Alfalfa fusarium resistance identification method based on sand culture inoculation system

Abstract

The invention belongs to the technical field of plant disease detection, and particularly relates to an alfalfa fusarium resistance identification method based on a sand culture inoculation system. The invention provides an alfalfa fusarium resistance identification method based on a sand culture inoculation system. The alfalfa fusarium resistance identification method comprises the following steps: pre-culturing sand culture seedlings before inoculation; performing fusarium inoculation treatment on the sand culture seedlings; grading the disease conditions of the sand culture seedlings inoculated with fusarium, counting the morbidity of the sand culture seedlings, counting the number of diseased plants at each stage after the disease condition grading, and calculating disease condition indexes by combining corresponding stages of the disease condition grading; and evaluating the resistance of the medicago sativa to fusarium root rot according to the disease index. According to the alfalfa fusarium resistance identification method provided by the invention, the problems that the identification period is long and symptoms are inconvenient to observe due to the fact that an identification method of alfalfa fusarium resistance germplasm is easily influenced by environmental variation, soil microorganism interference and non-uniform pathogen distribution can be solved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease detection, and particularly relates to a method for identifying resistance to Fusarium acetifer in alfalfa based on a sand culture inoculation system. Background Art

[0002] Alfalfa, one of the world's most important high-quality legumes, is known as the "king of forages" for its high yield, rich nutrition, good palatability, and strong nitrogen-fixing capacity, and holds significant value in the livestock industry. However, the continued expansion of alfalfa cultivation areas, the extension of planting periods, and the intensification and mechanization of cultivation have introduced another risk to the grass industry: alfalfa root rot. Once infected, root rot, particularly caused by Fusarium spp., is difficult to control and has become a major factor impacting the development of the alfalfa industry, often referred to as the "cancer" of alfalfa.

[0003] After the root rot pathogen infects the roots, the root vascular bundles turn brown and rot, leaving only fibrous tissue. The leaves above the ground turn yellow and wither, and the mortality rate of the whole plant reaches 30% to 60%. In severe cases, the yield is reduced by 30% to 40%.

[0004] When alfalfa root rot occurs, the crude protein content of the alfalfa plant decreases, and due to root damage, the plant's cold tolerance is weakened, leading to a significant increase in winter mortality. To reduce the yield and quality losses of alfalfa caused by root rot, it is necessary to cultivate new disease-resistant alfalfa germplasm. Therefore, it is necessary to identify Fusarium resistant alfalfa germplasm.

[0005] Currently, identification of Fusarium-resistant alfalfa germplasm relies primarily on natural field disease detection or conventional soil inoculation methods. However, these methods are susceptible to environmental variability, soil microbial interference, and uneven distribution of pathogens, resulting in long identification cycles and inconvenient symptom observation. Therefore, the development of a Fusarium-resistant identification method is urgently needed. Summary of the Invention

[0006] To address the issues of existing methods for identifying Fusarium medicago resistance, which are susceptible to environmental variation, soil microbial interference, and uneven distribution of pathogens, resulting in long identification cycles and inconvenient symptom observation, the present invention aims to provide a method for identifying Fusarium medicago resistance based on a sand culture inoculation system. To achieve this objective, the present invention employs the following technical solutions.

[0007] The present invention provides a method for identifying resistance of Fusarium acetifer to alfalfa based on a sand culture inoculation system, comprising the following steps: Pre-culture before inoculation of sand-cultured seedlings: The sand-cultured seedlings include sand-cultured seedlings and / or sand-cultured cuttings after seed germination; wherein, the pre-culture before inoculation of the sand-cultured seedlings after seed germination is obtained by using 16-26 mesh quartz sand as a cultivation matrix, adopting a matrix-type positioning sowing method, and laying alfalfa seeds at intervals on the cultivation matrix for cultivation; the pre-culture before inoculation of the sand-cultured cuttings is obtained by using 16-26 mesh quartz sand as a cultivation matrix, selecting soil-cultured alfalfa plants as cuttings, adopting a matrix-type positioning cutting method, and then inserting the cuttings into the cultivation matrix for cultivation.

[0008] The sand-cultured seedlings are inoculated with Fusarium spp.

[0009] The sand-cultured seedlings inoculated with Fusarium were disease graded and their incidence rates were calculated. At the same time, the number of diseased plants at each disease grade was counted, and the disease index was calculated based on the corresponding disease grade. The resistance of alfalfa to Fusarium was then evaluated based on the disease index.

[0010] The present invention provides a method for identifying Fusarium resistance of alfalfa based on a sand culture inoculation system. The method for identifying Fusarium resistance of alfalfa provided by the present invention first pre-cultivates sand culture seedlings before inoculation: wherein the sand culture seedlings include sand culture seedlings and / or sand culture cuttings after seed germination; wherein the sand culture seedlings after seed germination are suitable for large-scale rapid screening of variety germplasm; and the sand culture cuttings are suitable for rapid verification of single plant materials. The sand culture seedlings are then inoculated with Fusarium; the sand culture seedlings inoculated with Fusarium are graded and their incidence rates are calculated. At the same time, the number of diseased plants at each level after disease grading is calculated, and the disease index is calculated based on the corresponding level of disease grading; and the resistance of alfalfa to Fusarium root rot is evaluated based on the disease index. The sand culture inoculation system-based method for identifying resistance of alfalfa to Fusarium spp. provided by the present invention uses quartz sand as a culture medium, pre-cultures sand culture seedlings and sand culture cuttings after seed germination, and directly inoculates and identifies the sand culture seedlings and sand culture cuttings after seed germination. The sand culture seedlings used in the present invention include sand culture seedlings and / or sand culture cuttings after seed germination, thereby dividing the sand culture inoculation system into two systems. Sand culture seedlings can be quickly and extensively cultured and identified for resistant varieties, thereby shortening the overall identification cycle, solving the problem of long identification time in conventional methods for identifying Fusarium spp.-resistant germplasm of alfalfa. At the same time, sand culture cuttings can accurately and quantitatively identify resistance of transgenic seedlings and superior germplasm, solving the problem that conventional methods for identifying Fusarium spp.-resistant germplasm of alfalfa can only identify by variety. The addition of quartz sand as a matrix during the sand culture process can create a good growth environment for the root system, which is beneficial to the growth of the alfalfa plant root system. The sandy matrix is ​​relatively loose, and sampling and observing the root system is convenient and quick, and the plant root system can be completely extracted. At the same time, the single sand can greatly reduce the influence of other bacteria in the soil on the accuracy of this experiment, thereby ensuring the singleness of the strain for Fusarium resistance identification. In addition, the identification method of alfalfa Fusarium-resistant germplasm in the existing technology will not be easily affected by environmental variations, soil microbial interference and uneven distribution of pathogens, resulting in long identification cycles and inconvenience in symptom observation.

