CN107870224A - A kind of field identification method for evaluating corn aspergillus flavus infection resistance ability - Google Patents

A kind of field identification method for evaluating corn aspergillus flavus infection resistance ability Download PDF

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CN107870224A
CN107870224A CN201711074734.4A CN201711074734A CN107870224A CN 107870224 A CN107870224 A CN 107870224A CN 201711074734 A CN201711074734 A CN 201711074734A CN 107870224 A CN107870224 A CN 107870224A
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aspergillus flavus
corn
aspergillus
identification method
spore
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齐耀程
丁舰舟
阮龙
左晓龙
钱益亮
任四海
王俊
张玮
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INSTITUTE OF TOBACCO ANHUI ACADEMY OF AGRICULTURAL SCIENCES
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    • G01N2333/38—Assays involving biological materials from specific organisms or of a specific nature from fungi from Aspergillus
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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Abstract

The present invention provides a kind of field identification method for evaluating corn aspergillus flavus infection resistance ability, comprises the following steps：1) it is inoculated with Aspergillus flavus on Cha Shi solid mediums；2) media surface aspergillus spore is scraped, is prepared into Aspergillus flavus spore suspension with distilled water, concentration is 1.0 × 106Individual/ml；3) aspergillus spore is inoculated with using graded syringe in 15~20 days after maize ear pollination, is inoculated with 0.5ml spore suspensions respectively at three positions of corncob upper, middle and lower, 45 degree of syringe needle is stabbed diagonally in corncob bract.4) by fruit ear onset area and aflatoxin B1 content to determine resistance capacity of the corn inbred line to Aspergillus flavus infection.Compared with prior art, the present invention relies on maize field growth natural environment, meets agricultural production rule, and qualification result is accurate, and stability is high, and health is had no adverse effects.

