Method for simultaneously evaluating smut resistance and brown streak resistance of sugarcane

Abstract

The invention discloses a method for simultaneously evaluating smut resistance and brown streak resistance of sugarcane, and belongs to the field of sugarcane disease resistance evaluation. The method comprises the following steps: (1) collecting teliospores of sugarcane smut and culturing sugarcane brown streak bacteria; (2) artificially inoculating sugarcane smut by adopting a seed soaking method, and inoculating sugarcane brown streak after the sugarcane smut is inoculated for 30-35 days and sugarcane seedlings grow to 25-35 cm; (3) evaluating the disease resistance of the sugarcane smut: when the sugarcane seedlings grow out and the first smut occurs, starting to investigate the seedling emergence plant number and the morbidity plant number of the sugarcane, and dividing the disease resistance grade according to the morbidity; evaluating the disease resistance of the sugarcane brown streak: investigating the morbidity of the sugarcane brown streak 7-10 days after inoculating the sugarcane brown streak, and dividing disease resistance grades according to the area of infected leaves. According to the method, the disease resistance evaluation results of the two diseases can be obtained at the same time through one-time testing, and technical support is provided for prevention and treatment of smut and brown streak disease of sugarcane, rapid screening of disease-resistant breeding and breeding of disease-resistant varieties.

Description

Technical Field

[0001] The invention relates to a technology for evaluating the disease resistance of sugarcane, and in particular to a method for simultaneously evaluating the resistance of sugarcane to smut and brown streak. Background Art

[0003] The selection and application of disease-resistant varieties is the most economical and effective measure to prevent and control sugarcane smut and brown streak. The evaluation of sugarcane smut and brown streak resistance is particularly important in disease-resistant breeding and is a key link in disease-resistant breeding. At present, the disease resistance evaluation of sugarcane smut and brown streak is carried out separately and independently, so to obtain the disease resistance results of the two diseases, they need to be done separately, which is time-consuming, labor-intensive and inoculated with materials. In addition, the existing disease resistance evaluation grade classification of sugarcane smut is too broad and not strict for the classification of sugarcane smut resistance, which is no longer suitable for sugarcane disease-resistant breeding at this stage. This low efficiency seriously affects the speed of disease-resistant breeding. If the two diseases of smut and brown streak can be evaluated in the same batch of test materials without interfering with or affecting each other, the defects of the disease resistance evaluation of the two diseases can be solved. In summary, the simultaneous disease resistance evaluation of sugarcane smut and brown streak has become an urgent requirement for current disease-resistant breeding. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems existing in the prior art and provide a method for evaluating the "double resistance" of sugarcane smut and brown streak disease. The present invention can obtain the disease resistance evaluation results of two diseases at the same time in one test, so as to provide technical support for the prevention and control of sugarcane smut and sugarcane brown streak disease and the rapid screening and breeding of disease-resistant varieties.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows:

[0006] A method for simultaneously evaluating sugarcane resistance to smut and brown streak disease comprises the following steps:

[0007] (1) Collection of winter spores of sugarcane smut and cultivation of sugarcane brown streak pathogen

[0008] S1. Collection of teliospores of sugarcane smut: fresh smut whips are collected from sugarcane where sugarcane smut occurs in a sugarcane planting plot, air-dried, and teliospores on each whip are scraped off to obtain teliospores of sugarcane smut for later use;

[0009] S2. Cultivation of sugarcane brown streak pathogen: inoculate sugarcane brown streak pathogen onto a PDA plate for activation, and wait for the colonies to grow spores before use;

[0010] (2) Sugarcane smut inoculation and sugarcane brown streak inoculation (first inoculate with smut and then with brown streak)

[0011] S1. Inoculation of sugarcane smut: artificial inoculation is performed by seed soaking method, the sugarcane smut winter spores obtained in step (1) S1 are prepared into a spore suspension, the seed buds of the sugarcane material to be tested are immersed in the spore suspension, and then germinated overnight, and then the sugarcane buds are planted in the substrate;

[0012] S2, sugarcane brown streak inoculation: add ddH2O to the PDA plate of sugarcane brown streak fungus cultured in S2 of step (1), scrape off the mycelium and adjust it into spore solution, 30-35 days after the sugarcane smut inoculation, when the sugarcane seedlings grow to 25-35 cm, spray the spore solution on the leaves of the sugarcane seedlings, and then cover the sugarcane seedlings with plastic film for 1-3 days;