[0011] The method for identifying resistance to Fusarium medicago provided by the present invention shortens the cycle of a common soil culture inoculation system from 60 to 80 days to 20 to 30 days by establishing a standardized sand culture inoculation system and combining it with multi-parameter quantitative evaluation indicators, thereby significantly improving the efficiency of resistance identification and facilitating symptom observation. It can solve the problem that the identification method of Fusarium medicago resistance germplasm is easily affected by environmental variation, soil microbial interference and uneven distribution of pathogens, resulting in a long identification cycle and inconvenient symptom observation.

[0012] This study utilizes a sand culture inoculation system to establish a standardized sand culture system with a three-pronged coupling of "substrate, nutrition, and environment." By controlling the quartz sand particle size to 16-26 mesh and the paving weight to 200g / 400g, precise simulation of the root development environment is achieved. An innovative dual-channel evaluation model, using sand-cultured seedlings and sand-cultured cuttings after seed germination, simultaneously detects both inherited and induced resistance, enhancing the biological representativeness of the screening results. This ensures a natural simulation of pathogen infection. A comprehensive resistance evaluation matrix, encompassing morphological and physiological indicators, was constructed to enable multidimensional quantitative analysis of resistance phenotypes.

[0013] This method offers significant advantages over traditional soil culture methods: it shortens the experimental cycle and avoids interference with soil microbial communities. The identification of sand-cultured seedlings is suitable for large-scale resistance screening in alfalfa germplasm banks, while the identification of sand-cultured cuttings is suitable for applications such as verifying individual transgenic material and screening for high-quality disease-resistant strains.

[0014] Furthermore, the porosity of the cultivation matrix on which the alfalfa seeds are laid is 40%.

[0015] Furthermore, the alfalfa seeds are laid on the cultivation substrate, and a nutrient solution is added to the cultivation substrate for cultivation; when the seeds sprout to 1 cm, they are moved into a culture medium with a light intensity of 16 hours at 24°C, a dark intensity of 8 hours at 22°C, and a light intensity of 4200 lx for cultivation for 5 days to grow new leaves, thereby obtaining sand-cultured seedlings after the seeds germinate.

[0016] Furthermore, the alfalfa seeds are laid on the cultivation substrate in a standardized layout of 150 seeds per box in a pattern of 10 seeds per row×15 rows.

[0017] Furthermore, after the cuttings are inserted into the cultivation medium, nutrient solution is added for cultivation; one day after the cuttings are inserted, the sand-cultured cuttings are harvested and moved into a medium with a light intensity of 16h / 24°C, a dark intensity of 8h / 22°C, and a light intensity of 4200lx for cultivation for 15 days, so that the sand-cultured cuttings grow new stems, leaves and roots.

[0018] Furthermore, the nutrient solution is 1 / 2 Hoagland nutrient solution.

[0019] Furthermore, the cuttings are implanted on the cultivation substrate in a standardized layout of 40 rows / box constructed in a pattern of 5 plants / row×8 rows.

[0020] The cuttings are the first nodes of branches of alfalfa plants with a stem base diameter of 2.0 mm.

[0021] Furthermore, the disease index is negatively correlated with the resistance of alfalfa to Fusarium.

[0022] Among them, the corresponding levels of the disease classification are divided into 0~5 levels, which are set according to the degree of root rot of the sand-cultured seedlings after inoculation with Fusarium. The degree of root rot of the sand-cultured seedlings after inoculation with Fusarium is positively correlated with the corresponding level of the disease classification.

[0023] Furthermore, when the whole plant of the sand-cultured seedlings after inoculation with Fusarium is healthy and there is no root rot, the corresponding level of the disease grading is level 0; when the degree of root rot of the sand-cultured seedlings after inoculation with Fusarium is ≤25%, the corresponding level of the disease grading is level 1; when the degree of root rot of the sand-cultured seedlings after inoculation with Fusarium is 26%~50%, the corresponding level of the disease grading is level 2; when the degree of root rot of the sand-cultured seedlings after inoculation with Fusarium is 51%~75%, the corresponding level of the disease grading is level 3; when the degree of root rot of the sand-cultured seedlings after inoculation with Fusarium is ≥76%, the corresponding level of the disease grading is level 4; when the whole plant of the sand-cultured seedlings after inoculation with Fusarium is completely necrotic, the corresponding level of the disease grading is level 5.

[0024] Furthermore, the calculation formula of the disease index is as follows: Incidence rate = (number of diseased plants / total number of plants) × 100%; In the above formula, the unit of incidence is 100%; the number of diseased plants refers to the alfalfa plants with root rot symptoms in the treatment group; the total number of plants refers to the total number of alfalfa plants in the treatment group; Disease index = ∑ (number of diseased plants at each level × corresponding level) / (total number of plants × highest level) × 100; In the above formula, the number of diseased plants at each level refers to the number of diseased alfalfa plants under the grading standard; the corresponding level refers to the disease grading level 0 to level 5; the total number of plants refers to the number of all alfalfa plants in the treatment group; the highest level refers to the disease level 5.

