A method for rapid identification of resistance of litchi downy mildew to cyazofamid

Abstract

The present invention discloses the identification of nucleotide point mutations in the cytochrome b protein-related gene PlCyt b of litchi downy mildew pathogen and its application in monitoring resistance to the fungicide cyazofamid, and specifically discloses a molecular detection method and special primers for identifying the resistance of the 660-position nucleotide mutation of the PlCyt b gene of litchi downy mildew pathogen to cyazofamid. The molecular detection method provided by the present invention has high sensitivity, good stability, and short detection cycle, and can be used for high-throughput monitoring of the frequency of resistance genes of litchi downy mildew pathogens to the fungicide cyazofamid in the field and the occurrence and development of resistance.

Description

Technical Field

[0001] The present invention relates to the identification of nucleotide point mutations on cytochrome b protein-related gene PlCyt b of litchi downy mildew pathogen and its application in the detection of fungicide cyazofamid resistance, and specifically discloses a molecular detection method and special primers for rapid identification of nucleotide point mutations in PlCyt b gene of litchi downy mildew pathogen and its resistance to cyazofamid, belonging to the technical field of molecular biology. Background Art

[0002] Phytophthora litchi Chen ex Ko et al., classified as Oomycota, Peronosporales, Peronosporaceae, Phytophthora, is an important pathogenic oomycete in litchi production. Lychee downy mildew can infect young shoots, flower spikes, young fruits, fruit stalks and mature fruits of litchi, causing flower and fruit drop and rot, among which the fruit is the most harmful, seriously affecting the yield and quality of litchi. At present, there are 68 types of fungicides registered for the prevention and control of litchi downy mildew in my country, and the active ingredients mainly include single-site fungicides such as metalaxyl, azoxystrobin, fluthiazolinone and oxadiazine. However, with the irrational and frequent use of these fungicides, many plant pathogenic oomycetes have developed serious resistance to the above fungicides. Therefore, major international agrochemical companies have been committed to developing new fungicides with new mechanisms of action and no positive cross-resistance with existing fungicides on the market, for the prevention and control of plant pathogenic oomycete diseases such as litchi downy mildew and management of drug resistance.

[0003] Cyazofamid is a phenylimidazole fungicide developed by Ishihara Sangyo Co., Ltd. of Japan that has high activity against plant blight and downy mildew caused by oomycetes. This fungicide is a new type of low-toxic fungicide with good protective activity and certain systemic therapeutic activity. It has the advantages of long-lasting effect, resistance to rain erosion, safe and convenient use, etc. Cyazofamid targets the mitochondria of pathogens and can bind to the Qi site in the mitochondrial cytochrome bc1 complex, inhibiting the electron transfer from heme b Hto quinone-semihydroquinone, thereby inhibiting the synthesis of ATP and achieving the effect of killing pathogens. Cyazofamid obtained pesticide registration in my country in 2013, and is mainly used for the prevention and control of diseases such as tomato late blight, potato late blight, litchi downy mildew, cucumber downy mildew, grape downy mildew, ornamental chrysanthemum downy mildew, Rosaceae ornamental flower downy mildew, watermelon blight, and Chinese cabbage clubroot. At present, the Fungicide Resistance Raction Committee (FRAC) has classified its resistance risk as medium to high, and it is necessary to pay attention to the risk management of resistance during use. This laboratory obtained a litchi downy mildew strain with a high level of resistance to cyazofamid through the method of agent domestication, and some high-level resistant strains had a high survival fitness, indicating that the resistance risk of litchi downy mildew to cyazofamid is medium to high. Therefore, when using cyazofamid to control plant pathogenic oomycete diseases, it is necessary to strengthen resistance monitoring and provide timely early warning for the occurrence of cyazofamid resistance, so as to guide the scientific use of cyazofamid, delay the development of resistance, and extend the service life of the agent.

[0004] Conventional methods for detecting fungicide resistance include: mycelium growth rate method, mycelium dry weight determination method, spore germination determination method and agar spreading method. However, these methods all require the isolation and purification of pathogens, and then the pathogens are inoculated onto drug-containing culture media or onto living plants or tissues treated with fungicides, and the results can only be investigated after a certain period of time. Using conventional methods, when the frequency of drug-resistant strains in the field is greater than 1%, they can be detected (for example, if there is a 95% probability of detecting a 1% frequency of drug-resistant strains, the sample size required for detection needs to be greater than 300), so it has the disadvantages of low sensitivity, large workload, and long test cycle.

[0005] With the rapid development of molecular biology technology and the continuous expansion of its application scope, restriction enzyme PCR and allele-specific PCR molecular technology have begun to be used in the detection of pathogen resistance. Compared with traditional detection methods, it not only saves detection time and improves work efficiency, but also enhances the sensitivity of detection. The detection frequency is 10 -5 ~10 -4 , is more suitable for detecting low-frequency resistance genes and is also regarded as an ideal method for early diagnosis of resistance in the field. Therefore, molecular detection technology will play an increasingly important role in the sustainable management system of diseases. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a protein related to the resistance of litchi downy mildew to cyazofamid and its encoding gene, as well as its application as a marker for screening litchi downy mildew resistance to cyazofamid, in the identification of litchi downy mildew resistance to cyazofamid.

