Fusarium-specific gene segments and their use in rapid detection of fusarium species

Abstract

The present application provides a Fusarium-specific gene segment and its use in rapid detection of Fusarium species. Specifically, the present application provides a Fusarium-specific segment. Further, the present application also provides a primer pair for detecting Fusarium species, which amplifies the Fusarium-specific segment of the present application. Still further, the present application also provides the use of the Fusarium-specific segment or primer pair of the present application in detecting Fusarium species. Through the Fusarium-specific segment of the present application, different species within the Fusarium genus can be accurately distinguished, which can be applied in the field of plant quarantine to achieve rapid and accurate detection of Fusarium species.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, in particular to the field of fungal molecular detection, and more particularly to a Fusarium-specific gene segment and its use in rapid detection of Fusarium species. TECHNICAL BACKGROUND

[0002] Fusarium was formally named by German scholar Link in 1809 (Link, H. F., (1809), Gesellschaft Naturforschender Freunde zu Berlin, Magazin 3(1): 3-42), and its taxonomic research has spanned three centuries. As one of the most important plant pathogenic fungal genera in the world, Fusarium not only causes serious diseases of crops, but also can directly infect humans and livestock (Leslie, J. F. & Summerell, B. A., The Fusarium Laboratory Manual). According to statistics, Fusarium fungi can almost infect all major food crops, and at least 81 of the 101 most economically valuable crops are their hosts. This type of pathogen can cause a variety of diseases during the whole life cycle of the plant, including seed rot, seedling blight, root rot, stem rot, ear rot (including grain rot), canker, systemic wilt, and leaf spot, etc.

[0003] Currently, the identification of Fusarium species mainly relies on morphological characteristics, biological properties and phylogenetic analysis. However, due to the significant cross between different species in phenotypic characteristics (such as pathogenicity, toxin synthesis ability), it is difficult to achieve accurate taxonomic definition by traditional methods alone (Moretti, A. N., (2009), Zbornik Matice Srpske za Prirodne Nauke, 117, 7-13).

[0004] With the application of phylogenetic concepts in the classification of Fusarium, the problems faced by traditional taxonomy (such as relying on morphological characteristics and reproductive ability) have been gradually solved. Due to its high stability and being unaffected by the growth stage of the strain, the DNA sequence-based molecular marker technology has become the core method for species identification. Currently, the molecular identification of Fusarium is mainly achieved by analyzing the sequence similarity of multiple specific gene regions.

[0005] The sequences most commonly used to distinguish species in the genus *Fusarium* include: a portion of the genome encoding translocation elongation factor 1-α (tef) (Wulff, EG et al., (2010), *Environmental Microbiology*, 12, 649-657), β-tubulin (tub2) (O'Donnell, K. et al., (1998), *Proceedings of the National Academy of Sciences of the United States of America*, 95, 2044-2049), calmodulin (O'Donnell, K. et al., (2000), *Mycoscience*, 41, 61-78), and internal transcribed spacers (ITS1 and ITS2) within ribosomal repeats (Waalwijk, C. et al., (1996), *Mycologia*, 88, 361-368 and O'Donnell, K. & Cigelnik, E., (1997), *Molecular Phylogetics*). andevolution, 7, 103-116), and intergenic spacer regions (IGS) (Yli-Mattila, T. & Gagkaeva, T. in Molecular Identification of Fungi (eds Youssuf Gherbawy & Kerstin Voigt) 159-177 (Springer Berlin Heidelberg, 2010)). Not all sequences are equally effective for all species. For example, ITS regions do not function well in distinguishing closely related species in the genus Fusarium, for example... Fusarium avenaceum, Fusarium arthrosproioides , Fusarium tricinctum , F. sporotrichioides and F. Fusarium langsethiae Species complexes and their close relatives (O'Donnell, K et al., (2000), Mycoscience, 41, 61-78 and Yli-Mattila et al., (2002), Mycological Research, 106, 655-669). Furthermore, β-tub2 has been reported in the Fusarium solani species complex (…). Fusarium solaniIt does not function well in the species complex (Sampietro, DA et al., (2010), Fungal Biology, 114, 74-81).

