Screening method of sclerotiniose inhibiting material for brassica juncea

Abstract

The invention discloses a method for screening sclerotiniose inhibition materials for brassica juncea, and belongs to the technical field of sclerotiniose inhibition of brassica juncea. The method specifically comprises the steps that brassica juncea germplasm resource materials are collected; planting a mustard type rape germplasm resource material; collecting overground tissue materials of each single plant of each germplasm material 3 for insurance storage; field collection and activation of sclerotinia sclerotiorum: selecting third-generation new edge hyphae in the same period from inoculated sclerotinia sclerotiorum hyphae; and simulating biological fumigation: putting the sample into liquid nitrogen, quickly freezing and grinding, inoculating third-generation new edge new hyphae of sclerotinia sclerotiorum to one side with the substrate, transferring 0.15 g of ground rape aboveground tissue sample powder to one side without the culture medium in a culture dish, and when the crushed sample is added, the size of the sample area is in direct proportion to the addition amount. According to the method, different mustard type rape germplasm resource overground tissues have different inhibition effects on sclerotinia sclerotiorum, the inhibition effect of the overground tissues on sclerotinia sclerotiorum is measured by adding overground tissue broken samples, and then an inhibition material with a better sclerotinia sclerotiorum inhibition effect is screened out.

Description

Technical Field

[0001] The present invention belongs to the technical field of screening materials for suppressing Sclerotinia sclerotiorum in Brassica juncea, and relates to a method for screening materials for suppressing Sclerotinia sclerotiorum in Brassica juncea. Background Art

[0002] Sclerotinia sclerotiorum is a major disease of rapeseed, which is transmitted through contact between diseased plants and healthy plants. The pathogen produces hyphae at the diseased site and then infects the plants. It can infect throughout the growth stage of rapeseed, and is most severe during the flowering and fruiting period. With the change of farming systems, the increase in fertilizer application, and the promotion of high-yielding varieties, the trend of Sclerotinia sclerotiorum is increasing. At present, soil is mainly treated with chemical fumigants. However, while chemical fumigants kill harmful soil pathogens, they also have an adverse impact on the soil microecology.

[0003] Currently, the control of Sclerotinia sclerotiorum mainly relies on chemical control, but the control effect is not ideal, and it will kill beneficial microorganisms in the soil, damage the soil structure. Moreover, the long-term and large-dose use of chemical drugs causes environmental pollution and other problems. In the breeding of rapeseed varieties, excessive attention has been paid to the high yield of varieties while ignoring their resistance to Sclerotinia sclerotiorum. At present, no variety with excellent resistance to Sclerotinia sclerotiorum has been selected. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for screening materials for suppressing Sclerotinia sclerotiorum in Brassica juncea, and the screened materials for suppressing Sclerotinia sclerotiorum have a significant effect on suppressing Sclerotinia sclerotiorum in rapeseed.

[0005] The technical solution adopted by the present invention: A method for screening materials for suppressing Sclerotinia sclerotiorum in rapeseed, comprising the following steps:

[0006] Step 1: Collect germplasm resource materials of Brassica juncea: Collect 200 germplasm resource materials of Brassica juncea from all over the country;

[0007] Step 2: Plant germplasm resource materials of Brassica juncea: Use 200 germplasm resource materials of Brassica juncea and plant them in the experimental base. Each Brassica juncea strain is planted in a 1-row plot with a row length of 4 m, a row spacing of 40 cm, and a bed spacing of 50 cm. The direct seeding method is used for planting. After thinning and final singling, 30 rapeseed plants are retained in each row, and 2 rows of protection rows are planted around.

[0008] Step 3: Sampling: When the rapeseed is at the initial flowering stage, collect 3 above-ground tissue materials of each single plant of each germplasm material, store the obtained leaf samples in liquid nitrogen in a fresh-keeping box, and bring them back to the laboratory for storage in a -80°C refrigerator for later use;

[0009] Step 4, Field collection and activation of Sclerotinia sclerotiorum: The strain of Sclerotinia sclerotiorum causing sclerotinia disease is the Sclerotinia sclerotiorum collected from the main rapeseed production areas. The sclerotia of Sclerotinia sclerotiorum are rinsed with sterile water and then soaked in 70% alcohol for 5 minutes, rinsed 2 to 3 times with sterile water, left to dry overnight on filter paper, and finally placed in a petri dish containing PDA solid medium. The petri dish is incubated upside down at 22°C. After the mycelia grow to cover the petri dish, on the ultra-clean workbench, a sterilized inoculation loop is used to pick the mycelia at the edge of the colony and transplanted into another petri dish containing PDA solid medium for subculture. When the mycelia grow to the edge of the petri dish, inoculation on a new PDA is considered as one generation. The inoculated Sclerotinia sclerotiorum mycelia are selected from the newly grown marginal mycelia of the third generation at the same time.

