Method for efficiently screening tomato fusarium wilt resistant variety

Abstract

The invention discloses a method for efficiently screening tomato fusarium wilt resistant varieties. Relates to the technical field of vegetable disease-resistant breeding. Comprising the following steps: a, preparing a culture medium; b, sterilizing the culture medium and test equipment; c, disinfecting the tomato seeds; d, placing the disinfected tomato seeds on a culture medium, and culturing; e, activating tomato fusarium oxysporum on a potato dextrose agar culture medium; f, inoculating fusarium oxysporum; and g, after co-culture, identifying the fusarium wilt resistant tomato rootstock germplasm material by investigating disease indexes. The method can replace a tomato variety living body screening test, and screening of tomato fusarium wilt resistant germplasm materials can be completed in the tissue culture process (bottle). The screening time of the tomato fusarium wilt-resistant rootstock is greatly shortened, the test intensity of scientific researchers is reduced, meanwhile, the interference of an external bad environment is overcome, and the accuracy of screening the tomato fusarium wilt-resistant rootstock is improved.

Description

Technical Field

[0001] This invention relates to the field of vegetable disease resistance breeding technology, and more specifically to a method for efficiently screening tomato wilt resistant varieties. Background Technology

[0002] Fusarium wilt is a persistent soil-borne fungal disease caused by Fusarium oxysporum f.sp. Lycopersici (FOL). This pathogen can survive for many years in the soil and in diseased plant debris, and is highly contagious. Therefore, how to control Fusarium wilt has become a global focus.

[0003] Breeding varieties highly resistant to Fusarium wilt has become one of the fundamental ways to solve the Fusarium wilt disease in tomatoes.

[0004] Currently, the conventional screening method for tomato rootstocks resistant to Fusarium wilt is mainly the pathogenicity test of live seedlings. This method primarily utilizes tomato plants and requires the plants to reach a certain size before it can be implemented. However, when inoculating the roots of tomato rootstocks with Fusarium wilt, it is difficult to rule out interference from adverse environmental conditions or contaminating bacteria in the soil / substrate that could affect the growth of the seedlings. Therefore, this method has a certain degree of inaccuracy. To rapidly cultivate Fusarium wilt-resistant tomato rootstocks, there is an urgent need for a fast, economical, convenient, and reliable method to solve this technical challenge.

[0005] Therefore, whether an efficient method can be provided for screening tomato wilt-resistant varieties to overcome the above-mentioned technical deficiencies is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a method for efficiently screening tomato varieties resistant to Fusarium wilt. This method involves inoculating the plant with Fusarium wilt pathogen during tissue culture (using tools such as tissue culture bottles), thereby screening for a rapid, economical, convenient, and reliable method for selecting Fusarium wilt-resistant tomato rootstocks.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for efficiently screening tomato varieties resistant to Fusarium wilt includes the following steps:

[0009] a. Prepare the culture medium;

[0010] b. Sterilized culture media and experimental equipment;

[0011] c. Disinfect tomato seeds;

[0012] d. Place the sterilized tomato seeds on a culture medium and incubate them;

[0013] e. Activate the tomato wilt pathogen on potato dextrose agar medium;

[0014] f. Inoculate with Fusarium wilt pathogens;

[0015] g. After co-cultivation, tomato rootstock germplasm materials resistant to Fusarium wilt were identified by investigating the disease index.

[0016] Preferred: Step a culture medium: 1 / 2 MS medium and potato dextrose agar (PDA); wherein, 1 / 2 MS medium contains: potassium nitrate 950 mg, potassium dihydrogen phosphate 85 mg, ammonium nitrate 825 mg, magnesium sulfate 185 mg, calcium chloride 220 mg, boric acid 6.2 mg, potassium iodide 0.83 mg, manganese sulfate 22.3 mg, zinc sulfate 8.6 mg, sodium molybdate 0.25 mg, cobalt chloride 0.025 mg, copper sulfate 0.025 mg, ferrous sulfate 27.8 mg, inositol 100 mg, glycine 2 mg, thiamine hydrochloride 0.1 mg, nicotinic acid 0.5 mg, pyridoxine hydrochloride 0.5 mg, sucrose 30,000 mg, agar 7,000 mg, and disodium EDTA 37.3 mg.

