Specific identification method of a new pathogen of rice smut and application thereof

Abstract

The application discloses a specific identification method of a new pathogenic fungus of rice smut and application thereof. The identification method comprises the following steps: obtaining a specific sequence according to Moesziomyces antarcticus; designing a specific primer; treating a sample to be detected to obtain DNA in the sample to be detected; taking the DNA in the sample to be detected as a template, and performing PCR amplification on the specific primer; and determining whether the pathogenic fungus Moesziomyces antarcticus exists in the sample to be detected by analyzing whether a specific fragment of the Moesziomyces antarcticus exists in the PCR amplification product. The application provides an accurate detection technology for rice smut, and reduces sequencing cost, sequencing time, and labor cost of sequence comparison and analysis. In combination with enrichment of the target fungus, more than 90% of miscellaneous bacteria in a sample with a complex microbial background are excluded, and the sensitivity of the enrichment step to the target fungus is improved by more than 90%.

Description

Technical Field

[0001] This invention relates to the field of rice grain smut pathogen technology, and in particular to a specific identification method for a new rice grain smut pathogen and its application. Background Technology

[0002] Rice grain smut is the leading disease in hybrid rice seed production. In years with mild outbreaks, yield reduction is typically 5%-10%, while in severe cases, it can lead to reductions of 30%-60%. Rainy weather during the flowering and grain-filling stages of hybrid rice seed production exacerbates the disease's spread. Therefore, controlling rice grain smut is crucial. Detection of the pathogen is a vital aspect of rice grain smut control, and simple detection techniques can be widely applied to all stages of hybrid rice seed production for effective disease control.

[0003] Rice grain smut is generally believed to be caused by *Tilletia barclayana* (Bref.) Sacc & Syd. However, the inventors of this technology have discovered a new pathogen causing rice grain smut—*Moesziomyces antarcticus*. Furthermore, four consecutive years of sampling surveys have proven that *Moesziomyces antarcticus* has been the main pathogen causing rice grain smut in recent years. To date, research on *Moesziomyces antarcticus* has focused on the development and utilization of sugar alcohol and lipid metabolites, with no reports on its pathogenicity in rice. Currently, there is no specific identification method for this bacterium; conventional microbial molecular identification methods, such as PCR amplification of conserved sequences like ITS, 18S, and 16S, followed by sequencing and comparison, are generally used for identification, which is costly, time-consuming, and labor-intensive. Therefore, we have developed the present invention, namely, a specific identification method for the novel pathogen of rice grain smut, *Ustilago maydis*, for the detection of rice grain smut caused by *Ustilago maydis*. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] In view of the above-mentioned new discoveries and the shortcomings of the prior art, the purpose of this invention is to provide a specific identification method for a new pathogen of rice grain smut and its application, which can largely eliminate the interference of non-target bacteria, enrich and quickly detect the presence of the new pathogen of rice grain smut in samples with complex microbial backgrounds (including seeds, plants, biological tissues and soil samples, etc.).

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the main technical solution adopted in this invention includes specialized primers for the specific identification of new pathogens of rice grain smut, which consist of the following primer pairs:

[0008] MaUNS-F: tcaagagcctttgtcctcgg;

[0009] MaUNS-R: atgtcgtggcgagcatcgt.

[0010] Preferably, the application of specialized primers in the specific identification of new pathogens of rice grain smut.

[0011] On the other hand, this invention discloses a specific identification method for a new pathogen of rice grain smut, comprising the following steps:

[0012] (1) Based on the Antarctic smut fungus, a specific sequence SEQ ID NO:1 was obtained through biological analysis;

[0013] (2) Design specific primers by selecting a suitable segment within the sequence SEQ ID NO:1, including the specific primers as follows:

[0014] MaUNS-F: tcaagagcctttgtcctcgg;

[0015] MaUNS-R: atgtcgtggcgagcatcgt;

[0016] But it is not limited to this primer pair.