[0013] (3) Establishment of disease resistance evaluation system for sugarcane smut and sugarcane brown streak

[0014] S1. Evaluation of sugarcane smut resistance: When the sugarcane seedlings grow out and the first smut occurs, the number of sugarcane seedlings and the number of infected plants are investigated and the incidence rate is calculated; the disease resistance level is divided according to the incidence rate, level 1 high resistance: 0-2.50%, level 2 resistance: 2.51-5.00%, level 3 medium resistance: 5.01-7.50%, level 4 susceptible: 7.51-10.00%, level 5 high susceptible: greater than 10.00%;

[0015] S2. Evaluation of sugarcane brown streak disease resistance: The incidence of sugarcane brown streak disease was investigated 7-10 days after inoculation, and the disease resistance level was divided according to the infected leaf area. Level 1 high resistance means no lesions on the leaves, and level 2 resistance means lesions occupy 10% of the leaf area.

[0016] <10%, level 3 moderate resistance: lesions occupy 11% to 25% of the leaf area, level 4 moderate sensitivity: lesions occupy 26% to 50% of the leaf area, level 5 susceptible: lesions occupy 50% to 75% of the leaf area, level 6 highly susceptible: lesions occupy >75% of the leaf area.

[0017] Furthermore, in S1 of step (1), the sugarcane smut teliospores are stored in a drying oven at room temperature for later use, and the humidity of the drying oven is lower than 30%.

[0018] Furthermore, in step (1) S2, after the bacterial colonies grow spores, the PDA plate with the spores is placed in a 4° C. refrigerator for storage for later use.

[0019] Furthermore, in step (2) S1, the concentration of the spore suspension is 1×10 7 Teliospores / mL.

[0020] Furthermore, in step (2) S2, the concentration of the spore solution is 1×10 7 Spores / mL.

[0021] The beneficial effects of the present invention are as follows:

[0022] The present invention effectively solves the defects of separate disease resistance evaluation of sugarcane smut and sugarcane brown streak, such as time-consuming, labor-intensive, and inoculated materials-consuming, and the current smut resistance evaluation has too broad an equivalent division and low evaluation efficiency, and the two disease evaluation processes do not interfere with each other and do not affect the evaluation results. The present invention obtains the disease resistance evaluation results of sugarcane smut and sugarcane brown streak in one test, greatly improving the efficiency of disease resistance breeding, and providing technical support for preventing and controlling sugarcane smut and brown streak and improving the efficiency of disease resistance breeding. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0024] Example 1

[0025] A method for simultaneously evaluating sugarcane resistance to smut and brown streak disease comprises the following steps:

[0026] (1) Collection of winter spores of sugarcane smut and cultivation of sugarcane brown streak pathogen

[0027] S1. Collection of sugarcane smut teliospores

[0028] Collect fresh smut whips (teliospores) from sugarcane with sugarcane smut in a sugarcane planting plot, naturally air-dry at room temperature for 7 days, scrape off the teliospores on each whip to obtain sugarcane smut teliospores, collect the sugarcane smut teliospores in a plastic bag, store them in a drying oven at room temperature (humidity is less than 30%), and set them aside;

[0029] S2. Culture of sugarcane brown streak pathogen

[0030] The sugarcane brown streak pathogen collected from the experimental base of Guangxi Academy of Agricultural Sciences (73 meters above sea level, 22°51′ north latitude, 108°14′ east longitude) and stored in the refrigerator was inoculated onto a new PDA plate for activation culture. After the colonies grew spores, they were placed in a 4°C refrigerator for long-term use.

[0031] (2) Sugarcane smut and sugarcane brown streak inoculation

[0032] S1. Sugarcane smut inoculation

[0033] Artificial inoculation was carried out by seed soaking method, and 1×10 7 The spore suspension of teliospores / mL was prepared, and the seed buds of the sugarcane material to be tested were soaked in the spore suspension for 15 min, then placed in a large barrel for germination overnight, and potted in pots filled with substrate the next day, with 40 buds per pot;

[0034] S2. Sugarcane brown streak disease inoculation

[0035] Add ddH2O to the PDA plate cultured with sugarcane brown streak pathogen, scrape off the mycelium and adjust the spore concentration to 1×10 7 spores / mL of spore liquid, 32 days after the sugarcane smut inoculation in step (2) S1, when the sugarcane seedlings grow to about 30 cm (a critical window period for inoculating brown streak pathogen), the spore liquid is sprayed on the leaves of the sugarcane seedlings with a spray bottle to inoculate the sugarcane brown streak pathogen, and then the inoculated sugarcane seedlings are covered with a plastic film for 2 days and then uncovered; the incidence of brown streak pathogen is investigated 10 days after the sugarcane brown streak pathogen inoculation.

