CN113736696B - Acid bacterium delftia and application thereof in preventing and treating plant diseases - Google Patents
Acid bacterium delftia and application thereof in preventing and treating plant diseases Download PDFInfo
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Abstract
The invention discloses a Delftia tsuruensis and application thereof in preventing and treating plant diseases. In particular to a quenching bacterium, i.e. Delftia acidovorans HY-27 strain, which is deposited in the Guangdong province collection center for microorganism strains at 21/7 in 2021 and has the deposit number of GDMCC No.61820. The quenching strain can efficiently degrade quorum sensing signal molecules and utilize the quorum sensing signal molecules as a unique carbon source for growth, and has obvious and rapid degradation effect on OHL quorum sensing signal molecules. Therefore, quorum sensing signal molecules can be degraded to block the quorum sensing communication of the microbial population and inhibit the expression of pathogenic factors of pathogenic bacteria, so that the purpose of preventing and treating diseases is achieved, and the quorum sensing signal molecules have great application potential in the aspect of preventing and treating the harm of pathogenic bacteria which depend on AHLs mediated pathogenic bacteria. Can reduce the abuse problem of chemical pesticides and provides a new way and a new method for biological control of diseases.
Description
Technical Field
The invention belongs to the technical field of plant disease control. More particularly, relates to a acid bacterium delbrueckii and application thereof in preventing and treating plant diseases.
Background
Quorum Sensing (QS) is a system in which a microbial population undergoes a change in its physiological and biochemical properties during its growth due to an increase in population density, indicating a small number of bacterial cells or characteristics not possessed by a single bacterial cell. The quorum sensing system of bacteria is involved in the regulation of many biological functions, including the production of pathogenic agents from pathogenic bacteria of animals and plants. Acyl Homoserine Lactones (AHL) are key signal molecules for regulating quorum sensing systems, and include N- (3-oxohexanoyl) -L-homoserine lactone (N- (3-oxohexanoyl) -L-homoserine lactone, OHHL), N- (3-oxooctanoyl) -L-homoserine lactone (N- (3-oxooctanoyl) -L-homoserine lactone, OOHL), N- (3-oxodecanoyl) -L-homoserine lactone (N- (3-oxodecanoyl) -L-homoserine lactone, odDHL), isovaleryl-homoserine lactone (Isovaleryl-homoserine lactone), carboxylated acyl-homoserine lactones (carboxyl-AHL), aryl-homoserine lactone (aroyl-homoserine lactone) and coumaroyl-homoserine lactone (p-coumaroyl-hsyl-L), and the like. Recent studies have shown that different classes of Quorum-sensing enzymes (enzymes) capable of degrading AHL exist in different organisms, including bacteria and eukaryotes. Expression of AHL degrading enzyme in AHL dependent pathogenic bacteria and transgenic plant can inhibit the accumulation of QS signal molecule effectively, block pathogenic bacteria and raise plant resistance.
Depending on the signal molecules and sensing mechanisms synthesized by bacteria, QS systems can be divided into three representative types: gram-negative bacteria generally use Acyl Homoserine Lactone (AHL) molecules as signal factors, gram-positive bacteria generally use oligopeptide molecules (Al P) as signal factors, and many gram-negative and positive bacteria can produce a signal factor of furoylboronic acid diester (AI-2). While the way to interfere with Quorum sensing systems by inhibiting the mechanism of synthesis, accumulation, monitoring, or enzymatic degradation or modification of signal molecules is called Quorum Quenching (QQ).
Soft rot Erwinia sp is a common pathogenic bacterium in plants, and virulence factors secreted by the soft rot Erwinia sp are all influenced by quorum sensing, and agrobacterium tumefaciens-mediated quorum sensing causes transfer of Ti plasmids of plants, so that the plants die; on corn plants, maize wilt bacilli regulated by the EsaI/Esar QS system can produce a large amount of toxic capsular polysaccharide, which in turn leads to the development of microtubule cachexia and leaf blight etc. (crystal, zhang, etc.. Quorum sensing quenching and its application in biological control [ J ]. China science and technology Explorer 2015 (6): 399-399.). A large number of researches show that quorum sensing is an important mechanism causing host diseases, so in recent years, biological disease control is carried out by taking an interference quorum sensing system as a target, and the search for stable and efficient quenching and sterilization becomes a great hot point of the current researches. For example, patent document CN 109077066B discloses that an AHLs quenched bacteriophagus rhodococcus has significant degradation activity to quorum sensing signal molecules AHLs, which indicates that strains in nature have important effects on quorum quenching enzymes. Therefore, new AHLs quenching bacteria need to be researched, and more efficient biocontrol bacteria selection is provided for preventing and treating pathogenic bacteria depending on AHLs.
