Universal primer for rapidly detecting fusarium species and application thereof

Abstract

The invention provides a universal primer for rapidly detecting fusarium species and application of the universal primer. Specifically, the invention provides a primer for detecting fusarium species, and the primer is used for amplifying a fusarium specific section. Furthermore, the invention also provides an application of the universal primer of the fusarium species in detecting the fusarium species. By means of the universal primer of fusarium, different species in fusarium can be accurately distinguished, the universal primer can be applied to the field of plant quarantine, and rapid and accurate detection of fusarium species is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, in particular to the field of fungal molecular detection, more particularly to a universal primer for rapid detection of Fusarium species and use thereof. TECHNICAL BACKGROUND 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 fungi 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, this genus of fungi can infect almost all major food crops, and at least 81 of the 101 most economically valuable crops are their hosts. This pathogen can cause a variety of diseases throughout the 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 disease, etc.

[0002] 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).

[0003] 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 lack of influence from the developmental stage of the strain, 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.

[0004] The sequences most commonly used to differentiate among Fusarium species include: a portion of the genomic sequence encoding the elongation factor 1 -alpha (tef) (Wulff, E. G. et al., (2010), Environmental microbiology, 12, 649-657), beta-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), internal transcribed spacers in the ribosomal repeat region (ITS1 and ITS2) (Waalwijk, C. et al., (1996), Mycologia, 88, 361-368 and O'Donnell, K. & Cigelnik, E., (1997), Molecular phylogenetics and evolution, 7, 103-116), and the intergenic spacer (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, the ITS region does not work well to differentiate among closely related species of Fusarium, such as Fusarium avenaceum , Fusarium arthrosproioides, Fusarium tricinctum, F. sporotrichioides and F. Fusarium langsethiae the F. solani species complex and its close relatives (O'Donnell, K et al., (2000), Mycoscience, 41, 61-78 and Yli-Mattila et al., (2002), Mycological Research, 106, 655-669). In addition, beta-tub2 has been reported to not work well in the F. solani species complex (Sampietro, D. A. et al., (2010), Fungal Biology, 114, 74-81). Fusarium solani ​

[0005] 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

[0006] 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.

[0007] In this regard, the present invention provides at least primers targeting Fusarium-specific gene regions and their use in the rapid detection of Fusarium species.

[0008] Specifically, the present invention includes, but is not limited to, the following: In one aspect, the present invention provides primers for the specific detection of Fusarium species, wherein the primers target Fusarium-specific regions selected from SEQ ID NO: 29-42.

[0009] In one aspect, the primers of the present invention target Fusarium-specific regions selected from SEQ ID NO: 29, 30, 33, 34, 37, 41 or 42.

[0010] In one aspect, the primer of the present application targets a Fusarium specific segment selected from the group consisting of SEQ ID NO: 30, 34, 37, 41 or 42, preferably, the primer targets a Fusarium specific segment selected from the group consisting of SEQ ID NO: 37.

[0011] In one aspect, the primer of the present application targets a Fusarium specific segment selected from the group consisting of SEQ ID NO: 37.

[0012] In one aspect, the primer of the present application is a degenerate primer. and / or, the degenerate primer is 18-24 bp in length and has a GC content of 40-60%. and / or, the degenerate primer has a Tm value gradient controlled within ±2°C. and / or, the degenerate primer has no more than 3 base combinations at the degenerate base positions.

[0013] In one aspect, the primer of the present application is a degenerate primer.

[0014] In one aspect, the degenerate primer of the present application is 18-24 bp in length and has a GC content of 40-60%.

[0015] In one aspect, the degenerate primer of the present application has a Tm value gradient controlled within ±2°C.

[0016] In one aspect, the degenerate primer of the present application has no more than 3 base combinations at the degenerate base positions.

