Wheat scab resistance identification method

Abstract

The invention relates to the technical field of plant resistance identification, and particularly discloses a wheat scab resistance identification method which is technically characterized by comprising the following steps: S1, sowing wheat varieties in a field identification garden, respectively taking the wheat varieties as high-resistance, medium-resistance, medium-susceptibility and high-susceptibility different varieties, and performing field water and fertilizer management in the whole growth period of the wheat varieties, a wheat scab single-flower instillation inoculation method is used as a contrast; s2, preparing a PDA culture medium; s3, manufacturing a PDA flat plate; s4, propagating and activating gibberellic disease bacteria; s5, preparing a fusarium graminearum spore suspension; s6, preparing straws; s7, preparing short stalks with bacteria; s8, wheat scab germ inoculation; and S9, investigating the morbidity of the inoculated wheat ears, counting the number of morbidity spikelet and the total number of spikelet, and calculating the morbidity spikelet rate and the average severity. The method provided by the invention is simple in process, overcomes the defect of large disease difference among individuals when the method in the prior art is adopted, and is high in accuracy.

Description

Technical Field

[0001] The invention relates to the technical field of plant resistance identification, and in particular to a method for identifying wheat fusarium head blight resistance. Background Art

[0002] Wheat is one of the three major grain crops in my country, with many varieties (lines), wide distribution range, and large planting area. In the middle and lower reaches of the Yangtze River, wheat production is seriously affected by Fusarium head blight, which is gradually spreading to the Huanghuai wheat region.

[0003] Fusarium head blight is a highly destructive fungal disease worldwide, which has a serious impact on the yield and quality of wheat. In production, chemical agents (such as 50% carbendazim wettable powder, 25% cyproconazole suspension, 43% tebuconazole suspension, etc.) are mainly used for prevention and control. Studies have shown that carbendazim residues can be detected in wheat grains after different times and dosages of carbendazim. The large-scale use of chemical pesticides has caused ecological environmental pollution, air pollution, pesticide residues and other problems, seriously threatening the health of humans and animals. Planting resistant varieties is considered to be the safest and most effective way to prevent and control wheat fusarium head blight.

[0004] In the process of wheat fusarium head blight resistance breeding, disease resistance identification is required for targeted screening. At present, the commonly used methods for fusarium head blight resistance identification include natural disease identification method, single flower drip method, spore suspension spray method and soil surface diseased wheat grain inoculation method. However, these methods in the prior art have the following defects: 1. The process is cumbersome; 2. The disease incidence varies greatly between individuals; 3. The accuracy is low.

[0005] To this end, the present application provides a method for identifying resistance to wheat fusarium wilt after research to solve the above problems. Summary of the invention

[0006] The purpose of the present invention is to solve the technical problems raised in the above-mentioned background technology and provide a method for identifying resistance to wheat fusarium wilt.

[0007] The above-mentioned purpose of the present invention is achieved like this:

[0008] A method for identifying wheat fusarium head blight resistance comprises the following steps:

[0009] S1. Sow wheat varieties in the field identification nursery and classify them as highly resistant, moderately resistant, moderately susceptible and highly susceptible varieties, and manage field water and fertilizer well throughout their growth period. Use the single flower drip inoculation method for wheat fusarium as a control;

[0010] S2, prepare PDA culture medium;

[0011] S3, PDA tablet production;

[0012] S4, reproduction and activation of Fusarium fusiformis;

[0013] S5, preparing a spore suspension of wheat fusarium spores;

[0014] S6, preparing straw;

[0015] S7, preparing short stalks with bacteria;

[0016] S8, inoculation of wheat fusarium rust pathogen;

[0017] S9. Investigate the disease situation of the inoculated wheat ears, count the number of diseased spikelets and the total number of spikelets, and calculate the diseased spikelet rate and average severity.

