Primer and method for rapidly detecting and identifying pathotype of plasmodiophora brassicae based on PCR (Polymerase Chain Reaction) technology

Abstract

The invention relates to the technical field of plant pathogen identification, and provides primers and a method for rapidly detecting and identifying pathotypes of plasmodiophora brassicae based on a PCR (Polymerase Chain Reaction) technology. Comprising an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 1 and SEQ ID NO. 2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3; any one of the upstream primer and the downstream primer forms a primer. Identification of different pathotypes of plasmodiophora brassicae is rapidly completed through the PCR technology, and especially, the pathotype of plasmodiophora brassicae to cruciferae crops is rapidly and accurately identified through the difference of PCR amplification results; the identification result is accurate and repeatable, the operation process is simplified, the detection time is reduced, and the operation threshold is low.

Description

Technical Field

[0001] This invention belongs to the field of plant pathogen identification technology, and in particular relates to primers and methods for rapid detection and identification of pathogenic types of *Plasmodiophora* based on PCR technology. Background Technology

[0002] Clubroot fungi are obligate parasites of plant roots, causing clubroot disease in cruciferous crops (such as rapeseed, Chinese cabbage, and other cruciferous vegetables). Different physiological races of clubroot fungi exist, with significant differences in pathogenicity among them. Highly pathogenic races can cause severe disease in rapeseed and all cruciferous vegetables, while weakly pathogenic races only cause severe disease in Chinese cabbage-type cruciferous vegetables (such as Chinese cabbage and bok choy) and Chinese cabbage-type rapeseed. However, they cause very mild disease in Brassica oleracea-type cruciferous vegetables (such as kale and head cabbage), and almost no disease in Brassica oleracea-type rapeseed. Accurate identification of the pathogenic races of clubroot fungi in farmland allows for control of clubroot disease through adjustments to cruciferous crop variety distribution or precise chemical control, thereby increasing economic, social, and ecological benefits.

[0003] Internationally, there are two clubroot fungus identification systems: Williams and the European ECD. These systems, respectively, use artificial inoculation of 4 and 15 differential hosts to accurately identify the physiological races of clubroot fungus based on the disease incidence on each host. Completing this identification requires collecting a large number of clubroot fungi and cultivating a large number of differential hosts, resulting in a significant workload. The time required for identifying differential hosts from sowing to surveying varies from 45 to 60 days, which is lengthy. The growth and disease incidence of differential hosts require strict environmental conditions such as temperature and humidity, making the process difficult and reproducible. Obtaining or maintaining the purity of differential host seeds is also challenging, presenting a high technical barrier. While accurate identification of physiological races has some value in basic research, it has limited practical application in guiding clubroot disease control.

[0004] Currently, there are no reports on using PCR technology to identify different pathogenic serotypes of *Plasmodiophora* in cruciferous crops. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides primers and methods for rapid detection and identification of pathogenic types of *Plasmodiophora stearothermia* based on PCR technology, aiming to solve the problems mentioned in the background art.

[0006] In a first aspect, the present invention provides primers for rapid detection and identification of pathogenic types of *Plasmodiophora stearothermiae* based on PCR technology, including an upstream primer and a downstream primer, wherein the nucleic acid sequences of the upstream primer are shown in SEQ ID NO. 1 and SEQ ID NO. 2, and the nucleic acid sequence of the downstream primer is shown in SEQ ID NO. 3; The upstream primer and the downstream primer together form a primer set.

[0007] Furthermore, the primer set includes: The first primer set comprises an upstream primer having the nucleic acid sequence shown in SEQ ID NO. 1 and a downstream primer having the nucleic acid sequence shown in SEQ ID NO. 3; The second primer set consists of an upstream primer having the nucleic acid sequence shown in SEQ ID NO. 2 and a downstream primer having the nucleic acid sequence shown in SEQ ID NO. 3.

[0008] Furthermore, the application of primers for rapid detection and identification of pathogenic types of *Plasmodiophora* based on PCR technology in identifying the pathogenicity of *Plasmodiophora* to cruciferous crops, including rapeseed and cruciferous vegetables.