[0017] In one aspect, the primer sequence of the present application is selected from the group consisting of: SEQ ID NO: 43 and SEQ ID NO: 44; SEQ ID NO: 45 and SEQ ID NO: 46; SEQ ID NO: 51 and SEQ ID NO: 52; SEQ ID NO: 53 and SEQ ID NO: 54; SEQ ID NO: 59 and SEQ ID NO: 60; SEQ ID NO: 67 and SEQ ID NO: 68; or SEQ ID NO: 69 and SEQ ID NO: 70.

[0018] In one aspect, the primer sequence of the present application is selected from the group consisting of: SEQ ID NO: 45 and SEQ ID NO: 46; SEQ ID NO: 53 and SEQ ID NO: 54; SEQ ID NO: 59 and SEQ ID NO: 60; SEQ ID NO: 67 and SEQ ID NO: 68; or SEQ ID NO: 69 and SEQ ID NO: 70.

[0019] In one aspect, the primer sequences of the present application are SEQ ID NO: 59 and SEQ ID NO: 60.

[0020] In another aspect, the present application provides use of the primers of the present application for detecting Fusarium species, preferably, the Fusarium species are selected from the group consisting of the ones shown in Table 1.

[0021] 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 comparing the sequencing result 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).

[0022] In one aspect, the amplification product is sequenced using Sanger sequencing.

[0023] In another aspect, the present application provides use of the primers of the present application for analyzing the composition and relative abundance of Fusarium species in a sample, preferably, the Fusarium species are selected from the group consisting of the ones shown in Table 1.

[0024] In another aspect, the present application analyzes the composition and relative abundance of Fusarium species in a sample by a high-throughput sequencing platform.

[0025] In one aspect, the present application detects Fusarium species by PCR amplification using the primers.

[0026] 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.

[0027] In one aspect, the reaction conditions for the PCR amplification of the present application are 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 60 s, 33 cycles, and 72℃ extension for 10 min.

[0028] In one aspect, the present application detects PCR amplification products by agarose gel electrophoresis.

[0029] In one aspect, the present application analyzes Fusarium species composition and relative abundance in a sample by applying the primers of the present application in a high-throughput sequencing platform.

[0030] In one aspect, the Fusarium species of the present application is selected from the group consisting of

[0031] In one aspect, the Fusarium species of the present application is selected from the group consisting of Fusarium oxysporum , Fusarium acuminatum , Fusarium proliferatum , Fusarium fujikuroi , Fusarium lunulosporum , Fusarium noneumartii , Fusarium madaense, Fusarium martii, Fusarium mucidum, Fusarium terricola or Fusarium aberrans.

[0032] 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 (especially, based on the discovery of Fusarium species-specific gene segments, primers targeting the specific gene segments were designed), by designing single primer pairs targeting the molecular markers, not only can Fusarium be specifically identified, but also different species within Fusarium can be further specifically and accurately distinguished, while existing technologies usually need multiple primers to distinguish species in Fusarium. The DNA molecular markers and their targeting primers of the present application can be applied in the field of plant quarantine to achieve rapid and accurate detection of Fusarium species. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 : Design process of screening Fusarium-specific segment scheme.

[0034] Figure 2 : Display of the specific detection results of primer 1 on Fusarium species.

[0035] Figure 3 : Display of the specific detection results of primer 2 on Fusarium species.

[0036] Figure 4 : Display of the specific detection results of primer 3 on Fusarium species.

[0037] Figure 5 : Display of the specific detection results of primer 4 on Fusarium species.

[0038] Figure 6: shows the results of specific detection of Fusarium sp. by primer 5.

[0039] Figure 7 : shows the results of specific detection of Fusarium sp. by primer 6.

[0040] Figure 8 : shows the results of specific detection of Fusarium sp. by primer 7.

[0041] Figure 9 : shows the results of specific detection of Fusarium sp. by primer 8.

[0042] Figure 10 : shows the results of specific detection of Fusarium sp. by primer 9.

[0043] Figure 11 : shows the results of specific detection of Fusarium sp. by primer 10.

[0044] Figure 12 : shows the results of specific detection of Fusarium sp. by primer 11.

