Specific detection primer and kit for corynespora cassiicola and application thereof

Abstract

This invention belongs to the field of plant pathogen detection technology, specifically relating to specific detection primers, kits, and applications for *Cercospora cf. flagellaris*. Using the detection primers or kits of this invention, specific detection of *Cercospora cf. flagellaris* can be achieved, while also exhibiting good versatility across strains within the same species. Furthermore, sensitivity experiments show that the detection system and method established in this invention achieve a minimum detection concentration of 0.012 pg·µL for *Cercospora cf. flagellaris*. ‑1 This invention provides a detection system for soybeans exhibiting purple blotch symptoms in the field. This system can rapidly and accurately identify whether soybean plants carry the pathogen *Cercospora phytoalexinus*. Therefore, the detection primers and kit provided by this invention have excellent application prospects.

Description

Technical Field

[0001] This invention belongs to the field of plant pathogen detection technology, specifically relating to specific detection primers, kits and their applications for *Cercospora phytoalexinus*. Background Technology

[0002] Soybean purple seed stain (PSS) and soybean leaf blight (CLB), caused by the fungus *Cercospora cf. flagellaris*, have become major biological stressors affecting global soybean production. This pathogen has a wide host range and infects multiple sites, with the most significant damage occurring on seeds and leaves. After infection, purple seed stain causes irregular purplish-brown to dark purple patches on the seed coat; in severe cases, the seeds turn completely purple, severely impacting their commercial grade and market value. Leaf blight manifests initially as brown to reddish-brown lesions with yellow halos around the edges. As the disease progresses, the lesions merge and expand, causing premature leaf senescence and abscission, resulting in economic losses of up to 30% in epidemic years.

[0003] Soybeans play a crucial role in my country's food security and agricultural economic structure, and the early identification and precise intervention of related diseases have become an urgent need to ensure the stable development of the industry. Therefore, the development of a rapid and accurate detection technology system for *Cercospora phytoalexinus* has significant scientific value and application prospects for improving disease early warning capabilities and control efficiency.

[0004] Currently, detection methods for pathogenic fungi of the genus *Cercospora* are mainly divided into two categories: morphological identification and molecular biological detection. Morphological methods are based on the isolation and culture of pathogens, combined with colony morphology, conidiophores, and spore characteristics for identification. Although this method is the traditional identification standard, its process is cumbersome, time-consuming, and has significant limitations in identifying similar species, and is easily influenced by subjective experience. Molecular detection techniques, on the other hand, are characterized by high specificity and high sensitivity, and are gradually becoming the mainstream technical approach. Conventional PCR methods based on specific primers already exist, but their sensitivity is relatively low; technologies such as LAMP and RPA have been applied in the detection of *Cercospora sojina*, possessing the potential for rapid screening, but they are difficult to use for quantitative analysis and also pose a risk of aerosol cross-contamination.

[0005] Therefore, the development of a highly specific qPCR detection system based on new genetic loci aims to provide effective technical support for the dynamic monitoring and precise management of diseases related to Cercospora phytoalexinus. Summary of the Invention

[0006] To address the technical problems in the prior art, this invention provides specific detection primers, kits, and applications for Cercospora phytoluene. Using the detection primers or kits of this invention, specific detection of Cercospora phytoluene can be achieved, while also exhibiting good versatility among intraspecific strains.

[0007] In a first aspect, the present invention provides primers for the specific detection of Cercospora cf. flagellaris, wherein the target gene detected by the primers is Ccf11137, the nucleotide sequence of which is shown in SEQ ID NO.1, and the primers include a forward primer Ccf-F1 and a reverse primer Ccf-R1, the sequence of which is shown in SEQ ID NO.2, and the sequence of which is shown in SEQ ID NO.3.

[0008] This application provides a novel specific detection target gene Ccf11137 for Cercospora cf. flagellaris, and develops corresponding quantitative detection primers based on this target gene.

[0009] Secondly, the present invention also provides a kit for detecting Cercospora phytoalexina, the kit comprising the aforementioned forward primer Ccf-F1 and reverse primer Ccf-R1.

[0010] Furthermore, the concentrations of the forward primer Ccf-F1 and the reverse primer Ccf-R1 are 8-12 μmol·L⁻¹, respectively. -1 Preferably, the concentrations are 8 μmol·L⁻¹. -1 9 μmol·L -1 10 μmol·L -1 11 μmol·L -1 12 μmol·L -1 .

