Method for rapidly identifying anti-cantile leaf virus disease of cucumber through cucumber cotyledon injection inoculation

Abstract

The invention discloses a method for rapidly identifying cucumber anti-tile-leaf virus disease through cucumber cotyledon injection inoculation, which comprises the following steps: (1) taking cucumber diseased leaf tissues with typical tile-leaf virus disease symptoms, adding a PBS (phosphate buffer solution), grinding, and squeezing out juice; (2) disinfecting cucumber seeds, accelerating germination at constant temperature, sowing and culturing until cotyledons are completely expanded; (3) dividing cucumber seedlings into groups A and B; (2) injecting the juice obtained in the step (1) into intercellular spaces on the backs of two cotyledons of the group A cucumber seedlings by using a sterile injector until the whole cotyledons are filled with the juice; injecting the PBS buffer solution into intercellular spaces on the backs of two cotyledons of the group B cucumber seedlings until the whole cotyledons are filled with the PBS buffer solution; and (4) culturing: according to the phenomena of mottling, floral leaf, deformation and plant dwarfing of new leaves of the A group of cucumbers, judging whether the cucumbers are sensitive or resistant to the cantile leaf virus disease, and the phenomena do not occur in the B group of cucumbers. The method is simple to operate, easy to control the inoculation amount, small in leaf damage, good in repeatability, small in error and high in stability.

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease resistance breeding, and in particular relates to a method for rapid identification of cucumber resistance to walnut virus disease by injection inoculation of cucumber cotyledons, and belongs to the technical field of agriculture. Background Art

[0002] Cucumber (Cucumis sativus L.) is an important economic vegetable crop cultivated worldwide. my country is the country with the largest cucumber cultivation area and the highest total output in the world. Therefore, cucumber production occupies an extremely important position in my country's vegetable production.

[0003] Cucumber Vava leaf virus disease causes deformed leaves, curled edges, twisted veins, and mottled yellow-green leaves; the overall growth of the plant is slow, and the ability to set fruit is poor; when the disease is severe, the edges of the leaves curl up into a "hemispherical shape" and the growth point disappears. The disease can be found throughout the growth period, and the incidence rate of susceptible varieties is over 80% or even 100%, seriously affecting its yield, quality and commercial value. Vava leaf virus disease has become a major new epidemic disease that currently harms cucumber production.

[0004] Wavar leaf virus disease is a systemic disease caused by combined infection of Watermelon silver mottle virus (WSMoV) and Cucumber green mottle mosaic virus (CGMMV). It is characterized by serious harm, strong pathogenicity, and difficulty in prevention and control. Viral diseases are known as the "cancer" of plants, and there is currently no specific drug. At present, Wavar leaf virus disease has become a key disease restricting the development of the cucumber industry.

[0005] Breeding disease-resistant cucumber varieties is the most economical and effective measure, and the identification and screening of disease-resistant germplasm resources requires a set of artificial inoculation disease resistance identification methods at the seedling stage that can accurately reflect the resistance level of cucumber materials, and the operation is required to be simple, fast, reliable, accurate, and reproducible. At present, the main methods for mechanical friction transmission of cucumber virus diseases are juice friction inoculation, spray gun inoculation, and direct inoculation of diseased tissues. The juice friction inoculation method has cumbersome operation steps, large errors in the inoculation amount of each plant, and it is difficult to keep the friction force and friction area consistent, resulting in inconsistent cotyledon damage and inconsistent inoculation amount. Although the spray gun inoculation method is efficient, it is difficult to operate, and the equipment is expensive and the test cost is high. The direct inoculation method of diseased tissues is suitable for inoculation of small samples or when there are few diseased tissue materials and the virus is unstable. It cannot be inoculated with a large number of samples, the infection rate is low, and the amount of virus inoculated in different plants is inconsistent. The method of natural disease in the field, due to the complexity and diversity of field toxins, it is impossible to clearly identify the toxins that play a major role, the repeatability of identification is limited, and the consistency and objective stability of the inoculation results cannot be guaranteed.