At present, the prevention and treatment measures adopted at home and abroad for agricultural diseases are mainly chemical prevention and treatment, however, a large amount of chemical pesticides are used, so that a series of serious problems such as environmental pollution, ecological balance destruction, pathogenic bacteria drug resistance, food safety and the like are caused, and in addition, the abuse of antibiotics also causes the generation of microbial drug resistance. Therefore, the search for new and effective prevention strategies is urgent. And the group quenching is a new disease control strategy, and a new idea is opened for developing novel green safe disease control measures in the future. In the current international research on the prevention and treatment technology of microbial diseases, the search for stable and efficient quenching and sterilizing is the leading edge and hot spot of the world.
Disclosure of Invention
The invention aims to solve the technical problem of researching a novel AHLs quenching bacterium, namely Delftia closterium, provides more efficient biocontrol bacterium choices for controlling pathogenic bacteria depending on AHLs and plays a role in controlling plant diseases.
The first purpose of the invention is to provide a quorum quenching bacterium acidovorax Delftia (Delftia acidovorans) HY-27 strain capable of degrading quorum sensing signal molecules AHLs with high efficiency.
The second purpose of the invention is to provide the application of the Delftia acidovorans HY-27 strain in degrading quorum sensing signal molecules AHLs or preventing pathogenic bacteria causing AHLs mediated diseases.
The third purpose of the invention is to provide a method for preventing and treating the disease of pathogenic bacteria depending on AHLs by using the colony quenching bacterium, namely acid bacterium Delftia acidovorans, and a degrading bacterium agent and a biocontrol bacterium agent.
The above purpose of the invention is realized by the following technical scheme:
the invention uses soil samples collected from river sources as microbial sources, and obtains the quenching bacteria of a degradable quorum sensing signal molecule OHL through manual screening, separation, purification and identification. Through morphological characteristics, physiological and biochemical characteristics and 16S rDNA phylogenetic analysis of the strain, the identification bacterium HY-27 is Delftia acidovorans (Delftia acidovorans) and is preserved in the microbial strain preservation center of Guangdong province in 21 months at 2021, the preservation number is GDMCC No.61820, and the preservation address is No. 59 building 5 of Michelia Torrens 100, guangzhou city.
The strain HY-27 is streaked on an LB solid culture medium and placed in a biochemical incubator at 28 ℃ for constant-temperature culture for 48 hours, observation and recording are carried out periodically, and morphological characteristics are observed. The strain HY-27 is in an LB solid plate, and has the advantages of large colony diameter, wetness, translucency, irregular edge and a little-point protrusion in the center. When the strain HY-27 grows in an LB liquid culture medium, the strain is diffusively turbid, the strain changes from milky white to yellowish along with the prolonging of culture time, and the strain grows well at the temperature of 28 ℃. The strain HY-27 is observed in a field emission scanning electron microscope image, and the somatic cells are in a short rod shape and have terminal flagella.
The physiological and biochemical characteristics of the strain HY-27 are as follows: gram-negative bacteria, aerobic bacteria, and negative reactions in a gelatin liquefaction test and a starch hydrolysis test; the oxidase test and the catalase test are positive, the optimal growth temperature is 30 ℃, and the optimal pH is 7.0.
The acid-eating bacterium Delftia acidovorans (Delftia acidovorans) strain HY-27 provided by the invention has a remarkable degradation effect on quorum sensing signal molecule OHL. After the strain HY-27 is cultured for 12h and 24h, sampling is carried out, the degradation rate of the strain HY-27 after 12h of OHL 12 is detected by HPLC to reach 21.6 percent, the degradation rate after 24h can reach 53.6 percent, and the OD of the corresponding strain HY-27 600 The values are 0.137 and 0.202, respectively. The results show that the degradation of OHL is in positive correlation with the growth of the strain HY-27, and the strain HY-27 can degrade the OHL as a unique carbon source and utilize the growth of the OHL as the unique carbon source. Meanwhile, the strain HY-27 has good application potential in the aspect of preventing and treating plant diseases mediated by a population signal molecule OHL.