[0011] Furthermore, the kit also includes DNA polymerase, fluorescent dye, dNTPs, and Mg. 2+ and buffer solution.

[0012] Thirdly, the present invention also provides the application of the primers or the kits described herein in the detection of Cercospora phytoalexinus.

[0013] Fourthly, the present invention also provides a qPCR method for detecting *Cercospora phytoalexinus*, the method comprising the step of performing qPCR amplification on the genomic solution of the target using the primers or the kit described above.

[0014] Further, the step includes taking 1-2 μL of the DNA solution of the target sample and adding Hieff UNICON. ® Advanced qPCR SYBR Master Mix 8-12 μL, 8-12 μmol·L -1 0.3-0.5 μL each of the forward primer Ccf-F1 and the reverse primer Ccf-R1, 0.5-1.5 μL of DMSO, and bring the volume to 19-22 μL using RNA-Free H2O for qPCR amplification.

[0015] Further, the step includes taking 2 μL of the DNA solution of the target sample and adding Hieff UNICON. ® Advanced qPCR SYBR Master Mix 10 μL, 10 μmol·L -1 0.4 μL each of the forward primer Ccf-F1 and the reverse primer Ccf-R1, 1 μL of DMSO, and RNA-Free H2O were added to bring the volume to 20 μL for qPCR amplification.

[0016] Furthermore, the qPCR amplification program is as follows: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, 60℃ extension for 10 s, for 40 cycles; the melting curve is set to 65℃ for 5 s, 95℃ extension followed by cooling to 0.5℃ for storage.

[0017] Compared with the prior art, the present invention has the following beneficial technical effects.

[0018] This invention provides a specific target gene Ccf11137 for the detection of *Cercospora cf. flagellaris*, and develops corresponding quantitative detection primers and a real-time quantitative PCR method based on this target gene. The detection system established by this invention can not only achieve specific identification of *Cercospora cf. flagellaris* at the intergeneric level, but also effectively cover the general detection needs among different species within the *Cercospora* genus. Sensitivity test results show that the minimum detection concentration for *Cercospora cf. flagellaris* by this method can reach 0.012 pg·µL. -1 The system exhibits extremely high detection sensitivity. Further validation was performed using soybean grains showing symptoms of purple blotch in the field. The results showed that the detection system constructed in this invention can quickly and accurately identify whether soybean samples carry *Cercospora phytolaccos*, providing a reliable tool for the early diagnosis and precise monitoring of this pathogen, and demonstrating broad application prospects. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below.

[0020] Figure 1 The target regions of primers Ccf-F1 / Ccf-R1 and their sequence alignments across different species are shown.

[0021] Figure 2 The results show the qPCR amplification of DNA from 13 pathogenic bacteria using primers Ccf-F1 / Ccf-R1.

[0022] Figure 3 The results of PCR amplification of DNA extracted from six strains of Phytolacca acinosa using primers Ccf-F1 / Ccf-R1.

[0023] Figure 4 The amplification results and standard curves of Cefuroxime aureus DNA at different concentration gradients using primers Ccf-F1 / Ccf-R1 are plotted; where A is the melting curve, B is the amplification curve, and C is the standard curve.

[0024] Figure 5 Symptoms of soybean purple spot disease and qPCR test results. Detailed Implementation

[0025] The embodiments of the technical solution of the present invention will now be described in detail with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0026] Example 1

[0027]

[0028] Table 1. Species genomic information used for homology clustering analysis

[0029] .

[0030] Example 2

[0031] Primers Ccf11137-qPCR-F1 / R1 (hereinafter referred to as Ccf-F1 / Ccf-R1) were designed based on polymorphic sequence regions in the target gene Ccf11137. The targeted regions of the primers and the sequence alignment results between different species are as follows: Figure 1 As shown.

[0032] Based on the above-mentioned target genes, primers were designed to establish a quantitative PCR detection method. Genomic DNA from four Cercospora species (C. cf. flagellaris, C. cf. sigesbeckiae, C. fagopyri, and C. kikuchii) and eight major soybean pathogens (F. equiseti, F. oxysporum, Diaporthe longicolla, Alternaria alternate, Colletotrichum truncatum, Rhizoctonia solani, Pythium ultimum, Phytophthora sojae) and one Pseudocerospora kaki was used as templates to verify the specificity of the detection system.