Description

A kind of field identification method for evaluating corn aspergillus flavus infection resistance ability
Technical field
The present invention relates to agricultural technology field, Field inoculation method and evaluation aspergillus flavus-resistance more particularly to corn aspergillus flavus The authentication method of mould infection ability.
Background technology
China is corn planting big country, and maize growth process and the climate resources of planted in different ecological areas differ greatly.Huang-Huai-Hai Rain hotsync during corn maturation, feature are temperature height, high humidity, and fruit ear is subject to aspergillus flavus in grouting and dehydration Pollution, easily accumulates a large amount of aflatoxin in field, influences corn quality, endanger human and livestock health.Aflatoxin (Aflatoxin, abbreviation AF) is the general name of a kind of compound with similar structure, is aspergillus flavus (Aspergillus Flavus), the aspergillus such as aspergillus parasiticus (Aspergillus parasiticus), collection honeybee aspergillus (Aspergillus nomius) There is the secondary metabolic product of bioactivity, toxin out separated at present is respectively designated as aflatoxin caused by bacterium 12 kinds of B1, B2, G1, G2 etc., wherein B1 toxicity is most strong, and the change that all toxicant Poisonings found so far are most strong Carcinogenic substance is learned, its toxicity is far above cyanide, organic agricultural chemicals and arsenide, is to cause one of liver cancer, main pathogen of nasopharyngeal carcinoma, I class carcinogens are classified as by international cancer research institution (IARC).Food and feed structure of the aflatoxin to corn for raw material Into serious potential safety hazard, with the expansion of maize sown area, the outburst of aspergillus flavus disease may make grain security by disaster Property consequence.
Aspergillus flavus is weak parasite, is not easy spontaneous infection under complete corn kernel natural conditions, often occurs together in disease pest Infringement.Field corn fruit ear Aspergillus flavus inoculation common method is by the fruit during aspergillus spore suspension and maize growth Fringe contact causes its infection morbidity, and inoculation technique is divided into two kinds of nocuity and non-nocuity.The inoculation technique of nocuity includes knife The method of drawing, draw point method and row's skill of handling needles etc..Non- nocuity inocalation method include aspergillus spore suspend liquid top injection, sprinkling filigree and Silk channel inoculation etc..Henry, W.B compared Georgia and two kinds of state of Mississippi inoculation skill in 2010 with experiment Art (side pin and knife are drawn) and different vaccination rate.Side pin technology is used in the research of the state of Mississippi.The side skill of handling needles, which uses, is equipped with No. 14 The tree mark rifle of syringe needle is inoculated with the fruit ear after pollinating seven days, and pin is inserted in fruit ear through bract, and injection aspergillus spore hangs Supernatant liquid.Method is drawn using knife in the research in Georgia area, uses the common paring knife of a 6.4cm blade.Fruit ear is pollinated 14 days afterwards, aspergillus spore suspension is dipped with vaccinating lancet, vaccinating lancet is inserted in bract, and this technology would generally damage 3~5 Seed.
External Field inoculation method differs greatly in aspergillus flavus inoculation time, fruit ear position and vaccination ways etc., hair Sick effect is totally different, and repeatability is bad, has a strong impact on scientific research accuracy.It is yellow bent that such inocalation method is difficult to accurate quantification inoculation Mould spore, later stage statistical result also lack accuracy.The inoculation position of some inocalation methods be usually in fringe tip area of liability, under The infection in portion is often ignored.And such as filigree the methods of spraying is even more that can not directly act on seed, it is difficult to have it is obvious directly Pathogenic relation.The domestic research in laboratory research corn kernel mechanism of causing a disease and resistance mechanism is more, field research method The inoculation method of Primary Reference peanut, have not yet to see the complete feasible corn aspergillus flavus Resistence research method of report.
The content of the invention
In order to overcome domestic and international the deficiencies in the prior art, improve field research corn aspergillus flavus infection resistance morbidity stability and Accuracy, efficiently identify the resistance capacity of corn germplasm.The invention provides a kind of simple, efficient detection corn aspergillus flavus to resist The authentication method of sexuality, i.e. 3 spiral inocalation methods：Maize ear pollination after use graded syringe, on corncob, in, Inoculating spores suspension is distinguished at lower three positions, and syringe needle is stabbed diagonally in corncob bract, punctures single seed, spiral inoculation, makes Three vaccination downward spirals are equally spaced.Used technical scheme is：
1) Aspergillus flavus (numbering is inoculated with Cha Shi solid mediums：3.4410, protected purchased from China General Microbiological strain Hide administrative center), 30 DEG C are cultivated 7 days, and Aspergillus flavus spore is produced after growth；
2) Aspergillus flavus spore is eluted using sterile purified water, is prepared into Aspergillus flavus spore suspension, concentration 1.0 × 106/ml；
3) maize ear was pollinated in 15~20 days, in daily same time period, using graded syringe by aspergillus spore 45 degree of suspension is stabbed diagonally in corncob bract, it is desirable to is punctured seed, is rotated inoculation respectively at three positions of corncob upper, middle and lower 0.5ml spore suspensions.
4) each corn inbred line harvests whole fruit ears, the onset area that measurement Aspergillus flavus is infected on the 40th day in inoculation simultaneously To distinguish rank.
5) after corn ear dries, sampled centered on each vaccination to ambient radiation, each vaccination samples 4g, often Individual fruit ear samples 12 ± 0.1g.The quantitative detection of aflatoxin B1 is carried out with comminuted powder.Compared with prior art, it is of the invention The beneficial effect of " 3 spiral inocalation methods " is：
