Method for rapidly screening biocontrol bacteria of citrus huanglongbing and application of biocontrol bacteria

Abstract

The invention relates to the technical field of plant disease prevention and control, in particular to a method for rapidly screening biocontrol bacteria of Candidatus Liberobacter asiaticum and application of the method. The rapid screening method comprises the following steps: mixing an Lcr-GFP bacterial suspension and a to-be-screened strain CFS according to a volume ratio of 1: 1-17: 3, incubating for more than 3 h, detecting fluorescence intensity when the excitation wavelength is 474-490 nm and the emission wavelength is 533-549 nm, and screening the biocontrol bacteria with the fluorescence quenching rate being greater than or equal to 10%. According to the present invention, the screened bacterial strain Paenibacillus edorifer has the inhibition effect on Lcr-GFP, and has the inhibition effect on Clas colonization in the in vivo detection of HLB infected plants, such that the Lcr-GFP inhibition effect can be provided, and the Lcr-GFP inhibition effect can be provided; therefore, the method for rapidly screening the biocontrol bacteria can effectively identify and screen the biocontrol bacteria with remarkable inhibitory activity on Clas, and has the biocontrol potential of HLB prevention and control.

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease prevention and control, and particularly relates to a method for rapidly screening citrus Huanglongbing biocontrol bacteria and an application thereof. Background Art

[0002] Citrus Huanglongbing (HLB) is a Candidatus Plant diseases caused by Liberibacter spp. seriously threaten the sustainable development of the global citrus industry. Candidatus Liberibacter asiaticus (Clas) is currently the most serious pathogenic strain.

[0003] Biological control refers to the use of one organism to fight against another organism in order to achieve the purpose of preventing and controlling diseases. It has the characteristics of high efficiency, safety, and environmental friendliness, and has attracted the attention of researchers at home and abroad. Biocontrol bacteria can effectively protect plants from disease invasion by competing, inhibiting or killing pathogenic microorganisms. Because of its high efficiency, safety and environmental friendliness, it has become an important means of biological control. The current methods for screening biocontrol bacteria include the inhibition zone method and the high-throughput screening method based on fluorescent labeling. Both methods rely on the pure culture of pathogens. Although studies have shown that in the presence of auxiliary bacteria, Stenotrophomonas maltophilia Clas can grow rapidly in LG medium under FLMAT-1 co-culture conditions. After the culture medium was fed to psyllids, Clas was successfully inoculated back into citrus plants and induced HLB symptoms. However, the titer of Clas was very low (Ct>33.0), and pure culture of Clas has not been possible to date. Therefore, screening for Clas biocontrol agents requires a target strain that can replace Clas.

[0004] Liberbacter crescens BT-1 ( Lcr ) is the only member of the genus Liberibacter that can be cultured in pure culture. Its genome sequence is highly homologous to Clas and it is a key model organism for pathogenic Liberibacter species. Lcr The main screening methods for Bacteria are the inhibition zone method and the live-dead cell staining method. Lcr Compared to common strains, the strain's growth cycle is longer, making it difficult to form dense, dense colonies. Furthermore, the strain's activity is easily affected by external factors. These two methods have low success rates, hindering efficacy evaluation. Therefore, developing efficient and rapid screening methods for HLB biocontrol agents is particularly urgent. Summary of the Invention

[0005] In view of the above technical problems, the present application provides a method for rapidly screening citrus Huanglongbing biocontrol bacteria and application thereof, which can effectively identify and screen biocontrol bacteria with significant antibacterial activity against Clas.

[0006] The technical solution of the present application is as follows: The present application provides a method for rapidly screening citrus Huanglongbing biocontrol bacteria, which comprises the following steps: Lcr The GFP bacterial suspension is mixed with the to-be-screened bacterial strain CFS at a volume ratio of 1:1-17:3 for incubation for more than 3 h, and the fluorescence intensity is detected at an excitation wavelength of 474-490 nm and an emission wavelength of 533-549 nm, so as to screen the biocontrol bacteria with a fluorescence inhibition rate of ≥10%. Lcr The solvent of the GFP bacterial suspension is PBS buffer or TBS buffer.

