixed culture group (HH) of antagonistic bacteria and pathogens and the single culture group of pathogens (HBY) was performed to screen for antibacterial volatile metabolites of the antagonistic bacterium RQH1. Thresholds were set as VIP > 1.0, FC ≥ 1.5, and FC ≤ 0.67, with a P-value < 0.05, resulting in 41 differentially expressed metabolites. Figure 22As shown, among these volatile metabolites, substances comprising more than 10% include three categories: aromatics, hydrocarbons, and esters. In addition, there are 11 other categories: heterocyclic compounds, alcohols, ketones, aldehydes, terpenes, amines, nitrogen-containing compounds, phenols, other compounds, acids, and halogenated hydrocarbons. The volcano plot was obtained by taking the logarithm base 2 for the fold differences among the 41 metabolites and taking the absolute value of the logarithm base 10 for the p-value. (See figure...) Figure 23 As shown, there are 35 upregulated metabolites and 6 downregulated metabolites. Upregulated metabolites include the 14 categories mentioned above, while downregulated metabolites include esters, ketones, aldehydes, aromatic hydrocarbons, and others.

[0090] Based on the differential metabolite analysis results, four antibacterial volatile metabolites of RQH1 were identified from 35 upregulated metabolites: 4-methyl-heptane, (1.alpha.,2.alpha.,3.beta.)-1,2,3-trimethyl-cyclohexane, (3-methyl-2-butenyl)-benzene, and nonanoic acid.

[0091] III. Summary (1) The crude extract of RQH1 sterile fermentation broth extracted with three organic solvents had no inhibitory effect on HBY3-5, the pathogen of konjac soft rot.

[0092] (2) The antibacterial effect of the sterile fermentation broth of antagonistic bacteria RQH1 on HBY3-5 was determined. It was found that the antibacterial effect of the undiluted sterile fermentation broth of antagonistic bacteria RQH1 at different culture times was higher than that of the three diluted fermentation concentrations. The antibacterial activity of the sterile fermentation broth without high-temperature treatment at 48 h of culture was higher than that at 12 h, 24 h and 72 h of culture. The sterile fermentation broth treated with high temperature did not show any antibacterial activity.

[0093] (3) Using the pathogenic bacterium HBY3-5 as the target, the antibacterial effect of RQH1 against bacteria was determined by the two-part plate confrontation culture method and the filter paper disc method. The results showed that RQH1 antagonistic bacteria had an antibacterial effect against the pathogenic bacterium HBY3-5.

[0094] (4) The clustering, swimming and twitching movements of pathogenic bacteria HBY3-5 were measured by RQH1 antagonistic bacteria, and it was found that the antagonistic bacteria RQH1 had a significant effect on all three movements.

[0095] (5) Through GC-MS and differential metabolite analysis of RQH1 volatile metabolites, a total of 41 differential metabolites were obtained. Among them, 35 differential metabolites with an upregulation trend were substances whose production increased in RQH1 after double culture of RQH1 and HBY3-5. These included 14 types of substances, among which there were 4 antibacterial volatile metabolites, namely 4-methyl-heptane, (1.alpha.,2.alpha.,3.beta.)-1,2,3-trimethyl-cyclohexane, (3-methyl-2-butenyl)-benzene, and nonanoic acid.

[0096] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A high performance antagonistic bacteria (Bacillus sp.) RQH1, characterized by: Bacillus amyloliquefaciens The strain is preserved in China Center for Type Culture Collection, the preservation number is CCTCC NO. M 2023719, the preservation address is Wuhan, Wuhan University, China, and the preservation time is May 10, 2023. ​ 2. The high-performance antagonistic bacteria RQH1 according to claim 1, characterized in that: The strain has broad-spectrum antibacterial activity against konjac soft rot pathogen, and at least has significant inhibition effect on pathogenic bacteria K. oxytoca HBY3-5 and pathogenic fungus F. solani.

3. The high-performance antagonistic bacteria RQH1 according to claim 2, characterized in that: The strain has at least one plant growth-promoting function selected from the group consisting of phosphorus solubilization, potassium solubilization, and nitrogen fixation.

4. The high-performance antagonistic bacteria RQH1 according to claim 2, characterized in that: The strain can produce at least one volatile antibacterial metabolite selected from the group consisting of 4-methyl-heptane, (1.alpha.,2.alpha.,3.beta.)-1,2,3-trimethyl-cyclohexane, (3-methyl-2-butenyl)-benzene, and nonanoic acid.

5. The high-performance antagonistic bacteria RQH1 according to any one of claims 1-4 and its application in the prevention and control of konjac soft rot.

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