[0007] In order to achieve the object of the present invention, the technical solution of the present invention is as follows:

[0008] One object of the present invention is to provide a protein related to cyanazofamid resistance to litchi downy mildew, which is a protein shown in 1) or 2) below:

[0009] 1) A protein consisting of the amino acid residue sequence of SEQ ID NO: 8 in the sequence listing;

[0010] 2) A protein derived from SEQ ID NO: 8 in the sequence listing, wherein one or more amino acid residues are substituted and / or deleted and / or added and which is related to the cyazofamid resistance function.

[0011] Among them, the cyazofamid resistance-associated protein of litchi downy mildew fungus has phenylalanine at the 220th amino acid, which is sensitive to cyazofamid. When the 220th amino acid from the N-terminus mutates to leucine, the litchi downy mildew fungus shows resistance to cyazofamid.

[0012] In one embodiment of the present invention, the sequence of the resistance-associated protein (cytochrome b protein) of litchi downy mildew is as shown in SEQ ID NO:8. When the 220th amino acid from the N-terminus of SEQ ID NO:8 is phenylalanine (X=Phe), litchi downy mildew is sensitive to cyazofamid. When the 220th amino acid from the N-terminus of SEQ ID NO:8 is leucine (X=Leu), litchi downy mildew is resistant to cyazofamid.

[0013] The present invention also provides a gene encoding a protein associated with cyazofamid resistance.

[0014] The cDNA nucleotide sequence of the coding gene is shown in 1), 2) or 3) below:

[0015] 1) the nucleotide sequence of SEQ ID NO: 9 in the sequence listing;

[0016] 2) a DNA molecule that can recombine with the DNA sequence described in 1) under stringent conditions and encodes a protein related to cyazofamid resistance function;

[0017] 3) A DNA molecule having a similarity of 90% or more to the nucleotide sequence of SEQ ID NO: 9 in the sequence listing and encoding a protein related to cyazofamid resistance function.

[0018] In the coding gene, the 660th nucleotide from the 5' end is T, which is a genotype sensitive to cyazofamid.

[0019] When the 660th nucleotide from the 5' end is G, litchi downy mildew fungus shows resistance to cyazofamid.

[0020] In one embodiment of the present invention, the encoding gene is as shown in SEQ ID NO: 9, and the 660th nucleotide from the 5' end of SEQ ID NO: 9 is T (k=t), which is a genotype sensitive to cyazofamid.

[0021] When the 660th nucleotide from the 5′ end of SEQ ID NO: 9 is G (k=g), the litchi downy mildew fungus exhibits resistance to cyazofamid.

[0022] The expression cassette, recombinant expression vector or transgenic recombinant bacteria containing the above-mentioned coding gene also fall within the protection scope of the present invention.

[0023] Another object of the present invention is to provide the use of cytochrome b protein of litchi downy mildew pathogen or its encoding gene in identifying drug resistance of litchi downy mildew pathogen.

[0024] In one embodiment of the present invention, by detecting whether the 220th amino acid from the N-terminus of the cytochrome b protein of litchi downy mildew pathogen is changed from phenylalanine to leucine, if the 220th amino acid is changed from phenylalanine to leucine, its resistance to cyazofamid is improved; wherein, the cytochrome b protein of litchi downy mildew pathogen is the above-mentioned protein.

[0025] By detecting that the 660th nucleotide from the 5' end of the gene encoding the cytochrome b protein of litchi downy mildew pathogen is G, if the 660th nucleotide is G, its resistance to cyazofamid is improved; the gene encoding the cytochrome b protein of litchi downy mildew pathogen is the encoding gene shown.

[0026] Another object of the present invention is to provide a primer set for detecting or assisting in detecting whether there is a mutation site in the PlCyt b gene in litchi downy mildew pathogen, which is composed of the DNA molecules described in SEQ ID NO: 6 and SEQ ID NO: 7 in the sequence table.

[0027] The application of the primer set in identifying the drug resistance of litchi downy mildew also belongs to the protection scope of the present invention; the drug resistance refers to the resistance of litchi downy mildew to cyazofamid.

[0028] The present invention also provides a method for detecting or assisting in detecting whether there is a mutation site in the PlCyt b gene in litchi downy mildew pathogen, comprising the following steps:

[0029] Using the genomic DNA of the litchi downy mildew strain to be tested as a template, PCR amplification is performed using the primer pair shown in SEQ ID NO: 6 and SEQ ID NO: 7. If SEQ ID NO: 6 and SEQ ID NO: 7 can amplify a 528 bp band, then the PlCyt b gene in the litchi downy mildew pathogen to be tested has or is a candidate for a mutation site.

