Stripe disease inoculation identification method

Abstract

The invention relates to the technical field of agriculture, in particular to a stripe disease inoculation identification method which comprises the following steps: inoculating a stripe disease cake to a barley seedling leaf, fixing a water storage material on the stripe disease cake, culturing at 18-26 DEG C for 40-56 hours, and identifying the pathogenicity of the stripe disease on the barley leaf. According to the method provided by the invention, the speed of infecting the barley leaves by the stripe pathogens can be obviously increased, the condition that the infection time is too long by using a sandwich method is improved, the working efficiency is improved, and the method is of great significance for identifying the stripe disease resistance of the barley and identifying the pathogenicity of the stripe pathogens.

Description

Technical Field

[0001] The invention relates to the technical field of agriculture, and in particular to a stripe disease inoculation and identification method. Background Art

[0002] Barley is an annual herb of the genus Hordeum in the Poaceae family. It can be grown at different latitudes and altitudes because of its early maturity, drought resistance, barrenness resistance, salt resistance, and short growth period. Barley has important applications in staple food production, beer brewing, animal husbandry development, and health care product research and development. With the continuous development of cultivation technology and the continuous cultivation of high-quality varieties, barley production continues to increase. However, with the large-scale annual planting of barley, the continuous increase in the area of ​​continuous cropping, and changes in the natural environment, diseases such as barley stripe, powdery mildew, and fusarium head blight are prevalent, resulting in serious damage to barley yield and quality. Among them, barley stripe disease is the most serious.

[0003] Since stripe disease has a complex pathogen system and may show different symptoms at different growth stages, this increases the difficulty of its identification. At present, the stripe disease infection identification method mainly uses the "sandwich" method. Although it meets the needs of infection identification, the inoculation process of this method is complicated, the infection time is long, the seed germination rate during the inoculation process is low, the field emergence is uneven, and the problem of missing seedlings is serious. At the same time, phenotypic identification needs to be carried out in the field, which is greatly affected by the environment.

[0004] Therefore, there is an urgent need to prepare a stripe disease inoculation identification method with a short infection period. Summary of the invention

[0005] In order to improve the identification efficiency, the present invention provides a stripe disease inoculation identification method. The identification method provided by the present invention can significantly accelerate the speed of stripe disease fungi infecting barley leaves, improve work efficiency, and is of great significance for the identification of barley's resistance to stripe disease and the identification of the pathogenicity of stripe disease fungi.

[0006] A stripe disease inoculation identification method comprises the following steps: Inoculate the stripe disease cake onto the leaves of barley seedlings, then fix the water storage material on the stripe disease cake, and culture at 18℃~26℃ for 40h~56h before identifying the disease grade. The disease level identification is divided into five levels: Level 1: no disease; Level 3: the site of inoculation of pathogens turns yellow-brown for one week; Level 5: the site of inoculation of pathogens expands 0.2cm~0.5cm to both ends; Level 7: the site of inoculation of pathogens expands 0.5cm~1.0cm to both ends; Level 9: the site of inoculation of pathogens expands >1.0cm to both ends.

[0007] The stripe disease inoculation identification method provided by the present invention can be inoculated on the leaves of barley seedlings with 2 leaves and 1 heart, and the disease can occur in 6 to 7 days, with a short disease cycle, thereby improving the identification efficiency. The surrounding environmental factors of the method provided by the present invention can be controlled, and sufficient disease occurrence can be ensured. At the same time, the inoculation method is simple, the infection time is short, and the inoculation efficiency is high, thus laying a foundation for large-scale barley stripe disease infection identification tests.

[0008] Furthermore, the barley seedling leaves are barley seedling leaves with 2 leaves and 1 heart.

[0009] Furthermore, the inoculation position of the stripe pathogen cake on the leaves of the barley seedlings is the base of the second leaf from the bottom to the top of the barley seedlings.

[0010] Furthermore, the water storage material is fixed by wrapping the barley leaves covering the water storage material with tin foil.

[0011] Furthermore, the stripe pathogen is inoculated onto the leaves of barley seedlings by a needle puncture method: the side of the stripe pathogen cake with the colonies is attached to the base of the second leaf from bottom to top of the barley seedling leaves, and a sterile needle is used to pierce the bacterial cake and the leaves.

[0012] The present invention uses the seedling stage needle puncture method to identify the disease level and is divided into five levels, level 1: no disease; level 3: the needle punctures the leaf part, and the wound appears yellow-brown for a week; level 5: the needle punctures the leaf part, and the wound expands 0.2cm~0.5cm to both ends; level 7: the needle punctures the leaf part, and the wound expands 0.5cm~1.0cm to both ends; level 9: the needle punctures the leaf part, and the wound expands more than 1.0cm to both ends.

