Method for accurately evaluating resistance of corn variety to fusarium verticillium ear rot

Abstract

The invention belongs to the field of crop disease resistance identification. The invention discloses a method for accurately evaluating the resistance of a corn variety to fusarium verticillium ear rot. The method comprises the following steps: inoculating corn ears by adopting a needling inoculation method, harvesting, threshing and grinding the ears in a harvesting period, measuring the content of fumonisins in grains, and evaluating the resistance of a corn variety to fusarium verticillium ear rot through the content of fumonisins in the grains. According to the method, the resistance of the corn variety to the fusarium verticillium ear rot can be evaluated more accurately, and the corn variety harmful to human and livestock is eliminated, so that the food safety is guaranteed.

Description

Technical Field

[0001] This invention belongs to the field of crop disease resistance identification. Background Technology

[0002] Ear rot of corn is one of the major diseases affecting corn, and it can occur during the later stages of corn growth and storage. Fusarium is the main pathogen causing ear rot, with Fusarium verticillatum being the dominant pathogen. Fusarium verticillatum infection of corn ears not only reduces corn yield, but also produces fumonisin in the kernels. This toxin has organ toxicity, neurotoxicity, immunotoxicity, and carcinogenicity. It affects not only the spleen, liver, heart, and kidneys of chicks, but also reduces the reproductive performance of livestock, causing pulmonary edema syndrome in pigs and leukomalacia in equines. It can also cause esophageal cancer, colon cancer in humans, and liver cancer in rats, seriously impacting human and animal health.

[0003] Breeding and utilizing disease-resistant varieties is the most economical, effective, and environmentally friendly way to control maize ear rot and its fungal toxin contamination. However, according to the "Agricultural Industry Standard - Technical Specification for Identification of Maize Disease and Insect Resistance Part 8: Fusarium Ear Rot," the evaluation of maize varieties' resistance to Fusarium rot primarily shows resistance and moderate resistance, with very few highly susceptible varieties. For example, in 2024, our team identified the resistance of approximately 2,500 maize materials to Fusarium rot, of which only 1.19% were susceptible, and no highly susceptible varieties were found. In maize production, the problem of excessive fumonisin content frequently occurs. Li Lina et al. measured the fumonisin content in 67 maize samples, and 28% of the samples exceeded the limit. Therefore, traditional evaluation methods cannot eliminate maize varieties that pose a risk of fumonisin contamination in production. Summary of the Invention

[0004] To address the inability of traditional resistance evaluation methods to eliminate maize varieties posing a risk of fumonisin contamination in production, the inventors have developed a precise method for assessing the resistance of maize varieties to Fusarium wilt. This method enables accurate assessment of Fusarium wilt resistance in maize materials, laying the foundation for breeding resistant varieties and reducing fumonisin contamination levels in maize.

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

[0006] Plant the corn material to be identified and carry out normal field management such as watering, weeding and fertilization.

[0007] Among them, the preferred variety is planted in 2 rows, with a row length of 5m, a row spacing of 0.6m, a plant spacing of 0.25m, and double seeds sown. Thinning is carried out at the 3-5 leaf stage.

[0008] Furthermore, *Fusarium verticillatum* was cultured in the dark at 25–28°C on PDA medium. Once the colonies had fully colonized the culture dishes, an appropriate amount of sterile water was added. The colonies were then spread onto the surface of the medium using a spreader, allowing the conidia to dissipate into the sterile water. The solution was filtered through double-layered sterile gauze to obtain a concentration of 5 × 10⁻⁶. 5 ~5×10 6 Prepare a conidial suspension of 1 conidia / mL for later use.

[0009] Preferably, the spore suspension concentration is 2×10⁻⁶. 6 per mL.

[0010] Furthermore, 7-10 days after silking, a needle-punching inoculation method is used to puncture the outer husk of the female ear with a hollow metal needle. When the metal needle penetrates the grain, the conidial suspension is injected onto the ear, with 2 mL injected per plant.

[0011] Furthermore, during the harvest period, all inoculated female ears are collected according to the variety, and dried until the grain moisture content is below 14%. The female ears are then threshed, and the threshed grains are mixed evenly. A portion of the threshed grains is then ground into powder using a high-speed grinder. Three samples are taken from each variety for grinding.