1. implementation condition of the present invention is natural environment, the process of Aspergillus flavus infection fruit ear is met big by natural environment influence Field test requirements document.
2. the committed step of corn material inoculation pathogenic process all has regulation and control standard, aspergillus spore suspension is such as inoculated with Measure controllable, inoculation position is reasonably distributed, and Aspergillus flavus time of infection is identical after inoculation, and onset condition is natural environment, meets base Because of the oranon that type is unique difference.
3. this method test repeatability is good, the onset area of same corn inbred line ensure that experiment weight without significant difference Existing property, reduces systematic error.
4. this method effectively prevent the position seed of fruit ear upper, middle and lower three because water content difference causes infect anti-to corn The erroneous judgement of ability, onset area and content of toxins are calculated using triadic mean value method.
5. on the one hand this method can prevent top spore suspension from being flowed out from the inoculation hole of bottom, inoculating spores content is caused Difference；On the other hand be advantageous to accurate measurement onset area, avoid neighbouring vaccination bacterium circle from mutually overlapping mutually and cause area parameters to miss Difference, judge the resistance capacity of corn by accident.
6. this method is seed injury type inocalation method, start to be inoculated with after grain endosperm is formed after 15 days, now endosperm Moisture is high, and aspergillus spore can be bred rapidly under preference temperature using seed nutritional ingredient, therefore the spore being inoculated with The spore quantity of suspension can be reduced to 1.0 × 10 compared to other method6Individual/ml.
7. in terms of aflatoxin content two under the onset grade and unit quality grain that fruit ear onset area determines altogether With resistance capacity difference in size of the corn inbred line to Aspergillus flavus for determining different genotype.
Brief description of the drawings
Fig. 1 is morbidity design sketch of three kinds of inoculation methods to same corn inbred line, wherein (a) figure is top injection inoculation Method；(b) figure is row's pin inocalation method；(c) figure is 3 spiral inocalation methods.
Fig. 2 is 3 spiral inocalation methods to different resistant corn materials morbidity stability comparison diagram.
Embodiment
Present embodiment is the complete procedure identified from corn inbred line inoculation aspergillus spore to resistance capacity, wherein relating to And the method and deriving method arrived is all in protection domain.
The aspergillus flavus inoculation method miospore suspension concentration parameter optimization of embodiment 1
The present embodiment is intended to inquire into the most suitable spore suspension concentration that corn ear inoculation Aspergillus flavus spore causes to fall ill. Experiment process is as follows：1) streak inoculation Aspergillus flavus (3.4410) on Cha Shi solid mediums；2) 28 DEG C culture 7 days after, will Media surface aspergillus spore distillation water elution, filtering are prepared into Aspergillus flavus spore suspension.Set five spores dense Gradient is spent, concentration is followed successively by 1.0 × 104Individual/ml, 1.0 × 105Individual/ml, 1.0 × 106Individual/ml, 1.0 × 107Individual/ml, 1.0 × 108Individual/ml.3) aspergillus spore is inoculated with using syringe, in corncob upper, middle and lower three in 15~20 days after maize ear pollination Individual position, each 0.5ml of spore suspension of 3 spiral inoculation various concentrations, 45 degree of syringe needle are stabbed diagonally in corncob bract, pierced Break single seed.4) selecting three self-mating systems, susceptible material PI143, for common resistant material to close 17, height is anti-as experimental subjects Material is H152.5) resistance capacity that corn kernel spreads to Aspergillus flavus circle is determined by fruit ear Aspergillus flavus infection area.
In the present embodiment, control spore suspension maximum concentration 1.0 × 108Individual/ml, this is that conventional spore is reported in document Concentration, because this experiment uses injury formula inoculation method, i.e., to stab 1 seed as cost to improve the propagation of aspergillus spore Condition, therefore aspergillus spore concentration when can suitably reduce inoculation.
As shown in table 1, after being inoculated with 40 days, high anti-material H152 shows stabilization under various concentrations gradient, substantially in 1 grade of water It is flat, the high anti-characteristic of performance.Susceptible material PI143 is 1.0 × 1044.1 grades are shown as during individual/ml concentration, is raised with concentration, and 1.0×1066 grades are reached during individual/ml concentration.The corn material of common resistance closes 17 1.0 × 107Individual/ml and 1.0 × 108 During individual/ml concentration, because inoculating spores radix is larger, the susceptible rank of corn inbred line of its common resistance is excessive, covers it The true resistance against diseases of medium resistance, and 1.0 × 106Under individual/ml inoculum density, 3.7 grades are reached, middle water resistant can be embodied It is flat.In summary, 1.0 × 106Under individual/ml inoculum density, the resistance against diseases of the self-mating system of each resistance rank can obtain Preferably embody.
Influence of the different aspergillus spore suspension concentrations of table 1 to the susceptible rank of corn inbred line
2 different corn inbred lines of embodiment are identified Aspergillus flavus infection resistance capacity
The present embodiment is intended to inquire into different corn inbred lines in field test and Aspergillus flavus infection resistance capacity is identified.Experiment Process is as follows：
1) streak inoculation Aspergillus flavus (3.4410) on Cha Shi solid mediums；
2) 28 DEG C culture 7 days after, by aspergillus spore with distillation water elution, filtering be prepared into Aspergillus flavus spore suspension Liquid, it is 1.0 × 10 to prepare concentration6Individual/ml；
