A method for rapid identification of litchi downy mildew pathogen resistance to pyraclostrobin

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 fungicide pyraclostrobin, and specifically discloses a molecular detection method and special primers for identifying the resistance of litchi downy mildew pathogen PlCyt b gene 98 nucleotide or 682 nucleotide mutation to pyraclostrobin. 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 and the occurrence and development of resistance of litchi downy mildew pathogen to fungicide pyraclostrobin in the field.

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 fungicide pyraclostrobin resistance detection, specifically discloses a molecular detection method and special primers for rapid identification of nucleotide point mutations on PlCyt b gene of litchi downy mildew pathogen and its resistance to pyraclostrobin, 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, cyazofamid and dimethomorph. 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] Azopyrim is a new type of oomycete inhibitor developed by BASF, mainly used to control Phytophthora and downy mildew on crops such as potatoes, grapes, cucumbers and peppers. Azopyrim is a triazolopyrimidine fungicide that can bind to the Qo-distal site (Qo-distal) on the cytochrome bc1 complex (complex III) in mitochondria to block the electron transfer of the respiratory electron transport chain. The latest research results show that azopyrim is a dual-site fungicide that can bind not only to the Qo-distal site on complex III, but also to the Qi-site. Therefore, the Fungicide Resistance Action Committee FRAC classifies it as a QioI fungicide. Molecular docking results show that the binding mode of azopyrim to the cytochrome bc1 complex is different from that of traditional QoI and QiI fungicides, so it is believed that there is no cross-resistance between this agent and commonly used oomycete inhibitors such as myclobutanil, cyazofamid, and fenoxam-butyl. In 2019, pyraclostrobin obtained pesticide registration in my country. It can be used in combination with dimethomorph for the prevention and control of diseases of litchi, grapes, potatoes, tomatoes, cucumbers and peppers. The main targets of prevention and control include litchi downy mildew, grape downy mildew, cucumber downy mildew, potato late blight, pepper blight and other diseases. The current Fungicide Resistance Raction Committee (FRAC) has classified its resistance risk as medium to high, requiring resistance risk management. This laboratory obtained a litchi downy mildew strain with a high level of resistance to pyraclostrobin through the method of drug domestication, and some high-level resistant strains had a high survival fitness, indicating that the resistance risk of litchi downy mildew to pyraclostrobin is medium to high. Therefore, when using pyraclostrobin to control plant pathogenic oomycete diseases, it is necessary to strengthen resistance monitoring and provide timely early warning for the occurrence of pyraclostrobin resistance, so as to guide the scientific use of pyraclostrobin, 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 pyraclostrobin and its encoding gene, as well as its application as a screening marker for the resistance of litchi downy mildew to pyraclostrobin in the identification of the resistance of litchi downy mildew to pyraclostrobin.

[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 resistance to pyraclostrobin 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: 11 in the sequence listing;

[0010] 2) A protein derived from SEQ ID NO: 11 in the sequence listing, wherein one or more amino acid residues are substituted and / or deleted and / or added to the amino acid residue sequence of SEQ ID NO: 11 and associated with the pyraclostrobin resistance function.

[0011] Among them, the protein related to resistance to pyraclostrobin of litchi downy mildew pathogen has a genotype sensitive to pyraclostrobin in which the 33rd amino acid is serine and the 228th amino acid is aspartic acid. When the 33rd amino acid from the N-terminus mutates to leucine or the 228th amino acid mutates to asparagine (the mutation site exists alone), the litchi downy mildew pathogen shows resistance to pyraclostrobin.

[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:11. When the 33rd amino acid at the N-terminus of SEQ ID NO:11 is serine (Xaa=Ser) and the 228th amino acid is aspartic acid (Xaa=Asp), the litchi downy mildew is sensitive to pyraclostrobin. When the 33rd amino acid from the N-terminus of SEQ ID NO:11 is leucine (Xaa=Leu) and / or the 228th amino acid is asparagine (Xaa=Asn), the litchi downy mildew is resistant to pyraclostrobin.

[0013] The present invention also provides a gene encoding a protein related to pyraclostrobin 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: 12 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 the pyraclostrobin resistance function;

[0017] 3) A DNA molecule having more than 90% homology with the nucleotide sequence of SEQ ID NO: 12 in the sequence listing and encoding a protein related to the function of mefenamic acid resistance.

[0018] In the coding gene, the 98th nucleotide from the 5' end is C and the 682nd nucleotide from the 5' end is G, which is a genotype sensitive to pyraclostrobin.

[0019] When the 98th nucleotide from the 5' end is T and / or the 682nd nucleotide from the 5' end is A, the litchi downy mildew pathogen exhibits resistance to mefenamic acid.