[0036] (3) Establishment of disease resistance evaluation system for sugarcane smut and sugarcane brown streak

[0037] S1. Evaluation of sugarcane smut resistance

[0038] When the sugarcane seedlings grow out and the first smut disease occurs, the number of sugarcane seedlings and the number of diseased plants are investigated. The investigation lasts for 6 months and the incidence rate is calculated. The disease resistance level is divided according to the incidence rate. Level 1 high resistance: incidence rate 0-2.50%, level 2 resistance: incidence rate 2.51-5.00%, level 3 medium resistance: incidence rate 5.01-7.50%, level 4 susceptible: incidence rate 7.51-10.00%, level 5 highly susceptible: incidence rate greater than 10.00%;

[0039] S2. Evaluation of resistance to sugarcane brown streak disease

[0040] The incidence of sugarcane brown streak disease was investigated 10 days after inoculation of sugarcane brown streak disease. The disease resistance level was divided according to the infected leaf area, among which level 1 highly resistant was no lesions on the leaves, level 2 resistant was lesions occupying less than 10% of the leaf area, level 3 moderately resistant was lesions occupying 11%-25% of the leaf area, level 4 moderately susceptible was lesions occupying 26%-50% of the leaf area, level 5 susceptible was lesions occupying 50%-75% of the leaf area, and level 6 highly susceptible was lesions occupying more than 75% of the leaf area.

[0041] The method of Example 1 was used to simultaneously evaluate the "double resistance" to smut and brown streak of 29 sugarcane varieties, 80 plants of each variety were tested, and the test results are shown in Table 1.

[0042] Table 1

[0043]

[0044]

[0045] In order to verify that the two disease evaluations do not interfere with each other and do not affect the evaluation results during the evaluation process of the present invention, the present invention conducted a separate evaluation of resistance to smut as shown in Comparative Example 1, and a separate evaluation of resistance to brown streak as shown in Comparative Example 2. The methods of Comparative Example 1 and Comparative Example 2 were used to evaluate the disease resistance of smut (Comparative Example 1) and brown streak (Comparative Example 2) of 29 sugarcane varieties in Table 1, respectively. 80 plants were tested for each variety. The test results are shown in Table 2.

[0046] Comparative Example 1

[0047] A method for evaluating sugarcane resistance to smut, comprising the following steps:

[0048] (1) Collection of sugarcane smut teliospores

[0049] Collect fresh smut whips (teliospores) from sugarcane with sugarcane smut in a sugarcane planting plot, naturally air-dry at room temperature for 7 days, scrape off the teliospores on each whip to obtain sugarcane smut teliospores, collect the sugarcane smut teliospores in a plastic bag, store them in a drying oven at room temperature (humidity is less than 30%), and set them aside;

[0050] (2) Sugarcane smut inoculation

[0051] Artificial inoculation was carried out by seed soaking method, and 1×10 7 The spore suspension of teliospores / mL was prepared, and the seed buds of the sugarcane material to be tested were soaked in the spore suspension for 15 min, then placed in a large barrel for germination overnight, and potted in pots filled with substrate the next day, with 40 buds per pot;

[0052] (3) Evaluation of sugarcane smut resistance

[0053] When the sugarcane seedlings grow out and the first smut disease occurs, the number of sugarcane seedlings and the number of diseased plants are investigated. The investigation lasts for 6 months and the incidence rate is calculated. The disease resistance level is divided according to the incidence rate: level 1, highly resistant: incidence rate 0-2.50%, level 2, resistant: incidence rate 2.51-5.00%, level 3, moderately resistant: incidence rate 5.01-7.50%, level 4, susceptible: incidence rate 7.51-10.00%, level 5, highly susceptible: incidence rate greater than 10.00%.

[0054] Comparative Example 2

[0055] A method for evaluating sugarcane resistance to brown streak disease comprises the following steps:

[0056] (1) Cultivation of sugarcane brown streak pathogen

[0057] The sugarcane brown streak pathogen collected from the experimental base of Guangxi Academy of Agricultural Sciences (73 meters above sea level, 22°51′ north latitude, 108°14′ east longitude) and stored in a refrigerator was inoculated onto a new PDA plate for activation. After the colonies grew spores, they were placed in a 4°C refrigerator for long-term use.

