CN109001385A - The indoor appraising method and its application of a kind of peanut pod to Aspergillus flavus infection resistance - Google Patents
The indoor appraising method and its application of a kind of peanut pod to Aspergillus flavus infection resistance Download PDFInfo
- Publication number
- CN109001385A CN109001385A CN201810616100.5A CN201810616100A CN109001385A CN 109001385 A CN109001385 A CN 109001385A CN 201810616100 A CN201810616100 A CN 201810616100A CN 109001385 A CN109001385 A CN 109001385A
- Authority
- CN
- China
- Prior art keywords
- peanut pod
- peanut
- aspergillus flavus
- pod
- infection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/02—Food
Landscapes
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of peanut pods to the indoor appraising method of Aspergillus flavus infection resistance and its application in screening aspergillus flavus infection resistance groundnut germplasm.The indoor appraising method of a kind of peanut pod to Aspergillus flavus infection resistance, it is characterised in that the following steps are included: (1) selects aspergillus flavus strain；(2) selection, sterilizing and rehydration of peanut pod；(3) peanut pod is inoculated with Aspergillus flavus: being inoculated with healthy peanut pod with Aspergillus flavus spore suspension；(4) it cultivates: dark culture 7 days in the incubator after inoculation；(5) the peanut pod gradient of infection classification after cultivating；(6) peanut pod infection by Aspergillus flavus index is calculated；(7) resistance class is evaluated.Operation of the present invention is simple, easy to implement, can efficiently, the Aspergillus flavus infection resistance of accurate, batch identification peanut pod, be suitble to batch to screen the groundnut germplasm of Resistance to Aspergillus flavus invasion.It lays the foundation for research peanut pod shell to Aspergillus flavus infection resistance mechanism and breeding aspergillus flavus infection resistance peanut varieties.
Description
Technical field
The present invention relates to a kind of peanut pods to the indoor appraising method of Aspergillus flavus infection resistance and its in screening aspergillus flavus-resistance
Application in mould infecting peanut germ plasm resource.
Background technique
Generated secondary metabolite aflatoxin is always that flower is restricted in world wide after Aspergillus flavus infection peanut
An important factor for raw production, after eating aflatoxin-contaminated grain, can cause person poultry poisoning and induced hepatocellular carcinoma, and China
It is more serious one of the country of aflatoxin contamination again.Cultivating aspergillus flavus resisting kind is to solve aspergillus flavus and aspergillus flavus
Endotoxin contamination is most effective, economic and safest method, and establishes the identification of a fast and accurately peanut aspergillus flavus infection resistance
Method is conducive to the breeding for accelerating aspergillus flavus resisting peanut varieties.
Peanut pod shell is the first road barrier that peanut resists Aspergillus flavus infection, is played during peanut aspergillus flavus infection resistance
Important role.Currently, being had not been reported about peanut pod shell to the Resistance Identification of Aspergillus flavus infection and correlative study, this hair
The indoor appraising method of bright described peanut pod aspergillus flavus infection resistance and its application will be suitable for sieving efficiently, accurately, in batches
Aspergillus flavus infection resistance groundnut germplasm is selected, valuable materials is provided to aspergillus flavus resistance mechanism for research peanut pod shell, is conducive to
The peanut varieties of breeding aspergillus flavus infection resistance.
It is reported at present mainly to have following three method about infection resistance identification method of the peanut to Aspergillus flavus.1)
Peanut is identified by Field inoculation peanut plant to the resistance of Aspergillus flavus infection, the method is long qualification cycle, and needs a large amount of
Manually, the pollution vulnerable to microorganism in environment；2) by detecting aspergillus flavus using the method for weighing after artificial infection peanut son's benevolence
The degree infected, the method is although easy to operate, but since different peanut varieties are by after Aspergillus flavus infection, protein, oil-containing
Amount, oleic acid, linoleic content will appear different degrees of variation, cause the content results error of its Aspergillus flavus larger, difficult
To identify the resistance level of peanut after Aspergillus flavus infection well；3) invading for peanut son benevolence Aspergillus flavus is counted by artificial observation
Quantity and degree are contaminated, peanut son benevolence accurate can be identified to the resistance of aspergillus flavus.The method infects degree to aspergillus flavus
It is classified less, if be directly used in peanut pod Aspergillus flavus infection Resistance Identification, it is different can not accurately will to infect degree
Peanut pod classification, therefore the present invention establishes 9 grades of classification standards of Aspergillus flavus infection peanut pod for the first time, and it is yellow to calculate peanut pod
Aspergillus infection index simultaneously carries out evaluation of resistance.
Summary of the invention
The research of Aspergillus flavus infection resistance is not yet carried out at present for peanut pod shell, the purpose of the present invention is intended to provide one
Indoor appraising method and its application of efficient, the accurate peanut pod of kind to Aspergillus flavus infection resistance, invade peanut aspergillus flavus resisting
The breeding material of dye carries out batch screening, is research peanut pod shell to Aspergillus flavus infection resistance mechanism and breeding aspergillus flavus infection resistance
Peanut varieties lay the foundation.
To achieve the goals above, scheme adopted by the present invention is: a kind of peanut pod is to Aspergillus flavus infection resistance
Indoor appraising method, it is characterised in that the following steps are included: (1) selects aspergillus flavus strain；(2) selection, sterilizing of peanut pod
And rehydration；(3) peanut pod is inoculated with Aspergillus flavus: being inoculated with healthy peanut pod with Aspergillus flavus spore suspension；(4) it cultivates:
Dark culture 7 days in the incubator after inoculation；(5) the peanut pod gradient of infection classification after cultivating；(6) it is yellow bent to calculate peanut pod
Mould infestation index；(7) resistance class is evaluated.
