A method for identifying or tracing detection of xanthomonas citri, reagent and application

Abstract

The application discloses a method, reagent and application for identifying or tracing citrus canker bacteria. The method comprises the following steps: obtaining seven gene sequences of the to-be-tested citrus canker bacteria, performing multi-locus sequence typing analysis on the seven gene sequences, determining alleles and numbering, concatenating the allele numbers of the seven genes to serve as a sequence type, comparing the sequence type with a reference strain sequence type, and obtaining tracing information of the to-be-tested strain; the reference strain is a citrus canker bacteria with known tracing information; the seven genes are XAC_RS07980, XAC_RS21550, copB, egl, fliL, hrpA and pliY. The seven housekeeping genes suitable for multi-locus sequence typing of the citrus canker bacteria are used for tracing detection, different geographical source strains can be distinguished at high resolution, and the method has important significance for strain regional research, evolution, transmission, disease prevalence and prevention and treatment. The method can be used for identifying and tracing the citrus canker bacteria.

Description

Technical Field

[0001] This application relates to the field of citrus canker pathogen detection, and in particular to a method, reagent, and application for the identification or traceability of citrus canker pathogen. Background Technology

[0002] Citrus fruits are important tropical and subtropical fruits, widely distributed in more than 140 countries and regions worldwide, and are the world's largest fruit producer. Citrus canker is one of the most damaging diseases to citrus production worldwide and is an important target for plant quarantine both domestically and internationally.

[0003] The pathogen causing citrus canker is *Xanthomonas citri* subsp. *citri*. This pathogen has multiple lineages and subspecies. Based on differences in geographical distribution and pathogenicity, it is classified into five lineages: A, B, C, D, and E. Among them, lineage A, originating in Asia, is the most invasive, affects the most citrus varieties, and has the widest range of damage. Research shows that lineage A itself also exhibits variation, for example, lineage A* and lineage A... W The two variant strains originated from different regions. Furthermore, existing studies on strain differentiation show that pathogenic bacteria from different regions, such as strain A, exhibit widespread differentiation.

[0004] Currently, although various methods such as microscopic diagnosis, serological diagnosis, PCR diagnosis, and DNA probe diagnosis are available for detecting and diagnosing different strains of citrus canker pathogens, source tracing and research on citrus canker pathogens are still relatively lacking. Summary of the Invention

[0005] The purpose of this application is to provide a new method, reagent, and application for the source identification or detection of citrus canker pathogens.

[0006] The following technical solution is adopted in this application:

[0007] One aspect of this application discloses a method for identifying or tracing the source of citrus canker pathogen. The method includes obtaining the sequences of seven genes from a citrus canker pathogen strain to be tested; performing multi-site sequence typing analysis on the sequences of the seven genes to determine and number the alleles of each gene; concatenating the allele numbers of the seven genes in sequence as the sequence type of the citrus canker pathogen strain to be tested; and comparing the sequence type of the citrus canker pathogen strain to be tested with the sequence type of a reference strain to obtain identification or tracing information of the citrus canker pathogen strain to be tested. The reference strain is a citrus canker pathogen strain with known tracing information, and the sequence type of the reference strain is the sequence type obtained by concatenating the allele numbers of the seven genes of the reference strain in the same order as the citrus canker pathogen strain to be tested. The seven genes are XAC_RS07980, XAC_RS21550, copB, egl, fliL, hrpA, and pliY. In this application, the traceability information includes, but is not limited to, the lineage and origin of the *Citrus canker* strain. This application obtains the identification or traceability information of the *Citrus canker* strain to be tested by comparing the sequence type of the *Citrus canker* strain to be tested with the sequence type of a reference strain. For example, the identification or traceability information of the reference strain whose sequence type is closest to that of the *Citrus canker* strain to be tested is assigned to the *Citrus canker* strain to be tested.

[0008] This application employs seven genes—XAC_RS07980, XAC_RS21550, copB, egl, fliL, hrpA, and pliY—for multi-site sequence typing (MLST) analysis of the citrus canker pathogen. This high-resolution method enables the identification of the pathogen, tracing its geographical origin, and is significant for studying the regional diversity of the pathogen, as well as understanding its evolution, transmission, and disease prevalence. Using the method described in this application for the identification and tracing of the citrus canker pathogen can effectively improve the control of citrus canker.

[0009] In one implementation of this application, the sequence type is obtained by tandemly connecting the allele numbers of the seven genes in the order XAC_RS07980-XAC_RS21550-copB-egl-fliL-hrpA-pliY.

[0010] In one implementation of this application, the method for tracing and detecting the citrus canker pathogen further includes population structure analysis. The population structure analysis includes treating a group containing two or more sequence types as a clonal complex; during clustering, defining an ancestral sequence type in a clonal complex and using the ancestral sequence type as the basis for the evolution of other sequence types; connecting each sequence type with the ancestral sequence type by a straight line, the length of which indicates the closeness of the homology; thereby obtaining the population structure of the citrus canker pathogen strain to be tested and the reference strain.

[0011] In one implementation of this application, during population structure analysis, sequence types with only one allele number difference are called single-site variations, those with two allele number differences are called two-site variations, and sequence types that are not classified into any clonal complex are defined as single-site variations.

[0012] In one implementation of this application, the method for identifying or tracing the source of citrus canker pathogen further includes splicing the sequences of seven genes in the order XAC_RS07980-XAC_RS21550-copB-egl-fliL-hrpA-pliY to obtain the analytical sequence, and constructing a phylogenetic tree using the analytical sequences of the citrus canker pathogen strain to be tested and the reference strain.

[0013] In one implementation of this application, the phylogenetic tree is constructed using the maximum likelihood method and / or the adjacency method.

[0014] In one implementation of this application, the method for identifying or tracing the source of citrus canker pathogen further includes whole-genome SNP site clustering analysis of the strain to be tested and the reference strain.

[0015] In one implementation of this application, the whole-genome SNP site clustering analysis includes tandemly connecting the SNP sites of the citrus canker pathogen strain to be tested and the reference strain in the same order to serve as the analysis sequence of each strain, and constructing a phylogenetic tree based on the analysis sequence of the whole-genome SNP sites of each strain.

[0016] It should be noted that the identification or source tracing method for citrus canker pathogens in this application can obtain identification or source tracing information of the citrus canker pathogens to be tested through multi-site sequence typing analysis. Population structure analysis, phylogenetic tree of the analyzed sequences obtained by seven gene tandems, and whole-genome SNP site cluster analysis are all just further verifications of the accuracy of multi-site sequence typing analysis and source tracing detection from different perspectives. In the embodiments of this application, the MLST analysis results of the tested strains are basically consistent with the results of population structure analysis, phylogenetic tree of the analyzed sequences obtained by seven gene tandems, and whole-genome SNP site cluster analysis; indicating that the identification or source tracing detection method based on seven-gene MLST analysis in this application can effectively distinguish citrus canker pathogen strains from different geographical origins.

[0017] It should also be noted that the genome-wide SNP clustering analysis in this application can also be used independently for the tracing and identification of citrus canker pathogens. Furthermore, theoretically, identification and tracing based on genome-wide SNPs are more accurate, which is significant for resolving technical international trade disputes and overcoming technical trade barriers. This application is the first to use genome-wide SNPs for the identification and tracing of citrus canker pathogens, demonstrating innovation. In terms of ease of operation and cost, this application's seven-gene-based identification and tracing of citrus canker pathogens is more advantageous, meeting the needs of routine inspection and quarantine as well as the basic research needs for tracing and regional analysis of citrus canker pathogens.

[0018] Another aspect of this application discloses a reagent for identifying or tracing the source of citrus canker pathogen, comprising a primer combination for amplifying seven genes of citrus canker pathogen. The seven genes in this application are specifically XAC_RS07980, XAC_RS21550, copB, egl, fliL, hrpA, and pliY.