[0009] Secondly, the present invention provides a method for rapid detection and identification of pathogenic types of *Plasmodiophora stearothermia* based on PCR technology. Using the genomic DNA of the sample to be tested as a template, PCR amplification is performed using a primer set. After the reaction, the amplification products are subjected to agarose gel electrophoresis to observe whether specific bands appear. The PCR amplification reaction system consisted of: 0.5 μL DNA polymerase, 2.5 μL 10× Buffer, 0.5 μL each of 10 μM upstream and downstream primers (total 1 μL), 2 μL 2.5 mM dNTPs, 0.5 μL sample DNA, and 18.5 μL ddH2O. Separate reaction systems were prepared for the first and second primer sets. The nucleic acid sequences of the upstream primer are shown in SEQ ID NO. 1 and SEQ ID NO. 2, and the nucleic acid sequences of the downstream primer are shown in SEQ ID NO. 3; any one of the upstream primers and the downstream primers constitute a primer set.

[0010] Furthermore, the primer set includes: The first primer set comprises an upstream primer having the nucleic acid sequence shown in SEQ ID NO. 1 and a downstream primer having the nucleic acid sequence shown in SEQ ID NO. 3; The second primer set consists of an upstream primer having the nucleic acid sequence shown in SEQ ID NO. 2 and a downstream primer having the nucleic acid sequence shown in SEQ ID NO. 3.

[0011] Furthermore, the PCR amplification reaction process is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 34 cycles; 72℃ extension for 5 min.

[0012] Furthermore, the amplification products were subjected to agarose gel electrophoresis, and the results were observed as follows: (1) When the first primer set amplifies to a band of 988 bp and the second primer set amplifies to a band of 625 bp, the detected root-root bacterium belongs to a highly pathogenic strain. (2) If the first primer set amplifies to 510 bp and the second primer set amplifies to 625 bp without a bright single band, then the detected root-root bacterium is a weakly pathogenic strain. (3) When the first primer set amplifies to a band of 510 bp and the second primer set amplifies to a band of 625 bp, the detected root rot bacteria belong to a mixed infection of strong and weak pathogenic strains.

[0013] Furthermore, the sample is a clubroot fungus, extracted from typical clubroot tissue formed after disease in cruciferous crops.

[0014] The present invention has the following beneficial effects: (1) Internationally, there are two identification systems for clubroot fungi: Williams and the European ECD. First, these systems require the collection of a large number of typical clubroot tissues, which are then inoculated into 4 and 15 cruciferous differential hosts, respectively. Identification is then performed based on the disease status of the differential hosts, resulting in a large workload, complex operation, and long time requirement. Second, the growth and disease status of the differential hosts are strictly affected by environmental factors such as temperature and humidity, leading to unstable reproducibility. Finally, the seeds of the 19 differential hosts are difficult to obtain or preserve for a long time, and their purity after long-term cultivation is also difficult to guarantee. PCR technology can be used to quickly identify different pathogenic types of clubroot fungi, especially by using the differences in PCR amplification results to quickly and accurately identify the pathogenicity of clubroot fungi to cruciferous crops. The identification results are accurate and reproducible, and the operation process is simplified, reducing detection time and lowering the operational threshold.

[0015] (2) This method uses a primer set consisting of two sets of upstream and downstream primer pairs, which has high specificity and can accurately distinguish between strong and weak pathogenic types of clubroot bacteria and mixed infection types. It can not only identify the pathogenic type of clubroot bacteria after crop disease, but also detect the genomic DNA of microorganisms in the soil before crop sowing, so as to identify the pathogenic type of clubroot bacteria in the field in advance, and guide farmers to prevent clubroot disease by planting disease-resistant varieties or adjusting crop types (for example, replacing Chinese cabbage with cabbage in areas where weakly pathogenic clubroot bacteria are distributed), thereby achieving the purpose of reducing production costs, reducing pesticide use, and improving the quality of agricultural products; this method is suitable for multi-regional and multi-sample testing needs. Attached Figure Description

[0016] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures: Figure 1 This is the agarose gel electrophoresis result of the sample to be tested in Example 3 of the present invention, where M is the DNA Marker.

[0017] Figure 2 These are the observation results of strong and weak pathogenic root-knot bacteria provided in Example 3 of the present invention, where M stands for DNA Marker. Detailed Implementation

[0018] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0020] This invention provides primers for rapid detection and identification of pathogenic types of *Plasmodiophora stearothermia* based on PCR technology, including an upstream primer and a downstream primer. The nucleic acid sequences of the upstream primer are shown in SEQ ID NO. 1 and SEQ ID NO. 2, and the nucleic acid sequence of the downstream primer is shown in SEQ ID NO. 3. Any upstream primer and downstream primer form a primer set.

