Soybean mosaic virus notsv and a method for detecting the same by using pcr

Abstract

The application relates to a soybean mosaic virus NotSV and a PCR detection primer and method thereof. A new pathogen of the Caulimoviridae soymovirus is identified on the Nothapodytes sp. of Nothapodytes of the Nothapodytes sp. by the applicant. Since there is no detection method for the pathogen, specific detection primers / probes are designed according to the whole genome sequence of the pathogen, a method for detecting the pathogen is established, the virus species of the soybean mosaic virus is enriched, the host range of the virus is expanded, and the detection range of the virus infection related diseases of important medicinal raw materials (such as the Nothapodytes sp.) is expanded.

Description

Technical Field

[0001] This application belongs to the field of plant disease diagnosis and relates to a method for detecting pathogens in plants of the Cornaceae family. Background Technology

[0002] *Nothapodytes nimmoniana*, also known as stinky horsewood or stinky false firewood tree, is a plant belonging to the genus *Nothapodytes* in the family Cornaceae. It is mainly produced in India, Sri Lanka, Japan, and other regions, and in my country, it is only distributed in Taiwan. *Nothapodytes nimmoniana* and other plants in the genus *Nothapodytes* are high-quality raw materials for extracting the anticancer drug camptothecin and its derivatives. Among all plants found to contain camptothecin, *Nothapodytes nimmoniana* has the highest camptothecin content. Due to the extremely high demand for camptothecin, the population of *Nothapodytes nimmoniana* is declining sharply, and it has been listed as a sensitive species in some countries. In my country, it is listed as a very small population. In recent years, there have been cases of artificial introduction and rapid propagation of *Nothapodytes nimmoniana* and its cultivation in Guangdong, Yunnan, and Fujian provinces of my country. The introduction and domestication of plants has important production significance, but the process is often extensive and lacks targeted inspection and quarantine measures, leading to the introduction of some new viruses. Therefore, it is necessary to conduct virus testing on newly introduced plants of the genus *Pseudodra*, such as *Crispy Branch*, to determine their growth status and pathogen-carrying status. Summary of the Invention

[0003] Soymovirus belongs to the family Caulimoviridae. The particles of this genus are spherical, 50 nm in diameter, and contain a single, non-covalently closed circular double-stranded DNA (dsDNA) genome. The genome contains 7-8 open reading frames (ORFs), and its genome composition is very different from that of closely related genera. The host range of the genus *Soybean Mottle Virus* is very narrow, with only seven known viruses in this genus: *Cestrum yellow leaf curling virus* (CmYLCV), *soybeanchlorotic mottle virus* (SbCMV), *peanut chlorotic streak virus* (PCSV), *water chestnut soymovirus 1* (WCSV1), *Malva-associated soymovirus 1* (MaSV1), *Hibiscus soymovirus* (HSV), and *Blueberry red ringspot virus* (BRRV).

[0004] In 2023, multiple *Nothapodytes soymovirus* plants suspected of being infected with a viral disease were discovered at the Subtropical Plant Introduction and Acclimatization Garden of the Fujian Provincial Institute of Plant Research. The affected plants exhibited obvious mosaic mottling symptoms on their leaves. Through high-throughput RNA-seq sequencing combined with bioinformatics, PCR, and sequence alignment, the pathogen was identified as a novel virus belonging to the genus *Nothapodytes*. In the context of this application, this virus is referred to as *Nothapodytes soymovirus* (NotSV). After full-length amplification and sequencing, the genome of this novel virus is a circular dsDNA, 6677 bp in length, and its sequence is shown in SEQ ID NO.1. Using ORF Finder software, the viral genome was predicted to contain 7 open reading frames (ORFs): ORF1 encodes a hypothetical polypeptide of 34 amino acids, ORF2 encodes a hypothetical polypeptide of 24 amino acids, ORF3 encodes a 17.6 kDa protein (160 aa), ORF4 encodes a 47.6 kDa capsid protein (433 aa), ORF5 encodes a 71.9 kDa polymerase (reverse transcriptase + RNase H) (654 aa), ORF6 encodes a 16 kDa translation activator (145 aa), and ORF7 encodes a 35.8 kDa mobile protein (325 aa).

[0005] NotSV is a novel DNA virus identified by the applicant that infects plants of the genus *Notis* such as *Cercis spp.*. This virus affects the growth of *Notis* plants, and there are currently no reports on detection methods or primers for this virus. This invention designs five pairs of primers based on the virus's nucleotide sequence for specific detection, and establishes a PCR method for detecting NotSV using these designed specific primers, providing an effective tool for the detection, diagnosis, and control of this virus.

[0006] One of the objectives of this invention is to provide a PCR detection primer and method for a novel soybean mottle virus (Soybean mottle virus) of the genus *Soybean mottle virus*, *Soybean mottle virus*.

[0007] An isolated organism comprising nucleotides selected from: nucleotides or fragments thereof shown in sequence listing SEQ ID No: 1, or nucleotides complementary to the nucleotides shown in sequence listing SEQ ID NO: 1.

[0008] The organism in question is a Soymovirus; the nucleotide is DNA.

[0009] An isolated nucleotide comprising a nucleotide selected from: the nucleotide shown in SEQ ID NO: 1 or a fragment thereof; a nucleotide complementary to the nucleotide shown in SEQ ID NO: 1; the nucleotide shown in SEQ ID NO: 2 or a fragment thereof; and a nucleotide complementary to the nucleotide shown in SEQ ID NO: 2.