[0030] In the PCR amplification, the annealing temperature of the primer pair SEQ ID NO: 6 and SEQ ID NO: 7 is 60°C.

[0031] In the method, the mutation site refers to the 660th nucleotide from the 5' end of the PlCyt b gene in the litchi downy mildew pathogen being G, thereby causing the 220th amino acid from the N end of the cytochrome b protein of the litchi downy mildew pathogen to change from phenylalanine to leucine.

[0032] In the method, the mutated cytochrome b protein of litchi downy mildew pathogen is as shown in SEQ ID NO:8 in the sequence list, wherein the 220th amino acid from the N-terminus of SEQ ID NO:8 is leucine (X=Leu); the mutated PlCyt b gene in litchi downy mildew pathogen is as shown in SEQ ID NO:9, wherein the 660th nucleotide from the 5' end of SEQ ID NO:9 is G (k=g).

[0033] The present invention also provides application of the above method in identifying drug resistance of litchi downy mildew pathogen; the drug resistance refers to the drug resistance of litchi downy mildew pathogen to cyazofamid.

[0034] The litchi downy mildew pathogen having the mutation site has or is a candidate for having drug resistance.

[0035] Another object of the present invention is to provide a mutation site of the PlCyt b gene or protein in litchi downy mildew pathogen.

[0036] The present invention provides a mutation site of the PlCyt b gene in the litchi downy mildew pathogen, where the 660th nucleotide from the 5' end of the genome sequence of the PlCyt b gene of the litchi downy mildew pathogen is G. The 660th nucleotide from the 5' end of the genome sequence of the PlCyt b gene is G, that is, the 660th nucleotide from the 5' end in SEQ ID NO:9.

[0037] The present invention provides a mutation site of the cytochrome b protein in the litchi downy mildew pathogen, wherein the 220th amino acid from the N-terminus of the cytochrome b protein in the litchi downy mildew pathogen is leucine. The 220th amino acid from the N-terminus of the cytochrome b protein is leucine, which is the 220th amino acid from the N-terminus in SEQ ID NO:8.

[0038] The application of any of the above mutation sites in identifying drug resistance of litchi downy mildew also falls within the protection scope of the present invention; in the above application, the litchi downy mildew pathogen with the mutation site has or is a candidate for drug resistance. The drug resistance refers to the resistance of litchi downy mildew pathogen to cyazofamid.

[0039] The molecular detection method provided by the present invention has high sensitivity, good stability and short detection cycle. It can be used to monitor the frequency of resistance genes of litchi downy mildew fungi to the fungicide cyazofamid in the field in a high-throughput manner and the occurrence and development of resistance, thereby achieving early warning of resistant diseases, guiding timely adjustment of the prevention and control strategy of litchi downy mildew disease, promoting scientific use of drugs, delaying the occurrence and development of drug resistance, and playing an important role in the sustainable management system of diseases.

[0040] The method provided by the present invention for detecting point mutations of litchi downy mildew pathogens that produce resistance to cyazofamid can perform high-sensitivity, simple and rapid molecular detection of resistant strains, and timely understand the dynamics of drug resistance development, which is of great significance for formulating reasonable disease management plans and effectively controlling the development of drug-resistant diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Comparison of amino acid sequences of sensitive and resistant strains of litchi downy mildew (F220L); In the figure, A, the PlCyt b gene pattern of litchi downy mildew and the position of the F220L point mutation on the gene; B, the amino acid sequence comparison of PlCyt b protein of litchi downy mildew cyazofamid-sensitive and resistant strains. HN10-1, GDSZ-3, FJZZ-11 and FJFZ-34 are sensitive strains; GDSZ-3-1, GDSZ-3-2, FJZZ-11-1, FJZZ-11-2, FJFZ-34-1 and FJFZ-34-2 are resistant strains.

[0042] Figure 2 Figure 2 is the binding mode of cyazofamid and PlCyt b protein of litchi Downy Phytophthora before and after mutation (F220L); in the figure, A, amino acids interacting with cyazofamid and PlCyt b protein; B, binding posture and binding energy of cyazofamid and PlCyt b protein; C, amino acids interacting with cyazofamid and F220L-PlCyt b protein; D, binding posture and binding energy of cyazofamid and F220L-PlCyt b protein.

[0043] Figure 3 Screening of AS-PCR primers and optimal annealing temperature for F220L point mutation; in the figure, A, F220L-F1; B, F220L-F2; C, F220L-F3; D, F220L-F4.

[0044] Figure 4 F220L point mutation AS-PCR detection of resistant strains of litchi downy mildew (60℃); in the figure, HN10-1, GDSZ-3, FJZZ-11 and FJF1-34 are sensitive strains; GDSZ-3-1, GDSZ-3-2, FJZZ-11-1, FJZZ-11-2, FJFZ-34-1 and FJFZ-34-2 are resistant strains. DETAILED DESCRIPTION

[0045] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0047] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0048] The above strains are only the materials used in the examples of the present invention. In fact, when applying the screening markers described in the present invention, any commercially available oomycete strains can be used.

