Method for rapidly identifying corn fusarium graminearum grain rot resistance

Abstract

The invention relates to the technical field of maize germplasm disease resistance screening, in particular to a method for rapidly identifying maize fusarium graminearum grain rot resistance. The invention relates to a method for rapidly identifying the resistance of corn fusarium graminearum to grain rot. The method comprises the following steps: (1) mixing fusarium graminearum with a nutrient solution to obtain a fusarium graminearum solution; and (2) inoculating the fusarium graminearum liquid to a corn milk-ripe stage female ear, culturing, observing the infection condition after the culture is completed, and judging whether to resist diseases or not according to the disease level classification standard of the corn ear rot. According to the method, the identification of the ear grain rot resistance of fusarium graminearum can be completed in a laboratory incubator, the method can be operated throughout the year, the environment is controllable, the influence of other factors can be eliminated, a result can be obtained 8 days after inoculation, and the identification method is efficient and reliable.

Description

Technical Field

[0001] The invention relates to the technical field of disease resistance screening of corn germplasm, and in particular to a method for rapidly identifying resistance to Fusarium graminearum ear rot of corn. Background Art

[0002] Fusarium graminearum can infect a wide range of crops, including wheat, corn, rice, and barley, and is an important pathogenic fungus that threatens global food security. Fusarium graminearum can invade crops from the roots, stems, leaves, or ears, causing a decrease in crop yields. When invading from the ears, it often causes food safety problems because this type of fungus can synthesize and secrete a variety of toxins during the invasion process, contaminating the grains, and causing poisoning incidents after consumption by humans and animals.

[0003] Corn Fusarium graminear ear rot is a disease caused by Fusarium graminear, which mainly harms corn ears, resulting in reduced corn yield and quality. The symptoms of the disease include the top of the ear turning pink, pink to grayish white hyphae growing between the kernels, and in severe cases, the entire ear or part of the kernels rotting. Fusarium graminear prefers to grow and reproduce in a humid environment, especially in the grooves left by earworms or corn borers. In addition, the disease is also affected by temperature, humidity and insect bites, and the severity of the disease varies.

[0004] Identification of corn ear rot is an important part of corn resistance identification. The existing manual identification technology requires the cultivation of Fusarium graminearum first, the preparation of a certain concentration of spore suspension, and the inoculation of the spore suspension on the corn cobs 7 to 10 days after the corn silking. After the corn matures, the husks are peeled off to observe the resistance of the cobs. The existing method requires waiting for the corn to grow and mature, which takes a long time, and is affected by the corn growing season and natural environment such as temperature, air humidity and other pests and diseases, and the identification efficiency is low.

[0005] Therefore, how to provide a convenient and quick method for identifying corn ear rot resistance and solve the technical problems of low efficiency and long time of corn ear rot resistance identification methods in the prior art is an issue that technical personnel in this field urgently need to solve. Summary of the invention

[0006] The object of the present invention is to provide a method for rapidly identifying resistance to Fusarium graminearum ear rot of corn.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for rapidly identifying resistance to Fusarium graminearum ear rot of corn, comprising the following steps:

[0009] (1) mixing a spore suspension of Fusarium graminearum with a nutrient solution to obtain a Fusarium graminearum bacterial solution;

[0010] (2) The bacterial liquid of Fusarium graminearum is inoculated into female ears of corn at the milky stage, and then sealed in a ziplock bag and placed in an incubator for 8 to 10 days. After the culturing is completed, the infection situation is observed, and whether the female ears of corn are disease-resistant is determined according to the classification standard of corn ear rot disease.

[0011] Preferably, the final concentration of the Fusarium graminearum solution in step (1) is 4 to 6×10 5 spores / mL.

[0012] Preferably, the inoculation amount of the Fusarium graminearum solution in step (2) is 3 to 6 mL per corn ear.

[0013] Preferably, the culture temperature in step (2) is 27-29°C.

[0014] Preferably, the humidity of the culture in step (2) is 86-94%.

[0015] Preferably, during the culture period of step (2), the cells are cultured in a dark box and in light alternately for 12-12 hours.

