CN105602855B - A kind of aspergillus sydowii with broad-spectrum pesticide degradation characteristic - Google Patents
A kind of aspergillus sydowii with broad-spectrum pesticide degradation characteristic Download PDFInfo
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Abstract
It is categorized to be named as aspergillus sydowii the invention discloses one plant of fungi PA F-2 with broad-spectrum pesticide degradation characteristic（Aspergillus sydowii）, bacterial strain is preserved in Wuhan University's China typical culture collection center, address by September 7th, 2015：The Chinese Wuhan Wuhan Universitys, preserving number are：CCTCC NO.M2015513.The aspergillus sydowii PA F-2 of what the present invention filtered out can degrade 13 kinds of pesticides administer in the water body environment of pollution by pesticides, have very high application value in the comprehensive treatment of tackling and the pollution of current industrial place composite pesticide.
Description
Technical field
The invention belongs to field of environmental biotechnology, and in particular to one plant of fungi with broad-spectrum pesticide degradation characteristic is poly- more
Aspergillus PA F-2.
Background technology
Pesticide is widely used, and while bringing crop yield rapidly and efficiently to agricultural production, also gives water body, soil
Serious pollution is caused with atmospheric environment, also adds the product safety problem of agricultural product.Currently used pesticide reparation side
Method is that stove burns, the methods of thermal desorption and chemical treatment are repaired difficult but there are costly, be easy to cause secondary pollution etc.
Situation.Using can be using organic agricultural chemicals as energy substance in water body and soil, and it be degraded to nontoxic or less toxic chemical combination
The biological restoration that the biology (predominantly microorganism) of object is established, is acknowledged as a kind of efficient, safe and pollution-free and cheap
Repair mode, meanwhile, because rich and varied, widely distributed, adaptable, the easy domestication of the type of microorganism in the environment is easily prominent
The features such as change, has preferable advantage in current pesticide is biological prosthetic.
It is usually present compound pollution by pesticides, and pesticide variety diversification in China current industrial place, and current research
Obtained highly effective pesticide degrading microorganism is mostly specificity, i.e., only to a kind of pesticide or a kind of pesticide (such as organic phosphates or
Person's pyrethrin pesticide) degradation occurs, there are apparent defects in the industrial sites soil remediation of composite pesticide pollution.Together
When, the microorganism only with bioregulator degradation is in composite pesticide contaminated site, due to other a variety of toxic organic agricultures
Medicine lacks tolerance, thus cannot achieve the efficient degradation to pesticide.Meanwhile organic agricultural chemicals has enantiomer more, these have
The organic matter of chiral enantiomer structure usually will produce selective environmental behaviour in the environment, resolution in the environment and home to return to meeting
Very big difference is generated, and bioactivity also tends to have differences.Microorganism with broad-spectrum pesticide degradation characteristic, both can be right
Multiple Pesticides keep tolerance, moreover it is possible to while using Multiple Pesticides as energy substance, to Multiple Pesticides progress degradation, however this
The report of aspect is seldom.
Invention content
The first purpose of the invention is to provide one kind begging the new plant area periphery of agrochemicals Co., Ltd from Hubei Province Qi Zhouzhen
It screens and obtains in plant rhizosphere, Non-rhizosphere soil sample and water sample, to a variety of organic phosphates, organochlorine class, pyrethroids class and benzene oxygen class etc.
Pesticide has the fungi of height endurability and efficient degradation：Aspergillus sydowii (Aspergillus sydowii) PA F-2, the bacterium in
September is preserved in China typical culture collection center, address on 7th within 2015：The Chinese Wuhan Wuhan Universitys, preserving number are
CCTCC NO.M2015513。
The biological property of the bacterial strain mainly has：Growth cycle of the bacterium in liquid and solid fermentation culture medium is longer.