[0021] In some embodiments, the primer set includes: The first primer set comprises an upstream primer having the nucleic acid sequence shown in SEQ ID NO. 1 and a downstream primer having the nucleic acid sequence shown in SEQ ID NO. 3; The second primer set consists of an upstream primer having the nucleic acid sequence shown in SEQ ID NO. 2 and a downstream primer having the nucleic acid sequence shown in SEQ ID NO. 3.

[0022] In some embodiments, primers for rapid detection and identification of pathogenicity of *Plasmodiophora* based on PCR technology are used to identify the pathogenicity of *Plasmodiophora* to cruciferous crops, including rapeseed and cruciferous vegetables.

[0023] In some embodiments, the present invention provides a method for rapid detection and identification of pathogenic types of *Plasmodiophora stearothermiae* based on PCR technology. Using the genomic DNA of the sample to be tested as a template, PCR amplification is performed using a primer set. After the reaction, the amplification products are subjected to agarose gel electrophoresis to observe whether specific bands appear. The PCR amplification reaction system was as follows: 0.5 μL DNA polymerase, 2.5 μL 10× Buffer, 0.5 μL each of 10 μM upstream and downstream primers (total 1 μL), 2 μL 2.5 mM dNTPs, 0.5 μL sample DNA, and 18.5 μL ddH2O. Separate reaction systems were prepared for the first and second primer sets. The nucleic acid sequences of the upstream primer are shown in SEQ ID NO. 1 and SEQ ID NO. 2, and the nucleic acid sequences of the downstream primer are shown in SEQ ID NO. 3; any upstream primer and downstream primer constitute a primer set.

[0024] In some embodiments, the primer set includes: The first primer set comprises an upstream primer having the nucleic acid sequence shown in SEQ ID NO. 1 and a downstream primer having the nucleic acid sequence shown in SEQ ID NO. 3; The second primer set consists of an upstream primer having the nucleic acid sequence shown in SEQ ID NO. 2 and a downstream primer having the nucleic acid sequence shown in SEQ ID NO. 3.

[0025] In some embodiments, the PCR amplification reaction process is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 34 cycles; 72℃ extension for 5 min.

[0026] In some embodiments, the amplification products are subjected to agarose gel electrophoresis, and the results are observed as follows: (1) When the first primer set amplifies a band of 988 bp and the second primer set amplifies a band of 625 bp, the detected root-root bacterium belongs to a highly pathogenic strain. (2) If the first primer set amplifies to 510 bp and the second primer set amplifies to 625 bp without a bright single band, then the detected root-root bacterium is a weakly pathogenic strain. (3) When the first primer set amplifies to a band of 510 bp and the second primer set amplifies to a band of 625 bp, the detected root rot bacteria belong to a mixed infection of strong and weak pathogenic strains.

[0027] In some embodiments, the sample is a clubroot fungus, extracted from typical clubroot tissue formed after disease in cruciferous crops; Specifically, spindle-shaped root ulcer tissue was extracted and isolated from the roots of cruciferous crops after infection with Plasmodium floccosum.

[0028] Example 1: This example illustrates the enrichment of dormant spores of *Plasmodiophora*, specifically the preparation of genomic DNA from 17 different *Plasmodiophora* disease samples (numbered BS-1, BS-2...BS-17).

[0029] Specifically: ① Select typical clubroot tissue from the diseased sample, thaw it, remove any unaffected excess parts, rinse it clean, and cut 2-5g into a mortar; ② Add 2-4ml of sterile water and grind it into powder, filter it through 8 layers of gauze and rinse it with a small amount of sterile water, finally squeeze the gauze firmly, pour the filtrate into a 50ml sterile round-bottom centrifuge tube, and centrifuge at 2500g for 15min; ③ Discard the supernatant, add 30-40ml of sterile water to the precipitate, and shake it upside down to resuspend the precipitate in water, centrifuge at 2500g for 15min; ④ Repeat the above steps once; ⑤ Discard the supernatant; add 5-8ml of... After vortexing with 50% sucrose solution, resuspend the precipitate in the sucrose solution and centrifuge at 1930g for 10min; ⑥ Carefully transfer the middle and upper layers of liquid into another clean 50ml sterile conical centrifuge tube using a pipette or directly, add 40-50ml of sterile water, and mix by inverting and shaking, then centrifuge at 2500g for 15min; ⑦ Repeat steps 5-6 once; ⑧ Discard the supernatant, and store the obtained purified dormant spore precipitate of *Plasmodiophora* at -20℃ for later use.

