CN114350741B - Method for high-flux determination of activity of citrus canker pathogen bactericide - Google Patents
Method for high-flux determination of activity of citrus canker pathogen bactericide Download PDFInfo
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Abstract
The invention provides a method for measuring activity of a citrus canker bactericide at high flux, and belongs to the technical field of plant protection. The method comprises the steps of respectively incubating a bactericide to be detected with recombinant and wild citrus canker fungus liquid, and measuring fluorescence intensity of an incubation system to obtain Ra and Rb; incubating a blank control with recombinant and wild citrus canker fungus liquid respectively, and measuring fluorescence intensity of an incubation system to obtain Rc and Rd; the inhibition ratio was calculated according to the formula shown in formula I. The invention tracks the activity of Xcc-eYFP in the screening process of the bactericide by detecting the fluorescence signal of Xcc-eYFP, and can be used for high-throughput determination of the activity of various bactericides and compound bactericides with different compound proportions. The method of the invention is adopted to measure the inhibition rate of four single agents and three compound bactericides, and the result shows that compared with the conventional inhibition zone method, the method of the invention has the advantages of rapidness and high efficiency, and the detection result is also very accurate.
Description
Technical Field
The invention belongs to the technical field of plant protection, and particularly relates to a method for measuring activity of citrus canker bactericide in a high throughput manner.
Background
Citrus canker is caused by the gram negative bacterium Xanthomonas citriSubsp (Xcc), one of the most serious bacterial diseases in the global citrus industry. Pathogens of citrus canker pathogens are classified by differences in host range and pathogenicity into XccA, xccAw, xccA x, xccB and XccC pathogenicity strains. Wherein XccA strain has strong pathogenicity and wide host range, and is distributed in a plurality of citrus producing areas in the world.
Chemical control is the primary method of controlling citrus canker, copper fungicides are the most common field application fungicides. Xcc has created resistance to copper and environmental pollution problems due to abuse of copper bactericides. Screening and applying the novel bactericide and the compound bactericide is an alternative strategy for reducing the application dosage of the copper bactericide. The traditional bactericide screening method mainly comprises a test tube dilution method and a diffusion method. With the development of a plurality of fungicide types and compounds, the conventional fungicide screening method is difficult to meet the requirements of fungicide high-throughput screening.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for measuring the activity of citrus canker bactericides with high throughput, which can be used for measuring the activity of a large amount of bactericides and compound bactericides with high throughput.
The invention provides a method for measuring the activity of a citrus canker bactericide in a high throughput manner, which comprises the following steps:
Incubating the bactericide to be detected with recombinant and wild citrus canker fungus liquid respectively, and measuring the fluorescence intensity of the co-incubation system to obtain Ra and Rb respectively;
incubating a blank control with recombinant and wild citrus canker fungus liquid respectively, and measuring fluorescence intensity of an incubation system to obtain Rc and Rd respectively;
calculating inhibition rate according to a formula shown in a formula I, wherein the inhibition rate is used for representing the activity of the bactericide;
The Ra is the fluorescence intensity of a co-incubation system of the bactericide to be detected and the recombinant citrus canker fungus liquid; the Rb is the fluorescence intensity of the co-incubation system of the bactericide to be detected and the wild citrus canker; rc is the fluorescence intensity of a co-incubation system of a blank control group and recombinant citrus canker pathogen; the Rd is the fluorescence intensity of a co-incubation system of a blank control group and wild citrus canker bacteria;
inhibition (%) =1- (Ra-Rb)/(Rc-Rd) ×100% formula I;
the recombinant citrus canker pathogen comprises a recombinant plasmid; the recombinant plasmid is inserted with a coding gene of a traceable fluorescent protein label;
The excitation wavelength and emission wavelength of the fluorescence intensity of the co-incubation system are determined to be compatible with the traceable fluorescent protein tag.
Preferably, after the inhibition rate is calculated, the method further comprises: and constructing and obtaining a dose response curve by taking the inhibition rate as a Y axis and the concentration of the bactericide as an X axis, and calculating the minimum inhibitory concentration according to the dose response curve.
Preferably, the traceable fluorescent protein tag comprises eYFP, YFP or GFP.
Preferably, the backbone plasmid of the recombinant plasmid comprises a vector of the PBBR-MCS series for broad host protein expression.
Preferably, the effective viable count of the recombinant citrus canker bacteria in the recombinant citrus canker bacteria liquid is 1 multiplied by 10 8～1×109 cfu/ml.
Preferably, the co-incubation time is 6 hours; the temperature of the co-incubation was 28 ℃.
The invention also provides application of the method in screening citrus canker bactericides.