Liquid fermentation medium shaking table shaken cultivation reaches exponential phase in 48 hours, culture reaches stationary phase in 5 days, and zymotic fluid is clear
Clearly, hypha,hyphae forms green or bottle green is spherical；On solid PDA medium, the bacterium bacterium colony size is irregular, and edge is white
Color villiform, the aobvious green in ripe bacterium colony center, aerial hyphae and Ji Sheng mycelia are flourishing, easy picking.Bacterium colony is irregular cycle, table
Face is coarse, flat protuberance, matt；Semi-moist is easily provoked；Opaque white color, edge are green annular；Bacterium colony is larger, edge
It is irregular.It observes under an optical microscope, mycelia is in more carefully netted, and diameter is about 0.15 μm；Spore color is brown, and shape is
Circle, diameter are about 0.10 μm；Sporophore is elongated, and uniform diameter, branch is apparent, forked acute angle branch, and branch is not symmetrical.
Genomic DNA is extracted from the pure culture of bacterial strain aspergillus sydowii PA F-2, with the Internal Transcribed Spacers rDNA
(ITS) the specific primer I TS1/ITS4 of sequence obtains the amplified production of 543bp length, amplified production is through surveying by PCR amplification
Sequence company carries out sequencing, and sequence is as shown in SEQ ID NO.1.Sequence in sequence and GenBank is subjected to BLAST ratios
It is right, the results showed that the bacterial strain and aspergillus sydowii (Aspergillus sydowii) homology are very high, and similarity reaches
99.6%.It is determined as aspergillus sydowii in combination with Physiology and biochemistry test and Observation On The Morphology interpretation of result, the bacterial strain
(Aspergillus sydowii)。
The broad-spectrum pesticide degradation characteristic of the bacterial strain：In solid plate test experience, bacterial strain PA F-2 can be with 13 kinds
Current common pesticide and intermediate synthetic, organophosphor：Metrifonate, glyphosate, DDVP；Organochlorine：Paraquat；Pyrethroids class：
Effective cypermethrin, decis, Fenpropathrin, Biphenthrin；Amides：Acetochlor；Fragrant phenoxy carboxylic acid esters：Smart quinoline standing grain
Spirit；Benzene oxygen class：2,4-D, phenoxy benzoic acid；Phenyl ring class：O-chlorphenol；As solid medium sole carbon source, only nitrogen source
Or phosphorus source (containing the pesticide of nitrogen as only nitrogen source, contain the pesticide of P elements as unique phosphorus source) and sole carbon
Normal growth on the culture medium of nitrogen source or carbon phosphorus source, and biomass is high, growth rate is very fast.
In liquid shaking bottle experiment, the bacterial strain is with benzene oxygen class pesticide 2,4-D, organophosphorus insecticide metrifonate, pyrethroids class
Pesticide effective cypermethrin is in the culture medium of only carbon source, and growth rate is very fast, and degradation rate is high.It is complete in 48 hours cultivating
Degradable 2,4-D cultivates degradable effective cypermethrin in 5 days, is more than 80% to the degradation rate of metrifonate in 14 days.
Therefore, second object of the present invention is to provide applications of the aspergillus sydowii PA F-2 in terms of degrading pesticide, this 13
Kind pesticide is organophosphor：Metrifonate, glyphosate, DDVP；Organochlorine：Paraquat；Pyrethroids class：Effective cypermethrin, bromine cyanogen chrysanthemum
Ester, Fenpropathrin, Biphenthrin；Amides：Acetochlor；Fragrant phenoxy carboxylic acid esters：Quizalotop-ethyl；Benzene oxygen class：2,4-D, benzene
P-methoxybenzoic acid；Phenyl ring class：O-chlorphenol.
Due to considerably less in the research of broad-spectrum pesticide degrading microorganism both at home and abroad at present, what the present invention filtered out can drop
Solve water body environment improvement, tackling and current industrial field of the aspergillus sydowii PA F-2 in pollution by pesticides of 13 kinds of pesticides
There is very high application value in the comprehensive treatment of ground composite pesticide pollution.
Description of the drawings
Fig. 1 is aspergillus sydowii PA F-2 spores under an optical microscope and sporophore figure；
Fig. 2 is aspergillus sydowii PA F-2 locations drawing in the Internal Transcribed Spacers rDNA (ITS) unrooted phylogenetic tree；
Fig. 3 is aspergillus sydowii PA F-2 with organo-chlorine pesticide paraquat, organophosphorus pesticide metrifonate and DDVP and chrysanthemum
Ester pesticides effective cypermethrin is the growing state figure on the solid plate of energy substance；
Fig. 4 is in response to metrifonate of the trend surface analysis method analysis aspergillus sydowii PA F-2 under different temperatures and pH value condition
Degradation rate figure.