[0025] Furthermore, a resistance grade is set based on the disease index, and the resistance grade is used to further evaluate the resistance of alfalfa to Fusarium. The larger the disease index, the higher the corresponding resistance grade, and thus the lower the resistance of alfalfa to Fusarium.

[0026] Among them, when the disease index is ≤0.20, the corresponding level of the resistance grading is level 1, and the corresponding alfalfa has high resistance to Fusarium; when the disease index is 0.20~0.40, the corresponding level of the resistance grading is level 2, and the corresponding alfalfa has medium resistance to Fusarium; when the disease index is 0.40~0.60, the corresponding level of the resistance grading is level 3, and the corresponding alfalfa has medium resistance to Fusarium; when the disease index is greater than or equal to ≥0.60, the corresponding level of the resistance grading is level 4, and the corresponding alfalfa has high resistance to Fusarium.

[0027] Furthermore, the variety of the alfalfa seeds and the alfalfa plants is Pastoral.

[0028] Furthermore, the Fusarium is Fusarium oxysporum.

[0029] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a method for identifying Fusarium resistance of alfalfa based on a sand culture inoculation system. The method for identifying Fusarium resistance of alfalfa provided by the present invention is pre-culture of sand culture seedlings before inoculation: wherein the sand culture seedlings include at least one of sand culture seedlings and sand culture cuttings after seed germination; then the sand culture seedlings are inoculated with Fusarium; the sand culture seedlings inoculated with Fusarium are graded and their incidence rates are counted, and at the same time, the number of diseased plants at each level after the disease grading is counted, and the disease index is calculated based on the corresponding level of the disease grading; then the resistance of alfalfa to Fusarium root rot is evaluated according to the disease index. The present invention uses quartz sand as a culture medium, pre-cultures sand culture seedlings and sand culture cuttings after seed germination, and directly inoculates Fusarium on the sand culture seedlings and sand culture cuttings after seed germination and identifies them. The sand culture inoculation system-based method for identifying resistance of alfalfa to Fusarium spp. provided by the present invention uses quartz sand as a culture medium, pre-cultures sand culture seedlings and sand culture cuttings after seed germination, and directly inoculates and identifies the sand culture seedlings and sand culture cuttings after seed germination. The sand culture seedlings used in the present invention include sand culture seedlings and / or sand culture cuttings after seed germination, thereby dividing the sand culture inoculation system into two systems. Sand culture seedlings can be quickly and extensively cultured and identified for resistant varieties, thereby shortening the overall identification cycle, solving the problem of long identification time in conventional methods for identifying Fusarium spp.-resistant germplasm of alfalfa. At the same time, sand culture cuttings can accurately and quantitatively identify resistance of transgenic seedlings and superior germplasm, solving the problem that conventional methods for identifying Fusarium spp.-resistant germplasm of alfalfa can only identify by variety. The addition of quartz sand as a matrix during the sand culture process can create a good growth environment for the root system, which is beneficial to the growth of the alfalfa plant root system. The sandy matrix is ​​relatively loose, and sampling and observing the root system is convenient and quick, and the plant root system can be completely extracted. At the same time, the single sand can greatly reduce the influence of other bacteria in the soil on the accuracy of this experiment, thereby ensuring the singleness of the strain for Fusarium resistance identification. In addition, the identification method of alfalfa Fusarium-resistant germplasm in the existing technology will not be easily affected by environmental variations, soil microbial interference and uneven distribution of pathogens, resulting in long identification cycles and inconvenience in symptom observation.

[0030] The method for identifying resistance to Fusarium medicago provided by the present invention shortens the cycle of the common soil culture inoculation system from 60 to 80 days to 14 to 30 days by establishing a standardized sand culture inoculation system and combining it with a multi-parameter quantitative evaluation index. This significantly improves the efficiency of resistance identification and facilitates symptom observation. It can solve the problem that the identification method of Fusarium medicago resistant germplasm is easily affected by environmental variation, soil microbial interference, and uneven distribution of pathogens, resulting in a long identification cycle and inconvenience for symptom observation. Among them, the "multi-parameter quantitative evaluation index" refers to the statistical sand culture time, the incidence rate of sand culture seedlings after inoculation with Fusarium, and the disease index.

[0031] 2. The method for identifying resistance of Fusarium acetifer to alfalfa based on a sand culture inoculation system provided by the present invention also has the following advantages: (1) Experimental efficiency is significantly improved: Shortened cycle: Through standardized sand culture system and dynamic environmental control, the entire sand culture seed germination and inoculation experiment only takes about 14 days, and the entire sand culture cutting seedling inoculation experiment only takes about 30 days. The traditional soil culture inoculation method requires more than 60 days, and the efficiency is significantly improved; accelerated phenotypic manifestation: based on light-temperature coupling control and optimization of blue-red light ratio, plant metabolism and pathogen infection process are promoted, and interference factors are eliminated: the sand culture system avoids interference from soil microbial communities through quartz sand sterilization and soilless environment control; dual-mode verification: sand culture seedlings and sand culture cutting seedlings after seed germination are used simultaneously, and dual-channel data cross-validation is used to reduce the false positive / negative rate.

[0032] (2) Cost savings and ease of operation: Low material cost: Quartz sand can be sterilized at high temperature and reused ≥5 times, and the cost of a single experiment is low; low technical threshold: through sequential inoculation technology and standardized operating procedures, it can be implemented without complex equipment.

[0033] (3) Advantages of matrix transparency and separability: Low adsorption characteristics of quartz sand: Quartz sand with a mesh size of 16~26 and a particle size of 0.6mm~1.2mm is used. Its surface is smooth and non-sticky. When the plant is taken out, the sand particles can fall off naturally without strong washing. The root system is relatively intact. The traditional soil culture method is easy to break when only the roots are taken; non-destructive sampling: The loose structural characteristics of the sand culture matrix allow the roots and sand particles to be separated by gentle vibration, avoiding mechanical damage and ensuring that the natural state of the root morphology is preserved.