Therefore, the invention provides an application of acid bacterium Delftia aciclor (Delftia acidovorans) in degrading quorum sensing signal molecules AHLs, controlling pathogenic bacteria causing AHLs mediated diseases or controlling plant diseases causing AHLs mediated diseases.
The invention also provides application of the Delftia aciclodans (Delftia aciclodans) in preparation of preparations for degrading quorum sensing signal molecules AHLs, preventing and treating pathogenic bacteria mediated by AHLs or preventing and treating plant diseases depending on AHLs mediated pathogenic diseases.
Preferably, the AHLs include not only conventional AHLs such as: n- (3-oxohexanoyl) -L-homoserine lactone (N- (3-oxohexanoyl) -L-homoserine lactone, OHL), N- (3-oxooctanoyl) -L-homoserine lactone (N- (3-oxooctanoyl) -L-homoserine lactone, OOHL), and N- (3-oxodecanoyl) -L-homoserine lactone (N- (3-oxodecanoyl) -L-homoserine lactone, odDHL), and also include some new specific AHLs, such as: isovaleryl-homoserine lactone (Isovaleryl-homoserine lactone), carboxylated acyl-homoserine lactones (carboxyl-AHLs), aryl-homoserine lactone (Aryl-homoserine lactone) and coumaroyl-homoserine lactone (p-coumaroyl-HSL).
The invention also provides application of the Delftia acidovorans HY-27 strain in degrading quorum sensing signal molecules AHLs, preventing and treating pathogenic bacteria mediated by AHLs or preventing and treating plant diseases mediated by AHLs.
The invention also provides application of the Delftia acidovorans HY-27 strain in preparation of preparations for degrading quorum sensing signal molecules AHLs, preventing and treating pathogenic bacteria mediated by AHLs or preventing and treating plant diseases depending on AHLs mediated pathogenic diseases.
The invention provides a method for preventing and treating pathogenic bacteria diseases depending on AHLs, which treats crops by Delftia acidovorans (Delftia acidovorans) or bacterial liquid thereof.
Preferably, the acid bacterium Delftia acidovus (Delftia acidovorans) is the acid bacterium Delftia acidovus HY-27 strain.
More preferably, the crops are inoculated or sprayed with the liquid of the acid bacterium Delftia HY-27.
The invention also provides a degrading bacterial agent capable of degrading the quorum sensing signal molecules AHLs.
Preferably, the degrading bacteria agent contains Acidovorax Delftia (Delftia acidiovarans) HY-27 strain or bacteria liquid thereof.
The invention also provides a biocontrol microbial inoculum for pathogenic bacteria depending on AHLs or plant diseases caused by the pathogenic bacteria.
Preferably, the pathogenic bacteria that are pathogenic in dependence on AHLs comprise dicamba (Dickeya), pectobacterium (peobacterium), and/or Pseudomonas aeruginosa.
More preferably, the biocontrol microbial inoculum contains a Delftia acidovorans HY-27 strain or a bacterial solution thereof.
The most suitable culture medium for preparing the bacterial liquid of the Delftia acivosa HY-27 strain is Luria-Bertani (LB) culture medium, and the formula is as follows: 10.0g/L of tryptone, 5.0g/L of yeast extract, 10.0g/L of sodium chloride, pH 6.8-7.2 and sterilization at 121 ℃ for 15-25 min.
The invention has the following beneficial effects:
the research of the invention discovers that acid-eating bacterium Delftia acidovorans (Delftia acidovorans) has better degradation activity for quorum sensing signal molecule OHL, has stable and obvious degradation effect, and provides a new way and method for biological prevention and treatment of AHLs mediated pathogenic bacteria.
The invention provides a Delftia acidovorans (Delftia acidovorans) HY-27 strain with obvious degradation activity on quorum sensing signal molecules OHL, which is an environment-friendly strain, can grow by using OHL as a unique carbon source, has stable strain activity, can reach 53.6% in degradation rate after 24 hours, and has effective and obvious biocontrol effect on potato soft rot and white radish soft rot by using the HY-27 strain. Has great application potential in the aspects of quenching population, preventing and treating pathogenic bacteria and plant diseases which depend on AHLs mediated diseases. The invention not only can reduce the use of chemical pesticides, but also provides a new way for gradually replacing chemical control with biological control.
Drawings
FIG. 1 is a colony morphology of strain HY-27 on LB medium.
