CN105219654A - The aspergillus flavus strain of aflatoxin and the application in aflatoxin pollution of peanuts biological control thereof are not produced in one strain - Google Patents
The aspergillus flavus strain of aflatoxin and the application in aflatoxin pollution of peanuts biological control thereof are not produced in one strain Download PDFInfo
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Abstract
The invention belongs to field of environmental biotechnology, be specifically related to a strain and do not produce the aspergillus flavus strain of aflatoxin and the application in aflatoxin pollution of peanuts biological control thereof.The described aspergillus flavus strain called after Aspergillus flavus NAFFHN396 not producing aflatoxin, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 28th, 2015, does is preserving number CGMCC? No:10927.What the present invention obtained a strain aflatoxin composite variety genetically deficient does not produce malicious aspergillus flavus strain, this bacterial strain has significant restraining effect to the strong Output of toxin producing malicious aspergillus flavus strain, biocontrol microorganisms source is provided in the biological control in field for not producing malicious Aspergillus flavus from now on, this bacterium is separated from China, peanut has good colonization ability, have potential field competitive edge, produce malicious Aspergillus flavus infecting peanut to suppression, the aflatoxin contamination reduced in peanut is significant.
Description
Technical field
The invention belongs to field of environmental biotechnology, be specifically related to a strain and do not produce the aspergillus flavus strain of aflatoxin and the application in aflatoxin pollution of peanuts biological control thereof.
Background technology
Aflatoxin is a kind of very important mycotoxins of harm China food safety.At occurring in nature, some farm crop, comprise corn and peanut easily infects by Aspergillus flavus, cause seed to suffer the pollution of aflatoxin; Other plant, as nuts is also easily subject to the pollution of aflatoxin.The toxicity of aflatoxin is very strong, it is the carinogenicity mycotoxins that toxicity known is at present the strongest, it has extremely strong murder by poisoning to the liver of humans and animals and central nervous system, long-term low dose is taken in can cause the variation of embryo's deformity, gene and chromosomal variation, bring out the cancer such as primary hepatocarcinoma, cancer of the stomach, and disposable a large amount of absorption can cause acute poisoning even dead.
Peanut is bloomed on the ground geocarpy, and this biological characteristics makes pod during peanut growth, be vulnerable to infecting of Aspergillus flavus in soil and gather in the crops front aflatoxin contamination, and especially damaged pod is more easily infected by Aspergillus flavus.The technical measures of prevention and control aflatoxin pollution of peanuts comprise the peanut varieties utilizing aspergillus flavus resisting or drought resisting, the envrionment conditions improved cultivation step, control results and storage, but the aspergillus flavus resisting peanut varieties of high yield and high quality available both at home and abroad is so far few, the effect of cultivation prevention and control often reduces because of such environmental effects, adopt physics and chemistry method to carry out detoxification to contaminated product, not only cost is high but also effect is also undesirable.Biological prevention is utilized to be eliminate or reduce one of endotoxin contamination, the important technology approach ensureing peanut edible safety.
At present, the most successful means of biological prevention of aflatoxin contamination utilize atoxigenic aspergillus flavus strain.Since the nineties in 20th century, do not produce malicious Aspergillus flavus and obtain certain application in the crop aflatoxin contamination prevention and control such as peanut, corn and cotton, use at corn field and Peanut Fields and corn, peanut aflatoxin can be made to reduce 74.3%-99%.Not toxigenic bacterium strain is used in field not only can reduce the front endotoxin contamination of results, also can reduce the endotoxin contamination of the rear storage period of results.Utilizing in the world and not producing the most successful country of malicious flavus biological and ecological methods to prevent plant disease, pests, and erosion is the U.S., have also been obtained certain application in Australia.But on the whole, the biological control of aflatoxin pollution of peanuts is due to the restriction of many factors, and production application is still very limited, and in China almost or a blank.
Summary of the invention
A strain is the object of the present invention is to provide not produce the aspergillus flavus strain of aflatoxin.
Another object of the present invention is the application of aspergillus flavus strain in aflatoxin pollution of peanuts biological control providing a strain not produce aflatoxin.
For achieving the above object, the technical solution adopted in the present invention is:
The aspergillus flavus strain of aflatoxin is not produced in one strain, it is characterized in that, this Strain Designation is Aspergillus flavus NAFFHN396, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 28th, 2015, depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, its preserving number is CGMCCNo:10927.
The colonial morphology of above-mentioned Aspergillus flavus NAFFHN396 is: after 30 DEG C of dark culturing 7d, on CYA substratum, Aspergillus flavus NAFFHN396 is the bacterium colony of diameter 6.5cm, and mycelia is filbert, conidium tawny, bacterium colony surface powdery; On AFPA solid medium, the bacterium colony back side is bright orange.
