CN101608211A - Indoor detection method for resistance of peanuts to infection of Aspergillus flavus - Google Patents
Indoor detection method for resistance of peanuts to infection of Aspergillus flavus Download PDFInfo
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- CN101608211A CN101608211A CNA2009101593465A CN200910159346A CN101608211A CN 101608211 A CN101608211 A CN 101608211A CN A2009101593465 A CNA2009101593465 A CN A2009101593465A CN 200910159346 A CN200910159346 A CN 200910159346A CN 101608211 A CN101608211 A CN 101608211A
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Abstract
A kind of indoor detection method of peanut aspergillus flavus infection resistance, it comprises the steps: the harvesting peanut seed drying to water content 8～10%, it is 0.05～1 * 10 that aspergillus flavus strain is made into concentration
8The spore suspension of individual/mL; To make seed moisture content return to 15～20% with the ordinary method sterilization and with rinsed with sterile water in dry peanut seed; Peanut seed adopts kind of horizontal girdling of skin and the even boring method creation wound in the vertical cotyledon of seed diameter maximum cross section, is used for Aspergillus flavus and infects; Filter paper put into to add described spore suspension behind the culture dish wetting, to immerse 3～5min in the spore suspension through the peanut seed after the above-mentioned processing, seed takes out the back and the surface is blotted on the filter paper that is placed on described culture dish with aseptic filter paper, cover the ware lid subsequently and place incubator to cultivate 5～6 days, culture condition is 28～30 ℃ of humidity 80～90%, humidity; According to infection Aspergillus flavus in peanut seed surface and the hole as a result antagonism estimate.
Description
Invention field
The present invention relates to agricultural technology field, relate in particular to a kind of indoor quick, efficient detection method of peanut aspergillus flavus infection resistance.
Background technology
Peanut is one of five big oil crops in the world, also is important oil crops of China and cash crop.Aflatoxin is that flavus is infected the secondary metabolite that produces after the peanut, can make the mankind and multiple animal bring out experimental liver cancer, is the strongest chemical carcinogen of having found at present.It is the principal element that influences China's peanut production, processing and trade that flavus is infected the aflatoxin contamination that peanut causes.95% severity of aflatoxin pollution of peanuts that is used for home consumption that even more serious is is far above the peanut of outlet, and domestic consumer's health is constituted a serious threat, and this all becomes the limiting factor of China's peanut industry long term growth.
Flavus belongs to Ascomycetes, Eurotiale, Aspergillus, and also the someone will it classify the Moniliales of deuteromycetes as.Mycelium is made of the branch mycelia of many complexity, mycelia tool tabula.By the conidiophore that aerial hyphae forms, ultimate swelling is the top capsule, produces many stigmas on the capsule of top, and stigma has the branch of individual layer and bilayer, and the stigma top the spherical conidium that generates string.A large amount of conidial generations make whole bacterium colony present yellow-green colour.
Under field conditions (factors), aflatoxin mainly contains aflatoxin B1, B2, G1 and G2, finds this toxoid meta-bolites in animal body later on again, as aflatoxin M 1, M2, GM1 and P1 etc.The toxicity of aflatoxin is one of poisonous substance of kind of mycotoxin toxic maximum surplus in the of known 100, and is wherein the highest with aflatoxin B1 output especially, toxicity is maximum, carinogenicity is the strongest.Experiment shows that the male white rat medium lethal dose is 7.2 milligrams/kilogram, and female is 7.9 milligrams/kilogram.The report that causes human poisoning because of the edible food that goes mouldy is all arranged both at home and abroad.Acute aflatoxicosis incident once took place once as West India in 1974.Behind the corn that the resident has eaten mouldy (Aspergillus flavus infection), there are 397 people that acute poisoning hepatitis takes place, dead 106 people.Also have dog in batch that ascites and jaundice take place, how dead in 2～3 weeks.The symptom of this toxin poisoning is mainly heating, vomits, is off one's feed, and occurs jaundice then, and ascites can appear in weight person.Experimentation on animals shows, takes in for a long time and all can bring out liver cancer after lower concentration aflatoxin or short-term are taken in high density.