FIG. 2 is a scanning electron micrograph of cells of the strain HY-27.
FIG. 3 is a phylogenetic tree analysis diagram of strain HY-27.
FIG. 4 is a graph showing the measurement of OHL degradation activity by the strain HY-27 (CK is a blank control without addition of quenching sterilization).
FIG. 5 is an HPLC chart of the strain HY-27 for OHL degradation.
FIG. 6 is the biological control effect of the strain HY-27 on potato bacterial soft rot.
FIG. 7 is a diagram showing the biological control effect of the strain HY-27 on bacterial soft rot of white radish.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
The media and reagents used in the following examples are as follows:
LB medium: tryptone (Tryptone) 10.0g; 5.0g of Yeast extract (Yeast extract); 10.0g of sodium chloride (NaCl); 1000mL of distilled water; the pH was 7.2.
Basal salts Medium (MSM) ammonium sulfate (NH) 4 ) 2 SO 4 2.0g; magnesium sulfate heptahydrate (MgSO) 4 7H2O 0.2g; calcium chloride dihydrate CaCl 2 ·2H 2 0.01g of O; ferrous sulfate heptahydrate FeSO 4 ·7H 2 0.001g of O; disodium hydrogen phosphate dodecahydrate Na 2 HPO 4 ·12H 2 O1.5 g; potassium dihydrogen phosphate KH 2 PO 4 1.5g; 1000mL of distilled water; the pH was 6.5.
Basic culture medium (MM) ammonium sulfate (NH) 4 ) 2 SO 4 2.0g; dipotassium hydrogen phosphate K 2 HPO 4 10.5g; magnesium sulfate heptahydrate (MgSO) 4 ·7H 2 0.2g of O; anhydrous calcium chloride CaCl 2 0.01g; ferrous sulfate FeSO 4 0.005g; manganese chloride MnCl 2 0.002g; potassium dihydrogen phosphate KH 2 PO 4 4.5g; 1000mL of distilled water; the pH was 6.5.
The above culture medium is sterilized in autoclave (121 deg.C, 20 min) before use. If the preparation of the corresponding solid medium is required: 1.5% (w/v) agar powder was added to the liquid medium.
N-acyl homoserine lactone signal molecule: n- (3-oxohexanoyl) -L-homoserine lactone (OHL, 3OC6 HSL) was purchased from Shanghai Yanghi Biotech Co., ltd. X-gal was purchased from Sigma reagents. Analytical grade ethyl acetate, chromatographic grade methanol, chromatographic grade acetonitrile, chromatographic grade propanol were purchased from Dingguo corporation.
The potato, the radish and other materials used in the experiment are purchased from the vegetable and fruit market of southern China agriculture university.
EXAMPLE 1 isolation and screening of Strain HY-27
1. Soil sample collection:
collecting a plurality of soil samples from a river source, taking back the samples, and storing the samples in an ice warehouse at 4 ℃ for refrigeration for later use.
2. Enrichment culture of the strain:
taking a soil sample collected from a river source as a microbial source, and taking OHL as a unique carbon source for enrichment culture. Sterilizing a 250mL triangular flask after 50mL basal salt medium (MSM), cooling, and adding OHL mother liquor (with acetonitrile as solvent and concentration of 100 mM) to a final concentration of 5 μ M; then 5g of soil samples were added. After culturing at 30 ℃ and 200rpm for 7 days, the cells were inoculated into 50mL MSM at a final OHL concentration of 10. Mu.M in an amount of 10%. After culturing for 7d under the same conditions, the cells were inoculated with 10% of the inoculum size and transferred to 50mL MSM with a final OHL concentration of 20. Mu.M, and then cultured for 7d under the same conditions. The above operation was repeated until the OHL concentration was increased to 160. Mu.M.
3. Separating and purifying the strain:
the separation and purification of the strain are carried out by dilution, plate coating and plate drawing.
And taking out 1mL of MSM culture medium fermentation liquor obtained in the last culture and carrying out gradient dilution. Taking 1mL of final MSM culture medium fermentation liquor, and diluting the final MSM culture medium fermentation liquor to 10 concentration gradient by using fresh MSM culture medium -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 Of (2)And (3) uniformly coating 100 mu L of fermentation liquid from fermentation liquids with different concentration gradients on an LB solid plate culture medium respectively, and culturing at 30 ℃ until a large number of bacterial colonies appear. Selecting a plate culture medium with proper colony density, selecting single colonies with different colony morphologies on the plate, streaking and purifying the single colonies on an LB solid plate culture medium for multiple generations until the single colonies are separated. After the purified single colony was cultured overnight in LB liquid medium at 30 ℃ and 200rpm, it was stored in a refrigerator at-80 ℃ by the glycerol stock keeping method, and its quenching activity against OHL was confirmed by further experiments.
