CN108587928A - Malicious Aspergillus flavus and its application are not produced in a kind of low back mutation - Google Patents
Malicious Aspergillus flavus and its application are not produced in a kind of low back mutation Download PDFInfo
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- CN108587928A CN108587928A CN201810449015.4A CN201810449015A CN108587928A CN 108587928 A CN108587928 A CN 108587928A CN 201810449015 A CN201810449015 A CN 201810449015A CN 108587928 A CN108587928 A CN 108587928A
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Abstract
The present invention relates to one kind not producing malicious Aspergillus flavus and its application, more particularly to malicious Aspergillus flavus and its application are not produced in a kind of low back mutation.Aspergillus flavus provided by the invention is specially aspergillus flavus (Aspergillusflavus).The bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 10th, 2017, and (abbreviation CGMCC, address are：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC No.14122.A kind of malicious Aspergillus flavus that do not produce of low back mutation of the present invention can be applicable in biological control, specifically can be applicable to the malicious aspergillus flavus strain growth of inhibition production.The more common probability for not producing malicious aspergillus flavus strain back mutation production poison of malicious Aspergillus flavus back mutation production poison that do not produce of the low back mutation of the present invention significantly reduces, and can apply well in the biological control of the malicious aspergillus flavus of production.
Description
Technical field
The present invention relates to one kind not producing malicious Aspergillus flavus and its application, more particularly to malicious Huang is not produced in a kind of low back mutation
Aspergillus and its application.
Background technology
Aflatoxin (Aflatoxin, AFT) is mainly by aspergillus flavus (Aspergillus flavus) and aspergillus parasiticus
(A.parasiticus) secondary metabolite generated is a kind of extremely toxic substance, aflatoxin B1 toxicity maximum and generation
Most one of strong carcinogen in boundary.Aspergillus flavus is widely present on soil, plant and fruit, and vitality is indomitable, and peanut is most to hold
One of the crops of easy infection Aspergillus flavus.Aspergillus flavus pollution can cause peanut quality decline, quality safety by very big shadow
It rings.
Aspergillus flavus bacterium pollution control in field mainly peanut late growth stage and drying storage and transport process in, mainly have with
Lower method：In peanut pod development later stage, ensure that soil moisture is suitable first, avoid arid before harvesting cause kind of skin rupture and
Increase Aspergillus flavus infection chance.Secondly, avoid farming that pod is caused to damage in fruiting period and shell development phase.Third is being received
Pod is dried after obtaining in time, water content is made to be less than 5% rapidly.Select the new peanut variety etc. of tool resistance.But due to aspergillus flavus
The viability of bacterium is strong, and the spore of generation can resist a variety of severe natural conditions, cannot thoroughly avoid the infection of Aspergillus flavus.
At present both at home and abroad about the report for not producing malicious Aspergillus flavus is detached from soil, the U.S., which has been approved by, not to produce
The Aspergillus flavus of poison is used for the biological control of aflatoxin.Studies in China is only limitted to inhibit the malicious Aspergillus flavus life of production in laboratory
Long report has no and studies the molecule mechanism of not toxigenic bacterium, does not also carry out related field experiment research and practical application
Effect is reported.Since mould breeds in the natural environment, the ratio of mutation is high, some atoxigenic Aspergillus flavus may send out
Raw back mutation, production is malicious again.Therefore, it is necessary to select to cultivate it is primary do not secrete the Aspergillus flavus of aflatoxin, and grind
Study carefully its mutation mechanism, the bacterial strain of low back mutation is selected to be used for the biological control of aflatoxin.
Invention content
To solve the shortcomings of the prior art, the present invention provide a kind of low back mutation do not produce malicious aspergillus flavus strain and
It is applied.
Aspergillus flavus provided by the invention is specially aspergillus flavus (Aspergillus flavus).The bacterial strain is in 2017
Being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 10, (abbreviation CGMCC, address are：Beijing
The institute 3 of city Chaoyang District North Star West Road 1, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC No.14122.
