Bacillus GB-1 and application thereof in prevention and treatment of AHLs-dependent pathogenic pathogenic bacteria

Abstract

The invention discloses bacillus (Bacillus sp.) GB-1. The preservation number of the bacillus (Bacillus sp.) GB-1 in Guangdong Microbial Culture Collection Center is GDMCC NO: 65601. The bacillus (Bacillus sp. The strain GB-1 has a good effect of degrading quorum sensing signal molecules OHHL, the quorum quenching effect of the strain is stable and remarkable, a new way and method are provided for biological control of AHLs-mediated pathogenic bacteria, the strain has huge application potential, the use of chemical pesticides can be reduced, and the cost is reduced. A new way is provided for replacing chemical prevention and control with biological prevention and control, and the application prospect is good.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial degradation, and specifically relates to a strain of Bacillus ( Bacillus sp.) GB-1 and its application in preventing and controlling pathogens that rely on AHLs to cause disease. Background Art

[0002] In modern agricultural production, the main means of preventing and controlling crop diseases and pests is the use of chemical pesticides. The extensive use of chemical pesticides and antibiotics has led to the development of bacterial resistance, while ecological diversity, food safety and other issues have become increasingly prominent, so there is an urgent need to develop new green control strategies.

[0003] Quorum sensing (QS) is a communication mechanism between microbial cells. Microorganisms secrete signal molecules and sense changes in population density by monitoring the concentration of extracellular signal molecules. When a critical density value is reached, the signal molecules will activate genes that express specific traits in bacterial cells. These genes are mostly related to important biological functions of microorganisms. Studies have shown that the quorum sensing system is involved in regulating a variety of biological functions, such as the expression of pathogen virulence factors, the formation of biofilms, and the production of antibiotics.

[0004] There are many types of signal molecules, such as N-acyl homoserine lactone signal molecules (AHLs), diffusible signal factors (DSFs), oligopeptides (AIPs), etc. Among them, AHLs are the most intensively and detailed type of signal molecules studied to date. They are widely present in a variety of Gram-negative bacteria. In pathogenic bacteria mediated by AHLs, the pathogens continuously synthesize and secrete AHLs into the extracellular space. When the density of the pathogenic bacteria population continues to increase, the concentration of AHLs in the environment also increases. When the pathogenic bacteria detect that the AHLs in the environment have reached a certain concentration, some genes related to pathogenicity begin to express, such as if the Glebacillus ( Pectobacterium ) hydrolase synthesis gene, Pseudomonas aeruginosa ( Pseudomonas aeruginosa )The synthetic gene of pyocyanin, etc.

[0005] Quorum sensing is a widespread phenomenon in microorganisms, including many plant pathogens, such as Pectobacterium carotovoranum, the pathogen of soft rot. Pectobacterium carotovorum ) and Dickie's bacterium ( Dickeya zeae) and so on. Pectobacterium carrotus is an important pathogen of bacterial soft rot of many economic crops. It can infect a variety of temperate, subtropical crops and ornamental plants. The bacterial soft rot caused by Pectobacterium carrotus occurs widely. It has occurred and caused serious damage in many countries such as Lithuania, Japan, Germany, Argentina, New Zealand and China. It has become a global disease and has led to a reduction in the yield of related crops, causing significant economic losses. Pectobacterium carrotus synthesizes and secretes a series of cell wall degrading enzymes to promote the destruction of plant tissues. It infects the host plant and eventually causes symptoms related to soft rot in the host plant. Studies have shown that the synthesis of hydrolases of Pectobacterium carrotus is strictly regulated by the quorum sensing system. In addition to Pectobacterium carrotus, the plant pathogenic fungus Dickie's ( Dickeya zeae )EC1 also has quorum sensing phenomenon, and its signal molecule is 3OC6HSL, which is related to its motility and bacterial virulence.