[0017] (3) Process the sample to be tested to obtain the DNA in the sample to be tested. Using the DNA in the sample to be tested as a template, perform PCR amplification with the primers in step (2). Analyze whether there is a specific fragment of Moesziomyces antarcticus in the PCR amplification product to determine whether the sample to be tested contains the target pathogen Moesziomyces antarcticus.

[0018] Preferably, in step (3), the sample to be tested includes colonies, bacterial solutions, seeds, plants, biological tissues, or soil samples.

[0019] Preferably, step (3) of processing the sample to be tested includes enrichment, and the enrichment method includes the following steps:

[0020] Grind the sample to be tested in sterile water, mix well, and then spread the mixture onto solid culture medium HFJ. Incubate in a constant temperature incubator at 25℃-30℃ for 24-72 hours. Once colonies grow, proceed to the next step of the experiment.

[0021] Alternatively, grind the sample to be tested in sterile water, add the mixture to liquid culture medium HFJY, and then incubate in a shaker at 25℃-30℃ and 150-220rpm for 24-72h to obtain the bacterial solution for the next experiment.

[0022] The HFJ culture medium contains no nitrogen source and, by mass percentage, comprises: 0.4%-1% carbon source, 0.2%-0.6% calcium carbonate, 0.01%-0.05% magnesium sulfate, 0.01%-0.03% potassium dihydrogen phosphate, and 1.2% agar powder, with a pH of 6.5-7.0. This culture medium also contains antibiotics that inhibit bacteria but not fungi. The carbon source is one or more of sucrose, glucose, arabinose, mannitol, and sorbitol.

[0023] The HFJY culture medium contains no nitrogen source and, by mass percentage, comprises: 0.4%-1% carbon source, 0.2%-0.6% calcium carbonate, 0.01%-0.05% magnesium sulfate, and 0.01%-0.03% potassium dihydrogen phosphate, with a pH of 6.5-7.0. The medium also contains antibiotics that inhibit bacteria but not fungi. The carbon source is one or more of sucrose, glucose, arabinose, mannitol, and sorbitol.

[0024] Preferably, the mass ratio of the sample to be tested to sterile water is 1:1-4.

[0025] Preferably, for cultures in solid culture medium HFJ, DNA templates are prepared by scraping mixed colonies or by picking single colonies;

[0026] For bacterial culture in liquid medium HFJY, DNA templates are prepared using the bacterial culture or the concentrated bacterial cells after centrifugation.

[0027] Preferably, the rapid DNA template preparation method includes the following steps:

[0028] Mix the bacterial cells or bacterial solution with KOH solution, place the resulting mixture in a boiling water bath for 10-20 minutes, and then centrifuge to collect the bacterial cells.

[0029] Preferably, the KOH concentration in the mixture is 0.1M-0.5M.

[0030] (III) Beneficial Effects

[0031] The beneficial effects of this invention are as follows: Based on the inventors' discovery that the main pathogen of rice grain smut in recent years is a novel pathogen, *Moesziomyces antarcticus*, this invention develops a highly efficient and specific detection method targeting the specific sequence SEQ ID NO: 1 of *Moesziomyces antarcticus*. The enrichment step of this invention further improves the detection sensitivity of the target species in samples with complex microbial backgrounds, excluding more than 90% of other contaminating bacteria and increasing the number of target bacteria. Under efficient and rapid DNA template preparation conditions, the PCR efficiency of this specific fragment is high, with a minimum detectable copy number of approximately 100 in a 20 μl reaction system, reaching the scope of production applications. This invention can be used for quarantine, early warning and control of rice grain smut, and related research.

