Specific detection target psrrp8 of soybean phytophthora and detection primer and application thereof

Abstract

The application discloses a specific detection target PsRrp8 of soybean phytophthora, a DNA sequence of the specific detection target PsRrp8 is shown as SEQ ID NO. 1, and further provides a primer combination for detecting the soybean phytophthora, the primer combination comprises a forward primer and a reverse primer, a sequence of the forward primer PsRrp8-qPCR-F1 is shown as SEQ ID NO. 2, and a sequence of the reverse primer PsRrp8-qPCR-R1 is shown as SEQ ID NO. 3. P. sojae The minimum DNA concentration of the soybean phytophthora (Phytophthora sojae) detectable by using the detection primer combination provided by the application is 0.332 pg*muL ‑1 , and the quantitative detection technology has high sensitivity. The application can be successfully applied to the detection of the soybean phytophthora in a plant in a phytophthora root rot disease field, can rapidly detect the soybean phytophthora, and provides a new technical means for early warning of diseases.

Description

Technical Field

[0001] This invention belongs to the field of plant pathogen detection technology, specifically involving the specific detection target PsRrp8 of Phytophthora in soybean, its detection primers, and applications. Background Technology

[0002] Soybean Phytophthora ( Phytophthora sojae Phytophthora root rot is the main pathogen causing soybean root rot, continuously causing significant economic losses in major producing areas worldwide. This bacterium primarily infects the soybean root system and stem base, leading to plant wilting and even complete plant death; in severely affected fields, yield losses can reach up to 50%. Therefore, developing a highly sensitive and specific rapid detection system is of significant scientific and industrial value for achieving early warning and precise control of soybean root rot.

[0003] Currently, the detection methods for *Phytophthora indicum* mainly fall into three categories: morphological identification, immunological detection, and molecular biological techniques. Traditional morphological identification relies on pathogen isolation and culture followed by microscopic observation, a cumbersome procedure (requiring 5-7 days), susceptible to contamination, and with a low isolation success rate (<60%). While immunological detection (such as ELISA) shortens the detection time (2-3 hours), its specificity is poor, and it is less effective against closely related species (such as *Phytophthora indicum*). P. infestans Cross-reactivity exists. In molecular detection techniques, the loop-mediated isothermal amplification (LAMP) technology established by Dai et al. can complete detection within 1 hour, suitable for rapid field screening, but it cannot achieve quantitative analysis of pathogens. While the microfluidic chip detection system developed by Chen et al. achieves multiplex detection, the equipment is expensive and requires specialized operators. Currently, the most commonly used real-time quantitative PCR (qPCR) technology, although capable of quantification, is prone to cross-reactivity with existing primers (such as those based on the Ypt1 or Cox1 genes) in closely related species, and aerosol contamination in the laboratory environment can interfere with the detection results.

[0004] Therefore, there is an urgent need to develop highly specific PCR detection systems based on new targets to overcome the limitations of existing technologies. Summary of the Invention

[0005] To address the issues of low sensitivity and limited specificity in existing biological detection methods for Phytophthora soybeanis, this invention provides a novel method for detecting Phytophthora soybeanis (…). Phytophthora sojae ) Specific detection target PsRrp8 and its detection primers and applications.

[0006] In a first aspect, the present invention provides a specific detection target PsRrp8 for Phytophthora soybeanis, the DNA sequence of which is shown in SEQ ID NO.1.

[0007] Secondly, the present invention provides a primer combination comprising a forward primer and a reverse primer, wherein the sequence of the forward primer PsRrp8-qPCR-F1 is shown in SEQ ID NO.2, and the sequence of the reverse primer PsRrp8-qPCR-R1 is shown in SEQ ID NO.3.

[0008] Thirdly, the present invention provides a kit for detecting soybean Phytophthora, the kit comprising 10 μmol·L⁻¹ -1 The detection solution of the primer combination.

[0009] Fourthly, the present invention provides the application of the primer combination or the kit described herein in the detection of Phytophthora in soybean.

