Sclerotinia rot and clubroot resistance evaluation method for colza

Abstract

The invention discloses a colza sclerotiniose clubroot resistance evaluation method, and relates to the technical field of stress-resistant colza screening and evaluation. Comprising the following steps: 8-10 days after rape seedling emergence, diluting the plasmodiophora brassicae spore concentration to 2 * 10 < 6 > / mL to 3 * 10 < 6 > / mL by using sterile water, then mixing a plasmodiophora brassicae spore suspension and a sclerotinia sclerotiorum hypha suspension in equal proportion, and enabling the bacterial suspension liquid to slowly flow down along plant stems and be immersed into root soil along the positions 0.5-1 cm away from the basal parts of rape plant stems; the method comprises the following steps of: culturing at the constant temperature of 25 DEG C and the relative humidity of more than 90% in the presence of light for 16 hours and darkness for 8 hours alternately, observing the symptoms of roots, basal stems or petioles after 30 days, carrying out disease grading of the clubroot and the sclerotiniose, then calculating disease indexes according to the grading and the number of diseased plants at each stage, and further evaluating the resistance grades of the sclerotiniose and the clubroot according to the disease indexes. According to the evaluation method disclosed by the invention, disease occurrence conditions can be created under indoor conditions, so that sclerotinia sclerotiorum and plasmodiophora brassicae infect hosts at the same time, the evaluation time can be shortened, and meanwhile, the defect that the resistance evaluation of different diseases in different growth periods of the rape cannot truly reflect the resistance level of the hosts is overcome.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of resistance screening and evaluation of oilseed rape, and particularly relates to an evaluation method for resistance to sclerotinia stem rot and clubroot of oilseed rape. BACKGROUND

[0002] Sclerotinia stem rot of oilseed rape, also known as stem rot and rotten stem disease, is caused by Sclerotinia sclerotiorum, which harms multiple parts of oilseed rape such as stems, leaves, flowers and silique. Sclerotinia stem rot of oilseed rape not only leads to yield reduction, but also causes the oil content of oilseed rape to decrease sharply, which seriously affects the yield and quality of oilseed rape.

[0003] Clubroot of oilseed rape is a disease caused by Plasmodiophora brassicae. Clubroot of oilseed rape occurs in all oilseed rape planting areas. The disease has a high incidence rate and can cause complete loss of oilseed rape in severe cases, which has a great impact on oilseed rape production.

[0004] Sclerotinia stem rot and clubroot of oilseed rape have become two major diseases affecting oilseed rape production. The main strategy for their control is to plant disease-resistant varieties combined with improved cultivation management measures and timely chemical control. Therefore, the breeding, evaluation, screening and application of varieties resistant to sclerotinia stem rot and clubroot, especially those resistant to both diseases, are particularly important for reducing the harm of sclerotinia stem rot and clubroot of oilseed rape. Disease resistance evaluation can screen crop varieties with strong disease resistance, reduce the occurrence of diseases and pests during planting, reduce the amount of pesticide application, ensure the safe production and stable supply of food, and also become an essential auxiliary means for disease-resistant breeding. The application of plug seedling for disease resistance evaluation facilitates intensive operation and can screen a large number of materials at the same time, thus accelerating the breeding process of disease-resistant varieties.

[0005] Traditional methods for evaluating the resistance of oilseed rape to sclerotinia stem rot and clubroot require covering the whole growth period of oilseed rape, from the seedling stage to the mature stage, to obtain the final resistance results. The whole process is time-consuming, labor-intensive and costly in terms of field test resources, which seriously restricts the efficiency of disease-resistant material screening and the progress of breeding. Moreover, there are essential differences in the key time nodes for resistance evaluation of the two diseases. Clubroot resistance evaluation is concentrated in the seedling stage to the rosette stage, while sclerotinia stem rot resistance evaluation requires pathogen inoculation in the early flowering stage to the full flowering stage, and resistance investigation is delayed to the mature stage. This time mismatch makes it impossible to simultaneously implement resistance evaluation of the two diseases in the same material and growth cycle, which cannot truly and accurately reflect the comprehensive resistance level of the tested varieties / materials to sclerotinia stem rot and clubroot, directly affecting the accuracy and effectiveness of breeding of oilseed rape varieties with resistance to both diseases.