The invention provides a method for measuring the activity of a citrus canker bactericide in a high throughput manner, wherein the bactericide to be measured is respectively incubated with recombinant and wild citrus canker bacteria liquid, and the fluorescence intensity of an incubation system is measured to respectively obtain Ra and Rb; incubating a blank control with recombinant and wild citrus canker fungus liquid respectively, and measuring fluorescence intensity of an incubation system to obtain Rc and Rd respectively; and calculating the inhibition rate according to a formula shown in a formula I, wherein the inhibition rate is used for representing the activity of the bactericide. The inactive Xcc-eYFP caused fluorescence quenching, with a change in fluorescence intensity compared to the control Xcc-eYFP. The invention can track the activity of Xcc-eYFP in the screening process of the bactericide by detecting the fluorescence signal of Xcc-eYFP, and can be used for high-throughput determination of the activity of various bactericides and compound bactericides with different compound proportions. The method of the invention is adopted to measure the activity and inhibition rate of four single agents and three compound bactericides, and the result shows that compared with the conventional inhibition zone method, the method of the invention has the advantages of rapidness and high efficiency, and the detection result is more accurate.
Drawings
FIG. 1 is a schematic diagram of a sample adding mode of a ninety-six pore plate measured by an enzyme-labeled instrument; wherein 1, 2, 3,4,5, 6, 7, 8, 9, 10, 11, 12 are 12 concentration gradients of the bactericide;
FIG. 2 shows the fluorescence intensity of Xcc and Xcc-eYFP under LUYOR-3145RG irradiation after 6h incubation expressed at 12 concentration gradients of 30% copper (SC);
FIG. 3 is a graph showing the change in fluorescence intensity of Xcc-eYFP in 30% copper-king sc 12 concentration gradients as determined by a multifunctional microplate reader;
FIG. 4 is a graph showing the measurement of the change in fluorescence intensity of Xcc-eYFP in 12 concentration gradients among four bactericides using a multifunctional microplate reader;
FIG. 5 is a graph showing the results of evaluating resistance of bactericide-treated Hamlin orange leaves to citrus canker by needle punching inoculation; wherein, a, the citrus canker disease condition of the Hamlin orange leaves pretreated by different bactericides is evaluated by a needling inoculation method; b. calculating the incidence rate by comparing the incidence spots of the needle-punched fungus Hamlin leaf citrus canker with a control group; C. calculating the bacterial concentration in the Hamlin orange by a fluorescence counting method; x is 1.2 percent of octylamine acetate AS, C is 33 percent of Chunlei quinoline copper SC, W is 30 percent of copper king SC, R is 20 percent of copper rosinate EW;
FIG. 6 shows the results of verification of the inhibition ratio of 1.2% octylamine acetate AC of non-copper-containing bactericides to 3 copper-containing bactericides, wherein a. The inhibition ratio of three different compounding ratios of the three compounding agents is calculated; b, calculating the antibacterial rate of a compound proportion of 1.2% of octreotide acetate AS and 30% of copper SC; 1.05%: the antibacterial rate of the single-dose 1.2% of the octylamine acetate AS is 1.24%: the bacteriostasis rate of single agent 30% of copper sulfate SC is 67.5%: the antibacterial rate is the antibacterial rate when the optimal synergistic ratio of the compound agent reaches 1:1; x is 1.2 percent of octylamine acetate AS, C is 33 percent of Chunlei quinoline copper SC, W is 30 percent of copper king SC, R is 20 percent of copper rosinate EW.
Detailed Description
The invention provides a visual high-flux method for measuring the activity of a citrus canker bactericide, which comprises the following steps: incubating the bactericide to be detected with recombinant and wild citrus canker fungus liquid respectively, and measuring the fluorescence intensity of the co-incubation system to obtain Ra and Rb respectively; incubating a blank control with recombinant and wild citrus canker fungus liquid respectively, and measuring fluorescence intensity of an incubation system to obtain Rc and Rd respectively; calculating inhibition rate according to a formula shown in a formula I, wherein the inhibition rate is used for representing the activity of the bactericide;
The Ra is the fluorescence intensity of a co-incubation system of the bactericide to be detected and the recombinant citrus canker fungus liquid; the Rb is the fluorescence intensity of the co-incubation system of the bactericide to be detected and the wild citrus canker; rc is the fluorescence intensity of a co-incubation system of a blank control group and recombinant citrus canker pathogen; the Rd is the fluorescence intensity of a co-incubation system of a blank control group and wild citrus canker bacteria;
inhibition (%) =1- (Ra-Rb)/(Rc-Rd) ×100% formula I;
the recombinant citrus canker pathogen comprises a recombinant plasmid; the recombinant plasmid is inserted with a coding gene of a traceable fluorescent protein label; the excitation wavelength and emission wavelength of the fluorescence intensity of the co-incubation system are determined to be compatible with the traceable fluorescent protein tag.