[0010] Fifthly, the present invention provides a qPCR method for detecting Phytophthora in soybean, comprising the following steps: taking 1-2 μL of the DNA solution of the target organism, 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 PsRrp8-qPCR-F1 and the reverse primer PsRrp8-qPCR-R1, 0.5-1.5 μL of DMSO, and adjust the volume to 19-22 μL using RNA-Free H2O for qPCR amplification. The sequence of the forward primer PsRrp8-qPCR-F1 is shown in SEQ ID NO.2, and the sequence of the reverse primer PsRrp8-qPCR-R1 is shown in SEQ ID NO.3.

[0011] In some embodiments, 2 μL of the DNA solution of the target sample is taken and Hieff UNICON is added. ® AdvancedqPCR SYBR Master Mix 10 μL, 10 μmol·L -1 0.4 μL each of the forward primer PsRrp8-qPCR-F1 and the reverse primer PsRrp8-qPCR-R1, 1 μL of DMSO, and RNA-Free H2O were added to bring the volume to 20 μL for qPCR amplification.

[0012] In some embodiments, the qPCR amplification program is as follows: 95°C pre-denaturation for 30 s, 95°C denaturation for 5 s, 60°C extension for 10 s, for 40 cycles; the melting curve is set to 65°C for 5 s, followed by 95°C extension and then cooling to 0.5°C for storage.

[0013] Compared to existing technologies, this invention provides a highly reliable and specific molecular detection target, PsRrp8, as well as a qPCR primer combination and qPCR detection technology system for specifically detecting this target, PsRrp8. The detection primer combination provided by this invention is used to detect *Phytophthora sojae* (…). P.sojae The lowest detectable DNA concentration is 0.332 pg·µL. -1 This indicates that the quantitative detection technology has high sensitivity. This invention can be successfully applied to the detection of Phytophthora in plants affected by Phytophthora root rot in fields, enabling rapid detection of Phytophthora in soybeans and providing a new technical means for early disease warning. Attached Figure Description

[0014] 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.

[0015] Figure 1 The target regions of primers F1 / R1 and their sequence alignments across different species are shown.

[0016] Figure 2 The results are qPCR amplification of DNA from 13 pathogens using primers F1 / R1.

[0017] Figure 3 The results of qPCR amplification of DNA extracted from 10 strains of Phytophthora soybean using primers F1 / R1 are shown.

[0018] Figure 4 The amplification results and standard curves of *Phytophthora indicum* DNA at different concentration gradients using primers F1 / R1 were plotted.

[0019] Figure 5 The results show the field symptoms of soybean root rot and the results of qPCR detection; where AB: negative; CG: positive (C: Ct = 34.5; D: Ct = 30.0; E: Ct = 27.3; F: Ct = 26.0; G: Ct = 21.9). Detailed Implementation

[0020] 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.

[0021] Example 1

[0022] This invention utilizes soybean Phytophthora (… P.sojaeThe whole genome protein sequences of 13 soybean pathogens, including 13 causal fungi and 8 other Phytophthora species, were entered into OrthoFinder v2.4.0 software for clustering, resulting in 61,339 homologous proteomes. Analysis revealed that 106 of these homologous proteomes contained exactly one homologous protein in each species, and 19 Phytophthora genes in the soybean population showed no sequence polymorphism within the species. Subsequently, PsRrp8 was selected as a candidate target from genes with abundant polymorphism among different species. The nucleotide sequence of this target is shown in SEQ ID NO.1.

[0023]

[0024] Example 2

[0025] Primers PsRrp8-qPCR-F1 / R1 (hereinafter referred to as F1 / R1) were designed by selecting polymorphic sequence regions in the PsRrp8 gene. The target region of this primer pair and the sequence alignment between different species are as follows. Figure 1 As shown.