[0030] Example 2: This example illustrates the extraction of genomic DNA from dormant spores of *Plasmodiophora stenosum*. The genomic DNA of *Plasmodiophora* was extracted using the High-Efficiency Plant Genomic DNA Extraction Kit (DP350) from Tiangen Biotech (Beijing) Co., Ltd., specifically as follows: ① Add 100-300 µl of sterile water to the purified dormant spores of *Plasmodiophora* from Example 1 and vortex to mix. ② Transfer 100-200 µl to a 2 ml centrifuge tube, add 0.2 g of zirconium oxide beads, 400 µL of buffer FGA and 6 µL of LRNase A (10 mg / ml), vortex for 2 min, and incubate at room temperature for 5 min. ③ Add 130 µL of buffer LP2 and vortex for 1 min. ④ Add 100 µL of lysozyme (100 mg / ml), heat in a 37°C water bath for 30 min, and invert the tube several times. ④ Centrifuge at 12000 rpm for 5 min, and transfer the supernatant to a new centrifuge tube; ⑤ Add 1.5 times the volume of buffer LP3, and immediately vortex thoroughly for 15 sec. A flocculent precipitate may appear at this point; ⑥ Add the solution and flocculent precipitate from the previous step to an adsorption column CB3 (place the adsorption column in a collection tube), centrifuge at 12000 rpm for 30 sec, discard the waste liquid, and place the adsorption column CB3 in the collection tube; ⑦ Add 600 µL of wash buffer PW to the adsorption column CB3, centrifuge at 12000 rpm for 30 sec, discard the waste liquid, and place the adsorption column CB3 in the collection tube; ⑧ Repeat step ⑦; ⑨ Place the adsorption column CB3 back into the collection tube, centrifuge at 12000 rpm for 2 min, and discard the waste liquid; place the adsorption column CB3 at room temperature for several minutes to thoroughly dry any remaining wash buffer in the adsorption material; ⑩ Transfer the adsorption column CB3 to a clean centrifuge tube, and add 50 µL of buffer solution to the center of the adsorption membrane. Add µL ddH2O, let stand at room temperature for 2–5 min, centrifuge at 12000 rpm for 2 min, and collect the solution in a centrifuge tube.

[0031] Example 3: This example illustrates the amplification of specific primers for the genomic DNA of *Plasmodiophora stylosa*, specifically as follows: Using known highly and weakly pathogenic *Plasmodiophora* species (highly pathogenic *Plasmodiophora* 230308-1 isolated from Fuchun Town, Wuyuan County, identified as race 4 by Williams system; weakly pathogenic *Plasmodiophora* 240307-6 isolated from Xiangtang Town, Nanchang County, identified as race 9 by Williams system) whole genomic DNA as positive control templates, and genomic DNA of the test samples (17 different *Plasmodiophora* disease samples in Example 1) as detection templates, the PCR amplification reaction system was as follows: DNA polymerase 0.5 μL; 10×Buffer 2.5 μL; 10 μM upstream and downstream primers 0.5 μL each, totaling 1 μL, of which the first primer set: SEQ ID NO: 1 + SEQ ID NO: 3, and the second primer set: SEQ ID NO: 2 + SEQ ID NO: 3, with each primer set prepared separately; 2.5 mM dNTPs 2 μL; sample DNA 0.5 μL; ddH2O 18.5 μL; The nucleic acid sequences of the upstream primers are shown in SEQ ID NO. 1 and SEQ ID NO. 2. SEQ ID NO.1: 5'-CCGAACCGTACTTGGCTGAA-3'; SEQ ID NO.2: 5'-GGAAACGGTGCTGAACGC-3'; The nucleic acid sequence of the downstream primer is shown in SEQ ID NO. 3; SEQ ID NO.3: 5'-AGTTCGCTGCGTTCTTCATC-3'.

