CN105219654B - One plant of aspergillus flavus strain for not producing aflatoxin and its application in aflatoxin pollution of peanuts biological control - Google Patents
One plant of aspergillus flavus strain for not producing aflatoxin and its application in aflatoxin pollution of peanuts biological control Download PDFInfo
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Abstract
The invention belongs to field of environmental biotechnology, and in particular to one plant of aspergillus flavus strain for not producing aflatoxin and its application in aflatoxin pollution of peanuts biological control.The aspergillus flavus strain for not producing aflatoxin is named as Aspergillus flavus NAFFHN396, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 28th, 2015, and preserving number is CGMCC No:10927.The present invention one plant of aflatoxin composite variety gene delection of acquisition does not produce malicious aspergillus flavus strain, the bacterial strain has significant inhibiting effect to the Output of toxin for producing malicious aspergillus flavus strain by force, biocontrol microorganisms source is provided not produce biological control of the malicious Aspergillus flavus in field from now on, the bacterium is isolated from China, there is preferable colonization ability on peanut, with potential field competitive advantage, to inhibiting to produce malicious Aspergillus flavus infecting peanut, the aflatoxin contamination reduced in peanut is of great significance.
Description
Technical field
The invention belongs to field of environmental biotechnology, and in particular to one plant of aspergillus flavus strain for not producing aflatoxin and its
Application in aflatoxin pollution of peanuts biological control.
Background technology
Aflatoxin is a kind of critically important mycotoxin for endangering China's food security.In nature, some agricultures
Crop, including corn and peanut are easy to be infected by Aspergillus flavus, and seed is caused to be polluted by aflatoxin；Other plant,
As nut fruits are also easy to be polluted by aflatoxin.The toxicity of aflatoxin is very strong, is that the toxicity that is currently known is most strong
Carcinogenicity mycotoxin, there is extremely strong murder by poisoning, long-term low dose to take the photograph for liver and central nervous system to humans and animals
Enter to cause the variation of embryo's deformity, gene and chromosomal variation, induce the cancers such as primary carcinoma of liver, gastric cancer, and it is disposable a large amount of
Intake can cause acute poisoning even dead.
Peanut is bloomed geocarpic on the ground, and it is yellow that this biological characteristics makes pod be vulnerable to during peanut growth in soil
Infecting for Aspergillus and harvest preceding aflatoxin contamination, especially damaged pod is more easy to be infected by Aspergillus flavus.It is anti-
Control the technical measures of aflatoxin pollution of peanuts including the use of aspergillus flavus resisting or the peanut varieties of drought resisting, improve cultivation step,
The environmental condition of control harvest and storage, but the aspergillus flavus resisting peanut varieties pole of domestic and international available high yield and high quality so far
Few, cultivating the effect of prevention and control often reduces because of such environmental effects, is carried out to contaminated product using physics and chemical method
Detoxification, not only of high cost but also effect are also undesirable.It is to eliminate or reduce endotoxin contamination, guarantee peanut using biological prevention
One of important technology approach of edible safety.
Currently, the most successful means of the biological prevention of aflatoxin contamination are to utilize atoxigenic Aspergillus flavus
Strain.Since the 1990s, malicious Aspergillus flavus has not been produced in the crops aflatoxin contamination prevention and control such as peanut, corn and cotton
Certain application is obtained, corn, peanut aflatoxin can be made to reduce 74.3%-99% in corn field and Peanut Fields application.Field
Between apply not toxigenic bacterium strain can not only reduce harvest before endotoxin contamination, can also reduce harvest after store during endotoxin contamination.Generation
Using the malicious most successful country of aspergillus flavus biological and ecological methods to prevent plant disease, pests, and erosion is not produced it is the U.S. in boundary, certain application has also been obtained in Australia.But it is total
It is seen on body, due to the limitation of many factors, production application is still extremely limited for the biological control of aflatoxin pollution of peanuts, and
China almost or a blank.
Invention content
The purpose of the present invention is to provide the aspergillus flavus strains that one plant is not produced aflatoxin.
The aspergillus flavus strain for not producing aflatoxin it is another object of the present invention to provide one plant is in aspergillus flavus
Application in endotoxin contamination biological control.
For achieving the above object, the technical solution adopted in the present invention is：
One plant of aspergillus flavus strain for not producing aflatoxin, which is characterized in that the Strain Designation is Aspergillus flavus
NAFFHN396 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 28th, 2015,
Depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preserving number are CGMCC No:10927.
