Rapid identification method for resistance of muskmelon root rot

Abstract

The invention belongs to the technical field of plant disease resistance identification, and particularly relates to a rapid identification method of muskmelon root rot resistance, which comprises the following steps: S1, expanding propagation of pathogenic bacteria and preparation of spore suspension; s2, culturing muskmelon inoculums: after accelerating germination of muskmelon seeds, culturing the muskmelon seeds until the radicle length is 1-2.5 cm; s3, inoculating and culturing; s4, investigating disease conditions in a grading manner and calculating a disease index DI; s5, evaluating disease resistance. According to the method, the identification stage is advanced from the seedling stage needing long-time growth to the radicle stage, culture is carried out under the optimal condition, the disease development time is shortened from 25-40 d to 7 d, the single-batch identification time can be shortened by 55-65%, and the processes of resistant germplasm resource screening, filial generation identification and breeding are effectively accelerated. Besides, a grading standard based on root direct affected symptoms is established, interference of non-specific symptoms of the overground part caused by environmental pressure can be avoided, the accuracy of resistance evaluation is improved, and a more reliable basis is provided for a breeder to select parents and offspring.

Description

Technical Field

[0001] This invention belongs to the field of plant disease resistance identification technology, specifically relating to a rapid identification method for resistance to melon root rot. Background Technology

[0002] Melon root rot is one of the most serious diseases affecting the melon industry in my country. Currently, the main pathogens reported for melon root rot in my country include Fusarium solani (…). Fusarium solani ) and Cannosum ( Monosporascus cannonballus Among the various diseases, root rot caused by Fusarium solani is more prevalent in all producing areas of China, causing significant economic losses to melon production. Due to the difficulty in controlling this disease, screening for disease-resistant breeding materials and selecting disease-resistant varieties have become the most economical and effective ways to control melon root rot.

[0003] Currently, the commonly used method for identifying resistance to root rot in melons requires greenhouses or polytunnels and is limited by climate conditions, only allowing for testing during the spring and autumn seasons when temperatures are suitable for disease occurrence (April-June and September-November). The specific procedure is as follows: melon seedlings are first cultivated in plug trays. During the seedling stage, they are pulled out and their roots are washed. The pathogen is then inoculated using a spore-soaked root method. The seedlings are then transplanted back into the plug trays for subsequent seedling management. Because the disease spreads slowly after pathogen infection, a disease grading survey is usually conducted 25-40 days after inoculation (thin-skinned melons show symptoms earlier, while thick-skinned melons show symptoms later). The survey primarily uses symptoms on the above-ground leaves as the basis for judgment, and the entire identification cycle lasts 1.5-2 months.

[0004] In practice, existing methods for identifying resistance to root rot in melons have the following problems: (1) It is strictly limited by season and environment. It relies on specific climatic conditions and facilities such as greenhouses and polytunnels, and cannot achieve continuous identification all year round and in all weather conditions, which is not flexible and versatile enough; (2) The operation process is complicated. The management of seedling raising, transplanting, seedling moisture retention, watering, and pest control requires a lot of manpower, material resources and planting space, resulting in a large consumption of resources; (3) The disease progression is significantly affected by external temperature fluctuations, and the disease cycle is not constant (generally 25-40 days), resulting in poor repeatability and comparability of the identification results; (4) The identification criteria are relatively one-sided. The classification is based solely on the observation of symptoms of the above-ground stems and leaves, ignoring the initial pathological characteristics of the roots as the directly affected organs. This can easily lead to misjudgment of resistant and susceptible materials, especially the lack of accuracy in distinguishing moderately resistant materials. (5) The experimental cycle is too long. The entire process from pathogen culture, seedling cultivation, inoculation to investigation takes 1.5-2 months, which seriously restricts the scale and speed of screening resistant germplasm resources and delays the process of disease-resistant melon breeding. Summary of the Invention

[0005] The purpose of this invention is to provide a rapid identification method for resistance to root rot in melons, in order to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A rapid method for identifying resistance to root rot in melons includes the following steps: S1. Propagate the pathogen and prepare a spore suspension. The activated pathogen was cultured on PDA medium at 28°C for 10-12 days. Conidia were washed away with sterile water, and a 1×10⁻⁶ solution was prepared. 5 -1×10 7 spores / mL spore suspension; S2. Cultivating melon inoculum After germination, melon seeds are cultured until the radicle length is 1-2.5cm. S3. Inoculation and Culture Cut off 2-3mm of the root tip of the melon embryo to cause root damage, then soak it in a suspension of pathogenic spores for 15-25 minutes, remove it and place it in a culture dish lined with moistened absorbent cotton and filter paper, cover the dish and seal it, and transfer it to a constant temperature culture at 25-30℃ for 6-8 days. S4. Conduct a graded survey of disease incidence and calculate the disease severity index (DI). S5. Disease resistance evaluation The disease resistance grading criteria are as follows: .