[0007] As an embodiment, the calculation method of the fluorescence inhibition rate is: inhibition rate = (blank group fluorescence value - sample group fluorescence value) / blank group fluorescence value x 100%.

[0008] As an embodiment, the biocontrol bacteria with an inhibition rate of ≥10% are screened.

[0009] As an embodiment, the Lcr The solvent of the GFP bacterial suspension is TBS buffer.

[0010] As an embodiment, the Lcr The GFP bacterial suspension is prepared by diluting the initial bacterial solution with OD 600 1.0 by 10-100 times.

[0011] As an embodiment, the volume ratio is 1:1-3:1.

[0012] As an embodiment, the excitation wavelength is 488 nm, and the emission wavelength is 535 nm.

[0013] As an embodiment, the to-be-screened bacterial strain is cultured and fermented at 26-30℃, and the supernatant is collected by centrifugation to obtain the to-be-screened bacterial strain CFS.

[0014] As an embodiment, the culture is in NB culture medium.

[0015] As an embodiment, the centrifugation temperature is 2-8℃, and the centrifugation speed is 8000 g-12000 g.

[0016] The present application also provides the application of the biocontrol bacteria obtained by the method in the prevention and control of citrus Huanglongbing plants.

[0017] Compared with the prior art, the present application has the following beneficial effects: The present application is labeled by GFP Liberbacter crecens BT-1 strain ( Liberbacter crecens BT-1 / pBBR1-S-GFP, Lcr -GFP) suspension of bacteria to construct a rapid screening method of biocontrol bacteria of pathogenic strain Clas, in the strain screened by the rapid screening method of the present application, the strain Paenibacillus odorifer shows inhibition effect on Lcr -GFP, and the inhibition effect is significantly higher than that of various antibiotics such as tetracycline, streptomycin sulfate, and spectinomycin. Moreover, the present application further verifies that the screened strain Paenibacillus odorifer has an inhibitory effect on the accumulation of Clas in the leaves of HLB plants. The results show that the rapid screening method of biocontrol bacteria of the present application can effectively identify and screen biocontrol bacteria with significant antibacterial activity against Clas. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 For Liberbacter crescens BT-1 fluorescent labeling, wherein A is Liberbacter crescens BT-1 wild type strain ( Lcr ), B is Liberbacter crescens BT-1-GFP strain ( Lcr -GFP), and C is the pBBR1-S-GFP vector map; Figure 2 For Lcr -GFP bacterial suspension preparation of the best solvent; Figure 3 For detecting Lcr -GFP bacterial suspension excitation wavelength / emission wavelength, wherein A is the excitation wavelength, and B is the emission wavelength; Figure 4 For screening biocontrol bacteria system optimization results, wherein A: 100 μL Lcr -GFP and 100 μL CFS; B: 125 μL Lcr -GFP and 75 μL CFS; C: 150 μL Lcr -GFP and 50 μL CFS; D: 170 μL Lcr -GFP and 30 μL CFS; E: 180 μL Lcr -GFP and 20 μL CFS; Figure 5 For detection results of inhibition rate of the strain to be screened; Figure 6 For treatment effect of biocontrol bacteria Paenibacillus odorifer on HLB plants, A is a leaf sampling schematic diagram, and B is a qPCR detection result; Figure 7 For Lcr -GFP biocontrol bacteria screening flowchart; Figure 8 Flow chart of the biocontrol bacteria validation experiment. DETAILED DESCRIPTION

[0019] The present invention provides a method for rapidly screening citrus Huanglongbing biocontrol bacteria. The method comprises the following steps: Lcr -GFP bacterial suspension was mixed with the strain CFS to be screened at a volume ratio of 1:1-17:3 and incubated for more than 3 h. The fluorescence intensity was detected at an excitation wavelength of 474-490 nm and an emission wavelength of 533-549 nm. The biocontrol bacteria were screened based on a fluorescence inhibition rate ≥10%.