[0045] Figure 13 : shows the results of specific detection of Fusarium sp. by primer 12.

[0046] Figure 14 : shows the results of specific detection of Fusarium sp. by primer 13.

[0047] Figure 15 : shows the results of specific detection of Fusarium sp. by primer 14. DETAILED DESCRIPTION

[0048] In order to make the technical scheme of the present application better understood by those skilled in the art, the technical scheme of the present application will be described clearly and completely below in conjunction with specific embodiments. It should be pointed out that the following detailed description is exemplary and only a part of the embodiments of the present application, but not all the embodiments.

[0049] Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative efforts should belong to the scope of protection of the present application.

[0050] Unless otherwise defined, 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 belongs. The experimental materials used in the embodiments of the present application are all conventional experimental materials in the art, and can be purchased through commercial channels. The experimental methods without detailed conditions are carried out according to the conventional experimental methods or according to the operation instructions recommended by the suppliers.

[0051] In summary, the present embodiment is achieved by the following innovative steps: (1) based on multiple alignment of the whole genome sequences of 335 Fusarium species, screening for genus-specific nucleic acid fragments (300-1200 bp) at the junction of conserved and variable regions, which are not conserved in other fungal genomes; (2) designing degenerate primer pairs based on the multiple sequence alignment results, ensuring that the primer annealing sites are highly conserved within the Fusarium genus; (3) establishing a standardized detection process: after extracting the total DNA of the sample to be tested, specific PCR amplification is performed, and specific bands are verified by agarose gel electrophoresis; (4) Sanger sequencing of the amplification products of positive samples, and species-level identification by comparison with the self-constructed Fusarium-specific fragment database; (5) for environmental samples, it can be expanded to a high-throughput sequencing platform to accurately analyze the composition and relative abundance of Fusarium species in complex samples. The present application breaks through the limitations of traditional detection techniques, and can complete the specific identification of the Fusarium genus through a single PCR reaction, with the advantages of high detection sensitivity, strong specificity, convenient operation, low cost, etc., providing an efficient technical solution for clinical diagnosis, agricultural disease monitoring and environmental microbial research.

[0052] In one embodiment of the present application (for example, see Figure 1 ), the design process of the Fusarium genus-specific segment screening scheme can include the following steps: I. Phylogenetic alignment of Fusarium multi-species whole genome Input data set: integrate the whole genome FASTA files of 335 Fusarium species and their phylogenetic tree files (Newick format), where the phylogenetic tree is constructed based on a 2,722 gene multi-site analysis.

[0053] Alignment process configuration: multiple whole genome sequence alignment is performed by Progressive Cactus v2.0.3 using the progressive alignment algorithm guided by the phylogenetic tree. In the evolverFusariumSpp.txt configuration file, define the genome file path and phylogenetic topology structure, and set the reference genome as Fusarium graminearum (strain PH-1). Fusarium graminearum

[0054] Result output: generate a whole genome collinearity alignment result file (.hal format), and convert it to a multiple sequence alignment format (MAF) using the cactus-hal2maf module in the Cactus tool chain, and set the genome block size to 1 Mb to optimize the calculation efficiency.

[0055] II. Identification of conserved regions and screening of candidate fragments ​(1) Sliding window scan: Based on the MAF file of 335 species whole genome alignment, using Python script (window parameters: window width 18-25 bp, step 1 bp), the conservation score of each window (Conservation Score≥0.95) was calculated, and all 18-25 bp micro-conserved elements (MCE) were screened. The number of mismatched bases between different species in the same MCE was allowed to be ≤3.

[0056] (2) Intergenic segment selection: Identify double MCE structure with interval 300-1200 bp, use BedTools v2.30.0 to extract the sequence of the corresponding genomic region, and exclude the region with repeat sequence and transposon element annotation.

[0057] (3) Fusarium specificity-data filtering: Extract the nucleic acid sequence of the candidate segment fragment, perform homologous sequence search on the genome database covering 1508 non-Fusarium fungi, and filter the specific fragments that are only conserved in Fusarium and do not exist in other fungi.

