LAMP primer composition of fluthiapyrone-resistant genotype G699V Phytophthora coriacea and its application

Abstract

The present application relates to a LAMP primer combination for detecting the fluoxetine-resistant genotype G699V of Phytophthora truncatum and its application. The present application provides a LAMP primer combination for detecting the fluoxetine-resistant genotype G699V of Phytophthora truncatum. The primer combination comprises the nucleotide sequences shown in SEQ ID NOs. 1 to 4. The primer combination has strong specificity and good accuracy. When used to detect fluoxetine-resistant strains of Phytophthora truncatum, the primer combination can quickly, conveniently, and accurately identify strains resistant to the fluoxetine fungicide.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to a LAMP primer composition for detecting the fluazifop-resistant genotype G699V Phytophthora coriacea and its application. Background Art

[0002] Phytophthora colocasiae belongs to the phylum Oomycota, class Oomycetes, orders Peronosporales, family Pythiaceae, and genus Phytophthora. Taro blight, caused by Phytophthora colocasiae, is the most devastating disease of taro in cultivation; it has become a major constraint on taro production in taro-growing countries, resulting in yield losses of 25-30%. The pathogen also causes postharvest tuber rot, leading to severe storage losses. Symptoms of taro blight appear as small, water-soaked spots that increase in size and number over time. Under favorable environmental conditions, the disease quickly spreads throughout the field, causing leaves to wilt. Severely infected taro fields in Southeast Asia and other regions have experienced crop losses exceeding 30%. Currently, fungicides are the primary control agents for plant diseases caused by Phytophthora colocasiae. Fluthiazolinone is a new fungicide with a new chemical structure developed by DuPont in the United States. Its target protein is oxysterol-binding protein (OSBP), and it exhibits excellent biological activity against downy mildew and phytophthora.

[0003] Pathogens are extremely numerous in nature. When the proportion of drug-resistant individuals in a population reaches 3%, it can cause an epidemic of drug-resistant diseases, leading to the failure of drug control. Traditional methods for detecting resistant strains mainly involve isolating and culturing pathogens, then culturing them on drug-containing plates, and identifying drug resistance based on the inhibitory effect of the drug on mycelial growth. This method has a long detection cycle, taking up to 1 week or even several weeks from isolation to identification, and there is contamination from foreign bacteria during the pathogen cultivation process. In recent years, with the development of nucleic acid-related molecular detection technology, PCR technology has provided the advantages of rapid, sensitive, and accurate detection of drug resistance in plant pathogens. However, the detection requires expensive laboratory instruments and a cumbersome electrophoresis process, which takes a long time and is costly, and cannot meet the needs of rapid detection. In addition, the identification process requires exposure to a large number of toxic and hazardous reagents, posing a major safety hazard to laboratory operators.

[0004] Loop-mediated isothermal amplification (LAMP) is a novel constant-temperature in vitro nucleic acid amplification technology invented by Japanese scholar Notomi et al. It is widely used in genetic diagnosis of diseases in animals, plants, etc. The principle of this technology is: 4 specific primers are designed for the 6 regions of the target gene, and a chain displacement DNA polymerase is used to keep the temperature under isothermal conditions (about 65°C) for 30-60 minutes to complete the nucleic acid amplification reaction. Hydroxynaphthol blue (HNB) is a metal ion indicator. The color change principle of LAMP-HNB is: HNB combines with magnesium ions to make the initial color of the reaction system violet. As the reaction proceeds, Mg 2+ Hydroxynaphthol blue reacts with the precipitated pyrophosphate ions to form a magnesium pyrophosphate precipitate. The loss of magnesium ions by hydroxynaphthol blue causes the system to turn sky blue, while the unreacted system remains violet. The most significant feature of the LAMP method is its constant-temperature amplification, eliminating the need for expensive instruments such as cyclers and gel imaging systems. The amplification reaction is extremely rapid, typically completing within an hour. Results can be visually determined, resulting in high specificity, ease of operation, and suitability for the rapid identification and detection of pathogenic mutant genotypes. This technology is rarely reported on for the detection of drug-resistant genotypes in plant pathogens. Currently, there are no reports of LAMP rapid molecular identification of the fluazifop-resistant genotype G699V of Phytophthora truncatula, both domestically and internationally. Summary of the Invention

[0005] Based on this, it is necessary to provide a LAMP primer combination and kit with strong specificity and good accuracy for detecting the fluazifop-resistant genotype G699V of Phytophthora corylifolia, and use it in the detection of fluazifop-resistant Phytophthora corylifolia.