[0010] Step 5, Simulated biological fumigation: Select each sample of the above-ground tissue of the germplasm resources stored in an -80°C refrigerator, soak it in 75% ethanol for 3 - 5 minutes to kill and remove the microorganisms on the leaf surface, then rinse it 5 times with sterile water to remove the residual alcohol on the leaf surface. Place the sample in liquid nitrogen for quick freezing and grinding, and inoculate the newly grown marginal mycelia of the third generation of Sclerotinia sclerotiorum on one side with the substrate. Transfer 0.15 g of the ground powder of the above-ground tissue sample of rapeseed to the side without medium in the petri dish. When adding the ground sample, the size of the sample area is proportional to the added amount. Avoid contacting the medium during placement. The area of the Sclerotinia sclerotiorum lesion is calculated using the formula S = πr 2 where r represents the radius of the lesion plaque, in cm, and π is taken as 3.14; Seal the petri dish with a sealing film, then place it in an incubator at 22°C for constant temperature incubation for 24 hours, and then measure the area of the Sclerotinia sclerotiorum lesion plaque. Take the average value after 3 repetitions;

[0011] Step 6, According to the average value of the measured area of the Sclerotinia sclerotiorum lesion plaque, screen out the sclerotinia disease inhibitory materials with better inhibitory effects on sclerotinia disease. The inhibitory materials with an average value less than 10 cm 2 are the sclerotinia disease inhibitory materials of Brassica juncea.

[0012] Advantages of the present invention: The present invention utilizes the different inhibitory effects of the above-ground tissues of different Brassica juncea germplasm resources on the sclerotinia disease pathogen (Sclerotinia sclerotiorum). By adding the ground samples of the above-ground tissues, measuring their inhibitory effects on Sclerotinia sclerotiorum, and further screening out the inhibitory materials with better inhibitory effects on sclerotinia disease. Description of the Drawings

[0013] Figure 1 It is a schematic diagram for screening the resistance to sclerotinia disease of Brassica juncea; among them, the upper half of each petri dish is inoculated with Sclerotinia sclerotiorum on MS medium, and the lower half has no medium; Figure A is the control, without adding ground tissue samples; Figure B shows the green tissue ground sample as a highly resistant germplasm material (0.15 g); Figure C shows the green tissue ground sample as a low-resistant germplasm material (0.15 g);

[0014] Figure 2Frequency distribution diagram of the fumigation effect of sclerotinia rot on the above-ground tissue crushed samples of Brassica juncea germplasm resources; among them, the numbers on the X-axis represent the lesion size of Sclerotinia sclerotiorum, with the unit of cm2; the Y-axis represents the number of materials; three Brassica juncea germplasm resources materials used for field trials are marked on the graph. The name of the highly resistant material is: Hherucic2, the name of the moderately resistant material is: DafangqingYC, and the name of the lowly resistant material is: CehenggaoYC. Detailed implementation method

[0015] Research shows that at present, the control of sclerotinia rot mainly relies on chemical control, but the control effect is not ideal, and it will kill beneficial microorganisms in the soil, damage the soil structure, and long-term and high-dose use of chemical drugs will cause environmental pollution and other problems. In the breeding of rapeseed varieties, excessive attention has been paid to the high yield of varieties while ignoring their resistance to sclerotinia rot. At present, no variety with excellent resistance to sclerotinia rot has been selected. The use of biofumigation technology can be a better method for sclerotinia rot and can also provide a new idea for its control, specifically as the method in Example 1.

[0016] Example 1: A method for screening sclerotinia rot inhibitory materials for Brassica juncea, comprising the following steps:

[0017] Step 1: Collect Brassica juncea germplasm resources materials: Collect 200 Brassica juncea germplasm resources materials from all over the country;

[0018] Step 2: Plant Brassica juncea germplasm resources materials: Use 200 Brassica juncea germplasm resources materials and plant them in the experimental base. Each Brassica juncea strain is planted in a 1-row plot with a row length of 4m, a row spacing of 40cm, and a bed spacing of 50cm. The direct seeding method is used for planting. After thinning and finalizing the seedlings, 30 rapeseed plants are retained in each row, and 2 rows of protective rows are planted around.