[0020] In one embodiment of the present invention, the encoding gene is as shown in SEQ ID NO: 12, the 98th nucleotide from the 5' end of SEQ ID NO: 12 is C (y=c) and the 682nd nucleotide from the 5' end is G (r=g), which is a genotype sensitive to pyraclostrobin.

[0021] When the 98th nucleotide from the 5' end of SEQ ID NO: 12 is T (y=t) and / or the 682nd nucleotide from the 5' end of SEQ ID NO: 12 is A (r=a), the litchi downy mildew pathogen exhibits resistance to mefenamic acid.

[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 33rd amino acid from the N-terminus of the cytochrome b protein of litchi downy mildew pathogen is changed from serine to leucine or whether the 228th amino acid from the N-terminus is changed from aspartic acid to asparagine, if the 33rd position is changed from serine to leucine or the 228th position is changed from aspartic acid to asparagine, its resistance to pyraclostrobin is improved; wherein, the cytochrome b protein of litchi downy mildew pathogen is the above-mentioned protein.

[0025] By detecting that the 98th nucleotide from the 5' end of the coding gene of the cytochrome b protein of the litchi downy mildew pathogen is T or the 682nd nucleotide from the 5' end is A, if the 98th nucleotide is T or the 682nd nucleotide is A, its resistance to pyraclostrobin is improved; the coding gene of the cytochrome b protein of the litchi downy mildew pathogen is the coding 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:3 and SEQ ID NO:6 in the sequence list or the DNA molecules shown in SEQ ID NO:7 and SEQ ID NO:10 in the sequence list.

[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 pyraclostrobin.

[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] Taking the genomic DNA of the litchi downy mildew strain to be tested as a template, PCR amplification is performed using the primer pairs shown in SEQ ID NO:3 and SEQ ID NO:6 or SEQ ID NO:7 and SEQ ID NO:10. If SEQ ID NO:3 and SEQ ID NO:6 can amplify a 794 bp band or SEQ ID NO:7 and SEQ ID NO:10 can amplify a 505 bp band, then the PlCyt b gene in the litchi downy mildew pathogen to be tested has a mutation site or a candidate has a mutation site.

[0030] In the PCR amplification, the annealing temperature of the primer pair SEQ ID NO: 3 and SEQ ID NO: 6 is 64.1°C, and the annealing temperature of the primer pair SEQ ID NO: 7 and SEQ ID NO: 10 is 60°C.

[0031] In the method, the mutation site refers to the 98th nucleotide from the 5' end of the PlCyt b gene in the litchi downy mildew pathogen being T or the 682nd nucleotide from the 5' end being A, thereby causing the 33rd amino acid from the N-terminus of the cytochrome b protein of the litchi downy mildew pathogen to change from serine to leucine or the 228th amino acid from the N-terminus to change from aspartic acid to asparagine.

[0032] In the method, the mutated cytochrome b protein of litchi downy mildew pathogen is as shown in SEQ ID NO:11, wherein the 33rd amino acid from the N-terminus of SEQ ID NO:11 is leucine (X=Leu) and / or the 228th amino acid is asparagine (X=Asn), and the mutated PlCyt b gene in litchi downy mildew pathogen is as shown in SEQ ID NO:12, wherein the 98th nucleotide from the 5' end of SEQ ID NO:12 is T (y=t) and / or the 682nd nucleotide from the 5' end of SEQ ID NO:12 is A (r=a).

[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 pyraclostrobin.

[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, wherein the 98th nucleotide from the 5' end of the genome sequence of the PlCyt b gene of the litchi downy mildew pathogen is T or the 682nd nucleotide from the 5' end is A, and the two nucleotide mutations exist alone.

[0037] The two mutation sites of the cytochrome b protein in the litchi downy mildew pathogen provided by the present invention are that the 33rd amino acid from the N-terminus of the cytochrome b protein in the litchi downy mildew pathogen is leucine or the 228th amino acid from the N-terminus is asparagine, and the two amino acid mutations exist separately.

[0038] The mutation site refers to the mutation of the 98th nucleotide from the 5' end to T or the mutation of the 682nd nucleotide from the 5' end to A in the PlCyt b gene in the litchi downy mildew pathogen relative to the genotype sensitive to pyraclostrobin. This causes the 33rd amino acid from the N-terminus of the cytochrome b protein of the litchi downy mildew pathogen to change from serine to leucine or the 228th amino acid from the N-terminus to change from aspartic acid to asparagine.

[0039] The application of any of the above mutation sites in identifying the 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 pyraclostrobin.

