Specific molecular marker of Picea sicaria and detection method of specific molecular marker
By designing specific primer compositions and PCR amplification and sequencing technology, the rapid and accurate identification of spruce rust bacteria was solved, and 100% identification accuracy was achieved, avoiding missed detection.
Patent Information
- Application Number
- CN202510356685.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-25
- Publication Date
- 2025-05-13
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Figure BDA0005327544090000081 
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Abstract
Description
Technical Field
[0001] The invention relates to the field of microorganisms, and in particular to a specific molecular marker of Picea picea and a detection method thereof. Background Art
[0002] The spruce rust belongs to Fungi, Basidimycota, Pucciniomycetes, Pucciniales, Chrysomyxaceae, Chrysomyxa. The disease was first reported in North America and is currently mainly distributed in the United States and Canada. It can cause broom-shaped lesions on spruce, as well as trunk deformation, ulceration, growth loss, top death or breakage, and tree death. At the same time, the disease affects the growth of spruce and is susceptible to secondary infection by other pests and pathogens. The infection rate of spruce rust is extremely high, reaching 23-29%, and is increasing year by year in North America. In view of its great destructiveness to spruce, the dominant tree species in the world's major forests, this species has been listed as a key quarantine target by my country, the European Union, the United Kingdom, Argentina, Ukraine, Russia and other countries.
[0003] At present, the classification and identification of spruce rust pathogens at ports are mainly carried out through morphology. However, due to the complex life history and wide host range of spruce rust pathogens, as well as the spore stages with different morphologies (sexual spores, rust spores, summer spores, winter spores and basidiospores), conventional morphological screening and identification are difficult and prone to misdetection and omission. At the same time, this species has the characteristics of transparasitism, completing the summer spore and winter spore stages on plants of the genus Urtica, and completing the sexual organ and rust spore stages on plants of the genus Picea. The differences in the host range of each stage are very obvious, making it difficult to identify the fungus. This fungus can persist in the branches and bud tissues of spruce.
[0004] Spruce is the dominant tree species in my country's forests, with a national stock of about 697 million cubic meters. It is mainly distributed in Tibet, Xinjiang, Sichuan, Qinghai, Gansu, Greater and Lesser Khingan Mountains in Northeast China, Changbai Mountain and other places. Once the spruce rust fungus is introduced into my country, it is expected to affect spruce woodlands, plantations and ornamental spruce trees, leading to reduced tree growth and related ecological losses, which will pose a great threat to my country's ecological security. At present, the entry port inspection and quarantine system has not yet formulated standards related to the quarantine identification method of this species, especially the lack of methods related to molecular screening. Therefore, it is of great urgency and practicality to formulate a quarantine identification method for spruce rust fungus. Summary of the invention
[0005] The technical problem to be solved by the present invention is how to quickly and accurately identify Chrysomyxa arctostaphyli and effectively prevent misdetection and missed detection of the fungus.
[0006] In order to solve the above technical problems, the present invention firstly claims to protect a primer composition for detecting Picea picea.
[0007] The primer combination for detecting spruce rust fungus claimed in the present invention consists of primer pair A and primer pair B; the primer pair A consists of primer ITS4-BRf and primer LR5; the primer pair B consists of primer ITS5 and primer ITS4BR.
[0008] The primer ITS4-BRf is any single-stranded DNA of the following A1) or A2):
[0009] A1) a single-stranded DNA as shown in SEQ ID No. 1 of the sequence listing;
[0010] A2) a single-stranded DNA fragment having more than 85% identity with the single-stranded DNA fragment defined in A1);
[0011] The primer LR5 is any single-stranded DNA of the following B1) or B2):
[0012] B1) the single-stranded DNA shown in SEQ ID No. 2 of the sequence listing;
[0013] B2) a single-stranded DNA fragment having more than 85% identity with the single-stranded DNA fragment defined in B1);
[0014] The primer ITS5 is any single-stranded DNA of the following C1) or C2):
[0015] C1) a single-stranded DNA as shown in SEQ ID No. 3 of the sequence listing;
[0016] C2) a single-stranded DNA fragment having more than 85% identity with the single-stranded DNA fragment defined in C1);
[0017] The primer ITS4BR is any single-stranded DNA of the following D1) or D2):
[0018] D1) the single-stranded DNA shown in SEQ ID No. 4 of the sequence listing;
[0019] D2) A single-stranded DNA fragment having 85% or more identity with the single-stranded DNA fragment defined in D1).