[0032] This invention can specifically amplify the specific band of *Moesziomyces antarcticus*, a novel pathogen of rice grain smut, providing an accurate detection technique for this disease. It eliminates the need for sequencing and sequence comparison analysis, two steps required for conventional strain identification, saving over 90% of detection costs (based on a market price of 15 yuan per sample for DNA sequencing). It also reduces sequencing time and the associated labor costs for sequence comparison analysis. Combined with target bacteria enrichment, it eliminates over 90% of contaminating bacteria in samples with complex microbial backgrounds (such as seeds, plants, biological tissues, and soil samples), increasing the sensitivity of the enrichment step to the target bacteria by over 90%. Furthermore, the rapid DNA template preparation step further reduces the overall detection process time and DNA extraction costs. Attached Figure Description

[0033] Figure 1 The image shows the detection of the SEQ ID NO:1 fragment characteristics in 32 strains using primers MaUNS-F and MaUNS-R.

[0034] Figure 2 This image shows the detection of DNA templates from 32 strains using PCR validation with primers ITS1 and ITS4.

[0035] Figure 3 A graph showing the amplification efficiency of MaUNS-F and MaUNS-R primers.

[0036] Figure 4 Electrophoretic images of yellow rice and Y58s rice seeds detected using MaUNS-F and MaUNS-R primers. Detailed Implementation

[0037] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below through specific embodiments.

[0038] The invention of this application will be further illustrated by the following embodiments, but this does not limit the scope of this application.

[0039] Unless otherwise stated, the experimental methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in related fields such as molecular biology, biochemistry, analytical chemistry, cell culture, and recombinant DNA technology.

[0040] Unless otherwise specified in the specific embodiment description, all materials and reagents used in the following examples are commercially available.

[0041] Since this invention is based on the inventor's discovery that *Moesziomyces antarcticus* is a novel pathogen of rice smut (currently unreported domestically and internationally), there is currently no specific detection technology for this pathogen. The conventional molecular identification method for microorganisms involves PCR amplification of conserved sequence fragments such as ITS, 16S, and 18S, followed by sequencing of the amplified products and comparative analysis of the sequencing results to determine the species of the tested strain. This conventional conserved sequence amplification method can amplify the target fragment (band on gel electrophoresis) from almost all microorganisms, lacking species specificity. Results can only be obtained through sequencing and bioinformatics analysis (this method requires sequencing and comparative analysis of all or most microbial strains in the sample to exclude many non-target pathogens or common epiphytes and endophytes, resulting in high sequencing costs and a large workload). This invention can enrich new pathogens of rice grain smut, improve detection sensitivity, and identify the target pathogen simply by PCR amplification of specific fragments (manifested as specific bands in gel electrophoresis or strong signals in quantitative fluorescent PCR, etc.), without the need for sequencing and comparative analysis, saving more than 90% of the overall detection cost and reducing the workload of comparative analysis.

[0042] Based on the detailed genome map obtained from de novo sequencing (Illumina Novaseq 2D + Nanopore 3D) of the pathogenic bacterium Moesziomyces antarcticus Ma2-3 isolated from us, a specific sequence SEQ ID NO:1 was obtained through bioinformatics analysis, as shown below:

[0043]

[0044] Specific primers were designed by selecting an appropriate segment within the sequence SEQ ID NO:1, and PCR amplification was used to obtain a specific fragment of *Moesziomyces antarcticus*, the pathogen causing rice grain smut. The presence of the target pathogen was determined by PCR amplification analysis of the sample to be tested to identify the presence of the *Moesziomyces antarcticus* specific fragment.

[0045] The primers designed in this application are:

[0046] MaUNS-F: tcaagagcctttgtcctcgg, (SEQ ID NO: 2).

[0047] MaUNS-R: atgtcgtggcgagcatcgt, (SEQ ID NO: 3).