[0017] Potato glucose agar medium: 200g potato, 20g glucose, 18g agar, 1000mL water.

[0018] Preferred method: Step b: Sterilize culture medium and test equipment: Sterilize culture medium and test equipment at 121℃ for 30 min.

[0019] Preferred method: Step c, disinfecting tomato seeds, specifically involves: placing tomato seeds in a 15mL centrifuge tube, adding 75% alcohol, shaking for 30 seconds, rinsing with sterile water, adding 5% sodium hypochlorite and 0.1% Tween, shaking in the dark for 15 minutes, and then rinsing with sterile water 3 times, 1 minute each time.

[0020] Preferred method: Step d specifically involves using tweezers to pick up the sterilized tomato seeds onto 1 / 2 MS medium and culturing them for 3 to 5 days at 25–28°C and 12 h light / d.

[0021] Preferred: Step e is the step of activating Fusarium wilt: Fusarium wilt is cultured on potato dextrose agar medium. After the Fusarium wilt has filled the medium, a 5 mm diameter Fusarium wilt mycelium cake is used for inoculation in step f.

[0022] Preferred method: Step f: The inoculation environment is: in a tissue culture bottle.

[0023] Preferred method: Step g co-cultivation: 7 days under cultivation conditions of 25-28℃ and 12h light / d;

[0024] The disease severity index was specifically determined according to the disease severity grading standards: Grade 0: No symptoms; Grade 1: Slight browning of the roots; Grade 2: Obvious browning of the stem base; Grade 3: Wilting of the stem base, causing the plant to collapse; Grade 4: Severe wilting of the entire plant, leading to death.

[0025] Formula for calculating the disease index:

[0026]

[0027] Based on the criteria for classifying disease resistance in the population: High resistance: 0 < disease index < 5; Disease resistance: 5 < disease index < 20; Disease tolerance: 20 < disease index < 40; Disease susceptibility: disease index > 40, tomato rootstock germplasm materials resistant to tomato wilt were identified.

[0028] The present invention also provides the application of any of the above methods in agricultural production.

[0029] Preferred: For tomato breeding or germplasm.

[0030] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a method for efficiently screening tomato wilt-resistant varieties, and the technical effects achieved are as follows:

[0031] 1. By directly using seed identification, the resistance of tomato germplasm to Fusarium wilt can be identified during tissue culture. Compared with conventional identification, this saves time and effort by eliminating the time spent cultivating tomato plants.

[0032] 2. This allows for the identification of tomato resistance to Fusarium wilt without being limited by the growing season, enabling identification work to be carried out in any season. Furthermore, it allows for the screening of a large number of germplasm resources in a short period of time.

[0033] 3. It is not affected by other bacteria, nor will it produce the same phenomenon as the root irrigation inoculation method where inconsistent inoculation intensity occurs due to external environmental factors, thus causing identification errors.

[0034] 4. This method is consistent with the resistance performance of known resistant rootstocks to tomato wilt in the field. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0036] Figure 1 The attached figure shows the experimental results of the screening method provided by the present invention, including CFH-27 and TMS results.

[0037] Figure 2 The attached figure shows the experimental results of the screening method provided by the present invention, including Provence and MM results.

[0038] Figure 3 The attached figure shows the experimental results of the screening method provided by the present invention, specifically the results for CFH-11 and CFH-25.

[0039] Figure 4 The attached figure shows the experimental results of the screening method provided by the present invention, including CFH26 and the positive results. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] This invention discloses a method for efficiently screening tomato varieties resistant to Fusarium wilt.

[0042] All raw materials and equipment not mentioned in the examples are commercially available, and all unmentioned operating procedures are standard steps and will not be described in detail.

[0043] Example 1

[0044] A method for efficiently screening tomato varieties resistant to Fusarium wilt includes the following steps:

[0045] a. Prepare the culture medium;

[0046] b. Sterilized culture media and experimental equipment;

[0047] c. Disinfect tomato seeds;

[0048] d. Place the sterilized tomato seeds on a culture medium and incubate them;

[0049] e. Activate the tomato wilt pathogen on potato dextrose agar medium;

[0050] f. Inoculate with Fusarium wilt pathogens;

[0051] g. After co-cultivation, tomato rootstock germplasm materials resistant to Fusarium wilt were identified by investigating the disease index.