[0049] In the text, "resistant strains" refer to litchi downy mildew fungi that are resistant to the fungicide cyazofamid; "sensitive strains" refer to litchi downy mildew fungi that are sensitive to the fungicide cyazofamid.

[0050] The litchi downy mildew strains used in the following examples are: strains HN10-1, GDSZ-3, FJZZ-11 and FJFZ-34 are sensitive strains, and strains GDSZ-3-1, GDSZ-3-2, FJZZ-11-1, FJZZ-11-2, FJFZ-34-1 and FJFZ-34-2 are resistant strains.

[0051] The above strain HN10-1 was collected from Haikou City, Hainan Province, China; GDSZ-3 was collected from Shenzhen City, Guangdong Province, China; FJZZ-11 was collected from Zhuzhou City, Fujian Province, China; FJFZ-34 was collected from Fujian City, Fujian Province, China; GDSZ-3-1 and GDSZ-3-2 were obtained by domesticating GDSZ-3 with drugs under laboratory conditions; FJZZ-11-1 and FJZZ-11-2 were obtained by domesticating FJZZ-11 with drugs under laboratory conditions; FJFZ-34-1 and FJFZ-34-2 were obtained by domesticating FJFZ-34 with drugs under laboratory conditions. All strains were identified as litchi downy mildew pathogens by existing morphological and molecular biological methods. The above strains were all verified for drug resistance by colony growth assay.

[0052] Example 1: Induction of resistant mutants of Lychee Downy Phytophthora to cyazofamid

[0053] Sensitive strains of litchi downy mildew pathogen HN10-1, GDSZ-3, FJZZ-11 and FJFZ-34 were used as parents for the induction test of resistant mutants.

[0054] V8 medium: V8 vegetable juice 100 mL, CaCO3 1.4 g, agar powder 15 g, distilled water to 1 L, moist heat sterilization at 121℃ for 20 min.

[0055] The experimental steps are as follows:

[0056] 1) The parent of the litchi downy mildew pathogen was pre-cultured on V8 medium at 25°C for 7 days, and bacterial cakes with a diameter of 5 mm were obtained, 150 pieces / dish;

[0057] 2) Inoculate the bacterial cake with the mycelium side facing downward on a medium containing 0.05 μg / mL cyazofamid and culture at 25°C in the dark for 10 days;

[0058] 3) After new hyphae grow around the bacterial cake, inoculate the newly grown hyphae onto a V8 medium without a drug for expansion culture, then take the bacterial cake according to step 1) and inoculate it onto a medium containing 0.1 μg / mL of cyazofamid for further culture;

[0059] 4) Repeat step 3), and the drug concentration in the culture medium is gradually increased to 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL. Until the strain that can grow normally on the 1 μg / mL drug-containing medium and remains resistant after five generations on the blank V8 medium is screened, it is the cyazofamid-resistant mutant.

[0060] Example 2: Sensitivity of Lychee Downy Phytophthora to Cyazofamid

[0061] The resistant strains of litchi downy mildew were GDSZ-3-1, GDSZ-3-2, FJZZ-11-1, FJZZ-11-2, FJFZ-34-1 and FJFZ-34-2; the sensitive strains of litchi downy mildew were HN10-1, GDSZ-3, FJZZ-11 and FJFZ-34.

[0062] V8 medium: V8 vegetable juice 100 mL, CaCO3 1.4 g, agar powder 15 g, distilled water to 1 L, moist heat sterilization at 121℃ for 20 min.

[0063] The experimental steps are as follows:

[0064] 1) Cyazofamid was prepared with dimethyl sulfoxide (DMSO) as solvent at a concentration of 10 4 μg / mL mother solution. The sensitivity of sensitive strains of litchi downy mildew to cyazofamid was determined by diluting the cyazofamid mother solution with DMSO into working solutions of corresponding concentrations. The final concentrations of cyazofamid in the drug-containing medium were 0.002μg / mL, 0.004μg / mL, 0.008μg / mL, 0.01μg / mL, 0.02μg / mL and 0.04μg / mL, respectively. The sensitivity of resistant strains of litchi downy mildew to cyazofamid was determined by diluting the cyazofamid mother solution with DMSO into working solutions of corresponding concentrations. The final concentrations of cyazofamid in the drug-containing medium were 0.1μg / mL, 1μg / mL, 5μg / mL, 10μg / mL and 20μg / mL, respectively. Salicylic acid (SHAM) with a final concentration of 100μg / mL was used throughout the experiment to inhibit the bypass oxidation pathway of the pathogen.

[0065] 2) Cool the sterilized V8 medium to 45°C, add 100 μL of the corresponding drug working solution to 100 mL of V8 medium and mix well, pour the drug-containing medium into a culture dish with a diameter of 9 cm, use SHAM treatment as the blank control, and set up 3 technical replicates for each concentration of drug-containing medium.