The colonial morphology of above-mentioned Aspergillus flavus NAFFHN396 is：After 30 DEG C of dark culturing 7d, on CYA culture mediums, Huang Qu
Mould NAFFHN396 is the bacterium colony of diameter 6.5cm, and mycelia is filbert, conidium yellowish-brown, bacterium colony surface powdery；In AFPA
On solid medium, the bacterium colony back side is bright orange.
Utilize primer (CaMF and CaMR) the amplification Aspergillus flavus NAFFHN396 of the special calcium protein gene design of aspergillus flavus
DNA, and carry out sequence analysis, analysis result is shown, special calcium protein gene sequence length is 750bp, using BLAST points
Sequence and GenBank databases are compared analysis method, it is found that the affiliation of the bacterial strain and Aspergillus flavus is closest, together
Source property is up to 99% or more.The CYA solid mediums that Aspergillus flavus NAFFHN396 is cultivated 7 days are vaccinated with using the extraction of methanol method,
Extracting solution is measured using high performance liquid chromatography (HPLC), test result is shown：Aspergillus flavus NAFFHN396 does not produce yellow song
Mould toxin.
Using aflatoxin composite variety and its upstream and downstream primer that totally 30 gene nucleotide series design, to yellow bent
The DNA of mould NAFFHN396 carries out PCR amplification, and PCR amplification result is shown in that Fig. 3, the result of Fig. 3 are shown：The aflatoxin of the bacterium
12 genes of cluster deletion (the 5th~16 gene) are synthesized, the atoxigenic mechanism of Aspergillus flavus NAFFHN396 is because in DNA water
On flat, aflatoxin synthesizes 12 genes of cluster deletion, and aflatoxin synthesis is caused to be interrupted.
Applications of the above-mentioned Aspergillus flavus NAFFHN396 in aflatoxin pollution of peanuts biological control.
The present invention is by Aspergillus flavus AF2202 malicious with strong production atoxigenic Aspergillus flavus NAFFHN396 with 1：1 ratio is mixed
It cultivates 7 days on CYA solid mediums and shelled peanut after splice grafting kind, as a result shows respectively：Atoxigenic Aspergillus flavus
NAFFHN396 is 94.1% to the inhibiting rate of toxigenic bacterium Output of toxin on CYA, to toxigenic bacterium Output of toxin on shelled peanut
Inhibiting rate significantly reduces the yield of the aflatoxin of the Aspergillus flavus AF2202 of strong production poison up to 98%.
Beneficial effects of the present invention are as follows：The present invention one plant of aflatoxin composite variety gene delection of acquisition does not produce malicious Huang
Aspergillus strain, the bacterial strain have significant inhibiting effect to the Output of toxin for producing malicious Aspergillus flavus by force, malicious yellow bent not produce from now on
Biological control of the mould in field provides biocontrol microorganisms source, which is isolated from China, has on peanut and preferably colonizes energy
Power has potential field competitive advantage, and to inhibiting to produce malicious Aspergillus flavus infecting peanut, the aflatoxin reduced in peanut is dirty
Dye is of great significance.
Description of the drawings
Fig. 1 is colonial morphologies and conidium form of the Aspergillus flavus NAFFHN396 on CYA culture mediums.
Fig. 2 is the color at the bacterium colony back sides of the Aspergillus flavus NAFFHN396 on AFPA culture mediums.
Fig. 3 is the PCR amplification result that Aspergillus flavus NAFFHN396 does not produce strain system calcium albumen with other.
Fig. 4 is the phylogenetic tree of Aspergillus flavus NAFFHN396.
Fig. 5 is the HPLC measurement charts of Toxic extraction liquid on solid CYA culture mediums after Aspergillus flavus NAFFHN396 grows 7d
Spectrum.
Fig. 6 is the expansion of aflatoxin composite variety upstream gene and partially synthetic cluster gene in Aspergillus flavus NAFFHN396
Increase result.
Specific implementation mode
For a better understanding of the present invention, with reference to the embodiment content that the present invention is furture elucidated, but the present invention
Content is not limited solely to the following examples.