A kind of indoor appraising method of peanut pod to Aspergillus flavus infection resistance, the specific steps are as follows:
(1) it selects aspergillus flavus strain: choosing one plant of aspergillus flavus strain (infection ability is strong) and be inoculated in PDA culture medium, 30
DEG C culture 7 days after, the tween water for being 0.1% with the mass concentration of sterilizing (is exactly the water-soluble of the tween that mass concentration is 0.1%
Liquid) spore is rinsed, conidium is collected, and conidium is resuspended, obtains aspergillus spore suspension and (connect as aspergillus spore
Kind suspension is spare)；
(2) selection, sterilizing and rehydration of peanut pod: choosing peanut pod, (current year harvests the health of dryness in the sun, mature
It is full, unabroken), the soil on peanut pod surface is rinsed well with flowing water, is placed in sterile beaker；In biology
In safety cabinet, the NaClO solution that the mass concentration of sterilizing is 1% is added into beaker, impregnates 10-13min, outwells NaClO
After solution, peanut pod aseptic water washing 3 times (from starting to sterilize to the time control finished with aseptic water washing in 14-16
Minute), peanut pod is poured into sterile petri dish；Water content 20-25% (quality %) after making peanut pod rehydration；
(3) peanut pod is inoculated with Aspergillus flavus: taking 1mL that step is added aspergillus spore suspension prepared by step (1)
(2) in the sterile petri dish in equipped with peanut pod (sample), gently rotating sterile petri dish enables spore to be uniformly attached to pod
Peanut pod is put to gap is kept between peanut pod, mutually not closely in fruit surface；
(4) it cultivates: the culture dish equipped with the peanut pod for being inoculated with Aspergillus flavus in step (3) is placed in and (is carefully placed in)
Bottom contains in the finishing box of moisture, covers case lid, is placed in dark culturing 7 days in 30 DEG C of constant incubator；
(5) the peanut pod gradient of infection classification after cultivating: peanut pod surface Aspergillus flavus degree of adhesion is pressed, by peanut
Pod gradient of infection carries out 1-9 grades of classifications；
(6) peanut pod infection by Aspergillus flavus index (MOI) is calculated:
According to formula: peanut pod infection by Aspergillus flavus index=Σ (the number of sick fruits at different levels × relative disease grade represents numerical value)/(adjust
Look into the total number of fruits × highest level typical value) × 100, calculate the infection by Aspergillus flavus index of each peanut pod (sample)；
Wherein, the number of sick fruits at different levels are the total quantity of the infected peanut pods at different levels in 1-9 grades of peanut pod gradient of infection；
Relative disease grade represents numerical value as the typical value of peanut pod gradient of infection at different levels, respectively 0,1,2,3,4,5,6,7,8；Investigation is total
Fruit number is peanut pod total quantity in a culture dish；Typical value at the highest level is peanut pod gradient of infection grade highest level
Typical value 8；
(7) it evaluates resistance class: resulting final score being calculated according to peanut pod infection by Aspergillus flavus index M OI, will be spent
Raw germ plasm resource be divided into highly resistance, in resist, middle sense and high sense aspergillus flavus.
Aspergillus flavus strain described in step (1) (infection ability is strong) is aspergillus flavus strain AF2202 (Scientia Agricultura Sinica
Oil crops research institute of institute).
Conidium is resuspended described in (1) in step are as follows: adjustment spore concentration is 2 × 106A spore/mL.
It is controlled from starting to sterilize to the time finished with aseptic water washing at 14-16 minutes in step (2).
The quality of peanut pod is 30g (i.e. in aspergillus spore suspension and culture dish in sterile petri dish in step (3)
The proportion of peanut pod is 1mL:30g).
Step makes relative humidity 80-90% in finishing box in (4).
1-9 grades in step (5) are as follows:
9 grades: conidium 100% covers peanut pod surface, and the shell of peanut pod is all in green, it is seen that thick and solid
Spore layer, thick and solid spore layer account for peanut pod surface area up to 91%~100% (note: herein comprising 91%；Also contain herein
100%), recording the corresponding typical value of the grade is 8；
8 grades: conidium 100% covers peanut pod surface, and the shell of peanut pod is all in green, it is seen that thick and solid
Spore layer, thick and solid spore layer account for peanut pod surface area up to 71%~91% (note: herein comprising 71%；91% is free of herein,
It is 7 less than the corresponding typical value of the grade 91%), is recorded；
7 grades: conidium 100% covers peanut pod surface, and the shell of peanut pod is all in green, it is seen that thick and solid
Spore layer, thick and solid spore layer account for peanut pod surface area up to 51%~71% (note: herein comprising 51%；71% is free of herein,
It is 6 less than the corresponding typical value of the grade 71%), is recorded；
6 grades: conidium 81%~100% covers peanut pod surface, and (note: including 81% herein；Also contain herein
100%), the shell of peanut pod is largely in green, it is seen that thick and solid spore layer, thick and solid spore layer account for peanut pod surface
It accumulates up to 31%~51% (note: herein comprising 31%；51% is free of herein, i.e., less than 51%), recording the corresponding representative of the grade
Value is 5；
5 grades: conidium 61%~81% covers peanut pod surface, and (note: including 61% herein；81% is free of herein,
I.e. less than 81%), the shell of part peanut pod is as it can be seen that thicker spore layer accounts for peanut pod surface area up to 11%~31%
(note: including 11% herein；31% is free of herein, i.e., is 4 less than the corresponding typical value of the grade 31%), is recorded；
4 grades: conidium 41%~61% covers peanut pod surface, and (note: including 41% herein；61% is free of herein,
I.e. less than 61%), the shell of most of peanut pod is as it can be seen that thicker spore layer accounts for peanut pod surface area up to 6%~11%
(note: including 6% herein；11% is free of herein, i.e., is 3 less than the corresponding typical value of the grade 11%), is recorded；
3 grades: conidium 21%~41% covers peanut pod surface, and (note: including 21% herein；41% is free of herein,
I.e. less than 41%), the shell of most of peanut pod is as it can be seen that thicker spore layer accounts for peanut pod surface area up to 0%~6%
(note: including 0% herein；6% is free of herein, i.e., is 2 less than the corresponding typical value of the grade 6%), is recorded；
2 grades: conidium 0%~21% covers peanut pod surface, and (note: including 0% herein；21% is free of herein, i.e.,
Less than 21%), for the shell of most of peanut pod as it can be seen that without thick and solid spore layer, recording the corresponding typical value of the grade is 1；
1 grade: peanut pod health, the shell of whole peanut pods is as it can be seen that recording the corresponding typical value of the grade is 0.