Because Aspergillus flavus is infected food crop, the toxin of its generation can cause the person poultry poisoning and bring out liver cancer, can so how, just avoid its harm? at first to control conditions such as the required temperature of Aspergillus flavus growth and breeding, humidity and oxygen, avoid infecting of Aspergillus flavus.Here the most significant method is the water content of control grain, promptly in time dries behind the grain harvest, reduces its water content (be lower than 30% as the paddy water content, corn is lower than 12.5% etc.).The peanut that the most easily goes mouldy should also be noted that the complete of its shell.Next is Aspergillus flavus have been taken place infect, and should in time take appropriate measure to solve.As be peanut, then should in time choose mould grain, mould grain can not be eaten again; As be rice, because of toxin exists only in the top layer, should in water, wash by rubbing with the hands clean before eating; As be corn, because of toxin is stored in embryo portion, answer backing for several times to take off embryo; As be edible oil, should add appropriate bases, make the hydrogenation of six carbocyclic lactone of toxin open the water miscible tonka bean camphor sodium salt of formation, can discard.The damaging effect of aflatoxin has caused generally attention both domestic and external.World Health Organization's regulation in 1966, the highest the limiting the quantity of of aflatoxin is 30 microgram/kilograms in the food.The highest the limiting the quantity of of China's regulation is: (unit: microgram/kilogram) corn, peanut oil, peanut and goods thereof are 20; Rice and other edible oil are 10; Other grain, bean food are 5, but baby's milk powder substitute must not detect.
Studies show that Aspergillus flavus is infected peanut seed, its kind skin plays important provide protection.Peanut kind skin integrity is very favourable to improving resistance, and the kind skin composition and the flavus resistance of peanut are closely related.The peanut seed aspergillus flavus infection resistance at first requires to have intact pod shell and plants skin.Micro-and submicroscopic is observed and is shown, there is bigger difference in the structure of planting skin between anti-sense kind, it is the thicker cell walls of kind leatherware of resistant variety, combination closely between epidermic cell, the palisade cell layer is closeer and thick, and crack and hole are less etc., and the kind chrotoplast wall of susceptible variety is thinner relatively, iuntercellular is in conjunction with more loose, and crack and pore are bigger.Conventional flavus is infected method and has shortcomings such as time of infection is long, infection rate is low, cost height.The main substantially shortcoming of domestic existing peanut varieties aspergillus flavus infection resistance indoor appraising method is that length consuming time, efficient resistance low, that be unfavorable for peanut seed resource in enormous quantities is identified.
Summary of the invention
The purpose of (goal of the invention) present method is to identify by the different peanut varieties resource being carried out aspergillus flavus infection resistance, solve problems such as current Aspergillus flavus is infected cost height in the testing process, time of infection is long, efficient is low, realize the evaluation of rapid detection peanut aspergillus flavus infection resistance, for Screening and Identification aspergillus flavus resisting peanut seed resource lays the foundation.
(technical scheme) the invention provides a kind of indoor quick, efficient detection method of peanut aspergillus flavus infection resistance, and it comprises the steps:
The harvesting peanut seed drying is to water content 8～10%, and it is 0.05～1 * 10 that aspergillus flavus strain is made into concentration
8The spore suspension of individual/mL;
Make seed moisture content return to 15～20% with the ordinary method sterilization and with rinsed with sterile water in the drying peanut seed; Peanut seed adopts kind of horizontal girdling of skin and the even boring method creation wound in the vertical cotyledon of seed diameter maximum cross section, is used for Aspergillus flavus and infects;
Filter paper put into to add described spore suspension behind the culture dish wetting, to immerse 3～5min in the spore suspension through the peanut seed after the above-mentioned processing, seed takes out the back and the surface is blotted on the filter paper that is placed on described culture dish with aseptic filter paper, cover the ware lid subsequently and place incubator to cultivate 5～6 days, culture condition is 28～30 ℃ of humidity 80～90%, humidity;
According to infection Aspergillus flavus in peanut seed surface and the hole as a result antagonism estimate.
Detection method of the present invention requires peanut seed to have certain freshness, the peanut moisture content of kernels is 8～10% and the timely fresh seeds of natural air drying or normal temperature machine dry in results back when requiring Aspergillus flavus to infect, in addition, it is 28-30 ℃ that Aspergillus flavus is infected the needed envrionment temperature of peanut, and ambient moisture is the ambient moisture of 80-90%.