[0034] (4) Advantages of pathological observation: Visualization of the infection process: After inoculation with Fusarium, pathological characteristics such as hyphae attachment, root surface browning, and vascular bundle infection can be directly observed through the transparent sand culture system, supporting infection timeline tracking; a transparent observation window can be embedded in the side wall of the sand culture box, combined with a laser confocal microscope, to achieve microscopic observation of living root systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The results are a comparison of the plants of the treatment group and the control group after inoculation of Fusarium 14 days in the sand culture cutting seedlings of the present invention; wherein, Figure 1 A in the figure is the treatment group, and the three samples are parallel samples; Figure 1 B in the figure is the control group, and the three samples are parallel samples.

[0036] Figure 2The root systems of the sand-grown cutting seedlings inoculated with Fusarium spp. for 14 days were compared between the treatment group and the control group; Figure 2 A in the figure is the treatment group, and multiple samples are parallel samples; Figure 2 B in the figure is the control group, and multiple samples are parallel samples.

[0037] Figure 3 This is the state of the sand-cultured cutting seedlings during cutting in the present invention, wherein the multiple samples are parallel samples.

[0038] Figure 4 This is the 14-day growth state of the sand-cultured cutting seedlings of the present invention, wherein the multiple samples are parallel samples.

[0039] Figure 5 This is the state of seedlings being laid in sand culture in the present invention, wherein the multiple samples are parallel samples.

[0040] Figure 6 This is the growth state of the seedlings in sand culture for 7 days in the present invention, and the multiple samples are parallel samples.

[0041] Figure 7 This is the root status of the seeds in sand culture for 7 days in the present invention, and the three samples are parallel samples.

[0042] Figure 8 The results are compared between the seed sand culture inoculation with Fusarium for 5 days and the control group, wherein: Figure 8 A in the figure is the treatment group, and the three samples are parallel samples; Figure 8 B in the figure is the control group, and the three samples are parallel samples. DETAILED DESCRIPTION

[0043] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0044] Example 1 A method for identifying Fusarium spp. inoculated in alfalfa seeds in a rapid sand culture, comprising the following steps: (1) Seed preparation: The alfalfa seeds were cleaned, disinfected and sterilized, cultured in a double-layer filter paper plate in a dark environment at 4°C, and vernalized for 1 day to obtain vernalized alfalfa seeds.

[0045] Among them, the variety of alfalfa seeds is Pastoral, which was purchased from Claw (Beijing) Ecological Technology Co., Ltd.

[0046] The method of cleaning, disinfection and sterilization includes the following steps: First, rinse with sterile water twice, 2 minutes each; then wash with 75% alcohol by mass for 5 minutes; then rinse with sterile water twice, 2 minutes each; then wash with 20% sodium hypochlorite by mass for 20 minutes; finally, rinse with sterile water until clear.

[0047] (2) Preparation of sand culture medium box: 16-mesh quartz sand was used as the cultivation medium, and was rinsed with sterile water three times to remove impurities on its surface, and then sterilized by high pressure. The sterilized quartz sand was placed in a sand culture box to form a sand culture medium box.

[0048] Among them, the specifications of the sand culture box are 10cm×6cm×4cm.

[0049] During the seed germination stage, 200g of quartz sand is placed in each sand culture box. Gradual weight increase creates differentiated root development spaces, serving as the cultivation medium. The thickness of the cultivation medium during the seed germination stage is precisely controlled at 3.0cm ± 0.2cm, and the porosity is maintained at 40%, ensuring a dynamic balance between oxygen permeability and water retention.

[0050] (3) Seed germination: Using matrix positioning sowing technology, a standardized layout of 150 seeds per box was constructed in the mode of 10 seeds per column × 15 columns. The prepared alfalfa seeds after vernalization were spread flat in the sand culture matrix box. Figure 5 1 / 2 Hoagland nutrient solution was used for water and fertilizer management. After 2 days of light growth, the alfalfa seeds sprouted new buds and entered the germination period. After the seeds in the germination period germinated, the sand culture seedlings were obtained after the seeds germinated. Figure 6 Among them, the sand-cultured seedlings after seed germination are referred to as sand-cultured seedlings for short.

[0051] During the germination period, the daily replenishment rate for sand-cultured seedlings should be 60% of the saturated water holding capacity of the cultivation medium. During the initial stage, wrap the sand culture medium box with plastic wrap to seal it and reduce water loss, thereby achieving micro-environmental humidity control. For the first three days after alfalfa seeds sprout, maintain a high relative humidity of 95% within the sand culture medium box. Starting from the fourth day after the alfalfa seeds sprout, remove the plastic wrap from the sand culture medium box daily and ventilate it for two hours to reduce the humidity gradient within the sand culture medium box.

[0052] Among them, the humidity gradient decrease refers to a daily humidity drop of ≤5%.

[0053] The formula for 1 / 2 Hoagland nutrient solution is shown in Table 1.

[0054] Table 1 1 / 2 Hoagland nutrient solution formula

[0055] Note: When preparing 1 / 2 Hoagland nutrient solution, dissolve each chemical separately before mixing. Store the prepared stock solution in a brown bottle in the dark. To prepare the nutrient solution, dilute Stock Solutions A and B by 200-fold, dilute Stock Solution C by 1000-fold, and dilute Stock Solution D (iron salt) by 200-fold. Stock Solution A is Liquid A, Stock Solution B is Liquid B, Stock Solution C is Liquid C, and Stock Solution D (iron salt) is Liquid D (iron salt). " / " indicates no mass.