The thickness of above-mentioned thick and solid spore layer is about 2mm.
Indoor appraising method of a kind of peanut pod as described above to Aspergillus flavus infection resistance, it is characterised in that be applied to
It screens in aspergillus flavus infection resistance groundnut germplasm.
The present invention carries out moisturizing dark culture after being inoculated with peanut pod with aspergillus flavus strain, by observing Aspergillus flavus infection peanut
A situation arises for Aspergillus flavus in the quantity and peanut pod of pod, and the degree of Aspergillus flavus infection peanut pod is divided into 9 grades, is calculated
Peanut pod infection by Aspergillus flavus index simultaneously carries out evaluation of resistance.
The beneficial effects of the present invention are: it is easy to operate, easy to implement, it efficient, accurate, batch can identify peanut pod
Aspergillus flavus infection resistance is suitble to batch to screen the groundnut germplasm of Resistance to Aspergillus flavus invasion.The present invention is to peanut aspergillus flavus resisting
The breeding material infected carries out batch screening, invades for research peanut pod shell Aspergillus flavus infection resistance mechanism and breeding aspergillus flavus resisting
Dye peanut varieties lay the foundation.
Detailed description of the invention
Fig. 1 is 9 grades of classification figures of peanut pod gradient of infection in the present invention after Aspergillus flavus infecting peanut pod.1-9 in figure
Respectively represent 1 grade -9 grades in peanut pod gradient of infection grade scale of symptom.
Specific embodiment
Below with reference to examples of implementation, the present invention is described in further detail, but embodiments of the present invention are not limited to
This.
A kind of indoor appraising method of peanut pod to Aspergillus flavus infection resistance, the specific steps are as follows:
(1) aspergillus flavus strain is selected: with the strong aspergillus flavus strain AF2202 (Chinese Academy of Agricultural Sciences of high yield poison, infecting potential
Oil crops research institute) it is inoculation bacterial strain；Aspergillus flavus strain AF2202 is inoculated in PDA culture medium, 30 DEG C of dark culturings 7
It, with sterile mass concentration is that 0.1% Tween 80 liquid (tween water) collects conidium, and mitogenetic spore is resuspended when inoculation
Son, adjustment spore concentration are 2 × 106A spore/mL (i.e. modulation conidial suspension), obtains aspergillus spore suspension
(spare as aspergillus spore inoculation suspension)；PDA culture medium can be used existing；
(2) selection, sterilizing and rehydration of peanut pod: health, mature and plump, the nothing of selection current year harvest dryness in the sun are broken
The peanut varieties (such as: 16-61731 pod, Inst. of Oil Crops, Chinese Academy of Agriculture) of damage are with flowing water by peanut pod
The silt on fruit surface cleans up, and is placed in sterile beaker, in Biohazard Safety Equipment (beaker is placed in Biohazard Safety Equipment), to
The NaClO solution that the mass concentration of sterilizing is 1% is added in beaker, impregnates 10-13min (most preferably immersion 12min), during which between
It has a rest and shakes beaker peanut pod surface is made sufficiently to sterilize, outwell aqua sterilisa (i.e. NaClO solution) afterwards with aseptic water washing 3 times, from
Start sterilizing to finished with aseptic water washing time control at 14-16 minutes, peanut pod is poured into accordingly number it is sterile
In culture dish；Water content 20-25% (quality %) after making peanut pod rehydration；
(3) peanut pod is inoculated with Aspergillus flavus: taking 1mL that step is added aspergillus spore suspension prepared by step (1)
(2) in the sterile petri dish in equipped with peanut pod (sample) (quality of peanut pod is 30g), sterile petri dish is gently rotated
So that spore is uniformly attached to pod surface, puts peanut pod to a fixed gap is kept between peanut pod, mutually not closely；
(4) it cultivates: the culture dish equipped with the peanut pod for being inoculated with Aspergillus flavus in step (3) being carefully placed in bottom and is contained
Have in the finishing box of moisture, cover case lid, make relative humidity 80-90% in finishing box, is placed in 30 DEG C of constant incubator
Dark culturing 7 days；
(5) the peanut pod gradient of infection classification after cultivating: peanut pod surface Aspergillus flavus degree of adhesion is pressed, by peanut
Pod gradient of infection (V) is divided into 9 grades, i.e., 1~9 grade classification:
9 grades: conidium 100% covers peanut pod surface, and the shell of peanut pod is all in green, it is seen that thick and solid
Spore layer, thick and solid spore layer account for peanut pod surface area up to 91%~100% (note: herein comprising 91%；Also contain herein
100%), recording the corresponding typical value of the grade is 8；
8 grades: conidium 100% covers peanut pod surface, and the shell of peanut pod is all in green, it is seen that thick and solid
Spore layer, thick and solid spore layer account for peanut pod surface area up to 71%~91% (note: herein comprising 71%；91% is free of herein,
It is 7 less than the corresponding typical value of the grade 91%), is recorded；
7 grades: conidium 100% covers peanut pod surface, and the shell of peanut pod is all in green, it is seen that thick and solid
Spore layer, thick and solid spore layer account for peanut pod surface area up to 51%~71% (note: herein comprising 51%；71% is free of herein,
It is 6 less than the corresponding typical value of the grade 71%), is recorded；
6 grades: conidium 81%~100% covers peanut pod surface, and (note: including 81% herein；Also contain herein
100%), the shell of peanut pod is largely in green, it is seen that thick and solid spore layer, thick and solid spore layer account for peanut pod surface
It accumulates up to 31%~51% (note: herein comprising 31%；51% is free of herein, i.e., less than 51%), recording the corresponding representative of the grade
Value is 5；
5 grades: conidium 61%~81% covers peanut pod surface, and (note: including 61% herein；81% is free of herein,