[0010] A primer or probe that is specific to or complementary to a nucleotide selected from: the nucleotide or fragment thereof shown in SEQ ID NO: 1; or a nucleotide complementary to the nucleotide shown in SEQ ID NO: 1.

[0011] The primers or probes are selected from the nucleotides shown in the sequence listing SEQ ID NO: 15-26.

[0012] The use of the primers or probes in diagnosing plant virus infections, wherein the plant is one or more of the following species: Nothapodytes pittosporoides, Nothapodytes collina, Nothapodytes nimmoniana, Nothapodytes obscura, Nothapodytes obtusifolia, and Nothapodytes tomentosa.

[0013] Those skilled in the art understand that although a specific primer pair is used for the detection of newborn organisms in specific embodiments, this primer pair is merely exemplary and should not be construed as limiting the scope of protection of this application. Those skilled in the art, based on textbooks and the principle of nucleotide sequence complementarity (e.g., *Molecular Cloning: A Laboratory Manual*, J. Sambrook & MR. Green, 2017; p. 450 "Designing PCR Primers Using Primer3 Plus"; Chapter 13 "Preparation of Labeled DNA Probes, RNA Probes, and Oligonucleotide Probes"), are familiar with the principles of primer amplification of target sequences, the principles of probe binding to target sequences, and the design principles of primers and probes. Various primer / probe design software programs are available in the prior art, such as Primer Premier, Oligo7, and BeaconDesigner. When those skilled in the art know the genome sequence of an organism or its functional fragments (such as ORFs), they can obtain specific primer or probe sequence and structural information.

[0014] In a preferred embodiment of the present invention, organism-specific primers can be designed based on the following principles:

[0015] 1) The primers are 15 bp to 30 bp in length (including the endpoints, the same below);

[0016] 2) The G+C content in the primers is 40% to 60%;

[0017] 3) There cannot be four consecutive complementary bases within or between the primers;

[0018] 4) The Tm value of the primer is 55±5℃;

[0019] 5) The length of the amplification product obtained by the primers is 300 bp to 1500 bp;

[0020] 6) The primers are complementary to nucleotides selected from the following:

[0021] -The nucleotide or fragment thereof shown in SEQ ID NO: 1;

[0022] -A nucleotide complementary to the nucleotide shown in SEQ ID NO: 1;

[0023] According to some embodiments, the uses of the primers or probes described herein are also provided. In some embodiments, the uses of the primers or probes of this application are provided in the diagnosis of viral diseases (such as mosaic, mottle, and leaf curl) in plants of the genus *Pseudodra* (e.g., *Crispy Branch*).

[0024] No infection of other plant families by this new organism has been found so far. However, the possibility of it infecting other plant families cannot be ruled out.

[0025] The use of the primers or probes in identifying pathogens, wherein the identification refers to: determining whether the pathogen to be tested is the isolated organism; or determining whether the isolated organism is present in the sample.

[0026] The use of the primers or probes in the preparation of a pathogen detection device, wherein the pathogen is the isolated organism; and the detection device is in the form of a reagent and / or a chip.

[0027] A detection device comprising the aforementioned primers or probes; the detection device is in the form of reagents and / or chips.

[0028] A method for detecting pathogens, comprising the steps of:

[0029] 1) Provide the sample to be tested; the sample to be tested is derived from plants of the genus *Pterocarya*.

[0030] 2) Obtain the nucleotides of the sample to be tested; the nucleotides are DNA;

[0031] 3) Contact the nucleotides of the sample to be tested or their amplification products with the aforementioned primers or probes;

[0032] 4) Determine whether the aforementioned isolated nucleotides are present.

[0033] The beneficial effects of this invention are as follows: The inventors have identified a novel pathogen of the genus *Soybean Mottle Virus* in plants of the Cornaceae family, which is reported for the first time. Since there is no specific detection method for this pathogen, this application designs specific detection primers / probes based on the pathogen's complete genome sequence and establishes a method for detecting this pathogen. This enriches the variety of viruses in the *Soybean Mottle Virus* genus, expands the host range of viruses in this genus, and also expands the detection range of virus-related diseases in important medicinal raw materials (such as *Corydalis yanhusuo* twigs of the Cornaceae family), effectively protecting sensitive plants and extremely small populations. Attached Figure Description

[0034] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0035] Figure 1 Symptoms of brittle leaf mosaic disease: illustration.

[0036] Figure 2 : Genome structure of NotSV, a mottled virus of crisp soybean.

[0037] Figure 3 Phylogenetic trees were constructed based on the polymerase nucleotide sequences of NotSV and 19 viruses most similar to NotSV. Phylogenetic analysis was performed using MEGA X software with maximum likelihood statistics. The bootstrap method (1000 replicates) was used to assess phylogenetic progressions, which are shown below the branches.

[0038] Figure 4 Phylogenetic tree constructed based on the amino acid sequences of NotSV and 19 viral polymerases most similar to NotSV.

[0039] Figure 5 Establishment of the NotSV detection method. Lanes 1 to 6 represent the fragments amplified in *Pterocarya stenoptera* samples by the following six primer pairs: NotsoyV1261F / NotsoyV2185R, NotsoyV3306F / NotsoyV4037R, NotsoyV4026F / NotsoyV4723R, NotsoyV4427F / NotsoyV4844R, NotsoyV4822F / NotsoyV5189R, and NotsoyV4472F / NotsoyV5773R; M: DNA molecular weight marker. Detailed Implementation

[0040] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0041] Example 1. Identification and sequencing of pathogens

[0042] In 2023, the inventors discovered *Nothapodytes nimmoniana*, a plant exhibiting symptoms of mosaic mottling disease, in Fujian Province, China. Figure 1 ).