4. Strain screening:
the degrading bacteria were screened using a reporter strain CF11 (Agrobacterium tumefaciens).
Taking out the strain to be screened from a refrigerator at the temperature of-80 ℃, marking on an LB solid culture medium flat plate for activation, and culturing for 24 hours in an incubator at the temperature of 30 ℃. And (3) selecting a single colony, inoculating the single colony to an LB liquid culture medium, and culturing overnight at the temperature of 30 ℃ and the speed of 200rpm to obtain a bacterial liquid. And activating the strain to be screened by using an LB solid culture medium and preparing a bacterial liquid. 1 OD 600 The cells were inoculated into 1mL of MSM medium containing OHL as the sole carbon source at a concentration of 40. Mu. Mol/L. After 1mL of the reaction mixture was obtained, it was transferred to a 2mL centrifuge tube and incubated at 30 ℃ at 200 r/min. After 24h, 5. Mu.L of the mixture was spotted onto the top of MM agar strips (pH 6.5), followed by spotting a row (about 13-18 spots) of the bacterial solution of the reporter strain CF-11 directly below the mixture. The treated MM agar strips are placed in an incubator at 28 ℃ and are incubated for 24 hours in the dark, and the result is observed.
As a result, when the reporter strain detects the presence of OHL in the environment, the beta-galactosidase gene begins to express and release beta-galactosidase to the environment. The colorless compound X-gal (5-bromo-4-chloro-3-indole-beta-D-galactoside) in MM agar strips can be enzymolyzed by beta-galactosidase to galactose and a dark blue substance 5-bromo-4-indigo, and the colonies of the whole reporter strain can be changed into blue by 5-bromo-4-indigo. OHL belongs to a small molecular substance, can diffuse in the agar strip, and the diffusion distance is in direct proportion to the concentration. Therefore, the content of OHL in the sample can be judged according to the distance from the top of the reported strain to turn blue on the agar strip.
The results show that when the reaction mixture at the top of the MM agar strips contains OHHL, the lower reporter strain turns blue, the longer the reporter strain turns blue the more the reaction mixture content is, whereas, when no AHLs are contained in the reaction mixture, the lower reporter strain does not turn blue. According to the principle, a strain which can stably and efficiently degrade OHL is obtained by screening and is named as HY-27.
EXAMPLE 2 identification of Strain HY-27
This example carried out morphological identification, 16S rDNA phylogenetic analysis and physiological and biochemical identification for quenched HY-27, and identified the strain as acid bacterium Delftia acidovorans. The method comprises the following specific steps:
1. and (3) colony morphology characteristics: the quenching strain HY-27 colony has the advantages of large colony diameter, wetness, translucency, irregular edge and a little-point protrusion in the center in an LB solid plate. As shown in fig. 1; when the strain grows in an LB liquid culture medium, the strain is diffusively turbid, the strain turns to yellowish from milky white along with the prolonging of the culture time, and the strain grows well at the temperature of 28 ℃.
2. Morphological characteristics of the thallus: as shown in FIG. 2, the bacterial cells were short rod-shaped and had terminal flagella.
3. 16S rDNA identification and phylogenetic analysis: the genome of the strain HY-27 was used as a template, and the bacterial 16S rDNA universal primer was used for PCR amplification, and the product after PCR purification was trusteed to Weiying Jie based (Shanghai) trade Limited for sequencing. The length of the 16S rDNA sequence of the strain HY-27 is 1438bp. The sequence obtained by sequencing by logging in the NCBI database was subjected to similarity alignment using BLAST, and the result showed that the similarity of the 16S rDNA sequence of the strain HY-27 and the acid bacterium Delftia acidovorans (Delftia acidovorans) was 99% as shown in FIG. 3.
In conclusion, the strain HY-27 is identified as Delftia acivosa (Delftia acidovarans), and is deposited in 21.7.2021 in Guangdong province of microbial cultures collection center with the deposit number of GDMCC No.61820 and the deposit address of No. 59 building 5 of Michelia Tokoro 100, guangzhou City.