[0006] Therefore, how to provide a method for simultaneously evaluating the resistance of oilseed rape to clubroot and sclerotinia stem rot is a technical problem that needs to be solved by those skilled in the art. SUMMARY

[0007] The purpose of this invention is to provide a method for evaluating resistance to both Sclerotinia stem rot and clubroot in rapeseed, which overcomes the shortcomings of the inability to simultaneously evaluate resistance to the two diseases and to truly reflect the dual resistance of varieties / materials.

[0008] To achieve the above objectives, in a first aspect, the present invention provides a method for evaluating resistance to both sclerotinia stem rot and clubroot in rapeseed, comprising the following steps: 1) 8-10 days after rapeseed emergence, dilute the concentration of clubroot spores with sterile water to 2×10⁻⁶. 6 3 × 10⁻³ cells / mL 6 Mix the spore suspension of *Plasmodiophora stearothermiae* with the mycelial suspension of *Sclerotinia sclerotiorum* in equal proportions, and then mix the mixture with the mycelial suspension at a distance of 0.5-1 cm from the base of the rapeseed stem, allowing the mycelial suspension to slowly flow down the stem and soak into the soil around the roots. 2) Culture at a constant temperature of 25℃ and relative humidity of over 90%, with alternating light and darkness for 16 hours and 8 hours, observe the symptoms on the roots, stem base, or petioles after 30 days, classify the incidence of clubroot and sclerotinia disease, calculate the disease index based on the classification and the number of diseased plants at each level, and then evaluate the resistance level of sclerotinia and clubroot based on the disease index.

[0009] Furthermore, the specific method for preparing the *Plasmodiophora* spore suspension is as follows: Collect clubroot tissue from rapeseed plants affected by clubroot disease in the field. Chop 10g-20g of clubroot tissue, add 50mL-80mL of sterile water and pulverize. Filter through a 200-mesh filter. Centrifuge the filtrate at 3100rpm for 15min, discard the supernatant, redissolve the precipitate in 50mL of sterile water, and centrifuge at 3100rpm for 10min. Repeat this step several times. Discard the supernatant, add 5mL of 50% sucrose solution to the precipitate, mix well, and centrifuge at 3100rpm for 10min. Add 30mL of sterile water to the supernatant, centrifuge at 3100rpm for 10min, discard the supernatant, redissolve the precipitate in 30mL of sterile water, and centrifuge at 3100rpm for 10min. Finally, dissolve the precipitate in 5mL of sterile water, count the precipitate, and refrigerate for later use.

[0010] Furthermore, the specific method for preparing the *Sclerotinia sclerotiorum* mycelial suspension is as follows: Transfer the mycelial blocks of *Sclerotinia sclerotiorum* to PDA agar plates and incubate for 2 days at 22°C-25°C. Then transfer them to new petri dishes for further activation. After 3 days of activation, collect four 100mL samples from the edge of the *Sclerotinia sclerotiorum* colonies. -1 Add to PDB medium and culture at 22°C-25°C and 200 rpm for 4 days with shaking. Homogenize for 30 seconds to prepare mycelial suspension. Adjust the final concentration to 400x magnification and observe 8-12 mycelial segments per field of view.

