CN106596492A - Method for identifying infection resistance of peanuts to aspergillus flavus and application thereof - Google Patents
Method for identifying infection resistance of peanuts to aspergillus flavus and application thereof Download PDFInfo
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- CN106596492A CN106596492A CN201611234338.9A CN201611234338A CN106596492A CN 106596492 A CN106596492 A CN 106596492A CN 201611234338 A CN201611234338 A CN 201611234338A CN 106596492 A CN106596492 A CN 106596492A
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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Abstract
The invention discloses a method for identifying infection resistance of peanuts to aspergillus flavus. The method comprises the following steps: inoculating prepared aspergillus flavus spore suspension into healthy and aseptic peanut seeds; meanwhile, using a Czapek's medium as a reference product, and designing a special culture device; calculating the infection index of the aspergillus flavus according to the area of a mold ring to evaluate the infection resistance of a seed sample to the aspergillus flavus. The method has the advantages of convenience in operation, short time, low cost and high reliability; in an operation process, no complicated extraction process is needed, so that direct and long-time contact of a human body with the aspergillus flavus is reduced, and the physical health of an operation experimenter is benefited; the method is suitable for being popularized widely, and particularly applied to rapid and large-scale screening of peanut varieties having aspergillus flavus infection resistance.
Description
Technical field
The invention belongs to agricultural technology field, and in particular to one cultivate peanut the authentication method to Aspergillus flavus infection resistance and its
Using.
Background technology
Aflatoxin mainly has the secondary metabolite that aspergillus flavus and aspergillus parasiticus are produced, and is broadly divided into B1、B2、M1、
M2、G1And G2Deng 12, in being present in soil, animals and plants and various nuts, it is particularly easy to pollute peanut, corn, rice, soybean, little
The grain oil products such as wheat.Aflatoxin is that mycotoxicosis are especially big, mould to the very prominent class of human health risk
Verticillium toxin.Aspergillus flavus, as the prime producer of aflatoxin, is control product from the dip-dye of product Sources controlling aspergillus flavus
The effective control means of middle aflatoxin content.
Peanut eats as the important grain and oil crop of China, or directly, or for producing edible oil, Rapid identification flower
The raw resistance that aspergillus flavus is contaminated, it is significant safely for effective control peanut products.Peanut is identified in prior art
It is, artificial infection aspergillus flavus after culture appropriate time, to use thin-layered chromatography to the method that the resistance that aspergillus flavus is contaminated generally is adopted
Or the aflatoxin content in high effective liquid chromatography for measuring sample, the method testing cost is high and the consuming time is long；Again
Person is exactly artificial infection aspergillus flavus, after culture appropriate time, observes the quantity that peanut is contaminated by aspergillus flavus, the method operating error
Larger, above two method is unfavorable for Rapid identification.
The content of the invention
Authentication method and its application cultivated peanut to Aspergillus flavus infection resistance that the present invention is provided, solves prior art
In, with the aflatoxin content in thin-layered chromatography or high effective liquid chromatography for measuring sample, the method testing cost is high,
And it is long to expend the time；Solve the problems, such as directly to observe peanut larger by the method operating error of aspergillus flavus dip-dye quantity simultaneously.