For the biological characteristics explanation of preservation fungi：
The fungi is preserved in China typical culture collection center, and preserving number is CCTCC NO.M2015513.Address：In
The Wuhan state Wuhan Universitys, Classification system：Aspergillus sydowii, Chinese are：Aspergillus sydowii, the bacterium is in 2015
On September preservation in 7,.
Specific implementation mode
By following detailed description combination attached drawing it will be further appreciated that the features and advantages of the invention.The implementation provided
Example is only the explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.
【Embodiment 1】The screening of broad-spectrum pesticide degradation bacteria
Experiment soil sample and water sample pick up from and beg the new plant area periphery plant rhizosphere of agrochemicals Co., Ltd in Hubei Province Qi Zhouzhen
With Non-rhizosphere soil sample, water sample also picks up from 5 discharge outlet in the plant area, after soil sample and water sampling, return laboratory be immediately placed in-
20 DEG C of preservations, to kill insect in soil and plant tissue and some viruses.It is more in 4 DEG C of defrostings after processing 10 hours
Remaining soil sample is placed in 4 DEG C of refrigerators and preserves.10g soil samples or 10mL water samples are taken, the liquid enriched medium of 100mL sterilizings is added
(containing sterile glass beads) (enrichment culture based component：Peptone 10g/L, NaCl1.0g/L, KH2PO31.0g/L, pH=7),
150rpm vibrates 7 days.After cultivating 1 week, the culture solution 10mL in enriched medium is taken, is separately added into containing 50mg/L metrifonate
(TR), in the enriched medium of malathion (MA), paraquat (PA), lindane (Lin) and effective cypermethrin (β-CP), then at
It is cultivated 1 week under mutually synthermal and oscillating condition.Mode successively is tamed according to concentration 50,100,200,400 and 500mg/L, until most
After high concentration, according to dilution spread flat band method, 1mL supernatants is taken to be diluted to 10-5, take wherein 10-3,10-4,10-53 dilution ladders
200 μ L of solution under degree, respectively using 5 kinds of pesticides as MSM (Minimal Salt Media) basal medium of sole carbon source
(medium component：NH4N031.0g/L, MgS04·7H200.5g/L, (NH4)2S040.5g/L, KH2P040.5g/L,
NaCI0.5g/L, K2HP041.5g/L, pH=7) it is coated on culture medium, each processing repeats 3 tablets, then by tablet
28 DEG C of insulating box cultures are placed in, the bacterial strain quantity on each tablet is recorded after 5 days, filter out micro- life with efficient degradation characteristic
Object.It crosses on solid MSM culture mediums isolated purebred special degradation bacteria.
For growing state of the degradation bacteria filtered out in taking pesticide as the MSM culture mediums of only carbon source, then connect
Kind is in 13 kinds of common pesticide (organophosphors：Metrifonate, glyphosate, DDVP；Organochlorine：Paraquat；Pyrethroids class：Efficient chlorine cyanogen
Pyrethroids, decis, Fenpropathrin, Biphenthrin；Amides：Acetochlor；Fragrant phenoxy carboxylic acid esters：Quizalotop-ethyl；Benzene oxygen
Class：2,4-D, phenoxy benzoic acid；Phenyl ring class：O-chlorphenol) be carbon source, nitrogen source or phosphorus source solid MSM tablets (500mg/
L), cultivate 5 days for 28 DEG C.
Table 1:Growing state of the broad-spectrum pesticide degradative fungi on solid plate
Note："+" can grow apparent bacterium colony on tablet after indicating culture 5 days, " (+) " indicates bacterium colony growth but biology
It measures relatively low
Can be seen that according to fig. 3 with 1 growing state of table PA F-2 can selected test 13 kinds of common pesticides and in
Between synthetic tablet on normal growth, and with different pesticides be only carbon source, nitrogen source phosphorus source and carbon nitrogen phosphorus source condition
Under, which still has very high degradation capability, and biomass under the conditions of different disposal is also very high, the result
Namely confirm efficient degradation ability of the bacterial strain to Multiple Pesticides.
