Method for rapidly identifying living parasitic powdery phytopathogen

Abstract

The invention discloses a method for rapidly identifying in-vivo parasitic powdery plant pathogens, and relates to the technical field of molecular identification of plant pathogenic fungi, and the method comprises the following steps: 1, collecting samples such as plant leaves or stems with in-vivo parasitic pathogens such as powdery mildew and rust disease; 2, establishing a PCR amplification system; 3, adding an amplification template (powder pathogen) and carrying out PCR amplification; 4, separating, purifying and sequencing PCR products or sequencing molecular cloning transformants; and 5, carrying out nucleic acid database BLAST search on the sequencing sequence, and rapidly identifying the classification of living parasitic pathogen species causing plant diseases. The method can effectively avoid the technical bottleneck and cost expenditure that living parasitic fungi cannot be subjected to in-vitro culture to obtain pure culture thalli or an expensive trace DNA extraction kit is used for extracting genomes, and has important reference significance and practical value for rapid identification of living parasitic pathogenic bacteria capable of generating powder, such as powdery mildew and rust germs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology identification technology, and particularly relates to a method for rapidly identifying living parasitic powdery plant pathogenic fungi. BACKGROUND

[0002] The occurrence and prevalence of living parasitic fungal diseases such as powdery mildew and rust usually cause the quality and yield of crop products to decrease, and the disease in the seedling stage seriously affects the growth and development of crops and even causes the plant to die, which finally leads to a serious decline in economic benefits. Therefore, timely and accurate identification of living parasitic fungal diseases of plants is an important prerequisite for taking effective prevention and control measures.

[0003] At present, the existing identification methods of living parasitic fungal diseases have the following problems: 1. Existing research reports show that the identification of pathogenic fungi is mainly through the diagnosis of disease symptoms, the isolation and purification culture of pathogenic fungi, the verification of Koch's postulates, and then the observation of morphological characteristics of the isolated and purified pathogenic fungi to identify the species of the pathogenic fungi. The living parasitic powdery pathogenic fungi on plants may be a mixture, and these living parasitic pathogenic fungi cannot be isolated and purified on culture medium, but can only be subcultured on host plants, so that the corresponding pure culture or single spore inoculation culture is relatively cumbersome and difficult to obtain.

[0004] 2. The identification of fungal species based on morphological characteristics of fungal pathogens requires higher professional level and morphological classification experience. In order to more accurately understand the species classification of pathogenic fungi, universal primers such as ITS1 / ITS4 or specific primers for a specific species are generally used to amplify ribosomal internal transcribed spacer (ITS) or other specific molecular marker fragments for sequencing and molecular identification. [3] This is a great challenge for living parasitic powdery mildew and rust fungi. Some research reports have extracted genomic DNA from the powdery material collected from the living parasitic pathogenic fungi for PCR amplification by washing the diseased samples and collecting the powdery material of the living parasitic pathogenic fungi, but a large amount of collection of these powdery materials may contain a considerable amount of other epiphytic microorganisms, and the extracted genomic DNA actually comes from a mixed bacterial population, which brings inaccurate interference and even failure to the subsequent sequencing or identification. In order to avoid the pollution caused by the large amount of collection of powdery material, the use of trace DNA extraction kit can to some extent avoid such genomic pollution, but the trace extraction genomic kit is relatively expensive, and the cost of pathogen identification is high.

[0005] 3. Based on the above status quo, Fu Rongtao et al. reported a method suitable for rapid identification of plant pathogenic fungi. The method needs to carry out ultraviolet sterilization on the sample with spore-producing pathogens and prepare spore suspension by picking up spore pathogens with a dissecting needle, wherein the ultraviolet sterilization is basically ineffective for fungal spores and time-consuming (30 min), and even if the residual bacteria treated by ultraviolet sterilization still exist a certain abundance of DNA, the corresponding fragments in the primer binding region will still be amplified during PCR amplification. In addition, the preparation of spore suspension by picking up with a dissecting needle cannot guarantee that all spores are derived from the same pathogenic fungus, and the preparation volume is too large, time-consuming and relatively complicated. For non-living parasitic spore-producing fungi, it is relatively easy to separate and purify culture, and it is necessary to extract the genome of the obtained pure culture to carry out molecular identification.

