CN109694900B - Indoor rapid identification method for corn disease resistance - Google Patents
Indoor rapid identification method for corn disease resistance Download PDFInfo
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Abstract
The invention belongs to the technical field of Aspergillus flavus ear rot resistance of corn, and discloses an indoor rapid identification method for corn disease resistance, which comprises the following steps: A. preparing an aspergillus flavus spore suspension; B. preparing a sterilized sample of the corn variety to be tested; C. respectively inoculating the aspergillus flavus spore suspension on each seed of each disinfected tested corn sample, and culturing at constant temperature to obtain a corn variety infected with diseases; D. and (3) determining the average disease grade of each corn variety infected with diseases by using the susceptible varieties with the disease grade of 9 as a control, and dividing resistance according to the average disease grade to realize the identification of the resistance of the Aspergillus flavus ear rot of the corn. The invention has the characteristics of short identification period, low identification cost and accurate result.
Description
Technical Field
The invention belongs to the technical field of identification of resistance to Aspergillus flavus ear rot of corn, and particularly relates to an indoor rapid identification method for resistance to corn diseases.
Background
The maize ear rot is a maize disease closely related to human health. Corn production occurs throughout the world. The fungal complex of this disease can produce several mycotoxins (mycotoxins), such as aflatoxins and fumonisins, which affect human and animal health.
The aspergillus flavus (aflavis) in the aspergillus not only harms various grains such as corn and the like, but also produces toxic metabolite aflatoxin, which causes poisoning of people, livestock and poultry. Medical research shows that long-term consumption of food containing low-concentration aflatoxin is considered to be the main cause of liver cancer, gastric cancer, intestinal cancer and other diseases.
The corn ears and the corn kernels can be damaged, and the symptoms are that the damaged ears or the corn kernels have yellow, yellow-green and yellow-brown mildew layers, namely mycelium, conidiophore and conidia of pathogenic bacteria. The diseased grain is dull, not full, rotten, and empty and deficient inside, and is often filled with interlaced hypha and spores. The bracts of the diseased part of the fruit cluster are often penetrated by dense hypha and are adhered together to be stuck on the fruit cluster and not easy to peel off.
Once the corn is infected with the disease, the problem of aflatoxin accumulation caused by the infection of the corn can cause food pollution and feed pollution, and the aflatoxin enters a human body through a food chain, so that the human body is diseased. The excessive toxin in the corn causes the feed manufacturers to be reluctant to purchase local corn as feed processing raw materials, so that the commodity rate and the price of the local corn are low. Currently, pharmacological control is largely ineffective against this disease. Therefore, planting disease-resistant varieties is the most economic and effective way to solve the diseases. The screening of disease-resistant varieties requires simple, easy and accurate resistance identification technology.
However, the existing technology for identifying resistance to maize aspergillus flavus ear rot is generally a field artificial inoculation technology, and by means of inoculating maize varieties in a field planting condition, seeds are injured by a needle with bacteria in a spinning period of maize ears, and resistance level investigation is carried out for identification when the ears are mature and harvested; the defects of the prior art are that corn varieties need to be planted in the field, the workload is large, the cost is high, the period is long, and the identification result is greatly influenced by the weather conditions.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide an indoor rapid identification method for corn disease resistance.
The technical scheme adopted by the invention is as follows:
an indoor rapid identification method for corn disease resistance comprises the following steps: A. preparing an aspergillus flavus spore suspension; B. preparing a sterilized sample of the corn variety to be tested; C. respectively inoculating the aspergillus flavus spore suspension on each seed of each sterilized corn sample to be tested, and culturing at constant temperature to obtain a corn variety infected with diseases; D. and (3) determining the average disease grade of each corn variety infected with diseases by using the susceptible variety with the disease grade of 9 as a control, and dividing the resistance according to the average disease grade to realize the identification of the resistance to the Aspergillus flavus ear rot of the corn.
Further, the step a comprises: inoculating aspergillus flavus strain on a PDA flat plate, culturing aspergillus flavus spores at constant temperature, and mixing sterile water with the aspergillus flavus spores to obtain aspergillus flavus spore suspension.
Further, the constant temperature in the step A is 20-25 ℃; the number of Aspergillus flavus spores in the Aspergillus flavus spore suspension is 1 × 1051X 10 per mL7one/mL.