【Embodiment 2】The identification of bacterial strain
By the inoculation of purifying in solid PDA medium (ingredient：Potato 200g/L, glucose 20g/L, agar 20g/
L, pH=7) colony morphology characteristic of the observation bacterial strain on solid plate after culture 5 days, then a little bacterium colony of picking is aobvious in optics
The microstructure of micro- microscopic observation bacterial strain, and record the morphological feature of bacterial strain：Mycelia is in more carefully netted, and diameter is about 0.15 μm；
Spore color is brown, and shape is circle, and diameter is about 0.10 μm；Sporophore is elongated, and uniform diameter, branch is apparent, forked sharp
Angle branch, branch be not symmetrical.
The physicochemical property identification of bacterial strain is predominantly detected utilization of carbon source and nitrogen source and is tested using Physiology and biochemistry.Pass through charting
As a result, the carbon source and nitrogen source utilization ratio of bacterial strain are high, there is H2S generations and Starch Hydrolysis ability etc..
Table 2：The carbon source and nitrogen source of fungi PA F-2 utilizes experiment
Note："+" indicates test positive result after cultivating 5 days
Fungi ITS rDNA sequencings include that bacterial strain DNA is extracted, ITS rDNA amplification in vitros (use ITS universal primers
ITS1 and ITS4) and gene sequencing.It is sequenced to obtain sequence results by genome company, sequence is passed through as shown in SEQ ID NO.1
BLAST gene order comparisons are carried out in software analysis, the database for merging and bringing NCBI into, as shown in Figure 2.
【Embodiment 3】Bacterial strain analyzes the degradation rate of metrifonate, 2,4-D and effective cypermethrin
Using liquid MSM culture mediums, the final concentration that is added after three kinds of pesticides is set as 100mg/L, before measurement, by this bacterial strain
It is inoculated in PDA slant mediums and cultivates, then 28 DEG C of activation cultures 72 hours take 1mL sterile waters, be added in inclined-plane, will be oblique
The spore of face fungi and mycelia, which are scraped, forms spore suspension in sterile water, with 3% volume ratio (initial inoculation spore content
109A/mL, Sheng Wuliang <0.1g) it is added in culture medium.By all cultures based on the 28 DEG C of shaking table oscillations of shaking table 150rpm rotating speeds
It cultivates 5 days (metrifonate experimental group need to cultivate 14 days), measures PA F-2 bacterial strain dry weights in every group, use efficient liquid phase chromatographic analysis
Method (HPLC) measures degradation rate of the bacterial strain to metrifonate, 2,4-D and effective cypermethrin pesticide.
Table 3：The degradation rate and dry weight of three kinds of typical pesticides of fungi PA F-2 couple
It observes to obtain by list data and experimental phenomena, the degradation rate of PA F-2 couple 2,4-D are very high, and culture 48 is small
The biomass of Shi Hou, fungi have reached peak, and 2,4-D is degradable by this bacterial strain in culture medium；To effective cypermethrin
Secondly, after culture 5 days, culture medium clarification, biomass reaches highest to degradation rate, and pesticide therein has also been completely degraded；
Since the toxicity of metrifonate pesticide is higher, and more toxic DDVP is easily converted under aqueous conditions, therefore for
The growth of this bacterial strain and degradation generate certain influence, but after culture 14 days, PA F-2 to the degradation rate of metrifonate still
It has been more than 80%, and biomass also has increase compared to inoculation primary quantity.
【Embodiment 4】Bacterial strain analyzes the most suitable degradation condition of metrifonate
Select temperature, metrifonate concentration and pH value as 3 variables of research, temperature gradient selects 26,28 and 30 DEG C, enemy
A concentration of 50,100 and 200mg/L of hundred worms, pH value are set as 6.0,7.0 and 8.0, use the Box- of design expert softwares
Behnken methods analyze influence of the 17 kinds of various combinations of 3 variables to PA F-2 degradation metrifonate efficiency.