[0043] Using mirVana TM Total RNA was extracted from symptomatic leaves using a microRNA (miRNA) Isolation kit (Ambion, Thermo Fisher Scientific, Waltham, MA, United States). Total RNA was purified using an RNA Clean XP Kit (Beckman Coulter, Brea, CA, United States) and an RNase-Free DNase Set (QIAGEN GmbH, Hilden, Germany). Ribosomal RNA was removed using a RiboZero Magnetic Kit (Epicentre, Lucigen, Middleton, WI, United States), and cDNA libraries were constructed using the TruSeqRNA Sample Prep Kit (Illumina, San Diego, CA, United States). Paired-end sequencing was then performed on the Illumina HiSeq X platform.

[0044] Sequencing yielded 77,896,020 reads. After removing low-quality reads, initial assembly was performed using CLC GenomicsWorkbench 6.0.4 (Qiagen, Valencia, CA, United States), followed by reassembly using CAP3 software, resulting in 11,834 contigs. BLASTx was used to align these contigs with the NCBI Non-Redundant (Nr) database (E-value < 1e-5). The results showed 46 contigs ranging from 231 bp to 3411 bp with high similarity to several members of the soybean mottle virus genus. This indicates that the plants exhibiting mosaic mottle symptoms on crisp branches carry a soybean mottle virus.

[0045] To identify the characteristics of soybean mottle virus (HTS) in crisp branches, the inventors designed viral genome amplification primers based on the contiguous sequences of HTS and the end-to-end structural characteristics of circular DNA viral genomes (Table 1). A strategy combining full-length amplification and segmented overlapping amplification was employed, supplemented by nested PCR in key regions to enhance specificity and sensitivity. Seven overlapping fragments were amplified using Ex Taq (TaKaRa, Dalian, China). All obtained PCR fragments were cloned into the pMD19 vector (Takara) for bidirectional sequencing. Three positive clones were sequenced from each PCR fragment. The complete viral genome sequence was then obtained by splicing.

[0046] Table 1. Primer pairs used for NotSV genome cloning

[0047]

[0048] Note: F” / R” indicates nested primers

[0049] The target virus has a genome sequence length of 6,677 nt (SEQ ID NO: 1) and is a typical circular DNA virus. BLASTn analysis showed that the complete sequence of this virus has the highest similarity to several soybean mottle virus genera, with a nucleotide similarity of 75.15%-75.25%. The inventors speculate that the identified virus is a novel soybean mottle virus genus, and have tentatively named it Nothapodytes soymovirus (NotSV) (GenBank accession number PZ295459, not previously published in this application).

[0050] Genome analysis of NotSV (a soybean mottle virus) revealed that it possesses seven open reading frames (ORFs) and exhibits typical genomic structure characteristics of the soybean mottle virus genus. The genome encodes seven proteins / peptides: ORF1 encodes a 34-amino acid putative polypeptide (nt: 189–294), ORF2 encodes a 24-amino acid putative polypeptide (nt: 592–667), ORF3 encodes a 17.6 kDa protein (nt: 694–1177), ORF4 encodes a 47.6 kDa coat protein (nt: 1216–2518), ORF5 encodes a 71.9 kDa polymerase (reverse transcriptase + RNase H) (nt: 2641–4606), ORF6 encodes a 16 kDa translation activator (nt: 4593–5031), and ORF7 encodes a 35.8 kDa mobile protein (nt: 5488–6466). Figure 2 ).

[0051] Phylogenetic analysis was performed using maximum likelihood statistics (1000 bootstrap replicates) in MEGA 7 software. Sequence alignment was performed using ClustalW. Phylogenetic trees were generated based on the polymerase nucleotide and amino acid sequences of NotSV and 19 viruses from 8 genera of the Caulimoviridae family that are most similar to NotSV. Apple mosaic virus (AMV) of the Ilarvirus genus was used as a distant relation control. Phylogenetic trees constructed based on polymerase amino acid sequences show that NotSV clusters with seven members of the genus Soymovirus, including Blueberry Red Ringspot Virus (BRRV), Soybean Chlortic Mottle Virus (SbCMV), Peanut Chlorotic Streak Virus (PCSV), Cestrum Yellow Leaf Curling Virus (CmYLCV), Water Chestnut Soymovirus 1 (WCSV1), Malva-Associated Soymovirus 1 (MaSV1), and Hibiscus Soymovirus (HSV), indicating that NotSV is closely related to the genus Soymovirus. The phylogenetic tree constructed based on nucleotides is consistent with the amino acid phylogenetic tree. NotSV clusters in the same cluster as members of the soybean chlorotic mottle virus genus and on the same branch as blueberry red ring spot virus BRRV, indicating that NotSV is most closely related to BRRV. Figure 3 ,4).

[0052] The International Committee on Taxonomy of Viruses (ICTV) uses the following threshold criteria for classifying the genus *Soybean Chlorotic Mottle Virus*: (1) host range; (2) a nucleotide sequence difference of more than 20%, meaning a nucleotide sequence similarity of less than 80% with the polymerases of other viruses within the genus is considered a new virus. *Soybean Chlorotic Mottle Virus* has a relatively narrow host range. This invention is the first to discover that viruses in this genus can infect plants of the *Pseudolarix* genus, such as *Cephalotaxus fortunei*, thus expanding the host range of *Soybean Chlorotic Mottle Virus*. Simultaneously, the nucleotide and amino acid sequences of the NotSV polymerase were compared pairwise with those of seven members of the *Soybean Chlorotic Mottle Virus* genus. The results showed that the NotSV polymerase had the highest similarity to BRRV, at 71.37% and 68.18%, respectively (Table 3). The polymerase nucleotide sequence difference was 28.63%, clearly exceeding the threshold criteria for classifying the genus *Soybean Chlorotic Mottle Virus*. In conclusion, NotSV can be identified as a new virus within the *Soybean Chlorotic Mottle Virus* genus.