With existing peanut aspergillus flavus-resistance mould infect authentication method different be, the present invention adopts horizontal girdling of kind of skin and vertical cotyledon planar radial bore mode to carry out Aspergillus flavus to infect, not only accelerated flavus and infected process, the more important thing is that can reach peanut kind skin and seed benevolence inside carries out resistance simultaneously and identify purpose.
According to a preferred embodiment of the present invention, the peanut seed of described results is dried to water content 9～10%.
According to a preferred embodiment of the present invention, after the described peanut seed resource results, utilize flowing of the irradiation of 7～9 days sun and air,, dryly afterwards preserve under the room temperature if meet rainy weather then utilize drying machinery in 35～40 ℃ of scopes, to quicken moisture and distribute with moisture evaporation in the pod.
According to a preferred embodiment of the present invention, described aspergillus flavus strain in czapek's solution in electro-heating standing-temperature cultivator 28-30 ℃ cultivate 7 days after, being made into concentration at Bechtop with sterilized water is 0.05-1 * 10
8The spore suspension of/mL.
According to a preferred embodiment of the present invention, described czapek's solution comprises SODIUMNITRATE 3g, dipotassium hydrogen phosphate 1g, sal epsom (MgSO
47H
2O) 0.5g, Repone K 0.5g, ferrous sulfate 0.01g, sucrose 30g, agar 20g, deionized water (ddH
2O) be settled to 1000mL.
According to a preferred embodiment of the present invention, with described drying peanut seed with 0.8% HgCl
2Solution sterilization 6～8min, and with rinsed with sterile water 3～4 times.
According to a preferred embodiment of the present invention, described culture condition is 29～30 ℃ of humidity 85～90%, humidity.
(invention effect) advantage of the present invention is: reduced Aspergillus flavus and infected restriction by peanut kind skin, shorten time of infection 2～3 days, reduce Aspergillus flavus spore suspension usage quantity 30%～40%, improve detection efficiency, reduced the Pollution risk of Aspergillus flavus, especially at the aspergillus flavus infection resistance Rapid identification of peanut seed resource in enormous quantities to the testing staff.
Embodiment
After examination peanut seed resource results, utilize the irradiation of 7～9 days sun and flowing of air with moisture evaporation in the pod, if meet rainy weather then utilize drying machinery in 35～40 ℃ of scopes, to quicken water-dispersion and send to water content 8～10%, preserve under the room temperature;
It is 0.05～1 * 10 that aspergillus flavus strain (Aspergillus Flavus) is made into concentration with sterilized water
8The spore suspension of individual/mL;
With the drying peanut seed with 0.8% HgCl
2Solution sterilization 6～8min, and with rinsed with sterile water 3～4 times, seed moisture content returns to 15～20%.Peanut seed adopts kind of horizontal girdling of skin and the even boring method creation wound in the vertical cotyledon of seed diameter maximum cross section, is used for Aspergillus flavus and infects;
121 ℃ of autoclaving culture dish and filter paper, filter paper put into to add spore suspension behind the culture dish wetting, to handle back peanut seed and immerse 3～5min in the spore suspension, take out seed and seed is shown and blot with aseptic filter paper, place on the filter paper of culture dish, cover ware lid and place incubator to keep 80～90% humidity, 28～30 ℃ to cultivate 5～6 days;
According to infection Aspergillus flavus in peanut seed surface and the hole as a result antagonism estimate.
Preferred embodiment
Experiment material:
Peanut material: high aspergillus flavus infection resistance peanut seed J11 and high sense flavus kind golden flower 1012;
Strains tested and cultivation: aspergillus flavus strain is preserved for this laboratory.Bacterial strain is at czapek's solution (SODIUMNITRATE 3g, dipotassium hydrogen phosphate 1g, sal epsom (MgSO
47H
2O) 0.5g, Repone K 0.5g, ferrous sulfate 0.01g, sucrose 30g, agar 20g, deionized water (ddH
2O) be settled to 1000mL.) in electro-heating standing-temperature cultivator 28-30 ℃ cultivate 7 days after, being made into concentration at Bechtop with sterilized water is 0.05-1 * 10
8The spore suspension of/mL.
Experimental technique:
To according to infection Aspergillus flavus in aforesaid method gained peanut seed surface and the hole as a result antagonism estimate.
The different authentication method aspergillus flavus resisting of table 1 identification result is (2006) relatively
By finding out in the table 1,2, improving technology in the time spent, identify and all be better than conventional authenticate technology aspect used spore suspension usage quantity of each kind and the accuracy rate.