[0011] Furthermore, the criteria for classifying the severity of the disease are as follows: (1) Criteria for classifying clubroot disease: Grade 0: Normal root system, no swelling symptoms; representative value 0; Grade 1: Only a few small swellings are observed on the fibrous roots and lateral roots; no diseases or tumors are found on the main root; the representative value is 1. Grade 2: Two or more fibrous roots or lateral roots are swollen; or the diameter or length of the "tumor" is greater than 0.5cm and less than 1cm; or the main root is swollen and the length or diameter of the "tumor" is less than 0.5cm; the representative value is 2. Grade 3: Multiple "tumors" appear on the fibrous roots and lateral roots, or the diameter or length of the "tumor" is greater than 1cm, or the length or diameter of the "tumor" part of the main root is greater than 0.5cm and less than 1cm, with a representative value of 3; Grade 4: Significant swelling of the main root, with the "tumor" portion being longer or larger than 1 cm in diameter; the representative value is 4.

[0012] (2) Sclerotinia rot grading criteria: Level 0: Normal, asymptomatic; representative value 0; Grade 1: The base of the stem is water-soaked and not rotten; the representative value is 1. Grade 2: The base of the stem and the petiole near the ground are water-soaked, with a large number of mycelia attached; the representative value is 2; Level 3: The base of the stem and the petiole near the ground are wet and rotten, with a large amount of mycelium attached, or the leaves are yellowed, rotted and fall off; the representative value is 3.

[0013] Furthermore, the formula for calculating the disease index is as follows: Disease Index (DI) = 100 × ∑(number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × highest representative value); The classification criteria for resistance levels of sclerotinia stem rot and clubroot are as follows: High DI ≤ 5.0, Medium DI 5.0 < DI ≤ 15.0, Low DI 15.0 < DI ≤ 30.0, Low DI 30.0 < DI ≤ 40.0, Medium DI 40.0 < DI ≤ 50.0, High DI > 50.0.

[0014] Traditional methods for evaluating resistance to sclerotinia stem rot and clubroot in rapeseed require time and effort, starting from the seedling stage and continuing until maturity. Clubroot resistance evaluation is concentrated at the seedling and budding stages, while sclerotinia stem rot resistance evaluation involves inoculation from early to full flowering, followed by surveys at maturity. The evaluation of resistance to both diseases cannot be synchronized, failing to truly reflect the dual resistance of varieties / materials. This invention creatively utilizes the soil-borne nature of both sclerotinia stem rot and clubroot, and establishes a method for evaluating dual resistance to both diseases in rapeseed. The inventors unexpectedly discovered that creating disease occurrence conditions indoors and simultaneously infecting the host with *Sclerotinia stem rot* and *Cladosporium brassicae* significantly shortens the evaluation time and simultaneously reflects the resistance of rapeseed to both clubroot and sclerotinia stem rot, thus truly reflecting the dual resistance of materials / varieties. This overcomes the deficiency in existing technologies where resistance to clubroot and sclerotinia stem rot cannot be evaluated at the same time. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0016] Appendix Figure 1 Images showing symptoms inside pots of highly susceptible materials to sclerotinia stem rot; Appendix Figure 2 Symptoms inside the pot of a highly susceptible plant material for clubroot disease; Appendix Figure 3 Symptoms of clubroot-susceptible and sclerotinia-resistant materials compared to those of infected materials; Appendix Figure 4 This is a diagram of materials exhibiting resistance to sclerotinia stem rot, clubroot, and various other diseases. Detailed Implementation

[0017] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0018] Example 1 Using Deyou 6 (susceptible to clubroot and sclerotinia stem rot) as a control variety, we evaluated the resistance of Nanyou 1958R, Huashuang 5R, and Zhongyou 821 to both sclerotinia stem rot and clubroot.

[0019] 1. Rapeseed sowing: Prepare vegetable seedling substrate with a moisture content of 70% in pots. Soak 150 seeds of each rapeseed variety in warm water, select plump seeds for sowing, 10 seeds per pot, cover the surface with a film to retain moisture, and place in the dark at 25°C for 36 hours. After that, remove the film and cultivate under normal light. Set up 5 replicates for each variety.