[0066] 3) After pre-culturing litchi downy mildew on a V8 plate for 4 days, a bacterial cake with a diameter of 0.5 cm was punched along the edge of the colony using a borer, and the mycelium surface was inoculated downward on the drug-containing and control culture medium plates prepared in step 2), and the plates were placed in a 25°C incubator in the dark for incubation. The colony diameter was measured after 4 days.

[0067] 4) The colony diameter was measured by the cross method, and the inhibition rate of each concentration of the agent on the mycelial growth of the test strain was calculated based on the colony diameter. The inhibition rate was then converted into a probability value (Y), and the concentration of the agent was converted into a logarithmic value with a base of 10 (X). The virulence regression equation Y = a + bX and the correlation coefficient R of the tested litchi downy mildew strain were respectively obtained using the DPS data processing software. 2and effectively inhibited the concentration of EC 50 value and calculate the resistance multiple.

[0068] Resistance multiple = EC of resistant strain 50 EC / susceptible strains 50

[0069] The results are shown in Table 1:

[0070] Table 1 Sensitivity of tested litchi downy mildew strains to cyazofamid

[0071] strain Virulence regression equation <![CDATA[EC 50 ]]> <![CDATA[R 2 ]]> HN10-1 Y=8.63+1.67X 0.0066 0.9765 GDSZ-3 Y=9.04+1.91X 0.0077 0.9849 FJZZ-11 Y=9.72+2.10X 0.0057 0.9943 FJFZ-34 Y=9.63+2.05X 0.0055 0.9792 GDSZ-3-1 / >20 / GDSZ-3-2 / >20 / FJZZ-11-1 / >20 / FJZZ-11-2 >20 / FJFZ-34-1 / >20 / FJFZ-34-2 / >20 /

[0072] The sensitivity of litchi downy mildew to cyazofamid was tested and the results showed that the average EC values ​​of litchi downy mildew sensitive strains HN10-1, GDSZ-3, FJZZ-11 and FJFZ-34 were 50 was 0.006375 μg / mL, while the EC of the resistant strains GDSZ-3-1, GDSZ-3-2, FJZZ-11-1, FJZZ-11-2, FJFZ-34-1 and FJFZ-34-2 50 Greater than 20 μg / mL, significantly higher than the EC of sensitive strains 50 As shown in Table 1.

[0073] Example 3: Discovery of mutation sites of the PlCytb gene and cytochrome b protein in Phytophthora litchii

[0074] 1. Strain culture

[0075] 1. Strains: The resistant strains are GDSZ-3-1, GDSZ-3-2, FJZZ-11-1, FJZZ-11-2, FJFZ-34-1 and FJFZ-34-2; the sensitive strains are HN10-1, GDSZ-3, FJZZ-11 and FJFZ-34.

[0076] 2. Methods:

[0077] Strain culture: V8 medium (V8 vegetable juice 100mL, CaCO3 1.4g, agar powder 15g, distilled water to 1L, 121℃, sterilized for 20min) was used to culture litchi downy mildew at 25℃. The pre-cultured litchi downy mildew pathogens sensitive and resistant strains were inoculated on V8 medium covered with cellophane, cultured in the dark at 25℃ for 4 days, collected mycelium, frozen in liquid nitrogen and stored at -80℃ for genomic DNA extraction.

[0078] 2. Extract genomic DNA of sensitive and resistant strains of litchi downy mildew respectively:

[0079] 1) Take an appropriate amount of mycelium frozen in liquid nitrogen, grind it into powder with a mortar and pestle, and place it in a 2 mL centrifuge tube;

[0080] 2) Add 800 μL of 3% CTAB solution (preheated at 65°C) to the centrifuge tube, and then place in a 65°C water bath for 1 hour, mixing by inversion for 15 minutes;

[0081] 3) Add equal volumes of phenol:chloroform:isoamyl alcohol (25:24:1) to the centrifuge tube, mix well, and centrifuge at 12000g for 10 min at room temperature;

[0082] 4) Pipette 600 μL of supernatant into a new 2 mL sterile centrifuge tube, add an equal volume of chloroform:isoamyl alcohol (24:1), place on a 3D shaker at 75 rpm, room temperature, for 10 min;

[0083] 5) Centrifuge at 12000g for 10 min at room temperature, take 400 μL of supernatant into a new centrifuge tube, add 0.6 times the volume of isopropanol, and place in a -20°C refrigerator for 30 min;

[0084] 6) Centrifuge at 12000g, 4°C, 10 min, remove the supernatant and keep the precipitate, which is DNA;

[0085] 7) Add 800 μL of 75% ethanol to the centrifuge tube and tap the tube wall to wash the DNA;

[0086] 8) 7500g, 4°C, 5 min, centrifuge to remove supernatant, and dry under reduced pressure to remove alcohol;

[0087] 9) Add 30-50 μL ddH2O to the tube containing DNA, measure the DNA concentration and purity, and store at -20°C for later use.