[0018] Furthermore, the specific method of step S2 is:

[0019] Peel fresh potatoes and weigh 200g, cut into small pieces, add 1000mL distilled water and boil. After the potatoes are soft, filter with 6 layers of gauze to remove the residue, then pour the filtrate into a cooking pot, and then add 20g glucose and 10g agar powder in turn, stir, and after complete dissolution, make the filtrate volume up to 1000mL, and divide it into 250mL conical flasks, about 100mL per bottle, sterilize in a high pressure sterilizer at 121℃ for 25min, and then place at room temperature for use.

[0020] Furthermore, the method for making the PDA tablet in step S3 is:

[0021] In the cleanroom, place the sterilized PDA in a microwave oven to heat and melt it. On a sterile clean bench, pour the liquid PDA into sterile culture dishes, 15-20 mL per dish, to make a PDA plate, solidify it, and set it aside.

[0022] Furthermore, step S4 specifically includes:

[0023] (1) On a sterile clean bench, after the inoculation needle is heated to red and cooled, the mycelium is picked from the edge of the wheat fusarium spores strains PH-1, F0980, and F1213 in sequence, and inoculated in the middle of the PDA plate, respectively, and marked, sealed with a sealing film, and placed in an incubator at 25°C and cultured in the dark for 3-5 days;

[0024] (2) Observe whether the three fusarium spore-forming strains mentioned above are contaminated and whether the mycelium grows normally;

[0025] (3) The three normally growing Fusarium fusarium strains were reactivated; a 0.5 cm punch was used to punch bacterial cakes on the edges of the hyphae of wheat Fusarium fusarium strains PH-1, F0980, and F1213 in sequence; one bacterial cake of each Fusarium fusarium strain was selected and inoculated in the middle of the PDA plate, marked, sealed with sealing film, and placed in an incubator at 25°C in the dark for 3-5 days.

[0026] Furthermore, step S5 specifically includes:

[0027] (1) Select 30 g of fresh mung beans, add distilled water and boil for 15 min, filter with double-layer gauze to remove the residue, and then dilute the filtrate to 1000 mL, divide it into 250 mL conical flasks, measure 150 mL into each bottle, sterilize at 121°C for 15 min, and let it cool for later use;

[0028] (2) In a sterile clean bench, use a 0.5 cm diameter puncher to punch the edges of the mycelium of the three fusarium spores on the PDA plate in turn, mark them, take 5 cakes in each conical flask, inoculate 2 flasks of each fusarium spores, mark them, and then shake-cultivate them at 25° C. and 180 rpm for 72 hours, then filter with double-layer gauze to remove the residue, and obtain a spore suspension of wheat fusarium spores;

[0029] (3) Observe the number of conidia in the spore suspensions of three types of Fusarium fusiformis pathogens using a hemocytometer, repeating this process three times;

[0030] (4) Dilute the spore suspensions of the three wheat fusarium spores respectively and adjust the concentration of the spore suspension to 1×10 5 CFU·mL －1 , mix the three kinds of fusarium spore suspensions in equal proportions to make a mixed spore suspension for later use.

[0031] Furthermore, the method for preparing straw in step S6 is:

[0032] Collect the wheat straw that matured in the previous year, remove the leaves, ears and roots, leaving only the stems, and store them in a cool and dry place; take out the stored straw, split it vertically, and cut it into short stalks of 1 cm in length, sterilize it at 121℃ for 20 minutes, then place it on a clean workbench, cool and dry it, and set it aside.

[0033] Furthermore, the specific method for preparing the bacterial short stalks in step S7 is:

[0034] (1) In a sterile clean bench, place the short stalk in a mixed spore suspension, shake and incubate at 25° C. and 180 rpm for 24 h to prepare a short stalk of bacterial spores for later use;

[0035] (2) Place a 0.5 cm cake of strain F1213 in the middle of the PDA medium, take an appropriate amount of short stalks and spread them evenly on the medium, seal it with a sealing film, and place it in an incubator at 25°C in the dark for 3-5 days. When the mycelium covers the entire culture dish, the short stalks of mycelium are prepared and set aside.

