In-vitro identification method of cabbage black spot resistance

Abstract

The invention relates to the technical field of plant resistance identification, in particular to an in-vitro identification method for cabbage black spot resistance. The in-vitro identification method disclosed by the invention comprises the following steps: (1) suspending conidia generated after dark culture of alternaria alternata by using sterile water to obtain spore suspension of the alternaria alternata; and (2) inoculating the spore suspension to the in-vitro leaves of the cabbage, carrying out dark culture on the in-vitro leaves of the cabbage obtained by inoculation, investigating and recording the disease condition and calculating the disease index according to the disease severity of the cultured in-vitro leaves, and completing the evaluation of the black spot resistance of the in-vitro cabbage leaves according to the disease index. According to the method provided by the invention, the incidence rate of the inoculated cabbage in-vitro leaves is stable, and the method is easy to implement and simple and convenient to operate. The method can be used for quickly and accurately identifying the black spot resistance of the cabbage variety, and can effectively solve the problem of quickly identifying the black spot resistance of the cabbage variety in the scientific research, breeding and production of the cabbage.

Description

Technical Field

[0001] The present invention relates to the technical field of plant resistance identification, and particularly relates to an in vitro identification method for the resistance of cabbage to Alternaria brassicae Background Art

[0002] Alternaria brassicae causes Alternaria leaf spot of cabbage, which mainly affects the leaves, petioles, flower stalks and seed pods of cabbage. The typical symptoms are that at the initial stage of the disease on the leaves and petioles, small black spots appear, and then expand into dark brown round spots with a yellow halo. When the humidity is high, black mold will appear on the disease spots. The disease spots on the petioles of the stem are longitudinal strips, also accompanied by black mold. On the seed pods, dark brown fusiform disease spots appear, affecting seed setting and seed quality, and there is a mold of conidia of the pathogen.

[0003] Alternaria leaf spot of cabbage has become one of the main diseases affecting the quality of cabbage. Currently, chemical agents such as mancozeb and chlorothalonil are commonly used for prevention and control, but long-term or improper use is likely to cause pathogen drug resistance, environmental pollution and health hazards.

[0004] Therefore, it is crucial to formulate comprehensive prevention and control measures and breed disease-resistant varieties. And quickly and accurately identifying the resistance characteristics of cabbage varieties to Alternaria leaf spot has become an urgent issue for researchers to solve. Summary of the Invention

[0005] The purpose of the present invention is to provide an in vitro identification method for the resistance of cabbage to Alternaria brassicae. The operation method provided by the present invention is simple, the incidence rate of the in vitro leaves of cabbage after inoculation is stable, and it can quickly and accurately identify the resistance of cabbage varieties to Alternaria leaf spot. Through the treatment of the present invention, the problem of quickly identifying the resistance of cabbage varieties to Alternaria leaf spot in cabbage scientific research, breeding and production can be effectively solved.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides an in vitro identification method for the resistance of cabbage to Alternaria brassicae, including the following steps:

[0008] (1) Preparation of spore suspension

[0009] The conidia produced after the Alternaria brassicae is cultured in the dark are suspended with sterile water to obtain a spore suspension of Alternaria brassicae;

[0010] (2) Inoculation of pathogenic bacteria

[0011] After inoculating the spore suspension onto the in vitro leaves of cabbage, the in vitro leaves of the inoculated cabbage are cultured in the dark, and the disease condition is investigated and recorded according to the severity of the disease on the in vitro leaves after culture, the disease index is calculated, and based on the disease index, the resistance evaluation of the in vitro cabbage leaves to Alternaria leaf spot is completed.

[0012] Preferably, in the step (1), the dark culture time of Alternaria brassicae is 7 - 15 d, and the temperature is 25 - 30 °C.

[0013] Preferably, in the step (1), the concentration of the spore suspension is 5 - 15×10 4 spores / ml.

[0014] Preferably, in the step (2), the inoculation amount of the spore suspension is 200 - 300 μl / leaf.

[0015] Preferably, in the step (2), the dark culture temperature of the excised leaves of the inoculated cabbage is 25 - 30 °C, the humidity is 90 - 95%, and the time is 60 - 84 h.

[0016] Preferably, the method for grading the disease severity in the step (2) is as follows:

[0017] Grade 0: No obvious lesions;

[0018] Grade 1: The lesion area accounts for ≤ 5% of the leaf area;

[0019] Grade 3: 5% < the lesion area accounts for ≤ 25% of the leaf area;

[0020] Grade 5: 25% < the lesion area accounts for ≤ 50% of the leaf area, and the leaf is slightly yellowed;

[0021] Grade 7: 50% < the lesion area accounts for ≤ 75% of the leaf area, and the leaf is significantly yellowed;

[0022] Grade 9: 75% < the lesion area accounts for ≤ 100% of the leaf area, and the leaf is severely yellowed.

[0023] Preferably, the disease index in the step (2) is calculated using the following formula:

[0024] Disease index = [Σ (disease grade × number of plants with the disease at that grade) / (highest disease grade × total number of plants surveyed)] × 100.