[0048] In the embodiments of this application, the sample to be tested can be a colony, bacterial solution, seed, plant, biological tissue, soil sample, etc. The method for enriching the target pathogen in the sample is as follows: The sample is placed in sterile water (sample to sterile water ratio is 1:1-4), ground, and after mixing, the mixture is spread onto a solid culture medium HFJ (the culture medium contains no nitrogen source and, by mass percentage, contains: carbon source 0.4%-1%, calcium carbonate 0.2%-0.6%, magnesium sulfate 0.01-0.05%, potassium dihydrogen phosphate 0.01-0.03%, agar powder 1.2-2%, pH 6.5-7). 0; The culture medium also contains antibiotics that can inhibit bacteria but not fungi; The carbon source is one or more of sucrose, glucose, arabinose, mannitol and sorbitol. It is incubated statically in a constant temperature incubator at 25℃-30℃ for 24-72h. Once colonies grow, it can be used for the next experiment; Alternatively, the mixture after grinding the sample is added to liquid culture medium HFJY (the above formula is without agar powder), and then incubated in a shaker at 25℃-30℃ and 150-220rpm for 24-72h. The resulting bacterial solution can be used for the next experiment.

[0049] In this application, for cultures on solid culture medium HFJ, mixed colonies can be scraped to prepare DNA templates for PCR specificity testing according to the present invention, or single colonies can be picked to prepare DNA templates for PCR specificity testing. For bacterial suspensions on solid culture medium HFJY, the bacterial suspension or centrifuged and concentrated bacterial cells can be used directly to prepare DNA templates. A rapid method for preparing DNA templates is as follows: Mix bacterial cells or bacterial suspensions with KOH solution to achieve a final KOH concentration of 0.1M-0.5M. After boiling in a water bath for 10-20 minutes, centrifuge to collect the bacterial cells. Diluting with solution or undiluted solution can be used as a DNA template for PCR; alternatively, a commercial fungal DNA kit can be used to extract and prepare the DNA template.

[0050] Example 1

[0051] For the specificity verification of sequence SEQ ID NO:1, a pair of primers was designed to verify 32 strains. The primers designed in this application are:

[0052] MaUNS-F: tcaagagcctttgtcctcgg, (SEQ ID NO: 2).

[0053] MaUNS-R: atgtcgtggcgagcatcgt, (SEQ ID NO: 3).

[0054] In addition to the rice grain smut pathogens from 5 different locations (notes in Table 1), 27 other strains were used for validation as controls. These 27 control strains belong to 24 species, including 10 species of smut fungi from 6 genera (among which Moesziomyces aphidis is a close relative of Moesziomyces antarcticus) and 14 other species of plant pathogens and epiphytes, as shown in Table 1.

[0055] Table 1. 32 strains used for sequence specificity verification

[0056]

[0057] After obtaining 32 bacterial colonies using a rapid DNA template preparation technique, 1 μL of template was used for PCR reaction with a standard Taq polymerase. PCR program A was as follows:

[0058] (1) Denaturation at 95℃ for 2 min;

[0059] (2) Denaturation at 95℃ for 20 seconds;

[0060] (3) Annealing at 60℃ for 20 seconds

[0061] (4) Extend at 72℃ for 1 min

[0062] [Steps (2) - (4) 35 cycles]

[0063] (5) Extend the process at 72℃ for 5 minutes.

[0064] Electrophoresis and gel imaging were performed using a 0.8% agarose gel, and the results are as follows: Figure 1 The bacterial species in the swimming lanes are shown in Table 1.

[0065] from Figure 1 The results show that only the target pathogen strain could amplify a specific band (specific sequence), while the other 27 control strains did not have this specific band. Figure 1 The bottom band of the 32 strain samples was a primer dimer (which is not within the scope of this study), including the closely related species Moesziomyces aphidis, which did not have this band, indicating good specificity.

[0066] Example 2

[0067] To exclude in Example 1 Figure 1 The absence of specific bands in the other 27 control bacteria was not a problem with the DNA template. PCR amplification was performed using the same DNA template with ITS1 and ITS4 primers. PCR program B was as follows:

[0068] (1) Denaturation at 95℃ for 2 min;

[0069] (2) Denaturation at 95℃ for 20 seconds;

[0070] (3) Annealing at 56℃ for 20 seconds

[0071] (4) Extend at 72℃ for 1 min

[0072] [Steps (2) - (4) 35 cycles]

[0073] (5) Extend the heat at 72℃ for 5 minutes.