[0058] (2) Sugarcane brown streak disease inoculation

[0059] The sugarcane material buds to be tested were potted in pots filled with substrate, with 40 buds in each pot; ddH2O was added to the PDA plate cultured with sugarcane brown streak pathogen, and the mycelium was scraped off to adjust the spore concentration to 1×10 7 When the sugarcane seedlings grow to about 30 cm, the spore solution is sprayed on the leaves of the sugarcane seedlings with a spray bottle to inoculate the sugarcane brown streak disease. The inoculated sugarcane seedlings are then covered with plastic film for 2 days and then uncovered. The incidence of brown streak disease was investigated 10 days after the inoculation of sugarcane brown streak disease.

[0060] (3) Evaluation of resistance to sugarcane brown streak disease

[0061] The incidence of sugarcane brown streak disease was investigated 10 days after inoculation of sugarcane brown streak disease. The disease resistance level was divided according to the infected leaf area, among which level 1 highly resistant was no lesions on the leaves, level 2 resistant was lesions occupying less than 10% of the leaf area, level 3 moderately resistant was lesions occupying 11%-25% of the leaf area, level 4 moderately susceptible was lesions occupying 26%-50% of the leaf area, level 5 susceptible was lesions occupying 50%-75% of the leaf area, and level 6 highly susceptible was lesions occupying more than 75% of the leaf area.

[0062] Table 2

[0063]

[0064]

[0065] By comparing the data in Table 2 with the data in Table 1, it can be seen that there is no difference in the evaluation results of sugarcane smut and sugarcane brown streak disease when the simultaneous evaluation method of the present invention is used and the separate independent evaluation method is used, which proves that in the evaluation process of the present invention, the evaluation of the two diseases does not interfere with each other and the evaluation results are accurate.

[0066] Comparative Example 3

[0067] A method for simultaneously evaluating the resistance of sugarcane to smut and brown streak, based on Example 1, the timing of inoculation of sugarcane brown streak is changed, specifically, 25 days after the inoculation of sugarcane smut, spore liquid is sprayed on the leaves of sugarcane seedlings with a spray pot to inoculate sugarcane brown streak, and the other steps are the same as Example 1. The method of Comparative Example 3 is used to simultaneously evaluate the "double resistance" of smut + brown streak of the first 10 sugarcane varieties in Table 2, and 80 plants of each variety are tested. The test results are shown in Table 3.

[0068] Table 3

[0069]

[0070] Comparison of the data in Table 3 with the data in Table 2 shows that the inoculation timing of sugarcane brown streak disease in the present invention is changed, and the inoculation date of brown streak disease is advanced to 25 days after the inoculation of sugarcane smut disease. The evaluation results are significantly different from those of the separate independent evaluation methods, which proves that if brown streak disease is not inoculated at an appropriate time, inoculation too early will cause serious interference between the evaluations of the two diseases, resulting in inaccurate evaluation results.

[0071] Comparative Example 4

[0072] A method for simultaneously evaluating the resistance of sugarcane to smut and brown streak, based on Example 1, the timing of inoculation of sugarcane brown streak is changed, specifically, 38 days after the inoculation of sugarcane smut, spore liquid is sprayed on the leaves of sugarcane seedlings with a spray pot to inoculate sugarcane brown streak, and the other steps are the same as Example 1. The method of Comparative Example 4 is used to simultaneously evaluate the "double resistance" of smut + brown streak of the first 10 sugarcane varieties in Table 2, and 80 plants of each variety are tested. The test results are shown in Table 4.

[0073] Table 4

[0074]

[0075] Comparison of the data in Table 4 with the data in Table 2 shows that the inoculation timing of sugarcane brown streak disease in the present invention is changed, and the inoculation date of brown streak disease is delayed to 38 days after the inoculation of sugarcane smut disease. The evaluation results are significantly different from those of the separate independent evaluation methods, which proves that if brown streak disease is not inoculated at an appropriate time, inoculation too late will cause serious interference between the evaluations of the two diseases, resulting in inaccurate evaluation results.