[0006] In summary, the method for rapidly identifying living parasitic powdery plant pathogenic fungi of the present application prepares a living parasitic pathogenic fungus powdery suspension, which is prepared in situ by rapidly resuspending and mixing a small amount of PCR system component solution. The amplification is carried out by high-efficiency amplification of high-fidelity enzyme PrimeSTAR Taq with low template concentration sensitivity, which can amplify the DNA molecular fragments required for sequencing or cloning from a small amount of pathogenic sample, avoiding the use of expensive micro-volume genomic extraction kit and the risk of base mutation caused by PCR amplification. At the same time, it avoids the risk of genomic contamination caused by extracting the genome of a large amount of living parasitic powdery pathogen and saves the cost and time of extracting the genome and preparing the powdery amplification template suspension. These characteristics and advantages of the present application greatly accelerate the rapid identification of living parasitic powdery fungal pathogens of plants in the field, and strive for the best window period for the precise prevention and control of such diseases. SUMMARY

[0007] The present application provides a method for rapidly identifying living parasitic powdery plant pathogenic fungi, which aims to solve the technical problems in the prior art such as complex identification process, long time consumption, high cost of some reagent kits, and the need for experiment operators to have rich experience in fungal morphological identification.

[0008] The technical solution of the present application is as follows: A method for rapidly identifying living parasitic powdery plant pathogenic fungi, comprising the following steps: 1. Sample collection and treatment: Collect plant leaf or stem samples with living parasitic pathogens such as powdery mildew and rust from the field or field, and independently package the samples with a self-sealing bag after sampling to avoid cross contamination of pathogenic fungi between samples.

[0009] 2. The establishment of a rapid and efficient PCR amplification system: The ITS4 / PM5, ITS4 / ITS5 or ITS1 / ITS4 primers were used to amplify the spores of powdery mildew and rust fungi by PCR, and the PCR amplification system was as follows: 40 μL, 1 μL of each primer, 25.5 μL of dH2O, 4 μL of dNTP, 8 μL of 5×PrimeSTAR Taq Buffer, 0.5 μL of PrimeSTAR Taq, and 1×10 4 ~1×10 5 The concentration of the spores of the parasitic pathogen must be limited, because too high or too low concentration will affect the PCR amplification efficiency.

[0010] 3. Adding a template and PCR amplification: 1-2 μL of the PCR amplification system without the template was dropped onto the diseased leaf lesion using a sterile tip, and then the spore suspension was added back into the PCR amplification system after resuspension and mixing. The PCR amplification conditions were as follows: 98℃ pre-denaturation for 15 min, 98℃ denaturation for 10 sec, 55℃ annealing for 30 sec, 72℃ extension for 1 min, 40 cycles of amplification, 72℃ final extension for 5 min, and 16℃ temporary storage.

[0011] 4. Separation and purification of the PCR amplification product, sequencing or sequencing of the transformed clone: After the PCR amplification product was separated and detected by 1% agarose gel electrophoresis, the target band was cut and recovered for direct sequencing or sequencing of the transformed clone after cloning. After electrophoresis, the gel was stained to observe the target band, which was cut and recovered according to the instructions of the gel recovery kit. The PCR product was directly sent to a biological company for Sanger sequencing, or the PCR product was cloned into a vector, and the recombinant plasmid was extracted from the bacterial liquid to identify the positive transformant, and the cloned fragment was sequenced.

[0012] 5. BLAST search of the sequencing sequence of the parasitic pathogen to quickly identify the species classification: According to the sequencing result file, the sequence of the sequencing fragment was arranged, and a BLAST online search was performed in the NCBI nucleic acid database. According to the comparison result, the pathogenic species classification of the sequencing sample was quickly identified. The ITS sequence was usually determined as the same species (species) with the longest matching length, the highest score, and a sequence consistency of 99%-100% in the database record strain; if the ITS sequence comparison showed more than 99% consistency in multiple species, other commonly used primers for fungal identification were used for amplification and sequencing, and multiple gene sequence analysis was used for further identification.

[0013] Further, the sample collected in step 1 is fresh and has typical symptoms of parasitic diseases and the pathogen has a powdery substance.

[0014] Further, the sample of step 1 is collected from the fresh symptom of the living parasitic disease and the pathogen with powder, and then detected or temporarily stored at 4℃ for standby.

[0015] Further, the PCR amplification condition in step 3 is 98℃ pre-denaturation for 15 min, 98℃ denaturation for 10 sec, 55℃ annealing for 30 sec (the annealing temperature can be adjusted according to the primer Tm value), 72℃ extension for 1 min (specifically, 1 kb / min), 40 cycles of amplification, and 72℃ final extension for 5 min (optional) and temporary storage at 16℃.

[0016] For the two types of pathogens of powdery mildew and rust, each type includes a plurality of species at the genus classification chronological level, and since the two types of pathogens are living parasitic pathogens, they cannot be cultured in vitro, so it is difficult to achieve traditional separation, purification and identification. Molecular level identification usually requires extraction of genomic DNA followed by PCR amplification. For fungi that can be cultured in vitro, a large amount of pure culture can be obtained in the culture medium for genomic DNA extraction. However, it is difficult to obtain a large amount of pure culture of powdery mildew and rust living parasitic pathogens, so expensive micro-genomic extraction kits are usually used to extract genomic DNA. Therefore, the present application prepares a powder suspension in situ for living parasitic pathogens on plants, establishes a spore PCR amplification system using a low template concentration sensitive high-fidelity Taq DNA polymerase, directly amplifies DNA fragments for pathogen identification from living parasitic powdery pathogens, which not only reduces the cumbersome steps of DNA extraction, but also saves the cost of using expensive DNA extraction kits, while greatly reducing the risk of PCR amplification mutation, ensuring the accuracy of the amplified DNA fragments.