[0006] In practical applications, Fusarium ( ) Fusarium Distinguishing Fusarium species from their closely related species presents significant challenges. Due to the high degree of similarity in their genetic backgrounds and minimal differences in morphological characteristics, traditional identification methods (such as morphological observation or culture characteristic analysis) often fall short of accurate identification. While existing identification techniques based on multiple molecular markers (such as multi-site sequencing) can improve accuracy, they are cumbersome, costly, and time-consuming, making them unsuitable for large-scale testing. Therefore, in the fields of plant quarantine and disease control, there is an urgent need to develop a high-resolution detection technology based on a single DNA marker to achieve rapid and accurate identification and classification of Fusarium species. Summary of the Invention

[0007] To address the technical bottlenecks in existing Fusarium molecular identification techniques—namely, the identification ambiguity caused by insufficient interspecies polymorphism based on ITS sequences, the increased experimental costs (requiring 3-5 primer pairs for conventional methods) and operational complexity (multiple rounds of PCR and data analysis taking >24 hours) resulting from combined detection of multiple conserved genes, and the lack of a high-throughput detection system adapted to environmental samples—this invention proposes an innovative solution. By constructing a novel molecular marker system, this invention achieves (1) broad-spectrum coverage of single primer pairs (compatible with at least 335 Fusarium species), (2) precise identification efficiency of a single PCR reaction (genus / species dual specificity), and (3) cross-platform scalability for detection scenarios (compatible with traditional electrophoresis detection and high-throughput sequencing analysis of environmental samples). Specifically, this invention innovatively constructs a Fusarium genus-specific gene segment marker method based on whole-genome evolutionary characteristics, further identifying Fusarium genus-specific gene segments, and developing an integrated detection method that combines single-strain identification and complex environmental sample analysis by identifying these Fusarium genus-specific gene segments, providing a new generation of standardized tools for medical fungal identification, crop pathogen monitoring, and environmental microbiome research.

[0008] In this regard, the present invention provides at least a Fusarium-specific gene segment, primers targeting said gene segment, and their use in the rapid detection of Fusarium species.

[0009] Specifically, the present invention includes, but is not limited to, the following:

[0010] In one aspect, the present application provides a Fusarium-specific segment, which is between 2 micro-conserved elements (MCEs) in a genomic sequence and has a length of 300-1200 bp, wherein the MCE has a length of 18-25 bp, and the number of mismatched bases between the same MCEs in different Fusarium species is less than or equal to 3; wherein the Fusarium-specific segment is not a region of repeat sequence and transposable element annotation.

[0011] In one aspect, the Fusarium-specific segment of the present application has a length of 481-1053 bp, and / or the MCE has a length of 18 bp.

[0012] In one aspect, the Fusarium-specific segment of the present application has a length of 481-1053 bp.

[0013] In one aspect, the Fusarium-specific segment of the present application has a length of 508-1053 bp.

[0014] In one aspect, the Fusarium-specific segment of the present application has a length of 678, 840, 481, 810, 554, 1053, 794, 569, 762, 851, 697, 665, 999 or 508 bp. Preferably, the Fusarium-specific segment has a length of 840, 1053, 762, 999 or 508 bp.

[0015] In one aspect, the MCE of the present application has a length of 18 bp.

[0016] In another aspect, the present application provides a method for identifying a Fusarium-specific segment, comprising the following steps:

[0017] (1) performing whole genome alignment on Fusarium species;

[0018] (2) screening micro-conserved elements in the genomic sequences of the Fusarium species;

[0019] (3) extracting the inter-genomic interval segment between 2 micro-conserved elements; and

[0020] (4) performing cross-fungus genus-specific verification on the inter-genomic interval segment extracted in step (3) to screen segments with amplification signals only in the Fusarium genus;

[0021] wherein the microconserved unit has a length of 18-25 bp and the number of mismatched bases between different Fusarium species is less than or equal to 3 within the same microconserved unit; the intergenic spacer has a length of 300-1200 bp and is a region that is not annotated as a repeat sequence and a transposable element; preferably, the intergenic spacer has a length of 481-1053 bp and / or the microconserved unit has a length of 18 bp.