[0006] Currently, there is a lack of a simple, rapid and accurate method to evaluate the resistance of different cucumber germplasm resources to Cucumis sativus virus disease. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for rapidly identifying cucumber resistance to walnut leaf virus disease by injection inoculation of cucumber cotyledons.

[0008] The technical solution of the present invention is summarized as follows:

[0009] A method for rapidly identifying cucumber resistance to walnut virus disease by injection inoculation of cucumber cotyledons comprises the following steps:

[0010] (1) Take the cucumber leaf tissue with typical symptoms of walnut virus disease, add 0.01M, pH=7.2 PBS buffer, grind it in a mortar, and filter and squeeze out the juice through double-layer gauze;

[0011] (2) The cucumber seeds were sterilized with a 10% sodium phosphate tribasic aqueous solution, rinsed with clean water, placed in a culture dish lined with filter paper, and germinated at a constant temperature of 28° C. to 30° C. When the radicle grew to 0.5 to 1 cm, they were sown in a seedling tray filled with sterilized vermiculite and cultured at 26 to 28° C., 16 h light / 8 h dark conditions, until the cotyledons were fully expanded;

[0012] (3) Dividing the cucumber seedlings obtained in step (2) into group A and group B; injecting the juice obtained in step (1) into the intercellular spaces on the back of two cotyledons of the cucumber seedlings in group A with a sterile syringe until the entire cotyledon is filled; injecting 0.01 M pH=7.2 PBS buffer into the intercellular spaces on the back of two cotyledons of the cucumber seedlings in group B with a sterile syringe until the entire cotyledon is filled;

[0013] (4) Cultivate in the dark at 26-28°C for 24 h, then in the light for 16 h and in the dark for 8 h for 10-15 d; judge whether the cucumbers in Group A are susceptible or resistant to the disease based on whether the new leaves are mottled, mosaic, deformed, or the plants are dwarfed. Group B does not show mottled, mosaic, deformed, or dwarfed symptoms.

[0014] Advantages of the present invention:

[0015] (1) Through cotyledon injection inoculation, the inoculation amount is easy to control during the inoculation process, and the damage to the leaves is small. It can ensure the standardization of the operation of cucumber materials in different batches and different operators in the inoculation identification, and the accuracy, comparability, consistency and stability of the results. The identification cycle is short, the inoculation success rate is 100%, and it is more suitable for the inoculation of cucumber seedlings.

[0016] (2) Inoculate cucumber cotyledons with a syringe, and directly judge the resistance of cucumber materials / varieties to walnut leaf virus disease based on the symptoms of the new leaves after inoculation. This judgment method is simple, rapid, accurate and reliable, and can effectively avoid the problems of unstable symptoms, inaccurate results and poor repeatability caused by different degrees of leaf damage and different inoculation amounts in other inoculation methods. The inoculation solution is directly injected into the mesophyll cells, which is easy to observe and can avoid missed connections.

[0017] (3) Compared with the existing inoculation method, the inoculation time of the method of the present invention is greatly advanced. The disease of the cucumber seedlings can be investigated 10 days after inoculation, and the disease resistance of the cucumber materials can be graded. A set of grading standards suitable for evaluating the resistance of cucumber to Vulgaris virus disease has been established, which plays an important role in the breeding of cucumbers resistant to Vulgaris virus disease and the mining of disease-resistant genes. The method has the advantages of simple operation, easy control of inoculation amount, little damage to leaves, good repeatability, small error and high stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Photo of cucumber seedlings at the cotyledon flattening stage before inoculation with Vava virus.

[0019] Figure 2 These are photos taken after inoculation with Wavala virus; treatment 1 was taken 8 days after injection with Wavala virus; treatment 2 was taken 11 days after injection with Wavala virus.

[0020] Figure 3 These are photos taken after injection of 0.01M pH 7.2 PBS buffer; CK1 was taken 8 days after injection of 0.01M pH 7.2 PBS buffer, and CK2 was taken 11 days after injection of 0.01M pH 7.2 PBS buffer. DETAILED DESCRIPTION

[0021] The present invention takes the cucumber variety Jinyou 619 as a commercial cucumber variety resistant to Wagner leaf virus disease and Jinyou 180 as a commercial cucumber variety susceptible to Wagner leaf virus disease as an example. The present invention discloses these two cucumber varieties to enable those skilled in the art to better understand the present invention, but does not limit the present invention. Other varieties of cucumbers can also be used in the present invention.