[0058] (4) Fusarium specificity-experimental verification: Design primers for the nucleic acid sequence of the candidate segment fragment, perform PCR on Fusarium and non-Fusarium based on the primers, and all Fusarium species have bands, while non-Fusarium species have no bands.

[0059] (5) Align database to determine Fusarium species: Perform Sanger sequencing on the DNA that can appear PCR bands, and align with the database of 335 Fusarium-specific fragments to distinguish Fusarium species.

[0060] The following is an exemplary specific embodiment combined with actual operation: Example 1 Multi-species whole genome phylogenetic alignment Input dataset: Integrate the whole genome FASTA file and the phylogenetic tree file (Newick format) of 335 Fusarium species (see Table 1), where the phylogenetic tree is constructed based on a multi-locus analysis of 2,722 genes. Alignment process configuration: Perform multiple whole genome alignment by Progressive Cactus v2.0.3 using the progressive alignment algorithm guided by the phylogenetic tree. Define the genome file path and phylogenetic topology in the evolverFusariumSpp.txt configuration file, and set the reference genome as Fusarium graminearum (F. graminearum) strain PH-1. Fusarium graminearum Execution command:

[0061] Execution command: cactus. / js. / evolverFusariumSpp.txt. / evolverFusariumSpp.hal --maxMemory 64G --logFile cactus.log Result output: Generate whole genome collinearity alignment result file (.hal format), convert to multiple sequence alignment format (MAF) using cactus-hal2maf module in Cactus tool chain, set genome chunk size to 1 Mb to optimize computing efficiency: cactus-hal2maf. / js evolverFusariumSpp.hal evolverFusariumSpp.maf.gz --refGenome Fusarium_graminearum_genome.fasta --chunkSize 1000000 Example 2 Identification of conserved regions and screening of candidate fragments (1) Sliding window scanning: Develop a Python script based on the MAF file (window parameters: window width 18-25 bp, step size 1 bp) to count the base conservation score (Conservation Score ≥ 0.95) in each window, and screen out 18-25 bp micro-conserved elements (MCE). The number of mismatched bases between adjacent MCEs is allowed to be ≤ 3. Table 1 below is 14 pairs of MCE.

[0062] Table 1 Sequence information of 14 pairs of MCE

[0063] (2) Interspace section selection: Identify double MCE structures with an interval of 300-1200 bp, extract the corresponding genomic interval using BedTools v2.30.0, and exclude repetitive sequences (RepeatMasker v4.1.2) and transposable element annotation regions.

[0064] (3) Cross-species specificity verification: Design virtual primers (Primer3 v2.5.0, parameters: Tm = 58 ± 2°C, GC% = 40-60%) for candidate sections, perform cross verification in a genome database (NCBI RefSeq Fungi Release 210) covering 1508 non-Fusarium fungi through electronic PCR (e-PCR, EMBOSS v6.6.0), and screen sections with amplification signals only within the Fusarium genus (allowing ≤ 2 mismatches at the 3' end of the primer). After multiple rounds of screening, 14 Fusarium genus-specific fragments were obtained (Fusarium-specific fragments 1-14) Fusariumspecific signature tags, FuST), the sequence of which is shown in Table 2.

[0065] Table 2 14 Fusarium species-specific signature tags

[0066]

[0067]

[0068]

[0069] Example 3 High-precision degenerate primer design and system verification (1) Target screening and primer design Based on the 14 Fusarium-specific nucleic acid marker target segments (length range 481-1053 bp) obtained from the previous whole genome alignment, a multi-sequence joint analysis strategy was used: Clustal Omega 1.2.4 was used for multi-sequence alignment, and regions with a conservation rate of >95% in each marker were selected as primer design sites. Degenerate primers were designed using Primer Premier 5.0 software, with the following parameters: primer length 18-24 bp, GC content 40-60%, Tm value gradient controlled within ±2°C. The degenerate base rules were introduced: no more than 3 base combinations (such as R=A / G, Y=C / T) at each degenerate site. The inventors obtained 14 pairs of primers with gradient annealing temperatures (52-60°C) according to the specific target segments 1-14 in the table (see Table 3). The degenerate codes in the primer sequences and the nucleotides they represent are shown in Table 4 below.