Application of the malicious Aspergillus flavus in biological control is not produced in a kind of low back mutation of the present invention.
A kind of malicious Aspergillus flavus that do not produce of low back mutation of the present invention is inhibiting to produce answering during malicious aspergillus flavus strain is grown
With.
Specifically, a kind of malicious Aspergillus flavus that do not produce of low back mutation of the present invention is inhibiting to produce malicious aspergillus flavus strain growth
The application of microbial inoculum.
More specifically, the microbial inoculum is set as the aspergillus spore suspension of low back mutation.
What the present invention was reached has the beneficial effect that：
The present invention's does not produce malicious Aspergillus flavus in addition to having 27 point mutation sites, and there is also 3 deletion mutations.Due to yellow bent
The mutation rate of mould is high, therefore the mutation of general point there is a possibility that be mutated again, becomes back mutation and replys production again
Poison, and the possibility that deletion mutation generates back mutation is relatively low, it is malicious yellow that aspergillus flavus of the invention belongs to not producing for low back mutation
Aspergillus.
Malicious Aspergillus flavus is not produced in the low back mutation of the present invention, which or not to be unfolded to be colonized in the soil with the Aspergillus flavus of production poison
Competition, and not produce malicious Aspergillus flavus adaptable in the soil for low back mutation provided by the invention, has preferable raw
It deposits and fertility, infection of the Aspergillus flavus of production poison to crops and storage crops can be mitigated, significantly reduce crops
The content of aflatoxin, has the biological pesticide for developing into control aflatoxin contamination in product and storage crops
Great potential ensures that the health of consumers in general has great importance for ensureing the safety of susceptible agricultural product.
Description of the drawings
Fig. 1 is the Aspergillus flavus bacterium colony being separately cultured in soil.
Fig. 2 is the Fluorescence Identification observation chart of aspergillus flavus bacterium colony, wherein：(a) malicious aspergillus flavus bacterium colony fluoroscopic examination figure is not produced；
(b) malicious aspergillus flavus bacterium colony fluoroscopic examination figure is produced.
Fig. 3 is M1, M2 and D1 culture bacterial strain clone's number and generation spore quantity comparison diagram.
Fig. 4 is the rice aflatoxin content HPLC detection curve figures for not producing malicious Aspergillus flavus.
Fig. 5 is the aflatoxin content HPLC detection curve figures of the malicious Aspergillus flavus rice of production.
Fig. 6 is M1, M2 and D1 aspergillus flavus AflR gene promoter DNA cloning result electrophoresis patterns, wherein：M is molecular weight
Label.
Fig. 7 is that the gene promoter areas Aspergillus flavus AflR M1 and the alignment of D1 promoter regions scheme.
Fig. 8 is that the gene promoter areas Aspergillus flavus AflR M2 and the alignment of D1 promoter regions scheme.
Fig. 9 is to use and be not used M1 spore peanut aflatoxin changes of contents figures.
Specific implementation mode
For ease of it will be understood by those skilled in the art that the present invention, With reference to embodiment to the present invention into advance one
The explanation of step, but be not construed as limiting the invention.
Test material
Rice is purchased from common supermarket of Qingdao City；
PDA solid mediums and LB agar mediums；
PDA solid mediums：Potato 200g, glucose 20g, 15~20g of agar, tap water 1000ml；
LB agar mediums：Tryptone 10g, yeast extract 5g, NaCl10g, pH 7.0,20g/L agar；
Aflatoxin ELISA kit:The Nanjing bio tech ltd Sen Beijia；
Taq archaeal dna polymerases：Beijing bio tech ltd Kang Run Cheng Ye；
Agar：The Shanghai bio tech ltd Suo Laibao；
Primer sequence synthesizes and PCR product purification kit：Sangon Biotech (Shanghai) Co., Ltd.；
Microplate reader：Supreme Being agrees Tecan 50；
High performance liquid chromatography：Agilent Agilent-1200；
Divide spore machine:The still Electronic Science and Technology Co., Ltd. BFQ-100 of Nanjing thousand；
Bacterium source：It detaches and obtains in the soil of Qingdao of Shandong province Laixi City peanut producing region.