[0040] The molecular detection method provided by the present invention has high sensitivity, good stability and short detection cycle, and can be used to monitor the frequency of resistance genes of litchi downy mildew pathogens to the fungicide pyraclostrobin in the field and the occurrence and development of resistance in a high-throughput manner, 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.

[0041] The method provided by the present invention for detecting point mutations of litchi downy mildew pathogens that produce resistance to pyraclostrobin can perform high-sensitivity, simple and rapid molecular detection of resistant strains, and timely understand the dynamics of drug resistance, 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

[0042] Figure 1 Comparison of amino acid sequences of sensitive and resistant strains of litchi downy mildew (S33L). A. PlCyt b gene pattern of litchi downy mildew and the location of the S33L point mutation on the gene; B. Comparison of amino acid sequences of PlCyt b proteins of litchi downy mildew sensitive and resistant strains of pyraclostrobin. HN10-1, HN10-2, HN10-3 and GX10-1 are sensitive strains; HN10-1-1, HN10-1-2, HN10-2-1, HN10-2-2, HN10-3-1, HN10-3-2, GX10-1-1 and GX10-1-2 are resistant strains.

[0043] Figure 2Comparison of amino acid sequences of sensitive and resistant strains of litchi downy mildew (D228N). A. PlCyt b gene pattern of litchi downy mildew and the location of the D228N point mutation on the gene; B. Comparison of amino acid sequences of PlCyt b proteins of litchi downy mildew sensitive and resistant strains of pyraclostrobin. HN10-1, HN10-2, GDSZ-3, GX10-1 and FJ10-1 are sensitive strains; FJ10-1-1, FJ10-1-2, GX10-1-3 and GX10-1-4 are resistant strains.

[0044] Figure 3 Figure 2 shows the binding mode of pyraclostrobin and PlCyt b protein of litchi Downy Phytophthora before and after mutation (S33L)A. Amino acids interacting with pyraclostrobin and PlCyt b protein; B. Binding posture and binding energy of pyraclostrobin and PlCyt b protein; C. Amino acids interacting with pyraclostrobin and S33L-PlCyt b protein; D. Binding posture and binding energy of pyraclostrobin and S33L-PlCyt b protein.

[0045] Figure 4 Figure 2 shows the binding mode of pyraclostrobin and PlCyt b protein of litchi Downy Phytophthora before and after mutation (D228N)A The amino acids interacting with pyraclostrobin and PlCyt b protein; B The binding posture and binding energy of pyraclostrobin and PlCyt b protein; C The amino acids interacting with pyraclostrobin and D228N-PlCyt b protein; D The binding posture and binding energy of pyraclostrobin and D228N-PlCyt b protein.

[0046] Figure 5 AS-PCR primers and optimal annealing temperature were screened for S33L point mutation: S33L-F1; BS33L-F2; CS33L-F3.

[0047] Figure 6 Screening of AS-PCR primers and optimal annealing temperature for D228N point mutation; A is D228N-F1; B is D228N-F2; C is D228N-F3.

[0048] Figure 7 The S33L point mutation was detected by AS-PCR to detect the resistant strains of litchi downy mildew (64.1℃); HN10-1, HN10-2, HN10-3, GX10-1 and FJ10-1 were sensitive strains; HN10-1-1, HN10-1-2, HN10-2-1, HN10-2-2, HN10-3-1, HN10-3-2, GX10-1-1 and GX10-1-2 were resistant strains.

[0049] Figure 8D228N point mutation AS-PCR was used to detect resistant strains of litchi downy mildew (60℃); HN10-1, HN10-2, GDSZ-3, GX10-1 and FJ10-1 were sensitive strains; FJ10-1-1, FJ10-1-2, GX10-1-3 and GX10-1-4 were resistant strains. DETAILED DESCRIPTION

[0050] 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.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0052] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0053] 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.

[0054] In the text, "resistant strains" refer to litchi downy mildew fungi that are resistant to the fungicide pyraclostrobin; "sensitive strains" refer to litchi downy mildew fungi that are sensitive to the fungicide pyraclostrobin.

[0055] The litchi downy mildew strains used in the following examples are: strains HN10-1, HN10-2, HN10-3, GX10-1, GXBL-4, GDSZ-3 and FJ10-1 are sensitive strains, and strains HN10-1-1, HN10-1-2, HN10-2-1, HN10-2-2, HN10-3-1, HN10-3-2, GX10-1-1, GX10-1-2, GX10-1-3, GX10-1-4, FJ10-1-1 and FJ10-1-2 are resistant strains.