[0019] In one implementation of this application, the primer set includes a first primer set for amplifying the XAC_RS07980 gene, a second primer set for amplifying the XAC_RS21550 gene, a third primer set for amplifying the copB gene, a fourth primer set for amplifying the egl gene, a fifth primer set for amplifying the flil gene, a sixth primer set for amplifying the hrpA gene, and a seventh primer set for amplifying the pliY gene; the upstream and downstream primers of the first primer set are the sequences shown in Seq ID No. 1 and Seq ID No. 2, respectively; the upstream and downstream primers of the second primer set are the sequences shown in Seq ID No. 3 and Seq ID No. 4, respectively; the upstream and downstream primers of the third primer set are the sequences shown in Seq ID No. 5 and Seq ID No. 6, respectively; the upstream and downstream primers of the fourth primer set are the sequences shown in Seq ID No. 7 and Seq ID No. 8, respectively; the upstream and downstream primers of the fifth primer set are the sequences shown in Seq ID No. 9 and Seq ID No. 10, respectively; and the upstream and downstream primers of the sixth primer set are the sequences shown in Seq ID No. 11 and Seq ID No. 8, respectively. The sequence shown in No. 12; the upstream and downstream primers of the seventh primer set are the sequences shown in Seq ID No. 13 and Seq ID No. 14, respectively;

[0020] Seq ID No.1: 5'-CTGCGTACCGAACTTAAGACCCTCA-3'

[0021] Seq ID No.2: 5'-ACGCTCGAACACGTCGGACT-3'

[0022] Seq ID No.3: 5'-GCAGCATTGACCGCAACCCT-3'

[0023] Seq ID No.4: 5'-GGTTTCCAGCGCCAACTGTTC-3'

[0024] Seq ID No.5: 5'-TCTCAGGCCGCACCCATCGAC-3'

[0025] Seq ID No.6: 5'-CGGTTGGTCAGCAGTACCTCGTA-3'

[0026] Seq ID No.7: 5'-CCAATGGTGCTGCTGACACAGGT-3'

[0027] Seq ID No.8: 5'-TCCGCCTGCCGATCCTGTGGGAA-3'

[0028] Seq ID No.9: 5'-CACTTCTTGCCGGTCTCGCTGGT-3'

[0029] Seq ID No.10: 5'-AGCATTCCCCTGGAGCAGACT-3'

[0030] Seq ID No.11: 5'-GCAAGATCACCCCGGACAACGAGGA-3'

[0031] Seq ID No.12: 5'-TTGCTGCACAAGCGCTCCGAT-3'

[0032] Seq ID No.13: 5'-CGGACACAAAGCTTGCCTGAAA-3'

[0033] Seq ID No. 14: 5'-GGTGCGAACAACAATAAGACGATT-3'.

[0034] In one implementation of this application, the reagents further include citrus canker-specific primers, wherein the upstream and downstream primers of the citrus canker-specific primers are the sequences shown in Seq ID No. 15 and Seq ID No. 16, respectively.

[0035] Seq ID No.15: 5'-CGCCATCCCCACCACCACCACGAC-3'

[0036] Seq ID No. 16: 5'-AACCGTCCAATGCCATCCACTTCA-3'.

[0037] It should be noted that the key to the identification and tracing reagent for *Citrus canker* pathogen in this application lies in the use of primers for amplifying seven genes, followed by sequencing of the amplified products to obtain the sequences of the seven genes, facilitating subsequent multi-site sequence typing analysis. The *Citrus canker*-specific primers are primarily used to identify whether the test strain is *Citrus canker*. It is understood that if the test strain is confirmed to be *Citrus canker*, the *Citrus canker*-specific primers may not be used. It is also understood that the sequences shown in Seq ID No. 1 to Seq ID No. 14 are merely primers for amplifying the seven genes used in one implementation of this application. Under the inventive concept of this application, it is not excluded that more primers can be designed for these seven genes, as long as they can effectively determine the alleles of the seven genes; no specific limitations are made here.

[0038] Another aspect of this application discloses the application of seven genes or DNA barcodes based on seven genes in the identification or traceability of citrus canker pathogens.

[0039] Another aspect of this application discloses the application of reagents for detecting seven genes or DNA barcoding based on seven genes in the preparation of identification reagents or source tracing reagents for citrus canker pathogens. The seven genes in this application are XAC_RS07980, XAC_RS21550, copB, egl, fliL, hrpA, and pliY.

[0040] It should be noted that the seven genes used in this application are all known genes of *Citrus canker*. The key to this application is that research has found these seven genes to exhibit regional differences while remaining highly conserved; therefore, they can be used for the source tracing and detection of *Citrus canker*. Furthermore, the seven genes in this application have high conservation, thus enabling the design of DNA barcodes based on these seven genes for the identification of *Citrus canker*. It is understood that when the DNA barcodes of this application cover all seven genes, the identification of *Citrus canker* has higher accuracy and effectiveness; however, considering both testing costs and ease of use, it is also possible to select only one or a few genes to design DNA barcodes.

[0041] The beneficial effects of this application are as follows:

[0042] This application employs seven housekeeping genes suitable for multi-site sequence typing analysis of citrus canker pathogens to identify and trace the pathogens' origins. This method enables high-resolution differentiation of citrus canker pathogen strains from different geographical origins, which is of great significance for the study of regional diversity, evolution, transmission, and epidemic trends of citrus canker pathogens, as well as for the prevention and control of citrus canker disease. Attached Figure Description

[0043] Figure 1 This is an electrophoresis image of the specific PCR amplification product of the citrus canker pathogen isolated and identified in the embodiments of this application;

[0044] Figure 2 These are electrophoresis images of seven PCR amplification products from embodiments of this application;

[0045] Figure 3 This is a source distribution map of the 94 strains of citrus canker pathogens in the embodiments of this application;

[0046] Figure 4 This is a population genetic relationship diagram of the citrus canker pathogen under the shared 5 / 7 gene standard in the embodiments of this application;

[0047] Figure 5 This is a UPGMA clustering phylogenetic tree of 94 strains of citrus canker pathogens in the embodiments of this application;

[0048] Figure 6 This is an NJ phylogenetic tree based on 7 gene tandem sequences in the embodiments of this application;

[0049] Figure 7 This is the phylogenetic tree of wgSNP strains of citrus canker pathogens in the embodiments of this application. Detailed Implementation

[0050] The present application will be further described in detail below through specific embodiments. The following embodiments are only for further illustration of the present application and should not be construed as limiting the present application.

[0051] Example

[0052] I. Materials and Methods

[0053] 1. Test materials

[0054] Sixty-two tissue samples of citrus canker disease collected from different regions and preserved by the Shenzhen Customs Animal and Plant Quarantine Technology Center include: 17 samples from Guangxi, 4 samples from Fujian, 12 samples from Jiangxi, 2 samples from Hunan, 3 samples from Yunnan, 12 samples from Zhejiang, 10 samples from Guangdong, and 2 samples from Sichuan. The 62 samples consist of diseased leaves, fruits, and branches, and were isolated and identified for further research.

[0055] In addition, the tested strains also included 20 strains of citrus canker pathogens obtained from domestic and foreign sources. The strain information is shown in Table 1.

[0056] Table 1. Twenty strains of *Citrus canker* obtained from different regions at home and abroad.