[0022] In one aspect, the intergenic spacer of the present application has a length of 481-1053 bp and / or the microconserved unit has a length of 18 bp.

[0023] In one aspect, the intergenic spacer of the present application has a length of 481-1053 bp.

[0024] In one aspect, the intergenic spacer of the present application has a length of 508-1053 bp.

[0025] In one aspect, the intergenic spacer of the present application has a length of 678, 840, 481, 810, 554, 1053, 794, 569, 762, 851, 697, 665, 999 or 508 bp. Preferably, the intergenic spacer has a length of 840, 1053, 762, 999 or 508 bp.

[0026] In one aspect, the microconserved unit of the present application has a length of 18 bp.

[0027] In one aspect, the base sequence of the 2 microconserved units of the present application is selected from the following:

[0028] SEQ ID NO: 1 and SEQ ID NO: 2;

[0029] SEQ ID NO: 3 and SEQ ID NO: 4;

[0030] SEQ ID NO: 5 and SEQ ID NO: 6;

[0031] SEQ ID NO: 7 and SEQ ID NO: 8;

[0032] SEQ ID NO: 9 and SEQ ID NO: 10;

[0033] SEQ ID NO: 11 and SEQ ID NO: 12;

[0034] SEQ ID NO: 13 and SEQ ID NO: 14;

[0035] SEQ ID NO: 15 and SEQ ID NO: 16;

[0036] SEQ ID NO: 17 and SEQ ID NO: 18;

[0037] SEQ ID NO: 19 and SEQ ID NO: 20;

[0038] SEQ ID NO: 21 and SEQ ID NO: 22;

[0039] SEQ ID NO: 23 and SEQ ID NO: 24;

[0040] SEQ ID NO: 25 and SEQ ID NO: 26; or

[0041] SEQ ID NO: 27 and SEQ ID NO: 28.

[0042] In one aspect, the base sequence of the two microconserved units according to the present application is as follows:

[0043] SEQ ID NO: 3 and SEQ ID NO: 4;

[0044] SEQ ID NO: 11 and SEQ ID NO: 12;

[0045] SEQ ID NO: 25 and SEQ ID NO: 26; or

[0046] SEQ ID NO: 27 and SEQ ID NO: 28.

[0047] In one aspect, the Fusarium-specific segment according to the present application is selected from the group consisting of SEQ ID NO: 29-42.

[0048] In one aspect, the Fusarium-specific segment according to the present application is selected from the group consisting of SEQ ID NO: 30, 34, 41 and 42.

[0049] In one aspect, the multiple alignment of whole genome sequences in step (1) of the method according to the present application is performed by using a phylogeny-guided progressive alignment algorithm.

[0050] In one aspect, the reference genome is set to Fusarium graminearum (Fg) strain PH-1 in the multiple alignment of whole genome sequences in step (1) of the method according to the present application. Fusarium graminearum

[0051] ​In one aspect, in step (2) of the method of the present application, a sliding window scan is performed to screen micro-conserved elements in Fusarium species genome sequences.

[0052] Specifically, in step (2) of the method of the present application, the sliding window scan is performed by developing a Python script based on the MAF file with window parameters of 20 bp window width and 5 bp step size, and counting the conservation score (≥ 0.95) of each window to screen 18-25 bp micro-conserved elements (MCEs) with ≤ 3 mismatched bases between adjacent MCEs.

[0053] In one aspect, in step (3) of the method of the present application, double MCE structures with a 300-1200 bp interval are identified, and the corresponding genomic intervals are extracted using BedTools v2.30.0, and regions annotated with repetitive sequences (e.g., by RepeatMasker v4.1.2) and transposable elements are excluded.