[0020] In the present invention, based on Lcr The genome sequence is highly homologous to Clas, and the present invention constructs GFP fluorescent marker Liberbacter crecens BT-1 strain ( Liberbacter crecens BT-1 / pBBR1-S-GFP, Lcr -GFP), Lcr -GFP is kanamycin-resistant and green fluorescence can be clearly observed under a handheld UV lamp with excitation / emission wavelengths of 440 / 550 nm (Luyor-3415RG, Shanghai, China) Lcr The present invention will Lcr -GFP resuspension preparation Lcr -GFP bacterial suspension. As an embodiment, 5-6 weeks of growth and scattered distribution of GFP bacteria were collected from kanamycin-resistant BM7 agar medium. Lcr -GFP colonies were placed in sterile centrifuge tubes and resuspended in solvent to make Lcr -GFP bacterial suspension. Different solvents will affect Lcr -GFP activity, in the present invention, the Lcr -The solvent of GFP bacterial suspension is PBS buffer or TBS buffer, ensuring Lcr -GFP bacterial suspension has little fluorescence quenching within 5 hours; TBS buffer is preferred. Lcr OD of GFP bacterial suspension 600 OD 600 The initial bacterial solution of 1.0 is diluted 10 to 100 times, and the dilution multiple is preferably 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times or 90 times. Lcr-GFP bacterial suspensions are susceptible to external factors, causing fluorescence quenching. In the present invention, the excitation wavelength is selected to be 474-490 nm and the emission wavelength is selected to be 533-549 nm for fluorescence intensity detection. When the excitation wavelength is higher than 490 nm and the emission wavelength is lower than 533 nm, the fluorescence intensity is prone to jump during detection and even exceeds the maximum reading range of the instrument. When the excitation wavelength is lower than 474 nm and the emission wavelength is higher than 549 nm, the fluorescence intensity reading is lower during detection. Preferably, the excitation wavelength is 488 nm and the emission wavelength is 535 nm.

[0021] In the present invention, strains to be screened are selected from -80°C and cultured and fermented at 26-30°C to activate and propagate them, inducing the production of antimicrobial substances during the culture process. NB medium is a basic universal medium that can be used to cultivate a variety of heterotrophic bacteria. To accommodate the cultivation of a variety of strains and to minimize the impact of additional components on subsequent fluorescence quenching, the strains to be screened are cultured using NB medium. After fermentation, the fermentation broth is centrifuged to collect the supernatant (CFS) of the strains to be screened, containing various antimicrobial substances. In the present invention, the centrifugation is performed at 2-8°C, 8000-12000 g for 10-30 min; in one embodiment, the centrifugation is performed at 4°C, 10,000 g for 15 min.

[0022] In the present invention, the Lcr -GFP bacterial suspension is mixed and incubated with the strain CFS to be screened for 3 hours, and the fluorescence intensity is detected at the excitation wavelength and emission wavelength, and the biocontrol bacteria are screened by the fluorescence inhibition rate. In one embodiment, the strain to be screened with an inhibition rate ≥10% is the biocontrol bacteria. In the present invention, the inhibition rate can be obtained according to the degree of fluorescence quenching, and the inhibition rate calculation method is: inhibition rate = (blank group fluorescence value-sample group fluorescence value) / blank group fluorescence value × 100%. The degree of fluorescence quenching is related to the content of effective ingredients in the strain metabolites, and the NB culture medium used to culture the strain to be tested will also have spontaneous fluorescence within a specific wavelength range, affecting the accuracy and stability of the test results. Therefore, a sufficient amount of fluorescent bacteria is required in the mixed incubation to facilitate reading of higher fluorescence values. In the present invention, multiple Lcr -The volume ratio of GFP bacterial suspension to CFS of the strain to be screened, the accuracy and stability of the test results are evaluated according to the inhibition rate, Lcr - The mixed incubation of the GFP bacterial suspension and the CFS of the strain to be screened at a volume ratio of 1:1-17:3 is conducive to the accurate detection of subsequent fluorescence quenching; the preferred volume ratio is 1:1-3:1, and more preferably 3:1.