[0033] The primer composition used for the qPCR detection method against *Cercospora phytoalexina* consists of a forward primer Ccf-F1 and a reverse primer Ccf-R1. The primer sequences are as follows:

[0034] Ccf-F1: 5'-GTTTGGATGCAAGCTCACGG-3' (SEQ ID NO. 2);

[0035] Ccf-R1: 5'-GCCAGAACAGCTGCTCCTAT-3' (SEQ ID NO. 3).

[0036] Genomic DNA of the tested strain, HieffUNICON, was amplified using the designed primer combination Ccf11137-qPCR-F1 / R1. ®Advanced qPCR SYBR Master Mix 10.0 μL, forward and reverse primers (10 μmol·L⁻¹) -1 Add 0.4 μL of each of the following: genomic DNA, 2.0 μL of DMSO, and 1.0 μL of RNA-free H2O to a final volume of 20.0 μL.

[0037] The qPCR reaction program was as follows: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, and 60℃ extension for 10 s, for 40 cycles; the melting curve was set to 65℃ for 5 s, followed by 95℃ extension and then cooling to 0.5℃ for storage. After the qPCR reaction was completed, data analysis was performed in BioRad CFX Manager.

[0038] The results are as follows Figure 2 As shown in the left figure, the green curve indicates that Ccf-F1 / R1 specifically amplified a single product from the DNA of *Cercospora phytoalexinus*, a 123 bp DNA fragment with a melting curve Tm value of 77.4 °C. No effective amplification was observed in any of the other tested strains or the negative control. This result demonstrates that the primer combination designed based on the target gene Ccf11137 exhibits high specificity for the target species.

[0039] Example 3

[0040] To verify the intraspecific universality of primers Ccf-F1 / Ccf-R1, six *Cercospora phytorecitri* strains from different geographical origins were selected and named CF-1 to CF-6, respectively. Genomic DNA was extracted from these strains and used as templates. Enzyme-free sterile water was used as a negative control for PCR amplification. The source information of the six *Cercospora phytorecitri* strains is shown in Table 2.

[0041] Table 2. Sources of the 6 tested Phytolacca acinosa strains

[0042] ;

[0043] Genomic DNA of the tested strain was amplified using the designed primer combination Ccf-F1 / R1: 12.5 μL of 2 × Rapid Taq Plus Master Mix (Dye Plus) and upstream and downstream primers (10 μmol·L⁻¹). -1 Add 1.0 μL of each of the following: genomic DNA and RNA-free H2O to a final volume of 25.0 μL.

[0044] The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 35 cycles of 95℃ denaturation for 30 s, 60℃ annealing for 10 s, and 72℃ extension for 20 s; final extension at 72℃ for 7 min; and storage at 12℃. After the reaction, the amplified products were verified by agarose gel electrophoresis.

[0045] The results are as follows Figure 3 As shown, all six *Cercospora phytoalexinus* strains were able to amplify the expected bands using primers Ccf-F1 / Ccf-R1, while the negative controls showed no amplification. These results indicate that Ccf-F1 / Ccf-R1 has good intraspecific universality in the corresponding target species.

[0046] Example 4

[0047] To evaluate the sensitivity of the real-time fluorescence PCR detection system based on the target gene Ccf11137, the detection limit of *Cercospora phytoalexinii* genomic DNA was analyzed. The initial concentration was 10 ng·µL. -1 Genomic DNA of *Cercospora phytoalexina* was serially diluted 10-fold to obtain a concentration of 10 ng·µL. -1 1 ng·µL -1 100 pg·µL -1 10 pg·µL -1 1 pg·µL -1 and 100 fg·µL -1 A series of templates were used for qPCR amplification.

[0048] Genomic DNA of *Cercospora phytoalexinus* at different concentrations was amplified using the designed primer combination Ccf-F1 / R1: HieffUNICON ® Advanced qPCR SYBR Master Mix 10.0 μL, forward and reverse primers (10 μmol·L⁻¹) -1 Add 0.4 μL of each of the following: genomic DNA, 2.0 μL of DMSO, and 1.0 μL of RNA-free H2O to a final volume of 20.0 μL.

[0049] The qPCR reaction program was as follows: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, 60℃ extension for 10 s, for 40 cycles; the melting curve was set to 65℃ for 5 s, followed by 95℃ extension and then cooling to 0.5℃ for storage. After the PCR reaction was completed, data analysis was performed in BioRad CFX Manager.