[0053] Example 2. Establishment of a detection method for NotSV in crisp soybean branches

[0054] 1. Primer design:

[0055] Based on the NotSV virus whole genome sequence, primers for NotSV detection were designed using Primer Premier 6 software. The primer design sites included any fragment at any position in the NotSV full-length genome sequence (SEQ ID No. 1).

[0056] The primer design principles are:

[0057] 1) The primers specifically bind to any fragment of the NotSV genome;

[0058] 2) Primer length is 15 bp to 30 bp;

[0059] 3) The G+C content is between 40% and 60%;

[0060] 4) There cannot be four consecutive complementary bases within or between primers;

[0061] 5) The Tm value of the primers is 55±5°C;

[0062] 6) The length of the amplified product is 300 bp to 1500 bp.

[0063] Exemplary detection primers designed based on the above design principles include:

[0064] Table 2. Exemplary detection primers designed

[0065]

[0066] 2. The detection method is as follows:

[0067] Using the crisp branch samples used for high-throughput sequencing as materials, and taking a pair of Notsoy V4472F and Notsoy V5773R with amplified partial sequences as examples, a detection method for NotSV was established.

[0068] Take 0.1g of crisp branch leaves and grind them thoroughly in liquid nitrogen. Use the TIANGEN Universal Genomic DNA Extraction Kit (DP605) to extract DNA from the leaves. Refer to the instructions for specific steps. Dissolve the extracted DNA in 30 μL DEPC ddH2O and store at -80℃ for later use.

[0069] The concentration of DNA was determined using Nanodrop 2000. PCR was performed using 1000 to 2000 ng of DNA as a template. The PCR reaction system consisted of: 1 to 2 μL of DNA template, 1 μL of dNTPs (2.5 mmol / L each), 0.5 to 1 μM of forward and reverse primers, 0.25 μL of DNA polymerase (5 U / μL), 2.5 μL of 10× DNA polymerase buffer, and ddH2O to a final volume of 25 μL.

[0070] PCR reaction procedure: 94-95℃ pre-denaturation for 3 min; 94-95℃ denaturation for 30 s, 50-60℃ annealing for 30 s; 72℃ extension for 45 s to 3 min, 32 cycles; 72℃ final extension for 10 min. 5 μL of PCR product was subjected to 1% agarose gel electrophoresis.

[0071] 5 μl of PCR product was analyzed using a 1% agarose gel electrophoresis. The results are shown in the figure. Figure 5 Using six primer pairs—NotsoyV1261F / NotsoyV2185R, NotsoyV3306F / NotsoyV4037R, NotsoyV4026F / NotsoyV4723R, NotsoyV4427F / NotsoyV4844R, NotsoyV4822F / NotsoyV5189R, and NotsoyV4472F / NotsoyV5773R—PCR amplified target fragments of 924 bp, 731 bp, 697 bp, 417 bp, 367 bp, and 1301 bp in NotV1-infected crisp branch samples. Sequencing experiments further confirmed that the amplified bands were NotSV fragments.

[0072] Table 3. Sequence homology between NotSV and Soymovirus-associated viral polymerases

[0073] unit:%

[0074]