[0006] The specific technical solutions are as follows:

[0007] A LAMP primer combination for detecting Phytophthora truncatula with a fluazifop-resistant genotype G699V, the primer combination comprising a forward inner primer FIP with a nucleotide sequence as shown in SEQ ID NO.1, a reverse inner primer BIP with a nucleotide sequence as shown in SEQ ID NO.2, a forward outer primer F3 with a nucleotide sequence as shown in SEQ ID NO.3, and a reverse outer primer B3 with a nucleotide sequence as shown in SEQ ID NO.4.

[0008] A kit for detecting the fluazifop-resistant genotype G699V of Phytophthora truncatula, comprising the above-mentioned primer combination.

[0009] In one embodiment, the kit further comprises one or more of a DNA extraction reagent, other reagents required for LAMP amplification, and a visual indicator.

[0010] A method for identifying the resistance of Phytophthora truncatula to fluazifop, comprising detecting whether the genotype of Phytophthora truncatula is the fluazifop-resistant genotype G699V.

[0011] The fluazifop-resistant genotype G699V is a gene in the ORP1 (g7517) in the Phytophthora truncatula genome, in which the nucleotide at position 2165 at the 5' end is mutated from G to T, causing the amino acid at position 699 of the encoded protein to mutate from Gly to Val.

[0012] In one embodiment, the method comprises the following steps:

[0013] Extracting DNA of Phytophthora truncatula to be tested;

[0014] Using the DNA as a template, the primer combination is used to perform a LAMP amplification reaction; and

[0015] The result of the LAMP amplification reaction is judged.

[0016] In one embodiment, the judgment is performed by visual observation, and a visual indicator hydroxynaphthol blue needs to be added to the reaction system. After the reaction is completed, the color of the amplified product changes from violet to sky blue, and it is judged that the taro phytophthora to be tested is the fluthiazolinone-resistant genotype G699V, indicating that the taro phytophthora to be tested is resistant to fluthiazolinone; the color of the amplified product is violet, and it is judged that the taro phytophthora to be tested is the non-fluthiazolinone-resistant genotype G699V, indicating that the taro phytophthora to be tested is non-fluthiazolinone-resistant.

[0017] In one embodiment, the agarose gel electrophoresis test is performed, and if there are ladder bands in the electrophoresis result, it is judged that the taro fungus to be tested is the fluthiazolinone-resistant genotype G699V, indicating that the taro fungus to be tested is resistant to fluthiazolinone; if there are no ladder bands in the electrophoresis result, it is judged that the taro fungus to be tested is the non-fluthiazolinone-resistant genotype G699V, indicating that the taro fungus to be tested is non-fluthiazolinone-resistant.

[0018] In one embodiment, the LAMP reaction system is 25 μl, including 3.5 μl 10 mM dNTP Mixture, 2.5 μl 10×Isothermal Amplification Buffer II, 1.5 μl 100 mM MgSO4 solution, 0.5 μl 40 μM FIP primer, 0.5 μl 40 μM BIP primer, 0.5 μl 10 μM F3 primer, 0.5 μl 10 μM B3 primer, 1 μl 8000 U / ml Bst 3.0 DNA Polymerase, 2 μl hydroxynaphthol blue disodium salt, 1 μl template DNA, and ddH2O is added to 25 μl.

[0019] In one embodiment, the reaction conditions of the LAMP are 61° C. for 60 min, and inactivation at 80° C. for 10 min.

[0020] Use of the primer combination, the kit, or the method for rapidly identifying resistance of Phytophthora truncatula to fluazifop-p-butylpyrrolidone in any of the following:

[0021] a. Application in the detection of resistance of Phytophthora truncatula to fluazifop-p-butylpyrrolidone.

[0022] b. Used in assisting the screening of Phytophthora truncatulae that is resistant to fluazifop-pirfenidone.

[0023] c. Application in detecting the fluazifop-resistant genotype G699V of Phytophthora truncatula.

[0024] The invention discloses an application of the G699V mutation site of the ORP1 protein of Phytophthora truncatum in detecting the resistance of Phytophthora truncatum to fluazifop-acetone. Compared with the sensitive strain, the amino acid at position 699 of the ORP1 protein of the Phytophthora truncatum resistant strain to fluazifop-acetone is mutated from Gly to Val.