The invention provides an authentication method cultivated peanut to Aspergillus flavus infection resistance, comprises the following steps：
Step 1, after aspergillus flavus strain is activated, elutes aspergillus spore, is prepared into aspergillus spore suspension, standby；
Step 2, selects the peanut seed of health, peels off, removes the peel, and is then comminuted into powder, 40 mesh sieves is crossed, by the flower for obtaining
Fecula is soaked in 3-5min in the ethanol that volume fraction is 75%, and then aseptic water washing is clean, finally drains away the water, and is planted
Subsample, it is standby；
Step 3, by following structure fabrication culture apparatus：The culture apparatus includes culture dish, around the culture dish
Inwall is provided with dismountable ring support, and the ring support is positioned over the culture dish inner bottom part, the ring support
On be overlapped with horizontal transparent waffle slab, the transparent waffle slab is the network structure with some square lattices, and each
The length of side of square lattice is equal；
Step 4, weighs the seed sample of certain mass under aseptic condition, in being laid in culture dish, the seed in culture dish
Sample central authorities be added dropwise aspergillus spore suspension, seed sample be located at transparent waffle slab lower section, and with transparent waffle slab it
Between leave certain distance；Then by culture apparatus in 25-30 DEG C of light culture 3-7 days, until sample sets mould circle is grown, by system
The quantity of square lattice relative with sample sets mould circle on transparent waffle slab is counted, the area of sample sets mould circle is calculated
A1, and recorded；
Step 5, to be equal to the Czapek's medium of seed sample quality described in step 4 as control, operation and step 4
It is identical, differ only in：With Czapek's medium culture aspergillus flavus, until control group mould circle is grown, then by counting transparent
The quantity of the square lattice relative with control group mould circle on waffle slab, calculates the area C1 of control group mould circle, and gives
To record；
Step 6, as a result judges, by infection by Aspergillus flavus index F1 dip-dye resistance of the seed sample to aspergillus flavus is evaluated：
Wherein, infection by Aspergillus flavus index is calculated as follows：F1=A1/C1.
Preferably, it is 5% using volume fraction in step 1 in authentication method of the above-mentioned peanut to Aspergillus flavus infection resistance
Acetum wash-out aspergillus spore.
Preferably, in authentication method of the above-mentioned peanut to Aspergillus flavus infection resistance, in step 1, aspergillus spore concentration is
105Individual/ml.
Preferably, in authentication method of the above-mentioned peanut to Aspergillus flavus infection resistance, in step 3, each square lattice
The length of side is 2-4mm.
Preferably, in authentication method of the above-mentioned peanut to Aspergillus flavus infection resistance, in step 3, per 25 square lattices
One block plaid of composition, the common edge of two neighboring block plaid is disposed as redness.
Preferably, in authentication method of the above-mentioned peanut to Aspergillus flavus infection resistance, also including aflatoxin detecting step,
Specially：
Step a, the culture apparatus that step 4 grows sample sets mould circle is placed under the ultraviolet light of 365nm, observes sample sets
The fluorescent ring size of the aflatoxin that aspergillus flavus produces, by counting square lattice relative with fluorescent ring on transparent waffle slab
The quantity of son, calculates the area A2 of fluorescent ring, and is recorded；
Step b, the culture apparatus that step 5 grows control group mould circle is placed under the ultraviolet light of 365nm, observes control group
The fluorescent ring size of the aflatoxin that aspergillus flavus produces, by counting square lattice relative with fluorescent ring on transparent waffle slab
The quantity of son, calculates the area C2 of fluorescent ring, and is recorded；
Step c, as a result judges, produces dip-dye of the index F2 evaluation seed samples to aspergillus flavus by aflatoxin and resists
Property：
Wherein, aflatoxin produces index and calculates as follows：F2=A2/C2.
Present invention also offers above-mentioned peanut has Huang to the authentication method of Aspergillus flavus infection resistance in quick, batch screening
Aspergillus contaminates the application in the peanut varieties of resistance.
Compared with prior art, peanut of the invention has following beneficial effect to the authentication method of Aspergillus flavus infection resistance
Really：
1st, by the size of mould circle, and by peanut seed sample and it is best suitable for the Czapek's medium that aspergillus flavus grows
On the aspergillus flavus bacterium colony size that grows compare, calculate infection by Aspergillus flavus index, infection by Aspergillus flavus index is bigger, then illustrate the peanut
Kind is lower to the resistance that aspergillus flavus strain grows.
2nd, by the size of fluorescent ring, and by peanut seed sample and it is best suitable for the Czapek's medium that aspergillus flavus grows
On the aspergillus flavus bacterium colony size that grows compare, calculate aflatoxin and produce index, it is less that aflatoxin produces index, then say
The bright peanut varieties grow to aspergillus flavus strain and can largely suppress aspergillus flavus metabolism to produce aflatoxin.