[0026] Based on this new detection target, primers were designed to establish a quantitative PCR detection method, and nine major pathogens collected from soybeans were used. P.sojae Soybean Phytophthora, Pythium ultimum Ultimate rot, Fusarium oxysporum Fusarium oxysporum, F. solani Fusarium solani, F. equiseti Fusarium equisetifolium F. graminearum Fusarium graminearum, Rhizoctonia solani Rhizoctonia solani, Diaporthe longicolla Soybean stem rot fungus, Colletotrichum truncatum Anthrax bacillus and three other Phytophthora fungi ( P. infestans Phytophthora, P. capsici Phytophthora capsici, P. nicotianae (Tobacco Phytophthora). Used as a template to verify the specificity of the detection target.

[0027] The primer combination used in the qPCR detection method consists of the forward primer PsRrp8-qPCR-F1 and the reverse primer PsRrp8-qPCR-R1. The primer sequences are as follows:

[0028] PsRrp8-qPCR-F1: 5'-CGAAGCCTTTGACCCCCG-3' (SEQ ID NO.2)

[0029] PsRrp8-qPCR-R1: 5'-GAACTGCAATACCATCACCA-3' (SEQ ID NO. 3).

[0030] Genomic DNA of the tested strain, Hieff UNICON, was amplified using the designed primer combination PsRrp8-qPCR-F1 / R1. ® Advanced qPCR SYBR Master Mix 10.0 μL, forward primer PsRrp8-qPCR-F1 and reverse primer PsRrp8-qPCR-R1 (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.

[0031] 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.

[0032] The results are as follows Figure 2 As shown, PsRrp8-qPCR-F1 / R1 could only specifically amplify a melting curve with a melting temperature (Tm value) of 84.0℃ from *Phytophthora sojae* DNA, while other non-*Phytophthora sojae* strains and the negative control did not produce amplification. This indicates that the primer combination designed based on the new detection target PsRrp8 can achieve *Phytophthora sojae* (… P.sojae ) specific detection.

[0033] Example 3

[0034] To ensure that the specific amplification of Phytophthora soybeanis by primers F1 / R1 is species-wide, genomic DNA was extracted from 10 Phytophthora soybeanis strains from different geographical origins as templates, and enzyme-free sterile water was set as a negative control for qPCR amplification.

[0035] Genomic DNA of the tested strain, Hieff UNICON, was amplified using the designed primer combination PsRrp8-qPCR-F1 / R1. ® Advanced qPCR SYBR Master Mix 10.0 μL, forward primer PsRrp8-qPCR-F1 and reverse primer PsRrp8-qPCR-R1 (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.

[0036] 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.

[0037] The results are as follows Figure 3 As shown, primers F1 / R1 effectively amplified all 10 strains of Phytophthora soybeana, with Ct values ​​ranging from 20 to 30 and melting temperatures (Tm values) of 84.0℃. In contrast, the negative control showed no amplification, indicating that the target primers have good intraspecific universality for the amplification of Phytophthora soybeana.

[0038] Example 4

[0039] To evaluate the soybean Phytophthora () P.sojae The sensitivity of the real-time fluorescence PCR detection system established using the new detection target PsRrp8 was tested using 100 ng·µL. -1 The genomic DNA of Phytophthora soybeanis was serially diluted 10-fold to prepare 100 ng·µL solutions. -1 10 ng·µL -1 1 ng·µL -1 100 pg·µL -1 10 pg·µL -1 1 pg·µL -1 g and 100 fg·µL -1 qPCR amplification was performed after seven gradient templates.

[0040] Genomic DNA of different concentrations of *Phytophthora beanense* strains was amplified using the designed primer combination PsRrp8-qPCR-F1 / R1: Hieff UNICON ® Advanced qPCR SYBR Master Mix 10.0 μL, forward primer PsRrp8-qPCR-F1 and reverse primer PsRrp8-qPCR-R1 (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.

[0041] 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.