I.e. less than 81%), the shell of part peanut pod is as it can be seen that thicker spore layer accounts for peanut pod surface area up to 11%~31%
(note: including 11% herein；31% is free of herein, i.e., is 4 less than the corresponding typical value of the grade 31%), is recorded；
4 grades: conidium 41%~61% covers peanut pod surface, and (note: including 41% herein；61% is free of herein,
I.e. less than 61%), the shell of most of peanut pod is as it can be seen that thicker spore layer accounts for peanut pod surface area up to 6%~11%
(note: including 6% herein；11% is free of herein, i.e., is 3 less than the corresponding typical value of the grade 11%), is recorded；
3 grades: conidium 21%~41% covers peanut pod surface, and (note: including 21% herein；41% is free of herein,
I.e. less than 41%), the shell of most of peanut pod is as it can be seen that thicker spore layer accounts for peanut pod surface area up to 0%~6%
(note: including 0% herein；6% is free of herein, i.e., is 2 less than the corresponding typical value of the grade 6%), is recorded；
2 grades: conidium 0%~21% covers peanut pod surface, and (note: including 0% herein；21% is free of herein, i.e.,
Less than 21%), for the shell of most of peanut pod as it can be seen that without thick and solid spore layer, recording the corresponding typical value of the grade is 1；
1 grade: peanut pod health, the shell of whole peanut pods is as it can be seen that recording the corresponding typical value of the grade is 0；
6) peanut pod infection by Aspergillus flavus index (MOI) is calculated:
According to formula: peanut pod infection by Aspergillus flavus index=Σ (the number of sick fruits at different levels × relative disease grade represents numerical value)/(adjust
Look into the total number of fruits × highest level typical value) × 100, calculate the infection by Aspergillus flavus index of each peanut pod (sample)；
Wherein, the number of sick fruits at different levels are the total quantity of the infected peanut pods at different levels in 1-9 grades of peanut pod gradient of infection；
Relative disease grade represents numerical value as the typical value of peanut pod gradient of infection at different levels, respectively 0,1,2,3,4,5,6,7,8；Investigation is total
Fruit number is peanut pod total quantity in a culture dish；Typical value at the highest level is peanut pod gradient of infection grade highest level
Typical value 8；
(7) it evaluates resistance class: resulting final score is calculated according to peanut pod infection by Aspergillus flavus index (MOI), it will
Groundnut germplasm 0≤MOI < 25 are highly resistance groundnut germplasm；25≤MOI < 50 be in anti-groundnut germplasm；50≤MOI<
75 be middle sense groundnut germplasm；The high sense groundnut germplasm of MOI >=75 (i.e. by groundnut germplasm be divided into highly resistance, in resist, in
Sense and high sense aspergillus flavus).
Embodiment 1
(1) aspergillus flavus strain is selected: with the strong aspergillus flavus strain AF2202 (Chinese Academy of Agricultural Sciences of high yield poison, infecting potential
Oil crops research institute) it is inoculation bacterial strain；Aspergillus flavus strain AF2202 is inoculated in PDA culture medium, 30 DEG C of dark culturings 7
It, with sterile mass concentration is that 0.1% Tween 80 liquid collects conidium, and conidium is resuspended when inoculation, adjusts spore
Concentration is 2 × 106A spore/mL obtains aspergillus spore suspension (spare as aspergillus spore inoculation suspension)；
(2) selection, sterilizing and rehydration of peanut pod: selection groundnut germplasm HP17,16-61731,16-61722,
16-61534 (Inst. of Oil Crops, Chinese Academy of Agriculture) is material to be tested (i.e. peanut varieties), height sense groundnut germplasm
16-61594 (Inst. of Oil Crops, Chinese Academy of Agriculture) is control.Each germ plasm resource chooses 60 health, full, nothing
Damaged peanut pod is test sample, is cleaned up the silt on pod surface with flowing water, packing to 3 it is numbered
In beaker, 20 pods of each beaker, the mass concentration of each beaker sterilizing is molten for 1% NaClO in Biohazard Safety Equipment
Liquid, during which interval, which shakes beaker, makes peanut pod surface uniformly contact NaClO solution, after sterilizing 12 minutes (i.e. immersion 12min),
NaClO solution is outwelled, with aseptic water washing 3 times, pod is poured into the sterile culture accordingly numbered by extra water in the cup of falling dry combustion method
In ware.From the water content for starting to sterilize to the time control finished is rinsed with water at 15 minutes or so, after making peanut pod rehydration
For 20-25% (quality %), in favor of infecting for aspergillus spore；
(3) peanut pod is inoculated with Aspergillus flavus: the aspergillus spore suspension of step (1) preparation is added in step (2)
In culture dish equipped with sample (quality of peanut pod is 30g), 1mL Aspergillus flavus spore suspension is added in each culture dish
(2×106A spore/mL), shaking culture dish makes spore uniformly be attached to pod shell surface, puts pod with sterilizing tweezers, makes pod
Gap is kept between fruit, mutually not closely；
(4) it cultivates: the culture dish equipped with the peanut pod for being inoculated with Aspergillus flavus in step (3) being carefully placed in bottom and is contained
Have in the finishing box of moisture, cover case lid, make relative humidity 80-90% in finishing box, 30 DEG C dark culturing 7 days；
(5) the peanut pod gradient of infection classification after cultivating: each peanut pod adheres to according to pod surface in culture dish
Aspergillus flavus bacterium amount, carry out gradient of infection classification identification referring to grade scale, and record.In color No. 1 No. 01 culture dish in another name for Sichuan Province
Peanut pod infect grade 1 grade 0,2 grades 4,3 grades 8,4 grades 2,5 grades 2,6 grades 3,7 grades 1,8 grades 0,9 grades
0.It is all to be recorded in this way for examination peanut pod, specifically it is shown in Table 1；
(6) according to peanut pod infection by Aspergillus flavus index (MOI) calculation formula, the infection of each material to be tested is calculated separately
Index (MOI).
