Sclerotinia rot and clubroot resistance evaluation method for colza

Abstract

The invention discloses a colza sclerotinia and clubroot resistance evaluation method which comprises the following steps: inoculating two pathogenic bacteria of sclerotinia and clubroot at the same time by adopting a bacterial suspension soil injection mode at the seedling stage of colza, observing the symptoms of roots, stem bases or petioles after inoculating for a period of time, carrying out disease grade division on clubroot and sclerotinia, and determining the resistance of clubroot and sclerotinia. Calculating disease indexes, and evaluating sclerotiniose and clubroot resistance grades according to the disease indexes. Based on the characteristic that sclerotiniose and clubroot are both soil-borne diseases, the sclerotiniose and clubroot resistance evaluation method for colza is established in combination with disease occurrence and epidemic, disease occurrence conditions can be established under indoor conditions, sclerotinia sclerotiorum and plasmodiophora brassicae infect hosts at the same time, evaluation time can be shortened, and the evaluation accuracy is improved. Meanwhile, the defect that the host resistance level cannot be really reflected when different disease resistance evaluations are carried out in different growth periods of the rape is avoided.

Description

Technical Field

[0001] The invention relates to the technical field of agriculture, and in particular to a method for evaluating rapeseed resistance to sclerotinia and clubroot. Background Art

[0002] Rapeseed sclerotinia, also known as stem rot or stalk rot, is a disease caused by Sclerotinia sclerotiorum that harms multiple parts of rapeseed, including stems, leaves, flowers, and siliques. Rapeseed sclerotinia not only leads to a reduction in rapeseed yield, but also causes a sharp drop in rapeseed oil content, seriously affecting the yield and quality of rapeseed.

[0003] Rapeseed clubroot is a disease caused by Brassica rapa. Rapeseed clubroot occurs in all rapeseed growing areas. The incidence of rapeseed clubroot is high, and severe cases can cause rapeseed to fail to harvest, which has a great impact on rapeseed production.

[0004] Rapeseed sclerotinia and clubroot have become the two major diseases that affect rapeseed production. Their control strategies are mainly based on planting disease-resistant (tolerant) varieties combined with improved cultivation management measures and timely chemical control. Therefore, the selection, evaluation, screening and application of varieties resistant (tolerant) to sclerotinia and clubroot, especially those resistant to sclerotinia and clubroot, are particularly important to reduce the harm of rapeseed sclerotinia and clubroot. Disease resistance evaluation can screen out crop varieties with strong disease resistance, reduce the occurrence of diseases and pests during planting, reduce the amount of pesticide application, and ensure the safe production and stable supply of grain. It has also become an indispensable auxiliary means for disease-resistant breeding. The application of plug tray seedlings for disease resistance evaluation is convenient for intensive operation, and a large number of materials can be screened at the same time, accelerating the process of disease-resistant variety selection.

[0005] Traditional methods for evaluating rapeseed's resistance to Sclerotinia sclerotinia and clubroot are time-consuming (results can only be obtained from the seedling stage to the mature stage), labor-intensive (rapeseed sowing and field pathogen inoculation will consume a lot of manpower), and the evaluation is not synchronized (evaluation of clubroot resistance is concentrated in the seedling and bud stages; evaluation of Sclerotinia sclerotinia resistance is inoculation from the early flowering stage to the peak flowering stage, and investigation is conducted in the mature stage), and cannot truly reflect the resistance of varieties / materials. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a method for evaluating rapeseed with resistance to Sclerotinia sclerotinia and clubroot disease.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0008] Provided is a method for evaluating rapeseed for resistance to sclerotinia and clubroot, comprising the following steps:

[0009] During the rapeseed seedling stage, the bacterial suspension is injected into the soil and two pathogens, Sclerotinia rot and clubroot, are inoculated at the same time. After a period of inoculation, observe whether the roots are swollen, whether the base of the stem or the petiole is wet rot or white hyphae are attached or wilting. According to the symptoms, the incidence level of clubroot and Sclerotinia rot is divided, the disease index is calculated, and the resistance level of Sclerotinia rot and clubroot is evaluated based on the disease index.