[0025] Preferably, the evaluation criteria for the resistance of excised cabbage leaves to Alternaria brassicae in the step (2) are as follows:

[0026] High resistance (HR): Disease index = 0.10 - 11.11;

[0027] Resistant (R): Disease index = 11.12 - 33.33;

[0028] Moderately resistant (MR): Disease index = 33.34 - 55.55;

[0029] Susceptible (S): Disease index = 55.56 - 77.77;

[0030] High-sensitivity (HS): disease index = 77.78 - 100.

[0031] Preferably, the black spot pathogen is Alternaria brassicicola.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention provides an in vitro identification method for the resistance of cabbage to black spot. Using the in vitro leaves of cabbage seedlings at the four-leaf and one-heart stage as the inoculation object, the third or fourth true leaf of the cabbage seedlings is inoculated by the spraying method and the acupuncture method. The inoculation method is simple and convenient; the inoculated cabbage seedlings are cultured under moisturizing conditions at a constant temperature for 72 hours, and then the disease index can be investigated, which is characterized by convenience and rapidity. Brief Description of the Drawings

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0035] Figure 1 It is a schematic diagram of the disease grading of cabbage black spot. Detailed Embodiments

[0036] The following will describe in detail the technical solutions provided by the present invention in combination with the embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0037] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0038] Example 1

[0039] An in vitro identification method for the resistance of cabbage to black spot, the specific steps are as follows:

[0040] (1) Preparation of cabbage seedlings

[0041] Select 20 cabbage materials and number them (see Table 1). Each material requires 10 replicates for the identification of black spot resistance;

[0042] Use plug trays for seedling raising. Mix 50% carbendazim wettable powder into the substrate (Danish Pindstrup 0 - 6mm peat soil seedling raising substrate) at a ratio of 1:1000. After mixing the sterilized substrate (vermiculite: peat moss: sterilized soil = 1V:1V:2V), fill it into 50-hole plug trays;

[0043] Sow 1 - 2 seeds per hole. After sowing, water thoroughly, cover with a layer of non - woven fabric to keep moisture, and lift the non - woven fabric in time after emergence;

[0044] Thin out the seedlings and fill in the gaps after all the seeds have germinated to ensure one seedling per hole, and keep them until they reach the four - leaf and one - heart stage for standby;

[0045] (2) Preparation of spore suspension

[0046] For large - scale propagation and standby of the purified Alternaria brassicicola, use the streaking method or the mycelial block method. The specific operations are as follows:

[0047] Streaking method: For the purified Alternaria brassicicola, add sterile water, scrape the conidia on the surface of the culture medium with a sterile spreading rod, filter the conidia through two layers of sterilized lens paper, streak on a new PAD medium with a sterile inoculation loop. After the spores grow into mycelia, use a sterilized scalpel to take a small piece of agar block containing mycelia and place it at the center of a new PAD medium, and incubate it in the dark at 28 °C in a constant - temperature incubator for 15 days.

[0048] Mycelial block method: For the purified Alternaria brassicicola, use a sterilized scalpel to take a mycelial block with a diameter of 0.5 cm at the edge of the colony, and place it evenly in 5 positions (up, down, left, right, and middle) on fresh PDA. After the mycelial block grows new mycelia, pick the new mycelia and transfer them into fresh PDA medium, and incubate it in the dark at 28 °C in a constant - temperature incubator for 15 days.

[0049] Add sterile water to the plate of the propagated PDA black spot pathogen (Alternaria brassicicola), scrape the conidia on the surface of the culture medium with a spreading rod, filter the conidia through two layers of lens paper, and use a hemocytometer to measure the concentration of the spore suspension, and adjust it to 1×10 5 spores / ml for in - vitro leaf surface spraying inoculation.

[0050] (3) Preparation of in - vitro leaves

[0051] Take the 3rd or 4th true leaf of the cabbage seedlings prepared in step (1). Take 10 true leaves for each material, with 3 replicates, a total of 30 true leaves. Take the leaves from the same part of the cabbage seedlings of different materials, wipe the leaf surface with cotton soaked in 75% alcohol (for disinfection and removing the wax powder on the leaf surface to facilitate the attachment of the bacterial liquid), and dry them for standby;

[0052] Place the leaves in a culture dish (90×15 mm) and put a filter paper (diameter 85 mm) at the bottom.

[0053] (4) Inoculation of pathogens

[0054] Spray the spore suspension prepared in step (2) onto the filter paper at the bottom of the petri dish first to moisten it, and use the needle hole of a syringe (1 ml) to pierce 4 symmetrical needle holes on both the left and right sides of the leaf prepared in step (3). Then inoculate the detached leaf, spraying 200 μl of the spore suspension on each leaf, covering the lid and sealing it with a sealing film.