Such as color No. 1 No. 01 sample in another name for Sichuan Province, the typical value of the number of sick fruits of each grade and its corresponding grade is substituted into formula meter
Infestation index MOI is calculated, then MOI=(the 0*0+4*1+8*2+2*3+2*4+3*5+1*6+0*7+0* of color No. 1 No. 01 sample in another name for Sichuan Province
8)/(20*8) * 100=34.38, and so on, calculate another name for Sichuan Province color No. 1, each sample of 16-61731,16-61722,16-61534
Infestation index's (being shown in Table 1)；
(7) it according to the infestation index MOI of each material to be tested in step (6), carries out groundnut germplasm resistance class and comments
Valence, another name for Sichuan Province is No. 1 color, 16-61731,16-61722,16-61534 infestation index is between 25~50, be accredited as in resist
Groundnut germplasm.
Table 1 peanut seed resource (HP17,16-61731,16-61722,16-61534) Resistance Identification result
Note: peanut pod infection by Aspergillus flavus index abbreviation infestation index.
Embodiment 2
(1) aspergillus flavus strain is selected: with the strong aspergillus flavus strain AF2202 (Chinese Academy of Agricultural Sciences of high yield poison, infecting potential
Oil crops research institute) it is inoculation bacterial strain；Aspergillus flavus strain AF2202 is inoculated in PDA culture medium, 30 DEG C of dark culturings 7
It, with sterile mass concentration is that 0.1% Tween 80 liquid collects conidium, and conidium is resuspended when inoculation, adjusts spore
Concentration is 2 × 106A spore/mL obtains aspergillus spore suspension (spare as aspergillus spore inoculation suspension)；
(2) selection, sterilizing and rehydration of peanut pod: selection groundnut germplasm 16-61730,16-61714,16-
61639,16-61689 (Inst. of Oil Crops, Chinese Academy of Agriculture) is material to be tested, height sense groundnut germplasm 16-
61594 (Inst. of Oil Crops, Chinese Academy of Agriculture) are control.Each germ plasm resource chooses 60 health, full, nothing is broken
The peanut pod of damage is test sample, is cleaned up the silt on pod surface with flowing water, packing to 3 numbered burnings
In cup, 20 pods of each beaker, the NaClO that the mass concentration of sterilizing is 1% is added in each beaker in Biohazard Safety Equipment
Solution, during which interval, which shakes beaker, makes peanut pod surface uniformly contact NaClO solution, and after sterilizing 12 minutes, it is molten to outwell NaClO
Liquid is drained water extra in beaker, pod is poured into the sterile petri dish accordingly numbered with aseptic water washing 3 times.From the beginning of
Sterilize water content 20-25% (matter of the time control for being rinsed with water and finishing at 15 minutes or so, after making peanut pod rehydration
Measure %), in favor of infecting for aspergillus spore；
(3) peanut pod is inoculated with Aspergillus flavus: the aspergillus spore suspension of step (1) preparation is added in step (2)
In culture dish equipped with sample (quality of peanut pod is 30g), 1mL Aspergillus flavus spore suspension is added in each culture dish
(2×106A spore/mL), shaking culture dish makes spore uniformly be attached to pod shell surface, puts pod with sterilizing tweezers, makes pod
Gap is kept between fruit, mutually not closely；
(4) it cultivates: the culture dish equipped with the peanut pod for being inoculated with Aspergillus flavus in step (3) being carefully placed in bottom and is contained
Have in the finishing box of moisture, cover case lid, make relative humidity 80-90% in finishing box, 30 DEG C dark culturing 7 days；
(5) the aspergillus flavus bacterium amount that each peanut pod in culture dish adheres to according to pod surface, referring to grade scale into
Row gradient of infection classification identification, and record.As 16-61730 No. 01 culture dish in peanut pod infect grade 1 grade 0,2
Grade 0,3 grades 0,4 grades 0,5 grades 10,6 grades 9,7 grades 0,8 grades 0,9 grades 1.It is all to be pressed for examination peanut pod
This method record, is specifically shown in Table 2；
(6) according to peanut pod infection by Aspergillus flavus index (MOI) calculation formula, the infection of each material to be tested is calculated separately
Index (MOI).
Such as No. 01 sample of 16-61730, the typical value of the number of sick fruits of each grade and its corresponding grade is substituted into formula meter
Infestation index MOI is calculated, then MOI=(the 0*0+0*1+0*2+0*3+10*4+9*5+0*6+0*7+1* of 16-61730 No. 01 sample
8)/(20*8) * 100=58.13, and so on, calculate each sample of 16-61730,16-61714,16-61639,16-61689
Infestation index's (being shown in Table 2)；
(7) it according to the infestation index MOI of each material to be tested in step (6), carries out groundnut germplasm resistance class and comments
Valence, the infestation index of 16-61730,16-61714,16-61639,16-61689 are accredited as middle sense between 50~75
Groundnut germplasm.