[0056] The above strains HN10-1, HN10-2 and HN10-3 were collected from Haikou, Hainan Province, China; GX10-1 and GXBL-4 were collected from Beiliu, Guangxi, China; GDSZ-3 was collected from Shenzhen, Guangdong Province, China; FJ10-1 was collected from Ningde, Fujian Province, China; HN10-1-1 and HN10-1-2 were obtained by acclimating HN10-1 with drugs under laboratory conditions; HN10-2-1 and HN10-2-2 were obtained by acclimating HN10-2 with drugs under laboratory conditions ; HN10-3-1 and HN10-3-2 were obtained by taming HN10-3 with pesticides under laboratory conditions; GX10-1-1, GX10-1-2, GX10-1-3 and GX10-1-4 were obtained by taming GX10-1 with pesticides under laboratory conditions; FJ10-1-1 and FJ10-1-2 were obtained by taming FJ10-1 with pesticides 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.

[0057] Example 1: Sensitivity of Lychee Downy Phytophthora to Azopyram

[0058] The mutants resistant to litchi downy mildew are HN10-1-1, HN10-1-2, HN10-2-1, HN10-2-2, HN10-3-1, HN10-3-2, GX10-1-1, GX10-1-2, GX10-1-3, GX10-1-4, FJ10-1-1 and FJ10-1-2; the strains sensitive to litchi downy mildew are HN10-1, HN10-2, HN10-3, GX10-1, GXBL-4, GDSZ-3 and FJ10-1.

[0059] 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.

[0060] The experimental steps are as follows:

[0061] 1) Azopyrad was prepared with dimethyl sulfoxide (DMSO) as solvent at a concentration of 10 4μg / mL mother solution. The sensitivity of sensitive strains of litchi downy mildew to pyraclostrobin was determined by diluting the pyraclostrobin mother solution with DMSO into working solutions of corresponding concentrations. The final concentrations of pyraclostrobin in the drug-containing medium were 0.04μg / mL, 0.08μg / mL, 0.20μg / mL, 0.40μg / mL, 0.8μg / mL and 1μg / mL, respectively; The sensitivity of resistant strains of litchi downy mildew to pyraclostrobin was determined by diluting the pyraclostrobin mother solution with DMSO into working solutions of corresponding concentrations. The final concentrations of pyraclostrobin in the drug-containing medium were 5μg / mL, 10μg / mL, 20μg / mL, 40μg / mL and 100μg / mL. Salicylic acid (SHAM) with a final concentration of 100μg / mL was used throughout the experiment to inhibit the bypass oxidation pathway of the pathogen.

[0062] 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.

[0063] 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.

[0064] 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. 2 and effectively inhibited the concentration of EC 50 value and calculate the resistance multiple.

[0065] Resistance multiple = EC of resistant strain 50 EC / susceptible strains 50

[0066] The results are shown in Table 1:

[0067] Table 1 Sensitivity of tested litchi downy mildew strains to pyraclostrobin

[0068] strain Virulence regression equation <![CDATA[EC 50 ]]> <![CDATA[R 2 ]]> HN10-1 Y=1.82+2.27X 0.158 0.997 HN10-2 Y=1.32+2.21X 0.231 0.952 HN10-3 Y=1.74+2.41X 0.179 0.983 GX10-1 Y=1.37+1.62X 0.144 0.977 GXBL-4 Y=1.01+1.94X 0.315 0.984 GDSZ-3 Y=6.00+2.19X 0.351 0.996 FJ10-1 Y=1.18+1.68X 0.190 0.966 HN10-1-1 / >100 / HN10-1-2 / >100 / HN10-2-1 / >100 / HN10-2-2 / >100 / HN10-3-1 / >100 / HN10-3-2 / >100 / GX10-1-1 / >100 / GX10-1-2 / >100 / GX10-1-3 / >100 / GX10-1-4 / >100 / FJ10-1-1 / >100 / FJ10-1-2 / >100 /

[0069] The sensitivity of litchi downy mildew to pyraclostrobin was tested and the results showed that the average EC values ​​of the sensitive strains of litchi downy mildew HN10-1, HN10-2, HN10-3, GX10-1, GXBL-4, GDSZ-3 and FJ10-1 were 50 was 0.224 μg / mL, while the EC of the resistant strains HN10-1-1, HN10-1-2, HN10-2-1, HN10-2-2, HN10-3-1, HN10-3-2, GX10-1-1, GX10-1-2, GX10-1-3, GX10-1-4, FJ10-1-1 and FJ10-1-2 50 Greater than 100 μg / mL, significantly higher than the EC of sensitive strains 50 As shown in Table 1.

[0070] Example 2: Discovery of mutation sites of the PlCytb gene and cytochrome b protein in Phytophthora litchii

[0071] 1. Strain culture

[0072] 1. Strains: The resistant strains are HN10-1-1, HN10-1-2, HN10-2-1, HN10-2-2, HN10-3-1, HN10-3-2, GX10-1-1, GX10-1-2, GX10-1-3, GX10-1-4, FJ10-1-1 and FJ10-1-2; the sensitive strains are HN10-1, HN10-2, HN10-3, GX10-1, GXBL-4, GDSZ-3 and FJ10-1.