[0074] ITS 1: 5'-tccg tagg tgaa cctg cgg-3', (SEQ ID NO: 4).

[0075] ITS 4: 5'-tcct ccgc ttat tgat atgc-3', (SEQ ID NO:5)

[0076] Using the same electrophoresis detection method, the results are as follows: Figure 2 The bacterial species in the swimming lanes are shown in Table 1.

[0077] from Figure 2The results showed that the DNA templates of all 32 strains could amplify the fragment between ITS1 and ITS4 (this fragment varies in length among different strains; in this batch of strains, the fragment size was approximately 350bp-800bp, and the bottom band was a primer dimer, which was not within the scope of this study). This result proves that the DNA templates of all 32 strains were normal. Figure 1 and Figure 2 The results showed that MaUNS-F and MaUNS-R could amplify specific bands of the new pathogen of rice grain smut, Moesziomyces antarcticus.

[0078] Therefore, this primer can be used to perform rapid pathogen-specific detection of the new pathogen of rice grain smut, Moesziomyces antarcticus.

[0079] Example 3

[0080] To assess the amplification efficiency of MaUNS-F and MaUNS-R primer pairs for *Moesziomyces antarcticus* template amounts, PCR amplification was performed at different template amounts. Concentrations on the order of 10^6 mg / mL of cells were used. 3 10 4 10 5 10 6 10 7 and 10 8 Moesziomyces antarcticus liquid bacterial samples were processed using a rapid DNA template preparation technique. The sample was treated in a boiling water bath for 15 min, and then 1 μL of the DNA template was dissolved according to the original bacterial volume for PCR reaction. The total reaction volume was 20 μL. This corresponds to approximately 0.1, 1, 10, 100, 1000, and 10000 cells per cell in the PCR reaction system, respectively. The reaction procedure was the same as described above.

[0081] Figure 3 To detect the amplification efficiency of MaUNS-F and MaUNS-R primers.

[0082] from Figure 3The results showed that a specific band of *Moesziomyces antarcticus*, the pathogen of rice grain smut, could be effectively amplified when the cell count in a 20 μl PCR reaction system was between 100 and 10,000 (band brightness was not directly proportional to the cell count, which may be related to the DNA template processing method, but does not seem to affect the minimum detection sensitivity). Although a count of 10 cells was barely detectable, the band was not very obvious. Therefore, the minimum detectable copy number of this invention may be less than 100 cells. Whole-genome sequence analysis showed that this fragment was a single copy. Based on experience in the field, a specific detection method that can detect a template count of 100 copies can be used for routine pathogen detection.

[0083] Example 4

[0084] Two seeds from each of the two rice varieties, Huanghuazhan and Y58s, harvested in the current season, were placed in a 1.5 ml centrifuge tube containing 400 μl of sterile water and ground. 100 μl of the ground mixture was spread onto HFJ culture dishes containing solid culture medium (the remaining mixture was stored at -20℃ and designated as "Huanghuazhan-Z" and "Y58s-Z" respectively). The mixture was then incubated at 28℃ for 72 hours. Mixed colonies from 1 / 4 of the surface of each Huanghuazhan and Y58s culture dish were scraped and dissolved in KOH solution (using centrifuge tubes) to achieve a final KOH concentration of 0.1 M. The mixture was then placed in a boiling water bath for 10-20 minutes (simultaneously, the remaining mixture, designated "Huanghuazhan-Z" and "Y58s-Z", was used to prepare the same DNA template). The mixture was then centrifuged at 12000 rpm for 5 minutes, the supernatant was discarded, and the DNA template (i.e., the DNA template for the seed samples to be tested) was obtained by dissolving the supernatant in sterile water. A DNA template from *Moesziomyces antarcticus* Ma2-3 strain was used as a positive control, and sterile water (ddH2O) as a negative control. PCR was performed using primers MaUNS-F and MaUNS-R, T3Super PCR Mix, in a 20 μl reaction volume, according to PCR program A. Electrophoresis detection was performed as described previously. The results are as follows. Figure 4 .