[0054] In one aspect, in step (4) of the method of the present application, virtual primers are designed for the candidate segments (e.g., using Primer3 v2.5.0 with parameters of Tm = 58 ± 2°C and GC% = 40-60%), and cross-validation is performed in a database of 1508 non-Fusarium fungal genomes (e.g., NCBI RefSeq Fungi Release210) by e-PCR (e.g., e-PCR, EMBOSS v6.6.0) to screen segments with amplification signals only within the Fusarium genus (e.g., allowing ≤ 2 mismatches at the 3’ end of the primers).

[0055] In yet another aspect, the present application provides a primer pair for detecting or identifying a Fusarium species, which amplifies the Fusarium-specific segment of the present application.

[0056] In one aspect, the primer pair sequence of the present application is selected from the following:

[0057] SEQ ID NO: 43 and SEQ ID NO: 44;

[0058] SEQ ID NO: 45 and SEQ ID NO: 46;

[0059] SEQ ID NO: 47 and SEQ ID NO: 48; or

[0060] SEQ ID NO: 49 and SEQ ID NO: 50.

[0061] In yet another aspect, the present application provides use of the primer pair of the present application in detecting or identifying Fusarium species and / or analyzing the composition and relative abundance of Fusarium species in a sample, preferably the Fusarium species is selected from the group consisting of the species as shown in Table 1.

[0062] In one aspect, the use of the present application comprises the steps of performing sequence amplification using a primer pair targeting the Fusarium-specific segment, sequencing the amplification product, and aligning the sequencing results with a set of Fusarium species-specific sequences (e.g., the set of sequences disclosed in https: / / figshare.com / articles / dataset / 1475__14__ / 29598650?file=56381243).

[0063] In one aspect, the present application sequences the amplification product using Sanger sequencing.

[0064] In yet another aspect, the present application provides use of the primer pair of the present application in detecting or identifying Fusarium species and / or analyzing the composition and relative abundance of Fusarium species in a sample, preferably the Fusarium species is selected from the group consisting of the species as shown in Table 1.

[0065] In one aspect, the present application detects Fusarium species by PCR amplification using a primer pair.

[0066] In one aspect, the reaction system for the PCR amplification of the present application is 25 μL, including 2x mix 12.5 μL, DNA 2 μL, primer F and R each 1 μL, ultrapure water 8.5 μL.

[0067] In one aspect, the reaction condition for the PCR amplification of the present application is 95 °C pre-denaturation for 5 min, 95 °C denaturation for 30 s, 56 °C annealing for 30 s, 72 °C extension for 60 s, 33 cycles, 72 °C extension for 10 min.

[0068] In one aspect, the present application detects the PCR amplification product by agarose gel electrophoresis.

[0069] In one aspect, the present application determines the Fusarium species by sequencing the PCR amplification product and aligning the sequencing results with a set of Fusarium species-specific sequences.

[0070] In one aspect, the present application sequences the PCR amplification product using Sanger sequencing. Preferably, the raw data is filtered by Phred≥30 quality control.

[0071] In one aspect, the present application analyzes the composition and relative abundance of Fusarium species in a sample by applying the primer pair of the present application in a high-throughput sequencing platform.

[0072] In one aspect, the Fusarium species of the present application is selected from the group consisting of

[0073] In one aspect, the Fusarium species of the present application is selected from the group consisting of Fusarium oxysporum , Fusarium Fusarium acuminatum , Fusarium proliferatum , Fusarium fujikuroi , Fusarium lunulosporum , Fusarium noneumartii , Fusarium madaense, Fusarium martii, Fusarium mucidum, Fusarium terricola or Fusarium aberrans.

[0074] In one aspect, the Fusarium species of the present application is selected from the group consisting of Fusarium oxysporum , Fusarium Fusarium acuminatum , Fusarium proliferatum , Fusarium fujikuroi , Fusarium lunulosporum , Fusarium noneumartii or Fusarium madaense.