[0075] NotSV genome (SEQ ID NO:1)6677bp

[0076] 1 CGGACCTGAC TTTACATTTC ATTATAGTTT ATTAAAAGAT AAAGACGGAC CGGACTTGAA

[0077] 61 GTTACAATTA AATGTATCTT ATATTTATTT CAGTTACAGA CTCGAAAACG ACATAAAGAA

[0078] 121 AAAGTTTGAA AATGACATAA AGTAAAAGTA AAGTTGATGC TGACATTTCT GCTGCTTTAG

[0079] 181 TCATTTTTAT GGGGCCTACA TGTCTGATGC TGTTTCTTCG ATCATTACTT CCTCTGTGGA

[0080] 241 ATCGTAACTA CTGCTTTCTT CACAATATTT ACAACTGTGT CCTGGCACTC TTCTAATCTT

[0081] 301 TTTGCATATT TTCTTTTGAA TATCTTATGG GAGACCTGGT ACTTGATTGC TGAGAAGTAT

[0082] 361 TTTTTCGAAA TCCATGAATA CCTTCTCTTG TTCTGGACTG ATAGTGGTTG ATGATTTTGA

[0083] 421 ATATATCAAA CAATTTTCAG ATTTATAATT TACTCTGATA TTTGCTGTCT GATTTTGCAA

[0084] 481 ATGAAATACT ACTCCTGAAA ATAAAGTAGA AATTCTTTCT TCCATCTTCT CCTCCTTCAA

[0085] 541 ATCTTGGTCT TTTCCTAAAT TTATTAGCTA AACATTGTTC AATTACAATT TATGAACTA

[0086] 601 CCATTTGAAA TCTTTGTTAT AGCGAGAAGC GCACCAAAAA TCTGGTATCA GAGCCAAGAA

[0087] 661 ANNOUNCEMENT AAAAAAATCA ANNOUNCEMENT ATTATGGCCG

[0088] 721 CONNECTIONS AAGAAATAAATATATTAAAACAAAAATATATEACCATATA

[0089] 781 EXERCISES GGATACCTAA ACATATTATA TCTAGTAATG ACCTATTTT GGAACCTATA

[0090] 841 TGCTCTCAAA AAGAAGAATT ATTTCCTCAT TTATATCATA CTTTAAATCC TCAACTAAAT

[0091] 901 PHOTATTATTG CHATTAGG CHARACTER AAACACATTA ATAAACAAAAA CLOCKTAGE

[0092] 961 CONTACT CONTACTCC CGCTATTAAT CCTACT CONTACT WINDOW AGAAAAACAAA

[0093] 1021 GTAAAGAAT CITY AATAAAAAAT CTATAGAAC AACGATGTGTTACTATTCAA

[0094] 1081 CONNECTION AACTTTAAAAATAAAAGTAAAGAATCAA ATTTATTTATTTAAAAAAAAT

[0095] 1141 AAATCTAGTC AACCATTTTG GTTCAAAAAT AGAATGAA TTAGAGGATCAATAGAAG

[0096] 1201 ATTAAAAAAATATTTATTAC ATTATTAAAAAAATTATTGAAAATTATTGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA.