[0085] The test results showed that the sterile water negative control did not contain the target band, while the Ma2-3 strain was positive and contained the target band. The target band was also detected in both *Moesziomyces antarcticus* and *Moesziomyces var. chinensis* seeds after enrichment culture, while the target band was not detected in the unenriched ground mixtures "*Moesziomyces var. chinensis*-Z" and "*Moesziomyces var. chinensis*. This example demonstrates that the present invention can enrich and detect extremely low levels of the novel pathogen *Moesziomyces antarcticus* in very small sample quantities (2 rice seeds).

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. sequence list <110> Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences <120> A specific identification method for a new pathogen of rice grain smut and its application <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 5080 <212> DNA <213> Artificial Sequence <400> 1 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tacgaccgta tccaccgaga ttcgatgtat gggggatttg 900 acgtactagt tgcttacgcc gatggtgatc accttgccct cggaacgcag ctccttgagc 960 gcctcccaca gatgcttgcg accctcgggg ccagaggtag gcgtgtggat caggaagcag 1020 tccacgtagc ctgcacaatc acgcgaagga aacatcagta taccacgttg cggctctgat 1080 gtaatgtggc gaatggacag cttaccgtca aggttgatct tgttgaccga ctcgcgcagc gacgccagcg tctcctcctg cgtcttggag gagcgatga tcttggtcgt cacaaacacg tcgcgacgcg acgagtgcgt ctcgaggcac catgcacgca ccgcatcgcc caccctacgt 1260 cccgcaccaa cctcgtcagt caaagtcatt tttgcaacgt gtagacgtgc acacttactc cttttcgttt tcgtagtact gagccgagtc ctacagccac acgagcagga gtgattcacc 1380. ggtcagtacg ccatctcaag aatagaagc agagatccac gtacaatgtg gcggtatccc gtcttgagtg cggtggtgac cgacttgacc gtcacgtcgg tggggctctt gtacacgcca aagccgagca cgggcatgtc cacgccctcg cccagcggat acgtcgactg cagagtcagc ttgacggtca ttttgaacaa gtgatcagaa tgttggttct rich aggatggggt ggagttggta ggaagaaga gcaaagcaag ggaccgtgcg ctcgatccca cttcgcctcc actcttggac cctggcgaag cgccaaattc cgcactcggc ctgcatgact tggtcagccg ccgcctcgct 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acctgcgctt agccccgcag cctgtgcacc cgctggaggt 2640 gccgcttggg caggctggcc atttttgcct ccctcctgtc cggatcgaac cgcatcgcag 2700 cgctggcaag tgggcagggc cgtgtggcac ttcatgtgtt ttgtgttcgt aaaggcgacg 2760 agtgtgttgc accacgcgat tggctgctgt ggctgctcag gatggtagag caccaccacg 2820 ctatcaccca tccccgtagt ccttggcttt ctgcggtccg aagtgctgag tgaggttcca 2880 gtgggctgga tccccaacgg cttgttgggc ggatcgggat gtcgggacat gcagaaggtg 2940 aagttgcgga tggcaagcca gccgaagctt tgggcgtgtg cctccatcgg gtcctgcacc 3000 accgcattgg ctgcacccga atacaccagg cccagcaagt atgccatcgc aagcgcaaac 3060 gacgatgctc gccacgacat cctactcaat cttcccagag ccagtactgc aggtccaacg 3120 aaaagcttgg ctggtcttga gatggcgtcg ggaaagctga aagaacgatg