[0032] The PCR amplification reaction process was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 34 cycles; 72℃ extension for 5 min.

[0033] 5 μL of each PCR amplification product was aspirated onto a 1% agarose gel for electrophoresis. The sample loading order was BS-1(a), BS-1(b), BS-2(a), BS-2(b) ... BS-17(a), BS-17(b) (a represents the first primer set, b represents the second primer set). Electrophoresis was performed at 120V for 30 min, and then the agarose gel was observed under ultraviolet light. Among them, strongly and weakly pathogenic *Plasmodiophora* bacteria served as positive control groups, and ddH2O served as a blank control group. The observation results are as follows: Figure 2 As shown, specifically: (1) When the first primer set amplifies a band of 988 bp and the second primer set amplifies a band of 625 bp, the detected root-root bacterium belongs to a highly pathogenic strain. (2) If the first primer set amplifies to 510 bp and the second primer set amplifies to 625 bp without a bright single band, then the detected root-root bacterium is a weakly pathogenic strain. (3) When the first primer set amplifies to a band of 510bp (with or without a 988bp band) and the second primer set amplifies to a band of 625bp, the detected root rot bacteria belong to a mixed infection of strong and weak pathogenic strains. The agarose gel electrophoresis results of the samples to be tested are as follows: Figure 1 As shown, compared with the positive control group, the results showed that BS-2, BS-3, BS-7, BS-9, BS-10, BS-12, BS-14, and BS-17 were identified as highly pathogenic strains; BS-1, BS-4, BS-5, BS-6, BS-8, BS-11, BS-15, and BS-16 were identified as weakly pathogenic strains; and BS-13 was not identified.

[0034] Example 4: This embodiment illustrates a method for identifying the pathogenicity of different types of *Cladophora* bacteria by artificially inoculating *Brassica napus* variety Fengyou 737 and *Chinese cabbage* improved variety Qingza 3. Specifically: ① Take typical clubroot tissue from 17 collected clubroot disease samples, remove unaffected parts, weigh 240g of each, add appropriate amount of water to homogenize, and finally add sterile water to make up to 1.2L for later use; ② Place sterile seedling substrate into plastic boxes, make 20 holes (2cm in diameter and 2cm deep) in each box, and prepare 6 boxes for each disease sample; ③ Sow 3 boxes of Fengyou 737 and improved Qingza No. 3 for each disease sample, and sow 2-3 seeds in each hole; ④ After homogenizing the clubroot tissue, pour 10ml of the mixture evenly into the sowing hole, and finally cover with sterile seedling substrate and place in a suitable environment for growth.

[0035] Disease index was assessed 50 days post-inoculation. The disease grading criteria for clubroot are as follows: Grade 0: normal root system, no tumors; Grade 1: small tumors on lateral roots; Grade 3: swollen taproot, diameter less than twice that of the stem base; Grade 5: swollen taproot, diameter 2-3 times that of the stem base; Grade 7: swollen taproot, diameter 3-4 times that of the stem base; Grade 9: swollen taproot, diameter more than 4 times that of the stem base, or blackening of the swollen roots. Disease index = [Σ(number of infected plants at each grade × grade) / (total number of plants surveyed × highest disease grade)] × 100. Resistance evaluation criteria: Highly resistant (HR), disease index ≤ 5; resistant (R), 5 < disease index ≤ 10; moderately resistant (MR), 10 < disease index ≤ 20; moderately susceptible (MS), 20 < disease index ≤ 30; susceptible (S), 30 < disease index ≤ 50; highly susceptible (HS), disease index > 50. If both rapeseed and Chinese cabbage are identified as S or HS, then *Plasmodiophora stylosa* is a highly pathogenic strain; if rapeseed is identified as HR or R, and Chinese cabbage as S or HS, then *Plasmodiophora stylosa* is a weakly pathogenic strain.

[0036] The results of comparing the strong and weak pathogenicity of *Cladophora* strains in *Brassica napus* 'Fengyou 737' and *Chinese cabbage* improved variety 'Qingza 3' are shown in Table 1. The results show that BS-2, BS-3, BS-7, BS-9, BS-10, BS-12, BS-14, and BS-17 are strong pathogenic strains; while BS-1, BS-4, BS-5, BS-6, BS-8, BS-11, BS-13, BS-15, and BS-16 are weak pathogenic strains. This indicates that the results of artificial inoculation identification are consistent with the identification results of Example 3.