[0076] In summary, in the evaluation method of the present invention, the necessary condition for inoculating brown streak disease is to inoculate 30-35 days after sugarcane smut inoculation. Smut disease develops later after inoculation. Too early inoculation of brown streak disease, such as in comparative example 3, will cause the leaves of sugarcane seedlings to die easily, affecting the onset of smut disease, and smut disease may not develop. However, if brown streak disease is evaluated separately, there is no need to worry about the death of sugarcane seedlings (death is a high-susceptibility level of brown streak disease). If brown streak inoculation is late (after 35 days), as in comparative example 4, it takes 7-10 days for brown streak to develop, and generally 10 days is the best time for investigation. However, it is already 45 days later, when the early symptoms of smut have already appeared in the early stage of onset. This affects the development of smut whips, and the other characteristic of smut is that the leaves become smaller and the plants become smaller or taller (different varieties have differences), which affects the accuracy of the investigation and evaluation results of brown streak. Therefore, the step of inoculation during the window period of "30-35 days after inoculation of sugarcane smut" was changed, and the simultaneous evaluation of sugarcane smut and sugarcane brown streak was unsuccessful.

[0077] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make some modifications and improvements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be based on the definition of the claims.

Claims

1. A method for simultaneously evaluating sugarcane resistance to smut and brown streak disease, characterized in that: The steps include: (1) Collection of winter spores of sugarcane smut and cultivation of sugarcane brown streak pathogen S1. Collection of teliospores of sugarcane smut: fresh smut whips are collected from sugarcane where sugarcane smut occurs in a sugarcane planting plot, air-dried, and teliospores on each whip are scraped off to obtain teliospores of sugarcane smut for later use; S2. Cultivation of sugarcane brown streak pathogen: inoculate sugarcane brown streak pathogen onto a PDA plate for activation, and wait for the colonies to grow spores before use; (2) Sugarcane smut and sugarcane brown streak inoculation S1. Sugarcane smut inoculation: artificial inoculation is performed by seed soaking method, the winter spores of sugarcane smut are prepared into a spore suspension, the seed buds of the sugarcane material to be tested are immersed in the spore suspension, and then germinated overnight, and then the sugarcane buds are planted in the substrate; S2. Sugarcane brown streak inoculation: Add ddH2O to the PDA plate cultured with sugarcane brown streak pathogen, scrape off the mycelium and adjust it into spore solution. 30-35 days after the sugarcane smut inoculation, spray the spore solution on the leaves of the sugarcane seedlings, and then cover the sugarcane seedlings with plastic film for 1-3 days; (3) Establishment of disease resistance evaluation system for sugarcane smut and sugarcane brown streak S1. Evaluation of sugarcane smut resistance: When the sugarcane seedlings grow out and the first smut occurs, the number of sugarcane seedlings and the number of infected plants are investigated and the incidence rate is calculated; the disease resistance level is divided according to the incidence rate, level 1 high resistance: 0-2.50%, level 2 resistance: 2.51-5.00%, level 3 medium resistance: 5.01-7.50%, level 4 susceptible: 7.51-10.00%, level 5 high susceptible: greater than 10.00%; S2. Evaluation of sugarcane brown streak disease resistance: The incidence of sugarcane brown streak disease was investigated 7-10 days after inoculation of sugarcane brown streak disease. The disease resistance level was divided according to the infected leaf area, among which level 1 highly resistant means no lesions on the leaves, level 2 resistant means lesions occupying less than 10% of the leaf area, level 3 moderately resistant means lesions occupying 11%-25% of the leaf area, level 4 moderately susceptible means lesions occupying 26%-50% of the leaf area, level 5 susceptible means lesions occupying 50%-75% of the leaf area, and level 6 highly susceptible means lesions occupying more than 75% of the leaf area.

2. The method for simultaneously evaluating sugarcane resistance to smut and brown streak disease according to claim 1, characterized in that: In S1 of step (1), the sugarcane smut teliospores are stored in a drying oven at room temperature for later use, and the humidity of the drying oven is lower than 30%.

3. The method for simultaneously evaluating sugarcane resistance to smut and brown streak disease according to claim 1, characterized in that: In step (1) S2, after the bacterial colonies grow spores, the PDA plate with the spores is placed in a 4° C. refrigerator for storage for later use.

4. The method for simultaneously evaluating sugarcane resistance to smut and brown streak disease according to claim 1, characterized in that: In step (2) S1, the concentration of the spore suspension is 1×10 7 Teliospores / mL.

5. The method for simultaneously evaluating sugarcane resistance to smut and brown streak disease according to claim 1, characterized in that: In step (2) S2, the concentration of the spore solution is 1×10 7 Spores / mL.

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