Table 2 peanut seed resource (16-61730,16-61714,16-61639,16-61689) Resistance Identification result
Embodiment 3
(1) aspergillus flavus strain is selected: with the strong aspergillus flavus strain AF2202 (Chinese Academy of Agricultural Sciences of high yield poison, infecting potential
Oil crops research institute) it is inoculation bacterial strain；Aspergillus flavus strain AF2202 is inoculated in PDA culture medium, 30 DEG C of dark culturings 7
It, with sterile mass concentration is that 0.1% Tween 80 liquid collects conidium, and conidium is resuspended when inoculation, adjusts spore
Concentration is 2 × 106A spore/mL obtains aspergillus spore suspension (spare as aspergillus spore inoculation suspension)；
(2) selection, sterilizing and rehydration of peanut pod: selection groundnut germplasm 16-61602,16-61600,16-
61601,16-61598 (Inst. of Oil Crops, Chinese Academy of Agriculture) is material to be tested, height sense groundnut germplasm 16-
61594 (Inst. of Oil Crops, Chinese Academy of Agriculture) are control.Each germ plasm resource chooses 60 health, full, nothing is broken
The peanut pod of damage is test sample, is cleaned up the silt on pod surface with flowing water, packing to 3 numbered burnings
In cup, 20 pods of each beaker, the NaClO that the mass concentration of sterilizing is 1% is added in each beaker in Biohazard Safety Equipment
Solution, during which interval, which shakes beaker, makes peanut pod surface uniformly contact NaClO solution, and after sterilizing 12 minutes, it is molten to outwell NaClO
Liquid is drained water extra in beaker, pod is poured into the sterile petri dish accordingly numbered with aseptic water washing 3 times.From the beginning of
Sterilize water content 20-25% (matter of the time control for being rinsed with water and finishing at 15 minutes or so, after making peanut pod rehydration
Measure %), in favor of infecting for aspergillus spore；
(3) peanut pod is inoculated with Aspergillus flavus: the aspergillus spore suspension of step (1) preparation is added in step (2)
In culture dish equipped with sample (quality of peanut pod is 30g), 1mL Aspergillus flavus spore suspension is added in each culture dish
(2×106A spore/mL), shaking culture dish makes spore uniformly be attached to pod shell surface, puts pod with sterilizing tweezers, makes pod
Gap is kept between fruit, mutually not closely；
(4) it cultivates: the culture dish equipped with the peanut pod for being inoculated with Aspergillus flavus in step (3) being carefully placed in bottom and is contained
Have in the finishing box of moisture, cover case lid, make relative humidity 80-90% in finishing box, 30 DEG C dark culturing 7 days；
(5) the aspergillus flavus bacterium amount that each peanut pod in culture dish adheres to according to pod surface, referring to grade scale into
Row gradient of infection classification identification, and record.As 16-61602 No. 01 culture dish in peanut pod infect grade 1 grade 0,2
Grade 0,3 grades 0,4 grades 0,5 grades 0,6 grades 0,7 grades 1,8 grades 8,9 grades 11.It is all to be pressed for examination peanut pod
This method record, is specifically shown in Table 3；
(6) according to peanut pod infection by Aspergillus flavus index (MOI) calculation formula, the infection of each material to be tested is calculated separately
Index (MOI).
Such as No. 01 sample of 16-61602, the typical value of the number of sick fruits of each grade and its corresponding grade is substituted into formula meter
Infestation index MOI is calculated, then MOI=(the 0*0+0*1+0*2+0*3+0*4+0*5+1*6+8*7+11* of 16-61602 No. 01 sample
8)/(20*8) * 100=93.75, and so on, calculate each sample of 16-61602,16-61600,16-61601,16-61598
Infestation index's (being shown in Table 3)；
(7) it according to the infestation index MOI of each material to be tested in step (6), carries out groundnut germplasm resistance class and comments
Valence, the infestation index of 16-61602,16-61600,16-61601,16-61598 are accredited as high sense peanut 75 or more
Germ plasm resource.
Table 3 peanut seed resource (16-61602,16-61600,16-61601,16-61598) Resistance Identification result
Above-described embodiment is preferrred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other are any without departing from made simplification, substitution, combination, change, modification under Spirit Essence of the invention, should be
The substitute mode of effect, is all included in the scope of protection of the present invention.
Claims (10)
1. a kind of peanut pod is to the indoor appraising method of Aspergillus flavus infection resistance, it is characterised in that the following steps are included: (1) is selected
Select aspergillus flavus strain；(2) selection, sterilizing and rehydration of peanut pod；(3) peanut pod is inoculated with Aspergillus flavus: with Aspergillus flavus
Spore suspension is inoculated with healthy peanut pod；(4) it cultivates: dark culture 7 days in the incubator after inoculation；(5) peanut after cultivating
The classification of pod gradient of infection；(6) peanut pod infection by Aspergillus flavus index is calculated；(7) resistance class is evaluated.