[0025] Compared with the prior art, this application has the following beneficial effects:

[0026] This study discovered that fluazifop-resistant strains of Phytophthora truncatula exhibit resistance to fluazifop-resistant oxathiapiprolin through a mutation in the amino-terminal codon for amino acid 699, GGA (Gly) to GTA (Val), in the oxysterol-binding protein. Based on this finding, the applicants have provided a novel mutation site for detecting fluazifop-resistant oxathiapiprolin in Phytophthora truncatula.

[0027] Furthermore, the present application designed and optimized primers to perform corresponding mutations at position 699 of the oxysterol-binding protein of Phytophthora truncatula, placing the point mutation at the 3' end of F2, and finally optimized a set of LAMP primer combinations that can specifically identify the fluazifop-resistant genotype G699V of Phytophthora truncatula. By specifically recognizing 6 independent regions on the target sequence through two pairs of primers, the specificity is greatly improved compared to the 2 independent regions of the target sequence recognized by ordinary PCR primers, and the probability of false positives is also reduced.

[0028] Furthermore, combining the newly discovered fluoxetine-resistant genotype G699V of Phytophthora truncatum with LAMP technology allows for rapid identification of fluoxetine-resistant Phytophthora truncatum. This method, under isothermal conditions, enables rapid, convenient, efficient, highly specific, and sensitive detection of fluoxetine-resistant strains of Phytophthora truncatum G699V. It does not require complex and expensive instrumentation, making it ideal for on-site detection of resistant strains. This method has important practical implications for accurately diagnosing fluoxetine-resistant strains, timely understanding the development of resistance populations, guiding scientific drug use, reducing costs, and minimizing environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Growth of fluazifop-resistant and sensitive strains on plates containing 0.1 μg / mL fluazifop-resistant;

[0030] Figure 2 Sequencing results of fluazifop-resistant and sensitive strains;

[0031] Figure 3 Growth of fluazifop-resistant strains obtained by transferring resistance genes into sensitive wild-type strains on 0.1 μg / mL fluazifop-resistant plates; LT1534 and EV are sensitive strains, while ΔPcorp1-6, ΔPcorp1-24, and ΔPcorp1-203 are fluazifop-resistant strains obtained by transferring resistance genes into sensitive wild-type strains.

[0032] Figure 4 The upper middle image shows the color development of LAMP assays for fluazifop-resistant strains of Phytophthora truncatum (Pt. truncatum) and other pathogens. M: DL2000 DNA Marker; 1: CK; 2, 3: Ly20-2-3 and Ly20-5-5, susceptible strains of P. truncatum; 4, 5: MQ20-1-2 and MQ20-1-4, resistant strains of P. truncatum at other mutation sites; 6, 7: LT1534, Phytophthora capsici, and LZ-2, Phytophthora litchii; 8, 9, 10, 11, 12: SC19-1, SC19-2, SC19-3, SC19-6, and SC19-7, strains of P. truncatum with the fluazifop-resistant genotype G699V. The figure below shows the agarose gel electrophoresis images of LAMP detection of Phytophthora truncatum to fluazifop-resistant strains, fluazifop-resistant strains, other resistant genotype strains and other pathogens. DETAILED DESCRIPTION

[0033] To make the above-mentioned objects, features, and advantages of the present application more clearly understood, the specific embodiments of the present application are described in detail below with reference to the accompanying drawings. The following description sets forth many specific details to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways than those described herein, and those skilled in the art can make similar improvements without violating the scope of the present application. Therefore, the present application is not limited to the specific embodiments disclosed below.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0035] The term "resistant strain" refers to a strain of Phytophthora coriacea that is resistant to fluazifop-TKI. "Susceptible strain" refers to a strain of Phytophthora coriacea that is sensitive to fluazifop-TKI, and also refers to a wild-type strain.

[0036] Plant pathogenic microorganisms develop resistance under long-term stress from pesticides, and discovering the mechanism by which they develop resistance is of great significance for the prevention and control of plant diseases. In a study of plant disease resistance, the present application discovered that a strain of Phytophthora truncatula that is resistant to fluthiazolinone is one in which the ORP1 (g7517) gene in the Phytophthora truncatula genome mutates from G to T at the 2165th nucleotide from the 5' end, causing a mutation in the 699th amino acid codon of the protein it encodes, from GGA (Gly) to GTA (Val). This is of great significance for studying the resistance mechanism of pathogens and for disease prevention and control.

[0037] The genome of Phytophthora truncatula is available under the accession number GWHAOTV00000000 of the National Genome Data Center, Beijing Genomics Institute, Chinese Academy of Sciences, or the accession number JAGKQI0000000000 in the NCBI database.