By culture in 1 week, PA F-2 were measured in 17 groups of experimental groups respectively to the degradation rate of metrifonate.It is tied according to measuring
Fruit, highest metrifonate degradation rate be 75.3%, the condition of appearing in be set as 28 DEG C, 50mg/L metrifonate concentration, pH value 6.0
Experimental group in, as shown in Figure 3.Variance is obtained according to encoding factors：
Y=67.74+3.27X1+0.27X2-13.05X3+5.48X2X3-10.27X1 2-13.53X3 2
Y indicates degradation rate (%), X1Indicate temperature, X2Indicate metrifonate concentration, X3Indicate pH value.
SEQUENCE LISTING
<110>Wuhan University
<120>A kind of aspergillus sydowii with broad-spectrum pesticide degradation characteristic
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 543
<212> DNA
<213>Aspergillus sydowii（Aspergillus sydowii）PA F-2
<400> 1
ggtttccgca gtacgggctg tctccgggcg ccacctccca cccgtgtata cctaacactg 60
ttgcttcggc ggggaacccc ctcgggggcg agccgccggg gactactgaa cttcatgcct 120
gagagtgatg cagtctgagt ctgaatataa aatcagtcaa aactttcaac aatggatctc 180
ttggttccgg catcgatgaa gaacgcagcg aactgcgata agtaatgtga attgcagaat 240
tcagtgaatc atcgagtctt tgaacgcaca ttgcgccccc tggcattccg gggggcatgc 300
ctgtccgagc gtcattgctg cccatcaagc ccggcttgtg tgttgggtcg tcgtcccccc 360
cgggggacgg gcccgaaagg cagcggcggc accgtgtccg gtcctcgagc gtatggggct 420
ttgtcacccg ctcgactagg gccggccggg cgccagccga cgtctccaac catttttctt 480
caggttgacc tcggatcagg tagggatacc cgctgaactt aagcatatca ataagcggag 540
gaa 543
Claims (2)
1. a kind of fungi, it is characterised in that：The fungi is the aspergillus sydowii that preserving number is CCTCC NO.M2015513
（Aspergillus sydowii）PA F-2 bacterial strains.
2. application of the fungi described in claim 1 in terms of degrading pesticide, it is characterised in that：The pesticide refers to enemy hundred
Worm；Effective cypermethrin；2,4-D.
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| CN107164234B (en) * | 2017-05-19 | 2020-03-17 | 福建农林大学 | Fungus with strong weed suppression effect screened from passion fruit rhizosphere soil |
| CN107090412B (en) * | 2017-05-23 | 2019-10-18 | 北京市农林科学院 | It is a kind of degrade Biphenthrin aspergillus fumigatus and its application |
| CN108060106A (en) * | 2018-02-02 | 2018-05-22 | 北京科技大学 | A kind of separating screening method of metrifonate degradation bacteria strains |
| CN108384732B (en) * | 2018-02-09 | 2021-03-02 | 上海海洋大学 | Rhodobacter sphaeroides for reducing trichlorphon toxicity and application thereof |
| CN108485989B (en) * | 2018-03-26 | 2020-02-18 | 江南大学 | Method for repairing cadmium-polluted soil by using aspergillus polymyxa and black nightshade |
| CN108265010B (en) * | 2018-03-26 | 2020-03-06 | 江南大学 | Method for repairing cadmium-polluted soil by using aspergillus versicolor and Indian mustard |
| CN110438012B (en) * | 2019-08-05 | 2021-10-26 | 四川大学 | Aspergillus sakazakii H-1 for producing anthocyanin and application thereof |
| CN112680360B (en) * | 2021-01-11 | 2021-10-15 | 青海师范大学 | Aspergillus sydowii and application thereof in promoting plant growth and preventing and treating plant diseases |
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| CN102911875A (en) * | 2012-04-27 | 2013-02-06 | 河南省科学院生物研究所有限责任公司 | Preparation method for multifunctional soil remediation microbial inoculum |
| CN103333807A (en) * | 2013-07-04 | 2013-10-02 | 福州大学 | Aspergillus fumigatus strain for degrading pyrethroid pesticide |
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| CN104087517A (en) * | 2014-07-03 | 2014-10-08 | 环境保护部华南环境科学研究所 | Fungus capable of degrading phthalic acid ester and pesticide simultaneously and application thereof |
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