[0022] The technical solution of the present invention will be systematically and completely described below in conjunction with specific embodiments of the present invention. The following embodiments may enable those skilled in the art to understand the present invention more comprehensively, but do not limit the present invention in any way.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0024] Unless otherwise specified, the materials, reagents, etc. used in the following examples can be obtained from commercial channels or institutions.

[0025] Example 1

[0026] A method for rapidly identifying cucumber resistance to walnut virus disease by injection inoculation of cucumber cotyledons comprises the following steps:

[0027] (1) According to the proportion, take 1 g of cucumber (Jinyou 180) diseased leaf tissue with typical symptoms of WSMoV (containing only highly pathogenic strains of WSMoV and CGMMV), add 10 mL of 0.01 M, pH = 7.2 PBS buffer, grind in a mortar, and filter and squeeze out the juice with double-layer gauze;

[0028] (2) soaking cucumber seeds (Jinyou 619 and Jinyou 180, marked) in a 10% sodium phosphate aqueous solution for 20 min for disinfection, rinsing with clean water, placing in a culture dish lined with filter paper, germinating at a constant temperature of 28° C. (or any value between 28 and 30° C., such as 28.5° C., 29° C., 29.5° C. or 30° C.), and sowing in a seedling tray filled with sterilized vermiculite when the radicle grows to 0.5 to 1 cm, placing at 26° C. (or 27° C. or 28° C.), 16 h light / 8 h dark conditions, until the cotyledons are fully expanded;

[0029] (3) Dividing the cucumber seedlings obtained in step (2) into group A and group B; injecting the juice obtained in step (1) into the intercellular spaces on the back of two cotyledons of the cucumber seedlings in group A with a sterile syringe until the entire cotyledon is filled; injecting 0.01 M pH=7.2 PBS buffer into the intercellular spaces on the back of two cotyledons of the cucumber seedlings in group B with a sterile syringe until the entire cotyledon is filled;

[0030] (4) Cultivate in the dark at 26°C (or any temperature between 26°C and 28°C, such as 26.5°C, 27°C or 28°C) for 24 h, then culture in the light for 16 h and in the dark for 8 h for 10 d (or any day between 10 d and 15 d, such as 11 d, 12 d, 13 d, 14 d, 15 d); judge whether the cucumbers in Group A are susceptible or resistant to Wagwa virus disease based on whether the new leaves are mottled, mosaic, deformed, or the plants are dwarfed. The results showed that the one in Group A that was susceptible to Wagwa virus disease was Jinyou 180; the one in Group A that was resistant to Wagwa virus disease was Jinyou 619; in Group B, neither Jinyou 619 nor Jinyou 180 showed any signs of mottled, mosaic, deformed, or dwarfed.

[0031] (When cultivating, use insect-proof nets to protect against seeds that are infected with poison or other viral diseases such as aphids, thrips, and whiteflies that may affect the final result)

[0032] The experiment was repeated three times, with 10 plants in each replicate. The cucumber variety 'Jinyou 180', which is highly susceptible to Vulgaris virus disease, and the cucumber variety 'Jinyou 619', which is highly resistant to Vulgaris virus disease, were used as test materials, and 0.01M, pH=7.2 PBS buffer was inoculated as a control.

[0033] Example 2

[0034] The cucumber varieties / resources listed in Table 1 were used as test materials for testing. The steps were as follows:

[0035] (1) According to the proportion, take 1 g of cucumber (Jinyou 180) diseased leaf tissue with typical symptoms of WSMoV (containing only highly pathogenic strains of WSMoV and CGMMV), add 10 mL of 0.01 M, pH = 7.2 PBS buffer, grind in a mortar, and filter and squeeze out the juice with double-layer gauze;

[0036] (2) Soak cucumber seeds (cucumber varieties / resources listed in Table 1, marked) in a 10% sodium phosphate aqueous solution for 20 min for disinfection, rinse with clean water, place in a culture dish lined with filter paper, and germinate at a constant temperature of 28° C. (or any value between 28 and 30° C., such as 28.5° C., 29° C., 29.5° C. or 30° C.). When the radicle grows to 0.5 to 1 cm, sow in a seedling tray filled with sterilized vermiculite, and culture at 26° C. (or 27° C. or 28° C.) under 16 h light / 8 h dark conditions until the cotyledons are fully expanded.