[0019] Step 3: Sampling: When the rapeseed is at the initial flowering stage, collect 3 above-ground tissue materials of each single plant of each germplasm material. Store the obtained leaf samples in liquid nitrogen and in a fresh-keeping box, and bring them back to the laboratory and store them in a -80°C refrigerator for later use;

[0020] Step 4, Field collection and activation of Sclerotinia sclerotiorum: The strain of Sclerotinia sclerotiorum causing sclerotinia rot is the Sclerotinia sclerotiorum collected from the main rapeseed production areas. The sclerotia of Sclerotinia sclerotiorum are rinsed with sterile water and then soaked in 70% alcohol for 5 min, rinsed 2 to 3 times with sterile water, left to dry overnight on filter paper, and finally placed in a petri dish containing PDA solid medium. The petri dish is incubated upside down at 22 °C. After the mycelia grow to cover the petri dish, on the ultra-clean workbench, a sterilized inoculation loop is used to pick the mycelia at the edge of the colony and transplanted into another petri dish containing PDA solid medium for subculture. When the mycelia grow to the edge of the petri dish, inoculation on a new PDA is regarded as one generation. The inoculated Sclerotinia sclerotiorum mycelia are selected from the newly grown marginal mycelia of the third generation in the same period;

[0021] Step 5, Simulated biological fumigation: Select each sample of the above-ground tissue of the germplasm resources stored in a -80 °C refrigerator, soak it in 75% ethanol for 3 - 5 min to kill and remove the microorganisms on the leaf surface, then rinse it 5 times with sterile water to remove the residual alcohol on the leaf surface. The sample is quickly frozen and ground in liquid nitrogen, and the newly grown marginal mycelia of the third generation of Sclerotinia sclerotiorum are inoculated on one side with the substrate. 0.15 g of the powdered above-ground tissue sample of rapeseed is transferred to the side without medium in the petri dish. When adding the ground sample, the size of the sample area is proportional to the added amount. When placing, avoid contacting the medium. The area of the Sclerotinia sclerotiorum lesion is calculated using the formula S = πr 2 where r represents the radius of the lesion plaque, with the unit of cm, and π is taken as 3.14; The petri dish is sealed with a sealing film and then placed in an incubator at 22 °C for constant temperature incubation for 24 h, and then the area of the Sclerotinia sclerotiorum lesion plaque is measured. The experiment is repeated 3 times and the average value is taken;

[0022] Step 6, According to the average value of the measured area of the Sclerotinia sclerotiorum lesion plaque, select the sclerotinia rot inhibitory materials with better inhibitory effects. The inhibitory materials with an average value less than 10 cm 2 are the sclerotinia rot inhibitory materials of Brassica juncea.

[0023] To illustrate the effects of the present invention, the specific experiment is as follows:

[0024] Experimental materials: The experimental materials are 200 Brassica juncea germplasm resource materials collected by this research group from all over the country. The pathogen causing sclerotinia rot (Sclerotinia sclerotiorum) was collected and purified by this research group from the main rapeseed production areas in Guizhou.

[0025] Planting of experimental materials: 200 germplasm resources of Brassica juncea collected from all over the country for this study were planted in the teaching experimental base of Guizhou University (106°40′38.154″ E, 26°24′14.515″ N, altitude 1100 m) in October 2022. Each Brassica juncea strain was planted in a single row plot with a row length of 4 m, a row spacing of 40 cm, and a bed spacing of 50 cm. Direct seeding was used for planting. After thinning and final singling, 30 rape plants were retained in each row, and two rows of protective rows were planted around.

[0026] Sampling: When the rape was at the early flowering stage, aboveground tissue materials of 3 individual plants of each germplasm material were collected. The obtained leaf samples were stored in liquid nitrogen in a fresh-keeping box and then taken back to the laboratory and stored in a -80 °C refrigerator for later use.

[0027] Field collection and activation of Sclerotinia sclerotiorum: The sclerotinia strain used in this study was collected by the research group from the main rapeseed production areas in Guizhou. The sclerotia were rinsed with sterile water and then soaked in 70% alcohol for 5 min, rinsed 2 - 3 times with sterile water, left to dry overnight on filter paper, and finally placed on a PDA solid medium plate. The petri dish was incubated upside down at 22 °C. After the mycelia grew to cover the petri dish, on the ultra-clean workbench, a sterilized inoculation loop was used to pick the mycelia at the edge of the colony and transplanted into another petri dish containing PDA solid medium for subculture. When the mycelia grew to the edge of the petri dish, it was inoculated onto a new PDA as one generation. To ensure the viability of the inoculated mycelia, the inoculated sclerotinia mycelia were selected from the newly grown edge of the third-generation mycelia at the same time.