[0020] In a second aspect, the present invention claims a method for preparing the primer composition, comprising the steps of individually packaging each primer in the primer composition of claim 1.
[0021] In a third aspect, the present invention claims the use of the primer composition, which is as follows a) or b) or c):
[0022] a) preparing a test kit for assisting the detection of Picea picea;
[0023] b) preparing a test kit for assisting in the identification of Picea picea;
[0024] c) Identify or assist in identifying whether the tested fungus is Picea spruce rust pathogen.
[0025] In a fourth aspect, the present invention claims a kit for identifying or assisting in the identification of spruce rust pathogen.
[0026] The kit for identifying or assisting in identifying spruce rust pathogen claimed in the present invention may include the primer combination described above; and may also include DNA polymerase, 10×PCR buffer and / or dNTP.
[0027] In a fifth aspect, the present invention claims protection for the use of the kit in identifying or assisting in identifying whether a sample to be tested contains spruce rust pathogen.
[0028] In a sixth aspect, the present invention claims a method for assisting in identifying whether a test bacterium is spruce rust fungus, comprising the following steps: extracting genomic DNA of the test bacterium, performing PCR amplification using the primer pair A and the primer pair B in the primer composition of claim 1 to obtain a PCR amplification product, sequencing the amplification product, and making the following judgment based on the sequencing result:
[0029] (1) If the sequencing result of the PCR amplification product obtained by using the primer pair A has an identity of ≥99% with the nucleotide sequence shown in SEQ ID No. 5, and the sequencing result of the PCR amplification product obtained by using the primer pair B has an identity of ≥97% with the nucleotide sequence shown in SEQ ID No. 6, then the test fungus is Puccinia picea;
[0030] (2) If the sequencing result of the PCR amplification product obtained using the primer pair A has an identity of less than 99% with the nucleotide sequence shown in SEQ ID No. 5, or the sequencing result of the PCR amplification product obtained using the primer pair B has an identity of less than 97% with the nucleotide sequence shown in SEQ ID No. 6, then the test fungus is not Puccinia picea.
[0031] In the seventh aspect, the present invention claims protection for a method for assisting in identifying whether the test bacterium is Puccinia picea, comprising detecting whether the genomic DNA of the test bacterium contains the LSU sequence and the ITS sequence of Puccinia picea. If the genomic DNA contains the LSU sequence and the ITS sequence, the test bacterium is Puccinia picea; if the genomic DNA does not contain the LSU sequence or the ITS sequence, the test bacterium is not Puccinia picea.
[0032] The nucleotide sequence of the LSU sequence is shown as SEQ ID No. 5 in the sequence listing or has at least 99% identity with SEQ ID No. 5 in the sequence listing.
[0033] The nucleotide sequence of the ITS sequence is shown as SEQ ID No. 6 in the sequence listing or has at least 97% identity with SEQ ID No. 6 in the sequence listing.
[0034] In the above method, the fungus to be tested is a fungus of the genus Chrysomyxa.
[0035] Furthermore, the bacteria to be tested may be parasitic bacteria. The bacteria to be tested may be isolated from plants of the genus Picea or Urtica.
[0036] The test bacteria in the embodiment of the present invention can specifically be at least one of Chrysomyxa arctostaphyli, Chrysomyxa succinea, Chrysomyx arhododendri, Chrysomyxa zhuoniensis, Ascocalyx berenice, Phytophthora cactorum, Cenangium ferruginosum or Penicillium italicum.