[0070] Table 3 Information of 14 pairs of primers

[0071] Table 4 Degenerate codes and the nucleotides they represent

[0072] (2) Construction of experimental verification system The 14 pairs of primers in Table 3 were used to amplify the genomic DNA of 7 Fusarium species randomly selected from the 14 Fusarium species-specific signature tags (FuST) (Table 2) and 1 other fungus (Aspergillus niger) as a negative control. 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 other fungus (Aspergillus niger) as a negative control.Epicoccum nigrum (7-19) were used to verify the effectiveness of the primers. The results showed that the primer pairs corresponding to target fragments 1, 2, 5, 6, 9, 13, and 14 were specific to Fusarium and could be used to detect Fusarium species, and the primer pairs corresponding to target fragments 2, 6, 9, 13, and 14 were more stable in detecting the above-mentioned 7 Fusarium bands and could be more advantageous in determining the accuracy of Fusarium species (see Figure 2 ).

[0073] To verify the accuracy, we selected the most stable No. 9 primer pair to perform PCR amplification on 15 Fusarium strains (covering 11 pathogenic species such as Fusarium oxysporum and Fusarium graminearum) and 8 other fungi, and performed Sanger sequencing to verify the accuracy of the Fusarium species.

[0074] Sample preparation: The test strains included 15 standard Fusarium strains (covering 11 pathogenic species such as Fusarium oxysporum and Fusarium graminearum) and 8 non-Fusarium strains. The strains were cultured in potato dextrose agar (PDA) medium at 25°C, and after the colonies grew and matured, the mycelium was collected, washed twice with sterile water, ground into powder in liquid nitrogen, and the DNA was extracted with 2% CTAB buffer. The DNA sample was detected by 1% agarose gel.

[0075] (4) PCR amplification of target fragments: The PCR 25μL reaction system included 2×mix 12.5μL, DNA 2μL, primers F and R each 1μL, and ultrapure water 8.5μL; the PCR reaction conditions were: 95°C pre-denaturation for 5min, 95°C denaturation for 30s, 56°C annealing for 30s, 72°C extension for 60s, 33 cycles, and 72°C extension for 10min.

[0076] (5) Analysis of detection results: 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 Sunway Biotech), and the raw data were filtered by Phred≥30 quality control.

[0077] (6) Verification result: through system verification, the primer pair (Fus-9F: 5'-AATGAGGGCATTGCCTTC-3'; Fus-9R: 5'-CGTAKGARTGACCCATCT-3') corresponding to the target fragment 9 showed the best performance. All 15 strains of Fusarium could amplify a single bright band of 762 bp, and 8 strains of non-Fusarium control strains had no amplification product. Further, the PCR amplification product was subjected to first-generation Sanger sequencing, and the sequence obtained by sequencing was compared with the Fusarium-specific sequence set disclosed in https: / / figshare.com / articles / dataset / 1475__14__ / 29598650?file=56381243, so as to accurately distinguish 15 strains of Fusarium species (Table 5).

[0078] Table 5 Effectiveness and accuracy of No. 9 primer in identifying Fusarium species

[0079] "√" indicates that the PCR amplification product appears, and "X" indicates that the PCR amplification product does not appear Example 4 High-throughput detection technology for Fusarium community in environmental samples (1) Multifunctional primer design: based on the genus-specific degenerate primer designed in the early stage, the P5 / P7 flow cell anchor adapter compatible with the Illumina sequencing platform was integrated through 5' end extension strategy, realizing the functional integration of amplification primer and sequencing adapter. This design enables the simultaneous completion of target fragment amplification, sample multiplex labeling and sequencing library construction in a single PCR reaction.