[0013] Furthermore, the culture conditions after the stripe pathogen cake is inoculated onto the leaves of barley seedlings are: culture at 22° C. for 48 hours.

[0014] Furthermore, the light cycle of the culture in which the stripe pathogen cake is inoculated onto the leaves of the barley seedlings is alternating between 12 hours of light and 12 hours of darkness.

[0015] Furthermore, the solid culture medium is a PDA solid culture medium.

[0016] Furthermore, the water storage material is made of cotton.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The stripe pathogen cake provided by the present invention needs to be cultured at 18°C ​​to 26°C for 40h to 56h for infection, and the disease can occur after 6d to 7d. Compared with the "sandwich" method, not only the activity of the stripe pathogen is improved, but also the disease cycle is shortened. In addition, the method provided by the present invention can be inoculated on the leaves of barley seedlings, which improves the identification efficiency compared to the phenotypic identification of the "sandwich" method at the heading stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is the process of making bacterial cakes for stripe pathogen. In the figure, A is a diseased leaf; B is the isolation of pathogens; C~D are the purification and propagation of pathogens; E is the colony of stripe pathogens; and F is the making of bacterial cakes.

[0020] Figure 2 This is the process of stripe pathogen inoculation. In the figure, A is the leaf to be inoculated; B is the bacterial cake covering the leaf; C is the bacterial cake and leaf pierced with a sterilized needle; D is the bacterial cake covered with a cotton ball containing 70% deionized water; E is the cotton ball and bacterial cake wrapped in tin foil.

[0021] Figure 3 The standard for classifying disease levels of barley stripe disease inoculated by needle prick at the seedling stage is provided.

[0022] Figure 4 The "sandwich" infection process of barley seeds; in the figure, A is the isolated and purified stripe disease colony; B is the culture medium full of stripe disease hyphae; C is the barley seeds inoculated between two mushroom cakes full of hyphae.

[0023] Figure 5 The figure shows the changes of lesions on resistant and susceptible varieties after inoculation with stripe pathogen. In the figure, A1-A6 are the lesions on the resistant variety “NOIREMAROC” at 0 d, 3 d, 6 d, 9 d, 12 d and 20 d after inoculation; B1-B6 are the lesions on the resistant variety “Zaoshu No. 1” at 0 d, 3 d, 6 d, 9 d, 12 d and 20 d after inoculation; C1-C6 are the lesions on the susceptible variety “Jia-HY-460-6R” at 0 d, 3 d, 6 d, 9 d, 12 d and 20 d after inoculation; D1-D6 are the lesions on the susceptible variety “Jia-HY-101-6R” at 0 d, 3 d, 6 d, 9 d, 12 d and 20 d after inoculation. DETAILED DESCRIPTION

[0024] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0025] Example 1: A stripe disease inoculation and identification method.

[0026] Prepare the culture medium: Take 40.1g of potato dextrose agar medium solid powder, add 1000ml of distilled water, heat to boil to dissolve, sterilize in a 121℃ high pressure steam sterilizer for 20min, cool to 55℃ and pour into the plate.

[0027] Preparation of stripe disease pathogen: Figure 1 As shown in the figure, the barley leaves with streak disease stored in the laboratory were rinsed with distilled water, and 5 mm × 5 mm squares were cut from the lesions, as shown in the figure. Figure 1 As shown in A, the cells were treated with 75% alcohol for 30 seconds and 4g / 100mL sodium hypochlorite for 20 seconds, rinsed with sterile water three times, dried on sterile filter paper, and then inoculated on PDA solid culture medium, as shown in Figure 1 As shown in B, 3 pieces of diseased tissue were cultured in a 22°C dark constant temperature biochemical incubator for 4 days. Figure 1 As shown in C.

[0028] Preparation of stripe disease cakes: Use a hole puncher to punch holes at the edge of the above colony, collect the cakes, and use a bacterial inoculation needle to inoculate the cakes into new PDA culture medium. Inoculate one cake into each culture medium. Figure 1 As shown in D, continue to culture in a 22°C dark constant temperature biochemical incubator for 7 days. Figure 1 As shown in E, holes are then punched at the edge of the colony to obtain several 5 mm diameter streak bacteria cakes, such as Figure 1 As shown in F.

[0029] Stripe disease inoculation: Select barley seedlings with 2 leaves and 1 heart for inoculation. Figure 2 As shown in A.