[0016] Preferably, the corn ear rot disease classification standard in step (2) is: Level 1: the diseased area accounts for 0% to 5% of the total area of ​​the corn ear;

[0017] Level 2: The diseased area accounts for 6% to 15% of the total area of ​​the corn ear;

[0018] Level 3: The diseased area accounts for 16% to 35% of the total area of ​​the corn ear;

[0019] Level 4: The diseased area accounts for 36% to 55% of the total area of ​​the corn ear;

[0020] Level 5: The diseased area accounts for 56% to 100% of the total area of ​​the corn ear.

[0021] Preferably, the nutrient solution in step (1) comprises the following components: 20-30 mg / L gibberellin and 3-5 ml / L 1-methylcyclopropene.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This method can be used to identify resistance to Fusarium graminearum ear rot in a laboratory incubator. It can be operated throughout the year, and the environment is controllable. The influence of other factors can be excluded. Results are available 8 days after inoculation, and the identification is efficient and reliable.

[0024] The identification method of the present invention uses an isolated female ear of corn at the milky stage for identification, and determines the resistance of the corn variety by the infection degree of the pathogen (the proportion of the pathogen infection area to the female ear of corn). However, previous experiments have found that if the female ear of corn is directly inoculated with a spore suspension of Fusarium graminearum, the corn kernels are soft and lack nutrition because they are isolated milky ears, which will cause the kernels to be quickly infected, affecting the identification effect in the later stage. The present invention adds a nutrient solution to the spore suspension, which can effectively prolong the activity time of the isolated corn, thereby improving the stability and accuracy of the identification result.

[0025] In the nutrient solution prepared by the present invention, both gibberellin and 1-methylcyclopropene are plant growth regulators, which play an important role in delaying the senescence of isolated plant tissues. The addition of the nutrient solution can keep the isolated corn ears in a normal growth state, so that the real ear and kernel rot resistance of the isolated ears can be identified. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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 embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0027] Figure 1 This is the identification result of the method of comparative example 1 in experimental example 1.

[0028] Figure 2 This is a standard chart for classifying the disease levels of corn ear rot. DETAILED DESCRIPTION

[0029] The present invention provides a method for rapidly identifying resistance to Fusarium graminearum ear rot of corn, comprising the following steps:

[0030] (1) mixing a spore suspension of Fusarium graminearum with a nutrient solution to obtain a Fusarium graminearum bacterial solution;

[0031] (2) The bacterial liquid of Fusarium graminearum is inoculated into female ears of corn at the milky stage, and then sealed in a ziplock bag and placed in an incubator for 8 to 10 days. After the culturing is completed, the infection situation is observed, and whether the female ears of corn are disease-resistant is determined according to the classification standard of corn ear rot disease.

[0032] In the present invention, the culturing time in step (2) is preferably 8 days.

[0033] In the present invention, the final concentration of the Fusarium graminearum solution in step (1) is 4 to 6×10 5 spores / mL; preferably 5×10 5 spores / mL.

[0034] In the present invention, the inoculation amount of the Fusarium graminear liquid in step (2) is 3-6 mL / corn ear; preferably 4-5 mL / corn ear; and more preferably 5 mL / corn ear.

[0035] In the present invention, the culture temperature in step (2) is 27-29°C, preferably 27°C.

[0036] In the present invention, the humidity of the culture in step (2) is 86-94%; preferably 87-93%; more preferably 88-92%; and more preferably 90%.

[0037] In the present invention, during the culture period of step (2), dark box and light culture are alternately performed for 12-12 hours; the culture is sealed culture.

[0038] In the present invention, the corn ear rot disease level classification standard in step (2) is: Level 1: the diseased area accounts for 0% to 5% of the total area of ​​the corn ear;

[0039] Level 2: The diseased area accounts for 6% to 15% of the total area of ​​the corn ear;

[0040] Level 3: The diseased area accounts for 16% to 35% of the total area of ​​the corn ear;

[0041] Level 4: The diseased area accounts for 36% to 55% of the total area of ​​the corn ear;

[0042] Level 5: The diseased area accounts for 56% to 100% of the total area of ​​the corn ear.