[0075] The present study has completed whole genome sequencing and integrated analysis of 335 Fusarium species, covering 81% of the total known species of Fusarium. This is the first nearly complete genus-level genomic dataset in the field of fungi, which is significantly better than existing research. Based on this data resource, we successfully developed new DNA molecular markers (in particular, Fusarium species-specific gene segments, primers designed for the specific gene segments), which can not only specifically identify Fusarium, but also further specifically and accurately distinguish different species within the Fusarium genus by designing single primer pairs targeting the molecular markers. The existing technology usually needs multiple primers to distinguish the species in the Fusarium genus. The DNA molecular markers and their targeting primers of the present application can be applied to the field of plant quarantine to achieve rapid and accurate detection of Fusarium species. BRIEF DESCRIPTION OF DRAWINGS

[0076] Figure 1 : Design process of screening Fusarium-specific segment scheme.

[0077] Figure 2 : Display of the results of primer 1 for specific detection of Fusarium species.

[0078] Figure 3 : Display of the results of primer 2 for specific detection of Fusarium species.

[0079] Figure 4 : Display of the results of primer 3 for specific detection of Fusarium species.

[0080] Figure 5 : Display of the results of primer 4 for specific detection of Fusarium species. Detailed Implementation

[0081] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0082] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0083] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0084] In short, this implementation method is achieved through the following innovative steps: (1) Based on multiple alignment of the whole genome sequences of 335 Fusarium species, genus-specific nucleic acid fragments (300-1200bp) are screened at the boundary between conserved and variable regions. This segment is not conserved in the genomes of other fungi; (2) Based on the results of multiple sequence alignment, degenerate primer pairs are designed to ensure that the primer annealing sites are highly conserved within the Fusarium genus; (3) A standardized detection process is established: after extracting the total DNA of the sample to be tested, specific PCR amplification is performed, and the specific bands are verified by agarose gel electrophoresis; (4) The amplification products of positive samples are subjected to Sanger sequencing, and species-level identification is achieved by comparing with the self-constructed Fusarium genus-specific fragment database; (5) For environmental samples, it can be extended to a high-throughput sequencing platform to achieve accurate analysis of the species composition and relative abundance of Fusarium in complex samples. This invention breaks through the limitations of traditional detection technologies, enabling Fusarium genus-specific identification through a single PCR reaction. It boasts advantages such as high detection sensitivity and specificity, convenient operation, and low cost, providing an efficient technical solution for clinical diagnosis, agricultural disease monitoring, and environmental microbiology research.

[0085] In one embodiment of the invention (for example, see...), Figure 1 In the design process of screening Fusarium-specific segments, the following steps may be included:

[0086] I. Phylogenetic comparison of whole genomes of multiple species in the genus Fusarium

[0087] Input dataset: Whole genome FASTA files of 335 Fusarium species and their phylogenetic tree file (Newick format), which was constructed based on a 2,722-gene multilocus analysis.

[0088] Alignment pipeline configuration: Multiple whole genome sequence alignment was performed by Progressive Cactus v2.0.3 using the phylogeny-guided progressive alignment algorithm. The paths of genome files and the phylogenetic topology were defined in the evolverFusariumSpp.txt configuration file, and the reference genome was set as F. graminearum (strain PH-1). Fusarium graminearum

[0089] Result output: Whole genome collinear alignment result files (.hal format) were generated, and converted to multiple sequence alignment format (MAF) using the cactus-hal2maf module in the Cactus tool chain, with a genome block size of 1 Mb to optimize computational efficiency.

[0090] II. Conserved region identification and candidate fragment screening

[0091] (1) Sliding window scanning: Based on the MAF file of the whole genome alignment results of 335 species, a Python script was used (window parameters: window width 18-25 bp, step 1 bp) to calculate the conservation score (Conservation Score≥0.95) of each window, and all 18-25 bp micro-conserved elements (MCE) were screened, allowing no more than 3 mismatched bases between different species in the same MCE.