[0030] ① After washing the base of the second barley leaf with distilled water, stick the side of the streak disease cake with the colony to the base of the second leaf, and pierce the cake and the leaf with a sterile needle. Figure 2 B and Figure 2 As shown in C.

[0031] ② Dip an alcohol cotton pad with a diameter of 5 mm into an appropriate amount of sterile water so that the alcohol cotton pad has a water content of 70% by mass, and cover the bacterial cake with it. Use 8 mm × 20 mm tin foil to fix the streak disease bacterial cake and the moistened alcohol cotton pad on the leaf. Figure 2 D and Figure 2 As shown in E.

[0032] ③ After culturing in an incubator with a temperature of 22℃, a light intensity of 8000Lux, and a light cycle of 12h / 12h alternating light and dark for 48 hours, remove the tin foil, alcohol cotton and bacterial cake. Be careful not to scratch or break the leaves during this process to identify the disease level.

[0033] The disease level identification is divided into five levels: Level 1: no disease; Level 3: the inoculated site turns yellow-brown for one week; Level 5: the inoculated site expands 0.2cm~0.5cm to both ends; Level 7: the inoculated site expands 0.5cm~1.0cm to both ends; Level 9: the inoculated site expands more than 1.0cm to both ends. For specific phenotypes, see Figure 3 shown.

[0034] Example 2: A stripe disease inoculation and identification method.

[0035] Prepare the culture medium: Take 40.1 g of potato dextrose agar medium solid powder, add 1000 ml of distilled water, heat and boil to dissolve, sterilize in a 121°C high pressure steam sterilizer for 20 min, cool to 55°C and pour into a plate.

[0036] Preparation of stripe disease pathogen: After rinsing the barley leaves with stripe disease spots stored in the laboratory with distilled water, cut out 5mm×5mm squares from the spots, treat them with 75% alcohol by volume for 30s and 4g / 100mL sodium hypochlorite for 20s, rinse them three times with sterile water, dry them on sterile filter paper, inoculate them on PDA solid culture medium, 3 diseased tissues per dish, and culture them in a dark constant temperature biochemical incubator at 18℃ for 2d.

[0037] Preparation of streak disease cakes: Use a hole puncher to punch holes at the edge of the above-mentioned colony, inoculate the bacteria cakes into new PDA culture medium with an inoculation needle, inoculate one bacteria cake into each culture medium, continue to culture in a dark constant temperature biochemical incubator at 18℃ for 5 days, then punch holes at the edge of the colony to obtain several streak disease cakes with a diameter of 5 mm.

[0038] Inoculation of stripe disease pathogens: Select barley seedlings with 2 leaves and 1 heart for inoculation.

[0039] ① After washing the base of the second barley leaf with distilled water, stick the side of the streak disease cake with the colonies to the base of the second leaf, and pierce the cake and the leaf with a sterile needle.

[0040] ② Dip an alcohol cotton pad with a diameter of 5 mm in an appropriate amount of sterile water so that the mass fraction of the alcohol cotton pad's water content is 70%, cover the bacterial cake with it, and use 8 mm × 20 mm tin foil to fix the streak disease bacterial cake and the moistened alcohol cotton pad on the leaves.

[0041] ③ After culturing for 40 hours in an incubator with a temperature of 18°C, a light intensity of 8000Lux, and a light cycle of 12h / 12h alternating light and dark, remove the tin foil, alcohol cotton, and bacterial cake. Be careful not to scratch or break the leaves during this process to identify the disease level.

[0042] The disease level identification is divided into five levels: Level 1: no disease; Level 3: the site of inoculation of pathogens turns yellow-brown for one week; Level 5: the site of inoculation of pathogens expands 0.2cm~0.5cm to both ends; Level 7: the site of inoculation of pathogens expands 0.5cm~1.0cm to both ends; Level 9: the site of inoculation of pathogens expands more than 1.0cm to both ends.

[0043] Example 3: A stripe disease inoculation and identification method.

[0044] Prepare the culture medium: Take 40.1 g of potato dextrose agar medium solid powder, add 1000 ml of distilled water, heat and boil to dissolve, sterilize in a 121°C high pressure steam sterilizer for 20 min, cool to 55°C and pour into a plate.

[0045] Preparation of stripe disease pathogen: After rinsing the barley leaves with stripe disease spots stored in the laboratory with distilled water, cut out 5mm×5mm squares from the spots, treat them with 75% alcohol by volume for 30s and 4g / 100mL sodium hypochlorite for 20s, rinse them three times with sterile water, dry them on sterile filter paper, inoculate them on PDA solid culture medium, 3 diseased tissues per dish, and culture them in a dark constant temperature biochemical incubator at 26℃ for 6d.