[0036] Furthermore, the specific method of inoculating wheat fusarium scaber in step S8 is:

[0037] (1) When the wheat of different varieties bloomed, wheat plants with good growth and 4-6 spikelets blooming in the middle of the ear were selected and marked with colored adhesive tape of different colors, 3 cm below the base of the lower neck of the ear;

[0038] (2) Choose to inoculate in the evening and spray the marked wheat ears with water to keep them moist before inoculation;

[0039] (3) Inoculate the spore stalks and hyphae stalks into the glume of the middle spikelet of the wheat ear respectively; select the fifth spikelet from the lowest side of the marked wheat ear, gently pull open the glume on one side with the left hand, use tweezers in the right hand to grab a short stalk with bacteria, and gently put it into the glume. Release the left hand and use its own elastic force to clamp the short stalk with bacteria. The inoculation is completed, and the bag is sealed with a plastic bag for 3 days. After taking out the bag, mist it to keep it moist for one week.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] 1. The method of the present invention is simple in process, overcomes the defect of large differences in disease incidence between individuals when using the existing technology method, and has high accuracy;

[0042] 2. When a large number of new wheat varieties (lines) are identified through inoculation in the field, the method of the present invention simulates the natural disease process in the field through the short stalk inoculation method and the mycelium inoculation method, and quantitative inoculation without wounds is more efficient, which is conducive to the accurate identification of the comprehensive ability of wheat to resist infection and expansion, and the identification results are more reliable and convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is a flow chart of a method in an embodiment of the present invention. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0045] The implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0046] Reference Figure 1 As shown, the following are preferred embodiments provided by the present invention.

[0047] Embodiment: The embodiment of the present invention provides a method for identifying resistance to wheat scab, and the specific implementation steps are as follows:

[0048] Step 1. In mid-to-late October 2023, sow wheat varieties Sumai No. 3, Yangmai 158, Huaimai 20 and Aikang 58 in the field identification nursery as high-resistant, medium-resistant, medium-susceptible and highly susceptible varieties. Carry out field water and fertilizer management throughout the entire growing period, and use the single flower drip inoculation method of wheat fusarium as a control.

[0049] Step 2, prepare PDA culture medium: peel fresh potatoes, weigh 200g, cut into small pieces, add 1000mL distilled water and boil. After the potatoes are soft, filter with 6 layers of gauze (absorbent gauze, density 21*32) to remove the residue, pour the filtrate into a cooking pot, and then add 20g glucose and 10g agar powder in turn (agar powder is difficult to dissolve and easy to become sticky, so it should be added slowly over low heat with stirring in small amounts), stir, and after it is completely dissolved, make the filtrate volume to 1000mL, and divide it into 250mL conical flasks, about 100mL per bottle, sterilize in a high pressure sterilizer at 121℃ for 25min, and then place it at room temperature for use.

[0050] Step 3: In a clean room, heat the sterilized PDA in a microwave oven until it melts (it is best if the solution becomes clear). On a sterile clean bench, pour the liquid PDA into sterile culture dishes, 15-20 mL per dish, to make a PDA plate, solidify it, and set aside.

[0051] Step 4: Reproduction and activation of fusarium spores:

[0052] 1. On a sterile clean bench, heat the inoculation needle and let it cool, pick the mycelium from the edges of wheat fusarium spores strains PH-1, F0980, and F1213, and inoculate them in the middle of the PDA plate (the PDA plate is quickly preheated and dried on the flame outside the alcohol lamp), mark it, seal it with a sealing film, and place it in an incubator at 25°C in the dark for 3-5 days;

[0053] 2. Observe whether the three strains of fusarium spores are contaminated and whether the mycelium grows normally;

[0054] 3. Transfer and activate the three normally growing Fusarium spp. strains again. Use a 0.5 cm puncher (burn red with an alcohol lamp and let cool) to punch the edges of the mycelium of wheat Fusarium spp. strains PH-1, F0980, and F1213 with bacterial cakes in turn (after punching one strain, disinfect the puncher with 75% alcohol again, burn red with an alcohol lamp and let cool). Pick one bacterial cake from each Fusarium spp. strain and inoculate it in the middle of the PDA plate (the outer side of the PDA plate is quickly preheated and sterilized on the flame outside the alcohol lamp), mark it, seal it with a sealing film, and place it in an incubator at 25°C and culture it in the dark for 3-5 days.