Further, the step B includes: and (3) sterilizing each seed of each corn variety to be tested, cleaning with sterile water, and draining with sterile filter paper to obtain a plurality of sterilized corn variety samples to be tested.
Further, the disinfection treatment in the step B comprises the step of soaking each kernel of each corn variety to be tested with alcohol and sodium hypochlorite in sequence.
Further, in the step B, the mass fraction of the alcohol is 75%, and the mass fraction of the sodium hypochlorite is 3%; soaking each seed of each tested corn variety in alcohol for 30 s; each seed of each corn variety tested was soaked with sodium hypochlorite for 3 min.
Further, the specific steps of the step C are as follows: sucking quantitative aspergillus flavus spore suspension liquid to wet the sterilization filter paper to obtain the wet filter paper; and (3) putting each seed of the corn sample to be tested on the wet filter paper, and culturing at constant temperature to obtain the corn variety infected with diseases.
Further, the constant temperature culture temperature in the step C is 20-25 ℃; c, wetting a piece of sterilized filter paper to suck 5mL of the aspergillus flavus spore suspension; the number of kernels placed per wet filter paper was 10.
The invention has the beneficial effects that: the indoor rapid identification method for the corn disease resistance adopts an indoor identification mode, so that the cost of land cost and cultivation management cost for field identification to be planted in the field is avoided; the identification period of the identification method is 1 week, and compared with the field identification which needs 4 months for one season, the time period is greatly reduced; the identification method disclosed by the invention is used for culturing in an indoor constant-temperature incubator, so that inaccurate results caused by insufficient morbidity and the like due to unstable field climate conditions are avoided; the method can greatly reduce a large amount of aspergillus flavus produced by field identification and reduce the pollution to the environment. The method saves the cost of manpower and material resources, is simple to operate, and has more stable and credible identification result. Therefore, the method has the characteristics of short identification period, low identification cost and accurate result.
Detailed Description
The present invention is further illustrated below with reference to specific examples.
An indoor rapid identification method for corn disease resistance comprises the following steps: A. preparing an aspergillus flavus spore suspension; B. preparing a sterilized sample of the corn variety to be tested; C. respectively inoculating the aspergillus flavus spore suspension on each seed of each sterilized corn sample to be tested, and culturing at constant temperature to obtain a corn variety infected with diseases; D. and (3) determining the average disease grade of each corn variety infected with diseases by using the susceptible variety with the disease grade of 9 as a control, and dividing the resistance according to the average disease grade to realize the identification of the resistance to the Aspergillus flavus ear rot of the corn.
Further, the step a comprises: inoculating aspergillus flavus strain on a PDA flat plate, culturing aspergillus flavus spores at constant temperature, and mixing sterile water with the aspergillus flavus spores to obtain aspergillus flavus spore suspension.
Further, the constant temperature in the step A is 20-25 ℃; the number of Aspergillus flavus spores in the Aspergillus flavus spore suspension is 1 × 1051X 10 per mL7one/mL.
Further, the step B includes: and (3) sterilizing each seed of each corn variety to be tested, cleaning with sterile water, and draining with sterile filter paper to obtain a plurality of sterilized corn variety samples to be tested.
Further, the disinfection treatment in the step B comprises the step of soaking each kernel of each corn variety to be tested with alcohol and sodium hypochlorite in sequence.
Further, in the step B, the mass fraction of the alcohol is 75%, and the mass fraction of the sodium hypochlorite is 3%; soaking each seed of each tested corn variety in alcohol for 30 s; each seed of each corn variety tested was soaked with sodium hypochlorite for 3 min.
Further, the specific steps of the step C are as follows: absorbing quantitative aspergillus flavus spore suspension to wet the sterilized filter paper to obtain wet filter paper; and (3) putting each seed of the corn sample to be tested on the wet filter paper, and culturing at constant temperature to obtain the corn variety infected with diseases.
Further, the constant temperature culture temperature in the step C is 20-25 ℃; c, wetting a piece of sterilized filter paper to suck 5mL of the aspergillus flavus spore suspension; the number of kernels placed per wet filter paper was 10.