[0037] In an eighth aspect, the present invention claims a method for assisting in identifying whether a sample to be tested contains spruce rust fungus, comprising the following steps: extracting genomic DNA of the sample to be tested, performing PCR amplification using the primer pair A and the primer pair B in the primer composition of claim 1 to obtain a PCR amplification product, sequencing the amplification product, and making the following judgment based on the sequencing result:
[0038] (1) If the sequencing result of the PCR amplification product obtained by using primer pair A is ≥99% identical to the nucleotide sequence shown in SEQ ID No. 5, and the sequencing result of the PCR amplification product obtained by using primer pair B is ≥97% identical to the nucleotide sequence shown in SEQ ID No. 6, then the sample to be tested contains Puccinia picea;
[0039] (2) If the sequencing result of the PCR amplification product obtained using primer pair A has an identity of less than 99% with the nucleotide sequence shown in SEQ ID No. 5, or the sequencing result of the PCR amplification product obtained using primer pair B has an identity of less than 97% with the nucleotide sequence shown in SEQ ID No. 6, then the sample to be tested does not contain spruce rust.
[0040] In the ninth aspect, the present invention claims protection for a method for assisting in identifying whether a sample to be tested contains Picea picea, comprising detecting whether the genomic DNA of the sample to be tested contains the LSU sequence and the ITS sequence of Picea picea, if the genomic DNA contains the LSU sequence and the ITS sequence, then the sample to be tested contains Picea picea; if the genomic DNA does not contain the LSU sequence or the ITS sequence, then the sample to be tested does not contain Picea picea.
[0041] The nucleotide sequence of the LSU sequence is shown as SEQ ID No. 5 in the sequence listing or has at least 99% identity with SEQ ID No. 5 in the sequence listing.
[0042] The nucleotide sequence of the ITS sequence is shown as SEQ ID No. 6 in the sequence listing or has at least 97% identity with SEQ ID No. 6 in the sequence listing.
[0043] In the above method, the sample to be tested may be a plant tissue with disease spots.
[0044] In the above method, the reaction conditions for PCR amplification are as follows: 94°C for 3 min; 94°C for 30 s, 55°C for 30 s, 72°C for 70 s, 35 cycles; 72°C for 10 min, and stored at 4°C.
[0045] In the present invention, the term "identity" refers to the similarity of nucleic acid sequences measured using homology search sites on the Internet, such as the BLAST webpage of the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastn as a program, setting the Expect value to 0.0, the Max target Sequences to 100, the Short queries to Automatically adjust word size and other parameters to improve results for short queries, the Word size to 28, the Max matches in a query range to 0, the Match / Mismatch Scores to 1, -2, the Gap Costs to Linear, all Filters to Lowcomplexity regions, the Mask to Mask for lookup table only, and performing a search, the identity of a pair of nucleic acid sequences can be calculated, and then the value (%) of the identity can be obtained.
[0046] In the present invention, the identity ≥ 99% may be 99% or 100% identity.
[0047] In the present invention, the identity ≥ 97% may be 97%, 98%, 99% or 100% identity.
[0048] Experiments have shown that the primer composition for detecting Puccinia picea provided by the present invention can simply, quickly and effectively identify whether the strain to be tested is Puccinia picea with an accuracy of 100%, which is of great significance for the quarantine detection or identification of Puccinia picea. DETAILED DESCRIPTION
[0049] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.
[0050] The experimental methods in the following examples are all conventional methods unless otherwise specified.
[0051] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0052] Information about spruce rust
[0053] 1) Scientific name, Latin name and taxonomic status
[0054] Chinese name: Picea picea
[0055] English name: spruce broom rust
[0056] Scientific name: Chrysomyxa arctostaphyli
[0057] Taxonomic status: Fungi, Basidimycota, Pucciniomycetes, Pucciniales, Chrysomyxaceae, Chrysomyxa.
[0058] 2) Distribution
[0059] North America: Canada, United States.
[0060] 3) Host
[0061] It can harm Arctostaphylos spp., Arctostaphylos uva-ursi, Picea spp., European spruce (Picea abies), Engelmannii spruce (Picea engelmannii), white spruce (Picea abies), black spruce (Picea mariana), Sitka spruce (Picea pungens), red spruce (Picea rubens) and Sitchensis spruce, etc.