In the invention, the bactericide to be tested comprises a single bactericide or a compound bactericide; the type of the bactericide is not particularly limited in the present invention, and the activity of the conventional citrus canker bactericide in the art can be measured by the method of the present invention. In the implementation process of the invention, the bactericide to be detected comprises one or more of a cinerea water aqua (Aqueous solutions, AS), a chunlei-quinoline copper suspending agent (Suspension concentrate, SC), copper sulfate SC and 20% copper resinate aqueous emulsion (Emulsion in water, EW).
In the present invention, the blank is preferably a solvent; the volume of the blank control is the same as that of the bactericide to be tested.
In the invention, the original bacteria of the recombinant citrus canker bacteria liquid are citrus canker bacteria, and the citrus canker bacteria are preferably xanthomonas citri subspecies. In the present invention, the backbone plasmid of the recombinant plasmid preferably includes a vector of the PBBR-MCS series, more preferably PBBR-MCS 5, which is expressed by a broad host protein. In the present invention PBBR-MCS 5 is a broad host protein expression plasmid capable of stable expression in a variety of microorganisms. In the invention, the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO.2, and specifically comprises the following steps:
accttcgggagcgcctgaagcccgttctggacgccctggggccgttgaatcgggatatgcaggccaaggccgccgcgatcatcaaggccgtgggcgaaaagctgctgacggaacagcgggaagtccagcgccagaaacaggcccagcgccagcaggaacgcgggcgcgcacatttccccgaaaagtgccacctggcggcgttgtgacaatttaccgaacaactccgcggccgggaagccgatctcggcttgaacgaattgttaggtggcggtacttgggtcgatatcaaagtgcatcacttcttcccgtatgcccaactttgtatagagagccactgcgggatcgtcaccgtaatctgcttgcacgtagatcacataagcaccaagcgcgttggcctcatgcttgaggagattgatgagcgcggtggcaatgccctgcctccggtgctcgccggagactgcgagatcatagatatagatctcactacgcggctgctcaaacctgggcagaacgtaagccgcgagagcgccaacaaccgcttcttggtcgaaggcagcaagcgcgatgaatgtcttactacggagcaagttcccgaggtaatcggagtccggctgatgttgggagtaggtggctacgtctccgaactcacgaccgaaaagatcaagagcagcccgcatggatttgacttggtcagggccgagcctacatgtgcgaatgatgcccatacttgagccacctaactttgttttagggcg
actgccctgctgcgtaacatcgttgctgctgcgtaacatcgttgctgctccataacatcaaacatcgacccacggcgt
aacgcgcttgctgcttggatgcccgaggcatagactgtacaaaaaaacagtcataacaagccatgaaaaccgccac
tgcgccgttaccaccgctgcgttcggtcaaggttctggaccagttgcgtgagcgcatacgctacttgcattacagttta
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gcaaggcgacaaggtgctgatgccgctggcgattcaggttcatcatgccgtttgtgatggcttccatgtcggcagaat
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gcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactca
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cccttcggcgctcaccatacagaggctgccacaggcgagtgggatgaggtgcaatcgggtctgcgggcagccga
cgcccccccacccaccatgcgcgtggctgtcactgccgcgcggccgccgcgcgccaagccggcgccgcgacg
acgtgctgcgcaaccctccgacgcttcggaattcaagcttatggtgagcaagggcgaggagctgttcaccggggtg
gtgccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgat
gccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtga
ccaccttcggctacggcctgcagtgcttcgcccgctaccccgaccacatgaagcagcacgacttcttcaagtccgcc
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gaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcc
tggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatca
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cccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagacccc
aacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctg
tacaagtgaagctttacccatacgatgttccagattacgcttgatctagagcggccgccaccgcggtggagctccaat
tcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgtta
cccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgccct
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aatcagctcattttttaaccaataggccgactgcgatgagtggcagggcggggcgtaatttttttaaggcagttattggt
gcccttaaacgcctggtgctacgcctgaataagtgataataagcggatgaatggcagaaattcgaaagcaaattcga
cccggtcgtcggttcagggcagggtcgttaaatagccgcttatgtctattgctggtttaccggtttattgactaccggaa
gcagtgtgaccgtgtgcttctcaaatgcctgaggccagtttgctcaggctctccccgtggaggtaataattgacgatat
gatcatttattctgcctcccagagcctgataaaaacggtgaatccgttagcgaggtgccgccggcttccattcaggtc
gaggtggcccggctccatgcaccgcgacgcaacgcggggaggcagacaaggtatagggcggcgaggcggcta
cagccgatagtctggaacagcgcacttacgggttgctgcgcaacccaagtgctaccggcgcggcagcgtgaccc
gtgtcggcggctccaacggctcgccatcgtccagaaaacacggctcatcgggcatcggcaggcgctgctgcccg
cgccgttcccattcctccgtttcggtcaaggctggcaggtctggttccatgcccggaatgccgggctggctgggcgg
ctcctcgccggggccggtcggtagttgctgctcgcccggatacagggtcgggatgcggcgcaggtcgccatgccc
caacagcgattcgtcctggtcgtcgtgatcaaccaccacggcggcactgaacaccgacaggcgcaactggtcgcg
gggctggccccacgccacgcggtcattgaccacgtaggccgacacggtgccggggccgttgagcttcacgacgg
agatccagcgctcggccaccaagtccttgactgcgtattggaccgtccgcaaagaacgtccgatgagcttggaaag
tgtcttctggctgaccaccacggcgttctggtggcccatctgcgccacgaggtgatgcagcagcattgccgccgtgg
gtttcctcgcaataagcccggcccacgcctcatgcgctttgcgttccgtttgcacccagtgaccgggcttgttcttggc
ttgaatgccgatttctctggactgcgtggccatgcttatctccatgcggtagggtgccgcacggttgcggcaccatgc
gcaatcagctgcaacttttcggcagcgcgacaacaattatgcgttgcgtaaaagtggcagtcaattacagattttcttta
acctacgcaatgagctattgcggggggtgccgcaatgagctgttgcgtaccccccttttttaagttgttgatttttaagtc
tttcgcatttcgccctatatctagttctttggtgcccaaagaagggcacccctgcggggttcccccacgccttcggcgc
ggctccccctccggcaaaaagtggcccctccggggcttgttgatcgactgcgcggccttcggccttgcccaaggtg
gcgctgcccccttggaacccccgcactcgccgccgtgaggctcggggggcaggcgggcgggcttcgccttcgac
tgcccccactcgcataggcttgggtcgttccaggcgcgtcaaggccaagccgctgcgcggtcgctgcgcgagcct
tgacccgccttccacttggtgtccaaccggcaagcgaagcgcgcaggccgcaggccggaggcttttccccagaga
aaattaaaaaaattgatggggcaaggccgcaggccgcgcagttggagccggtgggtatgtggtcgaaggctgggt
agccggtgggcaatccctgtggtcaagctcgtgggcaggcgcagcctgtccatcagcttgtccagcagggttgtcc
acgggccgagcgaagcgagccagccggtggccgctcgcggccatcgtccacatatccacgggctggcaaggga
gcgcagcgaccgcgcagggcgaagcccggagagcaagcccgtagggcgccgcagccgccgtaggcggtcac
gactttgcgaagcaaagtctagtgagtatactcaagcattgagtggcccgccggaggcaccgccttgcgctgcccc
cgtcgagccggttggacaccaaaagggaggggcaggcatggcggcatacgcgatcatgcgatgcaagaagctg
gcgaaaatgggcaacgtggcggccagtctcaagcacgcctaccgcgagcgcgagacgcccaacgctgacgcca
gcaggacgccagagaacgagcactgggcggccagcagcaccgatgaagcgatgggccgactgcgcgagttgc
tgccagagaagcggcgcaaggacgctgtgttggcggtcgagtacgtcatgacggccagcccggaatggtggaag
tcggccagccaagaacagcaggcggcgttcttcgagaaggcgcacaagtggctggcggacaagtacggggcgg
atcgcatcgtgacggccagcatccaccgtgacgaaaccagcccgcacatgaccgcgttcgtggtgccgctgacgc
aggacggcaggctgtcggccaaggagttcatcggcaacaaagcgcagatgacccgcgaccagaccacgtttgcg
gccgctgtggccgatctagggctgcaacggggcatcgagggcagcaaggcacgtcacacgcgcattcaggcgtt
ctacgaggccctggagcggccaccagtgggccacgtcaccatcagcccgcaagcggtcgagccacgcgcctat
gcaccgcagggattggccgaaaagctgggaatctcaaagcgcgttgagacgccggaagccgtggccgaccggc
tgacaaaagcggttcggcaggggtatgagcctgccctacaggccgccgcaggagcgcgtgagatgcgcaagaaggccgatcaagcccaagagacggcccgaggg.