The primer (CaMF and CaMR) utilizing flavus special calcium bar protein gene to design increases the DNA of Aspergillus flavus NAFFHN396, and carry out sequential analysis, analytical results shows, special calcium bar protein gene sequence length is 750bp, BLAST analytical method is adopted sequence and GenBank database to be compared, find that the sibship of this bacterial strain and Aspergillus flavus is closest, homology is up to more than 99%.Utilize methyl alcohol method to extract and be vaccinated with the CYA solid medium that Aspergillus flavus NAFFHN396 cultivates 7 days, utilize high performance liquid chromatography (HPLC) to measure extracting solution, test result shows: Aspergillus flavus NAFFHN396 does not produce aflatoxin.
Utilize the primer of aflatoxin composite variety and upstream and downstream thereof totally 30 gene nucleotide series design, pcr amplification is carried out to the DNA of Aspergillus flavus NAFFHN396, pcr amplification the results are shown in Figure 3, the result display of Fig. 3: the aflatoxin composite variety of this bacterium lacks 12 genes (5th ~ 16 genes), the atoxigenic mechanism of Aspergillus flavus NAFFHN396 is because on DNA level, aflatoxin composite variety lacks 12 genes, causes aflatoxin to synthesize and interrupts.
The application of above-mentioned Aspergillus flavus NAFFHN396 in aflatoxin pollution of peanuts biological control.
Atoxigenic Aspergillus flavus NAFFHN396 is produced malicious Aspergillus flavus AF2202 to cultivate 7 days respectively on CYA solid medium and Semen arachidis hypogaeae after the combined inoculation of 1:1 ratio with strong by the present invention, result shows: atoxigenic Aspergillus flavus NAFFHN396 is 94.1% to the inhibiting rate of toxigenic bacterium Output of toxin on CYA, Semen arachidis hypogaeae reaches 98% to the inhibiting rate of toxigenic bacterium Output of toxin, significantly reduces strong output of producing the aflatoxin of the Aspergillus flavus AF2202 of poison.
Beneficial effect of the present invention is as follows: what the present invention obtained a strain aflatoxin composite variety genetically deficient does not produce malicious aspergillus flavus strain, this bacterial strain has significant restraining effect to the strong Output of toxin producing malicious Aspergillus flavus, biocontrol microorganisms source is provided in the biological control in field for not producing malicious Aspergillus flavus from now on, this bacterium is separated from China, peanut has good colonization ability, there is potential field competitive edge, produce malicious Aspergillus flavus infecting peanut to suppression, the aflatoxin contamination reduced in peanut is significant.
Accompanying drawing explanation
Fig. 1 is the colonial morphology of Aspergillus flavus NAFFHN396 on CYA substratum and conidium form.
Fig. 2 is the color at the bacterium colony back side of Aspergillus flavus NAFFHN396 on AFPA substratum.
Fig. 3 is that Aspergillus flavus NAFFHN396 does not produce the pcr amplification result of strain system calcium bar albumen with other.
Fig. 4 is the phylogenetic tree of Aspergillus flavus NAFFHN396.
Fig. 5 is that the HPLC of Toxic extraction liquid on the solid CYA substratum after Aspergillus flavus NAFFHN396 grows 7d measures collection of illustrative plates.
Fig. 6 is the amplification of aflatoxin composite variety upstream gene and partial synthesis cluster gene in Aspergillus flavus NAFFHN396.
Embodiment
In order to understand the present invention better, illustrate content of the present invention further below in conjunction with embodiment, but content of the present invention is not only confined to the following examples.
Embodiment 1: the separation of bacterial strain, screening and identification
(1) peanut kernel is utilized 70% ethanol surface sterilization 30s, utilize 1wt%NaClO to soak seed benevolence 2min subsequently, sterilized water washs 3 times, control dry peanut benevolence, is evenly arranged in Semen arachidis hypogaeae in aseptic plastic culture dish, 10/ware, space is kept between seed benevolence, not adjacent; After utilizing parafilm film to wrap in the culture dish that seed benevolence is housed, be carefully positioned over bottom and add in the box of moisture, build case lid, cultivate 7d for 30 DEG C;
(2) utilize the tweezers of sterilizing to dip the yellow-green colour conidium that a small amount of seed benevolence produces, fully suspend in containing the Tween-20 of 0.1wt% and after serial dilution, get 10
-4dilution conidial suspension coating PDA is dull and stereotyped, cultivate 2d for 30 DEG C, single bacterium colony of growth is got and is transferred to fresh CYA and AFPA solid medium center a little, substratum is nature pH, cultivate until bacterium colony covers with flat board for 30 DEG C, obtain the bacterium colony of purifying, the form of bacterium colony is: cultivate 7d on CYA solid medium after, NAFFHN396 is the bacterium colony of diameter 6.5cm, mycelia is filbert, conidium tawny, bacterium colony centre of surface young pilose antler shape, edge powdery, bacterium colony and conidial form are shown in Fig. 1; On AFPA solid medium, the bacterium colony back side is bright orange, sees Fig. 2.