[0088] 3. Amplification of the cytochrome b gene PlCyt b of sensitive strains of litchi downy mildew

[0089] Primers PlCyt bF (5'-ATGAGATGGAATAAAAAATCATTATT TGCGGTTGT-3', SEQ ID NO: 1) and PlCyt bR (5'-TTATTTTTTATTAATATTATAACGTACTAAATAAGTTTCT-3', SEQ ID NO: 2) were used to amplify the full-length PlCyt b gene encoding litchi downy mildew.

[0090] PCR reaction system (20 μL reaction system): 2×Taq PCR Starmix 10 μL, PlCyt bF 1 μL (10 μM), PlCyt bR 1 μL (10 μM), genomic DNA 100 ng (1 μL), ddH2O to 20 μL. PCR reaction program is as follows: 94.0℃ for 3 min; then 94.0℃ for 30 s, 55℃ for 30 s, 72.0℃ for 80 s, 35 cycles; finally 72.0℃ for 10 min; stored at 12℃.

[0091] The PCR product was sequenced. The cytochrome b protein PlCyt b gene of the litchi downy mildew resistant strain is 1155 bp in length, has no introns, and has a sequence as shown in SEQ ID NO:9. The 660th nucleotide from the 5' end of SEQ ID NO:9 is G (k=g), encoding an amino acid sequence as shown in SEQ ID NO:8, and the 220th amino acid at the N-terminus of SEQ ID NO:8 is leucine (X=Leu).

[0092] 4. Sequence Alignment

[0093] The full length of the PlCyt b gene of the resistant strains GDSZ-3-1, GDSZ-3-2, FJZZ-11-1, FJZZ-11-2, FJFZ-34-1 and FJFZ-34-2 and the sensitive strains HN10-1, GDSZ-3, FJZZ-11 and FJFZ-34 were sequenced. The sequence was analyzed and compared by DANMAN software, and the mutation type F220L was found in the cyazofamid-resistant strains. The specific results are as follows:

[0094] In the sensitive strain, the 660th nucleotide from the 5' end of the genomic sequence of the gene Plcyt b is T, that is, the 660th nucleotide from the 5' end of SEQ ID NO:9 is T (k=t), and the corresponding Plcytb protein has a phenylalanine (Phe) at the 220th amino acid from the N-terminus, that is, the 220th amino acid from the N-terminus of SEQ ID NO:8 is phenylalanine (X=Phe), while the 660th nucleotide from the 5' end of the genomic sequence of the gene Plcyt b in the resistant strains GDSZ-3-1, GDSZ-3-2, FJZZ-11-1, FJZZ-11-2, FJF1-34-1 and FJF1-34-2 is G, that is, the 660th nucleotide from the 5' end of SEQ ID NO:9 is G (k=g) ( Figure 1 A), the 220th amino acid from the N-terminus of protein Plcyt b is leucine (Leu), that is, the 220th amino acid from the N-terminus of SEQ ID NO: 8 is leucine (X=Leu) ( Figure 1 B).

[0095] The results showed that the resistant strain had a F220L mutation type on the PlCyt b protein, and a point mutation from T to G occurred at the 660th position from the 5' end of the genome. Analysis of the sequencing peak line graph showed an obvious single peak, and this base change also caused the mutation of phenylalanine at position 220 to leucine.

[0096] SEQ ID NO:8 (wherein X is F or L):

[0097]

[0098]

[0099] SEQ ID NO:9 (wherein k=g or t):

[0100]

[0101] Example 4: Verification of resistance-related mutation sites in litchi downy mildew pathogen

[0102] 1. Sequence Alignment

[0103] The amino acid sequence of the PlCyt b protein of litchi downy mildew was sequenced in our laboratory. The crystal structure in the protein data bank (PDB) was searched using the Protein BLAST tool of NCBI. In the search results, the sequence identity between the crystal structure 3L75 and the PlCyt b protein reached 53.75%, and 3L75 contained a heme substrate structure. Therefore, the 3L75 structure can be used as a template for constructing the PlCyt b structure.

[0104] 2. Homology Modeling

[0105] Based on the template protein three-dimensional structure file and sequence alignment file, the structural similarity was inferred from the sequence similarity, and the Modeller v9.19 program was used to perform homology modeling on the PlCyt b protein to obtain a reasonable three-dimensional structural model of the target protein, and the protein model was optimized by molecular mechanics.

[0106] The structure of the PlCyt b protein model was optimized using the Amber14 force field. The optimization process was carried out in two steps: first, 2000 steps of steepest descent optimization were performed, and then 2000 steps of conjugate gradient method were used to further optimize the structure. The final result was used as the model for subsequent analysis.

