CN1556391A - Peanut aspergillus flavus anti aflatoxin production preperty identification method - Google Patents
Peanut aspergillus flavus anti aflatoxin production preperty identification method Download PDFInfo
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- CN1556391A CN1556391A CNA2003101116825A CN200310111682A CN1556391A CN 1556391 A CN1556391 A CN 1556391A CN A2003101116825 A CNA2003101116825 A CN A2003101116825A CN 200310111682 A CN200310111682 A CN 200310111682A CN 1556391 A CN1556391 A CN 1556391A
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Abstract
The method includes following five steps: (1) sterilizing and rehydrating surface of peanut seed; (2) inoculating strain of aspergillus flavus to healthy peanut; (3) cultivating inoculated seed in incubator for seven days; (4) distilling toxin liquid of aspergillus flavus by using methyl alcohol after baking and grinding; (5) adding ammonia-mercury solution, measuring fluorescence value so as to determine toxin and evaluate grade of resistance. The invention authenticates resistance of peanut against toxin of aspergillus flavus, being suitable to screen breeding material in batch mode. Authenticating cost is low.
Description
Technical field
The present invention relates to one cultivates peanut Aspergillus flavus is produced the method that malicious resistance is identified.
Background technology
Aspergillus flavus produces malicious resistance and is meant that peanut kind benevolence after being subjected to having the Aspergillus flavus (Aspergillus flavus) that produces the aflatoxin ability and aspergillus parasiticus bacterium (A.parasiticus) and infecting, can suppress the characteristic of above-mentioned fungi generation aflatoxin.Have the peanut varieties that produces malicious resistance and can significantly reduce aflatoxin contamination level in the peanut products.Identify that at present method that aspergillus flavus produces malicious resistance is by detecting aflatoxin content after the artificial infection, the method that detects toxin is thin-layered chromatography (being called for short TLC) and high pressure lipuid chromatography (HPLC) (abbreviation HPLC).TLC and HPLC have complicated operation, sample detection expense height, characteristics high to equipment requirements, and the resistance that can not effectively be applied in the peanut breeding research is identified.
Summary of the invention
At above-mentioned deficiency, the object of the present invention is to provide a kind of simple to operate, cost is low, the aspergillus flavus that is suitable for peanut breeding research produces malicious resistance authentication method.
To achieve these goals, technical scheme of the present invention is: screening has high yield poison ability and shows stable aspergillus flavus strain; Optimize artificial inoculation conditions and aflatoxin extracting method; On fluorospectrophotometer, measure fluorescent value with standard aflatoxin B1 sample preparation variable concentrations solution, set up the typical curve of toxin concentration and fluorescent value; Measure the fluorescent value of artificial infection peanut sample; Calculate aflatoxin B1 content and estimate resistance with typical curve.
Aspergillus flavus produces malicious resistance authentication method, and its feature is realized by following steps: 1) peanut seed surface sterilization and rehydration; 2) peanut seed inoculation: inoculate healthy peanut with aspergillus flavus strain; 3) cultivate: cultivated 7 days in incubator the inoculation back; 4) extract aflatoxin: through baking, grinding, again with methanol extraction aflatoxin liquid; 5) toxin determination: add the ammonia mercury solution, on fluorospectrophotometer, measure fluorescent value, calculate aflatoxin B1 content and estimate the resistance grade with the typical curve between aflatoxin B1 and the fluorescent value.
The concrete steps of this method are as follows:
1) peanut seed surface sterilization and rehydration: get healthy peanut seed and place sterile petri dish, in Biohazard Safety Equipment, continue to spray the surface of the seed 1-1.5 minute with 70% alcohol, the fine rotation double dish makes the surface of the seed sterilization evenly, outwell alcohol and impurity, again with alcohol-pickled 5 minutes of same concentrations, and then with aqua sterilisa flushing 3 times, be controlled in 30 minutes from the beginning surface sterilization to the time that flushing finishes, making the water cut after the peanut seed rehydration is 20-30%.