[0097] 1261 ACATTTGGAG MULTIPLE MULTIPLE MULTIPLE TTTGGAG MULTIPLE MULTIPLE

[0098] 1321 AAAATGGATG AAGGTCCTAG TTATTCTAAA AAAGAGAAG ATATACCAACCCTCTAAT

[0099] 1381 TATCAAGAAC AAGHAGAGA TAGTTGAA AATCGATGGA AAAGATAGAAATTGGGAA

[0100] 1441 TTTCAATCAG GATACCCTGA ATTTCAAGAA TACTTATA AACAAGGTTATTTAAA

[0101] 1501 CONNECTIONS CONNECTIONS CONNECTIONS ACTCCTCATA CONNECTIONSACCTATTACT

[0102] 1561 GGAAATGTAG GTCCAGAAGG AATATTAAAT TTAGATTGTG TAAAAAATGGAAAAAGTT

[0103] 1621 TTAAAAGCAAT GGATGGCTAA ACAAGCTTTA ATGATCCAAA TTGAGAAGTTTAAAAAAT

[0104] 1681 ATGGACAATC NATIONAL NEIGHBORHOOD AAATAAAAA CTTCAGGAATGTTTTTTCGAT

[0105] 1741 SEQUENCE STATATIONS TYPE AATTTTGCAA CONTACTATTATTG

[0106] 1801 SEQUENCE CCAAAATTTA TCAAGTTC SEQUENCES TQUATCHATTACTCA

[0107] 1861 AAATCCGATA GTTGROUP REPEAT AAATGGGTT TAAATCAAATCCTTATGT

[0108] 1921 AATATGTGTG ATTTTGCAGA ATTTGCATGT CAATATAGTA GTTATTATTATAGATTACCA

[0109] 1981 GTAACCGAAA GAAATATATT TCTAATCATT TTCGTAAACA AATTACCTTATCCTCTTAAT

[0110] 2041 CHANNEL CHAINAGTT TCHROUND CHANNEL GTAAAGAAG CHANNEL CHAINCHACKTT

[0111] 2101 GGCGGAGTAA CHANGE AAAAGATTAT ATCTTACK AATGTAAAAAAAAATATATA

[0112] 2161 AAAAGTCAAA TACATGGAGC CACGCAATGT TGTGATAAAT TTTCCGATATACCACAAAAA

[0113] 2221 TTTGGATGTA GACCTTTTCG GGGGGAAAGT CGTTATAAAAA ATAAGAAATAATTAAAGAAA

[0114] 2281 CHANNEL CCTATAAACA CHANNEL STORM AAGGAAAAAAAAAACCA

[0115] 2341 TACAAAGAC CATGGAATTC TAAATATAGA AAAAAAAATT TTAAAAGAAAAAATAGAAAA

[0116] 2401 GAAATAAAAA ATAAAAATTA TTGTCCTACA GGAAAAAAAA ATGCCAATGTTGGATATGTC

[0117] 2461 AAGAAGAAGG ACATTATGCT AATGAATGTC CAAATAAAAA TAAAAGTAATAAAAAAGTAA

[0118] 2521 AAGTAAAATT ACTACAATAT ATGAGTAGTA TTAATCTAGA ACCTATAGAAGATATAGACA

[0119] 2581 TAAGTGAAAA TGAAAGTATA TATTGTTTAG AAACAACTTC AGAATCTTCAGATTTTGAAA

[0120] 2641 ATGAGTCAGA ATCAGAATAG TACTTTTATT AAAATCAAAA TAGCCCAAAAATAATTCTA

[0121] 2701 GCTTATATAG ATACAGGAGA TTCTTTATGT TTATTACAAG AAAAATAATTTACCAAAAACT

[0122] 2761 TTTTGGAAAG AATTAAAGAA ACCTATAAAA ATAACAATAG CAGATAAAAGAATTCTAGAA

[0123] 2821 ATAAATAAAG TTGCTTTATT AATAACCATA GTAATAAGAA ATAAAAAATTCTTAGTACCT

[0124] 2881 ACAATATATC AATTTAATGC AGGAGTACCT ATGATTATAG GAAATAATTTTCTTAGATTA

[0125] 2941 TATTATCCAT TTTGTCAATA TTTAACTTAT ATCACTCTTA AATGTCCTCAACTCAGTAAT

[0126] 3001 CAAAAACAAG AAATAATTAA AATTCCGATA TATCATTCTT TTAAACTAAAAATAATAAGA

[0127] 3061 CAAATATGTT CCATAATAAA TAATTTAGAA GAAGAAATGT TAATTAAAAATATAGAAGAA

[0128] 3121 AAATTACAAG AAAGATTTTC TAATGATCTT TTAGGAAATA AAAATACTAATGAAGAATTA

[0129] 3181 ATAGAAATAA AGCTTAAAGA CCCCTTACAA GAAGTAAATG TACCTAATAATATCCCTTAT

[0130] 3241 TCAGAAAAGG ATATCATTGA ATTTAAAAAA GATATGGATG ATTTAATAGAAAAAAGAGTT

[0131] 3301 ATTAGACCTA GTAATAGTCC TCACTCAGCA CCAGCATTTT ATGTTAATAATCATGCTGAA

[0132] 3361 GAAAAAAGAG GAAAAAGAAG AATAGTTATT AATTATAAAA AAATGAATGAAGCTACAATA

[0133] 3421 GGAACTCCAA AAACATTACC AAGAAAAGAT TATATAATAA ATAAATTAAAAGGGAAAAAAA

[0134] 3481 TGGTTTTCCA CCTTAGATGT TAAATCAGCA TATTGGCAAC TAAGATTACATGAGAATACA

[0135] 3541 AAACCATTAA CAGCTTTTAC TTTTCCACCACACCACAT ATGAATGGAATTCACCA

[0136] 3601 TAGTTTA SQUARECC NATIONAL SEASONAL TTAGTTAA SQUARE

[0137] 3661 TTAGTAAATATATATATATATTATTAGTATAAATATTCTGAAAAATAAA

[0138] 3721 ATTACKACC ATTAAGATGT TTAAAATAATTAATAGAT GTCAAAAAATTAGATTT

[0139] 3781 TTATCCCAGC CAAAAGCTAT TATTGCAAAA GAAAAAATAG ATTTTAGGATTAAATATT

[0140] 3841 TCTTCAGGAG AATTATTTT ACAAAAACAT ATTGTTGAAA AATTAAATTTATTTCCTGAT

[0141] 3901 AAAATAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATAGAAATATAGAAATAGAAAAATAGAAAAATAGAAA