[0057] strain number source Separately host varieties time 470679 1 Hong Kong, China pomelo 1963 470680 1 India lemon 1988 470681 1 India NA 1948 470682 1 India orange 1988 470683 1 India Lemon 1989 470686 1 Brazil lemon 1980 470687 1 Brazil Lemon (Tahitian lemon) 1980 470689 1 Brazil Lemon 1982 470690 1 New Zealand NA 1949 470691 1 New Zealand Tangerine 1957 470692 1 New Zealand lemon 1957 470693 1 New Zealand Sweet orange 1957 470694 1 Thailand Lemon 1992 470695 1 Zimbabwe Sweet orange 1992 470696 1 Japan NA 1982 QB-2003 2 Jiangxi NA 2003 QB-2004 2 Jiangxi NA 2004 QB-2005 2 Zhejiang NA 2005 QB-2006 2 Guangdong NA 2006 QB-2007 2 Guangxi NA 2007

[0058] In Table 1, "Source" represents the unit that provided the strain, where "1" represents the National Collection of Plant Pathogenic Bacteria (NCPPB) in the United Kingdom, "2" represents the Institute of Animal and Plant Quarantine of the Chinese Academy of Inspection and Quarantine Sciences, and "NA" represents that the host species of the strain is unknown.

[0059] 2. Reference strain

[0060] Thirty-one published whole genome sequences of *Citrus canker* strains were downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). Detailed information is shown in Table 2. This example uses strain X.citripv.citristr.306 (NC_003919.1) as a reference.

[0061] Table 2 Information on 31 Citrus canker pathogen strains with registered whole genome sequences obtained from the database.

[0062]

[0063]

[0064] In Table 2, NA represents strains whose information is unknown.

[0065] 3. Main reagents and culture media

[0066] (1) Reagents

[0067] Agar powder, peptone, yeast extract, and sodium chloride were all purchased from Thermo Fisher Scientific; Marker II DNA Ladder was purchased from Shanghai Solarbio Biotechnology Co., Ltd.; DL1000 DNA Marker and PrimeSTAR Max DNA Polymerase were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.; Bacterial DNA Extraction Kit (DP302) was purchased from Tiangen Biotech Co., Ltd.; ddH2O.

[0068] 50×TAE (pH 8.5): Tris Base 54g, Na2EDTA·2H2O 37.2g, acetic acid 57.1mL, bring to a final volume of 1L with ddH2O, store at room temperature, and dilute to 1:1 with ddH2O before use.

[0069] (2) Culture medium

[0070] LB liquid medium: Add 10.00g tryptone, 5.00g yeast extract, and 5.00g sodium chloride to 1L ddH2O. Stir until completely dissolved, then dispense 10mL into glass test tubes.

[0071] LB solid medium: Add 16.00g of agar powder to 1L of LB liquid medium and dispense into Erlenmeyer flasks.

[0072] All the above culture media were autoclaved at 121℃ for 20 minutes and then prepared for use.

[0073] 4. Major Instruments and Equipment

[0074] PCR instrument C1000 Touch Thermal Cycler (Bio-Rayet Biomedical Products (Shanghai) Co., Ltd.), Autoclave SQ510C (Chongqing Yamato Technology Co., Ltd.), Electronic Balance MP5002 (Shanghai Hengping Scientific Instruments Co., Ltd.), Ultrasonic Cleaner KQ-250DE (Kunshan Ultrasonic Instruments Co., Ltd.), Water Bath DK-8D (Shanghai Jinghong Experimental Equipment Co., Ltd.), Incubator DHP-9272 (Shanghai Yiheng Scientific Instruments Co., Ltd.), Shaker SHKE436HP (Thermo Fisher Scientific, USA), Metal Bath K30 (Hangzhou Aosheng Instruments Co., Ltd.), Micro UV Spectrophotometer NanoDrop 2000 (Thermo Fisher Scientific (China) Co., Ltd.), Benchtop Micro Centrifuge 18 Beckman Coulter, Inc. (USA), Electrophoresis apparatus DYY-11, DYY-12; Beijing Liuyi Instrument Factory, ChampGel 5000 gel imaging system; Beijing Saizhi Venture Technology Co., Ltd.

[0075] 5. MLST analysis software

[0076] Primer synthesis and sequencing for each gene were completed by BGI Genomics Co., Ltd. (Shenzhen); whole-genome resequencing was outsourced to Sangon Biotech (Shanghai) Co., Ltd. The analysis software used for data processing in this experiment included: ART 2.5.8 for sequence data generation, Bwa 0.7.17 for sequence alignment, Samtools 1.9 for format conversion, Chromas 2.6.6 for viewing sequencing peaks, Bioedit 7.0.5.3 for sequence organization, DNAMAN 7.0.2.176 for sequence organization, DNAstar 6.1 for sequence organization, PHYLOViZ 2.0 for data analysis, MEGA 10.0.1 for sequence organization, DnaSP 5.0 for data processing, and Primer Primier 5.0 for primer design.

[0077] 6. Experimental Methods

[0078] (1) Isolation, identification and preservation of citrus canker pathogens

[0079] Pathogen isolation and purification: Select 2-3 single lesions from fresh diseased material using the streak plating method, and cut off approximately 2 mm of each lesion. 2 Square sections of tissue at the junction of diseased and healthy tissue were sterilized by standing in 75% alcohol for 30 seconds, then rinsed three times in sterile water to remove excess alcohol. The tissue was then transferred to a 1.5 mL sterile EP tube, homogenized with a sterile pipette tip, and allowed to stand for 20 minutes. Bacterial isolation was performed on LB agar using the streak plate method, and the culture was incubated upside down at 28°C for 2–3 days. Colony growth was observed and recorded periodically to review the bacterial isolation results. Pale yellow, smooth, round, raised suspected single colonies were picked and purified three times on LB agar using the streak plate method.

[0080] Molecular biological identification: Samples were detected using primers specific to Citrus canker pathogens. The upstream and downstream primers of the primers specific to Citrus canker pathogens are the sequences shown in Seq ID No. 15 and Seq ID No. 16, respectively.

[0081] Seq ID No.15: 5'-CGCCATCCCCACCACCACCACGAC-3'

[0082] Seq ID No. 16: 5'-AACCGTCCAATGCCATCCACTTCA-3'.

[0083] Using laboratory-preserved *X. citri* subsp. *citri* strain 470679 as a positive control and *X. fuscans* subsp. *aurantifolii* strain 470685 as a negative control, the DNA of the tested strains was amplified according to the following PCR system, with an expected product size of 581 bp. The PCR reaction system used in this example was 25 μL: 10×Taq reaction buffer 2.50 μL, dNTPs (2.5 mmol / L each) 2.40 μL, forward primer (10 μmol / L) 1.00 μL, reverse primer (10 μmol / L) 1.00 μL, Taq DNA polymerase (5 U / μL) 0.25 μL, DNA template 1.00 μL, ddH₂O 16.85 μL, totaling 25 μL.

[0084] The PCR reaction conditions were: 95℃ for 10 min; then 35 cycles were performed: 95℃ for 30 s, 60℃ for 30 s, 72℃ for 1 min; after the cycle, 72℃ for 10 min; and then the system was left to standby at 10℃.

[0085] PCR product detection: After adding GelRed TM Electrophoresis was performed on a 1.5% agarose gel (1×TAE Buffer) to detect PCR amplification products. The amplified bands were photographed using a UV gel imaging system to verify whether the size of the amplified bands was consistent with that of the target bands.

[0086] Sequencing alignment: The PCR amplification products were sent to a sequencing company for sequencing, and the obtained sequences were submitted to the NCBI BLAST toolbar (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) for alignment to verify whether the isolated strain was Xanthomonas citri subsp. citri.

[0087] Short-term storage: Pure culture plates can be temporarily stored in a refrigerator at 4°C by sealing them with plastic wrap for about 30 days.