3rd, by arranging special culture apparatus, it is easy to quick result of calculation, increases the convenience of operation；By will be yellow bent
Mould infestation index, aflatoxin produce index and combine, the common resistance for evaluating peanut seed, as a result relatively reliable, accuracy
It is high, it is to avoid other fungus growth pollution results.
4th, by eluting spore using acetic acid so that aspergillus spore fast-growth and can be produced in acid condition
Substantial amounts of aflatoxin, reduces the incubation time of aspergillus flavus, and increases the reliability of experimental result.
5th, the method for the present invention is simple to operate, with low cost, go out result rapidly and complicated carrying is not needed in operating process
Take process, reduce direct, the prolonged contact of human body and aspergillus flavus, be conducive to that operator's is healthy, be suitable for big
Scope is promoted.
Description of the drawings
Fig. 1 is the structural representation of culture apparatus of the present invention；
Fig. 2 is the position relationship citing view in culture apparatus of the present invention between transparent waffle slab and mould circle.
Description of reference numerals：
1st, culture dish, 2, ring support, 3, transparent waffle slab, 4, mould circle.
Specific embodiment
With reference to specific embodiment, the present invention is described in detail, but should not be construed as the restriction of the present invention.It is following
The test method of unreceipted actual conditions in embodiment, generally operates according to normal condition, due to being not related to inventive point, thus it is not right
Its step is described in detail.
When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range
Any one numerical value can select between point and two end points.Unless otherwise defined, the present invention used in all technologies and
Scientific terminology is identical with the meaning that those skilled in the art of the present technique are generally understood that.Except the concrete grammar used in embodiment, equipment,
Outside material, according to those skilled in the art to the grasp of prior art and the record of the present invention, can also use and this
Any method of the similar or equivalent prior art of method, equipment described in inventive embodiments, material, equipment and material come real
The existing present invention.
Strain source：
Aspergillus flavus strain adopts existing commercially available aspergillus flavus strain, with aspergillus flavus strain in following examples
As3.2890 and aspergillus flavus As3.4408, is purchased from Institute of Microorganism, Academia Sinica (CGMCC).
Authentication method and its application cultivated peanut to Aspergillus flavus infection resistance that the present invention is provided, including following enforcement
Example：
Embodiment 1
One authentication method cultivated peanut to Aspergillus flavus infection resistance, specifically includes following steps：
Step 1, aspergillus flavus As3.2890 is yellow bent using the acetum wash-out that volume fraction is 5% Jing after slant activation
Mould spore, is prepared into aspergillus spore suspension, standby, and preferred aspergillus spore concentration is 105Individual/ml；
Step 2, selects the peanut seed of health, peels off, removes the peel, and is then comminuted into powder, 40 mesh sieves is crossed, by the flower for obtaining
Fecula is soaked in 3min in the ethanol that volume fraction is 75%, and for disinfection, then aseptic water washing is clean, finally drains
Moisture, obtains seed sample, standby；
Step 3, by following structure fabrication culture apparatus：The structure of the culture apparatus as shown in Figure 1-2, including culture dish
1, dismountable ring support 2 is provided with around the inwall of the culture dish 1, and the ring support 2 is positioned over the culture
The inner bottom part of ware 1, is overlapped with horizontal transparent waffle slab 3 on the ring support 2, the transparent waffle slab 3 be with it is some just
The network structure of square grid, and the length of side of each square lattice is equal, is 2mm, constitutes per 25 square lattices
One block plaid, the common edge of two neighboring block plaid is disposed as redness, is easy to count grid quantity.
