Method for selecting and remaining celery verticillium wilt-resistant seed plants

Abstract

The invention discloses a method for selecting and remaining celery verticillium wilt-resistant seed plants. Celery breeding experimental materials which grow consistently are cultivated in a plug seedling mode, the accuracy of disease resistance identification results is guaranteed, and the limitation of seasonal temperature conditions is overcome; a root dipping method is adopted for inoculation, the dosage of pathogenic bacteria liquid is small, and the inoculation effect is good; tall Utah is selected as a high-sensitivity contrast variety for disease resistance identification, when the disease state reaches 3-4 levels, the disease resistance difference among the varieties is fully expressed, and the selected disease-resistant material is most accurate; the 1 / 4-1 / 3 part of the vascular tissue of the main root of the celery is longitudinally cut downwards, so that the browning of the tissue is conveniently observed to judge the illness state, and the injury degree of the incision is not fatal; root dipping is carried out by using a compound liquid medicine of Menta.fludioxonil, copper hydroxide and indatin. Naphthalene. Cyhalothrin, incision healing and new root generation are promoted, so that the selected disease-resistant seed plants are used for seed reproduction, and the disease-resistant breeding efficiency of celery can be remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant breeding, and in particular relates to a method for selecting celery strains resistant to Verticillium wilt. Background Art

[0002] Celery (Apium graveolens L. var. dulce) is a biennial vegetable of the genus Apiaceae. It is rich in nutrition and has multiple pharmacological functions. It is known as a good vegetable and medicine and is grown in all parts of my country. In the past two or three decades, celery has developed rapidly in my country, with the planting area and consumption increasing continuously. It has become one of the most important vegetable crops in my country. With the expansion of celery planting area and the increase in planting times, the damage caused by various diseases and insects caused by continuous cropping has become increasingly serious. Among them, celery wilt is one of the most serious diseases, which is very difficult to prevent and control, causing great losses to celery production in various places.

[0003] Celery Verticillium wilt is caused by Fusarium oxysporum f.sp.apii (FOA), which initially manifests as browning of the root vascular tissue, followed by gradual necrosis of the roots and rhizomes, causing the leaves above ground to be unable to obtain sufficient nutrients from the root system, resulting in stunted growth, chlorosis, and ultimately plant death. The disease was first discovered in the United States in 1909 (Ryke, 1935), and soon became the most serious disease in major celery producing areas in the United States, and gradually spread to other parts of the world (Correl et al., 1985). It was first discovered in Zhuhai, Guangdong, around 2005 in my country (Chen Yuanfeng et al., 2008), and has now spread to most of my country's celery producing areas and posed a threat. At present, four physiological subspecies of FOA have been found in various parts of the world (Epstein et al., 2017; WO 2022 / 136652 A1), among which physiological subspecies 2 (FOA-R2) is the most common and is also the main pathogen of celery wilt in my country.

[0004] In the prior art, generally, when observing celery with Verticillium wilt, the leaves are found to be yellowing after being planted in the field. After confirming that the celery is infected with Verticillium wilt, the roots are observed. However, when the leaves turn yellow, Verticillium wilt has seriously affected the normal growth of the celery plants. It is possible to observe the roots simply to identify whether the plants are disease-resistant, but it is not possible to keep them for seed. Therefore, this method is not suitable for selecting seed plants.

[0005] In addition, in the prior art, the plants investigated and studied on Verticillium wilt are all planted in the field. In this case, the plants are greatly affected by field fungi and become sick quickly. Although the identification of Verticillium wilt in diseased plants has little impact, it has a great impact on the selection of seed plants.

[0006] People have long recognized that breeding disease-resistant varieties is the most economical, effective and environmentally friendly technical approach to crop disease prevention and control. The United States began to conduct research on celery Verticillium wilt resistance breeding in the 1970s, and has bred a number of disease-resistant varieties for production, with relatively ideal results (Quiros, 1993). At present, there are no reports on celery Verticillium wilt resistance breeding and disease resistance identification in my country.