[0088] For long-term storage: Add 200 μL of glycerol to a 2 mL cryovial and sterilize. Pick a purified single colony and place it in 5 mL of LB liquid medium. Incubate at 28°C and 120 rpm for 20 h. Add 800 μL of bacterial suspension to each cryovial, vortex to mix, flash freeze in liquid nitrogen, and store at -80°C for later use.

[0089] (2) Strain activation and DNA extraction

[0090] Strain activation: The preserved strain was activated on LB medium using the streak plate method and incubated upside down in a 28°C incubator for 2-3 days for later use.

[0091] DNA extraction of the strain: Genomic DNA of the citrus canker pathogen was extracted according to the instructions of the bacterial DNA extraction kit. The concentration of the extracted DNA was determined by NanoDrop 2000 and then stored in a -20°C freezer for later use.

[0092] (3) Selection of housekeeping genes and primer design

[0093] By combining simulated data using ART (https: / / www.niehs.nih.gov / research / resources / software / biostatistics / art / ) + Bwa-0.7.17 (https: / / sourceforge.net / projects / bio-bwa / ) + Samtools-1.9 (https: / / sourceforge.net / projects / samtools / files / samtools / ) software and comparing the single nucleotide polymorphisms of the whole genome sequences of Xanthomoans and X. citri subsp. citri, and combining this with the MLST analysis system for Gram-negative bacteria reported in the literature, relatively conserved but with some degree of local variation housekeeping genes were screened.

[0094] Using Primer5 and Oligo7 with the whole genome sequence of strain 306 as a reference, amplification primers for each gene were designed. Initially, multiple pairs of amplification primers were designed for each sequence. Fifteen X. citri subsp. citri strains were randomly selected for amplification pre-experiments to screen out the primer pair with the highest amplification efficiency. At the same time, gradient PCR was used to select the annealing temperature that was most similar to the primers and yielded the best amplification results. The designed primers were then submitted to the company for synthesis.

[0095] (4) Amplification and sequencing of housekeeping genes

[0096] The extracted bacterial DNA was diluted to approximately 100 ng / μL and used as a template for PCR amplification. Seven gene fragments from all strains were amplified by PCR and sequenced using a PrimeSTAR Max Premix (2×) high-fidelity polymerase system. The PCR reaction volume used in this section was 25 μL, including: 12.50 μL PrimeSTAR Max Premix (2×), 0.50 μL Forward primer (10 μmol / L), 0.50 μL Reverse primer (10 μmol / L), 1.00 μL DNA template, and 10.50 μL ddH2O, for a total of 25 μL.

[0097] The PCR reaction conditions were: 98℃ for 5 min; then 35 cycles were performed: 98℃ for 10 s, 62℃ for 5 s, and 72℃ for 3 s; after the cycle, the temperature was 72℃ for 5 min; and then the system was left to standby at 10℃.

[0098] PCR product detection and sequencing: After adding GelRed TM Electrophoresis was performed on a 1.5% agarose gel (1×TAE Buffer) to detect PCR amplification products. Images were taken using a UV gel imaging system to verify that the amplified bands matched the target band size. Each successfully amplified sequence was then bidirectionally sequenced.

[0099] (5) Sequence alignment analysis

[0100] Multilocus sequence typing: Manual proofreading was performed using the SeqMan plugin of DNAstar to check the forward and reverse sequencing results of each gene, removing sequences with poor sequencing quality at both ends. Chromas was used to view the sequence peak diagram and correct sequencing errors. Then, Bioedit software was used to compare the forward and reverse sequences to ensure that a single, high-quality dataset was obtained. If there were discrepancies between the forward and reverse sequencing results, repeated sequencing was performed, and the results with consistent forward and reverse sequencing were selected for analysis. The bidirectional sequences were assembled using SeqMan to obtain unidirectional accurate sequences. Fragments were extracted from the 31 downloaded whole genome sequences for each of the 7 genes. The processed sequences were imported into the NRDB (Non-redundant database) module of the MLST (Multilocus Sequence Typing) online analysis website (https: / / pubmlst.org / ) to analyze, confirm, and number the alleles, obtaining the allelic profile for each housekeeping gene. The seven gene allele numbers were tandemly linked in the order XAC_RS07980-XAC_RS21550-copB-egl-fliL-hrpA-pliY to obtain the sequence type (ST) of each strain.

[0101] Population structure analysis: The strain sequences were grouped and clustered using PHYLOViZ software (Francisco et al., 2012) based on the eBURST and UPGMA algorithms to clarify their genetic evolutionary relationships. A group containing two or more ST types was considered a clonal complex (CC). In eBURST clustering, a Founder (ancestral ST type) was defined within the CC, serving as the basis for the evolution of other ST types. These ST types were connected by a straight line, with the length of the line representing the degree of homology. ST types differing by only one allele number were called single-locus variants (SLV); those differing by two allele numbers were called double-locus variants (DLV). ST types not belonging to any clonal complex were defined as singletons.

[0102] Allelic diversity analysis: DnaSP and START software were used to calculate the G+C content (%), number of polymorphic sites, average pairwise nucleotide differences (π, dN / dS), and the ratio of nonsynonymous substitutions to synonymous substitutions for seven genes. G+C% reflects gene stability during evolution. π reflects the polymorphism level of each gene locus. The value of π ranges from 0 to 1; a larger value indicates higher gene polymorphism and greater variability. The dN / dS ratio reflects whether a gene is under external selection pressure. dN / dS > 1 indicates positive selection pressure (also known as natural selection pressure); dN / dS = 1 indicates neutral selection; and dN / dS < 1 indicates negative selection pressure (also known as purifying selection).

[0103] The sequences of the seven genes were assembled in the order XAC_RS07980-XAC_RS21550-copB-egl-fliL-hrpA-pliY, and Clustal W alignment was performed using MEGAX software. Phylogenetic trees were then constructed using the maximum likelihood (ML) and neighbor-joining (NJ) methods, with 1000 iterations of expansion. The developmental relationships between STs were analyzed using the Kimura two-parameter model.

[0104] (6) Cluster analysis of wgSNP strains of Citrus canker pathogen

[0105] Forty-nine strains of *Citrus canker* were selected, and DNA was extracted and its concentration determined. Sequencing required a DNA concentration above 50 ng / μL, and the total amount of DNA provided should be greater than 2 μg. The *Citrus canker* DNA was sent to Shanghai Sangon Biotech Co., Ltd. Using the complete genome sequence of strain 306 as a reference genome, whole-genome shotgun sequencing (WGS) was used to sequence the constructed PE (Paired-End) library at 2 × 150 bp using the Illumina Hiseq×Ten sequencing platform. The steps are as follows:

[0106] Data evaluation and quality control: The raw sequencing data were evaluated using FastQC. Then, Trimmomatic was used for quality trimming to ensure that the obtained data was valid and accurate.

[0107] Sequence alignment: Following the design workflow recommended by GATK Best Practice, the valid sample data were first aligned to the reference genome REF (strain 306) using BWA. Then, SAMtools was used to convert the alignment results into their correct format, sort them, and perform statistical analysis. Finally, GATK's MarkDuplicates tool was used to label repetitive sequences. Based on the alignment results, analyses were performed on duplicate reads and the distribution of insert size. BEDTools was used for depth coverage statistical analysis.

[0108] SNP detection: The HaplotypeCaller algorithm of GATK is used to analyze the genotypic differences between each sample and the reference genome. The analyses of different samples are merged and integrated to obtain the variation information of the samples and perform statistics.

[0109] Phylogenetic analysis: The neighbor-joining (NJ) algorithm in TreeBest software was used to construct a phylogenetic tree based on the SNPs of each sample to study the population structure of 50 X. citri subsp. citri strains (including reference strain 306).