Embodiment 1
1, strain separating is screened
0-20cm soil samples are taken after harvesting peanut, add sterile water fully shaking after ten minutes, and the centrifugation of 2000r room temperatures takes
Clear 20ul is coated on PDA culture medium.30 DEG C of constant temperature incubations, choose that colony diameter 3-7cm, initially then band yellow becomes after 10-14h
For yellow green, it is old after colour-darkening, it is flat or have radial rill, reverse side it is colourless or with brown, separate good Aspergillus flavus
It falls, is placed in 1.5ml centrifuge tubes, label is spare.In this screening, primary dcreening operation bacterium colony figure obtains altogether as shown in Figure 1, in this screening
1200 plants of aspergillus flavus strain.
2, fluorescence secondary screening and identification
The Aspergillus flavus spore tentatively chosen is crossed in PDA culture medium and is cultivated, after 30 DEG C of culture 14d, in darkroom
Bacterium colony is observed under 365nm ultraviolet lamps, because aflatoxin fluoresces in the UV lamp, as shown in Fig. 2, can be used for distinguishing
Production poison and atoxigenic Aspergillus flavus.2 plants are obtained after this secondary screening and does not produce malicious aspergillus flavus strain, are respectively labeled as not producing malicious yellow bent
Trichoderma strain M1 (hereinafter referred to as M1) and malicious aspergillus flavus strain M2 (hereinafter referred to as M2) is not produced, 1 is screened from the malicious aspergillus flavus strain of production
A production poison aspergillus flavus strain D1 (hereinafter referred to as D1).
Embodiment 2
The strain culturing of M1, M2 and D1 and production poison identification
The culture of M1 bacterial strains：It takes 250g rice to be placed in stainless steel disc, adds 100ml water, with aluminium-foil paper seal disc mouth, 120
DEG C moist heat sterilization 15min, is cooled to room temperature.The spore for taking aspergillus flavus strain takes 100ul uniformly to apply with the sterile aqueous suspensions of 200ul
It is distributed in LB culture mediums, 37 DEG C of culture 3-4d wait for that bacterium colony becomes bottle green, after generating apparent conidium, are scraped with steril cell
Knife scrapes the conidium of Aspergillus flavus from the surface of culture medium, is resuspended in 5ml sterile waters, fully shaking mixing, sterile
Under the conditions of be uniformly added in the rice solid medium sterilized, with 4 layers sterilize sterile gauze cover stainless steel discs, 30 DEG C
13-14d is cultivated in incubator.
It collects and covers with the rice of Aspergillus flavus, a small amount of growth is taken to have the rice of Aspergillus flavus, after weighing, add 10 times of volumes
Sterile water, fully homogenate carries out gradient dilution to 1 to without apparent granular substance to suspension in homogenizer：105, take 20ul
Aspergillus flavus suspension after dilution is spread evenly across in PDA culture medium, and 30 DEG C are inverted culture 7-10d, carry out bacterium colony counting.
The bacterial strain of same method culture M2 and D1.
It takes M1, M2 and D1 of identical weight to carry out bacterium colony culture and counts, remaining rice is placed in a point sample introduction for spore machine
Mouthful, Aspergillus flavus spore is collected, spore total amount is obtained.
As shown in Figure 3, after culture the bacterial strain of M1, M2 and D1 clone it is several between without significant difference, illustrate production poison and do not produce poison
For the growth rate and proliferative speed of aspergillus flavus strain without significant difference, significant difference is also not present in sporulation quantity.