[0050] like Figure 4 As shown, the detection system for *Cercospora phytoalexinus* uses template concentrations ranging from 10 ng·µL. -1 Up to 1 pg·µL-1 All six gradients achieved stable amplification (e.g. Figure 4 (See Figure A in the image), where the Ct values ​​are 15.07, 18.25, 21.56, 24.90, 28.54, and 32.06 respectively (e.g., ...). Figure 4 (The amplification curve in Figure B). Use this to plot a standard curve (e.g., Figure 4 (See Figure C). The linear relationship between the logarithm of DNA concentration (X) and the Ct value (Y) is: Y = -3.404X + 28.50 (R² + 28.50²). 2 =0.9994). The lowest detectable concentration of this system is 0.012 pg·µL. -1 The lowest detectable concentration.

[0051] The above results indicate that the real-time fluorescence PCR detection system established based on the new target gene Ccf11137 has high sensitivity.

[0052] Example 5

[0053] To verify the effectiveness of the above detection system in detecting the presence of *C. foetida*, a pathogen of soybean plants, healthy seeds of the Nannong 47 variety and purple-spotted soybean seeds from different origins—Heinong 143, Heinong 531, Nannong 47, Wansu 051, and Gongxiadou 13—were selected, and the seed coat genome was extracted for detection. Soybean genomic DNA carrying *C. foetida* was amplified using the designed primer combination Ccf-F1 / R1: Hieff UNICON ® Advanced qPCR SYBR Master Mix 10.0 μL, forward and reverse primers (10 μmol·L⁻¹) -1 Add 0.4 μL of each of the following: genomic DNA, 2.0 μL of DMSO, and 1.0 μL of RNA-FreeH2O to a final volume of 20.0 μL.

[0054] The qPCR reaction program was as follows: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, 60℃ extension for 10 s, for 40 cycles; the melting curve was set to 65℃ for 5 s, followed by 95℃ extension and then cooling to 0.5℃ for storage. After the PCR reaction was completed, data analysis was performed in BioRad CFX Manager.

[0055] The results are as follows Figure 5 The results showed that when testing soybean grains exhibiting symptoms of purple blotch (Heinong 143, Heinong 531, Nannong 47, Wansu 051, and Gongxiadou 13), the above-mentioned qPCR system could effectively and specifically detect infection with *Cercospora phytolaccosum* (see [link to qPCR results]). Figure 5 (Ct value in the middle table).

[0056] Subsequent pathogen isolation, culture, and phylogenetic identification of soybean grains that tested positive by qPCR yielded results consistent with the detection conclusions, further confirming the reliability of the detection system. This indicates that the real-time fluorescent PCR detection system constructed based on the target gene Ccf11137 can rapidly and accurately identify whether soybean plants carry the pathogen *Cercospora phytoalexinus*.

[0057] Unless otherwise specifically stated, the numerical values ​​set forth in these embodiments do not limit the scope of the invention. In all examples shown and described herein, any specific value should be interpreted as merely exemplary and not as a limitation, unless otherwise specified; therefore, other examples of exemplary embodiments may have different values.

Claims

1. Primers for the specific detection of Cercospora cf. flagellaris, characterized in that, The target gene detected by the primers is Ccf11137, the nucleotide sequence of which is shown in SEQ ID NO.

1. The primers include a forward primer Ccf-F1 and a reverse primer Ccf-R1, the sequence of which is shown in SEQ ID NO.2, and the sequence of which is shown in SEQ ID NO.

3.

2. A kit for detecting *Cercospora phytoalexinus*, characterized in that, The kit includes the forward primer Ccf-F1 and the reverse primer Ccf-R1 as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The concentrations of the forward primer Ccf-F1 and the reverse primer Ccf-R1 are 8-12 μmol·L⁻¹. -1 .

4. The reagent kit according to claim 3, characterized in that, The kit also includes DNA polymerase, fluorescent dye, dNTPs, and Mg. 2+ and buffer solution.

5. The use of the primers of claim 1 or the kits of any one of claims 2-4 in the detection of Cercospora phytoalexinus.

6. A qPCR method for detecting *Cercospora phytoalexinus*, characterized in that, The method includes the step of performing qPCR amplification on the genomic solution of the target object using the primers of claim 1 or the kits of any one of claims 2-4.

7. The method according to claim 6, characterized in that, The qPCR amplification program is as follows: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, 60℃ extension for 10 s, for 40 cycles; the melting curve is set to 65℃ for 5 s, 95℃ extension followed by cooling to 0.5℃ for storage.

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