[0110] II. Results and Analysis

[0111] 1. Isolation and identification of citrus canker pathogens

[0112] Sixty-two suspected citrus canker samples from different citrus varieties in eight major citrus-producing areas (Guangxi, Fujian, Jiangxi, Hunan, Yunnan, Zhejiang, Guangdong, and Sichuan) were initially isolated using culture media and typical colony observation, yielding 43 suspected isolates. These 43 suspected isolates were then amplified by PCR and sequenced using primers specific to citrus canker pathogens. The sequences obtained were compared with those of *Xanthomonas citri* subsp. *citri* already registered on NCBI, showing a similarity of 99% or higher. These results confirm that all 43 suspected isolates were indeed *Xanthomonas citri* pathogens. Partial electrophoresis results of the PCR amplification products are shown below. Figure 1 As shown, Figure 1 In the diagram, lane M is the Marker II DNA Ladder, + is the positive control, - is the negative control, N is the blank control, and lanes 1 to 13 are the PCR amplification products of the 13 suspected strains.

[0113] This experiment collected 62 suspected citrus canker samples and isolated and identified 43 citrus canker pathogens, with an isolation rate of 69.35%. The experiment found that the likelihood of isolating suspected pathogens from fresh lesions was generally higher, while the isolation rate was lower on lesions with high corking in the later stages of the disease, older leaves, or leaves collected a long time ago. Furthermore, during the pathogen isolation process, the growth rate was slower than some other fungi in the first two days, and typical suspected colonies were generally observed after 2-3 days of incubation. Thereafter, the suspected colonies grew rapidly, expanding to a diameter of 2-3 mm on the 4th-5th day after incubation.

[0114] This experiment involved sampling sites across a wide area, covering eight major citrus-producing regions. It also collected diverse varieties, including 13 different cultivars from five citrus fruit families: sweet orange, grapefruit, lemon, mandarin orange, and tangerine. The isolated strains are representative and can serve as material for subsequent research on the genetic diversity of *X. citri* subsp. *citri*. Table 3 shows the 43 isolated and identified *X. citri* strains, their origins, and hosts.

[0115] Table 3 Information on the strains of *Citrus canker* isolated.

[0116]

[0117]

[0118] 2. Amplification and sequencing of seven genes

[0119] Based on the principles of MLST gene selection, and by comparing the whole-genome polymorphism sites of *Xanthomonas citri* subsp. *citri* and referring to literature reports, 14 genes with a certain degree of conservation were initially screened, and strains from different sources were sequenced. Some genes were discarded because their conservation was too high and they were not suitable for intraspecific typing of *Xanthomonas citri*. Finally, seven gene loci with relatively high resolution (4123 bp in total) were identified to represent the whole genome sequence of *Xanthomonas citri* for MLST analysis. Multiple primer pairs were designed for the candidate genes, and PCR reactions were used for screening. Reaction conditions were optimized, and primers for the seven conserved genes were determined. These primers could amplify all tested strains with high product yields. The annealing temperature was set to 62℃, and 35 cycles of amplification were performed. The amplification products of the seven genes XAC_RS07980, XAC_RS21550, copB, egl, fliL, hrpA, and pliY were 714bp, 431bp, 648bp, 514bp, 475bp, 668bp, and 673bp, respectively, and were used for subsequent bidirectional sequencing. The amplification results of some strains are shown below. Figure 2 As shown in the table. Detailed information on the seven conserved genes and primers is provided in Table 4. The sequence information of all seven gene fragments used in this experiment has been uploaded to GenBank.

[0120] Table 4. Functions and primer sequences of seven genes.

[0121]

[0122]

[0123] Figure 2 In the lanes, A represents XAC_RS07980, B represents XAC_RS21550, C represents copB, D represents egl, E represents fliL, F represents hrpA, and G represents pliY; lanes 1, 4, 7, 10, 13, 16, and 19 represent the tested strain 470679; lanes 2, 5, 8, 11, 14, 17, and 20 represent the tested strain ZJwz-a1; lanes 3, 6, 9, 12, 15, 18, and 21 represent the tested strain QB-2003; lane M represents the DL1000 DNA Marker, with values ​​from top to bottom of 1000bp, 700bp, 500bp, 400bp, 300bp, 200bp, and 100bp.

[0124] 3. Multi-site sequence typing analysis

[0125] (1) Results of multi-site sequence typing

[0126] MLST analysis was performed on a total of 94 citrus canker pathogens, including the 20 strains listed in Table 1, the 43 strains identified in this experiment, and the 31 strains downloaded from the NCBI database. The 94 strains originated from 11 countries or regions, including China, the United States, India, New Zealand, Brazil, Zimbabwe, Reunion Island, Martinique, Thailand, Japan, and Argentina. Domestic strains were the most numerous, totaling 58 strains, accounting for approximately 61.70% of the total. These domestic strains originated from 10 regions, including Guangxi, Jiangxi, Guangdong, Zhejiang, Sichuan, Hunan, Chongqing, Fujian, Hong Kong, and Yunnan.

[0127] All seven gene fragments from all strains were amplified by PCR and sequenced. After proofreading and correction using software such as DNAstar, single forward-oriented FASTA sequences of the seven genes for each strain were obtained. The prepared sequences were imported into NRDB. Using the corresponding sequence of whole-genome sequencing strain 306 as a reference, 94 strains of *Citrus canker* were randomly divided into two groups: one group of 31 strains as reference strains and the other group of 63 strains as test strains. Allele numbering and ST-type determination were performed. The results are as follows: Figure 3 As shown in Tables 5 and 6. Figure 3The MLST analysis shows the origin distribution of 94 citrus canker pathogens. The left figure shows the different national origins of all tested strains, and the right figure shows the different provincial / municipal / autonomous region origins of Chinese strains. The results showed that all tested strains had 4, 3, 4, 7, 3, 5, and 5 alleles for the seven genes XAC_RS07980, XAC_RS21550, copB, egl, fliL, hrpA, and pliY, respectively.

[0128] Statistical analysis revealed that the 94 strains of citrus canker pathogens were classified into 19 sequence types, as shown in Tables 5 and 6. The ST type with the largest number of strains was ST-8, containing 28 strains, including some strains from the United States, China, and Japan, accounting for 29.8% of the total strains. This was followed by ST-3 with 13 strains and ST-16 with 11 strains, accounting for 13.8% and 11.7% respectively, all isolated from China. The 58 strains isolated from China were divided into 9 STs, with ST-8 being the dominant sequence type, containing 24 strains from Zhejiang, Guangdong, and parts of Guangxi. Furthermore, ST-16 mainly consisted of strains from Jiangxi and Fujian, while ST-3 included strains from Yunnan, Sichuan, and another part of Guangxi. All strains from Brazil were classified into ST-1, and strains isolated from New Zealand were separately classified into ST-6. Four strains isolated from India were divided into four different sequence types: ST-7 470682, ST-13 470681, ST-14 479680, and ST-17 470683. Another strain isolated from the United States, A... w Strains of sequence type AW13, AW14, AW15, AW16, AW12879, T×160197, and T×160042 were separately classified into sequence type ST-4. Based on the analysis of the isolation source, most strains isolated from different countries have different ST types. In summary, there is a rich diversity of sequence types among strains from different regions, and even among strains from the same isolation source, there are differences in ST types.

[0129] Table 5. Sequence types of citrus canker pathogens

[0130]

[0131]

[0132] Table 6. Distribution of allele numbers of citrus canker pathogens

[0133]

[0134] (2) Population structure analysis

[0135] Based on the shared 5 / 7 gene standard, strain sequences were grouped and clustered according to different genotypes using PHYLOViZ software based on the eBURST and UPGMA algorithms. The results are as follows: Figure 4 and Figure 5 As shown. Figure 4 This is a population genetic relationship diagram of citrus canker pathogens under the shared 5 / 7 gene standard. In the diagram, each square represents a ST, bright green represents core ST, and light green represents secondary core ST. The size of the numbers in the diagram is positively correlated with the number of strains. Figure 5 UPGMA clustering phylogenetic tree for 94 strains of citrus canker pathogen, where light colors represent core type ST, and the length of the color patch is positively correlated with the number of strains.