[0006] Quorum quenching (QQ) is a new disease control strategy based on the quorum sensing phenomenon. It refers to interfering with quorum sensing by inhibiting the synthesis and monitoring of signal molecules by microorganisms or by enzymatic degradation and modification of signal molecules, making it difficult for pathogenic bacteria to express genes related to pathogenicity, thereby reducing the pathogenicity of pathogenic bacteria and preventing and controlling diseases.

[0007] In the process of using quorum quenching to prevent and control diseases, it will not affect the growth of microorganisms and will not cause selection pressure. It can avoid the drawbacks of pesticides and antibiotics that easily cause pathogens to resist drugs, and open up new ideas for the development of new environmentally friendly plant disease prevention measures in the future. It is expected to become a new method of green and safe prevention and control of microbial diseases. Summary of the invention

[0008] In view of the shortcomings of existing pathogenic bacteria biological control technology, the present invention provides a Bacillus sp. ( Bacillus sp.) GB-1, which was deposited in Guangdong Microbiological Culture Collection Center on December 10, 2024, with the deposit number GDMCC NO:65601, and the deposit address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0009] In order to achieve the above purpose, the present invention adopts the following technical solutions: 1. Soil was collected from the Panax notoginseng planting base in Wenshan City, Yunnan Province, and added to MSM liquid culture medium containing N-(3-oxohexanoyl)-L-homoserinelactone (OHHL). Multiple strains with the ability to degrade AHLs were obtained by continuous enrichment culture, separation and purification. The degradation ability of strains to AHLs was detected by high performance liquid chromatography (HPLC), and finally the strain GB-1 with the strongest degradation ability was selected for subsequent experiments; 2. Identification of strain GB-1 (1) Morphological characteristics of strain GB-1: The colonies on the LB solid plate are round, convex, light yellow, moist, opaque, and have neat edges; strain GB-1 is a Gram-positive bacterium.

[0010] (2) Molecular identification The bacterial genomic DNA extraction kit was used to extract the genomic DNA of strain GB-1. The extracted genome was used as a template and 16SrDNA bacterial universal primers were used for PCR amplification. After the PCR product was sequenced, the sequencing results were compared with the sequences on NCBI. Combined with the morphological characteristics and molecular identification results, the strain was finally identified as Bacillus ( Bacillus sp.).

[0011] 3. The present invention conducted sensitivity analysis of different antibiotics on the strain GB-1. The strain GB-1 had a resistance to ampicillin (Amp) of more than 200 μg / mL, a resistance to kanamycin (Kan) and chloramphenicol (Cm) of 25 μg / mL, a resistance to streptomycin (Str) of 6.25 μg / mL, and a resistance to gentamicin of 3.125 μg / mL.

[0012] 4. Bacillus GB-1 has good degradation activity for quorum sensing signal molecules. After 48 hours, the degradation rate of 0.4 mM N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) was above 94.5%.

[0013] The present invention has the following beneficial effects: The Bacillus GB-1 screened and obtained by the present invention can be used to control pathogenic bacteria that rely on AHLs to cause disease. The Bacillus GB-1 provided by the present invention has great application potential in controlling the harm of pathogenic bacteria mediated by AHLs, and provides a new approach and method for biological control of plant diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The colony morphology of strain GB-1 cultured on LB solid medium for 1 day; Figure 2 This is the Gram staining result of strain GB-1; Figure 3 is the phylogenetic tree of 16SrDNA of strain GB-1; Figure 4 This is a graph showing the growth of strain GB-1 in different antibiotics; Figure 5 This is a graph showing the degradation activity of strain GB-1 on OHHL; Figure 6 This is the standard curve of OHHL HPLC detection; Figure 7 This is the degradation curve of strain GB-1 to OHHL; Figure 8 The disease occurrence of strains GB-1+LB, Pcc+LB, and Pcc+ GB-1 after inoculation into white radish for 24 hours; Fig. 9 This is the disease occurrence 24 hours after strains GB-1+LB, Pcc+LB, and Pcc+GB-1 were inoculated into potato tubers. DETAILED DESCRIPTION