The different authentication method aspergillus flavus resisting of table 2 identification result is (2007) relatively
Claims (8)
1. the indoor detection method of a peanut aspergillus flavus infection resistance, it comprises the steps:
The harvesting peanut seed drying is to water content 8～10%, and it is 0.05～1 * 10 that aspergillus flavus strain is made into concentration
8The spore suspension of individual/mL;
Make seed moisture content return to 15～20% with the ordinary method sterilization and with rinsed with sterile water in the drying peanut seed, on peanut seed, create wound;
Filter paper put into to add described spore suspension behind the culture dish wetting, to immerse 3～5min in the spore suspension through the peanut seed after the above-mentioned processing, seed takes out the back and the surface is blotted on the filter paper that is placed on described culture dish with aseptic filter paper, cover the ware lid subsequently and place incubator to cultivate 5～6 days, culture condition is 28～30 ℃ of humidity 80～90%, temperature;
According to infection Aspergillus flavus in peanut seed surface and the hole as a result antagonism estimate.
2. the method for claim 1 is characterized in that, the peanut seed of described results is dried to water content 9～10%.
3. method as claimed in claim 1 or 2, it is characterized in that, after the described peanut seed resource results, utilize the irradiation of 7～9 days sun and flowing of air with moisture evaporation in the pod, if meet rainy weather then utilize drying machinery in 35～40 ℃ of scopes, to quicken moisture and distribute, dryly afterwards preserve under the room temperature.
4. the method for claim 1 is characterized in that, the method for described creation wound comprise adopt kind of the horizontal girdling of skin with at the even boring method in seed diameter maximum vertical cotyledon cross section.
5. method as claimed in claim 1 or 2 is characterized in that, described aspergillus flavus strain in czapek's solution in electro-heating standing-temperature cultivator 28-30 ℃ cultivate 7 days after, being made into concentration at Bechtop with sterilized water is 0.05-1 * 10
8The spore suspension of/mL.
6. method as claimed in claim 3 is characterized in that described czapek's solution comprises SODIUMNITRATE 3g, dipotassium hydrogen phosphate 1g, sal epsom (MgSO
47H
2O) 0.5g, Repone K 0.5g, ferrous sulfate 0.01g, sucrose 30g, agar 20g, deionized water (ddH
2O) be settled to 1000mL.
7. the method for claim 1 is characterized in that, with described drying peanut seed with 0.8% HgCl
2Solution sterilization 6～8min, and with rinsed with sterile water 3～4 times.
8. the method for claim 1 is characterized in that, described culture condition is 29～30 ℃ of humidity 85～90%, temperature.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2009101593465A CN101608211A (en) | 2009-07-10 | 2009-07-10 | Indoor detection method for resistance of peanuts to infection of Aspergillus flavus |
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| CNA2009101593465A CN101608211A (en) | 2009-07-10 | 2009-07-10 | Indoor detection method for resistance of peanuts to infection of Aspergillus flavus |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102603391A (en) * | 2012-03-30 | 2012-07-25 | 广西宇鑫生物科技有限公司 | Compound amino acid organic fertilizer and production method of compound amino acid organic fertilizer |
| 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 |
| CN105340603A (en) * | 2015-11-09 | 2016-02-24 | 山东省花生研究所 | Aspergillus flavus infection method for peanut seeds in field |
| 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 |
| CN108796033A (en) * | 2018-07-03 | 2018-11-13 | 苏州良辰生物医药科技有限公司 | A kind of contamination method of biological tissue |
| 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 |
-
2009
- 2009-07-10 CN CNA2009101593465A patent/CN101608211A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102603391A (en) * | 2012-03-30 | 2012-07-25 | 广西宇鑫生物科技有限公司 | Compound amino acid organic fertilizer and production method of compound amino acid organic fertilizer |
| CN103114124A (en) * | 2013-02-06 | 2013-05-22 | 杨庆利 | Method for aflatoxin infection of peanuts |
| CN105340603A (en) * | 2015-11-09 | 2016-02-24 | 山东省花生研究所 | Aspergillus flavus infection method for peanut seeds in field |
| 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 |
| CN108796033A (en) * | 2018-07-03 | 2018-11-13 | 苏州良辰生物医药科技有限公司 | A kind of contamination method of biological tissue |
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Application publication date: 20091223 |