Step 4, weighs the seed sample of 15g under aseptic condition, in being laid in culture dish 1, the seed sample in culture dish 1
Central authorities be added dropwise 0.2ml aspergillus spore suspension, seed sample be located at transparent waffle slab 3 lower section, and with transparent grid
The distance of 3-6mm is left between plate 3；Then by culture dish in 25-30 DEG C of light culture 3-7 days, until mould circle 4 is grown, referred to as
Sample sets mould circle, now, sample sets mould circle is relative about 3 with transparent waffle slab, by count on transparent waffle slab 3 with sample
The quantity of the relative square lattice of product group mould circle, calculates the area A1 of sample sets mould circle, and is recorded；
In addition, culture apparatus is placed under the ultraviolet light of 365nm, sample sets mould circle (i.e. sample sets aspergillus flavus) is observed
The fluorescent ring size of the aflatoxin that aspergillus flavus produces, by counting square relative with fluorescent ring on transparent waffle slab 3
The quantity of grid, calculates the area A2 of fluorescent ring, and is recorded；
Additionally, record cultivates quality W2 of seed sample after quality W1 of front seed sample and culture；
It should be noted that the edge of the mould circle can not contact culture dish, so as to ensure the reliability of measurement result.
Step 5, using the Czapek's medium of 15g as control, operation is identical with step 4, differs only in：Cultivated with Cha Shi
Base culture aspergillus flavus, until control group mould circle is grown, then by counting relative with control group mould circle on transparent waffle slab 3
Square lattice quantity, calculate the area C1 of control group mould circle, calculate the area C2 of fluorescent ring, and remembered
Record, specifically includes：
Seed sample Czapek's medium described in 15g is laid in culture dish 1, the central authorities of seed sample in culture dish 1
The aspergillus spore suspension of 0.2ml is added dropwise, seed sample is located at the lower section of transparent waffle slab 3, and between transparent waffle slab 3
Leave the distance of 3-6mm；Then by culture dish in 28 DEG C of light cultures 3-7 days, until grow mould circle 4, referred to as control group mould
Circle, now, control group mould circle is relative about 3 with transparent waffle slab, by the transparent waffle slab 3 of statistics with control group mould circle
The quantity of relative square lattice, calculates the area C1 of control group mould circle, and is recorded；In addition, by culture apparatus
Under being placed in the ultraviolet light of 365nm, the fluorescence of the aflatoxin of the aspergillus flavus generation of mould circle (i.e. control group aspergillus flavus) is observed
Circle size, by the quantity of square lattice relative with fluorescent ring on the transparent waffle slab 3 of statistics, calculates the area of fluorescent ring
C2, and recorded.
Step 6, as a result judges, by infection by Aspergillus flavus index F1, aflatoxin index F2 and aspergillus flavus quality are produced
Index F3, evaluates dip-dye resistance of the seed sample to aspergillus flavus：
Wherein, infection by Aspergillus flavus index is calculated as follows：F1=A1/C1；
Aflatoxin produces index and calculates as follows：F2=A2/C2；
Aspergillus flavus performance figure is calculated as follows：F3=(W2-W1)/W1.
Contaminate evaluation of resistance：F1 illustrates that the height of specimen aspergillus flavus resisting is contaminated between the sensitive range of 0.001-0.600
Growth, illustrates that the low aspergillus flavus resisting of the sample contaminates growth between the sensitive range of 0.601-1.000；
F2 illustrates the generation of the height of specimen aspergillus flavus resisting toxin between the sensitive range of 0.001-0.600,
The generation of the low aspergillus flavus resisting toxin of the sample is illustrated between the sensitive range of 0.601-1.000；
F3 illustrates that the height of specimen aspergillus flavus resisting is contaminated, in 0.501- between the sensitive range of 0.001-0.500
Illustrate that the low aspergillus flavus resisting of the sample is contaminated between 1.000 sensitive range；
F1, F2 are between 0.01-0.600, F3 between 0.001-0.500, illustrate the peanut varieties to aspergillus flavus
Resistance probability is contaminated 100%；There is any two of which between the sensitive range that respective height aspergillus flavus resisting is contaminated, explanation
The peanut varieties are to the dip-dye resistance probability of aspergillus flavus more than 90%；Have wherein any one in respective height aspergillus flavus resisting
Between the sensitive range of dip-dye, illustrate the peanut varieties to the dip-dye resistance probability of aspergillus flavus more than 80%.
