An indoor high-efficiency identification method for chinese cabbage alternaria blight induced by alternaria brassicae

Abstract

The application discloses an indoor high-efficiency identification method of Chinese cabbage black spot induced by Leptosphaeria maculans, and comprises the following steps: S1, Chinese cabbage seedling culture; S2, preparation of spore suspension; S3, inoculation of test materials; and S4, culture and investigation of the test materials. Compared with the prior art, the application has the advantages of significantly shortened identification period, realized high-efficiency screening, high controllability of environment, high repeatability of results, reduced risk of misjudgment, saved resource cost and consistent green and sustainable agricultural development direction.

Description

Technical Field

[0001] This invention relates to the field of technology, specifically to an efficient indoor identification method for black spot disease of Chinese cabbage induced by Alternaria brassicae. Background Technology

[0002] Chinese cabbage (Brassica campestris L.ssp. Pekinensis) belongs to the Brassicaceae family and is an important vegetable crop in my country with an annual output value exceeding 100 billion yuan. In the past 10 years, the annual planting area of ​​Chinese cabbage in my country has ranged from 1.733 million to 1.867 million hectares. 2 With an output of approximately 80-100 million tons, it plays a crucial role in stabilizing market supply and prices.

[0003] Black spot disease is a global disease caused by fungi of the genus *Alternaria*, primarily affecting cruciferous vegetables such as Chinese cabbage, kale, and cauliflower. It can infect vegetables throughout their entire growth cycle, spreading rapidly and causing yield losses of 20%–50% in epidemic years. With changes in cultivation practices and continuous cropping in advantageous production areas, black spot disease has become the fourth most prevalent epidemic disease in major Chinese cabbage producing regions, after soft rot, downy mildew, and viral diseases, severely hindering the development of the Chinese cabbage industry. The pathogen can reproduce and infect at temperatures ranging from 2 to 35°C, exhibiting strong environmental adaptability and surviving in the soil for 3–10 years in diseased plant debris, making control extremely difficult. Traditional chemical control methods are costly and pose problems such as environmental pollution and pesticide residues, failing to meet the demands of green agriculture. Therefore, conducting black spot disease resistance breeding work in Chinese cabbage and developing highly resistant varieties is the most fundamental and cost-effective way to solve the black spot disease problem. However, there is a lack of resources resistant to black spot disease. No materials that are completely immune to the pathogen of black spot disease have been found among the cultivated species of Brassica genus in the Brassicaceae family. It is crucial to continue to explore Chinese cabbage germplasm resources that are highly resistant to black spot disease.

[0004] A precise and stable system for identifying and evaluating resistance to black spot disease is the technical foundation for exploring and utilizing resistant germplasm resources. Although preliminary explorations of indoor resistance identification techniques for black spot disease have been conducted both domestically and internationally in recent years, they have mainly focused on *Alternaria brassicae*, while common black spot pathogens include three main categories: *Alternaria brassicae*, *Alternaria brassicicola*, and *Alternaria japonica*. Studies have shown that black spot disease in Chinese cabbage is mainly caused by *Alternaria brassicae* and *Alternaria brassicicola*, with different optimal germination temperatures for their conidia: 17–20℃ and 25–30℃, respectively. With changing consumption patterns, year-round cultivation of Chinese cabbage has increased the incidence of black spot disease caused by *Alternaria brassicae*. Therefore, exploring the inoculation age, spore concentration, and inoculation method of *Alternaria brassicae* on experimental materials, and developing an efficient inoculation and identification method for black spot disease, has significant scientific and practical application value.

[0005] Existing techniques for identifying resistance to black spot disease in Chinese cabbage suffer from several bottlenecks, including long cycles, strong environmental dependence, low inoculation efficiency, and unstable phenotypic interpretation. Traditional field inoculation at the mature plant stage requires 60-90 days to complete a full identification cycle, and is greatly affected by seasonal, climatic, and disease prevalence fluctuations. This leads to problems such as cross-infection by non-target pathogens, and interference from wind, rain, and extreme temperatures on lesion development, resulting in poor repeatability and a high risk of misdiagnosis. Furthermore, field identification requires significant land, pesticides, and irrigation facilities, resulting in high resource costs, which is inconsistent with the development of green and sustainable agriculture. Therefore, there is an urgent need to establish a standardized, reproducible, high-throughput, and efficient indoor identification method for black spot disease in Chinese cabbage induced by *Alternaria brassicae*. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an efficient indoor identification method for black spot disease of Chinese cabbage induced by Alternaria brassicae.