[0017] In summary, compared with the existing technology, the beneficial effects of the present application are: 1. Only a small amount of spores is needed for PCR amplification, without the need for genomic DNA extraction, saving time and reagent cost; 2. The powder spore suspension template is prepared in situ by rapid resuspension and mixing of the micro-PCR system component solution, which is convenient and fast; 3. PCR amplification uses low template concentration sensitive high-fidelity enzyme PrimeSTAR Taq for efficient amplification, which avoids the risk of failure or base mutation of general fidelity Taq PCR amplification from trace amounts of pathogenic samples for DNA molecule fragments required for sequencing or cloning; 4. The trace amount of powder required for amplification template avoids the risk of genomic contamination caused by large-scale collection of living parasitic powdery pathogens for genomic extraction; 5. The established PCR amplification system is suitable for different primers (which can be universal primers or species-specific primers), and the PCR amplification conditions are suitable for the powdery template of any fungal group. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Flowchart of the rapid identification process of the powdery plant pathogen of the present application.

[0019] Figure 2 In the present application, a is the agarose gel electrophoresis result of the spore PCR product of the Xanthium strumarium powdery mildew pathogen (lane 1 uses P3, ITS5 as primers, lane 2 uses PM5, ITS4 as primers), b is the agarose gel electrophoresis result of the TA clone transformant PCR identification after the spore PCR amplification of the Xanthium strumarium powdery mildew pathogen in the present application (lanes 2-8 use P3, ITS5 as primers, lanes 9-14 use PM5, ITS4 as primers).

[0020] Figure 3 In the present application, a is the agarose gel electrophoresis result of the spore PCR product of the Coriaria sinica powdery mildew pathogen, b is the agarose gel electrophoresis result of the TA clone transformant PCR identification after the spore PCR amplification of the Coriaria sinica powdery mildew pathogen in the present application.

[0021] Figure 4 In the present application, a is the agarose gel electrophoresis result of the spore PCR product of the Coriaria sinica rust pathogen, b is the agarose gel electrophoresis result of the TA clone transformant PCR identification after the spore PCR amplification of the Coriaria sinica rust pathogen in the present application.

[0022] Figure 5 In the present application, a is the agarose gel electrophoresis result of the spore PCR product of the Viburnum dilatatum, Sonchus oleraceus, Koelreuteria paniculata, Vicia sepium powdery mildew pathogen (lanes 1-4 use ITS1, ITS4 as primers, lanes 5-8 use PM5, ITS4 as primers).

[0023] Figure 6 In the present application, a is the agarose gel electrophoresis result of the Viburnum dilatatum (lanes 1-4), Sonchus oleraceus (lanes 5-9), Koelreuteria paniculata (lanes 10-15), Vicia sepium (lanes 16-21) powdery mildew pathogen spore PCR (using ITS1, ITS4 as primers) amplification TA clone transformant PCR identification.

[0024] Figure 7 In the present application, a is the agarose gel electrophoresis result of the Viburnum dilatatum (lanes 1-4), Sonchus oleraceus (lanes 5-9), Koelreuteria paniculata (lanes 10-15), Vicia sepium (lanes 16-21) powdery mildew pathogen spore PCR (using PM5, ITS4 as primers) amplification TA clone transformant PCR identification. DETAILED DESCRIPTION