[0056] (4) Constructing a 16-hour light period / 8-hour dark period photoperiod system: When the new sprouts of alfalfa seeds grow to 1 cm, they are moved into a light incubator with a light intensity of 16 h / 24 ° C, 8 h / 22 ° C, and a light intensity of 4200 lx and cultured for 5 days to grow new leaves and obtain sand culture seedlings. Figure 7 .

[0057] (5) Preparation of inoculum: Fusarium was inoculated into PDB liquid culture medium and cultured on a shaker at 27°C and 150 rpm for 14 days to obtain Fusarium spore liquid.

[0058] The Fusarium species mentioned is Fusarium oxysporum, a common cause of alfalfa root rot. This species is described in the literature "Jia Lei, He Jiepu, Wu Chongbin, et al. Isolation, Identification, and Pathogenicity Analysis of the Pathogen Causing Root Rot of Alfalfa in Hohhot [J / OL]. Acta Grasslandiae Sinica, 1-17 [2025-08-06]. 1935.006." This species is identical to the Fusarium oxysporum described in the literature. The applicant also possesses the same species and guarantees that it will be made available to the public within 20 years from the date of application. The College of Grassland Science at Qingdao Agricultural University can provide this information to the public upon request.

[0059] The preparation method of PDB liquid culture medium is as follows: 200g potatoes, 20g glucose, add distilled water to prepare 1000mL.

[0060] (6) Infection of sand-cultured seedlings with fungi: Adjust the concentration of Fusarium spores in PDB liquid medium to 5 × 10 6 The spores / mL were used to obtain a spore suspension. The blood cell pool of the blood cell counting chamber was of the 25×16 type.

[0061] Take the sand culture seedlings obtained in step (4) and divide them into treatment groups Figure 2 A and control group Figure 2 B, 75 plants in each group.

[0062] Inoculate the sand-cultured seedlings in the treatment group with 20 mL of spore suspension in the sand-cultured substrate boxes. Add 20 mL of sterile water to the sand-cultured seedlings in the control group. After inoculation, the sand-cultured substrate boxes in both groups were placed in a light incubator with a light intensity of 4200 lx and a light intensity of 16 h / 24°C, 8 h / 22°C, and 4200 lx for 7 days. When the leaves and roots of the sand-cultured seedlings showed signs of yellowing, dwarfing, or root blackening and rot, the incidence rates were recorded and photographed.

[0063] (7) Calculation of incidence rate: The disease of the sand-cultured seedlings in the treatment group and the control group were graded according to the proportion of the lesion area of ​​the sand-cultured seedlings after inoculation with Fusarium spp., and their incidence rates were calculated.

[0064] Among them, the corresponding levels of disease grading are divided into 0 to 5 levels, and the specific standards are as follows: Level 0: The whole plant is healthy and disease-free, and there is no root rot.

[0065] Level 1: Root rot degree ≤ 25%.

[0066] Level 2: Root rot degree 26%~50%.

[0067] Level 3: Root rot degree 51%~75%.

[0068] Level 4: Root rot degree ≥76%.

[0069] Level 5: The entire plant is completely necrotic.

[0070] Among them, the degree of root rot refers to the scope of tissue death caused by pathogen infection.

[0071] The formula for calculating the incidence rate is as follows: Incidence rate = (number of diseased plants / total number of plants) × 100%; In the above formula, the unit of incidence is 100%; the number of diseased plants refers to the alfalfa plants in the treatment group that showed root rot symptoms; the total number of plants refers to the total number of alfalfa plants in the treatment group. Among them, alfalfa plants refer to sand-grown seedlings.

[0072] (8) Calculation of disease index: The disease index of the treatment group was calculated according to the following formula: Disease index = ∑ (number of diseased plants at each level × corresponding level) / (total number of plants × highest level) × 100; In the above formula, the number of diseased plants at each level refers to the number of diseased alfalfa plants according to the grading standard; the corresponding grade refers to the disease grade 0 to 5; the total number of plants refers to the number of all alfalfa plants in the treatment group; and the highest grade refers to disease grade 5. Alfalfa plants refer to sand-grown seedlings.

[0073] (9) The data of the average seedling length, average root length, incidence rate and disease index of the sand-cultured seedlings in the above-mentioned treatment group and control group were statistically analyzed, and the resistance of alfalfa to Fusarium was graded according to the size of the disease index. The specific resistance grades of alfalfa to Fusarium are as follows:

[0074] Level 1: Disease index ≤ 0.20, corresponding to high resistance of alfalfa to Fusarium.

[0075] Level 2: 0.20<Disease index≤0.40, corresponding to moderate resistance of alfalfa to Fusarium.

[0076] Level 3: 0.40<Disease index≤0.60, corresponding to moderate resistance of alfalfa to Fusarium.

[0077] Level 4: Disease index > 0.60, corresponding to high susceptibility of alfalfa to Fusarium.

[0078] The results are shown in Table 2.

[0079] Table 2 Data statistics of sand-cultured seedlings 7 days after inoculation with Fusarium

[0080] As shown in Table 2, after the sand-cultured seedlings were cultured for 7 days, they were inoculated with Fusarium. Obvious symptoms appeared 5 days after inoculation. Compared with the control group, the plants in the treatment group were short, with yellow stems and leaves, root rot and shorter roots. The average seedling length was 2.3 cm, the average root length was 3.4 cm, the incidence rate was 93.5%, and the disease index was 0.4328. According to the disease index, its resistance level to Fusarium was identified as level 3, indicating that its resistance to Fusarium was moderate.

[0081] Example 2 A method for identifying alfalfa cuttings by rapid sand culture inoculation of Fusarium spp. comprises the following steps: (1) Preparation of sand culture cuttings: The soil-cultured mother plants were strictly controlled within a 30-day growth period, and branches with a diameter of 2.0 mm were selected as cutting sources. Cuttings were cut using a double-section differential cutting technique, with the upper incision perpendicular to the branch axis and the lower incision at a 45-degree angle. Leaf buds were selected from the third node in the budding stage.