[0136] Figure 4 and Figure 5 The results showed that all tested strains were divided into two groups and one singleton. Group 1 contained most of the tested strains, totaling 15 ST types; Group 2 contained three ST types: ST-15, ST-17, and ST-18. ST-15 was an Aw type strain from the United States, ST-17 was an A* type strain isolated from India, and ST-18 was an A type strain isolated from Thailand. The singleton contained one sequence type, ST-4, which was an Aw type strain from the United States. Different STs connected by line segments within a group were defined as a CC. Figure 4 and Figure 5 The results show that in Group 1, apart from two independent STs (ST-7 and ST-14), the remaining tested strains formed two CCs, CC1 and CC2: CC1 consists of 11 STs, including strains isolated from China, New Zealand, and Japan; CC2 consists of 2 STs, including strains isolated from the United States, Brazil, Argentina, Zimbabwe, and other regions. Among the CCs, the Founder, also known as the core ST, is considered the origin of the development of other connected STs; the core ST of CC1 is ST-8. Although the Chinese citrus canker pathogen has multiple STs with a very wide geographical distribution, the population clustering analysis shows that all of them are distributed in CC1, suggesting they may have evolved from the same ST type.

[0137] Analysis of host varieties revealed that the *Citrus canker* strains used in this study were isolated from 13 different varieties of five citrus fruits: sweet orange, grapefruit, lemon, mandarin orange, and tangerine. Cluster analysis showed that strains in the CC1 core type ST-8 were isolated from 10 different varieties, and the 11 *Citrus canker* strains in CC2 were also isolated from five different hosts. The ST typing results indicate that strains isolated from the same host do not necessarily share the same ST type, while strains from different host varieties can be classified under the same ST type. Furthermore, no clear pattern was found between ST type and host variety. Therefore, the ST typing of *Citrus canker* is not significantly related to the host variety.

[0138] (3) Allelic diversity analysis

[0139] The resolution of the selected genes was verified using DnaSP and START software to evaluate the G+C content (%), number of polymorphic sites, mean difference (π) between pairs of sequences, and ratio of nonsynonymous to synonymous substitution (dN / dS) of seven housekeeping genes from 94 X. citri subsp. citri strains. The results are shown in Table 7.

[0140] Table 7. Results of allele diversity analysis of seven housekeeping genes.

[0141]

[0142]

[0143] Table 7 shows that the G+C percentage of the seven housekeeping genes ranged from 61.34% (egl) to 66.67% (XAC_RS07980), which is similar to the 64.6% G+C percentage of the whole genome sequence of strain 306. This indicates that these seven genes are highly conserved, relatively stable during evolution, and representative in reflecting phylogenetic relationships among strains. The π value reflects the degree of polymorphism at a gene locus; the closer the value is to 1, the greater the variability of the gene; conversely, the closer it is to 0, the more conserved the gene. The π values ​​of the seven housekeeping genes in this study ranged from 0.00039 (pliY) to 0.00246 (copB), all significantly less than 1, indicating a low allele mutation rate. The dN / dS ratio indicates whether the selected gene is under external selection pressure; a ratio > 1 indicates natural selection pressure, while a ratio < 1 indicates purifying selection pressure. Generally, a dN / dS ratio < 1 for housekeeping genes indicates they are under purifying selection. The dN / dS ratios of the seven housekeeping genes selected in the experiment were all less than 1, indicating that these seven housekeeping genes were subjected to purification selection pressure. In particular, the dN / dS ratios of the genes ×AC_RS07980 and fliL were 0, indicating that these genes were under stable selection and were less affected by external selection pressure.

[0144] The sequences of seven genes were assembled in the order XAC_RS07980-XAC_RS21550-copB-egl-fliL-hrpA-pliY. After alignment using MEGAX software and Clustal W, ML and NJ phylogenetic trees were constructed to analyze the phylogenetic relationships between ST types. The NJ phylogenetic tree based on the tandem sequences of the seven genes is shown below. Figure 6 As shown.

[0145] The results showed that both had the same topological structure, which was largely consistent with the goeBURST analysis. The phylogenetic tree showed that all strains could be divided into two major lineages, corresponding to Group 1 and Group 2 in MLST, respectively. The NJ tree showed that ST-7 (470682) and CC1 had high homology, while ST-13 (479681) was genetically more distant from other strains in CC1. This differed slightly from the eBURST clustering analysis results. The reason for this discrepancy might be that the selected genes had higher conservation. eBURST clusters allele numbers, while the NJ tree performs phylogenetic analysis based on sequence alignment. When clustering closely related strains, the differences between allele numbers are more significant than the differences caused by fewer polymorphic sites in a 4123bp sequence.

[0146] 4. Cluster analysis results of wgSNP of Citrus canker pathogen

[0147] Using the whole genome sequence of strain 306 as a reference genome, 49 geographically representative strains were screened using the WGS method and the Illumina Hiseqxten sequencing platform. These strains were resequencing, and SNP sites were screened by alignment. The results showed that the sequencing depth of the 49 citrus canker pathogens reached 200×, and a total of 6655 SNP sites were discovered. Among these, there were 4361 transition sites (C←→T, G←→A) and 2294 transversion sites (C←→A, G←→T, C←→G, A←→T), with a ratio of approximately 1:0.53. These SNP sites from each strain were tandemly arranged in the same order to obtain a FASTA sequence. These equal-length FASTA sequences were input into TreeBeST software, and a phylogenetic tree was constructed based on whole-genome SNP sites (wgSNP) using the NJ method to analyze the genetic diversity of citrus canker pathogens. The results are as follows: Figure 7 As shown. Figure 7 Phylogenetic tree of 49 strains of wgSNP, the pathogen of citrus canker.

[0148] Based on the wgSNP clustering results, the 50 strains (including reference strain 306) can be divided into 8 branches according to their phylogenetic relationships: Branch 1 includes strains 470681 and 470682, both isolated from India, which is the largest root branch and is more distantly related to other strains; Branch 2 includes 3 Indian strains (470680, CF80, 470683) and 1 Thai strain (470694); Branch 3 consists of 4 strains isolated from New Zealand; Branch 4 includes 4 Brazilian strains (REF, 470686, 470687, 470689) and 1 Zimbabwean strain (470695); strains isolated from China... The strains were divided into four branches: Branch 5 included some strains from Guangxi, one strain from Jishui, Jiangxi (Jxjs), one strain from Yunnan (YNxp), and one strain from Sichuan (SC-1); Branch 6 included seven strains from multiple origins including Zhejiang, Guangdong, Guangxi, Hunan, Fujian, and Jiangxi; Branch 7 consisted of four strains from Guangxi (Gxlb-2, Gxlb-3, Gxlb-4, Gxlb-10) and one strain from Hong Kong (470679); and Branch 8 included all strains from Zhejiang, two strains from Jiangxi (QB-2003, Jxgz), one strain from Guangdong (GD-2), and one strain from Japan (470696). The results show that strains isolated from India, Thailand, and other regions clustered at the root of the dendrogram, consistent with current findings regarding the origin of the disease. Similarly, the experimental results confirmed that the citrus canker pathogen in China has genetic polymorphism. Strains isolated from the same production area may differ due to different collection sites, such as the Guangxi strain, while strains isolated from different production areas may also have high similarity and cluster together, such as Branch6.