In the present invention, the traceable fluorescent protein tag preferably comprises eYFP, YFP or GFP. In the invention, the nucleotide sequence of the coding gene of the eYFP is shown as SEQ ID NO.1, and specifically comprises the following steps:
atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgcagtgcttcgcccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaag.
The eYFP tag protein is enhanced yellow-green fluorescent protein eYFP, the excitation wavelength is 513nm, the emission wavelength is 227 nm, and the eYFP tag protein is prepared from wild yellow-green fluorescent protein YFP through amino acid mutation and codon optimization.
In the invention, the effective viable count of the recombinant citrus canker bacteria in the recombinant citrus canker bacteria liquid is preferably 1 multiplied by 10 8～1×109 cfu/ml. In the invention, the bactericides to be tested are diluted by gradient concentration, and each bactericide to be tested is respectively incubated with the recombinant citrus canker fungus liquid. In the invention, the volume ratio of the bactericide to be detected and the blank control to the recombinant and wild citrus canker fungus liquid is preferably 1:1.
In the present invention, the time of the co-incubation is preferably 6 hours; the temperature of the co-incubation is preferably 28 ℃.
In the invention, the fluorescence intensity of the co-incubation system is preferably measured by a multifunctional enzyme-labeled instrument; the multifunctional enzyme-labeled instrument is preferably a Spark TM multifunctional enzyme-labeled instrument; the excitation wavelength of the multifunctional enzyme-labeled instrument is preferably 485nm, and the emission wavelength is preferably 532nm.
In the present invention, inactive Xcc-eYFP resulted in fluorescence quenching, with a change in fluorescence intensity compared to the Xcc-eYFP of the blank. The invention can be used for high-throughput determination of the activities of various bactericides and compound bactericides with different compound proportions by detecting the fluorescence signal of the Xcc-eYFP and tracking the activity of the Xcc-eYFP in the screening process of the bactericides.
In the invention, after the inhibition rate is calculated, a dose response curve is constructed by taking the inhibition rate as a Y axis and the concentration of the bactericide as an X axis, and the lowest inhibitory concentration is calculated according to the dose response curve, and the concentration of the screened medicament corresponding to the bottom point of the fluorescence value is the lowest inhibitory concentration (MIC, and the lowest concentration of the medicament corresponding to the time when the inhibition rate reaches the maximum).
The invention also provides application of the method in screening citrus canker bactericides.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention.
Example 1
Bacterial strain, growth conditions of plants:
the Xcc strain was selected from the Xanthomonas citri strain 306 (NCBI ID: 190486) and the homologous isolate.
All bacterial strains were kept in 15% glycerol and in a refrigerator at-80 ℃. Coli was cultured in beef extract peptone medium (LB) at 37 ℃. The Xcc strain was recovered and cultured on Nutrient Broth (NB) medium and Nutrient Agar (NA) plates at 28 ℃. The antibiotic concentration used in this example was gentamicin (20. Mu.g/mL). Citrus plants hamlin orange (Citrus sinensis (l.) Osbeck) were grown in a greenhouse with 16h light, 8h darkness, 28/26 ℃ temperature cycle and 80% humidity. Fully expanded young leaves, approximately 1 month old, were used.
Construction of the eYFP plasmid:
Plasmids were constructed as described in the previous study. Briefly, the eYFP coding region was cloned into the pBBR1-MCS5 plasmid to germinate the broad host range vector pBBR1-eYFP. The construct of pBBR1-eYFP was introduced into DH 5. Alpha. E.coli competent cells and then selected on LB plates containing 20. Mu.g/ml gentamicin. Plasmid DNA extracted from E.coli transformants was reintroduced into Xcc competent cells using electroporation. Xcc conversion was performed using GeneP μ lser Xcell (Bio-Rad, USA) system under the following conditions: cuvette 1mm, voltage 2400V; capacitance 25. Mu.F. The reconstituted Xcc-eYFP strain was spread on NA plates containing 20. Mu.g/ml gentamicin. Using ND-1000 (Bio-Rad, USA) measures DNA concentration. All constructed plasmids were confirmed by PCR and restriction. Meanwhile, the Xcc-eYFP strain can be confirmed and observed by a hand-held lamp (Luyor-3415 RG, shanghai). The fluorescent marker bacteria with stable genetic expression can be observed visually by using the instrument, and if the fluorescent marker bacteria are compared with a control group plate, obvious green fluorescence can be observed, the successful conversion can be judged.