Example 1
An indoor rapid identification method for corn disease resistance comprises the following steps: A. preparing an aspergillus flavus spore suspension; B. preparing a sterilized sample of the corn variety to be tested; C. respectively inoculating the aspergillus flavus spore suspension on each seed of each sterilized corn sample to be tested, and culturing at constant temperature to obtain a corn variety infected with diseases; D. and (3) determining the average disease grade of each corn variety infected with diseases by using the susceptible variety with the disease grade of 9 as a control, and dividing the resistance according to the average disease grade to realize the identification of the resistance to the Aspergillus flavus ear rot of the corn.
Example 2
An indoor rapid identification method for corn disease resistance comprises the following steps: A. preparing an aspergillus flavus spore suspension; B. preparing a sterilized sample of the corn variety to be tested; C. respectively inoculating the aspergillus flavus spore suspension on each seed of each sterilized corn sample to be tested, and culturing at constant temperature to obtain a corn variety infected with diseases; D. and (3) determining the average disease grade of each corn variety infected with diseases by using the susceptible variety with the disease grade of 9 as a control, and dividing the resistance according to the average disease grade to realize the identification of the resistance to the Aspergillus flavus ear rot of the corn.
The step A comprises the following steps: inoculating aspergillus flavus strain on a PDA flat plate, culturing aspergillus flavus spores at constant temperature, and mixing sterile water with the aspergillus flavus spores to obtain aspergillus flavus spore suspension.
The PDA in the invention is a potato glucose agar culture medium (200 g of potato, 15g of glucose, 18g of agar powder and 1000mL of water), aspergillus flavus spores are cultured after aspergillus flavus strains are plated on a PDA plate, and the aspergillus flavus spores are used as a subsequent inoculation step.
Example 3
An indoor rapid identification method for corn disease resistance comprises the following steps: A. preparing an aspergillus flavus spore suspension; B. preparing a sterilized sample of the corn variety to be tested; C. respectively inoculating the aspergillus flavus spore suspension on each seed of each sterilized corn sample to be tested, and culturing at constant temperature to obtain a corn variety infected with diseases; D. and (3) determining the average disease grade of each corn variety infected with diseases by using the susceptible variety with the disease grade of 9 as a control, and dividing the resistance according to the average disease grade to realize the identification of the resistance to the Aspergillus flavus ear rot of the corn.
The step A comprises the following steps: inoculating aspergillus flavus strain on a PDA flat plate, culturing aspergillus flavus spores at constant temperature, and mixing sterile water with the aspergillus flavus spores to obtain aspergillus flavus spore suspension.
The PDA in the invention is a potato glucose agar culture medium (200 g of potato, 15g of glucose, 18g of agar powder and 1000mL of water), aspergillus flavus spores are cultured after aspergillus flavus strains are plated on a PDA plate, and the aspergillus flavus spores are used as a subsequent inoculation step.
The constant temperature in the step A is 20 ℃; the number of Aspergillus flavus spores in the Aspergillus flavus spore suspension is 1 × 105one/mL.
Example 4
An indoor rapid identification method for corn disease resistance comprises the following steps: A. preparing an aspergillus flavus spore suspension; B. preparing a sterilized sample of the corn variety to be tested; C. respectively inoculating the aspergillus flavus spore suspension on each seed of each sterilized corn sample to be tested, and culturing at constant temperature to obtain a corn variety infected with diseases; D. and (3) determining the average disease grade of each corn variety infected with diseases by using the susceptible variety with the disease grade of 9 as a control, and dividing the resistance according to the average disease grade to realize the identification of the resistance to the Aspergillus flavus ear rot of the corn.
The step A comprises the following steps: inoculating aspergillus flavus strains on a PDA (personal digital assistant) flat plate, culturing aspergillus flavus spores at constant temperature, and mixing the aspergillus flavus spores with sterile water to obtain an aspergillus flavus spore suspension.
The PDA in the invention is a potato glucose agar culture medium (200 g of potato, 15g of glucose, 18g of agar powder and 1000mL of water), aspergillus flavus spores are cultured after aspergillus flavus strains are plated on a PDA plate, and the aspergillus flavus spores are used as a subsequent inoculation step.
The constant temperature in the step A is 23 ℃; the number of Aspergillus flavus spores in the Aspergillus flavus spore suspension is 1 × 107One per mL.