[0007] Furthermore, in step S1, the concentration of the spore suspension is 1×10⁻⁶. 6 spores / mL.

[0008] Furthermore, in step S2, the length of the melon radicle is 1.5-2cm.

[0009] Furthermore, in step S3, the soaking time is 20 minutes, and the sample is transferred to a constant temperature culture at 28°C for 7 days.

[0010] Furthermore, in step S4, the grading criteria for the radicle grading investigation are as follows: .

[0011] Furthermore, in step S4, the formula for calculating the disease index DI is as follows:

[0012] Compared with the prior art, the present invention has the following beneficial effects: (1) Short identification cycle and significantly improved efficiency. This invention advances the identification stage from the seedling stage, which requires a long growth period, to the radicle stage, and cultivates the seedlings under optimal conditions. The disease development time is shortened from 25-40 days to 7 days, and the identification time for a single batch can be shortened by 55-65%, which effectively accelerates the screening of resistant germplasm resources, identification of hybrid offspring, and breeding process.

[0013] (2) It is not limited by season or space and can achieve standardized and high-throughput identification. The laboratory constant temperature culture completely gets rid of the constraints of natural climate, and the culture dish occupies very little space. It does not need to rely on greenhouses or other facilities, and can achieve standardized synchronous identification in different locations 365 days a year. The experimental conditions are uniform, the results are highly comparable, and it is convenient to carry out large-scale, high-throughput germplasm resource resistance evaluation.

[0014] (3) Simple operation and significant cost savings. This invention eliminates the complex steps of seedling raising, transplanting, and long-term field management in traditional methods. It only requires seed germination, inoculation, and petri dish cultivation, which significantly reduces manpower, material resources, land and management costs, lowers the technical threshold, and makes it easier to promote and apply in various breeding units and laboratories.

[0015] (4) The identification results are more direct, accurate and sensitive. This invention establishes a grading standard based on direct root damage symptoms. The root responds to pathogen infection earlier and more directly than the aboveground parts, which can avoid interference from non-specific symptoms of the aboveground parts caused by environmental stress. At the same time, it is helpful to distinguish intermediate types of materials such as moderately resistant and moderately susceptible, which improves the accuracy of resistance evaluation and provides breeders with a more reliable basis for selecting parents and offspring.

[0016] (5) Good repeatability and stability. Key environmental factors such as temperature, humidity and inoculation concentration can be precisely controlled in the laboratory, eliminating the unavoidable errors in field trials. The experimental results between different batches and different operators are highly consistent, which greatly improves the reliability and persuasiveness of scientific research data. Attached Figure Description

[0017] Figure 1 This is a flowchart illustrating the rapid identification process for melon root rot resistance according to the present invention. Figure 2 This is a grading standard diagram for muskmelon root rot disease according to the present invention; Figure 3 The growth of highly resistant and highly susceptible melon varieties 40 days after seedling root dipping inoculation with the pathogen; Figure 4 This study investigated the growth of highly resistant and highly susceptible melon varieties 7 days after inoculation with bacterial solution during the radicle stage. Detailed Implementation

[0018] The technical solution of this invention patent will be clearly and completely described below. Obviously, the described embodiments are some embodiments of this invention, but not all embodiments.

[0019] Example 1 A rapid method for identifying resistance to root rot in melons includes the following steps: S1. Propagate the pathogen and prepare a spore suspension. Activated pathogens ( Fusarium solani The conidia were isolated from the melon base of Wumao Farm, Wuming District, Nanning City, Guangxi Province, and preserved at the Guangxi Academy of Agricultural Sciences. They were cultured on PDA medium at 28℃ for 10-12 days, and the conidia were washed off with sterile water to prepare 1×10⁻⁶ samples. 6 spores / mL spore suspension; S2. Cultivating melon inoculum Soak the melon seeds in clean water for about 6 hours, then take them out and rinse them twice with clean water. Then place the seeds in a petri dish lined with moistened absorbent cotton and filter paper, cover the dish, put it in a white transparent plastic bag, tie the plastic bag, and place it in an incubator at 31℃ until the radicle grows to 1-2.5cm, then use it for inoculation with pathogens. S3. Inoculation and Culture Cut off 2-3 mm of the root tip of the melon embryo to cause root damage, then soak it in a suspension of pathogenic spores for 20 minutes, remove it and place it in a petri dish lined with moistened absorbent cotton and filter paper, cover the dish and seal it, and transfer it to a constant temperature of 28℃ for 7 days. S4. Conduct a graded survey of disease incidence and calculate the disease severity index (DI). A disease grading survey was conducted on all tested melon materials, and the severity of the disease was divided into 6 levels (see Table 1 and...). Figure 1 Record the number of plants at each level in order to calculate the disease index; Table 1 Grading Standards for Melon Root Rot

[0020] ; S5. Disease resistance evaluation Resistance levels are classified based on the Disease Indication (DI) score. The disease resistance grading criteria are as follows: .