3) aspergillus spore is inoculated with using syringe, in corncob upper, middle and lower three in 15~20 days after maize ear pollination Individual position, each 0.5ml of spiral inoculating spores suspension, 45 degree of syringe needle are stabbed diagonally in corncob bract, puncture single seed.
4) the significant corn inbred line of Resistant Difference 12 is selected to be used as test material；
5) each corn inbred line collects morbidity fruit ear on the 40th day in inoculation, and measurement Aspergillus flavus infects circle area, and measuring and calculating is not Bacterium circle with self-mating system fruit ear averagely infects rank.Decision method is as follows, and the vaccination bacterium circle average area S of fruit ear three is 0<S≤ 0.20cm2It is denoted as " 1 "；The vaccination average area S of fruit ear bacterium circle three is 0.20<S≤0.79cm2It is denoted as " 2 "；Fruit ear bacterium circle three connects Kind point average area S is 0.79<S≤1.77cm2It is denoted as " 3 "；The vaccination average area S of fruit ear bacterium circle three is 1.77<S≤ 3.14cm2It is denoted as " 4 "；The vaccination average area S of fruit ear bacterium circle three is 3.14<S≤4.91cm2It is denoted as " 5 "；Fruit ear bacterium circle three connects Kind point average area S is 4.91<S≤+ ∞ is denoted as " 6 ".
6) corn ear is sampled centered on each vaccination to ambient radiation, and each vaccination samples 4g, each fruit ear Sample 12 ± 0.1g.Adsorb (ELISA) method using solid-phase enzyme-linked immune, by aspergillus flavus resisting toxin B1 antibody and enzyme-labelled antigen, The competitive immunoreaction of determined antigen and the Catalytic color reaction of enzyme detect the content of aflatoxin B1.
As shown in table 3, corn Field inoculation Aspergillus flavus, content and the onset grade of aflatoxin B1 are in extremely significantly just It is related.Different corn inbred line Resistance to Aspergillus flavus invasion relatively in, Shen 135, the onset grade highest of PI143 self-mating systems, H152, the onset grade of red 340 self-mating system Aspergillus flavus are minimum.According to documentation standards, the content of aflatoxin B1 is more than 20 μ G/g is the corn inbred line that production poison is heavier and seriously contaminates.Therefore, the corn inbred line of different resistance capacities can be passed through Bacterium circle area is infected tentatively to screen, and infecting bacterium circle average area approximation or infecting the equal self-mating system of bacterium circle rank to pass through Aflatoxin B1 content accurately distinguishes, and the method is applied to the screening of the disease-resistant self-mating system of crop field high flux corn.
The disease-resistant partition of the level table of the corn inbred line of table 2
State of an illness rank 0~1 1~2 2~3 3~4 4~5 6
Morbidity radius (cm) 0~0.25 0.25~0.5 0.50~0.75 0.75~1.00 1.00~1.25 >1.25
Onset area (cm2) <0.20 0.79 1.77 3.14 4.91 >4.91
The inoculation method difference corn inbred line Resistant Difference analysis of the same race of table 3
The top injection inoculation method of embodiment 3, row's pin inocalation method and 3 spiral inocalation methods implement comparison in difference
The present embodiment is intended to contrast infect effect of three kinds of Aspergillus flavus inoculation methods to corn inbred line, and optimization obtains most Good inoculation method.
Method one, top injection are non-nocuity inocalation method, are the inoculum concentration phase for ensureing three kinds of methods in the present embodiment Together, the 15th day after fruit ear pollination, by aspergillus spore suspension that 1.5ml concentration is 1.0 × 106/ml from top filigree Positive middle part is injected into bract, avoids damage to top seed.
Method two, row's pin inocalation method are that it is 1.0 × 106/ml to dip concentration using syringe needle the 15th day after fruit ear pollination Aspergillus spore suspension, inoculation is needled into fruit ear seed, 3 pins of continuous equidistant thorn on same vertical line, as row's pin Inoculation feature, it is desirable to each pin punctures 1 seed.
Method three, 3 spiral inocalation method methods as described in Example 2 are inoculated with.
Three kinds of inoculation methods form morbidity effect as shown in figure 1, (a) figure is top injection inoculation method, and the method is non-wound Evil property inocalation method, the top few seed for showing as corn ear are infected, typically by filigree silk position fall ill, and face Smaller, the poor repeatability of product.(b) figure is row's pin inocalation method, and this method causes wound by puncturing a seed, increases beneficial to aspergillus spore Grow, but because inoculum concentration is limited, the morbidity bacterium circle of fruit ear is small, and occurring degree is low, easily covers true morbidity effect, therefore not The resistance against diseases with quantitative description corn inbred line can effectively be embodied.(c) figure is 3 spiral inocalation methods, and this method is on single fruit fringe Divide the inoculation of three positions, prominent phenotypic advantage is that the bacterium circle at three positions is extended fully without overlapping, can obtained really averagely Bacterium circle area, reproducible, deciding grade and level is accurate.
Fig. 2 shows that 4 kinds of different 3 spirals inoculation aspergillus flavus sequela situations of resistant corn kinds confrontation are relatively stable.
Table 4 shows, what 3 spiral inocalation methods can substantially identify 5 kinds of corn inbred lines infects rank, filters out to Huang The best H152 self-mating systems of Aspergillus resistance.
Finally drawn a conclusion with reference to table 4, Fig. 1 and Fig. 2, the corn inbred line of 3 spiral method inoculations is than top injection inoculation Method and row's pin stab inoculation are easier to identify the resistance capacity of corn germplasm, and this method is simple to operate, efficient, and experiment is accurate Property and repeatability it is higher.
The influence that the method for the different vaccination Aspergillus flavus of table 4 infects to corn inbred line