Example 5
An indoor rapid identification method for corn disease resistance comprises the following steps: A. preparing an aspergillus flavus spore suspension; B. preparing a sterilized sample of the corn variety to be tested; C. respectively inoculating the aspergillus flavus spore suspension on each seed of each sterilized corn sample to be tested, and culturing at constant temperature to obtain a corn variety infected with diseases; D. and (3) determining the average disease grade of each corn variety infected with diseases by using the susceptible variety with the disease grade of 9 as a control, and dividing the resistance according to the average disease grade to realize the identification of the resistance to the Aspergillus flavus ear rot of the corn.
The step A comprises the following steps: inoculating aspergillus flavus strain on a PDA flat plate, culturing aspergillus flavus spores at constant temperature, and mixing sterile water with the aspergillus flavus spores to obtain aspergillus flavus spore suspension.
The PDA in the invention is a potato glucose agar culture medium (200 g of potato, 15g of glucose, 18g of agar powder and 1000mL of water), aspergillus flavus spores are cultured after aspergillus flavus strains are plated on a PDA plate, and the aspergillus flavus spores are used as a subsequent inoculation step.
The constant temperature in the step A is 25 ℃; the number of Aspergillus flavus spores in the Aspergillus flavus spore suspension is 1 × 106One per mL.
Example 6
An indoor rapid identification method for corn disease resistance comprises the following steps: A. preparing an aspergillus flavus spore suspension; B. preparing a sterilized sample of the corn variety to be tested; C. respectively inoculating the aspergillus flavus spore suspension on each seed of each sterilized corn sample to be tested, and culturing at constant temperature to obtain a corn variety infected with diseases; D. and (3) determining the average disease grade of each corn variety infected with diseases by using the susceptible variety with the disease grade of 9 as a control, and dividing the resistance according to the average disease grade to realize the identification of the resistance to the Aspergillus flavus ear rot of the corn.
The step A comprises the following steps: inoculating aspergillus flavus strain on a PDA flat plate, culturing aspergillus flavus spores at constant temperature, and mixing sterile water with the aspergillus flavus spores to obtain aspergillus flavus spore suspension.
The PDA in the invention is a potato glucose agar culture medium (200 g of potato, 15g of glucose, 18g of agar powder and 1000mL of water), aspergillus flavus spores are cultured after aspergillus flavus strains are plated on a PDA plate, and the aspergillus flavus spores are used as a subsequent inoculation step.
The constant temperature in the step A is 25 ℃; the number of Aspergillus flavus spores in the Aspergillus flavus spore suspension is 1 × 106one/mL.
The step B comprises the following steps: sterilizing each seed of each corn variety to be tested, cleaning with sterile water, and draining with sterile filter paper to obtain a plurality of sterilized corn variety samples to be tested; the disinfection treatment in the step B comprises the step of soaking each seed of each corn variety to be tested with alcohol and sodium hypochlorite in sequence; in the step B, the mass fraction of the alcohol is 75%, and the mass fraction of the sodium hypochlorite is 3%; soaking each seed of each tested corn variety in alcohol for 30 s; each seed of each corn variety tested was soaked with sodium hypochlorite for 3 min.
Example 7
An indoor rapid identification method for corn disease resistance comprises the following steps: A. preparing an aspergillus flavus spore suspension; B. preparing a sterilized sample of the corn variety to be tested; C. respectively inoculating the aspergillus flavus spore suspension on each seed of each sterilized corn sample to be tested, and culturing at constant temperature to obtain a corn variety infected with diseases; D. and (3) determining the average disease grade of each corn variety infected with diseases by using the susceptible variety with the disease grade of 9 as a control, and dividing the resistance according to the average disease grade to realize the identification of the resistance to the Aspergillus flavus ear rot of the corn.
The specific steps of the step C are as follows: absorbing quantitative aspergillus flavus spore suspension to wet the sterilized filter paper to obtain wet filter paper; and (3) putting each seed of the corn sample to be tested on the wet filter paper, and culturing at constant temperature to obtain the corn variety infected with diseases.
The temperature of constant temperature culture in the step C is 20 ℃; c, wetting a piece of sterilized filter paper to suck 5mL of the aspergillus flavus spore suspension; the number of kernels placed per wet filter paper was 10.