[0010] Further, the preparation method of the root knot fungus spore suspension is specifically as follows:

[0011] Collect the root swelling tissue of rapeseed with clubroot in the field, chop 10g-20g of the root swelling tissue, add 50mL-80mL sterile water to crush, filter with a 200-mesh filter, centrifuge the filtrate at 3100prm for 15min, discard the supernatant, redissolve the precipitate in 50mL sterile water, centrifuge at 3100prm for 10min, repeat this step several times; discard the supernatant, add 5mL of 50% sucrose solution to the precipitate, mix well, centrifuge at 3100r / min for 10min, add 30mL sterile water to the supernatant, centrifuge at 3100r / min for 10min, discard the supernatant, redissolve the precipitate in 30mL sterile water, centrifuge at 3100r / min for 10min, finally dissolve the precipitate in 5mL sterile water and refrigerate for use.

[0012] Furthermore, the preparation method of the mycelial suspension of Sclerotinia sclerotiorum is specifically as follows:

[0013] The mycelial blocks of Sclerotinia sclerotiorum were transferred to PDA medium plates and activated incubated in a 22℃-25℃ incubator for 2 days, and then transferred to new culture dishes for activation. A bacterial plate was made at the edge of the Sclerotinia sclerotiorum colony after 3 days of activation, and 4 plates of 100 mL were used. -1 Add to PDB medium, shake and culture at 22°C-25°C and 200 rpm for 4 days, homogenize for 30 seconds to make a mycelial suspension, and adjust the final concentration to 8-12 mycelium segments / field of view under a 400x microscope.

[0014] Furthermore, the rapeseed was inoculated 8-10 days after emergence; the concentration of dormant spores of the reserved root knotweed fungus was counted with a hemocytometer, and the spore concentration was diluted to 2×10 6 / mL-3×10 6 / mL; Mix the diluted root-knot fungus spore suspension with the sclerotinia mycelium suspension, absorb the bacterial suspension, and place it 0.5cm-1cm from the base of the rapeseed plant stem, allowing the liquid to slowly flow down the plant stem and soak into the soil at the root.

[0015] Further, the spore suspension of root knotweed fungus and the mycelium suspension of sclerotinia sclerotiorum are mixed in equal proportions.

[0016] Furthermore, the inoculated plants were maintained at a constant temperature of 25° C., a relative humidity of more than 90%, and cultured alternately with 16 h of light and 8 h of darkness.

[0017] The beneficial effects of the present invention are:

[0018] Based on the characteristics that both sclerotinia and clubroot are soil-borne diseases, the present invention establishes a rapeseed sclerotinia and clubroot disease concurrent resistance evaluation method in combination with the occurrence and prevalence of the diseases. The disease occurrence conditions can be created under indoor conditions, so that sclerotinia and brassica clubroot can infect the host at the same time, which can shorten the evaluation time and truly reflect the concurrent resistance of materials / variety. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a picture of the symptoms in the pot of the highly susceptible sclerotinia rot material in the test example;

[0020] Figure 2 This is a picture of the symptoms in the pot of the highly susceptible clubroot material in the test example;

[0021] Figure 3 Symptom diagram of the materials susceptible to clubroot and sclerotinia in the test example;

[0022] Figure 4 This is a picture of the material that is resistant to sclerotinia and clubroot and shows various resistances in the test examples. DETAILED DESCRIPTION

[0023] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0024] Test example

[0025] With Deyou No. 6 (susceptible to clubroot and sclerotinia) as the control variety, Nanyou 1958R, Huashuang 5R and Zhongyou 821 were evaluated for resistance to both sclerotinia and clubroot.

[0026] 1. Rape sowing: Put a vegetable seedling medium with a water content of 70% in a pot for later use. Soak 150 seeds of each rape variety in warm water, select full seeds for sowing, 10 seeds per pot, cover the surface with film to keep moisture, place in the dark at 25℃ for 36 hours, remove the film, and cultivate under daily light. Set 5 replicates for each variety.