(3) colony inoculation of purifying step (2) obtained is in 30mlPDA nutrient solution, after 30 DEG C of cultivation 5d, hypha body is taken out, filter paper is controlled solid carbon dioxide to divide, put into mortar, pour the abundant grind into powder of liquid nitrogen into, utilize CTAB method to extract mycelia DNA, utilize the concentration of spectrophotometric determination mycelia DNA, weaker concn is to 20ng/ μ l; With reference to the nucleotide sequence design primer (CaMF of Aspergillus flavus Calmodulin gene, 5 ' TTTTGCATCATGAGTTGGAC3 ', CaMR, 5 ' GARTWCAAGGAGGCCTTCTC3 ') pcr amplification is carried out to mycelia DNA extraction liquid, add each 1 μ l of CaMF and CaMR, the mycelia DNA extraction liquid 2 μ l of 10XTaqbuffer2 μ l, 10pmol, 1UTaqDNApolymerase1 μ l, 2.5mMMgCl in the system of 20 μ l
21 μ l, 2mmol/LdNTPs1 μ l, benefit aqua sterilisa to 20 μ l; Amplification condition is: amplification condition is: 94 DEG C of 3min sex change; 94 DEG C of 30s, 51 DEG C of 30s, 72 DEG C of 1min, 35 circulations; 72 DEG C of 7min extend.Amplified production is electrophoresis on 1% sepharose, pcr amplification the results are shown in Figure 3, Takara sepharose test kit is utilized to reclaim object fragment, be connected on carrier pMD-18T, transformed competence colibacillus cell DH5 α, select positive colony and carry out sequencing, sequencing results shows: special calcium bar protein gene sequence overall length is 750bp, BLAST analytical method is adopted sequence and GenBank database to be compared, find that the sibship of this bacterial strain and Aspergillus flavus is closest, homology is up to more than 99%.Adopt MEGA program to carry out phylogenetic evolution analysis to this bacterium, and drawing system grow tree, as shown in Figure 4.
Be Aspergillus flavus NAFFHN396 by this Strain Designation, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 28th, 2015, depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, its preserving number is CGMCCNo:10927.
(4) Aspergillus flavus NAFFHN396 is inoculated on fresh CYA solid medium, after 30 DEG C of cultivation 7d, utilize punch tool decentering 1cm, 2cm, 3cm gets mycelia block respectively, be placed in centrifuge tube, rifle head is utilized to smash mycelia block to pieces, add the methyl alcohol of 1.5ml, ultrasonication 15min, 12, the centrifugal 5min of 000rpm/min, draw supernatant, after utilizing the filter of 0.45 μm to filter, filtrate collection is measured in bottle in the tool lid of 1.8ml, the mensuration utilizing HPLC to carry out aflatoxin (utilizes the reverse post of C18, moving phase is 45% methanol/water, flow velocity is 0.7ml/min), sample size is 10 μ l, (appearance time of aflatoxin standard substance B2 is 10.8min to quantified by external standard method, the appearance time of B2 is 13.4min), the content of calculation sample aflatoxin, Fig. 5 is shown in by the HPLC result collection of illustrative plates of aflatoxin, as can be seen from Figure 5, Aspergillus flavus NAFFHN396 10.8 and 13.4min place all without peak value, show that this bacterial strain does not produce aflatoxin, the content of aflatoxin is 0ng/g.
(5) according to the special primer (respectively in table 1) of aspergillus flavus toxin composite variety and upstream and downstream thereof totally 30 gene design, the method of step (3) is utilized to carry out pcr amplification to Aspergillus flavus NAFFHN396DNA, the amplification of portion gene is shown in Fig. 6, and the result of Fig. 6 describes: the aflatoxin composite variety on this bacterium genome lacks 12 genes (5th ~ 16 genes).Exactly because the atoxigenic mechanism of Aspergillus flavus NAFFHN396 is on DNA level, aflatoxin composite variety lacks 12 genes, causes aflatoxin to synthesize and interrupts.