[0020] 2. Preparation of clubroot spore suspension: Ten days after rapeseed sowing, clubroot tissue of rapeseed in Xindu District, Chengdu was collected. After rinsing with running water and drying, 20 grams of clubroot tissue was chopped, 80 mL of sterile water was added, and the mixture was crushed with a juicer. The mixture was filtered through a 200-mesh nylon filter and the filtrate was transferred to a clean 50 mL centrifuge tube. The tube was centrifuged at 3100 rpm for 15 min, the supernatant was discarded, and the precipitate was redissolved in 50 mL of sterile water. The tube was centrifuged at 3100 rpm for 10 min. This step was repeated twice. Discard the supernatant, add 5 mL of 50% sucrose solution to the precipitate, mix well, centrifuge at 3100 rpm for 10 min, carefully transfer the supernatant to a sterile 50 mL centrifuge tube using a pipette, add 30 mL of sterile water, centrifuge at 3100 rpm for 10 min, discard the supernatant, redissolve the precipitate in 30 mL of sterile water, centrifuge at 3100 rpm for 10 min, finally dissolve the precipitate in 5 mL of sterile water, count using a hemocytometer, and dilute to obtain a spore concentration of 3 × 10⁻⁶. 6 per mL.

[0021] 3. Preparation of *Sclerotinia sclerotiorum* mycelial suspension: *Sclerotinia sclerotiorum*, isolated from rapeseed stems after inoculation for field disease resistance evaluation in Pengzhou, Chengdu, and stored at 4°C, was used. A 6mm diameter mycelial block was transferred to the center of a new PDA medium plate and activated at 23°C for 2 days. The plate was then transferred to a new petri dish for further activation. After 3 days, mycelial discs (Φ6mm) were removed from the edge of the *Sclerotinia sclerotiorum* colonies, and four discs were placed in 100mL of medium. -1 Add to PDA liquid culture medium, shake and culture at 23°C and 200 rpm for 4 days, homogenize for 30 seconds to prepare mycelial suspension, and finally adjust the concentration to 10 mycelial segments per field of view under a 400x microscope.

[0022] 4. Inoculation: Eight days after rapeseed emergence, mix diluted clubroot spore suspension and Sclerotinia sclerotiorum mycelial suspension in equal proportions. Use a pipette to draw up the mycelial suspension and apply it 0.5cm along the base of the rapeseed stem. -1 At a depth of cm, slowly press out the bacterial suspension, allowing it to flow slowly down the plant stem and soak into the root soil. Inoculate each plant with 1 mL of bacterial suspension.

[0023] 5. Management and investigation: Place the inoculated plants in a plant culture rack, maintain a constant temperature of 25°C and a relative humidity of over 90%, and alternate between 16 hours of light and 8 hours of darkness, and conduct investigations periodically.

[0024] During the investigation, the plants were uprooted, and the roots and above-ground parts were observed separately. Seven days after inoculation, a control variety (Deyou 6) was selected for investigation. The presence of water-soaked lesions or white mycelium on the stem base or petioles was observed, and the presence of dormant spores of clubroot fungus was examined under a microscope to determine the success of inoculation. If inoculation was successful, all tested varieties were investigated 30 days later. The presence of root swelling, wet rot at the stem base or petioles, and white mycelium were observed. Based on the symptoms, the severity of clubroot and sclerotinia rot was classified, and the disease index was calculated. The resistance level was evaluated based on the disease index.

[0025] The criteria for classifying the severity of disease are as follows: (1) Criteria for classifying clubroot disease: Grade 0: Normal root system, no swelling symptoms; representative value 0; Grade 1: Only a few small swellings are observed on the fibrous roots and lateral roots; no diseases or tumors are found on the main root; the representative value is 1. Grade 2: Two or more fibrous roots or lateral roots are swollen; or the diameter or length of the "tumor" is greater than 0.5cm and less than 1cm; or the main root is swollen and the length or diameter of the "tumor" is less than 0.5cm; the representative value is 2. Grade 3: Multiple "tumors" appear on the fibrous roots and lateral roots, or the diameter or length of the "tumor" is greater than 1cm, or the length or diameter of the "tumor" part of the main root is greater than 0.5cm and less than 1cm, with a representative value of 3; Grade 4: Significant swelling of the main root, with the "tumor" portion being longer or larger than 1 cm in diameter; the representative value is 4.