[0038] Therefore, one embodiment of the present application provides the application of the G699V mutation site of the ORP1 protein of Phytophthora truncatula in the detection of the resistance of Phytophthora truncatula to fluazifop-butyl ketone. Compared with the sensitive strain, the 699th amino acid of the ORP1 protein in the strain of Phytophthora truncatula resistant to fluazifop-butyl ketone mutates from Gly to Val.

[0039] The nucleotide sequence of the ORP1 gene (susceptible strain of Phytophthora truncatula) is shown in SEQ ID NO.5:

[0040]

[0041] The amino acid sequence of ORP1 protein (Phytophthora colocasiae sensitive strain) is shown as SEQ ID NO.6 below:

[0042] MQALQDAQQRFNDLVNNDWPERVPVESMPDYDPTYMKEGFLQKKGQRLKGWKRRWFVCDGRTLSYYISRKDRKPNAVIPLEGCTVQDGGLSETWNSPRIYLTDPATGIMYCLSAEEGIVVTQWLDVLRVAVARVNNGRAATDSNAASSSQSSSNRTRQHTRTQAQRLPSSSDDEDTRAHLKRAASLGPSQARTTTLKSASSAVGSSSTGSSTSNGEGKRATRMTSAPSAVTSSSSNTQPIQHHRVHRTKTQRLPTTISLENELSHGLDVLEALLCGHSATGSSSSIRNHVVFRPIGAVNGVLRSIGTDSSSGKQYARASVVLPVSSEVVAILLADHARRAEWDVHFPQSSHVATFDDATDLVHLSSGSFAQIQQTKPIVAPHVAAAACALCAAFLSGATSWEALLTAMVYAAAVGGIVSSIDYSALTTPRDLVILRHVRESAAPDSQDSSDDKSVDELGQSVALILEKSVVNELKPVVSGVVRAHVGLSGWLLEPVDSGHATLATYITDLDMKGWLSPTTRQSFLLSRLDCVSVLSEYVNQAHLCGSELGFGGGLDEDGEGEFETRSVGYEDTSEASGLGDIVDGESSAIFHPKTYMRGMMPLPSGGLKLIDKEIAKKQGGVVKDVIKSAGAKILEGKSAVSLSLPVRIFEPRTNLERVCDLMLYAPTFLNVAHAQNDALERFKYVVTFAVAGLHHSI GQLKPFNPILGETFQSTLNDGTDVSCEHTSHHPPISNFQFTGEKYSIAGFVLWHASMSVKSNAMLNTNKGPVRVTFPDAEGLPGTTIEYNLPYLQIGGLLWGDRTVDIMGNMMFEDKKNRLQCELRLNPD AKSGMGGMFSSSKTPTDSLRGVILDTSVSPPREICDVSGSWLHDLVFGNKTYWSINKFQSGYMVPFPEDKILASDSRHREDLHYLAAGDLDESQEWKVKLEVLQRADRKARLDGRRPNHWSFRSSAGH.

[0043] The underlined areas represent mutation sites.

[0044] One embodiment of the present application further provides a LAMP primer combination for detecting the fluazifop-resistant genotype G699V Phytophthora coriacea, comprising: a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, and a reverse outer primer B3. The nucleotide sequences of the primers are as follows:

[0045] FIP (F1c+F2):

[0046] 5'-GACTGGAACGTTTCACCCAGGA-GGACTGCATCACAGTATCGT-3';

[0047] BIP(B1c+B2):

[0048] 5'-CCACCACCCGCCTATCAGTAAC-AGCACGAAACCGGCAATC-3';

[0049] F3: 5'-TACGTGGTGACCTTCGCT-3';

[0050] B3: 5'-TGACGCTCATACTGGCATG-3'.

[0051] Through sequence analysis of Phytophthora truncatula, this application designs a LAMP primer combination that can specifically distinguish different resistance genotypes. Based on LAMP technology, the resistance genotype G699V of Phytophthora truncatula to fluazifop-butylpyrrolidone can be quickly and accurately identified.

[0052] Furthermore, one embodiment of the present application provides a kit comprising the above primer combination.

[0053] In a specific example, the kit may further include one or more of a DNA extraction reagent, other reagents required for LAMP amplification, and a visual indicator. The visual indicator may be selected from hydroxynaphthol blue, PicoGreen, SYBR Green I, or calcein, preferably hydroxynaphthol blue. The reagents required for LAMP amplification also include an isothermal amplification buffer, dNTPs, MgCl2, and HCl. 2+ , Bst DNA polymerase, etc.