[0015] The technical scheme of the present invention is further described in detail below in conjunction with specific embodiments and drawings, but the present invention is not limited to the following technical scheme. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. Ordinary technicians in this field can refer to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of the present invention to implement; The culture medium formula in the following examples is as follows: MSM liquid culture medium (1 L): (NH4)2SO42.0g, CaCl2·2H2O0.01g, FeSO4·7H2O0.001g, Na2HPO4·12H2O1.5g, MgSO4·7H2O0.2g, KH2PO41.5g, pH6.5; LB liquid medium: 5.0 g yeast extract, 10.0 g peptone, 10.0 g sodium chloride, 1000 mL deionized water, pH 7.2, sterilized at 121°C for 20 min; LB solid medium: add 17-20 g agar powder per 1 L liquid medium.

[0016] Example 1: Isolation and identification of strain GB-1 1. Isolation and screening of strain GB-1 Soil from the Panax notoginseng plantation in Wenshan City, Yunnan Province was collected and enriched and cultured with acyl homoserine lactones (AHLs) as the sole carbon source. 50 mL of basal salt medium (MSM) was placed in a 250 mL conical flask for sterilization. After cooling, OHHL mother solution (methanol or acetone as solvent) was added under sterile conditions to make the final concentration of OHHL 10 μM. At the same time, 5 g of soil sample was added. After culturing at 30 ° C and 200 rpm in a shaking table for 7 days, the inoculation volume was transferred to 50 mL MSM medium with an OHHL concentration of 40 μM at a rate of 10%. After culturing under the same conditions for 7 days, the inoculation volume was transferred to 50 mL MSM medium with an OHHL concentration of 80 μM at a rate of 10%. The culture was continued for 7 days, and so on, and the OHHL concentration was continuously increased to 160 μM. The culture solution of the last concentration was diluted 10 times to 1×10 -7 Then spread it on LB solid culture medium and culture it upside down at 30°C; after a single colony grows on the plate, pick up a single colony and streak it multiple times to purify it until a single colony is isolated.

[0017] Using the reporter strain Bacillus violaceus ( Chromobacterium violaceum )CV026 screened strains capable of degrading OHHL (when exogenous AHLs signal molecules appeared, the strains could produce purple bacteriocin), picked the above single colony and inoculated it into LB liquid culture medium, cultured it overnight at 30°C and 200rpm to obtain a bacterial solution, added OHHL methanol solution to 500μL of the bacterial solution with a final concentration of 10μM, incubated it at 28°C for 24h, centrifuged it to obtain the supernatant, and filtered the supernatant through a 0.22μm filter membrane to obtain a cell-free supernatant; took the activated purple bacillus CV026 bacterial solution, inoculated it into LB semi-solid culture medium (agar content 0.2-0.7%) at a rate of 1%-2%, mixed it well and poured it into an Oxford cup plate, removed the Oxford cup after solidification, added 100μL of the cell-free supernatant into the agar plate wells, and cultured it at 28°C for 24-48h. The degradation of OHHL was determined based on the generation and size of the purple ring around the holes, and the strain with the best OHHL degradation effect was selected based on the experimental results and named the degrading bacteria GB-1.

[0018] 2. Identification of strain GB-1 (1) Morphological identification Strain GB-1 was inoculated on LB solid plates and inverted for 24 h at 30°C. The colonies were round, slightly convex, creamy yellow, moist, opaque, with neat edges and smooth surfaces (see Figure 1 ); Gram staining of strain GB-1 showed blue color, indicating that the bacteria were Gram-positive bacteria (see Figure 2 ); (2) 16S rDNA molecular biological identification The bacterial genomic DNA extraction kit (Solebo) was used to extract the genomic DNA of strain GB-1. The genomic DNA of strain GB-1 was extracted according to the kit instructions. The extracted genome was used as a template and 16SrDNA bacterial universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1429R: 5'-GGTTACCTTGTTACGACTT-3') were used for PCR amplification. The PCR product was entrusted to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The 16SrDNA sequence measured by the strain was compared and analyzed in the GenBank database using BLAST, and related sequences with higher homology were selected to construct a phylogenetic tree and analyze the evolutionary relationship using MAGE5.0 software. The results are shown in Figure 3 The 16SrDNA sequence of the strain GB-1 isolated and purified by the present invention is similar to Bacillus sp.(in:Bacteria) strainXA15-41MH769066.1 has a homology of 99% and is the closest in evolution; its morphological characteristics and scanning electron microscopy observation characteristics are also most similar to those of Bacillus, so strain GB-1 was identified as Bacillus ( Bacillus sp.).