[0023] The present invention also provides a method for rapidly screening citrus Huanglongbing biocontrol bacteria, wherein the biocontrol bacteria obtained is used for preventing and controlling citrus Huanglongbing plants.

[0024] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available. If specific conditions of use are not specified, they are usually carried out under conventional conditions or under conditions recommended by the company.

[0026] Example 1 Construction and optimization of biocontrol bacteria screening system 1. Fluorescently labeled Liberbacter crecens BT-1 strain ( Lcr -GFP) because Lcr The activity of α-terminal β ... Lcr The transformation method was modified as follows: 5-6 week-old cells were collected from BM7 agar medium (13.7 mM α-ketoglutaric acid, 55 mM N-carbamoylmethylethanesulfonic acid, 67 mM potassium hydroxide, 15% fetal bovine serum, 30% modified Grace's insect cell medium, 1.5% (wt / vol) agar powder, pH 6.8). Lcr Transfer the colony (indicated by A in Error! Reference source not found.) to a 1.5 mL sterile centrifuge tube, add 800 μL of 0.5% xanthan gum (wt / vol) or 10% glycerol (vol / vol) to resuspend and wash the bacteria, centrifuge at 12,000 rpm for 3 min at room temperature, discard the supernatant, repeat the wash once, and add an appropriate amount of sterile water to resuspend the bacteria (OD 600=0.5~0.8) are competent cells; 200~400 μL of competent cells were taken into a pre-chilled 2 mm electroporation cuvette (Bio-Rad, Catalog No.165-2086, USA), and 2 μg of plasmid DNA (pBBR1-S-GFP, carrying a kanamycin resistance selection marker, constructed by a third party company) was added and mixed. After ice bathing for 20 min, electroporation was performed using an electroporation system (Bio-Rad Gene Pulser Xcell™, Serial No.617BR12551, USA) with the parameters set to 2.6 kV, 25 μF, and 200 Ω. Immediately after the electroporation, 1 mL of BM7 liquid medium was added to the electroporation cuvette and the entire volume was transferred to a 15 mL centrifuge tube. BM7 liquid medium was continued to be added to the final volume of 5 mL. After recovery at 28°C and 150 rpm for 16 h, 100-200 μL of the bacterial liquid was spread on the BM7 resistance (50 mg / L kanamycin) culture dish and cultured at 28°C. After 15 days, green fluorescent colonies were observed under a fluorescence microscope. At the same time, the fluorescent colonies were sequenced and identified to confirm that the fluorescent-labeled strain was Lcr strain. Successfully constructed GFP marker Liberbacter crecens BT-1 strain ( Liberbacter crecens BT-1 / pBBR1-S-GFP, Lcr- GFP) is kanamycin-resistant, and green fluorescence can be clearly observed under a handheld UV lamp with an excitation wavelength / emission wavelength of 440 / 500 nm (Luyor-3415RG, Shanghai, China). Lcr Colonies, such as Figure 1 shown.