It should be noted that all mould circles, fluorescent ring in statistic procedure 4 and step 5 (including sample sets and control group)
Size when, be the cumulative of several square lattice areas, wherein, the edge position of mould circle and fluorescent ring accounts for
According to area more than its place square lattice area half when, then by complete square lattice areal calculation；If little
When the half of its place square lattice area, then calculate for 0 by area.
It should be noted that the culture dish for selecting is the circular culture dish of a diameter of 10cm.
It should be noted that step 4, step 5 should be identical with the incubation time in step 6.
In the method for embodiment 1,5 samples of same peanut varieties are determined, calculate its infection by Aspergillus flavus index F1, Huang Qu
Mould toxin produces index F2 and aspergillus flavus performance figure F3, and as a result as shown in table 1, F1, F2 or F3 of different sample determinations is equal
It is closer to, the invention for illustrating the present invention has good stability.
The aspergillus flavus of the different peanut samples of table 1 contaminates Resistance Identification result
| Sample number into spectrum | F1 | F2 | F3 | Contaminate resistance probability |
| 1 | 0.5578 | 0.5331 | 0.1944 | It is high |
| 2 | 0.5545 | 0.5314 | 0.2023 | It is high |
| 3 | 0.5425 | 0.5310 | 0.2022 | It is high |
| 4 | 0.5467 | 0.5312 | 0.1890 | It is high |
| 5 | 0.5468 | 0.5225 | 0.1910 | It is high |
Embodiment 2
One authentication method cultivated peanut to Aspergillus flavus infection resistance, specifically includes following steps：
Step 1, aspergillus flavus As3.4408 is yellow bent using the acetum wash-out that volume fraction is 5% Jing after slant activation
Mould spore, is prepared into aspergillus spore suspension, standby, and preferred aspergillus spore concentration is 105Individual/ml；
Step 2, selects the peanut seed of health, peels off, removes the peel, and is then comminuted into powder, 40 mesh sieves is crossed, by the flower for obtaining
Fecula is soaked in 5min in the ethanol that volume fraction is 75%, and for disinfection, then aseptic water washing is clean, finally drains
Moisture, obtains seed sample, standby；
Step 3, by following structure fabrication culture apparatus：The structure of the culture apparatus as shown in Figure 1-2, including culture dish
1, dismountable ring support 2 is provided with around the inwall of the culture dish 1, and the ring support 2 is positioned over the culture
The inner bottom part of ware 1, is overlapped with horizontal transparent waffle slab 3 on the ring support 2, the transparent waffle slab 3 be with it is some just
The network structure of square grid, and the length of side of each square lattice is equal, is 2mm, constitutes per 25 square lattices
One block plaid, the common edge of two neighboring block plaid is disposed as redness, is easy to count grid quantity.
Step 4, weighs the seed sample of 10g under aseptic condition, in being laid in culture dish 1, the seed sample in culture dish 1
Central authorities be added dropwise 0.2ml aspergillus spore suspension, seed sample be located at transparent waffle slab 3 lower section, and with transparent grid
The distance of certain 3-6mm is left between plate 3；Then by culture dish in 25-30 DEG C of light culture 3-7 days, until mould circle 4 is grown,
Referred to as sample sets mould circle, now, sample sets mould circle is relative about 3 with transparent waffle slab, by counting on transparent waffle slab 3
The quantity of the square lattice relative with sample sets mould circle, calculates the area A1 of sample sets mould circle, and is recorded；
In addition, culture dish is placed under the ultraviolet light of 365nm, the aspergillus flavus of sample sets mould circle (i.e. sample sets aspergillus flavus)
The fluorescent ring size of the aflatoxin of generation, by square lattice relative with fluorescent ring on the transparent waffle slab 3 of statistics
Quantity, calculates the area A2 of fluorescent ring, and is recorded；
It should be noted that the edge of the spore circle can not contact culture dish, so as to ensure the reliability of measurement result.