2. a kind of peanut pod according to claim 1 exists to the indoor appraising method of Aspergillus flavus infection resistance, feature
In, the specific steps are as follows:
(1) it selects aspergillus flavus strain: choosing one plant of aspergillus flavus strain and be inoculated in PDA culture medium, 30 DEG C after culture 7 days, with going out
The mass concentration of bacterium is 0.1% to spit warm water washing spore, collects conidium, and conidium is resuspended, obtains aspergillus flavus spore
Sub- suspension；
(2) selection, sterilizing and rehydration of peanut pod: choosing peanut pod, with flowing water by the soil on peanut pod surface
It rinses well, is placed in sterile beaker；In Biohazard Safety Equipment, the mass concentration that sterilizing is added into beaker is 1%
NaClO solution, after outwelling NaClO solution, peanut pod is poured into peanut pod in sterile petri dish with aseptic water washing 3 times；
(3) peanut pod is inoculated with Aspergillus flavus: taking 1mL to be added in step (2) aspergillus spore suspension prepared by step (1)
In sterile petri dish equipped with peanut pod, gently rotating sterile petri dish enables spore to be uniformly attached to pod surface, puts
Peanut pod is to gap is kept between peanut pod, mutually not closely；
(4) it cultivates: the culture dish equipped with the peanut pod for being inoculated with Aspergillus flavus in step (3) being placed in bottom and contains moisture
In finishing box, case lid is covered, is placed in dark culturing 7 days in 30 DEG C of constant incubator；
(5) the peanut pod gradient of infection classification after cultivating: peanut pod surface Aspergillus flavus degree of adhesion is pressed, by peanut pod
Gradient of infection carries out 1-9 grades of classifications；
(6) peanut pod infection by Aspergillus flavus index (MOI) is calculated:
According to formula: peanut pod infection by Aspergillus flavus index=Σ (the number of sick fruits at different levels × relative disease grade represents numerical value)/(investigation is total
Fruit number × highest level typical value) × 100, calculate the infection by Aspergillus flavus index of each peanut pod (sample)；
Wherein, the number of sick fruits at different levels are the total quantity of the infected peanut pods at different levels in 1-9 grades of peanut pod gradient of infection；Relatively
Sick grade represents numerical value as the typical value of peanut pod gradient of infection at different levels, respectively 0,1,2,3,4,5,6,7,8；Investigate the total number of fruits
For peanut pod total quantity in a culture dish；Typical value at the highest level is the generation of peanut pod gradient of infection grade highest level
Tabular value 8；
(7) it evaluates resistance class: resulting final score being calculated according to peanut pod infection by Aspergillus flavus index M OI, by peanut species
Matter resource be divided into highly resistance, in resist, middle sense and high sense aspergillus flavus.
3. a kind of peanut pod according to claim 2 exists to the indoor appraising method of Aspergillus flavus infection resistance, feature
In aspergillus flavus strain described in step (1) is the aspergillus flavus strain AF2202 of Inst. of Oil Crops, Chinese Academy of Agriculture.
4. a kind of peanut pod according to claim 2 exists to the indoor appraising method of Aspergillus flavus infection resistance, feature
In conidium is resuspended described in (1) in step are as follows: adjustment spore concentration is 2 × 106A spore/mL.
5. a kind of peanut pod according to claim 2 exists to the indoor appraising method of Aspergillus flavus infection resistance, feature
In from starting to sterilize to the time control finished with aseptic water washing at 14-16 minutes in step (2).
6. a kind of peanut pod according to claim 2 exists to the indoor appraising method of Aspergillus flavus infection resistance, feature
In the quality of peanut pod is 30g (i.e. peanut in aspergillus spore suspension and culture dish in sterile petri dish in step (3)
The proportion of pod is 1mL:30g).
7. a kind of peanut pod according to claim 2 exists to the indoor appraising method of Aspergillus flavus infection resistance, feature
In step makes relative humidity 80-90% in finishing box in (4).
8. a kind of peanut pod according to claim 2 exists to the indoor appraising method of Aspergillus flavus infection resistance, feature
In 1-9 grades in step (5) are as follows:
9 grades: conidium 100% covers peanut pod surface, and the shell of peanut pod is all in green, it is seen that thick and solid spore
Layer, thick and solid spore layer account for peanut pod surface area up to 91%~100%, and recording the corresponding typical value of the grade is 8；
8 grades: conidium 100% covers peanut pod surface, and the shell of peanut pod is all in green, it is seen that thick and solid spore
Layer, thick and solid spore layer account for peanut pod surface area up to 71%~91%, and recording the corresponding typical value of the grade is 7；
7 grades: conidium 100% covers peanut pod surface, and the shell of peanut pod is all in green, it is seen that thick and solid spore
Layer, thick and solid spore layer account for peanut pod surface area up to 51%~71%, and recording the corresponding typical value of the grade is 6；
6 grades: conidium 81%~100% covers peanut pod surface, and the shell of peanut pod is largely in green, it is seen that thick
Real spore layer, thick and solid spore layer account for peanut pod surface area up to 31%~51%, and recording the corresponding typical value of the grade is
5；
5 grades: conidium 61%~81% covers peanut pod surface, and the shell of part peanut pod is as it can be seen that thicker spore
Layer accounts for peanut pod surface area up to 11%~31%, and recording the corresponding typical value of the grade is 4；
4 grades: conidium 41%~61% covers peanut pod surface, and the shell of most of peanut pod is as it can be seen that thicker spore
Sublayer accounts for peanut pod surface area up to 6%~11%, and recording the corresponding typical value of the grade is 3；
3 grades: conidium 21%~41% covers peanut pod surface, and the shell of most of peanut pod is as it can be seen that thicker spore
Sublayer accounts for peanut pod surface area up to 0%~6%, and recording the corresponding typical value of the grade is 2；
2 grades: conidium 0%~21% covers peanut pod surface, and the shell of most of peanut pod is as it can be seen that without thick and solid spore
Sublayer, recording the corresponding typical value of the grade is 1；
1 grade: peanut pod health, the shell of whole peanut pods is as it can be seen that recording the corresponding typical value of the grade is 0.
9. a kind of peanut pod according to claim 8 exists to the indoor appraising method of Aspergillus flavus infection resistance, feature
In the thickness of above-mentioned thick and solid spore layer is about 2mm.