[0027] 2. Lcr Preparation of -GFP bacterial suspension Lcr The activity of -GFP may be affected by the solvent. Lcr -GFP bacterial suspension. Collect 5-6 weeks of growth from kanamycin-resistant BM7 agar medium. Lcr -GFP colonies were transferred to a 1.5 mL sterile centrifuge tube, and 1 mL of solvent was added to resuspend and wash the bacteria. Centrifuge at 7000 g for 10 min at 22°C and discard the supernatant. Repeat the wash three times and add appropriate amount of solvent to adjust the concentration to OD 600=1.0, the bacteria suspension was prepared again by diluting 100 times with solvent. The solvents for screening were as follows: 0.85% NaCl, 0.9% NaCl, 1xPBS (8.006 g / L NaCl, 0.201 g / L KCl, 0.272 g / L KH2PO4, 2.865 g / L Na2HPO4·12H2O, pH 7.2~7.4), 1xTBS (2.4 g / L Tris, 8.8 g / L NaCl, pH 7.0), 10 mM MgCl2, sterile water, and all were sterilized before use. The experimental results are shown in Table 1. Figure 2 Lcr -GFP strain has strong and stable fluorescence in 1xPBS and 1xTBS buffers, and the fluorescence quenching is less within 5 hours, indicating that the two buffers have little effect on the activity of the strain, especially 1xTBS buffer, so it is determined to prepare Lcr- GFP bacteria suspension with 1xPBS or 1xTBS as the solvent.

[0028] 3. Lcr -Detection wavelength of GFP bacteria suspension Take Lcr GFP bacteria suspension in 96-well black enzyme plate, 200 μL / well, a total of 3 counting wells. Use full-wavelength multifunctional enzyme label instrument to detect fluorescence intensity to determine the best detection wavelength. Wavelength setting: excitation wavelength Ex 430~560 nm; emission wavelength Em 451~601 nm. The results are shown in Table 2. Figure 3 Lcr -GFP has the maximum fluorescence intensity at 488 nm / 512 nm (excitation / emission) wavelength. Since Lcr -GFP strain is susceptible to external factors and causes fluorescence quenching phenomenon, the fluorescence intensity change at 512 nm emission wavelength is extremely significant, in order to avoid large errors in the detection results, the final determination of the excitation wavelength is 474-490 nm, the emission wavelength is 533-549 nm to detect the fluorescence intensity, the best excitation wavelength is 488 nm, and the emission wavelength is 535 nm.

[0029] 4. Culture and fermentation of strains to be screened Take a small amount of glycerol bacteria to be screened from -80℃ and streak on NB solid medium, and incubate at 28℃. The next day, pick single colonies in 1 mL NB liquid medium and incubate at 28℃, 200 rpm for 16 h to obtain seed liquid. Inoculate the seed liquid into 10 mL NB liquid medium at 10% inoculation amount, and incubate at 28℃, 200 rpm for 48 h to induce the production of antibacterial substances. Centrifuge at 10000g at 4℃ for 15 min to collect the cell-free supernatant of the strain to be screened (CFS).

[0030] ​​5. Lcr -Optimization of CFS screening system for GFP bacterial suspension and strains to be screened Made with 1× TBS buffer Lcr -GFP bacterial suspension maintained high activity within 5 hours. In order to clarify the difference between the strain supernatant (CFS) and Lcr The optimal ratio of -GFP bacterial suspension incubation is Paenibacillus odorifer The strain was used as an example to optimize the biocontrol bacteria screening system. Paenibacillus odorifer strain CFS and Lcr -GFP bacterial suspension ratio experimental group, A: 100 μL Lcr -GFP and 100 μL CFS; B: 125 μL Lcr -GFP and 75 μL CFS; C: 150 μL Lcr -GFP and 50 μL CFS; D: 170 μL Lcr -GFP and 30 μL CFS; E: 180 μL Lcr -GFP and 20 μL CFS, set up the corresponding blank group. Detect the fluorescence intensity at 488 nm / 535 nm wavelength, detect once every 15 min, and continue to detect for 8 hours. Finally, calculate the ratio of each group. Paenibacillus odorifer Strain CFS pair Lcr The inhibition rate of -GFP was calculated as follows: inhibition rate = (fluorescence value of blank group - fluorescence value of sample group) / fluorescence value of blank group × 100%.