[0062] 4) Symptoms
[0063] In spruce, the first symptom is a whitening of the needles in summer. The awakening of dormant buds produces a prominent, dense arbuscular mass. The arbuscular mass is present in all seasons, with yellow-green needles that produce foul-smelling sex spores under the epidermis. Rust spores are then produced, giving the arbuscular mass an orange-yellow color. The needles then die and fall off in the fall, and the remaining arbuscular mass dies in the winter. Leaves of non-rust-caused conifer witches' broom remain their normal dark green color year-round; only a few needles fall off. Sometimes cankers, spindle-shaped swellings, and secondary arbuscular mass forms on the trunk and side branches. The tree loses vigor, and dieback, dead branches, and dead trees are common. In rare cases, more than 25% of the trees in a spruce forest are infected; fewer than 1% of the trees produce arbuscular mass.
[0064] On bearberry, rust is most noticeable in late spring, producing purplish-brown leaf spots with clusters of orange-brown, waxy teliospores on the underside of the leaves. In late spring, large numbers of sexual spores are produced on the current year's needles of spruce. The pathogen infects the bark and outer wood of the trunk and branches. Rust spores are produced in the summer and infect bearberry A.uuaursi. The teliospores produced on bearberry can infect spruce in the summer.
[0065] 5) Transmission routes
[0066] The rust spores of this pathogen are spread by air currents and can be spread over long distances during the transportation of wood.
[0067] 6) Observation of the morphology of pathogens
[0068] Winter spores: short and round, with rounded ends, smooth walls, colorless, no surface decoration, uniform thickness of 1 to 1.5 μm, and size of 13 to 18 μm × 23 to 64 μm.
[0069] Rust spores: long pear-shaped, spindle-shaped, orange-yellow, beaded, with columnar nodules on the wall, no smooth spots, size 16-25μm×23-35μm.
[0070] Basidiospore: size 7.5~8μm×8.5~9.5μm.
[0071] Example 1. Design and synthesis of primers
[0072] Through preliminary experiments and sequence comparisons, it was found that LSU and ITS sequences are relatively conservative within the spruce rust fungus species, but have a certain degree of variability between species. They are good barcode genes for distinguishing and identifying spruce rust fungus.
[0073] According to the LSU sequence (as shown in SEQ ID No. 5 in the sequence list) and the ITS sequence (as shown in SEQ ID No. 6 in the sequence list) of the spruce rust fungus, two pairs of primers were designed, which were named primer pair A and primer pair B. Primer pair A is composed of primer ITS4-BRf and primer LR5 for the LSU sequence, and primer pair B is composed of primer ITS5 and primer ITS4BR for the ITS sequence.
[0074] The primer sequences are as follows:
[0075] Primer ITS4-BRf: 5'-GGACCATGTACAAGTCTGTTGA-3' (SEQ ID No. 1 in the sequence listing);
[0076] Primer LR5: 5'-TCCTGAGGGAAACTTCG-3' (SEQ ID No. 2 in the sequence listing);
[0077] Primer ITS5: 5'-GGAAGTAAAAGTCGTAACAAGG-3' (SEQ ID No. 3 in the sequence listing);
[0078] Primer ITS4BR: 5'-TCAACAGACTTGTACATGGTCC-3' (SEQ ID No. 4 in the sequence listing).
[0079] Primer ITS4-BRf, primer LR5, primer ITS5 and primer ITS4BR were synthesized respectively.