Embodiment 1：Separation, screening and the identification of bacterial strain
(1) peanut kernel is utilized into 70% ethyl alcohol surface sterilization 30s, impregnates seed benevolence 2min followed by 1wt%NaClO,
Sterile water washing 3 times, drains shelled peanut, shelled peanut is uniformly arranged in sterile plastic culture dish, 10/ware, between seed benevolence
Gap is kept, not closely；After culture dish equipped with seed benevolence is wrapped using parafilm films, it is carefully positioned over bottom addition moisture
Finishing box in, cover case lid, 30 DEG C of culture 7d；
(2) the yellow green conidium generated in a small amount of seed benevolence is dipped using the tweezers of sterilizing, is containing 0.1wt%'s
After fully suspending and be serially diluted in Tween-20,10 are taken-4The conidial suspension of dilution is coated with PDA plate, 30 DEG C of trainings
2d to be supported, the single bacterium colony of growth is taken and is transferred to fresh CYA and AFPA solid mediums center a little, culture medium is nature pH,
Until bacterium colony covers with tablet, the form of the bacterium colony purified, bacterium colony is for 30 DEG C of cultures：7d is cultivated on CYA solid mediums
Afterwards, NAFFHN396 is the bacterium colony of diameter 6.5cm, and mycelia is filbert, conidium yellowish-brown, bacterium colony centre of surface young pilose antler shape, side
Edge powdery, bacterium colony and conidial form are shown in Fig. 1；On AFPA solid mediums, the bacterium colony back side is bright orange, sees Fig. 2.
(3) in the colony inoculation for the purifying for obtaining step (2) to 30ml PDA culture solutions, after 30 DEG C of culture 5d, by bacterium
Clusters are taken out, drained on filter paper, are put into mortar, pour into liquid nitrogen and be fully ground into powder, and mycelia is extracted using CTAB methods
DNA utilizes the concentration of spectrophotometric determination mycelia DNA, diluted concentration to 20ng/ μ l；With reference to Aspergillus flavus calmodulin base
Nucleotide sequence design primer (CaMF, 5 ' TTTTGCATCATGAGTTGGAC3 ', CaMR, 5 ' of cause
GARTWCAAGGAGGCCTTCTC3 ') PCR amplification is carried out to mycelia DNA extracting solutions, 10XTaq is added in the system of 20 μ l
2 μ l of buffer, each 1 μ l of CaMF and CaMR of 10pmol, 2 μ l of mycelia DNA extracting solutions, 1 μ l of 1UTaq DNA polymerase,
2.5mM MgCl21 μ l, 1 μ l of 2mmol/L dNTPs, aqua sterilisa is mended to 20 μ l；Amplification condition is：Amplification condition is：94℃
3min is denaturalized；94 DEG C of 30s, 51 DEG C of 30s, 72 DEG C of 1min, 35 cycles；72 DEG C of 7min extend.Amplified production is solidifying in 1% agarose
Electrophoresis on glue, PCR amplification result are shown in Fig. 3, recycle target fragment using Takara Ago-Gel kits, are connected to carrier
On pMD-18T, transformed competence colibacillus cell DH5 α select positive colony and carry out sequencing, and sequencing results are shown：Special calcium
Protein gene sequence overall length is 750bp, and sequence and GenBank databases are compared using BLAST analytic approach, hair
Now the affiliation of the bacterial strain and Aspergillus flavus is closest, and homology is up to 99% or more.The bacterium is carried out using MEGA programs
Phylogenetic evolution analysis, and drawing system development tree, as shown in Figure 4.
It is Aspergillus flavus NAFFHN396 by the Strain Designation, is preserved in Chinese microorganism strain on May 28th, 2015
Preservation administration committee common micro-organisms center, depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation
Number be CGMCC No:10927.
(4) Aspergillus flavus NAFFHN396 is inoculated on fresh CYA solid mediums, after 30 DEG C of culture 7d, using beating
Hole device takes mycelia block, is placed in centrifuge tube respectively from center 1cm, 2cm, 3cm, smashs mycelia block to pieces using pipette tips, is added 1.5ml's
Methanol, ultrasonication 15min, 12,000rpm/min centrifuge 5min, draw supernatant, are filtered using 0.45 μm of filter
Afterwards, the tool lid that filtrate is collected in 1.8ml measures in bottle, and the measurement that aflatoxin is carried out using HPLC is (reversed using C18
Column, mobile phase are 45% methanol/water, flow velocity 0.7ml/min), sample size is 10 μ l, quantified by external standard method (aflatoxin mark
The appearance time of quasi- product B2 is 10.8min, and the appearance time of B2 is 13.4min), the content of sample aflatoxin is calculated, it is yellow
The HPLC result collection of illustrative plates of aspertoxin is shown in Fig. 5, from fig. 5, it can be seen that Aspergillus flavus NAFFHN396 is at 10.8 and 13.4min
All without peak value, show that the bacterial strain does not produce aflatoxin, the content of aflatoxin is 0ng/g.