[0031] The experimental results are as follows Figure 4 As shown, 5 groups of different proportions in the present invention Paenibacillus odorifer The inhibition rates of the strains were all greater than 15%. The results showed that Lcr -GFP bacterial suspension and the CFS volume ratio of the strain to be screened is 1:1-17:3, which can accurately screen the biocontrol bacteria. Lcr -GFP bacterial suspension system, Paenibacillus odorifer Strain CFS pair Lcr -GFP had the most significant inhibition rate, so the best biocontrol bacteria screening system was determined to be Lcr The ratio of -GFP bacterial suspension to CFS of the strain to be screened was 3:1, and the total volume was 200 μL.

[0032] Test Example 1 Screening of biocontrol bacteria According to the screening system optimized by biocontrol bacteria in Example 1, the collected strains CFS to be screened were mixed with LcrThe GFP bacterial suspension (solvent is TBS buffer) is mixed uniformly at a ratio of 3:1, and the fluorescence intensity is detected immediately using a full-wavelength multifunctional microplate reader at 488 nm / 535 nm wavelength. Figure 7 The strain numbers and species categories of the strains to be screened are shown in Table 1.

[0033] Table 1 Strain numbers and species categories of strains to be screened

[0034] The substances, modes of action and action times of CFS of different strains may be different. In order to determine the time at which the biocontrol bacteria fully exert the inhibitory activity, detection is performed immediately after mixing, detection is performed every 2 min in the first stage, and detection is performed every 15 min in the second stage, and detection is performed for 5.5 hours. After detection, the inhibitory effect of the biocontrol bacteria is calculated according to the fluorescence quenching degree, and the formula is as follows: inhibition rate = (fluorescence value of blank group - fluorescence value of experimental group) / fluorescence value of blank group x 100%.

[0035] The experimental results are shown in Table 2. Figure 5 Most of the CFS of the strains tend to be stable after being incubated with GFP for 3 hours, so the incubation time is set to 3 hours. In addition, among the screened strains to be detected, only Lcr Paenibacillus odorifer Lcr shows an inhibitory effect on GFP. The results show that the method for rapidly screening biocontrol bacteria can screen strains with an inhibitory effect on GFP. Lcr Paenibacillus odorifer , and the inhibitory effect is significantly higher than that of many antibiotics such as tetracycline (Tet, 12.5 mg / L), streptomycin sulfate (Strep, 100 mg / L) and spectinomycin (Spec, 15 mg / L).

[0036] Test Example 2 The same as Test Example 1, except that the collected CFS of the strains to be screened is mixed uniformly with the GFP bacterial suspension (solvent is TBS buffer) at a ratio of 1:1. Lcr

[0037] The experimental results show that, among the screened strains to be detected, only Paenibacillus odorifer Lcr shows an inhibitory effect on GFP. The results show that the method for rapidly screening biocontrol bacteria can screen strains with an inhibitory effect on GFP. Lcr Paenibacillus odorifer , and the inhibitory effect is significantly higher than that of many antibiotics such as tetracycline, streptomycin sulfate and spectinomycin.​​​​​​

[0038] Test Example 3 Same as Experiment 1, except that the collected strains to be screened CFS were mixed with Lcr -GFP bacterial suspension (solvent is PBS buffer) was mixed evenly at a ratio of 3:1.

[0039] The experimental results showed that among the screened strains tested, only Paenibacillus odorifer Show Lcr -GFP inhibition. The results show that the method of rapid screening of biocontrol bacteria of the present invention can screen out Lcr -GFP suppressive strains Paenibacillus odorifer , and its inhibitory effect is more significant than that of tetracycline, streptomycin sulfate, spectinomycin and other antibiotics.