Step 5, using the Czapek's medium of seed sample quality described in 10g as control, operates, difference identical with step 4
It is only that：With Czapek's medium culture aspergillus flavus, until control group mould circle is grown, then by counting on transparent waffle slab 3
The quantity of the square lattice relative with control group mould circle, calculates the area C1 of control group mould circle, calculates fluorescent ring
Area C2, and recorded, specifically include：
Seed sample Czapek's medium described in 10g is laid in culture dish 1, the central authorities of seed sample in culture dish 1
The aspergillus spore suspension of 0.2ml is added dropwise, seed sample is located at the lower section of transparent waffle slab 3, and between transparent waffle slab 3
Leave the distance of 3-6mm；Then by culture dish in 25-30 DEG C of light culture 3-7 days, until growing mould circle 4, referred to as control group is mould
Bacterium circle, now, control group mould circle is relative about 3 with transparent waffle slab, by count on transparent waffle slab 3 with control group mould
The quantity of the relative square lattice of circle, calculates the area C1 of control group mould circle, and is recorded；In addition, by culture dish
Under being placed in the ultraviolet light of 365nm, the aflatoxin that the aspergillus flavus of control group mould circle (i.e. control group aspergillus flavus) produces is observed
Fluorescent ring size, by the quantity for counting square lattice relative with fluorescent ring on transparent waffle slab 3, calculate fluorescent ring
Area C2, and recorded.
Step 6, as a result judges, by infection by Aspergillus flavus index F1 and aflatoxin index F2 is produced, and evaluates kind of an increment
Dip-dye resistance of the product to aspergillus flavus：
Wherein, infection by Aspergillus flavus index is calculated as follows：F1=A1/C1；
Aflatoxin produces index and calculates as follows：F2=A2/C2.
Contaminate evaluation of resistance：F1 illustrates that the height of specimen aspergillus flavus resisting contaminates growth between 0.01-0.600,
Illustrate that the low aspergillus flavus resisting of the sample contaminates growth between 0.601-1.000；
F2 illustrates the generation of the height of specimen aspergillus flavus resisting toxin, between 0.601-1.000 between 0.01-0.600
Illustrate the generation of the low aspergillus flavus resisting toxin of the sample.
It should be noted that mould circle, the face of fluorescent ring (including sample sets and control group) in statistic procedure 4 and step 5
The calculation of product size is with embodiment 1.
It should be noted that the culture dish for selecting is the circular culture dish of a diameter of 10cm.
It should be noted that step 4 should be identical with the incubation time in step 5.
In the method for embodiment 2,5 samples of same peanut varieties are determined, calculate its infection by Aspergillus flavus index F1 and Huang
Aspertoxin produces index F2, and as a result as shown in table 2, the F1 or F2 of different sample determinations are closer to, and illustrates the present invention's
Invention has good stability.
The aspergillus flavus of the different peanut samples of table 2 contaminates Resistance Identification result
Embodiment 3
One authentication method cultivated peanut to Aspergillus flavus infection resistance, specifically includes following steps：
Step 1, aspergillus flavus As3.4408 is yellow bent using the acetum wash-out that volume fraction is 5% Jing after slant activation
Mould spore, is prepared into aspergillus spore suspension, standby, and preferred aspergillus spore concentration is 105Individual/ml；
Step 2, selects the peanut seed of health, peels off, removes the peel, and is then comminuted into powder, 40 mesh sieves is crossed, by the flower for obtaining
Fecula is soaked in 5min in the ethanol that volume fraction is 75%, and for disinfection, then aseptic water washing is clean, finally drains
Moisture, obtains seed sample, standby；
Step 3, by following structure fabrication culture apparatus：The structure of the culture apparatus as shown in Figure 1-2, including culture dish
1, dismountable ring support 2 is provided with around the inwall of the culture dish 1, and the ring support 2 is positioned over the culture
The inner bottom part of ware 1, is overlapped with horizontal transparent waffle slab 3 on the ring support 2, the transparent waffle slab 3 be with it is some just
The network structure of square grid, and the length of side of each square lattice is equal, is 2mm, is easy to count grid quantity.