[0007] To solve the above-mentioned technical problems, the technical solution provided by the present invention is: an efficient indoor identification method for black spot disease of Chinese cabbage induced by *Alternaria brassicae*, comprising the following steps:

[0008] S1: Chinese cabbage seedling cultivation: Select plump seeds and disinfect them. Sow the treated seeds in seedling trays containing sterilized substrate. Manage the seedlings according to routine procedures until they have four true leaves. Cut three true leaves as inoculation materials.

[0009] S2: Preparation of spore suspension: Alternaria brassicae was inoculated onto potato dextrose agar (PDA) medium and cultured for 7 days at 25°C under 8 hours of light and 16 hours of darkness. Colonies were scraped into pure water, filtered through 5 layers of gauze, centrifuged and the supernatant was discarded. The bacterial cells were then resuspended in sterile water and the spore concentration was adjusted to a suitable value.

[0010] S3: Inoculation of test material: Place the true leaves cut in step S1 into a plastic box lined with moist filter paper, and inoculate the spore suspension prepared in step S2 using a suitable inoculation method;

[0011] S4: Test material culture survey: After inoculation, the sample was kept in the dark and moist for 1 day, then transferred to an artificial climate incubator and cultured at a constant temperature at a suitable temperature. Disease survey was conducted and the disease index (DI) was calculated. Disease resistance level was classified according to the disease index.

[0012] Furthermore, the disinfection process described in step S1 is as follows: place the seeds in warm water at 55°C, stir continuously for 15 to 30 minutes, remove them, rinse with cold water, and air dry.

[0013] Furthermore, in step S1, the seedling tray is a 50-cell tray, with one seedling left in each cell to ensure consistent growth of the test materials.

[0014] Furthermore, in step S2, the suitable spore concentration is 1×10⁻⁶. 5 Before adjusting the spore concentration by resuspending the bacterial cells, the initial spore concentration was determined using a hemocytometer. The diluent was sterile water with 2% Tween-20 added.

[0015] Furthermore, in step S3, the suitable inoculation method is to puncture the leaf epidermis with a needle and then drip the spore suspension onto the punctured area.

[0016] Furthermore, in step S4, the suitable culture temperature is 25°C, the culture equipment is an artificial climate incubator, and the lesion expansion process is observed after constant temperature culture for 3 to 5 days. The final disease investigation is completed on the 7th day.

[0017] Furthermore, in step S4, the criteria for classifying the severity of the illness are as follows:

[0018] Immune (I): Disease index = 0;

[0019] High resistance (HR): 0.1-11.1;

[0020] Disease resistance (R): 11.12-33.33;

[0021] Mid-range antibody (MR): 33.34-55.56;

[0022] High ISO (HS): 55.56-77.77;

[0023] Disease (S): >77.78.

[0024] Furthermore, the disease index (DI) is calculated using the following formula: Disease Index = [∑(Number of diseased plants at each level × Number of representatives at each level) / (Total number of plants surveyed × Highest level)] × 100. The disease survey adopts a grading standard of 0, 1, 3, 5, 7, and 9 levels, specifically:

[0025] Level 0: No obvious symptoms of infection;

[0026] Grade 1: Inoculated leaves show small brown spots, without chlorotic spots;

[0027] Grade 3: Inoculated leaves have chlorotic spots less than 3 mm in size and no mold layer;

[0028] Grade 5: Inoculated leaves develop chlorotic spots larger than 3 mm, with very little mold, and the spots do not merge into patches;

[0029] Grade 7: Inoculated leaves develop chlorotic spots larger than 5 mm, with very little mold, and the spots merge into patches;

[0030] Level 9: Inoculated leaves show patches of disease, with obvious mold.