[0007] References:

[0008] 1. Ryker TC. Fusarium yellows of celery. Phytopathology, 1935, 25(5): 578-600;

[0009] 2. Correll JC, et al. Identification of Fusarium oxysporum f.sp.apii on the basis of colony size, virulence, and vegetativecompatibility. Phytopathology, 1986, 76(4): 396-400;

[0010] 3. EpsteinL,.et al.Races of the celery pathogen Fusarium oxysporumf.sp.apii are Polyphyletic.Phytopathology, 2017, 107(1): 463-473;

[0011] 4.WO 2022 / 136652 A1,2022,06,30;

[0012] 5. Chen Yuanfeng, et al. Biological characteristics of the pathogen of celery Verticillium wilt and screening of control agents. Proceedings of the 2008 Annual Academic Conference of the Chinese Society of Plant Pathology, Beijing: China Agricultural Science and Technology Press, 2008: 626-631;

[0013] 6. QuirosCF. Celery Breeding program at the department of vegetable crops, University of California, Davis. Hortscience, 1993, 28(4): 250. Summary of the invention

[0014] The purpose of the present invention is to provide a method for selecting disease-resistant seed plants for celery wilt resistance breeding, which is simple to operate, has a wide variety capacity, is accurate in selection, and has a high survival rate, and can greatly improve the breeding efficiency.

[0015] In order to achieve the above object, the technical solution of the present invention is as follows:

[0016] A method for selecting celery strains resistant to Verticillium wilt comprises the following steps:

[0017] (1) Preparation of celery materials: The highly susceptible celery variety Tall Utah was used as a control variety. All commercially available and preferred celery variety seeds participating in the identification of Verticillium wilt resistance and the selection of disease-resistant strains were sown in a flat-bottomed seedling tray filled with a sterilized seedling medium and cultivated according to conventional seedling cultivation methods. When the celery seedlings grew two true leaves, strong seedlings with basically the same growth were selected and transplanted into 50-hole plug trays respectively, and continued to be cultivated for 30-35 days. During the seedling cultivation process, corresponding control measures were taken according to the seasonal temperature characteristics to maintain the ambient temperature between 18°C ​​and 25°C to facilitate the growth and development of the seedlings. 100 plug tray seedlings were prepared for each participating variety.

[0018] (2) Preparation of inoculation solution: Severely yellowed typical Verticillium wilt susceptible plants were collected from celery production fields in advance, and the pathogens were isolated and purified from the diseased plants by tissue separation and culture. The isolated and purified pathogens were identified by microscopic morphology and molecular identification, and it was determined to be Fusarium oxysporum f.sp.apii (abbreviated as FOA). Then, the physiological subspecies identification primers were used for molecular detection, and it was further confirmed that it was the physiological subspecies 2 (FOA-R2) of FOA. The pathogen was propagated and cultured using PD liquid medium, filtered and centrifuged after 7 days, and a high-concentration FOA-R2 pathogen spore mother liquid was collected, which was then diluted with sterile water and counted using a hemocytometer. The concentration of the diluted spores was 10 7 FOA-R2 bacterial suspension with a concentration of cells / ml.

[0019] (3) Root inoculation: After 30-35 days of cultivation of celery seedlings in plug trays, a small number of weak seedlings were eliminated, and strong seedlings were selected and pulled out. The seedling medium on the root system was shaken off, and then the prepared 10 7 The roots of the seedlings should be dipped into the bacterial suspension with spores / ml. The roots should be dipped thoroughly and evenly, and the roots of the seedlings should be completely immersed in the bacterial suspension. Shake for 8-10 seconds, take out, drain the clear water, and re-implant in a 32-hole plug tray to continue culturing.