[0080] (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, a total of 5 cycles) realized the specific amplification of target fragments; the second stage (95℃ 30s, 65℃ 45s, 72℃ 1min, a total of 25 cycles) completed the index sequence annealing and adapter ligation. Through gradient annealing optimization, the primer dimer formation rate was reduced to <0.5%.

[0081] (3) Magnetic bead standardization library preparation: AMPure XP magnetic beads (0.8x volume) were used for product purification, the fragment distribution (main peak 200-350 bp) was detected by Agilent 2100 bioanalyzer, and the multi-sample libraries were mixed at equal molar concentration after Qubit 4.0 quantification.

[0082] (4) Illumina paired-end sequencing and bioinformatics analysis: 2x150 bp paired-end sequencing was performed on the NovaSeq 6000 platform. Raw data was quality filtered by Fastp (v0.23.2) (Q20≥95%, reads with N content >5% were removed). Effective data was aligned to the self-constructed Fusarium-specific fragment reference database by BWA-MEM (v0.7.17). The coverage depth of target fragments in each sample (≥30x for effective detection) was calculated by SAMtools (v1.15), and finally the relative abundance was calculated by MetaPhlAn3 algorithm (confidence threshold≥0.95).

[0083] Table 1335 Fusarium strains and genome information overview

Claims

1. A primer for specifically detecting Fusarium species, characterized by, The primer targets a Fusarium-specific segment selected from the group consisting of SEQ ID NOs: 29-42.

2. The primer according to claim 1, characterized in that, The primer targets a Fusarium-specific segment selected from the group consisting of SEQ ID NOs: 29, 30, 33, 34, 37, 41 or 42.

3. The primer of claim 1, wherein The primer targets a Fusarium-specific segment selected from the group consisting of SEQ ID NOs: 30, 34, 37, 41 or 42, preferably the primer targets a Fusarium-specific segment selected from the group consisting of SEQ ID NO:

37.

4. The primer of claim 1, wherein The primer is a degenerate primer; and / or, the degenerate primer has a length of 18-24 bp and a GC content of 40-60%; and / or, the degenerate primer has a Tm value gradient controlled within ±2°C; and / or, the degenerate primer has no more than 3 base combinations at the degenerate base site.

5. The primer of any one of claims 1-4, wherein, The primer sequence is selected from the group consisting of: SEQ ID NO: 43 and SEQ ID NO: 44; SEQ ID NO: 45 and SEQ ID NO: 46; SEQ ID NO: 51 and SEQ ID NO: 52; SEQ ID NO: 53 and SEQ ID NO: 54; SEQ ID NO: 59 and SEQ ID NO: 60; SEQ ID NO: 67 and SEQ ID NO: 68; or SEQ ID NO: 69 and SEQ ID NO:

70.

6. The primer of claim 5, wherein The primer sequence is selected from the group consisting of: SEQ ID NO: 45 and SEQ ID NO: 46; SEQ ID NO: 53 and SEQ ID NO: 54; SEQ ID NO: 59 and SEQ ID NO: 60; SEQ ID NO: 67 and SEQ ID NO: 68; or SEQ ID NO: 69 and SEQ ID NO:

70.

7. The primer according to claim 6, characterized in that, The primer sequence is SEQ ID NO: 59 and SEQ ID NO:

60.

8. Use of the primer according to any one of claims 1-7 in detecting or identifying Fusarium species, preferably the Fusarium species is selected from the group consisting of as shown in Table 1.

9. Use of the primer according to any one of claims 1-7 in analyzing the composition and relative abundance of Fusarium species in a sample, preferably the Fusarium species is selected from the group consisting of as shown in Table 1.

10. Use according to claim 8, characterized in that, The composition and relative abundance of Fusarium species in a sample is analyzed by a high-throughput sequencing platform. The composition and relative abundance of Fusarium species in a sample is analyzed by a high-throughput sequencing platform.

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