(5) it (is shown in Table respectively according to aspergillus flavus toxin composite variety and its upstream and downstream special primer that totally 30 genes design
1) PCR amplification, is carried out to Aspergillus flavus NAFFHN396DNA using the method for step (3), the amplification of portion gene is shown in figure
6, Fig. 6 result illustrates：Aflatoxin synthesis 12 genes of cluster deletion (the 5th~16 gene) on the bacterium genome.
Exactly because the atoxigenic mechanism of Aspergillus flavus NAFFHN396, on DNA level, aflatoxin synthesizes 12 bases of cluster deletion
Cause causes aflatoxin synthesis to be interrupted.
1 specific primer sequences of table
| Gene | Sense primer | Downstream primer |
| C1 | CGTTCCAGTAGTTCGTATCG | CATCGTAAACGTTGACACAG |
| C2 | TCGCCTTGTTCTCGCTATAC | ACACCTGATAGCGAGAGTTC |
| C3 | GCGATCTGTAACACTACACA | GCCATACGATTCCCAAGTCT |
| norB-cypA | GTGCCCAGCATCTTGGTCCA | AGGACTTGATGATTCCTCGTC |
| aflT | ATGACATGCTAATCGACGAG | AGGCGCATGCTACGGATC |
| pksA(pksL) | ACTTTGAGGGCGTTCTGTGC | CTTTCGGTGGTCGGTGATTC |
| nor1 | AGCACGATCAAGAGAGGCTC | GATCTCAACTCCCCTGGTAG |
| fasA(hexA) | TCCTATCCAGTCCACCTCGTA | CACATCTTTGTCTTGCCCGC |
| fasB(hexB) | ACAATCGAATGACAACACTGC | CCACCGAATCCACTACCTACA |
| aflR | ATGGTCGTCCTTATCGTTCTC | CCATGACAAAGACGGATCC |
| aflJ | CTTCAACAACGACCCAAGGTT | AGATGAGATACACTGCCGCA |
| adhA | CCTCGTGGGAGAGCCAAATC | GGAGCAAGAAGGTTACAGCG |
| estA | CGATGGGACTGACGGTGATT | ACCACGCCGCTGACTTTAT |
| norA | GTGTTCGTGTGTCGCCCTTA | GTCGGTGCTTCTCATCCTGA |
| ver1 | CATCGGTGCTGCCATCGC | CCTCGTCTACCTGCTCATCG |
| verA | CCGCAACACCACAAGTAGCA | AAACGCTCTCCAGGCACCTT |
| avnA | GCGATAGAACTGACAAAGGCA | GAATGAGTCTCCAAAGGCGAG |
| verB | TTCAGTGACAAAGGTCTTCGC | GGCAGCGTT ATTGAGCATCT |
| avfA | ATTCAAATCCTCGTTCGGTCG | TAGCCCGTTGGTTGTGTTCC |
| omtB | ACAGACGATGTGGGCAAACG | ACGCAGTCCTTGTTAGAGGTG |
| omtA | CAGGATATCATTGTGGACGG | CTCCTCTACCAGTGGCTTCG |
| ordA | AAGGCAGCGGAATACAAGCG | ACAAGGGCGTCAATAAAGGGT |
| vbs | AACGAGCAGCGTAAGGGTCT | TCAGCCAGAGCATACACAGTG |
| cypX | GGAGCCTACCATTCGCAACA | GGCTTTGACGAACAGATTCCG |
| moxY | TGCTACTGGAACGAAGACCG | CGACGACAACCAAACGCAA |
| ordB | GCTGCTACTGGAATGAAGACC | ATGCGACGACAACCAAACG |
| hypA | CGCAAGACGGCAGAGATACT | GCTCCTTCAGTTCCACACCA |
| nadA | TGACGAGGCCTGCGAGCTGT | AAGCCTCTTCAGAACGGTCA |
| hexA | TGTCCTCACCTCTGGCGTAT | AGACCAACCACTCTTATGGGC |
| glcA | AGACACAGTCATCGCCTGTT | GGTGCGAATAGGTGCAGGTA |
| sugR | TCAGCTGAAGCGCTCGAGAG | GTATTGCCGCACTATGTATG |
Embodiment 2：Applications of the Aspergillus flavus NAFFHN396 in aflatoxin pollution of peanuts biological control
By the Aspergillus flavus AF2202 of atoxigenic Aspergillus flavus NAFFHN396 and production poison 30 on PDA solid mediums
After DEG C culture 7d, 0.1wt%Tween-20 aqueous suspension conidiums, blood counting chamber counts conidial quantity, will not produce
Toadstool 1*10 consistent with the adjusting of the concentration of toxigenic bacterium4, ratio 1:1, it is seeded in CYA solid mediums after mixed in equal amounts
Centre, or it is inoculated into the peanut kernel surface of surface sterilization, seed benevolence is fully shaken, conidium is made uniformly to be attached on seed benevolence
Surface, while setting up the CYA solid mediums for being only inoculated with AF2202 or peanut kernel as a contrast, 30 DEG C of moisturizing culture 7d.