[0073] 2. Methods:

[0074] 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.

[0075] 2. Extract genomic DNA of sensitive and resistant strains of litchi downy mildew pathogen respectively:

[0076] 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;

[0077] 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;

[0078] 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;

[0079] 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;

[0080] 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;

[0081] 6) Centrifuge at 12000g, 4°C, 10 min, remove the supernatant and keep the precipitate, which is DNA;

[0082] 7) Add 800 μL of 75% ethanol to the centrifuge tube and tap the tube wall to wash the DNA;

[0083] 8) 7500g, 4°C, 5 min, centrifuge to remove supernatant, and dry under reduced pressure to remove alcohol;

[0084] 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.

[0085] 3. Amplification of the cytochrome b gene PlCyt b of sensitive strains of litchi downy mildew

[0086] Primers PlCyt bF (5'-ATGAGATGGAATAAAAAATCATTATTTGCGGTTGT-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.

[0087] 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℃.

[0088] 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: 12, wherein the 98th nucleotide from the 5' end of SEQ ID NO: 12 is C (y=c) and the 682nd nucleotide from the 5' end is G (r=g), encoding an amino acid sequence as shown in SEQ ID NO: 11, wherein the 33rd amino acid at the N-terminus of SEQ ID NO: 11 is serine (X=Ser) and the 228th amino acid is aspartic acid (X=Asp).

[0089] SEQ ID NO:11:

[0090]

[0091] The 33rd amino acid from the N-terminus of SEQ ID NO: 11 is X = S or L, and the 228th amino acid from the N-terminus is X = D or N.

[0092] SEQ ID NO:12

[0093]

[0094]

[0095] The 98th nucleotide from the 5' end of SEQ ID NO: 12, y=c or t, and the 682nd nucleotide from the 5' end, r=g or a.

[0096] 4. Sequence Alignment

[0097] The full length of the PlCyt b gene of the resistant strains HN10-1-1, HN10-1-2, HN10-2-1, HN10-2-2, HN10-3-1, HN10-3-2, GX10-1-1, GX10-1-2, GX10-1-3, GX10-1-4, FJ10-1-1 and FJ10-1-2 and the sensitive strains HN10-1, HN10-2, HN10-3, GX10-1, GXBL-4, GDSZ-3 and FJ10-1 were sequenced. The sequences were analyzed and compared by DANMAN software, and two separate resistance mutation types, S33L and D228N, were found in the pyraclostrobin-resistant strains. The specific results are as follows:

[0098] In the sensitive strain, the 98th nucleotide from the 5' end of the genomic sequence of the gene PlCyt b is C (the 98th nucleotide from the 5' end of SEQ ID NO: 12 is C (y = c) and the 682nd nucleotide from the 5' end is G (r = g)), and the 33rd amino acid from the N-terminus of the corresponding PlCyt b protein is serine (Ser) (the 33rd amino acid from the N-terminus of SEQ ID NO: 11 is serine (X = Ser) and the 228th amino acid from the N-terminus is aspartic acid (X = Asp)), while the 98th nucleotide from the 5' end of the genomic sequence of the gene PlCytb in the resistant strains HN10-1-1, HN10-1-2, HN10-2-1, HN10-2-2, HN10-3-1, HN10-3-2, GX10-1-1 and GX10-1-2 is T (the 33rd amino acid from the N-terminus of SEQ ID NO: 11 is serine (X = Ser) and the 228th amino acid from the N-terminus is aspartic acid (X = Asp)). The 98th nucleotide from the 5' end of NO:12 is T (y=t) and the 682nd nucleotide from the 5' end is G (r=g)), the 33rd amino acid from the N-terminus of protein PlCyt b is leucine (Leu) (the 33rd amino acid from the N-terminus of SEQ ID NO:11 is leucine (X=Leu), and the 228th amino acid from the N-terminus is aspartic acid (X=Asp) Figure 1 ); the 682nd nucleotide from the 5' end of the PlCyt b gene of the sensitive strain is G (the 98th nucleotide from the 5' end of SEQ ID NO:12 is C (y=c) and the 682nd nucleotide from the 5' end is G (r=g)), and the corresponding protein PlCyt b protein has aspartic acid (Asp) at the 228th amino acid from the N-terminus (the 33rd amino acid from the N-terminus of SEQ ID NO:11 is serine (X=Ser) and the 228th amino acid is aspartic acid (X=Asp)); while the genomic sequence of the gene PlCyt b in the resistant strains FJ10-1-1, FJ10-1-2, GX10-1-3 and GX10-1-4 has A at the 682nd nucleotide from the 5' end (the 98th nucleotide from the 5' end of SEQ ID NO:12 is C (y=c) and the 682nd nucleotide from the 5' end is G (r=g)). The 98th nucleotide from the 5' end of NO:12 is C (y=c) and the 682nd nucleotide from the 5' end is A (r=a)), the 228th amino acid from the N-terminus of protein PlCyt b is asparagine (Asn) (the 33rd amino acid from the N-terminus of SEQ ID NO:11 is serine (X=Ser) and the 228th amino acid is asparagine (X=Asn)) Figure 2 ).