[0019] Example 2: Antibiotic sensitivity analysis of strain GB-1 The resistance of GB-1 screened from the rhizosphere soil of Panax notoginseng was screened and determined. Several commonly used antibiotics, chloramphenicol (Chl), streptomycin (Str), ampicillin (Amp), kanamycin (Kan), gentamicin (Gen), and clindamycin (Cli) were selected, and the following 8 concentrations were set: 0μg / mL, 3.175μg / mL, 6.25μg / mL, 12.5μg / mL, 25μg / mL, 50μg / mL, 100μg / mL, and 200μg / mL. There were 3 replicates for each concentration. The seed solution cultured overnight was inoculated into LB liquid culture medium containing the corresponding final concentration of antibiotics at an inoculum size of 1%, and the OD was measured after culturing in a constant temperature shaker at 30°C and 200rpm for 24 hours. 600 The growth of bacteria in LB medium containing different concentrations and different antibiotics was observed. The results are shown in Figure 4 The results showed that the strain GB-1 had a resistance to ampicillin (Amp) of more than 200 μg / mL, a resistance to kanamycin (Kan) and chloramphenicol (Cm) of 25 μg / mL, a resistance to streptomycin (Str) of 6.25 μg / mL, and a resistance to gentamicin of 3.125 μg / mL.

[0020] Example 3: Degradation activity of strain GB-1 on OHHL GB-1 bacterial solution was obtained by overnight culture at 30°C and 200rpm. The OHHL methanol solution was added to 500μL bacterial solution with a final concentration of 10μM or 100μM. After incubation at 28°C for 24h, the supernatant was centrifuged and filtered through a 0.22μm filter to obtain a cell-free supernatant. Take the activated purple Bacillus CV026 bacterial solution, inoculate it in LB semi-solid medium (agar content 0.2-0.7%) at a rate of 1%-2%, mix it and pour it into the Oxford cup plate. After solidification, remove the Oxford cup, add 100μL of cell-free supernatant to the agar plate wells, and observe the experimental results after incubation at 28°C for 24-48. The degradation of OHHL was judged based on the generation and size of the purple circle around the hole. CK is a blank control without GB-1; The experimental results are as follows Figure 5 As shown in the figure, purple circles appeared around the holes in the CK experimental group, indicating that the sample contained OHHL; no purple circles appeared around the holes in the GB-1 experimental group, indicating that the sample did not contain OHHL, that is, OHHL had been completely degraded by GB-1. The results showed that strain GB-1 has the activity of degrading OHHL ( Figure 5 ).

[0021] Example 4: Detection of the OHHL-degrading activity of strain GB-1 using HPLC.

[0022] Strain GB-1 was cultured overnight in LB liquid medium to obtain bacterial suspension. 10 bacterial cells with OD values ​​were taken (volume V = 1 / OD 600 The bacterial solution was centrifuged at 4000 rpm for 10 min and the supernatant was discarded, which was an OD 600 The culture medium without bacteria was used as the control. The culture medium was cultured at 30℃ and 200rpm. The culture medium at 0h, 8h, 16h, 24h, 32h, 40h and 48h was taken and the residual amount of OHHL was determined by HPLC.