[0021] Example 2 2.1 Seedling stage root soaking inoculation Existing methods were used to inoculate melon seedlings with root rot pathogens through root soaking. Fusarium solaniThe bacteria were isolated from the melon base of Wumao Farm in Wuming District, Nanning City, Guangxi Province, and preserved at the Guangxi Academy of Agricultural Sciences. The bacterial concentration was 1×10⁻⁶. 6 After 40 days (spores / mL), the growth of the highly resistant melon variety 'Kewang Sweet No. 3' (sold by Langfang Kelong Seed Co., Ltd.) and the highly susceptible variety 'Shankemi No. 5' (sold by Shandong Kefeng Seed Industry Co., Ltd.) is shown in the figure. Figure 3 .

[0022] Depend on Figure 3 It can be seen that the highly resistant melon variety 'Kewang Sweet No. 3' is healthy, while the highly susceptible variety 'Shanke Honey No. 5' shows obvious symptoms of root rot and the plant dies.

[0023] 2.2 Inoculation during the radicle stage The method described in Example 1 was used to soak the bacterial solution during the radicle stage ( Fusarium solani The bacteria were isolated from the melon base of Wumao Farm, Wuming District, Nanning City, Guangxi Province, and preserved at the Guangxi Academy of Agricultural Sciences. The bacterial concentration was 1×10⁻⁶. 6 Seven days after inoculation (spores / mL), the growth of the highly resistant melon variety 'Kewang Sweet No. 3' and the highly susceptible variety 'Shanke Honey No. 5' was observed. Figure 4 .

[0024] Depend on Figure 4 It can be seen that the highly resistant muskmelon variety 'Kewang Sweet No. 3' showed good health, while the highly susceptible variety 'Shanke Honey No. 5' showed obvious symptoms of browning and rotting of the root and stem, and the plants were stunted. Therefore, the results of the radicle soaking inoculation method of this invention are consistent with those of the existing seedling root soaking inoculation method for identifying muskmelon disease resistance, indicating that the method of this invention can replace the existing method for identifying muskmelon root rot resistance.

[0025] In summary, this invention advances the identification stage from the seedling stage, which requires a long growth period, to the radicle stage, and cultivates the plants under optimal conditions. This shortens the disease development time from 25-40 days to 7 days, and reduces the identification time per batch by 55-65%, effectively accelerating the screening of resistant germplasm resources, identification of hybrid offspring, and the breeding process. Furthermore, this invention establishes a grading standard based on direct root damage symptoms, avoiding interference from non-specific symptoms in the aboveground parts caused by environmental stress, improving the accuracy of resistance evaluation, and providing breeders with a more reliable basis for selecting parents and offspring.

[0026] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A rapid identification method for resistance to root rot in melon, characterized in that, Includes the following steps: S1. Propagate the pathogen and prepare a spore suspension. The activated pathogen was cultured on PDA medium at 28°C for 10-12 days. Conidia were washed away with sterile water, and a 1×10⁻⁶ solution was prepared. 5 -1×10 7 spores / mL spore suspension; S2. Cultivating melon inoculum After germination, melon seeds are cultured until the radicle length is 1-2.5cm. S3. Inoculation and Culture Cut off 2-3mm of the root tip of the melon embryo to cause root damage, then soak it in a suspension of pathogenic spores for 15-25 minutes, remove it and place it in a culture dish lined with moistened absorbent cotton and filter paper, cover the dish and seal it, and transfer it to a constant temperature culture at 25-30℃ for 6-8 days. S4. Conduct a graded survey of disease incidence and calculate the disease severity index (DI). S5. Disease resistance evaluation The disease resistance grading criteria are as follows: 。 2. The rapid identification method for muskmelon root rot resistance according to claim 1, characterized in that, In step S1, the concentration of the spore suspension is 1×10⁻⁶. 6 spores / mL.

3. The rapid identification method for muskmelon root rot resistance according to claim 1, characterized in that, In step S2, the length of the melon radicle is 1.5-2cm.

4. The rapid identification method for resistance to root rot in melon according to claim 1, characterized in that, In step S3, the soaking time is 20 minutes, and the sample is transferred to a constant temperature culture at 28°C for 7 days.

5. The rapid identification method for resistance to root rot in melon according to claim 1, characterized in that, In step S4, the grading criteria for radicle grading are as follows: 。 6. The rapid identification method for resistance to root rot in melon according to claim 1, characterized in that, In step S4, the formula for calculating the disease index DI is as follows: 。

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