Example 8
An indoor rapid identification method for corn disease resistance comprises the following steps: A. preparing an aspergillus flavus spore suspension; B. preparing a sterilized sample of the corn variety to be tested; C. respectively inoculating the aspergillus flavus spore suspension on each seed of each sterilized corn sample to be tested, and culturing at constant temperature to obtain a corn variety infected with diseases; D. and (3) determining the average disease grade of each corn variety infected with diseases by using the susceptible variety with the disease grade of 9 as a control, and dividing the resistance according to the average disease grade to realize the identification of the resistance to the Aspergillus flavus ear rot of the corn.
The specific steps of the step C are as follows: absorbing quantitative aspergillus flavus spore suspension to wet the sterilized filter paper to obtain wet filter paper; and (3) putting each seed of the corn sample to be tested on the wet filter paper, and culturing at constant temperature to obtain the corn variety infected with diseases.
The constant temperature culture temperature in the step C is 22 ℃; c, wetting a piece of sterilized filter paper to suck 5mL of the aspergillus flavus spore suspension; the number of kernels placed per wet filter paper was 10.
Example 9
An indoor rapid identification method for corn disease resistance comprises the following steps: A. preparing an aspergillus flavus spore suspension; B. preparing a sterilized sample of the corn variety to be tested; C. respectively inoculating the aspergillus flavus spore suspension on each seed of each sterilized corn sample to be tested, and culturing at constant temperature to obtain a corn variety infected with diseases; D. and (3) determining the average disease grade of each corn variety infected with diseases by using the susceptible variety with the disease grade of 9 as a control, and dividing the resistance according to the average disease grade to realize the identification of the resistance to the Aspergillus flavus ear rot of the corn.
The specific steps of the step C are as follows: absorbing quantitative aspergillus flavus spore suspension to wet the sterilized filter paper to obtain wet filter paper; and (3) putting each seed of the corn sample to be tested on the wetting filter paper, and culturing at constant temperature to obtain the corn variety infected with diseases.
The constant temperature culture temperature in the step C is 25 ℃; c, wetting a piece of sterilized filter paper to suck 5mL of the aspergillus flavus spore suspension; the number of kernels placed per wet filter paper was 10.
Example 10
An indoor rapid identification method for corn disease resistance comprises the following steps:
the preparation method of the bacterial source comprises the following steps: inoculating the stored Aspergillus flavus strain on PDA plate, culturing at constant temperature of 25 deg.C for 5d, washing Aspergillus flavus spore with sterile water after the Aspergillus flavus strain grows over the plate, counting with blood counting plate under microscope, diluting the bacterial liquid to 1 × 106And each ml is ready for use.
Treatment of a test corn sample: selecting full, consistent and healthy-appearance seeds for disinfection treatment, soaking the seeds in 75% alcohol for one minute, then soaking the seeds in 3% sodium hypochlorite for three minutes, finally cleaning the seeds with distilled water for three times, and placing the seeds on sterilized filter paper to drain water for later use.
The inoculation method comprises the following steps: placing a layer of sterilized filter paper at the bottom of a culture dish, sucking 5mL of spore suspension to uniformly wet the filter paper, placing sterilized seeds on the wetted filter paper in the culture dish, uniformly placing 10 seeds in each dish, covering the dish with a cover, and placing the dish in a constant-temperature incubator for culture at 25 ℃. The photographs were observed after 7 days and the disease was recorded.
In addition, the step C of the invention can also carry out inoculation in the following mode, a PDA flat plate inoculated with the strain is adopted to replace sterilization filter paper, or a corn kernel sample to be tested is soaked in the aspergillus flavus bacterial liquid and then placed on the sterilization filter paper, or the corn kernel sample is placed on the sterilization filter paper and then the bacterial liquid is sprayed on the kernel for inoculation, and 3 methods can cause the kernel to be attacked.
And (3) determining the average disease grade of each corn variety infected with diseases by using the susceptible variety with the disease grade of 9 as a control, and dividing the resistance according to the average disease grade to realize the identification of the resistance to the Aspergillus flavus ear rot of the corn.
Comparative example
Contrast setting: using the susceptible variety as a control, and beginning to investigate the morbidity of the test sample when the control morbidity reaches a grade 9 standard.
The disease grade judgment standard is as follows: as in table 1 below.