[0055] Step 5: Prepare wheat fusarium spore suspension:

[0056] 1. Select 30g of fresh mung beans, add distilled water and boil for 15min, filter with double-layer gauze to remove the residue (degreased gauze, density 21*32), then adjust the filtrate to 1000mL, divide it into 250mL conical flasks, measure 150mL per bottle, sterilize at 121℃ for 15min, and let it cool for later use;

[0057] 2. In a sterile clean bench, use a 0.5 cm diameter puncher to punch the edges of the PDA plate mycelium of the fusarium spores ...

[0058] 3. Use a blood cell counting plate to observe the number of conidia in the spore suspensions of the three types of Fusarium fusiformis pathogens, and repeat 3 times;

[0059] 4. Dilute the three wheat fusarium spore suspensions separately and adjust the concentration of the spore suspension to 1×10 5 CFU·mL －1 , mix the three kinds of fusarium spore suspensions in equal proportions to make a mixed spore suspension for later use.

[0060] Step 6: Prepare straw: Collect the wheat straw that matured in the previous year, remove the leaves, ears and roots, leaving only the stems, and store them in a cool and dry place. In early April 2024, take out the straw, split it vertically, and cut it into short stalks of 1 cm long, sterilize it at 121℃ for 20 minutes, and then place it on a clean bench to cool and dry for later use.

[0061] Step 7: Preparation of short stalks with bacteria

[0062] 1. In a sterile clean bench, place the short stalks in a mixed spore suspension and shake at 25°C and 180 rpm for 24 hours to make short stalks of bacterial spores (short stalk inoculation method) for later use.

[0063] 2. As in step 4.3, place a 0.5 cm bacterial cake (strain F1213) in the middle of the PDA medium, take an appropriate amount of short stalks and spread them evenly on the medium, seal it with a sealing film, place it in an incubator at 25°C, and culture it in the dark for 3-5 days. When the mycelium covers the culture dish, the short stalks of mycelium are made (mycelium inoculation method) and set aside.

[0064] Step 8. Inoculation of wheat fusarium rust: In early April 2024, when the wheat varieties Sumai No. 3, Yangmai 158, Huaimai 20 and Aikang 58 are in bloom in each identification plot, select wheat plants with good growth and 4-6 spikelets in the middle of the wheat ears and mark them with colored adhesive tape of different colors (mark 3 cm below the base of the lower neck of the ear, the mark is conspicuous and easy to observe and count).

[0065] Choose the evening time for inoculation and spray the marked wheat ears with water to keep them moist before inoculation.

[0066] Inoculate the spore stalks and hyphae stalks in the husk of the spikelet in the middle of the wheat ear. Select the fifth spikelet from the lowest side of the marked wheat ear, gently pull open the husk on one side with your left hand, use tweezers in your right hand to grab a short stalk with bacteria, and gently put it into the husk. Release your left hand and use its own elasticity to clamp the short stalk with bacteria. The inoculation is completed, and the bag is sealed with a plastic bag for 3 days. After taking the bag out, mist it to keep it moist for one week. Set up a control group, and use the single flower drip method (marked with different colors of adhesive tape) to compare and analyze whether the results of the short stalk inoculation method and the hyphae inoculation method are reliable. Inoculate 20 ears with each of the three methods.

[0067] Step 9. 21 days after inoculation, investigate the disease status of the inoculated wheat ears, count the number of diseased spikelets and the total number of spikelets, and calculate the diseased spikelet rate and average severity.

[0068] As shown in Table 1 below, the test results of the short stalk inoculation method, mycelium inoculation method and single flower drip method are shown.