[0054] One embodiment of the present application also provides a method for identifying the resistance of Phytophthora truncatula to fluazifop, comprising detecting whether the genotype of Phytophthora truncatula is the fluazifop-resistant genotype G699V.

[0055] Among them, the fluazifop-resistant genotype G699V is a mutation of the 2165th nucleotide from the 5' end of ORP1 (g7517) in the genome of Phytophthora truncatula from G to T, resulting in a mutation of the 699th amino acid of the encoded protein from Gly to Val.

[0056] Because existing detection of drug resistance has the disadvantages of being time-consuming, labor-intensive, costly, and having low accuracy. This application combines LAMP technology with research on drug resistance mechanisms to detect Phytophthora truncatula with the fluthiazolinone-resistant genotype G699V. This identification method is simple, rapid, low-cost, highly sensitive, and highly specific, greatly improving detection efficiency. It does not require complex and expensive instruments and can better meet the requirements of on-site detection of resistant strains. It has important practical significance for the management, monitoring, and early warning of drug resistance epidemics of Phytophthora truncatula.

[0057] Specifically including steps a to c:

[0058] Step a: extracting DNA of the Phytophthora truncatula to be tested.

[0059] Step b: using the extracted DNA as a template, performing a LAMP amplification reaction using the above-mentioned LAMP detection primer combination;

[0060] Step c: judging the result of the LAMP amplification reaction.

[0061] In a specific example, a visual indicator hydroxynaphthol blue is added to the LAMP reaction system. During the LAMP reaction process, DNA polymerase performs polymerization, changes the number of protons and thus changes the pH value. A negative reaction result is displayed as violet, and a positive reaction result turns sky blue. Therefore, after the reaction is completed, the color change of the reaction system is used to determine whether it is the fluazifop-resistant genotype G699V of Phytophthora truncatum, indicating that the Phytophthora truncatum to be tested is resistant to fluazifop-resistant genotype G699V. The violet color indicates that the test result is negative, and it is the non-fluazifop-resistant genotype G699V of Phytophthora truncatum, indicating that the Phytophthora truncatum to be tested is non-fluazifop-resistant.

[0062] In one specific example, if a ladder-like band is detected in the agarose gel electrophoresis result, the taro Phytophthora truncatula strain is determined to be of the flutriafol-resistant genotype G699V, indicating that the taro Phytophthora truncatula strain is resistant to flutriafol. If a ladder-like band is not detected in the electrophoresis result, the taro Phytophthora truncatula strain is determined to be of the non-flutriafol-resistant genotype G699V, indicating that the taro Phytophthora truncatula strain is non-flutriafol-resistant.

[0063] This method is virtually unaffected by the large amounts of exogenous DNA and impurities present in the reaction mixture, eliminating the need for sample DNA purification. DNA extracted directly from diseased tissue can be used for rapid testing, significantly improving detection accuracy. Furthermore, conventional PCR detection methods require gel electrophoresis, which can easily cause product diffusion and become a major source of laboratory aerogel contamination. The LAMP reaction, on the other hand, only needs to be performed in a constant-temperature water bath, and the results can be directly determined by color changes, eliminating the need for expensive instrumentation and the tedious electrophoresis process, increasing the application value of resistance monitoring in agricultural production.

[0064] In one specific example, the LAMP reaction system is 25 μl, including 3.5 μl 10 mM dNTP Mixture, 2.5 μl 10×Isothermal Amplification Buffer II, 1.5 μl 100 mM MgSO4 solution, 0.5 μl 40 μM FIP primer, 0.5 μl 40 μM BIP primer, 0.5 μl 10 μM F3 primer, 0.5 μl 10 μM B3 primer, 1 μl 8000 U / ml Bst 3.0 DNA Polymerase, 2 μl hydroxynaphthol blue disodium salt, 1 μl template DNA, and ddH2O is added to 25 μl.

[0065] In a specific example, the reaction conditions of LAMP are 61° C. for 60 min, and inactivation at 80° C. for 10 min.

[0066] The above-mentioned LAMP amplification reaction system and LAMP reaction conditions are the optimal reaction conditions screened in this application. Those skilled in the art can also adjust them according to conventional means in the relevant technical field.

[0067] The LAMP primer combination, the kit, or the method can be used for rapid and reliable identification of the fluoxetine-resistant genotype G699V of Phytophthora truncatum, or for detecting the resistance of Phytophthora truncatum to fluoxetine, or for assisting in screening Phytophthora truncatum that is resistant to fluoxetine.