[0080] Example 2: Establishment of a method for detecting and quarantine spruce rust
[0081] 1. Total DNA extraction
[0082] Take 0.1 g of the sample to be tested (suspected pathogens or plant tissue containing pathogens), add it to a mortar, grind it into powder in liquid nitrogen, and transfer it to a 1.5 ml centrifuge tube. Add 300μL~500μL CTAB buffer (containing 0.1g proteinase K) to a centrifuge tube, mix well, and place in a 65℃ water bath for 1h; centrifuge at 13000g for 5min~10min, and retain the supernatant; add 500μL chloroform:isoamyl alcohol (volume ratio of 24:1) and mix well, centrifuge at 13000g for 5min~10min, and retain the supernatant; add another 500μL chloroform:isoamyl alcohol (volume ratio of 24:1) and mix well, centrifuge at 13000g for 5min~10min, and retain the supernatant; add 1mL isopropanol and mix well, place at -70℃ for 1h, or -20℃ overnight; centrifuge at 13000g for 30min, and DNA precipitate can be seen; wash the DNA precipitate with 70% ethanol and dry at room temperature; dissolve the DNA with 30μL~50μL Tris-EDTA buffer and set aside.
[0083] 2. PCR reaction system and parameters
[0084] 2.1 PCR reaction system
[0085] Reagent name Sample volume (μL) 10× PCR buffer 2.5 2.5mmol / L dNTP 2.0 10μmol / L upstream primer 1.0 10μmol / L downstream primer 1.0 5U / μL Taq DNA polymerase 0.2 10ng / μL template DNA 2.0 Double distilled water 16.3 Total volume 25.0
[0086] 2.2 PCR reaction conditions: 94℃3min; 94℃30s, 55℃30s, 72℃70s, 35cycels; 72℃10min, store at 4℃.
[0087] 3. After completing step 2, sequence the PCR amplification product and make the following judgments based on the sequencing results:
[0088] (1) If the sequencing result of the PCR amplification product obtained by using primer pair A is ≥99% identical to the LSU sequence of Puccinia picea (as shown in SEQ ID No. 5), and the sequencing result of the PCR amplification product obtained by using primer pair B is ≥97% identical to the ITS sequence of Puccinia picea (as shown in SEQ ID No. 6), then the sample to be tested is Puccinia picea or contains Puccinia picea.
[0089] (2) If the sequencing result of the PCR amplification product obtained using primer pair A is less than 99% identical to the LSU sequence of Puccinia picea (as shown in SEQ ID No. 5), or the sequencing result of the PCR amplification product obtained using primer pair B is less than 97% identical to the ITS sequence of Puccinia picea (as shown in SEQ ID No. 6), then the sample to be tested is not Puccinia picea or does not contain Puccinia picea.
[0090] Wherein, the identity is the identity of the nucleic acid sequence measured using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastn as a program, setting the Expect value to 0.0, the Max target Sequences to 100, the Short queries to Automatically adjust word size and other parameters to improve results for short queries, the Word size to 28, the Max matches in a query range to 0, the Match / Mismatch Scores to 1, -2, the Gap Costs to Linear, all Filters to Lowcomplexity regions, the Mask to Mask for lookup table only, and performing a search, the identity of a pair of nucleic acid sequences can be calculated, and then the identity value (%) can be obtained.
[0091] Example 3: Identification of whether the test bacteria is spruce rust
[0092] The basic information of the 9 tested strains is shown in Table 1. Among them, there were 2 strains of spruce rust pathogens and 7 species and 7 strains of non-spruce rust pathogens.
[0093] Among them, Ascocalyx berenice with strain number CBS110.36, Phytophthora cactorum with strain number CBS231.30, Coniferous twig dieback endophyte with strain number CBS111.24 and Penicillium italicum with strain number CBS719.73 are deposited in the Westerdijk Biodiversity Institute of the Netherlands (formerly the Netherlands Collection of Fungal Cultures, CBS) (website: https: / / eco-ri.nl / en / facilities / cbs-knaw-collections-and-databases), and the public can obtain them from the collection.
[0094] The spruce rust fungus with strain numbers AFTOL-ID 442 and 503CHA_PCG_NO1, the succinic rust fungus with strain number HMJAU 8397, the rhododendron rust fungus with strain number NEFU-P578, and the Chrysomyxa zhuoniensis with strain number BJFCR00521 are respectively preserved in the Mycological Specimens of Jilin Agricultural University, the Mycological Specimens of Beijing Forestry University, etc., and the public can obtain them from the center.