[0025] As Figure 1 shown, a method for rapid identification of a living body parasitic spore fungus pathogen of Chinese artichoke, comprising the following steps: 1. Collecting plant leaf samples with powdery mildew or rust disease in the wild; 2. Preparing and collecting the spore suspension of the Chinese artichoke powdery mildew (or rust) pathogen: using a sterile white tip to suck 1-2 μL of PCR system mixture, dropping it on the diseased leaf lesion, mixing the spores with the mixture with the tip, then sucking and adding it to the amplification PCR system and mixing; 3. Establishing a PCR reaction system: using fungal universal primers ITS4 / PM5 (or ITS4 / ITS5) to perform PCR amplification on the Chinese artichoke powdery mildew (or rust) pathogen spores. The PCR amplification system is: 1 μL of each primer, 25.5 μL of dH2O, 4 μL of dNTP, 8 μL of 5×buffer, 0.5 μL of PrimeSTAR, and 1×10 4 ~ 1×10 5 4. The PCR amplification conditions are: 98℃ pre-denaturation for 15 min, 98℃ denaturation for 10 sec, 55℃ annealing for 30 sec, 72℃ extension for 1 min, 40 cycles of amplification; 72℃ final extension for 5 min, and 16℃ temporary storage; 5. The amplification product is subjected to agarose gel electrophoresis to detect the presence or absence of the amplification product and the fragment size, and the PCR product is directly sequenced or the transformant with positive bacterial liquid after cloning is sequenced; as Figures 3-4 shown, the agarose gel electrophoresis results of the PCR products of different pathogen spores; 6. Sequencing data processing and analysis: the sequencing data of the PCR product is searched and located using the reverse complementary sequence of the 3' part of the primer, and the bidirectional sequencing sequence is spliced using the DNAMAN software; the cloning sequence is located using the PCR amplification primer sequence, and the cloning fragment sequencing sequence is obtained after removing the vector sequence and the primer sequence; 7. Sequencing sequence comparison and preliminary identification of the pathogen: the sequencing sequence is analyzed by the BLAST online comparison tool in NCBI, and the pathogen causing the Chinese artichoke powdery mildew (or rust) is preliminarily identified according to the scoring, coverage, and consistency of the comparison results. According to the comparison results, the corresponding sequences of the species with a consistency greater than 99% and the related species reported in the literature are downloaded, and the classification status of the pathogen species is determined through phylogenetic analysis.

[0026] ​The pathogenic bacteria identification process through the above-mentioned implementation case method can realize the rapid molecular identification of the powdery pathogen which cannot be cultured in vitro by living parasitism, and avoids the cumbersome steps of extracting genomic DNA and the cost of reagent kits. The application has important reference significance and practical value for the rapid identification of living parasitic pathogenic bacteria such as powdery mildew and rust fungi which can produce powdery substances.

Claims

1. A method for rapidly identifying living parasitic powdery plant pathogens, characterized in that: The following steps are involved: Step 1. Sample collection and processing: Collect samples of living parasitic pathogens of powdery mildew or rust, and temporarily seal the collected samples in ziplock bags for future use; the samples may be leaves, stems, or other parts of plants; Step 2. Establishment of PCR amplification system: PCR amplification of powdery mildew and rust fungi using universal fungal primer pairs ITS4 / PM5, ITS4 / ITS5, or ITS1 / ITS4. A 40 μL PCR amplification system includes 1 μL of upstream and downstream primers, 25.5 μL of dHO, 4 μL of dNTPs, 8 μL of 5× PrimeSTAR Taq Buffer, and 0.5 μL of PrimeSTAR Taq. The concentration of live parasitic pathogen powdery spores is 1×10 4 ~1×10 5 / mL; Step 3. Add template and PCR amplification: Use a sterile pipette tip to draw 1-2 μL of the PCR amplification system mixture without the powdered template, drop it onto the diseased leaf lesions, resuspend and mix in situ, draw up the powdered spore suspension, add it back to the PCR amplification system and mix again; PCR amplification conditions are: pre-denaturation at 98°C for 15 min, denaturation at 98°C for 10 sec, annealing at 55°C for 30 sec, extension at 72°C for 1 min, amplification for 40 cycles; final extension at 72°C for 5 min, and temporary storage at 16°C; Step 4. Isolate, purify, and sequence the PCR amplified products or molecularly clone and sequence the transformants: After PCR amplification products are detected and separated by 1% agarose gel electrophoresis, the PCR products can be sequenced directly or by molecular cloning and then selecting transformants for sequencing: After PCR products are separated by electrophoresis, the gel is stained and the observed target bands are cut. The purified PCR products are directly subjected to Sanger sequencing, or the purified PCR products are cloned and cloned. From transformants that test positive by PCR, cloned fragments on the recombinant plasmid are extracted and sequenced; Step 5. Rapid identification of species classification by BLAST search of sequencing sequences of pathogens of living parasitic powdery matter: Arrange the sequences of the sequencing fragments according to the sequencing result files, perform BLAST online search in the NCBI nucleic acid database, compare the sequences of the sequencing fragments with the search results, and identify the species classification of the pathogens of the sequenced samples based on the comparison results.

2. The method for rapid identification of living parasitic powdery plant pathogens according to claim 1, characterized in that: The sample collected in step 1 is a sample with typical symptoms of fresh parasitic diseases on living organisms and the pathogen contains powdery matter.

3. The method for rapid identification of living parasitic powdery plant pathogens according to claim 2, characterized in that: In the step 1, the samples of living organisms with typical fresh parasitic disease symptoms and pathogens containing powdery substances are collected and tested immediately or temporarily stored at 4° C. for future use.

4. The method for rapid identification of living parasitic powdery plant pathogens according to claim 1, characterized in that: The PCR amplification system and amplification reaction procedure in step 3 also include the in-situ preparation of a spore suspension of a living parasitic plant pathogen powder.

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