The rice sample of culture aspergillus flavus strain is taken, is fully ground in mortar after dry, adds the methanol of 10ml：Water (1：
1) aflatoxin is extracted, the aflatoxin content in rice sample is detected using high performance liquid chromatography.Take M1, M2 and D1
It carries out aflatoxin content and measures i.e. production poison identification, obtain shown in result table 1 and Fig. 4 Fig. 5：
Table 1 produces the testing result of each aflatoxin of malicious Aspergillus flavus D1
By table 1 and Fig. 4, Fig. 5 as it can be seen that the various aflatoxin contents detection in M1 and M2 samples is zero, it was demonstrated that M1
Aflatoxin is not generated in growth course with M2, and D1 can generate a large amount of AFB1 and AFG1 in growth course, contain
Amount is respectively 13.8881ng/ul and 8.6018ng/ul.
Embodiment 3
M1, M2 and D1 bacterial strain DNA sequence analysis
M1 bacterial strain DNA sequence analysis：The Aspergillus flavus spore detached on a small quantity is taken to be placed in ceramic mortar, liquid nitrogen grinding is destroyed
1mlTE- glucose solutions are added in epispore, fully wash mortar, and recycling solution is placed in centrifuge tube, and 1ml NaOH/ are added
SDS solution, mixing, ice bath 10min add 1ml renaturation solution, 4 DEG C of centrifugation 10min of 12000r to take 500ul supernatants, the bodies such as addition
Long-pending phenol/chloroform (24:1), mixing, 4 DEG C of 12000r centrifuge 5min, carefully draw supernatant, add the absolute ethyl alcohol of 2 times of volumes, ice
Upper precipitation 10min, 12000r room temperatures centrifuge 10min, abandon supernatant, and precipitation is washed with the ethyl alcohol of 500ul75%, drying at room temperature 5mi
Afterwards, with the sterile water dissolution Aspergillus flavus genomic DNAs of 100ul.
It is the promoter DNA sequence that template uses PCR amplification AflR genes using Aspergillus flavus DNA, the primer sequence is：
F:5'-CTCATGCAGGTGCTAAAGA-3'；
R:5'-GCACAACTCGTACAGCTAT-3'；
PCR amplification condition is 94 DEG C of pre-degeneration 5min, then 94 DEG C of 40S, 57 DEG C of 40S, 72 DEG C of 40S, 30 cycles.2%
Agarose gel electrophoresis detects PCR product, carries out sequencing after PCR product recycling and sequence analysis, electrophoretogram are as shown in Figure 6.
Same method carries out DNA sequence analysis to M2 and D2 bacterial strains.
PCR and the sequencing results are carried out as shown in Fig. 6, Fig. 7, Fig. 8 to M1, M2 and D1, the AflR gene promoters with D1
Sub-district alignment, M2 is with the presence of 15 site point mutation, and M1 is in addition to having 27 point mutation sites, there is also 3 deletion mutations,
The DNA sequence dna of missing 46,4 and 3 bit bases respectively.Since the mutation rate of Aspergillus flavus is high, the mutation of general point exists
The possibility being mutated again becomes back mutation and restores production poison again, and deletion mutation generate the possibility of back mutation compared with
It is low, therefore selected biocontrol bacterial strain of the M1 bacterial strains with 27 points and 3 deletion mutations as aflatoxin, and by the bacterium
Strain preservation, deposit number are CGMCC NO.14122, and the deposit date is on May 10th, 2017, preservation Classification And Nomenclature was aspergillus flavus
(Aspergillus flavus), depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center.