[0031] With the above structure, the present invention has the following advantages: the identification cycle is significantly shortened, achieving efficient screening: compared with the traditional identification cycle of 60 to 90 days required for inoculation of mature plants in the field, the present invention uses the detached true leaves of Chinese cabbage seedlings at the four-leaf stage for inoculation, and only 3 to 5 days are needed in an artificial climate chamber to observe a stable and typical lesion expansion process. The final disease investigation is completed on the 7th day. The entire process from sowing to data output can be completed within 20 days, which greatly improves the screening efficiency of disease-resistant individual plants or lines in the breeding population. It is particularly suitable for large-scale germplasm resource screening, F2 large-scale population segregation analysis and gene mapping research.

[0032] The invention offers strong environmental controllability, high result repeatability, and reduced risk of misjudgment: The entire process is conducted in a sterile substrate and a constant temperature and humidity artificial climate incubator, effectively avoiding multiple interferences commonly encountered in the field, preventing cross-infection by non-target pathogens, eliminating the non-specific promoting or inhibiting effects of wind, rain, and extreme temperatures on lesion development, preventing secondary infections caused by insect vector transmission and mechanical damage, ensuring that all tested materials are in a consistent growth state, meeting the accuracy requirements for disease resistance identification in national experiments, and verifying through repeated experiments that the results of indoor inoculation identification and field phenotypic identification of black spot disease are highly consistent.

[0033] Saving resource costs and aligning with the direction of green and sustainable agricultural development: This invention adopts a tray seedling raising + detached leaf inoculation mode. Each material only requires 3 seedlings and 3 leaves to complete three biological replicates. The material consumption of a single experiment is only 1 / 10 to 1 / 20 of that of field experiments. It does not require investment in large areas of land, pesticides, and irrigation facilities, and can be carried out at any time of year, breaking seasonal restrictions. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the morphological characteristics of Alternaria brassicae, the pathogen of Alternaria.

[0035] Figure 2 This is a schematic diagram illustrating the indoor disease severity classification of black spot disease in Chinese cabbage.

[0036] Figure 3 This is a diagram illustrating the severity levels of black spot disease on mature cabbage leaves in the field.

[0037] Figure 4 This is a schematic diagram illustrating the disease incidence of artificial inoculation with antiviral materials using different inoculation methods.

[0038] Figure 5 This is a schematic diagram illustrating the disease incidence of artificial inoculation with antiviral materials using different inoculation methods.

[0039] Figure 6 This is a schematic diagram showing the disease development of artificially inoculated materials with different culture temperatures to induce disease. Detailed Implementation

[0040] The present invention will now be described in further detail with reference to the accompanying drawings.

[0041] Combined with appendix Figure 1 This invention, based on a combination of extensive preliminary experimental data and years of field observation results, clarifies and solidifies the optimal combination of artificial inoculation parameters suitable for Alternaria brassicae infecting Chinese cabbage, specifically including the following steps:

[0042] S1: Cabbage seedling cultivation

[0043] Select plump seeds for disinfection treatment. The disinfection method is as follows: place the seeds in 55℃ warm water and stir continuously for 15-30 minutes. After removal, rinse with cold water and air dry. This disinfection treatment can prevent the seeds from carrying other pathogens, avoiding affecting the health of the plants and the accuracy of disease resistance identification results. Sow the treated seeds in 50-cell trays filled with sterilized substrate, leaving one seedling per cell to ensure uniform growth of the test materials. Perform routine seedling management. When the seedlings grow to the four true leaf stage, cut three true leaves as inoculation material. Three true leaves can be used for three inoculation replicates to ensure the stability and accuracy of the experiment.

[0044] S2: Preparation of spore suspension

[0045] Alternaria brassicae was inoculated onto potato dextrose agar (PDA) medium as follows: Using a 6mm perforator, colonies of Alternaria brassicae were ablated from the edge of the PDA medium and transferred to fresh PDA medium. The medium was incubated at 25°C for 7 days under 8h light / 16h dark conditions until the colonies completely covered the PDA medium. Colonies were scraped directly into pure water using a spatula, filtered through 5 layers of gauze into 15ml centrifuge tubes, centrifuged, and the supernatant was discarded. The cells were then resuspended in sterile water. The initial spore concentration was determined using a hemocytometer, and the concentration was adjusted to an appropriate value of 1×10⁻⁶ using sterile water diluted with 2% Tween-20. 5 spores / mL. Experiments have verified that this concentration is the optimal spore concentration for Alternaria brassicae to induce black spot disease in Chinese cabbage. It can effectively distinguish between resistant and susceptible materials, and will not cause excessive yellowing or tissue rot of detached leaves of resistant materials due to excessive concentration, thus affecting the judgment of the disease.