[0020] (4) Cultivation after inoculation: After inoculation, monitor the temperature and humidity of the substrate in the plug tray at any time, take corresponding measures according to changes in weather conditions, maintain a temperature above 25°C and a moist state, provide shade and cool down during high temperature periods in high temperature seasons, and use geothermal lines for automatic temperature control in low temperature seasons. Other management is carried out according to conventional methods.

[0021] (5) Disease resistance investigation and evaluation: 40-60 days after inoculation, the incidence of Verticillium wilt in the Tall Utah variety was investigated. A sharp knife was used to cut obliquely downward from one side of the root-stem junction into the 1 / 4-1 / 3 position of the main root column vascular tissue. Then the blade was adjusted to the angle and cut downward longitudinally. The severity of browning of the vascular tissue on the cut surface was observed, which was used as the grading standard for Verticillium wilt:

[0022] Grade 0, no browning symptoms;

[0023] Grade 1: brown spots are small and sporadic, accounting for less than 5% of the cut surface area;

[0024] Grade 2: brown spots occupy 5% to 50% of the cut surface tissue area and are dark brown;

[0025] Grade 3: brown spots occupy more than 50% of the cut surface tissue area, and rotten necrotic tissue appears;

[0026] Level 4: The vascular tissue of the rhizome is basically necrotic, and the plant turns yellow, dries up and dies.

[0027] The best time to conduct the survey is when the disease severity of most strains of the comparison variety Tall Utah reaches level 3-4.

[0028] The investigation is based on one variety, and the disease severity of each celery plant is investigated one by one, arranged in grades, and records are kept.

[0029] (6) Selection and propagation of disease-resistant strains: Select asymptomatic disease-resistant strains with a disease grade of 0. A maximum of 20 strains of one variety will be selected, with priority given to strains with typical characteristics. Plant them separately in a solar greenhouse for wintering and vernalization. Before planting, three commercially available agents, namely, a suspension of fine-arm pyrimidine, copper hydroxide wettable powder and indole, naphthalene and rutin root powder, are used to prepare a mixed liquid for root dipping: the suspension of fine-arm pyrimidine uses a dosage of 11% of the total effective ingredient, the dosage is 2g / L, the wettable powder of copper hydroxide uses a dosage of 77% of the effective ingredient, the dosage is 2g / L, and the indole, naphthalene and rutin root powder uses a dosage of 0.02% of potassium indole and sodium naphthaleneacetate, and 0.04% of brassinolide, the dosage is 1g / L. The purpose of root dipping is to control the infection of pathogens, promote wound healing and the generation of new roots, and is conducive to the survival of the selected seed plants after planting in the solar greenhouse. In the spring of the following year, the disease-resistant seed plants that have been overwintered in the solar greenhouse are transplanted to the seed production field, and a 100-mesh nylon net room is used to isolate the single plants before flowering, and houseflies are released in the net room to assist in pollination and seeding. Seeds are collected from individual plants after they mature in late July to early August.

[0030] (7) Verification of the effect of disease-resistant strain selection: The seeds obtained from disease-resistant strains are sown, cultured after inoculation, and evaluated for disease resistance according to the above-mentioned methods. The disease resistance performance is compared with the results of the previous round of investigation to verify the effect of disease-resistant strain selection.

[0031] Advantages of the present invention:

[0032] 1. Using a sterilized seedling medium to cultivate celery plug seedlings as experimental materials for identification of resistance to Verticillium wilt, compared with previous methods of cultivating celery seedlings in the field using soil, can not only effectively improve the uniformity of experimental materials and root soil conditions, and ensure the comparability and accuracy of the results of the Verticillium wilt resistance survey; but also the density of plug seedlings is higher than that of soil-grown seedlings, and they can be carried and easily managed manually. Because it is easier to control the temperature and humidity of plug seedlings, the test time is no longer limited by seasonal temperature changes; furthermore, plug seedlings are easy to sample and observe, and the roots are easy to clean and operate experimentally. In addition, from the perspective of the time of onset, plug seedlings have a slower onset rate than field seedlings, which gives more time to seed selection workers, and it is easier to select enough disease-resistant seed strains in one seedling cultivation.