[0052] To further optimize the technical solution:

[0053] Step a: Culture media: 1 / 2 MS medium and potato dextrose agar (PDA); wherein, 1 / 2 MS medium contains: potassium nitrate 950 mg, potassium dihydrogen phosphate 85 mg, ammonium nitrate 825 mg, magnesium sulfate 185 mg, calcium chloride 220 mg, boric acid 6.2 mg, potassium iodide 0.83 mg, manganese sulfate 22.3 mg, zinc sulfate 8.6 mg, sodium molybdate 0.25 mg, cobalt chloride 0.025 mg, copper sulfate 0.025 mg, ferrous sulfate 27.8 mg, inositol 100 mg, glycine 2 mg, thiamine hydrochloride 0.1 mg, nicotinic acid 0.5 mg, pyridoxine hydrochloride 0.5 mg, sucrose 30,000 mg, agar 7,000 mg, and disodium EDTA 37.3 mg.

[0054] Potato glucose agar medium: 200g potato, 20g glucose, 18g agar, 1000mL water.

[0055] Step b: Sterilize the culture medium and test equipment: Sterilize the culture medium and test equipment at 121℃ for 30 minutes.

[0056] Step c, disinfecting tomato seeds, involves placing the tomato seeds in a 15mL centrifuge tube, adding 75% alcohol, shaking for 30 seconds, rinsing with sterile water, adding 5% sodium hypochlorite and 0.1% Tween, shaking in the dark for 15 minutes, and then rinsing three times with sterile water for 1 minute each time.

[0057] Step d specifically involves using tweezers to pick up the sterilized tomato seeds and place them on 1 / 2 MS medium (with the lid on), and then culturing them at 25–28°C under 12h light / d conditions for 3–5 days.

[0058] Step e: Activation of Fusarium wilt pathogen: Culture Fusarium wilt pathogen on potato dextrose agar medium. After the Fusarium wilt pathogen has fully grown on the medium, take a 5 mm diameter Fusarium wilt fungal cake for inoculation in step f.

[0059] Step f: The inoculation environment is: in a tissue culture bottle.

[0060] Step g: Co-culture: 7 days under conditions of 25-28℃ and 12h light / d;

[0061] The disease severity index was specifically determined according to the disease severity grading standards: Grade 0: No symptoms; Grade 1: Slight browning of the roots; Grade 2: Obvious browning of the stem base; Grade 3: Wilting of the stem base, causing the plant to collapse; Grade 4: Severe wilting of the entire plant, leading to death.

[0062] Formula for calculating the disease index:

[0063]

[0064] Based on the criteria for classifying disease resistance in the population: High resistance (HR): 0 < disease index < 5; Disease resistance (R): 5 < disease index < 20; Disease tolerance (T): 20 < disease index < 40; Disease susceptibility (S): disease index > 40, tomato rootstock germplasm materials resistant to tomato wilt were identified.

[0065] The identification results are shown in Table 1 below:

[0066] Table 1 Disease index and disease resistance evaluation of different tomato varieties

[0067] variety Disease index Disease resistance CFH-27 1.67 High resistance TMS150(TMS) 6.25 Disease resistance Provence 70.90 Illness MoneyMaker (MM) 72.32 Illness CFH-11 35.71 Disease resistant CFH-25 34.38 Disease resistant CFH-26 25.00 Disease resistant good 45.00 Illness

[0068] Table 1 shows that CFH-27 and TMS150 are disease-resistant varieties.