[0068] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0069] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0070] The main biological materials involved in the following examples are as follows:

[0071] The NTC of Phytophthora truncatula to fluazifop-p-acetone, fluazifop-p-acetone-sensitive strains of Phytophthora truncatula (Ly20-2-3, Ly20-5-5), Phytophthora capsici (LT1534), Phytophthora litchii (LZ-2), resistant genotype G699V strains (SC19-1; SC19-2; SC19-3; SC19-6; SC19-7) and Phytophthora truncatula resistant strains at other mutation sites (MQ20-1-2, MQ20-1-4) were all derived from strains isolated from the field in our laboratory. The resistant strains were also isolated from the field and preserved in our laboratory.

[0072] Example 1: Mutation of the 699th amino acid codon at the amino terminus of the ORP1 protein from GGA (Gly) to GTA (Val) confers resistance to fluazifop-p-acetylcholine in taro mold.

[0073] The inventors collected and obtained Phytophthora truncatula strains Ly19-5, Ly20-8 and Ly20-9 from the fields in Longyan, Fujian Province. After being tested with the fluthiazolinone agent, they were identified as strains resistant to fluthiazolinone. The specific steps are as follows: the bacterial cakes of Phytophthora truncatula strains (Ly19-5, Ly20-8 and Ly20-9) were inoculated on a V8 medium plate containing 0.01 μg / mL fluthiazolinone, cultured in the dark at 25°C for 5 days, and observed whether they could grow normally. The strains that could grow normally were determined to be resistant strains ( Figure 1 ), calculated the resistance multiple and extracted DNA to clone the target gene, and sequence verification showed that the ORP1 gene in the genome of these strains mutated, and the 2165th nucleotide from the 5' end mutated from G to T, causing the 699th amino acid codon of the ORP1 protein it encoded to mutate from GGA (Gly) to GTA (Val), as shown in Figure 2. Figure 2 As shown. The drug sensitivity test found that these strains all showed high resistance to fluthiazolinone. Among them, the average EC 50 The value is 5.2639×10 -4 μg / ml, while the EC of resistant strains (obtained oxathiapiprolin-resistant genotype G699V-resistant strains SC19-1, SC19-3, and SC20-6) 50 The values ​​were 0.2902μg / ml~1.4687μg / ml, and the average EC of resistant strains 50 The value was 0.7239 μg / ml, and the average resistance multiple was 1376 times.

[0074] Furthermore, a mature and stable strain of pepper phytophthora was transformed using a genetic transformation system to prove that the resistance gene can cause drug resistance. Specifically, through CRISPR / Cas9 knockout technology, the 2165th nucleotide of the ORP1 gene of the resistant strain of taro phytophthora was mutated from G to T, causing the 699th amino acid codon of the encoded ORP1 protein to mutate from GGA (Gly) to GTA (Val). The obtained transformant strain of taro phytophthora was inoculated on a V8 medium plate containing 0.01μg / mL fluthiazolinone, and the mycelium was able to grow normally. Figure 3 As shown, it shows that after the resistance gene is transferred into the sensitive wild-type strain, a resistant strain is obtained, which is resistant to fluazifop-ethyl, proving that the resistance gene is related to causing resistance.

[0075] Example 2 Design of LAMP reaction primer composition

[0076] The inventors of this application discovered that fluazifop-resistant strains of Phytophthora corymbosum primarily harbor a mutation in the amino acid codon GGA (Gly) to GTA (Val) at position 699 of the oxysterol-binding protein (ORP1) that confer fluazifop-resistant strains. However, conventional methods for detecting this site typically involve cloning the gene and then sequencing it for identification, which is complex and costly. Therefore, the present application designed primers based on the mutation in the amino acid codon GGA (Gly) to GTA (Val) at position 699 of the oxysterol-binding protein (ORP1) using the website Primer Explorer (V5) at http: / / primerexplorer.jp / e / . Finally, the mutation site was located at the 3' end of the forward inner primer FIP, and LAMP experiments were carried out using DNA of fluazifop-resistant strains of Phytophthora coriacea, fluazifop-resistant strains, other resistant genotype strains and other pathogens as templates, and an LMAP primer combination that can specifically identify the fluazifop-resistant genotype G699V strain of Phytophthora coriacea was selected.

[0077] The specific primer sequences are as follows:

[0078] FIP (SEQ ID NO. 1):

[0079] 5'-GACTGGAACGTTTCACCCAGGAGGACTGCATCACAGTATCG T -3',;

[0080] BIP (SEQ ID NO. 2):

[0081] 5'-CCACCACCCGCCTATCAGTAACAGCACGAAACCGGCAATC-3';

[0082] F3 (SEQ ID NO. 3):

[0083] 5′-TACGTGGTGACCTTCGCT-3′;

[0084] B3 (SEQ ID NO. 4):

[0085] 5′-TGACGCTCATACTGGCATG-3′.