Claims (7)

1. a kind of field identification method for evaluating corn aspergillus flavus infection resistance ability, it is characterised in that be inoculated with using 3 spirals Method is inoculated with, to identify capacity variance of the different corn inbred lines in Resistance to Aspergillus flavus invasion.
2. a kind of field identification method of corn aspergillus flavus infection resistance ability, it is characterised in that comprise the following steps：
1) it is inoculated with Aspergillus flavus on Cha Shi solid mediums, culture is to producing a large amount of Aspergillus flavus spores；
2) aspergillus spore is washed with deionized water de-, is prepared into Aspergillus flavus spore suspension；
3) aspergillus flavus is inoculated with fruit ear seed by 3 spiral inocalation methods, passes through fruit ear onset area and aflatoxin B1 Content determines the aspergillus flavus resistance level of corn inbred line.
3. a kind of field identification method of corn aspergillus flavus infection resistance ability according to claim 1 and 2, its feature exist In 3 spiral inocalation methods are：Graded syringe is used after maize ear pollination, at the position of corncob upper, middle and lower three Inoculating spores suspension, syringe needle are stabbed diagonally in corncob bract respectively, are punctured single seed, spiral inoculation, are made three vaccinations Downward spiral is equally spaced.
A kind of 4. field identification method of corn aspergillus flavus infection resistance ability according to claim 2, it is characterised in that institute State different corn inbred lines female fringe pollinate the 15th~20 day in carry out identical standard inoculation.
A kind of 5. field identification method of corn aspergillus flavus infection resistance ability according to claim 2, it is characterised in that institute Aspergillus spore suspension concentration is stated as 1.0 × 105~1.0 × 106Individual/ml.
A kind of 6. field identification method of corn aspergillus flavus infection resistance ability according to claim 2, it is characterised in that institute State corn ear and carry out Resistance Identification evaluation within the 40th~45 day in each inoculation Aspergillus flavus spore suspension.
A kind of 7. field identification method of corn aspergillus flavus infection resistance ability according to claim 2, it is characterised in that with The aspergillus flavus resisting Toxin producing C of the aflatoxin B1 content evaluation corn inbred line of unit mass seed powder.
CN201711074734.4A 2017-11-03 2017-11-03 A kind of field identification method for evaluating corn aspergillus flavus infection resistance ability Pending CN107870224A (en)

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
CN109694900A (en) * 2019-02-19 2019-04-30 四川省农业科学院植物保护研究所 A kind of indoor rapid identification method of maize diseases resistance
CN110132963A (en) * 2019-04-26 2019-08-16 安徽省农业科学院烟草研究所 A kind of method of Rapid identification waxy corn germplasm
CN111748603A (en) * 2020-08-06 2020-10-09 东北农业大学 Artificial inoculation identification method for stem basal rot resistance of corn variety

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CN101603073A (en) * 2009-07-10 2009-12-16 山东省花生研究所 Field identification method for peanut aspergillus flavus infection resistance
CN105695556A (en) * 2016-02-18 2016-06-22 深圳市依波特科技有限公司 Screw inoculation method

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109694900A (en) * 2019-02-19 2019-04-30 四川省农业科学院植物保护研究所 A kind of indoor rapid identification method of maize diseases resistance
CN109694900B (en) * 2019-02-19 2022-05-06 四川省农业科学院植物保护研究所 Indoor rapid identification method for corn disease resistance
CN110132963A (en) * 2019-04-26 2019-08-16 安徽省农业科学院烟草研究所 A kind of method of Rapid identification waxy corn germplasm
CN111748603A (en) * 2020-08-06 2020-10-09 东北农业大学 Artificial inoculation identification method for stem basal rot resistance of corn variety
CN111748603B (en) * 2020-08-06 2023-07-14 东北农业大学 Method for identifying stem rot resistance patient of corn variety by artificial inoculation

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