[0040] Test Example 4: Verification of biocontrol bacteria Further verification Paenibacillus odorifer The therapeutic effect of the strain on HLB plants, using the biocontrol bacteria screened in Test Example 1 Paenibacillus odorifer Bacterial suspension (OD 600 =1.0) were used to conduct an in vivo antagonistic experiment on lemon plants infected with HLB (Ct<25). The experimental flow chart is shown in the figure. Figure 8 9 HLB lemon plants were selected and divided into 3 groups. The blank group was not treated. The other two groups were inoculated on the leaves of the plants using injection inoculation method and spray inoculation method respectively. Paenibacillus odorifer The injection inoculation method involves injecting the entire leaf with 1-2 mL per leaf; the spray inoculation method involves spraying 1-2 mL on both sides of the leaf. For injection inoculation, leaves were not re-inoculated after each sampling, whereas for spray inoculation, plants were re-inoculated after each sampling. Leaf samples were collected before inoculation and on days 7, 14, 21, 30, 45, and 60 after inoculation. Total DNA was extracted from the leaves, and Clas titer was determined by qPCR. The primer sequences used for qPCR are shown in Table 2, the qPCR reaction system is shown in Table 3, and the qPCR reaction procedure is shown in Table 4.

[0041] Table 2 Primer sequence information used for qPCR detection

[0042] Table 3 qPCR detection reaction system

[0043] Table 4 qPCR reaction procedure

[0044] With the extension of Clas strain infection time, the Clas titer in the leaves of infected plants is higher and higher, and the Ct value detected by qPCR is also reduced accordingly. Therefore, the treatment effect of biocontrol bacteria on HLB is evaluated by the Ct value change of the leaves of infected plants, and the larger the ΔCt is, the stronger the inhibition effect is. Among them, the Ct value change (ΔCt=Ct 45 -Ct0) in the detection of leaves collected on the 45th day after inoculation reflects the inhibition effect, as shown in Figure B, compared with the blank group, the Clas increment in the leaves of the injection inoculation group and the spray inoculation group is less at 45 days after inoculation, which indicates that Figure 6 the strain has an inhibitory effect on the accumulation of Clas in the leaves of plants. Paenibacillus odorifer

[0045] The above is only an embodiment of the present application, and does not limit the patent scope of the present application, and any equivalent structure or equivalent process transformation using the content of the present application specification, or direct or indirect application in other related technical fields, are also included in the patent protection scope of the present application.​

Claims

1. A method for rapidly screening citrus Huanglongbing biocontrol bacteria, characterized in that: The method comprises the following steps: Lcr -GFP bacterial suspension and the strain to be screened CFS were mixed and incubated at a volume ratio of 1:1-17:3 for more than 3 hours, and the fluorescence intensity was detected at an excitation wavelength of 474-490 nm and an emission wavelength of 533-549 nm. The biocontrol bacteria were screened with a fluorescence inhibition rate of ≥10%; Lcr The solvent of the -GFP bacterial suspension is PBS buffer or TBS buffer.

2. The method according to claim 1, characterized in that The fluorescence inhibition rate was calculated as follows: inhibition rate = (blank group fluorescence value - sample group fluorescence value) / blank group fluorescence value × 100%.

3. The method according to claim 1, characterized in that described Lcr The solvent of the -GFP bacterial suspension is TBS buffer.

4. The method according to claim 1, wherein described Lcr -GFP bacterial suspension was measured at OD 600 It is prepared by diluting the initial bacterial solution of 1.0 10 to 100 times.

5. The method according to claim 1, wherein The volume ratio is 1:1 to 3:

1.

6. The method according to claim 1, wherein The excitation wavelength is 488 nm, and the emission wavelength is 535 nm.

7. The method according to claim 1, characterized in that The strain to be screened is cultured and fermented at 26-30°C, and the supernatant is collected by centrifugation to obtain the strain to be screened CFS.

8. The method according to claim 7, characterized in that The culture was carried out in NB medium.

9. The method according to claim 7, characterized in that The centrifugation temperature is 2-8°C, and the centrifugation speed is 8000 g-12000 g.

10. Use of the biocontrol bacteria obtained by the method according to any one of claims 1 to 9 in the prevention and control of citrus Huanglongbing plants.

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