Visual high-throughput determination of fungicide inhibition rate:
33% Chunleiquiz copper SC, 1.2% octylamine acetate AS, 30% copper king SC and 20% copper rosinate EW were dissolved in 10mM MgCl 2 solution at the following mass concentrations and diluted to 12 concentration gradients respectively (Table 1).
TABLE 1
Mother solutions of 20% copper rosinate EW and 33% Chunleiquiz copper SC, 0.5mg/ml2% neomycophenolate AS and 0.03mg/ml30% copper master batch were prepared at concentrations of 0.03mg/ml and 0.25mg/ml, respectively. Three composite bactericides were then formulated at different volume ratios (table 2).
TABLE 2
Mu.l of the bactericide and 100. Mu.l of Xcc-eYFP bacterial suspension at a concentration of 1X 10 9 cfu/ml were each added to a 96-well plate (black 96-well plate, flat bottom). Wild-type Xcc and Xcc-eYFP without bactericide were used as controls. Each treatment was repeated three times. After incubation for 6h at 28 ℃. Fluorescence quenching of Xcc-eYFP in different concentrations of germicides was observed under hand-held lamps (Luyor-3415 RG, shanghai) and the Xcc-eYFP fluorescence intensity was measured by a multifunctional enzyme label (TECAN) at excitation wavelength 485 nm/emission wavelength 535 nm. The inhibition ratio was calculated as follows:
Inhibition (%) =1- (Ra-Rb)/(Rc-Rd) ×100%
Ra in the formula is the fluorescence intensity of the co-incubation system of the bactericide to be detected and the recombinant citrus canker fungus liquid; rb is the fluorescence intensity of the co-incubation system of the bactericide to be detected and the wild citrus canker; rc is the fluorescence intensity of a co-incubation system of the blank control group and the recombinant citrus canker pathogen; rd is the fluorescence intensity of the co-incubation system of the blank group and the wild type citrus canker. (shown in FIG. 1). Setting the inhibition rate as a (Y axis) and the concentration of the bactericide as an (X axis) in software GRAPHPAD PRISM, constructing a dose response curve, and calculating the Minimum Inhibitory Concentration (MIC) of the medicine according to the response curve. Three replicates were run.
Results
In order to realize the fluorescent visualization effect of the inhibition rate of the bactericide, firstly, the copper foil with better control effect on citrus canker in a copper preparation is selected. After the incubation was completed, the bacteriostatic effect was evaluated by observing fluorescence quenching of Xcc-eYFP in different concentrations of 30% copper-king SC under a hand-held lamp (Luyor-3415 RG, china). Neither the biocide nor the wild-type Xcc produced a fluorescent signal, but the Xcc-eYFP fluorescent intensity gradually increased as the biocide concentration gradually decreased. (FIG. 2). This is consistent with the result of the change in the Xcc-eYFP fluorescence values measured by the multifunctional microplate reader. (FIG. 3). Thus, we developed a high throughput assay in 96 well format using Xcc-eYFP to determine the activity of bactericides on Xcc-eYFP. Fluorescence intensities of Xcc-eYFP at four different bactericides at different concentrations were measured by a multifunctional microplate reader. As a result, the four bactericides have an inhibiting effect on the citrus canker. According to a dose response curve of the bacteriostasis rate and the concentration of the bactericide, the lowest bacteriostasis concentration (MIC) of the medicine is found when the bacteriostasis rate reaches the maximum: 1.2% Xin Junan acetate AS 128mg/mL, 33% Chunleiq copper SC 16mg/mL, 30% copper king SC 32mg/mL and 20% copper resinate EW 2mg/mL. So, according to the MIC of the medicine, the activity of 4 bactericides on Xcc is 20% copper resinate EW >33% spring mine copper quinoline SC >30% copper king SC >1.2% Xin Junan acetate AS (figure 4).
To verify the above results, the resistance of the bactericide-treated hamlin orange leaves to citrus canker was evaluated using a needle punching inoculation method. The results indicated that the control group of leaves had significant symptoms (a in FIG. 5) compared to the bactericide-treated leaves. Statistical analysis showed that Xcc-eYFP was significantly higher in both disease severity and bacterial numbers in the water treatment leaves than in the bactericide treatment leaves. Furthermore, as the virulence of the bactericide increases, the severity of the disease and the number of bacteria gradually decrease (b and c in fig. 5). These results indicate that the Xcc-eYFP assay can accurately evaluate the activity of the fungicide on Xcc, providing important reference data for field medications.