2) peanut seed inoculation: will sterilize and rehydration after peanut seed draw with aseptic scalper and break in the seed coat, add 1 milliliter of Aspergillus flavus spore suspension of preparing in advance, the rotation double dish makes the spore can be evenly attached to the surface of the seed.
3) cultivate: after the inoculation double dish is placed the plastic casing of adding a cover, add the aqua sterilisa of 2-3 millimeters deep in the plastic casing, making the interior relative humidity of box is 80-90%, plastic casing is put into 30 ℃ constant incubator and is cultivated 7 days.
4) extract aflatoxin: the alcohol with concentration 70% in Biohazard Safety Equipment sprays above-mentioned the surface of the seed through inoculated and cultured, put into 110 ℃ of baking oven bakings 1 hour then, the shape of claying into power after the cooling in Biohazard Safety Equipment, change in 250 milliliters the triangular flask, 100 milliliters of the methanol solutions of adding concentration 55%, in speed is vibration 30 minutes on the shaking table that changes of per minute 60, and extract contains aflatoxin.
5) toxin determination: get 10 milliliters of above-mentioned aflatoxin extracts,, get filtrate 20 microlitres through the filter paper filtering of sterilization, add 4 milliliters of ammonia mercury solutions, fully mixing is got 1 milliliter in cuvette, colorimetric on fluorospectrophotometer is calculated toxin B1 content with calibration curve method.Typical curve is gone up at fluorospectrophotometer (LS55) with U.S. Sigma company product aflatoxin B1 standard model and is set up typical curve such as Fig. 1.
6) evaluation of resistance standard: the toxin concentration of high anti-peanut varieties is 5.0 micrograms/below the gram, in anti-kind content of toxins be 5.1-15.0 microgram/gram, the susceptible variety content of toxins is 15.1-40.0 microgram/gram, high sense kind content of toxins is 40.1 micrograms/more than the gram.
The present invention can identify effectively that under comparatively simple experiment condition the aspergillus flavus of peanut varieties produces malicious resistance, especially be fit to breeding material is done screening in batches, simple to operate, aflatoxin content in can the peanut seed of quantitative test after artificial infection, the resistance cost of expert testimony reduces more than one times, and the resistance appraisal cost is cheap.
Description of drawings
Fig. 1 is that (model: LS55) go up the canonical plotting of setting up, the related coefficient between fluorescent value and the aflatoxin content is 0.9972 at fluorospectrophotometer with the aflatoxin B1 standard model in the present invention.
Embodiment
AF2202 is the inoculation bacterial strain with high yield poison aspergillus flavus strain.
Get healthy peanut seed and place sterile petri dish, continue to spray the surface of the seed 1 minute with 70% alcohol in Biohazard Safety Equipment, the fine rotation double dish makes the surface of the seed sterilization evenly, outwells alcohol and impurity, again with alcohol-pickled 5 minutes of same concentrations, and then with aqua sterilisa flushing 3 times.Be controlled in 30 minutes from the beginning surface sterilization to the time that flushing finishes, suitably regulate rehydration time according to the different cultivars seed size, the water cut after the assurance peanut seed rehydration is beneficial to infecting of Aspergillus flavus spore at 20-30%.The flushing aseptic scalper in back that finishes scratches peanut kind skin, adds 1 milliliter of Aspergillus flavus spore suspension, and the rotation double dish makes the spore can be evenly attached to the surface of the seed.
After the inoculation double dish is placed the plastic casing of adding a cover, add the aqua sterilisa of a small amount of (water layer thickness 2-3 millimeter) in the plastic casing, guarantee that the relative humidity of subenvironment is 80-90%.The constant incubator that plastic casing is put into 30 ℃ was cultivated 7 days.