[0142] 3961 CAAGGTTTCT TAAAAAATCT AGCAAAAGCT AGACGTCCCT TACAGAAAAAATTATCAGAA

[0143] 4021 AAAGTCCCTT GGACTTGGAC TGATAAAGAC AGCAGATATG TTCAGGAGTTAAAAAGCATCT

[0144] 4081 TGTATAAATC TACCCAGATT ATACAATGCT AATATTCAGG ATTTATTAATAGTTTCTACT

[0145] 4141 GATGCCAGCA ATGGTCATTG GGGAGCTATT ATGACAGCAA TACCTCAAGAAATATTGCAA

[0146] 4201 CONTACT ACAAAAATAA CATCHAATT CATCATTCAAA ATTCATTCAAATTCAGCAG

[0147] 4261 TTATTACCTACCTT TCAAACCTA CLAIM CCAATACCCAGTGGTACC

[0148] 4321 TTTACTGATA CAGAAACAAG ATACCCTATA CACCATCTAG AAACCCTTGCAGCAATTCGA

[0149] 4381 ACCTTCAGAA AATGGAAAAT TGATTACTA CCAAAACCTT TTATTTAAAACCGATTCC

[0150] 4441 AAATACCTAT CTGGATTCTT AAAATATAAC ATCAAAGCCA ACTACAATCAAGGACGATTA

[0151] 4501 ATTAGATGGC AGCTAGATT AAATCAGTAT GATTACAAGG TATTTATTAGAGTACA

[0152] 4561 CHATAATTTTG PROCESS TTTQACGA PARTGGAAAAG AGCAATAAAGATTGCAAA

[0153] 4621 ATCAATTCA GCAAAAAGAA ATCAGATCC ACCAAAAAT GAGAAATTGAAGGATTGC

[0154] 4681 AAAATGATT AAAAATTTTG AAAGAACAT TTTCAGTGCT AFTERATCCTAAAAG

[0155] 4741 AAATTACK AGAAAAATT CHAPTER TTGATCAAAT NEW CHAPTER

[0156] 4801 SECTION GAAAGCTTTT TATGTTATTYPECATCHATTATG

[0157] 4861 AATGGCATAA WEATHERING CHANGE AATGGCATAA CHAPTER ATTTGA

[0158] 4921 GTATAGAAGA AGCCCAGCAA GCTTTAAAAA ATAGTCAATT AGCCAATATTGTCCAACAAG

[0159] 4981 AAAAACCAGT ACGAAGATGT TTTGGAGAAY CTCAGGGAAG ACGATAAGGCTATA

[0160] 5041 ATACTGCCAG AAGCCTCTAT AAATATGCAT CAAACTCTCC TGTTTGAGGGGAGCGAAAAT

[0161] 5101 TTAAATAGA AAACCTTTGT GTTTTATAAA CTCTTTCGAG TTTTTTCAGATCTTTTGATC

[0162] 5161 TATTTCGAGT TAGTCGTGAG GTAATACTCT GGACGACACC CCAACATAATTTCATCAACA

[0163] 5221 QUESTIONNAIRE AAATTTTCTGCGTTTTCAAAA

[0164] 5281 CONTINUOUS WINDOW WINDOW QUOTE CONNECTEDGTTCAttachment

[0165] 5341 AGCATGTTAT AGTTATAATG ATATATGTTT AGAAAGCATG TTATAATCATGAATCATTTA

[0166] 5401 ACTAGATCGT ATTGATTAT ACGATTTTAG TATAAACA GGTACGAGCACGTTCAACTG

[0167] 5461 TGTTTTCAAA CACGGTGTGC AACTACAATG GAAAATACTA GAGAAAACAATCAAAATA

[0168] 5521 AAAGATATA ATATTGAAGA ATTTTTCGAT ATAAATACAAA ATAAAACTAGTTCTTAT

[0169] 5581 TATGGTTTAG ATTTCAAGT AGAAAGATCT AAAATAAAAG ATATAGAAAAACAAGGAAAAA

[0170] 5641 ATTACKTAA AAGATGGTCA NATIONAL ATCCCTAAAT TACTAATGTTGAAAAAAT

[0171] 5701 CONNECTED CONNECT ASSOCIATE CONNECTED WIND AAATATACTTCTCCHGGGG

[0172] 5761 CAAATAGCCT TATCACTTAT HELP CAAATTTCAG AAAGACTTGATAAAATAAAA

[0173] 5821 GTACKET TACKETTARD HELP CTATTCATTARDET

[0174] 5881 AGCACCTATT CCGCTATAGA TACTCCAATA GATTTAGCAG TTATAGACATAGAATAATA

[0175] 5941 TCTAAAAATC CTAAAAAAG AATTTTAGGA ATTTAAAAG GAAATCTTAATATCCAGTT

[0176] 6001 ATTAAATTTA ATTACKTTTT GCC ATTCCATTACAAAAATTTATTAGTCAG

[0177] 6061 TSEQUENCE SEQUENCE ATTTSEQUENCE SERIES TGAAACAGGTSEQUENCCA

[0178] 6121 CTTAGE CTTATGTAGE NATIONGCC CTAIN GTCATCAGETAGE .

[0179] 6181 AAACAAAAG ATTACKGRAPH CTTATTTAAC AAACAAACTCHAPTER GTG

[0180] 6241 ATTTTAGG AAAATAACAA AATACTTTTA ATTACKTATTCCAAAAACTTTA

[0181] 6301 CATTATAAG AACCTAAATT ATTAGATCT ACAAGTTCTA CATTATTTAAAAACCACCT

[0182] 6361 AACTTACATC ATAAAGAAAAATAAAGAAAATCAATAAATAAATAAAATTACAATCAA

[0183] 6421 ATAATAGAAT CONNECTIONS ASSISTANT TTAAATAAC CONNECTIONS ATCC

[0184] 6481 TTAACAGGAG AGTTTGATAC ATATTTATAG ACATGGAACT CGGACTTCAAAACATATTAC

[0185] 6541 AATATAACTC AATAACGGAC CTGACTTTAC ATTTCATTAT AGTTTTAAAAGATAAAGA

[0186] 6601 CGGAACGGAC TTGAAGTTAC AATTAAATGT AACAGACTCG AAAACGACATAAAGAAAAAG

[0187] 6661 TTTGAAAATG ACAAAAA

[0188] NotSV polymerase gene (SEQ ID NO:2)