[0082] Among them, the soil-cultured mother plant is the soil-cultured alfalfa plant, which is a high-quality disease-resistant alfalfa single-plant potted seedling that has been screened for Fusarium.

[0083] The cuttings were the first nodes of alfalfa plants with a stem base diameter of 2.0 mm.

[0084] (2) Preparation of sand culture medium box: 16-mesh quartz sand is used as the cultivation medium. It is rinsed three times with sterile water to remove surface impurities and then sterilized by autoclaving. Each sand culture box is filled with 400g of quartz sand, creating a differentiated root development space through gradient weight gain.

[0085] It should be noted that the thickness of quartz sand during the sand culture cutting seedling cultivation stage is precisely controlled at 3.5cm±0.2cm, and the porosity of the sand particles is maintained in the range of 40% to ensure a dynamic balance between oxygen penetration and water retention.

[0086] Among them, the specifications of the sand culture box are 10cm×6cm×4cm.

[0087] (3) Cultivation of sand-grown cuttings: Using matrix positioning cutting technology, a standardized layout of 40 rows / boxes is constructed in a pattern of 5 plants / row × 8 rows. Figure 3 Each box is used for cuttings. The lower end of the cutting is inserted obliquely into the quartz sand matrix, with the upper end of the cutting facing the outside. Then, 1 / 2 Hoagland nutrient solution is added. In the early stage of sand culture cuttings, the sand culture matrix box is wrapped with plastic wrap to seal it and reduce water loss, so as to achieve micro-environment humidity control.

[0088] The sand-cultured seedlings were moved into a light incubator with a light intensity of 4200±300lx and a light intensity of 16h / 24℃ and 8h / 22℃ for 15 days to allow them to grow new leaves and roots. Figure 4 .

[0089] (4) Preparation of inoculum: Fusarium was inoculated into PDB liquid culture medium and cultured on a shaker at 27°C and 150 rpm for 14 days to obtain an inoculum spore solution.

[0090] (5) Infection of cutting seedlings with fungi: The concentration of Fusarium spores was adjusted to 5 × 10 6 Spores / mL were used to obtain a spore suspension. The specification of the hemocytometer was 25×16.

[0091] Take the sand culture cuttings obtained in step (3) and divide them into a treatment group and a control group, with 20 seedlings in each group. Add 40 mL of spore suspension to the sand culture matrix box of the sand culture cuttings in the treatment group for inoculation. Add 40 mL of sterile water to the sand culture matrix box of the sand culture cuttings in the control group. After inoculation, the sand culture matrix boxes of the above two groups were placed in a light incubator with a light intensity of 16h / 24℃, 8h / 22℃, and 4200±300lx for 14 days. When the leaves of the sand culture cuttings turned yellow and the rhizomes showed brown to black with rot and softening symptoms, the incidence data were counted and recorded by taking photos. Figure 2 .

[0092] (6) Calculation of incidence rate: The percentage of lesion area of ​​sand culture cutting seedlings after inoculation with Fusarium spp. in the treatment groups was compared. Figure 1 A and control group Figure 1 The sand-cultured cuttings of B were graded for disease and their incidence rates were calculated.

[0093] Among them, the corresponding levels of disease grading are divided into 0 to 5 levels, and the specific standards are as follows: Level 0: The whole plant is healthy and disease-free, and there is no root rot.

[0094] Level 1: Root rot degree ≤ 25%.

[0095] Level 2: Root rot degree 26%~50%.

[0096] Level 3: Root rot degree 51%~75%.

[0097] Level 4: Root rot degree ≥76%.

[0098] Level 5: The entire plant is completely necrotic.

[0099] The formula for calculating the incidence rate is as follows: Incidence rate = (number of diseased plants / total number of plants) × 100%; In the above formula, the unit of incidence is 100%; the number of diseased plants refers to the alfalfa plants in the treatment group that showed root rot symptoms; the total number of plants refers to the total number of alfalfa plants in the treatment group. Among them, alfalfa plants refer to sand-grown cuttings.

[0100] (7) Calculation of disease index: The disease index of the treatment group was calculated according to the following formula: Disease index = ∑ (number of diseased plants at each level × corresponding level) / (total number of plants × highest level) × 100; In the above formula, the number of diseased plants at each level refers to the number of diseased alfalfa plants according to the grading standard; the corresponding grade refers to the disease grade 0-5; the total number of plants refers to the number of all alfalfa plants in the treatment group; and the highest grade refers to disease grade 5. Alfalfa plants refer to sand-grown cuttings.

[0101] (8) The data of the average seedling length, average root length, incidence rate and disease index of the sand culture cuttings in the above treatment group and the control group were statistically analyzed, and the resistance of alfalfa to Fusarium was graded according to the disease index. The specific resistance grades of alfalfa to Fusarium are as follows:

[0102] Level 1: Disease index ≤ 0.20, corresponding to high resistance of alfalfa to Fusarium.

[0103] Level 2: 0.20<Disease index≤0.40, corresponding to moderate resistance of alfalfa to Fusarium.

[0104] Level 3: 0.40<Disease index≤0.60, corresponding to moderate resistance of alfalfa to Fusarium.

[0105] Level 4: Disease index > 0.60, corresponding to high susceptibility of alfalfa to Fusarium.

[0106] The results are shown in Table 3.

[0107] Table 3 Statistics of cutting seedlings 14 days after inoculation with Fusarium

[0108] As shown in Table 3, the sand-cultured cuttings began to grow roots 14 days later, with an average rooting rate of 92.8%. Figure 8 The incidence rate was 87.4%, and the disease index was 0.3978. According to its disease index, its resistance level to Fusarium was identified as level 2, indicating that its resistance to Fusarium was moderate.

