Method for inoculating phomopsis sesamarium through stem cutting and application of phomopsis sesamarium

Abstract

The invention provides a method for inoculating sesame stem blight germs by cutting stems, which comprises the following steps of: preparing sesame seedlings and the germs, removing new leaves at the top ends of the sesame seedlings by using sterile scissors during inoculation, reserving three pairs of lower true leaves with consistent internode length, sucking fresh fungus cakes by using a sterile pipetting suction head, fixing the pipetting suction head with the fungus cakes to the top ends of the stems, and inoculating the sesame seedlings with the germs by using the sterile pipetting suction head. Spraying sterile water once in the morning and evening every day to ensure the attack humidity of pathogenic bacteria; and recording the morbidity and disease index of each group two weeks after inoculation. The inoculation method can be used for rapidly and stably identifying the pathogenicity difference of pathogenic bacteria, and can be applied to the identification of the stem blight resistance of sesame varieties.

Description

Technical Field

[0001] The invention belongs to the technical field of sesame pathogen inoculation, and relates to an inoculation method and application of sesame stem blight pathogens in living sesame stems. Background Art

[0002] Sesame is an important oil crop in my country. Sesame stem blight is one of the main fungal diseases in sesame production, which seriously threatens the yield and quality of sesame. Sesame stem blight is caused by infection with Macrophomina phaseolina, an opportunistic pathogen with a host range of more than 500 plant species. Microsclerotia carried on diseased remains, soil and seeds can become the source of primary infection in the following year.

[0003] At present, the inoculation methods for sesame stem blight pathogen include the toothpick method, the root dipping method with bacterial suspension, and the root irrigation method with bacterial solution. These methods have problems such as not being easy to cause disease, inconsistent disease severity, long disease cycle, and inability to directly observe the disease severity of the root system. They cannot meet the existing inoculation needs. Therefore, there is a need to improve the existing sesame stem blight pathogen inoculation methods. Summary of the invention

[0004] In view of the problems existing in the existing sesame stem blight inoculation, the present invention provides a method for inoculating sesame stem blight by cutting the stems. The inoculation technology can quickly and stably identify the pathogenicity differences of the pathogens, and the method can be applied to the identification of sesame varieties resistant to stem blight.

[0005] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0006] A method for inoculating sesame stem blight pathogens by cutting stems, comprising the following steps:

[0007] (1) Sesame seedling preparation

[0008] The field soil and the nutrient soil are mixed in proportion, the soil is sterilized by high temperature and high pressure, the sesame seeds are disinfected by sodium hypochlorite, then washed with sterile water, and dried on filter paper for later use; sesame seeds are sown in nutrient pots, cultured under light conditions, and seedlings with inconsistent growth are removed after sowing; when the sesame seeds grow to have 4 pairs of complete leaves and 1 pair of heart leaves, the sesame seedlings with consistent height and growth are retained for inoculation;

[0009] When sesame seeds are disinfected, 2.5% sodium hypochlorite is used for disinfection for 15 minutes; the weight ratio of field soil to nutrient soil is 3:1, and the soil is sterilized at 121°C for 2 hours.

[0010] (2) Preparation of pathogens

[0011] The sesame stem blight pathogen was activated on PDA plates and cultured at 28°C in the dark for 2 days;

[0012] (3) Vaccination method

[0013] Use sterile scissors to remove the new leaves at the top of the sesame seedlings, keep the internodes of the same length and the three pairs of true leaves at the bottom, use a sterile pipette tip to take fresh mushroom cakes, directly fix the pipette tip with mushroom cakes to the top of the stem, and spray with sterile water once in the morning and evening every day;

[0014] (4) Evaluation method

[0015] Two weeks after inoculation, the incidence rate of each treatment, the number and length of stem nodes where the lesions extended longitudinally were recorded, and the disease index of each treatment was calculated.

[0016] The pathogenicity of sesame stem blight pathogen is divided into three levels according to the disease index: 0-30% for weak pathogenicity strains, 31%-70% for moderate pathogenicity strains, and 71%-100% for strong pathogenicity strains.

[0017] The calculation formula of disease index is: disease index = Σ(number of diseased plants × representative value) / (total number of plants × representative value of the most severe disease level) × 100.

[0018] The inoculation method is applied in the resistance identification and grading of sesame stem blight.

[0019] Beneficial effects of the present invention

[0020] (1) Compared with the conventional root irrigation method with bacterial liquid and toothpick inoculation method, which have the problems of unstable disease development and long disease development period after inoculation, the advantages of the present invention are that the inoculum is easy to prepare, the inoculation technology is simple, the disease development rate after inoculation is high, reaching 100%, and the disease development period is short. The pathogenicity of the pathogen can be identified within two weeks after inoculation, and plants with strong pathogenicity, medium pathogenicity and weak pathogenicity can be distinguished.