Step 4, weighs the seed sample of 10g under aseptic condition, in being laid in culture dish 1, the seed sample in culture dish 1
Central authorities be added dropwise 0.2ml aspergillus spore suspension, seed sample be located at transparent waffle slab 3 lower section, and with transparent grid
The distance of certain 3-6mm is left between plate 3；Then by culture dish in 25-30 DEG C of light culture 3-7 days, until mould circle 4 is grown,
Referred to as sample sets mould circle, now, sample sets mould circle is relative about 3 with transparent waffle slab, by counting on transparent waffle slab 3
The quantity of the square lattice relative with sample sets mould circle, calculates the area A1 of sample sets mould circle, and is recorded；
It should be noted that the edge of the spore circle can not contact culture dish, so as to ensure the reliability of measurement result.
Step 5, using the Czapek's medium of seed sample quality described in 10g as control, operates, difference identical with step 4
It is only that：With Czapek's medium culture aspergillus flavus, until control group mould circle is grown, then by counting on transparent waffle slab 3
The quantity of the square lattice relative with control group mould circle, calculates the area C1 of control group mould circle, and is recorded, tool
Body includes：
Seed sample Czapek's medium described in 10g is laid in culture dish 1, the central authorities of seed sample in culture dish 1
The aspergillus spore suspension of 0.2ml is added dropwise, seed sample is located at the lower section of transparent waffle slab 3, and between transparent waffle slab 3
Leave the distance of 3-6mm；Then by culture dish in 25-30 DEG C of light culture 3-7 days, until growing mould circle 4, referred to as control group is mould
Bacterium circle, now, control group mould circle is relative about 3 with transparent waffle slab, by count on transparent waffle slab 3 with control group mould
The quantity of the relative square lattice of circle, calculates the area C1 of control group mould circle, and is recorded.
Step 6, as a result judges, by infection by Aspergillus flavus index F1, evaluates dip-dye resistance of the seed sample to aspergillus flavus：
Wherein, infection by Aspergillus flavus index is calculated as follows：F1=A1/C1.
Contaminate evaluation of resistance：F1 illustrates that the height of specimen aspergillus flavus resisting contaminates growth between 0.01-0.600,
Illustrate that the low aspergillus flavus resisting of the sample contaminates growth between 0.601-1.000.
It should be noted that the size of mould circle (including sample sets and control group) in statistic procedure 4 and step 5
Calculation is with embodiment 1.
It should be noted that the culture dish for selecting is the circular culture dish of a diameter of 10cm.
It should be noted that step 4 should be identical with the incubation time in step 5.
In the method for embodiment 3,5 samples of same peanut varieties are determined, calculate its infection by Aspergillus flavus index F1, as a result
As shown in table 3, the F1 of different sample determinations is closer to, and the invention for illustrating the present invention has good stability.
The aspergillus flavus of the different peanut samples of table 3 contaminates Resistance Identification result
| Sample number into spectrum | F1 | Contaminate resistance probability |
| 1 | 0.6025 | It is low |
| 2 | 0.6132 | It is low |
| 3 | 0.6100 | It is low |
| 4 | 0.6089 | It is low |
| 5 | 0.6101 | It is low |
, but those skilled in the art once know basic creation although preferred embodiments of the present invention have been described
Property concept, then can make other change and modification to these embodiments.So, claims are intended to be construed to include excellent
Select embodiment and fall into having altered and changing for the scope of the invention.
Obviously, those skilled in the art can carry out the essence of various changes and modification without deviating from the present invention to the present invention
God and scope.So, if these modifications of the present invention and modification belong to the scope of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to comprising these changes and modification.