Embodiment 4
Prevention practices
The peanut of normal field planting, waits for peanut Post flowering, and before gynophore is buried, separation acquisition is applied around every plant of peanut
M1 spore suspension 5ml, control group only applies sterile water 5ml, normal fertilising and watering.Harvest peanut after, take respectively using and
The peanut sample for not applying M1 spore suspensions, methanol is used after crushing：Aqueous solution (1：1) aflatoxin is extracted, using ELISA's
Method measures the content of the aflatoxin in peanut.The peanut of harvest extracts aflatoxin after room temperature preservation half a year,
ELISA method measures the content of aflatoxin.The results are shown in Figure 9.As seen from Figure 9, after using the harvesting peanut of M1 spores
Aflatoxin is lower than the content of the Aflatoxin in Peanut byHigh of unused M1 spores.After storing half a year, M1 Aspergillus flavus is used
The content of the peanut aflatoxin of spore increases to 9Ppb, and content is relatively low；And aspergillus flavus after not used peanut storage half a year
The content of toxin has reached 34Ppb, has been over limitation.Prove that M1 Aspergillus flavus can be used for the aflatoxin dirt of peanut
The biological prevention and control of dye.
The embodiments of the present invention described above are not intended to limit the scope of the present invention.It is any in the present invention
Spirit and principle within made by modifications, equivalent substitutions and improvements etc., should be included in the claim protection model of the present invention
Within enclosing.
Claims (5)
1. malicious Aspergillus flavus is not produced in a kind of low back mutation, it is characterised in that：Preservation Classification And Nomenclature is aspergillus flavus
(Aspergillus flavus), deposit number are CGMCC NO.14122.
2. application of the malicious Aspergillus flavus in biological control is not produced in a kind of low back mutation according to claim 1.
3. applying according to claim 2, the growth for inhibiting the malicious aspergillus flavus strain of production specifically can be applicable to.
4. a kind of malicious Aspergillus flavus that do not produce of low back mutation is inhibiting to produce malicious aspergillus flavus strain growth according to claim 1
The application of microbial inoculum.
5. application according to claim 4, it is characterised in that：The microbial inoculum is set as the aspergillus spore of low back mutation
Suspension.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112609021A (en) * | 2021-01-13 | 2021-04-06 | 中国农业科学院农产品加工研究所 | Aspergillus flavus RPA primer, kit, and aspergillus flavus detection method and device |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009032696A2 (en) * | 2007-08-31 | 2009-03-12 | The United States Of America, As Represented By The Secretary Of Agriculture | A water dispersible formulation for delivery of biocontrol fungi to reduce aflatoxin |
| US20120183507A1 (en) * | 2011-01-19 | 2012-07-19 | Dorner Joe W | Non-Toxigenic Strains of Aspergillus Flavus for Control of Aflatoxin Contamination in Crops |
| CN103509723A (en) * | 2013-09-25 | 2014-01-15 | 中国农业科学院农产品加工研究所 | Aspergillus flavus producing no aflatoxin and application thereof |
| CN107142217A (en) * | 2016-12-06 | 2017-09-08 | 青岛至成生物技术有限公司 | A kind of biological pesticide for being used to reduce crops aflatoxin content |
-
2018
- 2018-05-11 CN CN201810449015.4A patent/CN108587928A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009032696A2 (en) * | 2007-08-31 | 2009-03-12 | The United States Of America, As Represented By The Secretary Of Agriculture | A water dispersible formulation for delivery of biocontrol fungi to reduce aflatoxin |
| US20120183507A1 (en) * | 2011-01-19 | 2012-07-19 | Dorner Joe W | Non-Toxigenic Strains of Aspergillus Flavus for Control of Aflatoxin Contamination in Crops |
| CN103509723A (en) * | 2013-09-25 | 2014-01-15 | 中国农业科学院农产品加工研究所 | Aspergillus flavus producing no aflatoxin and application thereof |
| CN107142217A (en) * | 2016-12-06 | 2017-09-08 | 青岛至成生物技术有限公司 | A kind of biological pesticide for being used to reduce crops aflatoxin content |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112609021A (en) * | 2021-01-13 | 2021-04-06 | 中国农业科学院农产品加工研究所 | Aspergillus flavus RPA primer, kit, and aspergillus flavus detection method and device |
| CN112609021B (en) * | 2021-01-13 | 2023-01-17 | 中国农业科学院农产品加工研究所 | Aspergillus flavus RPA primer, kit, and aspergillus flavus detection method and device |
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Application publication date: 20180928 |