[0027] 2. Preparation of root-knot fungus spore suspension: 10 days after rape sowing, rape root tissue with root knot disease was collected in Xindu District of Chengdu City, rinsed with running water and dried, 20 g of root swollen tissue was chopped, added with 80 mL of sterile water, crushed with a juicer, filtered with a 200-mesh nylon filter, and the filtrate was transferred to a clean 50 mL centrifuge tube, centrifuged at 3100 rpm for 15 min, the supernatant was discarded, the precipitate was redissolved in 50 mL of sterile water, and centrifuged at 3100 rpm for 10 min. This step was repeated twice. The supernatant was discarded, 5 mL of 50% sucrose solution was added to the precipitate, mixed, centrifuged at 3100 r / min for 10 min, the supernatant was carefully transferred into a sterile 50 mL centrifuge tube with a pipette, 30 mL of sterile water was added, centrifuged at 3100 r / min for 10 min, the supernatant was discarded, the precipitate was redissolved in 30 mL of sterile water, centrifuged at 3100 r / min for 10 min, and finally the precipitate was dissolved in 5 mL of sterile water, counted with a hemocytometer, and diluted to obtain a spore concentration of 3 × 10 6 Pieces / mL.

[0028] 3. Preparation of mycelial suspension of Sclerotinia sclerotiorum: Take a mycelial block with a diameter of 6 mm from the Sclerotinia sclerotia isolated from the sclerotia of rapeseed stems after inoculation in the field disease resistance evaluation in Pengzhou, Chengdu, and store it at 4°C. Move it to the center of a new PDA medium plate, activate it in a 23°C incubator for 2 days, and then transfer it to a new culture dish for activation and culture in the same way. After 3 days, take a bacterial plate (Φ6 mm) at the edge of the Sclerotinia colony and press 4 pieces of 100 mL -1 The mixture was added to PDA liquid culture medium and cultured at 23°C and 200 rpm with shaking for 4 days. Mycelial suspension was prepared by homogenization for 30 seconds using a homogenizer, and the final concentration was adjusted to 10 mycelial segments per field of view under a 400x microscope.

[0029] 4. Inoculation: 8 days after rapeseed emerges, mix the diluted root-knot fungus spore suspension and the sclerotinia mycelium suspension in equal proportions. Use a pipette to suck up the bacterial suspension and slowly press it out along the base of the rapeseed plant stem 0.5cm-1cm away. Let the bacterial suspension slowly flow down the plant stem and soak into the root soil. Inoculate 1mL of the bacterial suspension per plant.

[0030] 5. Management and investigation: Place the inoculated plants in a plant culture rack, maintain a constant temperature of 25°C, a relative humidity of more than 90%, and culture them alternately with 16 hours of light and 8 hours of darkness, and investigate them regularly.

[0031] During the investigation, the plants were uprooted and the roots and above-ground parts were observed separately. Seven days after inoculation, the control variety (Deyou No. 6) was selected for investigation to observe whether there were water-soaked spots or white hyphae attached to the stem base or petiole of the plant, and whether there were dormant spores of clubroot in the fibrous roots of the plant under a microscope to determine whether the inoculation was successful. If the inoculation was successful, all the tested varieties were investigated 30 days later to observe whether the roots were swollen, whether the stem base or petiole was wet rotted and white hyphae attached. According to the symptoms, the incidence levels of clubroot and sclerotinia were divided, and the disease index was calculated. The resistance level was evaluated based on the disease index.

[0032] The criteria for grading disease are as follows:

[0033] (1) Classification criteria for clubroot disease:

[0034] Level 0: normal root system, no swelling symptoms; representative value 0;

[0035] Level 1: Only a few small swellings appear on the fibrous roots and lateral roots, and no diseases or tumors are found on the main roots; the representative value is 1;

[0036] Level 2: More than two fibrous roots or lateral roots are swollen; or the diameter or length of the "tumor" is greater than 0.5 cm and less than 1 cm; or the main root is swollen, and the length or diameter of the "tumor" is less than 0.5 cm; the representative value is 2;

[0037] Level 3: Multiple "tumors" appear on the fibrous roots and lateral roots, or the diameter or length of the "tumor" is greater than 1 cm, or the length or diameter of the "tumor" part of the main root is greater than 0.5 cm and less than 1 cm. The representative value is 3;

[0038] Grade 4: The main root is obviously enlarged, and the length or diameter of the "tumor" part is greater than 1 cm; the representative value is 4.