[0026] (2) Sclerotinia rot grading criteria: Level 0: Normal, asymptomatic; representative value 0; Grade 1: The base of the stem is water-soaked and not rotten; the representative value is 1. Grade 2: The base of the stem and the petiole near the ground are water-soaked, with a large number of mycelia attached; the representative value is 2; Level 3: The base of the stem and the petiole near the ground are wet and rotten, with a large amount of mycelium attached, or the leaves are yellowed, rotted and fall off; the representative value is 3.

[0027] Among them, the symptom images of potted plants with highly susceptible sclerotinia rot are shown in the image. Figure 1 See the image below for symptoms of highly susceptible clubroot disease in potted plants. Figure 2 Symptoms of clubroot infection, sclerotinia infection, and infected materials are shown in the diagram. Figure 3 The image shows materials exhibiting resistance to sclerotinia stem rot, clubroot, and various other resistances. Figure 4 .

[0028] The formula for calculating the disease index is as follows: Disease Index (DI) = 100 × ∑(number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × highest representative value); The classification criteria for resistance levels of sclerotinia stem rot and clubroot are shown in Table 1.

[0029] Table 1. Criteria for classifying resistance levels to sclerotinia stem rot and clubroot.

[0030] The survey yielded the following results, as shown in Table 2.

[0031] Table 2 Results of the evaluation of the dual resistance of each variety

[0032] Example 2 Field trials were conducted on the above-mentioned varieties, namely Deyou 6, Nanyou 1958R, Huashuang 5R, and Zhongyou 821, to evaluate their resistance to sclerotinia stem rot and clubroot. The field trial process was a routine experiment. The results showed that the evaluation results were consistent with the resistance evaluation results of Example 1, but the evaluation time was extended from 40 days in Example 1 to more than 190 days.

[0033] Based on the characteristics that clubroot and sclerotinia rot are both soil-borne diseases, and combined with the epidemic pattern and transmission route of sclerotinia rot in rapeseed seedlings, this invention uses soil injection of bacterial suspension during the seedling stage to simultaneously inoculate both pathogens, thus shortening the evaluation time for co-resistance to clubroot and sclerotinia rot. At the same time, it avoids the drawback of conducting different disease resistance evaluations at different growth stages of rapeseed, which cannot truly reflect the level of co-resistance to the host.

[0034] The method of this invention is not only applicable to the evaluation of rapeseed's resistance to clubroot and sclerotinia stem rot, but also applicable to the evaluation of other cruciferous crops' resistance.

[0035] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0036] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for evaluating resistance to Sclerotinia sclerotiorum and Plasmo¬ diophora brassicae in Brassica napus, characterized in that, Comprising the following steps: 1) 8-10 days after rape seedling, inoculate pathogenic bacteria; first dilute the concentration of Plasmodiophora brassicae spores to 2x10 6 6 / mL-3x10 6 6 / mL with sterile water, then mix the spore suspension of Plasmodiophora brassicae with the mycelium suspension of Sclerotinia sclerotiorum at the same proportion, along the stem base of rape plant 0.5cm-1cm, make the bacteria suspension liquid slowly flow down along the stem of plant and immerse into the soil of root; 2) Constant temperature 25℃, relative humidity above 90%, 16h light, 8h alternating dark culture, 30d after observing the symptoms of root, stem base or petiole, making the incidence of root rot, sclerotinia disease grade division, then according to the grade division and the number of each level of disease strain to calculate the disease index, and then according to the disease index to evaluate the resistance grade of sclerotinia disease, root rot.

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