[0042] The results are as follows Figure 4 The concentration was 100 ng·µL. -1 10 ng·µL -1 1 ng·µL -1 100 pg·µL -1 10 pg·µL -1 1 pg·µL -1 g P.sojae The Ct values ​​of the genomic DNA were 17.28, 19.87, 23.43, 26.78, 30.14, and 33.61, respectively. Standard curves were plotted based on the amplification results of the first six template concentrations. Figure 4The linear relationship between the logarithm (X) of the soybean Phytophthora infestans DNA concentration and the Ct value (Y) was derived as: Y = -3.266X + 33.44 (R² = 0.9993). Based on this linear equation, a Ct value of 35 (the conventional detection threshold) corresponds to a DNA concentration of 0.332 pg·µL. -1 The lowest concentration that the system can detect indicates that the quantitative detection technique has high sensitivity.

[0043] Example 5

[0044] To verify that the primers designed for this target can effectively detect Phytophthora root rot in soybean plants in the field, an investigation was conducted on soybean plants with different degrees of disease. Genome samples were extracted and subjected to F1 / R1-qPCR detection. Genomic DNA of different concentrations of Phytophthora soybean strains was amplified using the designed primer combination PsRrp8-qPCR-F1 / R1: Hieff UNICON ® Advanced qPCR SYBR Master Mix 10.0 μL, forward primer PsRrp8-qPCR-F1 and reverse primer PsRrp8-qPCR-R1 (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.

[0045] 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.

[0046] As shown in Figure 5, soybean plants affected by Phytophthora root rot were effectively infected with Phytophthora soybeanis, and the detection results were consistent with the severity of the disease. Among them, browning of the stem base and upward expansion of lesions are typical symptoms of soybean Phytophthora root rot. The higher the severity of this symptom, the greater the pathogen load detected.

[0047] 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. The application of a primer combination for detecting the specific target PsRrp8 of Phytophthora soybeanis in the detection of Phytophthora soybeanis, characterized in that, The primer combination includes a forward primer and a reverse primer. The sequence of the forward primer PsRrp8-qPCR-F1 is shown in SEQ ID NO.2, the sequence of the reverse primer PsRrp8-qPCR-R1 is shown in SEQ ID NO.3, and the DNA sequence of the specific detection target PsRrp8 is shown in SEQ ID NO.

1.

2. The application of a kit for detecting the specific target PsRrp8 of Phytophthora in soybean in the detection of Phytophthora in soybean, characterized in that, The kit contains 10 μmol·L⁻¹ -1 The detection solution of the primer combination as described in claim 1.

3. A qPCR method for detecting *Phytophthora indicum*, characterized in that, The steps include: Take 1-2 μL of the DNA solution from the target sample, add 8-12 μL of qPCR SYBR Master Mix (8-12 μmol·L⁻¹). -1 0.3-0.5 μL each of the forward primer PsRrp8-qPCR-F1 and the reverse primer PsRrp8-qPCR-R1, 0.5-1.5 μL of DMSO, and adjust the volume to 19-22 μL using RNA-Free H2O for qPCR amplification. The sequence of the forward primer PsRrp8-qPCR-F1 is shown in SEQ ID NO.2, and the sequence of the reverse primer PsRrp8-qPCR-R1 is shown in SEQ ID NO.

3.

4. The method according to claim 3, characterized in that, Take 2 μL of the DNA solution from the target sample and add 10 μL of qPCRSYBR Master Mix (10 μmol·L⁻¹). -1 0.4 μL each of the forward primer PsRrp8-qPCR-F1 and the reverse primer PsRrp8-qPCR-R1, 1 μL of DMSO, and RNA-Free H2O were used to bring the volume to 20 μL for qPCR amplification.

5. The method according to claim 4, characterized in that, The qPCR amplification program is as follows: pre-denaturation at 95℃ for 30 s; denaturation at 95℃ for 5 s, extension at 60℃ for 10 s, for 40 cycles; the melting curve is set to maintain at 65℃ for 5 s, and after extension at 95℃, the temperature is lowered to 0.5℃ for storage.

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