[0092] (2) Intervals selection: Double MCE structures with intervals of 300-1200 bp were identified, and the corresponding genomic region sequences were extracted using BedTools v2.30.0, and regions with repetitive sequences and transposable element annotations were excluded.

[0093] (3) Fusarium-specificity-data filtering: The nucleic acid sequences of candidate segment fragments were extracted, and a homologous sequence search was performed on a database of 1508 non-Fusarium fungal genomes, and specific fragments that were only conserved within the Fusarium genus but did not exist in other fungi were filtered.

[0094] (4) Fusarium-specificity-experimental verification: Primers were designed based on the nucleic acid sequences of the candidate segment fragments, and PCR was performed on Fusarium and non-Fusarium based on the primers, with all Fusarium species having bands and no bands in non-Fusarium species.

[0095] ​(5) Database alignment to determine Fusarium species: Sanger sequencing of DNA that was able to produce a PCR band and alignment to a database of 335 Fusarium-specific fragments to distinguish between Fusarium species.

[0096] The following is an exemplary embodiment incorporating actual operations:

[0097] Example 1. Multi-species whole genome phylogenetic alignment

[0098] Input data sets: Integrate whole genome FASTA files and phylogenetic tree files (Newick format) for 335 Fusarium species (see Appendix 1), where the phylogenetic tree was constructed based on a 2,722 gene multi-locus analysis. Alignment pipeline configuration: Perform multiple whole genome alignment using Progressive Cactus v2.0.3 with a phylogenetic tree guided progressive alignment algorithm. Define the paths to the genome files and the phylogenetic topology in the evolverFusariumSpp.txt configuration file, and set the reference genome to Fusarium graminearum (strain PH-1). Fusarium Fusarium graminearum

[0099] Execute the command:

[0100] cactus. / js. / evolverFusariumSpp.txt. / evolverFusariumSpp.hal --maxMemory 64G --logFile cactus.log.

[0101] Result output: Generate whole genome collinearity alignment result files (.hal format), and convert to multiple sequence alignment format (MAF) using the cactus-hal2maf module in the Cactus tool chain, with a genome chunk size of 1 Mb to optimize computational efficiency:

[0102] cactus-hal2maf. / js evolverFusariumSpp.hal evolverFusariumSpp.maf.gz --refGenome Fusarium_graminearum_genome.fasta --chunkSize 1000000.

[0103] Example 2. Conserved region identification and candidate fragment screening

[0104] ​(1) Sliding window scanning: Python script was developed based on MAF file (window parameters: window width 18-25 bp, step 1 bp) to calculate the conservation score (Conservation Score ≥ 0.95) of each window, and to screen out 18-25 bp micro-conserved elements (MCEs) with ≤ 3 mismatched bases between adjacent MCEs. Table 1 below shows 14 pairs of MCEs.

[0105] Table 1 Sequence information of 14 pairs of MCEs

[0106]

[0107] (2) Spacer selection: Double-MCE structures with a spacer of 300-1200 bp were identified, and the corresponding genomic intervals were extracted using BedTools v2.30.0, excluding repetitive sequences (RepeatMasker v4.1.2) and transposable element annotation regions.

[0108] (3) Cross-species specificity verification: Virtual primers were designed for the candidate segments (Primer3 v2.5.0, parameters: Tm = 58 ± 2°C, GC% = 40-60%) and cross-verified in a database of 1508 non-Fusarium fungal genomes (NCBI RefSeq Fungi Release 210) by e-PCR (e-PCR, EMBOSS v6.6.0), and segments with amplification signals only within the Fusarium genus were selected (allowing ≤ 2 mismatches at the 3' end of the primer). After multiple rounds of screening, 14 Fusarium genus-specific fragments (FuST) were obtained, the specific sequences of which are shown in Table 2. Fusarium

[0109] Table 2 14 Fusarium genus-specific segments

[0110]

[0111]

[0112]

[0113]