[0086] Among them, the underlined base "T" at the 3' end of the forward inner primer FIP is the point mutation base of the fluazifop-resistant genotype G699V of Phytophthora coriacea.

[0087] Example 3 Specificity test of LAMP detection primers

[0088] In order to verify that the primer sequence that can identify the fluazifop-resistant genotype G699V of Phytophthora truncatula is specific, this application selected the NTC of Phytophthora truncatula to fluazifop-resistant genotype G699V, sensitive strains (Ly20-2-3, Ly20-5-5), Phytophthora capsici (LT1534), Phytophthora litchii (LZ-2), and resistant genotype G699V strains (SC19-1; SC19-2; SC19-3; SC19-6; SC19-7) of Phytophthora truncatula to fluazifop-resistant genotype G699V as test materials.

[0089] The DNA of the test strain was extracted using the CTAB method. The specific method is as follows: shake the bacteria with 10% V8 liquid, collect the mycelium in a mortar, grind it with liquid nitrogen, take an appropriate amount of the ground mycelium powder in a 2ml centrifuge tube, add 900μl 2% CTAB extraction solution and 10μl 1% β-mercaptoethanol, water bath in a 65℃ water bath for 1h, invert and mix, centrifuge at 12000rpm for 10min; take the supernatant to a new centrifuge tube, add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), invert and mix, centrifuge at 12000rpm for 15min; take the supernatant to a new centrifuge tube, add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), invert and mix, centrifuge at 12000rpm for 10min; take the supernatant to a new centrifuge tube, add 1 / 3 volume of 3mol / L Mix NaAC and 2 volumes of anhydrous ethanol, then cool at -20°C for more than 3-4 minutes; centrifuge at 12,000 rpm for 10 minutes and discard the supernatant; add 800 μl of 75% ethanol for washing; centrifuge at 12,000 rpm for 5 minutes, discard the supernatant, retain the DNA, dry it in an oven for 5 minutes, and add 50 μl of ddH2O to dissolve the DNA.

[0090] LAMP amplification was performed using the DNA of the test strain as a template using the primers in Example 1. The LAMP reaction system was 25 μl, including 3.5 μl Sangon 10 mM dNTP Mixture, 2.5 μl NEW ENGLAND Biolabs INC. 10× Isothermal Amplification Buffer II, 1.5 μl NEW ENGLAND Biolabs INC. 100 mM MgSO4 solution, 4 μl Sangon 5 M Betaine, 0.5 μl 40 μM FIP / BIP primer, 0.5 μl 10 μM F3 / B3 primer, 1 μl NEW ENGLAND Biolabs INC. 8000 U / ml Bst 3.0 DNA Polymerase, 2 μl Sigma Hydroxynaphthol Blue Disodium Salt, and 1 μl 50 ng / μl template DNA, and ddH2O was added to make up to 25 μl. The LAMP reaction conditions were 61°C for 60 min and inactivated at 80°C for 10 min.

[0091] After the LAMP reaction, if the color of the amplified product changes from violet to sky blue, the assay is considered positive, indicating that the tested Phytophthora truncatum is resistant to the fluazifop-acetone fungicide. Otherwise, the assay is considered negative, indicating that the tested Phytophthora truncatum is not resistant to the fluazifop-acetone fungicide. Alternatively, 2 μl of the amplified product can be electrophoresed on a 2% agarose gel. If the characteristic ladder-shaped bands of LAMP are observed, the assay is considered positive; if no amplified bands are observed, the assay is considered negative.

[0092] Observation of test results:

[0093] Visualization of detection results Figure 4 As shown: After amplification, the color of the strains of Phytophthora truncatum that are resistant to the fluthiazolinone fungicide can be observed to change from violet to sky blue, or the agarose gel electrophoresis detection shows the characteristic ladder-shaped band of LAMP. The color of the strains that are not resistant to the fluthiazolinone fungicide is still violet, or no amplified band is seen in the agarose gel electrophoresis. This shows that the LAMP detection primers and methods designed and optimized in this application have strong specificity. The LAMP primer combination can be used for the rapid and reliable identification of the fluthiazolinone-resistant genotype G699V of Phytophthora truncatum, providing a theoretical basis and technical support for the resistance monitoring and resistance assessment of Phytophthora truncatum, and has important theoretical guiding significance for the occurrence and spread of Phytophthora truncatum and resistance management in Chinese crops.