Cultivate the alcohol of in Biohazard Safety Equipment, using concentration 70% after 7 days and spray the shelled peanut surface, put into 110 ℃ of baking oven bakings 1 hour afterwards, the shape of claying into power after the cooling in Biohazard Safety Equipment then changes in 250 milliliters the triangular flask, 100 milliliters of methanol solutions that add concentration 55% are that aflatoxin was extracted in vibration in 30 minutes on the shaking table that changes of per minute 60 in speed.
Under fluorospectrophotometer (LS55), set up typical curve (as shown in Figure 1) with aflatoxin B1 standard model (U.S. Sigma company product).Getting 10 milliliters of aflatoxin extracts is filtering on the filter paper of sterilization.Get filtrate 20 microlitres, add 4 milliliters of ammonia mercury solutions, fully mixing.Get 1 milliliter in cuvette, in the fluorospectrophotometer colorimetric.Calculate toxin B1 content with calibration curve method.
Just estimate peanut varieties produces poison to aspergillus flavus resistance grade with content of toxins.The toxin concentration of high anti-peanut varieties is 5.0 micrograms/below the gram, in anti-kind content of toxins be 5.1-15.0 microgram/gram, the susceptible variety content of toxins is 15.1-40.0 microgram/gram, it is 40.1 micrograms/more than the gram that height is felt the kind content of toxins.
As stated above, the content of toxins under peanut varieties " Taishan pearl " inoculation condition is 2.7-3.8 microgram/gram, and average out to 3.2 microgram/grams reach high anti-level; Peanut varieties " 15048 " content of toxins is 23.6-36.8 microgram/gram, and average out to 30.6 microgram/grams are susceptible variety.
Claims (2)
1. aspergillus flavus produces malicious resistance authentication method, it is characterized in that being realized by following steps: 1) peanut seed surface sterilization and rehydration; 2) peanut seed inoculation: inoculate healthy peanut with aspergillus flavus strain; 3) cultivate: cultivated 7 days in incubator the inoculation back; 4) extract aflatoxin: through baking, grinding, again with methanol extraction aflatoxin liquid; 5) toxin determination: add the ammonia mercury solution, on fluorospectrophotometer, measure fluorescent value, calculate aflatoxin B1 content and estimate the resistance grade with the typical curve between aflatoxin B1 and the fluorescent value.
2. aspergillus flavus according to claim 1 produces malicious resistance authentication method, it is characterized in that its concrete steps are as follows:
1) peanut seed surface sterilization and rehydration: get healthy peanut seed and place sterile petri dish, in Biohazard Safety Equipment, continue to spray the surface of the seed 1-1.5 minute with 70% alcohol, the fine rotation double dish makes the surface of the seed sterilization evenly, outwell alcohol and impurity, again with alcohol-pickled 5 minutes of same concentrations, and then with aqua sterilisa flushing 3 times, be controlled in 30 minutes from the beginning surface sterilization to the time that flushing finishes, making the water cut after the peanut seed rehydration is 20-30%;
2) peanut seed inoculation: will sterilize and rehydration after peanut seed draw with aseptic scalper and break in the seed coat, add 1 milliliter of Aspergillus flavus spore suspension of preparing in advance, the rotation double dish makes the spore can be evenly attached to the surface of the seed;
3) cultivate: after the inoculation double dish is placed the plastic casing of adding a cover, add aqua sterilisa in the plastic casing, making the interior relative humidity of box is 80-90%, plastic casing is put into 30 ℃ constant incubator and is cultivated 7 days;