[0099] The results showed that the resistant strain had two separate mutation types on the PlCyt b protein, namely, a point mutation from C to T at the 98th position from the 5' end of the genome (the 98th nucleotide from the 5' end of SEQ ID NO:12 was T (y=t) and the 682nd nucleotide from the 5' end was G (r=g)) and a point mutation from G to A at the 682nd position (the 98th nucleotide from the 5' end of SEQ ID NO:12 was C (y=c) and the 682nd nucleotide from the 5' end was A (r=a)). Analysis of the peak line graph of the sequencing showed an obvious single peak, and the changes of these two bases also resulted in the mutation of serine at position 33 and histidine at position 228 to leucine and asparagine, respectively, that is, the 33rd amino acid at the N-terminus of SEQ ID NO:11 was serine (X=Ser) and the 228th amino acid was asparagine (X=Asn); or The 33rd amino acid from the N-terminus of NO:11 is leucine (X=Leu), and the 228th amino acid from the N-terminus is aspartic acid (X=Asp), which are two separate mutation types.

[0100] Example 4: Verification of resistance-related mutation sites in litchi downy mildew pathogen

[0101] 1. Sequence Alignment

[0102] 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 6ZFU and the PlCyt b protein reached 52.73%, and 6ZFU contained a heme substrate structure. Therefore, the 6ZFU structure can be used as a template for constructing the PlCyt b structure.

[0103] 2. Homology Modeling

[0104] 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.

[0105] 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.

[0106] 3. Molecular Docking

[0107] The constructed PLCyt b protein and PLCyt b-S33L 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 pyraclostrobin was obtained, hydrogenated, and the structure was optimized using the MOPAC program to calculate the atomic charge of PM3. 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.

[0108] IV. Results Analysis

[0109] 1) Interaction between pyraclostrobin and PlCyt b protein and its mutant S33L

[0110] In order to analyze the driving force of the binding of pyraclostrobin to PlCyt b protein and mutant S33L, the interaction mode of pyraclostrobin-protein was analyzed. Pyraclostrobin binds to the hydrophobic pocket composed of 12 amino acid residues including His15, Leu16, Tyr19, Gly32, Ser33, Gly36, Leu39, Val193, Leu197, His201, Phe220 and Asp228 ( Figure 3 A). Ser33 and pyraclostrobin form a hydrogen bond, which can stabilize the binding between pyraclostrobin and PlCyt b with an affinity of -4.98 kcal / moL ( Figure 3 B). However, after the S33L point mutation of the PlCyt b protein, pyraclostrobin binds to a hydrophobic pocket consisting of eight amino acid residues, namely Gly32, Leu33, Gly36, Ile37, Leu39, Val40, Val193 and Leu197 ( Figure 3 C), the number of residues involved in molecular recognition is reduced. In addition, after the S33L mutation, due to the influence of steric hindrance, the posture of pyraclostrobin changes, affecting the binding conformation of the molecule, resulting in a weakening of its binding ability with the target -3.66kcal / moL ( Figure 3 D), thereby making the PlCyt b-S33L (the 33rd amino acid from the N-terminus of SEQ ID NO: 11 is leucine (X=Leu) and the 228th amino acid from the N-terminus is aspartic acid (X=Asp)) mutant strain show resistance.