[0107] 3. Molecular Docking

[0108] The constructed PLCyt b protein and PLCyt b-F220L protein structures were subjected to molecular mechanics optimization and used as receptors for molecular docking. Molecular docking was implemented using the AutoDock4.2.6 software package. The structure of the substrate cyazofamid was obtained, hydrogenated, and the MOPAC program was used to optimize the structure and calculate the PM3 atomic charge. Finally, Autodock Tools 1.5.6 was used to process the structures of the ligand and receptor respectively, and the docking box was wrapped around the active site. The number of grid points in the XYZ direction was set to 25.13×25.13×25.13, and the grid spacing was The antimycin A bound to the Qi site was used as the center of the docking box, the number of dockings was set to 200, and the other parameters used the default values.

[0109] 4. Results Analysis

[0110] Interaction of Cyazofamid with PlCyt b protein and its mutant F220L

[0111] In order to analyze the driving force of the binding of cyazofamid to PLCyt b protein and mutant F220L, the interaction pattern of cyazofamid-protein was analyzed. Cyazofamid is located in a hydrophobic pocket composed of 10 amino acid residues including His15, Leu16, Tyr19, Gly32, Ser33, Gly36, Ile39, Leu197, His201 and Phe220 ( Figure 2 A). Phe220 and cyazofamid form a π-π covalent bond, which can stabilize the binding between cyazofamid and PLCyt b with an affinity of -5.37 kcal / mol ( Figure 2 B). However, after the F220L point mutation of the PlCyt b protein, cyazofamid binds to a hydrophobic pocket consisting of 10 amino acid residues, namely Gly32, Ser33, Gly36, Ile37, Leu39, Val40, Val190, Val193, Leu194 and Leu197 ( Figure 2 C), although the number of residues involved in molecular recognition has not decreased, after the F220L mutation, due to the influence of steric hindrance, the posture of cyazofamid has changed, affecting the binding conformation of the molecule, resulting in a weakening of its binding ability with the target -5.04kcal / mol ( Figure 2 D), thereby making the PlCyt b-F220L (the 220th amino acid from the N-terminus of SEQ ID NO: 8 is leucine (X=Leu)) mutant strain show resistance.

[0112] V. Conclusion

[0113] The molecular docking method was used to study the reasons why mutant F220L produces resistance. The molecular docking results showed that cyazofamid can bind to the catalytic active site of the protein. The mutation of Phe220 to Leu220 can change the binding mode of cyazofamid with the target, weaken the hydrophobic interaction, and reduce its binding ability with the active center, thereby making the PlCyt b-F220L mutant strain show drug resistance.

[0114] Example 5: Method for detecting mutation sites in litchi downy mildew pathogen

[0115] 1. Primer Design

[0116] The strain was the strain used in Example 1, and the primers were as shown in Table 2. Allele Specific-PCR primers were designed according to the sequence of the mutant PlCyt b gene, the last base at the 3'-end of the forward primer was consistent with the mutated base, and in order to increase the specificity of the primer, mismatched bases A, T, G and C were introduced at the second base at the 3'-end of the primer, respectively; the reverse primers were as shown in Table 2.

[0117] Table 2. Primers used in AS-PCR detection of cyazofamid resistance in litchi downy mildew

[0118]

[0119] 2. Primer Screening

[0120] In this method, the last base of the upstream primer is consistent with the mutant, the penultimate base is also changed at the same time, and amplification is performed in combination with different annealing temperatures to improve primer specificity.

[0121] The PCR reaction system is as follows:

[0122] 20 μL PCR system: 2×Taq PCR Starmix 10 μL, F220L-F (1 / 2 / 3 / 4) 1 μL (10 μM), F220L-R 1 μL (10 μM), genomic DNA 100 ng (1 μL), and ddH2O to make up to 20 μL.

[0123] The PCR reaction conditions were as follows: 94°C for 3 min first; then 94°C for 30 s, 58-68°C for 30 s, 72°C for 40 s, 35 cycles; finally 72°C for 10 min; and stored at 12°C.

[0124] The results of PCR product amplification showed that ( Figure 3 ), primer F220L-F1 can amplify bands in both resistant and sensitive strains under the annealing temperature of 58-60℃, and cannot be used to distinguish between resistant and sensitive strains ( Figure 3A). Primers F220L-F2 / 3 / 4 can amplify obvious bands from resistant strains under 60°C annealing temperature conditions, but cannot amplify corresponding bands in sensitive strains ( Figure 3 B. Figure 3 C. Figure 3 D). Among them, primer F220L-F4 can still be used to distinguish resistant strains from sensitive strains at 61.8°C, and the optimal annealing temperature range is relatively wide. Considering the universality of primers, we selected primers F220L-F4 / F220L-R as AS-PCR primers, and 60°C was the annealing temperature. The amplification product of primer pair F220L-F4 / F220L-R is nucleotides 628-1155 in SEQ ID NO:9.