[0012] Further, 5g of powder was weighed from each sample and placed in a 50mL centrifuge tube. 25mL of 40% ethanol-water was added and mixed thoroughly. The mixture was then vigorously shaken on a shaker for 10 minutes, centrifuged at 4000 rpm for 5 minutes, and the supernatant was diluted with 1×PBS. The fumonisin content in the sample was then detected using a fumonisin (FB1) ELISA quantitative detection kit. The absorbance values ​​of the standard sample and the test sample were measured at 450nm using an ELISA reader, and the toxin content in the grains was calculated.

[0013] Furthermore, the resistance of maize varieties to Fusarium wilt was evaluated based on the toxin content in the kernels. The resistance evaluation criteria were as follows: toxin content ≤7.50 mg / Kg, highly resistant (HR); toxin content 7.51–10.00 mg / Kg, resistant (R); toxin content 10.01–12.50 mg / Kg, moderately resistant (MR); toxin content 12.51–15.00 mg / Kg, susceptible (S); toxin content >15.00 mg / Kg, highly susceptible (HS). Detailed Implementation

[0014] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0015] Planted in 2024 at the Gucheng Experimental Base in Dingxing County, Baoding City, Hebei Province. Each variety was planted in two rows, 5m long, with a row spacing of 0.6m and a plant spacing of 0.25m, using double-seed sowing. Two protective rows of various other varieties were placed around the perimeter. Field management, including watering, weeding, and fertilization, was normal after sowing.

[0016] Fusarium pseudovertebratum was cultured on PDA medium at 28°C in the dark for 7 days. After the colonies had fully colonized the culture dishes, an appropriate amount of sterile water was added to the dishes. The colonies were then spread on the surface of the medium using a spreader, allowing the conidia on the colonies to disperse into the sterile water. The conidia suspension was obtained by filtration through double-layer sterile gauze. The concentration of the conidia suspension was adjusted to 2 × 10⁻⁶ with sterile water. 6 Quantity / mL, for later use.

[0017] 7–10 days after the female ear spins silk, use the needle inoculation method to puncture the outside of the female ear bracts with a hollow metal needle. When the metal needle penetrates the grain, inject the spore suspension onto the ear, injecting 2 mL per plant.

[0018] Traditional resistance evaluation method: Before harvest, after removing the husks, investigate each ear individually. Evaluate the disease severity level based on the area infected by the pathogen on each ear: Level 1 disease affects 0%–1% of the ear area; Level 3 disease affects 2%–10% of the ear area; Level 5 disease affects 11%–25% of the ear area; Level 7 disease affects 26%–50% of the ear area; and Level 9 disease affects 51%–100% of the ear area. Calculate the average disease severity level for all ears of the same variety: Average disease severity level = ∑(Disease severity level × Number of plants) / Total number of plants. Evaluate resistance based on the average disease severity level. The resistance evaluation criteria are: Average disease severity level ≤ 1.5, Highly resistant (HR); Average disease severity level 1.6–3.5, Resistant (R); Average disease severity level 3.6–5.5, Moderately resistant (MR); Average disease severity level 5.6–7.5, Susceptible (S); Average disease severity level 7.6–9.0, Highly susceptible (HS).

[0019] Precise assessment method: All female ears were collected during the harvest season according to variety. After drying until the grain moisture content was below 14%, the ears were threshed. The threshed kernels were mixed evenly, and a portion was ground into powder using a high-speed grinder. Three samples were taken from each variety for grinding. 5g of powder was weighed from each sample and placed in a 50mL centrifuge tube. 25mL of 40% ethanol-water was added and mixed thoroughly. The mixture was vigorously shaken for 10 minutes, then centrifuged at 4000 rpm for 5 minutes. The supernatant was diluted with 1×PBS, and the fumonisin content in the sample was detected using a fumonisin (FB1) ELISA quantitative detection kit. The absorbance of the standard sample and the test sample was measured at 450nm using an ELISA reader, and the fumonisin content in the kernels was calculated. The resistance of maize varieties to *Fusarium oxysporum* ear rot was evaluated based on the toxin content in the kernels. The resistance evaluation criteria are as follows: toxin content ≤7.50 mg / Kg, high resistance (HR); toxin content 7.51~10.00 mg / Kg, resistance (R); toxin content 10.01~12.50 mg / Kg, moderate resistance (MR); toxin content 12.51~15.00 mg / Kg, susceptibility (S); toxin content >15.00 mg / Kg, high susceptibility (HS).