[0046] Preparation of streak disease cakes: punch holes at the edge of the above-mentioned colony with a hole puncher, inoculate the bacteria cakes into new PDA culture medium with an inoculation needle, inoculate one bacteria cake into each culture medium, continue to culture in a dark constant temperature biochemical incubator at 26℃ for 9 days, then punch holes at the edge of the colony to obtain several streak disease cakes with a diameter of 5 mm.

[0047] Inoculation of stripe disease pathogens: Select barley seedlings with 2 leaves and 1 heart for inoculation.

[0048] ① After washing the base of the second barley leaf with distilled water, stick the side of the streak disease cake with the colonies to the base of the second leaf, and pierce the cake and the leaf with a sterile needle.

[0049] ② Dip an alcohol cotton pad with a diameter of 5 mm in an appropriate amount of sterile water so that the mass fraction of the alcohol cotton pad's water content is 70%, cover the bacterial cake with it, and use 8 mm × 20 mm tin foil to fix the streak disease bacterial cake and the moistened alcohol cotton pad on the leaves.

[0050] ③ After culturing for 56 hours in an incubator with a temperature of 26℃, a light intensity of 8000Lux, and a light cycle of 12h / 12h alternating light and dark, remove the tin foil, alcohol cotton and bacterial cake. Be careful not to scratch or break the leaves during this process to identify the disease level.

[0051] The disease level identification is divided into five levels: Level 1: no disease; Level 3: the site of inoculation of pathogens turns yellow-brown for one week; Level 5: the site of inoculation of pathogens expands 0.2cm~0.5cm to both ends; Level 7: the site of inoculation of pathogens expands 0.5cm~1.0cm to both ends; Level 9: the site of inoculation of pathogens expands more than 1.0cm to both ends.

[0052] Comparative Example 1: like Figure 4 As shown, the infection of barley seeds is carried out based on the "sandwich" method. The specific conditions are as follows: the barley seeds are placed between two PDA culture media covered with mycelium and need to be placed at 6°C for 20 days for infection. The "sandwich" culture medium is a PDA solid culture medium. The components of the PDA solid culture medium are: 20g agar powder, 20g glucose, 200g peeled potatoes, and 1000mL sterile water pH 7.0.

[0053] Examples 1 to 3 were used to study the stripe disease inoculation identification method, and the "sandwich" method infection of Comparative Example 1 was used as a control. Since the results of Examples 1 to 3 were similar, for the convenience of subsequent discussion and reference, the results of Example 1 shall prevail. The relevant test methods and test results are as follows: 1. The change process of lesions of different disease-resistant and susceptible varieties infected with stripe pathogenic bacteria.

[0054] Barley disease-resistant varieties: Noire Maroc, Early 1, and susceptible varieties: Jia-HY-460-6R, Jia-HY-101-6R were selected, and the stripe pathogen was inoculated according to the method in Example 1 above, and the changes in lesions were observed at 0 d, 3 d, 6 d, 9 d, 12 d and 20 d after inoculation.

[0055] The Noire Maroc and HY-460-6R are recorded in "Huo Qingping. Physiological and molecular mechanisms of coordinated adaptation of barley roots and leaves to alkaline stress [D]. Inner Mongolia University for Nationalities, 2024".

[0056] The results are as follows Figure 5As shown, in Example 1, about one week after inoculation, brown-yellow stripe lesions began to appear at the inoculation site of the barley leaves; after 10 days, the growth rate of the lesions was different according to the difference in resistance and sensitivity between varieties, and the susceptible varieties had obvious differences compared with the resistant varieties; about two weeks, the difference in lesions between the susceptible varieties and the resistant varieties was the greatest; about three weeks, the lesions basically stopped growing and did not change.

[0057] 2. Investigation on the incidence levels of stripe pathogens in different barley varieties.

[0058] 1. Test methods The disease-resistant varieties of barley Noire Maroc, Zaoshu No. 1, Mengbi Mai No. 3, the susceptible varieties of Jia-HY-460-6R, Jia-HY-101-6R, Hua 30 and the medium-resistant variety of Mengbi Mai No. 5 were selected and inoculated with stripe pathogens according to the methods in the above Example 1 and Comparative Example 1. When the disease was fully developed, the barley disease grade was investigated according to the relevant grade standards. The "sandwich method" disease grade investigation is as follows:

[0059] The "sandwich method" disease grade survey is divided into five levels: level 1 immune: non-susceptible plants; level 3 highly resistant: incidence rate <5%; level 5 moderately resistant: incidence rate 5%~15%; level 7 susceptible: incidence rate 15%~40%; level 9 highly susceptible: incidence rate >40%.