[0095] The method established in Example 2 was used to identify whether the 9 test strains were spruce rust pathogens, and the experimental results were as follows:
[0096] PCR amplification was performed on 9 test strains using primer pair A and primer pair B, and the PCR products were detected by electrophoresis. The gel electrophoresis diagram showed that the amplified products of all test strains contained two bands of 755 bp and 595 bp.
[0097] The amplified products of the nine test strains were sequenced, and the sequencing results were compared with the LSU sequence and ITS sequence of spruce rust. The results showed (see Table 1) that the sequencing results of the PCR products of the test strains numbered AFTOL-ID 442 and 503CHA_PCG_NO1 using primer pair A were 100% and 100% identical to the LSU sequence of spruce rust, respectively, and the sequencing results of the PCR products using primer pair B were 100% and 99% identical to the ITS sequence of spruce rust, respectively. It can be seen that the primer pair A and the primer pair B were used to perform PCR amplification on the test strains with strain numbers AFTOL-ID 442 and 503CHA_PCG_NO1, respectively. The sequencing results of the amplified products were ≥99% identical to the LSU sequence and ITS sequence of spruce rust fungus. According to the judgment criteria in Example 2, it was identified as spruce rust fungus; the strain numbers were HMJAU 8397, NEFU-P578, BJFC The sequencing results of the PCR products of the test strains R00521, CBS110.36, CBS231.30, CBS111.24 and CBS719.73 obtained using primer pair A were 93.60%, 98%, 97%, 83.9%, 65%, 85.5% and 83.4% identical to the LSU sequence of spruce rust pathogen, respectively. The sequencing results of the PCR products obtained using primer pair B were 88.91%, 92.56%, 89.14%, 84.9%, 68%, 83.6% and 76.8% identical to the ITS sequence of spruce rust pathogen, respectively. Obviously, when primer pair A and primer pair B were used to perform PCR amplification on the test strains numbered HMJAU 8397, NEFU-P578, BJFC R00521, CBS110.36, CBS231.30, CBS 111.24 and CBS 719.73, respectively, the sequencing results of the amplified products were all <99% identical to the LSU sequence or ITS sequence of spruce rust pathogen, and according to the judgment criteria in Example 2, they were identified as non-spruce rust pathogen.
[0098] In summary, according to the method for detecting spruce rust fungus and the dedicated primers provided by the present invention, 9 test strains were identified, and the accuracy of the identification results was 100%.
[0099] Table 1. Basic information of the tested strains and the identity of their PCR amplification products with LSU and ITS sequences
[0100]
[0101]
Claims
1. A primer combination for detecting Puccinia picea, comprising a primer pair A and a primer pair B; the primer pair A comprises a primer ITS4-BRf and a primer LR5; the primer pair B comprises a primer ITS5 and a primer ITS4BR; The primer ITS4-BRf is any single-stranded DNA of the following A1) or A2): A1) a single-stranded DNA as shown in SEQ ID No. 1 of the sequence listing; A2) a single-stranded DNA fragment having more than 85% identity with the single-stranded DNA fragment defined in A1); The primer LR5 is any single-stranded DNA of the following B1) or B2): B1) the single-stranded DNA shown in SEQ ID No. 2 of the sequence listing; B2) a single-stranded DNA fragment having more than 85% identity with the single-stranded DNA fragment defined in B1); The primer ITS5 is any single-stranded DNA of the following C1) or C2): C1) a single-stranded DNA as shown in SEQ ID No. 3 of the sequence listing; C2) a single-stranded DNA fragment having more than 85% identity with the single-stranded DNA fragment defined in C1); The primer ITS4BR is any single-stranded DNA of the following D1) or D2): D1) the single-stranded DNA shown in SEQ ID No. 4 of the sequence listing; D2) A single-stranded DNA fragment having 85% or more identity with the single-stranded DNA fragment defined in D1).
2. A method for preparing the primer combination according to claim 1, comprising the step of individually packaging each primer in the primer combination according to claim 1.