[0149] The 49 strains of citrus canker pathogens selected in this study were isolated from 12 host varieties. The wgSNP clustering results showed that strains from different host varieties could be classified into the same branch, and strains from the same host variety did not show a regular clustering phenomenon. This further indicates that the genetic diversity of citrus canker pathogens is not significantly related to its host variety.

[0150] 5. Comparative Analysis of MLST Classification and wgSNP Method

[0151] Both MLST and wgSNP are generative techniques that rely on library construction. The richer the known strain resources, the higher the accuracy of the analysis results. The wgSNP and MLST generative results of 49 *X. citri* strains showed similarities between the two methods, indicating rich genetic diversity among different countries and regions. Both methods showed clustering based on geographical origin, but neither showed significant host specialization. MLST divided 50 identical strains into 14 STs, while wgSNP yielded 8 branches. The ST types and branches were largely corresponding, suggesting that the seven genes XAC_RS07980, XAC_RS21550, copB, egl, fliL, hrpA, and pliY can basically reflect the genetic variation of the entire genome. In both methods, strains isolated from India were located at the root of the phylogenetic tree, indicating that strains from other regions may have evolved from these strains. This aligns with the current consensus that *X. citri* subsp. *citri* originated in Southeast Asia, specifically India.

[0152] The eBURST algorithm used in MLST focuses more on the evolutionary relationships between different ST types of the strain, classifying ST types that are SLVs into the same CC and defining the ancestral ST type to predict the genetic evolution of the pathogen. Compared with wgSNP, the MLST-based source tracing detection of citrus canker only sequences 7 gene loci, has a shorter experimental cycle and lower cost, and is easier to promote in production.

[0153] III. Conclusion and Discussion

[0154] This study collected citrus canker samples from various domestic producing areas, including citrus, oranges, pomelos, and lemons, from different hosts. Citrus canker pathogens were isolated, and combined with citrus canker pathogens from other domestic and international sources, housekeeping genes suitable for MLST typing of citrus canker pathogens were screened. An MLST typing scheme for X. citrisubsp. citri was established, and cluster typing of the tested citrus canker pathogens was performed to explore the population genetic relationships of strains from different sources. Simultaneously, whole-genome resequencing technology was used to discover SNP information of the citrus canker pathogen genome. The evolutionary relationship of domestic and international citrus canker pathogens was analyzed using the wgSNP method, and the typing results of the MLST method were compared. This provides data reference and theoretical support for accurately assessing the source and transmission risk of citrus canker pathogens, developing differentiated quarantine measures, improving monitoring capabilities, controlling citrus canker at its source, and guiding safe citrus production. Therefore, by obtaining the sequences of seven genes from the citrus canker pathogen strain to be tested, performing multi-site sequence typing analysis on the sequences of the seven genes, determining and numbering the alleles of each gene, and then concatenating the allele numbers of the seven genes in sequence as the sequence type of the citrus canker pathogen strain to be tested, the sequence type of the citrus canker pathogen strain to be tested can be compared with the sequence type of the reference strain to obtain the identification and source tracing information of the citrus canker pathogen strain to be tested.

[0155] 1. Conclusion

[0156] (1) In this study, 62 suspected citrus canker samples from 13 different citrus varieties from eight major citrus producing areas, namely Guangxi, Fujian, Jiangxi, Hunan, Yunnan, Zhejiang, Guangdong and Sichuan, were isolated and identified. After isolating the pathogens, 43 strains of citrus canker pathogens were identified by observing typical colonies, amplifying and sequencing with specific primers and comparing the results.

[0157] (2) Seven housekeeping genes suitable for MLST analysis of citrus canker pathogen were obtained by screening using the bacterial MLST method: XAC_RS07980, XAC_RS21550, copB, egl, fliL, hrpA, and pliY. These genes are under stable selection and have a certain degree of gene diversity, which can be used for MLST analysis, thereby realizing the source detection of citrus canker pathogen.

[0158] (3) The MLST scheme established in this study has high resolution. The 94 strains of citrus canker pathogens from both domestic and foreign sources were divided into 19 ST types, which were classified into 2 groups and 1 singleton, namely type A, type A, and type A. w and A* type, A wGroup 1 defines two clonal complexes, both of which contain foreign strains. The CC1 strain originates from Asia and Oceania, while the CC2 strain originates from the Americas and Africa. Therefore, foreign strains of citrus canker may have different origins. All nine STs from China are distributed in CC1. Although Chinese citrus canker pathogens have multiple STs and a very wide geographical distribution, they may have evolved from the same ST type.

[0159] (4) After resequencing of 49 strains of citrus canker, 6655 SNP sites were obtained. Cluster analysis divided 50 strains into 8 branches closely related to their geographical origin, which was basically consistent with the MLST typing results.

[0160] (5) In MLST typing and wgSNP cluster analysis, strains with the same geographical origin can mostly cluster together. Strains with similar geographical locations are also closely related after clustering, indicating that the evolutionary variation of citrus canker pathogen may be related to its geographical origin. Strains isolated from different host varieties can be classified into one category, indicating that the genotype of citrus canker pathogen does not show obvious host specialization.

[0161] 2. Discussion

[0162] In this study, the isolation rate of *Citrus canker* was approximately 69.35%. The success rate of isolating suspected bacteria was generally higher on fresh canker lesions, while the isolation rate was lower on diseased leaves with highly corky lesions in the later stages of the disease, older leaves, or leaves collected a long time ago. Therefore, in field surveys, priority should be given to collecting fresh, unopened samples with small lesions and isolating the bacterial strain promptly. If immediate isolation is not possible, the samples can be temporarily stored in a cool, ventilated place or at -4°C to prevent mold growth. During bacterial isolation, the disinfection time with 75% alcohol must be carefully controlled; too long a time will kill the target bacteria, while too short a time will not effectively kill surface bacteria, thus inhibiting the growth of the target colony. Furthermore, *Citrus canker* was also isolated from diseased leaves collected in orchards shortly after pesticide application. This indicates that orchard management should focus on prevention before the onset of canker, as post-onset pesticide application is unlikely to achieve complete control of the pathogen.

[0163] MLST typing revealed that, geographically, the Chinese strains used in the experiment covered 10 provinces and municipalities across China, demonstrating a wide geographical span and some regional representativeness. ST typing classified the 58 strains into 9 ST types, confirming the rich polymorphism of *Citrus canker* in my country. However, eBURST and UPGMA clustering results showed that all 9 STs were distributed within CC1, indicating a strong correlation between strains from different regions. Regarding host varieties, *Citrus canker* strains were isolated from 13 different varieties of 5 citrus fruits: sweet orange, grapefruit, lemon, mandarin orange, and tangerine. Based on ST typing results, strains isolated from the same host did not necessarily share the same ST type, while strains from different host varieties could be classified under the same ST type. Furthermore, no clear pattern could be found between ST types and host varieties. Therefore, we conclude that ST typing of *Citrus canker* is not significantly related to host variety. In conclusion, the citrus canker pathogen in my country may have evolved from a single ST type. Influenced by environmental and human factors in different regions, the pathogen has exhibited different genetic characteristics during its evolution.

[0164] A w A* and A* are two variants of the A strain. The A* strain primarily infects lemon and is distributed in Southwest Asia, including Iran and India. Studies have shown that the distribution range of the A* type has gradually expanded in recent years, showing a trend of spreading to Southeast Asia. Although there are currently no reports of this strain occurring in my country, lemon is cultivated in Yunnan, Hainan, and Guangdong provinces. Therefore, it is necessary to strengthen the inspection and quarantine of this harmful organism and promptly cut off the spread of the pathogen. Strain 470694, isolated from lemon in Thailand, showed a closer phylogenetic relationship to Indian strain 470683 (A*) in both MLST clustering and wgSNP analysis, suggesting it should be of type A*. However, this strain is still registered as type A in the NCPPB, failing to update its classification in a timely manner. This ambiguous strain information can affect the accuracy of the analysis results. In genetic diversity analysis, strains with complete and clear information regarding isolation location, time, and host should be selected as research subjects.