[0094] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0095] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art could make several modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the patent in this application shall be based on the appended claims, and the specification and drawings may be used to interpret the claims.

Claims

1. A LAMP primer combination for detecting the fluazifop-resistant genotype G699V Phytophthora coroansis, characterized in that: The fluazifop-resistant genotype G699V is a gene in the ORP1 of the Phytophthora corylifolia genome in which the nucleotide at position 2165 at the 5' end is mutated from G to T, causing the amino acid at position 699 of the protein encoded by the gene to mutate from Gly to Val. The primer combination includes a forward inner primer FIP with a nucleotide sequence as shown in SEQ ID NO.1, a reverse inner primer BIP with a nucleotide sequence as shown in SEQ ID NO.2, a forward outer primer F3 with a nucleotide sequence as shown in SEQ ID NO.3, and a reverse outer primer B3 with a nucleotide sequence as shown in SEQ ID NO.

4.

2. A kit for detecting the fluthiapyrone-resistant genotype G699V Phytophthora truncatula, characterized in that: The fluazifop-resistant genotype G699V is a gene in the ORP1 of the Phytophthora corylifolia genome in which the nucleotide at position 2165 at the 5' end is mutated from G to T, causing the amino acid at position 699 of the protein encoded by the gene to mutate from Gly to Val. The kit comprises the primer combination described in claim 1.

3. The kit according to claim 2, wherein The kit further comprises one or more of a DNA extraction reagent, other reagents required for LAMP amplification, and a visual indicator.

4. A method for identifying resistance of Phytophthora truncatula to fluazifop, comprising detecting whether the genotype of Phytophthora truncatula is fluazifop-resistant genotype G699V; The fluazifop-resistant genotype G699V is a gene in the ORP1 of the Phytophthora corylifolia genome in which the nucleotide at position 2165 at the 5' end is mutated from G to T, causing the amino acid at position 699 of the protein encoded by the gene to mutate from Gly to Val. The method comprises the following steps: Extracting DNA of the tested Phytophthora truncatula; Using the DNA as a template, performing a LAMP amplification reaction using the primer combination of claim 1; and The result of the LAMP amplification reaction is judged.

5. The method according to claim 4, characterized in that The judgment is performed by visual observation, and a visual indicator hydroxynaphthol blue needs to be added to the reaction system. After the reaction is completed, the color of the amplified product changes from violet to sky blue, and it is judged that the taro phytophthora to be tested is the flutriafol-resistant genotype G699V, indicating that the taro phytophthora to be tested is resistant to flutriafol; the color of the amplified product is violet, and it is judged that the taro phytophthora to be tested is the non-flutriafol-resistant genotype G699V, indicating that the taro phytophthora to be tested is non-flutriafol-resistant.

6. The method according to claim 4, characterized in that The agarose gel electrophoresis test showed that the electrophoresis result showed the presence of ladder bands, indicating that the taro fungus to be tested was the flutriafol-resistant genotype G699V, indicating that the taro fungus to be tested was resistant to flutriafol; the electrophoresis result showed no ladder bands, indicating that the taro fungus to be tested was the non-flutriafol-resistant genotype G699V, indicating that the taro fungus to be tested was non-flutriafol-resistant.

7. The method according to any one of claims 4 to 6, characterized in that The LAMP amplification reaction system is 25 μl, including 3.5 μl 10 mM dNTP Mixture, 2.5 μl 10×Isothermal Amplification Buffer II, 1.5 μl 100 mM MgSO4 solution, 0.5 μl 40 μM FIP primer, 0.5 μl 40 μM BIP primer, 0.5 μl 10 μM F3 primer, 0.5 μl 10 μM B3 primer, 1 μl 8000 U / ml Bst 3.0 DNA Polymerase, 2 μl hydroxynaphthol blue disodium salt, 1 μl template DNA, and ddH2O is added to 25 μl; The reaction conditions of the LAMP amplification reaction are 61° C. for 60 min and inactivation at 80° C. for 10 min.

8. Use of the primer combination according to claim 1, the kit according to claim 2 or 3, or the method according to any one of claims 4 to 7 in any of the following: a. Application in the detection of resistance of Phytophthora truncatula to fluazifop-p-butylpyrrolidone; b. Application in assisting the screening of Phytophthora truncatulae resistant to fluazifop-pirfenidone; c. Application in detecting the fluazifop-resistant genotype G699V of Phytophthora truncatula.

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