Example 3 detection of OHL degrading Activity by Delftia acidovorans HY-27
This example uses a reporter strain (Agrobacterium tumefaciens) CF11 to examine the effect of strain HY-27 on OHL degradation.
The strain HY-27 is activated by an LB solid medium plate, and the plate is placed in an incubator at 28 ℃ for 48h. A single colony was picked and inoculated into a liquid LB medium, and cultured overnight at 28 ℃ and 200rpm to obtain a bacterial solution. Obtaining OD 6oo HY-27 cells with a value of 1.0 were inoculated into 1mL of MSM mineral salt medium containing only OHL as a sole carbon source. The concentration of OHL in the reaction system was 40. Mu. Mol/L. The reaction mixture is incubated under suitable conditions. Then the reaction mixture is sampled with the bacterial solution of the reporter strain Agrobacterium tumefaciens CF 11.
Meanwhile, bacillus thuringiensis subsp. israelensis B23 known to quench ohl was used as a positive control, and Escherichia coli (Escherichia coli) not having activity of quenching ohl was used as a negative control. The petri dish, on which the reaction mixture and the reporter strain had been spotted, was then placed in a 28 ℃ incubator in the dark, and after 24h of incubation, the experimental results were observed and photographed.
The results of the experiment are shown in fig. 4, where the diffusion distance on the OHHL agar strip is proportional to its concentration. Therefore, the content of OHL in the sample can be predicted according to the distance of the agar strip reporting the bacterial strain to turn blue from the top. In fig. 4, CK and negative control e.coli have the same OHHL diffusion distance, i.e. the OHHL content in the reaction mixture is similar; the positive control B-23 showed substantially no bluing with the reporter strain on the HY-27 agar strip of the experimental group, indicating that OHL had been degraded. Indicating that the strain HY-27 has quenching activity as well as B23.
Example 4 detection of Effect of Strain HY-27 on OHL degradation by HPLC
The AHLs signal used in this implementation is OHHL, and by referring to relevant references, the following table 1 is used to determine the more accurate and stable High Performance Liquid Chromatography (HPLC) conditions for OHHL:
TABLE 1 OHL High Performance Liquid Chromatography (HPLC) conditions
| HPLC | Waters e2695 |
| Chromatographic column | Kinetex EVO C18 reverse phase chromatography column (250. Mu. M.times.4.6 mm. Times.5 μm) |
| Column temperature | 30℃ |
| Mobile phase | Methanol: water = 30: 70 (v: v) |
| Flow rate of flow | 0.5mL·min -1 |
| Detecting wavelength | 210nm |
| Sample size | 20μL |
The OHL signal molecules are subjected to HPLC analysis under the conditions, the signal molecules have stable peaks, sharp peak types and proper peak retention time, and are about 6 min.
Activating the strain HY-27 frozen at-80 ℃ by using an LB solid plate, culturing at 30 ℃ for 48h, selecting a single colony on the plate, inoculating the single colony on a liquid LB culture medium, and culturing overnight at 30 ℃ and 200rpm to obtain a bacterial liquid. Centrifuging HY-27 bacteria liquid with a certain volume (V =1/OD 600) at 4000rpm for 10min, discarding the supernatant to obtain OD 600 Inoculating thallus to 20M1 MSM liquid culture containing OHLIn the medium (final concentration 0.2 mM), the culture was incubated at 30 ℃ and 200rpm and the samples were taken periodically. Determination of OD of mixture samples sampled at different times 600 The growth of the strain is shown, and the degradation of the strain to the signal molecule is shown by measuring the residual quantity of the OHL signal molecule by using HPLC.
Samples were taken after 12h and 24h of culture. HPLC detection results are shown in FIG. 5, in which the strain HY-27 has a degradation rate of 21.6% after OHHL 12h, and has a degradation rate of 53.6% after 24h, and the OD of the corresponding strain HY-27 600 The values are 0.137 and 0.202, respectively. Experiments preliminarily show that the strain HY-27 can degrade OHL as a unique carbon source and utilize the growth of the OHL as the unique carbon source.
Example 5 biocontrol Effect of Strain HY-27 on OHL disease-dependent plant Soft rot
In this example, the biocontrol effect of strain HY-27 on pathogenic bacteria dependent on OHL was studied by taking plant pathogenic bacteria, pectiobacter carotovorum subsp.