Using the aflatoxin on 1 step of embodiment (4) the method extraction CYA solid mediums；Aspergillus flavus poison in peanut kernel
Element extracting method be：Alcohol is sprayed to the peanut kernel surface of inoculation in draught cupboard, then in 110 DEG C of oven for baking
1h fully inactivates the Aspergillus flavus in seed benevolence, then grinds sample using high speed disintegrator in draught cupboard, thereafter with reference to national standard
NY/T1286-2007 method extraction peanut kernel in aflatoxin (methanol aqueous solution of 55% volumetric concentrations of 50ml,
The petroleum ether of 15ml 100%), and concentrate, reversed C18 post separations, post-column derivation, in excitation wavelength 365nm, wavelength of fluorescence
440nm is detected, quantified by external standard method, calculates the content of sample aflatoxin, and acquisition does not produce malicious Aspergillus flavus NAFFHN396 to production
The inhibiting rate of the content of toxins of toadstool AF2202.Aspergillus flavus NAFFHN396 is on CYA to the inhibiting rate of toxigenic bacterium Output of toxin
It is 94.1%, to the inhibiting rate of toxigenic bacterium Output of toxin up to 98% on shelled peanut, the aspergillus flavus of strong production poison can be significantly reduced
The yield of the aflatoxin of bacterium AF2202.Aspergillus flavus NAFFHN396 provides for Aspergillus flavus field biological control from now on
The source of biocontrol microorganisms, the bacterium are isolated from China, have preferable colonization ability on peanut, have the competition of potential field excellent
Gesture, to inhibiting to produce malicious Aspergillus flavus infection peanut, the aflatoxin contamination reduced in peanut is of great significance.
Obviously, above-described embodiment be only intended to clearly illustrate made by example, and not limitation to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And the obvious variation or change therefore amplified
It moves within still in the protection domain of the invention.
Claims (2)
1. one plant of aspergillus flavus strain for not producing aflatoxin, which is characterized in that the Strain Designation is Aspergillus flavus
NAFFHN396, preserving number are CGMCC No:10927.
2. applications of the Aspergillus flavus NAFFHN396 in aflatoxin pollution of peanuts biological control described in claim 1.
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| CN107142217B (en) * | 2016-12-06 | 2019-12-31 | 青岛至成生物技术有限公司 | Biopesticide for reducing aflatoxin content of crops |
| CN107245453B (en) * | 2017-06-02 | 2020-06-09 | 中国农业科学院农产品加工研究所 | Aflatoxin-producing aspergillus flavus and application thereof in degrading aflatoxin |
| CN107177516A (en) * | 2017-07-07 | 2017-09-19 | 福建农林大学 | A kind of aspergillus flavus avirulent strain and its application of preventing and treating aspergillus flavus pollution |
| CN109913375B (en) * | 2019-04-03 | 2020-11-10 | 吉林大学 | Aspergillus flavus without producing toxin, composition containing aspergillus flavus and application of aspergillus flavus |
| CN110074140B (en) * | 2019-05-28 | 2020-06-09 | 山东省花生研究所 | Biocontrol microbial inoculum for producing aspergillus flavus, preparation method and application thereof |
| CN110973163B (en) * | 2019-12-18 | 2021-04-09 | 吉林大学 | Liquid microbial inoculum for biologically preventing, controlling and spraying aflatoxin |
| CN113355246B (en) * | 2021-06-16 | 2022-05-24 | 中国农业科学院油料作物研究所 | Aspergillus flavus SX0104 without producing toxicity and application thereof |
| CN113322189B (en) * | 2021-06-16 | 2022-03-11 | 中国农业科学院油料作物研究所 | Aspergillus flavus HuBXY33 without producing toxicity and application thereof |
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