[0109] Comparative Example 1 A soil culture inoculated Fusarium acetalicum identification method comprises the following steps: (1) Inoculum preparation: The Fusarium acetosporum isolated from the diseased plants in the alfalfa planting area of ​​Inner Mongolia was selected as the dominant strain. The strain was transferred to a PDA plate and cultured at 25°C for 4 days. A 6 mm diameter bacterial cake at the edge of the colony was cut and inoculated into the wheat bran medium. The culture was continued for 7 days. The wheat bran culture was washed with sterile water and filtered through four layers of gauze to obtain the spore suspension. The concentration was adjusted to 5 × 10 6 pieces / mL.

[0110] (2) Seed treatment and seedling cultivation: Alfalfa seeds were soaked in 3% sodium hypochlorite for 2 minutes, disinfected with 75% ethanol for 30 seconds, and rinsed with sterile water 5 times.

[0111] Fill the trays with nutrient soil, sow seeds, and incubate in a greenhouse at 25°C and 35% humidity. Seedlings are inoculated when they have three compound leaves. Using sterilized soil, environmental exposure after transplanting can lead to the recolonization of soil microbial communities such as bacteria and actinomycetes. These microbes compete with Fusarium for nutrients or secrete antibacterial substances, significantly inhibiting the pathogen's infection efficiency and interfering with the accuracy of resistance assessment.

[0112] Among them, nutrient soil: nutrient soil: vermiculite: perlite = 3:2:1 are mixed according to the mass ratio of 1:1.

[0113] (3) Inoculation operation: Soak the roots of the seedlings in a solution with a concentration of 5×10 6 The cells were placed in a spore suspension of 500 ml / mL for 30 minutes, and then transplanted into nutrient pots in sterilized nutrient soil, with 5 plants per pot, repeated 3 times, and inoculated with a highly susceptible alfalfa variety as a susceptible control and a highly resistant variety as a resistant control.

[0114] Among them, the height of the nutrient pot is 10 cm and the diameter of the nutrient pot is 10 cm.

[0115] (4) Management after inoculation: Place the susceptible control and resistant control plants in step 3 in a 25°C greenhouse with 12 hours of light per day, keep the substrate moist, and water once a day.

[0116] (5) Disease investigation and analysis: 35 days after inoculation, the total soil culture period was 65 days, which was significantly longer than the 30-day period of sand culture. Disease investigation was conducted on the soil-cultured alfalfa seedlings after inoculation, and the disease index was calculated. The resistance level was divided into 0-4 levels according to the disease index.

[0117] The inoculated soil-grown alfalfa seedlings refer to the susceptible control and disease-resistant control plants in step 3.

[0118] The disease index is abbreviated as DI and is calculated according to the following formula: Disease index = ∑ (number of diseased plants at each level × corresponding level) / (total number of plants × highest level) × 100; In the above formula, the number of diseased plants at each level refers to the number of diseased plants under the grading standard; the corresponding level refers to the disease grading level 0 to level 4; the total number of plants refers to the number of all alfalfa plants in the treatment group; the highest level number refers to the disease level 4.

[0119] The method for identifying Fusarium spp. inoculated in soil culture provided in this comparative example suffers from the problems of complex soil microorganisms and a long soil culture cycle. Compared with Comparative Example 1, the method for identifying Fusarium spp. inoculated in alfalfa cuttings by rapid sand culture provided in Example 1 and the method for identifying Fusarium spp. inoculated in alfalfa cuttings by rapid sand culture provided in Example 2 do not suffer from the problems of complex soil microorganisms and a long soil culture cycle.

[0120] Comparative Example 2 A method for identifying Fusarium truncatum inoculated by hydroponic culture, comprising the following steps: (1) Seed preparation: Soak alfalfa seeds in 3% sodium hypochlorite for 2 minutes, sterilize with 75% ethanol for 30 seconds, rinse with sterile water five times, and sow in sterilized hydroponic containers. Under hydroponic conditions, the roots are suspended in the nutrient solution and lack the support of soil or gravel substrate, which makes the seedlings prone to lodging and affects the stability of the pathogen infection process.

[0121] (2) Reproduction of pathogen inoculum: The Fusarium spp. isolated from the diseased plants in the alfalfa planting area of ​​Inner Mongolia was selected as the dominant strain, and the strain was transferred to PDA solid medium and cultured at 25°C for 7 days. A 6 mm diameter bacterial cake was cut from the edge of the colony grown on PDA solid medium for 5 days and transferred to PDB liquid medium. The culture was shaken at 25°C and 150 r / min for 4 days, centrifuged at 4000 r / min for 10 min, and the supernatant was discarded. The mycelium was rinsed with sterile water 3 times, 200 mL of sterile water was added, and the suspension was allowed to stand for 24 h. The spore suspension was filtered through four layers of gauze and the concentration was adjusted to 5×10 6 This process requires repeated preparation of PDA / PDB culture medium, multiple centrifugation steps, and sterile manipulation. This process requires frequent use of consumables and equipment, increasing the cost per treatment by over 40% compared to sand culture methods. Furthermore, the process involves multiple independent steps, such as centrifugation and sterile filtration, requiring the operator to possess specialized microbiology laboratory training. A single preparation takes ≥5 hours, significantly longer than preparing the sand culture inoculum, and has a low tolerance for operational errors. Consumables include centrifuge tubes and gauze; equipment includes a centrifuge and shaker.

[0122] (3) Pathogen inoculation: Add 50 mL of sterile water to each dish. When the seedlings grow to 3 compound leaves, evenly pour the spore suspension to the roots. The inoculation amount for each plant is 1 mL.

[0123] (4) Control of hydroponic conditions: Temperature: constant at 26°C, photoperiod 12h / 12h, light intensity 3000lx, humidity: maintain relative humidity at 80% to prevent the substrate from drying out. Observe root system changes daily after inoculation, continue cultivation for 14 days, and record the incidence of disease.