3. The use of the primer composition according to claim 1 is as follows a) or b) or c): a) preparing a test kit for assisting the detection of Picea picea; b) preparing a test kit for assisting in the identification of Picea picea; c) Identify or assist in identifying whether the tested fungus is Picea spruce rust pathogen.
4. A kit comprising the primer combination according to claim 1.
5. Use of the kit according to claim 4 in identifying or assisting in identifying whether a sample to be tested contains spruce rust fungus.
6. A method for assisting in identifying whether a test bacterium is spruce rust fungus, comprising the following steps: extracting genomic DNA of the test bacterium, performing PCR amplification using the primer pair A and the primer pair B in the primer composition of claim 1 to obtain a PCR amplification product, sequencing the amplification product, and making the following judgment based on the sequencing result: (1) If the sequencing result of the PCR amplification product obtained by using the primer pair A has an identity of ≥99% with the nucleotide sequence shown in SEQ ID No.5, and the sequencing result of the PCR amplification product obtained by using the primer pair B has an identity of ≥97% with the nucleotide sequence shown in SEQ ID No.6, then the test fungus is Puccinia picea; (2) If the sequencing result of the PCR amplification product obtained using the primer pair A has an identity of less than 99% with the nucleotide sequence shown in SEQ ID No. 5, or the sequencing result of the PCR amplification product obtained using the primer pair B has an identity of less than 97% with the nucleotide sequence shown in SEQ ID No. 6, then the test fungus is not Puccinia picea.
7. A method for assisting in identifying whether a test bacterium is spruce rust fungus, comprising detecting whether the genomic DNA of the test bacterium contains an LSU sequence and an ITS sequence of spruce rust fungus, if the genomic DNA contains the LSU sequence and the ITS sequence, the test bacterium is spruce rust fungus; if the genomic DNA does not contain the LSU sequence or the ITS sequence, the test bacterium is not spruce rust fungus; The nucleotide sequence of the LSU sequence is as shown in SEQ ID No. 5 in the sequence listing or has at least 99% identity with SEQ ID No. 5 in the sequence listing; The nucleotide sequence of the ITS sequence is shown as SEQ ID No. 6 in the sequence listing or has at least 97% identity with SEQ ID No. 6 in the sequence listing.
8. The method according to claim 6 or 7, characterized in that: The fungus to be tested is a Chrysomyxa fungus.
9. A method for assisting in identifying whether a sample to be tested contains spruce rust fungus, comprising the following steps: extracting genomic DNA of the sample to be tested, performing PCR amplification using the primer pair A and the primer pair B in the primer combination of claim 1 to obtain a PCR amplification product, sequencing the amplification product, and making the following judgment based on the sequencing result: (1) If the sequencing result of the PCR amplification product obtained by using primer pair A is ≥99% identical to the nucleotide sequence shown in SEQ ID No. 5, and the sequencing result of the PCR amplification product obtained by using primer pair B is ≥97% identical to the nucleotide sequence shown in SEQ ID No. 6, then the sample to be tested contains Puccinia picea; (2) If the sequencing result of the PCR amplification product obtained using primer pair A has an identity of less than 99% with the nucleotide sequence shown in SEQ ID No. 5, or the sequencing result of the PCR amplification product obtained using primer pair B has an identity of less than 97% with the nucleotide sequence shown in SEQ ID No. 6, then the sample to be tested does not contain spruce rust.
10. A method for assisting in identifying whether a sample to be tested contains spruce rust fungus, comprising detecting whether the genomic DNA of the sample to be tested contains an LSU sequence and an ITS sequence of spruce rust fungus, if the genomic DNA contains the LSU sequence and the ITS sequence, the sample to be tested contains spruce rust fungus; if the genomic DNA does not contain the LSU sequence or the ITS sequence, the sample to be tested does not contain spruce rust fungus; The nucleotide sequence of the LSU sequence is as shown in SEQ ID No. 5 in the sequence listing or has at least 99% identity with SEQ ID No. 5 in the sequence listing; The nucleotide sequence of the ITS sequence is shown as SEQ ID No. 6 in the sequence listing or has at least 97% identity with SEQ ID No. 6 in the sequence listing.