10. a kind of indoor appraising method of the peanut pod as claimed in claim 1 or 2 to Aspergillus flavus infection resistance, feature
It is to be applied in screening aspergillus flavus infection resistance groundnut germplasm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810616100.5A CN109001385A (en) | 2018-06-15 | 2018-06-15 | The indoor appraising method and its application of a kind of peanut pod to Aspergillus flavus infection resistance |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810616100.5A CN109001385A (en) | 2018-06-15 | 2018-06-15 | The indoor appraising method and its application of a kind of peanut pod to Aspergillus flavus infection resistance |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109001385A true CN109001385A (en) | 2018-12-14 |
Family
ID=64601120
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810616100.5A Pending CN109001385A (en) | 2018-06-15 | 2018-06-15 | The indoor appraising method and its application of a kind of peanut pod to Aspergillus flavus infection resistance |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109001385A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109750080A (en) * | 2019-03-01 | 2019-05-14 | 山东省花生研究所 | A kind of method of the anti-fruit rot germ plasm resource of quick screening peanut |
| 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 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1556391A (en) * | 2003-12-30 | 2004-12-22 | 中国农业科学院油料作物研究所 | Peanut aspergillus flavus anti aflatoxin production preperty identification method |
| CN101608211A (en) * | 2009-07-10 | 2009-12-23 | 山东省花生研究所 | Indoor detection method for resistance of peanuts to infection of Aspergillus flavus |
| 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 |
| 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 |
| CN106596492A (en) * | 2016-12-28 | 2017-04-26 | 商丘市农林科学院 | Method for identifying infection resistance of peanuts to aspergillus flavus and application thereof |
-
2018
- 2018-06-15 CN CN201810616100.5A patent/CN109001385A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1556391A (en) * | 2003-12-30 | 2004-12-22 | 中国农业科学院油料作物研究所 | Peanut aspergillus flavus anti aflatoxin production preperty identification method |
| CN101608211A (en) * | 2009-07-10 | 2009-12-23 | 山东省花生研究所 | Indoor detection method for resistance of peanuts to infection of Aspergillus flavus |
| 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 |
| 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 |
| CN106596492A (en) * | 2016-12-28 | 2017-04-26 | 商丘市农林科学院 | Method for identifying infection resistance of peanuts to aspergillus flavus and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 李光星 等: "花生抗黄曲霉侵染研究初报", 《福建农业科技》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109750080A (en) * | 2019-03-01 | 2019-05-14 | 山东省花生研究所 | A kind of method of the anti-fruit rot germ plasm resource of quick screening peanut |
| CN109750080B (en) * | 2019-03-01 | 2022-01-25 | 山东省花生研究所 | Method for rapidly screening peanut fruit rot resistant germplasm resources |
| 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 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11970728B2 (en) | Method for indoors and rapidly identifying the resistance of wheat to black point disease caused by Bipolaris sorokiniana | |
| Macer | Resistance to eyespot disease (Cercosporella herpotrichoides Fron) determined by a seedling test in some forms of Triticum, Aegilops, Secale and Hordeum | |
| CN106613288A (en) | Rapid identification method for broad bean resistance to Acyrthosiphon pisum | |
| CN109001385A (en) | The indoor appraising method and its application of a kind of peanut pod to Aspergillus flavus infection resistance | |
| Sujatha et al. | Screening of tomato genotypes for root knot nematode (Meloidogyne incognita Kofoid and White Chitwood) | |
| Akhtar et al. | Comparison of methods of inoculation of Xanthomonas oryzae pv. oryzae in rice cultivars | |
| Katayama et al. | Pear (Pyrus species) genetic resources in Iwate, Japan | |
| CN113693033A (en) | Method for evaluating resistance of rice variety to southern rice black-streaked dwarf disease based on artificial inoculation | |
| CN101608211A (en) | Indoor detection method for resistance of peanuts to infection of Aspergillus flavus | |
| CN106119337A (en) | A kind of Rapid identification peanut varieties method to Aspergillus flavus resistance | |
| Dita Rodriguez et al. | Practical guidelines for early screening and field evaluation of banana against Fusarium wilt, Pseudocercospora leaf spots and drought | |
| Gradziel et al. | Resistance to brown rot disease in peach is determined by multiple structural and biochemical components | |
| CN102410950B (en) | Callosobruchus maculates standard sample and preparation method thereof | |
| CN102487630B (en) | Identification method of special type pathogenicity of fusarium oxysporum sesame | |
| CN107821342A (en) | A kind of multifibres amount, the breeding method of anti-nuclear polyhedrosis silkworm | |
| CN108359708A (en) | A method of by being inoculated with excised leaf Rapid identification plant root Disease Resistance | |
| CN101173213A (en) | Mutagenesis breeding method for high-fat dunaliella salina | |
| CN110100685B (en) | Method for identifying disease resistance of peanut net blotch by conidium inoculation | |
| CN112592956A (en) | Method for identifying resistance to soybean anthracnose | |
| CN106244632A (en) | A kind of extracting method of sesame wilt germs toxin | |
| Vannacci et al. | SEED TRANSMISSION OF FUSARIUM OXYSPORUM FSP BASILICI IN SWEET BASIL | |
| Barritt et al. | Red raspberry breeding in Washington with emphasis on fruit rot resistance | |
| Cousins et al. | A technique for screening grape germplasm for resistance to Meloidogyne incognita | |
| Venkataramanamma et al. | Screening of chickpea germplasm/genotypes against Fusarium wilt of chickpea under field and artificial condition | |
| CN108184368A (en) | The method for improving percentage of seedgermination |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Liao Boshou Inventor after: Kang Yanping Inventor after: Jiang Huifang Inventor after: Yan Liying Inventor after: Lei Yong Inventor after: Huaidongxin Inventor after: Qiu Xike Inventor after: Luo Huaiyong Inventor after: Yu Bolun Inventor before: Kang Yanping Inventor before: Liao Boshou Inventor before: Jiang Huifang Inventor before: Lei Yong Inventor before: Yan Liying Inventor before: Wan Liyun Inventor before: Huaidongxin Inventor before: Qiu Xike |
|
| CB03 | Change of inventor or designer information |