Secondly, in order to screen the copper-containing compound bactericide, the proportion of the copper preparation is reduced. We selected a non-copper containing biocide 1.2% octreotide acetate AC compounded with 3 copper containing biocides (table 2). With the gradual increase of the volume ratio of 1.2% of the octisamine acetate AC to 30% of the copper sulfate SC, the synergistic effect is enhanced, and the synergistic effect is strongest when the volume ratio reaches 1:1. Notably, when 20% copper roside EW, 33% chun-myc-quinoline copper SC, and 1.2% octreotide acetate AC were compounded, the synergy did not significantly change with increasing volume ratio (a in fig. 6). When the volume ratio of 1.2% of the octhlamide acetate AC to 30% of the copper sulfate SC is 1:1, the inhibition rate of the composite bactericide is up to 67.75%, which is more than 1.50% and 1.24% of that of two independent bactericides. The calculation shows that the application amount of the compound agent to the copper bactericide can be reduced to 55.8 percent (b in fig. 6) under the same inhibition rate. Thus, the data indicate that the Xcc-eYFP assay can be used to screen for composite bactericides, reducing the use of copper bactericides.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, according to which one can obtain other embodiments without inventiveness, these embodiments are all within the scope of the invention.
Sequence listing
<110> Gannan university of teachers and students
<120> Method for high throughput determination of citrus canker pathogen bactericidal activity
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aaaagtgcca cctggcggcg ttgtgacaat ttaccgaaca actccgcggc cgggaagccg 240
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aaccgccact gcgccgttac caccgctgcg ttcggtcaag gttctggacc agttgcgtga 960
gcgcatacgc tacttgcatt acagtttacg aaccgaacag gcttatgtca actgggttcg 1020
tgccttcatc cgtttccacg gtgtgcgtcc atgggcaaat attatacgca aggcgacaag 1080
gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc 1140
agaatgctta atgaattaca acagttttta tgcatgcgcc caatacgcaa accgcctctc 1200
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cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca 1380
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gatggggttc agccgactgc agatcgtggg gtgtctccgc ctgccggcgg ccccctggat 1620
ggcttgcccg ctcggcggac gatgtcccgg acccggctgc catctccccc tgccccctca 1680
cctgcgttct cggcgggcag cttcagtgac ctgttacgtc agttcgatcc gtcacttttt 1740
aatacatcgc tttttgattc attgcctccc ttcggcgctc accatacaga ggctgccaca 1800
ggcgagtggg atgaggtgca atcgggtctg cgggcagccg acgccccccc acccaccatg 1860
cgcgtggctg tcactgccgc gcggccgccg cgcgccaagc cggcgccgcg acgacgtgct 1920
gcgcaaccct ccgacgcttc ggaattcaag cttatggtga gcaagggcga ggagctgttc 1980
accggggtgg tgccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg 2040
tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca 2100
ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttcggc tacggcctgc 2160
agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc 2220
ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc 2280
gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg 2340
acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca 2400
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tcactctcgg catggacgag ctgtacaagt gaagctttac ccatacgatg ttccagatta 2700
cgcttgatct agagcggccg ccaccgcggt ggagctccaa ttcgccctat agtgagtcgt 2760
attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta 2820
cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg 2880
cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg aaattgtaag 2940
cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca 3000
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cccttaaacg cctggtgcta cgcctgaata agtgataata agcggatgaa tggcagaaat 3120
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cccattcctc cgtttcggtc aaggctggca ggtctggttc catgcccgga atgccgggct 3660
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gcttggaaag tgtcttctgg ctgaccacca cggcgttctg gtggcccatc tgcgccacga 4020
ggtgatgcag cagcattgcc gccgtgggtt tcctcgcaat aagcccggcc cacgcctcat 4080
gcgctttgcg ttccgtttgc acccagtgac cgggcttgtt cttggcttga atgccgattt 4140
ctctggactg cgtggccatg cttatctcca tgcggtaggg tgccgcacgg ttgcggcacc 4200
atgcgcaatc agctgcaact tttcggcagc gcgacaacaa ttatgcgttg cgtaaaagtg 4260
gcagtcaatt acagattttc tttaacctac gcaatgagct attgcggggg gtgccgcaat 4320