[0069] Table 1 Statistical table of test results of short stalk inoculation method, mycelium inoculation method and single flower drip method

[0070]

[0071]

[0072] Note: HR: highly resistant; MR: moderately resistant; MS: moderately susceptible; HS: highly susceptible.

[0073] The short stalk inoculation method, mycelium inoculation method and single flower drip inoculation method were treated with different inoculation methods. The diseased ear rate reached more than 95% on the 21st day of inoculation, and the fusarium smut inoculation was effective. The paired sample T test method was used to statistically analyze the experimental data of the short stalk inoculation method, single flower drip method, mycelium inoculation method and single flower drip method, and the results are shown in Table 2.

[0074] Table 2 Analysis of experimental data of single flower drip inoculation method, short stalk inoculation method and mycelium inoculation method

[0075]

[0076] Note: HR: highly resistant; MR: moderately resistant; MS: moderately susceptible; HS: highly susceptible.

[0077] The experimental results show that there is no significant difference in the results obtained by the short stalk inoculation method and the single flower drip method, there is no significant difference in the results obtained by the mycelium inoculation method and the single flower drip method, and the resistance levels of the different varieties after inoculation and identification using the three inoculation methods are consistent, indicating that the inoculation and identification results of the short stalk inoculation method and the mycelium inoculation method are feasible.

[0078] Through the experimental results and analysis of this method, it can be concluded that there is no significant difference in the results of inoculation of wheat fusarium rust by the short stalk inoculation method, mycelium inoculation method and single flower drip method. However, when a large number of new wheat varieties (lines) are identified through field inoculation, the short stalk inoculation method and mycelium inoculation method simulate the natural disease process in the field, and quantitative inoculation without wounds is more efficient, which is conducive to the accurate identification of the comprehensive ability of wheat to resist infection and spread, and the identification results are more reliable and convenient.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for identifying wheat fusarium head blight resistance, characterized in that: The following steps are involved: S1. Sow wheat varieties in the field identification nursery and classify them as highly resistant, moderately resistant, moderately susceptible and highly susceptible varieties, and manage field water and fertilizer well throughout their growth period. Use the single flower drip inoculation method for wheat fusarium as a control; S2, prepare PDA culture medium; S3, PDA tablet production; S4, reproduction and activation of Fusarium fusiformis; S5, preparing a spore suspension of wheat fusarium spores; S6, preparing straw; S7, preparing short stalks with bacteria; S8, inoculation of wheat fusarium rust pathogen; S9. Investigate the disease situation of the inoculated wheat ears, count the number of diseased spikelets and the total number of spikelets, and calculate the diseased spikelet rate and average severity.

2. A method for identifying wheat scab resistance according to claim 1, characterized in that: The specific method of step S2 is: Peel fresh potatoes and weigh 200g, cut into small pieces, add 1000mL distilled water and boil. After the potatoes are soft, filter with 6 layers of gauze to remove the residue, then pour the filtrate into a cooking pot, and then add 20g glucose and 10g agar powder in turn, stir, and after complete dissolution, make the filtrate volume up to 1000mL, and divide it into 250mL conical flasks, about 100mL per bottle, sterilize in a high pressure sterilizer at 121℃ for 25min, and then place at room temperature for use.

3. A method for identifying wheat scab resistance according to claim 1, characterized in that: The method for making the PDA tablet in step S3 is: In the cleanroom, place the sterilized PDA in a microwave oven to heat and melt it. On a sterile clean bench, pour the liquid PDA into sterile culture dishes, 15-20 mL per dish, to make a PDA plate, solidify it, and set it aside.