[0043] In the present invention, the nutrient solution in step (1) comprises the following components: 20-30 mg / L of gibberellin and 3-5 ml / L of 1-methylcyclopropene;

[0044] Preferred are 20 mg / L gibberellin and 5 ml / L 1-methylcyclopropene.

[0045] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] Example 1

[0047] A method for cultivating Fusarium graminearum comprises the following steps:

[0048] (1) Isolation and identification of pathogens from corn ears infected with Fusarium graminearum. Use 5% sodium hypochlorite and water at a dilution ratio of 1:10 to disinfect the surface of the infected corn kernels. After 2 minutes, remove the corn kernels, rinse them twice with sterile water, dry the surface moisture of the seeds on sterile paper, and place 3 seeds equidistantly in an acidified potato dextrose agar (PDA) medium (adjust the pH of the medium to 5.0 with 0.1 mol / L hydrochloric acid). Culture in the dark at 26°C. After 4 days, obtain the purified hyphae of Fusarium graminearum from the edge of the colony, transfer to a new acidified PDA medium, and culture in the dark at 26°C for 10 days until the hyphae cover the surface of the medium.

[0049] (2) Using mung bean culture medium as spore production medium, 20 grams of mung beans were placed in 1 liter of distilled water and boiled for 30 minutes. The mung bean soup was filtered through two layers of gauze and sterilized to obtain mung bean culture medium. The mycelium cultured in the culture dish of step (1) was inoculated into the sterilized mung bean culture medium, and cultured on a shaker at 25°C (155 rpm) for 10 days. The culture solution was filtered through gauze to obtain a spore suspension. The concentration was checked with a hemocytometer under a microscope, the spore concentration was determined, and the spore concentration was diluted to the inoculation concentration (500,000 spores / mL).

[0050] Example 2

[0051] A method for rapidly identifying resistance to Fusarium graminearum ear rot of corn, comprising the following steps:

[0052] (1) Prepare nutrient solution: Use water as solvent and add the following components at the following concentrations: 30 mg / L gibberellin and 3 ml / L 1-methylcyclopropene.

[0053] (2) The corn Fusarium graminearum was cultured in the laboratory, and a spore suspension (prepared by the method of Example 1, with a concentration of 500,000 spores / mL) was prepared. The spore suspension was then mixed with a nutrient solution (volume ratio of 5:1) to obtain a Fusarium graminearum bacterial solution, which was inoculated into a corn ear and cultured for 10 days. The inoculation site was one end of the corn ear, and the inoculation amount was 5 mL / corn ear. The bag was then sealed with a sealed bag. The culture temperature was 27°C, the culture humidity was 86%, and the dark box and light culture were alternated for 12-12 hours during the culture period, and the corn ear was kept moist. After the culture was completed, the infection was observed, and the disease resistance of the corn kernels was determined according to the corn ear rot disease level classification standard.

[0054] Among them, the classification standards for corn ear rot disease levels are:

[0055] Level 1: The diseased area accounts for 0% to 5% of the total area of ​​the corn ear;

[0056] Level 2: The diseased area accounts for 6% to 15% of the total area of ​​the corn ear;

[0057] Level 3: The diseased area accounts for 16% to 35% of the total area of ​​the corn ear;

[0058] Level 4: The diseased area accounts for 36% to 55% of the total area of ​​the corn ear;

[0059] Level 5: The diseased area accounts for 56% to 100% of the total area of ​​the corn ear.

[0060] Example 3

[0061] A method for rapidly identifying resistance to Fusarium graminearum ear rot of corn, comprising the following steps:

[0062] (1) Prepare nutrient solution: Use water as solvent and add the following components at the following concentrations: 25 mg / L gibberellin and 4 ml / L 1-methylcyclopropene.