[0046] S3: Inoculation of test materials

[0047] The true leaves cut in step S1 were placed in a plastic box lined with moist filter paper. The spore suspension prepared in step S2 was inoculated using a suitable inoculation method, which involved puncturing the leaf epidermis with a needle and then dripping the spore suspension onto the puncture site. Comparative experiments showed that this inoculation method, compared to spray inoculation and in vivo inoculation, resulted in rapid and stable disease development, high differentiation between resistance and susceptibility phenotypes, reduced the likelihood of misjudging disease severity, and was convenient, reproducible, and suitable for large-scale disease resistance screening of germplasm resources.

[0048] S4: Investigation of Experimental Material Cultivation

[0049] After inoculation, the test materials were kept in darkness and kept moist for 1 day, which could be achieved by covering them with a black plastic film. Afterward, they were transferred to an artificial climate incubator and cultured at a constant temperature of 25℃. 25℃ is the optimal culture temperature for *Alternaria brassicae* infection of Chinese cabbage. At this temperature, the lesion expansion rate of resistant materials is significantly lower than that of susceptible materials, the lesion morphology is stable, and it is easy to measure and statistically analyze. Temperatures that are too high or too low may interfere with the stable expression of the disease phenotype on detached leaves. The lesion expansion process was observed during 3–5 days of constant temperature culture. On the 7th day, a final disease survey was conducted, and the disease index (DI) was calculated. Disease resistance levels were determined based on the disease index.

[0050] The disease severity assessment used a 0, 1, 3, 5, 7, and 9-level grading system, specifically:

[0051] Level 0: No obvious symptoms of infection;

[0052] Grade 1: Inoculated leaves show small brown spots, without chlorotic spots;

[0053] Grade 3: Inoculated leaves have chlorotic spots less than 3 mm in size and no mold layer;

[0054] Grade 5: Inoculated leaves develop chlorotic spots larger than 3 mm, with very little mold, and the spots do not merge into patches;

[0055] Grade 7: Inoculated leaves develop chlorotic spots larger than 5 mm, with very little mold, and the spots merge into patches;

[0056] Level 9: Inoculated leaves show patches of disease, with obvious mold.

[0057] The disease index (DI) is calculated as follows: Disease index = [∑(number of diseased plants at each level × number of representatives at each level) / (total number of plants surveyed × highest level)] × 100.

[0058] The disease resistance level is classified according to the disease severity index as follows:

[0059] Immune (I): Disease index = 0;

[0060] High resistance (HR): 0.1-11.1;

[0061] Disease resistance (R): 11.12-33.33;

[0062] Mid-range antibody (MR): 33.34-55.56;

[0063] High ISO (HS): 55.56-77.77;

[0064] Disease (S): >77.78.

[0065] The specific embodiments of the present invention will be described in detail below:

[0066] 1. Materials and Methods

[0067] Test materials

[0068] The test materials for the inoculation identification system experiment were F904, a variety highly susceptible to black spot disease, and Qiulv 75, a resistant variety, identified by the Chinese cabbage research group of the Vegetable Research Institute of Tianjin Academy of Agricultural Sciences after years of field investigation. The test materials for germplasm resource identification and evaluation were the backbone germplasm resources of Chinese cabbage selected and preserved by the Chinese cabbage research group of the Vegetable Research Institute of Tianjin Academy of Agricultural Sciences over many years (Table 1). The test strain was Alternaria brassicae strain ACCC37296 provided by the Agricultural Microbiology Center of the China Association for the Preservation and Management of Microbial Culture Collections. Figure 1 ).