[0111] 2) Interaction between pyraclostrobin and PlCyt b protein and its mutant D228N

[0112] In order to analyze the driving force of the binding of pyraclostrobin to PlCyt b protein and mutant D228N, the interaction mode of pyraclostrobin-protein was analyzed. Pyraclostrobin binds to the hydrophobic pocket composed of 14 amino acid residues including His15, Leu16, Tyr19, Thr21, Leu25, Phe29, Gly30, Ser33, Leu200, His201, Ser205, Phe220, Lys227 and Asp228 ( Figure 4 A). Asp228 and pyraclostrobin form a hydrogen bond, which can stabilize the binding between pyraclostrobin and PlCyt b with an affinity of -5.94 kcal / moL ( Figure 4 B). However, after the D228N point mutation of the PlCyt b protein, pyraclostrobin binds to a hydrophobic pocket consisting of nine amino acid residues, namely His15, Leu16, Ile17, Tyr19, Ser33, Phe220, Tyr221, Tyr 225 and Asn228 ( Figure 4 C), the number of residues involved in molecular recognition is reduced. In addition, after the S33L mutation, due to the influence of steric hindrance, the posture of pyraclostrobin changes, affecting the binding conformation of the molecule, resulting in a weakening of its binding ability with the target -4.94kcal / moL ( Figure 4 D), thereby making the PlCyt b-D228N (the 33rd amino acid at the N-terminus of SEQ ID NO: 11 is serine (X=Ser) and the 228th amino acid is asparagine (X=Asn)) mutant strain show resistance.

[0113] V. Conclusion

[0114] The molecular docking method was used to study the reasons why mutants S33L and D228N produce resistance. The molecular docking results showed that pyraclostrobin can bind to the catalytic active site of the protein and bind near the heme. The mutation of Ser33 to Leu33 and / or Asp228 to Asn228 can change the binding mode of pyraclostrobin with the target, reduce the number of surrounding amino acid residues involved in recognition, weaken the hydrophobic interaction, and reduce its binding ability with the active center, thereby making the PlCyt b-S33L / D228N mutant strain show drug resistance.

[0115] Example 4: Method for detecting mutation sites in litchi downy mildew pathogen

[0116] 1. Primer Design

[0117] 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.

[0118] Table 2. Primers used in AS-PCR detection of litchi downy mildew pathogenic fungus to pyraclostrobin

[0119]

[0120]

[0121] 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.

[0122] The PCR reaction system is as follows:

[0123] 20 μL PCR system: 2×Taq PCR Starmix 10 μL, S33L / D228N-F (1 / 2 / 3) 1 μL (10 μM), S33L / D228N-R 1 μL (10 μM), genomic DNA 100 ng (1 μL), and ddH2O added to 20 μL.

[0124] 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.

[0125] The results of PCR product amplification showed that primers S33L-F2 and F3 could not distinguish resistant strains from sensitive strains when the annealing temperature was 58-68℃ ( Figure 5 B / C). S33L-F1 could only amplify a 794 bp band from the resistant strain at an annealing temperature of 64.1°C, but could not amplify a band from the sensitive strain ( Figure 5 A). Primers D228N-F2 and F3 cannot distinguish resistant strains from sensitive strains at annealing temperatures of 58-68°C ( Figure 6 B / C). D228N-F1 could only amplify a 505 bp band from the resistant strain at an annealing temperature of 60°C, but could not amplify a band from the sensitive strain ( Figure 6A). The above results show that the primer pairs S33L-F1 / S33L-R and D228N-F1 / D228N-R can detect the S33L and D228N resistance sites, respectively. Therefore, the primer pairs S33L-F1 / S33L-R (64.1°C) and D228N-F1 / D228N-R (60°C) were selected as AS-PCR primers to detect the above mutation sites. The amplification product of primers S33L-F1 / S33L-R is nucleotides 64-816 in SEQ ID NO: 12; the amplification product of primers D228N-F1 / D228N-R is nucleotides 643-1155 in SEQ ID NO: 12.

[0126] Detection of mutation sites

[0127] The genomic DNA of the litchi downy mildew pathogen to be tested is used as a template, and PCR amplification is performed with primer pairs S33L-F1 / S33L-R and D228N-F1 / D228N-R, and then agarose gel electrophoresis is performed. If a fragment can be amplified from the genomic DNA of the strain, the strain is determined to be a resistant strain. If a fragment cannot be amplified, the tested litchi downy mildew strain is determined to be a sensitive strain.

[0128] The PCR amplification system and reaction conditions were consistent with those in the experiment, the annealing temperature of the primer pair S33L-F1 / S33L-R was 64.1°C, and the annealing temperature of the primer pair D228N-F1 / D228N-R was 60°C.

[0129] The strains to be tested for primer pair S33L-F1 / S33L-R are: resistant strains HN10-1-1, HN10-1-2, HN10-2-1, HN10-2-2, HN10-3-1, HN10-3-2, GX10-1-1 and GX10-1-2; sensitive strains HN10-1, HN10-2, HN10-3, GX10-1 and FJ10-1.

[0130] The strains to be tested for the primer pair D228N-F1 / D228N-R are: resistant strains FJ10-1-1, FJ10-1-2, GX10-1-3 and GX10-1-4; sensitive strains HN10-1, HN10-2, GDSZ-3, GX10-1 and FJ10-1.