[0063] (2) The corn Fusarium graminearum was cultured in the laboratory, and a spore suspension was prepared (prepared by the method of Example 1, with a concentration of 500,000 spores / mL), and then the spore suspension was mixed with a nutrient solution (volume ratio of 6:1) to obtain a Fusarium graminearum bacterial solution, which was inoculated into a corn ear and cultured for 9 days. The inoculation site was the middle of the corn ear, and the inoculation amount was 6 mL / corn ear. The bag was then sealed, and the culture temperature was 29° C. The culture humidity was 94%, and there was no light during the culture period, and the corn ear was kept moist. After the culture was completed, the infection was observed, and the disease resistance of the corn kernels was determined according to the corn ear rot disease level classification standard.

[0064] Among them, the classification standards for corn ear rot disease levels are:

[0065] Level 1: The diseased area accounts for 0% to 5% of the total area of ​​the corn ear;

[0066] Level 2: The diseased area accounts for 6% to 15% of the total area of ​​the corn ear;

[0067] Level 3: The diseased area accounts for 16% to 35% of the total area of ​​the corn ear;

[0068] Level 4: The diseased area accounts for 36% to 55% of the total area of ​​the corn ear;

[0069] Level 5: The diseased area accounts for 56% to 100% of the total area of ​​the corn ear.

[0070] Example 4

[0071] A method for rapidly identifying resistance to Fusarium graminearum ear rot of corn, comprising the following steps:

[0072] (1) Prepare nutrient solution: Use water as solvent and add the following components at the following concentrations: 20 mg / L gibberellin and 5 ml / L 1-methylcyclopropene.

[0073] (2) The corn Fusarium graminearum was cultured in the laboratory, and a spore suspension (prepared by the method of Example 1, with a concentration of 500,000 spores / mL) was prepared. The spore suspension was then mixed with a nutrient solution (volume ratio of 4:1) to obtain a Fusarium graminearum bacterial solution, which was inoculated into a corn ear and cultured for 8 days. The inoculation site was one end of the corn ear, and the inoculation amount was 3 mL / corn ear. The bag was then sealed, and the culture temperature was 28°C, the culture humidity was 90%, and the dark box and light culture were alternated for 12-12 hours during the culture period, and the corn ear was kept moist. After the culture was completed, the infection was observed, and the disease resistance of the corn kernels was determined according to the corn ear rot disease level classification standard.

[0074] Among them, the classification standards for corn ear rot disease levels are:

[0075] Level 1: The diseased area accounts for 0% to 5% of the total area of ​​the corn ear;

[0076] Level 2: The diseased area accounts for 6% to 15% of the total area of ​​the corn ear;

[0077] Level 3: The diseased area accounts for 16% to 35% of the total area of ​​the corn ear;

[0078] Level 4: The diseased area accounts for 36% to 55% of the total area of ​​the corn ear;

[0079] Level 5: The diseased area accounts for 56% to 100% of the total area of ​​the corn ear.

[0080] Comparative Example 1

[0081] A method for rapidly identifying resistance to Fusarium graminearum ear rot of corn, comprising the following steps:

[0082] (1) Thresh the corn to be tested for resistance, keep the culture dish moist with sterile filter paper and sterile water, and place the corn kernels to be tested in the culture dish.

[0083] (2) Cultivate Fusarium graminearum in the laboratory and prepare a spore suspension (prepared in Example 1, with a concentration of 5×10 5 spores / mL), and then inoculate the prepared spore suspension on the corn kernels in the culture dish with an inoculation amount of 1 mL / corn kernel.

[0084] (3) Seal the culture dish, culture at 25°C, keep it moist during the culture period, and alternate between dark and light culture for 12-12 hours. Observe the infection of the corn kernels by Fusarium graminearum 5 days after inoculation; the determination method is: if the corn kernels after culture are almost completely rotten, the corn kernels are covered with hyphae, and there are pink products, then it is determined to be a susceptible material; if the corn kernels after culture have no obvious signs of rot, almost no hyphae growth, no pink products or lighter pink products, then it is determined to be a disease-resistant material.

[0085] Experimental Example 1

[0086] The disease resistance of corn varieties Y32(R), D39(R), YML1218(S), and Y46(S) was identified using the method of Example 4. The results are shown in Table 1. Note: R: resistant; S: susceptible. Figure 2 The experiment was repeated three times and the average value was taken.