[0114] Example 3 High-precision degenerate primer design and system verification

[0115] (1) Target screening and primer design

[0116] ​Based on 14 Fusarium-specific nucleic acid marker target regions (481-1053 bp in length) obtained from previous whole-genome alignment, a multiple sequence joint analysis strategy was adopted: multiple sequence alignment was performed using Clustal Omega 1.2.4, and regions with conservation >95% within each marker were selected as primer design sites. Degenerate primers were designed using Primer Premier 5.0 software, with the following parameters set: primer length 18-24 bp, GC content 40-60%, and Tm value gradient controlled within ±2℃. Based on the specific target regions 2, 6, 13, and 14 in Table 2, the inventors initially obtained 4 primer sets with gradient annealing temperatures (52-60℃) (see Table 3).

[0117] Table 3. Primer information for 4 pairs

[0118]

[0119] (2) Construction of experimental verification system

[0120] Sample preparation: The tested strains included 7 standard strains of Fusarium ( ). Fusarium oxysporum (Strain number 12-60-1) Fusarium acuminatum (Strain number 1-380-3) Fusarium proliferatum (Strain number 14-77-1) Fusarium fujikuroi (Strain number 13-65-1) Fusarium lunulosporum (Strain number 106) Fusarium noneumartii (Strain number 20) Fusarium madaense (Strain number 74) and 1 non-Fusarium fungus ( Epicoccum nigrum (Strain No. 7-19)). Culture and DNA extraction of the test strains: All strains were cultured in potato dextrose agar (PDA) medium at 25°C. After the colonies matured, the hyphae were collected, washed twice with sterile water, ground into powder in liquid nitrogen, and DNA was extracted with 2% CTAB buffer. The DNA samples were then tested using 1% agarose gel.

[0121] (3) PCR amplification of the target fragment: The PCR 25μL reaction system includes 2×mix 12.5μL, DNA 2μL, primers F and R 1μL each, and ultrapure water 8.5μL; the PCR reaction conditions are: 95℃ pre-denaturation for 5min, 95℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 60s, 33 cycles, and 72℃ extension for 10min.

[0122] (4) Detection result analysis: The amplification products were subjected to 1% agarose gel electrophoresis, and the length of the amplification products is shown in Table 1. The positive amplification products were purified by AMPure XP magnetic beads, and bidirectional Sanger sequencing was performed using an ABI 3730xl sequencer (Shanghai Biomedicine). The raw data were filtered by Phred≥30 quality control.

[0123] (5) Verification results: After system verification, the primer pairs in Table 3 can produce amplification products of 7 Fusarium species, and one non-Fusarium fungus cannot produce amplification products (see Figure 2 ). Further PCR amplification products were subjected to first-generation Sanger sequencing, and the sequences obtained by sequencing were compared with the Fusarium-specific sequence set disclosed in https: / / figshare.com / articles / dataset / 1475__14__ / 29598650?file=56381243, thereby accurately distinguishing 7 Fusarium species (Table 4).

[0124] Table 4 Effectiveness and accuracy of primer identification of Fusarium species

[0125]

[0126] "√" indicates the presence of PCR amplification products, and "×" indicates the absence of PCR amplification products.

[0127] Example 4 High-throughput detection technology for Fusarium community in environmental samples

[0128] (1) Multifunctional primer design: Based on the genus-specific degenerate primers designed in the early stage, the P5 / P7 flow cell anchor adapters compatible with the Illumina sequencing platform were integrated through 5' end extension strategy, realizing the functional integration of amplification primers and sequencing adapters. This design enables simultaneous completion of specific target segment amplification, sample multiplex labeling and sequencing library construction in a single PCR reaction.

[0129] (2) One-step multiplex PCR amplification-anchor ligation: HotStart Taq DNA polymerase (2.5 U / μL) and dNTPs (200 μM) system were used, and two-stage amplification program was set: the first stage (95℃ 5min; 95℃ 30s, 58℃ 45s, 72℃ 1min, 5 cycles) realized specific target segment specific amplification; the second stage (95℃ 30s, 65℃ 45s, 72℃ 1min, 25 cycles) completed index sequence annealing and adapter ligation. Through gradient annealing optimization, the primer dimer formation rate was reduced to <0.5%.