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gcgcggctcc ccctccggca aaaagtggcc cctccggggc ttgttgatcg actgcgcggc 4500
cttcggcctt gcccaaggtg gcgctgcccc cttggaaccc ccgcactcgc cgccgtgagg 4560
ctcggggggc aggcgggcgg gcttcgcctt cgactgcccc cactcgcata ggcttgggtc 4620
gttccaggcg cgtcaaggcc aagccgctgc gcggtcgctg cgcgagcctt gacccgcctt 4680
ccacttggtg tccaaccggc aagcgaagcg cgcaggccgc aggccggagg cttttcccca 4740
gagaaaatta aaaaaattga tggggcaagg ccgcaggccg cgcagttgga gccggtgggt 4800
atgtggtcga aggctgggta gccggtgggc aatccctgtg gtcaagctcg tgggcaggcg 4860
cagcctgtcc atcagcttgt ccagcagggt tgtccacggg ccgagcgaag cgagccagcc 4920
ggtggccgct cgcggccatc gtccacatat ccacgggctg gcaagggagc gcagcgaccg 4980
cgcagggcga agcccggaga gcaagcccgt agggcgccgc agccgccgta ggcggtcacg 5040
actttgcgaa gcaaagtcta gtgagtatac tcaagcattg agtggcccgc cggaggcacc 5100
gccttgcgct gcccccgtcg agccggttgg acaccaaaag ggaggggcag gcatggcggc 5160
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gcacgcctac cgcgagcgcg agacgcccaa cgctgacgcc agcaggacgc cagagaacga 5280
gcactgggcg gccagcagca ccgatgaagc gatgggccga ctgcgcgagt tgctgccaga 5340
gaagcggcgc aaggacgctg tgttggcggt cgagtacgtc atgacggcca gcccggaatg 5400
gtggaagtcg gccagccaag aacagcaggc ggcgttcttc gagaaggcgc acaagtggct 5460
ggcggacaag tacggggcgg atcgcatcgt gacggccagc atccaccgtg acgaaaccag 5520
cccgcacatg accgcgttcg tggtgccgct gacgcaggac ggcaggctgt cggccaagga 5580
gttcatcggc aacaaagcgc agatgacccg cgaccagacc acgtttgcgg ccgctgtggc 5640
cgatctaggg ctgcaacggg gcatcgaggg cagcaaggca cgtcacacgc gcattcaggc 5700
gttctacgag gccctggagc ggccaccagt gggccacgtc accatcagcc cgcaagcggt 5760
cgagccacgc gcctatgcac cgcagggatt ggccgaaaag ctgggaatct caaagcgcgt 5820
tgagacgccg gaagccgtgg ccgaccggct gacaaaagcg gttcggcagg ggtatgagcc 5880
tgccctacag gccgccgcag gagcgcgtga gatgcgcaag aaggccgatc aagcccaaga 5940
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Claims (6)
1. A method for high throughput determination of citrus canker fungicide activity, comprising the steps of:
Incubating the bactericide to be detected with recombinant and wild citrus canker fungus liquid respectively, and measuring the fluorescence intensity of the co-incubation system to obtain Ra and Rb respectively;
incubating a blank control with recombinant and wild citrus canker fungus liquid respectively, and measuring fluorescence intensity of an incubation system to obtain Rc and Rd respectively;
calculating inhibition rate according to a formula shown in a formula I, wherein the inhibition rate is used for representing the activity of the bactericide;
The Ra is the fluorescence intensity of a co-incubation system of the bactericide to be detected and the recombinant citrus canker fungus liquid; the Rb is the fluorescence intensity of the co-incubation system of the bactericide to be detected and the wild citrus canker; rc is the fluorescence intensity of a co-incubation system of a blank control group and recombinant citrus canker pathogen; the Rd is the fluorescence intensity of a co-incubation system of a blank control group and wild citrus canker bacteria;
inhibition (%) =1- (Ra-Rb)/(Rc-Rd) ×100% formula I;
The recombinant citrus canker pathogen comprises a recombinant plasmid; the recombinant plasmid is inserted with a coding gene of a traceable fluorescent protein label; the traceable fluorescent protein tag is eYFP, and the nucleotide sequence of the encoding gene of the eYFP is shown as SEQ ID NO. 1;
The excitation wavelength and emission wavelength of the fluorescence intensity of the co-incubation system are determined to be compatible with the traceable fluorescent protein tag.
2. The method of claim 1, further comprising, after calculating the inhibition ratio: and constructing and obtaining a dose response curve by taking the inhibition rate as a Y axis and the concentration of the bactericide as an X axis, and calculating the minimum inhibitory concentration according to the dose response curve.
3. The method of claim 1, wherein the backbone plasmid of the recombinant plasmid comprises a broad host protein expression PBBR-MCS set vector.
4. The method according to claim 1, wherein the effective viable count of the recombinant citrus canker bacteria in the recombinant citrus canker bacteria liquid is 1x 10 8~1×109 cfu/ml.
5. The method of claim 1, wherein the co-incubation time is 6 hours; the temperature of the co-incubation was 28 ℃.
6. Use of the method of any one of claims 1-5 for screening citrus canker bactericides.
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