[0028] Simulated biological fumigation: For each germplasm resource aboveground tissue sample stored in a -80 °C refrigerator, it was soaked in 75% ethanol for 3 - 5 min to kill and remove the microorganisms on the leaf surface, then rinsed 5 times with sterile water to remove the residual alcohol on the leaf surface. The sample was quickly frozen in liquid nitrogen and ground. The newly grown edge mycelia of the third generation of Sclerotinia sclerotiorum were inoculated on one side with the substrate. 0.15 g of the ground rape aboveground tissue sample powder was transferred to the side without the culture medium in a petri dish (9 cm in diameter). To ensure the inhibitory effect, when adding the ground sample, the size of the sample area was proportional to the added amount, and contact with the culture medium was avoided during placement. The area of the Sclerotinia sclerotiorum lesion was calculated using the formula S = πr 2 (where r represents the radius of the lesion plaque in cm, and π is taken as 3.14). The petri dish was sealed with a sealing film to ensure no air circulation inside and outside, and then placed in a 22 °C incubator for constant temperature incubation for 24 h, after which the area of the Sclerotinia sclerotiorum lesion plaque ( Figure 1 ) was measured. The experiment was repeated 3 times and the mean value was taken.

[0029] Field test verification method for fumigation effect: In this study, the biofumigation performance of selected mustard lines was evaluated. In June 2023, germplasms were planted in the field in Guiding (E107°9′20.516″, N26°40′30.432″), with 10 rows for each material, 4 m in row length, and 50 plants in each row. In September 2023, the above-ground tissues of each mustard germplasm resource material were harvested at the initial flowering stage. Subsequently, in the sclerotinia sclerotiorum nursery in Guiding, the harvested tissue of each germplasm resource material was mechanically ground and applied to the soil of a separate rapeseed sclerotinia sclerotiorum nursery plot at a depth of 10 cm. At the end of September 2023, the Brassica napus variety YY50 was planted on the same plot, as well as a control plot without mustard tissue. At the seed maturity stage in May 2024, the number of plants infected with sclerotinia sclerotiorum in each plot was recorded.

[0030] Analysis of test results: Analysis of the screening results of the fumigation effect of Brassica juncea: The variation range of the lesion area of Sclerotinia sclerotiorum inhibited by 200 Brassica juncea germplasm resources was relatively large ( Figure 2 ), and the variation range was 9.92 cm 2 - 24.72 cm 2 , with an average area of 17.86 cm 2 , and a standard deviation of 17.86 cm 2 .

[0031] Analysis of the verification results of the field test on the fumigation effect: One germplasm resource material of Brassica juncea that is highly resistant (Hherucic2), moderately resistant (DafangqingYC), and resistant (ChenggaoYC) to Sclerotinia sclerotiorum was selected and planted in the Sclerotinia sclerotiorum disease nursery in Guiding County, Guizhou Province (E107°9′20.516″, N26°40′30.432″) for the verification work of the fumigation effect. In this study, the antibacterial efficacy of different biofumigation materials was systematically evaluated through field tests (Table 1). The test data showed that the Hherucic2 treatment group exhibited the strongest biofumigation activity, and its prevention and control effect was significantly better than that of other treatment groups. Specifically, among the 1497 tested plants, only 84 plants showed symptoms of Sclerotinia sclerotiorum infection, and the disease incidence rate was 5.61%. In contrast, in the DafangqingYC treatment group with moderate biofumigation efficacy, the number of infected plants among 1533 samples increased to 149, and the infection rate was 9.72%, an increase of 73.26% compared with the Hherucic2 group. In the ChenggaoYC treatment group with the lowest antibacterial activity, 211 diseased individuals were detected among 1512 samples, and the disease incidence rate increased significantly to 13.96%, an increase of 148.84% compared with the Hherucic2 group. It is worth noting that the control group without biofumigation treatment showed the highest disease susceptibility. Among 1512 samples, 262 plants were infected with Sclerotinia sclerotiorum, and the incidence rate was as high as 17.33%, an increase of 209.27% compared with the Hherucic2 group. Multiple comparisons showed that there were significant differences in the disease incidence rates among the treatment groups (p < 0.01) (Table 1).

[0032] Table 1 Results of the field test on the antibacterial effect of Sclerotinia sclerotiorum

[0033]

[0034] 1 : Different letters represent significant differences at the p = 0.01 level.