[0130] (3) Magnetic bead standardization library preparation: product purification was performed using AMPure XP magnetic beads (0.8x volume), fragment distribution was detected by Agilent 2100 bioanalyzer (main peak 200-350 bp), and multiple sample libraries were mixed at equal molar concentration after Qubit 4.0 quantification.

[0131] (4) Illumina double-end sequencing and bioinformatics analysis: 2x150 bp double-end sequencing was performed on the NovaSeq 6000 platform, and the raw data was subjected to quality filtering (Q20≥95%, and reads with N content >5% were rejected) by Fastp (v0.23.2). Effective data was aligned to the self-constructed Fusarium-specific fragment reference database by BWA-MEM (v0.7.17). The coverage depth of specific target segments in each sample (≥30x for effective detection) was calculated by SAMtools (v1.15), and finally the relative abundance (confidence threshold≥0.95) was calculated by MetaPhlAn3 algorithm.

[0132] Table 1335 Fusarium Strains and Genome Information Overview

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

Claims

1. A Fusarium-specific segment, characterized in that, The *Fusarium*-specific region is located between two microconserved units in the genome sequence and has a length of 300-1200 bp, wherein the microconserved unit has a length of 18-25 bp, and the number of mismatched bases between the same microconserved unit is less than or equal to 3 between different *Fusarium* species; wherein the *Fusarium*-specific region is not a region annotated with repetitive sequences and transposon elements, and the *Fusarium*-specific region is selected from SEQ ID NO: 30, 34, 41 and 42.

2. The Fusarium-specific segment according to claim 1, characterized in that, The base sequences of the two microconserved units are shown below: SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 25 and SEQ ID NO: 26; or SEQ ID NO: 27 and SEQ ID NO:

28.

3. A method for identifying Fusarium-specific segments, characterized in that, Includes the following steps: (1) Perform whole-genome alignment of Fusarium species; (2) Screening for microconserved units in the genome sequences of Fusarium species; (3) Extract the genomic spacer segment between two microconserved units; and (4) Perform cross-fungal genus-specific verification on the genomic spacer segments extracted in step (3) and screen for segments that have amplification signals only within the Fusarium genus; The microconserved units are 18-25 bp in length, and the number of mismatched bases between the same microconserved units is less than or equal to 3 among different Fusarium species; the genomic spacer segments are 300-1200 bp in length and are not regions annotated with repetitive sequences or transposon elements; the Fusarium-specific segments are selected from SEQ ID NO: 30, 34, 41 and 42.

4. The method according to claim 3, characterized in that, The base sequences of the two microconserved units are shown below: SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 25 and SEQ ID NO: 26; or SEQ ID NO: 27 and SEQ ID NO:

28.

5. A primer pair for detecting or identifying species of the genus *Fusarium*, characterized in that, The primer pair amplifies the Fusarium-specific region according to claim 1, wherein the primer pair sequence is selected from the following: SEQ ID NO: 43 and SEQ ID NO: 44; SEQ ID NO: 45 and SEQ ID NO: 46; SEQ ID NO: 47 and SEQ ID NO: 48; or SEQ ID NO: 49 and SEQ ID NO:

50.

6. The use of the Fusarium-specific region according to claim 1 in the detection or identification of Fusarium species.

7. The use according to claim 6, characterized in that, The process includes the following steps: using primer pairs targeting specific regions of the Fusarium genus for sequence amplification, sequencing the amplified products, and comparing the sequencing results with a Fusarium genus species-specific sequence set to determine the Fusarium genus species.

8. The use according to claim 6, characterized in that, The Fusarium species mentioned are selected from the Fusarium species listed in Appendix 1.

9. The use of the primer pair according to claim 5 in the detection or identification of Fusarium species.

10. The use according to claim 9, characterized in that, The Fusarium species mentioned are selected from the Fusarium species listed in Appendix 1.

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