tggaagatga 3180 gctgagagaa gagaggtgat agcgggaaaa ggttgggctg gtatagtctc gcgagcaaag 3240 gtgggaggcg gcacagatga aggtcgtact gccccaccag ctttccggtc gaggcttcat 3300 atgaaagcat ggccacacga gcttcagccc gccactgcat agtcacaaca atgacaggac 3360 gccgctttga tattacaccc acatgcgcgc ctcggggcta aactcatttc gccacgcgca 3420 attgtagccc tgtcggtacg tcatcagttg gcgccaagcg atgcaaacag gcgacccccc 3480 ctgggccccg cttcccatct ccgcatcaac atcctcaaac tcactgcatc cagcacagat 3540 cttgagcttg aacgagaaag atatgcaaag ttgacacata tcgataggat gggttgatgt 3600 gaaagtgtgc agatccggcg gaggccacgc gagtgccgtc tgggcgtgtg agagctattc 3660 cgagcttgag ctggagctgg aaatggagct agagctggaa ctggactgcc tcgagctgcc 3720 actttgctgt gcgagacggg aagatctcag ctgctgttcg gtctgctcga ctctttctcc 3780 gcgcagtttc tggatggcat ccgaagaggt gaggttgttt tcgacaatgt gctcgtgcac 3840 ctgttccgag gtatcgtgta cgcgcggaaa ctcgaagatc tcgatggcat cctcggcacc 3900 tgcagccact tcctcctcca ggtcacgtcg agcgagattg cgagttccgc gagccagacg 3960 ttggacgcc gaaggtggt ttccagctgg catggcggct atgccagcca ggcagctcgt gacgagga gaaaggaca cgagcgatcc agatcgcctc atgattgcct cgctcgaatg taagaatgt ttcgaagcca atgacggacg atgtttctga aaggaagcca tgagcccacg aggagatggc cgtggtcagg tgttcttatc cactcgaagg ggaacagttc gccggcggca cggcatcgca atcccggcgg cggcatcgca gtgacttctc cggcatcagcagcgcaag gtcggcattg aaatggtggg agggacggcg ctgtggatcc ttctcgcaag tcgcggcgtc 4320. tttgtggcag ggttgcgggt gcgaatccac gcctcgtcgc ttggtgctca gcccacatgc 4380. attcacgggc tctcatttcg gattcagccc acctgctgtg ttggtggaga acgtggggtt 4440 gtggattgca cagccatgat accgactggg ccgcagcatc tcacatagct ttggcactca agccacggcc agccgcacag gttggcaccg tcgctactgc gacatggttg caggagacaa gtcggtggtg accctgagaa ttttcctatt tgtagaagtc gagggtgtaa cattatctca aaaaattgca tcccaatatc ggggcggacg gccggagtcg cggtccggtg ggtccggaa 4680. actccaccgc ttggcgagta tttgctgcag cgaagtttgg cggtcgaggc taggtcgccg 4740 aatgcccgcg ggtgcggctc gtgcgggcct gctcgcccgc actgagaacg caccgaaagc 4800 gtcgacgctt gagcagcagc agcctgtcag gacgctccta ttaaagcagc gatcccatga 4860 gccgggggtt gtgattgttt ttccctcccc aacctaaacc ctcttggaca tttcatctgg 4920 ctgccaggca cgccctttgc ttccgcatcc acgcaagcac atctacgcac gcatcatcga 4980 caaatcattc gaaagaacgt agcgacgcac acaactccag tcatgtcgac atccgaggta 5040 gcatgcgaaa agacgccgtc gacgtcgtcg ttgggcctct 5080 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 tcaagagcct ttgtcctcgg 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <400> 3 atgtcgtggc gagcatcgt 19 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <400> 4 tccgtaggtg aacctgcgg 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 tcctccgctt attgatatgc 20

Claims

1. A specific primer for the specific identification of *Moesziomyces antarcticus*, characterized in that, It consists of the following primer pairs: MaUNS-F: tcaagagcctttgtcctcgg; MaUNS-R: atgtcgtggcgagcatcgt.