[0165] Most Chinese strains differ significantly from those from abroad. Research indicates that four strains from India were classified into different ST types, located at the root of the phylogenetic tree in cluster analysis. This aligns with the prevailing view that Southeast Asia-India is the origin of the citrus canker pathogen, as canker has a long history in all citrus-producing regions of India, and its pathogen exhibits rich genetic diversity. Similarly, 16 strains from the United States were classified into three different ST types. It has been reported that three different virulent strains have been identified in the United States. Therefore, further strengthening of quarantine measures for citrus canker is necessary. Even though the pathogen is widely distributed, its different origins and genetic diversity lead to variations in pathogenicity. It is recommended that this pathogen be included as a quarantine pest in future revisions to the list of quarantine pests for imported plants. Citrus canker pathogens, as quarantine plant pathogens, have exhibited rich genetic diversity throughout their long evolutionary history. The MLST (Multiple Scale for Detection and Labelling) method can be used to label citrus canker pathogens from different production areas around the world, providing a reliable scientific basis for customs import and export quarantine.

[0166] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.

Claims

1. A method for identifying or tracing the source of citrus canker pathogen, characterized in that: The process includes obtaining the sequences of seven genes from the citrus canker pathogen strain to be tested, performing multi-site sequence typing analysis on the sequences of the seven genes, determining and numbering the alleles of each gene, cascading the allele numbers of the seven genes in sequence as the sequence type of the citrus canker pathogen strain to be tested, comparing the sequence type of the citrus canker pathogen strain to be tested with the sequence type of a reference strain, and obtaining identification or source information of the citrus canker pathogen strain to be tested. The reference strain is a citrus canker pathogen strain with known source information. The sequence type of the reference strain is obtained by tandemly connecting the allele numbers of the seven genes of the reference strain in the same order as the citrus canker pathogen strain to be tested. The seven genes are XAC_RS07980, XAC_RS21550, copB, egl, fliL, hrpA, and pliY.

2. The method according to claim 1, characterized in that: The sequence type is obtained by tandemly connecting the allele numbers of the seven genes in the order XAC_RS07980-XAC_RS21550-copB-egl-fliL-hrpA-pliY.

3. The method according to claim 1, characterized in that: It also includes population structure analysis, which includes treating a group containing two or more sequence types as a clonal complex; during clustering, defining an ancestral sequence type in a clonal complex and using the ancestral sequence type as the basis for the evolution of other sequence types; connecting each sequence type with the ancestral sequence type by a straight line, the length of which indicates the closeness of the homology; thereby obtaining the population structure of the tested citrus canker pathogen strain and the reference strain.

4. The method according to claim 3, characterized in that: Sequence types with only one allele number difference are called single-site variations, those with two allele number differences are called two-site variations, and sequence types that are not classified into any clonal complex are defined as single-site variations.

5. The method according to claim 1, characterized in that: It also includes splicing the sequences of seven genes in the order XAC_RS07980-XAC_RS21550-copB-egl-fliL-hrpA-pliY to obtain the analytical sequences, and constructing a phylogenetic tree using the analytical sequences of the tested citrus canker pathogen strain and the reference strain.

6. The method according to claim 5, characterized in that: The phylogenetic tree is constructed using the maximum likelihood method and / or the neighbor-joining method.

7. The method according to any one of claims 1-6, characterized in that: It also includes whole-genome SNP clustering analysis of the tested citrus canker pathogen strain and the reference strain.

8. The method according to claim 7, characterized in that: The whole-genome SNP clustering analysis includes tandemly connecting the SNP sites of the tested citrus canker pathogen strain and the reference strain in the same order to form the analysis sequence of each strain, and constructing a phylogenetic tree based on the analysis sequence of the whole-genome SNP sites of each strain.

9. A reagent for identifying or tracing the source of citrus canker pathogen, characterized in that: The primer combination includes amplification of seven genes of citrus canker pathogen, namely XAC_RS07980, XAC_RS21550, copB, egl, fliL, hrpA and pliY.

10. The reagent according to claim 9, characterized in that: The primer set includes a first primer set for amplifying the XAC_RS07980 gene, a second primer set for amplifying the XAC_RS21550 gene, a third primer set for amplifying the copB gene, a fourth primer set for amplifying the egl gene, a fifth primer set for amplifying the fliL gene, a sixth primer set for amplifying the hrpA gene, and a seventh primer set for amplifying the pliY gene. The upstream and downstream primers of the first primer set are the sequences shown in Seq ID No. 1 and Seq ID No. 2, respectively; The upstream and downstream primers of the second primer set are the sequences shown in Seq ID No. 3 and Seq ID No. 4, respectively; The upstream and downstream primers of the third primer set are the sequences shown in Seq ID No. 5 and Seq ID No. 6, respectively; The upstream and downstream primers of the fourth primer set are the sequences shown in Seq ID No. 7 and Seq ID No. 8, respectively; The upstream and downstream primers of the fifth primer set are the sequences shown in Seq ID No. 9 and Seq ID No. 10, respectively; The upstream and downstream primers of the sixth primer set are the sequences shown in Seq ID No. 11 and Seq ID No. 12, respectively; The upstream and downstream primers of the seventh primer set are the sequences shown in Seq ID No. 13 and Seq ID No. 14, respectively; Seq ID No.1: 5'-CTGCGTACCGAACTTAAGACCCTCA-3' Seq ID No.2: 5'-ACGCTCGAACACGTCGGACT-3' Seq ID No.3: 5'-GCAGCATTGACCGCAACCCT-3' Seq ID No.4: 5'-GGTTTCCAGCGCCAACTGTTC-3' Seq ID No.5: 5'-TCTCAGGCCGCACCCATCGAC-3' Seq ID No.6: 5'-CGGTTGGTCAGCAGTACCTCGTA-3' Seq ID No.7: 5'-CCAATGGTGCTGCTGACACAGGT-3' Seq ID No.8: 5'-TCCGCCTGCCGATCCTGTGGGAA-3' Seq ID No.9: 5'-CACTTCTTGCCGGTCTCGCTGGT-3' Seq ID No.10: 5'-AGCATTCCCCTGGAGCAGACT-3' Seq ID No.11: 5'-GCAAGATCACCCCGGACAACGAGGA-3' Seq ID No.12: 5'-TTGCTGCACAAGCGCTCCGAT-3' Seq ID No.13: 5'-CGGACACAAAGCTTGCCTGAAA-3' Seq ID No. 14: 5'-GGTGCGAACAACAATAAGACGATT-3'.

11. The reagent according to claim 9 or 10, characterized in that: It also includes citrus canker-specific primers, the upstream and downstream primers of which are the sequences shown in Seq ID No. 15 and Seq ID No. 16, respectively; Seq ID No.15: 5'-CGCCATCCCCACCACCACCACGAC-3' Seq ID No. 16: 5'-AACCGCTCAATGCCATCCACTTCA-3'.

12. Application of seven genes or DNA barcodes based on seven genes in the identification or traceability of citrus canker pathogens, wherein the seven genes are XAC_RS07980, XAC_RS21550, copB, egl, fliL, hrpA and pliY.

13. The application of reagents for detecting seven genes or DNA barcoding based on seven genes in the preparation of reagents for identifying or tracing the source of citrus canker pathogen, wherein the seven genes are XAC_RS07980, XAC_RS21550, copB, egl, fliL, hrpA, and pliY.

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