[0037] (3) injecting the juice obtained in step (1) into the intercellular spaces on the back of two cotyledons of the cucumber seedling obtained in step (2) using a sterile syringe until the juice fills the entire cotyledon;

[0038] (4) Cultivate in the dark at 26°C for 24 h, then cultivate in the light for 16 h and in the dark for 8 h for 15 d; investigate and grade the disease status of the cucumber seedlings according to the following grading standards:

[0039] Level 0: no symptoms;

[0040] Level 1: A few chlorotic spots appear on the inoculated leaves or the leaves on both sides of the main vein are asymmetrical, and the leaves are normal or the symptoms are not obvious;

[0041] Level 2: The heart leaves have clear veins or a few leaves have slight mosaic, but the shape is normal;

[0042] Level 3: Most leaves are mosaic, and a few leaves are deformed or obviously wrinkled;

[0043] Level 4: Most leaves are severely mosaic, new leaves or some leaves are wrinkled, deformed, or the plant is slightly dwarfed;

[0044] Level 5: Almost all leaves are severely mosaic, obviously deformed, and fern-like;

[0045] Disease index (DI) = ∑[(disease level × number of plants at that level) / (total number of plants investigated × 5)] × 100;

[0046] The disease resistance classification criteria for strain groups are:

[0047] High resistance (HR), 0 <DI≤15;

[0048] Disease resistance (R), 15 <DI≤35;

[0049] Medium resistance (MR), 35 <DI≤55;

[0050] Susceptible (S), 55DI≤75;

[0051] High sensitivity (HS), 75 <DI。

[0052] According to the results of the disease investigation, the disease resistance of 10 cucumber varieties / resources was evaluated according to the disease classification standard and disease index. The highly susceptible variety Jinyou 180 and the highly resistant variety Jinyou 619 were used as controls.

[0053] In this experiment, the results of cucumber variety / resource identification are shown in Table 1. Among the 10 cucumber varieties / resources identified, 1 variety was found to be highly resistant (HR); 2 varieties were resistant (R); and 7 were highly susceptible varieties or resources.

[0054] Table 1 Identification results of resistance to Cucumber leaf virus disease in 10 cucumber varieties / resources

[0055]

Claims

1. A method for rapid identification of cucumber resistance to walnut virus disease by injection inoculation of cucumber cotyledons, characterized in that The following steps are involved: (1) Take the cucumber leaf tissue with typical symptoms of walnut virus disease, add 0.01M, pH=7.2 PBS buffer, grind it in a mortar, and filter and squeeze out the juice through double-layer gauze; (2) The cucumber seeds were sterilized with a 10% sodium phosphate tribasic aqueous solution, rinsed with clean water, placed in a culture dish lined with filter paper, and germinated at a constant temperature of 28° C. to 30° C. When the radicle grew to 0.5 to 1 cm, they were sown in a seedling tray filled with sterilized vermiculite and cultured at 26 to 28° C., 16 h light / 8 h dark conditions, until the cotyledons were fully expanded; (3) Dividing the cucumber seedlings obtained in step (2) into group A and group B; injecting the juice obtained in step (1) into the intercellular spaces on the back of two cotyledons of the cucumber seedlings in group A with a sterile syringe until the entire cotyledon is filled; injecting 0.01 M pH=7.2 PBS buffer into the intercellular spaces on the back of two cotyledons of the cucumber seedlings in group B with a sterile syringe until the entire cotyledon is filled; (4) Cultivate in the dark at 26-28°C for 24 h, then in the light for 16 h and in the dark for 8 h for 10-15 d; judge whether the cucumbers in Group A are susceptible or resistant to the disease based on whether the new leaves are mottled, mosaic, deformed, or the plants are dwarfed. Group B does not show mottled, mosaic, deformed, or dwarfed symptoms.

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