[0035] In summary, the present invention discloses a method for screening materials for inhibiting Sclerotinia sclerotiorum in Brassica juncea. By collecting above-ground tissue samples of 200 Brassica juncea germplasm materials from all over the country, after grinding them, the Sclerotinia sclerotiorum (Sclerotinia sclerotiorum) was fumigated in a sealed culture medium. After 24 hours of treatment, the antibacterial effects of different materials were measured. That is, the larger the lesion, the worse the antibacterial effect, and the smaller the lesion, the better the antibacterial effect. The growth of Sclerotinia sclerotiorum after fumigation treatment was inhibited to varying degrees, indicating that using Brassica juncea tissue materials as fumigation materials can be used to control Sclerotinia sclerotiorum. At the same time, one planting material with good, medium, and poor antibacterial effects was randomly selected (among all the screened materials, the materials with good antibacterial effects were: the average value X of the Sclerotinia sclerotiorum lesion area ≤ 11.8 cm 2, The materials with poor antibacterial effect are: the average value X of the sclerotinia disease lesion area ≥ 23.9 cm 2 , The materials with medium antibacterial effect are: 11.8 cm 2 <the average value X of the sclerotinia disease lesion area < 23.9 cm 2 ), Their field sclerotinia disease inhibitory effects were measured by planting in the sclerotinia disease nursery. The results showed that the antibacterial effects of the three materials were consistent with the screening results, verifying the accuracy of this method and proving that the screened materials can be used for the prevention and control of rapeseed sclerotinia disease.

Claims

1. A screening method for materials inhibiting Sclerotinia sclerotiorum in Brassica juncea, characterized in that, It includes the following steps: Step 1, Collect Brassica juncea germplasm resources: Collect 200 Brassica juncea germplasm resources from all over the country; Step 2, Plant Brassica juncea germplasm resources: Use 200 Brassica juncea germplasm resources and plant them in the experimental base. Each Brassica juncea strain is planted in a 1-row plot with a row length of 4 m, a row spacing of 40 cm, and a bed spacing of 50 cm. The direct seeding method is adopted. After thinning and final thinning, 30 rapeseed plants are reserved in each row, and 2 rows of protective rows are planted around; Step 3, Sampling: When the rapeseed is at the initial flowering stage, collect the above-ground tissue materials of 3 individual plants of each germplasm material. Store the obtained leaf samples in liquid nitrogen and in a fresh-keeping box, and then bring them back to the laboratory and store them in a -80°C refrigerator for later use; Step 4, Field collection and activation of Sclerotinia sclerotiorum: The sclerotinia strain of Sclerotinia sclerotiorum is collected from the main rapeseed production areas. Wash the sclerotia of Sclerotinia sclerotiorum with sterile water and soak them in 70% alcohol for 5 min, wash them 2 to 3 times with sterile water, place them on filter paper to dry overnight, and finally put them into a petri dish containing PDA solid medium. Invert the petri dish and culture it at 22°C. After the mycelia grow over the petri dish, on the ultra-clean workbench, use a sterilized inoculation loop to pick the mycelia at the edge of the colony and transplant them into another petri dish containing PDA solid medium for subculture. When the mycelia grow to the edge of the petri dish, inoculate them on the new PDA as one generation. The inoculated Sclerotinia sclerotiorum mycelia are selected from the newly grown marginal new mycelia of the third generation in the same period; Step 5: Simulate biological fumigation: Select each sample of the above-ground tissue of the germplasm resources stored in a -80°C refrigerator, soak it in 75% ethanol for 3 - 5 minutes, then rinse it 5 times with sterile water, quickly freeze and grind the sample in liquid nitrogen, and inoculate the third-generation newly emerging marginal hyphae of Sclerotinia sclerotiorum on one side with the substrate. Transfer 0.15 g of the ground powder of the above-ground tissue sample of rapeseed to the side without the culture medium in the petri dish. When adding the ground sample, the size of the sample area is proportional to the added amount. Avoid contacting the culture medium during placement. The area of the Sclerotinia sclerotiorum lesion is calculated using the formula S = πr 2 where r represents the radius of the lesion plaque, in cm, and π is taken as 3.14; Seal the petri dish with parafilm, then place it in an incubator at 22°C for constant-temperature culture for 24 hours, measure the area of the Sclerotinia sclerotiorum plaque, and take the average value after 3 replicates; Step 6. According to the average value of the measured area of the Sclerotinia sclerotiorum plaque, if the average value is less than 10 cm 2 the inhibitory material is the inhibitory material for Sclerotinia sclerotiorum of Brassica juncea.

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