[0037] Table 1. Comparison of identification results between Fengyou 737 and improved Qingza No. 3

[0038] In summary, this invention enables rapid identification of different pathogenic serotypes of *Plasmodiophora stearothermiae* using PCR technology. In particular, it allows for rapid and accurate identification of the pathogenic serotypes of *Plasmodiophora stearothermiae* to cruciferous crops by analyzing the differences in PCR amplification results. The identification results are accurate and reproducible, and the operation process is simplified, reducing detection time and lowering the operational threshold.

[0039] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Primers for rapid detection and identification of pathogenic types of *Plasmodiophora* based on PCR technology, characterized in that: It includes an upstream primer and a downstream primer, the nucleic acid sequences of the upstream primer are shown in SEQ ID NO. 1 and SEQ ID NO. 2, and the nucleic acid sequence of the downstream primer is shown in SEQ ID NO. 3; The upstream primer and the downstream primer together form a primer set.

2. The primers for rapid detection and identification of pathogenic types of *Plasmodiophora* based on PCR technology as described in claim 1, characterized in that: The primer set includes: The first primer set comprises an upstream primer having the nucleic acid sequence shown in SEQ ID NO. 1 and a downstream primer having the nucleic acid sequence shown in SEQ ID NO. 3; The second primer set consists of an upstream primer having the nucleic acid sequence shown in SEQ ID NO. 2 and a downstream primer having the nucleic acid sequence shown in SEQ ID NO.

3.

3. The application of the primers for rapid detection and identification of pathogenic types of *Plasmodiophora* based on PCR technology as described in claim 1 or 2 in identifying the pathogenicity of *Plasmodiophora* to cruciferous crops, characterized in that: The cruciferous crops mentioned include rapeseed and cruciferous vegetables.

4. A method for rapid detection and identification of pathogenic types of *Plasmodiophora* based on PCR technology, characterized in that: Using the genomic DNA of the sample to be tested as a template, PCR amplification was performed using a primer set. After the reaction, the amplification products were subjected to agarose gel electrophoresis to observe whether specific bands appeared. The PCR amplification reaction system was as follows: 0.5 μL DNA polymerase, 2.5 μL 10× Buffer, 0.5 μL each of 10 μM upstream and downstream primers (total 1 μL), 2 μL 2.5 mM dNTPs, 0.5 μL sample DNA, and 18.5 μL ddH2O. The nucleic acid sequences of the upstream primers are shown in SEQ ID NO. 1 and SEQ ID NO. 2, and the nucleic acid sequences of the downstream primers are shown in SEQ ID NO. 3; the upstream primer and the downstream primer together constitute the primer set.

5. The primers for rapid detection and identification of pathogenic types of *Plasmodiophora* based on PCR technology as described in claim 4, characterized in that: The primer set includes: The first primer set consists of an upstream primer having the nucleic acid sequence shown in SEQ ID NO. 1 and a downstream primer having the nucleic acid sequence shown in SEQ ID NO. 3; The second primer set consists of an upstream primer having the nucleic acid sequence shown in SEQ ID NO. 2 and a downstream primer having the nucleic acid sequence shown in SEQ ID NO.

3.

6. The method as described in claim 5, characterized in that: The PCR amplification reaction process is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 34 cycles; 72℃ extension for 5 min.

7. The method as described in claim 6, characterized in that: The amplification products were subjected to agarose gel electrophoresis, and the results were observed as follows: (1) When the first primer set amplifies to a band of 988 bp and the second primer set amplifies to a band of 625 bp, the detected root-root bacterium belongs to a highly pathogenic strain. (2) If the first primer set amplifies to 510 bp and the second primer set amplifies to 625 bp without a bright single band, then the detected root-root bacterium is a weakly pathogenic strain. (3) When the first primer set amplifies to a band of 510 bp and the second primer set amplifies to a band of 625 bp, the detected root rot bacteria belong to a mixed infection of strong and weak pathogenic strains.

8. The method as described in claim 7, characterized in that: The sample was a clubroot fungus, extracted from typical clubroot tissue formed after disease in cruciferous crops.

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