[0021] (2) The present invention can achieve rapid and accurate identification of the pathogenicity of large quantities of sesame stem blight pathogens based on the differences in disease resistance and susceptibility of sesame varieties, and can be applied to the batch identification and screening of sesame germplasm resources resistant to stem blight. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 . Colony morphology of sesame stem blight pathogen grown on PDA plates for 2 days;

[0023] Figure 2 .Schematic diagram of the cut stem inoculation method;

[0024] Figure 3 . Comparison of pathogenicity of Coccidioides phaseoli strains with strong, medium and weak pathogenicity on the 14th day after inoculation;

[0025] Figure 4.Comparison of the incidence of strong and weak pathogenic Phaseolus vulgaris strains on the 17th day after inoculation. DETAILED DESCRIPTION

[0026] The specific implementation methods of the present invention are further described in detail below in conjunction with the examples. Unless otherwise specified, the instruments and equipment involved in the examples are all conventional instruments and equipment; the raw materials involved are all commercially available conventional raw materials; and the test methods involved are all conventional methods.

[0027] Example 1 A method for inoculating sesame stem blight pathogens by cutting stems

[0028] The method comprises the following steps:

[0029] (1) Planting of sesame seedlings

[0030] The test plant is the sesame variety Zhengzhi 13. Select plump sesame seeds, disinfect them with 2.5% sodium hypochlorite for 15 minutes, rinse them with sterile water 3 times, and dry them on filter paper for later use. Mix field soil and nutrient soil in a ratio of 1:3, sterilize them in an autoclave at 121℃ for 2 hours, and put them in nutrient pots after drying. After sowing 15 seeds evenly in each nutrient pot, cover the sesame seeds with sieved fine soil. After sowing, sesame is cultivated at 30℃, 16 hours of light and 8 hours of darkness, and watered 2 to 3 times a week.

[0031] During the growth period of sesame, weak seedlings were removed and 6 to 8 seedlings were planted in each nutrient pot. About 40 days after sowing, sesame had grown 4 pairs of true leaves and 1 pair of new leaves, and it could be used for inoculation test.

[0032] (2) Cultivation of sesame stem blight pathogen

[0033] Transfer the sesame stem blight pathogen stored at -80℃ to a PDA plate and culture it at 28℃ in the dark for 2-3 days for later use. Figure 1 ).

[0034] Figure 1 AH in the figure represent different strain numbers: A: 2012-35; B: 2012-224; C: 2014-36; D: 2014-4; E: 09-38; F: 2012-64; G: 2014-111; H: 2014-31.

[0035] (3) Inoculation of sesame stem blight pathogen

[0036] Select sesame seedlings with uniform growth, remove the top two new leaves with sterile scissors, and keep the lower stem node 2 cm and the lower three pairs of true leaves ( Figure 2 A).

[0037] Use a sterile pipette tip (inner diameter 6mm) to take a fresh bacterial cake from the edge of the sesame stem blight colony and fix the pipette tip on the top of the stem to ensure that the bacterial cake fits tightly to the incision at the top of the stem ( Figure 2 B).

[0038] Figure 2 In the figure, A: retain 3 pairs of true leaves and the top of the stem; B: fix the pipette tip with the bacterial cake to the incision.

[0039] Each plant corresponding to the sesame stem spot blight pathogen is a treatment group, and a control group is set up at the same time. The control group is not inoculated after stem cutting. Each treatment is repeated 3 times, with 6 to 8 seedlings in each repetition. The control group is not inoculated after stem cutting. Spray with sterile water once in the morning and evening every day. On the second day after inoculation, remove the pipette tip and observe the longitudinal expansion of the lesion.

[0040] (4) Evaluation method

[0041] Two weeks after inoculation, the number of diseased plants in each treatment, the length of longitudinal extension of the lesions and the number of stem nodes were recorded, and the incidence rate and disease index of each treatment were calculated.

[0042] Example 2 Pathogenicity and resistance identification of sesame stem blight pathogen

[0043] With reference to the disease classification standards for stem blight in the seedling stage in the Technical Specifications for the Prevention and Control of Sesame Stem Blight (NY / T 2043-2011), the disease classification standards for the cut stem inoculation method were formulated, as shown in Table 1. At the same time, the occurrence of the disease was counted.

[0044] Incidence rate = number of diseased plants / total number of plants × 100%;

[0045] Disease index = Σ(number of diseased plants × representative value) / (total number of plants × representative value of the most severe disease level) × 100;

[0046] Table 1. Disease classification standards for sesame stem blight by stem cutting inoculation

[0047]

[0048] On the 14th day after the sesame was inoculated with the stem blight pathogen of Example 1, the incidence of each strain and the control was investigated, the incidence was statistically calculated, and the disease index was calculated. According to the disease index range, the pathogenicity of the sesame stem blight pathogen was divided into 3 levels: 0-30% was identified as a weak pathogenicity strain, 31%-70% was identified as a medium pathogenicity strain, and 71%-100% was identified as a strong pathogenicity strain. The pathogenicity identification results of each strain are shown in Table 2.