[0033] 2. Use 10 7 Root inoculation can be carried out by dipping the roots in FOA-R2 suspension with spores / ml, which can greatly save the amount of bacterial solution while ensuring the inoculation effect and work efficiency, and the root inoculation is uniform.

[0034] 3. Select the highly susceptible variety Tall Utah as the comparison variety, and conduct disease resistance investigation and identification when the disease severity begins to become serious (level 3-4). The differences in disease resistance between different varieties and between individual plants will be fully demonstrated, which is convenient for analysis and comparison, and it is easy to accurately identify disease-resistant materials.

[0035] 4. Use a sharp knife to cut a vertical section downward at 1 / 4-1 / 3 of the main root's pith vascular tissue. This allows you to accurately judge the condition by observing the browning of the vascular tissue without causing fatal damage. After special wound healing treatment, it will easily recover and be used as a disease-resistant breeding material for the next step of breeding.

[0036] 5. Select three kinds of agents, namely, seed dressing agent, fine armor, chlorpyrifos suspension, copper hydroxide wettable powder and indole, naphthalene and lychee rooting powder, and prepare them in combination according to the experimental optimized concentration, and use them for root dipping treatment of celery seedlings with large incisions on the rhizomes. Fine armor, chlorpyrifos suspension is the best agent screened out from a variety of soil-borne fungal disease control agents in the present invention; copper hydroxide has a significant effect on preventing soft rot bacteria from infecting celery wounds; indole, naphthalene and lychee can promote wound healing and rooting at the same time. Using this compound liquid to dip the roots is the key to whether the disease-resistant strains can survive after planting. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with specific examples, and the advantages and features of the present invention will become clearer as the description proceeds. However, the specific experimental methods involved in the following examples are all conventional methods or implemented under the conditions recommended by the manufacturer's instructions unless otherwise specified.

[0038] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can all be purchased from the market.

[0039] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0040] Example

[0041] (1) Selection and sowing and seedling raising of celery varieties:

[0042] Twenty celery self-pollination materials, including Tall Utah, T01-1, K01-6, G01-11, G01-20, B04-1, R05-14, R05-20, T06-11, F07-11, A08-11, S08-15, Q09-12, L10-5, S222, S318, S405, S427, S428 and S430, were selected as experimental materials for identification of celery resistance to Verticillium wilt and selection of seed plants. Utah is a commercially available celery variety, T01-1, K01-6, G01-11, G01-20, B04-1, R05-14, R05-20, T06-11, F07-11, A08-11, S08-15, Q09-12, L10-5, S222, s318, S405, S427, S428 and S430 are commercially available celery varieties preferred offspring self-pollination materials, the public can obtain these materials from the Celery Research Group of the Vegetable Research Institute of Tianjin Academy of Agricultural Sciences for repeating the relevant experiments of the present invention. It has been verified through preliminary experiments that Tall Utah is a celery variety that is highly susceptible to Verticillium wilt.

[0043] Seeding and seedling raising: Seeds of all commercially available and preferred celery varieties participating in the identification of Verticillium wilt resistance and the selection of disease-resistant strains are sown in flat-bottomed seedling trays filled with sterilized seedling substrates and cultivated according to conventional seedling raising methods. When the celery seedlings grow two true leaves, select strong seedlings with basically the same growth momentum, transplant them into 50-hole trays, and continue to cultivate for 35 days. During the seedling raising process, take corresponding control measures according to the seasonal temperature characteristics to maintain the ambient temperature between 18°C ​​and 25°C to facilitate the growth and development of seedlings. Prepare 100 tray seedlings for each participating variety.