TABLE 1 evaluation of disease status for Aspergillus flavus resistant ear rot in maize
| Grading of disease conditions | Description of the invention |
| 1 | The disease area accounts for 0 to 1 percent of the total area of the seeds |
| 3 | The disease area accounts for 2 to 10 percent of the total area of the seeds |
| 5 | The disease area accounts for 11 to 25 percent of the total area of the seeds |
| 7 | The disease area accounts for 26 to 50 percent of the total area of the grains |
| 9 | Area of diseaseAccounts for 51 to 100 percent of the total area of the grains |
Kernels from 26 maize varieties tested were placed in petri dishes 7 days after inoculation and the results of the survey are given in table 2 below. As can be seen from the results in Table 2 below, the disease grade of 26 varieties is 3.9 at the lowest and 9.0 at the highest, and the resistance of different corn varieties can be fully expressed according to the variety resistance division standard.
Wherein the average disease grade is the ratio of the sum of the disease grades of all the kernels of the test corn sample of each variety to the number of all the kernels of each variety.
Table 226 maize variety indoor inoculation identification Aspergillus flavus average disease level result
Specific resistance level divisions are shown in table 3 below.
TABLE 3 resistance level partitioning of maize against resistance to ear rot
| Average disease level | Resistance to |
| ≤1.5 | High resistance to Highly Resist (HR) |
| 1.6～3.5 | anti-Resistant (R) |
| 3.6～5.5 | anti-Moderatey Resister (MR) |
| 5.6～7.5 | Susceptible (S) |
| 7.6～9.0 | High sensitivity Highly Susceptable (HS) |
Therefore, the method uses the susceptible variety as a contrast, and adopts the indoor rapid identification method, so that the indoor identification is realized, and the time period is greatly reduced; the error of inaccurate result caused by insufficient morbidity and the like due to unstable weather conditions is avoided; and simultaneously reduces the pollution to the environment.
The present invention is not limited to the above-described alternative embodiments, and various other forms of products can be obtained by anyone in light of the present invention. The above detailed description should not be taken as limiting the scope of the invention, which is defined in the claims, and which the description is intended to be interpreted accordingly.
Claims (2)
1. An indoor rapid identification method for corn disease resistance is characterized in that: the method comprises the following steps:
A. preparing an aspergillus flavus spore suspension: inoculating aspergillus flavus strain on a PDA flat plate, culturing aspergillus flavus spores at the constant temperature of 20-25 ℃, mixing sterile water with the aspergillus flavus spores to obtain aspergillus flavus spore suspension, wherein the number of the aspergillus flavus spores in the aspergillus flavus spore suspension is 1 multiplied by 1051X 10 per mL7Per mL;
B. sterilizing each seed of each corn variety to be tested, cleaning with sterile water, draining with sterile filter paper to obtain a plurality of sterilized corn variety samples to be tested, wherein the sterilizing treatment comprises the steps of soaking each seed of each corn variety to be tested in alcohol for 30s, and then soaking in a sodium hypochlorite solution for 3 min;
C. absorbing quantitative aspergillus flavus spore suspension to wet the sterilized filter paper to obtain wet filter paper; placing each seed of a corn sample to be tested on wetting filter paper, and culturing for 7 days at a constant temperature of 20-25 ℃ to obtain a corn variety infected with diseases;
wherein the volume of the aspergillus flavus spore suspension absorbed by wetting a piece of sterilized filter paper is 5 mL; the number of seeds placed on each piece of wet filter paper is 10;
D. and (3) determining the average disease grade of each corn variety infected with diseases by using the susceptible variety with the disease grade of 9 as a control, and dividing the resistance according to the average disease grade to realize the identification of the resistance to the Aspergillus flavus ear rot of the corn.
2. The indoor rapid identification method of corn disease resistance according to claim 1, characterized in that: and in the step B, the mass fraction of the alcohol is 75%, and the mass fraction of the sodium hypochlorite is 3%.
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| CN107870224A (en) * | 2017-11-03 | 2018-04-03 | 安徽省农业科学院烟草研究所 | A kind of field identification method for evaluating corn aspergillus flavus infection resistance ability |
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| 全基因组关联分析筛选玉米籽粒黄曲霉菌抗性基因;高蕾;《中国优秀博硕士学位论文全文数据库(硕士) 农业科技辑》;20190115;第1-29页 * |
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