[0124] (5) Disease investigation and grading: 14 days after inoculation, the disease of the inoculated alfalfa hydroponic seedlings was investigated, the disease index was calculated, and the resistance levels were divided into 0 to 4 levels according to the disease index.

[0125] The disease index is abbreviated as DI in English and is calculated according to the following formula: Disease index = ∑ (number of diseased plants at each level × corresponding level) / (total number of plants × highest level) × 100; In the above formula, the number of diseased plants at each level refers to the number of diseased plants under the grading standard; the corresponding level refers to the disease grading level 0 to level 4; the total number of plants refers to the number of all alfalfa plants in the treatment group; the highest level number refers to the disease level 4.

[0126] The hydroponic inoculation method of Fusarium provided in this comparative example has the problems of poor root support, high experimental cost and low operability.

[0127] Compared with Comparative Example 2, the identification method for alfalfa cuttings rapidly inoculated with Fusarium in sand culture provided in Example 1 and the identification method for alfalfa cuttings rapidly inoculated with Fusarium in sand culture provided in Example 2 do not have the problems of poor root support, high experimental cost and low operability.

[0128] In summary, the sand culture inoculation system provided by the present invention addresses the long cultivation period of Fusarium spp. in soil culture inoculation and reduces the presence of microorganisms in the system. It also addresses the poor root support, high experimental costs, and low operability issues of hydroponic inoculation. The sand culture inoculation system innovatively utilizes gravel as a cultivation mechanism, resulting in a low contamination risk, easy root observation, and a short experimental period, providing a new inoculation system for disease-resistant alfalfa breeding.

[0129] It should be noted that when the present invention involves a numerical range, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes a preferred embodiment.

[0130] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once the basic inventive concepts become known, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A method for identifying resistance to Fusarium acetifer in alfalfa plant based on a sand culture inoculation system, characterized in that: The steps include: Pre-cultivation of sand-cultured seedlings before inoculation: The sand-cultured seedlings include sand-cultured seedlings and / or sand-cultured cuttings after seed germination; wherein the pre-cultivation of the sand-cultured seedlings after seed germination before inoculation is achieved by using 16-26 mesh quartz sand as a cultivation matrix, adopting a matrix-type positioning sowing method, and laying alfalfa seeds at intervals on the cultivation matrix for cultivation; the pre-cultivation of the sand-cultured cuttings before inoculation is achieved by using 16-26 mesh quartz sand as a cultivation matrix, selecting soil-cultured alfalfa plants as cuttings, and adopting a matrix-type positioning cutting method, and then cultivating the cuttings after inserting the cuttings into the cultivation matrix; Inoculating the sand-cultured seedlings with Fusarium spp. The sand-cultured seedlings inoculated with Fusarium were disease graded and their incidence rates were calculated. At the same time, the number of diseased plants at each disease grade was counted, and the disease index was calculated based on the corresponding disease grade. The resistance of alfalfa to Fusarium was then evaluated based on the disease index.

2. The method for identifying resistance of Fusarium acetifer to alfalfa based on a sand culture inoculation system according to claim 1, characterized in that: The porosity of the cultivation matrix on which the alfalfa seeds are laid is 40%.

3. The method for identifying resistance of Fusarium acetifer to alfalfa based on a sand culture inoculation system according to claim 1, wherein: The alfalfa seeds are laid on the cultivation substrate, and a nutrient solution is added to the cultivation substrate for cultivation; when the seeds sprout to a height of 1 cm, the seeds are moved to a cultivation medium under conditions of 16 hours of light at 24°C, 8 hours of darkness at 22°C, and a light intensity of 4200 lx for cultivation for 5 days to grow new leaves, thereby obtaining sand-cultured seedlings after the seeds germinate.

4. The method for identifying resistance of Fusarium acetifer to alfalfa based on a sand culture inoculation system according to claim 3, wherein: The alfalfa seeds are laid on the cultivation substrate in a standardized layout of 150 seeds per box in a pattern of 10 seeds per row×15 rows.

5. The method for identifying resistance of Fusarium acetifer to alfalfa based on a sand culture inoculation system according to claim 1, wherein: After the cuttings are inserted into the cultivation medium, nutrient solution is added for cultivation; one day after the cuttings, the sand-cultured cuttings are harvested and moved into a medium with a light intensity of 16 hours at 24°C, 8 hours at 22°C, and 4200 lx for cultivation for 15 days, so that the sand-cultured cuttings grow new stems, leaves, and roots.

6. The method for identifying resistance of Fusarium acetifer to alfalfa based on a sand culture inoculation system according to any one of claims 3 or 5, characterized in that: The nutrient solution is 1 / 2 Hoagland nutrient solution.

7. The method for identifying resistance of Fusarium Medicago sativae based on a sand culture inoculation system according to claim 5, characterized in that: The cuttings are inserted into the cultivation substrate in a standardized layout of 40 rows / box in a pattern of 5 plants / row×8 rows; The cuttings are the first nodes of branches of alfalfa plants with a stem base diameter of 2.0 mm.

8. The method for identifying resistance of Fusarium Medicago sativae based on a sand culture inoculation system according to claim 1, characterized in that: The disease index is negatively correlated with alfalfa's resistance to Fusarium; Among them, the corresponding levels of the disease classification are divided into 0~5 levels, which are set according to the degree of root rot of the sand-cultured seedlings after inoculation with Fusarium. The degree of root rot of the sand-cultured seedlings after inoculation with Fusarium is positively correlated with the corresponding level of the disease classification.

9. The method for identifying resistance of Fusarium Medicago sativae based on a sand culture inoculation system according to claim 1, characterized in that: The variety of the alfalfa seeds and the alfalfa plants is Pastoral.

10. The method for identifying resistance of Fusarium Medicago sativae based on a sand culture inoculation system according to claim 1, characterized in that: The Fusarium is Fusarium oxysporum.

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