[0044] (2) Preparation of inoculation solution

[0045] In advance, typical Verticillium wilt susceptible plants with severe yellowing were collected from celery production fields. The pathogens of diseased plants were isolated and purified by tissue separation and culture. The isolated and purified pathogens were identified as Fusarium oxysporum f.sp.apii (abbreviated as FOA) through microscopic morphology and molecular identification. Then, the physiological race identification primers were used for molecular detection, which further confirmed that it was the physiological race 2 (FOA-R2) of FOA. The pathogen was propagated and cultured using PD liquid medium. After 7 days, it was filtered and centrifuged to collect the high-concentration FOA-R2 pathogen spore mother liquid, which was then diluted with sterile water and counted with a hemocytometer. The dilution component spore concentration was 10 7 FOA-R2 bacterial suspension with a concentration of cells / ml.

[0046] (3) Root inoculation

[0047] After 35 days of cultivation of celery seedlings in plug trays, a small number of weak seedlings were eliminated, and strong seedlings were selected and pulled out. The seedling medium on the root system was shaken off, and then the 10 prepared seedlings were used for each test variety. 7 The roots of the seedlings were dipped into the suspension of spores / ml for inoculation. The roots were required to be dipped thoroughly and evenly. The roots of the seedlings were completely immersed in the suspension and shaken for 8-10 seconds. The seedlings were taken out to drain the water and re-planted in a 32-hole tray for further cultivation. 96 strains of each test variety were inoculated.

[0048] (4) Culture after inoculation

[0049] After inoculation, monitor the temperature and humidity of the substrate in the plug tray at any time, take corresponding measures according to the weather conditions, maintain the temperature above 25℃ and keep it moist, shade and cool down during the high temperature period in the high temperature season, and use the geothermal line for automatic temperature control in the low temperature season. Other management is carried out according to the conventional methods.

[0050] (5) Disease resistance investigation and evaluation

[0051] 50 days after inoculation, we first investigated the incidence of Verticillium wilt in the Tall Utah variety. We used a sharp knife to cut obliquely downward from one side of the root-stem junction into the 1 / 4-1 / 3 position of the main root column vascular tissue, then adjusted the blade angle and cut downward longitudinally to observe the severity of browning of the vascular tissue on the cut surface, which was used as the grading standard for Verticillium wilt:

[0052] Grade 0, no browning symptoms;

[0053] Grade 1: brown spots are small and sporadic, accounting for less than 5% of the cut surface area;

[0054] Grade 2: brown spots occupy 5% to 50% of the cut surface tissue area and are dark brown;

[0055] Grade 3: brown spots occupy more than 50% of the cut surface tissue area, and rotten necrotic tissue appears;

[0056] Level 4: The vascular tissue is basically necrotic and the plant dies.

[0057] The best time to conduct the survey is when the disease severity of most strains of the comparison variety Tall Utah reaches level 3-4.

[0058] The investigation is based on one variety, and the disease status of each celery plant is investigated one by one, arranged in grades, and records are kept.

[0059] (6) Results of the disease-resistant strain survey

[0060] When the disease was severe in the high-susceptibility comparison variety Tall Utah, the 20 celery varieties tested were investigated, and the results are shown in Table 1. It can be seen that there are obvious differences in resistance to Verticillium wilt between different varieties, and there are also obvious differences in resistance to Verticillium wilt between different plants in the same variety. All the comparison varieties Tall Utah and G01-11 were infected, with more than 95% of the plants being diseased at level 2 or above, and more than 80% of the plants being seriously diseased at level 3 or above, showing overall severe susceptibility; G01-20 and A08-11 had less than 10 asymptomatic plants, and the proportion of seriously diseased plants at level 3 or above was 26-40%, showing overall severe susceptibility; T01-1 and S318 had 17 and 27 asymptomatic plants, and the proportion of seriously diseased plants at level 3 or above was 10-20%; S222 and S428 had no symptoms. There are 46 and 48 plants, and only 1 to 2 plants are seriously diseased at level 3 or above, showing strong disease resistance overall; while 12 materials including K01-6, B04-1, R05-14, R05-20, T06-11, F07-11, S08-15, Q09-12, L10-5, S405, S427, and S430 have more than 50 asymptomatic plants, and none of them are seriously diseased at level 3 or above, indicating that they have excellent disease resistance. Moreover, there are very obvious differences in the resistance of different plants of the same variety to Verticillium wilt, which makes it possible to select a certain number of disease-resistant plants from a variety group.