[0069] In summary, this invention directly utilizes seed identification, enabling the identification of tomato wilt-resistant rootstocks during tissue culture. Compared to conventional resistant variety screening tests, it eliminates the tedious, time-consuming, and labor-intensive process of cultivating tomato plants. Typically, resistant variety screening tests require large-scale laboratory facilities and experimental sites. This invention's identification of wilt-resistant tomato varieties is not limited by the growing season or location. Furthermore, it allows for the screening of a large number of resistant germplasm resources in a short time. It is not affected by other contaminating fungi and avoids the inconsistencies in inoculation intensity caused by fluctuations in external environmental factors, as seen in root drenching inoculation methods, which can lead to identification errors. The resistance performance of this method is consistent with that of known resistant and susceptible varieties in the field against tomato wilt.

[0070] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0071] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for efficiently screening tomato varieties resistant to Fusarium wilt, characterized in that, Includes the following steps: a. Prepare the culture medium; b. Sterilized culture media and experimental equipment; c. Disinfect tomato seeds; d. Place the sterilized tomato seeds on a culture medium and incubate them; e. Activate the tomato wilt pathogen on potato dextrose agar medium; f. Inoculate with Fusarium wilt pathogens; g. After co-cultivation, tomato rootstock germplasm materials resistant to Fusarium wilt were identified by investigating the disease index.

2. The method as described in claim 1, characterized in that, Step a: Culture media: 1 / 2 MS medium and potato dextrose agar (PDA); wherein, 1 / 2 MS medium contains: potassium nitrate 950 mg, potassium dihydrogen phosphate 85 mg, ammonium nitrate 825 mg, magnesium sulfate 185 mg, calcium chloride 220 mg, boric acid 6.2 mg, potassium iodide 0.83 mg, manganese sulfate 22.3 mg, zinc sulfate 8.6 mg, sodium molybdate 0.25 mg, cobalt chloride 0.025 mg, copper sulfate 0.025 mg, ferrous sulfate 27.8 mg, inositol 100 mg, glycine 2 mg, thiamine hydrochloride 0.1 mg, nicotinic acid 0.5 mg, pyridoxine hydrochloride 0.5 mg, sucrose 30,000 mg, agar 7,000 mg, and disodium EDTA 37.3 mg. Potato glucose agar medium: 200g potato, 20g glucose, 18g agar, 1000mL water.

3. The method as described in claim 2, characterized in that, Step b: Sterilize the culture medium and test equipment: Sterilize the culture medium and test equipment at 121℃ for 30 minutes.

4. The method as described in claim 3, characterized in that, Step c, disinfecting tomato seeds, involves placing the tomato seeds in a 15mL centrifuge tube, adding 75% alcohol, shaking for 30 seconds, rinsing with sterile water, adding 5% sodium hypochlorite and 0.1% Tween, shaking in the dark for 15 minutes, and then rinsing three times with sterile water for 1 minute each time.

5. The method as described in claim 4, characterized in that, Step d specifically involves using tweezers to pick up the sterilized tomato seeds and place them on 1 / 2 MS medium. The seeds are then cultured at 25–28°C under 12 h light / d conditions for 3–5 days.

6. The method as described in claim 5, characterized in that, Step e: Activation of Fusarium wilt pathogen: Culture Fusarium wilt pathogen on potato dextrose agar medium. After the Fusarium wilt pathogen has fully grown on the medium, take a 5 mm diameter Fusarium wilt fungal cake for inoculation in step f.

7. The method as described in claim 6, characterized in that, Step f: The inoculation environment is: in a tissue culture bottle.

8. The method as described in claim 7, characterized in that, Step g: Co-culture: 7 days under conditions of 25-28℃ and 12h light / d; The specific criteria for assessing the severity of illness are as follows: According to the severity grading standards: Level 0: Asymptomatic; Grade 1: Slight browning at the roots; Grade 2: Obvious browning at the base of the stem; Grade 3: Wilting at the base of the stem, causing the plant to collapse; Grade 4: Severe wilting of the entire plant, leading to death. Formula for calculating the disease index: According to the criteria for classifying population disease resistance: High resistance: 0 < disease index < 5; Disease resistance: 5 < disease index < 20; Disease tolerance: 20 < disease index < 40; Disease susceptibility: Disease index > 40, indicating the identification of tomato rootstock germplasm materials resistant to tomato wilt.

9. The application of the method according to any one of claims 1 to 8 in agricultural production.

10. The application as described in claim 9, characterized in that, Used for tomato breeding or germplasm.

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