4. A method for identifying wheat scab resistance according to claim 1, characterized in that: Step S4 specifically includes: (1) On a sterile clean bench, after the inoculation needle is heated to red and cooled, the mycelium is picked from the edge of the wheat fusarium spores strains PH-1, F0980, and F1213 in sequence, and inoculated in the middle of the PDA plate, respectively, and marked, sealed with a sealing film, and placed in an incubator at 25°C and cultured in the dark for 3-5 days; (2) Observe whether the three fusarium spore-forming strains mentioned above are contaminated and whether the mycelium grows normally; (3) The three normally growing Fusarium fusarium strains were reactivated; a 0.5 cm punch was used to punch bacterial cakes on the edges of the hyphae of wheat Fusarium fusarium strains PH-1, F0980, and F1213 in sequence; one bacterial cake of each Fusarium fusarium strain was selected and inoculated in the middle of the PDA plate, marked, sealed with sealing film, and placed in an incubator at 25°C in the dark for 3-5 days.

5. A method for identifying wheat scab resistance according to claim 4, characterized in that: Step S5 specifically includes: (1) Select 30 g of fresh mung beans, add distilled water and boil for 15 min, filter with double-layer gauze to remove the residue, and then dilute the filtrate to 1000 mL, divide it into 250 mL conical flasks, measure 150 mL into each bottle, sterilize at 121°C for 15 min, and let it cool for later use; (2) In a sterile clean bench, a hole puncher with a diameter of 0.5 cm was used to punch the edges of the mycelium of the three fusarium spores on the PDA plate in turn, and the cakes were marked. Five cakes were placed in each conical flask, and two flasks were inoculated with each fusarium spores. The flasks were marked, and then shaken at 25° C. and 180 rpm for 72 hours. The spores were then filtered with double-layer gauze to remove the residue, and a suspension of wheat fusarium spores was obtained. (3) Observe the number of conidia in the spore suspensions of three types of Fusarium fusiformis pathogens using a hemocytometer, repeating this process three times; (4) Dilute the spore suspensions of the three wheat fusarium spores respectively and adjust the concentration of the spore suspension to 1×10 5 CFU·mL －1 , mix the three kinds of fusarium spore suspensions in equal proportions to make a mixed spore suspension for later use.

6. A method for identifying wheat scab resistance according to claim 1, characterized in that: The method for preparing straw in step S6 is: Collect the wheat straw that matured in the previous year, remove the leaves, ears and roots, leaving only the stems, and store them in a cool and dry place; take out the stored straw, split it vertically, and cut it into short stalks of 1 cm in length, sterilize it at 121℃ for 20 minutes, then place it on a clean workbench, cool and dry it, and set it aside.

7. A method for identifying wheat scab resistance according to claim 1, characterized in that: The specific method for preparing the bacterial short stalks in step S7 is: (1) In a sterile clean bench, place the short stalk in a mixed spore suspension, shake and incubate at 25° C. and 180 rpm for 24 h to prepare a short stalk of bacterial spores for later use; (2) Place a 0.5 cm cake of strain F1213 in the middle of the PDA medium, take an appropriate amount of short stalks and spread them evenly on the medium, seal it with a sealing film, and place it in an incubator at 25°C in the dark for 3-5 days. When the mycelium covers the entire culture dish, the short stalks of mycelium are prepared and set aside.

8. A method for identifying wheat scab resistance according to claim 1, characterized in that: The specific method of wheat fusarium rust inoculation in step S8 is: (1) When the wheat of different varieties bloomed, wheat plants with good growth and 4-6 spikelets blooming in the middle of the ear were selected and marked with colored adhesive tape, 3 cm below the base of the lower neck of the ear; (2) Choose to inoculate in the evening and spray the marked wheat ears with water to keep them moist before inoculation; (3) Inoculate the spore stalks and hyphae stalks into the glume of the middle spikelet of the wheat ear respectively; select the fifth spikelet from the lowest side of the marked wheat ear, gently pull open the glume on one side with the left hand, use tweezers in the right hand to grab a short stalk with bacteria, and gently put it into the glume. Release the left hand and use its own elastic force to clamp the short stalk with bacteria. The inoculation is completed, and the bag is sealed with a plastic bag for 3 days. After taking out the bag, mist it to keep it moist for one week.

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