[0189] 1 ATGAGTCAGA ATCAGAATAG TACTTTTATT AAAATCAAAA TAGCCCAAAA AATAATTCTA

[0190] 61 GCTTATATAG ATACAGGAGA TTCTTTATGT TTATTACAAG AAAATAATTT ACCAAAAACT

[0191] 121 TTTTGGAAAG AATTAAAGAA ACCTATAAAA ATAACAATAG CAGATAAAAG AATTCTAGAA

[0192] 181 ATAAATAAAG TTGCTTTATT AATAACCATA GTAATAAGAA ATAAAAAATT CTTAGTACCT

[0193] 241 ACAATATATC AATTTAATGC AGGAGTACCT ATGATTATAG GAAATAATTT TCTTAGATTA

[0194] 301 TATTATCCAT TTTGTCAATA TTTAACTTAT ATCACTCTTA AATGTCCTCA ACTCAGTAAT

[0195] 361 CAAAAACAAG AAATAATTAA AATTCCGATA TATCATTCTT TTAAACTAAA AATAATAAGA

[0196] 421 CAAATATGTT CCATAATAAA TAATTTAGAA GAAGAAATGT TAATTAAAAAA TATAGAAGAA

[0197] 481 AAATTACAAG AAAGATTTTC TAATGATCTT TTAGGAAATA AAAATACTAA TGAAGAATTA

[0198] 541 ATAGAAATAA AGCTTAAAGA CCCCTTACAA GAAGTAAATG TACCTAATAA TATCCCTTAT

[0199] 601 TCAGAAAAGG ATATCATTGA ATTTAAAAAA GATATGGATG ATTTAATAGA AAAAAGAGTT

[0200] 661 ATTAGACCTA GTAATAGTCC TCACTCAGCA CCAGCATTTT ATGTTAATAA TCATGCTGAA

[0201] 721 GAAAAAAGAG GAAAAAGAAG AATAGTTATT AATTATAAAA AAATGAATGA AGCTACAATA

[0202] 781 GGAACTCCAA AAACATTACC AAGAAAAGAT TATATAATAA ATAAATTAAA AGGGAAAAAA

[0203] 841 TGGTTTTCCA CCTTAGATGT TAAATCAGCA TATTGGCAAC TAAGATTACA TGAGAATACA

[0204] 901 AAACCATTAA CAGCTTTTAC TTTTCCACCA CAACAACATT ATGAATGGAA TTCATTACCA

[0205] 961 TTAGGTTTAA AACAAGCACC AGGAATATTT CAGAATTTTA TGAATAAACA ATTACAAGGA

[0206] 1021 TTACKTAGTATATATTAGTATATATATTTATTCTGAAAAAAATAAA

[0207] 1081 ATTACKACC ATTACKGATGT TTAAAATAATTAATAGAT GTCAAAAAATTAGATTT

[0208] 1141 TTATCCCAGC CAAAAGCTAT TATTGCAAAA GAAAAAATAG ATTTTAGGATTAAATATT

[0209] 1201 TCTTCAGGAG AAATTATTTT ACAAAAACAT ATTGTTGAAA AATTAAATTTATTTCCTGAT

[0210] 1261 AAAATAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATATAGAA TTTTAGAAAATTAGAAATAGAAAAAAAAAAAAATAGAAA

[0211] 1321 CAAGGTTTCT TAAAAAATCT AGCAAAAGCT AGACGTCCCT TACAGAAAAAATTATCAGAA

[0212] 1381 AAAGTCCCTT GGACTTGGAC TGATAAGAC AGCAGATATG TTCAGGAGTTAAAAAGCATCT

[0213] 1441 TGTATAAATC TACCCAGATT ATACAATGCT AATATTCAGG ATTTATTAATAGTTTCTACT

[0214] 1501 GATGCCAGCA ATGGTCATTG GGGAGCTATT ATGACAGCAA TACCTCAAGAAATATTGCAA

[0215] 1561 CONTACT ACAAAAAATA CATCHCAG CTAAATCAAA ATTCATTCAAAGATTCAGCAG

[0216] 1621 TTATTCAATA CAAAAACCTT TCAAAACCTA GAAAAATTAA CCAAATATACCAGTGGTACC

[0217] 1681 TTTACTGATA CAGAAACAAG ATACCCTATA CACCATCTAG AAACCCTTGCAGCAATTCGA

[0218] 1741 ACCTTCAGAA AATGGAAAAT TGATTTACTA CCAAAACCTT TTATTTTAAAAACCGATTCC

[0219] 1801 AAATACCTAT CTGGATTCTT AAAATATAAC ATCAAAGCCA ACTACAATCAAGGACGATTA

[0220] 1861 ATTAGATGGC AGCTAGAATT AAATCAGTAT GATTACAAGG TATTATTAATTAAGAGTACA

[0221] 1921 GATAATTTTG CAGCAGATAC TTTGACTCGA GAATGGAAAG AGCAA.

[0222] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A separated organism, characterized in that, It contains nucleotides selected from the following: The nucleotide or fragment thereof shown in SEQ ID NO: 1; or a nucleotide complementary to the nucleotide shown in SEQ ID NO:

1.

2. The isolated organism according to claim 1, characterized in that, The organisms mentioned are microorganisms.

3. An isolated nucleotide, characterized in that, Selected from at least one of the following nucleotides: The nucleotide or fragment thereof shown in SEQ ID NO: 1; Nucleotides complementary to the nucleotide shown in SEQ ID NO: 1; The nucleotide or fragment thereof shown in SEQ ID NO: 2; Nucleotides complementary to the nucleotide shown in SEQ ID NO:

2.

4. A primer pair, characterized in that, It is selected from the following nucleotides: nucleotides complementary to the nucleotides shown in SEQ ID NO: 1 or SEQ ID NO:

2.

5. The primer pair according to claim 4, characterized in that, The primer pair is selected from any of the following primer pairs: The primer pairs shown in SEQ ID NO: 17 and 18; The primer pairs shown in SEQ ID NO: 19 and 20; The primer pairs shown in SEQ ID NO: 21 and 22; The primer pairs shown in SEQ ID NO: 23 and 24; The primer pairs shown in SEQ ID NO: 25 and 26; The primer pairs shown in SEQ ID NO: 27 and 28.

6. The use of the primer pair according to any one of claims 4 or 5 in the diagnosis of plant virus infections, characterized in that, The plant is a species of the genus *Pseudorasporium*; the species of the genus *Pseudorasporium* include any one or a combination of *Mabirium macranthum*, *Pseudorasporium thunbergii*, *Pseudorasporium odoratum*, *Pseudorasporium scaber ... and *Pseudorasporium pubescens*.

7. The use of the primer pair according to any one of claims 4 or 5 in the identification of pathogens, characterized in that, The identification refers to: determining whether the pathogen to be tested is the organism isolated as described in claim 1 or 2; or determining whether the organism isolated as described in claim 1 or 2 exists in the sample.

8. The use of the primer pair according to claim 4 or 5 in the preparation of a pathogen detection device, characterized in that, The pathogen is the isolated organism as described in claim 1 or 2; the detection device is in the form of a reagent or a chip.

9. A detection device, characterized in that, It comprises the primer pair as described in any one of claims 4 or 5; the detection device is in the form of a reagent or a chip.

10. A method for detecting pathogens, comprising the steps of: 1) Provide a sample to be tested; the sample is derived from plants of the Cornaceae family; 2) Obtain the nucleotides of the sample to be tested; the nucleotides are DNA; 3) Contact the nucleotides of the sample to be tested or their amplification products with the primer pair according to any one of claims 4 or 5; 4) Determine whether the isolated nucleotides as described in claim 3 exist.

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