The strains HY-27, Z3-3, E.coli and B23 were activated on LB solid medium plates and cultured at 30 ℃. After 48h, single colonies on the plates were picked, inoculated into liquid LB medium, and cultured overnight at 30 ℃ and 200rpm to OD 600 About 1.5. Among them, bacillus thuringiensis subsp. israelensis B23 is a strain known to have a degrading effect on OHHL (Dong Y, xu J, li X, ethyl. AiiA, an enzyme that activates the amino-carbonyl-amino-lactone, a quantitative-sensing signal and a specific of the virus of Erwinia carotovora [ J]Proceedings of the National Academy of Sciences,2000,97 (7): 3526-3531), E.coli is a known strain not having OHL degradation. In a biocontrol experiment, four experimental groups will be set up: z3-3+ LB, Z3-3+ E.coli, Z3-3+ B23 and Z3-3+ HY-27. Wherein, the experimental group Z3-3+ LB is a blank control group, Z3-3+ E. Coli is a negative control group, and Z3-3+ B23 is a positive control group.
Fresh potatoes and white radishes are selected as experimental materials respectively, and the materials are washed with distilled water. The potatoes and radishes were cut into slices having a thickness of about 0.3 cm. The bacterial suspension Z3-3 was mixed with bacterial suspensions B23, E.coli and HY-27 and liquid LB medium at a ratio of 1.5 mu L of the mixed bacterial liquid of each experimental group is respectively inoculated to the center of the treated experimental material. Adding sterile wet cotton for moisturizing, sealing with a preservative film, placing in an incubator at 28 ℃ for culturing, and observing the result after 36 hours.
The results are shown in fig. 6 and fig. 7, in the biocontrol experiments of potatoes and white radishes, the disease degree of the potatoes and white radishes in the experimental group Z3-3+ LB and Z3-3+ E. Coli is serious, and the disease spot area is larger than that of the potatoes and white radishes in the experimental group Z3-3+ B23 and Z3-3+ HY-27. Namely, the symptom of the soft rot disease is obviously reduced when the HY-27 and the pathogenic bacteria are inoculated together compared with the symptom when the pathogenic bacteria are inoculated independently. Experimental results show that the quenching strain HY-27 has a relatively obvious biological control effect on soft rot caused by Z3-3.
The experimental result shows that the strain HY-27 has effective and obvious biological control effect on potato soft rot and white radish soft rot caused by the Pectobacterium carotovorum subsp.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (6)
1. Acid bacterium dalfot specially for degrading quorum sensing signal molecules AHLsDelftia acidovorans) HY-27 strain is deposited in Guangdong province culture Collection of microorganisms at 21 st 7/2021 with the strain preservation number of GDMCC No.61820.
2. The acid bacterium delftia as claimed in claim 1 (a)Delftia acidovorans) The HY-27 strain is applied to degrading quorum sensing signal molecules AHLs, preventing pathogenic bacteria mediated by the AHLs or preventing plant diseases depending on AHLs mediated pathogenesis.
3. The acid bacterium delftia as claimed in claim 1 (a)Delftia acidovorans) The HY-27 strain is used in preparation of preparations for degrading quorum sensing signal molecules AHLs, preventing and treating pathogenic bacteria mediated by AHLs, or preventing and treating plant diseases dependent on AHLs mediated pathogenic diseases.
4. A method for preventing and treating AHLs-dependent pathogenic bacteria, which comprises treating crops with the Acidovorax defei HY-27 strain or its bacterial liquid as claimed in claim 1.
5. A degrading bacterial agent capable of degrading quorum sensing signal molecules AHLs, which is characterized by comprising the acidovorax delbrueckii HY-27 strain or a bacterial solution thereof as claimed in claim 1.
6. A biocontrol microbial inoculum for pathogenic bacteria causing AHLs or plant diseases caused by the pathogenic bacteria, which is characterized by comprising the acidovorax defueli HY-27 strain as claimed in claim 1 or a bacterial solution thereof.
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| CN108102991A (en) * | 2018-03-07 | 2018-06-01 | 山东戴盟得生物科技有限公司 | One plant of Bei Laisi bacillus and its application in watermelon blight is prevented |
| CN112646753A (en) * | 2021-01-21 | 2021-04-13 | 华南农业大学 | Klebsiella aerogenes and application thereof |
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