[0069] Table 1. Germplasm resources of 53 Chinese cabbage backbone lines tested

[0070]

[0071] 1.2 Test Methods

[0072] 1.2.1 Cultivation of Chinese cabbage seedlings

[0073] Select plump seeds, disinfect them, and sow them in 50-cell trays filled with sterilized substrate. The seedlings were managed in the glass greenhouse of the Wuqing Base of the Vegetable Research Institute of Tianjin Academy of Agricultural Sciences. When the seedlings grew to the four true leaf stage, an exploratory test of the artificial inoculation identification system for black spot disease in Chinese cabbage seedlings was conducted.

[0074] 1.2.2 Preparation of spore suspension

[0075] Alternaria brassicae colonies grown on potato dextrose agar (PDA) were ablated at the edges using a 6mm perforator and transferred to fresh PDA medium. The colonies were cultured for 7 days at 25°C with 8 hours of light followed by 16 hours of darkness. Once the PDA medium was fully covered, colonies were scraped directly into pure water using a spatula, filtered through five layers of gauze, and transferred to 15ml centrifuge tubes. After centrifugation, the supernatant was discarded, and the cells were resuspended in sterile water. The concentration of the spore suspension was measured using a hemocytometer, and the concentration was diluted to the experimentally determined concentration with sterile water (with 2% Tween-20 added).

[0076] 1.2.3 Effects of different inoculation methods on the disease index of black spot disease resistant materials

[0077] With 1×10 5The resistant materials were inoculated with a spore suspension concentration of spores / mL. Four inoculation methods were used to treat the experimental materials: seedling spray inoculation, seedling drip inoculation, detached leaf spray inoculation, and detached leaf drip inoculation. The resistant materials were sown in the same 50-cell tray, with 25 cells per material and 1 seedling per cell. The results were repeated 3 times. After inoculation, the materials were kept in the dark and moist for 1 day, covered with black plastic film, and cultured at 25°C in an artificial climate culture room. Normal seedling management was implemented. Disease incidence was investigated and statistically analyzed on days 3 and 7, and the disease index (DI) was calculated.

[0078] 1.2.4 Effect of different bacterial inoculation concentrations on the disease index of black spot disease resistant materials

[0079] Sterile water was used as a negative control, 1×10 3 cells / mL, 1×10 4 cells / mL, 1×10 5 cells / mL, 1×10 6 Different spore suspensions at different inoculation concentrations per mL were used as experimental groups. The resistant and susceptible materials were inoculated using the in vitro leaf drip method. The resistant and susceptible materials were sown in the same 50-well tray, with 25 cells per material treatment and 1 seedling per cell. The inoculation was repeated 3 times. After inoculation, the materials were kept in the dark and moist for 1 day, covered with black plastic film, and cultured at 25°C in an artificial climate incubator. Normal seedling management was implemented, and dynamic records and observations were recorded every 1 day. On the 3rd day, the disease situation was investigated and the disease index (DI) was calculated.

[0080] 1.2.5 Effect of different culture temperatures on the disease index of black spot disease resistant materials

[0081] With 1×10 5 The resistant and susceptible materials were inoculated with a spore suspension concentration of spores / mL. The test materials were treated using the in vitro leaf drop method. The resistant and susceptible materials were sown in the same 50-well tray, with 25 cells per material and 1 seedling per cell. The results were repeated 3 times. After inoculation, the materials were kept in the dark and moist for 1 day, covered with black plastic film, and cultured in an artificial climate incubator at 10℃, 15℃, 25℃, and 35℃. The seedlings were managed normally, and dynamic observations were conducted every 1 day. On the 7th day, the disease index (DI) was calculated.

[0082] The severity grading of black spot disease in Chinese cabbage is based on Wang Fengmin's method from 2007, divided into grades 0, 1, 3, 5, 7, and 9. Figure 2 Grade 0: No obvious infection symptoms; Grade 1: Small brown spots on inoculated leaves, no chlorotic spots; Grade 3: Chlorotic spots less than 3 mm on inoculated leaves, no mold layer; Grade 5: Chlorotic spots greater than 3 mm on inoculated leaves, with very little mold layer, the spots do not merge into patches; Grade 7: Chlorotic spots greater than 5 mm on inoculated leaves, with very little mold layer, the spots merge into patches; Grade 9: Patches of disease merge on inoculated leaves, mold layer is obvious.