[0061] Table 1 Number of plants with Verticillium wilt disease in various levels among the tested celery varieties (96 plants inoculated for each variety)

[0062]

[0063] (6) Selection and propagation of disease-resistant strains

[0064] Select asymptomatic disease-resistant strains with a disease level of 0 for seed propagation. A maximum of 20 strains are selected for one variety, and strains with typical characteristics of the variety are selected first. Plant them in a solar greenhouse according to the variety for overwintering cultivation and vernalization. Before planting the strains, use three commercially available agents, namely, a suspension of 11% of total active ingredients for the suspension of 11% of the suspension of 11% of the suspension of 2g / L of copper hydroxide, a formulation of 77% of active ingredients for the suspension of 2g / L of copper hydroxide, a formulation of 1g / L of indole, naphthalene, and rutin root powder, a formulation of 0.02% of potassium indole and sodium naphthaleneacetate, and 0.04% of brassinolide, and a formulation of 1g / L of indole, naphthalene, and rutin root powder. The purpose of root dipping is to control the infection of pathogens, promote wound healing and the generation of new roots, and facilitate the survival of the selected strains after planting in the solar greenhouse. In the spring of the following year, the disease-resistant plants that have been overwintered in the solar greenhouse are transplanted to the seed production field. Before flowering, they are isolated by a 100-mesh nylon net room, and houseflies are released in the net room to assist in pollination and seed setting. After the seeds mature in early August, the seeds are collected from the individual plants.

[0065] (7) Verification of the effect of disease-resistant strain selection

[0066] A series of seeds obtained from individual disease-resistant strains, together with the original seeds of the highly susceptible comparison variety Tall Utah, were sown and raised as described above. 64 robust seedlings from each individual plant were selected for inoculation, and the disease resistance was investigated and evaluated after 50 days of cultivation. The disease resistance performance was compared with the results of the previous round of investigation to verify the effect of the selection of disease-resistant strains.

[0067] Table 2 Number of celery plants with different levels of Verticillium wilt disease in the offspring population of disease-resistant celery plants (64 plants were inoculated for each offspring population)

[0068]

[0069] From the results in Table 2, we can see that selecting disease-resistant strains from a celery variety for breeding can effectively improve its disease resistance level, with significant results.