Claims (7)
1. an authentication method cultivated peanut to Aspergillus flavus infection resistance, it is characterised in that comprise the following steps：
Step 1, after aspergillus flavus strain is activated, elutes aspergillus spore, is prepared into aspergillus spore suspension, standby；
Step 2, selects the peanut seed of health, peels off, removes the peel, and is then comminuted into powder, 40 mesh sieves is crossed, by the peanut powder for obtaining
3-5min in the ethanol that volume fraction is 75% is soaked in, then aseptic water washing is clean, finally drains away the water, and obtains kind of an increment
Product, it is standby；
Step 3, by following structure fabrication culture apparatus：The culture apparatus includes culture dish (1), around the culture dish (1)
Inwall be provided with dismountable ring support (2), and the ring support (2) is positioned over the culture dish (1) inner bottom part, institute
State and horizontal transparent waffle slab (3) is overlapped with ring support (2), the transparent waffle slab (3) is with some square lattice
The network structure of son, and the length of side of each square lattice is equal；
Step 4, weighs the seed sample of certain mass under aseptic condition, in being laid in culture dish (1), plant in culture dish (1)
Subsample central authorities be added dropwise aspergillus spore suspension, seed sample be located at transparent waffle slab (3) lower section, and with transparent grid
Plate leaves certain distance between (3)；Then by culture apparatus in 25-30 DEG C of light culture 3-7 days, until growing sample sets mould
Circle, by the quantity of square lattice relative with sample sets mould circle on the transparent waffle slab (3) of statistics, calculates sample sets mould
The area A1 of bacterium circle, and recorded；
Step 5, to be equal to the Czapek's medium of seed sample quality described in step 4 as control, operates and step 4 phase
Together, differ only in：With Czapek's medium culture aspergillus flavus, until control group mould circle is grown, then by counting transparent network
The quantity of the square lattice relative with control group mould circle in panel (3), calculates the area C1 of control group mould circle, and gives
To record；
Step 6, as a result judges, by infection by Aspergillus flavus index F1 dip-dye resistance of the seed sample to aspergillus flavus is evaluated：
Wherein, infection by Aspergillus flavus index is calculated as follows：F1=A1/C1.
2. authentication method of the peanut according to claim 1 to Aspergillus flavus infection resistance, it is characterised in that in step 1, profit
With the acetum wash-out aspergillus spore that volume fraction is 5%.
3. authentication method of the peanut according to claim 1 to Aspergillus flavus infection resistance, it is characterised in that in step 1, it is yellow
Aspergillus spore concentration is 105Individual/ml.
4. authentication method of the peanut according to claim 1 to Aspergillus flavus infection resistance, it is characterised in that in step 3, often
The length of side of individual square lattice is 2-4mm.
5. authentication method of the peanut according to claim 4 to Aspergillus flavus infection resistance, it is characterised in that in step 3, often
25 square lattices constitute a block plaid, and the common edge of two neighboring block plaid is disposed as redness.
6. authentication method of the peanut according to claim 1 to Aspergillus flavus infection resistance, it is characterised in that also including yellow bent
Mould Mycotoxin identification step, specially：
Step a, the culture apparatus that step 4 grows sample sets mould circle is placed under the ultraviolet light of 365nm, and observation sample sets are yellow bent
The fluorescent ring size of the aflatoxin of mould generation, by counting square lattice relative with fluorescent ring on transparent waffle slab (3)
The quantity of son, calculates the area A2 of fluorescent ring, and is recorded；
Step b, the culture apparatus that step 5 grows control group mould circle is placed under the ultraviolet light of 365nm, and observation control group is yellow bent
The fluorescent ring size of the aflatoxin of mould generation, by counting square lattice relative with fluorescent ring on transparent waffle slab (3)
The quantity of son, calculates the area C2 of fluorescent ring, and is recorded；
Step c, as a result judges, produces index F2 by aflatoxin and evaluates dip-dye resistance of the seed sample to aspergillus flavus：
Wherein, aflatoxin produces index and calculates as follows：F2=A2/C2.
7. the peanut according to any one of claim 1-6 is sieved to the authentication method of Aspergillus flavus infection resistance in quick, batch
The application in the peanut varieties of resistance is contaminated in choosing with aspergillus flavus.
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| CN111979293A (en) * | 2020-08-28 | 2020-11-24 | 河南省农业科学院 | Indoor accurate identification method for resistance of peanut kernels to aspergillus flavus infection |
| CN112285071A (en) * | 2020-09-01 | 2021-01-29 | 河南工业大学 | Experimental method for using paeonol as aspergillus flavus bacteriostatic agent |
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