[0087] Table 1 Survey results of corn ear rot

[0088] Disease area Disease grade Y32 4.7％ 1 D39 3.2％ 1 YML1218 45.2％ 4 Y46 66.7％ 5

[0089] It can be seen from the contents recorded in Table 1 that the method of the present invention can accurately identify the disease resistance levels of different varieties, and the time required is short, and the identification results can be obtained within 8 days.

[0090] The disease resistance of Y32(R), D39(R), YML1218(S), and Y46(S) was further identified using the method of Comparative Example 1 to verify the accuracy of the method of the present invention. Figure 1 Note: DAI: Days after inoculation, R: resistant, disease-resistant; S: susceptible, disease-susceptible.

[0091] The results are as follows Figure 1 : On the first day after inoculation, there was no significant difference between the resistant and susceptible materials; on the second day after inoculation, the bacterial plaques around the grains of the susceptible materials began to grow, and the unique pink product of Fusarium graminearum began to deepen; on the third day after inoculation, the susceptible materials began to rot, and hyphae began to grow around the grains; on the fourth day after inoculation, the grains of the susceptible materials further rotted, the Fusarium graminearum plaques around the grains were larger, and the pink product was more; on the fifth day after inoculation, the grains of the susceptible materials were almost completely rotten, and the corn grains were covered with hyphae. However, the bacterial plaques around the grains of the resistant materials did not grow significantly, the pink product was lighter, and the grains showed no obvious signs of rot.

[0092] The experimental results show that the grains of the Y32 and D39 groups are almost completely rotten, the corn grains are covered with hyphae, and there are pink products, which are disease-resistant materials. The YML1218 and Y46 groups have no signs of rot, almost no hyphae growth, and have lighter pink products, which are susceptible materials. This is consistent with the resistance of the germplasm material itself, which is also consistent with the identification results of the identification method provided by the present invention. From the culture results of the 5th day, it can be seen that the D39 variety has the strongest resistance and the least pink product production, followed by Y32 (R), and finally YML1218 (S) and Y46 (S). However, the method of comparative example 1 can only determine which variety has disease resistance, but cannot accurately determine which variety has the disease resistance level.

[0093] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for rapidly identifying resistance to Fusarium graminearum ear rot in corn, characterized in that: The steps include: (1) mixing a spore suspension of Fusarium graminearum with a nutrient solution to obtain a Fusarium graminearum bacterial solution; (2) The bacterial liquid of Fusarium graminearum is inoculated into female ears of corn at the milky stage, and then sealed in a ziplock bag and cultured for 8 to 10 days. After the culture is completed, the infection situation is observed, and whether the female ears of corn are disease-resistant is determined according to the classification standard of corn ear rot disease.

2. The method according to claim 1, characterized in that The final concentration of the Fusarium graminearum solution in step (1) is 4 to 6×10 5 spores / mL.

3. The method according to claim 1, characterized in that The inoculation amount of the Fusarium graminearum liquid in step (2) is 3 to 6 mL per corn ear.

4. The method according to claim 1, characterized in that The culture temperature in step (2) is 27-29°C.

5. The method according to claim 1, characterized in that The humidity of the culture in step (2) is 86-94%.

6. The method according to claim 1, characterized in that During the culture period of step (2), the dark box and light culture are alternated for 12-12 hours.

7. The method according to claim 1, characterized in that The corn ear rot disease classification standard in step (2) is as follows: Level 1: the diseased area accounts for 0% to 5% of the total area of ​​the corn ear; Level 2: The diseased area accounts for 6% to 15% of the total area of ​​the corn ear; Level 3: The diseased area accounts for 16% to 35% of the total area of ​​the corn ear; Level 4: The diseased area accounts for 36% to 55% of the total area of ​​the corn ear; Level 5: The diseased area accounts for 56% to 100% of the total area of ​​the corn ear.

8. The method according to claim 1, characterized in that The culture in step (2) is sealed culture.

9. The method according to claim 1, characterized in that: The nutrient solution in step (1) comprises the following components: 20-30 mg / L of gibberellin and 3-5 ml / L of 1-methylcyclopropene.

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