[0083] The disease index is calculated using the following formula:

[0084] Disease index = [∑(number of diseased plants at each level × number of representatives at each level) / (total number of plants surveyed × highest level)] × 100

[0085] The disease resistance level classification criteria based on the disease severity index are shown in Table 2 below.

[0086] Table 2. Disease Index and Resistance Level Standards for Black Spot Disease in Chinese Cabbage

[0087]

[0088] 1.2.6 Evaluation of Black Spot Disease Resistance of Chinese Cabbage Backbone Line Germplasm Resources

[0089] Fifty-three Chinese cabbage backbone germplasm resources were selected and disinfected according to the method in 1.2.1. They were then sown in 72-cell trays in an artificial climate chamber, with 111 seedlings per material. When the Chinese cabbage seedlings grew to the stage of four true leaves, leaves from 75 plants of the same size from each material were selected for germplasm resource identification and evaluation using the established artificial inoculation disease resistance identification system. After inoculation, the plants were kept in darkness and moist for 1 day, covered with black plastic film, and managed normally during the seedling stage. Disease surveys and disease index calculations were carried out on the 3rd day of cultivation.

[0090] In addition, 36 seedlings from each material were selected and planted in the field for natural inoculation. Disease resistance at the mature stage was investigated for the 53 Chinese cabbage germplasm resources, and the disease index was calculated.

[0091] The disease severity classification for mature plants was based on the area method, with disease severity assessed on a scale of 0, 1, 3, 5, and 7 according to leaf symptoms. Figure 3 Grade 0: No obvious infection symptoms; Grade 1: Lesions cover less than 5% of the total leaf area; Grade 3: Lesions cover less than 6%-10% of the total leaf area; Grade 5: Lesions cover less than 11%-50% of the total leaf area; Grade 7: Lesions cover more than 51% of the total leaf area. The disease index calculation formula is the same as that for the seedling stage.

[0092] 1.3 Data Analysis

[0093] The data were organized using Excel software, and the significance of the differences was analyzed using Duncan's new multiple range method in SPSS 20.0 software.

[0094] 2 Results and Analysis

[0095] 2.1 Effects of different inoculation methods on the disease index of black spot disease resistant materials

[0096] To investigate the disease incidence of HB4 and HB7 tested materials, surveys were conducted on detached leaves on day 3 after inoculation, and on in vivo plants on days 3 and 7 after inoculation. Disease indices were statistically calculated. Results were found (Table 3). Figure 4In contrast, inoculation of detached leaves with plants resulted in rapid disease development, with stable and clear resistance phenotypes appearing as early as day 3. In contrast, inoculation of plants resulted in slower disease development, with smaller lesions and less pronounced differences in resistance between day 3. By day 7, the disease index stabilized and a clear resistance phenotype emerged. Compared to drip inoculation, spray inoculation resulted in more lesions, and the convergence of adjacent lesions could lead to misjudgments of disease severity. Considering ease of operation and repeatability, drip inoculation of detached leaves is more suitable for large-scale disease resistance screening of germplasm resources.

[0097]

[0098] Figure 4 The left side shows disease-resistant material, and the right side shows disease-susceptible material; A: Spraying method on the 3rd day of plant growth; B: Spraying method on the 7th day of plant growth; C: Drip grafting method on the 3rd day of plant growth; D: Drip grafting method on the 7th day of plant growth; E: Spraying method on detached leaves on the 3rd day of plant growth; F: Drip grafting method on detached leaves on the 3rd day of plant growth.

[0099] 2.2 Effects of different bacterial inoculation concentrations on the disease index of black spot disease resistant materials

[0100] On day 3 after inoculation using the detached leaf drip method, the disease incidence of HB4 and HB7 samples was investigated, and the disease index was calculated. The results showed (Table 4, ...). Figure 5 When the bacterial concentration is 1×10 3 At a spore concentration of 1 × 10⁶ / mL, no lesions appeared in either resistant or susceptible material. As the bacterial concentration increased, the disease index of both materials increased accordingly. 5 At a spore concentration of 1 × 10⁻⁶ / mL, the disease index of resistant material reached 5.8, classifying it as HR, while the disease index of susceptible material HB7 reached 92.9, classifying it as HS, showing a significant difference. As the bacterial concentration increased, when the bacterial concentration was 1 × 10⁻⁶ / mL... 6 At a spore concentration of 1 × 10⁶ / mL, the disease index increases accordingly. However, excessively high concentrations lead to excessive selection pressure, exacerbating yellowing of detached leaves from resistant materials and accelerating tissue decay in detached leaves from susceptible materials, thus affecting disease assessment. Therefore, a suitable inoculation concentration of 1 × 10⁶ / mL is recommended. 5 At a spore / mL level, it can effectively distinguish resistant and susceptible materials without affecting the normal development of the disease.