2. The application of the specific primers described in claim 1 in the specific identification of Moesziomyces antarcticus.

3. A specific identification method for the Antarctic smut fungus *Moesziomyces antarcticus*, characterized in that, Includes the following steps: (1) Based on the Antarctic smut fungus, a specific sequence SEQ ID NO:1 was obtained through biological analysis; (2) Design a set of specific primers by selecting a suitable segment within the sequence SEQ ID NO:1, wherein the specific primers are: MaUNS-F: tcaagagcctttgtcctcgg; MaUNS-R: atgtcgtggcgagcatcgt; (3) Process the sample to be tested to obtain the DNA in the sample to be tested. Using the DNA in the sample to be tested as a template, perform PCR amplification with the primers in step (2). Analyze whether there is a specific fragment of Moesziomyces antarcticus in the PCR amplification product to determine whether the sample to be tested contains the target pathogen Moesziomyces antarcticus.

4. The specific identification method for Moesziomyces antarcticus as described in claim 3, characterized in that, in step (3), the sample to be tested includes colonies, bacterial solutions, seeds, plants, biological tissues or soil samples.

5. The specific identification method for *Moesziomyces antarcticus* as described in claim 3, characterized in that, Step (3) involves processing the sample to be tested, including enrichment. The enrichment method includes the following steps: Grind the sample to be tested in sterile water, mix well, and then spread the mixture onto solid culture medium HFJ. Incubate in a constant temperature incubator at 25℃-30℃ for 24-72 hours. Once colonies grow, proceed to the next step of the experiment. Alternatively, grind the sample to be tested in sterile water, add the mixture to liquid culture medium HFJY, and then incubate in a shaker at 25℃-30℃ and 150-220rpm for 24-72h to obtain the bacterial solution for the next experiment. The HFJ culture medium contains no nitrogen source and, by mass percentage, comprises: 0.4%-1% carbon source, 0.2%-0.6% calcium carbonate, 0.01%-0.05% magnesium sulfate, 0.01%-0.03% potassium dihydrogen phosphate, and 1.2% agar powder, with a pH of 6.5-7.

0. This culture medium also contains antibiotics that inhibit bacteria but not fungi. The carbon source is one or more of sucrose, glucose, arabinose, mannitol, and sorbitol. The HFJY culture medium contains no nitrogen source and, by mass percentage, comprises: 0.4%-1% carbon source, 0.2%-0.6% calcium carbonate, 0.01%-0.05% magnesium sulfate, and 0.01%-0.03% potassium dihydrogen phosphate, with a pH of 6.5-7.

0. The culture medium also contains antibiotics that inhibit bacteria but not fungi. The carbon source is one or more of sucrose, glucose, arabinose, mannitol, and sorbitol.

6. The specific identification method for *Moesziomyces antarcticus* as described in claim 5, characterized in that, The mass ratio of the sample to be tested to sterile water is 1:1-4.

7. The specific identification method for *Moesziomyces antarcticus* as described in claim 5, characterized in that, For cultures on solid culture medium HFJ, DNA templates can be prepared by scraping mixed colonies or by picking single colonies. For bacterial culture in liquid medium HFJY, DNA templates are prepared using the bacterial culture or the concentrated bacterial cells after centrifugation.

8. The specific identification method for *Moesziomyces antarcticus* as described in claim 7, characterized in that, The rapid DNA template preparation method includes the following steps: Mix the bacterial cells or bacterial solution with KOH solution, place the resulting mixture in a boiling water bath for 10-20 minutes, and then centrifuge to collect the bacterial cells.

9. The specific identification method for *Moesziomyces antarcticus* as described in claim 8, characterized in that, The KOH concentration in the mixture is 0.1M-0.5M.

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