[0020] Table 1. Resistance of different maize varieties to Fusarium wilt of the ear.

[0021]

[0022] Note: Data in the table are mean ± standard error. Different lowercase letters after the data in the same column indicate significant differences (P<0.05).

[0023] The resistance of 34 maize varieties to Fusarium wilt was evaluated using two different methods. The conventional resistance evaluation showed differences in the average disease severity among the 34 varieties, but all were either resistant or moderately resistant. Xianyu 335 had the highest average disease severity at 4.0, indicating moderate resistance (MR) to Fusarium wilt. The other 33 varieties had average disease severity ranging from 1.6 to 3.3, all showing resistance (R). The precise evaluation method showed that 10 of the 34 maize varieties (29.41%) were highly resistant to Fusarium wilt; 11 were resistant (32.35%); 6 were moderately resistant (13.89%); 3 were susceptible (8.82%); and 4 were highly susceptible (11.76%).

[0024] Comparing the two evaluation methods revealed that the traditional method for evaluating resistance to Fusarium wilt cannot eliminate maize varieties with high production risks; however, the precise evaluation method can eliminate varieties with high fumonisin content in kernels after Fusarium wilt infection, thereby reducing the harm of fumonisin to humans and animals.

Claims

1. A method for accurately assessing the resistance of maize varieties to Fusarium wilt, characterized in that, Includes the following steps: (1) Planting and field management of the maize material to be identified; (2) Preparation of spore suspension; (3) Inoculation with pathogens; (4) Harvesting, drying and grinding: After harvesting all female ears according to variety during the harvest season, dry them until the grain moisture content is less than 14%, then thresh the female ears. After mixing the threshed grains evenly, take out a portion and grind it into powder using a high-speed grinding mill. Take 3 samples of each variety for grinding. (5) Fumonisin determination: The content of fumonisin in the grain was detected by using a fumonisin (FB1) ELISA quantitative detection kit; (6) Resistance evaluation: The resistance of maize varieties to Fusarium wilt is evaluated based on the toxin content.

2. The method as described in claim 1, characterized in that, The specific method for step (2) is as follows: Fusarium pseudovertebratum is cultured in the dark at 25-28 ℃ on PDA medium. After the colony has fully grown in the culture dish, an appropriate amount of sterile water is added to the dish. The colony is then spread on the surface of the medium using a spreader, allowing the conidia on the colony to fall into the sterile water. The conidia suspension is obtained by filtration through double-layer sterile gauze. The concentration of the conidia suspension is adjusted to 5 × 10⁻⁶ with sterile water. 5 ~5×10 6 Quantity / mL, for later use.

3. The method as described in claim 1, characterized in that, The specific method of step (3) is as follows: 7-10 days after silking, use the needle inoculation method to puncture the outside of the female ear husk with a hollow metal needle. When the metal needle pierces the grain, inject the spore suspension onto the ear, injecting 2 mL per plant.

4. The method as described in claim 1, characterized in that, The specific method for step (5) is as follows: Weigh 5 g of sample powder into a 50 mL centrifuge tube, add 25 mL of 40% ethanol-water mixture, shake vigorously for 10 min, centrifuge at 4000 r / min for 5 min, dilute the supernatant with 1×PBS, and then use a fumonisin (FB1) ELISA quantitative detection kit to detect the fumonisin content in the sample. Detect the absorbance of the standard sample and the sample to be tested at 450 nm using an ELISA reader, and calculate the toxin content in the grains.

5. The method as described in claim 1, characterized in that, The specific resistance evaluation criteria for step (6) are as follows: toxin content ≤ 7.50 mg / Kg, high resistance (HR); toxin content 7.51~10.00 mg / Kg, resistance (R); toxin content 10.01~12.50 mg / Kg, moderate resistance (MR); toxin content 12.51~15.00 mg / Kg, susceptibility (S); toxin content > 15.00 mg / Kg, high susceptibility (HS).

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