[0039] (2) Sclerotinia sclerotinia grading standards:

[0040] Level 0: normal, asymptomatic; representative value 0;

[0041] Level 1: The base of the stem is water-soaked but not rotten; the representative value is 1;

[0042] Level 2: The stem base and petiole near the ground are water-soaked, with a large number of hyphae attached; the representative value is 2;

[0043] Level 3: The stem base and petioles near the ground are wet rot, with a large amount of hyphae attached, or the leaves turn yellow or rot and fall off; the representative value is 3.

[0044] The disease index calculation formula is as follows:

[0045] Disease index (DI) = 100 × ∑ (number of diseased plants at each level × representative value of each level) / (total number of plants surveyed × highest representative value);

[0046] Sclerotinia and clubroot resistance classification standards:

[0047] Resistance level Disease index (DI) High resistance DI≤5.0 Zhongkang 5.0<DI≤15.0 Low resistance 15.0<DI≤30.0 Low sense 30.0<DI≤40.0 Medium 40.0<DI≤50.0 High Sensitivity DI>50.0

[0048] The following results were obtained from this test case investigation:

[0049]

[0050] At the same time, field evaluation of resistance to sclerotinia and clubroot was carried out. The results showed that the evaluation results of this test case were consistent with the field resistance evaluation results, but the evaluation time was shortened from the original more than 190 days to 40 days.

[0051] According to the characteristics that both clubroot and sclerotinia are soil-borne diseases, combined with the epidemic law and transmission route of sclerotinia in the rapeseed seedling stage, the present invention simultaneously inoculates two pathogens by injecting a bacterial suspension into the soil in the seedling stage, thereby shortening the evaluation time for concurrent resistance to clubroot and sclerotinia; and avoiding the disadvantage that different disease resistance evaluations are performed at different growth stages of rapeseed and the level of host concurrent resistance cannot be truly reflected.

[0052] The method of the invention is not only applicable to the evaluation of rapeseed's resistance to clubroot and sclerotinia, but also applicable to the evaluation of resistance of other cruciferous crops.

[0053] 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 present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0054] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for evaluating rapeseed resistance to Sclerotinia sclerotiorum and clubroot disease, characterized in that: The following steps are involved: During the rapeseed seedling stage, the bacterial suspension is injected into the soil, and both Sclerotinia rot and clubroot pathogens are inoculated at the same time. After a period of inoculation, the symptoms of the roots, stem bases or petioles are observed, the incidence levels of clubroot and Sclerotinia rot are classified, the disease index is calculated, and the resistance levels of Sclerotinia rot and clubroot are evaluated based on the disease index.

2. The method for evaluating rapeseed resistance to Sclerotinia sclerotiorum and clubroot disease according to claim 1, characterized in that: Pathogens are inoculated 8-10 days after rapeseed emerges; the spore suspension of root-knot fungus is mixed with the mycelium suspension of sclerotinia sclerotiorum, and the liquid is slowly flowed down along the stem of the rapeseed plant at 0.5cm-1cm from the base and soaked into the soil at the root.

3. The method for evaluating rapeseed resistance to Sclerotinia sclerotiorum and clubroot disease according to claim 2, characterized in that: Count the dormant spores of the reserved root knot fungus and dilute the spores to a concentration of 2 × 10 6 / mL-3×10 6 Pieces / mL.

4. The method for evaluating rapeseed for resistance to Sclerotinia sclerotiorum and clubroot disease according to claim 2, characterized in that: The spore suspension of root knot fungus and the mycelium suspension of sclerotinia sclerotiorum are mixed in equal proportions.

5. The method for evaluating rapeseed for resistance to Sclerotinia sclerotiorum and clubroot disease according to claim 1 or 2, characterized in that: The inoculated plants were maintained at a constant temperature of 25°C, a relative humidity of more than 90%, and cultured alternately with 16 h of light and 8 h of darkness.

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