[0055] (5) Management after inoculation

[0056] The detached cabbage leaves inoculated with the pathogen are incubated in the dark with humidity maintained in a biochemical incubator, with the conditions set as: temperature 28 °C, humidity 90%. Observe the disease occurrence on the leaves after 72 h. The grading of disease severity is shown in Figure 1 .

[0057] (6) Investigation and evaluation

[0058] Investigate the disease occurrence on the detached cabbage leaves and classify them according to the diseased leaf area:

[0059] Grade 0: No obvious lesions;

[0060] Grade 1: The lesion area accounts for ≤ 5% of the leaf area;

[0061] Grade 3: 5% < the lesion area accounts for ≤ 25% of the leaf area;

[0062] Grade 5: 25% < the lesion area accounts for ≤ 50% of the leaf area, and the leaf turns slightly yellow;

[0063] Grade 7: 50% < the lesion area accounts for ≤ 75% of the leaf area, and the leaf turns significantly yellow;

[0064] Grade 9: 75% < the lesion area accounts for ≤ 100% of the leaf area, and the leaf turns severely yellow;

[0065] Calculate the disease index DI (disease index):

[0066] Disease index = [Σ (disease grade × number of plants with the disease at that grade) / (highest disease grade × total number of investigated plants)] × 100.

[0067] Based on the disease index, complete the evaluation of the resistance of detached cabbage leaves to Alternaria brassicae. The evaluation criteria are:

[0068] High resistance (HR): Disease index = 0.10 - 11.11;

[0069] Resistant (R): Disease index = 11.12 - 33.33;

[0070] Moderately resistant (MR): Disease index = 33.34 - 55.55;

[0071] Susceptible (S): Disease index = 55.56 - 77.77;

[0072] Highly susceptible (HS): Disease index = 77.78 - 100.

[0073] The identification results are shown in Table 1.

[0074] Table 1

[0075]

[0076]

[0077] According to the characteristics that the infection route of black spot disease is through natural orifices or wounds, combined with its epidemic law and transmission route, the present invention can quickly and accurately identify the anti - black - spot disease characteristics of cabbage varieties during large - area promotion by artificially inoculating the in - vitro leaves of cabbage at the four - leaf - one - heart stage.

[0078] The incidence rate of the in - vitro leaves of cabbage after inoculation by the method of the present invention is stable, the method is easy to implement, and the operation is simple.

[0079] The above - mentioned is only the preferred implementation mode of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An in vitro identification method for resistance to black spot disease of cabbage, characterized in that: The following steps are involved: (1) Preparation of spore suspension The conidia produced by dark culture of the black spot pathogen are suspended in sterile water to obtain a spore suspension of the black spot pathogen; (2) Inoculation of pathogens After the spore suspension is inoculated into the detached leaves of cabbage, the detached leaves of cabbage obtained by the inoculation are cultured in the dark, and the disease severity on the detached leaves after the culture is investigated and recorded, and the disease index is calculated. Based on the disease index, the resistance of the detached cabbage leaves to black spot disease is evaluated.

2. The in vitro identification method according to claim 1, characterized in that In the step (1), the dark culture time of the black spot pathogen is 7 to 15 days, and the temperature is 25 to 30°C.

3. The in vitro identification method according to claim 1, characterized in that The concentration of the spore suspension in step (1) is 5 to 15×10 4 Spores / ml.

4. The in vitro identification method according to claim 1, characterized in that The inoculation amount of the spore suspension in step (2) is 200-300 μl / leaf.

5. The in vitro identification method according to claim 1, characterized in that: The in vitro cabbage leaves obtained by inoculation in the step (2) are cultured in dark at a temperature of 25 to 30° C., a humidity of 90 to 95%, and a time of 60 to 84 hours.

6. The in vitro identification method according to claim 1, characterized in that: The classification method of disease severity in step (2) is: Level 0: no obvious lesions; Level 1: The lesion area accounts for ≤5% of the leaf area; Level 3: 5%<lesion area accounts for ≤25% of leaf area; Level 5: 25%<lesion area accounts for ≤50% of leaf area, leaves are slightly yellowed; Level 7: 50%<lesion area accounts for ≤75% of leaf area, and leaves are obviously yellowing; Level 9: 75% < lesion area accounts for ≤100% of leaf area, and leaves are severely yellowed.

7. The in vitro identification method according to claim 1, characterized in that: The disease index in step (2) is calculated using the following formula: Disease index = [Σ(disease level × number of diseased plants at that disease level) / (highest disease level × total number of investigated plants)] × 100.

8. The in vitro identification method according to claim 1, characterized in that: The evaluation criteria for the resistance of the detached cabbage leaves to black spot disease in step (2) are: High resistance (HR): disease index = 0.10-11.11; Disease resistance (R): disease index = 11.12-33.33; Medium resistance (MR): disease index = 33.34-55.55; Susceptible (S): disease index = 55.56-77.77; High sensitivity (HS): Disease index = 77.78~100.

9. The in vitro identification method according to claim 2, characterized in that: The black spot pathogen is Alternaria brassicicola.

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