[0125] 3. Detection of mutation sites

[0126] The genomic DNA of the litchi downy mildew pathogen to be tested was used as a template, and PCR amplification was performed with primer pair F220L-F4 / F220L-R, and then agarose gel electrophoresis was performed. If a 528 bp fragment could be amplified from the genomic DNA of the strain, the strain was determined to be a resistant strain. If the fragment could not be amplified, the litchi downy mildew pathogen strain tested was determined to be a sensitive strain.

[0127] The PCR amplification system and reaction conditions were consistent with those in the experiment, and the annealing temperature was 60°C.

[0128] The strains to be tested are: sensitive strains HN10-1, GDSZ-3, FJZZ-11 and FJF1-34; resistant strains GDSZ-3-1, GDSZ-3-2, FJZZ-11-1, FJZZ-11-2, FJF1-34-1 and FJF1-34-2.

[0129] The results showed that primers F220L-F4 / F220L-R could only amplify the target fragment from the resistant strain, but could not amplify the band from the sensitive strain. Therefore, using primers F220L-F4 / F220L-R for PCR amplification at an annealing temperature of 60°C can effectively detect the resistance of litchi downy mildew to cyazofamid ( Figure 4 ).

[0130] Therefore, the primer pair F220L-F4 / F220L-R can be used to effectively detect the resistance of litchi downy mildew to cyazofamid.

Claims

1. A protein related to resistance of litchi downy mildew to cyazofamid, which is a protein consisting of the amino acid residue sequence of SEQ ID NO: 8 in the sequence table; wherein, The 220th amino acid from the N-terminus of SEQ ID NO: 8 is leucine.

2. The gene encoding the protein related to the resistance of litchi downy mildew to cyazofamid according to claim 1.

3. The coding gene according to claim 2, characterized in that: The cDNA nucleotide sequence of the encoding gene is shown in the nucleotide sequence of SEQ ID NO: 9 in the sequence listing; wherein the 660th nucleotide from the 5' end of SEQ ID NO: 9 is G.

4. An expression cassette, a recombinant expression vector or a transgenic recombinant bacterium containing the coding gene according to claim 2 or 3.

5. Application of cytochrome b protein of litchi downy mildew pathogen or its encoding gene in identifying resistance of litchi downy mildew pathogen to cyazofamid; in, By detecting whether the 220th amino acid from the N-terminus of the cytochrome b protein of litchi downy mildew pathogen is changed from phenylalanine to leucine, if the 220th amino acid is changed from phenylalanine to leucine, the resistance to cyazofamid is improved; wherein the cytochrome b protein of litchi downy mildew pathogen is the protein according to claim 1; By detecting that the 660th nucleotide from the 5' end of the coding gene of the cytochrome b protein of the litchi downy mildew pathogen is G, if the 660th nucleotide is G, the resistance to cyazofamid is improved; the coding gene of the cytochrome b protein of the litchi downy mildew pathogen is the coding gene shown in claim 3.

6. A primer set for detecting or assisting in detecting whether there is a cyazofamid resistance-related mutation site in the cytochrome b gene of litchi downy mildew pathogen, consisting of the DNA molecules shown in SEQ ID NO: 6 and SEQ ID NO: 7 in the sequence table.

7. Use of the primer set shown in claim 6 in identifying drug resistance of litchi downy mildew; the drug resistance is the resistance of litchi downy mildew to cyazofamid; by detecting whether the 220th amino acid from the N-terminus of the cytochrome b protein of litchi downy mildew is changed from phenylalanine to leucine, if the 220th amino acid is changed from phenylalanine to leucine, its resistance to cyazofamid is improved; wherein, The cytochrome b protein of litchi downy mildew pathogen is the protein described in claim 1.

8. A method for detecting or assisting in detecting whether there is a cyazofamid resistance-related mutation site in the cytochrome b gene of litchi downy mildew pathogen, comprising the following steps: The genomic DNA of the litchi downy mildew pathogen to be tested is used as a template, and PCR amplification is performed using the primer pair shown in SEQ ID NO: 6 and SEQ ID NO:

7. If SEQ ID NO: 6 and SEQ ID NO: 7 can amplify a 528 bp band, then the cytochrome b gene in the litchi downy mildew pathogen to be tested has a mutation site or a candidate has a mutation site; the mutated litchi downy mildew pathogen cytochrome b protein is shown in SEQ ID NO: 8 in the sequence table, wherein, The 220th amino acid from the N-terminus of SEQ ID NO: 8 is leucine; the mutated cytochrome b gene in the litchi downy mildew pathogen is shown in SEQ ID NO: 9 in the sequence listing, wherein the 660th nucleotide from the 5' end of SEQ ID NO: 9 is G.

9. The method according to claim 8, characterized in that In the PCR amplification, the annealing temperature of SEQ ID NO: 6 and SEQ ID NO: 7 is 60°C.

10. Use of the method of claim 8 or 9 in identifying the drug resistance of litchi downy mildew; the drug resistance is the resistance of litchi downy mildew to cyazofamid.

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