[0049] Table 2. Comparison of pathogenicity of sesame stem blight pathogens

[0050]

[0051] As can be seen from Table 2, the stem cutting inoculation method can ensure that the pathogens are 100% pathogenic, and the disease situation in each treatment group is consistent, which can clearly distinguish the pathogenicity of the pathogens in different treatment groups. The disease index values ​​of the tested strains range from 2.50 to 93.75, which can clearly distinguish the weak pathogenicity strains, the medium pathogenicity strains and the strong pathogenicity strains.

[0052] Figure 3 The figure shows a comparison of the pathogenicity of highly pathogenic, moderately pathogenic and weakly pathogenic Coccidioides phaseoli strains 14 days after inoculation. From left to right in the figure, they are the weakly pathogenic strains 2012-224, 2012-35, 2014-36, 2014-4; the moderately pathogenic strains 2012-64, 09-38; and the highly pathogenic strains 2014-31, 2014-111.

[0053] It can be seen that for the strain identified as weakly pathogenic, the lesions did not extend longitudinally or extended to the first node of the plant, and the leaves were not infected; for the strain identified as moderately pathogenic, the lesions extended longitudinally through the second node of the plant, or just extended to the third node, but some lower leaves remained green; for the strain identified as highly pathogenic, the disease progressed rapidly, the lesions extended longitudinally to below the third node, the leaves withered and fell off, the stems were covered with small black spots, which were the conidiophores and microsclerotia of sesame stem blight pathogen, and the plants dried up and died.

[0054] Figure 4 This is a comparison of the incidence of strong and weak pathogenicity of Phaseolus vulgaris strains on the 17th day after inoculation. The weak pathogenicity strain 2014-36 is on the left, and the strong pathogenicity strain 2014-111 is on the right. It can be seen that the pathogenicity difference between the strong and weak pathogenicity strains is very significant. On the 17th day after inoculation, the infection of the strong pathogenicity strain caused the whole sesame seedling to dry up and die, while the infection of the weak pathogenicity strain caused the stems above the first stem node to become ill, and the lesions did not continue to expand downward. Therefore, the cut stem inoculation method can be used to efficiently identify the pathogenicity of sesame stem blight pathogens.

[0055] In addition, according to the disease classification standard of sesame stem blight cut-stem inoculation method (Table 1), a resistance classification standard for sesame stem blight was formulated: grade 0 is resistant variety, grade 1 is medium-resistant variety, grade 2 is susceptible variety, and grades 3 and 4 are highly susceptible varieties. The method of Example 1 can also be used to identify sesame stem blight resistant varieties and susceptible varieties, and realize rapid resistant / susceptible resource screening of a large number of sesame germplasm resources.

Claims

1. A method for inoculating sesame stem blight pathogens by cutting stems, characterized in that: The method comprises the following steps: (1) Sesame seedling preparation The field soil and the nutrient soil are mixed in proportion, the soil is sterilized by high temperature and high pressure, the sesame seeds are disinfected by sodium hypochlorite, then washed with sterile water, and dried on filter paper for later use; sesame seeds are sown in nutrient pots, cultured under light conditions, and seedlings with inconsistent growth are removed after sowing; when the sesame seeds grow to have 4 pairs of complete leaves and 1 pair of heart leaves, the sesame seedlings with consistent height and growth are retained for inoculation; (2) Preparation of pathogens The sesame stem blight pathogen was activated on PDA plates and cultured at 28°C in the dark for 2 days; (3) Vaccination method Use sterile scissors to remove the new leaves at the top of the sesame seedlings, keep the internodes of the same length and the three pairs of true leaves at the bottom, use a sterile pipette tip to take fresh mushroom cakes, directly fix the pipette tip with mushroom cakes to the top of the stem, and spray with sterile water once in the morning and evening every day; (4) Evaluation method Two weeks after inoculation, the incidence rate of each treatment, the number and length of stem nodes where the lesions extended longitudinally were recorded, and the disease index of each treatment was calculated.

2. The inoculation method according to claim 1, characterized in that When disinfecting sesame seeds, use 2.5% sodium hypochlorite for 15 minutes.

3. The inoculation method according to claim 1, characterized in that The weight ratio of field soil to nutrient soil was 3:1, and the soil was sterilized at 121°C for 2 hours.

4. An application of the inoculation method according to claim 1 in identifying the pathogenicity of sesame stem blight pathogen, characterized in that: Two weeks after the pathogen inoculation, the disease situation of each group was investigated and the disease index was calculated. The pathogenicity of sesame stem blight pathogen was divided into three levels according to the disease index: 0-30% for weak pathogenicity strains, 31%-70% for moderate pathogenicity strains, and 71%-100% for strong pathogenicity strains.

5. The use according to claim 4, characterized in that The calculation formula of the disease index is: disease index=Σ(number of diseased plants×representative value) / (total number of plants×representative value of the most severe disease level)×100.

6. Use of the inoculation method according to claim 1 in the identification and classification of resistance to sesame stem blight.

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