[0101]

[0102] Note: Different lowercase letters after the data in the table indicate significant differences (P < 0.05).

[0103] Note: Figure 5 The left side contains disease-resistant materials, and the right side contains disease-susceptible materials; A:10 3 Spore / mL inoculation concentration; B: 10 4 Spore / mL inoculation concentration; C: 10 5Spore / mL inoculation concentration; D: 10 6 Spore / mL inoculation concentration;

[0104] 2.3 Effect of different culture temperatures on the disease index of black spot disease resistant materials

[0105] With 1×10 5 The bacterial suspension with spores / mL was inoculated using the in vitro leaf drop method. After inoculation, the test materials were placed at 10℃, 15℃, 25℃, and 35℃. Disease incidence of HB4 and HB7 was investigated on the 3rd day. Results showed (Table 5, ...). Figure 6 At 10℃, the disease index of the resistant material HB4 was 0.7, with a disease grade of HR, while the disease index of the susceptible material HB7 was 12.9, with a disease grade of R. As the temperature increased, the disease indices of both materials rose. At 25℃, the disease index of HB4 increased to 5.6, and the disease index of HB7 reached 92.0, indicating a significant difference in resistance and susceptibility between the two materials. When cultured at 35℃, both materials exhibited varying degrees of transpiration and decay in their detached leaves, making accurate disease grade determination difficult. This demonstrates that temperature significantly affects the development of disease caused by *Alternaria brassicae*, with 25℃ being the optimal temperature for effectively distinguishing between resistant and susceptible materials. Dynamic monitoring of lesion expansion in resistant and susceptible materials revealed that at 25℃, the lesion expansion rate in resistant materials was significantly lower than that in susceptible materials, and the lesion morphology was stable, making measurement and statistical analysis easier. Excessively high or low temperatures may interfere with the stable expression of the disease phenotype in detached leaves. Considering the practical situation, 25℃ should be selected as the standard culture temperature for subsequent experiments to ensure the accuracy and reproducibility of the inoculation results.

[0106] Table 5. Effects of different inoculation temperatures on black spot disease inoculation.

[0107]

[0108] Note: Different lowercase letters after the data in the table indicate significant differences (P < 0.05).

[0109] Note: Figure 6 The material on the left is resistant, and the material on the right is susceptible; A: 10℃ incubation temperature; B: 15℃ incubation temperature; C: 25℃ incubation temperature; D: 35℃ incubation temperature;

[0110] 2.4 Identification and Evaluation of Black Spot Disease Resistance of Chinese Cabbage Backbone Line Germplasm Resources

[0111] Table 6. Identification of black spot disease resistance in Chinese cabbage germplasm resources.

[0112]

[0113] In further verification experiments, optimized inoculation methods, inoculation concentrations, and culture temperature conditions were used to repeatedly test the disease incidence of 53 Chinese cabbage backbone germplasm materials. The results showed a high degree of consistency between indoor inoculation identification and field phenotypic identification of black spot disease, with the disease index fluctuation range controlled within 12%, demonstrating the good accuracy and stability of the identification system.

[0114] Combining the results of indoor artificial inoculation and field identification (Table 6), the resistance of 53 backbone germplasm materials to black spot disease was identified using Alternaria brassicae. Two highly resistant materials and 11 highly susceptible materials were selected, which accumulated an important material foundation for breeding Chinese cabbage resistant to black spot disease.

[0115] The present invention and its embodiments have been described above. This description is not restrictive, and the actual structure is not limited thereto. In conclusion, if those skilled in the art, inspired by this description, design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the scope of protection of this invention.