[0060] The barley Noire Maroc and Jia-HY-460-6R are recorded in "Huo Qingping. Physiological and molecular mechanisms of coordinated adaptation of barley roots and leaves to alkaline stress [D]. Inner Mongolia University for Nationalities, 2024"; the barley Mengbi Mai No. 5 is recorded in "Ma Yu, Lv Ersuo, Batu, et al. Analysis and application evaluation of the adaptability of the new barley variety Mengbi Mai No. 5 to high and stable yields [J]. Xinjiang Agricultural Sciences, 2023, 60(11): 2727-2734."; Wheat No. 3 is recorded in "Zhang Fengying, Bao Haizhu, Shi Youguo, et al. Study on the fertilizer and density effects of the new barley variety Mengbi Mai No. 3 [J]. Journal of Northern Agriculture, 2016, 44(06): 1-4."; the flower 30 is recorded in "Chen Jinyue, Zhang Mengchao, Xiong Lidong, et al. Effects of 137Cs-γ ray irradiation on germination and seedling growth of barley varieties [J]. Agricultural Science and Technology Communications, 2021, (11): 61-63+214.".

[0061] 2. Test results The results of identification of barley stripe disease in resistant and susceptible barley varieties by needle-prick method and sandwich method at seedling stage are as follows: Table 1 Identification results of barley stripe disease infected by different inoculation methods

[0062] Through investigation, it can be known that the infection time of the "sandwich" method takes 20 days and the inoculation time is long. The disease cycle is long, and generally obvious symptoms are shown in the heading stage, that is, it takes about 2 months from inoculation to disease investigation. The acupuncture method can be used for inoculation at the barley seedling stage, which is the 2-leaf 1-heart stage of the barley seedling. The disease can occur 1 week after inoculation. After 3 days of inoculation, the lesions basically no longer change, and the final disease investigation can be carried out. From the inoculation results in Table 1, compared with the "sandwich method", the seedling stage acupuncture method is convenient for inoculation, the onset time is short, the onset is sufficient, and the disease investigation is more efficient.

[0063] It is worth noting that the present invention uses stripe pathogens to infect barley leaves for disease grade identification. The stripe pathogens can be existing stripe pathogens or pathogens isolated from plants carrying stripe pathogens.

[0064] Although preferred embodiments of the present invention have been described, further variations and modifications may be made to these embodiments once those skilled in the art understand the basic inventive concepts.

[0065] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A stripe disease inoculation and identification method, characterized in that: The steps include: Inoculating the stripe disease cake onto the leaves of the barley seedlings, wherein the stripe disease cake inoculation position on the leaves of the barley seedlings is the base of the second leaf from the bottom to the top of the barley seedlings, and then fixing the water storage material on the stripe disease cake, culturing for 40h to 56h at 18°C ​​to 26°C, a light intensity of 6000Lux to 10000Lux, and a photoperiod of 12h / 12h alternating light and dark, then removing the water storage material and the stripe disease cake, and conducting disease grade identification; The disease level identification is divided into five levels: Level 1: no disease; Level 3: the site of inoculation of pathogens turns yellow-brown for one week; Level 5: the site of inoculation of pathogens expands 0.2cm~0.5cm to both ends; Level 7: the site of inoculation of pathogens expands 0.5cm~1.0cm to both ends; Level 9: the site of inoculation of pathogens expands more than 1.0cm to both ends.

2. The stripe disease inoculation and identification method according to claim 1, characterized in that: The water storage material is fixed in the following manner: the barley leaves covering the water storage material are wrapped with tin foil.

3. The stripe disease inoculation and identification method according to claim 1, characterized in that: The stripe pathogen is inoculated onto the leaves of barley seedlings by a needle piercing method: the side of the stripe pathogen cake with the colonies is attached to the base of the second leaf from the bottom to the top of the barley seedling leaves, and a sterile needle is used to pierce the bacterial cake and the leaves.

4. The stripe disease inoculation and identification method according to claim 1, characterized in that: The culture conditions after the stripe pathogen cake is inoculated onto the leaves of the barley seedlings are: culture at 22° C. for 48 hours.

5. The stripe disease inoculation and identification method according to claim 1, characterized in that: The material of the water storage material is cotton.

Images