Table 1 specific primer sequences
| Gene | Upstream 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: the application of Aspergillus flavus NAFFHN396 in aflatoxin pollution of peanuts biological control
By atoxigenic Aspergillus flavus NAFFHN396 and produce poison Aspergillus flavus AF2202 on PDA solid medium 30 DEG C cultivate after 7d, 0.1wt%Tween-20 aqueous suspension conidium, the sporogenic quantity of blood counting chamber statistical, by not toxigenic bacterium 1*10 consistent with the concentration adjustment of toxigenic bacterium
4ratio is 1:1, the central authorities of CYA solid medium are seeded in after balanced mix, or be inoculated into the peanut kernel surface of surface sterilization, abundant shake seed benevolence, make conidium evenly be attached on the surface of seed benevolence, set up simultaneously and only inoculate the CYA solid medium of AF2202 or peanut kernel in contrast, 7d are cultivated in 30 DEG C of moisturizings.Adopt the aflatoxin on embodiment 1 step (4) described method extraction CYA solid medium, the extracting method of aflatoxin in peanut kernels is: to the peanut kernel surface sprinkling alcohol of inoculation in stink cupboard, subsequently at 110 DEG C of oven for baking 1h, Aspergillus flavus in abundant deactivation seed benevolence, in stink cupboard, utilize high speed disintegrator to grind sample subsequently, thereafter with reference to the aflatoxin (methanol aqueous solution of 50ml55% volumetric concentration in the method extraction peanut kernel of GB NY/T1286-2007, the sherwood oil of 15ml100%), and it is concentrated, reverse C18 post is separated, post-column derivation, at excitation wavelength 365nm, wavelength of fluorescence 440nm detects, quantified by external standard method, the content of calculation sample aflatoxin, obtain and do not produce malicious Aspergillus flavus NAFFHN396 to the inhibiting rate of the content of toxins of toxigenic bacterium AF2202.Aspergillus flavus NAFFHN396 is 94.1% to the inhibiting rate of toxigenic bacterium Output of toxin on CYA, and Semen arachidis hypogaeae reaches 98% to the inhibiting rate of toxigenic bacterium Output of toxin, significantly can reduce strong output of producing the aflatoxin of the Aspergillus flavus AF2202 of poison.Aspergillus flavus NAFFHN396 is that the biological control of Aspergillus flavus field will provide the source of biocontrol microorganisms from now on, this bacterium is separated from China, peanut has good colonization ability, there is potential field competitive edge, produce malicious Aspergillus flavus infection peanut to suppression, the aflatoxin contamination reduced in peanut is significant.
Obviously, above-described embodiment is only for the example done clearly is described, and the restriction not to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And therefore amplified apparent change or variation are still within the protection domain of the invention.
Claims (2)
1. the aspergillus flavus strain of aflatoxin is not produced in a strain, it is characterized in that, this Strain Designation is Aspergillus flavus NAFFHN396, and its preserving number is CGMCCNo:10927.
2. the application of Aspergillus flavus NAFFHN396 in aflatoxin pollution of peanuts biological control described in claim 1.
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| 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 |
| CN107245453A (en) * | 2017-06-02 | 2017-10-13 | 中国农业科学院农产品加工研究所 | One plant is not produced malicious aspergillus flavus and its application in terms of aflatoxin degradation |
| CN109913375A (en) * | 2019-04-03 | 2019-06-21 | 吉林大学 | A kind of Aspergillus flavus of not toxin producing, the composition containing it and its application |
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| CN110973163A (en) * | 2019-12-18 | 2020-04-10 | 吉林大学 | Liquid microbial inoculum for biologically preventing, controlling and spraying aflatoxin |
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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 |
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| CN107245453A (en) * | 2017-06-02 | 2017-10-13 | 中国农业科学院农产品加工研究所 | One plant is not produced malicious aspergillus flavus and its application in terms of aflatoxin degradation |
| 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 |
| CN109913375A (en) * | 2019-04-03 | 2019-06-21 | 吉林大学 | A kind of Aspergillus flavus of not toxin producing, the composition containing it and its application |
| CN110074140A (en) * | 2019-05-28 | 2019-08-02 | 山东省花生研究所 | A kind of biocontrol agent, preparation method and application producing malicious aspergillus flavus |
| CN110973163A (en) * | 2019-12-18 | 2020-04-10 | 吉林大学 | Liquid microbial inoculum for biologically preventing, controlling and spraying aflatoxin |
| CN113322189A (en) * | 2021-06-16 | 2021-08-31 | 中国农业科学院油料作物研究所 | Aspergillus flavus HuBXY33 without producing toxicity and application thereof |
| CN113355246A (en) * | 2021-06-16 | 2021-09-07 | 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 |
| 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 |
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