Claims

1. A highly efficient indoor identification method for black spot disease of Chinese cabbage induced by *Alternaria brassicae*, characterized in that: Includes the following steps: S1: Chinese cabbage seedling cultivation: Select plump seeds and disinfect them. Sow the treated seeds in seedling trays containing sterilized substrate. Manage the seedlings according to routine procedures until they have four true leaves. Cut three true leaves as inoculation materials. S2: Preparation of spore suspension: Alternaria brassicae was inoculated onto potato dextrose agar (PDA) medium and cultured for 7 days at 25°C under 8 hours of light and 16 hours of darkness. Colonies were scraped into pure water, filtered through 5 layers of gauze, centrifuged and the supernatant was discarded. The bacterial cells were then resuspended in sterile water and the spore concentration was adjusted to a suitable value. S3: Inoculation of test material: Place the true leaves cut in step S1 into a plastic box lined with moist filter paper, and inoculate the spore suspension prepared in step S2 using a suitable inoculation method; S4: Test material culture survey: After inoculation, the sample was kept in the dark and moist for 1 day, then transferred to an artificial climate incubator and cultured at a constant temperature at a suitable temperature. Disease survey was conducted and the disease index (DI) was calculated. Disease resistance level was classified according to the disease index.

2. The method for high-efficiency indoor identification of black spot disease in Chinese cabbage induced by *Alternaria brassicae* according to claim 1, characterized in that: The disinfection process described in step S1 is as follows: place the seeds in warm water at 55°C, stir continuously for 15 to 30 minutes, remove them, rinse with cold water, and air dry.

3. The method for high-efficiency indoor identification of black spot disease in Chinese cabbage induced by *Alternaria brassicae* according to claim 1, characterized in that: In step S1, the seedling tray is a 50-cell tray, with one seedling left in each cell to ensure that the growth vigor of the test materials is consistent.

4. The method for high-efficiency indoor identification of black spot disease in Chinese cabbage induced by *Alternaria brassicae* according to claim 1, characterized in that: In step S2, the suitable spore concentration is 1×10⁻⁶. 5 The spore concentration was adjusted by resuspending the bacterial cells, and the initial spore concentration was detected by a hemocytometer. The diluent was sterile water with 2% Tween-20 added.

5. The method for high-efficiency indoor identification of black spot disease in Chinese cabbage induced by *Alternaria brassicae* according to claim 1, characterized in that: In step S3, the appropriate inoculation method is to puncture the leaf epidermis with a needle and then drip the spore suspension onto the punctured area.

6. The method for high-efficiency indoor identification of black spot disease in Chinese cabbage induced by *Alternaria brassicae* according to claim 1, characterized in that: In step S4, the suitable culture temperature is 25℃, the culture equipment is an artificial climate incubator, and the lesion expansion process is observed after constant temperature culture for 3 to 5 days. The final disease investigation is completed on the 7th day.

7. The method for high-efficiency indoor identification of black spot disease in Chinese cabbage induced by *Alternaria brassicae* according to claim 6, characterized in that: In step S4, the criteria for classifying the severity of the illness are as follows: Immune (I): Disease index = 0; High resistance (HR): 0.1-11.1; Disease resistance (R): 11.12-33.33; Mid-range antibody (MR): 33.34-55.56; High ISO (HS): 55.56-77.77; Disease (S): >77.

78.

8. The method for high-efficiency indoor identification of black spot disease in Chinese cabbage induced by *Alternaria brassicae* according to claim 7, characterized in that: The disease severity index (DI) is calculated using the following formula: Disease Severity Index = [∑(Number of diseased plants at each level × Number of representatives at each level) / (Total number of plants surveyed × Highest level)] × 100. The disease severity survey uses a 0, 1, 3, 5, 7, and 9-level grading standard, specifically: Level 1: No obvious symptoms of infection; Grade: Inoculated leaves have small brown spots, but no chlorotic spots; Grade: Inoculated leaves have chlorotic spots less than 3 mm in size and no mold layer; Grade 1: Inoculated leaves develop chlorotic spots larger than 3 mm, with very little mold, and the spots do not merge into patches; Grade 1: Inoculated leaves develop chlorotic spots larger than 5 mm, with very little mold, and the spots merge into patches; Grade 1: Inoculated leaves show patches of diseased spots that merge together, with a distinct mold layer.

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