4) extract aflatoxin: the alcohol with concentration 70% in Biohazard Safety Equipment sprays above-mentioned the surface of the seed through inoculated and cultured, put into 110 ℃ of baking oven bakings 1 hour then, the shape of claying into power after the cooling in Biohazard Safety Equipment, change in 250 milliliters the triangular flask, 100 milliliters of the methanol solutions of adding concentration 55%, in speed is vibration 30 minutes on the shaking table that changes of per minute 60, and extract contains aflatoxin;
5) toxin determination: get 10 milliliters of above-mentioned aflatoxin extracts, filter paper filtering through sterilization, get filtrate 20 microlitres, add 4 milliliters of ammonia mercury solutions, fully mixing is got 1 milliliter in cuvette, colorimetric on fluorospectrophotometer, calculate toxin B1 content with calibration curve method, typical curve is set up on fluorospectrophotometer with U.S. Sigma company product aflatoxin B1 standard model;
6) evaluation of resistance standard: the toxin concentration of high anti-peanut varieties is 5.0 micrograms/below the gram, in anti-kind content of toxins be 5.1-15.0 microgram/gram, the susceptible variety content of toxins is 15.1-40.0 microgram/gram, high sense kind content of toxins is 40.1 micrograms/more than the gram.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102331414A (en) * | 2011-08-29 | 2012-01-25 | 西南大学 | Aflatoxin B1 fluorescent sensitizer and applications thereof |
| CN102818740A (en) * | 2012-09-10 | 2012-12-12 | 广东省农业科学院作物研究所 | Identification method of resistance to aspergillus flavus infection of peanuts and application thereof |
| CN103114124A (en) * | 2013-02-06 | 2013-05-22 | 杨庆利 | Method for aflatoxin infection of peanuts |
| CN105230376A (en) * | 2015-11-12 | 2016-01-13 | 青岛友诚高新技术有限公司 | Method of field infected peanuts |
| CN106119337A (en) * | 2016-06-27 | 2016-11-16 | 山东省农业科学院生物技术研究中心 | A kind of Rapid identification peanut varieties method to Aspergillus flavus resistance |
| CN109001385A (en) * | 2018-06-15 | 2018-12-14 | 中国农业科学院油料作物研究所 | The indoor appraising method and its application of a kind of peanut pod to Aspergillus flavus infection resistance |
| RU2783882C2 (en) * | 2018-12-07 | 2022-11-21 | Оил Кропс Рисёч Инститьют, Чайниз Экедеми Оф Агрикалчерал Сайенсиз | Method for assessment of toxin-forming capability of aflatoxigenic strain |
-
2003
- 2003-12-30 CN CN 200310111682 patent/CN1267724C/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102331414A (en) * | 2011-08-29 | 2012-01-25 | 西南大学 | Aflatoxin B1 fluorescent sensitizer and applications thereof |
| CN102331414B (en) * | 2011-08-29 | 2014-06-11 | 西南大学 | Aflatoxin B1 fluorescent sensitizer and applications thereof |
| CN102818740A (en) * | 2012-09-10 | 2012-12-12 | 广东省农业科学院作物研究所 | Identification method of resistance to aspergillus flavus infection of peanuts and application thereof |
| CN102818740B (en) * | 2012-09-10 | 2014-06-25 | 广东省农业科学院作物研究所 | Identification method of resistance to aspergillus flavus infection of peanuts and application thereof |
| CN103114124A (en) * | 2013-02-06 | 2013-05-22 | 杨庆利 | Method for aflatoxin infection of peanuts |
| CN105230376A (en) * | 2015-11-12 | 2016-01-13 | 青岛友诚高新技术有限公司 | Method of field infected peanuts |
| CN106119337A (en) * | 2016-06-27 | 2016-11-16 | 山东省农业科学院生物技术研究中心 | A kind of Rapid identification peanut varieties method to Aspergillus flavus resistance |
| CN109001385A (en) * | 2018-06-15 | 2018-12-14 | 中国农业科学院油料作物研究所 | The indoor appraising method and its application of a kind of peanut pod to Aspergillus flavus infection resistance |
| RU2783882C2 (en) * | 2018-12-07 | 2022-11-21 | Оил Кропс Рисёч Инститьют, Чайниз Экедеми Оф Агрикалчерал Сайенсиз | Method for assessment of toxin-forming capability of aflatoxigenic strain |
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| Publication number | Publication date |
|---|---|
| CN1267724C (en) | 2006-08-02 |
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