[0070] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, other implementation methods can certainly be easily made by replacement or modification based on the technical contents disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A method for selecting celery strains resistant to Verticillium wilt, characterized in that: The method comprises the following steps: (1) Selection of celery materials and sowing and seedling raising: The highly susceptible celery variety TallUtah was used as a control variety, and seeds of all commercially available and preferred celery varieties participating in the identification of Verticillium wilt resistance and the selection of disease-resistant strains were sown in a flat-bottomed seedling tray filled with a sterilized seedling raising medium, and cultivated according to conventional seedling raising methods. When the celery seedlings grew two true leaves, the robust seedlings with basically the same growth were selected and transplanted into 50-hole trays respectively, and cultivated for another 30-35 days; (2) Preparation of inoculation solution: Severely yellowed typical Verticillium wilt susceptible plants were collected from celery production fields in advance, and the pathogen was isolated and purified from the diseased plants by tissue separation and culture. The pathogen was propagated using PD liquid medium. After 7 days, the solution was filtered and centrifuged to collect a high-concentration FOA-R2 pathogen spore mother solution, which was then diluted with sterile water and counted using a hemocytometer. The dilution yielded a spore concentration of 10 7 / ml FOA-R2 bacterial suspension; (3) Root inoculation: After 30-35 days of cultivation, select healthy seedlings and pull them out. Shake off the seedling medium on the root system. Then, dip the roots of the tested varieties into the prepared FOA-R2 bacterial suspension for inoculation. Then, replant them in 32-hole plug trays and continue to cultivate. (4) Cultivation after inoculation: After inoculation, monitor the temperature and humidity of the substrate in the plug tray at any time, take corresponding measures according to changes in weather conditions, maintain a temperature above 25°C and a moist state, provide shade and cool down during high temperature periods in high temperature seasons, and use geothermal lines for automatic temperature control in low temperature seasons. Other management is carried out according to conventional methods; (5) Disease resistance survey and evaluation: 40-60 days after inoculation, the incidence of Verticillium wilt in the Tall Utah comparison variety was first investigated. A sharp knife was used to cut obliquely downward from one side of the root-stem junction into the 1 / 4-1 / 3 position of the main root column vascular tissue. Then the blade was adjusted to make a vertical cut downward. The severity of browning of the vascular tissue on the cut surface was observed, which was used as the grading standard for Verticillium wilt. The best time to conduct the survey was when the disease severity of most of the comparison variety Tall Utah plants reached level 3-4. (6) Selection and propagation of disease-resistant seedlings: Select asymptomatic disease-resistant seedlings with a disease grade of 0 and plant them in a solar greenhouse for overwintering and vernalization according to their varieties. In the spring of the following year, transplant the disease-resistant seedlings that have overwintered and vernalized in the solar greenhouse to the seed production field. Use a 100-mesh nylon net to isolate individual plants before flowering, and release houseflies in the net to assist pollination and seed setting. Collect seeds from individual plants after the seeds mature in late July to early August. (7) Verification of the effect of disease-resistant strain selection: The seeds obtained from disease-resistant strains are sown, cultured after inoculation, and evaluated for disease resistance according to the above-mentioned methods. The disease resistance performance is compared with the results of the previous round of investigation to verify the effect of disease-resistant strain selection.

2. The method according to claim 1, characterized in that Step (1) During the seedling raising process, the ambient temperature is maintained between 18°C ​​and 25°C, and 100 plug tray seedlings are prepared for each test variety.

3. The method according to claim 1, characterized in that In step (2), the isolated and purified pathogen is identified by microscopic morphology and molecular identification to be Fusarium oxysporum f.sp.Apii, abbreviated as FOA, and then molecular detection is performed using physiological subspecies identification primers to further confirm that it is physiological subspecies No. 2 of FOA, FOA-R2.

4. The method according to claim 1, characterized in that During the step (3) of root inoculation, the roots are required to be dipped thoroughly and evenly, and the roots of the seedlings are completely immersed in the bacterial suspension. The roots are shaken for 8-10 seconds and then taken out to drain the clear water.

5. The method according to claim 1, characterized in that The Verticillium wilt grading standard in step (5) is: Grade 0, no browning symptoms; Grade 1: brown spots are small and sporadic, accounting for less than 5% of the cut surface area; Grade 2: brown spots occupy 5% to 50% of the cut surface tissue area and are dark brown; Grade 3: brown spots occupy more than 50% of the cut surface tissue area, and rotten necrotic tissue appears; Level 4: The vascular tissue of the rhizome is basically necrotic, and the plant turns yellow, dries up and dies.

6. The method according to claim 1, characterized in that Before planting the seeds, three commercially available agents, namely, a mixed solution prepared using a suspension of azopyrite, copper hydroxide wettable powder, and indole, naphthalene, and brassinolide rooting powder, were used for root dipping treatment: the suspension of azopyrite was selected in a formulation with 11% total active ingredients and a dosage of 2 g / L; the wettable powder of copper hydroxide was selected in a formulation with 77% active ingredients and a dosage of 2 g / L; and the indole, naphthalene, and brassinolide rooting powder was selected in a formulation with 0.02% potassium indole and sodium naphthylacetate and 0.04% brassinolide content, and a dosage of 1 g / L.

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