[0023] Extraction method of OHHL: take 5 mL of culture medium into a 15 mL centrifuge tube, centrifuge at 4000 rpm for 5 min, add ethyl acetate to the supernatant, shake well, shake vigorously for 3 min, let stand, separate layers, extract twice, collect and combine the ethyl acetate phase, put it into a round-bottom flask, concentrate and evaporate to dryness at a constant temperature of 50°C, dissolve it with 2 mL of chromatographic grade methanol, filter it through a 0.22 μM organic filter membrane into a sample injection bottle, and use HPLC to determine the residual amount of OHHL.

[0024] HPLC detection conditions of OHHL: HPLC instrument model: Shimadzu high performance liquid chromatography LC-2030. Chromatographic column is Elite C 18Reversed phase column (SupersilAQ-C18, 250nm×4.60mm, 5μm). Flow rate: 0.5mL / min. Column temperature: 30℃. Mobile phase: acetonitrile: water = 30:70 (v:v). Detection wavelength: 210nm. Injection volume: 20μL.

[0025] Quality control: The standard curve was prepared by calibrating the standard substance using the external standard method according to the conventional method ( Figure 6 ).

[0026] Degradation rate calculation method: OHHL degradation rate (%) = [1-(A1 / A0)] × 100%, A1 is the concentration of difenoconazole in the culture medium after strain GB-1 treatment for 48 h, and A0 is the initial concentration of OHHL in the culture medium.

[0027] HPLC test results are as follows Figure 7 As shown in the figure, the degradation rate of OHHL in strain GB-1 was about 94.5% after 48 h of culture, while the natural degradation rate of OHHL in the control was less than 10% within 48 h.

[0028] Example 5: Study on the biocontrol effect of strain GB-1 on soft rot.

[0029] In this example, Pectobacterium carrotis subspecies ( Pectobacterium carotovorum subsp. carotovora ) Pcc was used as an example to study the biocontrol effect of strain GB-1 on pathogenic bacteria that rely on AHLs to cause disease; 1. Use white radish and potato as host materials, clean them, disinfect their surfaces with hypochlorous acid (90 mg / L), and cut them into slices with a thickness of 0.4 cm; 2. The bacterial suspension OD of strain GB-1 and strain Pcc were respectively 600 Adjust to 1, set up three experimental groups: GB-1+LB (GB-1:LB volume ratio = 1:9), Pcc+LB (Pcc:LB = 1:9), Pcc+GB-1 (Pcc:GB-1 = 1:9), and inoculate the three experimental groups in the center of the treated host materials. The inoculation volume is 5 μL, and each treatment is repeated 3 times. After inoculation, the host materials are placed in a 28℃ incubator for 24 h; The experimental results are as follows Figure 8 and Fig. 9 As shown in the results, in the biocontrol experiment of potato and white radish, the disease area of ​​the experimental group Pcc + LB was larger than that of the experimental groups GB-1 + LB and Pcc + GB-1, and the disease degree was more serious, that is, the symptoms of soft rot disease were significantly alleviated when the OHHL-degrading bacteria GB-1 was co-inoculated with pathogenic bacteria compared with the inoculation of pathogenic bacteria alone. The experimental results showed that the strain GB-1 had a significant effect on the soft rot of Pectobacterium carrotii subsp. Pectobacterium carotovorum subsp. carotovora) It has significant biocontrol effects on potato soft rot and white radish soft rot caused by Pcc.

[0030] Through the above case studies, it was found that strain GB-1 has good degradation activity against quorum sensing signal molecules AHLs, and has great application potential in preventing and controlling the harm of pathogenic bacteria mediated by AHLs, which adds new content to environmentally friendly plant disease control methods.

Claims

1. A strain of Bacillus ( Bacillus sp.) GB-1, and its deposit number in Guangdong Microbiological Culture Collection Center is GDMCC NO:65601.

2. Use of the Bacillus GB-1 according to claim 1 in preventing and controlling plant pathogens.

3. The use according to claim 1, characterized in that: Control of plant pathogens by degrading the quorum sensing signal molecule OHHL.

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