[0131] The results showed that the primer pair S33L-F1 / S33L-R could only amplify a 794 bp fragment from the resistant strain at an annealing temperature of 64.1°C, but could not amplify a band from the sensitive strain ( Figure 7). The primer pair D228N-F1 / D228N-R can only amplify a 505 bp fragment from the resistant strain at an annealing temperature of 60°C, but cannot amplify a band from the sensitive strain ( Figure 8 ).

[0132] Therefore, the primer pairs S33L-F1 / S33L-R and D228N-F1 / D228N-R can be used to effectively detect the resistant strains of litchi downy mildew pathogenic fungus to pyraclostrobin at 64.1°C and 60°C, respectively.

Claims

1. Application of cytochrome b protein of litchi downy mildew pathogen or its encoding gene in identifying drug resistance of litchi downy mildew pathogen; wherein, By detecting whether the 33rd amino acid from the N-terminus of the cytochrome b protein of litchi downy mildew pathogen changes from serine to leucine or whether the 228th amino acid changes from aspartic acid to asparagine, if the 33rd amino acid from the N-terminus changes from serine to leucine or the 228th amino acid changes from aspartic acid to asparagine, the resistance to pyraclostrobin is improved; wherein the mutated cytochrome b protein of litchi downy mildew pathogen is a protein composed of the amino acid residue sequence of SEQ ID NO: 11 in the sequence table, the 33rd amino acid from the N-terminus of SEQ ID NO: 11 is leucine and the 228th amino acid is aspartic acid; or, the 33rd amino acid from the N-terminus of SEQ ID NO: 11 is serine and the 228th amino acid is asparagine; By detecting that the 98th nucleotide from the 5' end of the coding gene of the cytochrome b protein of the litchi downy mildew pathogen is T or the 682nd nucleotide from the 5' end is A, if the 98th nucleotide is T or the 682nd nucleotide is A, its resistance to pyraclostrobin is improved; the nucleotide sequence of the coding gene of the cytochrome b protein of the litchi downy mildew pathogen after mutation is as shown in SEQ ID NO: 12 in the sequence table, the 98th nucleotide from the 5' end of SEQ ID NO: 12 is C and the 682nd nucleotide from the 5' end is A; or, the 98th nucleotide from the 5' end of SEQ ID NO: 12 is T and the 682nd nucleotide from the 5' end is G.

2. A method for detecting or assisting in detecting whether there is a mutation site in the cytochrome b gene in litchi downy mildew pathogen, and its application in identifying the drug resistance of litchi downy mildew pathogen, wherein the drug resistance is the drug resistance of litchi downy mildew pathogen to pyraclostrobin; the method comprises the following steps: Using the genomic DNA of the litchi downy mildew pathogen to be tested as a template, PCR amplification is performed using a primer pair consisting of a fragment shown in SEQ ID NO: 3 and a fragment shown in SEQ ID NO: 6, or a primer pair consisting of a fragment shown in SEQ ID NO: 7 and a fragment shown in SEQ ID NO:

10. If the primer pair consisting of a fragment shown in SEQ ID NO: 3 and a fragment shown in SEQ ID NO: 6 can amplify a 794 bp band, or the primer pair consisting of a fragment shown in SEQ ID NO: 7 and a fragment shown in SEQ ID NO: 10 can amplify a 505 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 mutation site refers to the 98th nucleotide from the 5' end of the cytochrome b gene in the litchi downy mildew pathogen being T or the 682nd nucleotide from the 5' end being A, thereby causing the 33rd amino acid from the N-terminus of the cytochrome b protein of the litchi downy mildew pathogen to change from serine to leucine or the 228th amino acid from the N-terminus to change from aspartic acid to asparagine; The mutated litchi downy mildew pathogen cytochrome b protein is shown in SEQ ID NO: 11, wherein: The 33rd amino acid from the N-terminus of SEQ ID NO: 11 is leucine and the 228th amino acid is aspartic acid; or, the 33rd amino acid from the N-terminus of SEQ ID NO: 11 is serine and the 228th amino acid is asparagine; the cytochrome b gene in the litchi downy mildew pathogen after mutation is as shown in SEQ ID NO: 12, wherein the 98th nucleotide from the 5' end of SEQ ID NO: 12 is C and the 682nd nucleotide from the 5' end is A, or, the 98th nucleotide from the 5' end of SEQ ID NO: 12 is T and the 682nd nucleotide from the 5' end is G.

3. The use according to claim 2, characterized in that: In the PCR amplification, the annealing temperature of SEQ ID NO: 3 and SEQ ID NO: 6 is 64.1°C, and the annealing temperature of SEQ ID NO: 7 and SEQ ID NO: 10 is 60°C.

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